THESE
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Michigan State ,
University

 

 

 

This is to certify that the

dissertation entitled

Relationship Between Experimental Angiotensin II Induced
Hypertension and Activation of the Enddhelin System in
Male Rats: Effects of Salt Intake

presented by
Jennifer Rebecca Ballew

has been accepted towards fulfillment
of the requirements for

Ph.D. degree“. Pharmacology and
Toxicology

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Date 8/5//';ZOCDO

 

MS U is an Affirmative Action/Equal Opportunity Institution 0- 12771

 

 

 

PLACE IN REFURN BOX to remove this checkout from your record.
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RELATIONSHIP BE
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RELATIONSHIP BETWEEN EXPERIMENTAL ANGIOTENSIN II INDUCED
HYPERTENSION AND ACTIVATION OF THE ENDOTHELIN SYSTEM IN
MALE RATS: EFFECTS OF SALT INTAKE
By

Jennifer Rebecca Ballew

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

2000

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ABSTRACT

RELATIONSHIP BETWEEN EXPERIMENTAL ANGIOTENSIN II INDUCED
HYPERTENSION AND ACTIVATION OF THE ENDOTHELIN SYSTEM IN
MALE RATS: EFFECTS OF SALT INTAKE

By

Jennifer Rebecca Ballew

The overall objective of my thesis project was to determine the role of
endothelin-1 in the salt-sensitivity of angiotensin II induced hypertension. The
relationship between these two peptide hormones has been well documented,
but the specific mechanisms through which they interact have yet to be fully
elucidated. The main experimental strategy | employed in this thesis project was
measurement of hemodynamic and renal responses to endothelin-l receptor
antagonists in vivo. This approach was supplemented by use of in vitro studies
of vascular contractility. The data presented in this thesis Show that selective
ETA receptor blockade alone normalizes mean arterial pressure in angiotensin II
induced hypertension, particularly under conditions of high salt intake. This
presents a strong case that endothelin-1 acting at ETA receptors is an important
mechanism of the salt-sensitivity of angiotensin II induced hypertension.
Conversely, my data further demonstrate that selective ETB receptor activation

decreases mean arterial pressure in angiotensin II induced hypertension,

especially under high salt conditions. The potential clinical implications of my
data are that combined blockade of both ETA and ETB receptors will be less

effective at decreasing blood pressure than selective ETA receptor antagonism,

especially in people with salt-sensitive hypertension.

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Acknowledgments

In the slightly more than five years I have spent here at Michigan State
University as a graduate student in the Department of Pharmacology and
Toxicology, it has become my considered opinion that the best scientific work
results from collaborative efforts. This thesis project was the outcome of my

own hard work and the input, advice, and assistance of many other people.

First I wish to express my utmost love, appreciation, respect and
admiration for my thesis advisor, Dr. Greg Fink. From the commencement of
this project, Greg has encouraged me to think creatively and individually, even
when my ideas and opinions were in contrast to his. He helped me retain my
enthusiasm for science through the most trying times, and for this I am
profoundly grateful. Greg, you have been a true mentor and friend to me.

Thank you.

I would like to thank and acknowledge the other members of my thesis
guidance committee: Dr. Susan Barman, Dr. Jim Galligan, Dr. Stephanie Watts,
and Dr. Donna Wang. It has been a privilege to work with such distinguished
scientists. I thank Jim in particular for his humor, encouragement, discretion,

and commitment to graduate education.

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I am deeply grateful to Dr. Veronica Maher and Dr. Justin McCormick, co-
directors of the Michigan State University College of Osteopathic Medicine’s
Medical Scientist Training Program. Drs. Maher and McCormick have
demonstrated, time and again, their faith in my abilities and their dedication to

helping me receive the best possible scientific training.

I owe a great debt of gratitude to my fellow co-workers, Barbara Grant,
Dawna Dylewski, and Evan Brown, without whom this thesis project could not
have been completed in such an efficient manner. It has been my sincere
pleasure to work and laugh with all of you on a daily basis, and I am truly

appreciative of our friendly working environment.

Special thanks are due to Dr. John Thornburg, Dr. Bill Atchison, and Dr.
Keith Lookingland. Dr. Thornburg allowed me the opportunity to continue seeing
patients while working on this thesis and went above and beyond the call of duty
to make sure I stayed up-to-date in my clinical training. Dr. Atchison helped me
juggle medical school and graduate school requirements, and frequently and
humorously reminded me how to keep scientific politics in perspective. Dr.
Lookingland enthusiastically recruited me to this training program and helped me

get started in my graduate school career.

vi

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I offer loving thanks to my mother, Anne Marie Ballew, and my father,
Julius R. Ballew, whose high expectations kept me on a challenging upward
path, and whose love and encouragement convinced me to pursue my academic
goals. I am grateful to my brother, Jamie Ballew, for believing in my aptitudes
and for offering motivation whenever possible. Thanks also to my parents-in-
law, Jim and Bethel Lanz, and my grandmother-in-law, Helen Jameson, for their
continuing and abiding faith in my career goals and their countless prayers on

my behalf.

My thankful appreciation goes to my friends, Kaisa Johnson, Tiffany
Lasky, Mike O’Neill, Coleen Warriner, and Fran Wolber, as well as to my uncle
and aunt, Jim and Dianne McPharlin. The seven of you have formed my long-
distance support group and I can always count on each of you to listen, offer
advice, make me laugh, and comfort me during rough times. Thanks also for

being so much fun!

Lastly, and most importantly, I want to thank my husband, Mark Lanz. Mark has
been a constant and unconditional source of love, support, encouragement, and
energy to me. He has helped me keep my sense of humor throughout this
ordeal and I am eternally grateful to him. Thank you, Mark, for gracing my life
with your presence and your astonishing ability to keep long-term goals in sight.

I love you.

vii

LIST OF TABLES
LIST OF FIGURES

LIST OF ABBREVIA

Chapler1: INTROE

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TABLE OF CONTENTS

LISTOF FIGURES
LIST OF ABBREVIATIONS..................................................

Chapter1: INTRODUCTION..............................................

I. Hypertension................................... . .

Epidemiology...........................'.ffj.'.'."..'."..'.'Z.'."..'."..'.".327...".III...

Etiology.
Hemodynamics

Mechanisms of HypertenSIon
Pressure-Natriuresis...

Salt Sensitivity...

Treatment and Prevention

II. Renin-Angiotensin System...
Effects of Angiotensin II .
Angiotensin II Induced Hypertensron
Angiotensin II and Pressure-Natriuresis

Salt Sensitivity of Angiotensin II Induced HypertenSIon........'.'......'.. ..

Ill. Endothelin...
Biosyntheosis... . . .
Sites of Generation and Secretion
Endothelin Receptors...
Endothelin Signal Transduction Mechanisms
Endothelin Receptor Antagonists...

Endothelin and the Renin-Angiotensin System...

V. Hypothesis...
Specific Aim I
Specific Aim II...

viii

.3 Z

.xii

.xiii

.xix

ooo'xi

12
.....13
...14

15

.....17
......17
......18
.....18

......20
...21

Cardiovascular Physiology of EndothelIn ..23

Renal Physiology of Endothelin...........................................
Endothelin and Essential Hypertension.......................

.25
.27

.31

......35
...35

I

 

Chapter); GENEIh

Chapter 3

Acacia I

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Chrom:
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Metals-:I
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Statstc.

Prelimin
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Efficacy N
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Chapter 2:

Chapter 3:

Chapter 4:

GENERAL METHODS37
Animals. . 37
Surgical Procedures .. 37
-Arterial and Venous Cathetenzatron 38
-Surgical Uninephrectomy” .. ... ...39
-Deoxycortisosterone Acetate Implantation . .. ... ... ..39
Chronic Rat Maintenance and Measurements. 40
HemodynamicMeasurements... 40
MetabolicMeasurements... 41
Isolated Tissue Bath Measurements... 42
StatIstIcalAnalysrs 43
Preliminary Studies... ...45
Efficacy of PD156707 Tested In ”ET- 1 Induced Model of”
Hypertension... ............45
Efficacy of PD156707 TeStedm In DOCA-Salt Model 6r”
Hypertension... . 49
Rapid Termination of Endothelin Infusion . ...52
Autonomic Blockade and PD156707... 57
Effect of PD156707 on Angiotensin II Induced
Hypertension” ....59
Efficacy of ABT-627 TeS-ted. in Endothelin 1 Induced Model of
Hypertension... . ..64
Eff cacres of ABT-627 and Minoxidil Tested in DOCA-Salt Model of
Hypertension... 67
Effect of ABT-627 on Angrotensm WII Induced
Hypertension... ... . .70
Effect of ABT-627 on Acute Sarafotoxrn 6c Dose ResponSe
Curve... ..........73
Effect of ABT-627 on Acute Endothelin 1 Dose Response
Curve... ......76
Effect of A-192621 on Acute Sarafotoxm 6c Dose Response
Curve... .........79
Effect of A-192621 on Acute EndotheIIn 1 Dose Response
Curve... .. ...........82
Efficacy of A-182086 Tested in ET-1 Induced Model of”
Hypertension... 85
Role of ET. Receptors in Experimental Angiotensin II Induced
Hypertension in RatsBB
Introduction89
Methods91
Results94
DIscussmn101

 

Chapter 5;

Chapter 6

Chapter 7-

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Chapter 5:

Chapter 6:

Chapter 7:

Chapter 8:

Role of Endothelin ETB Receptor Activation in Angiotensin II
Induced Hypertension: Effects of Salt Intake... 105

IntroductIon 106
Methods108
Results111
DIscuSSIon123

Characterization of the Antihypertensive Effect of a Thiazide
Diuretic in Angiotensin II Induced
HypertenSIon ........130

lntroductron131

Methods133
Results136
DIscuSSIon144

Effects of Salt Intake and Angiotensin II on Vascular
Reactivity to Endothelin-1... .....148

IntroductIon149
Methods............... .........150
Results ....154
DISCUSSIon 161

General Discussion and Conclusions...... .168

Ang II and ET-1 can each cause hypertension. ...169
Salt intake contributes to severity of Ang II and ET-1 induced
hypertension... . ............169
ET- 1 mediates some of the cardiorenal effects of Ang

II. .... .170
Ang II induced hypertensron is the beSt model to Study the”
interaction between Ang II and ET—1.. ... .. .171.
ET-1 receptor antagonists reveal ET—1 mediated effects In vivo and
invitro... . ..171
ET—1, especially under conditions of increased salt intake is
required for the maintenance of experimental Ang II induced
hypertension... . . .. .. ..1 72
Ang II induced hypertenSIon depends primarily on the actions of
ET—1 on ETA receptors, and this effect Is greater under conditions
of high salt intake... 176

 

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The initial hypotensive effect of ETA receptor blockade in Ang II
induced hypertension is not due to a diuretic
effect............. 179

are receptor aetiia'Iiéfi 5555:5555 ii 153.1553 '
hypertensron ....180
Conclusions....................................... 184

xi

 

Chapter 3

Tabe Z

 

Tate 3

Chapter 3

Table 3.1:

Table 3.2:

Chapter 7

Table 7.1:

Chapter 8

Table 8.1:

LIST QF TABLES

Comparison of the change in mean arterial pressure
(mmHg) over time with endothelin-1 infusion

cessation and injection of P0156707... .55

Comparison of the change in heart rate (bpm) over
time with Endothelin-1 infusion cessation and

injection of PD156707...... ”......56

Mean arterial pressure in rats given chronic Ang II

infusion (5 ng/mIn)159

Novel findings from this thesis on the vascular and
renal effects of Ang II and salt intake and the

activation of ETA and ETB receptors.

xii

Chapter 3

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Chapter 3

Figure 3.1:

Figure 3.2:

Figure 3.3:

Figure 3.4:

Figure 3.5:

Figure 3.6:

Figure 3.7:

Figure 3.8:

Figure 3.9:

Figure 3.10:

List of Figures

Effect of PD156707 on endothelin—1 induced hypertension
in high saltrats” ..........47

Effect of PD156707 on endothelin-1 induced hypertension
in normal saltrats ...48

Effect of PD156707 on DOCA-salt hypertension... .51

Effect of termination of endothelin-1 infusion on mean
arterial pressure (mm Hg) in endothelin-1 induced
hypertension... ......54

Daily mean arterial pressures (mmHg) in high and normal
salt rats in the presence and absence of angiotensin II ..... 61

Changes in mean arterial pressure (mmHg) between daily
control pressures and pressures averaged over 35-50
minutes post injection of PD156707 in high salt rats ......... 62

Changes in mean arterial pressure (mmHg) between daily
control pressures and pressures averaged over 35-50
minutes post injection of PD156707 in normal salt rats ..... 63

Hemodynamic response to infusion of endothelin-1 (ET-1)
at 8 pmol/kglmin over a 72 hour time period prior to, during,
and after administration of the ETA selective antagonist,
ABT-627, 2 mg/kg/day in drinking water... ...66

Hemodynamic responses of DOCA-salt rats, 4-5 weeks post
DOCA-Salt treatment, to ABT-627 and Minoxidil... ..69

Changes in mean arterial pressure (mmHg) with increasing
doses of ABT-627 in the presence and absence of
angiotensin II in high salt rats... ....72

xiii

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Figure 3.11:

Figure 3.12:

Figure 3.13:

Figure 3.14:

Figure 3.15:

Chapter 4

Figure 4.1:

Figure 4.2:

Hemodynamic response to acute bolus i.v. injection of
sarafotcxin 6c (1-500 pmollkg) in rats on high salt intake..75

Hemodynamic response to acute bolus i.v. injection of
endothelin-1 (0—500 pmollkg) in rats on high salt intake... .78

Hemodynamic responses to acute bolus i.v. injections of
sarafotcxin 6c (doses = 62.5 to 500 pmollkg) in rats on high
salt intake at various time points after receiving the ETB
selective receptor antagonist A-192621 at 12 mglkg/day I. v.
injection... .. .. 8.1

Hemodynamic responses to acute bolus i.v. injections of
endothelin-1 (doses = 62.5 to 500 pmollkg) in rats on high
salt intake at various time points after receiving the ETB
selective receptor antagcnist, A-192621 at 12 mglkg/day I. v
injection... ...84

Hemodynamic response to infusion of endothelin-1 (8
pmollkglmin) over a 72 hour time period prior to, during, and
after administration of the non-selective ET-1 receptor
antagonist, A-182086 (12 mglkglday) i.v. injection... ..89

Line plots show hemodynamic responses to infusion of
angiotensin II (Ang II) at 5.0 nglmin and administration of
ABT-627 at 2 mglkglday in rats on either high sodium (top
panel) or normal sodium (bottom panel) intake... ...95

Line plots show hemodynamic responses to bolus
intravenous injection of ABT-627 at 2 mglkg in rats on high
sodium (top panel) or normal sodium (bottom panel)
intake... ...97

xiv

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Figure 4.3:

Figure 4.4:

Figure 4.5:

Chapter 5

Figure 5.1:

Figure 5.2:

Figure 5.3:

Effects of acute intravenous injection of angiotensin II (Ang
II) on mean arterial pressure in rats on high sodium intake,
before and after administration of ABT-627 at 2 mglkg/day
for 72 hours... ...98

Line plots Show water balance responses in milliliters per
day (mls/day) to infusion of angiotensin II (Ang II) at 5.0
nglmin and administration of ABT—627 at 2 mglkg/day in rats
on either high sodium (top panel) or normal sodium (bottom
panel) intake... ......99

Line plots Show sodium balance responses in
milliequivalents per day (mEq/day) to infusion of angiotensin
II (Ang II) at 5. 0 nglmin and administration of ABT-627 at 2
mglkglday in rats on either high sodium ”(top panel) or
normal sodium (bottom panel) intake... .. ..100

Line plots show hemodynamic responses to infusion of
angiotensin II (Ang II) at 5. 0 nglmin and administration of A—
192621 at 24 mglkglday”. In rats on either high sodium or
normalsodiumintake... ................112

Line plots Show water balance responses in milliliters per
day (mls/day) to infusion of angiotensin II (Ang II) at 5.0
nglmin and administration of A-192621 at 24 mglkg/day in
rats on either high sodium or normal sodium intake ........ 113

Line plots show sodium balance responses in
milliequivalents per day (mEq/day) to infusion of angiotensin
II (Ang II) at 5.0 nglmin and administration of A-192621 at
24 mglkg/day in rats on either high sodium or normal
sodium intake... ......1 14

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Figure 5.5:

Figure 5.6:

Figure 5.7:

Figure 5.8:

Chapter 6

Figure 6.1:

Figure 6.2:

Line plots Show hemodynamic responses to bolus intra-
arterial injection of A-192621 at 24 mglkg in rats on high
sodium or normal sodium intake... ....1 15

Line plots Show hemodynamic responses to infusion of
angiotensin II (Ang II) at 5. 0 nglmin and administration of A-
182086 at 24 mglkg/day”. In rats on either high sodium or
normalsodiumintake... . . . . ......118

Line plots show water balance responses in milliliters per
day (mIS/day) to infusion of angiotensin II (Ang II) at 5.0
nglmin and administration of A-182086 at 24 mglkg/day in
rats on either high sodium or normal sodium intake ........ 119

Line plots Show sodium balance responses in
milliequivalents per day (mEq/day) to infusion of angiotensin
II (Ang II) at 5.0 nglmin and administration of A-182086 at
24mglkg/day in rats on either high sodium or normal sodium
intake... ....120

Line plots show hemodynamic responses to bolus intra-
arterial injection cf A-182086 at 24 mglkg in rats on high
sodium or normal sodium intake... .. .. ...121

Hemodynamic responses to infusion of angiotensin II (Ang
II) at 5. 0 nglmin and administration of trichlormethiazide at
10 mglkg/day In rats on 8 mEq/day sodium intake or on 2
mEq/dayscdiumintake... .137

Water balance responses in milliliters per day (mls/day) in
response to infusion of angiotensin II (Ang II) at 5.0 nglmin
and administration of trichlormethiazide at 10 mglkg/day in
rats on 8 mEq/day sodium intake or 2 mEq/day sodium
intake... ....138

 

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Chapter 7

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Figure 6.3:

Figure 6.4:

Figure 6.5:

Chapter 7

Figure 7.1:

Figure 7.2:

Figure 7.3:

Sodium balance responses in milliequivalents per day
(mEq/day) in response to infusion of angiotensin II (Ang II)
at 5. 0 nglmin and administration of trichlormethiazide at 10
mglkg/day In rats on 8 mqulday sodium intake or 2
mEq/daysodiumintake... . 139

Hemodynamic responses to bolus intravenous injection of
trichlormethiazide at 10 mglkg in rats on 8 mEq/day sodium
intake or on 2 mEq/day sodium intake... ....140

Blood volumes in milliliters in rats on 8 mEq/day sodium
intake or on 2 mEq/day sodium intake on control day 2, and
Ang II infusion days 5, 10, and 14... ...142

Concentration dependent contraction to endothelin—1 (ET-1)
in control, A-192621 (ETB selective receptor antagonist,
30nM) incubated, ABT-627 (ETA selective receptor
antagonist, 30nM) incubated, and A-182086 (ETA/3
nonselective receptor antagonist, 30nM) incubated superior
mesenteric artery of the rat... 155

Concentration dependent contraction to endothelin-1 (ET-1)
in control and angiotensin II (Ang II) incubated superior
mesenteric artery of the rat... 156

Line plots Show mean arterial pressure (MAP) responses to
infusion of angiotensin II at 5.0 nglmin or phenlyephrine at
2uglmin for two hours in rats on either high (circles) or
normal (triangles) sodium intake... ....158

xvii

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Chapter 8

Figure 8.1:

Concentration dependent contraction to endothelin-1 (ET-1)
and phenylephrine (PE) in superior mesenteric artery from
normotensive control and hypertensive angiotensin II (Ang
II) infused rats on normal and high salt intake... 160

A model representing the inter-relationships between salt-
intake, Ang II, and ET-1 in the pathogenesis of
hypertension... 174

xviii

ACE
ANS

 

ACE
ANS
BP
BV
Ca++
cAMP
cGMP
CNS
CO
DAG
DOCA-salt
E050
ECE
ET
ET-1
ET-2
ET-3
HR
IP3
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VSMC

ABBREVIATIONS

angiotensin converting enzyme
autonomic nervous system
blood pressure

blood volume

calcium

cyclic adenosine monophosphate
cyclic guanlyl monophosphate
central nervous system

cardiac output

diacylglycerol
decxycorticostercne acetate-salt
effective concentration 50
endothelin converting enzyme
endothelin

endothelin-1

endothelin-2

endothelin-3

heart rate

inositol 1,4,5-triphosphate
intracellular calcium
intra-arterial

intraperitoneal

intravenous

NG-nitro-L-arginine methyl ester
NG-monomethyI-L-arginine
mean arterial pressure
norepinephrine

nitric oxide

nitric oxide synthase

one kidney-one clip
prostacyclin

protein kinase C
phospholipase A2
phospholipase C
renin-angiotensin-system
sarafotcxin 6c

subcutaneous

spontaneously hypertensive rat
total peripheral resistance
two-kidney-one-clip

vascular smooth muscle cell

xix

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Chapter 1
INTRODUCTION

I. Hypertension

At the present time, heart disease and stroke are the first and third leading
causes of death, respectively, in the United States. Each year, 43% of all deaths
in this country are attributed to cardiovascular disease (Whelton et al., 1994).
These cardiovascular killers present an enormous public health problem by
generating a financial burden of more than $259 billion in direct and indirect costs
(NI-ILBI, 1997). High blood pressure is one of the most important modifiable risk
factors for cardiovascular disorders such as myocardial infarction, stroke,
congestive heart failure, end-stage renal disease, and peripheral vascular
disease (Burt et al., 1995). Hypertension is somewhat arbitrarily defined by the
Sixth Report of the Joint National Committee on Detection, Evaluation, and
Treatment of High Blood Pressure as the finding of an average systolic blood
pressure >140 mmHg, or a diastolic blood pressure >90mmHg, or current
treatment with an antihypertensive medication. The objective of identifying and
treating hypertension is to reduce the risk of cardiovascular disease and its

associated morbidity and mortality (JNC-Vl, 1997).

Epidemiology
Hypertension affects nearly 50 million Americans (Burt et al., 1995). The

prevalence of hypertension increases with age, so that an estimated 50 percent

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of the United States population over 60 years of age have elevated blood
pressure readings (Cushman, 1994). Two-thirds of the entire hypertensive
population (69%) are aware of their diagnosis, half (53%) are taking medication
to lower their blood pressure, and only a quarter of these people (27%) are able
to maintain good blood pressure control (Burt et al., 1995). Hypertension
management is particularly problematic in population subgroups, including
African Americans, Hispanics, and Native Americans, in whom both awareness
and treatment of high blood pressure continues to be suboptimal (Semchenko et
al., 1998). Epidemiological studies have consistently shown a higher incidence
of hypertension in African Americans compared with Caucasian Americans,
regardless of age, although the difference is most pronounced at younger ages.
The Hispanic population has lower hypertension prevalence rates than those of
both non-Hispanic blacks and whites (Flack and Yunis, 1993).

Women and men share many of the same risk factors for hypertension, despite
the male predominance of this disorder. Hypertension is less common among
women than men up to 65 years of age in Caucasians and up to 45 years of age
for African Americans. In the older age groups, prevalence rates are actually
slightly higher in women than men. In all age groups, prevalence rates are
higher among black women than white women, and black women have greater
reductions in morbidity and mortality with antihypertensive treatment than white

women (LaCroix, 1993).

Tne US Depa-"I"
Wrgflmg blOOC

year 2000 {DHH

Etiology

One great
heterogeneous Ir
mgr door: press.
byix'fensI-on EJI
33.59 is Know a
ciagnosls of esse

h

mafierrulln
-'5 Us

a?» n
.ch aways d,

'.v’I':‘-.-a7*.s. 1994'

The US Department of Health and Human Services has established a goal of
controlling blood pressure in at least 50% of the hypertensive population by the
year 2000 (DHHS, 1991).

Etiology

One great difficulty in studying hypertension is that the disease process is
heterogeneous in its etiology. In nearly 95% of affected people, the cause of
high blood pressure is unknown, a condition referred to as essential
hypertension. Elevated blood pressure that can be attributed to a definable
cause is known as secondary hypertension (Deshmukh et al., 1998). The
diagnosis of essential hypertension is one of exclusion in that it is the only option
left after ruling out the potential causes of secondary hypertension, which are
almost always due to an alteration in renal function and/or hormone secretion
(Williams, 1994). Secondary hypertension is often curable with correction of its
cause.
Essential hypertension is more a description than a diagnosis since it indicates
only one particular physical finding, elevated blood pressure, rather than a cause
for the finding. The difficulty in identifying a cause lies in the fact that multiple
systems play a role in regulating arterial pressure, including the peripheral and/or
central nervous (Brody et al., 1987), renal (Guyton, 1991), endocrine (Ely et al.,
1997), and circulatory systems (Safer and London, 1987). The complex interplay

of these systems with one another further complicates the issue.

There l5 5

 

5,159 s.‘gn:.‘." r'.

metres of hype

   

to he dIsorc'er ; S
tims {Deshmkf‘

comes from the u:

 

Nth-etch et al 13

It Is most I.-
‘essental hype'te
deeds especal,

p'es M
e .atlon that l

’IQNI.
5,, ‘5 Value to l

There is also a clear genetic component to hypertension, as demonstrated
by the significantly higher rate of elevated blood pressure among first-degree
relatives of hypertensives than the general population. Additionally, concordance
for the disorder is much greater between monozygotic twins than for dizygotic
twins (Deshmukh et al., 1998). Further support for a genetic role in hypertension
comes from the uneven distribution of hypertension among racial groups
(Whelton et al., 1994; Burt et al., 1995).

It is most likely that several different underlying defects are responsible for
“essential hypertension” in different subpopulations of patients and that these
defects, especially when mixed with environmental stressors, result in the clinical
presentation that is known as essential hypertension. It is therefore of great
scientific value to understand the intricate relationships between the various

systems that work to control blood pressure.

Hemodynamics

Blood pressure (BP) is the product of cardiac output (CO) and total
peripheral resistance (TPR). Likewise, CO is the product of cardiac stroke
volume (SV) and heart rate (HR). These five functional parameters depend on
the heart to supply the pumping pressure, blood vessel tone to determine the
systemic resistance, and the kidneys to regulate intravascular volume. It is
important to keep the role of the kidneys in mind because renal fluid excretion
alone can completely return blood pressure to normal levels by reducing

intravascular volume. Therefore, the maintenance of chronic hypertension

requres renal pa
hype'tenspn a'e

Hemodym
m..-rd a filgl‘. CO
HCWEI'ef. as the '
amai shdes tie
Incease in TPR .
mwyfiaf'llc pr:
99-31% VaSCgfar

”5559C (Cofema'

Hechanisms of H)

The nerve]
In tIe hyperfine-or
‘5 lesrlbec abori-
rless accompane :l

3‘-'

:zcchL‘lQ TPR l5 3

 

 

requires renal participation even when the initial factors responsible for the
hypertension are external to the kidney (Deshmukh et al., 1998).

Hemodynamic studies performed in the initial stages of hypertension
found a high CO and normal TPR during periods of rest (Lund-Johansen, 1994).
However, as the hypertension progresses to a chronic state, both human and
animal studies have demonstrated a reduction in CO accompanied by an
increase in TPR (Lund-Johansen, 1989). Therefore the characteristic
hemodynamic profile of a chronic hypertensive subject is normal blood flow with
elevated vascular resistance, suggesting that regional blood flow is generally not

impaired (Coleman and Hall, 1993).

Mechanisms of Hypertension

The peripheral vasculature is one of the most extensively studied systems
in the hypertension field because of its obvious relationship to blood pressure.
As described above, an increase in TPR is sufficient to increase blood pressure
unless accompanied by a decrease in CO. The most common mechanism of
increasing TPR is direct vasoconstriction. Several neural, endocrine, and
paracrine factors function as vasoconstrictors, including norepinephrine (NE),
angiotensin II (Ang II), endothelin-1 (ET-1), vasopressin, insulin, and various
cytokines (Webb and Strachan, 1998). There is also evidence that TPR is
increased by remodelling and growth of the vascular smooth muscle itself,
resulting in decreased lumen size and therefore higher pressure through the

vessel (Alexander and Griendling, 1993).

The sympathetic nervous system (SNS) acts as both an initiator and a
facilitator of chronic hypertension (lzzo, 1999). Acute stimulation of SNS control
centers causes an increase in systemic blood pressure by producing arteriolar
and venous constriction. The SNS acts to maintain blood pressure through a
complex system of interactions with other systems. Activation of a-adrenergic
receptors in the vasculature causes a rise in TPR, while stimulation of a-
adrenergic receptors in the kidney produces volume expansion via renal
vasoconstriction and sodium retention. Activation of BI-adrenergic receptors in
the heart increase heart rate, and in the kidney stimulates release of renin,
thereby activating the renin-angiotensin-system (RAS), as discussed in the next
section. Experimental models have shown that lesions in the anteroventral third
ventricle, posterior hypothalamus, and area postrema result in decreased blood
pressure, thereby indicating the pressor effects of these brain regions (Wyss,
1999). Additional feedback neural control of blood pressure is from sensory
nerve endings in the carotid sinuses and aortic arch, the baroreceptors, which
relay blood pressure status to the brain (Chapleau, 1993).

The kidney plays a role in most forms of hypertension and a defect in
renal function is almost definitely involved in the pathogenesis of essential
hypertension (Kaplan, 1998a). Furthermore, the course of hypertension often
causes renal damage, leading to a circular relationship in which a kidney defect
leads to hypertension, which in turn further damages the kidney, and thus causes
more severe hypertension. An alteration in renal function which causes a

change in sodium and water output in response to arterial pressure shifts is

present is al: to

natvureSIs is d s

  
 

Pressure-Natl'iu

Under nor'
keney's excetor
Icme and a retu
frs‘. mmsed by (

‘ 990 COWley and

IOU cream

5X33;- F
4%“
Kaplan. 193
in m
~371th

.aflia .

