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ENHANCED ADRENERGIC SENSITIVITY OF MESENTERIC
VEINS COMPARED TO ARTERIES AND ITS RELATION TO
CALCIUM UTILIZATION AND HANDLING

presented by
Alexandra Hlavacova

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5/08 K./Proj/Acc&Pres/ClRC/DateDueindd

ENHANCED ADRENERGIC SENSITIVITY OF
MESENTERIC VEINS COMPARED TO ARTERIES AND
ITS RELATION TO CALCIUM UTILIZATION AND
HANDLING

BY

Alexandra Hlavacova

A DISSERTATION

Submitted to
Michigan State University
In partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY
Department of Pharmacology and Toxicology

2008

ABSTRACT

ENHANCED ADRENERGIC SENSI11VITY OF MESENTERIC VEINS
COMPARED TO ARTERIES AND ITS RELATION TO CALCIUM UTILIZATION
AND HANDLING
By
Alexandra Hlavacova

Mesenteric venous (MV) capacitance contributes to blood pressure
regulation. MV are more sensitive to the constrictor effects sympathetic nerve
stimulation and exogenous norepinephrine (NE) than mesenteric arteries (MA).
The focus of this dissertation was to clarify the mechanism responsible for
enhanced venous adrenergic reactivity.

My studies showed that there are differences in the contribution of a-
adrenergic receptor (ozAR) subtypes in NE-induced constrictions of MA and MV.
While a1ARs alone mediate NE-induced constriction of MA, GzAR and a1AR
functionally interact to potentiate NE-induced constrictions in MV. Previous
studies showed that a1oARs are the main functional o1AR subtype in MV. I
showed that the achR subtype is the constrictor GzAR in MV.

The mechanisms responsible for the interaction between am- and
achR were investigated using heterologous receptor expression techniques and
HEK-293 cells. I found that GzARS do not co-localize with the amAR nor do
quRs facilitate trafficking of a1 DARs to the plasma membrane. A positive control
showed that BzAR increased amAR trafficking to the membrane. Calcium

imaging experiments were performed to test, whether functional interaction

between am- and 02AR occurs in HEK-293 cells. While [32AR increased calcium

responses to NE in quR — expressing HEK-293 cells, azARs did not increase
calcium signals caused by amAR activation, suggesting that the functional
interaction between G1DAR and azAR is specific for veins. Furthermore,
immunocytochemical analysis on arterial and venous. myocytes revealed that MV
and MA express azARs. These results suggest that factors other than differential
receptor expression are required for the a1AR-02AR interaction.

Receptor interaction was investigated in pressurized MV in vitro by
measuring calcium signaling. NE responses were inhibited by the calcium
channel blockers, nifedipine and gadolinium. In the presence nifedipine of
gadolinium the ozAR antagonist yohimbine did not inhibit NE-induced
constrictions, suggesting that OzAR in MV couples to calcium influx. My data also
indicate that MV have less efficient intracellular calcium buffering mechanisms
than MA. Therefore, the additional calcium influx in MV contributes to enhanced
venous reactivity to NE. Taken together, 02AR contribute to increased MV
reactivity to NE by promoting influx of extracellular calcium; this mechanism is
not present in MA. In addition, MA but not MV efficiently buffer intracellular
calcium and this further enhances venous NE reactivity. Overall, 02AR in veins
might prove to be a more selective venous target in hypertension, where
increased sympathetic nerve activity increases the tone of veins and produces
blood redistribution .from veins to arteries. In addition, less efficient calcium
buffering of veins might be very important in physiological situations such as

postural changes, where abrupt venous filling could produce frequent syncopes.

To my parents, who taught me the value of perseverance and gave me the
confidence to pursue my dreams.

ACKNOWLEDGMETS

This dissertation is the final stage of four years of intensive work.
Without a great deal of support and advice of multiple parties involved, I would
have not been able to complete it.

First of all, I would like to thank my advisor Dr. Galligan for his great
mentoring and support during this journey. While he was always there to give
advice, he helped me to become more independent in pursuing scientific
endeavors. In addition, I would like to thank the rest of my Graduate Committee:
Dr. Stephanie W. Watts, Dr. Gregory D. Fink and Dr. William S. Spielman. I very
much appreciate your time and willingness to serve on my committee, as well as
your guidance in steering my project into the right direction.

I would also like to acknowledge the help of colleagues in Dr.
Galligan’s laboratory: Hui, Stacie, Hua, Jimwoo, Dima, Xiaochun, Vino and Hui
who have not only been great colleagues, but also friends.

I would like to thank American Heart Association for awarding me a
Predoctoral fellowship that provided me with financial support during my studies
at Michigan State University.

Last, but not least, I would like to acknowledge my parents and Redo

who made all of this happen. Thank you!

TABLE OF CONTENTS

Chapter 1: Introduction ..................................... '. .................................. 1

1. Regulation of blood pressure by autonomic nervous system........
Parasympathetic control of blood pressure and vascular tone...

Sympathetic control of blood pressure and vascular tone... ..

01-th

SNS innervation of arteries and veins and functional differences...

2. Role of veins and arteries in hemodynamics ............................... 9

Arteries in hemodynamics ................................................................. 14

3. o-Adrenoreceptors (GAR) — effectors of sympathetic nervous
system (SNS)..... ................................................................................... 15

a1AR signaling and calcium coupling 16
a1AR regulatron18
physiological role ofa1AR21
02AR signaling and calcium couplin923
(12AR regulation26
physiological role ofuzAR28
aAR in the mesentericvasculature.............................................30

4. Receptor cross-talk ............................................................... 32
General concept and examples of receptor dimerization ................. 33
Receptor dimerization in cardiovascular regulation and disease ....... 36

Dimerization of oAR...... 37

vi

Dimerization — independent receptor cross-talk 41
Pathways involved in 01-02AR cross talk... 45
5. Non-adrenergic non-cholinergic regulation of vascular tone.......47

Sensory innervation of blood vessels ....47
Vasoactive agents 48
Myogenic regulation of vasculature...... 49

6. Biochemistry of the regulation of vascular tone ........................ 52

Regulation of vascular contractility... 52
Regulation of vascular tone by modifying Ca2+ sensitivity. ............... 53
Modulation of Ca2+ sensitivity by azAR... ...56
Regulation of Ca2+ desensitization... 58

Chapter 2: Hypothesis and specific aims ............................................. 59

Chapter 3: Functional Interaction Between «1- and az-Adrenoreceptors in
Mesenteric Veins Does not Require Heterodimer Formation ................... 63

lntroductron65
Materials and methods 70
Results 79
Discusswn .108

Chapter 4: Differential Calcium Utilization between Mesenteric Arteries and
Veins during Alpha-Adrenergic Stimulation ........................................ .118

Introduction.................. .121

Materials and methods .127
Results .......130

vii

 

Discussion...

Chapter 5: Differential Calcium Handling between in Murine Mesenteric
Arteries and VeIns ..............................

Introduction
Materials and methods
Results...............
DISCUSSIOI'I

Chapter 6: General discussion and conclusions ..................................

Overall relevance of studies...... ....
a1-azAR interaction in mesenteric veins......
Lack of interaction in HEK293 cells............

and veins...
Physiological implications of GzAR in mesenteric veins... ..
Differences in calcium utilization between arteries and veins .......
Overall conclusions and implications.

Proposed model of aAR signaling In MA and MV..

References ......................................................................................

viii

.145

.157

.160
.165
.170
.187

195

196

.197
.201
Differences in the expression of aAR between mesenteric arteries
.207
.209
.211
. .220
.. .224

226

Chapter 1:

Table 1.

Chapter 3:

Table 2.

Chapter 5:

Table 3.

Table 4.

LIST OF TABLES

Not all veins are equal. Counduit veins including vena cava or
femoral vein exhibit different properties in mediating responses to
sympathetic nervous system than capacitance veins. Table
depictstypical capacitance and conduit veins..............................12

Source and working dilutions of the primary and secondary
antibodies used in oAR staining in arterial and venous SMCs and in

Decay phase of NE constriction is longer and composed of more
components in MV compared to MA. Decay of constriction was fitted
by standard exponential equation and time constants of each
component were recorded173

Effect of SERCA blockade on the tone and NE-mediated responses
in MA and MV. MV responded to thapsigargin exposure with strong
constriction. NE responses after thapsigargin incubation were
reduced in MV by 61%, but almost abolished in MA (reduced by
94%). In the presence of indirect inhibition of NCX-mediated Ca2+
export by ouabain, MA responded to thapsigargin with 10%
constriction. Ouabain potentiated NE responses of MA after
thapsigargin exposure, with constriction inhibited by only 38% after
thapsigargin exposure. 181

Chapter 3:
Figure 1.

Figure 2.

Figure 3.

Figure 4.

Figure 5.

Figure 6.

Figure 7.

Figure 8.

LIST OF FIGURES

Proposed dual mechanism for vascular sensitization. Circulation
research, 2007 (100)2121-129... .......55

Mesenteric veins are more sensitive to adrenergic stimulation
then arteries. Transmural stimulation of perivascular nerves
produced greater constriction in MV than in MA (A). Similarly,
concentration-response curve for exogenously applied NE is shifted
to the left in the MV(B) 80

Addition of indomethacin or nitro-L-arginine did not alter the
responses mediated by azAR agonist UK 14,304 in MV (A).
Despite the small effect produced by azAR agonist UK14,304 in the
MV, azAR antagonist yohimbine produced a significant rightward shift
in the NE concentration-response curve (B)... ...81

Effect of agAR antagonist in pressurized vessels. ldazoxan, an
azAR antagonist produced a significant rightward shift in the NE
concentration response curve in pressurized MV (8) but not in
pressurized MA (A) ........................................................................... 83

GzAR agonist potentiates a1AR — mediated responses in MV.
While UK14,304 did not affect the maximal response to PE in MV (A),
it enhanced PE - mediated constriction at low PE concentrations.
Similar effect was observed in the pressurized MV (B) ..................... 85

Effect of subtype selective azAR antagonist on NE-induced
constriction in MV. While no significant shift of the dose-response
curve was observed with BRL44408 (02A antagonist) (A) and imiloxan
(c123 antagonist) (B). MK912 (C) (02c antagonist) produced a similar
effect on NE concentration response curve as GzAR antagonist
yohimbine (Fig. 2B)86

Sensitivity to NE is not altered in achR KO mice. NE dose
response curve of the azoAR KO mice MV does not differ from WT
(A). ’Yohimbine still produces a rightward shift of the NE
concentration response curve in the KO mesenteric veins (B) ........ 88

Effect of subtype selective OzAR antagonist on NE responses in
the MV of CchR KO mice. While imiloxan (c123 antagonist) did not
affect the concentration response curve to NE in these KO MV, the
oZAAR antagonist BRL44408 produced inhibition of NE responses,
indicating enhanced function of this receptor in the KO mice ......... 89

Figure 9.

Figure 10.

Figure 11.

Figure 12.

lmmunocytochemical analysis of azAR expression in arterial
and venous SMCs. Pan-cadherin, the marker of plasma membrane
in arterial SMC (A) and venous SMC (B). GZAAR was detected in the
membrane of arterial SMC (C), while in venous SMC OZAAR staining
was detected both in the intracellular compartment and in the
membrane (D). OzBAR localized in intracellular organelles, likely
Iysosomes in arterial SMC in addition to the membrane (E). ongR
localized only intracellularly in the venous SMC (F). ochR was
found in the membrane of arterial SMC (G) and in venous SMC,
achR was expressed in the membrane with the majority of the
receptor pool localized intracellularly (H). The magnitude of ochR
expression was larger than the expression of the other two a2AR
subtypes. The blue staining in most cells represents nucleus. White
arrow points to receptor localized to the membrane... ...91

Expression of a1AR subtypes in MA and MV SMCs. o1AAR was
detected in plasma membrane and also in the nuclear membrane of
arterial SMC (A). In venous SMCs, this receptor localized
intracellularly. a1BAR was detected in the membrane of arterial SMCs
(C), while in the veins this receptor seemed to be expressed at low
levels intracellularly (D). Lower panel represents
immunocytochemical staining of omAR in arterial (E) venous (F)
SMCs. In the arterial SMC, this receptor localized to the plasma
membrane and subplasmalemmal compartment, whereas in the
venous SMC, it was mainly detected in the intracellular compartment.
However, some membrane staining of this receptor was also
detected in the venous SMC... ...92

Subcellular distribution of a1DARs in HEK-293 cells. A. 01 DAR
was localized predominantly intracellularly, with little co-localization
with the membrane marker pan-cadherin (A’ lower panel). B. omAR
cc-Iocalized with the endosomal marker EAA1 (B’). C. omAR also co-
localized with calnexin (C’), a marker for endoplasmic reticulum... 93

Relative distribution of omAR co-expressed with azAR subtypes
in HEK293 cells. Each panel represents individual 02AR subtypes
expressed in HA—oIDAR-expressing cell. Picture in the right panel
shows the overlay of the two images. Only one cell is visible in fig. A
as opposed to fig. A’, because transient transfection did not result
into 100% co-transfection. In this case, the second cell was
transfected only by azAAR. Data suggested that none of the 02AR
produced redistribution of amAR to the plasma membrane. Only
achR appeared to localize to the same cellular compartment as
a1DAR95

xi

Fig. 13.

Figure 14.

Figure 15.

Figure 16.

Figure 17.

Figure 18.

Localization of azAR in HEK293 cells. azAAR expressed in omAR—
expressing cells localized predominantly to the cell membrane (A)
and co-localized with the membrane marker pan-cadherin (A’).
Similarly, ozBAR was expressed mainly in the membrane (B), but also
intracellularly. Fig. 8’ depicts colocalization of angR with the
membrane marker pan-cadherin. Flag-achR was detected in the
intracellular compartment (0’) and little co-localization with pan-
cadherin was detected (C)97

BzAR co-expression enhances the targeting of a1oAR to the
cellular membrane (A). The two receptors co-localize when
expressed in the same cell (C). Figure E depicts co-localization of
amAR with pan-cadherin in a cell that also expresses BZAR... .98

MAR, but not ozARs, enhances the membrane distribution of HA
- tagged amAR. Histogram represents relative distribution of HA-
quR in the plasma membrane that was measured by the
luminescence surface expression assay. The effects of the three
GZAR subtypes on 01 DAR membrane expression are depicted in fig.
A. Only BzAR significantly enhanced the amount of 01 DAR localized in
the membrane(B)... ..99

NE treatment (1pM) of HA-amAR stable cells produces
oscillatory calcium response (A). Fig. 8 represents a typical
cellular calcium rise induced by the Ca2+ ionophore ionomycin.
lonomycin was employed to normalize the data due to non-
quantitative nature of our Ca2 fluorophore Fluo—4. Data were
normalized by calculating the area under the curve of NE response
and expressed as % of area under the curve of ionomycin
response101

azAR subtypes do not enhance calcium signaling mediated by
omAR. Calcium imaging of cells co-expressing GzAR subtypes with
amAR. Response to NE was expressed as a percentage of
ionomycin response for data normalization. None of the OzAR
subtypes significantly enhanced Ca2 responses of 01 DAR -
expressing cells. In contrast, BzAR was able to enhance the Ca2

responsesto NE(D)... .1..02

Norepinephrine causes calcium elevation in azAR — transfected
cells. Transiently transfected wild - type HEK293 cells with OzA- (A),
(123- (B) and achR (C) were treated with norepinephrine (1uM).
Traces represent individual cells and their calcium response to

xii

Figure 19.

Figure 20.

Chapter 4:

Figure 21.

Figure 22.

Figure 23.

Figure 24.

Relative distribution of a1.)- and BzAR in arterial and venous
SMCs. In the arterial SMCs, 01 D- and BzAR were found in the plasma
membrane and sub-plasmalemmal compartment (A and B). While we
did not perform an experiment to directly co-localize the two
receptors, there is strong possibility that they co-localize due to their
very similar distribution. [32AR co-Iocalized with pan-cadherin,
suggesting membrane distribution of this receptor in the arterial SMC
(D). In the venous SMC, om - and BZAR seemed to be expressed
mainly in the intracellular compartment, but also appeared on the
membrane. The two receptors seemed to be co-Iocalized (G) ....... 106

Propranolol, BzAR antagonist does not affect NE - mediated
responses in MV. 3uM propranolol did not produce any effect on NE
concentration — reponse curve (performed in pressurized
system)107

Intraluminal pressure alters the reactivity of MA and MV. The
E050 for NE in the pressurized MV (6mm Hg) was shifted to the right
compared to non-pressurized MV, while the maximal response has
been significantly enhanced by application of pressure (A). More
pronounced effect of pressure (60 mm Hg) has been observed in MA,
where E050 has been shifted 8-fold to the left. In addition, maximal
response to NE almost doubled in MA (B)... .....131

Gila proteins are not involved in NE — induced constriction of
MV. While yohimbine, the GzAR antagonist produced a right-ward
shift in the NE CRC (A), pre-treatment of MV with PTX (2ug/ml) for 2
hours at 37°C in oxygenated Krebs buffer did not affect NE CRC in
MV(B)... .. .. . ...132

NE responses in MA and MV are dependent on Ca2+ influx from
extracellular space. Cadmium chloride (IOOpM), the non- selective
Ca2+ channel blocker significantly attenuated NE responses in MA
(A). In MV, cadmium produced even larger inhibitory effect; NE
responses were abolished at low agonist concentrations and vessels
exhibited attenuated constriction at higher NE concentrations

L-type VOCC play a more important functional role in MA than
MV in adrenergic responses. Nifedipine (IuM) shifted the CRC to
NE to the right and significantly attenuated constriction to NE at
larger NE concentration (A). Nifedipine also produced a slight right-
ward shift of NE CRC in MV, although the inhibitory effect on venous
constriction did not reach statistical significance (B)... .135

xiii

Figure 25.

Figure 26.

Figure 27.

Figure 28.

Figure 29.

Figure 30.

SOC contribute to venous, but not arterial constriction to NE.
Gadolinium chloride (Gd3“) (10uM), blocker of Ca2*- induced Ca2+
release activated channels (CRAC) and SOC had no effect on NE
responses in MA (A). CRC to NE was shifted to the right in MV in the
presence of gadolinium (8). Although the inhibition was not
statistically significant, attenuation of NE responses suggest some
functional involvement of these channels in functional responses to

Effect of IP3 receptor antagonist 2-APB on NE - mediated
constriction of MA (A) and MV (B). 20uM 2-APB only slightly
attenuated responses to NE in MA. However, 50uM 2-APB
significantly attenuated constriction to NE in MA. 2-APB was much
less potent inhibitor of functional responses in the MV................137

NE - mediated constriction is more dependent on Ca2+ influx
than PE - induced constriction of MV. Responses to NE (1uM)
and PE (1 uM) were recorded before and after thapsigargin treatment
(1uM) (vessels were exposed to thapsigargin for 50min — time
required for MV to relax and stabilize the diameter). Depletion of
thapsigargin — sensitive Ca2 stores significantly reduced responses
to both NE and PE, although the % reduction of PE responses was
larger. .139

azAR and activation Ca2" channels in MV. Combined inhibition of
L—type VOCC and SOC by nifedipine and Gd3+ produced a significant
inhibition of NE responses in MV. When the 02AR antagonist
yohimbine was applied together with nifedipine and Gds“, NE CRC
was shifted slightly, but not Significantly to the Wright compared to
nifedipine and Gda‘+ alone. .. .. .140

Rho kinase pathway plays an important functional role in
adrenergic responses of both MA and MV. Rho kinase inhibitor
Y27632 (10pM) was continuously superfused over blood vessels and
NE responses of blood vessels were measured. Y27632 produced a
significant inhibition of NE - mediated constriction of both MA and
MV, indicating that enhanced sensitivity of MV to adrenergic
stimulation is not due to more prominent role or Rho pathway ...... 141

MA and MV exhibit similar Ca2+ sensitivity during NE- mediated
responses. MA and MV were incubated in buffer lacking Ca2+ in the
presence of EGTA (1mM). After 15 min wash that producing Ca2

depletion in both vessels, cumulative CRC to CaClz were constructed
in the presence of constant concentrations of NE; 0.3uM for MA and
0.03pM for MV, concentrations representing approximately the E050

xiv

Chapter 5:

for NE in these vessels. Both MA and MV exhibited bi-phasic
response to 0a0|2 and except at 1uM CaClz, the differences between
MA and MV were not statistically significant. Data are presented as
% of control response to the given NE concentration in Ca2+
containing buffer(2.5mM).....................................................144

Figure 31A. MV are more sensitive to NE than MA. E050 to NE is significantly

Figure 32.

Figure 33.

Figure 34.

shifted to the left in MV and maximal constriction is larger in MV. MA
were pressurized to 70mm Hg, while MV were exposed to pressures
of 8mm Hg. (A) Responses to adrenergic stimulation are longer-
lasting in MV than in MA (8). 30ml of 1uM NE solution was applied to
pressurized MA and MV to develop a stable constriction. Changes in
the vessel diameter including the wash-out period were recorded.
The decay phase of the constriction was significantly slower in MV
than in MA, p<0.005 (8)171

MV, but not MA develop a strong constriction in response to
SERCA blockade thapsigargin (1uM). Thapsigargin was applied
using microapplicator and responses of blood vessels to NE (1uM)
were recorder before and after thapsigargin exposure. Responses of
MV to thapsigargin were always larger than to NE and constriction to
thapsigargin decayed very slowly in MV (~45-55 min). Response to
NE was reduced by over 60% in MV after thapsigargin treatment (B)
(Table 1), but was almost abolished in MA (A). In contrast to MV, MA
did not contract in response to thapsigargin. Superfusion of
thapsigargin produced similar results than microapplication, but
response of MA produced a transient and oscillatory constriction that
was still much smaller than in MV, black trace, (0). This transient
constriction of MA was abolished in the absence of extracellular Ca2+
(grey trace) .Constriction to thapsigargin in MV consisted of two
components; the first component likely represented Ca2+ unloading
from SER, because this transient constriction was not abolished in
Ca2+ free media. However, in the absence of extracellular Ca2+,
sustained phase of constriction to MV was abolished, implicating the
dependence of this phase on Ca2+ influx (D)... ........174

SERCA blockade causes elevation of [Ca2‘]. in dispersed SMCs
from MA and MV. Representative traces of Ca2+ elevation during the
exposure to 1uM thapsigargin in arterial (A) and venous (B) SMC go
along with the data from contractility experiments, where thapsigargin
induced a sustained constriction in MV but not in MA... .177

Store-operated channels are coupled to constriction in MV, but
not in MA. Blood vessels were equilibrated in 0 extracellular Ca2+

XV

Figure 35.

Figure 36.

Figure 37.

Figure 38.

solution/1 mM EGTA for 15min. After this period, the vessels were no
longer able to respond to NE. Incremental increases of CaCI2
concentration up to 3uM produced a concentration-dependent
constriction in MV (B). In contrast, MA did not respond with a
constriction after 0 Ca2+ buffer was replaced with increasing
concentrations of CaClz (A) TRP01, the main component of $00 is
expressed in the membrane of both arterial and venous SMCs (0).
Application of 0d2+, Gd3* or La3+ did not inhibit the initial constriction
produced by thapsigargin in MV. However, the tonic phase of
constriction was significantly inhibited by La3+ and Gd3“, but not by
Cd2*(D)178

Ouabain potentiates constriction of MA and MV to tha igargin.
In the presence of indirect inhibition of NCX1-mediated Ca * efflux by
ouabain (0.5mM), MA constricted 10.251:0.7% when exposed to
thapsigargin. In addition, in the presence of ouabain, strong
oscillatory constriction developed in response to NE after
thapsigargin exposure (27% versus 2.5% in the absence of ouabain)
(A). Ouabain also increased the maximal constriction to thapsigargin
in MV. In addition, the decay phase of constriction was increased in
the presence of ouabain in MV (B). lmmunocytochemical staining of
freshly dispersed arterial and venous SMCs indicated the expression
of NCX in the membrane of both cells (C)... .....182

Inhibition of PMCA 4 affects Ca2+ homeostasis and potentiates
NE responses in MV. NE (1 in MV or 10uM in MA) was applied
before and at the end of caloxin 1b1 exposure that took 20min. MA
did not respond to caloxin 1b1 (A). In addition, response to NE has
not been affected by this PMCA pump inhibitor. In contrast,
application of caloxin 1b1 (200uM) by itself produced constriction in
MV. In the presence of caloxin 1b1, response to NE was enhanced
and the relaxation phase was also significantly prolonged in MV

Global distribution of disease burden attributable to six major
risk factors. WHO, 2003 (DALYs- disability — adjusted life
year) ........... 196

Model of the proposed adrenergic receptor signaling and
calcium coupling in venous smooth mucle cell (A). Activation of
cIAR by NE leads to production of IP3 and DAG. DAG activates
receptor - o erated channels (ROC, likely TRPC6) that results into
Na“ and Ca * influx. Ca2+ may either act on ryanodine receptors to
activate 0a2*-induced Ca2+ release (CICR). The accumulation of Na”
in small microdomains could reverse the activity of NCX, driving
additional Ca2+ influx and CICR, producing constriction. 02AR is also

xvi

activated by NE and couples to Ca2 influx through L-type voltage —
operated 032 channels (VOCC) and/or store operated channels
($00). $00 can also directly participate in vascular constriction.
During relaxation, Ca2 is taken up by plasma membrane Ca2 -
ATPase (PMZCA) and reverse mode NCX. The activity of NCX and
therefore Ca2 export is regulated by other transporters that regulate
Na gradient across plasma membrane, like the Na ++lK -ATPase.
Model of the proposed adrenergic receptor signaling and calcium
coupling in arterial smooth mucle cell (B). mAR in arteries activate
phospholipase C and production of lP3 and DAG. IP3 activates IP3
receptors on smooth endoplasmic reticulum, leading to 032 release
from the stores. DAG activates ROC, perhaps TRPC6, producing
Ca2 and Na influx in localized regions near the plasma membrane.
Whether this Ca2+ is sufficient to produce constriction remains to be
evaluated. However, increased concentration of Na causes NCX to
operate in its reverse mode, producing Ca2’r influx that directly
participates in arterial contraction. SOC refill the Ca2 stores when
Ca2 is depleted. However, they do not directly participate in arterial
constriction. During relaxation, Ca2 extrusion is very efficient and
NCX contributes to this process ............................................. 224

“Images in this dissertation are presented in color.”

xvii

2-APB
Ach
ATP
BAR
BP
cah-
CaM
Cd2+
CICR
CO

' COMT
CRC
DAG
DOCA
DOPA
EGTA
EPI
Gd"
HEK

HR

LIST OF ABBREVIATIONS

xviii

alpha adrenergic receptor
2-aminoethoxydiphenyl borate
acetyl choline

adenosine 5’ - triphosphate
beta adrenergic receptor

blood pressure

calcium

calmodulin

cadmium

calcium — induced calcium release
cardiac output
catechol-o-methyltransferase
concentration - response curve
diacyl glycerol
deoxycorticosterone acetate
L-dihydroxy — L — phenylalanine
ethylene glycol tetraacetic acid
epinephrine

gadolinium

human embryonic kidney

heart rate

IP;

K“

M3 receptors
MA
MAO
MAP
MAP kinase
MCFP
MLCK
MLCP
MV
Na+
NCX
NE
NET
NKA
NLA
NO
NSCC
PE
PIP2
PKC
PLC

xix

inositol triphosphate
potassium
muscarinic receptors 3
mesenteric artery
monoamine oxidase
mean arterial pressure
mitogen — activated protein kinase
mean circulatory filling pressure
myosin light chain kinase
myosin light chain phosphatase
mesenteric vein
sodium
sodium — calcium exchanger
norepinephrine *
norepinephrine transporter
sodium - potassium ATP-ase
nitro — L - arginine
nitric oxide
non — slective cation current
phenylephrine
phosphatidylinositol bisphosphate
protein kinase 0

phospholipase C

PM
PNS
PTX
RGS
ROC
RyR
SER
SHR
SMC
SNS
SOC
TPR

TRPC

VSMC

KO

plasma membrane
parasympathetic nervous system
pertussis toxin

regulators of G — protein signaling
receptor — operated channels
ryanodine receptor

smooth endoplasmic reticulum
spontaneously hypertensive rat
smooth muscle cell

sympathetic nervous system

store - operated channels

total peripheral resistance
transient receptor potential channel

vascular smooth muscle cell

wild type

knock - out

CHAPTER 1

Introduction

1. Regulation of Blood Pressure (BP) by the Nervous System.

Many studies stress the importance of neurogenic mechanisms in the
long-term regulation of BP (Morimoto et al. 2001,Kishi et al. 2001). Neural
control of BP is accomplished through the activity. of the autonomic nervous
system and sensory nerves that supply blood vessels. Two divisions of the
autonomic nervous system regulate BP: the sympathetic and parasympathetic
divisions. Both systems originate in nuclei within the central nervous system
(CNS) and give rise to preganglionic efferent nerve fibers that exit from the brain
stem or spinal cord. Sympathetic preganglionic fibers leave the CNS at the
thoracic and lumbar portions of spinal cord (thoracolumbar system), whereas
parasympathetic preganglionic fibers leave the CNS at the cranial and sacral
portions of the spinal cord (craniosacral system). Preganglionic fibers synapse
with the postganglionic neurons in sympathetic and parasympathetic ganglia.
Postganglionic nerves fibers innervate the target visceral organs. Although not
exclusively, sympathetic and parasympathetic nervous systems exhibit opposing
actions on the target visceral organs and the two systems work in a cooperative
and integrated manner to control organ function.

There are major differences in the anatomical organization of the
sympathetic and parasympathetic divisions of the autonomic nervous system.
Preganglionic sympathetic nerve fibers synapse with multiple postganglionic
neurons (with ratio up to 1:20) providing for more diffuse action of sympathetic
nerves on target tissues. There are two groups of sympathetic ganglia,

paravertebral chain ganglia perveltrabral ganglia. Sympathetic neurons in these

ganglia send their postganglionic axons to target tissues. Parasympathetic
postganglionic neurons are in ganglia embedded within the organs innervated.
In most organs, the ratio between preganglionic and postganglionic cells is 1:1.
Both SNS and PSN utilize acetylcholine (ACh) as the dominant
neurotransmitter in the preganglionic nerve fibers. However in the PNS, ACh is
released from postganglionic nerve fibers. Norepinephrine (NE) and epinephrine
(EPI) are the major neurotransmitters released by the postganglionic nerve fibers
in SNS with the exception of thermoregulatory sweat glands, where ACh

mediates the action of SNS.

PNS in blood pressure regulation. Parasympathetic fibers innervate
the heart and control heart rate and the strength of contraction. In the sinoatrial
(SA) node, each cardiac impulse is initiated by a spontaneous depolarization of
pacemaker cells. At threshold potential, this depolarization initiates an action
potential that is conducted through the atrial muscle fibers to atriovenricular (AV)
node. ACh decreases spontaneous depolarization by activating potassium (K‘)
currents in the SA node, delaying the time to threshold potential and therefore
heart rate. The strength of atrial contraction is also reduced by ACh via inhibition
of Ca2+ channel activity and reduction of cAMP levels. In addition, at larger ACh
concentration muscarinic receptors (M2) can directly couple to K“ channels,
producing a direct inhibition of atrial contraction. Owing to these inhibitory actions

together with systemic vasodilation described below, systemic administration of

small doses of ACh or cholinergic agonists produces a transient fall in BP and

bradycardia.

Parasympathetic control of vascular tone. While most vascular
beds lack parasympathetic innervation, but in those vessels receiving a
parasympathetic supply, Ach released from parasympathetic nerves causes
vasodilation. ACh activates muscarinic receptors, primarily the M3 subtypes
located on the endothelial cells of the vasculature. Activation of these receptors
activates Ca2*-calmodulin dependent activation of endothelial NO synthase
(eNOS), triggering NO production that diffuses to adjacent SMCs and
hyperpolarizes them. Vasodilatation may also ensue by the action of Ach on
sympathetic nerve endings, where it inhibits norepinephrine release. In instances
where endothelium is damaged, high concentrations of ACh can activate M3

receptors in vascular smooth muscle cells (SMCs) and produce vasoconstricticn.

SNS in blood pressure regulation. The SNS is continuously active
and the degree of its activity is constantly adapting to changes in the
environment. As described above, actions of SNS are more diffuse than actions
of PNS. For example, during the “flight of fight” response, all sympathetically —
innervated organs are affected simultaneously to cooperatively meet the
demands of organism during increased stress levels. In addition, increased SNS
promotes secretion of epinephrine (EPI) from adrenal medulla into the circulation,

producing an even more wide-spread effect of SNS activation. Three types of

sympathetic fibers (barosensitive, thermosensitive and glucosensitive
cardiovascular) innervate blood vessels, the heart, kidney and adrenal medulla
and contribute to BP regulation.

The SNS increases heart rate and contractility by activating BIAR and
to lesser extent BzARs. 83AR and aAR are also present in the heart, but role of
these receptors is not as dominant in the regulation of cardiac contractility. BARs
couple to Gs proteins and stimulation of the activity of adenylyl cyclase. This
results into accumulation of cAMP and activation of PKA. PKA affects function of
many cellular proteins, including ion channels by phosphorylation. Activation of
BARS accelerates the slow depolarization of SA node leading to faster rates to
reach threshold potential by the pacemaker cells, resulting into increased heart
rate. In addition, stimulation of BIAR increases contractile force; activation of Gs
can also directly activate the voltage-sensitive Ca2+ channels in the plasma
membrane of cardiac muscle, enhancing Ca2+ influx during contraction. Overall,
SNS activation increases cardiac output due to its positive chronotropic and
ionotropic effect on the heart. Together with the effects on the peripheral
vasculature (see below), SNS activation elevates BP.

EPI released from adrenal medulla directly increases cardiac heart rate
(HR) and contractility. EPI differentially regulates the tone of various vascular
beds, producing vasoconstriction in most resistance vasculature and veins while
producing vasodilatation of skeletal muscles.

Sympathetic control of vascular tone. NE and to lesser extent EPI

released from the sympathetic nerve endings and adrenal medulla are the major

circulating hormones regulating the vascular tone. The biosynthetic pathways for
catecholamine production are well understood and involve hydroxylation of the
amino acid tyrosine to the precursor L-dihydroxy — L — phenylalanine (DOPA).
This is the rate limiting step in the biosynthetic pathway. DOPA is decarboxylated
to form dopaminie that is taken up by storage vesicles in the nerve terminals.
Dopamine-B-hydroxylase then catalyses the production of NE in the vesicles. In
the adrenal medulla where EPI is the dominant catecholamine being released, an
additional step involves methylation of NE by phenylethanolamine-N-
methyltransferase. EPI is also stored in the granules of chromaffin cells in
adrenal medulla. Quantal release of neurotransmitters into synaptic cleft is
facilitated by the arrival of action potential that triggers Ca2+ influx into the nerve
terminal. Ca2+ facilitates the fusion of storage vesicles with axoplasmal
membrane and content of the vesicles including enzymes other proteins are
discharged to the synaptic cleft. However, NE is not the only transmitter released
by sympathetic nerves and role of ATP as a co-transmitter in sympathetic
regulation has been well established. The relative contribution of these two
transmitters in different vascular beds might provide for differential regulation of
their tone. While some studies suggest that sympathetic nerves supplying
mesenteric veins (MV) release equal or greater amount of ATP than mesenteric
arteries (MA) (Bobalova and Mutafova-Yambolieva 2001‘,Bobalova and
Mutafova-Yambolieva 20012), functional data indicate that MV constriction is
exclusively mediated by NE (Park et al. 2007). In MA a two-phasic neurogenic

constriction is observed in response to stimulation of sympathetic nerves. The

first phase is fast and involves activation of P2X receptors by ATP, whereas the
second phase has slower onset and is mediated by NE (Sneddon and Westfall
1984, von Kugelgen and Starke 1991).

Termination of NE signaling occurs by the combination of simple
diffusion and reuptake by the axonal terminals by norepinephrine transporter
(NET). NE can be also taken up by extraneuronal tissue by the low affinity
transporters OCT1 and OCT2. NE can be degraded by monoamine oxidase.
(MAO) and catechol-o-methyltransferase (COMT). However, metabolic
transformation of NE plays a minor role in signal termination such as the powerful
degradative pathway mediated by acetylcholine esterase in Ach signal
degradation.

The structure and degree of sympathetic innervation influences the
vessel responsiveness to sympathetic activation. SNS innervation varies with the
vessel size and vascular bed. In general, large elastic arteries are sparsely
innervated but as the vascular diameter decreases, the density of innervation
increases. This nature of the innervation has important implication in the
regulation of blood pressure. Because small arteries are the major contributor to
peripheral resistance, their high innervation density allows for rapid rise of blood
pressure under “flight or fight” conditions.

In addition to differences in the innervation between vessels of different
sizes, vascular beds appear to be regionally regulated by SNS. For example,
pharmacological treatment with the sympatholytic agent, clonidine, more

effectively inhibits sympathetic outflow to hepatomesentery circulation than

systemic circulation (Esler et al. 1992). In obesity, sympathetic outflow to skeletal
vasculature and kidney is increased, while it remains normal in the
hepatomesentery circulation and skin and even attenuated in the heart (Vaz et al.
1997). 2

It is not known, whether MA and MV are also differentially regulated by
SNS, but there is evidence that distinct sympathetic neurons project from inferior
mesenteric ganglia to these two vessels (Browning et al. 1999). In addition, the
nerve fibers of the perivenous plexus are less dense than of periarterial plexus
and their orientation also differs. Individual axons are oriented circumferentially
around veins, with larger density of vertical than horizontal fibers. In contrast, the
arterial plexus consists of axons arranged in net - like manner, with similar
density of horizontal and vertical fibers (Park et al. 2007). However, in contrast to
arteries where sympathetic fibers rarely cross the media-adventitia border,
sympathetic fibers penetrate deep into smooth muscle bundles of most veins
(Pang 2001 ).

Besides the anatomical differences, the electrophysiological properties
of the nerve fibers also differ between arteries and veins. Both arteries and veins
exhibit frequency-dependent vascular contraction. However, stimulation at equal
frequencies causes larger amount of NE release from nerve terminals in MV
compared to MA (Bobalova and Mutafova-Yambolieva 2001, Park et al. 2007).
This might be due to the fact that NE mediates all components of sympathetic
neurotransmission in the MV, whereas in MA, NE, ATP and neuropeptide Y

(NPY) mediate constriction (Westfall et al. 1996; Browning et al. 1999, Luo

2003). Furthermore, MV exhibit increased sensitivity compared to MA, with
greater vasoconstrictor at any stimulation frequency (Karim and Hainsworth
1976; Hottenstein and Kreulen 1987; Luo et al. 2003). This can not be attributed
solely to the fact that sympathetic nerves supplying-MV release more NE during
stimulation. As discussed below, veins are also more sensitive to exogenous
application of adrenergic agonists, implicating the postsynaptic signaling
differences in differential responsiveness of MA and MV to adrenergic stimulation
(Perez-Rivera et al. 2004, Luc et al. 2003). Circulating hormones or NPY
released form the sympathetic nerves and endothelial derived factors can

modulate the concentration of Ca2+ in SMCs or Ca2+ sensitivity of SMCs.

