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This is to certify that the
dissertation entitled

THE PRESENCE OF A LOCAL SEROTONERGIC SYSTEM IN
PERIPHERAL ARTERIES

presented by

Wei Ni

has been accepted towards fulfillment
of the requirements for the

Ph.D. degree in Pharmacology and Toxicology

 

 

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THE PRESENCE OF A LOCAL SEROTONERGIC SYSTEM
IN PERIPHERAL ARTERIES

Wei Ni

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

2006

ABSTRACT
THE PRESENCE OF A LOCAL SEROTONERGIC SYSTEM

IN PERIPHERAL ARTERIES

By
Wei Ni

Serotonin (5-hydroxytryptamine, 5—HT), originally discovered in the
intestinal tract and blood, has multiple functions in physiological and pathological
conditions. Increases in reactivity to 5-HT have been observed in a number of
different forms of hypertension models including DOCA-salt hypertensive rats,
spontaneously hypertensive rats and in human patients. However, whether there
is a local serotonergic system in peripheral arteries has not been studied. We
hypothesized that the existence of a local serotonergic system (including 5-HT
synthesis or metabolism, reuptake or release) in peripheral arteries and that this
local regulation of 5-HT is altered in hypertension.

We discovered: (1) the presence of essential enzymes for 5-HT synthesis
and metabolism in peripheral arteries; (2) peripheral arteries are capable of
taking up and releasing 5-HT; (3) the presence of a functional serotonin
transporter (SERT), which is the major protein responsible for 5-HT uptake; (4)
SERT expression is increased but arterial 5-HT uptake function is decreased in
mineralocorticoid hypertension (DOCA—salt hypertensive rats) and nitric oxide
synthase inhibited hypertension (LNNA hypertensive rats), but not in

spontaneously hypertensive rats compared to their non'notensive control.

Our studies showed that a serotonergic system exists in peripheral

arteries and may play a role in local control of total peripheral resistance.

DEDICATION

To my parents, Hengjin Ni and Suli Cai, who gave me life and encouraged me to

go after my dream.

ACKNOWLEDGEMENT

First of all, | wish to acknowledge my mentor Dr. Stephanie W. Watts, who taught

me to think, to speak, to write and to do research as a scientist. Thank you.

I would like to thank my committee members: Dr. Greg Fink, Dr. JR Haywood,
Dr. Marc Bailie and Dr. Keith Lookingland for their guidance, their challenges,

and their suggestions.

I am appreciated to be in Dr. Watts’s lab. I had great time to be with people
worked there. To my friend and classmate Dr. Keshari Thakali, thanks her for
being my good friend and the person I can always count on. To Janice
Thompson, Theo Irina Szasz, Jessica Diaz and Robert Burnett for their friendship

and support. I have learned so many things, science and life, from all of you.

At last, I would also like to thank my husband, Yue Huang, for his love and

unconditional supports!

TABLE OF CONTENT

List of Tables .............................................................................. x
List of Figures ............................................................................. xi
List of Abbreviations ................................................................... xv
Introduction ................................................................................... 1
l. Biosynthesis, storage, and metabolism of 5-HT .......................... 2
1. Biosynthesis ....................................................................... 2
A. Biochemistry .............................................................. 2
B. Location .................................................................... 2
C. Regulation ................................................................. 4
D. Pharmacology ............................................................ 6
2. Storage .............................................................................. 6
A. Location .................................................................... 6
B. Mechanisms ............................................................... 7
C. Regulation and Pharmacology ...................................... 8
3. Metabolism ......................................................................... 9
A. Biochemistry ............................................................. 9
B. Location ................................................................... 10
C. Regulation and Pharmacology ...................................... 10
II. Uptake and release of 5- HT ................................................... 11
1. The major component responsible for 5- HT uptake and release---
SERT ..................................................................................... 1 1
A. Molecular Biochemistry ................................................ 11
8. Location .................................................................... 13
C. Function and Physiology .............................................. 13
D. Pharmacology ............................................................ 15
E. Regulation ................................................................. 17
2. Other components responsible for 5-HT uptake and release---
Non-SERT dependent ............................................................... 20
Ill. 5-HT Function Mechanisms---- in General ................................. 21
1. Extracellular 5-HT functions (5-HT receptors) ............................ 21
2. Intracellular 5-HT functions .................................................... 21
IV. 5-HT and SERT in Physiological Cardiovascular System .............. 22
1. 5-HT in Physiological Cardiovascular System ............................ 22
A. Blood ........................................................................ 22
B Brain / Sympathetic Nervous System ............................... 23
C Heart ........................................................................ 23
D. Kidney ...................................................................... 24
E. Blood Vessels ............................................................ 24
F. Whole Body ............................................................... 26
2. SERT in the Physiological Cardiovascular System ...................... 26
A Pulmonary Vasculature ................................................ 26
B Platelet Function ......................................................... 27

vi

C. Cardiac Function ........................................................ 28

D. Other Cardiovascular Organs ........................................ 28
V. Serotonergic System in Cardiovascular Diseases ....................... 29
1. Serotonergic System and MI ................................................... 3O
2. Serotonergic System and PPH ............................................... 30
3. Serotonergic System and Hypertension .................................... 31
A. Free Circulating 5-HT Concentrations and 5-HT Receptors
Activation ................................................................................. 31
8. Uses of 5-HT Receptor Antagonists in the Treatment of
Hypertension ........................................................................... 33
C. High L-tryptophan Diet and Blood Pressure ....................... 35
D. TPH Function Inhibition and Blood Pressure ..................... 36
E. Other Evidence Supporting the Involvement of Serotonergic
System in Hypertension ............................................................ 37
F. An Unanswered Question --- The Existence of Local
Regulation of 5-HT in Peripheral Arteries ...................................... 38
Hypotheses ................................................................................ 39
Methods ..................................................................................... 42
l. Animal Uses ........................................................................ 42
II. Euthanasia .......................................................................... 42
III. Animal Models ..................................................................... 42
IV. BP Measurements ................................................................ 44
V. 5-HT Basal Level Measurement .............................................. 44
VI. TPH Activity Assay ............................................................... 45
VII. 5-HT Storage Assay .............................................................. 46
VIII. 5-HT Uptake Assay ............................................................... 46
IX. 5-HT Release Assay ............................................................. 46
X. 5-HIAA and 5-HT Measurement .............................................. 47
XI. Real Time RT-PCR ............................................................... 48
XII. lmmunohistochemistry ........................................................... 49
XIII. lmmunocytochemistry ........................................................... 50
XIV. Protein Isolation ................................................................... 51
XV. BCA Protein Assay ............................................................... 51
XVI. Western Blotting ................................................................... 52
XVII. Isolated Smooth Muscle Contractility Measurement ..................... 52
XVIII. Data Analysis and Statistics ................................................... 53
Results ....................................................................................... 55
Hypothesis #1 ............................................................................... 55
I.The Presence of 5-HT in Peripheral Arteries .................................... 55
II. The Presence of 5-HT Synthesis and Metabolism in Peripheral
Arteries ........................................................................................ 64

vii

1. The Existence of TPH1 mRNA and Protein in Peripheral Arteries... 64

2. The Activity of TPH in Peripheral Arteries .................................. 70
3. The Presence of AADC in Rat Aorta and Superior Mesenteric
Artery ...................................................................................... 73
4. The Presence of MAO A in Rat Aorta and Superior Mesenteric
Artery ...................................................................................... 73
5. The Activity of MAO A in Rat and Superior Mesenteric Artery ........ 81
III. The Lack of 5-HT Storage in Peripheral Artery ........................... 84
1. Investigation of Whether Rat Aorta Has Ability to Store 5-HT ........ 84
2. Investigation of “Serotonylated” 5-HT in Rat Aorta ....................... 89
IV. The Presence of Uptake and Release of 5-HT in Peripheral
Arteries ......................................................................................... 95
1. The Presence of SERT in Peripheral Arteries ............................. 95
2. The Presence of Active 5-HT Uptake in Peripheral Arteries ........... 100
A. 5-HT Uptake Time Course Study ..................................... 100
B. Investigation of SERT-dependent 5-HT Uptake .................. 106
C. Investigation of SERT-independent 5-HT Uptake ............... 110
3. The Presence of 5-HT Release in Peripheral Arteries ................... 125
Hypothesis #2 .................................................................................. 132
I. Measurement of SERT Expression in Aorta from Normotensive
and Hypertensive rats ........................................................................ 132
II. Comparison of the Basal Level of 5-HT in DOCA-salt Rats, LNNA
Rats and SHR with Their Normotensive Control Rats ............................ 135
III. 5-HT Uptake Comparison in DOCA—salt Rats, LNNA Rats and
SHR with Their Normotensive Control Rats ........................................... 138
IV. SERT Function in Contractility- Comparison of DOCA-salt Rats 145
with Normal Rats .............................................................................
Discussion ..................................................................................... 150
I.Biochemical Proof of the Presence of 5-HT and its Metabolite in
Peripheral Normal Arteries ................................................................. 150
II. Investigation of 5-HT Synthesis in Peripheral Normal Arteries ....... 151
III. Investigation of Metabolism of 5-HT in Peripheral Arteries ............ 153
IV. Investigation of Storage of 5-HT in Peripheral Arteries ................. 154
V. Investigation of Functional SERT in Peripheral Normal Arteries ..... 155
VI. Investigation of SERT-independent 5-HT Uptake Mechanisms in
Peripheral Arteries ............................................................................ 156

VII. Investigation of 5-HT Release Mechanisms in Peripheral Arteries. 158
VIII. Physiological Relevance of 5-HT Uptake in Peripheral Arteries and

Speculation ..................................................................................... 159
IX. Pathological Relevance of 5-HT Uptake in Peripheral Arteries and

Speculation ...................................................................................... 160
X. Basal 5-HT Concentrations in Aorta of DOCA-salt, LNNA

Hypertensive Rats and SHR .............................................................. 162

viii

XI. 5-HT Uptake Ability is Reduced in Aorta of DOCA—salt and LNNA
Hypertensive Rats, but not in SHR ..................................................... 164
XII. Other Arterial 5-HT-uptake Mechanisms in Hypertensive Animals. 167
XIII. The Effect of SERT Function on Arterial Contractility in

Hypertension .................................................................................... 166
XIV. Future Research ..................................................................... 169
XV. Limitations ............................................................................. 171

Conclusion ..................................................................................... 172

Reference ....................................................................................... 1 74

Bibliography .................................................................................. 193

Table 1.

Table 2.

Table 3.

Table 4.

Table 5.

Table 6.

LIST OF TABLES

The CT values in real time RT-PCR experiments detecting
the expression of tph1 and B-Z-microglobulin mRNA in rat
aorta and superior mesenteric artery ................................ 67

Quantification of 5—HIAA and 5-HT in A 5-HT storage study
in aorta from normal rats ............................................... 88

Quantification of 5-HIAA and 5-HT in A 5-HT storage study
in aorta from pargyline-treated rats .................................... 91

Quantification of 5-HIAA and 5-HT in A 5-HT storage study
in 5-HT—preloaded aorta from pargyline—treated rats ........... 93

Quantification of 5-HIAA and 5-HT in A 5-HT uptake study in
aorta from wild type and tph1-/- mice ................................ 109

Comparison of the effect of fluoxetine and fluvoxamine on
EC50 and threshold of 5—HT-induced contraction in aorta
from SHAM and DOCA rats ............................................. 149

Figure 1.

Figure 2.

Figure 3.

Figure 4.

Figure 5.

Figure 6.

Figure 7.

Figure 8.

Figure 9.

Figure 10.

Figure 11.

Figure 12.

Figure 13.

Figure 14.

LIST OF FIGURES

5-HT Biosynthesis and Metabolism Pathway ...................... 3
Top: Proposed Topology of Plasma-membrane SERT

Bottom: Mechanism of 5-HT Transporter .......................... 12
Diagram of Working Hypotheses ...................................... 41
Top: Chromatogram showing separation of 1 ng standards

using HPLC.

Bottom: Detection of basal levels of 5-HIAA and 5-HT in

thoracic aorta ................................................................ 56

Basal levels of 5-HIAA and 5-HT in peripheral arteries from
normal animals .............................................................. 58

Top: Representative of IHC using guinea pig ileum
myenteric plexus as positive control for 5-HT staining
Bottom: 5-HT IHC staining in rat aorta ............................... 61

5-HT staining in freshly dissociated rat aortic smooth muscle
cells ............................................................................. 63

A. Real time RT-PCR amplification curves of tph1 mRNA

expression in normal rat peripheral arteries ....................... 66
B. Real time RT-PCR dissociation curves of tph1 mRNA
expression in normal rat peripheral arteries ....................... 67

A. TPH staining in freshly dissociated rat aortic and superior
mesenteric artery smooth muscle cells

B. TPH IHC staining in rat aorta ...................................... 69
Quantification of 5-HIAA, 5-HT and 5-HTP in an in vivo TPH
activity study ................................................................ 72
Quantification of 5-HIAA, 5-HT and 5-HTP in A 5-HTP

uptake study ................................................................. 75
Chromatogram showing the measurement of 5-HTP in

tissues in An in vitro TPH activity study ............................. 77
Western analysis of AADC in rat peripheral arteries ............ 78

A. Western analysis examining MAO A in rat peripheral

xi

Figure 15.

Figure 16.

Figure 17.

Figure 18.

Figure 19.

Figure 20.

Figure 21.

Figure 22.

Figure 23.

Figure 24.

Figure 25.

Figure 26

Figure 27.

Figure 28.

Figure 29.

anefies
B. MAO A IHC staining in rat peripheral arteries .................

Quantification of 5-HIAA and 5-HT in A 5-HT uptake study in
arteries from normal rats .................................................

Quantification of 5-HIAA and 5-HT in A 5-HT uptake study in
arteries from pargyline-treated rats ..................................

Quantification of 5-HIAA and 5-HT in A 5-HT storage study
in arteries from normal rats ............................................

Quantification of 5-HIAA and 5-HT in A 5-HT storage study
in arteries from pargyline-treated rats ................................

Quantification of 5-HIAA and 5-HT in A 5-HT storage study
in 5-HT-preloaded aorta from pargyline-treated rats ...........

Western analysis examining 5-HT covalently bound protein
in rat aorta ....................................................................

Top: RT-PCR of SERT in rat aorta
Bottom: Western analysis of SERT in rat aorta, superior
mesenteric artery and mouse aorta ................................

SERT IHC staining in aorta from normal rat and mouse ......

SERT staining in freshly dissociated rat aortic smooth
muscle cells ..................................................................

Time course study of 5-HT uptake in normal rat superior
mesenteric artery ...........................................................

Time course study of 5-HT uptake in aorta from pargyline-
treated rats at 37°C (top) and room temperature (bottom) .....

Time course study of 5-HT uptake in superior mesenteric
artery from pargyline-treated rats at room temperature ........

Quantification of 5-HIAA and 5-HT in A 5-HT uptake study in
aorta from wild type and tph1-/- mice .................................

Effect of fluvoxamine and fluoxetine on 5-HT uptake in
peripheral arteries from pargyline-treated rats .....................

Effect of fluvoxamine on 5-HT uptake in aorta from

xii

80

83

86

88

91

93

94

97

99

102

103

105

107

109

112

Figure 30.

Figure 31.

Figure 32.

Figure 33.

Figure 34.

Figure 35.

Figure 36.

Figure 37.

Figure 38.

Figure 39.

Figure 40.

pargyline-treated wild type, SERT HET and SERT KO mice..

Quantification of 5-HIAA and 5-HT in A 5-HT uptake study in
peripheral arteries from normal rats in Na*-free PSS ............

A. Glyoxylic acid fluorescence in small mesenteric
resistance arteries from vehicle or 6-OHDA-treated rats

B. Quantification of 5-HIAA and 5-HT in A 5-HT uptake
study in peripheral arteries from 6-OHDA-treated rats ........

Effect of nisoxetine on 5-HT uptake in peripheral arteries
from normal rats ............................................................

Effect of corticosterone on 5-HT uptake in peripheral arteries
from normal rats ............................................................

Quantification of 5-HIAA and 5-HT in A 5-HT uptake study in
endothelium intact and denuded aorta normal rats ..............

Quantification of 5-HIAA and 5-HT in fluramines-induced 5-
HT release in aorta from pargyline-treated rats ...................

Quantification of 5-HIAA and 5-HT in (+)-fenfluramine-
induced 5-HT release in superior mesenteric artery from
pargyline-treated rats ......................................................

Effect of fluoxetine on (+)-fenfluramine-induced 5-HT
release in aorta from pargyline-treated rats .......................

Western analysis examining SERT expression in aorta from
hypertensive and normotensive rats .................................

Quantification of basal levels of 5-HIAA and 5-HT in aorta
from hypertensive and normotensive rats ..........................

Effect of fluvoxamine and fluoxetine on 5-HT uptake in aorta
from pargyline-treated SHAM(D) (Top) and DOCA rats
(Middle)

Comparison of total 5-HT uptake and SERT dependent 5-
HT uptake in aorta from SHAM(D) and DOCA rats (Bottom)...

xiii

114

116

119

120

122

124

127

129

131

134

137

140

Figure 41.

Figure 42.

Figure 43.

Effect of fluvoxamine and fluoxetine on 5-HT uptake in aorta

from pargyline-treated SHAMM (Top) and LNNA rats

(Middle)

Comparison of total 5-HT uptake and SERT dependent 5-

HT uptake in aorta from SHAMM and LNNA rats (Bottom)... 142

Effect of fluvoxamine and fluoxetine on 5-HT uptake in aorta

from pargyline-treated WKY (Top) and SHR rats (Middle)
Comparison of total 5-HT uptake and SERT dependent 5-

HT uptake in aorta from WKY and SHR rats (Bottom) .......... 144

Top: Effect of fluoxetine on a 5-HT concentration response

curve in aorta from SHAM and DOCA rats

Bottom: Effect of fluvoxamine on a 5-HT concentration

response curve in aorta from SHAM and DOCA rats ............ 148

xiv

LIST OF ABBREVIATIONS

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

5-HIAA 5-Hyd roxyindoacetic acid

5-HT 5-Hydroxytryptamine, serotonin
5-HTP 5—Hydroxytryptophan

AADC Amino acid decarboxylase
ACh Acetylcholine

Ang II Angiotensin II

AT 1 Angiotensin II receptor 1

BOA Bicinchoninic Acid

BH4 Tetrahydrobiopterin

BP Blood pressure

BSA Bovine serum albumin

CNS Central nervous system

DOCA Deoxycorticosterone acetate
08 Dissociation solution

DTI' Dithiothreitol

ET—1 Endothelin- 1

L-NNA Nm-nitro-L-arginine

MAPK Mitogen-activated protein kinase
MAO Monoamine oxidase

mCPP m-Chlorophenylpiperazine
(:I:)-MDMA 3,4-methylene-dioxymethamphetamine
MI Myocardial Infarction

 

 

XV

 

 

NE

Norepinephrine

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

NET Norepinephrine transporter

NOS Nitric Oxide Synthase

NSD 1015 3-Hydroxybenzylhydrazine

OCT Organic cation transporters

PBS Phosphate-buffered saline

PCPA Para-chlorophenylalanine

PE Phenylephrine

PKA Protein kinase A

PKC Protein kinase C

PP2A Protein phosphatase 2A

PPH Primary pulmonary hypertension
PSS Physiological salt solution

SBP Serotonin-binding protein

SERT Serotonin transporter

SHR Spontaneously hypertensive rats
SNPs Single Nucleotide Polymorphisms
SNS Sympathetic Nervous System
SSRIs Selective serotonin reuptake inhibitors
TBS Tris Buffered Saline

TBS-T Tris Buffered Saline + Tween
TPH Tryptophan hydoxylase

VMAT Vesicular monoamine transporter

 

 

 

 

TGs

Transglutaminases

 

 

 

TM Transmembrane domains
TPR Total peripheral resistance
WKY Wistar-Kyoto rats

 

 

 

 

xvii

 

Introduction:

Serotonin (5-hydroxytryptamine, 5-HT), as a neurotransmitter in the brain
and gastrointestinal tract, is involved in a wide variety of physiological functions
such as mood control, urine storage and voiding, the regulation of sleep and
body temperature, food intake and intestinal motility. 5-HT was first found as a
powerful vasoconstrictor over a century ago and has been recognized as an
arterial smooth muscle cell mitogen in the last 20 years (Nemecek et al., 1986).
The vascular effects of 5-HT are associated with many vascular diseases
including migraine (Buzzi et al., 2005), Raynaud’s phenomenon (Cooke JP and
Marshall JM, 2005), atherosclerosis (Hara et al., 2004) and were suggested to
contribute to liver tissue hypoperfusion following hepatic ischemia-reperfusion
(Murata et al., 2003). In the past decade, a strong argument for the role of 5-HT
and the serotonin transporter (SERT) in pulmonary hypertension has been built
(Marcos et al., 2003; Eddahibi et al., 1999; Eddahibi et al., 2002; Eddahibi et al.,
2000; MacLean et al., 2004; Morecroft et al., 1999). However, whether there is a
functional serotonergic system present in peripheral arteries and the roles and
mechanisms of circulating 5-HT in regulation of non-pulmonary, peripheral
arterial function and its contributions to diseases are not clear.

This section begins with an introduction of general background of 5-HT
and SERT, followed by a summary of the roles of 5-HT and SERT played in
physiological cardiovascular system and cardiovascular diseases, especially in

hypertension.

Biosynthesis, storage, and metabolism of 5-HT

1. Biosynthesis

A. Biochemistry: The essential amino acid tryptophan is the precursor for 5-HT.
Serotonin synthesis depends on the specific action and rate-limiting step of the
enzyme tryptophan hydroxylase (TPH; EC 1.14.164), which transfers a hydroxyl
group to the benzyl ring of tryptophan. Subsequent decarboxylation by amino
acid decarboxylase (AADC) results in formation of 5-HT (Figure 1). The structure
of 5-HT indicates it as a hydrophilic substance, which is preferably protonated at
physiological pH. Thus, 5-HT does not pass the lipophilic blood—brain barrier
and cell membrane readily.

B. Location: Over 95% of 5-HT in the body is synthesized in the
enterochromaffin cells of the intestine. 5-HT must be synthesized in the brain
because 5-HT cannot cross the blood—brain barrier. Those areas responsible for
synthesizing 5-HT are the clusters of cells in the midlinelraphe regions of the
pons and upper brainstem. Other sources of 5-HT include neuroendothelial cells
that line the lung, and a few other discrete sites (Cooper et al., 2003), some of
which are in the cardiovascular system. TPH mRNA and protein have been
detected in hamster heart (Slominski et al., 2002), and 5-HT synthesis has been
measured both in an HL-1 cardiomyocyte cell line, which represents adult
cardiomyocytes, and in neonatal rat ventricular cardiac myocytes (lkeda et al.,
2005; Cote et al., 2004). Thus, the cardiomyocyte provides a local source of 5-
HT in the heart. Both in vivo and in vitro experiments suggested a renal

formation of serotonin by decarboxylation of its amino acid precursor L-5-

5-HT Biosynthesis and Metabolism Pathway

/COOH
CH2CHNH2
©:§ Tryptophan
NH
Tryptophan
Hydroxylase
Rate Limiting /cooI-I
HchNHz
HO
\ 5-Hydroxytryptophan
(5-HTP)
NH
Amino Acid
Decarboxvlase
CH2CH2NH2

HO
\ 5-Hydroxytryptamine

(5-HT, Serotonin)

8

NH

MAO

Aldeh de
Dehy rogenase

<——

CH2COOH
HO
\ 5-Hydroxyindole Acetic Acid
NH (5-HIAA)
Figure 1.

hydroxytryptophan (L-5-HTP) in rat (Stier et al., 1985; Stier et al., 1984).
Serotonin synthesis has also been reported in adrenochromaffin cells of frogs
(Delarue et al., 1992). Recent studies have revealed TPH in new and
unexpected places such as the skin (Slominski et al., 2003). Sites of 5-HT
synthesis in the non-pulmonary peripheral vasculature have not yet been
identified, so our current understanding is that the systemic vasculature is
exposed to 5-HT through release of 5-HT by the platelet or to freely circulating 5-
HT.

C. Regulation: TPH is an extremely labile enzyme, which can be affected by
multiple factors. TPH uses Fe” as co-factor and Oz and tetrahydrobiopterin
(BH4) as co-substrates to hydroxylate tryptophan. Additionally, an in vitro study
suggested that TPH is an oxygen-inhibitable enzyme. The degree of TPH
inactivation is dependent on the partial pressure of oxygen to which the enzyme
is exposed and the temperature at which TPH is preincubated. TPH activity
declined in a linear fashion as the temperature increased from 0 to 45 °C (Kuhn
et al., 1979). The inactivation of TPH by oxygen could be recovered by
anaerobic incubation in the presence of dithiothreitol (DTT) and Fe2+ (Kuhn et al.,
1979). TPH activity can be irreversibly inactivated by nitric oxide (Kuhn and
Arthur, 1997). The phosphorylation of TPH by protein kinase A (PKA) and
CaZVcaImodulin-dependent protein kinase II results in increased TPH activity
(Kowlessur and Kaufman 1999). The phosphorylation site for PKA has been
reported at serine 58 (Kuhn et al., 1997) and at serine 58 and 260 for

Ca2*/calmodulin-dependent protein kinase ll-induced phosphorylation (Jiang et

al., 2000). The 14-3-3 proteins are a group of ubiquitous proteins that were first
discovered in the brain (Aitken et al., 1995, review). It has been reported that
the 14-3-3 proteins form a complex with phosphorylated rat brain TPH, thereby
increasing TPH enzymatic activity and inhibiting the protein phosphatase-1
induced dephosphorylation (Banik et al., 1997). It is interesting that, unlike
catecholamines, 5-HT synthesis by TPH is not inhibited by the end-product (5-
HT) nor by the major metabolite (5-hydoxyindoleacetic acid, 5-HIAA) (Cooper JR
et al., 2002). Thus, one way to study 5-HT synthesis is to inhibit AADC to
examine the accumulation of 5-HTP.