‘I.

«we: range Sun
' a

present is all forms of hypertension. This relationship, known as pressure-

natriuresis, is discussed further below.

Pressure-Natriuresls

Under normal circulation conditions, when blood pressure rises the
kidney’s excretion of sodium and water increases, resulting in reduced body fluid
volume and a return of blood pressure to the normal range. This phenomenon,
first proposed by Guyton et al. (1964), is known as pressure-natriuresis (Hall,
1990; Cowley and Roman, 1996; Kaplan, 1998b). A small increase in mean
arterial pressure produces a large increase in the rate of sodium and water
excretion, creating a steep slope of the curve relating these two variables (Hall,
1990; Kaplan, 1998b).

In many hypertensive patients, the indices commonly used to evaluate
renal function (glomerular filtration rate and renal plasma flow) are within the
normal range, suggesting that hypertension can develop in the absence of kidney
defects (Kaplan, 1998b). However, the relationship between renal sodium and
water excretion and mean arterial pressure is abnormal in all types of clinical and
experimental hypertension, indicating that some degree of renal dysfunction is
necessary for the development and maintenance of hypertension (Hall, 1990).
Under hypertensive conditions, the pressure-natriuresis curve is shifted to higher
pressures so that sodium balance can only occur at higher than normal blood
pressures. The mechanism of pressure-natriuresis is at least in part intrinsic to

the kidney, because it occurs in isolated perfused kidney preparations (Cowley

 

and Roman, 19'.
preSSLlre-nat'lw
nerwus System
p'essures by a“:
mdomne contra
Regardless of he
"at-areas theory
Seiafice and bioo
hyze'fensrcn to k.

1062‘

w.)

33” Sensitivity
Of all of I:

5 n‘

and Roman, 1996). Yet, factors external to the kidney also can influence
pressure-natriuresis. Release of vasoconstrictor factors or excess sympathetic
nervous system activation can shift the pressure-natriuresis relationship to higher
pressures by affecting glomerular filtration. Additionally, alterations in neural or
endocrine control of renal tubular function can precipitate such a shift.
Regardless of how systemic blood pressure is initially increased, the pressure-
natriuresis theory predicts that the relationship between sodium and water
balance and blood pressure must be shifted towards higher pressure for
hypertension to be maintained (Hall, 1990; Cowley and Roman, 1996; Kaplan,
1998).

Salt Sensitivity

Of all of the environmental factors implicated in the development of
hypertension, salt intake has generated the most attention and the most
controversy (Taubes, 1998). Several epidemiological studies, most notably the
lntersalt study of 1988, have shown a weak but significant relation between salt
intake and the development of hypertension (INTERSALT, 1988; Kaplan, 1993).
The available data suggests a threshold relationship between salt intake and
hypertension to the effect that those who ingest less than 50 mmol per day of
sodium do not develop hypertension and those who ingest more will develop
hypertension if they have other predisposing factors (Kaplan, 1993; Kotchen,
1999). Some researchers have suggested that chloride may play an important

role in hypertension development as well since over 95% of dietary salt is served

Inrreformfsl

 

showr that soc

  
  
  

associated it)?

Most rese
exz'ahs Why the
WT?“ elevated 58
Increase In blood
he sane lndzwdt.
West In cider t
mt: lowrenm sta

nsfiaency (GI,

in the form of sodium chloride (Weinberger, 1993). Sato et al. (1991) have
shown that sodium chloride loading in angiotensin II infused rats potentiates the
associated hypertension, whereas sodium citrate loading does.

Most researchers believe that a condition known as “salt sensitivity”
explains why the blood pressure of some individuals, but not others, increases
with elevated salt intake. Salt sensitivity is generally defined as a 10% or greater
increase in blood pressure after salt loading as compared to low-sodium intake in
the same individual (Weinberger, 1993). The prevalence of salt sensitivity is
highest in older, black, and/or diabetic hypertensive patients, as well as those
with low-renin status, increased sympathetic nervous activity, or renal
insufficiency (Guyton et al., 1964; Weinberger, 1993).

The exact etiology of salt-sensitive hypertension is not known. Guyton et
al. (1964) were among the first to suggest that hypertension may be a necessary
consequence of the kidney’s reduced ability to excrete a salt load. The resulting
sodium retention and consequent volume expansion produce a transient
increase in cardiac output (causing acute hypertension) that ultimately leads to
chronically increased peripheral vascular resistance (and therefore chronic
hypertension). This theory has been somewhat problematic to test in humans
because the onset of the increase in blood pressure is often difficult to determine,
but several experimental animal studies provide support for this variation over
time in hemodynamic response to salt loading (Campese, 1994).

Proposed renal causes of salt-sensitivity include defects in renal plasma

flow and increased tubular reabsorption of sodium. Potential neural mechanisms

 

Involve direct C'

 
 
  

levels have ai'sc"
m saft-resIstant
barorempt res
noreomephnne t
r gored rena: d:
l'lSu'llfl. the kale:
tare 3i: Men pr.
{Canoese 1994
Salt-sens :
Ire development

eq‘ ,, A.
ah. maggots as.

involve direct or indirect stimulation of the SNS by high salt intake. High salt
levels have also been shown to sensitize cardiopulmonary baroreceptor reflexes
in salt-resistant, but not salt-sensitive patients, suggesting a lack of appropriate
baroreceptor response in salt-sensitive people. There is evidence that increased
norepinephrine to dopamine ratios exist in salt-sensitive patients, suggesting that
reduced renal dopamine levels may contribute to salt-sensitivity. Additionally,
insulin, the kallikrein-kinin system, prostaglandins, and atrial natriuretic factor
have all been proposed to play a role in the salt-sensitivity of hypertension
(Campese, 1994).

Salt-sensitive hypertensives frequently show a greater propensity towards
the development of renal failure, left ventricular hypertrophy, microalbuminuria,
and metabolic abnormalities that may predispose them to cardiovascular
diseases (Campese, 1994). Ferri et al. (1998) have recently shown that salt-
sensitive hypertensives have elevated plasma levels of the endothelium—derived
substances endothelin-1, von Willebrand factor, and E-selectin. This finding
supports the theory that salt sensitivity is correlated with increased risk for

development of hypertension-related vascular damage.

Treatment and Prevention

Clinical management of hypertension is a challenge due to its
multifactorial nature. Prevention is most easily achieved through patient
education and lifestyle modifications, including weight loss, salt restriction,

increased potassium intake, decreased alcohol consumption, smoking cessation,

1O

and relaxation t

 

Desnrnukn et a
a firstllne treat
unsafeleveis p
acrenergfc rece;
sy‘rpat‘tolytlcs.
traders. and an
Graham, 1993 F
innitrtors are be
{Jackson and G;
”9911)! bang l!

‘2‘: ‘Y
‘9 ~ eat'nent of

and relaxation therapies (OMCC, 1996; JNC-V, 1997; Semchenko et al., 1998;
Deshmukh et al., 1998). These patient-controlled activities may also be used as
a first-line treatment of hypertension. If the blood pressure remains elevated to
unsafe levels, pharmacological treatments may be used, including diuretics, B-
adrenergic receptor antagonists, a-adrenergic receptor antagonists,
sympatholytics, angiotensin converting enzyme inhibitors, calcium channel
blockers, and angiotensin II receptor antagonists (Frishman, 1993; Gavras, 1993;
Graham, 1993; Perry, 1993; Puschett, 1993; Weir, 1993; Oates, 1996). Renin
inhibitors are being evaluated for possible use as antihypertensive agents
(Jackson and Garrison, 1996). Endothelin-1 receptor antagonists are also
currently being investigated as a promising class of novel therapeutic agents for

the treatment of hypertension (Webb and Strachan, 1998).

11

 

IL Renin-A!

l.

lt has lor

Integral rote In t'

oa‘ance In both I

 

experzmental by,
enzyme of the R
)uraglomerular ;
orreduced set C
$9,...”

“ s i! 'Ifi“
av» S. 099".

35”” n ~
alvte;.S;n I (A:-

ll. Renin-Angiotensin System

It has long been known that the renin-angiotensin system (RAS) plays an
integral role in the regulation of blood pressure as well as in electrolyte and fluid
balance in both normotensive individuals, and in a variety of forms of clinical and
experimental hypertension. Renin, an aspartyl protease, is the rate—limiting
enzyme of the RAS. It is secreted into the blood by the granular cells of the
juxtaglomerular apparatus of the kidney in response to decreased renal perfusion
or reduced salt delivery. Circulating renin cleaves the circulating preprohormone,
angiotensinogen, which is secreted by the liver, to produce a decapeptide called
angiotensin I (Ang I). This Ang I is in turn cleaved by angiotensin converting
enzyme (ACE) to produce an octapeptide called angiotensin II (Ang II). ACE is
located on the endothelial surface of pulmonary capillaries. Ang II is the primary
effector molecule of the RAS (Bader et al., 1994; Griendling and Alexander,
1994; Deshmukh, 1998). The rate of Ang II formation is inversely related to salt

intake because high salt levels inhibit renal release of renin.

Effects of Angiotensin II

There are at least two subtypes of Ang II receptors, denoted AT1 and AT2,
that have been cloned and characterized. These receptors belongs to the
superfamily of G-protein coupled receptors that have seven transmembrane
regions (Murphy et al., 1991; Sasaki et al., 1991). Ang II binding to the AT1

receptor results in a myriad of physiological events. In arterial smooth muscle, it

12

causes market:
giand, It acts tc
renal sodium re
noreplnephrme
:rrty mm a."
mt'actrlty and

causes growth a

 

 

 

 

{Gelsterfer et al .
dIre—c‘dy on the tu'

Deshmukh et al

Angiotensin ll In
lthas long

EVE-59p DVOgress

asei

causes marked vasoconstriction followed by a drop in blood flow. In the adrenal
gland, it acts to stimulate release of aldosterone which in turn acts to increase
renal sodium reabsorption. In the SNS, it facilitates the release of
norepinephrine to increase CO and TPR. In the brain, it stimulates sympathetic
activity, thirst and vasopressin secretion. It works in the heart to enhance
contractility and ventricular hypertrophy. Ang II is also a growth factor that
causes growth and remodelling of the vascular media, thereby increasing TPR
(Geisterfer et al., 1985; Robertson and Nicholls, 1993). In the kidney, Ang II acts
directly on the tubular cells to promote sodium and water reabsorption

(Deshmukh et al., 1998).

Angiotensin II Induced Hypertension

It has long been known that animals receiving daily infusions of Ang II will
develop progressive hypertension after a period of normotension (Koletsky et al.,
1966). When Ang II is administered parenterally to either humans or
experimental animals, the characteristics of the hypertension produced are
dependent on the amount given. Large doses of Ang II (>30 nglkglmin) result in
an acute pressor effect that occurs within seconds to minutes and returns to
normal within minutes of infusion cessation (Brown et al., 1981 ). This acute
pressor response is thought to be due to direct actions of Ang II on vascular
smooth muscle (Li et al., 1996). Conversely, small doses of the peptide (2-20
nglkglmin) take hours to days to produce an increase in blood pressure. This

slow pressor response persists for hours to days after stopping the infusion

13

(Brown et al .

 

Increased DIG
tone (Lult et a
Sludes
chronéc Infuse
potassium. ace
oontrols (Dowe
pctentated ate

the adrenergzc

(Brown et al., 1981). This chronic increase in blood pressure is accompanied by
increased production of aldosterone in some studies and increased sympathetic
tone (Luft et al., 1989).

Studies of vascular reactivity of mesenteric arteries from rats receiving
chronic infusions of low-dose Ang II have shown that contractile responses to
potassium, acetylcholine, and Ang II were not significantly changed compared to
controls (Dowell et al., 1996). However, the response to phenylephrine was
potentiated after chronic Ang II treatment, suggesting that Ang II may enhance

the adrenergic response in vivo.

Angiotensin II and Pressure-Natriuresis

Angiotensin II is one of the strongest modulators of pressure-natriuresis.
Long-term intravenous infusion of Ang II depresses the slope of the pressure-
natn'uresis curve and shifts it to higher pressures (Cowley and Roman, 1996).
Hall et al. (1990) have shown that dogs with intact renin-angiotensin systems,
under conditions of chronic increased salt intake, can maintain sodium balance
with only minor changes in blood pressure. However, in dogs under the same
conditions that also received continuous low dose intravenous infusions of Ang II,
large elevations in blood pressure were necessary to maintain sodium balance
as sodium intake was increased. These findings suggest that the RAS plays an
important role in the maintenance of normal blood pressure across a wide range

of sodium intakes and that an inability to suppress the RAS in response to

14

excess sodrur
and sense-qua

These
30595 of Ang
I‘COVl'fey anc
deaease be;
i815. Ang II E

her

excess sodium intake can lead to resetting of the pressure-natriuresis system
and consequently produce a chronic rise in blood pressure (Hall 1990).

These observations are softened by the report that, in rats, relatively large
doses of Ang II are needed to produce a shift in the pressure-natriuresis curve
(Cowley and Roman, 1996). Since these doses are still not sufficient to
decrease blood flow or renal interstitial hydrostatic pressure, it seems that, in
rats, Ang II acts to alter the pressure-natriuresis curve via enhanced renal tubular
reabsorption of sodium.

The role of Ang II in pressure-natriuresis has not yet been fully defined.

Salt Sensitivity of Angiotensin II Induced Hypertension

Blood pressure responses to Ang II are salt—dependent in that infusion of
Ang II at low doses causes hypertension in subjects with high salt intake but not
in subjects with normal salt intake (Muirhead et al., 1975; Robertson and
Nicholls, 1993; Haynes and Webb, 1998; Simon et al., 1998). Additionally, the
decrease in renal plasma flow seen in response to Ang II in subjects on a high
salt diet is normalized when the same subjects are switched to a low salt diet
(Sirvio et al., 1990; Robertson and Nicholls, 1993).

Hall et al. (1980) that the RAS plays a major role in maintaining steady-
state levels of arterial pressure and renal hemodynamics during chronic changes
in sodium intake. The mechanism by which Ang II chronically conserves sodium
is through increased renal sodium reabsorption. A decade later, Krieger et al.

(1989) showed that Ang II salt-dependent hypertension is consistently related to

15

Signrfioant Increases
and TPR. Ando et a
and hypertenswe e.“
parallel one another

In addition to I
reoent‘y reported the
ngrtglmin for two we
[Lombardi et al . 199
fiat/l and functiona:

' .4
"vi-9‘3) that favor so:

 

significant increases in fluid retention, blood volume expansion, cardiac output,
and TPR. Ando et al. (1990) have suggested, however, that the sodium retention
and hypertensive effects of Ang II salt-dependent hypertension do not always
parallel one another.

In addition to chronic infusion of low doses of Ang II, Lombardi et al. have
recently reported that transient exposure to high pressor doses of Ang II (435
nglkglmin for two weeks) results in the development of sustained hypertension
(Lombardi et al., 1999). This is purportedly due to structural (microvascular
injury) and functional (loss in intrarenal nitric oxide formation) changes in the

kidney that favor sodium retention.

16

m Endothelin
By the Mid:
endothelium-denve
otnrric oxide as a:
HICKEY et al. repor
was partied. sect.-
at In 1988 (Yanag

the most potent va

BiC’syrlthesis

The family

R3}, the snake I

MC). EEC—h ET IS
Seedfic precursor
Poélocig 1998. w"2

r

Ill. Endothelin

By the mid-1980s, cardiovascular researchers were searching for an
endothelium-derived constricting factor (EDCF) to counterbalance the discovery
of nitric oxide as an endothelium-derived relaxation factor (EDRF). In 1985,
Hickey et al. reported release of such an EDCF (Hickey et al., 1985). This factor
was purified, sequenced, and cloned in the monumental work of Yanagisawa et
al. in 1988 (Yanagisawa et al., 1988). The factor was named endothelin and is

the most potent vasoconstrictor yet known.

Biosynthesis

The family of endothelins (ET) consists of three isoforms (ET-1, ET-2, and
ET-3), the snake venom sarafotcxin (86c), and vasoactive intestinal contractor
(VIC). Each ET isopeptide is represented by a distinct gene that encodes a
specific precursor for the mature isoform (lnoue et al., 1989; lnagami et al., 1993;
Pollock, 1998; Webb et al., 1998). Each ET gene is transcribed and translated to
produce a 212 amino acid preproendothelin. A short secretory sequence is then
removed to form proendothelin, which is in turn acted on by a furin-like enzyme
to produce the inactive 38 residue peptide known as big ET. Endothelin
converting enzyme-1 (ECE-1 ), a neutral metalloendopeptidase, cleaves 17
amino acid residues off the C-terminal of big ET to form the final 21 amino acid
peptide, ET (lnagami et al., 1993; Webb et al., 1998; Takayanagi et al., 1998).

Each ET isoform contains two intrachain disulphide bridges linking paired

17

qsteine reSId.

Webb, 1998).

Sites of Gene
PM or
expression of
generation of
”MUM by?
SiStem. and
(Gray, 19335;
tier (How;
Expressed m

A . .
no: veer: Icer

Endothelin j
The GI

ha

fife been Ctr

or , | . ,

shawls;

s t
W. .
3-3 has

cysteine residues, thereby producing a semi-conical structure (Haynes and

Webb, 1998).

Sites of Generation and Secretion

ET-1 comprises 80% of total endothelin formation. Studies assessing the
expression of mRNA for preproendothelin—1 have shown that the major site of
generation of ET-1 is in endothelial cells (Webb et al., 1998). ET-1 is also
produced by the heart, kidney, posterior pituitary gland, the central nervous
system, and, to a very limited extent, human aortic vascular smooth muscle cells
(Gray, 1995). ET-2 is found in small amounts in endothelial cells, heart and
kidney (Howard et al., 1992; Plumpton et al., 1993). ET-3 is selectively
expressed in the endocrine, gastro-intestinal, and nervous systems. ET—3 has

not been identified in endothelial cells (Gray, 1995).

Endothelin Receptors

The endothelins act on at least two receptor subtypes, ETA and ETB, that
have been cloned, sequenced and characterized on the basis of their
pharmacology. The ETA receptor is preferentially activated by ET-1 and ET-2,
but ET-3 has very low affinity for this receptor subtype. ETB is a nonselective
receptor with practically equal affinity for all three isoforms of endothelin, as well
as for sarafotcxin 6c. ETA receptor mRNA is expressed most highly in the
vascular smooth muscle, aorta, heart, and kidney, but not in endothelial cells.

Conversely, mRNA for the ETB receptor is most highly expressed in cultured

18

endothelial cells an;

 

al.1993; Haynes 3'
The ET. and
snanng only a 70% 1
Structural features Ir
mooted receptors, II
and a relatively long
phospholipase C an
vasoconstrictors, ET
Night to be clue I:
at. 1993).
and

Mlnlfjaj

EXP-'ess

Subc;aSS
”my 0n Vasmlar

yeSGCoT‘IStnqunl a

was responsible to

endothelial cells and in smaller amounts in vascular smooth muscle (lnagami et
al., 1993; Haynes and Webb, 1998).

The ETA and ETB receptor subtypes appear to be functionally distinct,
sharing only a 70% sequence homology. However, both receptors have
structural features in common with the superfamily of rhodopsin-like G-protein-
coupled receptors, including seven hydrophobic membrane-spanning domains
and a relatively long extracellular N terminal. Both subtypes activate
phospholipase C and L-type calcium channels. In comparison to other
vasoconstrictors, ET is known to produce a prolonged contractile response that is
thought to be due to tenacious binding of the peptide to the receptor (lnagami et
al., 1993). Expression of ET receptors is increased by ischemia and cyclosporin,
and decreased by ET-1, Angll, and phorbol esters (Haynes and Webb, 1998).
The initial subclassiflcation of ET receptors held that the ETA receptor, located
mainly on vascular smooth muscle, was generally responsible for
vasoconstriction, and that the ETB receptor, located mainly on endothelial cells,
was responsible for vasodilation via nitric oxide and prostacyclin release (Pollock,
1998). Webb et al. showed that endogenous ET—1 confers basal constrictor tone
on vascular smooth muscle in humans through ETA receptor activation which is
modulated by endothelial cell NO-dependent ETB-mediated dilator tone (Webb et
al., 1998). However, it has recently been observed that some non-ETA receptors
mediate at least part of the vasoconstrictor actions of ET. Binding studies and
other in vitro experiments have shown that some vasculature contain ETB

receptor mediated constrictor elements (Bigaud et al., 1992; Clozel et al., 1992;

19

Cristal et a
of ET 5 ago

Icon).

It IS
Waugh ET
l‘I’eno. 193

U

Internatze;

Cristol et al., 1993). Additionally, other studies have demonstrated that infusion
of ETB agonists will elicit a hypertensive response in most species (Sakurai et al.,
1990).

It is believed that a substantial proportion of clearance of ET-1 occurs
through ETa receptor binding followed by receptor internalization (Haynes and
Webb, 1998; Webb and Strachan, 1998; Miyauchi and Masaki, 1999). The
internalized ET-1/ET3 receptor complex is sequestered into lysosomes where the
acidic environment facilitates ligand dissociation (Miyauchi and Masaki, 1999).
Antagonism of the ETB receptor subtype results in increased plasma
concentrations of ET-1, whereas blockade of ETA receptors does not. The
clearance function of ETB receptors contributes to the short half-life (~1 minute)

of ET-1 in the blood (Webb and Strachan, 1998).

Endothelin Signal Transduction Mechanisms

Binding of ET-1 to ETA or ETB receptors results in G-protein dependent
action of phospholipase C, leading to the rapid hydrolysis of the membrane
inositol phospholipid, phosphatidylinositol 4,5-bisphosphate, yielding cytosolic
inositol 1,4,5-triphosphate (IP3) and membrane-bound sn1,2-diacylglycerol
(DAG). IP3 causes a rapid increase in the intracellular concentration of calcium
through its release from intracellular stores, thereby activating Ca++ dependent
processes and leading to cell membrane depolarization and sustained
extracellular Ca+ influx. DAG activates protein kinase C, which in turn sensitizes

the contractile apparatus to the elevated levels of intracellular calcium. DAG also

20

aciiaes nudea’ 5
regulation Of 09”“[a

were 1998)

Endothelin Recep

Many pharrr
patwlany drugs t
adotional benefits
renal Impairment t'
orsuit. Several e

development. Sir

utinately prove tc
wt? be dissussed
P0156707

”O'I‘Depme Com;

rare shown this <

activates nuclear signalling mechanism that may have long-term effects on the
regulation of cellular growth and function (lnagami et al., 1993; Haynes and

Webb, 1998).

Endothelin Receptor Antagonists

Many pharmacological treatments for hypertension currently exist,
particularly drugs that function to counteract vasoconstriction. It is the potential
additional benefits of ET-1 receptor antagonists on vascular hypertrophy and
renal impairment that make the further refinement of these drugs a worthy
pursuit. Several endothelin receptor antagonists are currently under
development. Since it remains to be seen which of these antagonists will
ultimately prove to be of the most scientific value, only those used in this thesis
will be discussed herein.

PD156707 (Parke-Davis Pharmaceutical Research, Ann Arbor, MI) is a
non-peptide competitive antagonist for both ET receptor subtypes. Maguire et al.
have shown this compound to possess up to a 15000-fold greater selectivity for
the ETA receptor (subnanomolar affinity) compared with the ETB receptor
(micromolar affinity) in in vitro pharmacological experiments. This group also
demonstrated in functional experiments that PD156707 potently antagonized the
vasoconstrictor responses to ET-1 in isolated vascular preparations. An
additional interesting finding is that PD156707 proved to be a more effective

antagonist at lower concentrations than higher ones (Maguire et al., 1997).

21

 

Abbott Lab:
selecjjve receptor é

Porseror. A-127‘

 

(Opgenorth et al., ‘
high polenCy, craft-y I
Laboratories, 1998

ETA receptors {K = 3

 

ET t. receptors Col”)?
tare shown ART-62

In Isolated rat aorta

(

rt. “1
Aoastonal studies he

antagonism of ET-1 I
ill-192621 (Ab!

bIC-a‘ralia'ole nonpe~
ptl

Abbott Laboratories (Abbott Park, IL) has developed a nonpeptide ETA
selective receptor antagonist, A-127722, that is structurally distinct from
PD156707. A-127722 is the most potent ETA receptor antagonist yet reported
(Opgenorth et al., 1996). The active enantiomer of this racemate, ABT-627, is a
high potency, orally bioavailable and efficacious ETA receptor antagonist (Abbott
Laboratories, 1998). In vitro, ABT-627 has displayed very high affinity binding to
ETA receptors (K; = 34 pM), and a more than 1000-fold greater binding affinity to
ETA receptors compared with ETB receptors (K; = 63nM). Functional studies
have shown ABT-627 to dose-dependently inhibit ETA mediated vasoconstriction
in isolated rat aorta (Opgenorth et al., 1996; Abbott Laboratories, 1998).
Additional studies have shown this compound to be orally efficacious for in vivo
antagonism of ET-1 induced increases in MAP (Abbott Laboratories, 1998).

A-192621 (Abbott Laboratories, Abbott Park, IL) is a highly potent, orally
bioavailable nonpeptide selective antagonist of ETB receptors. A-192621 has
been shown to competitively antagonize the specific binding of [mu-labeled ET-1
to CHO cells transfected with human ETB receptors (KI = 8.8 nM) but is far less
potent in inhibiting binding to CHO cells transfected with human ETA receptors (K;
= 5.6 uM; Abbott Laboratories, 1998). In vivo, A-192621 inhibits the ETB-
receptor-mediated initial depressor response to intravenous sarafotcxin 60 (S6c,
an ETB receptor agonist; Abbot Laboratories, 1998). This blockade occurred
when A-192621 was administered either orally or intravenously. A-192621 did

not block ET-1 induced contractions in the rat aorta.

22

A482
bioavaia’ce
182386 has
labeted ET.
on CHO cej
7998} {n v:
Diessor res

Intrarenogs

Cardiovas.

Stud
m he Cara.
meaSE-d ‘4

W the RA

A-182086 (Abbott Laboratories, Abbott Park, IL) is a highly potent, orally
bioavailable nonpeptide mixed antagonist of both ETA and ETB receptors. A-
182086 has been shown to competitively antagonize the specific binding of [ml]-
labeled ET-1 to ETA receptors (K. of 0.2 nM) and to ETB receptors (KI of 1.23 nM)
on CHO cells transfected with the ET-1 receptor subtypes (Abbott Laboratories,
1998). In vivo, A—182086 inhibits both the ETA-receptor-mediated prolonged

pressor response and the ETB-receptor-mediated initial depressor response to

intravenous big ET-1 (Abbot Laboratories, 1998).

Cardiovascular Physiology of Endothelin

Studies with ET receptor antagonists have helped to define the role of ET
in the cardiovascular system. The cardiovascular effects of ET-1 include
increased vascular tone, increased mitogenesis, and activation of both the SNS
and the RAS. The marked pressor effect of ET-1 occurs mainly through an
increase in peripheral vascular resistance. Haynes and Webb have shown that
endogenous ET-1 generation and binding to ETA receptors contributes to the
maintenance of basal vascular tone in forearm resistance vessels of healthy
human subjects (Haynes and Webb, 1998). ET-1 produces a prolonged
vasoconstriction in the peripheral vasculature that lasts for up to 2 hours after the
infusion is stopped. This vasoconstriction leads to a slowly developing dose-
dependent decrease in blood flow through resistance vessels (Brunner, 1998;
Webb and Strachan, 1998). ET-1 has also been shown to produce

venoconstriction, most notably in the human dorsal hand veins in vivo (Webb and

23

St'acnan, ‘
vasodratio
1998). ind.-
pnysologr

As I
endogeno,
restated \
1938; We:
“are Silo“
tone Sofie

Elms-r31;

Strachan, 1998). Blockade of ET receptors in normotensive subjects produces
vasodilation accompanied by a drop in blood pressure (Haynes and Webb,
1998), indicating again that endogenous endothelin is involved in the
physiological maintenance of blood pressure.

As previously mentioned, recent studies suggest that overall effects of
endogenous ET-1 are due to a complex interplay between ETA receptor
mediated vasoconstriction and ETB receptor mediated vasodilation (Brunner,
1998; Webb and Strachan, 1998; Haynes and Webb, 1998). Webb and Strachan
have shown in human forearm vessels in vivo that the basal vascular constrictor
tone conferred by endogenous ET-1 via the ETA receptors is modulated by the
ETa mediated vascular dilator tone via NO release (Webb and Strachan, 1998).
This proposed interplay is of course further complicated by the reported
vasoconstrictor effects of ETB receptors (Sakurai et al., 1990).

ET-1 is also a potent mitogen that acts to produce hypertrophy of vascular
smooth muscle cells, as well as cardiac myocytes and fibroblasts (Webb and
Strachan, 1998; Haynes and Webb, 1998). ET-1 causes enhanced expression
of mRNA for the growth-promoting oncogenes, c-fos and c-myc, that play key
roles in the control of cell growth and proliferation (Force, 1998; Haynes and
Webb, 1998). The mitogenic effects of ET-1 are mediated by the ETA receptor

and may serve to amplify the vasoconstrictor effects of ET—1 (Force, 1998).

24

 

Renal Physiology.
ET-1 is syn'.
nesangzal and Inte
Immunonistocherr
ltégrer levels of ET

1998). Specnlcaity

 

greatest amount of t

Kidneys from
reeeptors. ET), rece

recta. artuate alteI'II
etal, 1995). ETB re
Multan collecting
IQtrey (Rubanyi ant

Mentor subtype ca

the malo’lty or the E

Renal Physiology of Endothelin

ET—1 is synthesized and released in the kidney by cells of renal tubular,
mesangial and interstitial origin, as well as in the renal vascular endothelium.
Immunohistochemical and gene expression studies have consistently found
higher levels of ET-1 in the renal medulla compared to the renal cortex (Pollock,
1998). Specifically, the inner medullary collecting duct cells are the sites of the
greatest amount of ET-1 gene expression.