2. Roles of veins and arteries in hemodynamics

Veins in hemodynamics. While a large amount ofiresearch has been
performed on the regulation of resistance vasculature, comparable studies of the
venous system are scarce. This deficit could be partly attributed to neglect on the
side of scientists, who ascribed a purely conduit role to the venous system. In
addition, monitoring venous pressures in vivo and venoconstriction in vitro poses
significant technical challenges. Despite the prevailing focus of the scientific
literature on the arterial side of the circulation, the role played by systemic veins
in human physiology as well as in certain pathophysiological conditions is

extremely important.

Veins and venules contain 60-75% of the total blood volume (Martin et
al. 1998). About half of this amount is stored in the small veins and venules of a
diameter less than 400 um (Monos et al. 1995). Small veins from the splanchnic
region are particularly critical in the regulation of blood distribution, because
nearly 40% of blood volume resides in this region (Greenway and Lister 1974).
The blood reservoir function of splanchnic bed is demonstrated by the fact that
hemorrhage of significant amount (20%) of blood volume does not alter MAP, HR
or 00. However, splanchnic blood volume is reduced (Greenway and Lister
1974, Pang 2001). This mechanism is also important during exercise, when
blood is rapidly transferred from the splanchnic region into cardiopulmonary
region in order to meet oxygen demand (Flamm et al. 1990). However, the most
significant blood reservoir is not necessarily the area that contains a significant
proportion of blood (area that regulates capacitance) but rather the area from
which a significant blood volume can be shifted in a fast and controlled manner.
Therefore the distribution of blood volume and the blood reservoir function are
not necessarily the same (Greenway and Lister 1974). The greatest blood
reservoirs are spleen, liver, large abdominal veins, subcutaneous venous plexus
and heart and lungs (Guyton et al. 1972).

The capacitance function of veins is related to their much smaller wall
to lumen ratio as well as much larger incremental distensibility at low pressure
ranges than arteries, resulting in much greater compliance (~30X). Although
there are studies investigating the contractile mechanisms of veins, the great

majority of those focus on larger caliber vessels like the saphenous or femoral

1O

veins (Crowley et al. 2002, MacLennan et al. 1997). These veins are not typical
capacitance vessels but rather counduit vessels. The function of these large
conduit veins greatly differs from the hemodynamic role of systemic and highly
compliant small diameter veins. Larger conduit veins are thin-walled, sparsely
innervated and exhibit differential sensitivity to baroreceptor reflex and
vasoconstrictor agents. It is believed that veins in the lower extremities do not
store significant amount of blood volume (Magder 1990) and veins from skeletal
muscle likely do not play a significant capacitance role owing to their low
responsiveness to sympathetic stimulation (Hainsworth et al. 1983). In contrast,
MV play a key role in controlling changes of vascular capacity (MV contain ~25%
of blood volume). In isolated canine MV preparation, elevation of intraluminal
pressure from O to 30mm Hg increases the lumen volume by 360% (Gaehtgens
and Uekennann 1971). Moreover, there are also differences in the distensibility
of the different size veins in the mesentery: in vivo studies in anesthetized rats
demonstrated that first-order venules are much more distensible than the larger
collecting veins (Lang and Johns 1987). However, these studies could not
differentiate between active tone and elastic properties of the vascular wall.
Table 1 depicts typical capacitance veins as well as conduit veins.

The SNS is the major regulator of venous capacitance (Deschamps
and Magder 1992). There are multiple control systems involved in SNS
regulation of the venous blood flow: these include local and systemic volume
receptors (Nijima 1977, Thrasher, et al. 1982) as well as arterial baroreceptors

(Brunner et al. 1988). When blood pressure is reduced, the resulting increase of

11

Table 1. Not all veins are equal. Counduit veins including vena cava or femoral
vein exhibit different properties in mediating responses to sympathetic nervous
system than capacitance veins. Table depicts typical capacitance and conduit
veins.

 

 

 

 

 

Capacitance Veins Conduit Veins
splanchnic veins femoral vein
mesenteric veins saphenous vein
vena cava
skeletal muscle veins

 

 

 

 

12

sympathetic outflow induces venoconstriction, arterial constriction, increased HR
and contractility. Most of the reflex change in the vascular capacitance is in the
splanchnic bed (Brunner et al. 1981). As discussed below, another regulatory
mechanism that does not involve neural reflex is myogenic activity of venous
SMCs

The capacity of the splanchnic bed to store blood has important
implications not only under physiological conditions. When splanchnic storage
capacity is chronically compromised, redistribution of blood from these vessels
can lead to pathophysiological consequences in the long term. From a
quantitative standpoint, capacitance of the systemic veins is the major
determinant of venous return to the heart. Under any given conditions under
equilibrium venous return equals 00; as a result of the increased preload in the
right atria, more forceful contractions of the myocardium occur (Starling’s law),
leading to increased 00 (Guyton et al. 1955) and MAP. Data from early studies
suggest that even very small changes in venous capacitance mediated by
baroreceptor reflex activation lead to significant increases in venous return and
CO (Heymans et al., 1958).

The above mentioned studies emphasize the importance of proper
regulation of venous tone in maintaining normal 00 and MAP. To better
understand the regulation of venous tone, we performed studies on mesenteric
capacitance veins with the aim to identify the differences in aAR signaling
between MA and MV that may contribute to enhanced adrenergic sensitivity of

MV. This mechanism might be important in hypertension, where increased SNS

13

activity increases the tone of the vasculature. However, veins might be
preferential target for this increased tone due to their sensitivity.

Arteries in hemodynamics. While veins contribute very little to TPR,
arteries are the major resistor of the circulation. There are major differences in
morphological properties of arteries and veins that are related to their differential
role in the circulation. As opposed to veins, increase in pressure only modestly
raises the radius of muscular arteries, maintaining the vascular resistance at
more constant level. Based on the Poiseuille’s law, the resistance of an individual
arterial segment is proportional to the fourth power of the radius and therefore
small arteries are the main contributor to vascular resistance. It has been
estimated that more than 50% of total vascular resistance resides in small
arteries of diameter <1OO um (Mulvany and Aalkjaer, 1990)

A large pressure drop has been detected in multiple vascular beds
including MA, skeletal muscle, cremaster and hamster cheek pouch indicating
the regulatory potential of these vascular beds (Mulvany and Aalkjaer 1990).
These arteries exhibit an intrinsic tone, indicating that they are not just a passive
resistor, but rather participate actively in the regulation of resistance (Stekiel et
al. 1986). Besides the SNS control of arterial resistance, tone of small arteries is
regulated by intravascular pressure (myogenic tone described in section below)
and also blood flow. Similarly to the large conduit arteries, blood flow relaxes the
tone of small arteries (Bevan and Joyce 1988).

TPR is increased in hypertension. This is accompanied by altered

structural characteristics of small arteries, characterized by increased media to

14

lumen ratio and reduced lumen. Studies suggest that the altered structural
characteristics involve autoregulatory mechanisms and can be perceived as an
adaptation of vascular wall to increased intravascular pressure produced by
hypertension (Guyton 1989). Neurohumoral factors that are altered in
hypertension can also directly promote proliferation of vascular SMCs, leading to
thickening of media in the resistance arteries.

Because regulation of arterial and venous tone determines TPR and
CO and therefore BP, it is very important to understand the mechanisms involved

in the regulation of the contractile state of both arteries and veins.

3. Agrenergjc recegtors are the main effectosz of §N_S._

There are two main families of adrenergic receptors that integrate the
signals of NE released from the sympathetic nerves and EPI released from
adrenal medulla: o- and B- adrenergic receptor families. ARs are 7-
transmembrane domain G-protein coupled receptors (GPCR) that are expressed
by different types of cells in the CNS and periphery (Guimaraes et al, 2001). oAR
have been divided into two families: the 01 and OzARS (McGrath et al, 1989).

Expression of aARs in blood vessels is not homogenous (Hrometz et
al. 1999, Chotani et al. 2004, Hussain et al. 1997) and vasoconstriction may be
mediated by a mixed population of oARs. The expression of oARs subtypes
differs across vascular beds as well as across different species (Rudner et al.
1999). Furthermore, there is some evidence that different oAR subtypes mediate

responses to nerve-released catecholamines (junctional receptors) as opposed

15

to circulating catecholamines (extrajunctional receptors) (Townsend et al. 2004;
Yang and Chiba, 2001). Moreover, the expression of aAR subtypes in vascular
beds is affected by aging (Rudner et al. 1999). In most blood vessels, the
predominant receptors mediating constriction are the GAR subtypes.

01-Adrenergic Receptor Signaling: Three 01AR subtypes have been
cloned and named 01A- (11 B-, aIDAR. These subtypes differ in their efficiency to
produce cellular responses; the 01 AAR and O1BARS are more efficient in
activating PLC compared to oIDAR (Schwinn and Johnston, 1995; Vazquez-
Prado and Garcia-Sainz 1996). These intrinsic properties of the receptor
subtypes might have important implications with regard to the differences in
adrenergic responsiveness observed between MA and MV.

The best characterized actions in the cardiovascular system related to
01AR activation include contraction, growth, proliferation of VSMCs and
increased cardiac contractility (Rudner et al. 1999; Piascik and Perez, 2001; Lin
et al. 2001). While aIARs can couple to several second messenger systems, the
predominant pathway is via pertussis toxin insensitive qu to phospholipase C
(PLC), leading to Pl turnover with the production of inositol triphosphate (IP3) and
diacylglycerol (DAG). DAG stimulates protein kinase C and IP3 acts on the IP3
receptor in endoplasmic reticulum to release intracellular [Ca2+] (Minneman et al.
1988). It has been established that following the Ca2“ release from intracellular
stores, aiARs also activate Ca2+ influx during receptor stimulation leading to a
more sustained Ca2+ increase (Piascik and Perez 2001). Besides L-type calcium

channels that may regulate this Ca2+ influx (Crowley et al. 2002; Villalba et al.

16

2007), oIARs activate non-selective cation channels (NSCC) via DAG (Helliwell
and Large 1997). It is not yet clear whether Ca2+ influx through this pathway is
triggered by store depletion of Ca2+ or whether it is activated by aIAR via
receptor-operated channels (ROC). Both (ROC) (Crowley et al. 2002; Facemire
and Arendshorst 2005) and store-operated channels (SOC) (Arnon et al.
2000;Lee, Rahimian et al. 2002) have been implicated in vascular constriction
mediated by CI1ARS. Recent studies suggest that at least some of the channels
that mediate Ca2+ entry belong to the family of transient receptor potential
canonical channels (TRPC).

NSCCs have been also detected in rat MA myocytes (Hill et al. 2006).
Transient receptor potential channel homologue 6 (TRPC6) has been identified
as the essential channel mediating the aIAR-activated NSCC current in the
vasculature (lnoue et al. 2001). A recent study has indicated, that a receptor-
accessory protein named Snapin physically interacts with diAAR and TRPC6
upon receptor stimulation and enhances Ca2+ influx through TRPC6 (Suzuki et
al. 2007). The authors suggested that the mechanism underlying this enhanced
Ca2+ influx mediated by snapin is due to enhanced insertion of TRPC6 into
plasma membrane upon aIAR activation. This leads to increased Ca2+ influx.

While there is strong evidence linking activation of GPCR to stimulation
of TRPC, it is not established whether these channels support constriction
directly. It has been proposed that during adrenergic stimulation Na+ entry
through NSCC could drive Na*—Ca2*-exchanger (NCX) -mediated Ca2+ entry in

small microdomains of the plasma membrane (PM) (Arnon et al. 2000). It has

17

been later found that TRPC6 can functionally couple to (NCX) by localized
increases of Na+ (Poburko et al. 2007). During resting membrane potentials, NCX
mediates Ca2+ extrusion. However, during the opening of NSCC by oARs, the
local increase of [Na"] in small microdomains drives the NCX into its reverse
mode of action, transporting Na+ out of the cell and importing Ca2+. NCX is
indeed becoming recognized for its role in facilitating Ca2+ influx during vascular
constriction (Yao et al. 1998). Studies have shown a great degree of co-
localization between NCX, SERCA, Na+-K+-ATPase (NKA) and $00 in small
microdomains of the cell (Arnon et al. 2000; Ambudkar 2006; Lynch et al. 2008)
allowing for efficient functional coupling between these Ca2+ handling proteins.

In addition to Ca2+ release and Ca2+ influx mediated by aIAR, studies
suggest that stimulation of this receptor also leads to Ca2+ sensitization that can
contribute to tonic contractions. This sensitization is dependent on PKC, Rho
kinase and tyrosine kinase (Crowley et al. 2002, Villalba et al. 2007, Dimopoulos
et al. 2007). In rabbit mesenteric artery, enhancement of Ca2+ sensitivity by PE is
dependent on tyrosine kinase activation while its is indepentent of PKC (Sasaki

etal1998)

Regulation of o1AR. Signaling of GPCR must be tightly regulated in
order to maintain functional specificity. oiARs, like other GPCR are regulated by
phosphorylation, transcription, trafficking and desensitization (Hawrylyshyn et al.
1980; Michelotti, 2000). While multiple mechanisms contribute to these

regulatory processes, receptor phosphorylation by G protein receptor kinases

18

(GRK) and subsequent recruitment of B-arrestins plays a very important role in
functional regulation of GPCR. Interaction of phosphorylated receptor with B-
arrestin uncouples the receptor from activation of G proteins and promotes
internalization of the receptor via clathrin-coated pits, resulting into removal of
activated receptor from the plasma membrane. All three aIAR have been shown
to undergo receptor desensitization upon repetitive exposure to NE (Vazquez-
Prado and Garcia-Sainz 1996).

There seems to be some functional specificity in receptor
desensitization mediated by GRK; multiple GRK isoforms are expressed in
cardiomyocytes that express aAR, BAR and ET receptors. While GRK2 seems to
be selectively targeting BIAR thereby modulating cardiac contractility, GRK3
seems to be more selective for oIAR, modulating growth and hyperthrophic
responses in the heart (Vinge et al. 2007).

The internalized receptor can be either dephosphorylated and recycled
or trafficked to the lysosomes for receptor downregulation (Krupnick and Benovic
1998). This is many times dependent upon the length of exposure to the agonist;
many receptors undergo significant downregulation only after hours of agonist
exposure. For example, 24h exposure to NE (2 uM) suppresses c113- and oIDAR
while 01 AAR expression is enhanced. This regulation occurs at the level of gene
expression and correlates with protein levels (Rokosh et al. 1996). These data
suggest that in situations where SNS activity is chronically elevated like in
hypertension, relative expression of individual subtypes might undergo dynamic

changes.

19

In recent years it is becoming more accepted that structurally diverse
ligands of a particular receptor induce ligand specific signaling. It is believed, that
different chemical structures of these compounds produce differential
conformation of the receptor, indirectly regulating'downstream signaling of the
receptor (chilka 2007). It has been shown that NE and EPI whose chemical
structure is closely related can induce a differential conformation of BzAR
(Swaminath et al. 2004), resulting into activation of different signaling pathways
in cardiomyocytes. EPI causes a rapid contraction as opposed to NE, where the
onset of maximal response is slower. In addition, EPI, but not NE, induces
signaling via Gi. It has been shown that following EPI stimulation, BzAR
undergoes a rapid phcphcrylation by GRK2 with subsequent dephosphorylation,
that are necessary for receptor recycling and Gi activation (Wang et al. 2008).
These data suggest that GRK can also function to fine tune receptor signaling.
This might have great relevance not only in adrenergic receptor regulation, but
also in modulating functional responses of other receptor families. Ligand-
selective regulation of receptor conformation has also great implications for
future drug development, where a specific conformation of receptor without any
unwanted side effects could be exploited by selective ligands that stabilize this
particular conformation.

Recently, a family of proteins named Regulators of G-protein Signaling
(RGS) was identified that bind Go-GTP and enhance the rate of GTP hydrolysis,
thereby helping to terminate receptor-mediated responses (Watson et al. 1996).

The importance of these regulatory proteins in Gq signaling is demonstrated in

20

the mice with RG82 deletion that develop a marked and sustained hypertension
with enhanced adrenergic contractility and attenuated responses to vasodilators
(Tang et al. 2003). Even though the prevailing believ about the etiology of
hypertension indicates that altered signaling 'of CNS and abnormal sodium
handling by the kidney are required to maintain elevated BP, the author of this
study suggests that the hypertension in RGS-l- mice likely ensues due to
increased peripheral vasoconstriction that can alter the blood pressure set point
(Tang et al. 2003).

RGS2 was shown to be activated by PKGI-a in nitric oxide (NO)-
dependent manner (Tang et al. 2003) and the mechanism by which RGSZ
attenuates Gq signaling likely involves inhibition of Ca2+ release from intracellular
stores as well as inhibition of capacitative Ca2+ entry during agonist stimulation
(Osei-Owusu et al. 2007). While more than 30 different RGS isoforms have been
identified (Hollinger and Hepler 2002), R682 has been f0und to specifically
interact with the third intracellular loop of diAAR, but not the closely related dig-
and oIDARs, suggesting a functional specificity in RGS-mediated inhibition
(Hague et al. 2005). Therefore, differences in the activity and expression of RGS
proteins between MA and MV might also affect the functional responses

mediated by GPCR agonists.
Physiological role of a1ARs in cardiovascular system. Due to a

lack of sufficiently sensitive subtype-selective ligands, the precise physiological

function and therapeutic potential of the individual subtypes have not been

21

elucidated. However, gene-disruption strategies were successfully applied to
generate strains with deficiencies of individual oAR subtypes. Acute
administration of NE or PE induces rapid raise of BP due to vasoconstricticn.
However, in mice lacking individual G1AR subtypes, this raise in BP is attenuated.
This indicates that all three subtypes contribute to the regulation of vascular tone
(Philipp and Hein 2004).

The oiAAR has been shown to play an important functional role mainly
in resistance arteries (Kong et al. 1994; Argyle and McGrath, 2000) and GIAAR
KO mice are hypotensive at rest (Rokosh and Simpson, 2002). The 013AR is
expressed in the peripheral arteries (Hrometz et al. 1999). However its role in
regulating vasoconstriction is controversial. Mesenteric arteries from the G1BAR
KO mice exhibit the same reactivity to phenylephrine as arteries from WT mice,
suggesting that this receptor doesn’t participate in agonist induced constriction
(Zuscik et al. 2001). Mice overexpressing G1BAR are characterized by
hypotension, autonomic failure and cardiac hypertrophy, further contradicting a
vasoconstrictor role of this receptor (Zuscik et al. 2001). However, this receptor
subtype mediates the constrictor actions of nerve-released NE (Townsend et al.
2004). A key role has been detected for (11 DAR in blood pressure control. G1DAR
KO mice which have a lower basal blood pressure then their WT counterparts.
Furthermore, this receptor plays a role in salt-induced hypertension, because
oioAR KO mice have a significantly attenuated BP response to salt-loading
(Hosoda et al. 2005). The role of oiDAR in hypertension development might be

also important with regard to MV. As discussed below, G1DAR is the main

22

subtype regulating the tone of MV and therefore it may play a role in attenuating
vascular capacitance that occurs in the developmental stages of salt-sensitive

hypertension.

02-Adrenergic Receptors: Similarly to a1ARs, three subtypes of
quRs have been cloned and characterized based on molecular, biochemical
and pharmacological approaches (a2A, 023, (:20). These receptors share about
50-60% identity, but the data suggest that there is no functional redundancy of
these three receptors. Initially, o2ARs were believed to be expressed only
prejunctionally to regulate neurotransmitter release. However, multiple studies
have shown, that OzARS are expressed postjunctionally, where they are involved
in a wide range of physiological processes. Aside from the regulation of
sympathetic tone and cardiovascular function, azARs are involved in lipid and
carbohydrate metabolism, platelet aggregation, analgeSia, gastrointestinal
motility and maintenance of fluid and electrolyte balance by the kidneys (Ruffolo
et al. 1993). All three receptors negatively couple to adenylate cyclase via
pertussis toxin sensitive Gi. However, under certain conditions, quRs mediate
increases of cAMP, implicating the activation of Gs (Ullrich et al. 1984; Jones et
al. 1991; Eason et al. 1992). In addition to inhibition of adenylate cyclase, CzAR
have been shown to activate receptor-operated K+ channels, inhibit voltage-
dependent Ca2+ channels and activate mitogen activated protein kinase cascade

(Richman and Regan 1998).

23

After aiARs were established as the main mediators of vascular tone
in response to NE, evidence started to accumulate that 02AR are also expressed
in vascular SMCs (Ruffolo, et al. 1986; Matthews et al. 1984; Daniel et al. 1991).
Many studies have indicated that GzAR plays a more prominent role in the
regulation of venous than arterial tone (Flavahan et al. 1984; Patel et al. 1981).
Even though arteries were found to express GzARS, their efficacy to produce
constriction was much smaller than for aIAR, indicating that they are functionally
uncoupled in arteries (Daniel et al. 1991). In addition to studies utilizing isolated
venous preparation, where a large OZAR reserve has been detected (Ruffolo
1986), studies in vivo also implied that postjunctional 02AR may play a more
prominent role in venous capacitance vessels (Patel et al. 1981). In addition,
activation of 02AR in vivo results into increased mean circulatory filling pressure
(MCFP), demonstrating its functional role in regulation of vascular capacitance
(Appleton et al. 1986). A

The main effect of ozAR activation is inhibition of adenylate cyclase via
Gi. However, whether this is the pathway producing vascular contractions
remains to be elucidated. Several studies suggest, that decreased cAMP
production is inadequate to explain all the actions elicited by OzAR stimulation
(Nakaki et al. 1983; Ulrich 1984). In porcine lateral vein, OzAR produces strong
constriction, but no change in the cAMP level is observed, indicating adenylate
cyclase — independent mechanism of vascular constriction (Wright et al. 1995).

Nonetheless, pressor responses mediated by ozAR are sensitive to pertussis

24

toxin which will inactivate Gm proteins (Boyer et al. 1983; Aburto, Jinsi et al.
1995)

Studies suggested that contraction mediated through azARs is not
dependent on intracellular Ca2+ release (Lepretre and Mironneau 1994), but
rather involves a pertussis toxin sensitive G protein and extracellular Ca2+ influx,
probably through voltage-gated calcium channels (VDCC) and (Ruffolo et al.
1984; Parkinson and Hughes). In the dog saphenous vein, stimulation of 02AR
leads to contraction and large increases in [Ca2+]; and myosin light chain (MLC)
phosphorylation (Aburto et al. 1993). The constriction is highly dependent on
PKC and DAG (Aburto et al. 1995). The mechanism by which GzAR activates
PKC involves activation of phospholipase D (PLD) that is converted to
phosphatidic acid followed by formation of DAG. DAG activates PKC and induces
contraction. The link between quR activation and PLD may be mediated by
tyrosine kinase (Uings et al. 1992); in rat fibroblasts, ozARs activate tyrosine
kinases via Gi (MacNulty et al. 1992). In addition, constriction mediated by GzAR
depends on activation of tyrosine kinase in saphenous vein or aorta (Jinsi and
Deth 1995; Carter and Kanagy 2002). In porcine palmar lateral vein, Src- tyrosine
kinase mediates constriction caused by azAR stimulation (Roberts 2001).

azAR-mediated constriction via tyrosine kinase might be also important
in NO synthase (NOS)—inhibition induced hypertension where reactivity of
vascular OzAR is enhanced, while aIAR-mediated constriction remains
unchanged (Kanagy 1997); quR-mediated constriction in these animals is more

dependent on Ca2+ influx through VDCC and involves activation of mitogen

25

activated protein kinase extracellular-signal regulated kinase kinase (MAP/ERK
tyrosine kinase) (Mukundan and Kanagy 2001; Carter and Kanagy, 2002).
Several other receptors including oiAR (Jinsi et al. 1996; Dessy et al. 1998), ET
receptors (Liu, et al. 1992) and angiotensin II receptors(Lassegue et al. 1991),
histamine receptors (Katoch and Moreland 1995) have been shown to constrict
blood vessels via this mechanism.

The Erk pathway is also a possible downstream target for tyrosine
kinase activation; MAP kinase can be activated by Src tyrosine kinase in Gi or
Gq-dependent manner (lgishi and Gutkind, 1998; Richman and Regan 1998). In
porcine palmar lateral vein, constriction caused by the azAR agonist, UK 14,304,
is inhibited by 80% in the presence of inhibitor of mitogen activated protein
kinase kinase (MEK), component of Erk transduction pathway. In addition,
inhibition of Src tyrosine kinase by PP2 prevents activation of Erk and
constriction to UK 14,304 in this vessel (Roberts 2001).

azAR regulation. Similarly to 01AR, OzARS are also subject to
regulation by phosphorylation, transcription and desensitization. Evidence
suggests, that desensitization of GzARS is regulated in a subtype selective
manner; the ochR being more resistant to desensitization by continuous
application of catecholamines compared to 02A and azBARs (Eason et al. 1992).
This is likely related to the fact that a2A- and ozaAR are subject to more extensive
phosphorylation by GRK after agonist exposure, compared to achR (Kurose and
Lefkowitz 1994). lnternalization of the three subtypes is dependent on the

presence of B-arrestins. The interaction between B-arrestins and the receptor

26

subtypes is also selective; B-arrestin 2 promotes internalization of both 023- and
achR whereas B-arrestin 1 only promotes ozBAR internalization. However,
neither of these B-arrestins significantly affected GZAAR internalization (DeGraff et
al. 1999). I

The third intracellular loop of OZAR has been shown to interact with B-
arrestins and differences between the receptors in this sequence might provide a
mechanism for selective regulation of desensitization (DeGraff et al. 1999). The
subtype-specific differences in this sequence may also partially explain
differences in trafficking. When the receptors are co-expressed in the same cells,
they differ markedly in their agonist-regulated targeting to the endocytic pathway
(Zastrow et al. 1992) and when the three receptors are expressed in HEK-293
cells, their cellular distribution also differs. While GZAAR and 023AR are
predominantly expressed in the membrane, achR seems to reside in the
intracellular compartment (von Zastrow et al. 1993). HoWever, the cellular
distribution seems to be dependent also on the cellular context; while in rat 1
fibroblasts, 0087 and rat kidney cells ochR accumulated in the endoplasmic
reticulum, it was efficiently trafficked to the plasma membrane in the P012 cells
(Hurt et al. 2000).

Temperature also regulates C12AR function. Even though same GzAR
subtypes might be expressed in the mesenteric vasculature, this regulatory
mechanism might have greater implication in the cutaneous vasculature due to
its role in thermoregulation. While reactivity of aIAR is attenuated in cold

conditions, cold temperatures have been shown to enhance reactivity of GzAR

27

(Flavahan et al. 1985). Studies suggested that UchR mediates the enhanced
responses to adrenergic stimulation under cold conditions (Chotani et al. 2000).
While ochR are silent 37°C, studies in transfected HEK293 have indicated that
cooling evokes redistribution of achR from Golgi compartment to the plasma
membrane, enabling this receptor to mediate responses to azAR stimulation
(Jeyaraj et al. 2001). The redistribution of this receptor to the membrane is
dependent upon the activation of Rho kinase (Bailey et al. 2004).

The dependence of achR-mediated constriction on Rho kinase
may have implications beyond the regulation of cutaneous blood vessels. As

described in Chapter 2, achR appears to mediate NE responses in MV.

Physiological role of azARs in cardiovascular system. In contrast
to the monophasic vasopressor responses caused by systemic administration of
aIAR agonists like phenylephrine, systemic administration of 02AR agonists
causes a bi-phasic response. Clonidine, a partial azAR agonist, causes a
transient rise in BP due to 02AR mediated constriction of peripheral blood
vessels. The transient vasopressor response is followed by a drop in BP to lower
than basal levels due to the activation of 02AR in the lower brainstem region that
directly inhibit sympathetic outflow.

Experimental evidence indicates that o2AARs are responsible for the
depressor effect mediated by clonidine. ozAAR KO mice lacks this
vasodepressor response (Link at al. 1996). This property makes (IzAAR a very

suitable target in hypertension therapy, where activating this receptor by agonists

28

like clonidine inhibits sympathetic outflow (Ruffolo et al. 1993). In addition to the
regulation of neurotransmitter release, 02AAR are expressed by endothelial cells,
where they mediate vasorelaxation by promoting NO production (Shafaroudi et
al. 2005). Based on the data from the OzAAR KO mice it is becoming more
accepted, that this subtype doesn’t play a functional role in vascular contraction.
The azBAR KO mice exhibits some hemodynamic differences compared to WT
mice. Clonidine doesn’t cause the initial BP increase in these animals and a
more pronounced fall in BP is observed compared to WT mice (Link et al. 1996).
Furthermore, the angR KO mice have an attenuated BP response to salt
loading, suggesting that this receptor plays a role in salt-induced hypertension
(Makaritsis et al. 1999). However, it is still to be elucidated whether the effect is
mediated by central, vascular or renal mechanisms. achR KO mice develop
hypertension in response to salt loading comparable to WT mice (Makaritsis et
al. 1999) and gross hemodynamic parameters are not affeCted by this receptor
deletion.

Even though a great amount of progress has been achieved in
characterizing the 02AR subtypes due to the availability of K0 models, it is
difficult to specify which 02AR acts in the periphery to mediate vasoconstrictor
responses. Although 023 and ago/SIRS are the most likely candidates for this
function, similar binding profiles of these two subtypes make it very difficult to
differentiate between them. GzBAR mediate pressor responses in anesthetized
rats (Docherty et al. 1998) and ochRs play a functional role in human

saphenous vein and also mediate cold-induced vasoconstriction (Gavin et al.

29

1997; Bailey et al. 2004). It has been suggested, that postjunctional oz). and
angRs play a more prominent role in arteries, while the achR might be
expressed especially in venous SMC (Docherty et al. 1998). However, nobody

has defined azARs in small capacitance vessels.

aAR distribution in mesenteric vasculature. As described in the
previous section, mesenteric vasculature is crucial in hemodynamic reglulation.
Distribution and involvement of different aAR subtypes has significant impact on
the regulation of this vasculature. In murine MA, only o1AR seems to mediate the
actions of NE (Perez-Rivera et al. 2004). Prazosin, an aIAR antagonist, causes a
parallel shift of the NE dose-response curve in this tissue. Studies with subtype-
selective antagonists suggested that 01 AAR and to a lesser extend the G1BAR,
mediate the NE responses in the MA (Martinez-Salas et al. 2007;Perez-Rivera,
dissertation). Furthermore, C11BAR subtype seems to play a more prominent role
in neurogenic constriction of MA (Perez-Rivera et al. 2005). Other studies have
suggested the preferential role of aIBAR in neurogenic constriction (Yang and
Chiba 2000).

In contrast, prazosin does not behave as a competitive antagonist of
NE mediated responses in the MV, suggesting the involvement of prazosin
resistant mechanisms, possibly GzARS in venous constriction (Perez-Rivera et al.
2007). Studies with aIAR-subtype selective antagonists in the MV indicated that
omAR plays the dominant role in PE-induced contraction, even though a slight

effect of C11BAR antagonist has also been observed (Perez-Rivera, dissertation).

30

The contractile role of (11 DAR has also been implied in the rat and mice aorta
(Tanoue et al. 2002), canine MV (Daniel et al. 1997) as well as in mice femoral
arteries (Zacharia et al. 2005).

It is interesting that despite differential functional involvement of the
three 01AR subtypes in MA and MV all three receptors were detected in MA and
MV by western blot using whole blood vessel homogenate. Furthermore, analysis
suggested that the three subtypes are expressed at similar level in MA and MV

(Perez-Rivera, dissertation).

GAR-mediated responses of MA and MV: Several studies have
implied, that isolated veins are more sensitive to exogenously applied NE
(Abdelmawla, et al. 1996; Perez-Rivera et al. 2004). Also, stimulation of
sympathetic nerves results into larger amplitude of depolarization with longer
duration in venous SMCs compared to arterial SMCs.(Hottenstein and Kreulen
1987). At any given frequency, the contraction in the MV is always left-shifted
relative to MA (Park et al. 2007; Luo et al. 2003).

Considering the differential function and embryonic origin of arteries
and veins, multiple independent mechanisms may underlie the enhanced
responsiveness of MV to adrenergic stimulation. Our preliminary data indicated
that indirect contribution of quR in MV and not in MA may enhance functional
responses in MV. Taken together with data from other studies that indicate the
role of ozAR in the enhancement of responses mediated by 01AR (Shepperson

1984; Haynes and Hill 1996; Daly et al. 1988; Wilson and Minneman 1991;

31

Reynen et al. 2000), we hypothesized that the cross-talk beween o1- and 02AR
may be a significant contributor to differences in the adrenergic responses
between MA and MV. While there is no evidence that the cross-talk between di-
and 02AR is based on heterodimer formation, the emerging concept of GPCR
heterodimerization as an ubiquitous process required for proper receptor
trafficking, signaling and enhancement of function provided an attractive target

for our hypothesis.
4. Receptor cross-tall

Early studies have suggested that the monomeric GPCR may not
completely explain GPCR function; when a chimera receptor composed of achR
and M3 muscarinic receptor, where the C-terminal portions were switched, was
transfected to COS-7 cells, no detectable binding for musCarinic or adrenergic
ligand was detected. However, when both chimeras were expressed together,
specific binding sites for the two ligands were detected, suggesting that perhaps
inter-molecular interaction between the two receptor chimeras alters the binding
pocket for ligand (Maggic et al. 1993). These initial observations have
revolutionized our perception about GPCR function and signaling, raising the
possibilities of existence of additional receptor phenotypes that can not be
simulated by expression of single receptor clones.

There is accumulating evidence, that signaling via a GPCR that

couples preferentially to one pathway may be affected by inputs from GPCR

32

coupling to other pathways (Maggi et al. 1980). Such interactions may affect
coupling specificity and efficacy, and therefore have important consequences for
physiological and pathophysiological processes. Interaction between receptors

can lead to a loss or enhancement of function (Werry et al. 2003).

Receptor heterodimerization. Interaction between GPCRs can lead
to a formation of heterodimers with pharrnacologically distinct properties from
monomeric receptors. For example, physical interaction between u and 6 opioid
receptors leads to a switch in receptor coupling from Gui/o to 60,, mediated
responses (Charles et al. 2003). In addition, heterodimerization may be required
for proper receptor transport and folding in order for the receptor to exert a
physiological function; for example the type R1 and type R2 GABA B receptors
are inactive as monomers when expressed alone in mammalian cells; it has been
observed that R1 is retained intracellularly as an immature glycoprotein when
expressed alone, whereas R2 is transported to the membrane efficiently but can
not bind GABA or mediate signaling. However, when co-expressed, the two
proteins reached the cell surface and were able to participate in functional
responses; in addition, cc-immunoprecipitation experiments revealed that these
two receptors physically interact (Jones et al. 1998; Kaupmann et al. 1998). The
fact that these two receptors were found to co-localize in the same brain region
and in addition the endogenous receptors could be co-immunoprecipitated from
the membrane preparation of brain cortex (Kaupmann et al. 1998) indicates that

heterodimerization is not a result of experimental artifact.

33

The significance of receptor heterodimerization in proper receptor
folding and transport has been also found to bear a great relevance in human
physiology. Coexpression of truncated CCR5 cytokine receptor that is not
trafficked to the plasma membrane caused the wild type CCR5 receptor to be
retained intracellularly, indicating that the wild type receptor heterodimerizes with
the tmncated receptor, leading to retention of the wilt type receptor in the
endoplasmic reticulum (Benkirane et al., 1997). Since the wild type CCR5
receptor is the main co-receptor for CD4 required for HIV entry, It has been
proposed that the dominant negative effect of the truncated receptor is
responsible for the slow onset of AIDS in patients who are heterozygous for this
mutation (Samson et al., 1996).

The rapidly accumulating data on receptor heterodimerization have
raised the possibility that dimerization might be an ubiquitous process which is
required for proper cellular expression of GPCR (Salahpour et al., 2004; thu et
al., 2006). Wlth the available data, however, it is hard to explain the existence of
a ubiquitous domain that is involved in receptor dimerization. Available literature
indicates that receptor dimers can be formed through the interaction in their N-
terminal domains, C-terminal domains as well as through transmembrane
domains. Some studies even suggest, that dimerization involves a combination
of the three mechanisms.

As an example, N-tenninal extracellular domain contributes to
dimerization of 0 family of receptors. The dimers in this family are connected by

disulfide bridges (mGluR, GABABR) (Tsuji et al., 2000; Romano et al., 1996).

Interactions through TM domains have also been observed, particularly TM1 and
TMVI and TMVII have been implicated in dimer formation (Hebert et al. 1996).
Even though interactions between TM domains might be harder to investigate,
employing a peptide corresponding to the TM domain suspected in interaction
may dissociate dimer formation. Using this approach, TMVI has been suggested
as the interface for the dimerization of BzAR (Hebert et al., 1996). Similarly, TMVI
and VII have been suggested to be involved dopamine receptor dimerization (Ng
et al. 1996). Heterodimerization can also be formed by interaction of the C-
terminal domains; GABABR1 and R2 have been shown to dimerize through the
C-terminus (Kaupmann et al., 1998).

Considering the implications of receptor dimerization in receptor
trafficking, signaling and pharmacological properties, it is very important to
understand its regulation. Studies suggest that heterodimerization can be either
increased, decreased on remained unchanged by the actioniof agonists (Bouvier
2001). As described above, heterodimerization is constitutive and is even
necessary for proper trafficking for certain receptors. These receptors usually
form dimers before they reach the plasma membrane in the endoplasmic
reticulum. In other cases agonists can increase the stability of receptor dimers.
Activation of B2AR by the agonist isoproterenol has been shown to stabilize
receptor dimer, while inverse agonist timolol shifted the equilibrium towards
monomeric receptor species (Hebert et al., 1996). In the case of 6-opioid
receptors, dimerization is decreased after exposure to agonist. However, this

process seems to be dependent upon receptor internalization; morphine that

35

does not induce receptor internalization has no effect on the balance between
monomers and dimers (Cvejic and Devi 1997).

