Table Of ContentAlpha1-Adrenergic Receptor Function and Stability in 1st Versus 3rd Order Mesenteric
Arteries and Veins of Normal Male Sprague Dawley Rats
by
Shawn Veitch
A Thesis
presented to
The University of Guelph
In partial fulfilment of requirements
for the degree of
Masters of Science
in
Biomedical Sciences
Guelph, Ontario, Canada
© Shawn Veitch, January, 2014
ABSTRACT
α -ADRENERGIC RECEPTOR FUNCTION AND STABILITY IN 1ST VERSUS 3RD
1
ORDER MESENTERIC ARTERIES AND VEINS OF NORMAL MALE SPRAGUE
DAWLEY RATS
Shawn Veitch Advisor:
University of Guelph, 2014 Dr. Ronald Johnson
Vascular branching facilitates blood flow changes and maintains cardiovascular
homeostasis. Vasomotor tone is partly maintained by sympathetic nerves, ensuring moment-to-
moment adjustments of net vascular tone. Norepinephrine (NE), phenylephrine and, sympathetic
neurogenic mediated contractility was evaluated in 1st and 3rd order rat mesenteric small arteries
(MA) and small veins (MV), exploring relative contributions from α-adrenergic receptors (α-
AR). α -AR subtype expression was quantified following NE exposure, focusing on microRNA-
1
30c activity. The results suggested that MV are more sensitive to AR activation than MA; α -
1
ARs predominantly regulate AR-mediated contractility, with functional evidence of α -AR
2
activity in 1st order MA and 3rd order MV; NE reduces α -AR mRNA expression in MV,
1
possibly related to venous relaxation at high agonist concentration, but does not involve miRNA-
30c activity; furthermore receptor isoform expression differs between 1st and 3rd order MA. This
represents another level of regulation for branched vascular beds, enabling more precise control
over vascular tone.
KEYWORDS: Branching, sympathetic nerves, norepinephrine, adrenergic receptors, microRNA
TABLE OF CONTENTS
INTRODUCTION .................................................................................................................................... 1
1.0 The Arterial and Venous System ..................................................................................... 1
1.1 Overview of vascular function ................................................................................ 1
1.2 Overview of vascular branching ............................................................................. 2
1.3 Comparison of arteries and veins ........................................................................... 4
1.3.1 Morphology ............................................................................................... 4
1.3.2 Contractility .............................................................................................. 5
1.3.3 Local metabolic control ........................................................................... 6
1.3.4 Innervation ................................................................................................ 7
2.0 Vascular smooth muscle cells (VSMCs ........................................................................... 9
2.1 Contraction of VSMCs ............................................................................................ 9
2.2 Relaxation of VSMCs ........................................................................................... 10
3.0 Sympathetic Nervous System Activity ........................................................................... 11
3.1 Overview of sympathetic nervous system ............................................................ 11
3.2 Norepinephrine ....................................................................................................... 12
3.3 Norepinephrine regulation of α-Adrenergic receptor mRNA ........................... 14
4.0 microRNA ............................................................................................................................. 15
4.1 miRNA structure and function .............................................................................. 15
4.2 miRNA biogenesis ................................................................................................... 16
4.3 miRNA involvement in cardiovascular biology and disease ............................ 17
Rationale ....................................................................................................................................... 19
CHAPTER 1 - CHARACTERIZATION OF α-ADRENERGIC RECEPTOR FUNTION IN 1ST
VERSUS 3RD ORDER MESENTERIC ARTERIES AND VEINS ...................................................... 22
Introduction ............................................................................................................................... 23
Materials and Methods ........................................................................................................... 25
Animals ........................................................................................................................... 25
Vessel isolation .............................................................................................................. 25
Assessment of vascular function ................................................................................. 26
Experimental protocols ................................................................................................. 27
Concentration contractile response studies ................................................... 27
Electrical field stimulation .............................................................................. 27
Data analysis .................................................................................................................. 28
Results .......................................................................................................................................... 29
General ............................................................................................................................ 29
Sympathetic neurogenic contractions in 1st versus 3rd order MA and MV ............ 30
Norepinephrine mediated contractions in 1st versus 3rd order MA and MV ......... 31
Phenylephrine mediated contractions in 1st versus 3rd order MA and MV ............ 32
Discussion .................................................................................................................................... 33
α-adrenergic receptor activity ....................................................................................... 34
Branching order differences ............................................................................................. 38
Conclusion ................................................................................................................................... 41
iii
CHAPTER 2 – CHARACTERIZATION OF α-ADRENERGIC RECEPTOR mRNA STABILITY
IN 1ST VERSUS 3RD ORDER MESENTERIC ARTERIES AND VEINS ........................................... 63
Introduction ................................................................................................................................. 64
Materials and Methods ........................................................................................................... 66
Sample collection .......................................................................................................... 66
Total RNA extraction ................................................................................................... 67
cDNA synthesis ............................................................................................................. 68
Real-time polymerase chain reaction (qPCR) ........................................................... 68
Data analysis .................................................................................................................. 69
Results .......................................................................................................................................... 69
Effects of NE exposure on α -AR mRNA and miR-30c expression in 1st and 3rd
1
order MA ........................................................................................................................ 69
Effects of NE exposure on α -AR mRNA and miR-30c expression in 1st and 3rd
1
order MV ........................................................................................................................ 70
Effects of NE exposure in arteries compared to veins ............................................. 70
Discussion .................................................................................................................................... 71
Conclusion ................................................................................................................................... 75
GENERAL DISCUSSION ....................................................................................................................... 95
REFERENCES ........................................................................................................................................ 105
iv
ACKNOWLEGEMENTS
I would like to send out my most sincere gratitude to my advisor, Dr. Ronald Johnson.
