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This is to certify that the
thesis entitled

THE EFFECTS OF CHRONIC ACTIVATION OF
ENDOTHELIN ETB RECEPTORS ON BLOOD
PRESSURE, VENOMOTOR TONE, NEUROHUMORAL
ACTIVITY, AND OXIDATIVE STRESS

presented by
Melissa Wei Li

has been accepted towards fulﬁllment
of the requirements for the

Ph.D. degree in Pharmacology and Toxicology_

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THE EFFECTS OF CHRONIC ACTIVATION OF
ENDOTHELIN ETB RECEPTORS ON BLOOD PRESSURE,
VENOMOTOR TONE, NEUROHUMORAL ACTIVITY, AND
OXIDATIVE STRESS

By

Melissa Wei Li

A DISSERTATION

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

DOCTOR OF PHILOSOPHY
Department of Pharmacology and Toxicology

2008

ABSTRACT
THE EFFECTS OF CHRONIC ACTIVATION OF ENDOTHELIN ETB
RECEPTORS ON BLOOD PRESSURE, VENOMOTOR TONE,
NEUROHUMORAL ACTIVITY, AND OXIDATIVE STRESS
By

Melissa Wei Li

Activation of endothelin ETB receptors (ETBRS) modulates blood pressure
by affecting several different physiological systems. The stimulation of ETBRS is
known to cause transient arterial relaxation, a marked increase in renal excretion
of sodium and water, and a clearance of endogenous endothelin-1 (ET-1). It is
reasonable to speculate that the depressor and natriuretic effects induced by
ETBR activation would cause blood pressure to decrease. Our lab, however,
revealed for the ﬁrst time that chronic activation (5 days) of ETBRS using
intravenous infusion of the ETBR selective agonist sarafotoxin 60 (86c) in rats
caused a seemingly paradoxical increase in arterial pressure (S6c-induced

hypertension).

I proposed the hypothesis that S6c-induced hypertension is mediated by
direct venoconstriction, increased sympathetic activity to the splanchnic region
mediated by elevated ROS levels in sympathetic ganglia, and increased release

of vasodilating neurotransmitters from sensory nerves.

Most studies of hypertension have focused on altered regulation of the
arterial vasculature. It is true that arterial resistance is elevated in hypertension.

However, there is also a decrease in venous capacitance. Unfortunately, there

have been few studies of the mechanisms causing constriction of veins in
hypertension. Since 36c is a selective venoconstrictor, venoconstriction may
mediate SGc-induced hypertension. Since the splanchnic veins are the main
capacitance region of the circulation, I focused on the effects of ETBR activation
in the splanchnic bed in my thesis project. In addition, celiac ganglionectomy
(CGX) attenuated SGc-induced hypertension, indicating that sympathetic
innervation participates in this hypertension model as well. My data also Show
that there was an increased neuronal, but not vascular, superoxide level in 86c-
induced hypertension. Systemic treatment of antioxidant (tempol) lowered
neuronal oxidative stress and blood pressure, suggesting that SGc—induced
hypertension is partially mediated by sympathoexcitation to the splanchnic
organs driven by increased oxidative stress in sympathetic ganglia. In ETBR-
deﬁcient transgenic (T 9) rats, ETBRS are not functional in endothelium, smooth
muscle, or renal tubules. The only place where ETBRS are functional is where
dopamine-B-hydroxylase is expressed. When CGX Tg wildtype (+/+) received

860, the increase in blood pressure was attenuated.

The results of my work provide strong evidence that SGc-induced
hypertension is mediated by increased splanchnic sympathetic activity at least
partially driven by increased neuronal superoxide level. Therefore, my studies
provide more information on how oxidative stress and sympathetic innervation

contribute to blood pressure regulation.

Copyright by
MELISSA WEI Li

2008

To my grandparents, Zhiqin Xiang and Zuoting Liu, for their endless love.
To my parents, Jinghua Liu and Jingxian Li, for their unconditional support.

To my husband, Zetian Mi, and my daughter Allie, for completing my life.

ACKNOWLEDGMENTS

I am truly fortunate to have had Dr. Fink as my thesis advisor. He is a
wonderful mentor, who cares about student training AND is good at student
training. I enjoyed every single meeting with him. No matter the talk was brief
(such as a short report for the research progress) or long (such as hours of
discussion for a public talk), I always got scientiﬁcally inspired. l have grown up
a lot as a scientist since I joined Dr. Fink’s lab. I am grateful to him for what I
Ieamed through his mentoring. If I was asked to mention a single thing which I
Ieamed from him and from which I could beneﬁt the most in my whole life, I

would say it’s his persistent passion for science.

Additionally, I would like to thank Dr. Haywood, Dr. Galligan, Dr. Watts,
and Dr. Wang, for serving as my committee members. They all gave me great

advice for my research.

Also, I would say thanks to all lab members, Helen (who’s also a close
friend and gave me valuable input), Barb, Andrew, Sachin, Hannah, and

Bridget. I enjoyed spending time with you all.

Thanks to my dearest husband, Zetian, without whose years-support, I
would not have achieved so much. He always encouraged me when I was
frustrated. He is my role model as a scientist, as well. Many thanks to my
parents, Jinghua Liu and Jingxian Li, who sacriﬁced a lot so that I could pursue

my dream of becoming a Ph.D. They were especially helpful since my daughter

vi

was born. Without their help, I can not imagine how I could handle my research

and family at the same time.

At last, but not the least, I want to say “thank you!” to my beautiful angel,

Allie. She makes me stronger as a mom and fearless of anything!

vii

TABLE OF CONTENTS

LIST OF FIGURES ............................................................................................... x
LIST OF ABBREVIATIONS ............................................................................ xvii
FREQUENTLY USED DRUGS AND CHEMICALS ......................................... xix
COMMONLY USED SOLUTIONS .................................................................... xx
CHAPTER 1: GENERAL INTRODUCTION ......................................................... 1
1. Hypertension as a disease .......................................................................... 2
2. Blood pressure regulation ........................................................................... 2
2.1 Humoral factors ................................................................................... 3
2.1.1 The endothelin system .................................................................. 3
2.1.1.1 Discovery and synthesis ........................................................ 3
2.1.1.2 Endothelin receptors .............................................................. 4
2.1.1.3 Endothelin-1 in physiological conditions ................................ 7
2.1.1.4 Endothelin in hypertension ..................................................... 9
2.1.2 Catecholamines .......................................................................... 10
2.1.3 Calcitonin gene-related peptide ................................................... 13
2.1.4 Others ......................................................................................... 15
2.2 Vascular function ............................................................................... 15
2.2.1 Arterial bed ................................................................................. 15
2.2.2 Venous bed ................................................................................. 16
2.2.3 Splanchnic bed — a critical bed for regulation of vascular

capacitance ................................................................................ 18
2.2.3.1 Veins and venules in the splanchnic bed ............................. 18
2.2.3.2 Differential sympathetic innervation, arteries vs. veins ........ 19
2.2.3.3 Increased splanchnic SNA in hypertension .......................... 21
3. Oxidative stress ......................................................................................... 22
3.1 Introduction ....................................................................................... 22
3.2 ROS in hypertension ......................................................................... 23
3.3 Approaches to measure oxidative stress .......................................... 25
4. SOC-induced hypertension ......................................................................... 27
4.1 Sarafotoxins ....................................................................................... 27
4.2 Pharmacology of 36c ......................................................................... 28

4.3 A novel hypertension model: chronic activation of endothelin type B
receptors by 86c ................................................................................. 29
CHAPTER 2: OVERALL HYPOTHESIS AND SPECIFIC AIMS ........................ 37

viii

CHAPTER 3: GENERAL METHODS ................................................................. 46

CHAPTER 4: CHARACTERIZATION OF CELIAC GANGLIONECTOMY
........................................................................................................................... 60

CHAPTER 5: NEURONAL REGULATION IN SGC-INDUCED HYPERTENSION .

........................................................................................................................... 84
CHAPTER 6: CHARACTERIZATION OF ENDOTHELIN B RECEPTOR-
DEFICIENT TRANSGENIC RATS ................................................................... 113
CHAPTER 7: SSC-INDUCED HYPERTENSION IN ENDOTHELIN B
RECEPTOR-DEFICIENT TRANSGENIC RATS .............................................. 132

CHAPTER 8: SENSORY INNERVATION IN SGC-INDUCED HYPERTENSION

......................................................................................................................... 153
CHAPTER 9: GENERAL CONCLUSIONS AND DISCUSSION ...................... 161
REFERENCES ................................................................................................. 175

LIST OF FIGURES
Some images in this dissertation are presented in color.

Figure 1: Factors regulating blood pressure. Details are discussed in
GENERAL INTRODUCTION .......................................................... 33

Figure 2: The proposed preteolytic processing pathway for conversion of
preproendothelin to endothelin—1. The preproform (203-amino
acid) ofET-1 is converted to 39-amino acid big ET-1 by dibasic
endopeptidase and carboxypeptidases. Big ET-1 is then
converted to 21-amino acid ET-1 by endothelin converting
enzymE. Many factors may stimulate or inhibit the expression
of ET—1 ............................................................................................ 34

Figure 3a: Blood distribution in various blood vessels. Over 75% of total
blood is stored in veins and venules ............................................... 35

Figure 3b: Blood volume distribution in various regions. About one third of
total blood volume is stored in the splanchnic bed .......................... 36

Figure 4: Overall hypothesis of the project. ETBR activation in the
Splanchnic bed increases venoconstriction directly, and
sympathetic nerve activity via increased ganglionic oxidative
stress. Both effects contribute to increased arterial blood
pressure. ETBR activation leads to enhanced CGRP release
from sensory nerves, which attenuates the blood pressure
increase by causing local vasodilation ............................................ 45

Figure 5a: Glyoxylic acid staining of mesenteric arteries. The blue
ﬂuorescence represents the presence of catecholamines
regulating blood pressure ............................................................... 76

Figure 5b: Glyoxylic acid staining of mesenteric veins. The blue
ﬂuorescence represents the presence Of catecholamines .............. 77

Figure 6: Tyrosine hydroxylase staining and calcitonin gene-related
peptide (CGRP) staining of mesenteric arteries. The green
ﬂuorescence shows the presence of tyrosine hydroxylase, a
key enzyme for catecholamine synthesis. The red
ﬂuorescence indicates the presence CGRP, a peptide
released from sensory neurons. The ﬁgures Show that CGX
effectively removed both sympathetic nerves and CGRP-
containing nerves rats ..................................................................... 78

Figure 7: Norepinephrine (NE) concentrations in splanchnic organs. NE
levels in splanchnic organs from CGX rats were Signiﬁcantly
lower than those from sham-operated rats, two weeks after the
surgery. *: Signiﬁcant difference from sham rats (P<0.05) ............... 79

Figure 8: NE concentrations in mesenteric arteries and veins. NE levels
in mesenteric arteries and veins from CGX rats were
signiﬁcantly lower than those from sham-operated rats, two
weeks after the surgery. *: Signiﬁcant difference from sham
rats (P<0.05) .................................................................................... 80

Figure 9: Constriction of mesenteric vessels in response to electrical
stimulation. The constriction in response to electrical
stimulation was virtually abolished in mesenteric vessels from
CGX rats .......................................................................................... 81

Figure 10: Norepinephrine concentrations in splanchnic organs from rats
with sham-operation, CGX, or CGX and IMGX. The animals
were sacriﬁced two weeks or twelve weeks after the
surgery/surgeries. *: Signiﬁcant difference from sham rats
(P<0.05) ........................................................................................... 82

Figure 11: Norepinephrine concentrations in mesenteric arteries and
veins from rats with sham-operation, CGX, or CGX and IMGX.
The animals were sacriﬁced two weeks or twelve weeks after
the surgery/surgeries. *: Signiﬁcant difference from sham rats
(P<0.05) .......................................................................................... 83

Figure12azThe depressor responses to ganglion blockade with
trimethaphan before (C1-C2, C=control), during (E1-E5,
E=infusion), after (R1-R3, R=Recovery) continuous S6c
infusion (5 pmollkg/min, iv) or vehicle into rats. Figure 1a
shows mean arterial pressure (MAP). MAP signiﬁcantly
increased during $6c-infusion period and it did not change with
vehicle infusion. *: a statistical difference (p<0.05) from the
control period .................................................................................. 100

Figure12b:ChangeS in MAP in S6c— or vehicle-infused animals with a
bolus injection of trimethanphan (15 mg/kg, iv), a ganglionic
blocker, during infusion period. MAP decrease was
signiﬁcantly bigger in S6c-treated rats than in vehicle-treated
rats on the fourth and the ﬁfth day. Numbers in parentheses
means the number of rats in each group. *: a statistical
difference (p<0.05) from control rats on the same day ................... 101

xi

Figure13azMean arterial pressure (MAP) in rats before (Cl-CZ,
=control), during (E1-E5, E=infusion), after (R1-R3,
R=r’ecovery) continuous 86c infusion (5 pmollkglmin, iv) or
vehicle. MAP signiﬁcantly increased during S6c-infusion period
and it did not change with vehicle infusion. *: a statistical

difference (p<0.05) from the control period ...................................... 102

Figure13b:Average (1: SEM) plasma norepinephrine (NE) concentrations
in rats receiving continuous S6c infusion (5 pmollkglmin, iv) or
vehicle according to the same protocol shown in Figure 1.
Plasma samples were collected on the second control day
(CZ), the third and the ﬁfth infusion days (E3 and E5), and
recovery day 2 (R2). Plasma NE levels did not increase during
S6c-infusion period, although MAP signiﬁcantly elevated ................ 103

Figure14: Average MAP before (day 1-2, control period), during (day 3-7,
infusion period), and after (day 8-10, recovery period)
continuous S6c infusion (5 pmollkglmin, iv) into celiac
ganglionectomized (CGX) and Sham-operated rats. MAP
signiﬁcantly increased during infusion period in sham-operated
rats. It increased signiﬁcantly only in the ﬁrst two days Of
infusion period in CGX-operated rats.*: a statistical difference
(p<0.05) from control period ............................................................ 104

Figure15a:Representative pictures showing superoxide levels in smooth
muscle cells from superior mesenteric arteries (SMA) and
veins (SMV) in control and SGc-infused rats, measured by
DHE. 02" levels, indicated by DHE ﬂuorescence intensity in
confocal images of smooth muscle cells, are higher in S6c-
treated rats than in sham rats. A, SMA from a sham rat; B,
SMA from a rat receiving 86c for one day; C, SMA from a rat
receiving 860 for ﬁve days; D, SMV from a sham rat; E, SMV
from a rat receiving 86c for one day; F, SMV from a rat
receiving S6c for ﬁve days. The scale bar in F applies to all
panels .............................................................................................. 105

Figure15szuantiﬁcation of mean ﬂuorescence intensity of smooth muscle
cells in SMA and SMV (n=7 in each group). Fluorescence of
smooth muscle cells was not signiﬁcantly greater in rats
receiving S6c for either one day or ﬁve days compared with
shams’ ............................................................................................. 106

Figure16: Superoxide levels in superior mesenteric arteries and veins
from control and $6c-infused rats, measured by lucigenin-
enhanced chemiluminescence. Rats were either treated with
$60 for 1 day or 5 days. Superoxide levels were not

xii

signiﬁcantly increased with S6c treatment. Basal superoxide
level was higher in veins than in arteries. *: a statistical
difference (p<0.05) from control rats ............................................... 107

Figure17azRepresentative pictures showing superoxide levels in inferior
mesenteric ganglion (IMG) neurons from control and S60-
treated rats. A, IMG from a sham rat; B, IMG from a rat
receiving S6c for one day; C, IMG from a rat receiving 860 for
ﬁve days; D, Comparison of mean ﬂuorescence intensity of
S6c-treated neurons to sham neurons (n=7 Sham rats; n=7
S6c-treated rats). The scale bar in C applies to all panels .............. 108

Flgure17b:Quantiﬁcation of mean ﬂuorescence intensity of sympathetic
neurons from S6C-treated and saline-treated rats (n=7 in each
group). The ﬂuorescence density was signiﬁcantly higher in
S6c-treated rats (one day or ﬁve day-infusion) than that in
control rats. Superoxide level increased in a time-dependent
manner. The level from rats with ﬁve day-infusion was higher
than that from rats with one day-infusion. *: Fluorescence of
IMG neurons was signiﬁcantly (P<0.05) greater in ganglia from
rats receiving 860 for both 1 day or 5 days compared with
shams’; #: Fluorescence of IMG neurons was signiﬁcantly
(P<0.05) greater in ganglia from rats treated with S6c for 5
days compared with those treated for 1 day .................................... 109

Figure18: Average MAP before (01-02, C=control), during (E1-E5,
E=infusion), and after (R1-R3, R=recovery) continuous S6c
infusion (5 pmollkglmin, iv) or vehicle into rats, while tempol
was given through drinking water (1 mmol/l). A third group of
animals were on S6c, while drinking normal distilled water.
Numbers in parentheses means the number of rats in each
group. MAP signiﬁcantly increased in S6c-infused rats without
tempol treatment. The rats with dual treatments of 86c and
tempol did not show a signiﬁcant increase in MAP. Tempol did
not change MAP in vehicle-treated rats. *: a statistical
difference (p<0.05) from control period ........................................... 110

Figure19a:Superoxide levels in inferior mesenteric ganglion (IMG)
neurons from control and S6c-treated rats. A, IMG from a
sham rat; B, IMG from a rat receiving S6c for one day; C, IMG
from a rat receiving S6c for one day while drinking tempol
water (1 mmol/l); The scale bar in C applies to all panels ............... 111

Figure19szuantiﬁcation of mean ﬂuorescence intensity of S6c-treated

smooth muscle cells to sham smooth muscle cells in SMA and
SMV (n=5 in each group). *: Fluorescence of IMG neurons was

xiii

signiﬁcantly (P<0.05) greater in ganglia from rats receiving S6c
alone compared with Shams’ .......................................................... 112

FigureZO: Mean arterial pressure of male and female WKY, Tg (+I+) and
T9 (SI/SI) rats. *2 signiﬁcant difference from WKY rats for each
gender; #: signiﬁcant difference from T9 (+I+) rats for each
gender; 1': signiﬁcant different from male Tg (+I+) rats .................... 124

Figure21: Plasma ET-1 concentrations in male and female WKY, Tg
(+I+) and T9 (sl/sl) rats. *2 signiﬁcant difference from WKY in
each gender; #: signiﬁcant difference from T9 (+I+) rats in
each gender; 1': signiﬁcant difference from male Tg (sllsl) rats ........ 125

Figure22: Glyoxylic acid staining of mesenteric arteries and veins from
T9 (sllsl), Tg (sll+), and Tg (+I+) rats. The blue ﬂuorescence
represents the presence of catecholamine ...................................... 126

Figure23azNE levels in mesenteric artery and vein from male WKY, Tg
(+I+), and T9 (SI/SI) rats. The concentration of NE in artery from
T9 (+I+) rats was signiﬁcantly higher than that from WKY rats ........ 127

Figure23szE levels in mesenteric artery and vein from male WKY, Tg
(+I+), and T9 (sllsl) rats. There was no Signiﬁcant difference
between any two groups for either blood vessel type ...................... 128

Figur024: NE levels in splanchnic organs from female WKY, Tg (sllsl),
and T9 (+I+) rats. No Signiﬁcant difference was shown
between any two groups for any organ. LK: lift kidney; RK:
right kidney; SI: small intestine ....................................................... 129

Figure25a:Comparison of frequency-response curves for neurogenic
constrictions of arteries from Tg (+I+), Tg (sllsl), and T9 (sll+)
rats. No signiﬁcant difference in frequency-dependent
response was shown ...................................................................... 130

Figure25b:Comparison of frequency—response curves for neurogenic
constrictions of veins from Tg (+I+), Tg (sllsl), and T9 (sll+)
rats. No signiﬁcant difference in frequency-dependent
response was shown ...................................................................... 131

Figure26: Mean arterial pressure (MAP) before (day 1-3), during (day 4-
10), after (day 11-14) continuous S6c infusion (30
pmollkglmin, sc.). MAP was Signiﬁcantly higher in T9 (SI/SI)
rats than in T9 (+I+) rats during the control period In Tg (+l)
rats. MAP signiﬁcantly increased upon S6c infusion in the ﬁrst
three days. It decreased signiﬁcantly on the ﬁrst two days of

xiv

the recovery period upon S6c removal. In Tg (sllsl) rats, MAP
did not change during the infusion or recovery period,
compared to the control period. Asterisks indicate a statistical
difference (p<0.05) from the last day of the control period .............. 146

Figure27: Heart rate (HR) before (day 1-3), during (day 4-10), after (day
11-14) continuous S6c infusion (30 pmollkglmin, so). In Tg
(+I) rats, HR signiﬁcantly decreased on the ﬁrst day of 86c
infusion. It increased signiﬁcantly on the ﬁrst two days of
recovery period upon 860 removal. In Tg (sl/sl) rats, HR did
not change during the infusion or recovery period, compared to
the control period. Asterisks indicate a statistical difference
(p<0.05) from the last day of the control period .............................. 147

FigureZB: Mean arterial pressure (MAP) during the control period (day 1-
3), tempol treatment period (day 4-6), tempol treatment with
S6c infusion (30 pmollkglmin, sc.) period (day 7-11), S6c
infusion period (day 12-13), and recovery period (day 14-17).
In Tg (+l) rats, MAP did not change with tempol treatment
alone. It signiﬁcantly increased during the whole period of S60
infusion while the rats were drinking tempol solution. It
decreased signiﬁcantly in the recovery period. In Tg (sllsl) rats,
MAP did not Change during the infusion or recovery period,
compared to the control period. Asterisks stand for a statistical
difference (p<0.05) from the last day of the control period.
Pound Signs represent signiﬁcant difference from the last day
of tempol treatment period. Cross signs indicate Signiﬁcant
difference from the last day of tempol treatment with S6C
infusion period ................................................................................ 148

Figure29: Heart rate (HR) during the control period (day 1-3), tempol
treatment period (day 4-6), tempol treatment with S6c infusion
(30 pmollkglmin, sc.) period (day 7-11), S6c infusion period
(day 12-13), and recovery period (day 14-17). In Tg (+I+) rats,
HR signiﬁcantly dropped on the ﬁrst day of S60 infusion (day
7). In Tg (sllsl) rats, HR did not change during the infusion or
recovery period, compared to the control period. Asterisk
stands for a statistical difference (p<0.05) from the last day of
control period. Pound sign represents a signiﬁcant difference
from the last day Of the tempol treatment period ............................. 149

Figure30azMean arterial pressure (MAP) before (day 1-3), during (day 4-
10), after (day 11-14) continuous S6c infusion (30
pmollkglmin, sc.) in Tg (+I+) rats. CGX Signiﬁcantly lowered
resting blood pressure. In CGX Tg (+I+) rats, MAP signiﬁcantly
increased upon S6c infusion on the ﬁrst two days, compared to

XV

the control period. Asterisks indicate statistical difference
(p<0.05) from the last day of the control period. Pound Signs
indicate statistical difference from intact Tg (+I+) rats on the
same day ......................................................................................... 150

Figure30szean arterial pressure (MAP) before (day 1-3), during (day 4-
10), after (day 11-14) continuous S6C infusion (30
pmollkglmin, so.) in T9 (sllsl) rats. CGX did not change resting
blood pressure of T9 (SI/SI) rats. There was no signiﬁcant
difference in MAP between T9 (+I+) and T9 (SI/SI) rats during
the infusion and recovery periods .................................................... 151

Flgure31: Heart rate (HR) before (day 1-3), during (day 4-10), after (day
11-14) continuous S6c infusion (30 pmollkglmin, SC.) in intact
and CGX Tg rats (+/+ and sl/sl). HR signiﬁcantly decreased
upon S6c infusion on the ﬁrst day in intact and CGX Tg (+I+)
rats, compared to the control period . In Tg (sllsl) rats, HR did
not change during the infusion or recovery period. Asterisk
indicate a statistical difference (p<0.05) from the last day of the
control period ................................................................................... 152

Figure32: Mean arterial pressure (MAP) before (day 1-3), during (day 4-
10), after (day 11-14) continuous S60 infusion (30
pmollkglmin, sc.) in neonatal capsaicin- and vehicle-treated
rats. The average resting blood pressure of capsaicin-treated
animals was higher than that of vehicle-treated ones. S6c
elevated MAP in a similar way in the two groups ............................. 159

Figure33: Heart rate (HR) before (day 1-3), during (day 4-10), after (day
11-14) continuous S6c infusion (30 pmollkglmin, so.) in
neonatal capsaicin- or vehicle-treated rats. The average
resting HR of capsaicin-treated animals was lower than that of
vehicle-treated ones. S6c reduced HR to a similar degree in
the two groups ................................................................................ 160

Figure34: Summary of the main ﬁndings of this research project ................... 167

xvi

BBB
CGRP
CGX
CO
CON
CVO
DOCA
ENS
EPI
ET
ETAR
ETBR
HR

IMG

NE

LIST OF ABBREVIATIONS

blood brain Barrier

calcitonin gene-related peptide
celiac ganglionectomy

cardiac output

Control

circumventricular organ
deoxycorticosterone acetate
enteric nervous system
epinephrine

endothelin

endothelin type A receptor
endothelin type B receptor
heart rate

inferior mesenteric ganglia
intraperitoneal

intravenous

mean circulatory ﬁlling pressure
mean arterial pressure
nicotinamide adenine dinucleotide
phosphate

norepinephrine

xvii

NO
NOS
02'
PBS
86c
sc

sl

SNS

VSMC

nitric oxide

nitric oxide synthase
superoxide anion

phosphate buffered saline
sarafotoxin 6c
subcutaneous

spotting lethal

sympathetic nervous system
tetrodotoxin

vascular smooth muscle cell

xviii

FREQUENTLY USED DRUGS AND CHEMICALS

Sarafotoxin 6c
Heparin

Ticarcillin
Enroﬂoxacin

Tylenol

Sodium pentobarbital
Dihydroethidium
Tetrodotoxin

Tempol

Norepinephrine

selective ETB receptor agonist
anticoagulant

antibiotic

antibiotic

pain reliever

anesthetic

oxidant sensitive ﬂuorogenic probe
sodium channel blocker
antioxidant

endogenous adrenal receptor agonist

xix

COMMONLY USED SOLUTIONS

Krebs’ physiological saline solution (1 L)

Sodium chloride 68.39
Potassium chloride 3.559
Calcium chloride 3.659
Magnesium chloride 2.459
Sodium phosphate, monobasic 1.659
Sodium bicarbonate 2.19

Glucose 2.09

0.1 M Phosphate buffered saline (1 L)

Monobasic monohydrate 3.859
Dibasic anhydrous 10.29
Sodium chloride 9.09
pH=7.4

CHAPTER 1

GENERAL INTRODUCTION

1. Hypertension as a serious disease

The deﬁnition of hypertension is systolic blood pressure of 140 mmHg or
higher, or diastolic blood pressure of 90 mmHg or higher, or both. In the US
alone, there are about 73 million people who are hypertensive. That is, almost
one in every three American adults has high blood pressure (American Heart
Association). Hypertension can damage the blood vessels and is associated with
other disease conditions, such as stroke, heart failure, heart attack, kidney
failure, and vision problems. Therefore, it is very important to study hypertension
and discover the causes of this disease. My research was designed to help
understand the causes of hypertension, and therefore ﬁnd improved treatments

for this serious disease.

2. Blood pressure regulation

To understand hypertension, we need to understand the physiology of
blood pressure regulation. As Figure 1 shows, mean arterial pressure (MAP) is
the product of cardiac output (CO) and total peripheral resistance (T PR). CO is
the product of stroke volume (SV) and heart rate (HR). SV is determined by both
ventricular preload and inotropy (force of cardiac contraction). And the former is
determined by blood volume and venous compliance. For TPR, it is mainly
determined by vascular structure and vascular function. Neurohumoral factors
are one of the most important groups of factors affecting blood pressure
regulation, Since the changes in these factors may alter renal sodium/water

excretion, venous compliance, inotroPY. HR, and TPR, thereby regulating MAP

through multiple pathways. Some of the factors regulating arterial blood pressure

are discussed below.