Because GPCR regulate multiple physiological functions of
cardiovascular system, receptor heterodimerization might have great implications
in modulating receptor responses in this system. Indeed, I31- and B2AR have
been shown to heterodimerize in intact cardiomyocytes and this interaction
resulted into increased ligand binding affinities for both subtype-selective ligands.
The altered binding properties were also accompanied by enhanced efficiency of
BAR agonist isoproterenol to stimulate intracellular response including cAMP
production and contractility. In addition, the heterodimerization between these

two receptors inhibited spontaneous activity of B2AR (Zhu et al., 2005).

Receptor heterodimerization in cardiovascular system. There is
also an example, how receptor heterodimerization may be involved in
cardiovascular disease. In patients suffering from preeclampsia it has been noted
that there is an overall increase of sensitivity to vasopressor angiotensin II.
Because the increased sensitivity was exclusive for angiotensin II, altered
signaling for angiotensin receptor (AT) has been proposed as the underlying
mechanism in this disease (Haller et al. 1989). Later studies have shown that
physical interaction between AT1 receptor and receptor for vasodepressor
bradykinin (B2) results into significantly enhanced responsiveness to angiotensin
II (AbdAIla, et al., 2000). Because increased AT1-82 heterodimer density and

angiotensin II signaling has been detected in the platelets and in omental vessels

from preeclamptic patients, it indicated that this heterodimer may at least partially
contribute to the enhanced angiotensin II responsiveness observed in this
disorder (AbdAlla et al., 2001). While the total amount of AT1 receptors did not
change during preeclampsia, the increased amount of heterodimers was due to

increased B2 expression levels.

Dimerization of GAR. Receptor dimerization seems to be ubiquitous
for GAR family members when these receptors are heterologously expressed.
While there are many reports on GAR and their involvement in receptor
dimerization, none of these reports relates this phenomenon to a physiological
situation and therefore its relevance needs to be further explored. Data indicate
that all three O1AR subtypes are able to form homodimers and homooligomers
when expressed in HEK293 cells (Vicentic et al., 2002; Uberti et al., 2003). In
addition, GAR have been shown to heterodimerize with other members of the
family and out of these, G1DAR provides a great example of how receptor
dimerization regulates its trafficking and pharmacology.

C11DAR is known to be poorly trafficked to the plasma membrane when
expressed in different types of cells; N-tenninal of the receptor has been shown
to contain a sequence that prevents it to be efficiently trafficked to the membrane
while supporting its retention in the endoplasmic reticulum (Hague et al., 2004).
G1BAR physically interacts with G1DAR and in addition to promoting membrane
expression of amAR, the interaction between (113- and G1DAR leads to formation

of a receptor entity with distinct pharmacological properties (Hague et al., 2005).

37

The functional responses of the dimer are stronger than responses of either
receptor alone and even though the membrane expression of G1DAR is
significantly enhanced upon co-expression with OIBAR as assessed by
luminometer surface expression assay, no high affinity binding sites for BMY
7378, the C11DAR selective antagonist, are detectable. This indicates that ligand
binding properties of G1DAR have been altered after it forms a heterodimer with
G13AR (Hague et al. 2006). In addition to G1BAR, O1DAR has also been detected
to heterodimerize with B2AR. Similarly to previous example, this interaction
affects receptor trafficking by promoting surface expression of (11DAR. Stimulation
of cells co-expressing these two receptors leads to increased functional
responses to NE (Uberti et al., 2005). However, the pharmacological properties
of C11DAR are not altered by this heterodimerization as opposed to G1BAR co-
expression.

G13AR also forms heterodimers with GIAAR, although the properties are
of the receptors are not altered by this heterodimerization. The functional
significance of this interaction remains to be elucidated (Uberti et al., 2003).

G2AR have also been also shown to form homodimers and oligomers
when expressed heterologously. G2BAR forms homodimers and a truncated
receptor mutant (G2BARm) that is retained in the endoplasmic reticulum confers
negative effect on surface trafficking of WT receptor, indicating a crucial role for
receptor dimerization in proper receptor trafficking to the plasma membrane. In
addition, the G2BARm also inhibited membrane trafficking of 02A- and GchR,

suggesting a role for heterodimerization between G23AR and the other G2AR

38

subtypes (Zhou et al., 2006). While the functional consequence for this homo-
and heterodimerization remains to be elucidated, if nothing else, this interaction
might be an important tool for receptors to be properly trafficked in order to exert
their function on the plasma membrane.

G2AAR has been shown to heterodimerize with G2CAR and this
interaction results to altered receptor desensitization properties; G2AAR
undergoes a rapid agonist-induced desensitization mediated by GRK
phosphorylation when expressed by itself (Eason and Liggett 1992). On the other
hand, G2CAR does not undergo receptor phosphorylation by GRK under similar
conditions, suggesting differences in the regulation of these receptors (Jewell-
Motz and Liggett 1996). However, formation of heterodimer between these two
subtypes results into attenuated phosphorylation of G2AAR by GRK and
decreased B-arrestin activation, suggesting functional implications in this
interaction (Small et al., 2006). This interaction might be important in the nerve
terminals, where these two receptors are believed to be co-expressed.
Considering the rapid desensitization of G2AAR when exposed to agonist, its role
in auto-inhibition would not be very effective on the sympathetic nerves that
exhibit a basal tone of firing and NE release if the receptor desensitized very
quickly upon NE exposure.

In addition to interactions occurring within the GAR family, several
studies reported cross-talk between G2-and BAR families. Since these receptor
types exhibit opposing action of adenylate cyclase activity, it might be expected

that if co-expressed in the tissue, they would antagonize each other when

39

activated by NE or EPI. However, several studies have shown that BAR and
G2AR can modulate each others pharmacology; in renal tissue stimulation of
G2AR results into decreased affinity of isoprenaline for BAR (Woodcock and
Johnston 1980) and in cerebral cortical slices it has been shown that stimulation
of BAR results into increased G2AR binding (Maggi et al. 1980). In addition, G2AR
co-stimulation can increase the responsiveness of BAR (Northam and Mcbley
1985). It has been recently shown that heterodimerization between BI- and
G2AAR may underlie some of the initial observations (Xu et al., 2003). The B1-
G2AR heterodimer has altered ligand binding properties compared to BIAR alone
and also exhibits a greater potency to stimulate adenylate cyclase.

Similarly to G1DAR, OchR has been shown to be localized
intracellularly when expressed in HEK293 cells. Screening of 25 related GPCR
revealed that only B2AR is able to enhance the membrane expression and
functionality of OchR (Prinster et al., 2006). This finding is interesting in the light
of data supporting heterodimerization between 010- and B2AR. In particular, our
studies (chapter 2) suggest that (12c and G1DAR are the main functional receptors
in MV.

In the murine MV, G2AR agonists such as clonidine and UK14304 do
not cause venoconstriction. Nonetheless, our studies suggest that G2AR is
involved in mediating the actions of NE in murine MV, even though its actions
might be indirect. While yohimbine, an G2AR antagonist, does not affect arterial
responses to NE, it produces a rightward shift of the dose-response curve to NE

in MV (chapter 2).

4O

The finding that G2AR blockade shifts the NE dose-response curve to
the right and G2AR agonists do not cause a significant venous constriction
suggests that G2AR may indirectly contribute to venous but not arterial
constriction. NE binds to G1 and G2AR and the G2AR may synergistically interact
with the O1AR in veins to enhance constriction to NE. In fact, a very early study
proposed a synergistic interaction between the two receptors in the saphenous
vein (Daly et al., 1988). Because nobody investigated the role of receptor
dimerization as a mechanism behind 01-02 synergistic interaction, the purpose of
a significant portion of this dissertation was to exclude/confirm the formation of

receptor heterodimer as a basis for this many times reported receptor interaction.

Dimerization independent receptor cross-talk. Initially, we tested
the hypothesis that synergistic interaction between (11- and G2AR is based on
heterodimer formation. However, after evaluation of the data that tested this
hypothesis, we have concluded that likely a different mechanism is responsible
for this functional receptor interaction. In the section below, examples of
interaction are presented that do not include formation of heterodimer.

Interactions between two receptors can occur via their intracellular
signal transduction pathways to produce a distinct functional effect (Werry et al.,
2003). Wnile there are many examples in the literature describing synergistic
effect produced by activation of two separate pathways, the molecular
mechanism is many times not clarified. There are multiple points, where two

transduction pathways can interact to produce a synergistic effect on mobilization

41

of Ca2+ that leads to enhanced response. Similarly to the interaction between
AT1 and 82 receptor in preeclamptic patients that contributes to disease
pathology, in many diseases including hypertension, interactions may contribute
to enhanced signaling and disease pathology. Elucidation of a functional
mechanism underlying receptor cross-talk may therefore reveal novel targets for
therapeutic interventions.

Multiple studies have demonstrated a potentiating interaction on [Ca2+]i
between Gui and qu coupled receptors. CXCR2 is a chemokine receptor that
preferentially couples to GGi (Damaj et al., 1996). When expressed in HEK-293
cells, activation of this receptor by its ligand interleukin-8 (IL-8) did not cause
Ca2+ elevation in the cells. However, following treatment with ATP that activates
endogenous P2Y1 and P2Y2 receptors in these cells, lL-8 was able to produce a
substantial Ca2+ elevation that did not seem to be dependent on influx,
suggesting enhanced mobilization of Ca2+ from intracellular stores (Werry et al.,
2002). While the molecular mechanism was not determined, the authors
proposed that GGq causes a reversible change on PLCB that reveals a binding
site for GBy. Another possible explanation for this finding is potentiation of PLC
activity.

Potentiation of PLC activity. There are multiple examples of how a
receptor cross-talk leads to potentiation of PLC activity. Treatment of HEK293
cells stably expressing M3 muscarinic receptors by carbachol leads to long-
lasting sensitization of PLC activity. Responses to purinergic stimulation,

carbachol and thrombin were significantly enhanced after carbachol pretreatment

42

for as long as 90min (Schmidt et al. 1996). Moreover, this potentiation was
observed even upon direct stimulation of G proteins by NH and GTva in
permeabilized cells. The study also revealed, that carbachol pretreatment leads
to increased substrate supply Ptdlns(4,5)P2 (PIP2 ) for PLC in addition to
increased PLC activity and this may be very important because limited amount of
substrate for PLC may limit its activity to produce IP3 and DAG. These data
indicate that PLC sensitization does not result from a direct coupling of receptors
to PLO-stimulating G proteins, but rather occurs downstream of G protein
activation.

While M3 receptors have been shown to activate both Gi and an
proteins in transfected cells (Offerrnanns et al. 1994), the enhanced PLC activity
by carbachol seems to be at least partially mediated by Gi proteins due to its
sensitivity to PTX treatment. However, Gq is also likely involved in increased
PLC activity observed after carbachol treatment because even in the presence of
PTX, increased accumulation of PLC substrate PIP2 was observed. Even though
inhibition of PKC did not affect acute PLC stimulation, a subsequent study has
shown that the long-term potentiation of PLC activity is also dependent on PKC
(Schmidt et al., 1998). In addition, only a combined treatment of PTX and
staurospcrine (PKC inhibitor) was able to abolish increased PIP2 substrate
supply for PLC, suggesting multiple mechanisms involved.

Potentiation of PLC activity might be also relevant for the interaction
between 01-02AR; PTX-sensitive GGi that are able to regulate PLC (Katz et al.,

1992; Jiang et al., 1996) are known to be activated by G2AR in the vascular

43

SMCs. PKC has also been suggested to be activated by G2AR in the vasculature
and play an important role in its mechanism of contraction (Aburto et al., 1995).

The increased PIP2 supply seems to be an attractive target for receptor
interactions, because continuous re-synthesis of this substrate seems to be
required for acute and sustained inositol phosphate signaling (Willars et al.,
1998). It is interesting that stimulation of Rho kinase may also increase the levels
of PIP2 by enhancing activity of PIP5- kinase (Chong et al., 1994) and Rho
kinase is a downstream target of many contractile GPCR including (11- and G2ARs
(Roberts et al., 2004; Tsai and Jiang 2006; Dimopoulos et al., 2007). Activation
of PIP2 by Rho kinase likely also involves activation of tyrosine kinases that work
in the same pathway. In HEK293 cells, the levels of Ptdlns(4,5)P2 are reduced by
inhibition of tyrosine kinase and this effect is similar to Rho kinase inhibition
(Rumenapp et al., 1998). Since many studies point to functional coupling
between G2AR and tyrosine kinases as well as Rho kinases (Carter and Kanagy
2002; Jinsi and Deth, 1995), increased PIP2 production as a mechanism
underlying 01-02AR synergistic interaction could also be considered.

While the enhanced PLC activity should lead to enhanced IP3
production in receptor cross-talk, IP3 levels are not necessarily elevated in a
synergistic cross-talk (Yeo et al., 2001). In certain cases, the enhanced Ca2+
mobilization could be due to sensitization of lns(1,4,5)P3 receptors. PKA can
sensitize these receptors through phosphorylation. This type of interaction has
been described in many cell types including parotid acinar cells and hepatocytes

(Bruce et al., 2002; Hajnoczky et al., 1993). This mechanism is likely relevant in

cells where Gs coupled receptors potentiate Gq-mediated responses, because
activation of PKA is dependent on activity of adenylate cyclase.

Activation of Ca2“ influx. In the previous examples, enhanced
mobilization of intracellular Ca2+ leading to enhanced responses seems to
underlie receptor synergism. However, potentiating interaction can also be based
on enhanced Ca2+ influx from extracellular space. For example, neuropeptide Y
sensitizes rat MA to responses mediated by several contractile receptors
including GAR; the tissue sensitization is partially mediated by increased Ca2+
influx through L-type calcium channels in the presence of neuropeptide Y due to
the sensitivity of the potentiation to nitredipine, the L-type calcium channel
antagonist (Andriantsitohaina and Stoclet 1988). In addition, some of the
reported 01-02AR synergistic interactions seem to be based on potentiation of

Ca2+ influx (see below).

Pathways involved in a1-G2AR cross talk. While several studies
documented 01-02AR synergistic interaction, they do not seem to point to a single
mechanism that would underlie this phenomenon. One study described 01-02
receptor interaction in cell line overexpressing G2ARs (Reynen et al., 2000), while
- other studies documented this interaction in tissue and native cells.

Chinese hamster lung fibroblasts (CHL) natively express O1AR,
although the specific subtype has not been characterized. When OzAAR was cc-
expressed in these cells, neither PE nor UK14304 were able to increase [Ca2*ji.

However, NE was able to cause concentration-dependent elevation of Ca2+. In

45

addition, when PE was co-applied with constant concentration of UK 14304,
dose-dependent Ca2" raise has been detected. This elevation of Ca2+ was
inhibited by both prazosin and rauwclscine (Reynen et al., 2000). However, the
mechanism behind this enhanced Ca2+ mobilization has not been investigated.
Similar interaction has also been described in neuronal cells where instead of
Ca2+ measurements the amount of 3H-inositol phosphates was quantified,
perhaps providing better mechanistic information about this synergism; in primary
glial cell cultures, PE caused only a fraction of inositol phosphates being
produced compared to NE. However, upon addition of UK14304, PE became
much more efficacious in inositol phosphates production (Wilson et al., 1991).
The enhanced inositol phosphate production perhaps leads to increased Ca2+
mobilization, as described by previous study.

Another, slightly different mechanism of interaction has been
suggested for G1— G2AR in the cauda epididymis of the guinea pig. In this tissue,
xylazine (G2AR agonist) did not produce a concentration-dependent constriction
like PE did. Similarly to the previous examples, addition of xylazine significantly
enhanced PE - mediated constriction. This enhanced response seems to be
mediated by Gi due to its sensitivity to PTX and seems to be dependent on Ca2+
influx through L-type calcium channels, because pre-incubation of the tissue with
L-type calcium channel blocker nifedipine attenuated the effect of xylazine. In
contrast to the previously described studies, xylazine did not potentiate
accumulation of inositol phosphates mediated by PE, suggesting that the

synergism between the two receptors is based on Gi-dependent potentiation of

Ca2+ influx instead of increased Ca2+ mobilization from the stores (Haynes and
Hill 1996). In fact, a similar mechanism has been described in rat tail artery; G2AR
did not affect Ca2+ mobilization from intracellular stores but was able to potentiate

Ca2+ influx through activation of calcium channels (Xiac and Rand 1989).

5. Non-adrenergic non-cholinergic regulation of vascular tone.

Sensory innervation of blood vessels. While SNS is the major
regulator of vascular tone, sensory nerves that also innervate blood vessels are
very important in the regulation of vascular homeostasis. Sensory nerves
function as afferent fibers collecting information about the environment and
carrying it back to the CNS. In addition, these fibers also have efferent function;
they may be activated locally to release vasoactive sensory neuropeptides
including substance P, NO, vasoactive intestinal peptide (VIP), ATP and
calcitonin gene-related peptide (CGRP) from the nerve endings (Holzer 1991;
Lundberg 1996). NO and CGRP are very potent vasodilators that antagonize the
effects of SNS; recently it has been noted that CGRP may play a greater role in
reduction of MA than MV tone mediated by perivascular nerve stimulation; the
arterial reduction of perfusion pressure in response to CGRP was at least
partially dependent on intact endothelium(Legros et al. 2007).

Substance P and ATP are involved in neuromodulation of sympathetic
and parasympathetic nervous system. Substance P can cause both vascular

contraction and relaxation (Whittle et al., 1989); it is interesting to note, that

47

substance P relaxes MA but constricts MV (Galligan et al., 2006). This may be
due to differential distribution of receptors between MA and MV, with contractile
NK-3 receptors expressed in the venous SMCs and NK—1 receptors coupled to

relaxation expressed in endothelial cells in arteries.

Substances modulating the vascular tone.

In addition to the above described mechanism regulating the vascular
tone, a variety of relaxing and contracting factors are released by the endothelial
cells located on the luminal side of the blood vessels. Vascular tone is
determined by the balance between vasoconstrictor and vasodilator stimuli that
directly affect [Ca2+], and Ca2+ sensitivity. The primary endogenous dilator is NO
whose production is induced by Ca2+- mobilizing agents and fluid shear stress
(Covers and Rabelink 2001). Other relaxing factors derived from endothelium
include prostaglandins and prostacyclins produced by the action of
cyclooxygenase. Activation of receptors in the endothelium can increase
production of these agents and G2ARs mediate vasorelaxation through the
release of NO (Nishina et al., 1999; Vanhoutte 2001).

The predominant vasoconstrictor agents released from the
endothelium include endothelin-1, angiotensin II, tromboxane A2 and
prostaglandin H2 (Ungvari and Koller 2000). Besides these well established
endothelium-derived vasoactive substances, there is a great number of

circulating hormones that contribute to short and long-term BP regulation.

48

Myogenic regulation of vascular tone

The contractile state of vascular smooth muscle primarily depends on
intracellular calcium concentration [Ca2+], that is modulated by vasoactive agents
and mechanical stimuli. In addition, the relationship between [Ca2+]I and vascular
tension can be modified by altering Ca2+ sensitivity of contractile proteins. This
mechanism also significantly contributes to regulation of vascular tone.

Regulation of vascular tone is very dynamic and besides circulating
vasoactive substances, intravascular pressure and flow also alter the local tone
of the vasculature. Intraluminal pressure alters membrane potential of vascular
SMS and this has a significant effect on vascular reactivity.

The signal transduction pathway mediating the effect of pressure on
vascular tone is complex. lntravascular pressure acts as mechanical force on the
vascular wall and distends it. The fact that tension of the SMCs inversely
correlates with the membrane potential has been discovered long time ago. It
has been found that stretching strips isolated from the guinea pig taenia coli
produced progressive depolarization and tension development compared to
unstretched preparations (Bulbring, 1954). Trachealis muscle cells progressively
depolarized with step increases in cell length (Coburn 1987). However, because
membrane potential came back to baseline despite maintained increase in cell
length, author proposed that depolarization occurred in response to stretch as

opposed to the maintained length itself. There is evidence that stretch-induced

49

non-selective cation channels play a role in depolarization of SMCs in response
to stretch (Davis et al., 1992). Depolarizing the cell to lower potentials leads to
activation of voltage-dependent calcium channels (Meininger and Davis 1992).
The ensuing rise in intracellular Ca2+ activates contractile machinery and induces
contraction. Increases in the intravascular pressures underlie the myogenic tone
that is very important in the local regulation of blood flow. Increasing
intravascular pressure leads to cell depolarization and development of the tone,
limiting the blood flow and preventing tissue overperfusion. Myogenic tone plays
a very important role in the resistance arteries and with decreasing lumen
diameter, myogenic tone is more prevalent.

Data on the local regulation of venous myogenic activity are not very
extensive compared to arteries. Early studies demonstrated the existence of
myogenic tone in special veins like the portal vein in the rat and small veins of
the bat wing (Johansson and Mellander 1975). One of the early studies detected
spontaneous depolarization in MV of guinea pig, indicating the existence of
myogenic tone in this tissue (Van Helden 1991). Some later studies suggested
that myogenic tone correlates with chronic intraluminal pressures and veins that
exhibit low pressures in vivo exhibit low degree of myogenic tone. For example,
in the human cephalic vein, no myogenic tone has been detected whereas larger
veins exposed to higher pressures in vivo like the saphenous veins were found to
exhibit myogenic activity (Szentivanyi et al., 1997; Berczi et al., 1992). In rat MV,
myogenic tone did not increase with stepped increases of intraluminal pressure

like in MA (Li et al., 2005).

50

Even though the extent of myogenic activity in veins is smaller than in
arteries, the significance of venous regulation by myogenic mechanism might be
still important; even a small increase in muscle tension due to pressure may
significantly attenuate the passive dilation of the highly distensible veins,
significantly attenuating the vascular capacity. Increased reactivity of capacitance
veins may contribute to arterial hypertension.

The myogenic activity of systemic veins is also very important from
physiological standpoint: when the venous pressure in the lower limbs increases
by the effect of gravitational force after standing up, activation of myogenic
activity prevents orthostatic intolerance. Conversely, attenuated myogenic activity
of systemic veins may contribute to hypotension and edema (Monos et al., 1995).

Because intravascular tone will alter the membrane potential and tone
of vascular SMCs, the reactivity of blood vessels will also differ under
pressurized and non-pressurized conditions. Physiologically, mice MA are
exposed to high pressures in vivo (60 mm Hg) while MV belong to low pressure
system. Therefore, the difference between the physiological intravascular
pressures and pressures of isolated vascular preparation is not very large in the
MV, but is large in MA. Therefore, studies of non-pressurized veins are likely to

be physiologically more relevant than studies of non-pressurized arteries.

51

6. Biochemistg of the regulation of vascular tone

Regulation of vascular contractility.

Regulation of vascular tone centers on the regulation of [Ca2+]i in the
vascular SMCs. Stimulation of blood vessels by vasoconstrictor agents ultimately
raises [Ca2+]i, leading to activation of Ca2*-calmodulin dependent protein kinase.
Ca2*-calmodulin dependent protein kinase then stimulates myosin light chain-
kinase (MLCK) which phoshorylates myosin light chains (Kamm and Stull 2001).
Activation of myosin ATPase activity by MLCK enables actin and myosin to
interact and generate tension.

Conversely, relaxation of SMCs is tightly coupled to dephoshorylation
of myosin light chain by myosin light chain phosphatase (MLCP). The activities of
both MLCK and MLCP are tightly regulated and while MLCK is activated by
calcium/calmodulin that directly links intracellular [Ca2+] to SMC contractility,
MLCP is activated by both vasodilator and vasoconstrictor stimuli and therefore
contributes to the Ca2*-independent regulation of vascular contractility. NO
activates guanylate cyclase which synthesizes cGMP and the resulting activation
of protein kinase G (PKG) directly activates MLCP (Surks et al., 1999).
Regulation of MLCP activity is not only important during vascular relaxation, but it
is considered the most important mechanism that influences Ca2+ sensitivity of

the contractile apparatus.

52

Regulation of vascular tone by modifying Ca2” sensitivity.

It has been established that the relationship between the extent Ca2+
elevation and tension development varies based on the alteration of Ca2+
sensitivity. Agonist-mediated elevation of Ca2+ produces greater tension at a
given Ca2+ than depolarization (Karaki 1989; Somlyo and Himpens 1989; Hirano
2007). Agonist stimulation initially increases MLC phosphrylation through
elevation of [Ca2+]i, but this is Immediately followed by decrease of MLCP
activity, leading to increases Ca2+ sensitivity (Somlyo and Somlyo 1994). These
two mechanisms cooperate in modulating vascular constriction.

Multiple second messengers mediate Ca2+ sensitivity. However, Rho
A, a Ras-related small monomeric GTPase is believed to play the most important
regulatory role (Hartshome et al., 1998). Activation of many GPCR and the
downstream stimulation of Rho - associated kinase has been shown to inhibit
MLCP by phosphorylation of myosin phosphatase target subunit 1 (MYPT1)
(Feng et al., 1999; Kitazawa et al., 2003). Multiple GPCRs regulate Ca2+
sensitivity by activation of Rho kinase; in vascular SMCs the receptor-dependent
activation of Rho kinase relies on activation of G12/13 and this pathway seems to
be independent of Gq mediated phosphorylation of MLC (Gohla et al., 2000).
Even though the role (3012/13 in activation of Rho A pathway has been well
established, recent data indicate that qui can also activate Rho kinase in PLC-

independent manner (Vogt et al., 2003). However, similar studies in vascular

53

SMCs need to be performed to confirm that this mechanism is relevant to the
regulation of vascular tone.

GAR induce large increases of [Ca2+]i and activation of Rho kinase
(Roberts, 2004; Tsai and Jiang 2006; Dimopoulosiet al., 2007). Activation of Rho
kinase by NE and some other contractile agents is regulated by phosphoinositide
3; kinase (PI3K) that acts upstream of Rho kinase (Wang et al., 2006). In
addition, src family of tyrosine kinases may be involved in activation of Rho
kinase (Nagao et al., 1999).

In addition to Rho kinase, PKC can also regulate Ca2+ sensitivity of
vascular SMCs. PKC is activated by DAG that is formed by activation of G
proteins, including Gi and Gq. It has been suggested, that PKC also enhances
Ca2+ sensitivity via its inhibitory action on MLCP (Masuo et al., 1994; lkebe and
Brozovich 1996). Wnile PKC has not been shown to phosphorylate MYPT1
directly as has been demonstrated for the Rho associated kinase, studies have
indicated that smooth muscle specific phosphoprotein CPI-17 acts downstream
of both Rho A and PKC activation to directly and potently inhibit MLCP (Kitazawa
et al., 2000). This suggests that CPI-17 might be a point of convergence for the
Rho and PKC pathways. Expression of CPI-17 seems to correlate with Ca2+
sensitivity and PKC-dependent constriction; vascular smooth muscle that exhibit
tonic activity also exhibit increased Ca2+ sensitivity and express larger amount of
CPI-17 than smooth muscle that exhibits phasic constriction (Woodsome et al.,
2001). Differences in the expression of CPI-17 between MA and MV could also

contribute to differential sensitivity to contractile agonists. In addition, changes in

the expression of CPI-17 might be relevant in hypertension, where enhanced

sensitivity to agonist stimulation has been noted. Increased vascular reactivity

 

 

 

Ca2+ channel

i I
"23 DAG Rho kinase

17

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Fig. 1. Proposed dual mechanism for vascular
sensitization. Circulation research, 2007 (100):121-129.

 

 

 

also occurs in
diabetes and
type 2 diabetic
mice have been
shown to have
increased level of
activated CPI-17
as well as RhoA
(Xie, Su et al.
2006).

The

signal

transduction involving Ca2+ sensitization is well coordinated to maintain signal

specificity. Under resting conditions, PKC, RhoA and ROK are distributed evenly

in the cytosol. However, activation or receptor can induce translocation of these

important regulatory proteins to the plasma membrane, contributing to the

efficacy of agonist-induced constriction (Taggart et al. 1999).

A recent study has proposed a new mechanism of Ca2+ sensitization in

SMCs that occurs within seconds of agonist stimulation. In rabbit femoral artery,

PE induced phosphorylation of MLC occurred at very similar time points as

phosphorylation of CPI-17, the main inhibitory protein of MLCP. This rapid

55

activation was dependent upon Ca2+ release from SR and also PKC. Activation
of CPI-17 together with MLCK synergistically increased MLC phosphorylaton,
resulting into constriction. This rapid Ca2+ sensitization was followed by slower
onset, Rho/PKC-dependent CPI-17 phosphorylation (Dimopoulos, et al. 2007).
Fig. 1 provides a diagram of pathways involved in the proposed dual inactivation
of MLCP (Taken from Circulation research, 2007 (100):121-129).

Rho kinase in arteries and veins. It is well established that regulation
of Rho kinase pathway plays an important role in agonist-induced constriction
and the degree of its involvement affects vascular constriction. A study has found
that inhibition of Rho kinase may produce greater reduction of agonist induced
constriction in veins than arteries (Robertson et al. 2007) suggesting that
perhaps Ca2+ sensitization mediated by agonist may play a greater role in
regulation of venous than arterial tone. If this also applies for small MV, it would
be also a possible contributor to their enhanced sensitivity to multiple

vasoconstrictors.

Modulation of Ca2+ sensitivity by G2AR. Besides directly stimulating
MLC, another mechanism by which G2AR can modulate vascular tone is
independent of MLC phosphorylation and involves increase in Ca2+ sensitivity
(Aburto et al. 1993). As discussed above, this can be mediated by PKC that is an
upstream target of CPI-17.

However, Ca2+ sensitization can be also mediated by tyrosine kinases

that play a role in G2AR-mediated constriction. Although the precise mechanism

by which tyrosine kinase elicits Ca2+ sensitization in SMCs has not been clarified,
studies suggest that src-family of tyrosine kinase are involved in the activation of
Rho kinase, resulting into inhibition of protein phosphatase (Nakao et al. 2002).
Even though Gi has been shown to activate tyrosine kinase and induce
constriction, role of other G proteins is also possible. In transfected HEK-293
cells, G12 protein activates Src tyrosine kinase that acts upstream of Rho kinase
(Nagao et al. 1999). While there is no report linking activation of G2AR to G12,
role of this protein in Ca2+ sensitization would be also plausible due to the
emerging role of this protein in Ca2+ sensitization.

The Ca2+ sensitization mediated by G2AR might also prove be
important in hypertension. In rats made hypertensive by NOS inhibition, G2AR
reactivity and Ca2+ sensitivity is enhanced. However, Ca2+ sensitization in these
animals is independent of tyrosine kinase activation and but rather involves PKC-
G (Carter and Kanagy 2003). Conversely, activation of PKC by non-adrenergic
agonists can enhance Ca2+ sensitivity (Jiang et al 1987). In dog MV, activation of
PKC ET-1 sensitizes the tissue to G2AR stimulation by UK 14,304 (Shimamotc et
al, 1995). Angiotensin II has also been shown to increase constrictions mediated
by G2AR in veins (Dunn et al. 1991). In addition, studies also indicate that CHAR
can also enhance the G2AR function in the saphenous vein (Daly et al. 1988),
cauda epididymis of the guinea pig (Haynes and Hill 1996) and rat tail artery
(Xiac and Rand, 1989). As will be discussed later, this phenomenon might also

occur in MV but not in MA, contributing to higher sensitivity of MV.

57

Regulation of Ca2+ desensitization. On the other hand, activation of
PKC and PKA in some SMCs seems to oppose RhoA/ROK pathway and
therefore involves Ca2+ desensitization. PKG is activated by cGMP that is
produced by guanylate cyclase and nitric oxide (NO). The exact mechanism by
which PKG inhibit Rho A has not been established but studies in vitro suggest
direct inactivation of RhoA by PKA and PKG phosphorylation (Somlyo et al.
2003). In addition, PKG also directly interacts with myosin phosphatase and this
interaction is required for cGMP-mediated activation of myosin phosphatase
(Surks et al. 1999). As discussed below, differences between MA and MV in the
functional GPCR and their modulation of Ca2+ sensitivity would also play an

important role in the differential regulation of arterial and venous tone.

Focus of my studies. While it has been well established that veins
are more sensitive to sympathetically - mediated stimulation than arteries, the
mechanism behind this enhanced sensitivity has not been explained. These
studies investigated the basic principles involved in the regulation of venous tone
at the level of SMCs, with the emphasis on G2AR contribution. Better
understanding of the receptors and their signaling pathways in the veins may
lead to increased awareness of processes than may be deregulated during

disease process such as hypertension.

58

CHAPTER 2

Hypothesis and Specific Aims

59

Overall hypothesis

The sympathetic nervous system is the main regulator of venous tone
and cardiac output. NE released from sympathetic nerves constricts MA and MV
by acting on GAR. However, MV are significantly more sensitive to the contractile
effects of NE compared to MA {Luo, 2003 #412}{Hottenstein, 1987 #433}{Perez—
Rivera, 2004 #330}. While multiple factors could underlie this enhanced
adrenergic sensitivity of MV, there are differences in the contribution of GAR
subtypes to NE-induced constrictions of MA and MV. In MV both 01- and G2AR
mediate NE-induced constriction while in MA G1AR only mediates constriction to
NE. G2AR potentiates the action of G1AR and receptor cross-talk could contribute
to enhanced adrenergic sensitivity of MV.

Using an integrative approach, my goal was to characterize the
mechanism of the functional interaction between (11- and G2AR that occurs in MV,
but not in MA. I hypothesized, that the interaction between these receptors
contributes to the increased calcium mobilization and adrenergic responsiveness
of MV compared to MA. To accomplish this goal, four specific aims were

addressed:
Sgcific aim #1: The overall goal of this aim was to test the hypothesis that

there is differential expression of C11- and G2AR subtypes in arterial and venous

myocytes obtained from murine mesenteric arteries and veins. Differential

6O

receptor expression would contribute to differences in the sensitivity of MA and
MV to adrenergic agonists.

Specific aim 1a: This aim tested the hypothesis that arterial and venous smooth
muscle cells (SMCs) express different GIAR subtypes. lmmunocytochemical
techniques were used to study receptor expression in freshly isolated murine
mesenteric arterial and venous SMCs.

Specific aim 1b: This aim tested the hypothesis that murine veins but not
arteries express G2ARs. Co-expression of both O1ARS and G2ARs in veins, but

not arteries, leads to receptor cross-talk in venous SMCs.

§9ecific aim E In this aim, I measured arterial and venous constriction in vitro
to investigate pharmacological interaction between 01 and G2ARs in intact MV
from mice. GAR subtype selective ligands were utilized to test the hypothesis
that G2AR synergistically interacts with G1AR. In addition, studies were performed
in MV of achR gene knockout (KO) mice to test for a functional role of GchR in
MV.

Sgcific aim #3: Interactions between (110- and G2ARs was investigated at the
molecular level, using heterologous receptor expression in HEK-293 cells. The
first hypothesis I tested was that the interaction is based on direct physical
interaction between the two receptors.

Specific aim 3a: G2AR subtypes were transiently co-transfected into GIDAR -
expressing HEK-293 cells and receptor co-localization experiments were

performed. In addition, (11- and G2AR co-expressing cells were treated with NE

61

and receptor trafficking was investigated. I also tested the hypothesis, that G2ARs
promote membrane trafficking of G1DAR.

Specific aim 3b: To test whether the functional interaction between (110- and
G2AR is occurs in HEK-293 cells, I used calcium imaging of HEK-293 cells co-
expressing (11D- and G2ARs.

gmcific aim # 4: Calcium dependent signaling mechanisms activated by of
GARs were investigated in intact MA and MV using a pressure myograph.
Dependence of functional responses on calcium stores and calcium channels
was assessed. Coupling of G2AR to calcium influx and its role in the sensitivity of

MV was investigated.

62

CHAPTER 3
Functional Interaction Between 01- and G2-Adrenoreceptors in

Mesenteric Veins Does not Require Heterodimer Formation

63

Abstract

Mesenteric veins (MV) are more sensitive to the contractile effects of
sympathetic nerve stimulation and G-adrenergic receptor (GAR) agonists than
mesenteric arteries (MA). We tested the hypothesis that MV co-express GIAR
and G2ARs and G2ARs interact directly with GIAR to increase adrenergic reactivity
of MV. We studied neurogenic and agonist-induced constrictions of MA and MA
in vitro and the subcellular distribution and function of GARs expressed in HEK-
293 cells in an effort to mimic MV receptor expression. Frequency response
curves (05-30 Hz) for neurogenic constriction and norepinephrine (NE)
concentration response curves (CRCs) were left shifted in MV compared to MA.
Yohimbine (G2AR antagonist) and MK912 (OchR antagonist) but not G2AAR or
G2BAR antagonists, produced rightward shifts in NE CRCs in MV but not MA. UK
14,304 (0.01-1 pM)(G2AR agonist) did not contract MA or MV but it potentiated
constrictions mediated by G1AR. NE constrictions in G2CAR knock-out (KO)
mouse MV were not different from wild type (WT) MV. lmmunocytochemistry
using dispersed MA and MV smooth muscle cells from WT mice revealed plasma
membrane expression of OzAAR and OchR. MV expressed G1DARS
predominantly in the intracellular compartment. In transfected HEK-293 cells,
G2ARs did not co-localize with GIDARS subcellularly and co-expression with
G2ARs did not alter O1DAR distribution. Co-expression with B2ARs increased
plasma membrane localization of C11DARS and enhanced calcium signaling

coupled to (11DARS. These data indicate that CID-OzARS do not interact directly to

enhance MV adrenergic reactivity. G2AR-linked intracellular signaling specific for

MV may account for enhanced adrenergic reactivity.

65

Introduction

Blood pressure regulation is complex and involves several interacting
systems including the sympathetic nervous system (SNS) which contributes to
regulation of body fluid volume and vascular tone (Guyenet, 2006). Sympathetic
nerves innervate arteries and veins and regulate their tone (Anderson et al.
1989). Vascular tone and structure of small arteries are the main determinants of
total peripheral resistance (TPR). Because veins store approximately 75% of
total blood volume (Martin et al. 1998), they are the main capacitance vessels in
the circulation. The storage capacity of veins is determined by structural
characteristics, but the SNS is the major regulator of venous tone (Pang 2001).
Since venous tone regulates capacitance, it directly affects blood return to the
heart and cardiac output (00) (Guyton 1955; Greenway and Lautt 1986).
Because blood pressure is a product of TPR and CO, regulation of venous tone
is critical for blood pressure control.

Despite the importance of venous tone in blood pressure control, veins
have not been studied as extensively as arteries. In addition, most studies used
large diameter veins (Gavin et al. 1997, Chen-Han Lee et al. 2002). The
hemodynamic functions of large conduit veins (vena cava, for example) greatly
differ from the functions of systemic, small diameter veins. Small veins,
particularly splanchnic veins, are the most important venous bed due to their
dense sympathetic innervation and high compliance (Pang 2001). These
characteristics have important implications in the pathophysiology of

hypertension, where reduction of venous capacitance occurs (Ferrario et al.

