IMPAIRED FUNCTION OF PREJUNCTIONAL ADENOSINE RECEPTORS ON PERIVASCULAR SYMPATHETIC NERVES IN SALT-SENSITIVE HYPERTENSION By Sutheera Sangsiri A DISSERTATION Submitted to Michigan State University In partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Pharmacology & Toxicology 2013 ABSTRACT IMPAIRED PERIVASCULAR ADRENERGIC NEUROTRANSMISSION BY THE PREJUNCTIONAL ADENOSINE RECEPTORS IN DOCA-SALT HYPERTENSION By Sutheera Sangsiri Hypertension is a major public problem that will affect more than 1.6 billion people worldwide by 2025. Essential hypertension is associated with sympathetic nerve hyperactivity and 50% of the patients are salt sensitive. Deoxycorticosterone acetate (DOCA)-salt rat model is a low rennin form of hypertension with transient salt and water retention and is a model of saltsensitive human hypertension. Norepinephrine (NE) and ATP are the major cotransmitters relased from perivascular sympathetic nerve varicosities supplying arteries and veins. NE and ATP regulate pheripheral arterial resistance and venous capacitance. Release of NE and ATP is precisely regulated by prejnctional autoreceptors. NE acts on the !2-adrenergic receptors (!2AR) to inhibit NE and ATP release and the !2AR function is impaired in DOCA-salt rats. ATP is hydrolyzed to adenosine which activates prejunctional adenosine A1 receptors (A1Rs) and A2ARs to modulate NE and ATP release. This work determined role of prejunctional A1Rs and A2ARs in adrenergic transmission. I also tested the hypothesis that prejunctional A1R and A2AR function is impaired in sympathetic nerves supplying mesenteric arteries and veins of DOCA-salt rats. Electricall-evoked NE release and constriction of blood vessel were measured in vitro by using amperometry and video microscopy. Drug effects on the kinetics of NE oxidtion currents were measured. My results show that prejunctional A1Rs couple to inhibition of NE release and A1R function is impaired in periarterial nerves of DOCA-salt rats. I also showed that prejunctional A2ARs couple to stimulation of adenosine transporter in perivenous sympathetic nerves and this receptor function is also compromised in DOCA-salt hypertension. These data suggests that the perivascular sympathetic nerve varicosities are an important target for the pathophysiology of salt sensitive hypertension. Furthermore, sympathetic autoreceptor dysfunction is not specific to !2-adrenergic receptors, but there is a more general disruption of prejunctional mechanisms controlling sympathetic neurotransmitter release. Finally, the data illustrate differential autoreceptor dysfunction in arteries and veins of DOCA-salt hypertension. ACKNOWLEDGEMENTS I would like to dedicate this honors dissertation to my advisor, Dr. James J. Galligan, in appreciation of him who always inculcates me to think critically in research, inspires me to take risks and instills me the belief that there is no limit to what I can accomplish. His passion, mentoring style and life have impressed the eager mind, who have had the privilege to work with him. No words can be expressed my gratitude. I will always be grateful to my committee members, Dr. Gregory M. Swain, Dr. Gregory D. Fink, Dr. Hui Xu, Dr. Colleen C. Hegg, for always challenging me to think out of the box, and for their guidance and support throughout my Ph.D. training. Especially, I would like to thank Dr. Hui Xu, and Dr. Gregory M. Swain for their patience and dedication for training and assisting me in tons of experiments. I also like to thank the Galligan lab (previous lab members: Dr. Hua Dong, Dr. Hui Wang, and Dr. Hong Zhao, and our present members: Dr. Xiaochun Bian, Loc Vinh Thang, Matthew J. Fhaner, Yogesh Bhattarai, Eileen Rodriguez-Tapia, Jasmina Jakupovic, Marion M. France, Leighann S. Jordan, Roxanne N. Fernandes, and David E. Fried) for their friendship, support and sense of humor, which makes me feel I was part of the team and makes everyday in the Galligan lab my impressive experience. ! "#! I am gratitude to the Royal Thai Government and Department of Pharmacology and Toxicology for giving me the opportunity to be a part of this program. This 5 year training has been truly inspirational and an invaluable experience of research, friendship, and love. Last but not least, I am deeply indebted to my family for their support and patience throughout these 5 years of the long-distance relationship, which has brought me to this honor achievement. ! "! TABLE OF CONTENTS LIST OF TABLES!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!ix LIST OF FIGURES!!!!!!!!!!!!!!!!!!!!!!!!!!!!!...x LIST OF ABBREVIATIONS!!!!!!!!!!!!!!!!!!!!!!!!!.xiii CHAPTER 1: General Introduction!!!!!!!!!!!!!!!!!!!!!!.1 1.1 Overview!!!!!!!!!!!!!!!!!!!!!!!!!!!!!..2 1.2 The autonomic nervous system!!!!!!!!!!!!!!!!!!!...3 1.3 Autonomic control of blood pressure!!!!!!!!!!!!!!!!!..9 1.3.1 Sympathetic control of arterial resistance!!!!!!!!!!!.9 1.3.2 Sympathetic control of venous capacitance!!!...!!!!!!10 1.3.3 Role of sympathetic supply on splanchnic circulation!!!!!..12 1.3.4 Long-term regulation of blood pressure!!!!!!!!!!!..12 1.4 Sympathetic neurotransmission!!!!!!!!!!!!!!!!!!!16 1.4.1 Sympathetic preganglion neurotransmission!!!!!!!!!.16 1.4.2 Sympathetic postganglion neurotransmission in focus on vascular system!!!!!!!!!!!!!!!!!!!!!!!!!..18 1.4.3 Adrenergic neurotransmission!!!!!!!!!!!!!!!..19 1.4.4 Neuropeptide Y (NPY) neurotransmission!!!!!!!!!!.28 1.4.5 Purinergic neurotransmission!!!!!!!!!!!!!!...!29 1.4.5.1 Adenosine 5’-triphosphate (ATP) neurotransmission.....31 1.4.5.2 "1-adrenoceptor-evoked postjunctional ATP release!.33 1.4.5.3 Pannexin1-facilitated cellular purine release!!!!!33 1.4.5.4 Contribution of pannexin-1 to neurogenic vascular contraction!!!!!!!!!!!!!!!!!!!.35 1.4.5.5 Beta-nicotinamide adenine dinucleotide (#-NAD) neurotransmission!!!!!!!!!!!!!.!!...39 1.4.5.6 Adenosine neurotransmission!!!!!!..!!!!..41 1.5 Modulation of sympathetic neurotransmitter release by prejunctional autoreceptors!!!!!!!!!!!!!!!!!!!!!!!!!!!49 1.5.1 The "2-adrenergic autoreceptors!!!!!!!!!!!!!!.50 1.6 Differential sympathetic regulation of arteries and veins!!!!!!!!...53 1.7 Hypertension!!!!!!!!!!!!!!!!!!!!!!!!!!!.58 1.7.1 Sympathetic overactivity in hypertension!!!!!!!!!!!62 1.7.2 Autoreceptor in hypertension!!!!!!!!!!!!!!!...62 1.7.3 DOCA-salt rat model of hypertension!!!!!!!!......!!...63 1.7.4 Amperometric measurement of norepinephrine!!!!!!!!67 ! "#! 1.7.5 Contribution of other catecholamines in amperometric detection of NE!!!!!!!!!!!!!!!!!!!!!!!!!!!.68 1.8 Perspectives!!!!!!!!!!!!!!!!!!!!!!!!!!!.70 1.9 Bibliography!!!!!!!!!!!!!!!!!!!!!!!!!!!..71 CHAPTER 2: Research specific aims and general methods!!!!!!!!!!84 2.1 Overall research goal!!!!!!!!!!!.!!!!!!!!!!!!.85 2.2 Specific aims!!!!!!!!!!.!!!!!!!!!!!!!!!!...87 2.3 Research design and methods!!!!!!!!!!!!!.!!!!!!.89 2.3.1 Specific aim 1!!!!!!!!....!!!!!!!!!!!!!!89 2.3.2 Immunohistochemistry protocol!!!!!!!!!!!!!!!.89 2.3.3 Specific aim 2!!!!!!!!!!!!!!!!!!!!!!!90 2.3.4 Protocol for measurement of vasoconstriction in vitro!!!!!!91 2.3.5 Specific aim 3!!!!!!!!!!!!!!!!!!!!!!!93 2.3.6 Protocol for amperometric measurement of NE!!!!!!!!..95 2.3.7 How to minimize unstable baseline?..............................................102 2.3.8 When is carbon fiber electrode no longer useful?..........................102 2.3.9 General methods!!!!!!!!!!!!!!!!!!!!!104 2.4 Ethical aspects of the proposed research!!!!!!!!!!!!.........105 2.4.1 Vertebrate animal subjects!!!!!!!!!!!!!!!!...105 2.4.2 Veterinary Care!!!!!!!!!!!!!!!!!!!!!..106 2.4.3 Euthanasia!!!!!!!!!!!!!!!!!!!!!!!..107 2.5 Bibliography!!!!!!!!!!!!!!!!!!!!!!!!!!....108 CHAPTER 3: Impaired function of prejunctional adenosine A1 receptors expressed by perivascular sympathetic nerves in DOCA-salt hypertension!!111 3.1 3.2 3.3 3.4 3.5 3.6 3.7 Abstract!!!!!!!!!!!!!!!!!!!!!!!!!!!..112 Introduction!!!!!!!!!!!!!!!!!!!!!!!!!!113 Materials and Methods!!!!!!!!!!!!!!!!!!!!!116 Results!!!!!!!!!!!!!!!!!!!!!!!!!!!...122 Discussion!!!!!!!!!!!!!!!!!!!!!!!!!!.146 Perspective!!!!!!!!!!!!!!!!!!!!!!!!!!154 Bibliography!!!!!!!!!!!!!!!!!!!!!!!!!..156 CHAPTER 4: DOCA-salt hypertension failed to enhance sympathetic NE clearance through A2A adenosine receptor activation!!!!!!!!!!!!!!!!...160 4.1 ! Abstract!!!!!!!!!!!!!!!!!!!!!!!!!!!..163 "##! 4.2 4.3 4.4 4.5 4.6 Introduction!!!!!!!!!!!!!!!!!!!!!!!!!!157 Materials and methods!!!!!!!!!!!!!!!!!!!!!165 Results!!!!!!!!!!!!!!!!!!!!!!!!!!!...171 Discussion!!!!!!!!!!!!!!!!!!!!!!!!!!.184 Bibliography!!!!!!!!!!!!!!!!!!!!!!!!!..190 CHAPTER 5: General discussion, perspectives, and future directions!!!!..195 5.1 5.2 5.3 5.4 5.5 ! Result summary!!!!!!!!!!!!!!!!!!!!!!!!196 General discussion!!!!!!!!!!!!!!!!!!!!!!..199 Therapeutic significance by modulation of perivascular sympathetic transmission!!!!!!!!!!!!!!!!!!!!!!!!!..205 Future directions!!!!!!!!!!!!!!!!!!!!!!!...207 Bibliography!!!!!!!!!!!!!!!!!!!!!!!!!..211 "###! LIST OF TABLES Table 1 Summary of differences between somatic and autonomic nervous system!....5 Table 2 Agonists and antagonists for "-adrenoceptors!!!!!!!!!!!!!!25 Table 3 JNC7 Blood Pressure Classification!!!!!!!!!!!!!!!!!!.59 Table 4 Agonists and antagonists for rat adenosine receptors!!!!!!!!!!...93 Table 5 Sources of primary and secondary antibodies and working dilutions for adenosine receptor staining!!!!!!!!!!!!!!!!!!!!!.120 Table 6 Analysis of concentration response curves for the effects of the A1R agonist, CPA, on norepinephrine oxidation currents recorded from the surface of mesenteric arteries and veins from sham and DOCA-salt rats!!!!!!..143 ! "#! LIST OF FIGURES Figure 1 Shematic illustration of the organization of the nervous system!!!!!!...4 Figure 2 Anatomical features of sympathetic and parasympathetic divisions of the autonomic nervous system!!!!!!!!!!!!!!!!!!!!!!.7 Figure 3 Differential attributions of peripheral blood volume, blood pressure, and vascular resistance in arteries, arterioles, capillaries, venules, and veins!...14 Figure 4 Volume-pressure relationships of arterial and venous compartments!!!..15 Figure 5 Schematic depiction of preganglionic sympathetic nerve transmission onto the postganglionic sympathetic nerve!!!!!!!!!!!!!!!!!!.17 Figure 6 Biosynthesis process of catecholamines!!!!!!!!!!!!!!!..21 Figure 7 Summary depiction of adrenergic neurotransmission for vasculature!!!.22 Figure 8 Schematic sketch of NE reuptake and metabolism!!!!!!!!!!.. 27 Figure 9 Schematic depiction of purinergic receptor structures!!!!!!!!!!30 Figure 10 Contribution of pannexin1 in neurogenic arterial contraction!!!!!!..36 Figure 11 Proposed model of pannexin1-induced purine release in mesenteric artery and vein!!!!!!!!!!!!!!!!!!!!!!!!!!!!!37 Figure 12 Purine chemical structures!!!!!!!!!!!!!!!!!!!!!40 Figure 13 Schematic representation of NE and ATP cotransmission for perivascular sympathetic nerve terminal!!!!!!!!!!!!!!!!!!!!!42 Figure 14 Neuronal ATP release and its degradation by guinea-pig vas deferens!!43 Figure 15 Summary diagram of adenosine receptor subtype signaling pathways!!.45 Figure 16 Differential sympathetic nerve distribution of arteries and veins!!!!!.56 Figure 17 Diagram illustrating lateral regulation for NE release and clearance of periarterial sympathetic nerves!!!!!!!!!!!!!!!!!!!.59 Figure 18 Ischemic heart disease (IHD) mortality rate in each decade of age vs usual systolic!!!!!!!!!!!!!!!!!!!!!!!!!!!!!..60 ! "! Figure 19 Stroke mortality rate in each decade of age vs usual systolic (A) and diastolic (B) blood pressure!!!!!!!!!!!!!!!!!!!!!!!!!61 Figure 20 Catecholamine structures!!!!!!!!!!!!!!!!!!!!!!69 Figure 21 Overall goals!!!!!!!!!!!!!!!!!!!!!!!!!!!.86 Figure 22 Redox reaction of norepinephrine!!!!!!!!!!!!!!!!!!..94 Figure 23 Recording chamber for amperometry!!!!!!!!!!!!!!!!!95 Figure 24 Carbon fiber microelectrode!!!!!!!!!!!!!!!!!!!!!97 Figure 25 Schematic diagram of the tip of a carbon-fiber microelectrode!!!!!!.98 Figure 26 Instrumental setup for amperometric NE detection!!!!!!!!!!..100 Figure 27 Amperometric detection of NE release by a mesenteric vein!!!!!!.101 Figure 28 Localization of A1Rs on periarterial and perivenous sympathetic nerves of sham and DOCA hypertensive rats !!!!!!!!!!!!!!!!!123 Figure 29 Localization of A2ARs on periarterial and perivenous sympathetic nerves of sham and DOCA hypertensive rats!!!!!!!!!!!!!!!!!.125 Figure 30 A2BRs on periarterial and perivenous sympathetic nerves from sham and DOCA hypertensive rats!!!!!!!!!!!!!!!!!!!!!!127 Figure 31 A3Rs does not express on periarterial and perivenous sympathetic nerves129 Figure 32 Representative traces of norepinephrine (NE) and adenosine inducedconstrictions of sham mesenteric arteries and veins!!!!!!!!!!131 Figure 33 Adenosine and CPA concentration-response curves (CRCs) for inhibition of neurogenic constrictions of sham, but not DOCA-salt mesenteric arteries..133 Figure 34 CPA and adenosine do not inhibit constrictions caused by phenylephrine ("1adrenergic receptor agonist) and ",#-MeATP (P2X receptor agonist)!!..135 Figure 35 Representative traces of CPA-mediated inhibition of NE oxidation currents and constrictions in sham MA!!!!!!!!!!!!!!!!!!!..137 Figure 36 CPA, but not CGS21680 inhibit norepinephrine (NE) oxidation currents in sham mesenteric arteries and veins!!!!!!!!!!!!!!!!..139 ! "#! Figure 37 Impaired A1R function in DOCA-salt mesenteric arteries but not veins!!141 Figure 38 Role of endogenous adenosine in adrenergic transmission of sham and DOCA-salt rats!!!!!!!!!!!!!!!!!!!!!!!!!!145 Figure 39 Localization of A2A adenosine receptors (A2ARs) on sympathetic nerves supplying mesenteric artery and vein of sham rats!!!!!!!!!!..172 Figure 40 CGS21680 enhanced NE clearance in mesenteric vein, but not artery of normotensive rats!!!!!!!!!!!!!!!!!!!!!!!!...174 Figure 41 NE transporters was involved in A2AR-enhanced NE clearance in sham mesenteric veins!!!!!!!!!!!!!!!!!!!!!!!!!.177 Figure 42 CGS21680 effects were antagonized by SCH58261!!!!!!!!!...178 Figure 43 CGS21680 revealed impaired prejunctional A2AR function of DOCA-salt mesenteric veins!!!!!!!!!!!!!!!!!!!!!!!!....181 Figure 44 Forskolin restored NE clearance mechanism of DOCA-salt mesenteric veins!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!.183 ! "##! LIST OF ABBREVIATIONS A1R A1 adenosine receptors A2AR A2A adenosine receptors A2BR A2B adenosine receptors A3R A3 adenosine receptors ACh Acetylcholine ANS Autonomic nervous system ATP Adenosine triphosphate AVP Arginine vasopressin Ca 2+ Calcium ion cAMP Cyclic adenosine monophosphate CGS21680 2-p-(2-Carboxyethyl)phenethylamino-5!-Nethylcarboxamidoadenosine hydrochloride hydrate CNS Central nervous system COMT Catechol-O-methyltransferase CPA N -cyclopentyl-adenosine DA Dopamine DAG Diacylglycerol DBP Diastolic blood pressure DMI Desipramine DOCA Deoxycorticosterone acetate ! 6 "###! DOPA Dehydroxyphenylalanine ENS Enteric nervous system GPCR G protein coupled receptor IMG Inferior mesenteric ganglia IP3 Inositol 1,4,5-triphosphate JNC7 Seventh report of the joint national committee on prevention, detection, evaluation, and treatment of high blood pressure KATP channel ATP-sensitive potassium channel MA Mesenteric artery mAChR Muscarinic acetylcholine receptor MAO Monoamine oxidase MCFP Mean circulatory filling pressure mmHg Millimeter mercury MA Mesenteric arteries MV Mesenteric vein Na + Sodium ion nAChR Nicotinic acetylcholine receptor NaCl Sodium chloride NE Norepinephrine NPY Neuropeptide Y OVLT Organum vasculosum lamina terminalis PLC Phospholipase C ! "#$! PNS Peripheral nervous system R Resistance SBP Systolic blood pressure SMG Superior mesenteric ganglia TH Tyrosine hydroxylase TPR Total peripheral resistance UK14304 5-Bromo-N-(2-imidazolin-2-yl)-6-quinoxalinamine,5-bromoN-(4,5-dihydro-1H-imidazol-2-yl)-6-quinoxalinamine, brimonidine VMAT Vesicular monoamine transporter !1-AR Alpha1-adrenergic receptor !2-AR Alpha2-adrenergic receptor "-NAD Beta-nicotinamide adenine phosphate ! "#! CHAPTER 1 GENERAL INTRODUCTION ! "! 1.1 Overview Adenosine is an ATP precursor and metabolite that is ubiquitous and that produces a wide variety of physiological responses (Olah et al., 2000; Tabrizchi et al., 2001). In the cardiovascular system, adenosine plays an important role in controlling cardiac and vascular function (Srinivas et al., 1998; Tabrizchi et al., 2001). Considerable progress has been made in understanding adenosine receptor subtypes and their signaling mechanisms in the cardiovascular system. However, due to the breadth effects of adenosine, our understanding about adenosine receptor function in the sympathetic nerves supplying arteries and veins is less well developed, particularly in hypertension. This issue is important in MA and MV because they are densely innervated by sympathetic nerves (King et al., 2007). Sympathetic nervous system control of the mesenteric circulation contributes significantly to blood pressure regulation by two mechanisms. Firstly, sympathetic nerves contribute to regulation of vascular resistance primarily in small MA (Fenger-Gron et al., 1995; Kreulen, 2003; Rothe, 1983). Secondly, sympathetic nerves regulate capacitance of MV, which provide about 60-70% of venous return to the heart (Kreulen, 2003; Martin et al., 1998; Pang, 2001). Understanding the contribution of adenosine and its receptors to control of the mesenteric circulation will help to understand the differential contributions of veins and arteries to blood pressure control and hypertension. This may also identify new drug targets for hypertension treatment. ! "! 