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2/05 cJClWQn.hdd-p.15

NEUROHUMORAL CONTROL OF SPLANCHNIC CIRCULATION
IN DOCA-SALT HYPERTENSIVE RATS
By

Hong Wang

A DISSERTATION

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

DOCTOR OF PHILOSOPHY
Department of Pharmacology and Toxicology

2005

ABSTRACT
NEUROHUMORAL CONTROL OF SPLANCHNIC CIRCULATION
IN DOCA-SALT HYPERTENSIVE RATS
By

Hong Wang

Most studies of the etiology of hypertension have focused on altered regulation of
the arterial vasculature. While arterial resistance is elevated in hypertension, there is also
a decrease in venous capacitance. Enhanced venous smooth muscle contractile activity
appears to be an important factor accounting for decreased systemic venous capacitance
in hypertension. There have been few studies of the mechanisms causing constriction of
veins in hypertension. The goal of my thesis work is to identify the mechanisms
responsible for increased venoconstriction in DOCA-salt model of hypertension.

Existing evidence suggests that there is both increased endothelin-1 (ET-1) and
sympathetically mediated venoconstriction in DOCA-salt rats. However, it is not clear if
this is due to increased ET-l levels and/or sympathetic activity in the Splanchnic
vasculature, the main capacitance bed of the circulation. My thesis work tested the
overall hypothesis that ET-1 levels and/or sympathetic nerve activity in the Splanchnic
vasculature was increased in DOCA-salt hypertensive rats.

The results of my work provide strong evidence that higher endothelial cell
formation of ET-1 is not likely responsible for enhanced ET-l mediated Splanchnic

venoconstriction in DOCA-salt rats in viva. When combined with the fact that venous

smooth muscle contractile responsiveness to ET-1 is not increased in DOCA-salt rats, the
combined data suggest that the ability of ET receptor antagonists to reduce
venoconstriction in DOCA-Salt rats in vivo results from actions of the drugs on a non-
vascular target, i.e. ET-l may exert indirect control of venous tone in vivo, for example,
via the modulation of sympathetic neurotransmission.

By estimation of nonhepatic Splanchnic norepinephrine Spillover rate, my work
provides direct evidence that there is increased Splanchnic sympathetic activity in
DOCA-salt hypertension, and altered local modulation of sympathetic neurotransmission,
including impaired presynaptic dZ-adrenoceptor function and inhibitory effect of ET-1 ,

contribute to sympathetic activation in this model of hypertension.

TABLE OF CONTENTS

LIST OF FIGURES ............................................................................................. viii
LIST OF ABBREVIATIONS ............................................................................... xii
CHAPTER 1: GENERAL INTRODUCTION ........................................................ 1
1. Hypertension .................................................................................................. 2
2. Regulation of blood pressure ......................................................................... 2
3. Determinants of blood pressure ..................................................................... 3
3.1 Arterial microvasculature ......................................................................... 3
3.1.1 Small arteries, arterioles and capillaries account for 90%
of vascular resistance .................................................................. 3
3.1.2 Regulation of microvascular resistance ....................................... 3
3.1.2.1 Myogenic regulation .............................................................. 4
3.1.2.2 Tissue metabolites .................................................................. 4
3.1.2.3 Endothelial cells are capable of releasing chemicals
that cause relaxation and constriciton of arterioles ................ 5
3.1.2.4 The sympathetic nervous system regulated blood pressure
and ﬂow by constricting the microvessels ............................. 5
3.2 Venous microvasculature ......................................................................... 6
3.2.1 Venous compliance and capacitance ........................................... 6
3.2.2 Methods to estimate venomotor tone in vivo ............................... 7
3.2.3 Factors regulating venous compliance ......................................... 7
3.2.3.1 Sympathetic nervous system .................................................. 8
3.2.3.1.] Synthesis, action and fate of norepinephrine at
sympathetic neuroeffector junction ............................ 8
3.2.3.1.2 Differential sympathetic neural control of
arteries and veins ........................................................ 9
3.2.3.1.3 SNS and venoconstriction .......................................... 9
3.2.3.2 Humoral Factors ................................................................... 10
3.2.3.3 Endothelin system ................................................................ 10
3.2.3.3.1 Isoforms and function .............................................. 10
3.2.3.3.2 ET-1 and venoconstriction ....................................... 11
3.2.4 Splanchnic veins and venules are the main vessels
contributing to total vascular capacitance .................................. 1 1
4. Vascular capacitance and compliance in Deoxycorticosterone
acetate (DOCA) -salt hypertensive rats ...................................................... 13
4.1 Deoxycorticosterone acetate (DOCA)-salt hypertensive rats ................ 13
4.2 Increased vascular resistance in hypertension ....................................... 13
4.3 Reduced total systemic and venous compliance .................................... 14
4.4 Factors mediating increased venomotor tone in hypertension ............... 15
CHAPTER 2: OVERALL HYPOTHESIS AND SPECIFIC AIMS ..................... 19

CHAPTER 3: GENERAL METHODS ................................................................. 27
1. Animals

1.1 Preparation of DOCA-salt and sham rats ................................................ 28

1.2 Surgery of rats used in Chapter 4 and 5 .................................................. 28

1.3 Surgery of rats used in Chapter 6 ............................................................ 29
2. Hemodynamic measurements ....................................................................... 30
3. Plasma ET-l measurement ........................................................................... 30
4. Plasma catecholamine measurement ............................................................. 31
5. Portal blood ﬂow measurement .................................................................... 31
6. Estimate of non-hepatic Splanchnic (NHS) spillover of NE ......................... 32
7. Radiotracer method ....................................................................................... 33

7.1 Radiotmcer infusion ................................................................................ 33
8. Immunofluorescence localization and quantiﬁcation of endothelial

ETB receptors ............................................................................................... 34
9. Statistical analysis of data ............................................................................. 35

CHAPTER 4: ENDOTHELIN-l CONCENTRATION AROUND THE
SPLANCHNIC VENOUS SMOOTH MUSCLE CELLS OF RATS IN THE

ESTABLISHED PHASE OF DOCA-SALT HYPERTENSION .......................... 37
Introduction ............................................................................................................ 38
Role of ET-1 in human hypertension and experimental models of
hypertension in animals ...................................................................................... 38
ET-l increases total peripheral resistance in DOCA-salt rats by contracting
arteries and arterioles .......................................................................................... 39
There is enhanced ET-l mediated venoconstriction in DOCA-salt
hypertension ........................................................................................................ 40
Part I ....................................................................................................................... 42
1) Total vascular ET-l content in mesenteric veins of DOCA-salt
hypertensive rats ........................................................................................ 42
2) Venous plasma ET-l concentration and net non-hepatic Splanchnic
release (nNHSR) of ET-1 ........................................................................... 43
Discussion for Chapter 4-Part I ........................................................................ 53
Part II ..................................................................................................................... 58
1) ETB receptor-mediated clearance of plasma ET-1 in the Splanchnic
bed of DOCA-salt hypertensive rats .......................................................... 58
2) Mechanisms of impaired ETB receptor-mediated clearance of plasma
ET-1 in the Splanchnic bed of DOCA-salt hypertensive rats ..................... 60
Discussion for Chapter 4-Part II ...................................................................... 73
Part III: ET-l synthesis in the Splanchnic vasculature of DOCA-salt rats ............ 80
Discussion for Chapter 4-Part III ..................................................................... 83

Part IV: Possible role of ETA receptor binding in the plasma clearance of ET-1

in the splanchnic bed of DOCA-salt rats ............................................................... 85
Discussion for Chapter 4-Part IV ..................................................................... 90
Discussion for Chapter 4 ........................................................................................ 93

CHAPTER 5: SYMPATHETIC NEUROTRANSMISSION IN THE
SPLANCHNIC BED OF EXTABLISHED DOCA-SALT HYPERTENSIVE

RATS ..................................................................................................................... 96
Introduction ............................................................................................................ 97
Autonomic innervation of the cardiovascular system .......................................... 97
Increased sympathetic nervous activity in human hypertension .......................... 97
Sympathetic nerves and adrenergic receptors in arteries of hypertensive
human subjects and animal models ...................................................................... 99
Sympathetic nerves and adrenergic receptors in veins of hypertensive
human subjects and animal models ...................................................................... 99
Causal mechanisms leading to sympathetic augmentation in hypertension
are poorly understood ........................................................................................ 100
Part I: NE release from the sympathetic nerves associated with the
splanchnic vasculature of anesthetized and conscious DOCA-salt rats ............... 102
1) Plasma NE concentration and NHS spillover of NE in anesthetized
and conscious DOCA-salt rats ................................................................. 102

2) Both the plasma NE level and estimated NHS spillover of NE are
signiﬁcantly increased in conscious DOCA-salt rats versus

sham rats .................................................................................................. 106
Discussion for Chapter 5-Part I ...................................................................... 113
Limitations ..................................................................................................... 1 16

Part II: Prejunctional a2-adrenoceptor inhibition of NE release in sympathetic
nerve endings associated with splanchnic vasculature in conscious

DOCA-salt rats ..................................................................................................... 1 17
Discussion for Chapter 5-Part II .................................................................. 127
Limitations ................................................................................................... 130

Part III: Neuronal NE uptake activity in the splanchnic vasculature of

anesthetized and conscious DOCA-salt rats ........................................................ 131
Discussion for Chapter 5-Part III ................................................................ 143
Limitation .................................................................................................... 144

Part IV: Interaction between prej unctional a2-adrenoceptor and neuronal
transmitter reuptake in DOCA-salt hypertensive rats .......................................... 146
Discussion for Chapter 5-Part IV ................................................................ 151

Part V: Modulation of sympathetic neurotransmission by endothelin-l in vivo

vi

in DOCA-salt hypertensive rats ........................................................................... 152
Discussion for Chapter 5-Part V ................................................................. 159

Discussion for Chapter 5 ...................................................................................... 160

CHAPTER 6: PRELIMINARY STUDIES ON SYMPATHETIC
NEUROTRANSMISSION AND ENDOTHELIN-l IN THE SPLANCHNIC
BED OF DOCA-SALT RATS IN THE EARLY DEVELOPMENTAL

STAGE ................................................................................................................. 166
Introduction .......................................................................................................... 167

Part 1: NE release and presynaptic a2-adrenoceptor ﬁmction in the sympathetic
nerves associated with the splanchnic vasculature of conscious DOCA-salt rats

in the early developmental stage .......................................................................... 168
Discussion for Chapter 6-Part I ................................................................... 176
Part 11: Net NHS release of ET-1 in conscious DOCA-salt rats in the early
developmental stage ............................................................................................. 178
GENERAL CONCLUSIONS AND DISCUSSION ............................................ 180
BIBLIOGRAPHY ................................................................................................ 186

vii

Figure 1:

Figure 2:

Figure 3:

Figure 4:

Figure 5:

Figure 6A:

Figure 6B:

Figure 7A:

Figure 7B:

Figure 8A:

Figure 8B:

Figure 9A:

Figure 98:

LIST OF FIGURES

Schematic representation of NE overﬂow from sympathetic nerves
to the circulation (NE spillover) ....................................................... 17

Schematic representation of overall hypothesis ................................ 25

Schematic representation of splanchnic circulation and the positions
of the arterial catheter and portal venous catheter ............................. 36

Vascular ET-l content of DOCA-salt and sham rats ........................ 47

Mean arterial pressure of anesthetized and conscious DOCA-salt
and sham rats ..................................................................................... 48

Aortic and portal venous plasma ET-l level in anesthetized
DOCA-salt and sham rats ................................................................. 49

Aortic and portal venous plasma ET-l level in conscious
DOCA-salt and sham rats ................................................................. 49

Net nonhepatic splanchnic release of ET-1 in anesthetized
DOCA-salt and sham rats ................................................................ 51

Net nonhepatic splanchnic release of ET-1 in conscious
DOCA-salt and sham rats ................................................................ 51

Mean arterial pressure responses to i.v. bolus selective ETB
receptor antagonist administration in anesthetized DOCA-salt
and sham rats ................................................................................... 62

Mean arterial pressure responses to i.v. bolus selective
ETB receptor antagonist administration in conscious DOCA-salt
and sham rats .................................................................................... 62

Effects of i.v. bolus selective ETB receptor antagonist
administration on aortic plasma ET-l level in anesthetized
DOCA-salt and sham rats ............................................................... 64

Effects of i.v. bolus selective ETB receptor antagonist
administration on aortic plasma ET-l level in conscious
DOCA-salt and sham rats ................................................................ 64

Figure 10A: Effects of i.v. bolus selective ETB receptor antagonist

viii

Figure 10B:

Figure 11A:

Figure 11B:

Figure 12:

Figure 13:

Figure 14:

administration on portal venous plasma ET-l level in
anesthetized DOCA-salt and sham rats .......................................... 66

Effects of i.v. bolus selective ETB receptor antagonist
administration on portal venous plasma ET-l level in
conscious DOCA-salt and sham rats ............................................... 66

Effects of i.v. bolus selective ETB receptor antagonist
administration on net NHSR of ET-1 in anesthetized
DOCA-salt and sham rats ............................................................. 68

Effects of i.v. bolus selective ETB receptor antagonist
administration on net NHSR of ET-1 in conscious
DOCA-salt and sham rats .............................................................. 68

Immunoﬂuorescence staining of vWF and ETB receptors
of cross sections of DOCA and sham small mesenteric

blood vessels .................................................................................... 70
Quantiﬁcation of the immunoﬂuorescence intensity of

ETB receptors ................................................................................... 72
Model for regulation and intracellular trafﬁcking of the

ET receptors ...................................................................................... 79

Figure 15A: Effects of selective ETB receptor antagonist, combined

Figure 15B:

Figure 16:

Figure 17:

Figure 18:

Figure 19:

ETA/B receptor antagonist and selective ETA receptor antagonist
on aortic plasma ET-l levels in anesthetized DOCA-salt and

sham rats ........................................................................................... 88

Effects of selective ETB receptor antagonist, combined ETA/B
receptor antagonist and selective ETA receptor antagonist on
estimated net NHSR of ET-1 in anesthetized DOCA-salt

and sham rats ................................................................................... 88
Aortic and portal venous plasma NE level in anesthetized
DOCA-salt and sham rats .................................................................. 108
Mean arterial pressure of anesthetized and conscious DOCA-salt
and sham rats ..................................................................................... 109
Estimated nonhepatic splanchnic spillover rate of NE in anesthetized
DOCA-salt and sham rats ................................................................ 110
Aortic and portal venous plasma NE level in conscious DOCA-salt
and sham rats ...................................................................................... 111

ix

Figure 20:

Figure 21:

Figure 22:

Figure 23:

Figure 24:

Figure 25:

Figure 26:

Figure 27:

Figure 28:

Figure 29:

Figure 30:

Figure 31:

Figure 32:

Figure 33:

Figure 34:

Estimated nonhepatic splanchnic spillover rate of NE in conscious

DOCA-salt and sham rats .................................................................. 112
Mean arterial pressure responses to i.v. bolus yohimbine
administration in conscious DOCA-salt and sham rats .................... 121
Effects of i.v. bolus yohimbine administration on aortic plasma
NE level in conscious DOCA-salt and sham rats ............................. 122
Effects of i.v. bolus yohimbine administration on estimated NHS
spillover rate of NE in conscious DOCA-salt and sham rats ............ 123
Mean arterial pressure responses to i.v. bolus clonidine administration
in conscious DOCA-salt and sham rats ........................................... 124
Effects of i.v. bolus clonidine administration on aortic plasma NE
level in conscious DOCA-salt and sham rats .................................... 125
Effects of i.v. bolus clonidine administration on estimated
NHS spillover rate of NE in conscious DOCA-salt and sham rats.. 126
Mean arterial pressure responses to i.v. bolus desipramine
administration in anesthetized DOCA-salt and sham rats ............... 135
Effects of i.v. bolus desipramine administration on aortic plasma
NE level in anesthetized DOCA-salt and sham rats ........................ 136
Effects of i.v. bolus desipramine administration on estimated NHS
fractional extraction of NE in anesthetized DOCA-salt and
sham rats .......................................................................................... 137
Effects of i.v. bolus desipramine administration on estimated NHS
spillover rate of NE in anesthetized DOCA-salt and sham rats ....... 138
Mean arterial pressure responses to i.v. bolus desipramine
administration in conscious DOCA-salt and sham rats ................... 139
Effects of i.v. bolus desipramine administration on aortic plasma
NE level in conscious DOCA-salt and sham rats ............................ 140
Effects of i.v. bolus desipramine administration on NHS ﬁ'actional
extraction of NE in conscious DOCA-salt and sham rats ................ 141
Effects of i.v. bolus desipramine administration on estimated NHS

spillover rate of NE in conscious DOCA-salt and sham rats ........... 142

Figure 35:

Figure 36:

Figure 37:

Figure 38:

Figure 39:

Figure 40:

Figure 41:

Figure 42:

Figure 43:

Figure 44:

Figure 45:

Figure 46:

Figure 47:

Figure 48:

Mean arterial pressure responses to i.v. bolus
yohimbine+desiprarnine administration in conscious DOCA-salt
and sham rats .................................................................................... 148

Effects of i.v. bolus yohimbine+desiprarnine administration on
aortic plasma NE level in conscious DOCA-salt and sham rats ...... 149

Effects of i.v. bolus yohimbine+desiprarnine administration on
estimated NHS spillover rate of NE in conscious DOCA-salt
and sham rats .................................................................................... 150

Mean arterial pressure responses to i.v. bolus A192621
administration in conscious DOCA-salt and sham rats ................... 155

Effects of i.v. bolus A192621 administration on aortic plasma
NE level in conscious DOCA-salt and sham rats ............................ 156

Effects of i.v. bolus A192621 administration on estimated NHS
spillover rate of NE in conscious DOCA-salt and sham rats ........... 157

Effects of i.v. bolus A192621 administration on plasma EPI
level in conscious sham rats ............................................................. 158

Schematic of splanchnic processing of tritiated NE (3H NE) .......... 165

Mean arterial pressure of conscious early DOCA-salt and sham
rats and MAP response to i.v. bolus yohimbine administration ...... 171

Aortic and portal venous plasma ET-l level in conscious early
DOCA-salt and sham rats ................................................................ 172

Estimated net nonhepatic splanchnic spillover rate of NE in
conscious early DOCA-salt and sham rats ....................................... 173

Effects of i.v. bolus yohimbine administration on aortic plasma
NE level in conscious early DOCA-salt and sham rats ................... 174

Effects of i.v. bolus yohimbine administration on estimated NHS
spillover rate of NE in conscious early DOCA-salt and sham rats..175

Schematic representation of experimental results ............................ 185

xi

LIST OF ABBREVIATIONS

DOCA ............................................................ deoxycorticosterone acetate
ET-l ............................................................... endothelin isoform 1

i.p ................................................................... intraperitoneal

i.v ................................................................... intravenous

MAP ............................................................... mean arterial pressure

SBP ................................................................ systolic blood pressure

DBP ................................................................ diastolic blood pressure

SNS ................................................................ sympathetic nervous system
SNA ................................................................ sympathetic nerve activity
VSMC ............................................................ vascular smooth muscle cell
NE .................................................................. norepinephrine

NA .................................................................. norepinephrine

EPI .................................................................. epinephrine

NO .................................................................. nitric oxide

MCFP ............................................................. mean circulatory ﬁlling pressure
vWF ................................................................ von Willebrand Factor

PBS ................................................................ physiological buffered saline
S6C ................................................................. sarafotoxin 6C

DHPG ............................................................. dihydroxylphenylglycol
MHPG ............................................................ 3-methoxy-4-hydroxylphenylglycol
DMI ................................................................ desipramine

xii

CHAPTER 1

GENERAL INTRODUCTION

1. Hypertension

Hypertension is defined as a sustained elevated blood pressure (systolic blood
pressure of 140 mmHg or greater and/or diastolic blood pressure of 90 mmHg or greater).
Hypertension is a major risk factor for stroke, heart failure, coronary artery disease, renal

failure and other conditions.

2. Regulation of blood pressure

Maintaining the homeostasis of blood pressure involves a complex interplay between
the nervous, endocrine, renal and cardiovascular systems. The autonomic nervous system
directly regulates the cardiovascular system by controlling vascular tone and cardiac
strength. The kidney controls sodium and water balance and thus the blood volume. The
endocrine system releases hormonal factors to act on the cardiovascular system, such as
the renin—angiotensin system, vasopressin, and epinephrine. The cardiovascular system
itself can regulate blood pressure by secreting a number of vasoactive factors including
endothelin-l, nitric oxide (NO), atrial natriuretic factor. These systems interact with each
other and form a complicated and delicate blood pressure controlling system.

Different mechanisms are responsible for the short-term and long-term control of
blood pressure. Short-term regulation (time frame of seconds to minutes) is dominated by
the baroreceptor mechanism. Changes in mean arterial pressure are sensed by the
baroreceptors and processed by the vasomotor centers in the medulla which differentially
regulate sympathetic and parasympathetic nervous system output. From the long-range

point of view (hours to days) the control of fluid balance by the kidney, adrenal cortex,

and central nervous system, with maintenance of a constant blood volume, is generally

considered to be of the greatest importance.

3. Determinants of blood pressure

Mean arterial pressure is determined by systemic vascular resistance and cardiac
output. Peripheral vascular resistance is regulated largely by the caliber of small arteries
and arterioles. Cardiac output depends on heart rate, cardiac contractility, and the amount
of blood returning to the heart, which in turn depends on venous and venular capacitance,

and blood volume.

3.1 Arterial microvasculature
3.1.1 The small arteries, arterioles, and capillaries account for 90% of vascular
resistance

The larger arteries have a low resistance to blood ﬂow and function primarily as
conduits. However, as arteries approach the organ they perfuse, they divide into many
small arteries both just outside and within the organ. These smallest of arteries and the
arterioles of the microcirculation constitute the resistance blood vessels; together they
regulate about 90% of the total vascular resistance. Contraction and relaxation of the
smooth muscle in the walls of small arteries and arterioles cause changes in vessel

diameter, which, in turn, inﬂuence blood ﬂow.

3.1.2 Regulation of microvascular resistance

The vascular smooth muscle cells around arterioles respond to a wide variety of

physical and chemical stimuli, altering the diameter and resistance of the microvessels.

3.1.2.1 Myogenic regulation

Vascular smooth muscle is able to contract rapidly when stretched and
conversely, to reduce actively developed tension when passively shortened. A vessel’s
ability to constrict when intravascular pressure is increased or dilate as the pressure is
decreased is called myogenic regulation. The most important beneﬁt of myogenic
regulation is that blood ﬂow and downstream pressure can be indirectly regulated when
the arterial pressure is too high or low for appropriate tissue perfusion. The myogenic
response is rapidly activated (within 2 seconds) by changes in arterial and venous
pressure. This allows rapid modiﬁcations in microvascular resistance before appreciable

changes in the tissue chemical and metabolic environment occur.

3.1.2.2 Tissue metabolites

Blood ﬂow is also governed by the metabolic activity of the tissue. Any
intervention that results in an 02 supply that is inadequate for the requirements of the
tissue prompts the formation of vasodilator metabolites. These metabolites are released
from the tissue and act locally to dilate the resistance vessels. Many substances have been
proposed as mediators of metabolic vasodilation, such as adenosine, lactic acid, CO; and

hydrogen ions.

3.1.2.3 Endothelial cells are capable of releasing chemicals that cause relaxation and
constriction of arterioles

The endothelium regulates local vascular tone through release of vasodilator (e. g.
NO) and vasoconstrictor substances. Endothelin-l (ET-1), synthesized by endothelial
cells, is a very potent vasoconstrictor. It causes arterial contraction by binding to ETA
receptors on the smooth muscle cells (More details on the endothelin system are provided

later).

3.1.2.4 The sympathetic nervous system regulates blood pressure and ﬂow by
constricting the microvessels

The sympathetic nervous system (SNS) plays a critical role in the maintenance of
physiological homeostasis in general, and arterial blood pressure in particular under basal
(resting) conditions and in response to acute stress. Post-ganglionic sympathetic neurons
innervating the heart and resistance vessels help control cardiac output, arterial blood
pressure and regional vascular conductance, thus ensuring the proper perfusion of vital
organs. Sympathetic nervous system stimulation of epinephrine release from the adrenal
medulla contributes importantly to the regulation of cardiovascular function as well as
energy metabolism.

Although the microvasculature uses local control mechanisms to adjust vascular
resistance based on the physical and chemical environment of the tissue and vasculature,
the dominant central regulatory system is the sympathetic nervous system. The arterial
pressure is monitored moment-to-moment by the baroreceptor system, and the brain

adjusts the cardiac output and systemic vascular resistance as needed via the sympathetic

and parasympathetic nervous systems. Sympathetic nerves communicate with the
resistance vessels and venous system through the release of norepinephrine onto the

surface of smooth muscle cells in vessel walls.

3.2 Venous microvasculature
3.2.1 Venous compliance and capacitance

Blood vessels are elastic, and they expand when the blood in them is under
pressure. The degree to which a distensible vessel expands when it is ﬁlled with ﬂuid is
determined by the distending pressure and its compliance. Compliance (c) is deﬁned by
the equation:

C = AV/ AP

Where AV is the change in volume and AP is the change in distending pressure.
The distending pressure is equal to the pressure inside the vessel minus the pressure
outside the vessel; this is called the transmural pressure. The more compliant a structure,
the greater the change in volume for a given transmural pressure change.

Venules are much thinner and contain much less elastin tissue than arterioles.
Because of these structural differences, venules are up to 60 times more distensible than
arterioles.

Vascular capacitance is the volume of blood contained in the vascular tree at a
given transmural distending pressure. The compliance of veins is far larger than any other
part of the systemic circulation. In addition, venules have a cross-sectional area that is far
larger than their arteriole counterparts. Consequently, venules have the capacity to store

3-4 times more blood than arterioles. Thus, venules are often described as the capacitance

section of the vascular bed and usually contain approximately 2/3 of the body’s total
blood volume. Vascular capacitance is therefore determined almost entirely by the

structure and smooth muscle constrictor activity of veins.

3.2.2 Methods to estimate venoconstriction in vivo

A variety of methods are available to estimate venoconstriction. The best
approach so far in experimental animals in vivo is by measurement of mean circulatory
ﬁlling pressure (MCFP). MCFP is deﬁned as the mean vascular pressure that exists after
a cessation in cardiac output and redistribution of blood, so that all pressures are the same
throughout the system (187). It represents the effective driving force for venous return of
blood to the heart. MCFP is affected by both blood volume and vascular capacitance
(almost entirely venous). A change in MCFP provides a uniquely useful index of a
change in overall venous smooth muscle tone if the blood volume is not concomitantly

changed.

3.2.3 Factors regulating venous compliance

The walls of veins contain variable amount of ﬁbrous connective tissue and smooth
muscle. Venous compliance thus is affected by 1) alterations in salt and water content of
the venous wall (172) and alteration in connective tissue and/or smooth muscle mass 2)
or contraction of smooth muscle in the vein wall (venoconstriction or increased
venomotor tone).

Venous tone in different vascular beds is regulated in different ways depending on

their nature (189, 244). Cutaneous venous tone is regulated by the thennoregulatory

system. Skeletal veins play an important role, especially during exercise, as a muscle
pump. Splanchnic venous tone largely contributes to mobilization of blood volume by

sympathetic nerve stimulation (82, 188).

3.2.3.1 Sympathetic nervous system
3.2.3.1.] Synthesis, action and fate of norepinephrine at sympathetic neuroeffector

junctions (Fig. 1)

Sympathetic nerves travel along arteries and veins and nerves are found in the

adventitia. Capillaries receive no innervation.

