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THESIS

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3 1293 018103

This is to certify that the

dissertation entitled

Pharmacologic, Physiologic, and Biochemical
Characterization of Epidermal Growth
Factor-induced Contraction in
Experimental Hypertension

presented by

Jennifer Anne Florian

has been accepted towards fulfillment
of the requirements for

PhD. degree in Phannacology/‘l'oxicology

ails

Major professor

Date 81m ! mi

MS U is an Affirmative Action/Eq ual Opportunity Institution

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1M chleflfi-p.“

PHAR

CHAR

IND[(

PHARMACOLOGIC, PHYSIOLOGIC, AND BIOCHEMICAL

CHARACTERIZATION OF EPIDERMAL GROWTH FACTOR-

INDUCED CONTRACTION IN EXPERIMENTAL HYPERTENSION
By

Jennifer Anne Florian

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

1999

Pl

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ABSTRACT

PHARMACOLOGIC, PHYSIOLOGIC, AND BIOCHEMICAL
CHARACTERIZATION OF EPIDERMAL GROWTH FACTOR-INDUCED
CONTRACTION IN EXPERIMENTAL HYPERTENSION

By

Jennifer Anne Florian

The purpose of the experiments described here was to characterize the contractile
response to a classic growth factor, epidermal growth factor (EGF), in arteries from
hypertensive rats. I have tested the hypothesis that the tyrosine kinase-dependent
signaling pathway utilized by EGF, is augmented in hypertension and, ultimately, results
in an enhanced contractile response to EGF. Elucidation of the role of tyrosine kinases is
crucial as tyrosine lcinases serve both vascular growth and contraction, both demonstrated
to be augmented in hypertension.

My experimental approach was designed to study the mechanism(s) by which
EGF stimulates contraction and to determine the dependence of this contractile response
on an elevation in blood pressure. The isolated tissue bath protocol was used to identify

and contractile response to EGF and to determine, pharmacologically, the signaling

 

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pathways stimulated by EGF to result in contraction. Biochemical assays were utilized to
investigate the mechanisms by which the enhanced contraction to EGF occurs.

The experiments conducted in this study indicated that the signal transduction
elements for EGF are enhanced in hypertension, resulting in an augmented contractile
response to EGF. Contraction to EGF in aorta from DOCA-salt rats was found to be
dependent on activation of the EGF receptor tyrosine kinase, MEK and L-type calcium
channels and persists in the presence of the endothelium. Biochemically, it was
demonstrated that there is a significant difference in mRNA levels for the EGF receptor
between aorta from sham and DOCA-salt rats and there was a trend for general tyrosine
kinase activity to be increased in vessels from DOCA-salt hypertensive rats. Contraction
to EGF did not appear until after a significant rise in systolic blood pressure and was
concurrent with a reduction in endothelium-dependent vasorelaxation, indicating that the
altered vascular reactivity is the result of the hypertension. The contraction to EGF
while, in part, was dependent on elevated systolic blood pressure, appears to be regulated
by other factors like mineralocorticoids. NO clearly inhibited EGF-induced MEK-
dependent contraction in the rat aorta but did not inhibit directly MEK activity. Together,
these findings more accurately define the role of EGF and tyrosine kinases in the
reactivity of vessels from hypertensive rats. Finally, these data provide evidence that
growth factors should be considered vasoconstrictors as well as growth modulators in

hypertension.

To my parents, Bill and Pat Florian,

who gave me roots and wings

iv

1 would I
support“? of mt
professionally-
t'ora better men!

I wish 10
Jim Gilligan. K
project and new

I would 1;
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Johns. Ron John:
of the most intell
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ACKNOWLEDGEMENTS

I would first like to thank my mentor, Dr. Stephanie Watts, who has always been
supportive of me and my career. She has taught me so many things, both personally and
professionally. Even though we had some ups and downs, I truly could not have asked
for a better mentor; you are my role model as a scientist. Thank you for everything.

I wish to thank the other members of my Guidance Committee, Drs. Greg Fink,
Jim Galligan, Kathy Gallo, and Ken Moore, who took the time to be interested in my
project and were always willing to help me in any way they could.

I would like to thank the coworkers in and out of the Watts lab over the years for
all of their help and friendship: Amber Russell, Amy Banes, Anne Dorrance, Doug
Johns, Ron Johnson, Barb Faubert and Yvonne Will and Jen Ballew. This group is some
of the most intelligent and humorous people I know. Who says scientists do not know
how to have a good time? I will truly miss all of you.

The love and interest of my family, especially my parents, Bill and Pat and my
brother and soon-to-be sister-in-law, Jeff and Stephanie (and buckaroo, too), has always
been a great support for me. I could not have done this without your generosity, both
emotionally and financially.

Finally, and most importantly. I thank my fiance and my best friend, David. From

the beginning, he has been there for me. His unending support has meant the world to me

and can he

to enjoy the

and can never truly be repaid. Thank you for teaching me not to take life so seriously and

to enjoy the ride.

vi

TABLE OF CONTENTS

LIST OF TABLES .......................................................................... xi
LIST OF FIGURES ........................................................................ xii
LIST OF ABBREVIATIONS ............................................................. xv
I. INTRODUCTION ........................................................................ l
A. Hypertension ........................................................................ 1

1. Prevalence .................................................................. l

2. Types of Hypertension .................................................... l

a. Primary Hypertension ............................................ 2

b. Secondary Hypertension ......................................... 2

3. Mineralocorticoid Hypertension ......................................... 3

a. Synthesis and Regulation of Mineralocorticoids ............. 3

b. Mechanism of Mineralocorticoid Action ...................... 4

B. Hypertension and Vascular Reactivity ............................................ 5

1. Vascular Contraction in Hypertension .................................. 5

2. Vascular Growth in Hypertension ....................................... 6

C. Protein Tyrosine Kinases .......................................................... 7

l. Nonreceptor Tyrosine Kinases ........................................... 7

2. Receptor Tyrosine Kinases ............................................... 7

3. Erk MAPK Pathway ...................................................... 8

D. Normotension and Vascular Reactivity .................................... 12

1. Vascular Contraction in Normotension ............................... 12

a. G protein—dependent Contraction .............................. 12

b. Tyrosine Kinase-dependent Contraction ...................... 13

c. Mechanisms of Tyrosine Kinase-dependent Contraction..l3

2. Vascular Growth in Normotension .................................... 14

E. Epidermal Growth Factor (EGF) ................................................ 15

1. Synthesis .................................................................. 15

2. EGF Related Ligands .................................................... 15

3. ErbB Receptors ........................................................... 16

F. EGF and Vascular Reactivity .................................................... 20

l. EGF and Vascular Growth .............................................. 20

a. EGF-stimulated Vascular Growth in Normotension ........ 20

vii

2. EGF and Vascular Contraction ......................................... 22
a. EGF-induced Vascular Contraction in Normotension. .....22
b. EGF-induced Vascular Contraction in Hypertension ...... 23
c. In Vivo Regulation of Blood Pressure by EGF ............. 24
G. Nitric Oxide (NO) and Vascular Function ..................................... 25
1. NO-mediated Effects in Normotension ............................... 25
2. N O-mediated Effects in Hypertension ................................ 25
3. Mechanisms of Inhibition by NO ...................................... 26
H. Hypothesis .......................................................................... 27
II. METHODS .............................................................................. 29
A. Animals ............................................................................. 29
B. Models of Hypertension .......................................................... 29
1. Deoxycorticosterone Acetate—Salt (DOCA-salt) ..................... 29
2. Goldblatt One Kidney- One Clip (lK-lC) ........................... 3O
3. Nm-Nitro-L-Arginine (L-NNA) ........................................ 3O
4 Wistar Kyoto (W KY) and Spontaneously Hypertensive Rats
(SHR) ...................................................................... 31 .
5. Measurement of Systolic Blood Pressure ............................. 31
C. Concentration-dependent Contraction Response Curves ..................... 32
1. Isolated Tissue Bath Protocol .......................................... 32
2. Concentration Response Curves to EGF Receptor Agonists ....... 33
3. Concentration Response Curves to AngII and 5-HT ................ 33
4. Effect of Endothelium on EGF-induced Contraction ............... 33
5. Effect of Signaling Inhibitors on EGF-induced Contraction ....... 34
D. Biochemical Assays ............................................................... 35
1. Measurement of EGF Receptor Messenger RNA .................. 35
2. Isolation of Proteins .................................................... 37
3. Biorad Protein Assay ................................................... 37
4. Pierce Tyrosine Kinase Assay .......................................... 38
5. MEK Assay .............................................................. 39
6. Irnmunoprecipitation of the EGF Receptor ........................ 4O
7. Western Analyses ...................................................... 41
E. Data Analysis ..................................................................... 42

b. EGF-stimulated Vascular Growth in Hypertension... . .....21

viii

III. EXPERIMENTAL RESULTS ...................................................... 44
Identification of Contractile Response to EGF in Aorta from Sham

A.

B.

and DOCA-salt Rats .............................................................. 44
Pharmacological Characterization of Contractile Response to EGF ....... 51
l. EGF Receptor Agonist-induced Contraction in Aorta from

Sham and DOCA-salt Rats ............................................. 51
2. Effect of PDO98059 on Contraction to AngII in Aorta from

Sham and DOCA-salt Rats ............................................. 52
3. Effect of Signaling Inhibitors on Maximal Contraction to

EGF in Aorta from DOCA-salt Rats ................................... 61
Biochemical Mechanisms for Contractile Response to EGF ................ 65
1. Measurement of EGF Receptor Messenger RNA in Aortic

Homogenate from Sham and DOCA-salt Rats ....................... 65
2. Measurement of EGF Receptor and ErbB-2 Protein Levels

in Aortic Homogenate from Sham and DOCA-salt Rats .......... 66
3. Measurement of Basal and EGF-stimulated Protein Tyrosine

Kinase Activity in Aortic Homogenate from Sham and

DOCA-salt Rats ......................................................... 71
Physiological Mechanisms for Contractile Response to EGF ............... 74
1. Contraction to EGF in Aorta from Sham and 1K- 1C Rats ......... 74
2. Contraction to EGF in Aorta from Sham and L-NNA Rats ........ 78
3. Contraction to EGF in Aorta from WKY and SHR Rats ........... 78
4 Changes in the Contraction to EGF During the Development

of DOCA-salt Hypertension ............................................ 83
5. Contraction to EGF in Aorta from Wistar and Wistar-Furth

Sham and DOCA-salt Rats ............................................. 98
6. Contraction to EGF in Aorta from Sham, Salt-Alone, and

DOCA-Alone Treated Rats ............................................. 99

Influence of the Endothelium on the Contractile Response to EGF ...... 107

1.

Effect of Endothelium on Contraction to EGF in Aorta

from Sham and DOCA-salt Rats ...................................... 107
Effect of SNAP on Contraction to EGF in Aorta from

DOCA-salt Rats ......................................................... 110
Changes in the Relaxation Response to ACh During the
Development of DOCA-salt Hypertension ........................ 113
Effect of L-NNA on Contraction to EGF in Endothelium-intact
Aorta from Sham and DOCA-salt Rats .............................. 120
Effect of the NO Donors SNAP and SN P on Constitutively
Activated MEK Protein ................................................ 123

ix

IV.

\"I.

IV.

VI.

DISCUSSION .................................................................. 126
Identification of Contractile Response to EGF in Aorta from Sham
and DOCA-salt Rats ............................................................ 127
Pharmacological Characterization of Contractile Response to EGF
in Aorta from DOCA-salt Rats ................................................ 128
1. Dependence on Calcium ............................................... 128
2. Independence of the Cyclooxygenase Pathway ..................... 130
3. Dependence on Tyrosine Kinases .................................... 130
a. EGF Receptor Tyrosine Kinase .............................. 130
b. Erk MAPK Pathway .......................................... 131
c. Effect of PDO98059 on Contraction to AngII ............. 132
Biochemical Mechanisms for Contractile Response to EGF .............. 133
1. Changes in ErbB Receptor Levels ................................... 133
a. ErbB-l Receptor Levels ...................................... 133
b. ErbB-2 Receptor Levels ...................................... 135
2. Enhanced Localization of ErbB Receptors .......................... 136
3. Changes in Protein Tyrosine Kinase Activity ...................... 138
Physiological Mechanisms for Contractile Response to EGF ............. 140
1. Dependence of the Contractile Response to EGF on
Blood Pressure .......................................................... 140
2. Independence of the Contractile Response to EGF on
Blood Pressure .......................................................... 142
3. Dependence of Vascular Reactivity on Mineralocorticoids ...... 143
Influence of the Endothelium on the Contractile Response to EGF ...... 145
1. Effect of NO on the Contractile Response to EGF ................. 145
2. Effect of NO on the Constitutively Activated MEK Protein. . . 147
a. NO Does Inhibit MEK Activity .............................. 147
b. NO Does Not Inhibit MEK Activity ........................ 148
3. Impact of NO on the Vasculature of Hypertensive Animals. . 149
CONCLUSIONS ................................................................ 15 l
BIBLIOGRAPHY ............................................................... 157

Table I:

Table 2:

Table 3:

 

 

 

LIST OF TABLES

Table 1: Systolic blood pressures for Sham, one kidney-one clip,
Nw-nitro-L-Arginine, Wistar-Kyoto and Spontaneously
Hypertensive rats ......................................................... 75

Table 2: Systolic blood pressures for Sham and DOCA-salt rats on
days 1, 3, 5, 7, 14, 21, 28 of DOCA-salt therapy ..................... 84

Table 3: Systolic blood pressure for sham Wistar, sham Wistar-Furth,
Wistar DOCA-salt, Wistar-Furth DOCA-salt, sham,
Salt-alone, and DOCA-alone rats .................................... 100

xi

Figure 1:

Figure 2:

Figure 3:

Figure 4

Figure 5:

Figure 6:

Figure 7:

Figure 8:

Figure 9

Figure 10:

Figure 11:

LIST OF FIGURES

Diagram of the EGF receptor signal transduction
pathway .................................................................... 11

Diagram of members of the ErbB receptor subfamily and
known ErbB receptor ligands ........................................... 19

Systolic blood pressures for 28 day Sham and DOCA-salt
rats .......................................................................... 46

Representative tracing of contractile response to EGF in aorta
from day 28 Sham and DOCA-salt rats ............................... 48

Contractile response curves to EGF and 5-HT in aorta from
Sham and DOCA-salt rats ................................................ 50

Contractile response curve to TGF-or in aorta from Sham and
DOCA-salt rats ............................................................ 54

Contractile response curve to Hb-EGF in aorta from Sham and
DOCA-salt rats ............................................................ 56

Effect of AT1 receptor antagonist losartan and MEK inhibitor
PD98059 on AngII-stimulated tyrosyl-phosphorylation of the

Erk MAPK proteins in cultured rat thoracic aorta smooth

muscle cells ................................................................ 58

Effect of PDO98059 on AngII-induced contraction in aorta
from Sham and DOCA-salt rats ......................................... 60

Effect of 4,5-dianilinophthalimide, AG1478, genistein,
PDO98059, diltiazem, and indomethacin on EGF-induced

contraction in aorta from sham and DOCA-salt rats ................ 64

Measurement of EGF receptor messenger RNA in aortic
Homogenate from Sham and DOCA-salt rats ........................ 68

xii

Figure 12

 

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Figure 12:

Figure 13:

Figure 14:

Figure 15:

Figure 16:

Figure 17:

Figure 18:

Figure 19:

Figure 20:

Figure 21:

Figure 22:

Figure 23:

Measurement of EGF receptor (ErbB-1) protein levels in aortic
Homogenate from Sham and DOCA-salt rats ........................ 70

Measurement of basal and EGF-stimulated tyrosine kinase
activity in aortic Homogenate from Sham and DOCA-salt
rats .......................................................................... 73

Contractile response curve to EGF in aorta from Sham and one
kidney-one clip rats ...................................................... 77

Contractile response curve to EGF in aorta from Sham
and L-NNA rats ........................................................... 80

Contractile response curve to EGF in aorta from Wistar-Kyoto
and spontaneously hypertensive rats ................................... 82

Sham and DOCA-salt contractile response curves to
EGF on days 1, 3, and 5 of DOCA-salt therapy ..................... 86

Sham and DOCA~salt contractile response curves to EGF on
days 7 and 14 of DOCA—salt therapy .................................. 88

Sham and DOCA-salt contractile response curves to EGF on
days 21 and 28 of DOCA-salt therapy ................................ 9O

Sham and DOCA-salt contractile response curves to EGF on
days 1 through 28 of DOCA-salt therapy ............................. 93

Scatter plot testing correlation between maximal contraction to
EGF and systolic blood pressure magnitude

on days 1 through 28 ..................................................... 95

Scatter plot testing correlation between maximal contraction to
EGF and systolic blood pressure magnitude on day 28 ............. 97

Contractile response curves to EGF in aorta from Wistar and
Wistar-furth Sham and DOCA-salt rats .............................. 102

xiii

thur:

Figure

Figure

Figure

FlElite

Figure 24:

Figure 25:

Figure 26:

Figure 27:

Figure 28:

Figure 29:

Figure 30:

Figure 31:

Figure 32:

Figure 33:

Figure 34:

Contractile response curves to EGF in aorta from Sham and

Salt-alone treated rats ................................................... 104
Contractile response curves to EGF in aorta from Sham and
DOCA-alone treated rats ............................................... 106
Contractile response to EGF in endothelium-intact aorta

from Sham and DOCA-salt rats ....................................... 109
Relaxation response curve to the NO donor SNAP in
EGF-contracted aorta from DOCA-salt rats ........................ 112
Sham and DOCA-salt relaxation response curves to

Acetylcholine on days 1, 3, and 5 of DOCA-salt therapy... . ....1 15
Sham and DOCA-salt relaxation response curves to

Acetylcholine on days 7 and 14 of DOCA—salt therapy ........... 117
Sham and DOCA-salt relaxation response curves to

Acetycholine on days 21 and 28 of DOCA-salt therapy ......... 119
Effect of L-NNA on the contractile response to EGF in
endothelium-intact aorta from Sham and DOCA-salt rats. . . . . . 122
Effect of NO donors on constitutively activated MEK

protein .................................................................... 125
Schematic diagram representing the traditional role of EGF

in the reactivity of vessels from hypertensive rats .................. 155
Schematic diagram representing the dual role of EGF in

the reactivity of vessels from hypertensive rats ..................... 157

xiv

 

BSA
cGllP
dCT P
DOC-salt
DOCA-sal:
4.5-dianiiit‘
DMEM
DhlSO
DNA

ET -1

Erk
GAPDH
GST
Grbl
GNRF
HhEGF
5-HT
JAK
INK
LANA
MAP
MAPK
MEK
MEK-2E

MKP. 1
lat
MICK
NDF
NE
NGF
N0
NCDC

ACE
Ang 11
ATP
BSA
cGMP
dCTP
DOC-salt
DOCA-salt
4,5.dianilio
DMEM
DMSO
DNA
ET- 1

Erk
GAPDH
GST
Grb2
GNRF
Hb—EGF
5-HT
JAK

JN K
L—NNA

LIST OF ABBREVIATIONS:

angiotensin l-converting enzyme

angiotensin II

adenosine 5’-triphosphate

bovine serum albumin

cyclic guanosine monophosphate

deoxycytidine triphosphate
deoxycorticosterone-salt

deoxycorticosterone acetate-salt
4,5-dianilinophthalimide

Dulbecco’s modified eagle’s medium
dimethylsulfoxide

deoxyribonucleic acid

endothelin- l

extracellular-signal regulated kinase
glyceraldehyde-phosphate dehydrogenase
glutathione S-transferase

growth factor receptor bound-2
guanine-nucleotide releasing factor

heparin binding-epidermal growth factor
5-hydroxytryptamine, serotonin

janus kinase

jun N -terminal kinase/stress-activated protein kinases
Nm-Nitro-L-arginine

mitogen activated protein

mitogen activated protein kinase

mitogen activated protein kinase kinase (MAPKK)
mitogen activated protein kinase kinase-2E (2 serines to
glutamates)

MAP kinase phosphatase-1

myosin light chain

myosin light chain kinase

neu differentiation factor

norepinephrine

nerve growth factor

nitric oxide

2-nitro-4-carboxypheny1-N, N -diphenylcarbamate

XV

NRT
lK-l
PCR
PDC
PE

PKC
PLC
RAE
RV.
RTl

SA]

W}

NRTK
1 K- 1 C
PCR

SOS
TGF-a

TBS-T
TPR
2K- 1C
WKY

nonreceptor tyrosine kinase

one kidney-one clip

polymerase chain reaction
platelet derived growth factor
phenylephrine

protein kinase C

phospholipase C
renin-angiotensin system
ribonucleic acid

receptor tyrosine kinase

reverse transcribed polymerase chain reaction
stress activate protein kinases
sodium dodecyl sulfate

src homology-2

src homology-3

Src homology-2 domain and collagen-like
spontaneously hypertensive rat
S—nitroso-N-acetylpenicillamine
sodium nitroprusside

son of sevenless

transforming growth factor-alpha
tris-buffered-saline
tris-buffered-saline + tween

total peripheral resistance

two kidney-one clip
Wistar-Kyoto

xvi

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each year t"
prevalent cat
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l hype

INTRODUCTION

A. Hypertension

1. Prevalence

It is estimated that nearly one million Americans die from cardiovascular disease
each year (Whelton et al., 1994). Hypertension, or high blood pressure, is the most
prevalent cardiovascular disease in the United States and The Joint National Committee
on comprehensive management of hypertension has defined hypertension as a sustained
systolic blood pressure greater than 140 mmHg and/or a sustained diastolic blood
pressure greater than 90 mmHg. Hypertension is a critical risk factor for other
cardiovascular diseases and, if left untreated, predisposes individuals to several target
organ diseases like congestive heart failure, stroke and kidney failure. Thus, the goal of
antihypertensive therapy is to reduce cardiovascular morbidity and mortality. Given
these facts, the importance of studying the mechanisms underlying hypertension is crucial
to developing therapeutic strategies to treat high blood pressure.

2. Types of Hypertension

Although millions of Americans are diagnosed with hypertension, the mechanism
by which the elevation in blood pressure occurs is uncertain. Individuals with
hypertension are classified as having either primary (essential) or secondary (non-

essential) hypertension.

Essential
(James et al.. 19*:
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a. Primary Hypertension
Essential or primary hypertension afflicts 15-20 percent of the human population
(James et al., 1990). There is no known cause of this form of hypertension, but studies
suggest that essential hypertension is genetically determined (Rapp, 1983; Ward, 1990).
Several investigators have used genetic linkage mapping to isolate chromosome regions
associated with blood pressure (Rapp and Deng, 1995; Bottger et al., 1996). In addition,
genetic models of hypertension have also been developed by repeated brother-sister
matings (Yamori and Swales, 1994). In 1963, Okamoto and Aoki derived the
spontaneously hypertensive rat (SHR) from selective breeding of Wistar rats. The
norrnotensive control strain most often paired with the SHR is the Wistar-Kyoto (WKY)
rat, also derived from the original strain of rats that produced the SHR.
b. Secondary Hypertension
Secondary hypertension is diagnosed when an underlying cause or external factor
can be identified. While patients with secondary hypertension make up only
approximately 10-20 percent of all cases of hypertension (Davis et al., 1977), it is
important to diagnose secondary hypertension as several causese can be readily corrected.
Animal models that mimic the pathogenesis of some forms of secondary hypertension
have been developed to examine the etiology of this disease. Renovascular models of
hypertension utilize renal artery constriction and/or unilateral constriction combined with

contralateral nephrectomy (Thurston, 1994). Chronic inhibition of nitric oxide synthase,

the enzyme re
pressure due

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the enzyme responsible for production of the nitric oxide, results in an elevation of blood
pressure due to increased peripheral resistance (Dananberg et al., 1993). Hypertension
can also be induced by chronic exposure to excess mineralocorticoids.

3. Mineralocorticoid Hypertension

a. Synthesis and Regulation of Mineralocorticoids

Primary aldosteronism, or mineralocorticoid excess, was first discovered and
characterized in the late 1950’s and is a well-known form of secondary hypertension of
familial origin and in response to excess production of aldosterone (Gordon et al., 1994).
In the glomerulosa of the adrenal cortex, the enzyme responsible for the synthesis of
aldosterone is aldosterone synthase or 18 methyl oxidase (Funder, 1994). Aldosterone, a
component of the renin-angiotensin system (RAS), is also produced locally in the
vasculature (Takeda et al., 1995). Mineralocorticoid synthesis is regulated primarily by
angiotensin II (AngII) via a negative feedback loop beginning with a decrease in arterial
blood pressure and activation of RAS. The substrate for renin, angiotensinogen,
circulates in the blood and is cleaved to produce angiotensin I. Angiotensin-converting
enzyme (ACE) is responsible for the conversion of angiotensin I to AngII. When
activated, AngII can act on the adrenal glomerulosa to increase aldosterone synthesis and
ultimately, raise blood pressure. In addition to systemically generated AngII, the
vasculature has been recognized to contain ACE and generate AngII (Okamura et al.,

1992; Rakugi et al., 1993). For several years, investigators have used exogenous

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mineralocorticoids, like aldosterone and deoxycorticosterone (DOC), to elevate blood
pressure in animals.
b. Mechanism of Mineralocorticoid Action

Mineralocorticoids act at renal mineralocorticosteroid receptors to cause sodium
and water retention (Kenyon and Morton, 1994). The mechanism of action for
mineralocorticoids includes sodium ion permeation of the epithelial mucosa] membrane
via sodium-hydrogen exchangers and activation of the sodium pump on the serosal
membrane. These events lead to enhanced sodium uptake, water retention and
ultimately, cellular volume expansion. Hypermineralocorticoidism is associated with
suppression of renin-angiotensin—aldosterone system due to sodium retention.