Recently, Bader and colleagues discovered that tph1 knock-out mice
exhibit only a minor reduction of steady state 5-HT levels in brain regions
(hippocampus and frontal cortex) with a lack of 5-HT in peripheral organs (gut,
blood and pineal gland), suggesting the existence of a second tph gene, which
they named tph2. The expression of tph1 is predominant in peripheral organs
such as the intestine, spleen and pineal gland and tph2 is present in brain
regions (Walther and Bader, 2003; Walther et al., 2003 a), which allows for
distinct sources of peripheral and central 5-HT. TPH1 and TPH2 are highly
homologous proteins exhibiting 71% of amino acid identity in human (Walther
and Bader, 2003). All the residues important for tryptophan, iron, BH4 and 14-3-
3 proteins binding are identical in TPH1 and TPH2 (Walther and Bader, 2003).
Consistently, both isoforms of TPH can be phosphorylated by Ca2*/calmodulin-
dependent protein kinase II and PKA. However, the N-terminus of the two

isoforms of TPH, which contain the regulatory domains, are quite different. The

KM values of the purified and recombinant TPH from carcinoid tumors and pineal
gland (TPH1) have been reported as 13 and 23 nM, respectively, while the TPH
isolated from brain stem (TPH2) exhibits a value of 142 uM (Kowlessur and
Kaufman, 1999), which suggests that the substrate of TPH, tryptophan, has
higher affinity for TPH1.

Pharmacology: Para-chlorophenylalanine (PCPA) is a potent, specific
and irreversible inhibitor of TPH activity both in vivo and in vitro, which drastically
reduces the 5-HT concentration in 5-HT neurons and terminals (Pandey et al.,
1983). Amphetamine analogs inhibit TPH activity potentially via production of
nitric oxide, superoxide and peroxynitrite (Kuhn and Geddes, 2000). The
interaction of 5-HT and these reactive oxygen species produces tryptamine-4,5-
dione, which rapidly and irreversibly inactivates TPH (Wrona and Dryhurst.,
2001). 3-Hydroxybenzylhydrazine (NSD 1015) is an AADC inhibitor, which has
been used in experiments to block the conversion of 5-HTP to 5-HT (Pandey et

aL,1983)

2. Storage

A. Location: In the periphery, the major 5-HT storage site is platelets.
Excluding platelets, the free circulating levels of 5-HT in plasma were reported as
15-120 nM, much lower than the levels of 5-HT in whole blood (uM) (Martin
1994). Other than playing a crucial role in 5-HT synthesis, enterochromaffin cells
are also a storage place of 5-HT (Nilsson et al., 1985). In the neuronal system,

5-HT is synthesized in the axon and stored in nerve terminals. Other 5-HT

storage sites including mast cells (Pihel et al, 1998), adrenal medullary cells
(Brownfield et al., 1985) and pinealocytes (Hayashi et al., 1999).

B. Mechanisms:

PM Platelets themselves do not synthesize 5-HT. However, they possess a
high efficacy SERT, enabling them to take up 5-HT from the gut and lung. As the
carrier and storage site of 5-HT, the platelets store 5-HT in dense, electron-
opaque granules and release it in a thrombotic event (Vanhoutte, 1991). Storage
of 5—HT in platelet granules requires active uptake of 5-HT from the cytoplasm by
vesicular monoamine transporter 2 (VMAT 2).

Transglutaminases (TGs) catalyze the calcium-dependent acyl transfer
between the y-carboxamide of a bound glutamine residue and the s-amine group
of a bound lysine, the primary amine group of a polyamine or possibly
monoamine, such as 5-HT (Zhang et al., 1998). Recently, Walther and his
colleagues reported that platelet intracellular 5-HT could be transamidated to
small GTPases by TGs during activation and aggregation of platelets, rendering
these GTPases constitutively active (Walther et al, 2003 b). This observation
suggested that 5-HT could be covalently bound to proteins and thus to be stored
in platelets.

Enterochromaffin Cells: Study of the subcellular localization of serotonin
immunoreactivity in rat enterochromaffin cells showed that 60% of 5-HT was
located in the dense cores of the secretory granules following uptake via VMAT

1. However, a significant amount of 5-HT (40%) was located in the cytoplasm,

outside of the secretory granules (Nilsson et al., 1985), which suggests two
different forms of 5-HT storage exist, at least in enterochromaffin cells.
New After being synthesized in neurons, 5-HT is taken up by VMAT 2 and
stored in synaptic vesicles. Different from other non-neuronal 5-HT storing cells
such as enterochromoffin cells, mast cells or platelets, soluble serotonin-binding
protein (SBP) has been found in serotonergic synaptic vesicles of central and
peripheral serotonergic neurons. This difference might come from different
origins of the cells. SBP appears to be neuroectoderm-specific, while
enterochromaffin cells are from endodermal origin, and mast cells and platelets
are derived from mesoderm (Gershon et al., 1983). SBP binds with 5-HT with
high affinity in the presence of Fe”. Two SBPs have been identified, with
molecular weights of 45 Kd and 56 Kd. Thus, SBP binds 5-HT and functions as
a storage protein for 5-HT in neurons (Gershon et al., 1983).
Mast Cells: Mast cells store serotonin and histamine together within large
secretory granules (Pihel et al, 1998). Moreover, two serotonin binding proteins
were found in mast cells which are different from the SBP found in brain (Tamir
etaL,1982)

Thus, from what has been discovered, 5-HT could be stored in vesicles or
bound to proteins in cells.
C. Regulation and Pharmacology: VMAT functions as a proton-amine
exchanger with a stoichiometry of two protons to one amine, which could be 5-
HT, dopamine, histamine etc. The electrochemical gradient across secretory

vesicle membranes (due to the activity of H+-ATPase) provides energy for the

transport of monoamines. As indicated above, two types of VMAT have been
discovered, VMAT 1 primarily in endocrine cells [enterochromaffin cells,
pinealocytes (Hayashi et al., 1999)] and VMAT 2 in neuronal cells and platelets.
The two VMATs differ in their ability to transport histamine, and in their sensitivity
to certain inhibitors. For example, reserpine blocks both, but tetrabenzine is
selective for VMAT2 (Peter et al. 1994). Recently, a study showed that the filling
of 5-HT in vesicles initiates an activation of Gaq protein (probably on vesicle
membrane), which by an unknown 5-HT receptor-independent mechanism
downregulates VMAT 2 function in platelets (Holtje et al., 2003). Because VMAT
function depends on the electrochemical gradient induced by H"-ATPase, any
chemical (such as concanamycin and bafilomycin), which inhibits H+-ATPase will
reduce VMAT function and therefore 5-HT storage in vesicles.

TGs transamidate 5-HT to proteins. Cystamine inhibits TGs function and
thus potentially reduces 5-HT storage in the form of 5-HT-transamidated

proteins.

3. Metabolism
A. Biochemistry:

Metabolism of 5-HT primarily occurs through actions of deamination by
monoamine oxidase (MAO) to form 5-hydroxyindole acetaldehyde, which in turn
is oxidized by aldehyde dehydrogenase to produce 5-HIAA (Figure 1).
Alternatively, 5-hydroxyindole acetaldehyde can be converted to 5-

hydroxytryptophol, but this pathway is considered insignificant. Other

metabolism pathways include transformation of 5-HT to melatonin. N—acetyl
transferase induces acetylation of the amine group of 5-HT to N-acetyl-5-HT.
Then, N-acetyl-5-HT is converted to melatonin by adding a methyl group (Cooper
JR et al., 2003).

B. Location:

MAO is an intracellular enzyme, found primarily in mitochondria. 5-HT
must be taken up inside a cell prior to being acted upon by MAO and both SERT
and norepinephrine (NE) transporter (NET) facilitate this uptake (Kawasaki and
Takasaki, 1987). Tissues or cells that contribute significantly to 5-HT metabolism
include the lung, intestine and endothelial cells of the arterial system, but any cell
that can take up 5-HT and possesses MAO has the potential to metabolize 5-HT.
In pineal gland, melatonin synthesis uses 5-HT as a precursor.

C. Regulation and Pharmacology:

Two isoforms of MAO have been found, MAO A and MAO B. MAO A is
more selective and has a much lower KM value (higher affinity with 5-HT) in
metabolizing 5-HT compared to MAO B. MAO A activity increased in rats with
low salt diet. This increased activity of MAO A was proved to be related to
angiotensin II receptor 1 (AT 1) activation due to increased angiotensin II levels
and an increased AT 1 receptor expression induced by low salt diet (De Luca
Sarobe et al., 2005). Glucocorticoid increased both MAO A mRNA and protein
expression via glucocorticoid receptor and Sp1 transcription factor function

(Manoli et al., 2005). Pharmacologically, MAO could be inhibited by pargyline.

10

Il. Uptake and release of 5-HT
5-HT is a charged molecule (protonated) in physiological condition, which
cannot cross lipid bilayer. SERT is the major protein responsible for uptake and
release of 5-HT, which transports 5-HT in either direction, depending on the
concentration gradient. SERT is important in that it plays a critical role in
regulating the function of the 5-HT receptors and the serotonergic system via

modulation of extracellular and intracellular cytoplasm 5-HT concentrations.

1. The major component responsible for 5-HT uptake and releaseu-SERT
A. Molecular Biochemistry:

SERT was cloned in 1991 by Hoffman and Blakely in the rat (Hoffman et
al., 1991; Blakely et al., 1991). Human SERT was cloned in placental
trophoblastic cells in 1993 (Ramamoorthy et al., 1993); the mouse SERT was
cloned in 1996 (Chang et al., 1996). Mouse SERT protein has an 88% homology
with the human SERT, compared to the rat SERT having 71% homology for the
human SERT. SERT proteins are positioned in the plasma membrane.
Hydropathy plots of the SERT protein, typically 630 amino acids long, have
suggested that the protein spans the bilayer 12 times (12 transmembrane
domains; TM) and that both amino and carboxy termini are intracellular (Figure 2
top). SERT possesses a large extracellular loop between TM3 and 4. This loop
has sites of glycosylation, important for the trafficking and stability of SERT
(Chen et al., 2002). In three sites of the protein, the amino terminus, the carboxy

terminus and the intracellular loop between TM8 and TM9, consensus sites for

11

Proposed Topology of Plasma-membrane SERT

Inhibitor
Interaction,
Cation
Dependence

‘IL ,1 1 OUT
. 112EI§5BILE§91<TII2
®2

The 12 membrane-spanning
human serotonin transporter

 
   
     

Substrate
lnteracti . n

 

 

 

] IN
COOH
o

Modified from http://intramural.nimh.nih.gov/research/lcs/research.html

Mechanism of 5-HT Transport
Na“, Cl'

  

OUT
I

 

 

IN

K‘” [H*]

 

5-HT

Figure 2.

12

phosphorylation by protein kinase C (PKC) and PKA exist (Ramammoorthy,
2002). TM1-TM3 are important for interactions with substrates such as 5-HT,
and TM11 and 12 are crucial for inhibitor interaction and dependence on cations.
Translated SERT protein is of a mass anywhere from 60-80 kDa, depending on
levels of glycosylation and phosphorylation. In 2000, it was reported that SERT
proteins homo-oligomerize (Kilic and Rudnick, 2000). The impact of
oligomerization on function is unclear (Schmid et al., 2001 a). Since this time,
SERT has been reported to hetero-oligomerize with myosin llA (Ozaslan et al.,
2003), NET (Kocabas et al., 2003), and the GABA transporter 1 (Schmid et al.,
2001 b).

B. Location:

SERT is widely distributed in central nervous system (CNS) and peripheral
sympathetic nervous system. SERT was also found in non-neural systems, such
as platelets (where 5-HT is stored), cells in gastrointestinal system (where 5-HT
is synthesized) and lung (where 5-HT is synthesized and inactivated) (Glilis et al.,
1982). Recently, a functional SERT was reported in osteoclast cells (Battaglino
et al., 2004).

C. Function and Physiology:

SERT, a sodium-dependent transporter, diminishes the function of 5-HT at
its extracellular cognate receptors by removing 5-HT from outside the cell and
bringing it back into a cell for metabolism via MAO A or vesicular repackaging.
The present model of SERT functioning (Figure 2 bottom) begins with

extracellular sodium binding to the carrier protein. 5-HT, in its protonated form,

13

then binds to the transporter and is followed by a chloride. This initial complex
creates a conformational change in the transporter protein: it revolves from facing
extracellularly to an inward-facing position where 5-HT and ions are released in
the cytoplasm.

Intracellular potassium binds to SERT to promote reorientation of SERT
for another cycle to the outside of the cell, at which time K+ is released. These
elements (Na‘, Cl', 5-HT, and K“) are thought to work in a single multifunctional
binding site. The driving force of the 5—HT transport across the plasma
membrane via SERT is dependent on the Na+ concentration gradient. Na+ ions
flow down their concentration gradient and 5-HT accumulates in the cells. The
energy to build Na+ concentration gradient comes from hydrolysis of ATP by
ATPase. ATP is utilized by Na*-K*-ATPase to actively transport 3 Na” ions out of
the cell and pump 2 K” ions into the cell. Therefore, 5-HT transport across
plasma membrane indirectly uses energy released by ATP hydrolysis. Thus, the
function of SERT is inhibited by metabolic inhibitors as well as by inhibitors of
Na”-K+ ATPase activity.

It is reasonably assumed that the function of 5-HT is terminated once
brought inside the cell. However, two laboratories have independently
demonstrated the necessity of uptake of 5-HT by SERT in mediating pulmonary
arterial smooth muscle proliferation to 5-HT (Marcos et al., 2003; Lee et al.,
2001). Additionally, a finding in the platelet supports the idea that 5-HT, once
intracellular, exerts physiological effects. Walther et al. demonstrated that 5-HT,

actively imported into a platelet by SERT, acts as a substrate for an enzyme

14

class of TGs to covalently modify RhoA or Rab 4 with the 5-HT moiety
(transamidation or ‘serotonylation’) (Walther et al., 2003 b). Serotonylation of
Rho A renders it constitutively active. Numerous other protein targets of TGs,
such as actin, have been identified (Griffin et al., 2002). Thus, intracellular 5-HT
has functional effects, which could be regulated by SERT by changing
intracellular 5-HT concentrations. While the platelet was the initial cell type in
which this was demonstrated, it is currently unknown whether intracellular 5-HT
can alter function in cell types of other cardiovascular tissues. It is possible that
vasoconstriction could be modulated by Rho A and actin serotonylation.
D. Pharmacology:

5-HT possesses high affinity (17.4 nM) for the rat and human SERT
derived from the brain (Rothman et al., 1999), and recognition of 5-HT occurs
within the first three transmembrane domains of the transporter. Drugs that
interact with transporter proteins can be divided into two basic classes: reuptake
inhibitors and substrate-type releasers. Reuptake inhibitors bind to
transporter proteins, but are not transported into cells themselves. These drugs
elevate extracellular 5-HT concentrations by blocking transporter-mediated
recapture of extracellular 5-HT. Substrate-type releasers bind to SERT proteins,
and subsequently are transported into cytoplasm. In neurons, releasers elevate
extracellular transmitter concentrations by a two-step mechanism: first they
promote efflux of transmitter by a process of VMAT-mediated exchange, and
then they increase cytoplasmic levels of transmitter by disrupting storage of 5-HT

in vesicles. The increased 5-HT in the cytoplasm by the second step provides

15

more 5-HT for release by transporter-mediated exchange. Because substrate-
type releasing agents must be transported into nerve terminals to promote
transmitter release, reuptake inhibitors may block the effects of releasers
(Rothman and Baumann, 2002). A variety of serotonin-selective reuptake
inhibitors (SSRls), such as fluoxetine (Prozac®), fluvoxamine (Luvox®), sertraline
(Zoloft®) and citalopram (Celexa®) are medications used for the treatment of
psychiatric disorders, including depression, panic disorder, and obsessive-
compulsive disorder (Gorman and Kent, 1999; Zohar and Westenberg, 2000).

In contrast to the SSRIs, which are commonly used drugs, there is only
one 5-HT releaser, (1)-fenfluramine (Pondimin®), that has been used clinically.
(i)-Fenfluramine and the more potent stereoisomer (+)-fenfluramine
(dexfenfluramine, Redux”) were highly effective anorexigens approved by the
Food and Drug Administration in 1996 for use in long-term medical management
of obesity. These drugs were widely prescribed until they were withdrawn from
the market in September 1997 due to reports of cardiac valvulopathy (Connolly et
al., 1997) and primary pulmonary hypertension (PPH) (Abenhaim et al., 1996).
(i)-Fenfluramine is metabolized in liver to (+)-norfenfluramine and (-)-
norfenfluramine. Most of the studies agree that fenfluramine and norfenfluramine
are SERT substrates and potent 5-HT releasing agents (Garattini, 1995). Other
5-HT releasers include the drug 3,4-methylene-dioxymethamphetamine Ki)-
MDMA or “ecstasy”] and the non-amphetamine piperazine derivatives m-
chloropheylpiperazine (mCPP), which is a major metabolite of the anti-

depressant trazodone (Otani et al., 1998).

16

E. Regulation:
Long-term regulation:

The SERT promoter in multiple species contains such basic elements as a
TATA-Iike, CREB, NFKB, P2, and SPI site (Chen and Reith, 2002;
Ramammoorthy, 2002; Blakely et al., 1998). Interestingly, an allele in the human
SERT promoter has been found, resulting in 14 (short or 5 allele) or 16 (long or I
allele) repeat elements, a difference of 44 base pairs and expression of an
mRNA species either 484 (s) or 528 (I) base pairs long (Heils et al., 1996).
Possession of the 5 allele is associated with a lower expression of SERT
(estimated at 1/3 of those possessing two I alleles), resulting in a reduced
capability to take up and release 5-HT. In the human, the $3 allele has been
associated with, but not proven to cause, an inability to handle stressful life
events (Murphy et al., 2004). Thus, a genetically-inheritable difference in alleles
suggests that humans, naturally, may express different amounts of SERT
throughout the whole body, including the cardiovascular system. Additionally, at
least 25 different Single Nucleotide Polymorphisms (SNPs) of SERT have been
reported for the human. The phenotypes, cardiovascular or otherwise, of these
SNPs are not known.

Acute regulation:

Phosphomlation / Dephosphorylation: When SERT proteins are artificially
expressed in cells, SERT activity is regulated in response to alterations in

calcium/calmodulin-dependent kinase, PKC, PKA, and protein kinase G (PKG)

17

activity; SERT bears sites for serine/threonine phosphorylation (Mlinar and
Corradetti, 2003; Ramamoorthy et al, 1998; Blakely et al., 1998). Intensive
studies were done to demonstrate alteration of SERT activity via
phosphorylation/dephosphorylation by two pathways, the PKG/p38 mitogen-
activated protein kinase (MAPK)Iprotein phosphatase 2A (PP2A) pathway
and the protein kinase C (PKC) pathway. PKG/p38 MAPK/PP2A activation
increases SERT activity by stimulating insertion of intracellular SERT into the cell
membrane (Blakely et al., 2005) and increasing total SERT activity (Zhu et al.,
2005). By contrast, PKC activation reduces SERT function by phosphorylating
SERT protein and causing translocation of SERT to the cytosol (Jayanthi et al.,
2005)

Data also support that 5-HT itself may regulate the activation status of the
SERT, independent of 5-HT receptors. Ramamoorthy et al. found that in
HEK293 cells transfected with SERT, which possessed no detectable 5-HT
receptors, 5-HT inhibited SERT phosphorylation and decreased the ability of
PKC activation-induced SERT activity reduction (Ramamoorthy et al, 1998;
Blakely et al., 1998). Preservation of SERT function by 5-HT makes logical
sense. Other SERT substrates, such as D-amphetamine and fenfluramine have
a similar inhibitory effect on PKC-mediated phosphorylation and internalization

(Ramamoorthy and Blakely, 1999).

5-HT2E, Receptor-related Regulation: 5-HT receptor-dependent SERT regulation

has been revealed. One study using 1C11 cells showed that 5-HT28 receptor

regulates SERT function differently in the presence and absence of 5-HT. In the

18

absence of external 5-HT, 5-HT28 receptor coupling to NO production ensures
PKG/p38 MAPK induced SERT phosphorylation and maximal 5-HT uptake
(Launay et al., 2006; Zhu et al., 2004). In the presence of 5-HT, the 5-HT2E3
receptor mediates PKC—dependent SERT phosphorylation, which reduces the
maximal velocity of 5-HT uptake (Launay et al., 2006). In addition, 5-HT2E3
receptor activation also reduces Na*-K*-ATPase activity by Na*-K*-ATPase
catalytic subunit phosphorylation. Because the basic function of Na*-K*-ATPase
is to maintain the high Na+ and K+ gradient across the plasma membrane, which
provides energy for 5-HT uptake by SERT, the impaired Na*-K*-ATPase function
also reduces SERT activity (Launay et al., 2006). The inhibition of SERT function
through 5-HT23 receptor activation is also supported by Dr. Maroteaux group’s in
vivo study. An acute agonist stimulation of 5-HT2B receptor triggers a transient
increase in plasma serotonin that is SERT-dependent and blocked by 5-HT28
receptor-selective antagonist or genetic ablation (Callebert et al., 2006). Altered
5-HT28 receptor expression and function have been reported in different
diseases, such as hypertension (Banes et al., 2003; Russell et al., 2002) and
pulmonary hypertension (Launay et al., 2002). It is possible that this changed 5-
HT23 receptor function will modify SERT function in diseases.

Membrane Choleeterel end Lipig Micredemeine: Depletion of membrane
cholesterol reduces SERT activity by changing the affinity of 5-HT for SERT and
a concomitant reduction of the maximal transport rate (Scanlon et al., 2001). The

function of SERT could be partially rescued by replacement of cholesterol, but

19

not by other sterols. A further study demonstrated that SERT has to be present
in cholesterol rich-lipid rafts (lipid microdomains) on the membrane to be
functional. An equilibrium may be found between an active raft-associated state
(in cholesterol rich-lipid rafts) and an inactive state residing outside lipid rafts in
plasma membrane-bound SERT. The detection of SERT-containing lipid rafts
are found in both intracellular and cell surface fractions suggesting that lipid rafts
association may be important for trafficking and targeting of SERT (Magnani et

aL,2004)

2. Other components responsible for 5-HT uptake and release-«Non-SERT
dependent

NET, dopamine transporter (DAT) and SERT (monoamine transporters)
belong to the Na*,Cl'—dependent gene family and share many similarities in
terms of function, mechanism and regulation. In the peripheral cardiovascular
system, 5-HT is taken up and released by adrenergic nerves (Kawasaki et al.,
1987). DA can be taken up by NET (Gu et al., 1994). It is possible that
monoamine transporters have the ability to act promiscuously and thus DAT and
NET might take up 5-HT.

Other candidates responsible for non-SERT uptake include the organic
cation transporters (OCT). The laboratory of Michael Gershon has demonstrated
a gastrointestinal upregulation of OCT-1I3 in SERT-deficient mice. Thus a
member of the OCT family is a reasonable candidate for alternative 5-HT uptake

(Chen et al, 2001). OCTs are promiscuous transporters, with substrates as
20

diverse as 5-HT, DA, NE, epinephrine, histamine, clonidine, and cimetidine.
Pharmacologically, OCTs are difficult to distinguish from one another but can be
distinguished from SERT by inhibition by corticosterone, O-methylisoprenaline

and levamisole (Martel and Azevedo, 2003; Horvath et al, 2003).

III. 5-HT Function Mechanisms-“- in General

5-HT has a wide variety of effects on almost all systems. Both intracellular
5-HT and extracellular 5-HT have functions in physiological and pathological
conditions.

1. Extracellular 5-HT functions (5-HT receptors):

Typically, 5-HT exerts its effects by activating 5-HT receptors. There are
seven families of 5-HT receptor (5-HT1-5-HT7), which have been identified in the
last 16 years and 15 subtypes have been cloned (Tierney 2001). Most 5-HT
receptors are G-protein coupled receptors, except the 5-HT3 receptor, which is a
ligand-gated ion channel. The 5-HT1B, 5-HT1D, 5-HT2A, 5-HT23, 5-HT3, 5-HT4, 5-
HT7 receptors are found in the cardiovascular and/or renal systems. The major
5-HT receptors responsible for the 5-HT-induced vasoactive effect are 5-HT1 and
5-HT2 receptors, while 5-HT7 receptor activation results in vessel relaxation.

2. Intracellular 5-HT functions:

It has been revealed during the past decade that the function of 5-HT is
not terminated once brought inside the cell. Two laboratories have
independently demonstrated the necessity of uptake of 5-HT by SERT in

mediating pulmonary arterial smooth muscle proliferation to 5-HT (Marcos et al.,

21

IV.

2003; Lee et al., 2001). Additionally, as stated above, a finding in the platelet
supports the idea that 5-HT, once intracellular, exerts physiological effects by
transamidating RhoA or Rab 4 (Walther et al., 2003 b). Moreover, Battaglino et
al. showed another exciting mechanism by which 5-HT could function
intracellularly. They suggested that intracellular 5-HT activates NF-kappa B to
enhance osteoclast differentiation and that the SERT inhibitor, fluoxetine could
cause bone mass decrease by inhibiting 5-HT uptake into osteoclast cells
(Battaglino et al., 2004). These studies showed new signal transduction
pathways supporting that intracellular 5-HT could exert its effects independent of
5-HT receptors. Thus, it is possible that in other cell types, such as vascular
smooth muscle or endothelial cells, intracellular 5-HT also has function and could

regulate vascular smooth muscle tone independent of 5-HT receptors.