Kidneys from all investigated species included both ETA and ETB
receptors. ETA receptors have been localized to the renal glomerulus, vasa
recta, arcuate arteries, and mesangial cells (Rubanyi and Polokoff, 1994; Clavell
et al., 1995). ETB receptors have been localized to the initial and terminal inner
medullary collecting duct, as well as the glomerulus and mesangial cells of the
kidney (Rubanyi and Polokoff, 1994; Clavell et al., 1995). Activation of either
receptor subtype causes renal vasoconstriction. In the rat, unlike dog or human,
the majority of the ET-1 mediated renal vasoconstrictor response is due to
activation of ETB receptors (Gurbanov et al., 1996). However, ET-1 shifts the
pressure-natriuresis relationship to higher pressure levels predominately via
activation of ETA receptors (Kassab et al., 1998). ET-1 constricts both afferent
and efferent arterioles equally, thereby causing reductions of renal plasma flow
and glomerular filtration rate (Haynes and Webb, 1998; Baylis, 1999). Several
lines of evidence suggest ET-1 plays a role in the pathogenesis of acute renal

failure following renal ischemia. In rats, administration of an ET-1 antibody prior

25

 

 

to clamping of thei
reduction ol renal
Masaki, 1999) A
bilateral OCduSIon
(Miyauchi and Mas

administration of Bt

 

renal failure (Brook!
In spite of ET
Suggests that ET-1
by direct actions on
vasopressm-stimuia
ETs receptors (Ede
50dlum by Inhibiting

asoenoing limb, an
Banks et al 1

998

and natriuretic effe-

§l3iltemlar filtratior

MET‘1 has a Cl;

to clamping of the renal artery prevents the increase of arteriolar resistance and
reduction of renal plasma flow and glomerular filtration rate (Miyauchi and
Masaki, 1999). Administration of the ETA receptor antagonist, BQ-123, prior to
bilateral occlusion of the renal arteries slows renal functional impairment
(Miyauchi and Masaki, 1999). Furthermore, at least two studies have shown that
administration of BQ-123 24-48 hours following renal ischemia can reverse acute
renal failure (Brooks et al., 1998; Miyauchi and Masaki, 1999).

In spite of ET-1’s potent vasoconstrictor effects, accumulating evidence
suggests that ET-1 has an important role in renal excretion of sodium and water
by direct actions on the renal tubules. ET-1 has been reported to inhibit arginine
vasopressin-stimulated water and chloride reabsorption in the collecting duct via
ETB receptors (Edwards et al., 1993). Additionally, ET-1 blocks reabsorption of
sodium by inhibiting the sodium-potassium ATPase in the proximal tubule, thick
ascending limb, and inner medullary-collecting duct (Haynes and Webb, 1998;
Banks et al., 1998; Plato and Garvin, 1999). ET-1 produces these potent diuretic
and natriuretic effects at doses too low to produce significant reduction of
glomerular filtration rate (Banks et al., 1998). Plato and Garvin have suggested
that ET-1 has a biphasic effect on sodium/water transport in the proximal tubule
by stimulating reabsorption at low doses of ET-1 and inhibiting reabsorption at
high doses of ET-1 (Plato and Garvin, 1999). These tubular effects of ET-1 also
occur with ETB selective receptor agonists and are not blocked by BQ-123,
indiwting that they are mediated by ETB receptors (Haynes and Webb, 1998).

These findings are supported by the report that ETB knockout mice develop

26

hypertenSlon seC€
studies have Sho‘
generation 01 ET'
reabsorption of 5‘

(Haynes and We.

Endothelin and

The multll
vascular hypertrr
activation of the
pathogenesis of

3-1 alone will r:

humans and ex:

hypertension secondary to renal sodium retention (Ohuchi et al., 1999). Other
studies have shown that under experimental hypertensive conditions, renal

generation of ET-1 is decreased, resulting in decreased tonic inhibition of tubular
reabsorption of sodium and water, and thereby leading to sodium retention

(Haynes and Webb, 1998).

Endothelin and Essential Hypertension

The multiple cardiovascular actions of ET-1 (increased vascular tone,
vascular hypertrophy, stimulation of the sympathetic nervous system, and
activation of the renin-angiotensin system) suggest a role for this peptide in the
pathogenesis of hypertension. It is well documented that short-term infusion of
ET-1 alone will produce a significant increase in mean arterial pressure in both
humans and experimental animals (Mortensen and Fink, 1990; Brunner, 1998).
Mortensen and Fink have further demonstrated that hypertension induced by
long-term ET-1 infusion in conscious rats is salt-dependent. In these studies,
only animals receiving 6 mEq per day of sodium experienced a significant
increase in MAP in response to ET-1 infusion at 5 pmollkglmin, whereas animals
receiving 2 mEq/day of sodium remained normotensive (Mortensen and Fink,
1992a). Cardiac output, stroke volume, water balance, and urinary sodium and
potassium excretion remained unchanged. Termination of the ET-1 infusion
resulted in rapid normalization of MAP. Therefore, ET-1 induced hypertension is

a salt-dependent model of hypertension. A connection between salt-sensitive

27

’T

 

hwenenSlon and
levels 0i ET" 'n s
tudles 9V6
nave prOduced var
only In caseS 0" 56

nought to be 5900'

 

1993) However. Ci
since ET-l Is thou;
decxycorticosteronr
correlation beMeer
and the level of sys
In vitro studies of IC
levels In spontanec
levels In nonnotens
However, polymer;

W bl00d presetirr

hypertension. Thzs

hypertension and ET-1 is further supported by the finding of increased plasma
levels of ET-1 in salt-sensitive human hypertensives (Ferri et al., 1998).

Studies evaluating plasma concentrations of ET-1 in human hypertension
have produced variable results. Circulating levels of ET-1 tend to be increased
only in cases of severe hypertension, and even then the increased levels are
thought to be secondary to impaired renal clearance of ET-1 (Haynes and Webb,
1998). However, circulating ET-1 may not reflect the role of ET-1 in hypertension
since ET -1 is thought to primarily act at the local tissue level. In rats with
decxycorticostercne acetate (DOCA)-salt induced hypertension, a significant
correlation between the concentration of immunoreactive ET-1 in vascular tissue
and the level of systemic blood pressure has been reported (Clozel et al., 1994).
In vitro studies of local mesenteric generation of ET-1 have shown increased
levels in spontaneously hypertensive rats (SHR) as compared to vascular tissue
levels in normotensive control Wistar-Kyoto (WKY) rats (Miyamori et al., 1991 ).
However, polymorphisms of the preproendothelin-1 gene are not co-segregated
with blood pressure for inbred salt-sensitive Dahl rats, a low-renin model of
hypertension. This finding indicates that changes in the levels of ET-1
expression per se may not affect the pathophysiology of this model of
hypertension (Haynes and Webb, 1998). Interestingly, human patients with ET-
secreting haemangioendotheliomas are generally hypertensive (Ruschitzka et
al., 1998) and a specific polymorphism (Lys198Asn) in the ET-1 gene is
associated with increased blood pressure in obese human patients (Tiret et al.,

1999). Other studies in hypertensive rats have shown reduced numbers of

28

ETJETa "3C9
res/113mm“l
The mi
amplify V8500
ETjreoeDlOr 5
model of hype
oells {Shanta a'
It IS cffil
oonfounorng e.‘
ofresstance vr
conduit vessefs
1998).
Expenme
drierent forms c
Ilbenension. 5L

”IDIIKIC. a me

ETA/ET; receptors in mesenteric vessels, but increased numbers of ETA
receptors in the cortex of the kidney (Brooks et al., 1998).

The mitogenic effects of ET-1 resulting in vascular hypertrophy may act to
amplify vasoconstrictor effects of ET-1. Sharifi and Schiffrin have shown that
ETA receptor antagonists act to normalize vascular structure in the DOCA—salt
model of hypertension, possibly through apoptosis of vascular smooth muscle
cells (Sharifi and Schiffrin, 1997).

It is difficult to interpret sensitivity to ET-1 in hypertension because of the
confounding effects of ET-1 induced vascular hypertrophy. In general, sensitivity
of resistance vessels to ET-1 is reduced and sensitivity of capacitance and
conduit vessels is increased under hypertensive conditions (Haynes and Webb,

1 998).

Experimental evidence suggests that the role of ET-1 may differ in
different forms of hypertension. In severe, salt-sensitive, low-renin models of
hypertension, such as the DOCA-salt, Dahl salt-sensitive and one-kidney one-
clip (1 K1 C, a model of renovascular hypertension in which one kidney is
removed and the contralateral renal artery is clipped) models, circulating and
tissue levels of ET-1 are increased (Haynes and Webb, 1998; Ruschitzka et al.,
1998; Schiffrin, 1998). Similarly, these models appear to be particularly sensitive
to the hypotensive effects of ET-1 receptor antagonists (Haynes and Webb,
1998; Ruschitzka et al., 1998; Schiffrin, 1998). In contrast, the endothelin system
does not appear to be augmented in spontaneously hypertensive rats (SHR), a

normal-renin model of hypertension. Furthermore, in two-kidney one-clip (2K1C,

29

 

a model of renc
contralateral kic
system Is not 3:
al., 1996) The:
receptor antago
”mnenson.
Two recs
Ir.- humans with I
blOOd flow respc
$816-ng regeptc
”Made of ET~‘

esma‘ hl’perte

lrteresnngly, the

We efiedlve at

Sim‘ga’ly. 1

a model of renovascular hypertension in which one renal artery is clipped and the
contralateral kidney is left intact) hypertension, a renin-dependent model, the ET
system is not activated and bosentan does not lower blood pressure (Sventek et
al., 1996). These strong experimental findings would appear to indicate that ET
receptor antagonism might be most effective in RAS-independent models of
hypertension.

Two recent studies have reported finding increased vascular ET-1 activity
in humans with essential hypertension. Cardillo et al. (1999) compared forearm
blood flow responses to an ETA selective receptor antagonist and an ETB
selective receptor antagonist, alone and in combination. They found that
blockade of ET-1 receptors produced a vasodilator response in patients with
essential hypertension, but not in normotensive controls. These results indicate
enhanced ET-1 mediated vasoconstrictor activity in the hypertensive subjects.
Interestingly, this study found that combined ETA and ETB receptor blockade was
more effective at producing vasodilation than ETA receptor antagonism alone.

Similarly, Taddei et al., ( 1999) also showed that combined ETA and ETB
receptor blockade caused a larger vasodilation in hypertensive patients
compared to normotensive controls. This group associated the difference
between groups with a decrease in tonic nitric oxide (NO) release. Taken
together, these studies in human hypertensive patients strongly support a role for

ET-1 in the pathogenesis of essential hypertension.

30

 

In addition
ET -I may contrib.

left venlncular hyp

 

 

In addition to its effects on the pathogenesis of essential hypertension,
ET-t may contribute to the complications of secondary hypertension, including

left ventricular hypertrophy, pre-eclampsia, and renal failure.

31

w. Endotheli

There app
I gulatton of bloc
ET-1 and Ang I l I
etal..1992. Brur
i losing Subpres:
neaitny rats (ch
sgnifirant nse In
predated an Incr
t=‘r.!>e.".ensive e‘fe

urine volume, or I

‘0 renal actions 0
Several gr
re;

IV. Endothelin and the Renin-Angiotensin System

There appears to be a connection between ET-1 and the RAS in the
regulation of blood pressure. It has been established that coadministration of
ET-1 and Ang II in rats will produce a synergistic rise in blood pressure (Yoshida
et al., 1992; Brunner, 1998). Yoshida et al. first showed this by continuously
infusing subpressor doses of ET-1 and Ang II, separately and in combination, in
healthy rats (Yoshida et al., 1992). Neither ET-1 nor Ang II alone produced a
significant rise in blood pressure, but the combination of these two agents
produced an increase of 32% compared to controls (lmai et al., 1992). This
hypertensive effect occurred without any changes in body weight, fluid retention,
urine volume, or urinary sodium excretion, indicating that this effect was not due
to renal actions of ET-1.

Several groups have reported that Ang II stimulates the generation and
release of ET-1 from endothelial cells (Emori et al., 1991; lmai et al., 1992; Webb
and Strachan, 1998; Brunner, 1998). lmai et al. were the first to show that Ang II
and arginine vasopressin immediately and dose-dependently induce expression
of preproendothelin-1 mRNA in multiple tissues, including endothelial cells (lmai
et al., 1992). More recently, Moreau and colleagues demonstrated that long-term
treatment with Ang II caused both elevated tissue levels of ET-1 and vascular
hypertrophy that was inhibited by ETA receptor blockade (Moreau et al., 1997).
Likewise, d’Uscio et al. showed that losartan, an AT1 receptor antagonist, inhibits

Ang II induced tissue increases of ET-1 (D’Uscio et al., 1995). Verapamil, a

32

 

 

mum channel {I
hatATI receptor
trough an actor
Angll -ET-1 Inte'
producton of ET-‘-

"$583118th 338085

 

ET-t production me
The flip Side
modulate the actlvl
Ireeased 2 Stem l
Illawaguchi et al..
moment admInIs
development of ET

All “ Concentrate

calcium channel blocker, did not prevent the increased ET-1 levels, suggesting
that AT1 receptor antagonists directly modulate tissue levels of ET-1, rather than .
through an action on systemic arterial pressure. A functional response of the
Ang II — ET-1 interaction was reported by Dohi et al (1992). Ang II stimulated
production of ET-1 potentiates norepinephrine-induced contractions in
mesenteric arteries of spontaneously hypertensive rats, indicating that vascular
ET-1 production may act to amplify the pressor effects of the RAS.

The flip side of this Ang II — ET-l relationship is that ET-1 is able to
modulate the activity of the RAS. Kawaguchi et al. reported that ACE activity is
increased 25-fold in the presence of ET-1 in cultured endothelial cells
(Kawaguchi et al., 1991). Similarly, Mortensen and Fink (1992b) showed that
concurrent administration of ET—1 and the ACE-inhibitor captopril prevented the
development of ET-1-induced hypertension. Surprisingly, an increase in plasma
Ang II concentration was not observed in rats receiving the same concentration
of ET-1 alone. These findings suggest the potential role of Ang II in ET-1-
induced hypertension is played out at a local tissue level, or that one peptide
increases the sensitivity of cardiovascular tissues to the other.

As discussed earlier, experimental evidence suggests that the role of ET-1
may differ in different forms of hypertension. In severe, salt-sensitive, low-renin
models of hypertension, circulating and tissue levels of ET—1 are increased and
these models appear to be particularly sensitive to the hypotensive effects of ET-
1 receptor antagonists (Schiffrin, 1998; Haynes and Webb, 1998; Ruschitzka et

al., 1999). In contrast, the endothelin system does not appear to be augmented

33

in normalrenin o

 

receptor antagon
hypertensron.

However, a

|
l

tat are hard to re:

administration of i4

d‘Uso'o et al that c

Increase of systolic

 

“ Induced hypertenl

fled the possum

n..}
h .
I'YI‘IEIIEDSIOTI may 3

Stud

yshowed that A
vasoconstriCtlon we'
antagonists (Rajago
leveiopment of Ang
anéxed ETA/ETB T95
that endogenous ET

in normal-renin or renin-dependent models of hypertension, indicating that ET
receptor antagonism might be most effective in RAS-independent models of
hypertension.

However, a handful of studies have investigated the effects of concomitant
administration of Ang II and ET receptor antagonists and have reported results
that are hard to reconcile with the above findings. It has been reported by
d’Uscio et al. that chronic ETA receptor antagonist therapy attenuates the
increase of systolic blood pressure and alterations of endothelial function in Ang
II induced hypertension (D’Uscio et al., 1997). Rajagopalan and colleagues
raised the possibility that many of the vascular effects seen in Ang II induced
hypertension may actually be mediated by endogenously expressed ET-1. Their
study showed that Ang II induced hypertension and its associated
vasoconstriction were prevented by concomitant adminstration of ETA receptor
antagonists (Rajagopalan et al., 1997). Herizi et al. reported that the
development of Ang II induced hypertension was totally prevented by bosentan,
a mixed ETA/ETB receptor antagonist, thereby further supporting the possibility
that endogenous ET-1 contributes to the cardiovascular effects of Ang II (Herizi
et al., 1998).

In vitro studies have reported that acute exposure to subcontractile
concentrations of Ang II potentiates vascular contractile responses to other
agonists (Henrion et al., 1992a). This action of Ang II has not been tested using
ET-1 as the second agonist, but the strong similarity in signaling mechanisms

used by Ang II and ET-1 in vascular smooth muscle (Tsuda et al., 1993) makes it

 

a likely possi on It,

have reported the

 

aortic strips from I
1998).

A recent st
In humans with es
s“Oiled that AT, I
95.891 013 corny)”.
hYDertensIon 3,,
vamnstrlctjgn |
1999), ms ,5 em
.eouoe the V3800

This tiWests

’96th anta’aon;

a likely possibility. Some studies using Ang II induced models of hypertension
have reported markedly attenuated vascular contractile responses to ET-1 in
aortic strips from Ang II infused rats (D’Uscio et al., 1997; Rajagopalan et al.,
1998).

A recent study highlights a potential critical link between Ang II and ET-1
in humans with essential hypertension (Ghiadoni et al., 2000),. This group
showed that AT1 receptor blockade with candesartan eliminates the vasodilating
effect of a combined ETA/ETB receptor antagonist in patients with essential
hypertension. Since it is known that endogenous ET-1 increases
vasoconstriction in essential hypertension (Cardillo et al., 1999; Taddei et al.,
1999), this is evidence that inhibiting the actions of Ang II at ATI receptors will
reduce the vasoconstrictor effect of ET-1 in humans with essential hypertension.

This thesis is the first body of work investigating the effect of ET-1

receptor antagonists on the salt-sensitivity of Ang II induced hypertension.

35

V. Hy
Tn

ET-l Sys‘

Intake Is
linemen.
9900gen

the 3"“A

vtf 'V

V. Hypotheses

The overall objective of this research project is to examine the role of the
ET-1 system in experimental Ang II induced hypertension. Our working
hypothesis is that the presence of ET-1, under conditions of increased salt
intake, is required for the maintenance of experimental Ang II induced
hypertension. This may occur either by Ang II stimulated production of
endogenous ET—1, or by an as yet unknown synergistic response resulting from
the actions of the two peptides. The following specific hypotheses will be

addressed:

I. In experimental Ang II induced hypertension, especially under conditions

of high salt intake:

A. ET-1 activates ETA receptors to increase blood pressure.

8. ET-1 activates ETg receptors to decrease blood pressure.

C. ET-1 affects blood pressure by modifying renal sodium and water
excretion.

D. ET-1 receptor antagonists will cause changes in blood pressure and
renal fluid excretion qualitatively similar to those produced by the

diuretic drug trichlormethiazide.

II. In experimental Ang II induced hypertension, ET-1 affects blood pressure:
A. As a result of changes in vascular reactivity to ET-1 produced by
angiotensin II, in vitro.
B. By altering ET-1 induced vascular contractility in a salt-dependent

manner.

37

a?“
i

Chapter 2
GENERAL EXPERIMENTAL METHODS

I. Animals

Male Sprague-Dawley rats (Charles River Laboratories, Portage, MI)
weighing 325-450 grams are used in these experiments. Upon arrival at our
facility, rats are maintained according to standards approved by the Michigan
State University All-University Committee on Animal Use and Care. All
experimental procedures are carried out in accordance with the “Guiding
Principles in the Care and Use of Animals” of the American Physiological
Society. Rats are acclimatized for at least two days prior to any surgical
procedures in clear plastic boxes with woodchip bedding. Rats are allowed
access to standard rodent chow (Teklad 22/5 Rodent Diet W 8640, Madison, WI)

and tap water ad libitum.

II. Surgical Procedures

All surgical procedures are performed after administration of pentobarbital
sodium (Nembutal’, Abbott Laboratories, N, Chicago, IL), 50 mglkg i.p., and
atropine sulfate (Sigma, St. Louis, M0), 0.2 mg, i.p. If necessary, anesthesia is
supplemented using methohexital sodium (Brevital®, Eli Lilly, Indianapolis, IN), 5-
‘IO mglkg, i.v. Before surgery is initiated, areas to be incised are shaved free of

fur and cleaned with disinfectant (Betadine®, Purdue Frederick Co., Norwlk, CT).

38

 

thong
CSTID
Inner it
3.933;.

aC‘Ia "I{

sang

edema
"e..“,

Normal body temperature is maintained during anesthesia by a water-heated pad
(Gorham-Rupp, Inc.).

A. Arterial and Venous Catheten'zation

The cannulae used in these procedures are constructed of polyvinyl
chloride (Tygon® Microbore) tubing with 4.5 cm silicone rubber tips (Dow
Corning” Silastic). The femoral vessels are exposed via a 2.0 cm incision in the
inner left hind leg of the rat. Small incisions are made in each vessel and
approximately 4.5 cm of the arterial and 7.0 cm of the venous catheters are
advanced through the vessels into the aorta and vena cava, respectively. The
larger arterial catheter is sutured to nearby leg muscle in a way that protects
against occlusion of the catheter during normal rat movement. Some rats are
additionally catheterized at the facial vein. This procedure involves a 1.0 cm
incision in the right side of the animal’s neck, just superior to the jugular pulse
point. The facial vein is cleared and catheterized using the same procedure
described above for the femoral vessels.

The free ends of the catheters are tunneled subcutaneously to the rat’s
head, where they are exteriorized by threading them through a stainless steel
spring tether. This tether is then anchored to the skull using jeweler's screws
and dental acrylic. After the rats have recovered from the anesthesia, they are
individually housed in stainless steel metabolism cages for the remainder of the
experimental protocol. A minimum of three recovery days is allowed before any

experiments are started.

39

 

,n’.
‘uuG

ureter
Is ere
BIS-313‘
35am

Some:

8. Surgical Uninephrectom y

The skin over the left lateral abdomenal wall is shaved and prepared with
an iodine antiseptic cleanser (Betadine‘a, Purdue Frederick 00., Nonivalk, CT). A
1.5 cm vertical incision is made through the skin and underlying muscle just
caudal to the rib age with scissors and tissue forceps. The renal vessels and
ureter are ligated with 4-0 suture silk (Ethicon‘m, Somerville, NJ). The left kidney
is exteriorized and excised with a number 10 scalpel blade (Bard-Parker“,
Becton-Dickinson, Franklin Lakes, NJ). The muscle layers and skin are
separately closed with 4-0 silk and 4-0 monofilament nylon suture (Ethicon®,
Somerville, NJ), respectively.

C. Deoxycorticosterone Acetate Implantation

A 3 x 1.5 cm rectangular area between the scapulae is shaved and
disinfected. Following a 1 cm lengthwise incision, a decxycorticostercne acetate
(DOCA) patch is implanted subcutaneously. DOCA implants are prepared by
mixing 1 part DOCA (Sigma, St. Louis, MO) with 2 parts silicone rubber (Dow
Corning”, Midland, MI) by weight, then curing overnight at room temperature.
Surgical placement of implants at 600 mglkg results in a dose of 200 mglkg
DOCA. Skin is closed with 40 nylon suture. Butorphanol tartate (Stadol‘m, Bristol
Laboratories, Princeton, NJ), 2 mglkg subcutaneous, is given post-operatively for
analgesia. Enrofloxacin (Baytril®, Bayer Corp., Shawnee Mission, KA), 5 mglkg
s.c., is administered daily for 3 days beginning the day of surgery for bacterial

prophylaxis.

4o

room wrtl

hater ant

Ih’lstlfig It
meaS-urer
OIEtreasm
Iterrain ;

Ill. Chronic Rat Maintenance and Measurements

The metabolism cages housing the rats are located in a climate-controlled
room with a 12-hour light-dark cycle. The rats are allowed access to distilled
water and either standard rodent chow (Teklad 22l5 Rodent Diet W 8640,
Madison, WI) or sodium-deficient rat chow (Teklad TD 170950, Madison, WI) ad
libitum. depending on the experimental protocol. The free end of the stainless
steel spring tether anchored to the rat’s skull is attached to a plastic hydraulic
swivel that will allow the instrumented rat to move freely in its cage without
twisting the catheters. This tethering spring also allows for cardiovascular
measurements and manipulations to be made without handling the rat, thereby
decreasing the amount of animal distress. Catheters are flushed daily to
maintain patency and the arterial catheters are filled with a heparinized sucrose

solution and occluded when not in use.

IV. Hemodynamic Measurements

Mean arterial pressure (MAP) and heart rate (HR) are measured via the
arterial catheter each morning between 8:00 and 11:00 am. The arterial
catheters are connected to external pressure transducers that have first been
zeroed at the level of the rat’s heart. The transducers are connected to digital
pressure monitors (Digi-Med Blood Pressure Analyzer, Micro-Med, Louisville,
KY, USA ) that output directly to a computerized data acquisition and storage
system (Digimed System Integrator, Micro-Med, Louisville, KY, USA). Data are

collected once every second for 20—30 minutes. The daily value is determined by

41

 

heav

trod

the average of the one-second recordings taken over the last five minutes of the

recording session.

V. Metabolic Measurements

Rats received drinking water in graduated cylinders that were refilled each
day, allowing for daily measurements of water intake. Rats were housed in
metabolism cages designed for urine collection, allowing for daily measurements
of urine output. Additionally, daily urine samples were collected and analyzed by
ion selective electrodes (Nova Electrolyte 16+ Analyzer, Nova Biomedical,
Waltham, MA, USA) for sodium, potassium, chloride, and creatinine levels as
indices of renal function. On control day 2 and experimental days 5, 10, and 14,
whole blood samples (1 cc) were collected by syringe via the arterial catheters,
placed on ice and analyzed by ion selective electrodes ((Nova Electrolyte 16+
Analyzer, Nova Biomedical, Waltham, MA, USA) for sodium, potassium, chloride,
BUN, and creatinine levels. Sodium and potassium excretion were calculated by
multiplying electrolyte concentration by daily urinary volume.

Plasma volumes were determined, via Evan’s Blue dye dilution,
approximately every fifth day of the experimental protocols. Evan’s Blue dye
binds to albumin in the plasma. The plasma volume measurement involved
collecting a 0.5 cc sample of blood by syringe from the rat’s arterial catheter
before injection of 0.2 cc Evan’s Blue dye into the catheter and collecting a
second 0.5 cc sample 10 minutes after injection of the dye. The samples were

then centrifuged and the plasma layer was collected. The plasma samples were

42

read
PW
sari;
resui
of the

and r

“a

II-"g-“Ij’l-

read on a fluorescent plate reader at 610 nm. The color detected was directly
proportional to the concentration of Evan’s Blue in the sample. The control
sample reading was subtracted from the post-injection sample reading and the
resulting number was compared to a standard curve, allowing for determination
of the total plasma volume. Blood volume was calculated from plasma volume

and hematocrit using the standard formula.

VI. Isolated Tissue Bath Measurements

Rats were killed (80 mglkg pentobarbital i.p.) and the superior mesenteric
arteries were dissected into helical strips. The endothelium was left intact.
Tissues were placed in physiological salt solution containing (mmol/l) 130 NaCl,
4.7 KCl, 1.18 KH2PO4, 1.17 MgSOa~7H20, 1.6 CaCL2-2H20, 14.9 NaHCOa, 5.5
dextrose, and 0.03 CaNa—EDTA. One end of the preparation was attached to a
glass rod and the other to a force transducer (model FT03, Grass Instruments,
Quincy, MA), and the strip was placed under optimum resting tension (600 mg,
as previously determined) and allowed to equilibrate for one hour. Muscle baths
were filled with warmed (37°C), aerated (95% 02 -5% C02) physiological salt
solution. Changes in isometric force were recorded on a polygraph (Grass
Instruments). After the hour of equilibration, arteries were challenged with a
maximal concentration of the dI-adrenergic receptor agonist phenylephrine (PE,
10 umoI/I). Tissues were then washed, and the status of the endothelium was
examined by observing arterial relaxation to the endothelium-dependent agonist
ACh (1 umol/l) in tissues contracted by a half-maximal concentration of PE (~10

nmolll).

43

 

 

toll

each I

VII.

The vessels then were incubated with increasing concentrations of PE (10‘
9 to 10*" M) followed by ET-1 (10'11 to 10‘7 M). The tissues were incubated with
each concentration for ~5 minutes before the next concentration was added. In
some preparations, superior mesenteric arteries were incubated with Ang II (10'10
M), A-192621 (an ETB selective receptor antagonist, 30nM, Abbott Laboratories,
Abbott Park, IL, USA), ABT-627 (an ETA selective receptor antagonist, 30 nM,
Abbott Laboratories, Abbott Park, IL, USA), or A-182086 (an ET NB non-selective
receptor antagonist, 30 nM, Abbott Laboratories, Abbott Park, IL, USA), for one

hour prior to the production of ET-1 dose response curves.

VII. Drugs

The ETA receptor antagonist, PD156707, was a generous gift from Parke-
Davis Pharmaceutical Research (Ann Arbor, MI). The ETA receptor antagonist,
ABT-627, the ETB receptor antagonist, A192621, and the non-selective ETA/3
receptor antagonist, A-182086, were generous gifts from Abbott Laboratories
(Abbott Park, IL). The Ang II and the thiazide diuretic, trichlormethiazide, were

obtained from Sigma Chemical Company (St. Louis, MO, USA).

