Michigan State
University

This is to certify that the
dissertation entitled

PHOSPHOINOSITIDE 3-KINASE UPREGULATION IN
HYPERTENSION: A REASON FOR ENHANCED ARTERIAL
CONTRACTION AND TONE

presented by

Carrie Annalice Northoott

has been accepted towards fulfillment
of the requirements for the

Ph.D. degree in Pharmacol and Toxiool

 

<l—wkw WWQ‘EéL

'Major Professor 5 Signature
gag/31003

Date

MSU is an Afiirmative Action/Equal Opportunity Institution

 

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PHOSPHOINOSITIDE 3-KINASE UPREGULATION m HYPERTENSION: A
REASON FOR ENHANCED ARTERIAL CONTRACTION AND TONE
By

Carrie Annalice Northcott

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

2003

PHOSPHC

REASC‘

 

 

 

 

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ABSTRACT

PHOSPHOINOSITIDE 3-KINASE UPREGULATION IN HYPERTENSION: A

REASON FOR ENHANCED ARTERIAL CONTRACTION AND TONE

By

Carrie Annalice Northcott

Hypertension is a disease characterized by enhanced arterial agonist-
induced contraction, reduced agonist-induced relaxation, spontaneous arterial
tone and smooth muscle cell remodeling. Phosphoinositide 3-kinase (Pl3-
kinase) is a multi-faceted enzyme involved in a variety of biological pathways and
an alteration in such a key enzyme may lead to the enhanced arterial contractility
and spontaneous tone observed in hypertension. Therefore I hypothesized that
spontaneous tone and agonist-induced contraction in hypertension is mediated
by an up-regulation of the PIS-kinase dependent signaling pathway. This, in turn,
ultimately links to an increase in intracellular calcium concentration through
opening L-type calcium channels, permitting arterial contraction. Elucidation of
the role of PIS-kinase in contraction in hypertension is vital to a better
understanding of the pathology of hypertension, as well as for utilization as a

potential therapeutic target.

Sponta

 

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contractile 9)
all calcium-'1'
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LY294402 ais
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assays were
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With significa'
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demonsvati:

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Spontaneous tone is driven by alterations in available calcium and p110
PIS-kinase subunits can directly activate L-type calcium channels. Isometric
contractile experiments revealed that LY294002, a Pl3-kinase inhibitor, inhibited
all calcium-induced spontaneous tone in aorta from hypertensive DOCA-salt,
LNNA, and SHR rats; aorta from respective normotensive rats displayed no tone.
LY294402 also eliminated spontaneous tone in mesenteric resistance arteries.
These studies suggested that there was an alteration in Pl3-kinase
protein/activity in arteries from hypertensive animals; therefore biochemical
assays were performed. p85a—associated Pl3-kinase activity in aorta from
DOCA-salt rats was 2-fold greater than sham. Western analyses revealed aorta
possessed p85a, p110a, p1 1013 and p1108 but not p1107 PIS-kinase subunits,
with significantly higher p1108 protein density in aorta from hypertensive DOCA-
salt, LNNA and SHR rats compared to their respective controls. Moreover,
immunohistochemical studies localized p1108 to aortic smooth muscle and
activity assays revealed elevated aortic p1108-associated activity in DOCA-salt
rats. LY294002 rightward shifted the enhanced contraction to norepinephrine in
aorta from DOCA-salt, LNNA and SHR rats compared to control animals,
demonstrating further physiological relevance of Pl3-kinase. LY294002 also
inhibited enhanced low-Mgz*-induced tone. Together, these data support an
increase in class I PIS-kinase protein/activity in the condition of hypertension.
lmportantly, this increase contributes to the enhanced contractility observed in

multiple models of hypertension.

DEDICATION

To

Gary. Peggy, Kim and Bill

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ACKNOWLEDGEMENTS

I would first like to thank my mentor Dr. Stephanie Watts. Thank you for
your precious time and seemingly endless supply of patience, assistance and
guidance. You have helped me grow as a scientist as well as a person. I will
always look up to you as an amazing role model! Thank you.

Furthermore, I would like to thank the members of my thesis committee,
Drs. Greg Fink, Jay Goodman, Alex Chen and Frank Brosius Ill. Thank you for
your interest in my project, valuable time and many suggestions. I hope you
enjoyed the yak milk.

I wish to thank the Watts’ laboratory for their help and friendship: Amy
Banes, Janice Thompson, Keshari Thakali, Zack Hickner, Chuck Smark, Hilary
Berlin and Wei Ni. Thanks for listening to me babble and for all of the good times
we have had, it has been fun! I look toward to the mischief we can get into in
the future.

I would also like to express my never-ending gratitude to all of my family,
especially my parents Gary and Peggy Taets. Thank you for teaching me to
never give up, among other things.

Finally, the best for last, thank you to my husband, Dr. Bill Northcott.
Thank you for enduring bruises, late night walks, patience, buying notecards at
all hours, reading manuscripts, for just being there when I needed you and for

when the day was done for loving me.

 

UST OF TA:
LIST OF FIG
UST OF A53]

I. INTROOU'
A Hyperte
B. Artena‘.
C. Pl3-kin

1. Sim
2. Upi:
3. Pro!
4. Ups
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7. PT

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TABLE OF CONTENTS

LIST OF TABLES ........................................................................................... ix
LIST OF FIGURES ......................................................................................... x

LIST OF ABBREVIATIONS ............................................................................ xv
I. INTRODUCTION ........................................................................................ 1
A. Hypertension ......................................................................................... 1
B. Arterial Dysfunction in Hypertension ...................................................... 5

C. Pl3-kinase ............................................................................................. 13

1. Structure of Pl3-kinase ...................................................................... 13

2. Lipid Kinase Activity ........................................................................... 19

3. Protein Kinase Activity ....................................................................... 20

4. Upstream and Downstream of Pl3-kinase ......................................... 23

5. Calcium and PI3-kinase ..................................................................... 24

6. Magnesium and PIS-kinase ............................................................... 27

7. PTEN (Phosphatase and Tensin Homolog ........................................ 29

D. Hypothesis ............................................................................................ 31

ll. METHODS .................................................................................................. 35

A. Animals ................................................................................................... 35

B. Models of Hypertension .......................................................................... 35

1. Mineralooorticoid Hypertension ......................................................... 35

2. Wistar—Kyoto (WKY) and Spontaneously Hypertensive Rats

(SHR) ................................................................................................ 36

3. Nw-nitro-L-arginine (LNNA) Hypertension .......................................... 36

4. Blood Pressure Measurements ......................................................... 39

C. Contractility ........................................................................................... 40

1. General Isolated Tissue Bath Protocol .............................................. 40

2. Spontaneous Tone and Inhibitors Protocol ........................................ 41

3. Agonist-stimulation of Pl3-kinase Protocol ........................................ 42

4. Calcium Protocol ............................................................................... 42

5. Magnesium Protocol .......................................................................... 43

6. Myograph Protocol ............................................................................ 44

D. Aortic Vascular Smooth Muscle Cell Culture ......................................... 44

E. Biochemical Assays ............................................................................... 45

1. Protein Isolation ................................................................................. 45

a. Whole Tissue Isolation ................................................................. 45

b. Calcium Channel Protein Isolation ............................................... 46

vi

 

 

5. lm.
F. Data A

I

III. RESULTE
A. Subhy;
upregc.

1. 8pc

2. PI3-
B.Subhyp
Channe
mOOeII
C- Subhy
I0caliz.
aorta Q
D'SUbh”
Utilize
Vascul
1. NE
2- M;
E SUbhy
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JO.
0) 'crrvma,
5'”-‘”Z'.:-§..

c. lmmunoprecipitation and Pl3-kinase Activity Assay Protein

Isolation ........................................................................................ 47

d. Vascular Smooth Muscle Cell Protein Isolation ............................ 47

e. BCA Protein .................................................................................. 48

2. Western Analyses ............................................................................. 49

a. Standard Western Blotting Protocol .............................................. 49

b. Calcium Channel Western Blotting Protocol ................................. 52

3. lmmunoprecipitation .......................................................................... 53

4. PI3-kinase Activity Assay ................................................................. 54

5. lmmunohistochemistry ...................................................................... 55

F. Data Analysis and Statistics .................................................................. 56

III. RESULTS ................................................................................................. 58
A. Subhypothesis #1: Pl3-kinase protein and/or its activity are

upregulated in aorta of DOCA-salt hypertensive rats. ........................... 58

1. Spontaneous tone and Pl3-kinase .................................................... 58

2. PI3-kinase Biochemistry ................................................................... 75

B. Subhypothesis #2: Pl3-kinase and L-type voltage gated calcium

channel interaction is altered in the aorta of the DOCA-salt rat

model of hypertension. .......................................................................... 94
C. Subhypothesis #3: Phosphatase and tensin homolog (PTEN) is

localized in vascular smooth muscle and is down-regulated in

aorta of the DOCA-salt rat. .................................................................... 106
D. Subhypothesis #4: Norepinephrine (NE) and magnesium (Mg"’*)

utilize the Pl3-kinase signaling cascade to elicit enhanced

vascular contraction in hypertension. .................................................... 106
1. NE and Pl3-kinase ............................................................................ 106
2. Mg” and Pl3-kinase ......................................................................... 111

E. Subhypothesis #5: PI3-kinase and its dependent signaling
pathways are up-regulated in multiple models of hypertension,
leading to enhanced vascular contraction and spontaneous tone

development. ........................................................................................ 128
1. Spontaneously Hypertensive Rat (SHR) model of hypertension ....... 128
a. Spontaneous tone and NE-induced contraction ............................ 129
b. Pl3-kinase biochemistry ................................................................ 136
2. Nm-nitro-L-arginine (LNNA) model of hypertension ........................... 143
a. Spontaneous tone, NE-induced contraction and Pl3-kinase ......... 143
b. PI3-kinase Biochemistry ................................................................ 150

F. Subhypothesis #6: Pl3-kinase functional alterations and
changes in protein are observed in the mesenteric resistance
arteries in the DOCA-salt model of hypertension ................................... 160
G. Time Course Studies ............................................................................. 168

vii

 

IV. DISCUSS
A Ration.
B, Idé-"ltf‘fil
C. Pl3-kf
D. PIS-kl".
E. Identifi

induce:
1. NE-H
2. Low-
F. Time C

V. SPECUU
VI. REFERE

VII. CURRIC

 

 

IV. DISCUSSION ............................................................................................ 191

A. Rationale ............................................................................................... 192

B. Identification of Pl3-kinase involvement in spontaneous tone ............... 193

C. PI3-kinase Biochemistry ........................................................................ 197

D. PI3-kinase and Ca2+ .............................................................................. 202
E. Identification of Pl3-kinase involvement in NE and Low Mg“-

induced contraction ............................................................................... 204

1. NE-induced Contraction .................................................................... 204

2. Low-MgZ*-induced contraction ........................................................... 206

F. Time Course Studies ............................................................................. 210

V. SPECULATION AND CONCLUSIONS ..................................................... 212

VI. REFERENCES ......................................................................................... 219

VII. CURRICULUM VITAE ............................................................................. 232

viii

Table I:

 

LIST OF TABLES

Table 1: Catalytic subunits, regulatory subunits, activators of PI3-
kinase, in vitro and in viva substrates and inhibitors of the
classes of PI3-kinase enzyme. ..................................................... 14

ix

Figure 1:

Eye 2:

Figure 3:
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Figure 5:

 

 

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:

LIST OF FIGURES

Representative tracing of spontaneous arterial tone in sham

and DOCA-salt rats ..................................................................... 9
Diagram of hypothesized downstream elements of PI3-

kinase in smooth muscle and potential outcomes ........................ 25
Diagram of working hypotheses ................................................... 33
Systolic blood pressures for WKY and SHR rats .......................... 37

Representative standard curve of Bovine Serum Albumin for
BCA protein analysis .................................................................... 50

Systolic blood pressure for 28 day Sham and DOCA-salt
rats ............................................................................................... 59

Representative tracing of spontaneous arterial tone in sham
and DOCA-salt rats ...................................................................... 61

Effect of LY294002 on spontaneous arterial tone in sham
and DOCA-salt rats ...................................................................... 63

Effect of increasing concentrations of LY294002 or Vehicle
on spontaneous tone in aorta from DOCA-salt and sham
rats ............................................................................................... 66

Effect of LY294002 and LY303511 on Akt and pAkt protein
in EGF-stimulated rat aortic smooth muscle cells ......................... 68

Effect of LY303511 on spontaneous arterial tone in aorta
from DOCA-salt and sham rats .................................................... 71

Effect of increasing concentrations of Wortmannin or
Vehicle on spontaneous tone in aorta from DOCA-salt and
sham rats ...................................................................................... 73

p85a-associated PI3-kinase activity, immunoprecipitation of
p850: and western analysis of p850: protein in aorta from
DOCA-salt and sham rats ............................................................. 76

figure 153
Figure 151
Figure I73
Figure 18:

Figure 19:

Figure 20:
Figure 21:
Figure 22:
Figure 23:
F'g‘dte 24:

Figure 25.

F’We 26:

 

Figure 14: Aortic protein from DOCA-salt and sham rats
immunoprecipitated with p85a and immunoblotted for p850,
p110a, p11013, p1107 and p1106 proteins .................................... 78

Figure 15: Western analyses of PDK and pPDK in aorta from DOCA-
salt and sham rats ........................................................................ 80

Figure 16: Western analyses of Akt and pAkt protein in aorta from
DOCA-salt and sham rats ............................................................. 82

Figure 17: Western analyses of p1108, p110a, p11OB and p1107 in
aorta from DOCA-salt and sham rats ........................................... 85

Figure 18: p1108-associated Pl3-kinase activity in aorta from DOCA-
salt and sham rats ........................................................................ 87

Figure 19: Aortic protein from DOCA-salt and sham rats
lmmunoprecipitated with p1106 antibody and
immunoblotted with antibodies against p1108, p110a,
p1108 and p1107. ......................................................................... 90

Figure 20: lmmunohistological studies of thoracic aorta from DOCA-
salt and sham rats probed with p1108 antibody ........................... 92

Figure 21: Representative tracing of spontaneous arterial tone in aorta
from DOCA-salt and sham rats exposed to Nifedipine ................. 95

Figure 22: Western analysis of pAkt in cells activated by EGF and
exposed to Diltiazem and LY294002 ............................................ 97

Figure 23: Effect of LY294002 on BayK8644-induced contraction in
aorta from DOCA-salt and sham rats ........................................... 99

Figur924: Effect of LY294002 on calcium chloride-induced
spontaneous tone in aorta from DOCA-salt and sham rats .......... 102

Figure 25: Western analyses of the a1c L-type calcium channel in
aortic protein from DOCA-salt and sham rats ............................... 104

Figure 26: Western analyses of PTEN and pPTEN in aorta from
DOCA-salt and sham rats ............................................................. 107

xi

Figure 27:

figure 28:

Figure 29:

Figure 30:

F@ure 31:
Figure 32'.
Fl9W9 33:
Figure 34;

Ewe 35:

Figure 3B:

t
I I'M
we :37;

F‘Q‘ure 38;

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Figure 27:

Figure 28:

Figure 29:

Figure 30:

Figure 31:

Figure 32:

Figure 33:

Figure 34:

Figure 35:

Figure 36:

Figure 37:

Figure 38:

Figure 39:

Effect of LY294002 on NE-induced contraction in
endothelium-denuded and endothelium-intact aorta from
DOCA-salt and sham rats ............................................................. 109

Effect of low Mg2+ PSS on arterial spontaneous tone in aorta
from DOCA-salt and sham rats .................................................... 112

Effect of LY294002 on spontaneous tone development in
aorta from DOCA-salt and sham rats incubated in normal
PSS and low Mg2+ PSS ................................................................ 114

Effect of high Mg2+ PSS on arterial spontaneous tone in
aorta from DOCA-salt and sham rats ........................................... 116

Effect of LY294002 on spontaneous tone development in
aorta from DOCA-salt and sham rats incubated in high Mg""’
PSS .............................................................................................. 119

p85a-associated PI3-kinase activity in aorta from DOCA-
salt and sham rats incubated in normal PSS or low Mg2+
P88 in isolated tissue baths ......................................................... 121

Effect of LY294002 on NE-induced contraction in aorta from
DOCA-salt and sham rats incubated in normal PSS or low
Mg2+ PSS ...................................................................................... 124
Effect of LY294002 on NE-induced contraction in aorta from
DOCA-salt and sham rats incubated in normal PSS or high
Mg2+ PSS ...................................................................................... 126
Spontaneous tone in SHR and WKY rats ..................................... 130

Effect of LY294002 on arterial spontaneous tone in SHR
and WKY rats ............................................................................... 132

Effect of LY294002 on NE-induced contraction in aorta from
SHR and WKY rats ....................................................................... 134

Western analyses of p1108, p110a, p11OB and p110] in
aorta from SHR and WKY rats ..................................................... 137

Western analyses of Akt and pAkt in aorta from SHR and
WKY rats ...................................................................................... 139

xii

figure 40:

Figure 42: ‘

figure 433
Figure 44:
Figure 453
Figure 453

Figure 471

 

 

 

Figure 40:

Figure 41:
Figure 42:

Figure 43:

Figure 44:

Figure 45:

Figure 46:

Figure 47:

Figure 48:

Figure 49:

Figure 50:

Figure 51:

Figure 52:

Figure 53:

Figure 54:

Western analyses of PTEN and pPTEN in aorta from SHR

and WKY rats ............................................................................... 141
Systolic blood pressures for LNNA and sham rats ....................... 144
Basal spontaneous tone in aorta from LNNA and sham rats ........ 146

Effect of LY294002 on arterial spontaneous tone in LNNA
and sham rats ............................................................................... 148

Effect of LY294002 on NE-induced contraction in aorta from
LNNA and sham rats .................................................................... 151

Western analyses of p1108, p110a, p110[3 and p110/ in
aorta from LNNA and sham rats ................................................... 153

Western analyses of Akt and pAkt in aorta from LNNA and
sham rats ...................................................................................... 156

Western analyses of PTEN and pPTEN in aorta from LNNA
and sham rats ............................................................................... 158

Effect of LY294002 on spontaneous tone in mesenteric
resistance arteries from DOCA-salt and sham rats ...................... 161

Western analyses of p850: and p1108 in mesenteric
resistance arteries from DOCA-salt and sham rats ...................... 164

Western analyses of Akt and pAkt in mesenteric resistance
arteries from DOCA-salt and sham rats ........................................ 166

Change in systolic blood pressure from Day 0 after 1, 3, 5,
and 7 days of DOCA-salt and sham treatment ............................. 169

Arterial spontaneous tone development in aorta from rats
after 1, 3, 5 and 7 days of DOCA-salt and sham treatment .......... 171

Effect of LY294002 on spontaneous tone in aorta from rats
after 1, 3, 5 and 7 days of DOCA-salt and sham treatment .......... 173

Effect of LY294002 on NE-induced contraction in aorta from
rats after 1 day of DOCA-salt and sham treatment ....................... 175

xiii

Figure 55: '

Figure 56:

Figure 57:

Figure 58'.

Figure 59;

Figure 60:

F’QUTE 61:

 

Figure 55:

Figure 56:

Figure 57:

Figure 58:

Figure 59:

Figure 60:

Figure 61:

Effect of LY294002 on NE-induced contraction in aorta from
rats after 3 days of DOCA-salt and sham treatment ..................... 177

Effect of LY294002 on NE-induced contraction in aorta from
rats after 5 days of DOCA-salt and sham treatment ..................... 180

Effect of LY294002 on NE-induced contraction in aorta from
rats after 7 days of DOCA-salt and sham treatment ..................... 182

Western analyses of protein isolated from aorta after 1, 3, 5,
7 and 28 days of DOCA-salt and sham rats with an antibody
against p850: ................................................................................ 184

Western analyses of protein isolated from aorta after 1, 3, 5,
7 and 28 days of DOCA-salt and sham rats with an antibody
against p1 108 ............................................................................... 186

Western analyses of protein isolated from aorta after 1, 3, 5,
7 and 28 days of DOCA-salt and sham rats with antibodies
against Akt and pAkt .................................................................... 188

Schematic diagram representing the dual role of PIS-kinase
in hypertension ............................................................................. 216

xiv

ME
um
Ah
kg”
N?

BCA

8P

mew
CO
DAG
OM80
DNA

DOCA

 

ACE
Ach
Akt
Ang II
ATP
BCA
Bcr
BP
Caz“
cdc42
CO
DAG
DMSO
DNA
DOCA
DTT
ECL
ECM
EGF

EGFr

LIST OF ABBREVIATIONS:

angiotensin-converting enzyme
acetylcholine

thymoma viral proto-oncogene 1
angiotensin II

adenosine 5'-triphosphate
bicinchoninic acid protein assay kit
breakpoint cluster region

blood pressure

calcium

cell division cycle protein 42
cardiac output

1 ,2-diacylglycerol

dimethyl sulfoxide
deoxyribonucleic acid
deoxycorticosterone acetate
dithiothreitol

enhanced chemiluminenscence
extracellular matrix

epidermal growth factor

epidermal growth factor receptor

XV

Em

6352

GAP

GAPOH

6.132

 

HCI
HEPES
HR

IGF-1

IRS

Erk
Gab2
GAP
GAPDH
Grb2
HCI
HEPES
HR
IGF-1
IP3

IRS
JAK

Ick
L-MMA
LNNA
Iyn
MABP/MAP
MAPK
MEKK
Mg2+
MLC

MLCK

extracellular signal regulated kinase
GRBZ-associated binding protein 2
GTPase activating protein

glyceraldehyde phosphate dehydrogenase
growth factor receptor-bound protein 2
hydrochloric acid

4-(2—hydroxyethyl) piperazine-1ethanesulfonic acid
heart rate

insulin-like growth factor-1

inositol (1,4,5) tris phosphate

insulin receptor substrate

janus kinase 1

lymphocyte-specific kinase

N(G) monomethyl-L-arginine
Nw-nitro-L-arginine

lck/yes-related novel protein tyrosine kinase
mean arterial blood pressure/mean arterial pressure
mitogen activated protein kinase

MAPK/Erk Kinase kinase

magnesium

myosin light chain

myosin light chain kinase

xvi

IIIIACI

NE

N0

 

 

 

III-1C
pAkt
PBS
PCR
PDGF
PDK

PE

PIB’kIR85(
PIP2

PKB

PKC
PLC
pMSF
P33

Pidlns

PTEN

Iac

MMAC1
NE

N0
1K-1 C
pAkt
PBS
PCR
PDGF
PDK
PE

Pl
Pl3-kinase
P|P2

PKB

PKC
PLC
PMSF
PSS
Ptdlns
PTEN

rac

mutated in multiple advance cancers 1
norepinephrine

nitric oxide

one kidney-one clip

phosphorylated Akt

phosphate buffered saline
polymerase chain reaction

platelet derived growth factor
3-phosphoinositide-dependent kinase
phenylephrine

phosphoinositide

phosphoinositide 3-kinase
phosphatidylinositol 4,5-bisphosphate
protein kinase B or

Related to A and C protein Kinase (RAC-PK)
protein kinase C

phospholipase C
phenylmethylsulfonyl fluoride
physiological salt solution
phosphatidylinositol phosphate
phosphatase and tensin homolog

small ras related G protein

xvii

ras
mo
ROS
38?
SDS
8H2
she
SVP
SHR

SHRSP

sm
SRE
STAT
SV
8*
TBS

TGFQ

TLC

 

 

 

ras
rho
ROS
SBP
SDS
SH2
shc
SHIP
SHR
SHRSP
sos
src
SRE
STAT
SV
syk
TBS
TBS-T
TGFa
TK
TLC

TPR

rat sarcoma oncogene homolog

ras homolog gene family

reactive oxygen species

systolic blood pressure

sodium dodecyl sulfate

src homology-2

src homology-2 domain and collagen-like
SH2-containing inositol phosphatase
spontaneously hypertensive rat

stroke prone spontaneously hypertensive rat
son of sevenless

Rous sarcoma oncogene

steroid response elements

signal transducer and activator of transcription
stroke volume

spleen tyrosine kinase

tris buffered saline

tris buffered saline-tween

transforming growth factor-alpha

tyrosine kinase

thin layer chromotography

total peripheral resistance

xviii

TPTE
TRIP

WKY

 

TPTE transmembrane phosphatase with tensin homolog
TRIP TPTE and PTEN homologous inositol lipid phosphatase
WKY wistar-kyoto

Zap 70 zeta-chain (T CR) associated protein kinase

xix

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A9,:
p'essure.
they have 1
28.2 9-5 are
form of me:
primary Cau
0f Contribut.
million Un ,1
Blood press
arteries as
determined
exerted Whey

the Pressure

 

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commonly re
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INTRODUCTION

A. Hypertension

Approximately 50 million Americans (age 6 and older) have high blood
pressure. Of those people with high blood pressure 31.6 °/o are unaware that
they have the condition, 27.4 °/o are on medication and are considered controlled,
26.2 % are on medication but don’t have it controlled and 14.8 % aren’t on any
form of medication. High blood pressure was listed on death certificates as the
primary cause of death of 44,619 Americans in 2000 and was listed as a primary
or contributing cause of death in approximately 118,000 of the more than 2.4
million United States deaths that year (American Heart Association, 2003).
Blood pressure is defined as a measurement of force applied to walls of the
arteries as the heart pumps blood through the body. Blood pressure is
determined by two readings: systolic blood pressure is the maximum pressure
exerted when the heart contracts, whereas diastolic blood pressure represents
the pressure in the arteries when the heart is resting between beats. High blood
pressure or hypertension is characterized by a patient having an average systolic
blood pressure > 140 mm Hg and/or an average diastolic blood pressure > 90
mm Hg. Sadly, high blood pressure has very few outward symptoms and thus is
commonly referred to as a silent killer. If left untreated, hypertension can
contribute to a variety of conditions including heart disease, stroke, myocardial

infarction and end-stage renal disease.

 

Bic
and total ;
product of

I
consists 0‘
(CO): TPF—
arteries a"

fourth pews

markedly ir

 

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layer Ultima'

 

 

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Changes IIo'
hemodi’nam.
One's bi00d I
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hiberr9nsi0r
secondary hy
one has no

apprOXImaleI)

Blood pressure (BP) is a product of two components, cardiac output (CO)
and total peripheral resistance (TPR): BP=CO x TPR. CO is determined by the
product of the stroke volume (SV) and the heart rate (HR): CO=SV x HR. TPR
consists of the mean arterial pressure (MAP) divided by the total cardiac output
(CO): TPR=MAPICO. TPR is determined by the caliber of small muscular
arteries and according to Poiseuille’s law, resistance varies inversely with the
fourth power of the blood vessel radius, so even a small decrease in the lumen
markedly increases resistance (lntengan and Schiffrin, 2000). Smooth muscle
cells contribute to the vessel caliber wig neuronal input and the endothelial cell
layer ultimately leading to the release vasodilator or vasoconstrictor compounds
in response to stimuli changing the diameter and flow of blood through the artery,
altering agonist-stimulated contraction, relaxation and development of
spontaneous arterial tone. In hypertension, there can be smooth muscle cellular
growth and/or remodeling that leads to a narrowing of the vessel, which in turn,
changes flow and resistance in the vessel and hence causes alterations in
hemodynamics. It is a combination of all the above components that determine
one’s blood pressure.

There are two categories in which one can be diagnosed with
hypertension, primary (otherwise known as essential or idiopathic) and
secondary hypertension. The clinical diagnosis of primary hypertension is when
one has no definable reason for the increase in blood pressure and

approximately 90 % of all diagnoses are of this type. The causes of essential

hypertens
processes
vesseis. rl
contnbute
and'orem
Cor
definable r
increase r'
name 3 f5
aldosteroni:
(Gordan ex
aldosrerOne
MinerajoCOr
type I adrer‘
mineralocorj
and are 99m
receptor, the
another Sterg
binds to SISr:
mineraloCOrT'
which reguh

 

hypertension are unknown, but it is suggested to be due to complex alterations in
processes in major biological and organ systems, including the heart, blood
vessels, nerves, hormones and kidneys. The numerous abnormalities that
contribute to elevation in blood pressure are thought to be genetic in their origin
and/or environmentally influenced (Messerli and Laragh, 2000).