1970; Ricksten et al. 1981; London et al. 1985). When splanchnic storage
capacity is reduced, redistribution of blood from these vessels increases venous
return to the heart (Greenway and Lautt 1986). Reduced venous capacitance is
accompanied by an increase in 00, which also occurs in pro-hypertensive
humans (Drukteinis et al. 2007). These findings point to the relevance of studies
investigating control of venous tone in blood pressure regulation.

Previous work revealed that MV are more sensitive to adrenergic
stimulation than arteries (Hottenstein and Kreulen 1987, Perez-Rivera et al.
2004, Luo 2003). This might be due to artery-vein differences in GARs in vascular
SMCs mediating contraction. Several studies indicated, that G2ARs plays more
prominent functional role in veins compared to arteries (Flavahan et al. 1984,
Ruffolo 1986, Patel et al. 1981). Studies from our laboratory indicated, that G2AR
indirectly contributes to venous constriction, whereas only G1AR mediates NE -
induced constriction of arteries. Several studies indicated that G2AR can exert a
synergistic effect on G1AR responses; functional interactions between (11- and
G2AR have been detected in cauda epididymis (Haynes and Hill 1996), primary
glial cell cultures (Wilson and Minneman 1991) and rat tail artery {Xiao, 1989
#660}. (11-AR and G2AR interactions also occur in heterologous expression
systems (Reynen et al. 2000).

a-ARs are G-protein coupled receptors (GPCRs) and there is
accumulating evidence, that signaling via a GPCR that couples preferentially to
one pathway may be affected by inputs from GPCR coupling to other pathways

(Milligan et al. 2006). The interaction between GPCR can lead to a loss or

67

enhancement of function (Werry and Vlfilkinson, 2003). In addition, GPCRs can
form heterodimers or heteromultimers with pharmacologically distinct properties
from monomeric receptors (Levac et al. 2002; Milligan et al. 2003). GARs can
also form heterodimers (Hague et al. 2004; Uberti et al. 2005; Hague et al. 2006).
lmportantly, the GmAR that mediates constrictor responses caused by
norepinephrine in MV (Perez-Rivera, 2007) is known to heterodimerize with other
receptors (Hague et al. 2005; Uberti et al. 2005). Localization of the G1DAR is
predominantly intracellular when the receptor is expressed alone in HEK-293
cells. B2AR as well as G13AR can form heterodimeric partners with O1DARS and
as a result of this interaction, G1DAR is targeted to the cell membrane more
efficiently, resulting in enhanced signaling mediated by the C11DAR (Uberti et al.
2004; Uberti et al. 2005).

In the present study we screened for the expression and functional
involvement of different G2AR subtypes in venous SMCs in order to identify the
native ct-AR subtypes relevant to this interaction. We also performed parallel
immunocytochemical analysis of GARs expressed in arterial SMCs to asses the
correlation of receptor expression with reactivity of MA and MV. In addition, we
used tissue from G2CAR KO mice to determine the effect of this receptor on
adrenergic sensitivity of MV. We tested the hypothesis that G2AR can increase
membrane trafficking of G1DAR by heterodimerizing with this receptor in MV and
HEK-293 cells. This mechanism could be very important in MV due to the
functional importance of O1DAR in MV. In addition, we performed calcium

imaging studies in transfected HEK-293 cells to test, whether the functional

68

interaction we observe in MV could be mimicked in HEK—293 cells that co-
express 01DAR and G2ARs. We predicted that this heterodimer functions as an
independent signaling unit with enhanced stimulus-response coupling compared

to (J1DAR signaling alone.

69

Materials and Methods:

Animals. 057/Bl6 male mice (25-30g) were purchased from Charles
River Breeding Laboratories (Portage, MI). Mice were maintained in the animal
care facility, according to the standards approved by the Michigan State
University Institutional Animal Care and Use Committee. Mice were individually
housed in clear plastic cages with access to standard chow (Harlanfl'eklad 8640

Rodent Diet) and tap water.

Heterozygotes for the neomycin-disrupted locus coding for OchR were
purchased from Jackson Laboratories, Bar Harbor, MA. These mice have been
previously characterized (Link, Stevens et al. 1995). As the colony expanded,
heterozygotes were bred and their offspring (9-12 weeks of age) WT and KO
mice were utilized in experiments. A previously published PCR protocol

(www.jacx.org) was followed to characterize the genotype 0f individual pups.

In Vitro Preparation of Mesenteric Arteries and Veins. Mice were
anesthetized, and the small intestine with its associated mesentery was removed
and placed in oxygenated (95% oxygen, 5% carbon dioxide) Krebs' physiological
saline solution of the following composition (millimolar): 117 NaCI, 4.7 KCI, 2.5
CaCl2, 1.2 MgCl2, 1.2 NaH2PO4 25 NaHCOs, and 11 glucose. A piece of the
intestine with associated vessels was removed and pinned flat in a silicone
elastomer-lined (Sylgard; Dow Corning, Midland, MI) petri dish. A section of
mesentery containing vessels close to the mesenteric border was cut out using

fine scissors and forceps. The preparation was transferred to a smaller silicone

70

elastomer—lined recording bath and pinned flat. Second- or third-order mesenteric
veins or arteries (100-200-pm diameter) were isolated for study by carefully
clearing away the surrounding fat tissue. The recording bath containing the
preparation was mounted on the stage of an inverted microscope (Olympus CK-
2; Tokyo, Japan) and superfused with warm (37°C) Krebs' solution at a flow rate
of 7 ml/min. All preparations were allowed a 20-min equilibration period during

which the vessels relaxed to a stable resting diameter.

Video Monitoring of Vessel Diameter. The output of a black- and-
white video camera (Hitachi model KP-111; Yokohama, Japan) attached to the
microscope was fed to a framegrabber card (Picolo, Euresys lnc., TX, USA).
mounted in a personal computer. The video images were analyzed in real-time
using Diamtrak Edge-tracking software (version 3.5, Diamtrak, Adelaide,
Australia), which tracks the distance between the outer edges of blood vessel in
the observation field' Changes in vessel diameter as small as 1 pm could be

resolved.

Transmural stimulation of perivascular nerves. Two Ag/AgCl
electrodes connected to a Grass Instruments stimulator (844) were placed
parallel to the longitudinal axis of MA and MV. Parameters for nerve stimulation
were the following: 60 stimuli, 0.5 ms duration of stimuli, frequency from 0.5, 1, 5,
10, 20 and 30 Hz and 150 V. Blood vessels were allowed to equilibrate for 15

min between each stimulus.

71

Antagonist Studies. After tissue equilibration, antagonists were
applied for additional 20 min to attain equilibrium before NE concentration-
response curves were generated. BRL44408 was used as an OzAAR antagonist;
BRL44408 pKi values for G2AAR, (123AR and G2CAR are 8.2, 6.2 and 6.8
respectively. lmiloxan was used as an G23AR antagonist: imiloxan pKi values for
G2AAR, G2BAR and G2cAR are 5.8, 6.9 and 6.0 respectively. MK912 was used as
an OchR antagonist; MK912 pKi values for G2AAR, (123AR and achR are 8.9,
8.9 and 10.2 respectively. All drugs were purchased from Sigma-Aldrich (St.
Louis, MO) and were added in known concentrations to the superfusing Krebs'
solution. A single agonist concentration-response curve was obtained from each
preparation. Because veins became sensitized to NE when two dose—response
curves were generated using the same tissue, a fresh vein was used to construct

control dose-response curves.

Data Analysis. Constrictions of blood vessels are expressed as a
percentage of the resting diameter. Half-maximal effective agonist concentration
(E050) and maximum response (Emax) were calculated from a least-squares fit of
individual agonist concentration-response curves using the following logistic

function from Origin 7.0 (Origin-Lab Corp., Northampton, MA):
Y= {[Emin - m...1/[1+(xIEC50)"]+E,.,.,,.,,

where Emin is the minimum response and was constrained to zero and

n is the slope factor. All data are expressed as mean :t S.E.M. Statistical

72

differences between groups were assessed by Student's two-tailed unpaired t

test.

HEK-293 cell culture and transfection. Tissue culture media and
related reagents were purchased from lnvitrogen (Carlsbad, CA). HEK-293 cells
were maintained in Dulbecco’s modified Eagle’s medium plus 10% fetal bovine
serum at 37°C, in a 5% CO2 incubator. Cell passage 25 to 30 was used in
experiments. For heterologous receptor expression cells were diluted to 105
cells/ml in complete medium and 800 pl of the cell suspension was transferred to
disposable electroporation cuvettes (Bio-Rad). 2 pg of plasmid DNA was added

to the cell suspension and electroporation was performed using a Gene Pulser
(Bio-Rad), employing the following parameters: resistance= °°, capacitance =

950 pFarads at 270 volts. After the procedure, cells were transferred to a 15 ml
Falcon tube and spun down at 3000 rcf for 3min. The cell pellet was
resuspended in tissue culture medium (see above). The transfection efficiency in

our experiments was 15%.

Plasmids. Human 02A, (123 and B2ARs were purchased from the cDNA
Resource Center at the University of Missouri-Rolla (www.cdna.org). The Red
express plasmid was purchased from Clontech (Mountain View, CA). Flag-
tagged human OchR was kindly provided from Dr. Maqsood A. Chotani (Ohio
State University). Human O1DAR and HA-tagged C11DAR were kindly provided by

Dr. Chris Hague (Emory University Medical School).

73

(11DAR stable cell line. HEK-293 cells were transfected by
electroporation as described. We used a hemagglutinin-tagged (HA) G1DAR
where the HA tag was attached to the N-terminal portion of the human (1.1DAR.
This plasmid contains the antibiotic resistance gene for geneticin, which was
used for selection of transfected cells. Cells were grown for 4 weeks and
geneticin-resistant colonies were isolated and grown in separate flasks. The
individual colonies were than tested by Western blot and lmmunocytochemistry
to screen for colonies with the highest receptor expression. To select the final
colony, the colonies with highest receptor expression were further subjected to
calcium imaging (see below) to identify the clones that yielded the largest NE-

induced calcium signal.

Isolation of Mesenteric Vascular Smooth Muscle Cells (VSMC)
and lmmunocytochemistry. MA and MV veins from male C57B6 mice (25-30 g)
were harvested and cells were isolated according to previously described
protocols (Jackson and Huebner, 1997). The blood vessels were cleaned of
surrounding fat and connective tissue in a cold dissociation buffer of the following
composition (millimolar): 140 NaCI, 5 KCI, 1 MgCl2, 10 HEPES and 10 glucose
(pH adjusted to 7.4). Tissues were than incubated with papain in the presence of
dithioerythrol (1 mg/ml) for 35 minutes at 37°C and this incubation was followed
by digestion in a collagenase (1.9 U/ml) lelastase (0.15 mg/ml) solution with
soybean trypsin inhibitor. Cells were allowed to attach to poly-D-lysine — coated

coverslips for 1 hour before they were fixed.

74

Table 2. Source and working dilutions of the primary and secondary antibodies
used in GAR staining in venous SMCs and in HEK293 cells.

 

IPrimary antibodies against GAR subtypes and cellular organelles

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Antigen Source lHost species IDilution

010 Santa Cruz, CAlgoat 1: 300

02,. Santa Cruz, CAlrabbit 1:400

G23 Santa Cruz, CAlrabbit 1:400

G23 lNeuromics, MN lrabbit 1:400

HA lSigma Mouse 1 :200

Flag IOncogene, MA lmouse 1:300

Pan-cadherin Abcam, MA Irabbit 1:400

EEA1 Abcam, MA mouse 1:400

Calnexin Abcam, MA mouse 1:400

Secondary Antibody
Host I

Source . Dilution Fluorophore

speCIes

goat lgG .lackson lmmunolaboratory rabbit 1:500 |Cy3

rabbit lgG Jackson lmmunolaboratory lgoat 1:500 |Cy3

mouse lgG Jackson lmmunolaboratory rabbit 1:200 IFITC

mouse IgG Jackson lmmunolaboratory lgoat 1 2400 |0y3

 

 

 

 

lmmunocytochemical analysis. VSMCs were allowed 3-4 hours
to attach to poly-D-lysine coated coverslips. Both HEK-293 and VSMCs were

fixed using Zamboni fixative for 20 minutes. After fixation, cells were incubated

75

with a blocking serum (4% serum in PBS; pH 7.4) to diminish nonspecific
binding. After overnight incubation with primary antibody (Table 2) at room
temperature in PBS containing 0.01% triton, cells were incubated for 1 h with
secondary antibodies (Table 1) at room temperature. In some experiments, the
nucleus was stained with 4'-6-diamidino—2-phenylindole (DAPI, Sigma-Aldrich, St.
Louis, MO) during the last 10 min of incubation with secondary antibody. Cells
were then washed in PBS and staining was visualized using a Nikon TE2000-U
inverted microscope. Photographs were taken using a SPOT Insight Color
Mosaic camera (Mager Scientific, Inc.) with Metalmaging Series software.
Controls with no primary antibodies were used to ensure that binding is specific.
In addition, antibodies were tested in transfected HEK-293 cells for their

selectivity against other G2AR subtypes.

Luminiscence surface expression assay. Transfected cells were grown
overnight in a tissue culture flask. In 48 hours, cells were plated into 15-mm
dishes at a concentration of 105 cells/ml. After 5 hours, cells were rinsed three
times with PBS and subsequently fixed with 4% paraforrnaldehyde in PBS for 30
min. After washing out the fixative with PBS, cells were incubated with blocking
buffer (2% non-fat dry milk in PBS). After 30 min incubation, primary antibody
conjugated to horseradish peroxidase (HRP) was added to the blocking solution
(Bethyl Laboratories, Montgomery, TX). The antibody was washed out after 1
hour by rinsing the cells three times with blocking buffer. 3,3’,5,5’-
tetramethylbenzidine (TMB) substrate kit (Vector Laboratories, Burlingame, CA)

was used to quantify HRP-conjugated to primary antibodies. The reaction was

76

stopped after 1 hour following addition of 50 ul of 1 N sulfuric acid. Absorbance
of yellow reaction product was measured at 450 nm using u-Quant plate reader
(Bio-Tek Instruments Inc.). The absorbance measured corresponds to the

amount of cell surface receptor.

Calcium imaging. Transfected cells were plated on round coverslips
at 5 x 104 cells/ml. 24 h post-transfection, coverslips were incubated in 1 ml of
OPTI-MEM, containing 10 pl of pluronic acid and 1 uM Fluo—4-AM (Molecular
Probes, lnvitrogen). Cells were incubated at 37°C for 45 min. The coverslip was
then mounted on the stage of confocal microscope (Leica TCS SL confocal
microscope, Leica Microsystems, Bannockburn, IL). Cells were continuously
superfused at room temperature with HEPES buffer of the following composition
(millimolar): 147 NaCl, 5 KCI, 1 MgCl2, 2 CaCl2, 10 HEPES (pH = 7.4) and after 5
min, drug treatments were begun. After obtaining 0t-AR receptor mediated
responses, a response to ionomycin (1 uM) was obtained and agonist responses
were normalized to the ionomycin response. F luo-4 was excited using an argon
laser and a wavelength of 488 nm. Emissions were collected in the range of
wavelengths between 480 and 520 nm. Images (512 x 512) were obtained every
3 seconds for 3 minutes. Each experiment consisted of 5 pre—drug (baseline)
images followed by drug application for 60 s (20 images) and washout for 35
images (105 3). Drug responses were quantitated by measuring the area under
delta fluorescence (response—baseline) curve. Responses of transfected cells

from one coverslip (typically 5-10 co-transfected cells) were averaged and three

77

coverslips containing cells from the same transfection were used as one

experimental sample. N values represent pooled data from one transfection.

78

Results

Neurogenic constrictions of MA and MV. MA and MV exhibited a
frequency-dependent constriction in response to transmural electrical stimulation;
however, MV exhibited greater sensitivity to stimulation than MA. The frequency
response curve in MV was to the left of that in MA, E050 of 2.7 :l: 1.3 Hz in MV
(n=4) and 7.5 :l: 0.9 Hz (n=4) in the MA. Also, MV reached a higher maximal
constriction than MA (32 :l: 3% constriction in W vs. 26 t 2% constriction in
MA)(Fig. 2A). Tetrodotoxin (TTX, 0.3 M), an antagonist of voltage-gated
sodium channels in neurons, abolished responses to electrical stimulation in MA

and MV confirming that the constriction was neurogenic.

G2AR do not mediate direct constriction of MV. Differences in the
responses of MA and MV to nerve stimulation could be due to differences in
transmitter release and/or and postjunctional signaling in VSMCs. We focused
our attention on GAR signaling in the VSMCs that mediate the effect of NE
released from sympathetic nerve endings. We found that NE, which activates (11-
and G2ARs, was more potent in contracting MV than MA, with E053 of 0.02
$0.004 BM in MV and 4 t 0.6 (M in MA (Fig. 2B), (P < 0.05), n=6. In contrast,
the G2AR agonist UK 14,304 did not contract MA or MV (Fig. 3A) (MA not
shown). We tested the possibility whether the lack of effect of G2AR agonist on
vascular contraction could be potentially due to opposing effects of G2ARs acting

in the endothelium to stimulate synthesis and release of vasodilators including

79

 

—O—Vein
45' —O-Artery
% _ 6
=3 30. (i? 9/
s %/ é/2
: /
3 15- /§
g o
/

 

 

I U

0 f0 20 30
Frequency [Hz]

B. 45- "0‘ Vein
—O—Artery

% contraction
—¥ 0.:
9 P
©2\PO—I\
50*
l—-.--I

 

o- /’
10"°1o"’10*"10'710“’ 1o*"1o4
Norepinephrine [M]

Fig. 2. Mesenteric veins are more sensitive to adrenergic stimulation then
arteries. Transmural stimulation of perivascular nerves produced greater
constriction in MV than in MA (A). Similarly, concentration-response curve for
exogenously applied NE is shifted to the left in the MV (8).

80

 

 

A. —o— Control
45- —o—- lndomethacin (10 pM)
..[j— NLA (100 1.1M)
.53 30-
‘6
E
g 15-
o /g
o\°
omezfifpégfiw—
10” 10'7 102
(UK 14,304) [M]
B.
- —0— Control
45 —o— Yohimbine (0. 3 11M)
/
.5 30- 2 /2
/ i/
s 15- i i/
0
.\° //
0.

 

 

10* 10'8 10'7 10‘ 10'5
(Norepinephrine) [Illl]

Fig. 3. Addition of indomethacin or nitro-L-arginine did not alter the responses
mediated by G2AR agonist UK 14,304 in MV (A). Despite the small effect
produced by G2AR agonist UK14,304 in the MV, G2AR antagonist yohimbine
produced a significant rightward shift in the NE concentration-response curve (B).

81

nitric oxide (NO) or prostacyclin. We used indomethacin (10 (M), an inhibitor of
cyclooxygenase, to block prostacyclin production and nitro-L-arginine

(100 uM), an inhibitor of nitric oxide synthase, to block NO production. Neither
of these enzyme inhibitors revealed a constriction response caused by UK
14,304 (n = 7, Fig. 3A). However, yohimbine (0.3 uM), the G2AR antagonist,
caused a 7-fold rightward shift in the NE concentration dose-response curve in
MV (Fig. 38); the control NE E053 was 12 i 2 nM while in the presence of
yohimbine this value was 80 i 10 nM (n = 6, P < 0.05). These data confirmed
previous findings that G2ARs do not directly couple to NE-induced constriction of
MV (Perez-Rivera et al. 2007). Yohimbine did not shift the NE CRC to the right in
the MA (not shown).

Effect of G2AR antagonists on NE responses in pressurized MA
and MV. Previous studies were performed in the non-pressurized system.
Because sensitivity of blood vessels is enhanced when they are pressurized, we
wanted to test the effect of G2AR antagonists in both MA and MV under
physiological pressures. Because application of pressure will alter the functional
responses of MA more than MV due to larger physiological pressures, by this
experiment we also wanted to rule out the possibility that under these conditions,
G2AR antagonist would be able to inhibit NE responses in MA just like in the MV.
Because studies indicated, that yohimbine can block GIARs we used idazoxan
Instead of yohimbine. ldazoxan is a G2AR antagonist, that it is structurally
unrelated to yohimbine and has higher selectivity for G2ARs. ldazoxan did not

produce a significant shift of the E030 for NE in MA (Fig. 4A). However, it

82

A- Artery -—o— Control

 

70. —o— lazoxan (10M)
60:
.5 50:
*5 40- g
g 30: T/gl 26
o 20.
0 .
/gi>
10'” 10 10 10‘ 10'5
(Norepinephrine) [M]

B. Vein —o- control
70- --o— ldazoxan (10M)
601 §

g 501 /+/ 0:0»
b 30«
g I
o 2011 +/<>
.\ 101/6 /
OJ

 

101° 10910210710 10‘"
(Norepinephrine) [NI]

Fig. 4. Effect of G2AR antagonist in pressurized vessels. ldazoxan, an G2AR
antagonist produced a significant rightward shift in the NE concentration
response curve in pressurized MV (8) but not in pressurized MA (A).

83

produced a similar rightward shift in the MV than yohimbine (Fig. 48) (0.03 vs 0.2
uM E053 for NE).

G2AR agonist potentiates responses mediated by G1AR. Based on
our data, we hypothesized that G2AR enhances the action of 01AR in the MV.
We tested the possibility whether stimulation of G2AR by UK 14,304 [0.1 uM] will
affect the responses mediated by G1AR in the MV. Concentration-response
curves were generated for NE and PE in the presence of UK 14,304. Activation
of G2AR did not potentiate the maximal response of MV to 01AR agonist PE (Fig.
5A). However, the results also indicated, that responses to lower concentration
of PE were potentiated by G2AR stimulation. This result further suggested that
G2AR might play a role in sensitizing the GAR in MV, at least in the lower agonist
concentrations, enhancing the responsiveness of MV. Finally, we performed
same experiment under pressurized conditions and UK 14,304 potentiated PE-
induced constrictions at low PE concentrations (Fig. 58). I

In order to determine the mechanism by which G2AR contribute to NE
induced constriction of MV we next attempted to identify the G2AR subtype that
mediates this response using subtype-selective G2AR antagonists. Neither the
OzAAR antagonist, BRL44408 (0.3 M, Fig. 6A) or the G23AR antagonist, imiloxan
(1 uM, Fig. 68) changed the NE concentration-response curve in MV. In contrast,
MK912, an OchR antagonist (0.01 IIM, Fig. 60) caused a six-fold right-ward shift
of the NE concentration—response curve. In these experiments the control NE
E053 was 7 i 1 nM while in the presence of MK912 this value was 40 i 9 nM (n =

6P<00$.

  
   

 

50-
g 40- f'tftiif’é‘é‘l‘in .M)
:5 3°r
b
m 20 -
8

1o-
: Phenylephn'ne
o o

10'” 10" 10'7 1o"5 10“ 10“

[Phenylephrine](M)

Pressurized system

 

 

- —o— UK14304 (0.10M) + PE
c .
.9. /
.‘é ‘ «I
P 10‘ /
2 i» i
3 - 0/
o\° o- O/
1E-9 ' ""1i=l-8 '
(Phenylephrine) [M]

Fig. 5. G2AR agonist potentiates GIAR — mediated responses in MV. While
UK14,304 did not affect the maximal response to PE in MV (A), it enhanced PE —
mediated constriction at low PE concentrations. Similar effect was observed in
the pressurized MV (8).

85

0t2A antagonist .
. _0_ Contra”: , 0‘23 antagonist
—o— BRL44408(300nM //¥ 50. —C:-|Co:1trol (1pM)
— — Ml oxan

Vi

/
._.._/_.__.____ MJZ W-.. __
10" 10 10710" 10" 1o 10'7 10"
(Norepinephrine) [M] [Norepinephrine] (M)

as
a

P
‘P

{\‘P

% contraction
A [a w
% contraction
-l N o:

O O

 

 

 

o
9

am antagonist
' —O— Control

. —O— MK 912 (Mnfig/ /$
o/ f

/.

10'9 10" 10'7 10‘6
[Norepinephrine] (M)

Fig. 6. Effect of subtype selective G2AR antagonist on NE-induced constriction in

MV. While no significant shift of the dose-response curve was observed with

BRL44408 (02A antagonist) (A) and imiloxan (G23 antagonist) (8). MK912 (0) (G23

antagonist) produced a similar effect on NE concentration response curve as

G2AR antagonist yohimbine (Fig. 28).

888

    

.3
9

% contraction
N
9

 

 

Functional responses of G23AR KO mice. To test the hypothesis that
G23AR is indeed responsible for the enhanced adrenergic sensitivity of MV, we
used tissues from G23AR KO mice in contractility experiments. We did not detect
any differences in NE concentration-response curves in MV from WT and G23AR
KO mice (Fig. 7A). In addition, yohimbine produced an 11-fold rightward in the
NE concentration response curve in MV from KO mice (Fig. 7B). This was
similar to the rightward shift caused by yohimbine in MV from WT mice (Fig. 38).
To test the possibility that other G2AR subtypes increased their function as a
result of physiological compensation to the loss of a functional G23AR, we utilized
MV from the KO mice to test the effect of the previously employed G2AR
antagonists on NE constrictions. Vln'Iile imiloxan (G23 antagonist) did not produce
significant effect on NE concentration response curve, the G2AAR antagonist
BRL44408 inhibited NE-induced constrictions of MV from G2CKO mice (n=4, Fig.
8). The maximal response to NE has also been suppressed (P<0.05) in these
KO MV.

lmmunocytochemical localization of GAR subtypes in VSMCs. To
correlate functional data with receptor expression, we used immunocytochemical
methods on freshly dispersed VSMCs from MA and MV to determine the
distribution of individual G1AR and G2AR subtypes. Fig. 9 depicts pan-cadherin
staining, a marker for the plasma membrane, in arterial (A) and venous (B)
SMCs. G2AARs were detected in the plasma membrane of both arterial (9C) and
venous SMCs (Fig. 9D). In contrast, G23AR did not localize to the plasma

membrane of venous SMCs and only intracellularly-localized receptors were

87

:h 0’
O O

% contraction
N
P

 

0.

- —o— achR KO mice

iii?

:3;

10"° 10* 10* 10'7 10* 10'5

60-
‘ —o— 300nM Yohimbine

40-

20-

 

0..

[Norepinephrine] (M)

0i2c KO mice
—0— control

"/i/gz“?

/

/i/2/2

 

10* 10* 10* 10'7 10* 10*

[Norepinephrine] (M)

Fig. 7. Sensitivity to NE is not altered in G23AR KO mice. NE dose response
curve of the G23AR KO mice MV does not differ from M (A). Yohimbine still
produces a rightward shift of the NE concentration response curve in the KO

mesenteric veins (8).

(12c KO mice

50 - -I- control
, —D— lmiloxan 1 [1M ,
40 —o— BRL44408 (0.3uM)

   

<75.
\
...\

% constriction
N
P

\\

 

\

10* 10* 10‘7 10* 10*
(Norepinephrine) [M]

Fig. 8. Effect of subtype selective G2AR antagonist on NE responses in the MV of
OchR KO mice. While imiloxan (G23 antagonist) did not affect the concentration
response curve to NE in these KO MV, the G2AAR antagonist BRL44408
produced inhibition of NE responses, indicating enhanced function of this
receptor in the KO mice

89

detected (Fig. 9F). We detected G23AR in intracellular compartment and in the
membrane of arterial SMC (Fig. 9E). The G23AR was expressed both in the
plasma membrane as well as in the intracellular compartment of venous SMC
(Fig. 9H). These data are consistent with pharmacological data that suggest that
G23ARs contribute to NE-induced constriction of MV from WT mice. G23AR was
also detected in the membrane of arterial SMCs (Fig. 90). With each experiment,
we performed controls with no primary antibodies. No staining was detected in
these controls. Contrary to what we expected, we detected expression of all
three G2AR subtypes in the arterial SMCs. When comparing the intensity of G2AR
staining between MA and MV, MV did not seem to express larger amount of
receptors.

Expression of (MARS. G1AARS mediate NE-induced constriction of MA
(Perez-Rivera) and lmmunocytochemistry revealed a strong expression of this
subtype in the plasma membrane and nuclear membrane of arterial SMCs (Fig.
10A). In the MV, this subtype was not expressed in the membrane, but in the
intracellular compartment (Fig. 108). We also detected G13AR in the membrane
of arterial SMCs (Fig. 100). This subtype mediates neurogenic constriction of
MA. GIDARs mediate NE-induced constrictions of MV and in venous SMCs, this
subtype localized predominantly to intracellular compartment. We also detected
strong immunostaining for G1DAR subtype in MA in the membrane and in the sub-
plasmalemmal compartment (Fig. 10F).

G1AR and G2ARs do not interact in HEK-293 cells. In order to begin

to probe the mechanism of (110-02AR interaction, we used stably and transiently

9O

-
,'

Pan-cad MA Pan-cad MV

‘ 10pm
OQBAR MV " dgAR MA d,,-AR MV

 

Fig. 9. 4 L ' ' analysis of G2AR expression in arterial and
venous SMCs. P-an cadherin, the marker of plasma membrane in arterial SMC
(A) and venous SMC (B). G2AAR was detected in the membrane of arterial SMC
(C), while in venous SMC G2AAR staining was detected both in the intracellular
compartment and in the membrane (D). G23AR localized in intracellular
organelles, likely Iysosomes in arterial SMC in addition to the membrane (E).
G23AR localized only intracellularly in the venous SMC (F). G23AR was found in
the membrane of arterial SMC (G) and in venous SMC, G23AR was expressed in
the membrane with the majority of the receptor pool localized intracellularly (H).
The magnitude of G23AR expression was larger than the expression of the other
two G2AR subtypes. The blue staining in most cells represents nucleus. White
arrow points to receptor localized to the membrane.

 

91

d,BAR MA G,BAR MV .

I (A) I 1m

3

G,DAR MA GmAR MV

 

Fig. 10. Expression of G1AR subtypes in MA and MV SMCs. GIAAR was
detected in plasma membrane and also in the nuclear membrane of arterial SMC
(A). In venous SMCs, this receptor localized intracellularly. G13AR was detected
in the membrane of arterial SMCs (C), while in the veins this receptor seemed to
be expressed at low levels intracellularly (D). Lower panel represents
immunocytochemical staining of G1DAR in arterial (E) venous (F) SMCs. In the
arterial SMC, this receptor localized to the plasma membrane and
subplasmalemmal compartment, whereas in the venous SMC, it was mainly
detected in the intracellular compartment. However, some membrane staining of
this receptor was also detected in the venous SMC

92

transfected HEK—293 cells. To test the hypothesis that OmAR forms a
heterodimer with G23AR, we first performed immunocytochemistry (ICC)
experiments to test for co-localization of these receptors in HEK-293 cells. As
has been previously reported, when C11DAR was expressed alone in HEK-293
cells, its localization was predominantly intracellular. We employed organelle—
specific markers to determine the cellular compartment where (11 DAR is localized.
As there was little co-localization with the plasma membrane-specific marker
pan-cadherin (Fig. 11A’), we used antibodies raised against the endosomal
marker, EEA1, and endoplasmic reticulum marker, calnexin, to determine the
intracellular compartment of O1DAR expression. Co-localization with these
markers suggested, that the 01DAR localized to the endosomes (Fig. 118’) and
endoplasmic reticulum (Fig. 110’). The HA-tag did not alter the localization of
G1DAR. These data are similar to the immunocytochemistry results from MV
SMCs where some G1DAR has been detected in the plasma membrane, but a
large fraction of total receptor pool has been also found intracellularly (Fig. 10F).
We next determined if co-expression with G2ARs would alter the
subcellular distribution of HA-GIDARs. G2AARs did not change the distribution of
the HA-C11DAR as the G2AAR was predominantly expressed in the membrane
whereas HA-GmAR remained in the intracellular compartment (Fig. 12A).
Similarly, G23AR was efficiently trafficked to the plasma membrane and there was
little subcellular localization with the HA-G1 DAR (Fig. 128). When FLAG-tagged

OchR was co-expressed in the HA-G1 DAR stable cells, it localized to the same

93

ISIIIII

G,DAR+cadherin G1DAR+EEA1 G,DAR+ca|nexin

 

Fig. 11. Subcellular distribution of G1DARS in HEK-293 cells. A. G13AR was
localized predominantly intracellularly, with little co-Iocalization with the
membrane marker pan-cadherin (A’ lower panel). B. G13AR co-Iocalized with the
endosomal marker EAA1 (8’). C. G1DAR also co-localized with calnexin (C’), a
marker for endoplasmic reticulum.

94

 

l 5 [till

Fig. 12. Relative distribution of (“MR co-expressed with G2AR subtypes In
HEK293 cells. Each panel represents individual G2AR subtypes expressed in
HA-GIDAR-expressing cell. Picture in the right panel shows the overlay of the two
images. Only one cell is visible in fig. A as opposed to fig. A’, because transient
transfection did not result into 100% co-transfection. In this case, the second call
was transfected only by G2AAR. Data suggested that none of the G2AR produced
redistribution of G13AR to the plasma membrane. Only GchR appeared to
localize to the same cellular compartment as G1DAR.

95

cellular compartments as HA-GmAR (Fig. 120) but HA-a1D-AR distribution was

not different from that seen in cells expressing only the HA-Cl1DAR. Both G2AAR
and G23AR were co-localized with the membrane marker pan-cadherin when
expressed in HA-G1DAR stable cells (Fig. 13 A’, 8’). In contrast, there was little
co-localization of Flag-G23AR with pan cadherin (Fig. 130’). As reported
previously, B2AR physically interacts with G1DAR in heterologous expression
systems (Uberti, Hague et al. 2005) and as a consequence of this interaction,
G1DAR is trafficked to the cell membrane more efficiently. Therefore, we utilized
the B2AR as a positive control for our receptor trafficking experiments. We found
co-localization of B2AR with G1DAR (Fig. 140) and that the C11DAR trafficked to the
plasma membrane more efficiently when co-transfected with the B2AR (Fig. 14A)
as indicated by co-localization of these receptors with the plasma membrane
marker pan-cadherin (Fig. 14E).

Quantitative assessment of cell surface expression of GARs. ICC
provides only a qualitative assessment of the relative subcellular distribution of
receptors. Therefore, we used a more quantitative assay to test the possibility
that co-expression with G2ARs enhances membrane expression of G1DARS. We
used a luminescence-based surface expression assay to quantitatively evaluate
the amount of HA-O1DAR expressed on the cell membrane when receptor was
expressed alone or when G2AR subtypes were co-expressed. Because the HA
tag was attached to the extracellular portion of the G1DAR we were able to
quantify membrane bound receptor. By this approach, we could use the highly

specific monoclonal antibodies against HA-tag. Histogram in Fig. 15 shows

O2BAR c Flag-G2CAR

ISIIm

+Pan-cadherin +Pan-cadherin +Pan-cadherin

 

Fig. 13. G2AAR expressed in GIDAR-expressing cells localized predominantly to
the cell membrane (A) and co-localized with the membrane marker pan-cadherin
(A'). Similarly, G23AR was expressed mainly in the membrane (8), but also
intracellularly. Fig. 8’ depicts colocalization of G23AR with the membrane marker
pan-cadherin. Flag-G23AR was detected in the intracellular compartment (0’)

and little co-localization with pan-cadherin was detected (0’).

97

oveflay

+pan—cadhenn

 

Fig. 14. B2AR co-expression enhances the targeting of G1DAR to the cellular
membrane (A). The two receptors co-localize when expressed in the same cell
(0). Figure E depicts co-localization of G13AR with pan-cadherin in a cell that also
expresses B2AR.

98

HA-G1D stable cells

 

 

 

 

 

 

8 0.25
I: 0.20
8
.2 0.15
,5 0.10
:E, 0.05
HA'OHD HAfl1D+02A HAfl1D+a28 HA'OHD'I'Olzc
3' HA-Gm - stable cells
. *

8 0.10

C 0.03“

a:

g 0.06- —r—

E 0.04-

:. 0.02-

—I

 

 

 

 

 

IVU

HA'G1D HA'G1D‘I'I32

Fig. 15. B2AR, but not G2ARs, enhances the membrane distribution of HA -
tagged G13AR. Histogram represents relative distribution of HA-G1DAR in the
plasma membrane that was measured by the luminescence surface expression
assay. The effects of the three G2AR subtypes on amAR membrane expression
are depicted in fig. A. Only B2AR significantly enhanced the amount of G13AR
localized in the membrane (8).

99

Luminescence data reflecting the surface expression of HA—GmAR when co-
expressed with each G2AR. None of the G2AR subtypes enhanced the surface
expression of HA-G1DAR, n=6. However, membrane expression of HA-(X10AR
increased significantly when this receptor was co-expressed with B2ARs, p<0.05
(Fig. 158).

Functional assessment of heterologously expressed GARs. We
next employed calcium imaging as a functional assay to test, whether the
functional interaction we observe between a1DAR and G2AR in MV could be
reproduced in HEK-293 cells stably expressing HA-G1DAR and transiently co-
transfected with individual G2AR subtypes. The Red Express plasmid was used to
mark the subset of cells transiently expressing G2ARs. NE (1 IIM) treatment of
HA-G1DAR expressing cells caused oscillatory calcium increases in individual
cells (Fig. 16A) and the frequency of these oscillations varied from 1 to 4 per
minute in different cells. We did not detect any difference between the functional
responses of WT and HA-tagged G1DAR. Because NE caused calcium
oscillations, we quantified this response by measuring the area under the curve
during the 1 minute NE application. After NE washout, a response to ionomycin
was obtained. The calcium response to ionomycin (10 pM) was sustained and
decayed only slowly (Fig. 168). On average, NE calcium responses were
approximately 15% of the ionomycin response. Fig. 17 depicts the magnitude of
NE responses as a percentage of total ionomycin response of HA-G1DAR-
expressing cells co-tranfected with the G2AR subtypes. The differences between

the amplitude of the responses between HA-G1D- and HA-Gm — G2AR co-

100

 

Norepinephrinev[1uM]

  

Time

    

A Baseline Fluorescence

[8*3]

lonomycin (1 IIM)

10 20 30 40 50 60 70
-2 Time (s*3)

A Baseline fluorescence

Fig. 16. NE treatment (1pM) of HA-(11DAR stable cells produces oscillatory
calcium response (A). Fig. 8 represents a typical cellular calcium rise induced
by the Ca2+ ionophore ionomycin. lonomycin was employed to normalize the data
due to non-quantitative nature of our Ca2+ fluorophore Fluo-4. Data were
normalized by calculating the area under the curve of NE response and
expressed as % of area under the curve of ionomycin response.