VIII. Statistical Analysis

Results are expressed as means i SEM. Mean values were compared
statistically using a one-way ANOVA followed by the protected least significant
difference test for post hoc comparisons. For in vivo data, within- and between-

group differences were analyzed using mixed-design ANOVA. Post hoc

44

 

oonpansc
Within grc
Inerence
Iaues we
Delanete
Results c

Slghlficar

comparisons between groups were performed by testing for simple main effects.
Within group comparisons were made using the protected least significant
difference test. For in vitro data, estimates of maximum response and E050
values were obtained from each concentration response curve using a four-
parameter logistic function. Other statistical procedures are described with the
Results of the individual studies. For all analyses, criterion for statistical

significance was a probability level of less than 0.05.

45

The
efen‘ve 2
Planned lI
P'G‘IIOusly

C .
Il- tartan

Chapter 3
DOSE-FINDING STUDIES

The purpose of the studies described in this chapter was to establish
effective doses and in vivo time courses of the ET—1 receptor antagonists that I
planned to use in the main part of my thesis project. These drugs had not been
previously well—characterized in vivo, especially in chronic dosing studies. The
first antagonist that was available to me for use in these studies was PD156707,
a potent but short-acting ETA selective receptor antagonist. I chose to study an
ETA selective receptor antagonist first because these receptors had been better
characterized than the ETB receptors, arterial constriction was known to be
regulated by ETA receptors, and at the onset of this project no selective ETB
receptor antagonists had been described in the literature. Subsequent
development by Abbott Laboratories of a long-acting and selective ETA
antagonist, ABT-627, and the first long-acting and selective ETa antagonist, A-
192621, allowed me to further investigate the potential long-term effects of ET-1
in Ang II induced hypertension. I chose to use A-182086, Abbott Laboratories
mixed ETA/ETB receptor antagonist, for my studies rather than the better
characterized mixed antagonist, bosentan, in order to study drugs as chemically
similar as possible. Unless otherwise noted, all studies were in normal
conscious male Sprague-Dawley rats chronically instnrmented for direct, daily
measurements of blood pressure and heart rate via catheterization of the femoral

artery, femoral vein, and facial vein, and housed as previously described in the

46

general met
DOCA-salt

2).

general methods (chapter 2). In two of the protocols, drugs were tested in
DOCA-salt rats prepared as described in the general methods section (chapter

2).

47

Ellertiven

Hypertenl

Rationale
P015670
{Maguire
We needs
enOugh tr
”flatten:

0639mm

a“: 90m:

aterial p

- ,J‘Ixoif

Effectiveness of PD156707 Tested in Endothelin-1 Induced Model of

Hypertension

Rationale: At the time this study was conducted, the ETA receptor antagonist,
PD156707, had been characterized mainly on the basis of in vitro pharmacology
(Maguire et al., 1997). Since our studies all involve whole animal experiments,
we needed to demonstrate that this drug would be both potent and efficacious
enough to suit our purpose. PD156707 was tested in an ET-l induced model of
hypertension. This model is both ET-1 and salt-dependent. We sought to
determine the efficacy, time course and dose range of the ETA receptor
antagonist that would produce an acute, dose-dependent decrease in the mean

arterial pressure in rats receiving ET-1 infusions and high salt intake.

Protocol: Some of the rats were maintained on high salt intake (n=8, ~8
mEq/day) and some were maintained on normal salt intake (n-8, ~2 mEq/day).
All rats received 8 pmollkglmin ET-1 over a 24 hour time period prior to
administration of the ETA receptor antagonist. Three doses of P0156707 (0.3,
0.03, and 0.003 mglkg) were then tested by injecting the drug into the facial vein
catheters of the rats (n = 4-6 rats per experimental group). BP and HR were

recorded from 10 minutes prior to until 60 minutes post-injection of P0156707.

Results: Infusion of ET-1 raised BP only in the high salt intake rats, producing a

control mean arterial pressure (MAP) of 144 mmHg in these rats compared to a

48

mntrol M
were no 1
(0 003 In
lowered
by repee
Dear. all
”Ilene:
he did;
allege-r
interest.
all Ol tl‘r

‘"9 $3?qu

control MAP of 106 mmHg in the normal salt intake rats (figures 3.1 & 3.2). There
were no signifimnt changes in BP with either the high (0.3 mglkg) or the low
(0.003 mglkg) dose of P0156707. However, the 0.03 mglkg dose of PD156707
lowered BP in high salt intake rats by approximately 25 mmHg, as demonstrated
by repeated measures ANOVA and Dunnetts multiple comparisons test, with
peak effect occurring between 30-50 minutes post-injection. Since the
hypertension in these high salt animals was induced by administration of ET-1,
the drop in MAP after injection of PD156707 clearly indicates that this dose of the
antagonist will reverse the actions of ET—1 mediated by ETA receptors in vivo.
Interestingly, the injection of PD156707 also produced a significant tachycardia in
all of the rats (data not shown). This response was seen with all three doses of

the drug that were tested.

49

ad‘s.
e1
2

1
1r

. 9
C023).
«OI-hut

3.1

figll'e

 

 

—e— 0.3 NGKG, n=5
—O—- 0.03 WEI/KG, n=5
—A—- 0.003 NEIKG, n=5

 

 

 

 

 

10

5 CamuNMP=1441e41

0 ‘Kl l- \l l
A fl: Ii \\

.5 4 ‘A A A % \

 

 

 

A MAP (mint-lg)
8
/
>/

 

 

 

 

 

 

 

 

 

451 k . l“ . ,
- " / C x>
20 lip—l NM” *
.25 * * * * *
m A __ .IL

5' 1s 30' 45' 60'
11NE(Mnures)

Figure 3.1 Effect of PD156707 on endothelin-1 induced hypertension in high salt
rats.

50

«WISE» p.332 9

 

 

+ 0.3 MGIKG, n=5
—0— 0.03 MGIKG, n=5
—A- 0.003 MGIKG, n=5

 

 

 

 

 

 

 

 

 

 

20 Control MAP = 107.4 i 2.9

15 I _ l
A 10 ~
E 5 l A A:
E 0 " ‘ ‘/A ”A
V O . / ’ ‘ (3;! ‘
<' '10‘ ' \b

-15 ‘.

-20 . . . . . . . e I . . . I

5' 15' 30' 45' 60'
TIME (Minutes)

F'QUI‘O 3.2 Effect of PD156707 on endothelin-1 induced hypertension in normal
salt rats.

51

Effectii

Rahm
repent

95:83. .,

k

by ea:

Effectiveness of P0156707 Tested in DOCA-Salt Model of Hypertension

Rationale: After demonstrating that PD156707 was efficacious in an acute, ET-
dependent model (see above), it was next desirable to test this drug in a well-
established chronic model of hypertension. Therefore, PD156707 was tested in
the decxycorticostercne acetate salt (DOCA-salt) model of hypertension. DOCA
is a mineralocorticoid that acts intracellularly in cells of the renal tubule to cause
sodium retention. This model is a chronic volume-expansion model and is RAS-
independent (Kenyon et al., 1994). DOCA-salt is considered to be the best
established model for studying the role of ET-1 in hypertension, as demonstrated
by upregulation of ET-1 in DOCA-salt rats and decrease in BP in response to ET-
1 antagonists in these animals (Brooks et al., 1998). We sought to determine the
dose of the ETA receptor antagonist that would produce an acute, dose-

dependent decrease in mean arterial pressure in DOCA-salt rats.

Protocol: Three doses of PD156707 (0.3, 0.03, and 0.003 mglkg) were tested
by injecting the drug into the femoral vein catheters of the rats (n = 4-6 rats per
experimental group). BP and HR were recorded from 10 minutes prior to until 60

minutes post-injection of PD156707.

Results: In the DOCA-salt model, PD156707 produced a dose-dependent

d9(3l‘ease in blood pressure (~12 mmHg) with a peak effect between 30-50

52

m,
mutes
r
epea‘e
0" k
’ ays a
\n m r
. r

T
ecen
pt:

minutes (figure 3.3). This was a significant drop in BP, as demonstrated by
repeated measures ANOVA and Dunnetts multiple comparisons test. Since ET-1
plays an established role in DOCA-salt hypertension, and PD156707 lowered BP
in this model, we conclude that the antagonist is efficacious at blocking ETA

receptors over the dose range used here.

53

«DIEEVn‘d‘S—Q

i 9% 3.3

in

 

 

+ 0.3 MGIKG, n=5
—0— 0.03 MGIKG, n=5
—A— 0.003 MGIKG, n=5

 

 

 

 

 

 

 

 

 

 

 

 

:2 Control MAP = 183.6 i 13,6 T
10 ., T T T
A AK
3 5 ‘ NM»). i A“ “H
V '5 _ \. j; “ e w ‘5
a .. , 4
-10 ~ ‘.» s‘
Q
-15 ‘ *
* *
.20 .4 * * * *
-25

 

 

 

5' 15' 30' 45' 60'
TIME (Minutes)

Figure 3.3 Effect of PD156707 on DOCA-salt hypertension.

Rapid '

Rationa
P0156]
1 indUCi
an: gor
ntagor
VESSUFE
lamina:
91' ESSuFe
We cops
(6033”

p
: l
en'eptOfs

Rapid Termination of Endothelin-1 Infusion

Rationale: In our first study, we showed that a moderate dose (0.03 mglkg) of
PD156707 was sufficient to produce a significant decrease in BP in rats with ET-
1 induced hypertension, but that this decrease was delayed 30-40 minutes after
antagonist injection. To demonstrate that this drop in BP was truly due to
antagonism of ET receptor activity, we examined the time course of blood
pressure fall after simply stopping the EM infusion. We hypothesized that
termination of the ET-1 infusion would produce an identical time course of blood
pressure fall to the administration of a moderate dose of PD156707 (0.03 mglkg).
We considered cessation of ET-1 infusion to be roughly equivalent to ET—1
receptor antagonism because of the known tenacious binding of ET-1 to its

receptors.

Protocol: All of the rats were maintained on high salt intake (n=8, ~8 mEq/day).
All rats received 8 pmollkglmin ET-1 over a 24 hour time period prior to
administration of the ETA receptor antagonist. At the onset of the timed
experiment, the ET—1 infusion was abruptly terminated. BP and HR were
recorded from 10 minutes prior to until 60 minutes after stopping the ET—1

infusion.

55

RESuh

ef‘m

~th

iecree
(zabie
Soppe
{22:39 1
antago

ET; rec

Results: Terminating ET-1 infusion resulted in a decrease in BP with a peak
effect between 30-50 minutes after stopping the ET-1 infusion (figure 3.4). This
decrease in BP closely paralleled the decrease seen after injection of PD156707
(table 3.1). An increase in HR was observed soon after the ET—1 infusion was
stopped, similar to the tachycardia observed after administration of PD156707
(table 3.2). These results support the hypothesis that the ETA receptor
antagonist, PD156707, acts primarily by preventing the actions of ET-1 at the

ETA receptor.

I I I I 1 1

:Ev GLJWQQLD. ..2LQHL4‘ :50:

HQUre ;

‘in‘. H»

 

+n=

 

 

 

.3
\l
O

.x
G
O

150 -

140 -

130 -

120 “

 

 

110

Mean Arterial Pressure (mm Hg)

OI

10'

j

20'

j

30' 40' 50' 60'

Time (minutes)

Figure 3.4 Effect of cessation of endothelin-1 infusion on mean arterial pressure
(mm Hg) in endothelin-1 induced hypertension.

57

 

O)
C?

; /&T/S/c%/3_/c°5/&>:’/8§/e>f/za/’ot log/C3 I .

145. 156.
143. 151.
140. 143.

1 140.
136. 136.
135. 134.

1 142.
132. 1431
131. 139.
133. 1371

1 132.
136. 135.
136. 137.

Table 1: Comparison of the change in mean arterial pressure (mmHg) over
time with endothelin-1 infusion cessation and injection of PD156707

 

58

344.

353.
362.1
383.
363.1
355.

397.

382.
37

366.1

 

389.

rate over
endothelin-1 infusion cessation and injection of PD156707

59

Autonomic Blockade and PD156707

Rationale: The unexpected tachycardia observed with both administration of
PD156707 and removal of exogenous ET—1 posed the question of whether or not
this response was a function of blocking ET-1 activity or baroreflex-mediated
sympathetic activation and parasympathetic withdrawal. To further investigate
this phenomenon, we utilized two drugs known to antagonize actions of the
autonomic nervous system (ANS) on the heart. Atropine is a competitive
antagonist for muscarinic receptors and thereby blocks parasympathetic nervous
system-mediated bradycardia (Brown and Taylor, 1996). Propranolol is a
nonselective competitive is-adrenergic receptor antagonist whose actions include
blockade of cardiac [31 receptors, resulting in decreased sympathetic actions on
HR (Hoffman and Lefi<owitz, 1996). We hypothesized that these drugs in
combination would abolish the tachycardic response seen upon administration of

PD156707.

Protocol: All of the rats were maintained on high salt intake (n=8, ~8 mEq/day).
At the onset of the timed experiment, atropine (0.02 mglkg) was injected via the
facial vein catheter. Five minutes later, propranolol (1 mglkg) was injected via
the same catheter. Five minutes after that, PD156707 (0.03 mglkg) was injected

as previously described in preliminary study I. BP and HR were recorded from

60

10 minutes prior to the atropine injection until 60 minutes post-injection of

PD156707.

Results: There was no increase in HR following PD156707 in the rats given
atropine and propranolol. This finding indicates that the tachycardia seen upon
blocking ETA receptors is autonomically mediated.

Conversely, PD156707’s hypotensive effect was unaffected by the presence of
the ANS antagonists. This suggests that the drop in blood pressure caused by

ETA receptor blockade is not significantly opposed by reflex cardiac stimulation.

61

The Effect of PD156707 on Angiotensin ll-Induced Hypertension

Rationale: Until the time of this study, no one had published an account of the
effects of an ETA receptor antagonist on previously established Ang II induced
hypertension. It is important to determine whether or not ET-1 plays a role in the
maintenance, as well as the initiation, of Ang II dependent hypertension.
Developing a time course for the action of ET-1 in Ang II dependent hypertension
would lead to a clearer understanding of the specific actions of this peptide in the
hypertension disease process. The current study examined this relationship with
the expectation that Ang II mediated hypertension gradually would become more
ET—1 dependent with time, reflected by an increasingly larger anti-hypertensive
response to the ETA antagonist. Further, we postulated that the role of ET-1

would be larger in rats on high versus normal salt intake.

Protocol: Some of the rats were maintained on high salt intake (n=8, ~8
mEq/day) and some were maintained on normal salt intake (n-8, ~2 mEq/day).
After recording BP and HR for two control days, Ang II was continously infused
i.v. at a rate of 5 nglmin for 15 days. Ang II was then removed for five recovery
days. On control day 1, and days 1, 5, 10, and 15 of Ang II infusion, and days 1,
3, and 5 of recovery, the rats received PD156707 at 0.03 mglkg, i.v. BP and HR
were recorded 10 minutes prior to until 60 minutes post-injection of PD156707.

Changes in MAP and HR to PD156707 were recorded 35-55 minutes after

62

antagonist injection, based on our preliminary results on the time-course of action

of this drug.

Results: Ang II increased MAP by ~22 mmHg over control MAPs in high salt
rats, but did not significantly change the MAPs in normal salt intake rats (figure
3.5).

ETA receptor blockade with PD156707 caused significant tachycardia (not
shown), but did not change BP before, during, or after chronic Ang II infusion in
rats on high salt intakes (figure 3.6). Injection of PD156707 in normal salt intake
rats did not produce changes in either HR (not shown) or BP (figure 3.7). These
data do not support an important role for ETA receptor activation in short-term
blood pressure control in rats with Ang II induced hypertension on either normal

or high salt intakes.

63

 

+ High salt intake, n=8
—O— Normal salt intake, n=8

 

 

 

.3
N
O

 

 

 

 

a

I Confirm Angll5nglmin Recovery

E 160 1 from

E 150 - Ang II

g 140 -

5 130~

.75 120 - w

a:

E 110 ‘ {KQWM’QJW

g 100 ‘

E 90 ,,,,,,,,,,,,,,,,,,,, T
C1 A1 A5 A10 A15 R2 R5

Protocol Day

Figure 3.5 Daily mean arterial pressures (mm Hg) in high and normal salt rats in
the presence and absence of angiotensin II.

 

 

Ang II 5nglmin

97 1

Recovery from
Ang II

.L

 

’15 20

E 15 ~

E Control
v 10 _

0.

§

5 r11
8:

c -5‘

.‘c‘

o '10 ‘

5 -15-

a:

E -20

 

 

 

 

 

T

 

 

T

 

 

 

C1 A1 A5 A10 A15 R1 R3 R5

Protocol Day

Figure 3.6 Changes in mean arterial pressure (mm Hg) between daily control
pressures and pressures averaged over 35-50 minutes post-injection of
PD156707 in high salt rats (n= 8).

65

 

20

 

Control Ang II 5nglmin Recovery from
15 1 Ang ll

1;]

 

 

 

 

 

%

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Mean Change in MAP (mmHg)
O

 

 

 

 

-20 . . , . . r . .
C1 A1 A5 A10 A15 R1 R3 R5
Protocol Day

Figure 3.7 Changes in mean arterial pressure (mm Hg) between daily control
pressures and pressures averaged over 35-50 minutes post-injection of
PD156707 in normal salt rats (n=8).

66

 

Effectiveness of ABT-627 Tested in Endothelin-1 Induced Model of

Hypertension

Rationale: At the time this study was conducted, the ETA receptor antagonist,
ABT-627, had been characterized mainly on the basis of in vitro pharmacology
(Opgenorth et al., 1996). Since our studies all involve whole animal experiments,
we needed to demonstrate that this drug would be both potent and efficacious
enough to suit our in vivo purposes. ABT-627 was tested in an ET-1 induced
model of hypertension. This model is both ET-1 and salt-dependent. We sought
to determine the efficacy, time course and dose of ABT-627 that would produce a
long-lasting decrease in the mean arterial pressure in rats receiving ET-1

infusions and high salt intake.

Protocol: The rats were maintained on high salt intake (n=3, 6 mEq/day). BP
and HR were measured for two control days (C1 and C2) before the drug
treatment period. All rats received 8 pmollkglmin ET-1 over a 24 hour time
Period prior to the concomitant administration of ABT-627 (2 mglkglday) for 24
hours. The antagonist was first administered as a bolus injection into the facial
vein catheters of the rats (n = 3 rats per experimental group) and then in the
drinking water. BP and HR were recorded each morning of the experiment, as

previously described. ET-1 infusion was continued for 24 hours after drug-

67

treatment was stopped and the rats were allowed 1 day to recover from the

infusion.

Results: Infusion of ET-1 raised BP significantly in all rats, producing a mean
arterial pressure (MAP) of 158 mmHg after the first day of ET-1 infusion in these
rats compared to an initial control MAP of 106 mmHg (figures 3.8). The 2
mglkglday dose of ABT-627 significantly lowered BP by approximately 37 mmHg,
as demonstrated by repeated measures ANOVA and Dunnetts multiple
comparisons test. Since the hypertension in these high salt animals was induced
by administration of ET-1, the drop in MAP after administration of ABT—627
clearly indicates that this antagonist will work to reverse the actions of ET—1 in

vivo.

68

 

 

+0

 

 

200

.3
on
O

.3
a
O

MAP (mm Hg)
:3 3‘
O O

.3
O
O

 

ET-1 infusion

 

 

ABT-

 

 

 

627

 

 

on
O

I

C1

Figure 3.8 Line plot shows hemodynamic response to infusion of endothelin-1
(ET-1) at 8 pmollkglmin over a 72 hour time period prior to, during, and after

I

C2

I

A1

I

A2
Protocol Day

I

A3

administration of the ETA selective antagonist, ABT-627, 2mg/kg/day in drinking
water. The horizontal bar from protocol day C1 to A2 depicts ET-1 infusion
period. The horizontal bar from protocol day 02 to A2 depicts ABT-627

administration. Mean arterial pressure (MAP) was significantly increased on day
C2 compared to day C1, and was significantly decreased on day A1 compared to

day C2.

69

 

 

we
a S;
W

01h

 

 

 

Effectiveness of ABT-627 and Minoxidil Tested in DOCA-Salt Model of

Hypertension

Rationale: After demonstrating that ABT-627 (2 mglkg/day) was efficacious in an
in vivo ET-dependent model (see above), it was next desirable to test this dose
of the drug in a well-established chronic model of hypertension. Therefore, ABT-
627 was tested in the decxycorticostercne acetate salt (DOCA-salt) model of
hypertension, as was done with PD156707 (see above). Since DOCA-salt is
considered to be the best established model of the role of ET-1 in hypertension,
we sought to determine whether the ETA receptor antagonist that would produce
a sustained decrease in MAP in DOCA-salt rats. To further elucidate the
mechanism by which ABT-627 acts to decrease BP in this salt—dependent model
of hypertension, I also tested a well-defined direct vasodilator, minoxidil, as a

positive control.

Protocol: Blood pressure and heart rate were recorded for two control days
before the rats received the antihypertensive drugs. ABT-627 (2 mglkglday)
was tested by administering a bolus injection of the drug into the femoral vein
catheters of the rats (n = 5 rats) followed by administration of ABT-627 at this
dose in the drinking water for five days. Minoxidil (200 mg/L) was administered
in the drinking water for five days (n=3). After the fifth day of drug treatment, the

BP and HR were recorded for two recovery days.

70

Results: In the DOCA-salt model, ABT—627 produced a significant and sustained
decrease in blood pressure (~40 mmHg) with a peak effect occurring within 24
hours (figure 3.9). Since ET-1 plays an established role in DOCA-salt
hypertension, and ABT-627 chronically lowered BP in this model, we conclude
that this antagonist is efficacious at this dose in lowering BP in this chronic model
of high blood pressure.

Similarly, minoxidil also significantly lowered BP in DOCA-salt rats (~50 mmHg)
for the duration of the drug treatment period (figure 3.9). The fact that the time
course of the hypotensive effect of ABT-627 resembled that of a known direct
vasodilator (minoxidil) suggests, but does not prove, that these agents may act

by similar mechanisms.

71

 

 

 

 

 

240
220 + ABT-627,2mglkglday

 

200 4
1801
160 4
140 1
120 -

MAP (mmHg)

 

 

 

 

 

100 . . . . s . . .
C1 A1 A5 R1

 

 

 

 

240
220 d
200 J
180 ~
160 -
140 -
120 .

Minoxidil, 20 mg! glday

 

MAP (mm Hg)

 

 

 

 

 

100 I I I a - I I I
C1 A1 A5 R1
Protocol Day

Figure 3.9 Line plots show hemodynamic responses of DOCA-salt rats, 4-5
weeks post implant, to 5 day administration of either ABT-627 or minoxidil. The
horizontal bar from CZ to A5 depicts period of drug administration. Mean arterial
pressure (MAP) was significantly lowered during the 5-day drug administration
period, A1 -A5, compared to day CZ for both drugs.

72

The Effect of ABT-627 on Angiotensin Il-lnduced Hypertension

Rationale: In a previous study, the ETA receptor antagonist, PD156707, did not
produce a change in blood pressure in rats with Ang II induced hypertension.
Since PD156707 is a short acting antagonist, it is possible that there was not
enough time for complete antagonism of all ET-1 actions affecting blood
pressure. Direct vasoconstriction by ET-1 takes 10-30 minutes to reach a
maximum, while other effects on blood pressure (eg., sympatho-excitation, fluid
volume changes, vascular remodelling) may take much longer to be expressed.
Therefore we next turned to a drug with a more prolonged duration of action at
ETA receptors. ABT—627 is highly potent, efficacious, orally bioavailable, and has

a half-life of over six hours in the rat (Abbott Laboratories, 1998).

Protocol: Rats were maintained on high salt intake (n=2 per dose, 6 mEq/day).
After recording BP and HR for two control days, Ang II was continuously infused
i.v. at a rate of 5 nglmin for 15 days. On days 5-10, the rats received the
selective ETA receptor antagonist ABT-627 in their drinking water at doses of 0.5,
1, 2, and 7 mglkg/day. BP and HR recordings were taken each day of the

protocol.

Results: ABT-627 produced a dose-dependent reduction of Ang II induced

hypertension in rats on high salt intake (figure 3.10). This response occurred

73

within 24 hours at doses 2 2 mglkglday. These results show that ABT-627 is

effective at chronically reversing Ang II induced hypertension and suggest an

important role for ETA receptor activation in Ang II induced hypertension.

74

 

 

 

 

 

     

 

 

 

 

 

 

 

 

+ 7 mglkglday, n=2
0.. 2 mglkglday, n=2
—v— 1 mglkglday, n=2
—v— 0.5 mglkglday, n=2
170
0 Ang"
‘5 160 .
g 150 q ABT-627 7"\
a. 14o~ / V
" V
E 130I Foo. .o
g l _ \ o
< 120 . ’5. iii-3 v\v
: , v~ .
3 110 P x.
5 100 . o/ ’
90.....--.........
C1 A1 A5 A10 A15

Protocol Day

Figure 3.10 Mean changes in mean arterial pressure (mmHg) with increasing
doses of ABT-627 in the presence and absence of angiotensin II in high salt rats.

75

Effect of ABT-627 on Acute Sarafotoxin-Gc Dose Response Curve

Rationale: ABT-627, a reported ETA selective receptor antagonist, is an
experimental compound that has not yet been fully characterized. It was
therefore desirable to demonstrate the receptor selectivity of this drug. To do so,
we utilized an ETB selective receptor agonist, sarafotcxin 6c ($60), to determine
whether ABT—627 inhibits either depressor or pressor responses to ETB receptor
activation. We hypothesized that ABT—627 would not shift the acute dose

response curve for S6c.

Protocol: On the first day of this study, 860 was injected (over time frames of a
few seconds) at progressively larger doses. This resulted in a depressor
response within a few seconds, followed by a sustained pressor response lasting
up to 30 minutes. Peak depressor and pressor responses were recorded for
each dose. This resulted in two dose response curves to S6c. After generation
of these curves, the rats received ABT-627 in their drinking water (~2 mglkglday)
for 72 hours. At this time, each rat again received 860 to produce a second set

of acute dose response curves.

Results: ABT-627 produced no shift in either the acute depressor or pressor

dose response curves for S60 (figure 3.11). Since S60 is a selective ETB

76

receptor agonist, we can conclude that ABT-627 at ~2 mglkglday does not block

ETB receptors mediating either decreases or increases in arterial pressure.

77

 

—O— Control Pressor Response, n=5
+ ABT-627 Pressor Response, n=5
—v— Control Depressor Response, n=5
+ ABT-627 Depressor Response, n=5

 

 

 

 

 

AMAP (mm Hg)
8

-10 - \

V

 

 

 

 

0 100 200 300 400 500 600
Dose of Sarafotoxin 6c (pmollkg)

Figure 3.11 Line plots show hemodynamic response to acute bolus IV injection
of sarafotcxin 60 (1-500 pmollkglml) in rats on high salt intake. MAP, mean
arterial pressure.

78

Effect of ABT-627 on Acute Endothelin-1 Dose Response Curve

Rationale: The previous experiment demonstrated that ABT-627 does not block
the blood pressure effects of the ETB receptor antagonist, 86c, at ETB receptors.
To further characterize the receptor selectivity and efficacy of this drug, it would
be desirable to test it against an ETA selective receptor agonist. Since no such
agonist exists, we opted to test ABT-627 against bolus injections of ET-1, which
activates both ETA and ETB receptors. We hypothesized that the depressor
effect of ET-1 (ETB mediated) would remain unchanged and the pressor effect

(ETA and ETB mediated) would be significantly reduced by ABT-627.

Protocol: On the first day of this study, larger bolus doses of ET-1 were injected
(over time frames of a few seconds) progressively. This resulted in two acute
dose response curves, as previously described for S6c (see above). After
generation of these curves, the rats received ABT-627 in their drinking water (~2
mglkglday) for 72 hours. At this time, each rat again received ET-1 to produce a

second set of dose response curves.

Results: ABT-627 produced no shift in the acute ETB mediated depressor dose
response curve for ET-1 (figure 3.12). These data, taken together with the 86c
data, further demonstrate that ABT-627 at ~2mglkglday does not block ETB

receptors. ABT-627 did reduce the acute ETA/ETB mediated pressor dose

79

response curve for ET-1. This finding, in the context of the above data, indicates
that ABT-627 is acting to block the ETA mediated portion of the pressor

response.

80

 

—O— Control Pressor Response, n=4
+ ABT-627 Pressor Response, n=4
—v— Control Depressor Response, n=4
+ ABT-627 Depressor Response, n=4

50 - L/%

 

 

 

 

 

AMAP (mm Hg)
N
O

 

\_\
-10 ~ ‘ '1'

 

 

 

 

0 100 200 300 400 500 600
Dose of Endothelin-1 (pmollkg)

Figure 3.12 Line plots show hemodynamic response to acute bolus IV injection
of endothelin-1 (OI-500 pmollkg) in rats on high salt intake. MAP, mean arterial
pressure.

81

Effect of A-192621 on Acute Sarafotoxin-6c Dose Response Curve

Rationale: A-192621, a reported ETB selective receptor antagonist, is an
experimental compound that has not yet been fully characterized (Abbott
Laboratories, 1998). It was therefore desirable to demonstrate the receptor
selectivity of this drug. To do so, we utilized an ETB selective receptor agonist,
sarafotcxin 6c (86c), to determine whether A-192621 inhibits either depressor or
pressor responses to the ETB receptor activation. We hypothesized that A-

192621 would shift both the pressor and depressor responses to $60.

Protocol: On the first day of this study, each rat was injected (over time frames
of a few seconds) with cumulative doses of 86c. This resulted in a depressor
response within a few seconds, followed by a sustained pressor response lasting
up to 30 minutes. Peak depressor and pressor responses were recorded for
each dose. This resulted in two dose response curves to S6c. After generation
of these curves, the rats received bolus intravenous injections of A-192621 (12
mglkg). Additional dose response curves were generated 1, 2, and 6 hours after

antagonist treatment.

