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ALTERATIONS OF SYMPATHETIC NEUROEFFECTOR MECHANISMS AND
THE UNDERLYING LOCAL MODULATORY MECHENISMS ASSOCIATED
WITH MESENTERIC ARTERIES AND VEINS IN DEOXYCORTICOSTERONE
ACETATE (DOCA)- SALT HYPERTENSIVE RATS

By

Min Luo

A DISSERTATION

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

DOCTOR OF PHILOSOPHY
Neuroscience Program

2003

Abstract

ALTERATIONS OF SYMPATHETIC NEUROEFFECTOR MECHANISMS AND
THE UNDERLYING LOCAL MODULATORY MECHENISMS ASSOCIATED
WITH MESENTERIC ARTERIES AND VEINS IN DEOXYCORTICOSTERONE
ACETATE (DOCA)- SALT HYPERTENSIVE RATS

By

Min Luo

The aim of my dissertation project was to test the hypothesis that alterations in
neurotransmitter release from sympathetic nerves and postsynaptic reactivity of
the vascular smooth muscle cells for the neurotransmitters are different in
arteries and veins from DOCA-salt hypertensive rats. These alterations are at
least partly due to the functional changes of the presynaptic aZ-adrenergic
receptors and to the effects of ET-1. The key ﬁndings of my research are as
follows:

1. The mechanisms underlying sympathetic neuroeffector transmission to
arteries and veins from normotensive rats are different as indicated by: i) different
neurotransmitters mediating sympathetic neuroeffector transmission to arteries
and veins. ii) Enhanced NE release relative to basal levels in veins compared to
anenes.

2. NE release is increased from sympathetic nerves to arteries and veins
from DOCA—salt hypertensive rats, which might be due to the impaired function of

presynaptic a2-adrenergic receptors and enhanced NE uptake activity.

The alterations in sympathetic neuroeffector mechanisms in arteries and veins in
DOCA-salt hypertension are also different as indicated by: i) In DOCA-salt
arteries, the presynaptic ETB receptors are activated which could also contribute
to the increased NE release. ii) In DOCA-salt veins, the postsynaptic ETa
receptors mediate a facilitatory effect on sympathetic neuroeffector transmission
which could contribute to the increased venomotor tone, whereas the facilitatory
effects mediated by postsynaptic ETA receptors observed in sham arteries were
removed in DOCA-salt arteries presumably due to the down-regulation of the ETA

receptors in DOCA-salt hypertension.

To my parents, (Pu-{Eng and' Quan-Qﬁng Luo for your uttermost love and' support

iv

Acknowledgments

This dissertation project was the outcome of my hard work and the input, advice,

and the assistance of many other people.

First, I wish to express my gratitude, respect and admiration for my mentor, Dr.
James J. Galligan. I thank Dr. Galligan for his support, understanding and his
patient training of me over the past four years. It was through his training I have
learned to independently write a manuscript, give a public presentation, design

an experiment and so on.

I next wish to thank and acknowledge the members of my dissertation guidance
committee: Drs. Greg Fink, David Kreulen, Keith Lookingland and Donna Wang. I
thank you all for your precious advice and endorsement for my dissertation
project. Particularly, I would like to thank Dr. Fink for providing me the
hypertensive and normotensive rats, Dr. Kreulen for teaching me to dissect the
inferior mesenteric ganglia and Dr. Lookingland for letting me use his HPLC

equipment and teaching me to conduct HPLC assays.

I would like to thank the Neuroscience Program for recruiting me and providing
me the excellent academic and extracurricular trainings. Particularly I would like
to thank Dr. Cheryl Sisk for supporting me over the last four years. I also thank
the American Heart Association Midwestern Afﬁliate for awarding me a pre-

doctoral fellowship (2001-2003).

Then, I thank Barb Grant from Fink’s lab for preparing most of the normotensive
and hypertensive rats, John Morris for helping me set up the HPLC equipment for
the ATP analysis and troubleshoot the problems I encountered, and Dr. Watts’s
lab for providing me some tissues from time to time. In addition, I would to thank
all those individuals who have helped me along the way. They are: my
colleagues in Galligan’s lab including the blood vessel group and the
gastrointestinal tract group, my classmates and friends: Jayme Mancini, Christina

Herden and James Otero.

I also would like to thank my American Friendship family: Hank Hanson and
Chris Boesen. I appreciate their friendship, support and endeavors to enrich my

experience of living in this country for the past years.

Finally I would like to acknowledge my husband Xiaopeng Li: without you, it

would be impossible for me to achieve this goal, and my parents Pu-Eng and

Guan-Ying Luo for their utterrnost love and support.

vi

TABLE OF CONTENTS

LIST OF TABLES ........................................................................... xiii
LIST OF FIGURES ............................................................................ xvii
LIST OF ABBREVIATIONS ................................................................. xxiv
CHAPTER 1: GENERAL INTRODUCTION ................................................ 1
1. Hypertension ................................................................................. 1
1.1 Essential vs. Secondary hypertension ....................................... 1
1.2. Etiology of Essential hypertension ........................................... 2
2. Hemodynamics of blood pressure .................................................... 3
3. Regulation of blood pressure ........................................................... 4

4. The role of sympathetic nervous system in long-term control of blood

pressure ........................................................................................ 6

5. Treatments of hypertension in humans .............................................. 9
6. Experimental hypertension models ................................................... 12
6.1. Genetic hypertension models ................................................ 12

6.2. Renal models of experimental hypertension ............................. 14

6.3. Neural models of hypertension .............................................. 15

vii

7. DOCA-salt hypertension .................................................................. 15

7.1. Salt intake and salt sensitivity ................................................ 15
7.2. Etiology of salt-sensitive hypertension ..................................... 16
7.3. Etiology of DOCA-salt hypertension ........................................ 18
8. The mesenteric vascular bed ............................................................ 20
8.1. Arterial system ................................................................. 20
8.2. Venous system .............................................................. 24

8.3. Difference in structural and mechanical properties between arteries

and veins .......................................................................... 26

9. Sympathetic innervation of arteries and veins .................................... 28

9.1. Sympathetic nerves innervate both mesenteric arteries and

veins ............................................................................... 28

9.2. Differential control of the sympathetic contraction of mesenteric

arteries and veins ................................................................ 31

10. Sympathetic neuroeffector mechanisms in arteries and veins ............ 32
10.1. Adrenergic transmission ..................................................... 34

10.2. Purinergic transmission ...................................................... 39

10.3. Co-transmission in the sympathetic nervous system ................. 41

10.4. NPY ............................................................................... 43

viii

11. Size dependency of sympathetic control of mesenteric blood vessels.“
11.1. Differences in sympathetic innervation ................................. 44
11.2. Differences in the proportion of co-transmission ..................... 44
11.3. Differences in cellular mechanisms underlying the P2X receptor-

mediated contraction ......................................................... 45

12. Local modulatory mechanisms for sympathetic neuroeffector
transmission ..................................................................................... 46
12.1. PrejunctionalaZ-adrenergic receptors on sympathetic nerves
modulates sympathetic nerve activity ..................................... 46

12.2 Altered function of peripheral catecholamine transporters may be

involved in the disturbances of the autonomic nervous system in

hypertension ..................................................................... 48

12.3. ET—1 modulates sympathetic nerve activity .............................. 50

13. ET-1 and its receptor subtypes ........................................................ 50
13.1. Synthesis and bioactivity ...................................................... 50

13.2. Signal transduction mechanisms ............................................ 51

13.3. ET-1 and DOCA—salt hypertension .......................................... 52

13.4. ET—1 modulates sympathetic nerve activity ............................... 54

14. Summary of the general introduction and issues to be resolved ........ 55

ix

CHAPTER 2: HYPOTHESES AND SPECIFIC AIMS .................................. 59

CHAPTER 3: Differential alterations in sympathetic neuroeffector

mechanisms in mesenteric arteries and veins in DOCA-salt hypertensive

rats ................................................................................................... 61
Introduction ............................................................................... 62
Methods ................................................................................... 66
Results .................................................................................... 71
Discussion ................................................................................ 84

CHAPTER 4: Impaired function of a2-adrenergic receptors on sympathetic

nerves associated with mesenteric arteries and veins in DOCA-salt

hypertension ..................................................................................... 91
Introduction ............................................................................... 92
Methods .................................................................................... 96
Results .................................................................................... 101
Discussion ............................................................................... 1 12

CHAPTER 5: Modulatory effects of ET receptor activation on sympathetic
neuroeffector transmission to mesenteric arteries and veins in DOCA-salt
hypertension .................................................................................... 1 18

Introduction .............................................................................. 1 19

Methods .................................................................................. 1 24

Results .................................................................................... 130
Discussion .............................................................................. 147
CHAPTER 6: GENERAL DISCUSSION AND CONCLUSIONS ................... 153

1. Differential sympathetic neuroeffector transmission to arteries and veins
from normotensive rats ...................................................................... 154
1.1 Different neurotransmitters mediating sympathetic neuroeffector
transmission to small mesenteric arteries and veins ................... 154

1.2. Enhanced NE release relative to basal levels in veins compared to
arteries ............................................................................. 155

1.3. Veins are more sensitive to nerve stimulation than arteries ........... 156

2. Alterations in sympathetic neuroeffector mechanisms in arteries and
veins in DOCA-salt hypertension ........................................................ 160
2.1. Alterations in sympathetic neuroeffector mechanisms in arteries in
DOCA—salt hypertension ....................................................... 160

2.2. Alterations in sympathetic neuroeffector mechanisms in veins in
DOCA—salt hypertension ....................................................... 164

2.3. Sympathetic nerve activity is increased in both arteries and veins in

DOCA—salt hypertension ....................................................... 167

xi

2.4. Differences in alterations of sympathetic neuroeffector transmission to

arteries and veins in DOCA-salt hypertension ........................... 169

3. Alterations in local modulatory mechanisms of sympathetic
neuroeffector transmission to arteries and veins in DOCA-salt hypertensive
rats ................................................................................................. 170
3.1. Alterations in prejunctional a2-adrenergic autoreceptors in arteries

and veins in DOCA—salt hypertensive rats ................................ 170

3.2. Alterations in modulatory effects mediated by ET receptors in arteries

and veins from DOCA—salt hypertensive rats ............................. 175

References .................................................................................... 187

xii

LIST OF TABLES

Table 3.1: Maximum response (Emax) and half maximum stimulation frequency

Table 3.2:

($50) in arteries and veins from sham and DOCA—salt rats. All data

are expressed as the mean 1 S.E.M and “n” values refer to the

number of animals from which the data were obtained. Windicates

significantly different from the $50 in sham and DOCA-salt veins (P <

0.05). #indicates signiﬁcantly different from the Emax in DOCA-salt
arteries and DOCA-salt veins (P<0.05) ..................................... 76

Emax and ECso values of the concentration-response curves for NE
and PE in arteries from sham and DOCA-salt rats. All data are
expressed as the mean _+_ S.E.M and “n” values refer to the number
of animals from which the data were obtained. There were no
difference in Emax and E050 between sham and DOCA—salt

arteries ............................................................................... 77

Table 3.3: Duration (seconds) of neurogenic responses caused by 5 and 10 Hz

stimulation of veins in the absence (Control) and presence of cocaine
(10 pM) and corticosterone (10 pM). All data are expressed as the
mean _t S.E.M and “n” values refer to the number of animals from

which the data were obtained. 'indicates signiﬁcantly different from

xiii

the duration in control sham and DOCA—salt veins at the same

stimulation frequencies (P < 0.05) ............................................ 78

Table 4.1: ATP released is not altered in DOCA-salt arteries. Data are the basal
ATP release, nerve stimulation-evoked release (stimulated release)
and evoked release normalized to basal levels (normalized release)
during the third stimulation period in sham and DOCA—salt arteries.
*indicates signiﬁcantly different from basal release (P < 0.05). There
were no differences between basal release and normalized release
between sham and DOCA—salt arteries. “1” is the correlation
coefﬁcient for the relationship between basal and stimulated release.

Both correlation coefﬁcients are different from zero (P < 0.05). . ....105

Table 4.2: NE release from pen-arterial and pen-venous nerves is increased in
DOCA—salt hypertension. Data are basal NE release, nerve
stimulation-evoked release (stimulated release), and evoked release
normalized to basal levels (normalized release) in sham and DOCA-
salt arteries & veins. * indicates signiﬁcantly different from basal
release (P < 0.05). # indicates signiﬁcantly different from normalized
release in sham counterparts (P < 0.05). sindicates signiﬁcantly
different from release in sham artery (P<0.05). “r” is the correlation
coefﬁcient for the relationship between basal and stimulated release.

All correlation coefﬁcients are different from zero (P < 0.05).........106

xiv

Table 5.1: Contractions caused by nerve stimulation and exogenous ATP from
sham & DOCA—salt arteries before and after the application of ET-1
(3 pM). All data are expressed as the mean 1 S.E.M and “n” values

refer to the number of animals from which the data were obtained.
*indicates signiﬁcantly different from the EC50 in control sham

arteries (p<0.05). ’indicates signiﬁcantly different from the Emax in

control sham arteries (p<0.05) ............................................... 142

Table 5.2: Contractions caused by exogenous ATP and NE from sham & DOCA-
salt arteries before and after the application of ET-1 (300 pM). All

data are expressed as the mean 1 S.E.M and “n” values refer to the

number of animals from which the data were obtained. *indicates

signiﬁcantly different from the EC50 in control sham arteries (p<0.05).
9 indicates signiﬁcantly different from the EC50 in control sham

arteries (p<0.05). ‘indicates signiﬁcantly different from the Emax in

control sham arteries (p<0.05) ............................................... 143

Table 5.3: Contractions caused by nerve stimulation from sham & DOCA-salt
arteries before and after the application of 860 (30 pM and 100 pM).
All data are expressed as the mean 1 S.E.M and “n” values refer to

the number of animals from which the data were obtained. 9

XV

indicates signiﬁcantly different from the 850 in control DOCA—salt

arteries (p<0.05) ................................................................. 144

Table 5.4: Responses to nerve stimulation and to exogenous NE from sham &
DOCA—salt veins before and after the application of ET-1 (3 pM). All
data are expressed as the mean _+_ S.E.M and “n” values refer to the
number of animals from which the data were obtained. *indicates

signiﬁcantly different from the EC50 in the control DOCA—salt veins

(p<0.05) ............................................................................ 145

Table 5.5: Responses to nerve stimulation and to exogenous NE from sham &
DOCA—salt veins before and after the application of $60 (30 pM). All
data are expressed as the mean 1 S.E.M and “n” values refer to the
number of animals from which the data were obtained. *indicates
signiﬁcantly different from the E050 in the control DOCA—salt veins

(p<0.05). 9 indicates signiﬁcantly different from the Emax in control

DOCA—salt veins (p<0.05) ..................................................... 146

xvi

Fig. 3.1:

LIST OF FIGURES

Comparison of frequency response curves for neurogenic contractions
of arteries and veins from sham and DOCA-salt rats. The frequency-
response curves from sham and DOCA—salt veins were shifted
signiﬁcantly to the left of those obtained from sham arteries. There was
no difference between the frequency-response curves from DOCA—salt

arteries, DOCA—salt veins and sham veins ................................... 79

Fig. 3.2: Contributions of a1-adrenergic and P2 receptor-mediated components

Fig. 3.3:

to neurogenic contractions of arteries from sham and DOCA-salt rats.
A: Frequency-response curves before and after sequential application
of prazosin and then PPADS in sham arteries. Prazosin alone had no
effect while PPADS greatly reduced contractions in sham arteries. B:
Frequency-response curves before and after sequential application of
prazosin and them PPADS in DOCA-salt arteries. Prazosin alone
greatly reduced contractions in DOCA-salt arteries while combined
application of prazosin and PPADS completely blocked the neurogenic

responses ............................................................................. 80

Frequency-response curves for neurogenic contractions in sham and
DOCA-salt veins before and after prazosin (0.1 pM) treatment.
Prazosin blocked the contraction caused by nerve stimulation in both

sham and DOCA—salt veins ....................................................... 81

xvii

Fig. 3.4: Comparison of contractions caused by adrenergic receptor agonists in
arteries from sham and DOCA-salt rats. Concentration-response
curves for NE from sham and DOCA—salt arteries were not different.
Concentration-response curves for PE were similar in arteries from

DOCA-salt and sham rats ......................................................... 82

Fig. 3.5: Concentration-response curves for UK 14,304 in arteries from sham and
DOCA-salt rats. UK14, 304 did not contract mesenteric arteries,
suggesting that org-adrenergic receptors are not expressed by

mesenteric blood vessels ......................................................... 83

Fig.4.1: Stimulation-dependent increase in ATP release from sham and DOCA-
salt arteries. ATP release caused by consecutive stimulations was
normalized to basal levels in arteries. Bars labeled Stim1, Stim 2, Stim
3, and Stim 4 represent release caused by the ﬁrst, second, third and
fourth stimulation periods respectively. In sham (A) and DOCA—salt
arteries (B), Stim1 and Stim2 were not different from each other and
Stim3 and Stim4 were not different from each other. * indicates
signiﬁcantly different from Stim2 (P < 0.05). Data are presented as
mean + SE and were analyzed by one-way analysis of variance with

Tukey—Kramer multiple comparisons test .................................... 107

xviii

Fig. 4.2: NE release was stable during consecutive periods of nerve stimulation.
NE release caused by consecutive stimulations was normalized to
basal levels in sham arteries (A), DOCA—salt arteries (B), sham veins
(C) and DOCA-salt veins (D). Bars labeled Stim1, Stim 2, and Stim 3
represent release caused by the ﬁrst, second, and third periods of
nerve stimulation, respectively. Stim1, Stim2, and Stim3 were not
different from each other in any preparation. Data are mean + SE and
were analyzed by one-way analysis of variance and Tukey-Kramer

multiple comparisons test ....................................................... 108

Fig. 4.3: a2-adrenergic receptors modulate ATP release in sham but not DOCA-
salt arteries. A: ATP release caused by nerve stimulation is compared
before and after yohimbine or UK 14,304 treatment in sham arteries.
*indicates signiﬁcantly different from release before yohimbine
treatment. #indicates signiﬁcantly different from release before UK
14,304 treatment. B: ATP release caused by nerve stimulation is
compared before and after yohimbine or UK 14,304 treatment in

DOCA—salt arteries. Data are presented as mean + SE ................ 109

Fig. 4.4A: NE release caused by nerve stimulation is potentiated by yohimbine

treatment in sham arteries. * indicates signiﬁcantly different from

release before yohimbine treatment. B: NE release caused by nerve

xix

stimulation was not altered by yohimbine treatment in DOCA—salt

arteries. Data are presented as mean + SE ............................... 110

Fig. 4. 5A: NE release caused by nerve stimulation is increased by yohimbine
and decreased by UK 14,304 in sham veins. * indicates signiﬁcantly
different from release before yohimbine treatment. # indicates
signiﬁcantly different from release before UK 14,304 treatment. B:
NE release caused by nerve stimulation was not altered by
yohimbine or UK 14,304 treatment in DOCA—salt veins. Data are

mean+SE ........................................................................ 111

Fig. 5.1A: Frequency-response curves caused by nerve stimulation from sham
arteries before and after the application of ET-1. Emax of the
frequency-response curves obtained after the application of ET-1 is
greater bigger than that obtained before the application of ET-1. B:
Frequency-response curves caused by nerve stimulation from
DOCA—salt arteries before and after the application of ET-1. There
was no difference in Emax and 850 between concentration-response
curves obtained before and after the application of ET-1. All data are
expressed as the mean + S.E.M and “n” values refer to the number of

animals from which the data were obtained .............................. 134

Fig. 5.2A: Frequency-response curves caused by nerve stimulation from sham

arteries before and after the application of ET-1. The Emax of the

XX

frequency-response curves obtained after the application of ET-1 is
greater compared with that obtained before the application of ET-1.
B: Frequency-response curves caused by nerve stimulation from
DOCA-salt arteries before and after the application of ET-1. There
was no difference in Emax and 850 between concentration-response
curves obtained before and after the application of ET-1. All data are
expressed as the mean + S.E.M and “n” values refer to the number

of animals from which the data were obtained ........................... 135

Fig. 5.3A: Frequency-response curves caused by nerve stimulation from sham
veins before and after the application of 86c. There was no
difference in Emax and $50 of the frequency-response curves
obtained before and after the application of 860. B: Frequency-
response curves caused by nerve stimulation from DOCA-salt veins
before and after the application of $60. The Emax of the frequency-
response curves obtained after the application of 860 is greater
compared with that obtained before the application of 86c. All data
are expressed as the mean + S.E.M and “n” values refer to the

number of animals from which the data were obtained ................ 136

Fig. 5.4A: Concentration-response curves for NE from sham veins before and

after the application of 86c. There was no difference in

concentration-response curves obtained before and after the

xxi

application of 86c. B: Concentration-response curves for NE from
DOCA-salt veins before and after the application of 86c. The ECso of
the concentration-response curves obtained after the application of
86c is smaller compared with that obtained before the application of
86c. All data are expressed as the mean + S.E.M and “n” values
refer to the number of animals from which the data were

obtained ........................................................................... 137

Fig. 5.5. Pictures taken from confocal ﬂuorescence microscopy showing ETB
receptors (Cy3 conjugated secondary antibody) and tyrosine
hydroxylase (FITC conjugated secondary antibody) are co-stained on
arteries from sham rats (A,B,C). Incubation with ETB receptor
antibody preincubated with control antigen (D) produces no staining
on the nerves ..................................................................... 138

Images in this dissertation are presented in color.

Fig. 5.6. Confocal images showing ETB receptors and tyrosine hydroxylase (TH)
are co-stained on arteries from DOCA—salt hypertensive rats (A, B,

C). Incubation with ETB receptor antibody preincubated with control

antigen produced no staining on the nerves (D) ........................ 139

Images in this dissertation are presented in color.

xxii

Fig. 5.7.

Fig. 5.8.

ETB receptors staining on veins from sham rats. The ETB receptor

staining at the smooth muscle cells (A) was different from TH staining

at sympathetic nerves (B). Omitting the TB receptor antibody (C) or
incubation with ETB receptor antibody preincubated with control

antigen (D) produces no staining on the smooth muscle cells.......140

Images in this dissertation are presented in color.

ETB receptors on veins from DOCA-salt rats. The ETB receptor

staining at the smooth muscle cells (A) was different from TH staining

on sympathetic nerves (B). Omitting the ETB receptor antibody (C) or
incubation with ETB receptor antibody preincubated with control

antigen (D) produced no staining on the smooth muscle cells ...... 141

Images in this dissertation are presented in color.

xxiii

on R
azR
ACE
CGRP
CNS
Cy3
DAG
DBH
DOCA
DOPA
EC50
EFS
EJC
EJP
Emax
ENaC
ET
FITC
HPLC

HR

LIST OF ABBREVIATIONS

a1-adrenergic receptor
a2-adrenergic receptor
Angiotensin Converting Enzyme
Calcitonin Gene-Related Peptide
Central Nervous System
lndocarbocyanine

sn1, 2-Diacylglycerol

Dopamine B—Hydroxylase
DeoxycortiCosterone Acetate
Dihydroxyphenylalanine
Half-maximal concentration
Electrical Field Stimulation
Excitatory Junction Current
Excitatory Junction Potential
Maximum Response

Epithelial Na+ channel
Endothelin

Fluorescein lsothiocyanate

High Pressure Liquid Chromatography

Heart Rate

xxiv

IMA
IMG
IMV
IP3
LDCV
MAO
MAP
MCFP
NGS
NE
NET
NLA
NO
NPY
OCT1
OCT2
OCTs
PBS
PE
P2R
RAS
RFLP

SCG

Inferior Mesenteric Artery
Inferior Mesenteric Ganglia
Inferior Mesenteric Vein
lnositol 1,4,5, -Triphosphate
Large Dense Core Vesicles
Monoamine Oxidase

Mean Arterial Pressure

Mean Circulatory Filling Pressure
Normal Goat Serum
Norepinephrine
Norepinephrine Transporter
AF-nitro—l-arginine

Nitric Oxide

Neuropeptide Y

Organic Cation Transporter 1
Organic Cation Transporter 2
Organic Cation Transporters
Phosphate Buffer Saline
Phenylephrine

P2 receptor
Renin-Angiotensin System
Restriction Fragment Length Polymorphism

Superior Cervical Ganglia

XXV

SDCV
SEJCs
3%
SHR
SNS

86a, 86b, SBc, 86d

TH
TPR
TTX
WKY
2K1C

1K1C

Small Dense Core Vesicles

Spontaneous Excitatory Junctional Currents
Half- maximal Frequency

Spontaneous Hypertensive Rat
Sympathetic Nervous System

Sarafotoxins 6a, Sarafotoxins 6b, Sarafotoxins 6c,
Sarafotoxins 6d

Tyrosine Hydroxylase

Total Peripheral Resistance

Tetrodotoxin

Wista r-Kyoto

Two-kidney, One clip

One-kidney, One clip

xxvi

Chapter 1

INTRODUCTION

1. Hypertension

Cardiovascular disease is the leading cause of death in western countries,
accountable for the annual death of nearly 1 million people in the United States.
Hypertension afﬂicts approximately 50 million Americans (Burt et al., 1995).
Although hypertension itself does not cause life-threatening symptoms, it is one
of the most important risk factors for cardiovascular diseases including
congestive heart failure, stroke, atherosCIerosis and renal failure (Burt et al.,
1995). These complications associated with hypertension are due to the fact that
its late stage hypertension can cause cardiac hypertrophy, endothelial

dysfunction and renal injury (Raij, 1998).

1. 1. Essentail vs. Secondary hypertension

Hypertension is a disease, which is difﬁcult to deﬁne. Nevertheless,
hypertension is a pathphysiological condition when systolic and diastolic
pressures are 3 140 mmHg and 90 mmHg, respectively (National Institute of
Health, 1997). One category of hypertension is secondary hypertension which
has identiﬁable causes such as endocrine disorders (aldosteronism), renal
diseases (renal failure, renal ﬁbrosis) and mechanical vascular abnormalities

(coarctation of the aorta). Hypertension of this kind can often be cured by

correcting its original cause (Lilly, 1993). 95% of hypertensive subjects fall into
the other category called essential hypertension which has unknown causes
(Lilly, 1993; Fink, 1998). Treatments mainly serve to control the elevation of
blood pressure rather than to cure the disease. The difﬁculty in ﬁnding the
causes for essential hypertension lies in the fact that essential hypertension is a
multifactorial disease with heterogenous etiology involving combinations of

abnormalities in the blood pressure regulating systems (Staessen et al., 2003).

1.2. Etiology of essential hypertension

There is considerable evidence suggesting that genetic factors play a role
in the incidence of essential hypertension. For example, the prevalence of
hypertension among the races is not evenly distributed with the higher incidence
in African Americans compared with Caucasian (Burt et al., 1995; Whelton et al.,
1994). There is also a familial aggregation of the disease demonstrated by the
signiﬁcantly higher rate of elevated blood pressure among the ﬁrst-degree
relatives of hypertensives than the general population and by greater
concordance between monozygotic twin than dizygotic twins (Burt et al., 1995;
Whelton et al., 1994). Genetic studies indicate that there are many genetic
alterations of the blood pressure controlling components (Hunt and Williams,
1994; Luft, 2002). The most established genetic alterations in the blood pressure
controlling components involve those regulating sodium and water balance such

as the epithelial Na+ channel (ENaC) of the renal collecting duct (Wamock,

1999) and kallikrein, the major enzyme for the synthesis of kinin which regulates
renal vascular tone (Katori and Majima, 1996).

It is generally believed that essential hypertension results from an
interaction of environmental and genetic factors (Hamet et al., 1998).
Environmental factors are the major contributors to modiﬁcation of genetic
inﬂuence as they can inﬂuence phenotype through the stimulation of gene
expression or via an interaction with gene products (Hamet et al., 1998). High
salt intake, stress and obesity, have been recognized to make an individual
susceptible to the development of hypertension or advance the clinical onset of
hypertension (Staessen et al., 2003). For example, a high salt intake alters the
expression of the renin gene, which ultimately will have an impact on blood
pressure level (Hamet et al., 1998). Furthermore, subjects with a family history
of hypertension have a greater increase in blood pressure when they are
exposed to mental and physical stresses than do subjects of normotensive
descent with matching basal blood pressure levels (Widgren et al., 1992).
Obesity, insulin resistance and dyslipidemia are metabolic conditions that appear
to be involved in the pathogenesis of essential hypertension as body weight and
blood pressure are often closely correlated based on the results from population

studies (Whyte, 1956; Harnet et al., 1998).

2. Hemodynamics of blood pressure
Arterial blood pressure is the product of total peripheral resistance (T PR)

and arterial blood volume. Peripheral resistance is mainly determined by arterial

tone whereas arterial blood volume is determined by cardiac output as well as
secretion of sodium and water (Guyton, 1991). Cardiac output is determined by
stroke volume and heart rate. While the heart provides the pumping pressure and
rate, venous return determines end-diastolic ﬁlling pressure and stroke volume
(Guyton, 1991). Venous return is determined by venomotor tone. Mean
circulatory ﬁlling pressure (MCFP) represents the pressure that drives venous
return and is an index of whole body integrated vascular tone and blood volume
(Yamamoto et al., 1980, Guyton, 1991 ).

As shown by hemodynamic studies on human and animals in the initial
stages of hypertension there is a high cardiac output and normal TPR (Lund-
Johansen, 1994). As the hypertension progresses to a chronic state, the
contribution to the rise of blood pressure by cardiac output is reduced whereas
that by TPR increases (Lund-Johansen, 1989; Coleman and Hall, 1993). Chronic
hypertension is therefore characterized by elevated resistance to blood ﬂow

through small arteries in most vascular beds (Folkow, 1982).

3. Regulation of blood pressure

Maintainance of normal blood pressure involves a complex interplay
among the nervous, endocrine, renal and cardiovascular systems.

The central nervous system controls blood pressure by adjusting the
autonomic outﬂow to the cardiovascular system. The paraventricular nucleus
(Kenney et al., 2003), area postrema (Hasser et al., 2000), rostral and caudal

ventral lateral medulla (Colombari et al., 2001) are involved through direct or

indirect connection with the autonomic nervous system. One well-known example
is the arterial baroreceptor reﬂex, a powerful and rapidly acting reﬂex serving as
a protective mechanism to maintain blood pressure homeostasis in the presence
of acute blood pressure changes (Dampney et al., 2002). The autonomic nervous
system directly regulates the cardiovascular system by primarily controlling
vascular tone and cardiac output (Dampney et al., 2002).

The kidney maintains sodium and water balance and controls the
intravascuclar blood volume through a mechanism called pressure-natriuresis
(McDonough et al., 2003). The kidney excretes more sodium and water when the
blood pressure rises so that blood volume is reduced and blood pressure returns
to the normal range (McDonough et al., 2003). Kidney function is regarded as a
long-term mechanism for blood pressure regulation (Hall et al., 1986). In
hypertension, pressure-natriuresis is desensitized and a greater rise of blood
pressure is required for kidney to execute this function (Guyton et al., 1972a).

There are various hormonal factors that act on the cardiovascular system
to regulate blood pressure including angiotensin II (Hall, 1991) and vasopressin
(Abboud et al., 1990). Angiotensin II causes vasoconstriction, aldosterone
release, sodium and water retention, and sympathoexcitation (Hall, 1991;
Ferrario, 1990). Vasopressin, also known as the antidiuretic hormone, causes
water retension (Abboud et al., 1990). The cardiovascular system can also
secrete a number of vasoactive factors such as endothelin (ET)—1, nitric oxide
(NO), and atrial naturetic peptide (ANP) (Shepherd and Katusic, 1991; Nicholls et

al., 1987; Luscher, 1990).

These above systems interact with one another, forming a delicate blood
pressure controlling mechanism. Hence, maintenance of blood pressure

homeostasis is an integrated function of those systems.

