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DISRUPTION OF NOREPINEPHRINE RELEASE AND CLEARANCE
MECHANISMS IN SYMPATHETIC NERVES ASSOCIATED WITH MESENTERIC
ARTERIES BUT NOT VEINS IN SALT-SENSITIVE HYPERTENSION

By

Hua Dong

A DISSERTATION

Submitted to

Michigan State University
In partial fulﬁllment of the requirements
For the degree of

DOCTOR OF PHILOSOPHY
Chemistry

2009

ABSTRACT

DISRUPTION OF NOREPINEPHRINE RELEASE AND CLEARANCE
MECHANISMS IN SYMPATHETIC NERVES ASSOCIATED WITH
MESENTERIC ARTERIES BUT NOT VEINS IN SALT-SENSITIVE

HYPERTENSION

By

Hua Dong

Norepinephrine (NE) is a vasoconstrictor neurotransmitter released from
sympathetic nerves that innervate the smooth muscle cells in arteries and veins.
Once released, NE diffuse across the junctional cleft, bind brieﬂy to receptor to
elicit smooth muscle contraction. The sympathetic nerves system regulates blood
pressure and its function is altered in animal models and human essential
hypertension. The altered function is associated with altered NE release.
However, little is known about the characteristics of local NE release from
periarterial and perivenous sympathetic nerves in normal and hypertensive
animals. I seek a better understanding of the functional differences between
sympathetic neurotransmission in mesenteric arteries (MA) and veins (MV), and
how this process is altered in hypertension. Deoxycorticosterone-acetate (DOCA)
salt rats were used as a model of salt-sensitive hypertension. Continuous
amperometry with a microelectrode measures the local concentration of
endogenous NE at the surface of a blood vessel as an oxidation current. Since

NE release is a multi-step process, one way the mechanisms can be probed is

by studying the temperature dependence of neurotransmitter release and
clearance.

The results of this dissertation study revealed important ﬁndings regarding
adrenergic neurotransmission in MA and MV from normotensive and DOCA-salt
hypertensive rats. 1) NE release and clearance from rat periarterial and ‘
perivenous sympathetic nerves are regulated differently. This difference is based
largely on the function of prejunctional (12 adrenergic receptors (azARs), NE
transporter (NET), calcium channels, but may also include different distribution of
vesicles that contain NE. NE release and clearance is regulated by azARs and
NET in MA but not in MV. 2) Adrenergic neurotransmission to MA is impaired in
DOCA-salt hypertension. The increased NE overﬂows and temperature
sensitivity in DOCA—salt MA is at least partially due to the impaired function of
azAR and NET. These changes are not due to the alterations in calcium handling
in the nerve terminal. 3) Oxidative stress alters sympathetic neurotransmission in
DOCA—salt hypertensive rats by impairing pertussis toxin-sensitive G proteins
which couple to GzAR at the nerve terminal. Chronic antioxidant treatment lowers
blood pressure and restores sympathetic nerve function at the neuroeffector
junction in DOCA—salt hypertensive rats.

Together, these results reveal the different supply of sympathetic nerves to
MA and MV may contribute to their different hemodynamic functions.
Furthermore, they reveal the importance of sympathetic nerve endings as a
therapeutic target and support a new mechanism for the beneﬁcial effect of

antioxidant treatment in salt-sensitive hypertension.

ACKNOWLEDGEMENTS

The research work conducted in this thesis was completed with the
guidance, support, and encouragement of many people.

First and foremost, I would like to thank my advisor, Dr. Greg M. Swain,
and my co-advisor Dr. James Galligan, for their continued support and guidance
throughout the course of my research. They offered valuable insight and direction
in guiding my research work. Under their excellent guidance, I learned how to
conduct research in the academic area. I appreciate all that they have done for
me during my PhD study.

I am indebted to the members of my committee, Dr. Gary Blanchard and
Dr. Gregory Fink, for their valuable suggestions, comments, and the time they
shared with me. In addition, I would like to thank my previous group members,
Dr. Jinwoo Park and Dr. Shihua Wang for their friendship and academic support
on my research.

My gratitude also goes to members of Dr. Swain’s lab: Dr. Hong Zhao, Dr.
Liang Guo, Dr. Doo Young Kim, Vernon Matthew Swope, Ayten Ay, Matthew
Fhaner, David Woodbury, Chen Qiu, Xingyi Yang, and Kelly Hutchins, for their
help and suggestions during my PhD study. I would also like to express my deep
appreciation to colleagues and friends from Dr. Galligan’s Lab: Dr. Hui Xu, Dr.
Xiaochun Bian, Hui Wang, Emmy Sangsiri, Vinoo Naidoo, and Dima Decker, for
their inspiring ideas and assistance. I was fortunate to have worked with these

talented researchers and students.

iv

Last but not the least, I owe my family much gratitude. I would like to thank
my parents, sister and husband for their unwavering encouragement,
understanding, and endless love along this journey. They were always there
whenever I needed them and their constant support was my strong crutch for the

completion of my PhD study.

TABLE OF CONTENTS
LIST OF TABLES ............................................................................... VIII
LIST OF FIGURES .................................................................................. IX

CHAPTER 1: GENERAL INTRODUCTION

1.1 Hypertension ................................................................................... 1

1.2 Blood pressure regulation .................................................................. 2
1.3 Sympathetic nervous system and hypertension ....................................... 3
1.4 Prejunctional regulation of neurotransmission ......................................... 9
1.5 Differential sympathetic innervations, mesenteric arteries vs. veins ......... 17
1.6 Oxidative stress and hypertension ................................................... 21
1.7 The DOCA-salt hypertension animal model ....................................... 24
1.8 Approaches to measure NE release ..................................... 25
1.9 Electrochemical detection of NE in vitro using microelectrodes .............. 27
1.10 Diamond and carbon ﬁber microelectrode ...................................... 30
1.11 Temperature as a tool to probe the SNS differences in arteries and veins.35
1.12 Research objectives and speciﬁc aims ............................................... 37

CHAPTER 2: EXPERIMENTAL SECTION

2.1 Boron doped diamond ﬁlm growth ................................................... 40
2.2 Diamond ﬁlm characterization ..................................................................... 41
2.3 Microelectrode preparation ............................................................... 43
2.4 Electrochemical measurement .................................................................. 44
2.5 Drug effect on NE detection ....................................................... 47
2.6 Normotensive and DOCA—salt hypertensive rats ................................. 48
2.7 Chronic antioxidant treatment for DOCA—salt hypertensive rats ................ 49
2.8 In vitro electrochemical measurement ................................................. 50
2.9 Chemical and drug application .......................................................... 54
2.10 Data analysis ................................................................................ 54

CHAPTER 3: DRUG EFFECTS ON THE ELECTROCHEMICAL DETECTION
OF NOREPINEPHRINE WITH CARBON FIBER AND DIAMOND
MICROELECTRODES

3.1 Introduction .................................................................................... 56
3.2 Results .................................................................................... 60
3.3 Discussion ..................................................................................... 77
3.4 Conclusion .............................................................................. 86

vi

CHAPTER 4: TEMPERATURE-RELATED DIFFERENCES IN SYMPATHETIC
NEUROEFFECTOR TRANSMISSION TO MESENTERIC ARTERIES AND
VEINS

4.1 Introduction .................................................................................... 89
4.2 Results .......................................................................................... 91
4.3 Discussion ..................................................................... 104
4.4 Conclusion .......................................................................... 1 17

CHAPTER 5: IMPAIRED ADRENERGIC NEUROTRANSMISSION TO
MESENTERIC ARTERIES BUT NOT VEINS IN DOCA-SALT HYPERTENSIVE
RATS

5.1 Introduction .................................................................................. 118
5.2 Results ........................................................................................ 121
5.3 Discussion ............................................................................. 133
5.4 Conclusion ................................................................................... 148

CHAPTER 6: 02' INTERACTS WITH PERTUSSIS TOXIN-SENSITIVE G-
PROTEINS TO DISRUPT a2 ADRENERGIC RECEPTOR FUNCTION
INSYMPATHETIC NERVES SUPPLYING MESENTERIC ARTERIES IN DOCA-
SALT HYPERTENSION

6.1 Introduction .................................................................................. 149
6.2 Results ........................................................................................ 149
6.3 Discussion ............................................................................. 151
6.4 Conclusion ................................................................................... 160
CHAPTER 7: CONCLUSIONS ..................................................................... 168

vii

LIST OF TABLES

Table 3.1 Drug Molecular Structure and Function ....................................... 59

Table 3.2 Summary of cyclic voltammetric AEm and Irene values for NE oxidation
in the absence of different drugs .............................................................. 83

Table 5.1 Q10 values for sham and DOCA—salt MA and MV with different
treatments ......................................................................................... 1 30

viii

LIST OF FIGURES

Figure 1.1 Sympathetic divisions of the autonomic nervous system .................. 4
Figure 1.2 Diagram of NE synthesis, release and clearance .......................... 10
Figure 1.3 Steps for NE synthesis ............................................................ 11
Figure 1.4 Metabolism of NE after release ................................................. 17
Figure 1.5 The structure of artery and vein ................................................ 19
Figure 1.6 Endogenous oxidant and antioxidant pathways ............................ 24
Figure 1.7 NE redox reaction .................................................................. 28
Figure 1.8 Representative functional groups that might exist on oxidized sp2
carbon surfaces ................................................................................... 32
Figure 1.9 Structure for diamond sp3 carbon .............................................. 34

Figure 2.1 SEM image of a Pt wire coated with a polycrystalline boron doped
diamond ﬁlm (left) and an expanded view of the microelectrode tip (right) ......... 41

Figure 2.2 Raman spectroscopy for diamond thin ﬁlm deposited on Pt wire, hex =
532 nm. Laser power = 30 mW. Integration time = 10 s .............................. 42

Figure 2.3 Diagram of the preparation of diamond and carbon ﬁber
microelectrode .................................................................................... 43

Figure 2.4 Cyclic voltammetric i-E curves for a diamond (left) and carbon ﬁber
(right) microelectrode in 1.0 mM K4Fe(CN)5 in 1 M KCI. Scan rate is 50 mV/s...46

Figure 2.5 Diagram of the experimental set up for in vitro NE release from
sympathetic nerve endings ..................................................................... 50

Figure 2.6 Video images of a mesenteric artery showing positions of the diamond
microelectrode, the stimulator (top left) and the change in artery diameter in
response to stimulation (top right). Temporal responses of the NE overﬂow
oxidation current (bottom red trace) and vessel diameter (bottom blue trace)
elicited by electrical stimulation ............................................................... 53

Figure 3.1 Cyclic voltammetric i-E curves for drugs at carbon ﬁber (P55, T650)
and diamond microelectrodes: (A) capsaicin, prazosin, UK 14,304 and
yohimbine, and (B) idazoxan, PPADS and cocaine at the P55 carbon ﬁber; (C)
capsaicin, prazosin, UK 14,304 and yohimbine and (D) idazoxan, PPADS and

ix

cocaine at the T650 carbon ﬁber; and (E) capsaicin, prazosin, UK 14,304 and
yohimbine and (F) idazoxan, PPADS and cocaine at the diamond microelectrode.
All curves were acquired in Krebs solution at a scan rate of 0.1 V/s. The
concentration of each drug was 0.1 mM .................................................... 64

Figure 3.2 Cyclic voltammetric i-E curves for NE at (A and B) P55 and (C and D)
T650 carbon ﬁber and (E and F) diamond microelectrodes with and without
capsaicin and prazosin. All curves were acquired in Krebs solution at a scan rate
of 0.1 V/s. The concentration of NE was 0.02 mM and the concentration of each
drug was 0.1 mM ................................................................................. 66

Figure 3.3 Plots of the cyclic voltammetric AEm and limiting current ratio, Iratio
(=laﬂﬁ.r drug/linmal), after drug exposure (capsaicin and prazosin) and washout at the
three microelectrode types. Statistical analysis was based on n = 3. *Represents
a signiﬁcantly different value from the control (before drug exposure) as
assessed using the standard t-test, P<0.05 ................................................ 69

Figure 3.4 Cyclic voltammetric i-E curves for NE at (A and B) P55 and (C and D)
T650 carbon ﬁber and (E and F) diamond microelectrodes with and without UK
14,304 and yohimbine. All curves were acquired in Krebs solution at scan rate of
0.1 V/s. The concentration of NE was 0.02 mM and the concentration of each
drug was 0.1 mM ................................................................................. 72

Figure 3.5 Plots of the cyclic voltammetric AEm and limiting current ratio, Iratio (=
lane, drug/linitial), after drug exposure (UK 14,304 and yohimbine) and washout at the
three microelectrode types. Statistical analysis was based on n = 3. *Represents
a signiﬁcantly different value from the control (before drug exposure) as
assessed using the standard t-test, P<0.05 ................................................ 76

Figure 3.6 Cyclic voltammetric i-E curves for NE at (A, B and C) P55 and (D, E
and F) T650 carbon ﬁber, and (G, H and l) diamond microelectrodes with and
without cocaine, idazoxan and PPADS. All curves were acquired in Krebs
solution at a scan rate of 0.1 V/s. The concentration of NE was 0.02 mM and the
concentration of each drug was 0.1 mM .................................................... 78

Figure 4.1 (A) NE oxidation currents recorded from a MA and MV in the absence
and presence of TTX. TTX completely blocked the stimulation evoked oxidation
current. (B) Pooled data from experiments illustrated in “A” showing the 'I'I'X
blocks stimulation evoked oxidation currents. *indicates signiﬁcantly different
from control (P < 0.05). (C) NE oxidation currents from a MA and MV in the
absence and presensive od CdCIz, a calcium channel blocker. (D) CdCl2
concentration response curve for inhibition of NE oxidation current evoked by
electrical stimulation. Veins were more sensitive to the inhibitory effects of CdC|2
compared to arteries. Data are mean :1: SEM ........................................... 93

Figure 4.2 NE oxidation currents recorded from a MA (A) and MV (B) at 37 °C

(black trace) and 28 °C (grey trace). The oxidation current continued to increase
in MA and MV at 37 oC throughut the stimulus train. At 28 °C the current began
to decline in MA while it was sustained in MV. The bar under the current traces
represent the period of nerve stimulation (60 pluses with a 0.5 ms pulse width).
(C) Plot of the temperature dependence of peak current amplitude in MA and
MV. The peak current amplitude in MV is more steeply temperature dependent.
(D) Q10 values for NE current amplitudes. Data are mean :I: S.E.M. * represents
signiﬁcant difference from MA, P < 0.05 .................................................... 96

Figure 4.3 Comparison of the time proﬁles of NE oxidation currents in MA and
MV at 37 and 28 °C. (A) 10% to 90% rise slope of NE oxidation current is greater
for MV compared to MA at 37 °C but not 28 °C, (B) The time to peak NE
oxidation current is signiﬁcantly greater at 37 than 28 °C for both MA and MV, (C)
the 10% to 90% decay time of NE oxidation current is much longer for MV than
for MA at both 37 °C and 28 °C. Data are mean :I: S.E.M. *indicates signiﬁcantly
different from MA; # indicates signiﬁcantly different from 37 °C, P < 0.05 ......... 98

Figure 4.4 Effect of the azAR blocker, idazoxan, NE oxidation current from MA
and MV at different temperatures. (A) NE oxidation current traces from a MA in
the absence and presence of idazoxan (1 pM) at 37 and 28 °C. At 37 °C,
idazoxan increases peak currents but the time to peak current is similar. At 28
00, idazoxan increases peak current and time to peak current as the current
amplitude increases throughout the train of stimulation in the presence of the
antagonist. (B) ldazoxan does not alter NE oxidation currents in MV at 37 or 28
°C. Bars under the current traces represent the period of nerve stimulation (60
pluses with a 0.5 ms pulse width). Pooled data for NE oxidation current at
different temperature with and without idazoxan for MA (C) and MV (D). Data are
mean :I: S.E.M.; *indicates signiﬁcant difference from control, P < 0.05 ........... 100

Figure 4.5 Time course of NE oxidation currents in MA and MV at 37 and 28 °C
with and without idazoxan. (A) 10 to 90% rise slope of NE oxidation current is
signiﬁcantly increased by idazoxan (1 (M) in MA but not MV at 37 °C and at 28
°C (B). (C) The 10 to 90% rise time of NE oxidation current was unaffected by
idazoxan at 37 °C in MA and MV but idazoxan increased the current rise time at
28 °C for MA but not MV (D). ldazoxan did not change the 10 to 90% decay time
of NE oxidation currents in MA or MV at 37 °C (E) and 28 °C (F). Data are mean
i SEM. and * represents signiﬁcant difference from control, P < 0.05 ........... 101

Figure 4.6 Effect of Gi/Go protein blocker, pertussis toxin (PTX, 3 ug/ml), on
inhibition of NE release in MA (A) and MV (B) at 37 and 28 °C. PTX increased
NE current in MA at both 37 and 28 °C but not in MV. azAR blocker, idazoxan (1
pM), increased NE current for MA in both PTX treatment group and control group
at both 37 and 28 °C. ldazoxan didn’t increase the current amplitude for MV in
either group at any temperature. Data are mean :I: SEM. and *indicates a
signiﬁcant difference between two groups, P < 0.05 ................................... 103

xi

Figure 4.7 Effect of cocaine (10 uM) on NE oxidation currents in MA and MV.
Representative recordings of oxidation currents in MA (A) and MV (B) at 37 and
28 °C. Bars under the current tracings represent the period of nerve stimulation
(60 pluses with a 0.5 ms pulse width). Cocaine increases peak oxidation currents
in MA at both temperatures. Oxidation currents in MV are unaffected by cocaine.
(C) Pooled data for effects of temperature and cocaine on oxidation currents in
MA and in MV (D). Data are mean :I: SEM. and *indicates a signiﬁcant
difference from control, P < 0.05 ............................................................ 105

Figure 4.8 Comparison of the time course for NE oxidation currents from MA and
MV at 37 °C and 28 °C with and without cocaine (10 (M). The 10 to 90% current
rise slope of NE oxidation current increased signiﬁcantly after treatment of
cocaine in MA at both 37 °C (A) and 28 °C (B). Cocaine did not affect the time
course of the current in MV at either temperature. (C) The 10 to 90% current rise
time was not affected by cocaine treatment for both MA and MV at 37 °C (C) but
increased at MA at 28 °C (D). The 10 to 90% decay time of the NE oxidation
current was prolonged in MA but not MV at 37 °C (E) and 28 °C (F). Data are
mean 1 SEM. and *indicates signiﬁcantly different from control, P < 0.05......106

Figure 4.9 Guanethdine concentration response curve for inhibition of NE current
in MA and MV at 37 °C. MV is more sensitive to the inhibition effect of
guanethidine. Data are mean :I: S.E.M ..................................................... 108

Figure 5.1 Representative recordings of in vitro continuous amperometric
measurement of NE oxidation currents from sham MA (A) and MV (B), and
DOCA-salt MA (C) and MV (D) at 37 °C (black trace) and 28 °C (grey trace). NE
oxidation currents were evoked by 10 Hz stimulus trains and the bar under the
current traces represents the period of nerve stimulation (60 pluses with a 0.5 ms
pulse width) ....................................................................................... 123

Figure 5.2 Temperature effects on NE maximum oxidation current evoked by
focal electric stimulation at MA (A) and MV (B) from sham and DOCA-salt rats.
The peak current amplitude in DOCA—salt MA is more steeply temperature
dependent. Data are mean :I: SEM. and * indicates signiﬁcantly different from
sham MA, P < 0.05 ............................................................................. 125

Figure 5.3 Comparison of the time course of NE oxidation current between sham
and DOCA-salt MA, and sham and DOCA-salt MV at 37 and 28 °C. (A) the 10 to
90% rise slope of NE oxidation current is signiﬁcantly lower in sham MA
compared to DOCA—salt MA, sham MV and DOCA—salt MV at 37 °C but not at 28
°C, (B) the 10 to 90% rise time of NE oxidation current is decreased signiﬁcantly
by temperature from 37 to 28 °C for all types of tissues with the sham MA has the
maximum decrease; and the rise time for sham MA is signiﬁcantly lower than
DOCA—salt MA, sham and DOCA—salt MV at 28 °C (C) the 10 to 90% decay time
of NE oxidation current was much less in sham MA than in DOCA-salt MA at 37
°C but not 28 °C; the current decay time was longer in sham and DOCA-salt MV

xii

than sham MA at both 37 °C and 28 °C. Data are mean :I: SEM. and *
represents signiﬁcantly different from 37 °C; # represents signiﬁcantly different
from sham MA, P < 0.05 ...................................................................... 127

Figure 5.4 Effect of azAR antagonist, idazoxan (1 pM), on temperature
dependent NE oxidation current at different temperatures. ldazoxan increased
peak NE current more than two fold for sham MA (A) and around 30% for DOCA-
salt MA at different temperatures (B). There was no change in NE current at
sham MV (C) and DOCA-salt MV (D) after idazoxan treatment. Data are shown
as mean 1 SEM. and * represents signiﬁcant different from control, P < 0.05
....................................................................................................... 132

Figure 5.5 Comparison of the time course of NE oxidation currents from sham
and DOCA-salt MA and MV at 37 and 28 °C with and without idazoxan. The 10 to
90% rise slope of NE oxidation current was signiﬁcantly increased after the
treatment of idazoxan for sham MA at both 37 °C(A) and 28 °C (B); idazoxan
increased 10 to 90 % current rise time signiﬁcantly only at 28 °C for sham MA (C
and D); the 10 to 90% decay time of NE current did not change after the
treatment of idazoxan at 37 °C (E) and 28 °C (F) for any type of tissues. Data
are mean :I: SEM. and * represents signiﬁcant difference from control, P < 0.05
....................................................................................................... 134

Figure 5.6 Effects of pertussis toxin (PTX, 3 ug/ml) on NE release at sham and
DOCA-salt MA and MV. After 2h tissue incubation in PTX, NE maximum current
was signiﬁcantly increased at sham MA (A) but not DOCA—salt MA, sham and
DOCA—salt MV (B, C and D). For comparison, we also blocked azAR function by
idazoxan for the control and PTX treatment groups. Both NE current and current
rise slope were increased signiﬁcantly by idazoxan for sham control and PTX
treatment groups (A) but the increase was very little in DOCA—salt MA control
group (B). There was no current increase in sham and DOCA—salt MV groups (C
and D). This represents that the function of Gi/Go which couples to azAR is
impaired in DOCA-salt MA and Gi/Go does not function in sham and DOCA-salt
MV as well as in sham MA. The current increased by idazoxan plus PTX was
signiﬁcantly greater than that caused by PTX alone for sham MA. Data are mean
:I: SEM. with * represents signiﬁcant difference, P < 0.05 ........................... 138

Figure 5.7 Effect of NE transporter blocker, cocaine (10 uM), on temperature
dependent NE oxidation current. Oxidation current was signiﬁcantly increased
from sham MA at every temperature (A) but only at low temperatures from
DOCA-salt MA (B). No current increase was observed at sham MV (C) and
DOCA-salt MV (D). Data are shown as mean i SEM. and * represents
signiﬁcant difference from control, P < 0.05 .............................................. 140

Figure 5.8 Comparison of the time course of NE oxidation currents from sham

and DOCA-salt MA and MV at 37 and 28 °C with and without cocaine. The 10 to
90% rise slope of NE oxidation current was increased signiﬁcantly after the

xiii

treatment of cocaine for sham MA at both 37 °C (A) and 28 °C (B) and for DOCA-
salt MA at 28 °C. Cocaine increased the 10 to 90 % current rise time signiﬁcantly
only at 28 °C for sham MA (C and D); the 10 to 90% decay time of NE current
was prolonged in sham MA at both 37 °C (E) and 28 °C (F); and it was prolonged
in DOCA-salt MA at 28 °C only. Data are mean 1 SEM. and * represents
signiﬁcant difference from control, P < 0.05 .............................................. 142

Figure 5.9 Concentration response curves for Ca channel blocker CdCl2 on NE
inhibition were not different between sham and DOCA—salt MA. Data are mean
:I:S.E.M ............................................................................................. 144

Figure 6.1 (A) Representative recordings of in vitro continuous amperometric
measurement of NE oxidation currents from sham and DOCA—salt MA before
(black trace) and after (grey trace) the blockade of presynaptic azAR using 1 uM
idazoxan. NE oxidation currents were evoked by 10 Hz stimulus trains and the
bar under the current traces represents the period of nerve stimulation (60 pluses
with a 0.5 ms pulse width). Statistical results for NE current (B) and 10-90% NE
current rise slope (C) from sham and DOCA-salt MA demonstrate that the NE
level is higher and 02AR function is impaired in DOCA—salt MA. Data are means i
SEM. with * represents signiﬁcant difference from control, and # represents
signiﬁcant difference from sham MA, P < 0.05 .......................................... 154

Figure 6.2 Mean arterial pressure (MAP) for chronic antioxidant treatment in vivo
study. MAP increased for three groups with time. It decreased in response to
chronic antioxidant treatment with apocynin (grey bar) and tempol (open bar)
compared to DOCA—salt controls, especially on day 18. Two-way ANOVA
indicates there is signiﬁcant difference for the MAP among three groups with time
(P < 0.05). Data are means i SEM. and * indicates signiﬁcant difference
compared to DOCA—salt controls by Newman-Keuls post hoc test, P < 0.05....156

Figure 6.3 Effects of chronic treatment of antioxidant, apocynin and tempol, on
the release of NE and prejunctional azAR function at the sympathetic nerve
endings. Representative traces of NE oxidation current from DOCA-salt control
(black), apocynin (dark grey) and tempol (light grey) groups before (A) and after
(B) the blockade of prejunctional azAR using 1 uM idazoxan. NE oxidation
currents were evoked by 10 Hz stimulus trains (60 pluses, 0.5 ms pulse width).
The maximum NE current (C) and 10-90% current rise slope (E) were
signiﬁcantly decreased in apocynin and tempol groups compared to DOCA-salt
controls. The differences in NE maximum current (D) and 10-90% current rise
slope (F) disappeared after the blockade of prejunctional azAR by idazoxan. This
indicates that the differences among three groups were partially caused by 02AR
and the function of azAR was restored by chronic apocynin and tempol treatment.
Data are mean :I: SEM. and * represents signiﬁcant difference from DOCA—salt
control by one-way ANOVA with Newman-Keuls post hoc test, P < 0.05 ........ 158

Figure 6.4 Effects of pertussis toxin (PTX, 3 uglml) on NE release at sham and

xiv

DOCA-salt MA. After 2h tissue incubation in PTX, NE maximum current and 10-
90% NE current rise slope were signiﬁcantly increased at sham MA (A and C) but
not DOCA-salt MA (B and D). For comparison, we also blocked azAR function by
idazoxan for the control and PTX treatment groups. Both NE current and current
rise slope were increased signiﬁcantly by idazoxan for sham control and PTX
treatment groups (A and C) but the increase was very little in DOCA—salt MA (B
and D). This represents that the function of Gi/Go which couples to OzAR is
impaired in DOCA-salt MA. The current increased by idazoxan plus PTX was
signiﬁcantly greater than that caused by PTX alone for sham MA. Data are
means :I: SEM. with * represents signiﬁcant difference from control, and #
represents signiﬁcant difference from PTX incubated MA, P < 0.05 ............... 161

Figure 7.1 Mesenteric artery and vein neuroeffector junctions in normotensive
rats. NE is released from sympathetic nerve terminals in response to nerve
stimulation and depolarization of the nerve terminal. NE bind with a1AR post
junctionally resulting in constriction of the smooth muscle cell. NE release in MA
is tightly regulated by prejunctional 02AR but not in MV, possibly due due to the
lower expression of Gi/Go proteins that couple to azAR. NE release is more
sensitive to calcium channel blockers in MV compared to MA. NE is cleared by
NET from the neuroeffector junction in MA but mainly by diffusion in MV; NET
function contributes to the temperature dependent difference in NE overﬂow
between MA and MV. It is possible that the readily releasable pool at the
sympathetic nerve endings is larger in MV but the reserve pool of vesicles is
further away from the active zone in perivenous compared to periarterial nerves.
These differences lead to the exceeding NE overﬂow at 37 °C and greater
temperature dependent sensitivity in NE overﬂow from MV than that from
MA ................................................................................................. 172

Figure 7.2 Mesenteric artery neuroeffector junctions in control and DOCA—salt rat.
In DOCA—salt hypertension, several changes occur to the sympathetic nerve
endings which affect neurotransmitter release. Calcium channel remain
functional, providing sufﬁcient Ca 2* inﬂux for vesicle fusion and neurotransmitter
release. However, the function of prejunctional azAR and NET are impaired in
DOCA-salt MA. NET contributes to the NE overﬂow temperature dependent
difference between DOCA—salt and sham MA. These differences all contribute to
the increased NE overﬂow at 37 °C and greater temperature dependent
sensitivity in NE overﬂow from DOCA-salt MA than that from sham MA ......... 175

Figure 7.3 Mesenteric artery neuroeffector junctions in DOCA-salt rat with (right)
and without (left) antioxidant treatment. There is increased ROS in the DOCA—salt
hypertensive rat MA nerve terminals. Increased ROS may target GJGO proteins
coupling to presynaptic OzAR in sympathetic nerve endings leading to impaired
neurotransmission and increased blood pressure. Chronic antioxidant treatment,
apocynin and tempol, lowers NE release, blood pressure and restores
sympathetic nerve function at the neuroeffector junction in DOCA—salt
hypertensive rats ................................................................................ 178

XV

CHAPTER 1
GENERAL INTRODUCTION

1.1 Hypertension

Hypertension is deﬁned as a sustained systolic blood pressure greater than
140 mmHg, and/or diastolic blood pressure over 90 mmHg [American Heart
Association]. Known as the “silent killer,” hypertension often produces few
obvious symptoms. However, uncontrolled high blood pressure can damage the
heart, eyes, kidneys, blood vessels, and the brain and can lead to congestive
heart failure, heart attack, kidney failure, or stroke. Hypertension occurs more
commonly among people over 35 and it is particularly prevalent in African
Americans, obese people, heavy drinkers and women who take birth control pills.
Every year, approximately 7.1 million deaths worldwide occur as a result of
hypertension related illness [1]. The goal of hypertension treatment is to lower
blood pressure and to protect the brain, heart, kidneys and other organs from
damage. Although the treatment rate is 75% or higher, less than 50% of
hypertensive patients achieve control of blood pressure to goal levels [2].

