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2/05 p:/C|RC/DateDuerindd-p.1

ENDOTHELIN A (ETA) AND ETB RECEPTOR INTERACTION IN ARTERIES
AND VEINS

BY

Keshari Maya Thakali

A DISSERTATION
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of
DOCTOR OF PHILOSOPHY
Department of Pharmacology and Toxicology

2006

ABSTRACT

ENDOTHELIN A (ETA) AND ETB RECEPTOR INTERACTION IN ARTERIES

AND VEINS

By

Keshari Maya Thakali

Veins maintain responsiveness while arteries lose responsiveness to the
vasoactive hormone endothelin-1 (ET-1) in situations of exposure to ET-1 (eg.
hypertension and experimental protocols). Thus, receptors for ET-1 — the
heptahelical ETA and El"l3 receptors - may function differently in arteries and
veins. While heptahelical receptors canonically interact with G-proteins in a 1:1
ratio, recent evidence suggests that receptor dimerization (hetero- or homo-) may
occur. Receptor dimerization can potentially affect a number of receptor
characteristics such as agonist affinity, potency and efficacy, as well as receptor
interaction. While ET A and ET 3 receptors are present on the smooth muscle of
rat thoracic aorta and vena cava, only vena cava have contractile ETA and Era
receptors as in aorta only ET A receptors couple to contraction. Previous studies
suggest that in vessels with functional populations of both ETA and El}, receptors,

ET8 receptors are capable of physically or pharmacologically altering ETA

receptor function. I hypothesized that ET A and ETl3 receptors can heterodimerize
in rat thoracic vena cava but not aorta and that there are functional

consequences of venous ETA and ETl3 receptor heterodimerization.

To test this hypothesis that ET A and ETI3 receptors heterodimerize in vena cava
but not aorta, I examined functional receptor interaction (vascular contractility),
biochemical receptor interaction (interaction via signal transducers) and physical
receptor interaction (receptor heterodimerization) of ETA and ET 3 receptors in rat
thoracic aorta and vena cava. Isolated contractility experiments demonstrated
that functional ETA and ET 3 receptor interaction occurred in vena cava but not
aorta as venous I':'I'A receptors were more sensitive to receptor blockade when
E‘l'l3 receptors were rendered dysfunctional either from desensitization or receptor
blockade. Biochemical signaling interactions via H202 signaling, p38 MAPK, Erk
MAPK, rho kinase, src and phosphatidyl inositol 3-kinase (PIS-K) do not mediate
functional venous ETA and El'.3 receptor interaction. Receptor co-localization of
ETA and ETB receptors on dissociated aortic and venous vascular smooth muscle
cells demonstrated that ET A and ET B receptors co-localized to the plasma
membrane of both aortic and venous vascular smooth muscle cells. These
results suggest that functional ETA and El'I3 receptor interaction is independent of
receptor location and may be an intrinsic property of veins and not due to

receptor heterodimerization.

ACKNOWLEDGEMENTS

I would first and foremost like to thank my parents, Renee and Aita Thakali, for
their continuous love and support. Morn, thank you for fostering my interest in
science. Dad, thank you for passing on your inquisitiveness and skepticism.
From you I Ieamed that with some Duct tape, superglue and the right set of tools,
anything can be fixed (even if it was not broken in the first place). Thank you
both for traveling around the world with me, expanding my horizons and molding
me into the independent individual I am today.

I would like to acknowledge my advisor, Stephanie W. Watts, PhD, for being the
most exceptional mentor a graduate student could ever dream of. Thank you for
taking the time to teach me both in the laboratory and the classroom. Thank you
for all of your countless hours spent reading my abstracts, manuscripts and
dissertation. Thank you for taking me to meetings and building up my scientific
network. Thank you (in advance) for all of the future opportunities I will have as a
direct result of the excellent training l have received under you mentorship.
Thank you for putting up with my practical jokes and my odd sense of humor.
Most of all, thank you for welcoming me into your family. I am constantly
humbled and amazed by everything you do (and have already accomplished)
and would like you to know that you will always be my role model.

Lastly, I would like to acknowledge my boyfriend, Zachary C. Taylor, MD. Thank
you for moving to “miserable” Michigan with me. Thank you for understanding
how important science is to me, but also thank you for reminding me that there
are other fun things in life, and those fun things typically involve bicycles and
puppy dogs. I love you and hope that you will always save a spot on the couch
for me and Kali Bear.

TABLE OF CONTENTS

LIST OF TABLES ................................................................................ viii
LIST OF FIGURES ............................................................................... ix
LIST OF ABBREVIATIONS ................................................................... xiii
I. INTRODUCTION .......................................................................... 1
A. The circulatory system .............................................................. 1
1. Blood vessel composition .................................................. 1
8. Function of arteries and veins ..................................................... 3
1. Determinants of blood pressure .......................................... 3
2. Mechanisms of blood pressure regulation ............................. 8
C. Role of veins in blood pressure regulation ................................... 10
D. Humoral control of blood pressure by circulating peptides 11
E. Endothelin ............................................................................ 12
1. ET-1 synthesis and degradation ....................................... 13
F. ET-1 signal transduction .......................................................... 17
1. ETA and ETB receptors are G-protein coupled receptors ......... 17
2. ETA and ETB receptor signal transduction ........................... 19
3. Reactive oxygen species signaling .................................... 24
G. Distribution of ETA and ETB receptors in blood vessels .................. 25
H. ETA and ETB receptor function in hypertension ............................. 30
I. ETA and ET; receptor “cross-talk? or interaction ............................. 31
J. Receptor dimerization .............................................................. 33
1. Physical means for receptor dimerization ........................... 33
2. Caveolae as cellular locations for receptor dimerization ........ 34
3. Functional consequences of receptor dimerization ............... 37
4. Can ETA and ETB receptor heterodimerization occur and
can heterodimerization affect ET receptor function? ................. 38
5. Significance of ETA and ETB receptor dimerization ............... 39
K. Hypothesis ........................................................................... 41
II. METHODS ............................................................................... 44
A. Isolated smooth muscle contraction ........................................... 44
1. ET-1 desensitization ...................................................... 45
2. ETa receptor desensitization ............................................ 45
3. Receptor antagonism studies ........................................... 45
4. H202 concentration response curves ................................. 46
B. Measurement of arterial and venous H202 production. ..46
C. Whole tissue immunohistochemistry .......................................... 47
D. Whole tissue protein isolation ................................................... 48
1. Cross-linking ............................................................... .48
E. Western blot analysis .............................................................. 49

receptor function. I hypothesized that ETA and EI'la receptors can heterodimerize
in rat thoracic vena cava but not aorta and that there are functional

consequences of venous EI'A and ET 3 receptor heterodimerization.

To test this hypothesis that ETA and ETB receptors heterodimerize in vena cava
but not aorta, I examined functional receptor interaction (vascular contractility),
biochemical receptor interaction (interaction via signal transducers) and physical
receptor interaction (receptor heterodimerization) of ETA and ETI3 receptors in rat
thoracic aorta and vena cava. Isolated contractility experiments demonstrated
that functional ETA and ETB receptor interaction occurred in vena cava but not
aorta as venous ETA receptors were more sensitive to receptor blockade when
ETl3 receptors were rendered dysfunctional either from desensitization or receptor
blockade. Biochemical signaling interactions via H202 signaling, p38 MAPK, Erk
MAPK, rho kinase, src and phosphatidyl inositol 3-kinase (PIS-K) do not mediate
functional venous ETA and EI'B receptor interaction. Receptor co-Iocalization of
ET, and ETl3 receptors on dissociated aortic and venous vascular smooth muscle
cells demonstrated that ETA and ETB receptors co-localized to the plasma
membrane of both aortic and venous vascular smooth muscle cells. These
results suggest that functional ETA and Er, receptor interaction is independent of
receptor location and may be an intrinsic property of veins and not due to

receptor heterodimerization.

ACKNOWLEDGEMENTS

I would first and foremost like to thank my parents, Renee and Aita Thakali, for
their continuous love and support. Morn, thank you for fostering my interest in
science. Dad, thank you for passing on your inquisitiveness and skepticism.
From you I Ieamed that with some Duct tape, superglue and the right set of tools,
anything can be fixed (even if it was not broken in the first place). Thank you
both for traveling around the world with me, expanding my horizons and molding
me into the independent individual I am today.

I would like to acknowledge my advisor, Stephanie W. Watts, PhD, for being the
most exceptional mentor a graduate student could ever dream of. Thank you for
taking the time to teach me both in the laboratory and the classroom. Thank you
for all of your countless hours spent reading my abstracts, manuscripts and
dissertation. Thank you for taking me to meetings and building up my scientific
network. Thank you (in advance) for all of the future opportunities I will have as a
direct result of the excellent training I have received under you mentorship.
Thank you for putting up with my practical jokes and my odd sense of humor.
Most of all, thank you for welcoming me into your family. I am constantly
humbled and amazed by everything you do (and have already accomplished)
and would like you to know that you will always be my role model.

Lastly, I would like to acknowledge my boyfriend, Zachary C. Taylor, MD. Thank
You for moving to “miserable” Michigan with me. Thank you for understanding
how important science is to me, but also thank you for reminding me that there
are other fun things in life, and those fun things typically involve bicycles and
PUPPY dogs. I love you and hope that you will always save a spot on the couch
for me and Kali Bear.

iv

TABLE OF CONTENTS

LIST OF TABLES ................................................................................ viii
LIST OF FIGURES ............................................................................... ix
LIST OF ABBREVIATIONS ................................................................... xiii
I. INTRODUCTION .......................................................................... 1
A. The circulatory system .............................................................. 1
1. Blood vessel composition .................................................. 1
B. Function of arteries and veins ..................................................... 3
1. Determinants of blood pressure .......................................... 3
2. Mechanisms of blood pressure regulation ............................. 8
C. Role of veins in blood pressure regulation ................................... 10
D. Humoral control of blood pressure by circulating peptides 11
E. Endothelin ............................................................................ 12
1. ET-1 synthesis and degradation ....................................... 13
F. ET-1 signal transduction .......................................................... 17
1. ETA and ETa receptors are G-protein coupled receptors 17
2. ETA and ETB receptor signal transduction ........................... 19
3. Reactive oxygen species signaling .................................... 24
G. Distribution of ETA and ETB receptors in blood vessels .................. 25
H. ETA and ETB receptor function in hypertension ............................. 30
I. ETA and ETB receptor “cross-talk” or interaction ............................. 31
J. Receptor dimerization .............................................................. 33
1. Physical means for receptor dimerization ........................... 33
2. Caveolae as cellular locations for receptor dimerization ........ 34
3. Functional consequences of receptor dimerization ............... 37
4. Can ETA and ETa receptor heterodimerization occur and
can heterodimerization affect ET receptor function? ................. 38
5. Significance of ETA and ETB receptor dimerization ............... 39
K. Hypothesis ........................................................................... 41
II. METHODS ............................................................................... 44
A. Isolated smooth muscle contraction ........................................... 44
1. ET-1 desensitization ...................................................... 45
2. ET a receptor desensitization ............................................ 45
3. Receptor antagonism studies ........................................... 45
4. H202 concentration response curves ................................. 46
B. Measurement of arterial and venous H202 production. ..46
C. Whole tissue immunohistochemistry .......................................... 47
D. Whole tissue protein isolation ................................................... 48
1. Cross-linking ............................................................... .48
E. Western blot analysis .............................................................. 49

1. SDS-polyacrylamide gel electrophoresis (PAGE)

(denaturing PAGE) ............................................................ 49
2. SDS-urea PAGE ............................................................ 49
3. Non-denaturing PAGE (NATIVE PAGE) ............................. 50
F. Co-immunoprecipitation ........................................................... 50
1. Traditional co-immunoprecipitation .................................... 50
2. True Blot® co-immunoprecipitation kit ................................ 51
3. Pierce ProFound® mammalian Co-IP kit ............................ 51
4. ExactaCruz® co-immunoprecipitation kit ............................ 52
5. Co-immunoprecipitation using Dynal® magnetic beads 53
G. Dissociation of vascular smooth muscle cells ............................... 53
H. Immunocytochemistry in freshly dissociated vascular smooth
muscle cells ..................................................................... 54
I. Data analysis and statistics ....................................................... 55
RESULTS ................................................................................. 57
A. Subhypothesis 1: Pharmacological receptor interaction in veins but
not arteries ........................................................................... 57
1. Non-specific ET,.,/ETl3 receptor desensitization ..................... 57
2. ET 8 receptor desensitization ............................................. 65
3. Does ETl3 receptor desensitization alter ET A receptor
function? ......................................................................... 65
4. Receptor interaction: contractility studies using receptor
antagonists ...................................................................... 7O
5. Section summary ........................................................... 74
B. Subhypothesis 2: Biochemical signaling interactions ...................... 75
1. Reactive oxygen species and ET -1-induced contraction 80
2. Signal transduction pathways ........................................... 83
3. Section summary ........................................................... 91
C. Subhypothesis 3: Physical endothelin receptor interaction 91
1. Antibody testing: dot blots ................................................ 92
2. Receptor co-Iocalization .................................................. 93
3. Co-immunoprecipitation of ETA and ET B receptors .............. 103
4. Section summary ......................................................... 107
DISCUSSION .......................................................................... 109
A. Rationale ........................................................................... 109
B. Differences in ET A and ETB receptor desensitization ................... 111
C. Functional ETA and ETl3 receptor interaction occurs in veins
but not arteries .................................................................... 1 14
D. Signaling pathway interactions may account for functional venous
ET A and ETl3 receptor interaction ............................................. 116
1. Reactive oxygen species signaling .................................. 117
2. ET-1 signal transduction ................................................ 120

E. Can ETA and ET B receptors physically interact via

vi

VI.

receptor heterodimerization? .................................................. 124
1. Co-immunoprecipitation of ETA and ET B receptors in rat

thoracic aorta and vena cava ............................................. 128
2. Receptor co-Iocalization ................................................ 129
F. Conclusions ....................................................................... 134
G. Speculation ........................................................................ 135
REFERENCES ........................................................................ 138
CURRICULUM VITAE ............................................................... 148

vii

 

Table 1.

Table 2.

Table 3.

Table 4.

LIST OF TABLES

Estimated EC50 values for Er-1-induced contraction in aorta and
vena cava in the presence of functional or SGc-desensitized ETB
receptors with and without ETA receptor antagonism ................. 69

Estimated EC,o values for ET -1-induced contraction in aorta and
vena cava and the presence and absence of functional ETl3
receptors with and without ETA receptor antagonism ................. 73

An abbreviated list of commercially available ETA and ETB receptor
antibodies and their antigenic sequences used for dot blot
analysis ........................................................................... 94

Different co-immunoprecipitation methods were performed in

several attempts to co-immunoprecipitate ETA and E'I'l3 receptors
from rat thoracic aorta and vena cava lysates ........................ 1 O8

viii

Figure 1.

Figure 2.

Figure 3.

Figure 4.

Figure 5.

Figure 6.

Figure 7.

Figure 8.

Figure 9.

Figure 10.

Figure 11.

Figure 12.

Figure 13.

Figure 14.

Figure 15.

LIST OF FIGURES

Rendered picture of arteries and veins ................................... 2

Histological cross-sections of rat thoracic aorta and thoracic
vena cava stained with a Masson trichrome stain 4

Alpha actin immunohistochemistry of rat thoracic aorta and
vena cava ......................................................................... 5

Western blot analysis of smooth muscle a-actin expression in rat
thoracic aorta and vena cava lysates ..................................... 7

Guyton-Coleman diagram of physiological factors that affect

pressure natriuresis and control blood pressure ........................ 9
Diagram of endothelin-1 (ET-1) biosynthesis ........................... 14
Effect of phosphoramidon, an endothelin converting enyzme
inhibitor, on big ET -1-induced contraction ............................... 16
Diagram of endothelin signal transduction .............................. 18
Diagram of ET A receptor signal transduction ........................... 20
Diagram of EI',3 receptor signal transduction ........................... 21

ET. and El’l3 receptor western blot analysis in rat thoracic vena
cava ............................................................................... 26

ETA and ETa receptor immunohistochemistry in rat thoracic aorta
and vena cava .................................................................. 27

ET-1 and 86c cumulative concentration response curves in rat
thoracic aorta and vena cava .............................................. 28

Electron micrographs of caveolae present on the plasma
membrane of aortic and venous vascular smooth muscle
cells ............................................................................... 36

ET -1 concentration response curves in rat thoracic aorta and vena
cava desensitized with ET-1 ............................................... 58

Figure 16.

Figure 17.

Figure 18.

Figure 19.

Figure 20.

Figure 21.

Figure 22.

Figure 23.

Figure 24.

Figure 25.

Figure 26.

Figure 27.

Figure 28.

ET -1 [1-31] concentration response curves in rat thoracic aorta
and vena cava desensitized with ET-1 ................................... 59

SSc-induced contraction of rat thoracic vena cava desensitized
with ET-1 or $60 ................................................................ 61

Norepinephrine-induced contraction of rat thoracic aorta and
vena cava was unaltered with ET-1 desensitization .................. 62

ET-t concentration response curves in the presence of ETA
receptor blockade or ET 3 receptor blockade in rat thoracic
vena cava desensitized with ET-t ......................................... 64

86c desensitization protocol and quantification of results in
rat thoracic vena cava ........................................................ 66

Comparison of ETA receptor blockade of ET-1 cumulative
concentration response curves in rat thoracic aorta with
functional EI'.3 receptors and desensitized ET B receptors 67

Comparison of ETA receptor blockade of ET-1 cumulative
concentration response curves in rat thoracic vena cava with
functional ETB receptors and desensitized Iz'l'l3 receptors 68

Comparison of ETA receptor blockade of ET-1 cumulative
concentration response curves in rat thoracic aorta with
functional ETB receptors and without functional ET B receptors 71

Comparison of ETA receptor blockade of ET-1 cumulative
concentration response curves in rat thoracic vena cava with
functional EI'l3 receptors and without functional ET 3 receptors 72

Exogenously added H202 contracts both rat thoracic aorta and
vena cava ........................................................................ 76

Basal H202 levels are significantly higher in rat thoracic vena
cava compared to aorta ...................................................... 77

ET -1 stimulates H202 production in rat thoracic vena cava but
not aorta .......................................................................... 78

ET -1-stimulated H202 production in vena cava is not concentration-
dependent and is reduced by either ETA receptor blockade or ET a
receptor blockade ............................................................. 79

Figure 29.

Figure 30.

Figure 31.

Figure 32.

Figure 33.

Figure 34.

Figure 35.

Figure 36.

Figure 37.

Figure 38.

Figure 39.

Figure 40.

Figure 41.

H202 is not involved in venous ET-1-induced contraction as
neither catalase nor PEG-catalase reduced venous
ET-1-induced contraction .................................................... 81

The catalase inhibitor 3-AT had no effect on maximal venous
ET -1 -induced contraction .................................................... 82

Effect of LY294002, a phosphatidyl inositol-3-kinase inhibitor,
on ET-t -induced contraction of rat thoracic aorta and
vena cava ........................................................................ 85

Effect of PD98059, an Erk MAPK, on ET-t -induced contraction
of rat thoracic aorta and vena cava ....................................... 86

Effect of PP1 (10 IIM), a src tyrosine kinase inhbitor, on ET-t -
induced contraction of rat thoracic aorta and vena cava 87

Effect of SB203580, a p38 MAPK inhbitor, on ET-1-induced
contraction of rat thoracic aorta and vena cava ........................ 88

Effect of Y27632, a rho kinase inhbitor, on ET-1-induced
contraction of rat thoracic aorta and vena cava ........................ 89

Effect of HA1077, a rho kinase inhbitor, on ET -1 -induced
contraction of rat thoracic aorta and vena cava ........................ 90

Dot blots testing ETA receptor antibodies from Alomone
Laboratories, Biodesign lntemational, Biogenesis and Fitzgerald
Industries ........................................................................ 95

Dot blots testing ETl3 receptor antibodies from Alomone
Laboratories, Biodesign lntemational and Biogenesis ............... 96

Projected confocal images of smooth muscle a-actin expression
in freshly dissociated aortic and venous vascular smooth muscle
cells ............................................................................... 98

Confocal images of ETA receptor, E'I'B receptor and cadherin
expression in freshly dissociated aortic vascular smooth muscle
cells ............................................................................... 99

Confocal images of ETA receptor, ETB receptor and cadherin
expression in freshly dissociated venous vascular smooth

xi

Figure 42.

Figure 43.

Figure 44.

Figure 45.

muscle cells ................................................................... 100

Confocal images of ETA receptor, ET 3 receptor and cadherin
expression in ET -1-stimulated freshly dissociated aortic
vascular smooth muscle cells ............................................ 101

Confocal images of ET A receptor, ETl3 receptor and cadherin
expression in ET-1-stimulated freshly dissociated venous
vascular smooth muscle cells ............................................ 1 02

Western blot analysis of ETA receptor expression and ETB receptor
expression in aorta and vena cava lysates ................................ 1 04

Example immunoprecipitation of ETl3 receptors from aortic and
venous lysates ........................................................................... 1 05

xii

 

AC
CAMP
CHO
DAG
DOCA
DZ
ET-1
ECL
ERK
FRET
GABA
GPCR
HEK
H202
IHC

JNK
MAP
MAPK
NEM
NE
02'

PE
PI3-K
PKA (or B or C)
PLC
ROS
860

LIST OF ABBREVIATIONS

adenylate cyclase

cyclic adenosine monophosphate
Chinese Hamster Ovary

diacyl glycerol
deoxycorticosterone acetate
dopamine 2 receptor

endothelin-1

enhanced chemiluminescence
extracellular regulated kinase (p42/44 MAPK)
Fluorescence Resonance Energy Transfer
y-aminobutyric acid

G-protein coupled receptor
human embryonic kidney
hydrogen peroxide
immunohistochemistry

inositol 1,4,5-triphosphate

c-jun N-terrninal kinase

mean arterial pressure

mitogen activated protein kinase
N-ethyl maleimide
norepinephrine

superoxide anion

phenylephrine

phosphotidyl inositol 3-kinase
protein kinase A (or B or C)
phospholipase

reactive oxygen species

sarafotoxin 6c, ET 3 receptor specific agonist

xiii

SDS sodium dodecyl sulfate
SEM

standard error of the mean
UNaV urinary sodium excretion

xiv

 

 

I. INTRODUCTION

A. The circulatory system

The circulatory system, consisting of arteries that distribute oxygenated blood to
tissues, and veins that return oxygen-poor blood to the heart for re-oxygenation,
is critical for providing tissues with nutrients and oxygen and removing waste
byproducts of tissue metabolism. Arteries and veins have different physiological
functions, and it is a goal of this dissertation to investigate how differences in

endothelin receptors determine arterial and venous function.

1. Blood vessel composition
Blood vessels are hollow organs comprised of three layers of cells (Figure 1). A
single layer of endothelial cells line the lumen of blood vessels and define the
intimal region of a blood vessel. The outermost layer of the blood vessel, the
adventitia, comprised primarily of fibroblasts, is a highly collagenous layer
providing structural integrity to vessels. Smooth muscle cells are present in the
medial layer of a blood vessel, between the intima and adventitia. The smooth
muscle cells of blood vessels are contractile cells that allow vessels to contract
and relax to various neuro-humoral stimuli. Between the layers of smooth
muscle are elastin fibers, which like the collagenous adventitia are critical for
structural support, but also provide elastic recoil to accommodate stretching of

the blood vessel. Recent studies suggest that in addition to medial smooth

Tunica adventitia

 

 

   

 

 

Carry blood away from Carry blood away from
the heartto organs organs and tissues
and tissues back to the heart

 

 

 

 

 

 

Flgure 1. Rendered picture of arteries and veins. The tunica intima is the
innermost layer of blood vessels. The tunica media, the middle layer, is where
contractile smooth muscles cells are located. The tunica adventitia is the

outermost layer, comprised primarily of collagen.

muscle cells, the adventitia may contribute to changes in vascular tone
(Laflamme ef al, 2006).Unique to veins are valves, which assist in returning blood

to the heart.

While the structure of blood vessels can be generalized by the presence of the
intima, media and adventitia, a closer histological analysis of the structure of
arteries and veins reveals several differences in arterial and venous structure.
Masson trichrome staining of histological sections of rat thoracic aorta and
thoracic vena cava, delineates the presence of smooth muscle (light pink),
collagen (blue staining in the adventitia), nuclei (dark purple) and elastin fibers
(bright red inbetween smooth muscle layers) (Figure 2). In aorta, multiple layers
of smooth muscle cells are clearly defined between the collagenous adventitia
and the single layer of endothelial cells lining the lumen of the blood vessel. The
vena cava is highly collagenous, with a single layer of endothelial cells lining the
intima. lmmunohistochemical analysis of smooth muscle a—actin expression
reveals the presence of smooth muscle in between elastin fibers in aorta and a

very thin layer of smooth muscle cells in vena cava (Figure 3).