His guidance, patience and expertise have been crucial during this program and have made the
completion of my work possible.
Additional thanks go out to the members of my Advisory Committee, Dr. Jonathan
LaMarre, Dr. Glen Pyle and Dr. Coral Murrant for their critical review, helpful advice and useful
suggestions during the course of my graduate studies.
I would also like to thank the members of the lab, Dr. Saad Enouri, Dr. Afrah Al-Najeer,
Dr. Monica Antenos and Dr. Tamas Revay for their continual technical support around the lab,
and assistance with statistical analysis.
Last but not least, I would like to thank my fellow graduate students for their genuine
conversations, and moral support.
v
DECLARATION OF WORK PERFORMED
I declare that with the exception of the items indicated below, all work reported in the
body of this thesis was performed by me.
Dr. Saad Enouri and Dr. Afrah Al-Najeer assisted with my experimental design and
statistical analysis for Chapter 1. Dr. Monica Antenos and Dr. Tamas Revay assisted with qPCR
and statistical analysis for Chapter 2.
vi
LIST OF TABLES
Table 1 Sympathetic neurogenic contraction maximum contraction (S ) in 1st
MAX
and 3rd order mesenteric arteries (MA) and veins (MV), in the absence
(control) and presence of the selective α - antagonist prazosin (100nM), or
1
the non-selective α-antagonist phentolamine (10µM) ...............................60
Table 2 Sympathetic neurogenic contraction stimulation frequency required to
achieve half the maximum contraction (S ), in 1st and 3rd order mesenteric
50
arteries (MA) and veins (MV), in the absence (control) and presence of the
selective α - antagonist prazosin (100nM), or the non-selective α-
1
antagonist phentolamine (10µM) ...............................................................60
Table 3 Norepinephrine (NE) contractile responses in 1st and 3rd order mesenteric
arteries (MA) and veins (MV) in the absence or presence of prazosin
(100nM), a selective α - antagonist, or phentolamine (10µM), a non-
1
selective α-antagonist. ................................................................................61
Table 4 Norepinephrine (NE) concentration required to achieve half maximal
contraction in 1st and 3rd order mesenteric arteries (MA) and veins (MV) in
the absence or presence of prazosin (100nM), a selective α - antagonist, or
1
phentolamine (10µM), a non-selective α-antagonist. ................................61
Table 5 Phenylephrine (PE) contractile responses in 1st and 3rd order mesenteric
arteries (MA) and veins (MV) in the absence or presence of prazosin
(100nM), a selective α - antagonist, or phentolamine (10µM), a non-
1
selective α-antagonist. ...............................................................................62
Table 6 Phenylephrine (PE) concentration required to achieve half maximal
contraction in 1st and 3rd order mesenteric arteries (MA) and veins (MV) in
the absence or presence of prazosin (100nM), a selective α - antagonist, or
1
phentolamine (10µM), a non-selective α-antagonist.. ..............................62
vii
LIST OF FIGURES
Figure 1 Frequency-response curves for sympathetic neurogenic constrictions in the
absence (control) and presence of prazosin (100nM) or phentolamine
(10µM), in 1st order and 3rd order mesenteric arteries ...............................42
Figure 2 Frequency-response curves for sympathetic neurogenic constrictions in the
absence (control) or presence of prazosin (100nM) or phentolamine
(10µM), in 1st order and 3rd order mesenteric veins ..................................44
Figure 3 Comparison of control frequency-response curves for sympathetic
neurogenic in 1st versus 3rd order mesenteric arteries and mesenteric veins
....................................................................................................................46
Figure 4 Effect of prazosin (100nM) or phentolamine (10µM), on norepinephrine
concentration-response curves in 1st order and 3rd order mesenteric arteries
....................................................................................................................48