There are two types of hypertension, essential (primary) and secondary
hypertension. About 90 to 95% of the hypertension is essential hypertension,
which arises from unknown causes. Secondary hypertension accounts for 5 to
10% of all hypertension cases and high blood pressure in these patients is a
result of other diseases, such as kidney disease or hormone secreting tumors.
Although the evidence shows that some forms of essential hypertension involve

primarily the sympathetic nervous system, mechanisms leading to increased

sympathetic nerve activity are poorly understood [3]. Therefore, it is important to
study hypertension and its connection to increased sympathetic nerve activity. My
research was designed to further understand the causes of hypertension which

may help develop improved treatments for this disease.

1.2 Blood pressure regulation

The mean arterial blood pressure (MAP) is the product of cardiac output
(CO) and total peripheral resistance (TPR). CO is the volume of blood being
pumped by the left ventricle of the heart per minute. CO is the product of heart
rate and stroke volume. TPR is the sum of the resistance to blood flow through all
peripheral blood vessels. TPR is determined largely by the diameter of small
arteries and arterioles. Humoral factors, the central and the peripheral nervous
systems, and kidney function are important factors regulating blood pressure.
Acute changes in blood pressure are regulated by the baroreﬂex arc, which also
contributes to the long-term blood pressure regulation [4]. Baroreﬂexes and
central signals from higher brain centers dominate the moment-to-moment
control of blood pressure through the sympathetic and parasympathetic nervous
system (SNS and PNS), are part of the autonomic nervous system [5]. The SNS
inﬂuences the distribution of blood throughout the body by constricting and
dilating the blood vessels. SNS activation can increase TPR and CO to increase
blood pressure. Effects of sympathetic activation on the kidney and the rennin-
angiotensin system can also inﬂuence the blood pressure regulation [6]. The

PNS can slow the heart and decrease blood pressure by activation of vagal

nerves. Other factors, such as the endothelin system, vascular function, and

circulating epinephrine all contribute to the blood pressure regulation.

1.3 Sympathetic nervous system and hypertension

1.3.1 Sympathetic nervous system (SNS)

The nervous system plays an essential role in short and long term blood
pressure regulation [7] and SNS innervating the splanchnic circulation are
particularly important [8]. SNS is the part of the autonomic nervous system. It, in
general, inhibits or opposes the physiological effects of the parasympathetic
nervous system, as in tending to reduce digestive secretions, speeding up the
heart, and contracting blood vessels [9]. The cell bodies of the preganglionic
sympathetic neurons are located in the intennediolateral gray matter of the spinal
cord, between levels T1 and L3 (see Figure. 1.1). After convergence and
divergence, most of the axons from these preganglionic neurons synapse with
postganglionic sympathetic neurons located within ganglia of the paravertebral
sympathetic chain, as well as within prevertebral ganglia. The synapse in the
sympathetic ganglion uses acetylcholine (ACh) as a neurotransmitter. ACh binds
with nicotinic receptors on the post synaptic neurons in sympathetic ganglia. The
synapse of the post-ganglionic neuron with the target organ uses norepinephrine
(NE), adenosine 5’-triphosphate (ATP) as neurotransmitters and neuropeptide Y
(NPY) as a neuromodulator [10]. Over the past several years, beta-nicotinamide
adenine dinucleotide (beta-NAD) was also found to be a neurotransmitter

released from the SNS [11]. The SNS is the primary short term regulator of blood

Dilates pupil

_ Decreases salivation
Brain Stem
_ Accelerates heartbeat
Cervical
Relaxes airways
Thoracic
Inhibits digestion
- Stimulates secretion of
Lumber EP and NE

 

Inhibits gut motility
And secretions

Relaxes urinary bladder

 

 

Figure 1.1 Sympathetic divisions of the autonomic nervous system

[http://www.goodpsych.com/storage/Sympathetic_Nervous_System__L.jp]

pressure regulation and plays an important role in long term blood pressure
regulations. Sympathetic nerves originating from the celiac and superior
mesenteric ganglia supply arteries and veins in the splanchnic circulation.
Sympathetic nerves travel along blood vessels near the adventitial medial border
where they form neuromuscular junctions with vascular smooth muscle cells [12-
14]. Quantitatively, sympathetic nerve activity is the most critical factor controlling
venoconstriction, especially in the splanchnic circulation [15, 16]. Increased
sympathetic activity produces vasoconstriction.

Enhanced SNS activity is involved in many cardiovascular diseases, such
as hypertension, and heart failure. Work by Esler demonstrated that NE release
from renal nerves is elevated in young borderline hypertensive patients and that
they also have altered spillover of central monoamine neurotransmitters from
subcortical regions of the brain [17]. Increased sympathetic neurotransmission
has also been documented in experimental hypertension models, such as
deoxycorticosterone acetate (DOCA)-salt hypertension [18] and spontaneous
hypertension in rats [19]. The blood level of the principal sympathetic
neurotransmitters, NE, is elevated in these models to suggest increased
neurotransmitter release from sympathetic nerves. Increased NE release in
hypertension indicates that alteration may take place at the sympathetic nerve

endings.

1.3.2 Neurotransmitters

NE. Among the neurotransmitters that are released by SNS, NE is the principle

neurotransmitter intimately regulate cardiovascular functions. And it is believed
that the NE: ATP ratio in a sympathetic vesicle may be as high as 50:1 [20, 21].
NE is a catecholamine that is secreted by the adrenal medulla and sympathetic
nerve endings to cause vasoconstriction, increase heart rate, blood pressure,
and the sugar level of the blood [9]. NE performs its action mainly by act on
adrenergic receptors on target cells. For example, NE bind with (11 adrenergic
receptor (a1AR) post junctionally resulting in constriction of the smooth muscle
cell. Many substances modulate NE release, some inhibiting it and some
stimulating it, for instance, NE can bind to presynaptic 02 receptors for the
inhibition for its release by negative feedback mechanisms [21]. Details for the
regulation of NE release and clearance will be introduced in section 1.4
(Prejunctional regulation of sympathetic neurotransmission). Numerous studies
have indicated that there is increased NE release from perivascular sympathetic
nerves and increased vasoconstriction in animal models of hypertension,
particularly salt sensitive hypertension which is associated with hyperactivity of

the sympathetic nervous system [22, 23].

ATP as a sympathetic neurotransmitter in the vasculature. ATP is a
multifunctional nucleotide, and plays an important role in cell biology as an
intracellular energy source [24]. In 1972, ATP was proposed as a
neurotransmitter in non-adrenergic, non-cholinergic nerves in the gut and bladder
[25]. It is an important signaling molecule in both the central and peripheral

nervous system. Activity-dependent release of ATP from synapses, axons and

glia activates P2 (including P2X and P2Y family) purinergic membrane receptors
that modulate intracellular calcium and cyclic AMP [26]. The P2X ionotropic
receptor subgroup comprises seven members (P2X1-P2X7) which are ligand-
gated Ca2+-permeable ion channels that open when bound to an extracellular
purine nucleotide [27]. The P2Y (P2Y1-P2Y15) receptors are G protein-coupled
receptors and modulate mainly intracellular calcium and cyclic AMP levels. The
level of ATP at nerve endings are primarily regulated by mitochondrial oxidative
phosphorylation which generates ATP from ADP [28]. Upon nerve stimulation,
ATP is released and then quickly degraded by ectonucleotidasesto to ADP and
ﬁnally to adenosine [29, 30]. Adenosine can activate the A1 receptor on the nerve
terminal to regulate neurotransmitter release via negative feedback.

For arteries, ATP (adenosine 5' triphosphate) and NE are co-transmitters
released by sympathetic nerves [31-33]. ATP is the dominant neurotransmitter in
small mesenteric arteries in normotensive rats; however, in arteries from DOCA-
salt hypertensive rats, NE is the major sympathetic neurotransmitter. As for the
vein, NE is the neurotransmitter mediating neurogenic contractions of mesenteric
veins from both sham and DOCA—salt rats [34]. This proﬁle differs somewhat in
guinea pig mesenteric arteries and veins where purinergic neurotransmission is
more prominent in veins [31]. NE activates postjunctional a adrenergic receptors
(aARs) in both arteries and veins [35]. However, there are differences in
receptors that react to the nerve-released NE exist between arteries and veins.
P2X receptors also play an important role besides aARs in regulating the arterial

tone, while only aARs regulate venous tone [36].

Neuropeptide Y (NPY) is released by perivascular sympathetic nerves..
NPY is a 36-amino-acid peptide neurotransmitter in the central and peripheral
nervous systems [37]. NPY is co-localized with NE in sympathetic nerves
supplying the cardiovascular system and known to be of importance for the
maintenance of blood pressure. It is released during high frequency sympathetic
nerve stimulation and has a direct vasoconstrictor action, varying in intensity
depending on the vascular bed examined [38]. The receptor protein that NPY
operates on is a G-protein coupled receptor. Five subtypes of NPY receptor have
been cloned and classiﬁed as Y1, Y2, Y3, Y4, Y5, and y6 based on their
molecular and pharmacological proﬁle [39]. Among them, Y1 and Y2 receptors
are involved in peripheral effects. NPY increases the constriction induced by NE
and ATP via the Y1 receptor post-junctionally [40] and inhibits NE release
through Y2 receptor pre-junctionally [41]. NPY levels are increased in human
diseases associated with sympathetic activation. For example, in human
hypertension, venoarterial plasma NPY level is elevated [38]. NPY content is
increased in nerves of the cerebral and mesenteric vasculature in spontaneously
hypertensive rats [42]. Also, there are increased plasma NPY levels in DOCA-

salt hypertension [43].

Beta-nicotinamide adenine dinucleotide (beta-MAD) as a sympathetic
neurotransmitter. Beta-NAD is a coenzyme found in all living cells. Until
recently, it was found to be a neurotransmitter released from sympathetic nerves
in blood vessels and urinary bladder with NE, ATP, and NPY upon nerve

stimulation [44]. Beta-NAD is released via a botulinum neurotoxin A-mediated

mechanism but not evoked either by the overﬂow of other neurotransmitters, e.g.
NE, or the neuropeptides, e.g., substance P, in canine mesenteric artery [11] .
The release of beta—NAD is sensitive to the voltage dependent calcium channel
and depends on synaptosomal-associated protein of 25 kDa (SNAP-25)
mediated exocytosis at a low level of nerve activation. It has found to work as an
inhibitory neurotransmitter that contributes to enteric inhibitory regulation of
visceral smooth muscles [45]. Since beta-NAD was just discovered to be a
member of neurotransmitter family in the past several years, no work showing its

function related with disease status, e.g. hypertension, has been altered.

1.4 Prejunctional regulation of sympathetic neurotransmission

Epinephrine is released from the adrenal medulla into the blood as a
hormone, while NE is the primary neurotransmitter for postganglionic
sympathetic adrenergic nerves where it is released from noradrenergic neurons.
It performs its action by being released into the synaptic cleft, where it acts on
adrenergic receptors, followed by the signal termination, either by degradation of
norepinephrine, or by re-uptake. Figure 1.2 shows a diagram of NE synthesis,

release and clearance.

1.4.1 NE synthesis
NE is synthesized from tyrosine as a precursor, and packed in to synaptic
vesicles. As shown in Figure 1.2 and 1.3, ﬁrst, the amino acid tyrosine (Tyr) is

transported into the sympathetic nerve axon. Tyr is converted to

Tyr Prejunctional

Receptor Diffuse into

1 Blood

 
  
   

 
 

Tyr

   

“If:
DOPA—*DA I ‘> NE—.—>°NE->
DD

 

Nerve
Terminal

 
 

i I Extraneuronal

I
U " Uptake
.1,

L1 ‘1 Neuronal Metabolized
+ Ca2+ Reuptake
(blocked by cocaine)

  

Tyr = tyrosine; TH = tyrosine hydroxylase; DD = DOPA decarboxylase;
DA = dopamine; DBH = dopamine B-hydroxylase; NE = norepinephrine

Figure 1.2 Diagram of NE synthesis, release and clearance

[www.cvpharmacology.com/norepinephrine.htm]

dihydroxyphenylalanine (DOPA) by tyrosine hydroxylase and this is the rate
limiting step for NE synthesis. DOPA is then converted to dopamine (DA) by
DOPA decarboxylase. Dopamine is transported into vesicles and then converted
to NE by dopamine B—hydroxylase (DBH). NE can be further converted by
phenylethanolamine-N-methyltransferase to epinephrine (EP), which is released

into the blood from the adrenal glands and carried throughout the body [46].

Tyrosine

(Tyr)

Dihydroxyphenylalanine
(DOPA)

Dopamine

(DA)

Norepinephrine

(NE)

Epinephrine

(EP)

CHZCHOOH
NH2
HO
Tyrosine Hydroxylase
HO
CH2CHOOH
NHZ
HO
Amino acid decarboxylase
HO

CHZCHZNHz

q

 

 

HO
i Dopamine B-hydroxylase

HO

I \ CHCHZNHZ

OH

HO l/

¢ Phenylethanolamine-N-methyltransferase
HO

CHCHZNHCH3

OH

Ci

HO

Figure 1.3 Steps for NE synthesis

II

I

1.4.2 NE release

After NE is synthesized inside the nerve axon, it is stored in vesicles and
then released by the nerve when an action potential travels down the nerve. An
action potential traveling down the axon depolarizes the membrane and causes
Ca2+ inﬂux to the nerve endings. The increased intracellular calcium causes the
NE vesicles to migrate to the axonal membrane and fuse with the membrane. NE
is then released through exocytosis along with other secondary neurotransmitters,
such as ATP, into the junctional cleft. NE then binds with post-junctional
adrenergic receptors to induce the smooth muscle cells (SMCs) constriction [47].
The store of NE in sympathetic nerve terminals is heterogeneous, with both large
(~100 nm diameter) and small (~50 nm diameter) vesicles. It is believed that both
vesicle populations participate in the release of NE [48]. There are several main
factors that regulate NE release including the calcium channels, presynaptic az-

adrenergic receptors and adenosine A1 receptors.

Ca” channels. There are two types of Ca2+ channels, voltage-dependent
calcium channel (VDCC) and ligand-gated Ca2+ channels [49]. VDCCs are
localized in the active zone and serve as one of the important mechanisms for
Ca2+ inﬂux into the cells, enabling the regulation of intracellular concentration of
free Ca2+ [50]. Neurotransmission from central and peripheral nerves depends on
the inﬂux of Ca” through VDCCs [51]. Around 50—100 [M of internal calcium
concentration is required for the synaptic vesicles to fuse with the plasma

membrane [52]. Vesicle fusion requires binding of Ca2+ with two proteins in the

vesicle membrane, synaptobrevinNAMP and synaptotagmin, and two proteins in
the synaptic membrane: soluble NSF attathed protein SNAP-25 and syntaxin
(which is closely associated with the VDCC) [53, 54]. There are several types of
VDCCs, including L-, N-, P/Q , R, and T types [55]. Among them, N- and FIG
type VDCCs play a more prominent role in neurotransmitter release.

In a sympathetic nerve varicosity, more than 98% of the small synaptic
vesicles are not releasable because they are tethered by synapsins to actin
ﬁlaments. To become releasable, a synaptic vesicle has to be mobilized from this
reserve pool, docking near a VDCC at the active zone, undenNent activation and
then triggered by Ca2+ for the exocytosis. All steps are Ca2+ independent [56, 57].
There is possibility that the transmitter release depends on two different nerve
terminal action potential-initiated Ca2+ signals: one extremely fast, local,
triggering signal, and the other slow, diffuse, regulatory signal. Near Ca2+
channels at the active zones are opened by the nerve terminal action potential
which leads to the transient rise of Ca2+ concentration >>100 pM. Ca2+ binds to
low afﬁnity receptors and then trigger exocytosis. After Ca2+ channels have
closed diffusion, it leads to a rise in global Ca2+ and Ca2+ binds to high afﬁnity
receptors and increases the availability of releasable vesicles at active zones [53].
Changes in the distribution of VDCCs may contribute to the altered synaptic

transmission in hypertension, however, few work has been done for that [58].

(lg-adrenergic receptor (dz-AR). Some of the NE released from the nerve

terminal binds to prejunctional azARs. OzARS are autoreceptors that couple to Gvo

I3

proteins and they play an important role in regulating NE release through
pertussis toxin sensitive negative feedback mechanisms [59, 60]. After activation
by NE, azARs decrease adenylate cyclase activity and cyclic adenosine 3”,-5’
monophosphate (cAMP) production, inhibit N- and P/Q-type Ca2+ channel
function, and activate pre-synaptic K" channels to inhibit NE release [61]. There
are 3 subtypes of 02-ARs: 02A, 023, and 020- Studies in mice revealed that both
the cm and 02c subtypes were required for normal presynaptic control of
transmitter release from sympathetic nerves in the heart and from central
noradrenergic neurons. The 02A subtype inhibits transmitter release at high
stimulation frequencies, whereas the 02c subtype modulates neurotransmission
at lower levels of nerve activity [62]. Some studies have shown that ATP release
is also regulated by GzAR in arteries and veins, however, this prejunctional
regulation of ATP are less marked than NE [21, 32, 63].

Previous work has demonstrated that the release of NE from sympathetic
nerves supplying MA is tightly regulated by presynaptic GzARS through negative
feedback [35, 60]. lmportantly, impaired presynaptic GzAR function is associated
with hypertension in animals [7, 23, 59, 64] and humans [65, 66]. However, it is
controversial whether or not the function of azAR is impaired in DOCA—salt rats.
Both impaired [23, 59] and unaltered [67] azAR autoreceptors in the isolated MA

have been reported.

Adenosine A1 receptors. The adenosine A1 receptor is a member of the

adenosine receptor group of Gi/o protein coupled receptors with adenosine as the

14

endogenous ligand [68]. The presynaptic adenosine A1 receptor is an important
autoreceptor that regulates ATP release [69, 70]. After released from the nerve
terminal, a large portion of ATP is enzymatically degraded rapidly to ADP, AMP,
and adenosine as the ﬁnal product, which activates the presynaptic A1 receptors
[60]. The A1 receptor is a Gila protein-coupled receptor and inhibits
neurotransmitter release by reducing Ca2+ inﬂux to the nerve terminal [70].
Similar as presynaptic quR, adenosine A1 receptor inhibit NE release by
decreasing adenylate cyclase activity and CAMP concentration, activating
presynaptic K+ channels and inhibiting N- and P/Q-type Ca” channels [71].

The function of prejunctional adenosine A1 receptors in hypertension
remains controversial. In a hypertension model in which rats are treated
chronically with the A1 receptor antagonist 1,3-dipropyl-8-sulphophenylxanthine
(DPSX), adenosine plays a regulatory role by inhibiting NE release and
maintaining blood pressure via an action at prejunctional A1 autoreceptors [72].
However, in the SHR (spontaneously hypertensive rat), the enhanced
noradrenergic neurotransmission is not due to defective modulation of
neurotransmission by adenosine [73]. The effects of adenosine on noradrenergic

neurotransmission in DOCA—salt hypertensive rat have not been studied.

1.4.3 NE metabolism and clearance
The actions of NE are carried out via the binding to adrenergic receptors. In
the mesenteric arteries and veins, NE acts primarily via 01 adrenergic receptor

(a1AR) to cause blood vessel constriction by triggering the speciﬁc second

massager pathways. The binding of NE to receptor depends on the
concentration of NE in the vicinity of the receptor. When the nerve stops
releasing NE, the concentration of NE in the junction decreases and NE will
leave the receptor. There are several mechanisms for NE to get cleared from the
junction: 1) most of the NE (around 90%) is transported back to the presynaptic
junction by the NE transporter (NET) for repackaging into the synaptic vesicles, 2)
some of the NE diffuses into capillaries and is carried out of the tissue by the
circulation, 3) some of the NE is metabolized by catechol-O-methytransferase
(COMT) and monoamine oxidase (MAO) with vanillylmandelic acid (VMA) as the
ﬁnal product of these pathways (as shown in Fig. 1.4), 4). a small portion of NE
(around 5%) is taken up by an extraneuronal uptake system and metabolized [74].

NET is a monoamine transporter that transports NE from the synapse back
to its vesicles for storage until later use [75]. It is consisted by 617 amino acids
and has 12 transmembrane domains. NET function is regulated by several
neurotransmitters, peptides, psychostimulant drugs, ionic environment,
nucleotides and various pathological conditions [76]. It can be blocked by
cocaine and therefore cocaine increases junctional NE concentrations by
blocking its reuptake and subsequent metabolism. The function of NET in
hypertension is not clear yet. Reduced reuptake has been observed to explain
the elevated NE in DOCA-salt hypertension and human essential hypertension
[77, 78]. However, unaltered neuronal reuptake of NE in DOCA-salt hypertensive
rats [79, 80] and enhanced NE neuronal uptake in chronic hypertension model

and spontaneously hypertensive rats arteries [81, 82] have also been reported.

I6

COMT

 

 

Norepinephrine > Normetanephrine
MAO MAO
OMT
Dihydroxymandelic acid C 4’ Vanillylmandelic acid

MAO = monoamine oxidase
COMT = catechol-O-methyltransferase

Figure 1.4 Metabolism of NE after release

1.5 Differential sympathetic innervations of mesenteric arteries

vs. veins

The splanchnic region consists of liver, spleen, pancreas, small and large
intestines. The splanchnic vascular bed holds arOund 33% of the total blood
volume [83]. It is richly innervated by sympathetic nerves. Therefore, sympathetic
control over the splanchnic vasculature may play an essential role in blood
pressure regulation. Mesenteric arteries and veins are considered very important
blood vessels in studies for the role of sympathetic nervous system in

hypertension.

1.5.1 Artery and vein structure

Arteries are blood vessels that carry oxygenated blood away from the

17

heart. As shown in Figure 1.5, an artery has three layers: an outer adventitital
layer, a muscular middle, and an inner layer of endothelial cells. The muscle in
the middle is elastic and strong and the inner layer is very smooth. According to
the size and function, arteries are divided into several types, the aorta, arteries,
arterioles and capillaries. The arteries deliver the oxygen-rich blood to the
capillaries where the exchange of oxygen and carbon dioxide occurs. The
capillaries then deliver the carbon dioxide and waste-rich blood to the veins for
transport back to the lungs and heart. Small arteries and arterioles are the main
resistance blood vessels involved in blood pressure regulation. These small
arteries are highly innervated by sympathetic nerves and respond to changes in
nerve activity by constriction or dilation. Arteries are resistance vessels and their
diameter changes lead to changes in total peripheral resistance and therefore,
blood pressure.

Veins are blood vessels that carry blood toward the heart. As shown in
Figure 1.5, a vein has a bigger lumen, and a thinner and less elastic smooth
muscle layer compared to the artery. According to their size and function, there
are several types of veins: superficial veins, deep veins, pulmonary veins,
systemic veins, and venules. Veins are referred to as capacitance vessels and
they contain 70% of the blood volume and 75% of which is in small veins and
venules [84]. Constrictions mediated by sympathetic nervous system of veins can
redistribute some of the stored blood from peripheral veins to the heart and then

into the arterial circulation and therefore lead to the cardiac output increase [85].

Vessel Characteristics

Connective Tissue w," j . _ , ,
Smooth Muscle .__- ‘13; ' -. ,.
Endothelium . -‘ ‘ “‘ “f?“ﬂ‘ﬁ”) ‘.
Muscular and 4.?'-‘.~.-;-1-.e.;‘-.',
elastic, thick

walled ' ' Artery

 
 
   

 
     
   

Thin walled with
smooth muscle,
flacid

 

Figure 1.5 The structure of artery and vein

[http:l/www.colorado.edu/intphys/Class/IPHY3430-200/013bloodpressure.htm]

1.5.2 Sympathetic innervation of arteries and veins

Sympathetic nerves are the most important regulator of vasoconstriction,
especially in the splanchnic circulation. Sympathetic nerve stimulation can
reduce intestinal blood volume by up to 60% [86]. The sympathetic nerves
projecting to the splanchnic bed originate from prevertebral and paravertebral

ganglia. The sympathetic neurons innervating arteries and veins are differentially

19

located in the ganglia and they show different physiological properties [87, 88].
Veins are more sensitive to the nerve stimulation and vasoconstrictors in both
physiological [89] and pathological conditions [34]. The relative contribution of
ATP and NE to neurogenic constriction varies depending on tissue type, age and
disease state of animal. Sympathetic nerve stimulation to veins induces a slow
contraction, primarily mediated by NE acting at 01 adrenergic receptors in rats.
On the other hand, in small arteries from rat, nerve stimulation evokes short
latency, fast contraction mediated mainly by ATP acting at P2X receptors [90] .
While in guinea pig and dog, NE is the primary neurotransmitter in mesenteric
arteries [47] and NE and ATP are the major neurotransmitters supplying
mesenteric veins [31]. In DOCA-salt hypertension, NE is the primary
neurotransmitter in both arteries and veins [34, 91], while in SHR model of
essential hypertension, the sympathetic tone to veins is elevated [92]. This
evidence supports the idea that there are fundamental differences in the

sympathetic neural control of arteries and veins.

1.5.3 Venous function in hypertension.

Arteries have been studied extensively due to their important role in the
blood pressure regulation. The role of veins in the development of hypertension
could also important. Small veins and venules in the splanchinic region store the
great majority of blood in the circulation and exhibit high active venoconstriction.
Sympathetic venoconstrictor activity is the most important determinant of

venomotor tone in splanchnic veins and the entire circulation [93]. The increase

20

of sympathetic nervous system activity could reduce vascular capacitance, which
can cause venous return to transiently exceed cardiac output, thereby increasing
central compartment blood volume and affects arterial blood pressure. Increased
vascular resistance and reduced venous capacitance in the splanchnic
circulation is found in established human and animal hypertension [94, 95]. The
decreased venous capacitance in hypertension may be due to increased
sympathetically mediated venoconstriction [96, 97]. This may be due to increased
sympathetic activity [98], increased efﬁciency of neurotransmission [96] or both. It
is possible that decreased venous capacitance, particularly in the splanchnic
circulation, precedes and contributes to human [99] and experimental

hypertension development [96].

1.6 Oxidative stress and hypertension

Oxidative stress results from an imbalance in the pro-oxidant-antioxidant
equilibrium in favor of the prooxidants, which can lead to potential oxidative
damage [100]. A number of diseases are associated with oxidative stress, such
as atherosclerosis, Parkinson's disease, heart failure, and Alzheimer's disease.
Reactive oxygen species (ROS) are ions or small molecules that include oxygen
ions, free radicals, and peroxides. The major ROS resulting from oxidative stress
are superoxide anion (02'), hydrogen peroxide (H202), hydroxyl radical (OH-)
and peroxynitrite (ONOO-). As shown in Figure 6, there are several pathways for
endogenous oxidant and antioxidant. 02' production is mediated by several

enzymes, such as nicotinamide adenine dinucleotide phosphate (NADPH)

21

oxidase and xanthine oxidase [101]. -O2‘ is very reactive and can be converted to
H202 either spontaneously or via superoxide dismutase (SOD). H2O2 is then
converted to H2O by and O2 by catalase or to OH- by the further oxidation by
transition metals: Fe and Cu [102].

Under physiological conditions, ROS are produced in a controlled manner
at low concentration and act as signaling molecules regulating the growth and
function of vascular smooth muscle cells [103]. Under pathological conditions,
increased ROS production leads to endothelial dysfunction, increased
contractility, and elevated sympathetic nervous system activity that all contribute
to vascular damage in cardiovascular diseases [104, 105]. Compelling evidence
supports the idea that an enhanced production of ROS and a decrease in the
antioxidant reserve in plasma and tissue contributes to hypertension in DOCA-
salt rats, the SHR [106], Dahl salt-sensitive rats [107], and angiotensin II
hypertension in rats [108]. ROS are also elevated in patients with salt-sensitive
hypertension and renovascular hypertension [109]. ROS play an important role in
the development of hypertension, which is largely due to -O2' excess and
decreased NO bioavailability in the vasculature and kidneys, and the ROS
mediated cardiovascular remodeling.

A major source of -O2‘ is NADPH oxidase. -O2' and NADPH oxidase
activity are elevated in the aorta of DOCA-salt rats, and in the aorta and cultured
vascular smooth muscle cell of SHRs [106]. In addition to the increased level of
NADPH oxidase, SOD activity was reduced in DOCA-salt rats which also leads to

increased levels of -O2' [106]. Inhibition of ROS generation with the NADPH

22

oxidase inhibitor, apocynin, and radical scavenging with antioxidants or SOD
mimetics decrease blood pressure and prevent development of hypertension in
most hypertension models [110-112]. For example, the SOD mimetic, tempol (4-
hydroxy-2,2,6,6-tetramethylpiperidinyloxy), lowers renal sympathetic nerve
activity, heart rate, and mean arterial blood pressue in DOCA—salt hypertensive
rats [113]. However, there is debate about whether or not tempol functions
through a NO dependent manner to inhibit NE release from sympathetic nerves
[105, 114]. There have been no studies focused on antioxidant treatment and NE
release from the sympathetic nerve endings in DOCA-salt hypertensive rats.
There are also other mechanisms through which ROS can be produced as
described earlier. These mechanisms contribute to the development of
hypertension, as well. ROS have been shown to function mainly through
oxidative modiﬁcation of proteins, especially Gyo proteins, and activation of
transcription factors that maintain vascular function and structure [115, 116]. ROS
can be measured by the lucigenin assay [117], dihydroethidium staining [118],
and cyanide-resistant oxygen consumption [119], etc. The role of ROS under
pathological conditions can also be accessed pharrnacologically using acute and
chronic antioxidant treatments, such as apocynin, tempol and N-acetyl-cysteine

[120].