101

P
I”

  
   

    

g G2AAdrenergic receptor g G 2BAdrenergic Receptor
5 40 o 0

Q B

3 30 2 30

c 2 2 C

'8 20 '5 20

E E

O 10 o 10

C C

.9 0 l. . . ...~ .9

°\° HA‘a1D HAfl1D+GZA °\° HA'CX1D HAfl1D+a23

   

 

  
   

    

C. D

g Gchdrenergic receptor E B2Adrenergic receptor
0 40 g

3 in

e 30 ‘2’

.E '5

g 20 E.

E 10 8

g . _ .2 ‘
-- o ‘ ‘ .\°

.\° HA3,1D HA.O,1D+O,2C “Amp Bz+HAmp

Fig. 17. G2AR subtypes do not enhance calcium signaling mediated by
G13AR. Calcium imaging of cells co-expressing G2AR subtypes with O1DAR.
Response to NE was expressed as a percentage of ionomycin response for data
normalization. None of the G2AR subtypes significantly enhanced Ca2+ responses
of G1DAR - expressing cells. In contrast, B2AR was able to enhance the Ca2+
responses to NE (D).

102

P
I”

 

 

 

 

 

 

 

60 aZAAR 75. QZBAR
t“? l §f°l
Q,
g 40: Mi 3 45- \
3 30' / i 3 . L
h i O 30.
S 20- é , l
E ' <1
<1 10- 2 . . 15.
0 15 30 6 2 1'5 ' 3'0
C_ Time Of NE application [min] Time of NE application [min]
so azc
s 5'"
c 40-
m .
f3 30
e .
o 20-
2 10‘
I]. II
<1 0‘ I fi #fi
0 ' 1'5 ' 30

Time of NE application [min]

Fig. 18. Norepinephrine produces calcium elevation in G2AR - transfected
cells. Transiently transfected wild - type HEK293 cells with 030,- (A), 023- (8) and
OchR (0) were treated with norepinephrine (1 uM) at three different time points.
Each data point represents average increase in fluorescence during NE
treatment from 6 cells.

103

expressing cell were not statistically significant, n=6. BzARs co-expressed in HA-
amAR stable cells potentiated NE-induced calcium responses, p<0.05 (Fig. 170).
To confirm that the GzAR subtypes are functional when hererologously expressed
in HEK293 cells, I performed calcium imaging experiments on HEK293 cells
transiently expressing individual G2AR subtypes. NE (1 pM) produced calcium
elevation in both 02A — and OzBAR expressing cells (Fig. 18A and B). While NE
produced calcium elevation in Flag-achR - expressing HEK293 cells, the
response was much smaller than for the other two receptor subtypes, likely due

to poor membrane expression of this receptor (Fig. 18C).

Implication of the interaction between am- and (MAR in the MV. Interaction
between these two receptors occurs in heterologus expression system so it might
be ubiquitous for tissues that co-express both receptors. In the MV, BzAR might
help with trafficking and function of (11 DAR. We employed immunocytochemistry
on dispersed SMCs to test for potential co-localization of these two receptors in
the SMCs. Indeed, we detected B2AR in the MV and its localization was
predominantly intracellular, with smaller amount of receptor localized to the
membrane (F ig.19). It appeared to be co-localized with the amAR (Fig. 19G).

We also detected BZAR in the arterial SMCs and it localized
predominantly to the plasma membrane and sub-plasmalemmal compartment
(Fig. 198). It strongly co-localized with the plasma membrane marker pan-
cadherin (Fig. 190). Even though we did not perform co-localization study of (32-
and amAR in the arterial SMCs, these two receptors localize to the same

compartments (Fig. 1 9A and B).

104

We also performed a functional study in the intact MV to test the effect
of potential a1D-BZAR interaction on adrenergic responses. We tested the effect
of propranolol, the BAR antagonist on NE-mediated responses in the MV. This
antagonist had no effect on the NE responses at 3 pM concentration in the

pressurized MV (Fig. 20).

105

Arterial SMC

 

   

Venous SMC

 

oveflay

 

Fig. 19. Relative distribution of am- and BZAR in arterial and venous SMCs.
In the arterial SMCs, am- and B2AR were found in the plasma membrane and
sub-plasmalemmal compartment (A and B). While we did not perform an
experiment to directly co-localize the two receptors, there is strong possibility that
they co-localize due to their very similar distribution. BzAR co-localized with pan-
cadherin, suggesting membrane distribution of this receptor in the arterial SMC
(D). In the venous SMC, am - and BZAR seemed to be expressed mainly in the
intracellular compartment, but also appeared on the membrane. The two
receptors

106

 

50~
: 40-
.2 .
E 30-
: .
m 20-
g .
o 10-
o\° 0"

Mesenteric veins
—I— Control

'—D- propranolol (3 M) *‘
” /t:r5

+/
if

1o"° 10'9 10‘8 10'7 10‘ 10‘"
(Norepinephrine) [M]

Fig. 20. Propranolol, BzAR antagonist does not affect NE - mediated
responses in MV. 3pM propranolol did not produce any effect on NE
concentration -— reponse curve (performed in pressurized system).

107

Discussion

MV play a major role in blood pressure regulation and l have shown
that tone in MV and MA are regulated differently by sympathetic nerves. This
conclusion is supported by data presented in this study where I showed
enhanced sensitivity of MV to the constrictor effects of sympathetic nerve
stimulation. Previous studies indicated, that nerve-mediated constriction is
inhibited by the P2X receptor antagonist PPADS in the arteries, but not in the
veins. In contrast, a1AR antagonist prazosin blocks this constriction in the MV,
but only inhibits responses in the MA (Park et al. 2007; Luo et al. 2003). These
studies imply the role of dARs as dominant receptors mediating the effects of
SNS in the MV, whereas P2X in addition to GAR mediate neurogenic responses
in MA. However, since MV is also more sensitive to exogenous application of NE
than MA, the present studies aimed to identify thepostsynaptic adrenergic
mechanisms that contribute to this enhanced reactivity of MV.

Because we were only able to detect the effect of dzAR antagonist
yohimbine on NE-mediated constriction and we did not see direct vasoconstrictor
responses to azAR agonist UK14,304, I performed experiments in the presence
of indomethacin and NLA to rule out the possibility that dzAR-mediated
constriction is concealed by the production of vasodilatory mediators NO and
prostaglandins by the concomitant activation of OZAR in the endothelium. Studies
suggest that activation of OzAR in the endothelium leads relaxation mediated by
NO release (Vanhoutte et al. 2001; Nishina et al.1999) and combined inhibition of

NO production and prostaglandings increases o-adrenergic reactivity (Dinenno et

108

al. 2004). However, in our experiments I did not detect any difference in
UK14304 mediated responses in the presence of the enzyme blockers.

I also performed some pilot experiments in the pressurized vessels
that more closely resemble the physiological conditions. Similarly to non-
pressurized vessels, under these conditions, 02AR antagonist still shifted the NE
concentration - response curve to the right in MV but not MA. Experiment with
structurally unrelated antagonist idazozan produced similar result than studies
with yohimbine, suggesting that the data are not produced by an experimental
artifact.

Even though 02AR in MV does not directly couple to vascular
constriction, my studies suggest that CleR in MV potentiates constrictions
mediated by a1AR and similar findings had not been observed in MA. I
hypothetized that the indirect and potentiating contribution of dzAR in MV might
be based on a direct protein-protein interaction between a1AR and azAR in the
MV.

Previous studies established that the G1DAR is the GAR subtype that
mediates NE induced constriction of MV (Daniel, Low et al. 1997, Perez-Rivera
2007). In order to begin to understand a possible interaction between on DAR and
azAR, I attempted to identify the azAR subtype expressed by MV. My first
studies used GzAR subtype-selective antagonists in an effort to inhibit NE
constrictions of MV. Results from these pharmacological experiments suggested
that OchR subtype is the main azAR contributing to NE induced constriction of

MV. This conclusion is supported by results from another study showing that the

109

achR mediates contractile responses to NE in veins (Gavin et al. 1997).
BRL44408 has 25 and 100-fold higher affinity for the aZAAR over the angR and
achR respectively making this drug a useful tool for identifying responses
mediated by the (IZAAR. However, antagonists for the C123AR (imiloxan) and the
achR (MK912) are only 10-20-fold more selective over the other subtypes. This
modest selectivity can limit their usefulness for receptor identification.
Nonetheless, the right-ward shift of the NE concentration-response curve
produced by MK912 very closely resembled the shift produced by yohimbine
while BRL44408 and imiloxan did not change the NE concentration response
curve at all. Based on these data, I concluded that the achR contributed to NE-
induced constriction of MV.

Because of the limited selectivity of some of the azAR antagonists I
sought alternative approaches to identification of the azAR subtype contributing
to NE-induced constriction of MV. In these additional studies I used MV from
achR KO mice. I predicted that the NE concentration response curve in MV
from these mice would be shifted to the right (reduced sensitivity to the
constrictor effects of NE) and that yohimbine would no longer change the NE
concentration response curve. However, contrary to what I predicted, MV from
the OchR KO animals did not exhibit attenuated sensitivity to adrenergic
stimulation. Furthermore, yohimbine still produced a rightward shift in the MV
from the KO mice. This finding could be attributed to physiological compensation
in the KO mice, where other azAR subtypes assume the function of the achR.

Indeed, there are many examples where gene knockout leads to upregulation in

110

the expression and function of other related gene products. In studies using
fermoral arteries from G1DAR KO mice, it was found that chloroethylclonidine, an
antagonist of o1BAR, became much more effective in inhibiting NE-mediated
contractile responses as it was in the WT. These authors concluded that there
was an increased functional role for the a1BAR in the O1DAR KO mice (Zacharia
et al. 2005). Another example’of physiological compensation in the adrenergic
receptor family has been reported for B1/[52AR KO mice that are characterized by
significant reduction of muscarinic receptor density in the heart. In addition, these
mice exhibit an exaggerated hypothensive response to stimulation by the B3AR
agonist CL316243, suggesting enhanced functionality of B3AR in these mice
(Rohrer et al. 1999). These studies demonstrate that disruption of one adrenergic
receptor subtype can result into enhanced function of other adrenergic receptor
subtype and therefore support the idea, that in the achR KO mice, other OzAR
subtypes may take on the function of achR. .

My pharmacological studies suggest that the achR is the functional
receptor G2AR subtype expressed by MV SMCs. However, data from the achR
KO mice suggest that other GzAR subtypes can also be expressed by these cells.
| next used immunocytochemical analysis on freshly dispersed venous SMC in
an effort to determine what a1AR and azAR are expressed by these cells. I
confirmed that G1DAR is expressed by MV SMCs and that most of this receptor is
not localized to the plasma membrane but instead this receptor is found
predominately in an intracellular compartment. If on DAR were to form a complex

with OchRS (heterodimer or heteromultimer), and this complex contributes to NE

111

induced constriction of MV, it would be predicted these two receptors would be
localized together in the plasma membrane where they would be available to
bind NE, and in subcellular compartments where the complex may be assembled
or stored. To test this prediction I used immunocytochemical methods to localize
azARs with ormARs in MV SMCs. I detected immunoreactivity for all three azARs
in MV SMCs and relatively little of this labeling was localized to the plasma
membrane. The order of the magnitude of GzAR subtypes expression was ochR
> QZAAR > azBAR. Another study has also shown that achR mRNA is expressed
at higher level than OzAAR mRNA in venous SMCs (Chotani et al. 2004). This
result is also consistent with our pharmacological data. The fact that we detected
localization of all three receptors to venous SMCs supports our suggestion that
another azAR subtype compensates for the loss of the achR in the KO mice by
upregulating its expression and function. We also performed parallel studies in
arterial SMCs. In contrast to what I predicted, we detected all three OZAR
subtypes in arterial myocytes. Although not exclusively, these receptors were
detected in the plasma membrane. All three a1AR subtypes were also detected in
the membrane of arterial myocytes, supporting their functional role in these
vessels; o1AAR was found to mediate responses to exogenously applied
adrenergic agonists, whereas a1BAR appeared to mediate constriction to nerve
stimulation in MA (Perez-Rivera et al. 2005) Even though no functional role for
amAR has been detected in MA, I detected this receptor in the membrane and

subplasmalemmal compartment of MA SMCs.

112

There is possibility that the antibodies directed against GZAR cross-
react with epitopes of other aAR subtypes. Therefore, we tested these antibodies
in transfected HEK293 cells against other oAR subtypes. Results from these
control experiments suggested, that the individual antibodies are selective for the
subtypes they are targeting, suggesting that they can be employed in detection of
receptors in the native system.

Our data in MV maintained in vitro and in acutely isolated MV SMCs
indicate that OgARS are localized to these cells and that one or more subtypes
can contribute to NE induced constrictions. However, because of limitations of
the pharmacological tools, antibody labeling and limited numbers of viable cells
that can be isolated from MV we next used heterologous receptor expression in
an effort to probe the mechanism by which OzARS can facilitate G1AR function.
Because previous studies in our laboratory suggested that (11 DAR is the main
subtype mediating adrenergic contractility in mouse MV, we created a HEK-293
cell line that stably expressed a hemagglutinin (HA)-tagged human amAR. The
HA tag was localized to the extracellular N-terminus of the (11DAR. Pilot studies
revealed that this epitope tag did not alter the function (increases in intracellular
calcium) and expression compared to the WT receptor. l was then able to
transiently transfect G2AR subtypes into this cell line.

I first tested the hypothesis that under appropriate conditions, the
a1DAR could form a heterodimer with azARs that would facilitate trafficking of the
G1DAR to the plasma membrane. This might be indicative of receptor

heterodimerization. When G1DAR was expressed by itself in HEK-293 cells,

113

relatively little was localized to the plasma membrane as was found in W SMCs.
In HEK-293 cells, most of the on DAR was localized the endosomes (as revealed
by co—localization with the endosomal marker, EEA1) and the endoplasmic
reticulum (as revealed by co-loalization with the ER marker, calnexin). When the
HA-ormAR cell line was transfected with OzARS we did not detect o1DAR
redistribution to the plasma membrane. This could also mean that only a small
fraction of the receptors form a heterodimer and this might not be detectable
using immunocytochemical methods. However, previous studies investigating
receptor heterodimerization between am- and BZAR indicated, that
immunocytochemical methods are sufficiently sensitive to detect changes in the
G1DAR distribution when co-expressed with BzAR (Uberti et al. 2005). In these
studies, visible overlap of expression between 010- and B2AR has been noted.
Indeed, by utilizing BZAR as a positive control in my studies, I detected
redistribution of amAR to the plasma membrane and the a1DAR and B2AR were
co-localized in the plasma membrane.

I used luminescence surface expression assay to more quantitatively
asses the amount of HA-amAR expressed in the membrane. By utilizing
monoclonal antibodies against HA-tag, l was able to selectively measure the
amount of membrane-bound receptor. This approached confirmed my findings
that surface expression of HA-omAR is not enhanced by co-expression with
OzARS subtypes. These findings go along with previously reported data that did
not support a role of 02AR subtypes in promoting amAR cell surface expression

(Uberti et al. 2005).

114

Even though the receptor trafficking data did not support the
hypothesis of receptor heterodimerization, we further speculated that functional
interaction between the receptors might not be dependent on physical interaction
between the two receptors and still might bedetectable in HEK-293 cells. We
performed calcium imaging studies in the HEK-293 cell system to screen for an
enhancement of quR signaling during quR co-expression. The magnitude of
the response has not been affected by co-transfecting quR subtypes to HA-
quR cell line. In these experiments, we used a red plasmid to mark transfected
cells and we found co-transfection efficiency of almost 90%. Therefore, it is
unlikely that the results are due to measurements in cells not transfected with
G2ARs. Because Fluo-4 is not a ratiometric dye and therefore absolute
magnitude of the response can not be quantified by this method, there is a
possibility that the functional interaction has been overlooked in our experiments.
In addition to the physiological calcium rise, calcium signal measured by Fluo-4 is
affected by fluorescence bleaching and cell loading. Normalization of data to
ionomycin can correct for some of these issues, even though there might also be
slight differences between cellular responses to ionomycin. However, the fact
that l detected the potentiating effect of the positive control BzAR on q1DAR
function in our experimental setting minimizes the possibility that technical
shortcomings prevented detection of a functional interaction between q1DARs
and quRs in HEK-293 cells.

Altogether, data from the heterologous system implied that differential

receptor expression between arteries and veins alone likely does not account for

115

the higher adrenergic sensitivity of MV to exogenously applied NE than MA.
Some additional factors, likely in the signaling pathway involving calcium
regulation might differ in the veins, leading to a more efficient stimulus-response
coupling. In fact, our findings from Western blot were similar to the current
immunocytochemistry results; all 3 q1AR subtypes were expressed in arteries
and veins at a similar level even though functional data implied a larger receptor
reserve in the veins (Perez-Rivera et al. 2004). Even though these data must be
cautiously interpreted, they contribute to a bigger picture we see about the
differential regulation of venous versus arterial tone; this view points us to the
direction of differential receptor coupling to intracellular signaling pathways
including differential calcium handling as opposed to differential receptor
expression alone. Indeed, my studies suggest that there are differences in Ca2+
' handling between MA and MV (chapter 3 and 4).

Even though I did not detect q1-quR interaCtion in HEK-293 cells, it
does not contradict the original finding that quRs sensitize q1AR-mediated
responses in MVs. Some additional mediators of the interaction not present in
the arteries or HEK-293 cells likely contribute to this interaction. Previously
reported studies with whole tissue and cell lines also detected functional
interaction between 01- and quR (Fukui et al. 2005). Even though I could not
detect it, cross-talk has also been described in heterologous expression system
between q1AR and OzAAR (Reynen et al. 2000). In these studies, NE by itself did
not raise Ca2+ in Chinese hamster lung fibroblasts that only express a1ARs. After

transfection with QZAAR, robust increase in intracellular Ca2+ was observed, that

116

was antagonized by both 01- and 02AR antagonists. In our studies, both 01- and
G2AR had to be transfected and perhaps the GAR subtype that the hamster lung
fibroblasts natively express might not be the a1DAR. However, in our case quR
was the most relevant to study because of its prominent functional role in the MV.

Another possible mechanism that might play a role in this proposed
functional interaction might be similar to the one described in cauda epididymis of
guinea pig. In this tissue, quR potentiated the influx of extracellular calcium
through L-type calcium channels when q1AR was co-activated (Haynes and Hill
1996), even though activation of quR itself did not cause a concentration-
dependent response. Similar mechanism has been detected in rat tail artery
(Xiao et al., 1989). Indeed, such mechanism could be relevant to MV, because
adrenergic responses of veins are very sensitive to Ca2+ channel blockade
(chapter 4). However, taking this route in investigating the receptor interactions,
it would not be pragmatic to use HEK293 cell as a model system. As opposed to
the contractile VSMCs, these cells are non-exCitable and lack the cellular
architecture and biochemical properties of the native SMCs. Therefore, further
functional studies on veins and venous SMCs are necessary to fully elucidate the

cellular pathway underlying this interaction.

117

CHAPTER 4

Differential Calcium Utilization between Mesenteric
Arteries and Veins during Alpha-Adrenergic Stimulation

118

ABSTRACT
Mesenteric veins (MV) are more sensitive to the constrictor effects of

norepinephrine (NE) than mesenteric arteries (MA). In MV UzARS indirectly
contribute to NE-induced constriction by enhancing constrictions caused by
q1AR activation. This interaction was not due to direct receptor interaction.
Therefore, we tested the hypothesis that quRs in MV couple to Ca2+ influx
thereby enhancing the constrictions mediated by a1AR. Agonist induced
constrictions of MA and MV (ZOO-300 mm outside diameter) were studied in vitro
using a pressure myograph. CdCl2 (100 (M), a subtype non-seletive Ca2+
channel blocker, inhibited NE—induced constriction in MA and MV. Nifedipine (1
pM, L-type Ca2+ channel blocker) blocked NE induced constriction in MA but had
little effect in MV. GdCI3 (10 (M), inhibitor of store—operated and stretch—
activated channels reduced NE constrictions in MV but not MA. 2-APB (20 and
50 (M), an |P3 receptor antagonist inhibited NE responses in MA but had little
effect in MV. Thapsigargin, which depletes intracellular Ca2+ stores by inhibiting
SERCA, constricted MV, but not MA, and inhibited NE responses in MA and MV.
Co-application of nifedipine and GdCI3 inhibited NE responses in MV. The quR
antagonist yohimbine produced no additional inhibition in the presence of
nifedipine and GdCI3 suggesting that azAR activate Ca2+ entry mechanisms. .NE
responses were attenuated to larger degree in MA than MV by the Rho kinase
inhibitor Y27632 (10 pM). Finally, Ca2+ response curves were obtained by
incubating tissues in Ca2+ free media and then adding back Ca2+ in graded
concentrations in the presence of NE (0.3 uM for MA and 0.03uM for MV). Ca2+

addition caused similar concentration-dependent constrictions in MA and MV.

119

These data indicate that differences in Ca2+ entry and buffering play a role in

differential regulation of arterial and venous tone.

120

INTRODUCTION

MV are significantly more sensitive to the constrictor effects of
norepinephrine (NE) than MA and this might be due to functional involvement of
quR in MV, but not in MA. quRs in MV indirectly contribute to NE-mediated
constriction. In addition, activation of 02AR enhances constrictions mediated by
q1AR (Perez-Rivera et al., 2007). Our studies indicated that this functional
interaction can not be reconstituted in HEK-293 cells co-expressing q1AR and
quRs indicating that additional intracellular messengers present in MV that are
activated by stimulation of quR are required for this functional synergism.

dAR signaling. q1ARs couple to stimulation of phospholipase C
(PLC) activity and production of diacylglycerol (DAG) and inositol triphosphates
(IP3) that acts on IP3 receptors in endoplasmic reticulum, leading to Ca2+ release.
Inhibition of |P3 receptors in small mesenteric arteries significantly attenuates
phenylephrine (PE)— induced constriction (Lamont and Wrer, 2004). Even
though q1AR can also activate Ca2+ influx in the vasculature through the
activation of transient receptor potential channel 6 (TRPC6) and reverse mode
Na*-Ca2*-exchanger (NCX), studies indicate that constriction mediated by quR
is even more dependent on Ca2+ influx from the extracellular space (Lepretre and
Mironneau, 1994). L-type Ca2+ channels are activated by quR in certain
preparations (Ruffolo and Nichols, 1988; Mukundan and Kanagy, 2001).
However, in rabbit saphenous vein quR-mediated contraction is almost entirely
dependent upon Ca2+ influx through non L-type Ca2+ channels (Aburto et al.,

1995). Even though Ca” influx was necessary for quR-mediated constriction in

121

this vessel, Ca2+ influx was not augmented during quR-mediated constriction.
Basal Ca2+ influx may be sufficient to induce constriction by quRs. Although the
specific channels mediating basal Ca2+ influx were not identified, it is possible
that store-operated channels (SOC) contribute to Ca2+ store refilling and perhaps
can play a functional role in quR signaling.

In many blood vessels, pressor responses mediated by G2AR are
sensitive to pertussis toxin (PTX), implicating the involvement of Gi/o proteins in
mediating vascular constriction (Boyer et al. 1983; Aburto et al. 1993). However,
inhibition of cAMP formation via a Gi dependent mechanism is not involved in
quR-mediated effects on vascular tone. While activation of adenylate cyclase by
forskolin produces a rightward shift in concentration-response curve to NE in
porcine palmar lateral vein, neither NE nor the quR agonist, UK 14,304, affect
basal levels of cAMP even though they constrict the veins (Wright et al., 1995).
Several studies have reported involvement of protein kinase C (PKC) in quR-
mediated constriction (Aburto et al. 1995, Carter and Kanagy, 2003). In canine
MV, PI3 kinase and PKC are involved in the membrane depolarization mediated
by quR (Yamboliev and Mutafova-Yambolieva, 2005). Depolarization of SMCs
by 02AR in this preparation is dependent on activation of non-selective cation
channels (NSCC) and chloride channels, but not on L-type voltage — operated
calcium channels (VOCC).

Mechanism of functional interaction between (11 — and azARs.
Several reports have documented a synergistic functional interaction between (11-

and quRs. Functional interaction between (11— and quR in MV could potentiate

122

NE-induced constriction, accounting for the enhanced adrenergic sensitivity of
MV. While some studies suggest increased accumulation of IP3 (Wilson and
Minneman, 1991) when the two receptors are simultaneously activated, other
studies indicate that quR may potentiate Ca2+ influx from the extracellular space,
leading to enhancement of mAR-mediated constriction (Haynes and Hill,
1996)(Xiao and Rand, 1989). In our studies, we were not able to detect
enhanced Ca2+ mobilization in HEK-293 cells when co-stimulating the two
receptors, suggesting that |P3 production was not been enhanced by co-
activation of 01 - and azARs in these cells. If stimulation of Ca2+ influx by G2AR
played a role in the interaction in MV, it is possible that we were not able to
detect this effect in HEK-293 cells due to absence of Ca2+ channels. While HEK-
293 cells express receptor operated channels (ROC) and SOC that are likely
composed of multiple TRPC channels (TRPC1, TRPC3, TRPC4 and TRPC6
have been detected (Wu et al., 2002, Bugaj et al., 2005), the L-type voltage —
operated Ca2+ channels have not been found in these cells (Berjukow et al.,
1996). In addition, the different composition of SOC and ROC in HEK-293 cells
may yield functional properties that differ from those in native venous SMC.
Activation of Ca2+ channels by receptors and second messengers might also be
differentially regulated in HEK-293 cells. Therefore, we used MV and venous
SMC to study the role of Ca2+ channels and their potential involvement in the 01 -
G2AR interaction.

Modulation of Ca” sensitivity by azAR. Another mechanism by

which quR can modulate vascular tone involves increases in the Ca2+ sensitivity

123

of contractile mechanisms in SMCs (Aburto et al. 1993, Carter and Kanagy,
2003). Increased Ca2+ sensitivity of the contractile proteins in MV could also
enhance the responses mediated by a1AR. It has been proposed that Ca2+
sensitization by OzAR may be mediated by tyrosine kinases or PKC; Studies
suggest that src-family of tyrosine kinase is involved in Rho kinase activation,
resulting into direct inhibition of myosin light chain phosphatase (MLCP) (Nakao
et al. 2002). MLCP dephosphorylates myosin light chain (MLC) and therefore
inhibits the interaction between actin and myosin that generates the tension in
SMCs. Indeed, in certain blood vessels like saphenous vein, palmar lateral vein
or rat aorta constriction mediated by quR is dependent on activation of tyrosine
kinases (Jinsi and Deth, 1995, Roberts, 2001, Carter and Kanagy, 2002).

PKC, a downstream target of CleR activation, can also inhibit the
action of MLCP (Masuo et al., 1994, lkebe and Brozovich, 1996) via its
phosphorylation of smooth muscle specific phosphoprotein CPI-17 (Kitazawa et
al. 2000). CPI-17 is a downstream target of Rho kinase and PKC and it can
directly and potently inhibit MLCP. Conversely, activation of PKC by non-quR
agonists can enhance Ca2+ sensitivity (Jiang et al. 1987). In dog MV, activation of
PKC by endothelin-1 or 12-O-tetradecanoyl-phorbol-13-acetate (TPA) sensitizes
the tissue to quR stimulation by UK14,304 (Shimamoto et al, 1995). Angiotensin
II also increases the efficacy of agonists acting at quRs in the veins (Dunn et al.
1991). Conversely, 02AR stimulation potentiates vascular responses to
angiotensin II and this interaction involves PLC/PKC/c—srclPl3K pathway

(Jackson et al. 2005). NADPH oxidase and superoxide activate Rho A/Rho

124

kinase that is also involved in the interaction downstream of PLC/PKC/c-src/PISK
pathway (Jackson et al. 2008). Therefore, modulation of PKC activity is also a
potential target for the functional (11- quR interaction. Activation of either Rho
pathway or PKC by quR may enhance Ca2+ sensitivity and constriction
mediated by a1ARs.

Because pressor responses mediated by quR are dependent on Gi/o, I
employed pertussis toxin (PTX) to investigate the involvement of this G protein in
the functional interaction. To investigate whether 02AR potentiates Ca2+ influx
during NE-mediated constriction, l utilized CdClz (Cd2*) the non-selective Ca2+
channel blocker, nifedipine - the L-type Ca2+ channel blocker and GdCI3 (Gd3") —
the blocker of stretch activated cation channels and SOC. To determine the
relative contribution of the IP3 receptor and release of Ca2+ from the stores in
arterial and venous constriction to NE, I used the IP3 receptor antagonist 2-
aminoethoxydiphenyl borate (2-APB). l hypothesized that if G2AR enhances Ca2+
influx during NE constriction and thereby enhances contractile responses of
q1AR, constriction to NE would be inhibited to smaller degree after intracellular
Ca2+ depletion by thapsigargin treatment that inhibits the smooth endoplasmic
reticulum Ca2+ ATP-ase (SERCA), as compared to PE that activates only a1AR.
To test for the potential differences in Ca2+ sensitivity between MA and MV
during NE — mediated responses, I employed the Rho kinase inhibitor Y272632
in both MA and MV to asses the relative contribution of Rho kinase pathway to
Ca2+ sensitization mediated by NE. I also performed concentration response

curves to CaCl2 in MA and MV in the presence of constant NE concentrations to

125

determine, whether there are any differences in Ca2+ sensitivity during NE —
mediated constriction.

Since Ca2+ signaling is highly dependent on membrane potential of
vascular SMCs, we utilized pressurized system. While contractility experiments
performed under non-pressurized conditions might not significantly affect the
Ca2+ signaling of MV because they are only exposed to low pressures in vivo, the
physiological pressures in mouse MA reach ~ 60mm Hg which would represents
a substantial change compared to pressure levels in non—pressurized arteries.

Therefore, the pressurized system was optimal for our experiments.

126

Materials and Methods

Animals. CS7/Bl6 male mice (25-309) were purchased from Charles
River Breeding Laboratories (Portage, MI). In the animal care facility, mice were
maintained according to the standards approved by the Michigan State University
All-University Committee on Animal use and Care. Mice were individually housed
in clear plastic cages with access to standard chow (Harlan/Teklad 8640 Rodent

. Diet) and tap water.

In Vitro Preparation of Mesenteric Arteries and Veins. Mice were
anesthetized, and the small intestine with its associated mesentery was removed
and placed in oxygenated (95% oxygen, 5% carbon dioxide) Krebs' physiological
saline solution of the following composition (millimolar): 117 NaCI, 4.7 KCI, 2.5
CaCl2, 1.2 MgCl2, 1.2 NaH2PO4 25 NaHCOa, and 11 glucose. A piece of the
intestine with associated vessels was removed and pinned flat in a silicone
elastomer—lined (Sylgard; Dow Corning, Midland, MI) petri dish. A section of
mesentery containing vessels close to the mesenteric border was cut out using
fine scissors and forceps. The preparation was transferred to a smaller silicone
elastomer-lined recording bath and pinned flat. Second- or third-order mesenteric
veins or arteries (100-200-pm diameter) were isolated for study by carefully
clearing away the surrounding fat tissue. The recording bath containing the
preparation was mounted on the stage of an inverted microscope (Olympus CK-

2; Tokyo, Japan) and superfused with warm (37°C) Krebs' solution at a flow rate

127

of 7 ml/min. All preparations were allowed a 20-min equilibration period during

which the vessels relaxed to a stable resting diameter.

Video Monitoring of Vessel Diameter. The output of a black- and-
white video camera (Hitachi model KP-111; Yokohama, Japan) attached to the
microscope was fed to a framegrabber card (Picolo, Euresys lnc., TX, USA)
mounted in a personal computer. The video images were analyzed real-time
using Diamtrak Edge-tracking software (version 3.5, Diamtrak, Adelaide,
Australia), which tracks the distance between the outer edges of blood vessel in
the observation field. Changes in vessel diameter as small as 1 pm could be

resolved.

Treatment with pertussis toxin. A piece of mesentery was placed in
a tube with 2pglml PTX in Krebs buffer. Tube was placed into 37°C incubator and
oxygenated. After two hours of incubation, tissue was pinned out and cleaned of
connective tissue as described above. Controls were subjected to the same

treatment without PTX.

Application of Y27632. Rho kinase inhibitor Y27632 was purchased
from Sigma-Aldrich (St. Louis, MO). Solution with final concentration of Y27632
was applied to the superfusing Krebs’ solution from a flow tube (0.3 mm tip
diameter) positioned directly over the MA or MV. Flow rate of this application

system was 0.5mllmin.

128

Data Analysis. Constrictions of blood vessels are expressed as
percentage constriction from the resting diameter. Half-maximal effective agonist
concentration (E050) and maximum response (Emax) were calculated from a least-
squares fit of individual agonist concentration-response curves using a logistic
function from Origin 7.0 (Origin-Lab Corp., Northampton, MA): Y = {[Emin -
Emax]/[1+(x/EC50)"]+Emax, where Emin is the minimum response and was
constrained to zero and n is the slope factor. All data are expressed as mean :I:
S.E.M. Statistical differences between groups were assessed by Student's two-
tailed unpaired ttest. In experiments testing for differences in Ca2+ sensitivity
between MA and MV in NE—mediated constriction, we first established the basal

vessel constriction to NE. Data are presented as a % of this control constriction

for MA and MV.

129

RESULTS

Pressure affects adrenergic responses of MA and MV.
Pressurization of MV to 7 mm Hg altered the NE concentration-response curve in
MV. The EC50 of pressurized MV was shifted to the right; the EC50 in the
pressurized MV was 0.05 :r: 0.01 (M vs 0.02 i 0.004 uM, (P < 0.01, n=9).
However, the Emax was increased in the pressurized MV from 41: 5% to 59
11% constriction (P<0.005, Fig. 21A).

Even greater differences were observed in MA (Fig. 218); when the
intravascular pressure was raised from 0 to 60mm Hg, the EC50 for NE was
significantly shifted to the left from 4 10.6 pM to 0.5 :l:0.1 uM, (P < 0.005, n=6). In
addition, the Emax was increased in pressurized MA from 25 12% to 44 j: 4% P
< 0.005). Under pressurized conditions, MV were still significantly more sensitive
(P<0.005) than MA to the constrictor effects of NE.

Pertussis toxin (PTX) does not change NE-induced constriction of
MV. Our previous studies indicated that 02AR indirectly participates in NE
responses in MV and based on our results we hypothesized that involvement of
second messengers and Ca2+ signaling pathways could underlie the observed
receptor interaction as opposed to direct receptor heterodimerization. To test the
involvement of GM, protein in NE-mediated constriction of MV, the vessels were
pre-incubated in 2 pg/ml PTX solution for 2 hours at 37°C. This treatment had no
effect on NE—induced constriction of MV (n = 4, Fig. 228).

Ca2+ channel blockade and NE responses in MA and MV. To test

the dependence of NE responses on Ca2+ influx in MA and MV, we used the non-

130

Arteries

 

80- —o— Non-pressurized
«I —o— Pressurized
r: 60-
.2 .
‘6'
m 40- ‘9
*3 . 9/
o\° 0‘ /. ,0
1o“° 10‘9 1o“3 10'7 10" 10“ 104
(Norepinephrine) [M]
Veins
80' —o— Non-pressurized
60' —o— Pressurized
.5 '. /"'
‘5 O
a . t/
O
.o /9/

 

o __...——--
1o“° 10'9 10‘8 10"7 10’5 10‘5
(Norepinephrine) [M]

Fig. 21. Intraluminal pressure alters the reactivity of MA and MV. The EC50
for NE in the pressurized MV (6mm Hg) was shifted to the right compared to non-
pressurized MV, while the maximal response has been significantly enhanced by
application of pressure (A). More pronounced effect of pressure (60 mm Hg) has
been observed in MA, where EC50 has been shifted 8-fold to the left. In addition,
maximal response to NE almost doubled in MA (B).

131

—0— Control

 

 

 

45. —o— Yohimbine (0.3 pM)
/
C
.2 30 /§ /§ %
in!
8
g / é/
c3,15- 1} §{/
0
.\° //
0.
10* 10‘i 10'7 10‘ 10‘
(Norepinephrine) [M]
—I— Control
45- —o— Pertussis toxin (2mglml)
: ><
.2 30-
13
g
g 15-
O
o\° II/
0" -8

10‘7 10‘ 10‘5
(Norepinephrine) [M]

Fig. 22. 6% proteins are not involved in NE — induced constriction of MV.
While yohimbine, the q2AR antagonist produced a right-ward shift in the NE CRC
(A), pre-treatment of MV with PTX (2pg/ml) for 2 hours at 37°C in oxygenated
Krebs buffer did not affect NE CRC in MV (B).

132

selective Ca2+ blocker Cdz“ (100 uM). Cd2+ inhibited constriction of both MA and
MV, indicating the dependence of NE constriction on Ca2+ influx. The effect of
Cd2+ was more pronounced in MV, where constriction was completely blocked at
low NE concentrations. At higher agonist concentrations, NE produced a slight
constriction that fell back again to 0 at 10 (M NE (n = 7, Fig. 23B). While Cd2+
inhibited NE-mediated constriction in MA, NE still caused a concentration—
dependent constriction in these vessels (n=5, Fig. 23A).

In MA, nifedipine significantly attenuated the response to NE at 1 and
3uM NE (P<0.05), (Fig. 24A, n = 7). In MV; the Emax has not been altered by
nifedipine, NE concentration— response curve (CRC) was slightly displaced to the
right, n=6 (Fig. 24B).

We next used Gd3+ (10 pM) to test the effect of SOC and stretch —
activated channels. In the MA, Gd3+ did not exhibit any effect on NE CRC, (n=3)
(Fig. 25A). However, in the MV, Gd3+ displaced the NE CRC to the right without

changing the Emax, n=5 (Fig. 258).

Decreased sensitivity to IP3 receptor blockade in MV. IP3 is
produced by the activation of PLC by Gq and liberation of |P3 and diacyl glycerol.
IP3 acts on the IP3 receptors on smooth endoplasmic reticulum in SMC, leading
to the activation of Ca2+ release from the stores (Minneman et al. 1988). To
determine the relative contribution of this pathway in adrenergic responses of MA
and MV, we used the IP3 receptor antagonist 2-APB (20 and 50 (M). At 20 uM,
only a slight inhibition of NE responses in MA was detected (Fig. 26A). However,

at 50 (M 2-APB, a right-ward shift of the NE CRC was observed, with significant

133

A. Arteries

 

701, —I— Control
: 60+ —o— ctIcr2 (100 pm
o .
g 40- “i
h .
1:5 30-
8 20-
o J - -—O
o\ 1: /i/Q Q
10'” 10‘8 10'7 _1o‘ 10“
(Norepinephrrne) [M]
Veins
8' :3? —A— Control
- -— —cocr 100 M
c + O 21 ll l/‘x‘
.2 5°: ‘
“g 40-
b 30-
g 201
o . C} %
g 10« /
0.