NE synthesis and storage take place in the vesicles of the varicosity. An action
potential causes an inﬂux of Ca++ into the nerve terminal, with subsequent fusion of the
vesicle with the plasma membrane and exocytosis of NE. NE is the main
neurotransmitter, although neuropeptide Y and ATP also are released (212) (245). The
transmitter then activates a— and B-adrenergic receptors in the membrane of the
postjunctional cell. NE that penetrates into these cells (a process termed uptake2) is
rapidly inactivated by catechol-O-methyltransferase to norrnetanephrine (NMN). In
some organs, the most important mechanism for termination of the action of NE in the
junctional space is active reuptake into the nerve (a process termed uptake 1) by high
afﬁnity transporters on the plasma membrane of sympathetic neurons. The amount of NE
released is tightly regulated by negative feedback mechanisms involving not only NE
itself (via (1.2-adrenergic receptors) but also many other mediators such as EPI, serotonin,

histamine and acetylcholine.

The effects of NE at the postjunctional level, i.e. vascular smooth muscle cells of
blood vessels and myocytes of the myocardium, involve B-adrenergic receptors, as well
as (11 - and a2-adrenergic receptors. While the former are involved in vasodilation ([32-
adrenergic receptors) and inotropy (Bl-adrenergic receptors), the latter mediate

vasoconstriction of the blood vessel wall.

3.2.3.1.2 Differential sympathetic neural control of arteries and veins

The sympathetic neurons innervating arteries and veins differ in their location in the
ganglia and in their electrophysiological properties (17), suggesting that there is a
differential sympathetic neural control of mesenteric arteries and veins (95). This
suggestion is supported by data from studies of the neuroeffector mechanisms in arteries
and veins. Intracellular electrophysiological studies of mesenteric veins showed that
sympathetic nerve stimulation produces a slow depolarization and contraction mediated
by norepinephrine acting at (xi-adrenergic receptors (93). In small mesenteric arteries and
arterioles, electrical stimulation evokes short latency, short-duration excitatory junction

potentials (EJPs) and contractions mediated by ATP acting at P2X receptors (77).

3.2.3.1.3 SNS and venoconstriction

Sympathetic nerves have the greatest quantitative importance in minute-to-minute
regulation of venoconstriction, especially in the splanchnic circulation (81) (216) (155).
Sympathetic nerve stimulation can reduce intestinal blood volume by up to 60%, leading

to a signiﬁcant redistribution of blood from the splanchnic veins. This change in volume

distribution can have a profound effect on overall cardiovascular functions including

blood pressure (81) (109) (65).

3.2.3.2 Humoral factors

There are many other inﬂuences on venous smooth muscle activity besides
sympathetic input. These include tissue metabolites, p02, pCO2, ions, osmolarity,
myogenic factors and circulating hormones. Veins also exhibit signiﬁcant myogenic tone
when distended by increased intraluminal pressure. Enothelium-derived vasodilators such
as prostanoids, NO affect venoconstriction, but in general their effects are less
pronounced than in arteries. A number of studies implicate endogenous ET-l in the
control of venomotor activity by binding to both ETA and ETB receptors on the smooth

muscle cells.

3.2.3.3 Endothelin system
3.2.3.3.] Isoforms and function

In 1988, Yanagisawa et al (267) discovered a 21-amino acid peptide,
subsequently named endothelin (ET), in the supernatant of cultured porcine aortic
endothelial cells. The ET family consists of three distinct peptides (ET-1, -2 and -3), all
with very similar structure (267). In addition, 31-residue ETs have also been identiﬁed
(113). ET-1 is the most important subtype in the vasculature (266). It comprised 21
amino acid residues with ﬁ'ee amino- and carboxy-terrnini. The frny cysteine residues of
this peptide form two intrachain disulﬁde bonds (between positions 1-15 and 3-11). Its

molecular weight was estimated to be 2492 from the sequence data.

10

ET-1 is one of the most potent vasoconstrictors known, being nearly 2 logs more
potent in this function than comparable amounts of norepinephrine (3). In addition to its
cardiovascular effects, ET-1 is involved in embryonic development (118),
bronchoconstriction (240), prostate grth (251), carcinogenesis (120), and

gastrointestinal (184, 252) (119) and endocrine function (185) (106).

3.2.3.3.2 ET-1 and venoconstriction

Evidence indicates that ET-1 is an important determinant of venous tone (23) (87)
(222) (268). Exogenous endothelin causes potent and sustained venoconstriction in
experimental animals (250) (171) and in humans (175) (88). This effect is mediated by
both ETA and ETB receptor subtypes, whereas arterial constriction to ET-1 is largely
through ETA receptor activation (27). Stimulation of ETB receptors on endothelial cells
can cause release of vasodilators that oppose venoconstriction (170) (219). In intact rats,
exogenous ET-1 is a particularly potent constrictor of the mesenteric/splanchnic
circulation, an effect mediated by both ETA and ETB receptors (73). On the other hand,
endogenous ET-l has only modest effects on basal, resting venoconstriction in
experimental animals (64) (104) or humans (88), as inferred from responses to
pharmacological antagonists. However, substantial evidence suggests a role for ET-1 in
the regulation of venoconstriction in response to cardiovascular stresses and in a variety

of disease states where decreased vascular capacitance plays a part (104) (210).

3.2.4 Splanchnic veins and venules are the main vessels contributing to total

vascular capacitance

ll

The splanchnic circulation consists of those vascular beds perfused by the celiac,
superior mesenteric and inferior mesenteric arteries. Smaller arteries branch off to supply
the various structures of the GI wall. The hepatic artery is a major branch of the celiac
artery that provides oxygenated blood to the liver. The stomach receives blood from
branches of the celiac artery. The celiac trunk also gives rise to splenic artery that
provides blood to the spleen. The superior mesenteric artery provides blood to the small
intestine, pancreas and the proximal region of the large bowel. The remainder of the
colon is perfused by the inferior mesenteric artery. The blood draining the vascular beds
perfused by the major splanchnic arteries empties into a system of veins that join to form
the portal vein. The portal vein ultimately collects the blood from the spleen and the G].
tract and delivers it to the liver where it arborizes into a series of smaller and smaller
vessels that ultimately deliver blood to the hepatic sinusoids. The liver receives a dual
blood supply. The portal vein drains the splanchnic circulation and provides 75% of the
total blood ﬂow. The hepatic artery provides the remaining 25%. In the hepatic
microcirculation, the partially deoxygenated blood carried by the low pressure, portal
vein joins the well oxygenated blood derived from the high pressure, hepatic artery.
Blood draining the liver empties into the hepatic vein, which subsequently ﬂows into the
inferior vena cava.

The splanchnic bed is a major reservoir of blood in the body. The organs perfused
by the splanchnic vasculature receive 25% of the cardiac output at rest. So the splanchnic
veins hold the largest amount of blood within the vascular system, i.e. the splanchnic
veins and venules are the main vessels contributing to total vascular capacitance.

Venoconstriction especially in this region can cause a huge change in the distribution of

12

blood, allowing more vital functions to be supported with the available cardiac output.
Factors regulating the venomotor activity in the splanchnic vasculature are critical in

determining active changes in total vascular capacitance.

4. Vascular capacitance and compliance in deoxycorticosterone acetate (DOCA) -
salt hypertensive rats
4.1 Deoxycorticosterone acetate (DOCA) - salt hypertensive rats
This is a hypertension model with suppressed renin activity due to water and
sodium retention. This method of inducing hypertension is important because it allows an
examination of the neural and hormonal contribution to hypertension secondary to
adrenal steroid excess, which may be relevant to understanding of essential hypertension

(200).

4.2 Increased vascular resistance in hypertension

The hallmark hemodynamic change in established hypertension is increased vascular
resistance. For the typical increase in resistance that increases arterial blood pressure by
30-50 mmHg, the arteriolar vasculature need constrict only 4-7%. In addition, there is
evidence of an increased incidence of temporary and, perhaps, permanent closure of
small arterioles, which would also elevate resistance. The vascular smooth muscle cells
of small arteries are hypertrophied, but hypertrophy of these cells around arterioles is
minor. Many factors contribute to increased vascular resistance in hypertension,

including alterations in neural and hormonal inputs and myogenic tone.

l3

4.3 Reduced total systemic and venous compliance

Because the hallmark hemodynamic change in established hypertension is
increased vascular resistance, it is not surprising that the common interest in the study of
blood pressure control is on arteries. However, in human subjects with established
hypertension, total systemic and venous compliance also are reduced (207) (198) (144).
In several models of experimental hypertension in animals, such as spontaneously
hypertensive rats, one and two-kidney Goldblatt renal hypertensive rats, and dogs made
hypertension by angiotensin H, aldosterone, and reduced renal mass (181), MCF P is
elevated even when blood volume and cardiac output are not increased, suggesting
decreased venous compliance. Decreased venous compliance is most marked in
peripheral vessels, and is particularly notable in splanchnic veins (166). An important
function of decreased venous capacitance and/or compliance in established hypertension
is to provide higher ﬁlling pressures to hypertrophied, less compliant cardiac ventricles in
order to maintain normal cardiac output (207) (198). Arterial vasoconstriction in the
splanchnic bed also contributes to decreased vascular capacitance by allowing passive
recoil of splanchnic veins. As the splanchnic circulation is the largest capacitance bed,
changes in mesenteric vasomotor tone will impact overall hemodynarnics including blood
pressure (81).

Reduced compliance of the extrathoracic venous system has also been
documented in DOCA—salt hypertensive rats (64). However, only few studies have
directly addressed venous capacitance function in DOCA-salt hypertension. Although
most investigators report that in established DOCA-salt hypertension, there is normal

cardiac output and increased total peripheral resistance (2 70), it is important to note that

14

hypertension develops fully even when total peripheral resistance is prevented from
increasing (97). This underlies the importance of cardiac output regulation in the

development of hypertension in this model.

4.4 Factors mediating increased venoconstriction in hypertension

Blood volume and capacitance of the venous system are the major determinants of
MCFP (263). In established hypertension, blood volume is either normal or reduced (198).
Thus, elevated MCFP in hypertension is caused by structural changes in the venous wall
(143), increased venous smooth muscle contractile activity, or both. Relative little is
known about the mechanisms responsible for venous changes in hypertension. Evidence
of structural abnormalities in veins exists for hypertensive humans (198) (143) and some
experimental models of hypertension, eg. spontaneously hypertensive rats (181). Recent
studies in my group showed that the passive distensibility and myogenic tone were
signiﬁcantly decreased in DOCA-salt small mesenteric veins, but not in small mesenteric
arteries compared to sham rats (253). Vascular remodeling was present in DOCA-salt
small mesenteric arteries and veins (253). Therefore, structural changes in the venous
wall contribute to the increased venoconstriction in DOCA-salt hypertension.

Increased sympathetic venoconstriction contributes to reduced venous
capacitance and compliance in established hypertensive human subjects and experimental
animals. Increased sympathetic tone to mesenteric veins was demonstrated in SHR and
rats with reduced renal mass. In DOCA-salt hypertensive rats, my lab found that ganglion
blockade caused a larger decline in the MCF P than in sham rats (64), suggesting

increased sympathetic venoconstriction in DOCA-salt rats. Both increased sympathetic

15

discharge rate and enhanced neuroeffector transmission can be contributing factors. A
major goal of my study is to identify the mechanism of increased sympathetic
venoconstriciton in DOCA-salt hypertension.

Because ET-1 is one of the most potent vasoconstrictors known in vivo, a
pathophysiological role for the peptide as a mediator of hypertension has been postulated.
ET-l has a prominent role in the pathogenesis of DOCA-salt hypertension (201). A
number of studies implicate that ET-1 is important in the control of venoconstriction by
binding to ETA and ETB receptors. Venoconstriction produced by endogenous ET-l
acting on ETA and ETB receptors is enhanced in DOCA-salt hypertension, since both
selective ETA antagonism and combined ETA/B antagonism are effective in decreasing
the MCFP of DOCA-salt rats (104). No signiﬁcant change occurs in sham rats. Another
major goal of my study is to identify the mechanism of enhanced ET-l mediated

venoconstriction in DOCA-salt hypertension.

16

Figure 1: Schematic representation of NE overﬂow from sympathetic nerves to the
circulation (NE Spillover). NE is removed from the circulation (plasma NE clearance)
through neuronal uptake (U 1) into sympathetic nerves and uptake into extraneuronal cells
(U2). DHPG is an intraneuronal metabolite of NE. Plasma DHPG levels are determined
mainly by MAO in sympathetic nerves, net leakage of NE from vesicles into the
axoplasm, reuptake of released NE, and extraneuronal O-methylation of DHPG catalyzed
by COMT. MHPG in plasma is derived from multiple sources, including 1) deamination
of NMN after its cellular uptake, 2) deamination of NMN after cellular uptake and
intracellular O-methylation of NE, 3) O-methylation of DHPG after its uptake from the
circulation, and 4) O-methylation of DHPG after its uptake from the interstitial ﬂuid but
before its entry into the circulation. In the presence of impairment of U1, production of
DHPG is reduced and that of MHPG is increased, although the majority of DHPG
production derives from NE leaking from the sympathetic vesicles, independent of NE
reuptake. MAO indicates monoamine oxidase; COMT, catechol-o-methyltransferase;
DHPG, dihydroxylphenylglycol ; MHPG, 3-methoxy-4-hydroxylphenylgiycol; NMN,

normetanephrine (192).

17

 

 
    
 

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NE transporter ' ' ' '

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spillover clearance

crrculatlon DHPG plasma
NE plasma cone. 0 .

DHPG
overﬂow ‘7 ‘7

 

 

 

CHAPTER 2

HYPOTHESES AND SPECIFIC AIMS

l9

Existing evidence suggests that there is both increased endothelin-1 and
sympathetically mediated venoconstriction in DOCA-salt rats. However, it is not clear if
venoconstriction is due to increased ET-l levels and/or sympathetic activity in the
splanchnic vasculature, the main capacitance bed of the circulation. The aim of my work
was to test the hypothesis that ET-1 levels and/or sympathetic nerve activity in the
splanchnic vasculature is increased in DOCA-salt hypertensive rats (as illustrated in

Fig. 2).

Specific Aim I

The focus of this speciﬁc aim was to test the hypothesis that ET-1 levels and/or
sympathetic activity in the splanchnic vasculature is increased in rats in the

established phase of DOCA-salt hypertension (4-5 week).

Specific Aim I-l: Previous studies in my lab have shown that venoconstriction produced
by endogenous ET-l acting on ETA and ETB receptors is enhanced in vivo in DOCA-salt
hypertension. The contractile responses of veins to ET -1, however, were unchanged in
vitro. Therefore, enhanced ET-l mediated venoconstriction in vivo could be due to
greater splanchnic vascular formation of ET-1 in DOCA-salt hypertension. This aim
tested the hypothesis that there was higher ET-l concentration around the splanchnic

venous smooth muscle cells of rats in the established phase of DOCA-salt hypertension.

Protocol I-a: I measured venous vascular ET-l content to determine if local

concentrations of ET-1 are higher in DOCA-salt rats.

20

Protocol I-b: Since most ET-1 in blood vessels is presumably located within endothelial
cells, I investigated further the ET-l levels affecting the splanchnic veins in vivo using
ET-l concentration in splanchnic venous blood as an index, which is presumed to be in
equilibrium with ET-l concentration in the interstitial ﬂuid of venous smooth muscle
cells. And I used the difference between inﬂowing arterial ET-l concentration and
outﬂowing portal venous ET-l concentration as an index of net non-hepatic splanchnic

release (nNHSR) of ET-1.

Protocol II-a: Plasma ET-l level is determined by a combination of factors and
clearance by endothelial ETB receptors is the most important mechanism for removing
ET-l from blood. The aim of this study was to determine if altered ETB receptor-
mediated plasma clearance of ET-1 contributes to circulating ET-l level and nNHSR of

ET-1 in DOCA-salt hypertension.

Protocol II-b: My functional studies suggest impaired ETB receptor-mediated plasma
clearance of ET-1 in the splanchnic bed of DOCA-salt rats. The aim of this study was to
identify the underlying mechanism by examining the expression of endothelial ETB
receptors in small mesenteric blood vessels of DOCA—salt and sham rats using double

label immunoﬂuorescence and cofocal microspy.

Protocol III: Endothelin converting enzyme is a rate-limiting step in ET-l generation.

The change in nNHSR of ET-1 due to inhibition of ECE should provide more direct

manifestation of ET-1 generation in the splanchnic bed. The aim of this study was to

21

estimate ET-l synthesis in the splanchnic vasculature using the difference between the
net NHSR of ET-1 in rats treated only with ETB receptor antagonist and that in rats

treated with both ETB receptor antagonist and ECE inhibitor as an index.

Protocol IV: There appears to be a relatively small amount of ET peptide in blood and
tissue in relation to the concentration of ET receptors. The binding of ET-1 to its
receptors is particularly tight and ET-l remains bound to ETA receptors even after their
internalization. The aim of this study was to test the possible role of ETA receptor

binding in the plasma clearance of ET-1 in the splanchnic bed of DOCA-salt rats.

Specific Aim I-2: There is substantial evidence for increased sympathetic input to the
cardiovascular system in DOCA-salt hypertension. The non-hepatic splanchnic
circulation accounts for ~50% of total peripheral sympathetic outﬂow in humans and
experimental animals. This aim was to test the hypothesis that there was increased
sympathetic activity to the splanchnic bed in conscious DOCA-salt rats using estimated

non-hepatic splanchnic NE spillover as an index.

Protocol 1: The aim of this study was to test the hypothesis that there was increased

plasma NE levels and estimate nonhepatic splanchnic spillover of NE in DOCA-salt

hypertensive rats.

Protocol II: Prejunctional az-adrenoceptors mediate strong inhibition of NE release from

sympathetic terminals. Changes in prej unctional regulation of sympathetic nerves

22

associated with veins in DOCA-salt rats remain to be ﬁrlly characterized. The aim of this
study was to test the hypothesis that prej unctional az-adrenoceptor inhibition of NE
release was dysﬁmctional in sympathetic nerve endings associated with splanchnic

vasculature in DOCA-salt hypertensive rats.

Protocol III: NET is critical in central modulation of sympathetic tone and autonomic
regulation of peripheral hemodynamics by reducing NE concentrations in the
neuroeffector junction and preventing the resultant deleterious effects on cardiovascular
tissues. The rate of spillover is also dependent on the adequacy of neuronal uptake. The
aim of this study was to test the hypothesis that neuronal NE reuptake was altered in the

splanchnic vasculature in DOCA-salt hypertensive rats.

Protocol IV: An interaction between reuptake and otz-receptors has been found to be
present in certain vascular beds so as to maintain a largely constant effector response.
The aim of this study was to test the hypothesis that the interaction between prejunctional
az-adrenoceptor and neuronal transmitter reuptake was altered in the splanchnic

vasculature in DOCA-salt hypertensive rats.

Protocol V: The modulatory effect of ET-1 on sympathetic neurotransmission has been
reported. The aim of this study was to test the hypothesis that the modulatory effect of
ET-1 on sympathetic neurotransmission was altered in the splanchnic vasculature in

DOCA-salt hypertensive rats.

23

Specific Aim II

Venous capacitance and compliance are reduced in established hypertension, but it is
uncertain whether this is a cause or a consequence of hypertension. Existing evidence
suggests that there is both increased endothelin-1 and sympathetically mediated
venoconstriction in early DOCA-salt rats. The focus of this specific aim was to test the
hypothesis that ET-1 levels and/or sympathetic activity in the splanchnic

vasculature was increased in the early stage of DOCA-salt hypertension (4-5 day).

Specific Aim “-1: I tested the hypothesis that sympathetic nerve activity was increased

in the splanchnic vasculature of early DOCA-salt hypertensive rats.

Specific Aim 11-2: I tested the hypothesis that there was impaired prejunctional a2-
adrenoceptor inhibition of NE release in the splanchnic bed of early DOCA-salt

hypertensive rats.

Specific Aim “-3: I tested the hypotheses that there was increased ET-l formation and

impaired ETB receptor mediated uptake activity in the splanchnic vasculature of early

DOCA-salt hypertensive rats.

24

Figure 2: Schematic representation of overall hypothesis. I tested the hypothesis that
there was increased ET-l levels and/or sympathetic nerve activity in the splanchnic
vasculature of rats in the established and early phase of DOCA-salt hypertension. In
speciﬁc aim I-l, protocol I tested splanchnic vascular and venous plasma ET-l levels. I
also used the portal venous aortic plasma ET-l concentration difference as an index of
nNHSR of ET-1. Protocol II focused on determining if altered ETB receptor mediated
plasma clearance of ET-1 contributed to circulating ET-l level and nNHSR of ET-1.
Protocol III aimed at estimating ET-l synthesis in the splanchnic vasculature using the
difference between the net NHSR of ET-1 in rats treated only with ETB receptor
antagonist and that in rats treated with both ETB receptor antagonist and ECE inhibitor as
an index. Protocol IV tested the possible role of ETA receptor binding in the plasma
clearance of ET-1 in the splanchnic bed. In Speciﬁc aim I-2, protocol I tested the
hypothesis that there was increased plasma NE level and estimated nonhepatic splanchnic
spillover of NE in DOCA-salt rats. Protocol II aimed at determining if there was impaired
prejunctional a2-adrenoceptor function in sympathetic nerve endings associated with
DOCA splanchnic vasculature that contributed to increased NE release. Protocol III
tested if there was altered neuronal NE reuptake in the DOCA splanchnic vasculature.
Protocol IV tested the existence of an interaction between prejunctional a2-adrenoceptor
function and NET activity. Protocol V tested the hypothesis that the modulatory effect of
ET-1 on sympathetic neurotransmission was altered in DOCA splanchnic vasculature.
Speciﬁc aim H tested some the above hypotheses in early stage of DOCA-salt

hypertensive rats.

25

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26

Blood
pressure

3 Cardiac output

I Cardiac filling

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I Mean circulatory
ﬂlllng pressure

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(Splanchnic, gut)

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Venous
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i vascular 1
ET content

 

CHAPTER 3

GENERAL METHODS

27

1. Animals

All experiments were performed in male Sprague-Dawley rats (Charles River
Laboratories) that weighed 175 to 225 grams at the beginning of the study. Until the time
of catheterization, rats were housed 2 or 3 per cage in a temperature and humidity
controlled room with a 12h on/12h off light cycle. Pelleted rat chow and water were

given ad libitum (8640 Rodent Diet; Harlan/'1‘ eklad).
1.1 Preparation of DOCA-salt and sham rats

The left kidney was exteriorized and removed under sodium pentobarbital anesthesia
(50 mg/kg, i.p.). DOCA-salt rats received a DOCA pellet (200 mg) implanted SC and
they received 1% NaCl and 0.2% KCl in their drinking water. Sham-operated rats (sham)
underwent uninephrectomy but drank tap water. Three weeks after surgery, arterial blood
pressure was measured using the tail cuff method. Rats with a mean systolic blood
pressure of >150 mmHg were considered hypertensive. Sham rats generally had a mean

systolic blood pressure 5 125mmHg.

1.2 Surgery of rats used in chapter 4 and 5

Approximately 4 weeks after DOCA implantation or sham surgery, catheters were
placed in each rat. Anesthesia was produced with sodium pentobarbital (30 to 50 mg/kg,
i.p.). Catheters with silicone rubber tips were inserted into the abdominal aorta via a
femoral artery. Another catheter was positioned in the portal vein via a side branch for
sampling blood (Fig. 3). A third catheter was placed into a femoral vein for drug
administration. The rats were allowed to recover on a heated pad for 30 min before the

start of study. In chronically instrumented rats, the ends of all 3 catheters were tunneled

28

subcutaneously to the neck, where they exited the rat inside a stainless steel spring
secured to the body with a harness around the rat’s thorax. Injections of enroﬂoxacin (5
mg/kg, i.v.) were given for bacterial prophylaxis. Rats were allowed to recover
consciousness on a heated pad under constant observation. They were then placed in
stainless steel metabolism cages under loose tethering allowing continuous access to all
catheters without handling or otherwise disturbing the rat. Enroﬂoxacin (5 mg/kg, i.v.)
was administered to all rats for a three day period after surgery. Vascular catheters were
ﬁlled with heparin saline solutions when not in use and ﬂushed daily. Experiments were
started after at least 4 days of surgical recovery, as previous work in my lab indicated that
normal food and saline intake in DOCA-salt rats typically did not recover to pre-surgery
amount until 4 days. Blood samples were taken (with or without drug intervention) with
at least a one day interval separating experiments. In both anesthetized and conscious
groups, blood samples were collected simultaneously from arterial, portal venous sites
into ice-chilled plastic tubes containing EDTA (or EGTA and reduced glutathione for NE
experiments) and centrifuged at 10,000g for 15 min at 4°C. Plasma was separated and

stored at —80°C until used.

1.3 Surgery of rats used in chapter 6

Catheterization and uninephrectomy were performed (as described earlier)
simultaneously on normal rats. Enroﬂoxacin (5 mg/kg, i.v.) was administered to all rats
for a three-day period after surgery. Vascular catheters were ﬁlled with heparin saline
solutions when not in use and ﬂushed daily. 4 days after surgery, rats were selected

randomly to receive DOCA pellet implantation SC or sham operation. Both DOCA-salt

29

and sham rats received 1% NaCl and 0.2% KCl in their drinking water. Blood samples
were collected simultaneously from arterial, portal venous sites for ET-l or NE, EPI
measurement on the 4‘“ day after DOCA or sham surgery. One drug intervention was

given on the 5th day and blood samples were taken again.

2. Hemodynamic measurements

Arterial pressure of anesthetized rats was determined by connecting the arterial
catheter to Powerlab data acquisition system (ADInstruments Inc., CO). In the conscious
group, arterial pressure was determined by connecting the arterial catheter to a low-
volume displacement pressure transducer (TXD-300, Micro-Med, Louisville, Kentucky,
USA) linked to a digital pressure monitor (BPA-200 Blood Pressure Analyzer, Micro-
Med), which derived systolic, mean and diastolic pressures and heart rate every 0.55
(sampling rate=1000Hz). All values were averaged minute-by-minute and saved using a

computerized data acquisition system (DMSI—200/4 system Integrator, Micro-Med).

3. Plasma ET-l measurement

ET-l content was measured by Quantitative Sandwich Enzyme Immunoassay
technique. It is a 5-hmy solid phase ELISA. A monoclonal antibody speciﬁc for ET—l has
been pre-coated onto a microplate (R&D Systems, Inc.). 100 pl standards/EDTA plasma
was pipetted into each well. The plate was covered and incubated for 1.5 hmys at room
temperature on a horizontal orbital microplate shaker set at 500 :1: 50 rpm. Any ET-l
present was bound by the immobilized antibody. Each well was washed ﬁny times to

eliminate any unbound substances. 200 pl enzyme-linked monoclonal antibody speciﬁc

30

for ET-1 was added to each well. The plate was covered and incubated for 3 hmys at
room temperature. Each well was again washed fmy times to remove any unbound
antibody-enzyme reagent. 200 pl enhanced luminol/peroxidc substrate solution was
added to each well. Incubation was for 30 min at room temperature on the benchtop.
Light was produced in proportion to the amount of ET-1 bound in the initial step. A
microplate luminometer was used to measure the intensity of the light omitted. Minimum
detectable concentration of ET-1 was 0.16 pg/ml. Some cross-reactivity and interference
was observed with hBig ET-2 (0.010%), pBig ET-39 (0.019%), hBig ET-38 (0.019%),
bBig ET-39 (0.018%), rBig ET-39 (0.020%), sarafotoxin (0.490%), hET-3 (7.8%) and
hET-2 (27.4%). Interassay coefﬁcients of variation is 7.1%. Intraassay coefficients of

variation is 2.1%.