B. Function of arteries and veins

1. Determinants of blood pressure
While these three layers of cells are common to both arteries and veins, how
these components are organized to function together likely determines

differences in arterial and venous function. Arteries typically have thicker layers

I. INTRODUCTION

A. The circulatory system

The circulatory system, consisting of arteries that distribute oxygenated blood to
tissues, and veins that return oxygen-poor blood to the heart for re-oxygenation,
is critical for providing tissues with nutrients and oxygen and removing waste
byproducts of tissue metabolism. Arteries and veins have different physiological
functions, and it is a goal of this dissertation to investigate how differences in

endothelin receptors determine arterial and venous function.

1. Blood vessel composition
Blood vessels are hollow organs comprised of three layers of cells (Figure 1). A
single layer of endothelial cells line the lumen of blood vessels and define the
intimal region of a blood vessel. The outermost layer of the blood vessel, the
adventitia, comprised primarily of fibroblasts, is a highly collagenous layer
providing structural integrity to vessels. Smooth muscle cells are present in the
medial layer of a blood vessel, between the intima and adventitia. The smooth
muscle cells of blood vessels are contractile cells that allow vessels to contract
and relax to various neuro-humoral stimuli. Between the layers of smooth
muscle are elastin fibers, which like the collagenous adventitia are critical for
structural support, but also provide elastic recoil to accommodate stretching of

the blood vessel. Recent studies suggest that in addition to medial smooth

Tunica intima
Tunica media

Tunica adventitia

 

 

   

 

 

Carry blood away from Carry blood away from
the heart to organs organs and tissues
and tissues back to the heart

 

 

 

 

 

 

Flgure 1. Rendered picture of arteries and veins. The tunica intima is the
innermost layer of blood vessels. The tunica media, the middle layer, is where
contractile smooth muscles cells are located. The tunica adventitia is the

outermost layer, comprised primarily of collagen.

muscle cells, the adventitia may contribute to changes in vascular tone
(Laflamme ef al, 2006).Unique to veins are valves, which assist in returning blood

to the heart.

While the structure of blood vessels can be generalized by the presence of the
intima, media and adventitia, a closer histological analysis of the structure of
arteries and veins reveals several differences in arterial and venous structure.
Masson trichrome staining of histological sections of rat thoracic aorta and
thoracic vena cava, delineates the presence of smooth muscle (light pink),
collagen (blue staining in the adventitia), nuclei (dark purple) and elastin fibers
(bright red inbetween smooth muscle layers) (Figure 2). In aorta, multiple layers
of smooth muscle cells are clearly defined between the collagenous adventitia
and the single layer of endothelial cells lining the lumen of the blood vessel. The
vena cava is highly collagenous, with a single layer of endothelial cells lining the
intima. lmmunohistochemical analysis of smooth muscle a-actin expression
reveals the presence of smooth muscle in between elastin fibers in aorta and a

very thin layer of smooth muscle cells in vena cava (Figure 3).

B. Function of arteries and veins

1. Determinants of blood pressure
While these three layers of cells are common to both arteries and veins, how
these components are organized to function together likely determines

differences in arterial and venous function. Arteries typically have thicker layers

Vena cava

H
AdventitialMedia?

 

Flgure 2. Histological cross-sections of rat thoracic aorta and thoracic vena cava
stained with a Masson trichrome stain. The three layers of a blood vessel are
clearly defined in the aorta (as indicated by arrows) but are not obvious in vena

cava.

Adventitia Media

mama-r:—

Vena cava

<——>
Adventitia/Media?

Flgure 3. Alpha actin immunohistochemistry of rat thoracic aorta and vena cava.

Arrows indicate smooth muscle a-actin antibody binding (the darker purple areas

in between elastin fibers in aorta, and the thin, subendothelial layer staining in

vena cava), delineating where smooth muscle is located aorta and vena cava.

of smooth muscle than veins and express more smooth muscle a-actin (a key
contractile protein) per microgram of total protein (Figure 4). Arterial smooth
muscle contraction or relaxation causes changes in arterial diameter, or tone. If
the circulatory system is thought of as an electrical circuit, arteries determine
resistance, while veins determine capacitance. Resistance is the impedance to
flow, thus increases in arterial tone (or decreases in arterial lumen diameter)
increase vascular resistance, and reductions in arterial tone (l.e. increases in
arterial diameter) decrease resistance. Capacitance is the ability to store charge,
and as functional capacitors, veins act as reservoirs for storing blood and contain

approximately 60-70% of the total blood volume.

Factors that determine arterial blood pressure include total peripheral resistance,
determined by the tone or contraction of arteries, and cardiac output, the volume
of blood pumped out of the heart per minute. Cardiac output is determined by
heart rate, how fast the heart contracts, and stroke volume, the volume of blood
ejected from the heart with each contraction. Elevated arterial blood pressure
can lead to organ damage, such as renal failure, stroke, myocardial ischemia and
myocardial infarction, while low blood pressure can cause insufficient organ
perfusion and organ damage due to hypoxia. Thus, it is of critical importance for
the body to maintain a homeostatic blood pressure that does not induce end-

organ damage, but allows for adequate organ perfusion.

Aorta Vena Cava

1234567891011. 1234567891011

40 ROM W40 kDA

 

(N=11)

 

a-actin den sitometry
(arbitrary units)

 

 

 

 

 

 

Vena Cava

Figure 4. Western blot analysis of smooth muscle a-actin expression in rat
thoracic aorta and vena cava lysates. Top: representative Western blots of a-
actin expression; bottom: quantitation of a-actin expression. Fifty micrograms of
protein (as measured by BCA protein assay) from aortic and venous lysates from
11 different rats (N=11) were loaded in each well, electrophoresed, transferred to
PVDF membrane and a-actin detected using a primary antibody specific for a-

actin and secondary antibody conjugated to horse radish peroxidase.

2. Mechanisms of blood pressure regulation
Acutely (from seconds to minutes), blood pressure is regulated by the
baroreceptor reflex. Changes in arterial pressure alter the firing rate of pressure-
sensitive baroreceptors located in the aortic arch and carotid sinus, signaling
regions in the brain (ie. rostral vental lateral medulla, RVLM) that control
sympathetic outflow to control the force and rate of cardiac contraction and
venous return to ultimately alter blood pressure. A more chronic mechanism to
control blood pressure is through regulation of blood volume through alterations
in natriuresis (Figure 5). Decreases in blood pressure activate the renin-
angiotensin system to increase sodium and water retention and increase blood
volume. Increased blood volume increases stroke volume and cardiac output,

thus increasing blood pressure (Boron and Boulpaep, 2003).

While our bodies have developed regulatory mechanisms for controlling blood
pressure, there are times when these systems fail to keep blood pressure
constant and high blood pressure can cause end-organ damage such as renal
failure, stroke, and heart failure. There many clinically approved
pharrnacotherapies for the treatment of hypertension, including diuretics, beta-
blockers, calcium channel blockers, angiotensin (AT1) receptor
blockers/angiotensin converting enzyme (ACE) inhibitors, etc., but typically
monotherapy is not adequate to maintain blood pressure at levels recommended
by The Seventh Report of the Joint National Committee on Prevention,

Detection, Evaluation, and Treatment of High Blood Pressure (JNC Vll). Since

 

 

 

 

 

 

 

 

 

 

Fluid
intake

 

 

 

Non—renal
fluid loss

 

 

 

I

I

 

 

Rate of change of extracellular fluid
volume

 

 

I

 

 

 

 

 

 

I...
l_ .......

 

 

 

 

 

 

 

 

 

 

 

 

ular
fluid volume
Arterial —9
pressure L $
if MAP
Blood
Total G—- I' I i n volume
peripheral
resistance f $
' Mean circulatory
Cardiac (I T Venous T ' .
T output return :I fllllng pressure

 

 

 

 

 

 

i

 

I Cardiac inotropy

 

 

 

 

 

 

T

 

[ Venoconstriction l

 

 

‘ I Vascular capacity lO—-—J

 

Flgure 5. Guyton-Coleman diagram of physiological factors that affect pressure

natriuresis and control

blood pressure, demonstrating how increased

venoconstriction can drive increases in cardiac output to ultimately increase

arterial pressure. MAP, mean arterial pressure; UNaV, urinary sodium excretion.

hypertension is a risk factor for cardiovascular disease and cardiovascular
disease is the number one cause of death in the United States, clearly a better
understanding of this disease and more effective treatments for hypertension are

needed.

C. Role of veins in blood pressure regulation

The majority of the studies to date on how vascular smooth muscle tone and
structure affect blood pressure have been performed in arteries, as they are
primarily responsible for controlling total peripheral resistance. As mentioned
previously, blood pressure can be increased by either increasing blood volume or
by increasing vascular resistance by vasoconstriction (narrowing of the arterial
lumen diameter) (Boron and Boulpaep, 2003). However, the mechanisms by
which chronically elevated blood pressure, I.e. hypertension, develop are more
complex than simply increasing blood volume or arterial vasoconstriction. During
the development of human essential hypertension, cardiac output increases but
not total blood volume. The increase in cardiac output is attributable to increased
effective blood volume or the blood volume relative to vascular capacitance, a
measure of venous compliance (or contractility) (Safar et al, 1975). Reduced
vascular capacitance or increased venous tone (decreased venous compliance)
is observed in some animal models of hypertension as well as human
hypertension (Ricksten et al, 1981). Specifically, in mineralocorticoid
hypertension, mean circulatory filling pressure (MCFP), an in vivo measure of

venomotor tone, is elevated suggesting that venous capacitance is reduced

10

(venous contractility is increased) in hypertension (Johnson et al, 2001; Palacios
et al, 1997). MCFP is measured when the heart is momentarily stopped and
pressure throughout the circulatory system equilibrates. The two physiological
determinants of MCFP are venous capacitance and blood volume, such that
either decreases in venous capacitance or increases in blood volume lead to
increases in MCFP. In human hypertension (Safar et al, 1975) and some animal
models of hypertension (Ricksten et al, 1981), total blood volume is not
increased, suggesting that the measured increase in MCFP is not driven by
changes in blood volume, but changes in venous tone. In hypertension, the
cause(s) of decreased vascular capacitance are still under investigation
(Johnson et al, 2001; Palacios et al, 1997). I contend that veins play an
important role in raising blood pressure by causing a shift of blood volume
from the abdominal cavity into the thoracic cavity, increasing venous

return to the heart and increasing cardiac output (Figure 5).

D. Humoral control of blood pressure by circulating peptides

The regulation of blood pressure is complex as multiple physiological processes
are involved, including the autonomic nervous system, the kidneys and
circulating hormones. These physiological processes do not operate
independently, but instead modulate each other’s activity and act in concert to
maintain blood pressure homeostasis. Circulating peptides involved in blood
pressure regulation include vasopressin, neuropeptide Y, angiotensin II,

bradykinin, the natriuretic peptides and endothelin-1 (ET-1). Vasopressin,

11

neuropeptide Y, angiotension II and ET-1 are all vasoconstrictors in mammals
and elevate blood pressure by increasing total peripheral resistance, while
bradykinin and the natriuretic peptides are vasodilators. However, these
peptides can also influence sympathetic nervous system activity and sodium and
water retention in the kidney. Synthesis and degradation of the various peptides
involved in blood pressure regulation are tightly controlled as these systems are
physiological compensatory mechanisms designed to maintain constant blood
pressure (Boron and Boulpaep, 2003). Likewise, dysfunction in the biosynthetic
pathways of any of these vasoactive peptides can cause significant fluctuations
in blood pressure. A number of cardiovascular diseases, including hypertension,
stroke and atherosclerosis involve alterations in the synthesis of vasoactive
hormones, thus understanding the pharmacological and physiological function
and regulation of these systems is paramount to understanding the

pathophysiology and treatment of these diseases.

E. Endothelin

Endothelin -1 (ET-1) is a potent endogenous vasoconstrictor and is a peptide in
the endothelin family of vasoconstrictor peptides discovered by Yanagisawa and
co-workers in 1988. The three endothelin isoforrns, encoded by separate genes,
- ET-1, ET-2 and ET-3 — vary in amino acid sequence and length. Endothelin
exerts its physiological response via binding to receptors, of which there are two,
the endothelin A (ETA) and the endothelin B (ETB) receptor; both ETA and El];

receptors are G-protein coupled receptors (GPCRs). The endothelin isoforrns

12

vary in their affinity for the endothelin receptors; ET-1 has higher affinity for ETA
receptors than ET-3, while ET-1 and ET-3 have similar affinities for the ETB
receptor (Kedzierski and Yanagisawa, 2001; Decker and Brok, 1998; Masaki,
2004). ET-1, ET-2 and ET-3 are present in plasma, but ET-1 is the only
endothelin found in the vasculature (Kedzierski and Yanagisawa, 2001). ET-1
binds both ETA and ETB receptors with similar affinities (Kr approximately 20 and
15 pM, respectively) (Lee et al, 1998). Another family of peptides, the
sarafotoxins 86b and 86c, isolated from asp (Atractaspis sp. of snake) venom
are structurally similar to the endothelins and are agonists at endothelin

receptors (Kloog and Sokolovsky, 1989; D'Orleans-Juste et al, 2003).

1. ET-1 synthesis and degradation
Biosynthesis of ET-1 initiates with transcription of the ET-1 gene that encodes for
preproET-1, a 212 amino acid peptide (Figure 6). PreproET-1 is cleaved to
proET-1 by a furin-like peptidase, and then proET-1 is cleaved to big ET-1 by a
subtilisin-like convertase. Big ET—1, the precursor to vasoactive ET-1, is a
substrate for the protease endothelin converting enzyme (ECE) (D’Orleans-Juste
et al, 2002). ECE, a metalloendoprotease that is structurally related to neutral
endopeptidase (NEP), occurs in three isoforrns: ECE-1, ECE-2 and EOE-3, of
which EOE-1 is predominantly physiologically relevant as EOE-3 specifically
forms ET-3 and knockout of EOE-2 has no developmental effects (D’Orleans-

Juste et al, 2003). There are four ECE—1 isoforrns: EOE-1a, ECE-1b, ECE-1c

13

ET-1 mRNA

I

Pre-proET-1
(212 amino acids)

*MMET-

1*

Big ET-1
(38 amino acids)

are all: as

(21 amino 1acids) *WETJU'M] ET-1[1-32]

CONTRACTION

Flgure 6. Diagram of endothelin-1 (ET-1) biosynthesis. Multiple enzymes are
capable of processing big ET-1 into different vasoactive endothelin peptides,
though ECE cleavage of big ET-1 is the classical pathway for ET-1 synthesis/big
ET-1 metabolism. ECE, endothelin converting enzyme; MMP, matrix

metalloprotease; NEP, neutral endopeptidase.

14

and ECE-1d due to four different promoters on a single gene (Yanagisawa ef al,
2000).The different ECE-1 isoforrn expression is due to alternative splicing and
the isoforrns differ in their N-terminal sequences and subcellular locations; ECE-
1a is located in the cytosol, nucleus and plasma membrane, ECE-1 b is located in
the cytoplasm, ECE-1c is located at the plasma membrane and ECE-1d is
present in several subcellular locations (Jafri and Ergul, 2003). ECE cleaves big
ET—1 (39 amino acids long) at the VaI21-Trp22 bond into active ET-1 (21 amino
acids long). Big ET-1 is also a substrate for chymase, which cleaves big ET-1 at
the Tyr31-Gly32 bond to form ET-1[1-31] (Nakano et al, 1997), which is vasoactive
in rat thoracic aorta (Kishi et al, 1998; Watts of al, 2002) and vena cava (Watts et
al, 2002). ET-1[1-31] can be proteolytically cleaved by NEP and/or ECE to form
ET-1 in some tissues (Kloog and Sokolovsky, 1989). In rat aorta,
phosphoramidon, a non-specific ECE/NEP inhibitor does not reduce ET-1[1-31]-
induced contraction, suggesting that in this vessel, ET-1[1-31] is not cleaved to
ET-1 to cause contraction (Kishi et al, 1998). Big ET-1 is also a substrate for
matrix metalloproteinase-2 (MMP-2) (Fernandez-Patron ef al, 1999). MMP-2 is
responsible for the digestion of extracellular matrix proteins including collagen,
laminin, elastin and fibronectin and cleaves big ET—1 at the Gly"“’-Leu33 bond to
form ET -1[1-32]. ET-1[1-32] is vasoactive and contracts rat mesenteric arteries
(Fernandez-Patron et al, 1999). In some tissues, NEP degrades ET-1
(D’Orleans-Juste et al, 2003; Kedzierski and Yanagisawa, 2001), though in rat
thoracic aorta and vena cava, the non-specific ECE/NEP inhibitor

phosphoramidon reduced big ET—1-induced contraction (Figure 7) suggesting

15

 

 

 

 

 

 

A. 100 . Aorta
+VehIcle
-D- Phosphorarnidon (10 (M)
c 75- ("=5
rte
mg
5’:
5.5. 5°“
22
d) :5.
n_ *
S 25‘
0- .
-12 -11 -1O -9 -8 -7 -l5
log big ET-1 [M]
B.
700 Vena Cava
-e-Vehicle
eooe -o-Phosphoramldon (10 pM)
c (N=5)
“1% 5(1)!
2 m
8,13 4004
£38 300
§3 ‘
Q :1.
0'2 MT
100-
o.
-12 -11 -10 -9 -8 -7 -6
logbig ET-1[M]

Flgure 7. Effect of phosphoramidon (10 IIM), an endothelin converting enyzme
inhibitor, on big ET-t-induced contraction of rat thoracic aorta (A) and vena cava

(B). Points represent means a: SEM for the number of animals (N) indicated in

parenthesis.

statistically significant differe

nce from Vehicle (p<0.05).

16

NE, norepinephrine; PE, phenylephrine. Asterisks represent a

that ECE and possible NEP are primarily involved in cleavage of big ET-1 into
vasoactive ET-1. Thus, the synthesis of ET-1 relies on several proteolytic
enzymes of which the activity and presence may differ between arteries and

veins.

F. ET-1 signal transduction
1. ETA and ETa receptors are G-protein coupled receptors

The ETA and ETB receptors are G-protein coupled receptors (GPCRs). GPCRs
couple to heterotrimeric G-proteins comprised of an a subunit and a By subunit
that are tightly bound together. The a subunit of the heterotrimeric G-protein
binds guanine nucleotides such as GDP and GTP and has intrinsic GTPase
activity. Ligand binding to the GPCR induces a conformational change in the
receptor and a subunit, allowing the active at subunit to hydrolyze GDP for GTP
and dissociate from the By subunit. Conventionally, the GTP-bound a subunit is
the primary mediator of GPCR signaling, but the dissociated By subunit is also
capable of activating downstream signaling cascades and eliciting cellular
responses. ET-1 activates multiple signaling pathways, the activation of which
may account for the multitude of physiological responses mediated by ET-1 in
vascular smooth muscle cells including, but not limited to vasoconstriction,
mitogenesis, Na“/H+ exchange, and anti-apoptosis (Kitamura et al, 1999; Decker
and Brok, 1998). The myriad of signaling pathways activated by ET-1 include,

but again are not limited to, phospholipase C B, phospholipase A2, phospholipase

17

 

  

Ii
A -

 

 

 

 

Gun 6012/13 Gui/Gas (36‘!
l I I \
PLCfi RhoA AC ERK,
I N I I ”Sim
IP3 DAG Rho Kinase PKA
\ J
V
CONTRACTION

Flgure 8. Diagram of endothelin signal transduction via ET, and ET B receptors.
AC, adenylate cyclase; DAG, diacyl glycerol; Erk, extracellular regulated kinase;
lPa, inositol 1,4,5-triphosphate; MAPK, mitogen activated kinase; Pl3-K,
phosphatidyl inositol 3-kinase; PLC, phospholipase C; PKA, protein kinase A;

PKC, protein kinase C.

18

D, the MAP kinases (ERK 1/2, JNK and p38), rho kinase, CAMP-mediated
signaling and tyrosine kinase-mediated signaling (specifically Src signaling)
(Kitamura ef al, 1999; Decker and Brok, 1998) (Figure 8).
2. ET, and ET; receptor signal transduction

The ETA and ETB receptors are capable of coupling to several G-proteins to
activate a number of signaling cascades (Figure 9, 10). The primary mechanism
for vascular ET-1-induced contraction is most likely via ETA and ETB receptors
coupling to Gag, a G-protein that activates phospholipase C B (PLCB). PLCB
hydrolyzes phosphatidylinositol 4,5-biosphophate (Ple) into inositol 1,4,5-
triphosphate (IP3) and sn1,2—diacylglycerol (DAG). IP3 binds to IP3 receptors in
the sarcoplasmic reticulum to cause release of intracellular calcium stores, while
DAG activates protein kinase C (PKC). ET-1 typically causes a biphasic
increase in intracellular calcium; the initial transient rise in intracellular calcium is
ng-mediated calcium release from intracellular stores, while the second
sustained rise in intracellular calcium occurs via PKG-mediated influx of

extracellular calcium (Kitamura et al, 1999).

When ETA and ETB receptors are over-expressed in cell lines, the receptors can
also couple to Go. and Gas, G-proteins that inactivate and active adenylate
cyclase, respectively. When human ET, and ETB receptors are expressed in
Chinese hamster ovary (CHO) cells, ET A receptors couple to Go... while ETB
receptors couple to Get. (Aramori and Nakanishi, 1992; Takagi et al, 1995). Get,3

and Go. increase and inhibit adenylate cyclase, respectively, affecting cAMP

19

ET, receptor:

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

NADPH
Gus Gag Gatzm oxidase
I
f AC 1' \ RIP" I

I CA1”? I \ Rho Kinase 1
IP DAG '
3 (“i
i PKA l l Myosin light H202

Caz. “fleas, “om PKG chaln phosphatase
H Intracellular stores

Influx of Vascular SI'I'IOOU'I

Vascular smooth
muscle growth

muscle growth extracellular Cal"

  

Vascular smooth muscle contraction<

W

Figure 9. Diagram of ET, receptor signal transduction. AC, adenylate cyclase;

 

cAMP, cyclic adenosine monophosphate; H202, hydrogen peroxide; IP3, inositol
triphosphate; 02’, superoxide anion; PLC, phospholipase C, PKA, protein kinase

A; (-), inhibition; 1, activation.

20

ETl3 receptor:

 

 

 

 

 

 

Got, 657 631;;13I
I \ fa \. l

I A? / \ chg RhoA

Ia...» Ep'E’SZMi'I’é Pram / \ i

Rho Kinase
I I ii=3 DAG

I P z l 1 H
K\ PKC
Cal: release from Myosin llght

. l, Intracellular stores 1 chain phosphatase

 

 

 

 

 

 

 

 

Vascular smooth Influx of
muscle growth extracellular Ca”

1, /
W
T" Z

> Vascular smooth muscle contraction <

 

 

Flgure 10. Diagram of ET a receptor signal transduction. AC, adenylate cyclase;

cAMP, cyclic adenosine monophosphate; DAG, diacylglycerol; ERK, extracellular

regulated kinase; H202, hydrogen peroxide; lPa, inositol triphosphate; JNK, c-Jun

N-terminal kinase; MAPK, mitogen activated kinase; 02‘, superoxide anion; PLC,

phospholipase C, PKA, protein kinase A; PKB, protein kinase B, PKC, protein

kinase c; (-), inhibition; 1. activation.

21

production and protein kinase A (PKA) activity. PKA activity in vascular smooth
muscle cells inhibits mitogenesis via inhibition of the ERK pathway and induces
vasodilation (Bornfeldt and Krebs, 1999). Thus, predicting PKA-mediated
responses depends on tissue expression of ET, and ETB receptor and receptor-
mediated G-protein coupling, factors which have not yet been determined in

blood vessels or smooth muscle cells.