4. The role of sympathetic nervous system (SNS) In long-term control of
blood pressure.

There is little disagreement that hyperactivity of the SNS occurs in the
early stages of hypertension as indicated by elevated plasma NE concentration,
increased total body NE spill over and increased sympathetic nerve trafﬁc (Julius
et al., 1988; Esler et al., 1989; lzzo, 1998). Increased SNS activity causes
increased arterial and venous constriction with a resulting increase in systemic
blood pressure. For example, activation of a—adrenergic receptors causes
increased vascular resistance and volume retention (through renal
vasoconstriction). Meanwhile, activaton of B-adrenergic receptors causes
increased cardiac output and renin release that consequently activate the renin-
angiotensin system (RAS), a mechanism known to reinforce SNS activity
(Ferrario, 1990). Accordingly, early hypertension is characterized by increased
cardiac output as a result of hyperactivity of cardiac sympathetic nerves (Julius et
al., 1988). Therefore, it is generally agreed that hyperactivity of the SNS is a
critical initiating factor for hypertension development. However, whether the SNS
plays a substantial role in long-term blood pressure control in humans remains

controversial (lzzo, 1998).

One view holds that the SNS plays little if any role in maintaining elevated
BP chronically (lzzo, 1998). The notion that SNS hyperactivity observed in earlier
phases of hypertension disappears chronically is based on two observations: 1)
during established hypertension cardiac output is often not increased whereas
increased vascular resistance accompanied by structural changes is the hallmark
abnormality. Therefore, during the chronic phase, structural changes such as
vascular hypertrophy instead of hyperactivity of the SNS are the sustaining force
for maintenance of high vascular resistance. 2) The plasma NE level is often
normal in established hypertension. For example, only about 30% of
hypertensive patients displayed plasma NE values greater than those found in

the normotensive population (Grassi, 1998).

An opposite view holds that there is sustained activation of the SNS in
chronic hypertension, which plays an important role in maintaining high blood
pressure (Mark, 1996; Leenen, 1999; lzzo, 1998). This notion is based on clinical
observations suggesting that the true hallmark abnormality in hypertension is a
dynamic blend of high cardiac output and systemic vascular resistance (lzzo,
1998). Whether cardiac output or vascular resistance is predominantly elevated
is dependent on the relative proportion of B-receptors (primarily cardiac) and or-
receptors (primarily systemic vascular) stimulation. For example, when
hypertensives are in the supine position there is a predominant stimulation of or-
receptors and elevated systemic resistance is the prevalent abnormality.

However, when they are at an upright posture elevated cardiac output is the

prevalent abnormality (lzzo et al., 1990). A declining function of B-receptors with
aging is also suggested to contribute to a seemingly decreased or normal cardiac
output as hypertension progresses in elderly patients (lzzo, 1998).

Although there are some reports that plasma NE level is normal in
established hypertension, others reported that men with untreated sustained
essential hypertension for more than 5 years showed increased supine venous
and arterial plasma catecholamines as well as an increased difference between
these two levels, indicating an increased sympathetic nerve activity in chronic
essential hypertension (Kjeldsen et al., 1981). Furthermore, plasma NE is not
always an appropriate means to assess SNS activity in the setting of increased
systemic BP as the level of circulating NE depends on not only the release of NE
from the nerve terminals, but also on plasma clearance of NE (Grassi, 1998). In
studies using arteriolar dilators to normalize BP for 6-8 weeks in essential
hypertension, plasma NE doubled (lzzo et al., 1987). These results suggest that
SNS hyperactivty present in established hypertension has been suppressed by
the chronic BP elevation and was unmasked when BP was lowered to normal
levels (lzzo et al., 1987). Therefore, unaltered plasma NE level may not
necessarily mean that SNS activity is not increased.

There is also evidence for increased SNS activity to speciﬁc organs in
chronic hypertension. For example, elevated cardiac and renal sympathetic
activity was demonstrated in patients with essential hypertension (Esler et al.,
1989). Increased peripheral sympathetic nerve ﬁring rates to the leg muscles in

humans has also been demonstrated (Grassi, 1998). These observations support

an ongoing role of the SNS in chronic hypertension. The strongest evidence
supporting an important role of the SNS in chronic hypertension perhaps is the
consistent antihypertensive effect exerted by adrenergic receptor blockers (van
Zwieten, 1996). For example, in young hypertensives, B-blockade alone is
extremely effective (Frisk-Holmberg et al., 1981). Furthermore, central
sympatholytic drugs or combined or and B adrenergic receptor blockade lower BP

in all forms of early and late essential hypertension (Campese et al., 1980; lzzo,

1998)

5. Treatments of hypertension in humans

As mechanisms underlying hypertension development are poorly
understood, rather than curing hypertension, current therapeutic approaches
serve to lower blood pressure and hopefully prevent other cardiovascular
diseases secondary to hypertension.

There are six classes of drugs used to treat hypertension: 1) diuretics
represendted by thiazides. Diuretics are recommended to be the ﬁrst drugs of
choice for initial antihypertensive therapy due to the affordability and efﬁcacy of
these drugs in treating hypertension (Gavras et al., 1997). The hypotensive
effects of diuretics are attributable to blood volume depletion as a result of
decreased sodium—reabsorption and increased water secretion in the kidney.
However, the exact mechanism underlying the long-term antihypertensive effect
of diuretics is unknown (Pepper, 1999). Diuretics also cause a number of

metabolic side effects including hypokalemia, dehydration and hyponatremia

(Pepper, 1999). 2) Calcium channel blockers. By blocking calcium entry into the
cytoplasm of vascular and cardiac smooth muscle cells, calcium channel
blockers decrease vascular tone, cardiac contractility and cardiac electrical
conductivity, leading to decreased peripheral resistance and cardiac output and
hence decreased blood pressure (Pepper, 1999). There are two subclasses of
calcium channel blockers: dihydropyridines (e.g., nifedipine, nicardipine) and
nondihydropyridines (e.g., verapamil and diltiazem). Dihydropyridines are more
potent as vasodilators, whereas the nondihydropyridines are more noted for their
cardiac effects (Pepper, 1999). 3) Angiotensin converting enzyme (ACE)
inhibitors represented by captapril. ACE inhibitors block the production and thus
the various cardiovascular actions of angiotensin II including vasoconstriction,
sympathoexcitation, and sodium and water retention. Accordingly, ACE inhibitors
lower blood pressure by decreasing peripheral resistance and blood volume.
ACE inhibitors are effective as monotherapy in about 50 percent of unselected
patients (Gavras et al., 1997). 4) Peripherally-acting sympatholytics act at the
level of sympathetic neuroeffector junction to inhibit sympathetic neuroeffector
mechanisms. For example, guanethidine and reserpine were used to treat
hypertension by depleting vesicular stores of NE. 5) Direct vasodilators including
nitro-compounds and hydralazine (Gavras et al., 1997). Nitro-compounds cause
vasodilation by stimulating cellular guanylate cyclase and hydralazine cause
vasodilation by inhibiting pharmacomechanical coupling as well as causing
membrane hyperpolarization (Kreye, 1984). The side effects of vasodilators

include reﬂex tachycardia and ﬂuid retention (Gavras et al., 1997). 6) Adrenergic

10

receptor blockers. There are several subclasses that block different subtypes of
the adrenergic receptors as follows:

A: B-adrenergic receptor blockers. One of the mechanisms by which [3-
adrenergic receptor blockers lower blood pressure is that they decrease cardiac
contractility and thus the cardiac output (Koch, 1981). B-adrenergic receptor
blockers also decrease the release of renin from juxtaglomerular cells in the
kidney (Karlberg, 1983). Most of the adverse effects of these drugs are caused
by the non-selective blockade of B-adrenergic receptors in various organs. For
example, adverse effects related to [31 blockade in the heart include bradycardia,
congestive heart failure, and cardiac conduction abnormalities, whereas those
related to 8; blockade include bronchospasm and sexual dysfunction (Gavras et
al., 1997). B: Non-selective a-adrenergic receptor blockers. They lower blood
pressure by blocking vascular or adrenergic receptors mediating vasoconstriction.
As the feedback inhibition of NE release from sympathetic nerve terminals
mediated by the prejunctional a2-adrenergic receptors were also blocked by the
non-selective adrenergic receptor blockers, they permit excessive release of NE.
This is why phentolamine, a non-selective adrenergic blocker, causes
palpitations and tachycardia. Hence, non-selective adrenergic blockers are used
primarily in the management of hypertension secondary to pheochromocytoma
(Frishman et al., 1999). C: Selective a1-adrenergic receptor blockers. Prazosin is
one example of this class. Selective a1-adrenergic receptor blockers usually do
not produce tachycardia and palpitation as they do not interfere with the

negative-feedback mechanism that controls the release of NE (Ram and Kaplan,

11

1979). Therefore, the 1993 report of the Joint National Committee included the
selective a1-adrenergic receptor blockers as candidates for monotherapy
(Frohlich, 1993). However, these drugs can cause substantial adverse effects
such as dizziness, orthostatic hypotension and sodium and water retention
(Gavras et al., 1997). D: a2-adrenergic receptor agonists. Clonidine, the (12
adrenergic receptor agonist has been used as centrally acting sympatholytic to
decrease sympathetic tone as they promote the inhibitory effects mediated by
prejunctional a2-adrenergic receptors (Mathew and Parker, 1971). Mental
depression is among the side effects caused by this class of drugs (Gavras et al.,

1997)

6. Experimental hypertension models

A number of animal models of hypertension have been developed to
address the role of these various blood pressure regulating systems in the
development of hypertension. Since the different models have a different
etiology, it is conceivable that the choice of a model signiﬁcantly inﬂuences the

outcome of experiments done using these different models.

6. 1. Genetic hypertension models
Genetic models of hypertension are produced by selective inbreeding of
normotensive strains of animals. They are characterized by spontaneous

elevation of blood pressure with aging (Kurtz et al., 1994). The speciﬁc

12

abnormalities that initiate genetic hypertension are unclear (Bohr and Dominizak,
1991).

The Spontaneous Hypertensive Rat (SHR), generated from the Wistar—
Kyoto (VVKY) normontensive strain, is the most widely used genetic strain of
hypertensive animals (Okamato and Aoki, 1963). Despite that SHRs are
relatively resistant to changes in sodium intake in the developmental stages of
hypertension, blood pressure in SHRs in established hypertension is augmented
by salt loading (Yamori et al., 1981). There is also evidence for an increase in
MCFP and cardiac output in the early stage of hypertension development in
SHRs although sodium and extracellular ﬂuid volume are not elevated (T rippodo
et al., 1981; Martin et al., 1998). Futherrnore, increases in MCFP and TPR are
maintained at the established stage of hypertension development in SHRs
(Martin et al., 1998).

The other commonly studied genetic model of hypertension in rats is the
Dahl salt-sensitive strain. The Dahl salt— sensitive and corresponding salt-
resistant strains were developed from normotensive Sprague-Dawley rats. In
contrast to the SHR, both genetic and environmental factors are involved in the
development of hypertension in the Dahl salt-sensitive strain (Friedman, 1988).
For example, increased dietary sodium is required for the rapid and full
development of the blood pressure elevation in Dahl salt-sensitive rats
(Friedman, 1988). The most established genetic alteration in Dahl salt— sensitive
strain involves the renin gene. For example, a restriction fragment length

polymorphism (RFLP) in the renin gene was found between Dahl salt-sensitive

13

and Dahl salt-resistant rats. Furthermore, the renin RFLP cosegregated with
blood pressure as the dose of the renin allele from Dahl salt-sensitive rat directly

correlates with the increment in blood pressure (Rapp et al., 1989).

6. 2. Renal models of experimental hypertension

Hypertension based on renal etiology is the most common form of
human secondary hypertension (Lilly, 1993). Therefore, renal models of
experimental hypertension are relevant to studies of the basis for renal-
dependent human hypertension. Renal models of hypertension use blockade of
renal blood ﬂow via stenosis of the renal arteries and /or reduction of renal mass.
The two-kidney, one clip (2K1 C) and the one-kidney, one clip (1K1C)
renovascular models of hypertension are the most commonly used models.
Hypertension in this model is initiated by stenosis of the renal artery (Goldblatt et
al., 1934), which is performed by a placing silver clip around the renal artery
causing a partial occlusion of blood ﬂow. In 2K1C, the renal artery supplying one
kidney is clipped and the contralateral kidney is left intact. In the 1K1C, the renal
artery supplying one kidney is clipped and the contralateral kidney is removed.
Renal renin levels and peripheral renin activity in 2K1C are elevated indicating an
important role for the renin-angiontensin system in the development of this form
of hypertension. In the 1K1C model, renin levels and peripheral activity are
reduced as the source of renin production is reduced. In the 2K1C model of
hypertension, there is an elevation of blood pressure that depends on an

increased TPR with a reduction in cardiac output, while the 1K1C model shows

14

an increase in TPR with a small increase in cardiac output (Thurston, 1994). In
both models there is an elevation in MCFP (Yamamoto et al., 1981; Edmunds et

aL,1989)

6.3. Neural models of hypertension

The central nervous system (CNS) is involved in regulating blood pressure
and hence has been implicated in the pathophysiology of hypertension. For
example, the CNS is involved in DOCA-salt hypertension (Gomez Sanchez,
1991) Speciﬁcally neural models of experimental hypertension with a genetic
basis include the stroke-prone spontaneous hypertensive rat models. There are
also models that involve surgical or pharmacological manipulations of critical
CNS centers for blood pressure regulation such as the paraventricular nucleus,
nucleus tractus solitarus or the rostral ventral medulla (Reis, 1981). Each of
these manipulations is designed to address the role played by each brain center
in the regulation of blood pressure. Models focusing on the role of peripheral
nervous system in blood pressure regulation can be produced by sinoatrial
denervation to remove baroreceptor reﬂex (Trindade and Krieger, 1984) and
degeneration of peripheral sensory nerve ﬁbers to remove afferent inputs to the

nervous system (Wang et al., 1998).

7. DOCA-salt hypertension

7. 1 Salt intake and salt sensitivity

15

As noted before, environmental and genetic factors contribute to the
incidence of essential hypertension. Salt intake and salt sensitivities are
examples of the environmental and genetic factors associated with hypertension,
respectively. Epidemiological studies have demonstrated a positive correlation
between dietary sodium intake and the prevalence of hypertension. For example,
the prevalence of hypertension in primitive societies with very low dietary salt
intakes is low (Page et al., 1974). Furthermore, studies performed in the 1980’s
on humans suggested a threshold relation between the amount of salt intake and
the incidence of hypertension (lntersalt, 1988; Kaplan, 1993). Those societies
with daily sodium intakes of less than 60 mmol fail to show a rise in arterial
pressure with aging (Kaplan, 1993).

The incidence of hypertension in individuals challenged with high salt
intake also depends upon whether those individuals are salt— sensitive (lntersalt,
1988 and Kaplan, 1993). Salt sensitivity of blood pressure is deﬁned as a 10% or
greater increase in blood pressure after salt loading compared with the blood
pressure at the time of low-salt intake (Weinberger, 1993). Salt-sensitivity is more
prominent in older, black people as well as in those with high sympathetic
nervous system activity or renal insufﬁciency (Guyton et al., 1964; Weinberger,

1993)

7.2 Etiology of salt sensitive hypertension

16

The exact etiology of salt- sensitive hypertension is unknown although it
seems to involve abnormalities in renal, neural, hormonal and genetic
mechanisms controlling blood pressure.

The kidneys have been suggested to play a central role in the
pathogenesis of sensitivity of blood pressure to salt intake. Renal causes of salt-
sensitivity include defects in renal plasma ﬂow and increased tubular
reabsorption of sodium (Campese, 1994; Hamet et al., 1998).

Potential neural mechanisms for high salt sensitivity involve stimulation of
the SNS by high salt intake. Studies suggests that salt-sensitive people lack the
appropriate baroreceptor response in responding to increase in blood pressure
as high salt levels have been shown to sensitize the baroreﬂex in salt-resistant,
but not salt-sensitive patients (T rimarco et al., 1991). This lack of baroreceptor
response is likely to be accountable for the blood pressure elevation in those
salt-sensitive patients when challenged with high salt intake. Hormonal factors
like the ANP (Melo et al., 2000), kallikrein-kinin system (Katori et al., 2001) and
renal dopamine (Jose et al., 2003) have all been considered to play a role in the
salt-sensitivity of hypertension.

Genetic studies showed that the effect of salt intake on blood pressure is
mediated through interactions with speciﬁc genetic loci (Hamet et al., 1998).
Those loci implicated in the genetic control of salt sensitivity include the renin
gene on rat chromosome 13 and the Gca gene at chromosome 2 which codes for

ANP receptor A (Hamet et al., 1998).

17

 

7. 3. Etiology of DOCA- salt hypertension

DOCA-salt hypertension is a model in which hypertension is produced by
administration of high levels of a mineralocorticoid (DOCA) and salt intake
accompanied by surgical uninephrectomy (Schenk and McNeill, 1992). The
mineralocorticoid promotes sodium and water re-absorption in the collecting
tubule of the nephron (Schenk and McNeill, 1992). It acts on the type 1-
adrenococorticosteroid receptor and enhances the permeability of renal tubules
to sodium via an amiloride—sensitive Na*-H+ exchanger and activation of a
serosal sodium pump (Schenk and McNeill, 1992).

Sodium retention and a subsequent volume expansion may contribute to
the initiation of hypertension although this is not likely to be the sole mechanism
by which blood pressure rises (Schenk and McNeill, 1992). Rather, increased
sodium levels can alter neurohonnonal mechanisms to contribute to the initiation
and/or maintenance of hypertension (Ferrario et al., 1987).

Altered neural mechanisms could include an attenuated baroreceptor
reﬂex and increased peripheral autonomic discharge to the cardiovascular
system. Sympathetic drive to the cardiovascular system is elevated as indicated
by increased circulating catecholamine levels (Bouvier et al., 1986) and
sympathetic nerve responses to various stimuli in DOCA-salt hypertensive
animals (Takeda et al., 1988a). Central catecholaminergic depletion attenuated
DOCA-salt hypertension as indicated by a normalization of blood pressure in
hypertensive animals injected with 6-OHDA intracerebroventricularly (Lamprecht

et al., 1977). Sympathetic nerve ﬁber activity has been shown to be elevated in

18

DOCA-salt treated rats as reﬂected in an enhanced splanchnic sympathetic
nerve output in response to electrical stimulation of the hypothalamus (T akeda,
1988 3).

Hormonal mechanisms including Angiotensin II, vasopressin,
catecholamines and ET-1 involved in control of autonomic function via peripheral
or central actions are altered in DOCA-salt hypertension (Schenk et al., 1992;
Schiffrin, 1998). Activity of the brain RAS is increased, which could increase
peripheral sympathetic discharges and vasopressin release (ltaya et al., 1986).
Vascular reactivity to vasopressin has been implicated to be enhanced in DOCA-
salt treated animals (Lariviere et al., 1988).

In DOCA-salt hypertensive animals, cardiac output and stroke volume
were signiﬁcantly elevated without concomitant increases in heart rate (Ueno et
al., 1988). It is likely that increased cardiac output is due to an expanded blood
volume and an augmented venous return (Ueno et al., 1988). Other studies of
DOCA-salt hypertension in dogs attribute the elevation in arterial pressure to an
increase in both total peripheral resistance and cardiac output (Schenk and
McNeill, 1992). In addition, DOCA-salt hypertension causes characteristic
changes in vascular structure that contribute to the overall pathology of the
DOCA-salt state (Schenk and McNeill, 1992)

Thus, this model is relevant to salt-sensitive human hypertension and
permits studies of changes in neural and hormonal mechanisms secondary to

adrenal steroid excess (Schenk and McNeill, 1992).

19

8. The mesenteric vascular bed

The splanchnic circulation receives 25% of cardiac output and is the
largest capacitance bed (Folkow, 1982 and Greenway, 1983). It consists of the
blood supply to the gastrointestinal tract, liver, spleen, and pancreas. The
splanchnic circulation is a hemodynamically important vascular region for its
contribution to peripheral resistance and its reﬂex capacitance control to secure
cardiac ﬁlling (Daugirdas, 2001). There are three major branches coming from
the abdominal aorta that supply blood the visceral organs: celiac, superior
mesenteric and inferior mesenteric artery. The celiac artery supplies the
stomach, spleen, duodenum and the liver (Rosenblum et al., 1997). The superior
mesenteric artery is the largest of all the aortic branches and carries over 10% of
the cardiac output. It supplies the small intestine and part of the large intestine
(Rosenblum et al., 1997). The mesenteric vascular bed originating from the
superior mesenteric artery consists of arcades of blood vessels with a gradient of
vessel caliber in which the largest blood vessels is most distal to (ﬁrst order) and
the smallest is most proximal to the intestinal wall (third or fourth order). The
inferior mesenteric artery supplies the caudal part of large intestine (Rosenblum

etaL,1997)

8. 1. The arterial system
Arterial structure: The arterial wall consists of three layers: the
innermost intima layer, the media layer and the outermost adventitia layer. The

intima is comprised of a single layer of endothelial cells embedded in an

20

extracellular matrix (Burton, 1954). The endothelial cells serve a dual function in
the control of vascular tone. They release relaxing factors such as nitric oxide
and adenosine and constricting factors such as endothelin (Rossi et al., 2001).
The media consists of smooth muscle cells and elastic lamina (Burton, 1954).
Elastic tissue allows the wall of an artery to be distended by the sudden thrust of
blood from the heart and then to contract again, which helps force the blood
fonlvard. Therefore, the elastic tissue tends to smooth pressure pulses and
lessen the velocity of the blood. The smooth muscle cells are innervated by
sympathetic nerve terminals which can cause constrictions of the smooth muscle
cells (Nilsson, 1985). Constrictions of the smooth muscle decrease the vessel
caliber and thereby increase intravascular pressure. In addition, the blood
vessels receive dense innervation from sensory neurons located in the dorsal
root ganglia (Wang et al., 1998). Sensory nerves release the vasodilator
neuropeptides calcitonin gene-releated peptide (CGRP) and substance P in
response to local stimuli (Wang et al., 1998). The adventitia is the most variable
layer, containing primarily loose connective tissues, nerves, and small nutrient
blood vessels in the larger arteries (Burton, 1954; Levick, 2000).

The actual composition of each of these layers varies with the type of
blood vessels. Large, conduit arteries such as superior mesenteric artery (with
internal diameter around 1.2 mm) are typically referred to as elastic arteries
because of their high ratio of elastic lamina to smooth muscle cells (Burton, 1954;
Levick, 2000). As branches of superior mesenteric artery are traced distally, the

relative amount of elastic tissue decreases and the relative amount of smooth

21

muscle increases in the media layer. At the level of vessels with internal diameter
around 150-200 pm, the resistance to blood ﬂow starts to become prominent
(Burton, 1954; Levick, 2000). The smallest arteries arterioles (<100 pm) have a
media layer composed almost entirely of smooth muscle and are particulariy
contractile. It is through the activity of the arterioles that blood is shunted from
one part of the circulation to another and hence they are perhaps the most
important governors of the peripheral resistance to blood ﬂow and therefore of

systemic blood pressure (Schiffrin, 1992).

Structural changes associated with hypertension: Hypertension is
associated with altered structure and mechanical properties of resistance arteries
(Schiffrin, 1992). Structural changes in vascular smooth muscle cells in
hypertension take up two forms: remodeling and growth. Remodeling is a
rearrangement of normal sized cells whereas growth is either an increased
smooth muscle cell mass due to increased protein synthesis (hypertrophy) or cell
replication (hyperplasia). As remodeling involves rearranging of existing cells and
extracellular matrix around a lumen without invoking a growth response, it leads

to increased media to lumen ratio, but not increased media width or cross

sectional area (Heagerty et al., 1993). In essential hypertension, the increased
media / lumen ratio of small arteries due to remodeling could contribute to
increased vascular resistance (Folkow, 1982; Aalkjaer et al., 1987). Although
remodeling occurs in arteries from SHRs, growth due to hyperplasia appears to

play a large role in the alteration of vascular structure (Heagerty et al., 1993).

22

Whether the altered vascular structure is a cause or a consequence of the
increased blood pressure is not clear. On the one hand, thickening of the
vascular wall could be a secondary adaptation produced by the elevation of
blood pressure as such changes in large arteries can be prevented by
antihypertensive treatment. On the other hand, altered structure could contribute
to blood pressure elevation by increasing vascular resistance. Studies on
mesenteric and renal arteries of SHR showed that wall thickening of the
vasculature occur at prehypertensive stage and were present even under
conditions where the blood pressure has been normalized. These observations
suggest that some structural alterations in the blood vessels observed in
hypertension are blood pressure independent and could be of etiological
importance in the initiation of hypertension (Lee and Smeda, 1985).

In DOCA-salt hypertension, morphological changes of the resistance
arteries occur within 2 weeks of elevation of blood pressure. These structural
changes include narrowing of the lumen and external diameters, and increases in
media width and media cross-sectional area. These structural changes were
found to correlate with an increase ET-1 mRNA (Schiffrin et al., 1996). In
addition, treatment of DOCA-salt hypertensive rats with the combined ETA/ETB
endothelin antagonist bosentan caused a complete regression of vascular
hypertrophy. The effects by bosentan were much less marked in lowering blood
pressure. These results suggest that in DOCA-salt hypertension over expression
of vascular ET-1 produces vascular hypertrophy independently of blood pressure

elevation (Schiffrin et al., 1995).

23

Structural changes induced by ET-1 are likely to be initiated by
vasopressin. A number of studies indicate that vasopressin is required for DOCA-
salt treatment to exert its vascular effects and vasopressin acts in part by
enhancing ET production. For example, vasopressin receptor antagonism
attenuated DOCA-salt induced increases in media-lumen ratio and expression of
the prepro ET-1 gene (Intengan et al., 1998.). These ﬁndings were consistent
with abnormal vasopressin levels and function in this model. Furthermore,
DOCA-salt treatment in the absence of endogenous vasopressin, for example in
homozygous vasopressin-deﬁcient Brattleboro rats, failed to alter vascular
structure, ET expression, or wall component stiffness and resulted in less blood

pressure elevation (Intengan et al., 1999).

8. 2. Venous system

The venous system has a similar architecture to that of the arterial
system. However, compared with arteries, the vessel wall of veins is much
thinner with lesser connective tissue and less layers of smooth muscle (Burton,
1954; Levick, 2000). Hence, veins have a greater lumen diameter, a smaller
media width, and consequently a smaller media-lumen ratio compared with their
corresponding arteries (Burton, 1954; Levick, 2000). Due to its structural
properties the venous system provides a signiﬁcant capacitance function (Monos
et al., 1995). 60-80% of the circulating blood volume is found in the systemic
veins and venules, particularly vessels with a caliber <400 pm (Hainsworsth,

1986). Vascular capacitance is deﬁned as the change in intravascular pressure

24

as a result of a unit change in intravascular volume (Guyton et al., 1972b). As
pressure in the right atrial is provided by blood redistrubted from the venous
system to the heart, a reduction in venous capacitance will shift blood from the
peripheral vascular beds towards the heart and increase right atrial pressure.
According to the Frank-Starling mechanism, the increased right atrial pressure
results in increased end diastolic ﬁlling pressure, myocardial stretch and more
forceful constriction. In this way, augmented venous return leads to higher stroke
volume and cardiac output (Guyton et al., 1972b).

In the splanchnic circulation, vascular capacitance is a function of vein
structure and venous smooth muscle activity (venomotor tone). There are a
number of factors that can affect venomotor tone including myogenic activity,
circulating and locally produced vasoactive hormones and innervation of veins by
the sympathetic nervous system. It is generally believed that the sympathetic
nervous system is the most important factor to regulate venomotor tone as it has
a constant and the greatest quantitative regulation of venomotor tone, especially
in the critical splanchnic circulation (Monos et al., 1995). Venomotor tone can be
assessed by measurement of MCFP, the pressure that exists across the entire
circulation when the heart is stopped. MCFP represents the major driving force
for venous return to the heart (Johnson et al., 2001 ).

Capacitance of the venous system in animals with established
hypertension is decreased (Safar and London, 1985, 1987). A reduction in
venous capacitance has therefore been implicated in the etiology of

hypertension. In SHR, Goldblatt 1K1C renovascular hypertensive rats, and

25

humans with essential hypertension, venous tone is elevated early in
development of hypertension and remains elevated into established hypertension
(Safar and London, 1987; Schobel et al., 1993, Yamamoto, et al., 1981; Martin et
al., 1998). It is not clear, however, whether increases in venous tone are an
adaptive response to maintain cardiac output in the presence of increased
resistance for ventricular ejection (Folkow, 1982; safar and London, 1987) or an

active initiating factor participating in hypertension development.

8. 3. Differences in structural and mechanical properties between
arteries and veins

Arteries and veins serve distinct functions in the circulation, i.e. resistance
vs. capacitance (Burton, 1954; Levick, 2000). These different functions result
from differences in their structural properties. Arterial walls are comprised of
substantial layers of smooth muscle providing efﬁcient adjustment to blood
pressure changes. As the muscle layer of venous wall is thinner compared with
arteries, veins are more capable of accommodating changes in blood volume
(Burton, 1954; Levick, 2000). Morphological studies on canine mesenteric artery
and vein found that secondary arteries contain a greater number of smooth
muscle cells within the media and have 5 to 10 times thicker smooth muscle
layer compared to the corresponding veins (Yamboliev et al., 2002). Moreover,
there is a tighter arrangement of smooth muscle cells in arteries as the arterial
smooth muscle cells have smaller intercellular gaps compared to veins. This

tighter arrangement suggests that arterial walls have closer intercellular coupling

26

and more efﬁcient production of force during muscle constriction, which provides
a greater mechanical resistance to blood ﬂow compared to veins (Yamboliev et
al., 2002). As veins have less smooth muscle cell, the wall tension or the
maximal force generated by constriction is much less compared with arteries
(Nilsson, 1985).

Despite that there is more total protein in the smooth muscle layer of
arteries compared with veins, the fractions of major contractile proteins such as
actin, myosin heavy and light chains, and calponin were found to be similar in
mesenteric artery and vein (Yamboliev et al., 2002). The only differences
observed were as follows: the arteries express a higher amount of the actin-
binding protein caldesmon than the .vein and a isoform of tropomyosin
predominates in the artery while the [3 isoform predominates in the vein
(Yamboliev et al., 2002). Although it remains to be determined whether such
differences in those accessory proteins account for the different function of
arteries and veins, thicker and more tightly assembled smooth muscle layer
rather than the proﬁle of the major contractile proteins is the likely cause for a
higher mechanical potential of the mesenteric artery compared to vein
(Yamboliev et al., 2002).

Descending down the mesenteric tree, there is a gradient of decreasing
wall thickness and increasing density of sympathetic innervation in arteries, but
not veins (Nilsson, 1985; Yamboliev et al., 2002). Furthermore, as the artery
becomes smaller, the contractile response to single electrical stimulation

becomes more rapid (Nilsson, 1985). This difference reﬂecting a differential

27

organization in neurotransmission with regard to the vessel size was much less
marked among the veins (Nilsson, 1985). Hence, compared to the corresponding
arteries small veins tend to have slower twitches (Nilsson, 1985). This difference
in contractile properties of arteries and veins perhaps underlie their different

hemodynamic function e.g., resistance vs. capacitance.

9. Sympathetic innervation of arteries and veins

9.1. Sympathetic nerves innervate both mesenteric arteries and veins

Sympathetic nerves directly innervate the cardiovascular system and have
a profound control over its function. Sympathetic nerves arise from para and
preverterbral ganglia. The paravertebral ganglia, also termed the sympathetic
chain or trunk, are a series of ganglia which lie in a line lateral and parallel to the
vertebral bodies of the spinal column. The ganglia are interconnected to each
other and extend from the base of the skull to the sacrum. Paravertebral ganglia
supply the thoracic organs (lung and heart), skin, sweat glands, head, neck and
skeletal muscle. The prevertebral ganglia are associated with the unpaired
visceral arteries and located in front of the vertebral column and cemented on the
aorta. They are divided into the celia ganglion, superior mesenteric ganglion and
inferior mesenteric ganglion and they innervate the blood vessels in splanchnic
circulation (Hsieh et al., 2000). Studies using retrograde tracing identiﬁed the
ganglionic origins of sympathetic nerves innervating the mesenteric artery and

vein at the ileal-cecal junction in the order of decreasing importance as follows:

28

celiac, superior mesenteric, paravertebral and inferior mesenteric ganglia (Hsieh
et al., 2000).

