THE ROLE OF INFLAMMATION AND OXIDATIVE STRESS ON ALPHA 2-ADRENERGIC RECEPTORMEDIATED INHIBITION OF CALCIUM CHANNELS IN DOCA-SALT AND OBESITY-RELATED
HYPERTENSION
By
Ryan Kwun-Yee Mui

A DISSERTATION
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of
Physiology - Doctor of Philosophy
2017

ABSTRACT
THE ROLE OF INFLAMMATION AND OXIDATIVE STRESS ON ALPHA 2-ADRENERGIC RECEPTORMEDIATED INHIBITION OF CALCIUM CHANNELS IN DOCA-SALT AND OBESITY-RELATED
HYPERTENSION
By
Ryan Kwun-Yee Mui
DOCA-salt and obesity-related hypertension are associated with inflammation and
sympathetic nervous system hyperactivity.

Prejunctional 2-adrenergic receptors (2ARs)

provide negative feedback to norepinephrine release from sympathetic nerves through
inhibition of N-type Ca2+ channels. Increased neuronal norepinephrine release in DOCA-salt and
obesity-related hypertension occurs through impaired 2AR signaling, however, the
mechanisms involved are still unclear. Mesenteric arteries are resistance arteries that receive
sympathetic innervation from the superior mesenteric and celiac ganglia (SMCG). We tested
the hypothesis that macrophages and oxidative stress impair 2AR-mediated inhibition of Ca2+
channels in SMCG neurons in hypertension development and maintenance in DOCA-salt
hypertensive rats and obesity-related hypertensive Sprague Dawley (SD) and Dahl salt-sensitive
(DahlS) rats. DOCA-salt treated rats developed severe hypertension. On a high fat diet (HFD), SD
rats developed obesity with mild hypertension while Dahl S rats developed obesity with severe
hypertension.

Only DOCA-salt hypertension was associated with increased vascular

macrophage accumulation in mesenteric arteries and increased SMCG mRNA levels of
monocyte chemoattractant protein-1 and tumor necrosis factor-. Impaired 2AR-mediated
inhibition of Ca2+ currents occurred in SMCG neurons from DOCA-salt hypertensive rats and to a
milder extent, in obese mildly hypertensive SD rats. This did not occur in SMCG neurons from

obese hypertensive DahlS rats. In DOCA-salt hypertension, 2AR dysfunction was not associated
with changes in receptor expression, GRK-mediated desensitization, or downstream signaling.
In DOCA-salt treated rats, macrophage depletion using clodronate-liposomes preserved 2AR
function in SMCG neurons and protected against hypertension development. Oxidative stress,
specifically thiol-oxidation, impaired 2AR-mediated inhibition of Ca2+ currents in SMCG
neurons and resulted in receptor internalization to the perinuclear cytoplasm in HEK-293T cells.
These results suggest that macrophage-mediated 2AR dysfunction may be more relevant in
states of mineralocorticoid-salt excess. Furthermore, oxidative stress may impair 2AR signaling
by promoting mechanisms of receptor internalization.

This thesis is dedicated to the many positive role models in my life, both past and present.
When you have the right people supporting you, anything and everything becomes possible.

iv

ACKNOWLEDGEMENTS

Most importantly, I would like to thank Dr. James Galligan for his endless patience,
support and guidance. I could not have asked for a better mentor and have learned so much
from his example. Thank you for taking a chance on me, for encouraging me to try even when
techniques, awards and fellowships seemed unattainable, and for believing in me even during
times that I doubted myself. This has made a huge difference in my personal and professional
development and I hope that I am able to provide the same quality of mentorship to students
of my own one day.
I would like to thank my committee members Dr. Julia Busik, Dr. Gregory Fink, Dr. Nara
Parameswaran, and Dr. Robert Wiseman. I am very grateful for all the constant feedback,
wonderful discussions, and help in making me think critically about my work.
I would like to thank all members of the Galligan laboratory, both past and present.
Most notable of all is Roxanne Fernandes, who partnered with me in many experiments and
also in life. This work would not have been as enjoyable and possible without her support and I
am so glad we got to do this together.
I would like to thank Dr. Gregory Fink for spearheading our program project grant. It has
been an absolute pleasure to have a role in this very large project and I am grateful for our
ongoing collaboration. Furthermore, Hannah Garver from his laboratory contributed to all the
animal model development and we could not have performed our studies without her. I would
also like to thank Dr. Richard Neubig who provided us with all the DNA constructs, HEK-293T
cells, and G1A1 cells used for these studies.

v

I would like to thank Dr. Justin McCormick as well as Bethany Heinlen from the DO/PhD
program. It is hard to believe that I met these two wonderful people over seven years ago, and I
am so appreciative for all of their support ever since. I cannot thank them enough for this once
in a lifetime opportunity.
Finally, I would like to thank all of my friends and family; the list of which is just too long
to individualize in this section. From Dr. Sebastien Hayoz, who sat with me often times after
hours when I was first learning patch clamp, to my parents, who always believed in me and
taught me the value of hard work. I thank you all for keeping me motivated and for all the
wonderful times we had and will continue to have together.

vi

TABLE OF CONTENTS

LIST OF TABLES .................................................................................................................................x
LIST OF FIGURES .............................................................................................................................. xi
KEY TO ABBREVIATIONS ................................................................................................................ xiv
CHAPTER 1: GENERAL INTRODUCTION ........................................................................................... 1
1.0 Human Hypertension: .......................................................................................................... 2
1.1 Blood Pressure Relationships ........................................................................................... 2
1.2 Hypertension: Definition and Conventional Treatment Strategies ................................. 3
1.3 Hypertension: Prevalence and Risk Factor-Related Trends ............................................. 5
1.4 Hypertension: Categorizing Beyond Primary and Secondary Classifications .................. 6
2.0 Blood Pressure Regulation via the Sympathetic Nervous System ....................................... 8
2.1 General Introduction to Sympathetic Blood Pressure Regulation ................................... 8
2.2 Central Regulation of Sympathetic Nerve Activity......................................................... 11
2.3 Effects of Renovascular SNA on Blood Pressure ............................................................ 15
2.4 Peripheral SNA at the Neuroeffector Junction .............................................................. 20
2.5 NE Synthesis, Release, Reuptake and Metabolism ........................................................ 21
2.6
2-Adrenergic Receptor ........................... 24
2.7 Physiological Relevance of 2AR Subtypes .................................................................... 26
2.8 Mechanisms of Prejunctional 2AR Regulation ............................................................. 28
3.0 Increased SNA in Human and Experimental Hypertension ............................................... 32
3.1 Increased SNA Human Hypertension ............................................................................. 32
3.2 Increased SNA in Experimental Hypertension ............................................................... 35
3.2.1 Enhanced SNA in Genetic Hypertension .................................................................... 35
3.2.2 Enhanced SNA in Salt and Mineralocorticoid Hypertension ...................................... 37
3.2.3 Enhanced SNA in Obesity-Related Hypertension ....................................................... 43
4.0 Inflammation and Oxidative Stress in Hypertension ......................................................... 47
4.1 Vascular Injury in Hypertension ..................................................................................... 47
4.2 Lymphocytes and Macrophages in Hypertension .......................................................... 49
4.2.1 Lymphocytes ............................................................................................................... 49
4.2.2 Macrophages .............................................................................................................. 50
4.2.3 Sympathetic Modulation of Macrophages ................................................................. 55
CHAPTER 2: RESEARCH AIMS AND HYPOTHESES .......................................................................... 57
1.0 Research Goals ................................................................................................................... 58
2.0 Overall Hypotheses ............................................................................................................ 59
3.0 Specific Aims ...................................................................................................................... 61

vii

CHAPTER 3: EXPERIMENTAL DESIGN AND METHODS .................................................................. 63
1.0 General Experimental Design ............................................................................................ 64
2.0 Materials and Methods...................................................................................................... 66
CHAPTER 4: ADVENTITIAL MACROPHAGE ACCUMULATION AND SMCG INFLAMMATION OCCUR
IN DOCA-SALT BUT NOT HIGH FAT DIET-INDUCED HYPERTENSION............................................. 74
1.0 Abstract .............................................................................................................................. 75
2.0 Introduction ....................................................................................................................... 76
3.0 Results ................................................................................................................................ 80
4.0 Discussion........................................................................................................................... 86
CHAPTER 5: ALPHA 2-ADRENERGIC RECEPTOR-MEDIATED INHIBITION OF CALCIUM CHANNELS
IN DOCA-SALT AND OBESITY-RELATED HYPERTENSION ............................................................... 90
1.0 Abstract .............................................................................................................................. 91
2.0 Introduction ....................................................................................................................... 93
3.0 Results ................................................................................................................................ 96
3.1 Retrograde Labeling and Recording from Dissociated SMCG Neurons in Culture ........ 96
3.2 Investigating2AR Function in DOCA-Salt Rats ............................................................. 99
3.3 Investigating 2AR Function in HFD-Fed Sprague Dawley (SD) Rats ........................... 103
3.4 Investigating 2AR Function in HFD-Fed Dahl Salt-Sensitive (DahlS) Rats................... 111
4.0 Discussion......................................................................................................................... 117
4.1 DOCA-salt hypertension ............................................................................................... 118
4.2 Obesity-related hypertension in SD and DahlS rats ..................................................... 120
CHAPTER 6: MACROPHAGES CONTRIBUTE TO HYPERTENSION DEVELOPMENT IN DOCA-SALT
RATS BY IMPAIRING ALPHA 2-ADRENERGIC RECEPTOR FUNCTION POSSIBLY VIA OXIDATIVE
STRESS-INDUCED RECEPTOR INTERNALIZATION ........................................................................ 125
1.0 Abstract ............................................................................................................................ 126
2.0 Introduction ..................................................................................................................... 128
3.0 Results .............................................................................................................................. 130
4.0 Discussion......................................................................................................................... 137
CHAPTER 7: ALPHA 2-ADRENERGIC RECEPTOR DYSFUNCTION IN HIGH FAT DIET-INDUCED OBESE
HYPERTENSIVE FEMALE SPRAGUE DAWLEY RATS ...................................................................... 141
1.0 Abstract ............................................................................................................................ 142
2.0 Introduction ..................................................................................................................... 143
3.0 Results .............................................................................................................................. 145
4.0 Discussion......................................................................................................................... 150
CHAPTER 8: GENERAL SUMMARIES/CONCLUSIONS AND DISCUSSION ...................................... 153
1.0 Mineralocorticoid-Salt Hypertension .............................................................................. 154
2.0 Obesity-Related Hypertension ......................................................................................... 156
3.0 Macrophages in DOCA-Salt and HFD-Induced Hypertension .......................................... 160
4.0 Limitations of Whole-Cell Approach ................................................................................ 163

viii

5.0

Other Considerations of 2AR Dysfunction in DOCA-Salt Hypertension......................... 165

REFERENCES ................................................................................................................................ 168

ix

LIST OF TABLES

Table 1. Taqman probes for genes assessed in the SMCG. .......................................................... 71

x

LIST OF FIGURES

Figure 1. Relationship between blood pressure, cardiac output, and peripheral vascular
resistance. ........................................................................................................................ 2
Figure 2. Arterial diameter and blood pressure is modulated by sympathetic nerve activity... .. 10
Figure 3. Central regulation of sympathetic nerve activity. ......................................................... 14
Figure 4. Enhanced SNA to the kidney increases blood pressure. .............................................. 17
Figure 5. Pressure-natriuresis relationships. ................................................................................ 19
Figure 6. NE synthesis, release, reuptake and metabolism in sympathetic neurons. .................. 23
Figure 7. Mechanism of negative feedback through the prejunctional 2-adrenergic receptor
(2AR). ............................................................................................................................ 25
Figure 8. GRK-dependent desensitization and internalization of ligand-activated 2AR. ........... 30
Figure 9. Macrophages contribute to enhanced SNA in hypertension. ....................................... 54
Figure 10. Hypotheses and specific aims of our study. ................................................................ 60
Figure 11. Experimental design. ................................................................................................... 65
Figure 12. DOCA-salt hypertension is associated with inflammation in SMCG and mesenteric
arteries. .......................................................................................................................... 81
Figure 13. Low and high adventitial macrophage infiltration in mesenteric arteries from DOCAsalt rats. .......................................................................................................................... 82
Figure 14. Hypertension development in Sprague Dawley (SD) rats fed a HFD for 6 months is not
associated with obesity or SMCG and mesenteric artery inflammation. ...................... 83
Figure 15. Obesity-related hypertension development in HFD-fed DahlS rats. ........................... 84
Figure 16. Obesity-related hypertension in HFD DahlS rats is not associated with inflammation
in SMCG and mesenteric arteries at 2 (top row), 4 (middle row), and 6 months (bottom
row). ............................................................................................................................... 85

xi

Figure 17. Immunohistochemical localization of sympathetic neurons in the SMCG (white
arrow)............................................................................................................................. 96
Figure 18. Detection of retrograde labeling with CTB-555 in SMCG section (A, B) and dissociated
SMCG neurons (C). ......................................................................................................... 97
Figure 19. Representative pure Ca2+ and Ba2+ currents recorded from SMCG neurons.. ............ 98
Figure 20. DOCA-salt hypertension is associated with impaired 2AR-mediated inhibition of Ca2+
channels in acutely dissociated mesenteric artery-projecting SMCG neurons. .......... 100
Figure 21. Impaired2AR-signaling in DOCA-salt hypertension does not involve changes in
receptor expression, receptor desensitization, or G-protein signaling to inhibit Ca2+
channels. ...................................................................................................................... 102
Figure 22. Effects of HFD on weight and blood pressure in SD rats. .......................................... 103
Figure 23. HFD-induced hypertension in non-obese SD rats (6 months) is not associated with
impaired 2AR-mediated inhibition of Ca2+ channels in acutely dissociated SMCG
neurons. ....................................................................................................................... 105
Figure 24. SMCG mRNA expression of 2AR and GRK subtypes are not changed after 6 months
of HFD in SD rats. ......................................................................................................... 106
Figure 25. HFD-induced hypertension in obese SD rats (12 months) is associated with impaired
2AR-mediated inhibition of Ca2+ channels in acutely dissociated SMCG neurons. ... 107
Figure 26. Effects of HFD on weight and blood pressure in obesity-prone (OP) and obesityresistant (OR) SD rats. .................................................................................................. 109
Figure 27. Effects of HFD on 2AR-mediated inhibition of Ca2+ channels in acutely dissociated
SMCG neurons from obesity-prone (left side) and obesity-resistant (right side) SD rats.
...................................................................................................................................... 110
Figure 28. Obesity-related hypertension development in HFD-fed DahlS rats. ......................... 111
Figure 29. Obesity-related hypertension in HFD-fed DahlS rats is not associated with changes in
current-voltage relationships, cell capacitance, or Ca2+ current inhibition in acutely
dissociated SMCG neurons at 2 (top row), 4 (middle row), and 6 months (bottom row).
...................................................................................................................................... 113

xii

Figure 30. Obesity-related hypertension in HFD-fed DahlS rats is not associated with impaired
2AR-mediated inhibition of Ca2+ channels in acutely dissociated SMCG neurons at 2
(top row), 4 (middle row), and 6 months (bottom row). ............................................. 114
Figure 31. Obesity-related hypertension in HFD-fed DahlS rats is not associated changes in
receptor expression in SMCG. ..................................................................................... 115
Figure 32. Obesity-related hypertension in HFD-fed DahlS rats is not associated changes in GRK
expression in SMCG. .................................................................................................... 116
Figure 33. Thiol-oxidation of cysteine residues. Oxidative modifications can affect protein
structure, function and localization. ............................................................................ 129
Figure 34. Clodronate-embedded liposome treatment attenuates adventitial macrophage
infiltration. ................................................................................................................... 130
Figure 35. Macrophage depletion with clodronate-liposomes (Clod) does not affect currentvoltage relationships and cell size versus PBS-liposomes (PBS). ................................. 131
Figure 36. Representative traces of Ca2+ current inhibition following macrophage depletion with
clodronate-liposomes (Clod) versus PBS-liposomes (PBS).. ........................................ 132
Figure 37. Macrophage depletion with clodronate-liposomes (Clod) preserves 2AR function
and protects against hypertension development in DOCA-salt rats versus PBSliposomes (PBS)............................................................................................................ 133
Figure 38. Oxidative stress impairs2AR function in SMCG neurons and G1A1 cells. .............. 135
Figure 39. Oxidative stress results in receptor localization to the perinuclear cytoplasm. ....... 136
Figure 40. Effects of HFD on body weight and blood pressure in female SD rats. ..................... 145
Figure 41. 4 months of HFD does not impair 2AR-mediated inhibition of Ca2+ channels in
acutely dissociated SMCG neurons.............................................................................. 147
Figure 42. 10 months of HFD impairs 2AR-mediated inhibition of Ca2+ channels by NE
activation in acutely dissociated SMCG neurons. ........................................................ 149

xiii

KEY TO ABBREVIATIONS

1AR- 1 adrenergic receptor
2A- 2 adrenergic receptor subtype 2A
2B- 2 adrenergic receptor subtype 2B
2C- 2 adrenergic receptor subtype 2C
2AR- 2 adrenergic receptor
Ach- Acetylcholine
ANG-II – Angiotensin II
AP- Area postrema
AT1R- Angiotensin 1 receptor
ATP- Adenosine triphosphate
BBB-Blood brain barrier
CRP- C-reactive protein
DahlS- Dahl salt-sensitive
DCT- Distal convoluted tubule
DOPA- Dihydroxyphenylalanine
eNOS- Endothelial nitrogen oxide synthase
GM-CSF- granulocyte-macrophage colony-stimulating factor
GppNHp- 5’-guanylyl imidodiphosphate
GPCR- G protein-coupled receptor
GRK- G protein-coupled receptor kinase
HA- Hemagglutinin
HBSS- Hank’s buffered salt solution

xiv

HFD- High fat diet
IML- Intermediolateral
ICAM-1- Intracellular adhesion molecule 1
JAM-1- Junctional adhesion modlecule-1
IL-6- Interleukin 6
M3 – Muscarinic 3 receptor
MCP-1- Monocyte chemoattractant protein-1
NADPH- nicotinamide adenine dinucleotide phosphate
NE- Norepinephrine
NET- Norepinephrine transporter
NFD- Normal fat diet
NO- Nitric oxide
NPY- Neuropeptide Y
NSF- N-ethyl-maleimide-sensitive factor (NSF)
NTS- Nucleus of the solitary tract
OP- Obesity-prone Sprague Dawley
OR- Obesity-resistant Sprague Dawley
OVLT- Organum vasculosum of the lamina terminalis
PPAR- Peroxisome proliferator-activated receptor
PVN- Paraventricular nucleus
RAAS- Renin-angiotensin-aldosterone system
RAS- Renin-angiotensin system
ROS- Reactive oxygen species
RVLM- Rostral ventrolateral medulla

xv

SD- Sprague-Dawley
SFO- Subfornical organ
SHR- Spontaneously hypertensive rat
SCG- Superior cervical ganglia
SMCG- Superior mesenteric and celiac ganglia
SNA- Sympathetic nerve activity
SNAP- Soluble N-ethyl-maleimide-sensitive factor attachment protein
SNAP-25- 25 kDa synaptosome-associated protein
SNARE- N-ethylmaleimide sensitive factor attachment protein receptor
SNS- Sympathetic nervous system
TNF- Tumor necrosis factor-
t-SNARE- Target membrane N-ethylmaleimide sensitive factor attachment protein receptor
TTX- Tetrodotoxin
v-SNARE- Vesicular membrane N-ethylmaleimide sensitive factor attachment protein receptor
WK- Wistar Kyoto

xvi

CHAPTER 1: GENERAL INTRODUCTION

1

1.0

Human Hypertension:
1.1

Blood Pressure Relationships

Blood pressure is a measurement of force exerted against the vascular wall of large
elastic arteries that is proportional to cardiac output and peripheral vascular resistance to blood
flow [1], as depicted in Figure 1. Cardiac output is the product of heart rate and stroke volume,
the latter of which is influenced by myocardial contractility, preload, and afterload [2]. Cardiac
output receives innervation from both the sympathetic (increases cardiac output) and
parasympathetic (decreases cardiac output) nervous system [3]. Peripheral vascular resistance
is contributed by small muscular resistance arteries, where resistance is inversely proportional
to radius to the fourth power [1]. The majority of blood vessels receive innervation only from
the sympathetic nervous system [3].

Figure 1. Relationship between blood pressure, cardiac output, and peripheral vascular
resistance. Increases in cardiac output and/or decreases in diameter of small resistance arteries
result in the accumulation of blood in large elastic arteries, which increases blood pressure.

2

1.2

Hypertension: Definition and Conventional Treatment Strategies

Definition: In humans, hypertension is defined in adults by systolic blood pressures
equal or greater to 140 mmHg and/or diastolic blood pressures equal or greater to 90 mmHg.
Hypertension development does not usually present with clinical symptoms [4], however, leads
to significant morbidity and mortality as a major risk factor for stroke, heart disease, and kidney
failure [5]. The risk of cardiovascular disease begins to increase as early as 115/75 mmHg [6, 7],
suggesting that there is benefit in reducing blood pressure even before hypertension begins.
Treatment: Hypertension is the leading cause of preventable death in the United States
[8] and therefore, requires aggressive diagnosis and treatment. Hypertension is the most
common chronic condition managed by health care providers [9]. Unfortunately, clinical
management success rates remain poor [10, 11] and novel approaches and therapeutic targets
are needed. Pre-hypertension (where blood pressures can range from 120/80-139/89 mmHg)
is typically managed by lifestyle modifications. Exercise, weight loss, and decreasing dietary salt
are commonly encouraged to delay or prevent hypertension development [9]. When
hypertension develops, treatment warrants pharmacological intervention, usually involving
more than one antihypertensive drug consisting either an angiotensin converting enzyme
antagonist (or angiotensin receptor blocker), a calcium channel blocker, or a diuretic [9].
Lifestyle modifications are equally important in hypertension management. Salt intake and
obesity can affect hypotensive therapy [12-14] and are therefore important therapeutic targets.
Resistant hypertension: A significant proportion of hypertensive patients are resistant
to conventional therapy. Resistant hypertension occurs when hypertension is sustained despite
the use of three anti-hypertensive drugs of different classes [15]. Resistant hypertension affects

3

5-30% of hypertensive patients [16] and furthers the risk of adverse cardiovascular-related
complications already associated with hypertension [17, 18]. Although universal therapeutic
recommendations are not yet determined, the addition of aldosterone antagonists in the
treatment of resistant hypertension appear to quite effective [19].

4

1.3

Hypertension: Prevalence and Risk Factor-Related Trends

The prevalence of human hypertension generally increases with age, typically beginning
after 25 years and becoming apparent after 50 years [20, 21]. Although hypertension
development is often regarded as a normal process of aging, the high prevalence of
hypertension cannot be explained only by an aging population. It is now clear that hypertension
is associated with common risk factors like high dietary salt intake and obesity [9, 22]. While the
clinical manifestations of hypertension become evident mainly in adulthood, factors that
contribute to its development may be rooted in childhood. Although hypertension cut-offs are
not clearly defined in children, higher blood pressures in childhood are able to predict
hypertension development and severity in adulthood [23-25]. Interestingly, the prevalence of
childhood and adolescent hypertension is on the rise and may be a attributed to the increased
consumption of processed foods and beverages, which leads to earlier exposure to high dietary
salt intake and obesity rates in children [26-31].

5

1.4

Hypertension: Categorizing Beyond Primary and Secondary Classifications
Primary versus secondary hypertension: The majority of human hypertension cases

(~95%) are classified as primary (essential, idiopathic) hypertension, where there is sustained
hypertension without an identifiable cause [9]. As a non-specific diagnosis, essential
hypertension is difficult to treat and often results in suboptimal management that may involve
inappropriate choice of anti-hypertension medication [32]. This is in contrast to secondary
hypertension (~5% of cases), where increased blood pressure arises due to an underlying and
often correctable medical condition [33]. Examples of secondary hypertension include
hyperaldosteronism, renal artery stenosis, thyroid disorders, pheochromocytoma, and Cushing
syndrome. Secondary hypertension can also result as a side effect from using medications such
as oral contraception, decongestants, steroids, and amphetamines [33].
Although hypertension is heterogeneous in nature, identifying subgroups of
hypertensive patients with common features may be useful in understanding the complex
mechanisms that underlie hypertension development and maintenance.
Inflammation and increased SNA: The most consistent findings in human hypertension
appear to be enhanced SNA and inflammation. With improvements in detection techniques, it
is now clear that the majority of hypertensive patients, especially during the development
phase, display enhanced SNA [34]. Inflammation is evidenced by levels of the inflammatory
marker C-reactive protein (CRP), which predictably increase from normotension to pre- and
overt hypertension, and identify those at risk of cardiovascular sequelae [35-38].
Salt-sensitivity: A significant subset of hypertensive patients is salt-sensitive. Although a
universally defined definition is still lacking, it is generally accepted that salt-sensitivity reflects

6

a 5-10% increase in blood pressure to salt intake [39]. Salt-sensitivity affects roughly half of all
essential hypertensive patients and a quarter of normotensive individuals, and may be due to
underlying genetic predisposition [40, 41].
Obesity: It is estimated that the majority of hypertension cases are attributed to obesity
[42, 43]. In humans, obesity is defined as body mass index greater or equal to 30 kg/m2.
Increases in body mass index is associated with hypertension development and blood pressure
significantly decreases with weight loss [42].
Low-renin and aldosterone-excess: A percentage of hypertensive patients (25-33%)
have low-renin hypertension [44, 45]. Of these, approximately 60-70% have resistant
hypertension [44]. Low-renin hypertension is higher among blacks and the elderly, and is
typically salt-sensitive [45]. Low-renin may be accompanied by high circulating levels of
aldosterone (a mineralocorticoid) [44, 46], which may be due to idiopathic autonomous
aldosterone hypersecretion [47, 48]. Primary hyperaldosteronism is the leading cause of
secondary hypertension and is evident in conditions like Conn’s syndrome, where aldosterone
is produced in excess by the adrenal gland [49].
The effects of sympathetic hyperactivity, inflammation, salt, obesity, low-renin status
and mineralocorticoids are all clinically relevant and are therefore exploited interventionally to
induce hypertension development in experimental animal models to better understand human
hypertension (see section 3- animal models for more detail).