1.2 The autonomic nervous system Organization of the nervous system is illustrated in Figure 1. The nervous system is divided into the central nervous system (CNS) and peripheral nervous system (PNS). The CNS is composed of the brain and spinal cord. The PNS is further divided into the somatic and autonomic nervous systems (ANS). Somatic nervous system is divided into the sensory and motor systems, which are responsible for sensation and body movement respectively. Nearly all visceral reflexes are mediated by the autonomic nervous systems. The differences between the somatic and autonomic nervous systems are summarized in Table 1. The ANS is divided into three divisions: i) the sympathetic nervous system, ii) the parasympathetic nervous system, and iii) the enteric nervous system (ENS). The ENS resides within the gastrointestinal tract and it is a selfcontained system as the disruption of connections between the CNS and the ENS does not alter substantially gastrointestinal function. The ENS is also supplied by sympathetic and parasympathetic nerves. Esophageal and stomach function are more dependent on sympathetic and parasympathetic innervation than the remaining portions of the gastrointestinal tract. ! "! Figure 1. Shematic illustration of the organization of the nervous system. ! "! Table 1. Summary of differences between somatic and autonomic nervous system. Somatic Nervous System Autonomic System Nervous The number of neurons in Single neuron from CNS to Two-neuron chain from efferent path effector organs CNS to effector organs Axon features Heavily myelinated axon Preganglionic neuron: Lightly myelinated axon Postganglionic neuron: Unmyelinated axon Neurotransmitter/receptor ACh/nAChR at neuron-target synapse ACh/mAChR (Parasym) NE/ ! or "-adrenoceptors (Sym) Target tissue Skeleton muscle Smooth muscle Cardiac muscle Glands Site of neurotransmitter Axon terminal release Effects on target tissue Excitatory only: contraction Varicosities terminals and axon muscle Excitatory or inhibitory Peripheral components Axon only found outside the CNS Preganglionic axons, ganglia, postganglionic neurons Summary of function Visceral functions (internal organ movement, and secretion, metabolism control) ! Posture, movement, and sensation "! Figure 2 illustrates the anatomical features of the efferent paths of the sympathetic and parasympathetic nervous systems. The sympathetic neurotransmission consisted of a preganglionic cholinergic fiber, and a postganglionic adrenergic fiber. The preganglionic sympathetic axon leaves the spinal cord at thoracic and lumbar (l1-l2) level to innervate the cell body of the postganglionic sympathetic nerves at the peripheral ganglia of the sympathetic chains, which are alongside of the spinal cord. The postganglionic fibers from lower thoracic and lumbar regions pass through the sympathetic chain ganglia and form synapses onto the postganglionic neurons within the prevertebral glanglia including celiac ganglion, superior and inferior mesenteric ganglia. One preganglionic fiber could form synapse to numerous postganglionic neurons with approximately 1:10 ratio allowing well-established coordination of the sympathetic tone from different spinal levels. The postganglionic sympathetic axons (unmyelinated) leave the ganglia and projects onto their particular effector organs. The parasympathetic preganglionic neurons reside within the brain stem and sacral spinal cord (Fig. 2). Unlike sympathetic chain ganglia that are close to the spinal cord, the parasympathetic ganglia lie close to or even reside in the effector target organs. Thus, the parasympathetic preganglionic axons are longer than that of sympathetic nervous system. The sympathetic and parasympathetic preganglionic axons release acetylcholine (ACh) onto the nicotinic-ACh receptor (nAChR) located on the postganglionic neurons. Once the postganglionic nerve is activated, it releases transmitter into effector organ to regulate the organ function. ACh is the major transmitter released from postganglionic parasympathetic nerves. Postganglionic ! "! sympathetic nerve releases a variety of neurotransmitter including NE, ATP/ !-NAD and NPY onto the postjunctional receptors to regulate the activity of that particular target organ. The proportion of NE, ATP, !-NAD and NPY varies in different sympathetic varicosities. The sympathetic and parasympathetic nervous systems are tonically active exerting peripheral regulation under central control to maintain homeostasis including blood pressure, heart rate and water intake in response to environmental changes (Kandel et al., 2000). Figure 2 Anatomical features of sympathetic and parasympathetic divisions of the autonomic nervous system. ISN, Inferior salivatory nucleus; DMNV, Dorsal motor nucleus of vagus; gl, gland; NA, Nucleus ambiguus; SSN, Superior salivatory nucleus. For interpretation of the references to color in this and all other figures, the reader is referred to the electronic version of this dissertation. (Barrett et al., 2012). ! "! Figure 2. (Cont’d) ! "! 1.3 Autonomic control of blood pressure Arterial pressure drives blood flow through the entire systemic circulation. Arterial blood pressure is determined by cardiac output and total peripheral resistance (Martin et al., 1998). Sympathetic nerve activation enhances heart rate and cardiac contractility leading to an increase in cardiac output and thereby increased arterial blood pressure. Conversely, parasympathetic nerves slow the heart and slightly affect peripheral resistance through release of the vasodilator nitric oxide. Parasympathetic nerves do not provide direct innervation to the vasculature. Under resting conditions, systemic arterioles are partially constricted by tonic sympathetic firing activity. Decreased sympathetic output leads to vasodilation (Mohrman et al., 2010b). Besides, small arteries and arterioles primarily contribute to peripheral resistance. Thus, blood flow through an organ is regulated by adjustments in internal diameter of small arteries and arterioles. The sympathetic nervous system regulates vascular resistance and vascular capacitance by chemical synapse formation, which is described later in details in sympathetic neurotransmission section. 1.3.1 Sympathetic control of arterial resistance Sympathetic nerves supply the adventitia of the arteries (Ekblad et al., 1984). The unmyelinated periarterial postganglionic sympathetic nerve varicosities release NE and ATP/!-NAD that acts on the "1-adrenergic and purinergic P2X receptors, respectively, on the arterial smooth muscle cells to mediate vascular contraction. Decreased arterial diameter enhances total peripheral resistance, hence decreases blood flow. Total peripheral resistance is a major contributor to arterial pressure. As the ! "! systemic organs are arranged in parallel and the vessels in individual organ are connected in series, total peripheral resistance could be determined by this equation: Where, TPR is total peripheral resistance, and R is resistance of each vascular bed as indicated. The resistance of small arteries and arterioles is the key determinants for total peripheral resistance as the capillary and venous resistance is small in comparison to arterial and arteriolar resistance (Fig. 3). 1.3.2 Sympathetic control of venous capacitance Veins are also very important for blood pressure control as 70% of the total blood volume resides in peripheral veins (Mohrman et al., 2010c). The term “compliance” depicts how much the blood volume changes upon a change in pressure. The volumepressure relationship in arteries is different from the venous compartment (Fig. 4). Veins have a relatively thin muscle layer and they are highly compliant. Therefore, venous pressures are relatively insensitive to normal volume changes. Arteries and arterioles have more muscular walls and are therefore less compliant than veins. This makes arterial pressure very sensitive to volume changes. It is important to note that small increase or decrease in venous compliance cause a significant amount of blood volume to shift to the heart increasing cardiac output and arterial blood volume. ! "#! Changes in cardiac output are transient however, and elevated arterial pressure is due largely to elevated blood volume in the low compliances arterial circulation. Thus, veins do make important contributions to normal regulation of blood pressure. Alterations in the factors that control venous compliance (sympathetic nerve activity for example) can then impact long-term blood pressure control and hypertension development (Fink et al., 2000; Safar et al., 1987; Simon et al., 1975; Simon et al., 1976). Although the veins acquire sympathetic-independent myogenic tone, they also receive considerable sympathetic innervation to regulate venous capacitance. Sympathetic nerves distribute deeply into the border of the adventitia and media of the veins. Sympathetic nerves release NE onto venous smooth muscle to mediate venous contraction through activation of !1-adrenergic receptors. The veins are more sensitive to neurogenic-(Nilsson et al., 1985) ,and NE-induced contraction than the arteries (Perez-Rivera et al., 2003). Elevated sympathetic nerve activity associated with decreased peripheral venous capacitance has been established in animal models and hypertensive subjects. The importance of this phenomenon is that when peripheral veins contract, blood is shifted from peripheral venous pool and enters the central pool (Vena Cava). This increased central venous volume tends to increase cardiac filling, and mean circulatory filling pressure (MCFP) (Fink et al., 2000; Perez-Rivera et al., 2003); hence, increased stroke volume and cardiac output (Perez-Rivera et al., 2003) via Starling’s law of the heart. This is extremely importance as increased cardiac output raises arterial blood pressure. ! ""! 1.3.3 Role of sympathetic supply on splanchnic circulation As the splanchnic circulation receives high blood flow and contains approximately one third of total blood volume, splanchnic vascular adjustment importantly affects overall cardiovascular hemodynamics. The sympathetic nervous system plays an important role in controlling splanchnic vascular tone and the postganglionic sympathetic nerves densely innervate mesenteric arteries and veins. Maximal neurogenic vasoconstriction of splanchnic vasculature leads up to 80% reduction in blood flow through splanchnic region and causes a huge blood mobilization from peripheral mesenteric veins to the central vena cava. The mesenteric arteries and veins are the target of the present study as they imitate the most important peripheral resistance and capacitance vessels respectively. 1.3.4 Long-term regulation of blood pressure There are a number of factors involved in long-term pressure regulation. Longterm pressure regulation is associated with blood volume control, and sodium handling, which affects plasma osmolality. Changes in mean arterial pressure can be accomplished by alteration of cardiac output and/or total peripheral resistance. Increased blood volume augments activity of sympathetic nerves supplying the heart, but not the kidney or lumbar regions. Increased plasma osmolality also influences longterm arterial pressure. Elevated osmolality by high-sodium intake is relevant to saltsensitive hypertension. A small increase in plasma osmolality induced by high-salt intake enhances muscle sympathetic nerve activity and plasma NE, as osmoreceptors in hypothalamus (organum vasculosum of the lamina terminalis (OVLT), supraoptic ! "#! nuclei, and median preoptic nuclei), which are highly sensitive to plasma osmolality alterations. The supraoptic nuclei also synthesize vasopressin, which is then transported to posterior pituitary gland. The significance of vasopressin is described later in DOCA-salt rat model of hypertension. Moreover, high-salt intake is positively + correlated with plasma Na levels and sympathetic nerve activity, which may contribute to increased regional sympathetic activity in humans (Farquhar et al., 2006) and animal models of salt sensitive hypertension (O’Donaughy et al., 2006). It could be argued that increased plasma osmolality may not be sustained under normal physiological conditions because high plasma osmolality activates the thirst reflex causing increased fluid intake to restore osmolality. However, increased plasma osmolality may be a major importance under pathologic condition including salt-sensitive hypertension. ! "#! Figure 3. Differential attributions of peripheral blood volume, blood pressure, and vascular resistance in arteries, arterioles, capillaries, venules, and veins (Mohrman et al., 2010a) ! "#! Figure 4. Volume-pressure relationships of arterial and venous compartments. !P, Pressure change; !V, volume change (Mohrman et al., 2010a) ! "#! 1.4 Sympathetic neurotransmission 1.4.1 Sympathetic preganglion neurotransmission The axon terminal of the preganglionic sympathetic nerve contains acetylcholine (ACh). ACh is synthesized from acetyl-Coenzyme A (acetyl-CoA) and choline with enzyme choline acetyltransferase as the catalyst. The substrate choline is transported + from extracellular fluid into the nerve terminal through the Na -dependent choline transporter. ACh is synthesized in the cytosol and is transported into the synaptic vesicles by the proton-driven vesicular-associated transporters (VAT). Once an action potential arrives, the voltage-gated Ca2+ channels are activated causing Ca2+ influx and exocytosis. Once released, ACh acts on the nicotinic cholinoceptors located on the cell body of postganglionic sympathetic nerves. One preganglionic neuron activates numerous of postganglionic neurons. The action of released ACh is shortly terminated by enzyme acetylcholinesterase into choline and acetate. Choline transporters capture choline back into the nerve terminal for further ACh synthesis (Fig. 5). Postganglionic sympathetic neurotransmission is described below. ! "#! Figure 5. Schematic depiction of preganglionic sympathetic nerve transmission onto the postganglionic sympathetic nerve. AcCoA: Acetyl-CoA, ACh: Acetylcholine, ChAT: Choline acetyltransferase, CHT: Choline transporter, VAT: Vesicle-associated transporter (Katzung, 2012). ! "#! 1.4.2 Sympathetic postganglion neurotransmission in focus on vascular system Postganglionic sympathetic nerves densely innervate the vascular system to regulate vascular tone and maintain blood pressure. Postganglionic sympathetic axons are nonmyelinated with 0.25-0.5 µm in diameter and surrounded by Schwann cell sheaths. Each axon contains periodic swellings known as “varicosities”, which have 0.52 µm in diameter every 3-5µm interval. The varicose regions are Schwann-free with fenestration. Varicosities contain a large number of synaptic vesicles, which is believed to be the sites for transmitter storage. Perivascular sympathetic varicosities make very close contact with smooth muscle cells. The junction between varicosities and the smooth muscle cells is ranged between 0.1-10 µm. A nerve impulse depolarizes a series of varicosities and causes the release of transmitters (Brock et al., 1993; Stjarne, 1989). There are three major transmitters that are costored and coreleased from periarterial sympathetic varicosities: NE, ATP, and NPY (Lamont et al., 2006). In certain specific vasculatures, sympathetic nerve could release other transmitters. For instance, dopamine is released from sympathetic nerves supplying renal and hepatic blood vessels. Epinephrine is released from sympathetic nerves innervating amphibian heart. ACh is released from the nerve supplying small arteries in cat skeleton muscle (Tsuru et al., 2002). Additionally, under continuous nerve stimulation, where enzyme dopamine !hydroxylase become saturated, it causes accumulation of newly synthesized dopamine available for release from sympathetic nerves (Soares-da-Silva, 1988). Release of sympathetic transmitters is triggered by a transient influx of Ca 2+ varicosity via voltage-sensitive Ca ! 2+ ion into nerve channel. Conversely, an increase in intra-varicosity "#! 2+ Ca concentration in from intraneuronal stores is likely to be ineffective for triggering transmitter release (Burgoyne et al., 1995). Among these postganglionic sympathetic transmitters, NE is the principal neurotransmitter released upon sympathetic nerve stimulation. 1.4.3 Adrenergic neurotransmission Synthesis of NE: The synthesis of NE is highly regulated and several enzymes are involved with this process (Fig. 6). NE and epinephrine are synthesized from tyrosine. Tyrosine is actively transported into the postganglionic sympathetic varicosity (Fig. 7). Tyrosine hydroxylase (TH) is the rate-limiting enzyme that catalyzes tyrosine hydroxylation to form dehydroxyphenylalanine (DOPA) using O2 and the cofactor tetrahydrobioterin (BH4). DOPA is decarboxylated by aromatic L-amino acid decarboxylase (DOPA decarboxylase) to produce dopamine. Dopamine is actively transported into synaptic vesicles via a vesicular monoamine transporter 2 (VMAT2). In the vesicle, NE is synthesized from dopamine by the membrane-associated enzyme dopamine !-hydroxylase. Most synthesized NE is leak from synaptic vesicular storage into the cytoplasm, but 90% of NE leakage is re-captured into synaptic vesicles by VMAT2. The other 10% of NE escapes the VMAT2 sequestration mechanism and is enzymatically degraded (Eisenhofer et al., 2004) (see removal, turnover, and metabolism of NE). Additionally, in adrenal medulla approximately 85% of NE is converted to epinephrine (E) by enzyme phenylethanolamine-N-methyltransferase using S-adenosylmethionine as a methyl donor. NE production varies with neuronal activity. In 1912 Elliot was first observed that long train electrical stimulation of splanchnic nerves ! "#! did not cause NE depletion of adrenal NE content. Later in 1950s, Holland and Schumann proposed that nerve stimulation caused compensatory increases in TH activity and catecholamine production (Zigmond et al., 1989). Generally, NE, epinephrine, dopamine, and DOPA (termed catechols) negatively regulate TH activity to stop further NE synthesis by reversibly competing with BH4 binding to the catalytic site of TH (Dunkley et al., 2004). However, numerous protein kinases and phosphatases modulate activity of TH. Phosphorylation augments enzyme affinity for the BH4 cofactor, hence increased TH hydroxylation rate (Dunkley et al., 2004) and diminishes the catechol negative feedback. It appears that TH activity is acutely increased by phosphorylation in response to elevated cAMP level and cellular depolarization (Zigmond et al., 1989). ! "#! Figure 6. Biosynthesis process of catecholamines (Katzung, 2012) ! "#! Figure 7. Summary depiction of adrenergic neurotransmission for vasculature. a) The essential amino acid tyrosine is captured into sympathetic axon for NE synthesis. To form NE, tyrosine is converted into dihydroxyphenylalanine (Dopa), and dopamine by tyrosine hydroxylase, and, aromatic amino acid decarboxylase respectively. b) Dopamine is next actively transported into a synaptic vesicle, where contains dopamine !-hydroxylase, and converted into NE and stored in the vesicle. c) Upon arrival of an action potential, vesicular exocytosis and NE release occurs. NE activates the postsynaptic receptors ("1, !1, !2) (d) leading to vascular contraction (f). e) NE also acts on the prejunctional receptors ("2, !2) to inhibit NE release. g) To terminate NE action, NE is removed from the neuroeffector junction by NET-mediated NE reuptake. h) After NE reentered into the axon terminal, NE is captured back into the vesicle via vesicular monoamine transporter 2 (VMAT2). i) Some NE escapes from VMAT reuptake and undergoes enzymatic metabolism by monoamine oxidase (MAO) within the nerve axon. j) degradation product dihydroxyphenylglycol is released into circulation. k) Partially, NE is uptake into postjunctional cell (uptake2) and degraded by catechol-O-methyl transferase into normetanephrine (Zhou, 2004). ! ! ""! Vesicular Storage of NE: In sympathetic axons, NE is stored in two types of synaptic vesicles: large, dense-core vesicles (Schwarzenbrunner et al., 1990)(~ 75-200 nm in diameter)(Lagercrantz, 1976) and small, dense-core vesicles < 50 nm diameter (Fried et al., 1985). In addition to NE, the large dense-core vesicles also contain a variety of neuropeptides, and ATP (Lagercrantz, 1976). It appears that different frequencies of sympathetic firing evoke different mixtures of neurotransmitter release. NE and ATP are release at low-frequency nerve stimulation, while the release of the other cotransmitter NPY peptide often occurs during period higher frequency of stimulation (Kennedy et al., 1997). Furthermore, the transmitter release under high 2+ frequency of sympathetic nerve firing appears to be Ca independent (Malpas, 2010). High frequency stimulation-induced multiple transmitter release causing activation of 2+ various receptors and second messengers. This results in a recruitment of Ca independent kinases and phosphorylation of transcription factors to modulate 2+ transmitter synthesis. Besides, there are other targets for Ca -independent phosphorylation including cytosolic and vesicular proteins responsible for vesicle docking and fusion (Wakade et al., 1995). In the adrenal medulla, NE and epinephrine are stored in chromaffin cells. Upon sympathetic nerve stimulation, chromaffin cells release ~80% epinephrine and 20% NE directly into the bloodstream. Release of NE: Once an action potential arrives at the presynaptic nerve 2+ terminal, the voltage-gated Ca channels located on the active zone change their 2+ conformation to the opening stage permitting Ca ! "#! 