Alpha1-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
α1-ADRENERGIC RECEPTOR FUNCTION AND STABILITY IN 1ST VERSUS 3RD
ORDER MESENTERIC ARTERIES AND VEINS OF NORMAL MALE SPRAGUE
DAWLEY RATS
Shawn Veitch
University of Guelph, 2014
Advisor:
Dr. Ronald Johnson
Vascular branching facilitates blood flow changes and maintains cardiovascular
homeostasis. Vasomotor tone is partly maintained by sympathetic nerves, ensuring moment-tomoment 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 (αAR). α1-AR subtype expression was quantified following NE exposure, focusing on microRNA30c activity. The results suggested that MV are more sensitive to AR activation than MA; α1ARs predominantly regulate AR-mediated contractility, with functional evidence of α2-AR
activity in 1st order MA and 3rd order MV; NE reduces α1-AR mRNA expression in MV,
possibly related to venous relaxation at high agonist concentration, but does not involve miRNA30c 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 α1-AR mRNA and miR-30c expression in 1st and 3rd
order MA ........................................................................................................................ 69
Effects of NE exposure on α1-AR mRNA and miR-30c expression in 1st and 3rd
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 (SMAX) in 1st
and 3rd order mesenteric arteries (MA) and veins (MV), in the absence
(control) and presence of the selective α1- antagonist prazosin (100nM), or
the non-selective α-antagonist phentolamine (10µM) ...............................60
Table 2
Sympathetic neurogenic contraction stimulation frequency required to
achieve half the maximum contraction (S50), in 1st and 3rd order mesenteric
arteries (MA) and veins (MV), in the absence (control) and presence of the
selective α1- antagonist prazosin (100nM), or the non-selective αantagonist 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 α1- antagonist, or phentolamine (10µM), a nonselective 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 α1- antagonist, or
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 α1- antagonist, or phentolamine (10µM), a nonselective 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 α1- antagonist, or
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
α1A-AR, α1B-AR and α1D-AR mRNA expression in 1st order mesenteric
arteries untreated (0 minutes) and following 15, 30 and 60 minutes of NE
(10-6M), as measured by RT-qPCR. .........................................................77
Figure 11
α1A-AR, α1B-AR and α1D-AR mRNA expression in 3rd order mesenteric
arteries untreated (0 minutes) and following 15, 30 and 60 minutes of NE
(10-6M), as measured by RT-qPCR ..........................................................79
viii
Figure 12
α1A-AR, α1B-AR and α1D-AR mRNA expression in 1st order mesenteric
veins untreated (0 minutes) and following 15, 30 and 60 minutes of NE
(10-6M), as measured by RT-qPCR ..........................................................81
Figure 13
α1A-AR, α1B-AR and α1D-AR mRNA expression in 3rd order mesenteric
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
α1A-AR mRNA, α1B-AR mRNA, α1D-AR mRNA and microRNA-30c
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
α1A-AR mRNA, α1B-AR mRNA, α1D-AR mRNA and microRNA-30c
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
α1A-AR mRNA, α1B-AR mRNA, α1D-AR mRNA and microRNA-30c
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
α1A-AR mRNA, α1B-AR mRNA, α1D-AR mRNA and microRNA-30c
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
CaCl2
Calcium chloride
CO
Cardiac output
CO2
Carbon dioxide
COX
Cyclooxygenase
CRCs
Concentration response curves
cAMP
Cyclic adenosine monophosphate
cGMP
Cyclic guanosine monophosphate
DAG
Diacylglycerol
EC50
Half maximum contraction
EMAX
Maximal contraction
x
EDHF
Endothelial-derived hyperpolarizing factor
EFS
Electrical field stimulation
EC
Endothelial Cells
ELAV
embryonic lethal abnormal vision
ET
Endothelin(s)
FRCs
Frequency response curves
GC
Guanylate cyclase
GDP
Guanosine diphosphate
GTP
Guanosine triphosphate
cGMP
Cyclic guanosine monophosphate
H2O2
Hydrogen peroxide
HPLC
High performance liquid chromatography
IP3
Inositol 1, 4, 5-trisphosphate
K+
Potassium ion
KCL
Potassium chloride
L-NAME
Nω-Nitro-L-arginine methyl ester hydrochloride
LDV
Large dense vesicles
MgCl
Magnesium chloride
MLC
Myosin light chain
MLCK
Myosin light chain kinase
MLCP
Myosin light chain phosphatase
MA
Mesenteric small arteries
MV
Mesenteric small veins
xi
Na+
Sodium ion
NaCl
Sodium chloride
NaH2PO4
Sodium phosphate
NaHCO3
Sodium bicarbonate
NE
Norepinephrine
NF1
Nuclear factor-1
NO
Nitric oxide
NOS
Nitric oxide synthase
nNOS
Neural nitric oxide synthase
eNOS
Endothelial nitric oxide synthase
NPY
Neuropeptide Y
PE
Phenylephrine
PIP2
phosphatidylinositol 4,5-bisphosphate
PKA
Protein kinase A
PKC
Protein kinase C
PKG
Protein kinase G
PLC
Phospholipase C
PPADS
Pyridoxal-phospate-6-azophenyl-2’,4’-disulfonic acid
RISC
RNA induced silencing complex
S50
Half-maximal response
SMAX
Maximal response
SLV
Small electron-leucent vesicles
Sp1
Sphingosine-1-phosphate
xii
SR
Sarcoplasmic reticulum
TPR
Total peripheral resistance
TTX
Tetrodotoxin
TTP
Tristetraprolin
UTR
Untranslated region
VOCCs
Voltage-operated Ca2+ channels
VR
Venous return
VSM
Vascular smooth muscle
xiii
INTRODUCTION
1.0 The Arterial and Venous System
1.1 Overview of vascular function
The vascular system is comprised of arteries, veins and capillaries, with most arteries
carrying oxygenated blood from the heart to peripheral tissues, and most veins returning
deoxygenated blood from the periphery back to the heart, maintaining cardiac filling pressure.
One exception exists within the pulmonary vasculature with pulmonary arteries carrying
deoxygenated blood from the heart to the lungs, and pulmonary veins carrying oxygenated blood
from the lungs to the heart [36].
Moment-to-moment control of the vascular system is necessary for the maintenance of
normal cardiovascular homeostasis. Arteries and veins are involved in the regulation of
hemodynamic variables such as venous return (VR), cardiac output (CO) and blood pressure
(BP). VR and CO are directly affected by arterial and venous resistance, venous compliance,
blood volume, heart rate and contractility [8]. BP is also affected by arterial and venous
contractility, following increases in total peripheral resistance (TPR) and VR respectively [20].
Arteries, specifically small arteries and arterioles, function as resistance vessels, maintaining
tight control of total peripheral resistance (TPR). Veins act as a blood reservoir, with the venous
system containing 60-70% of the total blood volume [39]. The majority of this blood is stored in
small veins (30%) and venules (45%) [40]. The arterial system stores significantly smaller
amounts of blood, with arteries containing about 18% and arterioles 3% of the total blood
volume [39]. This may explain why the cross sectional radius of veins is about 4x larger than
comparable arteries [36]. The venous system is about 30 times more compliant than the arterial
system [48,49], allowing veins to function as capacitance vessels, storing large amounts of
1
blood. Vascular compliance describes the ratio of the change in blood volume, to the change in
intraluminal distending pressure [44], and is therefore a measure of the distensibility of a blood
vessel. As a result, small changes in venous tone can cause a large change in venous return
altering both cardiac output and blood pressure.
It is well established that veins play an important role in the maintenance of normal
hemodynamics. Unfortunately, the majority of vascular research has focused on arteries with
limited numbers of studies evaluating venous function. The splanchnic vascular bed, specifically
the mesenteric circulation, is of particular importance when considering arterial and venous
function, as it stores approximately 25% of the total blood volume [40], with 80% of this blood
volume in the mesenteric veins [85]. Contractility of these veins results in a large increase in
blood returned to the heart, occurring during exercise, pregnancy and heart disease [63, 64, 82].
The easy accessibility of this vasculature renders it useful for study both in vitro and in vivo.
Knowledge of venous function is essential to understand complete vascular physiology, along
with the effects of drugs used to treat various cardiovascular diseases. As such, research in veins
is necessary.
1.2 Overview of vascular branching order
As blood is pumped out of the heart, it exerts an average pressure of 98 mm/Hg against
the walls of the aorta. In dogs, blood leaving the aorta supplies approximately 45,000 terminal
small arteries, giving rise to more than 20 million arterioles, with each arteriole branching into
about 80 capillaries [76]. Here, oxygenated red blood cells move through the capillaries in single
file, at a speed that is optimal for the exchange of gasses and nutrients. During this circuit, vessel
diameter progressively decreases, but the total cross sectional area of the arteries increases. This
2
is accompanied by a decreased pressure, increased resistance, all contributing to a reduction in
blood flow [76]. In the venous system, deoxygenated blood leaving the capillaries converges into
over 130 million venules, 73,000 small veins, and into the superior and inferior vena cava before
returning to the heart [76]. Average blood pressure is reduced from 12 to 3 mmHg from the
capillaries to the level of the vena cava, respectively. However, a reduction in total cross
sectional area and an increase in inner vessel diameter, results in the rapid transport of blood out
of the capillaries back to the heart [76].
The change in the blood flow, along with its properties relative to the vasculature, is of
interest as it presents the need for differing regulatory mechanisms varying across vessel type
and calibre. This is of particular interest in the splanchnic vasculature, specifically the mesenteric
small arteries (MA) and mesenteric small veins (MV), as this represents a highly branched
microvasculature, containing a large reservoir of blood to be mobilized. This branching property
is demonstrated in other vasculature beds, including batwing skeletal muscle [215]. Branching is
essential to regulate the flow of blood from the aorta, to the capillaries and back to the heart.
Given the large capacitance function of the mesenteric circulation, differential regulation of the
vasculature across branching order is critical to sustain integrated function and cardiovascular
homeostasis. Blood travels out of the main superior mesenteric artery into lower order (1st or 2nd)
mesenteric small arteries, followed by higher order (3rd or 4th) mesenteric arterioles, and into
capillaries. Blood then flows out of the tissue through higher order (3rd or 4th ) mesenteric
venules into lower order (1st or 2nd) mesenteric small veins and back into systemic circulation.
Arteries and arterioles have been included in studies examining branching order differences in
vascular function, which suggest possible regulatory differences. Under normal physiological
pressures, in situ, the vascular tone of mouse cremaster arteries increases with increasing
3
branching order [50]. In rats, the strength of stretch induced myogenic contractility increased
from 1st to 4th order MA [15]. Additional research in rat MA identified a significantly larger
contractile response to norepinephrine (NE) in 1st order MA compared to 4th order MA [16].
These results highlight changes in mechanisms regulating vascular tone across branching order.
Limited research has been performed in veins, identifying significant tone in microvascular
venules, caused by sympathetic neural activity. Shouka and Bohlen [164] induced haemorrhage
to decrease venular pressure, thus increasing sympathetic activity in rat MV. This suggested a
greater sensitivity to sympathetic activity in 1st order MV compared to 2nd and 4th order MV.
These apparent regional differences may be caused by variances in local metabolic control, or
the result of a greater dependence for SNS regulation between lower and higher order arteries
[12]. Further research exploring regulatory differences between lower and higher order arteries
and veins is required.
1.3 Comparison of arteries and veins
Distinct morphological and physiological differences exist regarding arterial and venous
function within the cardiovascular system. Arteries are under high pressure (resistance vessels)
and veins under low pressure (capacitance vessels) [194]. Identifying differences between
arteries and veins is important to advance our knowledge of cardiovascular physiology. As such,
analysis of venous function and regulation in comparison to that of arteries is required.
1.3.1 Morphology
Arteries and veins have functional differences, possibly attributed to structural
differences that exist. Both arteries and veins consist of three layers: tunica intima, tunica media
4
and tunica adventitia [36]. Arteries and veins have a tunica intima lined with endothelial cells,
and both have vascular smooth muscle cells (VSMCs) comprising the tunica media. Finally,
arteries and veins receive their nutrients from the vasa vasorum, located in the tunica adventitia
[36]. Aside from these similarities, the tunica media is thicker in arteries compared to its venous
counterpart [36], which is attributed to increased VSMC layering in arteries. Increased VSMC
layering enables arteries to handle greater pressure compared to veins. The amount of VSMCs in
the tunica media of veins, although not as thick as arteries, still plays a role in the maintenance of
venous tone.
Results obtained from contractile protein studies showed the total protein being greater in
arteries versus veins (1.4:1) on a mg protein/mg dry tissue basis. Differences were noticed
regarding expression of the thin filament binding proteins, proposed to play roles in modulations
of the actin-myosin coupling, ie. Calponin, caldesmon, tropomyosin (TM). There appeared to be
a tendency for an increase in calponin, which did not achieve significance [148]. There was a
significant increase in caldesmon in the artery (1.39:1) compared to vein [148]. Finally, there
appeared to be significantly more TM-α in both artery and vein than beta-actin; with veins
having less TM-α and more TM-β than artery [148]. These variances, along with those regarding
the relative size of the tunica media, may contribute to the functional differences identified
between arteries and veins.
1.3.2 Contractility
Various receptor-dependent agonists such as NE and receptor-independent agonists such
as potassium chloride (KCl) have demonstrated higher reactivity, through both potency and
maximum contractility, in veins compared to arteries [17,18]. Different responses to adrenergic
5
stimuli have been observed specifically in mesenteric and saphenous arteries and veins, with
veins demonstrating an increased potency (EC50; concentration of drug required to produce half
maximum contraction) to both NE and phenylephrine (PE) compared to arteries [17,18]. Mouse
MVs do not appear to desensitize to adrenergic stimuli to the same degree as the MA. This
suggests the possibility that veins might have a complement of adrenergic receptors not present
in arteries [35]. Additional differences have been identified in the presence of sarafotoxin 6c
(S6c), a selective ETB receptor agonist, causing contractility in mesenteric veins, having no
effect in mesenteric arteries [149]. Additionally, in contrast to arteries, veins appear to be
resistant to endothelin-1 (ET-1) mediated desensitization [150]. This may explain the higher
sensitivity of veins to endothelinergic receptor stimulation, and maintained contraction over time
in veins compared to arteries.
1.3.3 Local metabolic control
Metabolic control of small arteries and arterioles has been thoroughly explored, with
known differences existing among different vascular beds [34]. Depending on a tissues
metabolic needs, reduced or elevated tissue perfusion results in dilation or contraction of
resistance vessels, respectively [145]. Several mechanisms have been suggested for these
changes. Altered tissue PO2 results in oxidative phosphorylation, or the release of vasoactive
substances including prostanoids and leukotrienes [145-147]. Additional factors include;
ATP/ADP [151], or Pi [152]; decreased local pH caused by increased PCO2 [153]; increased
local K+ [154]; or local osmolarity [155]. These various metabolites may elicit their function
directly on VSMCs. They also locally modulate NE release from sympathetic nerve endings,
along with the sensitivity of adrenergic receptors [156]. Furthermore, larger arteries seem to be
6
much less sensitive to metabolic control [157]. The metabolic control of veins has been less
extensively studied. Blood in small veins appears to be equilibrated with tissue fluids, with
respect to its PO2, PCO2, pH, and adenosine and phosphate content [34]. Larger veins also
appear to be less sensitive to local metabolic control than smaller veins [34].