Figure 5 Effect of prazosin (100nM) or phentolamine (10µM), on norepinephrine
concentration-response curves in 1st order and 3rd order mesenteric veins
....................................................................................................................50
Figure 6 Comparison of control norepinephrine concentration response curves in 1st
versus 3rd order mesenteric arteries and mesenteric veins .........................52
Figure 7 Effect of prazosin (100nM) on phenylephrine concentration response
curves in 1st order and 3rd order mesenteric arteries ..................................54
Figure 8 Effect of prazosin (100nM) on phenylephrine concentration response
curves in 1st order and 3rd order mesenteric veins ......................................56
Figure 9 Comparison of control phenylephrine concentration response curves in 1st
versus 3rd order mesenteric arteries and mesenteric veins .........................58
Figure 10 α -AR, α -AR and α -AR mRNA expression in 1st order mesenteric
1A 1B 1D
arteries untreated (0 minutes) and following 15, 30 and 60 minutes of NE
(10-6M), as measured by RT-qPCR. .........................................................77
Figure 11 α -AR, α -AR and α -AR mRNA expression in 3rd order mesenteric
1A 1B 1D
arteries untreated (0 minutes) and following 15, 30 and 60 minutes of NE
(10-6M), as measured by RT-qPCR ..........................................................79
viii
Figure 12 α -AR, α -AR and α -AR mRNA expression in 1st order mesenteric
1A 1B 1D
veins untreated (0 minutes) and following 15, 30 and 60 minutes of NE
(10-6M), as measured by RT-qPCR ..........................................................81
Figure 13 α -AR, α -AR and α -AR mRNA expression in 3rd order mesenteric
1A 1B 1D
veins untreated (0 minutes) and following 15, 30 and 60 minutes of NE
(10-6M), as measured by RT-qPCR. ..........................................................83
Figure 14 microRNA-30c expression in 1st order MA, 3rd order MA, 1st order MV
and 3rd order MV, untreated (0 minutes) and following 15, 30 and 60
minutes of NE (10-6M), as measured by RT-qPCR ...................................85
Figure 15 α -AR mRNA, α -AR mRNA, α -AR mRNA and microRNA-30c
1A 1B 1D
expression in 1st versus 3rd order MA, untreated (0 minutes) and following
15, 30 and 60 minutes of NE (10-6M), as measured by RT-qPCR ...........87
Figure 16 α -AR mRNA, α -AR mRNA, α -AR mRNA and microRNA-30c
1A 1B 1D
expression in 1st versus 3rd order MV, untreated (0 minutes) and following
15, 30 and 60 minutes of NE (10-6M), as measured by RT-qPCR.. ..........89
Figure 17 α -AR mRNA, α -AR mRNA, α -AR mRNA and microRNA-30c
1A 1B 1D
expression in 1st order MA versus 1st order MV, untreated (0 minutes) and
following 15, 30 and 60 minutes of NE (10-6M), as measured by RT-qPCR
....................................................................................................................91
Figure 18 α -AR mRNA, α -AR mRNA, α -AR mRNA and microRNA-30c
1A 1B 1D
expression in 3rd order MA versus 3rd order MV, untreated (0 minutes) and
following 15, 30 and 60 minutes of NE (10-6M), as measured by RT-qPCR
....................................................................................................................93
ix
LIST OF ABBREVIATIONS
AR (s) Adrenergic receptor (s)
α-AR (s) Alpha adrenergic receptor (s)
AC Adenylate Cyclase
ADP Adenosine diphosphate
AMP Adenosine monophosphate
ATP Adenosine 5’-triphosphate
ATP-ase Adenosine 5’-triphosphataes
ARE (s) AU-rich element (s)
AUBP AU binding proteins
β-AR (s) Beta adrenergic receptor (s)
BK Bradykinin
Ca2+ Calcium ion
CaCl Calcium chloride
2
CO Cardiac output
CO Carbon dioxide
2
COX Cyclooxygenase
CRCs Concentration response curves
cAMP Cyclic adenosine monophosphate
cGMP Cyclic guanosine monophosphate
DAG Diacylglycerol
EC Half maximum contraction
50
E Maximal contraction
MAX
x
Description:3.3 Norepinephrine regulation of α-Adrenergic receptor mRNA . The venous system is about 30 times more compliant than the arterial system [48