Conversely, the diagnosis of secondary hypertension is when there is a
definable reason to the observed increase in blood pressure. Reasons for this
increase may be due to alteration in hormone secretion or renal function, to
name a few. One specific example of secondary hypertension is primary
aldosteronism. Primary aldosteronism was first clinically described in the 1950's
(Gordan at a/., 1994). It is a condition characterized by elevated circulating
aldosterone levels, which then leads to subsequent increases in blood pressure.
Mineralooorticoids (aldosterone and deoxycorticosterone) act through the renal
type I adrenocorticoid receptors and lead to antinatriuresis and kaliuresis. The
mineralocorticoid receptors at which the mineralocorticoids act are intracellular
and are generally located in the cytosol. Once the mineralocorticoid binds to the
receptor, the receptor dissociates from heat shock proteins and dimerizes with
another steroid receptor. The dimer then translocates to the nucleus where it
binds to steroid response elements (SRE) on deoxyribonucleic acid (DNA). The
mineralocorticoid activation of the renal receptors leads to cellular changes,
which result in salt and water retention by the kidney. Ultimately there is

suppression of the renin-angiotensin system due to the inhibitory effects of high

sodium 0"
to moduia‘
synthase '

aldosteror

 

 

 

 

 

the syntbe
control of .
There is a
pressure. ;
cleaves ang
on by angjc

 

IOCfeaSe BIC

sodium on renin secretion. Mineralooorticoids are known to act in other tissues
to modulate gene expression as well as exert non-genomic effects. Aldosterone
synthase or 18 methyl oxidase is the enzyme that specifies the synthesis of
aldosterone. Expression of angiotensin and elevated potassium levels stimulate
the synthesis of aldosterone in the glomerulosa of the adrenal cortex. The
control of aldosterone synthesis is largely regulated by angiotensin II (Ang II).
There is a negative feeback loop involving renal renin, responsive to arterial
pressure, B-adrenergic stimulus and Na” flux past the macula densa. Renin
cleaves angiotensin I from hepatic-derived angiotensinogen, which is then acted
on by angiotensin-converting enzyme (ACE) in the systemic vascular beds and
lung to produce Ang II. Ang II acts on the glomerulosa of the adrenal cortex to
increase aldosterone synthesis. Aldosterone promotes retention of sodium and
maintenance of blood volume and pressure. It is also known that aldosterone
secretion is controlled by changes in potassium ion concentration (Funder,
1994)

Excess secretion of mineralocorticoids can be mimicked in the
deoxycorticosterone acetate (DOCA)-salt rat, sheep, pig, and mouse and these
are thus used as experimental models of hypertension. DOCA-induced
hypertension was described 15 years prior to the isolation of aldosterone in rats
(Kenyon and Morton, 1994). The blood pressure of rats typically increases within
one week upon administration of DOCA-salt therapy and plateaus in 3-4 weeks

of therapy. Elevation of blood pressure is typically faster when the animals are

uninephfé
water (K8
Hy:

l
suppress ,
|
Hyperaidcl
tumor or t
|
inhibits the
actions of .
complex, re
drug treatrr
name a Iev.
one mainta
of hyperten'
Ongoing dev
more thorou
be Controllec

 

uninephrectomized and given high salt (1% sodium chloride) in their drinking
water (Kenyon and Morton, 1994).

Hypertension caused by hyperaldosteronism is associated with a
suppression of renin activity due to the retention of the sodium.
Hyperaldosteronism is treated by removal of a potential aldosterone-secreting
tumor or by treating with spironolactone, a potassium sparing diuretic, which
inhibits the binding of aldosterone to the mineralocorticoid receptor to inhibit the
actions of aldosterone. The treatment of primary hypertension usually is more
complex, requiring changes in lifestyle (diet, exercise, etc.) as well as therapeutic
drug treatment, including calcium channel blockers, B-blockers and diuretics, to
name a few. In the treatment of primary hypertension, there is evidence that if
one maintains a healthy diet and exercise, one may be able to prevent the onset
of hypertension and assist in lowering already elevated blood pressure. With
ongoing development of new drugs and treatments for high blood pressure and a
more thorough understanding to the causes, hypertension is a disease that can
be controlled thus preventing further detrimental health consequences. Hence,
the more we know about hypertension, the more we, as a population, will be able

to prevent it and treat those that are afflicted.

B. Arterlal dysfunctlon In hypertension
There are few “outward” indicators of hypertension. However, within the

vasculature there are marked changes that occur leading to the changes in TPR

that are observed. Hypertension is a disease that is characterized by enhanced
arterial agonist-stimulated contraction, reduced agonist-stimulated relaxation,
smooth muscle cell hypertrophy and/or eutrophy, and spontaneous arterial tone.
Multiple experimental models of hypertension, including spontaneously
hypertensive rats (SHR), DOCA-salt rats, 2-kidney 1 clip Goldblatt rats, 1-kidney
1 clip Goldblatt rats, renal wrap rats, Dahl-sensitive rats and Nm-nitro-L-arginine
(LNNA) rats to name a few, have been developed to study the development and
maintenance of the disease. Agonist-induced contraction, such as with the
agonists serotonin and norepineprhine (NE), is dramatically enhanced in multiple
forms of experimental hypertension (Collis and Vanhoutte, 1977; Watts at a/.,
1996; Kanagy, 1997). Acetylcholine-induced endothelium-dependent arterial
relaxation, mediated by nitric oxide (NO), is reduced in human essential
hypertension (Panza at a/., 1990) and in multiple forms of experimental
hypertension (Lockette at a/., 1986). These data suggest that in the condition of
hypertension that NO production may be diminished. This loss of NO may lead
to altered contractility and vascular smooth muscle cell growth.

Arteries consist of three layers: 1.) intima, which is the innermost layer
and consists of the endothelium in healthy tissue; 2.) media, which consists of a
tight spiral of smooth muscle cells embedded in proteoglycans and matrix
proteins, principally collagen, fibronectin and elastic fibers; and 3.) adventitia, a
layer of loose connective tissue which contains fibroblasts, lymphatics and

nerves which supply the outer ring of medial smooth muscle (Lindop, 1994;

Intertgar
processe
eutrophir
narrowing
the dei.
hypertens
clip Gold:
and Schiff:
of the me
increasing
lead to pg
rellovascm:
IDOCAj-sa
Sensitive fat
mOdels of
resWfiSlVerw

 

BDOInt of cm

I
2332)

 

 

Intengan and Schiffrin, 2000, Mulvany, 2002). In hypertension there are 2
processes that are involved in changing the structure of resistance arteries,
eutrophic and hypertrophic remodeling. Eutrophic remodeling involves a
narrowing of the outer diameter and lumen, however the cross-sectional area of
the media is not altered. Eutrophic remodeling is found in mild essential
hypertensive patients and the spontaneously hypertensive (SHR) and 2-kidney 1
clip Goldblatt hypertensive rat models of experimental hypertension (lntengan
and Schiffrin, 2000). Hypertrophic remodeling, in contrast, involves a thickening
of the media Wis cellular growth, thus also narrowing the lumen and again
increasing TPR. In the large arteries hypertrophy of smooth muscle cells may
lead to polyploidy. Hypertrophy is found in arteries from humans with
renovascular hypertension and pheochromocytoma, in deoxycorticosterone
(DOCA)-salt rats, 1-kidney 1 clip Goldblatt hypertensive rats and Dahl salt-
sensitive rat experimental models (Intengan and Schiffrin, 2000). In experimental
models of hypertension the hypertrophy and hyperplasia causes increased
responsiveness to pressor stimuli and can narrow the lumen, thus increasing the
TPR, which contributes further to an increase in blood pressure (Lindop, 1994).
Enhanced agonist-induced contraction is not as common a phenomenon in
human essential hypertension as it is in experimental models, however this is still
a point of contention among researchers (lntengan and Schiffrin, 2000; Mulvany,

2002)

 

further c
, |
tone vas:
describing
SM£(NI}
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may or rr:

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the condc

 

hypertensic
I0 aheratior
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genetically
WOmen Wit

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counterparts

SOOOIhehum

A phenomenon referred to as arterial spontaneous tone is thought to
further contribute to the maintenance of hypertension. Spontaneous
tone/vasomotion was first described in 1852 in intact animals by Jones
describing rhythmic changes in the bat wing and has been extensively studied
since (Nilsson and Aalkjaer, 2003). Spontaneous tone is defined as when the
artery contracts and relaxes on its own with no exogenous stimuli (Figure 1), that
may or may not have oscillatory actions in addition to the steady increase in
contraction. Spontaneous tone, or vasomotion, has been extensively studied in
the condition of hypertension. Tone development in the condition of
hypertension leads to “spontaneous” narrowing of the arteries, which then leads
to alterations in the TPR, which can further increase/propagate the condition of
hypertension. Thus, making it vital to understand the mechanism by which this
spontaneous tone develops. Enhanced tone has been observed in femoral
arteries from renal hypertensive rats, DOCA-salt hypertensive rats, rats
genetically predisposed to hypertension, essential hypertensive patients and
women with preeclampsia (Nilsson and Aalkjaer, 2003). The presence and
magnitude of spontaneous tone is variable in arterial tissues from hypertensive
animals.

Spontaneous tone in arteries from spontaneously hypertensive rats
(SHR), is significantly greater than their normotensive Wistar Kyoto (WKY) rat
counterparts (Sunano at a/., 1996). Tone in SHR aorta is also greater when the

endothelium layer is removed from the aortic preparation compared to when it is

Figure 1. Representative tracing of spontaneous arterial tone in endothelium-
denuded aorta from DOCA-salt and sham rats. Top tracing is aorta from a
normotensive sham rat and the bottom tracing is from a hypertensive DOCA-salt

rat. Tissues are under passsive tension for optimal force production.

 

 

Tension (mg)

Sham

200 mg

 

2 min

 

 

DOCA

10

ITone

er 3%.
sports
counts
NO soc
establis
Similarly
brachial
normoter
With 9881
decrease:
However,
amounts 0
”0 differem
from 3,,ng
Endotheliun

also IOUn d a

 

to no”Tloter

deI/elopmer

 

 

left intact. The calcium channel antagonist verapamil abolished tone, indicating
that calcium was a key component to development of spontaneous tone (Sunano
at a/., 1996). N(G) monomethyI-L-arginine (L-MMA) caused increases in active
spontaneous tone in aorta from endothelium-intact SHR rats which could be
counteracted by L-arginine, thus the endothelium attenuates tone by releasing
NO spontaneously (Sunano at a/., 1996). Basal formation of NO is reduced in
established hypertension of the SHR and renovascular hypertensive rats.
Similarly, in patients with essential hypertension, infusion of L-NMMA into the
brachial artery caused less vasoconstriction in hypertensives as compared to
norrnotensives, suggesting that basal formation of NO is also reduced in patients
with essential hypertension (Luscher, 1994). Moreover, if NO release is
decreased, this enables the increase in tone in arteries of hypertensive rats.
However, bioassays experiments using perfused SHR aorta reveal comparable
amounts of biologically active NO (Luscher, 1994) and Wang at al. (1999) found
no difference in spontaneous tone in endothelium-intact and denuded aortic rings
from angiotensin ll-infused hypertensive rats, suggesting that NO and the
endothelium may not be solely responsible for the maintenance of tone. They
also found an increase in superoxide anion in the hypertensive rats as compared
to normotensive rats which, by inactivating the endothelium-derived NO leading
to increase in calcium influx into the cells promoting spontaneous tone

development.

11

 

N
anenes
sensitlvli
1992: La
aortic str
from the I
(382’, sug
permeabi
characteri
and diiam
function c
hypertensi
inhibitor,
hypertensir
contractim
$anlects.

”Orepjnep.

 

 

Comparabjt—
Kah°nem 1

 

 

Numerous investigators have shown that spontaneous tone occurs in
arteries of hypertensive rats and this is due, at least in part, to altered calcium
sensitivity and/or handling in these tissues (Thompson et a/., 1987; Webb at a/.,
1992; Lamb etal, 1995; Pucci etal, 1995; Rapacon-Baker etal, 2001). Helical
aortic strips from SHR exhibited Ca2"-dependent myogenic tone, whereas aorta
from the WKY normotensive rats maintained stable resting tension to all levels of
Ca“, suggesting the observed intrinsic myogenic tone is due to increased
permeability to Ca2+ (Fitzpatrick and Szentivanyi, 1980). Myogenic tone is
characterized by constriction of a vessel after an increase in transmural pressure
and dilation of a vessel after a decrease in transmural pressure. Abnormal
function of voltage-dependent Ca2+ channels in arterial smooth muscle of
hypertensive patients was observed when a low concentration of the L-type Ca2+
inhibitor, nifedipine was added to arterial rings from normotensive and
hypertensive humans. Nifedipine caused a greater inhibition of calcium-induced
contraction in arteries from hypertensive subjects than the normotensive
subjects. However, the vasoconstrictor sensitivity and maximal responses to
norepinephrine (NE), serotonin (5-HT) and potassium chloride (KCL) were
comparable in arteries from normotensive and hypertensive humans (Hutri-
Kahonen, 1999). One mechanism I propose that alters spontaneous tone in the

condition of hypertension is an upregulation of the PI3-kinase signaling cascade.

12

C. Pl3-ki

Pl
d:scovere
antiphopl
(PDGFIS
ubiquitous
FIB-kinasi
stimuli. S.
in are pre
retrovirus-
Chickens ‘
Caenoma‘
lilesnan of

mnCt‘on (V.

 

I.§:

pi3~Ir
I

gene laminl

 

C. PI3-klnase

Pl3-kinase (also known as phosphatidylinositol 3-OH kinase) was first
discovered in the 19803 associated with middle T-antigen and pp60"'s'°, and with
antiphophotyrosine immunoprecipitates from platelet derived growth factor
(PDGF)-stimulated fibroblasts (Anderson and Jackson, 2003). Pl3-kinase is a
ubiquitous family of enzymes found in a wide variety of species and cell types.
PIS-kinase has been implicated in a variety of cellular responses to various
stimuli. Several of the important responses that PIS-kinase has been implicated
in are prevention of apoptosis in several cell types (Franke at a/., 1997), a
retrovirus-encoded Pl3-kinase was found to cause haemangiosarcomas in
chickens and to transform fibroblasts (Chang et a/., 1997), mutations in
Caenor/raba’itis e/egans Pl3-kinase gene caused a three-fold increase in the
lifespan of the adult (Morris at a/., 1996) and modulation of cardiac viability and

function (Vlahos at a/., 2003).

1. Structure of PIS-kinase

Pl3-kinase is a multifaceted enzyme, possessing both lipid and protein
kinase activity. Cloning of the catalytic subunits has led to organizing the multi-
gene family into three main classes based on their substrate specificity,

sequence homology and regulation (Table l). Class I and Class III PI3-kinases

13

Table I. Pl3-kinase subunits, a few of the proteins that are known to regulate
PIS-kinase, the in vitro and in vivo phosphatidylinositol phosphate (Ptdlns)
substrates of the respective Pl3-kinase subunits and the concentrations of the
inhibitors that effect the respective PIS-kinase subunits (Fruman at a/., 1998;

Vanhaesebroeck at at, 2001; Anderson and Jackson, 2003).

14

 

 

 

 

 

 

 

 

 

 

 

 

 

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15

are me
reg Jlatc
enzyme
00F sists
kno ivn e
(Varhaes
stud ed a
a 113 kifi'
least for.
regu aloe,
catal Itic
homc ogc
kinas; dc
Ras-t I'ldjr
legula fOry
lb ”3 Irina
the N'Iern

reSula: )ry ,

2003).

 

are made up of two subunits. One of the subunits plays primarily a
regulatory/adaptor role and the other maintains the catalytic function of the
enzyme. All the PIS-kinase catalytic subunits share a homologous region that
consists of a catalytic core domain (HR1; homology region 1) linked to HR2 [also
known as Pl kinase homology domain (PIK)] and a C2 domain (HR3)
(Vanhaesebroeck at a/., 2001). The class I enzymes are the enzymes most
studied and are found throughout the body. Class I Pl3-kinases are made up of
a 110 kilodalton catalytic subunit (referred to as or, B, 8 and y) (encoded by at
least four mammalian genes), as well as constitutively associated smaller
regulatory subunit that is either 50, 55, 85 or 101 kilodaltons in size. The
catalytic domains are composed of several modular domains, with four
homologous regions shared amongst most Pl3-kinase members; a catalytic lipid
kinase domain, a PI kinase domain, a 02 phospholipid binding domain and a
Res-binding domain, as well as an N-terminal domain which interacts with the
regulatory protein. The Class I Pl3-kinases are further divided into Class la and
lb PI3-kinases based on structure and mode of action. The Class lb p110y lacks
the N-terminal p85 binding site and instead associates with an unrelated
regulatory subunit, p101 (Vanhaesebroeck at a/., 2001; Anderson and Jackson,
2003)

p85a, the first regulatory subunit cloned, contains an N-terminalsrc
homology 3 (SH3) domain for binding proline-rich sequences in target proteins,

two src homology 2 (SH2) domains for interaction with phosphorylated tyrosine

16

reSId‘.
doma:
sizes I
Vanhal
derivec
devoid
compie)
distribut
2001: Ar
p85 regu
Suggests
intraceilul
Class ls l
Subunits 0
Stir
activity lea

to Specific

 

a p'Tl'r-X 7
Pl3—kinaSEI
bcaiIng I‘

 

residues on target proteins and a breakpoint-cluster-region (Bcr) homology
domain flanked by 2 proline-rich regions. These domains function as binding
sites for specific protein-protein interactions (Fruman, 1998; Krauss, 1999;
Vanhaesebroeck at at, 2001). Additional regulatory subunits (p50 and p55) are
derived from alternative splicing of p85a. The class lb regulatory subunit, p101 is
devoid of any known proteinzprotein interaction motifs and the p101/p1107
complex appears to be present only in mammals and displays restricted tissue
distribution, abundant only in white cells (Cantrell, 2001; Vanhaesebroeck at a/.,
2001; Anderson and Jackson, 2003). There is no evidence as of yet, that specific
p85 regulatory subunits isoforms pair with specific p110 isoforms. It is however,
suggested that different p85 subunits may associate with different subsets of
intracellular proteins (Fruman at a/., 1998). p110y, the catalytic subunit of the
Class lB Pl3-kinases and p1103, Class IA Pia-kinase, are activated by the G37
subunits of heterotrimeric G proteins.

Stimulation of almost every receptor that has associated tyrosine kinase
activity leads to Class IA PIS-kinase activation. Binding of the p85 SH2 domains
to specific tyrosine residues, with binding preferential to polypeptides containing
a p-Tyr-X1-X2-Met motif, within the receptor or signaling protein activates the
Pl3-kinase and recruits the cytosolic complex to the plasma membrane,
localizing it near the phosphatidylinositol phosphate (Ptdlns) substrate (Fruman
at a/., 1998). It is the phosphotyrosine binding that is thought to allow the

translocation of the cytosolic Pl3-kinase to the membrane. However,

17

nonty‘
dunng
as va
contrib.
membr.
demons
recruitm
scaffold
membrar

3) and IL-

 

 

nontyrosine-based recruitment mechanisms, such as the Janus kinases (JAKs),
during cytokine signaling, or Syk or ZAP70 during antigen-receptor signaling and
as various membrane proteins demonstrate constitutive association may
contribute to Pl3-kinase activation (Cantrell, 2001). Pl3-kinase recruitment to the
membrane is not solely done via receptors; there are a number of reports
demonstrating interactions between p85 and other adapters that lead to the
recruitment of PIS-kinase to the plasma membrane. For example, a signaling
scaffold including Shc, Grb2 and Gab2 recruits PIS-kinase to the plasma
membrane in cells activated by hematopoietic cytokines such as interleukin 3 (IL-
3) and IL-2 (Gadina stat, 2000; Gu stat, 2000).

Class II Pl3-kinases are approximately 170 kilodaltons and one of their
main features is a C-terminal C2 (CalB) domain that can bind in vitro to
phospholipids in a Ca2*-independent manner (MacDougall at at, 1995). A
Dmsophfla enzyme and three mammaliam isoforms of Class II Pl3-kinases have
been identified: Pl3K-C2a, Pl3KC2B and Pl3K-CZy (Fruman at at, 1998;
Vanhaesebroeck stat, 2001). Pl3K-C2y is predominately found in the liver,
whereas, both PI3K-C2a and Pl3K-CZB are ubiquitously expressed (Domin stat,
1997). Class II PIS-kinases can associate with the epidermal growth factor
receptor (EGFr) receptor in human carcinoma-derived A431 cells (Pl3K-02a and
Pl3K-CZB) utilizing Grb2 as an intermediary (Arcaro stat, 2000; Wheeler and
Domin, 2001), Pl3K-02a can be activated by insulin (Soos stat, 2001) and is

localized to clathrin-coated vesicles (Domin stat, 2000; Gaidarov, 2001).

18

Howey
appear
hnases
respon
Vanhae

hnases

PhOSphoi
Ptdijsiins
and Ptdl:
(although
nominally
Stimulus E

kinases he

 

 

 

1997; Film

”age “pic

 

However, little else is known about this class of PIS-kinases other than that they
appear to associated with the membrane fraction of cells. The class III Pl3-
kinases are suggested to be involved in intracellular trafficking processes and be
responsible for the cellular levels of PtdlnsSP (Fruman st at, 1998;
Vanhaesebroeck stat, 2001). Little else is known concerning the Class III Pl3-

kinases.

2- LipidfinaseAcchr

In general, PIS-kinases transfer a phosphate group to the 03 position of
phospholipids. The four currently known phosphorylated phosphoinositdes are:
Ptd(3)lns, Ptdlns(3,4)P2 and Ptdlns(3,4,5)P3 generated from Ptdlns, Ptdlns(4)P
and Ptdlns(4,5)P2, respectively, and the more recently identified Ptdlns(3,5)P2
(although not a direct Pl3-kinase product). Ptdlns(3,4,5)P3 and Ptdlns(3,4)P2 are
nominally absent in resting cells, however their levels rise rapidly in response to
stimulus and are thus thought to play second messenger roles. Class I Pl3-
kinases have the ability to phosphorylate Ptdlns, Ptdlns(4)P, Ptdlns(4,5)P2, and
Ptdlns(5)P. However in VII/0 the primary substrate is Ptdlns(4,5)P2, causing
increases in cellular levels of Ptdlns(3,4,5)P3 (Fruman stat, 1998). Class II Pl3-
kinases preferentially phosphorylate Ptdlns and Ptdlns(4)P, and under certain
conditions can phosphorylate Ptdlns(4,5)P2 to a limited degree (Domin at at,
1997; Fruman stat, 1998). Class III PIS-kinases phosphorylate only Ptdlns. Pl3-

kinase lipid products are not substrates for phopholipases and inhibition of the

19

prod.
respc
a dif‘e
leadut

1998z‘

-\

A
hnase?
IdSidna;
protein 5
and Emr,
CZupaHa

cells and

 

 

 

 

production of these lipids results in the inhibition of many acute cellular
responses. PIS-kinases transfer the phosphate to lipid head groups from ATP in
a different manner than the catalysis displayed by typical protein kinases, hence
leading to potential avenues for Pl3-kinase drug development (Fruman at at,

1998; Vanhaesebroeck stat, 2001).

3. Protein Kinase Activity

A review by Hunter (1995) was entitled “When is a lipid kinase not a lipid
kinase? When it is a protein kinase” and this applies to the complex enzyme of
PI3-kinase. Pl3-kinase has been found to not only have lipid kinase activity but
protein serine kinase activity as well as the ability to autophosphorylate (Stack
and Emr, 1994; Hunter, 1995; Wymann and Pirola, 1998; Walker stat, 1999;
Czupalla stat, 2003a; Czupalla stat, 2003b). Interferon-or treated lymphoid
cells and insulin stimulated insulin receptor substrate-1 (IRS-1) in adipocytes as
well as L6 muscle cells are directly phosphorylated by the p85/p110a Pl3-kinase
(Lam stat, 1994; Uddin stat, 1997; Pirola stat, 2003). Pirola at at (2003)
further demonstrated that Pl3-kinase mediated reduction in IRS proteins Via
different Pl3-kinase mediated mechanisms (insulin-induced reduction of IRS-1
was controlled by direct phosphorylation by PIS-kinase whereas IRS-2 reduction
occurred V/a Pl3-kinase activation of the mTor pathway) contributing to the
development of an insulin-resistant state in L6 myoblasts. These results

demonstrate the complexity with which Pl3-kinase can function. Further

20

ere
catal‘,
enzyn
(Leopc
regula‘.
regulat.
p1 10a c
contrast
phospho
autophos
activrty
regulates
at, 1997).

 

limited inh

Due
if”DIICafed
kinase (M

I
IA

alter the ac

lite mllOge:

 

 

evidence of PIS-kinases serine kinase ability is that manipulations within the
catalytic core of Pl3-kinase p110a and p110y make it possible to generate
enzymes that are active as protein, but no longer lipid kinases in Viva and in vitro
(Leopoldt stat, 1998; Pirola stat, 2001). Pl3-kinase also posses the ability to
regulate its own function Via p110a phosphorylating a Ser residue on the
regulatory subunits, therefore decreasing lipid kinase activity of the PI3-kinase
p110a dimer three- to seven fold (Carpenter stat, 1993; Dhand stat, 1994). In
contrast to the p110a PIS-kinase subunit, p1108 does not have the ability to
phosphorylated the regulatory subunit; it does however autophosphorylate. The
autophosphorylation activity of Pl3-kinase is hypothesized to regulate PIS-kinase
activity. In vivo and in vitro autophosphorylation of p1108 completely down-
regulates the PIS-kinase lipid kinase ability of the enzyme (Vanhaesebroeck st
at, 1997). The mechanism by which Pl3-kinase has protein kinase activity is still
being explored, as it is difficult to discern the two different kinase activities with
limited inhibitors.

Due to the dual protein and lipid kinase ability of Pl3-kinase it has been
implicated in several signaling pathways including mitogen activated protein
kinase (MAPK). Thus, an alteration in Pl3-kinase protein and/or activity may
alter the activation and function of the MAPK pathway. PIS-kinase is required for
the mitogenic effects of epidermal growth factor (EGF) and thrombin in human
airway smooth muscle proliferation (Stack and Emr, 1994). Additionally, Akt-3,

an isoform of Akt, is present and activated by Pl3—kinase in human vascular

21

inc
ceri-
inhii

200C

Illi'tlbitIOr
Survival .
mediated
Presence
DNA iragn
can 139 Sn“

IIlhlblilOl'l c

 

“@3ng
dihydrowle
intima,-O?

iRebsam en

 

smooth muscle cells (VSMC) and causes proliferation (Sandirasegarane and
Kester, 2001 ). Bovine aortic vascular smooth muscle cell (VSMC) migration was
increased when treated with extracellular matrix (ECM) proteins, but when the
cells were treated with the PIS-kinase inhibitor LY294002, migration was
inhibited, suggesting that Pl3—kinase plays a role in VSMC migration (Willis stat,
2000). The p110a isoform of Pl3-kinase is specifically required for EGF-
stimulated actin nucleation during lamellipod extension in breast cancer cells (Hill
et at, 2000). Hayashi et al. (1999) suggests that changes in the balance
between the Pl3-kinase/Protein Kinase B (PKB)/Akt pathway and the Erk/MAPK
pathways would determine phenotypes of visceral and vascular smooth muscle
cells. Activation of the PIS-kinase pathway by EGF results in cell survival and
inhibition of apoptosis and MAPK kinase (MEKK) inhibitors do not block the
survival effect that EGF provides in transforming growth factor 131 (TGF-B,)-
mediated apoptosis in the liver (Fabregat et al., 2000). Moreover, in the
presence of PI3-kinase inhibitors, the protective effect of EGF on cell viability,
DNA fragmentation, and caspase-3 activity is abolished. In hypertension, there
can be smooth muscle hypertrophy and eutrophy, which may be the result of
inhibition of apoptosis and smooth muscle cellular migration that could be Pl3-
kinase-mediated. Pl3-kinase is involved in steroid hormone 1a, 25-
dihydrovitamin Da —mediated vascular smooth muscle cell migration and
proliferation and migration of human pulmonary vascular smooth muscle cells

(Rebsamen stat, 2002; Goncharova stat, 2002). All these studies demonstrate

22

that
lipid
an in
throu.

migrat

hnase.
heterotr
activate
lead to ti
without tr.
measure
protein K
seriner’thrr
SUQQGSter

phOSphoip

 

  

and thUs ~

 

that Pl3-kinase is vital to a wide variety of functions and mediates it effects via its
lipid as well as protein kinase activities, ultimately having great potential for being
an important component in the development and maintenance of hypertension
through mediating vascular smooth muscle cell proliferation, contraction and

migration.