4. Plasma catecholamine measurement

Plasma NE and epinephrine were measured by radioenzymatic assay (REA) which
was based on the method described by Peuler and Johnson (177). The principle of this
technique is based on the conversion of NE and EPI to tritiated normetanephrine and
metanephrine in the presence of catechol-O-methyltransferase and tritiated S-
adenosylrnethionine as a labeled methyl donor. After puriﬁcation through a series of
organic extractions, tritiated normetanephrine is separated from the derivatives of
dopamine by oxidation with sodium periodate. Tritiated vanillin thus formed is counted

in a liquid scintillation spectrometer.

5. Portal blood flow measurement

31

After midline laparotomy, portal vein was exposed and lightly separated. Ultrasound
transit-time ﬂow probe (Transonic systems Inc. Ithaca, NY) was positioned around the
portal vein. Acoustic gel (Transonic systems Inc. Ithaca, NY) was applied in order to
achieve proper ultrasonic contact between probe and vessel and obtain satisfactory
acoustic sigrrai. During the experiments, probe was connected to a HT207 dual channel
ﬂow meter (Transonic Systems Inc.). Portal venous blood ﬂow (ml/min) was measured

after a 30 min stabilization period.

6. Estimate of non-hepatic splanchnic (NHS) spillover of NE

NHS NE spillover was estimated based on two assumptions: 1) Portal venous blood
ﬂow of DOCA-salt rats equals that of sham rats. 2) NHS fractional extraction (F X) of NE
(fractions of NE removed from plasma during passage through NHS organs) equals NHS

FX of epinephrine.

SR =[(NEo-NE,-)+ﬂVE,-x FX)] x Q x (1 -Hct)
NEiz concentration of NE in inﬂowing plasma (pg/ml)
NEG: concentration of NE in outﬂowing plasma (pg/ml)
Q: regional blood ﬂow
Hct: hematocrit
FX: fractional extraction of NE (i.e. fraction of NE removed from plasma during

passage through mesenteric organs)

Fractional extraction of epinephrine = (EPI,- — EPI,) / EPI,-

EPI,: concentration of EPI in inﬂowing plasma (pg/ml)

32

EPI,: concentration of EPI in outﬂowing plasma (pg/ml)

7. Radiotracer method

The radiotracer method allows the estimation of regional sympathetic nervous activity

from measurement of the organ-speciﬁc NE spillover rate.

7.1 Radiotracer infusion

Tritium-labeled NE ([3H]—NE; [levo-ring-2,5,6-3H]NE, 40-60 Ci/mmol, 1 mCi/ml,
PerkinElrner, MA) was diluted with 500 pl of 0.2 M acetic acid plus 50 it] of sodium
sulﬁte (100 mg/ml), 350 pl of reduced glutathione (6 mg/ml) qsp 10ml with 0.9% saline
and was infused at 27 til/min intravenously (femoral vein), i.e. 0.06 pCi/kg/min with the
pump for 90 min (rate at which blood pressure levels will not be modiﬁed in either sham
or DOCA-salt rats) into catheterized DOCA-salt or sham rats. The radiotracer infusion
was started at least 30 min before blood sampling to ensure attainment of steady-state
plasma [3H]-NE concentration. Arterial and portal venous blood samples were obtained
to measure [3H]-NE and endogenous NE. We are still working on the technique to
separate tritiated NE from its metabolites (mainly dihydroxylphenylglycol) with the help
of another lab. Ideally, timed collection of the [3H]-NE in the eluant leaving the HPLC
detection cell enables the separation. [3H]-NE can then be quantiﬁed by liquid
scintillation counting. Plasma concentrations of endogenous NE will be determined by

REA as described earlier.

33

8. Immunoﬂuorescence localization and quantification of endothelial ETB
receptors

Paraffin embedded sections of Methacarn (or known as Carnoy’s) (consists of 60%
methanol, 30% chloroform and 10% glacial acetic acid) ﬁxed mesenteric blood vessels
(including both superior mesenteric artery and vein and small mesenteric arteries and
veins from DOCA-salt and sham rats) were deparafﬁnized and rehydrated. Antigen
retrieval was performed to recover antigenicity by soaking the sections in Vector Labs
antigen unmasking solution and heating in a microwave for 2 minutes. After wash with
PBS, sections were incubated with rabbit anti-ETB receptor antibody (Alomone labs,
Jerusalem, Israel) at a dilution of l: 50in 0.01 M PBS with 1% Triton X-100 and sheep
anti-rat vWF antibody (Enzyme Research Laboratories, South Bend, IN) at a dilution of
1:200 in 0.01 M PBS with 1% Triton X-100 overnight at 4 °C in a humidiﬁed chamber.
Unbound primary antibody was washed away using phosphate buffered saline and after
washing, sections were incubated with cy3-conjugated goat anti-rabbit IgG (Jackson
Immunoresearch, PA) at a dilution of 1: 200 in 0.01 M PBS with 1% Triton X-100 and
cy5-conjugated donkey anti-sheep IgG (Jackson Immunoresearch, PA) at a dilution of
1:50 for 2h at room temperature in a humidiﬁed chamber. Unbound secondary antibodies
were washed away and sections were mounted with AquaPerm mounting medium
(Thermo Electron Corporation, PA). The slides were coverslipped and sealed with nail
polish. The slides were stored in dark at 4 °C until observed using a confocal ﬂuoresence
microscope.

The ﬂuorescence intensity of ETB receptors in endothelial cells was quantiﬁed using

Image J (software from NIH, downloaded from internet) using the ﬂuorescence staining

34

of von Willebrand Factor as an endothelial cell marker. The mean ﬂuorescence intensity
of ETB receptors from 3-9 individual endothelial cells was plotted and compared
between DOCA-salt and sham rats.

Images in this thesis are presented in color.

9. Statistical analysis of data

All data were given as mean i SEM. Differences in variables between rats were
compared by ANOVA followed by Bonferroni’s test to correct the error rate for multiple
comparisons. Differences in variables within rats (i.e. over time) were compared using
paired-t tests or repeated measures ANOVA followed by Bonferroni’s test. P5005 was

regarded as signiﬁcant.

35

arterial catheter Inferior vena cava

  

Celiac artery Hepatic artery

    
   

Hepatic vein

Large intestine

Figure 3: Schematic representation of splanchnic circulation and the positions of the

arterial catheter and portal venous catheter.

36

CHAPTER 4

ENDOTHELIN-l CONCENTRATION

AROUND THE SPLANCHNIC VENOUS SMOOTH MUSCLE CELLS OF

RATS IN THE ESTABLISHED PHASE OF DOCA-SALT HYPERTENSION

37

Introduction

Role of ET—1 in human hypertension and experimental models of hypertension in
animals

ET-1 is a powerful vasoconstrictor, has mitogenic properties, and induces
inﬂammatory responses. These effects contribute to altered vascular reactivity and to
vascular remodeling, important processes in blood pressure elevation. Therefore, a
pathophysiological role for the peptide as a mediator of hypertension has been postulated.
Unlike experimental models of hypertension, such as DOCA-salt hypertensive rats (127,
128), Dahl salt-sensitive rats (25), aldosterone-salt hypertensive rats (173), one kidney
one clip Goldbaltt hypertensive rats (226), stroke prone spontaneously hypertensive rats
(236), salt loaded spontaneously hypertensive rats (201, 236), in which ET-l has been
implicated to be important in the pathophysiology of hypertension, the role ET-l played
in a normal cardiovascular homeostasis and in mild essential hypertension in humans is
unclear. Plasma concentrations of ET-l-like immunoreactivity have been reported to be
normal or slightly elevated in patients with mild essential hypertension (204) (199).
However, increasing evidence suggests that ET-1 may be important in certain groups of
essential hypertensive patients, including severe and/or malignant hypertension (128),
pulmonary hypertension (76), hypertension in African Americans (49), salt-sensitive
hypertensives , low renin hypertension (129, 174), pre-eclampsia (83), and those with
obesity (129) and insulin resistance (271). In all of these conditions, plasma
immunoreactive ET levels are elevated and tissue ET-l expression is increased. Data
from clinical trials using combined ETA/B receptor blockers have already demonstrated

signiﬁcant blood-pressure-lowering effects (117). Thus, targeting the endothelin system

38

has important therapeutic potential in the treatment of hypertension, particularly by
contributing to the prevention of target organ damage and the management of

cardiovascular disease.

ET—l increases total peripheral resistance in DOCA-salt rats by contracting arteries
and arterioles.

ET-l has a prominent role in DOCA-salt hypertension (201). There is blood
pressure lowering upon administration of endothelin antagonists (130). Endothelin
receptor antagonists also regressed vascular grth and inﬂammation, and improved
endothelial dysfunction (130). DOCA-salt rats treated with endothelin antagonists were
protected from stroke and renal injury.

One important effect of ET-1 in DOCA-salt hypertension is to increase total
peripheral resistance by contracting arteries and arterioles (270). Most studies have
shown, however, that arterial plasma levels of ET-1 are not increased in DOCA-salt rats
(161, 162, 203, 223, 247). Nonetheless both preproendothelin-l (preproET-l) mRNA
expression and immunoreactive ET-l content of aorta and mesenteric arteries are
increased in DOCA-salt hypertensive rats (28, 127, 128, 203), suggesting that one
mechanism of ET-1 induced arterial constriction in hypertension is increased levels of the
peptide around arterial smooth muscle. A caveat to that conclusion is raised by ﬁndings
that contractions to ET-l are signiﬁcantly reduced in arteries of DOCA-salt rats when
compared with those of sham rats (103). Thus, increased local production of ET-1 by
arteries may be countered by decreased arterial responsiveness, and it remains unclear

how ET-l increases vascular resistance in DOCA-salt hypertension.

39

There is enhanced ET-l mediated venoconstriction in DOCA-salt hypertension.
The demonstration of the involvement of the endothelin system in the
development and maintenance of hypertension in DOCA-salt hypertensive rats has
stimulated investigations into a potential role of this system at the venous site of the
circulation, especially because in-vitro studies have consistently shown that the maximal
vasoconstrictor response to, and potency of, ET-1 in veins is greater compared to that in
corresponding arteries (23, 105) (190) (182). The venous system is an important, yet
infrequently considered part of the circulation. Hypertensive humans and experimental
animals also exhibit decreased systemic vascular capacitance (181, 198). This is
primarily the result of changes in the structure or vasoconstrictor activity of extra-
thoracic veins, especially those in the splanchnic bed (198). Reduced vascular
capacitance contributes to the hemodynamics of hypertension by contributing—along
with blood volume—to maintenance of “effective blood volume”, a critical determinant
of venous return of blood to the heart and thus cardiac output (198). In experimental
animals, information about venous tone in vivo can be derived ﬁom the MCF P (as
introduced earlier). Similar to vascular resistance, MCF P is increased in practically all
forms of hypertension (132) including DOCA-salt hypertensive rats (104). As blood
volume in hypertension is not increased but, in contrast, is usually slightly reduced, the
increase in MCF P in hypertension must be caused by a decrease in the compliance of the
venous system, which in turn may be a consequence of an increase in venous tone, of
structural venous changes or of the combination of these two effects (197). Endogenous

ET-l appears at least partly responsible for that augmented venoconstriction, since ET

40

receptor antagonists lower MCFP in DOCA-salt hypertensive but not nonnotensive rats

(104).

41

Chapter 4 - Part I
1) Total vascular ET-l content in mesenteric veins of DOCA-salt hypertensive rats

Rationale: Two possible mechanisms could explain increased ET-l mediated
venoconstriction: higher levels of the peptide around venous smooth muscle cells and/or
enhanced responsiveness of venous smooth muscle to ET-l. It has been shown that vena
cavae and small mesenteric veins from DOCA-salt hypertensive rats do not exhibit
increased reactivity to ET-1 in vitro (103, 254). Therefore, I hypothesized that higher ET-
1 mediated venoconstriction in DOCA-salt rats was caused by increased ET-l
concentrations around venous smooth muscle. First, I measured vascular ET-1 in the
superior and small mesenteric veins of hypertensive DOCA-salt and normotensive sham
rats. Since ET-1 is primarily synthesized de novo by vascular endothelial cells, I expected

vascular ET-l content to be representative of local vascular production of the peptide.

Experimental Procedures: Approximately 4 weeks after DOCA implantation or sham
surgery, rats were euthanized by sodium pentobarbital overdose (lOOmg/kg, i.p., Sigma,
MO) and segments of thoracic aorta, thoracic vena cava, abdominal vena cava were
removed and dissected free of fat. The complete mesenteric vascular bed was removed
and placed in a Petri dish full of ice cold Kreb’s solution. The small intestine was
stretched gently and pinned ﬂat. Superior mesenteric artery and vein, small mesenteric
arteries and veins (150-250 pm in diameter) were dissected free of fat and collected
separately. The tissues were snap-frozen in liquid nitrogen and stored at -80°C until
extraction of ET-1 was performed. On the day of extraction, tissues were homogenized

with a Polytron in lml 1M acetic acid containing 10 jig/ml pepstatin A (ICN

42

pharmaceuticals, Costa Mesa, CA) and immediately heated to 100°C for 10 min. After
chilling, the homogenate was centrifuged at 14,000g for 30 min at 4°C. The supernatant
was dried in a Speed-Vac and then reconstituted in 300 pl calibrator diluent (R&D
systems, Minneapolis, MN). The reconstituted sample was stored at -80°C until
measurement of ET-1 was performed. Vascular ET-l content was measured by
quantitative sandwich enzyme immunoassay technique. Cross reactivity of the antibodies

with some of the other peptides was mentioned in General Methods.

Results: ET-l content was normalized using tissue weight. I found that 1) consistent with
previous published result (28, 127, 128, 202), there was signiﬁcantly higher ET-l content
in aorta and superior mesenteric artery of DOCA-salt rats than sham rats; 2) ET-l content
was higher in small vessels than in larger vessels in both DOCA-salt and sham rats; 3)
there was no signiﬁcant difference in the ET-l content in veins of any size between

DOCA-salt and sham rats (Fig. 4).

2) Venous plasma ET—l concentration and net non-hepatic splanchnic release
(nNHSR) of ET-1

Rationale: Since most of the ET-1 in the blood vessels is presumably localized within
endothelial cells (72), I was not sure if my ﬁndings above indicate that the ET-l
concentrations at the surface of venous smooth muscle cells are the same in DOCA-salt
and sham rats. So I conducted additional experiments to measure portal venous plasma

ET-l level.

43

In contrast to circulating hormones, ET acts primarily as a local hormone in an
autocrine and paracrine fashion. Plasma concentrations are typically very low even in
pathological states (67). More importantly, ET has been shown to be secreted primarily
(80%) into the basolateral compartment (i.e., abluminally) and not into the apical
compartment(1uminally) (20%) (249). Thus, plasma concentrations may not be
representative of locally released ET-l at its site of action. However, the portal vein
drains venous blood from all of the splanchnic organs except the liver. Portal venous ET-
1 concentration therefore should reﬂect splanchnic venous ET-l concentration, which is
presumed to be in equilibrium with ET-l concentration in interstitial ﬂuid of vascular
smooth muscle cells. Therefore, studies on plasma ET-l levels are still helpful to
determine the direction of differences in locally released ET-l at its site of action
between DOCA-salt and sham rats. Because part of portal venous plasma ET-1 is derived
from arterial blood entering the splanchnic bed, it was also necessary to measure arterial
plasma ET-l level. Thus, I used the portal venous-aortic plasma ET-l concentration
difference as an index of net non-hepatic splanchnic release (NHSR) of ET-1. I presumed
also that most ET-1 in plasma is derived from vascular endothelial cells. The liver was
excluded from these experiments because of the difficulty of sampling blood from the
hepatic venous drainage in the rat. In addition, portal vein sampling prevents interference
of substantial hepatic extraction (35) with the measurement of splanchnic release of ET-

1.

Experimental Procedures: I did a preliminary study using anesthetized rats. Catheters

were inserted into the abdominal aorta, portal vein and femoral vein as introduced in

44

General Methods. Additional studies were performed in conscious rats because
anesthesia and acute surgery can affect hormone synthesis and release. In both
anesthetized and conscious groups, 0.5 ml blood samples were collected simultaneously
from arterial and portal venous catheters into ice-chilled plastic syringes and transferred
into ice-chilled plastic tubes containing EDTA. Blood samples were centrifuged at
10,000g for 5min at 4°C. Plasma was separated and stored at -80°C until assayed using

ELISA.

Results: The average mean arterial pressure (MAP) of DOCA-salt rats was signiﬁcantly
higher than that of sham rats in either anesthetized or conscious group (Fig. 5). The MAP
difference between anesthetized DOCA-salt and sham rats, however, was much smaller
than that between conscious DOCA-salt and sham rats.

Plasma ET-l concentration was signiﬁcantly higher in portal vein than in aorta in
anesthetized sham and DOCA-salt rats, indicating nNHSR of ET-1. However, there was
no signiﬁcant difference in portal venous plasma ET-l level between sham and DOCA-
salt rats or in aortic plasma ET-l level between the two groups (Fig. 6A). There was no
correlation between plasma ET-l concentrations and blood pressure levels of DOCA-salt
rats.

Plasma ET-l concentration also was signiﬁcantly higher in portal vein than in
aorta in conscious sham and DOCA-salt rats. However, there was no difference in portal
venous plasma ET-l level between conscious sham and DOCA-salt rats or in aortic

plasma ET-l levels between the two groups (Fig. 6B).

45

Both the aortic and portal venous plasma ET-l concentrations were signiﬁcantly
lower in conscious sham rats compared with anesthetized sham rats. In conscious DOCA-
salt rats, the portal venous plasma ET-l concentration was signiﬁcantly lower than that of
anesthetized DOCA-salt rats (Fig. 6A, 6B). The aortic plasma ET-l concentration was
lower in conscious DOCA-salt rats, but not quite signiﬁcant (P=0.066), compared with

anesthetized DOCA-salt rats.

The net NHSR of ET-1 was estimated using portal venous aortic plasma ET-l
concentration difference. There was no signiﬁcant difference in the nNHSR of ET-1
between DOCA-salt and sham rats in either anesthetized or conscious group (Fig. 7A,
7B). The basal nNHSR of ET-1 was signiﬁcantly lower in conscious sham rats than in
anesthetized sham rats. In conscious DOCA-salt rats, the nNHSR of ET-1 was lower, but

not signiﬁcantly so (P=0.05 7), compared with anesthetized DOCA-salt rats.

46

 

 

 

 

 

 

 

 

 

 

 

 

 

 

5 —sham(n=6)
‘ [:1 DOCA(n=6-13)
’8 I
54-
O
E
:53— I
0
‘E
O
02‘
Li .
1..
o :Uiﬂl l -.. *-..
«1.3-253‘“ 2' C .E C:
’5 as as :2 §§ 2 as
to o as t .
2 £2 82 - E“ c E
o “in ﬁg 3 g. o 7.1:
5 > “’> 0’ E 3"» E
.2 am e ..

Figure 4: Vascular ET-l content of DOCA-salt and sham rats. Bars indicate mean ET-l
content in blood vessels from sham and DOCA-salt rats. Brackets indicate SEM.

Asterisks indicate a signiﬁcant difference (P<0.05) between sham and DOCA-salt rats.

47

200

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

3‘ .#

I .

E 180 - sham (n=8-9) T

E. 160- :: DOCA (n=5-10)

0)

5 140 - *

g 120‘ r

3- 100 -

g 80.

E 60-

C

8 40 «

E 20 .

0 . .
anesthetized conscious

rats rats

Figure 5: Mean arterial pressure of anesthetized and conscious DOCA-salt and sham rats.
Bars indicate mean arterial pressure of anesthetized sham and DOCA-salt rats. Brackets
indicate SEM. Asterisks indicate a signiﬁcant difference (P<0.05) between sham and
DOCA-salt rats. # indicates a signiﬁcant difference (P<0.05) between anesthetized and

conscious DOCA-salt rats.

48

Figure 6:

A: Aortic and portal venous plasma ET-l level in anesthetized DOCA-salt and sham rats.
Bars indicate mean aortic or portal venous plasma ET-l concentration in anesthetized
sham and DOCA-salt rats. Brackets indicate SEM. Asterisks indicate a signiﬁcant

difference (P<0.05) between portal venous and aortic plasma ET-l concentration.

B: Aortic and portal venous plasma ET-l level in conscious DOCA-salt and sham rats.
Bars indicate mean aortic or portal venous plasma ET-l concentration in conscious sham
and DOCA—salt rats. Brackets indicate SEM. Asterisks indicate a signiﬁcant difference
(P<0.05) between portal venous and aortic plasma ET-l concentration. #3 indicate a

signiﬁcant difference (P<0.05) between conscious and anesthetized rats.

49

plasma ET—1 concentration (pg/ml)

plasma ET-1 concentration (pg/ml)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

6
— sham (n=9)
E DOCA(n=10) ,,
*
T
4 J
l
2 .
l
0 .
aorta portal vein
6
— sham (n=8)
:2 DOCA(n=5)
4 q
3* #v:
2 q T
# T
0 , .
aorta portal vein

50

Figure 7:

A: Net nonhepatic splanchnic release of ET-1 in anesthetized DOCA-salt and sham rats.
Bars indicate mean net nonhepatic splanchnic release of ET-1 in anesthetized sham and

DOCA-salt rats. Brackets indicate SEM.

B: Net nonhepatic splanchnic release of ET-1 in anesthetized DOCA-salt and sham rats.
Bars indicate mean net nonhepatic splanchnic release of ET-1 in conscious sham and
DOCA-salt rats. Brackets indicate SEM. Asterisk indicates a signiﬁcant difference

(P<0.05) between anesthetized and conscious sham rats.

51

 

 

 

 

 

 

 

 

 

- sham (n=9)
:1 DOCA (n=10)

 

 

 

 

— sham (n=8)
1:: DOCA (n=5)

 

 

 

 

 

 

 

2633 Em. *0 886.
2:20:93 28855: as:

u

2

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25333 033055: so:

 

 

 

52

 

 

Discussion for Chapter 4—Part I

1) ET-l content of mesenteric arteries and veins

ET-l content of blood vessels is determined by both local vascular production,
and reuptake and storage, of the peptide. These processes occur predominantly but not
exclusively in endothelial cells. Total vascular content of ET-1 thus represents a rough
index of local concentrations of the peptide available to cause vasoconstriction in viva.
Little is know about the status of tissue ET -1 in hypertensive patients. From gluteal
subcutaneous biopsies, Schiffrin et al demonstrated that the prepro-ET-l gene is
overexpressed in the endothelium of small arteries of moderate to severe hypertensive
patients, in contrast with mild hypertensive patients and normotensive subjects (202).
This may contribute to downregulation of endothelin receptors and lead to blunted
responses to ET-l, which has been demonstrated in isolated human small arteries from
hypertensive patients (89).

Measurements of splanchnic vascular ET-l content in my study produced novel
ﬁndings. First, on the basis of tissue weight, veins have higher ET-l content than arteries.
This may reﬂect a higher ratio of endothelial cells to other cell types in veins versus
arteries, or a greater ET-l synthetic capacity of venous compared to arterial endothelial
cells. However, more ET-l binding to venous smooth muscle ETA receptors and further
ground down into the supernatant remains to be a possibility too. Second, smaller blood
vessels had higher ET-l content than larger vessels. This may reﬂect the relative amount
of endothelial cells in my samples (especially in arteries), but also is consistent with an
important role for ET-1 in control of vascular resistance and capacitance, since these

physiological ﬁrnctions are mainly served by smaller sized vessels in the intact

53

circulation. Third, I found that vascular ET-l content was signiﬁcantly greater in DOCA-
salt rats only in the aorta and superior mesenteric artery. Signiﬁcantly increased ETA
receptor number and decreased contractile reponse to ET-l have also been found in
DOCA aorta and superior mesenteric artery (154, 254) (104). The combined results
suggest dysfunction of ETA receptors. Upregulation of the vascular endothelin system
may contribute to cellular processes associated with vascular remodeling and to increased
stiffness of conduit arteries in hypertension (142, 201).

Finally, I found that there was no signiﬁcant difference in ET-l content in veins
of any size between DOCA-salt and sham rats. This suggests that production of ET-1 by
venous endothelial cells is not increased in DOCA-salt hypertensive rats. This is
consistent with my previous ﬁnding of unchanged preproET-l mRNA level in mesenteric
veins in DOCA-salt rats (103). Although it has been found that cultured rat vascular
smooth muscle cells can synthesize/secret ET—1 in response to a variety of stimuli, it is
suggested (84) that in vivo expression of the gene for prepro ET-1 in SMC may depend
on the existing and/or past integrity of the blood vessel wall (e. g. endothelial
desquamation, phenotypic modulation of constituent SMC, and local production of
growth factors). It remains to be determined whether expression and synthesis of ET-1 by
VSMC represent a physiological and/or pathophysiological axis of regulation.

Steady-state vascular content of ET-1 , however, may not be an accurate indicator
of local formation of ET-1 around venous capacitance vessels. This is because released
ET-1 in vivo is subjected to rapid tissue binding, reuptake and metabolism. The kinetics

of ET-1 disposition are only beginning to be understood (35, 36).

54

2) ET-l level in venous blood and nNHSR of ET-1

Therefore, I conducted additional experiments designed to evaluate the rate of
ET-1 release by the non-hepatic splanchnic vasculature of normotensive and DOCA-salt
hypertensive rats in viva. Although ET-l release by endothelial cells is reported to be
polarized towards vascular smooth muscle (80%) compared to blood (20%) (249), I
reasoned that venous blood (portal vein) draining the splanchnic bed (except the liver)
would yield a reliable estimate of differences in ET-l production and release by
splanchnic vessels. Because part of portal venous plasma ET-1 is derived ﬁom arterial
blood entering the splanchnic bed, it was also necessary to measure arterial plasma ET-l
levels. Thus, in analogy to other studies (2, 24), I used the difference between arterial and
portal venous ET-l plasma concentrations as an index of net NHS release of ET-1.
Previous studies demonstrated that arterial plasma levels of ET-1 are not increased in
DOCA-salt rats. My results conﬁrmed this ﬁnding in both anesthetized and conscious
DOCA-salt rats (Fig. 6A, 6B). No studies have been reported on splanchnic venous
plasma ET-l level or nNHSR in this model of hypertension. I hypothesized that both
portal venous plasma ET-l concentration and nNHSR of ET-1 would be higher in
DOCA-salt rats compared to sham rats. This would indicate that the endothelial cells in
the splanchnic vasculature of DOCA-salt rats release more ET-1 in viva; and that venous
smooth muscle cells of the splanchnic bed are exposed to higher ET-l concentrations.

I found that the arterial plasma ET-l level is not signiﬁcantly higher in DOCA-
salt rats, i.e. similarly as in sham rats, plasma levels of circulating ET-1 in DOCA-salt
rats are too low to produce cardiovascular effects. The low plasma levels of ET-1 may be

attributable to rapid clearance of circulating ET-l (about 80% of the radioactivity was

55

cleared from the circulation within the ﬁrst minute of bolus injection of [1251] ET-l), a
slow rate of ET-1 production and/or preferential release of ET-1 to vascular smooth
muscles.

I also found that portal venous plasma ET-l level was signiﬁcantly higher than
aortic plasma ET-l level in both anesthetized hypertensive DOCA-salt rats and
normotensive sham rats, indicating net non-hepatic splanchnic release of ET-1 in both
groups of animals. However, there was no difference in portal venous plasma ET-l level,
or in nNHSR of ET-1 between the two groups. This provides the ﬁrst in viva evidence
that endothelial cells of the splanchnic vasculature of DOCA-salt rats do not produce
more ET-l compared to those of sham rats. However, as mentioned earlier, plasma ET-l
level is determined by a combination of factors including production rate, metabolism,
clearance and regional blood ﬂow. Blood ﬂows to the stomach, small and large intestines
are not affected by DOCA-salt treatment (97, 264), so disparities in regional blood ﬂow
are not likely to contribute to my ﬁndings. Some studies suggest that plasma ET levels
are also regulated by renal clearance of ET-1. Increased plasma ET levels could be
secondary to the impairment of renal clearance. It has been found that there is lower
urinary ET excretion in patients with salt-sensitive hypertension in which it has been
known that there is increased circulating ET level (235). However, opposite opinions also
exist suggesting that at least in human, plasma ET-1 and urinary ET-l represent two
distinct systems regulated by different control mechanisms. The ET-l normally present in
the urine is more likely to be of renal origin (210). In rats injected with 125I-ET-l, labeled
endothelin is detectable only in trace amount in the urine (5). The relationship between

plasma ET-1 and urinary ET-l has not been clariﬁed in DOCA-salt hypertensive rats.