2+ influx. High concentration of Ca in active zone induces the fusion of synaptic vesicle membrane and the presynaptic neural membrane, hence exocytosis occurs. Impulse frequency could modulate the transmitter release. The docked small vesicles in active zones is more likely to exocytose during low frequency stimulation, while the high frequency burst causes the release of large vesicle from random sites. Postjunctional action of NE at specific receptors: Once released, NE and cotransmitters diffuse across the neuroeffector junction and bind to their specific target receptors on the membrane of vascular smooth muscle (Fig. 7). NE activates the postjunctional !1-adrenergic receptors which couple to stimulation of the Gq/PLC/IP3 2+ 2+ pathway and Ca -induced Ca release from endoplasmic reticulum stores to mediate smooth muscle contraction. Furthermore, activation of the postjunctional !1-adrenergic receptors by NE also opens membrane pannexin-1 channels causing cellular ATP release to contribute to vascular contraction (see detail below). Prolonged exposure to NE causes desensitization in mesenteric veins, but not arteries. In addition to the postjunctional receptors, NE also acts on the prejunctional !2-adrenergic receptors providing negative feedback to the sympathetic nerve terminal to stop further transmitter release. Agonists and antagonists for adrenergic receptors are list in table 2. ! "#! Table 2. Agonists and antagonist for !-adrenoceptors !1-adrenoceptor !2-adrenoceptor Location Postjunctional site Prejunctional site Pharmacological action Mediate Inhibit transmitter release vasoconstriction Opens pannexin1 channel Agonist NE (non-selective) NE (non-selective) Phenylephrine Clonidine UK14304 Antagonist Prazosin Idazoxan Yohimbine Clearance and metabolism of NE: The action of NE is terminated mostly by the NE transporter (NET) reuptake (Fig. 7). Approximately 70% of released NE is recaptured back into the varicosities, and the remaining 30% is lost to extraneuronal transport and enzymatic metabolism (Eisenhofer et al., 2004). Figure.8 shows two distinct NE reuptake systems: uptake1 and uptake2. Uptake1 transporters consist of 12transmembrane domains and the reuptake mechanism is Na + - and Cl dependent. Uptake1 transporters have high affinity, but low capacity to NE and are predominantly located on the nerve terminal. Uptake1 is the main mechanism responsible for remove NE from the neuroeffector junction and their action is blocked by cocaine or tricyclic antidepressant desipramine (DMI). Conversely, uptake2 transporters are considered as ! "#! non-neuronal transporters because they are located on the postjunctional site (vascular + - smooth muscle cells, for example). Uptake2 transporters are Na -and Cl -independent and they have low affinity, but high capacity to NE. Besides, cocaine, and tricyclic antidepressants could not block the uptake2 system. Uptake 2 transporter could be blocked by the O-methylated metabolites of catecholamines (normethanephrine (NMN), and metanephrine) produced by COMT enzymes, and steroids (Eisenhofer, 2001). Once the uptake1 transporters transport NE into the nerve terminal, NE is mostly recaptured back into vesicles via vesicular monoamine transporter2 (VMAT2) and is available for another round of exocytosis. Sympathetic nerve terminal mitochondria contain monoamine oxidase (MAO), which metabolizes most NE not recaptured into synaptic vesicles. This intraneuronal metabolism pathway is also responsible for degradation of NE that leaks from synaptic vesicles. Some NE undergoes extraneuronal metabolism within the neuroeffector junction by catechol-O-methyltransferase (COMT) (Fig. 8). ! "#! Figure 8. Schematic sketch of NE reuptake and metabolism. Once an action potential arrives, the nerve releases NE onto the receptors on target cell. Then, NE is reuptake back into the nerve terminal via the uptake1 transporter, and is next reentered vesicular store via vesicular monoamine transporter2 (VMAT2). Escaped NE from transporter sequestration is metabolized by intraneuronal monoamine oxidase (MAO) and extraneuronal catechol-O-methyltransferase (COMT) (Eisenhofer, 2001). ! "#! 1.4.4 Neuropeptide Y (NPY) neurotransmission NPY is a small biologically active peptide (36 amino acids) found abundantly in the central and autonomic nervous systems (Reid, 2012). NPY is involved with regulation of numerous physiological processes, including thirst, appetite, blood pressure control, and energy metabolism. There are five subtypes of NPY receptors (Y1-Y5). All of them, except Y3, are GPCRs. Two subtypes of NPY receptors are important in the cardiovascular system: Y1 and Y2 receptors. In sympathetic neurotransmission, Y1 receptors are primarily located on the postjunctional site to mediate vascular contraction and potentiated NE-induced vasoconstriction. Y2 receptors express in both pre- and postjunctional sites. The prejunctional Y2 receptors couple to inhibition of NE release. NPY is synthesized in the soma, packed into vesicles, and transported to nerve varicosities (Stjarne, 1989). NPY and NE are co-stored in large, dense core vesicles in sympathetic nerves supplying the rat vas deferens (Fried et al., 1985). NPY is co-released with NE and ATP from sympathetic nerve endings and NPY is a potent vasoconstrictor in many vascular beds (Cowley-Jr et al., 2008), including the mesenteric circulation of the rat (Westfall, et al., ). The constrictor effects of NPY are mediated by Y1 receptors as demonstrated in the guinea pig ear artery (Morris, 1999). Additionally, NPY potentiates the constrictor effects of NE and ATP via a postjunctional action in rat vas deferens, mesenteric artery (Donoso et al., 1997a; Westfall et al., 1995), and rabbit femoral arteries (Ekblad et al., 1984). NPY is slowly cleared from the neuroeffector junction compared to other small molecule transmitters. This slow removal of NPY contributes to long duration of its effects. NPY probably removed by diffusion ! "#! and proteolysis by extracellular dipeptidyl peptidases 4 (DP4). DP4 is transmembrane glycoprotein consisted of 766 amino acid, which hydrolyzes post-proline bond of the Nterminus of NPY. 1.4.5 Purinergic neurotransmission In sympathetic neurotransmission, ATP and its breakdown products act at preand postjunctional purinergic receptors. Purines refer to as adenosine, ADP and ATP as they contain adenine base; pyrimidines are UDP and UTP (Ralevic et al., 1998). Both purines and pyrimidines mediate biological effect through purinergic receptors. Purinergic receptors are divided into two families: P1 and P2 receptors (P annotates purinergic or purine). P1 receptors are targets for adenosine, the final ATP degradation product. P1 receptors are metabotropic G-protein coupled receptors (Fig. 9). Adenosine receptors are further subdivided into A1, A2A, A2B, and A3 receptor subtypes. Details of adenosine receptors will be discussed later in adenosine signaling section. P2 receptors are targets for the nucleotides ATP, ADP, UTP, and UDP. There are 2 classes of P2 receptors: P2X receptors (ligand-gated ion channels) and P2Y receptors (G-protein coupled receptors) (Fig. 9). There are 7 P2X receptor subtypes (P2X1-7) and 5 P2Y receptor subtypes (P2Y1, P2Y2, P2Y4, P2Y6, and P2Y11). My work focused on ATP and adenosine as signaling molecules in peripheral sympathetic neurotransmission. ! "#! Figure 9. Schematic depiction of purinergic receptor structures. A) P1 metabotropic receptor (receptor for adenosine) is consisted of seven transmembrane domains. The second extracellular loop linked domain IV and V is critical for agonist/antagonist recognition. There are two subtypes of receptor for ATP: the ionotropic ligand-gated ion channel and metabotropic G protein-coupled receptor comprising two transmembrane domain (B), and the P2Y metabotropic receptor assembling seven transmembrane-spanning domains (C) (Burnstock, 2007). ! "#! 1.4.5.1 ATP neurotransmission The nucleotide adenosine 5’-triphosphate (ATP) is a key energy source in the cells. ATP is synthesized through mitochondrial electron transport chain and transported into the cytosolic compartment for further cellular energy utilization. ATP concentrations in the cytoplasm range between 2-5 milimolar (mM), and much higher in synaptic vesicles (up to 100 mM) (Burnstock, 2007). ATP and its degradation products (adenosine and ADP) are potent extracellular signaling molecules (Ralevic et al., 1998). ATP is a critical co-transmitter in the central and peripheral nervous systems, and the contribution of ATP as an excitatory neurotransmistter varies across tissues. In chromaffin cells from adrenal medulla, ATP is co-released with epinephrine. In sympathetic nerves supplying vas deferens, ATP is an important co-transmitter with NE and NPY. It is revealed that ATP was co-stored with NE in large-dense core vesicles with the ratio of 7:1 in bovine splenic nerves (Lagercrantz, 1976). Likewise, ATP is also released from non-neuronal cells that are important in pain signaling pathways. ATP released from damaged cells triggers the naked endings of peripheral C-fiber through the activation of ionotropic P2X receptors. Also, healthy non-neuronal cells (can you list some examples?) release ATP in response to sheer stress, stretch, osmotic swelling, and hypoxia (Burnstock, 2007). In the cardiovascular system, ATP signaling plays a crucial role during both normal physiological and pathological conditions including hypoxia-induced vasodilation, reactive hyperaemia, and hypertension. Periarterial sympathetic nerves supplying mesenteric artery co-release ATP and NE. ATP acts at ionotropic P2X1 ! "#! ionotrop receptors on arterial smooth muscle cells to cause short latency (a few milliseconds) and short duration (200 milliseconds) excitatory junction potentials (EJPs). EJPs are brief depolarizations of smooth muscle cells that are caused by cation influx through P2X channels (Demel et al., 2008b). EJPs are blocked by guanethidine or bretylium, drugs which also block NE release (Burnstock, 2007). These data support the conclusion that ATP and NE are co-stored in synaptic vesicles. Once the cotransmitter released, P2X activation by released ATP causes conformation change of + the transmembrane channel leading to increased channel permeability to Na and 2+ Ca . Influx of cations causes cellular membrane depolarization and activation of voltage-gated L-type Ca2+ channels. Ca2+ entry through the P2X1 receptor and the Ltype Ca2+ channel trigger and vascular contraction (Gitterman and Evans 2001). Neurogenic arterial contraction is biphasic response including rapid-transient and slow-sustained phases. The rapid phase arterial contraction is caused by the action of neuronally released ATP on the postjunctional inotropic P2X1 receptors. Meanwhile, NE-mediated contraction via the postjunctional !1-adrenergic receptors is slower due to G-protein coupling and the involvement of second messengers providing sustained arterial contraction phase (Fig. 10). In P2X1-null mice, the rapid contraction phase is absent in neurogenic arterial contraction. Furthermore, prazosin (!1-adrenoceptor antagonist) specifically attenuate the sustained contraction phase, without altering the transient rapid phase (Lamont et al., 2006). Additionally, Park and coworkers showed that PPAD (P2X antagonist) blocked arterial neurogenic contraction without affecting ! "#! NE release suggesting that ATP is required for mediating neurogenic arterial contraction. It should be noted that Park et al. performed a brief train of stimulation for 6 seconds, therefore they can only detect change in contraction amplitude, but they could not distinguish the biphasic contraction response. Conversely, Lamont group used long train of stimulation for 1 minute permitting them to detect the changes in biphasic neurogenic contraction response. 1.4.5.2 !1-adrenoceptor-evoked postjunctional ATP release In 1970s Bunstock proposed that ATP is coreleased with NE from postganglionic sympathetic varicosities (Burnstock, 1976) and it was later revealed by Starke and coworkers that the majority of nerve-evoked ATP release is come from non-neuronal site, where 32% of electrically-evoke ATP release is come from nerve varicosities, but the majority of 68% of released ATP is come from non-neuronal site, which could probably be smooth muscle cells. Non-neuronal ATP release by activation of the postjunctional !1-adrenoergic receptors is firstly reported in guinea-pig vas deferens (Kügelgen et al., 1991). The fundamental mechanism of !1-adrenoceptor-induced ATP release is involved with pannexin1 hemichannels (Dubyak, 2009). 1.4.5.3 Pannexin1-facilitated cellular purine release Pannexin is a novel protein family discovered in the past decade. Pannexin is structurally similar to the invertebrate gap junction protein “innexin”, and mammalian gap junction protein “connexin”, but pannexin does not form gap junctions. Pannexin ! ""! contains several extracellular glycosylation sites and it is likely that these glycosylations are the key component that prevents pannexin from forming gap junctions with adjacent cells (MacVicar et al., 2012). However, pannexin does form an unpaired hemichannel with extracellular space (Dubyak, 2009). Under basal physiologic condition, pannexin hemichannel remains in closing state, and changes in extra-or intra-cellular environment could induce pannexin protein into opening state leading to the release of intracellular signaling molecules onto extracellular compartment (Dʼhondt et al., 2009). There are three isoforms of pannexin proteins: pannexin1, pannexin2 and pannexin3. Pannexin1 and 3 form hexameric channels, and pannexin2 forms either heptameric or octameric channel. Pannexin proteins are synthesized and assembled in the ER and Golgi; then, they are transported and anchor into the plasma membrane. Transmembrane pannexin protein serves as a channel for transport of molecules between the intra- and 2+ extracellular compartments. Unlike connexin, pannexin activation is Ca -independent. Recent studies have provided a strong reason to assume that pannexin1 channel has permeability to ATP and contributes to cellular ATP release under physiological and pathological conditions. Pannexin1 protein is expressed on the membrane of vascular smooth muscle cells of middle cerebral artery (Burns et al., 2012), thoracodorsal artery (Billaud et al., 2011) and mesenteric artery (Galligan et al., unpublished data). Activation of the !1-adrenergic receptors located on smooth muscle leads to ATP release through pannexin1 channel and ATP-induced vascular contraction (Fig. 10-11). A panexin1 channel blocker and P2Y receptor antagonist prevented Pannexin-induced ATP release and ATP-induced contraction (Billaud et al., 2011). ! "#! 1.4.5.4 Contribution of pannexin-1 to neurogenic vascular contraction As shown previously that neuronal NE release could stimulate ATP release from vascular smooth muscle cells, the non-neuronal ATP could mediate synergistic arterial contraction along with NE. Postjunctional ATP release could activate neighboring P2X receptors and propagate neurogenic contractile response along the arteries (Fig. 10). However, venous contractile response is different from arterial contraction as perivenous varicosities contain different proportion of vasoactive transmitters. NE is the prominent transmitter to modulate venous contraction, but it is equivocal if ATP is coreleasd with NE from perivenous sympathetic varicosity. Role of pannexin channel in venous contraction response is undefined. Understanding the mechanism of pannexininduced purine release in the vein would clarify the debating issue about source of purine release in perivenous nerves. A further extension question would be about pannexin channel selectivity. Does pannexin channel selectively transport ATP or it provides a general transport for all purines? It has been proposed that purine release is linked to metabolic demands via local blood flow regulation. For instance, ATP is release from skeleton muscle during contraction; adenosine is released in the central nervous system (Dale et al., 2000; Frenguelli et al., 2003) during hypoxia. Does pannexin hemichannel plays a key role in the release of these cellular purines? ! "#! Figure. 10 Contribution of pannexin1 in neurogenic arterial contraction. 1) ATP released from sympathetic varicosity activates P2X inotropic receptor (P2X) on vascular smooth muscle cell (VSMC) mediating rapid arterial contraction. 2-4) Released NE acts on !1-adrenergic receptor (!1) producing sustained arterial contraction by two major mechanisms: !1-induced contraction (2) and !1-stimulated pannexin1 (pan1) causing ATP release onto extracellular space (3). Non-neuronal ATP activates remote P2X receptors on neighboring muscle cell propagating further contraction (4). Biphasic neurogenic contractile response is from Lamont and co workers’ work (Lamont et al., 2006) ! "#! Figure 11. Proposed model of pannexin1-induced purine release in mesenteric artery and vein. A) Periarterial sympathetic nerve release ATP and NE. ATP binds to P2X receptor and NE acts on !1-adrenergic receptor leading to vascular contraction. Activation of !1-adrenergic receptor induced pannexin1 trafficking into plasma membrane. Active pannexin1 transports purines (ATP and adenosine) from inside the cell into the neurovascular junction to further modulate sympathetic neurotransmission and induce vascular contraction. B) Similar phenomenon occurs in sympathetic nerve supplying mesenteric vein. Unlike periarterial nerve, NE is the major neurotransmitter released from perivenous nerve. The source of purine is mainly from smooth muscle cells ! ! ! A ! ! ! ! ! ! ! ! ! ! ! ! ! ! "#! ! Figure 11. (Cont’d) B ! "#! 1.4.5.5 Beta-nicotinamide adenine dinucleotide (!-NAD) neurotransmission !-NAD is two adenine nicotinamide nucleotides bonded together through their phosphate groups (Fig. 12). !-NAD has been recently proposed as an extracellular signaling molecule and to be a novel neurotransmitter. In the gastrointestinal tract, !NAD released by enteric nerves may provide inhibitory regulation of visceral muscle. This hypothesis is supported by data showing that the amount of !-NAD released by enteric nerve stimulation is 30-fold higher than the amount of ATP released from enteric nerves during the same stimulation period (Mutafova-Yambolieva et al., 2007). Recent evidence suggests that sympathetic nerves also release !-NAD along with NE, and ATP (Smyth et al., 2004; Smyth et al., 2006) in canine mesenteric arteries. Similar to sympathetic NE and ATP release, the release of !-NAD is also dependent on the Ntype Ca2+ channel as "-conotoxin GVIA prevents !-NAD release (Smyth et al., 2004). Further studies are needed to determine source(s), underlying mechanisms, and significance for health and disease of !-NAD signaling. ! "#! Figure 12. Purine chemical structures ! Beta-nicotinamide adenine dinucleotide (!-NAD) ! "#! 