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THE ROLE OF THE SLOW PRESSOR EFFECT OF ANGIOTENSIN II
IN THE DEVELOPMENT OF HYPERTENSION

By

Vyvian Janet Gorbea-Oppliger

A DISSERTATION

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

DOCTOR OF PHILOSOPHY

Department of Physiology

1994

ABSTRACT

THE ROLE OF THE SLOW PRESSOR EFFECT OF AN GIOTENSIN II IN THE
IN THE DEVELOPMENT OF HYPERTENSION

BY

VYVIAN JANET GORBEA-OPPLIGER

The slow pressor effect of angiotensin 11 (AH) is an important and
puzzling phenomenon. This effect develops at low plasma concentrations of
the peptide and causes a slowly-developing but ultimately large rise in blood
pressure. The recently developed selective, non-peptidergic, AT1 AH receptor
antagonists, losartan, and its active metabolite EXP 3174, were used to
investigate the mechanism of the slow pressor effect of A11 in a rat model of
hypertension. In this model, rats are chronically instrumented with arterial
and venous catheters. Experiments were performed to determine a dose of
All that was acutely (seconds to minutes) subpressor but ultimately produced
large elevations in blood pressure (days to weeks). This dose was utilized to
explore the role of the slow pressor effect of All in the development of
hypertension. Losartan effectively reversed the hypertension produced by
infusion of A11. The results indicate that there are two components to the
antihypertensive effect of losartan: a rapid effect due to blockade of AT1
receptors mediating the fast (seconds) AII pressor responses and a slower
effect (minutes to hours) due to blockade of other AT1 receptors.

Additional experiments using the sympatholytic drug clonidine were

performed to evaluate the neurogenic pressor activity in rats made

Vyvian Janet Gorbea-Oppliger
hypertensive by chronic infusion of a low dose of AH. The experiments
demonstrated that chronic intravenous infusion of AH (15 days) caused a
sustained hypertension that could be partially reversed by clonidine.

Clonidine did not affect arterial pressure in normotensive rats. These results,
and the findings of previous studies, indicate that the slow pressor effect of
AH is mediated in part by neurogenic mechanisms.

A final set of experiments was designed to test the following
hypothesis: if the hypertension observed during chronic infusion of AH is due
to circulating AH interacting with areas of the brain lacking a tight blood brain
barrier, then selective central administration of an AH antagonist should
reverse the hypertension. The AT1 antagonists, when administered
intracerebroventricularly (i.c.v.) at the level of the third ventricle, did not
attenuate the hypertension produced by AH. However, additional
experiments demonstrated that i.c.v. AT1 receptor antagonists do not gain
access to critical receptor sites where AH may act to produce hypertension.

Therefore, these experiments neither support nor refute the hypothesis.

Para losé y Elba, mis padres queridos.

Gracias por su ejemplo, apoyo y amor. . .

iv

ACKNOWLEDGMENTS

There are no words that could adequately express my appreciation to
the people who have helped me immensely throughout my graduate career.
To Dr. Gregory D. Pink and Dr. Robert B. Stephenson, thank you for your
encouragement, guidance, support, and friendship.

I would also like to thank Dr. Edward N. Robinson, Dr. John E.
Chimoskey, and Dr. Bari Olivier for their support and helpful advice while
serving on my thesis committee.

I am also grateful to Dr. William S. Spielman, Dr. William D. Atchison,
Dr. Nancy L. Kanagy, Dr. Luke H. Mortensen, Dr. Laurey R. Hanselman,
Claudette R. Buckingham, Sharon Shaft, Carmen Gear, Sharla Erbe, Gregg S.
Potter, Matthew G. Melaragno, and Renee L. Petit. I thank them for their
support, friendship and assistance.

To my parents and family Iosé V. Gorbea, Elba I. Gorbea, Ramona
Garcia vda. Padro, Ana E. Gorbea, Elba I. Gorbea, Larry D. Oppliger, and Kay
Oppliger, thank you for your love, example and support.

Finally, I wish to thank my husband and best friend Scott I. Oppliger
for his help, support and love. I thank God for his friendship, love, sense of

humor and understanding. Gracias. . .

TABLE OF CONTENTS

LIST OF TABLES ............................................. ix
LIST OF FIGURES ............................................. x
LIST OF ABBREVIATIONS ..................................... xiv
INTRODUCTION ..... . . . . . . ................................... 1

 

II.

1. Arterial and venous catheterizations ............ 19
2. Intracerebroventricular cannulation ............. 20

3. Catheterization for exposure of the dorsal
medulla ............................... 21
C. Wm .................... 21
D. W ...................... 22
E. Statistics ...................................... 22
F. m ....................................... 23
P ................................ 23

A. WWI:

W ................................ 23
1 Rationale ................................. 23

2. Protocol .................................. 24
3. Results .................................. 24
4. Discussion ................................ 25

3W

W ..................... 32
1. Rationale ................................. 32
2. Protocols ................................. 33
a. Efi‘ect of losartan on fast pressor response to
All .............................. 33
b. Efl'ect of Iosartan on angiotensin II induced
hypertension ....................... 34
3. Results .................................. 34
a. Effect of losartan on fast pressor response to
All .............................. 34
b. Efi‘ect of losartan on chronic All-induced
hypertension ....................... 35
4. Discussion ................................ 37
C. ‘ '
W ................ 72
1. Rationale ................................ 72
2. Protocol .................................. 73
3. Results .................................. 74
4. Discussion ................................ 75
D. WWIW
WW .............................. 84
1. Rationale ................................. 84
2. Protocol .................................. 85
3. Results .................................. 86
4. Discussion ................................ 86
E. WIN
5 . | I I] E B I 511 . I! E
m ........................................ 96
1. Rationale ................................. 96
2. Protocol .................................. 96
3. Results .................................. 98
4. Discussion ................................ 99
SUMMARY AND CONCLUSION ................................ 110
BIBLIOGRAPHY ............................................. 125
APPENDICES ............................................... 139
Appendix A ................................ 139

vii

Appendix B ................................ 140
Appendix C ................................ 141
Appendix D ................................ 142

viii

LIST OF TABLES

Table 1. Mean arterial pressure before and after losartan on days 2, 7,

and 12 of the chronic AH infusion (10 ng min"). .............. 139

Table 2. Mean arterial pressure before and after losartan on days 2, 7,

and 12 of the chronic AH infusion (4 ng min"). ............... 140

Table 3. Mean arterial pressure before and after losartan on days 2, 7, and 12

of the chronic AH infusion (2 ng min“). ..................... 141

LIST OF FIGURES

Figure 1. Mean arterial blood pressure and heart rate responses to chronic AII

infusion at 2 (n=8), 4 (n=5) and 10 ng-rnin’1 (n=6), i.v. ...........

Figure 2. Water balance and urinary sodium excretion responses to

chronic AH infusion at 2.0 (n=8), 4.0 (n=5) and 10.0 ng-rnin’1

Figure 3. Acute change in mean arterial pressure in response to All

infusions at 2.0 and 4.0 ng-rnin‘l .............................

Figure 4. Change in mean arterial pressure in response to bolus
injections of AH in vehicle control and losartan-treated group.

Figure 5. The effects of losartan (3 mg-kg", i.a.) on MAP during chronic

i.v. infusion of AH at 10 ng-min". ..........................

Figure 6. The effects of losartan (3 mg-kg“, i.a.) on heart rate during

chronic i.v. infusion of AH at 10 ng-min“. ....................

Figure 7. The effects of losartan (3 mg-kg", i.a) on water balance during

chronic iv. infusion of AH at 10 ng-min“. ....................

Figure 8. The effects of losartan (3 mg-kg", i.a.) on urinary sodium

excretion (UMV) during chronic iv. infusion of AH at 10 ng-min".

OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO

27

31

45

49

51

Figure 9. The effects of losartan (3 mg-kg“, i.a.) on MAP during chronic
i.v. infusion of AH at 4 ng-min". ........................... 55
Figure 10. The effects of losartan (3 mg-kg", i.a.) on heart rate during
chronic i.v. infusion of AH at 4 ng-min". ..................... 57
Figure 11. The effects of losartan (3 mg-kg", i.a.) on water balance
during chronic i.v. infusion of AH at 4 ng-min". ............... 59
Figure 12. The effects of losartan (3 mg-kg", i.a.) on urinary sodium
excretion during chronic i.v. infusion of AH at 4 ng-min“. ........ 61
Figure 13. The effects of losartan at (3 mg-kg", i.a.) on MAP during
chronic i.v. infusion of AH at 2 ng-min" ....................... 63
Figure 14. The effects of losartan (3.0 mg-kg“, i.a.) on HR during
chronic i.v. infusion of AH at 2.0 ng-min“. ................... 65
Figure 15. The effects of losartan (3 mg-kg“, i.a.) on water balance
during chronic i.v. infusion of AH at 2 ng-min". ................ 67
Figure 16. The effects of losartan (3 mg-kg", i.a.) on urinary sodium
excretion during chronic i.v. infusion of AH at 2 ng-min“. ........ 69
Figure 17. The effects of vehicle control on MAP during chronic i.v. infusion
of AH at 10 ng-min“ ...................................... 71
Figure 18. The effect of bolus injection of clonidine at a dose of
10 ug-kg" during AH infusion at 4.0 ng-min". ................. 81
Figure 19. The effect of bolus injection of clonidine at a dose of

10 ug-kg‘1 during infusion of vehicle (n=5). ................... 83

Figure 20. Blood pressure. responses to i.c.v. AH 149 ng before and

after i.c.v. EXP 3174. .................................... 91
Figure 21. The effects of acute i.c.v. EM” 3174 (1 pg in 2 pl of saline;

n=5) on MAP (top) and HR (bottom) during chronic i.v. infusion

of AH (4ng-min“). ....................................... 93
Figure 22. The effect of acute i.c.v. EXP 3174 (1 pg in 2 pl of saline; n=5) on

WB and UN,V during chronic i.v. infusion of AH (4ng-min"). ...... 95
Figure 23. A summary of MAP responses to rnicroinjections of 500 pg

and 500 ng AH into the area postrema (rising right bars). ........ 103
Figure 24. Change in MAP observed in response to 500 pg and 500 ng

AH into the area postrema before and after i.c.v. EXP 3174 (1 pg

in 2 pl of saline), and intravenous EXP 3174 (1 mg kg“). ......... 105
Figure 25. MAP responses to microinjection of 500 pg AH into the area

postrema of animals that received i.c.v. EXP 3174 after exposure

of the atlantooccipital membrane and prior to surgical incision of

the membrane . ......................................... 107
Figure 26. MAP responses to microinjection of 500 pg AH before (rising

right bars) and after (solid bar) an injection of hexamethonium

(HEX) (20mg-kg"). ...................................... 109
Figure 27. Factors involved in the pathogenesis of hypertension ........ 120
Figure 28. Schematic representation of the mechanisms by which AH

may be interacting at vascular smooth muscle cells (Vasc. Smooth

Ms.) and endothelium. ................................... 122

Figure 29. Schematic representation of the actions of AH at the level of the
area postrema. ......................................... 124
Figure 30. Summary flow chart showing various actions of AH in vascular

smooth muscle. ......................................... 142

xiii

ACE

ACEI

AII

AVP
BBB
CO

CSF

ECF

EDRF

i.a.
i.m.
i.p.
i.v.

i.c.v.

LIST OF ABBREVIATIONS

Angiotensin converting enzyme
Angiotensin converting enzyme inhibitors
Angiotensin I

Angiotensin H

Area postrema

Arginine vasopressin

Blood brain barrier

Cardiac output

Cerebrospinal fluid
Circumventricular organ
Extracellular fluid
Endothelium—derived relaxing factor
Hexamethonium

Heart rate

Intraarterial

Intramuscular

Intraperitoneal

Intravenous

Intracerebroventricular

Mean arterial pressure

Median eminence

xiv

NO

NOS

OVLT

PRA

RVH
RVLM

SEM

s.c.
SFO
SHR
UO

UNa

UNaV

ZKIC

Nitric oxide

Nitric oxide synthase inhibitors
Nucleus tractus solitarius
Organum vasculosum of the laminae terminalis
Plasma renin activity

Renin angiotensin system
Renovascular hypertension
Rostral ventro-lateral medulla
Standard error of the mean
Spinal cord

Subcutaneous

Subfornical organ

Spontaneously hypertensive rat
Urine output

Urinary sodium concentration
Urinary potassium concentration
Urinary sodium excretion
Urinary potassium excretion
Water balance

Two-kidney one-clip

XV

INTRODUCTION

Cardiovascular disease remains America's number one killer and a
major cause of disability. It is estimated that more than 930,000 Americans die
of cardiovascular disease each year, accounting for 43 per cent of all the
deaths in the United States of America (American Heart Association, 1993).
Hypertension, or high blood pressure, is the most common form of
cardiovascular disease, affecting one in three American adults, including more
than half the population over the age of 55 (American Heart Association,
1993). Therefore, it is important to gain a better understanding of the etiology
and mechanisms of this condition.

Hypertension may be defined according to its etiology. W
W is defined as blood pressure elevation without a
primary identifiable cause or etiology. It is a multiform disorder with a
variable natural history and prognosis. The characteristic hemodynamic
abnormality is a persistent elevahon of blood pressure associated with an
increase in total peripheral resistance, especially at the levels of the arteries
and arterioles. W consists of blood pressure elevation
with a primary identifiable cause or etiology such as unilateral or bilateral

kidney disease, primary aldosteronism (Cushing's syndrome), or renin

2
secreting tumor, among many other disorders (Kaplan et al., 1982).

Hypertension may also be defined by severity (NH-I Publication, 1993):

Category Systolic (mm Hg) Diastolic (mm Hg)
Stage 1 (Mild) 140-159 90-99

Stage 2 (Moderate) 160-179 100-109
Stage 3 (Severe) 180-209 110-119
Stage 4 (Very Severe) 2210 2120

Hypertension is a major risk factor for premature death and disability
due to the high number of individuals who are afflicted with this disease and
the problems associated with prolonged uncontrolled hypertension (Kaplan,
1982). For example, epidemiological studies suggest a continuous, positive
relationship between diastolic blood pressure (DBP) and systolic blood
pressure (SBP) and the long term risk of coronary heart disease, congestive
heart failure, and stroke (Starnler, 1991). Different causes for hypertension
have been proposed, such as altered body fluid homeostasis (Guyton et al.,
1974), increased activity of the sympathetic nervous system (Esler et al., 1977;
Robertson et al., 1979), altered vascular structure (Folkow, 1982), and
hormonal changes (Folkow, 1982). A hormonal system proposed to be a
pathogenic factor in clinical and experimental hypertension is the Lenin-
mgigtgnsjn system (RAS). Successful treatment of clinical hypertension with
angiotensin converting enzyme inhibitors (ACEI), drugs that inhibit the
formation of angiotensin H (AH), strongly suggests that abnormalities of the

RAS play a role in hypertension. In addition, circulating levels of renin and

3

AH are elevated in plasma and other tissues of some hypertensives. Thus,
examining the importance of the RAS in the development of hypertension is

crucial for understanding the pathogenesis of the disease.