2.1 Humoral factors
2.1.1 The endothelin system
2.1.1.1 Discovery and synthesis

In 1985, an endothelial cell-derived vasoconstrictor polypeptide was found
(Hickey, Rubanyi et al. 1985). Later the substance was isolated by Yanagisawa
et al. and it was named endothelin (ET), since it was originally found to be
generated by vascular endothelial cells (Yanagisawa, Kurihara et al. 1988). This
21-amino acid peptide has been shown to be one of the most potent endogenous
vasoconstrictors known. There are 3 family members of this group of mammalian
vasoactive peptides: ET-1, ET-2, and ET-3, which are encoded by 3 different
genes in human and rat tissues (lnoue, Yanagisawa et al. 1989). ET-2 is very
Similar to ET-1, but ET-3 is different from ET-1 at 6 out of 21 positions (Goraca
2002). There are two essential disulphide bonds in the structure of ETs: one
between amino acids 1 and 15, and the other between amino acids 3 and 11. As
Figure 2 Shows, the preproform of ET is a 203-amino acid peptide, from which
164 amino acids is cleaved by dibasic endopeptidase and carboxypeptidases.
generating 39-amino acid big ET (Seidah, Day et al. 1993; Xu, Emoto et al.
1994). Then big ET is converted to ET by endothelin converting enzyme (ECE)
(Xu, Emoto et al. 1994).

The ETs are generated by various tissues in vivo, including kidney, lung,

central nervous system, pituitary and peripheral endocrine tissues, and placenta

3

(Hemsen and Lundberg 1991); (Hemsen, Gillis et al. 1991; Stojilkovic and Catt
1992). ET-1 is the most important one among the three isoforrns, since it is the
main isoforrn in human cardiovascular regulation. It is the most ubiquitous and
powerful vasoactive peptide which can exert long-lasting constrictor effects on
blood vessels (Davenport 2002). Therefore, it is the best characterized among
the three. ET-1 is mainly generated and released continuously from the vascular
endothelial cells in vivo (Yanagisawa 1994). The released ET-1 induces vascular
blood vessels to constrict, therefore maintaining basal vascular tone (Haynes and
Webb 1994). Upon external stimuli, ET-1 is also released from WeibeI-Palade
bodies (the endothelium-speciﬁc storage granules), leading to additional
vasoconstriction (Russell, Skepper et al. 1998). Therefore, ET-1 acts primarily
as a paracrine hormone, as opposed to a circulating hormone (Levin 1995). To a
less extent, ET-1 is also produced by epithelial and smooth muscle cells (Miller,
Pelton et al. 1993), mast cells (Ehrenreich, Burd et al. 1992), mesanglal cells,
neurons, glial cells, and liver cells (Levin 1996). ET—2 is primarily synthesized in
the kidney and intestine. It has been reported to be a vasoconstrictor in human
blood vessels (Maguire and Davenport 1995). Mature ET—3 circulates in the
plasma (Matsumoto, Suzuki et al. 1994). Endothelial cells do not produce ET-3
and the principal source of ET-3 has not been identiﬁed yet. ET-3 is unique in

that it has different binding afﬁnities for the two endothelin receptors.

2.1.1 .2 Endothelin receptors
Two types of endothelin receptors have been cloned: endothelin receptor

type A (ETAR) (Aral, Hori et al. 1990) and endothelin receptor type B (ETBR)

(Arai, Hori et al. 1990; Sakurai, Yanagisawa et al. 1990). These two subtypes
Show 59% similarity in amino acid composition. Human and rat ETARS are 91%
similar. There is a 12% difference in ETBRS between human and rat (Davenport
2002). Both types are G protein-coupled rhodopsin-type receptors with 7
transmembrane domains (Sakurai, Yanagisawa et al. 1992; Alexander, Mathie et
al. 2007). The signal transduction pathways engaged upon activation of ET
receptors usually involve phospholipase C (PLC) (Bousso-Mittler, Kloog et al.
1989; Vigne, Lazdunski et al. 1989), phospholipase A2 (PLAz) (Bousso-Mittler,
Kloog et al. 1989; Stojilkovic, lida et al. 1991), phospholipase D (PLD) (MacNulty,
Plevin et al. 1990; Konishi, Kondo et al. 1991), or NaVH" exchange (Simonson,

Wann et al. 1989; Battistini, Filep et al. 1991; Kramer, Smith et al. 1991).

The dissociation rates of receptor-ligand complexes in the ET system are
isoform-dependent (Galron, Kloog et al. 1989; DeveSIy, Phillips et al. 1990) and
Species-dependent (Galron, Bdolah et al. 1991). These differences could be due
to different modes Of binding (Galron, Kloog et al. 1989). However, one unique
characteristic of ET binding is that the rate of dissociation is extremely slow in
various tissues. For example, the binding is almost irreversible in vascular
smooth muscle cells (VSMCS) from rats (Hirata, Yoshimi et al. 1988). ET
receptor activation induces long-lasting physiological effects, while the half-life of
circulating ET is very short (Anggard, Galton et al. 1989). This seeming paradox

is at least partially due to slow dissociation rate of ET from its receptors.

ETARS have the same binding afﬁnity for ET-1 and ET-2. On the other

hand, ET-3 has little or no afﬁnity for ETARS at physiological concentrations

5

(Sakurai, Yanagisawa et al. 1992). ETARS are mainly located at VSMCS and
cardiac muscle cells. They mediate vasoconstriction on ET—1 binding (Miller,
Pelton et al. 1993). They also mediate cellular proliferation as a growth factor

(Bobik, Grooms et al. 1990; Luscher, Oemar et al. 1993).

ETBRS have the same affinity for the three isofonns of ETS (Arai, Hori et
al. 1990; Sakurai, Yanagisawa et al. 1990). ETBRS are present on endothelial
cells, VSMCS, kidney, liver, etc. The activation of ETBRS has multiple functions.
Activation of receptors on vascular endothelium can produce vasodilation by the
release of endothelial cell derived vasodilators, such as nitric oxide and
prostaglandins (Wright and Fozard 1988; Warner, de Nucci et al. 1989; Rubanyi
and Polokoff 1994). Activation of ETBRS in the kidney leads to a marked
increase in renal excretion of sodium and water by inhibiting sodium reabsorption
in the renal tubules and water reabsorption in the collecting duct (Kohan, Padilla
et al. 1993; Plato, Pollock et al. 2000). Whereas, renal vasoconstriction is
induced by the stimulation of ETBRS in the rat (Clozel, Gray et al. 1992; Cristol,
Warner et al. 1993). In addition, ETBRS act as clearance receptors for
endogenous ET-1 (Ozaki, Ohwaki et al. 1995; Dupuis, Goresky et al. 1996;
Berthiaume, Yanagisawa et al. 2000). Recently, it has also been found that
ETBR activation constricts veins but only weakly constricts resistance arteries
(Moreland, McMullen et al. 1992; Gray, Lofﬂer et al. 1994; Johnson, Fink et al.
2002). Another in vitro study shows that ETBR activation leads to increased
oxidative stress in sympathetic ganglia (Dal, Galligan et al. 2004). Acute

activation of ETBRS in vivo induces a sustained blood pressure increase (Lau,

Galligan et al. 2006) and increased superoxide levels in sympathetic ganglia
(Dai, Galligan et al. 2004; Lau, Galligan et al. 2006). Therefore, the
multifunctional nature of ETBRS makes studying their roles in physiological and

pathological conditions complicated.

Although ETBRS are present in both arterial and venous vasculatures,
they are differentially expressed and functional in arteries and veins. For
example, both ETBR mRNA and protein densities are higher in veins than in
arteries (Watts, Fink et al. 2002). Furthermore, although ETBRS are present in
both arteries and veins, they are not functional in most arterial beds, including
large arteries and small arteries (Johnson, Fink et al. 2002; Watts, Fink et al.
2002). On the other hand, veins are constricted by $60, a selective ETBR
agonist, in a concentration-dependent manner, indicating that ETBRS are
functional in veins. Additionally, it has been shown that S6c-induced constriction
in ET-1 pretreated veins is the same as in untreated vessels, indicating that
venous ETBRS do not desensitize to ET-1 (T hakali, Fink et al. 2004). On the
contrary, ETARS on arteries and veins desensitize to ET—1. This may explain why
the hypertension is maintained while ETARS are densensitized to a increased
circulating ET-1 level in an animal model of hypertension (Nguyen, Parent et al.
1992; Fujita, Matsumura et al. 1995; Laurant and Berthelot 1996; Watts, Fink et
al. 2002).

2.1.1.3 Endothelin-1 in physiological conditions
ET-1 acts at multiple locations in biological systems under physiological

conditions. ET-1 participates in maintaining basal tone of vascular smooth

7

muscles (Haynes, Ferro et al. 1995). Resistance arteries and capacitance veins
are especially sensitive to ET—1 (Cocks, Broughton et al. 1989). Intravenous
administration of ET-1 induces a transient and fast vasodilation, followed by a
prolonged vasoconstriction (Yanagisawa, Kurihara et al. 1988), which in turn
elevates systemic blood pressure by increasing total peripheral resistance
(Rubanyi and Polokoff 1994). ET-3 can produce similar effects in a dose-
dependent manner (Mortensen, Pawloski et al. 1990). The initial vasodilation is
regulated through ETBRS on vascular endothelium by the release of endothelial
cell den'ved vasodilators (Wright and Fozard 1988; Warner, de Nucci et al. 1989;
Rubanyi and Polokoff 1994). ET-1-induced vasoconstriction iS mediated by both
ETARS and ETBRS located in VSMCS (Haynes, Strachan et al. 1995). ET-1 has
powerful positive inotropic effects on the heart in vitro (lshikawa, Yanagisawa et
al. 1988). Coronary exposure to ET-1 may result in ventricular arrhythmias and
myocardial ischemia (Ezra, Goldstein et al. 1989). A signiﬁcant amount of ET-1 is
generated by the collecting duct cells from inner medulla in the rat (Kohan and
Fiedorek 1991). Both acute and chronic intracerebroventricular administration of
ET-1 increases mean arterial blood pressure and heart rate through elevation of
central sympathetic activity (Ouchi, Kim et al. 1989; Matsumura, Abe et al. 1991;
Nishimura, Takahashi et al. 1991), indicating that ET-1 has a central pressor
action. ET-1 also plays a role in the peripheral nervous system. Studies have
demonstrated that ET binding sites are present in the baroreceptor and
chemoreceptor afferent pathways. Afferent activity of baroreceptors and

chemoreceptors can be affected by ETS (Spyer, McQueen et al. 1991). In human

arteries, endothelin-1 potentiates the effects of other vasoconstrictor hormones,
such as serotonin and norepinephrine, suggesting that ET-1 ampliﬁes the effects

of sympathetic nervous system activity (Yang, Richard et al. 1990).

2.1.1.4 Endothelin in hypertension

Plasma concentrations of ET-1 in experimental models of hypertension
are not increased unless malignant hypertension is present (Suzuki, Miyauchi et
al. 1990; Kohno, Murakawa et al. 1991). Elevated circulating ET levels have
been reported in some forms of human hypertension (Kohno, Yasunari et al.
1990; Shichiri, Hirata et al. 1990; Yoshibayashi, Nishioka et al. 1991). Several
experimental models of hypertension have shown activated endothelin system,
such as the deoxycorticosterone acetate (DOCA) salt hypertensive rats
(Lariviere, Day at al. 1993; Lariviere, Thibault et al. 1993; Li, Lariviere et al.
1994), DOCA salt-treated spontaneously hypertensive rats (SHR) (Schiffrin,
Lariviere et al. 1995), Dahl salt-sensitive rats (Doucet, Gonzalez et al. 1996),
and 1-kidney 1-clip Goldblatt hypertensive rats. The vasoconstriction caused by
ET-1 is more sensitive in hypertensive rats than in controls (T omobe, Miyauchi et
al. 1988; Suzuki, Miyauchi et al. 1990). In patients with essential hypertension,
an increased vasoconstrictor response to ET-1 was reported, indicating
enhanced vascular endothelin vasoconstrictor activity (Cardillo, Kilcoyne et al.
1999). In addition, ET-1-induced vasoconstriction of glomerular arterioles causes
a decrease in glomerular ﬁltration rate, renal plasma ﬂow, and sodium excretion,
as well as a signiﬁcant increase in renal vascular resistance (Lopez-Farre,

Montanes et al. 1989). Furthermore, in SHR rats less ET—1 is produced in the

renal medulla, where ET-1 mediates renal excretion of sodium and water through
ETBRS, resulting in water/sodium retention and hypertension (Kitamura, Tanaka
et al. 1989; Hughes, Cline et al. 1992). The results from experimental models are
consistent with those from patients with essential hypertension, since these
patients Show less urinary excretion of endothelin than control subjects. ECE
inhibitors are able to lower blood pressure in hypertensive rats (McMahon,
Palomo et al. 1991; McMahon, Palomo et al. 1993; Nishikibe, Tsuchida et al.
1993; Pollock and Opgenorth 1993; Douglas, Gellai et al. 1994; Veniant, Clozel
et al. 1994; Fujita, Matsumura et al. 1996). Endothelin receptor antagonists have
also been shown to have anti-hypertensive effects (Nishikibe, Tsuchida et al.
1993; Douglas, Gellai et al. 1994; Fujita, Matsumura et al. 1996). Moreover, data
from experiments in the dog indicate that the anti-endothelin therapies by ECE
inhibitors and ET receptor blockade are mediated by independent pathways,
Since the effects induced by different categories of agents are additive (Donckier,
Massart et al. 1997). Together these data indicate that ET-1 is involved in the

pathogenesis of hypertension.

2.1.2 Catecholamines

Catecholamines are a group of hormones widely distributed in mammalian
tissues. They include norepinephrine (NE), epinephrine (EPI), and dopamine.
EPI is released by the adrenal medulla and it performs its functions mainly as a
circulating hormone (Elliot 1905). EPI is a neurotransmitter of central nervous
system (CNS) (Hokfelt, Fuxe et al. 1974), as well as of autonomic nervous

system (Loewi 1936). A small portion of NE is released from adrenal medulla.

10

The majority of NE in the circulation is spillover from sympathetic nerves
innervating blood vessels (Silverberg, Shah et al. 1978). NE is the primary
neurotransmitter of mammalian sympathetic nervous system (Von Euler 1946).
Dopamine is a precursor of NE and EPI. It is localized with them in various
tissues (Bell 1983; Kopin 1985; Pierpont, DeMaster et al. 1985). It is an important
central transmitter (Carlsson, Fuxe et al. 1966). It is involved in peripheral

nervous regulation as well (Yorikane, Kanda et al. 1986; Bell 1987).

Tyrosine is the amino acid precursor for the biosynthesis of
catecholamines (Axelrod 1968). It is transported into sympathetic neurons or
chromafﬁn cells, and hydroxylated by thyrosine hydroxylase (TH), forming dopa
(Chirigos, Greengard et al. 1960). This step is rate-limiting in the synthesis of NE
and thus TH is critical in regulating NE synthesis. Then aromatic 1-amino acid
decarboxylase transformed dopa into dopamine through the reaction of
decarboxylation. Dopamine enters a chromafﬁn granule in adrenal medulla or a
vesicle in sympathetic neurons. Upon the entrance, dopamine is hydroxylated on
the IS position to form NE, by the enzymatic activity of dopamine-B—hydroxylase.
Finally, EPI is converted from NE by the enzyme phenylethanolamine
methyltransferase (Axelrod 1962).

As a critical sympathetic neurotransmitter, the majority of NE is
synthesized and stored in the vesicles of sympathetic nerves. Upon an action
potential, there is an inﬂux of Ca2+ into the nerve terminal. Then the vesicles
containing NE fuse to the plasma membrane and NE is released from the

vesicles by the process of exocytosis. The neurotransmitter then activates a- and

11

B-adrenergic receptors in the membrane of postjunctional cells. The termination
of NE is achieved in part by two uptake systems: uptake 1 and 2. For uptake 1,
the released NE in the junctional space is taken back into the nerves by high
afﬁnity transporters on the plasma membrane of sympathetic neurons. For
uptake 2, NE binds to postjunctional vascular smooth muscle cells, where it is
taken up by the corticosterone-sensitive extraneuronal monoamine transporter.
The amount of released NE is tightly regulated by not only NE itself (via 02-
adrenergic receptors on cell membrane of non-neuronal cells), but also many
other mediators such as EPI, serotonin , histamine and acetylcholine (El-Etri,
Ennis et al. 1999; Kulkarni, Opere et al. 2006). Normally, most of the NE
released by sympathetic nerves is taken up by the nerves where it is
metabolized. A small amount of NE, however, diffuses into the blood and
circulates throughout the body. With a high sympathetic nerve activation, the
amount of NE entering the blood increases Signiﬁcantly (Axelrod and Kopin

1969).

The release of sympathetic neurotransmitters, especially NE, is an index
of sympathetic nerve activity (Doyle and Smirk 1955; Wallin, Delius et al. 1973).
Measurement of NE spillover can be used for chronic studies of sympathetic
nervous system activity in awake animals (Sano, Way et al. 1989). Additionally,
this technique is feasible in human beings by the application of central venous
catheters (Esler, Jennings et al. 1984; Hasking, Esler et al. 1986; Esler, Jennings
et al. 1988). Although systemic NE spillover is very useful to indicate whole body

neural tone, it can not be used to predict local sympathetic function in an organ-

12

speciﬁc manner, since NE is not evenly distributed among all organs (Korner
1971). Therefore, regional sampling of NE is a more precise method to quantify
local sympathetic nervous activity (Robertson, Johnson et al. 1979). The actions
of NE on VSMCS and myocytes of the myocardium are through the activation of
B—adrenergic receptors, and a1- and q2-adrenergic receptors, resulting in
vasodilation, vasoconstriction, and inotropy, depending on receptors and

locations (Philipp, Brede et al. 2002; Brede, Nagy et al. 2003).

2.1.3 Calcitonin gene-related peptide

Capsaicin is a derivative of vanillyl amide (8-methyl-N-vanillyI-6-
nonenamide) with a molecular weight of 305.32. It is a primary ingredient in the
red hot chili peppers of the genus Capsicum (Holzer 1991). In the late 19th
century, Hogyes was the ﬁrst to report that the pungent action of an extract from
Capsicum was mediated through sensory nerves (Hogyes 1878). Later on,
capsaicin was shown to have a chronic blocking effect on sensory receptors
(Jancso 1968) and destroy some sensitive nerves at a very high concentration
(Jancso 1955). This group of nerves is termed “capsaicin-sensitive sensory
nerves” (CSSNS). They are usually C ﬁbers ( with unmyelinated axons) and A

ﬁbers (with thinkly myelinated axons) (Maggi 1993).

The cardiovascular system is widely innervated by CSSNS (Mazzocchi,
Malendowicz et al. 1992). CSSNS play important roles in blood pressure
regulation by releasing vasoactive neuropeptides and therefore modulating
peripheral resistance (Deng and Li 2005). Calcitonin gene-related peptide

(CGRP), is a major neurotransmitter in CSSNS (Bell and McDennott 1996). It is a

13

37-amino acid neuropeptide found by cloning of the calcitonin (CT) gene in 1983
(Rosenfeld, Mermod et al. 1983). CGRP is generated by alternative splicing of
the primary RNA transcript of the CTICGRP gene (Rosenfeld, Amara et al.
1981). There are two isoforms of CGRP: q-CGRP and B-CGRP. q-CGRP is
synthesized in neuronal tissues almost exclusively (Breimer, Maclntyre et al.
1988) and is widely distributed in the central nervous system and peripheral
nervous system in both animals and humans (Oh-hashi, Shindo et al. 2001). The
B-CGRP gene is believed to have arisen by gene duplication and these two
isoforms exhibit nearly identical biological activities (Wimalawansa 1996). CGRP
is synthesized in dorsal root ganglia where the cell bodies of sensory afferent
neurons reside. These sensory nerves terminate peripherally on blood vessels
and other tissues (Wimalawansa 1996). It is distributed at a higher density

around blood vessels than on the heart (VWmalawansa 1996).

CGRP is a very potent vasodilator (Brain, Williams et al. 1985). Studies
have shown that it is 100 to 1000 times more potent than substance P or
acetylcholine. It has been suggested that CGRP may play an important role in
regulating peripheral vascular tone and regional organ blood ﬂow under
physiological conditions (DiPette, Schwarzenberger et al. 1989). As to
pathological conditions, It has been shown that systemic administration of CGRP
lowers blood pressure in spontaneously hypertensive rats (Dipette and
WImalawansa 1995) and DOCA-salt rats (Supowit, Zhao et al. 1997) through
vasodilation. Therefore, CGRP may attenuate blood pressure increase as an

endogenous depressor substance. It plays a signiﬁcant role in the initiation,

14

development, and maintenance of hypertension (Deng and Li 2005).
Furthermore, ET-1 has been shown to induce the release of CGRP, most likely

through an ETAR-based mechanism (Wang and Wang 2004).

2.1.4 Others

There are many other hormones that are important in blood pressure
regulation. For example, the renin-angiotensin-aldosterone system (RAAS) plays
an important role in regulating blood volume and vascular resistance, which in
turn affects arterial pressure. Vasopressin (arginine vasopressin, AVP;
antidiuretic hormone, ADH) is a peptide hormone formed in the hypothalamus,
then transported via axons to, and released from, the posterior pituitary. It
regulates extracellular ﬂuid volume by affecting renal water excretion, leading to
a decrease in urine formation, an increase in blood volume, cardiac output, and
ﬁnally arterial pressure. On the other hand, some hormones favor blood pressure
decrease, such as arterial natriuretic peptide (ANP) and brain-type natriuretic
peptide (BNP). They not only dilate blood vessels, but also induce natriuresis and

diuresis.

2.2 Vascular function

Vascular resistance is determined by both the structure and the function of
blood vessels. Due to the differences between arteries and veins, each blood
vessel type is discussed separately. Additionally, the splanchnic region, a critical

bed in blood pressure regulation, is discussed.

2.2.1 Arterial bed

15

Arteries take blood away from the heart. Their walls are thick and enclose
a small lumen. Aorta and large arteries mainly serve to distribute blood, while the
small arteries and arterioles are the main vessels involved in blood pressure
regulation as well as blood ﬂow in various organs. These vessels are highly
innervated by autonomic nerves (especially sympathetic nerves), and respond to
changes in nerve activity and circulating hormones by constriction or
dilation. These vessels are referred to as resistance vessels. Changes in the
diameter of these resistance vessels lead to changes in total peripheral

resistance and blood pressure.

2.2.2 Venous bed

Veins take blood to the heart. Their walls are thin and enclose a large
lumen. They are not as elastic as arteries, because their walls contain mainly
connective tissue and less smooth muscle than arteries. Veins and venules
function as the primary capacitance vessels of the body, since most of the blood
is stored in these vessels. Both experimental results (De Michele, Cavallotti et al.
1991) and clinical data (Fitzpatrick, Hinderliter et al. 1986) show that altered
physiological functions of veins can contribute to the development and
maintenance of “arterial” diseases. Unfortunately, much less experimental
attention has been paid to veins, compared to arteries. Veins are usually viewed
as simple, passive “conduits” for return of blood to the heart. In fact, they are
complex physiological entities and important in overall function of the
cardiovascular system (Rothe 1983). Veins contain about 70% of the blood

volume, approximately 75% Of which is in small veins and venules (Rothe 1983)

16

(Figure 3a). As such, they are the most important blood reservoir in the

circulation.

Vascular compliance is deﬁned as “the ratio of a change in volume to the
concomitant change in transmural distending pressure” (Rothe 1993). It is a
quantitative measure of the elasticity of a vascular bed. Systemic venous
compliance is at least 30 times greater than that of arterial compliance in most
species of mammals, including rats and humans (Pang 2001). Vascular capacity
is the total blood volume held in the circulation at a speciﬁc pressure, as a sum of
unstressed and stressed blood volumes (Shoukas and Sagawa 1973). Vascular
capacitance is the relationship between contained volume and distending
pressure of a segment of the vasculature. Since it is a relationship, capacitance
can not be described by a simple number (Rothe 1993). Because the
compliance of veins is much larger than that of arteries, and the cross-sectional
area of venules is larger than that of arterioles, venules are frequently regarded
as the primary capacitance component Of the overall vascular tree. Therefore,
total vascular capacitance is almost entirely determined by the structural and
functional properties of veins. Constriction of veins can be very important in blood
pressure regulation, since it leads to a redistribution of some stored blood from
peripheral veins to the heart and then into the arterial circulation. AS a result, the
cardiac ﬁlling pressure and/or cardiac output increase (Halliwill, Minson et al.
1999) and increased arterial pressure occurs. There are several methods
available to measure total body venous tone (constriction degree of veins). Mean

circulatory ﬁlling pressure (MCFP), the effective driving force for venous return to

17

the heart, can be used as an index of venomotor tone and it is commonly used in
experimental work. MCFP is usually, but not always, increased in hypertension
(Martin, Rodrigo et al. 1998). The main factors determining MCFP are blood
volume, venous smooth muscle structure and constrictor tone (Yamamoto,
Trippodo et al. 1980). Safar et al. has indicated that blood volume is not
changed, or even reduced, in established hypertension (Safar and London 1987).
So either structural or functional changes of veins, or both, account for the

increased MCFP.

2.2.3 The splanchnic bed - a critical bed for regulation of vascular capacitance
The splanchnic region, including liver, spleen, pancreas, small and large
intestines, is the most important vascular bed to consider in studies concerning

the role of veins in hypertension.

2.2.3.1 Veins and venules in the splanchnic bed
The splanchnic vascular bed is very compliant, especially the veins. In
addition, the vein and venules in the splanchnic region account for most of the
active capacitance responses of the circulation (Greenway and Lautt 1986;
Rothe 1986; Rothe 1993). Moreover, this bed holds about 33% of the total blood
volume (Figure 3b) (Greenway and Lister 1974). So the splanchnic veins act as

the biggest modiﬁable reservoir of blood in the circulation.

Decreased venous capacitance associated with hypertension has been
documented in animal models (Fink, Johnson et al. 2000) and humans (Schobel,

Schmieder et al. 1993). This ﬁnding is especially evident in the splanchnic bed,

18

due to changes in smooth muscle activity (functional changes) and/or alterations
in connective tissue content and/or organization of vasculatures (structural
changes). The decrease in venous capacitance may lead to a blood shift from
peripheral vascular beds to arterial circulation (Ricksten, Yao et al. 1981). AS a
result, the blood pressure increases. Additionally, Miura reported that TPR, renal
vascular resistance, and hepatosplanchnic vascular resistance, were all
increased in the hepatosplanchnic bed before the occurrence of vascular
resistance elevation in other areas at the earliest stage of development in human
hypertension (Sugawara, Noshiro et al. 1997), indicating the important role of the
splanchnic circulation in blood pressure regulation and hypertension

development.

2.2.3.2 Differential sympathetic innervation: arteries vs. veins

The splanchnic bed is richly innervated, especially by sympathetic nerves.
Therefore, sympathetic control over the splanchnic vasculature may play a
critical role in blood pressure regulation. The compliance and capacitance of the
innervated splanchnic bed are altered in response to sympathetic stimulation or
inhibition. The splanchnic sympathetic nerves projecting to the splanchnic bed
arise from prevertebral and paravertebral ganglia. In rats, the cell bodies of a
great majority of sympathetic postganglionic neurons innervating the splanchnic
bed are located in two prevertebral ganglia: celiac ganglion and superior
mesenteric ganglion (T rudrung, Fumess et al. 1994; Hsieh, Liu et al. 2000;

Quinson, Robbins et al. 2001). These two ganglia are very close to each other in

19

the rat and are usually termed the “celiac plexus” (Chevendra and Weaver 1991;

Fumess, Koopmans et al. 2000).

Although sympathetic activation constricts both arteries and veins, the
sympathetic neurons innervating arteries and veins are differentially located in
the ganglia and they show different electrophysiological properties, indicating
there is a differential sympathetic neural control of mesenteric arteries and veins
(Browning, Zheng et al. 1999). These results are consistent with the data from
studies on the neuroeffector mechanisms in arteries and veins (Hottenstein and
Kreulen 1987). Sympathetic nerve stimulation to veins produces a slow
depolarization and contraction mediated by norepinephrine acting at 01-
adrenergic receptors. On the other hand, in small mesenteric arteries and
arterioles, electrical stimulation evokes short latency, short-duration excitatory
junction potentials and contractions mediated by ATP acting at P2X receptors

(Gitterrnan and Evans 2001).