1.3.4 Innervation
Studies have identified the splanchnic circulation, including MA and MV, being
innervated by both the sympathetic division of the autonomic nervous systems, and by spinal
sensory nerves [19]. This ensures tight control of the total blood volume stored within this
vasculature. Sympathetic nerve stimulation increases peripheral resistance, mobilizing up to twothirds of the blood stored in veins [20]. Local activation of sensory nerves evokes vasodilation;
the result of the NT released from these nerves [19]. Mediators of this vasodilation include NO
and calcitonin gene-related peptide [206, 207]. Sensory nerves can also be activated through
local reflexes [21].
Sympathetic nerve terminal density and anatomic location within MA differs from that of
MV. Nerve density in MV is less than that of MA, however the reduced number of VSMCs in
veins, leads to a greater relative sympathetic innervation of venous VSM [22]. In arteries
sympathetic nerves are restricted to the tunica media-adventitia border, with sympathetic
innervation throughout the tunica media in veins [22]. Vascular innervation of sympathetic
neurons appears to be species dependent [75]. Neurons innervating arteries and veins, including
the MA and MV, also appear to be separate [75]. Activation of the sympathetic nervous system
through direct nerve stimulation, or indirectly through reflex activation by baroreceptors and
chemoreceptors, suggest differential sensitivities between MA and MV [78, 79]. MA and MV
7
are also innervated by the inferior mesenteric ganglion, receiving innervation from distinct
neurons [80]. Taken together, MAs appear to be regulated by sympathetics differently than the
MV, with MV being more sensitive to sympathetic neurogenic stimulation than MA.
In vitro techniques that provide neurogenic stimulation of blood vessels, such as
electrical field stimulation or perivascular transmural nerve stimulation, are used to examine the
effects of sympathetic stimulation on contractile function of blood vessels. MA and MV respond
to sympathetic neurogenic stimulation in a frequency dependent manner. Veins are able to
contract at lower frequencies (1 – 2 Hz) than arteries (10-20Hz) [17, 23], suggesting an increased
sensitivity to sympathetic nerve activation in capacitance vessels, such as MV. As a result, small
changes in sympathetic activity could eject blood from the splanchnic vasculature into systemic
circulation, having little effect on arterial tone. In contrast, a large increase in sympathetic input
frequency would result in arterial and venous constriction, decreasing venous capacitance and
increasing arterial resistance. Differences regarding arterial and venous response to sympathetic
input may be due to several factors. One possibility is differences in neurotransmission at the site
of sympathetic innervation of vessels, or differences in postjunctional receptor populations of the
vessel [35]. With regards to the mesenteric vasculature, sympathetic neurogenic contractions are
caused by the release of NE, ATP and NPY in arteries, with NE predominating in veins [19, 23].
Whether this difference in neurotransmitter release affects the control of blood flow and volume
within the splanchnic circulation is unknown. Further exploration is required to identify
regulatory differences regarding sympathetic neurogenic stimulation between arteries and veins.
8
2.0 Vascular Smooth Muscle Cells (VSMCs)
Vascular smooth muscle cells are responsible for regulating vascular tone, ie., the overall
or net state of contraction and dilation of arteries and veins. Vascular tone describes the
prevailing diameter of a blood vessel relative to its diameter in the fully relaxed state [32].
Vascular tone is regulated by a variety of factors including sympathetic innervation, local and
circulating hormones, ions, or mechanical stimuli such as stretch-induced contractility [33, 34,
13]. Arterial and venous contractility are known to elevate blood pressure, by increasing vascular
resistance and venous return, respectively [37]. The sympathetic nervous system (SNS) exerts
significant control over vasomotor tone [38].
2.1 Contraction of VSMCs
Pharmacomechanical coupling through surface receptors [30] and electromechanical
coupling through changes in intraluminal pressure and VSMC stretch [31] are two main
regulators of VSMC contractility. Pharmacomechanical coupling describes the interaction
between any agonist and its receptor, and is independent of membrane potential. Key receptor
families include adrenergic, endothelinergic and angiotensin pathways. Electromechanical
coupling mechanisms include membrane depolarization activation of stretch-dependent ion
channels and voltage operated Ca2+ channels (VOCC) [31]. Both coupling mechanisms elicit
contractility by increasing intracellular calcium (Ca2+) through extracellular Ca2+ influx via
membrane channels, or the release of intracellular Ca2+ stores from the sarcoplasmic reticulum.
This occurs following the activation of voltage-operated Ca2+ channels and inositol 1,4,5triphosphate receptors (IP3), respectively [41,42]. Evaluation of pharmacomechanical coupling
will be the main focus of my thesis work, specifically the activity of alpha adrenergic receptors
9
(α-ARs), as they provide an important contribution to VSMC contractility following stimulation
of the peripheral sympathetic nervous system [28,29]. Additional mechanisms regulating
VSMCs will be covered throughout this review.
2.2 Relaxation of VSMCs
The vasodilator properties of the vasculature are not a focus of my thesis work. However
some points are important to note as the integrated effects of factors promoting contraction and
dilation of VSMCs determines net vascular tone, which is modulated moment-to-moment.
VSMC relaxation is caused by two primary mechanisms. Passive relaxation is associated with
the removal of intracellular Ca2+, and active relaxation is associated with the activation of cyclic
nucleotide-dependent signalling pathways in the presence of Ca2+ [1]. Active relaxation is
dependent upon signalling though both cAMP and cGMP dependent protein kinases [2].
Endothelial-derived factors including nitric oxide (NO) and prostacyclin play a critical
role in VSMC relaxation acting as upstream activators of relaxation pathways. Endothelial nitric
oxide synthase (eNOS) is Ca2+-dependent and activated by various stimuli such as acetylcholine
(ACh) and shear stress. eNOS converts the amino acid L-arginine to L-citrulline and NO in the
presence of molecular oxygen [5]. Once released, NO diffuses into adjacent VSMCs where it
activates guanylate cyclase (GC) increasing cGMP production [6]. This inhibits voltage-operated
Ca2+ channel activity decreasing intracellular Ca2+ following PKG activation [6]. Prostacyclin is
a prostanoid synthesized from arachidonic acid in endothelial cells by cyclooxygenase (COX)
[46]. Prostacyclin binds to prostacyclin receptors located on the plasma membrane of VSMCs,
activating adenylate cyclase, increasing cAMP, which activates PKA and produces VSMC
relaxation through a variety of downstream effects [7]. Various other vasodilators have been
10
identified to mediate VSMC relaxation such as endothelial derived hyperpolarizing factor;
however its signalling pathways are not currently clear and will not be considered in my thesis
work.
3.0 Sympathetic Nervous System Activity
As mentioned previously, the mesenteric vascular bed is innervated by sympathetic
neurons, producing contractility, and spinal sensory neurons, inducing vasodilation [19], with
arteries and veins being innervated by separate sympathetic and sensory neurons originating in
the peripheral sympathetic ganglia [20, 21]. The sympathetic nervous system is the most
important regulator of moment-to-moment vascular tone in arteries and veins.
3.1 Overview of the sympathetic nervous system
Sympathetic neurons originate in the spinal cord along with spinal nerves, between cord
segments T1 and L2. Each pathway from cord to tissue consists of preganglionic and
postganglionic sympathetic neurons [44]. Activation of the SNS induces the propagation of an
action potential, which travels to the postganglionic sympathetic nerve terminals, or nerve
varicosities [44]. The action potential depolarizes the nerve terminal, increasing membrane
permeability to calcium ions by the activation of VOCC. This is followed by the release of
neurotransmitters from preformed vesicles contained within the nerve axoplasm. Two types of
secretory granules have been identified in peripheral nerves; the small electron-lucent vesicles
(SLV) and large dense vesicles (LDV) [158, 159]. SLVs are about 50nm in diameter, containing
chemical transmitters such as NE, ACh, ATP, glutamate and GABA [158]. LDV are about
100nm in size, containing proteins and peptides such as neuropeptide Y, along with NE [158].
11
Following their release, neurotransmitters elicit physiological effects by binding to their cognate
receptors, located on the surface of VSMCs. Norepinephrine and ATP are the main
neurotransmitters released by sympathetic vesicles in MA [47, 165], with NE being the major
vasoconstrictor in MV [47]. Differences in neurotransmitter release patterns are of interest as
they can, in part, explain differences in arterial and venous function. Evaluation of the role of NE
in vascular contractility will be the main focus for my thesis research.
3.2 Norepinephrine
Synthesis of NE begins in the axoplasm of the terminal nerve endings of adrenergic nerve
fibers, but is completed inside secretory vesicles. NE is the main sympathetic neurotransmitter
released from the sympathetic perivascular nerves in the rat mesentery. Following its release, NE
binds to G-protein coupled α-AR and β-AR located on the VSM, exerting its physiological
effects [3]. Of these, only the α-adrenergic receptor (α-AR) appears to cause contractility
[28,29]. Pharmacological and molecular studies have identified 2 types of vascular α-AR
including α1-AR, α2-AR. Of these the α1-AR plays a crucial role in the regulation of vascular
tone. The α1-AR and α2-ARs can be further divided into 6 subtypes including α1A, α1B, α1D, α2A/D,
α2B, α2C [26, 27] located on VSMCs of various vessels. Approximately 95% of NE released
during stimulation of sympathetic nerve terminals does not activate receptors, and is transported
back into nerve terminals through an active transport process [44]. Norepinephrine may also
diffuse into capillaries and nearby tissues, or get destroyed by post junctional enzymes [44].
Binding of NE to α1-ARs activates membrane-bound phospholipase C (PLC), which
hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP2) to IP3 and diacylglycerol (DAG). IP3
stimulates intracellular Ca2+ release from the sarcoplasmic reticulum, by binding to the IP3
12
receptor, while DAG activates PKC [3]. PKC is an upstream regulator of numerous kinases
including MLCK, ERK1/2 and rho-kinases [3]. PKC also inhibits the activity of MLC
phosphatase (MLCP), which dephosphorylates MLC [3]. As such, contractile sensitivity to Ca2+
is enhanced by PKC. An increase in intracellular Ca2+ is brief, initiating contractility by a Ca2+calmodulin interaction, stimulating MLC phosphorylation [124]. The RhoA/Rho kinase pathway
also mediates VSMC contractility, inhibiting the dephosphorylation of MLCP as Ca2+
concentration begins to decline, maintaining VSMC force generation [44,124].
Postjunctional α2-AR activation by NE inhibits adenylate cyclase (AC) activity and
VOCC as well as stimulation of K+ channels. α2-ARs may couple to other intracellular pathways
involving Na+ K+ exchange and the activation of PLA2, PLC and PLD with the net result
promoting VSMC relaxation and constriction respectively [160]. α2-AR are also functionally
expressed in endothelial cells, causing relaxation of the rat aorta through NO production via NO
synthase [81]. This could reduce the effects produced by postjunctional α1-AR activation.
Activation of prejuctional α2-ARs inhibits N-, P- and Q-type of VOCC, resulting in a negative
feed-back modulation of NE release [161]. In rat mesenteric vessels, UK 14, 304, an α2-AR
agonist, and yohimbine, an α2-AR antagonist decreased and increased NE release, and
contractility, respectively, following sympathetic nerve stimulation [14, 38].
The adrenergic receptors, isoforms and numbers, represent a potentially important
regulator of vascular responsiveness following sympathetic nerve stimulation. Understanding α1AR subtype expression and function is important as the involvement of α1-AR subtypes involved
in the contractile responses to adrenergic agonists appears to differ within various vascular beds.
In rats, in vivo studies showed evidence for the significant role of α1D-AR in the regulation of
systemic arterial pressure [170]. In contrast, in vitro, the α1A-AR was identified as a predominant
13
subtype in rat mesenteric arteries [212]. The α1A-AR and α1D-AR are predominant in the renal
vascular bed [213], the α1D-AR subtype in rat cremaster muscle arterioles [173] and the α1A-AR
in rat femoral resistance arteries [214]. This being said, the understanding of α1-AR subtype
involved in the regulation of peripherial arterial resistance in rat is controversial and unknown in
veins. Potential differences may also exist regarding receptor subtype intracellular signalling
pathways [217].
In MA, the α1-AR has been shown to be the only receptor involved in NE-mediated
contractility [37], however both α1- and α2-ARs appear to cause contractility in MV [37]. It has
been suggested that α2-ARs may act in a modulatory capacity, sensitizing α1-ARs to NEmediated contractility [24,25]. Preliminary data generated in our laboratory by previous students
supports these statements. For the purpose of my thesis work, only the α1-AR will be considered,
however the α2-AR will be of some importance.
3.3 Norepinephrine regulation of α-AR mRNA
An alteration to catecholamine sensitivity in blood vessels has been observed in a number
of physiological and disease states, including pregnancy [63], hypertension [64], and congestive
heart failure [82]. Rat aortic strips became desensitized to NE-mediated effects following
prolonged exposure in vitro [65]. Additional observations in rabbit aortic strips following
catecholamine depletion with reserpine [66], or 6-hydroxy dopamine [67], resulted in an increase
in vascular sensitivity to NE in vitro. The molecular mechanisms regulating vascular sensitivity
to α-AR agonists are unclear. Observations have been made in non-vascular tissues providing a
possible explanation towards the regulation of α-AR. Both human platelets [68] and dispersed rat
parotid acinar cells [69] have demonstrated α-AR desensitization in response to agonist
14
exposure. Catecholamine induced reduction in the number of α-AR binding sites correlates with
a decrease in responsiveness to α-AR agonists [69]. Experiments in the rat salivary gland [70]
and brain [71] showed an increase in the number of α-AR following reserpine-induced
catecholamine depletion. α1-AR density on rabbit aortic smooth muscle also decreased following
several hours of catecholamine exposure [72]. Regulation of adrenergic receptor density on
VSMCs is not well understood, suggested to be a function of the relative rates of receptor
synthesis and degradation [73]. It may also involve the cycling of receptors between cell surface
and non-surface accessible sites [74]. Aside from these observations, little is known about the
effects of α1-AR stimulation on mRNA expression for that receptor. One study identified a timedependent decrease in α1-AR mRNA in rabbit aortic stem cells following NE treatment [59]. β2AR stimulation caused both a rapid cAMP-mediated increase in β2-AR mRNA transcription and
a slower (hours) decrease in mRNA level [83,84]. Understanding the relationship between α1AR stimulation and mRNA expression is of importance as it represents a potential therapeutic
target for diseases characterized by elevated catecholamine levels, such as hypertension and
heart failure.