1. W

The RAS plays an important role in the control of arterial pressure and
body fluid regulation. Renin, the rate-limiting enzyme of the RAS, is
produced primarily in the kidneys by specialized cells located in the media of
the renal afferent vessels. The renin-secreting cells are found primarily in
what is called the juxtaglomerular apparatus (Taugner and Hackental, 1989;
Barajas, 1979). The juxtaglomerular apparatus includes the most proximal part
of the afferent arteriole, the most distal part of the efferent arteriole, the
glomerular arterioles, the macula densa, and the interglomerular mesangium
(Barajas, 1979). Renin producing cells are not confined to the kidney: renin
has been identified in the brain (Unger et al., 1988), arteries (Dzau et al, 1987;
Thurston et al., 1975), heart (Lindpaintner et al., 1989), adrenal gland (Dzau et
al., 1987), and reproductive organs (Poisner et al., 1982, Pandey et al., 1984).
The possible role of the extra-renal tissue RAS remains to be defined;
however, evidence suggests that local production of renin is not the primary
determinant in the regulation of the activity of the enzyme (Kato et al., 1993).
For example, plasma renin of kidney origin is the major source of vascular
functional renin and the primary regulator of vascular AH formation (Kato et

al., 1993).

4

The only known substrate of renin is angiotensinogen, which is
synthesized and released into the plasma by the liver (for review Tewksbury,
1983; Menard et al., 1983; Gordon, 1983). Renin acts on angiotensinogen to
cleave off angiotensin I (AI). Circulating renin (plasma renin activity or PRA)
primarily determines the activity of the RAS and is used as the best index of
the general activity of the RAS. Conversion of A1 to AH occurs primarily
within the lung on the pulmonary endothelial surface by angiotensin
converting enzyme (ACE) (Ng and Vane, 1967; Ng and Vane, 1968). ACE is a
non-specific enzyme acting on other substrates such as bradykinin,
enkephalin, neurotensin, substance P, and luteinizing hormone releasing
hormone (Skidel and Erdos et al., 1985; Skidel et al., 1984; Erdiis and Skidel,
1987). AH is the main biologically active component by which the RAS exerts
its many effects. The accepted view is that AH acts as a blood-home hormone
in the regulation of vascular tone (arterial blood pressure) and fluid
homeostasis (Hall, 1986). Some effects of the hormone are: [1] release of
aldosterone from the adrenal cortex; [2] constriction of vascular and non-
vascular smooth muscle; [3] facilitation of sympathetic nervous system
activity; [4] release of arginine vasopressin (AVP); [5] stimulation of drinking
or thirst; and [6] functioning as a growth factor or growth modulator of
vascular smooth muscle and cardiac cells (Timrnermans et al., 1993; Duling,
1988). AH acts on two distinct membrane bound receptors (Chiu et al., 1989).
The receptors designated as AT, are inhibited by losartan, and those

designated as AT2 are inhibited by CGP 42112A and PD123177. The AT,

5
receptor is responsible for most of the physiological effects of AH. The

physiological and functional significance of the AT2 receptor remains to be
defined. However, recent evidence suggests a possible involvement of this
receptor in vascular smooth muscle angiogenesis and in brain ion channel
modulation (Timmermans et al., 1993).

The biochemical signalling pathways activated by AH binding to AT,
receptors have been studied extensively in cultured rat aortic smooth muscle
cells (Lasségue et al., 1990). AH activates several biochemical responses
comprised of phospholipase C mediated breakdown of the inositol
polyphospholipids (Brock et al., 1985) to generate inositol triphosphate and
diacylglycerol (Brock et al., 1985) and to mobilize intracellular calcium. A
second phase of AH activation is characterized by the accumulation of
diacylglycerol (for review see, Griendling et al., 1993), activation of protein
kinase C (for review see, Griendling et al., 1993 ), and phospholipase D-
mediated hydrolysis of phosphatidylcholine (Lassegue et al., 1991). The
increase in phospholipase(s) activity produced by AH initiates the formation
and release of prostaglandins and prostacyclin, and these autocoids are well-
known to stimulate adenylate cyclase activity (Peach, 1981). These
intracellular events are responsible for the known molecular actions of AH.

However, there are numerous effects that await additional investigation.

KW

The first experimental model that resembled human hypertension was

6
developed by Goldblatt et‘al., in 1934 (Goldblatt et al., 1934). This form of

hypertension, known as the two-kidney one-clip (2K1C) model, has
contributed to our understanding of how alterations in kidney function and
the RAS interact to induce hypertension. Two-kidney one-clip hypertension is
produced by the constriction of one renal artery by application of a silver clip
or another device over the artery, while the contralateral kidney is left
untouched. This mimics human renovascular hypertension (RVH) caused by
fibromuscular dysplastic renal artery disease and/ or atherosclerotic disease.
The mechanisms by which renal artery constriction produces elevations in
blood pressure are not known, but clearly the RAS is implicated. In
experimental animals, the development of renovascular hypertension has been
divided into phases related to RAS activity in an attempt to describe the
evolution of the disease (Robertson et al., 1987). Within minutes of renal
artery constriction or stenosis in experimental animals, a prompt rise in blood
pressure is observed. This rise in blood pressure parallels a rise in PRA and
plasma AH concentration. The hypertension during this phase can be relieved
by correction of the renal artery constriction (Robertson et al., 1987), removal
of the diseased kidney or treatment with ACEI (Rosenthal, 1993; Lee and
Baufox, 1991). The ensuing decrease in blood pressure is accompanied by a
fall in PRA and circulating AH, suggesting that during this phase the RAS
plays a significant role in the development of hypertension Within days or
weeks, depending on the experimental animal, a secondary phase occurs.

Most human patients with renovascular hypertension (RVH) are encountered

7
clinically during this phase. Blood pressure remains elevated while the PRA

and plasma AH concentration decrease towards normal. Nevertheless, the
hypertension can be relieved by correction of the renal artery stenosis
(Robertson et al., 1987), treatment with ACEI (Rosenthal, 1993), or an AH
antagonist (Timmermans et al., 1993). This disproportionate relationship
between blood pressure, PRA, and plasma AH concentration has been under
intense investigation. Nevertheless, several questions remain to be answered:
[1] how can drugs that inhibit the RAS be effective antihypertensive agents
when PRA and plasma AH concentration are not elevated? [2] how can the
RAS, specifically AH, cause hypertension when circulating levels of the
peptide are normal or only slightly elevated?

At this point, it should be noted that there also are other types of
hypertension where plasma RAS activity is not increased, yet ACEI and AH
antagonists are effective antihypertensive agents. For example, most humans
with essential hypertension, and the animal model of genetic hypertension, the
spontaneously hypertensive rat (SHR), do not exhibit excessive activity of the
circulating RAS (Kaplan, 1982; Bukenburg et al., 1991). Yet ACEI (Gavras et
al., 1978; Bukenburg et al., 1991) and AH AT, receptor antagonists (Tsunoda et
al., 1993; Timmermans et al., 1993; Wong et al., 1990c) are effective in

decreasing blood pressure in these forms of hypertension.

 

Three major hypotheses have been proposed to explain why drugs that

8
inhibit the RAS are effective antihypertensive agents in 2K1C hypertension,

RVH, SHR, and essential hypertension even in the absence of increased PRA
or plasma AH concentration.

W: ACE may have an additional antihypertensive effect unrelated
to inhibition of AH formation.

ACE has been shown, for example, to release vasodilatory
prostanoids (Galler et al., 1991), decrease bradykinin degradation (Carretero
and Scicli, 1991), and potentiate the actions of nitric oxide (endothelium
derived relaxing factor) (Goldschmidt et al., 1991). Recently, however, it has
been shown that the specific AH AT,-type receptor antagonist (losartan) lowers
blood pressure in the 2K1C hypertensive model in a manner equivalent to
ACE (T immermans et al., 1993), and that ACE do not lower pressure further
in animals pretreated with AT,-type receptor antagonists. Thus, it appears
that ACE lowers blood pressure by interference with the RAS and not by an

additional antihypertensive effect unrelated to inhibition of AH formation.

Hypothesis}: The ACE and AH antagonists antihypertensive efficacy results
from inhibition of various tissue renin-angiotensin systems.

The existence of local autocrine and paracrine RAS has been
demonstrated in a variety of tissues. Among the tissues proposed to have a
local RAS are blood vessels, kidney, adrenal gland, brain, and heart (Dzau,
1988). Activity of the RAS in these tissues is not always reflected by PRA or

plasma AH. Inhibition of local tissue AH production might then account for

9
the antihypertensive efficacy of ACE and AH antagonists in situations where

there are normal circulating levels of renin and AH. Some evidence supports
a role for tissue RAS in the development of hypertension (Nishimura et al.,
1992). However, other published reports do not support the tissue RAS as a
regulator of arterial blood pressure in hypertension (Kato et al., 1993;
Admiraal et al., 1993). Thus, the role of the tissue RAS in the development of

hypertension remains to be defined.

W There is an augmentation or increased responsiveness to the
"slow pressor effect" of AH.

Early studies in humans and experimental animals, using acute and
chronic exogenous infusions of AH as a model, showed that All raises blood
pressure via two distinct mechanisms (McCubbin et al., 1965; Dickinson and
Yu, 1967). One mechanism is the rapid (seconds to minutes) increase in
blood pressure (fast pressor effect) observed on acute intravenous
administration of large amounts of AH. This is widely accepted to be a result
of the actions of circulating AH on AT, receptors on vascular smooth muscle.
A less understood action of AH is the slowly developing increase in blood
pressure that is observed with long term (days to weeks) exogenous
administration of lower doses of AH - the so-called slow pressor effect of AH.
Regarding this later mechanism, the AH/ blood pressure relationship has been
investigated in normal human volunteers and in patients with renovascular

hypertension. Particularly revealing is the observation that in patients with

10
hypertension, for any level of circulating AH, there was a much higher level of

blood pressure than could be achieved by brief administration of AH in
normal human volunteers (Brown et al., 1979). In other words, long term but
very modest increases in plasma AH can produce a larger elevation in blood
pressure than is predicted based only on the fast pressor effect of AH. This
result suggests a possible role for the slow pressor effect of AH. If patients
exhibit increased responsiveness to this action of AH, even "normal" activity of
the RAS may be sufficient to cause hypertension gradually. A key experiment
testing this idea was reported by Li and Jackson (Li and Jackson, 1989).

N ormotensive control rats and SHR maintained normotensive by continuous
treatment with ACE were challenged with acute injections of AH and long
term infusions of the peptide. Responses to acute injections of AH did not
differ between SHR and normotensive rats, but SHR showed much larger
increases in blood pressure during chronic AH infusion. These data are the
first to demonstrate directly an enhanced responsiveness to the slow pressor
effect of AH in a model of hypertension. These findings, combined with the
ability of inhibitors of the RAS to lower mean arterial pressure (MAP) in the
SHR (Bukenburg et al., 1991; Wong et al., 1990a; Timmermans et al., 1993),
strongly implicate hyperresponsiveness to the slow pressor effect of AH in the
etiology of this form of hypertension. Impairment of the slow pressor effect
may represent an important mechanism of action of ACE and AH antagonist,
especially in forms of hypertension characterized by relatively normal PRA

and plasma AH concentration.

11
IV. WWII

The first illustration of the slow pressor effect was provided by
Dickinson and Lawrence in 1963 (Dickinson and Lawrence, 1963). They
showed that an initially subpressor dose of AH, a dose below the threshold for
the fast pressor effect of AH, produced a slowly progressive rise in arterial
pressure over one to three days when given chronically (1-10 days). Similar
responses have been reported using intravenous, subcutaneous and
intraperitoneal infusions of AH in dogs (McCubbin et al., 1963; Bean et al.,
1979; DeClue et al., 1978), rats (Fink et al., 1987; Li and Jackson, 1989), rabbits
(Yu and Dickinson, 1967), and man (Ames et al., 1965). Other known

vasoactive hormones appear incapable of eliciting a similar slow pressor effect.

A. ’o ‘19... u- in. n. o .l‘ . . .10- .! -., . any. 1.!

There are at least three possible explanations for the development of the
slow pressor effect of AH. The first proposed mechanism is sodium retention,
that is the inappropriate renal handling of salt and water which influences
blood pressure by increasing the extracellular fluid (ECF) volume. Increases
in ECF volume lead to an elevation of vascular volume causing an increased
venous return, an elevated cardiac output (CO), and an eventual rise in blood
pressure. Sodium retention may occur during the onset of the slow pressor
effect, since AH causes sodium retention by both a direct effect on the kidneys
(Ichikawa and Harris, 1991) and by an indirect stimulation of aldosterone

secretion (Brown et al., 1979). Evidence supports a role for sodium retention

12
as a mechanism for the development of the slow pressor effect of AH: studies

show that sodium loading augments the slow pressor effect, whereas sodium
depletion diminishes this effect (Davis 1975; Bianchi et al., 1968; Thurston and
Laragh, 1975; DeClue et al., 1978; Cowley and DeClue, 1976). Also, prevention
of sodium retention during combined administration of AH and salt prevent
hypertension development (Kriger,1990). Evidence against such a role for
sodium retention is the failure to observe significant urinary sodium retention
in animals receiving prolonged low dose-AH infusions (Kanagy et al., 1990),
and the failure to find significant increases in plasma aldosterone
concentrations in similar experiments (Kanagy et al., 1990).

The second explanation proposed for the development of the slow
pressor effect of AH involves new mechanisms. AH affects the brain and
autonomic nervous systems, and this interaction may be important in the
development of the slow pressor effect. Experiments with dogs suggest that
the brainstem is a major site within the central nervous system for the effect of
AH on blood pressure (Hilgenberg, 1969). Lesion studies thereafter
established that the area postrema (AP) — a brainstem Circumventricular
structure with high vascular permeability — is a site at which blood-borne AH
acts to produce a sympathoexcitatory effect resulting in an increased arterial
pressure (Ferrario et al., 1979). The importance of the AP for the slow pressor
effect of AH was suggested by studies demonstrating that AP ablation in rats
eliminated the slow pressor effect (Fink et al., 1987). AH also produces a

variety of peripheral sympathetic effects that may potentiate its pressor

13
actions. These effects include stimulation of the adrenal medulla and

sympathetic ganglia, facilitation of sympathetic ganglionic transmission,
potentiation of postganglionic neurotransmitter biosynthesis and release, and
inhibition of neurotransmitter reuptake (Brown et al., 1979). Evidence against
a role for sympathoexitation in the development of the slow pressor effect
arises from sympathectomy studies in the rat using 6-hydroxy-dopamine, a
neurotoxin (Li and Jackson, 1989; Ohnishi et al., 1987). Sympathectomy in the
rat did not prevent the slow pressor effect of AH from developing, suggesting
that intact sympathetic nerves are not essential for the slow pressor effect of
AH.

The third explanation proposed for the development of the slow pressor
effect of AH is vascular remodeling, hypertrophy and hyperplasia. These are
cardiovascular structural changes that occur at the level of the resistance
arteries and can contribute to the increase in total peripheral resistance found
in hypertensive individuals (Heagerty et al., 1993). Vascular remodeling refers
to refashioning or realignment of preexisting tissues, hypertrophy refers to an
increase in smooth muscle cell size, and hyperplasia refers to an increase in
smooth muscle cell number. Since all these changes can be caused by
chronically elevated arterial pressure, it is difficult to decide whether these
structural abnormalities are the cause or the effect of the hypertension
(Heagerty et al., 1993). The ability of AH to alter vessel structure has been
demonstrated in isolated perfused mesenteric vessels as a response to long

term administration of AH (Griffin et al., 1991; Dubey et al., 1992; Laporte and

14

Escher, 1992). This ability‘is not necessarily dependent on changes in pressure
(Griffin et al., 1991), but appears to depend on the growth promoting effects of
the peptide. This structural effect has the potential of developing a positive
feedback: the structural changes increase vessel resistance, thus increasing
pressure further and promoting additional vascular changes. There is
insufficient evidence available on the ability of AH to cause vascular structural
changes in vivo to accurately assess the role of this phenomenon in the slow
pressor effect of AH. The rapid reversibility of the slow pressor effect (Brown
et al., 1981), however, argues against a major contribution of altered vascular

structure.