7

2.0

Blood Pressure Regulation via the Sympathetic Nervous System
2.1

General Introduction to Sympathetic Blood Pressure Regulation

The sympathetic nervous system modulates both short and long term regulation of the
cardiovascular system without conscious control [50, 51]. Sympathetic neuroeffector
transmission increases blood pressure either by increasing cardiac output and by promoting
vasoconstriction of resistance arteries [52]. Increased peripheral vascular resistance is the
prominent hemodynamic disturbance in human hypertension as cardiac output is usually
relatively within normal limits, possibly due to parasympathetic compensation [53, 54].
Sympathetic regulation of the splanchnic circulation: The splanchnic circulation
provides the majority of the body’s vascular resistance through mesenteric resistance arteries.
Mesenteric resistance arteries are innervated by sympathetic neurons of the superior
mesenteric and celiac ganglion (SMCG) [55, 56], are not innervated by parasympathetic
neurons, and have profound influences on hemodynamics and blood pressure [57-60].
Sympathetic hyperactivity in the splanchnic circulation contributes to hypertension
development. Splanchnic denervation decreases blood pressure in normal [61] and
hypertensive rats [59, 60, 62, 63]. Furthermore, the density of sympathetic innervation to
mesenteric resistance arteries is similar to other vascular beds, such as cerebral resistance
vessels and femoral arterial beds [64, 65]. Therefore, investigating neurovascular mechanisms
of blood pressure regulation in the splanchnic circulation has been the focus of many
experimental hypertension studies and these approaches may have relevance in other vascular
systems that are more difficult to study.

8

Neurogenic hypertension: Evidence suggests that a large component of essential
hypertension development and maintenance is neurogenic [66]. The immediate effector
response occurs at the neuroeffector junction. For instance in resistance arteries,
depolarization of post-ganglionic sympathetic neurons causes the release of neurotransmitters
from nerve terminals onto vascular smooth muscle. Baseline sympathetic nerve activity
preserves vascular homeostatic tone while increased sympathetic firing rate increases blood
pressure by vasoconstriction [52], as depicted in Figure 2. It is well established that sympathetic
hyperactivity occurs in human essential hypertension as well as experimental hypertension
animal models [67, 68]. However, in order to study potential mechanisms that underlie
sympathetic alterations in hypertension, it is first important to understand how SNA is
modulated at different levels of regulation, including both the peripheral nervous system (PNS)
and central nervous system (CNS).

9

Figure 2. Arterial diameter and blood pressure is modulated by sympathetic nerve activity.
Higher frequency of sympathetic firing results in increased neuroeffector transmission,
resulting in the constriction of small resistance arteries and an increase in blood pressure.

10

2.2

Central Regulation of Sympathetic Nerve Activity

Sympathetic firing rate is largely determined by input received and processed by
circumventricular organs (CVOs) in the central nervous system (CNS) as well as through
autonomic reflexes. These are shown in Figure 3.
CVOs and autonomic regulatory centers: CVOs are specialized autonomic control
centers. They are extensively vascularized without a proper blood-brain barrier because they
monitor and respond to variations in systemic circulation composition, from basic blood
chemistry to hormone levels. CVOs include the subfornical organ (SFO), organum vasculosum of
the lamina terminalis (OVLT), and the area postrema (AP).
Sensory information received and interpreted by the CVOs provide feedback via distinct
neuroanatomical connections to autonomic regulatory centers in the hypothalamus and
brainstem. These centers include the paraventricular nucleus (PVN), the nucleus of the solitary
tract (NTS) and the rostral ventrolateral medulla (RVLM) [69]. Eventually, all transmitted
information reaches the RVLM, which is the main driver that increases sympathetic outflow.
RVLM neurons activate preganglionic sympathetic neurons, whose cell bodies are
mostly located in the intermediolateral (IML) nucleus of the thoraco-lumbar spinal cord. These
neurons project either directly to glands such the adrenal medulla, or to paravertebral and
prevertebral sympathetic ganglia, where they synapse onto post-ganglionic sympathetic
neurons. Post-ganglionic sympathetic neurons innervate target organs of the cardiovascular
and renovascular system [70], which are key players in blood pressure regulation.
A classic example of central mediated enhancement of SNA is hypernatremia, which can
result from increased salt consumption [71]. OVLT and SFO neurons contain osmoreceptors

11

that can detect changes in osmotic pressure via osmosis. Hyperosmotic challenges activate
these neurons. Increased glutamate and decreased GABA release activates neurons of the PVN
and subsequently the RVLM [71-74]. RVLM efferent neurons activate peripheral sympathetic
neurons, resulting in an increase in peripheral vascular resistance.
Autonomic Reflexes: Efferent sympathetic activity is also regulated by autonomic
reflexes. Although often described as short-term mechanisms of blood pressure regulation,
these reflexes can be modified to operate at higher blood pressures if hypertension is sustained
for a long period of time. These reflexes involve sensory input from afferent fibers of the cranial
nerves, which are then transmitted to the hypothalamus and brainstem [51].
Baroreceptor Reflex: The most classic example is the baroreceptor reflex. Baroreceptors
are sensory mechanoreceptors located on vagal and glossopharyngeal nerves that innervate
the carotid sinuses and aortic arch. Baroreceptors function to monitor and regulate acute
changes in blood pressure and are tonically sympathoinhibitory at rest. Increased blood
pressure from baseline stretches the vascular wall, resulting in increased baroreceptor sensory
transmission to the NTS. This Information is transmitted to the RVLM, which decreases output
to peripheral sympathetic neurons that innervate the heart and resistance arteries, allowing
blood pressure to decrease back to baseline [51]. Similarly, decreases in blood pressure from
baseline (such as with standing) reduces vascular stretch, decreasing baroreceptor sensory
input to the NTS, resulting in an increase in sympathetic output [51]. Baroreceptors can adapt
to prolonged changes in blood pressure by resetting their baseline response to operate at
higher blood pressures. Studies of the baroreceptor reflex by phenylephrine bolus delivery
(high pressure induction) and by inhalation of amyl nitrite (low pressure induction) suggest

12

impaired baroreceptor sensitivity in hypertensive individuals and even in normotensive
individuals that have a family history of hypertension [75, 76].
Chemoreceptor Reflex: Another autonomic reflex is the chemoreceptor reflex.
Chemoreceptors are also located in the carotid sinuses and aortic arch. Chemoreceptors
activate when O2 delivery and CO2 removal is reduced, which occurs as a result of poor tissue
perfusion when blood pressure falls (usually < 80 mmHg). Activated chemoreceptors transmit
signals to the RVLM, resulting in enhanced sympathetic outflow to the cardiovascular and
renovascular system [77, 78].

13

Figure 3. Central regulation of sympathetic nerve activity. Circumventricular organs, which
include the subfornical organ (SFO), organum vasculosum of the lamina terminalis (OVLT), and
area postrema (AP) monitor blood composition. This information is transmitted to regulatory
organs, which include the paraventricular nucleus (PVN), the nucleus of the solitary tract (NTS)
and the rostral ventrolateral medulla (RVLM). Information eventually reaches the RVLM, which
contains efferent neurons that synapse onto pre-ganglionic sympathetic neurons located in the
intermediolateral nucleus of the thoracolumbar spinal cord. Preganglionic sympathetic neurons
synapse onto post-ganglionic neurons in sympathetic ganglia that innervate cardiovascular and
renovascular systems to regulate blood pressure. Sympathetic nerve activity is also regulated
by autonomic reflexes. Baroreceptors and chemoreceptors in the carotid sinus and aortic arch
transmit sensory information regarding blood pressure changes to the NTS, resulting in effector
responses that attempt to restore blood pressure back to baseline.

14

2.3

Effects of Renovascular SNA on Blood Pressure

Renin-angiotensin-aldosterone system: Sympathetic regulation of renal function is
important in blood pressure control. The kidney is innervated by sympathetic nerves and renal
denervation attenuates increased blood pressure in the majority of salt-sensitive hypertension
models [79]. Vasoconstriction from enhanced SNA to the kidney has consequences on Na+ and
water balance through the renin-angiotensin-aldosterone system (RAAS), as shown in Figure 7.
Firstly, renal SNA to the proximal tubules activates 1ARs on renal tubular cells resulting
in increased Na+ and water reabsorption, and 1ARs on renal afferent arterioles, potentiating
vasoconstriction and reduced renal plasma flow [80, 81].
Secondly, Renal SNA also increases blood pressure indirectly through members of RAAS
[82, 83]. Juxtaglomerular (JG) cells within renal afferent arterioles are innervated by
sympathetic neurons. Enhanced SNA to JG cells activates 1-adrenrgic receptors (1AR),
resulting in release of the enzyme renin. Renin catalyzes the first step in the synthesis of
angiotensin II (ANG-II). Through the angiotensin type I (AT1) receptor, ANG II increases blood
pressure directly through vasoconstriction and by increasing Na+ and water reabsorption by
modulating ion transporter activity in the proximal tubule [84]. ANG-II also indirectly increases
blood pressure by enhancing aldosterone secretion from the adrenal cortex. Aldosterone
increases blood pressure through the cytosolic mineralocorticoid receptor by increasing Na+
and water reabsorption at the expense of K+ in the distal nephron. This is accomplished by
increasing apical permeability through upregulation of epithelial sodium channels (ENaC) and
by upregulating Na+/K+ ATPases in the basolateral membrane. Basolateral Na+/K+ATPase
upregulation keeps intracellular levels of Na+ low and K+ high, which sets up a gradient for more

15

Na+ (and water) reabsorption from the tubular lumen. K+ efflux into the tubule counterbalances
N+ entry, and is the reason why states of mineralocorticoid excess are often associated with
hypokalemia [82, 83].
The influences of RAAS on hypertension development extend beyond the renal system.
ANG-II has been implicated in enhanced neurotransmission both in the central and peripheral
nervous system. Staining for c-FOS shows that ANG-II infusion activates the PVN, SFO, and NTS
[85], and microdialysis studies show that intracerebroventricular administration of ANG-II
increases NE release in the hypothalamus [86], specifically in the PVN [87]. Furthermore, ANG-II
may also be increasing SNA through oxidative stress production by activating nicotinamide
adenine dinucleotide phosphate (NADPH) oxidases in CVOs, which contributes to hypertension
development [88]. In the periphery, ANG II has been shown to increase release of NE in
cardiovascular tissues [89-92] and EP from the adrenal gland [93].
In addition to the kidney, aldosterone increases blood pressure through Na+ and water
reabsorption in the colon [94], and also by mediating cardiovascular tissue injury through
inflammation and tissue remodeling [95, 96]. Finally, volume expansion from increased RAAS
further increases blood pressure through local vasoconstriction that occurs independently of
central control [97]. This local reflex is often overlooked, but can be quite powerful as seen in
the development of recurrent intradialytic hypertension in patients [98].

16

Figure 4. Enhanced SNA to the kidney increases blood pressure. Increased renal SNA
enhances renin release from juxtaglomerular cells of renal afferent arterioles, leading to
increased angiotensin II (ANG-II) production. ANG-II causes vasoconstriction, increases Na+ and
water reabsorption from the proximal tubule, and activates circumventricular organs in the
brain, which further increases SNA. ANGII also increases secretion of aldosterone from the
adrenal gland, which causes increased Na+ and water reabsorption in the distal nephron.
Volume expansion potentiates local vasoconstrictor responses that occur independent of
central control. Increases in SNA, total peripheral vascular resistance, and volume expansion all
combine to increase blood pressure.

17

Pressure-natriuresis: Renal Na+ and water reabsorption is regulated by renal perfusion
pressure. This pressure-natriuresis relationship prevents sustained fluctuations in blood
pressure. This occurs because increases in blood pressure elevate renal perfusion pressure,
which causes the kidneys to excrete more Na+ and water [99]. Dehydration returns blood
pressure back to baseline [78]. In early humans, this mechanism likely evolved for the opposite
scenario, where decreases in blood pressure from severe dehydration or blood loss enabled
survival by facilitating Na+ and water retention [99, 100]. This mechanism is mediated in large
by RAAS activity. JG cells secrete renin in response to a drop in renal perfusion pressure, which
is sensed by a decrease in vascular stretch by mechanoreceptors [99].
Blood osmolarity affects the pressure-natriuresis curve and Na+ intake is met with
compensatory Na+ clearance. JG cells regulate renin secretion also in response to feedback
received by neighboring macula densa (MD) cells of the distal convoluted tubule (DCT), which
contain chemoreceptors that sense tubular NaCl concentration. JC cells secrete renin when MD
cells sense low NaCl in the DCT [101]. Therefore, high salt intake (which increases DCT NaCl)
results in a left-shift in the pressure-natriuresis curve while low salt intake (which decreases
DCT NaCl) results in a right-shift in the pressure-natriuresis curve. Salt intake also affects the
slope of the pressure-natriuresis curve so that Na+ regulation can occur over a wider span of
blood pressures (Figure 8A).
Factors that determine the homeostatic set-point value of blood pressure are not
completely understood. However, it is conceivable that for hypertension to occur and be
maintained, this homeostatic set-point must be reset to operate at higher blood pressures. This
results in a right-shift in pressure-natriuresis [102] (Figure 8B). Interestingly, obesity also results

18

in a right-shift in pressure-natriuresis (Figure 8C) and it is suggested that this occurs due to
increased Na+ and water reabsorption as a result of activation of the sympathetic nervous
system and RAAS [103]. Salt-sensitive hypertension not only causes a right-shift in pressurenatriuresis, but also a decreases the slope [102] (Figure 8D). The steepness of the pressurenatriuresis curve reflects salt-sensitivity of blood pressure.

Figure 5. Pressure-natriuresis relationships. A: Salt affects slope. To maintain blood pressure
homeostasis, high dietary salt results in increased urinary Na+ and water excretion (RAAS
activity) and low dietary salt results in decreased Na+ and water excretion (RAAS activity). B: In
normotension, increases in blood pressure are accompanied by increases in urinary Na+ and
water excretion to maintain homeostasis. In hypertension, homeostatic set points are reset,
shifting the pressure-natriuresis curve to the right so that natriuretic responses now operate at
higher blood pressures. C: Obesity also causes a right-shift in pressure-natriuresis (SNA and
RAAS activity). D: Salt-sensitive hypertension causes a right-shift and flattening of slope.

19

2.4

Peripheral SNA at the Neuroeffector Junction

Mechanisms that contribute to enhanced sympathetic outflow also occur at sites of
neurotransmitter release, either via synapses between pre and post-ganglionic neurons or at
neuroeffector junctions between post-ganglionic neurons and non-neuronal target tissues.
During their course to the splanchnic circulation, pre-ganglionic sympathetic nerve fibers
originating in the IML column project to prevertebral ganglia like the SMCG and release
acetylcholine (Ach) onto nicotinic Ach receptors on post-ganglionic sympathetic neurons.
Postganglionic sympathetic neurons project to targets like mesenteric resistance arteries. Since
the sympathetic effector response is directly determined by neurotransmitter release and
action on target tissue, the neuroeffector junction represents that last regulatory level of SNA.
At the neuroeffector junction, neurotransmitters are released in a similar manner to
paracrine secretion, where target effector cells are located at greater distances (up to
thousands of nanometers) from nerve terminal release sites [104]. Neurotransmitters are
stored in vesicles contained in varicosities, which are swellings along sympathetic nerve
terminals that give the appearance of beads on a string [105]. Norepinephrine (NE) is the main
neurotransmitter released from post-ganglionic sympathetic neurons [51] and is co-released
with other sympathetic effector molecules like adenosine triphosphate (ATP) and neuropeptide
Y (NPY) [106]. The sympathetic effector responses of NE and ATP are similar, resulting in
vasoconstriction through 1/2 adrenergic receptors and P2X receptors, respectively. NPY
mediates vasoconstriction through NPY Y1 receptors, however, may serve a larger role in
regulating the extent of NE and ATP release [106-108].

20

2.5

NE Synthesis, Release, Reuptake and Metabolism

NE synthesis, release, reuptake, and metabolism all take place in postganglionic
sympathetic neurons. A simplified summary of these events are depicted in Figure 4.
NE synthesis: Synthesis of NE occurs first by the enzyme tyrosine hydroxylase, which
catalyzes the conversion of tyrosine to dihydroxyphenylalanine (DOPA) in the cytoplasm. The
enzyme DOPA decarboxylase then converts DOPA to dopamine, which is packaged into vesicles.
In vesicles, the enzyme dopamine -monooxygenase converts dopamine into NE [109].
Vesicular docking and release: These vesicles are then docked to the target membrane
in active zones, which are sophisticated compartments that contain all the constituents needed
for rapid exocytosis in response to electrical signals [110]. In brief, vesicular docking and fusion
to the presynaptic membrane is achieved through organized interaction between soluble Nethylmaleimide sensitive factor attachment protein receptors (SNAREs) found in vesicular
membranes (v-SNAREs) or target presynaptic membranes (t-SNAREs). The v-SNARE proteins
include synaptotagmin and synaptobrevin, and the t-SNARE proteins include syntaxin and 25
kDa synaptosome-associated protein (SNAP-25). Synaptotagmin is a Ca2+ binding/sensing and
membrane trafficking protein that first guides secretory vesicles to active zones at the
presynaptic membrane [111], in part, by interacting with SNAP-25 [112]. Once correctly
positioned, synaptobrevin binds syntaxin and SNAP-25, forming a “core SNARE complex” that
solidifies vesicular-to-presynaptic membrane interaction [113].
Exocytosis of neurotransmitters is achieved by the influx of Ca2+ ions during
depolarization due to the opening of voltage-gated N-type Ca2+ channels, which allows
intracellular concentrations to reach 1-300 M [110, 114, 115]. Exocytosis occurs because in its

21

Ca2+-bound state, synaptotagmin binds phosphatidylinositol 4,5-bisphosphate (PIP2) lipids in
the presynaptic membrane, leading to rapid membrane fusion, membrane penetration, and
neurotransmitter release [116].
NE reuptake and metabolism: When NE is released into the neuroeffector junction, its
action is limited in part by reuptake into the cytoplasm through the prejunctional NEtransporter (NET) and through degradation via catechol-O-methyltransferase [117, 118]. Once
returned to the cytoplasm, NE is either recycled back into vesicles or is metabolized by
monoamine oxidase (MOA) [118].
Enhanced neuroeffector transmission: An increase in neuroeffector transmission could
be achieved by mechanisms that increase NE synthesis and release or by mechanisms that
decrease NE reuptake and degradation. For instance, some studies suggest that impaired
neuronal uptake of NE contributes to enhanced sympathetic nerve activity in human essential
hypertension [119, 120].

22

Figure 6. NE synthesis, release, reuptake and metabolism in sympathetic neurons. A: Tyrosine
is converted into DOPA by tyrosine hydroxylase, which is converted into dopamine (DA) by
DOPA decarboxylase. DA is taken up into vesicles and converted into norepinephrine (NE) by
dopamine -monooxygenase. Synaptotagmin assists in transporting NE-containing vesicles to
active zones near the presynaptic membrane. Binding between synaptotagmin and SNAP-25
and synaptobrevin with SNAP-25 and syntaxin forms the “core SNARE complex”, solidifying
vesicular and presynaptic membrane interaction. B: Depolarization opens voltage-gated N-type
Ca2+ channels, resulting in Ca2+ influx. Ca2+ binds synaptotagmin and promotes its interaction
with phosphatidylinositol 4,5-bisphosphate (PIP2) in presynaptic membranes. This results in
rapid membrane penetration, fusion, and exocytosis. C: NE is exocytosed onto effector targets
like vascular smooth muscle. NE action is limited by reuptake back into the cytoplasm, which
occurs through the NE transporter (NET) and through degradation via catechol-Omethyltransferase (COMT). Once re-uptake occurs, NE is either repackaged into vesicles or
degraded by monoamine oxidase (MOA).

23

2.6

Negative Feedback Regulation Through the 2-Adrenergic Receptor

The -adrenergic receptor (2AR) regulates neurotransmitter release from nerve
terminals in the central and peripheral sympathetic nervous system [121, 122]. In

peripheral

tissues such as resistance arteries, sympathetic neuroeffector transmission results in blood
pressure elevation through the activation of postsynaptic 1-adrenregic receptors (1ARs) (and
to a lesser extent, 2ARs) on vascular smooth muscle cells. This potentiates intracellular Ca2+
release and activation of protein kinase C, resulting in vascular smooth muscle contraction
[123].
The extent of neuroeffector transmission is regulated by mechanisms of negative
feedback that include signaling through the prejunctional 2-adrenergic receptors (2AR), as
depicted in Figure 5A. The 2AR is an autoreceptor that couples via G proteins of the Gi/o
family to facilitate Gdimer-dependent inhibition of voltage-gated N-type calcium channels
[115, 124, 125], as depicted in Figure 5B. This mechanism prevents sustained increases in blood
pressure [126-129]. Importantly, prejunctional 2AR signaling provides negative feedback not
only to NE release, but also functionally similar neurotransmitters like ATP [130]. Furthermore,
mechanisms of 2AR signaling and negative feedback on neurotransmitter release are
conserved in adrenergic neurons of the CNS [131-133]. However, prejunctional 2AR signaling
at the neuroeffector junction represents the last line of defense to counteract enhanced
sympathetic output originating from the CNS and is the focus of the majority of our work.

24

Figure 7. Mechanism of negative feedback through the prejunctional 2-adrenergic receptor
(2AR). A: Activation of the 2AR by norepinephrine (NE) provides feedback inhibition to nerve
terminal NE release. By decreasing NE bioavailability in the neuroeffector junction, vascular
smooth muscle cells relax and blood pressure returns back to baseline. B: In its inactive state,
the 2AR associates with GDP-bound G-proteins at the membrane with sequestered
Gsubunits. In its inactive state, voltage-dependent Ca2+ influx occurs undisturbed, facilitating
NE release (top). When activated, G exchanges GDP for GTP, which releases G subunits that
directly inhibit voltage-gated N-type Ca2+ channels. This attenuates NE release (bottom).

25

2.7

Physiological Relevance of 2AR Subtypes

To date, three homologous subtypes of 2ARs have been identified and cloned; subtype
2A, 2B, and 2C. Disappointingly, pharmacological agents and ligands have not been selective
enough to fully investigate subtype-specific properties and function [134]. Fortunately, the use
of 2AR subtype-specific knockout mice has improved our understanding regarding the
physiological differences in how 2AR subtypes affect blood pressure regulation.
Knock out studies suggest that 2AR subtypes do not participate equally in hypertension
development. Evidence for this is supported in the subtotal nephrectomized mouse model of
salt-induced hypertension. Wildtype mice develop hypertension in response to chronic dietary
salt-loading similar to 2A and 2C knockout mice. However, 2A knockout mice start out with
higher baseline blood pressure and plasma catecholamine levels, and when salt-loaded,
develop hypertension at a faster rate (2 versus 4-5 weeks of salt treatment) than wildtype and
2C knockout mice. In contrast, mice heterozygous for 2B are resistant to hypertension
development despite chronic dietary salt loading [135, 136].
Feedback inhibition of NE release is associated mainly with 2A and 2C [133, 137-140].
Studies using 2AR subtype-specific knockout mice suggest that inhibition of NE release from
peripheral sympathetic nerves occurs mostly through 2A as significant increases in plasma and
urine NE levels occurred only in 2A knockout mice [139]. Transgenic rescue of 2A under in
2A/2C double knockout mice restores plasma NE levels comparable to wildtype mice [141]. In
contrast, 2C knockout mice have normal plasma and urine NE levels but have significantly
elevated plasma and urine epinephrine (EP) levels. Experiments performed in isolated
chromaffin cells suggest that only 2C is required for regulating EP release in the adrenal

26

medulla [139]. Tissue-specific contributions are also evident in the pancreas, where the
inhibition of insulin secretion in pancreatic islet cells is mediated mostly by 2A [142]. The
individual contributions of 2A and 2C autoregulation may also be dependent on physiological
conditions. Negative feedback through 2A is faster than 2C and is most influential at higher
frequencies of nerve stimulation [137], suggesting that tonic SNA may be regulated by 2C while
increased SNA may be regulated by 2A. While expression of all three subtypes can be detected
in postganglionic sympathetic neurons [133], in the neuroeffector junction, it suggested that
2B plays a larger role in vascular smooth muscle, mediating vasoconstriction like the 1AR
[140, 143].

27

2.8

Mechanisms of Prejunctional 2AR Regulation

Receptor internalization: Activity of the 2AR is regulated by ligand-occupied
desensitization, which results from receptor endocytosis [141, 144]. Gene knock-in strategies
using epitope-tagged receptors, such as hemagglutinin (HA)-tagged 2A, have made it easier to
monitor receptor trafficking and evaluate functional consequences of internalization. The
characteristics of 2A internalization and degree of receptor desensitization appear to be ligandspecific [144]. Clonidine and guanfacine are two 2AR agonists that have been used clinically to
treat hypertension [145]. Clonidine-occupied 2A internalizes faster and to a larger extent than
guanfacine-occupied 2A. This may be due to phosphorylation, which occurs to a higher degree
when the 2AR is activated by clonidine. Furthermore, unlike guanfacine-occupied 2A,
clonidine-occupied 2A internalizes as large puncta that localize predominantly in the
perinuclear cytoplasm. Taken together, these data may explain why clonidine potentiates a
larger and faster desensitization of 2AR-mediated inhibition of Ca2+ current in cultured
superior cervical ganglion (SCG) neurons than guanficine [144].
G protein-coupled receptor kinases: Receptor desensitization and internalization of
GPCRs in the active state is mediated through phosphorylation by members of the G proteincoupled receptor kinase (GRK) family. Phosphorylation of GPCRs results in G protein uncoupling
and arrestin-mediated receptor endocytosis [146-148]. Once internalized, receptors are either
recycled back to the plasma membrane or downregulated through lysosomal proteolytic
degradation [149]. These processes are depicted in Figure 6 using the 2AR as an example.
For the 2AR, GRK phosphorylation [150], arrestin binding [151], and G protein
activation [152] all occur within the third intracellular loop. GRK-induced desensitization may

28

also involve subtype-specific characteristics. GRK 2 and GRK 3, but not GRK 5 have been shown
to phosphorylate 2A and 2B. Agonist-occupied 2C does not undergo phosphorylation [153156]. Furthermore, 2AR subtypes have selective binding to different arrestin family members.
Internalization of 2B involves arrestin-2 and arrestin-3 while internalization of 2C involves
arrestin-3. By contrast, internalization of 2A occurs independently of these arrestins [157].
Relevance of GRK to hypertension: Most importantly for blood pressure control,
receptor internalization is an effective way of desensitizing the 2AR in postganglionic
sympathetic neurons. Concanavalin A treatment, which prevents receptor internalization,
reduces clonidine-induced 2AR desensitization of Ca2+ current inhibition to near completion in
SCG neurons [144]. Furthermore, upregulation of GRK 2 in the adrenal medulla dramatically
reduces negative feedback on catecholamine release through the 2AR [158]. Conditions that
alter GRK 2 expression may also affect 2AR signaling and blood pressure regulation. Noncanonical roles of GRK 2 involve the production of reactive oxygen species [159, 160] and GRK 2
is upregulated in inflammatory conditions that are associated with increased blood pressure
[161, 162]. Whether GRK 2 upregulation increases blood pressure via oxidative stress and/or by
increasing adrenergic drive through a reduction in 2AR signaling is not known.