Over the adventitial surface of the blood vessels, sympathetic nerves are
paravascular nerve bundles coursing along the longitudinal axis of the vessels.
Rather than innervating the blood vessels they travel along these nerve bundles
to reach and innervate other targets farther a long the blood vessels or to other
effector tissues. For example, paravascular nerves travel along mesenteric blood
vessels to supply the arterioles inside the gastrointestinal tract. Near the
adventitial-medial border nerve terminals are present as a perivascular plexus to
make close contact with vascular smooth muscle cells. This close neuromuscular
contact occurs at varicosities where nerve terminals are enlarged and
transmitters are stored. This is the site of sympathetic neuroeffctor transmission.
In mesenteric arteries and arterioles from rat and guinea pig, there is a close
apposition (<100 nm) between sympathetic varicosities and smooth muscle cells,
which demonstrates the ultrastructural morphology of a specialized
neuromuscular junction (Luff et al., 1987; Luff and Mclachlan, 1989). In terminal
arterioles with thin smooth muscle layer (1-2 smooth muscle cells) nearly every
smooth muscle cell is innervated by one varicosity. In larger arterioles and
arteries where the smooth muscle cell layer is thicker, only the smooth muscle
cells located at the adventitial/medial border are directly innervated (Luff et al.,
1987; Luff and Mc Lachlan, 1989). Smooth muscle cells on the blood vessels are
coupled through low resistance gap junctions, through which electrical and

chemical signals spread (Watts et al., 1994).

29

The specialized neuromuscular junctions were also observed in small
mesenteric veins of the guinea pig intestine with a similar frequency to that
occurring in mesenteric arteries (Klemm et al., 1993). The appearance and
arrangement of sympathetic nerves associated with arteries and veins are similar
although nerve density in veins is generally less than that in the adjacent arteries
(Nilsson, 1985). These structural data indicate that sympathetic nerves are
arranged and positioned to exert signiﬁcant control of the diameters of
mesenteric veins and arteries and arterioles.

Sympathetic nerves exert primary control over venomotor tone (Monos et
al., 1995). It is generally believed that sympathetic nerves have the greatest
quantitative importance in minute-to-minute regulation of venomotor tone
(Shoukas and Bohlen, 1990; Monos et al., 1995). Sympathetic nerve stimulation
can reduce intestinal blood volume by up to 60% leading to a signiﬁcant re-
distribution of blood from the splanchnic veins. In situ recordings of membrane
potential of mesenteric veins in anesthetized rats have shown that blockade of
neural input to the veins causes a membrane hyperpolarization (Lombard et al.,
1982), suggesting that there is tonic neural input causing smooth muscle
depolarization and constriction.

Sympathetic nerves also control arterial tone, particularly tone of the
small arteries (Nilsson, 1985). Nerve mediated constriction of arteries and veins
increases peripheral resistance (Ekas and Lokhandwala, 1980) and cardiac

output, respectively (Guyton et al., 1972b). Thus, by controlling the two functional

3O

parameters of blood pressure: peripheral resistance and cardiac output, the

sympathetic nervous system plays an important role in blood pressure regulation.

9. 2. Differential control of the sympathetic constriction of mesenteric
arteries and veins
As noted before, sympathetic innervation of arteries and veins is similar
with regard to the appearance and arrangement of the nerves on the blood
vessels. However, substantial evidence indicates that there is differential
sympathetic neural control of mesenteric arteries and veins. Firstly, studies
intended to determine the neuronal sources of sympathetic nerves innervating
the inferior mesenteric artery (IMA) and inferior mesenteric vein (IMV) suggested
that there are different groups of sympathetic neurons innervating arteries and
veins (Browning et al., 1999). The evidence includes: 1) retrogradely labeled
neurons were never found to project to both the IMA and the IMV (Browning et
al., 1999). Furthermore, neurons projecting to the IMA were located
predominantly in the central areas of the IMG whereas those supplying the IMV
were located more peripherally in IMG (Browning et al., 1999); 2) neurons
projecting to the IMV are larger than those to the IMA (Browning et al., 1999); 3)
although both groups of neurons ﬁre tonically, neurons projecting to the IMA
were more likely than those to the IMV to display inward rectiﬁcation when they
were hyperpolarized (Browning et al., 1999).
Secondly, the synaptic mechanism of neurotransmission may also differ

between arteries and veins. Although exogenous ATP and NE contract both

31

mesenteric arteries and veins, in studies using intracellular recording methods in
isolated mesenteric blood vessels from guinea pigs, it was shown that transmural
electrical stimulation of veins and arteries produced slow depolarizations
mediated by NE (Hottenstein and Kreulen, 1987). However in arteries, but not
veins, electrical stimulation evoked an excitatory junction potential (EJP, a short
latency and short duration membrane depolarization) mediated by ATP (Evans
and Surprenant, 1992).

Thirdly, veins are more sensitive to the vasoconstrictor effect of
sympathetic nerve stimulation than arteries as frequency-response curves for
nerve stimulation- induced constriction in veins are to the left of the curves from
arteries (Kreulen, 1986; Hottenstein and Kreulen, 1987). The threshold and half-
maximal frequencies for slow depolarization were also lower in veins compared
to arteries, which probably is due to the fact that resting membrane potentials of
venous smooth muscle cells are less negative than the arterial smooth muscle
cells (Kreulen, 1986). Therefore, venous smooth muscle cells are closer to
threshold for constrictions and it also may require less depolarization for veins to

contract than arteries.

10. Sympathetic neuroeffector mechanisms in arteries and veins
Sympathetic nerves control mesenteric vascular tone by releasing
vasoconstricting neurotransmitters to cause vascular constrictions. Upon the

propagation of action potential to nerve terminals a voltage-sensitive calcium

32

channel is activated to permit calcium entry, which initiates a cascade of events
eventually leading to the exocytosis of transmitters from synaptic vesicles (Fon
and Edwards, 2001). Sympathetic tone is reﬂected by the number of nerve
impulses traveling along the nerve terminals per unit time, namely the impulse
frequency. The higher the frequency, the more the transmitters will be released.
Studies investigating the discharge pattern of perivascular sympathetic nerve
ﬁbers in vivo have found that neuronal activity occurs in regular short bursts of
high frequency ﬁring rather than long sustained trains (Johnson and Gilbey,
1996). This result implies that electrical stimulation of sympathetic nerves with a
single train of relatively high frequency is more relevant to nerve impulses under
physiological conditions.

According to size and electron density of the core of the synaptic vesicles,
there are two major categories of synaptic vesicles in sympathetic nerve
terminals: large dense core vesicles (LDCV: external diameter 80-120 nm) and
small dense core vesicles (SDCV, some small vesicles may not have a dense
core). LDCVs mainly contain peptidergic neurotransmitters such as neuropeptide
Y (NPY) and are <20% of the vesicles in the nerve terminals (Stjarne, 2001).
Transmitter release from LDCV occurs at extrasynaptic sites and is triggered by
high frequency stimulation. SDCVs mainly contain NE and are > 80% of the
vesicles in the nerve terminals. Transmitter release from SDCV occurs at the
active zone of the prejunctional membrane of the nerve terminal and is triggered

by lower stimulation frequencies (Stjarne, 2001).

33

Sympathetic control of vascular tone also relies on the reactivity of the
postjunctional receptors for the transmitters. Reactivity of the postjunctional
receptors is determined by the number and binding afﬁnity of the receptors and
by their capacity to activate speciﬁc second messenger pathways (De

Champlain, 1990).

10. 1. Adrenergic transmission

Synthesis of NE: The ﬁrst step in the synthesis of NE is the conversion of
tyrosine to dihydroxyphenylalanine (DOPA) by tyrosine hydroxylase (TH)
(Thoenen, 1972). This is the rate-limiting step in the synthesis of NE. DOPA is
then decarboxylated to form dopamine. After taken up into the synaptic vesicles
by monoamine transporter, dopamine is converted to NE by side-chain
hydroxylation through the action of dopamine-B-hydroxylase (DBH). DBH is
localized within the synaptic vesicles where NE is stored and released via

exocytosis (Thoenen, 1972).

Metabolism of NE: Released NE is subject to several possible fates: 1)
metabolism including oxidative deamination by monoamine oxidase (MAO),
methylation by catechol o-methyltransferase and sulfoconjugation by phenol
sulfotransferase (Esler et al., 1990). 2) diffusion into the plasma. Of NE released
by sympathetic nerves throughout the body, perhaps 10-20% overﬂows into the
circulation (Esler et al., 1990). NE in the interstitial ﬂuid enters the circulation

passively by diffusion down a concentration gradient (Esler et al., 1990). 3)

34

reuptake into sympathetic nerves where it is either packaged into the synaptic

vesicles for re-release or metabolized by MAO (Esler et al., 1990).

Uptake of NE: There are two transporters for NE uptake with neuronal or
extraneuronal locations. NE uptake into the nerve terminals is performed by the
neuronal transporter, the so-called uptake-1 (Eisenhofer, 2001). The neuronal
transporter is located at the sympathetic nerve terminals and it has12 membrane-
spanning domains (Eisenhofer, 2001). Reuptake by this transporter is
responsible for rapid termination of NE action in the synaptic cleft, accounting for
almost 90% of the overall activity of the uptake system. Actions of this transporter
can be blocked by cocaine (Eisenhofer, 2001).

Extraneuronal NE transporters, the so-called uptake-2, are much more
diversely localized including vascular endothelium and smooth muscle cells. It
has been established that uptake-2 are organic cation transporters (OCTs),
including the classic corticosterone-sensitive transporter with a broad tissue
distribution and two other transporters (OCT1 and OCT2) localized to the liver,
kidney, and intestine (Eisenhofer, 2001). Important for clearance of NE from the
plasma, their activity is responsible for about 5% of total uptake (Eisenhofer,
2001). Hence, NE transporters (NET) function as part of integrated systems
where NE synthesis, release, uptake, and metabolism are regulated in a
coordinated fashion in response to the demands placed on the system (Esler et

aL,1988)

35

Assessment of sympathetic nerve activity: There are a number of
methods that have been used to assess sympathetic nerve activity. For example,
measurement of the changes in mean arterial pressure (MAP) and heart rate
(HR) after ganglionic blockade is used to estimate overall sympathetic nerve
activity (Deka-Starosta et al., 1989). Recording of renal or splanchnic nerve
activity provides a more direct measurement (Deka-Starosta et al., 1989). More
speciﬁcally, sympathetic nerve activity can be assessed at a regional level by
measurement of NE spill over rate (Esler et al., 1985) at an organ or tissue level
by measurement of NE content (Dae et al., 1989; Lorton, 1997), turnover rate
(T akahashi et al., 1993) and NE release using amperometry (Dugast et al.,
2002)

Tissue NE content generally reﬂects the steady state NE store in
sympathetic nerves. Decreased NE content suggests there is reduced NE store
that is due to an increased NE release. Therefore, tissue NE content is inversely
related to the activity of sympathetic nerves innervating the tissue (Lorton, 1997).
This is a simple one-time approach.

NE turnover rate can be measured by monitoring the decline of NE tissue
content over time after treatment with NE synthesis-blocking drugs, such as
or methylparatyrosine (McNamara and Murray, 2001). As NE synthesis is
stopped, NE content in the nerve terminals will only be affected by NE release.
Therefore, the rate of decline in NE content correlates with the transmitter
releasing activity of the sympathetic nerves. The faster the rates of decline in NE

content, the higher the sympathetic nerve activity (Takahashi et al., 1993).

36

NE released from sympathetic nerve terminals into the interstitial ﬂuid can
enter the venous circulation by diffusion down a concentration gradient. This is
called spillover of NE into circulation (Esler et al., 1988). NE spillover into the
venous circulation results in a higher plasma NE concentration in the venous
circulation compared to the arterial circulation. Hence, differences between these
two concentrations reﬂect the spillover rate. There are a number of factors that
can affect the spillover rate including regional blood ﬂow, activity of the uptake
systems, metabolism and diffusion ﬂow to the circulation. However, in general,
spill over rate is approximately proportional to the rate of sympathetic nerve ﬁring
(Esler et al., 1988). This relationship provides the experimental justiﬁcation for
the use of measures of regional NE spill over to plasma in the estimation of

regional sympathetic nervous activity (Esler et al., 1988).

Postjunctional action of NE: NE acts at metabotropic a— adrenergic
receptors on vascular smooth muscle cells to cause constriction. The
constrictions mediated by tar-adrenergic receptors are slowly developing and long
lasting as the receptors are G-protein coupled metabotropic receptors (Docherty,
1998). The intracellular mechanism involves the activation of Gq protein and
production of IP3 via a phosphoinositide pathway that leads to release of calcium
from the intracellular calcium store (Docherty, 1998).

There are two subtypes for a adrenergic receptors: a1 and a2 subtypes
(Docherty, 1998). The binding afﬁnities of NE for (11- adrenergic receptors were

much higher compared to a2- adrenergic receptors. For example, the binding

37

afﬁnities of dog mesenteric arteries and veins for a1-adrenergic receptors were
10 fold higher than for a2-adrenergic receptors (Shi et al., 1990). In addition, a
much higher density of a2-adrenergic compared to a1-adrenergic receptor
binding sites is required for a contractile response (Shi et al., 1990). A large
reserve for postjunctional vascular a1-adrenergic receptors, but not for a2-
adrenergic receptors might be accountable for the much higher maximum
responses produced by a1-adrenergic receptor agonists compared to a2-
adrenergic receptor agonists in canine saphenous vein (Ruffulo and Zeid, 1985).

In arteries, only a1-adrenergic receptors are functionally active, but in
veins a2-adrenergic receptors may also be functional. Ligand binding studies
demonstrated that both a1-and a2-adrenergic receptors are present on the
vascular smooth muscle cells of both mesenteric arteries and veins. However, in
mesenteric arteries, only a1-adrenergic receptors mediate NE- induced
constrictions. For example, prazosin, an a1-adrenergic receptor antagonist,
completely inhibited NE- induced constrictions in arteries (Shi et al., 1989).
Similarly, in isolated rat mesenteric resistance vessels prazosin was three orders
of magnitude more potent than yohimbine, the a2-adrenergic receptors
antagonist, in inhibiting NE-mediated calcium inﬂux, intracellular calcium release
and smooth muscle contraction in these vessels (Cauvin and Malik, 1984).
However, in mesenteric veins from dogs and cats, a2-adrenergic receptors also
mediate NE-induced constrictions. For example, dog mesenteric veins, but not
arteries responded to an a2-adrenergic receptor agonist (Shi et al., 1990). In

canine mesenteric veins, UK 14,304 and clonidine, the a2-adrenergic receptor

38

agonists, produced contractile responses (Shimamoto and Daniel, 1992; Shoji et
al., 1983). Phenylephrine (PE), an a1-adrenergic receptor agonist, was a more
potent agonist in the canine mesenteric artery than in the vein while UK 14,304
exhibited the opposite proﬁle of activity (Itoh and Rajfer, 1987).

Studies done on dog mesenteric artery and vein showed that the binding
afﬁnity for prazosin was lower in mesenteric veins compared with mesenteric
arteries whereas the maximum binding was similar, suggesting that the total
amount of a1-adrenergic receptors expressed on venous and arterial smooth
muscle cells is similar although the afﬁnity for the a1-adrenergic receptors is

lower in veins (Shi et al., 1989).

10. 2. Purinergic transmission

Metabolism: ATP plays an important role in sympathetic neuroeffector
transmission as a co-transmitter with NE. ATP is synthesized in mitochondria in
accordance with energy demands. Therefore, compared with NE synthesis, ATP
synthesis is perhaps much less tightly related to nerve activity. Hence, activity of
ATP as a neurotransmitter is mainly regulated by mechanisms responsible for
the termination of ATP actions. Nucleotidases that degrade ATP are a regulatory
mechanism for terminating the action of ATP as a neurotransmitter. There are at
least two kinds of nucleotidase mediating the degradation of ATP: membrane-
bound ecto-nucleotidase particulate and releasable nucleotidase soluble
(Westfall et al., 2002). Ecto-nucleotidase is localized at the nerve terminal

membrane and is constitutively active, whereas the release of soluble

39

nucleotidase is concomitant with ATP release and thus its action nerve activity-

dependent (Westfall et al., 2002).

Postjunctional action: ATP released from sympathetic nerve terminals
binds with its receptors localized at the smooth muscle cells to induce
constriction. Vascular receptors for ATP are P2 receptors, which are present on
both mesenteric artery and vein (Hirst and Jobling, 1989). There are two
subtypes for P2 receptors: P2X and P2Y. P2X receptors are ligand-gated
ionotropic receptors that are non-selectively permeable to cations including
sodium, potassium and calcium. P2X receptors mediate depolarization of smooth
muscle cells causing opening of voltage-dependent calcium channels and
provides an additional source of calcium inﬂux (Benham and Tsien, 1987). The
rise in the cytosolic level of calcium triggers the contraction of smooth muscle.
P2Y receptors are metabotropic G-protein coupled receptors, which mediate
constriction by inducing calcium release from intracellular calcium stores
(Benham and Tsien, 1987).

The receptor mechanism mediating ATP-induced contraction of smooth
muscle is different in arteries and veins. Constriction of the mesenteric artery
from rats caused by exogenously applied ATP was mediated by P2X receptors
(Galligan et al, 2001, Benham and Tsien, 1987, Gitterrnan and Evans, 2000),
whereas constrictions of the mesenteric vein from both rats (Galligan et al, 2001)

and guinea pigs (Mutafova-Yambolieva, 2000) were mediated by P2Y receptors.

40

10.3. Co-transmission in the sympathetic nervous system

ATP and NE are co-released from sympathetic nerves and mediate
vasoconstriction (Sneddon and Bumstock, 1984; von Kugelgen and Starke,
1985). The early evidence suggesting that ATP and NE are co-transmitters
released from sympathetic nerves was obtained from studies showing that
pretreatment with 6-hydroxydopamine to destroy sympathetic nerves abolished
the fast and slow phases of the constriction of the guinea pig vas deferens
mediated by ATP and NE, respectively (Fedan et al., 1981).

The notion that ATP and NE are stored and released from the same
synaptic vesicle originally came from studies showing that adrenal medulla
chromafﬁn cells released ATP and NEin a constant molar ratio indicating co-
release of ATP and NE from the same secretory granule (Todorov et al., 1996).
Studies of ATP and NE release from sympathetic nerve terminals innervating the
rat tail artery supported this notion. In those studies, NE release was measured
by continuous amperometry as well as by the slow depolarizations. ATP release
was measured by its postjunctional effects, EJPs. The evidence supporting co-
release includes: 1) activation of prejunctional B-adrenergic receptors increases
both electrically evoked ATP and NE release (Brock et al., 1997); 2) varying Ca2+
concentration and applying Ca2+ channel blockers results in similar effects on NE
and ATP release (Brock and Cunnane, 1999).

In contrast, studies on sympathetic neuroeffector transmission to the
smooth muscle cells of the guinea pig vas deference suggest that ATP and NE

are stored in separate synaptic vesicles and released via an independent

41

mechanism. Evidence supporting separate release mechanisms includes: 1) a
temporal disparity between ATP and NE release. The early transient twitch- like
constriction of the smooth muscle cells is mediated by ATP and reﬂects the early
and transient release of ATP, whereas the following tonic constriction represents
a later phase of NE release (Todorov et al., 1996). 2) Although prejunctional a2-
receptors seemed to regulate both ATP and NE release, they may be regulated
by different subsets of prejunctional a2-receptors as the reduction caused by 012
agonists, xylazine and clonidine, on ATP release was greater compared to that in
NE release (Westfall et al., 1996); 3) endogenously released NE had a greater
inﬂuence on its own release than on that of ATP as pretreatment with the 012
receptor antagonists idazoxan and yohimbine produced a substantial increase in
the overﬂow of NE and a lesser increase in the overﬂow of ATP (Westfall et al.,
1996); 4) NE release may be more dependent on calcium inﬂux through N-type
channels, whereas ATP release is coupled to calcium entry through P-type
channels. This conclusion is based on the results that the N-type calcium
channel antagonist omega-conotoxin reduced the electrical ﬁeld stimulation
(EFS)—evoked release of NE to a greater extent than ATP, while the P-type
calcium channel antagonist omega-agatoxin did the reverse (Westfall et al.,

1996)

ATP modulates NE release: In addition to its postjunctional action on

smooth muscle cells, ATP and its metabolites can modulate NE release by acting

at prejunctional P2 receptors on sympathetic nerve terminals. For example,

42

exogenous ATP enhanced synaptosomal NE release via P2X receptors and
inhibited NE release via P2Y receptors from cardiac sympathetic nerve terminals
(Sesti et al., 2002). ATP-induced release of NE depends on inﬂux of calcium from
the extracellular space through both voltage-gated calcium channels and

probably the P2X receptor itself (von Kugelgen et al., 1999).

10.4. NPY

It is known that NPY is localized in LDCVs of sympathetic nerve terminals
and can be released upon long duration nerve stimulation (Potter, 1988). Using
long trains of stimulation, NPY has also been shown to mediate part of the
contractile response to electrical ﬁeld stimulation (Phillips et al., 1998). However,
studies done by Gitterman showed that co-application of prazosin, the Q1-
adrenergic receptor antagonist and suramin, P2 receptor antagonist, abolished
neurogenic contractile responses in mesenteric artery of different sizes. These
results clearly demonstrated that NPY does not contribute to the constrictor
response in mesenteric vascular bed (Gitterman and Evans, 2001). Therefore,
NPY is more likely to function as a neuromodulator in mesenteric vascular beds

(Gitterman and Evans, 2001).

11. Size dependency of sympathetic control of mesenteric blood vessels

Comparative studies on sympathetic neuroeffector mechanisms in

mesenteric blood vessels with various sizes strongly suggest that there are size

43

related differences in several aspects of sympathetic neuroeffector transmission
including density of sympathetic innervation, proportion of ATP and NE
transmission and cellular mechanisms underiying ATP-induced smooth muscle

constriction.

1 1. 1. Difference in sympathetic innervation

In studies using TH and DBH to stain for the sympathetic nerves
innervating mesenteric blood vessels of different size it was found that as arteries
becomes smaller, the density of the sympathetic nerves increases (Nilsson,
1985). This gradient of sympathetic innervation may contribute to the differential
responses of those blood vessels to sympathetic nerve stimulation. For example,
responses to a single electrical stimulation are more rapid in the smaller artery
compared to the bigger artery (Nilsson, 1985). These differences were not
related to the properties of the vascular smooth muscle as responses to direct
electrical activation of the smooth muscle in all vessels were quite rapid.
Furthermore, the maximal neurogenic responses correlate with innervation

density, being greatest in the small arteries and least in the large arteries.

1 1.2. Difference in the proportion of co-transmission

It was shown that the relative roles of ATP and NE in mediating
sympathetic control of arterial tone in the rat mesenteric bed depend on the
diameter of the vessel. As the size of the blood vessel increases, the contribution

by ATP to sympathetic neuroeffector transmission decreases. For example, the

purinergic component of sympathetic transmission dominates in small-medium
arteries (3rd — 6th order) while constrictions in large arteries (1st order) are
almost entirely adrenergic (Gitterman and Evans, 2000). A similar proportion of
purinergic constriction has also been seen in other arteries when similar short
trains of stimulation were used (Evans and Cunnane, 1992; Warland and
Bumstock, 1987). The much smaller purinergic component of constriction in large
vessels is probably due to a lower density of sympathetic innervation in large
arteries (Luff and McLachlan, 1989) and a lower sensitivity of P2X receptors in

large mesenteric arteries (Gitterman and Evans, 2000).

1 1.3. Difference in cellular mechanisms underlying the P2X receptor
mediated smooth muscle contraction

Cellular mechanisms underlying P2X receptor-mediated constriction of
mesenteric arteries are different depending on the vessel size. In large arteries
P2X receptor mediated constriction is sensitive to the L-type calcium channel
blocker nifedipine (Bulloch et al., 1991; Omote et al., 1989). By contrast, in small
resistance arteries of the guinea-pig submucosa (Galligan et al., 1995) nifedipine
as well as cadmium, the non-selective calcium channel blocker, had no effect on
contractile responses mediated by P2 receptors. Furthermore, cyclopiazonic acid
which blocks calcium release from intracellular calcium store had no effect on
P2X receptor mediated responses in small arteries (Gitterman and Evans,
2000). These data suggest that for small arteries all the calcium required for

constriction enters directly though the P2X receptor channel whereas in large

45

arteries calcium entry through calcium channel and activation of intracellular
calcium stores are required for the P2X receptor mediated constriction

(Gitterman and Evans, 2000)

12. Local modulatory mechanisms for sympathetic neuroeffector

transmission

Sympathetic nerve activity is not exclusively dependent on impulse
frequency. As a matter of fact, transmitter-releasing activity of the sympathetic
nerves is under modulation by multiple autoreceptors and heteroreceptors
localized at the nerve terminal. These modulatory receptors can either enhance
or attenuate transmitter release during sympathetic stimulation (De Champlain,
1990). Together, these modulatory effects optimize sympathetic stimulation to
ensure that local sympathetic functions are integrated with those of other
circulating hormones under various conditions. Accordingly, altered sympathetic
drive in DOCA-salt hypertension could be due to altered local modulation on
sympathetic neuroeffector transmission to the vascular system (De Champlain,

1990)

12. 1. Prejunctional a2-adrenergic receptors on sympathetic nerves

modulate sympathetic nerve activity.

46

Prejunctional a2-adrenergic receptors located at the sympathetic nerve
terminals are autoreceptors. They are activated by released NE and inhibit
further NE release, thus mediating feedback inhibition of NE release (Langer,
1980). These receptors are metabotropic receptors coupled to a heterotrimeric
G-protein. Activation of prejunctional a2-adrenergic receptors causes
dissociation of the B, y subunits from the G-protein. The dissociated B, 7 subunits
interact directly with the pore-forming subunits of N- and PlQ-type calcium
channels, thus reducing the channels' open probability and inhibit calcium entry
into the cytoplasm. Inhibition of calcium entry eventually leads to inhibition of NE
release (Starke, 2001). Mechanisms other than modulation of calcium entry have
also been reported to underlie the inhibition of NE release by prejunctional a2-
adrenergic receptors. These potential mechanisms include regulation of
cytoplamic calcium homeostasis and interference with the transmitter releasing
process such as transporting and docking of the synaptic vesicles at the active
zone (Starke, 2001).

Studies on sympathetic neuroeffector transmission to guinea pig vas
deferens suggest that regulation of NE release by a2-adrenergic receptors is
frequency dependent. At lower frequencies (8 Hz), a2-adrenergic receptors
inhibit NE release and modulate the composition of the co-transmitters. As the
frequency of stimulation increases (above 8 Hz), the autoinhibition of the release
of NE is overridden and the amount of NE relative to ATP increases. Increased
release of NE caused an enhanced ATP mediated constriction via a

postjunctional mechanism. These results suggest that the prejunctional 012-

47

adrenergic receptors may function as a sensor that ”reads” the frequency of
action potentials and converts that information into discrete neurochemical
messages with varying proportions of co-transmitters. Some studies showed that
a2-adrenergic receptors also regulate ATP release (Westfall et al., 1996;
Bobalova et al., 2001a).

Increased sympathetic nerve activity in experimental hypertension
suggests that there are alterations in baroreﬂex function or in local mechanisms
modulating sympathetic neuroeffector transmission (De Champlain, 1990). Data
from several studies indicate that prejunctional a2-adrenergic receptor function is
impaired in hypertensive animals. For example, intravenously administered
yohimbine, an a2-adrenergic receptor antagonist, increased plasma NE levels in
sham normotensive, but not in DOCA-salt hypertensive rats (De Champlain,
1990). Furthermore, yohimbine potentiated nerve stimulation-evoked NE release
into the mesenteric vasculature in normotensive rats, but not in DOCA-salt
hypertensive rats (T suda et al., 1989). The increased NE release found in portal
vein and caudal artery of SHRs was due to a decrease in the functional activity of

prejunctional a2-adrenoceptor (Westfall et al., 1986).

12. 2. Altered function of peripheral catecholamine transporters may
be involved in disturbances of the autonomic nervous system In
hypertension (Eisenhofer, 2001).

In chronically hypertensive animals such as SHR, neuronal and

extraneuronal uptake of NE is enhanced in the blood vessel wall (Vanhoutte et

48

al., 1980). For example, the uptake of 3H-NE was greater in tail arteries from
SHR as compared to those from normotensive rats. Cocaine, an uptake-1
blocker, had a greater potentiating effect on responses to NE and electrical
stimulation in tail artery from adult SHR than in normotensive rats. The
magnitude of inhibition on uptake by cocaine was greater in SHR (Webb and
Vanhoutte, 1981). Furthermore, studies on young SHR showed that uptake of 3H
NE in tail arteries was signiﬁcantly higher compared to that in normotensive rats,
demonstrating an enhanced uptake of NE prior to the full development of
hypertension (Zsoter and Wolchinsky, 1983). Studies on mesenteric arteries (200
um) also showed that there was a 44% increase in neuronal NE uptake in
vessels from SHR in the established phaseof hypertension (Whall et al., 1980).
Studies on NE uptake in humans with essential hypertension yielded similar
results. For example, there was an enhanced leftward shift of the NE sensitivity
with cocaine in isolated subcutaneous resistance vessels from the hypertensives
suggesting an enhanced NE uptake in essential hypertension (Aalkjaer et al.,
1987). Studies on DOCA-salt hypertension, however, produced somewhat
different results. For example, there was no difference in uptake activity in
perfused caudal arteries between 3-week-DOCA-salt hypertensive rats and
normotensive rats (Longhurst et al., 1988). Yet, in vivo studies showed that
plasma NE level of DOCA-salt rats increased in the presence of neuronal uptake
blockade, clearly demonstrating that neuronal uptake of NE was not defective in

DOCA-salt hypertension (Drolet et al., 1989).

49

12.3. E T-1 modulates sympathetic nerve activity

In addition to changes in receptor subtypes and their responsiveness, some
neuromodulators including ET-1 may cause the alterations in sympathetic
neuroeffector mechanisms in DOCA-salt hypertensive animals (Rubanyi and

Polokoff, 1994).

13. ET-1 and its receptor subtypes

13.1 Synthesis and bioactivity

The endothelin family consists of three isoforrns of ETs: ET-1, ET-2 and
ET-3 and four sarafotoxins (86a, 86b, 86c and 86d, isolated from snake venom).
Endothelin converting enzyme-1, a metalloendopeptidease, cleaves the
precursor peptides of ET to form the ﬁnal 21 amino acid peptide, ET. ET-1
comprises 80% of total ET formation and is perhaps the most biologically active
member of the family. ET-1 in the vascular system is mainly synthesized and
secreted from the endothelial cells of the blood vessels.

Release of ET-1 appears to be directly connected to its de novo synthesis
as there is no evidence that ET-1 is stored in endothelial cells (Rubanyi and
Polokoff, 1994). The synthesis and activity of ET-1 is regulated by various factors
derived from endothelial cells and smooth muscle cells including vasoactive
substances, cytokines and growth factors (Rubanyi and Polokoff, 1994). For

example, in addition to inhibiting the activity of ET-1, endothelium-derived

50

vasodilator, NO, also inhibits production of ET-1. Smooth muscle cells also
release diffusible factors that suppress the production of ET-1 and/or proteases
associated with the degradation of ET-1 (Rubanyi and Polokoff, 1994).

Secretion of ET-1 is polarized as 80% of the total amount of ET-1 is
secreted toward the underlying smooth muscle cells (Rubanyi and Polokoff,
1994). The dynamics of synthesis and secretion of ET-1 are well-suited for ET-1
to be an important mediator of vasoconstriction. ET-1 produces prolonged
contractile responses. Because of its high vasoconstrictor potency and long-
Iasting actions, the continuous release of small amounts of ET could contribute to
the maintenance of vascular tone (Rubanyi and Polokoff, 1994). Other
cardiovascular effects of ET-1 include activation of both the SNS and RAS and

increased mitogenesis (Rubanyi and Polokoff, 1994).