23

SOD Catalase

lCu (II) 1 Fe (in

 

 

 

 

 

02 t '02 : H202 : H20 4' 02
Fenton
N0 reaction
NADPH oxidase l, v
Xanthine oxidase _
Lipoxygenase ONOO 'OH

Cycloxygenase

P-450 monooxygenase
Mitochondrial oxidative
Phosphorylation

Figure 1.6 Endogenous oxidant and antioxidant pathways

1.7 The DOCA-salt hypertension animal model

There are several hypertensive animal models of primary and secondary
hypertension. Rats are by far the most popular species in hypertension research,
including SHR, Dahl-salt sensitive, Deoxycorticosterone (DOCA) -salt, two-
kidney one-clip, and transgenic TGR (mRen2) 27 hypertensive models [121].
SHR is the most often used rat model, although it reﬂects only a rare subtype of
human hypertension, such as, primary hypertension that is inherited in a
Mendelian fashion.

The DOCA-salt hypertensive model is used to investigate the aspects of
salt-sensitive human hypertension. Deoxycorticosterone is a steroid hormone
that has mineralocorticoid activity and acts as a precursor to aldosterone. The

administration of DOCA, in combination with a high salt diet and unilateral

24

nephrectomy induces a low rennin form of hypertension [122]. Speciﬁcally,
administration of the mineralocorticoid affects Na“/H+ exchange and increases
the rate of Na+ reabsorption by kidney. High Na” increases water retention and
leads to increased blood volume and blood pressure. The DOCA—salt
hypertensive model is the only model in which rennin-angiotensin inhibition does
not decrease blood pressure, nor end-organ function. Studies have shown that
the periventricular areas of the hypothalamus in brain and the central
sympathetic and baroreceptor systems are crucial for the development of
hypertension in DOCA—salt ased sympathetic activity, elevated plasma NE
content, altered neural angiotensin II function, and altered baroreﬂex responses

have been found in DOCA—salt hypertension [77, 79, 123].

1.8 Approaches to measure NE release

NE overﬂow in the blood supply of tissues is an measure of sympathetic
nerve activity in both physiological and pathological conditions. The release of
NE has been widely studied in the heart, kidney and mesentery. There are
several widely used methods to detect NE from these tissues, including

mechanical bioassay, HPLC overﬂow technique and amperometry.

1.8.1 Mechanical bioassay
The phannacologically isolated noradrenergic component of nerve
stimulation induced smooth muscle contraction is often used to assess the

release of NE. Speciﬁcally, the tissue can be stimulated either by nerve

25

stimulation or drugs to release NE. NE binds with postjunctional adrenergic
receptors to induce contraction. Contraction amplitude is related to the amount of
NE released from sympathetic nerve ﬁbers. However, this technique can not
exclude the contribution of other neurotransmitters, such as ATP, to the smooth
muscle contraction. Furthermore, the measured contraction, which is a function
of NE concentration at the receptors, is often poorly correlated with the per pulse
release of NE and it is insensitive to subthreshold and saturating concentrations
of released NE. The resolution of this method is poor and can not measure NE

release from individual sites [124].

1.8.2 HPLC overﬂow technique

The nerve stimulation induced overflow of endogenous NE sampled either
by collecting the perfusate or microdialysis can be detected by HPLC coupled
with various detectors, such as electrochemistry, ﬂuorescence, UV-Vis, etc. This
technique is widely used to detect overﬂow of NE and ATP and their metabolites
[125, 126]. However, this technique measures NE released offset by clearance.
It reveals the average release of NE from multiple release sites at the nerve
ending, but not release from individual sites, thus it provides little information
about real time local release of NE from sympathetic nerves. The temporal

resolution is poor and the kinetics of NE release can not be reﬂected.

1 .8.3 Amperometry

Focal amperometric recording developed for dopamine measurement in

26

d...—

the brain has been successfully applied to NE release in mesenteric artery and
rat tail artery [127, 128]. The nerve stimulation induced NE oxidation current can
be recorded either by differential pulse amperometry or continuous amperometry.
After release, the juncitonal concentration of NE decreases with time due to
reuptake by the NET, extracellular metabolism and diffusion away. The
diffusional fraction is what is detected electrochemically. Therefore, the oxidation
current amplitude is proportional to the NE concentration in the vicinity of the
releasing sites. It is by far the best technique to study nerve- released NE.
Although this technique also measures NE released minus clearance and the
metabolites of NE, its high sensitivity allows measurement of NE in real time and
determine the time course of local release and clearance mechanisms. Details

will be described later.

1.9 Electrochemical detection of NE in vitro using

microelectrodes

Pioneered by Gonon and coworkers in 1984, electrochemical method with
a microelectrode was applied to detect dopamine released in the brain [129].
Since then, this technique has been widely used to study neurotransmitter
release kinetics in vitro and in vivo [127, 130-134]. Neurotransmitter can be
detected by continuous amperometry, differential pulse amperometry and fast
scan cyclic voltammetry. At a potential of 1V, NE can be detected by
electrochemical method, as co-transmitters ATP and NPY are non—electroactive.

NE can be detected as an oxidation current based on the following 2 electron / 2

27

L-InI—‘ﬂ - .- '

proton redox reaction:

”Dr”
HO

NE Quinone

 

Figure 1.7 NE redox reaction

In continuous amperometric detection, the potential of the working
electrode is adjusted to a value on the plateau of the voltammetirc curve (mass
transfer-limited region). The current that ﬂows between the recording and
counter electrodes is measured and its amplitude is related to the diffusive
overﬂow of NE from multiple neuroeffector junctions in the vicinity of the
recording electrode. Since the potential is held at a certain value, the background
current is low and therefore, the excellent temporal resolution and high sensitivity
provided by amperometry allows measurement in real time to be made. This
technique has been successfully applied to the measurement of neurotransmitter
from tissues and single cells, such as adrenal chromafﬁn cells [131, 135]. The
limit of detection of this technique can be as low as attomole and zeptomole for
the neurotransmitter measurement from single synaptic vesicle [136].

In differential pulse amperometry, a fixed potential is held at the working
electrode for most of the time, except for voltage pulses which are applied at
regular intervals. The current ﬂow is monitored prior to, and during the pulse. The
current ﬂow measured immediately prior to the pulse is subtracted from the

current measured during the pulse. The advantage of this technique is it can

28

remove “baseline drift”. [137, 138].

In fast scan cyclic voltammetry, a gradually increasing or decreasing
potential is applied at the working electrode with a high scan rate (> 100 V/s).
When the maximum or minimum desired potential is reached, the direction of the
sweep is reversed. The current ﬂow is measured during increases and
decreases in electrode potential. The occurrence of a signal peak at a particular
potential gives an indication of the type of substance present. At a high scan rate,
a relatively large background current is seen and smaller faradaic current can
only be detected by background subtraction. This technique has been applied to
measurements of dopamine, NE, serotonin, and ascorbic acid especially in the
central nervous system [134]. The advantage of this technique is that different
compounds can be distinguished by the position of the peak in the
voltammogram.

Most of the work to date using these methods with microelectrodes has
focused on neurotransmitter release and clearance in the central nervous and
peripheral nervous systems. There are several types of microelectrode, including
spherical shape, disk shape, cylindrical shape, and band shape of
microelectrode. Among them, disk and cylindrical shape of microelectrodes are
the most widely used types in the in vitro and in vivo neurotransmitter detection.
All types of microelectrode have a small tip dimension (less than 30 um diameter)
to allow measurement within a limited space, such as a single cell. Its small size
provides a spatial resolution of 10-100 pm. The background current of

microelectrode is much lower compared to the normal size microelectrode which

29

can enhance the signal to noise ratio (SIM and thus the sensitivity of the
detection. However, due to their small size, the faradaic current is also small and
therefore, the requirement for instruments with low noise is higher. Since
electrode capacitance is proportional to the area, the low capacitance of
microelectrode allows the applied potential to be changed rapidly and this is
extremely useful in the fast scan cyclic voltammetry. An important property of the
microelectrode is that its diffusion layer is larger than its size, so that the current
detected is under the steady state region, which is beneﬁcial to the sensitivity of

the detection.

1.10 Diamond and carbon ﬁber microelectrodes

A variety of materials can be used to fabricate microelectrodes. Nobel metal
electrodes (eg., platinum electrode) have been used; however, they are not
stable in biological environment [138]. Carbon ﬁber is the material that is often
employed. Carbon ﬁber is a material consisting of extremely thin ﬁbers about
0005-0010 mm in diameter and composed mostly of carbon atoms. The atomic
structure of carbon ﬁber is similar to that of graphite, consisting of sheets of
carbon atoms arranged in a regular hexagonal pattern, however, carbon ﬁber
may be turbostratic or graphitic, or have a hybrid structure with both graphitic and
turbostratic parts present. Carbon ﬁbers derived from Polyacrylonitrile (PAN) are
turbostratic, whereas carbon ﬁbers derived from mesophase pitch are graphitic
after heat treatment [139].

As shown in Figure 1.8, this kind of electrode material possesses a spz-

30

bonded carbon microstructure with an oxygenated surface, such as, carboxyl and
hydroxyl functional groups [140]. Carbon ﬁbers are resistance to drift when
exposed to biological tissue and are attractive for in vivo measurement due to
their small size and good conductivity. However, it is easy to get biomolecule
adsorption due to the presence of the It electron system and carbon-oxygen
functional groups. It is the oxidation/reduction reaction of the oxygenated
functional groups that give rise to the background faradaic features in the
voltammetry. The shifting of voltammetric background features of carbon ﬁber
microelectrode with variation in pH leads to changes in background-subtracted
voltammetric responses [141]. For in vivo experiments, the electrode’s response
to the hydronium ion is a particular concern because its voltammetric response
occurs over a broad range of potentials that overlap those of the catecholamines.
Simultaneous changes of the catecholamine concentration and pH occur in
biological tissue as a result of coupling respiration and energy production to pH
[142]

To remove surface oxide effects, a variety of surface pretreatments, such
as chemical derivation, heat treatment and washes by organic solution have
been developed. However, these pretreatments for carbon ﬁber are accompanied
by a loss in signal amplitude for some electroactive neurotransmitters such as
dopamine and NE [143]. Moreover, such surfaces absorb oxygen again over time
and the pH dependence eventually returns and incorporates oxygen with time.
Coating the carbon ﬁber with a ion-selective polymer, naﬁon, minimizes the

fouling effect but results in decreased sensitivity and response time of the

3]

electrode [144].

Pheno

o Carbony

Lacton

Carboxylic

o -Quinone

P -Quinone

 

Figure 1.8. Representative functional groups that exist on oxidized sp2 carbon

surfaces

Given the above described issues with sp2 carbon materials, it would be
good to have an electrode that has no hassle with the pretreatment to remove

the surface carbon-oxygen functional groups but is still responsive to the

32

analytes of interest. An oxygen-free boron-doped diamond electrode has the
potential to be very useful for the in vitro and in vivo measurement of
neurotransmitters. As shown in Figure 1.9, this kind of material consists of spa-
bonded carbon atoms in a tetrahedral arrangement with hydrogen terminated
surface. Its surface is hydrophobic, non-polar and chemically stable, low in
surface oxygen (< 0.02 atomic %) and reacts slowly with the environment (O2
/H2O). Diamond has important properties like extreme hardness, high thermal
conductivity, high chemical inertness, corrosion resistance and low
compressibility. Diamond can be applied in several areas, such as electronics,
thermal management, and optics [145]. Another very important application is for
electrochemistry.

The ﬁrst diamond electrode was developed in the mid 19808 [146].
Diamond naturally is a very good electrical insulator, however, it can be
converted to possess semimetal electronic properties by controlled doping with
impurities, such as boron. Boron is by far the widely used dopant since it has
small covalent radius, which lead to easy incorporated into substitutional sites
within the diamond without causing lattice distortion. Boron atom can substitute
the carbon atom during the thin-ﬁlm diamond growth [147]. It serves as an
electron acceptor and promotes p-type semiconductivity with acceptor activation
energy of 0.37 eV above the valence band. Valence band electrons are
promoted to the boron acceptors thermally, and therefore leave free electrons in
the dopant band and holes in the valence band to support current ﬂow. Electrical

conductivity of the electrode depends on the boron doping level, the grain

33

boundaries at the surface and the sp2 carbon impurity [148].

Comparing with oxygen-containing sp2 carbon electrodes, the hydrogen-
terminated boron doped diamond electrode exhibits weak adsorption by polar
molecules presumably because of the relative absence of polar carbon-oxygen
functional groups which promote strong dipole-dipole and ion-dipole interactions
with the adsorbate [148]. Therefore, diamond electrodes exhibit low background
current without the redox-active surface carbon oxygen functional groups and
this leads to improved signal to noise ratio (S/N). Boron doped diamond
electrode also possess other outstanding properties, such as, a wide working
potential window, pH independent background response, low limit of detection
and improved response precision and stability [149]. These properties make
boron doped diamond electrode a good choice when making measurements with
complex samples. It has been demonstrated that diamond microelectrode is
more stable when exposed to tissue for longer duration and the response
attenuation is much smaller. Thus, boron doped diamond electrodes have a very

wide application in the biological electrochemical study [150, 151].

 

Figure 1.9 Structure for diamond sp3 carbon

34

1.11 Temperature as a tool to probe the SNS differences in

arteries and veins

Temperature variation has been used to probe molecular mechanisms
controlling transmitter release and clearance in the central and peripheral
nervous systems. In their classic studies, Katz and Miledi used data from
temperature-dependence studies to propose that calcium entry during the falling
phase of an action potential in the motor nerve terminal initiated a multi-step
process that culminated in acetylcholine release at the neuromuscular junction
[152]. Temperature has large effects on enzyme kinetics, plasma membrane
state and synaptic transmission. As neurotransmitter release is a multi-step
process, it is not surprising that synaptic transmission would be markedly altered
by even small temperature changes. For example, small changes in temperature
can alter synaptic vesicle movements and can change the dynamics of vesicle
release responsible for synaptic plasticity in the central nervous system [153-155].
These studies showed treatments that increase synaptic efﬁcacy reduce the
temperature sensitivity of synaptic transmission. Increased synaptic efﬁcacy is
associated with an increased vesicle number and/or an increase in the content of
synaptic vesicles that are replenished after release or an increase in the
probability of release of the content of individual synaptic vesicles. It has been
shown that re-uptake of NE is reduced by moderate cooling in rat mesenteric
anenes[156]

There are also some studies focused on the effect of temperature on the

smooth muscle response to exogenous and endogenous NE [22-28]. For

35

example, the effect of decreasing temperature on vascular smooth muscle
responses to adrenergic agonists and neural activation has been shown. In
1968, Webb-Peploe and Shepherd [126, 157] reported that the vasoconstrictor
response to local cooling in dog cutaneous veins was dependent on an intact
sympathetic nervous system and was potentiated by a change in vascular
responsiveness to incoming neural impulses. With regard to the deep vessels, it
has been reported that cooling depressed the vasoconstrictor responses to
exogenous NE in the canine femoral veins, while it increased the responses to
electrical stimulation in canine mesenteric veins [158]. However, as for arteries, it
is reported that, there is an increase in potency of NE at lower temperature from
rabbit mesenteric artery [159] and decrease of NE from rat mesenteric artery
[156]. For both of them there is no change in efﬁcacy as measured by the
maximum smooth muscle contractile response during cooling. Thus, temperature
sensitivity would be a useful tool to probe physiological differences in either the
mechanisms controlling release and clearance of NE from periarterial and
perivenous nerves, or for the mechanisms regulating smooth muscle contraction.

However, almost no studies have been done related to temperature effects
in DOCA-salt hypertensive rats. As discussed before, we can hypothesize that
there are differences of NE release and blood vessel contractility between
normotensive and hypertensive rats, and also between artery and vein. And

temperature can be used as a tool to study these differences.

36

1.12 Research objective and specific aims

The sympathetic nervous system (SNS) plays an essential role in blood
pressure regulation and its activity is increased in hypertension. As circulating
levels of NE are related to SNS activity, studying the mechanisms of NE release
from sympathetic nerves can improve our understanding of the causes of
hypertension. Despite the prevailing focus on arteries, the role played by
systemic veins in the development of certain types of hypertension is also
important. The proposed study will focus on the DOCA-salt model of
hypertension in rats. DOCA—salt hypertension is also associated with increased
oxidative stress in the mesenteric vascular bed and this may contribute to
alterations in sympathetic nervous system function in hypertension. At the
vascular neuroeffector junction, prejunctional autoreceptor and NE transporter
regulate NE overﬂow. Alterations in the function of proteins which regulate
neurotransmitter release, such as the a2AR, would impair the negative feedback
pathway which controls NE release.

My research objective herein for this comprehensive study includes
characterizing changes in the adrenergic component of NE neurotransmission at
the neuroeffctor junction of mesenteric arteries and veins in DOCA—salt
hypertension using chronoamperometry with microelectrodes. I hypothesize
that there are artery-vein differences in NE release and clearance
mechanisms and these mechanisms are altered in DOCA-salt hypertensive
rats associated with increased reactive oxygen species. Since NE release is

a multi—step process, one way to test the hypothesis is to study the release and

37

clearance temperature-dependent difference. The proposed work will address the

hypothesis in the context of three speciﬁc aims:

Speciﬁc Aim 1: Study the differences of NE release and clearance
mechanisms between normotensive mesenteric arteries and veins.

1a. The NE current released from mesenteric arteries and veins will be measured
as a function of temperature. 1b. The function of prejuntional autoreceptor a2AR
will be tested to verify if this accounts for the artery-vein differences in
temperature sensitivity of NE transmission. 1c. NE transporter function will be

tested for the temperature dependent artery-vein NE transmission differences.

Speciﬁc Aim 2: Study how the mechanisms that regulate NE release and
clearance have been altered in DOCA-salt hypertensive rats.

2a. Test the hypothesis that 02 AR function is impaired in DOCA—salt rats and the
impaired a2AR function is related with the NE temperature dependent release
and clearance differences between normotensive and hypertensive arteries and
veins. 2b. Test the hypothesis that NET function is impaired in arteries and veins
of DOCA—salt rats and that NET activity contributes to artery-vein differences in

temperature sensitivity of NE signaling in tissues from sham and DOCA-salt rats.

Speciﬁc Aim 3: Test the hypothesis that the impaired function of

adrenergic neurotransmission is related with the increased oxidative stress

in DOCA-salt hypertensive rats.

38

3a. Effects of chronic antioxidant treatments (apocynin and tempol) on NE
release will be assessed by NE oxidation current measurement. 3b. Effects of
chronic antioxidant treatments on a2AR function will be accessed. 3c. The
function of G protein which couples to a2AR will be tested in normotensive and

DOCA—salt hypertensive rats.

39

CHAPTER 2

EXPERIMENTAL SECTION

2.1 Boron doped diamond film growth

Boron-doped diamond ﬁlms were grown on sharpened Pt wires (99.99%,
Aldrich Chemical, 76 pm diam) by microwave-assisted chemical vapor deposition
(CVD) [160-162]. Before growth, the etched wires were ultrasonically cleaned (5—
10 min) in acetone and ultrasonically seeded (30 min) in a diamond powder
suspension (5 nm particles, ca. 20 mg in 100 mL of ethanol, Tomei Diamond 00.,
Tokyo, Japan). The pretreated Pt wires were mounted horizontally on the top of a
quartz plate (10X10x1 mm) in the center of the reactor. Boron-doped diamond
ﬁlms were deposited from a 0.5% CH4/H2 (v/v) source gas mixture with 4 ppm of
diborane (0.1% B2H6 diluted in H2) added. All source gases were ultrahigh purity
grade (99.999%). During ﬁlm growth, the system pressure was 45 Torr, the
substrate temperature was approximately 750 °C (estimated by an optical
pyrometer), the microwave power was 400 W, and the total gas ﬂow was 200
standard cubic centimeters per minute (sccm). The deposition period was 10 h.
Under the above conditions, the nominal growth rate was estimated to be on the
order of 0.3 pm/h as the ﬁnal ﬁlm thickness was 3—5 pm. After the deposition, the

diameter of the cylindrical portion of the exposed electrode was ca. 80 pm [151].

40

2.2 Diamond film characterization
The boron-doped diamond electrode surface microstructure and
morphology were characterized by scanning electron microscopy (SEM) and

Raman spectroscopy.

2.2.1 SEM

SEM was used to check the surface morphology and the dimensions of
the electrode. It was performed with a JEOL 6400V electron microscope
equipped with LaBG emitter (Tokyo, Japan). The electrodes were attached to a
specimen stub by using silver conductive epoxy. As shown in Figure 2.1 (left), a
Pt wire was completely coated with a layer of polycrystalline boron-doped
diamond without noticable cracks or exposure of Pt. A higher magniﬁcation image
shown in Figure 2.1(right) demonstrates that the polycrystalline diamond consists

of well faceted crystallites with a diameter from 0.5 to 3 pm.

 

Figure 2.1 SEM image of a Pt wire coated with a polycrystalline boron-doped

diamond film (left) and an expanded view of the microelectrode tip (right)

4]

2.2.2 Raman spectroscopy

Raman spectroscopy was used to probe the ﬁlm microstructure. Raman
spectra were obtained using Raman 2000 instrument (Chromex, Albuquerque,
NM), equipped with a 50-mW Nd:YAG laser (532 nm line). The laser beam was
focused to a spot size of approximately 25 um resulting in an estimated power
density of ca. 150 kW/cmz. Spectra were collected using a 10-s integration time.
The spectrometer was calibrated using a single crystal diamond standard (100
orientation, Harris Diamond). Figure 2.2 shows a Raman spectrum for diamond
thin ﬁlm deposited on a Pt wire. An intense one-phonon diamond line is seen at
1332 cm'1 originating from the spa-bonded carbon structure [163-165]. There is
minimal scattering intensity observed between 1500 and 1600 cm", which arises
from amorphous nondiamond carbon (i.e., sp2 bonded carbon) impurity [151, 164,

166].

 

 

 

 

 

4000
1332 cm'1
/

”a? 3000
4.:
C
:3
O
O
V 2000-
.é‘
a) .
5
E 1000- M W

o i 560 $00 T 1500 ' zo'oo . 2500
Raman Shift (cm'1)

Figure 2.2 Raman spectrum for a diamond thin ﬁlm deposited on a Pt wire. hex:

532 nm. Laser power = 30 mW. Integration time = 10 s.

42

2.3 Microelectrode preparation

2.3.1. Preparation of the boron doped diamond microelectrode

The diamond-coated wire was attached to a copper wire with conductive
silver epoxy. As shown in Figure 2.3, electrodes were then insulated with
polypropylene [127]. Polypropylene is non-toxic to tissue and chemically
resistant to the isopropyl alcohol (IPA) used to clean the microelectrodes before
use. The resulting microelectrode was conically-shaped with an exposed length
of ca. 500 pm and a tip diameter of ca. 10 pm [151]. The microelectrode diameter
and length were be determined from SEM images. These dimensions were used
to calculate the electrode area. The geometric area of the conically-shaped
boron-doped diamond microelectrode was calculated using the following
equaﬁon:

A = Acone + Acylinder = ”2012 + UHZ "’ 27"“

where A is the electrode area (cmz), r is the cone base radius (cm), L is the
electrode length (cm), H is the ratio of the height of the cone and the radius of the
cone base.

‘—-‘ Carbon fiber
or Diamond
Tip ~ 500 um

g—Polypropylene
Pipet Tip

Silver Epoxy

 

 

' Epoxy

Copper wire

Figure 2.3 Diagram of the diamond and carbon ﬁber microelectrodes.

43

2.3.1. Preparation of carbon ﬁber microelectrode

A heat-treated (3000°C) pitch—base type (Textron Speciality) carbon ﬁber
with a diameter of ca. 35 um was inserted into a pipet tip and the tapered end
was heated using the coil of the micropipette puller (Model P-30, Sutter
Instrument, Novato, CA, USA). This softened the polypropylene and made a
conformal layer around the carbon ﬁber. The portion of the carbon ﬁber in contact
with the copper wire, and silver conductive epoxy CW2400 (Chemtronics,
Kennesaw, GA), was insulated with the polypropylene. The carbon ﬁber tip was
cut to a ca. 500 um length with a scalpel under a microscope. The resulting
cylindrical microelectrode was disk-shaped [166]. The microelectrode diameter
and length were determined from scanning electron microscopy (SEM) images.
These dimensions were used to calculate the electrode’s area. The geometric
area of the cylinder carbon ﬁber microelectrode was calculated by

A = Acylinder 4’ Adisk = 27th 1’ TITZ

where A is the electrode area (cmz), r is the electrode radius (cm) and L is the

electrode length (cm).

2.4 Electrochemical measurement

Cyclic voltammetry was used to determine the exposed area of the electrode,
to check the cracks and defects in the diamond layer, and to provide information
on the kinetics of heterogeneous electron-transfer reactions. The physical,
chemical and electronic properties of an electrode all inﬂuence the electrode

reaction kinetics and mechanisms for the redox system. All voltammograms were

44

obtained using a CHI 650 potentiostat (CHI system, Inc.). A three electrodes
system consisting of diamond or carbon ﬁber as a working electrode, carbon rod
as a counter electrode and a home-made Ag/AgCl was used as a reference
electrode. Solutions were deoxygenated with nitrogen for 15 min before a
measurement.

Potassium ferrocyanide, K4Fe(CN)6 is a good redox system to check the
electrode’s response because the apparent heterogeneous electron transfer rate
constant depends on surface cleanness, response sensitivity and stability due to
it’s inner-sphere redox mechanism [165]. Figure 2.4 shows the cyclic
voltammetric i-E curves of a diamond (left) and carbon ﬁber (right) microelectrode
in 1mM K4Fe(CN)6 in 1M KCI solution. Scan rate is 50 mV/s. ln cyclic
voltammetry, mass transport of the redox active molecule generally occurs solely
by diffusion. Steady state current are observed in the i-E curves for both diamond
and carbon ﬁber microelectrode. The EH2 value for diamond is about 292 mV vs.
Ag/AgCl, which is 20 mV more positive than that for carbon ﬁber microelectrode.
This reﬂects the slow electron transfer kinetics for diamond electrode [167].
These voltammograms indicate that the electrodes surfaces are clean, no
blemish or crack and the response of diamond toward K4Fe(CN)6 redox system

is similar to that for carbon ﬁber.

45

 

 

 

 

 

 

 

 

140 140
120: 1204
‘ 1004
100‘. 80+
A so. A i
g ‘ E 50.
v 60~ v j
*5 - E 40
g 40? g 20:
0 2°? 0 o-
0-l -20‘+
-204 4o~
04 '02 0:0 ' 0:2 ' 0:4 ' 0:6 ' 0:8 1 1.0 04 0.2 oio 0:2 0i4 oie ofe 1.0
Potential (V vs. Ag/AgCl) Potential (V vs. Ag/AgCI)

Figure 2.4 Cyclic voltammetric i-E curves for a diamond (left) and carbon ﬁber
(right) microelectrode in 1.0 mM K4Fe(CN)6 in 1 M KCI. Scan rate is 50 mV/s.

Exposed electrode area can also be determined from the measured mass
transport-limiting current. For the quasi-steady-state current, it holds that for the

carbon ﬁber microelectrode, the limiting current is given by

-cylinder _ an AD C

qss

 

rlnr

4Dt

1':
Whr
ee r2

 

For diamond electrode, it has a mixed cylinder and cone geometry. Thus
the limiting current for diamond electrode is given by

. _ ocylinder ocone

qss ss

And the conical term is given by

if"; = 4nFDCr(1+ qH”)

46

In the above equations, n is the number of electrons transferred per
equivalent, F is the Faraday constant, which is 96485 Clmol, A is the electrode
area (cmz), D is the diffusion coefﬁcient of the analyte (cmzls), C is the bulk
concentration of the analyte (mol/cm3), r is the cylinder or cone radius (cm), q =
0.30661, p =1.14466, and H is the aspect ratio hlr where h is height of the cone
(cm). The measurement period, t (s), is the time of the forward voltammetric scan.
The surface area of a cone is related to its aspect ratio according to the equation

A= nr2(H + 1)"2 [151] .

2.5 Drug effects on NE detection

For the drug effect studies with diamond and carbon ﬁber microelectrode
work described in Chapter 3, the following experimental protocol was followed.

Cyclic voltammetric measurements were made using P-55 (r = 10 um) and
T-650 (r = 8 pm) carbon ﬁber (Amoco, Greenville, SC), and boron-doped
diamond microelectrodes (r z 40 pm). All measurements were made in a
conventional, single-compartment glass cell using solutions purged for 10 min
with N2. A computer-controlled potentiostat (CHI-650, CH Instruments, Inc.,
Austin, TX) was used in a three-electrode conﬁguration to control the working
electrode potential and to measure the current ﬂow through the cell. A home-
made AglAgCl electrode (saturated KCI) was used as the reference and a large
area carbon rod served as the auxiliary electrode. The potential of the reference
was routinely checked against a standard SCE reference electrode in saturated

KCI (EM/Ago. = -45 mV vs. SCE). A potential scan rate of 0.1 V/s was used in all

47

the measurements and the potential range scanned was from -0.5 to 1.2 V.

For each new microelectrode, a cyclic voltammetric i-E in a Krebs’ buffer
solution (pH 7.4) was ﬁrst recorded followed by measurements in a Krebs’ buffer
solution containing NE (0.020 mM). Following these control measurements, the
electrochemical response for the drug (0.10 mM) in Krebs’ buffer was
investigated. Finally, after drug wash out with the buffer, cyclic voltammetric i-E
‘ curves in Krebs’ buffer and cyclic voltammetric i-E curves for NE were re-
recorded. At least three measurements were made for each drug using a new

microelectrode for each.

2.6 Normotensive and DOCA-salt hypertensive rats

All animal experiments were performed in accordance with the “Guide for
the Care and Usage of Laboratory Animals” (National Research Council). All
animal procedures were approved by the Institutional Animal Care and Use
Committee at Michigan State University. Adult male Sprague Dawley rats (250-
3009; Charles River Laboratories, Inc., Portage, MI) were uninephrectomized
and subcutaneous implanted of DOCA (200 mg kg") pellet under isoﬂurane
anesthesia. Post-operatively, the rats were given drinking water containing 1%
NaCI and 0.2% KCI. Normotensive controls (sham) to the hypertensive rats were
uninephrectomized but no DOCA pellet was implanted and these rats were
maintained on normal drinking water. Animals were housed two per cage with
access to food and water while being kept on a 12:12 h light-dark cycle in a room

with regulated temperature (22-24°C). Blood pressure was monitored by tail cuff

48

plethesmography and rats with systolic blood pressure 3150 mmHg were

considered hypertensive [8].