29

Figure 8. GRK-dependent desensitization and internalization of ligand-activated 2AR. When
activated by NE, 2AR dissociates from G proteins (1-2). This vacates the third intracellular loop
of 2AR enabling GRK2 phosphorylation (3). Arrestin binds the phosphorylated form of 2AR,
which prevents G protein-receptor re-association and results in receptor endocytosis (4). Once
internalized, 2AR undergoes lysosomal proteolytic degradation (down-regulation) or is
recycled back to the plasma membrane.

30

Receptor modification: Covalent protein modifications can also alter protein function.
Cysteine residues in particular, are important for proper 2AR signaling. G protein coupling to
the 2AR involves a conserved cysteine in the C-terminal region of Gi/o proteins. Treatment
with pertussis toxin (PTX) results in ADP-ribosylation of this cysteine, which impairs 2AR
signaling by uncoupling the receptor from its G protein [163]. Furthermore, treatment with
phenylmercuric chloride, which reacts with sulfhydryl groups on cysteine residues, significantly
decreases 2AR-ligand affinity [164]. Finally, the C-terminal cysteine of the 2AR is associated
with mechanisms of downregulation [165]. These results suggest that modifications to cysteine
residues on the G protein and receptor may affect 2AR signaling.

31

3.0

Increased SNA in Human and Experimental Hypertension
3.1

Increased SNA Human Hypertension

It is now well accepted that sympathetic hyperactivity contributes to hypertension
development, maintenance, and cardiovascular-related complications in humans [67, 68]. In
fact, sympathetic hyperactivity is also associated with prehypertension as well as normotension
with a positive family history of hypertension, suggesting a possible genetic component [166168]. Evidence of enhanced SNA in human hypertension studies has been mainly demonstrated
using techniques that include NE spillover and microneurography [34].
NE spillover: Due its relative ease and broad applicability, adrenergic drive has been
evaluated indirectly through measurement of plasma NE. The use of radiolabeled NE has
enabled the net quantification of enhanced NE spillover from nerve terminal release after
taking into account NE clearance from the blood [169, 170] and has validated numerous
observations that plasma NE increases in hypertensive versus normotensive subjects is due to
enhanced adrenergic drive [171]. Furthermore, the use of catheter placement has enabled the
detection of NE spillover in specific vascular regions that include the heart and kidney [169,
172]. Enhanced NE nerve terminal release in these organs occur in subjects with even just
borderline blood pressure elevation [173].
Microneurography: Enhanced SNA in human hypertension has also been determined
using microneurography, which involves the intra-neuronal recording of postganglionic
sympathetic nerve traffic to skeletal muscle [34]. Microneurography is a favored technique as it
is both direct and highly reproducible [34, 172], and is correlated with region-specific NE
spillover in the heart [174] and kidney [175]. When corrected for heart rate, muscle

32

sympathetic nerve traffic significantly increases as individuals progress from normotension to
pre-hypertension, from borderline hypertension to severe hypertension, and from severe
hypertension to resistant hypertension [176]. These results may suggest that increased
adrenergic drive precedes different stages of hypertension development.
Other techniques: Finally, it is worth mentioning that enhanced SNA has also been
studied with some success in humans through either heart rate monitoring, or device-assisted
approaches that target SNA. Heart rate, much like plasma NE is easy to measure, however it
may not be as easy to interpret. Heart rate alone cannot truly reflect adrenergic drive because
of parasympathetic (cholinergic) influences [3, 34]. This may explain why increased plasma NE
and muscle sympathetic nerve traffic does not always correlate with increased heart rate [172].
Therefore, caution should be used when interpreting heart rate as an index of adrenergic drive.
The contribution of enhanced SNA in human hypertension has most recently been
demonstrated using renal artery denervation in clinical studies and standard practice to treat
resistant hypertension. Earlier studies using catheter-based radiofrequency denervation of the
renal arteries showed success in sustainably decreasing blood pressure in patients with
resistant hypertension [177, 178]. Furthermore, renal denervation has been shown to decrease
plasma NE and muscle sympathetic nerve activity [179, 180]. However, newer investigations
designed to be more rigorous (SYMPLICITY HTN-3) [181] by including a larger sample sizes, a
blinded approach, and a sham control failed to show a decrease in blood pressure following
renal artery denervation [182]. Until more investigations are carried out, the benefits of renal
denervation remain unclear.

33

Limitations of human studies: Data from multiple human hypertension studies suggest
that increased SNA in hypertension involves increased postganglionic sympathetic nerve firing
and release of NE. Unfortunately, the mechanisms and precipitating factors that result in
enhanced adrenergic drive in human hypertension are not completely understood, largely
because essential hypertension is multifactorial in origin. Furthermore, there are only a handful
of techniques that are ethical for use in human subjects. Therefore, research investigations
have relied on animal models to study precipitating factors and underlying mechanisms that
contribute to enhanced SNA and hypertension in humans.

34

3.2

Increased SNA in Experimental Hypertension

A series of different experimental hypertension animal models can be useful in
understanding specific etiological factors that contribute to human hypertension development
and maintenance. Genetics, salt, mineralocorticoid, and obesity are all risk factors of
hypertension that involve increased sympathetic nerve activity in human hypertension.

3.2.1 Enhanced SNA in Genetic Hypertension
Many genetic rat models of hypertension were derived from variations of inbred strains.
These include the spontaneously hypertensive (SH) and dahl salt-sensitive (DahlS) rat.
The SH rat model: The SH rat is a derivative of Wistar Kyoto (WK) rats that develops
hypertension at 4-6 weeks of age even in the absence of experimental intervention [183] and
are prone to stroke-related death [184]. In the SHR model, hypertension development and
associated sequelae are more severe when put on diets consisting of high salt or fat [185, 186],
which is similar in human hypertension [187]. The SH rat has been useful in determining if
enhanced SNA participates in the initiation of essential hypertension. In young SH rats,
microdiaylsis probe measurements detect enhanced NE release in vascular beds of skeletal
muscle and subcutaneous fat [188], field stimulation of atrial preparations show enhanced
cardiac NE release [189], and sympathectomy protects against hypertension development
[190]. Increased NE release in young SH rats may be the result of prejunctional 2AR
dysfunction. In mesenteric arteries of young SH rats, electrical stimulation-evoked NE release is
enhanced versus WK rats. Furthermore, when mesenteric arteries are treated with the 2AR
antagonist yohimbine, NE release is sustained in young SH rats but increased in WK rats [191].

35

Furthermore, ganglionic blockade with high doses of hexamethonium (nicotinic Ach
receptor antagonist) results in a greater reduction in arterial pressure and renal SNA recordings
in SH versus WK rats [192].
Finally, enhanced SNA has also been suggested to occur centrally via inflammation in
the NTS, where expression of the leukocyte adhesion molecule JAM-1 is higher in SH rats versus
control WK rats [193].
The DahlS rat model: The DahlS rat is a derivative strain of Sprague Dawley (SD) rats
that has genetic predisposition to salt-sensitive hypertension associated with low renin
secretion. This is in contrast to Dahl-resistant (DahlR) rats, which do not develop salt-sensitive
hypertension or impaired renin secretion [194]. In the DahlS model, hypertension is achieved
even on high-normal (1%) and low (0.3-0.4%) salt diets [195], but occurs faster on diets that
higher in salt [196] and fat [197].
Early studies showed that hypertension development in DahlS rats involved impaired
barorereceptor reflex control. In DahlS rats, phenylephrine-induced increases in blood pressure
does not increase multifiber afferent aortic baroreceptor discharge [198] and dextran-induced
volume overload does not reduce splanchnic SNA [199] as effectively as DahlR rats.
Newer studies suggest that central sympathetic activation results from increased levels
of Na+ in cerebrospinal fluid (CSF). CSF Na+ levels are increased in high salt fed-DahlS rats [200],
which activates central sympathetic efferent regulatory regions. In this model, CSF Na+ is
contributed by dietary salt and defective transport of Na+ in CSF [201]. Central SNA may also be
enhanced by oxidative stress production, which occurs from salt loading and is increased in the
hypothalamus of high salt-fed DahlS rats [202]. Infusion of the antioxidant tempol into

36

intracerebral ventricles attenuates hypertension and renal SNA, as assessed by abdominal aorta
catheterization and renal nerve multifiber discharge recordings, respectively [202].
Hypertension in the DahlS rat may also involve enhanced nerve terminal release of NE
release. This was observed in early studies of salt-induced vasoconstriction, which was found
to be enhanced in high salt-DahlS versus DahlR rats. Interestingly, DahlR rats produced
vasodilation in response to salt loading [203]. Experiments measuring vascular resistance from
lumbar sympathetic nerve stimulation in perfused hindquarters show enhanced vasoconstrictor
response in DahlS rats that do not involve increased vascular reactivity to NE. This suggests that
hypertension in DahlS rats may involve impaired negative feedback to nerve terminal release of
NE [204]. It should be noted however that salt loading in DahlS rats does not increase
neurogenic mechanisms of enhanced renal SNA [205, 206] and therefore, enhanced
neuroeffector transmission in the DahlS may be organ-specific. Whether mechanisms that
increase nerve terminal release of NE involve impaired 2AR signaling is unknown.

3.2.2 Enhanced SNA in Salt and Mineralocorticoid Hypertension
In salt-sensitive hypertension, salt and water is retained, resulting in a rightward shift in
pressure-natriuresis that is contributed in part by enhanced SNA [207]. Salt-sensitivity can be
influenced by genetic factors, as seen in the DahlS rat, but also through acquired deficits that
interfere with excretory mechanisms of the kidney. Examples include sympathetic
hyperactivity, salt-loading, and mineralocorticoid excess [79].

37

Salt: High dietary salt is a risk factor for hypertension and blood pressure is proportional
to body Na+. Exactly how salt leads to the development of salt-sensitive hypertension is still
unclear but may involve mechanisms in the CNS and renal system. Hypernatremia from
increased salt consumption can activate osmosensory neurons in the OVLT and SFO, leading to
downstream activation of PVN neurons and subsequent activation of the RVLM neurons and
sympathetic efferents [72-74]. To some extent, this transient increase in blood pressure is
buffered by the kidney through pressure-natriuresis via decreased RAAS activity. However, the
homeostatic set-point of pressure-natriuresis can be reset, perhaps due to prolonged blood
pressure elevation from increased adrenergic drive. This may also occur through acquired or
pre-existing impairments in sodium excretion, leading to higher than normal Na+ and water
retention [208]. Volume expansion is also met with a regional vasoconstriction response that is
independent of central control, which further increases blood pressure [97]. For these reasons,
salt-loading has been commonly used to study increased SNA in salt-sensitive hypertension.
Aldosterone: As previously discussed, aldosterone contributes to increased blood
pressure mostly in the periphery by increasing Na+ and water reabsorption in the distal nephron
[82] and colon [94], and through cardiovascular tissue injury from inflammation, oxidative
stress production and tissue remodeling [95, 96, 209]. Activation of aldosterone-sensitive
neurons in the brain increases blood pressure, however, this occurs only with direct
aldosterone infusion into the cerebral ventricles [210, 211] and cannot be reproduced with
systemic infusion. This may be because aldosterone, despite its lipophilicity, does not easily
cross the blood brain barrier like other steroid hormones (corticosterone, progesterone,

38

estradiol, and testosterone) [212]. An indirect mechanism by which aldosterone may increase
SNA is through mechanisms that increase salt appetite [213, 214].
The effects of salt and mineralocorticoid signaling: Aldosterone and salt work
synergistically in the development of salt-sensitive hypertension, possibly via oxidative stress
production and damage to the cardiovascular and renovascular system. This may occur because
salt enhances MR signaling. This is evident in the salt-loaded DahlS rat where treatment with
the MR blocker eplerenone improves proteinuria and cardiac function [215]. However, this is
counterintuitive given that the DahlS rat is a model of low aldosterone. This can be explained by
salt-induced activation of MR signaling independent of aldosterone, which in salt-loaded DahlS
rats, occurs through Rac1, a member of the Rho family of GTPases [216] that has been
implicated in oxidative stress production through the activation of NADPH oxidase. NADPH
oxidases contribute oxidative stress by catalyzing the conversion of dioxygen to superoxide
[217]. Therefore, in hypertension, oxidative stress at least in in part, may occur through
mineralocorticoid signaling. Mechanisms of Rac1 activation in hypertension remain unclear, but
may be due to mechanical stretch in vascular endothelial cells, which increases in hypertension
[216, 218]. Rac1-induced activation of MR is also increased by aldosterone [219], which may
make hypertension more severe in models where salt loading is accompanied by
mineralocorticoid-excess. These findings are important and suggest that salt can enhance MR
signaling in hypertension, possibly via oxidative stress and is relevant even in models where
RAAS activity is low.
The DOCA-salt rat model: The study of salt and mineralocorticoid status in hypertension
has been mostly investigated using the deoxycorticosterone (DOCA, a mineralocorticoid

39

derivative of aldosterone)-salt hypertensive rat model. Here, DOCA administration (20-150
mg/kg) and 0.6-1% NaCl drinking water in uninephrectomized rats leads to the development of
low-renin hypertension [220]. Low-renin hypertension is commonly found in patients with saltsensitive hypertension, which account for about half of the cases of essential hypertension [40,
41, 221, 222]. Enhanced SNA has been studied extensively in DOCA-salt hypertension and
involves impairment of the baroreceptor reflex, enhanced brain renin-angiotensin system
(RAS), systemic oxidative stress production, enhanced lumbar, splanchnic and renal SNA, and
dysfunction of the prejunctional 2AR. Interestingly, many of these factors are salt-dependent,
suggesting that mineralocorticoid and salt act synergistically to enhance SNA.
Enhanced SNA in DOCA-salt hypertension begins with functional impairment of the
baroreceptor reflex, which can be detected even before hypertension develops. Activation of
the baroreceptor reflex using aortic depressor nerve stimulation generates reduced
sympathoinhibitory responses as measured via splanchnic nerve recordings in DOCA-salt rats
versus uninephrectomized (SHAM, control) rats after only 5 days of treatment. These deficits
are central as aortic depressor nerve stimulation initiates the baroreceptor reflex independent
of arterial baroreceptors [223].
Central mechanisms of enhanced SNA in DOCA-salt hypertension may result from
increased activation of RAAS and oxidative stress production in the brain. Although DOCA-salt
hypertension is characterized by low plasma renin and ANG II, increased levels of ANG II have
been detected in CSF [224]. In DOCA-salt hypertension, ANG II activation of AT1Rs is enhanced
in the SFO [225] and possibly other CVOs [226]. ANG II infusion has been shown to activate
neurons within the PVN, SFO, and NTS [85]. Furthermore, oxidative stress production may also

40

enhance central SNA. In DOCA-salt rats, superoxide levels in the hypothalamus are increased
and intracerebroventricular delivery of tempol (an antioxidant) decreases blood pressure and
renal SNA to a greater extent in DOCA-salt versus SHAM rats [227]. These effects do not occur
in DOCA-treated rats with access to normal water [227]. Plasma NaCl, which is mildly elevated
in DOCA-salt rats, may be sufficient to enhance sympathetic outflow. Nerve recordings show
significantly enhanced lumbar SNA in DOCA-salt versus SHAM rats. This effect does not occur in
DOCA-treated rats with access to normal drinking water [228].
Enhanced SNA has also been suggested by experiments using ganglionic blockade, celiac
renal denervation and celiac ganglionectomy. Ganglionic blockade by hexamethonium
decreases blood pressure to a greater extent in DOCA-salt versus SHAM rats [63, 227]. These
effects are not observed in DOCA-treated rats with access to normal water [227]. Renal
denervation can be achieved by renal vessel dissection followed by application of 10-20%
phenol in alcohol solution. The effects of renal denervation on the development of DOCA-salt
hypertension have been mixed. Renal nerve recordings performed in DOCA-salt rats show that
renal SNA is increased versus SHAM rats [229], and that renal denervation significantly reduces,
although not to completion, DOCA-salt induced elevation of blood pressure [229-231]. These
effects may be salt-dependent, as they do not occur in DOCA-treated rats with access to normal
drinking water [227]. Furthermore, enhanced renal SNA may involve inflammation and
oxidative stress. Increased levels of inflammatory mediators that include chemokine ligand,
monocyte chemoattractant protein-1 (MCP-1) and interleukin-6 (IL-6) in the kidney occur in
DOCA-salt hypertension and are significantly reduced by renal denervation [229]. Furthermore,
intracebroventricular administration of tempol lowers renal SNA to a greater extent in DOCA-

41

salt versus SHAM rats and DOCA-treated rats with access to normal water [227]. On the other
hand, in another study, renal denervation was not shown to reduce or delay DOCA-salt
hypertension development at any time point, suggesting that intact sympathetic innervation to
the kidney is not important in DOCA-salt hypertension [232]. Furthermore, a study performed
in mild DOCA-salt hypertension (50 mg/kg, 1% salt water, no nephrectomy) also showed that
renal denervation had no effect on DOCA-salt induced elevation of blood pressure versus nondenervated DOCA-salt rats [233]. Interestingly, using the same model, splanchnic denervation
through celiac ganglionectomy reduced the extent (although not fully) of DOCA-salt induced
elevation of blood pressure versus non-denervated DOCA-salt treated rats [59]. These results
suggest that at least in mild DOCA-salt hypertension, increased SNA may be regionally-specific
to the splanchnic circulation.
Finally, DOCA-salt hypertension is associated with functional impairment of
prejunctional 2AR signaling. In DOCA-salt rats, basal levels of plasma NE are increased [129,
227] and unchanged after administration of the 2AR-selective antagonist yohimbine [129].
Similarly, nerve stimulation-evoked NE release detected from mesenteric arteries using
amperometry show that NE release is enhanced in mesenteric arteries from DOCA-salt rats.
Furthermore, inhibition of NE release is blunted when artery preparations are treated with
yohimbine [234].

42

3.2.3 Enhanced SNA in Obesity-Related Hypertension
Obesity is an independent risk factor of hypertension and its increasing prevalence is
changing the overall cause of human hypertension. It is now estimated that more than half of
human hypertension cases are directly linked to obesity [42, 43]. Obese subjects have increased
levels of plasma NE [235] and increased muscle SNA [236] versus non-obese subjects. This
suggests that obesity, much like salt, contributes to hypertension by increasing SNA. The
development of obesity-related hypertension is multifactorial with some mechanisms are likely
conserved with other forms of hypertension, such as salt-sensitive hypertension. However,
great effort must be made to properly identify these similarities as well as potential differences
that may be used to develop more effective clinical treatment strategies. Experiments
performed in animal models suggest that obesity-related hypertension involves an increase in
SNA, RAAS activation, inflammation, and oxidative stress production [237].
The HFD-SD model: Obesity-related hypertension can be studied through high dietary
fat consumption in animals such as Sprague-Dawley (SD) rats. SD rats are not predisposed to
hypertension development and like humans, show individual variations.

In SD rats, a

moderately HFD (32% kcal fat, a typical Western diet) induces obesity that is associated with
mild hypertension (termed obesity-prone, OP) after 10 weeks of feeding. Remaining animals
exhibit normal body weight and blood pressure (termed obesity-resistant, OR) similar to SD rats
fed a normal fat diet (NFD, 11% kcal fat) [238]. This is characteristic of human obesity, which is
also bimodal in distribution [239]. Obesity-related hypertension in HFD-OP rats is associated
with an increase in vascular hypertrophy, plasma renin activity, circulating cholesterol, and lipid
peroxides versus HFD-OR rats and NFD-rats [238]. Lipid peroxides result from the oxidative

43

degradation of lipids and it is suggested that this might occur via increased lipolysis, secondary
to sympathetic activation [238, 240]. When carried out to 16 weeks of feeding, blood pressure,
plasma renin activity and lipid peroxide levels are still markedly elevated in HFD-OP rats versus
HFD-OR rats and NFD-SD rats. Furthermore, in HFD-OP rats, superoxide levels are significantly
increased in aortic rings versus HFD-OR rats and NFD-SD rats [240]. Given increased plasma
renin activity, it is suggested that SNA and ANG-II production through RAAS could be enhanced.
This is important as enhanced SNA increases renin release from JG cells and members of the
RAAS like ANG-II and aldosterone can enhance oxidative stress production [95, 96, 240, 241].
In a recent study, hypertension development in this model was suggested to occur
through inflammation-induced impaired baroreceptor regulation of renal SNA, as measured
from renal nerve recordings in response to changes in blood volume. 8 weeks of HFD (45% kcal
fat) resulted in obese and hypertensive rats with higher levels of TNF and IL-6 in the plasma
and kidney, and lower levels of urinary output and Na+ clearance. Furthermore, renal SNA
responses to the baroreceptor reflex initiated by phenylephrine (high pressure) and sodium
nitroprusside (low pressure) bolus infusion were attenuated in HFD-rats versus NFD-rats.
Interestingly, treatment of HFD-rats with the tacrolimus (an immunosuppressant) restored
renal SNA responses to the baroreceptor reflex. Tacrolimus also preserved blood pressure,
reduced inflammation, and improved urinary output and Na+ excretion [242]. Taken together,
these findings suggest that HFD and obesity may contribute to enhanced SNA (and possibly
RAAS activity) through chronic inflammation.
Approaches to study obesity and salt: Dietary salt and obesity are risk factors for
hypertension development [81, 243], and dietary salt and hypertension are also risk factors for

44

the development of obesity [244, 245]. Reductions in dietary salt improves blood pressure to a
greater extent in obese versus non-obese adolescents [246] suggesting the need to investigate
this relationship early in development. It has been suggested that mechanisms of salt-sensitivity
and obesity work synergistically to increase blood pressure by increasing SNA [81, 243], possibly
through insulin signaling [247], inflammation, and oxidative stress [81].
Strategies to investigate the effects and relationships of obesity and salt-sensitivity on
hypertension development can involve interventions that combine a HFD with high salt.
Interestingly, the combination of a high fat (32% kcal fat) and high salt (2-4%) diet in OP rats
accelerates the onset of hypertension, increases superoxide production in the aorta, increases
adipocyte size, and increases leptin production versus HFD containing normal salt (0.8%)-OP
rats [248]. However, the magnitude of hypertension was not affected by salt and may be
explained by a lack of salt-sensitivity in all SD rats. Therefore, a more suitable approach would
be to use a HFD in a salt-sensitive hypertension model, like the DahlS rat.
HFD-DahlS model: In the DahlS model, hypertension is achieved even on normal (1%)
and low (0.3-0.4%) salt diets, but occurs faster when given a diet that is high fat [197]. DahlS
rats on a low salt-HFD (0.3% salt, 45% kcal fat) for 8 weeks develop hypertension (that can be
detected as early as 2 weeks) associated with significantly increased weight and visceral
adiposity versus NFD (0.3% salt, 10% kcal fat) rats. These rats also develop hyperinsulinemia,
hyperglycemia, and higher urinary NE. Furthermore, replacing the low salt-HFD diet with a high
salt-HFD (8% salt, 45% kcal fat) for an additional 8 weeks worsens the degree of hypertension
and causes significant proteinuria [197], suggesting that salt aggravates mechanisms of
hypertension development in this model. This seems plausible as after 5 months of low salt

45

HFD-feeding, DahlS rats develop hypertension with proteinuria [249]. It has been suggested
that increased insulin in this model may be a reflection of enhanced SNA and that salt loading,
which causes insulin resistance even in NFD-DahlS rats, plays an important role [197, 247, 250].
Inflammation plays a role in this model. Four weeks on a normal salt-HFD (0.8% NaCl,
60% kcal fat) results in hypertension associated with significant weight gain, increased visceral
adiposity (without changes in leptin or cholesterol), and increased CD3+T-lymphocytes
accumulation in glomerular and medullary tissues versus low salt-NFD (0.4% NaCl, 14% kcal fat).
Treatment with the immunosuppressant mycophenolate mofetil attenuated hypertension
development and weight gain, reduced visceral adiposity, and prevented CD3+ T-lymphocyte
infiltration in the glomerulus [251].
Closely related to inflammation, oxidative stress also plays a role in this model. In a 20
week study, a low salt-HFD (0.3% NaCl, 35% kcal fat) not only induced hypertension with weight
gain in DahlS rats, but also increased aortic levels of superoxide versus low salt-NFD (0.3% NaCl,
5% kcal fat). Interestingly, although the DahlS rat is a model of low-renin/ANG-II, blockage of
the AT1 receptor with daily olmesartan attenuated HFD-induced hypertension and vascular
superoxide production, suggesting that ANG-II contributes to hypertension development in this
model through mechanisms that may involve oxidative stress [252].

46

4.0

Inflammation and Oxidative Stress in Hypertension
In all the animal models previously discussed, mechanisms of inflammation and

oxidative stress production appear to be common features that contribute to hypertension
development. Inflammation is associated with human hypertension. The inflammatory marker
CRP predictably increases as normotensive subjects develop prehypertension and are highest in
subjects with overt hypertension [35-38], which suggests that mechanisms of inflammation
may come into play before hypertension development [253].

4.1

Vascular Injury in Hypertension

Vascular protection against pressure overload: In addition to the mechanisms
previously discussed, vascular tissue injury can occur from pressure-overload. In arteries, layers
of vascular smooth muscle and adventitia provide mechanical support to the vascular
endothelial layer, which is in direct contact with friction caused by blood flow. Small changes in
blood flow occur all the time, from the pulsatile force of a beating heart to physiological
responses to for instance, exercise. Frictional force usually poses no threat to the endothelium,
which counteracts wall shear stress by releasing the potent vasodilator nitric oxide (NO) [254].
NO is made by the enzyme endothelial NO synthase (eNOS). Activation of muscarinic-3
(M3) receptors by Ach on smooth muscle of arteries (unlike the gut and bronchi) causes
dilation. Vasodilation requires eNOS. Mice lacking eNOS develop hypertension from impaired
Ach-induced relaxation [255]. In further support, the use of L-NAME, which inhibits NOS and
impairs NO production, is commonly used to induce experimental hypertension. Interestingly,
hypertension persists even once L-NAME treatment stops [256]. This may suggest that vascular

47

damage sustained from a reduction in NO may be robust enough to maintain high blood
pressure even when NO is restored.
Vascular injury and effects of ROS: Decreased NO bioavailability affects vascular
function and predisposes the endothelium to tissue injury during hypertension, where there is
sustained increase in mechanical loading from increasing blood pressure and flow. This affects
all layers of the blood vessel [257]. Evidence suggests that oxidative stress may play an
important role [257, 258]. Increased circumferential stretch increases oxidative stress
production in vascular smooth muscle and endothelium [259, 260]. ROS like superoxide can
decrease NO bioavailability by combining with NO to form peroxynitrite, which is a very strong
oxidizing agent [257, 261]. Vascular oxidative stress production in hypertension occurs through
NADPH oxidases [261] via Rac1 activation, possibly through mechanical stretch from vascular
endothelial cells during pressure-overload [216, 218]. Normal mechanisms that regulate levels
of ROS such as antioxidants like glutathione and enzymes like superoxide dismutase may be
insufficient in counteracting the increasing levels of oxidative stress [262].