The blood pressure of rats placed on DOC/DOCA therapy rises within a few days
and plateaus by three to four weeks. The elevation in blood pressure is faster and
ultimately higher if the rat is uninephrectomized and given a high salt (1% sodium
chloride) drinking solution (Kenyon and Morton, 1994). Sensitivity to DOCA-salt
therapy varies for different strains of rats. While Sprague-Dawley and Wistar rats are
sensitive to DOCA-salt therapy, the Wistar-Furth rat strain is relatively resistant to this
therapy (Sciotti and Gallant, 1987; Brunner, 1992). Because of these advantages, like
easily controlled progression of high blood pressure and strain sensitivity to DOCA-salt
treatment, we have chosen the DOCA-salt model of hypertension as the primary

experimental model of hypertension to conduct this research.

3. Hypertension
l. l'asrtti.
The pres~
resistance (TPR l.

and tasodilators.

 

associated with 31
vasoconstrictors; tl
possible cause of 1
changes in the str
responses to contra
receptor agontsts 11
9311311: been sl
h

53718118101] (C011

B. Hypertension and Vascular Reactivity

1. Vascular Contraction in Hypertension

The pressure of blood is determined by cardiac output and total peripheral
resistance (TPR). TPR is largely controlled through a balance between vasconstrictors
and vasodilators. Cardiovascular diseases like hypertension and atherosclerosis are often
associated with an increase in vascular tone or an imbalance favoring the actions of
vasoconstrictors; this increase in the tone of the vasculature has been postulated to be a
possible cause of high blood pressure. Enhanced vascular tone is the result of not only
changes in the structure of the blood vessel wall but also due to augmented pressor
responses to contractile agonists. For instance, contraction to several G protein-coupled
receptor agonists including serotonin or 5-hydroxytryptamine (5—HT) and norepinephrine
(NE) have been shown to be dramatically increased in multiple forms of experimental
hypertension (Collis and Vanhoutte, 1977; Watts et al., 1996; Kanagy, 1997). A study by
White et a1. (1996) found that contractile sensitivity to phenylephrine in vessels from
DOC-salt rats was dependent upon both the type of vessel used and the presence of the
endothelium. Decreases in contractile responses to both specific and nonspecific
activators of G proteins have also been observed. Contraction to AngII as well as
responses to phorbol dibutyrate, an activator of protein kinase C, were decreased in aortic
vessels from rats chronically treated with an inhibitor of nitric oxide synthase (Henrion et

al., 1996). Also, the sensitivity of isolated aortic strips from SHR was slightly depressed

to Ang H and the
response as that s
afunctional abr
conuactility obse

2. l'ascut'.

Smooth r
media of normal
10 the vessel. Vai
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to Ang H and the higher concentrations of AngII were required to produce a comparable
response as that seen in controls (Couture and Regoli, 1980b). These studies suggest that
a functional abnormality in the vasculature may be the reason for the enhanced
contractility observed in hypertension.

2. Vascular Growth in Hypertension

Smooth muscle is a primary cell type, in addition to collagen and elastin, in the
media of normal arteries, providing the vessel the ability to contract. Following damage
to the vessel, vascular smooth muscle undergoes a transition from a primarily contractile
phenotype to one of high synthetic ability (Majesky and Schwartz, 1990). Therefore,
another mechanism by which TPR and thus the pressure of the blood can be increased is .
through an encroachment of the blood vessel lumen by vascular smooth muscle cells.
Vascular smooth muscle cells of spontaneously hypertensive rats (SHR) exhibit higher
growth rates in response to fetal calf serum and to growth factors than vascular smooth
muscle cells of normotensive control rats (Hamada et al., 1990; Saltis and Bobik, 1992:
Saltis et al., 1993). Alternatively, while no differences in cell number (hyperplasia) were
observed between vascular smooth muscle cells from sham and Goldblatt two kidney-one
clip (2K-1C) hypertensive rats, cells from 2K-1C rats were hyperploidic and had
significantly more mean protein mass (both indicative of increased cell volume or

hypertrophy) (Owens and Schwartz, 1983). Recent studies suggest that protein tyrosine

kinases. 6111."

me enhanced

C. Protein T
1. N01
Prote:

tyrosine kina

cytoplasmic

NRTK super

including sh:

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‘leltuttatior

kinases, enzymes that phosphorylate tyrosyl-residues of proteins, may be a link between

the enhanced vascular growth rates and enhanced contractility observed in hypertension.

C. Protein Tyrosine Kinases

I. Nonreceptor Tyrosine Kinases

Protein tyrosine kinases are generally divided into two groups, nonreceptor
tyrosine kinases (NRTK) and receptor tyrosine kinases (RTK). Membrane-associated,
cytoplasmic and nuclear proteins including Src, Abl, FAK and the JAKs make up the
NRTK superfamily (Pawson, 1995). NRTKs have diverse effects on cell functions
including shape change, cell motility, signal transduction to the nucleus and negative
growth regulation (Hubbard et al., 1998). NRTKs contain no extracellular or
transmembrane domains but possess Src homology-2 (SH-2), SH-3, and pleckstrin
homology domains responsible for subcellular targeting and regulation of catalytic
activity (Hubbard et al., 1998).

2. Receptor Tyrosine Kinases

The RTK superfamily includes receptors for epidermal growth factor (EGF)
(figure 1), platelet-derived growth factor (PDGF), nerve growth factor (NGF), and insulin
growth factor (IGF) (Ullrich and Schlessinger, 1990). This class of tyrosine kinases is
responsible for transducing a signal to the inside of the cell to result in cell growth or

differentiation. RTK have an extracellular ligand binding domain, a transmembrane

domain- and a c
the absence 0‘
dimerization 811'
of the receptor
within the recet
and Grb2 tPaW
protein Grb2 Ct
iGhRFi SOS. p
etal- 1993). A
ultimately to th
regulated kinase
3. Erk .1.
The M.-
kinases thKS 1
and P14 km .\1

mmfl
“131013; pro“

domain, and a cytoplasmic domain with catalytic activity. Most RTKs are monomeric in
the absence of ligand. Upon ligand binding, the RTK is activated, leading to
dimerization and autophosphorylation of tyrosyl-residues within the cytoplasmic portion
of the receptor (Pawson, 1995). The autophosphorylation of cytosolic tyrosyl-residues
within the receptor generates binding sites for SH2 domain containing proteins like She
and Grb2 (Pawson, 1995) that allows these proteins to bind the receptor. The docking
protein Grb2 connects the EGF receptor with the guanine nucleotide releasing factor
(GNRF) SOS, promoting the conversion of unactivated ras-GDP to activated ras-GTP (Li
et al., 1993). Activation of ras allows for recruitment of Raf-1 (Wood et al., 1992) and
ultimately to the stimulation of the signaling cascade known as the extracellular signal
regulated kinase mitogen-activated protein kinase (Erk MAPK).

3. Erk MAPK Pathway

The MAPK superfamily of proteins kinases includes the c-jun-NH3-terrninal
kinases (JNKs or stress-activated protein kinases, SAPKs), p38 kinase and the Erks (p42
and p44 kDa MAPK). These kinases are involved in serine/threonine phosphorylation of
regulatory proteins and the activation of transcription factors involved in the regulation of
gene expression (Pulverer et al., 1991). The protein directly responsible for Erk MAP
kinase phosphorylation is mitogen activated protein kinase kinase, also known as MAPK

kinase/Erk kinase or MEK (Zheng and Guan, 1993). MAPK is directly activated by

MEK tMAPKl
and Goldsmith.

The mat
tyrosine and th:
dephosphorylat:
et al., 1997). F
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Upon ac
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MEK (MAPKK) via phosphorylation of critical threonine and tyrosine residues (Cobb
and Goldsmith, 1995) (figure 1).

The inactivation of the MAPK proteins occurs by dephosphorylation of the same
tyrosine and threonine residues. A variety of phosphatases have been implicated in the
dephosphorylation of the MAPKs and include MAP kinase phosphatase-1 (MKP-l) (Xu
et al., 1997), HVH2 (Guan and Butch, 1995) and the mitogen-activated phosphatase
PACl (Ward et al., 1994). Activation of the Erk MAPK pathway rests on the
phosphorylation of these proteins; the phosphorylation status of these proteins is a
balance between the current activation of kinases and phosphatases.

Upon activation, the Erk-MAPK pathway stimulates cell proliferation (Karpova et
al., 1997; Kelleher et al., 1995) and more recently has been implicated in vascular
contraction by growth factors (Zheng et al., 1997). One study has determined that while
total MAPK content is similar between thoracic aorta from normotensive rats and rats
made hypertensive via aortic ligation, tyrosine phosphorylated MAPK content was
greater in aorta from hypertensive rats as compared to shams (Tong et al., 1998). This
study also revealed that the specific MEK inhibitor PDO98059 reduced the blood pressure
of conscious hypertensive rats but did not affect blood pressure in sham rats. These data

suggest a role for tyrosine kinases in the regulation of vascular tone.

Figure 1. Diagram of the epidermal growth factor receptor (EGFR) signal
transduction pathway. Abbreviations: TK, tyrosine kinase; Grb2, growth factor
receptor bound-2; GNRF, guanine nucleotide releasing factor; SOS, son of
sevenless; Erk MAPK, extracellular regulated signal kinase mitogen activated
protein kinase; MEK, mitogen activated protein kinase kinase; MEKK, MEK

kinase.

10

 

 

 

 

r
E? \
KT W

Erk MAPK

l

Pathway
l
l

 

Cellular Response

11

D. Normotens

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Concentration c

D. Normotension and Vascular Reactivity
1. Vascular Contraction in Normotension
a. G-protein-dependent Contraction

Until recently, most studies suggested vascular contraction was mediated through
agonist activation of G-protein coupled receptors. The historical view of vascular
contraction was that it initiated by a hormone binding to a seven transmembrane domain
receptor, G-protein activation and recruitment of phospholipase C (PLC). PLC activation
produces 1,2-diacylglycerol, an activator of protein kinase C (Takai et al., 1979), and
inositol triphosphate leading to increased intracellular calcium levels and the activation of
myosin light chain kinase (MLCK). Ultimately, these events result in the
phosphorylation of the myosin light chain, formation of actin-myosin cross bridges with a
functional actinomyosin ATPase and smooth muscle contraction.

Vascular smooth muscle cell contractile activity is also modulated by the
concentration of free cytosolic calcium. In most smooth muscle cells, calcium enters the
cells from the extracellular fluid through calcium channels. Voltage-dependent calcium
channels in smooth muscle, mainly L-type and T-type, respond to changes in membrane
potential due to electrical or chemical stimuli (Yu and Bose, 1991). The major
mechanisms by which calcium mediates contraction is through intracellular calcium-
dependent myosin light chain phosphorylation, intracellular calcium-dependent

regulation of contraction by caldesmon and calponin, and calcium-dependent activation

12

of protein it
to a noyel c

lunases.

Tyi
role of th
phenylepi
inhibitedl
Salvo e[ ;
kinases '1
ohsen'ed
mCCI‘tCtnlg

St

of protein kinase C (Morgan and Suematsu, 1990). However, recent evidence has pointed
to a novel effector mechanism of smooth muscle contraction—the activation of tyrosine
kinases.
b. Tyrosine Kinase-dependent Contraction
Tyrosine kinase inhibitors have been used as pharrnacologic tools to examine the
role of this enzyme class in vascular contraction. Phasic and tonic contraction to
phenylephrine (PE) and NE, both G protein-coupled receptor agonists, was reversibly
inhibited by three different inhibitors of tyrosine kinases in vascular smooth muscle (Di
Salvo et al., 1993). Our laboratory has also provided support for the role of tyrosine
kinases in hormone-induced vascular contraction. 5-HT-induced contraction was.
observed to be dependent upon the Erk MAPK pathway in addition to classic signaling
mechanisms (phospholipase C and calcium channel activation) for the 5-HT2A receptor
(Florian and Watts, 1998).
c. Mechanisms of Tyrosine Kinase-dependent Contraction
Several laboratories have investigated the mechanism by which tyrosine kinases
stimulate contraction. One target is caldesmon. Caldesmon is a calmodulin- and actin-
binding protein of smooth muscle (Ngai and Walsh, 1984). In the absence of a stimulus,
caldesmon is unphosphorylated and capable of inhibiting actinomyosin ATPase activity
and thus contraction. However, upon phosphorylation of caldesmon, the inhibitory

activity is reduced allowing for activation of actinomyosin ATPase and the resultant

l3

contraction (Kg

 

 

kinase that can ;

 

and Hathaway, 1
phosphorylation i

and pulmonary a:

 

PE. or with fetal 0
19961. thus. 616’“
mpK path“’3§'- u

2, Vascular

Central to t
the Erk MAPK pd
illolloy et al., 19
tanscn'ption factor

SCVETHI SIUdieS ha

contraction (Ngai and Walsh, 1984). Studies have shown that MAPK is one protein
kinase that can phosphorylate caldesmon in smooth muscle (Childs et al., 1992; Adam
and Hathaway, 1993; Katoch and Moreland, 1995). Another possibility is the direct
phosphorylation of the myosin light chain, which was observed upon treatment of aortic
and pulmonary artery vascular smooth muscle with either a classic contractile agonist,
PE, or with fetal calf serum, known to contain several growth factors like EGF (J in et al.,
1996). Thus, elements of the contractile pathway can also serve as substrates for the Erk
MAPK pathway, ultimately, to result in contraction.

2. Vascular Growth in Normotension

Central to the proliferative process of vascular smooth muscle is the activation of
the Erk MAPK pathway by mitogens such as PDGF (Karpova et al., 1997) and Ang II
(Molloy et al., 1993) as the Erk MAPKs are responsible for the phosphorylation of
transcription factors like c-jun and c-myc that are crucial to cell growth (Blenis, 1993).
Several studies have examined protein tyrosine kinase expression and activity in the
vasculature. Vascular smooth muscle from rat aorta has been shown to express protein
tyrosine kinases (Srivastava, 1994) and to have high levels of tyrosine kinase activity (Di
Salvo et al., 1989). Importantly, this activity has been shown to be increased in response
to an acute elevation in blood pressure due to restraint, a moderate physical stress, or
AngII infusion (Xu et al., 1996). One growth factor whose mitogenic actions on the

vasculature has been extensively studied is EGF.

l4

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task

55'
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u‘

E. Epidermal Growth Factor (EGF)

1. Synthesis

In 1962, EGF was isolated from the mouse submaxillary gland (Cohen, 1962) and
since then has been reported to be produced in the kidney (Carpenter, 1979) and in
megakaryokytes (Ben-Ezra et al., 1990). EGF is produced in the kidney as a 1217 amino
acid prepro-EGF peptide that is anchored to the plasma membrane and cleaved by
proteolytic enzymes (Carpenter, 1979) to form a single chain peptide with a molecular
weight of approximately 6 Kda (Cohen, 1962). EGF and EGF-like growth factors
contain a 50 amino acid domain consisting of six cysteine residues that form three
disulfide bonds and a core arginine that stabilizes the orientation of the protein (Savage et
al., 1972). In normotensive humans, EGF is primarily stored in platelets with circulating
blood levels of EGF in the picomolar (40-50 pmol/L) range (Oka and Orth, 1983);
circulating levels of EGF have not been reported in hypertensive humans.

2. EGF Related Ligands

Since the discovery of EGF, several other EGF-related ligands have been
identified including transforming growth factor—alpha (TGF-a), heparin-binding EGF
like-growth factor (Hb-EGF), betacellulin, amphiregulin, and epiregulin. EGF, Hb-EGF,
and epiregulin have all been shown to stimulate proliferation of vascular smooth muscle
(Suithichaiyakul et al., 1990; Taylor etal., 1999; Higashiyama etal., 1991). Elucidating

other potential vascular effects, like contraction, of EGF is crucial as several of these

15

if

on.
cor.
too
1%
a

512

E

growth factors are produced within vascular wall. For instance, although Hb-EGF was
originally purified from macrophages (Higashiyama et al., 1991) and eosinophils (Powell
et al., 1993), messenger RNA (mRNA) for Hb-EGF was recently shown to be dose- and
time-dependently increased by hydrogen peroxide generating compounds (Che et al.,
1997) and by AngII (Temizer et al., 1992) in rat aortic smooth muscle cells. Hb-EGF
mRNA levels have also been demonstrated to be elevated in the left ventricle of SHR as
compared to WKY rats (Fujino et al., 1998). The vasoactive peptides AngII, endothelin-
1, whose cardiovascular actions are well established as well as oc-thrombin have been
found to stimulate transcript for epiregulin in rat aortic smooth muscle cells (Taylor et al.,
1999). Once released, EGF or EGF-like ligands can bind EGF (ErbB) receptors found on _
vascular smooth muscle (Saltis et al., 1995).

3. ErbB Receptors

The ErbB subfamily of receptor tyrosine kinases is made up of four different
members including EGF (ErbB 1) receptor, ErbB2, ErbB3 and ErbB4 (Wang et al., 1998)
(figure 2). The EGF receptor has homology with, and appears to represent, the normal
cellular form of the erb-Bl oncogene product (Downward et al., 1984). All ErbB
receptors are monomeric transmembrane glycoproteins consisting of an extracellular
ligand binding domain with two cysteine-rich clusters, a transmembrane domain, and a
cytoplasmic domain possessing ligand-activated tyrosine kinase activity and carboxy-

terminal residues (Wang et al., 1998). ErbB receptors are expressed in a wide variety of

16

 

tissues and are involved in cellular development and growth. Several studies indicate that
while all four receptors have similar structures, they differ greatly in ligand specificity
and kinase activity. The EGF receptor is capable of binding EGF, TGF-oc, Hb-EGF,
betacellulin, epiregulin and amphiregulin (Savage et al., 1972; Marquart et al., 1984;
Shoyab et al., 1989; Tzhar et al., 1994). ErbB-3 and ErbB-4 receptors bind isoforms of
Neu differentiation factor (NDF), also called heregulin (Plowman et al., 1993; Chang et
al., 1997; Zhang et al., 1997). No studies have identified a specific ligand for ErbB-2, but
it does appear to be the preferred partner for dimerization with the other ErbB receptors
and exhibits high basal tyrosine kinase activity (Karunagaran et al., 1996).

As with other receptor tyrosine kinases, the EGF receptor is activated upon
receptor hetero- and homodimerization. In the model proposed by Lemmon et a1. (1997),
EGF receptor dimerization requires the participation of two molecules of monomeric
EGF and involves the dimerization of a stable intermediate EGF-EGF receptor complex.
This dimerization allows the cytoplasmic tyrosine kinase domains to come into close
proximity, permitting autophosphorylation and activation of the receptors (discussed on
page 7 and 8). As with other receptors with intrinsic tyrosine kinase activity, EGF
receptor activation ultimately leads to activation of the tyrosine kinase-dependent

extracellular regulated kinase-mitogen activated protein kinase (Erk-MAPK) pathway.

17

Figure 2. Diagram of members of the ErbB receptor subfamily and known ErbB

ligands.

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Several studies have also suggested that the actions of several G protein-coupled
agonists are exerted through activation of the EGF receptor. Daub et a1. ( 1996)
demonstrated that endothelin-1 (ET-1) stimulated tyrosine phosphorylation of the EGF
receptor. Moreover, ET-l has been shown to synergistically enhance the mitogenic
activity of EGF in guinea pig airway smooth muscle and this enhancement was sensitive
to pertussis toxin inhibition (Fujitani and Bertrand, 1997). From these studies, one could
speculate that EGF receptors could profoundly affect the vasculature not only via direct
activation by EGF but also through indirect activation by agonists of G protein-coupled

receptors which are established as important modulators of TPR and thus blood pressure.

F. EGF and Vascular Reactivity

1. EGF and Vascular Growth

a. EGF-stimulated Vascular Growth in Normotension

Growth factors such as EGF act mitogenically in cells via activation of receptors
with intrinsic tyrosine kinase activity. As vascular smooth muscle can directly synthesize
and secrete Hb-EGF as well as be exposed to various mitogens through platelet
degranulation, studying the actions of these factors on normotensive smooth muscle is
critical for understanding changes in their effects on smooth muscle from hypertensive
animals. EGF induces mitogenesis in a number of cell types including rat kidney

fibroblasts (Lahaye et al., 1998), guinea pig airway smooth muscle (Fujitani and

20

Bertrand, 1997) and rat aortic smooth muscle (Ko et al., 1993). Transforming growth
factor-B (TGF-B), a different growth factor produced by vascular smooth muscle cells
and released by platelet degranulation (Assoian and Spom, 1986; Sarzani et al., 1989),
synergistically enhances the growth response to EGF by approximately 10-fold (Lahaye
et al., 1998; K0 et al., 1993). Moreover, the mitogenic response of smooth muscle to co-
treatment with the G protein-coupled receptor agonist endothelin-1 (ET-1) and was
significantly greater EGF (71% increase in relative mitogenic potency) than the response
to EGF alone (37 %) or ET-l alone (7%) (Fujitani and Bertrand, 1997).

Transactivation of the EGF receptor has also been shown to be another signaling
mechanism utilized by agonists of G protein-coupled receptors. Transactivation is the
process whereby the EGF receptor is activated, in the absence of EGF, by stimulation of
a different receptor. Several G protein-coupled receptor agonists including ET-l,
thrombin, bombesin, and carbachol have stimulated the phosphorylation and
transactivation of the EGF receptor (Daub et al., 1996; Daub et al., 1997). These studies
further suggest that EGF significantly impacts normal vascular growth not only directly
but also through cooperativity with other vascular modulators.

b. EGF-stimulated Vascular Growth in Hypertension

Generally, the role of peptide growth factors in the vasculature has been

considered largely mitogenic. Several studies indicate that aortic smooth muscle cells

from SHR have an enhanced growth response to EGF as compared to the normotensive

21

 

co

t.
1H

fV-t
m'

U?

3‘ ‘
\4

«(I

controls (Suithichaiyakul et al., 1990; Clegg and Sambhi, 1989; Bukoski et al., 1991).
Cells from SHR rats have amplified phosphoinositide catabolism, S6 kinase activation
and DNA synthesis, indicating that these cells are functionally more responsive than cells
from normotensive WKY rats (Scott-Burden et al., 1989). In addition, EGF can
potentiate the mitogenic effects of hormones like Ang II and vasopressin that are known
to affect the vasculature (Bagby et al., 1993; Mokashi et al., 1992). While it was
suggested that increased DNA synthesis in SHR cells was due to an increase in the
number of EGF receptors and therefore increased tyrosine kinase activity associated with
those receptors (Clegg and Sambhi, 1989), another study found no difference in the
number or affinity of EGF receptors between the SHR and WKY cells (Bukoski et al.,
1991). However, in a different study, it was demonstrated that there was an elevation in
EGF receptor levels in hypertensive Lyon rat aorta as compared to control levels
(Swaminathan et al., 1996). These studies suggest that the EGF signaling pathway that
utilizes tyrosine kinases may be amplified in hypertensive animals.

2. EGF and Vascular Contraction

a. EGF-induced Vascular Contraction in Normotension

Experimental evidence has demonstrated that EGF is a vasoconstrictor, capable of
stimulating contraction in multiple tissues. Berk et a1. (1985) was the first to demonstrate
that EGF (ECso =19 nmol/L) produced a maximal contractile response that was

approximately 40 % of the maximal response to AngII (ECso =6 nmol/L) in aortic strips

22

«h
an

er
«1
re

from normotensive rats. Studies have also indicated that EGF stimulates contraction in
rat mesenteric arteries, canine common carotid and mesenteric arteries, and in guinea pig
longitudinal and circular muscle (Muramatsu et al., 1985; Muramatsu et al., 1986; Yang
et al., 1992; Zheng et al., 1997). There appears to be tissue and species differences in the
mechanism by which EGF elicits contraction. EGF-induced contraction in rat mesenteric
arteries and guinea pig longitudinal muscle was sensitive to inhibition by the
cyclooxygenase inhibitor indomethacin (Muramatsu et al., 1985; Zheng et al., 1997)
while contraction of canine carotid artery to EGF was not reduced by indomethacin
(Muramatsu et al., 1986). Importantly, contraction to EGF in all of these vessels was
reduced by tyrosine kinase inhibitors. In addition to having a direct contractile action on .
rabbit aortic vascular smooth muscle, EGF pretreatment also potentiated the contractile
response to des-Arg9-bradykinin and or-thrombin (de Blois et al., 1992), indicating that
EGF is capable of stimulating contraction in tissues from rats with normal blood pressure.
b. EGF-induced Vascular Contraction in Hypertension

While several studies have examined the mitogenic responses of EGF in
hypertensive vascular smooth muscle cells, no studies have examined the contractile
response to EGF in experimental hypertension. However, in aortic rings from
hypercholesterolemic rabbits, a 22 % increase in maximal isometric tension in response
to EGF was observed in rats with modestly increased serum cholesterol levels (Merkel

and Bilder, 1992). In addition, an approximate 6-fold decrease in the EC50 for EGF

23

’73

hi

(51:5 to 8.3123 nmol/L) was reported between aortic rings from control and
hypercholesterolemic rabbits. Contraction to a different growth factor that also utilizes
the Erk MAPK pathway, PDGF, is enhanced, in terms of both potency and efficacy, in
aortic vessels from SHR as compared to WKY rats (Sauro and Thomas, 1993a, Sauro and
Thomas, 1993b). Moreover, basal and PDGF-stimulated tyrosine kinase activity was also
significantly increased in SHR aorta as compared to WKY aorta. Taken together, these
studies suggest the tyrosine kinase activity may be enhanced in hypertension and thus,
prompted us to examine the contractile response to EGF in vessels from hypertensive
rats.
c. In Vivo Regulation of Blood Pressure by EGF

Few studies have examined the in vivo hemodynamic effects of EGF in animals.
While EGF altered mean blood pressure by causing an initial pressor response followed
by a prolonged depressor response in conscious rats, an infusion of EGF only lowered
blood pressure in conscious monkeys (Keiser and Ryan, 1996). In contrast, medial
thickening of pulmonary arteries (100-200 um diameter) and a moderate elevation in
mean pulmonary arterial pressure were demonstrated in rats given intravenous infusion of

human recombinant EGF (125 pg/h) for one week (Gillespie et al., 1989).