2.7 Chronic antioxidant treatment of DOCA-salt hypertensive

rats

For the chronic antioxidant treatment work for DOCA-salt hypertension
described in Chapter 6, adult male Sprague Dawley rats (250-3009) were
uninephrectomized and implanted with radiotelemetry transmitters in their
femoral arteries as previous described [168]. An opening was cut in left thigh
area. A pocket was made by separating the skin and muscle in the abdomen
region. The radiotelemetry transmitter’s body was placed subcutaneously in the
abdomen area. The tubing of the transmitter was pulled to the left thigh and the
tip of the tubing was inserted into the abdominal artery through left femoral artery.
Every animal was placed in an individual cage above a heating pad for recovery
then was transferred to the telemetry animal room.

Rats were fed with either NaCI (control) alone or NaCI with either 2mM
apocynin (apocynin group) or 1mM tempol (tempol group). After a 5 days
recovery, transmitters were turned on for recording. The signals from transmitters
were monitored by radiotelemetry receivers (RFC-1, Data Sciences International)
which were connected to a data exchange matrix. Systolic blood pressure,
diastolic blood pressue, and mean arterial pressure were recorded every 10
minutes for two control days. Animals were then implanted with a DOCA-salt

pellet, and recordings were continued for the next 24 days. Data acquisition and

49

analysis were performed by custom software Dataquest ART 4.0 (Data Sciences

International).

2.8 In vitro electrochemical measurement

For the work described in Chapters 4 to 6, continuous amperometry with
diamond or carbon ﬁber microelectrodes combined with drug treatments of
tissues in vitro were used for the measurement of nerve stimulation induced
endogenous NE release from sympathetic nerve endings at mesenteric arteries
and veins from normotensive and DOCA-salt hypertensive rats. The whole setup

of the experiment is demonstrated in Figure 2.5.

Sprague—Dawley Rat Mesentery (small intestine)

  
 
     
  

Blood Vessel
Diameter
180-330 um

   

 

.10 m Diamtrak I Electrical

ch Stimulator (SO-80 V,
camera
Potentiostat t

10Hz, 60 pulses)

 

 
 

Oxygenated
Krebs' buffer

B

 

 

 

 

 

I ‘
F'°‘” ““95 1-3 mllmin emperature Controller
Temperature: 28 ~ 37°C
‘

 

 

Figure 2.5 Diagram of the experimental set up for in vitro NE release from

sympathetic nerve endings

50

2.8.1 Preparation of mesenteric arteries and veins

The sham and DOCA-salt rats were anesthetized with a lethal pentobarbital
injection (i.p., 50 mg). The entire mesenteric arcade was removed from the rat
and placed in an oxygenated (95% O2, 5% CO2) Krebs’ buffer solution of the
following composition (millimolar): 117 NaCl; 4.7 KCI; 2.5 CaCl2; 1.2 MgCl2; 25
NaHC03; 1.2 NaH2PO4, and 11 glucose. Secondary or tertiary MA and MV (150-
320 um) were separated from the surrounding connective tissue, isolated from
the intestine, stretched gently and pinned to the bottom of a small silicone
elastomer-lined chamber. The chamber was part of a single channel
temperature-controller system (Biosciences Tools, CA, USA), which regulated the
perfusate temperature. The chamber was mounted on the stage of an inverted
microscope (Olympus CK 2, USA) and superfused with Krebs’ solution (37 °C) at
a ﬂow rate of 1.5 ml/min. The ﬂow rate was controlled by a peristaltic pump
(Masterﬂex, Cole Parmer, USA). Tissues were perfused with Krebs’ buffer at 37

°C for 30 minutes before use.

2.8.2 Focal stimulation of perivascular nerves

Electrical stimulation of perivascular sympathetic nerves was accomplished
using a focal stimulating electrode composed of two parallel silver chloride-
coated wires, shielded with a glass capillary. The wires were connected to a
Grass Instruments stimulator (S88 Quincy, MA, USA). The stimulating electrode
was positioned on the blood vessel using a micromanipulator. Nerves were

stimulated using 60 stimuli at 10 Hz (stimulus duration, 0.5 ms; voltage, 50-80V).

51

2.8.3 Measurements of NE oxidation currents

The diamond or carbon ﬁber microelectrodes were immersed in distilled
isopropyl alcohol (IPA) prior to use for at least 15 min in order to clean the
electrode surface [169]. The microelectrode was afﬁxed to a micromanipulator
(MP-1, Narishige Instruments, Japan) for reproducible positioning along the
blood vessel surface. As shown in Figure 2.6, the exposed microelectrode was
positioned against the side of a MA or MV so that the NE ﬂux from nearby
neuroeffector junctions reached the entire surface. Such positioning also allowed
the microelectrode to move in concert with the blood vessel when it contracted
and relaxed, so as to maintain a constant electrode-vessel distance. The
microelectrode and the tissue were placed in the center of the chamber. A
platinum wire served as the counter electrode and a commercial “no leak’
Ag/AgCl electrode (3 M KCI, Cypress Systems Inc, USA) was used as the
reference. Electrochemical measurement of NE was made using an Omni 90
analog potentiostat (Cypress Systems Inc., USA). Continuous amperometric i-t
curves were recorded at a detection potential of 700 mV. At this potential, NE is
oxidized at a mass-transfer limited rate. An analog-to-digital converter (MiniDigi,
Axon Instruments, USA) and Axoscope software were used to collect the NE
signal from the analog potentiostat. The analog voltage output from the
potentiostat was low pass ﬁltered at the time constant of 200 ms (5 Hz) before
being digitized using and A/D converter at a sampling rate of 500 Hz. The data

were then stored on the computer hard drive for subsequent analysis.

52

Before stimulation

 

 

OH

OH
HO NH2 0 NH2 +
__.> + 2H + 26-
HO 0
NE NE-Quinone
Stimulation

 

'0 _
i I...“ = 10.4 pA
Current (pA) h—i
Diameter (pm)
30 um] Stimulation: 60 pulses at 10 Hz
Detection potential: + 700 mV

Figure 2.6 Video images of a mesenteric artery showing positions of the
diamond microelectrode, the stimulator (top left) and the change in artery
diameter in response to stimulation (top right). Temporal responses of the NE
overﬂow oxidation current (bottom red trace) and vessel diameter (bottom blue
trace) elicited by electrical stimulation.

2.8.4 Temperature Control

A TC-1 temperature controller (Biosciences Tools, CA, USA) was used to
regulate the perfusate temperature in the chamber with a precision of 0.2 °C.
Tissues were superfused with Krebs’ solution and equilibrated for 20 minutes at
37 °C. Then nerves were stimulated to evoke NE release at 37 °C. The
temperature was then progressively decreased from 37 to 28 °C (3 °C
decrements). At each temperature, NE oxidation currents were measured in the
absence and presence of drugs. Tissues were allowed to equilibrate at each

temperature for 10 min before making an electrochemical measurement.

2.9 Chemical and drug application

The Opti-MEM l Reduced Serum Medium was obtained from lnvitrogen
company (California, USA). All other chemicals and drugs were obtained from
Sigma-Aldrich Chemical company (St Louis, MO, USA). They were at least
reagent grade quality and used without additional puriﬁcation. Ultrapure water
(distilled, deionized, and ﬁltered over activated carbon, 17-18 Mn, Barnstead E-
Pure System) was used for solution preparation and cleaning. Agonists and
antagonists were added to the superfusing Krebs’ buffer for at least 20 minutes

before making an electrochemical measurement.

2.10 Data analysis

Analysis of NE i-t curves was performed using Clampﬁt 9.0, which is a part
of the pCLAMP 9.0 software package (Molecular Devices). Data are reported as

mean :I: SEM. with n values referring to the number of rats used for each study.

54

For comparisons of the NE peak current, 10-90% NE current rise slope, rise time,
and decay slope between MA and MV or between sham and DOCA-salt,
statistical signiﬁcance were assessed by the Student’s t-test. While the values
were compared using student paired t-test for the comparison before and after
drug treatment for the same type of tissue. The parameters of NE current
obtained at different frequencies were analyzed using a one-way ANOVA with
Newman-Keuls post hoc test or Student t-test for paired or unpaired data, as
appropriate. Blood pressure was analyzed with a two-way ANOVA and followed
by Newman-Keuls post hoc test at certain days. P < 0.05 was considered as

statistically signiﬁcant.

55

CHAPTER 3

Drug Effects on the Electrochemical Detection of NE
with Carbon Fiber and Diamond Microelectrodes

3.1 Introduction

Electroanalytical methods with carbon ﬁber microelectrodes have been
utilized for several decades now to measure electroactive neurotransmitters in
vitro and in vivo; molecules such as dopamine (DA), norepinephrine (NE) and
serotonin (5-HT) [134, 141, 170-173]. Two methods are routinely employed:
continuous amperometry (CA) and fast-scan cyclic voltammetry (FSCV). In both
methods, the electroactive neurotransmitter is measured as an oxidation or
reduction current with a magnitude that is proportional to the time-dependent ﬂux
to the electrode. The ﬂux depends on the balance between the rate of release
and the rate of any clearance mechanisms (e.g., reuptake) that might be
operative. CA provides excellent sensitivity and temporal resolution but lacks
qualitative information about the analyte being detected. In other words, at the
constant detection potential employed, all electroactive species present at the
electrode surface contribute to the current. In addition to good sensitivity and
temporal resolution, FSCV also provides qualitative information about the analyte
being detected based on differences in the oxidation or reduction potentials [141].

In recent years, these methods have been utilized to monitor
neurotransmitters in the peripheral nervous system, speciﬁcally, NE released

from sympathetic nerves innervating rat tail and mesenteric arteries [127, 128,

56

137, 174]. Sympathetic nerves project to the smooth muscle cells those makeup
blood vessels. Neuroeffector junctions are formed where these nerve endings
connect with the muscle cells and communication across these junctions is
accomplished by three neurotransmitters: adenosine 5'-triphosphate (ATP), NE
and neuropeptide Y (NPY). These neurotransmitters are stored in and released
from sympathetic nerves via Ca‘z-dependent exocytosis. Our group has been
using in vitro CA with a diamond microelectrode to study sympathetic neural
control mechanisms of arteries and veins [127, 166]. When using electrochemical
methods to investigate neurotransmission, drugs are often employed to probe
mechanisms of release and clearance, post-junctional responses, and to conﬁrm
that the current measured is associated with a particular signaling molecule. For
example, in studies of sympathetic neuroeffector transmission, yohimbine is often
used. This drug is an 02 adrenergic autoreceptor antagonist. a2 autoreceptors are
present at sympathetic nerve endings and function to mediate neurotransmitter
release via negative feedback [23]. In CA measurements, NE oxidation currents
will increase when this functional autoreceptor is blocked [91, 127, 131, 150, 166,
175].

When using drugs, it is necessary to verify that (i) the agent is not
electrochemically active in the potential region where the neurotransmitter is
detected and (ii) the drug does not attenuate the electrode response for the
neurotransmitter by rapid molecular adsorption and electrode fouling. It has been
reported in studies of dopaminergic neurotransmission that some drugs can

decrease the carbon ﬁber sensitivity for dopamine (DA) in vitro [176, 177].

57

 

Although few in number, previous studies have described the inﬂuence of some
drugs on the carbon ﬁber microelectrode response [133, 171].

We report herein on an investigation of the electroactivity of several
commonly used sympathetic neuroeffector agonists and antagonists at carbon
ﬁber and diamond microelectrodes. We also report how drug exposure affects
the electrode sensitivity for NE oxidation. The microelectrode response was
assessed using slow scan cyclic voltammetry and seven drugs were studied
(Table 3.1): prazosin, an 01 adrenergic receptor antagonist; yohimbine, an 02
adrenergic receptor antagonist ; idazoxan, an 02 adrenergic receptor antagonist;
UK 14,304, an (12 adrenergic agonist; cocaine, a norepinephrine transporter
blocker; PPADS, a P2X-purinoceptor antagonist; and capsaicin, a TRPV-1
receptor agonist [34, 91, 127, 178]. Along with the diamond microelectrodes, two
types of carbon ﬁber were investigated: a more microstructuralIy-ordered pitch-
base ﬁber (Thornel P-55) and a more microstructurally-disordered PAN-based
ﬁber (T hornel T-650). Slow—scan rather than fast-scan cyclic voltammetry was
used in these studies because the time scale of the measurements more closely
mimics those for CA, and the technique readily provides information about half-

wave potentials and oxidation currents.

58

Table 3.1. Drug Molecular Structure and Function

 

 

 

 

 

 

 

 

Redox
System Function Structure
OH
NE Neurotransmitter in sympathetic H NHZ
nervous system HO
H H CH3
Capsaicin Agonist for TRPV 1 channel I
H3C\O \ 013
O
O
. . /CH3
Cocaine Antagonist for NE transporter @ O
o
O
o / )
ldazoxan Antagonist for alpha-2 CE I1
O H
autoreceptor
CHO
H O \ / P03 N32
PPADS Antagonist for P2X purinergic I / O
/
H3C N N/N:©\
receptor
M038 803M
Prazosin

/(}]3

O
{5*
Antagonist for alpha-1 receptor \ / 0
M16 CH3
N O

\
/
NHz

 

59

 

Table 3.1. Drug Molecular Structure and Function (continued)

 

H
UK14,304A 'fth I TOTE]
gonlst or a p a- autoreceptor N
i /7 N/
N
Br

 

 

Yohimbine Antagonist for alpha-2

autoreceptor

 

 

3.2 Results

3.2.1 Norepinephrine Oxidation Current

Well-deﬁned cyclic voltammetric i-E curves were observed for NE in Krebs’
buffer at all three microelectrode types (100 mV/s). Steady-state or near steady-
state curves were observed with EH2 values of 213 i 4 mV for the P-55 carbon
ﬁber, 174 i 7 mV for the T-650 carbon ﬁber, and 370 i 10 mV for the diamond
microelectrode. The least positive E1/2 was seen for the more microstructurally-
disordered carbon ﬁber with a more positive value observed for the more ordered
ﬁber. Raman spectroscopic ID (1350 cm")/IG (1580 cm") band intensity ratios
conﬁrmed the degree of microstructural order. The results indicate that the
greater the fraction of exposed edge plane at the sp2 carbon electrodes, the

faster the electrode reaction kinetics are for this molecule [162, 179-181]. The

E1,2 for diamond was ca. 200 mV more positive than the values for the carbon

60

 

ﬁbers. This is because the NE oxidation reaction kinetics are generally more
sluggish at diamond. We suppose the sluggish kinetics are due to weak
adsorption on the H-tenninated, sp3-bonded carbon surface [150]. Unlike the
carbon ﬁbers, the diamond microelectrode (as prepared) possesses few carbon-
oxygen functionalities and no extended n-electron system. Dipole-dipole, ion-
dipole and n—rr interactions between polar molecules and the hydrogen-

terminated diamond surface are either very weak or non-existent.

3.2.2 Electrochemical Activity of the Drugs at Carbon Fiber and Diamond
Microelectrodes

Figure 3.1 shows cyclic voltammetric i-E curves for 0.1 mM concentrations
of each drug in Krebs’ buffer (100 mV/s). Data are presented for P-55 (Fig 3.1A
and B) and T-650 (Fig 3.10 and D) carbon ﬁber, and diamond (Fig 3.1E and F)
microelectrodes. Background voltammetric i-E curves in the absence of the
drugs are also presented, for comparison. For the P-55 carbon ﬁber, quantiﬁable
oxidation currents are apparent for yohimbine, prazosin and UK 14,304. The
nominal E1,2 or Ep/2 values were 779 i 12, 848 i 10 and 969 i 6 mV, respectively.
The peak or pseudo-limiting currents increased in the following order: yohimbine
< UK 14,304 < prazosin. The current variations are due either to a different
number of electrons transferred for each molecule and or to an
electrochemically-active area that is distinct for each. A low oxidation current is
seen for capsaicin with a nominal E1,2 of 639 i 16. Identical responses are seen

for idazoxan and cocaine with both exhibiting no electrochemical activity below

6]

900 mV. Above this potential though, there is anodic current associated with
oxidation of the drug. PPADS had no electrochemical activity at this electrode
over the entire potential range probed.

Quantiﬁable oxidation currents are also seen for yohimbine, prazosin and
UK 14,304 at the T-650 ﬁber. The nominal E1,2 values were 772 i 12, 870 1r 17
and 953 i 9 mV, respectively. The peak or pseudo-limiting oxidation currents
increase in the following order: yohimbine < prazosin < UK 14,304. This is
different from the order with the P-55 ﬁber. There is again a small oxidation
current for capsaicin starting at ca. 700 mV. As was the case for the P-55 ﬁber,
cocaine and idazoxan have no electrochemical activity below 900 mV. Above this
potential, there is net oxidation current for both. PPADS exhibited no
electrochemical activity over the entire potential range probed.
For the diamond microelectrode, well-deﬁned oxidation currents are apparent for
yohimbine, UK 14,304 and prazosin with nominal Em values of 713 i 13, 827 i 2
and 952 i 4 mV, respectively. The peak or limiting oxidation currents increase in
the following order: yohimbine < UK 14,304 < prazosin. A noteworthy difference
for diamond, as compared to the carbon ﬁbers, is the presence of two oxidation
peaks for yohimbine and prazosin. This could be due to a different reaction
mechanism at diamond or to the same reaction occurring at two distinctly-
different sites on the electrode. Oxidation current is seen for capsaicin albeit at a
signiﬁcantly lower magnitude than for the other three drugs. The nominal E1,2 was
624 i 21 mV. Similar responses are seen for cocaine and idazoxan with both

exhibiting no electrochemical activity below 1000 mV. Above this potential, there

62

is some net oxidation current. PPADS exhibited no electrochemical activity over

the entire potential range probed.

3.2.3 Effect of Capsaicin and Prazosin Exposure on Norepinephrine
Detection

Figure 3.2 shows cyclic voltammetric i-E curves for 0.020 mM NE, 0.020 mM NE
+ 0.10 mM capsaicin or prazosin, and 0.020 mM NE after drug washout for the P-
55 (Fig 3.2A and B), T-650 (Fig 3.20 and D) and diamond (Fig 3.2E and F)
microelectrodes. In all cases, the drug concentration was 5x greater than the NE
concentration. Comparison of Figure 3.2A and B reveals that exposure to
capsaicin caused no change in either the E12 or the limiting oxidation current for
NE at the more microstructurally-ordered carbon ﬁber. Figure 3.3A and B
summarize the change in half-wave potential, AE1,2, and limiting current ratio, Irauo,
for all three electrodes brought about by drug exposure. Capsaicin exposure
caused little change in the electrode response for NE. In the presence of the drug,
however, the NE oxidation current was totally suppressed (Fig 3.2A). This means
that an in vitro measurement of NE with this carbon ﬁber would be complicated in
the presence of this drug. Prazosin exposure produced no change in AE1,2 (10.8
i 6.9 mV) but the limiting current was reduced as Irena was 0.68 i 0.12” (Fig 3.3A
and B). In the presence of this drug, the NE oxidation current was nearly
abolished. This means that NE monitoring in the presence of this drug would be

problematic with this carbon ﬁber.

63

Figure 3.1. Cyclic voltammetric i-E curves for drugs at carbon ﬁber (P55, T650)
and diamond microelectrodes: (A) capsaicin, prazosin, UK 14,304 and
yohimbine, and (B) idazoxan, PPADS and cocaine at the P55 carbon ﬁber; (C)
capsaicin, prazosin, UK 14,304 and yohimbine and (D) idazoxan, PPADS and
cocaine at the T650 carbon ﬁber; and (E) capsaicin, prazosin, UK 14,304 and
yohimbine and (F) idazoxan, PPADS and cocaine at the diamond microelectrode.
All curves were acquired in Krebs solution at a scan rate of 0.1 V/s. The

concentration of each drug was 0.1 mM.

64

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65

Figure 3.2. Cyclic voltammetric i-E curves for NE at (A and B) P55 and (C and D)
T650 carbon fiber and (E and F) diamond microelectrodes with and without
capsaicin and prazosin. All curves were acquired in Krebs solution at a scan rate
of 0.1 V/s. The concentration of NE was 0.02 mM and the concentration of each
drug was 0.1 mM.

66

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67

The same general trends were observed for the T-650 carbon ﬁber (Fig 3.20 and
D). Exposure to capsaicin had only a minor effect on the electrode response for
NE. The electrode response was not signiﬁcantly affected by drug exposure in
terms of E1,2 (AE1,2= -11.3 i 2.7) but there was minor attenuation of the limiting
current (ham,= 0.86 i 0.02*) (Fig 3.3A and B). In the presence of the drug though
there was attenuation of the NE oxidation current but not total suppression as
was the case for the P-55 ﬁber. EH2 shifted by ca. 32 mV and Iratio decreased by
25%. Exposure to prazosin had a negligible effect on the NE voltammetric
response (AE1I2 = 4.3 i 1.2 and Iratjo = 0.88 i 0.08) (Fig 3.3A and B). In the
presence of the drug, the NE voltammetric response was signiﬁcantly attenuated
as E1,2 shifted positive by 45 mV and the limiting current decreased by 86%. A
well-deﬁned prazosin oxidation peak is evident at ca. 900 mV with no difference
in the current magnitude in the presence or absence of NE. This means that only

the drug is being oxidized at these positive potentials, and not NE.

For the diamond microelectrode, a well-deﬁned voltammetric response is
seen for NE before and after exposure to capsaicin (Fig 3.2E). There was little
change in either E1/2 or the limiting oxidation current after drug exposure and
washout (AE1,2 = 14.0 i 11.1 and Iratio = 0.94 i 0.03) (see Fig 3.3A and B). In the
presence of capsaicin, the 5,2 for NE oxidation shifted positive by some 200 mV
but the limiting current remained unchanged. This is different from the near total
suppression seen for the P-55 carbon ﬁber. A well-deﬁned voltammogram was

observed for NE prior to exposure to prazosin (Fig 3.2F). After drug exposure

68

:1 P5
A' = T65

 

 

 

 

- Diamond *
100 -
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A E 1!: (mV)
, .L , i re.
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j Capsaicin
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B I=P55
=T650
-Diamond
1.25 -
1.00 - *
*
0.75 -
i""° 0.50 -
0.25 -
0.00 . .
Capsaicin Prazosin

Figure 3.3. Plots of the cyclic voltammetric AE1,2 and limiting current ratio, Iratio
(=Ianerdmg/Iinitial), after drug exposure (capsaicin and prazosin) and washout at the
three microelectrode types. Statistical analysis was based on n = 3. *Represents
a significantly different value from the control (before drug exposure) as

assessed using the standard t-test, P<0.05.

69

and washout, there was a signiﬁcant positive shift in E12 and a slight attenuation
in the limiting current. AE1,2 increased by 83.2 i 14.3* and Irauo decreased to 0.78
i 0.11* (Fig 3.3A and B). In the presence of the drug, the NE oxidation current
was quite suppressed A low and slowly increasing oxidation current is seen for
NE between 400 and 800 mV (Fig 3.2F). The oxidation peak potential for the
drug was unaffected by the presence of NE but the oxidation peak current was
larger indicating that both the drug and NE undergo oxidation at these potentials.
Clearly, a measurement of NE would be complicated in the presence of prazosin

using this microelectrode.

3.2.4 Effect of Exposure to UK 14,304 and Yohimbine on Norepinephrine
Detection

Figure 3.4 shows cyclic voltammetric i-E curves for 0.020 mM NE, 0.020 mM NE
+ 0.10 mM UK 14,304 or yohimbine, and 0.020 mM NE after drug exposure and
washout using P-55 (Fig 3.4A and B), T-650 (Fig 3.4C and D) and diamond (Fig
3.4E and F) microelectrodes. A well-deﬁned cyclic voltammogram was observed
for NE before UK 14,304 exposure (Fig 3.4A). The EH2, ca. 200 mV, and limiting
current, ca. 5 nA, were not signiﬁcantly altered by drug exposure. For example,
AE1,2 and Iratio were -5.1 i 11.4 mV and 0.89 i 0.10, respectively (Fig 3.5A and B).
In the presence of UK 14,304, the NE voltammetric response was quite
suppressed though as E1,2 shifted positive by 140 mV and the limiting current
was reduced by 50%. As is seen in Fig 3.4A, a slowly increasing NE oxidation

current was recorded between 300 and 900 mV in the presence of the drug, at

70

which point drug oxidation commenced. The oxidation current for UK 14,304 in
the presence of NE was identical in position and magnitude to the current
response in the presence of just the drug. This indicates that only UK 14,304 is
being oxidized at these potentials and not NE. After exposure to yohimbine (Fig
3.43), the E112 for NE oxidation was unchanged (AE1,2 = 4.3 i 12.2 mV) but the
limiting current was signiﬁcantly reduced (lamo = 0.85 i 0.03*) (Fig 3.5A and B).
As was the case for UK 14,304, the NE oxidation current was suppressed in the
presence of yohimbine. For example, E1,2 shifted positive by over 140 mV and
the limiting current decreased by ca. 50%. A broadly increasing oxidation current
was observed for NE in the presence of the drug, as was the case for UK 14,304.
No steady-state limiting current was reached over this potential range and the
current was signiﬁcantly less than the control. The oxidation peak potential for
yohimbine was unchanged but the current magnitude increased in the presence
of NE indicating that both the drug and NE are oxidized at these potentials. Using
this carbon ﬁber, measurements of NE in the presence of these two drugs would
be complicated because of response attenuation.

A well-deﬁned cyclic voltammogram was seen for NE using the T-650
carbon ﬁber before exposure to UK 14,304 (Fig 3.40). After exposure of the T-
650 carbon ﬁber to UK 14,304, the E12, ca. 180 mV and limiting current, ca. 10
nA, were unchanged. For example, the nominal AE1,2 value was actually shifted
negative by -14.7 i 0.3* mV (easier to oxidize NE!) but Iratio remained statistically
unchanged at 0.94 i 0.11 (Fig 3.5A and B). In the presence of UK 14,304, the

oxidation current for NE was largely unaffected. The EH2 was shifted slightly

7]

Figure 3.4. Cyclic voltammetric i-E curves for NE at (A and B) P55 and (C and D)
T650 carbon ﬁber and (E and F) diamond microelectrodes with and without UK
14,304 and yohimbine. All curves were acquired in Krebs solution at scan rate of

0.1 V/s. The concentration of NE was 0.02 mM and the concentration of each
drug was 0.1 mM.

72

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positive but an unchanged limiting current of ca. 10 nA was reached prior to the
onset of drug oxidation at ca. 900 mV. Little change in the NE voltammetric
response was observed after yohimbine exposure and washout (Fig 3.4D). For
example, the nominal AE1,2 and Iraﬁo values were -4.3 i 9.5 mV and 0.97 i 0.11,
respectively (Fig 3.5A and B). In the presence of the drug, there was a positive
shift in the E12 and a decrease in the limiting current, but NE was detectable with
this carbon ﬁber. The E1,2 shifted positively by 38 mV and the limiting current
decreased by 38%. The onset of the oxidation current for yohimbine remained
the same in the presence of NE, ca. 800 mV, but the oxidation current was
slightly increased indicating that both NE and the drug are oxidized at these
positive potentials.

Exposure of the diamond microelectrode to UK 14,304 caused no change
in E112 or the limiting current (Fig 3.4E). For example, the nominal AE1,2 and Iratjo
values were 15.3 i 11.9 mV and 0.85 i 002*, respectively (Fig 3.5A and B). In
the presence of UK 14,304, the oxidation current response for NE was strongly
affected. A broadly increasing oxidation current, signiﬁcantly reduced in
amplitude compared to the control, was observed commencing at ca. 350 mV.
The current rise continued until 850 mV at which point oxidation of the drug
commenced. The oxidation peak position and current for UK 14,304 were
unchanged in the presence of NE indicating that only the drug is oxidized at
these positive potentials. After exposure to yohimbine and washout, a positive
shift in EH2 was observed with a signiﬁcantly decreased limiting current. For

example, the nominal AE1,2 and Iratio values were 37.0 :r 8.6 mV and 0.81 i 0.05*,

74

respectively (Fig 3.5A and B). In the presence of yohimbine, the NE oxidation
current was suppressed as a broadly increasing current was seen beginning at
350 mV that continued until ca. 650 mV, at which point oxidation of the drug
commenced. The NE oxidation current was signiﬁcantly less than the control until
600 mV was reached. The yohimbine oxidation peak potential was unchanged in
the presence of NE but the oxidation current magnitude was signiﬁcantly

increased indicating that both NE and the drug are oxidized.

3.2.5 Effect of Electrode Exposure to Cocaine, ldazoxan, and PPADS on
Norepinephrine Detection

Figure 3.6 shows cyclic voltammetric i-E curves for 0.02 mM NE, 0.02 mM
NE + 0.1 mM cocaine, idazoxan or PPADS, and 0.02 mM NE after drug washout
on P-55 (Fig 3.6A, B and C), T—650 (Fig 3.60, E and F) and diamond (Fig 3.6G, H
and I) microelectrodes. It is clear that neither cocaine, idazoxan nor PPADS are
electrochemically active at any of the three microelectrodes at potentials below
800 mV where NE is detected. Interestingly, diamond is the only microelectrode
at which a well-deﬁned oxidation peak is seen for PPADS with a nominal 5,2 of
881 d: 10 mV. lmportantly, for all three microelectrodes, the NE voltammetric
response in terms of E12 and the limiting current was largely unaffected by
PPADS, idazoxan and cocaine. Furthermore, NE could be reliably detected in the
presence of these drugs. There was no significant change in AE1,2 or decrease in
Iratio for any of the drugs at any of the three microelectrodes. The only major

difference for the three microelectrodes was the oxidation peak at 900 mV for the

75

A- = P55
a T65
10° ' - Diamond

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A5112 (mV) 5° '

, Tui LTl

 

UK 14304 YOhimbine

= P55
B . = T650
- Diamond
1.25 -

I rItIo

 

 

UK 14,304 Yohimbine

Figure 3.5. Plots of the cyclic voltammetric AE1,2 and limiting current ratio, Irauo (=
laﬂerdrug/Iinmal), after drug exposure (UK 14,304 and yohimbine) and washout at the
three microelectrode types. Statistical analysis was based on n = 3. *Represents
a significantly different value from the control (before drug exposure) as

assessed using the standard t-test, P<0.05.