 

101° 10° 10 10'7 I)”3 10“
(Norepinephrine)1 [M]

Fig. 23. NE responses in MA and MV are dependent on Ca” influx from
extracellular space. Cadmium chloride (100uM), the non — selective Ca2
channel blocker significantly attenuated NE responses in MA (A). In MV,
cadmium produced even larger inhibitory effect; NE responses were abolished at
low agonist concentrations and vessels exhibited attenuated constriction at
higher NE concentrations (B).

134

Arteries

 

70: —0— Control
601 —o— Nifedipine (1 IIM)
.3 5°: ,,
‘5 40' * ./.
m 1 /
i: 30- . SID/9‘0
: .
e\° 10' i“ /
ol 4 ’

10" 10" 10‘6 10“ 10“
(Norepinephrine) [M]

Veins

70' —-0— Control
04‘ —o— Nifedipine (1 UM)

t/ 7%

m

9.?

/ /§
/ /
/
1o"° 10*” 10" 10'7 10‘ 10‘
(Norepinephrine) [M]

Fig. 24. L-type VOCC play a more important functional role in MA than MV
in adrenergic responses. Nifedipine (1pM) shifted the CRC to NE to the right
and significantly attenuated constriction to NE at larger NE concentration (A).
Nifedipine also produced a slight right-ward shift of NE CRC in MV, although the
inhibitory effect on venous constriction did not reach statistical significance (B).

% constriction
-‘ N w -h
9.9

9.

 

 

9’.

135

A- Arteries —I— control

 

50' -a— GtICI3 (10uM)
: EC
'2 40" / _
4.0
U
N
‘3
o 20-
0 /
o\° ‘ fi’i/
O'mmgwm"

10'9 10" 1o" 10'6 10‘5
(Norepinephrine) [M]

Veins "" °°"t'°'

60- -—D-GdC13(10uM)
1-
: +4}.
'3 40‘ ‘5
~ 49/
8 20' /T/+
.\° 0. /r§:

 

10'9 10‘8 10'7 10“ 10'5
(Norepinephrine) [M]

Fig. 25. SOC contribute to venous, but not arterial constriction to NE. Gadolinium
chloride (Gd3*) (10pM), blocker of Ca2+ - induced Ca2+ release activated channels
(CRAC) and SOC had no effect on NE responses in MA (A). CRC to NE was shifted to
the right in MV'in the presence of gadolinium (B). Although the inhibition was not
statistically significant, attenuation of NE responses suggest some functional
involvement of these channels in functional responses to NE in MV.

136

Arteries
—I— Control

70 —o- 2-APB (20 uM)
60] —D- 2-APB (50 uM)
50-
40: i/i-+

30- / , -g ._
20:. é/é , /@ Q

10.
o.

% constriction

  

 

 

10“ 10"7 10’‘3 10‘ 10‘4
(Norepinephrine) [M]
Veins
—I— Control
70', _c— 2-APB (50 uM)
604 I~|

 

 

S 50-
's . /c§-§
'5 40-
m .
g 30- %
o I
°\° 20?

1o- /

oi /

10'“ 1o43 10'7 10‘6 10‘5
(Norepinephrine) [M]

Fig. 26. Effect of IP3 receptor antagonist 2-APB on NE - mediated
constriction of MA (A) and MV (B). 20pM 2-APB only slightly attenuated
responses to NE in MA. However, 50uM 2-APB significantly attenuated
constriction to NE in MA. 2-APB was much less potent inhibitor of functional
responses in the MV.

137

inhibition of maximal constriction, n=4. A different effect of 2-APB was observed
in the MV. 2-APB only started to produce some inhibitory effect in the MV at
50pM that was similar to the effect of 20uM in the MA (Fig. 26B). Higher
concentration of 2-APB was not employed due to potential non-specific effect of

this drug.

G2AR and activation of Ca2+ influx. In order to determine whether
activation of quR by NE couples to Ca2+ influx in MV, we utilized thapsigargin
(1 uM) to deplete intracellular Ca2+ stores. NE or PE was applied before and after
thapsigargin treatment and the % reduction from control response was analyzed
for both agonists. Thapsigargin produced a large constriction (57 :t 2 %) of MV
that deteriorated very slowly while thapsigargin was still present (~50 min). While
responses to PE were attenuated more than responses to NE (79 :t 5 for PE vs
57 :I: 13 % reduction for NE, n=6), there was not‘a statistically significant
difference between the two groups (Fig. 27).

Even though the difference between the magnitude of NE and PE
responses after thapsigargin treatment did not reach statistical significance, there
was a trend suggesting that NE induced constrictions are more dependent on
extracellular Ca2+ than PE induced constrictions of MV. Data presented above
indicate that NE induced constrictions of MV use both L-type VOCC and Gd3+-
sensitive Ca2+ channels. To test whether activation of GZAR by NE leads to
activation of these Ca2+ channels in MV, we tested the combined effect of Gd3+

(10 (M) and nifedipine (1 pM) in the presence or absence of azAR antagonist

138

100-

 

  

: 80'

o

‘8' 60' I
3

'o

o 404

h

\°

o 20*

 

 

 

 

 

Norepin'ephrine Phenylephrine

Fig. 27. NE — mediated constriction is more dependent on Ca” influx than
PE - induced constriction of MV. Responses to NE (1uM) and PE (1 (M) were
recorded before and after thapsigargin treatment (1 pM) (vessels were exposed to
thapsigargin for 50min - time required for MV to relax and stabilize the diameter).
Depletion of thapsigargin - sensitive Ca2+ stores significantly reduced responses
to both NE and PE, although the % reduction of PE responses was larger.

139

—I— Control
70_ -D- Nifedipine -I- GdCI2

      

 
 

 

 

  

. -o— Yohimbine + Nifedipine +GdCI2
60- \ I
,§ 504
c ' I
8 30': f
e\° 20-
10-
01 5 §/
10'9 10‘ 10'7 10" 10‘5
(Norepinephrine) [M]

Fig. 28. dzAR and activation Ca2+ channels in MV. Combined inhibition of L-
type VOCC and SOC by nifedipine and Gd3+ produced a significant inhibition of
NE responses in MV. When the 02AR antagonist yohimbine was applied together
with nifedipine and Gd3+, NE CRC was shifted slightly, but not significantly to the
right compared to nifedipine and Gd3 alone.

140

yohimbine (0.3 (M). Fig. 28 represents the combined effect of nifedipine and
Gd3+ on NE responses in MV, n=6. NE responses were significantly inhibited in
the presence of Gd3+ and nifedipine at 0.1 and 1pM NE. Yohimbine did not
further shift the NE CRC in the presence of Gd3+ and nifedipine.

Involvement of Rho kinase in adrenergic responses of MA and
MV. We hypothesized that increased involvement of Rho kinase in MV during
agonist — induced constriction might also play a role in the differences in
sensitivity to NE; increased activation of Rho kinase would increase Ca2+
sensitivity of contractile apparatus and therefore constriction. The Rho kinase
inhibitor Y27632 (10 (M) was continuously applied to blood vessels and NE CRC
were constructed for MA and MV. In MA, Y27632 produced a significant
attenuation of NE responses at all concentrations (Fig. 29A). The maximal
response to NE was inhibited by 49.11103 % from 34.414 to 17.0118 (n = 3).
Similarly, Rho kinase plays important functional role in venous responses to NE.
In the presence of Y27632, maximal response to NE was reduced by 24.5%:70

from 47.1:l:2.3 to 35.113, (Fig. 29B, n = 5).

Calcium sensitivity of NE - mediated responses in MA and MV.
To test for the differences in Ca2+ sensitivity between MA and MV during
adrenergic responses, we constructed cumulative CRC to Ca2+ in the presence
of constant concentration of NE, 0.03 [M for MV and 0.3 (M for MA
(concentration representing EC50 for NE in the two vessels). Initially, we

equilibrated the blood vessels in 0 Ca2+ solution/1mM EGTA for 15 min. This

141

Arteries

 

 

  
   

50: —-—Control

40- —o—Y27632(10I1M)
a ' I
E9 30: i
I; 20- 'i/
o .
e\° 10‘ i@.£§

o- 9 , 4
10' 10 10' 10 10 10
(Norepinephrine) [M]
Veins

50-
c I
2 40- f\
E .
b 30-
t":’ ' /
o 20- §
0 .
°\o 10- —-— Control

, —o— Y27632 (10 M)
o.

 

10* 10‘8 10‘7 10‘ 10‘
(Norepinephrine) [M]

Fig. 29. Rho kinase pathway plays an important functional role in
adrenergic responses of both MA and MV. Rho kinase inhibitor Y27632
(10pM) was continuously superfused over blood vessels and NE responses of
blood vessels were measured. Y27632 produced a significant inhibition of NE —
mediated constriction of both MA and MV, indicating that enhanced sensitivity of
MV to adrenergic stimulation is not due to more prominent role or Rho pathway.

142

initial treatment depleted intracellular Ca2+ in both blood vessels, because NE did
not evoke a constriction at the end of this incubation. Both vessels started to
constrict at Ca2+ concentration of 0.01 |JM (Fig. 30). We observed larger
response in MV compared to MA at 1 uM CaCl2 (p<0.05). However, differences
between the responses at higher CaClz concentrations were not statistically
significant. Both MA and MV reached 100% of the control response at 100 pM
CaCl2 (25 times lower than the Ca2+ concentration in standard Krebs’ solution).
With further increases of CaCl2, constriction in both MA and MV became larger
than the control suggesting increased Ca2+ sensitivity after the initial Ca2+

depletion.

143

.>

250- 0.3 LIM NE

 

200. Arteries

150-

100-

    

50-

  

 

% control constriction

0 0.01 0.1 0.3

 

3 10 30 100 3001000

 

   
   

1
Cacr2 (HM)
B.
0.03 uM NE
E 250!
-- Veins
-‘.:’ 200-
H
0)
g 150- 1;.
3 1°°' 53—;
s
.\° .....

 

0 0.010.1 0.3 1 3 10 30 100 3001000
CaCl2(uM)

Fig. 30. MA and MV exhibit similar Ca2+ sensitivity during NE — mediated
responses. MA and MV were incubated in buffer lacking Ca2+ in the presence of
EGTA (1mM). After 15 min wash that producing Ca2+ depletion in both vessels,
cumulative CRC to CaClz were constructed in the presence of constant
concentrations of NE; 0.3|JM for MA and 0.03pM for MV, concentrations
representing approximately the EC50 for NE in these vessels. Both MA and MV
exhibited bi-phasic response to CaClz and except at 1pM CaCl2, the differences
between MA and MV were not statistically significant. Data are presented as % of
control response to the given NE concentration in Ca2+ containing buffer
(2.5mM).

144

Discussion

Previous studies established that MV are more sensitive to
adrenergic stimulation than MA. The aim of the present study was to clarify the
differences in Ca2+ utilization between MA and MV during adrenergic stimulation
that may contribute to differential functional responses of these two vessels.
Because activation of quRs enhances constrictions caused activation of inR in
MV, we also tested the hypothesis whether quRs in MV couple to Ca2+ channels
and Ca2+ influx. This additional Ca2+ influx activated by 02AR could potentially
underlie the mechanism of synergistic interaction between 01 — and quR, as has
been previously described for other preparations including tail artery, cat nictating
membrane and cauda epididymis of the guinea pig (Xiao and Rand, 1989;
Haynes and Hill, 1996).

Pressure enhances the reactivity of MA but not MV. To study Ca2+
signaling in MA and MV, we utilized pressurized syStem due to the effect of
intraluminal pressure on membrane potential and reactivity of vascular SMCs.
Because MV are physiologically low pressure system, the difference in the
adrenergic reactivity between pressurized and non - pressurized MV was not as
large as the difference noted for MA. While the EC50 for NE was shifted ~2.5
times to the right in MV, the maximal response of MV has been significantly
increased. Therefore, the rightward shift does not represent a decrease in the
sensitivity of MV. As expected, we observed larger difference in the CRC for NE
between pressurized and non~pressurized MA. This result is plausible because

the difference in the pressure between pressurized and non-pressurized MA was

145

~ 60mm Hg as opposed to ~8mm Hg for MV. Stretch-activated channels in the
SMCs become more active when the intravascular pressure increases and acts
as mechanical force on the SMCs. This leads to increased cation influx and
lowering of the membrane potential that regulates the activity of multiple
channels. The decreased membrane potential enhances the blood vessels
reactivity.

To determine the potential role of Gyo prOteins in the 01 — 02AR
interaction, we used PTX treatment to inactive GM, and measured its effect on NE
responses in MV. Our findings indicate that other G proteins than Gi/o likely
mediate this interaction, since no effect of PTX on NE responses has been
detected while 02AR antagonists shifted the NE CRC curve to the right. This
result is somehow surprising as multiple studies indicate that pressor responses
in the vasculature mediated by 02AR involve activation of Gi/O (Nichols et al.,
1989; Aburto et al., 1995) and quR — mediated potentiation of q1AR signaling in
cauda epidymis involves PTX — sensitive proteins (Haynes and Hill, 1996). While
incomplete inactivation of Gyo proteins would prevent us from detecting an effect
of this inhibitor if Gyo proteins were involved, it is unlikely the treatment was
ineffective because it has been performed at high concentration of PTX for 2
hours. Same protocol has been shown to be effective in quinea-pig myenteric
neurons (Bian and Galligan, 2007). In addition, studies in our laboratory on rat
MA employing the same protocol (Stacie Demel, unpublished) have shown that
PTX is effective in inhibiting functional coupling of prejunctional azAR in DOCA-

salt rats. However, in SHAM rats this treatment did not result into complete

146

inactivation of Gi/oi Perhaps clue to larger Gyo, reserve in the SHAM animals.
These studies indicate that this protocol might not be universally effective and
perhaps this is related to the amount of the GVO, proteins in the given preparation.
In the study performed by Haynes and Hill, tissue was incubated for 24h in PTX -
containing Dulbecco’s modified eagle medium, although the concentration of
PTX was lower (200 ng/ml). This prolonged incubation would not have been
possible to perform with MV due to their fragility. To probe the involvement of
GVO, studies show that bolus injection of PTX in vivo 24 — 48 hours prior to the
actual experiment is an effective way to inactivate the proteins and perhaps this
would be a viable option for our future studies.

Role of Ca2+ influx in adrenergic responses of MA and MV. We
further investigated functional involvement of Ca2+ channels in NE induced
constriction of MA and MV. While MV seem to rely more on intracellular Ca2+
stores than MA (chapter 4), I predicted that quR in MV couples to a Ca2+
channel and thereby enhances the total Ca2+ elevation caused by NE. I also
predicted that I would detect differences in the Ca2+ channels involved in
adrenergic responses between MA and MV. The non-selective Ca2+ channel
blocker CdCl2 inhibited NE responses in MA and MV. However, the nature of the
inhibition differed between the two vessels; while CdCI2 produced concentration-
dependent inhibition of NE responses in MA, MV were more sensitive to CdCl2.
MV did not respond to NE in the presence of CdCl2 at low agonist concentrations
and even though we observed some constriction at higher NE concentrations,

the response was almost completely abolished at 10uM NE. These data suggest

147

that in the MA, CdClz blocks a Ca2+ entry mechanism that directly participates in
the GAR-mediated responses. However, an important mechanism of CdCIz
action in MV might be depletion of Ca2+ stores because there did not seem to be
a dose — dependent inhibition of NE responses in MV as in MA. Because the
time required to perform CRC in the vessels was ~1h and 45 min, it is possible
that capacitative Ca2+ entry was compromised leading to depletion of intracellular
stores and therefore inhibition of constriction. Cadmium has also been shown to
inhibit receptor — operated channels — the transient receptor potential channel 6
(TRPC6) that are activated by qAR stimulation and these channels might play
important functional role in vascular constriction of both MA and MV (lnoue et al.
2001)

However, CdCl2 not only blocks Ca2+ channels, it also blocks Ca2+
transporters. CdClz modulates the activity of Na*-Ca2*-exchanger (NCX) where it
competes with Ca2+ for import into the cell, thereby inhibiting Ca2+ influx (Trosper
and Philipson, 1983; Le et al. 2005). NCX works as a bidirectional exchanger;
while at resting membrane potentials its function is to maintain low Ca2+ levels by
pumping out Ca2+ it can also promote Ca2+ influx during constriction. This can be
mediated by coupling of NSCC TRPC6 to NCX, where localized increases of Na+
reverse Ca2+ transport producing Ca2+ influx. Studies in Chapter 5 indicate, that
NCX can mediate Ca2+ influx during GAR-mediated constriction in MA. Therefore,
it is possible that CdCl2 inhibited the NCX - mediated Ca2+ influx, inhibiting
constrictions in MA. In contrast, the role of NCX in NE-induced constrictions of

MV is not as prominent, although we can not exclude the possibility that reverse

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mode of NCX also contributes to Ca2+ influx in MV. Based on our current results,
MV are more sensitive to CdCI2 which because it has an additional site of action
in MV that is not present in MA. .

We tested the effect of nifedipine to asses the role of VOCC in NE-
induced constrictions of MA and MV. Nifedipine produced a greater inhibition of
NE induced constrictions in MA compared to MV. The role of L—type Ca2+
channels in NE constriction in arteries has been established previously
(Mukundan and Kanagy, 2001). In addition, these channels might play a greater
functional role in MA in response to sympathetic stimulation, because P2X
receptors are known to activate Ca2+ influx through these channels in MA in
response to ATP released from sympathetic nerves (Lagaud et al. 1996). In
contrast, only dAR are activated by sympathetic nerve stimulation in MV and P2X
receptors do not contribute to neurogenic constriction of MV (Luo et al. 2003;
Park et al. 2007).

Next I used GdCI3 to determine if NSCC contributes to NE constriction.
GdCl3 blocks the stretch-activated channels that are NSCC. GdCl3 had no effect
on NE responses of MA. However, in MV GdCl3 shifted the NE CRC to the right
suggesting some functional role of these NSCC in NE-mediated constriction of
MV. My studies indicate that GdCl3 blocks SOC entry in MV (Chapter 5) and also
other studies show, that GdCI3 is an effective blocker of SOC in other systems
(Trebak et al. 2002). On the other hand, GdCl3 might be less effective as a
blocker of receptor—operated Ca2+ channels (ROC), whereas 2-APB is more

potent inhibitor of this Ca2+ entry pathway (Trebak et al. 2002).

149

2-APB may exhibit dual inhibitory action in MV where it acts an IP3
receptor antagonist on the ER and therefore inhibits the Ca2+ release stimulated
by qu proteins (Minneman, 1988). In addition, the IP3 receptor is required for
conformational coupling of SOC and activation of SOC entry. In this model, IP3
receptors sense depletion of intracellular Ca2+ stores and transmit this message
to SOC via direct protein — protein interaction (Kiselyov et al. 1998). Therefore,
2-APB could prevent this interaction and activation of SOC. However, other
studies indicate that 2-APB can directly bind to and inhibit SOC (Dobrydneva and
Blackmore, 2001, Broad et al. 2001). In my studies, 2-APB did not abolish, but
significantly inhibited NE - stimulated responses in MA. GdCl3 had no effect on
NE induced constrictions of MA indicating no functional role of SOC in these
responses. Therefore, it is likely that the IP3 receptors are the only target for the
action of 2-APB in MA. An important question remains, however, about the role
or ROC since these channels are also inhibited by 2-APB and Cd”. A slightly
different result was obtained in rat MA, where application of 2-APB inhibits PE-
induced constriction by 95% (Lamont and Wler, 2004). While this inhibition was
greater than what we have observed (about 55% inhibition), it is possible that
species-dependent differences, as well as some differences between the
signaling of PE and NE could account for the differences in the potency of 2-APB
between our result and this study. 2-APB was much less effective in MV. This
result is unexpected as MV are dependent on intracellular Ca2+ stores for NE-
induced constriction. Even though thapsigargin treatment did not completely

abolish responses to NE and PE in MV, constriction was inhibited by more than

150

50%, suggesting that significant portion of the response is dependent on
thapsigargin - sensitive Ca2+ stores and the established mechanism of Ca2+
release from the stores mediated by Gq—coupled receptors involves IP3 —
dependent mechanism (Minneman, 1988). Therefore, one explanation of this
result would that IP3 receptors in MV are less sensitive to 2-APB, or perhaps
there is a larger IP3 receptor reserve on SER in the venous myocytes.
Considering that MV posses larger Ca2+ reserve in the stores than MA and it
takes longer for Ca2+ depletion in the absence of extracellular Ca”, it is possible
that the receptors regulating this Ca2+ release might exhibit differential sensitivity
to simulation in MA than MV. Another interpretation of this result would be that
there is a small contribution of IP3—mediated Ca2+ release required for NE
induced constriction of MV. Intracellular Ca2+ release could also be produced by
Ca2+-induced Ca2+ release (CIRC) by the action of Ca2+ on ryanodine receptors.
In this model, stimulation of qARs would lead to initial Ca2+ rise due to the
activation of Ca2+ channels (the yet unidentified Ca2+ channels or transporters —
NCX - blocked by Cd”), followed by CIRC. Another alternative explanation for
the low sensitivity to IP3 blockade in MV would be modulation of Ca2+ sensitivity
of the contractile apparatus (discussed below) as the main mechanism regulating
the vascular tone in MV. However, this mechanism is quite unlikely to be the
major pathway because of the large sensitivity of NE induced constrictions of MV
to Cd2+ and thapsigargin.

Another factor to consider when interpreting the data from MV is the

specificity of 2-APB action. Even though its effect was not as large as in the MA,

151

it still inhibited NE responses. Since SOC may directly participate in NE-induced
constriction of MV, perhaps the action of 2-APB might not be exclusively ascribed
to inhibition of IP3 receptors. The concentration used in these studies was at the
high end and therefore, we can not completely exclude the possibility that the
inhibition of NE was produced by the action of 2-APB on SOC. Similarly to the
result in MA, 2-APB could inhibit ROC component in MV. ROC that are
composed of TRPC6 are known to be activated by dAR via a DAG related
mechanism. Considering that CdC|2 also inhibits TRPC6 channels, the role of
these channels needs to be further explored. Utilization of the compound
SK&F96356, the more specific blocker for ROC should help to asses this
possibility. Unfortunately, current pharmacological tools do not allow for direct
identification of channels and their subunits that form SOC and ROC and
techniques such as direct electrophysiological measurements would only be able
to give a more specific answer about the composition‘and functional properties.
These channels might be composed of multiple TRPC homologues and different
composition yields different functional properties and sensitivities to channel
blockers. The specific properties of these channels in MV could perhaps be
exploited in the future for more selective targeting of venous SMCs with the goal
of increasing vascular capacitance.

Role of GzAR in Ca2+ influx. To determine whether NE induced
constrictions are more dependent on Ca2+ influx than PE responses in MV due to
functional coupling of quR to Ca2+ channels, I used thapsigargin to deplete

intracellular Ca2+ stores. I then compared the relative reduction of NE and PE

152

constrictions. My data indicate that NE responses in MV might be slightly more
dependent on Ca2+ influx, perhaps due to contribution of quR in NE but not PE
constriction. Because our studies suggested that both L-type VOCC and SOC
might contribute to NE-mediated responses, we tested the combined effect of the
two channel inhibitors on NE-induced constrictions in the absence and presence
of yohimbine. Combined nifedipine and GdCl3 treatment produced significant
inhibition of NE-induced constrictions. When yohimbine was applied together with
the channel blockers there was no additional inhibition of the NE constriction.
These data suggest that the indirect contribution of quR in MV may be based on
activation of Ca2+ entry. This type of mechanism would be consistent with results
from previous studies that suggest activation of L-type of Ca2+ channels as the
underlying mechanism enhancing contractility of O1AR by the action of quR
(Haynes and Hill, 1996, Xiao and Rand, 1989).

quR are regulated to larger degree by myogenic tone than inR
(Faber and Meininger, 1990). It is possible that activation of Ca2+ channels by
02AR would be subject to myogenic regulation, because activity of L-type Ca”
channels is dependent upon intravascular pressure and membrane potential. In
situations where intravascular pressure would be increased, increased activity of
L-type Ca2+ channels would be present. Release of NE by SNS would than
perhaps produce greater constriction due to increased functional coupling of
02AR to Ca2+ channels.

Ca2+ sensitivity of MA and MV during adrenergic stimulation.

Differences in Ca2+ sensitivity between MA and MV could also account for

153

differences in functional responses between the two blood vessels. Rho kinase
is the most important regulator of Ca2+ sensitivity and differential activation of this
pathway could also play a role in differential regulation of arterial and venous
tone. Indeed, a previous study suggested that inactivation of this pathway
produces greater inhibition of agonist induced constriction in veins than in
arteries (Robertson et al. 2007). However, these studies were not performed on
mesenteric vasculature. In addition, quR — mediated constriction in palmar
lateral vein as well as in some other blood vessels involves activation of Rho
pathway (Roberts, 2004), providing a testable hypothesis for quR action in MV.
Rho kinase might be more important in NE-induced constrictions of MA
compared to MV given the larger inhibition of functional response in MA. These
data suggest that more pronounced activation of Rho kinase pathway in MV
likely does not account for higher adrenergic responsiveness of MV. In these
studies, we did not test the possibility whether Rho kinase pathway is activated
by quR. However, the mechanism of inhibition of NE responses by Y27632 was
different than that produced by yohimbine. Rho kinase inhibition suppressed
maximal responses to NE, while yohimbine did not attenuate the maximal
constriction to NE. However, additional inhibitors of the Rho kinase pathway
should be tested because Y27632 is know to suppress some non-Rho kinase
mediated effects.

In addition, we obtained CaCI2 CRCs in the presence of a constant
concentration of NE to determine possible differences Ca2+ sensitivity during NE—

mediated constriction of MA and MV. Initially, the blood vessels were washed in

154

buffer in the absence of Ca2+, resulting into depletion of Ca2+ stores. After this
wash, MA and MV were no longer able to respond to NE due to Ca2+ depletion.
There were no substantial differences in CaClz sensitivity between MA and MV.
We observed a bi-phasic response in both MA and MV when NE was present;
there was an initial peak constriction at 0.3pM CaC|2 in MA and at 1 uM CaClz in
MV. After this initial peak, responses tapered off and starting at 10pM CaCl2,
responses were increasing in dose - dependent manner in both MA and MV. It is
interesting that at the larger Ca2+ concentration that was still smaller than
concentration in regular Krebs’ buffer, responses of both MA and MV were
significantly larger than control responses before the start of experiments.
Perhaps, this was due to increased Ca2+ sensitivity of contractile proteins after
the initial Ca2+ depletion.

Because the Ca2+ concentration in the buffer does not reflect the Ca2+
concentration in the cytosol, a direct relationship between Ca2+ sensitivity and
contractility in MA vs MV can not be established from this experiment. However,
initial Ca2+ depletion followed by small increases in Ca2+ likely affects Ca2+
storage in the SER and the magnitude of Ca2+ influx during NE stimulation. Our
experiments with Cd2+ provide direct evidence that both MA and MV are very
dependent on Ca2+ influx during adrenergic stimulation. It is likely, that Ca2+
influx and Ca2+ storage will be inhibited to similar degree in MA and MV by low
Ca2+ concentration in the buffer. Therefore, the fact that we did not observe any
major differences between MA and MV in NE responses at different Ca2+

concentrations suggests that there are no major differences in Ca2+ sensitivity of

155

Ca2+ - dependent excitation — contraction coupling between the two vessels.
Rather, differences in Ca2+ utilization appear to play a more important role in

differences in responsiveness to contractile agents.

Conclusions.

Overall, these data suggest that adrenergic responses of MA and MV
activate Ca2+ influx pathways but there are important differences in Ca2+ influx
mechanisms and utilization. In the MA, NE activates NCX and perhaps ROC, but
IP3 - mediated mechanism of Ca2+ release from the endoplasmic reticulum also
plays an important role in functional responses. In the MV, Ca2+ influx is crucial
and may be mediated by ROC and NCX due to high sensitivity of NE responses
to Cdz”. In contrast, MV may be more dependent on Ca2+ - induced Ca2+ release
as opposed to lP3 — mediated Ca2+ release due to low sensitivity to IP3 receptor
blockade. In addition, 02AR in MV may couple to Ca2+ influx, leading to greater
functional responses of MV when this receptor is activatedThese differences in
Ca2+ utilization might provide basis for differential kinetics of constriction between
the two blood vessels, with veins exhibiting faster rise of constriction to
adrenergic stimulation than arteries. In addition, differential Ca2+ utilization is
more likely to explain the differences in adrenergic sensitivity between MA and

MV as opposed to differential Ca2+ sensitivity of the two blood vessels.

156

CHAPTER 5

Differential Calcium Handling between in Murine
Mesenteric Arteries and Veins

157

ABSTRACT
Murine mesenteric veins (MV) are more sensitive to adrenergic

stimulation than mesenteric arteries (MA). The mechanism underlying this artery
vein difference was studied using MV presSurized to 8 mm Hg and MA to 60
mmHg in vitro. The norepinephrine EC50 value for MV constriction was 0.05 :I:
0.01 pM in MV and 0.5 :l:0.1 [JM in MA. We investigated differences in Ca2+
handling that might contribute to differences in MA vs MV adrenergic reactivity.
While both MA and MV constricted to NE and depolarizing agent KCI, MV
exhibited a slower relaxation than MA to both NE and depolarizing agent KCI. In
normal Ca2+ solution, thapsigargin (1 pM) caused a large phasic followed an a
smaller tonic constriction of MV. In Ca2*-free media, the tonic but not the phasic
constriction was abolished. The tonic constriction was inhibited by La” and Gd3+
but not Cdz“. Thapsigargin caused a small, transient constriction of MA with no
tonic response. This constriction was abolished in Ca2+ free buffer. After Ca2+
depletion, replenishment of Ca2+ caused a concentration - dependent
constriction of MV but not MA.

Oubain (0.5 mM) enhanced thapsigargin-induced constriction of MA
and MV. NE responses were inhibited by 61% in MV and by 94% (P<0.05) in
MA by thapsigargin. However, NE responses after in the presence of
thapsigargin and ouabain were abolished in MV but reduced in MA only by 38%,
indicating that facilitation of Ca2+ influx by ouabain after intracellular Ca2+
depletion is sufficient for NE to contract MA, but not MV. Caloxin 1b1 (200 (M) a

plasma membrane Ca2+-ATP-ase 4 (PMCA4) blocker did not afftect thapsigargin

158

responses in MA. Caloxin 1b1 potentiated and prolonged NE responses in MV.
Caloxin '1b1 did not affect NE responses in MA.

These data indicate that MV have larger Ca2+ stores than MA and
activation of SOC differentially affects the tone of MA and MV. Ca2+ removal after
constriction is more efficient in MA where NCX facilitates Ca2+ efflux. PMCA4 in
addition to NCX contribute to Ca2+ removal in MV. These differences may

contribute to increased reactivity of MV to NE.

159

INTRODUCTION

The mechanisms that regulate venous tone are incompletely
understood. While there are many studies focusing on the regulation of venous
tone, most of them use larger diameter conductance veins like the vena cava or
saphenous vein (Lee et al., 2001; Aburto et al., 1993). Small veins, particularly
the ones from splanchnic bed are the most important capacitance bed as they
store approximately 25% of total blood volume and are densely innervated by
sympathetic nerves. Increases in sympathetic nerve activity can rapidly shift
blood from the highly compliant splanchnic veins to the less compliant arteries
(Pang, 2001).

While the sympathetic nervous system (SNS) regulates tone in
mesenteric arteries (MA) and veins (MV), there are several functional differences
in the responses to SNS stimulation between the two vessels. SNS-induced
constriction of MV is mediated qrAR and 02ARS, while P2X1 receptors for ATP
and inR mediate constrictions in MA (Luo et al., 2003; Park et al. 2007; Perez-
Rivera et al., 2007).

Even though differential contribution of qARs subtypes in MA and MV
may contribute to the increased sensitivity of MV to adrenergic stimulation, our
studies indicate that differential receptor expression alone does not fully explain
the enhanced sensitivity of MV relative to MA. Previous studies indicate, that
both MA and MV equally express all three a1AR subtypes (Perez-Rivera,
unpublished). Furthermore, immunocytochemistry studies on freshly dispersed

vascular SMCs revealed that MA and MV express multiple GzAR subtypes at

160

comparable levels. Previous work in a heterologous expression system failed to
reveal an interaction between a1AR and a2AR as was observed in the MV
(Perez-Rivera et al., 2007), indicating that factors other than receptor expression
contribute to increased reactivity of MV compared to MA. MV are more sensitive
than MA to most other vasoconstrictors including ATP and endothelin—1 (ET-1),
suggesting a more general enhanced venous reactivity (Galligan et al., 2001; Xu
et al., 2007).

In this study, we attempted to identify differences in Ca2+ signaling
between MA and MV that may contribute to increased adrenergic sensitivity of
MV. Differences in Ca2+ influx and extrusion regulate vascular constriction. There
are two main Ca2+ efflux pathways in the vascular (SMCs): the high capacity Na+-
Ca2*-exchanger 1 (NCX1) and low capacity plasma membrane (PM) ATP-driven
Ca2+ pump (PMCA) (Schuh et al., 2003). Four isoforms of PMCA have been
identified and two isoforms (PMCA 1 and 4) are loCalized to vascular SMCs
(Schuh et al., 2003; Pande et al., 2006). In aortic SMCs, the NCX1 exchanger
accounts for ~90% of Ca2+ efflux released from intracellular stores (Lynch et al.,
2008). NCX is regulated by the Na*-K" —ATP-ase (NKA) via its influence on the
transmembrane Na+ gradient. The relative contribution of these two pumps in
Ca2+ export in MA and MV is not known.

NCX1 is a bi-directional transporter and while its action is crucial in
Ca2+ extrusion from the SMCs after constriction in addition to maintaining low
Ca2+ at resting membrane potentials, It can also facilitate Ca2+ influx during

receptor mediated constriction. qAR are known to activate transient receptor

161

potential channels 6 (TRPC 6) that carry the non-selective cation current (NSCC)
(lnoue et al., 2001). Opening of TRPC6 causes local accumulation of Na+ that
can reverse the NCX and facilitate Ca2+ influx during constriction (Poburko et al.,
2007). This suggests that NCX performs a dual function is SMCs, facilitating both
Ca2+ import and export. This dual role of NCX has been oonfirrned in mice
overexpressing NCX; the ventricular myocytes from these mice exhibit an
increased clearance of Ca2+ from cells in addition to prolonged time required to
reach peak [Ca2+]. (Yao et al., 1998).

To maintain intracellular Ca2+ concentration within a narrow range,
NCX and PMCA indirectly regulate activity of one another. If activity of NCX1 is
compromised by gene deletion of NKA alpha-2 isoforrn, PMCA activity is
increased (Lynch et al., 2008). Changes in the activities of these pumps affect
vascular tone. For example, receptor-mediated contraction is enhanced whereas
vasodilatation mediated by forskolin or SNP is inhibited in animals lacking NKA
q-2 isoforrn (Shelly et al., 2004). Similarly, differences in the level of expression
of PMCA lead to altered dynamics of Ca2+ handling and efflux that affect
responses to receptor stimulation (Liu et al., 1996).

Any residual Ca2+ spillover in SMCs is taken up into smooth
endoplasmic reticulum (SER) by smooth endoplasmic reticulum Ca2+ ATP-ase
(SERCA). SER store content is indirectly influenced by PMCA and NCX that
regulate cytosolic Ca2+ (Golovina et al., 2003). Selective inhibition of NKA (12

leads to local increases of Na+ in the cytoplasm and in turn local increases in

162

Caz“ by NCX. This results into increased Ca2+ uptake by SERCA and increased
store loading.

When SERCA is blocked, Ca2+ from the stores is unloaded leading to
Ca2+ depletion. This results into stimulation of store - operated channels (SOC)
that normally replenish the stores after Ca2+ is released (Leung et al., 2008).
However, if Ca2+ entering the cell is not taken up by SERCA due to its inhibition,
other pathways must remove this residual Ca2+. Even thought intracellular Ca2+ is
elevated in most types of blood vessels after SERCA blockade, it does not
always result into vascular constriction (Leung et al., 2008). Whether SOC
directly serve any functional purpose or whether their only purpose is to replenish
the stores is still a subject of controversy.

Since there is a large degree of interdependence and adaptability of
the various components of Ca2+ signaling to maintain Ca2+ homeostasis, to
handle this function more efficiently, expression of the NKA, NCX and SOC is
compartmentalized to small microdomains of the PM that overlie SER. However,
PMCA is more uniformly distributed in the PM (Juhaszova et al., 1996;
Ambudkar, 2006).

It is interesting that receptor-mediated constriction in mouse aorta is
potentiated in mice lacking the NKA q-2 isoforrn. In contrast, KCI-mediated
constriction is not affected in these KO mice (Shelly et al., 2004). The authors
suggested that increased Ca2+ load in these mice leads to increased release of
Ca2+ for any given inositol triphosphate (IP3) produced by receptor stimulation,

resulting into enhanced contraction. However, since KCI responses are mediated

163

predominantly by Ca2+ influx through voltage — operated calcium channels
(VOCC) with little utilization of Ca2+ stores, no difference in the responses
between WT and KO animals were detected.

These studies indicate how alterations in the Ca2+ handling processes
may affect vascular contractility. Differences in the Ca2+ handling between MA
and MV might also be contributing to enhanced functional responses of MV.
Therefore, we investigated the relative intracellular Ca2+ loads in MA and MV by
inhibiting SERCA. Our studies suggest that activation of SOC differentially affects
MA and MV. By utilizing inhibitors of the two Ca2+ efflux pathways PMCA and
NCX, differences in their relative contribution to relaxation and Ca2+ homeostasis
in MA and MV were assessed. These studies revealed that there are profound
differences in the way MA and MV handle Ca2+ and in addition to differences in
the functional receptors, this may be another important contributor to the

differential regulation of venous and arterial tone.

164

MATERIALS AND METHODS

Animals. C57/Bl6 male mice (25-309) were purchased from Charles
River Breeding Laboratories (Portage, MI). In the animal care facility, mice were
maintained according to the standards approved by the Michigan State University
All-University Committee on Animal use and Care. Mice were individually housed
in clear plastic cages with access to standard chow (Harlan/Teklad 8640 Rodent

Diet) and tap water.