V.” '!|' EE'I'II

It has become increasingly evident that blood-borne AH has major
effects on brain cardiovascular centers. The early observation by Dickinson
(Dickinson, 1965) that infusion of AH into the vertebral arteries of
unanesthetized rabbits produced rises in blood pressure larger than that
observed during intravenous infusion of the peptide was the key to the
discovery of the centrally mediated actions of AH. Since then many
contemporary investigators have shown that infusion of AH into the vertebral
arteries or intracerebroventricularly (i.c.v.) produces a large and rapid rise in
blood pressure at doses that are not effective when given intravenously
(Ferrario et al., 1970; Gildenberg et al., 1973; Breuhaus and Chimoskey, 1990).

Therefore, it is likely that AH can interact with the brain in some physiological

15

way, either acutely or chronically to elicit an increase in blood pressure. It is
not certain, however, that the mechanism of the hypertension caused by direct
administration of AH into the brain is the same as when circulating AH
concentrations are increased.

AH, like other peptides, cannot gain access into the brain tissue through
the blood brain barrier (BBB). A group of structures exists, however, called
the Circumventricular organs (CVO) which do not possess a normal BBB. The
CVO have a high capillary density, and a large number of endothelial
fenestrations which lack tight junctions, and they are in close proximity to the
brain ventricular system. Peptides such as AH can gain access to the brain
and evoke cardiovascular actions through direct actions with the CVO and
activation of neural pathways which originate from these structures. The
CVO implicated in cardiovascular regulatory functions include the subfornical
organ (SFO), the median eminence (ME), the AP, and the organum
vasculosum of the laminae terminalis (OVLT) (Saavedra, 1986; Mendelsohn et
al., 1984; Speth et al., 1985).

There are different approaches that have been used to elucidate the
sites in the brain where AH acts. The first approach is the localization of AH
receptor binding in the brain by autoradiographic and irnmunohistological
methods. A second approach involves removing or ablating the specific
tissues that may be involved in the actions of AH and then evaluating their
role in the development of hypertension. The third approach involves the

stimulation or antagonism of specific AH receptor subtypes that may be

16
crucial in the development of hypertension by AH. These approaches have

been used to investigate the actions of systemic AH in the brain and their
potential role in cardiovascular and volume homeostasis (Timmermans et al.,

1993).

VI.!' 'IIIllIil'

A primary goal of the current experiments is to further elucidate the
contribution of the slow pressor effect of AH to the development of
hypertension in a model of hypertension that depends exclusively on
circulating AH. This model of hypertension is commonly referred as the AH-
induced hypertension. In this model, small doses of AH infused chronically
(days to weeks) cause the development of hypertension in which the slow
pressor effect of AH plays a variable part. Hypertension is completely
reversible when the AH infusion is stopped. As mentioned earlier, the slow
pressor effect of AH has been demonstrated in the AH induced hypertensive
model in rats (Brown et al., 1981; Kanagy et al., 1990; for review Lever, 1993),
dogs (McCubbin et al., 1965), and rabbits (Dickinson and Lawrence, 1963). It
has been shown that AH-induced hypertension is not accompanied by
increases in daily water intake (Pawloski and Fink, 1990), and that AVP does
not contribute to the hypertension in this model (Cowley et al., 1981 ; Pawloski
et al., 1989). In addition, AH-induced hypertension in the rat is not necessarily
associated with a marked elevation in plasma AH concentration (Pawloski,

1990). This suggests: 1) that the animals may exhibit an increased

17

responsiveness to the slow pressor effect of AH; and that [2] even near normal
plasma AH concentrations are sufficient to produce hypertension.

The experiments in this projects were performed in instrumented
conscious rats that received chronic (days to weeks) infusions of AH. This
investigation will answer the following questions regarding the slow pressor
effect of AH: [1] Can the slow pressor effect of AH be reversed with AT, AH
receptor antagonists? [2] Can pharmacological blockade of the AT, receptors
cause a prolonged reversal of the hypertension produced by chronic
intravenous infusions of AH? [3] What is the contribution of the slow pressor
effect to the development of hypertension? As previously mentioned, there
are at least three possible explanations for the development of the slow
pressor effect of AH: sodium retention, nervous mechanisms, and vascular
structural changes. Previous experiments from Fink et al. (Fink et al., 1987)
support the idea that actions of AH on the brain are important in the
development of the slow pressor effect. Several questions will address this
hypothesis: [1] Is the slow pressor effect of AH mediated by AH acting on the
brain to increase sympathetic nervous system activity? [2] Can the receptors
in the brain be blocked selectively by AH antagonists and the hypertension
reversed? Answering these important questions will help elucidate the
mechanism of the slow pressor effect in the development of AH-induced
hypertension. Overall, these studies will contribute to an understanding of

the mechanisms by which AH elevates arterial blood pressure.

18
EXPERIMENTAL STUDIES

1. W
A. Animals

Male Sprague-Dawley rats (350-400 grams) were purchased from Sasco-
King (Madison, WI). The rats were housed in a light and temperature
controlled room and maintained in accordance with the Michigan State
University and National Institutes of Health animal care guidelines. Rats were
housed three per cage with free-access to tap water and standard rat chow
(Ralston-Purina) before catheterization surgery or other experimental
procedures. All the animals were surgically prepared and housed chronically
according to the protocols approved by the Michigan State University
Committee for Animal Use and Care. During the experiments, the rats were
housed in standard stainless-steel metabolism cages. The animals were
tethered to a swivel by a steel spring, one end of which was mounted to the
top of the cage and the other end of which was attached to the rat's head (as
described below). This setup allowed the rats free movement within the cage
and access to distilled water from a calibrated drinking tube and to a sodium
deficient rat chow (Teklad, Madison, WI). All the animals were euthanized

with an i.v. bolus injection of sodium pentobarbital at the end of each

experiment.

19
B. W
1. Arterial and venous catheterization

Rats were anesthetized with sodium pentobarbital (Nembutal’, Abbott
Laboratories, N.Chicago, IL), 45 mg-kg’1 i.p., and administered atropine sulfate
(Sigma, St.Louis, M0) 0.5 mg-kg’1 i.p., to suppress bronchial and salivary
secretions. The animals' body temperature was maintained with a water-
heated pad (Gorman-Rupp Inc, New York, New York). The animals were
shaved at the top of the head and the inner left leg. The areas were then
cleaned with 70% alcohol and scrubbed with BetadineO antiseptic (Purdue
Frederick Co., Norwalk, CT). The catheters were constructed of polyvinyl
chloride tubing ('l'ygon" Microbore; ID 0.020" X OD 0.60") with silicone
rubber tips (Silastic‘, Dow Corning, Midland, MI; ID 0.012" X OD 0.025";
arterial tip, 4.25 cm long; venous tip, 7 cm long). The catheters were placed
in the abdominal aorta and vena cava via the external iliac artery and vein.
The catheter placed in the abdominal aorta was used for recording arterial
blood pressure and heart rate and to draw blood samples. The catheter
placed in the posterior vena cava was used for infusion of drugs and sodium
chloride. Both catheters were tunneled subcutaneously to the top of the head,
where they were exteriorized and fed through a stainless steel spring (Small
Parts, Inc, Miami, FL). One end of the spring was attached to the skull with
dental acrylic (Dentsply International Inc, York, PA) and machine screws
(Small Parts, Miami, FL; Self Tapping # 00 x 3 / 16"), which served to anchor

the dental acrylic to the bone. The animals were allowed to recover from

20
surgery on the heated pad. Prophylactic antibiotics (Combiotic’, 20,000 U,

i.m., Pfizer, New York, New York) and analgesics (Stadol’, 1.0 mg-kg“, s.c.)
were given post-operatively. The animals received a continuous

(6 mmol-day‘) i.v. infusion of sodium chloride solution (6 mEq-day ") via an
external infusion pump (Harvard, South Nathick, MA) and were allowed

three days to recover post-surgery.

2. Intracerebroventricular cannulation

Animals were anesthetized with sodium pentobarbital (Nembutal‘,
Abbott Laboratories, N. Chicago, IL; 45 mg-kg", i.p.) and placed in a
stereotaxic device (David Koft Instrument, Tajunga, CA). The head of the rat
was realigned so the lambda-bregma plane was horizontal. A guide cannula
(20 gauge, Precision Guide", Franklin Lakes, NJ) was inserted 4 mm below the
skull surface through a hole made 1 mm posterior to the bregma and 1.5 mm
lateral to the midline. The cannula was held in place with dental acrylic
(Dentsply International Inc, York, PA) and two machine screws, which served
to anchor the dental acrylic to the bone. The animals were allowed to recover
from surgery on a heated pad. Prophylactic antibiotics (Combiotic’, 20,000 U
i.m., Pfizer, New York, NY) and analgesics (Stadol’, 1.0 mg-kg“, s.c) were
given post-operatively. Intracerebroventricular injections were made by
inserting 21-gauge stainless steel injectors (Small Parts, Miami, FL) into the
guide cannulas so they extended 1 mm beyond the tip. The injections were

given in a total volume of 2 pl. The i.c.v. injection site was confirmed by

21

multiple examinations of the pressor and dipsogenic responses to injection of

149 ng of AH (Sigma, St. Louis, MO).

3. Catheterization for exposure of the dorsal medulla

For these acute experiments rats were anesthetized with sodium
pentobarbital (Nembutal’, Abbott Laboratories, N. Chicago, IL; 45 mg-kg", i.p.)
and premedicated with atropine sulfate (Sigma, St. Louis, M0; 0.5 mg-kg") to
decrease bronchial secretions. The external iliac artery was cannulated using
PE-50 Intramedico tubing exteriorized through the back and connected to a
pressure transducer attached to a Grass Polygraph The change in MAP was
measured directly from the pulse wave of the blood pressure recordings. The
external iliac vein was cannulated using polyvinyl chloride tubing ('I‘ygon"
Microbore, Akron, OH) and was utilized for infusion of drugs. Each animal
was then placed and immobilized with its head in the nose-down position in a
stereotaxic frame (David Kopf Instrument, Tujunga, CA). The atlantooccipital
membrane was exposed through a dorsal midline incision. An incision was
made in this membrane that allowed access to the dorsal medulla in the
region of the AP such that a microinjection pipette could be stereotaxically

positioned.

C. HemmlmamicMeasutemems
A pressure transducer (Model P10EZ, Gould, Oxnard, CA) connected to

the arterial catheter was used to measure MAP and heart rate (HR). The

22
transducer was attached to a blood pressure monitor (Model BP2, Stiemke

Inc, New Orleans, LA). A polygraph (Model 73, Grass Instruments, Quincy,

MA) record of the pressure signal was made simultaneously.

D. W

Water intake (WI) in all the rats was determined utilizing a calibrated
drinking tube. The animals have access to distilled water ad libitum. Urine
output (U 0) was determined daily by collection of urine into a calibrated
measuring tube. Water balance (WB) was determined by subtracting UO from
W1. A sample of the collected urine was analyzed with a photometric
electrolyte analyzer (Model 43, Instrumentation Laboratories Inc, Lexinton,
MA) to determine urinary Na+ (UN,+) and I<+ concentration (Um). Urinary
sodium (U NaV), and potassium excretion (UKV) was determined by
multiplying the 24-hour urine volume by the sodium or potassium

concentration in the urine.

The majority of the experiments were designed for a mixed factorial
analysis of variance with repeated measures (Chrunch 4 Statistical Software,
Oakland, CA). Others were analyzed by a one-way analysis of variance for
repeated measures. In addition, analysis of contrasts was utilized in some
experiments. When comparing two independent samples, a students "t" test

was used. Most post-hoc analyses for specific differences between means

23

were performed using Dunnett's test for multiple comparisons. The statistical
analyses were performed utilizing a statistical software package (Crunch 4,

Crunch Software Corporation). A probability value of less than 0.05 (P<0.05)
was the criterion for statistical significance. Most data are expressed as mean

and standard error of the mean.

F. 12m
AH was obtained from Sigma Chemical Corp. (St. Louis, MO). Losartan

(formerly DuP 753, tradename COOZART“) and its active metabolite EXP 3174

were a generous gift from duPont de Nemours (W ilrnington, DE).

 

1. Rationale

The slow pressor effect is reported to occur at infusion rates of AH
below those necessary to elicit an acute rise in blood pressure (Dickinson and
Lawrence, 1963; McCubbin et al., 1965). The specific aim of this pilot study
was to determine the minimum infusion rate of AH necessary to elicit a
sustained hypertension in chronically instrumented rats without eliciting an
fast pressor effect. It was predicted that chronic hypertension would occur at
infusion rates of AH that do not increase MAP acutely (i.e. during the first

minutes of AH infusion).

24

2. Protocol '

Rats were placed on a 10-day i.v. infusion of AH (Days A1-A10 of the
AH infusion) at 2 ng-min", 4 ng~min'1 or 10 ng-min“. MAP, HR, WB, and
UMV were measured daily in all animals. For the chronic study rats were
monitored for three days prior to AH infusion (Days C1-C3 of control period),
during the 10-day AH infusion period, and for three days after the AH
infusion (Days R1-R3 of recovery period). For the acute study, changes in
MAP in response to AH infusions were recorded at multiple time points

during the first hour of AH administration.

3. Results

The hemodynamic responses to chronic i.v. infusion of AH at 2(n=6),
4 (n=5), and 10 (n=6) ng-min", are illustrated in Figure 1. The infusion rates
of 4 and 10 ng-rnin‘1 produced a significant rise in MAP that was sustained for
the duration of the 10-day AH infusion period (Al-A10). Cessation of the AH
infusion caused a return within 1-2 days of MAP to control levels. AH infused
at a rate of 4 ng-min“ i.v. was the lowest dose that significantly increased MAP
in this study. HR was not affected consistently by any dose of AH (Figure 1,
bottom). WB and UMV responses during the AH infusion period are
illustrated in Figure 2. WB was not affected consistently by any dose of AH.
UMV was not affected in groups receiving 2.0 or 10.0 ng-min“. However, in
the group receiving 4 ng-min", there was a decrease in UMV that was

significant on A1, A3, A6 and A9. The acute changes in MAP, in response to

25
chronic infusions of 2 and '4 ng-min“, are presented in Figure 3. The infusion

rate of 2 ng-rnin‘1 did not produce an acute rise (60 minutes) in MAP; the
infusion rate of 4 ng-min‘1 produced a significant rise in MAP at 40 and 60

minutes.

4. Discussion
AH infused at the rate of 4 ng-rnin‘l did not significantly increase MAP
acutely (seconds to minutes). However, when chronically infused it produced
a significant rise in MAP. This supports the prediction that chronic AH
induced hypertension will occur at infusion rates that do not increase MAP
acutely. This hypertension occurs without water retention and with only

modest, inconsistent reductions in renal sodium excretion.

26

Figure 1. Mean arterial blood pressure and heart rate responses to chronic AH
infusion at 2 (n=8), 4 (n=5) and 10 ng-rnin’1 (n=6), i.v. Infusions were started after
three control days (solid vertical line) and discontinued after 10 days of AH
infusion. Asterisks represent significant difference from control measurements
(p<0.05). Error bars represent S.E.M. C1-C3, control days; A1-A10, AH infusion

days; R1-R3, recovery days.