4W

There are a wide variety of proteins upstream and downstream of PI3-
kinase. A variety of substances, including cytokines, growth factors,
heterotrimeric G-proteins small GTP-binding proteins and tyrosine kinases
activate PI3-kinase. GTP-bound rat sarcoma oncogene homolog (ras) can also
lead to the recruitment and activation of the p110 subunit of Pl3-kinase with or
without the p85 regulatory subunit. One of the downstream processes utilized to
measure PI3-kinase activity is phosphorylation of Akt, otherwise known as
Protein Kinase B (PKB), or Related to A and C protein Kinase (RAC-PK), a
serine/threonine kinase. Ptdlns (4,5)P2 causes dimerization of Akt, which is
suggested to assist in Akt activation. Ptdlns(3,4,5)P3 binding to 3-
phosphoinositide-dependent kinases (PDK)-1 allows PDK-1 to phosphorylate
and thus activate Akt and its activation requires an intact pleckstrin homology
(PH) domain. The mechanism by which Ptd(3,4,5)P3 stimulates PDK-1 to
phosphorylate Akt is controversial, whether the binding inhibits auto-inhibition of

PDK-1 or allows localization of PDK-1 to the membrane is still being debated

23

(Centre
sense:
olapopl
hnase=
Sos. ar
synthes
Contract:
contract
bohne c
Ubdayo a
at, 1995

muscle c

Tr
phenome
handling ;
a" 1987;
kinase Su:
IMacrez
increaSe i-

Spontanec

 

(Cantrell, 2001). Once Akt is activated, it phosphorylates other proteins on their
serine/threonine residues. There are a variety of Akt targets including members
of apoptotic regulatory pathways. Some of the other downstream targets of PI3-
kinase (Figure 2) are PLCy, L-type calcium channels, PKC, Cdc42, rac, ras, rho,
$05, and MAPK to name a few. Activation of these proteins leads to protein
synthesis/cell cycle progression, cell survival, superoxide formation and
contraction. lmportantly, PIS-kinase has been implicated in smooth muscle
contraction of the canine basilar artery, (Yang stat, 2000a; Yang stat, 2001)
bovine carotid artery (Komalavilas stat, 2001), colonic smooth muscle cells
(lbitayo stat, 1998) and guinea pig gastric longitudinal smooth muscle (Zheng at
at, 1998). These studies all demonstrate that PI3-kinase is involved in smooth

muscle contraction.

5. Calcium and Pia—kinase

There are a plethora of crucial roles for calcium (Ca2*) in cells. One
phenomenon in smooth muscle cells that is due to altered Ca2+ sensitivity and/or
handling is spontaneous tone (Pucci stat, 1995; Lamb stat, 1995; Thompson at
at, 1987; Webb st at, 1992; Rapacon-Baker stat, 2001). All the Class I PI3-
kinase subunits associate with L-type Ca2+ channels and increase current directly
(Macrez stat, 2001) or Via PKC (Viard stat, 1999). The end result is an
increase in intracellular [Ca2+],. Studies have also solely implicated PKC in

spontaneous tone development (Komalavilas at at, 2001; Lamb at at, 1995;

24

Figure 2. Depiction of some of the hypothesized downstream elements of PI3-
kinase that may be present in vascular smooth muscle and the end result of
activation of these pathways (contraction, cell survival, superoxide formation and
protein synthesis/cell cycle progression). Abbreviations are as follows: Akt/PKB-
Protein Kinase B or Related to A and C protein Kinases (RAC-PK), cdc42-cell
division cycle protein 42, MAPK-mitogen activated protein kinase, PDK— 3-
phosphoinositide-dependent kinase, PKC- protein kinase C, PLC- phospholipase
C, PTEN- phosphatase and tensin homolog, ras- rat sarcoma oncogene

homolog, rho- sos- son of sevenless.

25

 

 

 

 

 

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Macrez stat, 2001). In vascular myocytes, Ang II stimulation of L-type calcium
channels and the associated increase in [Ca2+], is mediated through the PI3-
kinase p110y subunit (Viard stat, 1999; Macrez stat, 2001; Quignard stat,
2001). Pl3-kinase might also influence Ca2+ release through NO in the context of
mechanical stretch. In cardiac muscle, mechanical stretch activates Akt V/a PI3-
kinase (Petroff stat, 2001). After Akt activation, eNOS-catalyzed NO generation
enhances Ca2+ release from the sarcoplasmic reticulum. Akt also potentiates
neuronal L-type Ca2+ channel activation (Blair stat, 1999). The end result is an
increase in Ca2+. These data demonstrate a potential connection between PI3-
kinase mediated increases in calcium influx, possibly V/a L-type calcium channels
and increases in spontaneous tone. I will argue for a role of PIS-kinase in the

development and propagation of spontaneous tone development in hypertension.

6. Magnesium and El3-kinase

Magnesium (Mgz*) is a mineral required by every cell of the body and is
required for greater than 300 biochemical reactions including maintenance of
normal muscle and nerve function, heart rhythm, energy metabolism and protein
synthesis (Johnson, 2001; Facts About Dietary Supplements, 2001; Touyz,
2003). In the cardiovascular system intracellular [Mgz’*]l regulates contractile
proteins, modulates transmembrane transport of calcium, sodium, and
potassium, acts as a essential cofactor in activation of ATPases, and influences

DNA and protein synthesis (Rusch and Kotchen, 1994; Laurant and Touyz, 2000;

27

Touyz
wthli
1994:
induce
agonis
vascuie
Touyz,
ATPass
hnase
afiered
2000:\’
2001;lfl
44 199
Contract
Conannn-
of Pl3-k
mechann
kinase C
enhance:

anefiesfr

 

Touyz, 2003). Mg2+ concentrations are inversely proportional to blood pressure,
with hypomagnesemia being associated with hypertension (Rusch and Kotchen,
1994; Johnson, 2001; Laurant and Touyz, 2000; Touyz, 2003). Mg2+ deficiency
induces cardiovascular alterations such as elevated blood pressure, enhanced
agonist-mediated reactivity, attenuated responses to vasodilators and increased
vascular tone (Laurant and Berthelot, 1992; Laurant at at, 1997; Laurant and
Touyz, 2000; Touyz, 2003). Low MgZ*-utilization/activation of Ca2+, Na*/K"
ATPase, tyrosine kinases, protein kinase C (PKC), mitogen activated protein
kinase (MAPK), and Pl3-kinase signaling components have been implicated in
altered vascular tone and/or cellular growth (Touyz, 2003; Laurant and Touyz,
2000; Yang stat, 2001; Yang stat, 2000a; Yang stat, 2000b; Zheng st at,
2001; Wei stat, 2002; Bara and Guiet-Bara, 2001; Altura stat, 2001; Touyz at
at, 1998). Extracellular Mg“ deficiency, through Mg“ removal, induces
contraction of rat aorta, V/a the activation of MAPK, Pl3K and SH2 domain-
containing proteins (Yang stat, 2000; Yang stat, 2001). If Mg2+ is a modulator
of Pl3-kinase, the lower Mg2+ levels detected in hypertension may be one
mechanism that amplifies PIS-kinase activity. Low Mg?” may also activate Pl3-
kinase dependent pathways in aorta from normotensive animals eliciting
enhanced spontaneous tone and contraction, arterial dysfunctions observed in

arteries from hypertensive DOCA rats.

28

 

phosp
F’lCi-‘K.'.r
moietie
moduk
regulati
and ph
woponu
have be«
PTEN de
with U8'.
demonsr,
datPTEj
these finc

mOdulate:

will exam;

 

Ofanefle:

i I
ipertens

ac"Will. th

enhanCed :

 

7. P 1 EN (Ehasphatasa and Tansin Hamolgg)

PTEN is a unique tumor suppressor gene that encodes a dual-specificity
phosphatase (Li and Sun, 1997; Li stat, 1997; Stock st at, 1997) that controls
PIS-kinase activity. PTEN has the ability to remove the 3-phosphate from inositol
moieties and proteins phosphorylated by PIS-kinase (Dahia, 2000). PTEN
modulates cell cycle progression and cell survival in embryonic stem cells by
regulating Ptdlns (3,4,5)P3 and Akt signaling pathway (Sun stat, 1999). PTEN
and phosphorylated Akt, an indicator of PI3-kinase activity, are inversely
proportional in many primary leukemia and lymphoma samples and cell lines that
have been tested (Dahia stat, 1999). Wen stat (2001) provided evidence that
PTEN decreased tumor growth in VII/0 and prolonged survival in mice implanted
with U87MG glioma cells reconstituted with PTEN cDNA. These results
demonstrate that growth is inversely proportional to PTEN activity. The theory
that PTEN plays a role in apoptosis by regulation of Pl3-kinase is supported by
these findings. The mechanisms by which PTEN lipid phosphatase activity is
modulated, how it is targeted and its role in cell signaling are still being studied. I
will examine the possibility of the presence of PTEN in the smooth muscle cells
of arteries. I will also suggest that a small down-regulation of PTEN in
hypertension, if it is present in arteries, could lead to unopposed Pl3-kinase
activity, thus leading to a decrease in apoptosis, increase in cellular growth and

enhanced contraction during the condition of hypertension.

29

observe

 

In conclusion, Pl3-kinase is a diverse enzyme utilized in many different
pathways. If Pl3-kinase enzyme concentration or activity is altered in the
condition of hypertension due to increased expression or decreased control of
the enzyme, this may explain many of the cellular and functional alterations

observed in the condition of hypertension.

30

D: Hypothl

One
kinase funl
amounts 0'
substrates
suppressor
the activity c
cascades at
mediating ai

depicts the M

W

SPORE

Ubh 0thesi

of DOCA‘Salt I

D: Hypothesis

One question that remains and which I propose to address is how PI3-
kinase functions differently in the condition of hypertension. Endogenous
amounts of PI3-kinase may change the interaction of PI3-kinase with its
substrates and modify multiple outcomes. A potential mechanism is that a
suppressor of Pl3-kinase activity is reduced. Therefore, there is in increase in
the activity of PIS-kinase enzyme. PI3-kinase is involved in a variety of signaling
cascades and diverse biological processes and may play an important role in
mediating arterial contraction and spontaneous tone in hypertension. Figure 3

depicts the working hypotheses.

merall Hypothasis:

Spontaneous tone in a DOCA-salt rat model of hypertension is mediated
by an up-regulation of the PIS-kinase dependent signaling pathway. This, in turn,
ultimately links to an increase in intracellular calcium concentration through

opening L-type calcium channels, permitting arterial contraction.

Subhypgthasis #1: Pl3-kinase protein and/or its activity are upregulated in aorta

of DOCA-salt hypertensive rats.

31

is alte rec

Subhypotl

kinase sigr

regulated i

contraction .

ObSBNed If'l

hl'pertensim.

Subhypothasis #2: PI3-kinase and L-type voltage gated Ca2+ channel interaction

is altered in the aorta of the DOCA-salt rat model of hypertension.

Sgbhypothasis #3: PTEN is localized in vascular smooth muscle cells and is

down-regulated in aorta of the DOCA-salt rat.

Subhypathesis #4: Norepinephrine (NE) and magnesium (Mgz*) utilize the FIS-

kinase signaling cascade to elicit enhanced vascular contraction in hypertension.

h h i # : PI3-kinase and its dependent signaling pathways are up-
regulated in multiple models of hypertension, leading to enhanced vascular

contraction and spontaneous tone development.

Sgbhypathasis #6: Pl3-kinase functional alterations and changes in protein are

observed in the mesenteric resistance arteries in the DOCA-salt model of

hypertension.

32

Calcium
Channel
% v
C a2.
Figure 3. Depiction of working hypotheses. Abbreviations are as follows: Ca2*- 1
calcium, Mg"’*- magnesium, PIS-kinase- phosphoinositide 3-kinase, PTEN- + Co
Diractj

Phosphatase and tensin homolog and TK-tyrosine kinase.

33

Receptor

it» ii l'u-wwfi.
L.type mitts! 35:36:23
Calcium G-proteln coupled
Channel — //—.——-——-—- —— Receptor

CaZ+ TK/

1 Low Mg2+
85/ 110
p 9 ago

+ Contraction+
XLYZQ‘IOOZ

34

Animals

All anima
guidelines 01 Ml
housed in clear

access to standa

 

 

8. Models of PM
1. Mineralc
Male Spra
River (Portage.
isoflurane (lsoFli
During surgical p
under the animal,
arid a Silastice

deolit/Corticostero

MATERIALS AND METHODS

A. Animals

All animal procedures were followed in accordance with the institutional
guidelines of Michigan State University. Rats upon arrival to our facility were
housed in clear plastic boxes with wood chip bedding and allowed ad //b/'tum

access to standard rat chow (T eklad ®) and tap water.

B. Models of Hypertenslon
1. MineLekchrchcidfimedension

Male Sprague-Dawley rats (250-300 g) were purchased from Charles
River (Portage, MI) unless otherwise specified. Rats were placed under
isoflurane (lsoFloQ) anesthesia and shaved free of fur in the area of incision.
During surgical procedures the rats were kept warm by placing a heating pad
under the animal. The animals were uninephrectomized (flank incision, left side)
and a Silastic" (Dow Corning, Midland, MI) implant impregnated with
deoxycorticosterone acetate (DOCA; 200 mg/kg; Sigma Chemical Co., St Louis,
MO) was placed subcutaneously in the subscapular region. Postoperatively,
DOCA-salt rats were given a solution of 1 % NaCl and 0.2 % KCI for drinking.
Sham rats also received a uninephrectomy, but received no DOCA Silastic®
implant and drank normal tap water. All animals were fed standard rat chow and

had ad lib/tum access to food and water. The animals remained on the regimen

for four weeks (unless otherwise specified) prior to use.

35

To
contractili'
as descrit
3, 5 or 7 0

method as

2. fl
WK‘
Taconic Fe
systolic blc
higher than
normal rat (

3' N(1)

Male
(Indianapolis
water or Watt
MO) fer 14 d
On day 14 Ihi

lail Cliff memo

To examine the influence of blood pressure on changes in PIS-kinase and
contractility, time course experiments were performed. Surgery was performed
as described above. The animals remained on their respective treatments for 1,
3, 5 or 7 days, after which systolic blood pressures were taken using the tail cuff

method as described below.

2. Wiatar-Kyatg (WKY) and Spantanaogsly Hypafiansive Bats (SHR)

WKY (11-14 weeks old) and SHR (12 weeks old) rats were obtained from
Taconic Farms, Inc. (Germantown, NY). At the age we received the rats, the
systolic blood pressures of the SHR rats were consistently and significantly
higher than that of the WKY rats (Figure 4). The rats had aa’ lib/tum access to
normal rat chow and tap water. Systolic blood pressures were measured using

the tail cuff method described in section 4.

3. Nw-nitrQ-L-arginina (LNNA) ijpaflensign

Male Sprague Dawley rats were obtained from Harlan Laboratories
(Indianapolis, Indiana) (250-300 9). These animals received either normal tap
water or water supplemented with LNNA (0.5 g/L, Sigma Chemical Co., St Louis,
M0) for 14 days. The rats had ad tb/tum access to normal rat chow and water.
On day 14 the systolic blood pressure of these animals was measured using the

tail cuff method described in section 4.

36

Figure 4. Systolic blood pressures for Wistar Kyoto (WKY) (n=6) and 20
o.

Spontaneously hypertensive (SHR) rats (n=6). Columns represent the mean 0
L
value, where as the vertical lines represent the standard error of the mean. * g 150
o
Statistically significant difference (p<0.05) between WKY and SHR treatment at a
8 i5
groups. 2
.9 "'
F2
9.
U) 50
0

37

 

 

 

Systolic Blood Pressure

(mmHm

2001

 

 

 

 

N:
150-
y——=——.
100-
50-
0 vaY

38

 

4. Blood

Systolic ti

 

method using a

with wood shav;
pad and the rat
light was then p

minutes. This a

 

measurement of
the blood press
balloon transduc
Secured with

SphygmomanOrT
pulse pressure g
deflected to infra
rats, the sphygI

Whereas fOr the

DressU, e meaSL

4. Blaad Erasagra Meaagramants

Systolic blood pressures 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 then placed over the bucket. The rat was warmed for approximately 6
minutes. This allowed for vasodilatation of the tail artery, which facilitated the
measurement of the blood pressure. The rat was then placed in a restraint and
the‘ blood pressure cuff and balloon transducer was placed on the tail. The
balloon transducer was placed on the ventral side of the tail behind the cuff and
secured with tape. The blood pressure was measured utilizing a
sphygmomanometer in conjunction with the pulse transducer. After a stable
pulse pressure was obtained, the manual toggle on the sphygmomanometer was
deflected to inflate the cuff. To measure the blood pressure of the normotensive
rats, the sphygmomanometer pressure was set at approximately 200 mmHg,
whereas for the hypertensive rats it was set at 250 mmHg or higher. Three blood
pressure measurements were taken to obtain an average measurement. The
DOCA-salt and LNNA rat blood pressure measurements were taken at 2 weeks
of the study. The final DOCA-salt rat blood pressures were taken at 4 weeks or
at the end of their treatment. WKY and SHR systolic blood pressure
measurements were taken on the day of the experiment. The rat blood pressure

measurements for the time course experiments were take prior to surgical

39

 

procedure a
monitor any

course of the

C. Contra
1. Qfl
On the
pentobarbital
solution (PSS)
mmol/L Mgsc
mmol/L dextros
fat and conner
exceriment, the .
lUminaI face of
arterial Strip Was
attached to a Gra
Q“mill/1A) with

recordings using P

optimum resting Is.

to Briuilibraye far 0

procedure and then at the end of the given time point. These were done to
monitor any small change in pressure that may have occurred throughout the

course of the experiments.

C. Contractility Recordings

1. General laglated Tisaga Bath Protacol

On the day of the experiment rats were euthanized using 60 mg kg'1
pentobarbital (ip). Arteries were removed and placed in physiological salt
solution (P88) (103 mmol/L NaCl; 4.7 mmol/L KCL; 1.18 mmol/L KH2P04; 1.17
mmol/L MgS04-7H20; 1.6 mmol/L CaCl2-2H20; 14.9 mmol/L NaHCOa; 5.5
mmol/L dextrose, and 0.03 mmol/L CaNa2 EDTA). The arteries were cleaned of
fat and connective tissue and cut into helical strips. Depending on the
experiment, the endothelium was left intact or was removed by gently rubbing the
luminal face of these strips with a moistened cotton swab. One end of the
arterial strip was mounted onto a glass rod holder while the other end was
attached to a Grass‘D force-displacement transducer FI'OSC (Grass Instruments,
Quincy, MA) with 5.0 silk (George Tieman and Company, Plainview, NY). This
preparation was then placed into 10 ml tissue baths for isometric tension
recordings using PowerLab/s v.3.6 and Chart v.3.6.3/s software (Mountain View,
CA) and evaluated using a Macintosh computer. Strips were placed under
optimum resting tension (1,500 mg for aorta, determined previously) and allowed

to equilibrate for one hour. One arterial strip isolated from a normotensive rat

4O

Ii:
int
de
to

be:

to (
defi
EXOI

30m

was

there

and one arterial strip from a hypertensive rat was placed in the same bath,
thereby controlling for potential experimental variations. Tissue baths contained
warmed (37 °C), aerated (95 °/o 02/002) PSS. Administration of an initial
concentration of 1 x 10‘5 mol/L of the or,-adrenergic agonist, phenylephrine (PE),
was used to test arterial strip viability. The aortic strips had to contract to a
minimum of 500 mg, to be considered viable and for the experiment to continue.
Tissues were then washed to remove the PE and then tested for the functional
integrity of the endothelial cells. This was evaluated by testing endothelium-
dependent relaxation to acetylcholine (ACh) (1 x 10*3 mol/L) in strips contracted
to a half-maximal concentration of PE. Cumulative concentration curves were
performed to agonists. Antagonists, inhibitors or vehicle were incubated with the

vessels for one hour prior to experimentation.

2. Spgntanagga Tgne and Inhiaitgra Protaggl
Aortic strips from sham, DOCA-salt, WKY, SHR and LNNA rats were used

to examine the development of spontaneous tone. Spontaneous tone was
defined as a change in arterial tone when the arterial tissue independent of
exogenous stimulus. Figure 1 depicts spontaneous tone development in an
aortic strip isolated from a DOCA-salt rat (lower tracing) and the top tracing is
that of an aortic strip removed from a sham rat. Over the 30 minute period there
was a steady increase in contraction in the aortic strip from the DOCA-salt and

there was no exogenous agonist added. Only on isolated occasions would

41

 

spi

lor

rec

PIC

t0

COi

the

spontaneous tone be observed in aortic strips from a sham rats. Spontaneous
tone was monitored and LY294002 (20 umol/L), LY303511 (20 umol/L),
Nifedipine (50 nmoVL) or vehicle (DMSO and/or water) was added for 30 minutes
and the increase or decrease in tone that occurred during that time period was
recorded. Separate aortic strips were used to examine the cumulative addition of
Pl3-kinase inhibitors LY294002 and wortmannin. Concentration response curves
to LY294002 and wortmannin were generated by adding increasing
concentrations of vehicle, LY294002 (1x10'7- 3x10‘4 mol/L) or wortmannin (1x10'8
- 3x10“t mol/L) every 30 minutes and measuring changes in spontaneous tone

that occurred.

3. Aggnist-stimulatian 9f Pia-kinasa PLQIQQQI

Arterial strips were removed and placed in 10 ml tissue baths as stated
above. Norepinephrine (NE) (1x109 - 3x10‘5 moI/L) and BayK 8644 (1x10'10 -
3x1045 mol/L) were added in a cumulative fashion after a 1 hour incubation with
LY294002 (20 umol/L) or vehicle (OJ-0.2% DMSO). After addition, alterations in

agonist-induced contraction were recorded.

4. Calm
Thoracic aorta were removed, cleaned and placed in the tissue bath as
stated above. The aortic strips were first incubated in normal Ca2+ (1.6 mmol/L)

PSS challenged with PE (10'5 mol/L) and tested for endothelial status. Tissues

42

were washed and then incubated for 30 minutes in Ca2*-free buffer
supplemented with 1 mmol/L EGTA. The buffer was changed in the bath every
ten minutes, to allow for equilibration to the new buffer. Tissues were switched to
a Ca2*-free 0.03 mmol/L EDTA buffer, equilibrated for 15 minutes, changing
buffer every 5 minutes. LY294002 (20 umoI/L) or vehicle was then added for 15
minutes prior to addition of Ca2+. Ca2+ was added back to the bath in a
cumulative fashion (1x 10'6 -3x10'3 mol/L) with additions every 5 minutes.

Changes in spontaneous tone were recorded.

5. Magnasigm Prgtmol

Aortic strips from sham and DOCA-salt rats were used to examine the
effects of altered magnesium (Mgz") concentration on spontaneous tone and NE-
induced contraction. Separate aortic strips were incubated in PSS buffer
containing a low concentration of M92” (0.15 mmol/L), high Mg2+ (4.8 mmol/L), or
regular PSS (1.17 mmol/L Mg”) for 30 minutes. The buffer was changed every
10 minutes to permit equilibration. After the 30 minutes incubation changes in
spontaneous tone that occurred were recorded. LY294002 (20 umol/L) or
vehicle was incubated in the baths for 30 minutes prior to the addition of

increasing concentrations of NE (1 x10‘9 -3x10'5 moi/L).

43

Qer
The
Dfat

I
Iaye

6. Myograph Protocol

Small mesenteric resistance arteries (2 — 3 mm long, 150- 250 u diameter)
were dissected away from mesenteric veins under a light microscope and
mounted between two tungsten wires in a dual chamber wire myograph for
measurements of isometric force (University of Vermont Instrumentation Shop).
Arteries were bathed in 37 °C PSS aerated with 95 °/o 02/ 5 % C02. Tissues
equilibrated for 30 minutes with frequent changes of buffer prior to applying
optimal tension. Optimal tension (mesenteric resistance arteries: 400 mgs) was
applied by means of a micrometer and tissues equilibrated for 60 minutes before
exposure to a maximal concentration of PE (10‘5 mol/L). Spontaneous tone was
monitored and LY294002 (20 umol/L) or vehicle (0.1 % DMSO) was added for 30
minutes and the increase or decrease in tone that occurred during that time

period was recorded.

D. Aortic Vascular Smooth Muscle Cell Culture
Vascular smooth muscle cells were derived from the aorta of male
Sprague-Dawley rats. Aorta were excised in an aseptic manner, cleaned of fat,
connective tissue and cut into helical strips. The endothelium was removed by
gently rubbing the luminal face of these strips with a moistened cotton swab.
The strips were cut into small squares (2 x 2 mmm). These pieces of tissue were
placed lumen side down in a P-60 Corning culture dish (Corning, NY) and

layered with a small amount of serum-enriched media to keep the tissues moist

[medium consisted of DMEM with D-Glucose (4500 mg/liter), L-glutamine (1 %)
and HEPES buffer (25 mmol/L; GIBCO Life Technologies, Gaithersburg, MD)
containing fetal bovine serum (40 °/o v/v; Hyclone Laboratories, Logan UT) and
streptomycin (100 mg/ml)/penicillin (100 units/ml; GIBCO Life Technologies)].
Plates were placed in a 5 °/o 002 warming incubator maintained at 37 °C. Once
the tissues had attached to the plate (~ 18 hr), additional medium was added to
the dish. After ~1 week, a sufficient number of cells had migrated from the tissue
to reach confluency in the plate. Cells were trypsinized, seeded to T75 flasks
and fed with normal serum (10 °/o) DMEM. Cells were plated onto P-100 plates
and used when confluent between passages 2 and 9. With each new isolation
cells were positively stained for smooth muscle a-actin (Sigma Chemical,

St.Louis, MO); cultured rat fibroblasts did not stain with this antibody.

E. Biochemlcal Assays

1. Protein Isolation

a. Who/e 7729.909 Isolation

Rat thoracic aorta and mesenteric resistance arteries were removed from
the animal and placed in PSS and cleaned as described above. Tissues were
quick frozen and pulverized in a liquid nitrogen-cooled mortar and pestle and
solubilized in lysis buffer [0.5 mol/L Tris HCl (pH 6.8), 10 "/0 SDS, 10 °/o glycerol]
with protease inhibitors [0.5 mmol/L Phenylmethylsulfonyl fluoride (PMSF), 10

pg/ul aprotinin and 10 pg/ml leupeptin]. Homogenates were centrifuged (11,000 g

45

for 10 minutes, 4 °C) (small resistance arteries 11,000 for 20 minutes, 4 °C) and
supernatant total protein was measured using the Bicinchoninic Acid method

(BCA, Sigma Chemical Co., St. Louis, MO).

I). Membrane Protein Isolation

Thoracic aorta was removed from DOCA-salt and sham rats and cleaned
as stated above. The aorta was cut into helical strips and then further out into 2-
3 mm segments. The small segments were placed in 2 ml homogenizing
solution [2 mmol/L EDTA; 2 mmol/L EGTA; 250 mmol/L sucrose; 50 mmol/L
MOPS; 500 pg/ml leupeptin, antipain, and aprotinin; 10 mmol/L PMSF] in a
chilled glass dounce. The dounce was then placed in ice and the tissue was
ground with a Tissue Tearor (BioSpec Products, Inc., Bartlesville, OK) at speed
15 for 10 - 30 seconds. The tissues were dounced for 10 strokes. The sample
was then transferred to a centrifuge tube and place in a sonicator for 30 seconds
and vortexed to ensure mixing. The samples undenNent serial centrifugation;
3200 rpm, 10 minutes, 4 °C. supernatant was transferred to new tube; 8200 rpm,
10 minutes, 4 °C supernatant was transferred to an ultracentrifuge tube (filled to
top of tube with buffer) and centrifuged for 1 hour and 20 minutes, 48,000 rpm, 4
°C. The remaining pellet was resuspended in 50 pl homogenization buffer and

total protein was measured using the BCA protein assay.