In Vitro Preparation of Mesenteric Vessels. Mice were anesthetized,
and the small intestine with its associated mesenteric vessels was removed and
placed in oxygenated (95% oxygen, 5% carbon dioxide) Krebs' physiological
saline solution of the following composition (millimolar): 117 NaCl, 4.7 KCI, 2.5
CaCl2, 1.2 mM MgCl2, 25 mM NaHCOa, and 11 glucose. In some experiments
Ca” was omitted and the Ca2+ chelator EGTA (100 pM) was added to quench
residual Ca2+. A piece of the intestine with associated vessels was removed and
pinned flat in a silicone elastomer-Iined (Sylgard; Dow Corning, Midland, MI) petri
dish. A section of mesentery containing vessel close to the mesenteric border
(2"d or 3rd order vessel) was cleaned using fine scissors and forceps. MA and MV
were isolated for study by carefully clearing away the surrounding fat tissue.
Small piece of a vessel (1.5—2mm) was transferred to a recording chamber
containing Krebs-filled micropipettes. After the vessel was fastened to the glass
pipettes using ophthalmic sutures, the recording bath containing the preparation

was mounted on the stage of an inverted microscope (Olympus CK-2; Tokyo,

165

Japan) and superfused with warm (37°C) oxygenated Krebs' solution at a flow
rate of 7 ml/min. The vessels were than pressurized to reach intravascular
pressure of 60 mm Hg in MA and 7mm Hg in MV by adjusting the height of
Krebs’-filled syringe connected to the micropipette on which the vessel was
mounted. All preparations were allowed a 20-min equilibration period during

which the vessels relaxed to a stable resting diameter.

Video Monitoring of Vessel Diameter. The output of a black- and-
white video camera (Hitachi model KP-111; Yokohama, Japan) attached to the
microscope was fed to a framegrabber card (Picolo, Euresys lnc., TX, USA)
mounted in a personal computer. The video images were analyzed real-time
using Diamtrak Edge-tracking software (version 3.5, Diamtrak, Adelaide,
Australia), which tracks the distance between the outer edges of blood vessel in

the observation field' Changes in vessel diameter of 1 pm could be resolved.

Drug application. Drugs were applied in known concentrations by
addition to the superfusing Krebs’ solution or from a flow tube (0.3 mm tip
diameter) positioned directly over the MA or MV. Solution flowed through the
tube at 0.5 ml/min. All drugs were purchased from Sigma-Aldrich (St. Louis,
MO). Caloxin 1b1 was synthesized at the MSU Research Technology Support

Facility based on previously published sequence (Pande et al., 2006).

Data Analysis. Constrictions of blood vessels are expressed as
percentage constriction from the resting diameter. Half-maximal effective agonist

concentration (EC50) and maximum response (Emax) were calculated from a least-

166

squares fit of individual agonist concentration-response curves using a logistic
function from Origin 7.0 (Origin-Lab Corp., Northampton, MA): Y = {[Emrn -
Emax]l[1+(x/EC50)"]+Emax, where Emir. is the minimum response and was
constrained to zero and n is the slope factor. All data are expressed as mean i
S.E.M. Statistical differences between groups were assessed by Student's two-
tailed unpaired ttest. Determination of relaxation rate: Blood vessel diameter
was recorded during constriction to NE and KCI. For NE, the decay phase was
fitted by the non-linear regression function using Clam pfit and the time constanst
for individual relaxation segments were recorded. For KCI, linear regression was
used due to oscillations in vessel diameter during relaxation. The slope
representing vascular relaxation was used to estimate the rate of constriction
decline. Because MV desensitized after they reached peak constriction to KCI,

the desensitization phase was not considered in the calculations.

Isolation of Vascular Smooth Muscle Cells (SMCs). MA and MV
veins from male C57B6 mice (25-30 g) were harvested and cells were isolated
according to previously described protocol {Jackson, 1997 #15}. Briefly, the
blood vessels were cleaned of surrounding fat and connective tissue in a cold
dissociation buffer of the following composition (millimolar): (140 NaCI, 5 KCI, 1
MgCl2, 10mM HEPES and 10mM glucose, 10mM Bovine serum albumin, pH
adjusted to 7.4). Tissues were than incubated with papain in the presence of
dithioerythrol (1 mg/ml) for 35 minutes at 37°C and this incubation was followed
by digestion in a collagenase (1.9U/ml) lelastase (0.15mg/ml) solution with

soybean trypsin inhibitor (1mglml). Digested vessels were than washed twice to

167

remove residual enzymes using solution of the following composition (millimolar)
(135NaCl, 4KCI, 1MgCI2, 2CaCl2, 10HEPES, 10 glucose, pH adjusted to 7.4)

before they were dispersed using 1ml eppendorf pipette.

lmmunocytochemical analysis. Cells were allowed to attach to poly-
D-lysine — coated coverslips for 30min-1 hr after dispersion. SMCs were fixed
using Zamboni fixative for 30 minutes. After fixation, cells were rinsed in
phosphate buffered saline (0.1 M PBS, pH 7.4) and incubated with blocking
serum (4% serum in PBS; pH 7.4) to diminish nonspecific binding. After
overnight incubation with primary antibody (Table 1) at room temperature in PBS
containing 0.1% triton, cells were incubated for 1 h with secondary antibodies at
room temperature. Coverslips were then washed in PBS and staining was
visualized using a Nikon TE2000-U inverted microscope. Photographs were
taken using a SPOT Insight Color Mosaic camera (Mager Scientific, Inc.) with
Metalmaging Series software. Controls with no primary antibodies were used to
ensure that binding is specific. Rabbit antiserum for NCX was purchased from

Swant, Bellinzona, Swithzerland.

Calcium imaging. Dispersed SMCs were kept in a tube on ice before
they were used. Cells were used for up to 4hrs after isolation. 40 min prior to
calcium recording, cells were incubated in HEPES solution with 1pM Fluo-4 on
ice. 10min prior to recording, Soul of cell suspension was applied on top of a
coverslip mounted in a chamber on the stage of confocal microscope to give

sufficient time for cells to attach (Leica TCS SL confocal microscope, Leica

168

Microsystems, Bannockburn, IL). Cells were continuously perfused at room
temperature with HEPES buffer of the following composition (millimolar): 147
NaCI, 5 KCI, 1 MgCl2, 2 CaCl2, 10 HEPES, 10 Glucose pH = 7.4) and after 3 min
equilibration, dmg treatments were begun Fluo-4 was excited using an argon
laser and a wavelength of 488 nm. Emissions were collected in the range of
wavelengths between 450 and 500 nm. Images (512 x 512) were obtained every
3 seconds for 3 minutes. Each experiment consisted of 5 pre-drug (baseline)

images followed by thapsigargin (1 uM) application.

169

RESULTS

NE induced constriction of MA and MV. Concentration response
curves for NE in MV were left-shifted compared to MA; the EC50 for NE in MV
was 0.051001 uM compared to 0.5 10.1 uM in MA, (P<0.01, n=6). In addition,
the maximal constriction was significantly higher in MV than MA (59 1 2% vs. 44
1 4%, n=6, P<0.01, Fig. 31A).

MV exhibit slower relaxation than MA. NE induced constrictions in
MV were longer-lasting than MA (Fig. 313). The time necessary for the vessels
to relax was significantly different between MA and MV; 22751265 and
67811085 for MA and MV, respectively (P<0.01). When non-linear regression
was used to fit the decay phase of constriction in MA, it was best described by
two time constants, whereas in MV it was best described by 3 time constants.
The averaged time constants were much larger in MV than in MA (table 3). To
confirm that the decay of constriction was due to actual relaxation, we
continuously applied NE and recorded the diameter changes. Neither MA nor MV
desensitized to 1 (N NE, indicating that our results represent actual relaxation of
blood vessels (Fig. 31C).

To determine whether the longer relaxation time of MV is specific for
NE responses or if it represents more general mechanism of relaxation, the
relaxation rates of MA and MV to the depolarizing agent KCI (60 mM) were
compared. Maximal responses to KCI amounted to 50 1 2% in MA and 53 1 5%

in MV. However, during the application of KCI, MV desensitized after reaching

170

A. Pressurized system

—A— velns
60- Ar- arteries
5 /
'8 4o- 4 4'4
a / /
g 20 K A
:2 ' /. /
o\ A»

 

O

10'"1o"°1o" 10" 10'7 10‘5 10“
(Norepinephrine) [M]

 

 

3
B, '5 NE 11EM)
E 25-
.5
'5
g 0‘ o-
:3
2 -25-
g -50- ' Vein
*6 .
g _75. Fir—“fl.
2
.\°

Fig. 31A. MV are more sensitive to NE than MA. EC50 to NE is significantly
shifted to the left in MV and maximal constriction is larger in MV. MA were
pressurized to 70mm Hg, while MV were exposed to pressures of 8mm Hg. (A)
Responses to adrenergic stimulation are longer-lasting in MV than in MA (B).
30ml of 1pM NE solution was applied to pressurized MA and MV to develop a
stable constriction. Changes in the vessel diameter including the wash-out period
were recorded. The decay phase of the constriction was significantly slower in
MV than in MA, p<0.005 (B).

171

 

 

 

  

 

Norepinephrine (1 IIM)

E o . . Vein
‘: I, O Artery
.3 i
E 50-; 8 min
.9 ' l l———'|
o t
.E j:
c -1001:
u ,... ..-”:
3
B 450-
I:
L KCI(60mM)
o 100.....-

D. ‘5 i,
'3 75' I:
2’ MA
1.3 50- _. MV
1’
3 25_ '1.6 mlnI

(Fig. 31 continued) The decay of constriction to NE was entirely due to relaxation
and not due to desensitization, because neither MA nor MV desensitized when
NE was continuously applied (C). Similarly, when 30ml of 60mM KCI was
applied, the relaxation rate was significantly slower in the MV than in MA (D).
However, MV but not MA exhibited a small degree of desensitization during the
application of KCI that resulted into bi-phasic decay phase. Since the first phase
represented a combination of both desensitization and relaxation, the second
phase representing only vascular relaxation was taken into account when
relaxation rates were calculated.

172

Table 3. Decay phase of NE constriction is longer and composed of more
components in MV compared to MA. Decay of constriction was fitted by
standard exponential equation and time constants of each component were
recorded.
Decay constants of NE response

I time constant 1 (s) | time constant 2 (s)] time constant 3 (3)]
Arteries | 326511789 I 38.4911744 I
Veins | 297817044 L 13561455 I 65.791446 |

 

 

 

 

their maximal response to KCI, resulting in a biphasic decline of constriction (Fig.
31D). To estimate the rate of relaxation in MV, only the second phase of MV
relaxation curve was used. MA did not desensitize to KCI. The decline of
constriction was more complex for KCI than NE in that many vessels exhibited
oscillations of their diameter during relaxation, making it difficult to fit by non-
linear regression. Therefore, to get a rough estimate for comparative purposses, I
used linear regression to calculate the rate of constriction decline. The slope of
relaxation was significantly larger in the MA than MV; 2 10.1 in MA (n=6) versus
0.3 1 0.08 in MV (n=4), p<0.005.

Responses of MA and MV to SERCA blockade. Application of
thapsigargin (1 pM) alone caused a 57 1 2% constriction of MV, Fig. 323, n=12,
table 3. The vessel maintained a tonic constriction and relaxed very slowly to its
baseline after washout of thapsigargin (45-55 min). NE (1uM) responses in MV
were inhibited by 61% after thapsigargin treatment. In Ca2+ free medium,
thapsigargin caused a transient constriction of MV (Fig. 32C) but the tonic
constriction was abolished. In addition, I tested 01- and 10pM concentrations of
thapsigargin. These concentrations produced similar effect to 1pM thapsigargin

(not shown).

173

.>

 

 

 

 

 

 

 

 

a,B-MeATP
300‘ Mesenteric artery (1 I‘M)
A NE NE
3 25“ (10_IIMI Thapsigagin (1 uM) I1w_ .
a; 2°°‘ "T _ 7
g 150. 10min
(I ' I
'5 100-
50'
B.
300- Mesenteric vein
NE (1uM) Thapsigargin (1.1M) NE (mM)
E 250- 5:11
z? 200-
3
g 1504 V
w o
E 1001 10 mm
50-

 

Fig. 32. MV, but not MA develop a strong constriction in response to
SERCA blockade thapsigargin (1pM). Thapsigargin was applied using
microapplicator and responses of blood vessels to NE (1uM) were recorder
before and after thapsigargin exposure. Responses of MV to thapsigargin were
always larger than to NE and constriction to thapsigargin decayed very slowly in
MV (~45—55 min). Response to NE was reduced by over 60% in MV after
thapsigargin treatment (B) (Table 1), but was almost abolished in MA (A). In
contrast to MV, MA did not contract in response to thapsigargin.

174

 
   
 

 

 

Mesenteric artery

I- 1

a)
H

E
S 95- 6mm

c»

C 2+
:1: 35. 0 00a I1mlllIEGTA

8 O 2.5mM Ca2+

I—

0
°\ 75-

D.

‘ Mesenteric vein

0: 1
q.»

“2’
.5! 75-
'o g 0 mM CaClzl100uM EGTA
5, 50‘

c o 2.5 mM CaCl2
‘63 25- 1
o ':

h o. . .

 

 

(Fig. 32 continued) Superfusion of thapsigargin produced similar results than
microapplication, but response of MA produced a transient and oscillatory
constriction that was still much smaller than in MV, black trace, (C). This transient
constriction of MA was abolished in the absence of extracellular Ca2+ (grey trace)
Constriction to thapsigargin in 2MV consisted of two components, the first
component likely represented Ca2" unloading from SER, because this transient
constriction was 2not abolished in Ca2+ free media. However, in the absence of
extracellular Ca2 sustained phase of constriction to MV was abolished,
Implicating the dependence of this phase on Ca2 influx (D).

175

Thapsigargin did not constrict MA (Fig. 32A) but prolonged
thapsigargin application reduced NE (10 uM) constrictions by 94% (Fig. 32A,
Table 4), which was significantly more than in MV (P<0.05). However
constrictions of MA caused by the P2X1 receptor agonist, a,B-methylene ATP (1
1.1M) were unaffected by thapsigargin. a,B-methylene ATP-induced constrictions
of MA depend on extracellular Ca2+ entry (Gitterman and Evans, 2002). When
thapsigargin was applied by superfusion instead of microapplication, small
oscillatory constriction developed in MA. This response was abolished in the
absence of Ca2+ in the extracellular buffer (Fig. 32C). 10uM thapsigargin
produced same effect as 1uM thapsigargin (not shown). 2

To determine the direct effect of thapsigargin on [Ca2+] in individual
SMCs, we performed calcium imaging on freshly dispersed SMCs from MA and .
MV. Thapsigargin caused a transient increase in [Ca2+]: in MA SMCs followed by
small oscillations and a slightly elevated baseline (Fig. 33A). However, venous
SMCs responded to thapsigargin with a more sustained increase in [Ca2+] that
slowly declined back to baseline (Fig. 33B), paralleling the data from contractility
experiments. The Ca2+ elevation and decline in MV SMCs closely paralleled the
constriction and its decline in MV.

In certain blood vessels, activation of SOC by [Ca2+];

depletion is
coupled to constriction while in others increased SOC activity does not produce
constriction. We used Ca2+-free medium to deplete intracellular Ca2”r in order to

activate SOC. After a 10 min incubation in this buffer, we performed cumulative

concentration-response curve to CaClz. In MA, addition of CaClz produced little

176

A. Thapsigargin (111M)

 

 

 

 

 

 

   
   

 

o 301
g —arterialSMC
o 201
u _
g 104 .r 1mm 4'
S
E O-
4 xi

-10- ‘

B.
Thapsigargin (111M)

a, 30'
E
o 20-
o
(I)
2 10-
:3) 0 — Venous SMC
”- .
< 1mm

-10

Fig. 33. SERCA blockade causes elevation of [Ca2*]r in dispersed SMCs
from MA and MV. Representative traces of Ca2+ elevation during the exposure
to 1uM thapsigargin in arterial (A) and venous (B) SMC go along with the data
from contractility experiments, where thapsigargin induced a sustained
constriction in MV but not in MA.

177

Mesenteric artery

.p
‘P

w
c.’

% constriction
.1 N
O O
l l

 

0. 0.010.1 0.3 1 3 10 10100
CaCl2(uM)

Mesenteric vein

(«I -h
C O
1 l

% constri tion
N
‘3

 

 

0. 00101031 31010100
CaCl2(uM)

Fig. 34. Store-operated channels are coupled to constriction2 in MV, but not
in MA. Blood vessels were equilibrated in 0 extracellular Ca2 solution/1mM
EGTA for 15min After this period, the vessels were no longer able to respond to
NE Incremental increases of CaClz concentration up to 3pM produced a
concentration-dependent constriction in MV (B). In contrast, MA did not respond
with a constriction after 0 Ca2 buffer was replaced with increasing
concentrations of CaClz (A),

178

C- TRPC 1

 

10uM
Arterial SMC Venous SMC

 

 

 

 

D Thapsigargin (1pM)
' 60- 'i.
E E I Control
E 4.0- § f 0 Cadmium
‘3 E n Gadolinium
g * é o Lanthanium
:3, 20 * é
. *iét % ti
"0 1'0 2'0 3'0 4'0 5'0

Time (min)

(Fig. 34 continued) TRPC1, the main component of SOC is expressed in the
membrane of both arterial and venous SMCs (C). Application of Cd”, Gd3+ or
La3+ did not inhibit the initial constriction produced by thapsigargin in MV.
However, the tonic phase of constriction was significantly inhibited by La“ and
Gd3+, but not by Cd2+ (D).

179

or no constriction (Fig. 34A) while MV response with a robust concentration—
dependent constriction (Fig. 343).

TRPC1 may mediate capacitative Ca2+ entry during SOC stimulation
by SERCA blockade or after Ca2+ depletion(Brueggemann et al., 2006). Indeed,
TRPC1 seems to be the major component of SOC in many cell types including
vascular SMCs (Ambudkar et al., 2007). We performed immunocytochemistry on
freshly dispersed vascular SMCs from MA and MV to determine the distribution
of this channel. We detected a strong expression of TRPC1 in the membrane of
both MA and MV (Fig. 34C), indicating that MA also express this channel.

We further investigated the nature of the SOC in MV by employing
pharmacological tools. First, we tested the effect of Cd”, a non-selective Ca2+
channel blocker, on thapsigargin responses in MV. In the presence of Cd” (100
IIM) MV still responded to thapsigargin and the tonic phase of response has not
been abolished or inhibited. We also used GdCI3 (Gd3+) (10pM) blocker of stretch
- activated channels and SOC. In the presence of this cation, MV still responded
to thapsigargin with large constriction. However, the tonic phase of thapsigargin

response has been significantly attenuated (n=6), (Fig. 340).

MV are less efficient than MA in clearing intracellular calcium.
Slow relaxation rates after constriction in MV and the data obtained following
SERCA blockade led to hypothesis that MV do not clear intracellular Ca2+ as

efficiently as MA. To test this hypothesis, we performed experiments in the

180

Table 4. Effect of SERCA blockade on the tone and NE-mediated responses
in MA and MV. MV responded to thapsigargin exposure with strong constriction.
NE responses after thapsigargin incubation were reduced in MV by 61%, but
almost abolished in MA (reduced by 94%). In the presence of indirect inhibition of
NCX-mediated Ca2+ export by ouabain, MA responded to thapsigargin with 10%
constriction. Ouabain potentiated NE responses of MA after thapsigargin
exposure, with constriction inhibited by only 38% after thapsigargin exposure.

 

 

 

 

 

 

 

MA (% constriction) MV (% constriction)
Thapsigargin 0.27:0.27 57.31 :1 .88
NE control response 43.46t1.9 50.18i1.3
NE response after Thapsigargin 2.551123 19.65171
Thapsigargin+ouabain 1 02510.7 66.8:t1 .9
NE response after Thap + Oua 27.08119 7612.6
Ouabain 4.71:1 .6 3.9::1.7

 

 

 

 

 

181

A, Mesenteric artery
Thapsigargin (1 LlND
LIE (1 NM)
'4‘ . Thapsigargin

K " Thapsigargin + Ouabain
40'

17min

 

Reduction in Diameter (pm)

B Mesenteric vein

Thapsigargin (1 uM)

 

 

-75-" ziyfhapsigargin
Thapsigargin + ouabain

Fig. 35. Ouabain potentiates constriction of MA and MV to thapsigargin. In
the presence of indirect inhibition of NCX1-mediated Ca2+ efflux by ouabain
(0.5mM), MA constricted 10.25:I:0.7% when exposed to thapsigargin. In addition,
in the presence of ouabain, strong oscillatory constriction developed in response
to NE after thapsigargin exposure (27% versus 2.5% in the absence of ouabain)
(A). Ouabain also increased the maximal constriction to thapsigargin in MV. In
addition, the decay phase of constriction was increased in the presence of
ouabain in MV (B).

182

NCX

 

I 1OLIM |

Arterial SMC Venous SMC

 

(Fig. 35 continued) lmmunocytochemical staining of freshly dispersed arterial and
venous SMCs indicated the expression of NCX in the membrane of both cells
(C)-

183

presence of ouabain, blocker of the NKA. NKA inhibition causes intracellular Na+
accumulation inhibiting the Ca2+ extrusion function of NCX. We next tested the
hypothesis that more efficient Ca2+ removal by NCX contributes to the low
responsiveness of MA to thapsigargin. MA and MV were pre-treated with
ouabain (0.5 mM). Ouabain caused a small constriction of MA (Table 4) which
stabilized after 10 min. Thapsigargin (1 uM) applied to MA in the presence of
ouabain caused a sustained, 8-12% constriction (Fig. 35A, Table 4).Even though
the responses of MV to thapsigargin were strong and sustained, ouabain further
potentiated these responses, P<0.05 (Fig. 358, Table 4). As described above
thapsigargin blocks NE-induced constraction of MA. However, when MA were
exposed to ouabain and thapsigargin, NE caused large amplitude phasic
constrictions (Fig. 35A, Table 4). Ouabain did not potentiate the peak NE-
induced constriction of MV after thapsigargin treatment, but it did delay recovery
after NE and thapsigargin washout (Fig. 358, Table 4). We performed
immunocytochemical analysis of NCX in freshly dispersed SMCs from MA and
MV. We detected this protein the membrane of both SMCs (Fig. 35C).

Inhibition of PMCA4 pump in MA and MV. PMCA4 is the
predominant isoform expressed in vascular SMCs and caloxin1b1 inhibits this
enzyme (Pande et al., 2006). Caloxin1b1 (200 pM) did not constrict MA and
thapsigargin did not constrict caloxin1b1 pretereated MA. We next used
caloxin1b1 to test for the involvement of PMCA4 in clearing Ca2+ after NE (1 pM)
constriction. Caloxin 1b1 (200 uM for 10 min), did not alter the rate of decay of

the NE constriction after NE washout (n=3, Fig. 36A). In contrast, caloxin 1b1

184

constricted MV (n=4, Fig. 368) and potentiated NE-induced constractions in MV.
Also, caloxin1b1 treatment prolonged the recovery of NE-induced constriction

after NE washout (Fig. 368).

185

 

 

 

 

Mesenteric artery
300-
caloxin1b1 (zoouM)
g fltouM) ' NE (10pm
3 . —
.. 200-
a:
9 1 ’ '
a _ . J
- _ 8mm , "
100- r
B Mesenteric vein
NE1 M) caloxin1b1 NE+caloxin1b1
400-
"e"
a
a;
.5 200- /
o . :
. 16 min '4
0‘ I 'I

 

 

Fig. 36. Inhibition of PMCA 4 affects Ca2+ homeostasis and potentiates NE
responses in MV. NE (1 in MV or 10pM in MA) was applied before and at the
end of caloxin 1b1 exposure that took 20min. MA did not respond to caloxin 1b1
(A). In addition, response to NE has not been affected by this PMCA pump
inhibitor. In contrast, application of caloxin 1b1 (ZOOIJM) by itself produced
constriction in MV. In the presence of caloxin 1b1, response to NE was enhanced
and the relaxation phase was also significantly prolonged in MV (8).

186

DISCUSSION

MV are more sensitive than MA to the constrictor effects of NE.
Previous studies indicated differences in receptors mediating functional
responses between MA and MV where azARs indirectly contribute to NE
constriction in MV but not in MA (Perez-Rivera et al., 2007). However, in addition
to receptor differences there are additional mechanisms underlying this
pronounced sensitivity of MV to many vasoconstrictor agents. In these studies,
we studied the differences in Ca2+ clearance to asses its contribution to
differential regulation of arterial and venous tone.

MV exhibited a 10-fold higher sensitivity to adrenergic stimulation than
MA. and the rate of relaxation from NE - induced constrictions is slower in MV
than MA. Because MV also relaxed more slowly from constriction mediated by
KCI, the slow time course is probably due to differences in Ca” clearance rather
than slow dissociation of NE from a1AR or azAR in MV. In contrast to MA, MV
desensitized to application of 60 mM KCI and MV exhibited a bi-phasic relaxation
after KCI constriction. The fact that responses in MV are longer lasting than in
MA can be due to less efficient Ca2+ removal from cytosol after vascular
constriction. By employing specific drugs that interfere with Ca2+ handling we

investigated this possibility.

Ca2+ stores in MA and MV. Thapsigargin constricted MV but not MA.
The thapsigargin constriction in MV had two components; an early transient

constriction due to Ca2+ release from intracellular stores that persisted in the

187

absence of extracellular Ca2+ and a sustained constriction that was abolished in
the absence of extracellular Ca”, suggesting involvement of Ca2+ influx. It has
been proposed that Ca2+ influx during SERCA blockade is a good marker for
SOC in vascular SMCs (Leung, 2008). The sustained constriction caused by
thapsigargin was insensitive to Cdz“, so it is possible that Ca2+-release activated
Ca2+ channels (CRAC) or other SOC mediate the Ca2+ influx in MV during
SERCA blockade. Indeed, previous studies showed that Gd3+, but not Cdz“,
blocked SOC during SERCA blockade (Ma et al., 2000). While the early transient
constriction caused by thapsigargin was not altered in the presence of Gd3+ and
La3“, two cations that can block SOC, the tonic constriction of MV was inhibited
by these ions. Other studies showed that SERCA blockade in vascular SMCs
causes a large, transient increase in intracellular Ca2+ that corresponds to ER
Ca2+ unloading. This transient response is followed by plateau that represents the
balance between Ca2+ entry through SOC and Ca2+ efflux mediated by NCX,
PMCA and mitochondrial buffering (Golovina et al., 2003). SOC are opened by
store depletion (Golovina and Blaustein, 2000).

Calcium imaging results indicated, that arterial SMCs respond to
thapsigargin with a transient Ca2+ increase that returned to baseline faster than in
MV. These data indicate that MV posses a larger Ca2+ reserve in the SER. Also
the fact that constriction of MV to NE was not completely abolished after
thapsigargin incubation as it was in the MA (MV maintained ~40% constriction)

supports the idea of larger intracellular Ca2+ store in MV than MA.

188

Potential implications of larger Ca2+ reserve in enhanced
functional responses of MV. It is interesting to see that MV and MA do not
desensitize to continuous application of NE. However, MV, but not MA
desensitize to application of KCI. A profound difference is that during KCI-
mediated constriction, Ca2+ utilized in vessel constriction comes mostly from
extracellular stores through VOCC. In contrast, activation of receptors such as
aARs is coupled to both intracellular Ca2+ stores and Ca2+ influx pathways, with
Ca2+ stores likely contributing more to constriction in MV. In the light of our data it
is likely that MV poses larger intracellular Ca2+ reserve as MA. This might be a
consequence of the inefficient removal of cytosolic Ca2+ in MV to maintain
homeostasis. Therefore more Ca2+ is taken up by SERCA, increasing the SERs
Ca2+ content. In fact, studies suggest that if Ca2+ efflux pathways are inhibited by
knocking — out NKA alpha-2 isoform or by employing agents that inhibit NKA
alpha-2, SERCA activity and SER Ca2+ store loading are increased (Golovina et
al., 2003; Edwards and Pallone, 2007). The increased Ca2+ content in SER of MV
would cause increased Ca2+ release at any given level of |P3 generated
compared to MA, leading to a larger constriction. Indeed, it has been previously
proposed that enhanced Ca2+ content in stores results into larger Ca2+
mobilization and functional responses (Shelly et al., 2004).

Activation of SOC in MA and MV. Besides the profound difference in
the magnitude of constriction between MA and MV, an additional difference
between the responses of MA and MV to thapsigargin was the sustained

constriction in MV. The lack of a tonic constriction in MA could be explained by

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two separate mechanisms: 1) MA have more efficient Ca2+ buffering mechanisms
and/or 2) SOC activation in MA might not induce constriction as in MV. As NE-
induced constriction in MV are longer - lasting than in MA, the first mechanism
may be more likely. 1

Differences in Ca2+ buffering between MA and MV. To test the
hypothesis that Ca2+ efflux pathways are more efficient in MA, we used oubain to
block NKA causing indirect inhibition of NCX1 by altering the Na“ gradient.
Indeed, in the presence of ouabain, thapsigargin caused a sustained constriction
of MA, although this response was smaller than in MV. This result suggests that
Ca2+ efflux by the NCX contributes to quick removal of Ca2+ in MA. The fact that
mice lacking the NKA q-2 isoform exhibit diminished Ca2+ clearance by the NCX1
exchanger (Lynch et al., 2008) further supports our conclusion. Similarly,
transgenic mice overexpressing NCX1 have increased clearance of Ca2+ from the
cytosol (Yao et al., 1998). We also detected a potentiated response to
thapsigargin in the presence of ouabain in MV, suggesting that even though NCX
might not be as efficient in MV as in the MA, it contributes to Ca2+ removal. This
is further supported by immunocytochemical analysis, where NCX protein was
detected in membrane of both MA and MV. However, this type of analysis is not
quantitative and therefore differences in expression of NCX can not be excluded
as the contributor to differential Ca2+ clearance by NCX between MA and MV.

Differential role of SOC between MA and MV? While previous
results support increased Ca2+ extrusion in MA compared to MV, differences in

dynamics of Ca2+ removal can not fully account for differences in responses to

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SERCA blockade and SOC activation. Even though blockade of SERCA in most
vascular beds increases Ca2+ concentration, it is not always accompanied by
vascular constriction (Leung, 2008). Similar to our results, thapsigargin was
found to elevate Ca2+ concentration in rat MA, although it did not produce
constriction (Snetkcv et al., 2003). It is possible that SOC might be coupled to
constriction only in some blood vessels and MV but not MA are the ones that
exhibit this property. We detected TRPC1 - the main component of SOC in the
membrane of both MA and MV suggesting similar expression of channels
mediating SOC. SOC are activated by intracellular Ca2+ store depletion and it is
interesting that reintroduction of Ca2+ into the solution after Ca2+ depletion by
Ca2+ free buffer caused concentration—dependent constriction of MV but not MA.
However, after reaching a threshold concentration of Ca” (3 pM), constriction of
MV started to diminish. This was likely due to store replenishment and
diminished Ca2+ entry through SOC. These data further support a differential role
of SOC and Ca2+ handling between MA and MV. In fact, our previous studies
suggested that SOC may play some functional role in NE — mediated responses
of MV, but not MA.

Role of ch in Ca2+ influx in NE responses of MA and MV.
Thapsigargin- treatment abolished NE responses in MA. However when
thapsigargin and ouabain were both present, NE induced constrictions of MA
persisted. This result suggests that Ca2+ influx is sufficient to mediate NE-
lnduced constriction of MA even though Ca2+ stores are depleted. However, MV

did not respond to NE in the presence of ouabain, indicating that perhaps MV are

191

more dependent on intracellular Ca2+ than Ca2+ influx during aAR-mediated
constriction.

The constriction of MA to NE in the presence of ouabain after
thapsigargin treatment was likely mediated by Ca2+ influx through NCX. This
constriction was abolished in the absence of extracellular Ca”. Indeed, NCX is
recognized as a mediator of Ca2+ influx during agonist-stimulated constriction
(Poburko et al., 2007). NCX requires functional coupling with TRPC6, non-
selective cation channels activated by inRs (lnoue et al., 2001). Perhaps in the
absence of ouabain, NCX1 activity is not sufficient to support NE-mediated
constriction of MA when Ca2+ stores are depleted. However, in the presence of
ouabain, high intracellular Na+ concentration resulting from NKA inhibition and
activation of TRPC6 channels by aiARs potentiates NCX activity, driving Ca2+
influx. Ca2+ entry induces strong, oscillatory constriction as we have observed in
MA. Studies from mice overexpressing NCX1 further support the physiological
relevance of the bi-directional Ca2+ transport mediated by NCX1; the ventricular
myocytes from these mice exhibit an increased rise of intracellular Ca2+ during
constriction in addition to faster Ca2+ removal during relaxation (Yao et al., 1998).
Since we did not observe similar effect in the MV, it is likely that the NCX plays a
less prominent role in cAR-mediated constriction of the MV. Instead, constriction
of MV is more dependent on intracellular Ca2+ stores, likely mediated by Ca2+ -
induced Ca2+ release.

Role of PMCA4 in Ca” extrusion of MA and MV. We used an

inhibitor of the PMCA4 to asses the role of this Ca2+ efflux pathway in MA and

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MV. In contrast to the effect of ouabain in MA, thapsigargin did not constrict MA
in the presence of caloxin 1b1, a blocker of PMCA4. In addition, caloxin 1b1 did
not have any effect on NE—mediated constriction of MA. These results suggest
that PMCA4 does not play a major role in Ca” efflux in MA. While PMCA may
play dominant role in Ca2+ extrusion in certain SMCs like the uterine mycytes
(Shmigol et al., 1999), NCX accounts for the majority of Ca2+ removal in arterial
SMCs (Lynch et al., 2008).

Caloxin 1b1 by itself caused constriction and potentiated NE
responses in MV. In addition, it prolonged the recovery period after NE washout
in MV, implicating PMCA4 in Ca2+ buffering in MV. Inhibition of PMCA4 has also
been found to potentiate phenylephrine-mediated constriction in rat aorta (Pande
et al., 2006), suggesting that this transporter is active during agonist stimulation.
However, PMCA is active even during resting conditions in MV, maintaining low
Ca2+ levels. Overall, agonist induced constrictions of MV are very sensitive to
treatments that disrupt Ca2+ buffering systems MA are much more efficient in
Ca2+ buffering that may prevent constriction caused by Ca2+ accumulation.

Summary and conclusions. Altogether, our data indicate that there
are differences in Ca2+ handling between MA and MV during vascular
constriction and relaxation. Responses of MA to vasoconstrictors wash out more
quickly than in MV, because mechanisms responsible for clearing intracellular
Ca2+ are more efficient than in MV. NCX is not as efficient in MV in Ca2+ removal
and this is likely related to the function of other transporters in venous SMCs that

regulate Na+ gradient and NCX activity. Function of PMCA4 in addition to NCX

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compensates for the slower Ca2+ extrusion process in MV. There is a basal Ca2+
influx in MV, perhaps through SOC and PMCA4 significantly contributes to
maintenance of Ca2+ homeostasis during resting state. These differences might
have significant implications in the hemodynamics; quick regulation and
adjustments of arterial resistance are necessary to prevent hypoxia. If the time
required for resistance arteries to relax was not tightly regulated, detrimental
consequences could occur. On the other hand, the gradual filling of veins due to
slower relaxation of venous SMCs seems to be crucial in preventing abrupt filling
of the highly compliant veins. Fast filling of veins with large volume of blood
would likely lead to frequent and highly uncomfortable syncope.

In addition to Ca2+ removal, NCX also plays an important role in
functional responses in MA, facilitating Ca2+ influx during constriction. NCX might
also play a role in Ca2+ influx during constriction of MV, but activation of this
influx is not sufficient for functional responses; MV rely more on intracellular Ca2+
and posses larger Ca2+ store reserve and exhibit some dependence on SOC
during functional responses of MV. In contrast, activation of SOC in MA mainly
serves to replenish Ca2+ stores and activation of SOC is not coupled to vascular
constriction. Overall these differences in Ca2+ handling mechanism in addition to
differences in functional receptors likely contribute to larger reactivity of MV to

vasoconstrictor agents.

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Chapter 6.

General Discussion

195

General Discussion.
Overall relevance of studies

Cardiovascular disease (CVD) causes 17 million deaths per year

 

globally and is
Underwei ht

9 one of the most
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Unsafe water common causes

'High mortality of deaths in the

Blood pressure ' Low mortality

developed world

Tobacco . = If. Developed

Alcohol

 

(WHO, 2006).

0 25 50 75 100 125 150
Attributable DALY (0005)

Fig. 37. global distribution of disease burden attributable highly prevalent
to six major risk factors. WHO, 2003 (DALYs- disability —
adjusted life year). risk factor for CVD

Hypertension is

 

 

 

in the industrialized world. Increased longevity together with the prevalence of
contributing factors such as obesity, unhealthy diet and physical inactivity,
hypertension is becoming increasingly common health problem (Yusuf et al.,
2001). The proportion of global disease burden caused by hypertension is very
significant (Fig. 37, WHO, 2003) and it is estimated that 7.1million premature
deaths worldwide can be attributed to hypertension each year (WHO, 2006).
Hypertension along with atherosclerosis and thrombosis are the major
contributors to vascular dysfunction. Because vascular dysfunction compromises
organ function, it may cause the development of cerebrovascular disease,
ischemic heart disease and cardiac and renal failure. It many cases, these end-

point diseases might be preventable with antihypertensive therapy (MacMahon et

196

al., 1990). Studies from randomized clinical trials suggested that reduction of
blood pressure can attenuate the risk of stroke and coronary heart disease by 42
and 14%, respectively (Collins et al., 1990). Despite significant improvement in
the management of hypertension in the developed world, the cause of
hypertension remains unknown in 90-95% of cases. The remaining 5-10% cases
may be attributed to secondary hypertension due to kidney abnormalities, certain
cancers or congenital heart defects.

Sympathetic nervous system (SNS) and its regulation is a major focus in
hypertension research, because most hypertensive patients have elevated SNS
activity (Anderson et al., 1989, Schlaich et al., 2004), supporting a neurogenic
origin of hypertension. Elevated sympathetic tone can result from increased firing
rate of sympathetic nerves (Matsukawa et al., 1991), decreased function of the
NE transporter that leads to larger NE spillover (Schlaich et al., 2004) or
diminished baroreflex function so that arterial pressure increases are not
efficiently buffered (MAP) (Matsukawa et al., 1991). In addition, neurohumoral
factors such as angiotensin II, sodium and mineralocorticoids can affect SNS
activity (Perondi et al., 1992). A causal relationship between SNS overdrive and
hypertension is also demonstrated by the effectiveness of sympatholytic agents
like o- and B-adrenergic antagonists in lowering 8P (Goodman and Gillman,
2006).