13. 2. Signal transduction mechanisms

ET-1 causes constrictions of arteries and veins by binding with its
receptors on smooth muscle cells (Rubanyi and Polokoff, 1994). ET receptors
are found in the vascular wall, kidney, and heart, suggesting that ET-1 has a
widespread inﬂuence on cardiovascular function (Rubanyi and Polokoff, 1994).
There are two classes of receptors for ET-1 in the cardiovascular system. ETA
receptors are expressed by vascular smooth muscle cells and ETB receptors are
expressed by the endothelium and smooth muscle cells of some vascular beds

(Gellai et al., 1996; Rubanyi and Polokoff, 1994). The ETA and ETB receptor

51

subtypes are both G-protein coupled receptors with seven membrane-spanning
domains and a relatively long extracellular N terminal.

Activation of ET receptors on vascular smooth muscle cells stimulates
phospholipase C and thus the hydrolysis of phosphatidylinositol, which results in
the formation of inositol 1,4,5, -triphosphate (IP3) and SM, 2-diacylglycerol
(DAG). IP3 and DAG cause mobilization of calcium from intracellular calcium
stores. ET-1 also directly depolarizes the smooth muscle cells to activate
voltage-gated L-type calcium channels to permit calcium entry. Therefore, ET-1
induced vascular smooth muscle contraction involves the increase in cytoplasmic
calcium concentration by facilitaion of calcium inﬂux and mobilization of
intracellular calcium stores (Rubanyi and Polokoff, 1994).

ETB receptors in the endothelium mediate vasodilatation by stimulating
the production and release of vasodilator substances such as NO and
prostacyclin (Rubanyi and Polokoff, 1994). In veins and some large caliber
arteries, ETB receptors are expressed on the smooth muscle cells and mediate
vasoconstriction (Gellai, 1996; Johnson et al., 2002). Although the ETB receptors
expressed on the vascular smooth muscle cells are functionally different from
those expressed on the endothelium, no molecular distinction has ever been

made between these two receptors (Johnson et al., 1999).

13.3. E T-1 and DOCA-salt hypertension

Intravenous infusion of ET-1 causes elevation of systemic arterial blood

pressure in both humans and experimental animals (Mortensen and Fink, 1990;

52

Brunner, 1998). Blockade of ET receptors with acute intravenous administration
of the mixed ETA/ETB receptor antagonist TAK-O44 in normotensive subjects
caused a decrease in systemic blood pressure (Haynes et al., 1996). These
results suggest that ET-1 is involved in regulation of blood pressure.

ET-1 plays an important role in the development and maintenance of
DOCA-salt hypertension. This notion is supported by the observations that there
is overexpression of ET-1 in DOCA-salt hypertensive rats (Day et al., 1995) and
chronic treatment with a mixed ETA/ETB receptor antagonist decreased blood
pressure to a normal level (Doucet et al., 1996). The mechanisms underlying ET-
1’s role in DOCA-salt hypertension may be due to its potent and long-lasting
vasoconstricting effects.

ET-1 is a more potent constrictor of veins than arteries (Warner, 1990
and Leppaluoto, 1992; Johnson et al., 2002). This increased potency may be
related to the differences in expression of ET receptor subtypes and receptor
density (Johnson et al., 2002). For example, 860, the selective ETB receptor
agonist, produces constriction of mesenteric veins, but not the mesenteric
arteries from the rats, suggesting that functional ETB receptors are expressed on
smooth muscle cells of veins, but not arteries (Johnson et al., 2002). Studies in
vivo show that intravenous administration of ETs increase central venous
pressure, venous resistance and MCFP (Waite and Pang, 1992; Yamamato et
al., 1980). Studies in vitro show that ET-1 is found in the post arterial bed, i.e.,
capillaries and veins (Hemsen, 1991). In DOCA-salt hypertension, ET-1 induced

constriction was reduced in aorta, but maintained in vena cava (Watts et al.,

53

2002). Similar ﬁnding were obtained from studies on mesenteric blood vessels
where ET—1 induced constriction was decreased in mesenteric arteries, but
maintained in mesenteric veins from DOCA-salt rats (Johnson et al., 2002).
Studies showed that both an increase and decrease in venous pressure
was accompanied by an increase in ET-1 production, suggesting that ET-1 plays
a role in the long-lasting adaptations of veins to changes in blood volume
(Semeri et al., 1995). Exogenously applied ET-1 also caused increased MCFP in
rats (Waite and Pang, 1992). In humans, increased venous pressure caused by
blood volume expansion stimulates ET-1 production causing venoconstriction
and decreased venous compliance (Semeri et al., 1995). These results led to the
postulate that in DOCA-salt hypertension, elevated ET-1 production due to
increased venous pressure would cause prolonged venoconstriction as DOCA
and high salt cause sodium and water retention and volume expansion. This

could lead to enhanced cardiac output (Guyton et al., 1972b).

13.4. E T-1 modulates sympathetic nerve activity.

ET-1 and its receptors are present in the CNS (Nakagomi, et al., 2000),
sympathetic ganglia (Hemsen and Lundberg, 1991) and sympathetic nerve
terminals (Power et al., 1989). ET-1 modulates sympathetic neuroeffector
transmission to arteries (Zhang et al., 1996) and veins (Waite and Pang, 1992).
Its action can be prejunctional since ET receptors have been localized to
cholinergic nerve terminals in smooth muscle of the trachea (Femandes et al.,

1998) and at sympathetic nerve endings in human and porcine coronary arteries

54

(Power et al., 1989). ET-1 facilitated the purinergic components of
sympathetically mediated constrictions of rabbit saphenous artery (Mutafova-
Yambolieva and Radomirov, 1994). Furthermore, it was shown that ET-1
inhibited prejunctional NE release from sympathetic nerves and postjunctionalally
it increased the responsiveness of or adrenergic receptors to catecholamines in
rat mesenteric arteries (T abuchi et al., 1990). Therefore, ET can also act via a
postjunctional mechanism. Using isolated rat tail artery in vitro, superfusion of
ET-1 or 86c (ETB receptor agonist) with a subthreshold contractile concentration
signiﬁcantly reduced NE and ATP overﬂow caused by electric ﬁeld stimulation.
This effect was blocked by the selective ETB receptor antagonist, suggesting that
the ETB receptors may be important in prejunctional neuromodulation of
sympathetic tone by ET-1 to the blood vessels (Mutafova-Yambolieva and
Westfall, 1998).

The interactions between ET-1 and sympathetic neuroeffector
transmission is bi-directional. Transmitters released by sympathetic nerves can
also modulate ET—1’s vasoconstrictor effects. Studies on conscious rats have
shown that verapamil, which increases sympathetic nerve activity by decreasing
mean arterial pressure and activating the baroreceptor reﬂex, facilitated ET-1
induced venoconstriction. This facilitation was blocked by hexamethonium, the

nicotinic acetylcholine receptor antagonist (Waite and Pang, 1992).

14. Summary of the general introduction and issues to be resolved

55

Mechanisms of sympathetic transmission differ in arteries and veins.
Therefore, characterization of sympathetic neuroeffector mechanisms including
identifying the neurotransmitters as well as the postjunctional receptor subtypes
and reactivities for the transmitters in arteries and veins in mesenteric circulation
will further elucidate the difference in neural mechanisms controlling arterial and
venous tone. There is elevated activity of the SNS in DOCA-salt hypertension.
Despite that vascular peripheral resistance mainly resides in small arteries and
arterioles, the majority of studies have focused on medium and large arteries with
comparatively little research investigating small resistance arteries (Evans and
Surprenant, 1992; Phillips et al., 1998). As there is a vessel size- related
difference in mechanisms of sympathetic neuroeffector transmission to
mesenteric arteries (Nilsson, 1985; Gitterman and Evans, 2001), ﬁndings
obtained from studies on large and medium arteries are not applicable to the
small arteries and arterioles.

ATP is a co-transmitter of NE from sympathetic nerves associated with
the vascular system (Bumstock and Sneddon, 1985) and a few studies showed
that there are alterations in purinergic transmission associated with mesenteric
arteries in hypertension. For example, there was enhanced purinergic
transmission in mesenteric arteries from SHRs as EJPs evoked by single
electrical stimuli in mesenteric arteries from SHR are increased compared to
those from normal rats with similar passive electrical properties of the vascular
smooth muscle (Brock and Van Helden, 1995). Furthemmore, there was a

potentiated response to ATP in SHR (Naito et al., 1998). However, as no study

56

has been done to assess potential changes in purinergic component of the
sympathetic neuroeffector transmission to arteries from DOCA-salt hypertensive
animials it is unclear whether similar changes occur in DOCA-salt hypertension.

Although venomotor tone was known to be elevated in DOCA-salt
hypertensive rats (Fink et al., 2000), very little studies have been done on
changes in mechanisms of sympathetic control of venous tone in hypertension.
For example, there has been no direct assessment of changes in transmitter
release in mesenteric veins in DOCA-salt hypertension. Furthermore, no study
has been done to investigate the NE uptake activity of the sympathetic nerves
associated with mesenteric veins in DOCA-salt hypertension.

Data from several studies indicate that prejunctional a2-adrenergic
receptor function is impaired in hypertensive animals (De Champlain, 1990;
Moreau et al., 1995; Tsuda et al., 1989). Prejunctional a2—adrenergic receptors
associated with sympathetic nerves also regulate NE release in veins (Bobalova
and Mutafova-Yambolieva, 2001a; Foucart et al., 1987). The few studies of the
function of a2-adrenergic receptors associated with veins in hypertension,
however, have yielded controversial results. Increased NE release and
attenuated yohimbine effect on NE release occur in the portal vein of SHRs.
However, in the same study it was shown that in portal veins from DOCA-salt
hypertensive and 1K1C hypertensive rats there was not an increase in NE
release or an attenuation of the yohimbine effect on NE release (Westfall et al.,
1986). As the portal vein makes only a small contribution to total vascular

capacitance, this blood vessel may not be an appropriate model to study

57

s‘ an.“ “an
I ‘11

hypertension associated with changes in the function of peri-venous sympathetic
nerves.

ET-1 acting at ET receptors modulates sympathetic neuroeffector
mechanisms associated with arteries (Zhang et al., 1996) and veins (Waite and
Pang, 1992) via both a prejunctional and postjunctional mechanism. However, no
study has been done to determine whether ET receptors are localized at the
sympathetic nerves innervating the vascular system. Furthermore, it was shown
that veins produced a more potent and sensitve response than arteries to ET—1
(Johnson et al., 2002). In addition, functional ETB receptors are expressed on
smooth muscle cells of the veins, but not the arteries in mesenteric vascular bed
(Johnson et al., 2002). However, no study has been done to examine whether
the modulatory effects by subpressor ET—1 on sympathetic neuroeffector
transmission to mesenteric arteries and veins differ. As the most potent
vasoconstrictor substance known, ET-1 has been shown to play an important
role in the development of DOCA-salt hypertension (Day et al., 1995; Doucet et
al., 1996; Johnson et al., 2001). However, no study has been done to examine
whether the modulatory effects by subpressor ET-1 on sympathetic neuroeffector

transmission to arteries and veins alter in DOCA-salt hypertension.

58

Chapter 2

HYPOTHESES AND SPECIFIC AIMS

Overall hypothesis

Alterations in neurotransmitter release from sympathetic nerves and
postjunctional reactivity of the vascular smooth muscle cells for the
neurotransmitters are different in arteries and veins from DOCA-salt hypertensive
rats. These alterations are at least partly due to functional changes of the

prejunctional a2-adrenergic receptors and to the effects of ET-1.

DOCA-salt hypertension is a well-characterized model of experimental
hypertension which is associated with increased sympathetic nerve activity. This
model can be used to study the neurohormonal mechanisms secondary to adrenal

steroids excess in hypertension. This study has the following speciﬁc aims:
Speciﬁc aim 1: What are the alterations in neurotransmitter release from

sympathetic nerves and postjunctional reactivity to the transmitters in mesenteric

arteries and veins from DOCA-salt hypertensive rats?

59

Speciﬁc aim 2: Do prejunctional a2-adrenergic receptors modulate transmitter
release from sympathetic nerves in arteries and veins and are those modulatory

effects altered in DOCA-salt hypertension?

Speciﬁc aim 3: Does ET-1 modulate transmitter release from sympathetic
nerves and the postjunctional reactivity of arteries and veins to the
neurotransmitter and are those modulatory effects altered in DOCA-salt

hypertension?

60

CHAPTER 3

Differential alterations in sympathetic neuroeffector mechanisms in

mesenteric arteries and veins in DOCA-salt hypertensive rats

Min Luo‘, Margaret C. Hess‘, Gregory D. Fink1 '4, L. Karl Olsonz, Jennifer

Rogersz, David L. Kreulen2'3'4 Xiaoling Dai4 and James J. Galligan”
Departments of Pharmacology and Toxicology‘, Physiologyz, Ophthalmology and

Neurology3 and The Neuroscience Program“, Michigan State University, East

Lansing, MI 48824

This chapter is published in:

Autonomic neuroscience: Basic & Clinical. 2003, 104: 47-57.

61

INTRODUCTION

DOCA-salt hypertension is initiated, in part, by salt retention promoted
by treatment with the mineralocorticoid and uninephrectomy. Thus, this model is
relevant to salt-sensitive human hypertension and permits studies of the neural
and hormonal mechanisms associated with elevated blood pressure (Schenk and
McNeill, 1992). Changes in the function of the sympathetic nervous system occur
in DOCA-salt hypertension. Circulating NE and epinephrine levels are increased
in DOCA-salt hypertensive rats suggesting that there is increased release of NE
from sympathetic nerves and epinephrine from the adrenal gland in these
animals (Bouvier, et al., 1986). 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 NE than those obtained
from normotensive rats (Tsuda et al., 1989). Increased release of NE from peri-
arterial sympathetic nerves in DOCA-salt rats is due, at least in part, to a
disruption in the function of prejunctional a2-adrenergic autoreceptors (De
Champlain, 1990). 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 NE (Perry and Webb, 1988;
Longhurst et al., 1989; Suzuki et al., 1994) although other studies showed that
the sensitivity to adrenergic agonists did not change in caudal artery
(Herrnsmeyer et al., 1982) and the binding afﬁnity of a1-adrenergic receptors in

mesenteric arteries is reduced (Meggs et al., 1988).

62

The studies summarized above focused on sympathetic nerves and
adrenergic receptors in arteries. However, sympathetic nerves supply veins and
venomotor tone is controlled largely by sympathetic nerve activity (Monos et al.,
1995). In human hypertension and in SHRs, sympathetic neural input to veins is
increased. It has also been shown that venomotor tone is elevated in DOCA-salt
rats and this is largely due to the increased sympathetic tone in veins (Fink et al.,
2000). Detailed studies of the mechanisms underlying increases in sympathetic
input to veins in hypertension have not been done. These studies would be
important because the sympathetic neurons innervating arteries and veins differ
in their location in the ganglia and in their electrophysiological properties
(Browning et al., 1999), suggesting that there is differential sympathetic neural
control of mesenteric arteries and veins. 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 constriction mediated by NE
acting at 111-adrenergic receptors (Hottenstein and Kreulen, 1987; Van Helden,
1988). In small mesenteric arteries and arterioles, electrical stimulation evokes
short latency, short duration EJPs and constrictions mediated by ATP acting at
P2X receptors (Ramme et al, 1987; Evans and Surprenant, 1992; Gitterman and
Evans,2001)

Changes in neuroeffector transmission to veins in hypertension are
important because veins are more sensitive to the vasoconstrictor effects of

sympathetic nerve stimulation than arteries (Kreulen, 1986; Hottenstein and

63

Kreulen, 1987). Furthermore, 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 (Willems et al.,
1982), but these studies did not establish if there was increased release of NE or
if there was increased venous reactivity to NE. 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 a1-adrenergic receptor agonists, but
enhanced responses to nerve stimulation (Sudhir et al., 1990). This previous
work indicates that there are changes in sympathetic neuroeffector transmission
to veins in hypertension, but the details of these changes are unclear.

Identiﬁcation of the mechanisms controlling venomotor tone in
hypertension is important because venous compliance is reduced in hypertensive
subjects (Safar and London, 1987). Decreased venous compliance is most
marked in peripheral vessels, producing a redistribution of blood toward the heart
(Schobel et al., 1993). Decreased compliance of splanchnic veins is particularly
notable in hypertension (Nyhof et al., 1983) and, as the splanchnic circulation is
the largest capacitance bed, changes in mesenteric vasomotor tone will impact
overall hemodynamics (Greenway, 1983).

The data summarized above indicate that there is differential sympathetic
neural control of arteries and veins and that there maybe changes in sympathetic
neural control of both arteries and veins in hypertension. The present study used

an in vitro approach to examine sympathetic nerve-mediated constrictions of

64

 

._._.._3

1n

mesenteric arteries and veins from sham-operated normotensive (sham) and

DOCA-salt hypertensive rats in order to test this hypothesis.

65

METHODS AND MATERIALS

Animals

All experiments were done using Sprague-Dawley rats from Charles
River, Inc (Portage, MI). Upon arrival at the animal care facility, animals were
maintained according to standards approved by the Michigan State University
All-University Committee on Animal Use and Care. All experimental procedures
were carried out in accordance with the ”Guiding Principles in the Care and Use
of Animals" of the American Physiological Society. Rats were acclimatized for 2-
3 days prior to entry into any experimental protocol. Pelleted rat chow
(Harlan/Teklad 8640 Rodent Diet) and water were provided ad Iibitum. Rats were
housed in temperature and humidity controlled rooms with a 12 hours on / 12

hours off light cycle.

Preparation of DOCA-salt rats

Male Sprague-Dawley rats weighing 175-200 grams were anesthetized with
sodium pentobarbital (50 mg/kg i.p.). The skin over the left ﬂank (lateral
abdominal wall) was shaved and prepared with an iodine-based antiseptic. A 1.5
cm vertical incision was made through the skin and underlying muscle just caudal
to the rib cage. The left kidney was exteriorized and removed after ligation of the
renal artery, vein and ureter with 4-0 silk sutures. The muscle and skin layers
were closed separately with 4-0 silk and 4-0 monoﬁlament nylon sutures,

respectively. A 3 x 1.5 cm rectangle area between the shoulder blades of the

66

back was shaved and disinfected for s.c. DOCA implantation under a 1 cm
incision. The skin was closed with 4-0 nylon sutures. DOCA implants (600 mg/kg)
were prepared by mixing deoxycorticosterone acetate in silicone rubber resulting
in a giving dose of 200 mg/kg. Sham-operated rats underwent left kidney removal
only. Surgery was performed on a heated pad and rats recovered in a heated
box. Antibiotics (enroﬂoxacin, 5 mg/kg s.c.) and an analgesic (butorphanol
tartrate, 2 mg/kg, s.c.) were administered immediately after surgery. After
recovery the rats were housed under standard conditions for 4 weeks. DOCA-
implanted rats received standard pelleted rat chow and salt water (1 % NaCl +
0.2 % KCI) ad Iibitum. while sham rats received standard pelleted rat chow and
tap water ad Iibitum. Systolic blood pressure was measured using the tail-cuff
method four weeks after surgery. Rats with systolic blood pressure equal to or

higher than 150 mmHg were considered hypertensive.

Measurement of vasoconstriction in vitro
Rats were killed using a lethal pentobarbital injection (50 mg, i.p). The

small intestine was removed and placed in oxygenated (95% 02, 5% 002)
Krebs' solution of the following composition (mM): NaCl 117; KCI 4.7; CaCl2 2.5;
MgCl2 1.2; NaHCO3 25; NaH2PO4 1.2; glucose 11. The ileal segment was

placed in a petri dish and the mesentery was stretched gently and pinned ﬂat. A
section of mesentery close to the ileal wall was carefully cut free from the
intestine and the mesentery was transferred to a small silicone elastomer-lined

recording bath (2.5 ml volume). The arteries and veins (100 — 300 pm diameter)

67

were exposed by carefully cutting away surrounding adipose and connective
tissues. The recording bath containing the preparation was mounted on the stage

of an inverted microscope (Olympus CK-2) and superfused continuously with

warm (36 °C) Krebs' solution at a ﬂow rate of 7 ml/min. The output of a black and
white video camera (Hitachi, KP-111) attached to the microscope was fed to a
PCVision Plus frame-grabber board (Imaging Technology Inc., Wobum MA)
mounted in a personal computer. The sampling rate was 10 Hz and changes in
blood vessel diameter of 0.5 pm could be resolved. The video images were
analyzed using Diamtrak® software. The digitized signal was converted to an
analog output (DAC-O2 board, Keithley Metrabyte) and which was fed to an
analog to digital converter (Labmaster 125) and a second computer running
Axotape software (version 2.0, Axon Instruments, Inc. Foster City CA) for a
permanent record of changes in vein diameter. Analog signals were sampled at
33 Hz and data were stored on the computer hard drive for subsequent display

and analysis.

Drug application

Drugs were applied using a system of 3-way stopcocks so that the
superfusing Krebs’ solution could be changed to one containing a known
concentration of drug. The ﬂow rate was 9 ml/minute and it took one minute for
drugs to reach the tissue. NE, PE and ATP concentration- response curves were
constructed using non-cumulative addition of each agonist concentration. Each

agonist concentration was applied for 2 minutes and there was a 20 minute

68

interval between application of each agonist concentration. Tissues were
washed continuously with normal Krebs’ solution between each dose.
Antagonists were applied for a minimum of 20 minutes prior to testing their effect

on agonist- or nerve-mediated responses.

Transmural stimulation of perivascular nerves

Two parallel silver/silver chloride wire electrodes connected to a Grass
Instruments stimulator (S88) were placed perpendicular to the longitudinal axis of
mesenteric tissues. Parameters for nerve stimulation were: 30 stimuli, stimulus
duration 0.5 ms, frequency 0.2 — 30 Hz, voltage 60-140 volts. The neurogenic
origin of constrictions caused by electrical stimulation was veriﬁed in each
preparation by initially demonstrating that a constriction caused by 20 Hz
stimulation was blocked by tetrodotoxin (TTX, 0.3 pM). Preparations in which the
initial 20 Hz constriction was not blocked by 'l‘l'X were discarded. The peak
constriction and in some experiments the duration of vasoconstriction caused by
nerve stimulation was measured. Measurements were made using Axotape

software and on-screen cursors.

Statistical analysis
Agonist-induced constrictions were measured in pm and are expressed as
a percentage of the initial resting diameter of the blood vessel. For NE and PE

concentration-response curves, E050 and Emax were calculated from a least

69

squares ﬁt of individual agonist concentration-response curves using the

following logistic function (Origin 5.0 Microcal Software Inc., Northampton, MA):

Y = {(Ymin — Emax)/[1 + (X / ECso)m]} + Emax
Where “Ymin “ is the minimum response (constrained to 0), “Emax” is the
maximum response, “ECso” is the half maximal effective concentration and “m” is
the slope factor. For nerve stimulation frequency-response curves, the half
maximal effective stimulation frequency (S50) and Emax were calculated from
curves obtained in individual tissues using the following function (Origin 5.0
software):
Y = Emax X / (S50 + X)

Where “X” is the stimulation frequency tested and “y” is the peak response
amplitude. All data are expressed as the mean i S.E.M and “n” values refer to
the number of animals from which the data were obtained. Differences between
groups were assessed by the KruskaI-Wallis and Dunn’s multiple comparisons

tests and Students’ t-test.

Drugs

All drugs were obtained from Sigma Chemical Company (St. Louis, MO).

70

Results

General

Data were obtained from 68 sham rats and 74 DOCA rats. Mean systolic
blood pressure from sham and DOCA-salt rats was 118 1 1 mmHg and 179 1 3
mmHg, respectively (P < 0.05). The mean weight of sham rats was 462 1 6 g and
the mean weight of DOCA-salt rats was 322 1 9 g (P < 0.05). The inner diameter
of mesenteric arteries from sham and DOCA-salt rats was 141 1 7.4 pm, 149 1
8.0 pm (P > 0.05), respectively. The diameter of mesenteric veins from sham rats

was 214 1 9 pm and the diameter of mesenteric veins from DOCA-salt rats was

228 1 10 mm (P > 0.05).

Comparison of frequency response curves from sham and DOCA-salt
arteries and veins

Transmural stimulation caused frequency-dependent constrictions of
mesenteric arteries and veins (Fig. 3.1 ). There were no differences between the
frequency-response curves obtained in sham and DOCA-salt veins as the mean
Ema,x and $50 values obtained in these tissues were similar (Table 3.1).
However, frequency-response curves from sham and DOCA-salt veins were both
shifted to the left of the curves obtained from sham arteries (Fig. 3.1, Table 3.1).
The Emax obtained in DOCA-salt arteries was signiﬁcantly greater than that

occurring in sham arteries and there were no differences between the frequency

71

response curves obtained in veins and those obtained in DOCA-salt arteries

(Fig. 3.1, Table 3.1)

Increased adrenergic contribution to neurogenic constrictions in DOCA-
salt arteries

To test for contributions of ATP and NE to neurogenic constrictions in
mesenteric blood vessels, frequency-response curves in sham and DOCA-salt
arteries were constructed in the absence and presence of prazosin (0.1 pM). an
011- adrenergic receptor antagonist and PPADS (10 pM). a P2 receptor
antagonist. Prazosin blocked constrictions caused by exogenously applied NE
(10 pM, the response was 49 1 5% before prazosin treatment and 0 1 0% after
prazosin treatment, n=8), but it did not change the frequency-response curves in
sham arteries (Fig. 3.2A). The Emax was 19 1 5% before prazosin treatment and
14 1 2% after prazosin treatment (n=7, P > 0.05); the $50 value was 11 1 1 Hz
before prazosin treatment and 9 1 1 Hz after prazosin (n=7, P > 0.05).
Subsequent addition of PPADS completely blocked the neurogenic responses in
sham arteries (Fig. 3.2A). PPADS alone greatly reduced neurogenic constrictions
in sham arteries and therefore it was not possible to calculate Emax and $50
values reliably. However, before PPADS, 5, 10 and 20 Hz stimulation caused
constrictions of 8 1 2%, 20 1 6% and 26 1 4% respectively. After PPADS
treatment these values were 0.7 1 0.7%, 4 1 2% and 9 1 5% (n=5; P < 0.05 at

each stimulation frequency).

72

Prazosin markedly inhibited neurogenic responses in DOCA-salt arteries
(Fig. 3.2B). In these studies, the Emax was 25 1 2% before prazosin treatment
and 8 1 2% after prazosin treatment (n=6, P < 0.05); the S50 was 8 1 1 Hz before
prazosin treatment and 14 1 4 Hz after prazosin treatment (n=6, P > 0.05). The
response remaining in the presence of prazosin was blocked by combined
application of PPADS and prazosin (Fig. 3.28). PPADS alone had little effect on
responses in DOCA-salt arteries. In these studies, the control Emax was 34 1 5%
before PPADS treatment and 22 1 6% after PPADS treatment (n=6, P > 0.05);
the 850 was 7 1 2 Hz before PPADS treatment and 8 1 4 Hz after PPADS
treatment (n=6, P > 0.05). PPADS blocked P2 receptors in DOCA-salt arteries
as constrictions caused by ATP (0.1 mM) were blocked by PPADS. The control
ATP constriction was 43 1 9% before PPADS treatment and 13 1 3% after
PPADS treatment (P < 0.05). These data demonstrate that there was an increase
in the adrenergic contribution to neurogenic constrictions in DOCA-salt arteries
compared to that occurring in sham arteries.

When the frequency-response curves for nerve stimulation in the
presence of prazosin from sham and DOCA-salt arteries were compared, the
maximum response and half maximal frequency were found not to be different
(p>0.05). This result suggests that there is no change of purinergic component in

arterioconstriction in DOCA-salt arteries.

Neurogenic constrictions are blocked by prazosin in sham and DOCA-salt

veins

73

Similar studies to those described above were done in sham and DOCA-
salt veins and it was found that prazosin completely blocked neurogenic

constrictions in both types of tissues (Fig. 3.3).

Reactivity to a1-adrenergic and P2 receptor stimulation is not altered in
DOCA-salt arteries

The increased adrenergic component to the neurogenic response in
DOCA-salt arteries could be due to an increase in NE release or an increase in
reactivity of DOCA-salt arteries to the stimulatory effects of nerve-released NE.
To address this issue, concentration-response curves for NE, the 111-adrenergic
receptor agonist, PE, and the a2-adrenergic receptor agonist, UK 14,304, were
obtained in arteries from sham and DOCA-salt rats. NE and PE were
approximately equipotent in contracting sham and DOCA-salt arteries and the
Emax values were also similar in these tissues (Fig 3.4, Table 3.2). UK 14,304
(0.1-10 pM) did not contract sham or DOCA-salt arteries (Fig 3.5). Furthermore,
there was no difference between concentration-response curves for ATP
obtained in sham and DOCA-salt arteries. The ATP Emax value was 57 1 4% in
sham arteries (n=11) and this value was 471 5% in DOCA-salt arteries (n=8,
P>0.05). The ATP E050 was 0.11 0.02 pM in sham arteries and 0.2 1 0.08 pM in
DOCA-salt arteries (P>0.05). Taken together, these data indicate that 011-

adrenergic receptors mediate adrenergic vasoconstrictions in sham and DOCA-

74

salt mesenteric arteries and that there is no change in reactivity of DOCA-salt

arteries to a1-adrenergic or P2 receptor stimulation.

Inhibition of catecholamine uptake potentiates responses in DOCA-salt
veins

Cocaine (10 pM) and corticosterone (10 pM) treatment prolonged the
duration of the constriction caused by 5 and 10 Hz stimulation in DOCA-salt, but
not sham veins (Table 3.3). These frequencies were chosen for study as they

caused submaximal and maximum responses in sham and DOCA-salt veins.

75

Table 3.1: Maximum response (Emax) and half maximum stimulation frequency

(850) in arteries and veins from sham and DOCA-salt rats. All data are expressed

as the mean 1 S.E.M and “n” values refer to the number of animals from which

the data were obtained. ‘l’indicates signiﬁcantly different from the 850 in sham

and DOCA-salt veins (P < 0.05). #indicates signiﬁcantly different from the Emax

in DOCA-salt arteries and DOCA-salt veins (P<0.05).

 

 

 

 

 

Artery Vein
Sham (n=14) DOCA-salt Sham (n=22) DOCA-salt (n=24)
(n=13)
850 (Hz) 95 1': 1.21); 7.4 1 0.9 4.9 1 0.9 5.4 1 0.7
Emax (%) 21.9 i 3_1# 31.3 1 3.3 27.7 1 3.1 36.0 1 3.4

 

 

 

 

 

76

 

Table 3.2: Emax and ECso values of the concentration-response curves for NE
and PE in arteries from sham and DOCA-salt rats. All data are expressed as the
mean 1 S.E.M and “n” values refer to the number of animals from which the data
were obtained. There were no difference in Emax and E050 between sham and

DOCA-salt arteries.

 

 

 

 

 

 

 

 

Artery
NE Sham (n=9) DOCA-salt (n = 10)
ECso (11M) 5.4 1 2.5 4.7 1 1.0
Emax (%) 52 1 3.8 57 1 4.0
PE Sham(n = 6) DOCA-salt(n = 6)
E050 (11M) 1.5 1 0.8 3.2 11.1
Emax (%) 471 1.7 451 3.5

 

 

 

 

77

.v.

Table 3.3: Duration (seconds) of neurogenic responses caused by 5 and 10 Hz
stimulation of veins in the absence (Control) and presence of cocaine (10 pM)
and corticosterone (10 pM). All data are expressed as the mean 1 S.E.M and “n”
values refer to the number of animals from which the data were obtained.
*indicates signiﬁcantly different from the duration in control sham and DOCA-salt

veins at the same stimulation frequencies (P < 0.05).

 

 

 

 

 

Sham (n=3) DOCA-salt (n=4)
Stimulation Control Cocaine/ Control Cocaine/
Frequency corticosterone. corticosterone
5H2 60+9 51117 120151 271136-
10 Hz 58110 68120 7317 288117*

 

 

 

 

 

78

 

35
3O
25
20
15
1O

% Contraction

j

 

OOU‘I

 

—l:I- DOCA-salt vein (n=24)
—O—- Sham vein (n=22)

—I- DOCA-salt artery (n=13)
+ Sham artery (n=14)

/

+/’
4 / ’ (.7

/
/

10
Frequency (Hz)

100

Fig. 3.1: Comparison of frequency response curves for neurogenic constrictions

of arteries and veins from sham and DOCA-salt rats. The frequency-response

curves from sham and DOCA-salt veins were shifted signiﬁcantly to the left of

those obtained from sham arteries. There was no difference between the

frequency-response curves from DOCA-salt arteries, DOCA-salt veins and sham

veins.