Veins are more sensitive to vasoconstrictor effects of sympathetic nerve
stimulation than arteries under physiological conditions (Kreulen 1986) and
pathological conditions (Luo, Hess et al. 2003). Quantitatively speaking,
sympathetic nerve activity (SNA) is the most critical factor controlling
venoconstriction, especially in the Splanchnic circulation (Shoukas and Bohlen
1990; Ozono, Bosnjak et al. 1991). Sympathetic activation of the splanchnic
veins can reduce blood volume by up to 60%, redistributing blood from veins into
the heart and thereby increasing blood pressure (Karim and Hainsworth 1976;

Greenway 1983). The membrane potential of venous smooth muscle cells in

20

some hypertension models, such as SHRS, is relatively depolarized compared to
that in normotensive rats. This change is due to increased sympathetic tone to
veins in SHRS (Willems, Harder et al. 1982). Therefore the splanchnic SNA may

have more inﬂuence on veins than on arteries in blood pressure regulation.

2.2.3.3 Increased splanchnic SNA in hypertension

The sympathetic nervous system (SNS) plays an important role in
hypeitension development in animal models (Wyss 1993; Cabassi, Wnci et al.
2002) and in humans (Anderson, Sinkey et al. 1989; Weber 1993; Esler,
Rumantir et al. 2001; DeQuattro and Feng 2002; Zhu, Poole et al. 2005). The
SNS can elevate blood pressure by increasing cardiac output and peripheral
vascular resistance. It is generally found that sympathetic effects on the
cardiovascular system are augmented in hypertension (Nestel 1969; Egan, Panis
et al. 1987; Somers, Anderson et al. 1993; Esler and Kaye 1998). This could be
caused by increased sympathetic nerve density, high sympathetic nerve ﬁring
rates, epinephrine cotransmission, norepinephrine transporter dysfunction,
increased release of norepinephrine from the neuroeffector junction, increased
end organ responsiveness to sympathetic neurotransmitters, or a combination of
the above (Esler, Rumantir et al. 2001). Increased sympathetic
neurotransmission has been documented in experimental hypertension models,
such as DOCA-salt hypertension (Bouvier and de Champlain 1986) and
spontaneous hypeitension (Ekas and Lokhandwala 1981). Increased plasma
norepinephrine (NE) levels are sometimes measured in these models,

suggesting an enhanced neurotransmitter release from sympathetic nerves.

21

Clinically, antihypertensive drugs that reduce SNA may be the best treatment for
hypertensive patients with autonomic imbalance (Brook and Julius 2000).
Moreover, the SNS in the splanchnic bed is particularly important in blood
pressure regulation, since the splanchnic SNS innervates the majority of
resistance and capacitance vessels. The activation of the splanchnic SNS may
increase total peripheral resistance, reduce vascular capacitance, and elevate
blood pressure. Our lab and others show that blood pressure is Signiﬁcantly
lowered when most splanchnic sympathetic innervation is removed (Kregel and
Gisolﬁ 1989; King, Osborn et al. 2007). In humans, the removal of celiac plexus
was performed as a treatment for human essential hypertension before
antihypertensive medications were available (Heuer 1936; Martin 1938; Weiss

1939; Feet, Woods et al. 1940).

3. Oxidative stress
3.1 Introduction

Oxidative stress is a state where there is a disturbance in the prooxidant-
antioxidant balance in tissues in favor of prooxidant species, leading to potential
oxidative damage (Sies 1991). Reactive oxygen species (ROS) are a variety of
oxygen-containing molecules that are byproducts of cellular metabolic processes
under physiological conditions, including superoxide anion (02"), hydrogen
peroxide (H202), and hydroxyl ion (OH'). Superoxide production is mediated by
several enzymes including nicotinamide adenine dinucleotide phosphate
(NADPH) oxidase, xanthine oxidase, and nitric oxide synthase (NOS) (Wilcox

2002). In addition to direct effects, 02" affects tissues indirectly by generating

22

other ROS. It can be converted, via enzyme superoxides dismutase (SOD), into
H202, which is then converted into water by catalase and glutathione peroxidase,
or into OH' by interacting with reduced transition metals (T aniyama and

Griendling 2003).

3.2 ROS in hypertension

Under physiological conditions, ROS are present at low concentrations.
Originally, ROS were simply thought to be byproducts of some biological
reactions taking place in multiple cellular components such as mitochondria
(Balaban, Nemoto et al. 2005), peroxisomes (Schrader and Fahimi 2004), and
cytochrome P-450 (Gottlieb 2003; Gonzalez 2005). However, emerging evidence
has supported that ROS act as cellular signaling molecules (Zimmerman and
Davisson 2004; Mueller, Laude et al. 2005; Bedard and Krause 2007). They
achieve signaling functions by participating in phosphatase inhibition (Lee, Kwon
et al. 1998; Wu, Hardy et al. 2003; Goldstein, Mahadev et al. 2005; Kwon, Qu et
al. 2005), kinase activation (Griendling, Sorescu et al. 2000; Han, Kim et al.
2003; Furst, Brueckl et al. 2005), ion channel regulation (Hidalgo, Bull et al.
2004; Tang, Santarelli et al. 2004), and calcium signaling (Wang, Takeda et al.
1999; Cheranov and Jaggar 2006; Granados, Salido et al. 2006). Increased
ROS levels have been found in blood vessels, kidneys and other tissues in many
experimental models of hypertension, including DOCA-salt (Somers, Mavromatis
et al. 2000), Dahl salt-sensitive (Swei, Lacy et al. 1999), angiotensin II
hypertension (Kawada, lmai et al. 2002), and lead-induced hypertension (Vaziri,

Liang et al. 1999). ROS generation also is elevated in hypertensive patients

23

(Touyz and Schiffrin 2001). Under pathological conditions, ROS may impair
endothelium-dependent vasorelaxation (Somers, Mavromatis et al. 2000), induce
VSMC growth (Griendling, Minieri et al. 1994; Zafari, Ushio-Fukai et al. 1998),
elevate sympathetic nervous system activity (Campese, Ye et al. 2004), alter
renal function, and raise deposition of extracellular matrix proteins. All of the
above effects may contribute to pathophysiological changes, such as vascular
and organ damages, in a variety of cardiovascular diseases (Harrison 1997).
Accumulating evidence indicates that ROS play a role in the development and
maintenance of hypertension (T ouyz and Schiffrin 2004). Increased tissue ROS
levels appear to cause hypertension, because blocking ROS generation or
increasing their removal lowers blood pressure (Kopkan and Majid 2005;

Sullivan, Pollock et al. 2006; Baumer, Kruger et al. 2007).

About 20% of the oxygen consumed by the body goes to the nervous
system. Therefore, the nervous system is an important source of ROS. Emerging
evidence has shown that ROS plays an important role in neuropathogenesis of
cardiovascular diseases (Peterson, Sharma et al. 2006). Extensive research has
been carried out to discover the role of ROS in the central nervous system (CNS)
(Kishi, Hirooka et al. 2004; Lee, Shoji et al. 2004; Lu, Helwig et al. 2004; Wang,
Anrather et al. 2004; Gao, Wang et al. 2005; Peterson, Sharma et al. 2006).
Excessive production of ROS in the brain has been reported in many
experimental models of hypertension (Zimmerman and Davisson 2004). The
increased ROS levels in CNS may be crucial in the pathogenesis Of hypertension

(Griendling, Minieri et al. 1994; Zimmerman, Lazartigues et al. 2004). NAD(P)H

24

oxidase seems to be a major enzyme generating ROS in the brain. ROS plays an
important role as a key mediator in the central effect of angiotensin II on
cardiovascular regulation. Mechanisms of redox imbalance in CNS may be
involved in the pathogenesis of hypertension (Peterson, Sharma et al. 2006). In
addition, oxidative stress is important in the peripheral nervous system. Oxidative
stress has been shown to inhibit baroreceptor activity, contributing to
baroreceptor dysfunction in atherosclerosis (Li, Mao et al. 1996). Tempol, an
antioxidant, lowers renal sympathetic nerve activity, heart rate, and mean arterial
pressure in rats (Xu, Fink et al. 2001). The administration of diethyldithio—
carbamic (DETC), an SOD inhibitor, increased renal superoxide levels (Zou, Li et
al. 2001; Makino, Skelton et al. 2002) and peripheral sympathetic nerve activity
(Shokoji, Fujisawa et al. 2004). Moreover, increased neuronal superoxide levels
in an acute hypeitension model are partially due to direct ETBR activation in

sympathetic ganglia (Lau, Galligan et al. 2006).

3.3 Approaches to measure oxidative stress

ROS are highly reactive and relatively instable (half-life in seconds).
Therefore it is difﬁcult to measure ROS levels directly in biological systems.
Measurements of ROS are mainly performed by indirect methods. Usually some
“sensor” molecules are applied to target tissues. These sensors are oxidized by
ROS in various cellular elements, such as lipids, protein, or DNA (Freeman and
Crapo 1982; Pryor and Godber 1991; Hirota, Murata et al. 1999; Bowie and
O'Neill 2000). These oxidized sensors bring out luminescent or ﬂuorescent

signals to reﬂect the concentrations of ROS.

25

Lucigenin assay has been used extensively for 02" detection in many
biological systems (Griendling, Minieri et al. 1994; Guyton, Liu et al. 1996;
Rajagopalan, Kurz et al. 1996; Irani, Xia et al. 1997; Li, Zhu et al. 1998;
Skatchkov, Sperling et al. 1999), due to the access to intracellular sites, small
cellular toxicity, and 02" selectivity (T arpey and Fridovich 2001). This assay is
based on the mechanism that lucigenin (Luc2") can be reduced to LuCH-*, which
is then oxidized by 02" and elicit an unstable dioxetane component yielding light
during the spontaneous decomposition to its stable-state electronic conﬁguration
(Faulkner and Fridovich 1993). It has recently been shown that several
enzymatic systems in vitro may reduce Luc2" even without the presence of 02",
generating LucH-*, which is then oxidized automatically (Liochev and Fridovich
1997). Therefore lucigenin is able to be chemically active in a “redox cycle”
fashion. This is a major drawback in the use of this chemiluminescent substrate

in the detection of 02" (Liochev and Fridovich 1997).

Dihydroethidium (DHE) is a cell perrneant compound. It can be oxidized
by 02" with a two-electron transfer, resulting in the formation of the ﬂuorophore
ethidium bromide which is DNA-binding (Benov, Sztejnberg et al. 1998). The
reaction is relatively selective for 02", since the oxidations induced by other
oxidants, such as H202, ONOO', or hypochlorous acid, are minimal. The
evaluation of intracellular ﬂuorescent ethidium can be performed by ﬂow
cytometry (Carter, Narayanan et al. 1994). The oxidation can also be visualized
with digital imaging microﬂuorometry (Bindokas, Jordan et al. 1996; Li, Fink et al.

2003; Dai, Galligan et al. 2004; Lau, Galligan et al. 2006). Unlike lucigenin, DHE

26

does not generate “artiﬁcial” 02" through redox cycling. However, there are some
limitations to the application of DHE as a quantitative detector for the production
of 02" (Benov, Sztejnberg et al. 1998). One is that cytochrome c is also able to
oxidize DH E. This is critical when cytochrome c is released into the cytosol under
certain conditions (Green and Reed 1998). In addition, quantiﬁcation Of 02"
production using DHE is semi-accurate because of its capacity to increase rates
of 02" dismutation to H202 (Benov, Sztejnberg et al. 1998). Last, a control is
always required for comparison when the oxidation is evaluated by ﬂuorescent

images. Therefore, it is not a very convenient assay.

Other methods for 02" detection include reduction of ferricytochrome c
(Babior, Kipnes et al. 1973; Matsubara and Ziff 1986; Shingu, Yoshioka et al.
1989), adrenochrome Formation (Boveris 1984), cyanide-resistant oxygen
consumption (Hassan and Fridovich 1979), etc. Additionally, H202 can be
measured by horse radish peroxidase—linked assays (Boveris 1984),
dichloroﬂuorescein ﬂuorescence (Paul and Sbarra 1968), and intermediate

complex (Sies 1981 ).

4. S6c-induced hypertension
4.1 Sarafotoxins
Sarafotoxins are a family of peptides that show a high degree of similarity
in amino acid sequence of ETs. They were discovered in the venom of the Israeli
burrowing asp, Atractaspis engadensis (Kochva, Viljoen et al. 1982; Takasaki,
Tamiya et al. 1988). They are a group of four (sarafotoxin 6a, 6b, 6c, 6d) 21-

amino-acid peptides, with a similar structure to ETs’. The sequence similarity

27

between these two families varies from 52-67% (Kochva, Bdolah et al. 1993).
Both families have two disulﬁde bonds, a hydrophobic C-terminal, and three polar

charged side chains (Sokolovsky 1992).

Among sarafotoxins, sarafotoxin 6c (S6c), a member of sarafotoxin family
is very unique in that it is a selective agonist for ETBRS (Ambar, Kloog et al.
1989; Williams, Jones et al. 1991). 86c shows approximately the same binding
afﬁnity to ETBRS as ETS (Kloog, Bousso-Mittler et al. 1989; Wollberg, Bdolah et
al. 1991), while its binding afﬁnity to ETARS is much smaller than ET-1 and ET-2,
which are ETARS selective binding ligands (Galron, Kloog et al. 1989; Williams,
Jones et al. 1991). Therefore, S6c can be used as a selective ETBR agonist for

pharmacological studies.

4.2 Pharmacology of 860

Since S6c is a selective agonist for ETBRS, the pharmacological actions
induced by S6C are mainly mediated through ETBRS. S6c has been shown to
constrict isolated mesenteric veins in rats. 0n the other hand, S6c-induced
arterial constriction in mesenteric bed is negligible (Johnson, Fink et al. 2002).
Additionally, no constriction was demonstrated in isolated human mesenteric
arteries (Clozel, Gray et al. 1992). Therefore, in vitro results indicate that S6c is a

selective venoconstrictor.

Systemic in vivo pharmacology studies of 86c can not be performed in
humans, due to ethical issues. However, a scientist was accidentally bitten by a

Burrowing Asps Atractaspis engadensis, whose venom contains sarafotoxins,

28

during milking. He reported a rapid increase in blood pressure (Kumik, Haviv et
al. 1999). Several studies of local intravenous infusion of $60 to human hand
veins have been conducted (Strachan, Haynes et al. 1995; Strachan, Crockett et
al. 2000). All the data indicate that S6c causes veins to constrict in vivo although

it is not as potent as ET-1.

In early 90s, Clozel et al. performed short-term infusions of S6c into intact
rats and reported the following signiﬁcant ﬁndings (Clozel, Gray et al. 1992). 1)
Upon S6c infusion, there was a transient blood pressure decrease lasting a
couple of minutes after the infusion was begun. Then blood pressure was
increased for over 30 minutes. 2) S6c signiﬁcantly lowered mesenteric blood
ﬂow, indicating vasoconstriction in the splanchnic region. AS I mentioned earlier,
they also showed that 86c did not cause direct arterial constriction. 3) The
pressor effect was not due to ETBR activation in the central nervous system,
release of catecholamines from the adrenal medulla, or increased plasma levels
of ET—1. This is the earliest publication focused on S6c-induced cardiovascular
effects in whole animals. My data are quite consistent with the above results.
This earlier study provided critical support for the possibility of a hypertension
model produced by chronic infusion of S6c. My studies were among the ﬁrst to

characterize this new model.

4.3 A novel hypertension model: chronic activation of ETBRS by 86c
As discussed in the section above on endothelin, ETBR activation causes
different results at different locations. Global activation leads to a transient

depressor response by release of endothelial cell derived vasodilators, diuresis,

29

and natriuresis, all of which should produce a decrease in blood pressure
(Rubanyi and Polokoff 1994; Schiffrin 1998; Pollock, Allcock et al. 2000). To our
surprise, studies from our laboratory showed that ﬁve-day infusion (5
pmollkglmin, iv) of $60 into instrumented normal rats caused a marked and
sustained increase in mean arterial pressure (S6c—induced hypertension) (Fink,
Li et al. 2007). We believe venoconstriction contributes to S6c—induced
hypertension, because as described earlier, in vitro S6c has been shown to
constrict veins from most vascular regions but have little effect on most arteries
(Evans, Cobban et al. 1999; Johnson, Fink et al. 2002; Perez-Rivera, Fink et al.
2005; Savoia and Schiffrin 2007). Furthermore, chronic administration of other
relatively speciﬁc venoconstricting drugs also can increase arterial pressure
(Rothe 1983; Greenway and Lautt 1986; Hainsworth 1986; Mortensen, Pawloski
et al. 1990; Rothe 1993). Decreased venous capacitance has been documented
in animal models (Manning, Coleman et al. 1979; Ricksten, Yao et al. 1981;
Ackerrnann and Tatemichi 1983; Fink, Johnson et al. 2000) and in humans with
hypertension (Ulrych 1976; Mark 1984; Safar and London 1985). Veins in the
splanchnic region are probably the most important target for S60 and other
venoconstrictors, since they account for most of the active capacitance
responses of the circulation (Greenway and Lautt 1986; Rothe 1986; Rothe
1993). Veins in the splanchnic region hold about 33% of the total blood volume
(Greenway and Lister 1974) and maximal activation can reduce their volume up

to 60%, redistributing blood from the abdomen into the heart and thereby

30

increasing cardiac output and blood pressure (Karim and Hainsworth 1976;

Greenway 1983).

It is likely, however, that other mechanisms contribute to S6c-induced
hypertension. As reviewed earlier, the sympathetic nervous system plays an
important role in hypertension development in both animal models (Wyss 1993;
Cabassi, Vinci et al. 2002) and in humans (Anderson, Sinkey et al. 1989; Weber
1993; Zhu, Poole et al. 2005). My results show that partial removal of
sympathetic innervation to the splanchnic bed attenuates S6c-induced
hypertension, supporting the idea that sympathetic nerves are at least partially
involved in SGc-induced hypertension (Li and Fink 2005). Additionally, as
described above, increasing evidence has shown that ROS can function as
cellular signaling molecules (Bedard and Krause 2007). Increased tissue ROS
levels can cause hypertension, because blocking ROS generation or increasing
their removal lowers blood pressure (Kopkan and Majid 2005; Sullivan, Pollock et
al. 2006; Baumer, Kruger et al. 2007). Recent reports indicate that ETBR
activation increases oxidative stress in sympathetic ganglia in vitro (Dal, Galligan
et al. 2004) and in vivo (Lau, Galligan et al. 2006). Combined with the importance
of sympathetic nervous system, it is possible that ROS signaling could enhance
ganglionic neurotransmission and thereby increase sympathetic activity, although
the precise effects of increased ROS levels in sympathetic ganglia are unknown.
Therefore, it is reasonable to speculate that increased superoxide generation in

sympathetic ganglia is involved in S6c—induced hypertension.

31

My research was designed to identify mechanisms of hypertension caused
by sustained activation of ETBRS, with focuses on venous function, sympathetic

nerve activity, and oxidative stress in the splanchnic bed of the rat.

32

 

Mean Arterial Pressure

/"\

Cardlac Output * Total Perlpheral Resistance

/"\ /' ‘\

Stroke Volume * Heart Rate Arterlal Arterial

/ \ \Functlon Structure
Preload lnotropy
/’ '\ \ V

Blood Volume Venous Capacitance <———1 Neurohumoral Factors

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

T Renal Nafll-IZO < J

 

 

Figure 1: Factors mediating blood pressure. Details are discussed in GENERAL
INTRODUCTIONS.

33

 

 

 

 

 

 

 

 

 

 

 

 

Angiotensin II
Insulin

Glucose Promoter ET-1 gene Nitric Oxide

Hypoxra Prostacyclin
Vasopressin ANP

Cytokines l

Thrombin
Free Radicals Prepro-ET-1 (203 aa)

Di basic en dopepti dase
I Carboxypeptidase

Big EN (39 aa)

1 En dothelin convemng enzyme

K ET-l (21 aa) /

Endothelial Cell

 

 

Figure 2: The proposed preteolytic processing pathway for conversion of
preproendothelin to endothelin-1. The preproform (203-amino acid) of ET—1 is
converted to 39-amino acid big ET-1 by dibasic endopeptidase and
carboxypeptidases. Big ET-1 is then converted to 21-amino acid ET-1 by
endothelin conveiting enzyme (Rubanyi and Botelho 1991). Many factors may

stimulate or inhibit the expression of ET-1.

Vena Cava
3%

Aorta Arteries
...3% 10%

Arterioles
4%

   

Capillaries
4%

 
   

Veins Venules
50% 26%

Figure 3a: Blood distribution in various blood vessels. Over 75% of total

blood is stored in veins and venules.

35

Rest Splanchnic

0 Bed
23 /o 33%

   
 

Kidneys
1 %

Adipose
9%

   

Skin

2% Heart " Skeletal
Lungs
Channels 10% Muscle
5 A 17%

Figure 3b: Blood volume distribution in various regions. About one third of total

blood volume is stored in the splanchnic bed.

CHAPTER 2

OVERALL HYPOTHESIS AND SPECIFIC AIMS

37

Recently, our lab has published a new hypertension model: sarafotoxin
6c-induced hypertension (Fink, Li et al. 2007). When conscious rats received 860
intravenously for 5 days, there was a signiﬁcant increase in blood pressure and
the hypertension was sustained at that level during the infusion period. It is
believed that venoconstriction is involved in S6c-induced hypertension, because
in vitro S6c has been shown to constrict veins in most vascular regions but very
few arteries. Moreover, there is evidence indicating that ETBR activation
increases sympathetic nervous system activity. This action also may contribute to
SBc-induced hypertension. Recent data revealed that ETBR activation increases
oxidative stress in sympathetic ganglia. On the other hand, vasodilators released
from sensory nerves, such as CGRP may counterbalance the increase in blood
pressure. The objective of this work was to identify neural mechanisms
underlying S6c—induced hypertension. I proposed the hypothesis that $60-
induced hypertension is mediated by direct venoconstriction, increased
sympathetic activity to the Splanchnic region mediated by elevated ROS levels in
sympathetic ganglia, and increased release of vasodilating neurotransmitters

from sensory nerves. Figure 4 shows the overall hypothesis of the project.

Specific Aim I
Celiac ganglionectomy (CGX) was extensively used in my thesis project.
The purpose of this speciﬁc aim was to evaluate the effectiveness of CGX in

reducing the density and function of the splanchnic sympathetic innervation.

38

Speciﬁc Aim H: To test the hypothesis that CGX causes a nearly complete loss

of sympathetic innervation to mesenteric arteries and veins.

Protocol l-1: Glyoxylic acid staining allows the sympathetic nerves to be
visualized. Therefore, this technique was used to determine the distribution and

density of sympathetic nerves innervating mesenteric arteries and veins.

Protocol l-2: Another way to visualize sympathetic nerve density is tyrosine
hydroxylase staining, since sympathetic nerves contain tyrosine hydroxylase, a

key enzyme for the synthesis of catecholamines.

Protocol I-3: Norepinephrine (NE) concentrations in tissues, including vessels
and organs, were measured using high performance liquid chromatography as a

more quantitative index of sympathetic innervation density.

Speciﬁc Aim I-2: To test the hypothesis that contractile responses to sympathetic

nerve stimulation is Signiﬁcantly lowered by CGX in the Splanchnic vasculatures.

Protocol l: CGX effectiveness was evaluated at a functional level. The
sympathetic nerves on mesenteric arteries and veins from CGX rats were
stimulated by two parallel wire electrodes linked to an electrical stimulator. The
percentage of blood vessel constriction was measured as an indicator of

sympathetic neurotransmission.

39

Speciﬁc Aim II
The focus of this speciﬁc aim was to identify if sympathetic activity and
oxidative signaling in the splanchnic sympathetic ganglia are involved in S6c-

induced hypertension.

Speciﬁc Aim "-1: To test the hypothesis that S6c-induced hypertension is in part

by increased sympathetic nervous system activity.

Protocol I: Circulating NE level is an indicator of global sympathetic nerve activity
(SNA). So plasma NE concentrations were measured to determine if there is an

elevated SNA in S6c-induced hypertension.

Protocol II: CGX is an effective procedure to remove most splanchnic
sympathetic innervation. So it was used in S6c-induced hypertension to
determine if increased sympathetic activity to the splanchnic organs is a critical

mechanism causing hypertension in this model.

Speciﬁc Aim "-2: To test the hypothesis that reactive oxygen species levels in

neurons and vessels are increased with chronic activation of ETBRS.

4O

Protocol I: Dihydroethidium (DHE), a ﬂuorescent dye, was used to measure
superoxide levels in various tissues, including blood vessels and sympathetic

ganglia, after either 1 day or 5 days of S60 infusion.

Protocol II: Lucigenin chemiluminescence is a quantitative method to measure
superoxide concentrations. Again, a time course study was carried out to test if
increased levels of ROS occur in blood vessels in a time—dependent manner in

S6c-induced hypertension.

Speciﬁc Aim "-3: To test the hypothesis that increased superoxide levels in
sympathetic ganglia or blood vessels contribute to hypertension development in

S6C-induced hypertension.

Protocol I: Tempol (4-hydroxy 2,2,6,6,-tetramethyl peperidine 1-oxyl), a
superoxide dismutase mimetic, was used to test if increased blood pressure in
S6c-infused rats requires increased tissue levels of superoxide. Both
hemodynamic parameters and superoxide levels in sympathetic neurons were

evaluated.

Speciﬁc Aim Ill
Endothelin-B receptor (ETBR) transgenic spotting lethal rat model (T g
(sllsl)) is a unique animal model to study the roles of ETBRS in sympathetic

nerves. In this model, ETBRS are not functional in endothelium, renal tubular

41

cells, or vascular smooth muscles. They function only at the places where
dopamine-B-hydroxylase is expressed, including sympathetic nerves. These rats
are hypertensive. The aim of this speciﬁc aim was to discover the mechanisms
underlying the hypertension in T9 (SI/SI) rats, with a focus on the sympathetic
nerves. Since Tg (+I+) rats have 70 more copies of the ETBR gene than their
control strain, Wistar Kyoto (WKY) rats (the genetic background of these
transgenic rats) were also used to determine the differences between natural

wildtype and transgenic wildtype.

Speciﬁc Aim Ill—1: To conﬁrm others’ ﬁnding that the circulating ET-1 levels in T9

(SI/SI) are higher than in T9 (+I+) rats.

Protocol I: Endothelial ETBRS function as endogenous “clearance receptors” for
ET-1. In Tg (sllsl) rats, ETBRS are not functional in endothelium. Therefore,
circulating ET-1 in T9 (SI/SI) can not be cleared as much as in normal rats. An
increased ET-1 level has been reported by several labs. Blood pressure could be
increased by a higher ET-1 concentration through endothelin A receptor
activation. Therefore ET-1 in the circulation was measured by a quantitative

sandwich enzyme immunoassay.

Speciﬁc Aim Ill-2: Numerous studies have shown that elevated sympathetic

nerve activity may mediate hypertension. The goal of this speciﬁc aim was to test

42

The hypothesis that hypertension in T9 (SI/SI) rats is mediated by increased

sympathetic activity.

Protocol l a: Sympathetic nerve density was evaluated by glyoxylic acid staining

which makes sympathetic nerves visible.

Protocol l b: A quantitative way to determine sympathetic nerve density is to
measure norepinephrine (NE) concentrations in tissues. For this study, NE levels

were evaluated by high performance liquid chromatography.

Protocol II: Vasoconstrictor responses to the sympathetic nerve activation were
compared in tertiary mesenteric arteries and veins from T9 (sl/sl) and T9 (+/-i-)

rats.

Specific Aim IV
This speciﬁc aim was to test the hypothesis that SGc-induced hypertension

is caused at least in part by activation of ETBRs in sympathetic neurons.

Speciﬁc Aim IV-1: To test the hypothesis that 86c infusion causes hypertension

through activation of ETBRS in sympathetic neurons.

43

Protocol I: Hemodynamic parameters, including systolic blood pressure, diastolic
blood pressure, mean arterial pressure, pulse pressure, heart rate, and activity,

were compared in T9 (+I+) and T9 (SI/SI) rats, during 860 infusion.

Speciﬁc Aim lV-2: To test the hypothesis that ETBRS in the splanchnic
sympathetic nervous system play an important role in the development and the

maintenance of S6c-induced hypertension.

Protocol I: After a recovery from CGX for two weeks, hemodynamic responses

were compared in T9 (+I+) and T9 (SI/SI) rats as in Speciﬁc Aim IV-1.