MAP (mmHg)

HR (beats/min)

200

180

160

140

120

100

80

500

450

400

350

300

250

27

 

. 2 ng/min (n=6)
‘ 4 ng/min (n=5)
I 10m/min (n=6)

 

 

 

 

 

l l

l

Angiotensin II Infusion

 

l l l l l l l l l l

l

l
. rt
“m

 

C1 C2 C3

A1 A2 A3 A4 A5 A6 A7 A8 A9 A10

R1 R2 R3

 

 

L l

l

 

Angiotensin II Infusion

 

L l l l l l l 1 4L 1

l

 

l i

 

C1 C2 C3 A1 A2 A3 A4 A5 A6 A7 A8 A9 A10 R1

DA YS

Figure 1

R2 R3

 

 

26

Figure 1. Mean arterial blood pressure and heart rate responses to chronic AH
infusion at 2 (n=8), 4 (n=5) and 10 ng-rnin’1 (n=6), i.v. Infusions were started after
three control days (solid vertical line) and discontinued after 10 days of AH
infusion. Asterisks represent significant difference from control measurements
(p<0.05). Error bars represent S.E.M. C1-C3, control days; A1-A10, AH infusion

days; R1-R3, recovery days.

MAP (mmHg)

HR (beats/min)

200

180

160

140

120

100

80

500

450

400

350

300

250

27

 

 

. 2 ng/min (n=6)
‘ 4 ng/min (n=5)
I 10 nmnin (n=6)

 

 

 

 

l l

l l l

Angiotensin II Infusion

l l l l l J I l

 

4

. ‘i—
in.

l l

 

C1 C2 C3

A1 A2 A3 A4 A5 A6 A7 A8 A9 A10

R1 R2 R3

 

 

J l

 

l l

Angiotensin II Infusion

 

l l l l l l L l

 

l

l l

 

C1 C2

C3 A1 A2 A3 A4 A5 A8 A7 A8 A9 A10 R1

DA YS

Figure 1

R2 R3

 

 

28

Figure 2. Water balance and urinary sodium excretion responses to chronic AH
infusion at 2.0 (n=8), 4.0 (n=5) and 10.0 ng-rnin‘1 (n=6), i.v. Infusions were started
after three control days (solid vertical line) and discontinued after 10 days of AH
infusion. Asterisks represent significant difference from control measurements
(p<0.05). Error bars represent SEM. C1-C3, control days; A1-A10, AH infusion

days, R1-R3, recovery days.

WB (ml/da y)

UNa V (meq/da y)

30

25

20

15

10

29

Anmtensin II Infusion

 

 

 

 

I 2 ng/min (n=6)
A 4 ng/min (n=5) -
I 10 ng/min (n=6) "

 

l I l l 1 JM 1 l l l l

l

 

\:

V

l l

 

C1 C2

C3 A1 A2 A3 A4 A5 A8 A7 A8 A9 A1GR1 R2 R3

 

 

 

Angiotensin II Infusion

 

I l l l l l l l l l l

 

33A ’t’ti

 

l

 

l l

 

C1 C2

C3 A1 A2 A3 A4 A5 A8 A7 A8 A9 A10 R1 R2 R3

DA YS

Figure 2

 

 

30

Figure 3. Acute change in mean arterial pressure in response to AH infusions at
2.0 and 4.0 ng-min“. Asterisks indicate significant difference from control day

(C1) measurement (p<0.05).

MAP (mmHg)

180

160

A
A
O

A
N
O

100

80

31

 

ANGIOTENSIN II INFUSION
I 2 ng/min (n=6)
A 4 ng/min (n=5)

1

 

 

 

 

l l I I l l I

0' 10' 20' 30' 40' 50' 60'
Time (minutes)

Figure 3

5130 :1. -1 l, , A" :1. 01 - -. 30 -.-.o.' o 4.114. :1- ° !
Pressecfiffects
1. Rationale
This investigation was designed to test the following general

prediction: the antihypertensive action of losartan in AH hypertension
involves drug actions in addition to blockade of the fast pressor effect of AH.
To test this idea, the time course of the antihypertensive response to losartan
was characterized in rats receiving chronic intravenous infusions of AH and
compared to the time course of losartan blockade of the fast pressor effect of
AH.

The fast pressor effect (direct vasoconstriction) of AH is mediated via
the AT, receptor (Timmermans et al., 1993). The mechanism and the type of
receptor that mediates the slow pressor effect of AH are unknown. Recent
evidence, though, suggests that the AT, receptor is involved in the
development of the slow pressor effect (Smits et al., 1991). However, the time
course of the reversal of chronic AH-induced hypertension versus the fast
pressor effects of AH remains to be defined. Thus, the purpose of this
experiment was to characterize and compare the time course of losartan
blockade of the fast pressor response to AH with the time course of the
antihypertensive effect of losartan in rats receiving low-dose intravenous
infusions of AH. Several hypotheses were tested by this experiment: [1] If

AH is acting at vascular AT, receptors to produce a fast pressor effect, then

33

blockade of the vascular receptors with an AH AT, receptor antagonist
(losartan) will produce an acute blockade (seconds to minutes) of the fast
pressor effect, [2] If chronic AH infusion produces hypertension (AH-induced
hypertension) by acting on the AT,-type of AH receptors, then blockade of the
receptors with an AH AT, receptor antagonist (losartan) will produce a full
and prolonged reversal of the hypertension, and [3] If AH-hypertension is due
solely by vasoconstriction (fast pressor effect), then the time course of reversal

should be similar to reversal of the fast pressor effect.

2. Protocols
a. Effect of losartan on fast pressor response to All
Male Sprague-Dawley rats were prepared with chronic arterial and
venous catheters as described above. Rats received a bolus injection of AH (10
ng, i.v.) two times within 30 minutes prior to administration of losartan (3
mg-kg", i.v.; n=10) or vehicle control (5% dextrose, i.v.; n=10). Preliminary
experiments demonstrated that a 3 mg-kg“, i.v. dose of losartan produced a
maximal inhibition of pressor responses to AH without lowering MAP in
normal rats. Peak changes in MAP and HR, occurred within one minute after
injection of AH (fast pressor effect), were recorded before and at 5 minutes, 15
minutes, 30 minutes, 60 minutes, 1 hour, 2 hours, and 6 hours after losartan or
vehicle administration. After the first 24 hours, peak changes in MAP and HR

in response to AH were measured daily until they returned to control levels.

34
b. Effect of losartan on angiotensin II-induced hypertension

Male Sprague-Dawley rats received a continuous intravenous infusion
of one of three different doses of AH. In these rats, surgical recovery was
followed by 3 control days, 15 days of a continuous AH infusion (10 ng-min“,
n=10; 4 ng-min“, n=8; 2 ng-min“, n=8), and 2 recovery days. On days 2, 7,
and 12 of the AH infusion period, a bolus injection of losartan (3 mg-kg“, i.a.)
or vehicle control (5% dextrose, n=6) was given, and MAP and HR were
measured at 5 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 6 hours, 1
day and daily thereafter. WI, UO, WB, UMV and UKV were monitored daily

as described in the Methods section.

3. Results
a. Efl‘ect of losartan on fast pressor response to All

The responses of MAP to acute i.v. bolus injections of AH at 10 ng
before and after losartan (3 mg-kg", i.v.) or 5% dextrose (n=10) are illustrated
in Figure 4. In both groups there were no statistically significant changes in
resting MAP at any time. In rats that received losartan (Figure 4, lower
graph), there was a statistically significant inhibition (when compared to
vehicle control) of the fast pressor effect of AH by 5 minutes after losartan
administration. The magnitude of the blockade was not significantly different
between 5 minutes and 24 hours after losartan injection. Three days were
required for the fast AH pressor response to return to a level similar to that of

animals receiving only dextrose. In contrast, in animals receiving 5% dextrose

35
(Figure 4, upper graph), the fast effect of AH on MAP was unchanged at all

times after dextrose administration (compared to pretreatment pressor

responses).

b. Effect of losartan on chronic All induced hypertension

The time course of the antihypertensive effect of losartan in AH
hypertension was established in animals receiving three different doses of the
peptide.

The effects of administration of losartan during chronic AH infusion at
10 rig-min" on MAP are illustrated in Figure 5. Infusion of AH caused a
significant increase in MAP beginning by day 1 and lasting throughout the
15-day infusion period. Termination of the AH infusion caused an immediate
return (within hours) of MAP to the control period level.

Losartan lowered MAP on days 2, 7 and 12 of the AH infusion period.
The antihypertensive effect of losartan was characterized by both an acute and
a slowly developing decrease in blood pressure. On all three days, there was
a significant fall in MAP by 5 minutes after bolus injection of losartan, and
these depressor responses were not significantly different on days 2, 7, and 12
of AH infusion. The maximal blood pressure decrease after losartan injection,
however, was achieved 2-6 hours post-losartan injection on all three days.
The further decrease in MAP between 5 minutes and 24 hours was statistically
significant on all days. Recovery from the antihypertensive action of losartan

required 2-3 days. The heart rate response to bolus administration of losartan

O-xl

36
is shown in Figure 6. There was a significant tachycardia (presumed to be

caused by the baroreceptor reflex) following administration of losartan on
days 2, 7, and 12. WB and UMV were not affected consistently by losartan on
any day (Figure 7 and 8).

The results of administration of losartan during chronic AH infusion at
4 ng-min’1 on MAP are illustrated in Figure 9. Before losartan was given on
days 2, 7, and 12 MAP was significantly elevated above its pre-AH control
level. On days 2 and 12 of the AH infusion losartan did not produce a
significant acute (i.e., 5 minutes) decrease in MAP. On day 7, however, the
depressor response at 5 minutes was statistically significant. A slower decline
in MAP was observed, however, post-losartan administration: the decrease in
MAP between 5 minutes and 24 hours was significant on all three days.
Heart rate was not consistently altered by administration of losartan on days
2, 7 and 12 of the AH infusion (Figure 10). WB and UMV were not
consistently affected by administration of losartan (Figure 11 and 12).

The results of administration of losartan during chronic AH infusion at
2 ng-min'1 on MAP are illustrated in Figure 13. MAP was significantly
elevated, averaging +13 mmHg, on day 7 and day 12 of the AH infusion. No
significant acute fall (i.e. at 5 minutes) in MAP was observed after losartan
injection on any day. Losartan produced a slow decrease in MAP on days 2, 7
and 12 of the AH infusion. There were no significant changes in HR, WB or
UNaV (Figure 14-16).

The results of administration of vehicle (5% dextrose), utilized as a time

37
control, on MAP during chronic AH infusion at 10 ng-min‘1 are illustrated in

Figure 17. Blood pressure was significantly elevated throughout the AH
infusion and remained elevated after administration of 5% dextrose on days 2,
7 and 12 of the AH infusion . Vehicle injection did not cause significant

changes in HR, WB or UMV (data not shown).

4. Discussion

In these experiments three different infusion rates of AH were used in
an attempt to differentiate the fast and slow pressor effects of the peptide,
based on previous results indicating that the slow pressor effect requires lower
circulating concentrations of AH than the fast pressor effect (Brown et al.,
1981). In addition, the antihypertensive action of losartan was investigated at
three different time points during the AH infusion to test two possibilities:
tachyphylaxis to the fast pressor effect; and enhancement of the slow pressor
effect over time.

Losartan at a dose of 3 mg-kg" normalized MAP in rats made
hypertensive by chronic intravenous infusion of AH at all three infusion rates;
injection of vehicle did not affect MAP. This indicates that our model of AH
hypertension is caused solely by stimulation of AT, receptors. The same dose
of losartan also nearly abolished fast pressor responses to rapid, intravenous
injections of AH, confirming their well-known dependence on AT, receptor
activation (Timmermans et al., 1993). The time course for losartan's blockade

of the fast pressor effect differed significantly, however, from the time course

38
of the drug's antihypertensive action in AH hypertension. Fast pressor effects

were fully inhibited within five minutes of losartan injection, whereas
depressor responses in AH-hypertensive rats required hours to reach a
maximum. This suggests that reversal of the fast pressor effect of AH cannot
solely explain the ability of losartan to lower MAP in AH hypertension

In rats receiving the highest infusion rate of AH (10 ng-rnin"), losartan
injection produced both a rapid (within 5 minutes) and a slower (5 min to 6
hrs) decline in MAP. What is the cause of the slower fall in blood pressure?
In addressing this issue, it should be pointed out first that the typical time
course of antihypertensive drug action after parenteral dosing is a fairly rapid
attainment of a peak depressor effect followed by a slow, steady recovery
toward the initial pressure. Recovery is caused by clearance of active drug
from the biophase and the engagement of counter-regulatory pressure control
mechanisms, such as, sympathoexcitation via the baroreflexes, renal fluid
retention, and stimulation of the RAS. Part of the antihypertensive effect of
losartan in rats and man has been attributed to the formation of a carboxylic
acid metabolite, EXP 3174 (Wong et al., 1990d). Thus, it is tempting to
speculate that the slower component of the fall in MAP after losartan injection
is related to the gradual generation of EXP 3174, and a progressively more
complete blockade of vascular AT, receptors mediating the fast pressor
response. Measurement of fast pressor responses to acute AH injection in this
study indicates that this was not the case though, since inhibition of fast

responses peaked at 5 minutes after losartan injection and did not change in

39
magnitude for over 24 hours. We propose instead that the rapid fall in MAP

caused by losartan in AH hypertension was due to inhibition of the fast
pressor effect of AH, while the slower fall was caused by reversal of the slow
pressor effect.

If this proposal is correct, then it would appear that losartan decreased
MAP in rats receiving the lower infusion rates of AH (2 or 4 ng-min")
primarily by blocking the slow pressor effect, since little or no fall in pressure
was observed within 5 minutes after losartan administration in these rats.
This conclusion is consistent with previous data showing that the slow pressor
effect of AH is the predominant cause of hypertension in animals receiving
chronic, low-dose infusions of AH (Dickinson and Yu, 1967). It also is
consistent with findings that the antihypertensive action of parenteral losartan
is rapid in "high renin" models of hypertension, but much slower in "normal
renin" models like the SHR (Wong et al., 1990a).

There is abundant evidence that fast vascular responses to AH exhibit
tachyphylaxis (Peach, 1981), but that the slow pressor effect of the peptide
may actually increase during prolonged infusions (Bean et al., 1979). Thus, we
speculated that the relative contributions of the fast and slow pressor effects to
AH hypertension would change over time in the experiments reported here.
This did not appear to be the case, however, when the two effects were
quantitated based on the fast and slower components of the antihypertensive
effect of losartan. The magnitude of the acute (5 minute) depressor response

to losartan was not decreased significantly between days 2 and 12 of the AH

40

infusion in any group nor was the magnitude of the slower fall (5 minutes to
6 hours) in MAP after losartan increased between days 2 and 12 of AH
infusion. We conclude that the relative contribution of the fast and slow
pressor effects to AH hypertension depends primarily on the amount of AH
infused, and not on the duration of the infusion (at least between days 2 and
12 of AH infusion).