46

With
”1an

pIIBCe

o. lmmunopreoioitat/on ano’ P/3-k/nase Act/Vial Assay Protein Isolation

Whole aorta were cleaned of fat and connective tissue and snap frozen in
liquid nitrogen. Frozen samples were pulverized in a liquid nitrogen-cooled
mortar and pestle. The powder was resuspended in PIS-kinase buffer [20
mmol/L Tris, pH = 7.6; 10 % Glycerol; 1 °/o NP-40; 140 mmol/L NaCl; 2.5 mmol/L
CaClz; 1 mmol/L MgClz; 1 mmollL N33V04; 1 mmol/L Dithiothreitol (DTT); 1
mmollL PMSF] and placed in a microcentrifuge tube. The samples were placed
on ice for 30 minutes and vortexed every 5-10 minutes. Following incubation,
they were centrifuged at 14,000 rpm, 30 minutes at 4 °C. The supernatant was
removed and placed in a new tube and the pellet was discarded. BCA protein

analysis was performed to determine total protein isolated.

of Vascular Smooth Muscle Cell Protein isolation

Cells (P-100 plates) were switched to physiological salt solution (see
above) for 1 hour before the addition of agonist (final volume, 4 ml). At this same
time, antagonists or vehicle were added and equilibrated with tissues for 1 hour.
Examination after 1 hour indicates that the vehicle or treatments did not cause
the cells to lift off the plate or were not destroyed. Each dish was incubated
with one agonist concentration. EGF (10 nmol/L) or vehicle was added for 10
minutes to stimulate the EGF signaling cascade. After incubation, plates were
place on ice and the incubation buffer was aspirated. Cells were washed 3 times

(4 ml/wash) with phosphate buffered saline (PBS) containing sodium

47

 

To
”0)
inct
and
then

abSQ

orthovanadate as a tyrosine phosphatase inhibitor (10 mmol/L sodium
phosphate, 150 mmol/L NaCI, and 1 mmol/L sodium orthovanadate, pH 7.0).
Five hundred microliters of supplemented RIPA lysis buffer (50 mmol/L Tris HCI,
pH 7.5, 150 mmol/L NaCl, 2 mmol/L EGTA, 0.1 °/o Triton X-100, 1 mmol/L PMSF,
10 pg/pl aprotinin, 10 pg/pl leupeptin and 1 mmol/L sodium orthovanadate) was
added to each dish and cells were lysed with a rubber policeman. Lysate was
transferred into 1.5 ml centrifuge tubes and centrifuged at 14,000 x g for 10

minutes at 4 °C. The supernatant was aspirated from the pellet of cellular debris.

9. BOA Protein Assay

Bovine Serum Albumin (BSA) protein standard (Sigma, St. Louis, MO),
was utilized to make the standard curve to which the protein samples were
compared. The standard curve contained BSA of the appropriate concentration
(0, 2.5, 5, 10, 15 and 20 pg/pl), 5p| of the lysis buffer used to isolate the sample,
water (to a final volume of 100 pl) and 2 ml working reagent. Working reagent
consisted of 50 parts BCA and 1 part Copper (ll) Sulfate (Sigma, St. Louis, MO).
To determine the protein concentrations of samples, 5 pl protein supernatant
from each sample, 95 pl H20 and 2 mL working reagent was mixed and
incubated for 30 minutes at 37 °C. no C02; 2 replications per sample were done
and the protein concentration was an average of the two. The samples were
then analyzed on a DU® 640 spectrophotometer (Beckman, Fullerton, CA) at an

absorbance of 562 nm and a protein determination was determined by

48

 

prir
PT)
MA)

and _

comparing these values to the standard curve. Figure 5 illustrates a standard

curve output from the spectrophotometer.

2. Westsrn Analyses

a. Standard Westem Blotting Protocol

Lysate containing 4:1 in denaturing sample buffer [2.5 ml 1.0 mol/L Tris
pH 6.8, 2.5 ml 20 "lo Sodium Dodecyl Sulfate (SDS) 0.5 ml 0.1 % bromophenol
blue, 4.5 ml glycerol; 1 ml denaturing buffer and add 94 pl B-mercaptoethanol]
was boiled for 5 minutes and then separated on 7-10% SDS-polyacrylamide gels
(Running conditions: 7.5 cm gels; Laemli Running buffer: 90 g Tris base, 432 g
glycine, 15 9 SDS; and brought to 15 L volume with H20; 150-200 V until running
line is at end of plate). The samples were then transferred (Transfer buffer: 45 g
Tris base, 216 g glycine; 3 L methanol and brought to 15 L volume with H20; 100
V for 1 hour) to lmmobilon-P transfer membrane (0.45 pm, Millipore). Transfer of
rainbow molecular weight standards (Amersham Biosciences, Piscataway, NJ)
indicated proper transfer of proteins. Membranes were blocked for 3 hours [Tris-
buffer saline (TBS) (20 mmol/L Tris and 137 mmol/L sodium chloride; pH 7.6), 4
% chick egg ovalbumin, 2.5 °/o sodium azide]. Blots were probed overnight with
primary antibodies p85a (1:100), p110a(1:250), p11OB, p1107, p1105 (1:1000),
PTEN, pPTEN, Akt, pAkt , pPDK-1 (Ser 241)(1:1000; Cell Signaling, Beverly,
MA), PKB kinase/PDK 1 (1 :1000; BD Transduction Laboratories, San Diego, CA)

and smooth muscle a-actin (1:400; Oncogene, San Diego, CA) at 4 °C. Positive

49

Flgure 5. A representative standard curve illustrating the bovine serum albumin

(BSA) standards of 0 pg/pl, 2.5 pg/pl, 5 pg/pl, 10 pg/pl, 15 pg/pl and 20pg/pl.

Hi1 '. U L 1.; d l 0‘.

The samples are ran on a DU” 640 spectrophotometer (Beckman, Fullerton, CA)
at an absorbance of 562 nm. An r value of .97 or greater for the standard curve

was required to consider the test accurate. The samples are compared to this

  

standard curve to determine the protein concentration of the sample that was

tested.

  

LUPveftz

50

 

 

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51

 

oontrolsl
from BD '
was U87
from San
control Vi
NY). Srr
measure.
mmoll. T
inTBS. 7
lg. Horser
NJ or Anl
91100112
P1107, pl)
00. Blots
Finally, en

(AmefSham

a 5,5

Llsat

4.15% Tris.)

electricauy Se

controls for p110a was Jurkat cells; PKB kinase/ PDK1 was SW—13 Iysate (both
from BD Transduction laboratories, Palo Alto, CA); p11OB was K-562 cells; p1108
was U87MG cells; p1107 was U-937 cells; PTEN, was PTEN (FL) epitopes (all
from Santa Cruz Biotechnology, Inc., Santa Cruz, CA); and the pPTEN positive
control was EGF-stimulated A431 cells (Upstate Biotechnology, Lake Placid,
NY). Smooth muscle a-actin was used as a comparative smooth muscle cell
measure. Blots were rinsed in Tris buffered saline-Tween (T BS-T) (pH 7.6) (20
mmol/L Tris, 137 mmol/L sodium chloride and 0.1 °/o Tween-20), with a final rinse
in TBS. The blots were then incubated with secondary antibodies [Anti-mouse
lg, Horseradish Peroxidase (HRP)-linked; Amersham Biosciences, Piscataway,
NJ or Anti-rabbit lgG HRP-linked (Cell Signaling, Beverly, MA)] (p850: and
p110a1:2000 Anti-mouse; smooth muscle a-actin 1:5000 Anti-mouse; p11OB,
p110'y, p1106, Akt, pAkt, PTEN and pPTEN 1:2000 Anti-rabbit) for 1 hour at 4
°C. Blots were washed again using TBS-T and TBS as previously described.
Finally, enhanced chemiluminescence was performed with ECL” reagents

(Amersham Biosciences, Piscataway, NJ) to visualize the bands.

[7. Calcium Channel Westem Blotting Protocol

Lysate from calcium channel protein isolation (4:1 in denaturing loading
buffer) was incubated for 15 minutes in 37 °C water-bath and then loaded onto a
4-15% Tris-HCI gradient ready gel (Biorad, Hercules, CA). Proteins were

electrically separated on the gel (running conditions, 100 V- 180 V) and then

   

,A
._‘
/

 

Du
DH:

Afte

transferred (Transfer Buffer containing 10 % SDS; transfer conditions, 80 V,
constant voltage, 1 and 1/2 hours, 4 °C) to an lmmobilon-P membrane. The blot
was blocked overnight, 4 °C, in 10 °/o non-fat dry milk in TBS-T. Primary antibody
for the a1c subunit of the L-type calcium channel in 5 °/o non-fat dry milk in TBS-T
(1:1000; Alomone Labs, Jerusalem, Israel) was added and incubated overnight
at 4 °C. Blots were then washed, incubated in secondary [1:2000 Anti-rabbit lgG
HRP-linked (Cell Signaling, Beverly, MA)] at 4° C, washed again in TBS-T and

TBS and finally developed as stated above.

3. lmmgnsprscipitstion

Equal amounts of total protein from sham and DOCA-salt rat aortic Pl3-
kinase lysates were added to dolphin-nosed eppendorf tubes. Antibody (5 pl)
(p85a or p1108) and 1 ml PIS-kinase lysis buffer was added to the tubes and
placed on a rocker in the cold room overnight. The next morning, protein A
Agarose beads (70 pl, lnvitrogen, Carlsbad, CA) were added and the samples
were placed in the cold room and tumbled for 2-3 hours in order to attach the
protein bound to the antibody to the beads. AftenNards, the beads were pelleted
and the supernatant discarded (3,000 rpm, 3 minutes, 4 °C). The beads were
washed 2 times with 1 ml of buffers 1, 2 and 3 (Wash buffer 1: 1 % NP-40, 90%
Dulbecco’s Phosphate Buffered Saline (PBS); Wash Buffer 2: 100 mmol/L Tris
pH=7.6, 0.5 mollL LiCl; Wash Buffer 3: 1 mmol/L Tris pH 7.6, 100 mmol/L NaCl).

After the last wash loading buffer (1:1) [200 pl 4:1 denaturing buffer and 800 pl L-

53

Ripa bu
% Tnlor
protein
beads.

ready gr

remover
stated a
(p85o 8
[Wash h
moi/l Lir
With eac
DhOSpha
(lg-hydro
EGTA,

tempera.
Umol/L A
temperar
phospho

Wore Cer

Ripa buffer (50 mmol/L Tris-HCI, pH 7.5, 150 mmol/L NaCl 2 mmol/L EGTA, 0.1
% Triton X-100)] was added to the beads and boiled for 5 minutes to remove the
protein bound to the beads. The lysate was centrifuged (1 minute) to pellet the
beads. Lysate was loaded onto a 7% SDS gel or a 4-15% Tris-HCI gradient

ready gel and Western analysis was performed.

4. El3-kinsss Agnivity Asssy

This protocol was adapted from Kido et al (2000). Rat thoracic aorta was
removed from the animal and placed in PSS, cleaned and protein isolated as
stated above. The sample was immunoprecipitated with a PIS-kinase antibody
(p85a SH2 domain specific or p1105). The immuno complexes were washed
[Wash buffer 1: 1% NP-40, PBS; Wash buffer 2: 10 mmol/L Tris (pH 7.6), 0.5
mol/L LiCl; Wash buffer 3: 1 mmol/L Tris (pH 7.6), 100 mmollL NaCl] two times
with each buffer. The samples were resuspended in 20 pl of sonicated bovine
phosphatidylinositol (PI) (20 pg/sample; Sigma Chemical Co.) with 200 mmol/L 4-
(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (pH 7.1), 4 mmollL
EGTA, and 0.5 mmol/L sodium monophosphate for 6 minutes at room
temperature. The phosphorylation reaction began with the addition of 10 pl 250
pmol/L ATP containing 5 pCi of [WP] ATP and incubated for 6 minutes at room
temperature. The reaction was stopped by the addition of 15 pl of 4N HCI. The
phospholipids were extracted with 130 pl of CHCIalmethanol (1:1). Samples

were centrifuged 14,000 rpm, 1 minute, room temperature to separate the

54

sanrlel
Nesowri
CHCL3)
Whalma
polassiu
overnign
conespc
Persona

NIH imag

Connech
COmPOUl
Cryostat
With cold
around .
Burlingan
at room 1
blocked k
Vectastair

Laboralon

sample layers. The CHCI3 layer (35 pl) containing the product was then resolved
(resolving solvent: 49 ml CHSOH, 11.3 ml H20, 2 ml NH4OH, and lastly add 60 ml
CHCLS) on thin layer chromatography (TLC) plates (K6 Silica Gel 60 A;
Whatman). The samples were run on a pretreated TLC plate [(0.5 mM EDTA;
potassium oxalate, 1.95 mg/ 90 ml H20 and 40 % methanol) for 5 hours (or
overnight) and baked for 5 minutes at 95 °C] for 1 hour. Radiolabeled spots that
correspond with the PIS-monophosphate Pl(3)P were quantified using Bio-Rad”

Personal Molecular lmager FX system, Quantity One Bio-Bad ‘3’ Software and

NIH imaging Version 1.61 software.

5. lmmunshistoshsmistty

Aortic rings from sham and DOCA-salt rats were cleaned of fat and
connective tissue, placed in Tissue TekCryomold filled with Tissue-Tek O.C.T.
Compound and frozen on dry ice. Sections (8 pm) of aorta were cut on Bright
Cryostat Model OTF (Hacker Instruments, Inc, Fairfield, NJ). Slides were fixed
with cold acetone for ten minutes. After the slide dried a double circle was drawn
around the sections with an lmmEdge pen (Vector Laboratories, Inc.,
Burlingame, CA). Sections were incubated for 30 minutes in 0.3 % H202 in PBS
at room temperature and then rinsed 2 times in PBS. Aortic sections were
blocked for 1 hour at room temperature with 1.5% blocking serum in PBS from a
VSCtastain ABC (Avidin: Biotinylated enzyme Complex) kit, rabbit specific (Vector

Laberatories), p1106 primary (1 pg/ml in blocking serum) was added directly to

55

the sections
following de
secondary a
lollowed bi
humidified
and staine
QMmmr
Gill‘s For
(Vectamc
Photogra

Spot Ver

F. Data

D

0i anim;
Percent;
maximur
amrmi
in Prism!
'09) of m,
Utilizing th‘

quantified L

the sections and incubated overnight at 2 — 8 °C in a humidified chamber. The
following day, the slides were rinsed 3 times with PBS and then incubated with
secondary antibody for 30 minutes in a humidified chamber, rinsed again in PBS,
followed by a 30 minute incubation in ABC reagent at room temperature in a
humidified chamber. Sections were washed for a final time with PBS (3 times)
and stained with Peroxidase Substrate Kit DAB (3,3’-diaminobenzidine; Vector
Laboratories) for 2 minutes. Finally Vector Hematoxylin Nuclear Counterstain,
Gill’s Formula (Vector Laboratories) was performed. Slides were mounted
(Vectamount Mounting Medium) and viewed under a microscope for staining.
Photographs were take using a Spot Camera (Diagnostic Instruments, Inc.) and

Spot Version 3.3.2 for Mac OS software.

F. Data Analysis and Statistics

Data are presented as means 1: standard error of the mean for the number
of animals in parentheses. Contraction is reported as force (milligrams), as a
percentage of response to maximum contraction to PE, or as a percentage of
maximum contraction. EC50 values (agonist concentration necessary to produce
a half-maximal response) were determined using non-linear regression analysis
in Prism" version 3.0 and are reported as the mean of the negative logarithm (-
log) of the E050 value. Band density from Western analysis was determined
utilizing the NIH imaging Version 1.61 software. Pl(3)P radiolabeled areas were

quantified using the program Bio-Flad‘D Quantity One and NIH Imaging Software.

56

When com;
mdide co
analysis (l
pedomed

than or eqr

When comparing two groups, the appropriate Student’s t-test was used. For
multiple comparisons, an ANOVA followed by Least Significant Difference
analysis (LSD) and Student-Newman-Keul’s (SNK) post hoc tests were
performed using SAS version 8.2 statistical sofftware. In all cases, a p value less

than or equal to 0.05 was considered statistically significant.

57

A. Subhyp

in aorta

Th roug
hyoenens
the anima
sham rats

as repres

15.2

Sr
DOCA-s,-
lhis is or
kinase in
DOCA~se
sDonianer
(DOCA-3a
LY294002,
These dat.
dove/omen

suggeSted [)

RESULTS

A. Subhypothesis #1: Fla-kinase proteln and/or its activity are upregulated

in aorta of DOCA-salt hypertensive rats.

Throughout all experiments the systolic blood pressures (SBP) of the
hypertensive DOCA-salt rats (180 a: 3 mm Hg; N = 50, a representative sample of
the animals used) were significantly higher than the SBPs of the normotensive
sham rats (115 :l: 2 mm Hg; N = 50, a representative sample of the animals used)

as represented in Figure 6.

1- Wm

Spontaneous tone developed in endothelium-denuded aorta isolated from
DOCA-salt (Figure 7A; 2"d tracing) but not in sham rats (Figure 7A; 13‘ tracing and
this is quantified in 7B). LY294002 (20 pmol/L), a specific and reversible Pl3-
kinase inhibitor, significantly reduced spontaneous tone (Figure 8) in aorta from
DOCA-salt and not sham rats. Similarly, LY294002 (20 pmol/L) inhibited
spontaneous tone in aortic strips with the endothelium-intact from DOCA-salt rats
(DOCA-salt vehicle, +1.6:1.5% initial PE (10’5 moi/L) contraction vs. DOCA-salt
LY294002, -13.9:l:5.6 1.6::1.5% initial PE (10'5 mollL) contraction). LY294002
These data suggested that Pl3-kinase was a major contributor to the
development of spontaneous tone in the aorta of the DOCA-salt rat. It further

/suggested that there were differences in arterial Pl3-kinase, whether it is

58

 

Figure 6. Systolic blood pressures for a sampling of Day 28 Sham (n=50) and
Deoxycorticosterone Acetate (DOCA)-salt rats (n=50). Columns represent the
mean value, where as the vertical lines represent the standard error of the mean.

* Statistically significant difference (p<0.05) between sham and DOCA-salt

treatment groups.

59

Systolic Blood PresstJre
(mm HO)

 

N=50

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Figure 7. A: Representative tracing of spontaneous arterial tone in endothelium-
denuded aorta from DOCA-salt and sham rats. Tissues are under passsive
tension for optimal force production. B: Basal spontaneous tone differences in
aorta between sham and DOCA-salt rats. Data are presented as a percentage of
initial phenylephrine (PE) (10'5 moi/L) contraction and there was no difference in
this initial contraction between aortic strips from DOCA-salt and sham rats
(1475.8:675 mg vs. 1394.2:393 mg). Bars represent the mean spontaneous
tone development: SEM. * Statistically significant difference (p<0.05) between

sham and DOCA-salt treatment groups.

61

 

A

200 mol 2 min

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62

Figure 8. A: Representative tracing of spontaneous arterial tone in endothelium-
denuded aorta from DOCA-salt and sham rats. Tissues are under passsive
tension for optimal force production, vehicle (0.1% DMSO) or LY294002 (20
pmoI/L) was added and allowed to equilibrate for 1 hour. B: Effect of PIS-kinase
inhibitor LY294002 on spontaneous tone in endothelium-denuded rat aorta from
DOCA-salt and sham rats. Bars represent the mean change (A) in spontaneous
tone development in the presence of vehicle and LY294002: SEM. * Statistically

significant difference (p<0.05) between sham and DOCA-salt treatment groups.

63

Tnnninn Imm‘

h
8 am Vehicle

‘ - - A ‘ - - - - -—-- ‘ 4%!
w ' v—wv

W}

Sham LY294002 (20 pmollL) = A

 

 

 

 

    

Tension (mg)

 

LY294002 (20 pmoilL) :l'

 

DOCA
T Vehicle/LY294002 200 mg l 10 min

 

 

B 100

Contraction]
01 \l
<.= a”

N
3‘

Total A In Contraction
[% Initial PE (10'5 moi/L)

 

 

 

 

in amour
normotens
from shan
turther in
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agonist a
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t0 eplde
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“NaSe,
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by the I

in amount of PIS-kinase or increased activity of Pl3-kinase, between
normotensive and hypertensive rats. LY294002 had little to no effect on aorta
from sham rats, whereas it eliminated the spontaneous tone in the DOCA-salt rat
further implicating Pl3-kinase being altered in the condition of hypertension.
Increasing concentrations of LY294002 (10‘7- 3x10'4 moi/L) were added to aortic
strips in the tissue baths from DOCA-salt and sham rats in the absence of
agonist and spontaneous tone was monitored to ensure that the concentration
utilized in the experiments (20 pmol/L) was appropriate. LY294002 reduced
spontaneous tone in a concentration-dependent manner (Figure 9) in the aortic
strips from the DOCA-salt rats. The effect that LY294002 had on spontaneous
tone was reversible in all experiments, as spontaneous tone was restored upon
removal of LY294002 and return of PSS.

To determine biochemically that LY294002 had the ability to inhibit PI3-
kinase, Western analyses on vascular smooth muscle cells were performed. Rat
aortic primary smooth muscle cells, grown from rat aorta expiants, were exposed
to epidermal growth factor (EGF) in the presence or absence of LY294002 (20
pmol/L). Akt, a protein known to be downstream and phosphorylated by PI3-
kinase, and pAkt (the phosphorylated form) were examined. Figure 10
demonstrated that EGF induced an increase in Pl3-kinase activity as represented
by the increase in the pAkt protein levels. However, in the presence of

LY294002 this phosphorylation is eliminated, thus demonstrating that LY294002

65

Figure 9. A: Representative tracing of a LY294002 concentration response
curve (10‘7 to 3x10”1 moi/L) in endothelium-denuded aorta from DOCA-salt
hypertensive and sham normotensive rats. B: The effect of increasing
concentrations of LY294002 or vehicle (DMSO) on spontaneous tone in aorta
from DOCA-salt and sham rats. Data are presented as a percentage of the initial
phenylephrine (PE) (10'5 moi/L) contraction. Points represent means :l: SEM.

*p<0.05

66

Percentage PE

(1n'5 mnl/l \ n_._4__._ .~

 

 

A

 

 

 

 

A Sham 200 mQ'TS—min
O)
éL—————————
C
.9
2 DOCA
,9
-7 -6.5 -6 -5.5 -5 -4.5 -4 -3.5
log LY294002 (moi/L)
B
+ DOCA Vehicle
8 + DOCA LY294002
m =3 -I- Sham Vehicle
0. g +Sham LY294002
(D
38
c A
g g -20- * *
33 f
b -40-
'60 I l

 

 

 

-7 -6 -'5 it -3

Log LY294002
(mo l/L)

67

Figure 10. Western blot of Akt and pAkt proteins. pAkt is reflective of Pl3-
kinase activity, of rat aortic smooth muscle cells exposed to vehicle, LY294002

(20 pmol/L), and LY303511 (20 pmol/L) reel: (10 nmol/L).

68

Rat Aortic Smooth Muscle Cells

l3Akt +5. - ‘ .. *
Akt +M
EGF (10 nmol/L) - + I - + I - + I

 

LY294002 LY30351 1
(20 pmol/L) (20 pmol/L)

69

dor

ina
exa

LY:

(Fir
exp
LY3
a CC

aOii.

LYSI
inht
dope
dem
conc
lene
mUsc

mhihl

does function to inhibit Pl3-kinase activity. Total Akt levels were similar in all
samples.

To further ensure that LY294002 was acting selectively, l utilized an
inactive analog of LY294002 in out experiments, LY303511, as well as chose to
examine another PIS-kinase inhibitor, wortmannin. In contrast to LY294002,
LY303511 did not inhibit epidermal growth factor (EGF)-induced phosphorylation
of Akt, one of the substrates of Pl3-kinase, in cultured aortic smooth muscle cells
(Figure 10); total Akt protein density was similar in all samples. This was to be
expected if LY303511 was indeed an inactive analog of LY294002. Similarly,
LY303511 (20 pmol/L) failed to inhibit spontaneous tone (Figure 11), but caused
a contraction in the aorta from DOCA-salt rats without altering tone of sham
aorta. The cause of the contraction is unknown at this time. Due to the focus of
the studies being on Pl3-kinase, I chose not to further pursue the cause of the
LY303511-induced contraction in the aorta further. Wortmannin, another known
inhibitor of PI3-kinase, also inhibited spontaneous tone in a concentration-
dependent manner (Figure 12), similar to that observed with LY294002 further
demonstrating PI3-kinases involvement and potential up-regulation in the
condition of hypertension. I chose to use the Pia-kinase inhibitor LY294002 in
remaining studies due to the fact that wortmannin is also known to inhibit smooth
muscle myosin light chain kinase (MLCK), thus making data generated using this

inhibitor difficult to interpret (Davies et al, 2000).

70

Figure 11. Representative tracing of spontaneous arterial tone in endothelium-
denuded aorta from DOCA-salt and sham rats. Tissues are under passsive

tension for optimal force production, vehicle (0.1% DMSO) or LY303511 (20

pmoI/L) was added and allowed to equilibrate for 1 hour.

71

Vehicle

 

 

 

 

 
 
 

 

 

 

 

 

Sham
- - "“j—n— —
A D O C A Vehicle
U)
E
C
.5?) LY303511 (20 pmoi/L)
C
0)
F—
DOCA LY303511 (20 pmol/L)
A -_—_—
Vehicle/LY303511 800 mg I10 min

72

Figure 12. A: Representative tracing of a Wortmannin concentration response
curve (10'8 to 3x104 moi/L) in endothelium-denuded aorta from DOCA-salt
hypertensive and sham normotensive rats. B: The effect of increasing
concentrations of Wortmannin or vehicle (DMSO) on spontaneous tone in aorta
from DOCA-salt and sham rats. Data are presented as a percentage of the initial
phenylephrine (PE) (10'5 moi/L) contraction. Points represent means :1: SEM. *
Statistically significant difference (p<0.05) between DOCA-salt Vehicle and

DOCA-salt Wortmannin treatment groups.

73

Percentage PE
(10'5 moi/L) Contraction

>

U)
I:
m
3

DOCA

Tension (mg)

 

r 7 r r r T—r—r—
|-8 -7.5 -7 -6.5 -6 -5.5 -5 -4.5 -4 -3.5|
log Wortmannin (moVL)

 

 

+ DOCA Vehicle
+ DOCA Wortmannin
-I— Sham Vehicle

+ Sham Wortmannin

 

 

 

 

 

 

 

-'8 -'7 is is «r -3
Log Wortmannin
(moVL)

74

salt
13l
aor
2.8
p85
DO
wee
resr
the

FIS-
Pl3-
DI 1r
her
as c

the

of P
DPD
'ysal

(Flgu

2. Pia-kinase Bioshemistgr

Having established a functional change in PIS-kinase in aorta from DOCA-
salt hypertensive rats, I proceeded to measure PIS-kinase activity. As Figure
13A reveals, p85a associated PIS-kinase activity was significantly greater in
aorta from DOCA-salt rats as compared to sham (208% of sham; 5.71:0.9 vs.
2810.9 adjusted volume optical density x mmz, respectively). Equal amounts of
p850: protein were present in the immunoprecipitated aortic homogenates from
DOCA-salt and sham rats (Figure 13B) and this was confirmed with standard
western analyses (Figure 130). In order to determine which subunits were
responsible for this increase in Pl3—kinase activity, I used immunoprecipitation for
the p850: to determine what catalytic subunits were co-immunoprecipitated. This
Pi3-kinase subunit interaction (regulatory and catalytic subunit) is required for
PI3-kinase to be active. Figure 14 illustrates that p85a interacted with the
p110a, p1108 and possibly the p110[3 subunits. Moreover, it also appears that
there is more p1108 attached to p85a in the aortic iysates from the DOCA-salt rat
as compared to the sham, suggesting that p1108 may have an important role in
the increase in Pl3-kinase activity that is observed.

To examine how the increase in activity may affect proteins downstream
of Pl3-kinase, I measured PDK, the phosphorylated and active form of PDK,
pPDK, Akt and phosphorylated and active form of Akt, pAkt protein in aortic
lysates from DOCA-salt and sham rats. Similar protein expression of PDK, pPDK

(Figures 15 and 16) and Akt were found in the aorta from DOCA-salt and sham

75

Figure 13. A: p85a-associated PIS-kinase activity in aorta from hypertensive
DOCA-salt and normotensive sham rats. Pl(3)P was detected using thin layer
chromatography and quantified with Bio-Rad software. Bars represent mean
adjusted volume optical density x mm2 :l: SEM. B: lmmunoprecipitation of p850:
from aorta from hypertensive DOCA-salt and normotensive sham rats to confirm
equal amounts of protein loaded for Pl3-kinase assay. C: Western analyses of
p85a of aortic protein from DOCA-salt and sham rats. Bars represent mean
arbitrary densitometry units :1: SEM. * Statistically significant difference (p<0.05)

between sham and DOCA-salt treatment groups.

76

A

Pwswt—.»