1.4.5.6 Adenosine neurotransmission Adenosine is a purine nucleoside composed of adenine base and deoxyribose sugar (Fig. 12). Adenosine is produced intracellularly and extracellularly, but by different mechanisms (Latini and Pedata 2001). Intracellularly, adenosine is formed as AMP catabolism byproduct, which could be transported to extracellular space through the membrane via bidirectional nucleoside transporters. In the extracellular space, released nucleotides such as ATP are metabolized to adenosine by ecto-5’-nucleotidase (Cechova et al., 2008). Adenosine is released into extracellular space under physiologic and pathologic conditions. A large amount of adenosine is produced during hypoxia as cellular ATP consumption increases. When sympathetic nerves release ATP into the neuroeffector junction, ATP is rapidly degraded into ADP, AMP, and adenosine by membrane bound or soluble ecto-nucleotidases (Fig. 13). In the guinea-pig vas deferens, ATP released by sympathetic nerves is degraded by releasable nucleotidases (ATPDase and AMPase) as ATP metabolism profile is extremely low in the absence of nerve stimulation. Conversely, there is substantial ATP degradation during electrical nerve stimulation (Fig. 14) (Todorov et al., 1997). ! "#! Figure 13. Schematic representation of NE and ATP cotransmission for perivascular sympathetic nerve terminal. NE and ATP nucleotidases are released from nerve terminal. NE acts on !1-adrenoceptor, and ATP acts on P2X receptor in arteries or P2Y receptor in veins to elicit contraction. ATP undergoes sequentially metabolism to ADP, AMP, and adenosine (Ado) as the final degradation product via the action of releasable and extracellular membrane-bound nucleotidases. Releasable ATPDase exhibiting some similarity to ectonucleoside triphosphate/diphosphohydrolase (eNTPDase) converts ATP to ADP and ADP to AMP. Releasable AMPase exhibits similarity to ecto 5’ nucleotidase (E5’N) in converting AMP to adenosine. Ado and NE act on P1 (A1-adenosine) receptor and !2-adrenoceptor to inhibit further transmitter release. ! "#! Figure 14. Neuronal ATP release and its degradation by guinea-pig vas deferens. To study neuronally released ATP breakdown, 9 consecutive superfusates were collected during nerve stimulations (8 Hz 6s) with 10-s interval. Krbs: control sample of Krebs solution, PS: 10s immediately before nerve stimulation, S1-S6: 6 consecutive nerve stimulation, S7: 10s after nerve stimulation. A) Original HPLC chromogograms for 9 consecutive superfusates with nerve stimulation arranging from front to back B) Converted mean data showing. C-D) Similar experiment to A and B were performed in the absence of nerve stimulation (Todorov et al., 1997). A B C ! D "#! Adenosine receptor subtypes and signal transduction. Adenosine receptors are G protein-coupled receptors. There are four subtypes that are distinguished based on their ability to inhibit or stimulate adenylyl cyclase (AC) activity (Olah et al., 2000) and different agonist and antagonist binding affinity (Alexander et al., 1994; Ongini et al., 1996). The four subtypes of adenosine receptors are the A1, A2A, A2B, and A3 receptors (Fredholm et al., 2001; Klotz, 2000; Linden, 2001; Tabrizchi et al., 2001). Adenosine A1 receptors (A1Rs) couple to the inhibitory G proteins, Gi or Go (Linden, 2001; Ralevic et al., 1998) and receptor activation decreases cAMP due to adenylyl cyclase inhibition (Shen et al., 1995; Tabrizchi et al., 2001). A1Rs also activate phospholipase C (PLC) leading to an increase in inositol 1,4,5-triphosphate (IP3), 2+ diacylglycerol (DAG). IP3 stimulates Ca release from intracellular stores (eg. sarcroplasmic reticulum) (Ralevic et al., 1998) causing contraction in airway smooth + muscle (Abebe et al., 1998). A1R activation also opens K ATP channels in cardiac muscle (Liang, 1996; Liang, 1998), coronary arteries (Davie et al., 1999), and neurons 2+ (Andoh et al., 2006). Activation of A1R in the nervous system inhibits Ca currents resulting in suppression of neurotransmitter release (Fig. 15) (Ralevic et al., 1998; Storr et al., 2002). Adenosine A2ARs couple with Gs (Olah et al., 2000; Ralevic et al., 1998) to stimulate 2+ adenylyl cyclase and Ca channels. A2ARs also activate G(olf) (first identified in olfactory epithelium) in striatum to activate type III adenylyl cyclase (Herve et al., 1993; ! ""! 2+ Linden, 2001) (Fig. 15). A2BRs signal through Gs or Gq, which produce Ca mobilization and MAPK activation (Fig. 15). Activation of A3Rs signal through Gi and Gq proteins, which activate phospholipase C/D resulting in phosphorylation and desensitization of A1Rs (Linden, 2001; Tabrizchi et al., 2001). Figure 15. Summary diagram of adenosine receptor subtype signaling pathways (Klotz, 2007) !" ##### #!$ # '()*# ',# +)""# ,# "#!1# "# 3!45# ! !# /0# #!%!# '+)""# -.# ()*# #!%& # !#561# !# 12%0# " 12%0# "#! !#!1# !# 3!45# Adenosine in the cardiovascular system. In the heart, pharmacological and genetic data reveal a cardioprotective effect of adenosine during hypoxia (Auchampach et al., 1999; Lasley, 2005; Liang, 1996; Liang, 1998) via the activation of A1Rs, A2ARs, A2BRs and A3Rs on cardiac myocytes (Auchampach et al., 1999; Eckle et al., 2007; Lasley et al., 2007). In coronary arteries, activation of A2ARs and A2BRs enhances nitric oxide production (Olanrewaju et al., 2000) promoting vasodilation (Teng et al., 2008) to increase coronary blood flow (Talukder et al., 2003). This mechanism provides cardioprotection during ischemia. In the aorta, activation of A2ARs and A2BRs relaxes smooth muscle cells. In the renal vasculature, activation of A1Rs via Gi coupling causes renal afferent arteriole contraction, while activation of A2Rs via Gs coupling causes dilation of afferent arterioles (Welch, 2002). Endothelial cells also express A2AR and A2BR, and hypoxia modulates the expression of both receptor subtypes. Increased adenosine concentration during hypoxia reduces the high affinity A2AR expression, but conversely increases mRNA expression of the low affinity A2BR, which further promotes the release of an angiogenic factor, vascular endothelial growth factor (Feoktistov et al., 2004). The adenosine-induced effects are concentration dependent as low adenosine concentrations (micromolar range) activate A1Rs, while higher concentrations activate A1Rs and A2Rs. In MA, adenosine produces both relaxation and constriction. ! "#! Vasodilation of MA by adenosine is primarily mediated by A2Rs on smooth muscle cells, which link to KATP channels (Tabrizchi et al., 2001). Another possibility is that activation of A2Rs located on endothelial cells is associated with nitric oxide release. Activation of A3Rs reduces blood pressure in rats, but there is no evidence to indicate that A3R activation could elicit vasodilation (Tabrizchi et al., 2001). Furthermore, postganglionic sympathetic nerves innervating mesenteric blood vessels express A1Rs, which modulate sympathetic neurotransmission (Illes et al., 1988; Ralevic, 2000). Stimulation of adenosine receptors relaxes the portal vein of rats and saphenous and hind limb vein of dogs (Ralevic, 2000). Adenosine action on vascular tissues is especially important when oxygen demand is high and supply is low, as occurs in ischemia. In this situation, adenosine release is increased dramatically from cells and this can have a profound impact on blood vessel tone and blood flow (Tabrizchi et al., 2001). Although there is growing evidence revealing adenosine receptors subtypes and their functions in different blood vessels, characterization of these processes in MA and MV are not clearly described (Tabrizchi et al., 2001) particularly in hypertension. Therefore, further clarification of the role of adenosine in the mesenteric circulation is needed. Adenosine in sympathetic neurotransmission The sympathetic nervous system regulates blood pressure by regulating cardiac function and tone of resistance arteries and capacitance veins, particularly in the mesenteric circulation (King et al., 2007). Sympathetic nerves supply blood vessels at the adventitial-medial surface for arteries and they distribute deeper into the media for veins (Birch et al., 2008). Sympathetic ! "#! release of NE and ATP is precisely regulated by the prejunctional !2-autoreceptors and A1Rs (Demel et al., 2008a; Illes et al., 1988; Ralevic, 2000). NE acts at prejunctional !2-autoreceptors to inhibit co-transmitter release from sympathetic nerves. On the other hand, ATP is quickly terminated following its release by enzymatic degradation to ADP, AMP, and adenosine as a final product (Todorov et al., 1997; Todorov et al., 1999). Adenosine binds to prejunctional A1Rs to inhibit transmitter release. Evidence showed that chronic inhibition of a non-selective adenosine receptor antagonist, 1,3dipropyl-8-sulphophenylxanthine (DPSPX), increased blood pressure and purinergic and adrenergic neurotransmission (Guimaraes et al., 2003). This indicates that adenosine regulates blood pressure partly by acting at prejunctional adenosine receptors. Adenosine transporters Regarding sympathetic neurotransmission, adenosine could be formed by hydrolysis of released ATP or directly released from the postjunctional site. Under physiological condition, the major source of adenosine would be ATP degradation by ectoendonuclease in the neuroeffector junction. Conversely, during hypoxia, adenosine is probably released from the cells to modulate cell-surviving mechanisms (Frenguelli et al., 2003; Görlach, 2005). ! "#! 1.5 Modulation of sympathetic neurotransmitter release by prejunctional autoreceptors The release of sympathetic neurotransmitter is precisely regulated by various endogenous substances acting on a variety of prejunctional autoreceptors, which link to complex second messenger pathways (Brock et al., 1993). The autoreceptors are local machineries expressed on sympathetic varicosities to modulate the release of transmitters. Autoreceptors could facilitate/inhibit transmitter release or alter the transmitter synthesis (Brock, 1995). For instance, NE action on the prejunctional !2-AR inhibits further NE release, while circulating epinephrine action on the prejunctional "2AR facilitates the release of NE (Lennarstjarne et al., 1975). To study the function of the prejunctional autoreceptors, one may demonstrate that activation of the autoreceptor by adding selective exogenous agonist showing greater modulation of the release. Conversely, specific antagonist should alter transmitter release oppositely from that receptors being activated by the endogenous/exogenous agonist (Brock, 1995). For example, activation of prejunctional !2-AR by exogenous agonist UK-14304 or NE provides a negative feedback to sympathetic nerves resulting in a reduction of transmitter release and neurogenic contractile response. Conversely, blockade of !2-AR by its selective antagonist yohimbine causes an increase in NE release (Park et al., 2010) from periarterial sympathetic nerves. However, failure of an antagonist to alter transmitter may because the autoreceptors are not operative under experimental condition (Brock, 1995). ! "#! More interestingly, modulation of sympathetic transmitter release exhibits sophisticated cross-inhibitory regulation. Activation of prejunctional !2-AR inhibits both NE release (Park et al., 2010) and ATP-induced excitatory junction potential (EJP) (Demel et al., 2008b). Similarly, prejunctional adenosine A1R activation attenuates NE release (see chapter 3) and ATP-induced EJP response (Demel et al., 2008b). Voltage-gated Ca 2+ channels are the key component of neurotransmitter release. 2+ Increased intra-terminal Ca 2+ concentration via voltage-gated Ca 2+ trigger of transmitter release, while Ca -induced Ca2+ channel is the key release from intracellular stores seems to be ineffective mechanism for transmitter release (Burgoyne et al., 1995). Therefore, voltage-gated Ca 2+ channels are biological target for autoreceptors in modulating transmitter release. Here, a specific example of how the prejunctional !2adrenergic modulates sympathetic transmitter release is described. 1.5.1 The !2-adrenergic autoreceptors The prejunctional !2-adrenergic autoreceptors provide inhibitory modulation of transmitter release in both central and peripheral nerves. The prejunctional !2A/D-AR is the major autoreceptor providing negative feedback to stop sympathetic transmission (Guimaraes et al., 2001). The !2A subtype is cloned from human and pocrine tissue, which is slightly different from its homologous receptors cloned from rat, mouse, and guinea pig. The homologous of !2A subtype is named !2D and these two receptors are ! "#! simply referred to as !2A/D-AR (Guimaraes et al., 2001). Furthermore, !2C-AR also serves as an additional mechanism providing inhibitory modulation of transmitter release with different time course of expression from !2A/D-AR. The !2A/D-AR is operative immediately at birth, while the !2C-AR is become operative later in mice. The !2C-AR is a high affinity receptor; hence it regulates basal NE release. Conversely, the low affinity !2A/D-AR regulates transmitter release at higher sympathetic tone (Guimaraes et al., 2001; Philipp et al., 2002). Additionally, !2C-AR function is more prominent in peripheral, while !2A/D-AR is likely to be important in central nervous system (Guimaraes et al., 2001). The !2A/D and !2C-AR inhibit transmitter release by modulating voltage-gated Ca 2+ channels. The !2-AR inhibits L-type Ca2+ channels in rat retina (Dong et al., 2007), and N-type channels in sympathetic nerves (Schwartz, 1997). In sympathetic neurons, only !2A/D subtype, but not !2C contributes to inhibition of transmitter release. As !2-AR is coupled to inhibitory G-protein (Gi), it was first proposed that !2-AR might inhibit transmitter release by inhibiting adenylate cyclase activity. However, several studies found that neither forskolin nor phosphodiesterase inhibitors alter the effect of !2-AR-induced inhibition of NE release in various tissues leading to a conclusion that cAMP does not a link in presynaptic !2-AR (Starke, 1987). ! "#! Meanwhile, G protein !" subunits inhibit voltage-gated Ca 2+ channels resulting 2+ in inhibition of transmitter release, and this Ca channel inhibition is relieved by protein kinase C (PKC) (Herlitze et al., 2001; Swartz, 1993), which generally coupled to various prejunctional facilitatory autoreceptors including angiotensin AT1 receptors (Talaia et al., 2006), and prejunctional adenosine A2B receptors (Talaia et al., 2005). 2+ Phosphorylation of the voltage-gated Ca mediated inhibition of the Ca 2+ channels by PKC removes tonic G protein- channels. The !2-AR expression is not restricted to the prejunctional site as they also express on vascular smooth muscle and endothelial cells. The !2A/D, !2B, and !2C subtypes express on vascular smooth muscle cells mediating vasoconstriction (Kanagy, 2005), while !2-AR expressed by vascular endothelial cells couples to NO release and vasodilation in isolated coronary, renal and mesenteric arteries (Guimaraes et al., 2001). As the !2-ARs express in both pre-and post-junctional sites, it is equivocal to determine if the receptor agonist/antagonist effects on neurogenic contraction response is caused by pre- or post-junctional receptor activation. Continuous amperometry with microelectrode permit one to study sympathetic transmission by directly measuring transmitter release as an oxidation current proportional to concentration of transmitter release (See chapter2). This electrochemical technique provides unequivocal evidence of the autoreceptor function on transmitter release regardless of interference from the drug interacting at postjunctional !2-ARs. ! "#! 1.6 Differential sympathetic regulation of arteries and veins As arteries and veins obtain different hemodynamic functions, they receive differential sympathetic nerve innervation. Nonmyelinated sympathetic nerves form plexuses at border of tunica adventitia and media of arteries, while penetrate further down into media of veins suggesting closer contact between nerve varicosities and venous smooth muscle cells (Fig. 16A, C) than that in arterial bed (Fig. 16 B, D). Furthermore, glycosidic acid-induced fluorescent catecholamine data revealed that the veins have less sympathetic nerve fibers than the arteries with different nerve arrangement. The veins receive mostly circular nerve arrangement; while arteries receive both circular and longitudinal sympathetic innervation (Park et al., 2007). This differential nerve organization contributes to a unique “upstream” and/or “lateral” regulation of transmitter release and clearance at the neuroeffector junction (Stjarne, 1989) in periarterial, but not perivenous nerves (Fig. 17). Stärke and Stjärne proposed that transmitter release could be regulated by the autoreceptors expressed by remote varicosities (Stjarne, 1989). A train stimuli causes transmitter release from varicosities, accumulated, and diffusing to neighboring varicosities causing further activation of autoreceptors and transporters. The early evidence of upstream/lateral regulation was reported by Stjärne and Stjärne. They found that inhibition of ATP release from varicosities inside the patch was caused by activation of !2-autoreceptors outside the patch by exogenous agonist clonidine. It should be noted that !2-autoreceptors inside the patch were initially blocked by yohimbine (!2 antagonist) to prevent the receptor activation. Hence, the inhibition of ATP release observed inside the patch was caused ! "#! by the activation of neighboring !2-autoreceptors outside the patch (Stjärne et al., 1988). Furthermore, Park and coworkers also revealed differential kinetics of NE release and clearance of periarterial and perivenous sympathetic nerves. Periarterial is capable to inhibit NE release and remove NE more efficiently than that in perivenous nerves apparently at low frequency of nerve stimulation. This may because low frequency stimulation permits sufficient time for released transmitter to accumulate and diffuse to surrounding autoreceptors. They also revealed differential rising phase of NE oxidation current evoked by electrical nerve stimulation. Arterial NE current exhibited biphasic rise phase: initial rapid rise following by gradual rise. They proposed that the second phase (gradual rise) was influenced by the activation of prejunctional autoreceptors to inhibit NE release and this gradual rise was absent in venous NE current (Park et al., 2010). Furthermore, activation of !2-AR by exogenous agonist UK14304 causes a dramatic increase in NE release in periarterial nerves, while induces slightly increase in NE release in perivascular nerves. Additionally, !2-AR blockade by exogenous yohimbine increases NE release in periarterial, but not perivenous nerves (Park et al., 2007). Meanwhile, lateral NE clearance is also established predominantly in periarterial nerves. Cocaine, NE transporter blocker causes a remarkable increase in NE release in periarterial, but not perivenous nerves (Park et al., 2007). Therefore, it is logical to imply that upstream/lateral regulation provides higher efficiency to periarterial nerve to control release and removal of NE from neurovascular junction, which may account for smaller basal NE oxidation current measured by amperometry of periarterial nerves compared to perivenous nerves (Park et al., 2010; Park et al., 2007). Lack of ! "#! lateral regulation in perivenous nerves may be due to less sympathetic supply or/and less amount of transmitter release from varicosities to accumulate and diffuse to autoreceptors and/or transporters located on surrounding varicosities. Furthermore, arteries and veins may contain different transmitters in sympathetic varicosities and this issue is still controversial. In rat mesenteric bed, NE is likely to be a major transmitter released from perivenous nerves, while periarterial varicosities contain cotransmitter cocktail: ATP, NPY, and NE (Talaia et al., 2011). Donoso and coworkers demonstrated that blockade of NPY Y1, P2 purinergic, or !1-adrenergic receptors by specific exogenous antagonist reduces neurogenic vasomotor response and combination of antagonists for these three receptor completely abolish neurogenic response in rat mesenteric arteries (Donoso et al., 1997b). This is consistent with Park and coworkers’ work reporting that blockade of !1-adrenergic receptors by prazosin partially inhibited neurogenic arterial contraction, while prazosin abolished neurogenic venous contraction (Park et al., 2010) suggesting that NE is only major neurotransmitter released from perivenous sympathetic nerves. However, NPY and ATP seem to be cotransmitters along with NE in guinea pig mesenteric veins. Smyth et al showed colocalization of NPY and tyrosine hydroxylase-containing nerve fibers in both guineapig mesenteric arteries and veins. Additionally, NPY Y1 antagonist abolishes neurogenic venous contraction in NE-depleted veins. Moreover, blockade of purinergic receptors by PPAD and sumarin attenuates neurogenic contraction in NE-depleted veins (Smyth et al., 2000). These findings suggested that perhaps, perivenous sympathetic nerves also release ATP, but in only a small fraction and it does not ! ""! necessarily to plays an important role at the postjunctional site. Conversely, this small fraction of released ATP only acts prejunctionally to modulate cotransmitter release. Figure. 