Is the gradual decline in MAP after losartan administration caused by
delayed access of losartan (or EXP 3174) to the AT, receptors mediating the
slow pressor effect, or by an intrinsically slow reversal time for the effect
itself? Relevant to this issue are data on the time course of the fall in MAP
after terminating brief (minutes to hours) or long-term (days to weeks)
infusions of AH. After stopping brief infusions of pressor quantities of AH,
MAP returns to normal in a time period consistent with the plasma half-life of
AH (t, ,2 = ~10-20 seconds) (Morton, 1993). After stopping long-term infusions,
on the other hand, hours must elapse before MAP reaches normotensive
values (Brown et al., 1981; Smits et al., 1991). These results suggest even very
rapid blockade of AT, receptors mediating the slow pressor effect would cause
only a gradual fall in MAP; hence, our data do not provide evidence that
access of losartan to these receptors is slower than it is to the vascular
receptors involved in the fast pressor effect.

It is also noteworthy that the recovery times from losartan blockade of
fast pressor effects of AH, and from the drug's antihypertensive action in AH

hypertension, were virtually identical in these experiments (i.e. 2 to 3 days).

 

tot

sut

[8

Va

105

Sir

41
This result suggests a similarity in binding kinetics of losartan (or EXP 3174)

to the AT, receptors mediating the fast and slow pressor effects of AH. Two
subtypes of AT, receptors (AT, A and AT,,,) have been identified in the rat by
molecular cloning (T immermans et al., 1993), and their tissue distribution and
functional regulation are not identical (Timmermans et al., 1993). Our results,
though, do not provide any clear indication that the fast and slow pressor
effects are mediated by different subtypes of the AT, receptor.

The slow pressor effect of AH can require many days for full expression
(Brown et al., 1981), and several mechanisms may play a part in the
phenomenon (Lever, 1993). The ability of AH to shift the relationship between
arterial pressure and renal sodium excretion (toward lower sodium excretion)
has been proposed to be one such mechanism (Hall, 1986). Also, losartan has
been reported to possess potent diuretic properties (Burnier et al., 1993; Xie et
al., 1991) and was shown recently to reverse the effects of chronic AH infusion
on the "pressure natriuresis" relationship in rats (Van Der Mark and Kline,
1994). For these reasons, changes in renal sodium and water excretion in
response to losartan administration were monitored in the current study. No
evidence was found suggesting that enhanced renal fluid loss contributed to
the antihypertensive action of losartan in AH hypertension. However, normal
values for daily sodium excretion in the face of significantly lower MAP in
losartan-treated rats do confirm that the drug is able to shift the "pressure-
natriuresis" relationship in AH hypertension. Although there is evidence that

structural vascular changes caused by AH are involved in the slow pressor

42
effect (Lever, 1986; Lever,'1993), these may take weeks to be manifested and

thus were probably not a factor in our experiments. The relatively rapid
reversibility of AH hypertension by losartan further argues against a major
role for structural vascular alterations in our model.

Over the time course of these experiments, two other mechanisms are
more likely to have contributed to the slow pressor effect of AH. Schiffers et
al. (Schiffers et al., 1993) recently described a slowly developing increase in
tone in vascular strips exposed in vitro to AH for up to 3 days. The response
was distinct from the fast contraction produced by AH, required hours to be
expressed, and could be reversed within 0.5 hours by removing the AH. The
ability of losartan to inhibit this action of AH, however, was not tested. If a
slow increase in vascular tone of this type occurred in response to prolonged
increases in blood AH concentrations in viva, it could be an important
mechanism that mediates the slow pressor effect.

Another action of AH likely to be involved in the slow pressor effect is
augmentation of neurogenic vasoconstriction. This was the mechanism
originally proposed by Dickinson and colleagues to explain AH hypertension
in rabbits (Dickinson and Yu, 1967). Ferrario and his coworkers have
subsequently provided much additional supporting evidence for this concept
and have frequently reviewed the topic (Ferrario, 1983; Ferrario et al., 1972).
Actions of AH on the AP — a brainstem Circumventricular organ possessing
high concentrations of AT, receptors (Bennet and Snyder, 1976; Barnes et al.,

1992; Steckelings Muscha et al., 1992) and neurons activated by circulating AH

43
(Ferguson and Wall, 1992) -— appear particularly crucial to the neurogenic

effects of the peptide. In the rat model of AH hypertension, there is
substantial evidence for increased neurogenic pressor activity (Pawloski et al.,
1989; Gorbea-Oppliger and Fink, 1994; Luft et al., 1989), and ablation of the
AP was shown to virtually eliminate the slow pressor effect, without altering
the fast pressor effect of All (Fink et al., 1987). The ability of AH to "reset" the
baroreflex also depends on an intact AP (Matsukawa and Reid, 1990). Finally,
it was demonstrated recently that parenteral losartan (3 mg-kg, iv) rapidly
blocks AH activation of AP neurons (Lowes et al., 1993). Thus, inhibition of
actions of AH in the AP by losartan could play a key part in reversal of the
slow pressor effect.

In summary, losartan appears to lower MAP in AH hypertension in the
rat by at least one mechanism in addition to blockade of the fast pressor effect
of the peptide. The results of this study were interpreted to indicate losartan
also inhibits the slow pressor effect of AH. We suggest that blockade of the
slow pressor effect of AH is the primary mechanism of the drug's
antihypertensive action when circulating AH concentrations are not markedly

elevated.

Figure 4. Change in mean arterial pressure in response to bolus injections of
AH in vehicle control and losartan-treated group. The top graph presents the
animals that received 5% dextrose, and the lower graph presents the animals
that received losartan. The hashed bar is MAP. and the solid bar is the
change in MAP after bolus injection of AH at 10 ng. i.v. Asterisks indicate

significant difference (P<0.05) from vehicle control.

45

AMP

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46

Figure 5. The effects of losartan (3 mg-kg", i.a.) on MAP during chronic i.v.
infusion of AH at 10 ng-min“. The first and last solid bars represent
measurements taken during control and recovery periods. The solid bars on
days 2, 7, and 12 represent measurements taken before losartan. The open set
of bars on day 2, 7 and 12 represents measurements taken at 5 minutes, 15
minutes, 30 minutes, 1 hour, 2 hours, and 6 hours post-losartan. The hashed
bars on days 2, 7, and 12 represent measurements taken at 1 day, 2 days and 3
days post-losartan. Asterisks indicate significant difference (p<0.05) from

control (before losartan) measurements. Error bars represent SEM.

47

 

 

 

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48

Figure 6. The effects of losartan (3 mg-kg", i.a.) on heart rate during chronic
i.v. infusion of AH at 10 ng~min". The first and last solid bars represent
measurements taken during control and recovery periods. The sets of bars in
the middle represent measurements taken before and after the administration
of losartan during days 2, 7 and 12 (n=10) of the AH infusion. The ten bars
for each day represent measurement from control (solid) to 72 hours after
losartan bolus injection. Asterisks indicate significant difference (p<0.05) from

control (before losartan) measurements. Error bars represent SEM.

49

Ang'otensin ll Iriusion 10 ng’m'n

 

 

 

 

 

 

 

 

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50

Figure 7. The effects of losartan (3 mg-kg", i.a) on water balance during
chronic i.v. infusion of AH at 10 ng-min". The first and last solid bars
represent measurements taken during control and recovery periods. The sets
of bars in the middle represent measurements taken before and after the
administration of losartan during days 2, 7 and 12 of the AH infusion. The
four bars for each day represent measurement from control (solid bars; before

losartan) to 72 hours after losartan bolus injection. Error bars represent SEM.

 

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52

Figure 8. The effects of losartan (3 mg-kg", i.a.) on urinary sodium excretion
(UNaV) during chronic i.v. infusion of AH at 10 ng-min“. The first and last
solid bars represent measurements taken during control and recovery periods.
The sets of bars in the middle represent measurements taken before and after
the administration of losartan during days 2, 7, and 12 of the AH infusion.
The four bars for each day represent measurements from control (solid bars;
before losartan) to 72 hours after losartan bolus injection. Error bars represent

SEM.

 

 

 

 

 

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Figure 9. The effects of losartan (3 mg-kg“, i.a.) on MAP during chronic i.v.
infusion of AH at 4 ng-min". The solid set of bars in the middle represent
measurements taken before and after the administration of losartan on days 2,
7, and 12 of the AH infusion. The first solid bar on days 2, 7, and 12
represents measurements taken before losartan The open set of bars on days
2, 7, and 12 represents measurements taken at 5 minutes, 15 minutes, 30
minutes, 1 hour, 2 hours, and 6 hours post-losartan. The hashed bars on days
2, 7, and 12 represent measurements taken at 1 day, 2 days, and 3 days post-
losartan Error bars represent SEM. Asterisks indicate significant difference

(p<0.05) from control (before control) measurements.

55

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56

Figure 10. The effects of losartan (3 mg-kg", i.a.) on heart rate during chronic
i.v. infusion of AH at 4 ng-min". The solid set of bars in the middle represent
measurements taken before and after the administration of losartan on days 2,
7, and 12 of the AH infusion. The first solid bar on day 2, 7, and 12 represents
measurements taken before losartan. The open set of bars on days 2, 7, and
12 represents measurements taken at 5 minutes, 15 minutes, 30 minutes,

1 hour, 2 hours, and 6 hours post-losartan The hashed bars on days 2, 7, and
12 represent measurements taken at 1 day, 2 days, and 3 days post-losartan
Error bars represent SEM. Asterisks indicate significant difference (p<0.05)

from control (before control) measurements.

57

Inmsion 4 ng’m'n

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58

Figure 11. The effects of losartan (3 mg-kg“, i.a.) on water balance during
chronic i.v. infusion of AH at 4 ng-min“. The first and last solid bars represent
measurements taken during control and recovery period. The sets of bars in
the middle represent measurements taken before and after the administration
of losartan during days 2, 7, and 12 of the AH infusion. The four bars for each
day represent measurement from control (solid) to 72 hours after losartan
bolus injection. Asterisks indicate significant difference (p<0.05) from control

(before control) period measurements. Error bars represent SEM.

 

 

 

59

 

 

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Figure 12. The effects of losartan (3 mg-kg", i.a.) on urinary sodium excretion
during chronic i.v. infusion of AH at 4 ng-min“. The first and last solid bars
represent measurements taken during control and recovery period. The sets
of bars in the middle represent measurements taken before and after the
administration of losartan during days 2, 7, and 12 of the AH infusion. The
four barsfor each day represent measurement from control (solid bars; before

losartan) to 72 hours after losartan bolus injection. Error bars represent SEM.

 

 

 

 

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62

Figure 13. The effects of losartan at (3 mg-kg", i.a.) on MAP during chronic
i.v. infusion of AH at 2 ng-min". The solid sets of bars on the middle
represent measurements taken before and after the administration of losartan
on days 2, 7, and 12 of the AH infusion. The first solid bar on days 2, 7, and
12 represents measurements taken before losartan The open set of bars on
days 2, 7, and 12 represents measurements taken at 5 minutes, 15 minutes, 30
minutes, 1 hour, 2 hours, and 6 hours post-losartan. The hashed bars on days
2, 7, and 12 represent measurements taken at 1 day, 2 days, and 3 days post-
losartan Asterisks indicate significant difference (p<0.05) from control (before

losartan) measurements. Error bars represent SEM.

63

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Figure 14. The effects of losartan (3.0 mg-kg", i.a.) on HR during chronic i.v.
infusion of AH at 2.0 ng-min“. The solid sets of bars in the middle represent
measurements taken before and after the administration of losartan on days 2,
7, and 12 of the AH infusion. The first solid bar on days 2, 7, and 12
represents measurements taken before losartan. The open set of bars on days
2, 7, and 12 represents measurements taken at 5 minutes, 15 minutes,

30 minutes, 1 hour, 2 hours, and 6 hours post-losartan. The hashed bars on
days 2, 7, and 12 represent measurements taken at 1 day, 2 days, and 3 days
post-losartan Error bars represent SEM. Asterisks indicate significant

difference (p<0.05) from control (before losartan) measurements.

 

 

  

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66

Figure 15. The effects of losartan (3 mg-kg", i.a.) on water balance during
chronic i.v. infusion of All at 2 ng-min". The first and last solid bars represent
measurements taken during control and recovery period. The sets of bars in
the middle represent measurements taken before and after the administration
of losartan during days 2, 7, and 12 of the All infusion. The four bars for
each day represent measurement from control (solid) to 72 hours after losartan
bolus injection. Asterisks indicate significant difference (p<0.05) from control

(before losartan) period measurements. Error bars represent SEM.

67

 

- 72hrs Post-Losartan

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W 24hrs Post- Losartan

 

 

 

 

 

Angiotensin II Infusion 2 ng/min

 

 

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Figure 16. The effects of losartan (3 mg-kg", i.a.) on urinary sodium excretion
during chronic i.v. infusion of AH at 2 ng-min". The first and last solid bars
represent measurements taken during control and recovery period. The sets
of bars in the middle represent measurements taken before and after the
administration of losartan during days 2, 7, and 12 of the A11 infusion. The
four bars for each day represent measurement from control (solid) to 72-hours
after losartan bolus injection. Asterisks indicate significant difference (p<0.05)
from control (before losartan) period measurements. Error bars represent

SEM.

69

 

 

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_ 72hrs Post-Losartan

Control
////////// 24hrs Post- Losartan
W 48hrs Post-Losartan

 

 

 

Angiotensin II Infusion 2 ng/min

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70

Figure 17. The effects of vehicle control on MAP during chronic i.v. infusion
of All at 10 rig-min". The solid sets of bars in the middle represent
measurements taken before and after the administration of losartan on days 2,
7, and 12 of the AH infusion. The first solid bar on days 2, 7, and 12
represents measurements taken before losartan. The open set of bars on days
2, 7, and 12 represents measurements taken at 5 minutes, 15 minutes, 30
minutes, 1 hour, 2 hours, and 6 hours post-losartan. The hashed bars on days
2, 7, and 12 represent measurements taken at 1 day, 2 days and 3 days post-
losartan. Error bars represent SEM. Asterisks indicate significant difference

(p<0.05) from control (before losartan) measurements.

71

Ang'otmsin II ln'za’on 1o ng’m'n

 

 

 

 

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Figure 17

72
C: '1'. [I] E Ill.“ SI II]

Wigs:
1. Rationale

As previously mentioned, an important role for the RAS in the
pathogenesis of renovascular hypertension in man and experimental animals
is well established (Martinez-Maldonado, 1991). Temporal phases have been
described to identified the involvement of the RAS in the development of
renovascular hypertension. During the secondary phase, plasma A11 is
normal, or slightly elevated, but blood pressure can still be reduced by ACE
inhibitors or by relief of the renal artery stenosis. This ability of normal
plasma concentrations of All to support elevated blood pressures during
Phase II has been puzzling. The slow pressor effect of All has been postulated
to be responsible for the clear dependence of Phase II renovascular
hypertension on the RAS in the absence of substantially increased plasma AII
concentrations (Robertson et al., 1987; Lever, 1986).

As previously mentioned, one of the mechanisms associated with the
slow pressor effect of All is sympathoexcitation. Previous studies from our
laboratory, employing long-term intravenous infusions of low doses of All in
chronically instrumented rats, suggested that neural actions of All are a key
part of the slow pressor effect. Evidence included a gradually developing
augmentation of the depressor response to ganglion blockade in rats receiving
chronic AII infusions (Pawloski et al., 1989) and elimination of the slow

pressor effect by ablation of the AP, a brain region known to mediate some

73
central actions of circulating AII (Fink et al., 1987).