82

Results: A-192621 significantly inhibited both the the S6c mediated depressor
and pressor responses in a time-dependent manner (figure 3.13). The depressor
response, in particular, was almost completely eliminated for up to 6 hours after
A-192621 administration. Since 86c is a selective ETB receptor agonist, we can
conclude that A-192621 administered at 12 mglkg does effectively block ETB

receptors mediating either decreases or increases in arterial pressure.

83

 

—0— Control, n=7
-O- 1Hourn=5
”:1- 2Hourn=5
oA- 6Hour,n=5

 

 

 

40~

AMAP (mmHg)
or
O

 

 

 

62.5125.0 250.0 500.0

 

30
20 4

101 z:iéii33::2"“§i::i:::=ig
0 ........
.10.
-20.
-304 4
-40

AMAP (mmHg)

 

 

 

 

 

62.5125.0 250.0 500.0
Sarafotoxin 6c (pm ollkg)

Figure 3.13 Line plots show hemodynamic responses to acute bolus IV
injections of sarafotcxin 6c (doses = 62.5 to 500 pmollkg) in rats on high salt
intake at various time points after receiving the ETB selective receptor antagonist,
A-192621 at 12 mglkg iv injection. *Significant (p<0.05) difference in mean
arterial pressure from control value.

 

Effect of A-192621 on Acute Endothelin-1 Dose Response Curve

Rationale: The previous experiment demonstrated that A-192621 does
effectively block the blood pressure effects of the ETB receptor antagonist, 86c,
at ETB receptors. To further characterize the receptor selectivity and efficacy of
this drug, it was next be desirable to test it against an ETA selective receptor
agonist. Since no such agonist exists, we opted to test A-192621 against bolus
injections of ET-1, which activates both ETA and ETa receptors. We
hypothesized that the depressor effect of ET-1 (ETB mediated) would be
significantly inhibited and the pressor effect (ETA and ETB mediated) would be
slightly reduced by A-192621. Since we were primarily interested in effects of
this antagonist on the ETA mediated pressor response, we did not measure the

depressor responses in this study.

Protocol: On the first day of this study, each rat was injected (over time frames
of a few seconds) with cumulative bolus doses of ET-1. This resulted in two
acute dose response curves, as previously described for 860. Two hours after
generation of these curves, the rats received bolus intravenous injections of A-

192621 (24 mglkglday) to produce a second set of dose response curves to 860.

Results: A-192621 produced no shift in the acute ETA/6T3 mediated pressor

dose response curve for ET-1 (figure 3.14). This finding, in the context of

85

previously described data, indicates that A-192621 functions to selectively block

the ETB mediated portion of the pressor response.

 

 

 

 

 

 

 

+ COHtI'OI, n=5
..... DW1-2 Hour, ":5
50
4o -
a 1.
I ................... E]
E 30 d .......................
é a
i 20 .
E
4
10 I
0

 

 

 

62.51250 250.0 500.0

Endothelin-1 (pmollkg)

Figure 3.14 Line plots show hemodynamic response to acute bolus i.v. injection
of endothelin-1 (62.5 — 500 pmollkg) in rats on high salt intake, in the presence
and absence of A-192621.

87

Effectiveness of A-182086 Tested in Endothelin-1 Induced Model of
Hypertension

Rationale: At the time this study was conducted, the non-selective ETA/3 receptor
antagonist, A-182086, had been characterized mainly on the basis of in vitro
pharmacology (Abbott Laboratories, 1998). Since our studies all involve whole
animal experiments, we needed to demonstrate that this drug would be both
potent and efficacious enough to suit our in vivo purposes. A-182086 was tested
in an ET-1 induced model of hypertension. This model is both ET-1 and salt—
dependent. We sought to determine the efficacy, time course and dose of the
combined ETA/a receptor antagonist that would produce a sustained decrease in

the mean arterial pressure in rats receiving ET-1 infusions and high salt intake.

Protocol: The rats were maintained on high salt intake (n=3, 6 mEq/day). BP
and HR were measured for two control days (C1 and 02) before the drug
treatment period. All rats received 8 pmollkglmin ET-1 over a 24 hour time
period prior to the concomitant administration of A-182086 (24 mglkglday) for 24
hours. The antagonist was administered as two bolus intra-arterial injections (n =
3 rats per experimental group). BP and HR were recorded each morning of the
experiment, as previously described. ET-1 infusion was continued for 24 hours
after drug-treatment was stopped and the rats were allowed 1 day to recover

from the infusion.

88

Results: Infusion of ET-1 raised blood pressure in all rats, producing a control
mean arterial pressure (MAP) of ~150 mmHg on the first morning after initiation
of ET-1 infusion compared to a control MAP of 108 mmHg (figure 3.15). There
was no significant change in BP with administration of A-182086. Since the
hypertension in these high salt animals was induced by administration of ET-1,
and we have shown that a selective ETA receptor antagonist produces a fall in
MAP in this model of hypertension, the failure of MAP to decrease after injection
of A—182086 indicates that this antagonist is less effective at blocking the pressor
actions of ET-1 than selective ETA receptor antagonism alone. Since selective
ETB receptor antagonism causes elevated blood pressure in animals on high salt
intake (Pollock, 2000), this result with A-182086 suggests combined ETA and ETB
receptor antagonism will be less effective at lowering MAP in ET—1 dependent

forms of hypertension under high salt compared to normal salt conditions.

89

 

—o— n=9

 

 

 

 

200
ET-1 infusion

.3
on
O

 

A-1821086

. ~t

 

.3
a:
O

 

MAP (mm Hg)
.2: I
O O

.3
O
O

 

 

 

 

 

 

 

I I I

C1 C2 A1 A2 R1 R2
Protocol Day

Q
C

Figure 3.15 Line plot shows hemodynamic response to infusion of endothelin-1
(ET-1) at 8 pmollkglmin over a 72 hour time period prior to, during, and after
administration of the non-selective ET-1 receptor antagonist, A-182086
(24mglkglday) lV injection. The horizontal bar from protocol day C1 to A2
depicts ET-1 infusion period. The horizontal bar from protocol day 02 to A2
depicts A-182086 administration.

90

In summary, I was able to personally determine the appropriate dose,
maximal effect, receptor selectivity and time course of action for each of the
ET-1 receptor antagonists I used in this thesis project. Due to the lack of
effect of PD156707 in Ang II induced hypertension, likely due to its very
short plasma half-life, I decided not to pursue further studies with this drug.
The three ET-1 receptor antagonists from Abbott Laboratories were all
shown to have the requisite receptor selectivity and duration of action to be
useful in modulating blood pressure in my chronic in vivo studies. They also
had the additional benefit of being similar in chemical structure, allowing for

easier comparison of the three drugs.

91

Chapter 4

Role of ETA Receptors in Experimental Angiotensin II Induced

Hypertension in Male Rats

Jennifer R. Ballew and Gregory D. Fink
Department of Pharmacology and Toxicology, Michigan State University

East Lansing, MI 48824

This chapter submitted as a manuscript to:

Hypertension

92

INTRODUCTION

The renin angiotensin system (RAS) is an important factor in human
hypertension, even though most hypertensives do not exhibit elevated renin or
angiotensin II (Ang II) levels. Changes in sensitivity to the pressor actions of
Ang II may explain this anomaly. Animals receiving continuous small-dose
infusions of Ang II develop progressive hypertension (Ang II induced
hypertension) after a period of normotension (Koletsky et al., 1966). Our
laboratory, and others (Brown et al., 1979; Melaragno and Fink, 1996a), have
proposed that changes in sensitivity to this slow pressor effect of Ang II could
explain apparent “renin-dependent" forms of hypertension not associated with
marked RAS activation. One factor known to increase sensitivity to Ang II
induced hypertension is high salt intake (Muirhead et al., 1975; Simon et al.,
1998), but the mechanism is not established.

Our laboratory has previously reported that hypertension in animals
receiving continuous low dose infusions of endothelin-1 (ET-1) is also salt
sensitive (Mortensen and Fink, 1992a). Furthermore, there are several known
relationships between Ang II and ET-1. Ang II stimulates the ET-1 promotor to
cause the upregulation of ET-1 synthesis (Dohi et al., 1992; Imai et al., 1992;
Gray, 1995), ET-1 acts as an amplifier of the pressor effects of Ang II (Dohi et
al., 1992), and ET-1 partially mediates Ang II induced vascular hypertrophy
(D’Uscio et al., 1995). Most pressor actions of ET-1 are caused by activation

of the ETA subtype of ET-1 receptors (Haynes et al., 1996). Therefore, in the

93

present study I assessed the hypothesis that Ang II induced hypertension is
enhanced during high salt intake through stimulation of ETA receptors by

endogenous ET-1.

Methods

Male Sprague—Dawley rats (Charles River Laboratories, Portage, MI)
weighing 350-400 grams were used in these experiments. Upon arrival at our
facility, rats were maintained according to standards approved by the Michigan
State University All-University Committee on Animal Use and Care. All
experimental procedures were carried out in accordance with the “Guiding
Principles in the Care and Use of Animals” of the American Physiological
Society. Rats were acclimatized for at least two days prior to surgical
procedures in clear plastic boxes and were allowed access to standard rat
chow (Teklad 2215 Rodent Diet W 8640, Madison, WI) and tap water ad
libitum.
Surgical Procedures

All surgical procedures were performed after administration of
pentobarbital sodium (Nembutal®, Abbott Laboratories, N, Chicago, IL), 50
mglkg i.p., and atropine sulfate (Sigma, St. Louis, M0), 0.2 mg, i.p. If
necessary, anesthesia was supplemented using methohexital sodium
(Brevital‘b, Eli Lilly, Indianapolis, IN), 5-10 mglkg, i.v. At least two days prior
to initiation of the experimental protocol, rats were catheterized via the femoral
artery and vein as previously described (Melaragno and Fink, 1996a).

Chronic Rat Maintenance and Measurements
The metabolism cages housing the rats during the experiments were

located in a climate-controlled room with a 12-hour light-dark cycle. The rats

95

were allowed access to distilled water and sodium-deficient rat chow (Teklad
170950, Madison, WI) ad libitum, depending on the experimental protocol.
Half the rats were maintained on a daily sodium intake of 2 mEq/day (normal
salt intake), and the remaining half were maintained on 6 mEq/day (high salt
intake). All sodium chloride was delivered by continuous intravenous infusion.
Catheters were flushed daily to maintain patency and the arterial catheters
were filled with a heparinized sucrose solution and occluded when not in use.
Mean arterial pressure (MAP) and heart rate (HR) were recorded via the

femoral arterial catheter each morning of the protocol between 8:00 and 11:00
am. The arterial catheters were connected to external pressure transducers
(TXD-300; Micro-Med, Louisville, KY, USA) that were first zeroed at the level
of the rat’s heart. The transducers are connected to digital pressure monitors
(BPA-200 Blood Pressure Analyzer, Micro-Med, Louisville, KY, USA) that
output directly to a computerized digital pressure monitoring system. Data
were collected once every second for 15-30 minutes. The daily value was
determined by the average of the one-second recordings taken over the last
five minutes of the recording session.
Experimental Protocol

After blood pressure, heart rate and other variables were recorded for two
control days, Ang II was continuously infused i.v. at a rate of 5 nglmin for 15
days. On day 5, the rats received a one-time bolus injection of the selective
ETA receptor antagonist, ABT-627 (2 mglkg, i.v.; Abbott Laboratories, Abbott

Park, IL), followed by ABT-627 in their drinking water at a concentration (20

mg/L) to deliver an approximate dose of 2 mglkglday for days 5-10. The dose
of ABT-627 was chosen based on preliminary studies in which the antagonist
was tested against chronic pressor actions of ET—1 and Ang II (data not
shown). BP and HR recordings were taken continuously for one hour
immediately following the bolus injection of ABT-627 and then once each
subsequent day of the protocol. Urine (daily) and blood (control day 2 and
Ang II infusion days 5, 7, 10, 14) samples were collected and electrolyte and
metabolite concentrations were measured using ion selective electrodes
(Nova Electrolyte 16+ Analyzer, Nova Biomedical). Plasma volumes were
determined periodically by Evan’s Blue dye dilution, and blood volume was
calculated according to the standard formula. Daily electrolyte balance, water
balance, and other variables were calculated as previously described
(Melaragno and Fink, 1996a).

In a separate set of experiments, normal conscious male Sprague-Dawley
rats were catheterized and housed as described above. After recording BP
and HR for two control days, each rat received injections (over a time frame of
a few seconds) of increasingly large bolus doses of Ang II. Each dose was
separated by 10-15 minutes. After generation of this acute Ang II dose
response curve, the rats received ABT-627 in their drinking water (~2
mglkglday) for 72 hours. At this time, each rat again received Ang II to

produce a second acute dose response curve.

97

Statistical Analysis
Results are expressed as mean : SEM. For all data, within- and

between-group differences were analyzed using mixed-design ANOVA. Post
hoc comparisons were performed by testing for simple main effects. Criterion

for statistical significance was a probability level of less than 0.05.

98

Results
Mean Arterial Pressure and Heart Rate

As shown in figure 4.1, continuous i.v. infusion of Ang II (5 nglmin) induced
a signifimnt increase in MAP compared to control period values. No
significant change in MAP was observed in rats that did not receive Ang II.
The effect of Ang II was larger and occurred more rapidly in rats on high salt
intake (5-day average = 18 I: 3.1 mmHg) compared to rats on normal salt
intake (5—day average = 11 i 3.2 mmHg), but the difference between the two
groups was not statistically significant (p = 0.13). With chronic oral dosing in
rats on high salt intake receiving Ang II, ABT-627 (~2 mglkg/day) produced a
sustained reduction in MAP to levels not significantly different from that of
normotensive control rats within 24 hours. ABT-627 did not affect MAP in
normotensive control rats on high salt intake.

In rats on normal salt intake, ABT-627 also decreased MAP in rats receiving
Ang II, but the effect was significantly less (5—day average = -15 i 2.3 mmHg)
than in rats on high salt intake (5-day average = -22 i 2.1 mmHg; p = 0.0391).
Furthermore, in normal salt rats receiving Ang II, ABT—627 did not reduce MAP
to the level of normotensive control rats except on the first day of treatment.
Also, by day 5 of ABT-627 treatment, MAP in Ang II infused rats on normal salt
intake was no longer significantly less then pretreatment values. ABT—627 did

not reduce MAP in normotensive control rats on normal salt intake. ABT-627

99

 

+ High salt+ Ang II, n=9

 

 

 

 

 

 

MAP (mmHg)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

High salt —O—Highsaltcontrol,n=5
160
Angll infusion
14° ‘ ABT-627
120 I W
3o: *m
100 I
so -WW
C1 A1 A5 A10 A15 R2
+Normalsalt+Angll,n=9
Normalsalt —A—-Normalsaltcontrol,n=5
A 160
° Angll infusion
I
E 140 ‘ ABT-627
E
n- 120 '1
<
5 100 . g:
30 .a T . . . . a

 

 

 

 

 

C1 A1 A5 A10 A15 R2
ProtocolDay

Figure 4.1 Line plots show hemodynamic responses to infusion of angiotensin II
(Ang II) at 5.0 nglmin and administration of ABT-627 at 2 mglkglday in rats on
either high sodium (top panel) or normal sodium (bottom panel) intake. The
horizontal bar from protocol day A1 to A15 depicts Ang II infusion period. The
horizontal bar from protocol day A5 to A10 depicts ABT-627 administration. In
rats on high salt intake, MAP values on days A2-5 and A1345 were significantly
higher than the MAP average of days 01.2. In rats on normal salt intake, MAP
values on days A2-5 and A12-14 were significantly higher than the MAP average of
days C1-2. *Significant (p<0.05) difference in mean arterial pressure from
protocol day A5. + Significant (p<0.05) difference in mean arterial pressure
between normal salt + Ang II group and normal salt control group.

100

 

did not produce a significant change in heart rate in any of the four groups
studied (data not shown).

On day 5 of Ang II infusion, acute intravenous injection of ABT-627
gradually and significantly lowered MAP over a 1-hour time frame in
hypertensive Ang II infused rats, but had no significant effect in normotensive
controls (fig 4.2).

Pressor responses to acute bolus doses of Ang II were identical before and
after administration of ABT-627 (given in the drinking water, ~2 mglkglday for
3 days) (fig 4.3).

Metabolic Parameters

ABT-627 produced a slight retention of water in rats with Ang II induced
hypertension on normal salt intake, but did not affect water balance in any
other group (fig 4.4). Likewise, the ETA receptor antagonist produced slight
retention of sodium in rats with Ang II induced hypertension on normal salt
intake, but did not affect sodium balance in any other group (fig 4.5). In rats
on high salt intake with Ang II induced hypertension, a modest progressive
decrease in blood volume was observed across the duration of the protocol.
No changes in blood volume were seen in the other three groups (data not
shown). No significant changes in routine blood chemistries (e.g. plasma
sodium, potassium, glucose, osmolality, BUN, or creatinine concentrations)

were observed in any group throughout the protocol (data not shown).

101

‘5' 140
I
E 130
E
- 120
A
< 110
E
100
so
'5' 140
I
a 130
E 120
n.
110
<
E 100
so

 

_ +Highsalt+Angll,n-9
Hrghsalt —O—Highsaltcontrol,n=5

 

 

 

 

Normalsalt —A—Normalsaltcontrol,n=5

 

 

 

 

r

 

0 1O 20 30 40 50 60 70
+ Norm alsalt+ Ang II, n-9

 

 

 

 

 

 

 

 

 

0 10 20 30 40 50 60 70
Time (m lnutes)

Figure 4.2 Line plots show hemodynamic responses to bolus intravenous
injection of ABT-627 at 2 mglkg in rats on high sodium (top panel) or normal
sodium (bottom panel) intake. MAP, mean arterial pressure. *Significant
(p<0.05) difference from time of injection.

102

 

+ After ABT-627, n=5
—O— Control, n=5

 

 

 

 

A MAP (mmHg)
N OJ h 01 O) N as
O O O O O O O

 

Control resting MAP = 109 t 5 mmHg
ABT-627 resting MAP = 95 :l: 2 mmHg

A
O

 

 

O

 

0 20 4O 60 80 100 120
Ang II Dose (nglkg)

Figure 4.3 Effects of acute intravenous injection of angiotensin II (Ang II) on
mean arterial pressure in rats on high sodium intake, before and after
administration of ABT-627 at 2 mglkg/day for 72 hours.

103

 

+ High sa|t+ Ang II, n=9

 

 

 

 

 

 

High salt —0— High saltcontrol, n=5
50
Ang II infusion
40 ~
ABT-627
30 ~

20 4 gm M9

,,. W we

 

 

 

 

 

 

 

Water Balance (m lslday)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

C1 A1 A5 A10 A15 R2
2
“5 Normalsalt + Normalsalt+Angll,n=9
E —A— Normal salt control, n=5
m
— 50
E Ang II infusion
a 40 -
o ABT-627
:
2 30 ‘ *
a
m 20 1 §%
5
2
u 10 .
3
0-.
C1 A1 A5 A10 A15 R2

Protocol Day

Figure 4.4 Line plots show water balance responses in milliliters per day
(mls/day) to infusion of angiotensin II (Ang II) at 5.0 nglmin and administration of
ABT-627 at 2 mglkglday in rats on either high sodium (top panel) or normal
sodium (bottom panel) intake. The horizontal bar from protocol day A1 to A15
depicts Ang II infusion period. The horizontal bar from protocol day A5 to A1 0
depicts ABT-627 administration. *Significant (p<0.05) difference in water balance
from protocol day A5.

104

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

3‘ + High salt + Ang II, "=9
2 High salt —0— High salt control, n=5
0' 4
"5' Ang II infusion
11' 3 .
o A§T-627
C
2 2 .
fl
.. i\
E 1 «
.2
1:
o .
a) 0
C1 A1 A5 A10 A15 R2
3’ N ' It + Normal salt-r- Ang ll, ":9
E orma sa —A— Normal salt control, n=5
5' 4
“El Ang II infusion
3 3 , ABT-627
r:
2 2 ~
0
m *
E 1 « *
.2
u
i; 0+ H
C1 A1 A5 A10 A15 R2

Protocol Day

Figure 4.5 Line plots show sodium balance responses in milliequivalents per
day (mEq/day) to infusion of angiotensin II (Ang II) at 5.0 nglmin and
administration of ABT-627 at 2 mglkglday in rats on either high sodium (top
panel) or normal sodium (bottom panel) intake. The horizontal bar from protocol
day A1 to A15 depicts Ang II infusion period. The horizontal bar from protocol
day A5 to A10 depicts ABT-627 administration. *Significant (p<0.05) difference
in sodium balance from protocol day A5.

105

Discussion

Previous experiments show that ET-1 receptor blockade begun prior to
the start of Ang II infusion attenuates or eliminates Ang II induced
hypertension (D’Uscio et al., 1997; Rajagopalan et al., 1997; Herizi et al.,
1998). These results strongly support involvement of ET—1 in Ang II induced
hypertension. Earlier studies differ from the current one, however, in that they
all used higher rates of Ang II infusion (200 nglkglmin, so), used only rats on
normal salt intakes, and produced a more severe level of hypertension (+ 35-
85 mmHg over control levels) than observed here (+11-18 mmHg over control
levels). Further, no published studies addressed potential renal effects of ET-
1 receptor blockade in this model of hypertension.

The new findings in this study are: the selective ETA receptor antagonist,
ABT-627, entirely normalized blood pressure in rats with pre-existing Ang II
induced hypertension; the reduction in mean arterial pressure was larger and
longer-lasting in rats on high salt intake compared to rats on normal salt
intake; and changes in sodium and water excretion were not consistent with
the kidney being the primary target of the antihypertensive response to ABT-
627. I also showed that acute pressor responses to Ang II were unaffected by
ABT-627 treatment, indicating that this drug does not block Ang II receptors.
My results confirm that ET-1 acting at ETA receptors plays an important role in
Ang II induced hypertension, and further indicate that it’s role is magnified in

rats on high salt intake.

106

ET-1 is an accepted etiologic factor in several common models of
hypertension, most of which are salt-sensitive (Schiffrin, 1999). Although the
severity of the hypertension caused by Ang ll infusion in this particular study
was not statistically different between the high and normal salt intake groups,
Ang II infusion in the rat is well-known to be a salt-dependent model of
hypertension (Muirhead et al., 1975; Kanagy et al., 1990; And et al., 1991;
Csiky and Simon, 1997; Simon et al., 1998). Elevated salt intake may
increase the synthesis of ET-1, amplify the pressor actions of ET-1, or do both.
Results of published studies suggest no change in vascular ET-1 formation in
rats or humans receiving high salt intakes (Rajagopalan et al., 1997; Herizi et
al., 1998). Our laboratory has previously shown, however, that hypertension
produced by exogenous infusion of a low dose of ET-1 is markedly enhanced
by high salt intake (Mortensen and Fink, 1992a). Thus, salt most likely
amplifies the pressor actions of ET-1 rather than increasing synthesis and
release of the peptide in vascular endothelial cells.

Other investigators have reported that chronic Ang II infusion in rats
increased the formation of ET-1 in arteries (Imai et al., 1992; D'Uscio et al.,
1995; Moreau et al., 1997), although the effects of salt intake were not
examined. So tonic constriction of resistance arteries by endothelium-derived
ET-1 is a probable mechanism of elevated blood pressure in Ang II
hypertension. In this study I showed that ABT-627 significantly lowered mean
arterial pressure within one hour of bolus intravenous injection. This time

course is consistent with Ang II induced hypertension being dependent on ETA

107

receptor mediated arterial constriction. In addition to enhancing ET-1 release
from endothelial cells, Ang II could increase vascular reactivity to endogenous
levels of ET-1, although existing data do not support this hypothesis
(Rajagopalan et al., 1997; Herizi et al., 1998).

Reduction of sodium excretion by the kidney is a known cause of
hypertension development, and ET-1 has been shown to be an antinatriuretic
peptide under some circumstances (Kaasjager et al., 1997). A recent report
also demonstrated that chronic Ang II infusion in rats increases ET-1 gene
expression in the kidney (Barton et al., 1997; Barton et al., 1998). The current
study is consistent with an antinatriuretic role for ET-1 in Ang II induced
hypertension, since during ABT-627 treatment sodium excretion was
maintained constant while arterial pressure was lowered. The hypotensive
response to ABT-627, however, occurred rapidly and was not preceded by any
measurable increases in renal sodium or water excretion, or changes in
plasma electrolytes, or blood volume. Recent data of mine showed that a
thiazide diuretic was able to significantly reduce mean arterial pressure in rats
on high salt intake in the same protocol as described here (Ballew and Fink,
Chapter 6). But the antihypertensive response was delayed (1 -2 days) and
associated with negative salt and water balances. Thus, it is unlikely that
renal actions of ABT-627 caused the decrease in arterial pressure observed in
the hypertensive rats.

In summary, these data suggest that Ang II induced hypertension

depends primarily on increased arterial constriction produced by the actions of

108

ET-1 on ETA receptors, and that this effect is greater under conditions of
elevated salt intake. It is therefore conceivable that ETA selective receptor
antagonists will be effective antihypertensive agents in patients with salt-

sensitive forms of hypertension.

109

Chapter 5

Role of Endothelin ET; Receptor Activation in Angiotensin II Induced

Hypertension: Effects of Salt Intake

Jennifer R. Ballew and Gregory D. Fink
Department of Pharmacology and Toxicology, Michigan State University,

East Lansing, MI, 48823

This chapter submitted as a manuscript to:

American Journal of Physiology — Heart and Circulatory Physiology

110

Introduction

The overall goal of my research is to define the mechanisms responsible for
the salt-sensitivity of angiotensin II (Ang II) induced hypertension. Previous
work by others has implicated the endothelial derived peptide, endothelin-1
(ET-1), in the pathophysiology of Ang II induced hypertension (D’Uscio et al.,
1997; Rajagopalan et al., 1997; Herizi et al., 1998). Two receptor subtypes,
ETA and ETB, have been shown to mediate blood pressure effects of ET-1.
Activation of the ETA receptor can increase blood pressure by a variety of
mechanisms, including direct contraction of vascular smooth muscle and
alteration of the pressure-natriuretic function of the kidney (Haynes and Webb,
1998). Conversely, ETB receptor activation tends to lower blood pressure by
causing release of endothelial vasodilators and promoting renal loss of sodium
and water (Haynes and Webb, 1998). Both ETA selective and non-selective
ET-1 receptor antagonists have been shown to attenuate the development of
Ang II induced hypertension (D’Uscio et al., 1997; Rajagopalan et al., 1997;
Herizi et al., 1998), but the influence of salt intake was not investigated. I
recently reported that selective blockade of ETA receptors in rats with Ang II
induced hypertension caused a larger and more sustained fall in arterial
pressure when the rats were on a high versus a normal salt intake (Chapter 4).
I concluded that the salt sensitivity of Ang II induced hypertension is mediated
in part by endogenous ET-1 acting at ETA receptors. Recent studies showed

that a naturally occurring deletion of the ETB receptor gene (Gariepy et al.,

111

2000), and chronic blockade of ETB receptors with A-192621 cause salt-
sensitive hypertension in rats (Pollock, 2000). Therefore in this study I
conducted experiments to test the role of ETB receptors in the salt-sensitivity

of Ang II induced hypertension.

112

Methods

Animals: Male Sprague-Dawley rats (Charles River Laboratories, Portage, MI)
weighing 350-450 grams were used in these experiments. Upon arrival at our
facility, rats were maintained according to standards approved by the Michigan
State University All-University Committee on Animal Use and Care. All
experimental procedures were carried out in accordance with the “Guiding
Principles in the Care and Use of Animals” of the American Physiological
Society. Rats were acclimatized for at least two days prior to surgical
procedures in clear plastic boxes and were allowed access to standard rat
chow (Teklad 22/5 Rodent Diet W 8640, Madison, WI) and tap water ad
libitum.
Surgical Procedures: All surgical procedures were performed after
administration of pentobarbital sodium (Nembutal®, Abbott Laboratories, N,
Chicago, IL), 50 mglkg i.p., and atropine sulfate (Sigma, St. Louis, M0), 0.2
mg, i.p. If necessary, anesthesia was supplemented using methohexital
sodium (Brevital®, Eli Lilly, Indianapolis, IN), 5-10 mglkg, i.v. At least two
days prior to initiation of the experimental protocol, rats were catheterized via
the femoral artery and vein as previously described (Potter et al., 1997).
Chronic Rat Maintenance and Measurements: The metabolism cages
housing the rats during the experiments were located in a climate-controlled
room with a 12-hour light-dark cycle. The rats were allowed access to distilled

water and sodium-deficient rat chow (Teklad 170950, Madison, WI) ad libitum,

113

depending on the experimental protocol. Half the rats were maintained on a
daily sodium intake of 2 mEq/day (normal salt intake), and the remaining half
were maintained on 6 mEq/day (high salt intake). All sodium chloride was
delivered by continuous (24 hr/day) intravenous infusion. Catheters were
flushed daily to maintain patency and the arterial catheters were filled with a
heparinized saline solution and occluded when not in use.

Systolic, diastolic, and mean arterial pressures and heart rate were
recorded via the femoral arterial catheter each morning of the protocol between
8:00 and 11:00 am. The arterial catheters were connected to low-volume
displacement pressure transducers that were first zeroed at the level of the rat’s
heart. The transducers were connected to digital pressure monitors (Digi-Med
Blood Pressure Analyzer, Micro-Med, Louisville, KY) that provided input directly
to a computerized digital pressure monitoring system. Data were collected once
every second for 15-30 minutes. The daily value recorded was the average of
the one-second recordings taken over the last five minutes of the recording
session.