Another class of G-proteins activated by ET—1 is the 612113 family. In human
embryonic kidney (HEK)—293 cells, over-expressed ETB receptors couple to Gaia
(Kitamura et al, 1999) while in embryonic mouse fibroblasts microinjected with
plasmids encoding for the human ETA receptor, ETA receptors couple to 6012
(Gohla ef al, 1999). Intestinal smooth muscle cell ETA receptors couple to (3013
(Hersch et al, 2004), thus receptor-coupling to either Gan or Gaia may be cell-
specific as different cell types may have different complements of G-proteins.
60.12/13 signaling mediates a number of other physiological responses including
RhoA activation, regulating Na‘IH“ exchanger (NHE) activity, extracellular
regulated kinase (ERK) activity, c-Jun N-terminal kinase (JNK) activity and
nuclear factor IcB (NFIcB) activity (Kitamura et al, 1999; Gohla et al, 2000).
RhoA is a monomeric G-protein that activates rho kinase, a kinase that
phosphorylates and inactivates myosin light chain phosphatase (MYPT),
increasing calcium sensitivity and allowing smooth muscle contraction to occur at
concentrations of intracellular calcium that are typically sub-threshold for eliciting

contraction (Somlyo and Somlyc, 2003). RhoA can be activated by a number of

22

G-proteins including Gotq and Gan/13 (Gohla et al, 2000). Contractility studies
using isolated blood vessels suggest that vascular smooth muscle contraction to
ET-1 is partially mediated via RhoA and activation of the rho kinase pathway
(Hersch et al, 2004; Miao ef al, 2002). It is likely that ET A and ETB receptor
activation of the rho kinase pathway can occur via coupling to Getq and Gong/13.
Interestingly, cAMP and cGMP appear to inhibit the rho kinase pathway (Gohla et
al, 2000; Dong et al, 1998). Thus, ET-1 activation of rho kinase may occur
through several mechanisms, including direct activation of Gag by both ETA and
ETB receptors, direct activation of 6012/13 by ETA and ETB receptors, or indirectly
through ETB receptors, which via coupling to Go. decrease cAMP production.
However, in smooth muscle cells with functional ET, receptors, ETA receptor
coupling to Gas may reduce ETB receptor-mediated increases in rho kinase

activity.

The a subunits of G-proteins are not the only subunit responsible for GPCR
signaling; the dissociated By dimer is also involved in ET-1 signaling. The
cytoplasmic tail of the ETB receptor is involved in ET-1-induced activation of the
ERK1/2, JNK and p38 MAP kinases, likely through By signaling (Aquilla ef al,
1996). As mentioned previously, ETB receptors activate Goo, causing the a and
By subunits to dissociate. While the Ga. subunit decreases adenylate cyclase
activity, decreasing cAMP levels and PKA activity, the dissociated By subunit is
also capable of acting as a signaling molecule and can activate the protein

kinase B (PKB)IAkt pathway in sinusoidal endothelial cells (Liu ef al, 2003). ET-1

23

activation of the PKB/Akt pathway in rabbit basilar artery is coupled to ET-1-
induced contraction likely via activation of a MAPK pathway (Zubkov et al, 2000).
ETa receptor-mediated Got. signaling can indirectly activate PLCB via the By
subunit dimer (Decker and Brok, 1998). There are multiple signaling pathways
that ETA and ETB receptors activate, thus there is significant potential for
biochemical interactions of these signaling pathways in tissue populations with

both ETA and ETB receptors.

3. Reactive oxygen species signaling
Reactive oxygen species (ROS) such as superoxide anion (02') and hydrogen
peroxide (H202) are formed by EM activation of El', receptors in smooth muscle
cells (Li ef al, 2003a; Wedgwood ef al, 2001) and ET, receptors in endothelial
cells (Duerrschmidt at al, 2000). El', receptors on smooth muscle cells of
arteries and veins activate NADPH oxidase, an enzyme that generates 02'.
Superoxide is catalytically broken down into H202 via the action of superoxide
dismutase (SOD) of which there are 3: Cu/ZnSOD (extracellular and cytosolic)
and MnSOD (mitochondrial). H202 is broken down by the enzymes catalase and
glutathione peroxidase to form H20 and 0,. Both O,‘ and H202 can function as
signaling molecules and can induce vasoconstriction (02' and H202), vasodilation
(H202), and mitogenesis (02' and H202) (Wolin at al, 2001 ). Superoxide and H202
activate a number of signaling pathways that ET-1 activates and these ROS may
mediate a number of the physiological affects of ET-1. ET-1 administered

chronically to rats increased blood pressure by 18-20 mm Hg and increased renal

24

vascular resistance; tempol, a superoxide dismutase mimetic, reversed the
effects of ET-1 on both blood pressure and renal vascular resistance (Sedeek ef
al, 2003). These results suggest that the overall function of ET-t-stimulated ROS

is vasoconstrictive.

Li et al and other groups (Li et al, 2003a; Li et al, 2003b; Callera at al, 2003)
observed that ET-1 increases aortic and venous 0,; via NADPH oxidase and that
the ET, receptor mediates activation of NADPH oxidase. In DOCA-salt
hypertension, where aortic tissue ET-1 is elevated, O,‘ levels are elevated and
decreasing oxidative stress reduces blood pressure and improves hypertension-
related tissue damage such as endothelial cell damage. Guzik et al observed no
differences in basal superoxide levels in human saphenous vein and internal
mammary artery (Guzik at al, 2004). Interestingly, the source of 02' varied
between human arteries and veins. In human saphenous vein NAPDH oxidase
was the primary source for 02' generation, while the xanthinelxanthine oxidase
system generated 02' in human internal mammary arteries (Guzik at al, 2004). It
is possible that ET-1 may differentially affect the ROS production and

downstream signaling events in arteries and veins.

G. Distribution of ET A and ET; receptors in blood vessels
Both ETA and ETB receptors are present on vascular smooth muscle cells of
blood vessels (Figure 11, 12) and couple to smooth muscle contraction (Figure

13), while ETB receptors on endothelial cells couple to nitric oxide release.

25

ETA ET,

#I

120 kDa —> l». y . dimer?

 

 

dimer? ~ —-
--—-

--- 45 kDa _.
[:53 30 kDa _.

1o 10 + Competing

. a...
peptide

10 10 + Competing
peptide

Flgure 1 1 . El', and ET, receptor Western blot analysis in rat thoracic vena cava.
Proteins from venous lysates were separated using SDS-PAGE, and transferred
to PVDF. Membranes were probed with primary antibodies (1°) directed to ET,
(left lanes) and ET, receptors (right lanes) or primary antibody neutralized with
competing peptide (1° + competing peptide). Bands that are lighter in the
presence of competing peptide represent specific primary antibody binding. The

expected molecular weight of both the ET, and ET, receptor is around 45 kDA.

26

Aorta Vena cava

    

<
.2.
I—
Z

ET, Receptor

ADVENTITIA

\ i.“ .0
. I
ire ’5

    

Flgure 12. ET, and ET, receptor immunohistochemistry in rat thoracic aorta
(left) and vena cava (right). Tissues are oriented with adventitia on the left and
intima on the right. Black precipitate, as indicated by arrows, represents specific

primary antibody binding.

27

Aorta

.3"
§

 

(N=5-6)

is

   
  

Percentage PE/NE
(10 pM) Contraction
.8

 

  

 

 

Q-

-12 -11 -10 5 -i3
logET-t [M]

-l

U)

Aorta

F”

A
v

 

(Nfi)

Percentage PEINE
(10 pM) Connection

 

Ou—W

-12 -11 -1o -9 -s -7
IOQSGCIMI

 

 

 

 

 

 

 

 

 

700 Vena Cava
coo (N=5-6)

500.

400.

300.

200..

100..

0‘ I T U I
-12 -11 -1o -9 -8 -7 -45
IogET-1[M]

Vena Cava

7.5
(N=4)
501
25.
o. , ,
-12 -11 -10 -9 -e -7

I09 360 [Ml

Flgure 13. ET -1 (A) and 860 (B) cumulative concentration response curves in

rat thoracic aorta and vena cava. Points represent mean :1: SEM for the number

(N) of animals in parenthesis. ET-1, endothelin-1; NE, norepinephrine; PE,

phenylephrine; S6c, sarafotoxin 6c.

28

Endothelial ETB receptors also function as a clearance mechanism to remove
plasma ET-1 (Bohm et al, 2003). Endothelin receptors are present in a number
of other non-vascular tissues including renal collecting duct cells,
cardiomyocytes, hepatocytes, neurons, osteoblasts, keratinocytes and
adipocytes (Kedzierksi and Yanagisawa, 2001). On blood vessels, receptor
expression and coupling to contraction is dependent on the vascular bed and
species studied. ET, and ETB receptors that both participate in contraction are
present in rabbit jugular vein (Lodge ef al, 1995), rabbit saphenous vein (Lodge
at al, 1995), rat thoracic vena cava (Watts et al, 2002), rat superior mesenteric
vein (Claing et al, 2002), rat small mesenteric arteries (Adner ef al, 2001) and
hamster thoracic aorta (Lodge at al, 1995), while in other blood vessels such as
rat thoracic aorta (Watts of al, 2002; Lodge et al, 1995), rabbit carotid artery
(Lodge et al, 1995) and rat superior mesenteric artery (Claing et al, 2002) only
the ET, receptor mediates ET-1-induced contraction. A few of studies have
suggested that contractile ETB receptors are preferentially localized to veins,
while ET-1-induced contraction is mediated by ET, receptors on arteries
(Ekelund et al, 1994; Moreland et al, 1994). A recent study performed in human
forearm veins suggests that contractile ETB receptors on veins are an important
overlooked mechanism for increasing venous return and increasing blood ‘

pressure (Mitchell et al, 2004).

The presence of both ET, and ETB receptors on rat thoracic aorta and vena cava

has been confirmed using Western blot analysis (Watts of al, 2002; Figure 11)

29

and immunohistochemistry (Thakali et al, 2004; Figure 12). Both rat thoracic
aorta and vena cava contract to ET-1, a non-selective ET, and ETB receptor
agonist, while vena cava but not aorta contract to 86c, an ETa receptor agonist
(Figure 13). Why ETB receptors are present in rat thoracic aorta, but are not
coupled to contraction is not understood, but it is also possible that smooth
muscle contraction is not the correct endpoint for assessing ETB receptor activity

in rat thoracic aorta.

H. ET, and El'a receptor function in hypertension

The endothelin receptors present on vascular smooth muscle cells of arteries
and veins are important in blood pressure regulation. Systemic administration of
endothelin receptor antagonists reduce blood pressure in healthy, normotensive
people, suggesting that ET—1 is normally involved in control of vascular tone
(Haynes et al, 1996; Verhaar et al, 2000). The endothelin system also plays a
role in both human hypertension and several animal models of hypertension.
African American hypertensive patients have significantly higher plasma ET-1
levels than their normotensive counterparts and Caucasian hypertensive patients
(Grubbs et al, 2002). In DOCA-salt hypertension, aortic but not venous tissue
levels of ET-1 are elevated, and aorta but not vena cava from DOCA-salt
hypertensive rats exhibit reduced contraction to ET-1 (Watts of al, 2002). Recent
studies suggest that the ET, receptor is down regulated in human and animal
models of hypertension, leading to impaired ET-1 mediated arterial contraction,

while ETB receptor expression is not reduced (Reinhart et al, 2002; Telemaque—

3O

Potts et al, 2002). Giulumian et al reported that decreased Ca2+ influx, but not
reduced ET-1 binding to its receptors, could explain impaired contraction to ET-1
in the small coronary arteries of DOCA-salt rats, suggesting an uncoupling of the
ET-1 signaling mechanism (Giulumian ef al, 2002). Lodge et al (1995) and
Moreland et al (1996) suggest that ET, receptors mediate contraction on most
arteries and ET; receptors mediate contraction on most veins. Mitchell ef al
report that hand vein contraction to ET-1 is not reduced by an orally administered
ET, receptor antagonist (Mitchell ef al, 2004). Lack of arterial responsiveness to
ET-1 under high ET-1 conditions suggests that vasoconstrictive ET; receptors
present on veins may play an underestimated and underappreciated role in

regulating blood pressure.

I. ET, and ET; receptor “cross-talk” or interaction

As mentioned previously, there are vascular beds where both ET, and ET;
receptors mediate ET-1-induced contraction. Several reports also suggest
“cross-talk” between ET, and ET; receptors occurs, meaning that activation of
one receptor subtype alters the function of the other receptor subtype. In rabbit
jugular and saphenous veins and hamster aorta (vessels that have contractile
ET; receptors), ET-1-induced contraction is not reduced by ET, receptor
antagonism. However, when the ET; receptors on these vessels were
desensitized using S6c, an ET; selective agonist, ET-I-induced contraction was
sensitive to ET, receptor blockade. Interestingly, in rat aorta and rabbit carotid

artery (vessels that do not contract to ET; agonists), ET; receptor desensitization

31

using S6c did not alter ET, receptor blockade of ET-1-induced contraction
(Lodge at al, 1995). Lodge et al suggest that in rabbit jugular and saphenous
vein, ET-1 elicits contraction via both ET, and ET; receptors, but that under
normal conditions, ET, receptors are under receptor blockade by ET; receptors.
When ET; receptors are desensitized, ET, receptors are released from receptor
blockade and are able to participate fully in ET-1-induced contraction. ET, and
ET; receptor interaction has also been studied in the small mesenteric arteries
from rats, in which both ET, and ET; receptors are contractile (Adner et al,
2001), unlike the superior mesenteric artery from rat in which only ET, receptors
are responsible for ET-1-induced contraction (Lodge at al, 1995). In small
mesenteric arteries, desensitization of ET; receptors using 86c increased the
potency of ET-1 and the apparent affinity of FR139317, an ET, receptor
antagonist (Adner et al, 2001). Endothelin receptor interaction has also been
observed in mouse mesenteric veins but not arteries (Perez-Rivera et al, 2005),
renal afferent but not efferent arterioles (lnscho ef al, 2005), and pulmonary
arteries (Sauvageau et al, 2006). Thus, in tissues with mixed functional
endothelin receptor populations, pharmacological data suggests that ET;
receptors normally function to antagonize ET, receptors, but ET; receptor
desensitization releases ET, receptors from receptor blockade. Whether this
interaction between ET, and ET; receptors occurs at a physical or signaling level

is not known.

32

One possible explanation for differences between aortic and venous responses
to ET-1 is differences in G-protein complement in arteries and veins. To our
knowledge, a study comparing G-protein complement in arteries and veins has
not been performed to date. Adams et al compared gene expression of 4048
genes in macaque aorta and vena cava and observed that mRNA expression
was elevated in 68 out of 4048 genes in aorta compared to vena cava, with the
largest difference in mRNA expression occurring in a regulator of G-protein
signaling (RGS) 5 (Adams et al, 2000). Northern blot analysis suggests that a
similar pattern of increased RGS expression in aorta compared to vena cava
occurs in the rat (Adams of al, 2000). RGS proteins are GTPase-activating
proteins and function to inhibit GPCR signaling; specifically, RGSS binds and
inhibits Ga. and Gaq-coupled signaling (Zhou et al, 2001), two G-proteins
involved in ET-1 signaling. Over-expressed RGS5 inhibits ET-1-induced Ca2+
transients in HEK-293T cells, suggesting that RG85 can interfere with ET-1
signaling (Zhou et al, 2001). Higher RGSS expression in rat thoracic aorta
compared to vena cava may inhibit ET-1 signal transduction and specifically may

mask aortic ET; receptor-mediated signaling.

J. Receptor dimerization

1. Physical means for receptor dimerization
Conventionally, GPCR signaling is thought of as a ligand binding to its receptor
and the receptor coupling to a G-protein in a 1:1 ratio, causing a specific

response through effector molecules. However, recent investigations suggest

33

that a number of GPCRs are capable of homo— and heterodimerization (a
receptor dimerizing with an identical receptor or dimerizing with a different
receptor, respectively), altering the pharmacology of these receptors.
Heptahelical GPCRs, such as glutamate (Romano et al, 2001), angiotensin
(AbdAIla et al, 2001), B2 adrenergic (Hebert et al, 1996), and GABA; receptors
can form homo— and heterodimers (White et al, 1998). Dimerization has been
proposed to occur through N-terminal disulfide bonds such as in the case of the
Ca2+ sensing receptor (Goldsmith et al, 1999; Pace et al, 1999) and the
metabotropic glutamate receptor (Romano et al, 2001). Disulfide bonds in
residues cys101 and cys263 allow Ca2+ sensing receptor homodimerization, and
disruption of these disulflde bonds decreases agonist affinity for the receptors
and slows the kinetics of receptor activation and inactivation (Goldsmith ef al,
1999; Pace et al, 1999). Dimerization can also occur through interactions in the
C-terminus of receptors, as is the case for 6 opioid receptor homodimerization
(Cvejic and Devi, 1997). Coiled-coil interactions through leucine-zipper motifs
have also been proposed to facilitate receptor dimerization, such as in the case
of the GABA; receptor (Kammerer et al, 1999). The final mechanism by which
dimerization can occur is through transmembrane interactions as occurs with the
DZ dopamine receptor. Using truncated DZ receptor mutants in COS-7 and Sf9
cells, Lee et a! determined that for DZ dopamine receptors, transmembrane 4

was critical for receptor dimerization (Lee et al, 2003).

Z. Caveolae as cellular locations for receptor dimerization

Caveolae play an important role in receptor trafficking as internalization of
caveolae is inducible. The caveolin proteins (-1,2, and 3) function as scaffolding
proteins in the vasculature, caveolin-3 is muscle specific, while caveolin-1 (the
predominant caveolin) is required for caveolae formation and internalization.
Tyrosine phosphorylation of caveolin-1 by c-src is the signal required for
caveolin-1 to induce caveolae internalization (Stan, 2002). ET-1 activates a
number of signaling molecules, including c-src (Kodama ef al, 2003), thus if ET
receptors are localized to caveolae in aorta and vena cava, ET-1 could
potentially mediate receptor internalization into caveolae. Caveolin-1 may also
interact with filamin, an actin binding protein, suggesting that caveolae can

potentially affect smooth muscle contractility (Dreja et al, 2002).

Dreja et al have performed a number of experiments investigating the role of
caveolae in 5-hydroxytryptamine (5-HT)—induced contraction in rat tail artery.
They observed that 5-HT-induced contraction is dependent on caveolae as
cholesterol depletion (which depletes smooth muscle cell membrane caveolae
verified by electron microscopy) inhibits 5-HT-induced contraction. In the tail
artery, cholesterol depletion also inhibits ET-1-induced contraction (Dreja et al,
2002). Thus, in rat thoracic aorta and vena cava, caveolae (Figure 14) may
represent a cellular location for ET,/ET; receptor dimerization as well as a
mechanism for 1 receptor internalization and desensitization under ET-1-

stimulating conditions.

35

 

Flgure 14. Electron micrographs of caveolae (invaginations indicated by black

arrows) present on the plasma membrane of aortic and venous vascular smooth

muscle cells.

3. Functional consequences of receptor dimerization
A number of studies suggest that several receptor characteristics such as agonist
affinity, potency and efficacy as well as receptor internalization are affected by
receptor dimerization. In the case of GABA; receptor dimerization,
heterodimerization between the GABA;R1 and GABA;R2 receptors increases
agonist affinity 10-fold, as was observed by inhibition of radiolabeled GABA;
agonist binding by GABA and baclofen (Kaupmann et al, 1998). Agonist affinity
is not always affected by receptor dimerization - heterodimerization between the
B2 adrenergic receptor and the 6 and 1c opioid receptors does not affect ligand
binding, as ligand binding affinity for each heterodimer pair was similar to ligand
binding affinities in preparations with each receptor alone (Jordan et al, 2001),
suggesting that functional consequences of receptor dimerization vary for each
pairing. While ligand binding properties are unaffected by heterodimerization
between the B2 adrenergic receptor and the 6 and 1c opioid receptors, receptor
trafficking can vary, depending on the type of heterodimer formed. B2 adrenergic
receptor heterodimerized with the 6 opioid receptor undergoes agonist-mediated
internalization, while the B2/Ic receptor heterodimer does not undergo agonist-
mediated internalization (Jordan at al, 2001). Other receptor characteristics that
are potentially modifiable by receptor dimerization include agonist potency and
efficacy. With heterodimerization of the 6 and 1c opioid receptors, a synergistic
effect of dual agonist (DPDPE and U69593, 6 and 1c opioid receptor agonists,
respectively) binding leading to increased effector function was observed (Jordan

and Devi, 1999). When It and 6 heterodimers were expressed, 6 opioid receptor

37

antagonism increased potency and efficacy of II opioid receptor signaling, and
vice versa (Gomes et al, 2000). To date, much of work published on receptor
dimerization has occurred in cell lines in which receptors were artificially
expressed. Whether receptor dimerization functionally affects tissues - in

this case, ET receptors in arteries and veins - is an important question.

4. Can ET, and ET; receptor heterodimerization occur and can

heterodimerization affect ET receptor function?
Gregan et al demonstrated that ET, and ET; receptors heterodimerize in HEK-
293 cells over-expressing human ET, and ET; receptors, results that have since
been confirmed by Del and Galligan (2006). Short or prolonged incubation with
the non-specific agonist ET-1 had no effect on the ET,/ET; receptor heterodimer,
suggesting that heterodimer formation was constituitive. Interestingly, prolonged
incubation with an ET; receptor-selective agonist disrupted the ET,/ET; receptor
heterodimer, suggesting that with selective ET; receptor activation, the
heterodimer dissociates and ET; receptor internalization occurs (Gregan ef al,
2004). This study represents an excellent proof-of-principle study - ET, and ET;
receptors can heterodimerize. However, heterodimerization was assessed in a
highly artificial system and functional consequences of tissue ET, and ET;
receptor heterodimerization cannot be deduced from these over-expression
studies. Radioligand binding studies of ET, and ET; receptors in the rat anterior
pituitary gland demonstrated that ET; receptor agonists and antagonists did not

compete for K125’ET-1 binding unless ET, receptors were bound by an ET,

38

receptor antagonist. Thus, the authors suggest two models of ET, and ET;
receptor heterodimerization that explain their data. In the first model ET-1 acts
as a molecular bridge between the ET, and ET; receptors, inducing receptor
heterodimerization and in the second model ET, and ET; receptors
constituitively heterodimerize (Harada ef al, 2002). But like the proof-of-principle
study performed in HEK-293 cells, data from these radioligand binding studies
provide no information on the functional consequences of ET, and ET; receptor

heterodimerization in arteries and veins.

There is a wealth of data that highlights the importance of the endothelin system
in several animal models of hypertension and hypertension in humans, and
accordingly the endothelin system has been pursued as a potential drug target
for lowering blood pressure. However, if the endothelin receptors dimerize, this
could alter the pharmacology of each receptor and also alter the response to
drugs designed to interfere with the system. Thus, by understanding how ET
receptors interact, whether physically, pharmacologically or via biochemical
signaling, and determining if this interaction occurs in a similar fashion in arteries
and veins, rational drug therapies can be devised for treating endothelin-

dependent hypertension.

5. Significance of ET, and ET; receptor dimerization

Receptor dimerization is a fascinating field in pharmacology that could potentially

change the way tissues with different receptor populations are typically

39

characterized. This dissertation represents a foray into a phenomenon that has
not been highly described in whole tissue, as most of the work involving receptor
dimerization has been performed in cell lines over-expressing receptors of
interest. Thus, there are many questions as to how and if receptor dimerization
affects tissue or system function. In a number of human diseases, such as
systemic hypertension, pulmonary hypertension, coronary atherosclerosis,
myocardial ischemia and reperfusion and myocardial infarction, ET-1 plays a
pathogenic role. Understanding how the ET, and ET; receptors function
normally in blood vessels and under conditions of elevated ET-1 may provide
useful information on the treatment of such diseases and point here to an under-

appreciated role of the venous system in blood pressure regulation.

40

K. HYPOTHESIS

Veins maintain responsiveness while arteries lose responsiveness to the
vasoactive hormone endothelin-1 (ET-1) in situations of exposure to ET -1 (eg.
hypertension, experimental protocols). Thus, receptors for ET-1 - the
heptahelical ET, and ET; receptors — may function differently in arteries and
veins. While heptahelical receptors canonically interact with G-proteins in a 1:1
ratio, recent evidence suggests that receptor dimerization (hetero- or homo-) may
occur. Receptor dimerization can potentially affect a number of receptor
characteristics such as agonist affinity, potency and efficacy, as well as receptor
internalization. Aligned sequence analysis suggests that the ET, and ET; can
interact through N-terminal residues. ET, and ET; receptors are present on the
smooth muscle of arteries and veins and previous studies suggest that activated
ET; receptors are capable of physically or pharmacologically uncoupling ET,
receptors from receptor blockade. I hypothesized that ET, and ET; receptors
physically interact via recepter heterodimerization in veins but not arteries and
this heterodimerization functionally affects ET, and ET; receptor pharmacology.

To test this hypothesis I designed three subhypotheses:

Subhypothesis 1: To demonstrate a pharmacological interaction between ET,
and ET; receptors in rat thoracic vena cava but not aorta and to demonstrate that
activated ET; receptors pharmacologically uncouple ET, receptors from receptor

blockade in rat thoracic vena cava.

41

I expect that compared to arteries, veins will not desensitize to ET-1
because veins but not arteries have functional (i.e. contractile) ET;
receptors.