5-HT and SERT in Physiological Cardiovascular System

1. 5-HT in Physiological Cardiovascular System
A. Blood

Blood elements, particularly platelets, are a rich source of 5-HT. 5-HT is
taken up by SERT in platelets (Holmsen and Weiss, 1979) as platelets do not
synthesize 5-HT. The function of 5-HT in the blood is to promote platelet
aggregation and blood clotting. Although platelets contain 5-HT, they also
possess 5-HT2A receptors which, when activated, promote further platelet
activation and aggregation (Vanhoutte, 1991). Thus 5-HT2A receptor antagonists

such as ketanserin and sarpogrelate have proven effective anti-platelet/anti-

22

thrombotic agents. Other cells, which contain 5-HT, include mast cells and
macrophages.
B. Brain I Sympathetic Nervous System

Approximately 1—2% of body 5-HT is found in the brain. The central
effects of 5-HT on the cardiovascular system are complex. The effects of 5-HT
vary according to the site in which 5-HT is administered and the anesthetic status
of the animal. In general, in the rat, central injection of 5-HT increases blood
pressure (BP), although there are studies that suggest the opposite effect
(Callera et al.,1997). Moreover, the effects of central 5-HT in other species,
primarily cat and dog, are different from the rat in that 5-HT largely causes a
decrease in BP. It is clear that central 5-HT has the capability of altering BP and
appears to do so by interacting with both 5-HT1A and 5-HT2 receptors (Watts,
2005)
C. Heart

5-HT has pluripotent effects in the heart, outside of the well-documented
effects in the coronary arteries (Doggrell, 2003). Multiple receptors for 5-HT exist
in the heart, including those directly on cardiac myocytes and on the vagus and
sympathetic nerves. 5-HT stimulation of 5-HT3 receptors on the vagus nerves
accounts for the decrease in heart rate through activation of the BezoId-Jarisch
reflex. 5-HT can also act as a sympatholytic through activation of 5-HT1
receptors on sympathetic terminals, inhibiting NE release. However, 5-HT exerts
positive chronotropic effect in the rat through activation of 5-HT2A receptors,

activation of 5-HT4'receptors in the pig and human and 5-HT7 receptors in the cat

23

(Cote et al., 2004; Saxena and Villalon, 1991; Villalon et al., 1997). In isolated
cardiomyocytes, 5-HT also stimulates mitogenesis via the 5-HT23 receptor, which
is critical for development of the heart in the mouse (Nebigil and Maroteaux,
2001).
D. Kidney

Like the brain, the kidney has the ability to synthesize 5-HT from its
precursor tryptophan (Stier and Itskovitz; 1984; Stier et al., 1984; Hafdi et al.,
1996). 5-HT is also a mitogen in mesangial cells (Mene et al., 1991), but its most
profound effect in the kidney is to increase perfusion pressure by increasing
renovascular resistance through arterial contraction. This has been
demonstrated primarily in isolated perfused kidneys, although 5-HT is
vasodilatory in the renal vascular bed of the dog (Tian et al., 2002).

5-HT and dopamine play the opposite role in sodium retention in the
kidney. 5-HT causes sodium retention in the kidney, whereas dopamine
promotes natriuresis (Berndt et al., 2001). The precursors for 5-HT and
dopamine synthesis, L-5-HTP and L-DOPA, use the same transporter to enter
the proximal convoluted tubule, thereby compromising synthesis of the other
substance. Moreover, 5-HT and 5-HT1B receptor agonists stimulate the
sodium/phosphate cotransporter to decrease phosphate retention, potentially
contributing to renal failure (Hafdi et al., 1996).
E. Blood Vessels

Blood vessels, in particular arteries, serve an important function in

providing a controlled feed of blood to tissues through creation of a resistance to

24

blood flow or total peripheral resistance (TPR). TPR is defined by the reactivity
of small arteries and arterioles (5200 pm in humans), where resistance to blood
flow is determined by the calibre of the artery. This, in turn, is governed by the
size of the lumen based on (i) thickness of arterial wall; (ii) reactivity of the
smooth muscle and endothelial cells to neuronal neurotransmitters and
endogenous hormones; and (iii) concentration of vasoactive substances.

The effect of 5-HT on vasculature is complicated. Under normal
conditions, 5-HT2A receptor-mediated 5-HT-induced constriction in the systemic
circulation, while the endothelial 5-HT23 receptor relaxes pre-contracted rat aorta
(Villazon et al., 2002) and pig pulmonary artery (Glusa and Pertz, 2000). Studies
showed that subcontractile concentration of 5-HT interact synergistically with a-
adrenergic agonists and other vasoconstrictors including Ang II, histamine and
prostaglandin F2“ (Yildiz, 1998 for review). 5-HT—induced synergism with a
vasoconstrictor presents a more relative physiological role of 5-HT because of
the belief that free circulating 5-HT in plasma is low. Moreover, Cohen et al.
reported that in the rabbit saphenous vein, 5-HT enhances the release of NE by
stimulating prejunctional 5-HT1A receptors on the noradrenergic nerves (Cohen et
al., 1999), thus 5-HT may also regulate vascular smooth muscle tone by
modulating the sympathetic neurotransmission in blood vessels. 5-HT is also a
smooth muscle cell and endothelial cell mitogen, and, as in the event of
contraction, 5-HT can potentiate the mitogenic effect of other hormones

(Nemecek et al., 1986; McDuffie et al., 2000).

25

F. Whole body

When 5-HT is administered to a whole animal, the pattern of observed BP
changes varies based on species. In the rat, intravenous 5-HT elicits a classical
triphasic effect: an initial fast depressor (BezoId—Jarisch reflex via 5-HT3 receptor
activation), a pressor response (smooth muscle contraction via 5-HT2 receptor
activation) and a longer depressor response (probably activation of arterial 5-HT7
receptors, Dalton et al., 1996). It should be noted that, in other species, the
cardiovascular response to 5-HT is different: 5-HT (administered intravenously)
decreased BP in broiler chickens (Chapman and WIdeman, 2002), increased BP
in healthy calves (Linden et al., 1999) and increased BP in conscious sheep
(Nelson et al., 1987). Differences in these responses are probably explained by

different receptor expression, metabolism of 5-HT, uptake of 5-HT etc.

2. SERT in the Cardiovascular System

SERT functions as the major protein responsible for the transport of 5-HT
across membranes. It regulates the extracellular and intracellular 5-HT
concentrations and thus 5-HT functions. The study of SERT function in
cardiovascular system has been done by pharmacologically inhibiting SERT and
using genetically-altered mice.
A. Pulmonary Vasculature:

The physiological importance of SERT function is not well studied in many
cardiovascular-related organs. The best studied is the pulmonary vasculature.

In pulmonary endothelial and bovine smooth muscle cells, uptake of 5-HT via

26

SERT is necessary for 5-HT to function as a mitogen. Moreover, Morecroft et al.
demonstrated that citalopram and fluoxetine inhibited 5-HT-induced contraction
of pulmonary arteries isolated from the normal Sprague-Dawley rat (Morecroft et
al., 2005). In contrast, these agents potentiated contraction to 5-HT in pulmonary
arteries from Fawn-Hooded rats. The Fawn-Hooded rat is a strain with
genetically-impaired 5-HT storage and reuptake and, interestingly, is susceptible
to both pulmonary and systemic hypertension (Gonzalez et al., 1998). The
reason(s) for differences in the effects of the inhibitors in arteries from Sprague-
Dawley vs. Fawn-Hooded rats are not well understood. Citalopram also
potentiated contraction to 5-HT in the isolated intralobar pulmonary artery in an
endothelium-dependent manner in norrnoxic rats and endothelium-independent
manner in hypoxic rats (Wanstall et al., 2003). The fluramines (SERT substrate
and 5-HT releaser) also cause direct pulmonary vasoconstriction (Weir et al.,
1996; Desta et al., 1998) or potentiate vasoreactivity to other vasoconstrictors
(Barman and Isales, 1999). Collectively, these studies suggest that in the
pulmonary vasculature, SERT plays an important role in normal mitogenic and
contraction functions.

B. Platelet Function:

SERT inhibitors decrease platelet 5-HT content, inhibit thrombosis and
exacerbate bleeding (Maurer-Spurej, 2005) because of prevention of 5-HT
reuptake. However, an alternative argument has been proposed. Immediately
upon use of SSRIs, platelet 5-HT would not be depleted but extracellular 5-HT

would be higher because of preventing 5-HT reuptake, promoting a thrombotic

27

event (Kurne et al., 2004; lsbister et al., 2004). Thus, SERT inhibition may cause
dual effects in thrombosis.

The ability of promoter variants of SERT to influence function of the
cardiovascular system is illustrated by studies in platelets. Platelets from
humans which possess the L variant of the SERT promoter have a higher Vmax
than platelets from humans with no L variants (Greenberg et al., 1999).

C. Cardiac Function:

Compared to the tricyclic group of antidepressants, SERT inhibitors have
a decreased ability to induce cardiac arrthymias. However, citalopram has been
reported to increase the QT interval in humans (Kelly et al., 2004) and Pacher et
al. have published extensive work demonstrating the arrhythmogenic activities of
SERT inhibitors, specifically fluoxetine and citalopram, in the hearts of dogs,
rabbits, rats, and guinea pigs (Pacher et al., 1999; Pacher et al., 1998).

D. Other Cardiovascular Organs:

In the mouse adrenal medulla, SERT is required for stress-evoked
increases of catecholamine synthesis and Ang II AT2 receptor expression in the
adrenal gland (Armando et al., 2003).

The physiological function of SERT in other cardiovascular organs such as
the heart, peripheral vasculature, and kidney has not been thoroughly
investigated. One possible way to determine the role of SERT in cardiovascular
function is to examine the effect of removal or inhibition of SERT. A mouse,
which lacks a functional SERT was created by the group of Dr. Dennis Murphy.

Few studies on the physiological cardiovascular parameters in these animals

28

have been performed. Mice overexpressing SERT have recently been created.
Systemic arterial pressure was measured in these animals under anesthesia.
WIld type animals had a pressure of 76:34 mm Hg, while the SERT
overexpressing mice had a pressure of 82.9138 mm Hg. Similarly, heart rates
were not different between wild-type and SERT transgenic mice. These
measures may not reflect true blood pressure and heart rate due to the effects of
the anesthetic. Effects in the adrenal, kidney, brain and heart, as they pertain to
the cardiovascular system, were not noted, and thus questions as to the function

of SERT in these organs remain unanswered.

V. Serotonergic System in Cardiovascular Diseases

Evidence suggest that 5-HT plays roles in cardiac arrhythmias (5-HT4
receptor activation), coronary artery disease (raised blood 5-HT levels) and
vasospastic angina (increased transcardiac levels of 5-HT and supersensitivity of
the coronary artery to 5-HT) (Doggrell for review, 2003). The discoveries of
anorexigen-induced valvular heart disease (Connolly et al., 1997) and PPH
(Abenhaim et al., 1996) have driven the investigation of the association of 5-HT
in these diseases. A clinical study showed that the plasma concentration of 5-HT
and circulating levels of 5-HIAA are increased in PPH patients (Herve et al.,
1995; Kereveur et al., 2000). The loss of tph1 gene expression, and thus lack of
peripheral 5-HT was linked to a cardiac dysfunction phenotype (Cote et al.,
2003). This section focuses on 5-HT and SERT related cardiovascular diseases,

especially hypertension.

29

1. Serotonergic System and Myocardial Infarction (MI):

5-HT is normally released by activated platelets, causing vascular
contraction and enhanced platelet aggregation, which may lead to thrombus
formation and contribute to the pathogenesis of acute MI. On the other hand,
antidepressants, particularly the SSRls with high affinity for SERT, attenuate
platelet activation by depleting 5-HT storage in normal volunteers and in patients
with coronary artery disease (Hergovich et al. 2000; Serebruany et al., 2001).
More direct evidence is that the use of SSRIs with high affinity for the SERT is
associated with reduced odds of MI, and the extent of SERT inhibition among
SSRIs correlates with the degree of reduction in MI risk (Sauer et al., 2003).

The LL genotype of the SERT polymorphism is associated with a higher
risk of MI (Fumeron et al., 2002), SS genotype seems to have a protective role
against Ml, delaying the age of onset of the first episode (Coto et al., 2003). This
is in agreement with the evidence that inhibition of SERT reduced the risk for MI
(Sauer et al., 2003). Collectively, these studies confirmed that SERT activity (the
ability to regulate 5-HT concentration) is important in MI.

2. Serotonergic System and PPH:

5-HT and SERT have been reported to play an important role in the
pulmonary vascular smooth muscle hyperplasia and vascular remodeling and
have been associated with experimental hypoxic pulmonary hypertension and
human PPH. Consistently, the SERT inhibitors, citalopram and fluoxetine,
protect against hypoxic pulmonary hypertension (Marcos et al., 2003). Mice

lacking SERT (5-HTT") developed less hypoxia-induced right ventricular systolic

30

pressure increase and vascular remodeling than paired 5-HT'I"’+ controls
(Eddahibi et al., 2000). The LL genotype in SERT promoter was present in a
higher level in patients with PPH (Eddahibi et al., 2001) and chronic obstructive
pulmonary hypertension (Eddahibi et al., 2003).
3. Serotonergic System and Hypertension:

A finding common to experimental and genetic models of hypertension, as
well as in the hypertensive patient, is an increase in TPR. Although, 5-HT has
been recognized as a powerful vasoconstrictor for many decades, the
involvement of 5-HT in the etiology of hypertension is still controversial. The
following paragraphs lay out the evidence that support and refute the role of 5-HT
and SERT in hypertension.

A. Free Circulating 5-HT Concentration and 5-HT Receptors Activation
Evidence Refuting the Inant of the Serotonergic System in Hyeertension:
Free circulating plasma levels of 5-HT are relatively low (15-120 nmoI/L
compared with micromolar levels in whole blood, Martin, 1994). This free
circulating 5-HT is the 5-HT that would interact with the blood vessels, and thus it
has been argued that this is an insufficient level of 5-HT to activate 5-HT
receptors normally expressed (primarily 5-HT2A receptors on smooth muscle and
5-HT1 receptors in the endothelial cell). The role of the relatively newly
discovered vascular 5-HT7 receptor is less well understood.

Evidence Supporting the Involvement of the Serotonergic System in
Hyeertension: Enhanced 5-HT-induced arterial responses in hypertension has

been recognized as early as 1970. These effects have been observed as an

31

increased response to 5-HT including decreased threshold concentration of 5-HT
to induce contraction, increased potency (ECso) and efficacy in experimental
hypertension, including the mineralocorticoid (deoxycorticosterone acetate,
DOCA)—salt hypertension rats, spontaneously hypertensive rats (SHR), stroke
prone SHR, DahI-salt sensitive, 1 kidney-1 clip, nitric oxide synthase-inhibited
model of hypertension and even in coronary arteries from human patients
(Cohen et al., 1988; Collis and Vanhoutte, 1977; McGregor and Smirk, 1970;
Watts, 1998; Nishimura and Suzuki 1995; Golino et al., 1991). These changes in
hypertension have been defined as hyperresponsiveness or hyperreactivity.

The local and/or circulating level of 5-HT can be considered sufficient to
activate endogenous 5-HT receptors. The majority of 5-HT is stored in platelets.
Thrombotic events in which platelets aggregate can result in a high (micromolar)
local concentration of 5-HT (Holmsen and Weiss, 1979). Moreover,
subcontractile concentrations of 5-HT amplify arterial contraction to vasoactive
agonists such as Ang II, endothelin- 1 (ET-1) and NE, to name a few (Turla and
Webb, 1989; MacLennan et al., 1993; Yildiz et al., 1998). This is important as
low concentrations of 5-HT (nanomolar) are able to modify arterial contraction to
substances which control TPR (e.g. Ang II, ET-1 and NE).

Moreover, 5-HT has a significantly higher affinity for the 5-HT23 receptor
(KA =10 nmol/l), and this receptor is expressed in arterial smooth muscle.
Importantly, the arterial 5-HT23 receptor is expressed to a significantly greater
level in experimental hypertension and at least three 5-HT2.; receptor antagonists

have been reported to have antihypertensive effect and reduce vascular

32

reactivity to agonists in experimentally induced hypertension (Watts and Fink,
1999; Shingala and Balaraman, 2004). 5-HT1A and 5-HT7 receptor agonists were
suggested to have a role in the treatment of hypertension (Wainscott et al., 1996;
Doggrell, 2003) because 5-HT1A and 5-HT7 receptor activation are known to have
a vasodilatory effect.

Thus 5-HT has significant effects on the contractile state of vascular

smooth muscle directly and indirectly.

B. Uses of 5-HT Receptor Antagonists in the Treatment of Hypertension

Evidence Refuting the Involvement of the Serotonergic System in Hypertension:

 

There are a number of studies examining the effect of 5-HT receptor antagonists
in treating high BP which have had negative outcomes. A majority of this work
revolves around the use of ketanserin. This 5-HT2N20 receptor antagonist was
used as an effective antihypertensive strategy over two decades ago. Ketanserin
lowered BP of normal and hypertensive subjects, including humans, but BP
reduction was largely attributed to on adrenergic receptor blockade, not 5-HT2
receptor blockade (Cohen et al., 1983). Presently, ketanserin is used in a
population of women with severe pregnancy induced hypertension (van Schie et
al., 2002). Additional studies using 5-HT2A receptor antagonists that lacked
affinity for the a adrenergic receptors, such as cinanserin, did not lower BP in the
anaesthetized cat (McCall and Harris, 1987; Ramage, 1988). Lowering of BP in
anaesthetized SHRs by ketanserin or LY53857 seemed to be dissociated from 5-

HT2 receptor blockade (Docherty, 1989). In the hypertensive human (Stott et al.,

33

1988) and in SHRs (Gradin et al., 1985), use of ritanserin did not lower BP
(ritanserin lacks affinity for the 0:1 adrenergic receptor and has a high affinity for
the 5-HT2A receptor; http:l/pdsp.cwru.edulpdsp.asp). These studies suggest that
endogenous activation of 5-HT receptors is not important for maintaining
elevated BP.

Evidence Supporting the involvement of the Serotonergic Svstem in
Hypertension: Some other studies used 5-HT receptor antagonists as
antihypertensive therapy which dissociate the a adrenergic receptor blockade
caused by ketanserin from 5-HT2A receptor blockade or which demonstrate the
effectiveness of 5-HT receptor antagonists in lowering BP in the absence of
appreciable a receptor antagonism. This includes use of ketanserin in the SHR
(Balasubramaniam et al., 1993). One study (Wenting et al., 1984), published in
1984, demonstrated a reduction in high BP by ketanserin in humans by 22%.
This same concentration of ketanserin was not able to reduce a pressor
response to the a receptor agonist phenylephrine (PE), indicating that the
reduction in BP caused by ketanserin was independent of a adrenergic receptor
blockade. Consistent with the in vitro observation that the 5-HT23 receptor
antagonist, LY272015, reduces 5-HT—induced vasoconstriction in arteries from
DOCA-salt hypertensive rats, but not normotensive rats (Russell et al., 2002;
Watts et al., 1996). LY272015 also lowers blood pressure in hypertensive rats
but not normotensive rats (Watts and Fink, 1999). There are also specific forms

of hypertension that have been described as 5-HT dependent. This includes an

34

erythropoietin-driven model of hypertension (Azzadin et al., 1995) and

cyclosporine-induced hypertension (Krygicz et al., 1996).

C. High L-tryptophan Diet and Blood Pressure

Evidence Refuting the Involvement of the Serotonergic System Mvpertenm
Studies using L-tryptophan feeding in SHRs demonstrated a dose-dependent
decrease in BP (Sved et al., 1982), as did studies in DOCA-salt rats (Fregly et
al., 1987) and in humans (Woittiez et al., 1995). L-Tryptophan is the necessary
precursor for 5-HT synthesis and should elevate 5-HT synthesis through law of
mass action. Thus, if 5-HT endogenously increases BP, tryptophan should also
do so by virtue of increasing endogenous 5-HT. In the later study, salt intake
was reduced by tryptophan, suggesting an activity to decrease elevated BP.
Evidence Supporting the Involvement of the Serotonergic Svstem in
Hypertension: A number of studies suggest that tryptophan feeding, as a means
to increase 5-HT synthesis, may promote higher BP. SHRs given L-tryptophan in
their diet had an increased BP within 30 min of administration, and a maximal
elevation of BP 60 min post-administration (Howe et al., 2003). In a study
published in 1991, Ito et al. gave tryptophan to female stroke-prone SHRs prior to
mating, and discovered that the offspring of these mothers had a higher. BP, were
heavier and had a higher CNS load of 5-HT (Ito et al., 1991). These findings
suggest, but do not prove, that an increased 5-HT level was associated with a

higher systemic BP.

35

D. TPH Function Inhibition and Blood Pressure

Evidence Refutithhe Involvement of the Serotonergic Svstem in Hypertension:
Depletion of 5—HT by PCPA, an irreversible inhibitor of TPH, did not lower the BP
of SHRs (Collis and Vanhoutte, 1981).

Evidence Supporting the Involvement of the Serotonergic Svstem in
Hypertension: A few studies on removing or destroying 5-HT have demonstrated
a concomitant decrease of BP in hypertensive animals. In 1976, Buckingham et
al. (Buckingham et al., 1976) demonstrated that a single injection of 400 mg/kg of
body weight (intraperitoneally) of the TPH inhibitor PCPAME (p-
chlorophenylalaninemethyl ester) produced a fall in BP in DOCA-treated rats
ranging from 20—43 mmHg within the first day of injection, and kept BP
depressed for 8 days. PCPAME competes directly with TPH and binds
irreversibly to the enzyme, hence the long-lasting effects of PCPAME. In the
normal rat, a fall of 15—20 mmHg was observed only 8 days after injection of
PCPAME; there was no immediate fall in BP. No measurements of 5-HT were
made in plasma or arteries of the rats receiving PCPAME, and we propose that
part of the fall in BP was because 5-HT was depleted in the periphery. This idea
is supported by the findings that central 5-HT depletion by intracisternal injections
of the serotonergic neurotoxin 5,6-DHT (5,6-dihydroxytryptamine) did not alter
the course of development or magnitude of hypertension achieved in the DOCA-
salt rat (Myers et al., 1974) or SHRs (Browning et al., 1981), nor did 5,6-DHT
(i.c.v) alter the ability of PCPA to cause a reduction in BP (Buckingham et al.,

1976)

36

E. Other Evidence Supporting the Involvement of Serotonergic System in
Hypertension

In hypertension, platelet function and SERT activity are changed.
Opposite observations of how platelet function changes in hypertension have
been reported. A study in human essential hypertension indicated that platelets
in the early stages of essential hypertension display an overall increased
aggregation potential with an elevated cytosolic free calcium level (Taylor et al.,
1989). More recently, increased platelet aggregability was found in hypertensive
patients with carotid artery plaque, suggesting a further promotion of the
progression of arteriosclerosis in those patients (F usegawa et al., 2006).

However, other studies, such as in the stroke-prone SHR, showed that
aggregation of platelets from the hypertensive rat is decreased compared to
normotensive controls (Tomita et al., 1984). In DOCA-salt hypertensive rats,
platelet aggregation is decreased compared to normotensive controls (Umegaki
and lchikawa, 1993). The outcome of a majority of studies is that the content of
5-HT in platelets is decreased. In theory, these platelets, termed ‘exhausted’
(depleted due to overstimulation), have already been activated early in vivo due
to hypertension (Umegaki and lchikawa, 1993). In the human, platelets from
hypertensive subjects have a lower 5-HT content (Ding et al., 1994) and take up
less 5-HT than do platelets from normotensive patients (Kamal et al., 1984).

There are still some other hints suggesting that SERT function is changed
in hypertension. Plasma 5-HT concentration and the Vmax values for the 5-HT

uptake process were significantly higher in severely pre-eclamptic women,

37

compared with age and gestation-matched normal pregnant women and plasma
5-HT concentration was directly related to systolic and diastolic BP with severity
of the syndrome (Carrasco et al., 1998). Thus, it is conceivable that a changed
SERT activity and changed 5-HT concentration could happen in other types of

hypertension and related to BP increase or maintain BP at a high level.

F. An Unanswered Question ---- The Existence of Local Regulation of 5-HT
in Peripheral Arteries

The evidence presented suggests the ability of endogenous 5-HT to
modulate vascular smooth muscle tone and thereby to alter TPR and BP.

Any process changing 5-HT concentrations—such as synthesis,
metabolism, uptake and storage/release will therefore regulate 5-HT functions. It
is important to confirm/refute that peripheral arteries have local regulation of 5-

HT concentration and functions.

38

Hypotheses:

Hypothesis #1 (Figure 3):

A local serotonergic system, including synthesis, metabolism, storage, uptake

and release of 5-HT is present and functional in peripheral vasculature.

Hypothesis #2:

Changes in the localized serotonergic system contribute to the increase of blood

pressure in hypertension.

39

Figure 3.

Diagram of working hypotheses.

5-HlAA=5—Hydroxyindole acetic acid, 5-HT=5-hydroxytryptamine, 5-HTP=5-
hydroxytryptophan, AADC=amino acid decarboxylase, MAO A=monoamine

oxidase A, TPH= tryptophan hydroxylase.

40

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41

Methods:
I. Animal Uses

All animal procedures were followed in accordance with the institutional
guidelines of Michigan State University. Normal male Sprague-Dawley rats (225-
250 g) were purchased from Charles River (Portage, MI) or Harlan Industries,
Inc. (Indianapolis, IN). Male SHR and VWstar-Kyoto rats (WKY, 12 weeks) were
purchased from Taconic Farms, Inc. (Gerrnantown, NY). Normal male CS7BLI6
mice (20-24 g) were purchased from Charles River (Portage, MI). The SERT
targeted mutation mice and wild type male mice (C57BU6; 30-40 g) were
received from Dr. Dennis L. Murphy, National Institute of Mental Health. The
Tph1 -/- mice and wild type mice (C57BU6, 20-24 g) were received from Dr.
Michael Bader, Max Delbriick Center for Molecular Medicine (Germany). Until
surgery, the rats were kept in clear plastic boxes with free access to standard rat

chow (Teklad ®) and tap water.

II. Euthanasia:
Rats were anesthetized with pentobarbital (60 mg/kg, i.p.). Mice were

sacrificed by asphyxiation with carbon dioxide.

III. Animal Models:
Mineralocorticoig Hypertension:
Male Sprague-Dawley rats (250-300 9; Charles River, Portage, MI) were

anesthetized with isoflurane (IsoFlo®). Animals were uninephrectomized and a

42

Silastic® (Dow Corning, Midland, MI) implant impregnated with DOCA pellet (200
mg/kg) was placed subcutaneously on the back of the neck. Postoperatively,
rats were given a solution of 1% NaCI and 0.2% KCI for drinking. Sham rats
also received a uninephrectomy, but received no DOCA implant and drank
normal tap water. Animals were fed standard rat chow and had ad Iibitum
access to food and water. The animals remained on the regimen for four weeks

prior to use.

L-NNA Hypertension

Male Sprague-Dawley rats (250-300 g; Harlan, Indianapolis, IN) were
given tap water mixed with Nw—nitro-L-arginine (L-NNA, 0.5 g/L). The animals

were remained on the regimen for two weeks prior to use.