Blood pressure is a function of two variables that are controlled by
autonomic nervous system: total peripheral resistance (TPR) and cardiac output

(CO) (Medical Physiology, Boron). While TPR is a function of arterial tone and

197

resistance, CO is dependent on several variables: end-diastolic volume,
myocardial contractility and heart rate. Myocardial contractility and heart rate are
determined by both sympathetic (SNS) and parasympathetic division of
autonomic nervous system. End-diastolic volume represents the volume reached
by the ventricular chamber before contraction and is determined by venous
pressure. In turn, venous pressure is related to blood volume and tone of the
venous smooth muscle cells (SMC). The significant contribution of venous tone
to blood pressure regulation points to the importance of studies that investigate
their neural control mechanisms and signal transduction involved in contraction.
Reduction of venous capacitance occurs in hypertension (Edmunds, et
al., 1989, Ricksten et al., 1981; Ferrario et al., 1970; London et al., 1985)
accompanied by increased CO. Similar results have been obtained in
experimental models of hypertension, including 2-kidney, 1-clip hypertensive rats
(Edmunds et al., 1989) and spontaneously hypertensive rats (SHR) (Ricksten et
al., 1981). While CO is increased only transiently during hypertension
development and returns back to normal, the redistribution of blood from
capacitance veins to arteries will have profound effect on the hemodynamics of
the circulation. When increased tissue perfusion exceeds metabolic demands,
no metabolic gain for the tissue will be provided. However, damage to blood
vessels and organs may ensue. Therefore, autoregulatory mechanisms cause
the blood flow to tissues and organs to return to normal when high MAP tries to
increase the flow. These autoregulatory mechanisms convert any tendency of

high CO hypertension into high resistance hypertension (Guyton et al., 1989).

198

Studies suggest that the autoregulatory mechanisms are not mediated by
autonomic nervous system and mechanism that account for the increase in TPR
can include metabolic or myogenic responses to the increased perfusion
(Lombard et al., 1989; Folkow et al., ~1982). The increased TPR is the
mechanism responsible for the of high blood pressure in the more advanced
stages of hypertension (Ferrario et al., 1970; Smith et al., 1979; Cowley et al.,
1992).

Venous capacitance is determined by two main factors: 1) structure and
2) contractile activity of venous vascular smooth muscle cells (venomotor tone)
(Safar et al., 1985). It has been noted, that venous elasticity is reduced in
hypertension {Walsh, 1969 #302}. Even though structural characteristics of veins
play an important role in determining the capacity of veins for a given blood
volume, the regulation of venous tone might be more important with respect to
the etiology of hypertension. The measure of venomotor tone - mean circulatory
filling pressure (MCFP) - is an index of venous capacity for any given blood
volume (Yamamoto et al., 1983). A change in the MCFP reflects primarily a
change in the upstream distending pressure (veins and venules). Several
studies using various models of hypertension (2 kidney-1clip, SHR) have
provided evidence that MCFP is increased in the initial as well as more advanced
stages of this disease (Edmunds et al., 1989; Martin et al., 1998).

In addition to the role of veins in the etiology of hypertension, the
incidence of other venous diseases is very high. For example, orthostatic

intolerance that affects ~ 500,000 Americans results from inappropriate response

199

of the autonomic nervous system to the postural changes and may involve
inadequate regulation of venous tone. Other venous diseases that affect
significant proportion of population involve deep veins thrombosis and varicose
veins. 7

My studies investigated the mechanisms that are involved in the
regulation of venous tone, with the focus on comparative studies of oAR
signaling in MA and MV. While many previous studies indicated that veins are
more sensitive to sympathetic stimulation than arteries, the mechanism that
underlies enhanced venous reactivity has not been clarified. Because enhanced
sympathetic drive occurs in hypertension, veins would be affected more than
anenes.

While a goal in hypertension research is to detect its causes and
signaling mechanisms that are changed in the disease process, it is equally
important to characterize the basic principles that are involved in the regulation of
blood pressure under non-hypertensive conditions. Thorough understanding of
the basic regulatory mechanisms involved in the regulation of arterial and venous
tone will lead to better awareness of the mechanisms that may be deregulated in
disease state. Whether increased vascular tone can be causative of hypertension
or just a consequence of changes that occur during its’ etiology, control of
vascular tone is central to the regulation of blood pressure.

(:1 - azAR interaction .in MV. ozARs contribute to the regulation of
venous tone, while in the resistance vasculature only oiAR mediates constriction

caused by NE released from sympathetic nerves. l hypothesized that differential

200

receptor expression between MA and MV contributes to differences in adrenergic
reactivity between MA and MV. I found that G2AR in MV indirectly contributes to
NE— mediated responses. While direct activation of GzAR by OzAR agonist
UK14304 did not cause constriction in MV, stimulation of GzAR enhanced the
responses mediated by oiAR in MV. These initial findings suggested that the
synergistic effect of OzAR in MV might be an important contributor to venous
adrenergic sensitivity. l hypothesized that MA do not express 02AR and therefore
lack this synergistic mechanism in response to adrenergic stimulation. Since the
functional role of 01 DAR in MV has been established previously in our laboratory
and our current studies indicated, that ochR is the major GZAR subtype
contributing to the interaction in MV we focused specifically, although not
exclusively on interaction between these two receptors. In order to investigate
the molecular mechanism of this interaction, we utilized heterologous expression
system of HEK 293 cell in functional and expression studies because most of our
studies would have not been feasible in vascular smooth muscle cells (SMCs).
MV are very fragile and enzymatic isolation of venous SMCs yields small number
of cells that only survive for few hours after tissue dispersion. Another factor is
tissue culture of MV SMCs. If SMCs are maintained in a media containing serum,
they quickly dedifferentiate and loose their SMC phenotype.

Lack of <11 - G2AR interaction HEK-293 cells. There are multiple
advantages in employing HEK293 cell system in studies of protein interactions
and signaling. As opposed to primary culture of SMCs, they grow fast and are

easy to transfect. Control over the expression of proteins of interest provides a

201

great advantage over native system that has not been characterized in such a
great detail. However, overexpression of receptors in HEK293 cells is often
criticized for producing non-specific effects that would not occur at physiological
levels of expression. In my studies, I was able to demonstrate that interaction
only occurs between specific receptors, suggesting that certain functional and
structural specificity was maintained when the receptors were overexpressed.

In the light of the emerging concept of receptor heterodimerization as
ubiquitous process for efficient receptor trafficking, expression and functionality,
we predicted that the synergistic o1 — 02AR interaction involves heterodimer
formation. Most qAR subtypes have been shown to dimerize as well as
heterodimerize. Heterodimerization of omAR with (113- and 82AR has been well
documented in promoting receptor trafficking to the plasma membrane and
thereby enhancing functional coupling of this receptor. Although there are many
reports in the literature documenting synergistic interaction between 01 - and
02AR (Haynes and Hill, 1996, Xiao and Rand, 1989, Reynen et al., 2000), no
study investigated receptor dimerization as a potential mechanism underlying this
phenomenon.

We generated a stable cell line expressing hemaglutinin—tagged qioAR.
Because co-localization of two proteins is a good indication/prerequisite for their
physical association, we screened for receptor co-localization between om — and
the three ozAR subtypes. As our control, we utilized 82AR that is known to
heterodimerize with oiDAR. While co-expression of 82AR caused redistribution of

oioAR from intracellular compartment to the plasma membrane with strong co-

202

localization of the two receptors, immunocytochemical analysis did not suggest
similar effect of the three G2ARs on (11 DAR. Because some studies suggest that
dimer formation might be enhanced by agonist stimulation, we performed NE
treatment of co-expressing cells and studied redistribution of receptors after their
activation. NE treatment induced a profound internalization of o2A— and ongR,
but these two receptors did not co-intemalize with oiDAR. By employing ICC, we
were not able to detect internalization of am— and ochR, likely due to their
predominant intracellular expression. In contrast, 82AR internalization was
accompanied by co-internalization of oiDAR in small vesicles. Several receptors
that form heterodimers co-internalize as a heterodimer and therefore these
studies provided further evidence against the existence of heterodimer between
(110- and 02AR subtypes.

Lastly, we employed a more quantitative approach to study the effect of
GzAR on membrane expression of oiDAR. Luminometer surface expression
assay selectively quantified the amount of HA-tagged receptor on the cell
surface, providing an objective evaluation of receptor distribution. While 82AR
significantly enhanced membrane expression of oiDAR, we did not observe any
effect of OzARS on (11 DAR membrane expression. While these studies seemed to
suggest, that receptor dimerization might not be the underlying mechanism for
the functional interaction we observed in MV, we can not completely exclude this
possibility. Perhaps, the dimerization was below the detection limits of our
methods. Direct experiments involving receptor co-immunoprecipitation would be

required to exclude the possibility of receptor heterodimerization. In the light of

203

my data, however, it appeared to be more reasonable to investigate the receptor
interaction at the level of calcium signaling instead of performing biochemical
tests based on no evidence of physical interaction. I further performed functional
studies in HEK293 cells to determine, whether the functional interaction detected
in MV could be simulated in this system. This would allow us to further employ
this system in the investigations of the mechanism. However, even these studies
failed to detect the ai-ozAR functional synergism, while BzAR enhanced the
calcium signal mediated by (11 DAR.

Even though studies in the HEK-293 cells led to a series of negative
data, the information provided by these studies is very valuable in the context of
our studies. These experiments helped us to exclude several possibilities how
the functional interaction might occur. Based on these data, it is most likely that
the two receptors are not sufficient for the manifestation of the functional
synergism. Originally we hypothesized that 01- and GzAR form a single functional
entity that exhibits enhanced coupling to intracellular messengers and functional
responses. However, if this was the case we should be able to detect the
interaction in any cellular context, including HEK-293 cells. Ca2+ mobilization is a
good measure of adrenergically — mediated responses in both vascular SMCs
and in HEK293 cells. While both types of cells posses intracellular calcium stores
that can be activated in IP3 — dependent manner, there are differences in
adrenergically — mediated Caz“ utilization between the two cell types. While
oARs utilize intracellular Ca2+ through IP3 — dependent mechanism in vascular

SMCs, they also activate Ca2+ influx through multiple pathways that might not be

204

present in HEK293 cells. Even more importantly, ozAR seems to be more
dependent on Ca2+ influx than oiAR during vascular constriction and it was
shown to potentiate the signaling of oiAR (Xiao and Rand, 1989, Haynes and
Hill, 1996). Indeed, our studies seem to suggest that GzAR in MV also couples to
Ca2+ influx.

While HEK293 cells endogenously express Ca2+ channels including
receptor — operated and store operated channels (ROC and SOC), these
channels might differ from those activated by oAR in MV. SOC and ROC are
most likely composed of multiple TRPC channels (Bugaj et al., 2005).
Expression of TRPC1, TRPC3, TRPC4, TRPC6 has been detected in HEK293
cells (Shi et al., 2004, Garcia and Schilling, 1997). Combination of different
TRPC that determines the functional properties of SOC/ROC might be
characteristic for each cell type and tissue (Garcia and Schilling, 1997). This
conclusion is further reinforced by our experimental data that indicate that even
though both MA and MV express functional SOC (SERCA inhibition is an
established mechanism of SOC activation SERCA blockade increases
intracellular Ca2+ in both vessels), their properties and coupling to contraction
varies between MA and MV. Therefore, even though HEK293 cells express
multiple calcium channels, these channels might still poses different functional
characteristics than the ones expressed in MV. In addition, the accessory
proteins involved in regulation of these channels might differ in the two cellular
contexts. Furthermore, HEK293 cells do not seem to express endogenous L-type

VOCC that are present in excitable cells including vascular SMCs (Berjukow et

205

al., 1996) indicating that there are differences in expression of some important
Ca2+ regulatory proteins. Considering that our data indicate, that the L-type
VOCC might be a downstream target of G2AR in MV, presence of this channel
might be critical for the functional interaction.

How does expression of «AR relate to functional responses of MA
and MV? While we predicted that only MV but not MA express OzAR, our studies
did not confirm this hypothesis. We detected all three 02AR subtypes in the
SMCs of MA with pretty strong membrane localization. Since no functional role
for these receptors has been detected in vitro, these receptors might be
functionally uncoupled in MA as has been suggested by previous studies (Daniel
et al., 1991 ). On the other hand, caution must be taken when extrapolating data
from in vitro studies to in vivo studies. Other neurotransmitters and hormones are
present under in vivo conditions that may alter the responses of particular
receptor to its agonists. For example, signal transduction pathways activated by
angiotensin II synergistically interact with GzAR signaling, enhancing
vasoconstriction to angiotensin II (Gao et al., 2003, Jackson et al., 2005). Since
angiotensin II is present in the blood, it could also potentially modulate responses
of ozAR in the mesenteric vasculature under in vivo conditions.

In addition, coupling of OzAR but not 01AR to vascular constriction is very
sensitive to metabolic factors including pH. Carbon dioxide (C02) — mediated
acidosis was found to selectively inhibit ozAR-mediated constriction of skeletal '
muscle arteries and veins, while no effect on aiAR-mediated constriction was

observed (McGillivray-Anderson and Faber, 1990). While our experiments were

206

performed in oxygenated Krebs’ buffer, we can not completely exclude the
possibility that alterations in the extracellular environment during tissue removal
and preparation could have affected pH of the Krebs’ buffer — even small
changes of temperature itself lead to changes in pH. While these external
factors would be hard to control, they could also have a potential effect on our
functional studies of GzAR. It would be important to find out, whether changes in
ozAR signaling under in vitro conditions introduced by slight fluctuations of pH
are reversible or not. It would be also interesting to know, whether some
variability in the experimental conditions during tissue extraction and preparation
could have contributed to the variability in the responses to oz agonist UK 14304
in MV. While we did not observe constriction to UK 14,304 in many MV or the
constriction was negligible (< 5%), in certain MV we detected constriction as high
as 18%.

Differences in the expression of aAR between MA and MV. Overall,
data from immunocytochemistry experiments do not support increased 01— and
OzAR expression in MV as a basis for enhanced sensitivity of adrenergic
responses in MV. While previous studies suggested, that MV possess a larger
receptor reserve compared to MA due to their increased resistance to alkylating
agent phenoxybenzamine (Perez-Rivera et al., 2004), these results could also be
explained by more efficient stimulus - response coupling in MV compared to MA.
In addition, 01AR subtypes were expressed at larger levels in the membrane of

MA SMCs compared to MV SMCs, further suggesting that activation of smaller

207

amount of receptors increased constriction of MV. The oiAR is the dominant
functional adrenergic receptor in MA and MV.

Certainly, there are some limitations that have to be considered when
interpreting immunocytochemical data. While we tested the specificity of
antibodies for the GAR in transfected HEK-293 cells, the environment in primary
SMC is different. The amount of receptors in heterologous systems greatly
exceeds the amount expressed in native systems. Therefore, positive staining in
native systems requires higher sensitivity and specificity of primary antibodies
than in the heterologous systems. Therefore, even though our data suggest that
the antibodies we used are sensitive and selective in transfected HEK-293 cells,
they might not work as efficiently in native SMCs.

Differential effect of enzymes on the membrane of arterial and
venous SMCs and its effect on antibody staining. Even though the membrane
of venous SMCs was not as smooth as arterial SMCs after vascular digestion,
we could still detect equally strong membrane staining of certain proteins in the
membrane of venous SMCs as in arterial SMCs. For example, pan-cadherin
staining was equally bright in arterial and venous SMCs, even though the
membrane was not as smooth. In addition, TRPC1 staining was stronger in the
membrane of venous SMCs than arterial SMCs and NCX was detected at similar
level in both MA and MV SMCs. These data suggest that less intense membrane
staining of certain receptors in MV is likely not caused by damaged membrane

caused by the exposure to the enzymes used to prepare isolated SMCs.

208

achR KO mice. Because our initial pharmacological studies indicated
that UchR is the main functional G2AR in MV, we performed functional studies in
the MV of GchR KO mice. Based on our hypothesis we predicted that responses
in these MV would be right — shifted. In contrast, there was no difference in the
E050 and Emax between the WT and KO MV. As discussed in Chapter 2, the lack
of effect in these mice could be attributed to physiological compensation, that is
frequently observed in genetically — manipulated animals. There are also
examples how a deficiency in one adrenergic receptor subtype results into
upregulation of other adrenergic receptor. Studies utilizing femoral arteries from
showed that chloroethylclonidine, an oiaAR antagonist, was more effective in
inhibiting NE-mediated contractile responses in aiDAR KO mice than in WT
arteries. These authors concluded that there was an increased functional role for
the G1BAR in the aiDAR KO compared to WT mice (Zacharia, Hillier et al. 2005).
In the KO MV, yohimbine still caused the rightward shift in the CRC similar to WT
MV, suggesting that ozAR other than ochR mediates this inhibition. Data from
studies using subtype selective antagonists suggested, that ozAARs take over the
function of ochR in the KO mice.

Physiological implications of OzAR in MV. While the relative
contribution of 01- and 02AR in sympathetically - mediated contraction varies
between different vascular beds, studies have consistently found that NE
responses in systemic arteries are predominantly mediated by oiAR, whereas
ozAR mediate adrenergic responses in small arterioles and in veins. Even though

02AR is not the dominant functional receptor in MV, it enhances the function of

209

o1AR and its contribution to adrenergic responses and regulation of venous tone
might be important from physiological perspective. Moment — to - moment
adjustments in vascular capacitance are required to maintain homeostasis and
this is dependent on the integration of adrenergic and local metabolic and to
smaller degree myogenic determinants. It is possible that G2AR plays a
regulatory role in the MV, enhancing the function of oiAR when increased
venous tone is required to meet the demands of the circulation. Conversely,
ozAR function may be down-regulated under certain circumstances and in this
case, it would not enhance o1AR functionality, resulting into smaller effect of SNS
drive on venous SMCs tone. ozARs are more sensitive to metabolic and
myogenic regulation than oiAR (McGillivray-Anderson and Faber, 1990, Faber
and Meininger, 1990) and these factors could directly modulate venous
responsiveness to SNS by affecting functional coupling of ozAR. In addition,
C12AR exhibits differential sensitivity to SNS stimulation than 01AR and therefore
its activity might be dependent on the level of sympathetic drive. Studies suggest
that low frequency nerve stimulation preferentially constricts vessels where OzAR
mediates responses to NE (Ohyanagi et al., 1991). It is also interesting that in
our studies, 02AR was able to potentiate oiAR function only at low concentration
of PE, with no effect on maximal responses when concentration of PE was high.
Perhaps, ozAR enhances oiAR function only when stimulus from the sympathetic
nerves is very low. Since studies have confirmed, that SNS activity is elevated in
hypertension (Schlaich et al., 2004), perhaps targeting ozAR under these

conditions would be a useful and selective venous target in hypertension

210

treatment. However, more studies about the receptor subtypes in MV need to be
performed to develop a selective OQAR antagonist that does not act in the
brainstem.

Our studies addressed the functional responses of oARs to circulating
catecholamines. Additional studies are needed to address the contribution of
GzAR in venous responses to direct SNS stimulation to evaluate the physiological

significance of this receptor in mediating the actions of SNS in MV.

DIFFERENCES IN Ca“ U11LIZATION BETWEEN MA AND MV: ROLE
OF G2AR.

We further investigated Ca2+ signaling involved in NE responses to
identify the differences in Ca2+ utilization between MA and MV that could be
potentially attributed to ozAR signaling in MV, enhancing sensitivity of MV.
Differential dependence on Ca2+ from the stores and Ca2+ from extracellular
compartment can also affect the functional responses.

Role of azAR and Ca2+ influx in MV. Responses to PE were inhibited to
larger degree than NE responses after depletion of intracellular Ca2+ by
thapsigargin suggesting that contractile responses to NE that also involve
activation of GzAR in MV, utilize more extracellular Ca” than PE responses.
Because nifedipine and Gd3+ exhibited some inhibitory effect on NE responses in
MV, we tested the hypothesis whether the 02AR in the MV couples to Ca2+ influx
through these channels. Our studies suggested that in the presence of the two

inhibitors, ozAR antagonist yohimbine no longer significantly shifted the EC50 for

211

NE to the right in MV. This mechanism would go along the lines of previous
studies, where the potentiating effect of ozAR is based on activation of Ca2+
channels (Haynes and Hill, 1996, Xiao and Rand, 1989).

As mentioned above, 02AR are regulated to larger degree by myogenic
tone than 01AR (Faber and Meininger, 1990). It is possible that activation of Ca2+
channels by GzAR would be also a subject to myogenic regulation, because
activity of L-type Ca2+ channels is highly dependent upon intravascular pressure
and membrane potential. Increased pressure would lead to increased activity of
L-type Ca2+ channels. In addition, if G2ARs were stimulated, its coupling to L-
type Ca2+ channels would lead to enhanced functional responses under given

conditions.

Store-operated channels (SOC) in MA and MV. MV reacted very
sensitively to inhibition of smooth endoplasmic reticulum Ca2+ - ATPase
(SERCA), whereas only small and transient response was observed in MA. Our
studies indicated that two mechanisms are responsible for the differential
responses to SERCA blockade. I) MV posses larger Ca2+ reserve, while Ca2+
gets depleted very quickly in MA and II) SERCA blockade activates Ca2+ influx
through SOC and this influx is coupled to constriction in MV but not MA. The
increased Ca2+ reserve in MV can potentially influence the magnitude of
functional responses in MV. Studies suggest that when Ca2*'load in the stores is
enhanced there is an increased release of Ca2+ at any given level of IP3

produced. While we do not have appropriate data to attribute the enhanced

212

sensitivity of MV purely to this mechanism, it certainly contributes to functional
differences between MA and MV. The larger stores could also explain the fact,
why MV desensitize to KCI but no desensitization to NE is observed. While NE
responses in MV are dependent on Ca2+ stores (see below), KCI mediated
constriction depends on Ca2+ influx through VOCC. NE generates a large Ca2+
signal in venous SMC likely due to continuous refilling of the stores through SOC,
providing for continuous constriction. In contrast, Ca2+ influx during KCI exposure
is not sufficient to induce constant constriction of MV. However, in MA
constriction to KCI does not desensitize, suggesting that Ca2+ influx through
VOCC provides more powerful signal in MA than in MV. It is interesting that MV
were less sensitive to IP3 receptor inhibition by 2-APB than MA. Three
mechanisms might potentially explain it: I) the IP3 receptor is less sensitive in MV
or perhaps there is a greater IP3 receptor reserve on SER in MV. In addition, as
our studies suggest MV posses larger Ca2+ stores than MA and this could lead to
sufficient release of Ca2+ at a given IP3 produced even though the majority of IP3
receptors are blocked or II) MV do not utilize this pathway in adrenergic
responses. However, our studies with thapsigargin suggested that after depletion
of intracellular Ca2+ stores, only a fraction of control NE response is maintained.
This would be inconsistent with the idea that MV do not utilize intracellular Ca2+
stores. No other receptor that would be coupled to GPCR has been
characterized on SER. However, Ca2+ could be also released from SER by Ca2+ -
induced Ca2+ - release (CICR) by its action on ryanodine receptor. In this model,

oAR in MV could activate Ca2+ channels/transporters (large sensitivity of NE

213

responses to Cd”) and Ca2+ influx would activate CICR, providing for sustained
constriction. Lastly (Ill), involvement of messengers modulating the Ca2+
sensitivity of the contractile apparatus in MV may be the dominant mechanism
regulating the contractility of MV. We only performed few experiments to address
this issue (see below) and therefore more data are needed to make a decisive
. conclusion about this potential mechanism.

SOC are coupled to constriction in MV. The tonic but not the initial
phasic constriction to thapsigargin in MV was inhibited by SOC blockers Gd3+
and La3+ and in media lacking Ca2+. These results suggested that activation of
SOC in MV induces vascular constriction. While also MA express TRP channels
that contribute to SOC and SERCA blockade also increases intracellular Ca2+ in
MA likely through SOC activation, activation of these channels did not induce
constriction of MA even though [Ca2+]i was elevated. While SOC Ca2+ entry has

2+]: in the vasculature,

been consistently shown to induce increases in [Ca
activation of these channels by SERCA blockade did not consistently induce
constriction in systemic arteries and arterioles. Examples of vessels where
increased [Ca2+]; caused by SERCA blockade does not lead to vascular
constriction include rat MA, renal artery and coronary artery (Naganobu and Ito,
1994, Snetkov et al., 2003). It is particularly interesting, that in rabbit cerebral
arteriolar SMCs, SERCA blockade and KCI lead to similar increases in [Ca2+].

even though KCI and not SERCA blockade produce constriction {Flemming

2002 #2}.

214

The profound differences in the responses to SOC activation between
MV and MA and the above mentioned vessels may be based on physiological
and anatomical differences between the vessels. Perhaps in the MA, SOC are
functionally compartmentalized and serve to refill Ca2+ in endoplasmic reticulum,
without close localization and coupling to contractile proteins. In addition, MA
posses efficient mechanisms other than SERCA, that remove the accumulating
Ca2+ from the cytosol. In contrast, SOC may directly activate contractile proteins
in MV. In fact, we detected some functional role of SOC in NE responses in MV
but not MA, suggesting that activation of SOC also has functional implications in
sympathetic responses. However, more studies are required to explain the
heterogeneity in responses to SOC activation between MA and MV. Additionally,
it would be very interesting to find out the physiological implications of this
heterogeneous regulation of arterial and venous tone by SOC activation.

Bi-directional role of NCX in MA. Our studies indicate that NCX works
as bi-directional Ca2+ transporter in MA. It is more efficient in Ca2+ removal from
the cytosol in MA than in MV and also seems to play an important role in Ca2+
influx during NE—mediated constriction of MA. Even when thapsigargin-sensitive
Ca2+ stores were depleted, NE was still able to induce constriction of MA when
Ca2+ influx was potentiated by ouabain. This effect of ouabain was not observed
in MV, suggesting that intracellular Ca2+ stores are absolutely necessary for
adrenergic responses in MV.

Responses to NE were greatly inhibited by Cd2+ in both MA and MV,

although the kinetics of inhibition by Cd2+ differed between the two vessels. While

215

Cd2+ exhibited a dose—dependent inhibition of NE responses in MA, Cd”
abolished constriction to NE at NE concentrations that produced maximal
constriction in MV, suggesting the involvement of Ca2+—dependent mechanisms
inhibited by Cd2+ in MV. Cd2+ competes with Ca2+ for the same regulatory binding
sites on NCX, but with several orders of magnitude larger affinities (Le et al.,
2005). Inhibitory effects of Cd2+ on NE responses in MA and MV could be at least
partially attributed to inhibition of NCX. This is further supported by the fact
nifedipine does not fully account for the effect of Cd2+ (reduction of NE responses
was much smaller), and also SOC likely do not participate in NE constriction of
MA (no effect of Gd3+ in MA).

lmmunocytochemical analysis confirmed that NCX is expressed in the
membrane of MA. We also detected NCX in the membrane of venous SMCs.
These data further indicate that NCX plays an important functional role in MA and
MV (see below). NCX—mediated Ca2+ entry in MA might be an important
characteristic of functional responses in MA. Increased activity of reverse mode
NCX could enhance Ca2+ influx and increase TPR.

It is interesting that a recent study has proposed a role of NCX1 (the
dominant isoform expressed in the vasculature) in the etiology of salt-sensitive
hypertension. The selective inhibitor of NCX that preferentially blocks Ca2+ entry
mode (SEA0400) reduced blood pressure in salt-sensitive hypertension in rat. In
addition, mice heterozygous for NCX1 mutation resisted the development of salt-
sensitive hypertension while blood pressure in transgenic mice overexpressing

NCX1 was more salt-sensitive (lwamoto et al., 2004). The data suggested that

216

the NCX1 in the vasculature causes increased Ca2+ influx resulting in increased
vascular tone. This finding might also have clinical relevance, because the
plasma level of endogenous ouabain and other cardiotonic steroids is elevated in
large proportion (~50%) of hypertensive individuals (Manunta et al., 1999).
Ouabains are produced and stored by the cortex of adrenal gland (Laredo et al.,
1995). Endogenous ouabain has been shown to be identical to the plant —
derived ouabain and other structurally related endogenous ouabains have also
been identified. Among those are the cardenolides that include digoxin and
ouabain. The second group includes bufadienolides with 19-norbufalin,
telocinobufagin and marinobufagenin (Schoner and Scheiner-Bobis, 2007).
Increased NE overflow due to enhanced SNS activity together with enhanced
endogenous ouabain levels could greatly potentiate signaling of oAR in
hypertension.

Further studies are needed to determine the relative contribution of
NCX1 — mediated Ca2+ influx in MA and MV. Utilization of SEA0400 would help
to directly asses this. While our studies suggest that NCX plays an important
Ca2+ influx role in MA, it might aIso facilitate Ca2+ influx in MV. The studies by
lwamoto described above (NCX1 overexpression, salt—sensitive hypertension)
did not address the contribution of TPR and CO in hypertension development.
Since salt-sensitive hypertension models also exhibit increased venous tone and
decreased capacitance, it would be interesting to find out the role of NCX in

venous SMCs in the pre-hypertensive and hypertensive states.

217

Less efficient Ca2+ removal in MV than in MA. Our studies indicated
that responses of MV decay more slowly than in MA. While inhibition of the
plasma membrane Ca2+-ATPase 4 (PMCA4) did not affect constriction to NE nor
did it attenuate the relaxation in MA, it greatly potentiated constriction to NE in
MV and also prolonged the constriction decay phase. It is likely that NCX is the
dominant Ca2+ - clearance pathway in MA, whereas PMCA is likely involved in
Ca2+ homeostasis (inhibition of this transporter causes venous constriction) as
well as Ca2+ clearance after constriction in MV because of the less efficient Ca2+
efflux by NCX in MV. NCX has a large capacity for Ca2+ export, whereas PMCA
is the low capacity Ca2+ export system. Therefore, the relative contributions of
these two efflux pathways in MA and MV has the potential to explain, why

contractile responses of MV that reflect intracellular [Ca2+]:

decay more slowly in
MV. Even though the NCX more efficiently removes Ca2+ from the cytosol in MA,
this is not due to higher NCX expression in MA compared to MV. Perhaps,
differences in other transporters that influence the Na+ gradient across the
plasma membrane might differentially regulate the activity of NCX in MA and MV.

More efficient Ca2+ export mechanisms in MA compared to MV would
have great implications in human physiology. While the diameter of resistance
arteries must be regulated in a fast manner, such that if too long time was
required to relax arteries, it could potentially cause tissue ischemia, a slower rate

of Ca2+ removal and relaxation in MV could provide for gradual filling of veins

with blood instead of very large amount of blood being shifted to veins

218

instantaneously. Since veins are much more compliant than arteries, fast shifts of

large volumes of blood into the veins could cause syncope.

Ca2+ sensitivity of MA and MV. Our data did not indicate that the
contractile proteins of MV are more sensitive to Ca2+ than in MA. The CRC to
Ca2+ in the presence of constant NE concentration was almost identical in both
vessels. While the extracellular Ca2+ concentration does not reflect intracellular
Ca2+ levels, it is likely that after Ca2+ depletion, Ca2+ release and Ca2+ influx at
low Ca2+ concentrations are inhibited to similar degree in both vessels.
Therefore, these studies provided only indirect evidence that there are no major
differences in Ca2+ sensitivity. Ca2+ imaging studies could provide more
conclusive evidence against/for the existence of such mechanism.

Rho kinase. Our data indicate that Rho kinase plays an important
functional role in adrenergic responses of both MA and MV, perhaps even
greater role in MA due to larger inhibition of adrenergic responses by the inhibitor
in MA. Therefore, it is unlikely that the enhanced sensitivity of MV could be
attributed to an increased role of this pathway that leads to Ca2+ sensitization in
venous SMCs. Further studies should be performed in MA and MV from
hypertensive animals to asses the potential upregulation of Rho pathway in the
veins of hypertensive animals and its contribution to hypertension.

Role of altered Ca2+ sensitivity in hypertension. Ca2+ sensitization
and enhanced vascular contractility occur in hypertension. A recent study has

proposed an important role for (312/13 signaling and its downstream activation of

219

Rho kinase in salt-sensitive hypertension. While selective abrogation of (312113111
the smooth muscle cells did not alter basal blood pressure, it prevented the
development of DOCA—salt hypertension in these transgenic mice. On the other
hand, selective abrogation of Gq11 not only prevented hypertension development,
but also reduced basal blood pressure (WIrth et al., 2008). While these data
indicate a greater role for Go in regulation of blood pressure under basal
conditions, selective reduction of blood pressure under hypertensive conditions
by G12/13 abrogation provides a promising target for treatment of hypertension.
Indeed, studies suggest that independent of the cause of hypertension, activation
of the Rho kinase pathway seems to be the downstream signaling target in many
animal models and inhibition of this pathway can attenuate blood pressure

(Uehata et al., 1997, Seasholtz and Brown, 2004, Seko et al., 2003).

Overall Conclusions and Implications.

Studies described in this dissertation aimed to clarify the mechanism
behind the enhanced adrenergic sensitivity of MV. Multiple hypotheses were
tested and contributed to a better overall understanding of the differences in
adrenergic regulation of MA and MV.

a. azAR indirectly contributes to adrenergic responses in MV but
not MA and enhances the functional coupling of <1in Wnile direct
stimulation of quR does not induce constriction in MV, blocking this receptor
shifts the concentration - response curve to NE to the right. The ozAR subtype

mediating this interaction in WT mice is predominantly ochR subtype. In the

220

ochR KO mice, another 02AR subtype compensates for the loss of ochR
function.

b. The mechanism underlying the ui-azAR interaction likely does
not involve direct interaction between the two receptors through receptor
heterodimerization. No co-localization, co-trafficking or effect on the membrane
expression of oioAR has been detected in HEK-293 cells. In addition, stimulation
of the two receptors when co-expressed did not lead to the functional interaction,
suggesting that this synergism is specific for certain cellular phenotypes like the
MV and occurs at the level of intracellular messengers. While MA also express
OzAR subtypes, this interaction was not detected in these arteries.

-differential receptor expression does not account for the interaction and

differences in adrenergic sensitivity of MA and MV

-SMCs from MV express smaller amount of the GAR subtypes in the
membrane than MA, suggesting more efficient stimulus — response coupling of
oARs instead of increased receptor reserve as the underlying mechanism for
enhanced sensitivity of MV

c. uzAR promotes Ca2+ influx through L-type VOCC and SOC and
perhaps some other yet unidentified Ca2+ channel and this action facilitates
increased Ca2+ accumulation and contraction. This mechanism may underlie the
functional interaction between (11 — and C12ARS in MV

d. MV posses larger Caz“ reserve in the SER than MA. This can
lead to increased Ca2+ release from the endoplasmic reticulum at any given IP3

produced by the stimulation of qAR. MV are more dependent on intracellular

221

Ca2+ than MA. When the stores are depleted, potentiation of Ca2+ influx by NCX
facilitates responses to NE in MA, but not in MV. Intracellular Ca2+ stores are
necessary for NE response in MV. IP3 — independent CICR may also prove to be
important Ca2+ release mechanism in MV. 0

e. SOC are coupled to contractile responses in MV but not in MA.
SERCA inhibition activates SOC and this results into profound constriction of MV,
while the vascular tone in MA is unaffected. This may be attributed to differential
spatial organization of Ca2+ handling proteins between MA and MV, where SOC
can directly activate proteins of the contractile apparatus in the MV. While SOC
are present and their activation enhances intracellular Ca2+ in MA, they may be
more compartmentalized in the MA. In addition, SOC appear to play some
functional role in adrenergic responses of MV, but not MA.

f. MA posses more efficient alternative pathways to remove Ca2+
that enters the cell through SOC compared to MVAwhen SERCA in inhibited.
NCX contributes to Ca2+ removal in both MA and MV, although in MA, this
process is more efficient. These differences in Ca2+ handling contribute to the
differential responsiveness to SERCA blockade between MA and MV. However,
the more efficient Ca2+ removal by NCX in MA can not be attributed to increased
expression of NCX protein, because large expression of NCX was detected in
the plasma membrane of both arterial and venous myocytes.

-Responses to contractile agonist are longer lasting in W than MA,
further indicating less efficient Ca2+ efflux pathways in MV than MA. Not only

NCX, but also PMCA contributes to Ca2+ removal in MV. PMCA also contributes

222

to the maintenance in Ca2+ homeostasis during resting conditions in MV. Less
efficient Ca2+ removal could also contribute to increased Ca2+ accumulation

during agonist stimulation and enhanced functional responsiveness.

Altogether, these studies suggest that while there may not be major
differences in the expression of oAR between MA and MV, oAR in MV are more
efficiently coupled to contractile responses in vascular capacitance vessels.
Previous studies did not suggest that there are any major differences in the
contractile proteins between MA and MV (Yamboliev et al., 2002), indicating that
differences in signal transduction and Ca2+ handling are indeed the likely
candidates explaining the differences in vascular reactivity.

All five points described above could contribute to larger sensitivity of
MV than MA. When SNS activity is slightly elevated in pre-hypertensive subjects,
it may selectively target capacitance vasculature. Considering that GzAR
enhances oiAR signaling at low stimulation thresholds, ozAR antagonists may
selectively block an increase in venous tone when baseline SNS activity is

slightly elevated.

223

 

Venous Smooth Muscle Cell

Fig. 38A. Model of the proposed adrenergic receptor signaling and calcium
coupling in venous smooth mucle cell. Activation of a1AR by NE leads to
production of IP3 and DAG. DAG activates receptor - operated channels (ROC,
likely TRPC6) that results into Na and Ca2+ influx. Ca2 may either act on
ryanodine receptors to activate Ca2 -induced Ca2 release (CICR). The
accumulation of Na in small microdomains could reverse the activity of NCX,
driving additional Ca2 influx and CICR, producing constriction. azAR is also
activated by NE and couples to Ca2 influx through L-type voltage — operated
Ca2+ channels (VOCC) and/or store operated channels (SOC). SOC can also
directly participate in vascular constriction. During relaxation, Ca2 is taken up by
plasma membrane Ca2 - ATPase (PMCA) and reverse mode NCX. The activity
of NCX and therefore Ca2 export is regulated by other transporters that regulate
Na gradient across plasma membrane, like the Na /K -ATPase.

224

 

Arterial Smooth Muscle Cell

(Fig 38 continued). Model of the proposed adrenergic receptor signaling
and calcium coupling in arterial smooth mucle cell (B). oiAR in arteries
activate phospholipase C and production of |P3 and DAG IP3 activates IP3
receptors on smooth endoplasmic reticulum, leading to Ca2 release from the
stores. DAG activates ROC, perhaps TRPCG, producing Ca2 and Na influx in
localized regions near the plasma membrane Whether this Ca2 is sufficient to
produce constriction remains to be evaluated. However, increased concentration
of Na causes NCX to operate in its reverse mode, producing Ca2 influx that
directly participates in arterial contraction. SOC refill the Ca2 stores when Ca2

depleted. However, they do not directly participate in arterial constriction Durinig
relaxation, Ca2 extrusion is very efficient and NCX contributes to this process.

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