Because veins possess contractile ET; receptors to interact with ET,
receptors, I expect that venous ET-1-induced contraction will be
reduced by either ET, or ET; receptor antagonists and dual ET,/ET;
receptor antagonism will cause a greater inhibition than ET, or ET;

receptor antagonism alone.

Subhypothesis 2: To characterize biochemical signaling interactions between

ET, and ET; receptors in rat thoracic aorta and vena cava.

I hypothesize that reactive oxygen species signaling will differ between
arteries and veins because of the presence of contractile ET;
receptors on veins but not arteries.

I speculate that the presence of contractile ET; receptors on veins but
not arteries will mean that the signal transduction pathways activated
by ET-1 in veins will differ from those activated in arteries and

contribute to functional venous endothelin receptor interaction.

Subhypothesis 3: To demonstrate a physical interaction, Le. heterodimerization,

between ET, and ET; receptors in rat thoracic vena cava but not aorta.

I expect that single smooth muscle cell ET, and ET; receptor

immunocytochemistry of rat thoracic aorta and vena cava will

42

demonstrate that ET, and ET; receptors co-localize on the membrane
of venous vascular smooth muscle cells.

0 I expect that with ET-1 stimulation, venous ET, and ET;
receptors will internalize to a lesser degree than aortic ET,
receptors.

l posit that ET, and ET; receptors will co-immunoprecipitate in rat

thoracic vena cava but not aorta.

43

II. METHODS

A. Isolated smooth muscle contraction

Male Sprague Dawylet rats (225-250 9, Charles River Laboratories, Portage, MI)
were deeply anesthetized (until loss of eyelid reflex and withdrawal from painful
stimuli) with a pentobarbital injection (50 mg/kg, l.p). The thoracic aorta and
vena cava were removed and placed in warmed (37°C), oxygenated (95% O2,
5% CO2) physiological salt solution (PSS) of the following composition: 103 mM
NaCl; 4.7 mM KCI; 1.18 mM KH2PO4; 1.17 mM MgSO4-7H2O; 1.6 mM CaCl2-
ZH2O; 14.9 mM NaHCO;; 5.5 mM dextrose and 0.03 mM CaNa2 EDTA. Arteries
and veins were cleaned of fat and connective tissue and dissected into rings (34
mm) with the endothelial layer left intact. The rings were hung between wire
hooks with one end attached to a stationary glass rod and the other end attached
to a force transducer connected to a Grass polygraph for measurement of
isometric contractile force. Vessels were placed under optimum passive tension
(aorta: 4000 mg, vena cava: 1000 mg). Arteries and veins were equilibrated for
one hour in PSS prior to a wake-up challenge with a maximal concentration of
phenylephrine (PE) (10 (M) or norepinephrine (NE) (10 IIM), respectively. PE
was used to wake-up aorta to compare results to historical data, but was not
used to wake-up vena save as reproducible contractions to PE were unattainable
in our hands. Endothelial integrity was assessed by acetylcholine (1 uM)-
induced relaxation after contraction to PE (10 nM) or NE (10 (M) in aorta and

vena cava, respectively.

1. ET-1 desensitization
Aorta and vena cava were challenged with ET-1 (100 nM) for 15 minutes (when
aortic ET—1-induced contraction plauteaued). Then vessels were washed with
PSS for 90 minutes (5 minutes between washes), during which tone returned to
baseline. Vessels were allowed to rest (no washing) for 30 minutes before
performing either an ET-1 cumulative concentration response curve (10 pM —
100 nM), a norepinephrine concentration response curve (1 nM — 10 nM), or
challenged with a bolus of $60 (100 nM). If antagonists were used (atrasentan,
10 nM; 30-788, 100 nM), they were added during the 30-minute rest period
before performing ET-1 cumulative concentration response curves (10 pM — 100
nM).

2. ET; receptor desensitization
Vena cava were challenged with a bolus of the ET; receptor agonist 86c (100
nM, 15 minutes). Vena cava were washed with PSS for 15 or 30 minutes (5
minutes between washes) and then vena cava challenged again with S6c (100
nM). To investigate ET, receptor function in ET; receptor desensitized aorta and
vena cava, ET; receptors were desensitized with 86c (100 nM) and ET,
receptors blocked with atrasentan (10 nM) for one hour and then cumulative ET-
1 concentration response curves (10 pM - 100 nM) were performed.

3. Receptor antagonism studies
Aorta and vena cava were incubated with vehicle (0.0001% DMSO), ET,

receptor antagonist (atrasentan, 10 nM), ET; receptor antagonist (BO-788, 100

45

nM) or dual ET, and ET; receptor blockade (atrasentan + BQ-788) for one hour
and then ET-1 concentration response curves (10 pM — 100 nM) were performed.
4. H202 concentration response curves
Cumulative concentration response curves to exogenous H202 (1 uM- 1 mM)
were performed in rat thoracic aorta and vena cava. To determine if H202
mediated ET-1-induced contraction, aorta and vena cava were incubated with
catalase (2000 UlmL), PEG-catalase (163 and 326 UlmL), or 3-aminotriazole (50
mM) for one hour before performing cumulative concentration response curves to

ET-1 (10 pM — 100 nM).

B. Measurement of arterlal and venous H202 productlon

H202 production from rat aorta and vena cava was assessed using an Amplex
Red® Hydrogen Peroxide Assay kit (Molecular Probes, Eugene, OR). Three to
four millimeter segments of thoracic aorta and thoracic vena cava were dissected
from rats, cleaned of fat, connective tissue and blood and then incubated in 500
LII. of modified Kreb’s buffer containing (in mM): HEPES, 20; NaCl, 119; KCI, 4.6;
MgSO4 7H20, 1.0; Na,HPO,, 0.15; KH,P0,, 0.4; NaHCO,, 5, CaCl, 1.2;
dextrose, 5.5) at 37°C for one hour. Antagonists or vehicle were incubated
during the one hour equilibration. Buffer was carefully removed and 200 uL of
Amplex Red® reaction solution was added to the tissues. Tissues were
incubated with ET -1 (1, 10 and 100 nM) or ET-1 (100 nM) plus antagonists
[atrasentan (10 nM): ET, receptor antagonist, BQ-788 (100 nM): ET, receptor

antagonist or DDC (10 mM): SOD inhibitor] for 4 hours at 37°C. Li er al

46

previously determined that maximal ET-1-stimulated superoxide production
occurred after a four-hour incubation (Li et al, 2003a; Li et al, 2003b), thus I
chose to measure ET-1-stimulated H202 production after a four-hour incubation.
Then the Amplex Red® solution was transferred to a 96-well plate and
fluorescence emission was measured (excitation = 544 nm, emission = 584 nm)
on a SpectraMax Gemini plate reader (Molecular Devices, Sunnyvale CA) using
SoftMax®Pro software. A standard curve using known H202 concentrations was
performed with each experiment and was used to determine H202 concentration.
After each experiment, aortic and venous segments were homogenized and
protein concentrations determined using the bicinchoninic assay (BCA) for
protein determination. H202 production from rat aorta and vena cava is

represented as nmol/mg protein or as a percentage of control response.

C. Whole tissue immunohistochemistry

Cleaned rings of thoracic aorta and vena cava were formalin-fixed (10%)
overnight and paraffin-embedded at the histology lab at Michigan State
University. Sections (5 micron) were cut and placed on glass slides. Slides were
dewaxed (Z dips in xylenes; 3 minutes each), rehydrated (2 dips in 100%
ethanol, 2 dips in 95% ethanol, 2 dips in 90% ethanol, 2 dips in deionized water;
3 minutes each) and unmasked (boiled for 5 minutes in unmasking solution in the
microwave). Endogenous peroxidases were blocked by incubating sections for
30 minutes in 0.3% H202 (dissolved in phosphate buffered saline) at room

temperature and non-specific secondary binding blocked by incubating sections

47

for 1 hour in 1.5% blocking serum (for the species the secondary antibody was
raised in) at room temperature. Sections were incubated overnight (humidified
chamber, 4°C) with primary antibody [ET, antibody (5 ug/mL, Alomone
Laboratories), ET; antibody (5 uglmL, Alomone Laboratories)]. Sections were
also incubated with primary antibody quenched with five times the concentration
of competing peptide (solution made 2 hours before application). Sections were
incubated with secondary antibody for 30 minutes (humidified chamber, room
temperature) and with ABC reagent for 30 minutes (humidified chamber, room
temperature, Vector Laboratories). Staining was detected using the
diaminobenzidine peroxidase reagent (Vector Laboratories) and slides were
counterstained with hematoxylin. Slides were allowed to air dry, coverslips

mounted with Vectamount® and pictures taken with a Spot 2 digital camera on a

Leica light microscope.

D. Whole tissue protein isolation

Thoracic aorta and vena cava were snap frozen in liquid nitrogen, pulverized in a
liquid nitrogen-cooled mortar and pestle and solubilized in buffer (62.5 mM Tris,
pH 6.8, 2% SDS wlv, 10% glycerol vlv) with protease inhibitors (1 mM PMSF,
100 uglml aprotinin and 100 jig/ml leupeptin). Homogenates were centrifuged
(11,000 rpm for 10 minutes, 4°C) and supernatant collected. Total protein in the
supernatant was measured using the BCA method of protein measurement
(Sigma, St. Louis, MO). Samples were stored at -80 °C until use.

1. Cross-linking: Rat thoracic aorta and vena cava were isolated, dissected and

48

cleaned. Vessels were incubated with 5 mM disuccinimidyl tartrate (DST)
(dissolved in 20 mM HEPES pH 8.0, 1 hour, room temperature, end-over-end
mixing). The cross-linking reaction was stopped with 20 mM Tris (pH 7.5, 15
minutes, room temperature, with end-over-end mixing). Aorta and vena cava
were snap frozen in liquid nitrogen and homogenized with a HEPES lysis buffer
(20 mM HEPES pH 8.0, 150 mM NaCl, 1% SDS) according to the protein

isolation protocol mentioned above.

E. Western blot analysis

1. SDS-polyacrylamide gel electrophoresis (PAGE) (denaturing PAGE)

Fifty micrograms of protein (either aorta or vena cava lysates) was diluted in 4:1
denaturing sample buffer (250 mM Tris pH 6.8, 5% SDS wlv, 0.005%
bromophenol blue wlv, 45% glycerol v/v, 9.4% B-mercaptoethanol vlv), incubated
for 10 minutes at room temperature, separated on 10% SDS-polyacrylamide gels
and transferred (1 hour, 100 V) to lmmobilon P (PVDF) membranes. Membranes
were blocked overnight (Tris-buffered saline + 0.5% Tween (T EST), 5% nonfat
dry milk (Biorad), 0.025% sodium azide). Blots were probed overnight with
primary antibody [ET,: 1:200 dilution (raised in rabbit, Alomone Laboratories),
ET;: 1:200 dilution (raised in rabbit, Alomone Laboratories)] at 4 °C. rinsed in
TBS-T, with a final rinse in TBS, and incubated with secondary antibody (anti-
rabbit lgG, 1:1000 dilution) for 1 hour at 4 °C. After secondary incubation, blots
were rinsed in TBS and then incubated with ECL® reagents to visualize bands.

2. SDS-urea PAGE

49

A standard SDS-PAGE was performed except for the addition of 8M urea to the
standard 4:1 denaturing sample buffer, 10% SDS-acrylamide gels and the
Laemmli running buffer.

3. Non-denaturing PAGE (NATIVE PAGE)

A standard SDS-PAGE was performed except for the omission of SDS in 10%
acrylamide gels and the standard 4:1 denaturing buffer. B-mercaptoethanol was

also omitted from standard 4:1 denaturing buffer.

F. Co-immunoprecipitation

1 . Traditional co-immunoprecipitation

Cleaned thoracic aorta and vena cava were snap frozen in liquid nitrogen and
ground in a mild homogenization buffer (62.5 mM Tris pH 6.8, 2% SDS wlv, 10%
glycerol WM and freshly added peptidase inhibitors including 100 jig/ml aprotinin,
100 uglml leupeptin, 1 mM PMSF). Five hundred micrograms of protein (either
aorta or vena cava lysates) was incubated with 2 ug ET, or ET; receptor
antibody (Alomone Laboratories) for 2 hours at room temperature. Thirty
microliters of protein AIG beads (Santa Cruz) were incubated with
antibody/antigen complexes overnight at 4 °C with rocking. The beads were
washed 3 times with phosphate buffered saline (pH 7.4) buffer, with
centrifugation (1 minute, 2500 rpm) in between washes. Beads were boiled
(96°C) for five minutes in 2:1 sample buffer (plus 9.4% B-mercaptoethanol) and
centrifuged (1 minute, 2500 rpm). The supernatant containing the

immunoprecipitates were loaded and separated with 10% SDS-PAGE. Samples

50

were electrically transferred (1 hour, 100 V) to lmmobilon P (PVDF), blots
blocked overnight (T ris—buffered saline + 0.5% Tween (TBS—T), 5% nonfat dry
milk (Biorad), 0.025% sodium azide) and then incubated overnight (4°C) with ET,
and ET; receptor antibodies (1:200 dilution, Alomone Laboratories). Blots were
developed according to standard western blot protocols.

2. True Blot® co-immunoprecipitation kit

Rat thoracic aorta and vena cava were isolated, dissected and cleaned. Protein
was isolated with a modified lysis buffer (50 mM Tris pH 8.0, 150 mM NaCl, 1%
nonidet P-40 substitute) following the protein isolation protocol mentioned above.
Five hundred micrograms of aortic and venous lysates were pre-cleared with fifty
uL of TrueBIot rabbit lgG beads (3 min, 10,000 x 9). Five micrograms of antibody
(ET,, Alomone Laboratories; ET;, Alomone Laboratories; caveolin-1,
Transduction Laboratories) and pre-cleared lysates were incubated (2 hours,
room temperature). Fifty microliters of TrueBIot rabbit lgG beads was added to
the antibody-lysate complex and incubated overnight (4°C with end-cver-end
mixing). Beads were washed three times with lysis buffer and
immunoprecipitates recovered by boiling (10 minutes) in 2X reducing sample
buffer. Samples were then run through standard SDS-PAGE protocol and
developed using the TnieBlot anti-rabbit secondary antibody (1 :1000).

3. Pierce ProFound® mammalian Co-IP kit

One hundred microliters of antibody coupling gel was added to a spin cup
column and washed twice with phosphate buffered saline. Two micrograms of

ET; receptor antibody (Alomone Laboratories) was diluted in 100 uL of

51

phosphate buffered saline + sodium cyanoborohydride (5 mM) and incubated
overnight (4°C) with end-over-end mixing. The spin cup column was centrifuged
(1 min, 3000 x g) and quenching buffer plus sodium cyanoborohydride added to
the column for 30 minutes at room temperature with end-over-end mixing. The
antibody-coupled gel was washed 4 times with wash solution (with centrifugation:
1 min, 3000 x g). Lysates of rat thoracic aorta and vena cava were made using
the M-Per lysis buffer. Protein (1500 micrograms) from aortic and venous lysates
were added to the spin columns and diluted to a final volume of 300 (IL with
phosphate buffered saline. Columns were incubated overnight (4°C) with end-
over—end mixing. Columns were then washed 3 times with phosphate buffered
saline and immunoprecipitated proteins eluted off the column with three 50 BL
aliquots of elution buffer. The pH of samples was neutralized with Tris (1 M, pH
9.5) and samples ran through standard SDS-PAGE as mentioned above.

4. ExactaCruzO co-immunoprecipitation kit

The ExactaCruz® kit for homologous immunoprecipitation and western blotting
for antibodies raised in rabbit (Santa Cruz, sc-45043). Two micrograms of ET;
receptor antibody (Alomone Laboratories) was added to 50 BL of IP matrix
(beads) and incubated with end-over-end mixing (1 hour, 4°C). Beads were
washed twice with phosphate buffered saline (30 sec, 3000 rpm centrifugation).
Five hundred micrograms of aortic lysate was added to the antibody-bead
complex and incubated with end-over—end mixing (overnight, 4°C). Beads were
washed three times with phosphate buffered saline. lmmunoprecipitated proteins

were separated from beads by boiling (5 minutes, 96°C) in 2X denaturing sample

52

buffer and samples run through the standard SDS-PAGE protocol as described
above. lmmunoprecipitated proteins were detected (after primary antibody
binding) with a secondary antibody that came with the kit (1 :2000 dilution).

5. Co-immunoprecipitation using DynalO magnetic beads

Dynal magnetic rabbit IgG beads or protein A beads (60 ILL, Invitrogen) were
washed two times with phosphate buffered saline and then incubated with ET, or
ET; receptor antibody (2 pg, Alomone Laboratories; overnight, 4°C with end-
over-end mixing). Antibody bound beads were washed three times with
phosphate buffered saline and then cross-linked with DMP (100 mM, 30 minutes
at room temperature with end-over-end mixing). The cross-linking reaction was
stopped with the addition of Tris (50 mM, pH 7.5; 15 minutes with end-over-end
mixing). Cross-linked beads were washed three times with phosphate buffered
saline and then 500 pg of aortic or venous lysate was added to the beads (1 hour
with end-over-end mixing). Beads were washed three times with phosphate
buffered saline and then immunoprecipitated proteins were eluted off of the
bead-antibody complex by boiling for 5 minutes in 2X denaturing sample buffer.
Samples were then run through the standard SDS-PAGE protocol as described

above.

G. Dissociation of vascular smooth muscle cells
Rat thoracic aorta and vena cava were isolated, dissected and cleaned in chilled
dissociation solution containing (in mM): NaCl, 136; KCI, 5.6; M902, 1; Na2HPO4,

0.42; NaH2P04, 0.43; NaHCOa, 4.2; HEPES, 10; sodium nitroprusside, 8.72 and

53

bovine serum albumin, 1mg/mL (pH 7.4 with NaOH). After dissection, the whole
vena cava or 4-5 millimeters of aorta were cut into thin rings (as thin as possible)
and equilibrated at room temperature for 10 minutes in one milliliter of fresh
dissociation solution. Vessel pieces were incubated in one milliliter of an
enzymatic solution containing papain (26 UnitslmL) and dithiothreitol (1mglmL)
dissolved in dissociation solution for 45 minutes (at 37°C, with shaking). Then
vessel pieces were incubated in 1-2 mL of a second enzymatic solution
containing collagenase (1.95 Units/mL), elastase (0.15 mglmL) and soybean
trypsin inhibitor (1 mglmL) (aorta: 35 minutes, vena cava: 45 minutes with
shaking, 37°C) dissolved in dissociation solution. After the second enzymatic
digestion, the digestion solution was pulled off (leaving the tissue and cells in the
tube) and 4 mL of fresh, cold dissociation solution was added. Cells were placed
on ice for five minutes, the dissociation solution was discarded (carefully leaving
cells at the bottom of tube) and cells were rinsed again with 4 mL of fresh, cold
dissociation solution. The second wash of dissociation solution was gently
pipetted off, and cells were suspended and triturated (forcefully pipetted
approximately 10 times) in 0ptiMEM (plus sodium nitroprusside, 872 nM) to

dissociate vascular smooth muscle cells from the blood vessel matrix.

H. Immunocytochemistry in freshly dissociated vascular smooth muscle
cells
Two hundred microliters of freshly dissociated vascular smooth muscle cells

were placed on poly-lysine (50 ug/mL) coated coverslips (12 mm) and allowed to

54

adhere to the coverslips for 45 minutes (37°C, 4% CO2). Cells were fixed in
Zamboni’s fixative (4% paraforrnaldehyde, 15% picric acid, 0.1M phosphate
buffer pH 7.4, 20 minutes). Cells were permeabilized with Triton-X 100 (0.5%, 20
minutes) and incubated cells with lmageiT® signal enhancer (30 minutes, 37°C,
Invitrogen). Coverslips with cells were incubated with primary antibodies (ET,:
anti-sheep, Fitzgerald Industries; ET;: anti-rabbit, Alomone Laboratories; pan-
cadherin: anti-mouse, Sigma; 1:200 dilution in phosphate buffered saline, 0.5%
Triton-X 100) for 2 hours (37°C). Cells were then incubated with secondary
antibodies (Alexa555 anti-rabbit, 1:200; Alexa488 anti-sheep, 1:200; Alexa633
anti-mouse, 1:200; Invitrogen) for 1 hour (37°C). Coverslips were then mounted
on slides with ProFound® anti-fade mounting media (Invitrogen). Confocal
images were captured at the Center for Advanced Microscopy at Michigan State

University on a Zeiss confocal microscope.

I. Data analysis and statistics

Data was derived from 4-10 animals per group. Western blots densitometry was
performed using NIH image and normalized to smooth muscle ot-actin
expression. Contractility data is presented as mean :I: SEM, as a percentage of
the initial response to phenylephrine (10 IIM for arteries) or norepinephrine (10
BM for veins) for the number of animals indicated in parentheses. Agonist E050
values were calculated using a nonlinear regression analysis using the algorithm
[effect = maximum response I1 +(E050Iagonist concentration” (GraphPad Prism,

San Diego, CA). When clear maximal responses were not obtained, EC50 values

55

were considered estimates with the true EC50 value being equal to or greater than
the calculated value. When comparing concentration response curves, two-way
ANOVA with Bonferroni’s post hoc was performed. When comparing two groups
of estimated EC50 values, the appropriate Student’s f-test was used, and when
comparing EC50 values between three or more groups, one-way ANOVA with
Bonferroni’s post hoc test was performed. In all cases, a P value s 0.05 was
considered statistically significant. Some images in this dissertation are

presented in color.

56

III. RESULTS

Hypothesls: ET, and ET, receptors physically interact via receptor
heterodimerization in veins but not arteries and this heterodimerization

functionally affects ET, and ET, receptor pharmacology.

A. Subhypothesls 1: To demonstrate a pharmacological interaction between
ET, and ET, receptors in rat thoracic vena cava but not aorta and to demonstrate
that activated ET, receptors pharmacologically uncouple ET, receptors from

receptor blockade in rat thoracic vena cava.

1. Non-speclflc ET,/ET, receptor desensltlzatlon
Since veins but not arteries have functional, contractile ET, receptors, I
investigated the role of ET, receptor in ET-1 desensitization. Flgure 15 displays
lack of arterial contraction but partial venous contraction to ET-1 (10 pM — 100
nM) after desensitization with ET-1 (100 nM). Contraction to ET-1, given
cumulatively, after exposure to a concentration of ET-1 causing maximal
contraction (15 minutes, 100 nM, followed by 2 hours of washing) was abolished
in aorta (Flgure 15A), while in veins 36.3 a: 0.2% of maximal contraction to ET-1
remained (Flgure 158). Figure 16 displays ET, receptor-mediated contraction of
rat thoracic aorta and vena cava after ET-1 desensitization (100 nM, ). Aorta

were unresponsive to the ET, receptor agonist ET -1[1-31]

57

A. Aorta

 

+Control
-D- ET-1 (100 nM) desens.
(N=5)

5

Percentage PE
(10 I‘M) CODUBCIIOI’I
.8

i9

 

 

-12 -11 -10 -9 -8 -7 -l3
IogET-1 [M]

Vena Cava

 

-e- Control
.. -o- ET-1 (100 nM) desens.
(N=9)

 

1

Percentage NE
(10 pM) Contraction
a a a a a a s

 

 

 

-12 -11 -10 -9 36 -7 ~15
IogET-1[M]

Flgure 15. ET -1 (10 pM - 100 nM) concentration response curves in rat thoracic
aorta (A) and vena cava (B) desensitized with ET -1 (100 nM). Points represent
means a; SEM for the number of animals (N) indicated in parentheses. Asterisks
(*) indicate statistically significant differences from control responses (p<0.05).

ET-1, endothelin-1; NE, norepinephrine; PE, phenylephrine.

58

 

 

 

 

Aorta
75
+Control
-D- ET-1 (100 nM) desens.
g (N=4)
m.
a- m 50.
is
:0
6s
33 g 25.
o, *
-12 -11 ~10 -9 -8 -7 -6
log ET-1[1-31] [M]
' Vena Cava
500
-e-Control
-o- ET-I (100 nM) desens.
g 400. (N=4)
a) a 3m-
.88
go
93 2m:
8 3.
g a
100..
o.
-12 -11 -10 -9 -8 -7 -6

 

log ET-1[1-31] [M]

 

Flgure 16. ET -1[1 -31] (10 pM — 100 nM) concentration response curves in rat

thoracic aorta (A) and vena cava (B) desensitized with ET-1 (100 nM). Points

represent means a: SEM for the number of animals (N) indicated in parentheses.

Asterisks (*) indicate statistically significant differences from control responses

(p<0.05). ET-1, endothelin-1; NE, norepinephrine; PE, phenylephrine.