16 Differential sympathetic nerve distribution of arteries and veins. Sympathetic nerves supply arteries at border of adventitia and media (A), and penetrate deeper into tunica media of veins (B). Arteries are more densely innervated with sympathetic nerves (C) compared to veins (D). T, tunica; VSMC, vascular smooth muscle cell. ! ! ! &'! Figure. 17 Diagram illustrating lateral regulation for NE release and clearance of periarterial sympathetic nerves. The lateral regulation hypothesis proposed that NE released from one varicosity could diffuse to activate on remote varicosities to turn on autoinhibition negative feedback. Also, released NE from one site is removed by the NE transporters located on neighboring varicosities. This lateral inhibition is exclusive for periarterial (A), but not perivenous nerves (B). ! ! "#! 1.7 Hypertension Hypertension is an important public health problem. About 1 billion people worldwide had hypertension in 2000. This represents 30% of the global population which will be increased to 1.56 billion by 2025 (Patricia M Kearney, 2005). Hypertension causes approximately 7.1 million deaths worldwide each year (Chobanian et al., 2003). Hypertension is the key risk factor driving increased cardiovascular disease. The World Health Organization estimates that 62% of cerebrovascular disease and 49% of ischemia heart disease occurs in hypertensive patients with suboptimal blood pressure control (systolic blood pressure >115 mmHg)(Rodgers, 2002). Data from animal models of hypertension and from human studies have clearly shown that increased sympathetic activation contributes to hypertension development (Anderson et al., 1989; Schlaich et al., 2004). The Seventh Report of the Joint National Committee on Prevention, Detection, Evaluation, and Treatment of High Blood Pressure (JNC7, (Chobanian et al., 2003)) has defined essential hypertension as systolic/diastolic blood pressure over 140/90 mmHg. More importantly, elevated blood pressure is related to increased cardiovascular mortality risk. Every increment of 20/10mmHg systolic/diastolic pressure from the optimal blood pressure115/75 mmHg increases risks for ischemia heart disease and stroke by two and three fold, respectively (Fig. 18, 19). The stages for high blood pressure have been classified by JNC7 shown in table 3. ! "#! Table 3. JNC7 Blood Pressure Classification. SBP, systolic blood pressure; DBP, diastolic blood pressure (Chobanian et al., 2003) SBP/DBP Condition < 120/80 Normal 120-139/80-89 Prehypertension ! 140/90 Hypertension 140-159/90-99 >160/100 ! Hypertension stage 1 Hypertension stage 2 "#! Figure 18. Ischemic heart disease (IHD) mortality rate in each decade of age vs usual systolic. (A) and diastolic (B) blood pressure reported in the JNC 7. (Chobanian et al., 2003) ! "#! Figure 19. Stroke mortality rate in each decade of age vs usual systolic (A) and diastolic (B) blood pressure. (Chobanian et al., 2003) ')0*71&8*0)$%+)(& 29%*$:;5&<="*%#)1&>+"7&$;.&?@A&BC6& .-,% <6  &'(")*%+,&-%**.&/01""#01&&&&&& !"#!$%&'()*% /.!% +"#+$%&'()*% ,0% ,"#,$%&'()*% 1.% -"#-$%&'()*% /,% !% 0% .% /% "% /."% /0"% /,"% /!"% !"#$%&&'(")*%+,&-%**.&/01""#01&233456& 6(.)7/#8).(&*%(9# 2:*)&;<5#=>')*0(/#?%'7#&<,#@AB#CD"# .-,% !"#$%&'()*%+#!*)),#-./''0./# /.!% !"#!$%&'()*% +"#+$%&'()*% ,0% ,"#,$%&'()*% 1.% -"#-$%&'()*% /,% !% 0% .% /% "% +"% !"% $"% /""% //"% 1'0&*##$%&'()*%+#!*)),#-./''0./#23345"# ! "#! 1.7.1 Sympathetic overactivity in hypertension Chronic elevated sympathetic nerve activity is associated a variety of cardiovascular diseases including obesity, hypertension, sleep apnea (high prevalence in hypertension and stroke patients), and mental stress. A large-scale study has showed a significant correlation between work-place mental stress and hypertension development (Malpas, 2010). Regarding essential hypertension, it appears that sympathetic nerve activity is elevated in both animal models and human. 1.7.2 Autoreceptors in hypertension Data from animal models of hypertension and from human studies have clearly shown that increased sympathetic activation contributes to hypertension development (Anderson et al., 1989; Schlaich et al., 2004). Nevertheless, the mechanisms for sympathetic activation occurring in essential hypertension have not been conclusively determined (Mancia et al., 1999). One possibility is that activation of the sympathetic nervous system depends on the function of autoreceptors located on sympathetic nerve terminals (Demel et al., 2008a; Min Luo, 2004). Increasing evidence suggests that impairment of !2 autoreceptors, which provide negative feedback for sympathetic neuromodulation, contributes to development of hypertension (Bobalova et al., 2001; Demel et al., 2008a; Luo et al., 2003; Min Luo, 2004). However, it is not clear if impairment of A1 autoreceptor function is also involved in hypertension. ! "#! 1.7.3 DOCA-salt rat model of hypertension Administration of the mineralocorticoid deoxycorticosterone acetate (DOCA) in combination with a high salt diet and unilateral nephrectomy is used to induce hypertension in experimental animals (Pintoa et al., 1998; Schenk et al., 1992). This model is considered as a low rennin form of hypertension with salt and water retention, which is relevant to salt-sensitive human hypertension (Luo et al., 2003; Schenk et al., 1992). Although the DOCA-salt rat model of hypertension has been established for decades, one may question what mechanism leads to hypertension in this animal + model. Uninephrectomy decreases renal Na excretion. Chronic high-salt administration induces a sustained increase in plasma NaCl and osmolality, which activates neurons in the (O’Donaughy et al., 2006) organum vasculosum lamina terminalis (OVLT) and hypothalamus (Guyenet, 2006). That particular central activation drives + sympathoexcitation outflow. Moreover, negative alteration of renal Na excretion allows + DOCA-induced Na -retention leading to expansion of extracellular fluid and blood volume (Haack et al., 1977). However, volume expansion in DOCA-salt hypertension occurs during the first few weeks then returns to basal volume due to compensatory renal mechanisms. Yamamoto(Yamamoto et al., 1983) and Fink (Fink et al., 2000) found moderately increased blood volume in DOCA-salt hypertension during the early phase of DOCA-salt hypertension. O’Donaughy also proposed that mineralocorticoid; DOCA amplifies + Na signal to central network leading to augmented sympathoexcitation. In the absence of DOCA administration, the animals did not develop increased lumbar sympathetic nerve activity or elevated blood pressure under ! "#! unilateral nephrectomy and high-salt diet (O’Donaughy et al., 2006). Moreover, evidence suggested that vasopressin was elevated during increased plasma osmolality. Vasopressin (arginine vasopressin, AVP) is an antidiuretic polypeptide hormone produced by magnocellular secretory cells in hypothalamus, and stored and released from the posterior pituitary gland. AVP acts on the distal convoluted tubules and the medullary collecting duct to decrease water excretion. AVP could increase vascular resistance, as it is a potent vasoconstrictor. AVP is released in response to decreased blood volume or increased plasma osmolality. As small as 1% increase in plasma osmolality above the threshold could be detected by the osmoreceptor neurons in the hypothalamus causing AVP release. Selective inhibition of V1 vasopressin receptors significantly decreased plasma NaCl concentration, blood pressure, and lumbar sympathetic nerve activity (O’Donaughy et al., 2006). Crofton and co-workers also reported an increase in plasma vasopressin and urine vasopressin excretion in DOCAsalt rats early after DOCA-salt administration, which matched the timeline of elevated blood pressure of the animals (Crofton et al., 1979). Furthermore, administration of inactive synthetic vasopressin analogs: [l-deaminopenicillamine, 4- valine, 8-D-arginine] vasopressin or [l-(!-mercapto-!, !- cyclopentamethylenepropionic acid), 4-valine, 8-Darginine]vasopressin caused dramatically decreased in blood pressure in established DOCA-salt hypertension (Crofton et al., 1979). It should be noted that the animals used in Crofton experiments were Long-Evans strain and these rats were received 30mg/kg body weight DOCA and 1%NaCl drinking water. Moreover, blood pressure of DOCAsalt rats with genetic vasopressin deficiency were significantly lower than of those ! "#! DOCA-salt with normal synthesizing vasopressin (Kunes et al., 1989). Thus, vasopressin may partly account for development of DOCA-salt hypertension. Additionally, endothelin-1 (ET-1) level and mRNA expression are increased in DOCA-salt arteries (Day et al., 1995; Larivière et al., 1993; Watts et al., 2002). Endothelin (ET) is a local vasoconstrictor peptide released from vascular endothelial cells. There are three subtypes of ETs: ET-1, ET-2, and ET-3. Of these, ET-1 is the most potent vasoconstrictor (Larivière et al., 1993); hence it is logically implied that ET-1 is involved in pathophysiologic mechanism of hypertension (Deng et al., 1992). Two endothelin receptor subtypes have been cloned: ETA and ETB receptors. Similar to NEinduced response, the veins (rat vena cava) are more sensitive to ET-1 induced contraction that arteries (rat aorta) and ET-1 induced contraction mechanism may be impaired in DOCA-salt hypertension (Watts et al., 2002). Chronic administration of ETA antagonist could lower blood pressure of DOCA-salt hypertensive rats (Callera et al., 2006; Matsumura et al., 1995). Interestingly, elevation of ET-1 contributes to increased superoxide level in DOCA-salt rats (Callera et al., 2006; Li et al., 2003). Li and coworkers revealed that ET-1 and superoxide levels were elevated in DOCA-salt hypertension. Furthermore, ET-1 causes an increase in superoxide production in concentration (0.01-1 nM)-dependent manner in DOCA-salt carotid arteries and this effect is prevented by ETA antagonist (Li et al., 2003). More interestingly, Yu et al reported synergistic effect of a non-selective ET antagonist (bosentan) and vasopressin V1 antagonist on a reduction of total peripheral resistance and blood pressure of DOCAsalt hypertensive rats compared to single administration of bosentan. Vasopressin ! "#! antagonist would only reduce blood pressure when an ET-1 antagonist was also administered (Yu et al., 2001). These findings suggest that development of DOCA-salt hypertension is multifactorial associating with ET and vasopressin hormonal alterations. DOCA-salt–induced hypertension may involve in both brain-initiated sympathoexcitation and renal fluid retention. While Xu and co-workers found unaltered renal sympathetic nerve activity between DOCA-salt hypertensive and control rats (Xu et al., 2002), Takeda and Bunag directly illustrated elevated splanchnic sympathetic nerve activity in DOCA-salt hypertensive rats in basal and hypothalamic stimulation response (Takeda et al., 1980). Although this study was done in anesthetized rats, it is valuable finding to support the concept that increased sympathetic nerve activity, particularly splanchnic sympathetic nerves, is associated with hypertension development in DOCA-salt rats. Furthermore, De Champlain and Kopin groups reported elevated plasma catecholamine level as an indirect indicator for augmented sympathetic activity, in DOCA-salt hypertensive rats (Bouvier et al., 1986; Reid et al., 1975). Taken these data together, it is likely that there is a selective increase in splanchnic sympathetic nerve activity provided by the celiac ganglion in DOCA-salt hypertensive rats. The unchanged renal sympathetic nerve activity is because the celiac ganglion provides very slightly input to the kidney (Fig.1). On the other hand, it is possible that the alteration of sympathetic nerve activity may occur independently from the central network. For instance, the alteration of peripheral sympathetic transmission may occur due to disruption of the prejunctional control mechanism leading to increased transmitter released onto the postjunctional site. Previous students in our lab have shown that the disruption of adrenergic autoreceptor on postganglionic sympathetic ! ""! nerves supplying splanchnic circulation cause an increase in transmitter (ATP and NE) release. The present study focused on the postganglionic sympathetic neurotransmission at the level of the prejunctional purinoautoreceptor alterations leading to changes in transmitter release and clearance in both control and hypertensive animals. In addition to DOCA-salt rat model of hypertension, recently Kandlikar and coworkers have developed a mild DOCA-salt hypertensive rat. In this model the animals are received low dose of DOCA implantation (50 vs 200 mg/kg) without uninephrectomy (Kandlikar et al., 2011; Luo et al., 2004). The animal blood pressure is elevated gradually and the established arterial pressure is increased approximately 10-20 mmHg. They proposed that the small gradual increase in blood pressure permits one to study the casual mechanisms driving hypertension (Kandlikar et al., 2011). 1.7.4 Amperometric measurement of norepinephrine Amperometry with microelectrodes is an electrochemical technique adapted from voltammetry which is well characterized in chemistry laboratories for almost one century (Evanko, 2005). Amperometry was first introduced for ion chromatography and later it was implicated for detection of electroactive neurotransmitters including catecholamines (dopamine, norepinephrine, epinephrine) and ATP (Llaudet et al., 2005), which can be oxidized at the electrode surface at a given potential. The major breakthrough was when Adams and co-workers implanted carbon fiber microelectrode into rat. Catecholamines are oxidized to quinone and release electrons, which are transferred to the electrode surface and detected as an oxidation current (Mundorf et al., 2002). An ! "#! amperometric cell includes a carbon fiber microelectrode (working electrode), a platinum counter (auxiliary) electrode, and silver-silver chloride (Ag/AgCl) reference electrode. The direction of the electron flow depends on the properties of the neurotransmitter and can be controlled by potential applied to the working electrode. Generally, the potential of the working electrode is held sufficiently positive so that all released transmitters are oxidized. This will cause an accumulation of an oxidation current in response to a brief train of stimuli. The working electrode for transmitter detection is usually constructed from carbon fiber or diamond (Park et al., 2010; Park et al., 2007; Park et al., 2005). A reference electrode provides a fixed potential against which potential applied to the working electrode is measured and controlled. Platinum counter electrode is used to complete amperometric circuitry. Amperometric measurement produces transient electro-oxidation current that is proportional to the concentration of the neurotransmitter oxidized per unit time. Area under the curve is used to calculate the amount of released transmitter. The shape of oxidation current contains information about transmitter release, autoinhibition, and transmitter reuptake. 1.7.5 Contribution of other catecholamines in amperometric detection of NE As NE share similar structure with dopamine and epinephrine (Fig. 20), one may question that at the given potential 0.6 Volts, would the amperometric oxidation current results from the mixture of these three catecholamines? Although NE, dopamine and epinephrine become oxidizable at 0.6 Volts potential, epinephrine is exclusively released from adrenal medulla during stress response. Perivascular sympathetic nerves ! "#! do not contain enzyme phenylethanolamine N-methyltransferase to convert NE into epinephrine. However, dopamine could be released from sympathetic nerves supplying canine mesenteric blood vessels (Soares-da-Silva, 1988). Silva showed that prolonged sympathetic nerve depolarization with KCl causes dopamine release along with NE. Sustained nerve stimulation causes dopamine !