Odgm-—>»

IP: p850
C . .
Sham DOCA

p85a_> — —

DOCA

Sham DOCA

lB:p85a

Sham

IP: p850: lB: p850c

Adjusted volume

 

 

 

 

 

 

 

 

 

 

 

 

 

 

7 i-
(E 6 ”=8 __F—
x 5 W '1
g 4
g 3
o 2
E 1 .,_
a o
O Sham DOCA
9 2 N=4
.E fi—
3 15
2‘
E 100
'é 500
<
0
Sham DOCA
3 5000 N=‘r “l—
5 4000
2‘
9 3000
E 2000
< 1000
G Sham DOCA

77

 

Figure 14. lmmunoprecipitation (IP) with p85a antibody of aortic lysates from

hypertensive DOCA-salt and normotensive sham rats to examine which p110

subunits had the ability to interact with the p850r PIS-kinase subunit. Blots were
immunoblotted (iB) with antibodies against: A: p850r, B: p1 10a, C: p1108, D:

p11OB and E: p1107.

78

IP:p85a

 

IX
13: p850. cull-nu gun.“
Sham DOCA
[3
IB: p1100r """"". 7’"
Sham DOCA
(3
IB: p1108 _
Sham #56575:
I) _
IB: D1103 .- iii;
Sham 066A
E.
IB: p110'y
Sham DOCA

79

Figure 15. Western analyses of PDK and pPDK protein in aorta from
hypertensive DOCA-salt and normotensive sham rats. Bars represent mean

arbitrary densitometry units :t: SEM.

80

 

 

pPDK

 

 

 

 

 

 

PDK

 

 

 

 

1400-
1200-

.EED

 

600-
400‘
200-

G
nu
nu
1

252

m

IDEH(‘4”";;i; Gunny
fiF"!

pPDK->

81

Figure 16. Western analyses of Akt and pAkt protein in aorta from hypertensive
DOCA-salt and normotensive sham rats. Bars represent mean arbitrary
densitometry units: SEM. * Statistically significant difference (p<0.05) between

sham pAkt and DOCA-salt pAkt treatment group.

82

75001

 

N=12

 

 

 

 

 

 

 

g 6000-
'E
Akt—> --r 9—- :Z" 4000-
cu
pAkt—V - m 1% 3000—
Sham DOCA < 1500-
0

83

Sham DOCA

 

Akt

 

Sham

 

pAkt

rats. But, there was significantly lower pAkt protein density in aortic Iysate from
the DOCA-salt rats as compared to the sham (Figure 16). These data suggested
the enhanced PIS-kinase activity was probably not leading to enhanced
phosphorylation of Akt but, being possibly being funneled to an alternative
effector, possibly L-type calcium channels.

A profile of p110 subunits was next performed using western analyses.
The Class IA catalytic subunits p110a, p110(3 and p1108 but not p1107 were
present in the aorta of both DOCA-salt and sham rats, with p1105 being
significantly higher in the aorta from DOCA-salt as compared to sham rats
(p<0.05) (Figure 17). Confirmation of the specificity of the antibodies was
determined by examining a positive control for each antibody (for p1108, U-87
MG cells; for p110a, Jurkat cells; for p110B, K-562 cells; for p110y, U927 cells).
These data suggest that the increase in p85a associated PIS-kinase activity may
be due to the increase in the Class IA Pl3-kinase p1108 subunit, since it is the
only Pl3-kinase subunit that exhibited greater protein density in the aorta from
DOCA-salt as compared to sham rats.

To further investigate the Class IA Pl3-kinase p1106 subunits involvement
in the enhanced PIS-kinase activity observed in aorta of DOCA-salt rats, Pl3-
kinase activity assays were performed by immunoprecipitating for p1106 and
then measuring p1108-associated activity. Figure 18 reveals a significant
increase in p1106 associated PIS-kinase activity in the aorta from the DOCA-salt

rat as compared to the sham (158% of sham). p1105 appears to account for

84

Figure 17. Western analyses of protein isolated from aorta from DOCA-salt and
sham rats with antibodies for, A: p1105 (control=U-87 MG cells), B: p110a
(control: Jurkat cells), C: p11OB (control=K-562 cells), and D: p110y
(control=U937 cells). Bars represent mean arbitrary densitometry units a; SEM. *
Statistically significant difference (p<0.05) between sham and DOCA-salt

treatment groups.

85

i-saliand

5 1 SEM. '

alt

A

p1108—>

 

Sham DOCA U-87MG
Cells

p110a—h» _ c. -1

Sham DOCA Jurkat
Cells

 

w
c a
,1... ---- r I s
* 'é
Sham IOCA K-562 <

Cells

D

Sham DOCA U937

Cells

86

 

 

 

 

 

 

 

 

 

Sham DOC *

 

 

 

 

0 Sham DOCA

Figure 18. p1108-associated Pl3-kinase activity in aorta from hypertensive
DOCA-salt and normotensive sham rats. Pl(3)P was detected using thin layer
chromatography and quantified with NIH imaging software. Bars represent mean
arbitrary units :1: SEM. * Statistically significant difference (p<0.05) between sham

and DOCA-salt treatment groups.

87

nslve
lln laye!
agent met“

ween 5W

lP:p1108

 

 

 

 

 

4000*
:2 N:
5 3000-
2‘
$3 2000+ l
”1::
< 1000~
Sham
. a

Sham DOCA Sham DOCA

88

almost all of the enhanced Pia-kinase activity observed in the aorta from the
DOCA-salt rat. Further immunoprecipitation demonstrated that the p1106
antibody only recognized p1106 and not other p110 PIS-kinase subunits (Figure
19). p1108 had previously been hypothesized to solely be expressed in
hematopoietic cells. To ensure that the p1108 was in the vascular smooth
muscle, I performed immunohistochemical studies using the p1106 antibody on
aorta isolated from sham and DOCA-salt rats to determine where the subunit
was located, this is the same antibody used for PI3-kinase activity assays and
Western analyses. As revealed in Figure 20 there is p1106 specific staining in
the smooth muscle cell region (arrows) in the aortas of both the sham and
DOCA-salt rats (n=4). The aorta from the DOCA-salt rat had more intense
staining than that of the sham, supporting the Western protein data
demonstrating higher p1106 protein levels in the aorta from the DOCA-salt rat
(Figure 17A). These data support the hypothesis that PIS-kinase is upregulated
in terms of protein content as well as activity in the aorta of the DOCA-salt rat.
Thus I hypothesized that this increased activity leads to enhanced contractility in

the aorta from hypertensive DOCA-salt rats.

89

Figure 19. lmmunoprecipitation (IP) with p1106 antibody of aortic lysates from
hypertensive DOCA-salt and normotensive sham rats to examine if any of the
other p110 subunits could react to the p1106 antibody. Blots were
immunoblotted (IB) with antibodies against: A: p1108, 8: p1100c, C: p11OB and D:
p1107. Only aortic samples immunoblotted for p1106 showed positive staining

for the antibody, suggesting specificity for the p1108 antibody.

90

|P:p1108

l\
lB:p1108
Sham
fCfl
we B
lB: p1 100:
Sham
ancD
térg C
IB: p1 1013
Sham
[J
IBJH1OY
Sham

91

DOCA

Um r... .~.; .

DOCA Aorta

DOCA K-562
Cells

 
 

DOCA 0937
Cells

Figure 20. Representative pictures from immunohistochemical studies of
thoracic aortas (RA) from hypettensive DOCA-salt and normotensive sham rats.
8 pm sections of aorta were probed with no primary antibody (top left and bottom
left) or 1 ug/ml of p1108 antibody (top right and bottom right). The arrows
indicate the staining in the smooth muscle cell region of the section of those with

primary. Note those with no primary have little to no staining.

92

 

    
  
   

Sham RA
p1 105

DOCA RA

93

B- Subhypothesis #2: Pia-kinase and L-type voltage gated Ca“ channel
interaction is altered in the aorta of the DOCA-salt rat model of

hypertension.

The role of Caz“ in mediating arterial spontaneous tone has been
previously established. Nifedipine, an L-type Ca2+ blocker, inhibited spontaneous
tone in a manner similar to LY294002 (compare Figures 8A, 88 and 21). This
concentration of nifedipine was the minimum concentration that maximally
inhibited KCI-induced contraction in aorta (Florian and Watts, 1998). Diltiazem,
another L-type calcium channel inhibitor, was used to ensure that by inhibiting
the calcium channel I was not also blocking PIS-kinase. Figure 22 demonstrates
When rat aortic smooth muscle cells were activated with EGF (10 nmol/L), Akt,
Which is downstream of PIS-kinase, was phosphorylated and diltiazem did not
alter this activation. By contrast, LY294002 completely eliminated the
phosphorylation of Akt. To determine whether LY294002 could directly inhibit L-
tyne Ca2+ channels and thereby spontaneous tone, I examined the effects of

L\(294002 (20 pmol/L) on BayK8644-induced contraction; BayK8644 is a direct
L‘type channel agonist. In agreement with other investigators (Storm at a/.,
1 990; Watts at at, 1994), I observed an enhanced contraction to BayK8644 in
aOrta from the DOCA-salt rat. LY294002 (20 pmol/L) did not alter BayK8644-
inciuced contraction (Figure 23), indicating that LY294002 was not acting directly

to inhibit L-type Ca2+ channels. Finally, to link Ca2+, PIS-kinase and isolated

94

Figure 21. Representative tracing of spontaneous arterial tone in endothelium-
denuded aorta from DOCA-salt hypertensive and sham normotensive rats.
Tissues were under a tension for optimal force production, vehicle (ethanol) or

nifedipine (50 nmol/L) was added and allowed to equilibrate for 1 hour.

95

 

 

 

 

 

Vehicle 400 mg 10 min
Sham
- I.
Vehicle
DOCA W
Nifedipine (50 nmol/L)
Sham

 

 

 

Nifedipine (50 nmol/L)

 
 
 

DOCA

 

T“ “ ‘ “i“
-—

Vehicle/Nifedipine

96

Figure 22. Western blot examining pAkt, an indirect measurement of PIS-kinase
activity, of rat aortic smooth muscle cells exposed to vehicle, EGF (10 nmol/L),

Diltiazem (1 umol/L):1:EGF, LY294002 (20 umol/L) 1561:.

97

ll

Rat Aortic Smooth___ Muscle Cells

W’lkt —>, a»..- - __
EGF(10nmoI/L) - + - + - + - +

 

   

 

 

Diltiazem LY294002
(1 pmol/L) (20 pmol/L)

98

Figure 23. The effect of L-type voltage gated Ca2+ channel agonist BayK8644
and PIS-kinase antagonist LY294002 (20 umol/L) in endothelium-denuded rat
aorta from DOCA-salt and sham rats. LY294002 or vehicle was added to the
tissues 1 hour prior to cumulative addition of BayK8644. Data are presented as a
percentage of the initial phenylephrine (PE) (10'5 moVL) contraction. Points

represent means :1: SEM.

99

Percentage PE
(10'5 mol/L) Contraction

 

125

  
  
 

 

 

   

 

N= + DOCA Vehicle
100_ + DOCA LY 294002
-I— Sham Vehicle
75- +Sham LY294002
50-
25-
0 u
-10 -9 -8 -7 -6 -5
Log BayK8644
(mo VL)

100

tissue, all Caz” was removed from the isolated tissue bath and added back in
increasing concentrations in the presence of LY294002 (20 pmol/L) or vehicle
(0.1 % DMSO) and spontaneous tone development was monitored. LY294002
completely inhibited the development of spontaneous tone in aorta from DOCA-
salt rats, whereas vehicle-incubated aorta from hypertensive DOCA-salt rats
developed calcium-dependent spontaneous tone (Figure 24). Aorta from sham
rats did not develop spontaneous tone. Thus, these data demonstrate that PI3-
kinase utilizes extracellular Ca2+ to mediate enhanced spontaneous tone that is
observed in aorta from DOCA-salt rats.

Molero at al. (2001) have demonstrated an increase in L-type calcium
channel expression in membrane protein in small mesenteric arteries from
DOCA-salt rat as compared to the sham rats. This in combination with the
enhanced contractility to BayK8644 observed in aorta from DOCA-salt rats
(Figure 23) may explain the increase in spontaneous tone and suggest that the
enhanced contractility is due to increased levels of L-type calcium channels.
However, Western studies using aortic protein isolated from DOCA-salt and
sham rats demonstrated there was no significant difference between sham and
DOCA-salt rats in the 011C L-type calcium channel subunit (Figure 25). Thus,
these data demonstrate that it is not an increase in L-type Ca2+ channel protein
that is leading to the enhancement in spontaneous tone observed in aorta from

DOCA-salt rats. However, at the present time, it is unclear whether a change in

101

Figure 24. The effect of Pia-kinase antagonist LY294002 on Ca2*-induced
spontaneous tone in endothelium-denuded rat aorta from DOCA-salt and sham
rats. LY294002 (2O pmol/L) or vehicle (0.1% DMSO) was added to the tissues 1
hour prior to cumulative addition of calcium chloride. Data are presented as a
percentage of the initial phenylephrine (PE) (10'5 mol/L) contraction. Points
represent means :1: SEM. * Statistically significant difference (p<0.05) between

DOCA-salt vehicle and DOCA-salt LY294002 treatment groups.

102

 

100

 

 

N=4 + Doca Vehicle
5 + Doca LY294002
LIJ g 75. —l—Sham Vehicle
0. g * +Sham LY294002
‘1’ 8
.30 50-
5 Q
:6 *
(LE 25-
'0
Ca * 'k
0.
-7 -6 -5 -4 -3 -2
Log Calcium

Chloride (moVL)

103

Figure 25. Western analyses of protein isolated from aorta from DOCA-salt and
sham rats with antibodies for the 0:10 calcium channel subunit. Bars represent

mean arbitrary densitometry units :i: SEM.

104

 

 

 

 

 

 

40°01 N=7-8

331’ 3000- 1
C

D .

E? 2000-

9 ..

15:3 1000-

< d

0
Sham

 

Sham DOCA

105

Ca2+ channel activity, PIS-kinase, and/or a combination of both, is sufficient to

enable the development of arterial spontaneous tone.

C. Subhypothesis #3: PTEN (phosphatase and tensin homolog) is localized
in vascular smooth muscle cells and is down-regulated in aorta of the

DOCA-salt rat.

I next examined aortic homogenates for the presence of PTEN and its
inactive form, pPTEN. PTEN and pPTEN were both present in the thoracic aorta
of both DOCA-salt and sham rats (Figure 26) but the density of these proteins
was not different between the aorta of DOCA-salt and sham rats. Appropriate
positive controls were used to confirm antibody specificity. To my knowledge,

this was the first time that PTEN and pPTEN were localized to the arterial tissue.

D. Subhypothesis #4: Norepinephrine (NE) and magnesium (Mg’*) utilize
the PIS-kinase signaling cascade to elicit enhanced vascular contraction in

hypertension.

1. Norepinephrine and Pia-kinase

I examined the effect of LY294002 on NE-induced contraction, a
Contraction that is enhanced in DOCA-salt hypertension (Figure 27). LY294002

Shifted the NE-induced aortic contraction of sham and the DOCA-salt rats

106

Figure 26. Western analyses of PTEN and pPTEN in aorta from DOCA-salt
hypertensive and sham normotensive rats. Bars represent mean arbitrary

densitometry units a; SEM.

107

6000 1 N
5000 -
4000 1
3000 ‘
2000 -‘

Arbitrary Units

1000-

 

 

   

         
 

 

 

 

 

‘ , .\
. .
u a
.w I. .*"x"~
_ 1.3
. ”I,“

 
 

‘ .

 

 

 

 

Sham DO A
PTEN pPTEN

 

 

PTEN—> "'""" W
Sham DOCA

Sham DOCA

108

Figure 27. The effect of NE and PIS-kinase antagonist LY294002 (20 pmol/L) in
A: endothelium-denuded (-E) and B: endothelium-intact (+E) aorta from DOCA-
salt and sham rats. LY294002 or vehicle was added to the tissues 1 hour prior to
cumulative addition of NE. Data are presented as a percentage of the maximal
NE contraction. Points represent means 1 SEM. The values are the -|og EC50 of
the NE-induced contractions in the presence of LY294002. * Statistically
significant difference (p<0.05) between sham Vehicle and DOCA-salt Vehicle

treatment groups.

109

 

  
   
   

 

 

 

 

 

   

 

 

 

A 120 Rat Aorta -E
f N=5 -V— DOCA Vehicle
5 E 100- + DOCA LY294002
‘ g c -I- Sham Vehicle
5 5.3 80- —o- Sham LY294002
2 § 60-
g. s
(20 pmcllii' 2c; (”'3 40‘
e Z ,
f Dot: g 20- N54» LY294002
“om ' Sham: 72:01
I". O- IA.A _= J
MW" -10 -9 -8 -7 -6 -5 -4
mmm)., Log NE (mollL)
’ 1::.
he 409 E09
f B 120 Rat Aorta +E
' SW 'a' N=6 4— DOCA Vehicle
Valli? E 100.. . + DOCA LY294002
A'sa" g c -I-Sham Vehicle
.5 g 30. +Sham LY294002
2 in?
m E 60"
0’ o
9 o 401
E, LIJ
e Z 409 ECso Of
a 20‘ N51» LY294002
Sham: 6.6::0.1
0- W1.
-10 -9 -8 -7 -6 -5 -4

Log NE (mollL)

110

compared to vehicle treated control tissues in both endothelium-denuded (-E)
and endothelium-intact (+E) tissues, supporting a role for Pl3-kinase in NE-
induced contraction. Interestingly, the potency of NE in the presence of
LY294002, in aorta from DOCA-salt and sham rats incubated with LY294002 was
not significantly different {-|og E050 (M) sham-E=7.2:tO.1; DOCA-salt-E=7.2:1:O.1;
sham+E=6.6:O.1; DOCA-salt+E=6.4:O.1] (Figure 27A and 273). Thus, an
increase in Pl3-kinase activity may explain the enhanced arterial contractility to

NE observed in DOCA-salt hypertension.

2. Magnesium and BIS-kinase

Spontaneous tone developed in aorta isolated from DOCA-salt rats
(Figure 28A, 2"d and 4th tracing), with minimal tone development in the aorta from
sham rats (Figure 28A, 1"" and 3'‘1 tracing). Low Mg"’* PSS (0.15 mmol/L) induced
a Significant increase in spontaneous tone in aorta from both DOCA-salt and
Sham rats (Figure 28A and 28B), clearly visible in the aorta from DOCA-salt rats
(compare Figure 28A 2"d and 4"1 tracings). LY294002 (2O umol/L) Significantly
inhibited spontaneous tone in aorta from DOCA-salt rats incubated in both
normal and low Mgz” PSS compared to their respective vehicle controls (Figure
29 and 298). Converse to the increase in tone elicited by low Mg“, aortic strips
incubated in high Mg2+ PSS Showed reduced spontaneous tone with respect to
its vehicle control (Figure 30), albeit not to the same extent as when aortic strips

were incubated in LY294002. LY294002 (20pmol/L) inhibited spontaneous tone

lll

Figure 28. A: Representative tracing of spontaneous tone in endothelium-
denuded aorta from DOCA-salt and sham rats incubated in normal and low Mg“
PSS in isolated tissue baths. Tissues are under passive tension for optimal force
production. 8: Quantification of the effect of low Mg2+ PSS on spontaneous tone
in endothelium-denuded rat aorta from DOCA-salt and sham rats. Bars
represent the mean spontaneous tone development :1: SEM (N=18-28) (* p50.05

vs. Sham, # p_<_0.05 vs. DOCA PSS, 1 p_<_0.05 vs. Sham PSS).

112

lthelium-
and low ”9'
r optimal tar:
ltaneousl'ff
Bars

28) l' W

Sham

200 mg 5 min

- I ,. “nag
" ' '- . «at r of” .u .. -. -. n . n .- _ ..,..... .. w-...u~c-...omu.-.n.n “ml-to l,“ ........u..........a.......a4u.m..' I
.J ' r

F—“l
l Normal l

 

DOCA WWW

 

Sham

 

- ,__ .. . . ... .. W...“..mnmmu~«n-.om Iu-u mun-c -- M .. -‘- A Mr- _. :

,
I

 

DOCA

 

 

1] Low MET ,1

 

Normal PSS

"/0 Initial PE (10'5 mol/L)
Contraction

4

Wash -Low 1192+ /
Normal PSS

Wammflgz*l

Wash-LowMgz‘U
NormalPSS NormalPSS

Spontaneous Tone

 

50

40"“

30a

20—

10—

 

N=18-28

 

 

 

 

 

 

 

 

min“, .
.HUJHI .' .. o
2.7 ”0"“ .r' .‘g
.7. i.
r. .a; ._ 1
v.24 ~

 

 

Sham ocA
PSS Low M92+

 

 

113

Figure 29. A : Effect of LY294002 (20 pmoI/L) on spontaneous tone
development in endothelium-denuded aorta from DOCA-salt and sham rats
incubated in normal PSS and B: Low Mgz*(0.15 mmol/L). Bars represent the
mean spontaneous tone development :l: SEM (*p50.05 vs. Sham, 1' p50.05 vs.

DOCA)

114

Normal PSS

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

A A 50 N=3-28
% 25+ 1,
we 6 or __ [:1 __
33:3 -25‘
g ‘g -50-
:2 0 -75- ‘ ,
5 T_L
\0 -1OG
. ° Sham DOCA Sham DOCA
naneous m Vehicle LY294002 (20 pmol/L)
and shamai
. senif‘i
,repre B A 50 Low Mg‘?+ PSS
n ”(0.05.5 g N=5-28 4—
’ ’ g 25- r
m C
b .9 0. i _——
5 ‘3
i‘ E ’25‘
g 0 -50‘
s -75:
o\°
~100

 

 

 

 

Sham DOCA Sham
Vehicle LY294002 (20 pmoI/L)

 

 

115

Figure 30. Effect of high Mg2+ PSS incubation on spontaneous tone
development in sham and DOCA-salt rats. Bars represent the mean

spontaneous tone development :l: SEM (*p50.05 vs. Sham, T p50.05 vs. DOCA).

116

'ie
an

)5 vs. DOCA}

% Initial PE (10'5 mol/L)

High M92+ PSS

 

Contraction

 

N=7-28

_i:=_EJ__

 

 

 

i__l_

 

Sham DOCA Sham DOCA

 

 

Vehicle High M92+ P88

117

 

 

only a small amount further in aortic strips from DOCA-salt rats when incubated
in high Mg2+ PSS (Figure 31, the dark shaded area). Thus, Pl3-kinase does not
have the ability to elicit spontaneous tone when in the
presence of extracellular high Mg2+ to the same degree as with normal PSS,
possibly due to the high Mg2+ inhibition of calcium channels, thus already
inhibiting calcium dependent tone (Touyz, 2003). The effects of altered Mg2+
and/or LY294002 were reversible, as upon re-equilibration with normal PSS the
tissues returned to normal reactivity.

PIS-kinase activity assays were performed to determine if the increase in
low M92+ ~induced spontaneous tone that was LY294002- and thus likely Pl3-
kinase-dependent was reflected biochemically. Previous studies have
demonstrated that aorta immediately homogenized from DOCA-salt rats have
elevated PIS-kinase activity as compared to sham rats (Figure 11). In aortic
samples exposed to PSS, there was a trend for the increase in PIS-kinase
activity in aorta from DOCA-salt and sham rats incubated in PSS (Figure 32A,
compare shaded bars and Figure 328, compare radiolabeled Pl(3)P in 1“ and 3'6
lanes). When aortic strips were incubated in low Mg2+ there is also a trend for an
increase in Pl3-kinase activity, albeit not statistically significant, with values of
sham low Mg2+ Pl3-kinase activity now similar to that of values for aorta from
DOCA-salt rats incubated in PSS (Figure 32A and 328).

I last examined the effects of altered Mg2+ concentrations on NE-induced

contraction. Low Mgz“ significantly leftward shifted NE-induced contraction in

118

Figure 31. Effect of LY294002 (20 umol/L) on spontaneous tone development in
aorta from DOCA-salt and sham rats incubated in high Mg2+ (4.8 mmoVL) PSS.
The dark shaded area is the effect that LY294002 on high Mgzfiinduced
reduction of tone. Bars represent the mean spontaneous tone development a:

SEM (*p50.05 vs. Sham High M921, 1' p50.05 vs. DOCA Vehicle).

119

% Initial PE (10'5 mol/L)
Contraction

High Mg2+ PSS

 

 

 

 

 

 

 

 

 

 

 

25 N=5-7
0" __ —=—
-25 ___J
h.—
-50— * 1
~75

Sham DOCA Sham DOCA
Vehicle LY294002 (20 umol/L)

 

 

120

 

Figure 32. A: p85a-associated Pl3-kinase activity in aorta from DOCA-salt and
sham rats that were incubated in normal PSS or low Mg?” PSS in isolated tissue
baths. Bars represent mean arbitrary units :1: SEM (N=6-7). 8: Representative
results from Pl3-kinase activity. Spots represent radioactive product
corresponding to PIS-monophosphate from aortic homogenates from sham and
DOCA-salt rats incubated in normal PSS and low Mg2+ PSS in isolated tissue

baths.

121

Arbitrary Units

3000

2000-

1000-

 

6-7 [:3 PSS

B Low M92+

 

 

 

 

 

 

 

 

 

 

 

 

 

 

_. f .l
E.“ 'i“
(hf/1.1
i ' ..-'J r" .3...»
i' {if I. . A
jig-”xi

 

 

 

 

Sham DOCA

a "O I C

PSS Low Mg2+ PSS Low Mg2+
Sham DOCA

 

 

122

aorta from the sham. The NE-induced contraction in aorta from sham rats in low
M9” was similar to that of the DOCA-salt rats incubated in either PSS or low
Mg” (Figure 33A). No further leftward shift occurred when the aorta from DOCA-
salt rats was incubated in low Mg” (Figure 33A). Similar to previous studies in
which LY294002 corrected the enhanced contraction to NE in aorta from DOCA-
salt rats to that observed in sham, LY294002 (20 umol/L) shifted the NE-induced
aortic contraction of both the sham and DOCA-salt rats in the presence of low
Mg” compared to vehicle, resulting in similar potencies (Figure 338). When
comparing the LY294002-mediated inhibition of NE-induced contraction in aorta
from sham and DOCA-salt rats, there was no significant difference between the
aortic strips incubated in normal PSS compared to low Mg” PSS (Figure 33C).
Thus, low Mg” appears to elicit enhanced NE-induced contraction Via PI3-kinase
in aorta from sham rats and not in DOCA-salt rats.

High Mg” appears to have rightward shifted the NE-induced contraction in
the aorta from sham and DOCA-salt rats, however it was not a statistically
significant shift (Figure 34A). LY294002 (20 pmol/L) significantly rightward
shifted the NE-induced contraction in the aorta from both sham and DOCA-salt
rats incubated in high Mg” PSS, again implicating PI3-kinases involvement in
NE-induced contraction (Figure 348). When comparing the effects of LY294002
on NE-induced contraction in aorta in normal PSS compared to high Mg” PSS,

LY294002 significantly further rightward shifted the NE-induced contraction.

123

Figure 33. A: The effect of low Mg” on NE-induced aortic contraction in
endothelium denuded aorta from sham and DOCA-salt rats. B: The effect of the
PIS-kinase inhibitor LY294002 (20 pmol/L) (LY) on NE-induced contraction in
endothelium-denuded aorta from sham and DOCA-salt rats incubated in low
Mg”. C: A comparison of the effects of LY294002 (20 umol/L) on NE-induced
contraction in aorta incubated in low Mg” and normal PSS in isolated tissue
baths. Points represent means :f: SEM (N=4-7). The values are the —log EC50 :1:
SEM of the NE-induced contraction in the presence of normal PSS, low Mg”
and/or LY294002 (*p50.05 vs. Sham PSS, “p_<_0.05 vs. Sham Low Mg”, * p50.05

vs. DOCA Low Mg”).

124

>

120

 

100—
80-
60-
40~

Percentage Maximum
NE Contraction

to
‘.>

 

0

 

 

 

120

 

—L
000
<3?