59

after ET-1 exposure (Flgure 16A), while veins remained responsive to ET-1[1-
31] (though the response was significantly diminished) after ET-1 exposure

(Flgure 16B) (21.9 :I: 0.6% of maximum venous contraction to ET -1 ).

While the ET, receptor on arteries and veins desensitized in a qualitatively similar
manner, the ET, receptor on veins did not desensitize to ET-1 (Flgure 17A) or
the ET, agonist, 86c (Flgure 178) when a similar desensitization paradigm was
used (15 minute agonist challenge followed by 2 hour agonist washout). A bolus
concentration of 86c (100 nM) was administered because cumulative
concentration response curves to S6c were not reproducible. Contraction to a
maximal concentration of ET, receptor agonist S6c (100 nM) after EM (100 nM)
was not altered in veins (117 :I: 7% of norepinephrine contraction in control tissue
vs 118 a: 6% in ET -1 desensitized tissues, P> 0.05). Moreover, maximal venous
contraction to S6c after 86c pre-exposure (100 nM) was not altered (80 :I: 15% of
norepinephrine contraction in control tissues vs 87 :I: 12% in 86c desensitized
tissues, P > 0.05). Contraction to SSC (10 pM - 100 nM) was not observed in
aorta. These data suggest that the ET, receptor may account for maintained
venous responsiveness to additional ET challenges after desensitization, as the
ET, receptor remains functional (i.e. contractile) and responsive to ET-1 in the

desensitization time frame used.

Desensitization of the ET receptors after tissue exposure to ET-1 was specific to

60

A Vena Cava

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

175
-Control
150.. IZIET-1 (100 nM)desens.
c (N=4)
.9 125.
E g —r—
§§ 100.
C:
5 g 75-:
a) :1.
°- 2 50.
25.
0.
Contraction to 86c (100 nM)
Vena Cava
175
-Control
150- DSGC (100 nM)desens.
c (N=4)
.9125-
% ii
an r. .
C
63 75'
(I) 1
m e 50.
25.
0.
Contraction to S6c (100 nM)

Flgure 17. 86c (100 nM) -induced contraction of rat thoracic vena cava
desensitized with ET-1 (100 nM) (A) or 86c (100 nM) (B). Points represent
means a: SEM for the number of animals (N) indicated in parentheses. Asterisks
(*) indicate statistically significant differences from control responses (p<0.05).

ET-1, endothelin-1; NE, norepinephrine; PE, phenylephrine; 86c, sarafotoxin 6c.

61

A Aorta

 

 
   

 

150
-l-Control
125. -I:I-ET-1 (100 nM)desens.
g (N=3)
$8100-
CB
0
DIE
as 75-
6s
a g 50-
$-
0- I I
-10 -9 -8 -7 -6 -5 -4

 

 

 

 

 

 

'09 NE [W
B Vena Cava
125
-e-Control
-o-ET-1 (100 nM) desens.
c:100- (N=3)
.9
g g 751
b
a a
a 8
{is 501
d) :1.
O- o
z: 25,
-10 -9 - -7 -i3 -' -4
'09 NE [W

Flgure 18. Norepinephrine-induced contraction (1 nM - 10 BM) of rat thoracic

aorta (A) and vena cava (B) was unaltered with ET -1 (100 nM) desensitization.
Points represent means a: SEM for the number of animals (N) indicated in

parentheses. ET-1, endothelin-1; NE, norepinephrine; PE, phenylephrine.

62

the ET system (I.e. displayed homologous desensitization). Contraction to the ot-
adrenergic agonist norepinephrine was not reduced or rightward-shifted in control

or ET-t -desensitized aorta (Flgure 18A) or vena cava (Flgure 18B).

To determine which receptor subtype accounted for maintained venous ET -1-
induced contraction after desensitization, veins were desensitized to ET-1 as
described above and incubated with either vehicle, an ET, receptor antagonist
(atrasentan, 100 nM) or an ET, receptor antagonist (BO-788, 100 nM) for 30
minutes prior to performing ET -1 concentration response curves (10 pM — 10
nM). The ET, receptor antagonist 80-788 was unable to shift the remaining
contraction to ET-1 (EC50 (-log M) = 8110.1 in control tissues versus 81:01 in
80-788 incubated tissues), but did reduce maximal contraction to ET -1, though
this reduction was not statistically significant (Flgure 19B). However, the ET,
receptor antagonist atrasentan significantly reduced the remaining venous
contraction to ET-1 after desensitization (E05, (-log M) = 81:01 in control
tissues vs unmeasurable in atrasentan-incubated tissues, Flgure 19A). These
data suggest that the ET, receptor, along with small contributions by the ET,
receptor, is primarily responsible for continued venous responsiveness to ET-1.
These data also suggest that the presence of contractile ET, receptors in vena
cava but not aorta may alter ET, receptor pharmacology such that the ET,

receptor is less susceptible to receptor desensitizationfintemalization.

A. 175 Vena Cava

 

 

 

 

    

-e-Control
1504 -o-Atrasentm(100nM)
r: (N=5-6)
“1% 1254
2 (U
3.3 100.
g o
:0 75
£95 '
o s.
as w.
v t
25.
* *
0.
-12 -11 -10 -9 -8 -7 -6
log ET-1[M]
Vena Cava
B 175
' -e-Control
1501 -o-BQ~788(100nM)
(N=5-6)
125.

5

Percentage NE
(10 pM) Contraction
.3

 

 

 

-12 -11 -10 -9 -'8 -7 -6
logET-1 [M]

Flgure 19. ET -1 (10 pM - 100 nM) concentration response curves in the
presence of ET, receptor blockade (atrasentan, 100 nM) (A) or ET, receptor
blockade (BO-788, 100 nM) (B) in rat thoracic vena cava desensitized with ET-t
(100 nM). Points represent means a: SEM for the number of animals (N)
indicated in parentheses. Asterisks (*) indicate statistically significant differences

from control responses (p<0.05). ET -1, endothelin-1; NE, norepinephrine; PE,

phenylephrine.

2. ET, receptor desensltlzatlon
Since there are clear differences in desensitization between the ET, and ET,
receptors in vena cava, l next determined if the ET, receptor in vena cava did in
fact desensitize. The desensitization protocol was changed to alter the amount
of re-sensitization time (Is. change the number of washes, Flgure 20A). The
data in Flgure 208 demonstrates that S6c (100 nM, 15 minute) desensitization
followed by 15 minutes of re-sensitization (washing every 5 minutes) completely
abolished venous contraction to a subsequent challenge of 86c (100 nM). When
the re-sensitization time (and number of washes) was increased to 30 minutes (6
washes), venous contraction to subsequent challenges of 86c after 86c
desensitization were not different from the control contraction to $60 (100 nM,
Flgure 20B), suggesting that while venous ET, receptors do desensitize, they re-

sensitize in a significantly shorter time frame than desensitized ET, receptors.

3. Does ET, receptor desensltlzatlon alter ET, mceptor function?
I then investigated whether contractile ET, receptors could alter ET, receptor
function in both rat thoracic aorta and vena cava by desensitizing ET, receptors
with 86c and assessing ET, receptor function using the ET, receptor antagonist,
atrasentan. To ensure maximum ET, receptor desensitization, 36c (100 nM)
was incubated for one hour concurrent with ET, receptor antagonism

(atrasentan, 10 nM) and cumulative concentration response curves to ET-1 (10

65

A. . _
- Vena cava Example DOIygraph recordings.

1 86c (100 nM, 1 hr) k

3 washes (15 min re-sensitization)

 

l 360(100 nM,1hr) ,2 x

6 washes (30 min re-sensitization)

I 860(100 nM,1hr) k

Contraction to 86¢ (100 nM)

l

9

 

Percentage NE
(10 pM) contraction
.8 .8

.3

 

 

 

 

 

* #
Control 15 3'0
Re-sensitlzatlon time (minutes)

 

Flgure 20. 86c desensitization protocol (A) and quantification of results (B) in rat
thoracic vena cava. NE, norepinephrine. Asterisks represents a statistically

significant difference from control (p<0.05). # represents a statistically significant

difference from 30 minutes re-sensitization (p<0.05).

66

 

  
  
     

A. 175 Aorta
+Control
150- -I:I-Atrasentan(10nM)
N=4
mi 125. ( )
“-3
3.3 100.
$8 75
55:,
‘12 so.
251!
O.

 

 

 

 

-12 -11 .10... 3 -7 -6
logET-1[M]

B_ Aorta

-I- 86c desens.
-I:I- SGc desens. «I-
Atrasentan (10 nM)
(N=4)

 

Percentage PE
(10 pM) Contraction
.8 .8

.8

 

 

 

-12 -11 -10 -9 is 5! -6
logET-1[M]

Figure 21. Comparison of ET, receptor blockade (Atrasentan, 10 nM) of ET -1
cumulative concentration response curves in rat thoracic aorta with unbound ET,
receptors (A) and agonist-bound ET, receptors (B) (86c desens). Points
represent means a: SEM for the number of animals (N) indicated in parenthesis.

NE, norepinephrine; PE, phenylephrine.

67

A 700 Vena Cava

-e- Control
600, -o-Atrasentan (10 nM)
(N=4)

 

   

Percentage NE
(10 pM) Contraction

 

 

 

-12 -11 -10 59 -8 -7 -l
IOQET-IIMI

U)

B Vena Cava

 

-e- 86c desens.

500,, -o-SGC desens. +
Atrasentan (10 nM)
(Nd)

 
   

Percentage NE
(10 pM) Contraction

 

 

 

-12 -11 -10 -9 -8 37 -6
logET-HMI

Flgure 22. Comparison of ET, receptor blockade (Atrasentan, 10 nM) of ET-1
cumulative concentration response curves in rat thoracic vena cava with unbound
ET, receptors (A) and agonist-bound ET, receptors (8) (86¢ desens). Points
represent means a: SEM for the number of animals (N) indicated in parenthesis.

NE, norepinephrine; PE, phenylephrine.

68

Aorta

 

 

 

A.
Condition Aorta EC50 Aorta
(dog M) Fold shift
Control + ET, 8.2 3 2
Atrasentan (10 nM) receptors 7.7 '
86c (100 nM) - ET, 8.2 5 0
Atrasentan + 86c receptors 7.5 '
B. Vena cava
. . Vena cava Vena cava
0°"d't'°" EC, (4% M) Fold shift
Control + ET, 8 2 1 3
Atrasentan (10 nM) receptors 8.1 '
S6c (100 nM) - ET, 8.5 6 3,,
Atrasentan + 86c receptors 7.7 '

Table 1. Estimated E05, (-Iog M) values for ET-1-induced contraction in aorta (A)
and vena cava (B) in the presence of unbound or agonist-bound ET, receptors
with and without ET, receptor antagonism (atrasentan, 10 nM). Asterisks (*)
represents a statistically significant difference from + ET, receptors (p<0.05).

86c, sarafotoxin 6c.

69

pM — 100 nM) were performed without washing out 86c and the ET, receptor
antagonist. In rat thoracic aorta with unbound ET, receptors (control, not S6c-
desensitized), atrasentan (10 nM) caused a 3.2-fold rightward shift in ET-1-
induced contraction (Flgure 21A, Table 1). In aorta with agonist-bound ET,
receptors (SGc-desensitized aorta), atrasentan (10 nM) caused a 5.0-fold
rightward shift in ET-1-induced contraction (Figure 21 B). In vena cava with
unbound ET, receptors (control, not S6c-desensitized), atrasentan caused a 1.3-
fold rightward shift in ET-1-induced contraction (Flgure 22A, Table 1). In vena
cava with agonist-bound ET, receptors (SSc-desensitized), atrasentan caused a
63-fold rightward shift in ET-1-induced contraction (Flgure 228). These data
suggest that in vena cava, the presence of agonist-bound contractile ET,
receptors alters ET, receptor function such that ET, receptors are less

susceptible to receptor blockade.

4. Receptor Interactlon: contractlllty studies uslng receptor
antagonists
Receptor antagonism studies were also performed to investigate howfif ET,
receptors alter ET, receptor function. Cumulative ET-1 concentration response
curves were performed in the presence of vehicle, ET, receptor blockade
(atrasentan, 10 nM), ET, receptor blockade (BO-788, 100 nM) or ET, plus ET,

receptor blockade (atrasentan, 10 nM + BQ-788, 100 nM) and estimated

70

A. Aorta

-I- Vehicle
~0- Atrasentan (10 nM)
(N=4-5)

a

 

Percentage PE
(10 BM) Contraction
.8 .8

.3

 

o
I

 

V

-12 -11 -10 -9 43 -7 -6

 

 

 
 
  

 

 

log ET-t [M]
Aorta
8' 200
-I-BQ-788 (100 nM)
~D-Atrasentan (10 nM)
:150‘ +BQ-788(100nM)
w .9 (N=4-5)
.. a
E 0 100-
§ 3
a a
c 50-1
0' .
-12 -11 -10 -9 -8 -7 -6

log ET-1 [M]

Flgure 23. Comparison of ET, receptor blockade (Atrasentan, 10 nM) of ET-1
cumulative concentration response curves in rat thoracic aorta without ET,
receptor blockade (A) and with ET, receptor blockade (B) (BO-788, 100 nM).
Points represent means a SEM for the number of animals (N) indicated in

parenthesis. NE, norepinephrine; PE, phenylephrine.

71

A 800 Vena Cava

 

-e-Vehicle
700- -o-Atrasentan(10"M)
600. ("‘45)

Percentage NE
(10 pM) Contraction

100,

o. ,
-12 -11 -10 -9 -e -7 -6

log ET-1 [M]

 

 

 

B- Vena Cava

-e-BQ-788 (100 nM)

" -o-Atrasentan (10 nM)
+ 80-788 (100 nM)

(N=4—5)

 

l

 

1

Percentage NE
(10 pM) Contraction
assesses

 

‘P

 

I
_A

 

2 -11 -10 -9 -'8 -7 -l
IogET-1 [M]

U)

Figure 24. Comparison of ET, receptor blockade (Atrasentan, 10 nM) of ET-1
cumulative concentration response curves in rat thoracic vena cava without ET,
receptor blockade (A) and with ET, receptor blockade (B) (80-788, 100 nM).
Points represent means a; SEM for the number of animals (N) indicated in

parenthesis. NE, norepinephrine; PE, phenylephrine.

72

Aorta

 

 

 

A.
Condition Aorta EC,o Aorta
Hog M) Fold shift
Vehicle + ET, 8.2 6 7
Atrasentan (10 nM) receptors 7.3 '
BQ-788 (10 pM) Er 8.3
Atrasentan + BQ- ' 9 6.4
788 receptors 7.5
B. Vena cava
. . Vena cava Vena cava
C°"""'°" _ ECin (499 M) Fold shift _
Vehicle + ET, 8.1 2 3
Atrasentan (10 nM) receptors 7.8 '
80-788 (10 (IM) ET 8.5
- - B *
Atrasegteag + BO receptors 7.6 8.1

Table 2. Estimated EC50 (-log M) values for ET-1-induced contraction in aorta (A)
and vena cava (B) and the presence and absence of ET, receptor blockade, with
and without ET, receptor antagonism. Asterisks (*) represents a statistically

significant difference from + ET, receptors (p<0.05).

73

ECso values were calculated and compared between groups. In aorta without
ET, receptor blockade, atrasentan caused a 67-fold rightward shift in ET-1 -
induced contraction (vehicle vs atrasentan) (Flgure 23A, Table 2). In aorta with
ET, receptor blockade, atrasentan caused a 6.4-fold rightward shift in ET-1-
induced contraction (BO-788 vs 80-788 + atrasentan) (Flgure 238), which was
not different from the shift in the presence of functional ET, receptors. In vena
cava without ET, receptor blockade, atrasentan caused a 2.3-fold rightward shift
in ET -1-induced contraction (vehicle vs atrasentan) (Flgure 24A, Table 2).
When ET, receptors in vena cava were rendered non-functional with ET,
receptor blockade, atrasentan caused a 8.1-fold rightward shift in ET-1-induced
contraction (Flgure 24B), which was significantly greater than when ET,
receptors were not blocked, suggesting that functional ET, and ET, receptor

interaction occurs in vena cava, our model vein, but not aorta, our model artery.

5. Section summary
Through the use of contractility studies, I have demonstrated that functional,
contractile ET, receptors (present in vena cava but not aorta) are capable of
altering ET, receptor pharmacology. Desensitization experiments and receptor
antagonist experiments provide evidence for functional ET,/ET, receptor
interaction in veins but not arteries, suggesting that the rat thoracic vena cava is

an appropriate model to investigate ET,/ET, receptor heterodimerization.

74

B. Subhypothesls 2: To characterize biochemical signaling interactions

between ET, and ET, receptors in rat thoracic aorta and vena cava.

One possible explanation for functional endothelin receptor interaction in vena
cava but not aorta could be differences in endothelin receptor signal transduction
in arteries and veins. ET-I can also potentially signal through the production of
reactive oxygen species (ROS). ET-1 via ET, receptors stimulates NADPH
oxidase to increase superoxide production (Li et al, 2003; Loomis ef al, 2005).
Superoxide is a substrate for superoxide dismutase, which rapidly converts
superoxide into H202. Both superoxide and H202 have been reported to alter
vascular tone, causing both contraction and vascular bed depending on
experimental conditions and vascular beds studied (Clempus and Griendling,
2006). It is possible that differences in ROS production and signaling in arteries
and veins may explain why endothelin receptor interaction occurs in veins but not
arteries. Contractility studies were performed to investigate the signal
transduction pathways mediating ET-t-induced contraction in aorta and vena
cava and investigated the role of ROS in aortic and venous ET -1-induced
contraction. l determined if H202 was vasoactive in rat thoracic aorta and vena
cava, if ET-1 stimulated H202 production and if H202 mediated aortic and venous
ET-1-induced contraction. Also, to determine if signaling interactions can explain
functional venous ET ,IET , receptor interaction, cumulative concentration

response curves to ET -1 were performed in the presence of specific inhibitors of

75

 

 

 

150
+Aorta(+ E)
-l:I-Aorta(- E)
-e- Vena Cava (+ E)
w 8 (iv-10>
fi 8 100-
s s
Q) C
a e
c A
2
d)
1 q
es 5°
0- S
0:7 -6 -5 -4 -3 -2

I09 l"I202 [MI

Figure 25. Exogenously added H202 (1 uM — 1mM) contracts both rat thoracic
aorta and vena cava. Points represent means :1: SEM for the number of animals

(N) indicated in parenthesis. +E, endothelium intact; -E, endothelium denuded;

NE, norepinephrine; PE, phenylephrine.

76

 

(N=9-14) If
1.504 I
1.25-

[H 202] (nmol/mg protein)

 

 

 

 

 

 

 

 

 

Flgure 26. Basal H202 levels (nmol/mg protein) are significantly higher in rat
thoracic vena cava compared to aorta. Basal H202 production is significantly
reduced in aorta and vena cava by DDC (diethyldithiocarbamate, 10 mM), an
inhibitor of superoxide dismustase. Data are represented as nmol H202/mg of
total protein for the number (N) of animals indicated in parentheses. Asterisks (*)
represent a statistically significant difference from basal H202 levels (p<0.05),
pound signs (if) represent a statistically significant difference between aorta and

vena cava (p<0.05).

77

 

(N=9-14)

  
  

6

Tissue H202
(as a % control response)
3'3 $

 

 

 

 

 

 

C

 

 

 

 

Aorta
ET-1 (100 nM): .

Flgure 27. ET-1 (100 nM, 4 hour incubation) stimulates H202 production in rat
thoracic vena cava but not aorta. Data are represented as tissue H202
production as a percent of control H202 levels for the number (N) of animals
indicated in parentheses. Asterisks (*) represent a statistically significant

difference from -ET-1 (p<0.05).

78

Tissue H202
(as a % control response)

 

Figure 28. ET -1-stimulated H202 production in vena cava is not concentration-
dependent and is reduced by either ET, receptor blockade (atrasentan, 30 nM) or
ET, receptor blockade (100 nM). Data are represented as tissue H202
production as a percent of control H202 levels for the number (N) of animals

indicated in parentheses. Asterisks (*) represent a statistically significant

difference from basal H202 levels.

79

several signaling pathways, including rho kinase, p38 MAPK, Erk MAPK, src and
Pl3-K.

1. Reactive oxygen species and ET-1-lnduced contraction

Exogenously added H202 (1 IIM - 1 mM) contracted both rat thoracic aorta and
vena cava, but contracted veins much more robustly than arteries (Figure 25).
H202 levels were significantly higher in vena cava compared to aorta (vena cava:
1.26 a: 0.33 nmol/mg H202; aorta: 0.27 a: 0.07 nmol/mg H202) (Figure 26). ET-1
incubation (100 nM, 4 hour) increased venous but not aortic H202 levels (vena
cava ET -1: 154:17% control H202; aorta ET-1 94:13 % H202) (Figure 27). Both
ET, receptor blockade with atrasentan (10 nM) and ET, receptor blockade with
BQ-788 (100 nM) significantly reduced ET-1-stimulated H202 production in vena
cava (atrasentan: 95:30% control H202; BQ-788: 104:21% H202) (Figure 28).
To determine if H202 partially mediated ET-1-induced contraction, ET-1
cumulative concentration response curves were performed in the presence of
catalase or polyethyleneglwa (PEG) -catalase (which is supposedly more
membrane permeable because of the PEG moieties) to break down any ET-1-
stimulated H202 production. Catalase and PEG-catalase potentiated maximal
venous ET -1-induced contraction (Figure 29), suggesting that in veins, ET-1-
stimulated H202 may actually be vasodilatory to counter the vasoconstriction
elicited by ET-1. Cumulative ET-1 concentration response curves were then

performed in the presence of 3-aminotriazole (3-AT) (50 mM), a catalase

80

 

A 600 Vena Cava

 
  
     

 

 

 

 

 
  
 
  
     

 

 

 

+Control
Sm, -D-Catalase (2000 U/ITII)
(N=4)
as
23; 400-
8e
a s 300‘
Is:
as 2002
100‘
0- U I V
-12 -11 -10 -9 -8 -7 -6
Log ET-1 [M]
B- 800 Vena Cava
+Control v: e
7001 -o-PEG-Catalase
4 (1a; Ulml)
I: 600
“1.0 -I:I-PEG-Catalase
z§ 50,. (326U/ml)
0
or? d (N=4)
g g 400
a a 3001
0.0
5 200'
100'
912 -11 -10 -'9 is -'7 -6
Log ET-1 [M]

Figure 29. H202 is not involved in venous ET -1-induced contraction as neither
catalase (2000 U/ml) (A) nor PEG-catalase (163 and 326 U/ml) (B) reduced
venous ET-1-induced contraction. NE = norepinephrine, PEG-catalase =
polyethylene glycol catalase. Points represent means :1: SEM for the number (N)
of animals indicated in parentheses. Asterisks (*) represent a statistically

significant difference from control (p<0.05).

81

Vena Cava

 

-e- Control
-o-3-AT (50 mM)
(N=4)

l I

1

Percentage NE
(10 pM) contraction
assesses

L

 

:9

2 -11 -10 -9 T8 -7 -l
LogET-i [M]

 

 

U)

Figure 30. The catalase inhibitor 3-AT (50 mM) had no effect on maximal venous
ET -1-induced contraction. 3-AT = 3-aminotriazole, NE = norepinephrine. Points

represent means :I: SEM for the number (N) of animals indicated in parentheses.

82

inhibitor, in an attempt to potentiate/enhance ET-1-stimulated H202 levels.
Catalase inhibition with 3-AT did not alter aortic or venous ET -1-induced
contraction (Figure 30). These data suggest that in veins, ET -1 increases H202
production, but this H202 does not play a significant role in mediating venous ET -
1-induced contraction, but may actually be vasodilatory. In arteries, ET-1 does
not produce H202, thus it is unlikely that H202 mediates aortic ET-1-induced
contraction. Thus, I conclude that because reduction of venous H202 with
catalase did not inhibit ET -1-induced contraction, ET-t signaling via H202
production is not a likely mechanism responsible for venous endothelin receptor
interaction, but cannot extend my conclusions to ET -1 signaling through the

production of other ROS such as superoxide or hydroxyl radical.

2. Signal transduction pathways
Since ET -1 signal transduction through H202 production was not a likely
mechanism for explaining why venous ET, and ET, receptors functionally
interact, I examined the possibility that ET -1 activates differential signaling in
aorta and vena cava through other signaling pathways known to mediate
vascular contraction. I asked if inhibitors of p38 MAPK, Erk MAPK, src, rho
kinase and Pl3—K reduced aortic and venous ET-1-induced contraction. The
choice and concentration of inhibitors used was based on previous studies in our
laboratory that determined inhibitor specificity in cultured smooth muscle cells

(Watts, 1996; Banes et al, 1999; Florian and Watts, 1999; Northcott et al, 2002).