Specific Aim V
There is good evidence suggesting that the sensory nervous system plays
a role in blood pressure regulation. The focus of this speciﬁc aim was to

determine the involvement of sensory nerves in $6c-induced hypertension.

Speciﬁc Aim V-1: To test the hypothesis that the sensory nervous system plays a

role in $6c-induced hypertension.

Protocol l: Capsaicin-treated neonatal rats are characterized by loss of capsaicin-
sensitive sensory nerves. These rats were treated with S6c, followed by
hemodynamic measurements. The role of the sensory nervous system was

determined by the idea of “loss of function”.

   
   
   

   

.I II»

II .
/H\

1‘ Ganglion 02' . . 1 n4
ﬂ Vein Artery
123) TSNA l
Celiac Ganglionic Neuron
1 tccRP

 

[ArterialConstriction H Venous Constriction I

 

 

 

\

I ' Hypertension "J

Figure 4: Overall hypothesis of the project. ETBR activation in the splanchnic

 

 

bed increases venoconstriction directly, and sympathetic nerve activity via
increased ganglionic oxidative stress. Both effects contribute to increased arterial
blood pressure. ETBR activation leads to enhanced CGRP release from sensory
nerves, which attenuates the blood pressure increase by causing local

vasodilation.

45

CHAPTER 3

GENERAL METHODS

1. Animals

All animal protocols were approved by the All University Committee on

Animal Use and Care of Michigan State University.

1.1 Sprague-Dawley rats and WKY rats
Normal male Sprague-Dawley rats (300-325 9, Charles River, Portage,

MI, USA) or/and WKY rats (male: 300-325 9; female: 210-250 9, Charles River)
were used. Before the experiments, all rats were housed 2 or 3 per cage in a
temperature and humidity controlled room with a 12h on/12 off light cycle.
Pelleted rat chow (8640 Rodent Diet; Harlan/Teklad) and water were given ad

libitum.

1.2 Transgenic spotting lethal (T g (sllsl)) rats

Rats carrying transgenic gene of ETBRS whose expression is driven by
the human dopamine-li-hydroxylase (ml-l) promoter were generated. The
transgenic rats were crossed with the spotting lethal rats to breed transgenic
spotting lethal (T 9 (sllsl)) animals that carry functional ETBRS only regulated by
WH promoter, as previously described (Gariepy, Williams et al. 1998). Two pairs
of transgenic (T g) heterozygous ETBR-deﬁcient rats (two males and two
females, Tg (sll+)) were sent to Michigan State University from University of
Michigan animal facility. After each pair's mating, baby rats were born and
nursed for three weeks. The newborn rats were genotyped immediately post-
weaning by polymerase chain reaction on DNA extracted from tail biopsy
specimens. Experiments were started when the animals were approximately 12

weeks of age.

47

1. 3 Neonatal capsaicin-treated rats

Pregnant Wistar female rats (Charles River Laboratories Inc, Wilmington,
Massachusetts, USA) were ordered and housed. On the ﬁrst or second day after
birth, new born rats were injected with capsaicin (50 mg/kg, 5 mg/ml in 5%
ethanol, 5% Tween 80 in saline, subcutaneously). Control rats received vehicle
solution (5% ethanol, 5% Tween 80 in saline) of the same volume. The injections
were given while rats were anesthetized by ether. After nursing for three weeks,
male and female rats were separated and weaned. Male rats were used in the
experiments. Pelleted rat chow (8640 Rodent Diet; Harlan/Teklad) and water

were given ad libitum.

2. Vascular catheterization

Either sodium pentobarbital (30-50 ngg plus 0.4 mg atropine sulfate,
i.p.) or isoﬂurane was used for anesthesia. One polyvinyl catheter with a silicone
rubber tip was inserted into the abdominal aorta via a femoral artery for drawing
blood samples and hemodynamic measurements. A similar catheter was placed
into a femoral vein for drug administration. Free ends of the catheters were
passed through a stainless steel spring tether attached to a plastic harness ﬁxed
around the chest of the rat. Rats were allowed to recover consciousness on a
heated pad under constant observation. When they became conscious, they
were placed in individual cages allowing continuous access to both catheters
without handling or otherwise disturbing the rats. Acetaminophen was given for 3

days after the surgery to relieve surgical pain. Arterial lines were ﬁlled with a

48

heparin-saline solution and occluded when not in use. Rats were allowed at least

three days for recovery after the surgery before measurements were begun.

3. Radiotelemetry implantation

3.1 In intact rats

An opening was cut in left thigh area. A pocket was made by separating
the skin and muscle in the abdomen region. The rediotelemetry transmitter’s
body was placed subcutaneously in the abdomen area. The tubing of the
transmitter was pulled to the left thigh and the tip of the tubing was inserted into
the abdominal artery through left femoral artery. Every animal was placed in an
individual cage above a heating pad for recovery. Then they were transferred to
the telemetry animal room, where each cage was put on a radiotelemetry
receiver (RPC-1, Data Sciences lntemational). After a recovery for one week,

transmitters were turned on for recording.

3.2 In celiac ganglionectomized rats

Transmitter implantation was performed right after CGX was completed.
The body of the transmitter was placed in the abdominal cavity. Several sutures
were made to secure the position of the transmitter’s body. The catheter of the
transmitter was pulled subcutaneously to the opening in the left thigh area.
Catheterization was carried out in the similar way as introduced above in intact

animals. Animals rested ten days for recovery.

4. Hemodynamic measurements

4.1 By external blood pressure analyzer

49

Arterial pressure was determined by connecting the arterial catheter to a
low-volume displacement pressure transducer (TXD-300, Micro-Med, Louisville,
Kentucky, USA) linked to a digital pressure monitor (EPA-200 Blood pressure
Analyzer, Micro-Med), which measures systolic, mean, and diastolic pressures,
and heart rate every 0.5 second (sample rate = 1000 Hz). The transducer was
zeroed at mid-chest level of rats in a typical crouched posture and was calibrated
every day against a column of water. Hemodynamic parameters were recorded
for 30 minutes each morning between 10:00 AM - 12:00 PM without handling or
othenlvise disturbing the rat sitting quietly in its home cage. All data were
averaged minute-by-minute and saved using a computerized data acquisition

system (DMSl-400 System Integrator, Micro-Med).

4.2 By telemetry

The signals from transmitters were monitored by radiotelemetry receivers
(RPC-1, Data Sciences International) which were connected to a data exchange
matrix. Data, including systolic pressure, diastolic pressure, mean arterial
pressure, pulse pressure, heart rate, and activity, were collected every 10
minutes during the entire period of the experiments. Data acquisition and
analysis were performed by custom software Dataquest ART 4.0 (Data Sciences

International).

5. Minipump implantation
S6c solution was made and transferred into micro-osmotic pumps (Alzet,
Model 1007D). A small opening was made between the scapulae and a pocket

was opened for the subcutaneous implantation of the minipump with the delivery

50

port oriented towards the lower body. The drug was delivered at the rate of 30

pmollkglmin.

6. Celiac ganglionectomy (CGX)

The surgery was conducted on a heating pad to maintain the animals’
body temperature. The abdomen was opened through a midline laparotomy. The
small intestine was gently moved aside from the midline and covered with saline-
Soaked gauze. The celiac ganglion area was exposed. Cotton swabs were used
to rub off the fat covering the celiac plexus and main vessels (e9. aorta, celiac
artery, and mesenteric artery) in the vicinity. Visible nerves around the three
arteries were removed as completely as possible by stripping. The small intestine
was put back to the original position. A ticarcillin solution was applied to the
organs and the abdomen was closed in two layers with interrupted sutures. For
sham rats, the small intestine was moved out of the body. Only fat was rubbed
off and nerves stayed intact. Surgical recovery was monitored with the rats on a
heated table. After the rats awakened, they were fed with normal food and tap
water for one week during recovery from surgery. Acetaminophen was given for
3 days after the surgery to relieve surgical pain. We have Shown previously that
this procedure results in effective denervation of the splanchnic organs, as
indicated by > 90% depletion of norepinephrine content of liver, spleen and small
intestine, while causing only limited denervation Of the kidneys [King and Fink,

2007].

7. Inferior mesenteric ganglionectomy (IMGX)

51

The procedure was similar to that described for CGX. The only difference
was that inferior mesenteric ganglion, instead of celiac ganglion, was removed
using ﬁne scissors. Nerves around the ganglion and blood vessels were stripped

as well.

8. In vitro preparation of mesenteric vessels

Small intestine was removed and placed in oxygenated (95% O2, 5% CO2)
Krebs physiological saline. A segment of ileum with associated mesenteric
vessels was removed and pinned ﬂat in a dish. A section of third-order
mesenteric vessel was separated and transferred to a small recording bath
chamber and pinned ﬂat. Surrounding tissue around the vessel was cleaned by
ﬁne scissors and forceps. The chamber was mounted on the stage of an inverted
microscope (Olympus CK-2; Leco, St Joseph,vMichigan, USA). The vessel was
superfused with 37°C Krebs solution at a ﬂow rate of 7 ml/min for 20 min, and

allowed to relax to a stable resting diameter.

9. Video monitoring of vessel diameter

The output of a black-and-white video camera (Hitachi model KP-111;
Yokohama, Japan) attached to the microscope was fed to a PC Vision Plus
frame-grabber board (imaging Technology Inc., Woburn, MA) mounted in a
personal computer. The video images were analyzed using computer software
DiamTrack (Adelaide, Australia). The digitized Signal was converted to an analog
output (DAC-02 board; Keithley Megabyte, Tauton, MA) and fed to a chart
recorder (EZ Graph; Gould Inc., Cleveland, OH) for a record of vessel diameter.

Diameter changes as small as 1.8 pm could be resolved.

52

10.Transmural stimulation of perivascular nerves

Two parallel silver/silver chloride wire electrodes were positioned parallel
with the longitudinal axis of mesenteric vessels. The electrodes were linked to an
electrical stimulator (Grass Medical Instruments, $48). For each stimulation
frequency, a train of 30 stimuli were given, with stimulus duration of 0.5 ms and
at the voltages ranging from 70 to150 volts. The neurogenic nature of the
constriction induced by electrical stimulation was conﬁrmed by showing a
complete blockade of constriction at 20 Hz by applying tetrodotoxin (TTX, 0.3
pM). The tissue was discarded if the constriction at 20 Hz was not completely
blocked by 'lTX. The peak constriction at each given frequency was recorded.

Measurements were made using DiamTrack software.

11. Plasma sampling
Blood samples (0.5 ml or 1 ml, depending on the studies) were drawn
from the arterial or venous catheter over twenty-ﬁve pl of EGTAlglutathione
solution into ice-chilled plastic syringes and transferred to ice-chilled plastic
tubes. Blood samples were centrifuged at 10,000 g for 5 minutes at 4°C. Plasma

was separated and stored in a -80°C freezer for later assays.

12. Plasma catecholamine measurements
Plasma NE was measured by a radioenzymatic assay based on the
technique proposed by Peuler and Johnson (Peuler and Johnson, 1977). The
principle of this method is the conversion of NE and EPI to tritiated
norrnetanephrine and metanephrine in the presence of catechol-O-
methyltransferase and tritiated S-adenosylmethionine as a labeled methyl donor.

53

After puriﬁcation through a series of organic extractions, tritiated
normetanephrine was separated from the derivatives of dopamine by oxidation
with sodium periodate. Tritiated vanillin then was counted in a liquid scintillation

spectrometer.

13. DHE ﬂuorescence

Dihydroethidium (DHE), an oxidative ﬂuorescent dye, was used to
measure superoxide (02") levels in mesenteric arteries, mesenteric veins, and
inferior mesenteric ganglion (IMG) (Miller, Gutterman et al. 1998); (Millette, de
Champlain et al. 2000). Rats were sacriﬁced with pentobarbital sodium (ip).
Mesenteric vessels and IMG were removed from each rat. Blood vessels were
placed into oxygenated Krebs buffer at 4°C, dissected free of loosely adhering
tissue, and cut into 3- to 4-mm-wide ring segments. Unﬁxed frozen ring segments
were cut into 25-um-thick sections using a cryostat and placed on a glass slide.
DHE (5*10'6 moI/l) was topically applied to each tissue section. Slides were
incubated in a light-protected humidiﬁed chamber at 37°C for 60 minutes for
vessels and 45 minutes for IMG. Fluorescent images were observed with an
Olympus Fluoview laser scanning confocal microscope mounted on an Olympus
BW50WI upright microscope, equipped with krypton/argon lasers. A 488-nm
argon laser line was used to excite DHE ﬂuorescence, which were detected with
a 585-nm long-pass ﬁlter. Fluorescence was quantiﬁed by software Image J.
Individual cells (sympathetic neurons in inferior mesenteric ganglia and vascular
smooth muscle cells in blood vessels) were individually selected. The intensity of

red ﬂuorescence in the selected area was expressed as a number. The actual

intensity was the difference between the value of selected area and the value of
background. Five to ten cells were selected in each image. The average value of
all the selected cells represented the ﬂuorescent density of the target tissue in

one animal.

14. Lucigenin-enhanced chemiluminescence

Vascular 02" was quantiﬁed by lucigenin chemiluminescence. Isolated
vessel segments were cleaned and incubated for 30 minutes in modiﬁed Jude
Krebs buffer at 37°C in the presence of 10 mM diethyldithiocarbamate. Vessels
were transferred to small tubes which contained 5 uM lucigenin in modiﬁed
Krebs-HEPES buffer and incubated for 10 minutes at 37°C in the dark. After
incubation, tubes were put into a luminometer (T D-20e; Turner Designs,
Sunnyvale, CA). Luminescence measurements were integrated for 30S periods.
Ten repeated measurements were then averaged. After 10 cycles, the cell-
perrneant O2" scavenger tiron (10 mM) was added, and 15 more cycles were
read. The last 8 values, which were maximally reduced, were averaged. Data
were calculated as the change in the rate of luminescence per minute per
milligram of tissue of values before and after tiron and then converted to 02"

(nmol - min“ - mg tissue“) (Cifuentes, Rey et al. 2000); (Li, Fink et al. 2003).

15.G|yoxylic acid staining
Tertiary mesenteric arteries and veins were removed and placed in normal
PBS. After surrounding fat was trimmed off, blood was ﬂushed out with PBS (0.1
M, pH 7.4) through a 30 9a. hypodermic needle and syringe. Three mesenteric

arteries and three mesenteric veins were randomly selected in each rat and ﬁve

55

rats were used. Blood vessels were incubated in 2% glyoxylic acid solution (in 0.2
M phosphate buffer, pH=7) for 5 minutes at room temperature. Blood vessels
were then mounted on a microscope slide and placed in an oven (80°C) for 5
minutes. After being heated, blood vessels were mounted in mineral oil and
cover-slipped. Catecholamine ﬂuorescence was viewed by a ﬂuorescence
microscope (Nikon Eclipse TE 2000-U) equipped with a ﬁlter set, UV2E/C

(excitation ﬁlter, 340-380 nm and emission, 435-485 nm).

16. lmmunohistochemical staining of tyrosine hydroxylase and CGRP

Mesenteric arcades were pinned in a Sylgard-lined petri dish. A 30-gauge
hypodermic needle ﬁlled with PBS (PBS, 0.01 M, pH 7.2) was used to ﬂush blood
out of mesenteric vessels by cannulating the primary artery or vein. The tissues
were immersed in Zamboni ﬁxative (4% formaldehyde, 2% picric acid in 0.1 M
phosphate buffer, pH 7.4) at 4 °C for 24 hours. The tissues were then washed
three times at 10-min intervals with dimethyl sulfoxide followed by 3 washes at
10-min intervals with PBS. Arteries and veins were then dissected from
mesenteric fat and incubated overnight with primary antibody. We used a mouse
anti-tyrosine hydroxylase (TH) antibody (1:200 dilution in PBS, Calbiochem, San
Diego, Ca) to localize immunoreactivity for TH and a rabbit polyclonal anti-CGRP
antibody (1:200 in PBS, Chemicon, Temecula, CA) for CGRP. After incubation
with primary antibody, tissues were washed 3 times in PBS and then incubated
with Cy3 AfﬁniPure F(ab')2 Fragment Goat Anti-Mouse lgG (1:200 dilution in
PBS; Jackson Immunoresearch Laboratories, West Grove, PA) and goat anti-

rabbit ﬂuorescein isothiocyanate (1:40 dilution in PBS; Sigma-Aldrich, St. Louis,

M0) for 1 hour at room temperature. The tissues were washed 3 times with PBS
and then mounted on microscope slides and coverslipped using buffered glycerol

(pH 8.6). Fluorescent images were acquired by a confocal microscope.

17. Measurement of norepinephrine levels in mesenteric vessels

Mesenteric arcades were pinned in a Sylgard-Iined petri dish. A 30-gauge
hypodermic needle ﬁlled with PBS (PBS, 0.01 M, pH 7.2) was used to ﬂush blood
out of mesenteric vessels by cannulating the primary artery and vein. Fat around
vessels was cleaned. Three pieces of one-centimeter-Iong segments of tertiary
mesenteric arteries and veins were collected, put into 0.1N perchloric acid
solution, and stored at -80°C for later measurement. NE level was measured by
high performance liquid chromatography (HPLC) as previously described (Luo,
Hess et al. 2003). Brieﬂy, tissues were thawed and sonicated for membrane
disruption. Twenty-ﬁve pl supernatant was injected into a C-18 reverse-phase
analytical column, which was coupled to a single colorimetric electrode-
conditioning cell in series with dual-electrode analytical cells. The content of NE
was determined by comparing maximal heights to those of standards in the same
day. NE concentrations in vessels were expressed as NE (in picogram)/protein
(in grams). The protein content of blood vessels was measured by using folin

phenol reagent (Lowry, Rosebrough et al. 1951).

18. Norepinephrine measurement in the splanchnic organ tissues
The splanchnic organs were harvested, weighed, frozen, and stored at -
80°C. Perchloric acid was added to the collected tissues (0.1 M perchloric acid

solution, 2m|/0.59). Then the tissues were homogenized. The homogenate was

57

centrifuged at the rate of 12000 rpm for 15 minutes at 4°C. The supernatant was
collected. The supernatant was transferred into new tubes, followed by another
centrifuge at the same speed and temperature for 10 minutes. The ﬁnal
supernatant was collected by a syringe. About 0.3 ml of the ﬁnal supernatant was
placed in top compartment of a 30 KD ﬁltration tube and it was centrifuged twice
at 13000 rpm for 5 minutes at 4°C. The ﬁltrate was placed into a clean microtube
and NE content was measured by HPLC, as described in NE measurement in
mesenteric vessels. NE concentration in organs was expressed by NE (in

picogram)ltissue (in grams).

19. Plasma endothelin-1 measurement

Plasma ET-1 level was measured by a quantitative sandwich enzyme
immunoassay using an ELISA kit (Human Endothelin-1 Immunoassay,QETOOB,
R 8 D Systems, Minneapolis, MN). A 96-well microplate pie-coated with a
monoclonal antibody speciﬁc for ET-1 was provided in the kit. One hundred pl
standards or EDTA-plasma was pipetted into each well. The plate was then
covered and incubated for 1.5 hours at room temperature on a horizontal orbital
microplate shaker set at 500 rpm. Any ET-1 in the standards or samples was
bound by the monoclonal antibody. After the incubation, each well was washed
four times to eliminate any unbound substances. Two hundred pl enzyme-linked
monoclonal antibody speciﬁc for ET-1 was added to each well. The plate was
covered and incubated for 3 hours on the same shaker at room temperature.
Each well was again washed four times to remove any unbound antibody-

enzyme reagent. Then two hundred pl enhanced luminoI/peroxide substrate

58

solution was added to each well, followed by an incubation for 30 minutes at
room temperature on the benchtop. The microplate was wrapped by aluminum
foil for light protection. Light was produced in proportion to the amount of ET-1

bound in the initial step. A microplate luminometer was used to measure the

intensity of the light omitted.

20. Animal Euthanasia
Rats were euthanized by administration of sodium pentobarbital (100
mglkg, ip.), which is a commonly used method for euthanasia. This approach is
consistent with the recommendations of the Panel on Euthanasia of the

American Veterinary Medical Association.

21. Analysis of the data and statistics
Data were shown as means :I: standard error. A p value less than or equal
to 0.05 was considered statistically signiﬁcant. Different comparison methods
were used depending on the studies. The data were analyzed by GraphPad

lnStat and Prism. The details are described in each chapter.

59

CHAPTER 4

THE EFFECTS OF CELIAC GANGLIONECTOMY ON
SYMPATHETIC INNERVATION TO THE SPLANCHNIC ORGANS
IN THE RAT

60

INTRODUCTION

The splanchnic vascular bed (liver, kidneys, spleen, and small and large
intestines) holds about one third of the total blood volume (Greenway and Lister
1974; Greenway 1983). Therefore the splanchnic vasculature acts as the biggest
single reservoir of blood in the circulation. Small veins and venules are
considered as “capacitance vessels” since they are responsible for most of the
active capacitance responses of the circulation (Greenway and Lautt 1986;
Rothe 1986; Rothe 1993). A decreased venous capacitance in hypertension has
been reported in experimental models (Ferrario, Page et al. 1970; Smith and
Hutchins 1979) and humans (Schobel, Schmieder et al. 1993). This decrease in
venous capacitance will lead to a blood shift from peripheral vascular beds to the
heart and ultimately in part into the arterial circulation (Ricksten, Yao et al. 1981).
As a result, blood pressure increases. It has also been reported that
hepatosplanchnic vascular resistance is increased before the occurrence of
vascular resistance increases in other vascular beds during the development of
human hypeitension (Sugawara, Noshiro et al. 1997). This suggests that
increased arterial and venous tone in the splanchnic region may be important

factors causing hypertension.

The sympathetic nervous system plays an important role in hypertension
development both in animal models (Wyss 1993; Cabassi, Vinci et al. 2002) and
in humans (Anderson, Sinkey et al. 1989; Weber 1993; Zhu, Poole et al. 2005).
Generally, it is found that sympathetic effects on the cardiovascular system are

augmented in hypertension (Nestel 1969; Egan, Panis et al. 1987; Somers,

61

Anderson et al. 1993; Esler and Kaye 1998). This could be caused by increased
sympathetic nerve density, high sympathetic nerve ﬁring rates, epinephrine
contransmission, norepinephrine transporter dysfunction, increased release of
norepinephrine from the neuroeffector junction, increased end organ
responsiveness to sympathetic neurotransmitters, or a combination of the above

(Esler, Rumantir et al. 2001).

Stimulation of the sympathetic nerves induces arterial and venous
constrictions (Brooksby and Donald 1971; Fumess and Marshall 1974; Karim
and Hainsworth 1976; Ozono, Bosnjak et al. 1989). This sympathetic regulation
is very critical in cardiovascular hemodynamics by controlling resistance,
capacitance, blood ﬂow, and blood pressure in physiological conditions
(Mellander 1960; Brooksby and Donald 1971; Rothe 1983; Pang 2001) and
hypertension (Somers, Anderson et al. 1993; Johansson, Elam et al. 1999;
Schlaich and Esler 2003; Schlaich, Lambert et al. 2004). The vascular bed in the
splanchnic region, including arteries and veins, is densely innervated by the
sympathetic nervous system. Sympathetic nerve activity (SNA) is a very critical
factor controlling vasoconstriction in the splanchnic circulation (Shoukas and
Bohlen 1990; Ozono, Bosnjak et al. 1991). For example, a norepinephrine
Spillover study has shown that a big fraction of total body sympathetic outﬂow
goes to mesenteric organs (Aneman, Eisenhofer et al. 1996). Thus, increased
SNA to the splanchnic circulation may be a common component in the

development and maintenance of hypertension.

62

Sympathetic nerves project to the splanchnic blood vessels via the
prevertebral and paravertebral ganglia. In rats, the cell bodies of a great majority
of the sympathetic postganglionic neurons innervating the Splanchnic bed are
located in two prevertebral ganglia: the celiac ganglion and superior mesenteric
ganglion (Trudrung, Furness et al. 1994; Hsieh, Liu et al. 2000; Quinson,
Robbins et al. 2001). These two ganglia are very close to each other in the rat
and are usually termed “the celiac plexus” (Chevendra and Weaver 1991;
Fumess, Koopmans et al. 2000). Since sympathetic activity through the celiac
plexus is an important pathway in blood pressure regulation, a surgical removal
(celiac ganglionectomy, CGX) canhelp to reveal the roles of the splanchnic
sympathetic innervation in blood pressure regulation. In the study, I evaluated the
changes in the splanchnic sympathetic innervation and function after CGX in the

rat.

In addition, the sensory nerves and the sympathetic nerves are closely
localized in animals (Galligan, Costa et al. 1988; Lindh, Haegerstrand et al. 1988;
Davies and Campbell 1994) and humans (Levanti, Montisci et al. 1988; Del
Fiacco, Floris et al. 1992). The sensory nervous system plays important roles in
blood pressure regulation. Some vasoactive neuropeptides, such as calcitonin
gene-related peptide (CGRP), is released from some sensory nerves (Deng and
Li 2005). CGRP-containing sensory nerves terminate peripherally on blood
vessels at a higher density around blood vessels than on the heart

(Wimalawansa 1996).

63

Finally, since regeneration of sympathetic nerves has been reported after
ganglionectomy (Hill, Hirst et al. 1985; Yamada, Terayama et al. 2006), the
evaluation was performed at different points in time after the surgery. Other
experiments were carried out to discover the origin of the reinnervation that was

observed after CGX.

METHODS

Animals

Normal male Sprague-Dawley rats (300-325 9, Charles River, Portage,

MI, USA) were used in the experiments.

The following methods were used in current study.

Protocol

Celiac ganglionectomy (CGX);

Inferior mesenteric ganglionectomy (IMGX);

Glyoxylic acid staining;

Immunohistochemical staining of tyrosine hydroxylase and CGRP;
Measurement of norepinephrine levels in mesenteric organs;
Measurement of norepinephrine levels in mesenteric vessels;

In vitro preparation of mesenteric vessels;

Video monitoring of vessel diameter.

After CGX, rats were allowed a two-week-recovery. All experiments were

started after the recovery period.

Statistical analyses

Data were shown as means i standard error. Differences between groups

were evaluated by a two-way ANOVA followed by a post hoc test (Bonferronl’s).

A p value less than or equal to 0.05 was considered statistically signiﬁcant.

65

RESULTS
Sympathetic nerve staining

Both glyoxylic acid staining (Figure 5a and 5b) and tyrosine hydroxylase
staining (Figure 6) demonstrated that most sympathetic nerves were no longer
visible two weeks after CGX was performed. CGRP-containing sensory nerves
were absent as well (Figure 6). Figure 5 Shows that new nerves started to appear
on mesenteric arteries and veins ﬁve weeks after the surgery, and even more so

ten weeks later.

Norepinephrine (NE) content measurement

Figure 7 illustrates NE levels in the splanchnic organs, including kidneys,
liver, spleen, and small intestine. It shows that NE concentrations in all
splanchnic organs were signiﬁcantly decreased in CGX rats, compared to those

in sham rats, two weeks after the surgery.

Figure 8 shows NE content in mesenteric arteries and veins. The data

demonstrate that NE levels were signiﬁcantly lowered two weeks after CGX.

Electrical stimulation

Figure 9 shows contractile responses to transmural stimulation of
perivascular nerves on mesenteric vessels. The ﬁgure clearly demonstrates that

neurogenic constriction was largely abolished by CGX in both veins and arteries.

Nerve regeneration

Figure 5 Shows that reinnervation began around ﬁve weeks after CGX.
Reinnervation was also revealed by NE measurements (Figure 10 for splanchnic
organs, Figure 11 for mesenteric vessels). Sympathetic nerve density started to
increase in mesenteric vessels and most splanchnic organs. IMG was postulated
to be a possible origin of the reinnervation. The data showed that NE
concentrations in organs and vessels from CGX rats were not signiﬁcantly
different from those from CGX and IMGX rats, at the same time point, indicating

that IMG did not prevent the nerve regeneration following CGX.