Percentage Maximum
NE Contraction
o:
O
l

 

 

 

120

 

-1o -9 38 3 is -'5 -4
Log NE (moVL)

 

 

1004

Percentage Maximum 0
NE Contraction

 

N=4-7

 

 

 

-1o -9 -23 -'7 -6 .15 -4
Log NE (moVL)

-I— Sham PSS
"U-Sham Low Mg”
+ DOCA PSS

—V— DOCA Low Mg”

 

-Iog ECso (moi/L)
Sham PSS 7.7101
Sham Low Mg” 8.21:0.1"
DOCA PSS 8.21:0.1 "
DOCA Low Mg” 8.1:0.1"

 

 

 

-1o -9 13 37 is 35 -4
Log NE (moVL)
N=4—7

+ Sham Low Mg”
-D— Sham Low Mg” LY
+ DOCA Low Mg”
—v— DOCA Low Mg” LY

 

-|og E050 (moVL)
Sham Low Mg” LY 7.0::0.1'
DOCA Low Mg” LY 6.81:0.21

 

 

 

+Sham PSS LY
—l:}—Sham Low Mg2+ LY
+DOCA PSS LY

-V— DOCA Low Mg” LY

 

-Iog EC50 (moVL)
Sham PSS LY 7.0101
DOCA PSS LY 7.1:02

 

 

 

Figure 34. A: The effect of high Mg” (Hi) on NE-induced aortic contraction in
endothelium denuded aorta from sham and DOCA-salt rats. 8: The effect of the
PIS-kinase inhibitor LY294002 (20 pmol/L) (LY) on NE-induced contraction in
endothelium-denuded aorta from sham and DOCA-salt rats incubated in high
Mg”. C: A comparison of the effects of LY294002 (20 pmol/L) on NE-induced
contraction in aorta incubated in high Mg” and normal PSS in isolated tissue
baths. Points represent means :1: SEM (N=4-7). The values are the -log EC50 1:
SEM of the NE-induced contraction in the presence of normal PSS, high Mg”
and/or LY294002 (*p50.05 vs. Sham PSS, “p_<_0.05 vs. Sham High Mg”, 1 p_<_0.05

vs. DOCA High Mg”, * p50.05 vs. Sham LY294002 or DOCA LY294002).

126

>

120

 

Percentage Maximum
NE Contraction

A
#QCDO
a???

N=4-6

 

 

 

 

-9.5 -8.5 -7.5 3.5 35 -4'.5

Log NE (moVL)

 

Percentage Maximum
NE Contraction

N=4-6

 

-9

C 120

 

-l— Sham PSS

-D-Sham Hi Mg”
+ DOCA PSS

-V- DOCA Hi Mg”

 

-log EC£50 (moVL)
Sham PSS 7.71:0.1
Sham Hi Mg” 7.41:0.1
DOCA PSS 8.11:0.1‘
DOCA Hi Mg” 7.93:0.1

 

 

 

+Sham Hi Mg”
—El—Sham Hi Mg” LY
+DOCA Hi Mg”
-V—DOCA Hi Mg” LY

 

 

-log E050 (moVL)
Sham Hi Mg” LY 6.51:0.1'
DOCA Hi Mg” LY 6.71:0.2“

 

 

 

.5 -8.5”-7'.5 -6'.5 3.5 -4'.5
Log NE (moVL)

 

Percentage Maximum
NE Contraction

 

O
I

-9.

N=4-6L

 

 

 

 

5 -8.5 -7'.5 -6'.5 -5'.5 -4'.5
Log NE (moVL)

127

—I—Sham PSS LY
—D—Sham Hi Mg” LY
+DOCA PSS LY
—v— DOCA Hi Mg” LY

 

 

-log E050 (moVL)
Sham PSS LY 7.2:0.1*
DOCA PSS LY 7.01:0.1’

 

 

 

In conclusion, these data indicate that Pl3-kinase is not solely responsible
for NE-induced contraction, but activation of Pl3-kinase may alter NE-induced
reactivity. Similarly in a normotensive sham rat, low Mg”-activation of Pl3-kinase
may not have been sufficient to elicit a large enhancement in spontaneous tone,
but activation of the already enhanced PI3-kinase pathway and other signaling
pathways that are activated significantly increases arterial spontaneous tone in a
DOCA-salt rat. High Mg” has the capability to inhibit NE-induced contraction,
however only partially Via a PIS-kinase pathway, further implicating other

mechanisms involvement in NE-induced contraction and not solely PI3-kinase.

E. Subhypothesis #5: Fla-kinase and Its dependent slgnallng pathways are
up-regulated In multiple models of hypertenslon, leading to enhanced

vascular contraction and spontaneous tone development.

1. mn .nu=ol o-ertn iv '. HFI uni ofh 9: n i

SHR model of hypertension is a genetically based model of hypertension
and in the rats studied here the SBP of SHR (175 :I: 8 mm Hg; N=6) were
significantly higher than that of their normotensive WKY rat controls (114 :l: 3 mm

Hg; N=6) (Figure 4).

128

a. Spontaneous tone, NE -/'na’uced contract/on and P/3-lr/nase

Spontaneous tone did not appear to develop in the endothelium-denuded
aorta isolated from the hypertensive SHR or normotensive WKY rats in the
presence of vehicle (Figure 35). Interestingly, LY294002 (20 pmol/L), caused a
significant decrease in basal contraction (Figure 36) in the aorta from the SHR as
compared to the WKY, suggesting that there was some baseline tone present in
these arteries from the SHR, albeit the magnitude of the decrease was not
similar to what was observed in the DOCA-salt rats (compare Figure 36 and
Figure 8). These data suggest that PIS-kinase is important for basal
maintenance of contraction/tone in the SHR rats. It further suggests that there
were differences in arterial Pl3-kinase between the SHR and WKY.

l next examined the effect of LY294002 on NE-induced contraction in the
SHR model of hypertension. Similar to the DOCA-salt model NE-induced
contraction in aorta from SHR is significantly leftward shifted as compared to its
normotensive WKY control (Figure 37). In the presence of LY294002, the NE-
induced contraction was rightward shifted in the WKY and SHR compared to
vehicle treated control tissues (Figure 37). Interestingly the EC50 values of the
LY294002-incubated tissues were not significantly different (Figure 37). Thus, an
alteration in PIS-kinase may partially explain the enhanced arterial contractility to

NE observed in the SHR model of hypertension as well.

129

Figure 35. Basal spontaneous tone differences in aorta between WKY and SHR rats.
Data are presented as a percentage of initial phenylephrine (PE) (10'5 moVL)

contraction. Bars represent the mean spontaneous tone development: SEM.

130

 

 

 

 

N=5

 

AW

_ _
O 0
..i o.

cozombcoo
22: ea: me .525 o\..

 

-30

SHR

WKY

131

Figure 36. Effect of LY294002 (20 pmol/L), a PIS-kinase inhibitor, on

spontaneous tone in endothelium-denuded rat aorta from SHR and WKY rats.
Bars represent the mean change (A) in spontaneous tone development in the
presence of vehicle and LY294002 : SEM. * Statistically significant difference

(p<0.05) between WKY and SHR treatment groups.

132

 

LY294002

 

=5

N

 

 

 

30

_ _
0 0
2 1

.cozomzcoo

3.9: E: mm .35 «a.
cozomzcoo c_ < 38...

 

SHR

WKY

133

Figure 37. The effect of NE and PIS-kinase antagonist LY294002 (20 pmol/L) on
endothelium-denuded aorta from WKY and SHR rats. LY294002 or vehicle was
added to the tissues 1 hour prior to cumulative addition of NE. Data are
presented as a percentage of the maximal NE contraction. Points represent
means a: SEM. The values are the -log EC50 of the NE-induced contractions in
the presence of LY294002 or Vehicle. * Statistically significant difference

(p<0.05) between WKY Vehicle and SHR Vehicle treatment groups.

134

Percentage Maximum
NE Contraction

 

N=5

 

‘

 

+ SHR Vehicle
+ SHR LY 294002

-I— W KY Vehicle
+ W KY LY 294002

 

 

-log EC£50 (moVL)
WKY Vehicle 7.51:0.1
SHR Vehicle 7.81:0.1'
WKY LY294002 7.0:0.1

 

 

SHR LY294002 7.01:0.1

 

-95-a5-i5-65-§5-45
Log NE (mol/L)

135

 

b. P/3—lr/hase B/bchem/Zst/y

Examining PIS-kinase biochemically in the SHR model of hypertension,
there was no significant difference in the regulatory PIS-kinase subunit p85a
between aorta from SHR and WKY rats (Figure 38A). In aorta from both SHR
and WKY there were p1108 and p110a Class IA catalytic Pl3-kinase subunits,
with significantly higher p1106 protein in the aorta from the SHR as compared the
WKY (Figure 388 and 380). No p110y catalytic subunit was detected and the
p110|3 was difficult to discern in the aorta from both the WKY and SHR animals,
thus no decisive conclusion could be reached concerning p11OB (Figure 380).

I proceeded to examine the classical downstream pathway of PI3-kinase.
There was no significant difference in Akt protein levels in aorta from WKY and
SHR models of hypertension and unlike the DOCA-salt model of hypertension,
there was no significant difference in the pAkt protein levels between the aorta
from the SHR and WKY rats (Figure 39).

I last determined if there was PTEN and pPTEN protein in the aorta from
the SHR and WKY rats and if so, was it different in this model of hypertension.
There was PTEN and pPTEN present in the aorta from SHR and WKY animals
and it was not significantly different between the hypertensive and normotensive

animals (Figure 40).

136

Figure 38. Western analyses of protein isolated from aorta from WKY and SHR
rats with antibodies for, A: p85a, 8: p1108, C: p110a, and D: p110y. Bars
represent mean arbitrary densitometry units 3: SEM. * Statistically significant

difference (p<0.05) between WKY and SHR treatment groups.

137

3000- N

 

 

 

 

 

 

 

 

 

 

 

 

 

   

      
 

 

 

 

 

m u
A :E 2500 _T_
320004
”85“" "' -'
WKY SHR @1000-
< 500~
0
WKY
1000- N:
B é" 800-
3
P1105... a... ~__ 2‘ 600-
‘ m I
WKY SHR 3'3 4ooq
< 200-
O
WKY
C m 2000- N:
S 1500-
P1100,» _ 2" a
91000-
WKY SHR E
< 500‘
D G ‘i
'--— WKY SHR
91107...
WKY SHR U937
cells

138

Figure 39. Western analyses of Akt and pAkt protein in aorta from hypertensive
SHR and normotensive WKY rats. Bars represent mean arbitrary densitometry

units :1: SEM.

139

an
mauve-

ILL-mt

 

 

 

WKY SHR
pAkt

 

 

 

 

 

 

 

 

 

 

 

 

 

 

. .......fl...~, ..¥W..Ma§w%m .
R
l l H
t
k m
Y A
Y A K
K w
Am w
N
_ _ W n_U m 0
0 0
m m m m m
5 4 3 2 1

C

(D
3::
4...:

w.__n._<

140

Figure 40. Western analyses of PTEN and pPTEN in aorta from normotensive
WKY and hypertensive SHR rats. Bars represent mean arbitrary densitometry

units 2!: SEM.

141

tr

Ad)

1 000‘
500-

 

 

 

 

 

 

 

 

WKY SHR

 

 

 

 

 

PTEN

142

 

 

pPTEN

WKY SHR

 

pPTEN

 

2. Nw-nitro-L-arginine (LNNA) model of hypeflension

In the L-NNA model of hypertension a nitric oxide synthase inhibitor is
given in the drinking water of male Harlan rats for two weeks. This inhibits the
production of nitric oxide, which is thought to diffuse into the smooth muscle cell
where it stimulates cGMP production and leads to relaxation of the tissues
(Luscher, 1994). The SBP of LNNA rats (201 :t 8 mm Hg; N=7) were significantly

higher than that of their sham rat controls (121 :i: 3 mm Hg; N=9) (Figure 41).

a. Spontaneous tone, NE-Ihduced contraction and P/3-lr/hase

Spontaneous tone did develop in the endothelium-denuded aorta isolated
from the hypertensive LNNA and not in the normotensive sham rats. However, it
was not to the same extent as in the DOCA-salt model and it was not statistically
significant as compared to the sham rats (Figure 42). Even though during the
time period in which tone was monitored there was not a large apparent increase
in tone, there was some form of tone in the artery, because when the arteries
were incubated in LY294002 (20 pmol/L) there was a significant decrease in
basal contraction in the aorta from the LNNA as compared to the sham rats
(Figure 43). These data suggest that there was baseline tone present in these
arteries from the LNNA rats, in fact the inhibition was similar to that observed in
DOCA-salt rats (compare Figure 43 and Figure 8). Furthermore these data

suggest that PIS-kinase is important for basal maintenance of contraction/tone in

143

Figure 41. Systolic blood pressures for sham and N‘” -nitro-L-arginine (LNNA)
(n=7). Columns represent the mean value, where as the vertical lines represent
the standard error of the mean. * Statistically significant difference (p<0.05)

between sham and LNNA treatment groups.

144

1e (L236 250 _ N=7

rep’él'

a"
'I

Systolic Blood Pressure
(mmHg)
3
‘P

 

 

 

 

 

100 -
504
0
Sham

145

Figure 42. Basal spontaneous tone differences in aorta between sham and LNNA rats.
Data are presented as a percentage of initial phenylephrine (PE) (10'5 moVL)

contraction. Bars represent the mean spontaneous tone development: SEM.

146

 

 

 

 

 

LN NA

Sham

 

 

5
__
N
_
O
4

m _ J _

0000
321

cozomzcoo
3:05 we: mm .mas o\..

39.!

0
4..

147

Figure 43. A: Representative tracing of spontaneous arterial tone in
endothelium-denuded aorta from LNNA and sham rats. Tissues are under
passsive tension for optimal force production, vehicle (0.1% DMSO) or LY294002
(20 pmol/L) was added and allowed to equilibrate for 1 hour. 8: Effect of PI3-
kinase inhibitor LY294002 on spontaneous tone in endothelium-denuded rat
aorta from LNNA and sham rats. Bars represent the mean change (A) in
spontaneous tone development in the presence of vehicle and LY294002: SEM.
* Statistically significant difference (p<0.05) between sham and LNNA treatment

groups.

148

Total A in Contraction
[°/o Initial PE (10'5 moVL)

LY294002 (20 umoI/L)

vvv
— ' vv v

LY294002 (20 umol/L)

"rfi’vvv w —

 

Tension (mg)

Vehicle

 

Vehicle

4”

LNNA

 

 

100

Contraction]

Vehicle/LY294002 200 mg] 10 min

LY294002

 

\l
I O1

01
O

N
U!
l
p“;

N=5 *

222212
1 3453.:
J "‘9‘ S.
'i'
E

 

 

 

Sham

149

. .5 time;

‘. 1 - 7"“.

“‘N l ' . , _
, - all” .

. 30"?- 3, _; (I.

‘M"’ 'Iktffif‘tv'i‘ .._:
.- mm _ a

v4? . 'zr mid-._-
as?» - {1.
“get;

IL”-
' V

1:! .1: 4 ‘7‘, .Vki‘ ' _= "I!
n 5 .QL an}: ' , :‘3 I‘ .

_,_.
-

.. has“.

“4&3
LN NA

 

the LNNA rats as well as that there are differences in arterial Pl3-kinase between
the LNNA and sham rats.

I next examined the effect of LY294002 on NE-induced contraction in the
LNNA model of hypertension. Similar to the DOCA-salt and SHR models of
hypertension the NE-induced contraction in the aorta from LNNA is significantly
leftward shifted as compared to the normotensive sham control rats (Figure 44).
In the presence of LY294002, the NE-induced contraction was significantly
rightward shifted compared to vehicle treated controls in the aorta from the LNNA
and sham rats (Figure 44). Unlike the DOCA-salt and SHR models of
hypertension, the EC50 values, of the LY294002 incubated tissues, was still
significantly different between sham and LNNA (Figure 44). Thus, an alteration in
Pi3-kinase may only partially explain the enhanced arterial contractility to NE

observed in the LNNA model of hypertension.

[7. P/3-k/hase Biochemistry

Biochemically, the LNNA and sham rats have the Pl3-kinase subunits,
p85a, p1106, p110l3 and p110a present in the smooth muscle of the aorta, with
significantly higher protein levels of p1108 and p11OB in the aorta from LNNA as
compared to sham rats (Figure 45A - 450). There was no Class IB p1107 PI3-
kinase catalytic subunit in the aorta of either the LNNA or sham rats (Figure

45E).

150

Figure 44. The effect of NE and PIS-kinase inhibitor LY294002 (20 pmol/L) on
contraction in endothelium-denuded aorta from sham and LNNA rats. LY294002
or vehicle was added to the tissues 1 hour prior to cumulative addition of NE.
Data are presented as a percentage of the maximal NE contraction. Points
represent means a: SEM. The values are the -log EC50 of the NE-induced
contractions in the presence of LY294002 or Vehicle (* Statistically significant
difference (p<0.05) between sham Vehicle and LNNA Vehicle, * p<0.05 sham

LY294002 VS. LNNA LY294002 and " p<0.05 LNNA vs. LNNA LY294002).

151

 

120

 

 

 

 

 

 

 

 

 

E + LNNA Vehicle

3 100. +LNNA LY294002

.§ 8 -l—Sham Vehicle

g 33 30- +Sham LY294002

E E

§sah

E Cu: 40‘ -|og.ECw (moVL)

0 Z Sham Vehicle 80:01

5 20‘ LNNA Vehicle 8.210.1'

D. Sham LY294002 7.41.0.1
O LNNA LY294002 1710.1"
-10 -9 £3 -'7 -'6 -'5 -4

Log NE (moVL)

152

Figure 45. Western analyses of protein isolated from aorta from sham and
LNNA rats with antibodies for, A: p85a, 8: p1108, C: p110a, D: p11OB and E:
p110y. Bars represent mean arbitrary densitometry units 3: SEM. * Statistically

significant difference (p<0.05) between sham and LNNA treatment groups.

153

A

p85a_> .III- .-

p1105—>"—-w~

Sham
C
p1 106—I» .
a.
Sham
D

p110'y—>

Sham

Sham

Sham LNNA U

LNNA

LNNA

LNNA

LNNA

2 5‘
III ‘I

Arbitrary Units

Arbitrary Units

Arbitrary Units

ltS

Arbitrary Un

937 Cells

154

7000:
5000‘
3000:
1 000-

 

 

2500-
2000-
1 500-
1 000-

500*

 

 

 

 

 

 

 

 

 

Sham

In examining the classical downstream pathway of PIS-kinase, similar to the SHR
model, I found the no significant differences in the Akt and pAkt protein in the
aorta from the LNNA and sham rats (Figure 46). Thus, even though the
decrease in pAkt protein in the DOCA-salt model of hypertension is interesting, it
appears that this may be a model specific change and not a change induced
solely by an increase in blood pressure. Furthermore, what is interesting is that
there was no increase in pAkt observed in any model of hypertension tested,
which would have been expected with an upregulation of Pl3-kinase function
and/or protein. These data further support the hypothesis that in the condition of
hypertension, PIS-kinase may be utilizing other pathways than the classical Akt-
pAkt pathway that is traditionally associated with Pl3-kinase.

Another mechanism by which altered Pl3-kinase function and activity is
regulated is by alterations in the Pl3-kinase specific phosphatase PTEN. PTEN
and pPTEN protein was present in the aorta from the LNNA and sham rats,
however similar to the previous studies there was no significant difference in
protein levels of PTEN and the pPTEN in the aorta (Figure 47).

In conclusion, the LNNA and SHR models of hypertension, in general,
support previous findings in the DOCA-salt model of hypertension. Pl3-kinase is
involved in spontaneous tone and NE-induced contraction. PI3-kinase protein,
specifically the p1105 subunit, as well as the p110l3 in the LNNA model of

hypertension, are upregulated in these rat models hypertension and the

155

Figure 46. Western analyses of Akt and pAkt protein in aorta from hypertensive
LNNA and normotensive sham rats. Bars represent mean arbitrary densitometry

units :1: SEM.

156

5000q N“
4000‘ ._—r;_
a 3000‘
2000‘
1000‘

Units

Arbitra

 

 

 

 

 

 

Akt

 

34:-t !....x .1; I I II

Sham LNNA Sham LNNA
Akt pAkt

 

 

157

Figure 47. Western analyses of PTEN and pPTEN in aorta from normotensive
sham and hypertensive LNNA rats. Bars represent mean arbitrary densitometry

units 1 SEM.

158

5000 - N

4000-

 

3000-

 

2000-

Arbitrary Units

10001

    

 

 

 

 

 

   

0 "fig

Sham Sham LN
PTEN pPTEN

 

 

 

 

 

«ii-W“ W

Sham LNNA Sham LNNA
PTEN pPTEN

 

 

159

alterations observed with respect to PIS-kinase do not likely mediate their

function through the classical PI3-kinase signaling pathway of Akt and pAkt.

F. Subhypothesis #6: PI3-kinase functional alterations and changes in
protein are observed in the mesenteric resistance arteries in the DOCA-salt

model of hypertension.

Thus far, studies have utilized the aorta as the artery of choice because
there was a large amount of tissue and it was a place to characterize the
changes in PIS-kinase with relative ease. The aorta is a conduit artery and has
recently been found to play at least a small role in the maintenance of blood
pressure, due to changes in compliance in the aorta during the condition of
hypertension (Safar at a/., 1998; Salaymeh at a/., 2001). Even though the aorta
does play some role in hypertension, the aorta does not affect TRP to the extent
that resistance arteries do. Thus, l have chosen to examine the mesenteric
resistance arteries to determine if the alterations observed in the aorta also occur
in smaller arteries that have influence on blood pressure. Resistance arteries
approximately 240 microns in diameter were placed in a myograph for
measurements of isometric force. Spontaneous tone developed in several, but
not all, of the resistance arteries removed from the DOCA-salt rats (Figure 48A,
top tracing). Spontaneous tone did not develop in resistance arteries removed

from sham rats (data not shown). LY294002 (20 umol/L) significantly inhibited

160

 

Figure 48. A: Representative tracing of spontaneous arterial tone in
endothelium-denuded mesenteric resistance arteries from a DOCA-salt rat.
Tissues are under passsive tension for optimal force production, vehicle (0.1%
DMSO) or LY294002 (20 pmol/L) was added and allowed to equilibrate for 1
hour. 8: Effect of Pl3-kinase inhibitor LY294002 or vehicle on spontaneous tone
in endothelium-denuded rat aorta from DOCA-salt and sham rats. Bars
represent the fall of spontaneous tone (milligrams) in the presence of vehicle and
LY2940021 SEM. * Statistically significant difference (p<0.05) between sham and

DOCA-salt LY294002 treatment groups.

161

 

A
DOCA-Salt Mesenteric Resistance Artery

4o mgl m LY294002 (20 pmol/L)

     

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LY294002 (20 pmol/L) Vehicle

162

spontaneous tone in the resistance arteries from DOCA-salt rats as compared to
sham or vehicle treated arteries from DOCA-salt rats (Figure 48B). Albeit, the
inhibition of spontaneous tone that occurred did not have the magnitude that was
observed in the aorta from DOCA-salt rats (compare Figure 8 and Figure 48).
However, even small alterations in the radius in resistance arteries can play a
major role in blood pressure because it is the radius of arteries that mediates
total peripheral resistance.

To examine the resistance arteries biochemically, many resistance
arteries were removed from that sham and DOCA-salt animals. For each animal
the arteries were pooled and the protein was isolated for Western analyses.
Westerns revealed that there was no significant difference in the regulatory p850:
Class IA PI3-kinase subunit. However, similar to aorta, there was significantly
higher protein levels of the Class IA catalytic PIS-kinase subunit p1106 in the
resistance arteries from the DOCA-salt as compared to the sham (Figure 49).
Due to the limited amount of protein the other subunits were not examined.
There were also similar Akt and pAkt protein levels in the resistance arteries from
sham and DOCA-salt rats (Figure 50). These data demonstrate that several of
the characteristics observed in the aorta are similar in the resistance arteries
from DOCA-salt rats, suggesting that PIS-kinase function is altered in arteries
that have a functional influence on blood pressure maintenance, as well as in the

aorta.

163

Figure 49. Western analyses of protein isolated from mesenteric resistance
arteries from sham and DOCA-salt rats with antibodies for, A: p850: and B:
p1108. Bars represent mean arbitrary densitometry units 1 SEM. * Statistically

significant difference (p<0.05) between sham and DOCA-salt treatment groups.

164

 

 

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165

Flgure 50. Western analyses of Akt and pAkt protein in mesenteric resistance
arteries from hypertensive DOCA-salt and normotensive sham rats. Bars

represent mean arbitrary densitometry units 1 SEM.

166

 

N:

25.51%

 

°/o Alpha actin
Arbitrary Units

 

 

 

 

 

 

 

 

 

Sham DOCA

 

 

 

167

G. Time Course Studies

To determine when the alteration in PIS-kinase function and protein occur
in relationship to changes in blood pressure, time course studies were
performed. By day 5 of DOCA treatment the DOCA-salt rats had a significant
increase from day 0 in their blood pressure as compared to the time matched
sham animals (Figure 51). Spontaneous tone began to develop as soon as on
the 3rd day of treatment in the DOCA-salt rats, however this was not significant as
compared to the sham until the 5th day of treatment (Figure 52). LY294002 (20
pmol/L) inhibited the spontaneous tone that developed as early as Day 3 of
treatment (Figure 53) in the aorta of the DOCA-salt rats compared to the sham
rats.

To further examine when changes occur with respect to hypertension and
PIS-kinase, NE was added in a concentration dependent manner to aorta in the
presence of Vehicle or LY294002, after 1, 3, 5 and 7 days of treatment. There
was no significant difference in vehicle treated aorta from DOCA-salt and sham
rats on day 1 and LY294002, significantly rightward shifted the NE-induced
contraction in both aorta (Figure 54). On the 3rd day of treatment, the vehicle
treated aorta from the DOCA-salt rat was significantly leftward shifted as
compared to the corresponding vehicle treated sham (Figure 55). These data
suggest, that even before there was a significant increase in blood pressure,

there were changes in NE-induced contraction in aorta from DOCA-salt rats.

168

Figure 51 . Changes from Day 0 in systolic blood pressures 1, 3, 5 and 7 days
after sham and DOCA-surgery was performed on the rats. Columns represent
the mean change in systolic blood pressure from day 0 until end of the animals
respective treatment, where as the vertical lines represent the standard error of

the mean. * Statistically significant difference (p<0.05) between sham and

DOCA-salt treatment groups.

169

 

Change in Systolic Blood
Pressure (mmHg)

01
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Sham DOCA Sham DOCA Sham DOCA Sham DOCA

Day 1 Day 7

Day 3

Day 5

 

 

 

 

 

Figure 52. Basal spontaneous tone in aorta between sham and DOCA-salt rats
after 1, 3, 5 and 7 days of treatment. Data are presented as a percentage of
initial phenylephrine (PE) (10'5 moVL) contraction. Bars represent the mean
spontaneous tone development1 SEM. * Statistically significant difference

(p<0.05) between sham and DOCA-salt treatment groups.

171

 

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Day 7

 

 

 

Figure 53. Effect of PI3-kinase inhibitor LY294002 on spontaneous tone in
endothelium-denuded rat aorta from DOCA-salt and sham rats on Day 1, 3, 5
and 7 of treatment. Bars represent the mean change (A) in spontaneous tone
development in the presence of vehicle and LY2940021 SEM. * Statistically

significant difference (p<0.05) between sham and DOCA-salt treatment groups.

173

AER:
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Total A in Contraction
[% lni ial PE (10'5 mol/L)

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LY294002

 

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174

 

 

 

 

 

 

Figure 54. The effect of NE and PIS-kinase antagonist LY294002 (20 pmoVL) on
endothelium-denuded aorta from DOCA-salt and sham rats after 1 day of
treatment. LY294002 or vehicle was added to the tissues 1 hour prior to
cumulative addition of NE. Data are presented as a percentage of the maximal
NE contraction. Points represent means 1 SEM. The values are the -Iog EC50 of
the NE-induced contractions in the presence of LY294002 or Vehicle (' p_<_0.05 vs.

Sham Vehicle and “ p50.05 vs. DOCA Vehicle).

175

 

 

N:

Percentage Maximum
NE Contraction
o)
o
l

 

 

 

+ DOCA Vehicle
+ DOCA LY 294002

+ Sham Vehicle
—o— Sham LY294002

 

-Iog EC50 (moi/L)
Sham Vehicle 8.110.1
Sham LY294002 7.710.1 '
DOCA Vehicle 7.8102
DOCA LY294002 7.710.1 '

 

 

 

-23 -'7 -25 -5
Log NE (moi/L)

176

-4

 

 

Figure 55. The effect of NE and PI3-kinase antagonist LY294002 (20 umoI/L) on
endothelium-denuded aorta from DOCA-salt and sham rats after 3 days of
treatment. LY294002 or vehicle was added to the tissues 1 hour prior to
cumulative addition of NE. Data are presented as a percentage of the maximal
NE contraction. Points represent means 1 SEM. The values are the —Iog EC50 1
SEM of the NE-induced contraction in the presence of Vehicle or LY294002

(p_<_0.05 VS. Sham Vehicle).