83

Also, except for the rho kinase inhibitor Y27632, none of the inhibitors used
significantly reduced KCl-induced contraction (data not shown), a measure of
receptor-independent contraction, suggesting that these inhibitors (LY294002,
P098059, PP1, and 88203580) do not inhibit vascular contraction per se.
Inhibition of Pl3—K with LY294002 (20 pM) did not reduce aortic or venous ET-1-
induced contraction (Figure 31). Inhibition of src with PP1 (10 pM) did not
reduce aortic or venous ET-1-induced contraction (Figure 32). PD98059 (10
am, an inhibitor of Erk MAPK, also did not inhibit aortic or venous ET -1-induced
contraction (Figure 33). 88203580 (10 (M), an inhibitor of the p38 MAPK
pathway did not reduce venous ET -1-induced contraction, but significantly
reduced maximum aortic ET -1-induced contraction [vehiclez 153:8% PE (10 pM)
contraction; SB203580: 106:11%] (Figure 34). Inhibition of rho kinase with
Y27632 (10 pM) significantly reduced both maximum aortic and venous ET-1-
induced contraction [aorta vehicle: 106:5% (10 nM) PE contraction; aorta
Y27632: 59:7%; vena cava vehicle: 469:50% NE (10 (M) contraction; vena
cava Y27632: 272:39%] (Figure 35). While Y27632 has as higher affinity for rho
kinase, it is also capable of inhibiting PKC in some biochemical assays. Thus
ET -1 cumulative concentration response curves were performed in the presence
of another rho kinase inhibitor, HA1077. HA1077 (10 (1M) significantly reduced
aortic but not venous ET-1-induced contraction [aorta vehicle: 131 :1 1% (10 pM)
PE contraction; aortic HA1077: 92:11%; vena cava vehicle: 571:87% NE (10

pM) contraction; vena cava HA1077: 471 :67%] (Figure 36).

84

if

Aorta

 

Percentage PE
(10 pM) Contraction
a is I

5?

 

|
_L

o.
2 ~11

 

+ Vehicle
-D- LY294002 (20 pM)
(N =3)

 

33
log ET-1 [M]

-10 -9

Vena Cava

 

L
U)

 

...§§

assess

Percentage NE
(10 pM) Contraction

.5

 

a?

Figure 31.

2

-e- Vehicle
-o- LY294002 (20 pM)
(N=3)

 

 

   

 

-10 -'9 -'8
logET-I [M]

-11

 

Effect of LY294002 (20 (M), a phosphatidyl inositol-3-kinase

Inhibitor, on ET-1-induced contraction of rat thoracic aorta (A) and vena cava (8).

Points represent means : SEM for the number of animals (N) indicated in

parenthesis. NE, norepinephrine; PE, phenylephrine.

85

A Aorta

+Vehicle
-I:I- P098059 (10 pM)
(N=3)

 

Percentage PE
(10 pM) Contraction
$ 3.9

I?

 

 

 

 

 

 

 

-12 -11 -10 -9 -e -7 -6
IOQET-IIMI
B- Vena Cava
aoo
-e-Vehicle
700- -o-P098059(10 nM)
(N=3)
m g 600-
Z to 500.
SE
as 400.
8A
a 5:. 300-
0.0
I: 200-
100-
-12 -11 -1o -9 -§ 37 -6

 

log ET-1 [M]

Figure 32. Effect of P098059 (10 (M), an Erk MAPK, on ET -1-induced
contraction of rat thoracic aorta (A) and vena cava (8). Points represent means :

SEM for the number of animals (N) indicated in parenthesis. NE, norepinephrine;

PE, phenylephrine.

86

A. Aorta

 

200
+Vehicle
-I:l-PP1 (10 pM)
150.. (Nfi)

Percentage PE
(10 PM) CORITBCUOII
.8

    

 

 

 

.12 -11 -10 -9 13 -7 -l
IogET-1 [M]

U)

B. Vena Cava

-e-Vehicle
.. -o- PP1 (10 BM)
(N=4)

 

   

Percentage NE
(10 pM) Contraction

   

 

 

        

 

 

-12 -11 -10 -§ 13 -7 -6
log ET-1[M]

Figure 33. Effect of PP1 (10 (M), a src tyrosine kinase inhbitor, on ET-1-induced
contraction of rat thoracic aorta (A) and vena cava (8). Points represent means :

SEM for the number of animals (N) indicated in parenthesis. NE, norepinephrine;

PE, phenylephrine.

87

A Aorta

 

+Vehicle
-D-S8203580 (10 pM)
(N=6)

€93

  

Percentage PE
(10 pM) Contraction
3

39

 

 

 

 

 

 

 

-12 -11 -1o 39 -8 5 -6
logET-t [M]
B. VenaCava
800
-e-Vehicle
700- -o-SBZOBSBO(10 pM)
mg 600- (N=4)
Z
to 500.
6%:
E0 400-
ESE-.3001
(Lo
5200-
100-
0. i V r
-12 -11 -10 -9 -8 -7 -6

 

log ET-1 [M]

Figure 34. Effect of 88203580 (10 (M), a p38 MAPK inhbitor, on ET-1-induced
contraction of rat thoracic aorta (A) and vena cava (8). Points represent means :
SEM for the number of animals (N) indicated in parenthesis. NE, norepinephrine;

PE, phenylephrine. Asterisks (*) represent a statistically significant difference

from Vehicle (p<0.05).

88

.>

Aorta

-I- Vehicle
-D- Y27632 (10 pM)
(N=10)

if.

 

   
   

Percentage PE
(10 pM) Contraction
5.9 a $

93

 

 

 

0' I t
~12 ~11 ~10 -9 ~8 ~7 ~6
log ET~1 [M]

B- Vena Cava

§

 

-0- Vehicle
-o- Y27632 (10 pM)
(N=8)

. é

Percentage NE
(10 pM) Contraction
a a a

 

      

 

 

-12 -11 -10 -9 -8 -7 -1
IOQET-IIMI

U)

Figure 35. Effect of Y27632 (10 (M), a rho kinase inhbitor, on ET -1 -induced
contraction of rat thoracic aorta (A) and vena cava (8). Points represent means :
SEM for the number of animals (N) indicated in parenthesis. NE, norepinephrine;

PE, phenylephrine. Asterisks (*) represent a statistically significant difference

from Vehicle (p<0.05).

89

Aorta

.>
§

 

-I- Vehicle

-D-HA1077(10 uM)
(N=6)

Percentage PE
(10 pM) Contraction
:3 .8

I9

 

 

 

 

 

 

 

 

from Vehicle (p<0.05).

90

0- . r
-12 -11 -10 -9 ~8 - -15
logET-t [M]
B- VenaCava
800
-e-Vehicle
700" -o-HA1077(10IIM)
c 600- (N=4-6)
as g
83g 500a ] _ .
C
a!
$8 400,
a?”
c 200- i
100- . ‘.
_ I"
0- ‘ I I I
~12 ~11 ~10 ~9 ~8 ~7 ~6
IogET-1 [M]

Figure 36. Effect of HA1077 (10 BM), a rho kinase inhbitor, on ET -1-induced
contraction of rat thoracic aorta (A) and vena cava (8). Points represent means :
SEM for the number of animals (N) indicated in parenthesis. NE, norepinephrine;

PE, phenylephrine. Asterisks (*) represent a statistically significant difference

3. Section summary
l hypothesized that ET, and ET, receptor signaling pathway interactions might
explain functional ET, and ET, receptor interaction observed in veins but not
arteries. l determined that ET-1 stimulated the production of the vasoactive
reactive oxygen species H202 in veins but not arteries, but this H202 does not
mediate venous ET-1-induced contraction. Through the use of signal
transduction inhibitors, I also observed that rho kinase and p38 MAPK mediated
aortic ET-t-induced contraction and rho kinase mediated venous ET-1-induced
contraction. Within the context of these specific signaling pathways mediating
vascular contraction, ET-1 did not activate any clearly divergent pathways in
vena cava compared to aorta, suggesting that functional venous ET, and ET,
receptor interaction likely does not occur via activation of the specific signaling

pathways investigated or H202-mediated signaling.

C. Subhypothesis 3: To demonstrate a physical interaction, I.e.
heterodimerization, between ET, and ET, receptors in rat thoracic vena cava but

not aorta.

Recently, several G-protein coupled receptors have been reported to interact via
receptor dimerization, altering receptor function, trafficking and pharmacology
(Angers et al, 2002; Milligan, 2004; Maggio at al, 2005; Prinster at al, 2005).

Though ET, and ET, receptors can heterodimerize when they are co-transfected

91

in HEK293 cells (Gregan er al, 2004), it is not known whether ET, and ET,
receptors heterodimerize under physiological conditions, like when they are
normally expressed in blood vessels. Common approaches for determining if
receptors dimerize include receoptor co-immunoprecipitation, fluorescence
resonance energy transfer (FRET) between fluorescently labeled receptors, and
functional measures of receptor interaction (Milligan and Bouvier, 2005). l have
investigated functional ET, and ET, receptor interaction in Subhypotheses 1
and 2, and in Subhypothesis 3 I proposed to perform receptor co-
immunoprecipitation experiments and FRET analysis to determine if ET, and ET,
receptors physically interact via receptor heterodimerization in vena cava but not

aorta.

1 . Antibody testing: dot blots
The experiments in this section required the use of antibodies specific for either
ET, or ET, receptors. Because the ET, and ET, receptor are approximately 70%
homologous, it was necessary to verify antibody specificity. Dot blots were
performed using different antigenic sequences of ET, and ET, receptors and
various commercially available antibodies for ET, and ET, receptors (Figure 37
and 38, Table 3). I concluded that the most selective and sensitive antibody was
the Alomone ET, primary (raised in rabbit). The Alomone ET, primary antibody
(also raised in rabbit) was the next most sensitive primary antibody, but

unfortunately did recognize higher concentrations of ET, receptor epitopes. The

92

next most sensitive and selective antibody was the Fitzgerald ET, receptor
primary antibody (raised in sheep). For the Chemicon and Biogenesis ET, and
ET, receptor primary antibodies l was unable to detect any primary antibody
binding to any of the concentrations of antigenic peptides, as all of these primary
antibodies gave the same pattern of binding, suggesting nonspecific binding of
the anti-sheep secondary antibody used to detect primary antibody binding. I
chose to use the Alomone ET, and ET, receptor primary antibodies for co-
immunoprecipitation experiments and the Fitzgerald ET, and Alomone ET,

receptor primary antibodies for co-localization experiments.

2. Receptor co-locallzatlon
Western blot analysis and immunohistochemistry demonstrate that ET, and ET,
receptors are present in both rat thoracic aorta and vena cava. Because the
endothelium was not removed prior to vessel homogenization, I could not
determine from Western blots which cell types (smooth muscle cells, fibroblasts
or endothelial cells) contain ET, and ET, receptors. lmmunohistochemistry was
performed on 5-micron thick sections of aorta and vena cava but because I was
unable to distinguish single cells, I was unable to determine specifically which cell
types express these receptors. Thus, smooth muscle cells were freshly
dissociated from rat thoracic aorta and vena cava and adhered to coverslips.

Immunocytochemistry using primary antibodies directed to ET, and ET,

93

 

Antibody source Antigenic sequence Host species
Alomone Laboratories
ET, receptor NHNT ERSSH KDSMN rabbit
ET, receptor CEMLR KKSGM QIALN D rabbit
Biodesign International
ET, receptor Ac-SSHVE DFTPFP GTEFC- rabbit
Amide
ET, receptor KANDH GYDNF RSSNN sheep
Biogenesis
ET, receptor QEQNH NTERS SHK sheep
ET, receptor C-terminal peptide (proprietary sheep
sequence)
Fitzgerald Industries
ET, receptor QEQNH NTERS SHK sheep

Table 3. An abbreviated list of commercially available ET, and ET, receptor

antibodies and their antigenic sequences used for dot blot analysis.

94

,2 ng 20 ng_ 299 ng 2 ug COIIEIIPETING

. . PPTIDE-
. ,. _. Alomone ET,
AlomoneEJTo,a ' . 'I'. } _. '* Alomone ET,
Prima anti . . '.
ry y ‘ ' .- L ‘- ' ,1 . . Biodesign ET ,
g,- , Biodesign er,

Biodesign ET
Primary antibody

Biogenesis ET,
Primary antibody

Fitzgerald ET
Primary antibody

 

Flgure 37. Dot blots testing ET, receptor antibodies from Alomone Laboratories,
Biodesign International, Biogenesis and Fitzgerald Industries. Competing
peptides (2 ng - 2 ug) were spotted on nitrocellulose membranes and blots were
developed with different ET, receptor primary antibodies. The same pattern of
peptide dotting was used for the Biodesign ET,, Biogenesis ET, and Fitzgerald

ET, primaries as was used for the Alomone ET, primary antibody.

95

COMPETING

PEPTIDE:
2 2 2 2
.ng 0 ng 00.an ug Alomone ET,
PAlomone EbTo% AI ET
rimary anti y omone

o 0 fl "
Biodesign ET,
Biodesign ET,

Biodesign ET,
Primary antibody

Biogenesis ET,
Primary antibody

 

Flgure 38. Dot blots testing ET, receptor antibodies from Alomone Laboratories,
Biodesign International and Biogenesis. Competing peptides (2 ng - 2 ug) were
spotted on nitrocellulose membranes and blots were developed with different ET,
receptor primary antibodies. The same pattern of peptide dotting was used for
the Biodesign ET, and Biogenesis ET, primaries as was used for the Alomone

ET, primary antibody.

96

receptors and fluorescent secondary antibodies was performed to determine if
both ET, and ET, receptors are expressed by aortic and venous vascular smooth
muscle cells and if both ET, and ET, receptors co-Iocalize on venous vascular
smooth muscle cells. No primary antibody control experiments (i.e. cells just
stained with secondary antibodies) verified that the fluorescent images captured
represent binding of secondary antibodies to primary antibodies and no
background fluorescence from non-specific secondary antibody binding. Flgure
39 displays projected confocal images (I.e. 3-D images comprised of 6 micron
stacks captured from the top to the bottom of cells) of smooth muscle a-actin
expression on aortic and venous vascular smooth muscles cells. The high a-
actin expression present in the cells and elongated shape of the cells verifies that
the isolated cells are indeed vascular smooth muscle cells. To perform receptor
co-Iocalization experiments, cells were double-labeled with Fitzgerald .ET, and
Alomone ET, receptor antibodies (raised in sheep and rabbit, respectively).
Initially, confocal images were captured with singly (either ET, or ET, antibody)
labeled cells to confirm that there was no bleed-through or background emission
of one fluorescent secondary antibody in bandwidth of the second fluorescent
secondary antibody (data not shown). Cells were also labeled with a pan-
cadherin antibody to highlight the plasma membrane of the vascular smooth

muscle cells.

97

Aorta: Vena cava:
a-actin a-actin

 

Figure 39. Projected confocal images of smooth muscle a-actin expression in

freshly dissociated aortic (left) and venous (right) vascular smooth muscle cells.

98

Aortic vascular smooth muscle cells

ET, receptor ET, receptor

.____

10mm 10mm

   

cadherin overlay

10mm

 

Figure 40. Confocal images (6 um thick sections) of ET, receptor, ET, receptor
and cadherin expression in freshly dissociated aortic vascular smooth muscle
cells. Images are representative of aortic vascular smooth muscle cells from five
different rats. Yellow on the overlay image represents co-Iocalization of cadherin,

ET, receptors and ET, receptors.

99

Venous vascular smooth muscle cells

ETA receptor ET, receptor

*

'-
D

IOnm

'h

f:

3

10um
x;

   

cadherin overlay

10mm 10pm

 

Flgure 41. Confocal images (6 um thick sections) of ET, receptor, ET, receptor
and cadherin expression in freshly dissociated venous vascular smooth muscle
cells. Images are representative of venous vascular smooth muscle cells from
four different rats. Yellow on the overlay image represents co-Iocalization of

cadherin, ET, receptors and ET, receptors.

100

Aortic vascular smooth muscle cells
ET-1 stimulated
ET, receptor ET, receptor

10mm "———‘
IOllm

   

cadhefln oveflay

10ml)

10mm

 

Flgure 42. Confocal images (6 pm thick sections) of ET, receptor, ET, receptor
and cadherin expression in ET-1-stimulated (100 nM) freshly dissociated aortic
vascular smooth muscle cells. Images are representative of aortic vascular
smooth muscle cells from five different rats. Yellow on the overlay image

represents co-localization of cadherin, ET, receptors and ET, receptors.

101

Venous vascular smooth muscle cells
ET-1 stimulated

ET, receptor ET, receptor

IOum

 

cadherin overlay

10mm 'IOIIITI

 

Figure 43. Confocal images (6 pm thick sections) of ET, receptor, ET, receptor
and cadherin expression in ET-1-stimulated (100 nM) freshly dissociated venous
vascular smooth muscle cells. Images are representative of venous vascular
smooth muscle cells from four different rats. Yellow on the overlay image

represents co-localization of cadherin, ET, receptors and ET, receptors.

102

Flgure 40 shows that ET, and ET, receptors are expressed at the membrane of
aortic vascular smooth muscle cells. Flgure 41 shows that ET, and ET, (third
panel) receptors were expressed at the membrane of venous vascular smooth
muscle cells, with the white arrows indicating locations where ET, receptors and
ET, receptors co-localized in vena cava. In vena cava, there was significant
intracellular and membrane ET, receptor expression (Flgure 41). Aortic and
venous vascular smooth muscle cells were stimulated with EM (100 nM) during
the 45 minutes they were adhering to coverslips. ET-1 stimulation did not alter
the location of aortic or venous ET, or ET, receptors (Flgure 42, aorta and
Flgure 43, vena cava). ln vena cava, similar to basal conditions, significant ET,

receptor staining was present inside vascular smooth muscle cells.

3. Co-Immunopreclpltatlon of ET, and ET, receptors
Co-immunoprecipitation is another technique used to evaluate endothelin
receptor dimerization (Le. physical receptor interaction). If ET, and ET,
receptors dimerize in vena cava but not aorta, ET, and ET, receptors in venous
but not aortic lysates should co-immunoprecipitate. Traditional co-
immunoprecipitations were initially performed using protein AIG beads to
immunoprecipitate the ET, receptor using the Alomone ET, receptor antibody.
However upon immunoblotting with the Alomone ET, receptor antibody to
determine if the immunoprecipitation was successful or with the Alomone ET,

receptor antibody to determine if the ET, receptor co-immunoprecipitates with the

103

Aorta lysates Vena cava lysates

 

 

_. ..___._ h... . ._.. _.._..- .-.r__-z._.. __.___._ . . _ -
. -. I“ .‘ c.“ E: 5 E
. fl ‘

i_,qu . ‘ . 1 3f- '
" "" " w b -' <—50kba

8

an -—--—-—-., q.. _-._.—L

 

Aorta lysates Vena cava lysates

 

 

hf“.

m .. omw+5°k°a

‘. -.V + ‘4._, ,,.,

Flgure 44. Western blot analysis of ET, receptor expression (A) and ET,
receptor expression (8) in aorta (left) and vena cava lysates (right) from eleven
(11) different rats. The Alomone ET, receptor primary antibody (1 :200 dilution)
was used to detect ET, receptors and the Alomone ET, receptor primary

antibody (1 :200 dilution) was used to detect ET, receptors.

104

Aorta Vena cava

 

 

IP lys IP lys IP lys IP lys

Flgure 45. Example immunoprecipitation of ET, receptors from aortic and
venous lysates. ET, receptors were both immunoprecipitated and detected
(immunoblotted) with the Alomone ET, receptor antibody. IP, immunoprecipitate;

lys, lysate.

ET, receptor, immunoreactive bands for both the ET, and ET, receptor, which
typically resolve at 50 kDa (Flgure 44) were obscured by a 50 kDA heavy chain
antibody band from the immunoprecipitating antibody (Flgure 45). To avoid
detection of the contaminating band from the immunoprecipitating antibody, a
light chain-specific rabbit secondary antibody was then used for immunoblotting
after immunoprecipitation, but this secondary antibody did not recognize either

the Alomone ET, or ET, receptor antibodies.

Several co-immunoprecipitation kits were tried, including the Pierce Mammalian
colP kit, eBioscience True Blot, Santa Cruz ExactaBlot, and Dynal magnetic
protein A beads (Table 4) in a effort to prevent loading/detecting of the 50 kDA
heavy chain antibody band, however none of these kits successfully detected
ET, receptor immunoprecipitation or ET, and ET, receptor co-
immunoprecipitation. Cross-linking experiments were also performed to cross-
link ET, and ET, receptors in aortic and venous lysates with the hope that cross-
linked ET, and ET, receptors would migrate at a higher molecular weight
separating them from the immunoprecipitating antibody, however the degree of
protein degradation from aortic and venous lysates was too great to perform
Western blot analysis. Also, caveolar fraction were isolated by sucrose gradient
centrifugation, in an attempt to localize ET, and ET, receptors to caveolae, but

these fractions were too dilute to quantify ET, and ET, receptors.

106

4. Section summary
l hypothesized that ET, and ET, receptors would co-localize to the plasma
membrane in venous but not aortic vascular smooth muscle cells and that ET,
and ET, receptors would co-immunoprecipitate in venous but not aortic lysates.
Immunocytochemistry analysis demonstrated that ET, and ET, receptors co-
localized to the plasma membrane in both aortic and venous vascular smooth
muscle cells and that ET-1 stimulation did not qualitatively alter ET, and ET,
receptor localization in both aortic and venous vascular smooth muscle cells.
Receptor co-immunoprecipitation experiments were inconclusive as in every
immunoprecipitation technique employed, the immunoprecipitating antibody
masked detection of either the ET, or ET, receptor antibody. Based on our
receptor co-Iocalization experiments I concluded that ET, and ET, receptors co-
localize to the membrane in both aorta and vena cava, but I was unable to
determine if the receptors are within close enough proximity for receptor

heterodimerization to occur.

107

 

ColP Method: Successful? Problem with technique
. . Heavy chain IP antibody band
Traditional IF NO contamination
. Secondary antibody not
True Blot colP kit NO specific
Profound Mammalian NO Sample too diluted —
colP kit No signal detected
ExactaCruz homologous .
colP kit NO Did not work
Poor resolution of protein
NATIVE PAGE NO bands
. . Significant protein degradation
Receptor cross-linking NO during cross-linking
Dynal magnetic beads NO

Table 4. Different co-immunoprecipitation methods were performed in several

attempts to co-immunoprecipitate ET, and ET, receptors from rat thoracic aorta

and vena cava lysates.

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IV. DISCUSSION

A. Ratlonale

In the United States, approximately 30% of Americans have hypertension. A
small percentage of hypertensive patients have primary hypertension, in which a
single known gene is responsible for increasing blood pressure. However, most
patients are diagnosed with essential hypertension and there is no known
etiology for their elevated blood pressure. Anti-hypertensive drugs such as
calcium channel blockers, beta adrenergic receptor blockers, diuretics and
angiotension converting enzyme inhibitors and receptor blockers are available
but typically a combination of two or more of the above mentioned anti-
hypertensive drugs are required to achieve adequate decreases in blood
pressure as recommended by the Seventh Report of the Joint National
Committee on Prevention, Detection, Evaluation, and Treatment of High Blood
Pressure. Hypertension is a risk factor for many diseases including heart failure,
stroke, renal failure, etc. Thus, inadequate control of blood pressure leaves
patients at significantly higher risks of negative outcomes and increased risk of
death. Clearly, more effective treatments for controlling high blood pressure are

needed.

The endothelin system represents a potential target for anti-hypertensive therapy

that has not yet been aggressively pursued in the clinical arena. While

109

 

endothelin receptor antagonists are currently approved for the treatment of
pulmonary hypertension, they are not available for the treatment of systemic
hypertension. The mechanism of action of current anti-hypertensive drugs is
primarily to reduce arterial tone to reduce total peripheral resistance and blood
pressure. I posit that veins, specifically the endothelin receptors on veins, play
an important role in regulating and elevating blood pressure. I hypothesize that
the endothelin receptors, the ET, and ET, receptors, on veins interact via
receptor heterodimerization to alter the pharmacology of these receptors. I also
hypothesize that ET, and ET, receptors do not heterodimerize on arteries,
making the venous endothelin receptor system a unique target for anti-

hypertensive therapies.