56

Another mechanism that could inﬂuence venous plasma ET-l level and nNHSR
of ET-1 in DOCA-salt rats is altered ETB receptor-mediated uptake activity. For example,
enhanced endothelial cell production and release of ET-1 in the splanchnic vasculature of
DOCA-salt rats might not be apparent with my in viva measurements if there is also
increased removal of ET-1 from plasma by reuptake into endothelial cells. I tested this

possibility in my later studies.

3) Anesthetized versus conscious rats

My preliminary studies were performed on anesthetized rats after acute surgery for
catheter implantation. The MAP differences between DOCA-salt and sham rats were
much less than when they were conscious (probably reﬂecting the important role of the
sympathetic nervous system in regulation of blood pressure in DOCA-salt hypertension).
Further studies were then performed on conscious rats to avoid the effects of surgery and
anesthesia on sympathetic nerve activity, body ﬂuid status and hormone synthesis or
release. Rats also were in a normal crouched posture instead of lying on their back when
blood was sampled. This is a potentially signiﬁcant factor when considering vascular
capacitance function. I saw signiﬁcantly lower plasma ET-l concentration and nNHSR of
ET-1 in conscious rats, presumably reﬂecting less stress or anesthesia induced effects on

ET-l formation, metabolism, or uptake.

57

Chapter 4 - Part II

1) ETB receptor-mediated clearance of plasma ET-1 in the splanchnic bed of

DOCA-salt hypertensive rats

Rationale: Plasma ET—l level is determined by a combination of factors including
production rate, metabolism and clearance. Previous studies indicate that clearance by
endothelial ETB receptors is the most important mechanism for removing ET-l from
blood (19, 191, 213, 217).

ETB receptors are abundant on endothelial cells, vascular smooth muscles and
parenchymal cells of the lung and kidney. ET-1 is rapidly cleared ﬁom the circulation
mainly through the lungs (~80%), kidneys or liver, probably at least in part in a receptor-
mediated fashion like many other peptide hormones including angiotensin II and atrial
natriuretic peptide. The rapid clearance of ET-1 from the circulation may play a role in
protecting the systemic circulation from the vasoconstrictor actions of ET-1 (217). At the
low concentrations occurring physiologically, the major ET-l clearing sites appear to
reside in the lungs. When pulmonary binding capacity is saturated by ET-l , other
vascular beds may take over the function of clearing endothelin from the circulation
(217).

To test the possibility that a functional alteration in ETB receptors might affect the
venous plasma ET-l concentration and net nonhepatic splanchnic release of ET-1 in
DOCA-salt hypertension, I measured the aortic and portal venous plasma ET-l
concentrations and calculated portal venous-aortic plasma ET-l concentration difference

after selective ETB receptor blockade in both anesthetized and conscious rats.

58

Experimental Procedures: Catheters were inserted into abdominal aorta, portal vein and
femoral vein as introduced in General Methods. In the anesthetized group, rats were
allowed to recover ﬁom surgery on a heated pad for 30 min before blood sampling.
A192621, a selective ETB receptor antagonist (256), was then given (12 mg/kg, i.v.,
Abbott Laboratories, IL) and blood samples were collected again 30 min later. It has been
shown by previous studies in my lab (104) that this dose of A192621 is effective at
blocking ETB receptors and causes a signiﬁcant increase in MAP in DOCA-salt
hypertensive rats. The increase reaches maximum at 30 minutes after A192621
administration. In rats to be studied while conscious, blood samples (with and without

A192621 treatment) were taken with at least a one day interval separating experiments.

Results:

i. Effects of selective ETB receptor antagonist on MAP

Administration of A192621 caused a signiﬁcant increase in the mean arterial pressure
in both anesthetized DOCA—salt and sham rats (Fig. 8A). A192621 also caused a
signiﬁcant increase in the mean arterial pressure in conscious sham rats, but not in

conscious DOCA-salt rats (Fig. SB).

ii. Total ETB receptor mediated plasma clearance of ET-1

A192621 caused a signiﬁcant increase in the aortic plasma ET-l concentration in
both anesthetized (Fig. 9A) and conscious (Fig. 9B) sham and DOCA-salt rats. There was
no signiﬁcant difference in the aortic plasma ET-l concentrations between sham and

DOCA-salt rats after A192621 treatment under anesthesia or in the conscious state. The

59

magnitude of change in aortic plasma levels of ET-1 before and after treatment also was

not signiﬁcantly different between sham and DOCA-salt rats.

30 min after A192621 treatment, the portal venous plasma ET-l concentration was
signiﬁcantly and similarly increased in both anesthetized and conscious sham and

DOCA-salt rats (Fig. 10A, 10B).

iii. ETB receptor mediated plasma clearance of ET-1 in the NHS vasculature

After A192621 treatment, there was a signiﬁcant increase in the nNHSR of ET-1 in
anesthetized sham, but not anesthetized DOCA-salt rats (Fig. 11A).

In conscious animals, after ETB receptor antagonist treatment, nNHSR of ET-1 was
signiﬁcantly increased in sham rats, but remained unchanged in DOCA-salt rats (Fig.
11B).

Data from both anesthetized and conscious rats suggest impaired ETB receptor-
mediated clearance of plasma ET-1 in the splanchnic bed of DOCA-salt hypertensive

rats.

2) Mechanisms of impaired ETB receptor-mediated clearance of plasma ET-1 in

the splanchnic bed of DOCA-salt hypertensive rats

Rationale: Possible mechanisms underlying the decreased uptake activity of ETB
receptor speciﬁcally in the splanchnic vasculature of DOCA-salt rats include: a decrease
in overall endothelial cell function; a decreased density of endothelial ETB receptors; a

lower binding afﬁnity of ETB receptors for ET-l; or altered post-receptor ET-l

60

disposition mechanism. I tested the possibility that there were fewer ETB receptors in the

endothelial cells of DOCA-salt hypertensive rats.

Experimental Procedures: I examined the expression of ETB receptors in the
endothelial cells of cross sections from small mesenteric arteries and veins of DOCA-salt
and sham rats using double label immunoﬂuorescence and cofocal microscopy. Von
Willebrand Factor (vWF) was used as an endothelial cell marker and its distribution
pattern was used as an index of endothelial cell injury. The ﬂuorescence intensity of ETB
receptors in endothelial cells was quantiﬁed using Image J as an index of the total

number of endothelial ETB receptors. Images in this thesis are presented in color.

Results: There was no signiﬁcant change in the distribution pattern of vWF between
DOCA-salt and sham tissue. Although the average ﬂuorescence intensity of endothelial
ETB receptors was signiﬁcantly higher in sham small mesenteric veins than sham small
mesenteric arteries, there was no signiﬁcant difference between DOCA-salt and sham

tissues (Fig. 12, Fig. 13).

61

Figure 8:

A: Mean arterial pressure responses to i.v. bolus selective ETB receptor antagonist
administration in anesthetized DOCA-salt and sham rats. Bars indicate mean arterial
pressure before or after A192621 treatment in anesthetized sham and DOCA-salt rats.
Brackets indicate SEM. Asterisks indicate a signiﬁcant difference (P<0.05) between

mean arterial pressure before and after drug treatment.

B: Mean arterial pressure responses to i.v. bolus selective ETB receptor antagonist
administration in conscious DOCA-salt and sham rats. Bars indicate mean arterial
pressure before or after A192621 treatment in conscious sham and DOCA-salt rats.
Brackets indicate SEM. Asterisk indicates a signiﬁcant difference (P<0.05) between
mean arterial pressure before and after drug treatment. # indicates a signiﬁcant difference

(P<0.05) between DOCA-salt and sham rats.

62

mean arterial pressure (mmHg)

mean arterial pressure (mmHg)

200

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

_ sham (n=6) :-
160 . 1:1 DOCA (n=6) T
120 l T
80 -
4O -
0 . .
control A192621
280
q — sham (n=6)
240 [:1 DOCA (n=6)
200 t # T
T
160 w ..
120 ~
80 -
40 -
0
control A192621

63

Figure 9:

A: Effects of i.v. bolus selective ETB receptor antagonist administration on aortic plasma
ET-l level in anesthetized DOCA-salt and sham rats. Bars indicate mean aortic plasma
ET-l concentration before or after drug treatment in anesthetized sham and DOCA-salt
rats. Brackets indicate SEM. Asterisks indicate a signiﬁcant difference (P<0.05) between

mean aortic plasma ET-l concentration before and after drug treatment.

B: Effects of i.v. bolus selective ETB receptor antagonist administration on aortic plasma
ET-l level in conscious DOCA-salt and sham rats. Bars indicate mean aortic plasma ET-
1 concentration before or after drug treatment in conscious sham and DOCA-salt rats.
Brackets indicate SEM. Asterisks indicate a signiﬁcant difference (P<0.05) between
mean aortic plasma ET-l concentration before and alter drug treatment. # indicates a

signiﬁcant difference (P<0.05) between conscious and anesthetized rats.

aortic plasma ET-1 conc. (pg/ml)

aortic plasma ET-1 conc. (pg/ml)

 

30

 

 

 

 

 

 

 

 

 

 

 

 

A192621

 

 

 

 

 

 

* l

 

 

 

 

 

 

 

25 , — sham (n=9)
C: DOCA(n=10)
20 .
15 ~
10 .
5 .
0 ﬂ
control
30
25 , — sham (n=8)
E DOCA(n=5)
20 .
15 l
10 4
5 -1
a
0 ﬂ
control

65

A192621

Figure 10:

A: Effects of i.v. bolus selective ETB receptor antagonist administration on portal venous
plasma ET-l level in anesthetized DOCA-salt and sham rats. Bars indicate mean portal
venous plasma ET-l concentration before or after drug treatment in anesthetized sham
and DOCA-salt rats. Brackets indicate SEM. Asterisks indicate a signiﬁcant difference
(P<0.05) between mean portal venous plasma ET-l concentration before and after drug

treatment.

B: Effects of i.v. bolus selective ETB receptor antagonist administration on portal venous
plasma ET-l level in conscious DOCA-salt and sham rats. Bars indicate mean portal
venous plasma ET-l concentration before or after drug treatment in conscious sham and
DOCA-salt rats. Brackets indicate SEM. Asterisks indicate a signiﬁcant difference
(P<0.05) between mean portal venous plasma ET-l concentration before and after drug
treatment. #8 indicate a signiﬁcant difference (P<0.05) between conscious and

anesthetized rats.

66

portal venous plasma
ET-1 conc. (pg/ml)

portal venous plasma
ET-1 conc. (pg/ml)

 

30

 

— sham (n=9)
25‘ 1:: DOCA(n=10) . l

 

 

 

 

20-

15*

10:

 

 

 

 

 

 

 

 

 

 

 

control A192621

 

30

 

25 . — sham (n=8)
[:1 DOCA(n=5)

20*

 

 

 

 

154

10*

 

 

 

 

 

 

# #
0 ii I ,
control A192621

67

Figure 11:

A: Effects of i.v. bolus selective ETB receptor antagonist administration on net NHSR of
ET-1 in anesthetized DOCA-salt and sham rats. Bars indicate mean net non-hepatic
splanchnic ET-l release before or after drug treatment in anesthetized sham and DOCA-
salt rats. Brackets indicate SEM. Asterisk indicates a signiﬁcant difference (P<0.05)

between mean net non-hepatic splanchnic ET-l release before and after drug treatment.

B: Effects of i.v. bolus selective ETB receptor antagonist administration on net NHSR of
ET-1 in conscious DOCA-salt and sham rats. Bars indicate mean net non-hepatic
splanchnic ET-l release before or after drug treatment in conscious sham and DOCA-salt
rats. Brackets indicate SEM. Asterisk indicates a signiﬁcant difference (P<0.05) between
mean net non-hepatic splanchnic ET-l release before and after drug treatment. # indicates

a signiﬁcant difference (P<0.05) between conscious and anesthetized rats.

68

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

A19a621

 

 

 

 

 

control

69

 

 

 

 

 

 

 

 

 

 

 

 

1
r! e
.w
:llm
\0/ \I
)1 )5
9 I 8..
Em T m mm
1 t (
(A n A
mC w mC
m0 mo
SD SD
6 4 2 0 6 4 2

:EEE omega. EN

2833 $3.9 T._.m
o_c;ocm_aw 03305-5: «o:

oEcocmEm ozmaocéo: we...

Figure 12: Representative immunoﬂuorescence staining of vWF and ETB receptors of

cross sections of DOCA and sham small mesenteric blood vessels.

70

ETB receptor overlap

- - sun]

without primary antibody

1 f
1 Sum

cross section of sham small mesenteric vein

 

 

5pm

   

cross section of DOCA small mesenteric vein

71

 

8

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

- sham (n=7) a

.E‘ 40 - I: DOCA(n=6)
(I)
C
.9
.E T
s 30-
0
o
(I)
9
8 20 4
q:
C
(U
“s’

10 -

o l I

small mesen. small mesen.
arteries veins

Figure 13: Quantiﬁcation of the immunoﬂuorescence intensity of ETB receptors. Bars
indicate mean immunoﬂuorescence intensity of endothelial ETB receptors in sham and
DOCA-salt rats. Brackets indicate SEM. Asterisk indicates a signiﬁcant difference

(P<0.05) between different blood vessels in the same group.

72

Discussion for Chapter 4 — Part II
1) ET-l clearance by ETB receptors

ET-1 is rapidly removed from circulating blood, and current evidence suggests
that the ETB receptor is very important for that process. ET-l binds almost irreversibly to
the ETB receptor under physiological conditions (229). Direct transport of ETB receptor-
associated ET-l to lysosomes for degradation may serve as the main mechanism for
clearance of plasma ET-l (14). To determine whether altered endothelial ETB receptor
mediated uptake activity contributes to venous plasma ET-l level and nNHSR of ET-1, I
measured the portal venous plasma ET-l concentration and portal venous-aortic plasma
ET-l concentration difference in the presence of the selective ETB receptor antagonist
A192621.

ETB antagonist treatment signiﬁcantly increased the MAP in anesthetized
DOCA-salt and sham rats, possibly due to the blockade of plasma clearance of ET-1.
However, although ETB receptor antagonist treatment also signiﬁcantly increased the
MAP of conscious sham rats, it didn’t cause any signiﬁcant change in the MAP of
conscious DOCA-salt rats. This could be due to better physiological compensation, less
increase in plasma ET-l and/or decreased vascular response to ET-1 in conscious DOCA-
salt rats.

I found a signiﬁcant increase in portal venous-aortic plasma ET-l difference in
sham, but not DOCA-salt rats, after acute blockade of ETB receptors. Thus, in sham rats
there is signiﬁcant clearance of ET-1 from the splanchnic circulation through uptake
mediated by ETB receptors. The uptake activity of endothelial ETB receptors in the

splanchnic vasculature of DOCA-salt rats, however, was signiﬁcantly decreased (or even

73

absent). Interestingly though the whole body clearance of ET-1 mediated by ETB receptor
(predominantly pulmonary), which is reﬂected by the aortic plasma ET-l concentration
difference before and after A192621 treatment, was unchanged in DOCA-salt rats. These
novel results reveal a regionally speciﬁc defect in ETB receptor mediated plasma
clearance of ET-1 in the splanchnic vasculature of DOCA-salt rats.

Despite the absence of ETB receptor-mediated clearance of plasma ET-1 in the
splanchnic circulation of DOCA-salt rats, there is not an increase in the circulating ET-l.
It may reﬂect that the splanchnic bed plays a minor role in the plasma clearance of ET-1.
The relative role of splanchnic circulation in the plasma clearance of ET-1 can be tested
in the future by measuring the radiolabelled ET-l trapping by different tissues. Another
possibility is that there is less ET-l production in DOCA splanchnic vasculature.

Comparing the data from anesthetized rats and conscious rats, I found that the
change in aortic plasma ET-l level, and in nNHSR of ET-1 after ETB receptor blockade,
was greater in conscious versus anesthetized rats. This indicates that ETB receptor
mediated clearance of ET-1 was decreased in anesthetized and surgically prepared rats.
The decreased uptake activity of ETB receptors in anesthetized rats may be due to
desensitization of endothelial ETB receptors caused by higher circulating ET-l level, or to
a lower cardiac output (and tissue blood ﬂow) under anesthesia. However, data from both
conscious and anesthetized rats provide consistent evidence that the uptake activity of
endothelial ETB receptors in DOCA-salt rats is impaired in the splanchnic vasculature,

while remaining unchanged in the whole body.

74

One of the weaknesses of this study is that I didn’t measure the possible differential
inﬂuence of ETB receptor antagonist on the portal venous blood ﬂow of DOCA-salt and
sham rats. It was reported that A192621 didn’t cause any signiﬁcant change in the portal
venous blood ﬂow of normal rats (31). No similar studies have been done in DOCA-salt

hypertensive rats.

2) Mechanism and impact of reduced ETB receptor mediated plasma clearance of
ET-1

ETB receptors are localized in both endothelial cells and vascular (mainly
venous) smooth muscle cells. It is not clear if smooth muscle ETB receptors also play a
role in the clearance of plasma ET-l. So in my study, I only considered the possible
mechanisms contributing to the impaired uptake activity mediated by endothelial ETB
receptors.

Previous results indicate immunoreactive ETB receptor number tends to be higher
in vena cava compared to aorta, but no difference was found in the vena cavae of sham
versus DOCA-salt rats (254). However, that study did not distinguish between
endothelial and smooth muscle ETB receptors; these probably differ in their ability to
clear ET-l from the circulation. Possible differences between large and small veins also
were not considered. In my study, I found that there was no signiﬁcant difference in the
ﬂuorescence intensity of ETB receptors in the endothelial cells between DOCA-salt and
sham tissue. The lack of difference in the distribution pattern of vWF in the endothelial
cells between DOCA-salt and sham rats suggests the structure of the DOCA-salt

endothelium is intact. Western blot analyses from Dr.Watts lab showed that there was no

75

signiﬁcant difference in the protein level of ETB receptors between DOCA-salt and sham
small mesenteric blood vessels. Although that result represents the protein level of ETB
receptors in the entire blood vessel, the result is consistent with mine, i.e. both suggest
that the impaired ET B receptor-mediated uptake activity in the splanchnic bed of DOCA-
salt rats is not due to fewer ETB receptor on endothelial cells.

One of the limitations of this study is that it is difﬁcult to separately quantify the
ﬂuorescence intensity of ETB receptors in the membrane versus ETB receptors inside the
endothelial cells, which would, otherwise, give us an estimate of the relative number of
active ETB receptors and internalized, inactive ETB receptors. Unaltered total number of
ETB receptors in the endothelial cells of DOCA-salt rats doesn’t necessarily mean
unaltered number of active ETB receptors. It is possible that there are fewer active ETB
receptors in the endothelial cells of DOCA-salt rats and that contributes to the impaired
ETB receptor-mediated uptake activity. Theoretically, the cellular localization of ETB
receptors can be studied by taking different fractions of endothelial cells by
supercentriﬁrgation and testing the protein level of ETB receptors in different fractions
using western blot analysis. However, it may be hard techniqualy in terms of collecting
enough endothelial cells fractions.

In terms of other possible mechanisms underlying the decreased uptake activity of
ETB receptor speciﬁcally in the splanchnic vasculature of DOCA-salt rats, some
possibilities are:

l) a decrease in overall endothelial cell function
Previous studies compared the overall endothelial cell function between DOCA-

salt and sham small mesenteric blood vessels using acetylcholine induced vasorelaxation

76

(103). No signiﬁcant difference was found, suggesting that the overall endothelial cell
ﬁmction is unchanged in DOCA-salt small mesenteric blood vessels.
2) a lower binding afﬁnity of ETB receptors for ET-l
No radioligand binding assay has been done to provide direct evidence for the

binding afﬁnity of ETB receptors for ET-1 in DOCA-salt and sham rats. Functional
studies (103) showed that the dose response curve of small mesenteric veins to S6C, a
selective ETB receptor agonist, is not signiﬁcantly different between DOCA-salt and
sham rats, suggesting that the binding afﬁnity of ETB receptors for ET-1 is unchanged in
DOCA-salt rats.
3) altered post-receptor ET-l disposition mechanisms

It has been suggested (14) (Fig. 14) that both ETA and ETB receptors are rapidly
internalized upon agonist stimulation. This process is dependent on G-protein coupled
receptor kinases (GRKs), arrestin, dynamin, and clathrin (Agonist-activated receptors are
phosphorylated by GRKs, followed by binding of arrestin and initiation of receptor
internalization through a clathrin/dynamin-dependent mechanism). Internalized ETA and
ETB are directed to Rab 5 positive early endosomes. From this location, the two receptor
subtypes are targeted to different intracellular fates. The ETA receptor is directed to the
recycling compartment and subsequently reappears at the plasma membrane. The ETB
receptor is directed to lysosomes for degradation. That is suggested to be the mechanism
by which ETB receptor mediates the clearance of plasma ET-l. There may be something
wrong with one or multiple steps involved in the ETB receptor internalization pathway in

DOCA-salt rats that leads to the impaired uptake activity of ETB receptors.

77

In any case, the physiological signiﬁcance of reduced splanchnic ET-l plasma
clearance by ETB receptors is not known, i.e. it is uncertain if the decreased uptake
activity of ETB receptor in the splanchnic vasculature affects the ability of ET-1 to
control vascular capacitance in DOCA-salt rats. Further work is required to address this

question.

78

ETA/ETB

  
 

 

 
 
  
  

clathrin-
coated pits plasma
1 membrane
endosome . recycling
pathway

ETB \ETA
degrative recycling
pathway {Cf-:2— . mpartment

lysosome®

Figure 14: Model for regulation and intracellular trafﬁcking of the ET receptors (14)

79

Chapter 4 - Part III
ET-l synthesis in the splanchnic vasculature of DOCA-salt rats
Rationale: ET-1 is derived from preproendothelin-1. The translation of preproendothelin
mRNA results in the formation of a 212-amino acids preproendothelin peptide, which is
cleaved by a furin convertase (33) to the 38-amino acid peptide big ET-11-33 (91). Once
formed, big ET-1 is processed to ET-l 1.2] through cleavage of the Trp21-Va122 bond by
ET-converting enzyme-1 (ECE-l), which exists in 4 isoforms (a, b, c, and d) (259) and
by ECE-2 (205) (214) (208) (241) (48) and chymase (258). ECEs are localized in
endothelial (259) and smooth muscle cells (208) (153), cardiomyocytes (186) (186)
(114), and macrophages (69) (153). ECEs belong to the metalloprotease family (259),
share functional and structural similarity with neutral endopeptidases (NEPs) and Kell
blood group proteins (241) (48) (180), and are partially inhibited by phosphoramidon
(146). ECE-1 expression is regulated through protein kinase C-dependent mechanisms
(239), ETB receptors (160), the transcription factor ets-1(167), and cytokines (269).

Increased expression of ECE-1 has been found in failing human myocardium (156),
renal medullary area of DOCA-salt rats (96), but not in DOCA blood vessels (234). Even
though overexpression of ECE-1 itself does not lead to systemic hypertension (233), the
enzyme can be a crucial rate-limiting factor in the production of mature ET-1 in vivo.

It has been found that CGS compounds, which are vasopeptidase inhibitors that
simultaneously inhibit ECE and NEP, can dose-dependently increase plasma big ET-l
level, decrease the pressor effect of big ET-1 in conscious rats (47) and reduce plasma
ET-l to normal levels in rats with chronic heart failure (34). CGS compound has also

been found to reduce blood pressure in conscious unrestrained normotensive rats,

80

spontaneously hypertensive rats (The magnitude of the antihypertensive effects was
greater in SHRs than in normotensive rats) (4) and human without reﬂex increases in
heart rate (34).

In my studies mentioned in Chapter 4-part I and II, I measured vascular ET-l
content, plasma ET-l level before and alter blockade of the major process affecting
circulating ET-l level—ETB receptor mediated uptake, in order to get an estimate of the
formation of ET-1 by the splanchnic vasculature of DOCA-salt and sham rats. However,
since ECE is a rate-limiting step in ET-l generation, the change in nNHSR of ET-1 due
to inhibition of ECE should provide more direct manifestation of ET-1 generation in the
splanchnic bed. I ﬁrst treated my rats with ETB receptor antagonist to provide higher
basal level of ET-1. Then the rats were treated with a dual ECE/NEP inhibitor. The
difference between the net NHSR of ET-1 in rats treated only with ETB receptor
antagonist and that in rats treated with both ETB receptor antagonist and ECE inhibitor

can be used as an index of net NHS ET-l synthesis.

Experimental Procedures: Aﬁer cannulation surgery, rats were given A192621
(12mg/kg, i.v.) ﬁrst to block ET-l uptake and 30 minutes later, rats were treated with
CGSZ6393 (3mg/kg, i.v.), a dual ECE-1 and NEP inhibitor. Portal venous and aortic
blood samples were taken simultaneously 1 hmy later. Plasma ET-l levels were

measured.

Results: 30 min after surgery, the average MAP of sham rats was 87.8 :t 6.5 mmHg. It

was increased to 121.0 :1: 4.6 mmHg 30min after A192621 treatment. 60 min after

81

CGSZ6393 treatment, the average MAP was 89.0 :1: 6.9 mmHg. 30 min after surgery, the
average MAP of DOCA-salt rats was 119.6 :t 13.0 mmHg. It was increased to 163.3 :t
9.5 mmHg 30min alter A192621 treatment. 60 min after CGSZ6393 treatment, the
average MAP was 133.1 :t 12.7 mmHg. I didn’t ﬁnd, however, a signiﬁcant effect of
CGSZ6393 on the net NHSR of ET-1 in either DOCA-salt (nNHSR of ET-1 was 3.21 :t
0.9 pg/ml with ETB antagonist treatment and 4.83 i 1.3 pg/ml with ETB antagonist +
CGS 26393 treatment) or sham rats (nNHSR of ET-1 was 4.43 :t 1.1 pg/ml with ETB

antagonist treatment and 2.52 d: 1.3 pg/ml with ETB antagonist + CGS 26393 treatment).

82

Discussion for Chapter 4 - Part III
ECE is the rate-limiting step in ET-l generation. ECE inhibition is a very important
and attractive target for the inhibition of the ET system for treating cardiovascular
diseases where the ET system plays a pathogenic role, e. g. essential hypertension, chronic
heart failure (248). A number of non-selective and selective ECE-l inhibitors have been
identiﬁed and are currently in early-phase clinical studies. Results from early studies are

promising, particularly in essential hypertension.

In my studies, I found that ETB receptor antagonist signiﬁcantly increased the MAP
in both anesthetized DOCA-salt and sham rats, which was consistent with what I saw in
my previous studies. 60 min after ECE inhibition (i.e. 90 min after ETB receptor
blockade), the MAP in both DOCA-salt and sham rats were not signiﬁcantly different
from the basal level. It was not clear if this was due to the anti-hypertensive effect of the
ECE/NEP inhibitor I used or just the effect of ETB receptor antagonist on blocking the
clearance of ET-1 started to wear off. However, it is more likely to be the latter because
in my later studies, in vivo bolus injection of same dose of CGS 26393 failed to cause a

signiﬁcant decrease in the mean arterial pressure in conscious DOCA-salt rats.