The centrally acting sympatholytic drug clonidine has been shown to
cause a dramatic reduction in blood pressure in humans with renovascular
hypertension without lowering plasma renin activity (Mathias et al., 1983).
This result was interpreted to indicate a neurogenic basis for phase II
renovascular hypertension in humans (Mathias et al., 1983; Mathias et al.,
1987), and it may also suggest the operation of the slow pressor effect in such
patients. The current experiment is designed to determine whether clonidine
can reverse the slow pressor effect of All. The following prediction will be
tested: if the slow pressor effect of All is mediated by increased
sympathetically mediated pressor activity, then the slow pressor effect of AH

will be reversed by administration of a sympatholytic agent.

2. Protocol

The experimental protocols were started three days after catherization
of the rats. MAP and HR were recorded daily throughout the experimental
protocol for 10—30 minutes between 8:00 am. and noon by connecting the
exteriorized arterial line to a pressure transducer (Model P50, Gould, Oxnard,
CA) attached to a blood pressure monitor (Model BP2, Steimke Inc., New
Orleans, LA). UO, WI, UMV were monitored daily throughout the protocol.

One group of rats (n=5) received All for 15 days at a dose of 4 ng-min“,
and another group (n=4) received saline. Control period measurements were

obtained for 3 days prior to All or vehicle infusion. On days 2, 7, and 12 of

74
the All or saline infusion, clonidine hydrochloride at a dose of 10 ug-kg“, i.a.

was injected into each rat. MAP and HR were measured at 5 minutes, 15
minutes, 30 minutes, 1-hour, 2 hours, and 6 hours after clonidine. However,
since the greatest antihypertensive effect occurred between 2 and 6 hours,
those changes are reported. After 15 experimental days All, and vehicle
infusion were terminated, and 3 days of recovery period measurements were

made.

3. Results
Infusion of All caused a significant increase in MAP throughout the
15 day infusion period (Figure 18). There were no associated changes in HR,
WB or UN,V. Termination of A11 infusion caused an immediate return (within
24 hours) of MAP to the control period level. Rats not receiving AII showed
no significant change in any measured variable (Figure 19).

The result of bolus injections of clonidine at a dose of 10 jig-kg" into
animals receiving chronic intravenous infusion of All at 4 ng-rnin'l (n=5) are
illustrated in Figure 18. Clonidine lowered MAP on day 2 of the AH infusion.
period, and the decrease at six hours (-18:I:8 mm Hg) was statistically
significant (p=0.03). Similar results were obtained on day 7, when clonidine
had significantly decreased MAP (1615 mm Hg; p=0.05) by 6 hours.
Clonidine produced a fall in blood pressure on day 12 of the All infusion that
was statistically significant both at 2 (4813 mm Hg; p=0.03) and 6 hours

(4319 mm Hg; p=0.008) post-clonidine administration. MAP returned to pre-

75
clonidine values within 24‘hours after drug injection. HR was not consistently

affected by bolus injection of clonidine on days 2, 7, or 12 of the AH infusion.
WB and UMV also were not significantly affected by clonidine. In rats
receiving only saline vehicle (Figure 19), clonidine did not cause a significant
change in any variable on any of the three different days of administration.

4. Discussion

A variety of pharmacological tools have been employed in the past to
estimate the neurogenic component of arterial pressure regulation in
hypertension. Foremost among these are ganglion blockers and
a-adrenoreceptor antagonists. In experimental hypertension caused by a
chronic low-dose infusions of A11, both of these types of agents have been
shown to elicit an exaggerated fall in arterial pressure relative to that observed
in normotensive animals (Sato et al., 1991 ; Yu and Dickinson, 1971; Kutahira
et al., 1989; Luft et al., 1989). This has been taken to support the hypothesis
that AII-induced hypertension has a substantial neurogenic basis. A
complication inherent in this approach is that ganglion blockers and
a-adrenoreceptor antagonists lower blood pressure in normotensive animals
(Sato et al., 1991; Yu and Dickinson, 1971; Kutahira et al., 1989; Luft et al.,
1989). Therefore, their enhanced depressor activity in hypertensive animals
might be explained as a non-specific augmentation of vasodilation expected in
any hypertensive individual — the so-called "vascular amplification" property

of the hypertensive circulation (Korner et al., 1989)

76
Clonidine is an az-adrenoreceptor agonist which readily penetrates the

central nervous system and causes a decrease in sympathetic activity (Kooner
et al., 1991). This effect may result from stimulation of ozz-adrenoreceptors, or
from activation of recently characterized imidazoline-preferring receptors
(Bousquet et al., 1984; Ernsberger et al., 1984). Clonidine may act at several
brain regions to cause sympathoinhibition, but the rostral ventrolateral
medulla — where cell bodies of many bulbospinal neurons impinging on
sympathetic preganglion neurons are located — seems the most important
(Kooner et al., 1989; Sun et al., 1986). The drug lowers arterial pressure in
diverse types of clinical and experimental hypertension, but at lower doses has
little or no effect on pressure in normotensive individuals (Houston, 1981).
This property gives clonidine a distinct advantage over other sympatholytic
agents for identifying abnormalities of sympathetic function contributing to
the pathogenesis of hypertension as discussed above.

In the present experiment, a parenteral dose of clonidine was employed
which is known to cause sympathoinhibition and a depressor effect in some
forms of experimental hypertension in rats (Garty et al., 1990; Sannajust et al.,
1992). Injection of clonidine into conscious, undisturbed normotensive rats
failed to decrease arterial pressure (Figure 19), as has been reported previously
(Sannajust et al., 1992; Feng et al., 1992). In rats made hypertensive by
prolonged intravenous infusion of a low dose of All, clonidine consistently
reduced arterial pressure (Figure 18). The lack of HR changes after clonidine

suggests that the antihypertensive effect was due primarily to a fall in

77
vascular resistance. This finding supports earlier work indicating that low

amounts of blood-borne All can induce an increase in neurogenic
vasoconstrictor activity (Pawloski et al., 1989; Yu and Dickinson, 1971 ;
Dickinson and Yu, 1967), perhaps through actions on the AP (Fink et al.,
1987). It also provides a basis for understanding the ability of clonidine to
reverse renovascular hypertension without influencing plasma renin activity
(Mathias et al., 1983; Mathias et al., 1987). Prolonged exposure to modest or
occasional increments in plasma AII secondary to renal artery disease could
gradually evoke an increase in sympathetic nervous activity — an increase
suppressible by clonidine. In fact, elevated discharge frequency in muscle
sympathetic nerves was recently documented in humans with renovascular
hypertension (Miyajima et al., 1991). Angioplasty to relieve renal arterial
stenosis decreased both sympathetic activity and arterial pressure (Miyajima et
al., 1991).

What is the relative role of neurogenic pressor mechanisms in the slow
pressor effect of blood-borne AII? Strong evidence suggests other important
contributors. For example, alleviation of the fluid retaining properties of A11
prevents hypertension during chronic infusion of the peptide in dogs (Krieger
and Cowley, 1990). Also, numerous experiments have highlighted the
capacity of All to bring about structural vascular modifications able to
increase total peripheral resistance and arterial pressure (Lever, 1986). It is
likely that these, and perhaps other factors work in concert to produce the

slow pressor effect. As one possibility, it should be noted that both volume

78

retention and vascular hypertrophy would augment the vasoconstriction
caused by even a normal level of sympathetic nerve activity (Korner et al.,
1989). The relative importance of the different elements of the slow pressor
effect may vary between individuals, and/ or between different durations of
All exposure.

In the current experiment, no evidence was found that AII significantly
affected renal sodium and water excretion, consistent with previous similar
experiments in rats (Fink et al., 1987; Kanagy et al., 1990). However, the
animals studied here were already on a moderately high salt intake, which is
known to facilitate the slow pressor effect of A11 (Sato et al., 1991 ; Kanagy et
al., 1990; Ando and Fujita, 1990). Although clonidine can cause a modest
natriuresis and diuresis in rats (Miyajima et al., 1991), this action did not
appear to be of sufficient magnitude or duration in our study to influence
arterial pressure.

Chronic infusion of All has been shown to cause significant vascular
hypertrophy in rats within 10-12 days, even when arterial pressure was
prevented from increasing (Griffin et al., 1991). Such an action may have been
operative in raising arterial pressure in our study. But the observation that
the hypertension produced by AH was rapidly attenuated by clonidine and
was fully reversed within 24 hours of terminating the infusion, argues against
structural vascular change as the sole factor underlying the hypertension.

In summary, chronic intravenous infusion of a low dose of A11 into rats

caused a sustained hypertension that could be partially reversed by the

79
centrally acting sympatholytic drug clonidine. Clonidine did not affect arterial

pressure in normotensive rats. These results support previous studies
indicating that the slow pressor effect of circulating All is caused in part by

neurogenic mechanisms.

80

Figure 18. The effect of bolus injection of clonidine at a dose of 10 ug-kg’1
during AII infusion at 4.0 ng-min". The first and last solid bars represent
measurements taken during the control and recovery period when the rats
were only receiving saline infusion. The bars in the middle represent
measurements taken before and after the administration of clonidine on days
2, 7, and 12 of the A11 infusion (n=5). The two bars for each day on the MAP
and HR graphs represent measurement from control (solid) to 6-hours after
clonidine injection (see legend). The two bars for each day on the WB and
UMV graph represent measurements on the control day (solid) and 24 hours
after clonidine injection (see legend). WB=water balance (WI-UO);
UN_V=urinary sodium excretion. Asterisks indicate significant difference

(P<0.05) from control. Error bars represent SEM.

 

 

 

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82

Figure 19. The effect of bolus injection of clonidine at a dose of 10.0 jig-kg"
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1. Rationale

As previously mentioned, circulating All has several actions that result
from interaction of the peptide with known brain sites lacking a tight blood
brain barrier (Fink et al., 1987). Recent reports indicate that microinjection of
A11 into the AP — a midline CVO located at the floor of the fourth ventricle
on the dorsal surface of the medulla — results in a significant increase in
blood pressure (Lowes et al., 1993). In addition, the increase in blood pressure
can be blocked by intravenous administration of losartan. These data support
that All can act at AT, receptors in the AP to elicit its cardiovascular effects.
The slow pressor effect of All is also eliminated by area postrema ablation
(Fink et al., 1987), suggesting that the AT, receptors mediating the slow
pressor effect are located there.

The present experiment was designed to test the following hypothesis:
if the hypertension observed during chronic elevations of AH is due to
enhancement of the slow pressor effect in part by circulating AII interacting
with areas of the brain lacking a tight blood brain barrier, then selective
administration of an AII antagonist into the CSF should reverse the
hypertension. To test this hypothesis the ability of acute i.c.v. injection of an
AT, receptor antagonist to reverse hypertension in animals receiving chronic

low-dose AII infusion was tested. The active carboxylic metabolite of losartan,

85
EXP 3174, was utilized in these experiments because the possibility exists that

losartan is not metabolized in the CSF.

2. Protocol

The experimental protocols were started three days after catherization
of the rats. MAP and HR were recorded daily throughout the experimental
protocol for 10-30 minutes between 8:00 am. and noon by connecting the
exteriorized arterial line to a pressure transducer (Model P50, Gould, Oxnard,
CA) attached to a blood pressure monitor (Model BP2, Steimke Inc., New
Orleans, LA). UO, WI, UMV were monitored daily throughout the protocol as
described previously in the Methods section.

Two groups of male Sprague-Dawley rats were prepared with chronic
arterial and venous catheters as described in the Methods section Both
groups underwent i.c.v. cannulation two weeks before the experiments
(as described previously). Surgical recovery (4 days) was followed by 3
control days, 15 days of continuous AII infusion (4 ng-min") and 2 recovery
days. On days 2, 7, and 12 of the All infusion an i.c.v. injection of EXP 3174
(1 ug in 2 pl of saline, né) or vehicle control (2 ul saline, n= 2) was given;
MAP and HR were measured at 5 minutes, 15 minutes, 30 minutes, 1 hour, 2
hours,

6 hours, 1 day and daily after that. The dose of EXP 3174 used was
previously shown to block significantly the i.c.v. pressor and dipsogenic

responses to 149 ng AII i.c.v. (n= 4) (Figure 20).

86
3. Results

The result of i.c.v. EXP 3174 (1 ug in 2p] of saline; n=5) during chronic
AH infusion at 4 ng-min‘1 on MAP is illustrated in Figure 21. Infusion of AH
caused a significant increase in MAP throughout the 15—day infusion period
(Day 2, 130:1.7 mm Hg; Day 7, 137:6.6 mm Hg; Day 12, 14415.7 mm Hg;
P<0.01). Cessation of the AH infusion caused a return (within hours) of MAP
to control levels (Recovery, 9912.6 mm Hg).

EXP 3174 i.c.v. did not lower MAP on days 2, 7 and 12 of the ANG H
infusion period (Figure 21A). The HR response to i.c.v. EXP 3174 is shown in
Figure 218. Following administration of i.c.v. EXP 3174 there was no change
in HR. WB and UnaV were not affected consistently by i.c.v. EXP 3174 on
any day (Figure 22A and 228). In the group of animals receiving i.c.v. vehicle
(2 ul 0.9% NaCl; n=2) there was no changes in MAP and HR (data not

shown).

4. Discussion
The selective i.c.v. administration of AH antagonist was important
since the experiments were designed to assess the extent to which
hypertension was produced by an action of AH on the brain as opposed to an
action on the periphery. EXP 3174, the carboxylic metabolite of losartan, is a
selective, potent and long lasting AT, type of AH receptor antagonist. Since
the possibility exists that losartan is not metabolized in the CSF the metabolite

was employed in all the experiments described. EXP 3174 was administered

87
at a dose (1 ug in 2 pl of saline) shown to produce a long term (4 to 6 hours)

blockade of the pressor and dipsogenic effects of AH (Figure 20). Since the
peak antihypertensive effect of i.v. losartan in this model of hypertension was
observed at 2 to 6 hours after drug administration (Gorbea-Oppliger and
Fink, 1994), acute i.c.v. injection was deemed appropriate to test the
hypothesis. In addition, the antihypertensive effect of i.c.v. EXP 3174 was
investigated at three different time points during the 15-day AH infusion to
test the possibility of enhancement of the neurogenic component of the slow
pressor effect of AH over time.

Acute blockade of brain receptors responsible for the acute pressor and
dipsogenic effects of AH with i.c.v. EXP 3174 M affect the hypertension
produced by chronic low-dose AH infusion. This result is in contrast to the
prevention of this form of hypertension observed when the AP was
electrolytically ablated (Fink and Bruner, 1987). There are at least two
possibilities that could explain the failure of EXP 3174 to reverse the AH
induced hypertension. First, when EXP 3174 was injected into the CSF, it may
not have had access to the critical receptors at which blood borne AH acts to
produce the slow pressor effect. As previously mentioned, blood borne AH
has access to CVO such as the OVLT, the SFO and the AP. However, it
aPpfiars that some CVO are differentially accessible from the blood or the CSF.
Some CVO, such as the OVLT, are equally accessible from the blood or CSF,
since binding of blood borne AH was prevented by i.c.v. and/ or i.v. injection

Of All antagonist (Van Houten et al., 1983). On the other hand, binding of

88
radiolabelled AH, administered i.v. in the AP or the SFO was not prevented by

i.c.v. injection of an AH antagonist. Recently, Lowes et al. demonstrated that
i.v. losartan (3mg-kg") effectively blocked the pressor response to AII
microinjected into the AP (Lowes et al., 1993), suggesting that circulating
losartan can effectively reach the body of the area postrema and block AT,
receptors. It has been demonstrated that the ependymal lining at the CSF
interface differs between the different CVO (Brightman, 1975; Krisch and
Leonhardt, 1978). For example, the ependymal cells lining the SFO and the
AP are connected by tight junctions that may exclude the movement and
passage of compounds into the body of the CVO (Brightman, 1975; Krisch
and Leonhardt, 1978). The results of the present experiment suggest that EXP
3174, when administered into the CSF, may not enter the same brain sites as
blood borne AH. The AP and the SFO are two CVO that are sensitive to AH
and appear to have a greater accessibility from the blood side than from the
CSF. If circulating AH is acting on a critical receptor site in one of these
regions, i.c.v. EXP 3174 may not reach the critical receptor populations in a
sufficient concentration to block the effects of chronic i.v. AH.