48

4.2

Lymphocytes and Macrophages in Hypertension
Immune cell recruitment: Although commonly described in the pathogenesis of disease,

inflammation actually serves as a protective response to injury. Immune cells infiltrate into
sites of injury, attempt to eliminate the causative agent, and repair tissue damage. This process
involves the release of pro-inflammatory mediators, which in excess, can add to disease
progression and modify the microenvironment by changing cellular and protein function [253].
Immune cells such as lymphocytes and macrophages have been implicated in hypertension
development and maintenance in both humans and experimental animal models.

4.2.1 Lymphocytes
Lymphocytes play a role in adaptive immunity where inflammatory effector responses
involve specific antigen-receptor interactions that mostly occur after the initiation of innate
immunity [263]. T-lymphocytes mature in the thymus to become either CD8+ T-cells (cytotoxic)
or CD4+ T-cells (helper). CD4+ T-cells help other leukocytes mediate immune responses, such as
the activation of macrophages. CD8+ T-cells effectively eliminate cells by degranulation of
cytotoxic contents, by apoptosis through the Fas pathway, or by cytokine release [263, 264]. Blymphocytes undergo maturation in secondary lymphoid tissues such as the spleen and lymph
nodes. B-lymphocytes mediate humoral immune responses either as plasma cells, which
secrete antibodies against foreign antigens, or memory B cells, which release antibodies only
when antigens from a previous exposure are encountered [265].
Lymphocytes and hypertension: Lymphocytes have been implicated in human
hypertension. Mycophenolate mofetil is an immunosuppressant that suppresses T-lymphocyte

49

growth. Hypertensive patients taking mycophenolate mofetil for psoriasis or rheumatoid
arthritis (autoimmune conditions) develop significant reductions in blood pressure [266].
In animal models, T-lymphocytes appear to be more important than B-lymphocytes in
hypertension development. RAG1 knockout mice lack T and B-lymphocytes and are resistant to
DOCA-salt and ANG-II-induced hypertension, increased levels of vascular superoxide, and
vascular dysfunction. Adoptive transfer of T-lymphocytes (but not B-lymphocytes) into these
animals restores susceptibility to hypertension development, increased vascular superoxide
and vascular dysfunction [267]. Similarly in DahlS rats (4% salt diet), lymphocyte depletion via
targeted depletion of RAG1 attenuates lymphocyte infiltration in the kidney, proteinuria,
tubular and glomerular damage, and hypertension development [268]. Taken together, these
results suggest that T-lymphocytes are required for the development of hypertension and play
a role in oxidative stress and tissue damage.

4.2.2 Macrophages
Macrophages are phagocytes that are capable of secreting cytokines during innate
immune responses and during tissue repair [269]. Simplistically, once monocytes infiltrate into
tissues, they can differentiate into either M1 or M2 macrophages. M1 macrophages are
associated with pro-inflammatory oxidative stress production and release of cytokines such as
tumor necrosis factor- (TNF). M2 macrophages are associated with tissue repair and can
release anti-inflammatory cytokines such as transforming growth factor- (TGF). Macrophages
can fluctuate between M1 and M2 phenotypes [269]. Such adaptations may involve salt, which
attenuates the M2 phenotype. In vitro, salt has been shown to increase pro-inflammatory

50

cytokine production either through activation of mitogen-activated protein kinase [270] or by
attenuating IL-4 and IL-13 signals which promote the M2 phenotype [271]. The effect of salt on
macrophages is so profound that pro-inflammatory cytokine production occurs even in the
absence of stimulation [272]. This also affects T-lymphocytes, as salt prevents macrophage
suppression of T-lymphocyte proliferation [271].
Macrophages and human hypertension: Macrophages play an important role in
hypertension development and maintenance. In humans, circulating pre-activated monocytes
(as determined by in vitro stimulation with ANG-II) and macrophage-related markers (MCP-1,
soluble ICAM-1, E-selectin, GM-CSF) are increased in hypertensive versus normotensive
subjects [273-275].
Macrophages and Experimental hypertension: Animal studies have enabled a better
understanding of how macrophages contribute to increase SNA in the brain and peripheral
blood vessels. These mechanisms are summarized in Figure 9.
During inflammation, monocytes infiltrate into injured tissue via chemotaxis through
MCP-1 [269, 276]. An increase tissue macrophage infiltration is associated with the
development of hypertension in numerous models that include DOCA-salt [127, 277] and DahlS
hypertension [278]. Macrophage suppression attenuates hypertension development in these
models [127, 278]. In DOCA-salt and ANG-II-induced hypertension, blocking MCP-1 receptors on
macrophages with INCB3344 also prevents macrophage infiltration and protects against
hypertension development. This may suggest that tissue injury from DOCA-salt and ANG-II
treatment alone is insufficient in causing hypertension [279].

51

Macrophages and vascular injury: Unopposed pro-inflammatory cytokine release and
oxidative stress production from macrophages increases the severity of tissue injury.
Macrophage-derived ROS decreases NO bioavailability by reacting with NO (as previously
described) [257, 261]. Macrophage-derived TNFdecreases NO bioavailability directly by
reducing eNOS activity [280], and indirectly by activating NADPH oxidase [281], which further
enhance vascular oxidative stress production.

This promotes vascular dysfunction and

remodeling, which can increase vascular resistance. DOCA-salt treated mice that lack monocyte
colony stimulating factor (m-CSF) do not develop DOCA-salt related vascular remodeling,
dysfunction, or increased NADPH oxidase-derived superoxide in mesenteric arteries and are
protected from hypertension [282].
Macrophages and neuro-inflammation: Macrophages may also contribute to enhanced
SNA through mechanisms of neuro-inflammation [269, 283, 284].

Leukocytes in the blood

have limited access to the brain due to the blood brain barrier. Microglia are specialized
resident brain macrophages that are replenished by local proliferation and function mostly
independently of other immune cells [285, 286]. In hypertension induced by L-NAME and ANGII infusion, microglia release pro-inflammatory mediators in autonomic brain centers that are
associated with enhancement of SNA. Targeted microglial depletion using the transgenic
CD11b-diptheria toxin receptor (DTR) mouse decreases neuro-inflammation, glutamate
receptor expression in the PVN, and blood pressure in these models [285].
Furthermore, a recent study has demonstrated that hypertension is associated with proinflammatory status in the bone marrow, and that some of these immune cells extravasate into
the PVN to become microglial cells. Normotensive WK rats reconstituted with bone marrow

52

derived from SH rats develop hypertension that is associated with peripheral and central
inflammation. Specifically in the PVN, these animals had higher microglia number and percent
activation versus non-reconstituted WK rats. The authors of this study suggested that
extravasation may have occurred through the CSF, as CSF levels of the MCP-1 were elevated in
reconstituted rats [284]. This is supported by another study that found in SH rats, there is a
hypertension-related increase in junctional adhesion modlecule-1 (JAM-1) expression in the
brainstem, which enables recruitment of leukocytes to vascular endothelium [287]. Similarly,
hypertension was reduced in SH rats reconstituted with bone marrow from normotensive WK
rats and these animals had lower microglia number and percent activation in the PVN versus
non-reconstitued SH rats. [284]. In all rats, hypertension could be reduced by administering
minocycline, which is a brain-permeable antibiotic with anti-inflammatory properties, which
have also been effective in decreasing blood pressure in hypertensive patients [283, 284].
Macrophages and neuroeffector transmission: Finally, macrophage-related neuroinflammation may also contribute to enhanced SNA at the neuroeffector junction. We have
recently shown that in DOCA-salt hypertension, prejunctional impairment of 2AR signaling
involves macrophage-derived inflammation and oxidative stress as clodronate depletion of
macrophages reduces superoxide, preserves 2AR function, and protects against the late phase
of DOCA-salt hypertension development [127].

53

Figure 9. Macrophages contribute to enhanced SNA in hypertension. Microglia in the brain
secrete ROS and cytokines onto central autonomic regulatory regions that increase sympathetic
output to the kidney and resistance arteries, potentiating volume expansion (by increasing
Na+/H2O reabsorption) and vasoconstriction, respectively. This increases wall shear stress to the
endothelium and circumferential stretch to vascular smooth muscle and adventitial layers. To a
point, these effects can be buffered by the release of NO, which causes vasodilation. However,
prolonged stress and stretch increases ROS, which reduces NO bioavailability, resulting in
vascular dysfunction that makes the endothelium more susceptible to injury from pressureoverload. Tissue injury increases MCP-1 expression, which enables monocytes in circulation to
infiltrate via chemotaxis and differentiate into mature macrophages. Macrophages increase
ROS and cytokine production, which can be further aggravated by salt. As ROS and TNF levels
continue to rise, NO levels become depleted. Furthermore, the prejunctional 2AR becomes
impaired, resulting in enhanced NE release. These effects combine to increase vascular tone,
which leads to greater pressure-overload.

54

4.2.3 Sympathetic Modulation of Macrophages
Adrenergic drive also affects immune cell function. Lymphocytes and macrophages
express adrenergic receptors that can be activated by both NE and EP [288]. In general, it is
supported that activation of 1AR promotes pro-inflammatory responses while 2AR and 2AR
promote anti-inflammatory responses [288, 289]. However, the expression of adrenergic
receptors on immune cells is dynamic and their immunological influences may be dependent on
specific tissue environment. Therefore, it is important to investigate the effects of adrenergic
activation in the context of specific inflammatory conditions.
This is especially true for monocytes and macrophages, which respond rapidly to
inflammatory signals [288]. Activated macrophages increase synthesis and release of NE, which
contributes to inflammation and tissue damage in acute lung injury models. Interestingly,
antagonism of 2AR (RX 821002), but not 1ARs (prazosin) or ARs (metoprolol and ICI 118551)
significantly suppresses pulmonary immune cell infiltration, fibrin and hemorrhage. These
effects were reproduced by blocking tyrosine hydroxylase (-methyltyrosine) and could be
reversed by activating 2AR with UK14304 [290]. This suggests that activation of 2AR on
macrophages increases NE bioavailability and induces a pro-inflammatory phenotype.
Furthermore, the effects of adrenergic activation may be cell-type specific. WhereasAR activation favors CD8+ T-lymphocyte cell differentiation and enhances cytokine production
[291], in macrophages, proinflammatory status is suppressed by -ARs and enhanced by 2ARs
[292]. Consistent with this, activation of 2AR on macrophages leads to increased production of
TNF [293]. However, evidence also suggests that 2AR-specfiic activation can increase pro-

55

inflammatory cytokine production and release in macrophages even in the absence of proinflammatory stimuli [294].
Taken together, these data suggest that in macrophages, adrenergic drive in general,
may be pro-inflammatory, thus, supporting a possible interdependent relationship between
inflammation and enhanced SNA.

56

CHAPTER 2: RESEARCH AIMS AND HYPOTHESES

57

1.0

Research Goals
The goal of our research was to compare the effects of salt, mineralocorticoid, and HFD

on inflammation and hypertension development as it relates to prejunctional 2AR function.
Salt, mineralocorticoid, and obesity are all risk factors of hypertension. Activation of the
prejunctional 2AR results provides negative feedback to NE release from nerve terminals by
inhibiting Ca2+ channels and represents the last line of defense to counteract enhanced SNA in
hypertension. Macrophage-derived superoxide impairs signaling through the 2AR in DOCA-salt
hypertension, which is a model of inflammation, oxidative stress, and enhanced sympathetic
nerve activity. However, the mechanisms of this dysfunction remain unclear. It is also unclear if
similar deficits and inflammation occur in other types of hypertension, such as salt-sensitive
(with low mineralocorticoid) and obesity-related hypertension.
In our study, we evaluated inflammation and 2AR function in 3 models of
hypertension. The effects of mineralocorticoid and salt-sensitivity were studied using the
DOCA-salt rat. To study obesity-related hypertension, we induced hypertension and obesity
using a low-salt/HFD (0.3% salt, 60% kcal fat) in Sprague Dawley (DS) and Dahl salt-sensitive
(DahlS) rats. SD rats were selected because like humans, display great variability and are not
uniformly predisposed to developing salt-sensitive hypertension or obesity. By contrast, the
DahlS rat is genetically salt-sensitive, and like the DOCA-salt hypertensive rat, is a model of low
renin-hypertension.
Ultimately our goal was to perform the same experiments on a series of different animal
models in order to decipher specific or common etiological factors that contribute to human
hypertension development and maintenance.

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2.0

Overall Hypotheses
We will test the hypothesis that salt, mineralocorticoid, and high fat contribute to

hypertension development through increased inflammation and macrophage-associated
impairment of prejunctional 2AR-mediated inhibition of Ca2+ channels. We will also test the
hypothesis that the functional deficits lie at the receptor level and occur through oxidative
stress. Finally, we will test the hypothesis that macrophage depletion in these animal models
preserves 2AR function and attenuates hypertension development. Hypotheses as well as
specific aims 1-3 (which reflect chapters 4-6, respectively) are presented in Figure 10.

59

Figure 10. Hypotheses and specific aims of our study. Aim 1 is presented in Chapter 4 and
investigates if hypertension is associated with increased inflammation in SMCG tissue and
vascular macrophage infiltration in mesenteric arteries. Aim 2 is presented in Chapter 5 and
investigates if hypertension is associated with 2AR dysfunction in SMCG neurons. Aim 3 is
presented in Chapter 6 and investigates if macrophage depletion preserves2AR function and
attenuates hypertension and investigates how oxidative stress relates to 2AR dysfunction.

60

3.0

Specific Aims
Aim 1: To test the hypothesis that increased adventitial macrophage infiltration in

mesenteric arteries and an increase in MCP-1 and TNF expression in SMCG tissue is
associated

with

mineralocorticoid-salt

and

obesity-related

hypertension.

Vascular

macrophage infiltration will be determined by immunohistochemical localization of CD163 on
fixed whole-mounts of mesenteric arteries as an average of macrophage number per 1 mm2 of
tissue. Inflammation in SMCG tissues will be assessed by qRT-PCR for gene expression levels of
MCP-1 and TNF. In some animals, these experiments will be performed at different time
points to capture changes in inflammation that might occur during different developmental
stages of hypertension.
Aim 2: To test the hypothesis that impaired 2AR-mediated inhibition of Ca2+ channels
is associated with mineralocorticoid-salt and obesity-related hypertension. Whole-cell patch
clamp methods will be used to evaluate AR-mediated inhibition of Ca2+ channels, Ca2+
channel activity, current-voltage relationships, and cell membrane capacitance in acutely
dissociated SMCG neurons. In some animals, these experiments will be performed at different
time points to capture changes that might occur during different developmental stages of
hypertension. Levels of 2AR and GRK subtype mRNA expression will be evaluated by qRT-PCR
to verify if results occur due to changes in receptor expression or desensitization. GppNHp will
be used to evaluate Ca2+ channel inhibition independent of 2AR activation. Mechanisms of
receptor desensitization will be further evaluated using patch clamp and pharamocological
blockade of GRK.

61

Aim 3: To test the hypothesis that macrophage depletion preserves 2AR function and
attenuates hypertension development, and to test the hypothesis that oxidative stress
impairs 2AR function by promoting thiol-dependent receptor internalization. Liposomeencapsulated clodronate will be used to deplete animals of macrophages. Blood pressures will
be measured by the tail-cuff method. Macrophage depletion will be verified by
immunohistochemical localization of CD163 in whole-mounts of mesenteric arteries and by
spleen weight. Whole-cell patch clamp methods will be used to evaluate AR-mediated
inhibition of Ca2+ channels, Ca2+ channel activity, current-voltage relationships, and cell
membrane capacitance in acutely dissociated SMCG neurons. The effects of non-specific
oxidative stress (H2O2, t-BOOH) and diamide (thiol-specific oxidant) on 2AR function will be
evaluated using patch-clamp on SMCG neurons and G1A1 cells (HEK293-T cells that express
Ca2+ channels) co-transfected with 2AR and pertussis-insensitive (PTXi) Go (Cys351Gly). The
effects of oxidative stress on receptor localization will be evaluated by membrane colocalization of F-actin and 2AR in HEK293-T cells transfected with the 2AR.

62

CHAPTER 3: EXPERIMENTAL DESIGN AND METHODS

63

1.0

General Experimental Design
An algorithm of the experimental design used in these studies is depicted in Figure 10.

In brief, hypertension associated with inflammation and 2AR dysfunction will be further
explored by studies that involve macrophage-depletion, determine the level of dysfunction, and
determine how oxidative impairs 2AR function. In the absence of inflammation and 2AR
dysfunction, the next steps will depend on the degree of hypertension. In models with mild
hypetension, treatment time will increase to see if 2AR dysfunction occurs when hypertension
is prolonged and/or more severe. In models with more severe forms of hypertension, 2AR
function will be assessed at earlier time points to determine if 2AR dysfunction particpiates
more in the developmental phase of hypertension.
We will explore 2AR dysfunction further by determining if deficits lie at the level of the
receptor or through downstream signaling and investigate if oxidative stress impairs 2AR
function and localization.

64

Figure 11. Experimental design. We will ssess hypertension development and 2AR function
following administration of DOCA-salt or high fat diet. Experiments in models that do not
involve 2AR dysfunction will be repeated at earlier (for severe hypertension) or extended (for
mild hypertension) time points.

65

2.0

Materials and Methods
DOCA-Salt hypertensive rats: All animal use protocols were approved by the

Institutional animal Care and Use Committee of Michigan State University. Male SD rats
(Charles River Laboratories, Portage, MI) were delivered at 8 weeks of age and allowed to
acclimate for 5 days before surgical procedures. Rats were anesthetized via isofluorane
inhalation (0.5-2%) on a heating pad in dorsal recumbent position. The left kidney was
exteriorized and the renal artery, vein, and ureter were ligated with 5.0 silk before removing
the kidney. After uninephrectomy, some of the rats were given normal tap water ad libitum
(SHAM). The remaining rats received DOCA-salt treatment that consisted of a silastic pellet
containing 150 mg per 250 g bodyweight DOCA, which was implanted s.c in between the
scapulae and high salt/potassium drinking water (1% NaCl, 0.2% KCl) ad libitum. Following
surgery all rats were given penicillin (5mg/kg, i.m.) and carprofen (5 mg/kg, s.c.) and housed for
4 weeks on standard rat chow (Harlan/Tekland 8640 Rodent Diet).
Systolic blood pressure was measured in conscious rats using the automated CODA tailcuff system (Kent Scientific, Torrington, CT). Rats were restrained in holding chambers and
allowed to acclimate for 10 minutes before measurements were taken. Tail cuffs were inflated
to 250 mmHg and slowly deflated over a period spanning 15 seconds. Systolic blood pressure
was calculated by the average of 5 consecutive measurements using a volume recording sensor.
In some experiments, rats were depleted of macrophages using clodronate embedded
liposomes (ClodronateLiposomes, Amsterdam, The Netherlands), prepared as previously
described [295]. After uninephrectomy, rats were injected intravenously via the tail vein with
either clodronate embedded liposomes at 50 mg/kg of rat from a 5 mg/mL suspension

66

(clodronate-liposomes), or an equivalent volume of PBS containing empty liposomes (PBSliposomes). Thereafter, all rats were injected intraperitoneally once a week with clodronateliposomes at 25 mg/kg from a 5 mg/mL suspension, or an equivalent volume of PBS-liposomes
until the date of sacrifice.
Retrograde labeling:

For non-macrophage depletion studies, experiments were

performed on mesenteric artery-projecting SMCG neurons that were labeled with 1% cholera
toxin subunit B conjugated to Alex Fluor 555 (CTB-555, Thermo Fisher Scientific, Waltham, MA).
This procedure was done midway in the final week of DOCA-salt treatment. Rats were
anesthetized via isofluorane inhalation (0.5-2%) on a heating pad in dorsal recumbent position.
Following laparotomy, a loop of small bowel was exteriorized, spread, and secured by gauze
soaked in sterile pre-warmed HBSS. Three secondary branches of mesenteric arteries were
cleanly dissected from perivascular fat and encased in longitudinally cut silicone tubing. Once
placed around the artery, the tubing was sealed with silicone at both ends. Using a pipetteman,
30 L of CTB-555 was transferred directly onto arteries and allowed to incubate for 1 hour. The
tubing was then removed and washed with sterile HBSS. The bowel was undraped and returned
into the abdomen and moistened with 3 ml of HBSS. Rats received penicillin (5 mg/kg i.m.) and
carprofen analgesic (5 mg/kg, s.c.), and were housed for 4 days before the 4 week end point.
Immunohistochemistry was performed to determine labeling efficiency in the SMCG and in
fixed SMCG neurons. Retrogradely labeled SMCG tissue was fixed in in Zamboni fixative (4%
paraformaldehyde in 0.1 M phosphate buffer, pH 7.4) before embedding in O.C.T compound
(VWR, Randor, PA). 40 M sections were made using a HM 550 Cryostat and placed onto
superfrost plus microscope slides (Thermo Fisher Scientific, Waltham, MA). Slides were washed

67

with 0.1 M phosphate buffer (pH 7.4) and incubated in blocking buffer (0.1 M phosphate buffer,
4% donkey serum, 4% goat serum, 0.2% Triton X-100) for 1 hr at room temperature. Samples
were incubated overnight at 4 C in blocking buffer containing primary antibody against
tyrosine hydroxylase (TH) (BML-TZ1010-0050, Enzo Life Sciences, Farmingdale, NY). Samples
were washed with 0.1 M phosphate buffer and incubated in blocking buffer containing
secondary donkey anti-rabbit Alexa Fluor 488 (1:1000, Thermo Fisher Scientific, Waltham, MA)
for 1 hr at room temperature. Samples were washed with 0.1 M phosphate buffer and
mounted with mounting media containing DAPI (Thermo Fisher Scientific, Waltham, MA).
Images were taken using a Nikon TE2000-U inverted fluorescent microscope. SMCG neurons
were stained with the same protocol described above and imaged using an Olympus FluoView
FV1000 confocal laser scanning microscope.
HFD-induced hypertension: All animal use protocols were approved by the Institutional
animal Care and Use Committee of Michigan State University. Beginning at 3 weeks of age,
male Sprague-Dawley (SD) and Dahl salt-sensitive (DahlS) rats (Charles River Laboratories,
Portage, MI) were fed either a low salt-normal fat containing diet (NFD, 20 kcal% protein, 70
kcal% carbohydrate, 10 kcal% fat, 0.3% salt from Research Diets, #D12450J) or a low salt-high
fat containing diet (HFD, 20 kcal% protein, 20 kcal% carbohydrate, 60 kcal% fat, 0.3% salt from
Research Diets, #D12492) with access to tap water ad libitum for 6 months. Rats were weighed
once a week. Blood pressure was continuously monitored by radiotelemetery (TA11PA-C40,
DSI). This was accomplished by catheterization of the femoral artery with transmitter placed
subcutaneously in the inner thigh. Rats were housed in separation on top of a radiotelemetry
receiver (RPC-1, DSI) and blood pressure was continuously monitored through a data exchange

68

matrix and computer based data-acquisition system (Dataquest ART 4.36, DSI). Rats were
housed for 6 months. A separate batch of DahlS rats were analyzed at 2 and 4 months,
representing different stages of hypertension development. A separate batch of SD rats (Harlan
Laboratories, Haslett, MI) were analyzed after 12 months of feeding in order to allow more time
for obesity and hypertension to develop. A separate batch of obesity-prone (OP) and obesityresistant (OR) SD rats (Charles River Laboratories, Portage, MI) were analyzed after 6 months
weeks of feeding. A separate batch of female SD rats (Charles River Laboratories, Portage, MI)
were analyzed after 10 months of feeding in order to determine any sex-related effects.
Dissociated SMCG neurons: Rats were euthanized with sodium pentobarbital (50 mg/kg,
i.p.) or by decapitation after isofluorane inhalation (0.5-2%). SMCG tissues were removed,
dissected, cut into small pieces, and transferred into 3 mL of Earle’s balanced salt solution
(supplemented to 3.6 mg/mL glucose and 10 mM HEPES) containing 0.6 mg/ml collagenase D
(Roche, Indianapolis, IN), 0.4 mg/ml trypsin (Worthington, Lakewood, NJ), and 0.1 mg/ml
DNAse (Sigma, St. Louis, MO) at 35 oC for 1 hour in a shaking water bath. 6 mL of minimal
essential medium (MEM, Thermo Fisher Scientific, Waltham, MA), containing 10% fetal bovine
serum (FBS, Thermo Fisher Scientific, Waltham, MA), 1% penicillin/streptomycin (Thermo Fisher
Scientific, Waltham, MA), and 1% L-glutamine (Thermo Fisher Scientific, Waltham, MA) was
added to the reaction before dispersion using fire-polished Pasteur pipettes. Cells were
centrifuged twice at 70 x g, plated in MEM (containing 10% FBS, 1% penicillin/streptomycin)
onto poly-L-lysine coated 12 mm coverslips (VWR, Randor, PA), and stored in a humidified
incubator (95% air and 5% CO2) at 37 oC for 3-10 hours.