76

drug at diamond, and the increased current at this potential is the presence of NE.

The latter indicates that both the drug and NE are oxidized at these potentials.

3.3 Discussion

The key observations from this work are (i) the differential electrochemical activity
of some of the drugs at spz-bonded carbon ﬁber and spa-bonded diamond
microelectrodes, and (ii) the effect some of the drugs have on the voltammetric
response for NE depending on the microelectrode type and microstructure. The
ﬁrst aim of this work was to determine if any of the drugs exhibit electrochemical
activity at potentials less than 800 mV vs. Ag/AgCI; potentials where NE is
detected. For example, the E12 for NE oxidation at the two carbon ﬁbers is near
200 mV and at the diamond microelectrode is near 400 mV. Therefore,
signiﬁcant drug oxidation currents below 800 mV could pose an interference for
NE detection. Figure 3.1 shows that cocaine, idazoxan and PPADS exhibit little
electrochemical activity at either of the carbon ﬁbers or diamond at potentials
less than 800 mV. Similar conclusions have been reached previously for cocaine
[177]. Clearly, the electrooxidation kinetics for some drugs, like PPADS, are more
rapid at diamond based on the less positive E1/2. This is in contrast to the more
sluggish electrode kinetics for NE oxidation at diamond. This is a nice example
showing that not all redox reactions are kinetically slower at diamond as
compared to sp2 carbon ﬁber electrodes. Studies of the drug oxidation reaction
mechanisms and understanding the reasons for the differences in oxidation

reaction kinetics at the different electrodes will be the subject of future research.

77

Figure 3.6. Cyclic voltammetric i-E curves for NE at (A, B and C) P55 and (D, E
and F) T650 carbon ﬁber, and (G H and I) diamond microelectrodes with and
without cocaine, idazoxan and PPADS. All curves were acquired in Krebs
solution at a scan rate of 0.1 V/s. The concentration of NE was 0.02 mM and the
concentration of each drug was 0.1 mM.

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At this time, we speculate that the less positive Em for PPADS at diamond may
be due to more favorable interaction of the molecule with the hydrophobic sp3-
bonded carbon surface. lmportantly though, oxidation of PPADS, idazoxan and
cocaine at all three microelectrodes occurs well positive of the potentials used for
NE detection, so they present no direct interference. Furthermore, the
voltammetric response for NE oxidation, both in terms of the Em and limiting
current, was largely unaffected by exposure to any of these drugs. This is
apparent from the cyclic voltammetric curves presented in Figure 3.6 and the
data summary in Table 3.2. NE can be reliably detected with both carbon ﬁber
and diamond microelectrodes in the presence of any one of these drugs.

More signiﬁcant effects were seen for UK 14,304 and yohimbine in terms of drug
electrochemical activity and changes in the voltammetric response for NE. Both
drugs are electrochemically active at the carbon ﬁber and diamond
microelectrodes. A well-deﬁned oxidation peak is seen for UK 14,304 at all three
microelectrodes with an Em of ca. 950 mV, as shown in Figure 3.1. There were
no signiﬁcant differences in the electrode-reaction kinetics at any of the three
microelectrodes based on the similarity of the Em values. After drug exposure
and washout, the most positive shift in Em for NE oxidation was seen for the
diamond microelectrode. This is evident from the data in Figures 4 and 5, and
Table 3.3. At most though, Em shifted only by 15 mV and Irauo decreased only by
15%; both relatively minor changes. Greater adsorption of this drug on the low
oxygen, sp3-bonded carbon surface may be the reason for the positive shift in the

NE oxidation Em. lmportantly, the original NE voltammetric response could be

81

regained after a 20 min soak of the diamond microelectrode in clean 2-propanol.
This indicates that any kind of drug-surface interaction is relatively weak in
strength and is chemically reversible. For UK 14,304, the oxidation current for
NE in the presence of the drug was quite suppressed at the more
microstructurally-ordered carbon ﬁber (P-55) and diamond microelectrodes. The
NE oxidation current slowly increased with potential and never reached a steady
state prior to the onset of drug oxidation. The current magnitude in the presence
of the drug, even at potentials as positive of 0.6 V, was signiﬁcantly less for these
two microelectrodes than the values in the absence of the drug. Furthermore,
there was no additional current for NE oxidation at the potentials where the drug
is oxidized. These observations are consistent with there being a competitive
interaction between the drug and NE for sites on the surface where the oxidation
reaction can take place. The fact that the oxidation current maximum for the drug
was the same in the presence of NE suggests that, if there is competition for
sites, the drug preferentially interacts with these sites. These sites are apparently
not exclusively associated with the edge plane regions based on the data for the
more microstructurally-disordered carbon ﬁber (T-650). In the presence of UK
14,304 at this microelectrode, there was a slight shift in the NE oxidation E112 but
a stable limiting current was reached that was the same magnitude as that
recorded in the absence of the drug. This could mean that NE is preferentially
interacting with the edge plane sites while the drug is interacting more with the
basal plane sites of the sp2 carbon electrodes. Clearly, for this microelectrode,

sites are available for the oxidation of both compounds. For NE measurements in

82

Table 3.2. Summary of cyclic voltammetric AE1/2 and Iraﬁo values for NE oxidation
in the absence of different drugs.

*Represents a statistically different value as compared to the control (before drug
measurement) using Student t-test. P < 0.05. Iratio = laﬁerdrug/lmma..

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

P55 T650 Diamond
Drug I
AE1/2 (mV) I ratio AE1/2(mV) 1 ratio AEm (mV) I ratio
Capsaicin
4.8 i 7.6 0.99 i 0.02 -11.3 i 2.7 0,36 i 0.02 * 14.0 i 11.1 0.94 i 0.03
Prazosin
10.8 i 6.9 0.53 i 0,12 * 4.3 i 1.2 0.88 i 0.08 33.2 i 14.3 * 0.78 i 0.11
UK 14,304
-5.1 i 11.4 0.89 i 0.10 44,7 i 0.3 * 0.94 i 0.11 15.3 i 11.9 .035 i 0.02 *
Yohimbin I
4.3 i 12.2 0.35 i 0.03 * -4.3 i 9.5 0.97 i 0.11 37.0 i 8.6 0.31 i 005 *
Cocaine
2.0 i 9.2 135 i 013 * 41,3 i 23 * 1.23 i 0.23 -12.0 $7.1 0.99 $0.06
ldazoxan
-2.0 i 7.2 I034 _+_ 0.04 * -8.5 i 2.1 1.09 i 0.11 -7.3 i 7.9 0.39 i 0.03
PPADS I
0.7 i 4.8 I 1.00 i 0.06 , -220 i 8.6 0.99 i 0.03 0.7 i 4.7 0.99J_r 0.04

 

 

 

 

 

 

83

the presence of UK 14, 304, the T-650 carbon ﬁber would provide the best
performance.

For yohimbine, the most signiﬁcant changes in the NE voltammetric
response were also seen for the diamond microelectrode. The drug was
electroactive at all three microelectrode types with an Em between 0.7 and 0.8 V,
as seen in Figure 3.1. The least positive Em was observed for diamond,. This
means that the electrode reaction kinetics for this drug are most rapid at diamond,
possibly because of stronger molecular interaction with the surface. After drug
exposure and washout, the NE oxidation E1/2 for the two carbon ﬁbers was
unaffected but lmio for the more microstructurally-ordered carbon ﬁber (P-55) was
reduced by 15%. This is seen in Figure 3.5 and Table 3.3. In contrast, E"; for NE
oxidation at diamond was shifted signiﬁcantly positive and Irauo was reduced by
19%. In the presence of the drug, the trends for NE oxidation were similar to
those observed for UK 14,304. At the more microstructurally-ordered carbon ﬁber
(P-55) and the diamond microelectrode, the oxidation current for NE only
gradually increased with potential, never reaching a steady state prior to the
onset of drug oxidation. There was, however, additional current at both
microelectrodes for NE oxidation at the potentials where the drug is oxidized.
Again these observations may mean that there is competitive interaction between
the drug and NE for sites on the surface where the oxidation reaction can occur.
Again, these sites do not appear to be exclusively the edge plane regions based
on the data for the more microstructurally-disordered carbon ﬁber (T-650). In the

presence of yohimbine at this microelectrode, there was a slight shift in the NE

84

oxidation Em; and a limiting current was reached that was the same magnitude
as that recorded in the absence of the drug. For NE release measurements in the
presence of yohimbine, the T-650 carbon ﬁber would provide the best
performance.

The two drugs that produce the most complications for NE detection at all
three microelectrodes are capsaicin and prazosin. Capsaicin is easily oxidized at
all three microelectrodes near the potentials at which NE is detected. The
oxidation Em values were in the 0.55 to 0.65 V range with the least positive Em
seen for the microstructurally-disordered T-650 carbon ﬁber. After drug exposure
and washout, the Em and limiting current were largely unchanged from the
control values for all three microelectrodes, as seen in Figures 2 and 3, and
Table 3.3. The greatest reduction in ham of 14 % was seen for the T-650 carbon
ﬁber. These results indicate that any interaction of the drug with these three
microelectrodes is relatively weak and reversible. However, in the presence of
the drug, near total suppression of the NE oxidation current was seen for the
microstructurally—ordered P-55 carbon ﬁber. For the T650 carbon ﬁber and
diamond microelectrodes in the presence of the drug, the NE oxidation Eu;
shifted positive but nearly the same limiting current was reached. Prazosin was
also electrochemically active at all three microelectrodes with an E“; value
ranging between 0.82 to 0.87 V. After drug exposure and washout, the NE
oxidation Em shifted slightly positive but there was a signiﬁcant reduction in Irauo
of 32% for the P-55 carbon ﬁber. Small positive shifts in Em for NE oxidation

were seen for the T-650 carbon ﬁber and diamond microelectrodes after drug

85

exposure and washout with reductions in Iratio of 12 and 22%, respectively. In
contrast with the data for capsaicin, the results for prazosin indicate that any
interaction of the drug with these three microelectrodes is stronger and less
reversible. Most important, in the presence of the drug, the NE oxidation current
was nearly totally suppressed for all three microelectrodes.

Finally, two redox waves for NE were often observed at the diamond
microelectrodes with Em values of ca. 0.4 and 0.9 V. In contrast, only one redox
wave was seen at both carbon fibers. Our current supposition is that these two
peaks represent oxidation of NE at two distinct sites on the diamond
microelectrode. Not all diamond microelectrodes exhibit two peaks but the batch
used for this work did. The presence of the second peak seems to be connected
with the deposition conditions and post-deposition treatment. Ongoing work will

hopefully reveal the origin of this higher potential oxidation peak.

3.4 Conclusion

The key ﬁndings from the work can be summarized as follows:
1. The oxidation of PPADS, idazoxan and cocaine at all three
microelectrodes occurs well positive of the potentials used for NE detection, so
they present no direct interference. Furthermore, the voltammetric response for
NE oxidation, both in terms of the Em; and limiting current, was largely unaffected
by exposure to any of these drugs. NE can be reliably detected with both carbon
ﬁber and diamond microelectrodes in the presence of any one of these three

drugs.

86

2. The oxidation of UK 14,304 and yohimbine at all three microelecrodes
occurs at potentials positive of where NE is detection, so they present no direct
interference. The voltammetric response for NE oxidation at all three
microelectrodes was not signiﬁcantly affected by drug exposure. Minor shifts and
Em and reductions in limiting current were seen, but all effects were reversed by
soaking the microelectrodes in clean 2-propanol. In the presence of UK 14,304
and yohimbine, signiﬁcant response attenuation was seen for NE oxidation under
these test conditions at the P-55 and diamond micrelectrodes. Minor effects were
observed for the T-650 carbon ﬁber. For NE release measurements in the
presence of UK 14.304 or yohimbine, the T—650 carbon ﬁber provides the best
performance.

3. The two drugs that produce the most complications for NE detection at all
three microelectrodes are capsaicin and prazosin. Capsaicin was oxidized,
although low in current, at all three microelectrodes at the potentials where NE is
detected. Prazosin was not so it poses no direct interference. Exposure to both
capsaicin caused some minor alterations in the voltammetric response for NE but
these effects could be fully reversed by soaking the microelectrodes in clean 2-
propanol. Only partial response recovery was seen after 2-propanol soak.
Detection of NE in the presence of capsaicin is possible using all three
microelectrodes but near total suppression of the oxidation current is observed
for prazosin at all three. Measurements of NE release with any of the three
microelectrodes would be complicated in the presence of prazosin.

The results demonstrate that when drugs are used in electrochemical

87

studies of neuroeffector transmission, one should carefully assess the
electrochemical activity of the particular drug at the recording microelectrode and
how exposure to the drug affects the microelectrode response (i.e., sensitivity)
for the neurotransmitter of interest. 'Future work will involve studies of the

oxidation reaction mechanism of some of the electroactive drugs.

88

CHAPTER 4

TEMPERATURE-RELATED DIFFERENCES IN
SYMPATHETIC NEUROEFFECTOR TRANSMISSION TO
MESENTERIC ARTERIES AND VEINS

4.1 Introduction

Sympathetic nerves control arterial and venous tone. Arteries are
resistance while veins are capacitance vessels [87, 89]. The venous system
contains 70% of the total blood volume, with most of this in small veins and
venues. Mesenteric veins (MV) differ markedly from mesenteric arteries (MA) in
their contractile [8, 47] and electrical [88, 91] response to sympathetic nerve
stimulation.

There are major differences between MA and MV neuroeffector
transmission. First, adenosine 5’-triphosphate (ATP) and norepinephrine (NE) are
the primary neurotransmitters released by sympathetic nerves supplying small
MA [90], while NE is the primary vasoconstrictor released by perivenous nerves
in rat [31, 33, 34]. Second, excitatory junction potentials mediated by ATP acting
at P2X receptors are recorded from arteries but not veins [88, 89, 182] Third, the
depolarization produced by repetitive nerve stimulation is proportionally greater in
MV than MA [88]. Finally, NE release from sympathetic nerves in rat MV exceeds
release in MA even though the density of sympathetic nerves in MV is lower than
that in MA [47, 91].

The efﬁciency of neuromuscular transmission in blood vessels can be

affected by several factors that allow the strength of junctional communication to

89

be regulated. Prejunctional receptors are important in this regard [61]. The
prejunctional az-adrenergic receptors (azARs) mediate NE release as part of an
important negative feedback mechanism [29, 34, 91, 127, 183]. The role of
azARs in MA and MV is controversial. In rats, azARs regulate NE release
through negative feedback in MA more so than in MV [34, 91, 131]. However,
there is also work demonstrating that azAR-mediated neuromodulation plays a
greater role in canine MV than MA [32]. On the other hand, the termination of
transmitter actions at the neuroeffector junction depends on removal (NE
reuptake) and extracellular metabolism [74, 75]. The degree to which NE
clearance depends on these processes varies and may also vary between
arteries and veins [91, 124].

Temperature variation has been used to probe molecular mechanisms
controlling transmitter release and clearance in the central and peripheral
nervous systems [152]. At low temperature, the kinetics of reactions in
sympathetic nerve terminals are reduced (e.g., slow activation of Na+ channels or
depression of voltage-dependent Ca2+ channels) [124, 184]. Since NE release is
a multi-step process, one way to test the hypothesis that there are fundamental
differences in NE release and clearance mechanisms in arteries and veins is to
study the temperature-dependence of NE overflow.

Because NE can be oxidized via a 2 electron / 2 proton redox reaction, it
can be detected electrochemically as an oxidation current [129, 185], This is
accomplished using differential pulse amperometry or continuous amperometry

with carbon ﬁber microelectrodes to study NE release and clearance in blood

90

vessels maintained in vitro [130-133]. This technique measures the diffusive
overﬂow of NE from multiple neuroeffector junctions in the vicinity of the
recording electrode. The overflow depends on the mechanistic and kinetic
aspects of release and clearance. It is by far the best available method to study
the nerve stimulation-induced release of endogenous NE. Its high sensitivity and
fast response time allow measurements in real time to be made. In other words,
the time course of NE concentrations at the blood vessel surface can be probed
locally.

In this study, continuous amperometry was employed to investigate NE
levels in vitro at the adventitial surface of isolated rat MA and MV. A diamond
microelectrode was employed for detection due to its excellent response
sensitivity and reproducibility compared to a carbon ﬁber microelectrode [127,
167, 186]. The magnitude of the NE oxidation current detected is governed by
the complex interplay between release and clearance [124]. We used
temperature as a probe to test the hypothesis that NE release and clearance at

sympathetic neuroeffector junctions at rat MA and MV are differentially regulated.

4.2 Results

4.2.1 Sodium and Calcium Channel Function on NE Release at MA and MV
Electrical stimulation evokes neurotransmitter NE release from sympathetic

nerves and NE can be detected directly by electrochemical methods. The

oxidation current measured at 700 mV (vs. Ag/AgCl reference, 3M KCI) is directly

related to the concentration of endogenous NE at the blood vessel surface [127,

9]

128, 130]. After release from the nerve terminals, some NE molecules bind with
presynaptic receptors, some of them diffuse across the synaptic junction and
bind at speciﬁc receptors on the nearby vascular smooth muscle cells, and a
large faction gets cleared by neuronal reuptake. The remainder diffuses away
from the release sites. The latter is what is detected by the recording
microelectrode. Evidence that the current was caused by NE oxidation came
from measurements using a sodium channel antagonist, tetrodotoxin (TTX) [91].
In our study, we use 10 Hz for tissue nerve stimulation since it is at the half
maximum stimulation frequency response curve and thus more sensitive to
detect the differences between groups [91]. TTX (0.3 pM) blocked stimulation
evoked oxidation currents in arteries and veins (Fig. 4.1A).

We used the calcium channel blocker, CdClz, to inhibit NE release during
nerve stimulation 1B). Representative tracings show the inhibition caused CdClz
(30 uM) on NE release in MA and MV (Fig. 4.1B). The NE oxidation current was
inhibited by 50% in artery and by 75% in the vein. The release of NE in MV was
more sensitive to inhibition by CdClz compared to MA as the pECso in MV was 6.5

:1: 0.2 and in MA this value was 4.8 :l: 0.9 (P < 0.01).

4.2.2 Temperature Dependent NE Oxidation Currents from MA and MV

Figure 2A and B display representative NE oxidation current transients for MA
and MV at 37 and 28 °C, respectively. At 37 °C, the current amplitude for MV
(13.7 pA) is greater than that for MA (10.3 pA). At 28 °C, the current amplitude for

both MA and MV decreased to around 5 pA. The fractional decrease in current

92

Artery Vein Artery Vein

       

 
  
 

A (3 Control C C] Control
I TTX (0.3 uM) I CdCI2 (30 pM)
I
5 pA ‘ 5 pA
_' 5s _ 1;.
10 Hz 10 Hz 10 Hz
3 D
El Control (n = 4)
100 -TI'X(n=4) 1.2 El Artery(n=6)
E I E O Vein (n =10)
t
go” 8 0.8
3 8
o 0.50 g
'2 ‘° 04
N E .
é€0.25 * 2
* 0.0
0.00 Artery Vein '11 '10 -9 -8 -7 -6 -5 -4 -3

log [CdCIZ] (M)

Figure 4.1 (A) NE oxidation currents recorded from a MA and MV in the
absence and presence of TI'X. TTX completely blocked the stimulation evoked
oxidation current. (B) Pooled data from experiments illustrated in “A” showing
the 'ITX blocks stimulation evoked oxidation currents. *indicates signiﬁcantly
different from control (P < 0.05). (C) NE oxidation currents from a MA and MV in
the absence and presence of CdClz, a calcium channel blocker. (D) CdClz
concentration response curve for inhibition of NE oxidation current evoked by
electrical stimulation. Veins were more sensitive to the inhibitory effects of CdCIz

compared to arteries. Data are mean :I: S.E.M.

93

was greater for MV. Figure 1C is a plot of the maximum oxidation currents for MA
and MV as a function of temperature. There is a temperature dependence on the
current overﬂow from both vessel types but the temperature sensitivity is greater
for MV. At all temperatures, overﬂow at MV exceeded that at MA except at 28 °C.
At 37 °C, the NE current for MV is 1.6 times greater than for MA. The
temperature coefﬁcient Q10 is a measure of the rate of change of a biological or
chemical system as a consequence of increasing the temperature by 10° C. It

can be calculated by the following equation,

10
_ R2 T2_Tl
Q10 - (R1)

with R is the rate, the current amplitude in our case, and T is the temperature in

 

celsius degrees or kelvins. The calculated Q10 values for MA and MV were 2.0 :l:
0.2 and 3.1 :1: 0.4, respectively (P < 0.05). The larger 010 value indicates that NE
release and or clearance in MV have are more temperature sensitive than in MA.

The total charge passed during the NE measurement (Q), was calculated
by integrating the current/time proﬁle and is related to the number of electrons
transferred per molecule (n) and the number of moles of NE oxidized (N) through
Faraday's law, Q = nFN, where F is the Faraday constant (96,500 C moi"). The
number of NE molecules detected at 37°C in MA was 2.2 (:t 0.19) x 108 and 6.9
(:t 0.25) x 108 in MV. We plotted the number of NE molecules detected as the
function of the temperature for both MA and MV. The number of molecules

detected increased with the temperature and followed the same trend as the

94

peak current vs. temperature plots.

The current measured reflects NE overﬂow from nearby varicosities. During
the 6 s stimulation period, NE accumulates in the neuroeffector junction with a
fraction diffusing to the microelectrode. The increase in oxidation current is
related to the junctional concentration. As indicated above, the junctional
concentration is controlled by the amount of NE released during nerve
stimulation, offset by the amount cleared by neuronal reuptake or the amount
bound to nerve terminal or smooth muscle receptors. The rate of change of the
NE current reﬂects NE release kinetics off-set by the clearance kinetics [187].
The rates of NE release and clearance were quantitatively different for MA and
MV at the different temperatures. The representative curves shown in Fig. 4.2A
and B illustrate that the rise of the oxidation current for MV is much steeper than
that for MA at 37 °C. Furthermore, the rates of the current rise and decay time
were temperature sensitive. The 10 - 90% rise slope, rise time and decay time of
the NE oxidation current at 37 and 28 °C are shown in Fig. 4.3. The rate of
current rise was signiﬁcantly faster in NW than in MA at 37 °C. This difference
diminished at 28 °C. The peak NE current was larger in MV so although the rate
of rise was faster in MV than MA, the time to peak current was the same for both
blood vessels at 37 °C (Fig. 4.33). At 28 °C, NE oxidation currents peaked prior
to the end of the stimulation period so the current rise times in MA and MV were
signiﬁcantly less compared to that at 37 °C. Current decay times were longer in
MV compared to MA both temperatures. The current decay represents NE

clearance by re-uptake, metabolism and diffusion.

95

Artery Vein

   

 

 

 

 

 

 

 

 

 

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Art'ery véin
Temperature (°C)

Figure 4.2 NE oxidation currents recorded from a MA (A) and MV (B) at 37 °C
(black trace) and 28 °C (grey trace). The oxidation current continued to increase
in MA and MV at 37 °C throughout the stimulus train. At 28 °C the current began
to decline in MA while it was sustained in MV. The bar under the current traces
represent the period of nerve stimulation (60 pluses with a 0.5 ms pulse width).
(C) Plot of the temperature dependence of peak current amplitude in MA and
MV. The peak current amplitude in MV is more temperature dependent. (D) Q10
values for NE current amplitudes. Data are mean 1 SEM. * represents
significant difference from MA, P < 0.05

96

4.2.3 Effect of Temperature on the Regulation of NE Release by
Prejunctional a2 ARs

The azAR antagonist, idazoxan (10M), was employed to study its effect on the
temperature dependence of NE release from periarterial and perivenous
sympathetic nerves. ldazoxan increased oxidation currents recorded from MA at
both 37 and 28 °C. At 37 °C, the current increased in amplitude throughout the
period of stimulation in the absence and presence of idazoxan. However, at 28
°C in the absence of idazoxan, the current peaked and began to decay during the
stimulus train while in the presence of idazoxan the current continued to increase
thoughout the stimulus train (Fig. 4.4A). Although the oxidation currents were
smaller at 28 °C compared to 37 °C, idazoxan did not change the time course or
peak amplitude of the current in MV at either temperature (Fig. 4.43). There was
a 2-fold increase in the oxidation current for MA in the presence of the drug at all
temperatures (Fig. 4.40). ldazoxan didn’t change the Q10 for the MA peak current
(Table 4.1). ldazoxan did not change oxidation current amplitude at any
temperature in MV (Fig. 4.40).

Figure 4.5 shows the 10-90% rise slope (A,B), rise time (CD) and decay
time (E,F) for MA and MV at 37 and 28 °C in the absence (control) and presence
of idazoxan. ldazoxan increased the rate of rise of the oxidation current in MA,
but not MV, at 37 and 28 oC. The current rise time was not changed by idazoxan
in MA or MV at 37 °C; however, the current rise time was increased signiﬁcantly
by idazoxan in MA at 37 but not 28 °C (Fig. 4.5 CD). ldazoxan did not affect

current decay time in MA and MV at either temperature (Fig. 4.5 E,F).

97

   
 

        

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Figure 4.3 Comparison of the time profiles of NE oxidation currents in MA and
MV at 37 and 28 °C. (A) 10 to 90% rise slope of the NE oxidation current is
greater for MV compared to MA at 37 °C but not 28 °C, (B) The time to peak NE
oxidation current is signiﬁcantly greater at 37 than 28 °C for both MA and MV, (C)
the 10 to 90% decay time of NE oxidation current is much longer for MV than for
MA at both 37 °C and 28 °C. Data are mean t S.E.M. *indicates signiﬁcantly
different from MA; # indicates signiﬁcantly different from 37 °C, P < 0.05.

98

both 37 and 28 °C. At 37 °C, the current increased in amplitude throughout the
period of stimulation in the absence and presence of idazoxan. However, at 28
°C in the absence of idazoxan, the current peaked and began to decay during the
stimulus train while in the presence of idazoxan the current continued to increase
thoughout the stimulus train (Fig. 4.4A). Although the oxidation currents were
smaller at 28 °C compared to 37 °C, idazoxan did not change the time course or
peak amplitude of the current in MV at either temperature (Fig. 4.4B). There was
a 2-fold increase in the oxidation current for MA in the presence of the drug at all
temperatures (Fig. 4.4C). ldazoxan didn’t change the Q10 for the MA peak current
(Table 4.1). ldazoxan did not change oxidation current amplitude at any
temperature in MV (Fig. 4.40).

Figure 4.5 shows the 10-90% rise slope (A,B), rise time (CD) and decay
time (E,F) for MA and MV at 37 and 28 °C in the absence (control) and presence
of idazoxan. ldazoxan increased the rate of rise of the oxidation current in MA,
but not MV, at 37 and 28 °C. The current rise time was not changed by idazoxan
in MA or MV at 37 °C; however, the current rise time was increased signiﬁcantly
by idazoxan in MA at 37 but not 28 °C (Fig. 4.5 CD). ldazoxan did not affect

current decay time in MA and MV at either temperature (Fig. 4.5 E,F).

4.2.4 Function of Pertussis Toxin Sensitive G Protein in MA and MV
We used pertussis toxin (PTX) to examine the function of Gi/GO protein
which couples to the presynaptic ozAR. MA and MV were incubated in 3 pg/ml

PTX while control groups were incubated in OPTl-MEM medium [188]. The NE

99

I Control

Vein
I ldazoxan B .

  

 

   

 

 

 

 

37 °C
28°C
0 Artery D Vein
A 20 ’-t:1-Artery (n=21) 20 "-0- Control (n=22)
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8- 3
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28 30 32 34 36 38 ‘ ‘ ‘ ' ' ’
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emperature( ) Temperature( C)

Figure 4.4 Effect of the quR blocker, idazoxan, NE oxidation current from MA
and MV at different temperatures. (A) NE oxidation current traces from a MA in
the absence and presence of idazoxan (1 pM) at 37 and 28 °C. At 37 °C,
idazoxan increases peak currents but the time to peak current is similar. At 28
°C, idazoxan increases peak current and time to peak current as the current
amplitude increases throughout the train of stimulation in the presence of the
antagonist. (B) ldazoxan does not alter NE oxidation currents in MV at 37 or 28
°C. Bars under the current traces represent the period of nerve stimulation (60
pluses with a 0.5 ms pulse width). Pooled data for NE oxidation current at
different temperature with and without idazoxan for MA (C) and MV (D). Data are
mean 1: SEM; *indicates signiﬁcant difference from control, P < 0.05.

100

 

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Figure 4.5 Time course of NE oxidation currents in MA and MV at 37 and 28 °C
with and without idazoxan. (A) 10 to 90% rise slope of NE oxidation current is
signiﬁcantly increased by idazoxan (1 pM) in MA but not MV at 37 °C and at 28
0C (B). (C) The 10 to 90% rise time of NE oxidation current was unaffected by
idazoxan at 37 °C in MA and MV but idazoxan increased the current rise time at
28 °C for MA but not MV (D). ldazoxan did not change the 10 to 90% decay time
of NE oxidation currents in MA or MV at 37 °C (E) and 28 °C (F). Data are mean
1 SEM. and * represents significant difference from control, P < 0.05.