A second possibility that could explain the lack of effect of acute i.c.v.
EXP 3174 is that central receptors that mediate the pressor responses to
Chronic AH infusion are not of the AT, type or are of an AT, subtype not
antagonized by BMJ 3174. Several AT, receptor types have been identified
and Cloned in the rat (ATM, AT,,,, and ATm) (Timmermans et al., 1993). The

selectivity of losartan and/ or EXP 3174 for the subtype of AT, receptor is not

89
yet certain. Thus, it is possible to speculate that AH may be acting in a

subtype of AT, receptor that cannot be blocked by EXP 3174. Moreover, AH
receptors have been demonstrated intracellularly (Bandyopadhyay et al., 1988;
Sagiura et al., 1991), on nuclei (Re et al., 1984), and mitochondria (Goodfriend
et al., 1972) — these receptors may not be accessible to i.c.v. EXP 3174.
Furthermore, it has been reported that AH receptor complexes may be
internalized in brain tissues (Erickson et al., 1984). If a brain receptor
activated by chronic AH infusion was inaccessible to EXP 3174 due to
internalization of the receptor complex, or to an intracellular location, then
acute i.c.v. EXP 3174 would not be able to block the effects of AH in the brain.
In summary, acute i.c.v.EXP 3174 does not attenuate the hypertension
produced by chronic low dose infusion of AH. This may indicate that AH
does not act at brain sites to produce hypertension, or reflect an inability of
EXP 3174 to gain access to brain sites or the specific receptors at which AH

acts to produce hypertension.

90

Figure 20. Blood pressure responses to i.c.v. AH 149 ng before and after i.c.v.
EXP 3174. The hashed lines represent the blood pressure change in response
to AH before the i.c.v. EXP 3174. The solid bars represent the response to
individual bolus injections of i.c.v. AH at 5 minutes, 15 minutes, 30 minutes, 1
hour, 2 hours, 6 hours, and 1 days post-i.c.v. EXP 3174. The error bars
represent SEM. Asterisks represent significant difference from control (blood

pressure change to i.c.v. AH before i.c.v. EXP 3174).

91

 

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Figure 21. The effects of acute i.c.v. EXP 3174 (1 ug -2 ul“; n=5) on MAP (A)
and HR (B) during chronic i.v. infusion of AH (4ng-min“). The first and last
solid bars represent measurements taken during the control and recovery
periods. The solid set of bars on the middle represent measurements taken
before and after the administration of acute i.c.v EXP 3174 on days 2, 7, and 12
of the AH infusion. The first solid bar on days 2, 7, and 12 represents
measurements taken before i.c.v EXP 3174. The open set of bars on day 2, 7
and 12 represents measurements taken at 5 minutes, 15 minutes, 30 minutes, 1
hour, 2 hours, and 6 hours post-i.c.v. EXP 3174. The hashed bars on days 2, 7,
and 12 represent measurements taken at 1 day, 2 days and 3 days post-i.c.v.

EXP 3174. Error bars represent SEM.

 

 

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Figure 22. The effect of acute i.c.v. EXP 3174 (1 ug -2 ul"; n=5) on WB (top)
and UNaV (bottom) during chronic i.v. infusion of AH (4ng-min"). The first
and last solid bars represent measurements taken during the control and
recovery periods. The sets of bars in the middle represent measurements
taken before and after the administration of i.c.v. EXP 3174 on days 2, 7, and
12 of the AH infusion. The four bars for each day represent measurements
taken from control (solid) to 72 hours after i.c.v. EXP 3174 bolus injection.

Error bars represent SEM.

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1. Rationale

This series of experiments was aimed specifically at investigating if
acute i.c.v. EXP 3174 gains access to AT, receptors in the body of the AP.
Inaccessibility of E)? 3174 to brain sites where AH may be acting could
account, as mentioned previously, for the inability of i.c.v. EXP 3174 to reverse
AH-induced hypertension The hypothesis to be tested was the following: if
acute i.c.v. EXP 3174 gains access to AT, receptor sites located in the body of
the AP, then acute administration of EXP 3174 into the CPS should block the
pressor actions of AH microinjected into the AP. The AP was utilized to test
the hypothesis because it has been shown that the AP is necessary for the
maintenance of AH-induced hypertension (Fink et al., 1987). It is important
to note that the receptors mediating the fast pressor response to direct
microinjection of AH into the AP may not be the same as those involved in the

slow pressor effect of blood-home AH.

2. Protocol
Animals were surgically prepared as described in the Methods section
(B.3.). After surgical exposure of the dorsal medulla the AP was clearly
visualized, a microinjection glass micropipette (tip diameter < 20 um) was

connected to a 25 ul Hamilton microsyringe. The microinjection pipette was

97
backfilled with AH and positioned on the dorsal surface of the AP using a

micromanipulator. The microinjection needle was lowered 0.2-0.5 nun and AP
was injected. Two groups of rats received four microinjections of AH; one
group at a dose of 500 pg-IOOO n1" (n=8) and another at 500 ng-lOOO nl"(n=4).
The changes in MAP were evaluated with peak responses evaluated at 10—20
seconds after injection. Sufficient time between each dose (15 minutes) was
allowed for recovery to baseline MAP.

Two other groups of rats received two microinjections (15 minutes
apart) of AH into the AP before and after the i.c.v. injection of EXP 3174
(1 pg in 2 pl of saline). The doses of AH used for the two groups were 500
pg-IOOO nl’1 (n=6) and 500 ng-lOOO nl"(n=4). The change in MAP was
recorded at four time points after injection of EXP 3174. After the last
recording each rat received a systemic dose of EXP 3174 (1 mg-kg“, i.v.); the
change in MAP to microinjection of AH was recorded again at 15 and 30
minutes post-i.v. EXP 3174.

A third group of rats received i.c.v. injection of EXP 3174 (1 pg in 2 pl
of saline; n=4) before surgical exposure of the dorsal medulla and incision of
the atlantooccipital membrane. The rats were instrumented as described
above. The pressor responses to four microinjections of AH at a dose of
500 pg -1000 nl“ were performed allowing sufficient time for recovery of basal
MAP (15 minutes) between each microinjection.

The neurogenic component of the pressor responses to AH

microinjections was tested in a group of rats that received the ganglionic

98
blocker, hexamethonium (HEX) (20 mg-kg", i.v.; n=5). After two

microinjections of AH into the AP at doses of 500 pg -1000 nl", HEX was
administered i.v. (20 mg-kg"). The pressor response to 500 pg AH

microinjection was tested ten minutes after the i.v. injection of HEX.

3. Results

AH was microinjected into the AP at two doses of 500 pg (n=8; 500 pg
-1000nl) and 500 ng ( n=6; 500 ng -1000nl) at four separate times, 15 minutes
apart. The data summarized in Figure 23 show that significant and
reproducible changes in MAP occurred in response to 500 pg and 500 ng AH
microinjection (500 pg, 6.11 0.79 mm Hg, 7.3115 mm Hg, 6.1110 mm Hg,
7211.5 mm Hg; P<0.01; 500 ng, 17312.0 mm Hg, 15.8137 mm Hg, 12.0 13.3
mm Hg, 15.714.0 mm Hg; P<0.05).

In the second series of experiments (Figure 24) microinjection of AH
into the AP at doses of 500 pg (5710.3 mmHg, 5.3103 mmHg; P<0.001) and
500 ng (1414.0 mm Hg, 1715.4 mm Hg; P<0.05) produced significant pressor
responses. EXP 3174 i.c.v. gum attenuate the pressor responses to either
500 pg (610.6 mm Hg, 511.2 mm Hg; P=0.9) or 500 ng (218.0 mm Hg, 1915.2
mm Hg; P=0.1) of AH (Figure 24). In contrast, i.v. E)? 3174 (1 mg -kg", i.v.)
significantly attenuated the pressor responses to AH (500 pg, 0.21.5 mmHg,
0.210.1 mmHg; P<0.001; 500 ng, non-detectable response, 210.9 mmHg:
P<0.05).

The effect of i.c.v. EXP 3174 (1 ug in 2 ul; n= 4) administration before

99

exposure of the dorsal medulla and incision of the atlantooccipital membrane
on the AH microinjection pressor responses is shown in Figure 25. When
compared with the control responses to 500 pg AH into the AP (Figure 23A),
pre-treatment with EXP 3174 ding: significantly alter the response to AH
microinjected into the AP at a dose of 500 pg (613.0 mm Hg, 4.71.0.2 mm Hg,
7.0122 mm Hg, 4.7106 mm Hg; P=0.53).

The effect of the ganglionic blocker hexamethonium (HEX) in rats that
received microinjections of 500 pg AH into the area postrema is summarized
in Figure 25. A representative tracing for a rat that received the ganglionic
blocker HEX i.v. is illustrated on Figure 26 (bottom). HEX significantly
attenuated the change in MAP observed after microinjection of 500 pg AH into
the AP (n=-5; Pre-HEX 500 pg AH, 6.611.1 mm Hg; post-HEX 500 pg AH,

0.8105 mm Hg P=0.001).

4. Discussion

The data presented demonstrate that microinjection of AH (500 pg and
500 ng) into the AP resulted in a significant, consistent, and reproducible
increase in MAP (Figure 23 A and 23 B). These effects are specific to the AP
because microinjection of AH into adjacent areas, including the nucleus tractus
solitarius (NTS), did not result in similar cardiovascular responses (data not
shown). The volume of AH used for the acute microinjections (1000 nl) has
been previously reported to be optimal in that it allows maximal pressor

responses, but does not diffuse to adjacent areas such as the NTS (Lowes et

100

al., 1993). The blood pressure rise in response to microinjection A11 in the AP
was blocked by the ganglionic blocker, HEX This indicates that pressor
actions of AH were mediated primarily by activation of autonomic pathways
with subsequent increases in neurogenic vasoconstriction. In contrast, HEX
increased the pressor actions of i.v. AH (data not shown).

Acute i.c.v. injection of E)? 3174 $11;an affect the pressor responses
to the two different doses of AH microinjected into the AP- (Figure 23). In
contrast, when EXP 3174 was given i.v., pressor responses to microinjected AH
were blocked. This suggests that mugging EXP 3174 does enter the body of
the AP and effectively block the pressor effects of microinjected AH. Since
acute i.c.v. injections of EXP 3174 were performed after incision of the
atlantooccipital membrane, we considered the possibility that leakage of CSF
may have prevented i.c.v. EXP 3174 from reaching the AP. In order to test
this possibility i.c.v. EXP 3174 was administered before incision of the
atlantooccipital membrane and exposure of the dorsal medulla. This still did
not result in the blockade of the pressor responses to microinjected AH. These
results together suggest that acute i.c.v. EXP 3174 does not gain access to the
body of the AP when administered selectively into the CSF. Inaccessibility of
acute i.c.v. EXP 3174 to the AP might then explain why such injections did not
lower MAP in rats receiving chronic low-dose AH infusion (previous
experiment). Nevertheless, these experiments do not exclude the possibility
that circulating AH may not be acting at brain sites to produce the slow

pressor effect.

101
In summary, i.c.v. EXP 3174 did not block pressor responses to

microinjection of AH into the body of the AP. This suggests that acute i.c.v.
EXP 3174 does not gain access to some central sites where AH may act to

produce hypertension.

102

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Figure 23. A: A summary of MAP responses to microinjections of 500 pg AH
into the area postrema (rising right bars). B: A summary bar graph of MAP
responses to microinjections of 500 ng AH into the area postrema (solid bars).

Error bars represent SEM. “P<0.01, *P<0.05.

103

 

W ANGII500pg(n=8)

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Figure 23

 

104

Figure 24. Change in MAP observed in response to 500 pg (rising right bars) and
500 ng (solid bars) AH into the area postrema before and after i.c.v. EXP 3174
(lug-2 ul“), and i.v. EXP 3174 (1 mg kg“). Only i.v. injection resulted in
complete blockade of the responses to AH in the area postrema (last set of bars).

Error bars represent SEM. “P<0.01, *P<0.05.

- 500 ng ANG 11 into AP

’/////////////2 500 pg ANG II into AP

105

 

 

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Figure 24

106

 

 

 

Figure 25. Summary bar graph of MAP responses to microinjection of 500 pg AH
into the area postrema of animals that received i.c.v. E)? 3174 after exposure of
the atlantooccipital membrane (rising right bars) and prior to surgical incision of

the membrane (diagonal crosshatch bars). Error bars represent 1SEM.

107

 

Control500pginIoAP(n=8)

Pre-wa'thBP3174

500 pgintoAP(n=4)

........

 

 

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Figure 25

 

I
11.

108

Figure 26. A summary of MAP responses to microinjection of 500 pg AH before
(rising right bars) and after (solid bar) an injection of hexamethonium (HEX)
(20mg-kg“). Bottom: Typical BP tracing of one animal that received 500 pg AH

into the area postrema before an after an injection of HEX.

109

 

ANG II in AP 500 pg (n=5) - ANG II in AP
30 - ° Post-HEX (n=5)

 
  

Change in MAP (mmHg)
-\
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Figure 26

SUMMARY AND CONCLUSION

Following is a list of the major hypotheses proposed in the dissertation

with a brief summary of the experimental results obtained.

W: If AH is acting at AT, vascular receptors to produce the fast
pressor effect, then blockade of the vascular receptors with an AT, receptor
antagonist (losartan) will block the fast pressor effect.

The fast pressor effects of AH were blocked by the AT, receptor
antagonist losartan This confirms the well-known dependence of the fast
pressor effects of AH on AT, receptor activation. The fast pressor effects of
AH were inhibited fully within five minutes of losartan injection. The results
presented support the hypothesis that AH must be acting on AT, receptors to

mediate its fast pressor effect, as illustrated by the time course of its action.

W If chronic AH infusion produces hypertension by acting on the
AT, type of AH receptors, then blockade of the receptors with a selective AT,
receptor antagonist (losartan) will produce a full and prolonged reversal of the
hypertension.

Losartan at a dose of 3 mg-kg’1 normalized blood pressure in rats made
hypertensive by chronic intravenous infusion of AH. This suggests that our
model of AH induced hypertension is caused solely by stimulation of AT, type

AH receptors. The antihypertensive actions of losartan are prolonged, ranging

110

111

from 2 to 3 days in AH-induced hypertension.

W: If AH is acting only at the AT, receptors mediating the fast
pressor effect to produce chronic hypertension, then blockade of these
receptors during AH-induced hypertension will produce a decrease in blood
pressure with the same time course as blockade of the fast pressor effect.

The results presented do not support the hypothesis that AH is acting
only at receptors mediating the fast pressor effect to produce AH induced
hypertension. The time course of losartan's blockade of the fast pressor effect
differed significantly from the time course of the drug's antihypertensive
action in AH hypertension. This suggests that losartan lowers MAP by at least
one mechanism besides blockade of the fast pressor effect of the peptide. The
results are interpreted to suggest that losartan also inhibits the slow pressor
effect of AH, and that the slow pressor effect contributes significantly to AH
induced hypertension. It is the predominant hypertensive mechanism when

plasma AH concentrations are near normal.