69

Whole-cell patch clamp recordings. Whole-cell patch clamp was used to measure Ca2+
currents and 2AR-mediated inhibition of Ca2+ channels at room temperature (20-24 oC) using
an Axopatch 200A voltage clamp amplifier (Molecular Devices, Union City, CA) and a Digidata
1322A (Axon Instruments, Union City, CA) A/D converter. Schott 8250 capillary glass (World
Precision Instruments, Sarasota, FL) was used to generate recording electrodes (2-4 M) using
a P-97 micropipette puller (Sutter Instrument Co., Novato, CA). Electrodes were fire-polished
and filled with internal pipette solution that contained (in mM): CsCl 143, MgCl2 1, EGTA 11,
HEPES 10, MgATP 4, Na2GTP 0.3, adjusted to PH 7.2 with CsOH and 295 mosmol/kg with
sucrose. When appropriate, 5’-guanylyl imidodiphosphate (GppNHp, Sigma-Aldrich, St. Louis,
MO) was added to a final concentration of 500 M. The external recording solution contained
(in mM): NaCl 97, TEA-Cl 20, CsCl 4.7, CaCl2 5, MgCl2 1.2, HEPES 10, glucose 11, tetrodotoxin
(TTX) 0.00001, adjusted to pH 7.4 with CsOH and 310 mosmol/kg with CsCl. Drugs to evaluate
2AR function included NE, UK14304, and yohimbine and were manually administered into the
chamber. For GRK2 inhibition experiments, cells were pretreated with 30 M paroxetine (GRK2
inhibitor) or fluoxetine (control) in MEM for 50-60 minutes. For oxidative stress experiments,
cells were pretreated with 150 M H2O2, 150 M t-BOOH, or 50 M diamide in MEM for 30-40
minutes. Ca2+ current recordings were low-pass-filtered at 5 kHz. Data were evaluated as
maximal current inhibition and were analyzed using pCLAMP version 10.2 software (Molecular
Devices, Sunnyvale, CA).
qRT-PCR. Total RNA was extracted with the MagMax mirVana kit (Thermo Fisher
Scientific, Waltham, MA ) and reverse transcribed into cDNA with the High capacity RNA to
cDNA kit (Thermo Fisher Scientific, Waltham, MA). Taqman probes (Thermo Fisher Scientific,

70

Waltham, MA) for all assessed genes (Table 1) were used and were processed in the 7500 Fast
Real-Time PCR System (Thermo Fisher Scientific, Waltham, MA). Reactions were prepared with
Taqman Fast Advanced Master Mix (Thermo Fisher Scientific, Waltham, MA). Relative gene
transcript levels were calculated by the cycle threshold and normalized with endogenously
expressed GAPDH.

Table 1. Taqman probes for genes assessed in the SMCG.
Gene ID
Adr2a
Adr2b
Adr2c
Grk2
Grk3
Grk5
TNFa
MCP1
Gapdh

Assay ID
Rn00562488_s1
Rn00593312_s1
Rn00593341_s1
Rn00563688 m1
Rn00562822 m1
Rn00578086 m1
Rn01525859_g1
Rn00580555_m1
Rn01775763_g1

Immunohistochemical localization of macrophages and sympathetic nerve fibers.
Second order mesenteric arteries were carefully dissected from perivascular fat and fixed
overnight in Zamboni fixative (4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4).
Samples were washed with 0.1 M phosphate buffer (pH 7.4) and incubated in blocking buffer
(0.1 M phosphate buffer, 4% donkey serum, 4% goat serum, 0.2% Triton X-100) for 1 hour at
room temperature. Samples were incubated overnight at 4 C in blocking buffer containing
primary antibodies against CD163 (1:200; Cat #: MCA342R, BIORAD, Hercules, CA) and tyrosine
hydroxylase (TH) (BML-TZ1010-0050, Enzo Life Sciences, Farmingdale, NY). Samples were
washed with 0.1 M phosphate buffer and incubated in blocking buffer containing secondary

71

donkey anti-rabbit Alexa Fluor 594 (1:1000, Thermo Fisher Scientific, Waltham, MA) and goat
anti-mouse Alexa Fluor 488 (1:1000, Thermo Fisher Scientific, Waltham, MA) antibodies for 1
hour at room temperature. Samples were washed with 0.1 M phosphate buffer and mounted
with mounting media containing DAPI (Thermo Fisher Scientific, Waltham, MA). Images were
taken using an Olympus FluoView FV1000 confocal laser scanning microscope.
Transfection and 2AR localization in HEK-293T cells. All cells and DNA constructs were
kindly gifted to us by Richard Neubig. The human embryonic kidney (HEK-293T) cell line was
maintained in Dulbecco’s modified Eagle’s medium (DMEM, Thermo Fisher Scientific, Waltham,
MA), containing 10% FBS (Sigma-Aldrich, St. Louis, MO) and 1% penicillin/streptomycin (Thermo
Fisher Scientific, Waltham, MA) in a humidified incubator (95% air and 5% CO2) at 37 oC. Cells
were transfected with the porcine 2A adrenergic receptor in the pEGFP vector [296] using
Effectene (Qiagen, Valencia, CA) according to the manufacturer’s instructions 24 hours before
cells were treated with 150 M H2O2, 150 M t-BOOH, or 50 M diamide in DMEM (45
minutes). F-actin was stained with Alexa Fluor 594 phalloidin (Thermo Fisher Scientific,
Waltham, MA) and receptor membrane localization was evaluated using an Olympus FluoView
FV1000 confocal laser scanning microscope and by Image J using Pearson’s correlation
coefficient of co-localization. G1A1 are HEK 293 cells that stably express the 2B, 1, and 2
subunits of N-type Ca2+ channels that have been used to study 2AR-induced modulation of Ca2+
currents [297]. G1A1 cells were maintained in MEM (Thermo Fisher Scientific, Waltham, MA),
containing 10% fetal bovine serum (FBS, Thermo Fisher Scientific, Waltham, MA) and 1%
penicillin/streptomycin (Thermo Fisher Scientific, Waltham, MA) in a humidified incubator (95%
air and 5% CO2) at 37 oC. Cells were co-transfected with the 2A adrenergic receptor in the

72

pEGFP vector [296] and the pertussis toxin (PPTX)-insensitive murine Go (PTXi-Go) in the pCI
vector [298] using Effectene (Qiagen, Valencia, CA) according to the manufacturer’s instructions
24 hours before cells were treated with 150 M H2O2, 150 M t-BOOH, or 50 M diamide in
DMEM (45 minutes). All constructs were kindly gifted from Dr. Richard Neubig’s laboratory.
Statistical Analysis. All data are represented as mean  standard error of the mean (SE),
where n equals animal number or number of rats or cells from which all the data were
obtained. Data were analyzed with Prism software using the unpaired two sample-student’s ttest.

73

CHAPTER 4: ADVENTITIAL MACROPHAGE ACCUMULATION AND SMCG INFLAMMATION
OCCUR IN DOCA-SALT BUT NOT HIGH FAT DIET-INDUCED HYPERTENSION

74

1.0

Abstract

Macrophages are associated with mineralocorticoid, salt, and obesity-related
hypertension. We have previously shown that macrophage-associated neurovascular
inflammation occurs in mesenteric arteries of DOCA-salt hypertensive rats. However, it is
unclear if inflammation occurs in the SMCG that provides sympathetic innervation to
mesenteric arteries, or if this mechanism is relevant to other models of hypertension, such as
obesity-related hypertension. Therefore, we investigated if adventitial macrophage
accumulation in mesenteric arteries and SMCG inflammation were increased in DOCA-salt
hypertensive rats and high fat diet (HFD)-induced hypertensive Sprague Dawley (SD) and Dahl
salt-sensitive (DahlS) rats. DOCA-salt treatment resulted in hypertension development that was
associated with increased adventitial macrophage infiltration and SMCG inflammation. After 6
months, a HFD was associated with mild hypertension without significant weight gain in SD rats
versus normal fat diet (NFD)-fed rats. 6 months of HFD induced hypertension with significant
weight gain in DahlS rats versus NFD-fed rats, which became significant at 4 months and
continued to increase until the 6 month time point. A HFD was not associated with increased
adventitial macrophage infiltration and SMCG inflammation in SD and DahlS rats. These results
suggest that neurovascular inflammation in the splanchnic circulation may only be relevant to
states of mineralocorticoid-salt excess. These results may be explained by animal model
differences.

75

2.0

Introduction
Macrophages play an important role in human hypertension. Circulating pre-activated

monocytes and macrophage-related markers are increased in hypertensive subjects compared
to normotensive subjects [273-275]. Organs that are associated with hypertension
development include the peripheral vasculature, kidney and brain; organs all of which are
enriched with macrophages in many experimental models of hypertension [54, 299]. However,
the mechanisms that trigger monocyte recruitment and the role that macrophages play in the
pathogenesis of hypertension are still unclear. Studies using macrophage depletion strategies
suggest a large part involves pro-inflammatory cytokine and oxidative stress production [269].
In hypertension, vascular monocyte recruitment is largely dependent on chemotaxis via
the MCP-1/CCR2 system as blocking CCR2 (MCP-1 receptor) on macrophages attenuates
vascular macrophage accumulation and hypertension development caused by DOCA-salt and
ANG-II-infusion [279]. MCP-1 is upregulated during tissue injury and blood monocytes are on
constant patrol for endothelial damage [300, 301]. In hypertension, vascular tissue injury occurs
first in the endothelial layer, which is the only vascular layer that comes into direct contact with
blood flow. Normal increases in friction and wall shear stress are counteracted by endothelial
release of NO, which causes vascular smooth muscle relaxation to accommodate mild and
transient increases in blood pressure [255]. However, persistent increases in pressure or
decreases in NO bioavailability can cause endothelial dysfunction that makes the blood vessel
less flexible and more prone to injury [254, 257]. Enhanced SNA contributes to increased
mechanical loading and wall stress either directly by vasoconstriction or indirectly by activating
RAAS and increasing renal reabsorption of Na+ and water. Reductions in NO bioavailability are

76

mediated by ROS members like superoxide, which combines with NO to form peroxynitrite
[257, 261]. Enhanced vascular oxidative stress production occurs with prolonged mechanical
stretch to endothelial cells through Rac1 activation of NADPH oxidases [216, 218]. Therefore,
increases in intensity and duration from pressure-overload increases vascular ROS production,
depletes NO bioavailability, and makes blood vessels more prone to injury. Tissue injury
promotes upregulation of MCP-1, which recruits blood monocytes [279].
Macrophage accumulation and pro-inflammatory activation in vascular and neuronal
tissue play a significant role in the pathogenesis of hypertension. Macrophages contribute to
endothelial dysfunction and vascular remodeling that leads to an increase in vascular resistance
and stiffness, providing further support to tissue injury [302]. Macrophage-derived TNF and
superoxide deplete NO by reducing eNOS and activating NADPH oxidase, and reacting to form
peroxynitrite, respectively. This increases endothelial dysfunction, leading to tissue injury
progression and unopposed influence by endothelial-derived contracting factors such as
endothelin-1 [261, 280, 281, 303]. Macrophages also participate in flow-induced vascular
remodeling in hypertension. Remodeling involves not only the production of inflammatory
mediators, but also matrix metalloproteinases [304], which degrade extracellular matrix and
modulate cytokine and chemokine activity [305, 306]. Taken together, these results suggest
that macrophage-related endothelial dysfunction and vascular remodeling play a large role in
increasing vascular resistance.
We have recently shown that macrophage-derived superoxide contributes to
hypertension development in the DOCA-salt rat by impairing negative feedback of NE nerve

77

terminal release through the prejunctional 2AR in mesenteric arteries [127]. These results
suggest that macrophage-related neuro-inflammation play a role in modulating SNA.
Enhanced production of macrophage-derived inflammatory mediators are common
features of mineralocorticoid, salt-sensitive, and obesity-related hypertension. Under all of
these conditions, macrophage recruitment and activation appear to depend on peroxisome
proliferator-activated receptor (PPAR, glitazone receptor) [307, 308], which when activated by
metabolites of arachidonic (15-HETE) and linoleic (9-HODE, 13-HODE) acid, enter the nucleus to
potentiate gene transcription of inflammatory proteins [307, 309, 310]. Salt has been shown to
directly enhance mineralocorticoid signaling in macrophages through Rac1 activation, which
leads to activation of NADPH oxidases and enhanced oxidative stress production. This can occur
independently with either salt or aldosterone, but is enhanced in the presence of both [216,
219]. In obesity, leptin is increased and has been shown to enhance macrophage proliferation
and production of TNF and IL-6 [311].
Taken together, macrophages may play a role in mineralocorticoid, salt, and obesityrelated hypertension by affecting both vascular and neuronal tissue. Both can be investigated in
the splanchnic circulation, which provides most of the body’s vascular resistance through
mesenteric resistance arteries. Splanchnic denervation decreases blood pressure in normal [61]
and hypertensive rats [59, 60, 62, 63]. Mesenteric arteries are innervated by sympathetic
neurons of the superior mesenteric and celiac ganglion (SMCG) [55, 56]. Therefore, we
investigated if increased macrophage infiltration in mesenteric arteries and inflammation in the
SMCG was associated with hypertension caused by mineralocorticoid, salt, and obesity.
Mineralocorticoid-salt was investigated using the DOCA-salt rat model, which is a hypertension

78

model characterized by mineralocorticoid excess, salt-sensitivity, and low-renin hypertension.
We attempted to induce obesity-related hypertension in Sprague Dawley (SD) and Dahl saltsensitive (DahlS) rats through a low salt (0.3%) and high fat (60% kcal fat) diet. Unlike SD rats,
DahlS rats are associated with salt-sensitive and low-renin-hypertension. This enabled us to
study the effects of obesity in consideration of other hypertension-associated factors that
might be relevant to human hypertension.

79

3.0

Results
Effects of DOCA-salt treatment on blood pressure and inflammation. At the 4 week end

point, mean systolic blood pressure was significantly higher in DOCA-salt rats compared to
SHAM rats (Figure 12A). SMCG and mesenteric artery tissues were collected to evaluate
inflammation by qRT-PCR and macrophage infiltration, respectively. In the SMCG, levels of
monocyte chemoattractant protein-1 (MCP-1) and tumor necrosis factor- (TNF) mRNA were
significantly higher in DOCA-salt rats compared to SHAM rats (Figure 12B). In mesenteric
arteries, adventitial macrophage infiltration was evaluated by CD163 immunoreactivity and was
significantly higher in DOCA-salt rats compared to SHAM arteries (Figure 12, C and D).
Adventitial macrophages were in close proximity to perivascular sympathetic nerve fibers
identified by TH immunoreactivity (Figure 12C). Although adventitial macrophage infiltration
was generally increased in DOCA-salt hypertension, this occurred to variable degrees.
Mesenteric arteries displayed areas of low as well as high levels of adventitial macrophage
infiltration (Figure 13, A and B).

80

Figure 12. DOCA-salt hypertension is associated with inflammation in SMCG and mesenteric
arteries. A: Mean systolic blood pressure was increased in DOCA-salt rats compared to SHAM
rats; n = 7 rats/group. B: mRNA levels of monocyte chemoattractant protein -1 (MCP-1, left)
and tumor necrosis factor- (TNF, right) were higher in SMCG from DOCA-salt rats compared
to SHAM rats; n = 4 rats/group. C: Immunohistochemical labeling of CD163-positive
macrophages and tyrosine hydroxylase (TH)-positive sympathetic nerve fibers in whole-mounts
of mesenteric arteries. D: Mean adventitial macrophage number per mm2 was higher in
mesenteric arteries from DOCA-salt rats compared to SHAM rats; n = 7 rats/group. Data are
means  SE, *P < 0.05 vs. SHAM.

81

Figure 13. Low and high adventitial macrophage infiltration in mesenteric arteries from
DOCA-salt rats. Immunohistochemical labeling of CD163-positive macrophages and tyrosine
hydroxylase (TH)-positive sympathetic nerve fibers in whole-mounts of mesenteric arteries.
Mesenteric artery showing A: lower and B: higher degree of macrophage accumulation.

Effects of HFD on obesity, hypertension and inflammation in SD and DahlS rats. SD rats
fed a high fat diet (HFD) for 6 months did not develop significant weight gain but developed
mild hypertension compared to normal fat diet (NFD)-fed SD rats (Figure 14, A and B). DahlS
rats fed a HFD developed significantly higher weight gain and systolic blood pressure at 4 and 6
month endpoints compared to NFD-fed DahlS rats (Figure 15, A and B). SMCG and mesenteric
arteries were collected to evaluate inflammation by qRT-PCR and macrophage infiltration,
respectively. SMCG mRNA levels of MCP-1 and TNF were similar in NFD and HFD-fed SD rats
at 6 months (Figure 14C) and DahlS rats at 2 months (Figure 16A), 4 months (Figure 16D), and 6
months (Figure 16G). Similarly, macrophage accumulation in mesenteric arteries, as evaluated
by CD163 immunoreactivity, was similar regardless of diet in SD rats at 6 months (Figure 14, D
and E) and DahlS rats at 2 months (Figure 16, B and C), 4 months (Figure 16, E and F), and 6
months (Figure 16, H and I).

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Figure 14. Hypertension development in Sprague Dawley (SD) rats fed a HFD for 6 months is
not associated with obesity or SMCG and mesenteric artery inflammation. A: Body weight was
not increased in SD rats fed a HFD rats compared to NFD rats; n = 5 rats/group. B: Mean systolic
blood pressure was mildly increased in HFD rats compared to NFD rats; n = 5 rats/group. C:
mRNA levels of chemoattractant protein -1 (MCP-1, left) and tumor necrosis factor- (TNF-,
right) in SMCG tissue were similar in all rats; n = 3 rats/group. D: Immunohistochemical labeling
of CD163-positive macrophages and tyrosine hydroxylase (TH)-positive sympathetic nerve
fibers in whole-mounts of mesenteric arteries from HFD rats and NFD rats. E: Mean adventitial
macrophage number per mm2 was similar in mesenteric arteries from HFD rats and NFD rats; n
= 5 rats/group. Data are means  SE , *p<0.05.

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Figure 15. Obesity-related hypertension development in HFD-fed DahlS rats. A: Mean weight
was significantly increased at 4 and 6 months in HFD-DahlS rats compared to NFD-DahlS rats. B:
Mean systolic blood pressure was significantly increased at 4 and 6 months in HFD DahlS rats
compared to NFD DahlS rats. Data are means  SE, *P < 0.05, n = 5 rats/group.

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Figure 16. Obesity-related hypertension in HFD DahlS rats is not associated with inflammation
in SMCG and mesenteric arteries at 2 (top row), 4 (middle row), and 6 months (bottom row).
A, D, G: mRNA levels of monocyte chemoattractant protein -1 (MCP-1, left) and tumor necrosis
factor- (TNF, right) in SMCG tissue were similar in all rats; n = 3 rats/group. B, E, H:
Immunohistochemical labeling of CD163-positive macrophages and tyrosine hydroxylase (TH)positive sympathetic nerve fibers in whole-mounts of mesenteric arteries from HFD and NFD
DahlS rats. C, F, I: Mean adventitial macrophage number per mm2 was similar in mesenteric
arteries from HFD compared to NFD DahlS rats; n = 5 rats/group.

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4.0

Discussion
The main findings of this chapter are: 1) DOCA-salt treatment results in hypertension

that is associated with inflammation in mesenteric artery and SMCG tissue. 2) HFD induces mild
hypertension without significant weight gain in SD rats. 3) HFD induces hypertension with
significant weight gain in DahlS rats. 4) HFD-induced hypertension is not associated with
inflammation in mesenteric artery and SMCG tissue.
Accumulation of peripheral vascular macrophages contributes to hypertension by
mechanisms that are not fully understood [299]. Adventitial immune cell infiltration has been
suggested to occur bi-directionally, from both luminal (“inside-out”) and adventitial (“outsidein”) ends [304, 312]. Mechanisms of chemotaxis via MCP-1 may be important in hypertension
as blocking MCP-1 receptors reduces vascular macrophage accumulation and blood pressure
[279]. There are also interactions between vascular tissue injury, oxidative stress, and cytokine
production that can increase blood pressure [253, 313]. We found increased adventitial
macrophage accumulation in mesenteric arteries from DOCA-salt but not HFD-fed hypertensive
rats. There are differences in the animal models that can explain this result.
Firstly, monocyte recruitment in DOCA-salt rats may occur in response to vascular injury
sustained by accelerated hypertension development and excessive mineralocorticoid and salt.
The DOCA-salt hypertensive rat is a model of cardiovascular inflammatory and oxidative stress
[262] that is associated with significant endothelial dysfunction and arterial hypertrophy [314,
315]. This is commonly observed in animal models that develop severe hypertension [316] and
makes the blood vessel less flexible and more prone to injury [254, 257]. Early arterial
remodeling observed in this model suggests pronounced mechanical stress and strain to the

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arterial wall and endothelium [257, 316, 317] as well as stretch from pressure-overload which
enhances vascular production of superoxide, MCP-1, and endothelin-1 [259, 260, 318].
Designated blood monocytes specifically patrol for vascular damage, and have been observed
to display crawling behavior along the endothelium of mesenteric arteries. Following tissue
injury, these monocytes infiltrate through the endothelium, increase cytokine production, and
differentiate into macrophages [300, 301]. In addition, macrophages may be directed to the
adventitia from perivascular tissue. This may occur due to increased MCP-1 and ROS production
in the adventitia and media layers from pressure-overload [312]. For these reasons, the
accelerated rate and magnitude of DOCA-induced hypertension are likely to cause an increase
in vascular injury and macrophage infiltration.
In contrast, HFD-induced hypertension developed over a period of months, not weeks.
Therefore, vascular injury may be less severe in these models due to adaptations that are able
to preserve vascular wall structure during small and steady increases in vascular wall stress and
stretch. However, it is interesting that macrophage accumulation did not change between HFDSD rats and HFD-DahlS rats, considering the mild hypertension in SD rats versus more severe
hypertension in DahlS rats that was detected even at 4 months. This may be due to
observations that obesity in DahlS rats protects against obesity-related vascular dysfunction
[249], which occurs in other models like the SD rat [319]. This paradoxical advantage of obesity
may provide an additional level of protection against pressure-induced vascular injury in DahlS
rats that is not present in SD rats or DOCA-salt rats.
Furthermore, in DOCA-salt hypertension, vascular injury and monocyte recruitment may
be enhanced by elevated mineralocorticoid and salt. DOCA is a precursor for aldosterone

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synthesis. Aldosterone increases vascular macrophage accumulation [96, 320], possibly by
upregulating endothelial expression of intracellular adhesion molecule-1 [321]. Furthermore,
aldosterone enhances production of inflammatory mediators in arteries [313, 322-324] and
macrophages [313, 325]. In addition, salt enhances oxidative stress production through Rac1
activation of NADPH oxidase and these effects are augmented in the presence of aldosterone
[216, 219]. Therefore, DOCA-salt may provide additional feedforward support for vascular (and
neuronal, discussed below) tissue injury and monocyte recruitment that is not present in HFDinduced hypertension.
Secondly, while obesity is associated with vascular inflammation [326], it is possible that
in HFD-fed rats, macrophages accumulate more in mesenteric adipose tissue versus arteries.
Our macrophage analysis did not include perivascular fat, where inflammation is likely to be
increased due to continuous adipocyte turnover and expansion [327, 328]. Therefore, it is
possible that macrophages are retained in the adventitia of mesenteric arteries in DOCA-salt
hypertension while macrophages are retained in expanding perivascular fat in HFD-induced
hypertension. Furthermore, perivascular fat may also be limited in DOCA-salt hypertension due
to weight loss and reductions in perivascular fat size [329].
Finally, vascular macrophages were identified using CD163, which is a scavenger
receptor for the hemoglobin-haptoglobin complex expressed in mature tissue macrophages.
Activation of CD163 induces expression of pro-inflammatory cytokines, that include TNF-
[330]. We have previously shown that macrophage expression of CD163 in DOCA-salt rats
coincides with increases in TNF, p22phox and CD11b [127] which we did not measure in the

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present study. Therefore, we cannot rule out whether macrophage infiltration in obesityrelated hypertension involves a different subset of macrophages.
Similar to vascular inflammation, neuro-inflammation in the SMCG was increased in
DOCA-salt but not HFD-induced hypertensive rats. Inflammation was evidenced by an increase
in TNF and MCP-1 mRNA expression. These data support previous observations that
superoxide is increased in SMCG tissues of DOCA-salt hypertensive rats [331] suggesting that
sympathetic neuro-inflammation may be directly linked to activation of postganglionic
sympathetic neurons. Macrophage density in SMCG tissues in general is low compared to
mesenteric arteries (data not shown) and could not be objectively measured. An increase in
sympathetic ganglionic macrophages can be detected in severe neuronal injuries such as
complete nerve transection where there is extensive neuronal cell death [332]. The injury
sustained in hypertension development or maintenance is likely less severe. Nevertheless, this
makes it difficult to speculate the source and cause of increased TNF and MCP-1. It is possible
that these results may be due to tissue injury caused directly by mineralocorticoid-salt and/or
due to mineralocorticoid-salt induced pro-inflammatory activation of macrophages.
What is clear from these findings is that mesenteric artery and SMCG inflammation are
associated with DOCA-salt hypertension. The DOCA-salt hypertension model is associated with
enhanced SNA [220]. SMCG nerve fibers terminate in the adventitia, in close apposition to
macrophages. It is tempting to speculate that some form of neuro-immuno-vascular interaction
may contribute to hypertension development in this model. Whether vascular tone is increased
due to sympathetic modulation of macrophage activity or macrophage-directed modulation of
neuroeffector transmission, or both, remains to be determined.

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CHAPTER 5: ALPHA 2-ADRENERGIC RECEPTOR-MEDIATED INHIBITION OF CALCIUM CHANNELS
IN DOCA-SALT AND OBESITY-RELATED HYPERTENSION

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1.0

Abstract
DOCA-salt and obesity-related hypertension are associated with sympathetic nervous

system hyperactivity. Mesenteric arteries are resistance arteries that receive sympathetic
innervation from the superior mesenteric and celiac ganglia (SMCG) and have profound
influences on blood pressure. Prejunctional 2-adrenergic receptors (2ARs) provide negative
feedback to norepinephrine release from sympathetic nerve terminals through inhibition of Ntype Ca2+ channels. Increased neuronal norepinephrine release in DOCA-salt and obesityrelated hypertension occurs through impaired 2AR signaling, however, the mechanisms
involved are unclear. We tested the hypothesis that 2AR-mediated inhibition of Ca2+ channels
in SMCG neurons is impaired in DOCA-salt and obesity-related hypertension. DOCA-salt
hypertension was induced after 4 weeks of DOCA-salt treatment. Obesity-related hypertension
was induced using a high fat diet (HFD). Obesity-related hypertension developed in SD rats after
12 months of HFD. Obesity-prone, but not obesity-resistant SD rats developed obesity-related
hypertension after 6 months of HFD. DahlS rats developed obesity-related hypertension after 4
months of HFD. Whole cell patch clamp methods were used to record Ca2+ currents from SMCG
neurons maintained in primary culture. We found that 2AR-mediated inhibition of Ca2+
channels was impaired in DOCA-salt hypertension, and that these results were not due to
differences in receptor expression or downstream factors of signaling. We also found that
2AR-mediated inhibition of Ca2+ channels was impaired in obese mildly hypertensive SD rats
at 12 months and in normotensive obesity-resistant SD rats, but not in obese hypertensive
DahlS rats. These results suggest that enhanced SNA in hypertension include the neuroeffector

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junction, possibly via dysfunction of negative feedback through the 2AR. This is especially
relevant to mineralocorticoid-salt excess.