-hydroxylase become rate-limiting resulting in accumulation of dopamine available for release. Once release, dopamine could act on the prejunctional dopamine receptors providing the negative feedback to shut down sympathetic transmission. In my experiment, I used brief electrical-train stimuli (60 stimuli at 10 Hz frequency, 0.5 ms pulse duration). At this stimulation parameter, it would be hardly dopamine release. Figure 20. Catecholamine structures Dopamine ! Norepinephrine "#! Epinephrine 1.8 Perspectives Although there have been substantial advances in anti-hypertensive medication development and use, many patients do not respond well to these drugs or resist their use because of troubling side effects. Continued research directed at identifying new drug targets is needed. Clarifying the mechanism that leads to enhanced neurogenic vasoconstriction may open a door for a new avenue of anti-hypertensive drug development. ! "#! BIBLIOGRAPHY ! "#! BIBLIOGRAPHY Abebe W, Mustafa JS (1998). A1 adenosine receptor-mediated Ins(1,4,5)P3 generation in allergic rabbit airway smooth muscle. Am J Physiol Lung Cell Mol Physiol 275: 990997. Alexander SPH, Losinski A, Kendall DA, Hill SJ (1994). A comparison of A2 adenosine receptor-induced cyclic AMP generation in cerebral cortex and relaxation of precontracted aorta. British Journal of Pharmacology 111(1): 185-190. Anderson E, Sinkey C, Lawton W, Mark A (1989). Elevated sympathetic nerve activity in borderline hypertensive humans. Evidence from direct intraneural recordings. Hypertension 14: 177-183. Andoh T, Ishiwa D, Kamiya Y, Echigo N, Goto T, Yamada Y (2006). A1 adenosine receptor-mediated modulation of neuronal ATP-sensitive K channels in rat substantia nigra Brain Research 1124(1): 55-61. Auchampach JA, Bolli R (1999). Adenosine receptor subtypes in the heart: therapeutic opportunities and challenges. Am J Physiol Heart Circ Physiol 276: 1113-1116. Barrett K, Barman S, Boitano S, Brooks H (2012). Chapter 13. Autonomic Nervous System. In: Ganong's Review of Medical Physiology, Barrett KE BS, Boitano S, Brooks HL (ed), 24 edn. New York: McGraw-Hill. Billaud M, Lohman AW, Straub AC, Looft-Wilson R, Johnstone SR, Araj CA, et al. (2011). Pannexin1 regulates α1-adrenergic receptor− mediated vasoconstriction. Circulation Research 109: 80-85. Birch DJ, Turmaine M, Boulos PB, Burnstock G (2008). Sympathetic Innervation of Human Mesenteric Artery and Vein. Journal of Vascular Research 45: 323-332. Bobalova J, Mutafuva-Yambolieva VN (2001). Presynaptic α2-adrenoceptor-mediated modulation of adenosine 5' triphosphate and noradrenaline corelease: differences in canine mesenteric artery and vein. J of Autono Pharmacol 21: 47-55. Bouvier M, de Champlain J (1986). Increased basal and reactive plasma norepinephrine and epinephrine levels in awake DOCA-salt hypertensive rats. J Auton Nerv Syst 15: 191-195. Brock JA (1995). Chapter 5. Modulation of neurotransmitter release by autoreceptors. In: Powis DA, Bunn SJ (ed)^(eds). Neurotransmitter release and its modulation, 1 edn. Cambridge: Cambridge University Press. p^pp. ! "#! Brock JA, Cunnane TC (1993). Neurotransmitter release mechanisms at the sympathetic neuroeffector junction. Exp Physiol 78: 591-614. Burgoyne RD, Cheek TR (1995). Chapter 1. Mechanisms of exocytosis and central role of calcium. In: Powis DA, Bunn SJ (ed)^(eds). Neurotransmitter release and its modulation, 1 edn. Cambridge: Cambridge University Press. p^pp. Burns AR, Phillips SC, Sokoya EM (2012). Pannexin protein expression in the rat middle cerebral artery. J of Vasc Res 49: 101-110. Burnstock G (1976). Do some nerve cells release more than one transmitter? Neuroscience 1: 239-248. Burnstock G (2007). Physiology and pathophysiology of purinergic neurotransmission. Physiol Rev 87: 659-797. Callera GE, Tostes RC, Yogi A, I AC, Montezano, Touyz RM (2006). Endothelin-1induced oxidative stress in DOCA-salt hypertension involves NADPH-oxidaseindependent mechanisms. Clin Sci 110: 243-253. Cechova S, Venton BJ (2008). Transient adenosine efflux in the rat caudate–putamen. Journal of Neurochemistry 105: 1253-1263. Chobanian AV, Bakris GL, Black HR, Cushman WC, Green LA, Izzo-Jr JL, et al. (2003). Seventh report of the joint national committee on prevention, detection, evaluation, and treatment of high blood pressure. Hypertension 42: 1206-1252. Cowley-Jr AW, Michalkiewicz M (2008). Vasopressin and Neuropeptide Y. In: Izzo-Jr JL, Sica DA, Black HR (ed)^(eds). Hypertension Primer the Essential of High Blood Pressure Basic Science, Population Science, and Clinical Management, 4 edn. Philadelphia: Lippincott Williams and Wilkins. p^pp 70-73. Crofton JT, Share L, Shade RE, Lee-Kwon WJ, Manning M, Sawyer WH (1979). The importance of vasopressin in the development and maintenance of DOC-salt hypertension in the rat. Hypertension 1: 31-38. Dʼhondt C, Ponsaerts R, De Smedt H, Bultynck G, Himpens B (2009). Pannexins, distant relatives of the connexin family with specific cellular functions? BioEssays 31: 953-974. Dale N, Pearson T, Frenguelli BG (2000). Direct measurement of adenosine release during hypoxia in the CA1 region of the rat hippocampal slice. Journal of Physiology 526(1): 143-155. ! "#! Davie CS, Everitt DE, Standen NB (1999). Increase in the vasorelaxant potency of KATP channel opening drugs by adenosine A1 and A2 receptors in the pig coronary artery. European Journal of Pharmacology 383(2): 155-162. Day R, Larivière R, Schiffrin EL (1995). In situ hybridization shows increased endothelin-1 mRNA levels in endothelial cells of blood vessels of deoxycorticosterone acetate-salt hypertensive rats. Am J Hypertens 8: 294-300. Demel SL, Galligan JJ (2008a). Impaired Purinergic Neurotransmission to Mesenteric Arteries in Deoxycorticosterone Acetate-Salt Hypertensive Rats. Hypertension 52(2): 322-329. Demel SL, Galligan JJ (2008b). Impaired purinergic neurotransmission to mesenteric arteries in DOCA-salt hypertensive rats. Hypertension 52(2): 322-329. Deng LY, Schiffrin EL (1992). Effects of endothelin on resistance arteries of DOCA-salt hypertensive rats. Heart Circ Physiol 31: H1782-H1787. Dong C-J, Guo Y, Wheeler L, Hare WA (2007). Alpha2 adrenergic receptor–mediated modulation of cytosolic Ca++ signals at the inner plexiform layer of the rat retina. Invest Ophth Vis Sci 48: 1410-1415. Donoso MV, Brown N, Carrasco C, Cortes V, Fournier A, Huidobro-Toro JP (1997a). Stimulation of the sympathetic perimesenteric arterial nerves releases neuropeptide Y potentiating the vasomotor activity of noradrenaline: involvement of neuropeptide Y-Y1 receptors. Journal of Neurochemistry 69: 1048-1059. Donoso MV, Steiner M, Huidobro-Toro JP (1997b). BIBP 3226, suramin and prazosin identify neuropeptide Y, adenosine 5'-triphosphate and noradrenaline as sympathetic cotransmitters in the rat arterial mesenteric bed. J Pharmacol Exp Ther 282: 691-698. Dubyak GR (2009). Both sides now: multiple interactions of ATP with pannexin-1 hemichannels. Focus on “A permeant regulating its permeation pore: inhibition of pannexin 1 channels by ATP. Am J Physiol Cell Physiol 296: C235-C241. Dunkley PR, Bobrovskaya L, Graham ME, Nagy-Felsobuki EIv, Dickson PW (2004). Tyrosine hydroxylase phosphorylation: regulation and consequences. J Neurochem 91: 1025-1043. Eckle T, Krahn T, Grenz A, Köhler D, Mittelbronn M, Ledent C, et al. (2007). Cardioprotection by ecto-5'-nucleotidase (CD73) and A2B adenosine receptors. Circulation 115: 1581-1590. ! "#! Eisenhofer G (2001). The role of neuronal and extraneuronal plasma membrane transporters in the inactivation of peripheral catecholamines. Pharmacology & Therapeutics 91: 35-62. Eisenhofer G, Kopin IJ, Goldstein DS (2004). Catecholamine metabolism: a contemporary view with implications for physiology and medicine . Pharmacological Reviews 56: 331-349. Ekblad E, Edvinsson L, Wahlestedt C, Uddman R, Hakanson R, Sundler F (1984). Neuropeptide Y co-exists and co-operates with noradrenaline in perivascular nerve fibers. Regulatory Peptides 8: 225-235. Evanko D (2005). Primer: spying on exocytosis with amperometry. Nature Method 2: 650. Farquhar WB, Wenner MM, Delaney EP, Prettyman AV, Stillabower ME (2006). Sympathetic neural responses to increased osmolality in humans. Am J Physiol Heart Circ Physiol 291: H2181–H2186. Fenger-Gron J, Mulvany MJ, Christensen KL (1995). Mesenteric blood pressure profile of conscious, freely moving rats. Journal of Physiology 488(3): 735-760. Feoktistov I, Ryzhov S, Zhong H, Goldstein AE, Matafonov A, Zeng D, et al. (2004). Hypoxia modulates adenosine receptors in human endothelial and smooth muscle cells toward an A2B angiogenic phenotype. Hypertension 44: 649-654. Fink GD, Johnson RJ, Galligan JJ (2000). Mechanisms of increased venous smooth muscle tone in desoxycorticosterone acetate-salt hypertension. Hypertension 35: 464469. Fredholm BB, Jzerman API, Jacobson KA, Klotz K-N, Linden J (2001). International Union of Pharmacology. XXV. Nomenclature and Classification of Adenosine Receptors Pharmacol Rev 53(4): 527-552. Frenguelli BG, Llaudet E, Dale N (2003). High-resolution real-time recording with microelectrode biosensors reveals novel aspects of adenosine release during hypoxia in rat hippocampal slices. Journal of Neurochemistry 86: 1506–1515. Fried G, Terenius L, Hijkfelt T, Goldstein M (1985). Evidence for differential localization of noradrenaline and neuropeptideY in neuronal storage vesicles isolated from rat vas deferens. J Neurosci 5: 450-458. Görlach A (2005). Control of adenosine transport by hypoxia. Circulation Research 97: 1-3. ! "#! Guimaraes S, Morato M, Sousa T, Albino-Teixeira A (2003). Hypertension Due to Blockade of Adenosine Receptors. Pharmacol Toxicol 92: 160-162. Guimaraes S, Moura D (2001). Vascular adrenoceptors: an update. Pharmacol Rev 53: 319-356. Guyenet PG (2006). The sympathetic control of blood pressure. Nature Reviews: Neuroscience 7: 335-346. Haack D, Mijhring J, Mijhring B, Petri M, Hackenthal E (1977). Comparative study on development of corticosterone and DOCA hypertension in rats . Am J Physiol 233(5): F403-F411. Herlitze S, Zhong H, Scheuer T, Catterall WA (2001). Allosteric modulation of Ca2+ channels by G proteins, voltage-dependent facilitation, protein kinase C, and Cavβ subunits. PNAS 98: 4699-4704. Herve D, Levi-Strauss M, Marey-Semper I, Verney C, Tassin J-P, Glowinski J, et al. (1993). Golf and Gs in rat basal ganglia: possible involvement of Golf in the coupling of dopamine D1 receptor with adenylyl cyclase. Journal of Neuroscience 13(5): 2237-2248. Illes P, Jackisch R, Regenold JT (1988). Presynaptic P1-purinoceptors in jejunal branches of the rabbit mesenteric artery and their possible function. J Physiol 397: 1329. Kanagy NL (2005). α2-adrenergic receptor signalling in hypertension. Clinical Science 109: 431-437. Kandel ER, Schwartz JH, Jessell TM (2000). Principles of Neural Science. 4 edn. McGraw-Hill: New York. Kandlikar SS, Fink GD (2011). Mild DOCA-salt hypertension: sympathetic system and role of renal nerves. Am J Physiol Heart Circ Physiol 300: H1781-H1787. Katzung BG (2012). Chapter 6. Introduction to Autonomic Pharmacology. In: Basic & Clinical Pharmacology, Katzung BG, Masters SB, Trevor AJ (eds), 12 edn. New York: McGraw-Hill. Kennedy B, Shen GH, Ziegler MG (1997). Neuropeptide Y-mediated pressor responses following high-frequency stimulation of the rat sympathetic nervous system. The Journal of Pharmacology and Experimental Therapeutics 281: 291-296. King AJ, Osborn JW, Fink GD (2007). Splanchnic circulation is a critical neural target in angiotensin II salt hypertension in rats. Hypertension 50: 547-556. ! "#! Klotz K-N (2000). Adenosine receptors and their ligands. Naunyn-Schmiedeberg’s Arch Pharmacol 362: 382-391. Klotz KN (2007). Adenosine receptor pharmacology. In: Hasko G, Cronstein BN, Szabo C (ed)^(eds). Adenosine receptors: therapeutic aspects for inflammatory and immune diseases, 1 edn. Boca Raton: Taylor and Francis Group. p^pp 1-14. Kreulen DL (2003). Properties of the Venous and Arterial Innervation in the Mesentery. J. Smooth Muscle Res. 39(6): 269-279. Kügelgen I, Starke K (1991). Release of noradrenaline and ATP by electrical stimulation and nicotine in guinea-pig vas deferens. Naunyn-Schmiedeberg's Archives Of Pharmacology 344: 419-429. Kunes J, Nedvídek J, Zicha J (1989). Vasopressin and water distribution in rats with DOCA-salt hypertension. J Hypertens Suppl 7(6): S204-S205. Lagercrantz H (1976). On the composition and function of large dense cored vesicles in sympathetic nerves. Neuroscience 1: 81-92. Lamont C, Vial C, Evans RJ, Wier WG (2006). P2X1 receptors mediate sympathetic postjunctional Ca2+ transients in mesenteric small arteries. Am J Physiol Heart Circ Physiol 291: H3106-H3113. Larivière R, Thibault G, Schiffrin EL (1993). Increased endothelin-1 content in blood vessels of deoxycorticosterone acetate-salt hypertensive but not in spontaneously hypertensive rats. Hypertension 21: 294-300. Lasley RD (2005). New insights into adenosine-mediated myocardial protection. British Journal of Pharmacology 145: 701–702. Lasley RD, Kristo G, Keith BJ, Mentzer-Jr RM (2007). The A2a/A2b receptor antagonist ZM-241385 blocks the cardioprotective effect of adenosine agonist pretreatment in in vivo rat myocardium. Am J Physiol Heart Circ Physiol 292: H426-H431. Lennarstjarne, Brundin J (1975). Dual adrenoceptor-mediated control of noradrenaline secretion from human vasoconstrictor nerves: facilitation by beta-receptors and inhibition by alpha-receptor. Acta Physiol Scand 94: 139-141. Li L, Fink GD, Watts SW, Northcott CA, Galligan JJ, Pagano PJ, et al. (2003). Endothelin-1 increases vascular superoxide via endothelinA−NADPH oxidase pathway in low-renin hypertension. Circulation 107: 1053-1058. Liang BT (1996). Direct preconditioning of cardiac ventricular myocytes via adenosine A1 receptor and KATP channel. Am J Physiol 271 (5 Pt 2): H1769-1777. ! ""! Liang BT (1998). Protein kinase C-dependent activation of KATP channel enhances adenosine-induced cardioprotection. Biochem J 336 (Pt 2): 337-343. Linden J (2001). Molecular Approach to Adenosine Receptors: Receptor-Mediated Mechanisms of Tissue Protection. Annual Review of Pharmacology and Toxicology 41: 775-787. Llaudet E, Hatz S, Droniou M, Dale N (2005). Microelectrode biosensor for real-time measurement of atp in biological tissue. Anal Chem 77: 3267-3273. Luo M, Fink GD, Lookingland KJ, Morris JA, Galligan JJ (2004). Impaired function of alpha2-adrenergic autoreceptors on sympathetic nerves associated with mesenteric arteries and veins in DOCA-salt hypertension. Am J Physiol 286: H1558-H1564. Luo M, Hess MC, Fink GD, Olson LK, Rogers J, Kreulen DL, et al. (2003). Differential alterations in sympathetic meurotransmission in mesenteric arteries and veins in DOCAsalt hypertensive rats. Auton Neurosci 104(1). MacVicar BA, Thompson RJ (2012). Non-junction functions of pannexin-1 channels. Trends in Neurosciences 33(2): 93-102. Malpas SC (2010). Sympathetic nervous system overactivity and its role in the development of cardiovascular disease. Physiol Rev 90: 513-557. Mancia G, Grassi G, Giannattasio C, Seravalle G (1999). Sympathetic Activation in the Pathogenesis of Hypertension and Progression of Organ Damage. Hypertension 34: 724-728. Martin DS, Rodrigo MC, Appelt CW (1998). Venous tone in the developmental stages of spontaneous hypertension. Hypertension 31: 139-144. Matsumura Y, Fujita K, Miyazaki Y, Takaoka M, Morimoto S (1995). Involvement of endothelin-1 in deoxycorticosterone acetate-salt-induced hypertension and cardiovascular hypertrophy. J Cardiovasc Pharm 26: S456-458. Min Luo GDF, Keith J. Lookingland, John A. Morris, and James J. Galligan (2004). Impaired function of {alpha}2-adrenergic autoreceptors on sympathetic nerves associated with mesenteric arteries and veins in DOCA-salt hypertension. Am J Physiol Heart Circ Physiol 286: H1558-H1564. Mohrman DE, Heller LJ (2010a). Chapter 6. The Peripheral Vascular System. In: Cardiovascular Physiology, Mohrman DE, Heller LJ (eds), 7 edn. New York: McGrawHill. ! "#! Mohrman DE, Heller LJ (2010b). Chapter 7. Vascular Control. In: Cardiovascular Physiology, Mohrman DE, Heller LJ (eds). New York: McGraw-Hill. Mohrman DE, Heller LJ (2010c). Chapter 8. Central Venous Pressure: An Indicator of Circulatory Hemodynamics. In: Cardiovascular Physiology, DE M, LJ H (eds). New York: McGraw-Hill. Mundorf ML, Wightman RM (2002). Amperometry and Cyclic Voltammetry with Carbon Fiber Microelectrodes at Single Cells. Current Protocols in Neuroscience: 6.14.1116.14.22. Mutafova-Yambolieva VN, Hwang SJ, Hao X, Chen H, Zhu MX, Wood JD, et al. (2007). Beta-nicotinamide adenine dinucleotide is an inhibitory neurotransmitter in visceral smooth muscle. Proc Natl Acad Sci U S A 104: 16359-16364. Nilsson H, Ljung B, Sjöblom N, Wallin BG (1985). The influence of the sympathetic impulse pattern on contractile responses of rat mesenteric arteries and vein. Acta Physiologica Scandinavica 123: 303-309. OʼDonaughy TL, Brooks VL (2006). Deoxycorticosterone acetate–salt rats hypertension and sympathoexcitation driven by increased NaCl levels. Hypertension 47: 680-685. Olah ME, Stiles GL (2000). The role of receptor structure in determining adenosine receptor activity. Pharmacol Therapeut 85: 55-75. Olanrewaju HA, Mustafa SJ (2000). Adenosine A2A and A2B receptors mediated nitric oxide production in coronary artery endothelial cells. General Pharmacology 35: 171177. Ongini E, Fredholm BB (1996). Pharmacology of adenosine A2A receptors. Trends in Pharmacological Sciences 17: 364-372. Pang CCY (2001). Autonomic control of the venous system in health and disease: effects of drugs. Pharmacology & Therapeutics 90: 179-230. Park J, Galligan JJ, Fink GD, Swain GM (2010). Alterations in sympathetic neuroeffector transmission to mesenteric arteries but not veins in DOCA-salt hypertension. Auton Neurosci 152(1-2): 11-20. Park J, Galligan JJ, Fink GD, Swain GM (2007). Differences in sympathetic neuroeffector transmission to rat mesenteric arteries and veins as probed by in vitro continuous amperometry and video imaging. J Physiol 584(3): 819-834. ! "#! Park J, Show Y, Quaiserova V, Galligan JJ, Fink GD, Swain GM (2005). Diamond microelectrodes for use in biological environments. Journal of Electroanalytical Chemistry 583: 56-58. Patricia M Kearney MW, Kristi Reynolds, Paul Munter, Paul K Whelton, and Jiang He (2005). Global burden of hypertension: analysis of worldwide data. The Lancet 365(9455): 217-223. Perez-Rivera AA, Fink GD, Galligan JJ (2003). Increased reactivity of murine mesenteric veins to adrenergic agonists: functional evidence supporting increased α1adrenoceptor reserve in veins compared with arteries. The Journal of Pharmacology and Experimental Therapeutics 308: 350-357. Philipp M, Brede M, Hein L (2002). Physiological significance of alpha2-adrenergic receptor subtype diversity: one receptor is not enough. Am J Physiol Regulatory Integrative Comp Physiol 283: R287-R295. Pintoa YM, Paula M, Ganten D (1998). Lessons from Rat Models of Hypertension from Goldblatt to Genetic Engineering Cardiovascular Research 39(1): 77-88. Ralevic V (2000). Sympathoinhibition by adenosine A1 receptors, but not P2 receptors, in the hamster mesenteric arterial bed. European Journal of Pharmacology 387(3): 287293. Ralevic V, Burnstock G (1998). Receptors for purines and pyrimidines. Pharmacol Rev 50(3): 413-492. Reid IA (2012). Chapter 17. Vasoactive Peptides. In: Basic & Clinical Pharmacology, Katzung BG, Masters SB, Trevor AJ (eds). New York: McGraw-Hill. Reid JL, Zivin JA, Kopin IJ (1975). Central and peripheral adrenergic mechanisms in the development of deoxycorticosterone-saline hypertension in rats. Circulation Research 37: 569-579. Rodgers A (2002). The World Health Report 2002-Reducing risks, Promoting Healthy Life. Geneva, Switzerland: Wolrd Health Organization. Rothe CF (1983). Reflex Control of Veins and Vascular Capacitance. Physiological Reviews 63(4): 1281-1342. Safar ME, London GM (1987). Arterial and venous compliance in sustained essential hypertension. Hypertension 10: 133-139. Schenk J, McNeill JH (1992). The Pathogenesis of DOCA-Salt Hypertension. J Pharmacol Toxicol 27(3): 161-170. ! "#! Schlaich MP, Lambert E, Kaye DM, Krozowski Z, Campbell DJ, Lambert G, et al. (2004). Sympathetic Augmentation in Hypertension: Role of Nerve Firing, Norepinephrine Reuptake, and Angiotensin Neuromodulation. Hypertension 43: 169175. Schwartz DD (1997). Activation of alpha-2 adrenergic receptors inhibits norepinephrine release by a pertussis toxin-insensitive pathway independent of changes in cytosolic calcium in cultured rat sympathetic neurons. J Pharmacol Exp Ther 282: 248-255. Schwarzenbrunner U, Schmidle T, Obendorf D, Scherman D, Hook V, Fischer-Colbrie R, et al. (1990). Sympathetic axons and nerve terminals: the protein composition of small and large dense-core and of a third type of vesicles. Neuroscience 37: 819-827. Shen W-K, Kurachi