W: If the slow pressor effect of AH is mediated by increased
sympathetic pressor activity, then the slow pressor effect of AH will be
reversed by administration of a sympatholytic agent.

The sympatholytic agent clonidine caused a consistent decrease in blood
pressure in AH hypertension. The drug had no effect in normotensive animals

receiving a saline vehicle infusion. The results indicate that the slow pressor

112

effect of AH is caused in part by neurogenic mechanisms. This is consistent
with earlier work with the ganglionic blocker hexamethonium in this model of

hypertension (Pawloski, 1990).

W: If the hypertension observed during chronic i.v. infusion of AII
is due to circulating AH interacting with areas of the brain lacking a tight
blood brain barrier, then selective administration of an AH antagonist into the
brain should reverse the hypertension.

.It was shown that acute i.c.v. injection of E)? 3174, an active metabolite
of losartan, did not attenuate AH-induced hypertension. The results may
indicate that AH does not act on brain sites lacking a tight blood brain barrier
to produce hypertension or may reflect an inability of i.c.v. EXP 3174 to enter

brain sites at which AH acts to produce hypertension.

W: If acute i.c.v. E)? 3174 does gain access to AT, receptor sites
located in the body of the AP, then acute administration of i.c.v. EXP 3174 into
the CSF should block the fast pressor actions of AH microinjected into the
body of the AP.

The data presented demonstrated that i.c.v. EXP 3174 did not affect the
pressor responses to AH microinjected into the AP. The results suggest that
i.c.v. E)? 3174 does not gain access to the body of the AP when given into the
CSF. However, when E)? 3174 was administered parenterally, it did block

the pressor actions of AH microinjection, which suggests that parenterally

113
administered E)? 3174 does enter the body of the AP.

CONCLUSIONS

Clearly, AH is only one of many factors that may be involved in the
pathogenesis of hypertension (see Figure 27). But the success of ACEI for the
treatment of a wide spectrum of clinical hypertension has led some
investigators to propose a central role for the RAS in the pathogenesis of
hypertension. Thus, this dissertation was focused on the development of
hypertension in a model depending entirely on exogenously administered AH.
The recently developed AT, receptor antagonist, losartan, was a key tool used
to investigate the mechanisms by which AH raises blood pressure acutely and
chronically.

Plasma AH concentrations may be acutely elevated in situations such as
severe dehydration, severe sodium depletion, hypotension, and renovascular
stenosis. Under these circumstances, the concentration of AH may be such
that vascular vasoconstriction (fast pressor effect) is elicited, resulting in an
increase in total peripheral resistance and blood pressure. This action of AH is
mediated by interaction of the peptide with AT, receptors located in the
plasma membrane of vascular smooth muscle cells (Timmermans et al., 1993).
The signal transduction pathway consists of phospholipase C-mediated
breakdown of the inositol polyphospholipids (Griendling et al., 1993) to
generate inositol triphosphate and diacylglycerol, which leads to the

mobilization of calcium from intracellular stores (Griendling et al., 1993) (See

114
summary flow chart in Appendix D). Losartan effectively abolished the fast

pressor responses to rapid, intravenous injections of AH, confirming the
dependence of the fast pressor effect on AT, receptor activation (Timmermans
et al., 1993).

In most patients with essential hypertension and in some patients with
RVH, however, the circulating levels of renin and AH are not markedly
elevated. However, ACEI are effective in treating these forms of hypertension.
This paradox of an apparent dependence of the hypertension on the RAS
despite a lack of marked elevations of the peptide has led investigators to
speculate that AH raises blood pressure by mechanisms besides the fast
pressor effect. The slow pressor effect of AH is observed when smaller doses
of AH -— that do not result in a fast pressor effect — cause a gradual and
progressive increase in blood pressure. Thus, the slow pressor effect of AH
may be very important in the pathogenesis of hypertension. The mechanisms
responsible for this action of AH are not presently understood. Several
questions remain to be answered:

1. What receptor(s) is/ are activated by AH to produce the slow

pressor effect?

2. Where is AH acting to produce the slow pressor effect?

3. Why is the slow pressor effect slow?

4. What is the contribution of the slow pressor effect to the

development and pathogenesis of human essential and renovascular

hypertension?

115

The hypertension produced by chronic infusion of AH into conscious
rats was shown to be reversed by losartan in the current experiment. The
time course of reversal of the hypertension differed significantly from the time
course of reversal of the fast pressor effect of AH. This suggests that the
mechanism of action of losartan involves actions in addition to blockade of the
fast pressor effect. My results show that losartan reverses the slow pressor
action of AH. Thus, the slow pressor effect, whether primarily due to an
action on vascular smooth muscle, the brain, or the kidneys is caused by
activation of AT, receptors.

AH could act in the vascular smooth muscle through several different
mechanisms to elicit a slow pressor effect. For example, AH is known to cause
vascular hypertrophy, and this action is mediated via the AT, type of receptor
and is reversed or prevented by losartan (T irnmermans et al., 1993). As
previously mentioned, the rapid reversibility of AH-induced hypertension by
losartan argues against a major role for vascular hypertrophy in our model.
Other actions of AH could be taking place in the time frame of my
experiments. For example, AH induced vasoconstriction may result due to the
abnormal formation of nitric oxide (NO), a potent endothelium-derived
relaxing factor (EDRF), which is synthesized enzymatically from the amino
acid L-arginine. Many studies have shown that NO is important in the
control of basal blood vessel tone (Lowenstein et al., 1994; Sigmon and
Beierwaltes, 1993; Hu et al., 1994; Moncada et al., 1993). These studies

demonstrate that NO synthase (NOS) inhibitors, which inhibit the formation

116

of NO from its precursor, L-arginine, cause hypertension on chronic
administration to experimental animals (Sigmon and Beierwaltes 1994;
unpublished experiments from our laboratory). What is interesting is that the
hypertension elicited by the NOS inhibitors in conscious rats is reversed by
ACEI (Morton et al., 1993) and AT, receptor antagonists (Pollack et al., 1993).
These findings demonstrate a role for AH in the hypertension produced by
inhibition of NO synthase. Figure 28 presents one possible explanation for the
effectiveness of ACEI and AT, receptor antagonists during NO synthase
inhibition and a schematic representation of how NO may influence the RAS.
It is possible that NO under normal circumstances exerts a normal modulating
influence on the RAS (Pollock, 1993) and in the vascular smooth muscle it may
be a physiological antagonist of AH. This modulating effect inhibits the
vasoconstrictive actions of AH on vascular smooth muscle at the level of the
AT, receptor or intracellularly and results in the dominant vasodilatory effects
of NO. In addition, NO exerts vasodilatory actions of its own by activation of
other intracellular pathways (Moncada and Higgs, 1993). When these actions
of NO are inhibited, with NO synthase inhibitors, hypertension results due to
prevention of the vasodilatory actions of NO and a disinhibition of the
modulation of AH. Since NO is no longer present to modulate the actions of
AH, during blockade of NO synthase, vasoconstriction results. This may
explain why ACEI and AT, receptor antagonists are effective in reversing the
hypertension produced by NO synthase inhibitors. In circumstances were AH

is elevated, NO exerts a normal modulating effect on the vasoconstrictive

117

actions of AH. It is reasonable to speculate that in hypertension there may be

an abnormality of the NO system such that even smaller doses of AH result in

hypertension. The possible role of the NO system in the pathophysiology of
hypertension and the modulating role that NO may have on AH in vascular
smooth muscle remains to be elucidated.

Although effects of AH directly on blood vessels, or on renal sodium
and water handling, may contribute to the slow pressor effect, there is good
evidence indicating that the CNS plays a major role. An action of AH on the
CNS to influence blood pressure was first proposed by Bickerton and Buckley
(Bickerton and Buckley, 1961), based on cross perfusion experiments in which
the vascularly isolated head of the recipient dog was perfused entirely by
blood from a donor dog. AH injected into the donor dog raised blood
pressure in the body of both the donor and the recipient animal. Since the
only connection between the head and body of the recipient dog was neural,
the pressor effects in the recipient were mediated by an action of circulating
AH on the CNS. Since then, the literature on CNS effects of AH has grown
considerably (see Ferrario and Averill, 1991 for review). AH as a circulating
hormone may interact with various structures outside the BBB to influence
brain mechanisms that affect blood pressure, salt-appetite and thirst (Severs
and Daniel-Severs, 1973). In addition, an intrinsic and independent brain RAS
has been identified, and it can give rise to AH (Bunnemann et al., 1993), and
chronic, selective administration of AH into the brain, via the cerebroventricles

has been shown to cause hypertension (Fink et al., 1982).

 

Ill.

118
Studies by Dickinson and Lawrence (Dickinson and Lawrence, 1963)

showed that administration of AH into the vertebral arteries produced greater
pressor responses that did the same dose administered into the peripheral
circulation. These and other studies supported the medulla oblongata as a
primary site of action for CNS-pressor actions of AH (Joy and Lowe, 1970;
Scroop and Lowe, 1969). Further work identified the AP as the main
medullary structure in mediating neurally dependent pressor responses to
intravenous AH (Ferrario et al., 1979). Ferrario and co-workers performed
extensive investigations of the neuroanatomy, neurochemistry and physiology
of the AP (Ferrario et al., 1979). Their work confirmed the existence of
sympathoexcitatory neural pathways emanating from the AP (Ferrario et al.,
1979). Electrical stimulation of the AP was reported to result an increased
activity of bulbospinal sympathetic pathways (Ferrario et al., 1979; Barnes and
Ferrario, 1980) and direct injection of AH into the AP was shown to increase
blood pressure via neurogenic mechanisms (Lowes et al., 1993). Most critical
to the hypothesis being investigated here though was the finding that ablation
of the AP eliminated the slow pressor effect of AH in rats without altering the
fast pressor response (Fink et al., 1987)

Therefore, the latter experiments described in this dissertation (testing
hypothesis 4, 5, and 6) were predicated on the hypothesis that the slow
pressor effect of AH involves interaction of AH with AT, receptors in the AP.
Activation of these receptors by circulating AH results in excitation of

bulbospinal sympathoexcitatory pathways that synapse at the levels of the

119

rostral ventro-lateral medulla (RVLM) and cause in an increase in sympathetic
mediated activity to the vasculature, heart and kidney (Ferrario et al., 1979;
Barnes and Ferrario, 1980; see Figure 29). My results neither support nor
refute this hypothesis, however, since it was demonstrated that an AH
antagonist injected i.c.v. does not gain access to the AP. The i.c.v. injection
method then is not adequate to achieve one of my original goals, i.e. to
establish the importance of AT, receptors in the CNS in the slow pressor effect
of circulating AH. It is possible that this could be accomplished with direct
microinjections of AT, antagonists into the AP, but such experiments have not
yet been performed.

The RAS is an important hormonal pathway involved in cardiovascular
homeostasis of normotensive and hypertensive individuals. AH, the
biologically active component of the RAS, causes hypertension by activating
AT, receptors in target organs. The majority of my experiments were
concerned with elucidating the receptors that are activated by AH to produce
the slow pressor effect and potential sites where AH is acting to produce the
slow pressor effect. Further research should be aimed at characterizing the
importance of the slow pressor effect in experimental models of hypertension
where circulating renin and AH are not markedly elevated, at identifying the
site(s) at which AH acts to elicit the slow pressor effect, and to determine why

is the slow pressor effect of AH slow to develop.

120

Endothelin

 

Nitric OXide Genetic
Kidney
Salt . .
Anglotensm II
Obesity
Endothelial
Hyperinsulinemia Strucmfe

Age

Figure 27. Factors involved in the pathogenesis of hypertension

 

 

 

121

Figure 28. Schematic representation of the mechanisms by which AH may be
interacting at vascular smooth muscle cells (Vasc. Smooth Ms.) and
endothelium. Circulating AH interacts with the AT, type of AH receptors in
vascular smooth muscle cells and results in vasoconstriction. NO may
modulate the actions of AH at the level of the vascular smooth muscle AT,
receptor and be a physiological antagonist of AH. The endothelium may also
generate tissue AH that may interact with AT, receptors in the vascular
smooth muscle cell and NO may also modulate the actions of tissue generated
AH. Losartan, the prototype AT, receptor antagonist, binds to AT, receptors

and opposes the actions of AH.

1

 

Circulation

 

 

Figure 28

 

123

Figure 29. Schematic representation of the actions of AH at the level of the
area postrema. Circulating AH may interact with AT, receptors in the plasma
membrane of the AP or gain access to putative intracellular AT, receptors.
This results in activation of bulbospinal sympathoexcitatory pathways that
synapse in the rostral ventro- lateral medulla (RVLM), the spinal cord (SC),
and ultimately result in increased sympathetic activity of the heart, kidney or

vasculature.

 

124

 

 

 

Symp. Pathway

flymp. Pre-ganglionic
/©

Kidney <—
fi / Symp. Post-ganglionic

Vasculature

M‘

Heart

Figure 29

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APPENDICES

139

APPENDIX A

Mean arterial pressure before and after losartan on days 2, 7, and 12 of the
chronic AH infusion (10 ng‘min“). Asterisks represent significant difference

(P<0.05) from control measurement (Pre-losartan). :|:Represent significant

difference form pre-AH control blood pressme.

MAP (mm Hg)1SEM

 

 

 

 

 

 

 

 

 

162131 163171: 169191
12213": 13214": 14412":
11613“ 13215";- 13313“:
10913" 12316“ 12716“
10514" 11815“ 11315“
10314" 11015" 11317"
9914" 10715" 11217“
11313“ 11716" 11414"
12814“: 12616": 12714“:
14215": 14817“:j: 14615";

 

 

 

 

 

Pre-AII control blood pressure: 11412 mmHg

140

APPENDIX B

Mean arterial pressure before and after losartan on days 2, 7, and 12 of the
chronic AH infusion (4 ng-min"). Asterisks represent significant difference

(P<0.05) from control measurement (Pre-losartan).

 

 

 

 

 

 

 

 

 

14314 15116 13815
13212 13515“ 13316
12213“ 1281 “ 12813
1131 “ 12017" 12114“
1171 “ 11316“ 1121 "
11513“ 11416“ 11413“
11515“ 11716“ 11813“
1071 " 11719" 11113“
1221 “ 12218“ 11713"
12719“ 12915“ 12816“

 

 

 

 

 

141

APPENDIX C

Mean arterial pressure before and after losartan on days 2, 7, and 12 of the

chronic AH infusion (2 ng-min"). Asterisks represent significant difference

(P<0.05) from control measurement (Pre-Iosartan).

MAP (mm ngsm

 

 

 

 

 

 

 

 

 

12115 12615 12014
11915 12216 11815
11615“ 11716" 11116
1111 “ 11414" 11717
11115“ 11216" 11316
10614“ 11314“ 10913
10715" 11015" 1041 "
10213“ 1111 " 10513“
11114“ 11113“ 11516
11215“ 11316“ 10611“

 

 

 

 

 

142

APPENDIX D

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Ca+ Cellelifaatim
bkwl‘mzyrres
Cahadim Ni/Hhrfiput

Figure 30. Summary flow chart showing various actions of AH in vascular
smooth muscle.G,-protein, guanosine-triphosphate binding protein; PIP2,
phosphatidyl inositol 4,5-bisphosphate; IP3, inositol 1,4,5,-triphosphate; DAG,

diacylglyceride: SR, sarcoplasmic reticulum; C-kinase, protein kinase C.

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