101

current amplitudes were signiﬁcantly increased for MA at both 37 and 28 °C after
PTX treatment compared to control (Fig. 4.6A), but not for MV (Fig. 4.6 B). quR
blocker, idazoxan (1 pM), increased NE current for both the PTX treatment group
and the control group for MA at both 37 and 28 °C. There was no difference in
the current amplitude before and after idazoxan application for MV at either 37 or
28 °C. PTX didn’t change the 010 for the peak current for either MA or MV (Table
1). The current rise slope, rise time, and decay time after PTX treatment for MA
and MV follow the similar pattern as the data shown in Fig. 4.5. Speciﬁcally, PTX
increased the current rise slope from 1.4 1 0.2 to 2.1 1 0.4 pA/s (P < 0.05) at 37
°C and from 1.0 1 0.3 to 1.8 1 0.1 pA/s (P < 0.05) at 28 °C. PTX prolonged the
current decay time from 2.4 1 0.4 to 3.8 1 0.3 s (P < 0.05) at 28 °C but not at 37
°C. PTX didn’t change the current rise slope, rise time and decay time for MV and

current decay time for MA at either temperature.

4.2.5 Effect of Temperature on NE Reuptake

After release, approximately 90% of the NE is cleared from the neuroeffector
junction by the NET [74]. Cocaine (10 pM) was used for NET blockade. Cocaine
signiﬁcantly increased the maximum oxidation current at MA at all temperatures
with the largest increase of 80% at 37 °C (Fig. 4.7 A,C). Cocaine increased the
Q10 value for peak currents in MA from 2.0 1 0.2 to 2.7 1 0.3 (P < 0.05) (Table
4.1). Maximum currents in MV were not affected by cocaine at any temperature
(Fig. 4.7 3,0).

Cocaine increased the rate of current rise in MA at both 37 (Fig. 4.8 A) and

102

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Temperature (°C)

Figure 4.6 Effect of Gi/Go protein blocker, pertussis toxin (PTX, 3 ug/ml), on
inhibition of NE release in MA (A) and MV (B) at 37 and 28 °C. PTX increased
NE current in MA at both 37 and 28 °C but not in MV. quR blocker, idazoxan (1
11M), increased NE current for MA in both PTX treatment group and control group
at both 37 and 28 °C. ldazoxan didn’t increase the current amplitude for MV in
either group at any temperature. Data are mean :I: SEM. and *indicates a
signiﬁcant difference between two groups, P < 0.05.

103

28 °C (Fig. 4.88), with the greater increase at 37 °C. There was no change in the
rise slope for MV at any temperature in the presence of cocaine. Cocaine also
increased the total rise time of the oxidation current in MA at 28 °C but not in MV
(Fig. 4.8 D) Cocaine also prolonged current decay time in MA but not MV at 37

and 28 °C (Fig. 4.8 E and F).

4.2.6 Effect of Guanethidine on NE Release

In order to test if there is difference in the availability of NET in MA and
MV, guanethidine was employed. Guanethidine is taken up by NET to the nerve
endings [189]. It inhibits NE release and causes NE depletion by inhibition of the
Na‘ATPase dependent pump. Guanethidine inhibits NE in a concentration
dependent way, which is shown in Fig. 4.9. The release of NE in MV.was more
sensitive to inhibition by guanethidine compared to MA with the pECso for MV of

7.3 :l: 0.3 and for MA of 6.8 :t 0.08 (P < 0.01).

4.3 Discussion

4.3.1 Temperature-dependence of NE Oxidation Currents Recorded from
MA and MV

We veriﬁed that focal electrical stimulation of MA and MV evokes a
neurogenic oxidation current. This conclusion is supported by the observation
that the oxidation currents were blocked completely by the sodium channel
blocker, tetrodotoxin, indicating a requirement for axonal action potentials. We

also showed that the oxidation currents were inhibited in a concentration

104

A m, I Cocaine B

 

 

 

 

 

 

 

 

 

 

I 5 pA
M
10 Hz 10 HZ
Arte Vein
c 'y D
20 -—U—Control (n=9) 20 . .0. Control (n=9)
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Temperature (°C) Temperature (°C)

Figure 4.7 Effect of cocaine (10 11M) on NE oxidation currents in MA and MV.
Representative recordings of oxidation currents in MA (A) and MV (B) at 37 and
28 °C. Bars under the current tracings represent the period of nerve stimulation
(60 pluses with a 0.5 ms pulse width). Cocaine increases peak oxidation currents
in MA at both temperatures. Oxidation currents in MV are unaffected by cocaine.
(C) Pooled data for effects of temperature and cocaine on oxidation currents in
MA and in MV (D). Data are mean 1 SEM. and *indicates a signiﬁcant
difference from control, P < 0.05.

105

Figure 4.8. Comparison of the time course for NE oxidation currents from MA
and MV at 37 °C and 28 °C with and without cocaine (10 11M). The 10 to 90%
current rise slope of NE oxidation current increased signiﬁcantly after treatment
of cocaine in MA at both 37 °C (A) and 28 °C (B). Cocaine did not affect the time
course of the current in MV at either temperature. (C) The 10 to 90% current rise
time was not affected by cocaine treatment for both MA and MV at 37 °C (C) but
increased at MA at 28 °C (D). The 10 to 90% decay time of the NE oxidation
current was prolonged in MA but not MV at 37 °C (E) and 28 °C (F). Data are
mean :I: SEM. and *indicates significantly different from control, P < 0.05.

106

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Figure 4.9 Guanethdine concentration response curve for inhibition of NE current
in MA and MV at 37 °C. MV is more sensitive to the inhibition effect of
guanethidine. Data are mean :1: SEM.

108

dependent manner by the non-selective calcium channel blocker, CdClz. A novel
ﬁnding was that the oxidation currents recorded from MV were more sensitive to
CdCIz induced inhibition than those recorded from MA. This result is consistent
with the overall conclusion that regulation of NE release from periarterial and
perivenous nerves is different but the mechanism responsible for this difference
is not clear. Recent studies [190] have shown that may be differential calcium
channel subtype expression by periarterial and perivenous sympathetic nerves at
least in dog. Similar differences in calcium channel expression in rat MA and MV
might explain the increased CdClz sensitivity of NE release from perivenous
sympathetic nerves.

Temperature variation has been used to probe molecular mechanisms
controlling transmitter release and clearance in the central and peripheral
nervous systems [152]. As neurotransmitter release is a multi-step process, it is
not surprising that synaptic transmission would be markedly altered by even
small temperature changes. For example, small changes in temperature can
alter synaptic vesicle movements and can change the dynamics of vesicle
release responsible for synaptic plasticity in the central nervous system [153-155].
These studies revealed that treatments which increase synaptic efﬁcacy reduce
the temperature sensitivity of synaptic transmission. Increased synaptic efﬁcacy
is associated with an increased vesicle number and/or an increase in the content
of synaptic vesicles that are replenished after release. Thus, temperature
sensitivity would be a useful tool to probe physiological differences in the

mechanisms controlling release and clearance of NE from periarterial and

109

perivenous nerves.

We estimate that reducing the temperature from 37 to 28 °C, would
decrease the peak current by ~10% due to temperature-dependent changes in
the NE diffusion coefﬁcient [170, 191, 192]. However, NE oxidation currents
decreased by more than 40% in MA and MV when reducing the temperature
from 37 to 28 °C. This means that the decrease in current is caused primarily by
alterations in the NE release and clearance mechanisms rather than by reduced
diffusion.

Small arteries such as those studied here are resistance blood vessels that
deliver oxygenated blood to tissues. Small veins are capacitance blood vessels
which store blood and carry oxygen poor blood away from tissues. Based on
these different hemodynamic functions, it might be expected that arteries and
veins would be regulated differently by the sympathetic nervous system.
Furthermore, different subsets of sympathetic nerves supply MA and MV [87].
There are also functional differences between sympathetic nerves supplying MA
and MV. In small MA from rats, (S300 pm), like those studied here, ATP and NE
are co-transmitters [31, 33], while in rat mesenteric veins, NE is the dominant
neurotransmitter [34]. This proﬁle differs somewhat in guinea pig mesenteric
arteries and veins where purinergic neurotransmission is more prominent in veins
[31,47]

Several factors inﬂuence NE signaling: the amount of NE released,
binding to pre and post-junctional receptors, clearance by neuronal and

extraneuronal reuptake, diffusion, and metabolism [74, 124]. At 37 °C, peak NE

”0

currents were larger in MV compared to MA. However, there was a decline in the
peak NE current between 28 and 37 °C in MA at MV and this decline was larger
in MV compared to MA. At lower temperatures, altered ion channel function,
reduced enzyme activity and alterations in vesicle mobility and fusion with the
presynaptic membrane could all contribute to reduced NE release [124]. Each
could contribute to the different temperature dependence of NE overﬂow from MA
and MV. We investigated the function of sodium and calcium channels in MA
and MV (Fig. 4.1). NE oxidation currents were equally sensitive to Tl'X
suggesting that temperature-dependent changes in sodium channel function do
not make a major contribution to the difference in temperature sensitivity of MA
and MV. However, the release of NE in MV is more sensitive to the calcium
channel blocker CdClz compared to MA. Temperature dependent changes in
action potential propagation or calcium channel function are probably not a major
factor accounting for the temperature dependent NE current difference between
MA and MV. If this was the case, NE oxidation currents should be equally
affected by temperature reductions. Furthermore, studies in adrenal chromafﬁn
cells have shown that temperature dependent reductions in calcium signals can

not account for differences in vesicle mobilization [193].

4.3.2 Function of a2 ARs in MA and MV
Prejunctional azARs couple to Gyo proteins that link to the inhibition of
adenylate cyclase and N- and PlQ-type calcium channels and activation

potassium channels [61]. NE release from sympathetic nerves is partly

Ill

controlled by (12 ARs, which provide negative feedback to inhibit NE release. The
absence or inhibition of autoreceptor function would lead to greater NE release
and larger oxidation currents. Blockade of ozARs using idazoxan increased the
NE oxidation current at all temperatures for MA, but produced no change in
current amplitude for MV (Fig. 4.4). The rate limiting step for ozAR mediated
autoinhibition of NE release is the time required for sufﬁcient build up of the NE
concentration at the receptors during a train of stimulation [124]. In our study,
there was sufﬁcient time (Gs) for the accumulation of NE at ozARs, at least in MA,
as indicated by the increase in oxidation current caused by idazoxan through the
temperature range studied. It is also likely that sufﬁcient NE would be available
to activate prejunctional quRs on perivenous nerves but oxidation currents
recorded from MV were unaffected by idazoxan at any temperature. Sympathetic
nerves supplying MV do express functional azARs as exogenously applied azAR
agonists inhibit NE oxidation currents in these blood vessels [91]. GJGO protein
blockade by PTX failed to increase the NE current demonstrating the presynaptic
676., protein, which couples to GzARS, does not function as effectively in MV
compared to that in MA. However, the increase of NE current caused by PTX in
MA was smaller compared to the increase caused by idazoxan. The extra current
increase caused by idazoxan could be due to the blockade of the low afﬁnity
intrajunctional buffering sites, which bind NE and re-release it at a later time [124].
It may also be due to the low efficiency of the drug itself, or an insufﬁcient tissue
incubation period with PTX, since the effect of PTX inhibition on

neurotransmitters release is temperature and time dependent [194]. Nonetheless,

112

our work suggests that with the same PTX treatment, the function of GilGo
proteins in MV is not as effective as that in MA. Taken together, these data
suggest that NE release is not tightly regulated by endogenous NE acting
presynaptic ozAR in rat MV. It is possible that the prejunctional GzAR on
perivenous nerve endings is not accessible to endogenously released NE.
Alternatively, the lower density of sympathetic nerve ﬁbers supplying MV may
reduce the opportunity for lateral inhibition by NE released from adjacent
varicosities [124].

An interesting ﬁnding from the present work is that the increase in NE
oxidation current caused by idazoxan was augmented in MA at 28 °C. After
azAR blockade in MA, the peak NE current increased 83% at 37 °C but 102% at
28 °C; the current rise slope increased 69% at 37 °C but 122% at 28 °C. In
addition, at 28 °C, the NE oxidation current peaked and began to decline prior to
the end of the stimulus train but in the presence of idazoxan, the current
increased throughout the train. There are two potential explanations for these
results. First, sympathetic nerve endings, like other nerves, contain a readily
releasable pool and a reserve pool of synaptic vesicles. The reserve pool is
mobilized during trains of stimulation and helps to maintain NE release during
bursts of action potentials [124]. In our studies, a large amount of NE may be
released from the readily releasable and reserve pools of NE at 37 °C, but only a
small fraction binds to ozARs. This would produce minimal feedback inhibition.
However, at 28 °C, less NE is released from both pools of transmitter and a

larger fraction of NE binds to the ozAR producing more efﬁcient autoinhibition. It

113

is also possible that the presynaptic 02AR binding afﬁnity for NE is greater at 28
°C than at 37 °C. Previous work showed that cooling augments postjunctional
ozAR, but not o1AR, function on vascular smooth muscle in rat tail arteries and

canine veins [158, 195, 196].

4.3.3 Low Efﬁciency of NE Transporter in MV

NET is a monoamine transporter that transports around 90% of the NE
released from sympathetic nerves back into nerve terminals [75]. NET can be
blocked by cocaine and NET function is regulated by several neurotransmitters,
temperature, peptides, psychostimulant drugs, ionic environment, nucleotides
and various pathological conditions [76]. Vanhoutte and co-workers reported that,
in canine cutaneous veins, cooling reduced NE uptake [195] and Yamamoto et al.
[156] came to the same conclusion with rat mesenteric arteries. In our studies,
cocaine increased the peak NE current in MA but not MV at all temperatures
studied and cocaine increased the Q10 for the peak NE current in MA. The NET
expressed in HEK 293 cells has a Q10 value of 2.5 for transport of the ﬂuorescent
dye, ASP+ [197], so it would be anticipated that NET would function more
efﬁciently at 37 °C vs. 28 °C. This may be why the NE current increased
markedly at 37 °C where NET would be most effective at clearing NE. This
would account for the increased Q10 value for the peak NE current in the
presence of cocaine.

Cocaine also increased the current rise slope for MA at 37 and 28 °C

indicating that NET clears NE even as it is being released. However, Venton et

“4

a! monitored the actions of cocaine on dopamine release in the striatum of
anesthetized mice and they concluded that cocaine also enhanced dopamine
release by mobilizing a synapsin-dependent reserve pool of dopamine-containing
synaptic vesicles [198]. We can not exclude the possibility that the increased rise
slope of the NE current is partly due to increased NE release caused by cocaine.
The NE current rise slope, peak current or current decay were not altered in MV
at any temperature by cocaine indicating that NET does not make a major
contribution to NE clearance in rat MV even though NET expression can be
detected in these blood vessels [34]. Guanethidine inhibited NE release through
NET and therefore the complete inhibition of NE release by guanethdine in MV
(Fig. 4.9) also support that MV express NET, however, NET may not be
positioned to clear endogenously released NE or the low density of varicosities in
MV prevents lateral clearance by varicosities adjacent to those that release NE.
The NE current decay time was increased by cocaine in MA at 37 and 28
°C and cocaine treatment removed the difference in the temperature sensitivity of
NE currents between MA and MV (Fig. 4.8). This result indicates that NET is an
important factor accounting for MA vs. MV differences in temperature sensitivity.
Taken together, our study indicates that NET plays an important role in NE
clearance in MA but NE is mainly cleared by diffusion and possibly metabolism in

MV.

4.3.4 NE Vesicle Pools at Sympathetic Nerve Endings

llS

Sympathetic nerve endings, like other nerve endings, contain two vesicle
pools: the reserve pool and the readily releasable pool [124]. The readily
releasable pool is composed of docked vesicles that are depleted during high
frequency stimulation. The readily releasable pool is maintained by vesicle
recycling and reﬁlling from the reserve pool [199]. Our data show that at 28 °C,
the NE current in MA, but not MV, declines prior to the end of the stimulus train.
This may be due to a small readily releasable pool or slow mobilization of the
reserve pool at low temperature in individual varicosities. Ultrastructural studies
have revealed the sympathetic nerve varicosities can form specialized
neuromuscular junctions in submucosal arterioles and mesenteric veins [12-14].
These junctions have active zones with synaptic vesicle clusters and there is no
obvious difference between individual neuroeffector junctions or in junctional
density in arteries or veins. This suggests that the difference in temperature
sensitivity of peak NE oxidation currents is related to differences in release and
clearance mechanisms within individual neuroeffector junctions.

It is possible that the readily releasable pool is larger in MV but the reserve
pool of vesicles is further away from the active zone in perivenous compared to
periarterial nerves. Therefore, NE release from perivenous sympathetic nerves is
greater than from periarterial nerves at 37 °C, but at lower temperatures, the
transport rate of reserve vesicles to the release sites is slow, leading to a
reduction in NE release from perivenous nerves and the current is comparable to
that at MA. This proposal is consistent with data obtained in adrenal chromafﬁn

cells where it was shown that reﬁlling of the readily releasable pools is more

116

efﬁcient at physiological temperatures [193]. At room temperature, the release of
catecholamines from the reserve pool is delayed compared to that at 37 °C and
this difference is attributed to a decline in temperature-dependent mobilization of
the reserve pool of vesicles [200]. The rate of vesicle pool depletion is also
slowed at a synapse in the CNS at physiological temperatures due to an

acceleration of vesicle recruitment to release sites [201].

4.4 Summary and conclusions

Our work demonstrates that NE release and clearance from rat periarterial
and perivenous sympathetic nerves are regulated differently. This difference is
based largely on the function of prejunctional GzARS and NET but may also
include different function or expression of calcium channels. These differences
revealed the greater temperature sensitivity of NE overflow from sympathetic
nerves in MV compared to MA. It is possible that there are also MA-MV
differences in the content and recycling dynamics of NE containing vesicles in
periarterial and perivenous sympathetic nerves. These differences in
neuroeffector transmission may contribute to the different hemodynamic

functions of mesenteric arteries and veins.

117

CHAPTER 5

IMPAIRED ADRENERGIC NEUROTRANSMISSION TO
MESENTERIC ARTERIES BUT NOT VEINS IN DOCA-
SALT HYPERTENSIVE RATS

5.1 Introduction

The sympathetic nervous system innervating the splanchnic circulation
plays an important role in blood pressure regulation [7]. Mesenteric arterial
resistance vessels (MA) and mesenteric venous capacitance vessels (MV) in the
splanchnic circulation are particularly important [168]. MA and MV are densely
innervated by sympathetic nerves, which release norepinephrine (NE)
,adenosine—triphosphate (ATP) and neuropeptide Y (NPY) [202]. NE mediates a
constriction via binding with (nu-adrenergic receptors, while ATP causes
constriction by binding to P2X receptors on the postjunctional smooth muscle
cells. While NE and ATP are the main neurotransmitters for vasoconstriction in
MA, NE is the dominant neurotransmitter in MV in rats [31, 33, 203]. The venous
system contains m70% of the total blood volume, most in the small veins and
venules, and has been shown to be important in the development of
hypertension [95, 204, 205]. MV are also more sensitive to sympathetic activation
than MA and perivenous sympathetic nerves release more NE upon nerve
stimulation [88, 89].

The release of NE from peripheral nerves depends on the inﬂux of Ca”
through voltage dependent Ca2+ channels. Ca2+ stimulates migration, fusion and

exocytosis of synaptic vesicles [51]. After being released, 90% of the NE is

118

recaptured into the nerve terminal by the NE transporter (NET) [76].
Prejunctional regulation of NE release occurs via binding of NE to org-adrenergic
autoreceptors (ozAR) on sympathetic nerve endings [35]. It could also be
regulated by the ATP hydrolysis product, adenosine binding with prejunctional
adenosine A1 autoreceptors to inhibit NE release through negative feedback [29,
30]. Both of these receptors are coupled to pertussis-toxin sensitive second
messenger systems which inhibit neurotransmitter release in part by inhibiting
Ca2+ channels and in part by inhibiting adenylate cyclase [61]. Previous work
suggested that NE release in MA and MV is regulated differently by prejunctional
ozAR and adenosine A1 receptors.

Elevated sympathetic nerve activity which results in increased NE level
occurs in human hypertension and various animal models of hypertension, such
as deoxycorticosterone acetate (DOCA)-salt and spontaneously hypertensive
rats [18, 34, 75, 206]. However, the mechanisms that lead to impaired regulation
of NE in hypertension are not clear. The altered NE regulation in DOCA-salt
hypertension is closely related with the activity of sympathetic nerve ﬁring rate,
prejunctional Ca2+ channels, autoreceptors and NET uptake [59, 75, 79, 207].
However, the results with the function of these receptors in DOCA-salt rats are
controversial. For example, both impaired [23, 208] and unaltered [67]
prejunctional azAR in the isolated MA have been reported. Moreover, few
detailed studies of the mechanisms underlying increases in sympathetic input to
veins have been done in hypertension [8, 34, 209]. Since NE release and

clearance is a multi-step biochemical process, one manipulation to probe the

119

altered mechanisms of NE release and clearance in DOCA-salt hypertension is
through the study of the temperature dependence of NE overﬂow. Temperature
has large effects on enzyme kinetics, plasma membrane ﬂuidity, synaptic
transmission and vascular function [159]. lmportantly, temperature variation has
been used for many years as an experimental tool for probing the molecular
mechanisms underlying neurotransmitter release in the nervous system [152,
154,155,195]

Continuous amperometry with a carbon ﬁber microelectrode has been
used to detect the nerve stimulation-induced endogenous NE [127, 175, 187]. At
a potential of 500 mV, NE can be detected as an oxidation current, as oo-
transmitters ATP and NPY are non—electroactive at this potential. This technique
allows measurement of NE very near the release sites in real time and in
response to stimulation parameters that closely mimic endogenous sympathetic
nerve activity. The amount of NE measured at the microelectrode is determined
by the amount of NE released offset by reuptake and diffusion. And since the
microelectrode is so close to the NE release sites, NE release and clearance
kinetics can be studied. Similar observations have previously been made for NE-
induced electrochemical signals recorded at the adventitial surface of the rat tail
artery [128, 137].

In the present work, continuous amperometry with a carbon fiber
microelectrode was used to detect nerve stimulation induced endogenous NE
from sympathetic nerves supplying rat MA and MV in vitro. We used temperature

as a probe to study the release and clearance mechanisms of NE and how these

120

mechanisms are altered in DOCA-salt hypertensive rats. The study evaluated the
effect of prejunctional Ca2+ channel, azAR and NET blockade on NE release and
clearance in sham and DOCA-salt MA and MV. We tested the hypothesis that
impaired prejunctional azAR and NET are involved in the altered regulation of NE

in synaptic transmission in DOCA-salt hypertension.

5.2 Results

5.2.1 Sham and DOCA-salt rats

Mean systolic blood pressures for sham and DOCA-salt rats were 121 :l: 5
mmHg (n=56) and 198 j: 7 mmHg (n=52), respectively (P < 0.05). The mean
body weights of sham and DOCA-salt rats were 400 :I: 10 g and 321 :l: 12 g,

respectively (P < 0.05).

5.2.2 Temperature dependence of NE oxidation currents

Representative tracings of NE oxidation currents, elicited by 10 Hz trains of focal
nerve stimulation, at MA and MV from sham and DOCA-salt rats are shown in Fig.
5.1. The oxidation currents were normalized to the basal level before nerve
stimulation at 37 and 28 °C (Fig. 5.1). The current progressively increased during
the stimulation period of 6 s. During this period, multiple action potentials likely
occur that cause NE to build up in the neuroeffetor junction faster than NE can be
cleared by the NET. At 37 °C, the oxidation current recorded in DOCA-salt MA
was larger than that recorded from sham MA (13.1 pA vs. 10.2 pA). Furthermore,

the temporal rise in the oxidation current occurred faster in DOCA-salt MA than in

121

sham MA. At 28 °C, NE current from both sham and DOCA-salt MA decreased to
around 5 pA, and therefore the relative current decrease was greater in DOCA-
salt MA. However, there were no differences in the amplitude of oxidation
currents recorded for sham and DOCA-salt MV at any temperature. These data
are summarized in Fig. 5.2 A and B. There was a temperature dependent
increase in NE current in all blood vessels. Signiﬁcant differences in peak
oxidation current are seen at 37 and 34 °C for DOCA-salt MA compared to sham
MA. At 37 °C, the NE current in DOCA-salt MA was around 1.3 times greater
than sham MA while they were comparable at 28 °C, suggesting the overﬂow of
NE from DOCA-salt MA is more temperature-sensitive than in sham MA. The
temperature coefﬁcient, Q10, was the ratio of response amplitudes measured
over a 10 °C change in temperature. The calculated Q10 values are summarized
in Table 5.1. The Q10 values for DOCA-salt MA is signiﬁcantly greater than sham
MA (2.8 :l: 0.2 vs. 2.0 :I: 0.2, P < 0.05), which indicate that NE release and/or
clearance in DOCA-salt MA has greater temperature sensitivity than that for

sham MA.

5.2.3 Kinetics of NE oxidation currents at different temperatures

The measured oxidation current results from diffusional escape of some
fraction of the total NE molecules released at nearby neuroeffector junctions. The
early rise of current is controlled by the release kinetics (number of ﬁring events,
concentration of NE per firing event, etc.). The current decay is controlled largely

by the clearance kinetics (reuptake). The current transient near the peak is

122

Sham Artery Sham Vein
B. ,

   

A.
I 37 °C
I 28 °C
\ 5 pA
5 s
C. DOCA Artery D. DOCA Vein

   

Figure 5.1 In vitro continuous amperometric recordings of NE oxidation currents
from sham MA (A) and MV (B), and DOCA-salt MA (C) and MV (D) at 37 °C
(black trace) and 28 °C (grey trace). NE oxidation currents were evoked by 10 Hz
stimulus trains and the bar under the current traces represents the period of
nerve stimulation (60 pluses with a 0.5 ms pulse width).

123

controlled by a combination of the release kinetics offset by the clearance
kinetics. Current rising slope (rate of rise), rise time, and decay time have been
used to access the neurotransmitter release and clearance kinetics [91, 187].
The representative tracings in Fig. 5.1 illustrate that the rise of the oxidation
current for DOCA-salt MA is much faster than that for sham MA at 37 °C.
Furthermore, the rates of the current rise and decay time were altered by the
temperature. Fig. 5.3 shows plots of current rise slope (A), rise time (B), and
decay time (C) for MA and MV from control and DOCA-salt rats at 37 and 28 °C.

At 37 °C, the NE current rise slope in DOCA-salt MA was signiﬁcantly greater
than that in sham MA, but not at 28 °C. The current rise slope for both sham and
DOCA-salt MV was greater than sham MA at 37 °C but there was no signiﬁcant
difference in the rise slope between sham and DOCA-salt MV. There were no
signiﬁcant differences in the current rise time among these blood vessels at 37
°C. At 28 °C, the current rise time was decreased compared to that at 37 °C for
each type of the blood vessel, however, the rise time in sham MA is signiﬁcantly
lower compared to other types of blood vessels. The clearance of NE was
signiﬁcantly slower in DOCA-salt MA compared to sham MA at 37 °C, but not at
28 °C. There was no difference of NE clearance kinetics between sham and
DOCA-salt MV at either temperature. Together with the temperature dependent
NE peak oxidation currents, these data indicate that sympathetic

neurotransmission to MA is disrupted in DOCA-salt hypertension.

124

 

 

 

 

A “_ + Sham Artery ("‘20) A ‘4 ‘ -e— Sham Vein (n= 20)
g .3. DOCA Artery (n=20) <3 12 _ -C}— DOCA Vein (n=18) ‘
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Tempeth191°Cl Temperature (°C)

Figure 5.2 Temperature effects on NE maximum oxidation current evoked by
focal electric stimulation at MA (A) and MV (B) from sham and DOCA-salt rats.
The peak current amplitude in DOCA-salt MA is more steeply temperature
dependent. Data are mean :t SEM. and * indicates signiﬁcantly different from
sham MA, P < 0.05.

125

5.2.4 Prejunctional a2 adrenergic receptors function in DOCA-salt rats

The prejunctional Oz adrenergic receptor (02 AR) regulates NE release
from sympathetic nerves through a negative feedback mechanism [59, 60].
Therefore, we used the 02 AR antagonist, idazoxan (1 pM), to investigate
prejunctional (12 AR function in DOCA-salt MA and MV. As shown in Fig. 5.4,
idazoxan increased the peak oxidation current almost two-fold in sham MA at all
temperature, but this effect was not as prominent in DOCA-salt MA. ldazoxan
increased the peak NE current by 83% at 37 °C but 102% at 28 °C in sham MA
indicating that prejunctional ozAR can bind with NE more efﬁciently at 28 °C to
regulate NE release. Although there were signiﬁcant peak current increases in
DOCA-salt MA after ozAR blockade, the increases were much less than that
occurred at the same temperature in sham MA, e.g. 30% increase at 37 °C and
51% increase at 28 °C (Fig. 5.4A and B). The data suggest that prejunctional 02
ARs function is impaired in DOCA-salt MA. ldazoxan did not increase the NE
peak current signiﬁcantly in sham and DOCA-salt MV at any temperature (Fig.
5.40 and D). ldazoxan didn’t change the Q10 for peak current for any type of
blood vessel (Table 5.1).

Figure 5 shows the 10-90% rise slope (A,B), rise time (CD) and decay
time (E,F) for sham and DOCA-salt MA and MV at 37 and 28 °C in the absence
(control) and presence of idazoxan. ldazoxan increased rise slope of the current
in sham MA, but not DOCA-salt MA, and sham and DOCA-salt MV, at 37 and 28
°C. The current rise time was not changed by idazoxan in any blood vessel at 37

°C; however, the current rise time was increased signiﬁcantly by idazoxan in

126

Figure 5.3. Comparison of the time course of NE oxidation current between
sham and DOCA-salt MA, and sham and DOCA-salt MV at 37 and 28 °C. (A) the
10 to 90% rise slope of NE oxidation current is signiﬁcantly lower in sham MA
compared to DOCA-salt MA, sham MV and DOCA-salt MV at 37 °C but not at 28
°C, (B) the 10 to 90% rise time of NE oxidation current is decreased signiﬁcantly
by temperature from 37 to 28 °C for all types of tissues with the sham MA has the
maximum decrease; and the rise time for sham MA is signiﬁcantly lower than
DOCA-salt MA, sham and DOCA-salt MV at 28 °C (C) the 10 to 90% decay time
of NE oxidation current was much less in sham MA than in DOCA-salt MA at 37
°C but not 28 °C; the current decay time was longer in sham and DOCA-salt MV
than sham MA at both 37 °C and 28 °C. Data are mean :1: SEM. and *
represents signiﬁcantly different from 37 °C; # represents significantly different
from sham MA, P < 0.05.