Experimental Protocol: After blood pressure, heart rate and other variables were
recorded for two control days, Ang II was continuously infused i.v. at a rate of 5
nglmin for 15 days. On day 5, the rats began receiving intra-arterial injections of
the selective ETB receptor antagonist, A-192621 (12 mglkg; Abbott Laboratories,
Abbott Park, IL), or the non-selective ET-1 receptor antagonist, A-182086 (12
mglkg; Abbott Laboratories, Abbott Park, IL) every 12 hours during Ang II

infusion days 5-10 (total dose = 24 mglkglday). The dose of the antagonists was

114

shown to be efficacious in preliminary studies in which the antagonists were
tested against acute and chronic pressor actions of ET-1 and the selective ETB
receptor agonist, sarafatoxin 60 (data not shown). Blood pressure and heart rate
recordings were obtained for one hour immediately following the first injection of
ET-1 receptor antagonist and then once each subsequent day of the protocol.
Urine (daily) and blood (control day 2 and Ang II infusion days 5, 7, 10, 14)
samples were collected and electrolyte and metabolite concentrations were
measured using ion selective electrodes (Nova Electrolyte 16+ Analyzer, Nova
Biomedical). Plasma volumes were determined periodically by Evan’s Blue dye
dilution, and blood volume was calculated according to the standard formula.
Daily electrolyte balance, water balance, and other variables were calculated as
previously described (Potter et al., 1997).

Statistical Analysis: Results are expressed as means 1; SEM. Within group
differences were analyzed using a repeated measures ANOVA. Between
group differences were analyzed using a one-way ANOVA. Post hoc
comparisons were performed using the protected least significant difference
test. Criterion for statistical significance was a probability level of less than

0.05.

115

Results

ETB receptor antagonism using A-192621

As shown in figure 5.1, during the first five days of Ang II infusion MAP
rose significantly in rats on high salt intake (115 :l: 3 to 130 i 7 mmHg) but was
not significantly increased in rats on normal salt intake (110 :t 3 to 116 i 3
mmHg). The increment in MAP was significantly larger in rats on high salt intake.
MAP did not change over the same time period in rats not receiving Ang II.
There was no change in water balance (figure 5.2). However, in rats on normal
salt intake there was a significant sodium retention on day 1 of Ang II infusion
followed by a significant loss of sodium on day 3 (figure 5.3). No significant
changes occurred in rats on high salt intake.

After measurements were completed on day 5 of the Ang II infusion
period, each rat received a bolus intra-arterial injection of the ETB receptor
antagonist, A-192621 (12 mglkg), and MAP was recorded continuously for the
next hour. MAP rose significantly in all four groups (figure 5.4). In rats on high
salt intake the increase was significantly greater in control rats (41.8 i 4.1
mmHg) than in rats receiving Ang II (21.3 1r 2.6 mmHg). In rats on normal salt
intake the increase was significantly less in control rats (21.9 i 2.3 mmHg) than
in rats receiving Ang II (36.5 1: 4.2 mmHg). The increase in MAP observed in
rats on high salt intake was significantly greater than that seen in rats on normal

salt intake, regardless of whether they received Ang II.

116

 

+ High salt+ Ang II, n=9
—O— High salt control,n=5

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

High salt
200
.. Angll infusion
g 180 ~ .A-192621
E +++++++
E 160 . * * * * * * +
*
.. 1:
0- 140 - * *
<
5 120 - W \ffi
(5+ +++++
100 -
C1 A1 A5 A10 A15 R2
+ Normalsalt+AnglI,n=9
Normalsajt —A— Normalsaltcontrol,n=5
200
‘5 Angll infusion
I 180 4
E A-192621
160 ~
0- 140 - * * +
< * * * *
2 120 -
g: a: * +4.
100 - + +
C1 A1 A5 A10 A15 R2

Protocol Day

Figure 5.1 Line plots show hemodynamic responses to infusion of angiotensin II
(Ang II) at 5.0 nglmin and administration of A-192621 at 24 mglkglday in rats on
either high sodium or normal sodium intake. The horizontal bar from protocol
day A1 to A15 depicts Ang II infusion period. The horizontal bar from protocol
day A5 to A10 depicts A-192621 administration. *Significant (p<0.05) difference
in mean arterial pressure from control day 02. ‘Significant (p<0.05) difference in
mean arterial pressure from protocol day A5.

117

30

25

20

15

10

Water balance

Normal salt

30

25

20

15

Water balance

10

High salt

 

+ High salt-I- Ang II, n=9
—O—— High saltcontrol,n=5

 

 

 

 

Angll

infusion

 

 

  

A-192621

 

 

 

 

 

 

C1 A1

A5

A10

1,

A15 R2

 

 

+ Normal salt + Ang ll, n=9
—A— Normal salt control, n=5

 

 

Ang II

infusion

 

 

 

A-192621

 

 

 

 

 

 

A5

T f

A10
Protocol Day

1

Figure 5.2 Line plots show water balance responses in milliliters per day
(mlslday) to infusion of angiotensin II (Ang II) at 5.0 nglmin and administration of
A-192621 at 24 mglkg/day in rats on either high sodium or normal sodium intake.
The horizontal bar from protocol day A1 to A15 depicts Ang II infusion period.
The horizontal bar from protocol day A5 to A1 0 depicts A-192621 administration.
‘Significant (p<0.05) difference in water balance from protocol day 02.
‘Significant (p<0.05) difference in water balance from protocol day A5.

118

 

 

Na balance

Na balance

Normal salt

3

High salt

 

 

+ High salt + Ang II, n=9
—0— High salt control, n=5

 

 

 

Ang II

infusion

 

Pi

 

'A-192621

 

 

 

 

 

 

 

 

C1 A1

W

A5

A10

A15 R2

 

 

+ Normal salt+ Ang II, n=9
—A— Normal salt control, n=5

 

 

 

 

 

 

 

 

 

 

Ang II infusion
.. A-192621
1 *
*
C1 A1 A5 A10 A15 R2

Protocol Day

Figure 5.3 Line plots show sodium balance responses in milliequivalents per
day (mEq/day) to infusion of angiotensin II (Ang II) at 5.0 nglmin and
administration of A-192621 at 24 mglkglday in rats on either high sodium or
normal sodium intake. The horizontal bar from protocol day A1 to A15 depicts
Ang II infusion period. The horizontal bar from protocol day A5 to A10 depicts A-
192621 administration. *Significant (p<0.05) difference in sodium balance from
protocol day Cz. *Significant (p<0.05) difference in water balance from protocol

day A5.

119

 

 

+ High salt-r- Ang II, n=8

 

 

 

 

 

 

 

 

 

 

 

 

 

 

High salt —0— High saltcontrol,n=5
180
3 \LA-192621 bolus IA injection
1:
I 160 - * *
r:
E * * ...-'0‘.
E 140 1 * .tc’ * *
a:
n. of. g o o
E 120 * * * * * *
6
100 j
0 10 20 30 40 50 60
N I It + Normalsalt+ Ang ll,n=9
orma sa —A— Normalsaltcontrol,n=5
180
° A-192621 bolus IA injection
1 160 J * r:
v:
E . * * * *
E 140 ~ * * *
o. ...-... . . . . . .
< ‘ * ‘ r:
100 4
0 10 20 30 4O 50 60

Time (minutes)

Figure 5.4 Line plots show hemodynamic responses to bolus intra-arterial
injection of A-192621 at 24 mglkg in rats on high sodium or normal sodium
intake. MAP, mean arterial pressure. *Significant (p<0.05) difference from time
of injection.

120

During the next five days of Ang II infusion, A-192621 was administered to
all rats at an intra-arterial dose of 24 mglkglday. There was a significant
increase in MAP in all groups when compared to the pre-drug values recorded on
day 5 of Ang II infusion (figure 5.1). The increases in MAP during chronic A-
192621 treatment (difference between day 5 and day 10) were 23 i 8 mmHg in
high salt rats receiving Ang II, 24 i 5 mmHg in high salt control rats, 9 i 3 mmHg
in normal salt rats receiving Ang II, 14 i 4 mmHg in normal salt control rats. The
increases in MAP during chronic treatment with A-192621 were not significantly
different in any of the four groups. All four groups demonstrated a decrease in
water balance (figure 5.2) on the first day of A-192621 treatment but this change
was statistically significant only in high salt rats receiving Ang II. A similar
decrease in sodium balance was observed (figure 5.3) in all rats on the first day
of A-192621 treatment but this change was significant only in normal salt rats
receiving Ang II.

After stopping A-192621, MAP returned to pre-drug values over a period
of one to four days in all groups (figure 5.1) though recovery was faster in
animals on normal salt intake. Water balance increased on the first day after
stopping A—192621 in all groups except normal salt controls but this change was
significant only in high salt controls. There were no changes in sodium balance
in any of the four groups after A-192621 administration was terminated.

There were no significant differences in body weight between the four

groups (high salt Ang II, 405 i 8 9; high salt control, 393 :l: 5 9; normal salt Ang II,

407 :l: 7 9; normal salt control, 385 i 26 9). There were also no significant

121

differences in initial blood volume between the four groups (high salt Ang II, 55 :r
5 mllkg; high salt control, 53 i 4 mllkg; normal salt Ang II, 58 :l: 2 mllkg; normal
salt control, 57 i- 5 mllkg). Blood volume decreased significantly over the course
of the experiment, but to a similar degree in all four groups. There were no
significant changes in plasma sodium or BUN during the protocol, however

plasma potassium increased slightly but significantly in all four groups.

ETm receptor antagonism using A-182086

As shown in figure 5.5, during the first five days of Ang II infusion MAP
rose significantly in rats on high salt intake (109 i- 2 to 126 i 5 mmHg) and in
rats on normal salt intake (107 i 2 to 116 i 4 mmHg). The increment in MAP
was significantly larger in rats on high salt intake. MAP did not change over the
same time period in rats not receiving Ang II. There were significant no changes
in water balance (figure 5.6) or sodium balance (figure 5.7) over this time period.

After measurements were completed on day 5 of the Ang II infusion
period, each rat received a bolus intra-arterial injection of the ETA/a receptor
antagonist, A-182086 (12 mglkg) and MAP was recorded continuously for the
next hour. MAP decreased significantly in all groups, except normal salt control
rats, which showed an increase in MAP (figure 5.8). In rats on high salt intake
the decrease was significantly less in control rats (8.5 i: 1.6 mmHg) than in rats

receiving Ang II (23.8 :t 3.8 mmHg). In rats on normal salt intake, the Ang II

infused group showed a decrease of 12.4 d: 2.0 mmHg, whereas MAP actually

122

MAP (mmHg)

MAP (mmHg)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

+ High salt-i» Ang ll, ":9
High salt +H|9hsaltcontrol,n=5
160
Ang II infusion
* A-182086 * * * *
140 - * i: *
* *
*
120 ~
100 .
Ct A1 A5 A10 A15 R2
+ Normalsalt+Ang Il,n=9

Normal salt

160

140

120

100

 

+ Normal salt control, n=5

 

 

1

 

Ang II

Infusion

 

A-182086

 

 

 

MFW'P + + +

 

k
A\
A A
‘ A

 

 

C1

A1

A5

A10

Protocol Day

Figure 5.5 Line plots show hemodynamic responses to infusion of angiotensin II
(Ang II) at 5.0 nglmin and administration of A-182086 at 24 mglkglday in rats on
either high sodium or normal sodium intake. The horizontal bar from protocol
day A1 to A15 depicts Ang II infusion period. The horizontal bar from protocol
day A5 to A10 depicts A-182086 administration. *Significant (p<0.05) increase in
mean arterial pressure from control day C2. *Significant (p<0.05) increase in

mean arterial pressure from protocol day A5.

123

 

 

+ High salt + Ang II, n=9

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

High salt —O— High salt control, n=5
3 Ag II Infusion
: 25 ‘ A-182086
0 20 .
a
5 ‘5 ‘ a? it
0
1 ..
g o
5 _
C1 A1 A5 A10 A15 R2
+ Normalsalt+Angll,n=9
Normalsalt —A— Normalsaltcontrol,n=5
a, 30 =
0 Ang II Infusion
= 25 -
2 A-182086
a 20 .,
.o
3 15 ~
.. 10 {N}
3
5 ..
C1 A1 A5 A10 A15 R2

Protocol Day

Figure 5.5 Line plots show water balance responses in milliliters per day
(mlslday) to infusion of angiotensin II (Ang II) at 5.0 nglmin and administration of
A-182086 at 24 mglkg/day in rats on either high sodium or normal sodium intake.
The horizontal bar from protocol day A1 to A15 depicts Ang II infusion period.
The horizontal bar from protocol day A5 to A1 0 depicts A-182086 administration.
There were no significant changes in water balance across the length of the
protocol.

124

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

+ High salt-I» Ang II, n=9
High 53"; —O— High salt control, n=5
3
Ang II infusion
o A-182086
o 2 -
C
2
u
.o 1 .
fl
2
o ..
*
-1 -....H .......fi-
C1 A1 A5 A10 A15 R2
+ Normalsalt+Ang Il,n=9
Normalsalt ——A— Normalsaltcontrol,n=5
3
Ang II infusion
o
g 2 ~ A-182086
2
a:
.c 1 -
(U
z
o .
'1 T r r

 

 

 

 

 

 

 

C1 A1 A5 A10 A15 R2
Protocol Day

Figure 5.7 Line plots show sodium balance responses in milliequivalents per
day (mEq/day) to infusion of angiotensin II (Ang II) at 5.0 nglmin and
administration of A-182086 at 24mglkglday in rats on either high sodium or
normal sodium intake. The horizontal bar from protocol day A1 to A15 depicts
Ang II infusion period. The horizontal bar from protocol day A5 to A10 depicts A-
182086 administration. *Significant (p<0.05) difference in sodium balance from
protocol day C2.

125

 

—o— ngh salt + Ang II, n=9

 

 

 

 

 

 

 

 

 

 

 

 

ngh salt —O— ngh salt controI,n=5
180
3 A-182085 bolus IA Injection
3: 160 -
E
E 140 J
n.
<
120 .
5 .8 *
‘ *
100 l
r r r 1 8 * * *
0 1O 2O 30 4O 50 60
+ Normal salt-I- Ang II, n=9
Normalsalt —Ar— Normalsaltcontrol,n=5
180
f: 160 . \LA-182086 bolus IA Injection
E
E 140 -
a.
<
E 120 ~
100 4

 

 

 

0 1O 20 30 40 50 60
Tim 9 (m lnutes)

Figure 5.8 Line plots show hemodynamic responses to bolus intra-arterial
injection of A-182086 at 24 mglkg in rats on high sodium or normal sodium
intake. MAP, mean arterial pressure. *Significant (p<0.05) difference from time

of injection.

126

showed an increase of 3.8 i 0.5 mmHg in control rats. This difference was
statistimlly significant. In Ang II infused groups, the fall in MAP after
administration of A-182086 was significantly greater in rats on high salt intake.

During the next five days of Ang II infusion, A-182086 was administered to
all rats at an intra-arterial dose of 24 mglkg/day. There was a statistically
significant fall in MAP (9 i 3 mmHg) during this time period (compared to day 5
of Ang II infusion) only in Ang II infused rats on normal salt intake. There were
no significant changes in water (figure 5.6) or sodium balance (figure 5.7) during
A-182086 administration when compared to pre—drug values on day 5 of Ang II
infusion.

In normal salt rats receiving Ang II cessation of A-182086 treatment
resulted in a recovery of MAP to hypertensive levels 3 days later.

There were no significant differences in body weight between the four
groups (high salt Ang II, 403 i 12 9; high salt control, 397 i 8 9; normal salt Ang
II, 405 :t 9 9; normal salt control, 408 i 13 9). There were also no significant
differences in initial blood volume between the two high salt groups (high salt
Ang II, 56 :l: 3 mllkg; high salt control, 47 i 2 mllkg) or between the two normal
salt groups (normal salt Ang II, 45 i 3 mllkg; normal salt control, 45 i 3 mllkg).
However, initial blood volume was significantly larger in the high salt Ang II group
versus both normal salt groups. Blood volume decreased significantly over the
course of the experiment, but to a similar degree in all four groups. There were

no significant changes in plasma sodium, potassium or BUN during the protocol.

127

Discussion

It has been shown numerous times, and was confirmed in the current
study, that Ang II induced hypertension is potentiated by increased daily salt
intake (Muirhead et al., 1975; Kanagy et al., 1990; Csiky and Simon, 1997).
Although these earlier studies showed a larger degree of potentiation by high salt
than observed here, I used smaller increments in salt intake and lower infusion
rates of Ang II than employed previously. Nonetheless, Ang II infusion produced
a consistently greater rise in MAP in rats on high salt intake in these
experiments. An important goal of this study was to investigate the role of ETB
receptors in this phenomenon.

This is the first report on the effects of a selective ETB receptor antagonist
in Ang II induced hypertension. Previous studies have demonstrated that ETB
receptor activation opposes increases in MAP in some forms of hypertension,
including DOCA-salt rats (Matsumura et al., 2000; Pollock et al., 2000), and
humans with essential hypertension (Cardillo et al., 1999). Proposed
mechanisms have included both ETB mediated diuretic and natriuretic actions
(Pollock et al., 2000) and the release of endothelial cell derived vasodilators
(Cardillo et al., 1999). Therefore in this study I investigated the effects of acute
and chronic ETB, as well as combined ETA and ETB, receptor antagonism on
blood pressure and sodium and water balance in rats with Ang II induced
hypertension. I used changes in MAP one hour after acute intra-arterial injection

of ET-1 receptor antagonists as an index of the vascular component of ET-1

128

action. Since long-term changes in MAP require a shift in the renal pressure-
natriuresis relationship (Hall et al., 1980), I also assessed the effects of ET—1
receptor antagonism on renal sodium and water handling. The main new
findings of this study are that ETB receptor activation opposes Ang II induced
hypertension, high salt intake increases the contribution of ETB receptors to the
acute and chronic control of MAP, and the ETB receptor mediated component of
chronic Ang II induced hypertension is not modified by salt intake.

After five days of continuous Ang II infusion, ET-1 receptor antagonists
were injected intra-arterially and the resulting acute changes in MAP were
measured. Selective blockade of ETB receptors by A-192621 resulted in a
significant increase in MAP in all four groups of rats. This pressor response is
due to impaired release of endothelial vasodilators (nitric oxide, prostacyclin)
and/or increased ETA receptor stimulation, possibly due in part to diminished ET-
1 clearance (Haynes and Webb, 1998). The acute increase in MAP I observed
in control rats was larger under high salt conditions, consistent with earlier work
showing that A-192621 treated rats exhibit salt-sensitive hypertension (Pollock,
2000). This result suggests that high salt diet enhances ETB receptor mediated
vasodilation. Since the acute pressor response to A-192621 in high salt rats
receiving Ang II was significantly less than in high salt control rats, chronic Ang II
infusion appears to attenuate ETB receptor mediated vasodilation in this setting.
On the other hand, Ang II infusion augmented ETB receptor mediated
vasodilation in rats on normal salt intake. The mechanism for this salt-dependent

difference in Ang II effects on ETB receptor mediated vasodilation is unknown.

129

On day 5 of Ang II infusion, blockade of both ETA and ETB receptors with
A-182086 produced a significant acute decrease in MAP in all groups except
normal salt control rats. This change in MAP was presumably due to the net
effect of inhibiting both ETA mediated vasoconstriction and ETB mediated
vasodilation. I previously reported that injection of the selective ETA receptor
antagonist, ABT-627, in Ang II induced hypertension caused significant acute
falls in MAP that were of similar magnitude in rats on both high and normal salt
intake (Chapter 4). This indicates that ETA mediated vasoconstriction is
augmented equally by Ang II under conditions of normal and high salt intake.
This could be a consequence of either increased vascular ET-1 synthesis
(Rajagopalan et al., 1997; D’Uscio et al., 1998) or increased vascular reactivity to
endogenous ET-1 (Haynes and Webb, 1998). In normotensive control rats in this
study the acute changes in MAP produced by A-182086 were small and not
significantly different in rats on high versus normal salt intake. This confirms my
own and other reports that endogenous ET—1 exerts only modest net effects on
basal vascular tone in normotensive animals, regardless of salt intake (Haynes
and Webb, 1998; Chapter 4). Rats on high salt intake receiving Ang II showed a
larger fall in MAP to bolus injection of A-182086 than high salt controls,
presumably due to increased ETA receptor mediated vasoconstriction (D’Uscio et
al., 1997; Rajagopalan et al., 1997; Chapter 4) and decreased ETB receptor
mediated vasodilation in the Ang II infused rats (as discussed above). Rats on
normal salt intake receiving Ang II infusion also showed a larger acute fall in

MAP to A-182086 compared to normal salt controls. This effect is likely due

130

predominately to increased ETA receptor mediated vasoconstriction in Ang II
infused rats (D’Uscio et al., 1997; Rajagopalan et al., 1997; Chapter 4), since
ETB receptor mediated vasodilation is augmented by Ang II under normal salt
conditions (as discussed above). Taken together, these observations of acute
responses to ET—1 receptor antagonists indicate that ETA mediated
vasoconstriction plays an important role in Ang II induced hypertension at any
salt intake. Salt-sensitivity of this model though seems to be more dependent on
modulation of ETB mediated vasodilation.

Sustained alterations in MAP require a shift in the renal pressure-
natriuresis relationship, and ET-1 shifts the pressure-natriuresis relationship to
higher pressure levels predominately via activation of ETA receptors (Kassab et
al., 1998). ETB receptor activation generally causes natriuresis and diuresis
(Kohan et al., 1993; Clavell et al., 1995). Therefore during chronic dosing with
ET-1 receptor antagonists, I measured changes in sodium and water balance to
assess the specific contributions of ETA and ETB receptors to controlling renal
function in Ang II induced hypertension. Published reports show chronic Ang II
infusion increases renal ET-1 content (Barton et al., 1998), whereas the effect of
salt intake on renal ET-1 formation is controversial (Firth et al., 1995; Melo et al.,
1998; Morita et al., 1999).

During the first 24 hours of chronic ETB receptor blockade with A-192621,
MAP rose significantly and modest sodium and water loss occurred, presumably
via the pressure-natriuresis mechanism. Subsequently, however, sodium and

water balances were restored while MAP was elevated to a similar degree in all

131

four groups of rats. The fact that A-192621 did not have a large effect on salt
and water balance was reflected by a failure to observe significant changes in
blood volume or plasma electrolytes during the drug treatment period. Overall
these data show that A-192621 produces a similar chronic shift in pressure-
natriuresis to a higher pressure level in all groups. Thus Ang II does not change
the ETB mediated contribution to chronic blood pressure regulation.

During the first 24 hours of chronic combined blockade of ETA and ETB
receptors with A-182086, MAP fell slightly (but only significantly in normal salt
rats receiving Ang II) and modest sodium and water retention occurred (but only
significantly in high salt control rats), presumably via the pressure-natriuresis
mechanism. Subsequently, however, sodium and water balances were restored
while MAP remained significantly lower than pre-treatment values only in normal
salt rats receiving Ang II. A-182086 also produced no significant changes in
blood volume or plasma electrolytes during the drug treatment period. Overall
these data indicate that A-182086 produced a chronic shift in pressure-
natriuresis to a lower pressure level only in normal salt rats receiving Ang II
infusion.

The contribution of ET-1 to Ang II induced changes in the chronic
pressure-natriuresis relationship occurs through a balance of ETA (antinatriuretic)
and ETB (natriuretic) mediated actions. In a previous study (Chapter 4), I
showed that chronic blockade of ETA receptors alone mused a significant
decrease in MAP in Ang II induced hypertension that was well sustained over five

days in rats on high salt intake. In rats on normal salt intake, however, MAP fell

132

initially during ETA receptor blockade but then returned to pre-treatment levels by
day 5 of drug treatment. The present study suggests that the role of the ETB
receptor in pressure-natriuresis is accentuated under high salt conditions. This
may explain why the ETA antagonist, but not the combined ETA and ETB receptor
antagonist, produced a chronic fall in MAP in rats on high salt intake, and why
the combined antagonist was more effective at causing sustained MAP reduction
in rats on normal salt intake.

In summary, based on my results with ET-1 receptor antagonists, and the
work of others (D’Uscio et al., 1997; Rajagopalan et al., 1997; Herizi et al., 1998;
Chapter 4), I conclude that ET-1 plays a major role in the development of Ang II
induced hypertension, and that both vascular and renal actions of ET—1 are
important factors. Furthermore, my data indicate that some of the contributions
of ETA and ETB receptors in this model of hypertension are influenced by
changes in salt intake. ETA mediated vasoconstriction is increased by Ang II
infusion but is similar under conditions of high and normal salt intake. However,
ETB mediated vasodilation is increased by high salt intake and this mechanism is
significantly impaired by chronic Ang II infusion. Conversely, ETB mediated
vasodilation is enhanced by Ang II in rats on normal salt intake. The effects of
Ang II on ETA receptor mediated shift in the pressure-natriuresis relationship also
are not influenced by salt intake. On the other hand, ETB mediated natriuretic
effects are not influenced by Ang II infusion but are generally potentiated by high

salt intake. I conclude that together these actions of Ang II on ET-1 mediated

133

vascular and renal responses are important factors in the salt-sensitivity of Ang II

induced hypertension.

134

Chapter 6

Characterization of the Antihypertensive Effect of a Thiazide Diuretic in

Angiotensin II Induced Hypertension

Jennifer R. Ballew and Gregory D. Fink
Department of Pharmacology and Toxicology, Michigan State University,

East Lansing, MI, USA

This chapter has been submitted as a manuscript to:

Journal of Hypertension

135

INTRODUCTION

The antihypertensive efficacy of angiotensin converting enzyme inhibitors and
angiotensin AT1 receptor antagonists in forms of hypertension in which there is
no evidence of overactivation of the renin-angiotensin system suggests that
changes in sensitivity of cardiovascular tissues to angiotensin II could be
responsible. My research has focused on factors that can modify the long-term
responsiveness of the cardiovascular system to angiotensin II, using the model of
chronic (days to weeks) intravenous infusion of low doses of angiotensin II, is
angiotensin Il-induced hypertension. It is firmly established that increased daily
salt intake is one factor that causes a marked potentiation of angiotensin II-
induced hypertension (Muirhead et al., 1975; Kanagy et al., 1990; Ando et al.,
1991; Sato et al., 1991; Csiky et al., 1997; Simon et al., 1998), making this model
one of the many known salt-sensitive forms of hypertension.

Two different theories have been proposed to explain the salt-sensitivity of
angiotensin lI-induced hypertension. First, in some species angiotensin II causes
strong sodium and water retention by the kidney through direct and indirect (e.g.
aldosterone, vasopressin) mechanisms (Hall et al., 1980; Hall, 1986). One of
these indirect mechanisms may be stimulation of ETA receptors in the kidney, as
discussed in chapter 5. In a setting of elevated salt intake, infusion of
angiotensin II would cause a greater degree of sodium retention than under
conditions of normal salt intake. The resulting larger volume expansion would

lead to greater increments in cardiac output and arterial pressure (Krieger et al.,

136

1989). An alternative explanation is that under conditions of high salt intake, pre-
existing relative volume expansion causes a more marked activation of non-renal
pressor actions of angiotensin II than during normal salt intake. For example,
sympathoexcitatory (Sato et al., 1991) and direct vasoconstrictor (Csiky et al.,
1997) effects of angiotensin II are reported to be enhanced by high salt intake.
Another salt—sensitive, non-renal pressor action of Ang II recently described my
myself and other investigators (as discussed in chapters 4 and 5) is activation of
the vascular ET-1 system.

Diuretics are widely used antihypertensive drugs that, in part, lower arterial
pressure by facilitating renal salt and water excretion (Ramsey et al., 1994). One
question raised by the experiments with ET-1 receptor antagonists discussed in
chapters 4 and 5 is the relative contribution of renal and vascular effects of these
drugs to their cardiovascular actions. I hypothesized that if the renal actions of
the antagonists were primarily responsible for their antihypertensive effect, than
this effect should be slow in onset and preceded by (and strongly correlated with)
significant salt and water loss. However, the antihypertensive response to
diuretic drug treatment has not been directly tested in angiotensin II-induced
hypertension. Therefore, in the present study I examined the effects of the
thiazide diuretic trichlormethiazide (TCM) on arterial pressure and renal fluid
excretion in rats receiving chronic intravenous infusion of angiotensin II while on

fixed normal or high salt intakes.

137

METHODS

Male Sprague-Dawley rats (Charles River Laboratories, Portage, MI)
weighing 350-450 grams were used in these experiments. Upon arrival at our
facility, rats were maintained according to standards approved by the Michigan
State University All-University Committee on Animal Use and Care. All
experimental procedures were carried out in accordance with the “Guiding
Principles in the Care and Use of Animals” of the American Physiological
Society. Rats were acclimatized for at least two days prior to surgical
procedures in clear plastic boxes and were allowed access to standard rat
chow (Teklad 22/5 Rodent Diet W 8640, Madison, WI) and tap water ad
libitum.

Surgical Procedures

All surgical procedures were performed after administration of
pentobarbital sodium (Nembutal‘m, Abbott Laboratories, N, Chicago, IL), 50
mglkg i.p., and atropine sulfate (Sigma, St. Louis, M0), 0.2 mg, i.p. If
necessary, anesthesia was supplemented using methohexital sodium
(Brevitalo, Eli Lilly, Indianapolis, IN), 5-10 mglkg, i.v. At least two days prior
to initiation of the experimental protocol, rats were catheterized via the femoral
artery and vein as previously described (Potter et al., 1997).

Chronic Rat Maintenance and Measurements
The metabolism cages housing the rats during the experiments were

located in a climate-controlled room with a 12-hour light-dark cycle. The rats

138

were allowed ad libitum access to distilled water and sodium-deficient rat chow
(Teklad 170950, Madison, WI). Half the rats were maintained on a daily
sodium intake of 2 mEq/day (normal salt intake), and the remaining half were
maintained on 6 mEq/day (high salt intake). All sodium chloride was delivered
by continuous intravenous infusion. Catheters were flushed daily to maintain
patency and the arterial catheters were filled with a heparinized sucrose
solution and occluded when not in use.