6-OHDA Denervetion

Sympathetic neuronal denervation was induced by 6-hydroxydopamine (6-
OHDA) injections (McCafferty et al., 1997). Male Sprague-Dawley rats were
treated with four doses of 6-OHDA over 7 days (50 mg/kg on days 1 and 2 and
100 mg/kg on days 6 and 7; 0.1% ascorbic acid in physiological saline as vehicle
i.p.). Rats were euthanized (pentobarbital, 60 mg/kg i.p.), and arteries were
removed on day 8. Denervation was validated by glyoxylic acid staining in
mesenteric resistance arteries. Glyoxylic acid staining: Mesenteric resistance
arteries were removed. After fat was trimmed off, arteries were immersed into

glyoxylic acid (2%) for 5 min. Blood vessels were mounted on a microscope slide

43

and placed in an oven (100°C) for 5 min. The slides were removed and blood
vessels mounted in mineral oil and cover-slipped. Vessels were viewed using a
fluorescence microscope (Nikon LABOPHOT) and UV illumination (G-1A;
excitation, 546/10 nm; barrier filter, BA580). The absence of a fluorescent
network of staining of catecholamines in arteries confirms the effectiveness of 6-

OHDA in causing sympathetic denervation.

IV. BP Measurements:

Systolic BP of conscious rats were determined by the tail cuff method
using a pneumatic transducer. Briefly, the rat was placed in a plastic pail with
wood shavings covering the bottom. This pail was then placed on a heating pad
and the rat was contained in the bucket by a small metal cage. A warming light
was placed over the bucket. The rat was warmed for approximately 6 min. This
allowed vasodilatation of the tail artery, which facilitated the measurement of the
BP. The rat was placed in a restraint and the blood pressure cuff and balloon
transducer was placed on the tail and secured with tape. The BP was measured
utilizing a sphygmomanometer in conjunction with the pulse transducer. Three

blood pressure measurements were taken to obtain an average measurement.

V. 5-HT Basal Level Measurement

Aorta from normotensive and hypertensive animals were dissected,

cleaned and placed in 75 IIL of 0.05 mM sodium phosphate & 0.03 mM citric acid

44

buffer (pH 2.5) containing 15% methanol. Samples were frozen at -80°C at least

4 hours.

VI. TPH Activity Assay
In vivo 5-HT §vnthefi

Aorta, superior mesenteric artery and hypothalamus from NSD1015 (100
mg/kg, 30 min) or NSD1015 (100 mg/kg) + L-tryptophan methyl ester
hydrochloride (300 mg/kg, 60 min)—treated rats were dissected, cleaned in
physiological salt solution (PSS, 103 mM NaCl; 4.7 mM KCL; 1.18 mM KH2PO4;
1.17 mM MgSO4-7H20; 1.6 mM CaCl2-2H20; 14.9 mM NaHCO3; 5.5 mM
Dextrose, and 0.03 mM CaNazEDTA) and placed in tissue buffer [0.05 mM
sodium phosphate & 0.03 mM citric acid buffer (pH 2.5) containing 15%
methanol]. Samples were frozen at —80 °C until assay.
In vitro 5-HT synthesis

Aorta, superior mesenteric artery and dorsal raphe nucleus from normal
rats were dissected, cleaned in incubation solution [100 uM Fe(NH4)(SO4)2, 1
mM DTT, 50 mM Tris-HCI, pH 7.4]. Tissues were incubated in 1.5 ml plastic
centrifuge tubes with incubation solution plus 3250 U/ml catalase and 10 mM
NSD 1015 at 37 °C for 30 min. Then 200 uM BH4 and either H20 or 400 uM
tryptophan were added. After 40 min incubation, tissues were rinsed in drug-free

PSS, placed in 75 pL tissue buffer and saved at —80 °C until assay.

45

VII. 5-HT Storage Assay

At room temperature, naive or 5-HT-Ioaded (5-HT 1 uM, 15 min) aortae
from untreated or MAO A inhibitor pargyline (100 mg/Kg)-treated rats were
incubated with PSS in 1.5 ml plastic centrifuge tubes for 4 or 8 hours. Tissues
were then briefly dipped In drug-free PSS and placed in 75 uL tissue buffer.

Samples were frozen at —80 °C until assay.

VIII. 5-HT Uptake Assay:

At room temperature (unless otherwise specified), dissected and cleaned
arteries from normal or pargyline (100 mg/Kg)—treated animals were placed in
PSS in 1.5 ml plastic centrifuge tubes containing either vehicle or inhibitor for 30
min. 5-HT (1 uM) or vehicle (water) was then added for a varying amount of time
(time course studies) or for 15 min in experiments with inhibitor. Tissues were
then briefly dipped in drug-free PSS and placed in tissue buffer. Samples were
frozen at —80 °C until assay. In some experiments, the endothelium was removed

by gently rubbing the luminal face of the artery with a moistened cotton swab.

IX. 5-HT Release Assay:

At room temperature, dissected and cleaned arteries from normal or
pargyline (100 mg/Kg)-treated animals were placed in 100 uL PSS or SERT
inhibitor (diluted in PSS) in 250 uL microcentrifuge tubes for 30 min. Water or
(+)-norfenfluramine (1O uM), in some experiments (+)-fenfluramine (1, 10 uM),

was then added for 20 min. Tissues were taken out of the tubes and the PSS

46

solution in tube was saved on ice (PSS samples) and used to test 5-HT and 5-
HIAA release. Tissues were briefly dipped in drug-free PSS and placed in tissue
buffer (tissue sample, used to test 5-HT and 5-HIAA left in tissues). PSS

samples and tissue samples were frozen in -80 °C until assay.

X. 5-HIAA and 5-HT Measurement:

Samples were thawed, sonicated for 3 seconds and centrifuged for 30
seconds (10,000 g). Supernatant was collected and transferred to new tubes.
Tissue pellets were dissolved in 1.0 M NaOH and assayed for protein.
Concentrations of 5-HIAA and 5-HT in tissue supernatants and PSS samples
from 5-HT release assay were determined by isocratic High Performance Liquid
Chromatography (HPLC) coupled with electrochemical detection. Fifty
microliters of tissue supernatant or PSS sample was injected onto a C18 reverse
phase analytical column (Biosphere ODS, West Lafayett, IN) protected by a
precolumn cartridge filter. This column was coupled to a single coulometric
electrode conditional cell in series with dual electrode analytical cells (ESA,
Bedfore, MA). The conditioning electrode potential was set at 0.4V, while the
analytical electrodes were set at 0.12 and —0.31 V relative to the internal silver
reference electrodes. Amounts of 5-HIAA and 5-HT were determined by
comparing peaks areas in samples with those obtained from standards run the
same day, and reported as a concentration relative to protein content. The lower

limit of sensitivity for detection of 5-HIAA and 5-HT was 2-5 picogram/sample.

47

XI. Real Time RT-PCR:

Two-step RT—PCR was performed using a GeneAMP 7500 Real Time
PCR machine (Applied Biosystems, Foster City, CA). Total RNA from rat aorta
and superior mesenteric artery were isolated using the MELT Total RNA isolation
System Kit (Ambion, Austin, Taxes). Concentration of RNA was measured
spectrophotometrically (A260/A280). One microgram of total RNA was reverse
transcribed using a Taqman® reverse transcriptase kit (Applied Biosystems,
Foster City, CA, USA; buffer, MgCIz 5.5 mM, dNTP 500 uM, of each random
hexamer 2.5 uM, RNase inhibitor 0.4 pg/uL and MultiScribe Reverse
Transcriptase 1.25 ug/uL; 10 min hold at 25°C, 30 min hold at 48°C, 5 min hold
at 95°C). One microliter of this cDNA was taken through PCR using a SYBR®
Green Master Mix (Applied Biosystems, Foster City, CA, USA). PCR conditions
were: 95°C 10 min for AmpliTAQ® activation, 40 cycles of PCR (15 seconds
95°C, 60 seconds 60°C). Two sets of rat tph1 primer pairs were used. One of
the rat tph1 primer pair, rat [it-actin primer pair and B-2-microglobulin (B2m) pair
were purchased from Superarray (Frederick, MD). An alternative rat tph1 primer
pair was designed based on RefSeq Accession number: XM-341862.1 and was
synthesized by the Macromolecular Structures and Synthesis Facility at Michigan
State University. The rat tph1 primer fon~ard= GCC TGC TTT CTT CCA TCA
GT. The rat tph1 primer reverse= AGA CAT CCT GGA AGC 'ITG TGA. The
SERT primer forward= GGC CAG TAC CAC CGA AAC, SERT primer reverse =
CGG GGC AGA TCT TCC TCC ATA T.

48

CT values were derived as the threshold cycle at which product was first

detected and are reported as cycle numbers.

XII. lmmunohistochemistry:

Prepagtion of frozen sections: Tissues were frozen in OCT compound and
stored at -80°C until use. Sections were cut, cold acetone fixed, washed 3 times
with phosphate-buffered saline (PBS).

Prepaflttion of paraffin-embedded sections: Formalin-fixed, paraffin-embedded
rat thoracic aorta and superior mesenteric artery were washed twice in xylenes
and four times in 90% ethanol for 3 min each to dewax. Sections were
unmasked by microwaving them twice for 3 min in Vector Antigen Unmasking
Solution.

Endogenous peroxidase in fixed frozen sections or dewaxed, unmasked
paraffin-embedded section were blocked [0.3% H202 in phosphate-buffered
saline, PBS, for 30 min]. Sections were blocked for non-specific binding in PBS
containing 1.5% of competing serum. In a humidified chamber, samples were
incubated overnight with antibody (10 pg/mL, anti-TPH antibody, Sigma; 5 ug/mL
anti-SERT, C-20, Santa Cruz; 5 pg/mL, anti-5-HT YC5/45, Abcam, UK), antibody
neutrialized with 5-fold excess of competing peptide or blocking serum with
antibody. The remaining steps were carried out in accordance to the
manufacturer’s instructions (Vector Laboratories, Burlingame, CA, USA).
Sections were washed 3 times with PBS and incubated with a peroxidase-

conjugated secondary antibody (30 min, room temp). Samples were washed and

49

incubated with Vectastain® ABC Elite reagent (30 min, room temp) followed by
3,3’-diaminobenzidine (DAB)/H2O2. The reaction was stopped with washing,
sections were air dried, hematoxylin-stained, mounted and photographed using

an inverted Nikon microscope with a Spot digital camera.

XIII. lmmunocytochemistry:

Rat thoracic aorta and superior mesenteric artery were dissected and cut
into small rings in chilled dissociation solution [D8, containing (M): NaCI 0.137;
KCI, 0.0056; MgCl2, 0.001; Na2HPO4, 0.00042; NaH2PO4, 0.00042; NaHCO3,
0.0042; Na nitroprusside 2.6 mg/L and HEPES, 2.383 mglL; pH=7.4]. Small
vessel rings were incubated with enzymatic solution (containing papain, 26 U/ml
and DTT, 1 mg/ml) for 35 min and other enzymatic solution (containing, type II
collagenase 2.5 U/ml, elastase 0.15 mg/ml and soybean trypsin inhibitor, 1
mg/ml) for 45 min. We then draw off other enzymatic solution and gently added
3 ml of fresh DS and left on ice for 5 min, which was followed by draw off 08
without disturbance of the cells at the bottom. The cells were then triturated in
D8 containing Na nitroprusside (0.01 mM). One hundred uL of titrated cells were
put on polylysine coated coverslips and left in incubator (37°C, 5% CO2) for 30-
45 min. After the cells attached to coverslips, the DS was carefully removed and
the cells were fixed with Zamboni’s fixative (20 min). The cells were rinsed with
low-salt PBS twice (5 min each time) and cell membrane was perrneablized with
1% triton (5 min) followed by blocking serum incubation (5% serum, 20 min).

Cells were then incubated with primary antibody for 2 hours (37°C, anti-5-HT

50

antibody, anti-TPH antibody, 1:200, Sigma, anti-SERT antibody 1:200). Cells
were washed with low-salt PBS three times, followed by 1 hour incubation with
goat-anti-mouse Cy3 antibody or donkey-anti-goat Cy3 antibody (37°C, 1:1000,
Jackson ImmunoResearch). The coverslips were then mounted and
photographed using an inverted Nikon microscope or a confocal microscope with

a digital camera. Images in this dissertation are presented in color.

XIV. Protein Isolation:

Rat/mouse thoracic aorta and rat superior mesenteric artery were
removed from the animal and placed in PSS and cleaned as described above.
Arteries were quick frozen and pulverized in a liquid nitrogen-cooled mortar and
pestle and solubilized in lysis buffer [0.5 M Tris HCI (pH 6.8), 10% SDS, 10%
glycerol] with protease inhibitors [0.5 mM phenylmethylsulfonyl fluoride, 10 uglul
aprotinin and 10 ug/ml Ieupeptin]. Homogenates were centrifuged (11,000 g for
10 min, 4 °C). We utilized the Bicinchoninic Acid (BCA) method for protein

measurement (Sigma, St.Louis, MO). Samples were stored at —80 °C until use.

XV. BCA Protein Assay:

The bovine serum albumin (BSA) protein standard, consisting of a known
concentration of BSA, was utilized to make the standard curve to which the
protein samples were compared. The working reagent was made by mixing BCA

with Copper (II) Sulfate (50:1). To determine the protein concentrations of

51

samples, 5 pL protein from each sample, 95 pl H20 and 2 mL working reagent
were mixed and incubated for 30 min at 37 °C, no C02. The samples were
analyzed on a spectrophotometer at an absorbance of 562 nm and the protein

concentration determined by plotting these values on the standard curve.

XVI. Western Blotting:

Tissues homogenates (4:1 in denaturing sample buffer, boiled for 5 min)
were separated on SDS-polyacrylamide gels and transferred to Immobilon-P
membrane. Membranes were blocked for 3-4 hours (Tris Buffered Saline, TBS,
4% chick egg ovalbumin or 5% dry milk, 0.25% sodium azide). Blots were
probed overnight with primary antibody (4 °C), rinsed in TBS-Tween (TBS-T, pH
7.6) (20 mM Tris, 137 mM sodium chloride and 0.1% Tween-20), with a final
rinse in TBS (pH 7.6) (20 mM Tris and 137 mM sodium chloride) and incubated
with secondary antibody for 1 hour at 4 °C following with TBS-T and TBS

washes. Blots were incubated with ECL® reagents to visualize the bands.

XVII. Isolated Smooth Muscle Contractility Measurement:

The thoracic aorta was removed and placed in PSS. The aorta was
cleaned of fat and connective tissue and cut into helical strips. Aortic strips were
then mounted onto stainless steel rod holders and placed into 50 ml tissue baths
for isometric tension recordings using Grass polygraphs and transducers. Strips
were placed under optimum resting tension (1,500 mg for rat aorta, determined

previously) and allowed to equilibrate for one hour before exposure to

52

compounds. In experiments testing tissues from hypertensive animals, one
aortic strip isolated from a normotensive control and one aortic strip from a
hypertensive rat were placed in the same bath, thereby controlling for potential
experimental variations. Tissue baths contained warmed (37 °C), aerated (95%
02/002) PSS. Administration of an initial concentration of 10 pM PE was used to
test arterial strip viability; the strips must contract to a minimum of 500 mg for rat
aorta to be considered viable. To examine the status of the arterial endothelium,
tissues were contracted with a half-maximal concentration of PE (10-100 nM)
and once the contraction plateaued, the muscarinic agonist acetylcholine (ACh, 1
uM) was administered. The observation of a relaxation to ACh greater than 60%
of the PE (10-100 nM) -induced contraction was considered as endothelium-
intact. Cumulative concentration curves were performed to agonists.
Antagonists, inhibitors or vehicle were incubated with the vessels for one hour

prior to the experimentation.

XVIII. Data Analysis and Statistics:

Data are presented as means :I: standard error of the mean for the number
of animals in parentheses.

5-HIAA and 5-HT concentrations were quantified using standards run the
same day, and reported as a concentration relative to protein content.

Contractions are reported as force (milligrams) or as a percentage of
response to maximum contraction to PE. ECso values (agonist concentration

necessary to produce a half-maximal response) were determined using non-

53

linear regression analysis in Prism version 4.0 (San Diego, CA) and were
reported as the mean of the negative logarithm (-log) of the EC50 value (M).
These values represent the concentration necessary to produce a half-maximal
response in each tissue using the tissue’s own maximum response.

Band density quantitation in Western analyses was performed using NIH
Image (v.1.61). For each sample, the densities of the tested bands on Western
blotting are normalized to the density of the corresponding actin band.

When comparing two groups, the appropriate Student’s t-test was used.
When comparing 3 or more groups, an ANOVA followed by Newman Keuls post
hoc test was performed. In all cases, a p value less than or equal to 0.05 was

considered statistically significant.

54

Results

Hypothesis #1:
A local serotonergic system, Including synthesis, metabolism,
storage, uptake and release of 5-HT is present and functional In peripheral

vasculature.

We investigated the serotonergic system in peripheral arteries on the
following aspects: (1) presence of 5-HT; (2) 5-HT synthesis and metabolism; (3)
5-HT storage; (4) 5-HT uptake and release.

I. The Presence of 5-HT in Peripheral Arteries

Whether peripheral arteries contain endogenous 5-HT has not been
reported. We used three different experimental techniques, which provided the
same results suggesting the presence of 5-HT in peripheral arteries.

By using HPLC analyses, we detected the content of 5-HT and its MAO-A
metabolite 5-HIAA in aorta, superior mesenteric artery and carotid artery. Figure
4 shows a standard chromatogram (Top, a representative of all standard tracings
in data from HPLC) and a chromatogram of basal 5-HT and 5-HIAA in rat
thoracic aorta (bottom). The quantification of basal 5-HIAA and 5-HT levels in rat
aorta and superior mesenteric artery and mouse aorta are reported in Figure 5.

Figure 6 depicts the results of IHC experiments using a 5-HT antibody,

localizing 5-HT to rat aorta. The presence of 5-HT in peripheral arteries was

55

6 Mix Standard

:25.

1:7"

 

 

w 19.175

5-HIAA

awry

” { 7 3m 19.319
{r 5-HT

TII‘IET'FIBLE ST 0P

 

Basal Level 5-HIAAI5-HT in Rat Aorta

 

-I L———~— to .391
§' é 5-HIAA
ID

(.2 19.724
- rrrrerasu: srops'HT

 

Figure 4.

Top: Chromatogram showing separation of 1 ng standards using HPLC.

Bottom: Detection of basal levels of 5-HIAA and 5-HT in thoracic aorta.

5-HIAA = 5-hydroxyindoleacetic acid. 5-HT = 5-hydroxytryptamine.

56

Figure 5.

Basal levels of 5-HIAA and 5-HT in rat aorta (top), rat superior mesenteric artery
(middle) and mouse aorta (bottom) from normal animals.
5-HlAA=5—hydroxyindoleacetic acid, 5-HT=5-hydroxytryptamine, SMA=superior

mesenteric artery.

57

Rat Aorta

(N=7I

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Mouse Aorta

(N=4)

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58

supported by staining of the aorta, throughout the smooth muscle layers and
adventitia, with an antibody that recognizes 5-HT (Y05/45, Figure 6, bottom left).
Staining was significantly diminished when the antibody was pre-incubated with a
5-fold excess of 5-HT itself (Figure 6, bottom right). The dark precipitate formed
by DAB in serotonergic nerves of the guinea pig ileum myenteric plexus (Figure
6, top) was used as a positive control as this tissue contains a rich innervation of
serotonergic nerves (Galligan et al., 1986). To eliminate the possibility that the 5-
HT we observed in aorta section is actually in mast cells rather than in smooth
muscle cells, we used Giemsa’s Stain to test the existence of mast cells in rat
aorta. The aorta only rarely presented with recognizable mast cells while this
staining detected abundant mast cells in a human lymph node (data not shown).
Thus, it is unlikely that aortic 5-HT comes from mast cells.

Intracellular staining of 5-HT using the 5-HT antibody (goat anti-rabbit 5-
HT, Serotec, Raleigh, NC) and a fluorescent Cy3 secondary antibody in freshly
isolated aortic smooth muscle cells further confirmed the existence of
endogenous 5-HT in arterial smooth muscle (Figure 7, B). Using the same
exposure time, no fluorescence or very low fluorescence was observed in cells
with only secondary antibody incubation (Figure 7, C). The staining of a-actin

verified that the cells were smooth muscle cells (Figure 7, A).

59

Figure 6.

Top: Positive control for use of the 5-HT antibody (YC5/45, Abcam); guinea pig
ileum myenteric plexus contains identifiable 5-HT containing neurons marked
with the arrow.

Bottom: Immunohistochemical staining for 5-HT in aorta from normal rats. Aortic
section incubated with 5-HT primary antibody (left) compared with section
incubated with primary antibody prequenched with 5-HT. Arrows indicate the
placement of staining. Representative of 4 different experiments/rats.

5-HT = 5-hydroxytryptamine.

60

 

Presence of 5-HT in Rat Aorta

 

Guinea Pig lleum
Myenteric Plexus

 

POSItIve Controlfor 5- HT

Rat Aorta
5-HT Primary +,

    

5-HT‘ Prima

  

fig; ' '
fl“ ’V’
I" ' f -’
5}” ' Lumen

 

 

Figure 6.

61

Figure 7.

Freshly dissociated rat aortic smooth muscle cells stained with anti-a-actin
antibody (A), with anti-5-HT antibody (B) or secondary antibody alone (C).
Representatives of four separate experiments, each with a different rat.

5-HT = 5-hydroxytryptamine.

62

Presence of 5-HT in Freshly Isolated Rat
Aortic Smooth Muscle Cells

Rat Aorta

 

No primary

 

 

 

Figure 7.

 

63

II. The Presence of 5-HT Synthesis and Metabolism in Peripheral
Arteries

To investigate the synthesis and metabolism system in peripheral arteries,
we tested the existence and activity of enzymes that are important for serotonin
synthesis and metabolism: TPH, AADC and MAO A.
1. The Existence of TPH1 mRNA and Protein in Peripheral Arteries

We first investigated the existence of mRNA in peripheral arteries. Two
sets of rat tph1 primer pairs were used in our experiments. The first rat tph1
primer pair was purchased from Superarry (Frederick, MD). This primer was
designed according to RefSeq Accession #: XM_341862.1. We also designed
another rat tph1 primer. The sequences were rat tph1-L:
GCCTGCTI'TCTTCCATCAGT and rat tph1-R: AGACATCCTGGAAGCTTGTGA.
Figure 8 A. shows the amplification plots of tph1 and the housekeeping gene (3-2—
microglobulin (B2m) in rat aorta and superior mesenteric artery. CT values were
measured as the value at which measurable product was first observed in real
time RT-PCR. The CT values for tph1 and B2m in rat aorta and superior
mesenteric artery are reported in Table 1. Figure 8 B. shows the dissociation
curve for tph1 (both primers) in aorta and superior mesenteric artery. Only one
peak was observed in each of the dissociation curve, indicating the single
product of tph1 primer.

The translation of tph1 mRNA was confirmed by the immunocytochemistry
experiments in which we observed an expression of TPH protein in freshly

isolated aortic and superior mesenteric smooth muscle cells (Figure 9, top) using

64

Figure 8.

A. Real time RT-PCR amplification curves of tph1 mRNA expression in normal
rat aorta (top) and superior mesenteric artery (bottom) using tph1 primer
purchased from SuperArray® (left, Frederick, MD) or using tph1 primer designed
by us (right).

B. Real time RT-PCR dissociation curves of tph1 mRNA expression in normal
rat aorta and superior mesenteric artery using tph1 primer purchased from

SuperArray® (top, Frederick, MD) or using tph1 primer designed by us (bottom).

Table 1.

The CT values in real time RT—PCR experiments detecting the expression of tph1

and B-2-microglobulin mRNA in rat aorta and superior mesenteric artery.

B2m=8—2-microglobulin, tph=tryptophan hydroxylase, SMA=superior mesenteric

artery.

65

A.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

tph1
SuperArray
1.0e+001 . -. 1.06.00,
1-°e*°°° W" 1.0e+000 '
.59 ' to
E 1-09'003 251.012003
0 w ,. , 0
1.019004» " 1.06004
1.0e-oos~-~i——'— '1 .. « 1.064305 . , .
Rat Aorta Rat Aorta
107006 5 15 25 35 1-°°‘°°° 5 15 25 35
Cycles Cycles
tph1
S erArra tph1
1 08+001~~ up y 1,0e+001-- - .-
1 .Oe+000 1 .0e+000
1 .0e-001 1.0e-001
é 1.0e-002 0:: 1.09002
9 1.05003 9 105003
8 1.05004 8 '
1.05005 1-06-004
mm“ 1 .0e-005 ,‘
"°°°°° Rat 8m Ralt SMA l
- 1.0 005 -
1'06 007 5 15 25 35 e“ 5 15 25 35
Cycles Cycles

Figure 8.

66

tph1
SuperArray

Table 1.

Derivative

tph1

Derivative

Dissociation Curve

 

0.40

 

 

 

0.35 I

 

0.30

 

0.25

 

0.20

 

0.15

 

0.10

 

0.05

 

 

 

-0.00

 

 

 

 

 

 

 

 

-o.05 ‘
so 65 7o 75 so 85 90 90

Temperature

Dissociation Curve

l

0.40

 

l .
0.35 ., j .

 

0.30
0.25 .. “We - _-

0.20

 

 

0.15-E . g...-

 

0.10 Em.--“- - .. -____,-

0.05 -._c.

 

 

-0.00

 

 

 

 

 

 

-0.05 ‘
60 65 70 75 80 85 90 90

Temperature

Figure 8.

 

tph1 (SuperArray) tph1 B2m

 

Rat Aorta
SMA

24.922057
27.02:1.06

28.77::033 16.811025
30.351021 177020.32

 

67

Figure 9.

A. Freshly dissociated rat aortic smooth muscle cells (top) and superior
mesenteric arterial smooth muscle cells (bottom) stained with anti-TPH antibody.
Representatives of four separate experiments, each with a different rat.

B. Immunohistochemical staining of the TPH (Sigma) in smooth muscle between
cables of elastin/collagen in the rat aorta frozen section. Parallel sections were
incubated with antibody (left) or no primary antibody (right). Arrows indicate the
placement of staining.

TPH= tryptophan hydroxylase, SMA=superior mesenteric artery.