I began this study by choosing a model artery and a model vein to compare
endothelin receptor heterodimerization. I chose the rat thoracic aorta as a model
artery and the rat thoracic vena cava as a model vein. Though both of these
vessels are considered to be conduit vessels and do not directly determine
arterial resistance or venous compliance, both vessels (rat thoracic aorta and
vena cava) were large enough to provide adequate protein yields for biochemical
and functional assays to assess receptor heterodimerization. Western blot
analysis and immunohistochemical analysis demonstrated that rat thoracic aorta
and vena cava both express ET, and ET, receptors, suggesting that these

vessels were appropriate models for studying endothelin receptors (Flgures 11

110

and 12). Preliminary contractility experiments suggested that while rat thoracic
aorta express both ET, and ET, receptors, only ET, receptors couple to
contraction as ET, receptor agonists do not alter tone in aorta under basal tone
(Watts et al, 2002). Differently from rat thoracic aorta, in rat thoracic vena cava
both ET, and ET, receptors couple to contraction. This initial observation of a
difference in function of ET, and ET, receptors in arteries compared to veins
prompted the idea that perhaps the two receptor subtypes interact in veins

differently than in arteries.

B. leferences In ET, and ET, receptor desensltlzatlon

To enhance our understanding of the nature of a possible functional interaction
between ET, and ET, receptors in vena cava but not aorta, I performed
contractility studies examining ET, and ET, receptor-mediated contraction.
Thoracic aorta from DOCA-salt hypertensive rats display significantly reduced
contraction to ET-t when compared to their normotensive counterparts, while ET-
1-induced contraction was not different between vena cava from DOCA-salt
hypertensive rats and their normotensive counterparts (Watts 9! al, 2002). I
hypothesized that the difference in response to ET-1 in arteries and veins from
DOCA-salt hypertensive rats could be due to the presence of functional ET,
receptors that couple to contraction on veins but not on arteries. The first
contractility experiments to assess receptor desensitization were designed to

mimic the high levels of plasma ET-1 in DOCA-salt hypertension. Aorta and

111

vena cava were desensitized with a concentration of ET-1 that induced maximal
contraction, the ET-1 was washed out until tone returned to baseline, vessels
were challenged again with ET-1 and the contraction to ET-1 after desensitization
quantified. Aorta desensitized with ET-1 did not contract to an additional ET-1
challenge, while vena cava contracted with ET-1 were able to contract to
additional ET-1 stimulation, though the magnitude of contraction was significantly

reduced (Flgure 15).

l hypothesized that functional (contractile) ET, receptors on vena cava might
mediate the remaining venous contraction to ET -1 after ET-1 desensitization
since aorta (where only ET, receptors couple to contraction) completely
desensitize to ET -1. Through the use of specific receptor antagonists I
determined that the remaining ET -1 contraction in veins after ET-1
desensitization was mediated by ET, receptors (Flgure 19), suggesting that the
contractile ET, receptors on vena cava were not responsible for the remaining
contraction to ET-1 after desensitization. ET, receptor desensitization
experiments with the ET, receptor agonist sarafotoxin 60 (S60) demonstrated
that the venous ET, receptor does in fact desensitize (Flgure 20). However, the
resensitization time frame is much shorter than that of the ET, receptor,
highlighting an important difference between ET, and ET, receptor

desensitization and resensitization.

112

Classical G-protein receptor desensitization occurs through receptor
internalization, initiating with G-protein kinase (GRK) phosphorylation of the
cytoplasmic tail of the receptor, allowing B-arrestin binding and receptor
internalization via dynamin/clathrin-coated pits. Sequence analysis of the
cytoplasmic tails of the ET, and ET, receptors suggest that because of low
sequence homology in this region, ET, and ET, receptors may be
phosphorylated by different GRK isoforms and internalized differently. Freedman
et al demonstrated that when ET, and ET, receptors were overexpressed in
HEK293 cells, both receptors were primarily phosphorylated by GRK2,
suggesting that despite sequence differences, ET, and ET, receptor
desensitization is initiated by the same GRK (Freedman et al, 1997). However,
Bremnes at al observed that when ET, and ET, receptors were overexpressed in
CHO or COS cells, intracellular trafficking of the ET, and ET, receptors differed.
While both receptors where similarly internalized via an arrestin/dynamin-
dependent pathway, ET, receptors were recycled and reinserted into the plasma
membrane and ET, receptors targeted to Iysosomal compartments (Bremnes et
al, 2000), demonstrating that following internalization the fates of the ET, and
ET, receptor significantly differed. Since most of the experiments investigating
ET, and ET, receptor desensitization and internalization have been performed in
cells lines overexpressing ET, and ET, receptors, it is unknown if the conclusions
from these experiments correlate to ET, and ET, receptor trafficking in tissues

such as the rat thoracic aorta and vena cava. It is possible that ET, receptors in

113

veins are recycled and reinserted in the plasma membrane or there may be a
large pool of intracellular ET, receptors ready to be inserted in the plasma
membrane after desensitization and lysosomal degradation of ET, receptors.
Our desensitization experiments highlight differences between aortic and venous
ET, and ET, receptor function and suggest that the presence of a functional ET,
receptor (as is present on the vena cava) might alter the function of the ET,

receptor.

C. Functlonal ET, and ET, receptor Interactlon occurs In velns but not
arteries

To further test the hypothesis that functional ET, and ET, receptor interaction
occurs in veins but not arteries to alter the pharmacology of the receptors, I
designed contractility experiments utilizing selective ET, and ET, receptor
antagonists. In these experiments, the ability of an ET, receptor antagonist,
atrasentan, to shift ET -1-induced contraction was compared in aorta and vena
cava with unbound and agonist-bound ET, receptors (accomplished by
desensitization with S6c). I observed that in aorta, ET, receptor blockade
(determined by comparing EC50 values) was not significantly different whether
ET, receptors were unbound or agonist-bound (desensitized with $60) (Flgure
21). Differently from the aorta, in vena cava ET, receptor blockade was

quantitatively greater when ET, receptors were agonist-bound (S6c desensitized)

114

compared to when ET, receptors were unbound (Flgure 22), suggesting that ET,

and ET, receptors functionally interact in vena cava but not aorta.

As another way of assessing ET, receptor function (via atrasentan inhibition of
ET-1-induced contraction) in the presence and absence of ET, receptors, ET,
receptor blockade with the selective ET, receptor antagonist 80-788. In aorta,
the degree of ET, receptor-inhibited ET-1-induced contraction was not different
whether ET, were not blocked (vehicle incubated) or blocked (BO-788 incubated)
(Figure 23). However, in vena cava, ET, receptor antagonism inhibited ET-1-
induced contraction to a significantly greater extent when ET, receptors were
rendered blocked with BQ-788 (100 nM), compared to vehicle-incubated vena
cava without ET, receptor blockade (Figure 24). These data support the idea
that functional ET receptor interaction occurs in vena cava but not aorta. It was a
goal of this dissertation to understand why and how this receptor interaction

occurs in veins but not arteries.

Our studies are not the first to describe functional ET, and ET, receptor
interaction. Lodge at a/ initially described in rabbit saphenous vein and jugular
vein (vessels that have both contractile ET, and ET, receptors) a population of
ET, receptors that were only sensitive to ET, receptor blockade when ET,
receptors were either desensitized or antagonized (Lodge at al, 1995). In

vessels with only contractile ET, receptors, such as the rat aorta and rabbit

115

 

carotid artery, ET, receptor desensitization or inhibition had no effect on ET,
receptor function (Lodge at al, 1995). Adner at al observed in small mesenteric
arteries that ET, receptor desensitization increased ET -1 potency and the
apparent affinity of an ET, receptor antagonist (Adner at al, 2001). Receptor
“cross-talk" between the ET, and ET, receptors has also been described to occur
in rat trachea where ET, receptor or ET, receptor antagonism alone was not
sufficient to inhibit ET -1 -induced contraction, but dual ET, and ET, receptor
blockade significantly inhibited ET-1-induced contraction (CIozeI and Gray,
1995). Similar results were observed in human isolated bronchi (Fukuroda et al,
1996), rat mesenteric veins but not arteries (Claing at al, 2002), mouse
mesenteric veins but not arteries (Perez-Rivera et al, 2005), rat renal afferent but
not efferent arterioles (lnscho at al, 2005) and pulmonary arteries (Sauvageau at
al, 2006), suggesting that ET, and ET, receptor “cross-talk" or interaction may
occur in vessels that have both functional ET, and ET, receptors. Why some
vessels have both contractile ET, and ET, receptors and others only have
contractile ET, receptors (I would hypothesize veins and arteries, respectively) is

still unknown.

D. Signaling pathway Interactions may account for functlonal venous ET,
and ET, receptor lnteractlon
One possible explanation for how ET, and ET, receptors functionally interact in

vena cava but not aorta is that cross-talk or interaction between ET, and ET,

116

 

receptor-induced signal transducers could occur. The ET, and ET, receptors
activate multiple signal transduction pathways such as phospholipases (A, B and
D), MAPK kinases (Erk, JNK and p38), PKC, rho kinase and tyrosine kinases
such as src (Figures 8-10) via coupling to several G-proteins, including G, 6,,
G, and (5,2,3. ET-1 also increases intracellular calcium through opening of non-
selective cation channels, L-type voltage gated Ca2+ channels, and Ca2+ release
from intracellular stores (both lP,-sensitive Caz” channels and ryanodine-
sensitive Ca2+ channels) (Neylon, 1999). ET-1 also stimulates production of
reactive oxygen species (Li et al, 2003a; Thakali at al, 2005; Wedgwood et al,
2001), which can potentially induce signaling in addition to what is activated by
ET-1 itself. I envisioned in vena cava, which have both functional ET, and ET,
receptors, ET-1-activated signal transduction pathways coupling to contraction
would differ from aorta that only have functional ET, receptors. I also
hypothesized that differences in ET -1 signaling between aorta and vena cava
could explain how functional ET, and ET, receptor interaction occurs in vena

cava but not aorta.

1. Reactlve oxygen specles slgnallng
Several groups have reported that ET-1 via ET, receptors activates the
superoxide-producing enzyme NADPH oxidase (Banes-Berceli er al, 2005; Li et
al, 2003a; Loomis at al, 2005; Wedgwood ef al, 2001). Superoxide, the product

of NADPH oxidase activity, H202 generated by the catalytic reduction of

117

 

superoxide by superoxide dismutase, and hydroxyl radical are examples of ROS
that can alter vascular tone (Ardanaz and Pagano, 2006; Lyle and Griendling,
2006). Superoxide can directly constrict smooth muscle as superoxide
generating systems constrict endothelium-denuded arteries (Jin et al, 2004), and
can potentiate constriction by quenching nitric oxide, the endothelium derived
relaxing factor. H202 has been reported to induce both contraction and
relaxation, depending on experimental conditions, species studied and vascular
bed studied (Lucchesi er al, 2005; Gil-Longo and Gonzalez-Vazquez, 2005). |
hypothesized that ROS production would differ between aorta and vena cava
because of the different complement of functional ET receptors in these two
vessels. I chose to focus on the ability of ET-1 in aorta and vena cava to
stimulate production of H202 instead of superoxide for the following reasons: 1)
H202 is not a free radical like superoxide and is inherently more stable than
superoxide; 2) H20, has a larger diffusion radius compared to superoxide; and 3)
H202 can cross membranes. Thus, in aorta and vena cava I compared basal
levels of H202 production, ET-1-stimulated H202 production, the ability of
exogenously added H202 to alter vascular tone, and whether H202 mediates ET-

1-induced contraction.

When basal H202 levels were measured using the Amplex Red H202 assay, I

observed that basal levels of H20, production were significantly higher in vena

cava compared to aorta (Flgure 26). Measurable H202 levels were nearly

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A

 

completely reduced when vessels were incubated with diethyldithiocarbate (DDC
or DETC), an inhibitor of superoxide dismutase (the enzyme that catalyzes the
hydrolysis of superoxide to H202), suggesting that the primary source of H202 in
both aorta and vena cava was via the reduction of superoxide. In isolated tissue
bath experiments, exogenously added H20, contracted both rat thoracic aorta
and vena cava and the contraction in vena cava was much more robust than that
in aorta (when the contraction to H202 was normalized to maximal contraction to
an adrenergic agonist) (Flgure 25). I observed that ET -1 increased H20,
production in vena cava but not aorta (Flgure 27). While the ET-l-stimulated
H202 production in vena cava was not concentration-dependent (for the
concentrations investigated), inhibitors of ET, and ET, receptors significantly
reduced ET-1-induced H20, production, suggesting that ET-1-stimulated H202
production was not a non-specific effect of ET-1 (Flgure 28). Since ET-1
stimulated H202 production in vena cava and exogenous H20, constricted vena
cava l hypothesized that venous ET-1-induced contraction might be partially
mediated by ET -1-stimulated H202. Thus, removing or reducing endogenous
H202 with catalase should have reduced venous ET -1-induced contraction.
Contrary to what I expected, when vena cava were incubated with catalase or
PEG-catalase (the PEG moiety supposedly increases cell permeability),
maximum ET -1 -induced contraction was enhanced, suggesting that endogenous
H20, may mediate vasodilation in these vessels (Figure 29). In the presence of

3-aminotriazole, a catalase inhibitor, venous ET -1-induced contraction was not

119

 

altered from vehicle-incubated vena cava (Flgure 30). Collectively, these data
suggest that though ET-1 stimulates H202 production in vena cava and
exogenous H202 constricts vena cava, it is unlikely that ET-1-stimulated H20,

mediates venous ET -1 -induced contraction.

From these experiments I concluded that ET-1 signaling via H202 production was
not a likely mediator of ET, and ET, receptor functional interaction in vena cava.
It is possible that ET -1 activates signaling pathways through the production of
other reactive oxygen species such as superoxide or hydroxyl radical thus I may

have limited our conclusions by only investigating the actions of H202.

2. ET-1 slgnal transduction
Since ET -1-stimulated H202 signaling does not explain why functional ET, and
ET, receptor interaction occurs in vena cava, I chose to examine which specific
signal transducers were activated by ET, and ET, receptors that couple to
contraction in aorta and vena cava. I chose to investigate the involvement of p38
MAPK, Erk MAPK, src, rho kinase and PI3-K in mediating ET-1-induced
contraction in aorta and vena cava because these pathways are known to couple
to smooth muscle contraction (Somlyo and Somlyc, 2000; Hilgers and Webb,
2005), have been reported to mediate ET-t-induced contraction (Yamboliev at al,
2000; Zubkov et al, 2000; Miao ef al, 2002; Kawanabe at al, 2004, Kodama er al,

2003), and are important in arterial hyperresponsiveness in hypertension

120

 

(Northcott er al, 2002; Touyz, 2003; Kwon at al, 2004; Wehrwein at al, 2004).
While there are many reports investigating ET-1 signal transduction in the
literature, most of these reports recount ET-1 signaling in cells over-expressing
ET, and ET, receptors and cultured smooth muscle cell lines, I.e. they have
investigated ET-1 signal transduction under non-physiological conditions. Of the
reports regarding ET-1 signaling, different vascular beds have been studied,
different species of animals have been studied, and experimental protocols vary
enough such that a generalized statement summarizing ET receptor signaling in

the vasculature would likely be unfair to make.

To determine which signaling pathways mediated aortic and venous ET-1-
induced contraction, I compared the ability of specific signaling inhibitors to
reduce ET -1-induced contraction. I observed that pharmacological inhibition of
rho kinase significantly inhibited both aortic and venous ET-1-induced contraction
(Figure 35) and p38 MAPK inhibition marginally reduced maximal aortic ET-1-
induced contraction (Flgure 34). Inhibitors of Erk MAPK, src and Pl3-K did not
alter maximal aortic or venous ET-t-induced contraction (Flgures 31-33),
suggesting that activation of these pathways is not necessary for aortic or venous
ET-1-induced contraction. Using cultured aortic smooth muscle cells, our group
previously verified that the concentrations of inhibitors used caused selective
inhibition of the corresponding signaling molecule (Watts, 1996; Banes et al,

1999; Florian and Watts, 1999; Northcott et al, 2002) and except for the rho

121

 

kinase inhibitor Y27632, did not reduce non-receptor mediated contraction (KCI-
induced contraction, data not shown). Since the rho kinase pathway was
similarly involved in mediating ET-1-induced contraction in both aorta and vena
cava and p38 MAPK played only a minor role in aortic ET-1-induced contraction,
I chose not to further investigate specific ET, and ET, receptor signaling. I
concluded that ET -1-mediated signal transduction via Erk MAPK, src and Pl3-K
were not required for aortic and venous ET -1-induced contraction, thus these
pathways are likely not responsible for ET, and ET, receptor interaction in vena

cava.

I expected that since vena cava have both contractile ET, and ET, receptors and
aorta only have contractile ET, receptors, ET -1 -induced signaling and contraction
in vena cava would be mediated by several signaling transduction pathways that
would be distinctly different from ET, receptor mediated signaling and contraction
in aorta. However, of the signaling pathways investigated, both aortic and
venous ET-t-induced contraction was similarly dependent on rho kinase activity,
suggesting that signaling interactions through the rho kinase, p38 MAPK, Erk
MAPK, src and Pl3-K pathways are not likely responsible for mediating functional
venous ET receptor interaction. There are other mechanisms/signaling pathways
involved in smooth muscle contraction that I did not investigate. It is possible that
by only investigating a select number of signaling pathways, l have overlooked

other pathways that contribute to ET, and ET, receptor interaction in vena cava

122

 

w——~

or that there yet undiscovered pathways mediating ET, and ET, receptor

contraction in both aorta and vena cava.

An important mechanism of smooth muscle contraction that I chose not to
investigate was the increase in intracellular calcium. ET -1-induced contraction
and increases in intracellular calcium in dissociated rat mesenteric arteries and
veins was insensitive to L-type calcium channel blockade with nifedipine (Claing
et al, 2002), Supporting previous studies by Guilumian at al who demonstrated
that ET -1-induced contraction in small mesenteric arteries was dependent on
sarcoplasmic reticular ryanodine receptor activation and intracellular calcium
release (Giulumian at al, 2000). However, in porcine coronary arteries,
extracellular calcium influx mediated ET-1-induced contraction as voltage-gated
calcium channel blockers reduced ET -1-induced contraction (Kasuya at al, 1989),
suggesting that cellular mechanisms regulating ET -1-induced increases in
intracellular calcium vary depending on species and vascular bed studied. Claing
et al observed that ET, receptor activation increased intracellular calcium in
mesenteric vein smooth muscle cells but not in mesenteric artery smooth muscle
cells (Claing at al, 2002), suggesting that there may be differences in ET -1-
induced calcium signaling in arteries and veins because of the presence of both

contractile ET, and ET, receptors on veins.

123

 

l have preliminary evidence suggesting that H202-induced contraction in aorta
and vena cava differ in their dependence on extracellular Ca2+ influx. Vena cava
contract to H202 in the presence of L-type Ca2+ blockers and when Ca2+ is omitted
from PSS, while aortic contraction to H202 (after KCI contraction) is completely
inhibited when Ca'“ is removed from PSS (data not shown), suggesting that aorta
require extracellular Ca2+ influx for H202-induced contraction, while vena cava do
not. While it is likely the conclusions I can make regarding differences in Ca2+
handling in aorta and vena cava may only be limited to H202-induced contraction,
the source intracellular Ca“ (extracellular influx vs. release of intracellular stores)
may be an important difference between contraction of aorta and vena cava and
may provide an explanation to why functional ET, and ET, receptor interaction is

observed in vena cava but not aorta.

E. Can ET, and ET, receptors physlcally Interact vla' mceptor
heterodlmerlzatlon?

Another possible explanation for why I observed functional ET, and ET, receptor
interaction in vena cava but not aorta is that ET, and ET, receptors might
physically interact in vena cava via receptor heterodimerization. G-protein
receptor dimerization, originally proposed in the early 1980s, has recently
emerged as an important phenomenon that regulates the function and
pharmacology of G-protein coupled receptors. Several G-protein coupled

receptors have been reported to homodimerize (two receptors of the same

124

 

 

 

subtype physically interacting), heterodimerize (two receptors of different
subtypes physically interacting), and oligomerize (multiple receptors physically
interacting with each other). The best characterized example of receptor
homodimerization is that of B, adrenergic receptors (reviewed by Prinster et al,
2005). Receptor heterodimerization of GABA,1 and GABA,, receptors, (3,
adrenergic receptors and angiotensin (AT)1 receptors, taste receptors
(T1 R2” 1 R3; T1R1/T 1 R3), 6, adrenergic receptors and opioid (5 and x)
receptors, opioid receptors (M: and 6m), somatostatin receptors, purinergic
receptors and olfactory receptors have been investigated in depth (reviewed by
Prinster et al, 2005). Receptor dimerization can alter how receptors function by
altering downstream signal transduction events, receptor internalization, receptor
localization (specifically endoplasmic reticulum retention and membrane
trafficking), and receptor pharmacology (agonist affinity and efficacy) (Angers et
al, 2002; Milligan, 2004; Maggio et al, 2005; Prinster at al, 2005). Currently,
reports of G-protein coupled receptor dimerization are primarily limited to cells
systems overexpressing receptors. Heterologous overexpression systems are
excellent tools for assessing receptor dimerization because these systems can
be easily modified and manipulated to determine how GPCRs can physically
interact with each other. One caveat to using heterologous overexpression
systems is that these are immortalized cell lines, with significantly different
properties from the native cells or tissues where GPCRs would normally exist. In

the few studies investigating the physiological significance of receptor

125

 

 

 

dimerization, conclusions are typically based on indirect observations of receptor
function, thus the physiological significance of receptor dimerization has not yet
been fully explored and heterologous overexpression systems are currently the
best available tools for assessing GPCR dimerization. It is important to note that
of all available prescription drugs, approximately half of these rely on G-protein
coupled receptors to achieve their therapeutic effects, thus receptor dimerization

may be of clinical significance.

Like many other G-protein coupled receptors, the ET, and ET, receptors, when
over-expressed in HEK293 cells, can heterodimerize (Gregan et al, 2004).
Gregan er al demonstrated that while ET, and ET, receptor heterodimerization
was constitutive, prolonged ET, receptor activation caused the ET, and ET,
receptor heterodimer to dissociate, likely due to ET, receptor internalization
(Gregan et al, 2004). Dai at al confirmed that the ET, and ET, receptors co-
localize (suggesting that the ET, and ET, receptors heterodimerize) in an
independent study over-expressing human ET, and ET, receptors in HEK293
cells and that co-transfection of ET, and ET, receptors altered receptor
desensitization and trafficking (Dai and Galligan, 2006). While these studies by
Gregan er al and Dai at al demonstrate that in fact ET, and ET, receptors can
heterodimerize and colocalize, respectively, whether ET, and ET, receptors
heterodimerize in tissues, such as rat thoracic aorta or vena cava, and whether

receptor heterodimerization alters receptor or tissue function is still unknown.

126

 

 

 

The following approaches are generally accepted for assessing G-protein
coupled receptors dimerization: 1) there should be some measure of functional
receptor interaction (though personal interpretation of functional responses can
be highly variable); 2) the receptors should co-immunoprecipitate/co-localize; and
3) fluorescence resonance energy transfer (FRET) or another measure of
receptor proximity (such as BRET) should demonstrate that the receptors are
within close proximity to each other (Milligan and Bouvier, 2005). Taking these
guidelines in to consideration, I initially proposed to perform co-
immunoprecipitation experiments with the hypothesis that ET, and ET, receptors
would co-immunoprecipitate in venous but not aortic lysates. I also proposed to
perform FRET experiments with fluorescently labeled ET, and ET, receptors on
aortic and venous vascular smooth muscle cells to demonstrate that ET, and ET,
receptors were in closer proximity to each other in venous compared to aortic
vascular smooth muscle cells because of receptor heterodimerization. Both the
co-immunoprecipitation and FRET experiments relied on the use of antibodies to
selectively detect ET, and ET, receptors. Antibody selectivity can often be
questionable and since the sequences of the human ET, and ET, receptor are
approximately 60% homologous, I compared the selectivity of several
commercially available ET, and ET, receptor antibodies. Also, FRET
experiments required ET, and ET, receptor antibodies that were raised in

different species. I performed a series of dot blots on which increasing amounts

127

of antigenic peptides (which the different antibodies were raised against) were
spotted on a nitrocellulose membrane. Based on the dot blot results (Figures 37
and 38) I decided to use the Alomone ET, and ET, receptor primary antibodies
for co-immunoprecipitation experiments and the Fitzgerald ET, and Alomone ET,

receptor primary antibodies for co-Iocalization experiments.