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2.0

Introduction
Enhanced SNA plays an important role in hypertension development, maintenance, and

progression [333]. Central mechanisms of increased SNA have been studied extensively in the
majority of hypertension animal models. Sympathetic effector responses however, may be
determined by alterations in peripheral SNA [334] in vascular beds such as mesenteric
resistance arteries. Peripheral sympathetic nerve activity (SNA) increases in experimental
hypertension [335] including DOCA-salt hypertension [336], where uninephrectomy, the
mineralocorticoid derivative deoxycorticosterone (DOCA), and salt loading increase SNA and
blood pressure [337]. Sympathetic nerve terminal release of NE onto vascular smooth muscle
causes vasoconstriction. The prejunctional 2-adrenergic receptor (2AR) inhibits NE release by
coupling via Gi/o proteins to inhibit voltage-gated N-type calcium channels that control NE
release [125, 338]. This negative feedback regulates sympathetic tone and prevents sustained
increases in blood pressure [127, 129]. Of the three 2ARs, 2AAR and 2CAR couple to feedback
inhibition of NE release, with 2AAR being more predominant at higher levels of sympathetic
nerve activity [137].
Receptor desensitization and internalization of activated 2AR is mediated through
phosphorylation by members of the G protein-coupled receptor kinase (GRK) family.
Phosphorylation of the 2AR occurs through GRK 2 and GRK 3 but not GRK 5, resulting in G
protein uncoupling and arrestin-mediated receptor endocytosis [146-148, 153-156]. GRK 2 is
upregulated in inflammatory conditions that are associated with increased blood pressure [161,
162]. Whether GRK 2 upregulation increases blood pressure by reducing prejunctional 2AR
signaling is not known.

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Hypertension is associated with region-specific enhancement of SNA [233, 339].
Therefore, it is important to separately investigate mechanisms of SNA control in vascular beds
of specific organ systems. In DOCA-salt hypertension, peripheral mechanisms that enhance
neurotransmission include impaired inhibition of NE release caused by prejunctional 2AR
activation in mesenteric arteries [127, 340, 341]. Mesenteric arteries are major resistance
arteries with profound influences on hemodynamics and blood pressure. Mesenteric arteries
receive sympathetic innervation largely from the superior mesenteric and celiac ganglion
(SMCG). Enhanced splanchnic SNA contributes to hypertension and splanchnic denervation
decreases blood pressure in normal [61] and hypertensive rats [60, 62, 63]. We have recently
shown that prejunctional impairment of 2AR signaling in mesenteric arteries contributes to
the late phase of DOCA-salt hypertension development [127]. It is unclear if these results are
due to receptor impairment or downstream signaling, or if these deficits occur in other models
of hypertension, such as obesity-related hypertension. Blunted responses to the 2AR
antagonist, yohimbine, on plasma NE suggest 2AR impairment in obesity-association
hypertension in dogs [342], however it is not known if this is attributed to presynaptic or
central influences, or if NE release is augmented in splanchnic circulation.
Therefore, we tested the hypothesis that 2AR-mediated inhibition of Ca2+ channels in
SMCG neurons is impaired in DOCA-salt and obesity-related hypertension. Whole cell patch
clamp methods were used to record Ca2+ currents from SMCG neurons maintained in primary
culture in order to evaluate 2AR function and determinants of downstream signaling. DOCAsalt hypertension was induced by 4 weeks of DOCA-salt treatment. Obesity-related
hypertension was induced using a HFD at several time points in SD rats and DahlS rats.

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Investigations that use a series of different animal models to decipher specific and common
etiological factors of human hypertension may lead to a better understanding of human
hypertension and how to improve current treatments.

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3.0

Results
3.1

Retrograde Labeling and Recording from Dissociated SMCG Neurons in Culture

Isolation of SMCG neurons and retrograde labeling using CTB-555. The SMCG contains
a heterogeneous mixture of sympathetic neurons and satellite glial cells (Figure 17).
Sympathetic neurons were identified by TH but also by distinctively large and rounded nuclei
with prominent nucleoli compared to nuclei of satellite glial cells, which in general were smaller
and irregularly shaped. CTB-555 is retrogradely transported from mesenteric arteries to the cell
bodies of SMCG neurons. CTB-555 is detectable in a small percentage of SMCG neurons (Figure
18). Retrograde labeling efficiency was reached by 4 days post-labeling. CTB-555 was detectable
in a small percentage of dissociated SMCG neurons. Enzyme and mechanical dissociation
ensured that SMCG neurons were isolated away from satellite glial cells (Figure 18).

Figure 17. Immunohistochemical localization of sympathetic neurons in the SMCG (white
arrow). SMCG neurons were identified by tyrosine hydroxylase (TH, left image). These neurons
had large nuclei (middle image) with distinguishable nucleoli structures (asterisk). DAPI-staining
revealed many smaller nuclei, which corresponded to satellite glial cells. Merged image (right)
shows that satellite glial cells surrounded neuron cell bodies in the SMCG.

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Figure 18. Detection of retrograde labeling with CTB-555 in SMCG section (A, B) and
dissociated SMCG neurons (C). A: CTB-555 and tyrosine hydroxylase (TH) positive neurons can
be detected in low numbers in SMCG sections. These are SMCG neurons that innervate
mesenteric arteries. B: Retrograde labeling efficiency is reached at 4 days post-labeling; n=1-3
rats/group. C: CTB-555 can be detected in dissociated SMCG neurons that are positive for TH.
Enzyme and mechanical dissociation methods can isolate SMCG neurons away from satellite
glial cells (evidenced by a lack of surrounding satellite glial nuclei). Data are means  SE.

Verification of pure Ca2+ currents recorded from SMCG neurons. Na+ channels were
blocked by 10 nM TTX. K+ channels were blocked by 20 mM TEA-Cl and 143 mM CsCl. Ca2+
currents were detectable in SMCG neurons bathed in standard external solution (Figure 19A).
Ba2+ has higher affinity for Ca2+ channels that Ca2+, and therefore augments the magnitude of
current detected. Exchanging external solution that contained Ba2+ instead of Ca2+ significantly
increased currents recorded from SMCG neurons (Figure 19B). Addition of 1 mM CdCl to inhibit
Ca2+ channels completely attenuated currents (Figure 19C).

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Figure 19. Representative pure Ca2+ and Ba2+ currents recorded from SMCG neurons. A: Ca2+
currents recorded at -10, 0, and +10 mV. B: When standard external solution was exchanged for
external solution containing 10 mM BaCl2, current magnitude increased considerably. C: Ba2+
currents were attenuated when Ca2+ channels were inhibited with 1 mM CdCl.

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3.2

Investigating 2AR Function in DOCA-Salt Rats

Effects of DOCA-salt treatment on 2AR function in mesenteric artery-projecting SMCG
neurons. Whole-cell recordings were performed on acutely dissociated mesenteric arteryprojecting SMCG neurons identified by CTB-555 to evaluate 2AR function. Normalized Ca2+
current-voltage relationships and cell membrane capacitance were similar in all SMCG neurons
(Figure 20, A and B). Ca2+ current inhibition via activation of 2AR was evaluated using NE and
the 2AR-specific agonist UK14304 (each at 1 M) at +10 mV. Inhibition of Ca2+ current was
significantly reduced in SMCG neurons from DOCA-salt rats compared to SHAM rats (Figure
20D). Pretreatment of neurons with the 2AR antagonist, yohimbine (10 M, 1 min), blocked
Ca2+ current inhibition caused by NE (Figure 20E).

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Figure 20. DOCA-salt hypertension is associated with impaired 2AR-mediated inhibition of
Ca2+ channels in acutely dissociated mesenteric artery-projecting SMCG neurons. A:
Normalized Ca2+ current-voltage relationships were similar in all SMCG neurons; n = 22-25 cells.
B: Mean cell membrane capacitance was similar in all SMCG neurons; n = 33-36 cells. C:
Represent traces of Ca2+ current at baseline and when 2AR was activated by 1 M NE in SMCG
neuron from SHAM (left) and DOCA-salt (right) rat. D: Ca2+ current inhibition was impaired in
SMCG neurons from DOCA-salt rats when 2AR was activated by NE (1 M) (left) and UK14304
(1 M) (right); n = 9-11 cells. E: Pre-treatment with yohimbine (10 M) attenuated 2AR
function equally in all SMCG neurons; n = 7-9 cells. Data are means  SE, *P < 0.05.

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Mechanisms that impair 2AR function do not involve downstream signaling in DOCAsalt hypertension. We investigated if impaired 2AR signaling was due to differences in 2AR
expression and/or desensitization. mRNA levels of 2AR subtypes 2A, 2B, and 2C were equal in
SMCG samples from DOCA-salt and SHAM rats (Figure 21A). G protein-coupled receptor kinases
(GRKs) phosphorylate activated G protein-coupled receptors, resulting in arrestin-dependent
receptor desensitization [343]. mRNA levels of GRK 2, but not GRK 3 or GRK 5, were higher in
SMCG samples from DOCA-salt rats compared to SHAM rats (Figure 21B). In order to determine
if GRK 2 contributed to impaired 2AR signaling in DOCA-salt hypertension, we measured 2ARmediated inhibition of Ca2+ currents by NE (1 M) in SMCG neurons pretreated (50-60 min) with
the GRK2 inhibitor paroxetine (30 M) versus fluoxetine (control, 30 M). Paroxetine did not
increase 2AR-mediated inhibition of Ca2+ currents recorded from DOCA-salt neurons or SHAM
neurons compared to fluoxetine (Figure 21C). In order to investigate if impaired 2AR signaling
was due to a reduced ability of G-proteins to inhibit Ca2+ channels, we dialyzed the nonhydrolyzable GTP analog, 5’-guanylyl imidodiphosphate (500 M, GppNHp) directly into SMCG
neurons. GppNHp inhibited Ca2+ currents similarly in all SMCG neurons (Figure 21D).

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Figure 21. Impaired2AR-signaling in DOCA-salt hypertension does not involve changes in
receptor expression, receptor desensitization, or G-protein signaling to inhibit Ca2+ channels.
A: mRNA expression of 2AR subtypes 2A (left), 2B (middle), and 2C (right) were similar in
SMCG from DOCA-salt rats compared to SHAM rats; n = 4 rats. B: mRNA expression of G-protein
receptor kinase (GRK) subtype 2 (left), but not 3 (middle) and 5 (right), were increased in SMCG
from DOCA-salt rats compared to SHAM rats; n = 4 rats. C: Treating SMCG neurons with the
GRK2 inhibitor paroxetine (30 M) did not increase 2AR-mediated inhibition of Ca2+ channels
compared to fluoxetine (30 M); n = 6-8 cells. D: The non-hydrolyzable GTP analog, 5’-guanylyl
imidodiphosphate (GppNHp, 500 M) caused similar inhibition of Ca2+ channels in all SMCG
neurons; n = 6-8 cells. Data are means  SE, *P < 0.05.

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3.3

Investigating 2AR Function in HFD-Fed Sprague Dawley (SD) Rats

Effects of HFD on obesity and hypertension development in SD rats. SD rats fed a HFD
for 6 months developed mild hypertension without significant weight gain compared to NFD-SD
rats (Figure 22, A and B). We extended feeding time to 1 year in order to determine if increasing
the duration of a HFD led to hypertension with obesity. SD rats fed a HFD for 12 months
developed mild hypertension that was associated with significant weight gain compared to
NFD-SD rats (Figure 22, C and D).

Figure 22. Effects of HFD on weight and blood pressure in SD rats. A and B: 6 months of HFD
feeding does not cause weight gain but induces mild hypertension (increase in mean, systolic,
and diastolic pressure) compared to NFD rats; n=4-5 rats/group C and D: 12 months of HFD
feeding induces obesity with mild hypertension (increase in mean and diastolic pressure)
compared to NFD rats; n=5 rats/group. Data are means  SE, *P < 0.05.

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Effects of 6 months of HFD feeding on 2AR function in SMCG neurons isolated from
non-obese and mildly hypertensive SD rats. Normalized Ca2+ current-voltage relationships and
cell membrane capacitance were similar in all SMCG neurons regardless of diet (Figure 23, A
and B). Ca2+ current inhibition via activation of 2AR was evaluated using NE and the 2ARspecific agonist UK14304 (each at 1 M) at +10 mV. Inhibition of Ca2+ current was similar in all
SMCG neurons in non-obese mildly hypertensive SD rats fed a HFD for 6 months versus NFD
rats (Figure 23D). Pretreatment of neurons with the 2AR antagonist, yohimbine (10 M, 1
min), blocked Ca2+ current inhibition caused by NE (Figure 23E).

These results are not

associated with changes in 2AR or GRK mRNA expression (Figure 24, A and B).

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Figure 23. HFD-induced hypertension in non-obese SD rats (6 months) is not associated with
impaired 2AR-mediated inhibition of Ca2+ channels in acutely dissociated SMCG neurons. A:
Normalized Ca2+ current-voltage relationships were similar in all SMCG neurons; n = 24-26 cells.
B: Mean cell membrane capacitance was similar in all SMCG neurons; n = 26-29 cells. C:
Represent traces of Ca2+ current at baseline and when 2AR was activated by 1 M NE in SMCG
neuron from NFD (left) and HFD (right) rat. D: Ca2+ current inhibition was similar in all SMCG
neurons when 2AR was activated by NE (1 M) (left) and UK14304 (1 M) (right); n = 7-9 cells.
E: Pre-treatment with yohimbine (10 M) attenuated 2AR function equally in all SMCG
neurons; n = 7 cells. Data are means  SE, *P < 0.05.

105

Figure 24. SMCG mRNA expression of 2AR and GRK subtypes are not changed after 6
months of HFD in SD rats. A: mRNA expression of 2AR subtypes 2A (left), 2B (middle), and 2C
(right) were similar in SMCG tissues; n = 3 rats. B: mRNA expression of G-protein receptor
kinase (GRK) subtype 2 (left), 3 (middle) and 5 (right), were similar in SMCG tissues; n = 3 rats.

Effects of 12 months of HFD feeding on 2AR function in SMCG neurons isolated from
obese mildly hypertensive SD rats. Similar whole-cell patch clamp approaches were used to
determine whether a longer period of HFD feeding affected 2AR function. Normalized Ca2+
current-voltage relationships and cell membrane capacitance were similar in all SMCG neurons
regardless of diet (Figure 25, A and B). Ca2+ current inhibition via activation of 2AR was
evaluated using NE and the 2AR-specific agonist UK14304 (each at 1 M) at +10 mV. Inhibition
of Ca2+ current was reduced in SMCG neurons in obese mildly hypertensive SD rats fed a HFD
for 12 months versus non-obese normotensive NFD rats (Figure 25D). Pretreatment of neurons

106

with the 2AR antagonist, yohimbine (10 M, 1 min), blocked Ca2+ current inhibition caused by
NE (Figure 25E).

Figure 25. HFD-induced hypertension in obese SD rats (12 months) is associated with
impaired 2AR-mediated inhibition of Ca2+ channels in acutely dissociated SMCG neurons. A:
Normalized Ca2+ current-voltage relationships were similar in all SMCG neurons; n = 23-24 cells.
B: Mean cell membrane capacitance was similar in all SMCG neurons; n = 23-24 cells. C:
Represent traces of Ca2+ current at baseline and when 2AR was activated by 1 M NE in SMCG
neuron from NFD (left) and HFD (right) rat. D: Ca2+ current inhibition was reduced in SMCG
neurons from HFD-rats versus NFD rats when 2AR was activated by NE (1 M) (left) and
UK14304 (1 M) (right); n = 7-10 cells. E: Pre-treatment with yohimbine (10 M) attenuated
2AR function equally in all SMCG neurons; n = 6-7 cells. Data are means  SE, *P < 0.05.

107

Effects of 6 months of HFD feeding on weight, blood pressure and, 2AR function in
SMCG neurons isolated from obesity-prone (OP) and obesity-resistant (OR) rats. In order to
determine if 2AR dysfunction in 12-month HFD SD rats was due to age, obesity or prolonged
exposure to HFD, we performed a similar 6 month study using OP and OR-SD rats.
A HFD for 6 months induced mild hypertension with obesity in OP rats (Figure 26A) but
had no effect on weight gain and blood pressure in OR rats (Figure 26B).
Normalized Ca2+ current-voltage relationships and cell membrane capacitance were
similar in all SMCG neurons regardless of diet or susceptibility to obesity (Figure 27; A, B, F, and
G). Ca2+ current inhibition via activation of 2AR was evaluated using NE (1 M) at +10 mV. Ca2+
current inhibition was similar in all SMCG neurons regardless of diet in OP rats (Figure 27D).
Inhibition of Ca2+ current was reduced in SMCG neurons from HFD-OR rats versus NFD-OR rats
(Figure 27I). Pretreatment of neurons with the 2AR antagonist, yohimbine (10 M, 1 min),
blocked Ca2+ current inhibition caused by NE (Figure 27, E and J).

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Figure 26. Effects of HFD on weight and blood pressure in obesity-prone (OP) and obesityresistant (OR) SD rats. A and B: HFD induced obesity with mild hypertension (increase in
systolic blood pressure) in OP rats compared to NFD; n=5-6 rats. C and D: HFD had no effect on
weight or blood pressure in OR rats. N=4-5 rats/group. Data are means  SE, *P < 0.05.

109

Figure 27. Effects of HFD on 2AR-mediated inhibition of Ca2+ channels in acutely dissociated
SMCG neurons from obesity-prone (left side) and obesity-resistant (right side) SD rats. A and
F: Normalized Ca2+ current-voltage relationships were similar in all SMCG neurons; n = 14-17
cells. B and G: Mean cell membrane capacitance was similar in all SMCG neurons; n = 16-18
cells. C and H: Represent traces of Ca2+ current at baseline and when 2AR was activated by 1
M NE in SMCG neuron from NFD (left) and HFD (right) rat. D: Ca2+ current inhibition was
similar in all SMCG neurons from OP rats regardless of diet; n = 6-7 cells. I: Ca2+ current
inhibition was significantly reduced in SMCG neurons from HFD-OR rats versus NFD-OR rats; n =
8 cells. E and J: Pre-treatment with yohimbine (10 M) attenuated 2AR function equally in all
SMCG neurons; n =5-6 cells. Data are means  SE, *P < 0.05.

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3.4

Investigating 2AR Function in HFD-Fed Dahl Salt-Sensitive (DahlS) Rats

Effects of 2, 4, and 6 months of HFD feeding on body weight and hypertension. In order
to investigate the effects of HFD in a model that is salt-sensitive, we performed a 6 month study
in DahlS rats. DahlS rats fed a HFD developed significantly higher weight and blood pressure at
4 and 6 month endpoints compared to NFD-fed DahlS rats (Figure 28, A-D).

Figure 28. Obesity-related hypertension development in HFD-fed DahlS rats. A: Mean weight
was significantly increased at 4 and 6 months in HFD-DahlS rats compared to NFD-DahlS rats. BD: Mean, systolic, and diastolic blood pressure was significantly increased at 4 and 6 months in
HFD DahlS rats compared to NFD DahlS rats. Data are means  SE, *P < 0.05, n = 5 rats/group.

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Effects of 2, 4, and 6 months of HFD feeding on 2AR function in SMCG neurons
isolated from DahlS Rats. We investigated the effects of HFD on 2AR function in DahlS rats at
2, 4, and 6 month time points, which represent different stages of hypertension development.
All the data from patch clamp studies at these time points are presented in Figure 29 and Figure
30. Normalized Ca2+ current-voltage relationships and cell membrane capacitance were similar
in all SMCG neurons regardless of diet and time point (Figure 29; A, B, D, E, G, and H ). Ca2+
current inhibition via activation of 2AR was evaluated using NE and the 2AR-specific agonist
UK14304 (each at 1 M) at +10 mV. Inhibition of Ca2+ current was similar in all SMCG neurons
regardless of diet or time point (Figure 30; A, C, and E). Pretreatment of neurons with the 2AR
antagonist, yohimbine (10 M, 1 min), blocked Ca2+ current inhibition caused by NE (Figure 30;
B, D, and F). These results are not associated with changes in mRNA expression of 2AR (Figure
31; A, B and C) or GRK (Figure 32; A, B and C).

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Figure 29. Obesity-related hypertension in HFD-fed DahlS rats is not associated with changes
in current-voltage relationships, cell capacitance, or Ca2+ current inhibition in acutely
dissociated SMCG neurons at 2 (top row), 4 (middle row), and 6 months (bottom row). A, D,
G: Normalized Ca2+ current-voltage relationships were similar in all SMCG neurons; n = 17-28
cells. B, E, H: Mean cell membrane capacitance was similar in all SMCG neurons; n = 25-33 cells.
C, F, I: Represent traces of Ca2+ current at baseline and when 2AR was activated by 1 M NE in
SMCG neuron from NFD (left) and HFD (right) rat. Note: Data continued in Figure 30.

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Figure 30. Obesity-related hypertension in HFD-fed DahlS rats is not associated with impaired
2AR-mediated inhibition of Ca2+ channels in acutely dissociated SMCG neurons at 2 (top
row), 4 (middle row), and 6 months (bottom row). A, C, E: Ca2+ current inhibition when 2AR
was activated by NE (1 M) (left) and UK14304 (1 M) (right) was similar in all SMCG neurons; n
= 7-8 cells. B, D, F: Pre-treatment with yohimbine (10 M) attenuated 2AR function equally in
all SMCG neurons n = 6-7 cells. Data are means  SE.

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Figure 31. Obesity-related hypertension in HFD-fed DahlS rats is not associated changes in
receptor expression in SMCG. There was similar mRNA expression of 2AR subtype 2A (left), 2B
(middle) and 2C (right) in SMCG regardless of diet at A: 2 months, B: 4 months, and C: 6
months; n = 3 rats. Data are means  SE.

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Figure 32. Obesity-related hypertension in HFD-fed DahlS rats is not associated changes in
GRK expression in SMCG. There was similar mRNA expression of GRK subtype 2 (left), 3
(middle) and 5 (right) in SMCG regardless of diet at A: 2 months, B: 4 months, and C: 6 months;
n = 3 rats. Data are means  SE.

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4.0

Discussion
In the present study, we used whole-cell patch clamp to directly evaluate 2AR-

mediated inhibition of Ca2+ channels in dissociated SMCG neurons of DOCA-salt and HFDinduced hypertensive rats. The main findings are: 1) DOCA-salt hypertension is associated with
impaired 2AR function. 2) In SD rats, 6 months of HFD induces mild hypertension without
obesity that is not associated with impaired 2AR function. 12 months of HFD induces mild
hypertension with obesity that is associated with impaired 2AR function. 3) In obesity-prone
SD rats, HFD-induces mild hypertension with obesity that is not associated with impaired 2AR
function. In obesity-resistant SD rats, HFD does not cause hypertension or obesity but is
associated with impaired 2AR function. 4) In DahlS rats, HFD rapidly induces hypertension with
obesity that is not associated with impaired 2AR function.

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4.1

DOCA-salt hypertension
We found that 2AR-mediated inhibition of Ca2+ channels was significantly impaired in

DOCA-salt hypertensive rats. These results may explain previous findings that NE release is
increased in mesenteric arteries of DOCA-salt hypertensive rats [127]. We further showed that
deficits in 2AR signaling did not involve changes in receptor mRNA expression, Ca2+ channel
density or voltage-dependent activation, or G-protein inhibition of Ca2+ channels using GppNHp
activation. Taken together, these results suggest that impaired Ca2+ channel modulation is likely
due to changes in 2AR function.
The DOCA-salt rat is a model of neurogenic hypertension that results from enhanced
SNA. This is evidenced by impaired central regulation of the baroreceptor reflex [223], central
activation of CVOs through brain RAAS and oxidative stress [85, 225-227], salt-dependent
enhanced lumbar SNA [228], salt-dependent enhanced renal SNA [229-231] and enhanced
splanchnic SNA [59]. Taken together with our results, DOCA-salt hypertension is associated with
central and peripheral-driven mechanisms that enhance SNA. The purpose of prejunctional
2AR signaling is to counteract enhanced sympathetic outflow. We previously showed that
impaired 2AR signaling begins during the late phase of DOCA-salt hypertension (week 3-4)
[127]. Therefore, central mechanisms of enhanced SNA resulting from mineralocorticoid-salt
may precede peripheral 2AR impairment [337]. This suggests that prejunctional dysfunction of
2AR enhances the effects of central sympathetic activation to effector organs and contributes
to sustained increases in blood pressure in this model. This dual synergism may explain why
hypertension development in this model is so robust and rapid versus other models where
hypertension development is either milder or slower.

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While exploring mechanisms of receptor desensitization, we found that mRNA levels of
GRK2 were higher in SMCG from DOCA-salt hypertensive rats.

GRK2 is upregulated in

inflammatory conditions associated with hypertension and results in a loss of 2AR sympathoinhibitory function in the adrenal medulla [158, 162]. However, the GRK2 inhibitor paroxetine
did not improve 2AR-mediated Ca2+ current inhibition in SMCG neurons of DOCA-salt rats. This
suggests that GRK2-mediated 2AR desensitization may not be relevant in SMCG neurons, or
that due to its short half-life [344], prolonged incubation of neurons in the absence of agonist
resulted in GRK2 degradation beyond the detection limits of our assay.
Interestingly, our findings were associated with an increase in SMCG inflammation and
adventitial macrophage infiltration (presented in Chapter 4). However, whether inflammation
and macrophages impair 2AR function remains unknown (but are investigated in Chapter 6).