79

A Sham artery

35 - -0- Control (n=7)
-A- Prazosin (n=7)
30 ’ -v- Prazosin/PPADS (n=5)

25-
20.

% Contraction

 

 

DOCA-salt artery

' -D- Control (n=6)
—A— Prazosin (n=6)
--v— Prazosin/PPADS (n=4)

. EL&
_ ,/

i ei/ )‘

 

M

1 10
Frequency (Hz)

Fig. 3.2: Contributions of a1-adrenergic and P2 receptor-mediated components

to neurogenic constrictions of arteries from sham and DOCA-salt rats. (A)

Frequency-response curves before and after sequential application of prazosin

and then PPADS in sham arteries. Prazosin alone had no effect while PPADS

greatly reduced constrictions in sham arteries. (B) Frequency-response curves

before and after sequential application of prazosin and them PPADS in DOCA-

salt arteries. Prazosin alone greatly reduced constrictions in DOCA-salt arteries

while combined application of prazosin and PPADS completely blocked the

neurogenic responses.

80

Sham and DOCA-salt veins

 

 

 

35 _ —o— Sham (n=17)
—CI- DOCA-salt (n=19)
3O _ -O- Sham/Prazosin (n=10)
-I— DOCA-salt/Prazosin (n=9)
.5 25 ' £1; I?
§2°r ‘ I
815 .
O
°\° 10 L
5' E
O . . . . . “.1
0.1 1 10 100
FreqUency (Hz)

Fig. 3.3: Frequency-response curves for neurogenic constrictions in sham and
DOCA-salt veins before and after prazosin (0.1 pM) treatment. Prazosin blocked

the constriction caused by nerve stimulation in both sham and DOCA-salt veins.

81

Sham and DOCA-salt artery

-0- Sham PE (n=6)
70 _—D— DOCA-salt PE (n=6)
I -O- Sham NE (n=9)
60 _—I— DOCA-salt NE (n=10) ;

50-

O

% Contraction
to co 4:.
O O

_x
O
l '

 

 

0.1 1 10 100
Concentration (11M)

Fig. 3.4: Comparison of constrictions caused by adrenergic receptor agonists in
arteries from sham and DOCA-salt rats. Concentration-response curves for NE
from sham and DOCA-salt arteries were not different. Concentration-response

curves for PE were similar in arteries from DOCA-salt and sham rats.

82

- a

70 - —CI— Sham artery (n=6)

’ —I- DOCA-salt artery (n=6)
60 ' -O-Sham vein (n=3)
50 ’ +DOCA-salt vein (n=3)

4o-
30-
20+

% Contraction

'10- L
O. / \ D 9
0.1 1 10
[UK 14.304] (11M)

 

Fig. 3.5: Concentration-response curves for UK 14,304 in arteries from sham and
DOCA-salt rats. UK14, 304 did not contract mesenteric arteries, suggesting that 012

adrenergic receptors are not expressed by mesenteric blood vessels.

83

Discussion

Neurogenic responses in mesenteric arteries

NE and ATP are co-transmitters released by sympathetic nerves
associated with most arteries (Kennedy, 1996). Studies by Gitterman and Evans
(2001) showed that the P2 receptor antagonist, suramin, inhibits (GO—80%)
neurogenic constrictions of mesenteric arteries (< 300 um diameter) from
normotensive rats indicating that ATP is the dominant vasoconstrictor transmitter
in these vessels. This result was conﬁrmed in the present study where it was
shown that the neurogenic responses of sham mesenteric arteries (5 300 pm)
were greatly reduced by PPADS, a P2 receptor antagonist. My study also
showed that prazosin did not alter frequency-response curves from sham
arteries. However, co-application of prazosin and PPADS completely blocked
neurogenic responses in sham arteries. Therefore, NE makes only a minor
contribution to sympathetic neuroeffector transmission to small mesenteric
arteries in normotensive rats. Conversely, neurogenic constriction of small
mesenteric arteries from DOCA-salt rats were greatly reduced by prazosin
indicating that NE makes a major contribution to sympathetic neuroeffector
mechanisms in small mesenteric arteries from DOCA-salt hypertensive rats. This
is a novel ﬁnding and indicates that DOCA-salt hypertension is associated with a

change in neuroeffector mechanisms in small mesenteric arteries.

84

‘r‘ .1 (2.. m-1

In DOCA-salt arteries, the Emax caused by nerve stimulation was greater
than that occurring in sham arteries. This result suggests that sympathetic
neuroeffector mechanisms was potentiated in DOCA-salt arteries, which could be
due to increased transmitter release (a prejunctional mechanism) or increased
sensitivity of vascular smooth muscle to sympathetic neurotransmitters (a
postjunctional mechanism). My data show that concentration-response curves
for NE in DOCA-salt arteries were not different from those obtained in sham
arteries. This result differs from previously published work that showed increased
(111-adrenergic receptor reactivity of arteries from DOCA-salt rats (Perry and
Webb, 1988; Longhurst et al., 1989; Suzuki et al., 1994). Differences in the
methods used to assess vascular reactivity could account for the difference in
results obtained in my study and by these other investigators. Longhurst et al.
(1988) and Suzuki et al. (1994) studied changes in perfusion pressure of the
entire mesenteric vascular bed, whereas Perry and Webb (1988) measured
agonist-induced increases in isometric force of arterial strips. I measured
diameter changes in unpressurized blood vessels. However, the discrepancy
between the data from previous work and my own data may also be due to
differences in the size of the mesenteric arteries studied. Perry and Webb (1988)
studied large mesenteric arteries (1 mm o.d.) while Suzuki et al. (1994) studied
the main branches of the superior mesenteric arterial trunk (~0.5 mm o.d.).
Longhurst et al. (1988) studied perfusion pressure in the whole mesenteric

vascular bed and changes perfusion pressure would be affected by changes in

85

A . __.

the diameter of the larger mesenteric arteries. I studied small mesenteric
resistance arteries (<200 pm diameter). Gitterman and Evans (2001) have shown
that the relative proportion of purinergic and adrenergic contributions to
sympathetically-mediated arterial constriction changes with the size of the
mesenteric arteries. They showed that the adrenergic component increases and
the purinergic component decreases as the diameter of the artery increases. NE
is the principal vasoconstrictor in mesenteric arteries 3500 pm In diameter while
ATP is the predominant neurogenic constrictor in the small mesenteric arteries
(Gitterman and Evans, 2001). My study also showed that in small (<200 pm
diameter) mesenteric arteries, NE plays a minor role in sympathetic neuroeffector
mechanisms in normotensive rats. Therefore, reactivity of the 111-adrenergic
receptor in the smaller arteries may be unaffected by DOCA-salt hypertension
because NE is not normally the predominant vasoconstrictor transmitter in these
blood vessels. My data also show that concentration-response curves for ATP in
DOCA-salt arteries were not different from those obtained in sham arteries (see
also Galligan et al., 2001). Therefore, changes in sensitivity to nerve-released
ATP or NE are unlikely to account for the increased neurogenic constriction in
DOCA-salt arteries. It is also unlikely a2 adrenergic receptor upregulation in
small mesenteric arteries accounts for the increased neurogenic constriction in
DOCA-salt tissues. This conclusion is based on data showing that clonidine or
UK 14,304 did not contract small mesenteric arteries from sham or DOCA-salt

rats.

86

Previous studies showed that there is increased release of NE from
sympathetic nerves in DOCA-salt hypertension (T suda et al., 1989; de
Champlain, 1990). Although NE release was not measured directly in the present
study, the reduced NE content in DOCA-salt arteries (Luo et al., 2003) suggests
that there was increased release of NE from sympathetic nerves in DOCA-salt
rats leading to store depletion. This result and conclusion is similar to that
reported previously in studies of DOCA-salt rats (Crabb et al., 1980; Longhurst et
al., 1989). Increased NE release could contribute to the increased neurogenic
constrictions and also could stimulate increased NET expression in DOCA-salt
arteries. Though the mechanism underlying this increased adrenergic
neurotransmission remains unclear, dysfunction of a2-adrenergic autoreceptors
in DOCA-salt hypertension (T suda et al., 1989; de Champlain, 1990), which

under normal conditions inhibit NE release, might be a cause.

Neurogenic responses in mesenteric veins

This study is unique in that I examined sympathetic constrictions of veins
in order to assess changes that might be associated with hypertension. The
present study and previous work (Kreulen, 1986; Hottenstein and Kreulen, 1987)
showed that veins are more sensitive than arteries to nerve stimulation as
frequency-response curves in veins are to the left of those obtained in arteries.
This conclusion was based on the observation that the 850 was lower in sham
and DOCA-salt veins (~5 Hz) compared with sham arteries (~10 Hz). Studies on

sympathetic nerve activity in rat tail artery and veins show that the dominant ﬁring

87

1‘77“

frequencies are between 2 and 6 Hz (Johnson and Gilbey, 1998). Similarly, the
frequency of synaptic activity of presumed vasoconstrictor neurons in rat superior
cervical ganglia (SCG) is 3 Hz (McLachlan et al., 1997). Therefore, if the in vivo
frequency of nerve activity in sympathetic nerves associated with mesenteric
blood vessels is similar to that in tail vessels and in the SCG, then increases in
sympathetic nerve activity would produce greater constrictions of veins than
arteries. These data also suggest that increases in sympathetic nerve activity
would increase venous tone before changing arterial tone. The splanchnic
circulation holds one-third of total blood volume and this store resides largely in
veins (Greenway, 1983). As the sympathetic nervous system is the principal
mechanism for controlling venomotor tone (Monos et al., 1995), small increases
in sympathetic nerve activity would increase venous return and cardiac stroke
volume. Therefore, at low levels of sympathetic nerve activity, vascular-based
increases in systemic blood pressure could be due, in part, to an increase in
venomotor tone.

Neurogenic constrictions of veins from sham and DOCA-salt rats are
mediated by NE acting at aI-adrenergic receptors as they are blocked by
prazosin (Luo et al., 2003). Furthermore, NE-induced constrictions of veins are
mimicked by PE, an 111-receptor selective agonist, but not by the a2-receptor
selective agonists, clonidine or UK 14,304 (Luo et al., 2003). NE and PE
concentration-response curves from DOCA-salt veins were shifted to the right of
those obtained in sham veins (Luo et al., 2003). Similar decreases in NE

sensitivity occur in hand veins taken from human hypertensive subjects (Sudhir

88

et al., 1990). Decreased NE sensitivity could be due to downregulation of a1-
adrenergic receptors in DOCA-salt veins caused by the increased sympathetic
nerve activity known to occur in DOCA-salt hypertension (De Champlain, 1990;
Fink et al., 2000). However, I found that neurogenic responses were not different
between sham and DOCA-salt veins. Maintenance of nerve-mediated responses
in the presence of decreased postjunctional sensitivity could indicate that more
NE is released from nerves in DOCA-salt veins. This hypothesis was supported
by data showing that NE content was lower in DOCA-salt compared to sham
veins (Luo et al., 2003) as increased NE release would lead to store depletion.
In DOCA-salt veins, there was also an increase in NET activity as inhibition of NE
uptake by combined application of cocaine and corticosterone caused a leftward
shift in concentration-response curves to exogenous NE (Luo et al., 2003) and
prolonged neurogenic constrictions in DOCA-salt, but not sham veins. There
was also an increase in the amount of NET protein in DOCA-salt veins (Luo et
al., 2003). A similar upregulation of NE uptake also occurs in SHRs (Zsoter and
Wolchinsky, 1983) and in human hypertensive subjects (Aalkjaer et al., 1987).
Taken together these data suggest that there is increased release of NE from
sympathetic nerves in DOCA-salt veins, which results in lower levels of stored
NE and increased NET levels. Direct measurements of NE release in vivo and in

vitro will be required to substantiate this suggestion.

Summary and conclusions

89

In summary, NE mediates sympathetic neuroeffector mechanisms by
acting at (an-adrenergic receptors in mesenteric veins while ATP, acting at P2X
receptors, is the dominant vasoconstrictor transmitter in small mesenteric arteries
from normotensive rats. This result supports the proposal that there is differential
sympathetic neural control of arteries and veins (Dehal et al., 1992; Browning et
al., 1999). In DOCA-salt rats, neurogenic responses in mesenteric arteries are
potentiated due to Increased adrenergic neurotranmission. In mesenteric veins
from DOCA-salt rats there is no change in neurogenic responses, but there is a
decrease in responses to exogenous NE due to an increase in NET levels in
DOCA-salt veins (Luo et al., 2003). Therefore, it is likely that NE release from
perivascular sympathetic nerves in DOCA-salt hypertension is increased, which
stimulates an upregulation of NET levels in blood vessels (Luo et al., 2003). The
increased venomotor tone known to occur in DOCA-salt rats in vivo (Fink et al.,
2000) must be due to factors other than an intrinsic increase in reactivity of
mesenteric veins to neurally-released NE. Locally acting or circulating
substances are likely to interact with sympathetic nerves in the control of

venomotor tone in DOCA-salt hypertension.

90

Chapter 4

Impaired function of a2 adrenergic autoreceptors on sympathetic nerves

associated with mesenteric arteries and veins in DOCA-salt hypertension

Min Luo, Gregory D. Fink, Keith J. Lookingland, John A. Morris, James J.

Galligan

Department of Pharmacology & Toxicology and the Neuroscience Program

Michigan) State University, East Lansing, MI 48824 USA

This chapter was submitted as a manuscript to:

American Journal of Physiology: Heart and Circulatory Physiology

91

Introduction

There is increased activity of the sympathetic nervous system in DOCA-
salt hypertension (De Champlain, 1990; Schenk and McNeill, 1992). Evidence
supporting this conclusion includes: the presence of increased circulating
catecholamine levels in DOCA-salt hypertensive rats (De Champlain, 1990;
Foucart S., et al., 1987), greater decreases in arterial blood pressure caused by
ganglionic blockade with hexamethonium in DOCA-salt rats compared to sham
normotensive rats (Fink et al., 2000); and normalization of blood pressure in
DOCA-salt hypertensive rats following central catecholamine depletion after 6-
hydroxydopamine treatment (Lamprecht et al., 1977).

Increased sympathetic nerve activity in DOCA-salt hypertension suggests
that there may be alterations in the local mechanisms that modulate sympathetic
neuroeffector mechanisms (Schenk and McNeill, 1992; Westfall et al., 1986). 012
adrenergic autoreceptors located on sympathetic nerve terminals mediate
feedback inhibition of NE release (Langer, 1980). Data from several studies
indicate that prejunctional a2-adrenergic receptor function is impaired in
hypertensive animals. For example, intravenously administered yohimbine, an
012 adrenergic receptor antagonist, increased plasma NE levels in sham
normotensive, but not in DOCA-salt hypertensive rats (De Champlain, 1990;
Moreau et al., 1995). Furthermore, yohimbine potentiated nerve stimulation-
evoked NE release into the mesenteric vasculature in normotensive rats, but not

in DOCA-salt hypertensive rats (T suda et al., 1989). As nerve stimulation would

92

activate periarterial and perivenous nerves, the source of NE in these studies
was unclear.

ATP and NE are co.transmitters released by sympathetic nerves
associated with arteries (Bobalova and Mutafova-Yambolieva, 2001a; Bobalova
and Mutafova-Yambolieva, 2001 b; Bumstock et al., 1985; Gitterman and Evans,
2001). In normotensive rats, ATP is the dominant neurotransmitter in small
mesenteric arteries (Fink et al., 2000; Luo et al., 2003); however, in arteries from
DOCA-salt hypertensive rats NE is the major sympathetic neurotransmitter (Luo
et al., 2003). This increased adrenergic component in sympathetic neuroeffector
transmission to DOCA-salt arteries is not likely to be due to an increase of
postjunctional reactivity to NE as the dose-response curves for exogenous NE
and phenylepherine were not different between sham and DOCA-salt arteries
(Luo et al., 2003). Increased adrenergic transmission is, therefore, likely due to
increased NE release. In addition, it was shown that sympathetic
arterioconstriction was increased in DOCA-salt arteries (Luo et al., 2003). This
potentiation appears to be attributable to increased NE release as there was no
change in postjunctional reactivity of DOCA-salt arteries to exogenous NE and
ATP (Luo et al., 2003). Whether ATP release is altered in DOCA-salt arteries is
yet to be determined.

Sympathetic nerves exert primary control over venomotor tone (Langer,
1980) and NE is the neurotransmitter mediating neurogenic constrictions of
mesenteric veins from sham and DOCA-salt rats (Luo et al., 2003). Prejunctional

a2-adrenergic receptors associated with sympathetic nerves also regulate NE

93

release in veins (Bobalova and Mutafova-Yambolieva, 2001b; Foucart et al.,
1987). The few studies of the function of a2-adrenergic receptors associated with
veins in hypertension, however, have yielded controversial results. Increased NE
release and attenuated yohimbine effect on NE release occur in the portal vein of
SHRs. However, in the same study it was shown that in portal veins from DOCA-
salt hypertensive and 1K1C hypertensive rats there was not an increase in NE
release or an attenuation of the yohimbine effect on NE release (Westfall et al.,
1986). As the portal vein makes only a small contribution to total vascular
capacitance, this blood vessel may not be an appropriate model to study
hypertension associated with changes in the function of peri-venous sympathetic
nerves.

Veins are more sensitive to the vasoconstrictor effect of sympathetic nerve
stimulation than arteries as frequency—response curves caused by nerve
stimulation obtained from veins are to the left of those from arteries (Hottenstein
and Kreulen, 1987; Kreulen 1986; Luo et al., 2003). Accordingly, small increases
in sympathetic nerve activity would increase venous tone before changing arterial
tone. As 25% of the blood volume is in the splanchnic circulation, increases in
mesenteric venomotor tone will lead to an increase in venous return and cardiac
output. These changes will have a profound impact on overall hemodynamics
(Greenway, 1983). Therefore, mesenteric veins are an appropriate system to
study hypertension-associated changes in sympathetic nerve function.

Our previous studies showed that there is maintained in vitro neurogenic

venoconstriction in the presence of decreased postjunctional reactivity to

94

exogenously applied NE. These data indicate that there must be increased NE
release in mesenteric veins from DOCA-salt rats (Luo et al, 2003). In addition,
the steady state stores of NE in DOCA-salt arteries and veins were reduced,
presumably due to increased NE release in DOCA-salt rats (Luo et al., 2003).
Clearly, direct measurements of NE release in mesenteric arteries and veins are
needed to substantiate these suggestions.

The present study examined changes in sympathetic neurotransmitter
release in DOCA-salt hypertension and the function of a2-adrenergic receptors in
regulating ATP and NE release from periarterial nerves, and NE release from
perivenous nerves. 1 also assessed changes in a2-adrenergic receptor function

in DOCA-salt hypertension.

95

METHODS

Animals

See Chapter 3 Methods.

Preparation of DOCA-salt rats

See Chapter 3 Methods.

Tissue preparation
Rats were killed using a lethal sodium pentobarbital injection (50 mg, i.p).

The intestine was removed and placed in oxygenated (95% 02, 5% 002) Krebs'
solution of the following composition (mM): NaCl (117); KCI (4.7); Ca012 (2.5);
M9012 (1.2); NaHCO3 (25); NaH2PO4 (1.2); glucose (11) in a silicone elastomer-
lined petri dish. The mesenteric bed was stretched gently and pinned ﬂat.
Mesenteric arteries or veins from about 75% of the mesentery bed were
dissected out by carefully cutting away surrounding adipose and connective

tissues. Blood vessels were transferred to a small silicone elastomer—lined

recording bath (1.0 ml volume). The recording bath containing the preparation

was superfused continuously with warm (36 0C) Krebs' solution at a ﬂow rate of
7-8 ml/min. The preparation was equilibrated for 1 hour before beginning

experiments.

Drug application

96

Drugs were applied using a system of 3-way stopcocks so that the
superfusing Krebs’ solution could be changed to one containing a known
concentration of drug. It took one minute for drugs to reach the tissue. Agonists
or antagonists were applied for 30 minutes prior to testing their effects on

transmitter release.

Transmural stimulation of perivascular nerves

Two parallel silver/silver chloride wire electrodes connected to a Grass
Instruments stimulator (888, Quincy, MA) were used for transmural stimulation
(30 stimuli, stimulus duration 0.5 ms, frequency 10 Hz, voltage 120—140 volts).
We showed previously that 10 Hz causes maximum constrictions in mesenteric
arteries and veins (Luo et al., 2003). Two conditioning trains of stimulation were
applied at 20-minute intervals for ATP measurement and one conditioning train of
stimulation for NE measurement. Nerve-mediated ATP and NE release was
stable following the conditioning trains of stimulation (Bobalova and Mutafova-
Yambolieva, 2001a). The ﬂow of the superfusing solution was stopped during
nerve stimulation and the solution (1 ml) was quickly withdrawn from the bath by
using a 1 ml syringe. Flow was restarted immediately after sample withdrawal.
Superfusate was then processed for high performance liquid chromatography
(HPLC) measurement of ATP and NE. Basal levels of ATP or NE were

measured in superfusate samples collected before nerve stimulation.

97

Measurement of adenine nucleotides and adenosine by HPLC with
fluorescence detection

Our measurements of ATP release include ATP and its metabolites: ADP,
AMP and adenosine. Superfusate samples (500 pl) were analyzed using HPLC
in conjunction with ﬂuorescence detection (Levitt B, et al., 1984). Brieﬂy,
chloroacetaldehyde (25 pl) was added to superfusate samples, which were then

heated for 40 min at 80 °C in an oven (Baxter, Scientiﬁc Products) to produce the

ﬂuorescent derivative 1, N6-ethenopurine analogs. The derivatization reaction
was stopped by cooling the samples on ice. Separation of derivatized ATP,
ADP, AMP and adenosine was achieved using a C-18 column (Biophase ODS, 5
pm, 250 X 4.6 mm, Bioanalytical Systems) and a gradient system in which the
concentration of mobile phase buffer B was increased from 0 to 100% in 10 min
and maintained at 100% for 5 min followed by a decrease to 0% in 5 min. The
column then was re-equilibrated with 100% mobile phase A for 10 min. Mobile
phase A consisted of 0.1 mol/l KH2P04 (pH = 6.0) and mobile phase B consisted
of 0.1 mol/I KH2PO4 with 25% methanol (pH = 6.0). Fluorescence signals were
measured using a programmable ﬂuorescence detector (RF-10Axl, Shimadzu,
Japan) at an excitation wavelength of 300 nm and emission wavelength of 420
nm. Fluorescence signals were integrated using Gilson 712 software (Gilson
Medical Electronics, Middleton, WI). Individual adenine nucleotides and
adenosine were identiﬁed by retention times established using known standards.
The standard curves for each individual adenine nucleotide and adenosine were

constructed by using standards dissolved in Krebs’ solution and plotted using

98

peak height as dependent variables and the standard as the independent
variable. The amounts of individual adenine nucleotides and adenosine were
then calculated using linear standard curves. Total ATP overﬂow was a

summation of measured ATP plus its metabolites: ADP, AMP and adenosine.

Measurement of NE by HPLC with electrochemical detection

Aliquots of the collected superfusate (50 pl) were acidiﬁed with phosphoric
acid (pH = 2.6), ﬁltered through a 0.22 pM Cameo 3N syringe ﬁlter and injected
into an isocratic HPLC system equipped with a C—18 reverse-phase analytical
column (5-pm spheres, 250 x 4.6 nm, Biophase ODS, Bioanalytical Systems).
The HPLC column was coupled to a single coulometric electrode-conditioning cell
in series with dual-electrode analytical cells (ESA). The conditioning electrode
potential was set at +0.4 V; the analytical electrodes were set at +0.12 V and -
0.31 V, respectively, relative to the reference electrodes. The HPLC mobile phase
consisted of 1.0 mol/L phosphate-citrate buffer, pH = 2.65, with 0.1 mmol/L
EDTA, 0.0475% sodium octylsulfate, and 5% methanol. The amount of NE in the
samples was determined by comparing peak heights (determined by a Hewlett
Packard Integrator, Model 3393A) with those obtained from standards run on the

same day.

Statistical analysis
All data are expressed as the mean i S.E.M and “n" values refer to the

number of animals from which the data were obtained. Differences between

99

groups were assessed by analysis of variance and Kramer Tukey multiple
comparisons test or Students’ t—test using GraphPad InStat version 3.0 for
Windows 95 (GraphPad Software, San Diego CA). P < 0.05 is considered

signiﬁcant.

Drugs

All drugs were obtained from Sigma Chemical Company (St. Louis, MO).

100

RESULTS

Nerve-mediated ATP release was not changed in DOCA-salt compared with
sham arteries

ATP release caused by consecutive periods of nerve stimulation was
normalized to the basal release prior to nerve stimulation. Normalized ATP
release increased at the third stimulation after two conditioning stimulations and
there was a positive correlation between the basal release and stimulation-
induced ATP release caused by the third stimulation (Table 4.1). Normalized
ATP release caused by the third stimulation was not different from that caused by
the fourth stimulation (Fig.4.1). The normalized release caused by the third
stimulation was used as the release before drug treatment and that caused by
the fourth period of stimulation was used to assess release after drug treatment.
Basal ATP release prior to nerve stimulation and normalized ATP release were
not different between sham and DOCA-salt arteries (Table 4.1). TTX blocked
ATP release caused by nerve stimulation. The normalized release in sham
arteries before TTX treatment was 2.9 :1: 0.7 and after TTX treatment was 1.1 i
0.09 (n == 4, P < 0.05). The normalized release before TTX treatment in DOCA-
salt arteries was 4.7 :I: 1.5 and after TTX treatment was 0.6 i 0.1 (n = 5, P <

0.05).

Increased NE release in DOCA-salt arteries and veins

101

Nerve stimulation produced TTX-sensitive NE release in arteries and
veins. NE release caused by consecutive nerve stimulation was normalized to
the basal release prior to nerve stimulation. Normalized NE release was not
different during the ﬁrst, second and third stimulation (Fig.4.2). There was a
positive correlation between the basal levels and the stimulation-induced NE
release by the second stimulation in both arteries and veins (Table 4.2).
Therefore, normalized release caused by the second stimulation was taken as
the release before drug treatment and that caused by the third stimulation was
used to assess release after drug treatment. The normalized release in sham
arteries before TTX treatment was 1.6 :t 0.1 and after TTX treatment was 0.7 i
0.1 (n = 4, P < 0.05). The normalized release before TTX treatment in DOCA-
salt arteries was 4.1 :I: 1.4 and after TTX treatment was 0.7 :I: 0.2 (n = 4, P <
0.05). The normalized release in sham veins before TTX treatment was 4.6 :l: 0.3
and after TTX treatment was 0.7 :1; 0.3 (n = 4, P < 0.05). The normalized release
before TTX treatment in DOCA-salt veins was 4.4 :I: 0.7 and after TTX treatment
was 1.3 :t 0.5 (n = 4, P < 0.05). Basal NE release was not different between
sham and DOCA-salt arteries or between sham and DOCA-salt veins (Table
4.2). Normalized NE release caused by nerve stimulation was compared
between arteries and veins from sham and DOCA-salt hypertensive rats. The
normalized NE release was increased in both DOCA-salt arteries and veins
compared with their sham counterparts (Table 4.2). In addition, normalized NE

release in sham veins was greater than that in sham arteries (Table 4.2).

102

a2 adrenergic autoreceptor function is impaired in arteries from DOCA-salt
rats

To determine whether (12 adrenergic autoreceptors regulate ATP release,
the effects of the 012 adrenergic receptor antagonist, yohimbine (1 pM). and the
agonist, UK 14,304 (10 pM). on ATP release in arteries from sham rats were
studied. In sham arteries, yohimbine increased ATP release caused by nerve
stimulation whereas UK 14,304 decreased ATP release caused by nerve
stimulation (Fig. 4.3A). In DOCA-salt arteries, the normalized ATP release before
yohimbine treatment was not different from the release after yohimbine treatment
(Fig.4.3B). Similarly, ATP release before UK 14,304 treatment was not different

from the release after UK 14,304 treatment in DOCA-salt arteries (Fig. 4.38).

012 adrenergic autoreceptors associated with mesenteric arteries regulate
NE release and are impaired in DOCA-salt hypertensive rats

Yohimbine increased NE release caused by nerve stimulation in sham
arteries (Fig. 4.4A), but it did not alter evoked NE release in DOCA-salt arteries

(Fig. 4.43)

012 adrenergic autoreceptors are impaired in veins from DOCA-salt rats
We assessed the function of a2 adrenergic autoreceptors associated
with veins by examining the effect of yohimbine (111M) on NE release. NE release

caused by nerve stimulation before and after yohimbine treatment was compared

in sham and DOCA-salt rats. Yohimbine increased NE release in sham veins

103

(Fig. 4.5A), but failed to alter NE release in DOCA-salt veins (Fig. 4.5B).
Conversely, UK 14,304, decreased NE release in sham (Fig. 4.5A), but not

DOCA-salt veins (Fig.4.53).

104

Table 4.1: ATP released is not altered in DOCA-salt arteries. Data are the basal
ATP release, nerve stimulation-evoked release (stimulated release) and evoked
release normalized to basal levels (normalized release) during the third
stimulation period in sham and DOCA-salt arteries. *indicates signiﬁcantly
different from basal release (P < 0.05). There were no differences between basal
release and normalized release between sham and DOCA-salt arteries. “r” is the
correlation coefﬁcient for the relationship between basal and stimulated release.

Both correlation coefﬁcients are different from zero (P < 0.05).

 

 

 

 

 

 

 

Artery

Sham (n=18) DOCA-salt (n=19)
Basal release (ng /ml) 2.7 1 0.4 3.9 1 1.0
Stimulated release (ng/ ml) 6.9 1 1.0* 11.4 1 2.4*
Normalized release (fold increase) 2.9 1 0.3 4.1 1 0.5
r 0.59 0.71

 

 

 

 

105

Table 4.2: NE release from peri-arterial and peri-venous nerves is increased in
DOCA-salt hypertension. Data are basal NE release, nerve stimulation-evoked
release (stimulated release), and evoked release normalized to basal levels
(normalized release) in sham and DOCA-salt arteries & veins. * indicates
signiﬁcantly different from basal release (P < 0.05). # indicates signiﬁcantly
different from normalized release in sham counterparts (P < 0.05). mndicates
signiﬁcantly different from release in sham artery (P<0.05). “r" is the correlation
coefﬁcient for the relationship between basal and stimulated release. All

correlation coefﬁcients are different from zero (P < 0.05).

 

 

Artery Vein
Sham DOCA-salt Sham DOCA-salt
(n=17) (n=15) (n=16) (n=18)

 

Basal release 1331 1 243 2673 1 931 611 1 1001] 4771 83

(Pg/ml)

 

Stimulated 1705 1 284* 7148 1 1799* 1764 1 490* 4624 :1: 1850*

release (pg/ml)

 

 

 

Normalized 1.5 1 0.2 3.9 1 0.7# 2.9 1 0.51] 8.4 1 2.8#
release (fold

increase)

r 0.90 0.90 0.70 0.82

 

 

 

 

 

 

106

A Sham artery B DOCA-salt artery

8 ' (n=6) 8 ' (n=5)

 

 

 

 

Normalized ATP release (fold)
Normalized ATP release (fold)

Stim1 Stim2 Stim3 Stim4 Stim1 Stim2 Stim3 Stim4

Fig.4.1: Stimulation-dependent increase in ATP release from sham and DOCA-
salt arteries. ATP release caused by consecutive stimulations was normalized to
basal levels in arteries. Bars labeled Stim1, Stim 2, Stim 3, and Stim 4 represent
release caused by the ﬁrst, second, third and fourth stimulation periods
respectively. In sham (A) and DOCA-salt arteries (8), Stim1 and Stim2 were not
different from each other and Stim3 and Stim4 were not different from each
other. *indicates signiﬁcantly different from Stim2 (P < 0.05). Data are presented
as mean + SE and were analyzed by one-way analysis of variance with Tukey-

Kramer multiple comparisons test.