24

C01

1‘ I

as

i‘
«If

G. Nitric oxide and Vascular Function

1. NO-mediated Effects in Normotension

The endothelium takes part in the regulation of vascular tone by releasing
contracting and relaxing factors under basal conditions and when activated by stimuli. A
vasodilator released by the endothelium is endothelium-derived relaxing factor, identified
as nitric oxide (NO). Many studies have found that endothelial N 0 produced in response
to agonists reduces vascular contractility (Furchgott and Zawadzki, 1980; Matsumoto et
al., 1993; Ward and Angus, 1993; Doyle and Duling, 1997). NO-generating compounds
as well as the precursor to NO, L-arginine, inhibit vascular smooth muscle cell
proliferation (Garg and Hassid, 1989; Taguchi et al., 1993; Kariya et al., 1989). In
addition, NO donors have been demonstrated to inhibit not only the number of migrating
vascular smooth muscle cells but also the distance migrated (Sarkar et al., 1996).
Moreover, chronic pharmacological inhibition of NO production leads to elevated arterial
pressure, coronary microvascular remodeling and cardiac hypertrophy (Numaguchi et al.,
1995). From these studies, it can be postulated that NO may inactivate a component of
the signaling pathway that is activated during both growth and contraction. MEK may be
such a component.

2. NO-mediated Effects in Hypertension

Contrary to normal vessels, there appears to be an abnormality in endothelial

function in arteries from hypertensive animals. Acetylcholine-induced endothelium-

25

dependent arterial relaxation, mediated by NO, is reduced in human essential
hypertension (Panza et al., 1990) and in several forms of experimental hypertension
(Lockette et al., 1986). Intimal lesion formation as seen after balloon-induced arterial
injury, which destroys the functional endothelium, is due in part to vascular smooth
muscle growth and migration. Interestingly, in acute hypertension, MAPK proteins have
been shown to be upregulated in response to a rise in blood pressure (Xu et al., 1996).
These findings suggest that endothelial NO production may be diminished in
hypertension and that a loss of NO may lead to inappropriate smooth muscle cell growth
and contractility. The idea that NO may influence the overall activity of the MAPK
pathway in vascular smooth muscle is compelling given that some forms of vascular
disease are associated with endothelial dysfunction and excessive smooth muscle
proliferation.

3. Mechanisms of Inhibition by NO

Since the critical role of the endothelium in vascular relaxation was first described
by Furchgott and Zawadzki (1980), several attempts have been made to determine the
mechanisms by which NO inhibits cellular contraction and growth. Generally, NO is
thought to diffuse into the smooth muscle cell where it targets the heme portion of
soluble guanylate cyclase, stimulating cGMP production and relaxation of the tissue
(Murad, 1986). However, NO inhibits vascular function by non—cGMP mechanisms like

S-nitrosation or adenosine diphosphate (ADP)-ribosylation of proteins (Kanagy et al.,

26

61.1

El

1831 l

asso

1996). NO is known to form nitrosothiol bonds with cysteine residues in proteins (Duhe
et al., 1998). This mechanism of action, S-nitrosation of thiols, appears to be the case in
NO-induced inactivation of protein kinase C activity (Gopalakrishna et al., 1993). These
data suggest the possibility that in normal vessels, NO-related species produced by the
endothelium may reduce vascular contraction via a direct effect on MEK, while
hypertensive vessels demonstrating reduced endothelial function, have enhanced tyrosine

kinase activity and vascular reactivity.

H. Hypothesis

Because vascular growth and contraction are enhanced in vascular smooth muscle
from hypertensive animals and both growth and contraction have been shown to be
dependent upon the activation of tyrosine kinases, a multifaceted approach was utilized to
test the overall hypothesis and subhypotheses stated below:

I hypothesize that the activity of vascular smooth muscle tyrosine kinase(s)
associated with the EGF receptor is increased in response to stimulation by EGF in
hypertension, resulting in the observed increase in growth/mitogenesis as reported in the
literature and, as I propose to show, increased contractility. I tested the following

specific hypotheses in experimentally hypertensive rats:

27

h sis #1: EGF-induced contraction is enhanced in vessels from

hypertensive rats

W EGF-induced contraction is due to an increase in EGF
receptor density and/or tyrosine kinase activity associated with the EGF

receptor

W EGF-induced contraction is dependent upon an increase in

systolic blood pressure

mm NO inhibits MEK activity to reduce EGF-induced

contraction

28

MATERIALS AND METHODS
A. Animals
All animal procedures were followed in accordance with institutional guidelines
established by Michigan State University. When the rats arrived at our facility, they were
housed in clear plastic boxes with wood chip bedding, and allowed ad libitum access to

standard rat chow (T eklab) and tap water.

B. Models of Hypertension

1. Deoxycorticosterone Acetate-Salt (DOCA-salt) Model of Hypertension

Sprague-Dawley rats (225- 250 g) were purchased from Charles River (Portage,
MI) and Harlan Laboratories (Indianapolis, IN); Wistar and Wistar-Furth rats were
purchased from Harlan Laboratories. Under methoxyflurane (Metophanem, Mallinckrodt
Veterinary, Mundelin, IL) anesthesia, the area to be incised was shaved free of fur. The
rats body temperature was maintained during surgery by placing a heating pad under the
rat. The animals underwent uninephrectomy (flank incision, left side) and a Silastic"
(Dow Corning, Midland, MI) implant impregnated with DOCA (200mg/kg) was
implanted subcutaneously in the subscapular region. Sham rats were uninephrectomized
but did not receive the DOCA implant. After surgery, DOCA-treated rats received water
supplemented with 1.0% NaCl and 0.2% KCl; sham animals received normal tap water.

To examine the influence of either DOCA therapy alone or high salt therapy alone, some

29

Sprague-Dawley rats received the DOCA implant and normal tap water while other rats
received the high salt solution but not the DOCA implant. All animals were fed standard
rat chow and had ad libitum access to both food and water. After 1, 3, 5, 7, 14, 21 or 28
days, systolic blood pressures were measured using the tail cuff method described below.

2. Goldblatt One Kidney-One Clip (IK-I C) Model of Hypertension

Under a mixture of pentobarbital (NembutalQ‘, Abbott Laboratories, N. Chicago,
IL; 50 mg/kg, i.p.) and atropine (0.04 mg/kg, i.p.), the Sprague-Dawley rats (Charles
River) underwent right uninephrectomy and a solid silver clip (0.23 mm internal
diameter) was placed around the left renal artery. Sham rats were not uninephrectomized
nor did they receive the clip. After surgery, the lK-lC rats were given one analgesic .
dose of butorphanol tartate (Stadolf, Bristol Laboratories, Princeton, NJ ; 0.1mg i.m.).
Both lK-lC and sham rats were fed standard rat chow and had ad libitum access to food
and normal tap water. After 4 weeks, systolic blood pressures were measured using the
tail cuff method described below.

3. Nw-Nitro-L-Arginine (L-NNA) Model of Hypertension

Sprague-Dawley rats (250-300g, Harlan Laboratories) received either normal tap
water (sham) or tap water supplemented with L-NNA (0.5 g/L, Sigma Chemical Co., St.
Louis, M0) for 14 days. The rats had ad libitum access to normal rat chow. On day 14,

systolic blood pressures were measured using the tail cuff method described below.

30

WE

Ill

“'2

by

Jr-
1’,k

4. Wistar-Kyoto ( WKY) and Spontaneously Hypertensive Rats (SHR)

WKY and SHR (12 weeks old) rats were obtained from Charles River
Laboratories (Portage, MI). At twelve weeks of age, the systolic blood pressure of the
SHR rats is consistently and significantly higher than that of the WKY rats. The rats had
ad libitum access to normal rat chow. Systolic blood pressures were measured using the
tail cuff method described below.

5. Measurement of Systolic Blood Pressure

A rat was placed in a pail containing clean bedding that was placed directly on a
heating pad. A steel cage was placed over the rat to restrict movement by the rat. A
warming light was placed over the cage and the rat warmed for 5-6 minutes to
sufficiently vasodilate the tail artery of the rat. When the rat was sufficiently warm, the
rat was placed into a restrainer. A blood pressure cuff was slipped on the tail and the
balloon transducer was secured, using tape, to the ventral side of the tail behind the cuff.
After a stable pulse pressure was obtained, the manual toggle on the sphygmomanometer
was deflected to inflate the cuff around the tail. To measure the systolic blood pressure
of normotensive rats, the pressure was set for approximately 150-175 mmHg and for
hypertensive rats, the pressure was set for 200 mmHg or higher. This procedure was

repeated 3-4 times to obtain an average blood pressure for each rat.

31

d:

0.1.

.n
tic

C. Concentration-dependent Contraction Response Curves

1. Isolated Tissue Bath Protocol

On the appropriate day, rats were euthanized (80 mg kg:1 pentobarbital, i.p.) and
the thoracic aortae removed. Arteries were dissected into helical strips (0.25 x 1 cm) and
unless otherwise stated, the endothelial cell layer removed by rubbing the luminal side of
the vessel with a moistened cotton swab. Tissues were placed in physiologic buffer for
measurement of isometric contractile force using standard bath procedures.
Physiological salt solution contained (mmol/L): NaCl, 130; KCl, 4.7; KH2P04, 1.18;
MgSO4°7H20, 1.17; CaC12°2HzO, 1.6; NaHCO3, 14.9; dextrose, 5.5; and CaNazEDTA,
0.03. One end of the preparation was attached to a glass rod, the other attached to a force
transducer (FTO3, Grass Instruments, Quincy, MA, USA) and the strip placed under
Optimum resting tension (1500 mg for all tissues) and allowed to equilibrate for one hour.
Aortic strips from sham and hypertensive rats were placed in the same bath to ensure
both smooth muscle strips were exposed to the same environment. Muscle baths were
filled with warmed (37°C), aerated (95%02/5%C02) physiological salt solution.
Changes in isometric force were recorded on a Grass polygraph (Grass Instruments,
Quincy, MA). After an hour equilibration, arteries were challenged with a maximal
concentration of the a,-adrenergic receptor agonist phenylephrine (PE, 10 umol/L).

Tissues were washed and the status of the endothelium examined by observing arterial

32

relaxation to the endothelium-dependent agonist acetylcholine (1 umollL) in tissues
contracted by a half-maximal concentration of PE (approximately 10 nmol/L).

2. Concentration Response Curves to EGF Receptor Agonists

The EGF receptor agonists used in these studies were EGF, TGF-a and Hb-EGF.
Each agonist concentration (10 pmol/L-300 nmol/L) was incubated with the tissue for
approximately 10 minutes in order to allow the contraction to plateau before the next
concentration was added. The concentration range used for EGF in these studies is
physiologically relevant as normotensive human platelet-rich plasma concentrations of
EGF (approximately 45 pmol/L) have been reported in the low end of this range (Oka
and Orth, 1983).

3. Concentration Response Curves to Ang” and 5-H T

Aortic strips from sham and DOCA-salt rats were used to examine the cumulative
contractile response to 5-HT (lnmol/L- 3O umollL). Separate aortic strips were
pretreated with either vehicle (DMSO) or the MEK inhibitor PDO98059 (10 umollL) for
one hour prior to conducting a cumulative response curve to Ang II (0.1 nmol/L-300
nmol/L).

4. Effect of the Endothelium on EGF-induced Contraction

In experiments examining contraction to EGF in the presence of the endothelium
as well as contraction to EGF and relaxation to ACh during the development of DOCA-

salt hypertension, each sham and DOCA-salt rat aorta was cut into two strips and the

33

endothelium was removed from only one strip. The cyclooxygenase pathway is also
known to produce vasorelaxant substances like prostacyclin upon treatment with Ach.
To ensure any relaxation responses to ACh observed during the experiment were
specifically due to NO, the tissues were preincubated with the cyclooxygenase inhibitor
indomethacin (10 umollL). Relaxation to ACh was observed by precontracting the aortic
strip to a half-maximal contraction with PE and then conducting a cumulative response
curve to ACh (lnmol/L-lOumol/L). In experiments examining the effect of the nitric
oxide synthase inhibitor Nw-Nitro-L-Arginine (L-NNA) on EGF-induced contraction in
endothelium-intact rat aorta, vehicle (0.1% deionized water) or L-NNA (100 umol/L) was
allowed to equilibrate in the bath for one hour prior to conducting a cumulative response
curve to EGF. To examine the influence of exogenous NO on EGF-induced contraction,
endothelium-denuded DOCA-salt rat aorta was maximally contracted to EGF and then a
concentration response curve to the NO donor S-nitroso-N-acetylpenicillamine (SNAP,
10pmol/L—lOOnmol/L) was conducted.

5. Efiect of Signaling Inhibitors on EGF-induced Contraction

When examining the effects of signaling inhibitors on EGF-induced contraction,
vehicle (dimethylsulfoxide or DMSO, 0.1%) or inhibitor [4,5-dianilinophthalimide (10
umollL), PDO98059 (10 umollL), genistein (5 umollL), AG1478 (250 nmol/L and 1
umollL), diltiazem (1 umollL), indomethacin (10 umollL)] was added to the bath after the

tissue had maximally contracted to EGF (approximately 30nmol/L). The inhibitor was

34

allowed to incubate with the tissue for 30 minutes and the percent decrease in contraction

was calculated.

D. Biochemical Assays

1. Measurement of EGF Receptor Messenger RNA

Sham and DOCA-salt rats were anesthetized with sodium pentobarbital (80
mg/kg). Aortae were removed, cleaned of fat, snap frozen in liquid nitrogen and stored at
-70 degrees Celsius. RNA was extracted from aorta using the RNeasy mini kit. Samples
were lysed and homogenized using guanidinium isothiocyanate buffer. The sample,
combined with ethanol, was added to a RNeasy mini spin column (Qiagen; Valencia,
CA) and total RNA bound by centrifugation. The contaminants were eluted from the
column before the total RNA was eluted with water. RNA was quantified by
spectrophotometry.

RNA (1 ug) was subjected to first strand cDNA synthesis using oligo dT as a
primer. To complete this step, the sample was combined with the RNAse inhibitor
RNAsin, (20 units), dithiothreitol (0.1 mM) and Oligo dT (0.5 ug) and heated to 65
degrees Celsius and cooled to allow annealing. The annealed sample was combined with
AMV reverse transcriptase (100 units), dNTPs (500 uM), bovine serum albumin (1 ug),
and reverse transcriptase buffer. This sample was allowed to incubate for one hour at 42

degrees Celsius. Samples were then precipitated with linear acrylamide, ammonium

35

acetate (4 M) and ethanol. The cDNA pellets were washed with ethanol (80%, 100 ul)
and allowed to dry before being resuspended in Tris-EDTA (pH 7.4, 20 ul).

Polymerase chain reactions (PCR) were carried out on the cDNA sample (1 ug) to
amplify sections of the cDNA for the EGF receptor. PCR was also carried out to amplify
sections of the cDNA for GAPDH for standardization. Reactions were performed on a
Perkin Elmer Gene Amp PCR thermal cycler system (35 cycles), using Taq DNA
polymerase. 32P—dCT P was included in the PCR reaction to allow for quantitation and
phosphorimage analysis. Samples were resolved on vertical polyacrylamide gels, the
gels were dried and exposed to phosphorimage screen. The results are corrected for the
constitutively expressed gene GAPDH.

EGF receptor upstream primer (forward):

CAC GAA TTC CGA GGG AGT TTG TGG AAA ATT CTG
EGF receptor downstream primer (reverse):

CAC GGA TCC TGC ACT AGA T GC TGC TTG CT G AC
GAPDH upstream primer (forward):

TCC CT C AAG ATT GTC AGC AA
GAPDH downstream primer (reverse):

AGA TCC ACA ACG GAT ACA TI

36

2. Isolation of Proteins

Sham and DOCA-salt rat aorta were cleaned of fat and the endothelium and cut
into small pieces (1mm x 1mm). For the Pierce tyrosine kinase assay, the tissues were
placed in oxygen enriched physiologic salt solution (PSS) and exposed to either vehicle
(PSS) or EGF (10 nmol/L) for 10 minutes at 30°C. After the incubation, the tissue pieces
were washed three times with phosphate buffered saline. While placed on ice, tissue for
both the tyrosine kinase assay and the EGF receptor protein studies was homogenized in
homogenizing buffer (400ul) and allowed to settle for 10 minutes. The homogenate was
spun down for 10 minutes at 12000g. The lysate (approximately 200ul) was retained and
the pellet discarded. The protein content in each sample was then determined and _
equalized using the Biorad protein assay described below.

3. Biorad Protein Assay

The protein concentration of sham and DOCA-salt aortic lysate was determined
using the Biorad Protein Micro Assay (Biorad, Hercules, CA). Sham and DOCA-salt
lysate was diluted 10 fold in deionized water (10ul lysate and 90ul dH20). Eighty
microliters of the dilution was added to a test tube containing deO (720ul). Protein
standards (2.5, 5, 10, 20, 40 and 80 ug/ml protein) were prepared using 10 fold diluted
stock gamma-globulin (1mg/ml). The protein standards (800ul) were added test tubes.
Deionized water (800ul) was used as a blank. Biorad concentrated dye reagent (200ul)

was added to the test tubes. The tubes were vortexed and let sit for 5 minutes at room

37

temperature. After the incubation, tubes were vortexed again and the absorbance read
(595 OD) using a Beckman DU 640 spectrophotometer. Using the equation of the line
produced by the standard curve, protein concentrations in the aortic homogenate samples
were determined and the samples equalized to one protein concentration.

4. Pierce Tyrosine Kinase Assay

The ELISA based tyrosine kinase assay kit (kit 2) was purchased from Pierce
Chemical (Rockford, IL). This assay quantifies the activity of tyrosine kinases in a tissue
sample. The tyrosine kinase peptide substrate (50ul at 10ug/ml) was added to the
background, tyrosine kinase sample, and the tyrosine kinase control wells while 1%
bovine serum albumin (BSA, 50ul) was added to the standard curve, phosphatase, and the
autophosphorylation wells. The plate was covered and incubated for 30 minutes at
37°C. After the incubation, the wells were washed three times with tris buffered saline
(TBS, 250 ul) to remove excess tyrosine kinase substrate. ATP/MgCl2 buffer (40ul) was
added to all wells except the standard curve and phosphatase wells. 1% BSA (lOul) was
added to the background well. EGF-stimulated lysate from DOCA-salt rat aorta (lOul)
was added to the autophosphorylation well. The constitutively activated kinase GST-
MEK-ZE (lOul) was added to the tyrosine kinase control wells. Vehicle or EGF-
stimulated sham or DOCA-salt rat aorta lysate (lOul) containing the tyrosine kinase
sample was added to the appropriate tyrosine kinase sample wells. Stock

phosphopeptide, homogenizing buffer, deionized water and EGF-stimulated DOCA-salt

38

rat aorta lysate (lOul each) was added to the phosphatase well. For the standard curve
wells, homogenizing buffer (lOul), deionized water (30ul) and diluted phosphopeptide
(10ul; 0, 0.001, 0.005, 0.01, 0.05, and 0.1 ug/rnl) was added. The plate was covered and
incubated for 30 minutes at 30°C to allow the tyrosine kinases to phosphorylate the
substrate. After the incubation, the plate was washed three times with TBS (250ul).
Anti-phosphotyrosine (PY20)-horseradish peroxidase-labeled antibody (75ul, 1:500
dilution) which recognizes phospho-tyrosyl residues was added to all of the wells to bind
to the tyrosyl—phosphorylated peptide substrate. The plate was covered and incubated for
one hour at 37°C. The plate was washed three times with TBS (250ul). l-stepTM Turbo
TMB-ELISA substrate (lOOul) was added to the wells and incubated for 10 minutes at
room temperature on a shaker. In the presence of antibody-coated tyrosyl-
phosphorylated substrate, the TMB-ELISA substrate turns blue in color. To stop the
reaction, 1N H280, (lOOul) was added to the wells turning the medium yellow in color.
The absorbance of the plate wells was read on a microplate EL 340 reader (BIO-TEK
Instruments) at 405nm. Using the equation of the line for the standard curve, the activity
of tyrosine kinases contained in each sample was determined.

5. MEK Assay

The fusion protein GST-MEK-ZE (Dudley etal., 1995) is human MEK-l, with
serines 218 and 222 mutated to glutamate (E) and cloned into pGEX-2T to generate a

GST-fusion protein. Serines 218 and 222 are normally substrates for raf and must be

39

till

PE

S.)

ribs
.4“.

F0.

«Bl

phosphorylated in order to activate MEK. The acidic mutation (E) somewhat mimics the
charge imparted by the phosphate, and can partially active the enzyme. MEK-2E (0.5ug)
was added to an eppendorf tube containing assay dilution buffer and one of the following
treatments: deionized water (NO donor vehicle representing basal phosphorylation),
PD98059 (MEK inhibitor) vehicle (DMSO), PD98059 (10 umol/L), or the NO donors
SNAP (100 nmol/L) or SNP (1 nmol/L). The tubes were incubated in a shaking water
bath at 30°C for 30 minutes. After the incubation, inactive (unphosphorylated) MAPK
protein (1.4 ug; Upstate Biotechnology, Lake Placid, NY) and Magnesium/ATP solution
(500 umol/L cold ATP and 75 mmol/L magnesium chloride in assay dilution buffer) was
added to each tube and the mixture was incubated in the bath for another 30 minutes.
Following the second incubation. samples were subjected to western analysis as
described below.

6. Immunoprecipitation of the EGF Receptor

Equal amounts of protein (as determined using the Biorad microprotein assay)
from sham and DOCA-salt rat aortic lysate was added to eppendorf tubes. EGF receptor
antibody (5ul, Santa Cruz Biotechnology, Santa Cruz, CA) was added to the tubes and
placed on a rocker in the cold room overnight. The following morning, protein A/G
(30u1, Santa Cruz) beads and L-RIPA buffer were added to the tubes and tubes tumbled
for an hour in order to isolate the EGF receptor bound to the antibody. Afterwards.

protein A/G beads were pelleted and the supernatant discarded. The beads were washed

4O

three times with L-RIPA buffer and after the last wash, loading buffer (4: l) was added to
the beads and the boiled for 5 minutes to remove the EGF receptor from the beads. The
lysate was centrifuged to pellet the beads while the lysate was loaded on a 5% SDS gel
and western analysis performed.

7. Western Analyses

Lysate (4:1 in denaturing loading buffer, boiled 5 minutes) was loaded and
separated on a 5% denaturing SDS-polyacrylamide gel (7.5 cm gels, constant voltage of
100 V for approximately 3 hours). Proteins were electrically transferred to prepared
Immobilon-P membrane (100 V, constant voltage for one hour, 4°C). Transfer of
rainbow molecular weight standards separated along with the lysate samples indicated the
success of transfer. After transfer of proteins, gels were stained with Gelcode Blue
(Pierce, Rockford, IL) to view the consistency of protein loading between lanes.
Immobilon-P membranes were then blocked for 3-4 hours in Tris-buffer saline + Tween-
20 (0.1%, TBS-T) containing chick egg ovalbulmin (4 %) and sodium azide (0.025 %).
MEK assay blots were probed with activated MAPK antibody (1:20000, Promega,
Madison, WI). Goat polyclonal EGF receptor antibody or goat polyclonal ErbB-2
receptor antibody (1 ug/ml, Santa Cruz Biotechnology) was used to detect EGF receptor
and ErbB-2 receptor protein. All blots were incubated overnight (4°C). The following
morning, blots were washed 3 times with TBS-T (30 minutes, 5 minutes, 5 minutes) and

once with TBS (5 minutes). MEK assay blots were incubated with anti-rabbit (1:3000,

41

Zymed Laboratories, San Francisco, CA) and EGF/ErbB-2 receptor blots were incubated
with anti-goat antibody linked to horseradish peroxidase (1:2000, Santa Cruz
Biotechnology) for one hour and incubated with blots at 4°C. Blots were washed using
the same protocol as after the first antibody incubation. Enhanced chemiluminescence
using Amersharn reagants (Arlington Heights, IL) was performed on the blots to visualize

antibody-labeled bands.