Mean arterial pressure and heart rate were recorded via the femoral
arterial catheter each morning of the protocol between 8:00 and 11:00 am.
The arterial catheters were connected to external pressure transducers (TXD-
300; Micro-Med, Louisville, KY, USA) that were first zeroed at the level of the
rat’s heart. The transducers are connected to digital pressure monitors (BPA-
200 Blood Pressure Analyzer, Micro-Med, Louisville, KY, USA) that output
directly to a computerized digital pressure monitoring system. Data were
collected once every second for 15-30 minutes. The daily value was
determined by the average of the one-second recordings taken over the last
five minutes of the recording session.

Experimental Protocol

After blood pressure, heart rate and other variables were recorded for two
control days, Ang II was continuously infused i.v. at a rate of 5 nglmin for 15
days. On day 5, the rats received a one-time bolus injection of the diuretic,
trichlormethiazide (TCM; 10 mglkg, i.v.; Sigma Chemical Co., St. Louis, MO,

USA), followed by TCM in their drinking water at a concentration (250 mglL) to

139

deliver an approximate dose of 10 mglkglday on days 5-10 of Ang II infusion .
Blood pressure and heart rates were recorded continuously for one hour
immediately following the bolus injection of TCM and then once daily between
each subsequent day of the protocol. Urine samples were collected daily and
blood samples (1 ml) on control day 2 and Ang II infusion days 5, 7, 10, 14.
Urine electrolytes and routine blood chemistries were measured using ion
selective electrodes (Nova Electrolyte 16+ Analyzer, Nova Biomedical).
Plasma volumes were determined periodically from the 10 minute distribution
volume of Evan’s Blue dye, and blood volume was calculated as previously
described. Daily electrolyte balance, water balance, and creatinine clearance
were calculated as previously described (Potter et al., 1997).
Statistical Analysis

Results are expressed as mean i SEM. For all data, within- and
between-group differences were analyzed using mixed-design ANOVA. Post
hoc comparisons between groups were performed by testing for simple main
effects. Within group comparisons were made using the protected least
significant difference test. Criterion for statistical significance was a probability

level of less than 0.05.

140

Results

Body weights
There was no significant difference between mean body weights (i SEM)
of the four groups of rats in this study: normal salt control, 408 :t 10.1 9; normal

salt + Ang II, 406 :l: 6.9 9; high salt controls, 402 29.9 9; high salt + Ang II, 402 i
8.1 9.
Responses to Ang II infusion

Continuous i.v. infusion of Ang II at 5 nglmin over the first 5 days (AS-A10)
induced a significant increase in MAP in rats on high salt intake (figure 6.1). No
significant alterations in MAP over the same time period were observed in rats on
high salt but not receiving Ang II, or in either group of rats on normal salt intake
(figure 6.1). Heart rate (data not shown), water balance (figure 6.2) and sodium
balance (figure 6.3) were unchanged (compared to the values on control day 2)
in all groups during this period (figures 6.2 and 6.3).
Responses of MAP to Acute TCM Administration

After all daily measurements were completed on day 5, TCM (10 mglkg,
iv) was administered and MAP was monitored continuously for 1 hour (figure
6.4). No significant changes in MAP were seen in the two groups of rats on
normal salt intake . In control rats on high salt intake, TCM caused a small but

significant increase in MAP. In rats receiving Ang II and high salt intake, TCM

141

High Salt

 

 

+ High salt+ Ang II, n=9
—0— High saltcontrol, n=5

 

 

Ang II infusion

 

8%

 

 

D in retic

 

 

*

M

 

wt,

 

 

'7

C1 A1

Normal Salt

A 150
O
I
E 140
E
a 120
<
E
100
30
A 160
a
I
E 140
E
Q 120
<
5
100
80

1*

A5

A10

A15 R2

 

 

+ Norm alsalt+ Ang II, n=9
—A— Normal salt control, n=5

 

 

Ang II infusion

 

 

 

 

D in re tic

 

 

 

 

 

A5

‘7

A10

Protocol Day

Figure 6.1 Hemodynamic responses to infusion of angiotensin II (Ang II) at 5.0
nglmin and administration of trichlormethiazide at 10 mglkglday in rats on 8

mEq/day sodium intake or on 2 mEq/day sodium intake. The horizontal bar from
protocol day A1 to A15 depicts Ang II infusion period. The horizontal bar from
protocol day A5 to A10 depicts trichlormethiazide administration. MAP, mean
arterial pressure. MAP was significantly higher than on control day 2 on days
A5-R1 in rats on high salt intake receiving Ang II. *Significant (p<0.05) difference

in mean arterial pressure from protocol day A5.

142

 

 

 

 

+ High saIt+ Ang II, n=9

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

3 High salt —0— High salt control,n=5
E 24 ~ Ang II infusion
G
g 20 ‘ Diuretic
2 16 -
a
m
h 12 J
3
ru 8 7
s 4_

o -.....W

C1 A1 A5 A10 A15

A + Normalsalt+Ang Il,n=9
2 Normalsalt —A— Normalsaltcontrol,n=5
E 24 -
q, Ang II infusion
g 20 -
u Diuretic
T. 16 -
m
L 12 a
3
I!

8 _
3

4 _

c1 A1 A5 A115 A15

 

P re to c o I D a y
Figure 6.2 Water balance responses in milliliters per day (mlslday) in response
to infusion of angiotensin II (Ang II) at 5.0 nglmin and administration of
trichlormethiazide at 10 mglkg/day in rats on 8 mEq/day sodium intake or 2
mEq/day sodium intake. The horizontal bar from protocol day A1 to A15 depicts
Ang II infusion period. The horizontal bar from protocol day A5 to A10 depicts
trichlormethiazide administration. *Significant (p<0.05) difference in water
balance from protocol day A5.

143

 

 

+ High salt + Ang II, n=9
—O—— High salt control, n=5

 

High salt

 

 

Ang II infuslon

 

D lu re tlc

 

   

Na Balance (m quday]
N

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

-1 W
.. C1 A1 A5 A10 A15
:' +Normalsalt+Angll,n-9
E 3 Normalsa —-A——Normalsaltcontrol,n=5
o-

m Angllinfusion
E 2 - Diuretlc
0
0
r:
2 1‘ ¥¥
a
m
'0'“ o _
z
.1 --..-...*. .......
C1 A1 A5 A10 A15

Protocol Day

Figure 6.3 Sodium balance responses in milliequivalents per day (mEq/day) in
response to infusion of angiotensin II (Ang II) at 5.0 nglmin and administration of
trichlormethiazide at 10 mglkg/day in rats on 8 mEq/day sodium intake or 2
mEq/day sodium intake. The horizontal bar from protocol day A1 to A15 depicts
Ang II infusion period. The horizontal bar from protocol day A5 to A1 0 depicts
trichlormethiazide administration. *Significant (p<0.05) difference in sodium
balance from protocol day A5.

144

 

+ High salt + Ang II, n=7

 

 

 

 

 

 

 

 

 

 

 

 

 

 

High salt —0— High saltcontrol,n=5
160
3 \l/TCM bolus IV injection
I 140 -
E * * * *
E
"' 120 ~
0.
<
2
80 I 1 I r r v r I 1 r r r r
0 10 20 30 40 50 60
+ Normal salt + Ang II, n=9
Normalsalt —A— Normalsaltcontrol,n=5
160
g ¢TCM bolus IV injection
140 ~
E
E
" 120 d
:L
<
E 100 -
80 r r . r . w r

0 10 20 30 40 50 60
Time (minutes)

Figure 6.4 Hemodynamic responses to bolus intravenous injection of

trichlormethiazide at 10 mglkg in rats on 8 mEq/day sodium intake or on 2
mEq/day sodium intake. MAP, mean arterial pressure. *Significant (p<0.05)

difference from time of injection.

145

caused a slight decrease in MAP that achieved statistical significance by 45
minutes following drug injection.
Responses to Chronic TCM Administration

Chronic oral dosing with TCM at 10 mglkglday produced a significant fall
in MAP (compared to the day A5 value) in rats on combined Angll and high salt
intake (figure 6.1 ). This antihypertensive response required 72 hours to reach a
maximum. In contrast, MAP was unchanged by TCM treatment in all other
groups. After termination of TCM therapy MAP rose in rats on combined Ang II
and high salt intake, but remained significantly lower than the maximum MAP
achieved prior to the start of TCM treatment (day A5). Cessation of Ang II
infusion on day A15 of the protocol resulted in a return of MAP to normotensive
levels within 48 hours in this group of rats. Heart rate was unaffected by TOM
treatment in all groups (data not shown).

Water balance (figure 6.2) was significantly decreased (compared to the
day 5 value) during the first 24 hours after starting TCM treatment within all
groups, except for the control rats on high salt intake. But the absolute difference
in water balance between day A5 and day A6 (net water loss) was not
significantly different between groups (p=0.73). Similarly, TCM caused a sodium
loss (figure 6.3) during the first 24 hours that was significant (compared to day A5
values) only in the two groups on normal salt intake. Nonetheless, the absolute
difference is sodium balance between day A5 and day A6 (net sodium loss) was

not significantly different between any of the groups (p=0.55). Termination of

146

 

:1 High salt + Ang II, n=9
:1 High salt control, n=5
m Normal salt + Ang II, n=9
m2! Normal salt control, n=5

 

 

 

Blood Volume (mls)

 

CZ A5 A10 A14
Protocol Day

Figure 6.5 Blood volumes in milliliters in rats on 8 mEq/day sodium intake or on
2 mEq/day sodium intake on control day 2, and Ang II infusion days 5, 10, and
14. Body weights were not significantly different between groups.

147

TCM therapy caused no significant alterations in water or sodium balance in any
group.
Blood Volumes and Chemistries

Absolute blood volumes were not significantly different in rats on high salt
versus normal salt intake prior to the start of Ang II infusion (figure 6.5).
Furthermore, there were no significant changes in any group throughout the
remainder of the experiment. No differences in plasma sodium, potassium,
glucose, osmolality , blood urea nitrogen or creatinine clearance were found
between the groups prior to the start of Ang II infusion, and no significant

changes occurred in these measures throughout the study (data not shown).

148

Discussion

Ang II induced hypertension is known to be a salt-sensitive model
(Muirhead et al., 1975; Kanagy et al., 1990; Ando et al., 1991; Sato et al., 1991;
Csiky et al., 1997; Simon et al., 1998). In rats, however, Ang II infusion has not
been associated with significant renal sodium and water retention (Kanagy et al.,
1990; Heulsemann et al., 1985; Hu et al., 1998), a finding confirmed in the
present investigation. A possible implication is that sodium and water depletion
with diuretics would not be expected to lower MAP in this model, but this has
never been directly tested. The main new finding of this study is that treatment
with the thiazide diuretic TCM increased sodium and water excretion and lowered
MAP in rats receiving chronic Ang II infusion. Interestingly, this occurred only in
rats on high salt intake, but not in rats on normal salt intake, even though all
animals showed a similar diuretic response to TCM.

The traditional explanation for the antihypertensive effect of diuretics is an
initial fall in cardiac output secondary to a diuretic induced decrease in blood
volume, followed by an autoregulatory decline in total peripheral resistance
(Ramsey et al., 1994). Using the indicator dilution method, I was unable to
detect significant differences in blood volume between rats on the two salt
intakes used here, or a decline in blood volume in response to diuretic therapy.
This is not an atypical finding and probably indicates that only very small

changes in intravascular or other volumes are necessary to exert significant

149

hemodynamic effects (Hall, 1986). The differences in blood pressure response
to TCM, despite similar salt and water loss in the four groups, nonetheless
reinforce the accepted theory that volume depletion alone cannot fully account
for the antihypertensive response to diuretic treatment (Ramsey et al., 1994).

There is some evidence that direct vasodilation can contribute to the
antihypertensive action of diuretics (Ramsey et al., 1994). To test this possibility
I measured the initial blood pressure response to acute intravenous injection of
TCM. Only the rats on high salt intake receiving Ang II showed a significant fall
in MAP over the one-hour study period. However, this reduction in blood
pressure was small, and the maximal response to TCM was not observed until
72 hours after starting therapy. These data are not consistent with direct
vasodilation being an important component of the blood pressure response to
TCM.

Diuretics are known to be more efficacious at lowering blood pressure
when the normal renin response to salt and water loss is blunted (Ramsey et al.,
1994). Previous work showed that infusion of Ang II at a rate similar to the one
used here completely suppressed plasma renin (Hu et al., 1998). Thus, I
assume that in my model plasma Ang II concentrations were relatively constant
throughout the Ang II infusion period of the protocol. This would be expected to
augment the antihypertensive response to salt and water loss caused by TOM.

Nevertheless, in rats on normal salt intake, TCM did not lower MAP even when
compensatory changes in RAS activity were prevented by exogenous Ang II

infusion despite the occurrence of significant increases in salt and water

150

excretion. As discussed below, this finding may reflect the fact that the
antihypertensive effect of diuretics is primarily due to interruption of pressor
mechanisms dependent on both relative volume excess and adequate amounts
of Ang II. In rats on normal salt intake, this pressor mechanism did not appear to
be activated, since Ang II infusion at 5 nglmin did not increase MAP compared
to rats on normal salt intake only.

Rats on high salt intake alone did not become hypertensive or exhibit a fall
in MAP in response to TCM. On the other hand, rats on high salt intake receiving
Ang II infusion did become hypertensive and also demonstrated a significant
reduction in MAP during treatment with TCM. This confirms previous results
indicating the existence of pressor mechanisms whose activation requires the
presence of both Ang II and high salt intake (Sato et al., 1991; Csiky et al., 1997).
I hypothesize that salt and water depletion caused by TCM lowers blood
pressure by impairing activation of one or more of these mechanisms.

Sympathoexcitation is one mechanism that contributes to the chronic
pressor actions of Ang II (Fink, 1997). Furthermore, several lines of evidence
indicate that this effect is enhanced in animals on high salt intake (Sato et al.,
1991). A second potential pressor mechanism in Ang II induced hypertension is
activation of the vascular endothelin (ET-1) system. It has been reported that
Ang II can increase vascular preproendothelin-1 gene expression in vitro and in
vivo (Imai et al., 1992; D’Uscio et al., 1995; Moreau et al., 1997), and that ET-1
receptor antagonists slow the development of Ang II induced hypertension

(Moreau et al., 1997; Rajagopalan, 1997; Herizi, 1998). I recently showed that

151

the difference in chronic pressor effects of Ang II in rats on high versus normal
salt intake is due to greater activation of endothelin ETA type receptors during
high salt intake (Chapter 4). Although high salt intake alone does not increase
ET-1 levels in the vasculature (Schiffrin, 1995a; Schiffrin, 1995b), my previous
results suggest that the ability of Ang II to stimulate ET-1 gene expression may
be augmented by high salt intake. In light of these observations, I propose that
the salt and water loss caused by TCM may lower blood pressure by decreasing
the contribution of either the sympathetic nervous system or ET-1 to Ang I I
induced hypertension. Further research is required to distinguish between these

two possibilities.

152

Chapter 7

Effects of Salt Intake and Angiotensin II on Vascular Reactivity

to Endothelin-1

Jennifer R. Ballew, Stephanie W. Watts, and Gregory D. Fink
Department of Pharmacology and Toxicology, Michigan State University

East Lansing, Michigan, 48824

This chapter submitted as a manuscript to:

Journal of Pharmacology and Experimental Therapeutics

153

INTRODUCTION

Chronic infusion of angiotensin II (Ang II) is an experimental model of
“salt-sensitive” hypertension, i.e. infusion rates of Ang II that don’t affect arterial
pressure in animals on low or normal salt intake cause a significant rise in
pressure in animals on higher salt intakes (Muirhead et al., 1975; Kanagy et al.,
1990; Ando et al., 1991; Csiky and Simon, 1997). The endothelial cell derived
vasoconstrictor peptide endothelin-1 (ET-1) is now believed to play a key role in
this and several other salt-sensitive forms of hypertension (Schiffrin, 1999). In
rats on normal salt intake, chronic Ang II infusion is reported to cause both
increased vascular formation of ET-1 (Imai et al., 1992; D’Uscio et al., 1995;
Moreau et al., 1997), and potentiated pressor responses to the peptide (Yoshida,
1992). Furthermore, the development of hypertension is attenuated by
concomitant treatment with ET-1 receptor antagonists (Rajagopalan et al., 1997;
D’Uscio et al., 1997; Herizi et al., 1998). I hypothesized that ET-1 could also
participate in the salt-sensitivity of Ang II induced hypertension, because
administration of a selective ETA receptor antagonist to rats with Ang II induced
hypertension produced a larger and more sustained fall in arterial pressure when
animals were on high versus normal salt intake (Chapter 4). The goal of the
current investigation was to determine if vascular reactivity to ET-1 is

differentially affected by salt intake during chronic Ang II infusion.

154

METHODS

Animals: All animal procedures were carried out in accordance with
institutional guidelines established by Michigan State University. Male Sprague-
Dawley rats weighing 350-450 g were purchased from Charles River
Laboratories (Portage, MI). Upon arrival at our facility, rats were maintained
according to standards approved by the Michigan State University All-University
Committee on Animal Use and Care. All experimental procedures were carried
out in accordance with the “Guiding Principles in the Care and Use of Animals" of
the American Physiological Society. Rats were acclimatized for at least two days
prior to surgical procedures in clear plastic boxes and were allowed access to
standard rat chow (Teklad 22/5 Rodent Diet W 8640, Madison, WI) and tap water
ad libitum.

Isolated tissue bath protocol: Rats were killed (80 mglkg pentobarbital ip)
and the superior mesenteric arteries were dissected into helical strips. The
endothelium was left intact. Tissues were placed in physiological salt solution
containing (mmol/I) 130 NaCl, 4.7 KCI, 1.18 KHzPO4, 1.17 MgSO4-7H20, 1.6
CaCL2~2H20, 14.9 NaHCOa, 5.5 dextrose, and 0.03 CaNa-EDTA. One end of
the preparation was attached to a glass rod and the other to a force transducer
(model FT03, Grass Instruments, Quincy, MA), and the strip was placed under
optimum resting tension (600 mg, as previously determined) and allowed to
equilibrate for one hour. Muscle baths were filled with warmed (37°C), aerated

(95% 02 —5% 002) physiological salt solution. Changes in isometric force were

155

recorded on a polygraph (Grass Instruments). After the hour of equilibration,
arteries were challenged with a maximal concentration of the a1-adrenergic
receptor agonist phenylephrine (PE, 10 umoI/l). Tissues were then washed, and
the status of the endothelium was examined by observing arterial relaxation to
the endothelium-dependent agonist ACh (1 umol/l) in tissues contracted by a
half-maximal concentration of PE (~10 nmolll).

The vessels then were incubated with increasing concentrations of PE (10'
9 to 10‘5 M) followed by ET-1 (10'11 to 10'7 M). The tissues were incubated with
each concentration for ~5 minutes before the next concentration was added. In
some preparations, superior mesenteric arteries were incubated with Ang II (1 O"°
M), A-192621 (an ETB selective receptor antagonist, 30nM, Abbott Laboratories,
Abbott Park, IL, USA), ABT-627 (an ETA selective receptor antagonist, 30 nM,
Abbott Laboratories, Abbott Park, IL, USA), or A-182086 (an ET N3 non-selective
receptor antagonist, 30 nM, Abbott Laboratories, Abbott Park, IL, USA), for one
hour prior to the production of ET—1 dose response curves.

In vivo protocol: Normal male Sprague-Dawley rats were chronically
instrumented for direct, daily measurements of blood pressure (BP) and heart
rate (HR) via catheterization of the femoral artery and vein, as previously
described (Potter et al., 1997). Rats were maintained on either high salt intake
(n=12, 6 mEq/day) or normal salt intake (n=10, 2 mEq/day) for at least three days
prior to starting the experiment. Sodium intake was fixed by delivering sodium
chloride via continuous intravenous infusion to rats consuming a sodium-deficient

diet (Teklad, Madison, WI) and drinking distilled water.

156

Systolic, diastolic, and mean arterial pressures and heart rate were
recorded via the femoral arterial catheter each morning of the protocol between
8:00 and 11:00 am. The arterial catheters were connected to low-volume
displacement pressure transducers that were first zeroed at the level of the rat’s
heart. The transducers were connected to digital pressure monitors (Digi-Med
Blood Pressure Analyzer, Micro-Med, Louisville, KY) that output directly to a
computerized digital pressure monitoring system. Data were collected once
every second for 15-30 minutes. The daily value was the average of the one-
second recordings taken over the last five minutes of the recording session.

In chronic infusion experiments, after recording BP and HR for two control days,
Ang II was continuously infused iv at a rate of 5 nglmin for 7 days. Then the
animals were killed and the superior mesenteric arteries were extracted and used
in the isolated tissue bath protocol, as described above. In acute infusion
experiments, a separate group of rats on normal (n=5) or high (n=5) salt intake
received continuous infusion of either Ang II (5 nglmin) or PE (2 ug/min) over a
two hour time period. The following day, these same rats were given ABT-627 (2
mglkg, i.v., Abbott Laboratories, Abbott Park, IL ) one hour prior to the start of
Ang II or PE infusion.

Data Analysis: Estimates of maximum response and E050 values were
obtained from each concentration response curve using a four-parameter logistic
function. Results are expressed as means 1: SEM. For these results, mean
values were compared statistically using a one-way ANOVA followed by the

protected least significant difference test for post hoc comparisons. For in vivo

157

data, within- and between-group differences were analyzed using mixed-design
ANOVA. Post hoc comparisons between groups were performed by testing for
simple main effects. Within group comparisons were made using the protected
least significant difference test. Criterion for statistical significance was a

probability level of less than 0.05.

158

 

RESULTS

To confirm that contraction of superior mesenteric arteries to ET-1 is
mediated exclusively by ETA receptors, I tested the ability of three different ET-1
receptor antagonists to block contractions to ET-1 (figure 7.1 ). With A-192621,
an ETB selective receptor antagonist, there were no differences in ECso or
maximal response compared to untreated arteries (control: 1.5 :l: 0.3 x 10‘9 M and
133.3 i11%;A-1926212 1.2 i 0.3 x10‘9 M and 128 :11%). VVItI'l A—182086, an
ETA/3 receptor antagonist, there were no differences in maximal response
compared to untreated arteries (control: 133.3 3: 11%; A-182086: 148 :I: 10%).
However, the ECso value was significantly increased (control: 1.5 :I: 0.3 x 10'9 M;
A-182086: 6.5 i 0.7 x 10'9 M). Curve parameters could not be determined for
ABT-627 because the curve was extremely right-shifted.

To test the possibility that Ang II rapidly augments vascular reactivity to
ET-1, an ET-1 concentration response curve was generated in vessels pre-
incubated for one hour with a subcontractile concentration of Ang II (10‘10 M).
As shown in figure 7.2, the maximal response to ET-1 in animals exposed to
subcontractile levels of Ang II (132.4 i 17.8 % PE contraction) was not
significantly different from the maximal response in control rats (155.5 :t 12.6 %
PE contraction). There was no difference in E050 values between the two
groups.

To determine if pressor responses during short term exposure to Ang II in

rats on high or normal salt intake is influenced by endogenous levels of ET-1

159

 

—O— Control, n=8

+ A-182086 , 30 nM, n=5
+ ABT-627, 30nM, n=5
+ A-192621, 30 nM, n=5

 

 

 

 

 

 

 

175«
150—
C
3.12s
0
£100-
5 75.
0
g 50‘
o\° 25.
0-

 

 

 

log ET-1 [M]
Figure 7.1 Concentration dependent contraction to endothelin-1 (ET-1) in
control, A-192621 (ETB selective receptor antagonist, 30nM) incubated, ABT-627
(ETA selective receptor antagonist, 30nM) incubated, and A-182086 (ETA/3
nonselective receptor antagonist, 30nM) incubated superior mesenteric artery of
the rat. PE, phenylephrine (10“5 M). Values are mean SE; n= number of
animals. For A-192621, neither the E050 value nor the maximal response was
shifted. For A-182086, the EC50 value was significantly larger and the maximal
reSponse was not different than control. For ABT-627, curve parameters could
not be calculated.

160

 

—O— Control, n=7
+ Subthreshold Ang II, n=7

 

 

300

 

250 -
200 .
150
100

l

l

% PE Contraction
OI
O

 

 

 

 

log ET-1 (M)

Figure 7.2 Concentration dependent contraction to endothelin-1 (ET-1) in
control and angiotensin II (Ang II) incubated superior mesenteric artery of the rat.
PE, phenylephrine (1045 M). Values are mean SE; n= number of animals. There
were no statistically significant differences between the curves.

161

acting on ETA receptors, Ang II (5 nglmin, n=5) and PE (2 uglmin, n=5) were
infused into conscious rats. MAP was similarly and significantly increased in
both normal and high salt groups after two hours of infusion either of Ang II or PE
(figure 7.3). One day later, this protocol was repeated in the same rats one hour
after administration of the selective ETA receptor antagonist, ABT-627 (2 mglkg).
Treatment with ABT-627 alone did not significantly change MAP. Two hours
following the start of Ang II infusion in rats exposed to ABT-627, there was no
significant increase in MAP (figure 7.3). PE infused rats showed a significant
increase in MAP that was similar in magnitude to that observed in the absence of
ABT-627 treatment.

Chronic infusion of Ang II produced a significant elevation of MAP
compared to that seen in control rats regardless of salt intake (table 7.1). This
increase was significantly (p=0.0329) larger in animals on high salt intake than in
those on normal salt intake. As shown in figure 7.4, maximal responses of rat

superior mesenteric arteries to PE in Ang II infused rats on normal (132.6 1 16.9
% PE contraction) and high salt intake (90.6 i 11.5 % PE contraction) were not
significantly different from each other or their respective controls (100.9 i 9.4 and
134.7 :t 27.6 % PE contraction). There were no significant differences in the

E050 values between the four groups in response to PE.

As shown in figure 7.4, superior mesenteric arteries from Ang II infused
rats on normal salt intake had significantly increased maximum responses to ET-
1 (251.8 1: 28.6 % PE contraction) compared to controls (135.3 i 31.9 % PE

contraction; p=0.0495). Conversely, superior mesenteric arteries from Ang II

162

 

+ High saltcontrol,n=5

----- o----- High salt+ABT-627, n=5
+ Norm alsalt control, n=5

----- A----- Normal salt + ABT-627, n=5

 

 

 

160
Angiotensin II (5 nglm in)

140 a

120 —

MAP(mmHg)

 

100 .

 

 

 

Pro-infusion Post-infusion

 

160
Phenylephrine (2 uglm in)

140 _

120 4

MAP (mmHg)
zeal-at- st-

 

100 -

 

 

 

80 . .
Pre-infusion Post-infusion

Figure 7.3 Line plots show mean arterial pressure (MAP) responses to infusion
of angiotensin II at 5.0 nglmin or phenlyephrine at 2uglmin for two hours in rats
on either high (circles) or normal (triangles) sodium intake. Responses are
shown the day before (solid lines) and one hour after (dashed lines)
administration of ABT-627 (2mglkg). *Significant (p,0.05) difference in mean
arterial pressure from pre-infusion time point.

163

Table 1. Mean arterial pressure in rats given chronic Ang II (5nglmin) infusion

 

Pre—lnfusion of Ang II

Post-Infusion of Ang II

 

 

 

 

 

Normal salt control 105 :r 2 110 i 3
Normal salt+Ang II 115 :2 125 i5*
High salt control 114 i 2 121 j: 6
Highsalt+Angll 115 i4 136 i7*

 

 

 

Data are mean :t S.E.M. * indicates significantly different from pre-infusion

values (p<0.05)

164

 

 

——d>—— lliglt saiH:+ llrlg IL n:-6
-—<>—— lligltlsait CCInthIL n==6
+ Norm alsalt+ Ang II, n=6

 

 

 

 

 

 

 

 

 

 

 

 

c
o -—£>—— hlorun al:salt ccintrtih n==5
:3 3(30
0
“ .25l) -
‘- ‘-
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I“ 100 «
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i O
:2 5° Z.
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-11 -10 -9 -8 -7
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0
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ur
a 50 "
$ 0 ..
-9 -8 -7 -6 -5
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Figure 7.4 Concentration dependent contraction to endothelin-1 (ET-1) and
phenylephrine (PE) in superior mesenteric artery from normotensive control and
hypertensive angiotensin II (Ang II) infused rats on normal and high salt intake.
Values are mean SE; n= number of animals. For ET-1, there were no differences
in ECso values between groups. In normal salt rats, maximal responses to ET-1
were significantly greater in the group that received chronic angiotensin II (Ang II)
infusion. For PE, there were no significant differences in ECso values or maximal
responses between the groups.

165

infused rats on high salt intake had significantly decreased maximum response to
ET-1 (123.4 i 35.4 % PE contraction) compared to controls (245.6 i 41.7 % PE
contraction; p=0.0263). There was no significant difference in maximal
contraction to ET-1 between high salt and normal salt control groups (p=0.141).
There were no significant differences in the E050 values between the four groups

in response to ET-1.