107

   
    

 

 

 

1o _ Sham artery (n=5) 1o - DOCA-salt artery (n=5)

15 . g .

8 8 ~ :9, 8 -

a) _ a) ,

9 9

2 6 - 2 6 -

93 , 9 .

‘2‘ 4 - LE 4 -

'o , '0 .

E ‘15 '

E E - ..

Stim1 Stim2 Stim3 Stim1 Stim2 Stim3
C D

10 Sham vein (n=4) 10 - DOCA-salt vein (n=6)

 

 

 

1:: A .

9 3—3 8.

9 % 6'

w .

z ”2’ 4-

'O 'O

2i

«i E

E E l ,
2 3 o ..

Stim1 Stim2 Stim3 Stim1 Stim2 Stim3

Fig. 4.2: NE release was stable during consecutive periods of nerve stimulation.
NE release caused by consecutive stimulations was normalized to basal levels in
sham arteries (A), DOCA-salt arteries (B), sham veins (C) and DOCA-salt veins
(D). Bars labeled Stim1, Stim 2, and Stim 3 represent release caused by the
ﬁrst, second, and third periods of nerve stimulation, respectively. Stim1, Stim2,
and Stim3 were not different from each other in any preparation. Data are mean
+ SE and were analyzed by one—way analysis of variance and Tukey-Kramer

multiple comparisons test.

108

A Sham artery B DOCA-salt artery

:1 Before drug treatment (n=4)
_- After drug treatment (n=4)

:1:

a Before drug treatment (n=4)
' - After drug treatment (n=4)

.1.

O
_L
O

8..

m
, .

 

 

O)
V U V

 

 

Normalized ATP release (fold)
.b

Normalized ATP release (fold)
1;

ON
...

   

1

0
Yohimbine UK14,304 Yohimbine UK14,304

 

 

 

 

 

 

Fig. 4.3: a2-adrenergic receptors modulate ATP release in sham, but not DOCA-
salt arteries. A: ATP release caused by nerve stimulation is compared before
and after yohimbine or UK 14,304 treatment in sham arteries. *indicates
signiﬁcantly different from release before yohimbine treatment. #indicates
signiﬁcantly different from release before UK 14,304 treatment. B: ATP release
caused by nerve stimulation is compared before and after yohimbine or UK

14,304 treatment in DOCA-salt arteries. Data are presented as mean + SE.

109

A Sham artery B DOCA-salt artery

 

 

 

, n=5

8 - (n=5) A 8 ( )

€ . E

E» 9

m 6 ' g 6 "

a m

9 £2

9 9 4 _

F ”:3

'O

.3 9 2 -

(_U N

E E

g z 0 . .
Control Stim Stim w/Yoh Control Stim Stim w/Yoh

Fig. 4.4A: NE release caused by nerve stimulation is potentiated by yohimbine
treatment in sham arteries. *indicates signiﬁcantly different from release before
yohimbine treatment. B: NE release caused by nerve stimulation was not altered
by yohimbine treatment in DOCA-salt arteries. Data are presented as mean +

SE.

110

A Sham vein B DOCA-salt vein

 

 

 

 

E Before drug treatrr‘ent (n=1) :1 Before drug treatment (n=4)

20 l' -Aﬂ8f (11119 treatment (n=4) 20 _ _After drug treatment (n=4)
g f A .
:9, 16 t E 16 -
a I g .
o 12 ~ “I -
E) , g 12
Lu .
Z 8 - % 8 _
‘U _ '0 _
.3 93
‘5 4 - 1:6 4 '
E * # E
‘23 2 o

Yohirrbine UK14,304 Yohimbine UK14,304

Fig. 4.5A: NE release caused by nerve stimulation is increased by yohimbine and
decreased by UK 14,304 in sham veins. *indicates signiﬁcantly different from
release before yohimbine treatment. #indicates signiﬁcantly different from
release before UK 14,304 treatment. B: NE release caused by nerve stimulation
was not altered by yohimbine or UK 14,304 treatment in DOCA-salt veins. Data

are mean + SE.

111

DISCUSSION

Increased sympathetic tone in veins in hypertension

In human hypertension and in SHRs, sympathetic neural input to veins is
increased (Willems et al., 1982). Furthermore, the membrane potential of
venous smooth muscle cells in SHRs is depolarized compared to that found in
normotensive rats, a change that is due to increased sympathetic tone to veins in
SHRs (Willems et al., 1982). It has also been shown that venomotor tone is
elevated in DOCA-salt hypertensive rats and this is largely due to increased
sympathetic input to veins (Fink et al., 2000), which will cause decreased venous
compliance. In hypertension, decreased venous compliance occurs in peripheral
vessels and this change is most prominent in splanchnic veins causing
redistribution of blood toward the heart, leading to increased cardiac output
(Moreau et al., 1995). The shift of blood from the venous side of the circulation
to the arterial side will result in increased arterial blood pressure (Nyhof et al.,
1983). There are a number of changes that could account for increased
sympathetic tone to veins in hypertension. My study is the ﬁrst to directly
measure NE release caused by nerve stimulation in mesenteric veins from
hypertensive rats. I showed that more NE is released during nerve stimulation in
DOCA-salt veins than in sham veins. This result provides one potential
mechanism underlying the increased venomotor tone found in DOCA-salt

hypertensive rats (Fink et al., 2000).

112

We normalized nerve stimulation-induced ATP and NE release to their
basal levels to minimize differences in release that might be due to hypertension-
induced structural changes in blood vessels. For example, hypertrophy of the
smooth muscle of arteries occurs in DOCA-salt hypertension (Schenk and
McNeill, 1992). Therefore, it would be problematic for interpretation of the results
to normalize the stimulation-induced release to tissue weight or total protein. The
correlation between basal release and nerve stimulation-induced ATP and NE
release suggests that the basal release of ATP and NE is from nerve terminals.
This result also justiﬁes normalization of stimulation-induced release to that
occurring under basal conditions. Unaltered basal levels of ATP and NE in
arteries and veins from DOCA-salt hypertensive rats suggest that the overall
number of nerve terminals associated with mesenteric arteries and veins is not
changed in DOCA-salt rats. Therefore, the increased release most likely results
from the increased transmitter release per nerve terminal rather than from
hyperinnervation of those blood vessels. Upregulation in NE release from nerve
terminals in DOCA-salt rats differs from changes known to occur in SHRs where
it has been shown that there is an increased density of sympathetic nerve ﬁbers
in the vasculature (Cassis et al., 1985; Tabei et al., 1995).

There is more NE release from peri-venous nerves per stimulus
compared to pen-arterial nerves as NE release caused by nerve stimulation
normalized to basal levels is greater in veins than in arteries from sham rats.
This result could explain in part why veins are more sensitive to the contractile

effects of sympathetic nerve stimulation than arteries (Hottenstein and Kreulen,

113

1987; Luo et al., 2003). However, postjunctional factors could also contribute to
increased venous sensitivity to sympathetic neural input. For example, venous
smooth muscle cells have a less negative resting membrane potential compared
with the arterial smooth muscle cells (Hottenstein and Kreulen, 1987).

The greater increase of NE release in veins compared to arteries during
nerve stimulation may be related to the different functional properties of
sympathetic nerves innervating arteries and veins (Browning, et al., 1999). The
notion that sympathetic nerves innervating arteries and veins is different was
based on the following evidence: 1) in arteries, stimulation of sympathetic nerves
elicits excitatory junction potentials mediated by ATP followed by a slow
depolarization mediated by NE whereas in veins only slow depolarizations
mediated by NE occur (Evans et al., 1992; Hottenstein and Kreulen, 1987); 2)
sympathetic neurons innervating mesenteric arteries and veins differ in their
localization in prevertebral ganglia and in their electrophysiological properties
(Browning et al., 1999). My data showing a greater increase of NE release
during sympathetic nerve stimulation in veins compared with arteries provides
another important example of the difference in the functional properties of

sympathetic nerves supplying arteries and veins.

a2 adrenergic autoreceptors on peri-venous nerves are impaired in DOCA-

salt rats
Yohimbine increased NE release in sham, but not DOCA-salt veins,

whereas UK 14,304 decreased NE release in sham, but not DOCA-salt veins.

114

These results suggest that the function of prejunctional a2-adrenergic receptors
associated with mesenteric veins is impaired in DOCA-salt hypertensive rats.
This differs from previous data demonstrating integrity of the autoinbihitory
function of a2-adrenergic receptors in portal veins from DOCA-salt rats (Westfall
et al., 1986). This difference in results could be attributed to the difference in
vascular beds studied. Although the portal vein is linked to the mesenteric
circulation, it serves a much smaller capacitance function compared with

mesenteric veins and is less likely to be affected by hypertension.

a2 adrenergic autoreceptors on peri-arterial nerves regulate ATP release
and are impaired In DOCA-salt rats

Nerve stimulation with the parameters used in the present study produces
vasoconstriction that can be blocked by 'l'I’X (Luo et al., 2003) and ATP release
measured here was TTX-sensitive. Therefore, I concluded that ATP release
caused by electrical stimulation is nerve-mediated. ATP is a co-transmitter
released with NE in the vascular system (Bobalova et al., 2001a; Bobalova et al.,
2001b; Bumstock et al., 1985; Gitterman and Evans, 2001), particularly in small
mesenteric arteries where it plays a major role in mediating sympathetic
neurotransmssion (Fink et al., 2000; Luo et al., 2003). My study showed that 012-
adrenergic receptors regulate ATP release as yohimbine increased, whereas UK
14,304 decreased, ATP release in sham arteries. These results are consistent
with previous studies that showed that yohimbine (1 pM) increased the overﬂow

of ATP caused by electrical ﬁeld nerve stimulation (Bobalova et al., 2001b).

115

Taken together these data indicate that ATP and NE release, at least that caused
by 10 Hz stimulation, are regulated in a similar manner.

My study is the ﬁrst to directly measure nerve stimulation-induced ATP
release from sympathetic nerves in DOCA-salt hypertensive rats. My study
showed that ATP release is not altered in mesenteric arteries from DOCA-salt
rats. This result is consistent with studies on ATP release from sympathetic
nerves in the isolated kidney from SHRs where it was shown that ATP release
was not altered compared to that occurring in Wistar Kyoto rats (Bohmann et al.,
1995). In DOCA-salt arteries, a2 adrenergic receptor function is impaired as
yohimbine or UK 14,304 did not change ATP release, but this does not result in
an increase in ATP release. It is possible that changes in other mechanisms
regulating ATP release may compensate for impaired function of a2-adrenergic
receptors in hypertension (Cheung, 1989). For example, it has been shown that
the inhibitory effects of prejunctional prostaglandin E2 receptors on sympathetic
neurotransmitter release are enhanced in SHRs (Rump et al., 1990).

l have shown previously that ATP mediates neurogenic constrictions of
small mesenteric arteries from sham rats while both ATP and NE mediate
neurogenic constrictions in arteries from DOCA-salt rats (Luo et al., 2003). It
was concluded that the increased adrenergic component of neurogenic
constriction is due to increased NE release rather than an increase in
postjunctional reactivity as constrictor responses to NE or phenylepherine are not
different between sham and DOCA-salt arteries (Luo et al., 2003). The present

study showed directly that NE release caused by nerve stimulation was

116

increased in DOCA-salt arteries, further demonstrating an alteration of
sympathetic neuronal function in DOCA-salt hypertension. Impaired function of
prejunctional a2-adrenergic receptors is likely to account for increased NE
release in DOCA-salt arteries as NE release was increased when 112-adrenergic
receptors were blocked by yohimbine in sham arteries. This result is consistent
with studies showing a failure of yohimbine to increase plasma NE level (De
Champlain, 1990; Moreau et al., 1995) and nerve stimulation—induced NE
release by yohimbine in the mesenteric vasculature of DOCA-salt rats (Langer,

1980; Tsuda et al., 1989).

Summary and Conclusion

There is a greater increase of nerve stimulation-induced NE release in
mesenteric veins compared with arteries from sham rats, which indicates a
greater release of NE per nerve terminal. This result provides further support for
the hypothesis that sympathetic nerves with different functional properties
innervate arteries and veins. NE release caused by sympathetic nerve
stimulation is increased in mesenteric arteries and veins from DOCA-salt
hypertensive rats although ATP release is not altered in arteries. Prejunctional
a2-adrenergic receptors on sympathetic nerves regulate ATP release in sham
arteries and NE release in sham arteries and veins. Function of these receptors
is impaired in DOCA-salt arteries and veins, which would contribute to increased
nerve stimulation-induced NE release and increased peripheral arterial and

venomotor tone in DOCA-salt hypertension (Fink et al., 2000; Luo et al., 2003).

117

CHAPTER 5

Modulatory effects of ET receptor activation on sympathetic neuroeffector

transmission to mesenteric arteries and veins in DOCA-salt hypertension

118

Introduction

ET-1 modulates the activity of the sympathetic nervous system and it is a
potent vasoconstrictor (Rubanyi and Polokoff, 1994; Schiffrin, 2001). For
example, ET-1 has modulatory effects on the responses to sympathetic nerve
stimulation of various vascular systems including the rat mesenteric arteries
(Tabuchi et al., 1990), rabbit saphenous arteries (Mutafova-Yambolieva and
Radomirov, 1994) and dog mesenteric arteries (Zhang et al., 1996). There are
three potential mechanisms by which ET-1 acting at its receptors can modulate
sympathetic neuroeffector mechanisms. The ﬁrst mechanism is prejunctional
where ET-1 affects transmitter release. For example, ET-1 inhibits NE release
from rat and dog mesenteric arteries (Tabuchi et al., 1990; Zhang et al., 1996).
However, others showed that ET-1 could both inhibit and increase NE and ATP
release in isolated rat-tail artery and the effect is dependent on the concentration
of ET-1 (Mutafova-Yambolieva et al., 1998). Therefore, it remains controversial
whether the effect mediated by ET-1 on transmitter release is inhibitory or
facilitatory. The second mechanism is postjunctional where ET-1 changes the
postjunctional reactivity of the blood vessels to stimulation by neurotransmitters.
For example, ET-1, at subcontractile concentrations, potentiates contractile
responses to NE in human coronary arteries (Yang et al., 1990c), dog mesenteric
arteries (Zhang et al., 1996) and rat mesenteric arteries (Tabuchi et al., 1990).

The third is a mixed prejunctional and postjunctional mechanism by which ET-1

119

can both alter transmitter release and postjunctional reactivity to the
neurotransmitters (T abuchi et al., 1990; Zhang et al., 1996).

There are two subtypes of receptors for ET-1 in the cardiovascular
system. In arteries, ETA receptors are expressed on vascular smooth muscle
cells and ETB receptors are expressed on endothelium. ETA receptors mediate
vasoconstriction by releasing IP3 and mobilizing intracellular calcium. ETg
receptors in the endothelium mediate vasodilation by releasing NO and
prostacyclin (Rubanyi and Polokoff, 1994). In veins and in the aorta ETg
receptors are also localized to smooth muscle cells where they mediate
vasoconstriction (Moreland et al., 1992; Davenport et al., 1995; Johnson et al,
2000). This conclusion is supported by data showing that 86c, a selective ETB
receptor agonist, contracts mesenteric veins, but not arteries from rats,
suggesting that functional ETB receptors are expressed on smooth muscle cells
of mesenteric veins, but not arteries (Johnson et al., 2002). In addition, ET-1 is a
more potent constrictor of veins than arteries (Warner et al., 1990 and
Leppaluoto et al., 1992; Johnson et al., 2002). This increased potency may be
related to the differences in expression of ET receptor subtypes and receptor
density (Johnson et al., 2002).

Modulation by ET-1 on sympathetic neuroeffector transmission to the
vascular system changes in hypertension (T abuchi et al., 1990). For example, in
isolated mesenteric arteries from SHR and WKY rats, the enhancement of the
responses to NE by a subpressor dose of ET-1 was attenuated in SHR

compared to WKY rats. The inhibitory effect of ET-1 on NE release was also

120

attenuated in SHR compared to WKY rats (Tabuchi et al., 1990). A number of
studies suggest that ET-1 plays an important role in the development of DOCA-
salt hypertension. For example, there is overexpression of ET-1 in DOCA-salt
hypertensive rats (Day et al., 1995; Yu et al., 2002). ET-1 production in cultured
endothelial cells from DOCA-salt hypertensive rats was up-regulated (Takada et
al., 1996). Furthermore, chronic treatment with a mixed ETA/ETB receptor
antagonist decreased blood pressure to a normal level (Doucet et al., 1996). The
elevated sympathetic nerve activity in DOCA-salt hypertension could be due to
altered local modulation on sympathetic neuroeffector transmission to the
vascular system (De Champlain, 1990). Therefore, the role played by ET—1 in the
development of DOCA-salt hypertension could be due to its function as a potent
vasoconstrictor as well as a modulator for the sympathetic nervous system.
However, no study has been done to assess the potential alteration of ET’s
modulatory effects on sympathetic neuroeffector transmission to the vascular
system in DOCA-salt hypertension. My previous study demonstrated that
purinergic transmission dominates sympathetic neuroeffector mechanisms in
sham arteries whereas in DOCA-salt arteries there is increased adrenergic
transmission due to the increased NE release (Luo et al., 2003). It was also
demonstrated that sympathetic nerve activity associated with veins from DOCA-
salt rats is increased as indicated by increased NE release, which results in
decreased postjunctional reactivity to NE (Luo et al., 2003). Whether altered
modulation by ET-1 on sympathetic neuroeffector transmission to mesenteric

arteries and veins contribute to those alterations is unknown.

121

The present study tested the hypothesis that ET-1 modulates sympathetic
neuroeffector transmission to mesenteric arteries and veins and those
modulatory effects by ET-1 are altered in DOCA-salt hypertension. The following
studies were done to test the above hypothesis: 1) Measurement of the effect of
exogenous ET-1 on sympathetic neuroeffector transmission to arteries and veins;
nerve stimulation evoked constriction of arteries and veins were compared before
and after application of an ET receptor agonist. 2) Assess postjunctional effects
of ET-1. Responses of arteries and veins to exogenous ATP and NE were
compared before and after the application of ET receptor agonist. 3) The
postjunctional effect of ET-1 was subtracted from the overall effect in order to
determine the magnitude of the prejunctional effects of ET-1. As a selective ETA
receptor agonist is not available, the effect mediated by ETA receptor on
sympathetic neuroeffector mechanisms can be indirectly measured assessed by
comparing the effects of a by non-selective ET receptor agonist, ET-1, and the
effects of the selective ETB receptor agonist, 86c. The changes in the
modulatory effects associated with DOCA-salt hypertension can be studied by
comparing those effects on mesenteric arteries and veins from sham and DOCA-
salt rats.

There are ET binding sites on cardiac nerve terminals (Davenport et al.,
1989) and paravascular nerves (Power at al., 1989). However, those reports did
not determine whether those nerves were sympathetic nerves. Nor is there any
report with regard to the expression of ET receptors on sympathetic nerve

terminals innervating mesenteric blood vessels. ET binding sites are widely

122

distributed on the vascular smooth muscle cells of various blood vessels
including conductance and resistance arteries, arterioles and veins (Power et al.,
1989; Davenport et al., 1989). However, it is not clear which receptor subtype is
expressed on the vascular smooth muscle cells. Therefore, another goal of the
present studies was to identify the location of ET receptors in the mesenteric
vascular bed with regard to postjunctional or prejunctional location using
immunohistochemical methods. Ultrastructural studies have shown that
sympathetic nerve endings have close contact with smooth muscle cells of
arteries and veins (Luff et al., 1987). lmmunohistochemical studies have
localized the NE synthesizing enzymes: TH in perivascular nerves associated
with mesenteric veins and arteries (Fumess and Marshall, 1974). Co-staining of
ET receptors and TH, a marker for sympathetic nerves, would demonstrate if ET

receptors are localized on the sympathetic nerves.

123

Methods and materials

Animals

See Chapter 3 Methods.

Preparation of DOCA-salt rats

See Chapter 3 Methods.

Measurement of vasoconstriction in vitro
Rats were killed using a lethal pentobarbital injection (50 mg, i.p). The small

intestine was removed and placed in oxygenated (95% 02, 5% 002) Krebs'
solution of the following composition (mM): NaCl 117; KCI 4.7; Ca012 2.5; MgCI2
1.2; NaHCO3 25; NaH2P04 1.2; glucose 11. The ileal segment was placed in a

petri dish and the mesentery was stretched gently and pinned ﬂat. A section of
mesentery close to the ileal wall was carefully cut free from the intestine and the
mesentery was transferred to a small silicone elastomer-lined recording bath (2.5
ml volume). The arteries (4° and 5° order) and corresponding veins were
exposed by carefully cutting away surrounding adipose and connective tissues.
The recording bath containing the preparation was mounted on the stage of an

inverted microscope (Olympus CK-2) and superfused continuously with warm (36

oC) Krebs' solution at a ﬂow rate of 7 ml/min. The output of a black and white
video camera (Hitachi, KP-111) attached to the microscope was fed to a

PCVision Plus frame-grabber board (Imaging Technology Inc., Wobum MA)

124

mounted in a personal computer. The sampling rate was 10 Hz and changes in
blood vessel diameter of 0.5 pm could be resolved. The video images were
analyzed using Diamtrak® software. The digitized signal was converted to an
analog output (DAC—02 board, Keithley Metrabyte) and which was fed to an
analog to digital converter (Labmaster 125) and a second computer running
Axotape software (version 2.0, Axon Instruments, Inc. Foster City CA) for a
permanent record of changes in vein diameter. Analog signals were sampled at
33 Hz and data were stored on the computer hard drive for subsequent display

and analysis.

Drug application

Drugs were applied using a system of 3-way stopcocks so that the
superfusing Krebs’ solution could be changed to one containing a known
concentration of drug. The ﬂow rate was 8 ml/minute and it took one minute for
drugs to reach the tissue. NE and ATP concentration-response curves were
constructed using non-cumulative addition of each agonist concentration. Each
agonist concentration was applied for 2 minutes and there was a 20 minute
interval between application of each agonist concentration. Tissues were washed
continuously with normal Krebs’ solution between each dose. Agonists were
applied for a minimum of 30 minutes prior to testing their effect on agonist- or

nerve-mediated responses.

Transmural stimulation of perivascular nerves

125

Two parallel silver/silver chloride wire electrodes connected to a Grass
Instruments stimulator (S88) were placed perpendicular to the longitudinal axis of
mesenteric tissues. Parameters for nerve stimulation were: 30 stimuli, stimulus
duration 0.5 ms, frequency 0.2 - 30 Hz, voltage 60-140 volts. The neurogenic
origin of constrictions caused by electrical stimulation was veriﬁed in each
preparation by initially demonstrating that a constriction caused by 20 Hz
stimulation was blocked by TTX (0.3 pM). Preparations in which the initial 20 Hz
constriction was not blocked by TTX were discarded. The peak constriction and
in some experiments the duration of vasoconstriction caused by nerve stimulation
was measured. Measurements were made using Axotape software and on-

screen CU I'SOTS.

Immunohistochemical localization of ETB receptors in arteries and veins

1) Tissue ﬁxation: Mesenteric tissues were taken from sham and DOCA-
salt hypertensive rats. Blood was ﬂushed from blood vessels by injecting
phophate buffer saline (PBS) solution into the lumen of the blood vessels with a
30 G 1/2 inch needle. The tissues were ﬁxed with 4% paraforrnaldehyde for 10
minutes and washed with 0.01 M PBS 3 times and 10 minutes each time at room
temperature. Blood vessels were then completely dissected out from the
mesenteric fat and connective tissue.

2) Primary antibody incubation: Mesenteric arteries and veins from sham
and DOCA-salt hypertensive rats were divided into 4 groups by incubating with: i)

primary antibody for ETB receptor (Alamone Laboratories, Jerusalem, Israel;

126

raised from rabbit, kept at -20 °C and diluted to 1:200 in 0.01 M PBS (pH 7.2); ii)
primary antibodies for ETB receptor and TH (Calbiochem, San Diego, CA; raised
in mouse and diluted to 1:200 in 0.01 M PBS, pH 7.2); iii) primary antibody for
ETg receptor preincubated with control antigen for 1 hour; iv) no primary
antibody. The blood vessels were incubated with antisera for 24 hours at room
temperature.

3) Secondary antibody incubation: After washing with 0.01 M PBS 3 times
at 10 minutes each time, blood vessels were incubated in the secondary antibody
for 1 hour in a sealed humidiﬁed chamber. Each of the four groups was
incubated with two secondary antibodies from different species which bind to the
primary antibody for ETB receptors or TH: indocarbocyanine (Cy3) and
ﬂuorescein isothiocyanate (FITC) conjugated lgG (Jackson lmmunoresearch,
PA) diluted (1:200 and 1:40 respectively) in 0.01 M PBS containing 1% normal
goat serum (NGS).

4) Mounting and observation of the blood vessels under ﬂuorescence
microscope. After the blood vessels were washed with 0.01 M PBS 3 times and
10 minutes each time, they were mounted with buffered glycerol (pH 8.0) to
slides coverslipped for ﬂuorescence microscopy. Staining in the groups
incubated with primary antibody for ETg receptors was considered positive when
the other two control groups (no primary antibody or primary antibody
preincubated with control antigen) showed negative staining. For the group
incubated with primary antibodies for both TH and ETB receptors, negative cross

reactivity was assured as the group incubated with primary antibody for TH

127

(raised from mouse) alone does not demonstrate Cy3 (anti-rabbit) ﬂuorescence
and the group incubated with primary antibody for ETg receptors (raised from
rabbit) alone does not demonstrate FITC (anti-mouse) ﬂuorescence.
Furthermore, co-staining of TH and ETg receptors was examined using a
confocal microscopy (Leica Microsystems, Heidelberg, Germany). Images in this

dissertation are presented in color.

Statistical analysis

Agonist-induced constrictions were measured in pm and are expressed as a
percentage of the initial resting diameter of the blood vessel. For NE and ATP
concentration-response curves, E050 and Emax were calculated from a least
squares ﬁt of individual agonist concentration-response curves using the

following logistic function (Origin 5.0 Microcal Software Inc., Northampton, MA):

y = {(Ymin — Emax)/[1 + ( x/ ECso)m]} + Emax
Where “Ymin “ is the minimum response (constrained to 0), “Emax” is the maximum
response, “ECso” is the half maximal effective concentration and “m” is the slope
factor. For nerve stimulation frequency-response curves, the half maximal
effective stimulation frequency (S50) and Emax were calculated from curves
obtained in individual tissues using the following function (Origin 5.0 software):

y = EmaxX / ($50 + X)

where “X” is the stimulation frequency tested and y is the peak response

amplitude. All data are expressed as the mean 1 S.E.M and ”n” values refer to

128

the number of animals from which the data were obtained. Differences between

groups were assessed using Students’ t-test.

Drugs

All drugs were obtained from Sigma Chemical Company (St. Louis, MO).

129

Results

Modulatory effects of ET receptor activation on sympathetic neuroeffector

transmission to mesenteric arteries in DOCA-salt hypertension

1. E T-1 facilitated the nerve- mediated vasoconstriction of sham arteries,
but not DOCA-salt arteries.

To determine the overall effects mediated at ET receptors on sympathetic
neuroeffector transmission to arteries, responses to nerve stimulation from sham
and DOCA-salt arteries before and after the application of ET receptor agonists
were compared. ET-1 with a low concentration (3 pM) increased the Emax of the
frequency-response curves caused by nerve stimulation of sham arteries and
had no effects on that of DOCA-salt arteries (Fig. 5.1, Table 5.1). Similarly, ET-1
with subcontractile concentration (300 pM) increased the Emax of the frequency-
response curves caused by nerve stimulation obtained from sham arteries and
had no effects on that of DOCA-salt arteries (Fig. 5.2, Table 5.2). An ETB
receptor agonist, 860 (30 pM), had no effects on the nerve-mediated response of
arteries from sham and DOCA-salt rats (Table 5.3). However, when the
concentration of S6c increased to 100 pM, it decreased the Sso of the frequency-

response curves of DOCA-salt arteries, but not sham arteries (Table 5.3).

2. E T-1 decreased the reactivity to ATP and increased the reactivity to NE

of sham arteries, but not DOCA-salt arteries

130

To determine the postjunctional effects mediated by ET receptor on
sympathetic neuroeffector transmission to arteries, the reactivity of arteries from
sham and DOCA-salt rats to exogenous ATP before and after the application of
ET receptor agonists were compared. ET-1 (3 pM) increased the E050 of the
concentration-response curves for ATP from sham arteries, but had no effects on
that from DOCA-salt arteries (Table 5.1). Consistently, ET-1 with subcontractile
concentration (300 pM) increased the E050 of the concentration-response curves
for ATP from sham arteries, but had no effect on DOCA-salt arteries (Table 5.2).
Conversely, ET-1 (300 pM) decreased the E050 of the concentration-response
curves for NE from sham arteries, but not DOCA-salt arteries (T able 5.2). $60
(30 pM) had no effect on concentration-response curves for ATP of arteries from

either sham and DOCA-salt rats. The EC5o (mM) of the ATP concentration-

response curves from sham arteries before and after ET-1 application were 0.1 1
0.04 and 0.11 0.03, respectively (n=4, P>0.05). The Emax (%) of the
concentration-response curves from sham arteries before and after ET-1

application were 47.4 1 5.1 and 40.71 2.5, respectively (n=4, P>0.05). The EC50

(mM) of the concentration-response curves from DOCA-salt arteries before and
after ET-1 application were 0.45 1 0.2 and 0.3 1 0.1, respectively (n=6, P>0.05).
The Emax (%) of the concentration-response curves from DOCA-salt arteries
before and after ET-1 application were 49 1 9.9 and 34 1 5.7, respectively (n=6,

P>0.05).

131

Modulatory effects of ET receptor activation on sympathetic neuroeffector

transmission to mesenteric veins in DOCA-salt hypertension

1. 86c facilitated nerve-mediated vasoconstriction of mesenteric veins
from DOCA-salt hypertensive rats.

Responses to nerve stimulation from sham and DOCA-salt veins before
and after the application of ET receptor agonists were compared. ET-1 (3 pM)
had no effect on the response to nerve stimulation of veins (Table 5.4). 86c had
no effect on the maximum response to nerve stimulation of sham veins (Table
5.5, Fig. 5.3A), but increased the maximum response of DOCA-salt veins (Table
5.5, Fig. 5.3B). There was no difference in the Sso of the response to nerve

stimulation before and after the application of 86c (Table 5.5).

2. E T-1 and 860 increased the sensitivity of adrenergic receptors in
mesenteric veins from DOCA-salt hypertensive rats. The reactivity of veins to
exogenously applied NE before and after the application of ET receptor agonists
was compared in sham & DOCA-salt veins to determine their postjunctional
effects of ET receptors on veins. Both ET-1 (Table 5.4) and 86c decreased the
ECso of concentration-response curves to NE from DOCA-salt veins (Table 5.5
and Fig. 54.8) and had no effects on those of sham veins (Table 5.5 and Fig.

5.4.A).

132

Immunohistochemical staining of ETB receptors

Arteries from sham and DOCA-salt rats incubated with primary antibody
for ETB receptors demonstrate positive staining on sympathetic nerves as there
was co-staining from with primary antibodies for TH and ETB receptors. Arteries
incubated with primary antibody for ETg receptors preincubated with blocking
peptide or with no primary antibody demonstrated negative staining (Fig. 5.5 and
Fig. 5.6). Therefore, ETB receptors were localized to sympathetic nerves in
anenes.

Veins from sham and DOCA-salt rats incubated with primary antibody for
ETB receptors demonstrate positive staining on smooth muscle cells that is not
co-localized with TH staining. Veins from sham and DOCA-salt rats incubated
with primary antibody for ETB receptors preincubated with the blocking peptide or
with no primary antibody demonstrated negative staining (Fig. 5.7 and Fig. 5.8).
Therefore, ETB receptors were localized on the smooth muscle cells in veins.

Images in this dissertation are presented in color.