E. Data Analysis

Contractility data are presented as means 11: SEM as a percentages of the PE (10
umol/L or EC50)-, EGF- or the maximal contraction to EGF (100-300 nmol/L) for the
number of animals indicated in parentheses. Unpaired Student's t tests were used where
appropriate in comparing two groups responses (p< 0.05 considered statistically
significant). One way analysis of variance followed by Student Newman Kuels
comparisons test or Dunnett’s multiple comparions test was used when comparing three
or more groups. Using Prism software, agonist EC50 values, defined as the concentration
of EGF necessary to produce a half—maximal response, were calculated using a nonlinear
regression analysis and the algorithm [effect = maximum response/1 + (EC50/agonist
concentration)].

Western data and MEK activity data are represented in arbitrary densitometry

units. EGF receptor mRNA data are represented in arbitrary densitometry units

42

expressed as EGFR cDNA expression corrected for GAPDH cDNA expression.
Unpaired or paired Student's t tests were used where appropriate in comparing two
groups responses and ANOVA followed by a Tukey post hoc test was used when
comparing responses of three or more groups (p< 0.05 considered statistically
significant). Quantitation of band density was performed on a PowerMac 8100 computer
using the public domain NIH Image program (written by Wayne Rasband at the U. S.
National Institutes of Health and available from the Internet by anonymous ftp from
zippy.nimh.nih.gov or on floppy disk from NTIS, 5285 Port Royal Rd., Springfield, VA

22161, part number PB93-504868).

43

A. ldentificati
salt Rats

As rep
DOCA-salt ra
sham rats mti
sham rats (41;
dramatic conu
“as observed
from UOYmot
re.t‘tesenrath e
Sm hipfiflens
irorn day 28 I
not be calm”;
did COntract 1
aorta from D
Clln-e as seen

I

In DOC/APSE}

SUEEESI

RESULTS

A. Identification of Contractile Response to EGF in Aorta from Sham and DOCA-
salt Rats

As represented in figure 3, the systolic blood pressures (SBP) of the 28 day
DOCA-salt rats was significantly higher than the blood pressures of the normotensive
sham rats with values of 19217 mmHg and 117:4 mmHg, respectively. Normotensive
sham rats (412:8 g) weighed significantly more than the DOCA-salt rats (2631-9 g). A
dramatic contraction to EGF, 45¢7% of a maximal PE (10 umol/L)-induced contraction,
was observed in aortic tissue from rats after 28 days of DOCA-salt therapy while aorta
from normotensive sham rats displayed minimal contraction, 1_+_1%, to EGF. A.
representative tracing of EGF-induced contraction in aorta from day 28 sham and DOCA-
salt hypertensive rats is in figure 4. EGF contracted endothelium-denuded aortic strips
from day 28 DOCA-salt rats with a -log EC50 [mol/L] of 7.73:0.73; an EC50 value could
not be calculated for the sham rats (figure 5, top). The sham aortae were viable as they
did contract to PE (figure 4). It is important to point out that the contraction to EGF in
aorta from DOCA—salt rats is not merely a leftward-shift in the concentration response
curve as seen with 5-HT (figure 5, bottom), but the appearance of a contraction that was
not observed in aorta from normotensive rats. Because contraction to EGF was observed
in DOCA-salt aorta but was minimal in aorta from sham normotensive rats, these results

suggest that the response may be due to an upregulation

44

Figure 3. Systolic blood pressures for Day 28 Sham and Deoxycorticosterone
Acetate-Salt (DOCA-Salt) rats. Columns represent the mean and vertical lines
represent the standard error of the mean for the number of animals indicated in
parentheses. * Statistically significant difference (p<0.05) between sham and

DOCA-salt groups.

45

25C

20C

(mmHg)
61‘
n

100

Systolic Blood Pressure

50

250

 

0 0 0 0

O 5 0 5

2 1 1
BIEEV

0:5on 3065 0:996

DOCA-Salt

Sham

46

Figure 4. Representative tracing for epidermal growth factor (EGF)-induced
contraction in endothelium-denuded aorta from sham normotensive and DOCA-

salt hypertensive rats after 28 days of therapy.

47

3:95 seen 5390 65.83
_ _

 

 

 

 

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48

Figure 5. Concentration-dependent contraction to EGF in endothelium-denuded
sham normotensive and DOCA-salt hypertensive rat aorta after 28 days of therapy
(top). Concentration-dependent contraction of endothelium—denuded sham rat and
DOCA-salt aorta to 5-hydroxytryptamine (5-HT) (bottom). Points represent the
means and vertical bars the standard error of the mean for the number of animals
indicated in parentheses. * Statistically significant difference (p < 0.05) between

responses of sham and DOCA-salt rat aorta.

49

 

 

 

 

 

 

 

 

   

 

A -o-Sham (n=7)
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50

 

in the activity of one or more of the signal transduction components activated by the EGF

receptor in hypertensive rats.

B. Pharmacological Characterization of Contractile Response to EGF

I. EGF Receptor Agonist-induced Contraction in Aorta from Sham and DOCA-

salt Rats

Having determined that the contractile response to EGF is dramatically increased
in vessels from DOCA-salt hypertensive rats but not sham rats, we next examined the
signaling pathways utilized by EGF to result in aortic contraction. As the peptide TGF-a
is also capable of activating the EGF receptor, we tested contraction to TGF-or (10 .
pmol/L-300 nmol/L) in aortic strips from DOCA-salt and sham rats (figure 6). The
maximum contractile response to TGF-a was 30:7% of the maximal PE (10 umol/L)-
induced contraction (12111141 mg) in vessels from the DOCA-salt rats as compared to
3_-t_-3% PE-induced contraction (1023:94 mg) in aorta from normotensive sham rats.

Another agonist of the EGF receptor is Hb-EGF. As with TGF-a, Hb-EGF (10
pmollL- lOnmol/L) stimulated concentration-dependent contraction in aortic strips from
DOCA-salt hypertensive rats but not in sham normotensive rats (figure 7). Maximal Hb-
EGF-induced contraction was 28i9% of PE-induced contraction in aortic strips from
DOCA-salt rats; no contraction was observed in sham aorta. These experiments suggest

that the appearance of a contractile response is not specific to EGF, but rather the

51

signaling pathway utilized by the EGF receptor may be amplified in DOCA-salt
hypertension to result, ultimately, in contraction.

2. Efiect of PDO98059 on Contraction to Ang]! in Aorta from Sham and DOCA-

salt Rats

Because contraction to 5-HT and EGF, both activators of tyrosine kinases, was
increased in aorta from DOCA-salt hypertensive rats, it could be postulated that
contraction to all vasoconstrictors that activate tyrosine kinases is enhanced in vessels
from hypertensive rats. To gain insight to this question, I used the agonist Ang 11. Our
laboratory has previously demonstrated that Ang II is capable of activating the Erk
MAPK pathway and stimulating phosphorylation of the 44 kDa and 42 kDa Erk MAPK
proteins in vascular smooth muscle (figure 8). It should be noted that Erk MAPK
phosphorylation stimulated by Ang II was sensitive to inhibition by the MEK inhibitor
PDO98059. I next wanted to determine if contraction to Ang II is enhanced in
hypertensive vessels and if this contraction was mediated by the Erk MAPK pathway.
Maximal contraction to Ang II was not different between aorta from sham (71 :33% PE
contraction) and DOCA-salt rats (401-15%) (figure 9). Further, AngII-induced
contraction was not sensitive to inhibition by PDO98059 in aorta from sham rats

(54:25%) or DOCA-salt hypertensive rats (46i12%) (figure 9), suggesting that not all

52

Figure 6. Concentration-dependent contraction of endothelium-denuded sham
normotensive and DOCA-salt hypertensive rat aorta to transforming growth
factor-or (TGF-a). Points represent the means and vertical bars the standard error
of the mean for the number of animals indicated in parentheses. * Statistically
significant difference (p < 0.05) between responses of sham and DOCA-salt rat

aorta.

53

50

 

Percent PE (105 mol/L)
Contraction
B 8 8

—L
O
l

 

 

-o- Sham (n=6)
-D- DOCA-Salt (n=6)

 

 

 

 

 

log TGF-or [mol/L]

54

Figure 7. Concentration-dependent contraction of endothelium-denuded sham
normotensive and DOCA—salt hypertensive rat aorta to heparin binding epidermal
growth factor like-growth factor (Hb-EGF). Points represent the means and
vertical bars the standard error of the mean for the number of animals indicated in
parentheses. * Statistically significant difference (p < 0.05) between responses of

sham and DOCA-salt rat aorta.

55

Percent PE (105 mol/L)
Contraction

4O

 

 

 

 

 

—o—Sham (n=3) 3
-cr- DOCA-Salt (n=3) 2':
30-
:: ,2]
20-
* / __ --
* _
10- * __
0
-12 -11 -1o -9 -3

 

log HB-EGF [mol/L]

56

 

 

 

 

Figure 8. Effect of AT, receptor antagonist losartan (umol/L) and the MEK
inhibitor PD98059 (10 umollL) on Angiotensin II (10 nmol/L) stimulated tyrosyl-
phosphorylation of the Erk MAPK proteins in cultured rat thoracic aorta smooth
muscle cells. Representative of four experiments performed on cells derived from

four separate explants, each from a different rat. ND indicates nondetectable.

57

__ 93 + .2: F __ 92 + .23 .2: o.
8:83 558.. 8882 $888 __ mc< e_e_;e>

 

 

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58

Figure 9. Effect of the MEK inhibitor PD98059 (10 umol/L) on Angiotensin II-
induced contraction in endothelium-denuded aorta from sham normotensive and
DOCA-salt hypertensive rats. Points represent the means and vertical bars the

standard error of the mean for the number of animals indicated in parentheses.

59

 

 

Percent PE (105 mol/L)
Contraction

125

 

-o- Sham-Vehicle (n=5)

-0- Sham-PD98059 (n=5)

-I- DOCA-Salt-Vehicle (n=5)
-Cl- DOCA-Salt-PD98059 (n=5)

100-

75-

 

 

-11 -10 V b -8 -7
log Ang 11 [mol/L]

6O

 

 

 

vasoconstrictors that activate tyrosine kinases demonstrate enhanced vascular contraction
in aorta from DOCA-salt hypertensive rats. Moreover, these data further support the idea
that there may be an upregulation in the signaling pathway that is specific to EGF that is
responsible for the contraction observed in DOCA-salt hypertensive vessels.

3. Effect of Signaling Inhibitors on Maximal Contraction to EGF in Aorta. from

DOCA-salt Rats

In the next studies, aorta from DOCA-salt rats was maximally contracted to EGF
(100-300 nmol/L) and then a signaling inhibitor or vehicle was added for 30 minutes.
The effect of the signaling inhibitors on contraction to EGF in sham aorta was not
examined as a contraction was not consistently obtained. EGF binds to a plasma
membrane EGF receptor, activating the receptor’s intrinsic tyrosine kinase activity; thus,
the effect of the EGF receptor tyrosine kinase inhibitors 4,5-dianilinophthalimide (10
umollL) and AG1478 (250 nmol/L and 1 umollL) on EGF-induced contraction was
examined. This concentration of 4,5-dianilinophthalimide, a staurosporine derivative, is
documented to reduce significantly EGF—stimulated EGF-receptor autophosphorylation
(ICso value of 1-10 umollL) in serum-starved A431 cells (Buchdunger et al., 1994) and in
our laboratory, has minimal effects on phorbol diburtyrate-induced contraction (data not
shown). Contraction to EGF was markedly reduced (85¢14% reduction) in the presence
of 4,5-dianilinophthalimide (figure 10, top), indicating that EGF—induced contraction

requires the activation of the EGF receptor tyrosine kinase. Similarly, EGF-induced

61

contraction was also reduced by AGl478 (250 nmol/L and 1 umollL) in a concentration-
dependent manner (figure 10, top). Because EGF utilizes tyrosine kinases and
specifically the Erk-MAPK pathway in its mitogenic signal transduction pathway,
experiments were conducted to determine if the dual specificity kinase MEK (tyrosine
and threonine phosphorylation) is activated by EGF to cause contraction. The general
tyrosine kinase inhibitor genistein reduced contraction to EGF by 723; 14%, indicating
that EGF-induced contraction is, in part, mediated by tyrosine kinases. Moreover, EGF-
induced contraction was also inhibited 98i2% by the MEK inhibitor PDO98059 (10
umollL) (figure 10, top), demonstrating the importance of the dual-specificity kinase
MEK in EGF-induced vascular contraction in aorta from DOCA-salt hypertensive rats.
Vascular smooth muscle contraction is dependent upon an increase in intracellular
calcium; therefore, EGF-induced smooth muscle contraction was examined in the
presence of the L-type voltage-gated calcium channel inhibitor diltiazem (1 umol/L). A
9911% reduction in EGF-induced contraction was observed in the presence of diltiazem
(figure 10, bottom), suggesting that growth factor-induced contraction is also dependent
on calcium channels. Some reports in the literature suggest that EGF-induced contraction
is the result of activation of the cyclooxygenase pathway (Muramatsu et al., 1985);
therefore the effect of indomethacin, a cyclooxygenase inhibitor, on EGF-induced

contraction was examined. EGF-induced contraction in the presence of indomethacin (10

62

Figure 10. Averaged effects for the inhibition of maximal EGF-induced
contraction by vehicle (0.1% dimethylsulfoxide), the EGF receptor tyrosine
kinase inhibitors 4,5-dianilinophthalimide (4,5-dianilio., 10 umol/L) and AG1478
(250 nmol/L, 1 umollL), the general tyrosine kinase inhibitor genistein (5 umol/L)
the MEK inhibitor PDO98059 (10 umol/L) (top); Averaged effects for inhibition
of maximal EGF-induced contraction by the L-type calcium channel inhibitor
diltiazem (1 umol/L), and the cyclooxygenase inhibitor indomethacin (10 umol/L)
in DOCA-salt hypertensive rat aorta. Columns represent the means and vertical
bars the standard error of the mean for the number of animals indicated in
parentheses. * Statistically significant difference (p<0.05) between vehicle and

inhibitor-treated groups.

63

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umollL) was not different from vehicle alone (figure 10, bottom). This finding indicates
that at least in endothelium-denuded DOCA-salt rat aorta, EGF-induced contraction is not
dependent on activation of the cyclooxygenase pathway. Because EGF-induced
contraction is dramatically enhanced in DOCA-salt hypertensive vessels and is dependent
on tyrosine kinases, these findings further suggest that the EGF receptor signaling
pathway that utilizes tyrosine kinases and specifically the Erk MAPK pathway may be
amplified in DOCA-Salt hypertension and thus, responsible for the contractile response

to EGF.

C. Biochemical Mechanisms for Contractile Response to EGF

1. Measurement of EGF Receptor Messenger RNA Levels in Aortic Homogenate .

from Sham and DOCA-salt Rats

A molecular approach was taken to determine if EGF receptor expression was
different between aorta from sham and DOCA-salt rats. Messenger RNA (mRNA)
density for the EGF receptor was compared in aorta from sham and DOCA-salt rats after
extraction of tissue RNA, amplification through reverse transcribed-polymerase chain
reaction (RT-PCR) and analysis of EGF receptor cDNA levels by phosphorimage
analysis and corrected for GAPDH. Figure 11 represents the results of these experiments
and data are expressed in arbitrary densitometry units and defined as EGF receptor cDN A

expression corrected for GAPDH cDNA expression. While sham aorta did express

65

mRNA for the EGF receptor (1.87x10’5 i 0.49x10'5), a significantly greater amount of
EGF receptor mRNA was present in aorta from DOCA-salt rats (4.37x10’5 i 1.03x10'5).
These findings suggest that aorta from DOCA-salt rats could contain greater levels of
EGF receptor protein.

2. Measurement of EGF Receptor (ErbB-1) and ErbB—2 Receptor Protein Levels

in Aortic Homogenate from Sham and DOCA-salt Rats

We next determined if this increase in mRNA translated into an increase in EGF
receptor (ErbB-1) protein in aortic tissue. Immunoprecipitation followed by Western
analysis was utilized to detect differences in EGF receptor protein levels in aorta from
sham and DOCA-salt rats. As depicted in figure 12, the results from three separate
experiments were variable. While EGF receptor protein from DOCA-salt aorta was
greater than sham levels in one experiment, sham levels were greater in the following
experiments. Because consistent results for the EGF receptor were not attainable using
immunoprecipitation, experiments are currently being conducted using
immunohistochemistry to characterize differences in EGF receptor levels or in the
organization of receptors that might account for the observed contractile response to
EGF.

Because a quantifiable difference in EGF receptor (ErbB-l) levels could not be
detected between aorta from sham and DOCA-salt rats, we next wanted to determine if

the enhanced response to EGF was due to increased expression of a different EGF

66

Figure 11. EGF messenger RNA expression in aorta from sham normotensive
and DOCA-salt hypertensive rats. Columns represent mean and vertical lines
represent the standard error of the mean for the number of animals indicated in
parentheses. * Statistically significant difference (p<0.05) between sham and

DOCA-salt groups.

67

 

0.00006

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0.00004
0.00003
0.00002
0.00001

328298

(200 In. <0 .9 882.03
cemwm. xw <zoo mmwm

0.00000

68

Figure 12. EGF receptor protein expression in aorta from sham normotensive
and DOCA-salt hypertensive rats. Representative of three separate experiments
performed on aortic homogenate from three different rats. Columns represent

arbitrary densitometry units.

69

EGFR on 10/13/98

     

Arbitrary
Densitometry Units

EGFR on 10/23/98

Arbitrary
Densitometry Units

   

ham A-salt

EGFR on 10/27/98

 

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Arbitrary
Densitometry Units

 

 

 

 

 

Sham DOCA-salt

70

receptor. As stated in the introduction, the ErbB-2 receptor is the preferred partner for all
of the EGF receptor subtypes and has been shown to be overexpressed in rapidly
proliferating cells. Based on these facts, ErbB-2 protein levels were examined in aortic
lysate from sham and DOCA-salt rats. However, when the immobilon-P membranes
were probed with an antibody directed against the ErbB-2 receptor, no antibody bands
were detected in the same molecular weight range as the ErbB-2 receptor (data not
shown). Thus, the observed contraction to EGF is probably not due to increased
expression of the ErbB-2 receptor.

3. Measurement of Basal and EGF-stimulated Protein Tyrosine Kinase Activity

in Aortic Homogenate from Sham and DOCA-salt Rats

The fact that contraction to EGF, shown to be dependent on tyrosine kinases, was
apparent in aorta from DOCA-salt but not sham rats, suggests that tyrosine kinase activity
may be enhanced in DOCA-salt rat aorta. The Pierce Tyrosine Kinase Assay kit was
utilized to investigate basal (PSS)— and EGF- (100 nmol/L) stimulated protein tyrosine
kinase activity in sham and DOCA-salt rat aorta. Basal protein tyrosine kinase activity
was observed in both sham (0.028 t 0.010 ug/ml) and DOCA-salt aortic homogenate
(0.039 :9; 0.015 ug/ml) (figure 13). While there was a trend for EGF—stimulated (0.044 t
0.012 ug/ml) tyrosine kinase activity to be increased in aortic homogenate from DOCA-
salt rats, there was not a significant difference in tyrosine kinase activity when compared

to sham homogenate (0.027 i 0.007 ug/ml). Although these data suggest general

71

Figure 13. Effect of vehicle (physiologic salt solution) or EGF (100 nmol/L)
on general tyrosine kinase activity in aorta from sham normotensive and DOCA-
salt hypertensive rats. Columns represent the mean and vertical lines represent

the standard error of the mean for the number of animals indicated in parentheses.

72

 

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73

tyrosine kinase activity is not dramatically enhanced in DOCA-salt aorta, it should be
pointed out that this assay reflects total activity and not tyrosine kinase activity specific to
the EGF receptor signaling pathway. In as much, the activity of the EGF receptor
pathway may in fact be enhanced, however, this enhancement could be ‘diluted’ out

when examining total activity.

D. Physiological Mechanisms for Contractile Response to EGF

1. Contraction to EGF in Aorta from Sham and IK-I C Rats

Having observed that contraction to EGF was apparent in aorta from DOCA-salt
hypertensive rats but not sham normotensive rats, I next determined if this contractile
response to EGF occurred in other forms of experimental hypertension or was specific to
the DOCA-salt model of hypertension. Thus, I conducted studies investigating EGF-
induced contraction in aorta from sham normotensive and lK-lC hypertensive rats. 1K-
lC hypertensive rats were significantly more hypertensive than normotensive sham rats
(185;th mmHg and 1201-2 mmHg, respectively; Table 1). A significant increase in
EGF-induced maximal contraction (39.17% PIE-induced contraction) in aorta from lK-lC
hypertensive rats was observed (figure 14). EGF contracted lK-lC aortic strips with a -
log EC50 [mol/L] value of 8.801011. A small contraction to EGF (7:370) was observed

in aorta from normotensive sham rats with a -log EC50 [mol/L] of 8.621017.

74

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Figure 14. Concentration-dependent contraction of endothelium—denuded sham
normotensive and one kidney-one clip (lK-lC) hypertensive rat aorta to EGF.
Points represent the mean and vertical lines represent the standard error of the
mean for the number of animals indicated in parentheses. * Statistically

significant difference (p < 0.05) between responses of sham and lK-lC aorta.

76

 

 

 

 

 

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2. Contraction to EGF in Aorta from Sham and L-NNA Rats

We next used the L-NNA-induced model of experimental hypertension which
raises systolic blood pressure by inhibiting the enzyme nitric oxide synthase (NOS) and
thus, the production of the vasodilator nitric oxide (NO). Harlan Sprague-Dawley rats
were given L-NNA (0.5g/L) in their drinking water for 14 days. As represented in Table
l, the systolic blood pressure of the L-NNA-treated rats, 2131-9 mmHg, was significantly
elevated as compared to that of sham rats (130:6 mmHg). Aortic strips from the L-NNA
hypertensive rats displayed an increased contraction to EGF as compared to aorta from
sham rats (figure 15). The maximal contraction obtained from strips from L-NNA
hypertensive rats was 32¢5% of the maximal PE-induced contraction. A contraction to
EGF was also observed in aorta from sham rats (17il%). It is unknown why aorta from
these sham rats contracted to EGF.

3. Contraction to EGF in Aorta from WK Y and SHR Rats

To this point, all of the rats used in these studies have developed hypertension via
experimental therapy. Therefore, we next determined if EGF would cause contraction in
aorta from genetically hypertensive rats. Spontaneously hypertensive rats (SHR) and
their normotensive counterpart, Wistar-Kyoto rats (WKY), were purchased from Charles
River at twelve weeks old. At this time point, the systolic blood pressures of the SHR
rats, 15312 mmHg, was significantly and consistently higher than that of the WKY rats

11711 mmHg, (Table 1). As seen with other hypertensive rats, a profound contraction to

78

Figure 15. Concentration-dependent contraction of endothelium-denuded sham
normotensive and Nw-Nitro-L-Arginine (L-NNA) hypertensive rat aorta to EGF.
Points represent the mean and vertical lines represent the standard error of the
mean for the number of animals indicated in parentheses. * Statistically

significant difference (p < 0.05) between responses of sham and L-NNA aorta.

79

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log EGF [moi/L]

80

 

Figure 16. Concentration-dependent contraction of endothelium-denuded
Wistar-kyoto normotensive (WKY) and spontaneously hypertensive rat (SHR)
aorta to EGF. Points represent the mean and vertical lines the standard error of
the mean for the number of animals indicated in parentheses. * Statistically

significant difference (p < 0.05) between responses of WKY and SHR aorta.

81

 

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EGF was observed in aorta from SHR rats while minimal contraction was demonstrated
in aorta from WKY rats. Maximal contraction to EGF in aorta from SHR rats was
51:8% of the maximal PE contraction, as compared to 1214% for WKY rats (figure 16).
Taken together, these data suggest that the contraction to EGF is not specific to the
DOCA-salt model of hypertension but rather may be common to hypertension.

4. Changes in Contraction to EGF During the Development of DOCA-salt

Hypertension

Having established that an enhanced contraction to EGF is observable in aorta
from several different models of hypertensive rats but minimal in sham normotensive
rats, we next wanted to determine when this contractile response appears during the .
development of DOCA-salt hypertension. It is often speculated that enhanced vascular
reactivity is a primary cause of the rise in systolic blood pressure while others suggest
that the enhanced responsiveness is an effect of high blood pressure. Thus, sham and
DOCA-salt rats were sacrificed on days 1, 3, 5, 7, 14 and 21 of DOCA-salt therapy after
measurement of their systolic blood pressure in order to determine if the contraction to
EGF appears before or after a significant elevation in systolic blood pressure.

There were no differences in the systolic blood pressures or the contraction to
EGF between sham rats on any of the days used in this study. On days 1, 3 and 5, there
were no differences in the systolic blood pressures or the contraction to EGF in DOCA-

salt and sham rats (table 2 and figure 17). By day 7, there was a significant difference in

83

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Figure 17. Concentration-dependent contraction of endothelium-denuded sham
normotensive and DOCA-salt hypertensive rat aorta to EGF on days 1, 3 and 5 of
DOCA-salt therapy. Points represent mean and vertical lines the standard error of
the mean for the number of animals indicated in parentheses. Contraction
(milligrams) to phenylephrine (10 umollL): sham rats (day 1, 893153; day 3,
10501152; day 5, 990177); DOCA-salt rats (day 1, 850113; day 3, 980189; day

5, 1090130).