4.0 microRNAs
4.1 miRNA structure and function
MicroRNAs (miRNAs) are a class of endogenous non-coding RNAs, approximately 2025 nucleotides in length, which are potent regulators of gene expression. They appear to be
involved in the regulation of about one third of all mammalian genes [51]. MicroRNAs act at the
post-transcriptional level by binding to complementary sequences in the 3’-untranslated region
(3’UTR) of target mRNAs. This inhibits mRNA expression through one of two mechanisms; 1)
15
translational suppression or 2) induction of target mRNA degradation. Through these effects,
microRNAs are directly involved in regulating cellular phenotype, along with other various
biological processes such as differentiation, development, proliferation and tumorigenesis
[52,53,54]. miRNAs are also involved in the regulation of various receptors such as the β2adrenergic receptor [56], while playing a role in various cardiovascular diseases [60].
4.2 miRNA biogenesis
The generation of mature single-standed miRNAs, from long primary transcripts, or
primiRNAs is a multi-step process. primiRNAs are processed into small hairpin premiRNAs in
the nucleus through the activity of an enzyme complex consisting of Drosha and DiGeorge
Syndrome Critical Region 8 (DGCR). Pre-miRNAs are then exported into the cytoplasm. Here
the RNAse III enzyme Dicer [57] cleaves the loop region of the hairpin to generate a short
miRNA:miRNA duplex. One strand is degraded, leaving a mature single-stranded miRNA
molecule which becomes incorporated into the RNA-induced silencing complex (RISC) [55].
This is the site of interaction between the miRNA and its target mRNA.
Expression profiling of microRNAs through cloning and sequencing, or microarrays has
shown that multiple miRNAs are often expressed in specific cell types and tissues [208].
Different combinations of miRNAs are also expressed in different cell types and tissues, which
may co-ordinately regulate cell-specific target types [209]. Many known target genes of miRNAs
contain several miRNA binding sites, and the degree of translational repression may increase
exponentially with the number of miRNA binding sites, located in the 3’ UTR [209, 210].
Depending on the time of discovery, each miRNA has been labelled with a specific number. The
most abundant miRNA in rat carotid artery are miRNA-145, let-7, miRNA-125b, miRNA-125a,
16
miRNA-23 and miRNA-143, whereas those the rat heart are miRNA-1, let-7, miRNA-126,
miRNA-133, miRNA-26a, and miRNA-30c [211]. Limited studies have examined the role of
miRNAs on α-AR mRNA; however conserved genomic segments were identified using
bioinformatics programs, such as TargetScan, proposing possible regulatory involvement from
the miRNA-30 family [121].
4.3 miRNA involvement in cardiovascular biology and disease
Over the last decade, miRNAs have proven to be potent regulators of thousands of genes.
They appear to be pivotal regulators in normal development and physiology, as well as disease
pathophysiology [60]. miRNAs are highly expressed in the cardiovascular system [61]; however
their biological roles in the mammalian cardiovascular system have only been under
investigation since 2005 [62]. Studies have demonstrated miRNA involvement in both
cardiovascular development as well as cardiovascular disease [60]. With regards to receptors,
miRNAs appear to be involved in the regulation of both endothelin and estrogen receptor mRNA
expression [108,109]. They also interact with the adrenergic receptor family, regulating baseline
β2-AR expression and decreasing agonist-promoted down-regulation [56]. TargetScan identified
conserved sites within the 3’-UTR of α1-AR mRNA, suggesting the possibility that miRNA-30
a/b/c/d/e all potentially regulate α1-AR mRNA [121]. MicroRNA-30c is increased in aortic
tissues from patients with thoracic aortic dissection [162], and in hypoxia-induced pulmonary
vascular smooth muscle cells [163]. Regulation of the α1-AR mRNA is still largely unknown,
with regard to both transcriptional (transcription factors, etc.) and post-transcriptional regulators
such as miRNAs. As such, much work is required to elucidate the regulatory mechanisms
17
controlling α1-AR levels in arteries and veins in the presence of norepinephrine, and, in
particular, any roles played by miRNAs.
18
RATIONALE
Arteries and veins play an important role in cardiovascular homeostasis. The splanchnic
vascular bed, including the mesenteric arteries and veins, is the most significant vascular bed in
the body in terms of volume. This system of vessels makes important contributions to systemic
vascular blood pressure and capacitance, respectively. It is also highly branched, with blood flow
directly affected by the calibre of vessel and degree of vascular branching [76]. Limited research
has been conducted in arteries and veins, exploring the regional differences that exist within
various vascular beds. These studies demonstrated increased responses to sympathetic activity in
1st versus 4th order MA and MV [16, 164].
Control of vascular tone involves many factors acting in concert to maintain vascular
function. One of these factors, the sympathetic nervous system is a key regulator of arterial and
venous function in the splanchnic circulation. Norepinephrine, released from presynaptic
sympathetic nerve terminals, activates postjunctional α1- and in some cases α2- AR located on
vascular smooth muscle cells, causing vasoconstriction. It is generally understood that arteries
are less sensitive to adrenergic stimuli compared to their venous counterparts; however the origin
and underlying mechanism for these differences are still unclear. Arteries have a greater number
of layers of vascular smooth muscle cells compared to the veins, thickening the vessel wall. This
yields a greater nerve density, and relative sympathetic innervation of VSM in mesenteric veins
then that of mesenteric arteries [22]. Until recently little has been known about venous function
in health and disease. It is assumed that these differences in vessel morphology and innervation
contribute to differences in sympathetic mediated regulation of arterial and venous tone.
19
It is well known that sympathetic mediated contractions in the vasculature involve
activation of α-AR on the VSM. Understanding α-AR regulation in both arteries and veins is
important to further our knowledge of integrated vascular function. It may also identify potential
therapeutic targets for the management of cardiovascular diseases. Regulation of sympathetic
neurogenic vascular contractile function can occur through altered neurotransmission, changes in
VSMC receptor subtypes and densities, and changes in post-receptor intracellular events such as
α1-AR mRNA expression [59]. MicroRNAs are endogenous, small, non-coding RNA that
promote degradation or translational suppression of their target mRNA [60]. The involvement of
miRNAs in α1-AR mRNA regulation is also unknown; however β2-ARs are regulated by miRNA
let-7f [56]. MicroRNA-30c was shown to be upregulated in hypoxia-induced pulmonary vascular
smooth muscle cells [163]. TargetScan [121] identified potential target sites in α1-AR mRNA for
the microRNA-30 family. Taken together, a greater understanding of α1-AR regulation at the
functional and molecular level is necessary. The focus of my thesis will be on α1-AR function
and mRNA expression in 1st and 3rd order MA and MV.
20
Hypotheses
I hypothesize that lower order (1st) MA and MV will demonstrate a greater contractile
response to adrenergic stimuli compared to higher order (3rd) vessels. I also hypothesize that α1AR mRNA will be subject to miRNA-dependent regulation, and that the expression of these
mRNA and miRNA will be different between i) 1st and 3rd order vessels, and ii) between arteries
and veins.
Objectives
Two objectives were completed in order test these hypotheses:
1. Investigation of contractile differences in vitro to adrenergic stimulation in 1st versus 3rd
order MA and MV in normal male rats through exogenous application of adrenergic agonists
(norepinephrine and phenylephrine) and sympathetic neurogenic stimulation using electrical
field stimulation.
2. Investigation of specific miRNA activity (miRNA-30c) associated with α1-AR mRNA
expression in 1st versus 3rd order MA and MV of normal male rats, using time dependent
experiments.
21
CHAPTER 1
CHARACTERIZATION OF α-ADRENERGIC RECEPTOR FUNCTION IN 1st VERSUS
3rd ORDER MESENTERIC ARTERIES AND VEINS OF NORMAL MALE SPRAGUE
DAWLEY RATS
22
Introduction
The blood transports oxygen, nutritive substrates, metabolic waste, chemical messengers
and electrolytes to and from bodily tissues [76]. In order to maintain homeostasis of blood flow
in high pressure arteries, as well as low resistance veins, regional differences in morphology and
control of vascular tone are necessary. Both arteries and veins have similar gross vascular
composition, consisting of three layers; the tunica intima, tunica media and tunica adventitia. The
tunica media is populated by VSMCs with arteries displaying increased VSM layering when
compared to corresponding veins [36]. Differences in vascular morphology contribute, in part, to
functional specialization between arteries and veins, ie. resistance and capacitance properties,
respectively [85]. The highly distensible nature of the venous system enables a substantial
amount of total blood volume to be stored in the systemic veins and venules, particularly the
splanchnic circulation [13, 39].
Regulation of vascular tone also differs between the arteries and veins [32]. The
integrated vascular effects of neurogenic influences, intrinsic myogenic properties, and
circulating humoral influences and local endothelial derived factors [33, 34, 13] provides for a
highly modulated control of vascular tone and cardiovascular homeostasis. Neurogenic
influences are comprised of the local neural control mechanisms produced by the sympathetic
nervous system (SNS). Electrical field stimulation studies of isolated vessels have identified the
release of NE, ATP and NPY following peripheral SNS activation, in both arteries and veins [23,
45]. Interestingly the relative contribution from each of these neurotransmitters to contraction
varies from one blood vessel to another [23]. For the most part, NE and ATP are the main
neurotransmitters released by sympathetic nerves supplying MA [165]. NE is the major
23
constrictor released by perivascular sympathetic neurons in veins [174]. Meanwhile neurally
released NPY is responsible for the modulation of NE activity and possibly ATP [184].
The SNS produces potent vasoconstriction, exerting primary control over moment-tomoment changes in vascular tone [77]. Sympathetic vasoconstriction is attributed to activation of
α-adrenergic receptors (ARs), with recent studies identifying expression of α1A, α1B, α1D, α2B, α2D
–ARs in rat vascular smooth muscle cells [26, 27]. Morphology of vessels, as well as
neurohumoral and myogenic regulation of vascular tone, can vary with the size of vessels. As
such, blood vessel branching order also represents an important consideration regarding overall
integrated regulation of blood flow in a vascular bed and overall vascular homeostasis. As blood
leaves the heart it enters the aorta, which branches into progressively smaller arteries and
arterioles. As blood flows from the heart towards the peripheries, resistance to flow increases,
blood pressure is reduced and the total cross-sectional area is increased. These factors work
together to reduce the forward velocity of blood flow [76] to ensure optimal rate of flow through
the capillaries. Blood pressure continues to drop as blood is collected into post-capillary venules
and veins, with blood flow velocity increasing as it returns to the heart via the vena cava [76]. To
date, very few studies have explored differences in integrated function in the vasculature based
on branching order, with results varying. One study identified increased myogenic contractility
in 4th order versus 1st order, or with increasing branching order in, mesenteric arterioles [15].
Another study has suggested an increased response to NE in lower order mesenteric arterioles
compared to higher order arterioles [16]. Many studies have been conducted in the splanchnic
circulation as it is a large vascular bed, containing a large percentage of the total blood volume,
highly branched, and easy to access for study.
24
Adrenergic receptors represent a critical mechanism of control of vascular tone and of
overall integrated vascular function. Activation of the postjunctional AR on VSMC can be
characterized pharmacologically using exogenous application of receptor selective agonists and
antagonists to isolated tissue preparations in vitro. However, stimulation of peripheral
sympathetic nerves innervating blood vessels to produce contractions enables a more physiologic
evaluation of integrated control of the vasculature. The following study will use these two
experimental approaches to evaluate integrated vascular function in 1st versus 3rd order MA and
MV, with a focus on α1-AR activity. Based on previous findings in arteries and arterioles from
various vascular beds, I hypothesize that 1st order MA and MV will be more responsive to
sympathetic neurogenic input, and exogenously applied adrenergic agonists when compared with
3rd order mesenteric vessels.
Materials and Methods
Animals
Male Sprague-Dawley (SD) rats, 7-9 weeks of age, were individually housed in a room
that was humidity and temperature-controlled and maintained under a 12:12 hour light-dark
cycle. The rats were fed pelleted rat chow and water ad lib. All experiments were approved by
the Institutional Animal Care and Use Committee of the University of Guelph and conformed to
standards set forth by the Canadian Council of Animal care [15].
Vessel isolation
Rats were euthanized by an intraperitoneal injection of a lethal dose of pentobarbital (50
mg/kg; CEVA Santé Animal, Libourne, Fance). The small intestine and associated mesenteric
25
vessels were then removed and bathed in oxygenated (95%O2, 5%CO2) cold (4oC, pH 7.35-7.40)
Krebs physiological buffer solution containing in mmol/L: sodium chloride 117, potassium
chloride 4.7 (KCl), calcium chloride 2.5 (CaCl2), magnesium chloride 1.2 (MgCl), sodium
phosphate 1.2, sodium bicarbonate 25 and glucose 11. A section of mesenteric vessels were
separated and pinned flat into a silicone lined recording bath, containing cold Kreb’s solution. A
1st or 3rd order MA (1st-295.3±6.7 μm; 3rd-204.3±3.6 μm) or MV (1st-386.4±9.3 μm; 3rd273.1±8.5 μm) was then isolated with the use of a dissection microscope and fine surgical
instruments by clearing the surrounding connective tissue and adipose tissue.
Assessment of vascular function
The recording bath containing an isolated vessel was mounted on the stage of an inverted
microscope and connected to a system of 3 way stop cock syringes. Vessels were superfused
with warmed (37oC) oxygenated Krebs buffer solution for 20 minutes at a rate of 7 ml/min using
a peristaltic pump (Minipuls 3 peristaltic pump, Gilson, Inc. USA), allowing the vessel to
equilibrate and achieve a resting baseline diameter. Black and white images of vessels were
obtained by a video camera attached to the inverted microscope, which fed data to a PCVision
Plus frame-grabber board located in a personal computer. The digital signal generated was
converted into an analog output as described previously [16]. The outer diameter of the vessel
was tracked in real time using Diamtrak® computer software (Adelaide, Australia), which
enabled continuous measurements of vessels with a resolution of diameter change of 0.5µm.
Vessels were tested for viability before any experiments were conducted. Contractility
was assessed using potassium chloride (KCl; 80mM) and NE (10µM). Based on previous work
in the lab and published results [23], a vessel was considered viable for study if a minimum 25%
26
contraction to KCl and 20% contraction to NE were observed. In the total of 172 vessels isolated
for study, 16 veins and 21 arteries were discarded for failing viability testing with NE.