68

Presence of TPH Protein in Freshly Isolated
A. Arterial Smooth Muscle Cells

 

Rat
Aona

 

Rat
SMA

 

 

 

 

B. Presence of TPH Protein in Rat Aorta

 

Rat Aorta
TPH Primary Primary

Lumen

     

t
t 1 so»

 

 

 

 

Figure 9.
69

an anti-TPH monoclonal antibody (Sigma, St Louis, MO, USA) and a fluorescent
anti-mouse secondary antibody, which confirmed the existence of TPH protein in
arterial smooth muscle. Again, smooth muscle cells were confirmed by a-actin
staining (data not shown). No fluorescence or very low fluorescence was
pictured in cells without primary antibody incubation (data not shown). We also
did IHC experiments testing the expression of TPH in artery sections using the
same anti-TPH antibody. However, we did not get very convincing results.
Relatively darker DAB precipitates appeared in sections with primary antibody
incubation (Figure 9, bottom left) compared to control sections (Figure 9, bottom

right).

2. The Activity of TPH in Peripheral Arteries

To test whether the arterial TPH is functional, we measured 5-HTP levels
in peripheral arteries and in hypothalamus from NSD 1015 (100 mglKg, 30 min)-
treated rats. NSD 1015 is an inhibitor of AADC and blocks the conversion of 5-
HTP to 5-HT. Hypothalamic tissues, that contains terminals of central
serotonergic neuron terminals, were used as positive control for this experiment.
Using HPLC we found non-detectable 5-HTP in aorta, fairly low amount of 5-HTP
in superior mesenteric artery (003510.021 ng/mg protein) and 1612026 ng/mg
protein of 5-HTP in hypothalamus. In all three tissues from tryptophan (300
mglKg, 60 min) and NSD 1015-treated rats, we observed increases of 5-HTP

concentrations (Figure 10). We also tested the possibility of peripheral arteries

70

Figure 10.

Quantification of 5-HIAA, 5—HT and 5-HTP in aorta (top), superior mesenteric
artery (middle) and hypothalamus (bottom) in rats treated (i.p.) with AADC
inhibitor NSD 1015 (100 mglKg, 30 min) or NSD 1015 (100 mglKg, 30 min) +
tryptophan (300 mg/Kg, 60 min). Bars represent means :I: SEM for the number of
animals in parentheses. * p<0.05 compared to NSD1015 (alone)- treated animal.
5-HlAA=5-hydroxyindoleacetic acid, 5-HT=5-hydroxytryptamine, 5-HTP=5-
hydroxytryptophan, AADC=amino acid decarboxylase, SMA=superior mesenteric

artery.

71

A—‘l
N»

d

g protein)
9 .° .-‘
I? 09 9

OD
we

Rat Aorta

[:lNSD 1015
.NSD 1015+ tryptophan

(N=4)

 

Concentration(nglm

9
9,591!-

Concentration(nglm protein)
9 .° .° .°
'9 «P 9’ 9°

ogtfli [Ii

 

5-HIAA 5-HTP

Rat SMA

DNSD 1015
.NSD 1015
+tryptophan
(N=3-4)

 

i?

0|

.5

w

l

N

Concentration(nglmg protein)

 

 

 

 

 

 

5i

5-HIAA 5-HTP

5-I-IT

Rat Hypothalamus

DNSD 1015
.NSD 1015+tryptophan

(N=3)

 

 

 

 

 

 

 

 

5-I-IT

5-HIAA

5-I-ITP _
figure 10.

72

taking up 5-HTP. The concentration of 5-HTP increased in rat aorta and superior
mesenteric artery after incubation with exogenous 5-HTP (1 (M) for 30 minutes
(Figure 11).

We also tested TPH activity in in vitro experiments. In the presence of
AADC inhibitor, substrate and all the co-factors that TPH needs to synthesize 5-
HT, no 5-HTP accumulation in arteries (with or without tryptophan Figure 12, A,
B), a low level of 5-HTP (without tryptophan) and an increased 5-HTP peak (with
tryptophan) in our positive control -- raphe nucleus (Figure 12, C, D) were

observed.

3. The Presence of AADC in Rat Aorta and Superior Mesenteric Artery
Figure 13 shows the Western analysis of the expression of AADC protein

(Abcam, Cambridge, MA) in rat aorta and superior mesenteric artery. We used

rat adrenal medulla as a positive control. The bands in rat aorta and superior

mesenteric artery samples as well as adrenal medulla migrated at ~55kDa.

4. The Presence of MAO A in Rat Aorta and Superior Mesenteric Artery
Western analysis using an antibody specifically recognizing MAO A
protein (H-70, Santa Cruz, CA, USA) was performed in rat aorta and-superior
mesenteric artery whole tissue homogenate supernatant. This MAO A antibody
is a rabbit polyclonal antibody raised against amino acids 458-527 of MAO A of
human origin. Figure 14, top shows the bands in both rat aorta and superior

mesenteric artery samples migrated at ~ 70 kDa, consistent with our positive

73

Figure 11.

Quantification of 5-HIAA, 5-HT and 5-HTP in aorta (top) and superior mesenteric
artery (bottom) from MAO A inhibitor pargyline (100 mg/Kg)-treated rats after 20
minutes of vehicle or 5-HTP (1 uM) incubation. Bars represent means 1 SEM for
the number of animals in parentheses. * p<0.05 compared to vehicle incubation.
5-HIAA=5-hydroxyindoleacetic acid, 5-HT=5-hydroxytryptamine, 5-HTP=5-
hydroxytryptophan, MAO A=monoamine oxidase A, SMA=superior mesenteric

artery.

74

A
{‘3

SR

Concentration(nglmg protein)

 

.°.°.°.°.°."‘.'"‘
‘P

“1.67

In

 

Concentration(nglmg prote
O O O O A A A
'9 i‘ “B °.° ‘-? '9 1‘

Rat Aorta

DVehicle
.5-HTP 1 ”M 20 min

(N=4) l

 

 

 

 

 

 

5-HIAA 5-HT

Rat SMA

|:|Vehicle
.5-HTP
1 11M 20 min

(N=4) l

 

 

 

 

 

 

5-HIAA 5-i-IT 5-l-ITP

Figure 11.

75

Figure 12.

Chromatogram showing the measurement of 5-HTP in rat aorta (A: without
tryptophan; B: with tryptophan) and raphe nucleus (C: without tryptophan; D: with
tryptophan) samples incubated with NSD1015 and other cofactors for 5-HT
synthesis. Solid line: sample tracing. Dot line: 3 mix standards including 20 pL,
75pg/uL 5-HT, 5-HTP and 5-HIAA.

5-HlAA=5-hydroxyindoleacetic acid, 5-HT=5-hydroxytryptamine, 5-HTP=5-

hydroxytryptophan, SMA=superior mesenteric artery.

76

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AADC Protein Expression in Arteries

 

 

 

 

 

 

 

Rat Adrenal
Rat SMA Rat Aorta Medulla
Figure 13.

Western blot of AADC in homogenates from rat superior mesenteric artery and
aorta, where each lane represents a different rat. Adrenal medulla was used as

positive control.

AADC=amino acid decarboxylase. SMA=superior mesenteric artery.

78

Figure 14.

A: Western blot of MAO A in homogenates from rat superior mesenteric artery
and aorta, where each lane represents a different rat. Rat gut mucosa was used
as positive control.

B: Immunohistochemical staining of the MAO A (Santa Cruz Biotechnology) in
smooth muscle between cables of elastin/collagen in the rat aorta and superior
mesenteric artery paraffin embeded section. Parallel sections were incubated
with antibody (left) or no primary antibody (right). Arrows indicate the placement
of staining. Representatives of four separate experiments, each with a different
rat.

MAO A = monoamine oxidase A. SMA=superior mesenteric artery.

79

MAO A Protein Expression in Arteries

A.
F...—-- ...- —----‘

 

 

 

 

 

 

 

Rat Gut
Rat SMA Rat Aorta Mucosa
B-
MAO A Primary
Rat
Aona
Rat
SMA

 

 

 

 

Figure 14.

80

control, rat gut mucosa.

Immunohistochemical experiments using the same antibody localized the
MAO A protein to smooth muscle and the endothelial layer of the rat aorta and
superior mesenteric artery (Figure 14 B, bottom). The pictures on the right show
rat aorta and superior mesenteric artery sections incubated with secondary

antibody, but no primary antibody.

5. The Activity of MAO A in Rat and Superior Mesenteric Artery

To investigate whether MAO A is functional in peripheral arteries, we
compared 5-HT and 5-HIAA concentrations in peripheral arteries with in vitro
incubation with either exogenous 5-HT (1 1M, 15 min) or vehicle. We measured
a basal level of 5-HT and 5-HIAA in rat aorta (5-HT, 0.28:0.05 nglmg protein; 5-
HIAA, 1.38:0.09 nglmg protein, Figure 15 top) and in superior mesenteric artery
(5-HT, 0.32:0.16 nglmg protein; 5-HIAA, 1.210.08 ng/mg protein, Figure 15
bottom). After incubation with 5-HT (1 pM, 15 min), 5-HIAA concentrations were
increased to 6782048 nglmg protein and 7921.2 nglmg protein in rat aorta and
superior mesenteric artery respectively with minor change of 5-HT levels (rat
aorta, 0.43:0.07 nglmg protein; superior mesenteric artery, 1.210.18 nglmg
protein). Moreover, we confirmed that the metabolism of 5-HT in arteries was
mediated via MAO A by doing the same experiment in arteries from pargyline-
treated rats. We observed that 5-HIAA production was abolished and 5-HT
levels were increased in arteries from pargyline-treated rats (rat aorta, 5-HT=

0.92:0.07 nglmg protein, 5-HIAA= 00025100025 nglmg protein; superior

81

Figure 15.

Quantification of 5-HIAA and 5-HT in aorta (top) and superior mesenteric artery
(bottom) incubated with vehicle or 1 uM exogenous 5-HT for 15 minutes.
Arteries were from normal rats. Bars represent means :1: SEM for the number of
animals in parentheses. * p<0.05 compared to vehicle-incubated tissues.

5-HIAA = 5-hydroxyindoleacetic acid. 5-HT = 5-hydroxytryptamine.

SMA= superior mesenteric artery.

82

. ‘9

N“

0"

Concentration (nglmg protein)
1‘ 't’ ‘1’ ‘r ‘I'

O

 

Rat Aorta
I:IVehicle

.5-HT1 uM, 15min
(N=5)

r—ni

 

Concentration (nglmg protein)
<5 -.* N ‘1’ E 9' 1’ 1' 0." '9

 

 

5-I-IT

Rat SMA

* EJVehicle
.5-HT 111M 15 min
(N=4)

 

 

III a:

 

5-H'IAA 5-HT

Figure 15.

83

mesenteric artery, 5-HT= 22810.24 nglmg protein, 5-HIAA= 002510.008 nglmg
protein, Figure 16). VVItI'I exogenous 5-HT incubation, the arterial 5-HT levels
significantly increased with minor increase of 5-HIAA (rat aorta, 5-HT= 24910.37
nglmg protein, 5-HIAA= 001310.013 ng/mg protein; superior mesenteric artery,

5-HT= 4.021054 nglmg protein, 5-HIAA= 01310.05 nglmg protein, Figure 16).

III. The Lack of 5-HT Storage in Peripheral Artery
1. Investigation of Whether Rat Aorta Has Ability to Store 5-HT

Basal endogenous levels of 5-HT in rat aorta were reported as above.
Here we tested whether a peripheral artery has the ability to store more than
basal level of 5-HT. To test this idea, we incubated untreated-aorta and 5-HT-
loaded aorta from normal and pargyline-treated rats in PSS and compared 5-
HIAA and 5-HT content in rat aorta and PSS after 0, 4 or 8 hours. The 5-HIAA
and 5-HT we measured in PSS were released during 4 or 8 hours incubation
from rat aorta. The amount of 5-HIAA and 5-HT remaining in rat aorta after
incubation was also measured.

First we tested whether aorta would maintain endogenous 5-HT. We
observed that in aorta from normal rats, the 5-HT content was maintained in
aorta after 4 or 8 hours of PSS incubation (Figure 17 bottom, Table), while 5-
HIAA was not and was released to PSS (Figure 17 top, Table 2). We next tested
whether aorta could store more than basal level of 5-HT by comparing 5-HIAA
and 5-HT levels in aorta after 4 or 8 hours PSS incubation in exogenous 5-HT (1

11M, 15 min) preloaded-aorta with those at time 0 and right after exogenous 5-HT

84

Figure 16.

Quantification of 5-HIAA and 5-HT in aorta (top) and superior mesenteric artery
(bottom) incubated with vehicle or 1 11M exogenous 5-HT for 15 min. Arteries
were from rats treated with MAO A inhibitor pargyline (100 mg/Kg, i.p., 30 min).
Bars represent means 1 SEM for the number of animals in parentheses. *
p<0.05 compared to vehicle-incubated tissues-

5-HIAA = 5—hydroxyindoleacetic acid. 5-HT = 5-hydroxytryptamine.

SMA= superior mesenteric artery. MAO A = monoamine oxidase.

85

Aorta From Pargyline-treated Rats

"ESP

DVehicle
.5-HT1 11M 15 min

(N=4)

N
1

1"}:

  

Concentration (nglmg protein)
‘1’ 1‘ ‘1" ‘P

 

_ |'_'l

5-HIAA 5-i-IT

O

SMA From Pargyline-treated Rat

 

 

’E
35’ 91 EIVehicIe
33' .5-HT1 11M 15 min
237‘ (N=4)
36'
55- *
.5 4'
E 3-
E, 2-
E 1.
<3 o-
——-'-_ .
5-HIAA 5-HT

Figure 16.

86

Figure 17.

Top: Quantification of 5-HIAA and 5-HT released into PSS during 0, 4 or 8 hours
incubafion.

Bottom: Quantification of 5-HIAA and 5-HT remaining in tissues after 0, 4 or 8
hours incubation in PSS.

Time 0 represents no incubation (basal endogenous 5-HlAA and 5-HT levels).
Bars represent means i SEM for the number of animals in parentheses.

* p<0.05 compared to basal level (no incubation, time 0).
5-HlAA=5-hydroxyindoleacetic acid. 5-HT=5-hydroxytryptamine.

PSS=physiological salt solution.

Table 2.
Quantification of 5-HlAA and 5-HT released into PSS and remained in tissue
during 0, 4 or 8 hours incubation. Values reported as nglmg protein.

* p<0.05 compared to basal level (no incubation, time 0).

87

PSS

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

€1.4- Clo
E 1.2- .4 hours
a .8 hours
E 1.0- (N=6-8)
2’ 0.8-
‘E
.2 0.6-
E
‘E' 0.4-
a:
2 0.2-
o
0 o.c 5 HT
...... Rat Aorta
.s 0-5' :10
43 .4 hours
3. o_4. ”49:2." 8 hours
as» (N=6-8)
g: o-3 _T_ l ;
‘E ,
:2 0.2 /
e *
a , %
3 0.1 /
5 Z %
0 o.c . .5
5-HIAA 5-HT
Table 2. Figure 17.
0 4 Hours 8 Hours
PSS 5-HIAA 0 1.02:0.23* 1 .018:0.19*
5-HT 0 0.097:0.05* 0.05:0.006*
Rat 5-HIAA 0.27:0.05 04210.03" 0.085=0.01*
Aorta 5-HT 0.28:0.07 0.26:0.1 0.29:0.065

 

 

 

 

88

incubation. We rinsed and flushed aorta with fresh drug-free PSS to eliminate
exogenous 5-HT attached on the aorta wall. As shown in Figure 18, bottom and
Table 3, aortic 5-HIAA level was greatly increased by 15 minutes incubation of 1
uM exogenous 5-HT with a moderate increase of 5-HT in aorta. After 4 or 8
hours incubation with PSS, the majority of 5-HIAA was released to PSS with a
slight though significant decrease of 5-HT in aorta with time (Figure 18, top and
Table 3). We then took a further step to investigate whether aorta would store 5-
HT when 5-HT metabolism was inhibited. We did a similar experiment in aorta
from MAO A inhibitor, pargyline (100 mglKg, 30 min)-treated rats. After
incubation with exogenous 5-HT (1 (M, 15 min), the aortic 5-HT content
increased with no change of 5-HIAA (Figure 19, bottom, Table 4). However, this
is an acute accumulation of 5-HT, after 4 or 8 hours, 5-HT concentration
decreased by releasing both 5-HT and 5—HlAA from aorta to PSS (Figure 19, top,

Table 4).

2. Investigation of “Serotonylated” 5-HT in Rat Aorta

We tested whether 5-HT covalently bound to protein exists in peripheral
arteries by Western analysis. We separated rat aorta protein on a 12% gel by
molecular weight and blotted with a 5-HT antibody. Multiple bands were
visualized (Figure 20), suggesting the presence of serotonylated proteins in rat

aona.

89

Figure 18.

Top: Quantification of 5-HIAA and 5-HT released into PSS during 4 or 8 hours
incubation from 5-HT preloaded rat aorta (5-HT 1 (M, 15 min).

Bottom: Quantification of 5-HIAA and 5-HT in naive aorta (basal level) and in 5-
HT preloaded aorta after 0, 4 or 8 hours incubation in PSS.

Time 0 represents no incubation (basal endogenous 5-HIAA and 5-HT levels).
Bars represent means :l: SEM for the number of animals in parentheses.

* p<0.05 compared to basal level. # p<0.05 compared to 5-HT 1 uM incubation.
5-HlAA=5-hydroxyindoleacetic acid. 5-HT=5-hydroxytryptamine.

PSS= physiological salt solution.

Table 3.

Quantification of 5-HlAA and 5-HT in naive aorta and 5-HIAA and 5-HT released
to PSS or remained in 5-HT preloaded aorta after 0, 4 or 8 hours PSS incubation.
Values are reported as nglmg protein.

* p<0.05 compared to basal level. # p<0.05 compared to 5-HT 1 uM incubation.

90

PSS

 

 

 

 

3515.0- * * :IBasal
% , -5-HT1 uM 15 min
512'5' // -4 Hours
3100. .8 Hours
3 (N=6)
c 7.5
.2
*5 5.0.
E
8 2.5-
C
O
U 0.0

5-HIAA 5-HT

:5 Rat Aorta

L__lo

.5-HT1uM 15 min
.4 hours

-8 hours

(N=6)

 

 

 

 

r—1 - “it _#
5-HT
Figure 18.
4 Hours 8 Hours

 

 

13.08:1.72* 12.93:1.71*

 

5-HT 0 0 O 0

 

Rat 5-HIAA 1.38:0.09 6.78:0.48* 20210.03" 0.991045"

 

Aorta 5-HT 0.28:0.05 0.43:0.07 0.16:0.03” 0.14:0.01"

 

 

 

 

 

91

Figure 19.

Top: Quantification of 5-HIAA and 5-HT released into PSS during 4 or 8 hours
incubation from 5-HT preloaded (5-HT 1 (M, 15 min) aorta from pargyline-treated
rat.

Bottom: Quantification of 5-HlAA and 5-HT in aorta (basal level) and in 5-HT
preloaded aorta from pargyline-treated rats after 0, 4 or 8 hours incubation in
PSS.

Time 0 represents no incubation (basal endogenous 5-HIAA and 5-HT levels).
Bars represent means 1 SEM for the number of animals in parentheses.

* p<0.05 compared to basal level. # p<0.05 compared to 5-HT 1 uM incubation.
5-HlAA= 5-hydroxyindoleacetic acid. 5-HT= 5-hydroxytryptamine.

PSS: physiological salt solution. Points represent means i SEM.

Table 4.

Quantification of 5-HIAA and 5-HT in aorta and 5-HlAA and 5-HT released to
P88 or remained in 5-HT preloaded aorta from pargyline-treated rats after 0, 4 or
8 hours PSS incubation. Values are reported as nglmg protein.

* p<0.05 compared to basal level. # p<0.05 compared to 5-HT 1 uM incubation.

92

PSS

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

’E
'53 0'77 ... L—JBasal
g o 6- .5-HT 1 [1M 15 min
or 0 5. -4 Hours
% ' -8 Hours
5 0.4- 01:4)
.5 0.31 .
‘E'é *
b 0.2- *
8
c 0.1-
O
0 0.0
5-HIAA 5-HT
? Rat Aorta
'6 3.0! Do
£25 .5-HT1uM15 min
or ' -4 hours
’///. 8 h
g 2'0! - OUI‘S
g (N-4)
: 1.5-
.2
g 1.0-
C
8 0.5-
C
O
U o_c m
5-HIAA
Table 4. Figure 19.
0 5414;- 1.“M 4 Hours 8 Hours
mIn
PSS 5-HIAA o o 0.15:0.03* 0.46:0.16*
5-HT 0 o 0.19:0.03* o.22=o.os*
Rat 5-HIAA o 0.01:0.01 0.08:0.01 0.09:0.02
Acme 5-HT 0.92:0.07 2.49:0.37* 1.08:0.09" 0.47101"

 

93

No primary

 

Figure 20.

Western blot of 5-HT in homogenates from rat aorta. Representatives of three
separate experiments, each with a different rat. Left: Blot incubated with
secondary antibody with no exposure to primary antibody. Right: Blot incubated

with rabbit anti-5-HT antibody (Serotec, Raleigh, NC).

94

IV. The Presence of Uptake and Release of 5-HT in Peripheral Arteries
1. The Presence of SERT in Peripheral Arteries

The presence of SERT mRNA was demonstrated by real time RT-PCR.
Figure 21 top shows that one PCR product, obtained using primers developed
through Primer Express® software for the SERT encoding sequence, was
identified in cDNA made from rat aorta. No product observed in the lanes with no
reverse transcriptase suggesting no genomic contamination in our cDNA
samples. In Western analyses, an antibody directed toward the carboxyterminus
of the human SERT recognized one protein band of 74 kDa mass in
homogenates of rat aorta, mesenteric resistance arteries, mouse aorta and
mouse aorta sample immunoprecipitated with the same antibody (Figure 21,
bottom). IHC experiments were performed in rat aorta paraffin embedded and
mouse aorta frozen sections using an antibody that recognized the C-terminus
(C-20) of the SERT. The SERT was expressed in the smooth muscle and in the
endothelial cell layer and the black DAB precipitate did not appear in sections
incubated with antibody plus competing peptide (Figure 22, compare top and
middle panel). The adventitia of the vessels of rat aorta stained darkly, but this
was not competed off by competing peptide; thus this staining is likely
nonspecific. The bottom panel showed aorta sections incubated with Secondary
antibody but no primary antibody.

The presence of SERT in peripheral artery was further validated by
immunocytochemistry. We double-stained freshly isolated rat aortic smooth

muscle cell using SERT antibody (C-20) and a specific cell plasma membrane

95

Figure 21.

Top: Final product of real time RT-PCR for detection of SERT and GAPDH
mRNA in rat aorta. 1-4 are separate rats. RT=reverse transcriptase.

Bottom: Western blot of SERT in homogenates from rat aorta, rat superior
mesenteric arteries, mouse aorta homogenates and immunoprecipitated protein
(by SERT C-20 antibody, Santa Cruz Biothechnology) from a mouse aorta.
Representatives of at least four separate experiments, each with a different
rat/mouse.

SERT= serotonin transporter. SMA= superior mesenteric artery.

96

SERT mRNA Expression in Rat Aorta

 

 

SERT
1 2 3 4

 

 

GAPDH

1 2 3 4

 

 

SERT Protein Expression in Arteries

 

Rat Aorta Rat SMA

 

74”) mm~---

Mouse Aorta

 

74'kE) Ill-ll III-II

Homog. IP

 

 

 

Figure 21.
97

Figure 22.

Immunohistochemical staining of the SERT (Santa Cruz Biotechnology, C-20) in
smooth muscle between cables of elastin/collage in the rat aorta (left, paraffin
embedded sections) or mouse aorta (right, frozen sections). Parallel sections
were incubated with antibody alone (top), quenched with a 5X excess competing
peptide (middle) or no primary (bottom). Arrows indicate the placement of
staining. Representative of 4-6 different experiments (rats/mice).

SERT: serotonin transporter.

98

SERT Protein Expression in Arteries

 

Rat Aorta Mouse Aorta

 

 

 

 

Figure 22.
99

marker pan-cadherin antibody. By using confocal microscopy, we scanned a
single layer of smooth muscle cell. Overlaying the staining of the SERT antibody
and the pan-cadherin antibody, we observed that most of the SERT was

localized on plasma membrane (Figure 23).

2. The Characterization of Active 5-HT Uptake in Peripheral Arteries
A. 5-HT Uptake Time Course Study

Different tissues (conduit or small vessels) from normal or pargyline-
treated rats were used in 5-HT uptake time course studies.

Superior mesenteric arteries from normal rats were incubated in normal
PSS with 5-HT (1 nM) for 0, 15, 30 or 45 minutes in room temperature (25 °C).
Figure 24 demonstrates that arterial 5-HIAA content increased in a time-
dependent manner with a concomitant minor increase of 5-HT.

We also tested 5-HT uptake in a time course study using arteries from
pargyline-treated rats. The merits of using pargyline-treated rats are that we can
study 5-HT uptake directly and reduce changes in 5-HlAA. Rat aortae were
incubated in PSS with 1 uM pargyline and 1 uM 5-HT for 0, 15,30, 45, 60 and 90
minutes at room temperature (25 °C) and 37 °C (Figure 25). At room
temperature, aortae took up 5-HT in a time dependent manner and plateaued at
60 minutes with minimal changes of 5-HlAA. The maximal level of 5-HT in aorta
was 8.48:0.71 nglmg protein. However, at 37 °C, uptake plateaued after 30
minutes of exogenous 5-HT (1 uM) incubation with maximal aortic 5-HT level at

5.25:0.27 nglmg protein. After 15 minutes 5-HT (1 uM) incubation, aortic 5-HT

100

Figure 23.

Confocal image of freshly dissociated rat aortic smooth muscle cells double
stained with pan-cadherin antibody (Sigma) and SERT antibody (C-20, Santa
Cruz Biotechnology). Representative of cells from 6 different experiments (rats).

SERT= serotonin transporter.

101

 

 

SERT Protein Expression in Freshly
Isolated Rat Aortic Smooth Muscle Cells

Pan-cadherin

    
 

O
Overlay

Figure 23.
102

Rat SMA

|:IVehicle

.5-HT1pM 15min
MS-HT 111M 30min
24:2: 5-HT 1p.M 45min

(N=5-9)

    
 
    

.3
.°

*

 
 
  

Concentration (nglmg protein)
N

P

5-HIAA 5-HT

Figure 24.

Time course for 5-HT uptake in the rat superior mesenteric artery incubated with

5-HT (1 HM) in room temperature.

Bars represent means :1: SEM for the number of animals in parentheses.

* p<0.05 compared with time 0 (no incubation).