85

 

Percent PE (105 mol/L)
Contraction

4:.
O
L

co
‘3

 

+ Sham Day 1 (n=3)
-0— DOCA-Salt Day 1 (n=4)
+ Sham Day 3 (n=4)
—D— DOCA-Salt Day 3 (n=4)
+ Sham Day 5 (n=4)
-v— DOCA-Salt Day 5 (n=4)

01
‘1’

 

 

 

log EGF [mol/L]

86

Figure 18. Concentration-dependent contraction of endothelium-denuded
sham normotensive and DOCA-salt hypertensive rat aorta to EGF on days 7 (top)
and 14 (bottom) of DOCA-salt therapy. Points represent the mean and vertical
lines the standard error of the mean for the number of animals indicated in
parentheses. Contraction (milligrams) to phenylephrine (10 umol/L): sham rats
(day 7, 930155; day 14, 951144); DOCA-salt rats (day 7, 1075182; day 14,
673193). * Statistically significant difference (p < 0.05) between aortic responses

from sham and DOCA-salt Day 14 rats.

87

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§ 50- -Cl- DOCA-Salt Day 7 (n=4)
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‘3

*

Contraction
.h.
0

 

 

20-

Percent PE (105 mollL)

 

 

-12 '11 -‘IO -9 ‘8 _7 '6
log EGF [mol/L]

88

Figure 19. Concentration-dependent contraction of endothelium-denuded
sham normotensive and DOCA-salt hypertensive rat aorta to EGF on days 21
(top) and 28 (bottom) of DOCA-salt therapy. Points represent the mean and
vertical lines the standard error of the mean for the number of animals indicated in
parentheses. Contraction (milligrams) to phenylephrine (10 umol/L): sham rats
sham rats (day 21, 913+277; day 28, 8444-131); DOCA-salt rats (day 21, 770+56;
day 28, 799+130). * Statistically significant difference (p < 0.05) between aortic

responses from sham and DOCA-salt Day 28 rats.

89

Percent PE (105 mol/L)
Contraction

Percent PE (105 moIIL)
Contraction

O)
O

 

-o— Sham Day 21 (n=4)
-D- DOCA-Salt Day 21 (n=4)

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SBP between DOCA-salt and sham rats (15319 mmHg and 11618 mmHg, respectively)
as well as a modest but insignificant contraction to EGF in aorta from DOCA-salt rats
that was not seen in sham rats (figure 18, top). The appearance of a significant
contractile response to EGF in aortic strips from DOCA-salt hypertensive rats (57118%
of maximal PE-induced contraction) but not sham normotensive rats (0%) was observed
on day 14, a time point when SBP had been elevated for approximately one week (table
2, figure 18, bottom). On day 21 of DOCA-salt therapy, there was a significant
difference in SBP between DOCA-salt and sham rats and contraction to EGF in aorta
from DOCA-salt rats averaged 2915% of the maximal PE response as compared to
27125% for sham rats (figure 19, top). The large standard error value for the sham rats is
because the aorta from one rat had an aberrantly high response to EGF while the others
responded minimally. As contraction to EGF did not appear until after a significant rise
in blood pressure, these results suggest that the effects of EGF on vascular growth and
contraction may not contribute to the development of hypertension, but, if involved, may
support the chronic maintenance phase of elevated blood pressure.

Figure 20 depicts the contractile response curves to EGF in aorta from DOCA-salt
rats on days 1 through 28 of DOCA-salt therapy. When observed together, these data
suggested that the contractile response to EGF was an ‘all or none’ response that appears
after a significant elevation in systolic blood pressure. These data prompted me to

determine if there was a correlation between systolic blood pressure and the maximal

91

Figure 20. Concentration-dependent contraction of endothelium-denuded
Sprague-Dawley sham normotensive and DOCA—salt hypertensive rat aorta to
EGF on days 1 through 28 of DOCA-salt therapy. Points represent mean and
vertical lines the standard error of the mean for the number of animals indicated in

parentheses.

92

Percent PE (105 mol/L) Contraction

100

 

-o- Sham Day 28 (n=7)

-0- DOCA-Salt Day 1 (n=4)

80- + DOCA-Salt Day 3 (n=4)

-tx- DOCA-Salt Day 5 (n=4) 7

+ DOCA-Salt Day 7 (n=4)

-V- DOCA-Salt Day 14 (n- )

60‘ -I- DOCA-Salt Day 21 (n: )
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Figure 21. Scatter plot testing the correlation between systolic blood pressure
and the maximal response to epidermal growth factor [represented as a percent
phenylephrine (10 umollL)-induced contraction] in aorta from DOCA-salt rats on

days 1 through 28 of therapy. R2 value = 0.3596.

94

Maximum EGF Contraction

«_L—L—L
NO‘IV
anew

(Percent PE Response)

 

 

    

 

 

* P: 0.3596
I
00: '
I I
75- I I
50-
.I
25-
0‘ l l
75 125 175 225

Systolic Blood Pressure (mmHg)
Days 1 - 28

95

Figure 22. Scatter plot testing the correlation between systolic blood pressure and
the maximal response to epidermal growth factor [represented as a percent
phenylephrine (10 umol/L)-induced contraction] in aorta from DOCA-salt rats on

day 28 of therapy. R2 value = 0.2256.

96

 

 

 

12: 0.2256

 

 

125

_ _
0 5 0 5
O 7 5 2
1

Aomcoamom mm 2.85%
cozombcoo mom E:E_xm_2

250

200
Systolic Blood Pressure (mmHg)

Day 28

97

response to EGF. We first examined the correlation between the systolic blood pressure
of rats on days 1 through 28 of DOCA-salt therapy and the maximal response to EGF
where a significant correlation was observed (r2=0.3596) (figure 21). However, as
depicted in figure 22, once the DOCA-salt rats were hypertensive, the maximal response
to EGF was independent (r2: 0.2256) of the absolute degree of elevation in systolic blood
pressure. Because there was not a significant correlation between the absolute magnitude
of systolic blood pressure and EGF responsiveness, these data suggested that an elevation
in blood pressure alone was not the sole cause for the contractile response to EGF.

5. Contraction to EGF in Aorta from Wistar and Wistar-Furth Sham and DOCA-

salt Rats

Because contraction to EGF is observed in vessels from DOCA-salt hypertensive
rats only after a significant rise in blood pressure, we wanted to determine if increased
systolic pressure is required for EGF-induced contraction. We chose to use Wistar and
Wistar-Furth sham and DOCA-salt rats as Wistar rats become hypertensive on DOCA-
salt therapy while Wistar-Furth rats do not become hypertensive with the exact same
treatment (Sciotti and Gallant, 1987; Bruner, 1992). Table 3 depicts the systolic blood
pressures of the Wistar and Wistar-Furth rats. Wistar DOCA-salt rats (17619 mmHg)
had significantly higher blood pressures than Wistar shams (12014 mmHg). The
maximal contraction to EGF in aorta from Wistar DOCA-salt rats (3513% max PE-

induced contraction) was greater than the maximal contraction to EGF in aorta from

98

Wistar sham rats (413%) (Figure 23). Interestingly, the Wistar-Furth DOCA-salt group,
whose blood pressures (13416 mmHg) were not different from the Wistar-Furth sham
group (11213 mmHg) (Table 3), did contract to EGF, [3419% max PIS-induced
contraction (Figure 23)]. Aorta from the Furth sham group did not contract to EGF. The
results of these experiments further suggest that the contractile response to EGF is not
solely dependent on elevated blood pressure and that DOCA-salt therapy itself may also
be a stimulus for this enhanced response to EGF.

6. Contraction to EGF in Aorta from Sham, Salt-alone and DOCA-alone

Treated Rats

In order to determine the influence of DOCA and high salt on EGF-induced
contraction, Sprague—Dawley rats were placed on either DOCA therapy alone (200mg
DOCA, normal tap water) or high salt therapy (no DOCA, 1.0% NaCl + 0.2% KCI) for
four weeks. Rats placed on high salt therapy alone (15119 mmHg) did have
significantly higher blood pressures than that of shams (12814 mmHg) (Table 3).
Interestingly, rats on high salt, though hypertensive, did not demonstrate a contraction to
EGF (1016% as compared to 712% for aorta from sham rats), that was of the same
magnitude as that observed in aorta from DOCA-salt rats (figure 24). DOCA therapy
alone also significantly raised the systolic blood pressures of DOCA rats (156111
mmHg) as compared to sham rats (11311 mmHg) (Table 3). However, as seen in figure

25, the contractile response to EGF was only modestly increased, 1619% PB contraction,

99

 

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100

Figure 23. Concentration-dependent contraction of endothelium-denuded
Wistar and Wistar-furth sham normotensive and DOCA-salt hypertensive rat
aorta to EGF. Points represent the mean and vertical lines the standard error of
the mean for the number of animals indicated in parentheses. * Statistically
significant difference (p < 0.05) between aortic responses from Wistar sham and
Wistar DOCA-salt rats or between Wistar-Furth sham and Wistar-Furth DOCA-

salt rats.

101

01
O

 

 

 

      

 

 

-o— Wistar Sham (n=8)
A -o- Wistar DOCA-Salt (n=8)
d 40_ -l-Wistar-Furth Sham (n=9)
2 -Cl- Wistar-Furth DOCA-Salt (“:32
“g: 8
‘_ +6 30-
v as
e a
o
E o 20‘
a)
8
a)
n. 10-
O —— __—' I
-12 -11 -1o -9

log EGF [mol/L]

102

Figure 24.

Concentration-dependent contraction of endothelium-denuded

Sprague-Dawley sham normotensive and salt-alone treated hypertensive rat aorta

to EGF. Points represent the mean and vertical lines the standard error of the

mean for the number of animals indicated in parentheses.

103

 

100

 

 

 

 

 

 

 

 

-o- Sham (n=5)
3 -I- Salt-Alone (n=5) T 1- --
= 30‘ -o— DOCA-salt (n=4)
0
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L33 '5 >—<>—-3>
1— 6 60'I .
V cu
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6'3 20- .
‘4‘. a. _n_-r"- —- ‘
O ——- -... - . -
-12 -1 1 -10 _9 '8 _7

log EGF [mol/L]

104

Figure 25. Concentration-dependent contraction of endothelium-denuded
Sprague-Dawley sham normotensive and DOCA-alone treated hypertensive rat
aorta to EGF. Points represent the mean and vertical lines the standard error of
the mean for the number of animals indicated in parentheses. * Statistically
significant difference (p < 0.05) between aortic responses from sham and DOCA-

alone treated rats.

105

 

 

100

 

 

 

 

 

 

 

 

-o- Sham (n=4)
-I- DOCA-Alone (n=4) .- ,_
g 80- + DOCA-Salt (n=4)
'5 1’
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-12 -11 -10 -9 -8

log EGF [mol/L]

106

 

 

in aorta from DOCA-alone treated rats as compared to sham responses, 1i1%. Taken
together, these data provide evidence for the idea that an ‘absolute’ elevation in blood
pressure alone is not the only stimulus for contraction to EGF and that the combination of
high blood pressure as well as DOCA and salt treatment may be a stimulus enabling

aortic contraction to EGF.

E. Influence of the Endothelium on the Contractile Response to EGF

I. Effect of the Endothelium on Contraction to EGF in Aorta from Sham and

DOCA-salt Rats

With the understanding that endothelium-denuded aortic strips are not entirely
physiological, we examined the contraction to EGF in endothelium-intact aortic strips
from day 28 sham and DOCA-salt rats to determine if EGF-induced contraction persisted
in the presence of the endothelium. To ensure the endothelium was intact and functional,
ACh (1 umollL)-induced vascular relaxation was examined in aortic strips contracted
with PE (EC50 concentration). Relaxation was observed in aortic strips from both sham
rats (77i6%) and DOCA-salt rats (13_-+_-9%), suggesting that the endothelium of aorta
from DOCA—salt rats was intact and somewhat functional. As seen in figure 26,
contraction to EGF was observed in both endothelium-intact (15:10% maximal PE-
induced contraction) and endothelium-denuded (28:9%) aortic strips from DOCA-salt

rats. Interestingly, at higher concentrations of EGF (30- 300 nmol/L), endothelium-intact

107

Figure 26. Concentration-dependent contraction of endothelium-denuded and
endothelium-intact sham and DOCA-salt hypertensive rat aorta to EGF. Points
represent the mean and the vertical bars represent the standard error of the mean
for the number of experiments indicated in parentheses. * Statistically significant
difference (p < 0.05) between responses of endothelium-intact DOCA-salt rat

aorta and endothelium-denduded DOCA-salt rat aorta.

108

 

Percent PE (1 0"5 mol/L)
Contraction

50

 

-o-Sham + Endothelium (n=4)

-o-Sham - Endothelium (n=4)

-I- DOCA-Salt +Endothe|ium (n=4)

4°“ -0- DOCA-Salt -Endothe|ium (n=4)
*

*

(A)
O
1

N
O
1

 

 

 

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O
l

 

 

 

 

 

 

 

 

 

-12

log EGF [mol/L]

109

aortic strips from sham rats did contract, 12t2%, slightly while endothelium-denuded
sham aorta did not contract (figure 26). A significant difference in the contraction to
EGF (1-3 nmol/L) was observed between endothelium-denuded and endothelium-intact
aortic strips from DOCA-salt rats. These results indicate that a contraction to EGF in
aortic strips from DOCA-salt hypertensive rats does occur even in the presence of the
endothelium. In addition, it appears that the endothelium is still minimally functional
given the response to EGF in aorta from DOCA-salt rats is slightly blunted in the
presence of the endothelium.

2. Effect of the N0 Donor SNAP on Contraction to EGF in Aorta from DOCA-

salt Rats

As EGF uses the Erk MAPK pathway for cell signaling and I have demonstrated
that EGF-induced contraction is dependent on MEK and can be blunted in the presence of
the endothelium, the effect of exogenous NO on EGF-induced contraction was
determined. In these contractile studies, only denuded aortic strips from DOCA-salt
hypertensive rats (systolic blood pressure: 204:16 mmHg) were used because a
contraction was not attainable in aorta from sham normotensive rats. The NO donor
SNAP completely inhibited tyrosine kinase—dependent contraction to EGF (figure 27),
indicating that the vascular smooth muscle from DOCA-salt hypertensive rats is not
functionally resistant to the vasorelaxant effects of NO and that NO might inhibit

vascular contraction by inhibiting tyrosine kinase activity.

110

Figure 27. Effect of S-Nitroso-N-Acetylpenicillamine (SNAP) on percent
maximal EGF-induced contraction in endothelium denuded aorta from DOCA-
salt hypertensive rats. Points represent the mean and the vertical bars represent
the standard error of the mean for the number of experiments indicated in
parentheses. * Statistically significant difference (p<0.05) between the control

and SNAP treatment groups.

111

 

 

 

 

 

—0— Vehicle (n=4)
-Cl- SNAP (N=4)

 

 

150

q - u —
0 5 O 5
O 7 5 2

125-

cozombcoo umoan-u. 0w
3E_xm_2 Samson.

 

log SNAP (mol/L)

112

3. Changes in the Relaxation to ACh During the Development of DOCA-salt

Hypertension

The next series of experiments tested the hypothesis that endothelial N O normally
functions to inhibit tyrosine kinase activity, thereby reducing vascular contraction; and,
during the development of hypertension, reduced NO function allows for an increase in
tyrosine kinase activity and an exaggerated contraction to EGF. Aortic relaxation to the
endothelium- and NO-dependent relaxant agonist ACh and contraction to EGF (figures
17 — 20) were examined as DOCA-salt hypertension developed (table 2). To examine
the relaxation response to ACh, aortic strips were first pretreated with the
cyclooxygenase inhibitor indomethacin (10 umol/L) for one hour. PE was used to
produce a half-maximal contraction and after reaching a plateau, a cumulative response
curve to ACh was conducted. As with contraction to EGF on days 1, 3, and 5 of DOCA-
salt therapy, there were no differences in relaxation responses to ACh between sham and
DOCA-salt rats (figure 28). By day 7, blood pressures of DOCA-salt rats were
significantly higher than that of sham rats, however, ACh-induced relaxation was not
different between sham and DOCA-salt rats (figure 29, top). The potency of ACh to
induce relaxation was significantly shifted nearly 4-fold by day 14 (figure 29, bottom), a
time point when the DOCA-salt rats had been hypertensive for approximately one week
and demonstrate a profound contraction to EGF (figure 18). At the end of the four week

therapy, ACh reduced PE-induced contraction by only 29: 12% in aorta from DOCA-salt

113

Figure 28. Concentration-dependent relaxation of endothelium-intact sham
normotensive and DOCA-salt hypertensive rat aorta to acetylcholine (ACh) on
days 1, 3 and 5 of DOCA-salt therapy. Points represent the mean and the vertical
bars represent the standard error of the mean for the number of experiments

indicated in parentheses.

114

 

 

 

125

 

 

 

-o— Sham Day 1 (n=3)
8 -o- DOCA-Salt Day 1 (n=4)
"8 + Sham Day 3 (n=4)
9! 10°” -0- DOCA-Salt Day 3 (n=4)
E + Sham Day 5 (n=4)
8 -v— DOCA-Salt Day 5 (n=4)
A 75-
8
O
t“.
LU 50—
O. '..'
E g p
w \
g 25- "§\
0. \‘\
0'
0 "K:\\‘e~ .
-10 :7 _6 ~

log ACh [mol/L]

 

 

Figure 29. Concentration-dependent relaxation of endothelium-intact sham
normotensive and DOCA-salt hypertensive rat aorta to acetylcholine (ACh) on
days 7 (top) and 14 (bottom) of DOCA-salt therapy. Points represent the mean
and the vertical bars represent the standard error of the mean for the number of
experiments indicated in parentheses. * Statistically significant difference
(p<0.05) between the relaxation responses from sham and DOCA-salt Day 14

groups.

116

 

 

 

125

 

 

 

 

 

 

 

 

 

: -o- Sham Day 7 (n=4)
{72) -c1— DOCA-Salt Day 7 (n=4)
:5 1001
E
o
0
’5); 75.
O
E-l
LLI 50~
D.
1%
g 25-
a)
D.
0 I ' ' ' I
-10 -9 -8 -7 -6 -5 -4
log ACh [mol/L]
125
g -o— Sham Day 14 (n=8)
:5 -D- DOCA-Salt Day 14 (n=8)
g 100. *
c
o
O
8 75-
0
Hi
LU 50-
0.
1%
e 25-
a:
0.
O l I 1 I I
-10 -9 -8 -7 -6 ‘5 '4

log ACh [mol/L]

117

Figure 30. Concentration-dependent relaxation of endothelium-intact sham
normotensive and DOCA-salt hypertensive rat aorta to acetylcholine (ACh) on
days 21 (top) and 28 (bottom) of DOCA-salt therapy. Points represent the mean
and the vertical bars represent the standard error of the mean for the number of
experiments indicated in parentheses. * Statistically significant difference
(p<0.05) between the relaxation responses from sham and DOCA-salt Day 28

groups.

118

 

 

 

 

125

 

 

 

 

 

 

 

C -o-Sham Day 21 (n=2)
.8: -Cl- DOCA-Salt Day 21 (n=3)
g 100-
C
O
0
’3 75~
IO
0
9.
L” 50-1
D.
‘5';
g 25-
(D
D.
0 I I j I -I
-10 -9 -8 -7 -6 -5 -4
log ACh [mol/L]
125
g -o- Sham Day 28 (n=4)
33 -D- DO’DA-is‘alt Day 28 (n=4)
g 100.. * * * *
8 * * *
0
,3 75-
It)
0
B
I.” 50-
D.
8
g 25-
d)
D.
0 I T I I I
-10 -9 -8 -7 -6 -5 -4

log ACh [mol/L]

119

rats as compared to 80:7% in sham aorta (figure 30, bottom). These experiments indicate
that after blood pressure rises, a reduction in endothelium-dependent NO function is
concurrent with the appearance of a contraction to EGF. Moreover, these results suggest
that neither a reduction in endothelium-dependent relaxation or enhanced vasoconstriction
to EGF contribute to the elevation in blood pressure but rather may contribute to the
chronic maintenance of hypertension.

4. Efi‘ect of L-NNA on Contraction to EGF in Endothelium-intact Aorta from

Sham and DOCA-salt Rats

To examine the influence of endogenous NO on EGF-induced contraction,
endothelium-intact aortic strips from sham and DOCA-salt hypertensive rats were first
incubated with vehicle or the NO synthase inhibitor L-NNA (100 umol/L); afterwards, a
cumulative concentration response curve to EGF was conducted. L-NNA did not affect
EGF-induced contraction in aorta from sham rats (figure 31). There were no differences
in contraction to EGF between aorta from DOCA—salt rats in the presence or absence of
L-NNA (figure 31). However, it must be mentioned that the relaxation response to ACh
(l umollL) was significantly reduced in endothelium-intact aorta from DOCA-salt rats
[(71- 3% reduction in PE (EC50)-induced contraction)] as compared to responses from
sham aorta [(65i5 % reduction in PE (EC50)-induced contraction)]. Thus, while

exogenous NO was capable of inhibiting tyrosine kinase-dependent contraction,

120

Figure 31. Effect of the NO synthase inhbitor Nw-Nitro-L-Arginine (L-NNA
100 umol/L) on EGF—induced contraction in endothelium-intact aorta from sham
normotensive and DOCA-salt hypertensive rats. Points represent the mean and
the vertical bars represent the standard error of the mean for the number of

experiments indicated in parentheses.

121

 

 

 

 

(.0
0

Percent PE (105 moIIL)
Contraction
N
O

_L
O
I

-o- Sham Vehicle (n=4)
-0- Sham LNNA (n=4)
-I- DOCA-Salt Vehicle (n=4)

 

 

 

 

 

- -Ei- DOCA-Salt LNNA (n=4)
T"
4
4:“

l- \I'

/ll ‘L

“-___--—-.'___.
-12 -11 '10 '9 .18
log EGF [mol/L]

122

an enhancement in contraction to EGF was not observed with the addition of L-NNA.
This finding was not necessarily surprising as ACh-induced relaxation was significantly
reduced in aorta from DOCA-salt rats.

5. Eflect of the N0 donors SNAP and SNP on Constitutively Activated MEK

Protein

The MEK assay, which directly examines the activity of MEK in the absence of
agonist stimulation and other cellular proteins, was used to more directly observe the
effect of NO on MEK enzymatic activity. The MEK protein used in this study is human
MEK-1 that has been mutated at serines 218 and 222 to glutamate, rendering the enzyme
partially activated in the absence of phosphorylation. Figure 32 (top) is a representative
blot from a MEK assay. PD98059 (10 umollL) inhibited the ability of the active GST-
MEK-ZE protein to tyrosyl-phosphorylate the 42 kDa Erk MAPK (Erk-2) protein,
demonstrating that the activity of MEK in this assay can be inhibited. By contrast, a 30
minute incubation with the NO donors SNAP (100 nmol/L) and SNP (l nmol/L)
demonstrated little effect on the ability of MEK to phosphorylate the 42 kDa Erk MAPK
protein. On average, neither SNAP nor SNP significanly inhibited the activity of MEK
(figure 32, bottom). These data further refute the hypothesis that NO has the ability to

inhibit contraction through a direct inhibition of MEK.

123

Figure 32. Visualized blot (top) of the effect of SNAP and sodium nitroprusside
(SNP) on the phosphorylation of the 42 kDa mitogen activated protein kinase
(MAPK) by the mutant protein GST-mitogen activated protein kinase kinase-2E
(GST-MEK-2E). Averaged effects of SNAP and SNP on phosphorylation of Erk-
2 by MEK (bottom). Columns represent the mean and the vertical bars represent
the standard error of the mean for the number of experiments indicated in

parentheses.

124

 

 

ACIIVG MAPK Antibody
Arbitrary Densitometry Units

1615 5497 4624 6510

2151

 

 

 

 

 

 

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_n.
m m m n ..o. a o
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DISCUSSION

Augmented growth and contraction in vascular smooth muscle has been
associated with hypertension. As hypertension is the leading cardiovascular disease in
the United States, the development of therapeutic strategies to treat hypertension is
critical to preventing cardiovascular associated morbidity and mortality.

The purpose of the experiments described here was to investigate the functional
role of tyrosine kinases in contraction to EGF, a classical activator of tyrosine kinases, in
vessels from hypertensive rats. I hypothesized that the activity of vascular smooth
muscle tyrosine kinases associated with the EGF receptor is increased in response to
stimulation by EGF in hypertension, resulting in the observed increase in
growth/mitogenesis as reported in the literature and, as we proposed to show, increased
contractility. The findings of these experiments are relevant to the treatment of
hypertension as tyrosine kinases have been shown to serve both vascular growth and
contraction.

The isolated tissue bath system was used to identify and pharmacologically
characterize the contractile response to EGF in aortic vessels. As several mechanisms
could account for the observed contraction to EGF in hypertensive vessels, different
molecular approaches were utilized to identify potential mechanism(s) by which

contraction to EGF could be enhanced in hypertensive vessels. Previous studies lead me

126

to hypothesize that the activity of tyrosine kinases utilized by the EGF receptor is
upregulated upon a sustained elevation in blood pressure. In addition, endothelium-
dependent vasorelaxation has been shown to be diminished in some forms of
experimental hypertension and, at the same time, tyrosine kinase-dependent contraction is
increased in vessels. With these findings in mind, I hypothesized that NO normally
reduces the activity of MEK thereby constitutively inhibiting contraction, but the
development of hypertension leads to reduced NO function and results in increased MEK

activity and ultimately, enhanced contraction.