Experimental protocols
Concentration contractile response studies
All drugs were obtained from Sigma-Aldrich® (Oakville, Ontario, Canada), and were
added at known concentrations to the superfusing Krebs buffer solution. Norepinephrine (NE;
non-selective α1- / α2 - AR agonist) and Phenylephrine (PE; selective α1-AR agonist)
concentration response curves were constructed using cumulative addition of increasing log
doses of each agonist concentration (10-10M to 10-5M). Each log dosage was added to 30 mL of
the superfusing Krebs buffer solution, which was flowing at 7 ml/min, totalling approximately 4
minutes and 30 seconds for each dose. Prazosin (10-7M; selective α1-AR antagonist) [17, 23], or
phentolamine (10-5M; non-selective α1- / α2 - AR antagonist) [166] was applied to the tissue for
20 minutes before the agonist was applied, and throughout the remainder of the experiment.
Pretreatment with antagonist did not change resting vessel diameter.
Electrical field stimulation
Electrical field stimulation (EFS) (0.2-32 Hz, 0.1 ms pulse duration, 60 V, for 10
seconds) was applied by means of an insert with two platinum wire electrodes that were
positioned in parallel on both sides of the longitudinal axis of the tissue preparation (recording
bath) and were connected to a Grass S 88 electrical stimulator (Astro-Med, West Warwick,
USA). Tetrodotoxin (TTX; voltage-dependent sodium channel blocker; Alomone Ltd, Jerusalem,
Israel) verified that EFS contractility was neurogenic in origin, and not caused by direct
27
stimulation of VSMCs. Vessels were stimulated with a frequency of 10 Hz in the absence and
presence of TTX (0.3µM). Mesenteric vessels were retested, following a 20 minute washout
period from TTX, to ensure the return of neurogenic responses. Electrical field stimulation was
applied following a 10 minute washout period between increasing stimulation frequencies. In
experiments where antagonists were used, the drugs were applied for 20 minutes before the first
stimulation, and throughout the remainder of the experiment including washout periods between
successive electrical stimulations.
Data analysis
Concentration contractile response curves (CRC) were plotted as percent contraction of
the vessel against a log scale of agonist concentration. Frequency contractile response curves
(FRC) were plotted as percent contraction of the vessel against a log scale of stimulation
frequency (Hz). Percent contraction at each agonist concentration or stimulation frequency was
determined as the percentage reduction from the vessels resting initial baseline diameter.
(baseline diameter – minimum diameter)
% contraction =
x 100
(baseline diameter)
CRC and FRC data were fitted individually by a four-parameter logistic equation (sigmoidal fit):
Y = {(Emin-Emax)/[1 + (X-EC50)P]} + Emax
28
From these plots, the half-maximal effective agonist concentration or the half maximal
stimulation frequency (EC50 or S50) and maximum responses (Emax or Smax) were calculated. The
–log of the EC50 (pD2) was also calculated from the concentration contractile response studies.
The Shapiro-Wilk normality test was used to confirm that the collected data was normally
distributed. Differences between group means for Smax or Emax, and S50 or pD2 were assessed by
student’s two-tailed unpaired t-test. Assessing differences between more than two groups of
means was done using Analysis of Variance (ANOVA), followed by a Tukey’s post-hoc test. A
P value of <0.05 was considered statistically significant. Vessel response at each dose or
frequency tested was determined as change from the resting vessel diameter, and not maximal
vessel diameter achievable. Vessels studied were not dilated to their maximum diameter as they
were not pressurized or stretched in any manner. All contractile responses were expressed as
percentage contractility and not change in vessel diameter in µm based on previous accepted
(published) methods [18].
Results
General
Data were obtained from 116 normal male rats (mean weight of 329.5±3.7 g). From these
rats, 1st order MA (295.3±6.7µm) and MV (386.5±9.3µm) or 3rd order MA (204.3±3.6µm) and
MV (273.1±8.5µm) were isolated. 1st order MA had a significantly larger diameter compared to
3rd order MA (P<0.05). 1st order MV were also significantly larger in diameter compared to 3rd
order MV (P<0.05). Furthermore, 1st and 3rd order MV were significantly larger than MA of
similar order (P<0.05).
29
Sympathetic neurogenic contractions in 1st versus 3rd order MA and MV
In the first set of experiments, FRC were generated in 1st and 3rd order MA and MV. This
was done in the absence (control) and presence of prazosin, a selective α1-AR antagonist, or
phentolamine, a non-selective (α1, α2) α-AR antagonist. In 1st order MA, control responses
showed significantly greater SMAX (29.33 ± 2.26%) values when compared to values in the
presence of prazosin (11.69 ± 1.78%) and phentolamine (11.26 ± 1.09%; Figure 1A; Table 1;
P<0.05). Prazosin and phentolamine treated vessels generated similar results, regarding SMAX and
S50. 3rd order MA control responses demonstrated a significantly greater SMAX (31.29 ± 2.24%)
and S50 (12.46 ± 0.62 Hz) compared to both prazosin (15.75 ± 1.51%; 6.73 ± 1.31 Hz) and
phentolamine (9.47 ± 2.25%; 4.19 ± 1.32 Hz) treated vessels. Prazosin (15.75 ± 1.51%) treated
vessels also produced a significantly greater SMAX when compared to phentolamine groups (9.47
± 2.25%, Figure 1B; Table 1; P<0.05). 1st order MV control responses had a significantly greater
SMAX (25.59 ± 2.03%) and significantly lower S50 (2.12 ± 0.42 Hz) when compared to prazosin
(15.57 ± 3.10%; 12.47 ± 2.33 Hz) or phentolamine groups (10.36 ± 0.93%; 12.00 ± 2.69 Hz).
Prazosin groups also generated a significantly greater SMAX (15.57 ± 3.10%) when compared to
phentolamine groups (10.36 ± 0.93%; Figure 2A; Table 1; P<0.05). 3rd order MV control
responses produced a significantly greater SMAX (29.94±2.35%) and significantly lower S50 (2.39
± 0.41 Hz) when compared to prazosin (2.56 ± 0.54%; 11.64 ± 0.99 Hz) or phentolamine treated
vessels (3.24 ± 0.65%; 19.80 ± 1.33 Hz; Figure 2B; Table 1; Table 2; P<0.05). Finally, in 3rd
order MV, pre-treated prazosin vessels displayed a significantly lower S50 (11.64 ± 0.99 Hz),
when compared to phentolamine groups (19.80 ± 1.33 Hz; Figure 2B; Table 2; P<0.05).
Differences between 1st and 3rd order MA existed as 3rd order MA, pre-treated with
prazosin, had significantly larger SMAX values(15.75±1.51) compared to values from 1st order
30
MA (11.69±1.78; Table 1; P<0.05). Meanwhile, in 1st order MA, vessels pre-treated with
prazosin or phentolamine displayed a significantly higher S50 values (11.95 ± 2.95 Hz; 11.35 ±
2.28 Hz) compared to 3rd order MA values (6.73±1.31 Hz; 4.19±1.32 Hz; Table 2; P<0.05). 1st
and 3rd order MV also had some differences as 1st order MV revealed a significantly greater
SMAX in the presence of prazosin (15.57 ± 3.10%) or phentolamine (10.36 ± 0.93%) compared to
3rd order MV (2.56 ± 0.54%; 3.24 ± 0.65%; Figure 3A; Table 1; Table 2; p<0.05). Arterial and
venous differences also existed, as both 1st and 3rd order MV control vessels demonstrated a
significantly lower S50 (2.12 ± 0.42 Hz; 2.39 ± 0.41 Hz) when compared to 1st and 3rd order MA
(11.09 ± 0.95 Hz; 12.46 ± 0.62 Hz; Table 2; P<0.05). In the presence of prazosin or
phentolamine, 3rd order MA demonstrated significantly greater SMAX (15.75 ± 1.51 %; 9.47 ±
2.25 %) and lower S50 (4.19 ± 1.32 Hz; 6.73 ± 1.31 Hz), compared to 3rd order MV (2.56 ±
0.65%; 3.24 ± 0.54%; 11.64 ± 0.99 Hz; 19.80 ± 1.33 Hz; Table 1; Table 2; P<0.05).
Norepinephrine mediated contractions in 1st versus 3rd order MA and MV
Exogenously applied NE produced concentration-dependent contractions in 1st and 3rd
order MA (Figure 4) and MV (Figure 5) control vessels, with MV relaxing at maximum agonist
concentrations, and MA responses plateauing. In 1st and 3rd order MA and MV, minimal
contractility was observed at maximum NE concentrations in the presence of prazosin, while
phentolamine completely blocked NE-mediated contractility in all vessels. In 1st order MA,
control vessels produced a significantly larger EMAX (32.31 ± 2.25%) and PD2 (5.56 ± 0.05)
when compared to prazosin treated vessels (4.79 ± 2.60%; 3.38 ± 1.13). In 3rd order MA control
vessels also produced a significantly larger EMAX (25.87 ± 1.50%) and PD2 (5.37 ± 0.08) when
compared to prazosin treated vessels (2.50 ± 2.02%; 2.79 ± 1.14; Figure 4; Table 3; Table 4;
31
P<0.05). 1st order MV control vessels had a significantly greater EMAX (23.96 ± 1.03%) and
lower PD2 (7.18 ± 0.08) when compared to prazosin treated vessels (2.63 ± 0.86 %; 8.48±0.28).
3rd order MV control vessels exhibited a significantly larger EMAX (22.95 ± 1.05%) and PD2
(7.28 ± 0.19) when compared to prazosin treated vessels (1.19 ± 0.19%; 6.28 ± 0.31; Figure 5;
Table 3; Table 4; P<0.05). Within the range of NE concentrations tested, there were no
differences between control and phentolamine treated vessels in 1st or 3rd order MA or MV, as
contractility was completely blocked. With regards to branching order differences, NE generated
a significantly greater EMAX in 1st order MA (32.31 ± 2.25%) compared to 3rd order MA (25.87 ±
1.50% ; Figure 6A; Table 3; Table 4; P<0.05), with no apparent differences between 1st and 3rd
order MV (Figure 6B). Finally, 1st and 3rd order MA displayed a significantly greater EMAX
(32.31 ± 2.25%; 25.87 ± 1.50%) compared to 1st and 3rd order MV (23.96 ± 1.03%; 22.95 ±
1.05%; Table 3; P<0.05). 1st and 3rd order MV control vessels however, demonstrated
significantly greater pD2 values (7.18 ± 0.08; 7.28 ± 0.19) compared to 1st and 3rd order MA
(5.56 ± 0.05; 5.37 ± 0.08; Table 4; P<0.05)
Phenylephrine mediated contractions in 1st versus 3rd order MA and MV
Phenylephrine produced concentration-dependent contractions in 1st and 3rd order MA
(Figure 7) and MV (Figure 8). Prazosin completely blocked PE-induced contractions in both 1st
and 3rd order MV, however minimal contractility was observed at maximum PE concentrations
in 1st and 3rd order MA. In 1st order MA, PE control vessels had a significantly greater EMAX
(30.19 ± 1.64%) and PD2 (5.33 ± 0.10), compared to prazosin treated vessels (4.92 ± 3.50%; 3.58
± 1.21; P<0.05). 3rd order MA control vessels also displayed a significantly greater EMAX (33.55
± 1.51%) and PD2 (5.76 ± 0.10), when compared to prazosin treated vessels (2.76±1.34;
32
4.68±0.12; Figure 7; Table 5; Table 6; P<0.05). No differences were observed in the presence of
prazosin within the range of PE concentrations tested. A comparison of 1st vs 3rd order MA
indicates a significantly larger EMAX in 3rd order MA (33.55 ± 1.51%) compared to 1st order MA
(30.19 ± 1.64%; Figure 9A; Table 5; Table 6; p<0.05). No differences were observed between 1st
and 3rd MV (Figure 9B). Differences between MA and MV were also identified, with PE
producing a significantly greater EMAX in 1st and 3rd order MA (30.19 ± 1.64%; 33.55 ± 1.51%)
compared to MV (16.65 ± 1.68%; 14.42 ± 2.25%; Table 5; Table 6; p<0.05). PE also revealed a
significantly lower pD2 in 1st and 3rd order MA (5.33 ± 0.10; 5.76 ± 0.10) compared to 1st and 3rd
order MV (7.01 ± 0.36; 6.77 ± 0.20; Table 5; Table 6; p<0.05).
Discussion
The vascular constrictor effects produced by the sympathetic nervous system are
mediated by α-adrenergic receptors. Mesenteric arteries and veins, contribute important
resistance and capacitance functions to the systemic circulation, respectively. This system of
vessels is also highly branched, with known functional and morphological differences based on
vessel region [50]. In the current study, it was hypothesized that 1st order MA and MV would
demonstrate greater contractility in response to sympathetic neurogenic stimulation and
application of exogenous adrenergic agonists, when compared to 3rd order MA and MV.
Sympathetic neurogenic contractility was significantly reduced following pre-treatment with
prazosin (selective α1-AR antagonist), or phentolamine, (non-selective α1-/α2- AR antagonist),
when compared to responses in control vessels. Prazosin and phentolamine treatment affected
sympathetic neurogenic contractions differently in 1st versus 3rd order MA and MV. NE mediated
contractility was only observed at high agonist concentrations in the presence of prazosin, and
33
was completely blocked by phentolamine in 1st and 3rd order MA and MV. Phenylephrine
mediated contractility was also only observed at high agonist concentrations in 1st and 3rd order
MA in the presence of prazosin, completely blocking contractility in 1st and 3rd order MV. These
results present possible technical limitations, as the EMAX and PD2 values identified may not be
accurate. Even though contractility was detected, it cannot be assumed that this was the
maximum response, as it represents the maximum response within the range of agonist
concentration tested. Finally, NE-mediated contractility demonstrated a significantly larger
maximum response in 1st order MA versus 3rd order MA. Meanwhile, PE produced a
significantly larger maximum contraction and was more potent in 3rd order MA versus 1st order
MA.
α-adrenergic receptor activity
Sympathetic neurogenic stimulation generated FRCs of similar shape and magnitude in
1st versus 3rd order MA and MV. In MA, prazosin significantly reduced maximum contractions
to EFS in 1st and 3rd order MA when compared to control. Phentolamine further attenuated
contractility in 3rd order MA, while producing similar results in 1st order MA, compared to
prazosin treated vessels. Interestingly, in 3rd order MA, vessels pre-treated with prazosin or
phentolamine increased sympathetic nerve activation potency, when compared to control vessels.