5-HlAA=5-hydroxyindoleacetic acid. 5-HT= 5-hydroxytryptamine. SMA=superior

mesenteric artery.

103

Figure 25.

Time course for 5-HT uptake in the aorta from pargyline-treated rats incubated
with exogenous 5-HT (1 uM) at 37 °C (Top) or at room temperature (bottom).
Bars represent means :1: SEM for the number of animals in parentheses.

* p<0.05 compared with time 0 (no incubation).

5-HIAA= 5-hydroxyindoleacetic acid. 5-HT= 5-hydroxytryptamine.

104

A
h c» on O
I I I I

Concentration(nglmg protein)
N

Rat Aorta (37 °C)

DVehicle
.5-HT 1uM 15min
m5-HT 115M 30min
33:2 5-HT 1uM 45min
.5-HT 1.1M 60min . ..
.5-HT 1uM 90min * *
(N=4)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

O
I

A
.h m on O
J I I I

Concentration(nglmg protein)
N

 

 

 

 

 

 

 

5-HIAA 5-HT

Rat Aorta (Room Temperature)

EZIVehicle *
.5-HT 1p.M 15min

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

O
I

 

 

 

 

 

 

 

 

@5-HT 1uM 30min ,z,
.5-HT 111M 45min *
.5-HT 1uM 60min :3;
.5-HT 1uM 90min
(N=6-10) .1; £55
5-HIAA 5-HT
Figure 25.

105

levels are similar at room temperature and 37 °C incubation groups (room
temperate, 3.07:0.35 nglmg protein; 37 °C, 3.12:0.45 nglmg protein). Similarly,
superior mesenteric artery from pargyline-treated rats time-dependently take up
5-HT, which did not increase further after 60 minutes of 5-HT incubation in room
temperature (Figure 26). All of our following 5-HT uptake experiments incubated
tissues with 5-HT (1 (M) for 15 minutes at room temperature.

The real uptake of exogenous 5-HT into arteries was confirmed by using
tph1-/- mice. The tph1-/- mice expressed normal amounts of 5-HT in brain but
lack of 5-HT in periphery (Walther et al., 2003). The blank background of 5-HT in
these mice makes them a good model for studying 5-HT uptake in peripheral
arteries. Figure 27, table 5 compares the uptake of exogenous 5-HT in aorta
from untreated wild type and tph1-/- mice. It is clear that basal 5-HT and 5-HIAA
concentrations were significantly lower in aorta from tph1-/- mice. Upon
incubation with exogenous 5-HT, the level of 5-HIAA increase in mouse aorta is
much higher than the 5-HT increase in both wild type and tph1-/- mice, which is
similar to what we observed in rat arteries.

Thus, we concluded that 5-HT can be taken up by various peripheral

arteries and 5-HT metabolized to 5-HIAA quickly after it gets into arteries.

B. Investigation of SERT-dependent 5-HT Uptake

We next investigated whether the active 5-HT uptake in peripheral arteries

was mediated by SERT. We preincubated arteries (rat aorta and superior

106

Rat SMA (Room Temperature)

[ZIVehicle
V4 5-HT1uM 15min

"T’T’T 5-HT 1p.M 30min

A
C
I

  

 

 

 

 

 

 

 

 

 

 

 

 

N
I

 

 

 

 

 

 

 

 

 

 

Concentration (nglmg protein)

7//_ 5-HT 1.1M 45min y

5.. 525252 5-HT 1.1M 60min é
.5-HT 1.1M 90min 5% g

4- (N=4-12) : é
%

Z

Z.

 

 

 

 

 

 

 

 

   

8 5+th 5-HT

Figure 26.

Time course for 5-HT uptake in the superior mesenteric artery from pargyline-
treated rats incubated with 5-HT (1 (M) at room temperature.

Bars represent means :i: SEM for the number of animals in parentheses.

* p<0.05 compared to time 0 (no incubation).

5-HIAA= 5-hydroxyindoleacetic acid. 5-HT= 5-hydroxytryptamine.

SMA= superior mesenteric artery.

107

Figure 27.

5-HT uptake in the aorta from wild type (top) and tph1-l- mice (bottom) incubated
with exogenous 5-HT (1 11M, 15 min) at room temperature.

Bars represent means 1 SEM for the number of animals in parentheses.

* p<0.05 compared to vehicle.

5-HIAA= 5-hydroxyindoleacetic acid. 5-HT= 5-hydroxytryptamine.

TPH=tryptophan hydroxylase.

Table 5.

Quantification of 5-HIAA and 5-HT in aorta from wild type or tph1-l- mice
incubated with vehicle or exogenous 5-HT (1 11M, 15 min) at room temperature.
Values are reported as nglmg protein.

Bars represent means a: SEM for the number of animals in parentheses.

* p<0.05 compared to vehicle.

108

Wild*Type Mouse Aorta

 

 

 

 

 

 

 

 

 

 

 

 

€2.01 -
,9, DVehIcle
g .5-HT 11M 15 min
cents- (N=3-4)
B:
E. ..
c 1.0"
.2
.8
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o
o
c
o
o 0.0 ,
5-HIAA 5-HT
’5 2 0 TPH1 -I- Mouse Aorta
.g ' * EIVehicle
a .5-HT 1 uM 15 min
at 1 .5- (N=3-4)
Bi
5
c 1 .0"
.2 *
E
E 0.5. 1:1 1
o
o
5
o 0.0-
5-HIAA 5-HT
Table 5. Figure 27.
Wild Type Mouse Aorta TPH 1-/- Mouse Aorta
Vehicle 5-HT 111M 15min Vehicle 5-HT 111M 15min
5-HIAA 0.37:0.17 1.74:0.25* 0.0231002 1.5:0.40*
5-HT 0.49:0.10 0.85:0.19* 0.0831005 0.62:0.11"

 

 

 

 

 

109

mesenteric artery) with either vehicle or one of the SERT inhibitors fluvoxamine
(1 (M) or fluoxetine (1 (M) for 30 minutes prior to exposure to 5-HT (1 (M) for 15
minutes. The 5-HT levels in arteries incubated with 5-HT and SERT inhibitors
were significantly lower than in arteries only incubated with exogenous 5-HT
suggesting a SERT dependent 5-HT uptake in arteries. Neither fluoxetine (1
uM) nor fluvoxamine (1 uM) inhibited the entire uptake of 5-HT (Figure 28).
SERT-targeted mutation mice (SERT KO) were used in our study to test
the dependence of SERT in 5-HT uptake in peripheral arteries. Basal level of 5-
HlAA in aorta from pargyline-treated SERT KO mice were much lower compared
with WT mice with a minor change of basal 5-HT level (Figure 29, vehicle
incubation). Fluvoxamine inhibited 5-HT uptake in aorta from WT mice but not in
SERT HET or SERT KO mice (Figure 29). Total 5-HT uptake reduced in aorta

from SERT KO mice with no further inhibition of fluvoxamine pre-incubation.

C. Investigation of SERT-independent 5-HT Uptake

We tested whether there is a Na*-independent uptake because
monoamine transporters (SERT, NET and DAT) are Na“-K*-Cl'—dependent
transporters, which have the potential to take up 5-HT. The result showed that
there was a 5-HT uptake in aorta and superior mesenteric artery from pargyline-
treated rats in Na‘ free (Na+ was replaced isosmotically with N-methylglucamine)
- PSS with 1 uM exogenous 5-HT (15 min), though the total uptake was
significantly reduced compared to that in regular Na” containing PSS. SERT

inhibitor, fluoxetine or fluvoxamine, did not further reduce 5-HT uptake in aorta

110

Figure 28.

Effects of a 30-minute preincubation with SERT inhibitor fluvoxamine (1 11M) or
fluoxetine (1 (M) on 5-HT uptake (1 uM 5-HT incubation for 15 min) in aorta (top)
or superior mesenteric artery (bottom) from pargyline treated rats.

Bars represent means :1: SEM for the number of animals in parentheses.

* p<0.05 compared to vehicle. # p<0.05 compared to 5-HT (1 uM).

5-HlAA= 5-hydroxyindoleacetic acid. 5-HT= 5-hydroxytryptamine.

SERT= serotonin transporter. SMA=superior mesenteric artery.

111

 

 

Concentration(nglmg protein)

Concentration (nglmg protein)

0)

5’5"}?

‘
L

Rat Aorta

DVehicle
.Fluvoxamine 111M

 

'f’t’t‘i'm

A
L

 

EFluoxetine 1p.M
.5-HT 111M
.5-HT+Fluvoxamine *
E5-HT+Fluoxetine
(N=3-10) *#
5-HIAA 5-HT
Rat SMA
' |:|Vehicle *

 
  
 
    

.Fluvoxamine 111M
EFluoxetine 111M
.5-HT 111M
.5-HT+Fluvoxamine
E5-HT+Fluoxetine

(N=3-6)

 

 

O
I

 

Figure 28.

112

Figure 29.

Effects of a 30-minute preincubation with SERT inhibitor fluvoxamine (1 (M) on
5-HT uptake (1 uM 5-HT incubation for 15 min) in aorta from wild type (top),
SERT HET (middle) and SERT KO mice (bottom).

Bars represent means :i: SEM for the number of animals in parentheses.

* p<0.05 compared with vehicle.

5-HIAA= 5-Hydroxyindoleacetic acid. 5-HT= 5-hydroxytryptamine.

SERT= serotonin transporter.

113

 

E"
?

a? ‘1'

Concentration (nglmg protein)
O
15

Wild Type Mouse Aorta

DVehicle
.Fluvoxamine 1 uM
.5-HT1 uM
.5-HT-I-Fluvoxamine
(N=3-4)

 

P
‘P

N
.°

.3 .5
‘P ‘1'

Concentration (nglmg protein)
C
3r

 

......5 H5

5-HIAA 5-HT

SERT HET Mouse Aorta
CIVehicle
.Fluvoxamine 1 uM
.5-HT 1 uM
.5-HT-i-Fiuvoxamine
(N=3-4)

*

 
  
   
 

 

P
‘P

N
?

1"

'9

0

Concentration (nglmg protein)
ir

 

.°
1':

 

5-HT

SERT KO Mouse Aorta

DVehicle

.Fluvoxamine 1 11M

.5-HT 1 uM

.5-HT+Fluvoxamine
(N=3-4)

Figure 29.

 

5-HiAA

114

Figure 30.

Effects of a 30-minute preincubation with SERT inhibitor fluvoxamine (1 (M) or
fluoxetine (1 pM) on 5-HT uptake (1 uM 5-HT incubation for 15 min) in Na“ -free
PSS in aorta (top) or superior mesenteric artery (bottom) from pargyline treated
rats.

Bars represent means :1: SEM for the number of animals in parentheses.

* p<0.05 compared to vehicle. # p<0.05 compared to 5-HT (1 uM) uptake in
normal PSS (containing Na*).

5-HIAA= 5-hydroxyindoleacetic acid. 5-HT= 5-hydroxytryptamine.

SERT= serotonin transporter. SMA=superior mesenteric artery.

115

Concentration(nglmg protein)

Concentration(nglmg protein)

‘3

w
l

 

 

|:|Vehicle
Fluvoxamine 1 uM
E Fluoxetine 111M
.5-HT 1 uM
5-HT+Fluvoxamine
g 5-HT+Fluoxetine

.5-HT 111M with Na+
(N=6-10)

 

Rat SMA
5- .

I:IVehIcle
5_ .5-HT1uM

.5-HT1 ”M with Na+
4_ (N=4-6)
.l
21 .II'_
1-

r—I
5-HIAA

Rat Aorta

 

 

 

 

116

5-HT

Ill|||llllllllllllllllllllllll

 

 

Figure 30.

incubated in Na+ free- PSS (Figure 30).

We then tested whether this 5-HT uptake was SNS, NET and OCT-
dependent in peripheral arteries. To investigate whether uptake of 5-HT by the
artery was dependent on sympathetic nerves, we used 6-OHDA denervated rats.
Rats were injected with 6-OHDA during an 8-day protocol, and arteries procured
on day 8. Validation of denervation was verified by lack of glyoxylic acid-reacted
norepinephrine (Figure 31, top). While basal levels of 5-HT and 5-HIAA were
slightly but not significantly lower than in untreated animals, arteries were still
capable of taking up and metabolizing 5-HT as the concentrations of 5-HT and 5-
HIAA increased in the vessels exposed to 5-HT for 15 minutes. This was true for
all 3 arteries examined (Figure 31).

The NET inhibitor nisoxetine (100 nM) was unable to inhibit 5-HT uptake
(Figure 32). The OCTs inhibitor, corticosterone (1 uM) did not inhibit 5-HT
uptake in aorta but potentially could in superior mesenteric from pargyline-treated
rats (Figure 33).

Based on our observation, arterial 5-HT uptake is not dependent on SNS,
NET or OCT.

As the barrier between arterial smooth muscle and blood, endothelium
could also take up 5-HT. Uptake of 5-HT, at least in the superior mesenteric
artery appears to be largely independent of the endothelium as removal of this

cell layer did not alter 5-HT uptake or metabolism (Figure 34).

117

Figure 31.

A. Glyoxylic acid fluorescence in small mesenteric resistance arteries from
vehicle or 6-OHDA-treated rats. The white network is sympathetic nerve fibers.
B. 5-HT uptake in aorta, carotid artery and superior mesenteric artery from 6-
OHDA treated rats.

Bars represent means :1: SEM for the number of animals in parentheses.

* p<0.05 compared with vehicle.

5-HlAA=5-hydroxyindoleacetic acid. 5-HT= 5-hydroxytryptamine. SMA=superior

mesenteric artery.

118

 

A' Mesenteric

 

 

 

 

 

 

. - c0!" “
Artery Glyoxylic
Acid Staining Vehicle " 6-OHDA, Day 8
E 3.5- * RatAorta
% DVehicle
a 3.0- .NS;HT1p.M
a =
g 2.5- ( l
E 2.0-
C
;§ 1.5-
g 1.0-
C
§ 0.54 i
O
U .c
5-HIAA 5-HT
E 3.51 Rat SMA
33 DVehicle
a 3-0‘ * .5-HT 1 ”M
2’ 2.5- (N=4)
Bi
5 2 o-
s
g 1.5- *
‘6
i: 1.0-
C
8
0 0.5 - ,_..., -
5-HIAA 5-HT
351 Rat Carotid Artery
EIVehicle
3.01 .5-HT1uM

 

Concentratioin (nglmg protein)

9
>

2.5- (N=4)
2.0-
1.5-
1.0- ' .
0.5- _
[:1 Figure 31.
" 5-H'iAA 5-HT
1 19

Rat SMA

.h
J

|:]Vehicle
.Nisoxetine 100 nM
.5-HT 1 (1M

:1" , i 5-HT+Nisoxetine

w
I

 

Concentration (nglmg protein)
N

 

 

   

Figure 32.

Effects of a 30-minute preincubation with NET inhibitor nisoxetine (100 nM) on 5-
HT uptake (1 uM 5-HT incubation for 15 min) in aorta from normal rats.

Bars represent means 1: SEM for the number of animals in parentheses.

* p<0.05 compared with vehicle incubation.

5-HlAA=5-hydroxyindoleacetic acid. 5-HT=5-hydroxytryptamine.

NET=norepinepherin transporter. SMA=superior mesenteric artery.

120

Figure 33.

Effects of a 30-minute preincubation with OCT inhibitor corticosterone (1 (M) on
5-HT uptake (1 uM 5-HT incubation for 15 min) in aorta (Top) and superior
mesenteric artery (bottom) from pargyline-trated rats.

Bars represent means 1 SEM for the number of animals in parentheses.

* p<0.05 compared with vehicle incubation. #p<0.05 compared with 5-
HT+fluvoxamine+corticosterone.

5-HlAA=5-hydroxyindoleacetic acid. 5-HT=5-hydroxytryptamine.

OCT: organic cation transporter. SMA=superior mesenteric artery.

121

 

Concentration(nglmg protein)

in)

—‘
N
I

.3
O

m
J

h
J

Concentration(nglmg prote
N

Rat Aorta *#
7l___IVehicle *#
6 |]]]]]Ifl]Corticosterone 111M
Fluvoxamine-I-Corticosteron :
5.5-HT 1 uM
u 5-HT+Corticosterone
4.5-HT+Fluvoxamine+

   
   
  
 

  
   
 
     
     
     

N w
I I

 

Corticosterone
(N=7-8)
* ill
5-HIAA 5-HT
Rat SMA
|_—_|Vehicle *#

||]I]]I[|Corticosterone 1p.M
F luvoxamine+Corticosterone

5-HT1 M l
M 5-HT+ orticosterone
.5-HT+Fluvoxamine+

Corticosterone

(N=4)
5-HIAA I , 5- HT

L

 

 

 

_ 0"" II:

o
L

Figure 33.

122

Figure 34.

5-HT uptake (1 uM 5-HT incubation for 15 min) in endothelium-intact or
endothelium denuded superior mesenteric artery from normal rats.

Bars represent means :1: SEM for the number of animals in parentheses.

* p<0.05 compared to vehicle incubation.

5-HIAA= 5-hydroxyindole acetic acid. 5-HT= 5-hydroxytryptamine.

SMA=superior mesenteric artery.

123

 

Concentration (nglmg protein)

A

Concentration (nglmg protein)

.°

N

N

—‘L

P

P

N

N

P

 

Rat SMA (with Endothelium)

 

 

 

 

 

 

 

5.
DVehicle
.5-HT
0‘ (N=5-10)
.5-
o.
5.
G
Rat SMA (without Endothelium)
5' * DVehicle
.5-HT
0- (N=5-10)
.5-
.0-
5.. l l
0 . I 1 .
5-HIAA 5-HT

Figure 34.

124

3. The Presence of 5-HT Release in Peripheral Arteries

(+)-Fenfluramine and (+)-norfenfluramine are SERT substrates and 5-HT
releasers. We used these two drugs to test the release of 5-HT from arteries. To
enhance the signal of 5-HT, we used pargyline-treated animals to study 5—HT
release from arteries.

Figure 35 shows that (+)-fenfluramine concentration (1 uM or 10 11M, 20
min) dependently released endogenous 5-HT from rat aorta into PSS. Similar
amounts of 5-HT were released from rat aorta by incubation with 10 uM (+)-
fenfluramine (0.37:0.07 nglmg protein) and 10 11M (+)-norfenfluramine
(0.35:0.18 nglmg protein) for 20 minutes. The remaining 5-HT in aorta after
fluramines incubation were 0.56:0.12 nglmg protein and 0.74:0.22 nglmg
protein after (+)-fenfluramine or (+)-norfenfluramine-incubation, respectively.
Thus, the released 5-HT during fluramines incubation accounts for about 30% of
total endogenous 5-HT in rat aorta (Figure 35). The release of endogenous 5-HT
is not a vessel specific effect as we observed fenfluramine (10 11M, 20 min)
released endogenous 5-HT in superior mesenteric artery from pargyline-treated
rats (Figure 36). Fluoxetine (1 uM, 30 min), which potentiated basal 5-HT
release, did not inhibit (+)-fenfluramine (1 (M or 10 11M, 30 min)—induced 5-HT

release from rat aorta (Figure 37).

125

Figure 35.

(+)-Fenfluramine (1 11M or 10 (M) or (+)-norfenfluramine (10 11M) induced 5-HT
release from aorta obtained from pargyline-treated rats. Top: released 5-HIAA
and 5-HT during fluramine incubation. Bottom: remained 5-HIAA and 5-HT after
fluramine incubation.

Bars represent means :i: SEM for the number of animals in parentheses.

* p<0.05 compared to vehicle incubation.

5-HlAA=5-hydroxyindoleacetic acid. 5-HT=5-hydroxytryptamine.

PSS: physiological saline solution.

126

PSS

F 0.6 .
1 :IVehicle
. 0 -(+)-Fenfluramine1uM --
°- '57 -(+)-Fenfluramine 1011M
§§§(+)-Norfenfluramine 10uM
(N=3-8)

 

 

 

i323 i’ihi Fl

5-HIAA 5-HT

 

Rat Aorta

2.0
- [:IVehicle

.(+)-Fenfluramine
1 uM .
1%: (+)-Fenfluramine _T_
10uM ,
. .(+)-Norfenfluramine
' 10pM
(N=3-8)

.1.
0|

 

Concentration (nglmg protein)
0

 

 

 

0.5—MM

5-HIAA

 

 

127

Figure 36.

(+)-Fenfluramine (1O uM)-induced 5-HT release in superior mesenteric artery
from pargyline-treated rats. Top: released 5-HlAA/5-HT during (+)-fenfluramine
incubation. Bottom: remaining 5-HlAA/5-HT after (+)-fenfluramine incubation.
Bars represent means :1: SEM for the number of animals in parentheses.

* p<0.05 compared to vehicle incubation.

5-HIAA= 5-hydroxyindole acetic acid. 5-HT= 5-hydroxytryptamine.

PSS: physiological saline solution. SMA=superior mesenteric artery.

128

Concentration (nglmg protein)

.c .O
h OI
l J

I
00
I

 

 

.3 .3 N
0 0| O
l l J

Concentration (nglmg protein)
C
in

 

P
o

PSS

 

 

 

 

 

 

 

 

 

DVehicle *
.Fenfluramine 10 uM 20 min
(N=5-7)
5-HIAA 5-HT
Rat SMA
[:lVehicle
.Fenfluramine 10uM 20 min
(N=5-7)
l
r— , - ,
5-HIAA 5-HT

Figure 36.
129

Figure 37.

Effects of 30 minutes SERT inhibitor fluoxetine (1 11M) preincubation on (+)-
fenfluramine (10 (M) —induced 5-HT release in aorta from pargyline-treated rats.
Top: released 5-HlAA and 5-HT during fluramine incubation. Bottom: remaining
5-HIAA and 5-HT after (+)-fenf|uramine incubation.

Bars represent means :i: SEM for the number of animals in parentheses.

* p<0.05 compared to vehicle incubation.

5-HIAA=5-hydroxyindoleacetic acid. 5-HT=5-hydroxytryptamine.

PSS=physiological saline solution. SERT=serotonin transporter.

130

PSS

DVehicle *
.Fluoxetine1 uM
.Fenfluramine1 uM

A
2"

   
 
   
    
 

 

Concentration(nglmg protein)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

1,0. MFenfluramine1 0 uM f/
.Fenfluramine1 uM Z
+Fluoxetine % .
2m Fenfluramine 1 0 uM %
0,5. +Fluoxetine % .
(N=3-8) . é '
2
w, /
0.0- we. .. A
5-HIAA 5-HT
A Rat Aorta
é, 2-5 ~- Vehicle
25 ' Fluoxetine 1 pM
ozo Fenfluramine 1 (M
2’ 5:32 Fenfluramine 10 11M
3,15 l . Fenfluramine 111M
g. +Fluoxetine ,
3 1 0 .88 Fenfluramine 10 M r
‘5 ' +Fluoxetine :
*5 (N=3-8) i
3 0.5- E
= I
8 :
0.0- -
5-HIAA -HT

 

Figure 37.

131

Hypothesis #2:

Changes in the localized serotonergic system contribute to the

increase of blood pressure in hypertension.

We focused on comparing (1) SERT expression; (2) basal 5-HIAA and 5-
HT levels; (3) peripheral arteries 5-HT uptake ability; and (4) function of SERT in

modifying 5-HT—induced aortic contraction in normotensive and hypertensive rats.

i. Measurement of SERT Expression in Aorta from Normotensive and
Hypertensive rate

We used two experimental hypertensive rats model (DOCA-salt
hypertensive rats and LNNA-hypertensive rats) and one genetic hypertensive
model (SHR). The blood pressure of the animals we used in our study were SBP
[mmHg], DOCA= 197:6, SHAM(D)= 112:4, LNNA= 228:9, SHAM(L)= 128:2,
SHR= 172:7 and WKY= 121:3. Western blotting analysis using a SERT
antibody, which recognizes amino acids (516-630) mapping at the carboxy
terminus of the SERT protein (H115 antibody, Santa Cruz, CA) showed a single
band slightly lower than 75 kDa (Figure 38). The expression of SERT in aorta
from DOCA-salt rats was significantly elevated compared to SHAM rats
(SHAM(D), which paired with DOCA-salt hypertensive rats) (arbitrary unit,
normalized to a-actin expression, SHAM(D)= 2774:469, DOCA= 4374:125,
p<0.05). This upregulation of SERT protein expression was not caused by

increased transcription but increased translation of mRNA, as we observed no

132

Figure 38.

Western blotting analysis of SERT in aorta homogenates from SHAME», DOCA-
salt hypertensive rats, SHAM“, LNNA hypertensive rats, WKY and SHR.
Representative of three experiments/rats.

SERT= serotonin transporter.

SHAM“), = SHAM rats paired with DOCA-salt hypertensive rats.

SHAM(L,= SHAM rats paired with LNNA hypertensive rats.

133

 

mzm >x>> <22._ 23% <08 éz<zw

 

 

 

 

Figure 38.

Al

mox Sq
5.8-8

Hmmm
T

T
max mu

 

 

I, W?

134

difference in the delta CT value of SERT in SHAM and DOCA-salt samples in a
real time RT-PCR study [Delta CT(SERT CT- GAPDH CT) SHAM(D)= 1.75 :i: 0.1,
DOCA-salt = 2.45 :l: 0.13; p= 0.064; n = 6]. Similarly, SERT expression was also
upregulated in LNNA rat aorta (arbitrary unit, SHAMM (normotensive rats paired
with LNNA hypertensive rats) = 2074.3:291, LNNA= 3603:169, p<0.05).
However, we did not observe a significant difference in SERT aortic expression
between WKY and SHR. Figure 38 shows the representative picture from three

different experiments/rats.

ll. Comparison of the Basal Level of 5-HT in DOCA-salt Rats, LNNA Rats
and SHR with Their Normotensive Control Rats
We compared the basal level of 5-HT and its MAO A oxidized metabolite,
5-HIAA, in aorta from untreated (no pargyline treatment) normotensive and
hypertensive rats. Figure 39 demonstrates that 5-HT but not 5-HIAA
concentrations were significantly lower in aorta from DOCA-salt compared to
SHAM rats (5-HT [nglmg protein], SHAM(D)= 0.31:0.07 and DOCA= 0.17:0.03,
p<0.05). Both 5-HIAA and 5-HT concentrations in aorta were lower in LNNA rats
compared to their control normotensive rats (5-HlAA [nglmg protein], SHAM(L)=
0.73:0.16 and LNNA= 0.10:0.02, p<0.05; 5-HT [nglmg protein], SHAM=
0.55:0.16 and LNNA= 0.24:0.03, p<0.05). No differences were found in aortic

5-HIAA and 5-HT concentrations when comparing values from SHR to WKY.