127

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sham MA at 28 °C (Fig. 5C,D). ldazoxan did not affect current decay time in any
types of blood vessels at either temperature (Fig. 5E,F) suggesting that

prejunctional azARs do not contribute to NE clearance.

5.2.5 Function of pertussis toxin (PTX) sensitive G protein in DOCA-salt
rats

The azAR is GilGo protein coupled receptor. PTX was used to examine the
function of GilGo protein. Peak NE oxidation currents were signiﬁcantly increased
after PTX incubation at 37 and 28 °C for sham MA but not DOCA-salt MA, sham
and DOCA-salt MV (Fig. 5.6). After ozAR blockade by 1 uM idazoxan, the peak
currents increased for control and PTX treatment groups from sham MA (Fig.
5.6A). The peak current was increased by idazoxan only in the control group
from DOCA-salt MA at both temperatures, which is consistent with the result
shown in Fig. 5.4. There were no differences in the oxidation current before and
after idazoxan application for sham and DOCA-salt MV at either 37 or 28 °C. PTX
didn’t change the Q10 for the NE current for any type of blood vessel (Table 1).
The oxidation current rise slope, rise time and decay time after PTX incubation
were similar to the data shown in Fig. 5.5. PTX only increased the current rise
slope for sham MA from 1.4 i 0.2 to 2.1 i 0.4 pA/s (P < 0.05) at 37 °C and from
1.0 1 0.3 to 1.8 i 0.1 pA/s (P < 0.05) at 28 °C. The current rise time was
prolonged from 2.4 1 0.4 to 3.8 1 0.3 s (P < 0.05) at 28 °C but not at 37 °C for
sham MA. The current rise slope and rise time didn’t change for DOCA-salt MA

and sham and DOCA-salt MV. PTX didn’t change the current decay time for any

129

 

 

 

 

 

 

 

 

 

 

Q10 Sham Artery DOCA Artery Sham Vein DOCA Vein

Control 2.0 :l: 0.2 2.8 :l: 0.2 * 3.1 i 0.4 * 2.7 :t 0.2 *
ldazoxan 1.7 :I: 0.2 2.6 d: 0.3 3.3 i 0.7 3.0 :t 0.2
Cocaine 2.7 :t 0.3 # 2.3 :l: 0.4 3.0 :t 0.4 2.4 :l: 0.2
PTX 1.9 :r: 0.2 2.9 :I: 0.4 2.9 1: 0.4 2.5 :l: 0.3

 

Table 5.1 Q10 values for sham and DOCA-salt MA and MV with different
treatments. Data are mean 1: SEM. *indicates signiﬁcantly different from sham

MA; # indicates signiﬁcantly different from control, P < 0.05.

130

 

type of blood vessel.

5.2.6 NET Function in MA and MV from DOCA-salt rats

NE is cleared by NET and/or diffusion away from the junction. To test NET
function in DOCA-salt MA and MV, 1 measured the temperature dependent NE
current after the blockade of NET by cocaine (10 pM). Fig. 5.7 shows peak
oxidation currents before and after cocaine. Cocaine increased the NE current in
sham MA at all temperatures withaa maximum increase of 81% from 8.8 to 15.9
pA at 37 °C. The current was increased signiﬁcantly for DOCA-salt MA at 28 and
31 °C, while there was very little increase at 37 °C. NE current from sham and
DOCA-salt MV were not signiﬁcantly changed by cocaine at any temperature.
Cocaine signiﬁcantly increased the Q10 for sham MA and the difference in Q10
between sham and DOCA-salt MA disappeared (Table 1). This result indicates
that NET is one of the important factors that contribute to the different
temperature sensitivity of NE overﬂow between sham and DOCA-salt MA.

NE current rise slopes were increased by cocaine at 37 and 28 °C, with a greater
increase at 37 °C for sham MA (Fig. 5.8A and B). Cocaine increased the current
rise slope at 28 but not 37 °C for DOCA-salt MA. The current rise time was also
increased for sham MA at 28 °C. NET blockade didn’t affect the current rise
slope and rise time in MV from sham and DOCA-salt rats. Since cocaine inhibits
NET function, the remaining NE can only been cleared by diffusion which slows
NE clearance kinetics. So there was prolonged NE current decay time in sham

MA at 37 and 28 °C and in DOCA-salt MA at 28 °C. Cocaine did not

131

 

 

 

 

 

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Figure 5.4 Effect of azAR antagonist, idazoxan (1 pM), on temperature

dependent NE oxidation current at different temperatures. ldazoxan increased
peak NE current more than two fold for sham MA (A) and around 30% for DOCA-
salt MA at different temperatures (B). There was no change in NE current at
sham MV (C) and DOCA-salt MV (D) after idazoxan treatment. Data are shown

as mean 1 SEM. and * represents signiﬁcant different from control, P < 0.05.

132

change the current decay time in sham and DOCA-salt MV suggesting that NE

clearance from the neuroeffector junction in MV is mainly by diffusion.

5.2.7 Ca2+ channel function is not altered in DOCA-salt MA

Miledi and Katz ﬁrst discovered that neurotransmission was dependent on
Ca inﬂux into prejunctional terminals [51]. Depolarization-evoked entry of Ca2+
into nerve terminals plays a key role in triggering NE release during trains of
stimulation of sympathetic nerves [174]. The role of Ca2+ sensitivity on adrenergic
neurotransmission from sympathetic nerves was compared between sham and
DOCA-salt MA since the differences were all found in MA. CdClz, a Ca2+ channel
blocker, was applied y (106 — 10“ M) at 37 °C and caused a concentration-
dependent inhibition of peak NE amplitude (Fig. 5.9). CdClz concentration
response curves were not different between DOCA-salt and sham MA (pEC5o
was 5.2 1 0.3 and 5.0 i: 0.2, respectively, P > 0.05). CdC|2 (100 mM) completely

inhibited NE release.

5.3 Discussion

5.3.1 Temperature sensitive NE current in MA and MV from control and
DOCA-salt rats

NE, ATP and NPY are co-transmitters released from sympathetic nerve
endings supplying MA and MV. There is increased sympathetic nerve activity in
DOCA-salt hypertension, which is associated with elevated plasma NE levels
[34, 208, 210]. We, therefore, investigated the NE release and clearance

133

Figure 5.5 Comparison of the time course of NE oxidation currents from sham
and DOCA-salt MA and MV at 37 and 28 °C with and without idazoxan. The 10 to
90% rise slope of NE oxidation current was signiﬁcantly increased after the
treatment of idazoxan for sham MA at both 37 °C(A) and 28 °C (B); idazoxan
increased 10 to 90 % current rise time signiﬁcantly only at 28 °C for sham MA (C
and D); the 10 to 90% decay time of NE current did not change after the
treatment of idazoxan at 37 °C (E) and 28 °C (F) for any type of tissues. Data
are mean :1: SEM. and * represents signiﬁcant difference from control, P < 0.05.

134

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mechanisms and how DOCA-salt hypertension differentially alters transmission
to MA and MV. NE was measured in vitro as an oxidation current using
continuous amperometry. Both NE oxidation current and the elicited blood vessel
constriction were blocked completely by tetrodotoxin (TTX, 0.3 pM), a voltage-
gated sodium channel blocker. This demonstrates that NE release was nerve-
mediated. We also conﬁrmed that the drugs used in this study (e.g., idazoxan,
cocaine and CdClz) are electrochemically-inactive at carbon ﬁber microelectrodes
at the potential where NE is detected. Therefore, they do not inﬂuence the
reproducibility and stability of the NE measurement [186].

NE currents were temperature dependent for MA and MV from both sham
and DOCA-salt hypertensive rats. This could be due to the effect of temperature
on biological phenomena and, in particular, on enzyme kinetics, plasma
membrane state, and synaptic transmission [211]. Dunn and Mercier have
conﬁrmed that decreasing temperature reduced the number of quanta of
transmitter released per nerve impulses [212]. Our results indicated that at 37 °C,
nerve stimulation evoked greater NE overﬂow in DOCA-salt compared to sham
MA. However, this difference between sham and DOCA-salt MA decreased at
lower temperatures and was abolished at 28 °C. NE overﬂow in DOCA-salt MA
shows greater temperature sensitivity than sham MA. Increased NE overﬂow
could be due to increased release of NE from sympathetic nerve terminals,
impaired function of prejunctional autoreceptors, reduced clearance or
extracellular enzyme metabolism. The altered mechanisms of NE release and

clearance in DOCA-salt MA have a greater impact on NE regulation at 37 but not

136

28 °C. This point has been further conﬁrmed by the fact that a greater current rise
slope and prolonged decay time were observed for DOCA-salt MA, compared to
sham MA only at 37 °C, which illustrates a faster release and slower clearance of
NE regulation at DOCA-salt MA.

Dunn has pointed out in their work that the reduction in quantal content with
temperature is probably the result of reduced calcium inﬂux through voltage-
gated calcium channels in the synaptic terminals [212]. Calcium passage through
the pore-forming region of an open calcium channel has little temperature
sensitivity [213]. Morris and Clarke pointed out that this high temperature-
sensitivity of calcium channels resulted from coupling to multiple metabolic
events [214]. Because neurotransmitter release is closely related with the
function of calcium channels, we thus investigated the function of calcium
channels in sham and DOCA-salt MA. However, my data show that CdClz blocks
NE release equally well in sham and DOCA-salt MA at 37 °C. Herein, the
prejunctional differences related to NE release and clearance in sham and
DOCA-salt MA is not due to the function of calcium channels.

Despite the focus on arteries, the role played by veins in the development of
hypertension is important. Previous work has shown that there is increased
sympathetically-mediated venoconstriction in hypertension [96, 97]. Mean
circulatory ﬁlling pressure (MCFP) can provide a good estimate of overall
circulatory capacitance or compliance, properties determined for the most part by
the smooth muscle tone of veins when combined with measurements of blood

volume [215]. Although MCFP cannot be measured directly in humans, it is

137

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DOCA Artery

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Temperature (°C) Temperature (°C)

Sham Vein DOCA Vein

N

_A

NE Peak Current (pA) 0
NE Peak Current (pA) U
o- 6?

28 37 28 37
Temperature (°C) Temperature (°C)

Figure 5.6 Effects of pertussis toxin (PTX, 3 pg/ml) on NE release at sham and
DOCA-salt MA and MV. After 2h tissue incubation in PTX, NE maximum current
was significantly increased at sham MA (A) but not DOCA-salt MA, sham and
DOCA-salt MV (B, C and D). For comparison, we also blocked 02AR function by
idazoxan for the control and PTX treatment groups. Both NE current and current
rise slope were increased signiﬁcantly by idazoxan for sham control and PTX
treatment groups (A) but the increase was very little in DOCA-salt MA control
group (B). There was no current increase in sham and DOCA-salt MV groups (C
and D). This represents that the function of Gi/Go which couples to OzAR is
impaired in DOCA-salt MA and G-,IGo does not function in sham and DOCA-salt
MV as well as in sham MA. The current increased by idazoxan plus PTX was
signiﬁcantly greater than that caused by PTX alone for sham MA. Data are mean

:t SEM. with * represents signiﬁcant difference, P < 0.05.

138

increased in hypertension models, including DOCA-salt hypertension in rats
[216-218]. This may be due to increased sympathetic activity [98], increased
efﬁciency of neurotransmission [96] or both. My work indicates that there is no
change in NE neurotransmission in DOCA-salt compared to sham MV. Thus, the
difference in venous function is probably due to increased sympathetic activity at

postjunctional but not prejunctional sites.

5.3.2 Impaired prejunctional a: adrenergic receptors function in DOCA-salt
MA but not MV

The increased overﬂow of NE and current rise slope both suggest that
there may be alterations in the local mechanisms that modulate prejunctional NE
release. Previous work demonstrated that the release of NE from sympathetic
nerves supplying MA is tightly related with the regulation by prejunctional 02AR
through a pertussis toxin-sensitive negative feedback mechanism [35, 60].
lmportantly, impaired prejunctional ozAR function is associated with hypertension
in animals [7, 23, 59, 64] and humans [65, 66]. However, the function of quR in
DOCA-salt rats is controversial. Both impaired [23, 59] and unaltered [67] azAR
autoreceptors in the isolated MA have been reported. Our work indicates that
after the blockade of prejunctional ozAR with its antagonist, idazoxan, peak NE
current amplitude increased almost 2-fold in sham MA but much less increase
occurred in DOCA-salt MA at any temperature. This is consistent with studies
showing that another azAR antagonist, yohimbine, failed to increase the NE

levels in the DOCA-salt mesenteric vasculature [23, 208]. The current rise slope

139

_D_Sham Artery (n=9) p —C}— DOCA Artery (n=9)

 

 

 

 

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Figure 5.7 Effect of NE transporter blocker, cocaine (10 uM), on temperature
dependent NE oxidation current. Oxidation current was signiﬁcantly increased
from sham MA at every temperature (A) but only at low temperatures from
DOCA-salt MA (B). No current increase was observed at sham MV (C) and
DOCA-salt MV (D). Data are shown as mean :t SEM. and * represents
signiﬁcant difference from control, P < 0.05.

140

was lower in sham MA than DOCA-salt MA at 37 °C. This may be due to the
inhibition effect of prejunctional OzAR on the release of NE after NE concentration
at the neuroeffector junction accumulated to a certain degree. The current rise
slope increased after 62AR blockade signiﬁcantly in sham MA but not in DOCA-
salt MA indicating that NE release is regulated by prejunctional OzAR but this
regulation is impaired in DOCA-salt MA. However, the slow increase of the NE
current in sham MA could be due to slow diffusion of NE from the junction to the
electrode or NE diffusion could be delayed by NE binding with a low afﬁnity
binding site in or near the junction. There may be a low afﬁnity intrajunctional NE
binding site that does not play a role in activation of the smooth muscle but slows
NE diffusion and thereby maintains the NE concentration at near smooth muscle
receptors [124]. Furthermore, the effects of idazoxan could not be accounted for
changes in NE clearance as this agent had no effect on the current decay time,
which is consistent with previous work [91, 187]. 1 demonstrated previously that
the prejunctional ozAR response was augmented at 28 °C. After OzAR blockade
in sham MA, the peak NE current and the current rise slope increased more at 28
°C than that at 37 °C. In addition, the NE current reaches the maximum prior to
the end of the stimulus train at 28 °C. However, after OzAR blockade by idazoxan,
the current increased until the stimulation stops for sham MA. This also suggests
that 02AR inhibit NE release more efﬁciently at 28 oC. The enhanced 62AR
function at 28 °C could be due to a higher percentage of NE binding with ozAR,
or a higher binding coefficient [158, 195, 196]. In DOCA-salt MA, the current rise

time decreased very little from 37 to 28 °C and it failed to increase after ozAR

141

Figure 5.8 Comparison of the time course of NE oxidation currents from sham
and DOCA-salt MA and MV at 37 and 28 °C with and without cocaine. The 10 to
90% rise slope of NE oxidation current was increased signiﬁcantly after the
treatment of cocaine for sham MA at both 37 °C (A) and 28 °C (B) and for DOCA-
salt MA at 28 °C. Cocaine increased the 10 to 90 % current rise time signiﬁcantly
only at 28 °C for sham MA (C and D); the 10 to 90% decay time of NE current
was prolonged in sham MA at both 37 °C (E) and 28 °C (F); and it was prolonged
in DOCA-salt MA at 28 °C only. Data are mean :1: SEM. and * represents

signiﬁcant difference from control, P < 0.05.

142

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I Sham Artery (n=6)
1.25- A DOCA Artery (n=9)

0. 50-

Normalized NE Response

0

 

 

 

00 I I I I I I V _
-11 -1 -9 -8 -7 -6 -5 -4 -3 -2
log [CdClz]
Figure 5.9 Concentration response curves for Ca channel blocker CdClz on NE

inhibition were not different between sham and DOCA-salt MA. Data are mean

:tS.E.M.

144

blockade at 28 °C, suggesting that the NE release from sympathetic nerve
terminals is not tightly regulated by the OzAR. Furthermore, it is known that in
sham MA, ATP is the main neurotransmitter causing arterial constriction, while
NE is the dominant neurotransmitter in the DOCA-salt MA. It has been reported
that ATP in the sympathetic nerve terminal is easier to be depleted in DOCA-salt
MA [63]. Our data suggest that NE is resistant to depletion, which might be a
compensatory mechanism to maintain vascular tone in DOCA-salt MA.

Prejunctional GzARS couple to pertussis toxin (PTX) sensitive GJGO
proteins to inhibit release of neurotransmitter by opening K” channels, blocking
voltage-dependent Ca2+ channels and inhibiting adenylate cyclase. The impaired
function of prejunctional 62AR in DOCA-salt MA may be due either to receptor
down regulation or the impaired signaling pathway to the autoreceptors. We used
PTX to inhibit GJGO protein function. PTX failed to increase the NE current in
DOCA-salt MA demonstrating that the prejunctional GJGO protein which couples
to GzARS does not function effectively compared to that in sham MA. Taken
together, my data suggest that NE release is not tightly regulated by endogenous
NE acting presynaptic 62AR in DOCA-salt MA which may be due to the less
functional GilGo protein. This alteration may be related with the increased level
of reactive oxygen species which can cause protein and tissue damages [219].

In contrast to MA, there is neither signiﬁcant peak NE current increase,
nor the current rise slope increase after prejunctional ozAR blockade in sham and
DOCA-salt MV. This result is consistent with the study showing no signiﬁcant

increase in NE release after yohimbine application in portal veins from DOCA-salt

145

rats [59]. PTX treatment failed to induce the NE current increase in MV. These
data suggest that NE release is less regulated by prejunctional 62AR in MV,
although MV does express GzAR [91]. This may be due to the low density of
prejunctional GzAR, or the GzAR in MV is not close enough to the NE release
sites at the sympathetic nerve terminals. However, cooling did not augment the
function of prejunctional azAR in MV as compared to sham MA (Fig. 5.4). This
suggests it is more likely that the prejunctional (12AR in MV locate at some
distance away from the NE release sites. At 37 °C, there is more NE released
and a small portion can diffuse away to activate the prejunctional azAR, so the
peak NE current increases after 62AR blockade, though not signiﬁcantly.
However, at 28 °C, NE release is decreased and little NE can diffuse to activate
prejunctional GzAR. So no increase in peak NE current was observed in sham
and DOCA-salt MV although the binding of NE with prejunctional azAR became

more efﬁciently at low temperature.

5.3.3 Dysfunction of NET in DOCA-salt MA but not MV

NET removes NE from the neuroeffector junction once it is released from
the nerve terminal [220]. Reduced reuptake may explain the elevated NE in
DOCA-salt hypertension and human essential hypertension [77, 78]. However,
unaltered neuronal reuptake of NE in DOCA-salt hypertensive rats [79, 80] and
enhanced NE neuronal uptake in chronic hypertensionmodel and spontaneously
hypertensive rats arteries [81, 82] have been reported also. Our study

demonstrates that there is a dysfunction of NET and the reuptake of NE is

146

reduced in DOCA-salt MA. After block NET function, peak NE current increased
markedly at all temperatures with the greatest increase at 37 °C in sham MA.
This suggests that NET function is reduced at low temperature, which is
consistent with previous work showing that cooling can depress the
extraneuronal uptake of NE in vascular smooth muscle [156, 158]. The reduced
function of NET to increase NE amplitude might help to compensate for reduced
synaptic transmission at low temperature and thus maintain the NE concentration
at the smooth muscle receptors. However, the increased peak current and
prolonged current decay time were only seen at low temperature in DOCA-salt
MA after NET blockade suggesting the reduced function of NET. At 37 °C, there
were excessive NE released, so the dysfunctional NET uptake NE less efﬁciently.
While at lower temperature, there were less NE being released so NET can bind
with NE and function relatively efﬁcient to uptake NE. NET can clear NE before it
diffuses to the microelectrode and this leads to the increased current rise slope
for MA at 37 and 28 °C and increased current rise time at °C after NET blockade.
Cocaine also increased the Q10 for the peak NE current in sham MA which is
then similar to that for DOCA-salt MA. Thus, NET is one of the important factors
that accounts for the temperature dependent sensitivity of NE overﬂow but this
function is impaired in DOCA-salt MA.

There was no change in the peak NE current, current rise slope, current
rise time, and decay time after NET blockade in sham and DOCA-slat MV. This
could be due to either low density of NET at the nerve endings or NET is

localized far away from the NE release sites to clear NE, although MV does

147

express NET in sympathetic ganglia [34]. Our result reveals that NE clearance
seems to be mainly by diffusion instead of uptake through NET in sham and

DOCA-salt MV.

5.4 Conclusion

The present work demonstrates that, the overﬂow of NE display greater
temperature sensitivity in DOCA-salt MA than sham MA and NE transporter
account for this difference. Presynaptic (leR and NE transporter play a greater
role in regulating NE release and clearance at the neuroeffector junction in sham
MA than in MV. These mechanisms have been altered in DOCA-salt MA but not
MV. The elevated NE overﬂow from perivascular sympathetic nerves in DOCA-
salt MA is likely due to impaired GilGo protein coupled presynaptic org-adrenergic

autoreceptors, and NE transporters, but not presynaptic calcium channels.

148

CHAPTER 6

Oz' INTERACTS WITH PERTUSSIS TOXIN-SENSITIVE G-
PROTEINS TO DISRUPT (12 ADRENERGIC RECEPTOR
FUNCTION INSYMPATHETIC NERVES SUPPLYING
MESENTERIC ARTERIES IN DOCA-SALT
HYPERTENSION

6.1 Introduction

Under physiological conditions, reactive oxygen species (ROS) act as
signaling molecules regulating the growth and function of vascular smooth
muscle cells [103]. Enhanced production of ROS and a decrease in the
antioxidant reserve in plasma and tissues were reported in hypertensive animals
and human [116]. The major ROS resulting from oxidative stress are superoxide
anion, hydrogen peroxide, hydroxyl radical and peroxynitrite (02', H202, OH-,
ONOO-). It has been found that in the deoxycorticosterone acetate (DOCA)-salt,
the genetic SHR, the Dahl-salt sensitive and the angiotensin-induced models of
hypertension in rats, that a progressive increase of 02' production in vascular
and cardiac tissue occurs during the development of hypertension [106-108,
221]. 02' is at the origin of the cascade leading to the formation of most ROS. It
is a free radical species, which is usually short-lived due to its rapid reduction by
superoxide dismutase (SOD) to hydrogen peroxide and then catalyzed to H20
and Oz. [101].

NADPH oxidase is the major source of :Oz‘ in the vasculature [222]. 02'

and NADPH oxidase activity are increased in DOCA-salt hypertension including

149

in the sympathetic nervous system [106, 223]. There are functional changes in
the sympathetic nervous system in DOCA-salt hypertensive rat, including the
increased sympathetic nerve activity and enhanced release of norepinephrine
(NE) [18, 34]. ROS affect sympathetic nervous system function. H202 can
activate the protein kinase C pathway causing facilitation of NE release from
nerve terminals [224]. In addition, 02‘ can reduce the effect of NO, which
modulates NE release through a negative feedback mechanism [225]. A
previous study revealed that ROS can cause enhanced production of inositol
triphosphate and reduced production of cyclic GMP in cultured vascular smooth
muscle cells [101]. Nishida et aI revealed that a pertussis toxin-sensitive G
protein is the target of R08 [115]. However, Giniatullin et al showed that the
depressant effect of H202 on acetylcholine release was pertussis toxin-
insensitive, ruling out Gilo-protein dependent cascades [226]. In addition to the
increased level of NADPH oxidase, SOD activity was reduced in DOCA-salt rats
which can also cause increased levels of -O2' [106].

The effects of antioxidant treatment on ROS levels have been studied in
animal models of hypertension. Some studies have shown that inhibition of ROS
generation with apocynin (a NADPH oxidase inhibitor) and radical scavenging
with antioxidants or SOD mimetics, such as tempol (4-hydroxy-2,2,6,6-
tetramethylpiperidinyloxy), decrease blood pressure and prevent development of
hypertension in most hypertensive models [222, 227, 228]. However, other
studies have shown that acute treatment with antioxidants, such as ascorbic acid,

do not decrease blood pressure in hypertensive patients [229] or in DOCA-salt

150

rats [113]. Long-term treatment of hypertensive patients with ascorbic acid can
reduce systolic blood pressure [230]. In the adult hypertensive SHR model,
chronic treatment with the antioxidants, melatonin or N-acetyl-cysteine, were
found to reduce blood pressure, heart rate and circulating NE levels [120]. The
effect of tempol on NE secretion, sympathetic nerve activity, and blood pressure
regulation may be in part dependent and in part independent of NO [105, 113].

In DOCA-salt rats, there is increased plasma NE levels due, in part, to the
impaired function of prejunctional d2AR, which contributes to the increased
sympathetic nerve activity [23, 64]. Although the precise effects of increased
ROS in sympathetic nervous system are unknown, it is possible that ROS
signaling can impair the nerve terminal and thereby cause increased release of
NE. To test the hypothesis that ROS may raise sympathetic nervous system
activity and impair the function at the neuroeffctor junction, I evaluated the effects
of the NADPH oxidase inhibitor, apocynin, and SOD mimetic, tempol, on blood
pressure and NE release from DOCA-salt MA. I also examined the function of G
.70 protein, which couples to the prejunctional d2AR. NE release was measured
locally at the blood vessel surface in vitro using continuous amperometry with a
microelectrode. NE concentration changes with time were recorded as an

oxidation current in these studies.

6.2 Results

6.2.1 Sham and DOCA-salt rats

Mean systolic blood pressures for sham and DOCA-salt rats were 121 :t 5

151

mmHg (n=56) and 198 :I: 7 mmHg (n=52), respectively (P < 0.05). The mean
body weights of sham and DOCA-salt rats were 400 i: 10 g and 321 i 12 g,

respectively (P < 0.05)

6.2.2 Impaired function of prejunctional a2AR in DOCA-salt MA

NE is released from sympathetic nerve endings innervating MA and MV
[67]. Nerve stimulation-induced NE release can be detected as an oxidation
current by continuous amperometry with a carbon ﬁber microelectrode [127, 187].
The oxidation current measured reﬂects the extracellular concentration of NE at
the artery surface, which equals the amount of NE released offset by neuronal
reuptake and diffusion. There is increased NE release in DOCA-salt MA [34, 59].
Representative NE time profiles before and after a2AR blockade by idazoxan (1
uM) at sham and DOCA-salt MA are shown in Fig. 6.1A. The bars under the
traces represent the period of nerve stimulation. The focal nerve stimulation
produced signiﬁcantly greater NE overﬂow for DOCA-salt than for sham MA as
shown in Fig. 6.1 B. The rise of the current was also much steeper for DOCA-salt
MA as shown in Fig. 6.10 Previously, we showed that this is at least partially due
to the impaired o2AR function (Chapter 5). In order to examine the function of
o2AR, we applied the antagonist, idazoxan, and measured the NE current again.
The oxidation current was signiﬁcantly increased by ca. 79% in sham MA while
only ca. 25% in DOCA-salt MA. Similarly, the current rise slope was increased
signiﬁcantly by ca. 67% in sham MA but only ca. 22% in DOCA-salt MA.

ldazoxan itself does not contribute to the increase of the oxidation current [186].

152

So these results illustrate that the function of prejunctional o2ARs is impaired in

DOCA-salt MA.

6.2.3 Chronic apocynin and tempol treatment lowers blood pressure of
DOCA-salt rats

DOCA-salt rats were separated into three groups: DOCA-salt control
group, apocynin-treated group and tempol-treated group. Blood pressure was
recorded for 2 control days and 24 treatment days. There was a signiﬁcant
interaction between treatment and blood pressure (P < 0.05). The mean arterial
blood pressure (MAP) was plotted as a function of time: control, treatment day 18
and treatment day 24 in Fig. 6.2. No difference was found in MAP among three
groups in the control period. MAP for three groups increased over 24 treatment
days but it was signiﬁcantly lowered by apocynin and tempol treatment
compared to DOCA-salt control group, especially on day 18. However, by the
end of the study, the MAP from the apocynin and tempol group was not

signiﬁcantly different from that of the DOCA-salt group on day 24.

6.2.4 Apocynin and tempol treatment reduce NE release and restore a2ARs
function in DOCA-salt MA

Previous studies on the kinetics of neurotransmitter release have shown
that the release of ATP and NE is frequency dependent and at lower stimulation
frequency (up to 8 Hz), NE is released in very small amounts compared with

ATP [91, 231]. In other words, the portion of NE released from sympathetic

153

I control DOCA-salt

I idazoxan
v. -_I 5 M
5 s _./

 

 

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10 Hz
B c
18 * :m‘
* - Control (n=20) g *
2 1 :1 ldazoxan (n=20) I; #
3 # o. 2
g 12 o
c -
m
9 e
8 .9 1
w 2'5
2 c
9
3
O
Sham DOCA-salt Sham DOCA - salt

Figure 6.1 (A) Representative recordings of in vitro continuous amperometric
measurement of NE oxidation currents from sham and DOCA-salt MA before
(black trace) and after (grey trace) the blockade of presynaptic q2AR using 1 pM
idazoxan. NE oxidation currents were evoked by a 10 Hz stimulus train and the
bar under the current traces represents the period of nerve stimulation (60 pluses
with a 0.5 ms pulse width). Statistical results for NE current (B) and 10-90% NE
current rise slope (C) from sham and DOCA-salt MA demonstrate that the NE
level is higher and q2AR function is impaired in DOCA-salt MA. Data are means :t
SEM. with * represents significant difference from control, and # represents
signiﬁcant difference from sham MA, P < 0.05.