1. Co-lmmunopreclpltatlon of ET, and ET, receptors In rat thoracic
aorta and vena cava
Since I hypothesized that ET, and ET, receptors would heterodimerize in veins
but not arteries, I expected that ET, and ET, receptors would co-
immunoprecipitate in vena cava but not aorta. I initially immunoprecipitated the
ET, receptor using the Alomone ET, receptor antibody and asked if the ET,
receptor (detected with Alomone ET, receptor primary antibody) co-
immunoprecipitated with the ET, receptor. Unfortunately, contamination with the
50 kDa heavy chain of the immunoprecipitating antibody prevented detection of
either the ET, and ET, receptors as both of these proteins also resolve at
approximately 50 kDa. Since the traditional immunoprecipitation method was not
successful for determining if ET, and ET, receptors co-immunoprecipitate, I
tested several commercially available co-immunoprecipitation kits to attempt to
avoid detection of the immunoprecipitating antibody (Table 4). Unfortunately,

none of these kits/techniques were successful in detecting co-

128

immunoprecipitated ET, and ET, receptors, and in fact none of these kits were

successful in confirming/detecting a successful immunoprecipitation.

I also attempted to crosslink ET, and ET, receptors with the idea that if the
receptors were in close proximity of each other, cross-linking them would slow
their migration through a polyacrylamide gel. This would allow us to detect cross-
Iinked ET, and ET, receptors that should theoretically migrate at a higher
molecular weight than uncross-Iinked ET, and ET, receptors, resolving
immunoreactive bands for the ET, and ET, receptors separately from the
immunoprecipitating antibody band. However, after incubating both vessels and
lysates with cross-linkers, the degree of protein degradation was too high for
Western blot analysis. It is probable that I chose the inappropriate cross-linker
for cross-linking ET, and ET, receptors, as how ET, and ET, receptors physically
associate is still unknown. It is also possible that ET, and ET, receptor
heterodimerization may be an artifact of receptor overexpression in a cell line and

does not occur in blood vessels.

2. Receptor co-locallzatlon
In conclusion, our receptor co-immunoprecipitation experiments were
inconclusive as l was unable to either confirm or deny if ET, and ET, receptors
co-immunoprecipitate in vena cava but not aorta. Since the co-

immunoprecipitation experiments were inconclusive I next attempted receptor co-

129

localization and FRET experiments to determine if ET, and ET, receptors
physically interact via receptor heterodimerization in vena cava but not aorta.
Ideally I would have performed receptor co-localization and FRET analysis on
paraffin-embedded sections of rat thoracic aorta and vena cava to determine
where, in whole vessels, ET, and ET, receptors were located. However, the
collagen and elastin fibers of blood vessels autofluoresce at the wavelengths of
commonly used fluorescent secondary antibodies. l was primarily concerned
with the location of ET, and ET, receptors on the smooth muscle cells of aorta
and vena cava, as l have assumed that vascular smooth muscle cells are the cell
types where ET, and ET, receptors would heterodimerize and affect receptor
function. I enzymatically dissociated vascular smooth muscle cells from rat
thoracic aorta and vena cava, adhered the cells to coverslips and performed
immunocytochemistry experiments on fixed vascular smooth muscle cells. I
labeled the ET, and ET, receptors with the Fitzgerald ET, and Alomone ET,
receptor primary antibodies, respectively and labeled the plasma membrane with
an antibody for pan-cadherin (a membrane marker). Confocal images of 6
micron-thick sections of the whole smooth muscle cell (from the top to bottom)
were captured for comparison of the cellular location of ET, and ET, receptors

from aortic and venous vascular smooth muscle cells.

Surprisingly, I observed that in both aortic and venous vascular smooth muscle

cells, both ET, and ET, receptors co-localized at the plasma membrane (Flgures

130

40 and 41). I observed that ET, receptors in both aorta and vena cava were
primarily located at the membrane (co-localized with pan-cadherin staining),
while ET, receptors were found both at the plasma membrane and intracellularly.
The membrane marker, pan-cadherin, was present at the plasma membrane of
vascular smooth muscle cells, but was also present intracellularly. It is possible
that the intracellular presence of ET, receptors and pan-cadherin may be an
artifact of our immunocytochemistry protocol (perhaps resulting from fixation or
cell permeabilization) or may be due to antibody non-specificity (though our dot
blots suggest that the Alomone ET, receptor antibody is selective for its antigenic
sequence). Human ET, receptors overexpressed in HEK293 cells were primarily
expressed on the plasma membrane, while ET, receptors overexpressed in
HEK293 cells were present equally expressed on the plasma membrane and
intracellular compartments (Dai and Galligan, 2006). When both ET, and ET,
receptors were co-transfected in HEK293 cells, both receptors were present at
the plasma membrane, suggesting that ET, receptor expression was necessary

for membrane targeting of the ET, receptor (Dai and Galligan, 2006).

l was also interested in determining if ET, and ET, receptors in aorta and vena
cava internalize differently after ET-1 stimulation. I observed that stimulation of
aortic and venous vascular smooth muscle cells with ET-1 did not qualitatively
alter ET, or ET, receptor location, I.e. ET, and ET, receptors co-localized at the

plasma membrane in both aortic and venous vascular smooth muscle cells even

131

after ET-1 stimulation (Figures 42 and 43). Our observations are in agreement
with Gregan et al who observed that ET, and ET, receptors remained at the
plasma membrane even after 60 minutes of ET-1 stimulation in HEK293 cells co-
transfected with ET, and ET, receptors (Gregan at al, 2004). Interestingly,
Gregan et al observed that selective ET, receptor activation induced ET,
receptor internalization in co-transfected cells, suggesting that selective ET,

receptor activation disrupted the constitutive ET,/ET, receptor heterodimer.

There are other possible explanations for why I did not observe ET, and ET,
receptor internalization after ET-1 stimulation, the simplest but not necessarily
the correct explanation being that ET, and ET, receptors on vascular smooth
muscle cells do not internalize with ET -1 stimulation, though the contractile
response to ET-1 desensitizes. Immunocytochemistry experiments in freshly
dissociated vascular smooth muscle cells were performed in the presence of
sodium nitroprusside, a nitric oxide donor, and in the absence of Ca2+ to optimize
imaging conditions of the vascular smooth muscle cells. To mt knowledge, it is
unknown if the presence of nitric oxide or the absence of Ca2+ acutely affects ET,

and ET, receptor signaling, localization and internalization.
While immunocytochemistry results suggested that in both aorta and vena cava

ET, and ET, receptors co-localize to the plasma membrane of vascular smooth

muscle cells, the resolution (4000 — 7000 A) of conventional confocal microscopy

132

does not give any information about molecular receptor proximity. I proposed to
perform FRET experiments where one receptor subtype (for example the ET,
receptor) was labeled with a donor fluorophore and the other receptor subtype
(the ET, receptor) was labeled with an acceptor fluorophore and the donor and
acceptor fluorophores have overlapping excitation/emission spectra. If the two
receptors are within close enough proximity (within a nanometer range) (Wallrabe
and Periasamy, 2005), specific excitation of the donor fluorophore will cause the
donor fluorophore to emit energy that will in turn increase acceptor fluorescence
while quenching donor emission. Successful FRET experiments depend on
several factors including primary antibody binding efficiency, fluorophore dipole
alignment, distance between donor and acceptor fluorophores (which becomes
an issue when fluorescent secondary antibodies are used to detect primary
antibody binding) and spectral bleed through due to spectral overlap of donor and

acceptor fluorophores.

In immunocytochemistry experiments of dissociated aortic and venous vascular
smooth muscle cells I labeled the ET, receptor with the Alexa488® secondary
antibody and the ET, receptor with the Alexa555® secondary antibody. The
Alexa fluorophores are brighter than other fluorophores that emit at similar
wavelengths, are more photostable (resistant to bleaching) (Panchuk—Voloshina
ef al, 1999), and this pair of donor/acceptor fluorophores

(Alexa488®/Alexa555®) has been successfully used in FRET experiments

133

(Wallrabe and Periasamy, 2005). In our experiments with fluorescently labeled
ET, and ET, receptors on aortic and venous vascular smooth muscle cells, I did
not observe significant bleed through between the two fluorescent secondary
antibodies (Alexa488® and Alexa555®) or background fluorescence, but the
intensity of fluorescence was very low. Because of the low fluorescence intensity
from labeled ET, and ET, receptors, I concluded that FRET experiments were

not likely to be successful.

F. Conclusions

I hypothesized that ET, and ET, receptors heterodimerize in rat thoracic vena
cava but not aorta, and that receptor heterodimerization would alter ET, and ET,
receptor function in vena cava. l generated contractility data that suggests that
ET, and ET, receptors functionally interact in vena cava but not aorta. In vena
cava, when ET, receptors were rendered nonfunctional, either through receptor
desensitization or receptor blockade, ET, receptors were more susceptible to
receptor blockade, suggesting that in vena cava, ET, receptors modulate ET,
receptor function. I investigated whether signal transduction interactions could
explain why ET, and ET, receptors functionally interact in vena cava, but
concluded that signaling via the following pathways — H202, p38 MAPK, Erk
MAPK, src, PI3-K and rho kinase - were not responsible for ET, and ET,
receptor cross-talk in vena cava. Receptor co-localization experiments

suggested that ET, and ET, receptors co-Iocalize at the plasma membrane of

134

both aortic and venous vascular smooth muscle cells, suggesting that receptor
location does not govern functional receptor interaction. Unfortunately, other
methods of assessing physical ET, and ET, receptor interaction, such as oo-
immunoprecipitation experiments and FRET experiments did not provide any
conclusive results regarding the physical association and proximity of ET, and

ET, receptors in aortic and venous vascular smooth muscle cells.

G. Speculatlon

It is possible that ET, and ET, receptors, when over-expressed at artificially high
levels heterodimerize with alterations in receptor pharmacology, but when these
receptors are normally expressed in tissues, like vascular smooth muscle cells,
they do not heterodimerize or their physical location does not determine receptor
function. It is also possible that I relied too heavily on the current tools available
to assess receptor heterodimerization, specifically commercially available ET,
and ET, receptor antibodies. I expect that future development of better
antibodies for the rat ET, and ET, receptors and novel co-immunoprecipitation
protocols will allow FRET experiments and receptor co-immunoprecipitation
experiments to be performed to determine if ET, and ET, receptors do in fact

heterodimerize in tissues such as the rat thoracic vena cava.

Future experiments investigating how ET, and ET, receptors increase

intracellular calcium in arteries and veins are likely important for understanding

135

how functional ET, and ET, receptor interaction occurs in rat thoracic vena cava.
In general, there is a lack of data surrounding venous vascular biology and to our
knowledge, there has yet been a comprehensive comparison of calcium handling

in arteries and veins.

The use of ET, receptor deficient rats may be useful in determining if ET, and
ET, receptors heterodimerize in vena cava but not aorta. I have preliminary
contractility data that suggests that homozygous ET, receptor deficient rats do
not exhibit functional ET, and ET, receptor interaction, unlike their homozygous
wild type counterparts, confirming the idea that a contractile ET, receptor is
required for ET,/ET, receptor interaction. Western blot analysis suggests that
both homozygous ET, receptor deficient and ET, receptor wild type rats express
the ET, receptor In vena cava, but in the homozygous ET, receptor deficient rats,
the ET, receptor is nonfunctional and does not induce contraction. In the future,
immunocytochemistry experiments should be performed to determine cellular
localization of ET, and ET, receptors in vena cava and aorta of homozygous ET,

receptor deficient and ET, receptor wild type rats.

Receptor dimerization is a nascent field in receptor characterization and has
more recently been recognized as a phenomenon with potentially clinical
ramifications as many therapeutic drugs target G-protein coupled receptors.

Most accounts of receptor dimerization have been reported in cell lines

136

overexpressing tagged receptors. Only a few reports question whether receptor
dimerization is a physiological phenomenon with functional consequences. While
our data suggest that functional ET, and ET, receptor interaction in vena cava
does not appear to be dependent on receptor co-localization, only with improved
tools to assess receptor dimerization, like better ET, and ET, receptor antibodies
and novel co-immunoprecipitation approaches, can I actually determine if ET,

and ET, receptors heterodimerize when natively expressed in tissues.

137

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VI. CURRICULUM VITAE

Keshari M. Thakali

PERSONAL DATA
Name: Keshari Maya Thakali B445 Life Sciences Bldg
Born: 8/4/81 Dept of Pharmacology and Toxicology
Michigan State University
East Lansing, MI 48824
Phone: (517) 353-3900
Fax: (517) 353-8915
E-mail: thakalik@msu.edu
EDUCATIONAL BACKGROUND
1998-2002 University of Arizona, Tucson, A2

8.8. (Environmental Science, Minor: Chemistry)

2002-present Michigan State University
Doctoral candidate (Pharmacology and Toxicology)

Project Title: “Endothelin A (E TA) and E T3 receptor
interaction in arteries and veins"

TEACHING ACTIVITIES
2004-2005 Tutor for Medical Pharmacology (PHM 563)
Michigan State University
2005 Tutor for Veterinary Pharmacology (PHM 556)
Michigan State University
2005-2006 Lecturer, Introduction to Chemical Toxicology (PHM 450)

148

Reproductive Toxicology Section

Michigan State University

RESEARCH TRAINING

2000—2002 Lab Assistant: Environmental Microbiology Laboratory,
University of Arizona. Isolation and characterization of multi-
copper oxidases in Escherichia coli. Supervisor: Dr.
Christopher Rensing.

2002 Research Rotation: Cardiovascular Pharmacology

2002-present

Laboratory, Michigan State University. Endothelin receptor
desensitization in aorta and vena cava.
Supervisor: Stephanie W. Watts, PhD

Doctoral Dissertation Research: Cardiovascular
Pharmacology Laboratory, Michigan State University.
Endothelin A (ETA) and ET, receptor interaction in arteries
and veins.

Mentor: Stephanie W. Watts, PhD

PROFESSIONAL ACTIVITIES
American Heart Association

American Physiological Society:

Student member of the Women in Physiology Committee

American Society for Pharmacology and Experimental Therapeutics:

Student member of the ASPET Centennial Committee

Reviewer for American Journal of Physiology Heart and Circulatory Physiology

Reviewer for Journal of Pharmacology and Experimental Therapeutics

Reviewer for Naunyn-Schmiedeberg's Archives of Pharmacology

149

ACADEMIC AND PROFESSIONAL HONORS

2006 NIDDK Minority Travel Award

2005 Caroline tum Suden/Francis Hellebrandt Professional
Opportunity Award

2005 NIDDK Minority Travel Award

2004-present Ford Foundation Predoctoral Diversity Fellowship

2004 NIDDK Minority Travel Award

2004 Michigan State University Chronic Disease Initiative
Fellowship

2003 Merck New Investigator Travel Award

2002-2003 Michigan State University Competitive Doctoral Enrichment
Fellowship

PRESENTATIONS

ERA

Thakali K Rondelli C, Fink GD, Watts SW. Chymase—dependent processing of
big ET-1 in arteries but not veins. 60‘“ Annual Fall Conference of the Council for

 

High Blood Pressure Research in Association with the Council on Kidney in
Cardiovascular Disease, 2006 (San Antonio, TX).

Thakali K, Lauren Davenport, Fink GD, Watts SW. Pleiotropic effects of hydrogen
peroxide in arteries and veins from normotensive and deoxycorticosterone
acetate-salt hypertensive rats. Michigan Hypertension Workshop; 2005 (Kellogg
Biological Institute, Michigan State University).

Thakali K, Fink GD, Galligan JJ, Watts SW. Arteries and veins desensitize

differently to endothelin. Michigan Colloquium on Pharmacology, 2003
(University of Michigan).

150

 

Thakali K, Fink GD, Galligan JJ, Watts SW. Arteries and veins desensitize
differently to endothelin. Michigan Hypertension Workshop; 2003 (Kellogg
Biological Institute, Michigan State University).

POSTERS
Thakali K Galligan JJ, Fink GD, Watts SW. Pharmacological endothelin receptor

 

interaction occurs in veins but not arteries. 60‘” Annual Fall Conference of the
Council for High Blood Pressure Research in Association with the Council on
Kidney in Cardiovascular Disease, 2006 (San Antonio, TX).

Thakali K, Davenport L, Fink GD, Watts SW. Multiple signal transduction
pathways are involved in hydrogen peroxide-induced contraction in rat thoracic
aorta and vena cava. Experimental Biology 2006 (San Francisco, CA).

Thakali K, Fink GD, Watts SW. K” channels differentially modulate contraction to
hydrogen peroxide (H202) in arteries and veins; effects in deoxycorticosterone
acetate (DOCA)-salt hypertension. 59‘" Annual Fall Conference of the Council for
High Blood Pressure Research in Association with the Council on Kidney in
Cardiovascular Disease, 2005 (Washington, DC).

Thakali K, Rondelli C, Fink GD, Watts SW. Endothelin converting enzyme (ECE)
is present and functional in rat thoracic aorta and vena mva. The Ninth
lntemational Conference on Endothelin (E T-9), 2005 (Park City, UT).

Thakali K, Fink GD, Watts SW. Endothelin-1 (ET-1) increases hydrogen peroxide
(H202) in veins, but not arteries. Experimental Biology 2005 (San Diego, CA).

Thakali K, Smark CT, Rondelli CM, Fink GD, Watts SW. Reduced aortic but not
venous contraction to big Endothelin-1 (ET-1) in deoxycorticosterone acetate
(DOCA)-salt hypertension: the role of endothelin converting enzyme (ECE). 58‘"
Annual Fall Conference of the Council for High Blood Pressure Research in

151

 

Association with the Council on Kidney in Cardiovascular Disease, 2004
(Chicago, IL).

Thakali K, Fink GD, Watts SW. Increased blood pressure causes changes in
aortic endothelin-1 (ET-1) and norepinephrine (NE) contractility in
deoxycorticosterone acetate (DOCA) salt hypertension. 14‘" Annual Phi Zeta
Research Day, 2004 (College of Veterinary Medicine, Michigan State University).

Thakali K, Fink GD, Watts SW. Increased blood pressure causes changes in
aortic endothelin-1 (ET-1) and norepinephrine (NE) contractility in
deoxycorticosterone acetate (DOCA) salt hypertension. Experimental Biology
2004 (Washington, DC).

Thakali K, Watts SW. ET-1 contraction signaling in veins is independent of
extracellular hydrogen peroxide (H202). 13‘" Annual Phi Zeta Research Day,
2003 (College of Veterinary Medicine, Michigan State University).

Thakali K, Ni W, Li M, Fink GD, Watts SW. The anorexigen (+)
norfenfluramine as a pressor; enhanced response in hypertension. 57‘”
Annual Fall Conference of the Council for High Blood Pressure Research in

Association with the Council on Kidney in Cardiovascular Disease; 2003
(Washington, DC).

Thakali K, Watts SW. ET-1 contraction signaling in veins is independent of
extracellular hydrogen peroxide (H202). 57‘" Annual Fall Conference of the
Council for High Blood Pressure Research in Association with the Council on
Kidney in Cardiovascular Disease; 2003 (Washington, DC).

Thakali K, Fink GD, Galligan JJ, Watts SW. Arteries and veins desensitize

differently to endothelin. XVth Scientific Meeting of the Inter-American Society
of Hypertension; 2003 (San Antonio, TX).

152

 

PAPERS

Thakali K, Davenport L, Fink GD, Watts SW. Cyclooxygenase, p38 MAPK, Erk
MAPK, rho kinase and src mediate hydrogen peroxide-induced contraction of rat
thoracic aorta and vena cava. J Pharmacol Exp Ther. 2006 Sep 26; [Epub ahead
ofpnnfl.

Thakali K, Lau Y, Fink GD, Galligan JJ, Chen AF, Watts SW. Mechanisms of
hypertension induced by nitric oxide (NO) deficiency: focus on venous function. J
Cardiovasc Pharmacol 2006, 47(6):742-750.

Thakali K, Davenport L, Fink GD, Watts SW. Pleiotropic effects of hydrogen
peroxide in arteries and veins from normotensive and deoxycorticosterone

acetate-salt hypertensive rats. Hypertension. 2006, 47(3):482—487.

Thakali K, Demel SL, Fink GD, Watts SW. Endothelin-1 (ET-1) —induced
contraction in veins is independent of hydrogen peroxide (H202). Am J Physiol
Heart Circ Physiol. 2005, 289(3):H1115-1 1 12.

Ni W, Li W, Thakali K, Fink GD, Watts SW. The fenfluramine metabolite (+)-
norfenfluramine is vasoactive. J Pharmacol Exp Ther. 2004, 309(2):845-852.

Thakali K, Fink GD, Galligan JJ, Watts SW. Arteries and veins desensitize
differently to endothelin. J Cardiovasc Pharmacol. 2004, 43(3):387-393.

Grass G, Thakali K, Klebba PE, Thieme D, Muller A, Wildner GF, Rensing C.
Linkage between catecholate siderophores and the multicopper oxidase CueO in

Escherichia coli. J Bacteriol. 2004, 186(17):5826-5833.

Roberts SA, Weichsel A, Grass G, Thakali K, Hazzard JT, Tollin G, Rensing C,
Montfort WR. Crystal structure and electron transfer kinetics of CueO, a

153

multicopper oxidase required for copper homeostasis in Escherichia coli. Proc
Natl Acad Sci U S A. 2002, 5;99(5):2766—2771.

BOOK CHAPTERS
Thakali K, Galligan JJ, Fink GD, Watts SW. Arterial and venous function in
hypertension; a vascular focus. Comprehensive Hypertension. (in press).

ABSTRACTS
Thakali K Galligan JJ, Fink GD, Watts SW. Pharmacological endothelin receptor
interaction occurs in veins but not arteries. [Abstract] Hypertens. 48:2006.

 

Thakali K, Rondelli C, Fink GD, Watts SW. Chymase-dependent processing of
big ET-1 in arteries but not veins. [Abstract] Hypertens. 48:2006.

Rondelli C, Pervaiz MH, Watson RE, Thakali K Fink GD, Watts SW. Veins are
not arteries: a story of remodeling in hypertension. [Abstract] Hypertens.
48:2006.

 

Thakali K, Davenport L, Fink GD, Watts SW. Multiple signal transduction
pathways are involved in hydrogen peroxide-induced contraction in rat thoracic
aorta and vena wva. The FASEB Journal, Abstract #9043, 2006.

Thakali K, Fink GD, Watts SW. K“ channels differentially modulate contraction to
hydrogen peroxide (H202) in arteries and veins; effects in deoxycorticosterone
acetate (DOCA)-salt hypertension. [Abstract] Hypertens. 46: 842, 2005.

Thakali K, Rondelli C, Fink GD, Watts SW. Endothelin converting enzyme (ECE)

is present and functional in rat thoracic aorta and vena cava. The Ninth
lntemational Conference on Endothelin (E T-9), 2005 (Park City, UT).

154

Thakali K, Fink GD, Watts SW. Endothelin-1 (ET-1) increases hydrogen peroxide
(H202) in veins, but not arteries. The FASEB Journal, Abstract #6862, 2005.

Kayal A, Rondelli CM, Thakali K, Watson RE, Rovner AS, Fink GD, Watts SW.
Myosin heavy chain B isoform is more predominant in rat veins than arteries. The
FASEB Journal, Abstract #1135, 2005.

Thakali K, Smark CT, Rondelli CM, Fink GD, Watts SW. Reduced aortic but not
venous contraction to big Endothelin-1 (ET-1) in deoxycorticosterone acetate
(DOCA)-salt hypertension: the role of endothelin converting enzyme (ECE).
[Abstract]. Hypertens. 44: 562, 2004.

Ni W, Li M, Thakali K, FinK, GD, and_Watts SW.: The fenfiuramine metabolite (+)
norfenfluramine is vasoactive. Fund. Clin. Pharmacol. 18(S1):p.139, A38, 2004.

Thakali K, Fink GD, Watts SW. Increased blood pressure causes changes in
aortic endothelin-1 (ET-1) and norepinephrine (NE) contractility in
deoxycorticosterone acetate (DOCA) salt hypertension. The FASEB Journal, 19,
Abstract #2018, 2004.

Smark CT, Thakali K, Watts SW. Role of caveolae in endothelin-1 (ET-1)-
induced contraction in arteries and veins. Experimental Biology meeting
abstracts [accessed at http://www.biosis-select.orglfaseb/index.htrnl]. The
FASEB Journal, 19, Abstract #2044, 2004. '

Thakali K, Watts SW. ET-1 contraction signaling in veins is independent of
extracellular hydrogen peroxide. [Abstract]. Hypertens. 43: 1352, 2003.

Thakali K, Ni W, Li M, Fink GD, Watts SW. The anorexigen (+)-norfenfluramine

as a pressor; enhanced response in hypertension. [Abstract]. Hypertens. 42: 387,
2003.

155

 

Thakali K, Fink GD, Galligan JJ, Watts SW. Arteries and veins desensitize
differently to endothelin. XVth Scientific Meeting of the Inter-American Society of
Hypertension. 2003 (San Antonio, TX).

156

 

 

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