166

DISCUSSION

The overall goal of my research is to define the mechanisms responsible
for the salt-sensitivity of Ang II induced hypertension. Recent work by others
implicates ET-1 in the acute and chronic pressor effects of Ang II. Specifically, it
has been proposed that Ang II can interact with ET—1 in at least two ways. First,
Ang II has been shown to increase endothelial cell synthesis and release of ET-1
both in vitro (Emori et al., 1991; Dohi et al., 1992; Imai et al., 1992; Kohno et al.,
1992) and in vivo (Barton et al., 1997; Moreau et al., 1997; Lariviere et al., 1998;
Ferri et al., 1999). Second, Ang II administered in vitro potentiates the vascular
contractile response to other agonists (Henrion et al.,1992). Endogenous or
infused Ang II administered in vivo also augments contractile responses to other
agonists (Qui et al., 1994; Dowell et al.,1996). I recently demonstrated that
blockade of ETA receptors in rats receiving chronic intravenous infusions of Ang
II causes a larger and more sustained fall in arterial pressure when the rats were
on a high versus a normal salt intake (Chapter 4). This result led me to conclude
that ET-1 could participate in the mechanism of salt-sensitivity in Ang II induced
hypertension. The goal of the current studies was to test the hypothesis that the
salt-sensitivity of Ang II induced hypertension is due in part to amplification of the
vascular contractile effects of ET-1. The main new finding from this work is that
chronic infusion of Ang II alters vascular reactivity to ET-1 in a salt-dependent
fashion: in rats on normal salt intake maximum contractile responses to ET-1 are

increased, while in rats on high salt intake they are decreased.

167

In vitro studies have reported that short-term exposure (30-60 minutes) to
very low (subthreshold for direct contraction) concentrations of Ang II potentiate
vascular contractile responses to other agonists (Henrion et al., 1992a). The
mechanism of this effect is not fully elucidated, but probably involves activation of
protein kinase C in the vascular smooth muscle cell (Henrion et al., 1992b). This
action of Ang II has not been tested with ET-1 as the second agonist, but the
strong similarity in signaling mechanisms used by Ang II and ET-1 in vascular
smooth muscle (Tsuda et al., 1993) makes it a likely possibility. Furthermore,
there is evidence from chronic (6 day) in vivo experiments in rats that the pressor
actions of ET-1 are potentiated by concomitant exposure to Ang II (Yoshida et
al., 1992). Another study, though, failed to find such an effect during rapid, bolus
injections of the two agonists in the canine coronary circulation (Kiss et al.,
1998). My data do not support the idea that contractile effects of ET-1 are
amplified after short-term exposure to Ang II in vitro, at least in the superior
mesenteric artery of the rat. I observed no significant change in the
concentration-response curve to ET-1 in arteries pre-exposed for one hour to
Ang II.

Pressor responses, and changes in hindlimb vascular resistance, to acute
bolus injections of Ang II in vivo are reported to be reduced by prior blockade of
ET-1 receptors, especially when low doses of Ang II are administered
(Balakrishnan et al., 1996; Champion et al., 1998). This suggests that
physiological amounts of ET-1 may amplify the vascular contractile response to

Ang II in vivo. I performed experiments to investigate this phenomenon in rats on

168

 

normal and high salt intake. My results confirm that pressor responses to acute
(2 hour) exposure to low amounts of Ang II in vivo are significantly reduced by
prior blockade of ETA receptors. This effect appears to be specific for Ang II
since no change in the pressor response to phenylephrine infusion was seen
after administration of the ETA antagonist. One interpretation of these results is
that Ang II infusion for 2 hours stimulated the release of ET-1 from arterial
endothelial cells, and that this ET—1 accounted of the accompanying rise in
arterial pressure. Most studies, however, have failed to find evidence for ET-1
release by Ang II in short-term infusion protocols (Klein et al., 1995; Delemarre et
al., 1998), including an investigation in humans on differing levels of salt intake
(Ferri et al., 1999). Thus, I interpret the results to indicate that physiological
amounts of ET-1 acting at ETA receptors amplify the pressor actions of
exogenous Ang II. My data, however, do not provide any insight into the
mechanism of this interaction. It is notable though that this short-term effect was
observed in rats on both normal and high salt intake, and therefore is not likely to
alone explain the salt-sensitivity of chronic Ang II induced hypertension.

In a final experiment, I evaluated vascular reactivity to ET-1 in vitro in
superior mesenteric arteries from rats that received chronic (7 day) infusions of
Ang II. Interestingly, I found divergent results depending on whether the rats
were on normal or high salt intake. Rats on high salt intake became significantly
more hypertensive than rats on normal salt intake, as has been described many
times previously. Furthermore, maximal response of mesenteric arteries to ET-1

from these rats were significantly suppressed compared to responses in control

169

rats on high salt intake. Similar results have been reported by other investigators
using a model of chronic Ang II induced hypertension involving infusion of higher
doses of Ang II (200 nglkglmin, subcutaneously) in rats on normal salt intake
(Rajagopalan et al., 1997; D’Uscio et al., 1997). Arteries from those rats
exhibited a marked increase in preproET-1 gene expression and ET-1 peptide
content (Rajagopalan et al., 1997; D’Uscio et al., 1997), and there was a strong
negative correlation between reactivity to ET-1 and arterial peptide
concentrations (D’Uscio et al., 1997). It has been suggested that a decrease in
mesenteric response to ET-1 in vitro is a consequence of ETA receptor down-
regulation caused by chronic increase in ET—1 release (Nguyen et al., 1992).
Although I did not measure arterial content of ET-1 in my studies, I propose that
a similar mechanism could account for depressed maximal responses to ET-1 in
superior mesenteric arteries from rats in my study on high salt intake and Ang II
infusion.

Mesenteric arteries from rats receiving an infusion of Ang II for 7 days but
maintained on a fixed normal salt intake exhibited a significantly increased
maximum response to ET-1 in vitro. To my knowledge, this is the first report of
potentiation by exogenous Ang II of in vitro contractile responses to ET—1 in
vascular smooth muscle, although others have reported that Ang II amplifies the
bronchoconstrictor actions of ET-1 via a leukotriene-dependent pathway (Pitt and
Nally, 1999). As shown in figure 6.1, contraction of superior mesenteric arteries
to ET-1 is mediated exclusively through ETA receptors, and Ang II has been

reported to increase ETA receptor expression (Hatakeyama et al., 1994). Thus,

170

 

one potential explanation for my results is that chronic exposure to Ang II
increased ETA receptor number in superior mesenteric arteries in rats on normal
salt intake.

This still does not explain, however, why the effects of chronic Ang II
infusion on contraction of superior mesenteric arteries to ET-1 in vitro were
different in rats on high versus normal salt intake. My experiments do not
provide a definitive answer. I speculate that long-term exposure to Ang II can
upregulate both ETA receptor number and preproET-1 gene expression. Though
high salt intake alone does not stimulate vascular ET-1 formation (D’Uscio et al.,
1997; Barton et al., 1998; Ikeda et al., 1999), my data are consistent with the
idea that high salt intake plus Ang II may be a more effective stimulus to
preproET-1 gene expression and ET-1 synthesis than Ang II alone. One
possible mechanism could involve the effects of Ang II and high salt intake on
endothelial nitric oxide (NO) action. Long-term exposure to Ang II in vivo
(D’Uscio et al., 1997), and to high salt intake (Boegehold, 1995), are reported to
impair NO activity in resistance arteries. There is also evidence that increased
NO activity suppresses ET-1 formation in blood vessels (Boulanger and Liischer,
1990). The combination of Ang II and high salt could produce a larger increase
in vascular ET-1 synthesis than Ang II alone because of less NO-mediated
inhibition. In support of this idea, it has been shown that administration of the NO
synthase inhibitor, L-nitro—arginine methyl ester, to rats on normal salt intake
caused a highly significant potentiation of the chronic pressor responses to Ang II

infusion in rats (Melaragno and Fink, 1996). Finally, there is evidence that the

171

pressor (Mortensen and Fink, 1992) and vascular resistance (Grossman et al.,
1990) effects of ET-1 are increased by high salt intake. Therefore, enhanced
synthesis of ET-1 in blood vessels in rats on high salt intake receiving Ang II,
combined with some amplification of the pressor effect of ET-1 by high salt alone,
could explain the larger contribution of ET-1 to Ang II induced hypertension under
high salt conditions. Direct evidence for this theory needs to be obtained in
future experiments.

In summary, Ang II was shown to cause changes in pressor and vascular
contractile effects of ET-1 that are dependent on time of exposure to Ang II, and
on salt intake. During long-term infusion Ang II can increase both vascular
reactivity to ET-1 and ET-1 formation. Each of these mechanisms may
contribute to the dependence of Ang II induced hypertension on ETA receptor

activation, but high salt intake apparently shifts the balance toward the latter.

172

Chapter 8

General Discussion

A large proportion of the population of the modem westemized countries
will develop hypertension during their lifetimes (Kaplan, 1998c; Mohrman and
Heller, 1991). High blood pressure is now second only to upper respiratory
infections as an indication for office visits to physicians in the United States
(Woodwell, 1997). Hypertension is the most pervasive modifiable risk factor for
cardiovascular disease (Kannel and Wilson, 1999) and heart disease is the major
killer of people living in modernized societies. The high prevalence of
hypertension, it’s robust impact on the incidence of heart disease, and it’s
potential to be treated justify the continued quest of physicians, biomedical
researchers, and health officials to develop a better understanding of this
deceivingly complex medical disorder.

Hypertension is defined simply as a chronic elevation of arterial blood
pressure above 140/90 mmHg (JNC-V, 1997). Approximately 95% of diagnosed
cases of hypertension are attributed to essential hypertension, or increased
blood pressure without an obvious cause (Deshmukh et al., 1998). There are
some universally accepted contributing factors to essential hypertension
(Mohrman and Heller, 1991). Among these are environmental factors (stress,
smoking, high salt/ high fat diet, sedentary lifestyle), genetic influences, structural
changes in the heart and peripheral blood vessels, alterations in neural control of

blood pressure, and renal defects.

173

There are also several known hormonal regulators of blood pressure,
which include Ang II and ET-1. These two peptide hormones affect blood
pressure both individually and in tandem, under both acute and chronic

conditions.

Ang II and ET-1 can each cause hypertension

As was discussed in chapter 1, Ang II affects blood pressure by a myriad
of mechanisms, including direct vasoconstriction, activation of the sympathetic
nervous system, release of aldosterone from the adrenal gland, direct stimulation
of renal sodium and water retention, and cardiac and vascular hypertrophy. The
importance of Ang II as an etiologic factor in human hypertension is undisputed.

ET-1 modulates local vascular tone and structure by both ETB receptor
mediated release of vasodilating substances (namely NO and prostacyclin) from
the endothelial cells, and by ETA receptor mediated strong, long-lasting
constriction of the vascular smooth muscle cells. The renal actions of ET-1 are
more controversial and could serve to either increase or decrease arterial
pressure. Recent evidence strongly supports an important role for ET-1 in the

pathogenesis of human hypertension.

Salt intake contributes to severity of Ang II and ET-1 induced hypertension
It has long been established that infusion of Ang II at low doses causes a
more severe hypertension in subjects on high salt intake than in subjects on

normal salt intake (Muirhead et al., 1975; Kanagy et al., 1990; Simon, 1998).

174

 

The magnitude of the hypertension in this model is dependent on the rate of Ang
II infusion, the salt intake, and the duration of the infusion. In some studies, an
initially subpressor infusion rate caused a gradual rise in arterial blood pressure
after several days (refs). This salt sensitive model of hypertension was used in
most experiments in this thesis project. Although my protocols involved identical
rates and duration of Ang II infusion, the differences in blood pressure response
between rats on normal and high salt intake were not identical between studies,
even though the high salt rats always showed a larger pressor response. I
cannot fully account for this variable effect of salt intake, but possibilities include
variation between litters or seasonal effects.

It also has been clearly demonstrated that hypertension induced by
chronic ET—1 infusion is salt-dependent (Mortenson and Fink, 1990). While in
many other forms of salt-sensitive hypertension the role of endogenous Ang II in
blood pressure control is reduced, the contribution of ET-1 is enhanced.
Increased ET-1 plasma levels have been reported in human patients with salt-
sensitive hypertension, particularly African Americans and the elderly (Ferri et al.,
1998; Ergul et al., 1996). Furthermore, ET-1 is now an accepted etiologic factor
in several common salt-sensitive experimental models of hypertension, including
DOCA-salt, Dahl salt-sensitive, one-kidney-one—clip Goldblatt (1 K1 C) and chronic

renal failure (Schiffrin, 1999).

175

 

 

 

ET-1 mediates some of the cardiorenal effects of Ang II

As was discussed in chapter 1, Ang II stimulates the synthesis and
release of ET-1 from the endothelial cells and increases renal ET-1 formation.
Moreover, ET-1 amplifies the pressor effects of Ang II and partially mediates Ang
II induced vascular hypertrophy. ET-1 receptor blockade begun prior to the start
of chronic Ang II infusion prevents the development of Ang II induced
hypertension. Thus, Ang II and ET-1 may interact to increase blood pressure by

renal as well as vascular mechanisms.

Ang II induced hypertension is the best model to study the interaction
between Ang II and ET-1

The overall objective of my thesis project was to examine the interaction
between Ang II and ET—1 in salt-sensitive hypertension. Several experimental
models exist to study the role of the RAS in hypertension. The two-kidney-one-
clip Goldblatt model (2K1 C) is the best characterized renin-dependent model of
hypertension. Most studies show that ET-1 antagonists do not signifiwntly
affect blood pressure in this model (Sventek, 1996). A problem with using this
model for studying possible ET-1 mediated effects of Ang II on blood pressure is
that ET-1 can affect other components of the RAS besides Ang II. For example,
ET—1 activates ACE (Kawaguchi et al., 1991) and can decrease renin secretion
(Berthold et al., 1999). Additionally, I was interested in studying salt-sensitive

hypertension and blood pressure in this model is not affected by salt intake.

176

Therefore, I chose to use an experimental model that is both salt-dependent and

unequivocally caused by the actions of Ang II.

ET-1 receptor antagonists reveal ET-1 mediated effects in vivo and in vitro

The main experimental strategy I chose to study the interaction between
Ang II and ET-1 in salt-sensitive hypertension was measurement of
hemodynamic and renal responses to pharmacological antagonists in vivo. This
strategy was supplemented by use of in vitro studies of vascular contractility.
Other possible experimental approaches to addressing my overall hypothesis
could have included measurements of ET-1 peptide concentrations, preproET-1
gene expression, ECE activity, or ET-1 receptor expression in vasculature and
other target tissues in vivo and in vitro. Genetically manipulated strains of
rodents lacking key components of the ET-1 system also could have been
employed for in vivo and in vitro studies.

A potential weakness of my pharmacological strategy would be a lack of
either efficacy or selectivity of the ET-1 antagonists. I attempted to minimize
these possibilities by extensively characterizing the efficacies (acutely and
chronically) and selectivities of the antagonists l employed in my main studies.
One outcome of these preliminary studies was to employ an ETA selective
receptor antagonist in addition to PD156707 as an experimental tool. This
decision was based on concerns about the effectiveness of PD156707, probably

related to it’s very short plasma half-life. In the cases of ABT-627, A-192621

177

 

and A-182086, my preliminary data indicated these dnigs had adequate effects

and receptor selectivity for use in the in vivo studies.

ET-1, especially under conditions of increased salt intake, is required for
the maintenance of experimental Ang II induced hypertension

The data obtained in my thesis project is integrated with previous findings
in figure 8.1 to generate a new model representing the interrelationships between
salt-intake, Ang II, and ET-1 in the pathogenesis of hypertension. This model
focuses on actions of Ang II and ET-1 on the vasculature, and on the kidney.
Each peptide has other potential targets that could exert an influence on short- or
long-term control of blood pressure. These were not investigated in my studies
and therefore are not included in the model. My study, and the model, were
predicated on the assumption that minute-to-minute control of MAP is primarily
achieved by changing vascular smooth muscle tone, whereas long-term steady-
state blood pressure levels are determined by both vascular mechanisms and the
natriuretic and diuretic functions of the kidney (Hall, 1986). Accordingly,
inferences about ET-1 mediated vascular mechanisms were derived from both in
vitro contractile studies, and from acute responses to ET—1 receptor antagonists
in vivo. Similarly, inferences about ET-1 mediated changes in sodium and water
balance were derived from steady-state changes in blood pressure and sodium

excretion during chronic administration of ET-1 receptor antagonists.

178

 

179

 

180

The main new findings from my research that are incorporated into the model
are: vascular pressor effects mediated by ETA receptor activation are amplified
by Ang II, but not high salt intake; renal pressor effects mediated by ETA receptor
activation are amplified by the combination of high salt intake and Ang II;
vasodilation mediated by ETB receptor stimulation is amplified by high salt intake;
Ang II modifies the response to vascular ETB receptor activation in a salt-
dependent manner; and renal effects of ETB receptor activation are not
significantly influenced by Ang II (Table 8.1). These findings will be discussed in

more detail in the following sections.

181

182

 

Vascular Mechanisms

Renal Mechanisms

 

Ang II

Salt

Ang II

Salt

 

ETA
pressor

response

I (Tl)

-- (I)

II

 

 

ETB
depressor

response

 

ii

 

 

 

 

183

 

 

Ang II induced hypertension depends primarily on the actions of ET-1 on
ETA receptors, and this effect is greater under conditions of high salt intake

I chose to investigate first the effect of ETA receptor activation on Ang II
induced hypertension because this receptor subtype was the best characterized
at the onset of this thesis project. ETA receptors were known to be expressed
highly in the vascular smooth muscle cells and kidney, but not in endothelial cells
(lnagami et al., 1993; Haynes and Webb, 1998), suggesting an important role for
this receptor in acute and chronic blood pressure control. In vivo work in humans
demonstrated that endogenous ET-1 confers basal vasoconstrictor tone via ETA
receptor activation (Webb et al., 1998). Most importantly, studies in humans with
essential hypertension revealed increased ETA mediated vasoconstriction
compared to normotensive controls (Cardillo et al, 1999). In a later study, this
increased ETA mediated vasoconstriction was linked to activation of Ang II AT1
receptors (Ghiadoni et al., 2000).

Previous work by others showed that specific blockade of ETA receptors
significantly attenuated the development of Ang II induced hypertension (D’Uscio
et al., 1997; Rajagopalan et al., 1997). However, none of these earlier studies
addressed the potential effects of salt intake, or the effects of ETA receptor
activation on chronic sodium and water excretion.

In this thesis, I present the novel findings that selective ETA receptor
blockade lowered MAP in Ang II induced hypertension within one hour of drug

administration, whereas the dmg had no effect on blood pressure in rats not

184

receiving Ang II. This effect was not modified in rats on differing salt intakes. l
interpreted these results to indicate that chronic infusion of Ang II increased
vascular ETA receptor activation, independent of salt intake. In related studies
using a two-hour intravenous infusion protocol, pressor responses to Ang II were
significantly inhibited by prior blockade of ETA receptors in rats on both high and
normal salt intake. There are at least two possible mechanisms to explain these
data. Ang II could increase ET-1 tissue concentrations and/or increase vascular
reactivity to endogenous ET—1. The former has been demonstrated in earlier
studies of Ang II induced hypertension and was not directly evaluated in my
work. The latter mechanism was investigated further in a series of in vitro
vascular reactivity studies.

In rat superior mesenteric arteries, where I showed that ET-1 mediated
contraction is due entirely to ETA receptor activation, vascular contractile
responses to ET-1 were dramatically affected by chronic Ang II infusion. In
arteries from rats on high salt intake and Ang II, vascular contractile responses to
ET-1 were significantly reduced compared to high salt controls. Conversely, in
arteries from rats on normal salt intake and Ang II, vascular contractile responses
to ET-1 were significantly augmented compared to normal salt controls. Salt
alone did significantly affect ET-1 induced contractile responses. The precise
reasons for this differential response to Ang II are not known. Based on previous
work, chronic Ang II infusion has at least three important effects on the arterial
vasculature: increased ET-1 synthesis, increased ETA receptor number, and

decreased nitric oxide activity (Hatakeyama et al., 1994; D’Uscio et al., 1997;

185

Rajagopalan et al., 1997; D’Uscio et al., 1998). Thus, the expected ET-1
contractile response of arteries from Ang II infused rats would be the result of the
net effect of these three changes. Increased ET-1 content in arteries would be
expected to decrease contractile responses to exogenous ET-1 due to ETA
receptor downregulation (D’Uscio et al., 1997). Increased ETA receptor number
would be expected to increase ET-1 contractile responses. Diminished NO
activity would be expected to increase contractile responses to most agonists,
including ET-1. However, a long-term suppression of NO activity would also be
expected to increase arterial ET-1 synthesis (Boulanger and Li‘ischer, 1990).
High salt intake alone does not affect vascular ET-1 synthesis (Ikeda et al, 1995;
D’Uscio et al., 1997; Barton et al., 1998). The influence of high salt intake on
ETA receptor number is not known. However, high salt intake has been shown to
decrease NO activity in arterial resistance vessels (Boegehold, 1995). l
speculate that Ang II infusion during high salt intake shifts the balance of these
three vascular effects in favor of increased ET-1 synthesis, and thus diminished
vascular reactivity to exogenous ET-1 because of ETA receptor downregulation.
I further speculate that, under normal salt conditions, the balance of the effects
produced by Ang II favor increased ETA receptor number and diminished NO
mediated vasodilation. Thus, vascular responsiveness to ET-1 would be
increased. 1

Previous studies showed increased ET-1 synthesis in the kidneys of rats
with Ang II induced hypertension (Barton et al., 1997; Barton et al., 1998). In my

rats receiving long-term Ang II infusion, chronic treatment with ABT—627

186

 

completely normalized blood pressure within one day. This effect was well-
maintained over the 5 day treatment period in rats on high salt intake, but
gradually subsided over 5 days in rats on normal salt intake. In control rats on
either salt intake, chronic ETA receptor blockade had no effect on blood pressure
or sodium excretion. This suggests that chronic Ang II infusion shifts the
pressure-natriuresis relationship to a higher pressure level in part by activation of
renal ETA receptors and that this effect is greater on high salt intake. Consistent
with my results, in hypertensive Dahl salt-sensitive rats ETA receptor blockade
also caused a shift in the pressure-natriuresis relationship to a lower pressure
level (Kassab et al., 1998). This effect of chronic renal ETA receptor blockade
could be due to either inhibition of ETA receptor mediated renal hemodynamic
effects or to unopposed ETB receptor mediated natriuresis and diuresis. The
second possibility was addressed in additional studies in my thesis project, and
may provide an explanation for the increased chronic antihypertensive effect of

the ETA receptor antagonist under high salt conditions.

The initial hypotensive effect of ETA receptor blockade in Ang II induced
hypertension is not due to a diuretic effect

A comparison of the time course and antihypertensive effects of ETA
receptor blockade and thiazide diuretic treatment in Ang II induced hypertension
was performed to determine if the primary antihypertensive response to ETA
receptor blockade was caused by effects on the vasculature or the kidney. My

study of the antihypertensive effect of trichlormethiazide in Ang II induced

187

hypertension served as a positive control for the effects of ETA receptor
blockade. The effect of a diuretic drug had not been previously tested in Ang II
induced hypertension, and thus the time course and blood pressure effects of
this treatment were not known. The reduction in blood pressure resulting from
ETA receptor blockade occurred rapidly and was associated with a tendency for
increased sodium and water balance during the first 24 hours. This pattern of
response closely resembles that seen with chronic administration of minoxidil, a
direct vasodilator, to rats with Ang II induced hypertension (Melaragno and Fink,
1996b). On the other hand, the peak blood pressure lowering effect of the
thiazide diuretic required 72 hours and was preceded by a large loss of sodium
and water. Thus, it is clear that ETA receptor antagonism does not lower blood
pressure initially in Ang II induced hypertension via a diuretic effect on the

kidney.

ETB receptor activation opposes Ang II induced hypertension

In contrast to ETA receptor mediated events, ETB receptor activation tends
to decrease MAP by causing release of endothelial vasodilators and promoting
renal loss of sodium and water. Furthermore, recent studies showed that a
naturally occurring deletion of the ETB receptor gene (Gariepy et al., 2000) and
chronic blockade of ETB receptors cause salt-sensitive hypertension in rats
(Pollock, 2000). Previous reports demonstrated that ETB receptor activation
opposes increases in MAP in both human essential hypertension (Cardillo et al.,

1999) and in DOCA-salt rats (Pollock et al., 2000).

188

E

My thesis work was the first investigation of the effects of a selective ETB
receptor antagonist in Ang II induced hypertension. My findings, in agreement
with those from other models of hypertension, showed that ETB receptor
activation opposes the rise in MAP seen in Ang II induced hypertension.
Moreover, selective blockade of ETB receptors resulted in a significant increase in
MAP within minutes of the initiation of drug therapy (suggesting a vascular
mechanism) in all four groups of rats studied, regardless of salt intake or Ang II
infusion. It should be noted, however, that the increases in MAP were largest in
rats receiving high salt intake, suggesting high salt diet enhances ETB receptor
mediated vasodilation. The mechanism for this salt-dependent difference in ETB
mediated vasodilation is unknown. None of my in vitro vascular reactivity
experiments were designed to investigate ETB mediated vascular effects.

Future studies could be conducted to investigate the effects of S6c, the ETB
selective receptor agonist , on resistance arteries from rats maintained on
varying salt intakes. Possible explanations for an apparent increase in ETB
mediated vasodilation in rats on high salt intake include: increased ETB receptor
number or affinity for ET-1, increased vascular cyclooxygenase activity, and
increased vascular reactivity to either NO, PGI2, or other endothelial-derived
vasodilators.

An important new finding of my thesis research was that Ang II appears to
attenuate ETB receptor mediated vasodilation in rats on high salt intake, yet
augment this effect in rats on normal salt intake. The former effect plays a major

role in promoting hypertension in rats receiving chronic Ang II infusion and high

189

salt intake. Conversely, the ability of Ang II to increase ETB mediated
vasodilation probably plays a role in preventing hypertension in rats on normal
salt intake. The mechanism for this salt-dependent difference in the effects of
Ang II on ETB receptor mediated vasodilation is unknown. This could be
addressed in future studies by investigating the vascular contractile responses to
S6c of resistance arteries from rats receiving both chronic Ang II infusions and
varying salt intakes. Possible explanations for the effects of Ang II on ETB
receptor mediated vasodilation in high salt rats are that: combined effects of high
salt and Ang II to decrease NO activity in resistance arteries could impair ETB
mediated vasodilation, which is mediated in part by NO; or, combined effects of
high salt and Ang II could increase ET-1 synthesis, which would lead to
downregulation of ETB receptors. A possible explanation for the effects of Ang II
on ETB receptor mediated vasodilation in normal salt rats is increased ETB
receptor number or affinity. Ang II was shown to increase ETB receptor
expression in cardiac myocytes In vitro (Kanno et al., 1993).

Combined blockade of both ETA and ETB receptors with A-182086
produced a decrease in MAP that was presumably due to the net effect of
inhibiting both ETA mediated vasoconstriction and ETB mediated vasodilation.
Since the overall result of combined ET-1 receptor blockade was a slight drop in
blood pressure only in rats receiving chronic Ang II infusion, it can be assumed
that the ETA mediated vasoconstriction predominates over ETB mediated
vasodilation in Ang II induced hypertension. This is supported by my findings

that both the in vivo pressor and the in vitro vascular contractile effects are

190

exclusively due to ETA receptor activation. The acute depressor response to
combined ET-1 receptor blockade in rats receiving Ang II was presumably larger
in high salt rats than in normal salt rats because ETA mediated vasoconstriction
is similar regardless of salt intake, whereas ETB mediated vasodilation is

significantly greater in rats on normal salt intake.

 

Published studies show chronic Ang II infusion increases renal ET-1
content (Barton et al., 1998), while the effect of salt intake on renal ET-1
formation is controversial (Firth et al., 1995; Melo et al., 1998; Morita et al.,
1999). As discussed above, it is likely that renal actions mediated by ETA
receptors contribute to the sustained increase in MAP observed in rats with Ang
II induced hypertension. ETB receptor activation has been reported to cause

natriuresis and diuresis (Kohan et al., 1993; Clavell et al., 1995). My data

 

suggest that ETB receptor mediated natriuretic and diuretic effects are not
influenced by chronic Ang II infusion. This is somewhat surprising in view of
evidence that Ang II infusion increases renal ET-1 synthesis (Barton et al., 1998).
Other work indicates that high salt intake increases ETB receptor mediated
natriuretic and diuretic effects (Pollock, 2000; Gariepy et al, 2000), and my data
tend to support this idea. The mechanism of the effect is not known, but could
involve increased renal NO activity secondary to high salt intake (Schultz and
Tolins, 1993). Natriuretic responses elicited by ETB receptor stimulation are
mediated in part by NO (Hoffman et al., 2000). As noted earlier in this

discussion, increased ETB receptor mediated natriuretic and diuretic effects could

191

account for the relatively better antihypertensive efficacy of selective ETA

receptor antagonism in high versus normal salt rats.

Conclusions

Ang II induced hypertension is a well-established salt-sensitive model of
hypertension. ETA selective receptor antagonists have been shown to lower
blood pressure in salt-sensitive models of hypertension (Schiffrin, 1999) and to
prevent the development of Ang II induced hypertension (D’Uscio et al., 1997;
Rajagopalan et al., 1997). The data presented in this thesis show that selective
ETA receptor blockade alone normalizes MAP in Ang II induced hypertension,
especially under conditions of high salt intake, presenting a strong case that ET-1
acting at ETA receptors is an important mechanism of the salt-sensitivity of Ang II
induced hypertension.

Conversely, decreased ETB receptor function has been shown to increase
MAP in other models of hypertension (Pollock et al., 1999; Matsumura et al.,
2000; Cardillo et al., 2000). My thesis work was the first to show that this is also
true in Ang II induced hypertension. Furthermore, the depressor actions of ETB
receptor stimulation in the vasculature and the kidney are increased by high salt
intake. Thus, combined blockade of both ETA and ETB receptors is less effective
at lowering blood pressure than selective ETA receptor antagonism under
conditions of high salt intake. An implication of this conclusion is that selective
ETA receptor blockade should be more effective than combined ET-1 receptor

blockade at lowering blood pressure in salt-sensitive subgroups of human

192

 

hypertensive patients, such as African Americans and the elderly. Similarly,
combined ET-1 receptor antagonists should be most effective in subgroups that

are salt-resistant or on a low-salt diet.

 

193

 

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