This may be due to a technical limitation, as 3rd order MA control vessel contractile responses
did not plateau in the range of stimulation frequencies tested. In MV, prazosin significantly
reduced maximum contractions in 1st order MV, while completely blocking contractions in 3rd
order MV. Phentolamine decreased contractility in 1st order MV compared to responses in
prazosin treated vessels, and completely blocked contractility in 3rd order MV. It is important to
34
note that minor contractility was observed at maximum stimulation frequencies in 3rd order MV
in the presence of prazosin or phentolamine. This being said, sympathetic nerve activity had a
more potent effect in the presence of prazosin, compared to phentolamine. However the use of
higher stimulation frequencies, or lower antagonist concentrations is required to accurately
identify differences. Additionally, due to the relatively minor contractility observed, the EMAX
and PD2 values were calculated manually. Taken together, these findings suggest the primary
involvement of the α1-AR in 1st and 3rd order MA and MV, with possible contributions from the
α2-AR in 3rd order MA and 1st order MV. It has been determined that α1-ARs mediate direct
contractile responses in mouse MA, while α2-ARs contribute indirectly to NE-mediated
contractility in MV but not MA, in vitro [37]. This is assumed to be the result of an α2-ARmediated increase in α1-AR sensitivity [37]. Previously it was shown that neither clonidine nor
UK-14,304 (α2-AR selective agonists) constricted mesenteric arteries or veins from rats [17] or
mice [35]. UK-14,304 did potentiate PE-mediated contractility in mouse MV [37]. Rat tail
arteries displayed similar data, with UK-14,304 causing a leftward shift to methoxamine CRC
(α1-AR agonist) [185]. At the same time α1-AR and α2-AR both mediated contractility in rabbit
and dog saphenous veins [28, 29]. Functional postjunctional α2-ARs have also been identified on
rat cremaster arterioles and on human resistance vessels [90, 93], suggesting a possible species
difference. Here an inverse relationship between vessel diameter and magnitude of α2-ARmediated responses was observed [93].
Differences in the relative expression of α-ARs exist across arteries in many vascular
beds including; the mesenteric, splenic and renal vascular beds; all of which are regulated by
both α1- and α2- AR [118]. Differences have also been suggested to exist between arteries and
veins of the same vascular bed. Veins are more sensitive than arteries to the constrictor effects of
35
sympathetic nerve stimulation, and application of NE and PE [118]. The present study confirmed
this, as 1st and 3rd order MVs contracted at lower concentrations of NE and PE, demonstrating an
increased sensitivity to adrenergic stimuli than MA. This is displayed by significantly higher PD2
values (Table 4; Table 6). It is important to note that MV began to relax at high concentrations of
NE and PE, whereas MA responses remained constant. Mesenteric vein relaxation may have
been caused by a variety of factors, including; endothelial derived vasodilators (NO, EDHF,
prostaglandins), receptor desensitization or receptor internalization. The stable response
observed in MA may be the result of the system (ie. ligand + receptors) being maximally
occupied and activated, with no more reserve for contraction. These results contradict those
observed in mouse mesenteric vessels, where MA desensitized to adrenergic stimuli, attributed to
a complement of adrenergic receptors found in veins not arteries [35].
Interactions between α1- and α2-AR are common in heterologous receptor expression
systems [92]. Functional interactions have been proposed to exist in the rabbit isolated femoral
artery [168]. Contractile responses to PE were potentiated by prior exposure to UK-14 304, a
selective α2-AR agonist [168]. In mice, UK 14,304 did not constrict MA or MV but enhanced
constrictions and Ca2+ signalling mediated by α1-AR stimulation in MV [24]. Direct coupling of
α1- and α2-AR is assumed to occur in guinea pig cauda epididymis, and cultured rat glial cells, as
α1-AR-mediated responses were potentiated by xylazine, a selective α2-AR agonist [96,97]. This
functional interaction was found to be the result of α2-AR coupling through a Gi/o protein [96]. In
Chinese hamster lung fibroblasts, NE did not change intracellular [Ca2+] in cells expressing only
α1-AR. Intracellular calcium was increased by NE in those cells co-expressing α1-AR and α2AARs [28]. Interactions between adrenergic receptors and other receptors or systems have also
been identified. In the mouse atria, angiotensin II and bradykinin receptors were both affected by
36
post-junctional α2-AR activity [94]. Heterologous interactions or receptor cross-talk can result in
loss of contractile function (desensitization), as well as a gain or enhancement of contractile
function. α-adrenergic receptors are G-protein coupled receptors and signalling via one receptor
linked pathway may be affected by G-protein coupled receptors, coupled to other signalling
pathways [98]. Such interactions may affect coupling specificity and efficacy, and may be
important for physiological and pathophysiological implications.
Interactions between α1-AR and α2-AR may explain the differences observed in my
thesis research following NE and PE exposure, between MA and MV. It may also explain the
differences noticed following sympathetic nerve activation in the presence of prazosin or
phentolamine. This represents a limitation of this study as α2-AR activity was not directly
explored. Clonidine, a selective α2-AR agonist, could have been used to identify possible direct
effects of α2-AR activation in 1st versus 3rd order MA and MV. Yohimbine (selective α2-AR
antagonist) could confirm the selective activity of clonidine on α2-AR. Yohimbine could also be
used in EFS studies to determine contributions of NE induced contractions via activation of α2AR. However, pre-junctional α2-ARs present on SNS terminals regulate the release of NE
following EFS, thereby presenting challenges when using selective α2-AR agonists and
antagonists to explore post-junctional effects. Previous use of yohimbine, during sympathetic
nerve activation, resulted in a greater maximum contraction, in guinea pig MA and MV [93].
Experiments using lower concentrations of prazosin or phentolamine could address the
limitations mentioned previously, and may identify potential differences in α-AR activity. The
concentrations used in this study completely blocked NE and PE-mediated contractility. Lower
antagonist concentrations may permit VSM contractions at higher concentrations of NE, possibly
37
yielding a rightward shift in NE or PE CRC [37]. Yohimbine could also assist in investigating
NE activity on α2-AR.
It is noteworthy, that sympathetic nerve terminals in arteries and veins [23, 45] also
release ATP and NPY that can contribute to, and modify, EFS induced contractile responses. My
thesis research was restricted to the adrenergic system and did not evaluate these
neurotransmitters. The aforementioned may provide some explanation for the results obtained in
the presence of phentolamine in both 1st and 3rd order MA, following EFS. NE and ATP
significantly contribute to sympathetic neural responses in rabbit saphenous and jejunal artery,
and guinea-pig submucosal arterioles [110]. Opposing results were obtained in guinea pig
mesentery, indicating ATP and NPY activity in MV and not MA [23]. Future studies evaluating
ATP and NPY, using purinergic and NPY receptor antagonists, PPADs and NPY BIBP3226,
respectively, would be useful to decipher the activity of these neurotransmitters in rat MA and
MV, during EFS. Measuring neurotransmitter release patterns would also be useful in
characterizing sympathetic neurotransmission in the vessels used in my studies, but was not
included due to time and technical constraints with my research program. High performance
liquid chromatography (HPLC) techniques could analyze samples of the superfusate for ATP,
AMP, ADP, NPY and NE, before electrical stimulation (resting overflow) and during electrical
stiumulation (electrically evoked overflow) [174].
Branching Order Differences
The vasculature functions to maintain tight control of blood pressure and blood flow
between large and small arteries and veins. Changes in size and function of blood vessels from
arteries, arterioles, capillaries, venules and veins are known to be accompanied by differences in
38
regulatory mechanisms. These differences have not been thoroughly investigated, with most
studies focusing on arteries of a single branching order. Differences in phenylephrine mediated
contractility were identified between the rat thoracic aorta (TA) and abdominal aorta (AA), with
the TA demonstrating a more pronounced increase in IP3 formation and larger contraction than in
the AA [100]. In rat MA, sensitivity to transmural pressure induced contractions (myogenic
responses) increased with order of vessel generation, as 4th order MA demonstrated the greatest
contractions compared to other branching orders [15]. This is in accordance with the greater
importance of the smaller arterioles in the local regulation of resistance and blood flow to tissues
[15]. Additional differences were found in response to vasodilators, such as endothelium derived
hyperpolarizing factor (EDHF) and acetylcholine, as dilation increased with decreasing arterial
size in rat MA, attributed to an increased density of myoendothelial gap junctions [101, 103].
Research regarding regional differences in veins is limited. However in rats, 1st order MV are
more sensitive to sympathetic activity compared to 4th order MV [164].
In the current studies NE and PE generated differing CRC in MA, with NE resulting in a
greater maximum contraction in 1st order MA, while PE generated a greater maximum
contraction in 3rd order MA. Each of these agonists generated CRCs of similar shape and
magnitude in 1st versus 3rd order MV. Hilgers and De Mey, 2009 [16] had similar findings as
NE-mediated contractility was significantly greater in 1st order MA than 4th order MA. The
contradicting results produced by PE suggest a possible regulatory difference in α-AR mediated
contractility between 1st and 3rd order MA. As mentioned previously, α2-AR may provide direct
or indirect contributions to VSMC contractility. This may be through Gi/o proteins [96], or the
facilitation of α1-AR Ca2+ signalling by α2-AR activity [24]. α2-AR are also expressed by
endothelial cells, and are responsible for the release of vasodilator factors; primarily NO and to a
39
lesser extent EDHF and prostanoids [104]. This was attributed to the α2-AR-mediated dilation
observed in the rat aorta [105]. α2-AR activity may contribute to NE mediated contractility in 1st
order MA versus 3rd order MA. α2-AR activity may also produce greater dilatory effects in 3rd
order MA compared to 1st order MA, attenuating the overall contractile response. Removing the
endothelium pharmacologically and/or physically would eliminate contributions to net vascular
tone by vasodilatory effects produced by α2-AR, following NE treatment. While the release of
endothelial derived vasodilators, or constrictors, could contribute to net vascular tone, it was not
the focus of the current studies, albeit a limitation. Endothelial derived mediator release could be
evaluated pharmacologically by application of Nω-Nitro-L-arginine methyl ester hydrochloride (LNAME), a NOS inhibitor, and indomethacin, a COX inhibitor, inhibiting endothelial derived NO
and prostaglandin activity, respectively. The endothelium could also be mechanically denuded
via perfusion of distilled water through the vessel or a piece of hair through the lumen of the
vessel.
Differences in contractile responses to NE and PE in the current studies may involve
differences in receptor subtypes ie. α-AR subtypes. Both NE and PE have demonstrated affinities
for all α1-AR subtypes, however PE activation of α1B-AR resulted in submaximal contractionility
[106]. In mouse MV, PE CRC were right shifted only in the presence of BMY-7378, an α1D-AR
antagonist [24]. This study also showed a right shifted NE CRC in the presence of MK 912, a
selective α2C-AR antagonist [24]. In vivo studies, in rats, showed evidence for the significant role
of α1D-AR in the regulation of systemic arterial pressure [170]. Furthermore, studies using L767,314, a selective α1B-antagonist, illustrated that the α1B-AR is not involved in the PEmediated pressor response [171]. Finally, α1A-ARs mediate contractile responses to NE in rat
femoral resistance arteries [169]. Expression of α-AR subtype varies within vascular beds, with
40
the α1A-AR / α1D-AR predominant in the renal vascular bed [172], and the α1D-AR subtype in the
rat cremaster muscle arterioles [173]. The α1-AR subtypes involved in the regulation of
peripheral resistance and capacitance in the mesentery of rats is still unclear. The use of agonists
and antagonists specific for α1-AR subtypes would be useful for determining the effects
produced by each α1-AR isoform in the mesentery.
Conclusion
α1-ARs control vascular smooth muscle tone and thus modulates peripheral arterial
resistance and venous compliance. The α1-AR appears to be the primary adrenergic receptor
regulating contractility in1st and 3rd order MA and MV following sympathetic neurogenic
stimulation, as well as exogenous NE and PE treatment. There also appears to be possible
contributions from the α2-AR in 3rd order MA and 1st order MV. Whether or not this is the result
of a direct or indirect effect is unknown. Understanding the differences between arteries and
veins is essential for the progress of vascular physiology. Identifying differences regarding the
regional heterogeneity of a highly branched system of vessels is important, as they may improve
current treatment strategies for various cardiovascular diseases.
41
Figure 1. Frequency-response curves for sympathetic neurogenic constrictions in the absence
(control) and presence of prazosin (100nM) or phentolamine (10µM), in 1st order (A) and 3rd
order (B) mesenteric arteries. Data are presented as mean ± SEM from n rats. P<0.05, versus
prazosin treated groups; SMAX (*), S50 (†), versus phentolamine treated groups; SMAX (#), S50 (Ψ).
42
(A)
(B)
43
Figure 2. Frequency-response curves for sympathetic neurogenic constrictions in the absence
(control) or presence of prazosin (100nM) or phentolamine (10µM), in 1st order (A) and 3rd order
(B) mesenteric veins. Data are presented as mean ± SEM from n rats. . P<0.05, versus prazosin
treated groups; SMAX (*), S50 (†), versus phentolamine treated groups; SMAX (#),S50 (Ψ).
44
(A)
(B)
45
Figure 3. Comparison of control frequency-response curves for sympathetic neurogenic in 1st
versus 3rd order mesenteric arteries (A) and mesenteric veins (B). Data are presented as mean ±
SEM from n rats. P<0.05, 1st versus 3rd (*)
46
(A)
(B)
47
Figure 4. Effect of prazosin (100nM) or phentolamine (10µM), on norepinephrine
concentration-response curves in 1st order (A) and 3rd order (B) mesenteric arteries. Data are
presented as mean ± SEM from n rats. P<0.05, versus prazosin treated groups; EMAX (*), PD2 (†),
versus phentolamine treated groups; EMAX (#), PD2 (Ψ).
48
(A)
(B)
49
Figure 5. Effect of prazosin (100nM) or phentolamine (10µM), on norepinephrine
concentration-response curves in 1st order (A) and 3rd order (B) mesenteric veins. Data are
presented as mean ± SEM from n rats. P<0.05, versus prazosin treated groups; EMAX (*), PD2 (†),
versus phentolamine treated groups; EMAX (#), PD2 (Ψ).
50
(A)
(B)
51
Figure 6. Comparison of control norepinephrine concentration response curves in 1st versus 3rd
order mesenteric arteries (A) and mesenteric veins (B). Data are presented as mean ± SEM from
n rats. P<0.05, 1st versus 3rd; EMAX (*), PD2 (†).
52
(A)
(B)
53
Figure 7. Effect of prazosin (100nM) on phenylephrine concentration response curves in 1st
order (A) and 3rd order (B) mesenteric arteries. Data are presented as mean ± SEM from n rats.
P<0.05, versus prazosin treated groups; EMAX (*), PD2 (†), versus phentolamine treated groups;
EMAX (#), PD2 (Ψ).
54
(A)
(B)
55
Figure 8. Effect of prazosin (100nM) on phenylephrine concentration response curves in 1st
order (A) and 3rd order (B) mesenteric veins. Data are presented as mean ± SEM from n rats.
P<0.05, versus prazosin treated groups; EMAX (*), PD2 (†), versus phentolamine treated groups;
EMAX (#), PD2 (Ψ).
56
(A)
(B)
57
Figure 9. Comparison of control phenylephrine concentration response curves in 1st versus 3rd
order mesenteric arteries (A) and mesenteric veins (B). Data are presented as mean ± SEM from
n rats. p<0.05, 1st versus 3rd; EMAX (*),PD2 (†).
58
(A)
(B)
59
Table 1. Sympathetic neurogenic contraction maximum contraction (SMAX) in 1st and 3rd order
mesenteric arteries (MA) and veins (MV), in the absence (control) and presence of the selective
α1- antagonist prazosin (100nM), or the non-selective α-antagonist phentolamine (10µM).