177

 

 

Percentage Maximum
NE Contraction
O)
O
I

 

N:

 

 

  

 

+ DOCA Vehicle
+ DOCA LY294002
+ Sham Vehicle
+ Sham LY294002

 

 

 

 

 

 

 

l

-3 -7 .2 -5
Log NE (mollL)

178

—log EC,0 (moVL)
Sham Vehicle 7.810.1
Sham LY294002 7.5102 '
DOCA Vehicle 8210.1 *
DOCA LY294002 7.8102

 

-4

 

 

When incubated with LY294002 (20 pmoI/L) the sham and DOCA-salt were not
significantly different (Figure 55). On the 5th and 7th days of treatment, the aorta
from the DOCA-salt treated with vehicle was leftward shifted as compared to
sham vehicle (Figures 56 and 57). On the 5th day LY294002 rightward shifted
the sham and DOCA-salt with respect to their controls (Figure 56). On the 7th
day of treatment the LY294002 treated aorta from DOCA-salt rats were rightward
shifted compared to the DOCA-salt vehicle, but no shift occurred with respect to
the sham. There was also no significant difference between the sham vehicle,
sham LY294002 and DOCA LY294002 (Figure 57). These data suggest that
PIS-kinase may be altered in the aorta as soon as the 3rd day of DOCA-salt
treatment, however, due to the small numbers (N=5) of animals and that animals
had different progressions of their hypertension, it was difficult to definitively
determine Pl3-kinases involvement in the enhanced NE-induced contraction at
the early stages of hypertension.

When examining the induction of the DOCA-salt model of hypertension for
biochemical studies, there was a significant increase in SBP by Day 7 of
treatment (Figure 51). However, Western analyses revealed no significant
differences in p85a, (01108, or pAkt/Akt protein levels over the 1,3,5 and 7 days
of treatment (Figures 58, 59, and 60). Upon examining the significant changes in
Pl3-kinase mediated spontaneous tone, I would have expected to observe some
changes in the PI3-kinase subunits. I do realize that there have may have been

alterations in other PIS-kinase subunits, but I chose to examine the p1105

179

Figure 56. The effect of NE and Pl3-kinase antagonist LY294002 (20 umol/L) on
endothelium-denuded aorta from DOCA-salt and sham rats after 5 days of
treatment. LY294002 or vehicle was added to the tissues 1 hour prior to
cumulative addition of NE. Data are presented as a percentage of the maximal
NE contraction. Points represent means 1 SEM. The values are the -log E050 1
SEM of the NE-induced contraction in the presence of Vehicle or LY294002

(p_<_0.05 vs. Sham Vehicle, “p_<_0.05 vs. DOCA Vehicle).

180

 

Percentage Maximum
NE Contraction

 

 

0-
-10

N:

 

   

-+— DOCA Vehicle
+ DOCA LY294002

+ Sham Vehicle
+ Sham LY294002

 

-log EC50 (moi/L)
Sham Vehicle 7.910.1
Sham LY294002 7.4102 *
DOCA Vehicle 8210.1
DOCA LY294002 7.710.1 #

 

 

 

 

 

I l

-9 -i3 -7 -6 -5
Log NE (mollL)

181

.4

Figure 57. The effect of NE and Pl3-kinase antagonist LY294002 (20 umol/L) on
endothelium-denuded aorta from DOCA-salt and sham rats after 7 days of
treatment. LY294002 or vehicle was added to the tissues 1 hour prior to
cumulative addition of NE. Data are presented as a percentage of the maximal
NE contraction. Points represent means 1 SEM. The values are the -Iog EC50 of
the NE-induced contractions in the presence of LY294002 or Vehicle. The values
are the —Iog EC50 1 SEM of the NE-induced contraction in the presence of
Vehicle or LY294002 (' p50.05 vs. Sham Vehicle and ' p50.05 vs. DOCA

Vehicle).

182

Percentage Maximum
NE Contraction

Day 7

 

 

     

12° N_ + DOCA Vehicle
+ Sham Vehicle
60-
40‘ -Iog E050 (moi/L)
Sham Vehicle 7.9101
20_ Sham LY294002 7.810.1
DOCA Vehicle 8.110.1 "
0 DOCA LY294002 7.810.1

 

 

 

 

 

 

-10 -9 :8 -7 -5 -5 -4
Log NE (mollL)

183

Figure 58. Western analyses of protein isolated from aorta from sham and

DOCA-salt rats, after 1,3,5, 7 and 28 days of treatment, with antibody for p85a.

Bars represent mean arbitrary densitometry units 1 SEM.

184

p850c

 

75

01
C.’

Arbitrary Units
N
‘f‘

 

N=3-6

 

 

 

 

 

 

 

 

185

 

 

 

 

 

 

Figure 59. Western analyses of protein isolated from aorta from sham and
DOCA-salt rats, after 1,3,5, 7 and 28 days of treatment, with antibody for p1108.
Bars represent mean arbitrary densitometry units 1 SEM. * Statistically significant

difference (p<0.05) between sham and DOCA-salt treatment groups.

186

p1108

 

N=3-6

 

 

 

 

 

 

 

 

 

 

 

 

50

40-
20-

_
0
3

e15 1232.

 

Animal

Day

187

Figure 60. Western analyses of protein isolated from aorta from sham and
DOCA-salt rats, after 1,3,5, 7 and 28 days of treatment, with antibodies for Akt
and pAkt. Bars represent mean arbitrary densitometry units 1 SEM. * Statistically

significant difference (p<0.05) between sham and DOCA-salt treatment groups.

188

 

 

 

 

 

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pAkt/Akt

 

 

 

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N:

 

 

 

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215 2832
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189

subunit because I detected alterations in aorta from Day 28 DOCA-salt rats,
p850: because it is the main regulatory subunit and pAkt and Akt, with the thought
of the increase in cellular vascular smooth muscle, i would have expected an
increase in pAkt during the early stages of DOCA-salt hypertension development.
However, alterations in these signaling components may have been so small that
they were difficult to detect using Westerns. I also realize that PI3-kinase activity
may have been increased without a change in protein values and I did not do
Pl3-kinase activity assays. In conclusion, the alteration in PIS-kinase function
appeared as early as Day 3 of treatment, however these changes functionally
were not reflected in protein levels by using Western analyses. Thus, it appears
the changes in Pl3-kinase, NE-induced aortic contraction and blood pressure that
occurred went simultaneously in turn making it hard to discern if there was any

separation of the two.

190

 

DISCUSSION

Cardiovascular disease is the number one killer in the United States.
Over 50 million Americans and 1 billion people worldwide have hypertension with
hypertension being one of the major risk factors of cardiovascular disease.
Recently, the seventh report of the Joint National Committee on Prevention,
Detection, Evaluation and Treatment of High Blood Pressure was released. In
this report patients with systolic blood pressures between 120-139 mm Hg and
diastolic blood pressures between 80-89 mm Hg are now to be considered pre-
hypertensive and require health-promoting lifestyle modifications to prevent
cardiovascular disease. Patients with prehypertension are at increased risk for
progression to hypertension (Chobanian et al, 2003). Thus, it is imperative that
the scientific community more fully understand the pathogenesis of the condition
and develop further therapeutic strategies to treat hypertension.

Pl3-kinase plays a key role in cellular growth, apoptosis as well as being
implicated in modulating vascular contraction (Wymann at a/., 1998; Anderson at
a/., 1999; Rameh and Cantley, 1999; Cantrell, 2000; Coelho and Leevers, 2000;
Vanhaesebreck at al, 2001). PIS-kinase has been referred to as a signaling
“hub” in that many stimuli have Pl3-kinase in their signaling pathways and that
once they converge onto PI3-kinase, there are a variety of effectors that
ultimately lead to smooth muscle cell contraction, cell survival, superoxide
formation, protein synthesis and cell cycle progression (Figure 2). With this in

mind, I hypothesized that in the condition of hypertension there is an upregulation

191

 

in Pl3—kinase protein and/or activity that is responsible for enhanced PIS-kinase-
mediated growth, as the literature reports, as well as enhanced contraction and
spontaneous tone development in the condition of hypertension. The findings of
these experiments are relevant to the further understanding of the pathogenesis

of hypertension as well as provide new ways of potentially treating hypertension.

A. Rationale

The isolated tissue bath and myograph systems were used to examine
Pl3-kinases involvement in spontaneous tone and contraction in aorta and
mesenteric resistance arteries from hypertensive rats. To determine whether the
PIS-kinase mediated alterations in contraction and spontaneous tone were due to
alterations in protein and/or activity of PIS-kinase, I used a variety of biochemical
assays including: western analyses, lmmunohistochemistry, immunoprecipitation
and Pl3-kinase activity assays. If an alteration in PIS-kinase is vital in
spontaneous tone development, it would be reasonable to hypothesize that it is
also vital in agonist-mediated contraction. Thus, I hypothesized that the
enhanced NE-induced contraction observed in the aorta in hypertension was
mediated by PIS-kinase. Low [Mgb]. has been observed in the condition of
hypertension and I hypothesized that PIS-kinase was involved in the
enhancement of spontaneous tone observed and further examined NE-induced
contraction in the presence of altered Mg2+ concentrations. To demonstrate that

the alterations observed in hypertension, with respect to Pl3-kinase, were not

192

model dependent, or unique to aorta, I examined the aorta from SHR and LNNA
models of hypertension, as well as mesenteric resistance arteries from DOCA-
salt and sham rats. The mesenteric resistance arteries were used because
unlike the aorta, which is a conduit artery, the resistance arteries play a direct
role in modifying peripheral resistance. Finally, to further explore when the
alteration in PIS-kinase occurred in respect to the development of when blood
pressure elevation in DOCA-salt hypertension, time course studies were
performed to further elucidate when the changes in Pl3-kinase occur with respect

to the development of hypertension.

8. Identification of Pl3-klnase Involvement in Spontaneous Tone

The main hypothesis stemmed from the initial finding that LY294002, a
PI3-kinase inhibitor, eliminated spontaneous tone in aorta from a DOCA-salt and
had no effect on tone of the aorta from sham. Renal arterial spontaneous tone
has been observed in hypertensive patients, with resultant phasic changes in
renal perfusion that can be reduced by calcium channel blockade (Hollenberg
and Sandor, 1984; Hollenberg, 1987). These findings suggested an important
role for PI3-kinase in spontaneous tone development and indicate a potential
interaction with calcium, all leading to an effect that may have serious
physiological implications.

A pharmacological inhibitor must inhibit its respective target in a

concentration-dependent manner. To test this, I added increasing concentrations

193

of LY294002 to isolated tissue baths that had aorta in which spontaneous tone
had developed. LY294002 inhibited spontaneous tone in a concentration-
dependent manner. The concentration of LY294002, 20 umol/L, that was
primarily used throughout these thesis experiments was not the concentration
that elicited a maximal inhibition of spontaneous tone. LY294002 also has the
ability to inhibit casein kinase II (CK2) at concentrations used for these
experiments (Davies at a/., 2000). However, because of the lack of selective
CK2 inhibitors, it was not possible to control for this limitation. To ensure
inhibition of PI3-kinase another PIS-kinase inhibitor was used, wortmannin.
Wortmannin also inhibited spontaneous tone in a concentration dependent
manner. LY294002 was used for all further experiments due to the fact that
wortmannin has been reported to inhibit MLCK at the same concentration that it
inhibits PIS-kinase and MLCK is an important component of contraction (Davies
9131, 2000).

Finally, to ensure the specificity of LY294002, I used an inactive analog of
LY294002, LY303511. LY303511 has a single atom substitution from LY294002
and has no known actions on PIS-kinase and thus provided an excellent control
for evaluating possible nonspecific effects of LY294002 (Vlahos at a/., 1994).
LY303511 did not inhibit spontaneous tone, but it did elicit a contraction in the
DOCA-salt tissue and had no effect on the sham. The reason for this contraction

was unclear, but LY303511 did not inhibit PIS-kinase activity, as it did not reduce

194

 

EGF-induced phosphorylation of Akt. Interestingly, the contraction occurred only
in the aorta from DOCA-salt and not sham rats.

Ideally LY294002 would lower the blood pressure of hypertensive DOCA-
salt rats in vivo if indeed there was a true importance of Pl3-kinase in the
maintenance/development of hypertension. In trial experiments, LY294002,
when given acutely in vivo to conscious hypertensive DOCA-salt rats, lowered
blood pressure (data not shown) but it also lowered heart rate, making it difficult
to determine the role PIS-kinase in hypertension in the vasculature. Therefore, to
further examine the role of PIS-kinase in arteries that had a more relevant role in
the maintenance of TPR mesenteric resistance arteries were used. I realize that
this is still not the ideal, however subunit specific PIS-kinase inhibitors are not
available at this time and it is difficult to target a drug to solely the smooth muscle
of arteries. Pia-kinase is vital in growth and contraction in the heart, thus only
subunit specific drugs would be effective in potentially evaluating the role of PI3-
kinase in regulation of blood pressure (Vlahos at a/., 2003). Mesenteric
resistance arteries were isolated for studies of isometric tension recordings using
a myograph. The resistance arteries from sham rats did not develop tone and
the arteries from DOCA-salt rats displayed variable levels of spontaneous tone,
not to the same magnitude observed in aorta. Hence, the data for the resistance
arteries was reported as the fall to vehicle or LY294002 in milligrams. In
resistance arteries small changes in the diameter of the artery can potentially

lead to large changes on the TPR, thus altering blood pressure. When the

195

 

LY294002 was added to resistance arteries from DOCA-salt rats, the tone of the
arteries was significantly reduced. LY294002 had no effect on, nor did
spontaneous tone develop in resistance arteries and aorta from sham rats,
implicating the change with respect to PIS-kinase is found only those arteries in
hypertensive animals. These results further demonstrate that PIS-kinase is a key
component in spontaneous tone development in the resistance arteries as well
as aorta from DOCA-salt rats suggesting Pl3-kinase is playing a crucial role in
not only hypertension related growth but also contraction.

Two other rat models of hypertension, SHR and LNNA rats, and their
respective normotensive controls were used to demonstrate that upregulation of
PI3-kinase protein, PIS-kinase regulation of spontaneous tone and enhanced
Pl3-kinase-mediated NE-induced contraction in arteries from hypertensive
animals was not a model-dependent phenomenon. Arteries from the LNNA
model of hypertension developed a small amount of tone in the presence of
vehicle and when exposed to the PIS-kinase inhibitor LY294002, tone was
significantly inhibited, similar to what was observed in the DOCA-salt model of
hypertension. With respect to the SHR model of hypertension I did not observe a
significant increase in spontaneous tone while incubated with vehicle (DMSO),
unlike other studies in which spontaneous tone in arteries from SHR was
significantly greater than their normotensive WKY rat counterparts (Sunano st 31,
1996). However, the recording of spontaneous tone was made when the arteries

were exposed to vehicle (DMSO) and DMSO has the ability to inhibit

196

spontaneous tone. Tone did not develop in the aorta from SHR rats to the
magnitude, nor did LY294002 inhibit this tone to the magnitude, that it did in the
DOCA-salt model of hypertension. Enhanced myogenic activity was observed in
mesenteric arteries from SHR compared to WKY at 5 weeks of age, as
hypertension was developing, but it was not observed at 20 weeks when
hypertension was considered established, except at high transmural pressures
where SHR arteries were able to maintain tone and WKY rats were not (Izzard at
a/., 1996). These data suggest that the lower level of tone I observed in SHR as
compared to the DOCA-salt model may be because SHR animals of 12 weeks of
age were used and had established hypertension, and thus the spontaneous
tone may not have been of the same magnitude as when the animals were
younger. Interestingly, spontaneous tone did not develop in arteries from sham,
sham-LNNA, and WKY rats, nor did LY294002 have any effect on basal tone in
these animals, further suggesting a difference Pl3-kinase in aorta from
normotensive rats and hypertensive animals. In summary, Pl3-kinase plays a
major role in the maintenance of spontaneous basal tone in arteries from

hypertensive animals.

C: Pl3-klnase Biochemistry
Having established that there is functional change in Pl3-kinase in aorta
from hypertensive rats, I proceeded to examine PIS-kinase from a biochemical

standpoint with the hypothesis that there would be increased Pl3-kinase protein

197

and/or activity in the aorta from hypertensive rats. To assay for Pl3-kinase
activity, I used an antibody against p85a. I chose to use this antibody vs. a
general phosphotyrosine antibody as others have used because of the specificity
for PI3-kinase association provided by p85a. I realize by using this antibody that
the PIS-kinase activity assay only reflected upregulation of activity associated
with the class IA PIS-kinase family and that LY294002 at the concentration I used
had the ability to inhibit the class II PIS-kinase family as well. Because only the
p850: antibody was utilized to determine PIS-kinase activity, the activity assays
may have underestimated the total changes in PIS-kinase activity that may occur
in hypertension, neglecting the class II and class III Pl3-kinase families. There
were no significant differences in the p85a protein levels, but there was
significantly elevated PIS-kinase activity in aorta from DOCA-salt rats as
compared to sham. These data suggest the PI3-kinase mediated enhancement
of spontaneous tone observed may be due to this enhancement in class la PI3-
kinase activity observed.

This led me to inquire if the increase in activity was associated with an
increase in p110 protein density. l detected p85a, p110a, p1108 and p1106 in
the thoracic aorta of DOCA-salt, LNNA and sham rats, as well as p85a, p110a,
and p1105 in SHR rats. There was a significantly greater amount of p1108 in the
aorta of DOCA-salt, LNNA and SHR compared to their normotensive controls, as
well as a significantly greater p11OB in the LNNA model of hypertension. In

DOCA-salt and sham mesenteric resistance arteries p85a and p1108 were also

198

present, with significantly higher p1106 in the resistance arteries from the DOCA-
salt rats, demonstrating that the alteration observed in aorta is also present in
resistance vessels. These data are in contrast to the findings of Macrez at al.
(2001) in which p110a, p1108, p1107 were found, but no p1108 in rat portal vein
myocytes. The localization of p1108 to the aorta was unexpected because
p1 108 had been reported to be restricted to hematopoietic cells
(Vanhaesebroeck at a/., 1997). The upregulation of p1108 subunit density may
constitute a potential mechanism of the enhanced Pl3-kinase activity observed.
PI3-kinase activity assays in which the p1108 antibody was used in order to
examine specifically p1108 specific activity demonstrated an increase in the
p1106-mediated Pl3-kinase activity. Furthermore, immunohistochemical studies
revealed that p1108 catalytic Pl3-kinase subunit is indeed localized to vascular
smooth muscle in the aorta and more intense staining was observed in the aorta
from DOCA-salt rats as compared to sham, supporting the findings from western
analyses. In summary, these data show there is an increase in arterial Pl3-
kinase activity and protein, specifically p1105, in the condition of hypertension
and that this upregulation may explain the PIS-kinase-mediated spontaneous
tone that develops.

There may also be an alteration in the regulation of PIS-kinase activity
separate from a change in PIS-kinase density that causes the change in
contractility observed in hypertension. PTEN, a tumor suppressor gene, is down

regulated in certain types of cancer. Similar to cancer, growth of vascular

199

 

smooth muscle cells is clearly enhanced and dysregulated in hypertension. If
under the conditions of hypertension PTEN were reduced, this would allow for a
functional increase in activity of PIS-kinase. This is the first time, to the best of
our knowledge, that PTEN and pPTEN, the inactive form of PTEN, were localized
to the arteries. However, PTEN and pPTEN were not significantly different in the
aorta from DOCA-salt, LNNA and SHR as compared to their respective
normotensive control rats. It is unlikely, but possible, that there is an increase in
PTEN activity and I have not addressed PTEN activity specifically, only protein
levels. Alteration in phosphatase activity may not solely be because of one
phosphatase, but a combination of several phosphatases. SH2-containing
inositol phosphatase (SHIP), transmembrane phosphatase with tensin homology
(TPTE) PTEN homologous inositol lipid phosphatase (TRIP) and Jumpy are
three other recently discovered phosphatases that have the ability to inhibit PI3-
kinase actions by dephosphorylating the 03 position of phosphoinositide
products (Scharenberg at a/., 1998; Walker of al, 2001b; Wishart at a/., 2003).
Moreover, it is hypothesized that these phosphatases are limited to eliciting their
actions in specific regions of the cell, for example, TRIP is hypothesized to he
localized to the endoplasmic reticulum (Walker of a/., 2001b). Thus, if a specific
phosphatase is localized to the area in which Pl3-kinase regulates contraction
and the phosphatase is down-regulated in the condition of hypertension, this may
also be a reason for enhanced PIS-kinase mediated contraction. This is

assuming that it is a downstream product of PIS-kinase that is affecting

200

contraction. These phosphatases also only inhibit the lipid kinase action of PI3-
kinase and it may be that Pia-kinase is mediating the enhanced contraction in the
condition of hypertension via its protein kinase ability. However, this is purely
speculation and requires further investigation. To summarize, PTEN protein is
not altered in the aorta from hypertensive and normotensive animas, however,
phosphatases may be indeed altered in the condition of hypertension and only
further experimentation will reveal if this is a correct hypothesis.

There are many proteins downstream of PIS-kinase, however the classical
PI3-kinase downstream target is Akt, mediated through PDK. Phosphorylation of
Akt is the classical way to examine activation of PIS-kinase in the cell. Therefore,
I hypothesized the increase in PIS-kinase activity observed in aorta from DOCA-
salt rats compared to sham animals would be reflected in an increase in
phosphorylation of Akt in the condition of hypertension. There were no
significant differences in PDK or phosphorylated PDK in aorta between sham and
DOCA-salt rats, which was somewhat surprising, in light of the increase in Pl3-
kinase activity observed. There were also no significant differences between
total Akt protein levels in aorta from DOCA-salt, LNNA or SHR rats compared to
their respective normotensive controls. This experiment was necessary in order
to determine if it was indeed an increase in Pl3—kinase activity or simply more Akt
protein available to be activated. Surprisingly, there were significantly lower pAkt
levels in the aorta from the hypertensive DOCA-salt rats compared to the

normotensive sham rats, but in the LNNA and SHR models there was no

201

 

significant difference in pAkt. The decrease in pAkt in the DOCA-salt rats were
also surprising in the face of the fact that there is clearly an upregulation of PI3-
kinase activity in the aorta from the DOCA-salt rat. I would have also expected
an increase in pAkt in the other two models of hypertension as well if it was a
change to the increase in blood pressure. Even so, this led me to further
hypothesize that the increase in Pl3-kinase enzyme/activity was being directed
toward another PIS-kinase downstream pathway to lead to enhanced
spontaneous tone in the condition of hypertension, potentially acting on L-type

calcium channels.

D: PIS-kinase and Ca2+

In experimental and clinical hypertension, investigators have identified
defects in arterial Ca2+ handling resulting in inappropriately high basal Ca2+ levels
(Rusch and Kotchen, 1994). This is reflected in development of spontaneous,
non-agonist-induced arterial tone. Therefore, I hypothesized that this increase in
intracellular Ca2+ level was due, at least in part, to an alteration in Pl3-kinase
activity and/or protein, mediating its effects through L-type Ca“ channels. Altered
membrane depolarization, a main stimulus for Ca2+ current through voltage-gated
Ca2+ channels, is a critical parameter that may be also affected by PIS-kinase
that I did not address in these studies.

The findings of dramatically enhanced contraction to BayK8644 in aorta

from DOCA-salt rats supported previous observations in coarctation-hypertensive

202

rats, DOCA-salt hypertensive rats, LNNA and SHR compared to their respective
normotensive controls (Storm at a/., 1990; Watts of a/., 1994; Manso at a/.,
1999). These data suggested an upregulation of L—type calcium channel activity
in DOCA-salt rat hypertension. Recently, Molero at al. (2001) found an increase
in L-type Ca2+ channel expression in membrane protein from DOCA-salt
compared to sham rats in small mesenteric arteries. However, in the aorta I
found no significant difference in the a1c subunit of L-type Ca2+ channels,
suggesting that the increase in calcium may be due to increased specific activity
and not protein density. Viard at al. (1999) demonstrated that 687 dimers have
the ability to stimulate vascular L-type Ca"”' channels through PIS-kinase, which
adds support to the notion that PIS-kinase and L-type Ca2+ channels are linked
and G-protein coupled receptors have the potential to tap into this interaction.
Macrez at al. (2001) demonstrated that p110 subunits could directly associate
with L-type Ca2+ channels increasing the flux of Ca2+ through the channel. Thus,
if there is an alteration in PIS-kinase activity, this may further activate Ca2+
channels. PIS-kinase regulation of Ca2+ channels was strengthened by the
finding that LY294002 inhibited all Ca2*-induced spontaneous tone in the aorta
from DOCA-salt rats. At the present time, the mechanism by which PI3-kinase
activates L-type Ca2+ channels is unclear. Preliminary data using co-
immunoprecipitation has suggested a possibility for Pl3-kinase protein to directly
associate with the L-type Ca2+ channel (data not shown), providing a possible

mechanism in which these two may interact. Whether this is sufficient to enable

203

the development of arterial spontaneous tone and hyperresponsiveness
observed in hypertension remains to be seen. PIS-kinase plays a role in many
downstream elements that have modulatory effects on contractility in the
vascular smooth muscle, such as rhoA and its corresponding effects on MCLK
that may alter contraction in a Pl3-kinase dependent manner.

However PIS-kinase may be connected to Ca2+ channels, their interaction
has implications for the hyperresponsiveness to contractile agonists that is
characteristic in hypertension. Because Pl3-kinase is integral to signaling of
hormones involved in contraction and growth, an increase in Pl3—kinase activity

has significant implications for arterial function.

E. Identification of Pl3-klnase Involvement In NE and Low Mg’+ -lnduced

contraction
1. NE-indoooo Contraction

Class I Pl3-kinases are activated wa receptors with intrinsic tyrosine
kinase activity (EGFr, NGFr, PDGFr), receptor-associated tyrosine kinase activity
(JAK2, JAK1, FAK) and via G-protein coupled receptors (Thrombin,
Lysophosphatidic acid) (Wymann and Pirola, 1998). NE is an important
vasoactive neurotransmitter found throughout the body and is known to
contribute to hypertension. NE activates Pl3-kinase in vascular smooth muscle

and small arteries waa pertussis-sensitive G-protein receptor (Hu at a/., 1996;

204

Walker at a/., 2001a). Naito at a/. (1998) have demonstrated that the intact
mesenteric vascular bed of SHR rats show potentiated responses to NE as well
as to ATP as compared to tissue from WKY rats. NE also increased the
perfusion pressure in a concentration-dependent manner in the intact mesenteric
bed, where arteries with maximal responses were larger in the SHR as compared
to the WKY rats. l have already demonstrated that there is altered PI3-kinase
activity in aorta from DOCA-salt hypertensive rats. It is possible that the reason
that NE elicits its enhanced arterial contraction is the increase in PIS-kinase
activity in the condition of hypertension. LY294002 normalized NE-induced
contraction in aorta from DOCA-salt rats and SHR rats to that of the response of
their respective normotensive controls, indicating that it is an alteration in PI3-
kinase that is responsible for enhanced contraction to NE. In the presence of
LY294002 the NE-induced contraction was rightward shifted in the LNNA rats,
however not to the level of the sham control. This difference between the models
may be due to: 1.) LNNA animals only receive the treatment for 2 weeks,
whereas the DOCA-salt animals has been treated for 4 weeks and thus the
vascular remodeling in LNNA rats may not be to the degree that it is in the
DOCA-salt rats, and 2.) LNNA rats by the end of 2 weeks have blood pressures
of approximately 200 mm Hg compared to the 180 mm Hg of the DOCA-salt rats,
or 175 mm Hg of the SHR. In summary, PIS-kinase is responsible for part if not
all the enhanced NE-induced contraction observed in aorta in the condition of

hypertension, further implicating Pl3-kinase in being important not just in altered

205

growth observed in contraction, but playing a major role in altered contractility as

well.

2. Low Mg 2+-ing1;,gooo oontraotion

Arterial spontaneous tone and enhanced contractility are considered
commonly observed in experimental forms of hypertension. Magnesium
deficiency has been found in multiple experimental models of hypertension
(Touyz eta/., 1991; Wells and Agrawal, 1992; Rusch and Kotchen, 1994; Saito at
al. 1995). In humans, the occurrence of magnesium deficiency has been
debated, largely based on a disagreement about how to measure Mg2+ (Resnick
efai, 1984; Resnick atai, 1997; Sasaki eta/., 2000; Fox atai, 2001). However,
even small alterations in M9” may induce vascular changes, increasing the risk
for cardiovascular related conditions. Mgzfideficiency leads to spontaneous tone
and enhanced agonist-induced contraction (Laurant and Berthelot, 1992; Laurant
at a/., 1997; Laurant and Touyz, 2000; Touyz, 2003). PIS-kinase mediates
spontaneous tone and enhanced NE-induced contraction observed in aorta from
DOCA-salt hypertensive rats. I proposed to examine whether a decrease in Mg2+
activates the PIS-kinase signaling cascade, eliciting the increase in spontaneous
tone and enhanced vascular contraction observed in hypertension.