133

A Sham artery B DOCA-salt artery

 

 

 

 

50-
60 ' —0— Before ET-1 (n=4) _ “C'- Before ET-I (n=4)
50’ +AﬂerET-1 (MM 3 3 50- “FAME-1 (39M)
C b / C L
.2 f ‘/ Q ,9 40L
3 4° .' /o’9 E . .5
o\° 20- o\° 20'
1o: /;/10- /
04:11. /R/9 . . . OM’ ELJAUA L . .
1 0 1 10
Frequency(l-lz) Frequenqu-lz)

Fig. 5.1 A: Frequency-response curves caused by nerve stimulation from sham
arteries before and after the application of ET-1. Emax of the frequency-response
curves obtained after the application of ET-1 is greater bigger than that obtained
before the application of ET-1. B: Frequency-response curves caused by nerve
stimulation from DOCA-salt arteries before and after the application of ET-1.
There was no difference in Emax and 850 between concentration-response
curves obtained before and after the application of ET-1. All data are expressed

as the mean + S.E.M and “n” values refer to the number of animals from which

the data were obtained.

134

A Sham artery B DOCA-salt artery

 

 

 

 

60F—o—eeroesr-1(n=4) GOC-O-BeforeET-1 (n=4)
.+MerEr-1(300pM) 50F+AﬂerET-1(3001M
wi—
. c P
2.5.. 40*- .\3 (g) 40L
‘3 3°“ / E T
o .
o _ 0 20.
\° 20 3 /Q o\° I ‘79
0’,,,Q/? , 1.....1 L 0’111n/&é§11111 1
10 1 10
Framers/(Ft) Framers/(It)

Fig. 5.2 A: Frequency-response curves caused by nerve stimulation from sham
arteries before and after the application of ET—1. The Emax of the frequency-
response curves obtained after the application of ET-1 is greater compared with
that obtained before the application of ET-1. B: Frequency-response curves
caused by nerve stimulation from DOCA-salt arteries before and after the
application of ET-1. There was no difference in Emax and $50 between
concentration-response curves obtained before and after the application of ET-1.

All data are expressed as the mean + S.E.M and “n” values refer to the number

of animals from which the data were obtained.

135

fi“ ‘K‘E‘T‘C‘IT'LI

A Sham vein. B DOCA-salt vein

 

 

 

mr—O-Before$c(n=4) 60- —0-BeforeS$c(n=6)
i+AﬂerSSC(309M) . —I—After86c (30le)
c: 50f 50-
3. i g .
g 40- '43 40-
:3 ’ e .
a 3"- 5 '9 30* [/i—ii
o I - o ’
°\ 2°." .49 s 2° T / E‘QD
10» 5’? 10- .ﬁ/
0 ’ 41.1.1 . 14L 11111 . 4 44 0 m
0.1 1 10 0.1 1 10
Freqmin) Frequency(Hz)

Fig. 5.3 A: Frequency-response curves caused by nerve stimulation from sham
veins before and after the application of 86c. There was no difference in Emax
and $50 of the frequency-response curves obtained before and after the
application of S6c. B: Frequency-response curves caused by nerve stimulation
from DOCA-salt veins before and after the application of 86c. The Emax of the
frequency-response curves obtained after the application of 86c is greater
compared with that obtained before the application of 86c. All data are

expressed as the mean + S.E.M and “n” values refer to the number of animals

from which the data were obtained.

136

A Sham vein B DOCA-salt vein

 

 

60 - —o—BeforeSGc(n=8) 60--o—Bafoiesec(n=6)
501+AiterSGc (30pM) >+Nhar86c (301M
: - 5 c 50- \;
-‘-’ 40- 8/ \6 “2c": 40’
8 - 7‘ 2 ~ g~ £3
E 30» 5 III- / Q
n o >
10F 10.
o 0’ /
1E-3 0.01 0.1 1 153 0.01 0.1 1
NE (NW hEMVI)

Fig. 5.4 A: Concentration-response curves for NE from sham veins before and
after the application of S6c. There was no difference in concentration-response
curves obtained before and after the application of 86c. B: Concentration-
response curves for NE from DOCA-salt veins before and after the application of
86c. The ECso of the concentration-response curves obtained after the
application of 86c is smaller compared with that obtained before the application
of $60. All data are expressed as the mean + S.E.M and “n” values refer to the

number of animals from which the data were obtained.

137

51w

B

 

ETBR blocking peptide

Fig. 5.5: Pictures taken from confocal fluorescence microscopy showing ETB
receptors (ETBR, Cy3 conjugated secondary antibody) and TH (FITC conjugated
secondary antibody) are co-stained on arteries from sham rats (A, B, C).
Incubation with ETB receptor antibody preincubated with control antigen (D)

produces no staining on the nerves.

138

DOCA-salt arte

 

u

_' {I

.‘
o

‘
"‘1 t
.‘ \
A
\ ' o - ‘
. 0 . , .7 I

“r” . 4.31.4
.‘p- - ‘V '
s ‘riif. i'

ETBR blocking

t

 

Fig. 5.6: Confocal images showing ETB receptors and tyrosine hydroxylase (TH)
are co-stained on arteries from DOCA-salt hypertensive rats (A, B, and C).
Incubation with ETB receptor antibody preincubated with control antigen

produced no staining on the nerves (D).

139

Sham vein

No 1° ETBR antibody ETBR blocking
peptide

 

Fig. 5.7: ETB receptors staining on veins from sham rats. The ETB receptor
staining at the smooth muscle cells (A) was different from TH staining at
sympathetic nerves (B). Omitting the ETB receptor antibody (C) or incubation
with ETB receptor antibody preincubated with control antigen (D) produces no

staining on the smooth muscle cells.

140

.lmCA-—Salmﬂt

 

Fig. 5.8: Stain ETB receptors on veins from DOCA-salt rats. The ETB receptor
staining at the smooth muscle cells (A) was different from TH staining on
sympathetic nerves (B). Omitting the ETB receptor antibody (C) or incubation
with ETB receptor antibody preincubated with control antigen (D) produced no

staining on the smooth muscle cells.

141

I!

Table 5.1: Constrictions caused by nerve stimulation and exogenous ATP from
sham & DOCA-salt arteries before and after the application of ET-1 (3 pM). All

data are expressed as the mean 1 S.E.M and “n” values refer to the number of

animals from which the data were obtained. *indicates signiﬁcantly different from

the E050 in control sham arteries (p<0.05). ﬂindicates signiﬁcantly different from

the Emax in control sham arteries (p<0.05).

 

 

 

 

 

 

 

 

Sham arteries DOCA-salt arteries
ATP Control (n=7) W/ ET-1 Control (n=6) W/ ET—1

ECso (mM) 0.08 1 0.02 0.25 i 0.05: 0.2 1 0.04 0.3 1 0.06
Emax (%) 61.3 1 3.8 67.4 1 6.3 46.8 1 7.1 44.8 +_ 10.6

Nerve Control (n=4) W/ ET-1 Control (n=4) W/ ET-1
stimulation
850 (Hz) 7.1 1 1.3 5.3 1 0.5 11.11 0.6 9.91 2.3
Emax (%) 44.9 1 5.6 52.4 1 5.11] 41.31 2.6 40.51 4.2

 

 

 

 

 

142

 

Table 5.2: Constrictions caused by exogenous ATP and NE from sham & DOCA-
salt arteries before and after the application of ET-1 (300 pM). All data are

expressed as the mean 1 S.E.M and “n values refer to the number of animals
from which the data were obtained. ﬂindicates signiﬁcantly different from the

EC50 in control sham arteries (p<0.05). *indicates signiﬁcantly different from the

Emax in control sham arteries (p<0.05).

 

 

 

 

 

 

 

 

 

 

 

Sham arteries DOCA-salt arteries
ATP Control (n=5) W/ ET-1 Control (n=4) WI ET-1

E050 (mM) 0.05 1 0.02 0. 13 1 0.021] 0.18 1 0.09 0.16 1 0.06
Emax (%) 68.2 1 5.3 54.6 1 16.2 62.3 1 9.2 55 1 11.7

NE Control (n=4) W/ ET-1 Control (n=4) W/ ET-1
EC50 (pM) 5.3 1 2.6 1.0 1 0.3 1] 3.4 1 1.2 1.6 1 0.6
Emax (%) 61.5 1 10.7 60 1 10.9 59.3 1 9.3 52.1 1 6.2

Nerve Control (n=4) W/ ET-1 Control (n=4) W/ ET-1
stimulation
850 (Hz) 11.5 1 6.9 3.9 1 1.7 8.4 1 0.7 9.6 1 2.4
Emax (%) 29.2 1 2.5 53.4 1 7.9* 21 i 3-3 30 i 5-6

 

 

 

 

 

143

 

Table 5.3: Constrictions caused by nerve stimulation from sham & DOCA-salt

arteries before and after the application of 86c (30 pM and 100 pM). All data are

expressed as the mean 1 S.E.M and “n” values refer to the number of animals

from which the data were obtained. .1. indicates signiﬁcantly different from the S

in control DOCA-salt arteries (p<0.05).

50

 

 

 

 

 

 

 

 

Sham arteries DOCA-salt arteries
Nerve Control (n=4) W/ 86c (30 pM) Control (n=4) W/ 86c (30 pM)
stimulation
S50 (Hz) 6.8 1 1.5 15.8 1 6.8 8.21 2.1 9.91 0.7
Emax (%) 33 1 8.1 35 1 6.8 351 3.8 321 2.9
Nerve Control (n=4) W/ 86c (100 pM) Control (n=4) W/ S6c (100 pM)
stimulation
850(Hz) 5110.9 5311.1 10.2121 4.61211
Emax (%) 37 1 6.8 38 1 8.8 33.3 1 5.0 33.3 1 2.0

 

 

 

 

 

144

 

Table 5.4: Responses to nerve stimulation and to exogenous NE from sham &
DOCA-salt veins before and after the application of ET-1 (3 pM). All data are
expressed as the mean 1 S.E.M and “n” values refer to the number of animals

from which the data were obtained. *indicates signiﬁcantly different from the EC50

in the control DOCA-salt veins (p<0.05).

 

 

 

 

 

 

 

 

Sham veins DOCA-salt veins
NE Control (n=5) W/ ET-1 Control (n=4) W/ ET-1
E050 (pM) 0.0571 0.03 0.054 1 0.02 0.27 1 0.07 0.04 1 0.016:
Emax (%) 60 1 2.5 65 1 2.7 35.1 1 4.3 44.4 1 8
Nerve Control (n=4) W/ ET-1 Control (n=5) W/ ET-1
stimulation
350 (Hz) 5.9 1 4.0 4.31 1.9 4.9 1 0.8 4.5 1 0.7
Emax(%) 31117 33111 40111 4318

 

 

 

 

 

145

 

Table 5.5: Responses to nerve stimulation and to exogenous NE from sham 8.

DOCA-salt veins before and after the application of 86c (30 pM). All data are

expressed as the mean 1 S.E.M and “n” values refer to the number of animals

from which the data were obtained. *indicates signiﬁcantly different from the EC50

in the control DOCA-salt veins (p<0.05). 1} indicates signiﬁcantly different from

the Emax in control DOCA-salt veins (p<0.05)

 

 

 

 

 

 

 

 

Sham veins DOCA-salt veins
NE Control (n=8) W/ 86c (n=8) Control (n=6) W/ 860
ECSO()1M) 0.04 1 0.01 0.03 1 0.01 0.15 1 0.01 0.02 1 0.01*
Emax (%) 46 1 4.5 48 1 5.1 48 1 5.1 54 1 6.3
Nerve Control (n=4) W/ 860 Control (n=6) W/ 86c
stimulation
S50 (Hz) 3.2 1 1.4 6.1 1 0.8 3.4 1 1.6 2.2 1 0.6
Emax (%) 31 1 7.0 36 1 9.4 22.1 1 1.6 32.9 1 2.9 e

 

 

 

 

 

146

 

DISCUSSION

ETA receptors mediate facilitation of sympathetic vasoconstriction to sham
arteries.

A low concentration (3 pM) of ET-1 potentiated neurogenic responses to
nerve stimulation of sham arteries as it increased the Emax of the frequency-
response curves. ET—1 at a subcontractile concentration (300 pM) potentiated
responses to nerve stimulation in sham arteries as it increased the Emax of the
frequency-response curves to nerve stimulation. Furthermore, the potentiation
caused by ET-1 at 300 pM is greater than that by ET-1 at 3 pM. The potentiation
by ET-1 on contractile responses to sympathetic nerve stimulation is consistent
with the results obtained from other studies (Tabuchi et al., 1990; Mutafova-
Yambolieva and Radomirov, 1994; Zhang et al., 1996). For example, ET-1 (300
pM) enhanced the pressor responses to nerve stimulation of the rat mesenteric
arteries (Tabuchi et al., 1990). Yet, to exert facilitatory effect on the responses of
dog mesenteric arteries to sympathetic nerve stimulation, the concentration of
ET-1 was raised to 1 nM (Zhang et al., 1996). This result suggests that different
concentrations are required for ET-1 to exert potentiating effects on sympathetic
neuroeffector transmission to the mesenteric arteries from animals of different
species.

ET-1 (300 pM) also increased the reactivity of sham arteries to NE as it
decreased the E050 for NE. This result is similar to that reported in other studies

demonstrating a potentiating effect on the reactivity of mesenteric arteries to

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exogenous NE by ET-1 with similar concentrations (T abuchi et al., 1990; Zhang
et al., 1996). Studies done on human mammary arteries also demonstrated a
facilitation of the constrictions to NE by ET-1 with the same concentration (300
pM, Yang et al., 1990). Therefore, the facilitatory effect mediated by ET-1 on
sympathetic neuroeffector transmission to sham arteries is likely to be via the
postjunctional mechanism involving a potentitation of the reactivity of the
adrenergic receptors. Conversely, ET-1 decreased the reactivity of sham arteries
to ATP as it increased the E050 of the concentration-response curves for ATP.
This is different from the result obtained from studies using rabbit saphenous
artery that showed a potentiated effect on the responses to exogenous ATP by
ET-1 (Mutafova-Yambolieva and Radomirov, 1994). This discrepancy may be
related to the different vascular bed used. Taken together, these results indicate
that ET-1 has a differential modulatory effect on the reactivity of P2 and
adrenergic receptors. Furthermore, the facilitatory effect mediated by ET-1 on
postjunctional reactivity is mediated by ETA receptors as it was shown that there
was no functional expression of ETB receptors on the arterial smooth muscle
cells (Johnson et al., 2002). My study was unable to demonstrate positive
staining of ETg receptors on the arterial smooth muscle cells and any effect of
$60 on responses of arteries to exogenous ATP.

86c had no effect on the responses of sham arteries to nerve stimulation.
This result is similar to that obtained from other studies showing that the
modulatory effect caused by ET-1 on dog mesenteric arteries was mediated by

ETA receptors (Zhang et al., 1996). However, 86c potentiated the responses of

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DOCA-salt arteries to nerve stimulation as it decreased the S50 of the frequency-
response curves of DOCA-salt arteries caused by nerve stimulation. My study
also demonstrated positive staining for ETB receptors at the sympathetic nerve
terminal innervating the mesenteric arteries. Therefore, it appears that in DOCA-
salt arteries ETg receptors located at the sympathetic nerve terminal facilitate
sympathetic neuroeffector transmission to DOCA-salt arteries. Yet, ET-1 had no
effect on the vasoconstrictions of DOCA-salt arteries caused by nerve stimulation
as there was no difference in frequency-response curves caused by sympathetic
nerve stimulation from DOCA-salt arteries obtained before and after the
application of ET-1. Combined together, these results suggest that ETA receptors
must mediate an inhibitory effect on sympathetic neuroeffector transmission to
DOCA-salt arteries to counteract the facilitatory effect mediated by ETB
receptors. This notion is supported by other studies showing that ET-1 acting at
ETA receptors inhibited NE release from sympathetic nerves innervating
mesenteric arteries from dog (Zhang et al., 1996). Furthermore, ET-1 had no
effect on the postjunctional reactivity of DOCA-salt arteries to ATP and NE as
there was no difference in both concentration-response curves for NE and ATP
obtained before and after the application of ET-1. The disappearance of
modulatory effects by ET-1 on DOCA-salt arteries is likely due to the
desensitization of the ET receptors on the arterial smooth muscle cells from
DOCA-salt rats as indicated by a reduced ET-1 induced constrictions of
mesenteric arteries from DOCA-salt rats (Johnson et al., 2002). This decreased

reactivity is likely a result of the increased level of circulating ET-1 level in DOCA-

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salt hypertensive animals (Day et al., 1995;Yu et al., 2002). Hence, the
facilitatory effect by ET-1 on sympathetic neuroeffector transmission to sham
arteries with normal ET receptors suggest that increased ET-1 level would
enhance the sympathetic constriction of arteries. This effect would contribute to
the elevation of blood pressure, and likely occurs during the development of

DOCA-salt hypertension when the ET receptors are not yet desensitized.

ETB receptors mediate facilitation of sympathetic vasoconstriction of
DOCA-salt veins via a postjunctional effect.

ETg receptors are differentially expressed in arteries and veins as they are
expressed on the smooth muscle cells of the veins and on the sympathetic
nerves of the arteries. The present study used subcontractile concentrations of
ET-1 (3 pM) and $60 (30 pM) based on the results obtained from studies by
Johnson et al., (2002). These concentrations are also similar to endogenous
levels of ET-1 (Rubanyi and Polokoff, 1994). S6c potentiated the responses to
nerve stimulation of DOCA-salt veins, but not sham veins as it increased the Emax
of the frequency-response curves of DOCA-salt veins to nerve stimulation. 86c
also increased the reactivity of DOCA-salt veins, but not sham veins to NE as it
decreased the E050 of the concentration-response curves for NE. Similarly, ET-1
increased the reactivity of DOCA-salt veins to exogenously applied NE (Table
5.3.). Those results combined together suggest that ET-1 acting at ETg receptors
potentiates sympathetic neuroeffector transmission to veins from DOCA-salt rats

at least partly by increasing postjunctional reactivity to NE. However, ET-1 had

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no effects on nerve-mediated response of DOCA-salt veins. Possibly, inhibitory
prejunctional ETA receptors are activated by ET-1 in DOCA-salt veins to
counteract the facilitatory postjunctional ETg receptors. The reactivity of ET
receptors is maintained in DOCA-salt veins (Johnson et al., 2002), suggesting
that there is no functional change of the ET receptors per se. Therefore, the
emergence of a facilitatory effect on sympathetic neuroeffector transmission to
DOCA-salt veins by 86c can attribute to factors other than a functional change of
the ET receptors such as the coupling of the intracellular signaling pathways
downstream from the ET and adrenergic receptors.

Studies in vitro found that ET—1 was in the post arterial bed, i.e., capillaries
and veins (Hemsen and Lundberg, 1991) and ET-1 binding sites were distributed
in the veins (Power et al., 1989; Davenport et al., 1989). Studies in vivo showed
that intravenous administration of ETs increased central venous pressure,
venous resistance and MCFP (Waite and Pang, 1990; Yamamato et al., 1980). In
DOCA-salt hypertension, ET-1 induced constriction was reduced in rat aorta
(Watts et al., 2002) and mesenteric arteries (Johnson et al., 2002), but
maintained in vena cava (Watts et al., 2002) and mesenteric veins (Johnson et
al., 2002). The venous system plays an important role in blood pressure
regulation by serving a capacitance function. In DOCA-salt rats there is an
elevated sympathetic drive to the venous system as indicated by a greater
decrease of the MCFP by hexamethonium, a blocker of sympathetic ganglionic
transmission, on DOCA-salt rats compared to the control rats (Fink, et al., 2000).

Elevated sympathetic drive to the venous system would increase venous return

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and cardiac output, which will lead to elevated blood pressure (Guyton et al.,
1972b). Although ET-1 and the sympathetic nervous system control
venoconstriction (Waite and Pang, 1992), and ETA antagonist did no alter the
effect of hexamethonium on MCFP. Therefore endogenous ET-1 doesn’t
modulate sympathetic venoconstriction through ETA receptors (Fink et al., 2000).
This result is consistent with the result obtained from the present study
suggesting that ETB receptors located at the venous smooth muscle cells
mediating the facilitatory effect caused by ET—1 on sympathetic neuroeffector
transmission to the veins from DOCA-salt rats. Hence, the modulatory effects by
ET-1 acting at ETB receptors on sympathetic neuroeffector transmission to veins
indicated by the present study are likely to contribute to the MCFP elevation

observed in DOCA-salt rats (Fink et al., 2000).

Summary and Conclusion

The present study suggests that ET-1 acting at ETA receptors facilitate
sympathetic neuroeffector transmission to mesenteric arteries from sham rats,
which could raise blood pressure in the development of hypertension. This
facilitation by ET-1 was removed in DOCA-salt hypertension possibly due to the
desensitization of ETA receptors. My study also showed that ET-1 acting at ETg
receptors facilitate sympathetic transmission to veins from DOCA-salt rats via a
postjunctional mechanism. This facilitation is likely to contribute to the increased

MCFP observed in DOCA-salt rats (Fink et al., 2000).

152

CHAPTER 6

General discussion

153

1. Differential sympathetic neuroeffector transmission to arteries and

veins from normotensive rats

1.1. Different neurotransmitters mediating sympathetic neuroeffector

transmission to small mesenteric arteries and veins

My study showed that ATP is the dominant neurotransmitter mediating
sympathetic constriction of the small mesenteric arteries, whereas in mesenteric
veins NE is the transmitter mediating smooth muscle contraction. A recent study
using confocal ﬂuorescence microscopy to visualize Ca2+ transients in
perivascular nerves and adjacent smooth muscle cells from rat mesenteric small
arteries in response to electrical ﬁeld stimulation with both low (< 1 Hz) and high
frequencies (10 Hz) demonstrated that the local Ca” transients are due to
activation of P2X receptors by ATP released from sympathetic perivascular
nerves. Consistent with my results, these studies provided further evidence that
purinergic components mediate a component of sympathetic neuroeffector
mechanisms in small mesenteric arteries (Lamont and Wier, 2002; Lamont et al.,
2003). The purinergic component predominates during the early phases of a train
of stimulation (< 20 s) as in my study where the train length was 3 3. When the
stimulation persists beyond that duration and last for 3 minutes, a slowly rising
force and asynchronous propagating Ca2+ waves resulting from the release of

NE acting at a1-adrenergic receptors became dominant (Lamont et al., 2003).

154

Therefore, it appears that NE dominates sympathetic neuroeffector mechanisms
during the later part of a long train of stimulation. Yet, studies investigating the
discharge pattern of perivascular sympathetic nerve ﬁbers innervating the tail
artery from normal rats in vivo suggest that neuronal activity occurs in regular
short bursts of high frequency ﬁring rather than long sustained trains (Johnson
and Gilbey, 1996). Hence, prolonged impulses of sympathetic nerves are more
likely to occur under unique physiological or pathophysiogical conditions such as
essential hypertension when nerve activity is enhanced.

Results obtained from my studies suggest that: 1) as constrictions of small
arteries and arterioles are the major source of peripheral resistance, ATP plays a
role in controlling peripheral resistance and thus blood pressure regulation under
normal conditions. ATP causes a multitude of cardiovascular effects such as
positive inotropy in the heart, platelet aggregation, release of endothelial factors,
and growth stimulation of vascular smooth muscle cells (Eriinge, 1999); 2)
differential sympathetic neural control of arteries and veins (Dehal et al., 1992;
Browning et al., 1999), which may be one mechanism underlying the different
function performed by arteries and veins in mesenteric vascular bed (eg.

resistance vs. capacitance).

1.2. Enhanced NE release relative to basal levels in veins compared to arteries

My study showed that basal release of NE was strongly correlated to

stimulation-induced release of NE in arteries and veins, suggesting that basal

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release of NE comes from the nerve terminals. Other studies using continuous
amperometry demonstrated that there was spontaneous release of NE from
nerve terminals (Brock et al., 2000). These results suggest that basal release of
NE measured in my study was due to the spontaneous release of NE from
sympathetic nerve terminals. Furthermore, my study showed that there is a lower
basal release of NE in sham veins compared to sham arteries. This is consistent
with other reports that sympathetic innervation of veins is generally less than
arteries (Nilsson, 1985).

My study also showed that there was more NE release measured as
normalized release per impulse of nerve stimulation in veins compared to
arteries. This provides further evidence supporting the notion that sympathetic
nerves supplying veins and arteries are different (Kreulen, 1986; Hottenstein and
Kreulen, 1987). In addition, greater release of NE per impulse of nerve
stimulation in veins compared to arteries could partly account for the observation
that veins are more sensitive to nerve stimulation than arteries (Kreulen, 1986;

Hottenstein and Kreulen, 1987; Luo et al., 2003).
1.3. Veins are more sensitive than arteries to nerve stimulation.

As frequency response curves caused by nerve stimulation of veins are
shifted to the left of arteries, veins are more sensitive than arteries to nerve

stimulation as shown previously (Kreulen, 1986; Hottenstein and Kreulen, 1987).

The potential causes accountable for this phenomenon are discussed below.

156

In small mesenteric arteries, ATP is the dominant neurotransmitter
mediating sympathetic smooth muscle constraction whereas in mesenteric veins
NE is the transmitter mediating venous smooth muscle contraction. NE is about
20 fold more potent than ATP in contracting arteries (Luo et al., 2003). Given that
the concentrations of the neurotransmitter at the neuroeffector junction depend
the ﬁring frequency of the nerve terminals, it would require higher frequency to
release enough ATP to produce the constrictions of the same amplitude as that
caused by NE. Accordingly, by having ATP instead of NE as the dominant
transmitter under physiological conditions small mesenteric arteries have a
capacity to resist changes and ﬂuctuations in neural input compared to their
corresponding veins.

There is a greater release of NE per impulse in veins compared to arteries.
Studies by Nilsson (1985) showed that the density of sympathetic innervation of
mesenteric veins was less than arteries. Furthermore, innervation density is
associated with maximum responses to nerve stimulation as maximum
responses from veins are smaller than arteries (Nilsson, 1985). Therefore, the
greater release of NE per impulse in veins compared to arteries is not likely due
to the difference in innervation density of arteries and veins. Rather, it is due to
the difference in functional properties of sympathetic nerves innervating veins
and arteries. Different functional properties of sympathetic nerves innervating
arteries and veins are demonstrated by the following results.

Although it was reported that there is ATP release from sympathetic

nerves innervating mesenteric veins in responding to nerve stimulation (Bobalova

157

et al., 2001a; Bobalova et al., 2001b), it does not prove that ATP mediates
sympathetic neuroeffector transmission to mesenteric veins. It is possible that the
released ATP functions as a neuromodulator provided that it is released from the
synaptic vesicles. My studies demonstrated that in veins NE is the sole
neurotransmitter as prazosin completely blocked the venoconstriction caused by
nerve stimulation. This result is also consistent with other studies showing that
NE mediats slow depolarization of venous smooth muscle cells leading to the
contractions (Evans and Surprenant, 1992; Hottenstein and Kreulen, 1987; Luo
et al., 2003). However, in arteries from normotensive rats, ATP is the dominant
transmitter mediating sympathetic neuroeffector transmission. Sympathetic
neurons innervating mesenteric arteries and veins differ in their localization in
prevertebral ganglia and in their electrophysiological properties (Browning et al.,
1999). There is a greater postjunctional sensitivity to NE in veins compared to
arteries. This is supported by results showing that veins are more sensitive to
exogenous NE than arteries as the EC50 for NE from veins is smaller than that
from arteries (Luo et al., 2003). This greater sensitivity of veins to NE compared
to arteries indicates that there is a difference in the postjunctional mechanisms
mediating NE-induced smooth muscle contractions. This greater postjunctional
sensitivity to NE in veins could be due to the more depolarized membrane
potential of venous smooth muscle cells compared to arterial smooth muscle
cells (Kreulen, 1986) or more effective electrical coupling of venous smooth

muscle cells (Nilsson, 1985).

158

Studies on dog mesenteric artery and vein showed that the binding afﬁnity
for prazosin was lower in veins compared with arteries whereas the maximum
binding was similar, suggesting that the total amount of a1-adrenergic receptors
expressed on venous and arterial smooth muscle cells is similar whereas the
afﬁnity of the a1-adrenergic receptors is lower in veins (Shi et al., 1989).
Therefore, the greater postjunctional sensitivity to NE in veins is unlikely be due
to the differences in density or binding afﬁnity of the adrenergic receptors as the
afﬁnity is lower in veins. These results suggest that factors involved in the
intracellular signaling pathway downstream to the receptors play an important

role in determining venous sensitivity to adrenergic stimulation.

As veins are more sensitive than arteries to sympathetic nerve stimulation,
similar increases in sympathetic nerve activity would increase venous tone
before changing arterial tone. The splanchnic circulation holds one-third of total
blood volume and this store resides largely in veins (Greenway, 1983). As the
sympathetic nervous system is the principal mechanism for controlling venomotor
tone (Monos et al., 1995), small increases in sympathetic nerve activity would
increase venous return and cardiac stroke volume. Therefore, at low levels of
sympathetic nerve activity, vascular-based increases in systemic blood pressure

could be due, in part, to an increase in venomotor tone.

159

2. Alterations in sympathetic neuroeffector mechanisms in arteries

and veins in DOCA-salt hypertension

2.1. Alterations in sympathetic neuroeffector mechasnims in arteries in

DOCA-salt hypertension

2.1a. Unaltered basal release of ATP and NE in DOCA-salt arteries

The basal release of ATP and NE were correlated to nerve stimulation-
induced release of ATP and NE, respectively suggesting that basal release is
spontaneous release of those neurotransmitters. Many studies showed that there
is spontaneous release of ATP from sympathetic nerve terminals. For example,
spontaneous excitatory junctional currents (SEJCs) mediated by spontaneously
released ATP from sympathetic nerves were recorded in rat mesenteric arteries
(Astrand and Stjame, 1989). SEJCs and EJCs as a result of spontaneous and
evoked release of ATP, respectively from sympathetic nerves innervating the
guinea-pig vas deferens were identical in amplitude and time course (Brock and
Cunnane, 1988). Furthermore, spontaneous Ca2+ transients were observed in
smooth muscle cells of rat mesenteric small arteries and are caused by the
actions of spontaneously released ATP from sympathetic nerve terminals
(Lamont and Wier, 2002).

SEJCs produced by spontaneously released transmitters were unaffected
by TTX, suggesting that spontaneous release does not require action potentials

(Brook and Cunnane, 1988; Astrand and Stjame, 1989). Furthermore, my result

160

showing that basal NE release is not altered in DOCA-salt arteries despite
elevated sympathetic nerve activity supports the notion that basal release is
nerve activity independent. In addition, spontaneous release per nerve terminal
is likely to be derived from single quanta (Astrand and Stjame, 1989; Stjame et
al., 1994). Accordingly, the magnitude of spontaneous release per nerve terminal
would depend upon the quanta size and hence the overall spontaneous release
at the neuromuscular junctions would depend upon both the quantal size and
innervation density. My study showed that the basal release of ATP and NE are
not different between sham and DOCA-salt arteries. These results suggest that
the spontaneous release of ATP and NE and thus the innervation density of
mesenteric arteries is not altered in DOCA-salt hypertension assuming that
quantal size is not altered. Studies on mesenteric arteries from SHRs yielded
similar results as spontaneous release of ATP measured by EJPs was not

different between SHRs and WKYs (Brock and Van Helden, 1995).

2.1b. Increased nerve stimulation-induced release of NE, but not ATP

My study directly demonstrated that nerve stimulation-induced release of
NE was increased in DOCA-salt arteries compared to sham arteries, indicating
that there is an alteration of sympathetic neuronal function in DOCA-salt
hypertension. This result is consistent with a previous result obtained from our
laboratory showing that NE content was reduced in DOCA-salt arteries (Luo et
al., 2003). This result and conclusion is also similar to those reported previously

in studies of DOCA-salt rats (Crabb et al., 1980; Tsuda et al., 1989). For

161

example, signiﬁcant decreases in the NE content of the mesenteric artery, renal
artery and cardiac tissue were found in DOCA-salt rats (Crabb et al., 1980).
Electrical stimulation of sympathetic innervation caused a signiﬁcantly greater
release of NE into the mesenteric vasculature of DOCA-salt hypertensive rats
than in age-matched normotensive controls (Tsuda et al., 1989). As ATP release
was not altered in DOCA-salt arteries, increased NE release is likely to account
for the increased neurogenic contractions of smooth muscle in DOCA-salt
anenes.