67

DISCUSSION

Some global sympathetic denervation methods have been available for
several decades, such as 6-hydroxydopamine (6-OHDA) (Hokfelt, Jonsson et al.
1972; Evans, Heath et al. 1979) and guanethidine (Evans, Heath et al. 1979)
treatments. Both of the two techniques disrupt sympathetic innervation
systemically, without a Speciﬁc target. On the other hand, bilateral celiac
ganglionectomy (CGX) speciﬁcally removes most splanchnic sympathetic
neurons. As early as 1930s, CGX was performed as a treatment for human
essential hypertension (Heuer 1936; Martin 1938; Weiss 1939; Peet, Woods et
al. 1940). The surgery was reported to lower blood pressure, with a partial or
complete sympathetic blockade in 87% of the patients, indicating the involvement
of splanchnic sympathetic innervation in blood pressure regulation in humans.
Animal studies also suggest that CGX effectively lowers mean arterial pressure
in physiological and pathological conditions (Kregel and Gisolﬁ 1989; King,
Osborn et al. 2007). Therefore, the celiac plexus, including the celiac ganglion
and superior mesenteric ganglion, is a critical component of overall sympathetic
regulation of blood pressure. Removal of this plexus, i.e. CGX, can be used to
investigate the roles of splanchnic sympathetic nerves in cardiovascular

regulation.

In current study, I evaluated the effectiveness of CGX in denervating
splanchnic vessels and organs by studying structural changes of the nerves via
two histological methods, glyoxylic acid staining of catecholamine-containing

nerves and tyrosine hydroxylase (TH) staining. The vascular bed in the

68

splanchnic region was expected to show signiﬁcantly lower innervation density
after CGX. Consistent with the expectation, staining shows that the sympathetic
nerves innervating mesenteric vessels (arteries and veins) are almost completely
absent two weeks after CGX. The data are in accordance with other groups’
ﬁndings (Hill, Hirst et al. 1985; Galligan, Costa et al. 1988; Yamada, Terayama et
al. 2006). They all reported effective sympathetic denervation after small
intestines were extrinsically denervated by nerve crushing, CGX, or nerve
freezing. Although Hill claimed a partial degeneration after two weeks of the
surgery, Yamada showed no regeneration even ninety days after CGX. The

variability may be due to differences in denervation techniques.

In addition to qualitative examination by images, we quantiﬁed
sympathetic nerve density by measuring norepinephrine (NE) contents in the
splanchnic organs and mesenteric vessels via high performance liquid
chromatography (HPLC). Our data show that CGX signiﬁcantly lowered NE
concentrations in the organs, compared to those in organs from Sham-operated
rats. The results are consistent with previous studies Showing that chemical
sympathectomy, by 6-OHDA, or alpha-methyI-tyrosine, or a combination of both,
leads to a major NE content depletion in mice and rats (Williams, Peterson et al.
1981; Bellinger, Felten et al. 1989). Among all the splanchnic organs, the
decrease in NE levels was the greatest in the spleen. The data are concordant
with others showing that over 85% of splenic NE is lowered in rats by CGX
(Bellinger, Felten et al. 1989). Combined, the results indicate that there is an

extremely rich innervation by the sympathetic nerves in the spleen and CGX is

69

an effective surgery to denervate most sympathetic nerves in the splanchnic
organs, especially in the spleen. Similarly, there was an almost complete loss of
norepinephrine in mesenteric arteries and veins, indicating a marked decline in
sympathetic nerve density after CGX. Hence this study further conﬁrmed that
CGX is a successful procedure to remove most sympathetic innervation to the

splanchnic vascular bed.

The next step to validate the effectiveness of CGX was to look at the
sympathetic nerves at the functional level. Previous functional studies of CGX
mainly focused on single neurons (Hill, Hirst et al. 1985) or measuring
electrophysiological parameters (Marlett and Code 1979). I applied direct
electrical transmural stimulation to perivascular nerves on mesenteric vessels.
This method is more integrative in showing the effects of sympathetic
denervation on vasculature at the tissue, rather than the cellular, level. Our
results showed that two weeks after CGX, mesenteric veins and arteries did not
respond to electrical stimulation at a frequency as high as 10 Hz. A very small
constriction was observed at very high frequencies (20 and 30 Hz), which usually
do not occur under physiological conditions. Therefore, the functional study
indicates that CGX largely eliminates sympathetic activity to arteries and veins in

the splanchnic region at two weeks after surgery.

It is worth mentioning that CGX does not impair sympathetic input only to
the intestine, but also other splanchnic organs. For example, approximately 66%
of the sympathetic supply to kidneys originates from the paravertebral ganglia in

rats while 20% of originates from the prevertebral ganglia (Ferguson, Ryan et al.

70

1986; Sripairojthikoon and Wyss 1987; Chevendra and Weaver 1991). In
addition, numerous studies have reported that the renal innervation is critical in
many hypertension models (Liard 1977; Katholi, Naftilan et al. 1980; Vlﬁntemitz,
Katholi et al. 1980; Norman and Dzielak 1982; Osborn and Camara 1997; Jacob,
Clark et al. 2005). Therefore, a concern could be raised that any cardiovascular
effects of CGX are mainly due to renal denervation, since our data show that
some renal nerves are removed during the CGX. However, a colleague in our lab
has performed bilateral renal denervation (RDX) and CGX, separately, in
angiotensin (Ang) II salt hypertension (King, Osborn et al. 2007). The data
showed that RDX does not attenuate the blood pressure increase induced by
Ang II treatment, while CGX signiﬁcantly decreases Ang lI-mediated
hypertension. This study shows that CGX can have important cardiovascular

actions independent of any effects on renal sympathetic nerve activity.

In addition, the sensory nerves and the sympathetic nerves are closely
localized in animals (Galligan, Costa et al. 1988; Lindh, Haegerstrand et al. 1988;
Davies and Campbell 1994) and humans (Levanti, Montisci et al. 1988; Del
Fiacco, Floris et al. 1992). Our immunochemistry results revealed that calcitonin
gene-related peptide (CGRP)-containing sensory nerves were removed during
the CGX procedure. CGRP is a very potent vasodilator (Brain, Williams et al.
1985) and is mainly found in dorsal root ganglia where the cell bodies of sensory
afferent neurons exist. The afferent neurons terminate peripherally on blood
vessels and other tissues (Wimalawansa 1996). It has been suggested that

CGRP may play an important role in regulating peripheral vascular tone and

71

regional organ blood ﬂow under physiological conditions (DiPette,
Schwarzenberger et al. 1989; Vaishnava and Wang 2003). As to pathological
conditions, several labs have shown that systemic administration of CGRP
lowers blood pressure in spontaneously hypertensive rats (Dipette and
Wimalawansa 1995) and DOCA-salt rats (Supowit, Zhao et al. 1997). Therefore,
it is possible that partial removal of the sensory nerves in the splanchnic bed may
affect cardiovascular regulation after CGX. In order to test this idea, neonatal
capsaicin-treated rats were used. In this rat model, capsaicin-sensitive nerves
are destroyed right after birth (Gamse 1982; Holzer—Petsche and Lembeck 1984).
Our preliminary data (not shown here) indicate that CGX did not affect the overall
sensory nerve regulation of hemodynamic parameters in ETBR activation-
induced hypertension (Fink, Li et al. 2007), in spite of the partial absence of the
nerves. This could be due to upregulation of CGRP receptors or a higher binding

afﬁnity to these receptors after CGX.

Furthermore, attention should be paid to nerve regeneration after CGX,
Since sympathetic postganglionic neurons show regrth after transplantation
(Olson and Malmfors 1970). Also, after chemical or surgical sympathectomy,
regeneration of sympathetic postganglionic nerves innervating mesenteric
vessels has been reported (Hill, Hirst et al. 1985; Galligan, Costa et al. 1988;
Yamada, Terayama et al. 2006). Since nerve regeneration possibly leads to
reinnervation and return of neuroeffector transmission, it’s a critical issue to test if
the effectiveness of CGX is time-dependent. Therefore, I examined reinnervation

of the sympathetic postganglionic neurons after CGX. Glyoxylic acid staining

72

showed that two weeks after CGX sympathetic nerves were almost completely
removed. By ﬁve weeks, a small portion of the sympathetic innervation had
returned, while the density of innervation was almost fully restored by ten weeks.
Assay of tissue norepinephrine content also demonstrated that innervation levels
were higher in the splanchnic organs twelve weeks after CGX than after two
weeks. The results indicate a regeneration of sympathetic nerves after CGX.
Therefore, CGX is a time-sensitive procedure and experiments involving CGX
must be performed in a timely manner to avoid the confounding effects due to

nerve regeneration.

Sympathetic postganglionic nerves projecting to the splanchnic organs
originate from the celiac ganglion, superior mesenteric ganglion (celiac plexus),
and inferior mesenteric ganglion (IMG) (Hsieh, Liu et al. 2000; Quinson, Robbins
et al. 2001). While the celiac plexus provides most of the sympathetic supply to
the upper part of the digestive system, the IMG primarily innervates the lower
digestive tract (furness and Costa 1987). Reinnervation of the splanchnic organs
was observed in our CGX rats. A possible source of these nerves is the IMG.
Reinnervation of the sympathetic postganglionic nerves after CGX in mice has
been reported to be derived from the IMG (Yamada, Terayama et al. 2006).
Therefore I performed both CGX and inferior mesenteric ganglionectomy (IMGX)
in the rat to investigate the role of IMG in nerve regeneration after CGX.
Norepinephrine measurements two weeks after the surgeries showed that IMGX
and CGX together did not further lower norepinephrine concentrations in

splanchnic organs and mesenteric vessels, compared to those in the organs and

73

tissues of rats undergoing CGX alone. Similar results were observed twelve
weeks after the surgeries. Our results indicate that the IMGX is not a major
source of sympathetic reinnervation in the splanchnic organs after CGX in the
rat. More studies are necessary to discover the origin of the splanchnic

reinnervation after CGX in rats.

Prolonged diarrhea was reported after CGX in dogs (Dayton, Schlegel et
al. 1984). We did not observe any loose feces in our CGX or CGX and IMGX
rats, indicating CGX rats have normal intestinal water/electrolyte absorption. This
is consistent with other groups’ conclusions (T eitelbaum, Sonnino et al. 1993;
Sarr, Walters et al. 1994; Duininck, Libsch et al. 2003). The animals survived the
surgeries well and they showed no signs of illness. Previous studies also show
that bilateral splanchnic nerve section does not cause any changes in behavior,
food intake, abdominal fat, body weight, brown adipose tissue weight, abdominal
organ weight, plasma Ieptin concentration, or hypothalamic neuropeptide Y level,
compared to shams (Fumess, Koopmans et al. 2001). Additionally, a recent
study shows that sodium intake is not affected by CGX in rats fed 2% NaCl
(Osborn, Guzman et al. 2007).

Conclusions and limitations

Increasing evidence indicates that the sympathetic nervous system,
especially in the splanchnic region, may be critical in blood pressure regulation in
humans and animals (normotensive and hypertensive). A selective denervation
of sympathetic nerves in the splanchnic bed, such as CGX, would be a useful

procedure to determine the roles of the splanchnic sympathetic nervous system

74

in many physiological and pathological conditions. The current study
characterized CGX, with a focus on qualiﬁcation/quantiﬁcation of the splanchnic
sympathetic innervation at structural and functional levels. My results indicate
that CGX is an effective procedure to remove the sympathetic innervation to the
splanchnic bed within one month after the surgery. CGX, therefore, could be
used in any studies designed to determine if the splanchnic sympathetic nervous

system is involved in physiological or pathophysiological processes.

The current study, however, has several limitations. First of all, all the
experiments were in vitro studies. Some in vivo studies, such as monitoring
hemodynamic parameters (blood pressure, heart rate, total peripheral resistance,
etc.), could be performed in CGX rats to identify the cardiovascular effects in
conscious animals. Secondly, my data suggest that CGX is a time-sensitive
procedure and nerve regeneration may occur after CGX. Therefore, functional
studies, such as electrical stimulation, should be carried out to examine the
effectiveness of CGX at different points in time. It is expected that after a certain
period of time (maybe 2 months) following CGX vascular responses to electrical
stimulation is bigger than right after recovery. Additionally, blood pressure could
be monitored continuously after CGX to determine if sympathetic nerve
regeneration would affect blood pressure regulation. Last, the origin of nerve
regeneration after CGX remains unclear. A retrograde tracer could be used to

discover the speciﬁc source of reinnervating ﬁbers.

75

Sham Two weeks after CGX

   

Five weeks after CGX Ten weeks after CGX

 

Figure 5a: Glyoxylic acid staining of mesenteric arteries. The blue ﬂuorescence

represents the presence of catecholamines. The scale bar applies to all pictures.

76

Sham Two weeks after CGX

Five weeks after CGX Ten weeks after CGX

- 200 um
I

Figure 5b: Glyoxylic acid staining of mesenteric veins. The blue ﬂuorescence

 

 

represents the presence of catecholamines. The scale applies to all pictures.

77

CGRP TH Colocalization

Sham

40 pm

 

Figure 6: Tyrosine hydroxylase staining and calcitonin gene-related peptide
(CGRP) staining of mesenteric arteries. The green ﬂuorescence shows the
presence of tyrosine hydroxylase, a key enzyme for catecholamine synthesis.
The red ﬂuorescence indicates the presence CGRP, a peptide released from
sensory neurons. The ﬁgures Show that CGX effectively removed both

sympathetic nerves and CGRP-containing nerves.

78

 

 

 

 

 

 

 

 

1.2
1.0 r
i?
a — Sham (n=4)
3 0.8 - :2 CGX(n=4)
a
B
3 0.6 -
5
E 0.4 4
in
z
0.2 -
0.0 - ‘
96““ ®¢°€l 940 0606‘s“ 99‘06“
~06 “" 00
as:

Figure 7: Norepinephrine (NE) concentrations in splanchnic organs two weeks

after CGX. *: Signiﬁcant difference from sham rats (P<0.05).

79

 

 
    

 

 

 

 

400

E - Sham (n=6)
E 300 - E] CGX (n=6)
2

a

9

g 200-

B

O

>

O

_.I

m 100 a

z

 

 

 

Arteries Veins

Figure 8: Norepinephrine concentrations in mesenteric arteries and veins. NE
levels in mesenteric arteries and veins from CGX rats were signiﬁcantly lower
than those from Sham-operated rats, two weeks after the surgery. IMGX did not
further reduce sympathetic nerve density. *: Signiﬁcant difference from sham rats

(P<0.05).

80

 

       

' 4 . Sham Artery (n=4)’ *
3° ‘ I Sham Vein (n=4) r ‘ *
. U- CGX Artery(n=4) :k T
25 _ ft CGX Vein (n=4)~ / F
. * I l ‘
T j T
g 20: -
38— 15 " e s ("I 3|: *
g 10-
0 d
5 ..
o.

 

 

Frequency (Hz)

Figure 9: Constriction of mesenteric vessels in response to electrical stimulation.
The constriction in response to electrical stimulation was virtually abolished in
mesenteric vessels from CGX rats. *2 Signiﬁcant difference from the same type of

vessel in CGX rats (P<0.05).

81

 

.3
N

 
  
 
   
 

- Sham (n=4)

:3 CGX-2wk (n=4)

— CGXSIMGX-Zwk (n=5)
l:l CGX-12wk (n=5)

- CGX&IMGX-12wk (n=5)

NE Content (uglg tissue)
9 P P 7‘
«h 09 G O

P
N

 

 

0.0

 

. (we!
\ﬁﬁ“ 939‘“

\QO“?! \vvloeioéa1‘“‘“ 59‘33“

Figure 10: Norepinephrine concentrations in splanchnic organs from rats with
sham-operation, CGX, or CGX and IMGX. The animals were sacriﬁced two
weeks or twelve weeks after the surgery/surgeries. *: Signiﬁcant difference from

sham rats (P<0.05).

82

 

400

— Sham (n=4)

:3 CGX-Zwk (n=4)

300 - _ CGX-12wk (n=5)

I:l CGX&IMGX-12wk (n=5)

 

100 . 2.

NE Levels (ngg protein)
N
O
O

 

 

 

 

 

 

 

 

 

 

Artery Vein

Figure 11: Norepinephrine concentrations in mesenteric arteries and veins from
rats with sham-operation, CGX, or CGX and IMGX. The animals were sacriﬁced
two weeks or twelve weeks after the surgery/surgeries. *: Signiﬁcant difference

from sham rats (P<0.05).

83

CHAPTER 5

INCREASED SUPEROXIDE LEVELS IN GANGLIA AND
SYMPATHOEXCITATION ARE INVOLVED IN SARAFOTOXIN 6C-
INDUCED HYPERTENSION

INTRODUCTION

We recently reported that chronic activation of ETBRS using intravenous
infusion of the ETBR selective agonist, sarafotoxin 60 (86c), in rats causes an
increase in arterial pressure (SGc-induced hypertension) (Fink, Li et al. 2007).
This was surprising in light of the fact that the two best-described physiological
responses to ETBR activation—release of vasodilators from endothelial cells,
and increased renal sodium and water excretion— should lead to a fall in arterial
pressure (Rubanyi and Polokoff 1994; Schiffrin 1998; Pollock, Allcock et al.
2000). We believe venoconstriction contributes to S6c-induced hypertension,
because in vitro S6c has been shown to constrict veins from most vascular
regions but to have little effect on most arteries (Evans, Cobban et al. 1999;
Johnson, Fink et al. 2002; Perez-Rivera, Fink et al. 2005; Savoia and Schiffrin
2007). Furthermore, chronic administration of other relatively speciﬁc
venoconstrictors drugs also can increase arterial pressure (Rothe 1983;
Greenway and Lautt 1986; Hainsworth 1986; Mortensen, Pawloski et al. 1990;
Rothe 1993). Decreased venous capacitance has been documented in animal
models (Manning, Coleman et al. 1979; Ricksten, Yao et al. 1981; Ackermann
and Tatemichi 1983; Fink, Johnson et al. 2000) and in humans with hypertension
(Ulrych 1976; Mark 1984; Safar and London 1985). Veins in the splanchnic
region are probably the most important target for 86c and other venoconstrictors,
since they account for most of the active capacitance responses of the circulation
(Greenway and Lautt 1986; Rothe 1986; Rothe 1993). Veins in the splanchnic

region hold about 33% of the total blood volume (Greenway and Lister 1974) and

85

maximal activation can reduce their volume up to 60%, redistributing blood from
the abdomen into the heart and thereby increasing cardiac output and blood

pressure (Karim and Hainsworth 1976; Greenway 1983).

It is likely, however, that other mechanisms contribute to SGc—induced
hypeitension. Reactive oxygen species (R08) are a variety of oxygen-containing
molecules that are by-products of cellular metabolic processes under
physiological condition, including superoxide anion (02"), hydrogen peroxide
(H202), and hydroxyl ion (OH‘). Superoxide production is mediated by several
enzymes including nicotinamide adenine dinucleotide phosphate (NADPH)
oxidase, xanthine oxidase, and nitric oxide synthase (NOS) (Wilcox 2002).
Recently, increasing evidence has shown that ROS can function as cellular
signaling molecules (Bedard and Krause 2007). They achieve this signaling
function by participating in phosphatase inhibition (Lee, Kwon et al. 1998; Wu,
Hardy et al. 2003; Goldstein, Mahadev et al. 2005; Kwon, Qu et al. 2005), kinase
activation (Griendling, Sorescu et al. 2000; Han, Kim et al. 2003; Furst, Brueckl
et al. 2005), ion channel regulation (Hidalgo, Bull et al. 2004; Tang, Santarelli et
al. 2004), and calcium signaling (Wang, Takeda et al. 1999; Cheranov and
Jaggar 2006; Granados, Salido et al. 2006). Increased ROS levels have been
found in blood vessels, kidneys and other tissues in many experimental models
of hypertension, including renovascular (Lennan, Nath et al. 2001), DOCA-salt
(Somers, Mavromatis et al. 2000), Dahl salt-sensitive (Swei, Lacy et al. 1999),
and lead-induced hypeitension (Vaziri, Liang et al. 1999). ROS generation also is

elevated in hypertensive patients (T ouyz and Schiffrin 2001). Increased tissue

86

ROS levels can cause hypertension, because blocking ROS generation or
increasing their removal lowers blood pressure (Kopkan and Majid 2005;
Sullivan, Pollock et al. 2006; Baumer, Kruger et al. 2007). Recent reports
indicate that ETBR activation increases oxidative stress in sympathetic ganglia in
vitro (Dai, Galligan et al. 2004) and in vivo (Lau, Galligan et al. 2006). Although
the precise effects of increased ROS levels in sympathetic ganglia are not
known, it is possible that ROS signaling could enhance ganglionic
neurotransmission and thereby increase sympathetic activity. Therefore, we
tested the hypothesis that increased superoxide generation in vascular tissues or

sympathetic ganglia is involved in SSc—induced hypertension.

87

METHODS
Animals
Normal male Sprague-Dawley rats (300-325 9, Charles River, Portage,

MI, USA) were used in these experiments.

The following methods were used in current study.

Vascular catheterization;

Hemodynamic measurements by external blood pressure analyzer;

Celiac ganglionectomy (CGX);

Dihydroethidium ﬂuorescence;

Lucigenin-enhanced chemiluminescence;

Plasma sampling;

0 Plasma norepinephrine assay.

Protocols
S6c-infusion

For S6c-treated rats, saline vehicle (3.47 pl lmin) was infused
intravenously for two days (control period, C). Then the animals were treated with
S6c (5 pmollkglmin, iv) for ﬁve days (infusion period, E), followed by a three-day-
infusion of saline only (3.47 pl lmin, recovery period, R). Sham rats received
saline only (3.47 pl lmin) for ten consecutive days. Hemodynamic parameters

were measured every day.

Tn'methaphan study

88

Trimethaphan (15 mglkg, iv) was given daily to SGc-treated and sham
rats. The peak fall in blood pressure after trimethaphan injection (generally within

2-5 minutes) was recorded as an index of sympathetic pressor activity.

Plasma NE measurement
S6c-infused and sham rats were used in this study. Blood samples were
collected on the 2"Cl control day, the 3rd and the 5th day of infusion period, and the

2nd recovery day.

CGX study
Celiac ganglionectomy and sham-operation were performed 10 to 14 days
prior to beginning the study. Rats were then subjected to S6c infusion as

described above.

Superoxide studies in S6c-induced hypertension

For SBc—treated rats, saline (3.47 pl/min) was infused intravenously for two
days (control period, 0). Then the animals were treated with 86c (5 pmollkglmin,
iv). Tissues were collected after S6c infusion for one day or ﬁve days. Sham rats
received saline for seven consecutive days. Animals were sacriﬁced in pairs of

S6c-treated and saline-treated rats.

The effect of tempol treatment on S6c-induced hypertension

After the recovery from catheterizations of femoral artery and vein, and a
two-day control period, one group of rats were infused with saline while drinking
water containing tempol tempol (4-hydroxy 2,2,6,6,-tetramethyl peperidine 1-oxyl,

a superoxide dismutase mimetic, 1 mmol/L). Each water bottle was wrapped with

89

aluminum foil to protect the drug from light exposure. The tempol solution was
prepared fresh every day. The second group received 86c (5 pmollkglmin) and
the third group received 86c while drinking tempol solution. 86c and tempol were
removed after a 5-day—infusion in group 2 and 3, followed by saline infusion

(recovery period).

DHE staining in S6c-treated rats with tempol treatment

One group of rats was given water containing tempol (1 mmol/L)
immediately after catheterization surgery. After Six days of tempol treatment, rats
received 86c (5 pmollkglmin, iv) infusion for one day while they continued to
receive tempol. A second group of animals were infused with saline only (3.47
pllmin, iv) for seven days and did not receive tempol treatment. A third group of
rats was treated with saline for six days, then received 86c (5 pmollkglmin, iv) for
one day. For DHE staining, one animal from each group was sacriﬁced and

tissues were collected from all three rats and studied together.

Statistical analyses

The effect of CGX on MAP was evaluated by Student ttest compared to
the average of baseline MAP in intact rats. Within-group differences over time
were assessed by a one-way ANOVA and posthoc multiple comparisons with
Tukey’s test (GraphPad lnstat 3). Between-group differences were evaluated
using the protected least signiﬁcant difference test. Superoxide levels in lucigenin
studies were reported in the unit of nmollmin/mg protein. DHE staining was
quantiﬁed by taking readings from three to ﬁve individual sympathetic neurons or

vascular smooth muscle cells in each rat. The ﬂuorescence density of the

90

measured cells was the difference between the absolute number in arbitrary
ﬂuorescence units and the value of background. Control rats were used for
comparison. The ratio of ﬂuorescence density from treated rats to that from
controls was used for statistical comparisons. When comparing 2 groups, the
appropriate Student ttest was used. Analysis of variance followed by Tukey post
hoc test was performed when comparing 3 or more groups. In all cases, a P
value less than or equal to 0.05 was considered statistically signiﬁcant. A P<0.05

was considered Signiﬁcant. All of the results are presented as mean :t SE.

RESULTS
Acute ganglion blockade in SGc-induced hypertension

Figure 12a shows mean arterial pressure (MAP) changes during 5-day
$60 or vehicle infusion. MAP increased signiﬁcantly during S6c infusion while it
did not change in vehicle-treated rats (average of 123.8 :I: 1 vs 106.7 :I: 2 mmHg).
Figure 12b shows acute depressor responses to ganglion blockade with
trimethaphan. Infusion of S6c produced sustained hypertension, whereas no
changes in blood pressure were observed in vehicle-treated rats. No differences
in depressor responses to ganglion blockade were found in vehicle and S6c-
treated rats during the control period or during the ﬁrst 3 days of the infusion
period. However, on days 4 and 5 of the infusion period, the response to
trimethaphan was signiﬁcantly larger in SBc—treated rats than in vehicle-treated
control animals. Termination of 86c treatment was associated with a return of

trimethaphan responses to control period values.

91

Plasma norepinephrine levels in SGc-induced hypertension

Mean arterial pressure (MAP) increased signiﬁcantly in S6c-infused rats
and the increase was sustained during the infusion period. MAP did not change
in vehicle-treated animals (average of 127.4 :I: 1 vs. 99.6 1: 2 mmHg, Figure 13a).
Plasma norepinephrine concentrations (Figure 13b) did not change in either
vehicle-treated rats or $6c-treated rats relative to their respective control period
values, nor were differences between vehicle- and S6c-treated rats signiﬁcant at

any time during the study.

S6c-induced hypertension in celiac ganglionectomized rats

Figure 14 shows the effect of CGX on S6c-induced hypertension.
Ganglionectomy alone slightly decreased resting arterial pressure, although not
signiﬁcantly. The increase in pressure during S6c treatment, however, was
markedly less in CGX than in sham-operated rats. In fact, during the last three
days of S6c infusion, arterial pressure in CGX rats returned to values not

signiﬁcantly different from those measured during the control period.

Superoxide levels in mesenteric vessels of 86c-treated rats

DHE staining showed an apparent increase in superoxide level in smooth
muscle cells from mesenteric arteries and veins from rats receiving S6c,
compared to vessels from rats receiving saline only (Figure 15a), but the
difference was not statistically signiﬁcant (Figure 15b). Similarly, lucigenin-
enhanced chemiluminescence measurements (Figure 16) indicated that although

the mean values of superoxide levels were increased in blood vessels from the

92

rats treated with S6c, the differences were not statistically signiﬁcant. The basal

level of superoxide in veins was signiﬁcantly higher than in arteries.

Superoxide levels in inferior mesenteric ganglion of SGc-treated rats

Figure 17a shows DHE staining of inferior mesenteric ganglion (IMG) from
rats receiving saline for 7 days, or 1- or 5-day infusions of S6c. More intense
ﬂuorescence was observed in lMGs from rats treated with S6c for either 1 day or
5 days, compared to those treated with saline. In addition, more ﬂuorescence
was observed in IMG from rats receiving 86c for 5 days than in those receiving
S6c for 1 day. Quantitative analyses revealed that these differences were

statistically Signiﬁcant (Figure 17b).

Eﬁ'ects of tempol on arterial pressure and ganglion superoxide in SGc-treated rats

Figure 18 demonstrates that tempol treatment prevented a signiﬁcant rise
in blood pressure in rats receiving 86c, but had no effect in control rats. Figure
19a shows DHE ﬂuorescence in IMG of rats treated with saline, 86c, and 86c
plus tempol. Quantiﬁcation revealed that superoxide levels in IMG from rats
treated with 86c and tempol were signiﬁcantly lower than those in IMG from rats

treated with S6c alone (Figure 19b).