135

Figure 39.

Detection of basal concentrations of 5-HIAA and 5-HT in thoracic aorta from
SHAM“), rats, DOCA-salt hypertensive rats, SHAMM rats, LNNA hypertensive
rats, SHR and WKY rats.

Bars represent means:SEM for the number of animals in parentheses. * p<0.05
compared to appropriate normotensive control.

5-H|AA=5-hydroxyindoleacetic acid. 5-HT=5-hydroxytryptamine.

SHAM(D,= SHAM rats paired with DOCA-salt hypertensive rats.

SHAM(L,= SHAM rats paired with LNNA hypertensive rats.

136

DSHAM (D)
.DOCA
(N=6-7)

Rat Aorta

 

 

 

 

 

 

0. a an... .... c.
1 0

0 0 0 0
.5395 mEB$=ozEEoocoo

from

Figure 39.

 
 

5-i'iT

 

(N=5-6)

 

CISHAM (L)
- LNNA
DWKY
.SHR

(N=3-4)

 

 

 

Rat Aorta
Rat Aorta

 

W
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5-i-iiAA

137

lll. 5-HT Uptake Comparison in DOCA-salt Rats, LNNA Rats and SHR with
Their Normotensive Control Rats

Active 5-HT uptake was measured in aorta from pargyline-treated rats. By
inhibiting 5-HT metabolism, the observed changes in 5-HT concentrations in
aorta after incubation with exogenous 5-HT directly reflect the 5-HT uptake in
aona.

Sections of aorta from the same rat were incubated with vehicle, the
SERT inhibitor fluoxetine (1 uM), fluvoxamine (1 uM), 5-HT (1 uM), 5-HT (1 uM)
plus fluoxetine (1 uM) or 5-HT (1 uM) plus fluvoxamine (1 uM). Aorta were
incubated with inhibitor for 30 minutes before 5-HT was added (15 min
incubation). We measured 5-HlAA and 5-HT concentrations in aorta after
incubation. Aorta actively took up exogenous 5-HT while both SERT inhibitor
fluvoxamine and fluoxetine reduced this 5-HT uptake in aorta from all animals
studied (Figure 40,41 ,42).

To quantify total 5-HT uptake after incubation with exogenous 5-HT, we
subtracted the 5-HT concentration in aorta incubated with vehicle from that in
aorta incubated with exogenous 5-HT (5-HT 1 uM — vehicle, [nglmg protein],
SHAMm): 3.14:0.22, DOCA= 2.36:0.31, p<0.05; SHAM(._)= 3.50:0.39, LNNA=
2.51:0.25, p<0.05; WKY= 2.29:0.25 and SHR= 2.08:0.40, Figures 40,41,42,
bottom). These results suggest that aorta from DOCA and LNNA rats took up
less 5-HT compared to their normotensive rats while no difference existed
between aorta from SHR and WKY. Consistent with our previous experiments,

neither the SERT inhibitor fluoxetine (1 11M) nor fluvoxamine (1 uM)

138

Figure 40.

Effect of a 30-minute preincubation with SERT inhibitor fluoxetine (1 11M) or
fluvoxamine (1 uM) on 5-HT concentrations in aorta from pargyline-treated
SHAMM rats (top) and DOCA-salt hypertensive rats (middle).

Comparison of total 5-HT uptake and SERT dependent 5-HT uptake in aorta from
SHAM“), and DOCA-salt rats (bottom).

Bars represent means:SEM for the number of animals in parentheses.

* p<0.05 compared to vehicle incubation. ” p<0.05 compared to 5-HT (1 uM)
incubation.

5-HlAA=5-hydroxyindoleacetic acid, 5-HT=5-hydroxytryptamine.

SERT=serotonin transporter.

SHAM(D)=SHAM rats paired with DOCA-salt hypertensive rats.

139

 

Concentration(nglmg protein)

Concentration(nglmg protein)

Concentration(nglmg protein)

C)

"I
I

h
I

w
I

 

 

SHAM (D) Rat Aorta
|:]Vehicle
.Fluvoxamine 1uM
EFluoxetine 111M
5-HT 111M
’// 5-HT+FIuvoxamine
E5-HT+Fluoxetine

(N=6-11)

 

5-HIAA 5-HT

DOCA Rat Aorta
DVehicle
.Fluvoxamine 1 uM
EFluoxetine 1 uM
.5-HT 1 ”M .

 

 

3- .5-HT+FIuvoxamine
ES-HT+FIuoxetine *#
2- (N=7-13) *
1 -
c _ l‘l i? a
5-HIAA 5-HT
4. Rat Aorta 5-HT Uptake Comparison
DSHAMiD)
.DOCA
3. l (N=6-13)
p=0.056
2.

 

 

,, Ffl
I fll Figure40.

 

Total uptake Fluvoxamine Fluoxetine
140

Figure 41.

Effect of a 30-minute preincubation with SERT inhibitor fluoxetine (1 11M) or
fluvoxamine (1 (M) on 5-HT concentration in aorta from pargyline-treated
SHAMM rats (top) and LNNA hypertensive rats (middle).

Comparison of total 5-HT uptake and SERT dependent 5-HT uptake in aorta from
SHAM,” and LNNA rats (bottom).

Bars represent means:SEM for the number of animals in parentheses.

* p<0.05 compared to vehicle incubation. ’ p<0.05 compared to 5-HT (1 uM)
incubation.

5-HlAA=5-hydroxyindoleaoetic acid, 5-HT=5-hydroxytryptamine.

SERT= serotonin transporter.

SHAM(L,= SHAM rats paired with LNNA hypertensive rats.

141

-( at", protein)

‘-
atll II

A

 

c.

Concentration(nglmg protein)

Concentration(nglmg protein)

5-

l

4

 

   

 

5‘

w
I

'9

A
l

 

SHAMM Rat Aorta
DVehicle *
.Fluvoxamine 1 uM
EFluoxetine 1uM

.5-HT 1 “M
.5-HT+FIuvoxamine
E5-HT+FIuoxetine * .-
(N=8) *#

 
 
    
  

5-HIAA 5-HT
LNNA Rat Aorta
l:lVehicle
.Fluvoxamine 1 uM
EFluoxetine 1uM *
.5-HT 1 ”M

.5-HT+Fluvoxamine
55-HT+Fluoxetine

(N=8)

5-HIAA 5-HT
Rat Aorta 5-HT Uptake Comparison
.1; DSHAMiL)
.LNNA
* (N=8)

 

 

 

 

Total uptake Fluvoxamine Fluoxetine

142

Figure 41.

Figure 42.

Effect of a 30-minute preincubation with SERT inhibitor fluoxetine (1 nM) or
fluvoxamine (1 pM) on 5-HT concentration in aorta from pargyline-treated WKY
(Top) and SHR (Middle).

Comparison of total 5-HT uptake and SERT dependent 5-HT uptake in aorta from
WKY and SHR (Bottom).

Bars represent means:SEM for the number of animals in parentheses.

* p<0.05 compared to vehicle incubation. ‘ p<0.05 compared to 5-HT (1 11M)
incubation.

5-HlAA= 5-Hydroxyindoleacetic acid, 5-HT= 5-Hydroxytryptamine.

143

Concentration(nglmg protein) Concentration(nglmg protein)

Concentration (nglmg protein)

5. I:IVehicle

.?

‘r’

41 Rat Aorta 5-HT Uptake Comparison
3.

WKY Rat Aorta

5' EIVehicle
.Fluvoxamine 1uM
4' EFluoxetine 1p.M
.5-HT 111M
3- .5-HT+Fluvoxamine *
ES-HT+Fluoxetine

(N=4) *#

 

 

5-HIAA 5-HT

SHR Rat Aorta

.Fluvoxamine1 uM

Fluoxetine 1 uM

5-HT 1 ”M
.5-HT+F|uvoxamine *
E5-HT+FIuoxetine

(N=3)

 

 

5-HIAA 5-HT

IZIWKY
.SHR
(N=3-4)

 

 

1- [rim

Total uptake Fluvoxamine Fluoxetine
144

blocked the entire 5-HT uptake stimulated by exogenous 5-HT (Figures
40,41,42).

To quantify the SERT-mediated 5-HT uptake in aorta, we compared 5-HT
uptake in the presence and absence of fluvoxamine or fluoxetine. We subtracted
the 5-HT concentration (nglmg protein) in aorta after incubation with exogenous
5-HT plus SERT inhibitor (fluvoxamine or fluoxetine) from that of aorta only
incubated with exogenous 5-HT [5-HT — (5-HT+fluvoxamine) or 5-HT - (5-
HT+fluoxetine)]. This is a value we interpreted to reflect 5-HTT-dependent 5-HT
uptake. There was significantly lower SERT-mediated 5-HT uptake (calculated
from fluvoxamine inhibition) in DOCA (1.83:0.12 nglmg protein) compared to
SHAM“), (2.54:0.30 nglmg protein) and in LNNA (1.45:0.20 nglmg protein)
compared to its normotensive control (2.18:0.23 nglmg protein) while no
difference existed in SHR (1.05:0.32 nglmg protein) compared to WKY rats
(1.23:0.22 nglmg protein). Similar results were obtained from the calculation of
SERT-mediated 5-HT uptake by fluoxetine inhibition (nglmg protein, SHAM(D)=
1.92:0.12, DOCA=1.43:0.18, p=0.056; SHAM(L)= 2.71:0.25, LNNA= 1.91:0.20,

p<0.05; WKY= 0.89:0.33, SHR=1.10:0.47)

IV. SERT Function in Contractility- Compare DOCA-salt Rats with Normal
Rate

The next experiments determined whether increased expression and

decreased 5-HT uptake ability of the SERT in arteries possessed activity

sufficient to modify 5-HT—induced contraction. The model of DOCA-salt

145

hypertensive rats was used, as arteries from these rats are well known for their
super-sensitivity to the contraction effect of 5-HT. In aorta isolated from SHAM
and DOCA-salt hypertensive rats, cumulative 5-HT concentration response
curves were investigated in the presence of vehicle or one of two different SERT
inhibitors, fluoxetine or fluvoxamine. We used 1 uM of SERT inhibitors, which is
consistent with our 5-HT uptake study. Neither drug at the concentration of 1 uM
affected KCI -induced contraction (6-100 mM, data not shown). Figure 43 and
Table 6 describes the 5-HT concentration response curves in aorta from SHAM
and DOCA rats graphically and using pharmacological parameters, respectively.
We confirmed that aorta of DOCA-salt rats were hyperresponsive to 5-HT
compared with SHAM, evidenced by a small but significant decrease in the ECso
value of 5-HT and a decreased threshold concentration for contraction (Table 6).
Fluoxetine and, to a greater measure, fluvoxamine decreased the threshold of 5-
HT—induced contraction in aorta from DOCA-salt but not normotensive rats.
Similarly, the potency of 5-HT was significantly enhanced by fluvoxamine in aorta
from DOCA-salt rats (4.2-fold). Fluvoxamine also potentiated 5-HT-induced

contraction in aorta from SHAM rats.

146

Figure 43.

Effect of the SERT inhibitor fluoxetine (1 uM; top) and fluvoxamine (1 uM;
bottom) on 5-HT-induced contraction in aorta from SHAM and DOCA-salt rats.
Points represent means:SEM for the number of animals in parentheses.

* p<0.05 compared to vehicle incubation.

147

Rat Aorta

200- +Sham Control
-o- Sham Fluoxetine

+ DOCA Control

—‘L
0|
‘P
U
0
O
>
1"
i:
O
x
o
'1‘
:

(N=4-9)

 
    

Percentage PE (105 M) Contraction
3
?
Put—i

 

 

-1o -9 -8 7 -5 -5 $1 33.
Log 5-HT [M]

Rat Aorta

200' +Sham Vehicle
-o- Sham Fluvoxamine
+DOCA Vehicle
150- -v- DOCA Fluvoxamine

(N=4)

IOI'I

Contract

.8
O
‘P

at
?

 

Percentage PE (105 M)

 

O
L

 

-1o -9 5.

Log 5-HT [M]

Figure 43.
148

 

 

 

 

 

 

 

 

 

 

 

 

°°“"°' SHAM DOCA-salt
Threshold 6.92:0.07 8.00:0.16*
£050 5.90:0.08 6.13:0.09*
Fluoxetine SHAM DOCA-salt
Threshold 7.06:0.25 8.83:0.16*
£050 5.90:0.10 6.44:0.08*
Fluvoxamine SHAM DOCA-salt
Threshold 7.50:0.20" 8.50:0.28*
£050 6.43:0.21” 6.85:0.08*
Table 6.

Quantification of the threshold and EC50 in 5-HT-induced contraction in aorta
from normotensive and hypertensive rats. Values reported as the negative
logarithm of the value [M].

*indicates statistically significant differences (p<0.05) when compared to SHAM.
# indicates statistically significant differences (p<0.05) when compared to vehicle.
ECso = concentration of 5-HT eliciting a half-maximal contraction.

Threshold = lowest concentration at which contraction is observed.

149

Discussion:

Originally discovered in intestinal tract and blood, multiple functions of 5-
HT in physiological and pathological conditions have been revealed. The loss of
precise serotonergic system regulation (including 5-HT synthesis or metabolism,
reuptake or release and 5-HT receptor expression) in the brain leads to mental
disease. 5-HT is related to many peripheral vascular diseases but the role of a
local serotonergic system has not been studied. In this study, we investigated
the existence of a local serotonergic system in peripheral arteries and whether

this local regulation of 5-HT was altered in hypertension.

l. Biochemical Proof of 5-HT and its Metabolite in Peripheral Normal
Arteries

It was considered that peripheral arteries are only exposed to 5-HT from
blood. Whether peripheral arterial smooth muscle has endogenous 5-HT was
not known. The existence of endogenous 5-HT and its metabolite 5-HlAA is the
fundamental tenet of our hypothesis -- a local serotonergic system exists in
peripheral arteries.

Three different techniques were used in our experiments. All results
suggested the existence of 5-HT in peripheral arteries. Use of HPLC (Figure 4),
as opposed to a classic assay using measurement of [3H]5-HT uptake, was
critical, because we could measure both 5-HIAA and 5-HT in these assays. The
existence of 5-HT in smooth muscle is supported by 5-HT immunoreactivity in

smooth muscle layers and cytosol of freshly isolated smooth muscle cells that

150

are not exposed to blood, a potential source of extraneous 5-HT through blood
platelets. While arteries contain mast cells, it is unlikely that these cells make a
significant contribution to the 5-HT concentrations measured as few mast cells
could be identified. Mast cell populations have been reported as rich in the
coronary adventitia (Wolf et al., 1998), a site where there was in fact strong
staining for 5-HT in our study (Figure 6). Additionally, mast cells have been
found in femoral arteries (Mathiau et al., 1993). it is important to note that
measures of mast cells were not made in the carotid and superior mesenteric
arteries, and thus we cannot exclude the possibility that some of the 5-HT and 5-
HIAA measured came from mast cells in these particular arteries. The staining of
5-HT antibody in isolated aortic smooth muscle cells (Figure 7) further supported
that at least part of the 5-HT we measured using HPLC came from arterial
smooth muscle cells.

Thus, we quantified basal arterial endogenous 5-HIAA and 5-HT
concentration by HPLC measurements and localized 5-HT in endothelium and

peripheral arteries smooth muscle cells by immunostaining.

ll. Investigation of 5-HT Synthesis in Peripheral Normal Arteries
The presence of TPH is considered a hallmark of 5-HT synthesis. in
literature, studies investigating the serotonergic innervation of peripheral arterial
blood vessels were performed before the advent of PCR and antibody-based IHC

and Western analyses.

151

As depicted in Figure 1, TPH, AADC and MAO A are enzymes responsible
for each step of 5-HT synthesis and metabolism. The existence of these
enzymes is an essential requirement for synthesizing and metabolizing 5-HT in
peripheral arteries. We tried to identify the existence of TPH protein in peripheral
artery by Western analysis and lHC. However, due to technique limitations of the
antibodies, we were not able to visualize TPH protein in homogenates from rat or
mouse aorta in Western analysis. This could be due to degradation of TPH
protein in the procedure of protein isolation. The results from IHC experiments
were not convincing either. Similar results were observed in both frozen sections
(Figure 9) and paraffin embedded sections (pictures not shown) with relatively
darker staining in sections incubated with primary antibody compared to no
primary incubation. Thus, another experimental technique was necessary to
identify the existence of TPH protein. Pictures from immunocytochemistry
experiments showed a positive TPH antibody staining in freshly isolated aortic
smooth muscle cells. To lend support to this finding, we did real time RT-PCR
experiments to investigate the existence of tph1 mRNA. The results using two
different sets of tph1 primers suggested the presence of tph1 mRNA in peripheral
artery, which reinforced the result of existence of TPH protein in our
immunocytochemistry experiment. We followed these measures with an activity
assay.

The rationale of our TPH activity assay was that the measurement of the
accumulated 5-HTP in arteries, when AADC was inhibited by NSD1015, would

indicate that a functional TPH exists. The assumption of this experiment was

152

that peripheral arteries do not take up 5-HTP (the 5-HTP that we measured was
synthesized in arteries). Though we did observe a 5-HTP peak in arteries from
NSD1015 treated rats and an increased 5-HTP was observed in NSD1015 + 5-
HT synthesis substrate, tryptophan-treated rats, we cannot make the conclusion
that this 5-HTP was synthesized by the peripheral artery as we also observed
that peripheral arteries are capable of taking up 5-HTP (Figure 11). Thus, it is
important to do an in vitro experiment to test the function of TPH in arteries. As
we mentioned in the introduction, TPH is an extremely unstable enzyme, which
loses its function quickly due to exposure to oxygen. In our in vitro TPH activity
assay (Figure 12), we incubated isolated rat arteries with incubation solution, in
which we added the reducing agent ferrous ammonium sulfate and D'I'I' to
recover oxygen reduced TPH activity, added catalase to stabilize TPH, added 5-
HT synthesis cofactor BH4, added NSD1015 to inhibit the conversion of 5-HTP to
5-HT and in some of our experiments we added tryptophan. There was no
detectable 5-HTP in artery samples, while very low amount of 5-HTP was
observed in rat raphe samples. We have two explanations for our data. The
first, there is TPH expression in peripheral arteries. However, this TPH is
not functional, at least in normal animals. The presence of TPH, AADC and
BH4 (Gross et al., 1993) in arteries suggests the possibility of 5-HT synthesis. In
disease condition, TPH activity, BH4 production and Ca2+lca|modulin-dependent
protein kinase could change and TPH might be activated by these changes.
Evidence supporting this speculation is increased Ca2*/calmodulin-dependent

protein kinase activity in SHR (Boknik et al., 2001) and increased vascular gene

153

and protein expression of PKA in two-kidney, one clip (2K-1C) renal hypertensive
vs. normotensive two kidney (2K) rats (Callera et al., 2004), which might lead to
increased TPH activity. However, it was reported that in DOCA-salt hypertensive
rats, the arterial BH4 level was decreased. The reduction of BH4 minimized the
possibility that a functional TPH exists in peripheral arteries. The second
explanation for our data is that there is 5-HT synthesis in peripheral arteries
from normal rats. However, we are limited by the sensitivity of HPLC to

measure this level of 5-HTP.

Ill. Investigation of Metabolism of 5-HT in Peripheral Arteries
Uptake study in arteries from normal rats (Figure 15) showed that after 15
minutes of incubation with exogenous 5-HT, the concentration of 5-HIAA in
arteries was significantly higher than 5-HT, suggesting that 5-HT was taken up
into blood vessel and rapidly metabolized. This observation suggests that
peripheral arteries might play an important role in clearance of free

circulating 5-HT in blood.

IV. Investigation of Storage of 5-HT in Peripheral Arteries
We do not believe that peripheral arteries function like platelets as a “5-HT
sink”, which store and release 5-HT, because even in an extreme condition, in
which we preload 5-HT in aorta from pargyline-treated rats, we did not observe
that 5-HT could be kept in aorta. Peripheral arteries take up and metabolize 5-

HT but do not store 5-HT.

154

 

We also investigated the localization and the form of endogenous
intracellular 5-HT. Western analysis showed serotonin-modified proteins in
arteries. Moreover, numerous proteins were reported as the substrates of TC",
such as actin, myosin and troponin (Griffin et al., 2002). Whether this
modification is linked to a functional change is not known yet. Other 5-HT could
be packed in vesicles, as we observed fluramines-induced 5-HT release,
although we do not have direct evidence showing the existing of VMAT in

peripheral arteries.

V. Investigation of functional SERT in Peripheral Normal Arteries

We employed a multi-faceted approach in determining that peripheral
smooth muscle has the potential to synthesize SERT. Real time RT-PCR and
gel analyses of the final product after 35 cycles indicated the presence of one
product. Translation of this message was confirmed by successfully measuring
SERT protein product. Importantly, we confirmed immunohistochemically that a
majority of the SERT is located in smooth muscle. Endothelial cells were stained
positive for the SERT and this is consistent with previous findings (Lee et al.,
1986; Small et al., 1977; Brust et al., 2000).

Having demonstrated the presence of the SERT protein in arteries, we
determined if peripheral arteries had the ability to take up 5-HT. In a simple
assay (Figure 24), we demonstrated that 5-HT was taken up in a time-dependent
manner by observing increased 5-HIAA with time in arteries. Because MAO A is

an enzyme classically found in mitochondria, the increased arterial 5-HIAA after

155

exogenous 5-HT incubation suggested the ability of peripheral arteries to taken
up 5-HT. We proved that this uptake is SERT-dependent by pharmacologically

inhibition (Figure 28) and genetic ablation of SERT (Figure 29).

VI. Investigation of SERT-independent 5-HT Uptake Mechanisms in
Peripheral Arteries

We noticed that neither pharmacological inhibition nor genetic ablation of
SERT could abolish all the 5-HT uptake in arteries, which suggested that other
SERT-independent mechanisms are involved in 5-HT uptake.

One of the vessels we have used is the aorta, which contains sparse
innervation by the sympathetic nervous system. Uptake in this minimally
innervated tissue lends indirect support that it is the smooth muscle cells rather
than nerves, takes up 5-HT (Kawasaki and Takasaki, 1984; Kawasaki et al.
1987). Moreover, in arteries from animals that have been chemically denervated
or exposed to the NET inhibitor nisoxetine (Figure 32), uptake in all vessels
remained normal. While these experiments do not exclude the possibility that
sensory nerves could take up 5-HT, these data strongly support the idea that
uptake of 5-HT into the artery is nerve- independent. Smooth muscle is not,
however, the only arterial cell type that can take up 5-HT. Cultured endothelial
cells take up and metabolize 5-HT (Lee et al., 1986; Small et al., 1977; Brust et
al., 2000). By contrast, human fibroblasts appear to have a significantly lower
rate of 5-HT uptake and metabolism (Small et al., 1977). In our experiments, the

endothelial cell appears to make little contribution to 5-HT uptake in the whole

156

artery, though the SERT was located immunohistochemically to these same
cells. The lack of effect of endothelial cell and neuron removal on 5-HT uptake
may be because these two cell types do not contribute significantly and/or
sufficiently to the total mass and uptake capability of the artery. Thus, the
sensitivity of our assay (2—5 pg) may be insufficiently low, such that we were
unable to reveal uptake by the endothelial cell and sympathetic neuron. This is a
noted limitation of these studies.

NET, DAT and SERT are all Na+-dependent transporters and use the
energy provided by ATP. The uptake of 5-HT in Na+-free PSS demonstrated that
part of 5-HT that crosses the cell membrane does not depend on Na+ moving
downhill of the electrochemical gradient, which was built by Na“-K+ ATPase. We
do not know, whether all of the 5-HT that passes the cell membrane is energy
dependent, as in our experiment, arteries were incubated in a high concentration
(compared to intracellular 5-HT concentration) of exogenous 5-HT (1 nM, 15
minutes) solutions. Experiments depleting ATP would help us answer the
question whether the SERT-independent 5-HT uptake is active transport.

OCTs are non-specific cation transporters. Though it has been reported
that OCTs are upregulated in the gastrointestinal system in SERT-deficient mice
and they might play a role in 5-HT uptake, we did not observe the OCTs inhibitor
reducing 5-HT uptake in arteries from normal animals. We did not have a
successful experience in identifying OCTs in arteries by Western analysis. We
visualized multiple bands in our positive control as well as in our samples by

using the antibody and there was no competing peptide available to verify the

157

 

specificity. OCTs are not involved in 5-HT uptake in normal condition but we
could not exclude the possibility that in diseases or situations when the function

of SERT lost, OCTs might start to take up 5-HT as a redundant mechanism.

VII. Investigation of 5-HT Release Mechanisms in Peripheral Arteries

(+)-Fenfluramine and (+)-norfenfluramine (fluramines) are SERT
substrates and release 5-HT. The mechanism behind this effect is that
fluramines are taken up by SERT into cytoplasm and then act on VMAT to
release vesicular 5-HT. With the increased cytoplasmic 5-HT, SERT is reversed
and starts to transport 5-HT outside of cells (Rothman and Baumann, 2002). If
this is the only mechanism by which fluramines release 5-HT, we may conclude
that part of endogenous 5-HT is stored in vesicles in peripheral artery smooth
muscle. However, we do not yet have evidence to support the existence of the
vesicles. The release of 5-HT from arteries may have significant physiological or
pathological effect by changing the local 5-HT concentration.

Because fluramines need to be taken up into smooth muscle cells to
cause 5-HT release, SERT inhibition should reduce this effect. _ In our
experiment, fluoxetine itself (1 uM) caused increases of 5-HT in PSS (Figure 37)
during incubation, which is much higher than the amount of 5-HT' released by
fluramines. Thus, the inhibition effect of fluramines-induced 5-HT release by
fluoxetine could be masked by fluoxetine action. The observation of increased
5-HT in PS8 by fluoxetine suggests that there is a spontaneous release of 5-HT

in the peripheral artery. in normal conditions, there could be a balance between

158

5-HT release and uptake in peripheral arteries. The mechanism of this

spontaneous release is not clear, nor is the source of the intracellular 5-HT.