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4.2

Obesity-related hypertension in SD and DahlS rats
Blunted responses to the 2AR antagonist, yohimbine, on plasma NE suggest

presynaptic 2AR impairment in obesity-related hypertension in dogs [342]. However, these
studies have not been performed in rats and are unable to determine if enhanced NE release
occurs in the splanchnic circulation. Unlike DOCA-salt hypertension, the relationship between
obesity-related hypertension and 2AR function is not as straightforward and is not associated
with increased SMCG inflammation or adventitial macrophage infiltration (presented in Chapter
3). Interestingly, mild hypertension in obese SD rats was associated with 2AR impairment
while severe hypertension in obese DahlS rats was not associated with 2AR impairment. These
results may be explained by differences in animal model and by duration exposed to a HFD.
Obesity-related hypertension in SD rats: At 6 months, a HFD induced mild hypertension
without weight gain in SD rats that was not associated with 2AR dysfunction or differences in
receptor and GRK mRNA expression. This suggests that impaired 2AR signaling does not play a
role in the developmental phase of HFD-induced hypertension in SD rats.
However, increasing HFD exposure to 12 months resulted in the development of mild
hypertension with obesity that was associated with impaired 2AR-mediated inhibition of Ca2+
channels. This suggests that either obesity or prolonged exposure to HFD, or both, was
associated with 2AR dysfunction. Therefore, we performed the same 6 month study in OP-SD
rats, which are more susceptible to developing early obesity-related hypertension [238]. In
agreement with previous reports, 6 months of HFD induced obesity and mild hypertension in
OP-SD rats. However, 2AR dysfunction was not associated with the development of obesity-

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related hypertension in OP-SD rats. These results suggest that it may be the duration of
exposure to a HFD and not obesity per se that contributes to 2AR dysfunction in SD rats.
Interestingly, 6 months of HFD feeding in OR-SD rats resulted in 2AR dysfunction
despite a lack of increased body weight or hypertension. Taken together with our observations
that HFD-induced obesity in mildly hypertensive OP-SD rats was not associated with 2AR
dysfunction, it is tempting to speculate that obesity, at least in the short term may help
preserve 2AR function. High dietary fat induces oxidative stress production in adipose tissues
[345] and blood vessels [346] by upregulating NADPH oxidase. It is possible that in the initial
stages of obesity development, adipocytes surrounding blood vessels are able to counteract
ROS by shielding the blood vessel or through secretion of anti-inflammatory adipokines, such as
adiponectin. Adiponectin is recognized for its anti-inflammatory and insulin sensitizing
properties. On a HFD, OP but not OR-SD rats, develop glucose intolerance and insulin resistance
until about 6 months [347]. The anti-inflammatory effects of adiponectin activity correlate with
high glucose and insulin insensitivity, which include suppressing ROS production in blood
vessels [348]. Therefore, it is possible that OR-SD rats (and 2AR) are more susceptible to ROSinduced by a HFD, at least during the first 6 months of a HFD. However, more studies are
needed to substantiate this hypothesis.
The relevance of 2AR dysfunction in obesity-related hypertension development in SD
rats remains unclear. In SD rats, 2AR dysfunction detected after 12 months of a HFD was mild
compared to DOCA-salt hypertension, and did not worsen the severity of hypertension versus 6
months of a HFD diet. In fact, systolic blood pressure in 12 month HFD-fed SD rats was not
significantly different than NFD, suggesting that blood pressure may actually be improving.

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Therefore, it is possible that in chronically obese SD rats, 2AR dysfunction may develop only
after hypertension has established, as a mechanism that maintains blood pressure. This is
observed in the SH rat, where there is an age-dependent decline in adrenal 2AR expression
that coincides with impaired inhibition of nicotinic-evoked EP secretion [349].
Obesity-related hypertension in SD rats: HFD fed DahlS rats developed obesity-related
hypertension, which was detectable as early as 4 months. The degree of hypertension was
more severe than SD rats, resembling DOCA-salt hypertension. However, unlike DOCA-salt
hypertension, 2AR dysfunction was not detected in SMCG neurons from DahlS rats at any of
the time points studied. This suggests that 2AR dysfunction does not play a role in DahlS
obesity-related hypertension. This may reflect the lack of increased TNF and MCP-1 mRNA
expression in the SMCG and a lack of increased vascular macrophage accumulation in
mesenteric arteries. This suggests that neurovascular damage sustained in obesity-related
hypertensive DahlS rats is not as severe as DOCA-salt hypertensive rats. This may seem
counterintuitive as salt-induced hypertensive DahlS rats develop vascular and endothelial
dysfunction like DOCA-salt hypertensive rats, which occurs even in normotensive DahlS rats
[249, 350, 351]. However, we used a low salt/HFD in our study, and thus, the effects of salt are
less influential versus studies that used high salt to induce hypertension. Furthermore, dietinduced obesity in DahlS rats protects against vascular dysfunction [249], possibly because
obesity is unable to increase RAAS activity [249, 350]. This paradoxical advantage of obesity
may provide an additional level of protection against neurovascular injury that preserves 2AR
function in DahlS rats despite a degree of hypertension that is comparable to DOCA-salt rats.
Like DahlS rats, RAAS activity is also suppressed in the DOCA-salt rat. This suggests that 2AR

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dysfunction may be primarily caused by exogenous mineralocorticoid. This may also help
explain 2AR dysfunction observed in obese hypertensive SD rats (12 month HFD) as obesity
increases RAAS in SD rats [238].
Mechanisms of enhanced SNA in obesity-related hypertension: Experimental and
human obesity-related hypertension is associated with sympathetic hyperactivity [34, 352].
However, we suggest that these mechanisms differ from those in DOCA-salt hypertension.
Oxidative stress has been associated with sympathetic hyperactivity in obesity-related
hypertension. Adipose tissues may be releasing pro-inflammatory molecules that enhance
central sympathetic nerve activity [353, 354] as pro-inflammatory molecules can access CVOs
[355, 356]. Furthermore, obesity-related hyperleptinemia and insulinenemia can also directly
activate central mechanisms that increase SNA. Intravenous and intracerebroventricular
administration of leptin increases renal and lumbar sympathetic activity [357, 358] and insulin
infusion increases lumbar sympathetic nerve activity by increasing glutamatergic drive to the
rostral ventrolateral medulla [359].
Compared to SD rats, DahlS rats were more susceptible to the development of obesityrelated hypertension. In our 6 month studies, a HFD induced a mild increase in blood pressure
without significant weight gain in SD rats and pronounced hypertension with obesity in DahlS
rats, which was detected as early as 4 months. Although SD rats are commonly used to study
obesity and hypertension, only a subset of SD rats are susceptible to diet-induced obesityrelated hypertension [238]. In contrast, DahlS rats are genetically predisposed to salt-sensitive
hypertension [360], and mild increases in blood pressure occurs even on a low-salt (0.3%) diets
[195]. Furthermore, there is also evidence that leptin and insulin synergistically increase

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sympathetic outflow [361] and that in DahlS rats, insulin and leptin exaggerate obesity-induced
blood pressure responses to salt in central cardiovascular regulatory centers [197].
Interestingly, like obesity, salt-sensitive hypertension is associated with insulin
resistance [362]. Hyperinsulinemia occurs in the DahlS rat [363] and augments DOCA-salt
hypertension [364]. Therefore, there are common mechanisms of enhanced SNA in both saltsensitive and obesity-related hypertension, however, we suggest that they do not include
impairment of 2AR function in SMCG neurons.

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CHAPTER 6: MACROPHAGES CONTRIBUTE TO HYPERTENSION DEVELOPMENT IN DOCA-SALT
RATS BY IMPAIRING ALPHA 2-ADRENERGIC RECEPTOR FUNCTION POSSIBLY VIA OXIDATIVE
STRESS-INDUCED RECEPTOR INTERNALIZATION

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1.0

Abstract
DOCA-salt hypertension is associated with inflammation, oxidative stress, and

sympathetic nervous system hyperactivity.

Prejunctional 2-adrenergic receptors (2ARs)

feedback inhibit norepinephrine (NE) release from sympathetic nerves by coupling via Gi/o to
inhibit Ca2+ channels. Increased neuronal NE release in DOCA-salt occurs through impaired
2AR signaling; however, the mechanisms involved are unclear. Mesenteric arteries are
resistance arteries that receive sympathetic innervation from the superior mesenteric and
celiac ganglia (SMCG). We tested the hypothesis that macrophages impair 2AR-mediated
inhibition of Ca2+ channels in SMCG neurons obtained from DOCA-salt hypertensive rats. Whole
cell patch clamp methods were used to record Ca2+ currents from SMCG neurons maintained in
primary culture. Clodronate-liposomes were used to deplete animals of macrophages. In
Chapters 4 and 5, we showed that DOCA-salt treatment increased adventitial macrophage
accumulation in mesenteric arteries, increased SMCG mRNA levels of monocyte
chemoattractant protein-1 and tumor necrosis factor-, and impaired 2AR-mediated
inhibition of Ca2+ currents in SMCG neurons. In the present study, we show that macrophage
depletion preserves 2AR function and protects against hypertension development in DOCAsalt treated rats. Macrophages mediate inflammatory responses by oxidative stress production.
Oxidative stress is associated with adrenergic dysfunction and Gi/o are targets of oxidative
stress. We tested the hypothesis that oxidative stress impairs 2AR function and found that
oxidative stress impaired 2AR-mediated inhibition of Ca2+ currents in SMCG neurons.
Inhibition of 2AR-Gi/o coupling following pertussis toxin (PTX) treatment occurs due to ADPribosylation on the C-terminal cysteine (position 351) of Gi/o. It is not known if thiol-oxidation

126

of this residue results in similar uncoupling. Therefore, we tested the hypothesis that oxidizing
agents impair 2-AR function by thiol-oxidation of Cys351 on Go. The thiol-specific oxidant
diamide impaired 2AR function equally in G1A1 cells co-transfected with 2AR and PTXi-Go
(PTX-insensitive due to a Cys351Gly mutation). Oxidative modification can also affect protein
trafficking. We tested the hypothesis that thiol-oxidation affects receptor localization and
found that oxidative stress resulted in significant 2AR internalization to the perinuclear
cytoplasm in HEK-293T cells. These results suggest that macrophages contribute to the
development of DOCA-salt hypertension by impairing 2AR function through oxidative stressinduced receptor internalization.

127

2.0

Introduction
Inflammation and oxidative stress play a role in hypertension development and

sympathetic hyperactivity in salt-sensitive hypertension [127, 185, 202, 227, 365]. Reactive
oxygen species (ROS) and immune cell infiltration can be detected in mesenteric arteries early
in hypertension development [127, 366-368]. This is especially relevant in the DOCA-salt
hypertensive rat, which is a model of cardiovascular inflammation and oxidative stress [262].
Oxidative stress from peripheral inflammation contributes to centrally-mediated enhancement
of SNA [369], but can also increase peripheral sympathetic outflow by directly affecting NE
release from nerve terminals.
The prejunctional 2-adrenergic receptor (2AR) inhibits NE release by coupling via Gi/o
proteins to inhibit voltage-gated N-type calcium channels that control NE release [125, 338].
This negative feedback regulates sympathetic tone and prevents sustained increases in blood
pressure [127, 129]. In mesenteric arteries of DOCA-salt hypertensive rats, inflammation and
oxidative stress production enhance neuroeffector transmission, which contributes to
increased blood pressure [127, 370, 371]. In agreement with these findings, we showed that
DOCA-salt hypertension was associated with an increase in adventitial macrophage
accumulation (Chapter 4) and impairment of 2AR-mediated inhibition of Ca2+ channels in
SMCG neurons that did not involve 2AR expression, GRK-dependent desensitization, or
downstream signaling factors (Chapter 5). Therefore, the primary focus of this study was to
determine if macrophages contributed to DOCA-salt hypertension by impairing 2AR function in
SMCG neurons.

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Macrophages are major sources of oxidative stress, which can affect cell signaling and
neuroeffector transmission [313]. Oxidative protein modification can affect protein structure,
function and localization [372, 373]. Oxidative targets include amino acids like cysteine, whose
thiol groups are oxidation-sensitive [372], as depicted in Figure 33. Cysteine residues are
important for proper 2AR signaling. G protein coupling to the 2AR involves a conserved
cysteine in the C-terminal region of Gi/o proteins. Treatment with pertussis toxin (PTX) causes
ADP-ribosylation of this cysteine, resulting in 2AR-G protein uncoupling [163]. It is unknown if
cysteine oxidation impairs 2AR signaling in DOCA-salt hypertension. Therefore we tested the
effects of oxidative stress, specifically with respect to thiol-oxidation, on 2AR function and
localization in SMCG neurons and HEK-293T cells.

Figure 33. Thiol-oxidation of cysteine residues. Oxidative modifications can affect protein
structure, function and localization. Oxidation reactions are reversible with the exception of
the formation of sulfonic acid derivatives.

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3.0

Results
Effects of clodronate-liposomes on macrophage infiltration. Macrophage infiltration of

mesenteric arteries was evaluated by CD163 immunoreactivity, which was significantly higher
in mesenteric arteries from DOCA-salt compared to SHAM rats treated with PBS-liposomes.
Clodronate-liposomes prevented macrophage accumulation in mesenteric arteries from all rats
(Figure 34, A and B). The effectiveness of clodronate-liposomes in depleting macrophages was
also confirmed by a significant reduction in spleen weight compared to PBS-liposome treatment
(Figure 34C).

Figure 34. Clodronate-embedded liposome treatment attenuates adventitial macrophage
infiltration. A: Immunohistochemical labeling of CD163-positive macrophages and tyrosine
hydroxylase (TH)-positive sympathetic nerve fibers in whole-mounts of mesenteric arteries
from DOCA-salt rats and SHAM rats treated with clodronate-liposomes (Clod) or PBS-liposomes
(PBS). B: Mean adventitial macrophage number per mm2 was higher in DOCA-salt rats
compared to SHAM rats treated with PBS and significantly reduced in all rats treated with Clod;
n = 5-7 rats/group. C: Mean spleen weight was reduced in rats treated with Clod; n= 3-5
rats/group. Data are means  SE, *P < 0.05.

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Effects of macrophage depletion on 2AR function and blood pressure in DOCA-salt
rats. Normalized Ca2+ current-voltage relationships and cell membrane capacitance were
similar in all SMCG neurons (Figure 35, A and B). Peak current occurred at +10 mV in SMCG
neurons (Figure 35A). Ca2+ current inhibition via activation of 2AR was evaluated using NE and
UK14304 (each at 1 M) at +10 mV. In SMCG neurons from DOCA-salt rats treated with
clodronate-liposomes, Ca2+ current inhibition was significantly higher when 2AR was activated
with 1 M NE (Figure 37A) and UK14304 (Figure 37B) versus neurons from DOCA-salt rats
treated with PBS-liposomes. Pretreatment with yohimbine (10 M) reduced 2AR function to
similar levels in all SMCG neurons (Figure 37C). Clodronate-liposome treatment prevented
significant increases in systolic blood pressure in DOCA-salt treated. Mean systolic blood
pressure in DOCA-salt rats receiving clodronate-liposomes was comparable to SHAM rats
(Figure 37D).

Figure 35. Macrophage depletion with clodronate-liposomes (Clod) does not affect currentvoltage relationships and cell size versus PBS-liposomes (PBS). A: Normalized Ca2+ currentvoltage relationships were similar in all SMCG neurons; n = 19-23 cells. B: Mean cell membrane
capacitance was similar in all SMCG neurons; n = 27-36. Data are means  SE.

131

Figure 36. Representative traces of Ca2+ current inhibition following macrophage depletion
with clodronate-liposomes (Clod) versus PBS-liposomes (PBS). A: SHAM PBS-liposomes B:
DOCA PBS-liposomes C: SHAM clodronate-liposomes D: DOCA clodronate-liposomes.

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Figure 37. Macrophage depletion with clodronate-liposomes (Clod) preserves 2AR function
and protects against hypertension development in DOCA-salt rats versus PBS-liposomes
(PBS). A: When activated with NE (1 M), 2AR-mediated inhibition of Ca2+ channels in SMCG
neurons was higher in DOCA-salt rats treated with Clod compared to DOCA-salt rats treated
with PBS, but less than SHAM rats treated with Clod; n = 8-9 cells. B: When activated with
UK14304 (1 M), 2AR-mediated inhibition of Ca2+ channels in SMCG neurons was higher in
DOCA-salt rats treated with Clod compared to DOCA-salt rats treated with PBS, similar to SHAM
rats treated with Clod; n = 6-7 cells. C: Pre-treatment with yohimbine (10 M) attenuated 2AR
function equally in all SMCG neurons n = 5-6. D: Blood pressure was lower in DOCA-salt rats
treated with Clod compared to DOCA-salt rats treated with PBS; n = 5-7 rats / group. Data are
means  SE, *P < 0.05.

133

Effects of oxidative stress on a2AR function and localization. DOCA-salt hypertension is
associated with oxidative stress in celiac ganglia [331] and mesenteric arteries [127]. Therefore,
we investigated if oxidative stress impaired 2AR function. SMCG neurons were pretreated with
the non-specific oxidants H2O2 and t-BOOH (each at 150 M), and the thiol-specific oxidant
diamide (50 M) for 30-40 min prior to activating 2AR with NE (1 M). H2O2, t-BOOH and
diamide significantly reduced 2AR-mediated Ca2+ current inhibition in SMCG neurons (Figure
38A). ADP-ribosylation of Cys351 of the Go protein by PTX attenuates 2AR signaling [163].
The effects of Cys351 are currently unknown. Therefore, we wanted to determine if thioloxidation of Cys351 affected 2AR function. This was investigated in G1A1 cells transfected with
2AR and PTXi Go. The PTXi Go protein harbors a Cys351Gly mutation that does not affect
signaling [298]. Cells expressing the 2AR also expressed PTXi Go (Figure 38B). Diamide
treatment (50 M) significantly reduced 2AR-mediated inhibition of Ca2+ channels in cells
transfected with 2AR and PTXi Go (Figure 38C).
Protein oxidation can affect protein localization. Therefore, we investigated the effects
of oxidative stress on 2AR receptor localization by treating 2AR-transfected HEK-293T cells
with H2O2, t-BOOH or diamide for 40 min. In untreated cells, 2AR was predominantly localized
to the membrane as indicated by co-localization with F-actin (Figure 39, A and B). Oxidative
stress significantly reduced the fraction of membrane-bound receptor and increased the
fraction localized to the perinuclear cytoplasm (Figure 39, A and B).

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Figure 38. Oxidative stress impairs2AR function in SMCG neurons and G1A1 cells. A: 2ARmediated inhibition of Ca2+ channels was impaired in acutely dissociated SMCG neurons treated
with H2O2 (150 M), t-BOOH (150 M), and diamide (50 M); n = 7-10 cells. B: G1A1 cells cotransfected with 2AR and PTXiGo, which contains a Cys351Gly mutation. C: Diamide (50 M)
impairs 2AR-mediated inhibition of Ca2+ channels in G1A1 cells co-transfected with PTXi Go; n
= 3-6 cells. Data are means  SE, *P < 0.05 vs. untreated.

135

Figure 39. Oxidative stress results in receptor localization to the perinuclear cytoplasm. A:
Treatment of 2AR-GFP transfected HEK 293T cells with H2O2 (150 M), t-BOOH (150 M), and
diamide (50 M) resulted in receptor internalization from the membrane to the perinuclear
cytoplasm (left column). Alexa Fluor 594 phalloidin was used to stain F-actin and localize the cell
membrane (middle column). Membrane localization of 2AR was highest in untreated cells
(right column). B: Pearson’s correlation coefficient of co-localization between 2AR and
membrane F-actin decreased in 2AR-GFP transfected HEK 293T cells treated with H2O2, tBOOH, and diamide; n = 16-18 cells. Data are means  SE, *P < 0.05 vs. untreated.

136

4.0

Discussion
The main findings of this chapter are: 1) Macrophages contribute to DOCA-salt

hypertension by impairing 2AR function. 2) Thiol-oxidation impairs 2AR function in SMCG
neurons. 3) Cys351 of the Go protein does not participate in oxidative stress inducedinhibition of 2AR signaling in G1A1 cells. 4) Thiol-oxidation results in 2AR mislocalization to
the perinuclear cytoplasm in HEK-293T cells.
The DOCA-salt rat is a model of oxidative stress and inflammation [262]. Neuronal
superoxide levels are increased in celiac ganglia of DOCA-salt hypertensive rats [331]. In
agreement with these observations, we detected significantly higher mRNA expression of TNF
and MCP-1 in SMCG. SMCG neurons innervate mesenteric arteries, which together with
previous work, were found to be enriched with adventitial macrophages, TNF, and superoxide
[127]. These results suggest that peripheral vascular and neuro-inflammation from
macrophages may be important in the development of DOCA-salt hypertension. Macrophages
and oxidative stress are associated with sympathetic hyperactivity in DOCA-salt hypertensive
rats [127, 227]. A better understanding of these relationships may lead to improvements in the
clinical management of hypertension.
In the present study, we used whole-cell patch clamp to directly evaluate 2ARmediated inhibition of Ca2+ channels in dissociated SMCG neurons of DOCA-salts receiving
either clodronate-liposomes (to deplete macrophages) or PBS-liposomes (vehicle). We found
that 2AR-mediated inhibition of Ca2+ channels was significantly impaired in PBS-liposome
treated DOCA-salt rats versus SHAM rats. Furthermore, in DOCA-salt treated rats, we found
that macrophage depletion preserved 2AR function and protected against increases in blood

137

pressure. These results are in agreement with previous observations that macrophage-derived
superoxide impairs 2AR signaling [127].
Macrophages promote oxidative stress that contributes to vascular injury and
remodeling [374, 375], and we add to this list impaired 2AR function. In DOCA-salt
hypertension, antagonism of the MCP-1 receptor with INCB3344 attenuates vascular
macrophage accumulation and reverses hypertension [279]. This is in agreement with our
findings that macrophage depletion preserves blood pressure in DOCA-salt rats. Whether
macrophage depletion affects other mechanisms that contribute to DOCA-salt hypertension,
such as vasopressin, salt and endothelin-1 [337] remain to be determined. Interestingly, 2AR
activation in macrophages is associated with increased TNF production [293]. It is tempting to
speculate that increased NE release from nerve terminals through impaired 2AR function may
enhance macrophage pro-inflammatory capacities, suggesting possible neuro-immune
synergism that may contribute to hypertension. Therefore, our findings support the use of
therapeutic approaches that target macrophages in the clinical management of hypertension.
This may be relevant to patients with resistant hypertension, who often have salt-sensitivity,
low-renin and increased mineralocorticoid status [44]; all of which are hallmarks of the DOCAsalt hypertensive rat. This, in addition to the successful use of mineralocorticoid antagonists like
spironolactone in patients with resistant hypertension [376] demonstrates that the DOCA-salt
rat may be a suitable model to study human resistant hypertension.
Macrophages are major sources of oxidative stress that may affect cell signaling and
neuroeffector transmission by oxidative protein modifications that alter protein structure,
function and localization [313, 372, 373]. Oxidative stress has been implicated in modulating G

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protein-coupled receptor signaling [377, 378] and oxidative targets include amino acids like
cysteine, whose thiol groups are oxidation-sensitive to almost all forms of ROS [372, 379].
Cysteine residues are important for proper 2AR signaling. The most notable of these involves a
conserved cysteine in the C-terminal region of Gi/o proteins. Treatment with pertussis toxin
(PTX) causes ADP-ribosylation of this cysteine, resulting in 2AR-G protein uncoupling [163]. In
our study, non-specific oxidants (H2O2, t-BOOH) and the thiol-specific oxidant diamide impaired
2AR function in SMCG neurons similar to DOCA-salt challenge. However, diamide also
impaired 2AR signaling in G1A1 cells transfected with PTXi Go, which harbors a Cys351Gly
mutation. Therefore, these results suggest that oxidative modification of other cysteine
residues could be contributing to 2AR dysfunction. Cysteine modifications by phenylmercuric
chloride have been shown to decrease 2AR-ligand affinity [164] and the C-terminal cysteine of
the 2AR is associated with mechanisms of downregulation [165]. Additional studies are
needed to identify the cysteine residues responsible for oxidation-mediated 2AR dysfunction.
Furthermore, in 2AR-transfected HEK-293T cells, oxidative stressors that included
diamide caused receptor internalization to the perinuclear cytoplasm. Oxidation of proteins can
occur directly from ROS or through secondary oxidation processes like lipid peroxidation.
Oxidation of proteins is important in the pathological progression of disease because it is
associated with protein downregulation. Mechanisms of repair for oxidized proteins are not as
frequently reported and characterized in comparison to mechanisms that repair oxidized lipids
and nucleic acids [379]. Although there are disulfide reductases that can reduce oxidized
cysteines (before conversion to sulfonic acids) [380], it is generally accepted that oxidized
proteins are targeted for degradation by the proteasome system [381] or chaperone-mediated

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autophagy with incomplete degradation within lysosomes [382]. Therefore, it is possible that
oxidation-mediated internalization of the 2AR impairs 2AR signaling through mechanisms of
receptor downregulation/degradation.
Recently, there has been growing evidence supporting that some oxidative protein
modifications do not result in degradation, rather, occur in order to regulate cell signaling
[383]. Our findings are not in disagreement with this notion either. In fact, internalizationmediated downregulation is a natural mechanism that regulates 2AR signaling. Interestingly,
the perinuclear distribution of internalized 2AR in our studies bears a close resemblance to
2AR internalization following prolonged clonidine-activation [144]. Agonist-mediated receptor
internalization is an efficient method of receptor desensitization. Therefore, we suggest that
sulfhydryl-oxidation of cysteines to sulfenic acids may promote mechanisms of 2AR
desensitization by uncoupling 2AR from its G-protein through receptor internalization.
Oxidative stress has been shown to modulate signaling of the D1-dopamine receptor via Gprotein uncoupling [384] and this mechanism may be responsible for 2AR dysfunction in
DOCA-salt hypertension.

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CHAPTER 7: ALPHA 2-ADRENERGIC RECEPTOR DYSFUNCTION IN HIGH FAT DIET-INDUCED
OBESE HYPERTENSIVE FEMALE SPRAGUE DAWLEY RATS

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Abstract
In humans, blood pressure and hypertension prevalence tend to be higher in males than

females until the age of 65. Testosterone may promote mechanisms that contribute to
hypertension development while estrogen may provide protection against hypertension
development. Many experimental models suggest that hypertension induced by ANG-II, LNAME, mineralocorticoid-salt and obesity are more severe in males. We previously showed
(Chapter 5) that long term HFD feeding induces obesity-related to mild hypertension in male SD
rats that is associated with prejunctional 2AR dysfunction in SMCG neurons. Given the sexdifferences in susceptibility to hypertension development, we wanted to investigate if a HFD
induced obesity, hypertension, and impaired 2AR-mediated inhibition of Ca2+ channels in
SMCG neurons from female SD rats. Short term HFD feeding (4 months) did not cause weight
gain, hypertension or prejunctional 2AR dysfunction in female SD rats. Long term HFD feeding
(10 months) caused obesity, increased diastolic blood pressure and mild impairment of 2ARmediated inhibition of Ca2+ in SMCG neurons. The results of our short term study suggest that
SD female rats are more resistant to HFD-induced hypertension development than SD male
rats. The results of our long term study suggest that similar to SD males, SD females develop
HFD-induced obesity related to mild hypertension and 2AR dysfunction. It is not known if
these results are due to obesity itself or from prolonged exposure to a HFD.