CGS 26393 also failed to cause any signiﬁcant change in either the nNHSR of ET-1,
nor the aortic or portal venous plasms ET-l levels in either DOCA-salt or sham rats.
When combined with the fact of lack of signiﬁcant effect on MAP, both data suggest that

a higher dose of ECE inhibitor may be required in future studies.

However, another possibility exists. ECE-independent pathways also contribute to
ET-l production. In ECE-1 knockout mice, tissue levels of ET-1 are reduced by only

one-third (265). Indeed, chymase generates ET-l 1-2. (258). In addition, 2 novel ET-l 1-2.-

83

font

chit

forming enzymes, a non-ECE metalloprotease and a vascular smooth muscle cell
chymase, have been cloned. Therefore, activation of alternative ET—l production pathway

could also be used to explain the lack of change in the nNHSR of ET-1 in my study.

84

Chapter 4 - Part IV

Possible role of ETA receptor binding in the plasma clearance of ET—1 in the

splanchnic bed of DOCA-salt rats

Rationale: In contrast to other peptides, there appears to be a relatively small amount of
ET peptide in blood and tissue in relation to the concentration of ET receptors. Besides,
the binding of ET-1 to its receptors is particularly tight and ET-l remains bound to ETA
receptors even after their intemalization (67). These facts lead to the hypothesis that ETA
receptors may also function as a clearance receptor, and the binding of ET-1 to ETA
receptors in the vascular smooth muscle cells may affect the measurement of nNHSR of
ET-1. To test the possibility that ETA receptor binding contributes to the nNHSR of ET-
1, I compared the nNHSR of ET-1 after either combined ETA and ETB receptor
blockade with that after selective ETB receptor antagonism alone. The difference was
used as an index of the amount of ET-1 bound to ETA receptors in the splanchnic
vasculature of normotensive and DOCA-salt hypertensive rats. nNHSR of ET-1 in rats
treated with selective ETA receptor antagonist was also calculated to further identify the

role of ETA receptors in the clearance of plasma ET-l.

Experimental Procedures: Two sets of experiments were performed. Both were done in
anesthetized rats.

1) A182086, a mixed ETA/B receptor antagonist, was given (12 mg/kg, i.v., Abbott
Laboratories, IL) to rats 30 minutes after recovery from catheterization as described in
General Methods. Aortic and portal venous blood samples were collected before and l

hmy after drug treatment. The dose of A182086 was shown to be efﬁcacious in

85

preliminary studies, in which antagonist was tested against acute pressor actions of ET—1
(data not shown). Published studies also indicate that this dose is effective in blocking
both receptor subtypes in rats (256). The nNHSR of ET-1 after A182086 treatment was
compared with that after selective ETB receptor antagonist A192621 treatment. The
difference was used as an index of the amount of ET-1 bound to ETA receptors in the
splanchnic vasculature.

2) Atrasentan, a selective ETA receptor antagonist, was given (1mg/kg, i.v.) to rats 30
minutes after recovery from catheterization as described in General Methods. Aortic and
portal venous blood samples were collected before and 1 hmy after drug treatment. Aortic
and portal venous plasma ET-l levels were measured. The increase in aortic plasma ET-l
level and nNHSR of ET-1 caused by atrasentan was compared with those caused by

selective ETB antagonist and combined ETA/B antagonist.

Results:

1) A182086 treatment did not cause a signiﬁcant change in mean arterial pressure in
either DOCA-salt or sham rats (data not shown). A182086 caused a signiﬁcantly greater
increase in the aortic plasma ET-l concentration in both DOCA-salt and sham rats than
the selective ETB receptor antagonist. There was no signiﬁcant difference in the
magnitude of increase between DOCA-salt and sham rats (Fig. 15A). A182086 also
signiﬁcantly increased the net NHSR of ET-1 in both groups (Fig. 15B) and the

magnitude of increase was not signiﬁcantly different between the two groups.

2) Atrasentan signiﬁcantly decreased the MAP of DOCA-salt (from 120.3 i 7.9 mmHg

to 88.6 :t 8.0 mmHg). but not sham rats (from 101.5 at 6.8 mmHg to 117.7 d: 8.5 mmHg).

86

Atrasentan caused a small increase in the aortic plasma ET-l level and the nNHSR of
ET-1 in both DOCA-salt and sham rats (Fig. 15A, 15B). There was no signiﬁcant

difference in the magnitude of increase between the two groups.

87

Figure 15:

A: Effects of selective ETB receptor antagonist, combined ETA/B receptor antagonist
and selective ETA receptor antagonist on aortic plasma ET-l levels in anesthetized
DOCA-salt and sham rats. Bars indicate the magnitude of increase in aortic plasma ET-l
level with different drug treatments in anesthetized sham and DOCA-salt rats. Brackets
indicate SEM. Asterisks indicate a signiﬁcant difference (P<0.05) between different drug

treatments.

B: Effects of selective ETB receptor antagonist, combined ETA/B receptor antagonist
and selective ETA receptor antagonist on estimated net NHSR of ET-1 in anesthetized
DOCA-salt and sham rats. Bars indicate the magnitude of increase in net NHSR of ET-1
with different drug treatments in anesthetized sham and DOCA-salt rats. Brackets
indicate SEM. Asterisks indicate a signiﬁcant difference (P<0.05) between different drug
treatments. # indicates a signiﬁcant difference (P<0.05) between DOCA-salt and sham

rats.

88

magnitude of increase in net NHSR

 

 

 

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89

Discussion for Chapter 4 — Part IV

Combined ETA and ETB receptor blockade caused a signiﬁcant increase in aortic and
portal venous plasma ET-l concentrations in both DOCA-salt and sham rats. The
magnitude of that increase was roughly two times higher than that seen in rats with ETB
receptor blockade alone. This suggests that in both DOCA—salt and sham rats, a
signiﬁcant amount of ET-1 is removed from the circulation by ETA receptor binding and
is consistent with a previous hypothesis (67) that functional ETA receptors also act as
“clearance” receptors. Combined ETA and ETB receptor blockade also caused a much
higher increase in the net NHSR of ET-1 in both group of rats compared with the increase
caused by ETB receptor blockade alone, suggesting that ETA receptors also contribute to
the plasma clearance of ET-1 in the splanchnic circulation. Although selective ETB
antagonism caused a signiﬁcant higher increase in the nNHSR of ET-1 in sham rats than
DOCA-salt rats, there was no signiﬁcant difference in the magnitude of increase in the
nNHSR of ET-1 between sham and DOCA-salt rats after the treatment of combined
ETA/B antagonist. These suggest that there may be more ETA receptor binding in the
splanchnic bed of DOCA-salt rats.

To further understand the role of ETA receptors in the plasma clearance of ET-1, I
treated my DOCA-salt and sham rats with selective ETA receptor antagonist. What I
expected to see is a great increase in the aortic plasma ET-l concentration, representing
the huge amount of ET-1 bound to ETA receptors in the systemic circulation. And
ideally, the increase in aortic plasma ET-l concentration caused by ETA antagonist plus
the increase caused by ETB antagonist should be close to the increase caused the

combined ETA and ETB receptor antagonist. Surprisingly, in my study, I found that

90

selective ETA antagonist only caused a small increase in the aortic plasma ET-l
concentration in both DOCA-salt and sham rats. The magnitude of increase in aortic
plasma ET-l level with combined ETA and ETB antagonism is much greater than the
sum of increase in aortic plasma ET-l level with selective ETB and ETA antagonism,
suggesting that systemic plasma ET-l clearance by ETA receptors is different in the
presence and absence of ETB receptors.

In terms of the effect of selective ETA receptor antagonist on the nNHSR of ET-1 ,
similarly, I would expect to see a huge increase in the nNHSR of ET-1 in both DOCA-
salt and sham rats, representing the huge amount of ET-1 bound to ETA receptors in the
splanchnic vasculature. I also expected to see greater increase in DOCA-salt rats
compared with sham rats because there appears to be more ETA receptor bindings in
DOCA splanchnic bed according to this study. And again, surprisingly, selevtive ETA
receptor antagonist only caused a small increase in the nNHSR of ET-1 in both DOCA-
salt and sham rats. The increase is not signiﬁcantly greater in DOCA-salt rats. This
suggests that it is the same in the splanchnic vasculture that the plasma clearance of ET-1
by ETA receptors is different in the presence and absence of ETB receptors.

My data suggest that the effects of combined receptor antagonist were not consistent
with simple additive receptor blockade. Plasma ET-1 is not cleared ﬁ'om the circulation
via direct and additive activation of ETA and ETB receptors. One possible explanation
could be that there is an important interaction between ETA and ETB receptors in the
plasma clearance of ET- 1. For example, functional ETB receptors may have an inhibitory
effect on the clearance activity of ETA receptors. Findings in other in vitro studies have

also suggested the existence of an interaction between the ETA and ETB receptors

91

including vascular (both arteries and veins) (152) (131) and nonvascular preparations
(71) (70). The mechanism behind the proposed interaction between ETA and ETB
receptors is not understood but may involve interactions at the receptor level or through
second messengers (70). Biochemical studies using cells transfected with different
endothelin receptor subtypes to examine receptor and second-messenger interactions
should provide valuable insight into the mechanism behind the proposed receptor ”cross
talk”.

My studies suggest that there may be more ETA receptor binding in the splanchnic
bed of DOCA-salt rats. However, it is not consistent with previous in vitro studies in
other group (152) (154) showing that ETA receptor binding was reduced, whereas binding
afﬁnity was not altered in membranes of both large and small mesenteric arteries from
DOCA-salt compared with sham rats. The decrease in ETA receptor binding was not due
to reduced expression of ETA receptors, since protein levels were elevated in vessels ﬁom
DOCA-salt rats compared with sham rats (154). The ﬁnding‘that ETA receptor protein
was elevated while maximum binding was decreased indicates that some of the ETA
receptors are modiﬁed in a manner that will reduce binding. No similar studies have been
performed in mesenteric veins. Future in vitro studies in the ETA receptor binding using
mesenteric veins can provide basis for further comparison of ETA receptor binding in the

whole splanchnic vasculture between DOCA-salt and sham rats.

92

Discussion for Chapter 4

Previous studies in my lab have provided evidence for increased venoconstriction
in conscious DOCA-salt hypertensive rats using repeated measurements of blood volume
and MCFP, an in vivo index of venous smooth muscle contractile activity (64).
Endogenous ET-l appears at least partly responsible for that augmented venoconstriction,
since ET receptor antagonists lower MCF P in DOCA-salt hypertensive but not
normotensive rats (104).

Two possible mechanisms could explain increased ET-l mediated
venoconstriction: higher levels of the peptide around venous smooth muscle cells and/or
enhanced responsiveness of venous smooth muscle to ET-l. It has been shown that vena
cava and small mesenteric veins from DOCA-salt hypertensive rats do not exhibit
increased reactivity to ET-1 in vitro (103) (254). Therefore, it was logical to conclude
that higher ET-l mediated venoconstriction in DOCA-salt rats is caused by increased ET-
1 concentrations around venous smooth muscle. Measurement of preproET-l gene
expression — as an index of ET-1 synthesis —in small veins, however, revealed no
differences between sham and DOCA-salt rats (103). That ﬁnding does not support the
hypothesis that increased venomotor activity in DOCA-salt rats is caused by local
overproduction of ET-1 by veins.

Veins in DOCA-salt rats could be exposed to higher concentrations of ET-1 by
mechanisms other than increased venous endothelial cell synthesis. For example,
increased production and release into blood of ET-1 from upstream arterial endothelial
cells could affect venous smooth muscle cell activity directly (112), or provide “excess”

ET-l to be taken up into venous endothelial cells for subsequent release. To test these

93

possibilities, I made two kinds of measurements to estimate ET-l concentrations in the
biophase around venous smooth muscle cells: total vascular ET-l content and venous
plasma ET-l concentration. Measurements were made in the splanchnic vascular bed,
since that region represents the most important capacitance bed in the circulation. I also
estimated net non-hepatic splanchnic release of ET-1 by determining the difference
between ET-l concentrations in inﬂowing arterial and outﬂowing portal venous blood.
Plasma ET-l level is determined by a combination of factors including production rate,
metabolism and clearance. To test the possibility that a functional alteration in ETB
receptors might affect the venous plasma ET-l concentration in DOCA-salt hypertension,
I measured the portal venous-aortic plasma ET-l concentration difference after selective
ETB receptor blockade in both anesthetized and conscious rats. I also measured the portal
venous-aortic plasma ET-l concentration difference after combined ETA/B and selective
ETA receptor blockade in order to test the potential role of ETA receptor in the plasma
clearance of ET-1.

My studies found that neither ET-l content of venous capacitance vessels nor
portal venous plasma ET-l concentration is higher in DOCA-salt versus sham rats.
Furthermore, estimated nNHSR of ET-1 is not higher in DOCA-salt versus sham rats
even when major processes removing ET-l from the circulation are blocked. Collectively
the results provide strong evidence that higher endothelial cell formation of ET-1 is not
likely responsible for enhanced ET-l mediated splanchnic venoconstriction in viva.
When combined with the fact that venous smooth muscle contractile responsiveness to
ET-1 is not increased in DOCA—salt rats, the data suggest that the ability of ET receptor

antagonists to reduce venoconstriction in DOCA-salt rats in viva results from actions of

94

the drugs on a non-vascular target. I speculate that the enhanced venoconstrictor effect of
ET-1 could be mediated by an action of ET-1 on the sympathetic nervous system. A
number of studies have shown prejunctional modulation of sympathetic
neurotransmission by ET-1 in vitro (158), e. g. Intrarenal arterial infusion of ET-1
inhibited norepinephrine release induced by renal sympathetic nerve stimulation in
anesthetized dogs (147). Localization of ET-1 receptors on the nerve terminals have been
reported (60). Neurotransmission in sympathetic ganglia also can be modulated by ET-l
(238) (26). A previous study in my lab (104) found that pretreatment of DOCA-salt
hypertensive rats with an ETB receptor antagonist produced greater declines in MCF P
following ganglionic blockade than after ganglionic blockade alone. These ﬁndings
suggest that ET-1 acting at ETB receptors may provide an important contribution to
venoconstriction through modulation of sympathetic tone to the veins. The ETB
antagonist may have facilitated NE release from sympathetic nerve terminals on veins,
accounting for the signiﬁcantly larger response of MCFP to subsequent ganglion
blockade. This action presumably was more prominent in DOCA-salt rats because of
their higher pretreatment levels of endogenous vascular ET-l.

The decrease in venous capacitance in hypertension might be mediated by other
mechanisms, such as changes in venous wall structure, or other inﬂuences on venous
smooth muscle activity besides ET-1 and sympathetic input, such as reactive oxygen

species.

95

CHAPTER 5

SYMPATHETIC NEUROTRANSMISSION IN THE SPLANCHNIC BED OF

RATS IN THE ESTABLISHED PHASE OF DOCA-SALT HYPERTENSION

96

Introduction

1. Autonomic innervation of the cardiovascular system

The sympathetic nervous system plays an important part in regulating cardiovascular
homeostasis. Catecholarninergic neurons releasing NE are widely distributed in the brain,
but are located in particular in the medulla and pons (66). The hypothalamus, and parts of
the limbic system including the amygdala, receive projections from these brainstem
noradrenergic nuclei. Electrophysiologic and anatomical experiments carried out in
animals provide evidence of a connection between pressor noradrenergic hypothalarnic
and brainstem centers and sympathetic preganglionic neurons in the thoracolumbal cord
(99, 242).

Autonomic outﬂow from the brainstem is divided principally into sympathetic and
parasympathetic (vagal) branches. Efferent ﬁbers of sympathetic nerves travel along the
nerve column into sympathetic ganglia. These ganglia use acetylcholine as a
neurotransmitter where activity is transmitted to noradrenergic neurons which innervate
the heart, vasculature and many other organs of the body.

Celiac, superior mesenteric, and inferior mesenteric ganglia provide axons that are
distributed with the three major gastrointestinal arteries arising from the aorta. The celiac
ganglion innervates the stomach, liver, pancreas, duodenum, and the ﬁrst part of the
small intestine; the superior mesenteric ganglion innervates the small intestine; and the
inferior mesenteric ganglion innervates the descending colon, sigmoid colon, rectum,

urinary bladder, and sexual organs.

2. Increased sympathetic nervous system activity in human hypertension

97

Evidence drawn from a number of smyces, including electrophysiological and
neurochemical techniques, provides compelling evidence that overactivity of the
sympathetic nervous system is commonly present in younger patients with essential
hypertension. In borderline and established hypertension, nerve ﬁring rates in
postganglionic sympathetic ﬁbers passing to skeletal muscle blood vessels are increased.
There is also increased spillover of the sympathetic neurotransmitter NE from the heart
and kidneys, providing evidence of stimulated sympathetic outﬂow to these organs (1, 58,
80, 260). The increased cardiac and renal sympathetic nerve ﬁring provides a plausible
mechanism for the development of hypertension, through the regulatory inﬂuence of the
sympathetic nervous system on renin release, glomerular ﬁltration rate, and renal tubular
sodium reabsorption, and on cardiac growth and pump performance (51). The speciﬁc
causes of the increased sympathetic activity in essential hypertension remain largely
unknown, although genetic inﬂuences are evident and behavioral and lifestyle factors
appear to be involved. There is growing evidence of an underlying disturbance in CNS
monoarninergic control of sympathetic outﬂow (57), which may perhaps be the common
mediating mechanism of peripheral sympathetic activation with stress, obesity, and
physical inactivity. In hypertensive patients, NE spillover from the brain on average is
higher than in healthy subjects, as is the overﬂow into the internal jugular veins of
dihydroxylphenylglycol (DHPG, the intraneuronal metabolite of NE) and 3-methoxy-4-
hydroxylphenylglycol (MHPG, the extraneuronal metabolite of NE) (62). In patients
with an increased spillover of NE and its metabolites ﬁ'om the brain, peripheral

sympathetic activity is increased (62).

98

3. Sympathetic nerves and adrenergic receptors in arteries of hypertensive human

subjects and animal models

Changes in the function of the sympathetic nervous system occur in DOCA-salt
hypertension. Circulating norepinephrine and epinephrine levels are increased in DOCA-
salt hypertensive rats, suggesting that there is an increased release of norepinephrine from
sympathetic nerves and epinephrine from the adrenal gland in these animals (12). This
result is supported by studies done in vitro, where it was shown that stimulation of
sympathetic nerves associated with mesenteric arteries from DOCA-salt rats released
more norepinephrine than those obtained from normotensive rats (237). Increased release
of norepinephrine from periarterial sympathetic nerves in DOCA-salt rats is due, at least
in part, to a disruption in the function of prej unctional (12-adrenergic autoreceptors (29).
There are also postjunctional changes in arterial smooth muscle that result in enhanced
neurogenic constrictions in tissues from DOCA-salt rats. Large mesenteric arteries from
DOCA-salt rats are more sensitive to the contractile effects of exogenously applied
norepinephrine (133, 176), although other studies showed that the sensitivity to
adrenergic agonists did not change in caudal artery (90), and the binding afﬁnity of al -

adrenergic receptors in mesenteric arteries is reduced (149).

4. Sympathetic nerves and adrenergic receptors in veins of hypertensive human

subjects and animal models

Venoconstriction is controlled largely by sympathetic nerve activity (155). Changes in

neuroeffector transmission to veins in hypertension are important because veins are more

99

sensitive to the vasoconstrictor effects of sympathetic nerve stimulation than arteries

(116)(138)

In human hypertension and in spontaneously hypertensive rats (SHRs), sympathetic
neural input to veins is increased: Human forearm veins obtained as biopsy specimens
from normotensive and hypertensive subjects show several hypertension-associated
disturbances in neuroeffector transmission, including decreased contractile responses to
al-adrenergic receptor agonists, but enhanced responses to nerve stimulation (221). The
membrane potential of venous smooth muscle cells in SHRs is depolarized compared to
that found in normotensive rats. This change is due to increased sympathetic tone to veins
in SHRs (257). It has also been shown that hexamethonium caused a signiﬁcantly larger
decline in the MCFP in DOCA-salt than sham rats, suggesting that increased sympathetic
venoconstrictor activity is an important mediator of increased venoconstriction in DOCA-
salt hypertension. This previous work indicates that there are changes in sympathetic
neurotransmission to veins in hypertension, but the mechanism of these changes are

unclear.

5. Causal mechanisms leading to sympathetic augmentation in hypertension are

poorly understood

Although there is growing evidence that essential hypertension and many
hypertensive animal models are neurogenic (1, 80), and are initiated and sustained by
overactivity of the sympathetic nervous system, the precise causal mechanisms leading to

sympathetic augmentation in hypertension are still poorly understood. Among others,

100

possible mechanisms leading to sympathetic augmentation in hypertension include
increased sympathetic nerve ﬁring rate, increased NE release from the nerve terminals,
impaired prejunctional (12 adrenoceptor feedback inhibition, altered neuronal NE

reuptake (53, 193), and facilitation of NE release by neurohumoral factors such as ET-l.

It is important to identify the mechanisms since the increase in sympathetic activity
is a mechanism for both initiating and sustaining the blood pressure elevation.
Sympathetic nervous activation also confers speciﬁc cardiovascular risk. Stimulation of
the sympathetic nerves to the heart promotes the development of left ventricular
hypertrophy and contributes to the genesis of ventricular arrhythmias and sudden death.
Syrnpathetically mediated vasoconstriction in skeletal muscle vascular beds reduces the
uptake of glucose by muscle, and is thus a basis for insulin resistance and consequent

hyperinsulinemia(50) (107, 140).

101

Chapter 5 — Part I

NE release from the sympathetic nerves associated with the splanchnic vasculature

of anesthetized and conscious DOCA-salt rats.

1) Plasma NE concentration and NHS spillover of NE in anesthetized and conscious
DOCA-salt rats

Rationale: There is substantial evidence for increased sympathetic input to the
cardiovascular system in DOCA-salt hypertension (29). The splanchnic vasculature is
richly innervated by sympathetic nerve ﬁbers. Non hepatic splanchnic (NHS) circulation
accounts for ~50% of total peripheral sympathetic outﬂow in humans (39) and
experimental animals (2) (38). Sympathetic nerve stimulation can reduce intestinal blood
volume by up to 60%, leading to a signiﬁcant redistribution of blood from the splanchnic
veins. This change in volume distribution will have a profound effect on overall
cardiovascular functions including blood pressure (81) (65, 109). Net production of NE
by the NHS circulation has been documented in normotensive rats (74), but not in
DOCA-salt hypertensive rats.

In mesenteric veins of spontaneouly hypertensive rats or reduced renal mass
hypertensive rats, membrane potentials were less negative compared to veins in control
rats. Adrenergic blockade in hypertensive rats caused a hyperpolarization of venous
smooth muscle membrane potential to a level similar to that measured in sham rats (25 7).
Recent studies by my group also found more depolarized mesenteric venous smooth
muscle membrane potential in DOCA-salt rats than sham rats, and adrenergic blockade

hyperpolarized the venous smooth muscle membrane potential to a level similar to that

102

measured in sham rats. These data suggestithat in hypertensive rats there is a tonic
elevation of sympathetic input to mesenteric veins. Previous studies in my group (138)
showed that in both sham and DOCA rats, mesenteric veins are more sensitive than
arteries to nerve stimulation. Therefore increases in sympathetic nerve activity are likely
to have a larger effect on veins than arteries for a given change in nerve activity.
Although there was no signiﬁcant difference in the frequency response curves obtained
for contraction of DOCA and sham veins, DOCA veins were less sensitive to exogenous
NE. This suggests that the sympathetic nerves may release more NE to compensate for
the decrease in postjunctional sensitivity in DOCA-salt hypertension. DOCA-salt arteries
are sensitized to the contractile effects of nerve stimulation. This could be due to
increased transmitter release from sympathetic nerves or increased sensitivity of vascular
smooth muscle to sympathetic neurotransmitters. The observation that there is no
increase in sensitivity to exogenous NE in arteries indicates that there may be an increase
in transmitter release. This suggestion is supported by the result in arteries showing that
the increased response may be due to the combined contractile actions of nerve released
NE and ATP in DOCA arteries while ATP alone mediates contractions in sham arteries.
Although there is indirect evidence for increased sympathetic nerve activity to the
splanchnic circulation in DOCA-salt hypertensive rats (100, 145, 215), no direct
measurements of the net production of NE by the NHS circulation have been reported. In
my study, portal venous plasma NE concentration (N Epv), which should reﬂect
splanchnic venous NE concentration, was measured. I also used the difference in plasma

NE concentration between the portal venous blood and arterial blood (NEpv-NEa) as a

103

measure of net NHS release of NE, which is an index of splanchnic sympathetic nerve
activity.

Both the portal venous plasma NE level and the net NHS release of NE, however,
are just rough indice of splanchnic sympathetic nerve activity. NE is concurrently taken
up and released from tissue beds. As a consequence, tissue NE release and removal can’t
be assessed by measurements of plasma NE and arteriovenous differences alone.
Therefore, NHS NE spillover, which represents the rate of entry of locally released NE
into portal venous plasma, was estimated as introduced in General Methods based on two
assumptions: 1) Portal venous blood ﬂow of DOCA-salt rats equals that of sham rats
(264). 2) NHS fractional extraction (F X) of NE (ﬁ'actions of NE removed ﬁ'om plasma
during passage through NHS organs) equals NHS FX of epinephrine (13, 16, 22, 92).

Of the NE released by sympathetic nerves throughout the body, perhaps 10-20%
overﬂows into the circulation (Fig. 1). The bulk of the remainder is disposed of by
reuptake back into the nerves. NE escaping into the interstitial ﬂuid of individual organs
enters the circulation passively by diffusion down a concentration gradient. Possible
determinants of this rate of NE spillover include factors such as regional blood ﬂow and
the adequacy of neuronal NE uptake, but in general the overﬂow of NE is approximately
proportional to the rate of sympathetic nerve ﬁring. The existence of this proportionality
between the rate at which NE released from sympathetic nerves spills into the venous
drainage of individual organs and the rate of sympathetic nerve ﬁring provides the
foundation for the use of NE overﬂow measurements as an index of sympathetic nerve

ﬁring rates in inaccessible sites, such as the sympathetic nerves of splanchnic organs.

104

Experimental Procedures: Initial experiments were performed in 4-5 week DOCA-salt
and same age sham rats under urethane anesthesia (1.2 g/kg i.p.). Silicone rubber tipped
catheters were placed into abdominal aorta via a femoral artery and portal vein via a
small branch of the portal vein after laparotomy. 1 ml blood samples were collected
simultaneously from arterial and portal venous catheters. Plasma NE and epinephrine
were measured using a radioenzymatic assay. NHS spillover of NE was estimated as
introduced in General Methods. Sampling of blood from the portal venous and aortic sites
offers an advantage over conventional measurements in hepatic venous and arterial
plasma. A substantial proportion of total body sympathetic outﬂow is directed towards
splanchnic organs. The liver, however, removes approximately 57% and 32% of
circulating NE and EPI, respectively (42) (115). Sampling before the liver avoids the
interference of the efﬁcient hepatic extraction of NE on the observation of substantial
splanchnic organ NE spillover.

Further experiments were perfomed using conscious chronically tethered rats to avoid

the inﬂuence of anesthesia and surgical stress on plasma catecholamine levels.