SMAX (%)
MA
MV
1o
FRC
3o
1o
3o
Control
29.33±2.26(7) ab
31.29±2.24 (6) ab
25.59±2.03 (8) ab
29.94±2.35 (8) ab
Prazosin 100nM
11.69±1.78 (5)
15.75±1.51 (4)*†b
15.57±3.10 (5) *b
2.56±0.54 (4)
Phentolamine
10uM
11.26±1.09 (4)
9.47±2.25 (4)†
10.36±0.93 (4) *
3.24±0.65 (4)
Data are expressed as mean ± SEM; nd, not determined; n, number of rats. P<0.05, 1st versus 3rd
(*), MA vs MV (†), versus prazosin treated vessels (a), versus phentolamine treated vessels (b).
Table 2. Sympathetic neurogenic contraction stimulation frequency required to achieve half the
maximum contraction (S50), in 1st and 3rd order mesenteric arteries (MA) and veins (MV), in the
absence (control) and presence of the selective α1- antagonist prazosin (100nM), or the nonselective α-antagonist phentolamine (10µM).
S50
MA
MV
1o
FRC
3o
1o
3o
Control
11.09±0.95 (7)
12.46±0.62 (6) ab
2.12±0.42 (8)†ab
2.39±0.41 (8)†ab
Prazosin 100nM
11.95±2.95 (4)*
6.73±1.31 (4) †
12.47±2.33 (5)
11.64±0.99 (4) b
Phentolamine
10uM
11.35±2.28 (5)*
4.19±1.32 (4)†
12.00±2.69 (4)
19.80±1.33 (4)
Data are expressed as mean ± SEM; nd, not determined; n, number of rats. P<0.05, 1st versus 3rd
(*), MA vs MV (†), versus prazosin treated vessels (a), versus phentolamine treated vessels (b).
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 α1- antagonist, or
phentolamine (10µM), a non-selective α-antagonist.
EMAX (%)
MA
1o
NE
MV
3o
1o
3o
Control
32.31±2.25 (7)*†a
25.87±1.50 (7)†a
23.96±1.03 (7)a
22.95±1.05 (7)a
Prazosin 100nM
4.79±2.60 (4)
2.50±2.02 (5)
2.63±0.86 (5)
1.19±0.19 (5)
Phentolamine 10uM
nd (4)
nd (4)
nd (4)
nd (4)
Data are expressed as mean ± SEM. Emax, maximum contraction based on data fitted to the
logistic equation; nd, not determined; n, number of rats. P<0.05, 1st versus 3rd (*), MA vs MV (†),
versus prazosin treated vessels (a), versus phentolamine treated vessels (b).
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 α1- antagonist, or phentolamine (10µM), a non-selective α-antagonist.
PD2
MA
1o
NE
MV
3o
1o
3o
Control
5.56±0.05 (7) a
5.37±0.08 (7) a
7.18±0.08 (7)†a
7.28±0.19 (7)†a
Prazosin 100nM
3.38±1.13 (4)
2.79±1.14 (5)
8.48±0.28 (5)
6.28±0.31 (5)
Phentolamine 10uM
nd (4)
nd (4)
nd (4)
nd (4)
Data are expressed as mean ± SEM. PD2, the –log of the concentration required to achieve half the
maximum contraction; nd, not determined; n, number of rats. P<0.05, 1st versus 3rd (*), MA vs
MV (†), versus prazosin treated vessels (a), versus phentolamine treated vessels (b).
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 α1- antagonist.
EMAX (%)
MA
MV
1o
PE
3o
1o
3o
Control
30.19±1.64 (6)† a
33.55±1.51(6)*† a
16.65±1.68 (6)
14.42±2.25(7)
Prazosin 100nM
4.92±3.50 (4)
2.76±1.34 (4)
nd (4)
nd (4)
Data are expressed as mean ± SEM. Emax, maximum contraction based on data fitted to the
logistic equation; nd, not determined; n, number of rats. P<0.05, 1st versus 3rd (*), MA vs MV (†),
versus prazosin treated vessels (a), versus phentolamine treated vessels (b).
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 α1- antagonist.
PD2
MA
MV
1o
PE
3o
1o
3o
Control
5.33±0.10 (6) a
5.76±0.10 (6)* a
7.01±0.36(6)†
6.77±0.20(7)†
Prazosin 100nM
3.58±1.21 (4)
4.68±0.12 (4)
nd (4)
nd (4)
Data are expressed as mean ± SEM. PD2, the –log of the concentration required to achieve half the
maximum contraction; nd, not determined; n, number of rats. P<0.05, 1st versus 3rd (*), MA vs
MV (†), versus prazosin treated vessels (a), versus phentolamine treated vessels (b).
62
CHAPTER 2
CHARACTERIZATION OF α-ADRENERGIC RECEPTOR mRNA STABILITY IN 1st
VERSUS 3rd ORDER MESENTERIC ARTERIES AND VEINS OF NORMAL MALE
SPRAGUE DAWLEY RATS
63
Introduction
Vascular tone is one important determinant of cardiac output and blood pressure. Control
of vascular tone is largely mediated via neural pathways, activating α- and β- AR. Under normal
circumstances this activity is involved in the momen-to-moment control of cardiovascular
hemostasis. Arteries and veins work as a system, regulating TPR and VR respectively, with
arteries acting as resistance vessels, and veins as capacitance vessels, storing about 70% of total
blood volume. The splanchnic circulation receives nearly 25% of cardiac output, and contains
20% of total blood volume [39], with the majority of this blood stored in splanchnic veins. This
is attributed to large increases in VR, following minimal decreases in venous diameter [115].
Under normal conditions this function permits large changes in blood volume and blood
distribution, as occurs during exercise, haemorrhage or pregnancy. Chronic increases in VR may
however lead to cardiovascular complications [117].
A variety of factors determine the capacity for NE to affect arteries and veins. These
include relative α- and β- AR densities, AR affinities to NE, and plasma catecholamine
concentrations [39]. Adrenergic receptor density on VSMCs may be determined by the rates of
receptor synthesis and degradation [74]. It may also be a function of receptor cycling between
cell surface and non-surface accessible sites [74]. Catecholamine overexposure affects α- and βAR activity in several tissues, including VSMC [72-74]. This was observed when catecholamine
exposure decreased α1- and β2-AR density in aortic smooth muscle cells [59]. NE exposure was
then shown to decrease α1-AR mRNA expression [59]. The mechanisms behind this effect are
unknown.
64
Messenger RNA stability has recently been shown to be strongly influenced by RNARNA interactions, mediated by small RNAs [112]. Nearly half of all mammalian genes are now
predicted to be regulated by small noncoding RNAs [111,112]. Among this group are
microRNAs, a class of endogenous non-coding RNAs, approximately 20-25 nucleotides in
length, which serve as a potent regulator for gene expression [51]. MicroRNAs function at the
post-transcriptional level by binding to complementary sequences in the 3’-untranslated region
(3’-UTR) of target mRNAs. This inhibits mRNA expression either by blocking translation or by
signalling mRNA degradation, directly regulating cellular phenotype. Numerous studies have
shown miRNAs to be important regulators of various biological processes such as
differentiation, development and tumorigenesis [52-54].
MicroRNA activity has been associated with a variety of receptors systems. Estrogen
receptor-α activation with estradiol upregulates the expression of 26 miRNAs, and
downregulates the expression of 6 miRNAs in the mouse aorta [113]. Endothelin receptor-B
(ETB) has also been shown to be a target for miRNA-21 in gastric cancer, as cells treated with a
miRNA-21 inhibitor increased ETB protein production [114]. Minimal research has investigated
microRNA-dependent regulation of the adrenergic receptor family, with one study demonstrating
miRNA let-7f down regulation of β2-AR expression in mouse lung cells [56]. To date, no studies
have investigated miRNA activity on α1-AR mRNA. Computational algorithms can be used to
predict miRNA targets due to their base-pairing rules between miRNA and mRNA target sites,
location of 3’UTR binding sequence location and conservation of target binding sequences
within related genomes [126]. Online databases such as TargetScan v6.2
(http://www.targetscan.org) have identified conserved sites within the 3’-UTR of the α1-AR
gene, with high potential to represent target sites for the miRNA-30 family [121]. MicroRNA-
65
30c is of particular interest in this context as it is increased in various disease states including
thoracic aortic dissection, and pulmonary arterial hypertension [162, 163]. Understanding α1-AR
subtype expression between 1st and 3rd order MA and MV is important as α1-AR subtypes
involvement in the contractile responses to adrenergic agonists appears to differ within various
vascular beds.
The present study examined the effects of NE on α1-AR mRNA and miRNA-30c
expression, to identify a possible miRNA-dependent mechanism in vasomotor homeostasis. I
hypothesized that NE decreases α1-AR mRNA expression in MA and MV, in part through the
activity of miRNA-30c. Two experiments were completed towards addressing this hypothesis.
These included the effect of NE exposure on 1) α1A/B/D-AR mRNA expression and 2) miRNA30c expression.
Materials and Methods
Sample collection
1st and 3rd order MA and MV were obtained from the small intestine as described in the
methods section of Chapter 1. Once removed, 1 mg each of 1st and 3rd order arterial and venous
tissue were collected. The amount of tissue for study required five 1st order MA and 10 3rd order
MA, and 10 1st order MV and 15 3rd order MV. Vessels were submerged into cold Krebs buffer
solution, and all tissue surrounding the artery or vein was removed, while preserving the vessels
structural integrity. Cold buffer was changed every 5 minutes during dissection. Samples were
placed in RNAlater and were stored at -80oC. For time course experiments, untreated (0
minutes/control) vessels, and MA and MV superfused with norepinephrine (Sigma, Aldrich,
Oakville, Ontario, Canada) at a concentration of 10-6M for 15, 30 and 60 minutes, were
66
collected. NE was mixed with Krebs’ physiological buffer and maintained at 37oC and pH 7.4.
The NE solution was changed every 5 minutes, a procedure used previously in the lab.
Total RNA extraction
Total RNA containing small RNAs was isolated using the mirVANA™ miRNA
Extraction (Ambion, Austin, TX) kit. MA and MV samples were carefully removed from
RNAlater and placed in 300µl of lysis buffer. The tissue was homogenized with a plastic pestle
homogenizer, until all visible clumps were dispersed. 30µl of miRNA Homogenate Additive was
then added and left on ice for 10 minutes. 250µl of Acid-Phenol:Chloroform was added,
vortexed for 60 seconds, and centrifuged for 5 minutes at 10,000xg at 20oC. The upper aqueous
phase was then removed, being careful to not touch the bottom phase, and transferred to a fresh
tube. 1.25 volumes of 100% ethanol, maintained at room temperature, was added to the aqueous
phase. Lysate/ethanol mixture was pipetted onto a filter cartridge/collection tube, and was
centrifuged for 15 seconds at 10,000xg. Flow was discarded, and 700µl of Wash Solution 1 was
applied, then it was centrifuged for 5-10 seconds and 10,000xg. This was repeated twice using
500µl of Wash Solution 2/3. 50µl of elution solution, at 95oC, was then applied to the filter
cartridge and was centrifuged for 30 seconds at 10,000xg. The quantity and quality of total RNA
was assessed using the Nanodrop ND-1000 Spectrophotometer (NanoDrop Technologies Inc.,
Wilmington, DE, USA). A 260/280 ratio (RNA integrity) of 1.8-2.1 was deemed acceptable.
RNA samples were stored at -80oC.
67
cDNA synthesis
iScript™ cDNA Synthesis Kit (BioRad, Hercules, CA), and TaqMan MicroRNA Reverse
Transcription Kit (Applied Biosystems, Foster City, CA) were used to generate cDNAs for these
experiments. Different kits were required for mRNA and miRNA studies. Approximately 250 ng
RNA from each sample was added to a cDNA synthesis reaction using the iScript cDNA
Synthesis kit. Reactions were placed in a thermocycler, and were incubated for 5 minutes at
25oC, 30 minutes at 42oC and 5 minutes at 85oC. Approximately 50ng RNA from each sample
was added to a cDNA synthesis reaction using the Taqman®MicroRNA Reverse Transcription
Kit (Applied Biosystems) and appropriate RT primer pools for (Taqman® Rodent miRNA Array
A, Applied Biosystems). These included miRNA-30c and snoU6 as a housekeeping gene for
relative quantification. Reactions were placed in a thermal cycler (BioRad, Hercules CA) and
incubated for 30 minutes at 16oC, 30 minutes at 42oC and 5 minutes at 85oC. cDNA was stored at
-80oC until required.
Real-time polymerase chain reaction (qPCR)
The SsoFast™ EvaGreen® Supermix (Bio-Rad) and TaqMan® Universal PCR Mastermix
II with UNG (Applied Biosystems) were used to complete experiments 1 and 2. Once again
different kits were required for mRNA and miRNA studies. 25ng of cDNA was added to the
SsoFast™ EvaGreen® Supermix, with 500nM of each primer of interest. Primers used included
GAPDH forward (5’-GTCTTCACCACCACGGAGAAGGC-3’) and reverse (5’ATGCCAGTGAGCTTCCCGTTCAGC-3’); α1A-AR forward (5’GTAGCCAAGAGAGAAAGCCG-3’) and reverse (5’-CAACCCACCACGATGCCCAG-3’);
α1B-AR forward (5’-AGGGCCATCTCTGTGGGCCTGGTG-3’) and reverse (5’-
68
GATGGAGATGACCGTGGACAAGAC-3’); α1D-AR forward (5’CGTGTGCTCCTTCTACCTACC-3’) and reverse (5’-GCACAGGACGAAGACACCCAC-3’).
Reactions were pipetted into a 96 well plate and placed in a Bio-Rad CFX96 Real-Time PCR
System and incubated according to manufacturer’s instructions. An equal portion of cDNA from
each sample was added to Taqman® Universal PCR Mastermix II with UNG (Applied
Biosystems) with appropriate PCR primer pools (Taqman® Rodent miRNA Array A, Applied
Biosystems). Reactions were pipetted onto a 96 well plate and were placed in a Bio-Rad CFX96
Real-Time PCR System, and were incubated according to manufacturer’s instructions.
Data analysis
Data was analyzed using Biogazelle qBase+ software (Biogazelle NV, Zwijnaarde,
Belgium). A student’s t-test was used for comparison between groups, and one-way ANOVA,
followed by a Tukey’s post-hoc test, was used to examine the time-dependent effects caused by
NE exposure. All the results were expressed as mean ± standard error. P<0.05 was considered
statistically significant.
