ALTERATIONS IN THE BRAIN RENIN-ANGIOTENSIN SYSTEM IN A MODEL OF FETAL ...

221 Pages·2014·2.07 MB·English

by  Academic Computing

ALTERATIONS IN THE BRAIN RENIN-ANGIOTENSIN SYSTEM IN A MODEL OF
FETAL PROGRAMMING

BY

ALLYSON CATHERINE MARSHALL

A Dissertation Submitted to the Graduate Faculty of
WAKE FOREST UNIVERSITY GRADUATE SCHOOL OF ARTS AND SCIENCES
In Partial Fulfillment of the Requirements for the Degree of
DOCTOR OF PHILOSOPHY
Integrative Physiology and Pharmacology
May 2014
Winston Salem, North Carolina

Approved by:
Mark C. Chappell Ph.D., Advisor
Debra I. Diz, Ph.D., Advisor
Examining Committee:
Robert N. Taylor, M.D., Ph.D., Chair
Hossam A. Shaltout, Ph.D.
Lisa K. Washburn, M.D.
ACKNOWLEDGEMENTS
This dissertation is the product of the tremendous guidance and expertise of my
thesis advisors Drs. Mark C. Chappell and Debra I. Diz. You make an amazing
mentoring team, and I am so grateful for the opportunity to work with both of you. This
experience has shaped me as a scientist and person, and I am proud of the research we
produced. I would also like to thank members of my doctoral committee, Robert Taylor,
Lisa Washburn, and Hossam Shaltout. The direction and input you gave me during our
meetings was essential in shaping this project.
In addition, I would like to thank all the faculty, trainees, and technicians of the
Hypertension and Vascular Research Center. The center truly feels like a family, and
everyone here has helped me through sharing ideas, advice, supplies, or offering support.
To Ellen Tommasi and Nancy Pirro, thank you for your training and patience. I began
my first year with very little research experience and you helped me settle into the labs
and graduate school.
Lastly, I thank my family and friends. Mom, Dad, and Christine, thank you for
your constant support, love, and encouragement. This would have been impossible
without you.

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TABLE OF CONTENTS
PAGE
LIST OF FIGURES AND TABLES……………………………………………………...v
LIST OF ABBREVIATIONS…………………………………………………………….ix
ABSTRACT AND AIMS………………………………………………………………...xi
CHAPTER ONE: INTRODUCTION……………………………………………………..1
Fetal Programming and Developmental Plasticity
The Central Renin-Angiotensin System
Angiotensin Metabolism by Endogenous Peptidases
The Renin-Angiotensin System and Blood Pressure Control
Rationale and Aims
Literature Cited
CHAPTER TWO:
FETAL BETAMETHASONE EXPOSURE ATTENUATES THE
ANGIOTENSIN-(1-7)-MAS RECEPTOR AXIS IN THE DORSAL MEDULLA
OF ADULT SHEEP……………………………………………………………...43
Published in Peptides 44: 25-31, 2013.
CHAPTER THREE:
ANTENATAL BETAMETHASONE EXPOSURE IS ASSOCIATED WITH
LOWER CSF ANG-(1-7) AND INCREASE CSF ACE IN ADULT SHEEP…..70
Published in American Journal of Physiology: Integrative, Comparative, and
Regulatory Physiology 305: R679-688, 2013.
CHAPTER FOUR:
ENHANCED ACTIVITY OF AN ANGIOTENSIN-(1-7) NEUROPEPTIDASE
IN GLUCOCORTICOID-INDUCED FETAL PROGRAMMING…………….108
Published in Peptides 52C:74-81, 2013.

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CHAPTER FIVE:
EVIDENCE FOR AN ANGIOTENSIN-(1-7) NEUROPEPTIDASE
EXPRESSED IN THE BRAIN MEDULLA AND CSF OF SHEEP…………..135
Published in The Journal of Neurochemistry, 2014.
CHAPTER SIX: SUMMARY AND CONCLUSIONS………………………………..167
Summary of Findings
Fetal Programming and RAS Involvement
Localization and Specificity of ChP RAS Alterations
Ang-(1-7) Metabolism in the CSF
Brain Medullary Ang-(1-7) Peptidase Activity
Subcellular Localization and Peptidase Release
General Limitations of Studies
Concluding Statements
COPYRIGHT AND PERMISSION FOR PUBLISHED MANUSCRIPTS…………...199
SCHOLASTIC VITA…………………………………………………………………..201

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LIST OF FIGURES AND TABLES
CHAPTER TWO
Figure 1: Mas and AT1 Receptor Expression in the Dorsal Medulla
of 0.5- and 1.8-year Old Sheep…………………………..64

Figure 2: Validating Angiotensinogen Antibodies in Sheep: Ang I
Intact Angiotensinogen and Internal Angiotensinogen…..65

Figure 3: Ang I Intact Angiotensinogen is Lower in Dorsal Medulla
of BMX Offspring at 0.5-years and Higher at 1.8-years of
Age……………………………………………………….66

Figure 4: Angiotensin Peptide Content and Ratios in 0.5-year Old
Offspring…………………………………………………67

Figure 5: Angiotensin Peptide Content and Ratios in 1.8-year Old
Offspring…………………………………………………68