105

Results:
1) Neither the plasma NE level nor the estimated NHS spillover of NE is higher in
anesthetized DOCA-salt rats than sham rats

I found that plasma NE level in portal vein equals that in aorta in anesthetized
DOCA-salt and sham rats, indicating no net NHS production of NE. Furthermore, there
was no signiﬁcant difference in portal venous plasma NE concentration between DOCA-
salt and sham rats, and surprisingly, higher aortic plasma NE concentration in sham rats
(Fig. 16). This may be at least partly explained by the inhibitory effect of anesthesia on
sympathetic nerve activity, especially in DOCA-salt rats, reﬂected by the much lower
MAP in anesthetized DOCA-salt rats compared with that of conscious DOCA-salt rats
(Fig. 17). I estimated NHS spillover of NE and found no signiﬁcant difference between
anesthetized DOCA-salt and sham rats (Fig. 18).

Data from anesthetized rats suggest that the sympathetic nerve activity is not

increased either globally or in the splanchnic bed of DOCA-salt rats.

2) Both the plasma NE level (aortic and portal venous) and estimated NHS spillover
of NE are significantly increased in conscious DOCA-salt rats versus sham rats
There is signiﬁcantly higher aortic plasma NE level in conscious DOCA-salt rats than
sham rats (Fig. 19). Portal venous plasma NE level is also signiﬁcantly increased in
DOCA-salt rats (Fig. 19). DOCA-salt rats have signiﬁcantly greater estimated NHS
spillover of NE than sham rats (Fig. 20). But the aortic plasma NE level and NHS

spillover of NE in DOCA-salt rats are unrelated to arterial blood pressure and heart rate.

106

There is no signiﬁcant difference in the estimated fractional extraction of NE in the
splanchnic bed between DOCA-salt (approximately 42%) and sham rats (approximately
25%).

In both DOCA-salt and sham rats, there is signiﬁcant higher EPI level in the aortic
plasma than portal venous plasma (81 :i: 19 pg/ml in sham aortic plasma; 46 i 12 pg/ml in
sham portal venous plasma; 89 i 16 pg/ml in DOCA aortic plasma; 46 i: 7 pg/ml in
DOCA portal venous plasma). However, there is no signiﬁcant difference in either aortic

or portal venous plasma EPI level between DOCA-salt and sham rats.

107

 

 

 

 

 

 

 

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Figure 16: Aortic and portal venous plasma NE level in anesthetized DOCA-salt and
sham rats. Bars indicate mean aortic or portal venous plasma NE concentration in
anesthetized sham and DOCA-salt rats. Brackets indicate SEM. Asterisk indicates a

signiﬁcant difference (P<0.05) between sham and DOCA-salt rats.

108

re
8

 

 

 

— sham (n=4-5) .. #
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mean arterial pressure (mmHg)
01
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anesthetized rats conscious rats

Figure 17: Mean arterial pressure of anesthetized and conscious DOCA-salt and sham

rats. Bars indicate mean arterial pressure in anesthetized and conscious sham and DOCA-

salt rats. Brackets indicate SEM. Asterisk indicates a signiﬁcant difference (P<0.05)

between sham and DOCA-salt rats. # indicates a signiﬁcant difference between conscious

and anesthetized DOCA-salt rats.

109

500

 

 

 

 

 

 

E — sham (n=5)
\3 400- c: DOCA(n=7)
s

g 300. l

9

32‘

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I

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Figure 18: Estimated nonhepatic splanchnic spillover rate of NE in anesthetized DOCA-
salt and sham rats. Bars indicate estimated NHS spillover rate of NE in anesthetized sham

and DOCA-salt rats. Brackets indicate SEM.

110

500

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

— sham (n=22)
A [:3 DOCA(n=19)
E 400 - .
‘9 t
‘7 l
g 300-
8
IJJ
z 200 -
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8
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aorta portal vein

Figure 19: Aortic and portal venous plasma NE level in conscious DOCA-salt and sham
rats. Bars indicate mean aortic or portal venous plasma NE concentration in conscious
sham and DOCA-salt rats. Brackets indicate SEM. Asterisks indicate a signiﬁcant

difference (P<0.05) between sham and DOCA-salt rats.

111

 

200

 

 

 

 

 

 

 

 

 

 

 

 

E
E - sham (n=22)
E) [:1 DOCA(n=19)
I: 150 a "
:3 l
:5-
in
“2" 100 4
CD
I
Z
3 50 .
(0
E
'a
to
or
0 .

Figure 20: Estimated nonhepatic splanchnic spillover rate of NE in conscious DOCA-salt
and sham rats. Bars indicate mean estimated NHS NE spillover rate in conscious sham
and DOCA-salt rats. Brackets indicate SEM. Asterisks indicate a signiﬁcant difference

(P<0.05) between sham and DOCA-salt rats.

112

Discussion for Chapter S-Part I

Numerous experimental and clinical studies, using various approaches to evaluate the
release of sympathetic transmitters and to obtain sympathetic nerve microneurographic
recordings, have shown clearly that sympathetic system activity and reactivity is
increased in experimental models of hypertension, including DOCA-salt hypertensive
rats. Consistent with published results (12), I found increased aortic plasma NE level in
conscious DOCA-salt rats, suggesting systemic sympathetic hyperactivity in DOCA-salt
rats. What needs to be mentioned here is that I did similar studies in anesthetized rats and
I were not able to ﬁnd either increased arterial or portal venous plasma NE level in
DOCA-salt rats, although previous studies by de Champlain et a1 (12) found signiﬁcantly
higher baseline NE level in anesthetized DOCA-salt rats than in the respective
normotensive controls.

In essential hypertension, there is increased cardiac and renal NE spillover and
unchanged hepatomesenteric NE spillover (122) (54, 192, 194, 195). Increased cardiac
(68) and renal sympathetic tone (110, 111) has also been reported in DOCA-salt
hypertension. However, non-hepatic splanchnic NE spillover has never been measured
before. I estimated the spillover of NE from the splanchnic bed to portal venous plasma
and found it was signiﬁcantly increased in DOCA-salt rats compared with sham rats. This
difference was not a consequence of hemodynamic inﬂuences on transmitter washout to
the circulation, as my later studies showed that portal blood ﬂows were similar in the two
groups. There is also a signiﬁcantly increased portal venous plasma NE level in DOCA-
salt rats than sham rats. Since the portal vein drains venous blood from the splanchnic

organs, I used portal venous plasma NE concentration as an index of splanchnic

113

sympathetic nerve activity. As I expected, I saw signiﬁcantly increased portal venous
plasma NE level in DOCA-salt rats. The increased portal venous plasma NE level and
increased NHS NE spillover suggest that there is increased splanchnic sympathetic nerve
activity in DOCA-salt rats. In the future, total body NE spillover can be measured by the
radiotracer method (introduced in more detail later) and the ratio of NHS NE spillover to
total body NE spillover can be used as an index of the contribution of increased NE
spillover in the splanchnic bed to the increase in total body NE spillover in DOCA-salt
rats.

The origin of this sympathetic activation still remains largely unknown. NE
release from sympathetic nerve endings is modulated pre-junctionally by adrenergic
receptor mechanisms (125). Moreover, 80-90% of neuronally released NE is taken back
up by the sympathetic nerve endings through an active reuptake (reuptake l) mechanism
(55). NE release from the nerve terminal can be facilitated by other neurohumoral factors
like ET-l, Ang II and EPI. Thus, in addition to increased sympathetic nerve ﬁring,
changes in either or all of these modulatory mechanisms in DOCA-salt rats could
confound the interpretation of plasma NE level and regional spillover measurements. For
example, a decrease in 02 prej unctional inhibition of peripheral NE release would serve
to augment the amount of NE released per unit sympathetic nerve discharge and result in
an overestimation of SNS activity based on plasma NE spillover. It has been suggested
that an increased rate of sympathetic nerve ﬁring, faulty NE reuptake into the
sympathetic nerves of the heart and prejunctional augmentation of NE release by EPI

contribute to the increased spillover of NE from the kidneys and heart to plasma in

essential hypertension (193). Increased sympathetic nerve ﬁring rate to the skeletal

114

muscle has also been reported in DOCA-salt rats. It is well documented in the
spontaneously hypertensive rat that there is an increase in the density of sympathetic
innervation. This possibility in DOCA-salt hypertension is still under investigation by my
group. The increased arterial and portal venous plasma NE level in DOCA rats is not
likely due to the facilitation of sympathoneural NE release by EPI through stimulation of
prejunctional 0.2-adrenoceptors located at the sympathetic nerve terminals. As in my
study, although the aortic plasma EPI level was signiﬁcantly higher than portal venous
plasma EPI level in both DOCA-salt and sham rats, neither the aortic nor the portal
venous plasma EPI level in DOCA-salt rats is signiﬁcantly higher than that of sham rats.
This is not the same as de Charnplain’s result showing increased arterial EPI level in
DOCA-salt rats (12). It has been suggested by other lab that the enhancement of NE
release by EPI is absent in rat mesentery (196).

The increased portal venous plasma NE level in conscious DOCA-salt rats is not
likely due to less NE extraction by the mesenteric organs, as the estimated NHS
ﬁ'actional extraction of NE is the same in DOCA-salt and sham rats.

In my later studies, I tested some of the other possibilities contributing to
sympathetic nervous system activation in the splanchnic bed of DOCA-salt hypertension.

Sympathetic nerve system release of EPI from the adrenal medulla contributes
importantly to the regulation of cardiovascular function as well as energy metabolism.
Similar to what has been found in essential hypertension (122) (192, 193, 195, 209), there
appears to be normal adrenal medullary secretion of epinephrine in DOCA-salt rats, as
there was no signiﬁcant difference in the aortic plasma epinephrine level between

DOCA-salt and sham rats. However, the interpretation of plasma EPI levels as a measure

115

of secretion from the adrenal medulla is not straightforward given the possibility of
DOCA treatment related changes in clearance. Although the fractional extraction of
epinephrine I obtained from my study appears to be unchanged in DOCA-salt rats, tracer
methodology, i.e. infusion of tritium labeled epinephrine should also be employed to

study plasma epinephrine kinetics.

Limitations:

1) Estimated NE spillovers are also dependent on removal processes that intervene
between sites of release and measurement in the bloodstream (43). Because of highly
efﬁcient neuronal uptake, only a small fraction of NE released by sympathetic nerves

throughout the body escapes into the bloodstream. So spillover is not equal to release.

2) It is not possible to know from which sympathetic nerves in the splanchnic bed the
released NE is derived, and in what proportions. So my measurements did not directly
reﬂect release of NE from sympathetic nerves to splanchnic veins.

However, it has been shown in dog (7) and guinea pig (8) that release of
neurotransmitters in mesenteric veins exceeds release of neurotransmitters in mesenteric
arteries, although both vessels demonstrate similar density of sympathetic neuronal
terminals (218). In both sham and DOCA-salt rats, mesenteric veins are more sensitive
than arteries to the vasoconstrictor effect of sympathetic nerve stimulation, as frequency-
response curves caused by nerve stimulation obtained ﬁ'om veins are to the left of those
from arteries. Accordingly, small increases in sympathetic nerve activity would increase
venous tone before changing arterial tone and increases in sympathetic nerve activity are

likely to have a larger effect on veins than arteries for a given change in nerve activity.

116

Chapter 5 - Part II
Prejunctional aZ-Adrenoceptor Inhibition of NE Release in Sympathetic Nerve
Endings Associated with Splanchnic Vasculature in Conscious DOCA-Salt Rats

Rationale: Prejunctional az-adrenoceptors mediate strong inhibition of NE release from
sympathetic terminals, constituting an important autoregulatory system for sympathetic
tone. Several mechanisms by which this inhibition of NE release may be mediated have
been described. These include activation of inwardly rectifying K+ channels, inhibition of
N-type voltage-gated Ca++ channels, and acceleration of Na‘VH+ exchange (18).
Prejunctional regulation of transmitter release by orz-adrenoceptors is altered in some
experimental models of hypertension (21) (220). Either impaired (237) or unaltered (45)
org-adrenoceptor prejunctional negative feedback system in the isolated mesenteric artery
of DOCA-salt animals have been reported. Changes in prejunctional regulation of
sympathetic nerves associated with veins in DOCA-salt rats remain to be fully
characterized. Luo et al (137) showed that nerve stimulation caused a greater increase in
the release of NE over basal level in DOCA veins compared to sham veins. The (12-
adrenoceptor agonist UK14304 inhibited stimulation induced NE release in sham, but not
DOCA veins. So in vitro studies suggest that prej unctional aZ-adrenoceptors are
dysfunctional in DOCA veins. However, no studies have been reported that were
performed to directly measure NE release from the splanchnic bed in conscious rats. In
my study, I evaluated the functioning of the prejunctional org-adrenoreceptor negative
feedback mechanism controlling NE release by measuring the changes in NE overﬂow

following az-adrenoreceptor agonist or antagonist treatment.

117

Experimental Procedures: Experiments were performed in 4-5 week DOCA—salt and
same age sham rats. After sodium pentobarbital anesthesia (50 mg/kg, i.p.), silicone
rubber tipped catheters were placed into abdominal aorta via a femoral artery and portal
vein via a small branch of the portal vein after laparotomy. The third catheter was
inserted into a femoral vein. I set up the rats as introduced in the General Methods. The
rats were treated with either 0.2-adrenoceptor antagonist yohimbine (0.5 mg/kg, i.v.) or
agonist clonidine (10 pig/kg, i.v.) after at least frny days of recovery from anesthesia and
surgical stress. 30 min after drug administration, 1 ml blood samples were collected
simultaneously from arterial and portal venous catheters. Plasma NE and EPI were
measured using a radioenzymatic assay. NHS spillover of NE was estimated as

introduced in General Methods.

Results:
1) Effects of 0.2-adrenoceptor antagonist yohimbine
Acute treatment with yohimbine didn’t cause any signiﬁcant change in the MAP in
sham rats. Yohimbine caused a small, but signiﬁcant decrease in the MAP of DOCA-salt
rats (Fig. 21). Yohimbine signiﬁcantly increased the heart rate in both sham (from 369 at
14 to 405 d: 16 beats/ min) and DOCA-salt rats (from 363 :t 12 to 406 3c 14 beats/min).
Yohimbine signiﬁcantly increased aortic (Fig. 22) and portal venous plasma NE
concentrations in both sham and DOCA-salt rats (from 209 d: 23 to 467 i 76 pg/ml in
sham aortic plasma; from 226 :t 33 to 446 :1: 66 pg/ml in sham portal venous plasma;
from 339 :t: 63 to 589 :t 113 pg/ml in DOCA aortic plasma; from 315 i 50 to 568 d: 92

pg/ml in DOCA portal venous plasma). There was no signiﬁcant difference in the

118

magnitude of increase between DOCA-salt and sham rats (144 i 44% and 99 d: 47% in
sham and DOCA-salt aortic plasma; 79 :h 26% and 105 i 36% in sham and DOCA-salt
portal venous plasma). Yohimbine also signiﬁcantly increased the aortic and portal
venous plasma EPI concentrations in sham rats (from 73 :l: 22 to 236 :t 88 pg/ml in aortic
plasma; from 37 :t 11 to 104 i 34 pg/ml in portal venous plasma). However, yohimbine
didn’t cause any signiﬁcant change in either aortic or portal venous plasma EPI level in
DOCA-salt rats (from 104 :l: 32 to 105 i 27 pg/ml in aortic plasma; from 50 :t 12 to 65 :l:
15 pg/ml in portal venous plasma).

Yohimbine didn’t cause any signiﬁcant change in the estimated NHS fractional
extraction of NE in either group (from 25 i 11% to 42 i 11% in sham rats; from 42 i 4%
to 31 d: 6% in DOCA-salt rats). Yohimbine signiﬁcantly increased the NHS NE spillover

in sham rats, but not in DOCA-salt rats (Fig. 23).

2) Effects of aZ-adrenoceptor agonist clonidine

Acute treatment of clonidine didn’t cause any signiﬁcant change in the MAP and
heart rate of sham rats. Clonidine didn’t have a signiﬁcant effect on the heart rate of
DOCA-salt rats (from 372 i 18 beats/min to 361 i 20 beats/min) either. However,
clonidine signiﬁcantly decreased the MAP of DOCA-salt rats (Fig. 24).

Clonidine didn’t signiﬁcantly change the aortic plasma NE concentrations in either
sham or DOCA-salt rats (Fig. 25). Clonidine signiﬁcantly decreased the portal venous
plasma NE concentration only in sham (from 233 i 26 pg/ml to 140.5 :1: 35 pg/ml), but
not DOCA-salt rats (from 336 i 52 pg/ml to 354 i 119 pg/ml). Clonidine didn’t cause
any signiﬁcant change in plasma EPI levels in sham rats (from 62.2 :1: 11 to 130 i 35

pg/ml in aortic plasma; from 39 d: 8 to 72 d: 21 pg/ml in portal venous plasma). Clonidine

119

signiﬁcantly increased the portal venous plasma EPI level in DOCA-salt rats (from 87.6
d: 25 to 125.1 :1: 35 pg/ml in aortic plasma; from 46 :l: 10 to 73 :t 16 pg/ml in portal venous
plasma).

Clonidine didn’t cause any signiﬁcant change in the NHS FX of NE in either group
(from 18 i 20% to 39 i 10% in sham rats; from 38 i 7% to 30 :l: 7% in DOCA-salt rats).
Clonidine signiﬁcantly decreased the NHS NE spillover in sham, but not DOCA-salt rats

(Fig. 26).

120

- sham (n=8)
:1 DOCA (n=8)

 

200 t

I:

 

H20

150 «

 

100+

50~

mean arterial pressure (mmHg)

 

 

 

 

 

 

   

 

 

 

7

control yohimbine

Figure 21: Mean arterial pressure responses to i.v. bolus yohimbine administration in
conscious DOCA-salt and sham rats. Bars indicate mean arterial pressure before or after
yohimbine treatment in conscious sham and DOCA-salt rats. Brackets indicate SEM.
Asterisk indicates a signiﬁcant difference (P<0.05) between sham and DOCA-salt rats. &

indicates a signiﬁcant difference (P<0.05) between before and after drug treatment.

121

- sham (n=8)
1:3 DOCA (n=8)
800

 

——tao

600 .

 

400 - -—

 

 

200 ‘

arterial plasma NE oonc. (pg/ml)

 

 

 

 

 

 

 

 

 

 

 

0 . .
control yohimbine

Figure 22: Effects of i.v. bolus yohimbine administration on aortic plasma NE level in
conscious DOCA-salt and sham rats. Bars indicate mean arterial plasma NE
concentration before or after yohimbine treatment in conscious sham and DOCA-salt rats.
Brackets indicate SEM. Asterisk indicates a signiﬁcant difference (P<0.05) between
sham and DOCA-salt rats. &s indicate a signiﬁcant difference (P<0.05) between before

and after drug treatment.

122

_ sham (n=8)
1:1 DOCA (n=8)

 

300

250 «

200 «

150« _L

100 4

 

 

50-1

estimated NHS NE spillover (pg/min)

 

 

 

 

 

 

 

 

 

 

 

control yohimbine

Figure 23: Effects of i.v. bolus yohimbine administration on estimated NHS spillover rate
of NE in conscious DOCA-salt and sham rats. Bars indicate mean estimated NHS NE
spillover rate before or after yohimbine treatment in conscious sham and DOCA-salt rats.
Brackets indicate SEM. Asterisk indicates a signiﬁcant difference (P<0.05) between
sham and DOCA-salt rats. & indicates a signiﬁcant difference (P<0.05) between before

and after drug treatment.

123

 

250

 

- sham (n=8)
[:1 DOCA (n=9)

 

 

 

.42.

 

150+

 

100‘

mean arterial pressure (mmHg)

  

 

 

 

 

 

 

 

 

 

 

clonidine

Figure 24: Mean arterial pressure responses to i.v. bolus clonidine administration in
conscious DOCA-salt and sham rats. Bars indicate mean arterial pressure before or after
clonidine treatment in conscious sham and DOCA-salt rats. Brackets indicate SEM.
Asterisk indicates a signiﬁcant difference (P<0.05) between before and after drug
treatment. & indicates a signiﬁcant difference (P<0.05) between sham and DOCA-salt

rats.

124

— sham (n=9)
1:1 DOCA (n=10)

 

O)
O
O

‘I’

400 -

 

 

 

N
O
O

arterial plasma NE conc. (pg/ml)

 

 

 

    

 

 

 

 

 

 

control clonidine

Figure 25: Effects of i.v. bolus clonidine administration on aortic plasma NE level in
conscious DOCA-salt and sham rats. Bars indicate mean arterial plasma NE
concentration before or alter clonidine treatment in conscious sham and DOCA-salt rats.
Brackets indicate SEM. Asterisk indicates a signiﬁcant difference (P<0.05) between

sham and DOCA-salt rats.

125

- sham (n=9)
:3 DOCA (n=10)

 

 

 

 

 

E

\ 300-

3

'6

>

.2

'6. 200‘

(I)

UZJ l
w T

I

Z 100-

a .
(D

g i
‘63

‘1’ 0

 

 

 

 

 

 

 

 

control clonidine

Figure 26: Effects of i.v. bolus clonidine administration on estimated NHS spillover rate
of NE in conscious DOCA-salt and sham rats. Bars indicate mean estimated NHS NE
spillover rate before or after clonidine treatment in conscious sham and DOCA-salt rats.
Brackets indicate SEM. Asterisk indicates a signiﬁcant difference (P<0.05) between

before and after drug treatment.

126

Discussion for Chapter 5—Part II

Prejunctional aZ-adrenoceptors are activated by NE that is released upon neural
membrane depolarization thus limiting further release of neurotransmitters (126). Data
from several studies indicate that prejunctional 0.2-adrenoceptor function is impaired in
hypertensive animals. In my, study, I tested the hypothesis that there was impaired (:2-
adrenoceptor function in the systemic and splanchnic circulation in conscious DOCA-salt
rats in vivo by comparing the effects of (12-adrenoceptor antagonist and agonist on
plasma NE level and the estimated NHS NE release between DOCA-salt and sham rats.

I found that in sham rats, the administration of yohimbine produced no signiﬁcant
change in MAP despite the important increase in plasma NE level. The lack of change in
blood pressure could be due to the blockade of postjunctional (12-adrenergic receptors
that mediate vasoconstriction (243). In DOCA-salt rats, however, the decrease in blood
pressure induced by yohimbine could result from ampliﬁed postjunctional 02-
adrenoceptor blockade even there was a simultaneous increase in NE release.

Yohimbine signiﬁcantly increased the systemic plasma NE concentration in both
DOCA-salt and sham rats, suggesting that the systemic prejunctional 0.2-adrenoceptor
regulation of NE release is normal in DOCA-salt rats. However, yohimbine didn’t cause
any signiﬁcant change in the plasma EPI level in DOCA-salt rats, suggesting impaired
prejunctional (12-adrenoceptor regulation of EPI release from adrenal gland. My ﬁnding
is not consistent with previous studies by De Champlain J et a1 showing that
intravenously administered yohimbine increased plasma NE levels in conscious
normotensive but not DOCA-salt hypertensive rats (30, 157). However, they also found

decreased function of the prejunctional (12-adrenergic autoinhibitory mechanism at the

127

adrenal medulla in DOCA-salt rats, reﬂected by lack of signiﬁcant effect of yohimbine
on plasma EPI level (157).

In my study, I also found that yohimbine only signiﬁcantly increased the estimated
NHS NE spillover in sham, but not DOCA-salt rats, suggesting that there is impaired
prejunctional (12-adrenoceptor function in the splanchnic bed of DOCA-salt rats. This is
consistent with previous in vitro studies by Luo et al (137) showing that yohimbine,
increased NE release in sham, but not DOCA mesenteric arteries and veins. An
alternative explanation could be that under basal conditions the prejunctional 02-
adrenoceptors do not thave a dominanting inﬂuence on the transmitter release in the
splanchnic vasculature of DOCA-salt rats.

I also treated my rats with clonidine as an alternative way to look at 02-
adrenoceptor function. I found that clonidine only signiﬁcantly decreased the portal
venous plasma NE level in sham rats. It didn’t cause a signiﬁcant change in either arterial
or portal venous plasma NE level in DOCA-salt rats. Clonidine signiﬁcantly decreased
the estimated NHS spillover of NE in sham rats, but failed to cause any signiﬁcant
change in that of DOCA-salt rats. So data from the clonidine experiment also suggest that
there is impaired (12-adrenoceptor function in the splanchnic bed of DOCA-salt rats. In
another hypertension model, conscious male stroke-prone spontaneously hypertensive
rats (SPSHR), it was reported that i.v. administration of clonidine (5 ug) decreased
splanchnic nerve activity signiﬁcantly (136). The different effect of clonidine on the
splanchnic bed of DOCA-salt rats and SPSHR may be related to the different

hypertension mechanisms underlying these two hypertension models.

128

In conclusion, both yohimbine and clonidine studies suggest that in DOCA-salt rats,
there is impaired activity of the prejunctional (12-adrenergic negative feedback
mechanism regulating the release of NE from the sympathetic nerve ﬁbers associated
with the splanchnic vasculature under basal conditions. This regulatory mechanism also
seemed impaired at the level of the adrenal medulla under basal conditions. These
alterations could contribute at least in part to elevate the plasma levels of NE and net
NHS NE spillover in DOCA-salt rats. One possible mechanism underlying these
alterations could be (12-adrenoceptor desensitization, perhaps due to high basal
sympathetic activity in DOCA-salt rats (9). Impairment of 0.2 receptor mediated
prejunctional negative feedback mechanism because of reduced expression of the 02
receptor subtype A has been reported in spontaneously hypertensive rats (272).

(12-adrenoceptor mediated neuromodulation plays a greater role in veins than in
arteries(7). Quantitative differences in (12-adrenoceptor mediated neuromodulation in the
splanchnic blood vessels may participate to differing contributions of arteries and veins to
the control of blood ﬂow and volume distribution in splanchnic circulation. Besides the
impaired 0.2 inhibitory mechanism, enhanced sensitivity of facilitatory prej unctional 132
mechanisms have also been reported in DOCA-salt rats (11). There is an impairment of
the functional reciprocal balance between the a and B prejunctional receptors (1 1). Since
DOCA-salt hypertension seems to depend on the sympathetic nervous system, these
abnormalities may act together in facilitating the release of sympathetic
neurotransmitters, therefore sustaining an increased sympathetic tone and reactivity

which, in turn, may help develop and maintain DOCA-salt hypertension.

129

It is important to keep in mind, though, that with chronically increased NE release
into the neuroeffector junction that exceeds the capacity of the neuron to return NE to
prejunctional vesicles, there can be a net neuronal loss of NB. This loss is considered a
feature of severe progressive heart failure (40, 150). Tissue NE depletion has also been

reported in DOCA-salt hypertension (138) (94).

Limitation: It can be argued that yohimbine and clonidine are acting on the central
nervous system to produce the change in plasma NE level observed in normotensive rats,
and that the lack of effect of yohimbine and clonidine on splanchnic NE release in
DOCA-slat rats may reﬂect an alteration in central regulatory mechanisms. My study
does not permit one to distinguish between the central and peripheral effects of
yohimbine and clonidine. However, two studies carried out in cats (179) and in rabbits
(228) demonstrated that yohimbine, at a similar dose to the one used in my study, acts
mainly in the periphery to produce its increase in plasma NE levels. No similar data are

available for clonidine.

130

Chapter 5 - Part III

Neuronal NE uptake activity in the splanghnic vasculature of anesthetized and '

conscious DOCA-salt rats.

Rationale: Recent identiﬁcation of a syndrome of orthostatic intolerance (01) (an
autonomic disorder characterized by excessive tachycardia, minimal blood pressure
changes, and increased NE after assuming a standing position) resulting from a defect in
norepinephrine uptake has renewed clinical interest in enzymes and transporters involved
in the synthesis and metabolism of NE. A dominant negative mutation in human
norepinephrine transporter (NET) reduces its surface expression via heterologous
oligomerization (85), thus rendering a major ﬁ'action of the normal transporter inert and
reducing NE reuptake in affected heterozygotes (211).