154

nervous system may vary by stimulation frequency. Therefore, the effect of
antioxidant treatment on sympathetic nerve function was studied by endogenous
NE measurement at different stimulation frequencies. On the 24th day of the
chronic antioxidant treatment, the rats were sacriﬁced and the mesenteric tissue
was taken out for measurement. Representative NE oxidation current proﬁles
before and after o2AR blockade from the DOCA-salt, apocynin and tempol
groups are shown in Fig. 6.3 A and B. Apocynin and tempol decreased the peak
current signiﬁcantly at all stimulation frequencies with the most prominent
difference occurring at 2 and 5 Hz for apocynin and tempol treatment groups (Fig.
6.3C). And the current rise slopes, which were partially determined by
presynaptic o2AR function, were signiﬁcantly reduced by apocynin and tempol at
10 and 20 Hz, probably due to restored function of o2ARs.

ldazoxan (1 (M) was used to investigate the function of prejunctional
d2ARs. ldazoxan increased NE peak current 2-fold in the apocynin and tempol
treatment groups at stimulation frequencies 5 10 Hz and ca. 1.5 fold at 20 Hz.
Oxidation currents recorded for DOCA-salt MA were increased by idazoxan only
at stimulation frequencies 5 10 Hz with smaller increases than that occurring in
antioxidant treatment groups. ldazoxan failed to increase NE current at 20 Hz for
DOCA-salt MA. Oxidation current rise slopes were increased by idazoxan at
stimulation frequencies 5 10 Hz in apocynin and tempol treatment groups but
there was no change in the DOCA-salt group. Both the differences in peak
current and current rise slopes among three groups were abolished after the

blockade of o2AR. Taken together, these data support a role for oxidative stress

155

- DOCA-salt (n=13)
1.7:: Apocynin (n=8)
:1 Tempol (n=9)

 

 

 

   

165' l
g1 * * .
E 140- *f
E
a _ .,
g 115- 1
90. a" l I.“ .0-.. _

Control Day 18 Day 24

Figure 6.2 Mean arterial pressure (MAP) for chronic antioxidant treatment for
DOCA-salt rats in vivo study. MAP increased for the three groups with time. It
decreased in response to chronic antioxidant treatment with apocynin (grey bar)
and tempol (open bar) compared to DOCA-salt controls, particullaly on day 18.
Two-way ANOVA indicates there is signiﬁcant difference for the MAP among
three groups with time (P < 0.05). Data are means i SEM. and * indicates
signiﬁcant difference compared to DOCA-salt controls by Newman-Keuls post

hoc test, P < 0.05.

156

in the impairment of o2AR which leads to the increase in NE release and the rate
~of release in DOCA-salt MA. d2AR function was restored, at least in part, after

chronic antioxidant treatment at sympathetic nerve endings.

6.2.5 Impaired function of pertussis toxin sensitive G protein in DOCA-salt
MA

Previous studies revealed that ROS, mainly 02', produce membrane protein and
lipid damage disrupting intra and intercellular signaling [101]. Since the
prejunctional o2AR is a G-protein coupled receptor, it is possible that the
increased ROS target 6760 proteins and, therefore, cause impaired signaling
pathway to a2AR. To test this, I incubated sham and DOCA-salt MA in 3 pg/ml
pertussis toxin (PTX) to examine the function of GilGo protein [188]. Both the NE
peak current and current rise slope from PTX incubated sham MA were
signiﬁcantly increased compared to control incubated tissue (Fig. 6.4 A and C).
However, there was no difference in either the oxidation peak current or the
current rise slope in DOCA-salt MA after PTX incubation compared to control (Fig.
6.4 B and D). These observations demonstrate that the function of 676., protein
coupled to prejunctional o2AR is impaired in DOCA-salt MA. For comparison,
idazoxan (1 pM) was used to investigate the function of prejunctional o2AR in
control and PTX incubated tissues. Marked increases in both NE peak current
current and current rise slope were observed in sham incubated MA but very little
increase in DOCA-salt tissues (Fig. 6.4). However, the increase of current

caused by PTX is less than those caused by idazoxan alone and PTX plus

157

Figure 6.3. Effects of chronic antioxidant treatment, apocynin and tempol, on the
release of NE and prejunctional q2AR function at the sympathetic nerve endings.
Representative traces of NE oxidation current from DOCA-salt control (black),
apocynin (dark grey) and tempol (light grey) groups before (A) and after (B) the
blockade of prejunctional a2AR using 1 pM idazoxan. NE oxidation currents were
evoked by 10 Hz stimulus trains (60 pluses, 0.5 ms pulse width). The maximum
NE current (C) and 10-90% current rise slope (E) were signiﬁcantly decreased in
apocynin and tempol groups compared to DOCA-salt controls. The differences in
NE maximum current (D) and 10-90% current rise slope (F) disappeared after the
blockade of prejunctional a2AR by idazoxan. This indicates that the differences
among three groups were partially caused by a2AR and the function of o2AR was
restored by chronic apocynin and tempol treatment. Data are mean :I: SEM.
and * represents signiﬁcant difference from DOCA-salt control by one-way

ANOVA with Newman-Keuls post hoc test, P < 0.05.

158

NE current (pA)

rn

NE current rise slope (pA/s)

Control

 

Control

5 10
Frequency (Hz)

Control

Frequency (Hz)

 

 

20

 

 

B ldazoxan (1 pM)
#_ DOCA
Apocynin
Tempol
‘5 A 1* “t
p 131
i wwwﬂl‘n
Ss ‘ ~ *1. i 1'11]
I‘gwi‘EKCMIW

 

ldazoxan (1 pM)

- DOCA (n=10)
E11111 Apocynin (n=10)
1:1 Tempol (n=10)

_x

NE current (pA)

 

2 5 1o 20
Frequency (Hz)
F
ldazoxan (1 (M)
7;?
-DOCA(n=10) i7 *
# -Apocynin (n=10) ;; 5 iﬂ
1‘: :ITempoI (n=10) 3 a * 1‘1
0)
3 2 *
'C
E
g 1 * *
8 * *
Lu
2 2 5 1o 20
Frequency (Hz)

159

idazoxan. Taken together, these results suggest that the tissue incubation in the
medium itself did not change the function of presynaptic ozAR. It is the impaired
function of Gi/Go protein coupled to 02AR in the DOCA-salt MA that made the

difference in NE overﬂow compared to sham MA.

6.3 Discussion

The results conﬁrm previous observations regarding differences in
sympathetic neurotransmission to MA from sham and DOCA-salt rats. Speciﬁcally,
the results demonstrate that endogenous NE release from DOCA-salt MA,
evoked by nerve stimulation, is signiﬁcantly greater than that from sham MA [34].
The prejunctional autoreceptor regulates NE release through negative feedback
and it has been established that the function of prejunctional 02AR is impaired in
human hypertension [65, 66] and many animal hypertension models, including
SHR and DOCA-salt rats [7, 59]. It is therefore likely that the elevated NE
overﬂow and the increased rising slope of the current in DOCA-salt MA are
partially due to the impaired function of prejunctional azAR. The present study
was designed to investigate the role of oxidative stress on the elevated NE
release in DOCA-salt MA and if the sympathetic neurotransmission at the

neuroeffector junction is impaired by oxidative stress.

160

EA.

A Sham DOCA-salt

CD

   

<3 3
E E
5 E
0 O
E s
O. 0
Lu 0.
uJ
Z 2
C Sham D DOCA salt
7!? 3 * * g 3
E * e
g 2 3 2
o 2
a U)
“l 3
'3 1 E 1
E c
g ‘ “E
3 D
0 o O I
(F 90 Q0 6‘ (l- @0 4,0
0056" q ($91» 0* 0004‘ Q 19+ 19
\c \o 964’ * so
+‘ a“ + 6
<2" cf? <2" 00“

Figure 6.4 Effects of pertussis toxin (PTX, 3 ug/ml) on NE release at sham and
DOCA-salt MA. After 2h tissue incubation in PTX, NE maximum current and 10-
90% NE current rise slope were signiﬁcantly increased at sham MA (A and C) but
not DOCA-salt MA (B and D). For comparison, we also blocked azAR function by
idazoxan for the control and PTX treatment groups. Both NE current and current
rise slope were increased significantly by idazoxan for sham control and PTX
treatment groups (A and C) but the increase was very little in DOCA-salt MA (B
and D). This represents that the function of Gi/Go which couples to azAR is
impaired in DOCA-salt MA. The current increased by idazoxan plus PTX was
signiﬁcantly greater than that caused by PTX alone for sham MA. Data are
means is SEM. with * represents signiﬁcant difference from control, and #
represents signiﬁcant difference from PTX incubated MA, P < 0.05.

161

Many studies have raised the possibility that an enhanced production of
ROS, especially 02', may be involved in the development of hypertension [101,
116]. In addition to the effects of ROS directly on the vasculature [222],
augmented 02' production contributes to the development of hypertension also
through activation of the sympathetic nervous system [227]. Enhanced formation
of 02' results from increased NADPH oxidase activity, and reduced SOD activity
occurs in DOCA-salt rats [106, 228]. Inhibition of ROS generation with apocynin,
an NADPH oxidase inhibitor, and radical scavenging with antioxidants or SOD
mimetics, like tempol, can decrease blood pressure and prevent development of
hypertension in most hypertensive models [111, 227, 232]. In the adult SHRs,
chronic treatment with the antioxidants, melatonin or N-acetyl-cysteine, reduce
blood pressure in association with a reduction in heart rate and in circulating NE
[120]. It therefore likely that 02' level is increased in DOCA-salt rats and
antioxidant treatment can lower the NE release at the sympathetic nerve endings.
For that reason, we focused this study on the role played by antioxidant,
apocynin and tempol on the recovery effect in sympathetic neurotransmission in
the DOCA-salt hypertension, with speciﬁc attention to NE bioavailability.

My results conﬁrm that oxidative stress is involved in the changes that
occur in sympathetic neurotransmission in DOCA-salt hypertension. Speciﬁcally, i
show that chronic treatment of antioxidants, apocynin and tempol, reduced blood
pressure in a time dependent manner, especially at day 18. Although there was
no signiﬁcant difference of MAP from apocynin and tempol group compared to

DOCA-salt group at the end of the study, the chronic antioxidant treatment is a

I62

time course response and the fact that blood pressure for an extended period
would minimize chronic injury caused by accumulation of 02’ during the course of
the study. In order to verify that, at day 24, the effectiveness of antioxidant
treatment on sympathetic nerve was assessed by measuring dihydroethidium
(DHE) intensity, a marker of superoxide level in the sympathetic ganglia. The
DHE staining of inferior mesenteric ganglia was reduced by ca. 50% in apocynin
and tempol treated group compared to DOCA-salt control group [233]. This
conﬁrms that although the MAP didn’t change at the 24th day, the antioxidant
treatment lowered the superoxide level in DOCA-salt rats in the overall treatment
period. Furthermore, the nerve stimulation induced release of NE was
signiﬁcantly decreased in the apocynin and tempol groups while the function of
prejunctional GzAR was restored compared to DOCA-salt group. And we also
demonstrate that the function of pertussis toxin sensitive Gi/Go protein, which
could be the target of ROS [115, 234], is impaired in DOCA-salt rats. These
ﬁndings are in keeping with the hypothesis that the increased sympathetic nerve
activity may due in part to the elevated level of ROS and ROS may alter the
sympathetic neuroeffector junction in DOCA-salt hypertensive rats.

DOCA-salt rats have signiﬁcantly greater mRNA levels of the NADPH
oxidase subunit p22phox in the prevertebral sympathetic ganglia than do sham
rats, which suggests that NADPH oxidase is increased and is responsible for
increased 02‘ production and possibly contributes to increased blood pressure in
the DOCA-salt hypertensive rat [223, 228]. The mechanisms modulating the

NADPH oxidase activity in hypertensive rats is still unclear. Some studies

163

 

suggest that the local activated renin-angiotensin system and endothelin system
in DOCA-salt hypertensive rats might be involved in the higher levels of 02‘
production [235, 236]. Further studies are needed to verify this hypothesis. In
addition to the increased NADPH oxidase activity, a decreased CulZn SOD
activity appears to play an important role in the high level of aortic -Oz’ formation
in DOCA-salt rats [106]. Apocynin and tempol decreased ~02" level by different
mechanisms. Apocynin is known as an inhibitor of NADPH oxidase. However, a
recent study suggests that apocynin predominantly acts as an antioxidant which
works independent of NADPH oxidase [237]. So far, it is not known if this
alternative mechanism for apocynin works in the sympathetic neurons. In
addition, it is shown that long-term treatment of apocynin can reduce the 02'
level and blood pressure, but not the acute treatment in a short time [114].
Tempol works as a SOD mimetic to reduce the 02‘ level regardless of its origin.
Both long term and short term treatment of tempol can lower the blood pressure
in DOCA-salt rat [114].

The novel ﬁnding in my study is the beneﬁcial effect of chronic antioxidant
treatment on the sympathetic nerve function. It is well known that under
pathological conditions, ROS has deleterious effect on vasculature, such as
impair the endothelium dependent vasorelaxation [104], induce vascular smooth
muscle cell growth [238] and activate vasoconstrictory signal transduction
pathway [239]. l demonstrate that ROS also act via prejunctional mechanisms
that contribute to the elevated sympathetic nervous system activity in DOCA-salt

hypertensoin. The restored function of prejunctional 02AR by chronic apocynin

I64

 

and tempol treatment groups demonstrates that the impairment of azAR may
partially caused by the elevated level of ROS. One thing we need to point out
that although ROS was shown to increase the entry of Ca2+ into myocytes [116],
the prejunctional Caz+ channel function equally well in sham and DOCA-salt
hypertensive rats MA (Chapter 5). So ROS didn’t change the function of
prejunctional Ca2+ channel. Gi/Go are target protein of ROS [115] and it has been
revealed that oxygen radicals can cause protein damage and degradation [234],
as well as protein fragmentation and polymerization [240] by oxidizing the
sulfhydryl groups and methionine residues [241]. My study suggests that
impaired Gi/Go proteins, which may be caused by elevated ROS, is part of the
reason for the impaired function of prejunctional uzAR. However, I do expect that
after the PTX incubation for sham MA, the increase of NE current should at least
equal to the increase that caused by idazoxan, if not more than that (Fig. 6.4).
The greater current increase caused by idazoxan could be partially due to its
blockade of the low afﬁnity intrajunctional buffering sites which bind NE and re—
release it at a later time [124]. It could also be possible that PTX works less
efﬁciently on blocking Gi/Go proteins, or the tissue incubation in PTX was not
sufﬁcient enough [194]. Nonetheless, my work suggests that with the same PTX
treatment, the function of Gi/Go proteins in DOCA-salt MA is signiﬁcantly lower
than that in sham MA. In addition, there may be other vesicle regulating proteins
in the sympathetic nerve terminal which are sensitive to ROS. For example,
SNAP25, one of three essential fusion proteins, which mediates the ROS-

induced impairment of releasing machinery in the central nervous system [226],

I65

 

may also be a target of ROS in the sympathetic nervous system. Furthermore,
there is also study showing that not only are the proteins themselves potential
targets, but also interactions between molecules within a signaling complex may
be modiﬁed by R08 [222].

I do not exclude other possibilities that can lead to increased overﬂow of
NE in DOCA-salt MA, such as impaired function of NE transporter (Chapter 5). It
has also been published that nitric oxide (NO) inhibits NE release from
sympathetic nerves [242]. The reduction in the bioavailability of NO as a result of
elevated oxidative stress contributes to the increase in NE overﬂow from SHR
MA [225]. Many studies have shown that SOD mimetic, tempol, can
scavenge 02‘, increase the bioavailability of NO, which cause vasodilation and
thus reduce the blood pressure in SHR and angiotensin ll-infused hypertensive
rats [243, 244]. However, tempol can directly inhibit peripheral sympathetic
nerves, possibly by inhibiting sympathetic neuroeffector transmission; this effect
is NO-independent in DOCA-salt hypertensive rats [113]. Similar results have
been obtained by Campese and his coworkers [105]. My study supports this idea
by showing that tempol restored the function of prejunctional azAR which inhibits

NE release and thus decreased the peripheral sympathetic nerves activity.

6.4 Summary and conclusions

Our data herein indicate that the increased sympathetic overflow in
DOCA-salt MA is partially due to the impaired function of presynaptic OzAR.

Oxidative stress alters the sympathetic neurotransmission in DOCA-salt

166

hypertensive rats probably by impairing pertussis toxin-sensitive G proteins which
couple to GzAR at the nerve terminal. Chronic treatment with apocynin and
tempol reduced oxidative stress, lowered the mean arterial blood pressure and
the sympathetic overflow. Chronic antioxidant treatment also has the beneficial
effect on the sympathetic nerve function by restoring presynaptic OzAR function in
DOCA-salt MA and thus decreased the overﬂow of NE. Taken together, these
observations strongly suggest that oxidative stress in the cardiovascular system
constitutes an important mechanism in the development of hypertension and
support a new mechanism for the beneﬁcial effect of antioxidant treatment in

hypertension.

167

__.1._

CHAPTER 7

CONCLUSIONS

Sympathetic nerves innervating MA and MV play an important role in
blood pressure regulation. Alteration in sympathetic nerve function underlies
several types of hypertension including DOCA-salt hypertension. NE is the major
neurotransmitter in the sympathetic nervous system and elevated plasma NE
levels occur in DOCA-salt hypertension in rats. However, how hypertension
alters the mechanisms that regulate NE transmission is unclear. A major focus of
my thesis work was to test the hypothesis that there are artery-vein differences in
NE release and clearance mechanisms and these mechanisms are altered in
DOCA-salt hypertensive rats. I also proposed that alterations in sympathetic
neurotransmission are associated with oxidative stress. Continuous amperometry
with a microelectrode was used to monitor endogenous NE release elicited by
focal nerve stimulation, at the adventitial surface of MA and MV from sham and
DOCA-salt rats in vitro. Measurements were made as a function of temperature
from 28 to 37 °C. There are four major conclusions that can be drawn from the
results of this work.

I. The drugs used to study sympathetic neurotransmission can alter the
microelectrode response sensitivity and stability. The effects of drugs
on microelectrode response sensitivity and stability should be
ascertained before making in vitro or in viva electrochemical

measurements.

I68

 

Amperometry coupled with drugs was used to investigate the mechanisms
regulating NE release. It is, therefore, of importance to investigate if the drugs
are electrochemically active at the electrode potential where NE is detected and
if the drug attenuates the electrode response for NE by molecule adsorption and
electrode fouling. Carbon ﬁber (T 650 and P 55 type) and diamond
microelectrodes were used for this study. Results revealed that cocaine,
idazoxan and PPADS are electrochemically inactive at both microelectrodes at
the potentials used to detect NE, and drug exposure has little effect on the NE
oxidation current. On the other hand, exposure to capsaicin, prazosin, yohimbine
and UK 14,304 produced varying degrees of NE oxidation current attenuation at
both microelectrode types. When yohimbine and UK 14,304 are present, NE can
be detected but the oxidation current is reduced. Furthermore, detection of NE in
the presence of prazosin or capsaicin is complicated by the fact that both drugs
are oxidized near the potentials used for NE monitoring.

Carbon ﬁber and diamond microelectrode has sp2 and sp3 bonded carbon
surface microstructure, respectively. The inﬂuence of drugs on NE detection at
these two microelectrodes behaves differently due to the extent of absorption of

drug on the microelectrode surface. NE is favorable to react at the more

microstructurally-disordered material, like T-650 carbon ﬁber; and some drug (e.g.

PPADS, yohimbine) has more favorable interaction with microstructurally-ordered
material, such as P-55 carbon ﬁber and the hydrophobic sp3-bonded diamond.
Therefore, diamond possesses the superior properties over carbon ﬁber for

biochemical detection due to the weak adsorption of polar biomolecules and

169

 

contaminants; on the other hand, for NE measurement in the presence of drug,
especially those can strongly adsorb on the electrode surface, the T-650 carbon

ﬁber provides the best performance.

ll. NE release and clearance mechanisms are different in sympathetic

nerves associated with MA and MV.

Arteries and veins contribute differentially to the hemodynamics. My work
reveals that the release and clearance mechanisms of NE from MA and MV are
different. Speciﬁcally, 1) NE overﬂow from MV exceeds than that from MA, and it
behaves more temperature sensitive compared to MA, 2) NE release in MA is
tightly regulated by prejunctional 02AR but not in MV, possibly due due to the
lower expression of 6760 proteins that couple to azAR, 3) NE release is more
sensitive to calcium channel blockers in MV, 4) NE is cleared by NET from the
neuroeffector junction in MA but mainly by diffusion in MV; NET function
contributes to the temperature dependent difference in NE overﬂow between MA
and MV, 5) the possible differences may also exist in the content and recycling
dynamics of NE vesicles in periarterial and perivenous sympathetic nerves. The
key ﬁndings are shown in Fig. 7.1.

One interesting ﬁnding is that cocaine didn’t increase NE oxidation currents in
MV, however, guanethidine, a known inhibitor of NE secretion through NET,
decreased NE release more in MV than in MA. This may be explained by the
reduced opportunity for lateral inhibition by NE released from adjacent

varicosities due to the low density of sympathetic nerve ﬁbers supplying MV

170

(Stjarne and Stjarne, 1995). Future study may focus on Ultrastructural studies
comparing the subcellular structure of sympathetic varicosities using
transmission electron microscopy. These studies may provide insight into the
sympathetic nerve terminal, patterns of vesicle distribution, and possibly the NET
distribution at the nerve terminals [12, 13].

Although the azAR antagonist, idazoxan, failed to induce a signiﬁcant
increase in NE currents in MV, the azAR agonist, UK 14,304, inhibited NE
currents. This result supports the presence of functional prejunctional azARs in
MV [91]. It is possible that GzARS on perivenous nerve endings are not be
accessible to endogenously released NE. Alternatively there may be several
subtypes of prejunctional GZAR existing at MV, which can be activated by
different agonists or blocked by different antagonists. Future studies of
prejunctional azARs could focus on speciﬁc 02A, 023, 020, and 020 subtypes that
may selectively inhibit NE release at the sympathetic nerve endings in MV.

The differences in neuroeffector transmission may contribute to the different
hemodynamic functions of MA and MV. MV are more sensitive to the
vasoconstrictor effects of sympathetic nerve stimulation than MA. Reduced
regulation of NE release and clearance by prejunctional 02AR and NET may
maintain venous tone that leads to a redistribution of some stored blood from

peripheral veins to the heart and then into the arterial circulation.

171

Sham MA Sham MV

3p0J106|80J0§|N

 

 

 

Figure 7.1 Mesenteric artery and vein neuroeffector junctions in normotensive
rats. NE is released from sympathetic nerve terminals in response to nerve
stimulation and depolarization of the nerve terminal. NE bind with a1AR post
junctionally resulting in constriction of the smooth muscle cell. NE release in MA
is tightly regulated by prejunctional 02AR but not in MV, possibly due due to the
lower expression of GilGo proteins that couple to azAR. NE release is more
sensitive to calcium channel blockers in MV compared to MA. NE is cleared by
NET from the neuroeffector junction in MA but mainly by diffusion in MV; NET
function contributes to the temperature dependent difference in NE overflow
between MA and MV. It is possible that the readily releasable pool at the
sympathetic nerve endings is larger in MV but the reserve pool of vesicles is
further away from the active zone in perivenous compared to periarterial nerves.
These differences lead to the exceeding NE overﬂow at 37 °C and greater
temperature dependent sensitivity in NE overflow from MV than that from MA.

172

Ill. There is impaired regulation of NE transmission from sympathetic
periarterial nerves in DOCA-salt rats.

Several changes at the neuroeffector junction occur which alter adrenergic
neurotransmission to MA in DOCA-salt hypertension. This conclusion is based on
the following ﬁndings, 1) NE overﬂow is greater from DOCA-salt MA than sham
MA at 37 and 34 °C, and it is more sensitive to temperature change compared to
sham MA, 2) the function of prejunctional azAR is impaired in DOCA-salt MA; 3)
there is no difference in calcium channel function in terms of regulating NE
release between DOCA-salt and sham MA, 4) NET dysfunction accounts for the
slow clearance of NE at the vicinity of the sympathetic nerve junction in DOCA-
salt MA; NET contributes to the NE overﬂow temperature dependent difference
between DOCA-salt and sham MA, 5) there is no difference in NE release and
clearance mechanisms between DOCA-salt and sham MV. The key ﬁndings are
shown in Fig. 7.2.

There are many other factors which contribute to the probability and amount
of NE released, since transmission from individual varicosities involves the
release of variable size packages of transmitter onto different size receptor
patches [245]. One possibility for the increased NE release in DOCA-salt MA is
the increased NE bioavailability in the nerve terminal. Other possible
mechanisms include changes in the ability of NE to be stored in vesicles or the
distribution of readily releasable vesicles clocked at the sympathetic nerve
endings. Transmission electron microscopy would be able to show the altered

distribution or amount of the NE vesicles at the nerve endings in the future study.

173

Facilitation at the neuroeffctor junction occurs due to increased calcium in nerve
terminals in response to trains of stimulation [246]. I expected NE release to be
more sensitive to calcium channel blockers in DOCA-salt MA compared to sham
MA; however, this was not the case. There are L-, N-, P/Q , R, and T type
voltage dependent calcium channels [55], among which, N- and P/Q type play a
more prominent role in neurotransmitter release. Therefore, the function of
speciﬁc N- and PK) type calcium channels on NE release in DOCA-salt MA may
be included in the future study. Another future study targeting the impaired
function of NET with knock out animals will also provide insight into the
importance of adrenergic neurotransmission in hypertension.

The dysfunction of presynaptic azAR and NET but not calcium channels
contributes to the impaired adrenergic neurotransmission to MA but not MV in
DOCA-salt hypertensive rats. These altered mechanisms on NE release and
clearance maintain peripheral arterial tone when coupled with the increased
cardiac ﬁlling pressure and/or cardiac output caused by increased venous tone

causes sustained increases in arterial pressure.

174

DOCA-salt MA

J

epouoajeomlw

   

 

 

Figure 7.2 Mesenteric artery neuroeffector junctions in control and DOCA-salt rat.
ln DOCA-salt hypertension, several changes occur to the sympathetic nerve
endings which affect neurotransmitter release. Calcium channel remain
functional, providing sufﬁcient Ca 2+ influx for vesicle fusion and neurotransmitter
release. However, the function of prejunctional azAR and NET are impaired in
DOCA-salt MA. NET contributes to the NE overflow temperature dependent
difference between DOCA-salt and sham MA. These differences all contribute to
the increased NE overﬂow at 37 °C and greater temperature dependent
sensitivity in NE overﬂow from DOCA-salt MA than that from sham MA.

175

 

IV. Reactive oxidative stress impairs the sympathetic nerve function at the
neuroeffector junction and its effects contribute to high blood
pressure in DOCA-salt hypertensive rats.

ROS are involved in the development of hypertension, partly by increasing
peripheral and central sympathetic nerve activity [101]. However, the targets of
ROS in the nervous system and how they modulate neurotransmission in
hypertension remain unclear. My work demonstrated that increased ROS targets
GilGo proteins coupling to prejunctional GzAR in sympathetic nerve endings
leading to impaired neurotransmission and increased blood pressure. Chronic
antioxidant treatment lowers blood pressure and restores sympathetic nerve
function at the neuroeffector junction in DOCA-salt hypertensive rats. The key
ﬁndings are summarized in Fig. 7.3. This work reveals a new mechanism for the
beneﬁcial effect of antioxidant treatment in hypertension.

Oxygen radicals can cause protein damage and degradation [234], protein
fragmentation and polymerization [240] by oxidizing the sulfhydryl groups and
methionine residues [241]. ROS may cause damage to the GVO protein coupling
to azAR by chemical reaction. 02AR contains two cysteine residues, which are
connected by a disulﬁde bond and play a role in ligand binding [247]. Thiol group
in the cysteine residue can be oxidized by ROS [248], which may be the target of
ROS at Gi/o protein. Future work may include the chemical reaction study of how
ROS interact with the oxidizable amino acid in the Guo protein coupling to azAR.
In addition, there may be other vesicle regulating proteins in the sympathetic

nerve terminal which are sensitive to ROS. Such as, SNAP25, which mediates

176

 

the ROS-induced impairment of releasing machinery in the central nervous
system [226], may also be a target of ROS in the sympathetic nervous system.
Additional targets at nerve terminal may also include the NET, which is impaired
by ROS [249, 250]. If and how the impaired NET in DOCA-salt hypertensive
model is related to increased ROS level can be studied in the future.

ROS, however, may exert their cardiovascular effects through other pathways.
For example, ROS can reduce the production and/or availability of NO and
results in NO-mediated sympathoinhibitory effects [105]. Future work may also
focus on if and how NO in the mesenteric vasculature is decreased by ROS and
how this may alter NE release mechanisms in DOCA-salt hypertension.

Although my work suggests that chronic antioxidant treatment lowers
sympathetic nerve activity and blood pressure, the cause-effect relationship
between oxidative stress and hypertension remains controversial [251]. It may be
beneﬁcial to investigate the role of ROS at nerve endings and blood pressure
regulation using genetic models to locally deliver a superoxide dismutase gene

regulating oxidative stress in a speciﬁc target tissue.

177

Antioxidant treated
DOCAsthA

DOCA-salt MA

eponoejeomlw

   

 

 

Figure 7.3 Mesenteric artery neuroeffector junctions in DOCA-salt rat with (right)
and without (left) antioxidant treatment. There is increased ROS in the DOCA-salt
hypertensive rat MA nerve terminals. Increased ROS may target Gl/G0 proteins
coupling to presynaptic azAR in sympathetic nerve endings leading to impaired
neurotransmission and increased blood pressure. Chronic antioxidant treatment,
apocynin and tempol, lowers NE release, blood pressure and restores
sympathetic nerve function at the neuroeffector junction in DOCA-salt

hypertensive rats.

178

In conclusion, the results of this thesis work extend our knowledge of
adrenergic neurotransmission from periarterial and perivenous sympathetic
nerves, the role of adrenergic neurotransmission in blood pressure regulation,
and the role of ROS in altering NE release from sympathetic nerves and blood

pressure regulation.

179

 

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