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2.0

Introduction
In humans, hypertension prevalence and blood pressures in general tend to be higher in

males versus females independent of race or ethnicity [385]. This difference occurs until about
age 65 [386, 387] and may be explained by findings that age-associated blood pressure occurs
later and more rapidly in females (typically around age 50-60) compared to males [388]. While
sex differences in blood pressure are not large and are usually less than 10/5 mmHg
(systolic/diastolic), they nevertheless suggest that females may be less susceptible to
hypertension development in early adulthood before menopause.
Sex-differences in blood pressure are also evident in experimental models of
hypertension. Hypertension development is more severe in males versus females in ANG-II
induced hypertension [389], L-NAME-induced hypertension [390], mineralocorticoid-salt
hypertension [391, 392], and salt-induced DahlS hypertension [393]. These effects may be
attributed to estrogen. Aldosterone infusion and high salt induces higher blood pressure in
males that is associated with increased Fra-like activity (indicator of chronic neuronal
excitation) in the PVN. Interestingly, ovariectomy increases PVN Fra-like activity and attenuates
protection against hypertension development in female rats [391] .
However, estrogen is not able to explain all hypertension-related sex differences. In
HFD-induced hypertension, increased blood pressure appears to depend on testosterone.
Castrated but not ovariectomized rats are protected from HFD-induced hypertension.
Administration of exogenous testosterone restores susceptibility in castrated male rats and
augments hypertension development in ovariectomized female rats [394]. Hypertension
development in the SH rat also appears to be androgen-dependent [395, 396]. Urinary Na+

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excretion measurements suggest that this may involve impairment of pressure-natriuresis,
which occurs in male SH rats but not female or castrated SH rats. Testosterone
supplementation in castrated and ovarietomized SH rats attenuates protection against
impaired pressure-natriuresis [396].
Understanding sex differences in blood pressure may lead to a better understanding of
hypertension pathogenesis as well as mechanisms that might protect against hypertension
development. This could lead to the development of better therapeutics that are tailored
specifically to patient backgrounds. Therefore, we wanted to determine if females were
protected from 2AR defects in hypertension development. We selected to study sex-specific
effects using the HFD-induced obesity/hypertension model in SD rats (at 4 month and 10 month
time points).

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Results
Effects of HFD on obesity and hypertension development in female SD rats. A HFD did

not increase body weight or blood pressure at 4 months (Figure 40, A and B). We also
determined if increasing the duration of a HFD led to the development of obesity-related
hypertension. Female SD rats fed a HFD for 10 months did not develop increased systolic or
mean arterial blood pressure, but did develop increased diastolic pressure and body weight
gain compared to NFD-SD rats (Figure 40, C and D).

Figure 40. Effects of HFD on body weight and blood pressure in female SD rats. A and B: 4
months of HFD feeding does not increase body weight or blood pressure; n=5 rats/group. C and
D: 10 months of HFD feeding caused weight gain and an increase in diastolic pressure
compared to NFD rats; n=4-5 rats/group. Data are means  SE, *P < 0.05.

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Effects of 4 months of HFD feeding on 2AR function in SMCG neurons. Normalized Ca2+
current-voltage relationships and cell membrane capacitance were similar in all SMCG neurons
regardless of diet (Figure 40, A and B). Ca2+ current inhibition via activation of 2AR was
evaluated using NE and the 2AR-specific agonist UK14304 (each at 1 M) at +10 mV. Inhibition
of Ca2+ current was similar in all SMCG neurons (Figure 40D). Pretreatment of neurons with the
2AR antagonist, yohimbine (10 M, 1 min), blocked Ca2+ current inhibition caused by NE
(Figure 40E).

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Figure 41. 4 months of HFD does not impair 2AR-mediated inhibition of Ca2+ channels in
acutely dissociated SMCG neurons. A: Normalized Ca2+ current-voltage relationships were
similar in all SMCG neurons; n = 25-31 cells. B: Mean cell membrane capacitance was similar in
all SMCG neurons; n = 37-43 cells. C: Represent traces of Ca2+ current at baseline and when
2AR was activated by 1 M NE in SMCG neuron from NFD (left) and HFD (right) rat. D: Ca2+
current inhibition was similar in all SMCG neurons when 2AR was activated by NE (1 M) (left)
and UK14304 (1 M) (right); n = 5-8cells. E: Pre-treatment with yohimbine (10 M) attenuated
2AR function equally in all SMCG neurons; n = 5-6 cells. Data are means  SE.

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Effects of 10 months of HFD feeding on 2AR function in SMCG neurons. Normalized
Ca2+ current-voltage relationships and cell membrane capacitance were similar in all SMCG
neurons regardless of diet (Figure 42, A and B). Ca2+ current inhibition via activation of 2AR
was evaluated using NE and the 2AR-specific agonist UK14304 (each at 1 M) at +10 mV.
Inhibition of Ca2+ current was reduced when 2AR was activated by NE in SMCG neurons of
HFD-fed female SD rats (Figure 42D). Pretreatment of neurons with the 2AR antagonist,
yohimbine (10 M, 1 min), blocked Ca2+ current inhibition caused by NE (Figure 42E).

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Figure 42. 10 months of HFD impairs 2AR-mediated inhibition of Ca2+ channels by NE
activation in acutely dissociated SMCG neurons. A: Normalized Ca2+ current-voltage
relationships were similar in all SMCG neurons; n = 16-17 cells. B: Mean cell membrane
capacitance was similar in all SMCG neurons; n = 30 cells. C: Represent traces of Ca2+ current at
baseline and when 2AR was activated by 1 M NE in SMCG neuron from NFD (left) and HFD
(right) rat. D: Ca2+ current inhibition was reduced when 2AR was activated by NE (1 M) (left)
but not UK14304 (1 M) (right) in SMCG neurons of HFD-fed female SD rats; n = 5-6 cells. E:
Pre-treatment with yohimbine (10 M) attenuated 2AR function equally in all SMCG neurons;
n = 3-5 cells. Data are means  SE.

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4.0

Discussion
We wanted to investigate if 2AR impairment observed in obese hypertensive male SD

rats was sex-specific. Therefore, we performed a similar short term (4 month) and long term
(10 month) HFD study in female SD rats. The major findings of this study are: 1) A HFD induced
obesity and increased diastolic pressure only during longer exposure to a HFD. 2) Long term
HFD-induced obesity and increased diastolic blood pressure was associated with mild
impairment of 2AR-mediated inhibition of Ca2+ channels in SMCG neurons.
Before the age of 65, females appear to be less susceptible to higher blood pressure and
hypertension development versus males [385-387]. This has been largely attributed to female
sex hormones, which may be cardioprotective [397]. However, the prevalence of hypertension
and related sequelae in females may be changing due to increasing rates of obesity [398-400].
Obesity is a major risk factor for hypertension [401] and females are more prone to obesity
than males [398, 402]. In general, males tend to have higher SNA than premenopausal females
of comparable age [403, 404]. However, whether sex affects obesity-related enhancement of
SNA is still undetermined. Therefore, in the present study, we investigated the effects of HFD,
obesity, and hypertension on 2AR function in SD females.
In male SD rats, we previously showed that short term exposure to HFD resulted in mild
hypertension (mean arterial, systolic, and diastolic blood pressure) without significant weight
gain that was not associated with 2AR dysfunction in SMCG neurons. In the present study,
similar to male SD rats, short term HFD feeding in female SD rats did not increase body weight
or impair 2AR function. However, unlike male SD rats, short term HFD feeding in female SD
rats did not cause an increase in mean arterial, systolic or diastolic blood pressure. These

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results suggest that in the short term, females may be protected from HFD-induced
hypertension development. Protection against diet-induced hypertension in female rats has
been reported in several experimental models that include diets consisting of high fat [394] and
high fructose [405]. Interestingly, ovariectomy increases susceptibility to fructose-induced
hypertension, but not HFD-induced hypertension [394, 405]. This suggests that estrogen does
not play a role in HFD-induced hypertension. Furthermore, in HFD-induced hypertension,
castration protects against hypertension development and exogenous testosterone
administration restores susceptibility to hypertension in castrated male rats and augments
hypertension in ovariectomized females [394]. Therefore, in our study, SD female rats may be
protected from HFD-induced hypertension development because of lower levels of androgen
compared to SD male SD rats.
In male SD rats, we previously showed that long term exposure to a HFD leads to obesity
with mildly elevated mean arterial and diastolic blood pressure that is associated with 2AR
dysfunction (Chapter 5). In the present study we found that this was similar in female SD rats,
although to a milder degree. In female SD rats, only diastolic blood pressure increased and
2AR dysfunction was detected only when activated by NE and not the 2AR-specific agonist
UK14304. It should be noted that UK14304 mediated inhibition of Ca2+ channels tended to be
lower in SMCG neurons from HFD-fed rats versus NFD-fed rats (P=0.08) and may have become
significant if we increased cell number. It was less likely that NE was mediating a response
through other adrenergic receptors as the 2AR antagonist yohimbine attenuated the effects of
NE equally in all neurons. Therefore, we suggest that differences in NE-mediated inhibition of
Ca2+ channels are attributed to altered 2AR function.

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Evidence suggests that obesity-related impairment in autonomic control of the
cardiovascular system is sex-specific [406]. Although our study captures only a small picture of
total SNA, our results demonstrate that 2AR dysfunction may participate differently in obese
males and females following long term exposure to a HFD. We suggested that impaired 2AR
signaling did not play a role in the developmental phase of HFD-induced hypertension in male
SD rats because short term HFD-fed male rats were hypertensive without a change in 2AR
function. However, in female SD rats, we detected 2AR impairment as diastolic blood pressure
began to increase after long term exposure to a HFD. Therefore, it may be possible that in
female SD rats, 2AR dysfunction plays a role in hypertension development unlike males, where
2AR may play a role in hypertension maintenance during long term exposure to a HFD.
However, a more thorough analysis of 2AR function at different time points is needed to fully
substantiate this hypothesis.

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CHAPTER 8: GENERAL SUMMARIES/CONCLUSIONS AND DISCUSSION

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Mineralocorticoid-Salt Hypertension
The DOCA-salt hypertensive rat is characterized by cardiovascular inflammation,

oxidative stress, and enhanced SNA [220]. The majority of studies have focused on central
mechanisms that enhance sympathetic output. However, even enhanced activation of postganglionic sympathetic nerves from higher centers can be counteracted by the prejunctional
AR. Activation of the prejunctional AR provides immediate feedback inhibition to NE (and
ATP) release from nerve terminals. In this study, we show that DOCA-salt hypertension is
associated with functional impairment of the prejunctional 2AR.
DOCA-salt treatment resulted in severe hypertension that was associated with an
increase in neuro-inflammation in the SMCG, an increase in adventitial vascular macrophage
infiltration, and impaired 2AR-mediated inhibition of Ca2+ channels in SMCG neurons. Deficits
in 2AR signaling did not involve mechanisms of receptor expression, GRK-dependent
desensitization, or downstream signaling and likely involved receptor function.
Furthermore, macrophages contributed to this dysfunction as macrophage depletion in
DOCA-salt treated rats preserved 2AR-mediated inhibition of Ca2+ channels and protected
against hypertension development. Finally, oxidative stress, specifically thiol-oxidation,
impaired 2AR signaling by promoting receptor internalization to the perinuclear cytoplasm.
These results may suggest that macrophage-derived ROS impairs prejunctional 2AR signaling
by promoting mechanisms of receptor internalization and desensitization/degradation.
Common features of resistant hypertension include salt-sensitivity, low-renin and
increased mineralocorticoid status [44]. These are all hallmarks of the DOCA-salt hypertensive
rat [220]. This, in addition to the successful use of mineralocorticoid antagonists like

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spironolactone in patients with resistant hypertension [407] suggests that the DOCA-salt
hypertensive rat is a suitable model to study resistant hypertension. Furthermore, our results
suggest that there may be benefits of targeting oxidative stress and macrophage-derived
inflammation in human resistant hypertension either directly or as adjunctive therapy.
Conclusion: Macrophages contribute to DOCA-salt hypertension by impairing
prejunctional 2AR-mediated inhibition of Ca2+ channels in SMCG neurons through mechanisms
of oxidative stress-induced receptor internalization.

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2.0

Obesity-Related Hypertension
Unlike DOCA-salt hypertension, the development of obesity-related hypertension was

not clearly associated with macrophages and prejunctional 2AR impairment in mesenteric
arteries. This was consistently observed in both HFD-fed SD rats and also DahlS rats. We used
these rats specifically to determine how a HFD and obesity affect blood pressure regulation in
rats with different backgrounds. SD rats are not predisposed to hypertension development,
have normal RAAS responses, and like many humans, show great variability [408]. By contrast,
DahlS rats are predisposed to developing salt-sensitive and low-renin hypertension [409].
Studying the effects of obesity and HFD using different animal models is important because of
the variability in susceptibility and onset of obesity-related hypertension in humans.
On a high fat diet (HFD), SD rats slowly developed obesity with mild hypertension over
the course of a year while Dahl S rats developed obesity with severe hypertension by 6 months.
Obesity-related hypertension was not associated with neuro-inflammation in the SMCG or an
increase in adventitial macrophage infiltration in SD and DahlS rats. This may have been
attributed to compensatory mechanisms that could respond to gradual increases in blood
pressure (versus rapid onset like in DOCA-salt hypertension), thus, preventing tissue injury.
Mild but significantly impaired 2AR-mediated inhibition of Ca2+ currents occurred in
SMCG neurons from obese mildly hypertensive SD rats (male and female) after prolonged
exposure to HFD feeding (10-12 months). In male SD rats, this impairment did not augment the
severity of hypertension versus 6 months of HFD feeding, suggesting that 2AR dysfunction
may serve more in hypertension maintenance rather than development in this model. In female
SD rats, this impairment occurred at the same time as increased diastolic pressure, and may

156

play a role in hypertension development after prolonged exposure to a HFD. Impaired 2AR
function did not occur in DahlS rats at any of the time points studied. Prejunctional 2AR
dysfunction and enhanced NE release has been suggested to occur in salt-induced hypertensive
DahlS rat [204]. Therefore, the lack of 2AR dysfunction in our study may reflect the fact that
HFD, and not salt was used to induce hypertension.
There are several explanations to account for differences in hypertension and
susceptibility to 2AR dysfunction within animal models in our HFD study and in contrast to
findings from our DOCA-salt study. All diets used in the HFD study consisted of 0.3% salt. While
this is relatively low for the rat, it significant for DahlS rats, which have been shown to develop
increased blood pressure even on a 0.3-0.4% salt diet [195]. Therefore, salt, at least in part, may
explain the quicker onset and degree of hypertension developed in HFD-fed DahlS rats versus
HFD-SD rats. Furthermore, obesity increases RAAS activity [410], which likely occurred more in
SD rats considering DahlS rats are genetically predisposed to low-renin secretion [409]. From
our DOCA-salt study, 2AR dysfunction was associated with mineralocorticoid excess and it is
possible that aldosterone played a similar role in SD but not DahlS rats. Furthermore, despite a
more severe form of hypertension in obese DahlS rats, diet-induced obesity in DahlS rats
protects against vascular dysfunction [249], possibly because obesity is unable to increase RAAS
activity [249, 350]. This may protect against vascular injury, thus, explaining why macrophage
infiltration was not increased and why 2AR function was not altered in obese hypertensive
DahlS rats compared to DOCA-salt hypertensive rats.
The effects of obesity on 2AR function, unlike DOCA-salt, are complex and remain
elusive. This was further exemplified by our paradoxical findings that following 6 months of a

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HFD, OP-SD rats, which developed obesity and hypertension faster than unspecified SD rats,
were not associated with 2AR dysfunction while 2AR dysfunction occurred in non-obese
normotensive OR-SD rats. We speculate that in the short term, obesity may protect against
HFD-induced oxidative stress production [345], which we know from our DOCA-salt study,
impairs 2AR function. In obese OP rats, production of anti-inflammatory adipokines like
adiponectin may buffer ROS from blood vessels. Glucose intolerance and insulin resistance
occur in OP-SD but not OR-SD rats [347] and adiponectin suppresses vascular ROS production
during states of hyperglycemia and insulin-insensitivity [348].
However, what is clear from our combined studies is that hypertension alone is not
associated with vascular macrophage infiltration or 2AR dysfunction. The use of multiple
hypertension models is helpful in studying the contributions of selected precipitating factors to
specific pathological processes observed in human hypertension. Blood pressure-dependent
associations with 2AR dysfunction in our studies likely involved mineralocorticoid and salt, but
not obesity. The only association detected was between mild 2AR impairment and a HFD
(prolonged exposure in SD rats and short term exposure in OR-SD rats). Therefore, we suggest
that in obesity-related studies, careful attention should be placed on the choice of diet and
duration of exposure, and not just on the development (or lack) of obesity itself.
Conclusion: Unlike DOCA-salt hypertension, macrophage-associated impairment of
2AR-mediated inhibition of Ca2+ channels in SMCG neurons does not occur in obesity-related
hypertension. Prolonged HFD-feeding leads to obesity with mild hypertension and impaired
prejunctional 2AR function in male and female SD rats. In the SD rat, 2AR dysfunction is more
associated with a HFD than obesity, and in the short term, obesity may protect against HFD-

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induced 2AR impairment. A HFD causes obesity and severe hypertension development in
DahlS rats but does not alter prejunctional 2AR function in SMCG neurons. Therefore, DahlS
rats are more susceptible than SD rats to HFD-induced obesity-related hypertension; however,
the underlying mechanisms for this do not involve impairment of the prejunctional 2AR.

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3.0

Macrophages in DOCA-Salt and HFD-Induced Hypertension
It is important to appreciate that macrophage number alone does not provide adequate

information regarding the inflammatory status of macrophages, which is likely more important
for hypertension development. Macrophage depletion in our DOCA-salt studies showed that
macrophages contribute to hypertension development by impairing 2AR function. However,
macrophage depletion was not performed in our obesity-related studies because it was not
practical to administer clodronate-liposomes for the extended time it would take to develop
obesity-related hypertension in SD rats (months to a year in HFD-SD rats versus weeks in DOCAsalt rats). Therefore, we cannot rule out whether macrophages impaired 2AR dysfunction in
our aged obese and mildly hypertensive SD rats. Future experiments could involve
administering drugs that suppress macrophage-derived inflammatory mediators or inhibit MCP1 to clarify whether this relationship also exists in some forms of obesity-related hypertension.
Macrophages without question play a role in obesity-related disorders. Obesity is
associated with chronic low-grade inflammation and macrophages are the main leukocytes in
adipose tissue [310, 411]. However, it is reasonable that the pro-inflammatory status of
macrophages in DOCA-salt hypertension was more severe than obesity-related hypertension.
This may be explained not only by the rate of hypertension development which may cause
vascular damage in DOCA-salt rats, but enable time for compensatory mechanisms to protect
against vascular damage in slow developing obesity-related hypertension. Rather, the use of
mineralocorticoid and salt, which are precipitating factors that drive rapid hypertension
development in the DOCA-salt model, also directly promote pro-inflammatory polarization of
macrophages. Salt increases pro-inflammatory cytokine production by activating mitogen-

160

activated protein kinase [270] and by attenuating IL-4 and IL-13 signals that favor the M2
phenotype [271]. The effect of salt on macrophages is so profound that pro-inflammatory
cytokine production occurs even in the absence of macrophage stimulation [272]. Furthermore,
mineralocorticoid signaling also enhances pro-inflammatory polarization of macrophages [412,
413] as well as vascular macrophage recruitment [321]. Selective deletion of the MR from
macrophages protects DOCA-salt treated rats from hypertension and cardiovascular remodeling
[414]. Therefore we suggest that macrophages are likely relevant to both obesity-related and
DOCA-salt hypertension, however, macrophages may play a more dominant role, especially
with respect to adrenergic signaling when hypertension develops rapidly due to
mineralocorticoid-salt excess.
Our macrophage depletion study suggested that macrophages impair AR. Whether
this occurs directly or indirectly cannot be fully addressed from this approach alone. Although it
is well supported that macrophages play a role in DOCA-salt hypertension development [127,
279, 307, 415], T-lymphocytes have also been implicated as well [267]. RAG1 knockout mice
lack T and B-lymphocytes and are resistant to DOCA-salt hypertension, increased levels of
vascular superoxide, and vascular dysfunction. Adoptive transfer of T-lymphocytes (but not Blymphocytes) into these animals restores susceptibility to hypertension development, increased
vascular superoxide and vascular dysfunction [267]. Furthermore, lymphocyte suppression
using mycophenolate mofetil protects against the development of DOCA-salt hypertension
[416]. Our results do not suggest that T-lymphocytes do not play a role and we suggest that
they are likely just as important as macrophages in hypertension development. In general, Tlymphocytes play a role in adaptive immunity where inflammatory effector responses involve

161

specific antigen-receptor interactions that mostly occur after the initiation of innate immunity
[263]. Macrophages participate in both innate and adaptive immune responses. In adaptive
immunity, macrophages present antigens to CD4+ helper T-lymphoctes, which then activate
CD8+ cytotoxic T-lymphocytes [417]. Macrophages have also been shown to directly stimulate
and enhance cytotoxic capacities of CD8+ T lymphocytes [418]. On the other hand, CD4+ Tlymphocytes also modulate the pro-inflammatory status of macrophages, which largely forms
the basis of tissue transition from a pro-inflammatory state to one of repair [419]. Given the
interdependent functional relationship between macrophages and T-lymphocytes, it is
conceivable that suppression of one cell type would ultimately affect the other.

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4.0

Limitations of Whole-Cell Approach
In the present study, we used whole-cell patch clamp in order to directly evaluate 2AR-

mediated inhibition of Ca2+ channels in dissociated SMCG neurons of DOCA-salt and obesityrelated hypertension. Whole-cell approaches are used to study 2AR signaling in nerve
terminals [124, 125, 338, 420], which are too small for direct electrophysiological recordings.
While this approach is beneficial as it allows investigating pre-junctional 2AR responses
independent of post-junctional 2AR responses, our results need to be interpreted in light of
several considerations.
We evaluated 2AR function in the cell bodies of SMCG neurons while hypertensionrelated changes occur in nerve terminals. In our studies, we showed that SMCG inflammation
was associated with 2AR dysfunction in SMCG neurons. Sympathetic neurons in prevertebral
ganglia are affected in models where inflammation occurs in distal target tissues [421, 422].
However, we cannot be sure that our findings occur at the nerve terminal using data from our
assay alone. In DOCA-salt hypertension, these 2AR deficits have been verified in mesenteric
artery preparations using nerve stimulation and amperometric measurements of NE release
[127]. These experiments will be performed in our obesity-related hypertension models in the
near future.
Furthermore, analysis of 2AR function in dissociated SMCG neurons occurs hours after
tissues are removed from the rat and incubated in artificial supportive media. Therefore, the
damage sustained to the 2AR in our hypertension models likely needs to be severe or
permanent enough to be detected by our assay. It is possible that 2AR dysfunction occurs in
obesity-related hypertension but this impairment is able to recover when SMCG neurons are

163

cultured. Therefore we can only suggest that based on our results, 2AR dysfunction does not
seem to play a role in obesity-related hypertension development. Ultimately, these findings will
need to be verified using in vivo approaches.

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5.0

Other Considerations of 2AR Dysfunction in DOCA-Salt Hypertension
This work has largely focused on prejunctional 2AR function with applications on nerve

terminal NE release from SMCG neurons onto mesenteric arteries. It is possible that 2AR
dysfunction occurs in other tissues and systems that may also contribute to DOCA-salt
hypertension development.
Impairment of endothelial 2AR. Activation of endothelial 2ARs causes vasodilation
through increased NO release onto vascular smooth muscle [423, 424]. In rat mesenteric
arteries, this occurs independently of cAMP as treatment with forskolin (inhibitor of cAMP
production) does not attenuate endothelial 2AR-mediated NO release [425]. However, this
effect is inhibited by PTX treatment, suggesting that coupling to Gi/o proteins activates NO
synthase in the vascular endothelium [426, 427]. Activation of endothelial 2ARs has been
shown to reverse endothelial dysfunction during reperfusion injuries [428, 429] and may also
serve in counteracting increased vascular permeability from cytokine-related disruption of tight
junctions, which can leads to enhanced immune cell infiltration [430-433]. Therefore,
endothelial 2ARs provide a mechanism that counteracts vasoconstriction from post-junctional
1/2 AR activation and vascular immune cell infiltration. It is possible that like SMCG neurons,
2AR dysfunction might also occur in the endothelium of DOCA-salt treated rats, which would
additionally contribute to hypertension development/maintenance as well as vascular injury.
Impairment of central 2AR. Activation of central 2ARs (primarily through the 2A and
2C subtypes) results in sustained decreases in blood pressure associated with SNA suppression
[434, 435]. Interestingly, from antisense gene knock down studies, activation of the 2B subtype

165

appears to cause salt-sensitive hypertension [436]. However, 2A is the most abundant subtype
expressed in cardiovascular control centers [437] and likely plays a larger role than2B on SNA
regulation. Macrophage-associated neuro-inflammation occurs in the brain. This occurs via
activation of microglia, which are specialized brain macrophages that release pro-inflammatory
mediators in autonomic brain centers that are associated with increased glutamate-signaling
and enhanced of SNA [285]. Even though clodronate liposomes do not easily pass through the
BBB [438], we cannot rule out the possibility that DOCA-salt hypertension may also result from
microglial-mediated impairment of central 2ARs. Furthermore, it has been recently shown that
in hypertension, activated bone marrow precursors contribute to an increase in microglia
number and activation via MCP-1-mediated extravasation into the brain from CSF [284].
Therefore, it is possible that peripheral immune cells (which are susceptible to clodronateliposomes) may influence activity in CNS cardiovascular centers in DOCA-salt rats.
Prejunctional 2AR impairment on kidney function. The prejunctional 2AR also
regulates SNA to the kidney. Enhanced SNA to the kidney affects blood pressure through
increased renin release (enhancing RAAS) and reabsorption of Na+ and water. Although renal
SNA studies have yielded mixed results, some studies suggest that DOCA-salt hypertension
results from enhanced renal SNA that is associated with increased chemokine ligand, MCP-1
and interleukin-6 (IL-6) [229]. We did not consider the effects of 2AR dysfunction or
macrophage depletion on renal mechanisms of blood pressure regulation. However, these
mechanisms are important given that hypertension was induced specifically by administering
mineralocorticoid and salt.

Considering the profound renovascular macrophage-related

inflammation that occurs in DOCA-salt treated rats, it is also likely that clodronate treatment in

166

our study affected kidney function. Therefore, our findings that macrophage-depletion
protected against blood pressure in DOCA-salt treated rats are likely not attributed only to
decreased sympathetic drive to mesenteric arteries, and likely involve other organ systems that
include the kidney.

167

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