Augmentation of purinergic transmission to mesenteric arteries (Brock and
Van Helden, 1995) and vas deferens (Guitart et al., 2002; Naito et al., 1998)
occurs in tissues from SHRs. However, my data indicate that there was no
alteration in nerve mediated ATP release or postjunctional reactivity to ATP in
small mesenteric arteries from DOCA-salt rats. This conclusion is based on the
results showing that nerve stimulation-induced ATP release and reactivity to
exogenous ATP was not altered in DOCA-salt arteries. The discrepancy between
my result and the results obtained from studies on SHR is likely due to the
different hypertension model used for the studies. Nevertheless, this is the ﬁrst
study that has assessed ATP release from sympathetic nerve terminals in the

mesenteric vascular bed in DOCA-salt hypertension.

2.1c. Increased noradrenergic component of neurogenic constrictions in

arteries from DOCA-salt rats

162

My study showed that there is an increase in the contribution of NE to
sympathetic neuroeffector mechanisms in DOCA-salt arteries as prazosin-
induced inhibition of neurogenic constrictions was greatly increased in DOCA-salt
arteries. This is a novel ﬁnding, indicating that DOCA-salt hypertension is
associated with a change in the function of sympathetic nerves ﬁbers associated
with small mesenteric arteries.

Changes in postjunctional reactivity are not likely to contribute to the
increased adrenergic component as the response of DOCA-salt arteries to
exogenous NE is not altered. However, unaltered reactivity to exogenous NE does
not necessarily indicate that there is no change in afﬁnity or density of the
a1—adrenergic receptors. Postjunctional reactivity depends on not only the density
and afﬁnity of the receptors, but also the cellular capacities to activate the speciﬁc
second messenger pathways (De Champlain, 1990). It was shown that there is
dissociation between receptor density, afﬁnity and reactivity to exogenous agonists.
For example, there was increased density of mesenteric cad-adrenergic receptor
binding sites with a reduced afﬁnity in mesenteric arteries from DOCA-salt rats. The
increased density of a1-adrenergic receptors did not correlate with the vascular
responses to electrical nerve stimulation and exogenous agonists (Meggs et al.,
1988). Similarly, an augmented vascular sensitivity in mesenteric arteries from
DOCA-salt hypertensive rats to NE co-exists with unaltered adrenergic receptor
afﬁnity (Perry and Webb, 1988). These results suggest that additional factors
participate in the vascular response to adrenergic stimulation in this model including

an alteration in a post-receptor activation event.

163

It is also unlikely 012- adrenergic receptor upregulation in small mesenteric
arteries and direct activation of these receptors accounts for the increased
neurogenic smooth muscle contractions in DOCA-salt tissues. This conclusion is
based on data showing that clonidine or UK 14,304 did not directly contract small

mesenteric arteries from sham or DOCA-salt rats.

2.1d. Potentiated neurogenic constrictions in mesenteric arteries

Constrictions caused by exogenous ATP and NE were not altered in DOCA-
salt arteries, suggesting that the postjunctional reactivity to these
neurotransmitters are maintained. Therefore, the potentiated neurogenic
constrictions caused by nerve stimulation in DOCA-salt arteries compared to
sham arteries must result from increased NE release, as ATP release was not

altered.

Combined together, these data ﬁndings suggest that there is a potentiated
adrenergic transmission in arteries in DOCA-salt hypertension. These results
also conﬁrm the notion that there is increased sympathetic nerve activity in

DOCA-salt hypertension.

2.2. Alterations in sympathetic neuroeffector mechanisms in veins in
DOCA-salt hypertension

2.2a. Increased NE release in DOCA-salt veins

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There were maintained neurogenic constrictions in the presence of
decreased postjunctional reactivity and increased NE release. The maintained
neurogenic constriction is based on an increased release of NE from sympathetic
nerves associated with mesenteric veins from DOCA-salt hypertensive rats. NE
release from sympathetic nerves is increased according to the following
evidence: i) NE content was lower in DOCA-salt veins due to a depletion of
sympathetic NE stores (Luo et al., 2003); ii) direct measurements on the
superfusate obtained from the blood vessels showed that nerve stimulation-
induced NE release is increased in DOCA-salt veins. This result provides a
potential mechanism underiying the increased venomotor tone found in DOCA-
salt hypertensive rats (Fink et al., 2000). This was the ﬁrst study that directly
measured NE release in mesenteric veins and showed that NE release is
increased in veins from DOCA-salt hypertensive rats.

My study also demonstrated that the neurogenic constrictions of DOCA-
salt veins are maintained whereas a previous study from our laboratory showed
that there was decreased postjunctional reactivity to NE (Luo et al., 2003).
Considering that neurogenic constrictions depend on both the amount of
transmitter release and postjunctional reactivity. The increased nerve mediated
NE release demonstrated in my study provides direct evidence that sympathetic
nerve activity is elevated in DOCA-salt veins. Reduced postjunctional reactivity
results in the failure for the neurogenic constrictions of veins to increase despite

the increased NE release.

165

2. 2b. Increased NE uptake activity

My study demonstrated that NE uptake is increased in DOCA-salt veins,
which may occur in response to the increased NE release and hence provides a
compensatory mechanism to offset the potential for augmented postjunctional
actions of NE. This is the ﬁrst study that assessed NE uptake activity in
mesenteric vascular beds in DOCA-salt hypertension. As the decreased
postjunctional reactivity is reversed when NET uptake is blocked, it suggests that
the decreased postjunctional reactivity is due to the decreased availability of NE
as a result of enhanced NET uptake activity. Therefore, the increased NE release
results in enhanced NET uptake activity, which leads to decreased postjunctional
reactivity. Similar ﬁndings were obtained from studies on mesenteric and tail
arteries in SHRs (Webb and Vanhoutte, 1981; Whall et al., 1980; Zsoter and
Wolchinsky, 1983). However, studies on caudal arteries of DOCA-salt
hypertension indicate that there was no change in uptake activity (Longhurst et
al., 1988). In addition, in vivo studies suggest that neuronal uptake of NE was
not altered in DOCA-salt hypertension (Drolet et al., 1989). Therefore, it appears
that there are vascular bed-dependent alterations of NE uptake activity in DOCA-
salt hypertension.

The increased uptake of NE usually results from enhanced release
and/or synthesis of the neurotransmitter (Houchi et al., 1993), which could serves
as a compensatory mechanism for the increased NE release in DOCA-salt rats.
However, the precise mechanism underlying the enhanced NE uptake activity

remains to be elucidated. It could be due to a direct action of NE on NET or

166

through indirect mechanism. For example, it was shown that enzyme activities
such as Na*-K+-ATPase and MgZ+-activated ATPase are increased in mesenteric
arteries of hypertensive animals (Gardner and Laing, 1965; Oka and Angrist,
1967; Ooshima, 1973). Since NE uptake is activated by Na*-K*-and Mg”-
dependent ATPase, their enhanced activities could account for the increased NE

uptake in DOCA-salt hypertension.

2.3. Sympathetic nerve activity is increased in both arteries and veins from

DOCA-salt hypertensive rats.

2.3a. Increased NE release is due to the facilitated transmitter-releasing
process in DOCA-salt arteries and veins. 7

In my studies, the same nerve stimulation parameters were used to cause
neurogenic constrictions of sham and DOCA-salt arteries and veins. Therefore,
the increased NE release observed in DOCA-salt arteries and veins is not due to
increased ﬁring frequency of the sympathetic nerves associated with these blood
vessels which could occur in vivo. Instead, changes in the transmitter-releasing
process at the axonal terminal level must account for this phenomenon. These
changes could include: i) more effective electrical coupling due to changes in
electrical properties of the axonal plasma membrane; ii) increased NE synthesis
and storage; iii) increased size of the readily releasable pool of NE. These
changes could partly result from attenuated inhibitory effects by autoreceptors

and enhanced facilitatory effects by heteroceptors.

167

Consistent with the antihypertensive effects of drugs that block adrenergic
receptors, these results support the notion that NE plays an important role in the
pathogenesis of hypertension. Increased NE release in DOCA-salt arteries and
veins could contribute to the increased neurogenic constrictions of the blood
vessels. Increased NE release in arteries and veins from DOCA-salt
hypertensive rats also suggests that sympathetic nerve activity is increased
during the maintenance stage of hypertension. Thus, it favors the hypothesis that
sympathetic nervous system not only plays an important role in the initiation of

hypertension, but also the maintenance of DOCA-salt hypertension.

2. 3b. NE acts directly at a1 adrenergic receptors in mesenteric arteries

and veins from sham and DOCA-salt rats.

My study demonstrated that only 611-adrenergic receptors directly mediate
NE-induced constrictions of arteries and veins, which is consistent with other
studies (Shi et al., 1989; Cauvin and Malik, 1984). However, others have shown that
in canine mesenteric veins 112-adrenergic receptors contribute signiﬁcantly to NE-
induced constrictions (Shi et al., 1990; Shoji et al., 1983; Itoh et al., 1987). My study
cleariy demonstrated that direct activation of a2-adrenergic receptors by its
selective agonist, UK 14,304, does not cause signiﬁcant constriction of veins.
Therefore, if a2-adrenergic receptors are expressed by rat small mesenteric arteries
and veins, their function is not clear. The discrepancies between results obtained

from those studies and my study suggest that there are species-dependent

168

differences in the expression or function of the postjunctional a2-adrenergic

receptors.

2.4. Differences in alterations of sympathetic neuroeffector transmission to

arteries and veins in DOCA-salt hypertension

The major difference in alterations of sympathetic neuroeffector
transmission to arteries and veins are as follows: i) neurogenic constriction of
veins is maintained, but neurogenic constriction of arteries is potentiated from
DOCA-salt hypertensive rats; ii) postjunctional reactivity of DOCA-salt arteries is
not changed while in DOCA-salt veins postjunctional reactivity is decreased.

Although neurogenic constrictions of veins are not elevated, the increase
in nerve-mediated NE release in veins indicates that the activity of sympathetic
nerves associated with veins is increased. Therefore, sympathetic neuroeffector
transmission to mesenteric arteries and veins is potentiated in DOCA-salt
hypertensive rats. Nevertheless, the fact that neurogenic constrictions are
maintained in DOCA-salt veins, but potentiated in DOCA-salt arteries does
suggest that there is a temporal disparity in alterations of sympathetic
neuroeffector transmission to arteries and veins. This temporal disparity could
reﬂect the distinct role played by the venous and arterial circulation in the
development of DOCA-salt hypertension. There is no doubt that at the early
stage of DOCA-salt hypertension, sympathetic nerve activity is elevated (Julius et

al., 1988; Esler et al., 1989; lzzo, 1998). Considering that the decreased

169

reactivity of postjunctional 611-adrenergic receptors occurs secondary to the
increased transmitter release, at the early stage of hypertension development
when the receptor reactivity is not yet decreased neurogenic constrictions of
veins must be potentiated. The elevated neurogenic constrictions of veins would
in increased cardiac output that is known to occur in early stage of hypertension
(Lund-Johansen, 1994). Therefore, it also supports the notion that veins as the
capacitance vessels play an important role in the initiation of hypertension
whereas arteries providing the peripherial resistance are important in maintaining

the high blood pressure (Lund-Johansen, 1989; Coleman and Hall, 1993).

3. Alterations in local modulatory mechanisms of sympathetic
neuroeffector transmission to arteries and veins in DOCA-salt hypertensive

rats

3.1. Alterations in prejunctional adrenergic autoreceptors in arteries and

veins in DOCA-salt hypertensive rats

3. fa. Prejunctional adrenergic autoreceptors associated with mesenteric
arteries regulate ATP and NE release and are impaired in DOCA-salt
hypertensive rats.

My study demonstrated that a2-adrenergic receptors regulate ATP release.

This result is consistent with the results obtained from other studies showing that

170

yohimbine increased the overﬂow of ATP caused by electrical ﬁeld nerve
stimulation (Westfall et al., 1996; Bobalova and Mutafova-Yambolieva, 2001a).
The regulation by 112-adrenergic receptors of ATP release indicates that ATP and
NE release are regulated in a similar manner. However, the result that 612-
adrenergic receptors regulate both ATP and NE release does not provide
evidence supporting the notion that ATP and NE are stored in the same synaptic
vesicles. Release of ATP and NE may be differentially regulated by prejunctional
a2-adrenergic receptors. For example, the reduction of ATP release caused by
the 612 agonists, xylazine and clonidine, was greater than the reduction in NE
release (Westfall et al., 1996). Furthermore, pretreatment with the 612 receptor
antagonists, idazoxan and yohimbine, produced a substantial increase in NE
overﬂow and a lesser increase in ATP overﬂow (Westfall et al., 1996). Therefore,
although prejunctional 112-adrenergic receptors regulate both ATP and NE
release, they may be regulated by different subsets of prejunctional a2-
adrenergic receptors or the same a2-adrenergic receptors my couple to different

inhibitory mechanisms linked to NE and ATP release.

3. 1 b. Function of prejunctional adrenergic autoreceptors associated with
mesenteric arteries is impaired in DOCA-salt hypertensive rats.

My study demonstrated that the function of prejunctional adrenergic
autoreceptors associated with mesenteric arteries is impaired in DOCA-salt
hypertensive rats as yohimbine had inhibitory effects on NE release in sham

arteries, but not DOCA-salt arteries. This result is consistent with other studies

171

showing that prejunctional a2-adrenergic receptor function is impaired in
hypertensive animals. For example, intravenously administered yohimbine, an
a2—adrenergic receptor antagonist, increased plasma NE levels in sham
normotensive, but not in DOCA-salt hypertensive rats (De Champlain, 1990).
Furthermore, yohimbine potentiated nerve stimulation-evoked NE release into
the mesenteric vasculature in normotensive rats, but not in DOCA-salt
hypertensive rats (T suda et al., 1989). The increased NE release found in portal
vein and caudal artery of SHR rats was due to a decrease in the functional
activity of prejunctional a2-adrenergic receptors (Westfall et al., 1986).

As my study also showed that nerve stimulation-induced NE release was
increased in DOCA-salt arteries, it is possible that the impairment of the
prejunctional a2-adrenergic receptors is responsible for the increased NE release
in DOCA-salt arteries. Therefore, the impaired function of prejunctional a2-
adrenergic receptors could contribute to the increased sympathetic nerve activity
in experimental hypertension (De Champlain, 1990).

In DOCA-salt arteries, function of the prejunctional a2-adrenergic
receptors is impaired. However, ATP release is not increased in DOCA-salt
arteries, which suggest that ATP release is also regulated by other factors such
as the prejunctional P1-receptors (Langer, 1980). Alterations in those regulatory
factors may have counteracted the impaired function of a2-adrenergic receptors
in DOCA-salt hypertension to maintain ATP release at a normal level. For
example, changes in local mechanism modulating ATP release were proposed to

be considered in SHR (Cheung, 1989). Furthermore, the unaltered ATP release

172

compared to an increased NE release appears to favor the notion that ATP and
NE may not be co—released from the sympathetic nerve terminals (Westfall et al.,

1996), at least in mesenteric arteries from DOCA-salt rats.

3.10. Prejunctional adrenergic autoreceptors associated with mesenteric
veins are impaired in rats from DOCA-salt hypertension.

My study demonstrated that the function of prejunctional a2-adrenergic
receptors associated with mesenteric veins is impaired in DOCA-salt
hypertensive rats. This is the ﬁrst study that assessed the function of
prejunctional a2-adrenergic receptors in mesenteric veins in DOCA-salt
hypertension. The impaired function of a2-adrenergic receptors could account for

the increased NE release observed in DOCA-salt veins.

The mechanism underlying the impaired function of the a2-adrenergic
autoreceptor function remains to be elucidated. Attenuated function of the 612-
adrenergic autoreceptors will cause increased NE release, which in turn can
desensitize the receptors and lead to further NE release. Thus, a positive
feedback cycle forms and leads to the impairment of the a2-adrenergic
autoreceptor function (De Champlain, 1989). The impaired function of the 012-
adrenergic autoreceptors could also be due to the imbalance between two
prejunctional modulatory mechanisms: the inhibitory a2-adrenergic receptor and

the facilitatory B-adrenergic receptor as in DOCA-salt hypertension there co-

173

exists an attenuation of the a2-adrenergic receptor function and an enhancement

of B-adrenergic receptor function (De Champlain, 1990).

The impaired function of the prejunctional org-adrenergic receptors indicate
that there could be a down-regulation of receptor function, which could be due to
a decreased receptor density and/or receptor afﬁnity. There could also be a
functional impairment of the signal transduction pathways linked to the
prejunctional a2-adrenergic receptors. There are a few studies suggesting that
sodium ion affects the reactivity of prejunctional a2—adrenergic receptors. For
example, increased dietary sodium intake decreases the afﬁnity of brainstem a2-
adrenergic receptors (Gavras, 1986). Furthermore, Limbird et al. (1982) reported
that increased sodium concentration inhibits the binding of tat-adrenergic receptor
agonists in vitro. These results suggest that sodium loading can decrease the
responsiveness of a2-adrenergic receptors and this could be a mechanism by
which sodium loading contributes to hypertension development. Nevertheless,
little is known of the precise mechanisms by which sodium affects the reactivity

of 112-adrenergic receptors (Limbird et al., 1982).

In conclusion, prejunctional a2-adrenergic receptors on sympathetic
nerves regulate ATP release in sham mesenteric arteries and NE release in
sham arteries and veins. These receptors are impaired in DOCA-salt arteries and
veins, which might account for the increased NE release. The potentiated

neurogenic responses of arteries (Luo et al., 2003) and the increased venomotor

174

tone in DOCA-salt hypertensive rats (Fink, et al., 2000) could be attributable to
the increased NE release in arteries and veins, respectively. Therefore, DOCA-

salt hypertension is associated with an alteration of neuronal function.

3.2. Alterations in modulatory effects mediated by ET receptors in arteries

and veins from DOCA-salt hypertensive rats

3.2a. Alterations in modulatory effects mediated by ET receptors in arteries

from DOCA-salt hypertensive rats

1) ET-1 acting at postjunctional ETA receptors differentially modulates
postjunctional 611-adrenergic receptors and P2X receptors.

My study demonstrated that subcontractile ET-1 presumably acting at
postjunctional ETA receptors decreased the reactivity of sham arteries to ATP
and increased the reactivity to NE. This result indicates that ET-1 acting at ETA
receptors differentially modulates P2X receptors and 611-adrenergic receptors as
these two receptors mediate the constrictions caused by ATP and NE
respectively in arteries (Galligan et al., 2001; Luo et al., 2003). The differential
modulation mediated by ETA receptors is perhaps related to the different
intracellular signaling mechanisms linked to the P2X receptors and 611-adrenergic
receptors activation. For example, P2X receptors are ligand—gated ionotropic
receptors and non-selectively permeable to cations including calcium. The

depolarization of vascular smooth muscle cells mediated by P2X receptors also

175

cause opening of voltage-dependent calcium channels and provides an
additional source of calcium inﬂux (Benham and Tsien, 1987). The rise in the
cytosolic level of calcium triggers the contraction of smooth muscle. The 611-
adrenergic receptors are metabotropic G-protein coupled receptors. The
intracellular mechanism involves the activation of Gq protein and production of
IP3 via a phosphoinositide pathway that leads to release of calcium from the
intracellular calcium store (Docherty, 1998). Similarly, ETA receptors are also G-
protein coupled receptors. Activation of vascular ETA receptors stimulates
phospholipase C and subsequent hydrolysis of phosphatidylinositol, which
results in the formation of IP3 and DAG and calcium release from intracellular
stores (Rubanyi and Polokoff, 1994). Thus, ETA and a1-adrenergic receptors are
linked to the same intracellular pathway to induce vascular constrictions.
Therefore, pretreatment with subcontractile ET-1 acting at ETA receptors could
activate and prime the common intracellular signaling pathways, which leads to
the ampliﬁcation of the signals produced by the subsequent adrenergic receptors
activation. This could be the mechanism underiying the facilitatory effect of the
adrenergic receptor sensitivity by ET-1 acting at ETA receptors. This hypothesis
is supported by results obtained from studies on rabbit aorta (Henrion and Laher,
1993). These studies demonstrated that potentiation by subcontractile ET-1 (100
pM) of the sensitivity of the rabbit aorta to NE was unaffected by removal of the
endothelium or changes in Ca2+ entry, but was prevented by protein kinase C
inhibitors, suggesting that ET—1 increases the sensitivity to NE by activating

protein kinase C-dependent mechanisms. However, the mechanism by which the

176

~21: :3. .ﬁaa’

‘20!

ETA receptors decreased the sensitivity of P2X receptors is unknown. It may be
due to inhibition of the P2X receptors by molecules produced in response to the

ETA receptors activation.

2) ET-1 facilitates sympathetic neuroeffector transmission to sham, but not
DOCA-salt arteries, which could be due to the down-regulation of the
postjunctional ETA receptors in DOCA-salt hypertension.

My study demonstrated that ET-1 presumably acting at postjunctional ETA
receptors potentiated sympathetic neuroeffector transmission to sham arteries
and this effect was not observed in DOCA-salt arteries. Furthermore, ET-1
altered the postjunctional reactivity of sham arteries to ATP and NE and these
effects were not observed in DOCA-salt arteries. The disappearance of
modulatory effects by ET-1 on DOCA-salt arteries is likely due to the down-
regulation of the ETA receptors as indicated by a reduced ET-1 induced
constrictions of mesenteric arteries from DOCA-salt rats (Johnson et al., 2002). It
was also found that binding of endothelium denuded mesenteric artery to ET-1
and subsequent accumulation of IP3 induced by ET-1 application was
signiﬁcantly lower in DOCA-salt rats compared to normal rats, suggesting that
decreased ET receptor density results in decreased activation of ET receptors
(Nguyen et al., 1992). The down regulation of the ETA receptors is likely a result
of increased vascular ET-1 production in DOCA-salt rats (Lariviere et al., 1993;
Day et al., 1995;Yu et al., 2002). Hence, the result that ET-1 facilitates

sympathetic neuroeffector transmission to sham arteries infers that increased

177

ET-1 production would enhance the sympathetic constriction of arteries and
leads to blood pressure elevation. This would occur when the ET receptors are
not yet down regulated as down-regulation of the receptors occurs secondary to

increased vascular ET—1 production (Lariviere et al., 1993).

3) Facilitatory prejunctional ETg receptors are activated to overcome the
inhibitory prejunctional ETA receptors and likely contribute to the increased NE
release in DOCA-salt arteries.

My study demonstrated that 86c, the ETg receptor agonist, potentiated,
via a prejunctional mechanism, sympathetic neuroeffector transmission to
DOCA-salt arteries, but not sham arteries. This result suggests that prejunctional
ETg receptors mediate a facilitatory effect on transmitter release in DOCA-salt
arteries. This facilitatory effect mediated by prejunctional ETg receptors in DOCA-
salt arteries could be due to the functional activation of the receptors in DOCA-
salt hypertension. An alternative explanation is that the facilitatory effect is
unmasked in DOCA-salt arteries due to the impaired function of inhibitory
endothelial ETg receptors. This hypothesis is supported by the result showing
that indomethacin and NLA, the inhibitors for vasodilators produced by
endothelial ETg receptor activation, enhanced ET-1-induced constrictions of
sham arteries whereas constrictions of arteries from DOCA-salt rats were
unaffected by these two drugs (Johnson et al., 2002). This result, however, could

also simply reﬂect a generally impaired function of the endothelial cells in DOCA-

178

salt hypertension rather than a speciﬁc impairement of the endothelial ETg
receptors.

However, ET-1, the non-selective ETA and ETg receptor agonist, had no
effect on sympathetic neuroeffector transmission to DOCA-salt arteries. Taken
together, these results suggest that ETA receptors mediate an inhibitory effect
whereas ETB receptors a facilitatory effect on transmitter release in DOCA-salt
arteries. The notion that ET-1 acting at ETA receptors to inhibit transmitter
release is based on results obtained from a number of studies showing that in
normal animals ET—1 inhibits NE release from sympathetic nerve terminals in
mesenteric vascular bed (Zhang et al., 1996; Tabuchi et al., 1990) and non
vascular tissues including rat and guinea pig vas deference (Wiklund et al., 1990;
Wiklund et al., 1991) and stomach (Nakamura et al., 2003). Some of those
studies identiﬁed that the receptor subtype mediating this inhibitory effect of ET-1
is the ETA receptor (Zhang et al., 1996). Hence, although the effect of ET-1 on
co-transmitter release remains to be established, my study does support that in
sham arteries ETA receptors mediates an inhibitory effect on transmitter release
whereas in DOCA-salt arteries this inhibitory effect is overcome by a facilitatory
effect mediated by ETg receptors. The activation of a facilitatory prejunctional
ETB receptors leading to the disinhibition of transmitter release could in part

account for the increased NE release in DOCA-salt arteries.

179

3. 2b. Alterations in modulatory effects mediated by ET receptors in veins

from DOCA-salt hypertensive rats

1) ETg receptors mediate facilitation of sympathetic constriction of DOCA-
salt veins via a postjunctional effect.

My study demonstrated that ETg receptors mediate a facilitatory effect on
sympathetic neuroeffector transmission to DOCA-salt veins at least partly by
increasing the postjunctional reactivity to NE. It was shown that there is no
functional change of the postjunctional ETg receptors in veins from DOCA-salt
rats as the reactivity of DOCA-salt veins to 86c was not altered (Johnson et al.,
2002). Therefore, the facilitatory effect mediated by ETg receptors on
sympathetic neuroeffector transmission to DOCA-salt veins must be attributed to
factors other than functional change of the vascular ETB receptors.

The 611-adrenergic receptors and ETg receptors both are metabotropic
G-protein coupled receptors. Vascular constrictions caused by the 611-adrenergic
receptors and ETB receptors both involve production of IP3 via a phosphoinositide
pathway leading to release of calcium from the intracellular calcium store
(Docherty, 1998; Rubanyi and Polokoff, 1994). In DOCA-salt veins intracellular
signaling pathways linked to ETB and 611-adrenergic receptors activation could be
somehow coupled so that the activation of ETg receptors could lead to the
enhancement of the signal transduction associated with the 611-adrenergic

receptor activation. The coupling of these two signaling pathways seems to be a

180

change associated with DOCA-salt hypertension as the modulation did not occur
in veins from sham rats.

The facilitatory effect mediated by postjunctional ETg receptors in DOCA-
salt veins is also likely due to the unmasking of this effect by the impaired
function of inhibitory endothelial ETg receptors. Dysfunction of the endothelial
ETg receptors in DOCA-salt veins is demonstrated by the ﬁndings that NLA, the
blocker for vasodilators produced by endothelial ETg receptors activation,
enhanced S6c—induced constrictions of sham veins, but not DOCA-salt veins
(Johnson et al., 2002). This ﬁnding, however, could also simply reﬂect a
generally impaired function of the endothelial cells in DOCA-salt hypertension

rather than a speciﬁc impairement of the endothelial ETB receptors.

2) The facilitatory effect mediated by ETg receptors on sympathetic
neuroeffector transmission to DOCA-salt veins could contribute to the increased
MCFP.

Veins are more sensitive than arteries to ET—1 (Johnson et al., 2002).
Studies in vivo showed that intravenous administration of ETs increased central
venous pressure, venous resistance and MCFP (Waite and Pang, 1990;
Yamamato et al., 1980). ET-1-induced constriction of hand veins of human
subjects with essential hypertension is greater compared to normotensives
(Haynes et al., 1994). Furthermore, ET-1 induced- venoconstriction correlated
positively with mean arterial pressure in the hypertensive subjects, but not in the

normotensives. In DOCA-salt hypertension, ET-1 induced smooth mscule

181

 

contraction was reduced in rat aorta (Watts et al., 2002) and mesenteric arteries
(Johnson et al., 2002), but maintained in vena cava (Watts et al., 2002) and
mesenteric veins (Johnson et al., 2002). Furthermore, ET—1 exerts a greater
effect on venomotor tone in vivo in DOCA-salt rats compared to normal rats, and
these data are consistent with a role for ET—1 in DOCA-salt hypertension (Fink et
al., 2000)

In DOCA-salt rats there is an elevated sympathetic drive to the venous
system as indicated by a greater decrease of the MCFP via blockade of
sympathetic ganglionic transmission in DOCA-salt rats compared to the normal
rats (Fink et al., 2000). The elevated sympathetic drive and venomotor tone
observed in DOCA-salt rats (Fink et al., 2000) can attribute to the facilitatory
effects mediated by ETB receptors on sympathetic neuroeffector transmission to
veins demonstrated by my study. Similar to my result, it was shown that
sympathetically mediated venoconstriction was substantially potentiated by ET—1
in hypertensive, but not normotensive subjects (Haynes et al., 1994). The venous
system plays an important role in blood pressure regulation by serving the
capacitance function. Elevated sympathetic drive to the venous system would
increase venous return and cardiac output, which will lead to elevated blood

pressure (Guyton et al., 1972b).

3. 2c. Differential alterations of modulation by ET receptors on sympathetic

neuroeffector transmission to arteries and veins

182

 

My study demonstrated that ET receptors mediate a differential
modulation of sympathetic neuroeffector transmission to arteries and veins,
which lies in the following aspects: 1) ETg receptors are differentially expressed
in arteries and veins as they are expressed on the smooth muscle cells of the
veins and on the sympathetic nerves of the arteries. 2) In arteries, ET-1 acting at
ETA receptors facilitates sympathetic neuroeffector mechanisms in sham rats,
which could contribute to the elevation of peripheral resistance and hence
hypertension development. This facilitation by ET-1 was removed in DOCA-salt
hypertension possibly due to the down-regulation of the vascular ETA receptors in
the maintenance stage of DOCA-salt hypertension. In veins, ET-1 acting at
postjunctional ETg receptors facilitates sympathetic transmission in DOCA-salt
rats. This facilitation is likely to contribute to the increased venomotor tone

observed in DOCA-salt rats (Fink et al., 2000).

183

 

Sympathetic neuroeffector transmission to arteries

Sham artery ( DOCA artegr

/Sympathetic nerve terminal

 

9
.3"

 
 
   

 

Endothelium
4 \
p ? 9 a E ;.
ATP 111:5 ® ‘3
W s w v .....
. ET-1

 

 

 

 

 

0100.00... 0000.00

Smooth muséle cells

In sham arteries, ATP acting at P2X receptors functions as the dominant
neurotransmitter. NE release is inhibited by prejunctional 112-adrenergic
autoreceptors. ET-1 acting at postjunctional ETA receptors exerts an inhibitory
effect on P2 receptors and a facilitatory effect on a1-adrenergic receptors. The

latter effect could be the mechanism by which subcontractile ET-1 potentiated

sympathetic neuroeffector transmission to sham arteries.

184

 

In DOCA-salt arteries, NE release is increased due to the impaired
function of presynaptic a2-adrenergic receptors, which could account for the
increased adrenergic component in sympathetic neuroeffector transmission as
well as the potentiated sympathetic constriction. ATP release is maintained.
Postjunctional ETA receptors are down regulated possibly due to the increased
ET—1 production. A facilitatory effect mediated by prejunctional ETB receptors for
transmitter release is unmasked due to the impaired function of inhibitory
endothelial ETB receptors producing vasodilators, which could also contribute to

the increased NE release. Images in this dissertation are presented in color.

185

Sympathetic neuroeffector transmission to veins

Sham vein DOCA vein

       
   

/8ym pathetic nerve terminal

Endothelium

  

I00000000 000

 

Smooth muscle cells

In sham veins and DOCA-salt veins, NE acting at the (111-adrenergic
receptor is the neurotransmitter. NE release is increased in DOCA-salt veins,
which stimulates the up-regulation of NE uptake system. This increased NE
release is due to the impaired function of the prejunctional (112-adrenergic
autoreceptors. The postjunctional ETB receptors mediates a potentiating effect on
sympathetic transmission to DOCA-salt veins by increasing the postjunctional
reactivity to NE, which could contribute to the increased venomotor tone of

DOCA-salt veins. Images in this dissertation are presented in color.

186

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