VIII. Physiological Relevance of 5-HT Uptake in Peripheral Arteries and
Speculation
It has been argued that 5-HT plays an insignificant role in the physiological
control of blood vessel tone as uptake of 5-HT by platelets and/or adrenergic
nerves causes circulating levels of 5-HT to be low [estimated between 15—120
nM, Martin, 1994] and out of a range that can directly cause constriction. One
manner by which physiological, autocrine/paracrine concentrations of 5-HT may
affect vascular tone is by potentiating arterial contraction and mitogenesis to
other vasoactive substances (Szabo et al.,1991; Yildiz et al, 1998; Watanabe et
al. 2001). Thus, a local 5-HT system may play a significant role in affecting local
contractility and mitogenesis by regulating local 5-HT concentration. This could
not be discerned by measuring plasma concentrations of 5-HT.
Because we have observed 5-HT uptake and the physical presence of the
SERT in multiple arteries, this finding may be global in the body. Peripheral,
non-pulmonary arteries could then be added to the growing list of already
recognized sites of 5-HT uptake, including the brain, cerebral arteries (Scatton et
al., 1985; Amenta et al., 1985; Brust et al., 1996), pulmonary arteries (Eddahibi,
1999), gut (Chen et al., 2001), pituitary (Johns et al., 1982), airway smooth
muscle (Dodson et al., 2004) and cardiac myocytes (Sari and Zhou, 2003). The

uptake and metabolism of 5-HT in peripheral arteries could play a role in

159

reducing 5-HT concentrations to maintain a normal physiological 5-HT content in
free circulation. One can speculate that, dysfunction of this 5-HT uptake and
metabolism system in peripheral arteries would lead to increased free circulating

5-HT and diseases related to that.

IX. Pathological Relevance of 5-HT Uptake in Peripheral Arteries and
Speculation
The role of 5-HT and, more recently, its regulator, SERT, in hypertension
is controversial and intriguing. The enhancement of potency for 5-HT in inducing
vascular contraction or pressor responses in hypertension is striking. Increases
in reactivity to 5-HT have been observed in a number of different forms of
hypertension models including DOCA-salt hypertensive rats (Watts, 1998), SHR
(Nishimura and Suzuki, 1995), and in human patients (Golino et al., 1991). Many
mechanisms may contribute to this hyperresponsiveness such as changes in the
5-HT receptor signaling or changes in the circulating levels of 5-HT. It makes
logical sense that a change of the activity of the 5-HT regulator (SERT) would
change circulating 5-HT concentrations and thus change the responsiveness of
arteries to 5-HT. We compared SERT function by testing 5-HT uptake of aorta
from normotensive and hypertensive rats and tested whether changed in SERT
expression and function were common to hypertension by using three
hypertension models, the DOCA-salt rats, LNNA rats and SHR.
These three models of hypertension are different in terms of mechanisms

by which animals became hypertensive and the human hypertension they

160

resemble. DOCA-salt hypertension or mineralocorticoid hypertension resembles
the clinical situation of aldosterone excess. DOCA-salt hypertension is caused
by a surgical uninephrectomy followed by administration of a DOCA pellet and
excess salt (1% NaCl+ 0.2% KCI in water). Blood pressure then rises within a
few weeks into the hypertensive range. This is a sodium-dependent, low-renin
model. Non-sodium mechanisms have been suggested to play a role in the
development of DOCA-hypertension, including activation of sympathetic nervous
system and vasopressin activation. LNNA inhibits all isoforms (endothelial,
neuronal and inducible) of nitric oxide synthase (NOS), with a moderate
selectivity for the endothelial isoform (Takahashi et al., 1995). Established
hypertension was developed in two weeks after rats were given water with LNNA
(0.5 g/L). The loss of nitric oxide production directly leads to a reduced nitric
oxide-mediated vasodilation and superoxide “quenching” ability in blood vessels.
Other mechanisms involved in LNNA-induced hypertension include activation of
angiotensin converting enzyme (Charpie et al., 1997) and the AT1 receptor
(Verhagen et al., 2000) but not vasopressin (Loichot et al., 2000). The model of
LNNA hypertensive rats resembles a reduced bioavailability of nitric oxide in
hypertensive individuals (Kelm, 2003, review). SHR develops hypertension and
target organ damage similar to those seen in human essential hypertension. The
blood pressure of SHR is relatively sodium-independent (Griffin et al., 2001).
Therefore, investigation of the serotonergic system in these three models

would answer the question of whether there is a change of component of

161

serotonergic system in hypertension and whether this is a general change in all

hypertension.

X. Basal 5-HT Concentration in Aorta of DOCA-salt, LNNA Hypertensive
Rats and SHR
As we stated earlier the free circulating plasma 5-HT levels (15-120 nM)
(Martin, 1994) could not activate the 5-HT2A receptor (Ki= 100-3000 nM, PDSP Ki
Database), which is the major receptor mediating 5-HT-induced arterial
contraction in normotensive animals. However, many reports showed that
plasma 5-HT concentrations and arterial 5-HT receptors are changed in
hypertension. in essential hypertensive humans (Fetkovska et al., 1990) and
cyclosporine-induced hypertensive rats (Reis et al., 1999), reduced levels of
platelet 5-HT and increased plasma 5-HT concentration were measured.
Consistent with these reports, unpublished work from our lab determined that the
DOCA-salt rat platelet 5-HT was reduced (ng/ml of whole blood, SHAM(D)=
215.6:35.4, DOCA= 150.4:36.7) while free circulating 5-HT concentration was
increased (ng/ml whole blood, SHAM(D) =5.82:1.14, DOCA= 28.1:11.1). The
current observation of reduced basal 5-HT content in aorta from DOCA-salt rats
suggests that arteries function as a metabolism organ for 5-HT with decreased 5-
HT uptake ability in DOCA-salt hypertension. We speculate that the increased
free circulating 5-HT in DOCA-salt rats may result from decreased 5-HT storage
in and uptake by platelets and arteries. The increased extracellular free

circulating 5-HT in DOCA-salt hypertensive rats may have significant effects. 5-

162

HT has high affinity for the 5-HT23 receptor (Ki= 10 nM, Bonhaus et al., 1995),
and the expression and function of 5-HT23 receptor is upregulated in arterial
smooth muscle in DOCA-salt hypertensive rats (Banes and Watts, 2003; Watts,
2002). This receptor is activated endogenously to maintain the high blood
pressure of DOCA rats (Watts and Fink, 1999). Moreover, nanomolar
concentrations of 5-HT potentiate arterial contraction to ET-1 and NE (Watts,
2000). Thus, it is possible that increased free circulating 5-HT concentration
induces vasoconstriction, increases total peripheral resistance and thus elevates
blood pressure in DOCA-salt hypertensive rats.

Unfortunately, there are no literature reports of measurements of platelet
5-HT content or free circulating 5-HT concentration in the LNNA hypertensive
rats. Our observation of decreased basal aortic 5-HT and 5-HIAA concentration
suggests a changed SERT activity and changed level of circulating 5-HT in
LNNA rats. Similar to the DOCA-salt rat, 5-HT23 receptor expression and
function is increased in LNNA rats and is necessary for maintaining elevated
blood pressure (Russell et al., 2002). It is possible that these changed factors of
the serotonergic system in LNNA rats play roles in increasing and maintaining
blood pressure. Further studies need to be done to investigate the change of
platelet and free circulating 5-HT in LNNA rats.

Only two studies in 1985 investigated platelet 5-HT level in SHR. As
opposed to a decreased 5-HT in platelet in DOCA-salt hypertensive rats, SHR
have more platelets and similar platelet 5-HT levels in SHR compared to WKY

(Guicheney et al., 1985, a, b). Free circulating 5-HT in SHR has not been

163

reported. Our observation of similar basal 5-HT levels in aorta from SHR and
WKY was consistent with these reports in platelet. We are aware that the basal
level of 5-HT concentration is lower in WKY compared to Sprague-Dawley
normotensive rats. This may suggest an inherent difference in these strains. The
serotonergic system may have different degree of impact on blood pressure
regulation in WKY compared to Sprague-Dawley rats.

it is important to understand arterial function of SERT in hypertension not
only as a reflection of circulating 5-HT levels, but also because intracellular 5-HT
may have a function. Changing intracellular 5-HT concentration would in turn
change arterial contraction. Thus, our observation of an altered basal activity of
SERT in DOCA-salt and LNNA hypertension may have consequent pathological

results.

XI. 5-HT Uptake Ability was Reduced in Aorta of DOCA-salt and LNNA
Hypertensive Rats, but not in SHR
By normalizing 5-HT content to protein concentration and comparing the
change of 5-HT concentrations in nglmg protein, our results showed a decreased
total 5-HT uptake and a decreased SERT-dependent 5-HT uptake (Figure 40,
41) in aorta from DOCA-salt and LNNA hypertensive rats exposed to exogenous
5-HT, compared to that of aorta from their normotensive SHAM rats. We did not
observe differences in 5-HT uptake in aorta from SHR and WKY. Our results
suggest that SERT function was impaired in DOCA-salt and LNNA hypertensive

rats. The decreased total 5-HT uptake observed in DOCA-salt and LNNA

164

hypertensive rats may account for the decreased basal aortic 5-HT content in
both models and the increased free circulating 5-HT (DOCA-salt hypertension).
Possible explanations for the contradictory findings of an upregulated SERT
protein expression and a decreased SERT function in DOCA-salt and LNNA rats
includes different membrane/cytosol distribution of SERT in these SHAM and
hypertensive animals as well as post translational modification of SERT proteins.
Phosphorylation of SERT by p38 MAPK increases SERT function by stimulating
the insertion of intracellular SERT into the cell membrane and increasing total
SERT activity (Blakely et al., 2005). By contrast, PKC reduces SERT function by
phosphorylating SERT protein and causing translocation of SERT to the cytosol
(Jayanthi et al., 2005). Thus, it is possible that the total expression of SERT
increases in DOCA and LNNA hypertensive animals while the membrane fraction
or functional SERT actually decreases.

We did not observe a difference in basal aortic 5-HT content, 5-HT uptake
ability or SERT protein expression in aorta from SHR compared to WKY rats.
There are several factors that may contribute to the differences we observed.
First, as we discussed above, the strain is different (WKY vs. Sprague-Dawley).
Second, the systolic blood pressures of SHR (172:7 mmHg) were lower than
DOCA-salt rats (197:6 mmHg) and LNNA rats (228:9 mmHg). The change of
SERT expression and function may only happen in severe hypertension. Third, it
is possible that the deoxycorticosterone we used in our DOCA-salt hypertension
model causes the different SERT expression and function. However, Kulikov

reported that stimulation of mineralocorticoid receptors had no effect on SERT

165

radioligand binding density in rat midbrain (Kulikov et al., 1997). Furthermore,
the observation of impaired SERT function was also obtained in LNNA
hypertensive rats. Thus, it is unlikely that the change of SERT function in DOCA-
salt hypertension is from effects of mineralocorticoid.

More importantly, a correlation between 5-HT23 receptor expression and
plasma 5-HT or SERT function was reported recently (Callebert et al., 2006).
Callebert et al. observed significantly increased plasma serotonin levels in wild
type mice with upregulated 5-HT23 receptor after exposure to chronic hypoxia but
not in 5-HT23 receptor-l- mice. Acute treatment with 5-HT23 receptor agonist
induced a rapid SERT-and 5-HT23 receptor-dependent increase of plasma
serotonin levels, which suggests that 5-HT23 receptor activation inhibits SERT
function and thus less 5-HT is taken up. Upregulated 5-HT23 receptor expression
and function in DOCA and LNNA hypertension have been reported (Watts and
Fink, 1999, Watts, 2000; Russell et al., 2002). On the other hand, the 5-HT23
receptor was not involved in 5-HT-induced vasoconstriction in the hindquarters of
SHR (Calama et al., 2004), which suggests 5-HT23 receptor was not activated in
SHR as it was in DOCA-salt and LNNA hypertensive rats. Our results are
consistent with Calleberts’ report in that we observed an increased free
circulating 5-HT with increased 5-HT23 receptor function while decreased SERT
function in DOCA rats; increased 5-HT23 receptor function while decreased
SERT function in LNNA rats; no change of 5-HT23 receptor and normal SERT

activity in SHR. How the 5-HT23 receptor regulates SERT is not yet clear.

166

XII. Other Arterial 5-HT-uptake Mechanism in Hypertensive Animals

Similar to what we observed in normal animals, in arteries from
hypertensive rats, a maximal concentration of fluoxetine (1 [nM) and fluvoxamine
(1 uM) did not block the entire uptake stimulated by exogenous 5-HT. This
suggests that there are still other mechanisms by which 5-HT is transported into
aona.

it is important to note that we studied one concentration of exogenous 5-
HT at one time point in our active uptake study. Studies that examine 5-HT
uptake at various times and with a range of 5-HT concentrations need to be done
to compare SERT Km and Vmax values in aorta from DOCA-salt, LNNA
hypertensive rats and their SHAM control rats. However, this is difficult without
determination of other mechanisms for 5-HT uptake. Thus, kinetic studies have

to wait until these alternative mechanisms are discovered.

XIII. The Effect of SERT Function on Arterial Contractility in Hypertension
Fluoxetine and fluvoxamine differed in their effects in aorta from normal

rats, where a potentiation of 5-HT—induced contraction was observed with
fluvoxamine but not fluoxetine. Interestingly, Gruetter et al (Gruetter et al., 1992)
demonstrated nearly 10 years ago that 5-HT-induced contraction in aorta from
normal rats could be markedly potentiated by citalopram, another SERT inhibitor,
but only modestly by fluoxetine. Moreover, Cohen and Wiley (Cohen and Wiley,
1997) observed similar results in the relative inability of fluoxetine to potentiate

aortic contraction to 5-HT.

167

How SERT modifies arterial contraction is not clear. We may speculate
that the active uptake and metabolism of 5-HT in peripheral arteries is a
protective mechanism of the body to keep a normal level of free circulating 5-HT
and prevent blood pressure increases. When this uptake was inhibited by
fluoxetine/fluvoxamine, peripheral arteries lost the ability to clear environmental
5-HT, and were exposed to a relatively higher concentration of 5-HT. In
normotensive animals, 5-HT2A receptor-mediated 5-HT-induced contraction
would not be changed because of the relatively low affinity of 5-HT for 5-HT2A
receptor. The change of the amount of 5-HT by fluoxetine inhibition was not
enough to cause change in aortic contraction. In hypertensive animals, the
expression and function of 5-HT25 receptors (for which 5-HT has much higher
affinity) are upregulated. This change of local 5-HT concentration might be
sufficient to activate the 5-HT23 receptors to cause the potentiated contraction as
we observed. This speculation can also be applied to explain the potentiated
response to 5-HT in aorta from DOCA-salt rats compared to SHAM rats. The
impaired SERT function in aorta from DOCA-salt rats suggest a loss of an arterial
5-HT clearance mechanism in these animals, which might lead to elevated free
circulating 5-HT, vascular tone and blood pressure.

The alternative explanation of our observation is that fluoxetine and
fluvoxamine do not selectively inhibit SERT. Fluoxetine has fairly high affinity for
rat 5-HT2A receptor (Ki= 299:31 nM, Rothman et al., 2000) and might cause
inhibition of (+)-norfenfluramine-induced contraction. This is not consistent with

our observation of potentiation of 5-HT-induced contraction in aorta from DOCA-

168

salt rats. The affinity of fluvoxamine for 5-HT2A receptor is over 12,000 nM (no
affinity of fluvoxamine for 5-HT23 receptor has been reported). It is unlikely that
the concentration we used in our experiments (1 uM) inhibited 5-HT2A receptor
and 5-HT23 receptor.

The important message from these particular findings is that inhibition of
the transporter can affect contractility, and likely does so in a local manner. One
can also speculate that the change of serotonergic system could also happen in
arteries in which 5-HT plays a role in other pathophysiological states such as
migraine and coronary vasospasm, and this has certainly been supported by
findings in cerebral arteries (Amenta et al., 1985; Brust et al., 1996). With respect
to blood pressure regulation, the finding of a functional peripheral SERT is
potentially supported by findings that fluoxetine causes a pressor response in

conscious rats (Lazarigues et al., 2000).

XIV. Future Research

The finding of a local serotonergic system in peripheral arteries is exciting
and opens a new area of serotonin research. More studies need to be done to
further characterize this system.

Western analysis is the best way to quantify and compare protein
expression. Showing the direct evidence of the existence of TPH1 protein by
Western analysis is very important. A new TPH1 antibody has been reported
recently (Sakowski et al., 2006). This is the first antibody that allows the

differentiation of TPH1 and TPH2 upon immunoblotting, immunoprecipitation,

169

and immunocytochemical staining of tissue sections from brain (TPH2) and gut
(TPH1). Recombinant TPH1 and TPH2 protein should be used as positive
control in these future experiments. The activity of tryptophan hydroxylase is
enhanced by phosphorylation of Ser58, by cAMP dependent PKA and
Ca2*/calmodulin kinase II. Tryptophan hydroxylase (phospho 858) antibody is
also commercially available and worthy to try.

It is not yet known which proteins could be modified by intracellular 5-HT.
With the knowledge of actin and myosin as substrates for TGII and important
proteins for vasoconstriction, we could choose these proteins as candidates and
perform an immunoprecipitation experiment with 5-HT antibody. This experiment
would further elucidate which kind of protein could be modified by 5-HT.

The in vitro measurement of TPH activity in peripheral arteries is still an
ongoing project in our lab. It is possible that we could detect 5-HTP if we extend
the incubation time and/or adjust other conditions, such as increasing tryptophan
concentration. it is also worthwhile to test TPH activity in arteries in the
presence/absence of PKA or Ca2*/calmodulin-dependent protein kinase activator
and compare TPH activity in arteries.

Whether a change in the local 5-HT concentration, by the mechanisms
that we discussed in our studies, is sufficient to alter vascular tone is not clear.
To answer this question, we could use the SERT substrate and 5-HT releaser
(+)-fenfluramine to actively release endogenous 5-HT and test whether this is
sufficient to cause vasoconstriction or to potentiatie the effect of other

vasoconstrictors.

170

It is necessary to investigate SERT-independent 5-HT uptake
mechanisms. After elucidating these mechanisms, we should be able to

compare Km and Vmax of peripheral arterial SERT and brain SERT.

XV. Limitations

The original intention of this study was to determine the presence and
potential function of the SERT in peripheral arteries. We have not compared this
transporter, either kinetically, biochemically, or pharmacologically, to the classic
transporters in the brain and the lung. Such work will be important to determine if
these proteins could be potentially separate pharmacological targets. In our
studies we used the thoracic aorta in RT-PCR, Western, IHC, and contractility
assays. We are aware that the aorta is not a resistance artery and thus we
repeated some of these studies in the smaller artery, superior mesenteric artery,

which could be more similar to real resistance arteries.

171

Conclusions

Unlike previous serotonin research in vasculature, which focused on
serotonin receptor functions, we investigated whether peripheral arteries have
the ability to regulate 5-HT concentration locally, and possible roles of this
regulation in physiological and pathological conditions.

Overall, we demonstrated the presence of a functioning serotonergic
system in normal rat peripheral arteries. We proved the presence of the
essential components for 5-HT synthesis, such as TPH and AADC, but we were
limited by our technique to directly prove or disprove an active local peripheral
arterial 5-HT synthesis. The existence of uptake and metabolism of 5-HT was
clearly shown by observing increased 5-HIAA in isolated arteries from normal
animals incubated with exogenous 5-HT (1 (M, 15 min). Using arteries from
pargyline-treated rats, we studied the 5-HT uptake and metabolism mechanisms
and eliminated the change of 5-HT concentration caused by metabolism. A
basal- and fluramine-induced endogenous 5-HT release was observed, which
indicated the possibility of local increases of extracellular and, accordingly,
decreases of intracellular 5-HT concentration and as a consequence, changed 5-
HT function. The 5-HT uptake of peripheral arteries is partially mediated by
SERT, while other mechanisms involved in this process need to be further
elucidated. There is no 5-HT storage in peripheral arteries as we observed that
5-HT content reduced back to basal level in 5-HT-loaded aorta after 4 hours
incubation in PSS. Studies in arteries from hypertensive animals revealed a

decreased basal endogenous 5-HT and increased expression but impaired

172

function of the SERT in peripheral arteries. SERT modifies 5-HT-induced
contraction, and inhibition of the transporter is significantly enhanced in
mineralocorticoid hypertension.

Understanding the functions of the local serotonergic system in peripheral
arteries in physiological and pathological conditions will help us reveal the
etiology of 5-HT related peripheral vesicular diseases and find new targets for

drug development.

173

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BIBLIOGRAPHY
Personal Data
Name: Wei Ni B445 Life Science Building
Born: 1/13/1978 Department of Pharmacology and Toxicology
Michigan State University
East Lansing, MI. 48824-1317
Phone number: (517) 353-3900
Fax number: (517) 353-8915

Educational Background
1996-2001 Peking University Health Science Center

Beijing, P. R. China
B.S. (Pharmacology)

2002-present Michigan State University, East Lansing, Michigan
Doctoral candidate (Pharmacology and Toxicology)

Teaching Activities

2005-2006 Lecturer for Pharmacology (PHM) 450 class

Research Training

2001-2002 Hepatoprotective role of Ganoderma lucidum
polysaccharide against BCG-induced immune liver
injury in rats.
Research with Dr. Zhibin Lin and Dr. Guoliang
Zhang

2002-present The presence of a local serotonergic system in

peripheral arteries.
Doctoral thesis research with Dr. Stephanie W.
Watts, Michigan State Unversity

Membership in Academic Societies
American Society of Pharmacology and Experimental Therapeutics

American Heart Association
American Physiological Society
Society for Experimental Biology and Medicine

 

Awards

American Heart Association Predoctoral Fellowship-July,2004 to June,2006
ASPET Graduate Student Travel Award- Experimental Biology, 2005

ASPET Graduate Student Travel Award- Experimental Biology, 2006

MSU College of Veterinary Medicine Fellowship (Fall,2002; Fall,2003;
Spring,2004; Fall,2004)

MSU Graduate School Incentive Fellowship-2004

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MSU Phi Zeta Research Day Best Poster Presentation by a Ph.D. Student-
2004

MSU the Graduate Office Fellowship- Fall, 2005

MSU the Graduate School Dissertation Completion Fellowship- 2006

Papers
Zhang GL, Wang YH, Ni W, Teng HL and Lin ZB: Hepatoprotective role of

Ganoderma lucidum polysaccharide against BOG-induced immune liver injury in
mice. World J Gastroenterol. 2002 Aug; 8(4): 728-33.

Ni W, Li MW, Thakali K, Fink GD and Watts SW: The fenfluramine metabolite
(+)-norfenfluramine is vasoactive. J. Pharmacol. Exp. Ther., 2004, 309(2):845-
52.

Ni W, Thompson JM, Northcott CA, Lookingland K and Watt SW: The serotonin
transporter is present and functional in peripheral arterial smooth muscle. J
Cardiovasc Pharmacol., 2004, 43(6):770-781.

Ni W, Wilhelm CS, Bader M, Murphy DL, Lookingland K, Watts SW: (+)-
Norfenfluramine-induced arterial contraction is not dependent on endogenous 5-
hydroxytryptamine or 5-hydroxytryptamine transporter. J Pharmacol Exp Ther.,
2005, 314(3):953-60.

Ni W and Watt SW: 5-Hydroxytryptamine in the cardiovascular system: focus on
the serotonin transport (SERT). In press, Clin. Exp. Pharmacol. Physiol, 2006,
33(7):575-83.

Ni W, Lookingland, Watts SW: Arterial 5-HT transporter function is impaired in
DOCA and LNNA but not spontaneously hypertensive rats. Hypertension. 2006,
48(1):134-40.

Ni W, Lookingland K, Watts SW. Response to blood pressure in mutant rats
lacking the 5-hydroxytryptamine transporter. Hypertension. 2006

Abstract

Ni W, Zhan GL, Lu J, Teng HL, Bu XY, Lin ZB: Effect of ganoderma lucidum
extracts on the immune liver injury induced by BCG and the possible
mechanism. 3rd International Symposium on Hepatology, Hangzhou, China,
October 2001

Thakali K, Ni W, Li MW, Fink GD and Watts SW: The anorexigen (+)-
norfenfluramine as a pressor; enhanced response in hypertension. (Abstract)
57th Annual Fall Conference of the Conference of the Council for High Blood
Pressure Research, Washington DC, September 2003, Hypertension,
42(3),425.

194

Ni W, Li MW and Watts SW: Dual role of 5-HT2A receptor and 5-HT transporter
in (+)-norfenfluramine-induced aortic contraction. Experimental Biology Meeting,
Washington DC, 2004.

Ni W, Li MW, Thakali K, Fink GD and Watts SW: The fenfluramine metabolite
(+)-norfenfluramine is vasoactive. Fund. Clin Pharmacol 18(S1):p.139,A
3.7,2004

Watts SW, Ni W, Northcot CA, Lookingland K, Thompson J.: The serotonin
transporter in peripheral arteries. Fund. Clin Pharmacol 18(S1):p.139,A
3.7,2004 Serotonin club symposium-EPHAR Satellite in Porto 2004.

Ni W, Wilhelm C, Bader M, Lookingland K, Watts SW: (+)-Norfenfluramine-
induced arterial contraction is not dependent on 5-HT release: use of tryptophan
hydroxylase (TPH) 1 deficent mice. 58th Annual Fall Conference of the
Conference of the Council for High Blood Pressure Research, Chicago,lL,
October 2004, Hypertension, 44(4), 518.

Ni W and Watts SW: Peripheral arterial uptake and release of 5-HT.
Experimental Biology Meeting, San Diego, 2005.

Ni W, Lookingland K and Watts SW: 5-HT transporter (5-HTT) function is
impaired in DOCA-salt hypertensive rats but not spontaneously hypertensive
rats (SHR). 59th Annual Fall Conference of the Conference of the Council for
High Blood Pressure Research, Washington DC, September, 2005,
Hypertension, 46(4), 843.

Ni W, Lookingland K and Watts SW: Peripheral arteries take up but do not
concentrate 5-HT. Experimental Biology Meeting, San Francisco, 2006

NM, Szasz T, Lookingland K and Watts SW: The existence of a 5-HT synthetic
and metabolism system in rat peripheral arteries. 60th Annual Fall Conference
of the Conference of the Council for High Blood Pressure Research, San
Antonio, TX, October, 2006, Hypertension, 48(4), e81.

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