[Mgz”]. levels are lower in vascular smooth muscle cells of SHR vs. WKY
rats and serum and erythrocyte Mg2+ levels are lower in DOCA-salt SHR

compared to SHR (T ouyz at a/., 1991; Rusch and Kotchen, 1994). Extracellular

206

Mg” deficiency, through Mg2+ removal, induces contraction of rat aorta, via the
activation of MAPK, Pl3-kinase and SH2 domain-containing proteins (Yang at al.,
2000b; Yang at a/., 2001). l determined that low extracellular Mg2+
concentrations enhanced spontaneous tone in aorta from both sham and DOCA-
salt rats, a tone that was eliminated by LY294002, a PIS-kinase inhibitor. Pl3-
kinase alters Ca2+ flux and upregulation of Pl3-kinase enhances spontaneous
tone in a calcium-dependent manner (Viard er a/., 1999; Macrez at a/., 2001).
Intracellular [Ca2+], and Ca2+ uptake in DOCA treated rats is inhibited when oral
Mg2+ is given to DOCA rats, suggesting a role for Mg2+ in the handling of Ca2+ (Kh
at a/., 2000). Low extracellular Mg2+ levels initiate contraction in canine basilar
arterial smooth muscle cells via Ca2+ influx through voltage-gated Caz“ channels,
intracellular Ca2+ release and activation of PKC and PIS-kinase (Yang at al.,
2000b). Thus, low Mgzfiinduced enhancement of spontaneous tone in aorta from
DOCA-salt rats is likely due to Pl3-kinase stimulation potentially via PIS-kinase
activating L-type Ca2+ channels and increasing [Ca2+].. Conversely, high Mg2+
attenuated spontaneous tone in the aorta from DOCA-salt hypertensive rats,
confirming other studies (Laurant and Berthelot, 1992). Interestingly, Class II
PIS-kinases require Mg2+ to elicit their lipid kinase activity as evidenced by the
enzymes only being able to phosphorylate Ptdlns(4)P in the presence of Mg2+
(Arcaro at at, 2000). This is unlike Class I Pl3-kinases that do not require M9”
to elicit their lipid kinase actions. The mechanism by which altered [M921].

—mediates it effects on Pl3-kinase is unknown and is under investigation.

207

PIS-kinase activity is increased in aorta from hypertensive DOCA-salt rats
as compared to sham rats. If low Mg” does indeed activate or disinhibit PI3-
kinase in vascular smooth muscle, PI3-kinase activity should be increased in the
smooth muscle of the aorta. When endothelium-denuded aortic strips from
DOCA-salt and sham rats were incubated in normal PSS and low Mg” PSS,
there was a trend for increases in PI3-kinase activity caused by low Mg”
stimulation of Pl3-kinase. These data do not quantitatively repeat earlier studies
in which there was statistically significantly higher PI3-kinase activity in the
DOCA as compared to sham. However in the present experiment, several
differences in protocol may account for not observing a significant increase in
PIS-kinase activity and these are all variables that may have masked and/or
altered the PIS-kinase activity: 1.) tissues were incubated in altered salt
conditions, 2.) tension was pulled on the strips to achieve optimum length, 3.)
PIS-kinase protein isolation buffer had Mg” present, and 4.) more time elapsed
until protein isolation. Nonetheless, there was a clear trend of increased activity
in the arteries incubated in low Mg” in isolated tissue baths. The small increase
in spontaneous tone in aorta of the sham rats compared to the large increase
observed in the aorta from DOCA-salt rats, combined with the fact that the low
Mg” incubation demonstrated a trend for increased activity in both, suggests that
while low Mg” can activate PI3-kinase, Pl3-kinase is not the sole component that

mediates spontaneous tone.

208

In DOCA-salt rats, Laurant at a/. (1995) determined the blood pressure-
lowering effect of magnesium supplementation in DOCA-salt hypertension was
associated with lower in viva cardiovascular reactivity to NE and Ang II. Other
studies have shown that low extracellular levels of Mg” potentiated NE-induced
vasoconstriction in mesenteric arteries from SHR but not in WKY rats and altered
vasopressin-induced vascular contraction; high Mg” attenuated both
vasopressin- and NE-induced vasoconstriction (Laurant at a/., 1997). My studies
demonstrated hyperreactivity to NE-induced in aorta from sham rats incubated in
low Mg” PSS resulting in similar potency of aorta from DOCA-salt rats.
However, in aorta from DOCA-salt rats, there was no further hyperreactivity to
NE-induced contraction. LY294002 (20 pmoI/L) normalized the NE-induced
enhanced contraction in all aortic strips. These data further support the idea that
Pia-kinase is responsible for enhanced NE-induced contraction, similar to
previous studies and that PI3-kinase mediates the enhanced NE-induced
contraction in the presence of low Mg”. The lack of a further leftward shift in NE-
induced contraction in aorta from DOCA-salt rats by low Mg” indicates that more
than PIS-kinase must be stimulated to further shift the contraction in the aorta, or
that NE-induced activation of PI3-kinase in the DOCA-salt rats is already
maximal, and NE, unlike spontaneous tone, may depend on PIS-kinase activity to
a lesser degree. LY294002 further inhibited spontaneous tone only a negligible
amount in the presence of high extracellular Mg” levels as compared to vehicle.

NE-induced contraction in the presence of high Mg” further rightward shifted

209

contraction than that as compared to normal Mg”, supporting others that high
Mg” may inhibit Ca” channels and not solely Pl3-kinase (Touyz, 2003). In
summary, these data demonstrate clearly that low Mg” has the ability to utilize
and/or promote Pl3-kinase activation in aorta as demonstrated by the increase in
magnitude of spontaneous tone in aorta from DOCA-salt rats and the NE-

induced leftward shift in contraction in aorta from sham rats.

F. Time Course Studies

A common question asked when discussing alterations in the condition of
hypertension is, “Which comes first; does the change in Pl3-kinase cause the
increase in blood pressure, or is it the change in blood pressure that causes the
increase in Pl3-kinase?” One means to examine this question is to use time
course studies. These studies revealed a trend for LY294002-inhibitable
increase in spontaneous tone as early as the 3'd day of DOCA-salt treatment, at
which time there was no increase in blood pressure. By the 5‘h day of DOCA-salt
treatment there was a statistically significant increase in blood pressure, and
spontaneous tone that could be inhibited by LY294002. The enhanced NE-
induced contraction observed in aorta in DOCA-salt rats, appears to occur prior
to the significant increase in blood pressure (Day 3) and this shift can be inhibited
by LY294002, suggesting that this shift is a PIS-kinase mediated event.
However, no discemable changes occurred with respect to Pl3-kinase protein or

the classical pAkt Pi3-kinase signaling pathway. However, the alterations that

210

occurred may have been too small to detect using westerns, may have occurred
later in development and it may have just been an increase in PIS-kinase activity,
as opposed to protein density, although this seems unlikely. These data are also
confounded by the fact that hypertension in the rat does not develop on the exact
same time scale for every animal, thus the changes may have occurred at
slightly different time points. In addition, the changes that occur may have
happened during a time period in which I didn’t isolate protein, or do contractility
measurements. However, these data do suggest that spontaneous tone,
enhanced NE-induced contraction, Pl3-kinase function and the elevation in blood
pressure appear to occur just prior to the increase in blood pressure or at least

synergistically with the change in blood pressure.

211

SPECULATION AND CONCLUSIONS

The main focus of the work contained in this dissertation was to gain a
better understanding of potential alterations in PI3-kinase in the condition of
hypertension and how these alterations would effect arterial contraction with
respect to enhanced spontaneous tone and agonist-induced contraction. P13-
kinase is a promiscuous enzyme being involved in many cellular functions as well
an enzyme that is still being extensively studied and revealed to regulate new
actions in the cell.

In the condition of hypertension, there is smooth muscle cell growth,
enhanced contraction and spontaneous tone. The classical way in which PI3-
kinase is thought of is in with respect to cellular growth. However, I wanted to
examine a potential role for Pl3-kinase in eliciting enhanced agonist-induced
contraction and spontaneous tone in hypertension. Several investigators have
implicated PIS-kinase in having the ability to mediate contraction in arteries
(Yang et a/., 2000a; Komalavilas at a/., 2001; Yang at a/., 2001). Thus, it stood
to reason that the Pl3-kinase that was mediating enhanced growth might also be
contributing to the enhanced contraction observed in hypertension. Therefore, I
hypothesized that there was enhanced Pl3-kinase protein/activity in the condition
of hypertension that played vital roles in enhanced agonist-induced contraction
as well as in spontaneous tone development observed.

Ca” involvement in spontaneous tone in hypertension has been

previously established (Thompson at ai, 1987; Webb at a/., 1992; Lamb at a/.,

212

1995; Pucci at a/., 1995; Sunano at a/., 1996; Rapacon-Baker at a/., 2001). l
demonstrated that Pl3-kinase plays a major role in this Ca”-mediated
spontaneous tone. However, the mechanism by which Pl3-kinase activates this
increase in Ca” flux is still unknown. Pl3-kinase p110 subunits have the ability
to activate L-type calcium channels (Macrez at a/., 2001; Viard at a/., 1999).
However, due to the lack of an increase in the or, L-type calcium channel subunit,
these data suggest that PI3-kinase has the ability to make more channels
functional, or is initiating a longer opening of the channel. The exact mechanism
by which PIS-kinase alters L-type calcium channel function has yet to be
elucidated. However, ion flux studies in reconstituted phospholipid vesicles show
that phosphorylation of the or1 and [3 subunits can greatly increase the number of
functional Ca” channels after phosphorylation (Catterall, 2000). Pl3-kinase has
serine kinase function as well as lipid kinase function, thus it may be that PI3-
kinase phosphorylates L-type calcium channels in order to increase calcium flux
through the channel, possibly requiring direct interaction of PIS-kinase and the
calcium channel.

lmportantly, these experiments revealed an increase in Pl3-kinase activity,
at least partially via p1 106. It was a surprise that p1108 was present in vascular
smooth muscle, because p1108 was hypothesized to be preferentially in
hematopoietic cells. Recently, p1106 was detected in breast cancer cells and in
melanocytes and to play a major role in EGF-driven in vitro migration of breast

cancer cells, demonstrating that p1106 is found and functionally significant in

213

other nonhematopoietic tissue (Sawyer at a/., 2003). It is not apparent though
how different p110 isoforms play specific functional roles in cells. p110 isoforms
do have the capability to be differentially regulated. CD-28-mediated recruitment
of p11OB results in an increase in its lipid kinase activity, whereas recruitment of
p1105 results in a decrease in lipid kinase activity (Benistant at a/., 2000).
Moreover, in leukocytes under redox stress there is an interaction of Ras with
p1108 and p1108 but not with p110a, suggesting specific p110 targeting
(Vanhaesebroeck at a/., 2001). Recently, knock-in mice expressing a mutated
and catalytic dead p1105 were created, in turn demonstrating no p1105 activity

(Okkenhaug at a/., 2002). These mice provide another tool to examine the
cellular function and importance of the p1106 PIS-kinase subunit. In addition,
DOOO, a recently developed PIS-kinase p1106-specific inhibitor, was unable to
inhibit some of the classical downstream targets of PIS-kinase, Akt, Erk2, S6
kinase and GSK-3B, suggesting p1108 does not signal via the classical PI3-
kinase mediated pathways (Sawyer at a/., 2003). The leos of DOOO for the
p110 Pl3-kinase subunits, determined by in vitro lipid kinase assays, were
reported to be 0.33 umol/L and 7.7 umol/L for p1108 and p1107, respectively,
whereas inhibition of 50% could not be attained for p110a or p1 108 (Sawyer at
a/., 2003). These data suggest DOOO is relatively specific for p1108 and not the
other p110 subunits. Another inhibitor that may assist in delineating p1108
involvement in contraction is IC87114, a recently developed PI3-kinase p1108

specific inhibitor (Sadhu at at, 2003). All of the class 1A isoforms are capable of

214

producing the same lipids in vitro, however little is known concerning the serine
kinase specificity of the subunits, thus this may be one mechanism in which the
isoforms direct specific functions in cells. In summary, these data support the
role of p1106 in a non-classical signaling pathway, the presence of p1106 in
vascular smooth muscle and upregulation of related PIS-kinase activity and
protein in hypertension. Further experiments with p1105-specific inhibitors
and/or genetically altered animals will have to be performed to determine if p1106
is responsible for the enhanced contraction and spontaneous tone observed in
hypertension.

These experiments have also demonstrated PI3-kinases importance in
enhanced agonist-induced contraction, as evidenced by the NE-induced
contraction studies, in multiple models of hypertension. PI3-kinase also plays a
vital role in spontaneous tone development in resistance arteries as well as aorta
from hypertensive animals compared to sham. Low extracellular Mg”, a
phenomenon found in hypertension, appears to mediate its altered contraction at
least partially via PIS-kinase. Finally, the time course studies demonstrated that
the alterations in PIS-kinase dependent spontaneous tone and alterations in
contraction appear to go hand in hand with the development in hypertension.

In conclusion, PIS-kinase is not only important in growth regulation and
cellular migration, as l have demonstrated PI3-kinase also plays a vital role in
contraction in hypertension (Figure 61), potentially via upregulation of the p1108

subunit. Further investigation will reveal the mechanism by which Pl3-kinase

215

 

 

 

Figure 61. A schematic diagram depicting the novel role of PIS-kinase in
mediating enhanced contraction and spontaneous tone in arteries from
hypertensive rats. In addition to the often-documented role of PIS-kinase in
maintenance of growth, Pl3-kinase activity and protein are increased in the
condition of hypertension that ultimately lead to enhanced contraction and
spontaneous tone in aorta and resistance arteries, in turn leading to an increase

in total peripheral resistance and ultimately an increase in systolic blood pressure.

216

 

 

 

 

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217

regulations contraction, whether it is direct action of the lipid kinase activity or
protein kinase activity, whether specific Ptdlns traffic to specific proteins and
whether class II and class III also have involvement in altered contraction in
hypertension. Further research will also identify the potential therapeutic
capabilities of using PI3-kinase subunit specific inhibitors in the regulation of

hypertension, as well as in other diseases.

218

 

 

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231

EEESQNALDAIA

CURRICULUM VITAE

Carrie A. Northcott

Name: Carrie Annalice Northcott B445 Life Science Building

Born: 9/21/73

Department of Pharmacology and Toxicology
Michigan State University

East Lansing, MI 48824-1317

Phone: (517) 353-3900

Fax: (517) 353-8915

E-mail: taetscar@ msu.edu

W

1991 -1 993

1 993-1995

1 995-1 998

1 999-present

Black Hawk College, Kewanne, Illinois
A.S. (Agriculture Transfer)

University of Illinois, Champaign-Urbana, Illinois
B.S. (Agronomy)

University of Illinois, Champaign-Urbana, Illinois
M.S. (Crop Sciences)

Thesis Title: “Flow Cytometric Bioanalysis of
Environmental Contaminants.”

Michigan State University, East Lansing, Michigan
doctoral candidate (Pharmacology and Toxicology)

Project Title: “Phosphoinositide 3-kinase
upregulation in hypertension: a reason for enhanced
arterial contraction and tone. ”

W

1996 Teaching Assistant, Plant and Animal Genetics,
(NRES 220, ANSCI 220, CPSC 220)
University of Illinois

1997 Teaching Assistant, Applied Statistical Methods, (NRES 340,
ANSCI 340, AGE 340, CPSC 340)
University of Illinois

232

T
2002

2002

2002

2003

Carrie A. Northcott

VIT ’
Lecturer, Introduction to Chemical Toxicology (th 450)
Cardiovascular Toxicology Section
Michigan State University

Tutor for Medical Pharmacology(th 563)
Michigan State University

Tutor for Veterinary Pharmacology (th 556)
Michigan State University

Lecturer, Introduction to Chemical Toxicology (th 450)
Cardiovascular Toxicology Section
Michigan State University

W

1 993-1 995

1995-1998

1998-1999

1999

Lab Assistant: Molecular Cytogenetics Laboratory,
University of Illinois. Independent research projects and
assisted graduate students with research, organized files
and general laboratory maintenance. Supervisor: Dr. A.
Lane Rayburn

Research Assistant: Molecular Cytogenetics
Laboratory, University of Illinois. Determined effects of
agrichemicals on animal and plant genomes by flow
cytometry. Supervisor: Dr. A. Lane Rayburn

Visiting Research Specialist: Molecular Cytogenetics
Laboratory, University of Illinois. Examined protective
effects of potential anti-carcinogen, PCC, on DNA in bone
marrow and tissue culture cells. Supervisors: Dr. A. Lane
Rayburn, Dr. Michael Plewa and Dr. Bettina Francis

Research Rotation: Cardiovascular Pharmacology
Laboratory, Michigan State University. EGF-induced
arterial contraction in non-hypertensive diabetic rats.
Supervisor: Dr. Stephanie W. Watts

233

Carrie A. Northcott

W
1999-present Doctoral Thesis Research: Cardiovascular

Pharmacology Laboratory, Michigan State University.
Phosphoinositide 3-kinase upregulation in hypertension: a
reason for enhanced arterial contraction and tone. Mentor: Dr.
Stephanie W. Watts

P F l A T ITI

American Physiological Society (APS)

American Society for Pharmacology and Experimental Therapeutics
(ASPET)

Graduate Recruitment and Education Committee-ASPET

Society for Experimental Biology and Medicine (SEBM)

Reviewer for Hype/tension Journal

Reviewed ASPET 2003 Cardiovascular Best Paper Awards

Reviewed SEBM 2002 Travel Awards

Phi Theta Kappa Honor Society

W

1991- 1992 Henry County Farm Bureau Scholarship

1991- 1993 Academic Achievement Scholarship

1993 Outstanding Agriculture Transfer Student

1994-1995 0.8. Carmen Scholarship

1 996-1 998 Pioneer Fellowship

1996-1998 Environmental Toxicology Scholar

1997 Alumni Award for Student Travel

2001-present American Heart Association Predoctoral Grant

2001 SEBM Travel Grant Award

2001 Merck New Investigator Award

2002 ASPET Graduate Student Travel Award

2002 ASPET Cardiovascular Division Best Paper Award

2002 New Investigator Award for US. Fellows

2002 Sigma Xi Graduate Student Award

2003 IASH/National Heart Lung and Blood Institute Travel Award
PRESENTATIONS

Fall 1996 “The Effects of Herbicide Interaction on Chinese Hamster

Ovary Cells” -Poster presentation, American Society of
Agronomy Meetings, Indianapolis, IN

234

Carrie A. Northcott

W

Fall 1997

Spring 1997
Spring 1998

Spring 2000

Spring 2001

Summer 2001

Summer 2001

Fall 2001

Fall 2001

“Whole Cell Clastogenicity of Atrazine on Aquatic Plants” -
Oral presentation, American Society of Agronomy Meetings,
Anaheim, CA

“Flow Cytometric Monitoring of Aquatic Ecosystem
Subjected to Periodic Flooding” -Oral presentation, Illinois
Groundwater Consortium, Makanda, IL

“The Ability of Epidermal Growth Factor to Contract Summer
2000 Thoracic Aorta from Streptozotocin-Induced Diabetic
Rats” -Oral presentation, 1st year research rotation seminar;
Pharmacology Research Colloquium; Annual Phi Zeta
Research Day, Michigan State University, MI; and Michigan
Hypertension Meetings, Gull Lake, MI

“Epidermal Growth Factor-Induced Arterial Contraction; Lack
of Effect in Non-Hypertensive Diabetic Rats” —Poster
presentation, Experimental Biology, Orlando, FL

“Pl3-Kinase Inhibition Abolishes EGF-Induced and
Spontaneous Contraction in Aorta from Doca-salt
Hypertensive Rats” —Ora| presentation, Pharmacology
Research Colloquium, Detroit, MI

“Epidermal Growth Factor-Induced Arterial Contraction; Lack
of Effect in Non-Hypertensive Diabetic Rats” —Poster
presentation, Annual Phi Zeta Research Day. Michigan
State University, Ml

“Phosphoinositide 3-Kinase Inhibition Abolishes
Spontaneous and Epidermal Growth Factor-Induced Arterial
Contraction in Mineralooorticoid Hypertension” —Poster
presentation, Council for High Blood Pressure Research
2001 meetings, Washington DC.

“Pl3-Kinase; A Major Component of EGF-Induced and
Spontaneous Contractions in Aorta from Doca-salt
Hypertensive Rats?” — Oral presentation, Seminar, Michigan
State University, MI

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Carrie A. Northcott

P EN on ’

Spring 2002 “Increased Phosphoinositide 3-Kinase Activity as a Cause
for Enhanced Contractility in Deoxycorticosterone (DOCA)-
salt rat hypertension” -Poster and Oral presentation,
Experimental Biology, New Orleans, LA; Oral presentation-
Michigan Hypertension Meetings, Gull Lake, MI and
Pharmacology Research Colloquium, Toledo, OH

Fall 2002 “Phosphoinositide 3-Kinase and Calcium: Partners in
Spontaneous Tone?”-Poster presentation, Council for High
Blood Pressure Research 2002 meetings, Ortando, FL

Spring 2003 “Low Magnesium (Mg”) as an Activator of Arterial
Phosphoinositide 3-Kinase (PI3K) Resulting in Enhanced
Contractility in Deoxycorticosterone (DOCA)-salt
Hypertension” -Poster presentation, XVth Inter-American
Society of Hypertension Meetings 2003, San Antonio. TX

BALEBS
Toots, C.A,: The effects of herbicide interaction of Chinese hamster ovary cells.
Journal of Natural Resources and Die Sciences Education. 25:81 -84, 1996.

McMurphy, L.M, Biradar, D.P.. 1315.121}... and Rayburn, A.L.: Differential effects
of weathered fly ash and fly ash leachate on the maize genome. Archives of
Environmental Contamination and Toxicology. 31 :166-169. 1996.

Toots, C,A.. and Rayburn, A.L.: The clastogenic potential of herbicides found in
Illinois Groundwater III. Ill/halls Groundwater Consortium Proceedings. 6:219-
226, 1996.

Taets. CA. and Rayburn, A.L.: Coal fly ash exposure at agronomic levels does
not induce triploidy in the maize genome. Bulletin of Environmental
Contamination and Toxicology. 56:690-695. 1996.

loots, C.A,, and Rayburn, A.L.: Flow cytometric monitoring of aquatic

ecosystems subjected to periodic flooding. Ill/hols Groundwater Consortium
Proceed/ngs. 7:48-55, 1997.

236

Carrie A. Northcott
AP ’
Toots, CA, Aref, S. and Rayburn, A.L.: The clastogenic potential of triazine

herbicides found in potable water supplies. Environmental Health Perspectives.
106 (4)2197-201, 1998.

Rayburn, AL and Nogthoott, 9A.: Flow cytometric monitoring of aquatic
ecosystems subjected to periodic flooding. ill/hois Groundwater Consortium
Proceedings. 8:70-82, 1998.

Aref, S.. Kocheriginsky, M.. Noflhoon, CA, and Rayburn, A.L.: Analysis of nuclei
fluorescence histograms using non-linear functions or wavelets. Proceedings of
the Conference on Applied Statistics in Agriculture. 1 1 270-82, 1999.

Rayburn, A.L., Bouma. J., and Nonhoott, C,A,: Comparing the clastogenic
potential of atrazine with caffeine using Chinese hamster ovary (CHO) cells.
Toxicology Letters 1 21 :69-78, 2001.

We... Florian. J.A., Dorrance, A. and Watts. S.W.: Arterial epidermal
growth factor receptor expression in deoxycorticosterone acetate-salt
hypertension. Hypertension. 38(6):1337-41, 2001.

Watts, S.W.. Fink, G.D.. W and Galligan, J.J.: Endothelin-1-induced
venous contraction is maintained in DOCA-salt hypertension; studies with
receptor agonists. Bn’tish Journal ofPhannaco/ogy. 137:69-79, 2002.

Noohoon. C.A,, Poy. M.N.. Najjar, SM. and Watts. S.W.: PI3-kinase mediates
enhanced spontaneous and agonist-induced contraction in aorta of DOCA-salt
hypertensive rats. Circulation Research. 91 :360-369. 2002.

Li. L., Fink, G.D.. Watts, S.W., Nonhoott, 9A,, Galligan, J.J.. Pagano, P.J. and
Chen. A.F.: Endothelin-1 increases vascular superoxide via endothelinA-NADPH
oxidase pathway in low-renin hypertension. Circulation. 107:1053-1058, 2003.

Loberg. R.D., Northcott, CA, Watts, SW. and Brosius Ill, F.C.: Role of GSK-3
activity in arterial reactivity during DOCA-salt hypertension. Hmertensr‘on.
41:898-902. 2003.

Atkins, K.B.. Nogthoott, Q.A., Watts, SW. and Brosius III, F.C.: Effect of PPARg
ligands on hypertension and vascular smooth muscle marker expression.
Hypertension. Submitted, 2003.

Noohoon, CA. and Watts. S.W.: Low Mg” enhances arterial spontaneous tone
via Pl3K in DOCA-salt hypertension. Hmertensron. Submitted, 2003.

237

Carrie A. Northcott

W
Nonhcott, CA. Watts, SW. and Hsueh, W.: Vasoactive growth factors and

adhesion molecules. In: lzzo. J.L. Jr. and Black. H.R. eds. Hypertension Primer,
Third edition. Philadelphia, PA: Lippincott Williams 8. Wilkins; pp. 66-69. 2003

AB T T

Northcott CA. and Watts, S.W.: Epidermal growth factor-induced arterial
contraction: lack of effect in non-hypertensive diabetic rats. FASEB J. 15:A215.
203.8, 2001.

Watts, S.W., Northcott, CA. Luo. M.. Galligan, J.J.. and Fink, G.D.: Venous
smooth muscle retains responsiveness to endothelin in deoxycorticosterone
acetate salt hypertension. Hypertension. 38:P163. 519. 2001.

Noflhoon CA. and Watts. S.W.: Phosphoinositide 3-Kinase inhibition abolishes
spontaneous and epidermal growth factor-induced arterial contraction in
mineralocorticoid hypertension. Hypertension. 38:P19, 493, 2001.

Noohcott, CA, Poy. M.N., Najjar, SM. and Watts. S.W.: Increased
phosphoinositide 3-kinase (PI3-K) activity as a cause for enhanced contractility in
deoxycorticosterone (DOCA)-salt hypertension FASEBJ 16:A572. 444.8, 2002.

Loberg. R.D., Northoott, 9A.. Brosius, PC, and Watts. S.W.:
Phosphatidylinositidol 3-Kinase Mediated Vascular Hyperreactivity of Arteries
from DOCA Hypertensive Rats is Independent of Akt and GSK-3 Activity.
American Heart Association Council of High Blood Pressure Meetings. Abstract
Hypertension. 40:P201, 431. 2002.

Northoott, CA. and Watts, S.W.: Phosphoinositide 3-Kinase and Calcium:
Partners in Spontaneous Tone? American Heart Association Council of High
Blood Pressure Meetings. Hypertension. 40:P212. 432, 2002.

Francis, B.M., Northoott, CA. and Rayburn. A.L.: Lack of in vivo Genotoxicity of a
Dietary Soy Supplement. Society of Toxicology Meetings. pp. 122, 2003.

Northcott. QA. and Watts, S.W.: Low magnesium (Mg”) as an activator of
arterial phosphoinositide 3-kinase (PI3K) resulting in enhanced contractility in
deoxycorticosterone (DOCA)-salt hypertension. X Vth Inter-American Society of
Hypertension Meetings. Ap ril, 2003.

238

Carrie A. Northcott

AB TRA c n ’

Northcott, CA. and Watts, S.W.: Phosphoinositide 3-kinase (PI3-kinase)
involvement with spontaneous tone development in mesenteric resistance
arteries from deoxycorticosterone acetate (DOCA)-salt rats: is p110d the culprit?
American Heart Association Council of High Blood Pressure Meetings.
September. 2003.

Northcott. C.A.. Loberg. RD, and Watts, S.W.: Are rho kinase and
Phosphoinositide 3-kinase (PI3-kinase) cohorts in spontaneous tone
development in DOCA (deoxycorticosterone acetate)-salt rat hypertension?
American Heart Association Counc/l otHrgh Blood Pressure Meetings.
September, 2003.

239