93

DISCUSSION

The data presented here suggest that the sympathetic nervous system
contributes to $6c-induced hypeitension. For example, depressor responses to
the ganglion blocker trimethaphan became gradually larger during a 5-day S6c
infusion. This ﬁnding is generally accepted as evidence for elevated sympathetic
activity to the cardiovascular system (Biaggioni and Robertson 2002), although
other explanations are possible (Biaggioni and Robertson 2002; Degoute 2007).
Therefore, plasma norepinephrine levels were measured as another index of
sympathetic nervous system activity. However, plasma norepinephrine
concentrations were not elevated during 360 infusion. This could be due to the
insensitivity of this method for detecting increased sympathetic nervous system
activity (Aneman, Eisenhofer et al. 1996; Peaston and Weinkove 2004).
Alternatively, it could indicate that sympathetic activity is increased in a regionally
speciﬁc way in SSC-induced hypertension. As discussed in the Introduction, there
is evidence that venoconstriction participates in SBc—induced hypertension. Since
the splanchnic veins and venules are the main capacitance segments of the
circulation, we speculated that sympathetic nerves to the splanchnic vascular
bed may be activated in S6c—induced hypertension. The celiac plexus contains
the cell bodies of most sympathetic neurons innervating the splanchnic organs.
Therefore, surgical celiac ganglionectomy (CGX) was performed to determine if
sympathetic nerves in the splanchnic region play a role in blood pressure
responses to S6c infusion. The results showed that in celiac ganglionectomized

rats 86C increased blood pressure Signiﬁcantly for the ﬁrst two days of the ﬁve

day infusion period. But subsequently pressure returned to levels not signiﬁcantly
higher than pre-infusion control period values, despite continued 86c

administration.

Collectively, these ﬁndings support the idea that S6c-induced
hypertension is partially dependent on increased activity of the sympathetic
nerves (with cell bodies in the celiac ganglion plexus) supplying the splanchnic
organs. These nerves control smooth muscle activity in the arteries and veins of
the intestine, stomach, pancreas, mesentery, liver and other organs. They
account for a substantial fraction of norepinephrine released from the peripheral
sympathetic nervous system (Hottenstein and Kreulen 1987; Stjarne, Bao et al.
1994), and their activation leads to both increased systemic vascular resistance
and decreased capacitance (Mark 1984; Hainsworth 1986; Pang 2001). AS noted
earlier, activation of the splanchnic sympathetic nerves can redistribute blood
from compliant peripheral veins into the central circulation and thereby increase

blood pressure (Karim and Hainsworth 1976; Greenway 1983).

It’s not Clear why an inﬂuence of the sympathetic nervous system on blood
pressure in S6c-induced hypertension was only demonstrable 3 or more days
after starting S6c infusion. One possibility is that the early increase in blood
pressure is non-neurogenic and causes a baroreﬂex mediated reduction in
sympathetic activity until signiﬁcant resetting of the reﬂex occurs. This could be
tested by performing S6c infusion in animals with sino-aortic denervation.
Another question is: If splanchnic sympathetic nerve activity is increased during
86c infusion, why isn’t this reﬂected in elevated peripheral plasma concentrations

95

of norepinephrine? The likely explanation is that norepinephrine released from
splanchnic sympathetic nerves is efﬁciently cleared from the circulation by the
liver (Aneman, Eisenhofer et al. 1995; Eisenhofer, Aneman et al. 1995). This idea
could be tested using regional norepinephrine spillover measurements in $60-

infused rats.

Under physiological conditions, ROS are present at low concentrations
and act as Signaling molecules regulating the growth and function of vascular
smooth muscle cells (Rao and Berk 1992; Cosentino, Sill et al. 1994). Under
pathological conditions, ROS may impair endothelium-dependent vasorelaxation
(Somers, Mavromatis et al. 2000), induce vascular smooth muscle cell growth
(Zafari, Ushio-Fukai et al. 1998), elevate sympathetic nervous system activity
(Campese, Ye et al. 2004), alter renal function (Han, Lee et al. 2005), and cause
increased deposition of extracellular matrix proteins (Ha and Lee 2003). All of
these effects may contribute to vascular and organ damage, and other
pathophysiological changes, in a variety of cardiovascular diseases (Harrison
1997; Griendling 2004; Touyz and Schiffrin 2004; Brandes and Kreuzer 2005).
Accumulating evidence indicates that ROS play physiological and

pathophysiological roles in hypertension (T ouyz and Schiffrin 2004).

Most studies indicate that endothelin-family peptides stimulate ROS
generation in blood vessels and other tissues by acting on ETA subtype
receptors (ETARS) (Callera, Touyz et al. 2003; Ozdemir, Parlakpinar et al. 2006).

However, endothelin-induced superoxide production in sympathetic neurons in

vitro is mediated by ETBRS (Dai, Galligan et al. 2004). Furthermore, acute
activation of ETBRS in vivo in conscious rats produces increased superoxide
concentrations in sympathetic ganglia (Lau, Galligan et al. 2006). Therefore, we
tested the hypothesis that chronic infusion of 36c also would increase superoxide
in sympathetic ganglia. We made measurements in the inferior mesenteric
ganglion (IMG), a prevertebral ganglion providing innervation to splanchnic blood
vessels, small intestine, colon, rectum, etc. (Szurszewski 1981), because
ﬂuorescent imaging is easier in this ganglion than in the celiac plexus. In order to
assess a possible effect on blood vessels of chronic exposure to 86c in vivo,
superoxide levels in mesenteric arteries and veins also were determined.
Because evidence reported above suggested that the mechanisms of S6c-
lnduced hypeitension might differ during the early and later stages, we measured

superoxide levels in rats treated with S6c for 1 or 5 days.

Both DHE staining and lucigenin-enhanced chemiluminescence
measurements revealed that superoxide levels were not signiﬁcantly increased in
either arteries or veins from rats treated with S6c for 1 or 5 days. This result is
consistent with previous work (discussed above) indicating vascular superoxide
production in response to endothelin peptides is mediated by ETARS.
Interestingly, the basal level of superoxide in veins was signiﬁcantly higher than
in arteries, possibly due to a higher nitric oxide production in arteries which may
interact with superoxide to lower the level, compared to veins (Szasz, Thakali et
al. 2007). Lucigenin was not used to determine ROS levels in ganglia because

the method lacks sufﬁcient sensitivity for very small amounts of tissue. However,

97

DHE staining showed that superoxide levels were signiﬁcantly increased in
sympathetic ganglia from S6c—treated rats. Moreover, the increase was time-
dependent. That is, superoxide levels were higher in sympathetic neurons from

rats receiving 860 for 5 days versus 1 day.

With this evidence of increased ROS in sympathetic ganglia in S6c-
induced hypertension, we designed an experiment to address the cause-and-
effect relationship between increased oxidative ROS and elevated blood
pressure. Tempol, a superoxide dismutase mimetic (Schnackenberg, Welch et al.
1998); (Shokoji, Nishiyama et al. 2003), was used to test if increased blood
pressure in S6c-infused rats required increased tissue levels of superoxide. Our
results conﬁrmed previously published studies showing that tempol does not
affect blood pressure in normotensive rats (Sullivan, Pollock et al. 2006).
However, tempol attenuated blood pressure elevations in S6c-infused rats,
suggesting that increased tissue concentrations of superoxide contribute
importantly to S6c-induced hypeitension. To determine possible target tissues for
the actions of tempol, we measured superoxide levels in sympathetic neurons
from rats treated with S6c, with and without tempol. These results showed that
superoxide was lower in a prevertebral ganglion of rats treated with tempol
compared to those treated with S6c alone, although levels were not completely
normalized. These results are consistent with the idea that tempol ameliorated

S6c-induced hypertension by reducing ROS levels in sympathetic ganglia.

How do ROS in sympathetic ganglia affect post-ganglionic sympathetic
nerve activity? Although there is no direct evidence in sympathetic ganglia, in

98

other neuron types ROS have been Shown to increase cell excitability by
inhibiting calcium-activated potassium channel (Soto, Gonzalez et al. 2002;
Zucker and Gao 2005). Therefore, we hypothesize that in S6c-induced
hypertension, increases in superoxide content in the prevertebral sympathetic
ganglia cause higher post-ganglionic nerve discharge and constriction of
splanchnic blood vessels. We further hypothesize that tempol decreases blood
pressure in S6c-induced hypertension by reducing ROS levels in sympathetic
ganglia and thereby post-ganglionic sympathetic nerve activity. Previous studies
have shown that tempol is, in fact, sympathoinhibitory in hypertensive rats (Fujita,
Ando et al. 2007; Han, Shi et al. 2007). A caveat to this conclusion, however, is
that tempol also has other pharmacological effects which could attenuate
hypertension, for example, opening large conductance, calcium activated

potassium channels (Xu, Jackson et al. 2006).

In summary, the data presented here are consistent with the idea that
hypertension produced by chronic activation of ETBRS in rats is due to both
neurogenic and non-neurogenic mechanisms. The neurogenic mechanism has a
delayed onset and probably results from changes in sympathetic activity to the
splanchnic vascular bed. Although it is not known if chronic selective activation of
ETBRS occurs under normal circumstances, the model illuminates the possible
importance of both venoconstriction and the splanchnic sympathetic nerves in

the development of hypertension.

99

 

140

 

 

—O— Saline (n=6)
+ 86c (n=7)
3"
E 120 -
.5.
e 110 1
E
100 -
90 I 'lnfu'sion'period I

 

 

 

C1 C2 E1 E2 E3 E4 E5 R1 R2 R3
TIME (DAYS)

Figure 12a: The depressor responses to ganglion blockade with trimethaphan
before (01-02, C=control), during (E1-E5, E=infusion), after (R1-R3,
R=Recovery) continuous S6c infusion (5 pmollkglmin, iv) or vehicle into rats.
Figure 1a shows mean arterial pressure (MAP). MAP signiﬁcantly increased
during S6c-infusion period and it did not change with vehicle infusion. *: a

statistical difference (p<0.05) from the control period.

100

 

 

 

0
'3
as:
g -20
n.
E
.5 -40 -
0
3
9 .
3 -60 - * — Saline=6
D * 1:1 $6c=7

 

 

 

Infusion Period ]

 

r

C1 C2 E1 E2 E3 E4 E5 R1 R2 R3
Time (days)

Figure 12b: Changes in MAP in $6c- or vehicle-infused animals with a bolus
injection of trimethanphan (15 mglkg, iv), a ganglionic blocker, during infusion
period. MAP decrease was Signiﬁcantly bigger in SGc—treated rats than in
vehicle-treated rats on the fourth and the ﬁfth day. Numbers in parentheses
means the number of rats in each group. *: a statistical difference (p<0.05) from

control rats on the same day.

101

 

 

 

 

 

 

 

140
-O- Control (n=9)
-0— SSC (n=8)
130 .
E
E 120
E.
a.
< 110
E
100 i
90 Infusion period I
C1 C2 E1 E2 E3 E4 E5 R1 R2 R3
TIME (DAYS)

Figure 13a: Mean arterial pressure (MAP) in rats before (C1-C2, C=control),
during (E1-E5, E=infusion), after (R1-R3, R=recovery) continuous S6c infusion (5
pmollkglmin, iv) or vehicle. MAP signiﬁcantly increased during S6c-infusion
period and it did not change with vehicle infusion. *: a statistical difference

(p<0.05) from the control period.

102

 

 

 

 

 

 

 

 

 

500
a Control (n=9)
— 86c (M)
400 -
E.
5 300 -
Iii
2
E 200 -
in
3
a.
100 .
0
C2 E3 E5
Time Idavsl

Figure 13b: Average (1 SEM) plasma norepinephrine (NE) concentrations in rats
receiving continuous S6c infusion (5 pmollkglmin, iv) or vehicle according to the
same protocol shown in Figure 1. Plasma samples were collected on the second
control day (C2), the third and the ﬁfth infusion days (E1 and AE), and recovery

day 2 (R2). Plasma NE levels did not increase during S6c-infusion period,

although MAP signiﬁcantly elevated.

103

 

 

R2

 

 

 

 

 

 

 

140
-O- Control (n=6)
—0— CGX =6
130 -
a“:
E 120 -
E
(L
< 110 ‘
E
100 -
90 I 'Infu'sion'period I

c1 c2 E1 E2 E3 E4 E5 R1 R2 R3
TIME (DAYS)

Figure 14: Average mean arterial pressure (MAP) before (day 1-2, control
period), during (day 3-7, infusion period), and after (day 8-10, recovery period)
continuous S6c infusion (5 pmollkglmin, iv) into celiac ganglionectomized (CGX)
and sham-operated rats. MAP signiﬁcantly increased during infusion period in
sham-operated rats. It increased signiﬁcantly only in the ﬁrst two days of infusion

period in CGX-operated rats.*: a statistical difference (p<0.05) from control

period.

104

 

 

F
50 p m

Figure 15a: Representative pictures showing superoxide levels in smooth
muscle cells from superior mesenteric arteries (SMA) and veins (SMV) in control
and SGc-infused rats, measured by DHE. 02" levels, indicated by DHE
ﬂuorescence intensity in confocal images of smooth muscle cells, are higher in
S6c-treated rats than in sham rats. A, SMA from a sham rat; B, SMA from a rat
receiving 86c for one day; C, SMA from a rat receiving 86c for ﬁve days; D, SMV
from a sham rat; E, SMV from a rat receiving S6c for one day; F, SMV from a rat

receiving S6c for ﬁve days. The scale bar in F applies to all panels.

105

3.0

 

 

 

 

 

 

 

— Vehicle-treated (n=7)
2.5 q — 1-day-S6c-infuslon (n=7)
§ — 5-day-S6c-infusion (n=7)
§ 2.04
§
3 1.5-
u.
0
5 1.0-
2
O
0‘ 0.5-
0.0

 

Arteries Veins

Figure 15b: Quantiﬁcation of mean ﬂuorescence intensity of smooth muscle cells
in SMA and SMV (n=7 in each group). Fluorescence of smooth muscle cells was
not signiﬁcantly greater in rats receiving S6c for either one day or ﬁve days

compared with shams’.

106

 

 

 

 

 

 

 

 

E 0.25

.3

g - Vehche-treated (n=7)

9- - 1-day-S6c-lnfuslon (n=5)
‘5” 0-20 ‘ - 5-day-SSc-Infuslon (n=7)
3 *
E

3 0.15 .

E

5

vi

3 0.10 -

>

O

.I

O

E 0.05 -

x

2

O

‘3‘

fl) 0.00

Arteries Velns

Figure 16: Superoxide levels in superior mesenteric arteries and veins from
control and S6c-infused rats, measured by lucigenin-enhanced
chemiluminescence. Rats were either treated with S6c for 1 day or 5 days.
Superoxide levels were not signiﬁcantly increased with S6c treatment. Basal
superoxide level was higher in veins than in arteries. *2 a statistical difference

(p<0.05) from control rats.

107

 

Figure 17a: Representative pictures showing superoxide levels in inferior
mesenteric ganglion (IMG) neurons from control and S6c-treated rats. A, IMG
from a sham rat (the arrow points to a glial cell); B, IMG from a rat receiving 860
for one day; C, IMG from a rat receiving S6c for ﬁve days (the arrow points to a
neuron); D, Comparison of mean ﬂuorescence intensity of S6c—treated neurons
to sham neurons (n=7 Sham rats; n=7 S6c—treated rats). The scale bar in C

applies to all panels.

108

 

 

— Vehicle-treated (n=7) *#

a, 4 . :3 1-day-S6c-infusion (n=7)
3 _ 5-day-S6c-infusion (n=7)
§

2 3-

O

2

u.

O 2 -

.2

H

E

32 1 -

0

 

 

 

Figure 17b: Quantiﬁcation of mean ﬂuorescence intensity of sympathetic
neurons from S6c-treated and saline-treated rats (n=7 in each group). The
ﬂuorescence density was signiﬁcantly higher in S6c-treated rats (one day or ﬁve
day-infusion) than that in control rats. Superoxide level increased in a time-
dependent manner. The level from rats with ﬁve day-infusion was higher than
that from rats with one day-infusion. *: Fluorescence of IMG neurons was
signiﬁcantly (P<0.05) greater in ganglia from rats receiving S6c for both 1 day or
5 days compared with shams’; #: Fluorescence of IMG neurons was signiﬁcantly
(P<0.05) greater in ganglia from rats treated with S6c for 5 days compared with

those treated for 1 day.

109

 

-O- Sallne+Tempol (n=7)
-0— S6c+Tempol (n=8)
* *+ 36c Only (n=5)

.t
O
O

5

120 -

100 -

 

 

Mean Arterial Pressure (mmHg)

 

 

| Infusion period |
C1 C2 E1 E2 E3 E4 E5 R1 R2 R3
Time (days)

 

Q
0

Figure 18: Average (:1: SEM) mean arterial pressure (MAP) before (01-02,
C=control), during (E1-E5, E=infusion), and after (R1-R3, R=recovery)
continuous S6c infusion (5 pmollkglmin, iv) or vehicle into rats, while tempol was
given through drinking water (1 mmon). A third group of animals were on S6c,
while drinking normal distilled water. Numbers in parentheses means the number
of rats in each group. MAP signiﬁcantly increased in S6c-infused rats without
tempol treatment. The rats with dual treatments of 86c and tempol did not Show
a signiﬁcant increase in MAP. Tempol did not change MAP in vehicle-treated

rats. *: a statistical difference (p<0.05) from control period.

110

 

Figure 19a: Superoxide levels in inferior mesenteric ganglion (IMG) neurons
from control and SSc-treated rats. A, IMG from a sham rat; B, IMG from a rat
receiving S6c for one day; C, IMG from a rat receiving 86c for one day while

drinking tempol water (1 mmol/l); The scale bar in C applies to all panels.

111

 

- Vehicle-treated (n=5)
4 " :3 1-day-86c-lnfuslon (n=5)

— 1-day-S6c-lnfuslon whlle drlnklng tempol
water (n=5)
3 - *

J—

Relatlve Fluorescence

 

 

 

 

   

teams 1

 

 

 

Figure 19b: Quantiﬁcation of mean ﬂuorescence intensity of $6c-treated smooth
muscle cells to sham smooth muscle cells in SMA and SMV (n=5 in each group).
*: Fluorescence of IMG neurons was signiﬁcantly (P<0.05) greater in ganglia

from rats receiving S6c alone compared with shams’.

112

CHAPTER 6

CHARACTERIZATION OF ENDOTHELIN-B RECEPTOR-
DEFICIENT TRANSGENIC RATS

113

INTRODUCTION

Endothelin type B receptors (ETBRS) are present and functional in various
tissues, such as endothelial cells, vascular smooth muscle cells (VSMCS), kidney
and liver. Activation of ETBRS on vascular endothelium can produce vasodilation
through the generation of endothelial cell derived vasodilators, such as nitric
oxide and prostaglandins (Wright and Fozard 1988; Warner, de Nucci et al. 1989;
Rubanyi and Polokoff 1994). Activation of ETBRS in the kidney leads to a marked
increase in renal excretion of sodium and water by inhibiting sodium reabsorption
in the renal tubules and water reabsorption in the collecting duct (Kohan, Padilla
et al. 1993; Plato, Pollock et al. 2000). In addition, ETBRS act as endogenous
“clearance receptors” for ET-1 (Ozaki, Ohwaki et al. 1995; Dupuis, Goresky et al.
1996; Berthiaume, Yanagisawa et al. 2000). On the other hand, renal
vasoconstriction is induced by the stimulation of ETBRS in the rat (Clozel, Gray
et al. 1992; Cristol, Warner et al. 1993); (Clozel, Gray et al. 1992; Cristol, Warner
et al. 1993). Recently, it has also been found that ETBR activation contracts
veins from most vascular beds but only weakly constricts most non-renal
resistance arteries (Moreland, McMullen et al. 1992; Gray, Lofﬂer et al. 1994;
Johnson, Fink et al. 2002). ETBR activation also leads to increased oxidative
stress in splanchnic sympathetic ganglia (Dai, Galligan et al. 2004). Acute (2-
hour) activation of ETBRS in vivo induces a sustained blood pressure increase
(Lau, Galligan et al. 2006) and elevates oxidative stress level in sympathetic

ganglia (Dai, Galligan et al. 2004; Lau, Galligan et al. 2006). Therefore, the

114

multifunctional nature of ETBRS makes studying their roles in physiological and

pathological conditions complicated.

A mutation of the ETBR gene in humans causes Hischsprung’s disease,
which leads to aganglionic megacolon and pigment abnormalities (Puffenberger,
Hosoda et al. 1994). The spotting lethal (SI) rat (with a background related to the
WKY rat) is an animal model for human Hischsprung’s disease (Gariepy, Cass et
al. 1996; Gariepy, Williams et al. 1998). In this model, there is a natural 301 base
pair deletion which spans the exon 1-intron 1 junction in the ETBR gene. The
deletion leads to an abnormal ETBR mRNA lacking the coding sequence for the
receptor’s transmembrane domain. No expression of functional ETBRS is
detected in tissues from homozygous sllsl rats. The animals show intestinal
aganglionosis and color spotting. This phenotype is lethal and the rat usually dies
at the age of one month due to the intestinal defect. In order to rescue the (sllsl)
rats, the human dopamine-ii-hydroxylase (ml-l) promoter was used to direct
transgenic (Tg) expression of ETBR to colonizing precursors of the enteric
nervous system (Gariepy, Williams et al. 1998). The [ﬁH— ETBR transgene
compensates for deﬁciency of endogenous ETBRS in these rats. Therefore the
intestinal obstruction is prevented. This transgenic rescue generates Tg (sllsl)
rats with ETBRS that are expressed and functional in adrenergic tissues,
including the locus ceruleus, adrenal medulla, and sympathetic ganglia. Unlike in
normal animals, ETBRS are not functional in endothelium, renal tubules, or

vascular smooth muscles in T9 (sllsl) rats.

115

In Tg (+I+) rats, not only endogenous but extra copies of the ETBR gene
(by WH- ETBR transgene) are present. Therefore, WKY rats (the background of
these transgenic rats) were used as a more natural wildtype control to evaluate
strain differences. Gender differences have been reported in various
hypertension models (Mallik R. Karamsetty 2004; Reckelhoff 2005). Speciﬁcally,
gender differences in blood pressure regulation have been reported in T9 (sllsl)
rats (Sullivan, Pollock et al. 2006). The differences are possibly mediated by
disparities in plasma ET-1 in the circulation. In addition, the T9 (sllsl) rats are
reported to be salt-sensitive. That is, the animals show elevated blood pressure
on high salt diet (Gariepy, Ohuchi et al. 2000; Taylor, Gariepy et al. 2003).
Others, however, have reported that systolic blood pressure of T9 (sllsl) rats is
signiﬁcantly higher than that of controls even on normal salt diet (Elmarakby,

Dabbs Loomis et al. 2004).

The speciﬁc goal of current study was to characterize this unique rescued
transgenic rat model, with an emphasis on neurohumoral mediation (NE and ET-
1), gender differences (female vs. male), and strain differences (WKY vs. Tg
(+I+) vs. Tg (sllsl)) in hemodynamic regulation. The overall goal of the work was

to better understand the role of the ETBRS in arterial pressure regulation.

116

METHODS

Animals

Transgenic endothelin type B receptor-deﬁcient rats (Male: 300-325 9;

Female:

200—2259) and Vlﬁstar Kyoto (WKY) rats (with a related genetic

background to the transgenic rats) were used in these experiments. All rats were

bred at the animal facility of Michigan State University.

The following methods were used in current study.

Plasma endothelin-1 measurement;

Norepinephrine measurement in mesenteric vessels;
Norepinephrine measurement in organ tissues;
Glyoxylic acid staining;

In vitro preparation of mesenteric vessels;

Video monitoring of vessel diameter;

Transmural stimulation of perivascular nerves;

Hemodynamic measurement by radiotelemetry.

Statistical analyses

Differences were assessed by a 1-way ANOVA and posthoc multiple

comparisons with Tukey’s test (GraphPad Instat 3). A P<0.05 was considered

signiﬁcant. All of the results are presented as meaniSE.

117

RESULTS
Blood pressure

Continuous radiotelemetry recording shows (Figure 20), in each gender,
mean arterial pressure (MAP) in T9 (sllsl) rats was signiﬁcantly higher than that
in T9 (+I+) rats (Male: 124 :I: 2 vs. 114 :I: 1 mmHg; Female: 129 :I: 2 vs. 105 :l: 1
mmHg; P<0.05). In addition, MAP of male Tg (+I+) rats was higher than that of

females (P<0.05) and male WKY (P<0.05).

Plasma E T-1 concentration

Plasma ET-1 levels in T9 (sllsl) rats were higher than those in T9 (+I+)
animals for both genders (Male: 5.2 :I: 0.9 vs. 3.6 d: 0.1pg/ml; Female: 7.3 :I: 1.4
vs. 3.6 t 0.4 pg/ml; P<0.05) (Figure 21). Also, circulating ET-1 in male Tg (SI/SI)

rats was signiﬁcantly lower than in females (P<0.05).

Glyoxylic acid staining

Mesenteric tertiary arteries and veins from male animals were used for
glyoxylic acid staining. Figure 22 Shows sympathetic nerve distribution on the
vessels. There was no visible difference in nerve density among Tg (+I+), Tg

(sll+), and T9 (sllsl) rats, in either arteries or veins.

NE content measurements

In male rats (Figure 23a), NE concentration in T9 (+I+) arteries was higher
than that of male WKY (P<0.05). In female rats (Figure 23b), no signiﬁcant
difference was found in NE content of mesenteric arteries or veins among three

groups of rats.

118

Figure 24 shows NE concentrations in splanchnic organs, including
kidneys, liver, spleen, and small intestine, from female WKY, T9 (+I+), and T9

(sllsl) rats. There were no signiﬁcant differences between any groups.

Electrical stimulation

Mesenteric arteries and veins from male T9 (+I+), Tg (SV+), and T9 (sllsl)
were collected for this experiment. Figure 25a (for arteries) and Figure 25b (for
veins) Show that mesenteric vessels constricted in response to electrical
stimulation in a frequency-dependent manner. For each vessel type, there were
no differences in vasoconstrictor responses among the three groups of rats.
Additionally, as it has been shown previously (Luo, Hess et al. 2003), veins were

more sensitive than arteries to electrical stimulation.

119

DISCUSSION

For both male and female animals, resting blood pressure in T9 (sllsl) rats
was Signiﬁcantly higher than that in T9 (+I+) rats. This hypertension was also
reported by other labs in both genders of intact Tg (+I+) rats (Taylor, Gariepy et
al. 2003; Sullivan, Pollock et al. 2006) or sham-operated rats of DOCA-salt
hypertension model (Kawanishi, Hasegawa et al. 2007). However the data
remain controversial, since these animals were reported to be normotensive
elsewhere (Perry, Molero et al. 2001). Our results are consistent with those in
most research labs. More importantly, the blood pressures were measured by
radiotelemetry, while others performed hemodynamic measurements by tail-cuff
method, which does not monitor the highly dynamic nature of blood pressure as

accurately as telemetry (Kurtz, Grifﬁn et al. 2005; Kurtz, Grifﬁn et al. 2005).

My data conﬁrm the ﬁnding from other groups that there is a Signiﬁcant
increase in circulating ET-1 levels in T9 (sllsl) rats, compared to T9 (+I+) rats
(Peny, Molero et al. 2001; Sullivan, Pollock et al. 2006). So a higher ET-1 level in
the circulation may lead to a blood pressure increase via endothelin type A
receptor (ETAR) activation. In fact, it has been demonstrated that hypertension
induced by high salt diet in T9 (sllsl) rats can be completely abolished by ETAR
blockade, suggesting that the hypertension is caused entirely by endothelin

acting at ETARS (D'Angelo, Pollock et al. 2005).

Until now, most characterization studies of T9 (sllsl) rats have been
focused on the vascular and renal ET systems, while sympathetic innervation

has received little attention. The sympathetic nervous system, especially in the

120

splanchnic bed, plays an important role in hypertension development and
maintenance (Anderson, Sinkey et al. 1989; Weber 1993; Wyss 1993; Cabassi,
Wnci et al. 2002; Zhu, Poole et al. 2005); (Karim and Hainsworth 1976;
Greenway 1983). It is generally found that sympathetic effects on the
cardiovascular system are elevated in hypertension (Nestel 1969; Egan, Panis et

al. 1987; Somers, Anderson et al. 1993; Esler and Kaye 1998).