NET is critical in central modulation of sympathetic tone and autonomic
regulation of peripheral hemodynamics by reducing NE concentrations in the
neuroeffector junction (10) and preventing the resultant deleterious effects on
cardiovascular tissues. The amount and type of neurotransmitter removed at any given
site varies (37). NET removes not only residual NE but, a major portion of surplus
dopamine as well (20). Peripherally, NET is perhaps most important in the heart (59, 78).
In humans, approximately 92% of cardiac NE removal occurs via NET, with the
remainder eliminated by exraneuronal carriers and circulatory dissipation (40). So the
rate of spillover is also dependent on the adequacy of neuronal uptake, not solely on the
rate of NE release. At a given rate of sympathetic nerve ﬁring, the capacity for neuronal
reuptake of NE inﬂuences rates of NE overﬂow. This has been shown experimentally,

with electrical stimulation of the sympathetic nerve supply, in a range of individual

131

organs (108) (124, 261, 262) and for the body as a whole in the pithed rabbit (227). The
magnitude of the effect of pharmacological neuronal NE uptake (U 1) block on NB
overﬂow differs between different organs and tissues, depending on the importance of U1
in NE disposition in a particular site (6, 168). Typical neurochemical consequences of
neuronal NE reuptake impairment are facilitation of NE spillover ﬁom sympathetic
nerves to plasma, reduced clearance of NE from plasma by the body as a whole and by
individual organs, and alteration in the pattern of NE metabolites due to reduction in
neuronal metabolism and augmentation of extraneuronal metabolism (41, 53, 59, 79).
Neuronal uptake of NE was found to be normal in cardiac and vascular tissue in
young spontaneously hypertensive rats (SHR) (151) and increased in resistance arteries
of adult SHR (41 ). Impaired neuronal NE reuptake in lean patients with essential
hypertension perhaps is due to dysfunction of the NE transporter (193). In vitro studies
suggest reduced neuronal reuptake of NE in the heart of DOCA-salt rats partly due to
reduced NET 80 kD subunit protein level (255). Luo M et al (138) found that there was
an upregulation of NE transporter activity in the splanchnic vasculature of DOCA-salt
rats as neuronal and extraneuronal NE uptake blockers, cocaine and corticosterone,
prolonged neurogenic responses in veins from DOCA-salt but not sham rats. There was a
signiﬁcant increase in NET-immunoreactivity in arteries, veins and sympathetic ganglia
when compared to levels in the tissues from sham rats. No in vivo studies have been done
to study the functioning of NET and the relative importance of neuronal uptake in the
disposition of released NE in the splanchnic bed of DOCA-salt rats. It is likely that
norepinephrine release from perivascular sympathetic nerves in DOCA-salt hypertension

is increased (as I found in my study), which stimulates an upregulation of NET levels in

132

blood vessels. The latter may serve to reduce NE levels in the neuroeffector junction,
thereby providing a mechanism to partially offset the potential deleterious effects of
excess and unregulated NE in DOCA-salt rats. In addition, this adaptive response may
prevent the global neuronal depletion of NE.

To test if there is altered NET activity that contributes to plasma NE level in
DOCA-salt rats, I estimated the NHS spillover of NE and NHS FX of NE in the presence
of the neuronal uptake inhibitor, desipramine, in anesthetized and conscious DOCA-salt

and sham rats.

Experimental procedures: Initial experiments were performed in 4-5 week DOCA-salt
and sham rats under urethane anesthesia (1.2 g/kg, i.p.). Silicone rubber tipped catheters
were placed into abdominal aorta via a femoral artery and portal vein via a small branch
of the portal vein after laparotomy. The third catheter was inserted into a femoral vein. 30
min after recovery, the rats were treated with desipramine (2mg/kg, i.v.). 30 min later, 1
ml blood samples were collected simultaneously from arterial and portal venous
catheters. Plasma NE and epinephrine were measured using a radioenzymatic assay. NHS
spillover of NE was estimated as introduced in General Methods.

Additional studies were performed using couscious rats which were treated with
desipramine (2mg/kg, i.v.) after at least ﬁny days of recovery from anesthesia and

surgical stress.

Results: In anesthetized rats: Desipramine didn’t cause any signiﬁcant change in the

mean arterial pressure in either DOCA-salt (Fig. 27) (from 93.7 :1: 9.1 mmHg to 91.8 :t

133

6.1 mmHg) or sham rats (from 95.4 i 1.7 mmHg to 87.3 i 6.1 mmHg). Acute blockade
of neuronal NE uptake signiﬁcantly increased the aortic and portal venous plasma NE
level in both sham and DOCA-salt rats (Fig. 28) (from 567.2 :1: 89.9 to 824.3 i 75.8
pg/ml in sham aortic plasma; from 504.6 :1: 71.7 to 878.9 i 82.7 pg/ml in sham portal
venous plasma; from 370.0 d: 26.7 to 891.2 i 93.0 pg/ml in DOCA aortic plasma; from
458.9 d: 50.0 to 710.7 :1: 59.8 pg/ml in DOCA portal venous plasma). The magnitude of
increase was signiﬁcantly higher in the aortic plasma of DOCA-salt rats than in that of
sham rats. Desipramine caused a signiﬁcant decrease in the estimated NHS F X of NE
only in sham rats (Fig. 29). Desipramine didn’t cause a signiﬁcant change in the
estimated NHS spillover in either group (Fig. 30).

In conscious rats: Desipramine signiﬁcantly decreased the MAP in DOCA-salt rats
(Fig. 31) (from 164.3 i 5.2 mmHg to 154.2 :1: 6.1 mmHg), but not in sham rats (from
102.5 i: 2.2 mmHg to 108.8 :1: 3.2 mmHg). Desipramine caused a signiﬁcant increase in
the aortic plasma NE level in sham rats, but not DOCA-salt rats (Fig. 32). Desipramine
didn’t cause any signiﬁcant change in the estimated NHS FX of NE (Fig. 33) and the

estimated NHS NE spillover (Fig. 34) in either DOCA-salt or sham rats.

134

- sham (n=5-7)
[:1 DOCA (n=6-7)

 

 

-i..r-_ _.__._...-

.L
.h.
C

4...;
CN
CO

 

 

601

mean arterial pressure (mmHg)
CD
8 o

N
O

 

 

 

 

 

 

 

 

 

 

C

control DMI

Figure 27: Mean arterial pressure responses to i.v. bolus desipramine administration in
anesthetized DOCA-salt and sham rats. Bars indicate mean arterial pressure before or

after DMI treatment in anesthetized sham and DOCA-salt rats. Brackets indicate SEM.

135

- sham (n=5-7)
:3 DOCA (n=6-7)

 

1 200 —“

 

 

A
O
8 o
o O

600‘

400‘

200 r

arterial plasma NE cone. (pg/ml)

 

 

 

  

 

 

 

 

 

 

 

control [jun

Figure 28: Effects of i.v. bolus desipramine administration on aortic plasma NE level in
anesthetized DOCA-salt and sham rats. Bars indicate mean arterial plasma NE
concentration before or after DMI treatment in anesthetized sham and DOCA—salt rats.
Brackets indicate SEM. Asterisks indicate a signiﬁcant difference (P<0.05) before and

aﬂer drug treatment.

136

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

g 100
S
:3 _ sham (n=6)
g 80 t *' :1 DOCA (n=6)
0)
E
.9 5° 1 T
*6
t!
lit 40«
m
E
20 a
E
(0
.§
‘63 0
0)

control DMI

Figure 29: Effects of i.v. bolus desipramine administration on estimated NHS ﬁactional
extraction of NE in anesthetized DOCA-salt and sham rats. Bars indicate mean estimated
NHS NE fiactional extraction before or after DMI treatment in anesthetized sham and
DOCA-salt rats. Brackets indicate SEM. Asterisk indicates a signiﬁcant difference
(P<0.05) between sham and DOCA-salt rats. # indicates a signiﬁcant difference (P<0.05)

between before and after drug treatment.

137

600

 

 

500 j _ sham (n=5-8)
1:1 DOCA (n=6-7)

400 . I

300 - i

 

 

 

 

 

200 ~

100 *

estimated NHS NE spillover (pg/min)

 

 

 

 

 

 

 

 

 

conti'ol 0th

Figure 30: Effects of i.v. bolus desipramine administration on estimated NHS spillover
rate of NE in anesthetized DOCA-salt and sham rats. Bars indicate mean estimated NHS
NE spillover rate before or after DMI treatment in anesthetized sham and DOCA-salt

rats. Brackets indicate SEM.

138

_ sham (n=10)
E DOCA (n=8)

 

 

 

  

 

 

 

 

 

 

 

 

 

 

250
’6:
3E 200
E
9 3 .
3
a 150- I
2
o.
7:“
g 100‘
(0
6
a) ..
E 50

O I

control DMl

Figure 31: Mean arterial pressure responses to i.v. bolus desipramine administration in
conscious DOCA-salt and sham rats.Bars indicate mean arterial pressure before or after
DMI treatment in conscious sham and DOCA-salt rats. Brackets indicate SEM. #
indicates a signiﬁcant difference (P<0.05) between DOCA-salt and sham rats. Asterisk

indicates a signiﬁcant difference (P<0.05) between before and after drug treatment.

139

_sham (n=10)
1:: DOCA (n=8)

1 000

 

00
O
O
l
———+

 

O)

O

O
l

400 -

 

200 1

arterial plasma NE conc. (pg/ml)

 

 

 

 

 

 

 

 

 

 

DMI

Figure 32: Effects of i.v. bolus desipramine administration on aortic plasma NE level in
conscious DOCA-salt and sham rats. Bars indicate mean arterial plasma NE
concentration before or after DMI treatment in conscious sham and DOCA-salt rats.

Brackets indicate SEM. Asterisk indicates a signiﬁcant difference (P<0.05) before and

after drug treatment.

140

- sham (n=10)
[:1 DOCA (n=8)

 

 

 

60
g 50. T l
X
LL
uzr 40-
2’6
2 30a
8
a“; 20‘
.E
8 104

o

 

 

 

 

 

 

   

 

 

 

control DMI

Figure 33: Effects of i.v. bolus desipramine administration on NHS fractional extraction
of NE in conscious DOCA-salt and sham rats. Bars indicate mean estimated NHS NE
fractional extraction before or after DMI treatment in conscious sham and DOCA-salt

rats. Brackets indicate SEM.

141

_ sham (n=10)
12:21 DOCA (n=8)

 

 

 

 

  

E200

g .

3160-

9 l l
.9 ,
3120+

(I)

LU 1

2

g 801

z r

8

*5 404

.E .

‘17:

° 0

 

 

 

 

 

 

 

 

 

 

control DMI,

Figure 34: Effects of i.v. bolus desipramine administration on estimated NHS spillover
rate of NE in conscious DOCA-salt and sham rats. Bars indicate mean estimated NHS
NE spillover rate before or after DMI treatment in conscious sham and DOCA-salt rats.

Brackets indicate SEM.

142

Discussion for Chapter S-Part III

The lack of effects of desipramine on the NHS F X and spillover of NE in conscious
rats is not likely due to inadequate dose, as same dose signiﬁcantly increased the plasma
NE levels in both anesthetized DOCA-salt and sham rats. In the future, plasma DHPG,
the intraneuronal metabolite of NE, can be measured to conﬁrm that I gave my rats a
sufﬁcient dose of desipramine (decreased plasma DHPG is expected).

My results may suggest that neuronal uptake accounts for a very small proportion of
NE/EPI removal from the splanchnic circulation. However, the effects of
pharmacological neuronal uptake block on the rate of NE spillover, with systemic
administration of the blocking drug desipramine, is complicated by the fact that
sympathetic nerve ﬁring rates fall (55), presumably due to an action of the drug within
the central nervous system (53, 225). Decreased NE clearance after DMI administration
leads to increased levels of NE in the brain stem and to increased stimulation of
inhibitory 0.2a receptors, resulting in decreased sympathetic outﬂow. DMI-induced
sympathoinhibitory effects of increased NE in the brain stem can predominate over the
functional elevation of NE stimulation of peripheral targets (183). Attenuated baroﬂex
function has also been observed in DMI-treated rats (183). The attenuation in
sympathetic outﬂow and to suppression of the baroreﬂex obscure or prevent the
expression of the peripheral manifestations (such as the NHS NE spillover rate I was
looking for in my study) of impaired NE uptake. The change in NE spillover rate could
be a consequence of this fall in nerve ﬁring rate plus uptake blockade. Actually, it has
been reported that intravenous desipramine dose-dependently inhibited splanchnic

sympathetic nerve discharge (-64 +/- 3% after 4 mg/kg iv DMI) in urethane-anesthetized

143

rats (98). It has been suggested that after i.v. desipramine, blockade of neurotransmitter
reuptake increased NE concentrations at sympathoeffector junctions so as to offset the
fall in SNA, so that there was little change in NE spillover (44). Changes in systemic NE
concentrations will provide some insight into the magnitude of this effect in my
experiments. Unfortunately, currently there are no drugs available that only speciﬁcally
block neuronal uptake of NE and have no sympathoinhibitory effect.

A paper published in J Nucl Cardiol in 2003 showed an alternative way to evaluate
the activity of NET (75). In this study, the authors were interested in the NET activity in
the heart of heart failure patients. They injected I-123 metaiodobenzylguanidine (MIBG),
which is a guanethidine congener, into human subjects through an indwelling catheter
and the conjugate count images of the thorax were acquired using a gamma camera. The
images were entered into a dedicated computer for quantitation of I-123 MIBG. Activity
of NET can be assessed from uptake of I-123 MIBG because radiolabeled preparations of
MIBG have been shown to mimic uptake of tritiated NE into the storage vesicles by NET
and provides a marker for sympathetic nerve activity and activity of the transporter. A
similar experimental design could be used in my study. However, it is not clear if it
would be more difﬁcult to acquire satisfactory images from gut in animals compared with

from heart in human subjects.

Limitation: Alterations in the function of the NET could be a result of one or several
changes. For example, there could be a change in the amount of transporter protein
inserted in the membrane or there could be a change in the transport capacity of the

transporter or there could be a change in the way the protein is regulated by intracellular

144

signaling mechanism. The spillover measurement does not discriminate among the

various alternative cellular and molecular changes that might occur in the transporter.

145

Chapter 5 - Part IV
Interaction between Preiunctional 0.2-adrenoceptor and Neuronal Transmitter

Reuptake in DOCA-Salt Hypertensive Rats

Rationale: It has been reported previously that in the mesenteric vessels where the
effector response is normally influenced by the combined activity of az-adrenoceptors
and uptake, failure of one mechanism increases the activity of the other, so as to maintain
a largely constant effector response (163). An interaction between reuptake and a2-
receptors is present in the vas deferens of the mouse and rat, guinea-pig atrium, the
spleen and various blood vessels from the dog (135). Patients with genetic impairment of
the prejunctional (12c adrenergic receptor have augmented activity of the norepinephrine
uptake-1 transporter (75). The mechanism is not delineated but may be related to
increased NE substrate in the symaptic cleﬁ. No similar study has ever been done in the

splanchnic vasculature of DOCA-salt rats.

Experimental Procedures: Experiments were performed in 4-5 week DOCA-salt and
same age sham rats. After sodium pentobarbital anesthesia (50 mg/kg, i.p.) anesthesia
(1.2 g/kg i.p.), silicone rubber tipped catheters were placed into abdominal aorta via a
femoral artery and portal vein via a small branch of the portal vein after laparotomy. The
third catheter was inserted into a femoral vein. I set up the rats as introduced in the
General Methods. After at least fmy days of recovery from anesthesia and surgical stress,
the rats were treated with yohimbine (0.5mg/kg, i.v.), 10min later the rats were given

desipramine (2mg/kg, i.v.). 30 min after drug administration, 1 ml blood samples were

146

collected simultaneously from arterial and portal venous catheters. Plasma NE and EPI
were measured using a radioenzymatic assay. NHS spillover of NE was estimated as

introduced in General Methods.

Results: Yohimbine+DMI treatment didn’t cause any signiﬁcant change in the MAP of
either sham (Fig. 35) (from 101.2 1 3.0 to 94.2 i 2.8 mmHg) or DOCA-salt rats (from
164.1 :t 10.2 to 167.1 :t 4.6 mmHg). Yohimbine + DMI treatment caused a signiﬁcant
increase in the arterial plasma NE concentration in sham rats (Fig. 36). The increase was
greater than that caused by DMI and yohimbine treatment alone, but not signiﬁcantly so.
Yohimbine+DMI treatment didn’t cause any signiﬁcant change in the arterial plasma NE
concentration in DOCA-salt rats (Fig. 36). Yohimbine+DMI signiﬁcantly increased the
estimated NHS NE spillover in sham rats and the magnitude of increase was not
signiﬁcantly different than that caused by yohimbine and DMI alone. Yohimbine+DMI

didn’t cause any signiﬁcant change in the NHS NE spillover in DOCA-salt rats (Fig. 37).

147

- sham (n=4)
‘2 DOCA (n=4)

 

250

200 '

Hat-
-1

 

 

150-

100~

mean arterial pressure (mmHg)
01
O

 

 

 

 

    

 

 

 

 

 

C

control YOH+DM|

Figure 35: Mean arterial pressure responses to i.v. bolus yohimbine+desiprarnine

administration in conscious DOCA-salt and sham rats. Bars indicate mean arterial
pressure before or after YOH+DMI treatment in conscious sham and DOCA-salt rats.
Brackets indicate SEM. Asterisk indicates a signiﬁcant difference (P<0.05) between

DOCA-salt and sham rats.

148

— sham (n=6)
C: DOCA (n=4)

1 000

 

8004

600‘ I

400 ~

 

 

arterial NE conc. (pg/ml)

200-

 

 

 

 

 

 

 

 

 

 

control YOH+DM|

Figure 36: Effects of i.v. bolus yohimbine+desiprarnine administration on aortic plasma
NE level in conscious DOCA-salt and sham rats. Bars indicate mean arterial plasma NE
concentration before or after YOH+DMI treatment in conscious sham and DOCA-salt

rats. Brackets indicate SEM. Asterisk indicates a signiﬁcant difference (P<0.05) before

and after drug treatment.

149

 

 

 

- SHAM (n=6)
:3 DOCA (n=4)

 

 

 

 

 

 

0"
O
1

     

 

 

 

 

 

 

O

 

 

 

estimated NHS NE spillover (pglmllmin)

contiol YOH-‘t-DMI

Figure 37: Effects of i.v. bolus yohimbine+desiprarnine administration on estimated NHS
spillover rate of NE in conscious DOCA-salt and sham rats. Bars indicate mean estimated
NHS NE spillover rate before or after DMI treatment in conscious sham and DOCA—salt
rats. Brackets indicate SEM. Asterisk indicates a signiﬁcant difference (P<0.05) before

and after drug treatment.

150

Discussion for Chapter 5—Part IV

In my previous study, DMI alone didn’t cause any signiﬁcant change in the
estimated NHS NE spillover in either DOCA-salt or sham rats. In this study, after (12-
adrenoceptor blockade by i.v. yohimibine, NE spillover was increased in response to i.v.
DMI in sham rats. This may reﬂect the increased NET activity after the failure of
prejunctional a2 autoinhibiton. Besides, DMI has two distinct actions: it blocks neuronal
reuptake by direct actions on nerve endings and reduces SNA by actions of raised
intrasynaptic NE concentrations on inhibitory a2-adrenoceptors within the CNS.
Therefore, blockade of a2-adrenoceptors may block the central sympathoinhibitory effect
of desipramine. And subsequently, the increase in NE concentration at sympathoeffector
junctions would not be offset by a fall in SNA. In contrast to what happened in sham rats,
yohimbine+DMI didn’t cause any signiﬁcant change in the estimated NHS NE spillover
in DOCA-salt rats. This could be due to impairment of prej unctional a2-adrenoccptors
function in the splanchnic bed of DOCA-salt rats. It could also reﬂect the absence of an

important functional interaction between the two mechanisms.

151

Chapter 5 — Part V
Modulation of Sympathetic Neurotransmission by Endothelin-1 in vivo in DOCA-

Salt Hypertensive Rats

Rationale: I speculate based on the results of my endothelin study discussed earlier in
Chapter 4 that the enhanced venoconstrictor effect of ET-1 could be mediated by an
action of ET-1 on the sympathetic nervous system. A number of studies have shown
prejunctional modulation of sympathetic neurotransmission by ET-1 in vitro (158), e. g.
Intrarenal arterial infusion of ET-1 inhibited norepinephrine (NE) release induced by
renal sympathetic nerve stimulation in anesthetized dogs (147). This neuroinhibitory
effect of ET-1 was mediated through activation of ETB receptor mechanisms (224).
Localization of ET-1 receptors on the nerve terminals have also been reported (60).
Neurotransmission in sympathetic ganglia also can be modulated by ET-l (238).
Superoxide is elevated in prevertebral sympathetic ganglia in DOCA-salt hypertension,
and ET-1 is a potent stimulus for the elevation of superoxide levels in sympathetic
ganglia, an effect that may be mediated by the upregulation of ETB receptors (26).
Superoxide production evoked by ET-l may play a role in the increased sympathetic
excitability in DOCA-salt hypertension (26). Increased circulating ET-l level was found
in ganglionectomized DOCA-salt hypertensive rats (63). A previous study in my lab
(104) found that pretreatment of DOCA-salt hypertensive rats with an ETB receptor
antagonist produced greater declines in MCF P following ganglionic blockade than after
ganglionic blockade alone. These ﬁndings suggest that ET-1 acting at ETB receptors may
provide an important contribution to venoconstriction through modulation of sympathetic

tone to the veins. The ETB antagonist may have facilitated NE release from sympathetic

152

nerve terminals on veins, accounting for the signiﬁcantly larger response of MCFP to
subsequent ganglion blockade. This action presumably was more prominent in DOCA-
salt rats because of their higher pretreatment levels of endogenous vascular ET-l.
Furthermore, in hypertensive patients, ET-l has been shown to potentiate sympathetically
mediated venoconsctriction (87).

No studies have been performed to directly measure splanchnic NE release in
vivo to evaluate the effect of ETB receptor blockade on sympathetic input to the
splanchnic bed in DOCA-salt hypertensive rats. In my study, I treated rats with an ETB

receptor antagonist and measured aortic and portal venous plasma catecholamine levels.

Experimental Procedures: The selective ETB receptor antagonist, A192621 was given
(12 mg/kg, Abbott Laboratories, IL) via a femoral venous catheter to conscious DOCA-
salt and sham rats at least 4 days after recovery from catheterization as described earlier.
Blood samples were collected simultaneously from the arterial and portal venous
catheters 30 min after drug treatment. Plasma NE, EPI levels were measured using REA.

NHS spillover of NE was estimated.

Results: As I saw in my previous studies, A192621 signiﬁcantly increased the MAP in
conscious sham, but not conscious DOCA—salt rats (Fig. 38). A192621 caused a
signiﬁcant increase in the arterial plasma NE level in sham rats. A192621 tended to
decrease the arterial plasma NE level in DOCA-salt rats, however, the decrease was not
signiﬁcant (Fig. 39). The ETB receptor antagonist didn’t cause any signiﬁcant change in

the estimated NHS NE spillover in either DOCA-salt or sham rats (Fig. 40).

153

ETB antagonist treatment caused an approximately 5-7 fold increase in both the
aortic and portal venous plasma EPI levels in sham rats (Fig. 41). No signiﬁcant change

in either aortic or portal venous plasma EPI level was observed in DOCA-salt rats.

154

- sham (n=7)
I: DOCA (n=7)

 

 

 

  

 

 

 

 

 

 

 

 

 

 

250
a
E J
LS, 200 .. T
a
a. 150-
3 £
a
g 1oo~
t
to
g 50«
<1)
5
0

control ETB' antagonist

Figure 38: Mean arterial pressure responses to i.v. bolus A192621 administration in
conscious DOCA-salt and sham rats. Bars indicate mean arterial pressure before or after
selective ETB receptor antagonist treatment in conscious sham and DOCA-salt rats.
Brackets indicate SEM. Asterisk indicates a signiﬁcant difference (P<0.05) between
DOCA-salt and sham rats. £ indicates a signiﬁcant difference (P<0.05) between before

and after drug treatment.

155

- sham (n=8)
:1 DOCA (n=6)
60

 

500'

400' I

 

300
20d

1 00*

 

arterial NE cone. (pg/ml)

 

 

 

 

 

 

 

 

 

 

control ETB'antagonist

Figure 39: Effects of i.v. bolus A192621 administration on aortic plasma NE level in
conscious DOCA-salt and sham rats. Bars indicate mean arterial plasma NE
concentration before or aﬁer selective ETB receptor antagonist treatment in conscious
sham and DOCA-salt rats. Brackets indicate SEM. Asterisk indicates a signiﬁcant

difference (P<0.05) before and after drug treatment.

156

_ Sham (n=8)
[:3 DOCA (n=5)

 

 

 

 

 

A250

.9

E

320m

5 l
>

(_3 ,
3150-

CD

LIJ

Z

a) 100- T
I

Z

'8

*5 50-

.§

:5

0 o

 

 

 

 

 

 

 

 

 

control ETB antagonist

Figure 40: Effects of i.v. bolus A192621 administration on estimated NHS spillover rate
of NE in conscious DOCA-salt and sham rats. Bars indicate mean estimated NHS NE
spillover rate before or after selective ETB receptor antagonist treatment in conscious

sham and DOCA-salt rats. Brackets indicate SEM.

157

_ AEPI (n=9)
1: PEPl (n=9)

1200

 

_L
O
O
O

 

on
O
O

600 .

400-

 

plasma EPI conc. (pg/ml)

200‘

o —l_"l

control ETB antagonist

 

 

 

 

 

 

 

Figure 41: Effects of i.v. bolus A192621 administration on plasma EPI level in conscious
sham rats. Bars indicate mean arterial plasma EPI concentration before or after selective
ETB receptor antagonist treatment in conscious sham and DOCA-salt rats. Brackets

indicate SEM. Asterisks indicate a signiﬁcant difference (P<0.05) before and after drug

treatment.

158

Discussion for Chapter 5-Part V

A192621 didn’t cause any signiﬁcant change in the arterial plasma NE level in
DOCA-salt rats as it did in sham rats, suggesting that the global inhibitory effect of ET-1
on sympathetic nerve activity via ETB receptors is impaired in DOCA-salt rats, which,
may consequently suggest a relative ‘facilitatory’ effect of ET-1 on SNA. ETB receptor
antagonist treatment didn’t cause any signiﬁcant change in the estimated NHS NE
spillover in either DOCA-salt or sham rats, suggesting the lack of an inhibitory effect of
ET-1 on SNA in the splanchnic bed.

It is noteworthy that the ETB antagonist caused an approximately 5-7 times
increase in both the aortic and portal venous plasma EPI levels in sham rats, suggesting a
strong inhibitory effect of ET-1 on adrenal medullary secretion of EPI. This inhibitory
effect of ET-1, however, is missing in DOCA-salt rats because ETB antagonist treatment
didn’t cause any signiﬁcant change in either aortic or portal venous plasma EPI levels.
The modulatory effect of ET-1 on adrenal medullary EPI release has been reported before
in control rats (123). The same group also found that infusion of exogenous ET-l into an
isolated, perfused adrenal gland preparation resulted in an increase in the basal release of
NE and EPI in DOCA rats, suggesting the functional integrity of this modulatory effect in
DOCA-salt rats. The difference between their result and mine may reside in the fact that

my studies were perfomed in whole animals.