A. Identification of Contractile Response to EGF in Aorta from Sham and DOCA- .
salt Rats

A profound contraction to EGF was observed in arteries from experimentally
hypertensive rats that was not observed in arteries from sham rats. While the sensitivity
of vascular smooth muscle to vasoconstrictors like S-HT is enhanced in vessels from
DOCA-salt hypertensive rats, we observed the actual appearance of a dramatic
contraction to EGF in aortic strips from DOCA-salt hypertensive rats. Excessive
vascular smooth muscle cell growth/remodeling is also common to the DOCA-salt model
of hypertension used in this study. A significant increase in the total amount of vascular
DNA has been observed after 10 days of treatment with DOC-salt therapy as compared to

that from sham rats (Mangiarua et al., 1981). Taken together, these data indicate that

127

significant changes in the signaling pathway for EGF occur during the development of
hypertension that allow for the appearance of a contraction to EGF. Thus, EGF should

not be considered as only a modulator of vascular growth but also, of vascular reactivity.

B. Pharmacological Characterization of the Contractile Response to EGF in Aorta
from DOCA-salt Rats

1. Dependence on Calcium

With respect to the mechanism by which EGF-induced contraction occurs, it was
observed that EGF-induced contraction was dependent upon the activation of calcium
channels as the L-type voltage-gated calcium channel inhibitor diltiazem reduced EGF-
induced contraction. This finding is not necessarily surprising given the well-established
role of calcium in vascular contraction, but raises an interesting question as to how a
growth factor receptor couples to voltage-gated calcium channels. While EGF has been
shown to reduce depolarization-stimulated calcium uptake by voltage-gated
dihydropyridine-sensitive calcium channels in rat pituitary cells (Hinkle et al., 1993),
voltage-operated calcium channel currents in vascular smooth muscle cells isolated from
rabbit ear arteries were increased by PDGF (W ijetunge and Hughes, 1995). The increase
in calcium channel current in response to PDGF was shown to require tyrosine
phosphorylation as preincubation with the tyrosine kinase inhibitor genistein reduced

calcium channel currents. Thus, activation of the EGF receptor may cause

128

phosphorylation of voltage-dependent calcium channels and lead to increased channel
activity. Alternatively, a study by Peppelenbosch et al. (1991) has provided evidence for
another mechanism by which the EGF receptor could stimulate voltage—dependent
calcium channel activity. In this study, activation of the EGF receptor was demonstrated
to initially activate voltage-independent calcium channels allowing for a transient influx
of calcium in A431 cells. The influx of calcium resulted in the activation of calcium-
dependent potassium channels and membrane hyperpolarization, ultimately, leading to
the activation of hyperpolarization-sensitive calcium channels and calcium influx.
Finally, one study has shown that the activity of shark rectal gland sodium-potassium-
ATPase was significantly reduced when the catalytic subunit was serine/threonine
phosphorylated (Bertorello et al., 1991). If a serine/threonine protein kinase activated by
the EGF receptor is capable of phosphorylating and inhibiting the activity of this ATPase,
thereby preventing potassium efflux and causing membrane depolarization, then it could
be envisioned to be another mechanism by which the EGF receptor could couple to
voltage-dependent calcium channels. Taken together, these studies suggest that the EGF
receptor may be coupled to voltage-dependent calcium channels in vascular smooth
muscle and therefore could be utilized as a mechanism to elicit contraction. In support of
our demonstration of a profound contraction to EGF in hypertensive arteries, contractile
responsiveness to Bay K 8644, a L-type calcium channel agonist, is enhanced in arteries

from DOCA-salt rats (Watts et al., 1994). If calcium channel activity is increased in this

129

model of hypertension and the EGF-receptor utilizes this same channel or is abnormally
coupled to this channel in hypertension, then this may be one explanation for the
increased efficacy of EGF in vessels from DOCA-salt hypertensive rats. While
previously reported studies indicate that EGF is capable of stimulating calcium influx,
our findings extend these to demonstrate an important functional role for EGF-induced
calcium influx, that role being vascular contraction.

2. Independence of the C yclooxygenase Pathway

In contrast to the crucial role of calcium, activation of the cyclooxygenase
pathway does not appear to be a requirement for EGF-induced contraction in aorta from
DOCA-salt rats. The cyclooxygenase inhibitor indomethacin did not reduce EGF-
induced contraction in DOCA-salt rat aorta. This finding is in disagreement with studies
examining EGF-induced contraction in rat ileocolic and superior mesenteric arteries and
in guinea pig gastric longitudinal muscle (Muramatsu et al., 1985; Zheng et al., 1997).
Thus, it appears that activation of the cyclooxygenase pathway by EGF is species- and
tissue-specific.

3. Dependence on Tyrosine Kinases

a. EGF Receptor Tyrosine Kinase

Because vascular growth and contraction both depend upon the activation of

tyrosine kinases (Sauro and Thomas, 1993a; Zheng et al., 1997), we postulated that the

common link in these events was an upregulation in the activity of tyrosine kinases

130

associated with the EGF. The first tyrosine kinase to be activated by EGF is contained
within the EGF receptor. Our results using the EGF receptor tyrosine kinase inhibitors
4,5-dianilinophthalimide and AGl478 indicate that activation of the receptor tyrosine
kinase is required for EGF-induced contraction. The requirement of EGF receptor
activation for EGF-induced contraction is also supported by the finding that contraction
to TGF-ot and Hb-EGF, both capable of activating the EGF receptor, was enhanced in
strips from DOCA-salt rats.
b. Erk MAPK Pathway

MEK is a dual specificity kinase capable of activating Erk-MAPK proteins by
phosphorylating tyrosine and threonine residues (Zheng and Guan, 1994). MEK appears
to be critical for contraction in response to EGF as the MEK inhibitor PDO98059
dramatically reduced EGF-induced maximal contraction in aorta from DOCA-salt rats.
Genistein, a general tyrosine kinase inhibitor, also inhibited contraction to EGF, further
supporting a role for tyrosine kinases in vascular contraction. In a different study, aorta
from WKY and SHR displayed a small, graded contractile response to PDGF with aorta
from SHR achieving a slightly but significantly greater maximal isometric contraction
than the WKY rats (225:20 and 153:17 mg respectively) (Sauro and Thomas, 1993a;
Sauro and Thomas, 1993b). An upregulation of MAPK protein density or activity could
result in increased phosphorylation of contractile proteins. As discussed in the

introduction, caldesmon and myosin light chain are two contractile protein targets of

131

tyrosine kinases. The phosphorylation of either contractile protein results in vascular
contraction, thus, an increase in the activity of the Erk MAPK pathway and MEK could
result in an enhanced contraction to EGF or other activators of the Erk MAPK pathway.

c. Effect of PDO98059 on Contraction to Ang”

Alternatively, we determined that not all mitogens use the Erk MAPK pathway to
stimulate contraction and that not all vasoconstrictors demonstrate enhanced contractile
ability in the vasculature of hypertensive rats. Activation and phosphorylation of the Erk
MAPK pathway is known to occur upon treatment of vascular smooth muscle cells with
AngII (Molloy et al., 1993; Watts et al., 1998). However, even though AngII stimulates
Erk MAPK phosphorylation, we found this activation is dissociated from contraction as
PDO98059 did not inhibit AngII-induced contraction. Moreover, contraction to AngII in
aortic strips from DOCA-salt rats was not different from contractile responses of sham
rats, and this is in agreement with other studies (Couture and Regoli, 1980a). These data
would support the hypothesis that there is a change in the signaling pathway specific to
EGF, or at least to compounds that use a tyrosine kinase/Erk MAPK-dependent pathway
for vascular contraction, that allows for this profound contraction to occur in
hypertensive vessels.

From the pharmacological studies, it was determined that contraction to EGF was
apparent in aorta from DOCA-salt but not sham rats. EGF-induced contraction was slow

to develop and completely reversible. The contraction to EGF was shown to be

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dependent upon the activation of the EGF receptor, the Erk MAPK pathway, and calcium
channel activation but not the cyclooxygenase pathway. Finally, TGF-ot and Hb-EGF,
both activators of the EGF receptor, also stimulated contraction in aorta from DOCA-salt
but not sham rats, indicating that the appearance of a contractile response is not specific

to EGF but rather the signaling pathway utilized by the EGF receptor.

C. Biochemical Mechanisms for Contractile Response to EGF

1. Changes in ErbB Receptor Levels

a. ErbB—1 Receptor Levels

One mechanism by which an increase in contraction, specific for EGF, could
occur is by an upregulation in the number of EGF receptors used to mediate contraction
to EGF. I hypothesized that blood vessels from DOCA-salt rats have a greater number of
EGF receptors than vessels from sham rats and this increase in receptor number results in
the appearance of a contraction to EGF. Although significantly more mRN A for the EGF
receptor was detected in aorta from DOCA-salt rats as compared to sham aorta, a
quantifiable difference in actual EGF receptor protein was not detected when EGF
receptor protein was specifically isolated via irnrnunoprecipitation of aortic homogenate
from sham and DOCA-salt rats. In preliminary experiments, EGF receptor protein levels
were also examined using Western analysis of total aortic lysate. However, as with

experiments utilizing immunoprecipitation, two problems were encountered. In some

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instances, multiple proteins were recognized by the antibody for the EGF receptor.
Degradation products of the EGF receptor or glycosylated forms of the EGF receptor
could account for the multiple proteins that were recognized by the EGF receptor
antibody. It could also be that the antibody used in these experiments was not specific
for the EGF receptor and was nonspecifically binding to other proteins. In other cases,
no detectable differences in the amount of EGF receptor protein were observed. A
possible explanation for observing increased levels of mRNA for the EGF receptor but
not observing increased levels of the actual protein is a posttranslational modification of
the EGF receptor such that the protein is degraded before it becomes functional.

Variable results like these have also been reported in the literature. Although it .
has been suggested that increased DNA synthesis in SHR cells was due to an increase in
the number of EGF receptors and, therefore, increased tyrosine kinase activity associated
with those receptors (Clegg and Sambhi, 1989), another study found no differences in the
number or affinity of EGF receptors for EGF between the SHR and WKY cells (Bukoski
et al., 1991). In a different study, an increase in EGF receptor maximal binding without
increased binding affinity in the aorta of the Lyon hypertensive rat was observed as
compared to control values (Swaminathan et al., 1996). Although I did not directly
measure EGF receptor binding, it could be speculated that an increase in the number of
EGF receptors or the binding of EGF to EGF receptors could increase the activation of

the receptor tyrosine kinase and signal transduction pathway associated with the receptor,

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allowing for an augmented response to EGF. Alternatively, EGF receptor (ErbB-1)
levels may not increase, but rather, the levels of a different ErbB receptor subtype is
enhanced in lysate from DOCA-salt rats.
b. ErbB-2 Receptor Levels

In addition to ErbB-1, he ErbB receptor superfamily also includes ErbB-2, ErbB-
3 and ErbB-4 receptors (Wang et al., 1998). As EGF (ErbB-1) receptor levels may not
different between sham and DOCA-salt aorta, 1 next hypothesized that aorta from
DOCA-salt rats express higher levels of a different ErbB receptor than sham rats. The
observation that agonists specific to ErbB-1 (EGF and TGF-a) stimulated contraction in
aorta from DOCA-salt hypertensive rats helped to focus my attention on the ErbB-2
receptor for several reasons. First, ErbB-2 has no known ligand but does have high
intrinsic tyrosine kinase activity and is the preferred partner for the other ErbB receptors.
Second, amplification and overexpression of the ErbB-2 receptor causes constitutive
activation of ErbB-2, ligand-independent activation of the EGF receptor and inhibits the
down-regulation of ligand-activated EGF receptor as well as promotes the recycling of
the EGF receptor back to the cell surface (Worthylake et al., 1999). Finally, the ErbB-2
receptor was a likely candidate to be increased in hypertensive vessels as coexpression of
ErbB-l and ErbB-2 receptors occurs more often in ovarian cancer cells (47-68 % of
ovarian cancers) than in normal ovarian cells (9-18 % normal ovarian epithelial cells)

(Bast et al., 1998). However, western analysis did not reveal detectable levels of ErbB-2

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in aortic lysate from sham or DOCA-salt rats. These findings could be interpreted in two
ways. First, the western analysis protocol used to detect ErbB-2 receptor levels may not
have been sensitive enough to detect changes in the amount of ErbB-2 protein in aortic
homogenate. Alternatively, ErbB-2 may not be highly expressed in vessels from
hypertensive rats and therefore, does not play a role in EGF-induced contraction.
Because my results neither proved nor refuted the role of ErbB-2 in EGF-induced
contraction, future experiments are required to determine more accurately determine
whether or not ErbB-2 or other ErbB receptor subtypes (ErbB-3 and ErbB-4) play a role
in contraction to EGF.

2. Enhanced Localization of ErbB Receptors

Another explanation for why EGF receptor levels were not different between
aorta from sham and DOCA-salt rats is that the protein levels are the same and the levels
of another protein that associates with the EGF receptor is increased such that enhanced
EGF receptor signaling and contraction occurs in hypertensive vessels. One potential
protein is tenascin-C. Extracellular matrix proteins like tenascin-C, also known as
hexabrachion or cytotactin, are expressed in low levels in normal adult tissue but are
increased in actively remodeling or pathologic tissues (Erickson, 1989). Serum has been
shown to selectively regulate tenascin-C expression with out affecting the expression of
other matrix proteins (Sharifi et al., 1992). Extracellular matrix proteins not only help to

maintain the structure of the vessel but also have been shown to be responsible for

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differences in growth characteristics between SHR and WKY rats (Scott-Burden et al.,
1989). Strong tenascin-C immunohistochemical staining has been observed at major
branches of the aorta in WKY rats while SHR rats had markedly increased staining at the
same sites, sites which are exposed to significant mechanical stress (Mackie et al., 1992).
Tenascin-C contains EGF-like domains (Engel, 1989) and studies by Jones et al. (1997a)
suggest that EGF-dependent neointimal smooth muscle cell proliferation is modulated by
tenascin-C as tenascin-C expression was co-localized with EGF in obstructive lesions
from infant lung biopsies. I did not measure tenascin-C levels in aortic homogenate from
sham and DOCA-salt rats but an increase in tenascin-C expression could be postulated to
be one mechanism whereby contraction to EGF could be increased in DOCA-salt aorta.
While our studies did not demonstrate a change in EGF receptor or ErbB-2 proteins
between aortic homogenate from sham and DOCA-salt hypertensive rats, other studies
have demonstrated enhanced clustering and activation of EGF receptors through the
induction of tenascin-C (Jones et al., 1997b). Moreover, in rat pulmonary artery smooth
muscle cells, tenascin-C was required for EGF-dependent growth and the cooperativity
between tenascin-C and the EGF receptor was facilitated by the avB3 integrin receptor,
resulting in reorganization of the actin cytoskeleton and increased EGF receptor
clustering (Jones et al., 1997b). In addition to modulating EGF-stimulated vascular
growth, enhanced EGF receptor clustering through an upregulation in tenascin-C

expression could also be a mechanism by which the observed dramatic contraction to

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EGF occurs. The positioning of EGF receptors via tenascin-C into close proximity may
allow the receptors to be more readily activated in the presence of EGF and ultimately,
result in a greater contractile response to EGF. Taken together, these studies represent a
different mechanism by which EGF receptor signaling could be enhanced in the absence
of an increase in the actual number of EGF receptor. Further, these studies represent a
potential future area of investigation that will more fully examine the mechanism by
which contraction to EGF occurs in hypertensive vessels.

3. Changes in Protein Tyrosine Kinase Activity

The main hypothesis of these experiments was that the profound contraction to
EGF in aorta from DOCA-salt rats is the result of increased vascular tyrosine kinase
activity. While a significant difference was not observed, our studies revealed a trend for
general tyrosine kinase activity to be increased, both basally and in response to EGF, in
aortic vessels from DOCA-salt rats. In a different study, basal and PDGF-stimulated
tyrosine kinase activity was significantly increased in SHR as compared to WKY aorta
(Sauro and Thomas, 1993a; Sauro and Thomas, 1993b). Further, one study found that
although total MAPK content was similar between thoracic aorta from normotensive rats
and rats made hypertensive via aortic ligation, tyrosine phosphorylated MAPK content
was greater in aorta from hypertensive rats as compared to shams (Tong et al., 1998). It
was also demonstrated that the MEK inhibitor PDO98059 (5 mg, sc) reduced the blood

pressure of conscious hypertensive rats but did not affect blood pressure in sham rats,

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indicating that the activity of MEK specifically, may be increased in hypertension. One
reason for not observing a significant difference in tyrosine kinase activity between sham
and DOCA-salt rats could be that total aortic homogenate was used in the tyrosine kinase
assay. Although the activity of all tyrosine kinases within vascular smooth muscle may
not be significantly increased, the activity of tyrosine kinases associated specifically with
the EGF receptor like the EGF receptor itself or MEK may, in fact, be increased in aortic
cells from hypertensive rats, but this increase in activity is diluted out when the activity
of all tyrosine kinases is examined together. Further studies isolating specific proteins
like the EGF receptor and MEK are necessary to more accurately examine tyrosine
kinase activation in vascular smooth muscle from hypertensive rats, both basally and in
response to EGF.

From these biochemical studies, it has been determined that messenger RNA for
the EGF receptor is increased in aorta from DOCA-salt rats as compared to shams.
However, a quantitative difference between actual EGF receptor or ErbB-2 receptor
protein in aorta from sham and DOCA-salt rats was not observed when examined using
immunohistochemistry and Western analysis. Also, although there was a trend for
general tyrosine kinase activity to be increased in aorta from DOCA-salt rats above that
of shams rats, a significant difference was not obtained. These studies provide more
evidence for the idea that the signaling pathway utilized by the EGF receptor may be

enhanced in hypertension.

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D. Physiological Mechanisms for Contractile Response to EGF

1. Dependence of the Contractile Response to EGF on Blood Pressure

With respect to the dependence of EGF-induced contraction on blood pressure,
contraction to EGF was found to be profoundly increased in aortic vessels from not only
experimentally hypertensive rats-- DOCA-salt, lK-lC, and L-NNA models of
hypertension-- but also in vessels from genetically hypertensive rats (SHR) as well. It
also was demonstrated that contraction to EGF was significantly greater in aorta from
DOCA-salt rats after 14 days of therapy, a time point when the systolic blood pressure of
DOCA-salt rats was significantly elevated above that of sham rats. Further, a significant
correlation was observed between systolic blood pressure and the maximal contraction to
EGF. Sirniliarly, Mangiarua et al. (1981) reported that the blood pressure of Wistar rats
on DOC-salt therapy was slightly but significantly elevated by day 10. DNA synthesis
was markedly increased in arteries from DOC-salt rats as compared to shams by day 10
as well, but synthesis was similar between shams and DOC-salt rats at day 30, indicating
that increased synthesis occurs primarily in the first days of treatment. Together, these
studies suggest that during the initial development of hypertension, significant cellular
changes are occuring within the vascular smooth muscle and these changes may promote
or allow enhanced EGF receptor signaling and ultimately, vascular contraction.

Mechanical stress results in transient increases in blood pressure and if the vessel

is chronically exposed to this elevated blood pressure, the artery wall thickens, resulting

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in hypertension. It is known that arteries like the aorta and the carotid arteries remodel in
response to high blood pressure as well as demonstrate medial hypertrophy (enlarged size
of vascular smooth muscle cells) and hyperplasia (increased number of vascular smooth
muscle cells) of the arterial wall (Owens, 1989). Several investigators are examining
how elevated blood pressure is sensed by cells and some studies have indicated that
mechanical stress can induce intracellular signaling (Takahashi and Berk, 1996; Ishida et
al., 1996). In a study by Hu et al. (1998) it was demonstrated that PDGF Ot-receptors and
Erk-2 MAPK proteins were phosphorylated and activated, in the absence of ligand
activation, in response to vascular smooth muscle cell stretch. It has also been observed
that increases in artery pressure result in membrane depolarization (Harder, 1984) and .
phospholipase C activation (Matsumoto et a1. 1995). Further, Prewitt and co-workers
have found that elevating intraluminal pressure in isolated mesenteric arteries increases
the expression of proto-oncogene c-fos, known to involved in cellular growth of vascular
smooth muscle cells (Miriel et al., 1999). Alternatively, a depolarizing stimulus that
induced calcium influx, independent of ligand-receptor activation has been shown to
transactivate the EGF receptor (Zwick et al., 1999). Thus, it could be postulated that
increased stretch on the blood vessel or a depolarizing stimulus causing calcium influx
could also activate the EGF receptor and therefore, increase the activity of the signaling
pathway utilized by the EGF receptor. Together, these studies also suggest that increased

stretch on the blood vessel can result in enhanced intracellular signaling.

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2. Independence of the Contractile Response to EGF on Blood Pressure

The observation that contraction to EGF occurred in arteries from multiple forms
of experimental hypertension next lead me to hypothesize that contraction to EGF is
dependent upon an elevation in blood pressure. To address this question, Wistar and
Wistar-Furth rats were placed on DOCA-salt therapy as studies have determined Wistar
rats but not Wistar-Furth rats are sensitive to DOCA-salt therapy and hypertension. If
our hypothesis was correct, EGF would have stimulated contraction in hypertensive
Wistar DOCA-salt rats but not in normotensive Wistar-Furth rats on DOCA-salt therapy.
As expected, contraction to EGF occurred in aorta from Wistar DOCA-salt rats but not
Wistar shams. Surprisingly, EGF stimulated a virtually identical contractile response in
aorta from Wistar-Furth DOCA-salt rats, as that observed from Wistar DOCA-salt rats,
even in the absence of high blood pressure. Because contraction to EGF occurred in the
absence of high blood pressure in aorta from Wistar-Furth DOCA-salt rats, we next
wanted to address the influence of DOCA-alone and high salt-alone on contraction to
EGF. The therapies of DOCA alone or high salt alone on Sprague-Dawley rats both
produced a significant increase in blood pressure, but only DOCA therapy resulted in a
significant contraction to EGF. To further refute the total dependence of EGF-induced
contraction on blood pressure, a significant correlation was not observed between the
absolute degree of elevation in blood pressure and the maximal response to EGF. In a

different study, Ullian et al., (1997) found that AngII- and PE-induced contractions were

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potentiated in aortic rings from Wistar rats on DOCA—salt therapy but not in rings from
Wistar-Furth DOCA-salt rats. Together with our findings, these data suggest that DOCA
or mineralocorticoids, in general, may modulate specifically the response to EGF
independently of elevated blood pressure. However, one question remains; how do
mineralocorticoids regulate the contractile response to EGF?

3. Dependence of Vascular Reactivity on Mineralocorticoids

Abnormal vascular reactivity observed in SHR-stroke prone (SHRSP) has been
thought to be dependent on adrenal mineralocorticoids (Brunner and Webb, 1988). In
support of this concept, plasma aldosterone levels have been reported to be elevated in
older SHRSP (18 weeks) with malignant hypertension (Kim et al., 1992) and to play a
role in the development of renal injury in the remnant kidney model of chronic renal
failure (Greene et al., 1996). In the study by Greene et al., exogenous aldosterone
administration completely reversed the ability of combined enalapril and losartan
treatment to alleviate hypertension. In a different study, expression levels of vascular
mineralocorticoid receptor mRNA were found to be increased in 4 and 9 week old
SHRSP and also, plasma aldosterone levels were significantly increased in 9 week old
SHRSP when compared with age-matched WKY (Takeda et al., 1997). Further,
spironolactone, a mineralocorticoid receptor antagonist, treatment did not lower blood
pressure in SHRSP as compared to placebo-treated SHRSP but did demonstrate a

protective effect against cerebrovascular and renal lesions (Rocha et al., 1998). One

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speculative mechanism by which mineralocorticoids could regulate vascular reactivity to
EGF is if a mineralocorticoid response element (MRE) was contained within the
promoter region of the gene for the EGF receptor. Excess mineralocorticoids, shown to
be elevated in several experimental models of hypertension, could bind mineralocorticoid
receptors, translocate to the nucleus and bind to the MRE within the gene for the EGF
receptor, thereby promoting synthesis of the EGF receptor. Thus, mineralocorticoids
may play a role in regulating vascular reactivity to EGF via stimulating synthesis of the
EGF receptor. This idea is supported by the observation that mRNA levels for the EGF
receptor were increased in aorta from DOCA-salt rats but is in conflict with the results
demonstrating that the levels of EGF receptor protein were the same in sham and DOCA-
salt rats. Mineralocorticoids may also regulate tenascin-C expression which could, in
turn, modulate EGF receptor activity. Further experiments examining the influence of
mineralocorticoids on the EGF receptor and possibly proteins like tenascin-C that are
associated with the EGF receptor are required to more accurately determine the effect of
mineralocortoids on EGF signaling.