Results
Effects of NE exposure on α1-AR mRNA and miR-30c expression in 1st and 3rd order MA
Norepinephrine exposure had no effect on α1A/B/D-AR mRNA expression, in both 1st and
3rd order MA (Figure10, Figure 11). α1A-AR and α1D -AR mRNA expression was significantly
higher in 3rd order MA compared to 1st order MA at 0 minutes (untreated). This difference was
abolished following NE exposure (Figure 15A; Figure 15C; P<0.05). MicroRNA-30c expression
69
was unaffected by NE exposure, in 1st and 3rd order MA (Figure 14A; Figure 14B) and not
different in 1st versus 3rd order MA (Figure 15D).
Effects of NE exposure on α1-AR mRNA and miR-30c expression in 1st and 3rd order MV
In 1st order MV, α1B-AR and α1D -AR mRNA expression was significantly decreased
following 30 and 60 minutes of NE exposure, compared to control (Figure 12B; Figure 12C;
P<0.05). In 3rd order MV, α1B –AR mRNA expression was significantly decreased following 60
minutes of NE exposure (Figure 13B; P<0.05). α1D –AR mRNA expression was significantly
decreased following 30 and 60 minutes of NE exposure (Figure 13C; P<0.05). There were no
differences in α1A/B/D-AR mRNA expression in 1st order MV compared to 3rd order MV untreated
vessels (0 minutes). α1B-AR mRNA expression was significantly lower in 1st versus 3rd order
MV following 15 of NE exposure (Figure 16B; P<0.05). MicroRNA-30c expression was
unaffected by NE exposure, in 1st and 3rd order MV (Figure 14C; Figure 14D), and not different
1st versus 3rd order MV (Figure 16D).
Effects of NE exposure in arteries compared to veins
1st order MV, untreated vessels (0 minutes) demonstrated increased α1A-AR, α1B-AR and
α1D -AR mRNA expression, compared to 1st order MA untreated vessels (Figure 17A; Figure
17B; Figure 17C; P<0.05). There were no differences in α1A/B/D-AR mRNA expression between
3rd order MA and 3rd order MV (Figure 18). Finally, there were no differences in miRNA-30c
expression.
70
Discussion
The dynamic nature of the vasculature requires rapid changes in order to maintain
hemodynamics and appropriate cellular traffic. Norepinephrine, a potent vasoconstrictor, elicits
marked reductions in α1-AR and β2-AR mRNA expression in cardiac and aortic tissues [128,
129]. I postulated that NE induced reductions in α1-AR mRNA will occur in MA and MV,
possibly mediated in part at the level of mRNA stability involving microRNA activity.
MicroRNA-30c was selected to be investigated as it appears to be down regulated in pulmonary
aortic vascular smooth muscle cells, from rats with pulmonary arterial hypertension [130]. To
gain insight into the acute effects of NE on α1-AR mRNA and miRNA-30c expression levels,
MA and MV were superfused with NE for different times. As determined by qPCR, the
expression of α1B-AR and α1D-AR mRNA decreased in 1st and 3rd order MV in a time dependent
manner. These findings were not observed in MA, where NE treatment appeared to have no
effect on gene expression.
A variety of second messenger pathways are associated with the α1-AR, and may
participate in the regulation of α1B-AR and α1D-AR mRNA. Stimulation of the α1-AR causes
Ca2+ mobilization and increases IP3, DAG and prostaglandin levels [59]. In VSMCs,
prostaglandin E2 activates AC [131]. Phorbol-esters, a DAG mimetic, as well as cAMP decreases
the density of α1-AR in VSMCs [131,132], linked to the activation of protein kinase C and
protein kinase A respectively. Exposure to phorbol-esters, similarly to NE, also caused a rapid
(minutes) uncoupling of the receptor from IP3 turnover and Ca2+ mobilization [134-136]. The
nature of the effect of NE on α1-AR mRNA may be related to the uncoupling of the α1-AR from
a second messenger pathway. Prolonged exposure to cAMP analogues (or agents such as
forskolin, which directly stimulate AC) produced a loss of receptor binding sites that was
71
accompanied by substantial decreases in β2-AR mRNA levels [133]. As such, there are several
potential second messengers that might be involved in regulating α1-AR mRNA expression. It
would be of interest to compare the mechanisms by which α1- AR and β2-AR mRNAs are
regulated by agonists.
A disruption in the transcriptional regulation of the α1B-AR and α1D-AR, may underlie the
decreased expression of α1B-AR and α1D-AR mRNA. Recent studies revealed that chronic
stimulation of neonatal rat cardiomyocytes with NE leads to the repression of α1B-AR and α1DAR mRNA [137]. These changes were likely transcriptional in origin, and were followed by
changes in receptor protein by radioligand binding. Additional agonists including ET-1, and
prostaglandin F2α recapitulated these effects [137]. Faber and colleagues showed that plateletderived growth factor (PDGF-BB) suppress α1D-AR level (both protein and mRNA), while not
affecting α1A-AR and α1B-AR levels [138]. They later determined that this decrease occurred at
the level of transcription, as PDGF-BB was suggested to increase activator protein-2 (AP-2)
binding within the proximal promoter region [139]. Nuclear factor 1 (NF1) has been shown to
activate transcription of the α1B-AR gene following interaction with its P2 promoter in liver cells
[142]. They also demonstrated a reduced NF1 expression, paralleling a similar down regulation
of α1B-AR mRNA transcription [142]. Michelotti et al. [141] identified a methylation-dependent
disruption of Sp1 binding to GpC dinucleotides located in the proximal α1D-AR promoter [141].
An inverse correlation between transcriptional activity and methylation density exists, with
methyl-CpG now recognized as a gene-silencing signal [140]. The effect of NE on
transcriptional activity in VSMCs, is unknown. With the vast number of transcription factor (TF)
families, and the combinatorial control of genes by multiple TF [143], further study in this area is
required to understand NE mediated α1-AR mRNA regulation.
72
Effects mediated by NE on α1B-AR and α1D-AR mRNA may also occur at the level of
post-transcriptional control. Actinomycin D, a polypeptide that inhibits transcription, was
included in experiments to provide insight into the transcriptional versus posttranscriptional
nature of the mechanism behind NE mediated regulation of α1-AR mRNA expression [59]. With
the α1-AR mRNA being more stable in the absence of NE, it was assumed that the effects of NE
were likely due to decrease in the stability of α1-AR mRNA [59]. Pentamers or nonamers of
adenine (A) or uridine (U) RNA base pairs, located in the 3’-UTR region of mRNA, are termed
AU-rich elements (AREs) and play a role in regulating RNA stability [186]. They are important
cis elements that strongly affect transcript half-life under basal conditions and in response to
cellular activation [186, 187]. AREs act as binding sites for AU binding proteins (AUBPs),
which can stabilize or destabilize the transcript, depending on the protein involved. Several
AUBPs have been characterized which include tristetraprolin (TTP), AU-rich binding
Tristetraprolin destabilizes target transcripts by recruiting exosomes (a multi-protein complex
that may degrade RNA), promoting mRNA decay through decapping and deadenylation [188,
189]. AUF-1 is generally a destabilizing protein acting through a similar mechanism to TTP
[190]. AUF-1 has also demonstrated stabilizing abilities in vivo [190]. In contrast, ELAV
proteins, in particular Hu Antigen R (HuR) stabilize bound transcripts. This increased stability is
possibly caused by competing for ARE binding sites with destabilizing proteins, and physically
relocating transcripts from P-bodies to polysomes [191]. P-bodies are sites within a cell where a
high level of decay activity is present. The relative abundance of stabilizing and destabilizing
AUBPs is thought to determine the absolute level of decay of the transcripts [187,191].
Differences regarding AR expression between1st and 3rd order MA existed, with α1A-and
α1D-AR mRNA being expressed significantly higher in untreated (0 minutes) 3rd order MA
73
versus 1st order MA. This difference disappears following NE treatment; however some time
points appear to trend towards an increased expression. The only significant difference between
1st and 3rd order MV was the significantly higher expression of the α1B-AR in 3rd order MV
following 15 minutes of NE treatment. This suggests a more rapid decrease in α1B-AR mRNA in
1st order MV. Once again other time points appear to trend towards an increased expression.
Finally the differences detected between arteries and veins suggested an increased expression of
all α1-AR subtype mRNA in untreated (0 minutes) 1st order MV versus 1st order MA. This
difference was abolished following NE treatment, possibly the result of the decrease α1-AR
mRNA in 1st order MV. The large variance observed at various time points, in 1st and 3rd order
MA and MV presents a limitation of this study, possibly regarding the sample size used. Neither
a sample size calculation nor power analysis of the data was performed. Therefore, whether or
not I had enough power to pick up differences between means is unknown. I did however base
my sample size off previous student’s data from the lab.
MicroRNAs, a class of non-coding RNAs, function as repressors in all known animal
genomes [144]. To date, no studies have been performed investigating the relationship between
miRNAs and α1-AR mRNA. However, β2-AR mRNA expression is regulated by miRNA let-7f
[56]. TargetScan [http://www.targetscan.org] identified highly conserved binding sites within the
3’UTR of α1-AR mRNA for the miRNA-30 family. Here miRNA-30c was the chosen target, and
was shown to be expressed in all tissue examined, and not affected by NE treatment. The wide
variability displayed in the data collected suggests a possible biological limitation as miRNA
expression varies within tissues [116]. A possible technical limitation exists regarding
experimental technique, both at the vessel collection and during RT-qPCR, which presents an
74
area for possible variability. Also, as mentioned previously, the sample size used may have been
too small to detect differences.
MicroRNA-30c does not appear to be involved in the regulation of α1-AR mRNA in MA
or MV VSMCs. At the same time, Thoracic aortic dissection and hypoxia resulted in increased
miRNA-30c expression in aortic tissue and vascular smooth muscle cells, respectively [162,
163]. This provides evidence for the expression of miRNA-30c in VSMCs, under conditions
characterized by increased sympathetic activity. Additional experiments targeting the remaining
members of the miRNA-30 family, in the presence of NE, are required in order to rule out NEmediated miRNA effects on adrenergic receptor expression in mesenteric VSMCs. MicroRNA
microarray or deep sequencing analysis may be useful for further profiling miRNA expression
[162] in 1st and 3rd order MA and MV. This could identify any differentially expressed miRNA,
and possibly identify additional factors involved in NE-mediated decreases in α1-AR mRNA.
Moreover, bioinformatic analysis could organize differentially expressed genes and miRNAs
into hierarchial categories, such as NE upregulated and NE downregulated [162].
Conclusion
Norepinephrine exposure resulted a time dependent reduction in α1B-AR and α1D-AR
mRNA in 1st and 3rd order MV. Norepinephrine mediated regulation of α1-AR in 1st vs 3rd MV is
different from MA, and also appears to be subtype specific. The mechanisms behind these
observations are unknown, with microRNA-30c having no apparent involvement with NEmediated effects. Down-regulation of α1-AR may occur at the level of translation, during
posttranslational modifications of the receptor protein, or during cellular translocation of
receptors to the plasma membrane. Much emphasis has been placed on changes in transcriptional
75
regulation; however, changes in mRNA stability caused by microRNA activity may also affect
the transcriptional activity of α1-AR mRNA although the mechanism remains to be elucidated.
76
Figure 10. α1A-AR, α1B-AR and α1D-AR mRNA expression in 1st order mesenteric arteries
untreated (0 minutes) and following 15, 30 and 60 minutes of NE (10-6M), as measured by RTqPCR. Results are expressed as mean ± SE. * p < 0.05 versus control, n=4-6 for each treatment
time point.
77
A)
B)
C)
78
Figure 11. α1A-AR, α1B-AR and α1D-AR mRNA expression in 3rd order mesenteric arteries
untreated (0 minutes) and following 15, 30 and 60 minutes of NE (10-6M), as measured by RTqPCR. Results are expressed as mean ± SE. * p < 0.05 versus control, n=4-6 for each treatment
time point.
79
A)
B)
C)
80
Figure 12. α1A-AR, α1B-AR and α1D-AR mRNA expression in 1st order mesenteric veins
untreated (0 minutes) and following 15, 30 and 60 minutes of NE (10-6M), as measured by RTqPCR. Results are expressed as mean ± SE. * p < 0.05 versus control, n=4-6 for each treatment
time point.
81
A)
B)
C)
82
Figure 13. α1A-AR, α1B-AR and α1D-AR mRNA expression in 3rd order mesenteric veins
untreated (0 minutes) and following 15, 30 and 60 minutes of NE (10-6M), as measured by RTqPCR. Results are expressed as mean ± SE. * p < 0.05 versus control, n=4-6 for each treatment
time point.
83
A)
B)
C)
84
Figure 14. microRNA-30c expression in (A) 1st order MA, (B) 3rd order MA, (C) 1st order MV
and (D) 3rd order MV, untreated (0 minutes) and following 15, 30 and 60 minutes of NE (10-6M),
as measured by RT-qPCR. Results are expressed as mean ± SE. * p < 0.05 versus control, n=4-6
for each treatment time point.
85
A)
B)
C)
D)
86
Figure 15. A) α1A-AR mRNA, B)α1B-AR mRNA, C) α1D-AR mRNA and D) microRNA-30c
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. Results are expressed as mean ± SE. * p < 0.05 versus
control, n=4-6 for each treatment time point.
87
A)
C)
B)
D)
88
Figure 16. A) α1A-AR mRNA, B)α1B-AR mRNA, C) α1D-AR mRNA and D) microRNA-30c
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. Results are expressed as mean ± SE. * p < 0.05 versus
control, n=4-6 for each treatment time point.
89
A)
B)
D)
C)
90
Figure 17. A) α1A-AR mRNA, B)α1B-AR mRNA, C) α1D-AR mRNA and D) microRNA-30c
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. Results are expressed as mean ± SE. * p <
0.05 versus control, n=4-6 for each treatment time point.
91
A)
C)
B)
D)
92
Figure 18. A) α1A-AR mRNA, B)α1B-AR mRNA, C) α1D-AR mRNA and D) microRNA-30c
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. Results are expressed as mean ± SE. * p <
0.05 versus control, n=4-6 for each treatment time point.
93
B)
A)
C)
D)
94
GENERAL DISCUSSION
The physiological differences between arteries and veins cooperate to sustain tissue
supply of oxygen and nutrients, deliver various substances and return of deoxygenated blood
back to the heart. As blood flows, blood vessel number, diameter and total cross-sectional radius
changes, effecting blood flow velocity and mean blood pressure [76]. A number of vascular beds
have been studied within the body, with the splanchnic vasculature representing an important
active capacitance bed in the body, that shows vast branching within proximal arterioles and
venules, of which have known regulatory differences existing between lower and higher order
vessels [15, 16, 50, 164].
Maintenance of myocardial preload (myocardial filling volume) and afterload (systemic
vascular resistance) requires the integrated control of both venous capacitance and arterial
resistance, respectively [181]. Changes in venous capacitance is an important factor in the
control of VR and CO. Cardiac output must equal venous return, under all hemodynamic
conditions, indicating the reliance of cardiac output on venous return [8]. Reduced venous
capacitance can alter venous return, cardiac output and peripheral resistance and thus blood
pressure through auto-regulation [8]. Altered arterial resistance and venous capacitance has also
been reported in cardiovascular diseases including hypertension and congestive heart failure
[183].