159

Discussion for Chapter 5

Consistent with previous reports, I found increased SNA in DOCA-salt
hypertension. Two primary mechanisms may contribute to the increase in peripheral SNA
in DOCA-salt hypertension under resting conditions: 1) reduced tonic baroreﬂex
inhibition of ‘normal’ central SNS outﬂow; and 2) a primary increase in CNS-generated
sympathetic nerve discharge. Both arterial and cardiopulmonary baroreﬂexes tonically
inhibit central SNS outﬂow in humans (139). Thus one possibility is that this tonic
inhibition lessens in DOCA-salt hypertension, allowing progressively greater levels of
SNS activity to peripheral tissues (Rowe & Troen, 1980). It has been reported that the
arterial and/or cardiopulmonary baroreﬂex control of renal SNS activity was reduced in
DOCA-salt hypertension (230). In several clinical contexts, most notably cardiac failure
and essential hypertension, Esler and colleagues (62, 121) have demonstrated the possible
importance of projections of noradrenergic neurons to the forebrain in generating
elevated levels of peripheral SNS activity. Increased central nervous system NE turnover
(into the subcortical venous drainage) (reﬂecting increased noradrenergic activity) has
been reported in patients with primary hypertension. Increased overﬂow of endogenous
NE from paraventricular nucleus of the hypothalamus (PVH) was found in conscious
spontaneously hypertensive rats (178). However, both decreased hypothalamus NE
turnover (68) and unchanged overﬂow of endogenous NE from PVH nucleus (178) have
been reported in DOCA-salt rats, suggesting a reciprocal relationship between the
activities of central and peripheral noradrenergic ﬁbers (159). The reduced turnover of

NE in the hypothalamus may be secondary to the elevated blood pressure, reﬂecting an

160

attempt to decrease noradrenergic activity, a possibly compensatory change in actively
trying to counteract the rise in blood pressure in DOCA-salt rats (68).

My study is the ﬁrst to estimate NHS NE spillover in DOCA-salt hypertensive rats.
I found that the estimated NHS NE spillover was increased in DOCA-salt hypertensive
rats, suggesting increased splanchnic sympathetic nerve activity. This is of particular
importance as sympathetic nerve stimulation can enhance splanchnic venoconstriction,
reduce intestinal blood volume by up to 60%, leading to a signiﬁcant redistribution of
blood from the splanchnic veins. This change in volume distribution will have a profound
effect on overall cardiovascular functions including blood pressure (81) (65, 109).

The increased sympathetic nerve activity in DOCA-salt hypertension suggests that
there may be alterations in the local mechanisms that modulate sympathetic
neurotransmission. Possibilities include impaired neuronal reuptake, impaired
prejunctional (12-adrenoceptor function, facilitation of NE release by other neurohumoral
factors such as EPI, and ET-1. 1 designed a series of in vivo studies to test these
possibilities and found that there was impaired prej unctional (12-adrenoceptor function in
the splanchnic vasculature of DOCA-salt rats. This would serve to augment the amount
of NE released per unit sympathetic nerve discharge. ET-l has an inhibitory effect on
systemic NE release in sham rats. That inhibitory effect is missing in DOCA-salt rats
systemically. My results concerning the neuronal uptake activity in the splanchnic bed
are hard to explain because of the inﬂuence of the central sympathoinhibitory effect of
the neuroal uptake inhibitor I used on the regional sympathetic ouﬂow. However, the

result may also reﬂect that there is little neuronal uptake occuring in the splanchnic bed.

161

My studies have been focused on the sympathoadrenal system under basal (resting)
conditions. Experiments aimed at determining the functional integrity of NET and
prejunctional 0.2-adrenoceptor inhibition mechanism of DOCA-salt hypertension in face
of hyperactivation of the sympathoadrenal system (by stimuli such as haemorrhage) can
be performed in the future. These observations will be important considering that the
NET activity and the negative feedback mechanism are probably maximally activated
during the reﬂex hyperactivation of the sympathetic nervous system. These experiments
should help us gain insight into the augmented sympathoadrenal adjustments to acute
stress (e. g. haemorrhage) and possible DOCA-salt hypertension- related differences in
regional SNS regulatory mechanisms as well as epinephrine secretion from the adrenal
medulla compared with sham rats.

Plasma concentration of NE is commonly used as an index of SNA. Several
important caveats limit the validity of plasma NE levels as an index of sympathetic
“tone”. Multiple factors determine the proportionality between the rate of neuronal
release of NE and plasma NE concentration. Plasma NE concentration is determined by
both the rate of diffusion of NE from the various nerve junctions into the plasma (the
apparent release rate or spillover rate) and the rate of removal (clearance) from the
plasma by the processes of uptake, metabolism and urinary excretion. Thus, a rise in
plasma NE concentration could result from either an increase in spillover rate with no
change in clearance or a decrease in clearance with no change in spillover rate. In like
fashion, a decrease in plasma NE concentration could result from a decrease in spillover
rate and/or an increase in clearance. For example, normal circulating catecholarnines

were found in the presence of markedly increased muscular sympathetic nerve activity in

162

pulmonary arterial hypertension (246), further underscoring their limited usefulness as
indicators of sympathetic nervous system activity.

My estimate of NHS spillover of NE provides a better index of splanchnic regional
sympathetic nerve function. However, it is still an estimated value based on the two
assumptions mentioned in General Methods. One assumption is that there is no EPI
release from the splanchnic bed and the fractional extraction of NE equals that of EPI.
However, it has been reported by Esler et a1 (56) that at rest, epinephrine was released
from the heart in older men despite the fact that adrenal medullary secretion of
epinephrine was reduced. For this reason, I am developing the radiotracer methods
described by Esler et a1 (1979) (52) for the measurement of NE clearance and spillover
rate. Since both clearance of NE from the bloodstream and the rate of NE entry into the
bloodstream (spillover) determine plasma NE levels (spillover = clearance X arterial
plasma NE level), Esler et al used the speciﬁc activity (SA) of plasma [3 H] NE measured
during intravenous infusion of [3 H] NE to estimated NE clearance from and NE spillover
into the systemic circulation. Increased dilution of the tracer with endogenous NE from
inﬂowing to outﬂowing plasma reﬂects local release of transmitter and provides the basis
of NE spillover measurements (Fig. 42). I also did preliminary studies to test the second
assumption (Portal venous blood ﬂow is the same in sham and DOCA-salt rats) on which
my estimated NHS spillover in previous studies was based. I found that there was no
signiﬁcant difference in the portal blood ﬂow between DOCA-salt (18.6 i 3.56 ml/min,
n=3) and sham rats (25.78 :1: 3.13 m1/min, n=3), which was consistent with pusblished

results (97) (264), suggesting that DOCA treatment didn’t signiﬁcantly change the portal

163

blood ﬂow, and the increase in NHS spillover in DOCA-salt rats was not due to
decreased portal blood ﬂow.

In the future, radiotracer methods can be used in conscious chronically tethered rats
with different drug interventions to provide a more precise assessment of NHS spillover
of NE and a better guide to overall sympathetic nervous tone in the splanchnic bed of
DOCA-salt hypertensive rats. However, successful measurements of various plasma NE
metabolites (DHPG, MHPG) require separation and quantiﬁcation techniques feasible for

small volume samples, which is still a challenge for our group.

164

 

 

 

 

 

 

 

 

J’HNE __ [>3

H'NE extraction

Figure 42: Schematic of splanchnic processing of tritiated NE (3H NE). The majority of
[3 H] NE is removed from plasma via a clearance mechanism that involves neuronal
uptake. Within sympathetic nerves, [3 H] NE is metabolized into [3H]DHPG by
monoamine oxidase (MAO), with some subsequent release into the venous circulation

(192).

165

CHAPTER 6

PRELIMINARY STUDIES ON SYMPATHETIC NEUROTRANSMISSION AND

ENDOTHELIN-l IN THE SPLANCHNIC BED OF DOCA-SALT RATS IN THE

EARLY DEVELOPMENTAL STAGE

166

Introduction

The pathogenesis of essential hypertension is still uncertain, but genetic factors and
the sympathetic nervous system are likely to be involved. Sympathetic nerve activity and
hormonal circulatory control mechanisms, however, are affected by blood pressure itself.
Hence, early functional changes are best investigated in normotensive subjects at risk to
develop hypertension, such as normotensive offspring of hypertensive parents.

Neural adrenergic factors have long been hypothesized to be important in the
initiation of high blood pressure. Higher level of plasma NE (134) (102) and overall rate
of spillover of NE to plasma (61) have been found in offspring of hypertensive parents.
Although resting muscle sympathetic nerve activity (measured by microneurography)
appears to be normal (86), more pronounced increases in muscle sympathetic nerve
activity and blood pressure in response to mental stress was noticed in offspring of
hypertensive than in offspring of normotensive parents (164). The possible association
between predisposition to essential hypertension, plasma NE level and two
polymorphisms of the gene for tyrosine hydroxylase (Tetranucleotide TCAT repeat and
ValSlMet polymorphisms) were studied and the studied TH polymorphisms do not
appear to be associated with a family history of essential hypertension (102). However,
decreased baroreceptor sensitivity has been found in offsprings of hypertensive parents
(169).

To investigate the pathogenesis of DOCA-salt hypertension, I used DOCA-salt rats
in the early developmental stage (4-5 days). Fewer studies have been done compared with
human subjects. However, it has been found that central attenuation of baroreﬂex

precedes the development of hypertension in DOCA-salt treated rats (231).

167

Chapter 6 - Part I
NE release and Preiunctional aZ-Adrenoceptor function in the sympathetic nerves
associated with the splanchnic vasculature of conscious DOCA-salt rats in the early

developmental stage.

Rationale: Venous capacitance is reduced in established DOCA-salt hypertension, but it
is uncertain whether this is a cause or a consequence of hypertension. It has been found
that the baseline radial arterial distensibility and compliance were comparable in
offspring of hypertensive and normotensive parents (32). However, there were also
studies showing that there was increased carotid arterial stiffness in children with a
parental history of hypertension (148) and decreased brachial arterial compliance in still-
normotensive offspring of hypertensive parents (15). When the ﬁrnction of veins in the
early stage of hypertension was examined, venous distensibility was found to be
decreased in normotensive young men with hypertensive relatives (101). Previous studies
in my lab showed that MCFP is increased early (before blood pressure rose) in DOCA-
salt hypertension, and it is not due to intravascular blood volume expansion. This
indicates that structural and/or functional vascular alterations precede a distinct rise in
blood pressure, suggesting that venous changes could be a cause of elevated blood
pressure. Pharmacological studies in the early stage of hypertension in DOCA-salt rats
(7-14 days after DOCA implantation) demonstrated an increased MCFP dependent on
both endogenous ET-1 and sympathetic neurogenic venoconstriction. This suggests that
ET-1 and SNS contribute to increased venoconstriction early in the development of
DOCA-salt hypertension. The mechanisms are still unclear. No studies about the

reactivity to NE of blood vessles from early DOCA-salt hypertensive rats had been

168

performed. In this study I tested the hypotheses that 1) the enhanced sympathetic nerve
mediated venoconstriction occurring early during the development of DOCA-salt
hypertension could be attributable to increased NE release from the splanchnic bed. 2)

Impaired prejunctional (12-adrenoceptor function contributes to this increased NE release.

Experimental Procedures: All rats used in this study were uninephrectomized and
drank 1% NaCl/0.2% KCl solution throughout the protocol. After sodium pentobarbital
anesthesia (50 mg/kg, i.p.), silicone rubber tipped catheters were placed into abdominal
aorta via a femoral artery and portal vein via a small branch of the portal vein after
laparotomy. The third catheter was inserted into a femoral vein. I set up the rats as
introduced in the General Methods. After at least frny days of recovery from anesthesia
and surgical stress, hemodynamic measurements (HR, SBP, DBP, MAP) were made on
two control days (C1 and C2). After measurements were completed on day C2, rats either
received a DOCA patch (200mg/kg, s.c. DOCA) or sham implant surgery (SHAM) under
methohexital sodium (Brevital sodium) anesthesia (9 mg/kg, i.v.). Blood samples were
collected from arterial and portal venous catheters on the 4th day (A4) after DOCA or
sham surgery. On the 5th day (A5), rats were treated with prejunctional (12-adrenoceptor
antagonist yohimbine (0.5 mg/kg, i.v.) and arterial and portal venous blood samples were
taken 30 min after drug treatment. Plasma NE and EPI levels were measured using a
radioenzymatic assay and net NHS spillover of NE was estimated. The reason that I was
interested in what happened in rats 4 and 5 days after DOCA or sham implant surgery

was that based on my previous studies, the MAP stared to increase on the 4th day after

169

DOCA implantation. My goal was to evaluate the change in sympathetic

neurotransmission accompanying the increase in BP.

Results: I found that the mean arterial pressure was signiﬁcantly higher in DOCA-salt
rats (122.0 i 2.5 mmHg) at 4 days than sham rats of the same age (113.2 in 3.9 mmHg)
(Fig. 43). There was no signiﬁcant difference in the arterial plasma NE concentration
(Fig. 44) (365 :l: 40 pg/ml in sham; 411 i 110 pg/ml in DOCA) and the estimated NHS
NE spillover (Fig. 45) (119 i 39 pg/min in sham; 64 d: 29 pg/min in DOCA) between 4-
day sham and DOCA-salt rats.

Yohimbine caused a signiﬁcant decrease in the MAP in DOCA-salt (Fig. 43)
(from 120 d: 4 to 104.2 i 4 mmHg) but not sham rats, which was similar to what I saw in
established DOCA-salt and sham rats. Although yohimbine signiﬁcantly increased the
arterial plasma NE concentration only in sham rats (Fig. 46) (from 365 :l: 40 to 1113 :l:
210 pg/ml), it caused a signiﬁcant increase in the estimated NHS NE spillover in both
groups of rats (Fig. 47) (from 119 i 39 to 499 i 83 pg/min in sham, from 65 :t 33 to 326

at 51 pg/min in DOCA).

170

 

150

 

—0— sham (n=7)

‘40 ‘ —o— DOCA (n=8)

 

 

 

130 <
120 .
110 ~
100 ~
90 ~

mean arterial pressure (mmHg)

 

 

80‘

 

C1 C2 A4 A5 yohimbine

Figure 43: Mean arterial pressure of conscious early DOCA-salt and sham rats and MAP
response to i.v. bolus yohimbine administration. Dots indicate mean arterial pressure in
control days, 4 and 5 days after DOCA implantation (sham surgery) and MAP response
to yohimbine treatment. Brackets indicate SEM. # indicates a signiﬁcant difference
(P<0.05) between sham and DOCA-salt rats. Asterisk indicates a signiﬁcant difference

(P<0.05) before and after drug treatment.

171

800

 

 

 

 

 

 

E _ sham (n=7)
B: :3 DOCA (n=8)
9: 600 -
o
I:
8 l
uzJ 4oo~
m
E
w
r“;
o 200 ‘1
'13
o
co
0

 

 

 

 

 

 

 

Figure 44: Aortic and portal venous plasma ET-l level in conscious early DOCA-salt and
sham rats. Bars indicate mean arterial plasma NE concentration in conscious 4-day sham

and DOCA-salt rats. Brackets indicate SEM.

172

 

 

 

 

 

 

,5 200
E — sham (n=7)
3 160 _ [:1 DOCA (n=8)
53
>
g
E 120 .
{0
ur
Z
(I) 80 j I
I
z ,
8
*5 40 -
.§
8
0 .

 

 

 

 

 

 

 

Figure 45: Estimated nonhepatic splanchnic spillover rate of NE in conscious early
DOCA-salt and sham rats. Bars indicate meanestimated NHS NE spillover rate in

conscious 4-day sham and DOCA-salt rats. Brackets indicate SEM.

173

 

1600

 

 

 

 

 

 

 

 

 

 

 

 

Lg - Sham (n=7) *
\ [:1 DOCA(n=8)
53,1200 -
o’
C
8
LIJ 800 -
z
(B
E
3
.2
t
8 I
O .

control yohimbine

Figure 46: Effects of i.v. bolus yohimbine administration on aortic plasma NE level in
conscious early DOCA-salt and sham rats. Bars indicate mean arterial plasma NE
concentration before or after yohimbine treatment in conscious early sham and DOCA-
salt rats. Brackets indicate SEM. Asterisk indicates a signiﬁcant difference (P<0.05)

between before and after drug treatment.

174

 

on
O
O

 

 

 

 

 

 

E

E

\g — sham (n=7)

T: 600 . C: DOCA (n=8) o
2%

'a'. .
m

LIJ 400 -

Z

(I)

I

Z

'8 200 —

E i

a 0 ﬂ

 

 

 

   

 

 

 

control yohimbine

Figure 47: Effects of i.v. bolus yohimbine administration on estimated NHS spillover rate
of NE in conscious early DOCA-salt and sham rats. Bars indicate mean estimated NHS
NE spillover rate before or after yohimbine treatment in conscious early sham and
DOCA-salt rats. Brackets indicate SEM. Asterisks indicate a signiﬁcant difference

(P<0.05) between before and after drug treatment.

175

Discussion for Chapter 6-Part I
My studies suggest unchanged SNA both globally and in the splanchnic bed of
DOCA-salt rats at 4-day. Although yohimbine signiﬁcantly increased the arterial plasma
NE concentration only in sham rats, it caused a signiﬁcant increase in the estimated NHS
NE spillover in both groups of rats, suggesting that the function of 0.2-adenoceptor,
although unchanged in the splanchnic bed, is impaired systemically.

A hyperreactivity of the SNS has long been suspected in the development of
hypertension, but most studies have yielded inconsistent results. Besides the numerous
studies showing increased SNA mentioned in the Introduction, there are also studies
suggesting that there is no enhanced sympathetic activity or heightened cardiovascular
responsiveness among normotensive young adults who are farnilially predisposed to
essential hypertension (141). In addition to methodological problems, it is likely that the
activity of the SNS changes during the natural history of high blood pressure. For
example, as suggested by Noll et al. (165), it is likely that in normotensive individuals
with a genetic background for hypertension, muscle sympathetic nerve activity is
increased only in response to factors such as stress, becomes increased even under
unstimulated conditions in borderline hypertension, and eventually returns to normal
levels in established hypertension. Therefore, on the basis of my results and those of the
literature, it is possible that in the early stage of DOCA-salt hypertension, splanchnic
sympathetic nerve activity is normal under basal condition, maybe increased only in
response to factors such as stress, and becomes increased even under unstimulated

conditions in established hypertension.

176

Therefore, although my studies didn’t ﬁnd increased sympathetic nerve activity
either globally or in the splanchnic bed under baseline conditions, further studies testing
the response of SNS to certain stimuli should be performed, as dysfunction of this system
id more likely to occur during stress. It was found (165) that the activity of the
sympathetic nervous system and plasma norepinephrine levels were increased during
mental stress only in offspring of hypertensive parents, whereas the response to hypoxia
was similar in offspring of hypertensive and normotensive parents, suggesting an
abnormal regulation of the sympathetic nervous system to certain stressful stimuli in
offspring of hypertensive parents. On the basis of this study, the response of SNS to

various stimuli may need to be tested in the future.

177

Chapter 6 — Part II
Net NHS release of ET-1 in conscious DOCA-salt rats in the early developmental

m

Rationale: Increased plasma ET-l production has been demonstrated in young,
untreated, essential hypertensive patients with no signs of target-organ damage,
suggesting that ET may play a role in the early phases of development of hypertension in
some subsets of patients (206). In normotensive offspring of hypertensive parents,
handgrip exercise produced increased ET-l plasma levels in offspring of hypertensive
parents and resulted in a sustained ET-l release into the bloodstream during recovery
compared with offspring of normotensive parents (232). Mental stress signiﬁcantly
increased plasma endothelin level in offspring of hypertensive parents (165).
Pharmacological studies in the early stage of hypertension in DOCA-salt rats (7-14 days
after DOCA implantation) by my group demonstrated an increased MCFP dependent on
both endogenous ET-1 and sympathetic neurogenic venoconstriction. There are few
studies on the mechanisms contributing to enhanced ET-l mediated venoconstriction in
early DOCA-salt rats. It was found that there was no signiﬁcant difference in the
reactivity to ET-l between DOCA-salt and sham small mesenteric veins (Dr. Galligan).
To test the possibility that higher endothelial formation of ET-1 contributes to increased
ET-l mediated venoconstriction, I performed preliminary studies to measure aortic and
portal venous plasma ET-l level and estimated net NHS release of ET-1 in rats 4th day
after DOCA or sham surgery. As I mentioned before, ET-1 is a locally released vascular
regulator that at very low concentrations enhances contractions to norepinephrine and at

higher concentrations has potent direct vasoconstrictor properties. In experimental

178

animals, endothelin stimulates sympathetic outﬂow. If as I concluded from my previous
studies that ET-1 increased venoconstriction indirectly in DOCA-salt rats by modulation
of SNA, alterations in the local release of ET-1 could alter the activity and vascular
effects of the SNS. I also treated rats with selective ETB receptor antagonist A192621 on
the 5th day to see if the impaired ETB receptor mediated plasma clearance of ET-1 occurs

early in the development of hypertension.

Experimental Procedures: Methods similar to those described earlier in Chapter 6-Part

I were used to assess net NHS ET-l release.

Results: I was not able to continue the experiment after I collected samples from three

DOCA-salt and sham rats because of the lack of supply of A192621.

179

General Conclusions and Discussion

The work covered by this thesis is part of the project funded by NIH Program
Project Grant which compares the different hemodynamic regulation of arteries and veins
and aims at proving the novel idea that functional and/or structural changes in veins,
especially small capacitance veins and venules are important in the pathogenesis of
DOCA-salt hypertension. I examined the regulation of veins because the venous system
is an important inﬂuence on cardiac output, and has been reported to be abnormal in early
hypertension (46).

My work focused mainly on identifying the mechanisms by which endothelin-1
and sympathetic nervous system increase venomotor activity in DOCA-salt hypertension
in in vivo whole animal level.

Results from present studies suggest that (corresponding to numbers in the bottom
half part of Fig. 48):

A. ET-l concentration around venous smooth muscle cells is not increased in
DOCA-salt hypertensive rats. Evidence includes: 1. venous vascular ET-l content is
not increased in DOCA-salt rats; venous preproET mRN A level is not increased in
DOCA-salt rats; 2. there is not a higher portal venous plasma ET-l level and higher net
NHS release of ET-1 in DOCA-salt rats despite impaired ETB receptor mediated plasma
clearance of ET-1; 3. ETA receptors play an important role in the plasma clearance of
ET-1 both globally and in the splanchnic bed. Although I have indirect evidence for more
ETA receptor binding in the splanchnic bed of DOCA-salt rats, currently there is no

direct evidence to support this. I also found impaired ETB receptor mediated plasma

180

 

clearance of ET-1 in DOCA splanchnic vasculature, shown as number 4 in Fig. 48.
However, the physiological signiﬁcance is not clear.

B. Systemic sympathetic nerve activity and sympathetic drive to the
splanchnic bed are increased in DOCA-salt hypertensive rats. Evidence includes: 1.
plasma NE levels (both aortic and portal venous plasma) are signiﬁcantly increased in
DOCA-salt rats; the estimated NHS spillover of NE is signiﬁcantly increased in DOCA-
salt rats.

C. There is alteration in local modulation of sympathetic neurotransmission
in DOCA-salt hypertensive rats. Evidence includes: prejunctional a2—adrenoceptors
function is impaired in the splanchnic bed of DOCA-salt rats (shown as number 2 in Fig.
48); the global inhibitory effect of ET-1 on sympathetic nerve activity via ETB receptors
is impaired in DOCA-salt rats (shown as number 3 in Fig. 48). My efforts to identify the
functional integrity of NET (shown as ? in Fig. 48) and the interaction between
prejunctional inhibitory autoreceptor and neuronal uptake (shown as ? in Fig. 48) were
complicated by the complex properties of the neuronal uptake inhibitor I used. No better
drug choice is available currently.

Collectively my results provide strong evidence that higher endothelial cell
formation of ET-1 is not likely responsible for enhanced ET-l mediated splanchnic
venoconstriction in DOCA-salt rats in viva. When combined with the fact that venous
smooth muscle contractile responsiveness to ET-1 is not increased in DOCA—salt rats, the
combined data suggest that the ability of ET receptor antagonists to reduce

venoconstriction in DOCA-salt rats in vivo results from actions of the drugs on a non-

181

vascular target. i.e. ET-l may exert indirect control of venous tone in vivo, for example,
via the modulation of sympathetic neurotransmission.

Despite increasing evidence for an important contribution of neurogenic
mechanisms to DOCA-salt hypertension, the precise causal mechanisms leading to
sympathetic augmentation in DOCA-salt hypertension remain unclear. The present study
provides direct evidence that there is increased splanchnic NE spillover and suggests that
impaired prejunctional (12-adrenoceptor function and inhibitory effect of ET-1 both
contribute to splanchnic sympathetic activation in hypertension. My ﬁnding of an
increased sympathetic activity and altered local modulation of sympathetic
neurotransmission was obtained from estimated NHS NE spillover. Further studies using
the radiotracer method will provide more precise estimate of sympathetic nerve activity
and therefore further test the notion that splanchnic sympathetic overactivity plays an
important role in the underlying causes of DOCA-salt hypertension.

It is worth mentioning that studies of the roles of endothelin-1 and sympathetic
nervous system in the regulation of venoconstriction in DOCA-salt rats are complicated
by the fact that the effects of these two systems on the arterial system and kidney also
contribute signiﬁcantly to the high blood pressure. It has been found by my group that
changes in venous ﬁrnction may play a particularly important role in a novel model of
experimental hypertension in rats produced by chronic intravenous inﬁrsion of the
selective ETB receptor agonist sarafotoxin 6C (S6C), as actions of S6C on arteries
(endothelium-mediated vasodilation) and the kidney (marked diuresis and natriuresis)

should produce hypotension intead of hypertension. Studies of the two systems in this

182

model may help better elucidate the underlying important roles of the two systems in
mediating venoconstriction in the development and maintenance of hypertension.

1 excluded the liver from my experiments because of the difﬁculty of sampling
blood from the hepatic venous drainage in the rat and the potential interference of hepatic
ET-1 and NE extraction with the measurement of splanchnic release. However, it is
important to note that liver is an important capacitance vascular bed. As I mentioned
before, while adrenergic venoconstriction occurs in a number of vascular beds,
splanchnic venules account for nearly 50% of the blood that is mobilized to the systemic
circulation. Within the splanchnic vascular bed, the hepatic venules/veins represent the
major smyce of blood mobilization. The hepatic venous system contains about the same
amount of blood as the venules/veins in skeletal muscle and twice the volume stored in
the intestine. However, hepatic venules/veins can mobilize nearly 65% of their stored
blood during sympathetic activation, while their counterparts in skeletal muscle can
displace less than 30% of the stored blood. Indeed, the hepatic venous system mobilizes
an amount of blood following sympathetic activation that is equivalent to the combined
contributions of the intestine and skeletal muscle. The greater sensitivity of hepatic
venules/veins to sympathetic activation reﬂects either more adrenergic innervation, a
higher density of adrenergic receptors, or both. Therefore, future studies may need to take

the liver into account.

183

Figure 48: Schematic representation of experimental results, explained in detail in

General Conclusion and Discussion.

184

sham

 

  

  

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

d . ””5"“? deaminated
nora renergrc I‘
nerveterminal \ W metaboutes 1 Blood
\ NE' pressure
is. 15A. 494 }
\'~ ( " 4; f Cardiac output
_ ‘4/ 1
3><:> VSMC W l
O)
8 . == 0 EC I Mean circulatory
3 "mm" 9 (5 i ﬂlllng pressure
ET 1/ uptake NVE J
' c ' BLOOD
t bol ﬂ
mea Ism ME I Venous return
DOCA-salt (SplalichnIC. out)
Mm” deaminated [ Venous volume
Venous
capacltanee
u... I
I Venoconstrlctlon
5. 1
g production ~ .3 . EC t vascular
_ﬁ 3 A _1 ET content
-' it 1/ "puﬁitve .
metabolism ‘ ' 2 '1 BLOOD

 

185

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