From these studies it was determined that EGF-induced contraction is common to
several forms of experimental hypertension and that the contractile response did not
appear until after a significant elevation in blood pressure. A significant correlation was
observed between systolic blood pressure and the maximal response to EGF however, the

maximal response to EGF was independent of the absolute value of systolic blood

144

pressure. Interestingly, contraction to EGF occurred in aorta from Wistar-Furth rats that
were on DOCA-salt therapy but were not hypertensive. Minimal contraction to EGF was
observed in rats on DOCA therapy or high salt therapy, even though the rats were
hypertensive, indicating that an elevated systolic blood pressure alone is not the sole

cause for the contraction to EGF.

E. Influence of the Endothelium on the Contractile Response to EGF

Recent reports have focused on the importance of MAPK activation in the
regulation of smooth muscle cell growth and contraction (Zheng et al., 1997; Kelleher et
al., 1995). Erk MAPK proteins are activated by growth factors and hormones and this
activation can lead to the mitogenesis and proliferation of smooth muscle that is observed
in some vascular diseases. The phosphorylation status of the Erk MAPK proteins is a
balance between the current activation of kinases and phosphatases. I specifically
addressed the novel hypothesis that NO can affect the kinase MEK and thereby alter the
activity of the MAPK pathway.

1. Eflect of NO on the Contractile Response to EGF

Several reports have revealed that exogenous NO donors relax vascular smooth
muscle and our results are in agreement; the NO donor SNAP relaxed rat aortic tissues
contracted with EGF, the contraction of which was demonstrated to be dependent on the

activation of MEK. In addition, the contractile response to EGF was significantly

145

reduced in the presence of the endothelium. As the response to EGF in endothelium-
intact DOCA-salt strips was slightly attenuated, these findings suggest that the
endothelium is still somewhat functional and capable of reducing vascular contraction.
Interestingly, a concentration-dependent contraction to EGF was evident in endothelium-
intact aortic strips from sham rats but not in endothelium-denuded sham rat aorta. One
speculative possibility for the observed contraction to EGF in endothelium-intact aortic
strips from sham rats is the presence of endothelial EGF receptors which, when
stimulated, could stimulate the release of an endothelial derived contractile factor.
Because we demonstrated that EGF-induced contraction in aorta from DOCA-salt
hypertensive rats was significantly reduced by the MEK inhibitor PD98059, the finding
that NO inhibited EGF-induced contraction further supports the idea that N 0 may inhibit
tyrosine kinase/MEK activity to reduce vascular contraction. To the contrary, any
available endogenous NO, although reduced in this form of hypertension, did not reduce
EGF-induced contraction in aorta from DOCA-salt rats. Further, a significant reduction
in ACh-induced relaxation was observed only after a substantial rise in blood pressure
and was concurrent with the appearance of a profound contraction to EGF. Together,
these finding alternatively suggest that NO may not have a significant effect on MEK
activity. While the experiments examining the effect of NO on EGF-induced MEK-
dependent contraction are useful, they provide only indirect support the idea that NO

inhibits MEK activity.

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2. Effect of NO on the Constitutively Activated MEK Protein

To better link the functional event-- inhibition of MEK-dependent contraction by
NO-- and the biochemical event of interest-- inactivation of MEK by NO-- we used a
MEK assay to examine changes in MEK activity caused by NO. However, the maximal
concentrations of SNAP and SNP used to inhibit contraction did not affect the activity of
the fusion protein GST-MEK-ZE. These results can be interpreted in several ways.

a. N0 Does Inhibit MEK Activity

First, NO may inhibit MEK activity. NO completely inhibited tyrosine kinase-
dependent contraction to EGF. The possibility of NO directly affecting a kinase is
supported by several recent papers demonstrating that NO inhibits kinases including rat
janus kinase-2 (JAK-2) activity (Duhe et al., 1998), and MAPK activity (Kubo et al.,
1998). A different study found NO reduced the tyrosyl-phosphorylation of focal
adhesion proteins (Kaur et al., 1998). In a relevant and interesting paper by Estrada et al.
(1997), it was demonstrated that N O inhibited EGF receptor phosphorylation and tyrosine
kinase activity; however, there are two points that warrant attention. First, the cells used
in their study, EGFR-T17 fibroblast cell line, highly over express EGF receptor protein.
Second, extremely high concentrations of NO (in the millimolar range) were used to see
an effect by NO. While it may be possible in diseases like septic shock for the
concentration of NO to possibly reach such high levels, the combination of over

expressed EGFR and millimolar NO, begs the question- is this system truly

147

representative of a physiological state? Taken together, these recent studies indicate that
NO is capable of affecting the activity of protein kinases. One speculative mechanism by
which NO could inhibit MEK is the following. There is a cysteine residue near the
catalytic domain of MEK (Zheng and Guan, 1994) and thus it is possible that NO could
form an S-nitrosothiol bond with the cysteine residue, likely inactivating MEK. This
mechanism of action, S-nitrosylation of thiols, appears to be the case in NO-induced
inactivation of protein kinase C activity (Gopalakrishna et al., 1993) and the EGF
receptor tyrosine kinase (Estrada et al., 1997). Similarly, one study demonstrated NO
could inhibit the autokinase activity of J AK-2, most likely through oxidation of critical
dithiols to disulfides (Duhe et al., 1998).
b. N0 Does Not Inhibit MEK Activity
The other possibility is that NO does not affect MEK and this is more likely the
case. This position is supported by our demonstrating that endogenous NO did not inhibit
contraction to EGF and that NO did not significantly inhibit phosphorylation of the 42
kDa Erk MAPK protein in the MEK assay. Several possibilities exist to explain these
results. First, N 0 may really have no effect, either against mutation-activated or agonist-
activated MEK. The power of the MEK assay used in this study is that it examines the
direct effect of NO on an isolated and pre-activated MEK protein in the absence of agonist
stimulation and other cellular proteins, like phosphatases and cGMP, that may effect the

activity of MEK. The fact that the MEK inhibitor PD98059 but not the NO donors

148

reduced the activity (represented as a decrease in 42 kDa Erk MAPK phosphorylation) of
MEK indicates that NO probably does not inhibit MEK directly.

The finding that NO did not directly inhibit the activity of MEK was not
necessarily surprising as the majority of the effects of NO are mediated via cGMP-
dependent mechanisms (Murad, 1986). However, several studies have indicated that NO
can directly mediate hyperpolarization and relaxation of arterial smooth muscle (Tare et
al., 1990; Cohen et al., 1997). Both exogenous and endogenous NO have been shown to
activate directly calcium-dependent potassium channels in cell-free rabbit aortic
membrane patches without requiring cGMP (Bolotina et al., 1994). Also, NO has been
demonstrated to stimulate the activity of vascular sodium-potassium—ATPase activity,
independent of cGMP activation (Gupta et al., 1994). From these studies it could be
speculated that NO reduces EGF-stimulated vascular contraction not through inhibition of
tyrosine kinase activity but rather through hyperpolarization of vascular cell membranes.

3. Impact of NO on the Vasculature of Hypertensive Animals
The idea that NO may influence the overall activity of proteins, either kinases or
phosphatases, associated with the MAPK pathway in vascular smooth muscle is
compelling given that some forms of vascular disease are associated with endothelial
dysfunction and excessive smooth muscle proliferation. Acetylcholine-induced
endothelium-dependent relaxation, known to be mediated by NO, is reduced in human

essential hypertension (Panza et al., 1990) and in several forms of experimental

149

hypertension (Lockette et al., 1986), indicating that endothelial NO production may be
diminished. Alternatively, aorta from the SHR display increased tyrosine kinase activity
as compared to normotensive WKY (Sauro and Thomas, 1993a). Also in acute
hypertension, MAPK proteins have been shown to have higher activity in response to a
rise in blood pressure (Xu et al., 1996). From these findings, it can be speculated that
NO, under normal conditions, may inhibit tyrosine kinase activity, preventing smooth
muscle hyperplasia and hyperresponsiveness to contractile agonists. However, during
vascular disease associated with endothelial dysfunction, the loss of NO allows for
excessive and abnormal activation of the MAPK pathway leading to enhanced smooth
muscle growth and contration. These studies however, would suggest that this inhibition

was not due to a direct effect of NO on MEK activity.

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CONCLUSIONS

The main goal of the work contained in this dissertation was to gain a better
understanding of the pharmacologic and physiologic mechanisms by which EGF
stimulates vascular contraction in arteries from hypertensive rats. EGF, a growth factor
well established as an activator of tyrosine kinases, was used to examine differences in
contractile responses of arteries from sham normotensive and DOCA-salt hypertensive
rats. These experiments more fully defined the role of EGF and the Erk MAPK pathway
as modulators of vascular contraction in vessels from hypertensive rats.

Classically, EGF has been shown to mediate growth (figure 33) of vascular
smooth muscle cells from hypertensive animals. Several studies have demonstrated that
EGF-stimulated growth was enhanced in vascular smooth muscle cells from hypertensive
rats as compared to responses of vascular smooth muscle cells from normotensive rats
(Suithichaiyakul et al., 1990; Clegg and Shambi, 1989; Bukoski et al., 1991). Recent
studies have implicated tyrosine kinases in vascular contraction (Di Salvo et al., 1993;
Zheng et al., 1997). However, the role of EGF as a vasoconstrictor was less well defined.
While some studies have examined contraction to EGF in vessels from normotensive rats
(Berk et al., 1985), no studies had examined the contractile response of EGF in
hypertension. As vascular growth, known to be dependent on tyrosine kinases, is
enhanced in vascular smooth muscle, I hypothesized that contraction to EGF would also

be enhanced in vessels from hypertensive rats.

151

The experiments conducted in this study indicated that the signal transduction
elements for EGF are enhanced in hypertension, resulting in an augmented contractile
response to EGF. Contraction to EGF in aorta from DOCA-salt rats was found to be
dependent on activation of the EGF receptor tyrosine kinase, MEK and L-type calcium
channels and persists in the presence of the endothelium. Biochemically, it was
demonstrated that there is a significant difference in mRN A levels for the EGF receptor
between aorta from sham and DOCA-salt rats. Physiologically, contraction to EGF did
not appear until after a significant rise in systolic blood pressure and was concurrent with
a reduction in endothelium-dependent vasorelaxation, indicating that while the altered
vascular reactivity is at least in part the result of the high blood pressure. It was also
determined that the contractile response to EGF is in part dependent on elevated systolic
blood pressure, other factors like mineralocorticoids and the length of time that the
animals are hypertensive may also regulate the response to EGF in vessels from
hypertensive rats. Taken together, these findings have demonstrated that the signaling
pathway activated by EGF is significantly enhanced in hypertension and this
enhancement results in not only vascular growth but also vascular contraction in vessels
from hypertensive rats and this is represented in figure 34.

Importantly, it should be noted that the concentrations of growth factors used in
the studies were in a range for exerting physiological modifications on blood vessels that

could affect total peripheral resistance (Oka and Orth, 1983). This idea is especially

152

compelling given the knowledge that in humans, EGF is stored in platelets along with
other contractile agonists and mitogens like S-HT. As several studies have suggested the
actions of several G protein-coupled agonists are exerted through activation of the EGF
receptor (Daub et al., 1996; Fujitani and Bertrand, 1997), the potential exists for the EGF
receptor to dramatically modulate vascular reactivity. From these studies, one could
speculate that EGF receptors profoundly affect the vasculature not only via direct
activation by EGF but also through indirect activation by G protein-coupled receptors.
Thus, it can be envisioned that during a thrombotic event leading to platelet degranulation
and the release of platelet contents, the potential exists for synergistic action of these
vascular agonists on not only growth but also contraction.

Excessive vascular smooth muscle cell growth and hyperresponsiveness to
contractile agonists are known to contribute to elevated total peripheral resistance and
thus systolic blood pressure. From these findings, it could be postulated that the observed
enhancement in reactivity to EGF, both synthetically and functionally, could support the
chronic maintenance phase of hypertension. Further investigation may reveal the
molecular mechanisms by which this enhanced vascular reactivity to EGF occurs as well
as identify potential therapeutic targets to prevent the excessive vascular growth and

contraction associated with cardiovascular disease.

153

Figure 33. Schematic diagram representing the traditional role of EGF in the
reactivity of vessels from hypertensive rats. EGF stimulates growth to a greater
extent in hypertensive vessels resulting in increased total peripheral resistance and

ultimately, systolic blood pressure.

154

 

EGF

i

Tyrosine Kinases/
Erk MAPK Pathway

l

1 Growth

i

Total Peripheral
Resistance

i

Systolic Blood Pressure

155

Figure 34. Schematic diagram representing the novel, dual role of EGF on the
reactivity of vessels from hypertensive rats. In addition to demonstrating an
enhanced mitogenic response in vascular smooth muscle cells from hypertensive
rats, EGF stimulates a dramatic vascular contraction in aortae from hypertensive
rats, both proven to increase total peripheral resistance and ultimately, systolic

blood pressure.

156

EGF

i

Tyrosine Kinases/

Erk MAPK Pathway
Growth icontraction

\ /

Total Peripheral
Resistance

1

Systolic Blood Pressure

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BIBLIOGRAPHY

Adam LP, Hathaway DR. Identification of mitogen—activated protein kinase
phosphorylation sequences in mammalian h-caldesmon. FEBS Letters. 322:56-60, 1993.

Assoian R, Spom MB. Type B transforming growth factor in human platelets: release

during platelet degranulation and action on vascular smooth muscle cells. J. Cell Biol.
102:1217-1223, 1986.

Bagby SP, Kirk EZ, Mitchell LH, O’Reilly MM, Holden WE, Stenberg PE, Bakke PC.
Proliferate synergy of ANG II and EGF in porcine aortic vascular smooth muscle cells.
Am. J. Physiol. 2652F239-F249, 1993.

Bast RC, Pusztai L, Kems B-J, MacDonald JA, Jordan P, Daly L, Boyer CM,
Mendelsohn J, Berchuck A. Coexpression of the HER-2 gene product, pISSHER'z, and

epidermal growth factor receptor, pl7OEC’F‘R, on epithelial ovarian cancers and normal
tissues. Hybridoma. 17:313-321, 1998.

Ben-Ezra J, Sheibani K, Hwang DL, Lev-Ran A. Megakayocyte synthesis is the source
of epidermal growth factor in human platelets. Am. J. Pathol. 137:755-759, 1990.

Berk BC, Brock TA, Webb RC, Taubman MB, Atkinson WJ, Gimbrone MA, Alexander
RW. Epidermal growth factor, a vascular smooth muscle mitogen, induces rat aortic
contraction. J. Clin. Invest. 75: 1083-1086, 1985.

Bertorello AM, Aperia A, Walaas SI, Nairn AC. Phosphorylation of the catalytic subunit
of Na+-K+-ATPase inhibits the activity of the enzyme. Proc. Natl. Acad. Sci. USA.
88:11359-11362, 1991.

Blenis J. Signal transduction via the MAP kinases: Proceed at your own RSK. Proc.
Natl. Acad. Sci. 90:5889-5892, 1993.

Bolotina VM, Najibi S, Palacino JJ, Pagnao PJ, Cohen RA. Nitric oxide directly

activates calcium-dependent potassium channels in vascular smooth muscle. Nature.
368:850-853, 1994.

158

Bottger A, van Lith HA, Kren V, Krenova D, Bila V, Vorlicek J, Zidek V, Musilova A,
Zdobinska M, Wang J-M, van Zutphen B, Kurtz TW, Pravenec M. Quantitative trait loci
influencing cholesterol and phospholipid phenotypes map to chromosomes that contain

genes regulating blood pressure in the spontaneously hypertensive rat. J. Clin. Invest.
98:856-862. 1996.

Bruner CA. Vascular responsiveness in rats resistant to aldosterone-salt hypertension.
Hypertension. 20:59-66, 1992.

Bruner CA, Webb RC. Adrenal—dependent change in vascular reactivity in stroke-prone
spontaneously hypertensive rats. Hypertension 12:388-392, 1988.

Buchdunger E, Trinks U, Mett H, Regenass U, Muller M, Meyer T, McGlynn E, Pinna L,
Traxler P, Lydon N. 4,5-dianilinophthalimide: A protein-tyrosine kinase inhibitor with
selectivity for the epidermal growth factor receptor signal transduction pathway and
potent in vivo antitumor activity. Proc. Natl. Acad. Sci, USA. 91:2334-2338, 1994.

Bukoski RD, DeWan P, Bo J. Mechanism of the enhanced epidermal growth factor-
induced growth response of genetically hypertensive vascular myocytes. Circ. Res.
69:757-764, 1991.

Carpenter G, Cohen S. Epidermal growth factor. Ann. Rev. Biochem. 48:193-216,
1979.

Chang H, Riese DJ, Gilbert W, Stern DF, McMahan UJ. Ligands for ErbB-family
receptors encoded by a neuregulin-like gene. Nature 387:509-512, 1997.

Che W, Asahi M, Takahashi M, Kaneto H, Okado A, Higashiyama S, Taniguchi N.
Selective induction of heparin-binding epidermal growth factor-like growth factor by

methylglyoxal and 3-deoxyglucosone in rat aortic smooth muscle cells. J. Biol. Chem.
272:18453-18459, 1997.

Childs TJ, Watson MH, Sanghera 18, Campbell DL, Pelech SL, Mak AS.
Phosphorylation of smooth muscle caldesmon by mitogen—activated protein (MAP)

kinase and expression of MAP kinase in differentiated smooth muscle cells. J. Biol.
Chem. 267:22853-22859, 1992.

159

Clegg KB, Sarnbhi MP. Inhibition of epidermal growth factor-mediated DNA synthesis
by a specific tyrosine kinase inhibitor in vascular smooth muscle cells of the
spontaneously hypertensive rat. J. Hypertens. 7:S144-S145, 1989.

Cobb MH, Goldsmith B]. How MAP kinases are regulated. J. Biol. Chem. 270:14843-
14846, 1995.

Cohen RA, Plane F, Najibi S, Huk I, Malinski T, Garland CJ. Nitric oxide is the
mediator of both endothelium-dependent relaxation and hyperpolarization of the rabbit
carotid artery. Proc. Natl. Acad. Sci. USA 94:4193-4198, 1997.

Cohen 8. Isolation of a mouse submaxillary gland protein accelerating incisor eruption
and eyelid opening in the new-bom animal. J. Biol. Chem. 237: 1555-1562, 1962.

Collis MG, Vanhoutte PM. Vascular reactivity of isolated perfused kidneys from male
and female spontaneously hypertensive rats. Circ. Res. 41:759-767, 1977.

Couture R, Regoli D. Vascular reactivity to angiotensin and noradrenaline in rats
maintained on a sodium free diet or made hypertensive with desoxycorticosterone acetate
and salt (DOCA/SALT). Clin. Expt. Hypertension. 2:25-43, 1980a.

Couture R, Regoli D. Vascular reactivity to angiotensin and noradrenaline in
spontaneously and renal hypertensive rats. Clin. Expt. Hypertension. 2:45-63, 1980b.

Dananberg J, Sider RS, Grekin RJ. Sustained hypertension induced by orally
administered nitro-L-arginine. Hypertension. 1993;21:359-363.

Daub H, Weiss FU, Wallasch C, Ullrich A. Role of transactivation of the EGF receptor
in signalling by G-protein-coupled receptors. Nature. 379:557-560, 1996.

Daub H, Wallasch C, Lankenau A, Herrlich A, Ullrich A. Signal characteristics of G
protein-transactivated EGF receptor. EMBO J. 16:7023-7044, 1997.

Davis JO, Laragh JH, Selwyn A, eds. Hypertension: Mechanisms, Diagnosis, and
Management. New York: HP Publishing Co., Inc., 1977.

DeBlois D, Drapeau G, Petitclerc E, Marceau F. Synergism between the contractile

effect of epidermal growth factor and that of des-Arg9-bradykinin or of DL-thrombin in
rabbit aortic rings. Br. J. Pharmacol. 105:959-967, 1992.

160

Deng AY, Dene H, Rapp JP. Congenic strains for the blood pressure quantitative trait
locus on rat chromosome 2. Hypertension. 30:199-202. 1997.

Di Salvo J, Gifford D, Kokkinakis A. ATP- and polyphosphate-mediated stimulation of
pp60c-src kinase activity in extracts from vascular smooth muscle. J. Biol. Chem.
264:10773-10778, 1989..

Di Salvo J, Steusloff A, Semenchuk L, Satoh S, Kolquist K, Pfitzer G. Tyrosine kinase

inhibitors suppress agonist-induced contraction in smooth muscle. Biochem. Biophys.
Res. Commun. 190:968-974, 1993.

Downward J, Yarden Y, Mayes E, Scrace G, Totty N, Stockwell P, Ullrich A. Close
similarity of epidermal growth factor rceptor and v-erb-B oncogene protein sequences.
Nature 307:521-527, 1984.

Doyle MP, Duling BR. Acetylcholine induces conducted vasodilation by nitric oxide-
dependent and —independent mechanisms. Am. J. Physiol. 272:H1364-H1371, 1997.

Dudley DT, Pang L, Decker SJ, Bridges AJ, Saltiel AR. A synthetic inhibitor of the
mitogen-activated protein kinase cascade. Proc. N at’l. Acad. Sci. 92:7686-7689, 1995.

Duhe RJ, Evans GA, Erwin RA, Kirken RA, Cox GW, Farrar WL. Nitric oxide and thiol
redox regulation of janus kinase activity. Proc. Nat’l. Acad. 95: 126-131, 1998.

Engel J. EGF-like domains in extracellular matrix proteins: localized signals for growth
and differentiation? FEBS Lett. 251:1-7, 1989.

Erickson HP, Bourdon MA. Tenascin: an extracellular matrix protein prominent in
specialized embryonic tissues and tumors. Annu. Rev. Cell Biol. 5:71-92, 1989.

Estrada C, Gomez C, Martin-Nieto J, De Frutos T, Jimenez A, Villalobo A. Nitric oxide

reversibly inhibits the epidermal growth factor receptor tyrosine kinase. Biochem. J.
326:369-376, 1997.

Florian JA, Watts SW. Integration of mitogen-activated protein kinase kinase activation
in vascular 5-hydroxytryptamine2A receptor signal transduction. J. Pharrnacol. Exp. Ther.
284:346-355, 1998.

161

Fujino T, Hasebe N, Fujita M, Takeuchi K, Kawabe J, Tobise K, Higashiyama S,
Taniguchi N, Kikuchi K. Enhanced expression of heparin-binding EGF-like growth
factor and its receptor in hypertrophied left ventricle of spontaneously hypertensive rats.
Cardiovascular Research 38:365-374, 1998.

Fujitani Y, Bertrand C. ET-l cooperates with EGF to induce mitogenesis via a PTX-
sensitive pathway in airway smooth muscle cells. Am. J. Physiol. 2722C1492-Cl498,
1997.

Funder JW. Adreanl steroids. In: Swales J. D., ed. Textbook of Hypertesion. Oxford:
Blackwell Scientific Publications; 388-396, 1994.

Funder JW. Aldosterone, salt and cardiac fibrosis. Clin. Exp. Hyperten. 19:885-899,
1997.

Furchgott RF, Zawadzki JV. The obligatory role of endothelial cells in the relaxation of
arterial smooth muscle by acetylcholine. Nature 288:373-6, 1980.

Gan BS, McCannell KL, Hollenberg MD. Epidermal growth factor-urogastrone causes ,
vasodilatation in the anesthetized dog. J. Clin. Invest. 80: 199-206, 1987.

Garg UC, Hassid A. Nitric oxide-generating vasodilators and 8-Bromo-cyclic guanosine
monophosphate inhibit mitogenesis and proliferation of cultured rat vascular smooth
muscle cells. J. Clin. Invest. 83:1774-1777, 1989.

Gillespie MN, Rippetoe PE, Haven CA, Shiao R-T, Orlinska U, Maley BE, Olson JW.
Polyamines and epidermal growth factor in monocrotaline-induced pulmonary
hypertension. Am. Rev. Respir. Dis. 140:1463-1466, 1989.

Gopalakrishna R, Chen ZH, Gundimeda U. Nitric oxide and nitric oxide-generating
agents induce a reversible inactivation of protein kinase C activity and phorbol ester
binding. J. Biol. Chem. 268:27180-27185, 1993.

Gordon RD, Stowasser M, Klemm SA, Tunny TJ. Primary aldosteronism and other
forms of mineralocorticoid hypertension. In: Swales JD, ed. Textbook of Hypertension.

Oxford: Blackwell Scientific Publications; 865-892, 1994.

Greene EL, Kren S, Hostetter TH. Role of aldosterone in the remnant kidney model in
the rat. J. Clin. Invest. 98:1063-1068, 1996.

162

Guan K-L, Butch E. Isolation and characterization of a novel dual specific phosphatase,
HVH2, which selectively dephosphorylates the mitogen-activated protein kinase. J.
Biol. Chem. 270:7197-7203, 1995.

Gupta S, McArthur C, Grady C, Ruderman N. Stimulation of vascular N A+-K+-ATPase
activity by nitric oxide: a cGMP-independent effect. Am. J. Physiol. 2662H2146-H2151,
1994.

Hamada M, Nishio I, Baba A, Fukuda K, Takeda J, Ura M, Hano T, Kuchii M,
Masuyama Y. Enhanced DNA synthesis of cultured vascular smooth muscle cells from
spontaneously hypertensive rats. Differences of response to growth factor, intracellular
free calcium concentration and DNA synthesizing cell cycle. Atherosclerosis. 81: 191-

198, 1990.

Harder DR. Pressure-dependent membrane depolarization in cat middle cerebral artery.
Circ. Res. 55:197-202, 1984.