Y (1995). Mechanisms of adenosine-medated actions on cellular and clinical cardiac electrophysiology. Mayo Clin Proc 70: 274-291. Simon G, Pamnani MB, Dunkel JF, Overbeck HW (1975). Mesenteric hemodynamics in early experimental renal hypertension in dogs. Circulation Research 36: 791-798. Simon G, Pamnani MB, Overbeck HW (1976). Decreased venous compliance in dogs with chronic renal hypertension. Proc Soc Exp Biol Med 152: 122-125. Smyth L, Bobalova J, Ward SM, Keef KD, Mutafova-Yambolieva VN (2000). Cotransmission from sympathetic vasoconstrictor neurons: differences in guinea-pig mesenteric artery and vein. Autonomic Neuroscience 86: 18-29. Smyth LM, Bobalova J, Mendoza MG, Lew C, Mutafova-Yambolieva VN (2004). Release of beta-Nicotinamide Adenine Dinucleotide upon Stimulation of Postganglionic Nerve Terminals in Blood Vessels and Urinary Bladder. J Biol Chem 279(47): 4889348903. Smyth LM, Breen LT, Mutafova-Yambolieva VN (2006). Nicotinamide adenine dinucleotide is released from sympathetic nerve terminals via a botulinum neurotoxin Amediated mechanism in canine mesenteric artery. Am J Physiol Heart Circ Physiol 290: H1818-H1825. Soares-da-Silva P (1988). Evidence for dopaminergic co-transmission in dog mesenteric arterial vessels. Br J Pharmacol 95: 218-224. Srinivas M, Song Y, Shryock JC, Belardinelli L (1998). Miduturu Srinivas, Yejia Song, John C. Shryock, and Luiz Belardinelli*. Drug Development Reseach 45(3-4): 420-426. Starke K (1987). Presynaptic α-autoreceptors. Rev Physiol Biochem Pharmacol 107: 73-146. ! "#! Stjarne L (1989). Basic mechanisms and local modulation of nerve impulse-induced secretion of neurotransmitters from individual sympathetic nerve varicosities. Rev Physiol Biochem Pharmacol 112: 1-137. Stjärne L, Stjärne E (1988). Evidence that α2-agonists may inhibit sympathetic transmitter secretion by an effect 'upstream' of varicosities. Acta Physio Scand 133: 433-434. Storr M, Thammer J, Dunkel R, Schusdziarra V, Allescher H-D (2002). Modulatiory effect of adenosine receptors on the ascending and decending neural reflex responses of rat ileum. BMC Neuroscience 3(21). Swartz KJ (1993). Modulation of Ca2+ channels by protein kinase C in rat central and peripheral neurons: disruption of G Protein-mediated Inhibition. Neuron 11: 305-320. Tabrizchi R, Bedi S (2001). Pharmacology of adenosine receptors in vasculature. Pharmacol Therapeut 91: 133-147. Takeda K, Buñag RD (1980). Augmented sympathetic nerve activity and pressor responsiveness in DOCA hypertensive rats. Hypertension 2: 97-101. Talaia C, Morato M, Quintas C, Gonçalves J, Queiroz Gr (2011). Functional crosstalk of prejunctional receptors on the modulation of noradrenaline release in mesenteric vessels: a differential study of artery and vein. Eur J Pharmacol 652: 33-39. Talaia C, Queiroz G, Pinheiro H, Moura D, !alves JG (2006). Involvement of G-protein βγ subunits on the influence of inhibitory α2-autoreceptors on the angiotensin AT1-receptor modulation of noradrenaline release in the rat vas deferens. Neurochem Int 49: 698707. Talaia C, Queiroz G, Quintas C, Gonçalves J (2005). Interaction between adenosine A2B-receptors and α2-adrenoceptors on the modulation of noradrenaline release in the rat vas deferens: possible involvement of a group 2 adenylyl cyclase isoform. Neurochem Int 47: 418-429. Talukder MA, Morrison RR, Ledent C, Mustafa SJ (2003). Endogenous adenosine increases coronary flow by activation of both A2A and A2B receptors in mice. Journal of Cardiovascular Pharmacology 41(4): 562-570. Teng B, Ledent C, Mustafa SJ (2008). Up-regulation of A2B adenosine receptor in A2A adenosine receptor knockout mouse coronary artery. Journal of Molecular and Cellular Cardiology 44(5): 905-914. ! "#! Todorov LD, Mihalova-Todorova S, Westfall TD, Sneddon P, Kennedy C, Bjur RA, et al. (1997). Neuronal release of soluble nucleotidase and their role in neurotransmitter inactivation. Nature 387(1): 76-79. Todorov LD, Mihalova-Todorova ST, Bjur RA, Westfall DP (1999). Differential cotransmission in sympathetic nerve: role of frequency of stimulation and prejunctional autoreceptors. The journal of Pharmacology and Experimental Therapeutics 290: 241246. Tsuru H, Tanimitsu N, Hirai T (2002). Role of perivascular sympathetic nerves and regional differences in the features of sympathetic innervation of the vascular system. Jpn J Pharmacol 88: 9-13. Wakade AR, Przywara DA (1995). Calcium-independent modulation of neurotransmitter release. In: Powis DA, Bunn SJ (ed)^(eds). Neurotransmitter release and its modulation, 1 edn. Cambridge: Cambridge University Press. p^pp 200-213. Watts SW, Fink GD, Northcott CA, Galligan JJ (2002). Endothelin-1-induced venous contraction is maintained in DOCA-salt hypertension; studies with receptor agonists. Br J Pharmacol 137: 69-79. Welch WJ (2002). Adenosine A1 receptor antagonists in the kidney: effects in fluidretaining disorders. Current Opinion in Pharmacology 2: 165-170. Westfall TC, Yang CL, Curfman-Falvey M (1995). Neuropeptide-Y-ATP interactions at vascular sympathetic neuroeffector junction. J Cardiovasc Pharm 26: 682-687. Xu H, Fink GD, Galligan JJ (2002). Nitric oxide-independent effects of tempol on sympathetic nerve activity and blood pressure in DOCA-salt rats. Am J Physiol Heart Circ Physiol 283: H885-H892. Yamamoto J, Goto Y, Nakai M, Ogino K, Ikeda M (1983). Circulatory pressure-volume relationship and cardiac output in DOCA-salt rats. Hypertension 5: 507-513. Yu M, Gopalakrishnan V, McNeill JR (2001). Role of endothelin and vasopressin in DOCA-salt hypertension. Br J Pharmacol 132: 1447-1454. Zigmond RE, Schwarzschild MA, Rittenhouse AR (1989). Acute regulation of tyrosine hydroxylase by nerve activity and by neurotransmitters via phosphorylation. Ann Rev Neurosci 12: 415-416. ! "#! CHAPTER 2 RESEARCH SPECIFIC AIMS AND GENERAL METHODS ! "#! 2.1 Overall research goal Adenosine, a final ATP metabolite, is a signaling molecule in the cardiovascular system. Adenosine receptors are G-protein coupled receptors and there are four subtypes: A1, A2A, A2B, and A3 but receptors controlling arterial and venous tone, particularly in the mesentery, have not been identified. This is important because small mesenteric arteries (MA) and veins (MV) contribute to the resistance and capacitance functions of the peripheral circulation and identification of the adenosine receptor subtypes in this vascular bed is still unclear (Tabrizchi et al., 2001). Thus, my first goal is to identify the expression of adenosine receptors expressed by the sympathetic nerves supplying MA and MV. The second goal is to determine the function of prejunctional adenosine A1 and A2A receptors in modulating adrenergic neurotransmission. The last part of my research goal was to test the hypothesis that adenosine receptor dysfunction contributes to increased NE release from sympathetic nerves supplying MA and MV in deoxycorticosterone acetate (DOCA)-salt hypertensive rats. The overall goal is described in a summary diagram (Fig. 21). ! "#! Figure 21. Overall goals. Aim 1, Characterize adenosine receptors expressed by sympathetic nerves supplying MA and MV (#1). Aim 2, Determine the function of the prejunctional A1 receptors (A1) in modulating NE release; test if A1R dysfunction contributes to elevated NE release in DOCA-salt hypertension (#2). Aim 3, Determine the role of prejunctional Adenosine 2A receptors (A2A) in sympathetic transmission and tested if A2A receptor dysfunction contributes to elevated NE availability in DOCA-salt hypertension. !1, !1-adrenergic receptor; !2, !2-adrenergic receptor, ado, adenosine; NET, NE transporter; Pan1, pannexin-1; VSMC vascular smooth muscle cells. Solid lines indicate established mechanism. Dashed lines indicate equivocal mechanisms. ! "#! 2.2 Specific Aims Specific aim #1 Test the hypothesis that A1 and A2A adenosine receptors localize to sympathetic nerves innervating MA and MV (prejunctional site), while A1Rs are localized to venous but not arterial smooth muscle cells (postjunctional site). Specific aim 1a: Localize all adenosine receptor subtypes on the sympathetic nerves supplying mesenteric arteries and veins. Immunohistochemistry (IHC) for all adenosine subtypes (A1, A2A, A2B, and A3) and tyrosine hydroxylase, a marker for sympathetic nerve will be used to determine the co-localization of the prejunctional adenosine receptors on perivascular sympathetic nerves. The expression of the prejunctional adenosine receptors are not determined by PCR or western analysis, as these two techniques could not distinguish the pre-and postjunctional adenosine receptors. My strategy is to combine the IHC data with functional studies to reveal the role of adenosine in modulating sympathetic transmission. Specific aim 1b: Identify postjunctional adenosine receptors mediating MV constriction in vitro by using agonists and antagonists for adenosine receptor subtypes. ! "#! Specific aim #2 Examine the inhibitory modulation by the prejunctional A1R on sympathetic NE release and test the hypothesis that A1R dysfunction causes an increase in NE release in DOCA-salt hypertension. Specific aim 2a: Establish concentration response curves (CRCs) for adenosine receptor agonists for inhibition of neurogenic constrictions of MA and MV in vitro. Adenosine receptor antagonists, and adenosine transporter blocker are used for further receptor characterization. Specific aim 2b: Measure NE release and vasoconstriction in vitro using continuous amperometry with carbon-fiber microelectrode and video microscopy, respectively. Adenosine receptors on sympathetic nerves will be studied in isolated MA and MV in vitro. Adenosine receptor antagonist and adenosine transporter blocking agents are used to reveal role of endogenous adenosine. Specific aim #3 Determine role of the prejunctional A2A receptor in adrenergic transmission and test if the A2AR impaired function contributes to DOCA-salt hypertension. Specific aim 3a: test the hypothesis that activation of A2AR decreases NE availability in the neuroeffector junction by increasing the activity of the NE transporter. ! ""! Specific aim 3b: Test the hypothesis that A2AR receptor dysfunction causes an increase in neurogenic NE availability in the neuroeffector junction of DOCA-salt hypertension. Measurement of NE release and vasoconstriction in vitro are accomplished using amperometry and video microscopy respectively. A2AR agonists, and antagonists are used reveal the role of the A2AR in modulating adrenergic transmission and its alteration in DOCA-salt hypertensive rats. 2.3 Research design and methods 2.3.1 Specific aim 1 Establish the expression of adenosine receptor subtypes on sympathetic nerves innervating MA and MV (prejunctional) using immunohistochemistry (IHC). Rationale I use IHC to determine receptor localization by observing the colocalization of adenosine receptors and marker proteins for sympathetic nerves (TH). IHC data reveal the distribution of adenosine receptors in mesenteric blood vessels and their sympathetic nerve supply. The IHC preliminary results provide background for further functional studies. 2.3.2 Immunohistochemistry protocol Prejunctional receptor staining: MA and MV from DOCA-salt and sham rat were excised, cleaned of access fat and connective tissue in ice-cold 0.1M PBS. Vessels are perfused with cold saline to remove blood from the lumen. Tissues ! "#! were cut in1 cm lengths and fixed in Zamboni fixative for 2 hours on ice. Then, the tissues were washed 3x with 0.1M PBS and then incubated in 4% mixture of goat-sheep blocking serum for 1 hour. Then, the vessels were incubated with antibodies against particular adenosine receptor and TH to show localization of adenosine receptor and sympathetic nerve fibers, respectively. To control for non-specific binding, control vessels were incubated with no primary antibodies. To prevent contamination, the IHC for particular adenosine receptor subtypes were performed at different day. Next, the vessels were incubated with the secondary antibodies-conjugated to fluorescent markers. Specimens were viewed under a confocal microscope. 2.3.3 Specific aim 2 Determine if activation of the prejunctional A1 receptors inhibits NE release and test the hypothesis that A1R dysfunction causes an increase in NE release in DOCA-salt MA and MV. The pharmacological properties of adenosine receptors on sympathetic nerves will be studied in isolated MA and MV maintained in vitro. Neurogenic vasoconstriction and real time NE release are measured by using video microscopy and amperometry respectively. Rationale Although measuring nerve-mediated vasoconstriction in vitro is an indirect method to measure changes in NE release, this method is still useful for electro-inactive compounds (adenosine), which cannot be measured by amperometry (see below). Another benefit of this method is to screen the ! "#! pharmacology responses before doing amperometry, which is more challenging than measuring blood vessel diameter alone. 2.3.4 Protocol for measurement of vasoconstriction in vitro Tissue preparation: Rats were killed using a lethal pentobarbital injection (100 mg ip). The entire mesentery was removed and tertiary MA and MV (120-300 !m) are dissected out and transferred to a small recording chamber. The tissue was pinned flat with 50 !m diameter stainless steel pins. MA and MV were carefully cleaned from adipose and connective tissues under dissecting microscope. The Tissue was superfused continuously at flow rate of 3 ml/min with warmed (36oC), oxygenated (95% O2, 5% CO2) Krebs’ solution containing mM: 117 NaCl, 4.7 KCl, 2.5 CaCl2, 1.2 MgCl2, 25 NaHCO3, 1.2 NaHPO4, and 11 dextrose. The output of black and white video camera attached to the microscope was fed to a PCVision Plus frame-grabber board (Imagine Technology, Woburn, MA) mounted in a personal computer. The video images were recorded and analyzed using Diamtrak® software, which tracks the distance between the outer edges of the vessel in the observation field. The sampling rate was 10 Hz and changes in blood vessel diameter of 0.5 !m were resolved. The tissue was allowed to equilibrate for 30 minutes before beginning experiment. During this time, MA and MV relax to stable resting diameter. Drug application: Agonists are applied using a system three-way stopcock so that superfusing Krebs’ solution can be changed to one containing a known concentration of drug. It took 4 minutes for drugs to reach the tissue. Vessels ! "#! were first tested with maximum concentration of NE (10 !M for MA, 1 !M for MV) 3 times to verify stable responses. Then, agonist CRCs were constructed using cumulative addition for agonists that do not cause desensitization (e.g. NE, PE). For agonists that cause desensitization, a control experiment was done to determine recovery time (e.g. 15 minutes for ",#-me-ATP, or 45 min for CPA) and the CRCs were constructed using noncumulative addition of each drug concentration. Drugs were applied for 10 minutes and washed for optimum interval between applications of each agonist concentration with normal Krebs’ solution. Antagonists were applied for 20 minutes prior to testing their effect on agonist or nerve-mediated responses in the continued presence of antagonist. Transmural stimulation of perivascular nerves: This protocol was for measurement of contractile response mediated by nerve stimulation. Two wire electrodes were placed parallel to the longitudinal axis of the vessel under the Krebs’ solution level, but not touching the tissue. Parameters for nerve stimulation were 30 stimuli, stimulus duration of 0.5 ms, frequency 0.2-30 Hz and voltage of 60 V. The neurogenic origin of constrictions caused by electrical stimulation was verified in each preparation by initially demonstrating that constriction caused by 20-Hz stimulus was completely blocked by the Na+ channel blocker, tetrodotoxin (TTX, 0.3 !M). Preparations in which the 20-Hz constriction was not blocked by TTX were discarded. Peak constriction was measured using Diamtrak® software. ! "#! Table 4. Agonists and antagonists for rat adenosine receptors Subtypes A1 A2A A2B A3 A2A/A2B Selective Agonist CPA, CCPA, R-PIA Selective Antagonist DPCPX CGS 21680 SCH58261 - - IB-MECA BWA 1433, MRS 1523 NECA 8-p-sulfophenyltheophilline, ZM 241385 Data analysis: The frequency response curves (0.2-30 Hz) and CRCs were created and half maximal effective concentrations (EC50) were determined using a non-linear fitting routine and a logistic equation (Origin 8.0 software). Comparisons of tissues from control and DOCA-salt rats will be made. Statistical differences will be assessed using Student’s t-test and analysis of variance with P<0.05 indicating significance differences among treatment groups. 2.3.5 Specific aim 3 Determine the function of the prejunctional A2A receptors on sympathetic neurotransmission and test the hypothesis that dysfunction of prejunctional A2ARs contributes to increased NE release from sympathetic nerves in DOCA-salt hypertensive rats. Electrically-evoke NE release is measured by using NE amperometry. Kinetics of NE release and clearance are calculated. Rationale Continuous amperometry is used to detect catecholamine release from nerve endings in the central and peripheral nervous system (Brock et al., ! "#! 1997; Brock et al., 2000; Brock et al., 2004; Dugast et al., 2002; Dunn et al., 1999; Gonon et al., 1993; Park et al., 2007; Yavich et al., 2005). This technique takes advantage of the electroactive property of catecholamines, like NE. Amperometry detects small quantities of NE by measuring the oxidation current (Teschemacher, 2005) produced by the redox reaction (Fig. 22). This technique provides real-time information on the local concentration of NE near the neuroeffector junction in the vasculature. HPLC and radioligand detection are also used to measure NE release in response to nerve stimulation. However, these methods cannot measure NE release in real time and they require long periods of nerve stimulation to evoke sufficient NE release for detection. Our laboratory has successfully detected NE released from sympathetic nerve stimulation in rat mesenteric blood vessels using amperometry (Dong et al., 2009; Park et al., 2007; Park et al., 2006). Figure 22. Redox reaction of norepinephrine. ! "#! 2.3.6 Protocol for amperometric measurement of NE Preparation of recording chamber: A plastic chamber was designed for holding two electrodes (reference and counter electrodes) on each side of the chamber. A recording chamber was prepared by gluing a coverslip to the bottom of a customized plastic chamber (Fig.23) using superglue and let dry. The next day, the bottom of chamber was filled with Sylgard, vacuums the bubbles for 5 minutes, and let dry for 2-3 days at room temperature. Sylgard is non-toxic and broken coverslip is easily to replace. Figure 23. Recording chamber for amperometry. The chamber includes two holders for reference and counter electrodes (a,b), inlet at lower level (c), and outlet at higher level (d). The bottom of the chamber is made from ® clear glass cover slip and topped with clear silicone Sylgard . ! "#! Carbon fiber microelectrode preparation: The electrode was produced as previously described (Dong, 2009; Park et al., 2007). Briefly, the carbon fiber was insert into a propylene pipet tip (Dot Scientific, Michigan, #PF2411). Then, the electrode tip was carefully melted by the heated-puller machine (Sutter Instrument, Model P-30, CA, USA), and gently pulled by a flat-end forceps to create a slight angle (Fig. 24a) permitting a horizontal contact to the vessel. The carbon fiber tip was then cut to approximately 700µm length under a microscopy by using a glass scale. Each strip on the scale indicates 300 µm in width). Two ends of the silver wire were peeled off insulation (Fig. 24b,d). One end of the wire was connected to the carbon fiber by silver-epoxy glue (Fig. 24b, Fig. 25) and the other end was left for a connection with the potentiostat. The open gap between the pipet and silver wire is sealed with clear epoxy glue (Fig. 24c, Fig. 25). ! "#! Figure 24. Carbon fiber microelectrode. a) Carbon-fiber electrode tip is sealed with slight angle and cut into 700µm, b) Carbon fiber is connected to the silver wire by silver epoxy, c) Electrode gap is sealed with epoxy glue, d) the other end of the electrode was blared of insulation for potentiostat connection. ! "#! Figure. 