Therefore, the splanchnic sympathetic nervous system was studied to test
if the hypertension in T9 (sllsl) rats is mediated via sympathetic hyperinnervation.
This was accomplished by studying structural changes of the nerves via
histological method, glyoxylic acid staining of catecholamine-containing nerves.
Staining of sympathetic nerves innervating mesenteric arteries and veins showed
that the nerve density was the same in T9 (+I+) and T9 (sllsl) animals,
suggesting that the hypertension in T9 (sllsl) rats was not due to a difference in
the density of sympathetic innervation. Sympathetic nerve density also was
quantiﬁed by measuring norepinephrine (NE) contents in the splanchnic organs
(kidneys, liver, spleen, and small intestine) and mesenteric vessels via high
performance liquid chromatography (HPLC). The data revealed that the
sympathetic nerve densities in the splanchnic organs and mesenteric vessels
were comparable between T9 (+I+) and T9 (sllsl) rats. Furthermore, the results
from functional studies of sympathetic nerves were consistent with the data from
nerve density experiments. That is, the nerve responses to electrical stimulation

were similar in arteries and veins from these two groups of rats.

121

Mean arterial pressure of male Tg (+I+) rats was higher than that of male
WKY rats. One piece of evidence in my study could be an explanation: the
norepinephrine concentration in mesenteric arteries from male Tg (+I+) was
signiﬁcantly higher than that from WKY rats. Higher regional catecholamine level
may be an indicator of a higher sympathetic activity in that vascular region. The
activation of the sympathetic nerves innervating resistance arteries may lead to a

direct vasoconstriction and blood pressure increase (Weber 1993; Wyss 1993).

Additionally, the splanchnic sympathetic ganglia in T9 (+I+) rats ETBRS
have 70 more copies of the ETBR gene than normal WKY rats (Gariepy, Williams
et al. 1998). More experiments, such as chronic S6c infusion into WKY rats and
calcium imaging in celiac ganglionic neurons with ETBR activation, need to be
done to elucidate the differences in sympathetic ganglion function between WKY
and T9 (+I+) rats. If sympathetic neurons from T9 (+I+) rats respond to 86c more
than those from WKY, this transgenic rat strain may represent an interesting
model to study the effects of ETBRS in the sympathetic ganglia without the need

for exogenous activation of the receptors with S6c.

Gender differences in blood pressure regulation have been reported in this
rat model (Sullivan, Pollock et al. 2006). The data in current study support these
ﬁndings. In addition, in human saphenous veins, the binding capacity for ETARS
in males is higher than that in females (Ergul, Shoemaker et al. 1998). This could
help explain why the MAPs are comparable in T9 (sllsl) between the two
genders, even with a higher ET—1 concentration in the circulation in the females.

Therefore, ETAR densities need to be measured in T9 (sllsl) rats to test if

122

different receptor densities make a contribution to the difference in resting blood
pressure between the two genders. In the future, more research should be
carried out to study gender differences in T9 (sllsl) rats, either through surgical
approaches (oophorectomy or orchidectomy) or drug treatment (estrogen or

androgen or hormone blockers).

Collectively, my data Show that there are no differences in the splanchnic
sympathetic innervation at structural and functional levels between WKY, Tg
(+I+), and T9 (sllsl) rats, indicating a successful genetic rescue in T9 (SVsl). My
results conﬁrm the ﬁndings of others that plasma ET-1 levels are higher in both
male and female Tg (sllsl) rats. The main reason for the hypertension in T9 (sllsl)
rats seem to be augmented ETAR activation because of their high circulating
endothelin-1 level. Still, the possibility of the splanchnic sympathetic nervous
system’s involvement in T9 (sllsl) hypertension can not be excluded, since no
publications have shown studies of neural regulation in hemodynamic regulation
of these transgenic rats by means of direct neural blockade. CGX could be
performed in T9 (sllsl) rats to test the hypothesis that the splanchnic sympathetic
innervation contributes to the hypertension. If the results support that hypothesis,
more studies need to be done to reveal the relationship between ETAR activation

and the sympathetic nervous system in the splanchnic bed.

123

 

 

 

 

 

 

 

 

A — WKY(n=4/2)
£1 1:1 Tg (+I+) (n=7/8)
_ — Tg(sllsl)(n=6/7)

E 150 # #
2 *
3 ...
ﬂ
2 100 -
a.
E
h
E
< 50 -
C
a
O
E

o _ 15-1—53.1

Male Female

Figure 20: Mean arterial pressure of male and female WKY, Tg (+I+) and T9
(sllsl) rats. *: signiﬁcant difference from WKY rats for each gender; #1 signiﬁcant
difference from T9 (+I+) rats for each gender; 1': signiﬁcant different from male Tg

(+I+) rats.

124

 

10
— WKY(n=6I6)

Tg (+r+) (n=5/10)
3 ‘ — Tg (sllsl) (12110)

#
*

 

 

 

 

 

 

 

E

\

D

e

m

c

.2 #

H

is

b 6 ' *

c

O

O

5

O 4 r

‘-

I

[—

I.I.I

iii 2 -

E

in

g 1-..”;
0

 

Male Female

Figure 21: Plasma ET-1 concentrations in male and female WKY, Tg (+I+) and
T9 (sllsl) rats. *2 signiﬁcant difference from WKY in each gender; #: Signiﬁcant
difference from T9 (+I+) rats in each gender; 1': signiﬁcant difference from male

Tg (sllsl) rats.

125

Artery Vein

sI/sl

s|/+

+/+

200 pm

 

Figure 22: Glyoxylic acid staining of mesenteric arteries and veins from T9
(sllsl), Tg (sll+), and T9 (+I+) rats. The blue ﬂuorescence represents the

presence of catecholamine. The scale bar applies to all pictures.

126

 

300

* — MWKY(n=6)
A 250 - MT9(+/+)(n=5)
5 —T—— — M T9 (sllsl) (n=10)
8
2 200 -
D.
2’
g 150-
.2
O .
O 100 - r.
-l ‘1

50 - ,: . Ii

0 _ .‘ I. ,

 

 

 

 

 

 

Artery Vein
Figure 23a: NE levels in mesenteric artery and vein from male WKY, Tg (+/+),
and T9 (sllsl) rats. The concentration of NE in artery from T9 (+I+) rats was

signiﬁcantly higher than that from WKY rats.

127

300

 

 

 

 

 

 

   

 

- F WKY (n=6)

25o _ T I: FTg (+I+)(n=6)
E - FTg (sllsl) (n=5)
O
2 200 -
a.
2'
3 150-
i
O
6 100 -
_I
E

50 -

o _ v. I..

Artery Vein
Figure 23b: NE levels in mesenteric artery and vein from male WKY, Tg (+I+),
and T9 (sllsl) rats. There was no signiﬁcant difference between any two groups

for either blood vessel type.

128

 

700

 

 

 

- FWKY(n=6)

600 - l':l FTg (sllsl) (n=6)

'3 - FTg (+I+) (n=6)
5’, 500 - . ~
.0 ..3
a 400 -
E
m _
3 300
> . .
3 % :1
m 200 - if;
z
100 _ | i 1
, _ I

Liver LK RK SI Spleen

Figure 24: NE levels in splanchnic organs from female WKY, Tg (sllsl), and T9
(+I+) rats. No Signiﬁcant difference was shown between any two groups for any

organ. LK: lift kidney; RK: right kidney; SI: small intestine.

129

w
o

 

 

—" T9 (+I+) (n=5)
—<>- Tg (sllsl) (n=5)
—'v- Tg (sll+) (n=5)

N
U‘I
a

 

 

 

N
o
l

 

—b
U'I
l

Constriction (%)

 

 

 

54
0.
‘5 l I
0.1 1 10 100

Frequency (Hz)

Figure 25a: Comparison of frequency—response curves for neurogenic
constrictions of arteries from T9 (+I+), Tg (sllsl), and T9 (sll+) rats. No signiﬁcant

difference in frequency-dependent response was shown.

130

8

 

 

—O— Tg (+I+) (n=5)
—O- Tg (sllsl) (n=5)
—v— Tg (sll+) (n=5)

  
  

N
01
I

 

 

_. N
or o
1 l

Constriction (96)
3

 

 

 

5.
o.
'5 l 1
0.1 1 10 100
Frequency (Hz)

Figure 25b: Comparison of frequency—response curves for neurogenic
constrictions of veins from T9 (+I+), Tg (sllsl), and T9 (sll+) rats. No signiﬁcant

difference in frequency-dependent response was shown.

131

CHAPTER 7

MECHANISMS OF HYPERTENSION INDUCED BY ENDOTHELIN
B RECEPTOR (ETBR) ACTIVATION: STUDIES IN ETBR-
DEFICIENT RATS

132

INTRODUCTION

We have recently established a novel hypertension model: S6c—induced
hypertension (Fink, Li et al. 2007). In this model, endothelin type B receptors
(ETBRs) are chronically activated by the infusion of sarafotoxin 6C (860), a
selective ETBR agonist, into conscious rats. Most physiological responses
mediated by ETBRS cause decreased blood pressure (Rubanyi and Polokoff
1994; Schiffrin 1998; Pollock, Allcock et al. 2000), e.g. vasodilators released from
endothelial cells, and enhanced excretion of sodium and water by the kidneys.
Therefore, the mechanisms underlying S6c-induced hypertension are unclear.
However, because S6c constricts veins from most vascular regions but has much
less effect on arteries (Strachan, Haynes et al. 1995; Strachan, Crockett et al.
2000; Johnson, Fink et al. 2002; Alexander, Mathie et al. 2007), we hypothesized
that one mechanism of SGc-induced hypertension could be direct

venoconstriction.

Additionally, sympathetic nerve activity is the most critical factor controlling

venoconstriction, especially in the splanchnic circulation (Shoukas and Bohlen
1990; Ozono, Bosnjak et al. 1991). Sympathetic activation of the splanchnic

veins can reduce blood volume up to 60%, redistributing blood from veins into

the heart and thereby increasing blood pressure (Karim and Hainsworth 1976;

Greenway 1983). Therefore, it is interesting that ETBRS are found on the

sympathetic neurons (T akimoto, Inui et al. 1993; Pomonis, Rogers et al. 2001),

and can affect their function (T akimoto, Inui et al. 1993(Yamada, Kushiku et al.

133

1999; lsaka, Kudo et al. 2007). One effect of ETBR activation in sympathetic

ganglia is increased levels of reactive oxygen species (ROS). For example,
superoxide levels are increased in sympathetic neurons from prevertebral

ganglia (innervating the splanchnic organs) when they are treated with $60 in
vitro (Dai, Galligan et al. 2004). Furthermore, acute intravenous infusion of 86c
into conscious rats increases superoxide levels in prevertebral sympathetic
ganglia (Lau, Galligan et al. 2006). Because ROS can function as signaling
molecules in neurons and thereby affect neuronal activity (Dai, Galligan et al.
2004); (Zimmerman and Davisson 2004; Sun, Sellers et al. 2005; lnfanger,
Shanna et al. 2006), 86c could cause hypertension by increasing post-ganglionic
sympathetic activity. In fact we recently produced evidence that SGc-induced
hypertension depends in part on ROS-driven sympathetic activation to the
splanchnic region (Li, Dai et al. 2007). Nevertheless, previous work has not

allowed a clear delineation of the overall importance of ETBRS on sympathetic

neurons to S6c-induced hypertension.

In humans, a mutation of the gene of ETBR causes Hischsprung’s
disease, characterized by aganglionic megacolon and pigment abnormalities
(Puffenberger, Hosoda et al. 1994). The spotting lethal (SI) rat is a naturally
occurring animal model of human Hischsprung’s disease (Gariepy, Cass et al.
1996; Gariepy, Williams et al. 1998). In this rat model, there is a 301-bp deletion
which spans junction of the exon 1-intron 1 junction in the ETBR gene. The

deletion leads to the result of an abnormal ETBR mRNA lacking the coding

134

sequence for the receptor’s transmembrane domain. No functional ETBRS are
detected in tissues from homozygous sllsl rats. The animals Show intestinal
aganglionosis and color spotting. They usually die at the age of one month due to
congenital intestinal aganglionosis. In order to rescue the (SI/SI) rats, the human
dopamine-ﬂ-hydroxylase (till-l) promoter was used to direct transgenic (T 9)
expression of ETBR to colonizing precursors of the enteric nervous system
(Gariepy, Williams et al. 1998). The WH- ETBR transgene compensates for
deﬁciency of endogenous ETBRS in these rats. Therefore the intestinal
obstruction is prevented. This transgenic rescue generates Tg (sllsl) rats with
ETBRS that are expressed and functional in adrenergic tissues, including the
locus ceruleus, adrenal medulla, and sympathetic ganglia. Unlike in normal
animals, ETBRS are not functional in endothelium, renal tubules, or vascular
smooth muscles in T9 (sllsl) rats. Therefore, this unique animal model can be
used to investigate the roles of ETBRS in a tissue-speciﬁc manner. In Tg (+I+)
rats, not only endogenous but extra copies of ETBR gene (by IIIH- ETBR
transgene) are present. In previous sections of my thesis, I characterized S6c-
induced hypertension, including the effects on oxidative stress in blood vessels
and sympathetic ganglia; showed the effects of CGX on splanchnic nerve density
and function; and described basal blood pressure, and splanchnic sympathetic
nerve density and function in T9 (sllsl) and T9 (+I+) rats. Here I tested the
hypothesis that if S6c-induced hypertension is due in part to the activation of

ETBRS on the sympathetic neurons.

135

METHODS
Animals
Male transgenic ETBR-deﬁcient rats (300 — 325 g) were used in the

experiments.

The following methods were involved in this study.
0 Celiac ganglionectomy (CGX);
o Radiotelemetry implantation;
o Minipump implantation;

o Hemodynamic measurements by telemetry.

Experimental protocols
860 only treatment

This protocol was used for intact rats (one week after transmitter
implantation) and celiac ganglionectomized rats (three weeks after transmitter
implantation). Baseline data were recorded for three days as a control period.
S6c solution was infused using micro-osmotic pumps (Alzet, Model 1007D). The
drug was delivered at the rate of 30 pmollkglmin, so. After seven days, the pump

was removed and an additional four days of recording was obtained.

86c and tempol treatment

When the initial 860 infusion study was completed in normal rats,
transmitters were turned off. After ten days transmitters were turned on again,
and three days of control period data were collected. Then all rats were give

tempol (4-hydroxy 2,2,6,6,-tetramethyl peperidine 1-oxyl, a superoxide dismutase

136

mimetic) in the drinking water at a concentration of 1 mmolll. Each water bottle
was wrapped with aluminum foil for light protection. Tempol-containing drinking
water was freshly prepared every day. After the rats received tempol for three
days, 860 was infused for ﬁve days, as described above. Tempol treatment was
then terminated while 360 infusion continued for two additional days. Finally, S6c
infusion was stopped by removing the pumps and rats were monitored for a four

day recovery period.

Statistical analyses

The effect of CGX on MAP during the control period was evaluated by
Student ttest compared to the average of baseline MAP in intact rats. Between-
group differences over time was assessed by a 2-way ANOVA and posthoc
multiple comparisons with Bonferroni’s test. A P<0.05 was considered signiﬁcant.

All of the results are presented as meaniSE.

137

RESULTS
Eﬁects of S60 infusion in T9 (+/+) and T9 (-/-) rats

As shown in Figure 26, mean arterial pressure (MAP) was signiﬁcantly
higher in T9 (sllsl) rats (124 :I: 2 mmHg) than in T9 (+I+) rats (114 :I: 1 mmHg)
during the control period. During the ﬁrst three days of S6c treatment, MAP
increased signiﬁcantly in T9 (+I+) rats (~30 mmHg). Subsequently MAP
decreased to a level not signiﬁcantly higher than during the control period. When
S6c infusion was terminated, however, MAP fell signiﬁcantly below control period
values for two days. In Tg (sVsl) rats, S6c did not signiﬁcantly affect MAP at any
time during the infusion period; termination of infusion also was without effect.
Baseline heart rates (HR) were not different in T9 (+I+) (311 :I: 4 bpm) and T9
(sllsl) (318 :I: 4 bpm) rats (Figure 27). In Tg (+I+) rats, HR declined signiﬁcantly
on the ﬁrst day of S6c infusion, then gradually returned to pie-infusion values.
When S6c infusion was terminated, HR was signiﬁcantly increased on the ﬁrst
two days of the recovery period. The infusion of S6c had no effect on HR in T9

(SI/SI) rats.

Effects of tempol treatment on S6c-induced hypertension

Tempol treatment alone did not change MAP (Figure 28) or HR (Figure
29) in either Tg (sllsl) or T9 (+I+) rats. S6c infusion during tempol treatment
increased MAP in T9 (+I+) rats but not Tg (sllsl) rats. Ceasing tempol treatment
during continued S6c infusion did not affect MAP in either group of the rats. MAP

decreased signiﬁcantly in T9 (+I+) but not Tg (sllsl) rats when 86c was

138

withdrawn. Tempol treatment had no effect on basal HR in either Tg (+I+) or T9

(sllsl) rats, nor did it modify responses to S6c infusion (Figure 29).

Effects of celiac ganglionectom y ( CGX) on S6c-induced hypertension

In Tg (+I+) rats, CGX caused a signiﬁcant decrease in basal MAP
compared to sham-operated controls (Figure 30a; intact rats are the same as
shown in Figure 26). In T9 (sllsl) rats CGX also decreased basal MAP, but the
difference was not statistically Signiﬁcant (Figure 30b; intact rats are the same as
shown in Figure 28). 86c infusion increased MAP signiﬁcantly for only two days
in CGX Tg (+I+) rats, then MAP returned to levels no different from those of the
control period. Unlike in intact Tg (+I+) rats, termination of S6c infusion did not
affect MAP in CGX Tg (+I+) rats. 86c infusion in CGX Tg (sllsl) rats produced no
change in MAP throughout the study. CGX had no effect on basal HR in either

group of rats, not did it modify HR responses to S6c infusion (Figure 31).

139

DISCUSSION

The transgenic (T g) spotting lethal (sl) rat is a unique animal model to
study the roles of ETBRS in blood pressure regulation. Although heterozygous
ETBR-deﬁcient mice (Ohuchi, Kuwaki et al. 1999) and T9 (sllsl) mice (Kapur,
Sweetser et al. 1995) are available, their small size makes physiological studies
difﬁcult. On the other hand, in T9 (sllsl) rats (Gariepy, Williams et al. 1998)
numerous aspects of cardiovascular system function have already been
characterized (Gariepy, Ohuchi et al. 2000; Sullivan, Pollock et al. 2006). For our
purposes, a key attribute of these rats is their failure to express functional ETBRS
in endothelial cells, renal tubular cells, and vascular smooth muscle cells. But
because the “rescue” transgene is linked to a dopamine-B-hydroxylase promoter,
robust ETBR expression occurs in sympathetic neurons and other cells
expressing high levels of this gene. Thus they offer an ideal opportunity to isolate
cardiovascular effects of ETBR activation mediated by the sympathetic nervous
system. It is already known that Tg (sllsl) rats exhibit a pressor response to acute
activation of ETBRS with high doses of $60 (Pollock, Portik-Dobos et al. 2000).
The goal of the current study was to determine if chronic infusion of lower doses
of S6c would increase MAP in T9 (sllsl) rats, as a way of clarifying the role of the

sympathetic nervous system in this new model of hypertension.

The baseline MAP of T9 (sllsl) rats was ~ 10 mmHg higher than that of T9
(+I+) rats. This ﬁnding is consistent with the results of other studies (Gariepy,
Ohuchi et al. 2000; Sullivan, Pollock et al. 2006).There are several possible

reasons for the higher blood pressure in T9 (sllsl) rats. First, ETBRS function as

140

endogenous ET-1 “clearance receptors”, i.e. circulating ET-1 is removed from
blood by ETBRS located on endothelial cells. Tg (sllsl) rats lack this function, so
have higher circulating levels of ET-1 (Sullivan, Pollock et al. 2006). This could
cause elevated blood pressure through activation of ETA receptors (Elmarakby,
Dabbs Loomis et al. 2004). Second, hypertension in T9 (sllsl) rats is salt-
sensitive (Gariepy, Ohuchi et al. 2000; Elmarakby, Dabbs Loomis et al. 2004;
Ohkita, Wang et al. 2005) and is attenuated by diuretic therapy (Gariepy, Ohuchi
et al. 2000). Furthermore, selective knockout of ETBRS in the collecting duct of
the kidney results in hypertension (Ge, Bagnall et al. 2006). Thus, hypertension
in T9 (sllsl) rats may be due to renal sodium and water retention. Third, there is
limited evidence that increased sympathetic activity can contribute to
hypertension in T9 (sllsl) rats, at least during high salt intake (Ohkita, Wang et al.
2005). We did not investigate this question directly in the current study. But
increased sympathetic input to the splanchnic region via the celiac ganglion is
not critical for hypertension in T9 (sllsl) rats, since CGX reduced MAP to
approximately the same degree in T9 (+I+) and T9 (sllsl) rats. Finally, tempol
treatment also had no effect on blood pressure in hypertensive Tg (sllsl) rats.
This was somewhat surprising, since hypertension in T9 (sVsl) rats may be driven
by increased circulating ET-1 (Elmarakby, Dabbs Loomis et al. 2004), and
endothelin-dependent hypertension in one study was attenuated by tempol
treatment (Sedeek, Llinas et al. 2003). However, other work suggests that
oxidative stress has only a limited role in endothelin-dependent hypertension

(Elmarakby, Loomis et al. 2005; Sullivan, Pollock et al. 2006).

141

Early studies on cardiovascular responses to acute S6c infusion revealed
sympathetically mediated constriction of the mesenteric circulation (Clozel, Gray
et al. 1992). We have found evidence that sympathetic activation—especially in
the splanchnic region— secondary to increased ganglionic superoxide levels
contributes to chronic S6c-induced hypertension in Sprague-Dawley rats (Li, Dai
et al. 2007). Thus, we hypothesized that 860 would induce hypertension in T9
(sllsl) rats by activating ETBRS and increasing ROS levels in the sympathetic
ganglia. Two results from the current study suggest that this mechanism is not
operative in T9 (+I+) or T9 (sllsl) rats. First, contrary to what was observed in our
previous work in normal Sprague-Dawley rats (Li, Dai et al. 2007), tempol did not
attenuate S6c—induced hypertension in either T9 (+I+) or T9 (sllsl) rats. There are
at least two possible technical reasons that could explain this difference. One
reason may be the strain difference. The transgenic rats were derived from the
Wistar Kyoto (\NKY) strain, while all of our previous work was in Sprague-Dawley
animals. Another difference is that, in the current study, 860 was given by
subcutaneous infusion at a dose (30 pmollkglmin) calculated to produce similar
blood pressure changes to those we observed previously using intravenous
infusion (5 pmollkglmin). Thus it is possible that the strength of activation of
ETBRS differed in the two protocols (even though the increases in arterial
pressure were similar). Another possibility is that overexpression of the ETBR
transgene in the sympathetic neurons alters their ability to respond to $60 with

increased generation of ROS. Finally, plasma clearance ofET-1 is impaired in T9

142

(sVsl) rats, so it is possible that ganglionic ETBRS are downregulated by high

circulating concentrations of ET-1 in these rats.

A second piece of evidence arguing against an important effect of S6c on
the sympathetic activity is that although MAP increased signiﬁcantly during
chronic S6c infusion in T9 (+I+) rats, no change in MAP was observed in T9
(sllsl) rats. The obvious interpretation of this result is that in normal rats 86c must
be able to activate ETBRS in non-adrenergic tissue to cause chronic
hypertension. As discussed earlier, a possible site for this hypertensive effect of
S6c is vascular smooth muscle in veins. Stimulation of ETBRS in other most
tissues (endothelial cells, renal epithelial cells) should lower blood pressure.
However, another possible target is renal arterioles, where ETBR stimulation
produces direct vasoconstriction and reduced renal blood ﬂow (Clozel, Gray et al.
1992); (Gellai, DeWolf et al. 1994; Pollock, Jenkins et al. 2005). Nevertheless, it
also is possible that ETBRS on sympathetic neurons are simply non-functional in
T9 (+I+) and T9 (sllsl) rats, as discussed above. Data from experiments exploring
the effects of CGX on SGC-induced hypertension suggest that this may be the

case.

Our results demonstrated that when Tg (sllsl) rats were treated with S6c,
there was no change in blood pressure. So it seems that the sympathetic
activation is not involved in T9 (sllsl) rats upon 86c infusion, which was to our
surprise and quite contrary to our previous ﬁndings. Some unpublished results
(not shown here) from the Kreulen laboratory indicate that ETBRS are actually

not functional in the splanchnic sympathetic neurons of adult Tg (sllsl) rats. As

143

we noted earlier, circulating ET-1 level is high in T9 (sllsl) rats, so it is possible
that ETBRS are somehow downregulated by a high ET-1 concentration in the
circulation. Since ETBRS must be functional during the development of enteric
nervous system in T9 (sllsl) rats, it is possible that ETBRS may have a greater

role in SGC-induced hypertension in younger rats. This idea remains to be tested.

In additional experiments, we removed celiac ganglia (CGX) to further test
if the splanchnic sympathetic innervation is critical for S6c-induced hypertension.
The increase in blood pressure during S6c infusion was less in CGX T9 (+I+) rats
than that in intact Tg (+I+) rats, suggesting that the splanchnic sympathetic
nervous system makes a contribution to the hypertension. Interestingly, we also
found that MAP increase during the second half infusion period was less than
that in the ﬁrst half. Our previous data show that direct venoconstriction mainly
mediates SSc-induced hypertension in the ﬁrst 2-3 days, then neuronal regulation
gradually becomes more important. In support of this idea, current study
demonstrated that CGX attenuated S6c—induced hypertension in T9 (+I+) rats,
most markedly, in the later stage of the infusion period. Collectively, our previous
and present results are very consistent in showing that the splanchnic
sympathetic nerves are partially involved in S6c-induced hypertension, especially

during the maintenance phase.

Perspectives
Most studies on ETBRS have been focused on the receptor’s beneﬁcial
effects via the mechanisms of vasodilation. Our lab is one of the few groups that

have carried out in vivo experiments with a focus on the vasoconstrictor effects

144

induced by ETBR activation. Our results indicate that S6-induced hypertension is
indeed mediated through ETBR activation, since S6c does not change MAP in
T9 (sllsl) rats. Furthermore, the development and the maintenance of S6c-
induced hypertension are attenuated when the majority of the splanchnic
sympathetic innervation is removed. Accumulating evidence indicates that the
sympathetic nervous system (SNS) may be an important mediator in
experimental models and human hypertension. My data not only support the
above ﬁnding but suggest that the SNS in the splanchnic bed, at least partially
via ETBR activation, is a critical neural component in blood pressure regulation.
Therefore, ETBRS in the splanchnic SNS could be a therapeutic target for the

treatment of hypertension.

145

 

 

 

 

 

 

 

 

 

 

 

 

220
A Control $60 (30 pmollminlkg, sc) Recovery
P 200 -
I
E
j; 180 - —.-— MTg (+I+)(n=7)
'5 —O— MTg (sllsl)(n=6)
3 160 - * *
g *
a 140 '
'5
‘I'.’ -
< 120
5
g 100 -
80 - - . . . .
0 2 4 6 8 1O 12 14

Time (days)
Figure 26: Mean arterial pressure (MAP) before (day 1-3), during (day 4-10),

after (day 11-14) continuous 86c infusion (30 pmollkglmin, sc.). MAP was
signiﬁcantly higher in T9 (sllsl) rats than in T9 (+I+) rats during the control period
In T9 (+/) rats. MAP signiﬁcantly increased upon S6c infusion in the ﬁrst three
days. It decreased signiﬁcantly on the ﬁrst two days of the recovery period upon
86c removal. In Tg (sllsl) rats, MAP did not change during the infusion or
recovery period, compared to the control period. Asterisks indicate a statistical

difference (p<0.05) from the last day of the control period.