Henrion D, Dowell FJ, Levy BI, Michel J-B. In vitro alteration of aortic vascular
reactivity in hypertension induced by chronic NG-nitro-L-arginine methyl ester.
Hypertension. 28:361-366, 1996.

Higashiyama S, Abraham JA, Miller J, Fiddes JC, Klagsbrun M. A heparin-binding
growth factor secreted by macrophage-like cells that is related to EGF. Science 251:936-

939, 1991.

Hinkle PM, Nelson EJ, Haymes AA. Regulation of L-type voltage-gated calcium
channels by epidermal growth factor. Endocrinology 133:271-276, 1993.

Hu Y, Bock G, Wick G, Xu Q. Activation of PDGF receptor at in vascular smooth
muscle cells by mechanical stress. FASEB J. 121135-1142, 1998.

Hubbard SR, Mohammadi M, Schlessinger J. Autoregulatory mechanisms in protein-
tyrosine kinases. J. Biol. Chem. 273:11987-11990, 1998.

Ishida T, Peterson TE, Kovach NL, Berk BC. MAP kinase activation by flow in
endothelial cells. Role of [31 integrins and tyrosine kinases. Circ. Res. 79:310-316, 1996.

163

James GD, Baker PT. Human population biology and hypertension. In: Laragh JH,
Brenner BM, eds. Hypertension: Pathophysiology, Diagonsis and Management. New
York, NY: Raven Press Publishers, 137-145, 1990.

Jin N, Siddiqui RA, English D, Rhoades RA. Communication between tyrosine kinase
pathway and myosin light chain kinase pathway in smooth muscle. Am. J. Physiol. 271:
H1348-H1355, 1996.

Jones PL, Cowan KN, Rabinovitch M. Tenascin-C, proliferation and subendothelial
fibronectin in progressive pulmonary vascular disease. Am. J. Pathol. 150:1349-1360,
1997a.

Jones PL, Crack J, Rabinovitch M: Regulation of tenascin-C, a vascular smooth muscle
cell survival factor that interacts with the avBB integrin to promote epidermal growth
factor receptor phosphorylation and growth. J. Cell Biol. 1392279-293, 1997b.

Kanagy NL. Increased vascular responsiveness to or-adrenergic stimulation during NOS
inhibition-induced hypertension. Am. J. Physiol. 273:H2756—H2764, 1997.

Kanagy NL , Sarkar R, Watts SW, Webb RC. Non-cGMP, paracrine effects of nitric
oxide in the vasculature. In Sowers J .R. ed. Contemporary Endocrinology:
Endocrinology of the Vasculature. Humana Press; 37-47, 1996.

Kariya K, Kawahara Y, Araki S, Fukuzaki H, Takai Y. Antiproliferative action of cyclic
GMP-elevating vasodilators in cultured rabbit aortic smooth muscle cells.
Atherosclerosis. 80:143-147, 1989.

Karpova AY, Abe MK, Li J, Liu PT, Rhee JM, Kuo W-L, Hershenson MB. MEKl is
required for PDGF-induced ERK activation and DNA synthesis in tracheal myocytes.
Am. J. Physiol. 272: L558-L565, 1997.

Karunagaran D, Tzahar E, Beerli R, Chen X, Graus-Porta D, Ratzkin BJ, Seger R,
Hynes NE, Yarden Y. ErbB-2 is a common auxiliary subunit of NDF and EGF receptors:
implications for breast cancer. EMBO J. 15:254-264, 1996.

Katoch SS, Moreland RS. Agonist and membrane depolarization induced activation of
MAP kinase in swine carotid artery. Am. J. Physiol. 269: H222-H229, 1995.

164

Kaur K, Yao J, Pan X, Matthews C, Hassid A. NO decreases phosphorylation of focal
adhesion proteins via reduction of Ca in rat aortic smooth muscle cells. Am. J. Physiol.
2742Hl613-H1619, 1988.

Keiser J A, Ryan MJ. Hemodynamic effects of epidermal growth factor in conscious rats
and monkeys. Proc. Natl. Acad. Sci. 93:4957-4961, 1996.

Kelleher MD, Abe MK, Chao T-S, Jain M, Green JM, Solway J, Rosner MR, Hershenson
MB. Role of MAP kinase activation in bovine tracheal smooth muscle mitogenesis. Am.
J. Physiol. 268: L894-L901, 1995.

Kenyon, CJ, Morton 1]. Experimental models of hypertension. In: Swales J.D., ed.
Textbook of hypertension. Oxford: Blackwell Scientific Publications; 477-492, 1994.

Kim S, Tokuyama M, Hosoi M, Yamamoto K. Adrenal and circulating renin-angiotensin
system in stroke-prone hypertensive rats. Hypertension. 20:280-291, 1992.

K0 Y, Stiebler H, Nickenig G, Wieczorek AJ, Vetter H, Sachinidis A. Synergistic action
of angiotensin II, insulin-like growth factor-I, and transforming growth factor-B on
platelet-derived growth factor-BB, basic fibroblastic growth factor, and epidermal growth

factor-induced DNA synthesis in vascular smooth muscle cells. Am. J. Hypertens.
6:496-499, 1993.

Krymaskaya VP, Hoffman R, Eszterhas A, Kane S, Ciocca V, Panettieri RA. EGF
activates ErbB-2 and stimulates phosphtidylinositol 3-kinase in human airway smooth
muscle cells. Am. J. Physiol. 276:L246-L255, 1999.

Kubo T, Saito E, Hanada M, Kambe T, Hagiwara Y. Evidence that angiotensin II,
endothelins and nitric oxide regulate mitogen-activated protein kinase activity in rat
aorta. Eur. J. Pharrnacol. 347:337-346, 1998.

Kyriakis JM, App H, Zhang X, Banerjee P, Brautigan DL, Rapp UR, Avruch J. Raf-l
activates MAP kinase-kinase. Nature. 358:417-421, 1992.

Lahaye DHTP, Camps MGM, Van Erp PE, Peters PHJ, VanZoelen EJJ. Epidermal

growth factor (EGF) receptor density controls mitogenic activation of normal rat kidney
(NRK) cells by EGF. J. Cell. Physiol. 174:9-17, 1998.

165

Lemmon MA, Bu Z, Ladbury JE, Zhou M, Pinchasi D, Lax I, Engelman DM,
Schlessinger J. Two EGF molecules contribute additively to stabilization of the EGFR
dimer. EMBO J. 16:281-294,1997.

Li N, Batzer A, Daly R, Yajnik V, Skolnik E, Chardin P, Bar-Sagi D, Margolis B,
Schlessinger J. Guanine-nucleotide-releasing factor hSosl binds to Grb2 and links
receptor tyrosine kinases to Ras signalling. Nature 363:85-88, 1993.

Lockette W, Otsuka Y, Carretero O. The loss of endothelium-dependent vascular
relaxation in hypertension. Hypertension 8[Suppl II]:61-66, 1986.

Lopez-Ilasaca M. Signaling from G-protein-coupled receptors to mitogen-activated
protein (MAP)-kinase cascades. Biochem. Pharmacol. 56:269-277, 1998.

Mackie EJ, Scott-Burden T, Hahn AW, Kern F, Bernhardt J, Regenass S, Weller A,
Buhler FR. Expression of tenascin by vascular smooth muscle cells. Alterations in
hypertensive rats and stimulation by angiotensin 11. Am. J. Pathol. 1412377-388, 1992.

Majesky MW, Schwartz SM. Smooth muscle cell diversity in arterial wound repair.
Toxicol. Pathol. 18:554, 1990.

Mangiarua, E., N. Basso, P. Ruiz, and A. C. Taquini. Vascular structural changes in
DOC-salt hypertensive rats. Hypertens. 3: 3183-8186, 1981.

Marquardt H, Hunkapiller MW, Hood LE, Todaro GJ. Rat transforming growth factor
type 1: structure and relation to epidermal growth factor. Science 223:1079-101082,
1984.

Matsumoto T, Kinoshita M, Toda N. Mechanisms of endothelium-dependent responses

to vasoactive agents in isolated porcine coronary arteries. J. Cardiovasc. Pharmacol.
22:228-234, 1993.

Matsumoto H, Baron CB, Coburn RF. Smooth muscle stretch-activated phospholipase C
activity. Am. J. Physiol. 268:C458-C465, 1995.

Merkel LA, Bilder GE. Modulation of vascular reactivity by vasoactive peptides in
aortic rings from hypercholesterolemic rabbits. Euro. J. Pharmacol. 222:175-179, 1992.

166

Miriel VA, Allen SP, Schriver SD, Prewitt RL. Genistein inhibits pressure-induced
expression of c-fos in isolated mesenteric arteries. Hypertension 34:132-137, 1999.

Mokashi S, Severson DL, Hollenberg MD. Synergistic actions of epidermal growth
factor-urogastrone and vasopressin in cultured aortic A-lO smooth muscle cells. J. Cell.
Physiol. 152: 372-381, 1992.

Molloy CJ, Taylor DS, Weber H. Angiotensin II stimulation of rapid protein tyrosine
phosphorylation and protein kinase activation in rat aortic smooth muscle cells. J. Biol.
Chem. 268:7338-7345, 1993.

Morgan KG, Suematsu E. Effects of calcium on vascular smooth muscle tone. Am J.
Hypertens. 3:2918-2988, 1990.

Murad F. Cyclic guanosine monophosphate as a mediator of vasodilation. J. Clin.
Invest. 7811-5, 1986.

Muramatsu I, Hollenberg MD, Lederis K. Vascular actions of epidermal growth factor-
urogastrone: possible relationship to prostaglandin production. Can. J. Physiol.
Pharmacol. 63: 994-999, 1985.

Muramatsu I, Hollenberg MD, Lederis K. Modulation by epidermal growth factor-
urogastrone of contraction in isolated canine helical mesenteric arterial strips. Can. J.
Physiol. Pharmacol. 64:1561-1565, 1986.

Ngai PK, Walsh MP. Inhibition of smooth muscle actin-activated myosin Mg2"-ATPase
activity by caldesmon. J. Biol. Chem. 259: 13656-13659, 1984.

Numaguchi K, Egashira K, Takemoto M, Kadokami T, Shimokawa H, Sueishi K,
Takeshita A. Chronic inhibition of nitric oxide synthesis causes coronary microvascular

remodeling 1n rats. Hypertension 26: 957- 962, 1995.

Oka Y, Orth DN. Human plasma epidermal growth factor/B-Urogastrone is associated
with blood platelets. J. Clin. Invest. 72: 249-259, 1983.

Okamura T, Aimi Y, Kimura H, Murakami K, Toda N. Existence of renin in the
endothelium of human artery. J. Hypertens. 10:49-53, 1992.

167

Owens GK, Schwartz SM. Vascular smooth muscle cell hypertrophy and hyperploidy in
the goldblatt hypertensive rat. Circ. Res. 53:491-501, 1983.

Owens GK. Control of hypertrophic versus hyperplastic growth of vascular smooth
muscle cells. Am. J. Physiol. 7257:Hl755-H1765, 1989.

Panza J A, Quyyumi AA, Brush JE, Epstein SE. Abnormal endothelium-dependent
vascular relaxation in patients with essential hypertension. N. Engl. J. Med. 323:22-27,
1990.

Pawson T. Protein modules and signalling networks. Nature 373:573-580, 1995.

Peppelenbosch MP, Tertoolen LG, SW de Laa. Epidermal growth factor-activated
calcium and potassium channels. J. Biol. Chem. 266: 19938-19944, 1991.

Plowman GD, Green JM, Culouscou J-M, Carlton GW, Rothwell VM, Buckley S.
Heregulin induces tyrosine phosphorylation of HER4/pl80‘w3“. Nature 366:473-475,
1993.

Powell PP, Klagsbrun M, Abraham JA, Jones RC. Eosinophils expressing heparin-
binding EGF-like growth factor mRN A localize around lung microvessels in pulmonary
hypertension. Am. J. Pathol. 143:784-793, 1993.

Pulverer BJ, Kyriakis JM, Avruch J, Nikolakaki E, Woodgett JR. Phosphorylation of c-
jun mediated by MAP kinases. Nature 353: 670-674, 1991.

Rakugi H, Jacob HJ, Krieger LE, Ingelfinger JR, Pratt RE. Vascular injury induces

angiotensinogen gene expression in the media and neointima. Circulation 872283-290,
1993.

Rapp J. Genetics of experimental and human hypertension. In: Genert J, Kuchel O,
Hamet P, Cantin M, eds. Hypertension Physiopathology and Treatment. New York, NY:
McGraw-Hill Publishing Co; 582-598, 1983.

Rapp JP, Deng AY. Detection and positional cloning of blood pressure quantitative trait
loci: is it possible? Identifying the genes for genetic hypertension. Hypertension
25:1121-1128, 1995.

168

Rocha R, Chander PN, Khanna K, Zuckerman A, Stier CT. Mineralocorticoid blockade
reduces vascular injury in stroke-prone hypertensive rats. Hypertension 312451-458,
1998.

Rocha R, Chander PN, Zuckerman A, Stier CT. Role of aldosterone in renal vascular
injury in stroke-prone hypertensive rats. Hypertension 332232-237, 1999.

Saltis J, Bobik A. Vascular smooth muscle growth in genetic hypertension: evidence for
multiple abnormalities in growth regulatory pathways. J. Hypertension 10:635-643,
1992.

Saltis J, Agrotis A, Bobik A. Differences in growth characterisitics of vascular smooth
muscle from spontaneously hypertensive and Wistar-Kyoto rats are growth factor
dependent. J. Hypertens. 11: 629-637, 1993.

Saltis J, Thomas AC, Agrotis A, Campbell JH, Campbell GR, Bobik A. Expression of
growth factor receptors on arterial smooth muscle cells. Dependency on cell phenotype
and serum factors. Atherosclerosis 1182 77-87, 1995.

Sarkar R, Meinberg EG, Stanley JC, Gordon D, Webb RC. Nitric oxide reversibly
inhibits the migration of cultured vascular smooth muscle cells. Circ. Res. 782225-230,
1996.

Sarzani R, Brecher P, Chobanian AV. Growth factor expression in aorta of normotensive
and hypertensive rats. J. Clin. Invest. 8321404-1408, 1989.

Sauro MD, Thomas B. Tyrphostin attenuates platelet-derived growth factor-induced
contraction in aortic smooth muscle through inhibition of protein tyrosine kinase(s). J.

Pharmacol. Exp. Ther. 2672 1119-1125, 1993a.

Sauro MD, Thomas B. Decreased sensitivity of aorta from hypertensive rats to
vasorelaxation by tyrphostin. Life Sci. 53: 371-376, 1993b.

Savage CR, Inagami T, Cohen S. The primary structure of epidermal growth factor. J.
Biol. Chem. 247:7612-7621, 1972.

Schlessinger J, Ullrich A. Growth factor signaling by receptor tyrosine kinases. Neuron
9:383-391, 1992.

169

Sciotti V, Gallant S. Resistance to mineralocorticoid-induced hypertensive vascular
disease. Hypertension 102176-180, 1987.

Scott-Burden T, Resink TJ, Burgin M, Buhler FR. Extracellular matrix proteins:
differential influence on growth and biosynthesis patterns of vascular smooth muscle
cells from SHR and WKY rats. J. Cell Physiol. 1412267-274, 1989.

Sharifi BG, LaFleur DW, Pirola CJ, Forrester JS, Fagin JA. Angiotensin II regulates
tenascin gene expression in vascular smooth muscle cells. J. Biol. Chem. 267223910-
23915, 1992.

Shing Y, Christofori G, Hanahan D, Ono Y, Sasada R, Igarashi K, Folkman J.
Betacellulin: a mitogen from pancreatic Bcell tumors. Science 259:1604-1607, 1993.

Shoyab M, Plowman GD, McDonald VL, Bradley JG, Todaro GJ. Structure and function
of human amphiregulin: a member of the epidermal growth factor family. Science
243:1074-1076, 1989.

Srivastava AK. Protein tyrosine kinase activity in cultured vascular smooth muscle cells
(VSMC) from rat aorta. Int. J. Biochem. 26:547-550, 1994.

Stokoe D, Macdonald SG, Cadwallader K, Symons M, Hancock JF. Activation of raf as
a result of recruitement to the plasma membrane. Science 264: 1463-1467, 1994.

Suithichaiyakul T, Clegg KB, Sambhi MP. Selectively enhanced stimulation of DNA
synthesis by EGF in vascular smooth muscle cells from young and adult SHR. Clin. and
Exper. Hyper. Theory and Practice. A122307-316, 1990.

Swaminathan N, Vincent M, Sassard J, Sambhi MP. Elevated epidermal growth factor
receptor levels in hypertensive Lyon rat kidney and aorta. Clin. and Exper. Pharmacol.

Physiol. 23: 793-796, 1996.

Taguchi J, Abe J, Okazaki H, Takuwa Y, Kurokawa K. L-arginine inhibits neointimal
formation following balloon injury. Life Sci. 53:387-392, 1993.

Takahashi M and Berk BC. Mitogen-activated protein kinase (ERKl/2) activation by
shear stress and adhesion in endothelial cells. J. Clin. Invest. 98:2623-2631, 1996.

170

Takai Y, Kishimoto A, Kikkawa U, Mori T and Nishizuka Y. Unsaturated diacylglycerol
as a possible messenger for the activation of calcium-activated, phospholipid-dependent
protein kinase system. Biochem Biophys Res Commun. 91:1218-1224, 1979.

Takeda Y, Miyamori I, Yoneda T, Iki K, Hatakeyama H, Blair IA, Hsieh F-Y, Tadeda R.
Production of aldosterone in isolated rat blood vessels. Hypertension 25: 170-173, 1995.

Takeda Y, Miyamori I, Inaba S, Furukawa K, Hatakeyama H, Yoneda T, Mabuchi H,
Takeda R. Vascular aldosterone in genetically hypertensive rats. Hypertension 29:45-48,
1997.

Tare M, Parkington HC, Coleman HA, Neild TO, Dusting GJ. Hyperpolarization and

relaxation of arterial smooth muscle caused by nitric oxide derived from the endothelium.
Nature 346269-71, 1990.

Taylor DS, Cheng X, Pawlowski JE, Wallace AR, Ferrer P, Molloy CJ. Epiregulin is a
potent vascular smooth muscle cell-derived mitogen induced by angiotensin II,
endothelin-l, and thrombin. Proc. Natl. Acad. Sci. 96:1633-1638, 1999.

Ternizer DH, Yoshizumi M, Perrella MA, Susanni EE, Quertermous T, Lee M-E.
Induction of heparin-binding epidermal growth factor-like growth factor mRNA by

phorbol ester and angiotensin II in rat aortic smooth muscle cells. J. Biol Chem.
267:24892-24896, 1992.

Thurston, H. Experimental models of hypertension. In: Swales J .D., ed. Textbook of
hypertension. Oxford: Blackwell Scientific Publications; 477-493, 1994

Tong X, Velaverde V, Guan H, Yu C, Jaffa A, Nasjletti A. Contribution of mitogen

activated protein kinase to vascular contraction in hypertension. Hypertension 322591,
(P10), 1998.

Tzahar E, Levkowitz G, Karunagaran D, Yi L, Peles E, Lavi S, Chang D, Liu N, Yayon
A, Wen D, Yarden Y. ErbB-3 and ErbB-4 function as the respective low and high

affinity receptors of all neu differentiation factor/heregulin isoforms. J. Biol. Chem.
269:25226-25233, 1994.

Ullian ME, Islam MM, Robinson CJ, Fitzgibbon WR, Tobin ET, Paul RV. Resistance to
mineralocorticoids in Wistar-Furth rats. Am. J. Physiol. 272:H1454-Hl461, 1997.

171

Ullrich A, Schlessinger J. Signal transduction by receptors with tyrosine kinase activity.
Cell 61:203-212, 1990.

Wang L-M, Kuo A, Alimandi M, Veri MC, Lee C-C, Kapoor V, Ellmore N, Chen X-H,
Pierce JH. ErbB2 expression increases the spectrum and potency of ligand-mediated
signal transduction through ErbB4. Proc. Natl. Acad. Sci. 95:6809-6814, 1998.

Ward JE, Angus JA. Acute and chronic inhibition of nitric oxide synthase in conscious
rabbits: role of nitric oxide in the control of vascular tone. J. Cardiovasc. Pharmacol.
21:804-814. 1993.

Ward R. Familial aggregation and genetic epidemiology of blood pressure. In: Laragh
JH, Brenner BM, eds. Hypertension: Pathology, Diagnosis and Management. New
York, NY: Raven Press Publishers; 81-100, 1990.

Ward Y, Gupta S, Jensen P, Wartmann M, Davis RJ, Kelly K. Control of MAP kinase

activation by the rnitogen-induced threonine/tyrosine phosphatase PACl. Nature
367:651-654, 1994.

Watts, SW, Finta KM, Lloyd MC, Storm DS, Webb RC. Enhanced vascular
responsiveness to Bay K 8644 in mineralocorticoid- and N-nitro arginine-induced
hypertension. Blood Pressure. 3: 340-348, 1994.

Watts SW, Baez M, Webb RC. The 5-Hydroxytryptarr1ine2,3 receptor and 5-HT receptor
signal transduction in mesenteric arteries from deoxycorticosterone acetate-salt
hypertensive rats. J. Pharmacol. Exp. Ther. 277:1103-1113, 1996.

Watts SW, Florian JA, Monroe KM. Dissociation of angiotensin II-stimulated activation
of mitogen-activated protein kinase kinase from vascular contraction. J. Pharmacol. Exp.
Thera. 286:1431-1438, 1998.

Wijetunge, S. and A. D. Hughes. Effect of platelet-derived growth factor on voltage-
operated calcium channels in rabbit isolated ear artery cells. Br. J. Pharmacol. 115: 534-

538, 1995.

Whelton PK, He J, Klag MJ. Blood pressure in westemized population. In: Swales J .D.,
ed. Textbook of hypertension. Oxford: Blackwell Scientific Publications; 11-21, 1994.

172

White RM, Rivera CO, Davison CB. Differential contribution of endothelial function to

vascular reactivity in conduit and resistance arteries from deoxycorticosterone-salt
hypertensive rats. Hypertension 27: 1245-1253, 1996.

Wood KW, Sarnecki C, Roberts TM, Blenis J. ras mediates nerve growth factor receptor
modulation of three signal-transducing protein kinases: MAP kinase, raf—l, and RSK.
Cell 68:1041-1050, 1992.

Worthylake R, Opresko LK, Wiley HS. ErbB-2 amplification inhibits down-regulation
and induces constitutive activation of both ErbB-2 and epidermal, growth factor receptors.
J. Biol. Chem. 274:8865-8874, 1999.

Xu Q, Fawcett TW, Gorospe M, Guyton KZ, Liu Y, Holbrook NJ. Induction of mitogen-
activated protein kinase phosphatase-1 during acute hypertension. Hypertension 30: 106-
111, 1997.

Xu Q, Liu Y, Gorospe M, Udelsman R, Holbrook NJ. Acute hypertension activates
mitogen-activated protein kinases in arterial wall. J. Clin. Invest. 97: 508-514, 1996.

Yamori Y, Swales JD. The spontaneously hypertensive rat. Genetic Models of
Hypertension. Swales JD, ed. In: Textbook of Hypertension. Cambridge, MA.
Blackwell Scientific Publications. 447-454, 1994.

Yan M, Templeton DJ. Identification of 2 serine residues of MEK-1 that are
differentially phosphorylated during activation by raf and MEK kinase. J. Biol. Chem.
269:19067-19073, 1994.

Yang S-G, Saifeddine M, Hollenberg MD. Tyrosine kinase inhibitors and the contractile
action of epidermal growth factor-urogastrone and other agonists in gastric smooth

muscle. Can. J. Physiol. Pharmacol. 70:85-93, 1992.

Young M, Fullerton M, Dilley R, Funder J. Mineralocorticoids, hypertension, and
cardiac fibrosis. J. Clin. Invest. 9322578-2583, 1994.

Yu J, Bose R. Calcium channels in smooth muscle. Gastroenterology 10021448-1460,
1991.

173

Zhang D, Sliwkowski MX, Mark M, Frantz G, Akita R, Sun Y, Hillan K, Crowley C,
Brush J, Godowski PJ. Neuregulin-3 (NRG3): a novel neural tissue-enriched protein that
binds and activates ErbB4. Proc. Natl. Acad. Sci USA. 94:9562-9567, 1997.

Zheng C-F, Guan K-L. Activation of MEK family kinases requires phosphorylation of
two conserved Ser/T hr residues. EMBO J. 13: 1123-1131, 1994.

Zheng C-F, Guan K-L. Cloning and characterization of two distinct human extracellular
signal-regulated kinase activator kinases, MEKl and MEK2. J. Biol. Chem. 268211435-
11439, 1993.

Zheng X-L, Mokashi S, Hollenberg MD. Contractile action of ethanol in guinea pig
gastric smooth muscle: inhibition by tyrosine kinase inhibitors and comparison with the
contractile action of epidermal growth factor-urogastrone. J. Pharmacol. Exp. Ther. 2822
485-495, 1997.

Zwick E, Wallasch C, Daub H, Ullrich A. Distinct calcium-dependent pathways of
epidermal growth factor receptor transactivation and PYK2 tryosine phosphorylation in
PC12 cells. J. Biol. Chem. 274:20989-20996, 1999.

174