Figure 6: Correlation Between Mas and AT Receptor Expression
1
and Peptide Levels………………………………………69

CHAPTER THREE
Figure 1: Ang I Intact Angiotensinogen Protein Expression in
Choroid Plexus Tissue…………………………………...99

Figure 2: (Pro)renin Protein Expression in Choroid Plexus
Tissue...............................................................................100

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Figure 3: Peptidase Activity in Solubilized Fraction of Choroid
Plexus from Control and Betamethasone Exposed
Offspring………………………………………………..101

Figure 4: Peptidase Activity in Brush Border Enriched Fraction of
Choroid Plexus tissue…………………………………...102

Figure 5: Angiotensin Peptide Content in Choroid Plexus and
Cerebrospinal Fluid Samples…………………………...103

Figure 6: Ang I Intact Angiotensinogen Expression in Cerebrospinal
Fluid…………………………………………………….104

Figure 7: Analysis of Ang-(1-7) Metabolism in Cerebrospinal Fluid
of Control and BMX Offspring…………………………105

Figure 8: Saturation Curves for Ang-(1-7) and Ang II Metabolism by
PCMB Sensitive Enzyme in the Cerebrospinal Fluid…..106

Figure 9: Diagram of Potential Localization of RAS Components in
the Choroid Plexus and Cerebrospinal Fluid…………...107

CHAPTER FOUR:
Figure 1: PCMB and o-phenanthroline Abolish Ang-(1-7)
Metabolism……………………………………………..128

Figure 2: Cysteine Peptidase Inhibitors and Chelating Agents Reduce
Peptidase Activity………………………………………129

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Figure 3: Optimal pH for Peptidase Activity is 7.5 in Control and
Betamethasone Exposed Animals………………………130

Figure 4: Analysis of Competition or Inhibition of Angiotensin
Peptide for Peptidase Activity………………………….131

Figure 5: Mean Arterial Pressure and Peptidase Activity are Higher
in Betamethasone Exposed Animals……………………132

Figure 6: Proposed Schematic for the Role of the Neuropeptidase in
RAS Processing Pathways in Brain Medulla, Cerebrospinal
Fluid, and Choroid Plexus……………………………...133

Table 1: Specific Inhibitors do not Inhibit Peptidase Activity…..134

CHAPTER FIVE:
Figure 1: Ang-(1-7) Metabolizing Peptidase Activity in the Brain
Medulla and Cerebrospinal Fluid…………………….…158

Figure 2: Chelating Agents, but not Specific Inhibitors, Block
Peptidase Activity………………………………………159

Figure 3: JMV Inhibits Peptidase Activity at Subnanomolar
Concentrations………………………………………….160

Figure 4: Apparent Kinetic Constants for Ang-(1-7), Ang II, and Ang
I…………………………………………………………161

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Figure 5: Purified Peptidase Lacks Other Ang-(1-7) Metabolizing
Enzymes………………………………………………..162

Figure 6: UV HPLC Analysis of Angiotensin Peptides Incubated
with Peptidase…………………………………………..163
Figure 7: UV HPLC Analysis of Neuropeptides Incubated with
Peptidase………………………………………………..164

Table 1: Purification of Peptidase from Starting Material……….165

Tabe 2: Comparison of Peptidase Activity in Brain Medulla and
CSF………………………...…………………………...166

Table 3: Comparison of Metabolism Velocities for Angiotensins and
other Neuropeptides……………………………...…......167

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LIST OF ABBREVIATIONS

11β-HSD = 11β-hydroxysteroid dehydrogenase
ACE = Angiotensin converting enzyme
AM = Amastatin
Aogen = Angiotensinogen
Ang = Angiotensin
Ang I = [Asp1-Arg2-Val3-Tyr4-Ile5-His6-Pro7-Phe8-His9-Leu10]
Ang II = [Asp1-Arg2-Val3-Tyr4-Ile5-His6-Pro7-Phe8]
Ang-(1-7) = [Asp1-Arg2-Val3-Tyr4-Ile5-His6-Pro7]
Ang-(1-4) = [Asp1-Arg2-Val3-Tyr4]
APMA = aminophenylmercuric acetate
BBM = Brush border membrane
BM = Betamethasone
BMX = Betamethasone exposed
BRS = Baroreflex sensitivity
BS = Bestatin
BSC = Benzylsuccinate
ChP = Choroid plexus
ChP4 = Choroid plexus of the fourth ventricle
CHYM = Chymostatin
CSF = Cerebrospinal fluid
DEAE = Diethylaminoethyl sepharose
DTT = Dithiothreitol
EDTA = Ethylenediaminetetraacetic acid
EGTA = Ethylene glycol tetraacetic acid
GC = Glucocorticoid
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HPLC = High performance liquid chromatography
JMV-390 = N-[3-[(hydroxyamino) carbonyl]-1-oxo-2(R)-benzylpropyl]-L-leucine
LIS = Lisinopril
MAP = Mean arterial pressure
NEP = Neprilysin
NTS = Solitary tract nucleus
PCMB = P-chloromercuribenzoic acid
PRR = (Pro)renin receptor
RAS = Renin-angiotensin system
SDS = Sodium dodecyl sulfate
SEM = Standard error of the mean
TOP = Thimet oligopeptidase

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