146

 

500

 

 

   

 

 

 

 

 

 

 

 

 

Control 86c (30 pmollminlkg, sc) Recovery
450
.‘E‘
E 400 . + MT91+I+IIH=7I
g —0— MTg(sIlsl)(n=6)
3
‘5' 350 -
E
‘I'.’ 300 -
O
O
:I:
250 -
200 . . . , 97
0 2 4 6 8 10 12 14
Time (days)

Figure 27: Heart rate (HR) before (day 1-3), during (day 4-10), after (day 11-14)
continuous 86c infusion (30 pmollkglmin, sc.). In Tg (+l) rats, HR signiﬁcantly
decreased on the ﬁrst day of S6c infusion. It increased signiﬁcantly on the ﬁrst
two days of recovery period upon 86c removal. In Tg (sllsl) rats, HR did not
change during the infusion or recovery period, compared to the control period.
Asterisks indicate a statistical difference (p<0.05) from the last day of the control

period.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

220
E 200 -
E —O— M T9 (+I+) (n=6)
75 180 ~ —0— MTg (sllsl) (n=6)
5
m . # #
§ 160 f * * #
O. *
- 14o— - m
.2 4 O O . x
'5 WW 0 ‘0‘» e e e e
E 1204 -
5
g 100 -
80 r . . , , g r
o 2 4 6 8 1o 12 14 16
Time (days)

Figure 28: Mean arterial pressure (MAP) during the control period (day 1-3),
tempol treatment period (day 4-6), tempol treatment with 86c infusion (30
pmollkglmin, sc.) period (day 7-11), 86c infusion period (day 12-13), and
recovery period (day 14-17). In Tg (+l) rats, MAP did not change with tempol
treatment alone. It signiﬁcantly increased during the whole period of 86c infusion
while the rats were drinking tempol solution. It decreased signiﬁcantly in the
recovery period. In Tg (sllsl) rats, MAP did not change during the infusion or
recovery period, compared to the control period. Asterisks stand for a statistical
difference (p<0.05) from the last day of the control period. Pound signs represent
signiﬁcant difference from the last day of tempol treatment period. Cross signs

indicate signiﬁcant difference from the last day of tempol treatment.

148

 

500
Control

 

450 -

400 -

350 -

300 1

250 ~

Heart Rate (beats/min)

200 .

 

150 .

 

$60

Recovery

 

 

 

—.— M Tg +I+ (n=6)
-0— M Tg sllsl (n=6)

 

 

 

 

 

 

 

 

 

 

 

4 6 8 10
Time (days)

r

12

V I

14 16

Figure 29: Heart rate (HR) during the control period (day 1-3), tempol treatment

period (day 4-6), tempol treatment with 86c infusion (30 pmollkglmin, so.) period

(day 7-11), 86c infusion period (day 12-13), and recovery period (day 14-17). In

Tg (+I+) rats, HR signiﬁcantly dropped on the ﬁrst day of 86c infusion (day 7). In

Tg (sllsl) rats, HR did not change during the infusion or recovery period,

compared to the control period. Asterisk stands for a statistical difference

(p<0.05) from the last day of control period. Pound sign represents a signiﬁcant

difference from the last day of the tempol treatment period.

149

 

 

 

 

 
 

 

 

  

 

 

 

 

 

180
A Control 86c (30 pmollminlkg, sc) Recovery
U)
:I:
E 160 -
g * *
g -.— Intact Tg (+I+) (n=7)
3 140 * —O— cex Tg(+I+)(n=6)
2
n.
g 120 -
g
<
g 100 - S
0
E
80 r l I I I I
0 2 4 6 8 10 12 14

Time (days)
Figure 30a: Mean arterial pressure (MAP) before (day 1-3), during (day 4-10),
after (day 11-14) continuous 86c infusion (30 pmollkglmin, sc.) in T9 (+I+) rats.
CGX signiﬁcantly lowered resting blood pressure. in CGX Tg (+I+) rats, MAP
signiﬁcantly increased upon $60 infusion on the ﬁrst two days, compared to the
control period. Asterisks indicate statistical difference (p<0.05) from the last day
of the control period. Pound signs indicate statistical difference from intact Tg

(+I+) rats on the same day.

150

 

150
Control 36c (30 pmollminlkg, sc) Recovery

 

 

140 - —.— Intact Tg (sllsl)(n=7)
—O— cex Tg (sllsl)(n=6) ..

 

 

 

 

Mean Arterial Pressure (mmHg)

 

 

 

 

 

130 -
120 -
110
0 2 4 6 8 10 12 14
Time (days)

Figure 30b: Mean arterial pressure (MAP) before (day 1-3), during (day 4-10),
after (day 11-14) continuous 86c infusion (30 pmollkglmin, so.) in T9 (sllsl) rats.
CGX did not change resting blood pressure of T9 (sllsl) rats. There was no
signiﬁcant difference in MAP between T9 (+I+) and T9 (sllsl) rats during the

infusion and recovery periods.

151

 

600

 

 

 

 

 

 

 

 

 

Control 86c (30 pmollminlkg, sc)) Recovery
E 500 ' —O— M lntactTg (+I+) (n=7)
Tn —O— M Intact Tg(sllsl) (n=6)
g —V— M ccx Tg (+I+) (n=6)
% 400 _ —A— mccx Tg (sllsl)(n=7)
a
n:
1:
8
I 300 -
200 . . . . .
0 2 4 6 8 10 12 14
Day

Figure 31: Heart rate (HR) before (day 1-3), during (day 4-10), after (day 11-14)
continuous S6c infusion (30 pmollkglmin, so.) in intact and CGX Tg rats (+/+ and
sllsl). HR signiﬁcantly decreased upon S6c infusion on the ﬁrst day in intact and
CGX Tg (+I+) rats, compared to the control period . In Tg (sllsl) rats, HR did not
change during the infusion or recovery period. Asterisk indicate a statistical

difference (p<0.05) from the last day of the control period.

152

CHAPTER 8

SENSORY INNERVATION IN SGC-INDUCED HYPERTENSION

153

INTRODUCTION

There is good evidence suggesting that the sensory nervous system plays
a role in blood pressure regulation. As discussed in the GENERAL
INTRODUCTION, calcitonin gene-related peptide (CGRP) is a very potent
vasodilating neuropeptides. CGRP is primarily synthesized in dorsal root ganglia,
where sensory neuron bodies are located. These sensory neurons terminate on
many cardiovascular tissues, including blood vessels (Li and Wang 2003). CGRP
has been reported to innervate almost all vascular beds (Dipette and
VWmalawansa 1995; Wimalawansa 1996). Also, CGRP receptors are present
throughout the arterial system (Wimalawansa and Maclntyre 1988). CGRP
affects blood pressure regulation mainly by increasing water and sodium
excretion secondary to elevated renal blood ﬂow and glomerular ﬁltration rate
(Shekhar, Anand et al. 1991). Activation of CGRP receptors leads to reduced
vascular resistance in the kidney (Howden, Logue et al. 1988; Jager, Muench et
al. 1990) and relaxation of afferent arterioles in the glomeruli (Sanke, Hanabusa
et al. 1991). CGRP is released from perivascular sensory nerve terminals that
are capsaicin-sensitive (Holzer and Maggi 1998). A sensory-motor denervation
model is available in rats (Holzer-Petsche and Lembeck 1984). In this model, the
neonatal rats are treated with capsaicin and capsaicin-sensitive sensory nerves

are impaired right after birth. Thereby the release of CGRP is inhibited.

It has been shown that ET-1 induces CGRP release by acting on ETA
receptors (ETARS) (Wang and Wang 2004). In SGc-induced hypertension, since

ETBRs are mainly occupied by $60, the “clearance receptor’ function of ETBRS

154

to remove endogenous ET-1 from the circulation may be less effective.
Therefore, plasma ET-1 level may be increased and thereby causes the release
of CGRP through activation of ETARs. Neonatal capsaicin-treated rats were

used to explore the role of sensory nerves and CGRP in SSc—induced

hypertension.

155

METHODS
Animals

Male neonatal capsaicin-treated rats were used in this study.

The following methods were used in current chapter.
0 Hemodynamic measurements by telemetry;

o Minipump implantation.

Experimental protocol
SBc-infusion

Baseline data were recorded for three days as a control period. Then
animals were weighed for preparation of $60 solution. 360 solution was made
and transferred into micro-osmotic pumps (Alzet, Model 10070). A small opening
was made between the scapulae and a pocket was created for the subcutaneous
implantation of the mini-pump with the delivering site toward the lower body. The
drug was delivered at the rate of 30 pmollkglmin. After an infusion for seven
days, the pumps were taken out, followed by additional recording for four days,

as a recovery period.

Statistical Analyses
Due to the small sample size (n=2) of this study, the statistical analyses

were not performed.

RESULTS
Resting mean arterial pressure (MAP) in vehicle-treated rats was lower

than that in capsaicin-treated rats (Figure 32). With 86c treatment, MAP

156

increased in both groups, then it gradually dropped back to baseline level. Upon
the removal of S6c, MAP did not change compared to that of the last infusion

day.

Figure 33 shows that the resting heart rate (HR) in vehicle-treated rats
was higher than that in capsaicin-treated rats. HR decreased upon $60 infusion
and gradually increased. Statistical analyses were not performed on these data

because of the small number of animals studied.

157

DISCUSSION

The difference in resting pressure between the two groups of rats
suggests a successful capsaicin treatment, since it has been shown that sensory
nerves counterbalance the prohypertensive effects of several neuro-hon'nonal
systems to maintain normal blood pressure (Vaishnava and Wang 2003). In
capsaicin-treated rats, capsaicin-sensitive sensory nerves degenerate right after
birth. The release of some neuropeptides, such as CGRP, from sensory nerves,
is attenuated in capsaicin-treated rats, due to the neural degeneration. These
peptides are potent vasodilators as well as diuretic and natriuretic factors. As a

result of loss of these functions in capsaicin-treated rats, hypertension occurs.

The data suggest that sensory nerves do not play a major role in $60-
induced hypertension, since the increase in MAP during 86c infusion was the

same in vehicle- and capsaicin-treated rats.

HR results showed similar baroreceptor reﬂex mediated blood pressure
change in both animals. 80 the baroreﬂex was not impaired by neonatal

capsaicin treatment.

158

 

 

 

 

 
  

 

  

 

 

 

 

 

160
A Control 86c (30 pmollminlkg, sc) Recovery
12'
E, 140 -
g —Q— Vehicle-treated (me)
a -O— Cap-treated (n=2)
3 120 -
6'.
E
g 100 4
<
c
3
E 80 -

 

0 2 4 6 8 10 12 14
Time (days)
Figure 32: Mean arterial pressure (MAP) before (day 1-3), during (day 4-10),
after (day 11-14) continuous S6c infusion (30 pmong/min, sc.) in neonatal
capsaicin- and vehicle-treated rats. The average resting blood pressure of
capsaicin-treated animals was higher than that of vehicle-treated ones. $60

elevated MAP in a similar way in the two groups.

159

 

 

 

 

 

 

 

 

 

 

 

 

440 - Control 86¢ (30 pmollminlkg, sc) Recovery
A 420 -
.E
E
7, 400 -
iii
:3, 380 -
2
g 360 -
E
g 34° ‘ -.— Vehicle-treated (n=2)
-O— Cap-treated (n=2)
320 -
300 I . . . . .
0 2 4 6 8 10 12 14

Time (days)
Figure 33: Heart rate (HR) before (day 1-3), during (day 4-10), after (day 11-14)
continuous 36c infusion (30 pmollkglmin, sc.) in neonatal capsaicin- or vehicle-
treated rats. The average resting HR of capsaicin-treated animals was lower
than that of vehicle-treated ones. S6c reduced HR to a similar degree in the two

groups.

CHAPTER 9

GENERAL CONCLUSIONS AND DISCUSSION

161

A major focus of our laboratory is to test the hypothesis that veins mainly
located in the Splanchnic bed play an important role in blood pressure regulation.
In support of this idea our lab recently developed a novel experimental
hypertension model: SGC-induced hypertension (Fink, Li et al. 2007). In this
model, sarafotoxin 6C (S6c) is infused into conscious rats, and induces a
sustained hypertension by activating ETB subtype endothelin receptors. Previous
data from our lab and others indicate that SGC-induced hypertension could be
mediated by direct venoconstriction, but that activation of the sympathetic nervous
system (especially to the Splanchnic region) also may play a role (as reviewed
earlier). The main goal of my thesis project was to investigate neural mechanisms
underlying SGC-induced hypertension, using both in vivo and in vitro approaches.
The major conclusions from my research, and the results supporting them, are

summarized below.

Specific Aim I

The sympathetic nervous system is important in blood pressure regulation,
and regionally-Speciﬁc increases in the sympathetic nerve activity occur during
the development of hypertension. Therefore, the sympathetic activation in key
tissues may be an important cause of hypertension. Sympathetic neurons
innervating the splanchnic organs may be especially critical, because the
splanchnic vascular resistance is increased early in hypertension development.
In addition, sympathetically mediated decreases in the Splanchnic vascular

capacitance may account for the central redistribution Of blood volume that

162

 

occurs early in hypertension. The celiac ganglionic plexus contains the majority
of the sympathetic neurons that innervate the splanchnic organs and tissues.
Celiac ganglionectomy (CGX) involves surgical removal of the celiac ganglionic
plexus, and has been used by ourselves and others to study the roles of the
splanchnic sympathetic innervation in cardiovascular regulation. In the current
study I characterized the short-term (two-week) and long-term (ﬁve-week and
ten-week) effects of CGX in rats on the splanchnic sympathetic nerve structure
and function. In the Short-term, norepinephrine concentrations in whole
splanchnic organs and mesenteric arteries and veins were Signiﬁcantly
decreased by CGX. lmmunohistochemistry and glyoxylic acid staining Showed an
almost complete loss of the typical sympathetic innervation of mesenteric arteries
and veins. Constrictor responses of mesenteric arteries and veins to sympathetic
nerve stimulation were abolished by CGX. However, the effects of CGX were
time-dependent, Since Signiﬁcant regeneration of sympathetic nerves was
Observed ﬁve weeks after surgery. The inferior mesenteric ganglion had minimal
impact on this reinnervation process. Additionally, in vivo studies demonstrated
that the splanchnic sympathetic innervation is important in normal blood pressure
regulation, based on the ﬁnding that CGX Signiﬁcantly lowers resting blood
pressure in normal Sprague Dawley (SD) rats. TO summarize, CGX is an
effective means to impair sympathetic input to the splanchnic organs, but the

effect of the procedure is not permanent.

Speciﬁc Aim II

163

This study was performed to determine if SGC-induced hypertension is
caused by increased generation Of reactive oxygen species (ROS) and/or
activation of the sympathetic nervous system. The model employed was
continuous 5-day infusion of 86c into male Sprague-Dawley rats. No Changes in
superoxide anion levels in arteries or veins were found in hypertensive 36C-
treated rats. However, the superoxide levels were increased in sympathetic
ganglia from $6c-treated rats. In addition, superoxide levels in ganglia increased
progressively the longer the animals received 86c. Treatment with the anti-
oxidant tempol attenuated SGC-induced hypertension and decreased superoxide
levels in ganglia. Acute ganglionic blockade lowered blood pressure more in 86c-
treated rats than in vehicle-treated rats. Although plasma norepinephrine (NE)
levels were not increased in 86C hypertension, surgical ablation Of the celiac
ganglionic plexus, which provides most of the sympathetic innervation to the
splanchnic organs, signiﬁcantly attenuated hypertension development.
Collectively, the data suggest that a regional, as opposed to global, increase in
sympathetic nerve activity is involved in $6c-induced hypertension. SGc-induced
hypertension iS partially mediated by sympathoexcitation to the splanchnic

organs driven by increased oxidative stress in prevertebral sympathetic ganglia.

Speciﬁc Aim Ill
ETBR-deﬁcient transgenic (T g) spotting lethal (SI/SI) rats Show elevated
blood pressure, compared to T9 (+I+) control rats. The goal of this speciﬁc aim

was to characterize this rat model, with a focus on sympathetic innervation. An

164

increased plasma ET-1 level was found in T9 (sl/Sl) rats, suggesting a possible
pathway for blood pressure elevation through ETAR activation by ET-1 binding.
Sympathetic nerve density was evaluated by glyoxylic acid staining and no
difference was demonstrated between T9 (sllsl) and T9 (+I+) rats. NE
concentrations in mesenteric arteries and veins were not signiﬁcantly different
between the two groups of rats in both genders, except that NE level in Tg (SI/SI)
animals was higher than that in T9 (SI/SI) controls in mesenteric artery from male
rats. Similarly, no difference in NE concentrations was Shown in the splanchnic
organs. Finally, an electrical stimulation study was carried out as a functional test
of sympathetic inﬂuences on the splanchnic vascular bed. The vasoconstrictor
response to the sympathetic stimulation was comparable in Tg (sllsl) and T9
(+I+) rats. All the data suggest that hypertension in T9 (SI/SI) rats is not likely due
to differential sympathetic innervation to the splanchnic blood vessels. Therefore,
the hypertension is more likely caused by increased ET-1 concentration in the
circulation. Data from another group support this ﬁnding, since hypertension in
T9 (sl/SI) fed with high salt diet was completely abolished by ETAR blocker

(D'Angelo, Pollock et al. 2005).

Specific Aim IV

Since 86C constricts most veins, and other relatively selective
venoconstrictors can increase arterial pressure, we proposed that
venoconstriction is one mechanism Of S6c-induced hypertension. It has also

been found, however, that 86c infusion may increase sympathetic activity to the

165

splanchnic region by increasing oxidative stress in prevertebral ganglia. This
speciﬁc aim was to determine if SGc-induced hypertension is caused primarily by
direct constriction Of veins and/or activation of the sympathetic nervous system.
The model employed was a continuous 7-day infusion of 86C into male ETBR-
deﬁcient transgenic (T g) spotting lethal (sllsl) and control Tg (+I+) rats. I used
the Tg (SI/SI) rat because it does not express functional ETBRS in most tissues,
except for sympathetic neurons (and other tissues with high dopamine-8-
hydroxylase activity). S60 induced a Signiﬁcant blood pressure increase in T9
(+I+) rats, while BP was not changed in T9 (SI/SI) rats, suggesting that $60-
induced hypertension is mediated in part by direct venoconstriction. Treatment
with an antioxidant tempol did not affect the response to 86c in either strain. The
splanchnic sympathetic denervation by chronic celiac ganglionectomy
Signiﬁcantly attenuated SSC—induced hypertension in T9 (+I+) but not Tg (SI/SI)
rats. I conclude that 1) ETBRS on the sympathetic neurons of T9 (sllsl) and T9
(+I+) rats may function differently than those of wild-type rats, and 2) $60-
induced hypertension iS mediated by both direct venoconstriction and the

splanchnic sympathetic activation.

Speciﬁc Aim V

My limited preliminary data suggest that sensory innervation does not play
a major role in SSC-induced hypertension, based on the ﬁnding that there is no
difference in blood pressure responses to S6c infusion between capsaicin-

treated and vehicle-treated rats. Also, baroreceptor reﬂex is not impaired in

166

neonatal capsaicin-treated rats, since heart rate Signiﬁcantly decreased in

response to blood pressure increases in capsaicin-treated rats.

The above ﬁndings are summarized in Figure 34.

   

/

1 Ganglion 02'

@ TSNA

Celiac Ganglionic Neuron

. Artery T Ganglion O,-

T SNA

Celiac Ganglionic Neuron

 

 

|_Venous Constriction l lArterialConstriction]

 

 

V

l
[ Hypertension I

 

 

 

 

Figure 34: Summary Of the main ﬁndings of this research project.

167

GENERAL CONCLUSIONS

Taken together, my results provide evidence that supports the following

conclusions.

E TB receptor activation-induced hypertension is an important model of
hypertension.

SGC-induced hypertension is a unique hypertension model, because: a.
86C is a selective venoconstrictor and sec-induced activation of ETBRS leads to
hypertension. AS I mentioned earlier, hypertension has been viewed as an
“arterial” disease and most research has been on arteries while veins have
received much less attention. This new hypertension model shows that veins
could play an important role in blood pressure regulation; b. ETBRS are located
on sympathetic neurons, as well as vascular smooth cells. Stimulation of ETBRS
induces sympathetic activation. My studies, along with ﬁndings from other
groups, indicate that increased sympathetic nerve activity is involved in 86c-
induced hypertension. Therefore, $6c-induced hypertension is a model that

involves both vascular and neuronal mechanisms.

Increased splanchnic sympathetic nerve activity is a cause of
hypertension.

My studies support the idea that increased Splanchnic sympathetic nerve
activity (SNA) is a cause of hypertension. When celiac ganglia, a plexus
containing most neurons innervating the Splanchnic organs, is surgically removed

(CGX), $6c-induced hypertension is attenuated, indicating that Splanchnic nerve

168

activity partially mediates the hypertension. In addition, superoxide levels in
prevertebral sympathetic ganglia are higher in SBC-treated rats than in vehicle-
treated rats. So my results are consistent with other groups’ data, since many
studies have Shown that elevated oxidative stress cause increased SNA
(Campese, Ye et al. 2004). ROS have been shown to be involved in the
development of hypertension by increasing peripheral and central SNA. Possible
mechanisms are either nitric oxide (NO)-dependent where decreased
productions/availability Of NO leads to a reduction in NO-mediated
sympathoinhibitory effects (Campese, Ye et al. 2004; Zucker 2006; Zhang, Yu et
al. 2008), or NO-independent where tempol directly regulate calcium channel
and/or potassium Channel activities (Xu, Jackson et al. 2006; Zucker 2006; Chen,

Patel et al. 2007).

ROS, however, may exert their cardiovascular effects through a nonneural
pathway. For example, ROS induce oxidative damages in DNA, lipids, and
proteins in VSMCS and vascular endothelial cells, possibly resulting in vascular
dysfunction and endothelial dysfunction (Ballinger, Patterson et al. 2000).
Therefore, tempol does not lower blood pressure exclusively by decreasing SNA.

An approach to clarifying this issue is proposed in FUTURE STUDIES.

169

FUTURE STUDIES

It remains controversial if intravenously administered 86c exerts its effects
partially by binding to central ETBRS. On one hand, it is unlikely that blood-bome
$60 passes the blood brain barrier (BBB), Since its structural analogue ET-1
does not cross the capillary-endothelial junction due to the electrical polarity.
When mice were given a radioactive tracer with an unselective ETAR/ETBR
antagonist for in vivo ET receptor labeling, the radioactivity was very low in the
brain, indicating that the antagonist does not cross the BBB (Aleksic, Szabo et al.
2001). On the other hand, it has been Shown that ET-1 regulates P-glycoprotein
(a critical component of the BBB protecting the CNS from neurotoxic agents)
function by binding to ETBRS, but not ETARS (Hartz, Bauer et al. 2004). In
addition, Na/K/Cl cotransporter at rat brain capillary endothelial cells could be
activated by ET-1 and ET—3, suggesting a possible role of ETS in the BBB (Vigne,
Lopez Farre et al. 1994). Furthermore, circumventricular organs (CVOS) with an
incomplete BBB in the brain directly sense the concentrations of various
compounds, especially peptide hormones, in the bloodstream. These organs
could be possible action Sites of 86c to regulate blood pressure. Previous
results in our lab and others support this idea by the evidence of ETBR
presence in several key CVOS (Yamamoto, Suzuki et al. 1997). Additional
studies Should be carried out to elucidate the involvement of central ETBRS in
S6c-induced hypertension. For example, radio-labeled 860 could be
intravenously infused and radioactivity in the brain, especially in CVOS, could be

measured to determine if 860 binds to ETBRS in the CNS. If it is true, ETBR

170

antagonists could be administered to Speciﬁc CVOS in S6c-induced
hypertension, followed by hemodynamic studies, to verify if 86c regulates the

cardiovascular system at least partially by activating ETBRS in the brain.

In sympathetic ganglia from T9 (+I+) rats, there are 70 more copies of the
ETBR gene than normal WKY rats, the natural wildtype. My data demonstrate
that the resting blood pressure of WKY rats is Signiﬁcantly lower than that of Tg
(+I+) rats. More experiments, such as chronic 86c infusion into WKY rats and
calcium imaging in celiac ganglionic neurons with ETBR activation, need to be
done to elucidate the differences in the function of sympathetic ganglia between
WKY and T9 (+I+) rats. If sympathetic neurons from T9 (+I+) rats respond to 86c
more than those from WKY, this transgenic rat strain may represent an
interesting model to study the effects of ETBRS in sympathetic ganglia without

the need for exogenous activation of the receptors with $60.

In my thesis project, SGC-induced hypertension model was performed in
two ways. The model initially involved giving Sprague-Dawley (SD) rats 86C at
the rate of 5 pmollmin/kg intravenously and hemodynamic parameters were
measured by external blood pressure analyzer. In the second stage of the
project, the model was performed using osmotic minipumps delivering S6c (at
the rate of 30 pmollminlkg, so.) and a continuous hemodynamic measurement by
radiotelemetry. The animals in these experiments were mainly ETBR transgenic
and WKY rats. So more work should be done to calibrate the $6c-induced
hypertension model, induced by subcutaneous 86c administration and monitored

by telemetry. For instance, SD rats could be used to conﬁrm that Similar results

171

from WKY rats could be reproduced in SD rats. Similarly, oxidative stress studies
were performed in SD rats. Experiments in WKY rats Should be carried out to

determine if there is any strain difference.

In addition, alternative methods could be used to evaluate sympathetic
nerve activity (SNA) in the splanchnic bed in SGc-induced hypertension. Non-
hepatic splanchnic NE spillover, for example, is an accurate way to examine the

splanchnic SNA.

Although my data suggest that systemic antioxidant treatment lowers
neural superoxide level and arterial pressure, the cause-effect relationship
between oxidative stress and hypertension remains controversial (Pollock 2005).
A direct way to ﬁnd out the relationship between these two phenomena is to
locally deliver a gene regulating oxidative stress in a Speciﬁc target tissue. So for
S6c-induced hypertension, a virus vector carrying superoxide dismutase (SOD)
gene could be introduced into the celiac ganglia, followed by a hemodynamic
study with 86c infusion and superoxide evaluation. The advantage of this
procedure is that 02" level can be reduced in a more Speciﬁc way in the target
vascular region. If increased 02" in the celiac ganglion contributes to blood
pressure elevation in the SGC-induced hypertension model, increasing SOD
activity should have an antihypertensive effect. The rats treated with SOD gene
transfer and 86C Should Show a smaller increase in blood pressure than rats
treated with 86c and sham-vector. Superoxide concentrations in celiac ganglionic
neurons from rats given S6c should be lower in rats that are treated with SOD

gene transfer. This type of gene transfer study may help to elucidate the cause-

172

effect relationship between oxidative stress and hypertension. The limitations of
this method would be possible immune response to viruses and difﬁculty of

strictly localizing gene delivery.

It has been Shown that NO generation in various vasculatures is higher in
females than in males in humans (Herman, Robinson et al. 1997) and animals
(Hayashi, Fukuto et al. 1992). Renal NO synthase (NOS) activity is higher in
female Tg (SI/SI) rats than that in males (Neugarten, Ding et al. 1997; Taylor,
Gariepy et al. 2003). In addition, estrogen replacement therapy Signiﬁcantly
increased renal NOS levels in oophorectomized rats (Neugarten, Ding et al.
1997). On the other hand, androgen administration has been Shown to be
associated with increased atherosclerosis in animals (Herman, Robinson et al.
1997). Therefore, the above results indicate that female hormones play a
protective role in cardiovascular regulation, while male hormones may have
deleterious effects on the cardiovascular system. Possible mechanisms include
the effects of these hormones on renin-angiotensin system (Ellison, lngelﬁnger et
al. 1989; Gallagher, Li et al. 1999; Dubey, Oparil et al. 2002), on vasoconstrictors
(James, Sealey et al. 1986; van Kesteren, Kooistra et al. 1998), and on oxidative

stress (Dantas, TosteS et al. 2002; Reckelhoff 2005).

Gender differences should be studied further in the future. I used both
male and female transgenic rats in my studies, since gender differences in blood
pressure regulation have been reported in this rat model (Sullivan, Pollock et al.
2006). My preliminary data support these ﬁndings. In the future, more research

Should be carried out to study gender differences in T9 (sllsl) rats, either through

173

surgical approaches (oophorectomy or orchidectomy) or drug treatment
(estrogen or androgen or hormone blockers). Additionally, in human saphenous
veins, the density of ETARS in females iS higher than that in males (Ergul,
Shoemaker et al. 1998). So with the same ET-1 concentration in the Circulation,
ET-1 would induce more vasoconstriction in men than in women, which may lead
to a higher blood pressure in men. Therefore, ETAR density could be measured
in T9 (SI/SI) rats to test if the different receptor densities make a contribution to

the difference in resting blood pressure between the two genders.

174

 

#mﬁﬂ_—____

 

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Anderson, E. A., C. A. Sinkey, et al. (1989). "Elevated sympathetic nerve activity
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