Understanding the factors that contribute to the differential regulation of arterial and
venous function in relation to vessel branching order will enhance our understanding of
integrated vascular function. It will also provide insight on how differences in vessel regulation
can impart regional speciality of function to vascular beds, while contributing to the maintenance
of overall cardiovascular homeostasis. My thesis investigated α1-AR activity, and mRNA
95
stability, in MA and MV across branching orders ie., in 1st versus 3rd order vessels. The
experiments described, characterizing α1-adrenergic receptor function in 1st versus 3rd order MA
and MV, were designed to evaluate (i) differences in α1-AR mediated contractile function
between MA and corresponding MV, (ii) whether α1-AR activity changes based on vessel order
of generation, and (iii) expression of microRNA 30c, a miRNA with a predicted binding site on
α1-AR.
In Chapter 1, post-junctional α-AR mediated contractility was evaluated in 1st and 3rd
order MA and MV to NE, PE and EFS. I described the primary role of the α1-AR in 1st and 3rd
order MA and MV, with possible contributions from the α2-AR. This was demonstrated by pretreating vessels with the adrenergic receptor antagonists prazosin or phentolamine, which
completely blocked NE and PE mediated contractility. Results produced by sympathetic nerve
activation suggested the possibility of α2-AR activity in 3rd order MA and 1st order MV.
Meanwhile, pre-treatment with prazosin and phentolamine had similar effects on sympathetic
neurogenic contractility in 1st order MA, blocking contractility in 3rd order MV until maximum
stimulation frequencies. Conversely, sympathetic neurogenic-induced contractions of 2nd and 3rd
order rat MA were not changed in the presence of prazosin, whereas prazosin completely
abolished responses in MV [17,193]. In mice, only the α1-AR mediated direct contractile
responses in MA and MV, with indirect contributions from the α2-AR in MV only, in vitro,
presenting possible species differences [37].
In Chapter 2, NE effects on α1-AR subtype expression was evaluated over time in 1st and
3rd order MA and MV. The α1A- α1B- and α1D-AR subtypes all displayed similar expression levels
within all vessel types studied. Each α1-AR subtype mRNA was expressed higher in 1st order
MV versus 1st order MA. No significant differences were observed between 3rd order MA and
96
MV. These findings may be the result of higher order arteries and veins having a greater
dependence on other regulatory mechanisms, including local metabolic control. Veins are
known to be more sensitive to adrenergic stimuli when compared to arteries. Increased venous
sensitivity to adrenergic stimuli has functional relevance, as small changes in venous tone are
associated with larger changes in blood flow back to the heart [49]. The mesenteric bed has a
high level of sympathetic innervation, with a greater degree of sympathetic innervation in veins
given the reduced number of VSMC layers [192], possibly providing an explanation for the
increased venous sensitivity. This relationship was also observed in Chapter 1 however MA
demonstrated a larger maximum response to NE and PE compared to MV. This could be related
to the morphological differences that exist between MA and MV, with MA having increased
VSMC layering.
Chapter 1 revealed a stabilizing response to NE and PE in 1st and 3rd order MA, with
relaxation being observed at the highest concentrations in 1st and 3rd order MV. The use of higher
agonist concentrations would be required to confirm whether contractility does stabilize.
Moreover, Chapter 2 showed that acute periods of NE exposure reduced α1A- α1B- and α1D-AR
mRNA expression in 1st and 3rd order MV, with no differences observed in 1st or 3rd order MA.
Norepinephrine mediated decreases in α1-AR mRNA occurred in rabbit arterial SMC [59].
Further investigation will be required to the preliminary studies I conducted to clarify the
molecular effects mediated by NE in rat MA. It is also possible to assume that findings obtained
in one vascular bed may not directly apply to another. The function of veins and arteries of
various organs presents great vascular heterogeneity at a physiological level, and may
importantly reflect adaptive or protective mechanisms. For instance, skeletal muscle vessels
experience large pressure changes and develop myogenic reactivity which is greater than that
97
exhibited by other vessels, such as cerebral and mesenteric [216]. The absence of high distending
pressures and pulsatile flow conditions in the physiologic pressure range of most veins could
explain a reduced need for constitutive dilator release in these vessels. In contrast, arteries see
high pressures and as such need greater dilator release to counteract myogenic reactivity. For this
reason it would be of interest to explore the effects of increasing transmural pressure, on α-AR
activity following sympathetic nerve activation, and agonist application. It would also be of
interest to explore the effects of increasing pressure on NE-mediated effects on α-AR mRNA
expression. Longer exposure times may also be useful to help determine if this reduced
expression in MV is sustained, or if it returns to normal. Vessels obtained from heart failure rats
would shed some insight on chronic exposure of the vasculature to NE. Norepinephrinemediated decreases in α1-AR mRNA expression, evident after 1 hour of NE treatment, returned
to basal levels during the remaining 18 hours of exposure [59]. Under similar conditions in these
same cells, α1-AR density decreased gradually over 24 hours of NE treatment, and remained
depressed for 3 days, returning to basal levels once NE treatment finished [59]. Protein analysis
techniques, such as western blot, could discern whether the decrease in mRNA expression,
noticed in Chapter 2, is accompanied by a decrease in protein expression. Immunolocalization
techniques would also be useful to assess protein location within a cell, and determine whether it
is expressed in the cytosol or the membrane.
Many possible processes may mediate agonist-dependent regulation, including
desensitization. Desensitization is defined as the diminished response to a stimulus over time
despite continued or repeated stimulation, taking place within seconds to minutes of agonist
exposure [200]. Receptor uncoupling, following receptor phosphorylation [178], or receptor
sequestration from the cell surface, following a conformational change to the receptor [133], are
98
both associated with β-AR desensitization. Another possibility is receptor down-regulation,
defined as the loss of total receptor-binding sites in the cell with an accompanying loss in
effector stimulation. Receptor uncoupling and sequestration are rapid processes that take place
within seconds to minutes following agonist exposure, whereas receptor down regulation occurs
following a more prolonged stimulation (>1 hour) [133]. Murine mesenteric veins appeared to be
resistant to desensitization compared to arteries, which was contributed to a greater α-AR
receptor reserve [35], presenting another possible species differences.
Research investigating the relationship between vessel calibre and function is limited,
with none investigating differences regarding α1-AR subtype, or miRNA expression profiles.
Results from Chapter 1 described differences concerning vessel calibre, following NE and PE
treatment. NE exposure produced a greater maximum response in 1st order MA compared to 3rd
order MA. This may be the result of α2-AR mediated sensitization of the α1-AR, following coactivation of α1-AR and α2-AR in 1st order MA, increasing maximum contractility [37, 92]. The
observed differences may also be related to the release of vasodilators from the endothelium,
following α2-AR activation on endothelial cells, attenuating NE-mediated effects in 3rd order MA
[81]. These findings are in accordance with previous findings in 1st order MA and 4th order MA
from WKY rats [16]. Increased sympathetic activity also appears to produce a larger effect in 1st
order MV then in 4th order MV in rats [164]. Meanwhile, PE exposure produced a greater
maximum response and potency, in 3rd order MA compared to 1st order MA. This has also been
previously found with phenylephrine being significantly more potent in smaller (4th order) MA
compared with larger (1st order) MA, in rats [16]. Treatment of 1st order MA with high
concentrations of PE may have resulted in Ca2+ diffusion or transport through myoendothelial
cell junctions resulting in NO release, attenuating maximum response [177]. Inclusion of L-
99
NAME, resulted in a 2 fold increase to PE mediated contractility in MA, a response that was
abolished by disrupting the endothelium [176]. Hilgers and De Mey [16] pharmacologically
indicated that activation of arterial vascular smooth muscle cells can be accompanied by
endothelial vasomotor influences involving gap junctions and a balance between endotheliumderived NO and ET-1, and that this myoendothelial coupling is more prominent in distal
compared with proximal resistance arteries [16]. Chapter 2 went on to describe an increase in
α1A- and α1D-AR mRNA expression in 3rd order MA versus 1st order MA untreated vessels, with
no observable differences in 1st versus 3rd order MV untreated vessels. This agrees with the
increased maximum response observed following PE-mediated contractility in 3rd order MA
versus 1st order MA. During the cumulative addition of PE, vessels were exposed to the agonist
for 30 minutes during the duration of the experimental trial. With 3rd order MA demonstrating
increased α1-AR mRNA subtype expression compared to 1st order MA, it is possible that this
increased α1-AR mRNA expression correlates with increased α1-AR, resulting in an increased
reactivity to α1-AR agonists. It cannot be certain that increased α1-AR expression is responsible
for these observations. Unfortunately, results produced by sympathetic nerve activation, or
exogenous NE and PE treatment, in the presence of adrenergic antagonist, do not agree with this
assumption. This could have been caused by additional factors involved in sympathetic
neurogenic stimulation, including the non-adrenergic non-cholinergic neurotransmitters (ATP,
GABA, NPY, CGRP, etc).
My research did not investigate the relationship of arterial and venous myogenic
characteristics across differing sizes of mesenteric veins. It is still unknown if pressure induced
myogenic reactivity increases in mesenteric veins with the order of generation of the vessel as
has been found in mesenteric arteries [15]. It is clear that larger arteries and veins are much less
100
sensitive to metabolic control than smaller ones [157]. It is possible that vessels of higher order
require a more sensitive level of regulation than lower order vessels. Similar relationships exist
in rat MA vasodilator responses, with endothelial derived hyperpolarizing factor and
acetylcholine demonstrating increased dilation with decreasing vessel size [101, 103]. Taken
together, higher ordered vessels appear more sensitive to various vasoactive stimuli including;
pressure, metabolic factors and dilator factors. This is of interest considering the local
physiological importance of small arteries and veins. Small arteries and arterioles provide the
largest portion of resistance to blood flow in the entire mesenteric circulation [85,194]. Small
veins and venules provide important capacitance functions, storing 80% of the total blood
volume in the mesenteric circulation [85,194]. With ~20% of total blood volume stored within
the splanchnic venous bed [39], it is essential to have tight control over tone in these vessels
[85].
It is also possible that reactive oxygen species (ROS), generated during NE or PE
mediated contractility, played a role in the differences observed during functional studies.
Several studies have indicated that addition of super oxide radical, H2O2 or a hydroxyl radical
(OH·) generating system to vascular tissue in vitro causes contraction [201-204]. Another study
suggested that endothelium derived H2O2, superoxide and OH· are involved in NE-mediated
contractility [205]. The same study went on to indicate that endothelial NO scavenges ROS
generated during rat aortic ring contractions [205]. This was evident following a significant
right-ward shift to NE and PE CRC in the presence of hydroxyl radical scavengers (thiourea,
DMSO, mannitol and histidine), a shift that was several times les activity, generated from the
interaction of oxygen-derived free radicals with NO. In addition, superoxide radicals, hydrogen
peroxide and hydroxyl radicals generated in endothelial cells are involved, which augmented
101
NE-induced vasoconstriction of normotensive endothelium intact rat aortic rings [205]. The
relationship between ROS and NO presents possible regulatory differences between arteries and
veins of differing calibre.
Chapter 2 provided some preliminary data evaluating the mechanisms that could explain
decreasing α1A- α1B- and α1D-AR mRNA expression following NE exposure. MicroRNA-30c is
expressed in 1st and 3rd order MA and MV, and is unaffected by NE activity. The high variability
displayed by the RT-qPCR results presents a potential limitation. A biological variability may
account for this, as miRNA expression varies within tissues [116]. Examining the remaining
members of the miRNA-30 family may identify a post-transcriptional regulator of α1-AR
mRNA. Difficulty is presented by the activity of several second messengers, possibly involved in
modulating α1-AR mRNA including; IP3, DAG and prostaglandins [59]. This suggests the
possibility of Ca2+ and PKC as potential mediators in NE-mediated of α1-AR mRNA decrease
[195]. Still, recent studies using chronic stimulation of cardiomyocytes with NE leads to the
induction of α1A-AR mRNA, with concurrent repression of α1B-AR and α1D-AR mRNA [137].
These changes were not caused by altered mRNA stability, and were reflected in the binding
activity of [3H]prazosin to α1-AR at the protein level [137]. It was suggested that a
transcriptional mechanism may account for the subtype specific changes of the α1-AR mRNA
expression [181].
Inflammation can alter the vascular response to α1-AR activation, presenting a possible
cross talk between inflammatory mediators and α1-AR function and/or expression [196]. In
human monocytic cells, TNF-α and IL-β exposure increased α1A-AR mRNA expression, had no
effect on α1B-AR mRNA expression and decreased α1D-AR mRNA expression [199]. This same
study identified decreased α1B-AR and α1D-AR mRNA expression in human umbilical vein
102
endothelial cells [199]. Norepinephrine mediates cytokine expression and release. Endogenous
NE induced a time dependent increase of cardiac IL-6 mRNA after 1 hour of infusion [197]. In
macrophages, NE also appeared to regulate LPS-induced TNF-α production in an autocrine
fashion [198]. As such, it is possible that cytokines played a role in the decreased α1A-AR , α1BAR and α1D-AR mRNA expression in the current study, following NE exposure.
In summary, the findings presented in my thesis provided some insights into the regional
heterogeneity of mesenteric vessels, regarding α-AR function. Mesenteric arteries demonstrated
contradicting results with increased and decreased responses to NE and PE, in 1st versus 3rd order
MA respectively. This may be in part due to α2-AR activity in 1st order MA, or contributions
from endothelial derived vasodilators in 3rd order MA, in the presence of increasing
concentrations of NE. The inclusion of L-NAME or indomethacin, along with the physical
removal of the endothelium would assist in sorting out dilator contributions. Furthermore,
increasing concentrations of PE may have promoted endothelial derived NO release in 1st order
MA, attenuating response. Our results provide supporting evidence for an increased sensitivity
(PD2/S50) in 1st and 3rd order MV, with increased maximum responses in MA. This increased
sensitivity could be explained by the increased α1A- α1B- and α1D-AR mRNA expression in MV
compared to MA. Finally, the present thesis identified venous relaxation at high concentrations
of NE and PE. In MA however, the response plateaued. This may be explained by the downregulation in α1A- α1B- and α1D-AR mRNA expression following NE treatment, but no definitive
conclusions can be drawn. Future studies exploring the differences between α1- and α2- AR
function, as well as α1A- α1B- and α1D-AR function, in vessels varying in size, is required. This
would advance our knowledge of regional differences within highly branched vascular beds,
contributing to our understanding of integrated vascular function. Differences between vascular
103
function and calibre in certain cardiovascular diseases marked by altered sympathetic activity,
such as hypertension, are of relevance. This may reveal potential novel pharmacological targets
that could be of therapeutic value, in many clinical settings.
104
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