25 Schematic diagram of the tip of a carbon-fiber microelectrode. All items are not drawn to scale. Modified from (Mundorf et al., 2002). ! "#! Tissue preparation: Tertiary MA and MV were isolated, cleaned of adipose and connective tissue, placed into a small silicone chamber, and perfused with warmed (36oC) oxygenate (95% O2, 5% CO2) Krebs’ solution at flow rate 2.5 ml/min (Fig. 26A). Amperometric detection: Firstly, a platinum wire counter electrode and a commercial “no leak” Ag-AgCl (3 M KCl, model EE009, Cypress System Inc., USA) reference electrode were mounted in the chamber to complete the electrochemical circuit. Carbon fibers working electrode was briefly dipped into distilled isopropyl alcohol (Ranganathan et al., 1999) and affixed to a micromanipulator (MP-1, Narishige USA). The working electrode was connected to the electrode holder and mounted into the micromanipulator. The electrode was lower just above the surface of the blood vessel by using micromanipulator. All electrodes were then connected to potentiostat. The microelectrode was then adjusted the position parallel to the blood vessel so that NE flux from nearby release site was sensed by the electrode surface. The microelectrode was pressed gently against the vessel to enable the microelectrode to main contact with the vessel during stimulation-evoked constrictions (Fig. 27A,B). Amperometric measurements were made with an Omni 90 analog potentiostat (Cypress Systems Inc.), a mini digi analog-to-digital converter and a computer running Axon 9.0 (Axon Instruments, Union City, CA, USA). Data were obtained at 100 Hz sampling rate. An applied potential of 400 mV was used to detect NE current because this is approximate voltage for NE to be oxidized at masstransfer limited rate (Park et al., 2007). Current recording was low pass filtered at ! ""! a time constant of 200 ms (5 Hz) before being digitized using A/D converter at a sampling rate of 100Hz. Data were stored on the computer for further analysis (Fig. 26). Figure 26. Instrumental setup for amperometric NE detection. A, overall amperometry setup. B, Magnification part of carbon-fiber electrode positioned on the vessel and connected to the potentiostat (Dong, 2009). ! "##! Focal nerve stimulation: Short trains of electrical stimulation will be applied to trigger NE release using bipolar focal stimulation electrode positioned along the surface of the vessel. The electrode will be placed at a distance of 200 !m from the carbon fiber to minimize the stimulus artifact in the current recording (Fig. 29 A,B). Trains of 60 stimuli (0.3 ms pulse width, 60 V) at frequency of 0.5 to 20Hz will be used to stimulate transmitter release. Drug Application: Drugs (Table 5) will be added to the Krebs’ buffer and applied for 20 min before assessing their effects on nerve-mediated responses. Figure 27. Amperometric detection of NE release by a mesenteric vein. A, The vessel is gently cleaned off fat and connective tissue permitting electrode contact. A bipolar stimulating electrode (a) and a carbon fiber microelectrode (b) are positioned on the vessel surface. Nerve stimulation (ns) is applied to evoke NE release and contraction. C-D, NE oxidation current (C) and vasoconstriction (D) are detected in real-time. pA, pico ampere; s, second; µm, micrometer. ! "#"! 2.3.7 How to minimize unstable baseline? It is necessary to acquire stable baseline before beginning any amperometric experiment. Having stable baseline allow one to measure current amplitude and kinetics of the oxidation current accurately. Here are some suggestions when experiencing unstable baseline. 1. Check if the tubing is cracked especially in the pump connection areas. Cracked tubing causes unstable superfusing rate resulting in unstable baseline. 2. Check the solution volume in the recording chamber. Low level of solution in the chamber causes noise. I found that the solution should cover approximately one third of reference electrode to stabilize amperometric baseline. 3. Slow down the flow rate. Too fast flow rate causes noises, but too slow flow rate kills the tissue. My superfusing flow rate was set at 2.5 ml/minute. 4. Carbon-fiber electrodes that are “leaky” with poor seal around the carbon fiber can cause unstable baseline and alter the oxidation current characteristics. Switching to a new carbon electrode may be needed. 2.3.8 When is carbon fiber electrode no longer useful? Ionic composition in Krebs solution appears to deteriorate the surface of carbon fiber microelectrode. The oxidation current is found to decay over time after used for several days. The ! "#$! microelectrode may require reconditioning with purified isopropyl alcohol (distilled and kept with carbon powder to precipitate impurities) to improve microelectrode sensitivity. The control peak amplitude of NE oxidation current obtained from sham arteries and veins are 12.7±4.8 and 14.7±6.0 pA (n=53 and 48, respectively) and 13.3±3.8 and 17.3±4.6 pA from DOCA arteries and veins (n=19 and 20, respectively). Data are mean ± S.D. This electrically-evoked NE releases was performed under a brief train of 60 stimuli at stimulation parameter of 10 Hz frequency, 0.5 ms pulse duration, and 60-80 V intensity. These oxidation currents were measured by using carbon fiber electrode with 700± 150 µm in length, which equals to 2.5 blocks of glass scale (1block = 300 µm). Control peak current amplitude below the average range in part indicates loss of electrode sensitivity and the carbon electrode should be discarded. It should be noted that there are several factors that can determine the amplitude of oxidation current. Healthy isolated blood vessel is the key factor of getting a pleasurable oxidation current. A healthy vessel can be prepared by gently and fast cleaning of connective tissue and quickly incubating the tissue with oxygenated warm Krebs. Furthermore, short tip of carbon electrode detect smaller amount of released NE resulting in low oxidation current; hence small current may result from the tip of electrode is far below 700 µm. Moreover, the animal itself could be a problem. Some very hypertensive DOCA-salt blood vessels exhibit low current or probably no response to nerve stimulation as the nerves are dramatically damaged. Thus, to verify if the low oxidation current is caused by the less-sensitive electrode or an unhealthy vessel, one may have to compared the current response with the ! "#$! contractile response. The contractile response for sham arteries and veins are 20.9±5.4 and 33.6±8.2% (n=40 and 34, respectively) and for DOCA arteries and veins are 19.8±7.1 and 27.0±9.4% (n=15 and 14, respectively). The low oxidation current, but high contraction response may be another key indicator for discarding a carbon-fiber electrode. 2.3.9 General methods Animals: All animal procedures were approved by the Institutional Animal Care and Use Committee at Michigan State University. Upon arrival at the animal care facility, rats are acclimatized for 2-3 days in clear plastic boxes with wood chip bedding in temperature and humidity-controlled rooms with a 12:12 light-dark cycle. Rats are fed with pelleted rat chow (Harlan/Tekled 8640 Rodent Diet) and ad libitum water. DOCA-salt hypertensive rats: Male Sprague-Dawley rats (250-275g) were used for all of the studies. DOCA-salt and control (sham) rats were prepared as previously published procedure (Luo et al., 2003; Watts et al., 2002). Briefly, DOCA-salts rats received unilateral nephrectomy, Deoxycorticosterone acetate (DOCA) subcutaneous implantation, and 1%NaCl+0.2%KCl drinking water. Sham rats only undergo unilateral nephrectomy and received distilled drinking water. After 4 weeks of recovery, the animal’s mean arterial blood pressure was measured by tail-cuff method. The blood pressure above 150 mmHg is considered hypertensive. ! "#$! Data analysis: Data were reported as mean ± SE with ‘n’ values indicating the number of rats from which the data will be obtained. Concentration-response data were fitted using non-linear regression (Origin 8.0 or Graphpad Prism) in order to determine EC50 values. Statistical differences between treatment groups were determined using one- or two-way ANOVA, and Student’s t-test for paired or unpaired data, as appropriate. Differences were considered significance when P<0.05. 2.4 Ethical aspects of the proposed research To study the expression and functional properties of adenosine receptor in normotensive and hypertensive rats, the use of animal subjects is necessary for this project. Every attempt was made to reduce animal use and expense in accordance with AAALAC accreditation. 2.4.1 Vertebrate animal subjects Male Sprague Dawley rats (8-9 weeks, 250-275 g) were used in this study. Rats were obtained from Charles River Laboratories, Inc. Number of rats used in specific aim #1: 10 rats (5 sham, 5 DOCA) for immunohistochemistry (IHC). Tissue from 1 rat was used for staining all adenosine receptor subtypes from artery and vein. This will include both positive and negative controls. Experiments were done in triplicates. Number of rats used in specific aim #2: approximately 100 rats for contractility and amperometry studies (50 sham, 50 DOCA) ! "#$! Number of rats used in specific aim #3: approximately 100 rats for amperometry studies (50 sham, 50 DOCA) It is important to note that I work as part of a larger group of investigators who study DOCA-salt hypertension at Michigan State University. It is a policy of our group that all animals are shared amongst the group. As I only used some of the mesenteric blood vessels from each rat, I shared these animals with 4 other students. The numbers of rats indicated above were not for my studies only but these animals supported 3 other ongoing projects as well. This arrangement further reduces the number of rats used in our research group. Justification of animal use: The justification for the choice of using rats is that great amount of studies have been performed in rat. This provides a logical basis for prediction and interpretation of data. I reduced the number of rats used whenever it was possible. Moreover, I shared the subjects with our collaborators who also study hypertension using the same model to minimize the animal used. 2.4.2 Veterinary Care Animal husbandry was provided by University Laboratory Animal Resources (ULAR) staff under the guidance of supervisors who are certified as Animal Technologists by the American Health Association for Laboratory Animal Science (AALS). Animals were cared for and checked on everyday by members of Dr. Galligan’s laboratory. ULAR staff provided veterinary care. The animal care and use program conforms to the standards in The Guild for the Care and Use of Laboratory Animals DHEW pub. No. (NIH 78-23, Revised 1996). This includes ! "#$! periodic surveillance of animal facilities, review of all funded projects for human use of animals, and the appropriate use of surgical anesthesia, analgesics, and tranquilizers. 2.4.3 Euthanasia Euthanasia was performed by giving a high dose of pentobarbital (50 mg/kg, intraperitonial injection). This protocol is consistent with the recommendation of the panel on euthanasia of the American Veterinary Medical Association. ! ! "#$! BIBLIOGRAPHY ! "#$! BIBLIOGRAPHY Brock JA, Bridgewater M, Cunnane TC (1997). b-Adrenoceptor mediated facilitation of noradrenaline and adenosine 5'-triphosphate release from sympathetic nerves supplying the rat tail artery. British Journal of Pharmacology 120: 769-776. Brock JA, Dunn WR, Boyd NSF, Wong DKY (2000). Spontaneous release of large packets of noradrenaline from sympathetic nerve terminals in rat mesenteric arteries in vitro. British Journal of Pharmacology 131: 1507-1511. Brock JA, Tan JHC (2004). Selective modulation of noradrenaline release by a2adrenoceptor blockade in the rat-tail artery in vitro. British Journal of Pharmacology 142: 267-274. Dong H Disruption of norepinephrine release and clearance mechanisms in sympathetic nerves associated with mesenteric arteries but not veins in saltsensitive hypertension. Ph.D Dissertation, Michigan State University, EastLansing, 2009. Dong H, Wang S, Liu A, Galligan JJ, Swain GM (2009). Drug effects on the electrochemical detection of norepinephrine with carbon fiber and diamond microelectrodes. Journal of Electroanalytical Chemistry 632: 20-29. Dugast C, Cespuglio R, Suaud-Chagny MF (2002). In vivo monitoring of evoked noradrenaline release in the rat anteroventral thalamic nucleus by continuous amperometry. journal of Neurochemistry 82: 529–537. Dunn WR, Brock JA, Hardy TA (1999). Electrochemical and electrophysiological characterization of neurotransmitter release from sympathetic nerves supplying rat mesenteric ateries. British Journal of Pharmacology 128: 174-180. Gonon F, Bao JX, Msghina M, Suaud-Chagny MF, Stjarne L (1993). Fast and local electrochemical monitoring of noradrenaline release from sympathetic terminals in isolated rat tail artery. journal of Neurochemistry 60: 1251-1257. Luo M, Hess MC, Fink GD, Olson LK, Rogers J, Kreulen DL, et al. (2003). Differential Alterations in Sympathetic Neurotransmission in Mesenteric Arteries and Veins in DOCA-salt Hypertensive Rats. Autonomic Neuroscience 104(1). ! "#$! Mundorf ML, Wightman RM (2002). Amperometry and Cyclic Voltammetry with Carbon Fiber Microelectrodes at Single Cells. Current Protocols in Neuroscience: 6.14.11-16.14.22. Park J, Galligan JJ, Fink GD, Swain GM (2007). Differences in sympathetic neuroeffector transmission to rat mesenteric arteries and veins as probed by in vitro continuous amperometry and video imaging. Journal of Physiology 584(3): 819-834. Park J, Galligan JJ, Fink GD, Swain GM (2006). In vitro continuous amperometry with a dimond microelectrode coupled with video microscopy for simultaneously monitoring endogenous norepinephrine and its effect on the contractile response of a rat mesenteric artery. Analytical chemistry 78(19): 6756-6764. Ranganathan S, Kuo T-C, McCreery RL (1999). Facile preparation of active glassy carbon electrodes with activated carbon and organic solvents. Analytical chemistry 71: 3574-3580. Tabrizchi R, Bedi S (2001). Pharmacology of adenosine receptors in the vasculature. Pharmacology & Therapeutics 91: 133-147. Teschemacher AG (2005). Real-time measurements of noradrenaline release in periphery and central nervous system. Autonomic Neuroscience: Basic and Clinical 117: 1-8. Watts SW, Fink GD, Northcott CA, Galligan JJ (2002). Endothelin-1-induced venous contraction is maintained in DOCA-salt hypertension; studies with receptor agonists. British Journal of Pharmacology 137: 69-79. Yavich L, Ja¨ka¨la¨ P, Tanila H (2005). Noradrenaline overflow in mouse dentate gyrus following locus coeruleus and natural stimulation: real-time monitoring by in vivo voltammetry. Journal of Neurochemistry 95: 641-650. ! ! ""#! CHAPTER 3 IMPAIRED FUNCTION OF PREJUNCTIONAL ADENOSINE A1 RECEPTORS EXPRESSED BY PERIVASCULAR SYMPATHETIC NERVES IN DOCA-SALT HYPERTENSION ! """! 3.1 Abstract Increased sympathetic nervous system activity contributes to DOCA-salt hypertension in rats. ATP and norepinephrine (NE) are co-released from perivascular sympathetic nerves. NE acts at prejunctional !2-adrenergic receptors (!2-AR) to inhibit NE release and !2-AR function is impaired in DOCA-salt rats. Adenosine, an enzymatic ATP degradation product, acts at prejunctional A1 adenosine receptors (A1R) to inhibit NE release. We tested the hypothesis that prejunctional A1R function is impaired in sympathetic nerves supplying mesenteric arteries and veins of DOCA-salt rats. Electrically-evoked NE release and constrictions of blood vessels were measured in 6 vitro using amperometry and video microscopy. Adenosine and N -cyclopentyladenosine (CPA, A1R agonist) constricted mesenteric veins but not arteries. Adenosine and CPA (0.001-10 "M) inhibited neurogenic constrictions and NE release in mesenteric arteries and veins. DOCA-salt arteries were resistant to adenosine and CPA-mediated inhibition of NE release and constriction. The A2A adenosine receptor agonist, CGS21680 (0.01-0.1 µM) did not alter NE oxidation currents. We conclude that there are prejunctional A1Rs in arteries and both pre and postjunctional A1Rs in veins hence adenosine selectively constricts the veins. Prejunctional A1R function is impaired in arteries, but not veins, from DOCA-salt rats. Sympathetic autoreceptor dysfunction is not specific to !2-adrenergic receptors, but there is a more general disruption of prejunctional mechanisms controlling sympathetic neurotransmitter release. ! ""#! 3.2 Introduction !Adenosine is an ATP precursor and metabolite and an intercellular signaling molecule (Mark and Olah, 2001; Tabrizchi and Bedi, 2001). In the cardiovascular system, adenosine plays an important role in controlling cardiac output and vascular function (Tabrizchi and Bedi 2001; Shryock and Belardinelli, 1997). Considerable progress has been made in understanding adenosine receptor subtypes and their signaling mechanisms in the cardiovascular system. However, due to the breadth effects of adenosine, our understanding about adenosine receptor function in the sympathetic nerves supplying arteries and especially veins is less well developed, particularly in hypertension. This issue is important in mesenteric arteries and veins because they are densely innervated by sympathetic nerves (King et al., 2007). Sympathetic nerves supply blood vessels at the adventitial-medial border in arteries and they distribute more deeply into the media for veins (Pang, 2001; Birch et al., 2008). Sympathetic nervous system control of the mesenteric circulation contributes significantly to blood pressure regulation by two mechanisms. Firstly, sympathetic nerves regulate resistance in small mesenteric arteries (Kreulen, 2003; Rothe 1983; Fenger-Gron et al., 1995). Secondly, sympathetic nerves regulate capacitance of mesenteric veins which hold a large fraction of total blood volume and which provide 6070% of venous return to the heart (Martin et al., 1998; Pang, 2001). Venous constriction shifts blood volume from the high compliance capacitance veins to the low compliance resistance arteries causing an increase in arterial pressure (Fink, 2009). Adenosine receptors are G-protein coupled receptors (Mark and Olah, 2001; Morato et al., 2008). There are four types of adenosine receptor (A1 A2A, A2B, and ! ""#! A3)(Tabrizchi and Bedi, 2001; Fredholm et al., 2001) that are distinguished based on their ability to inhibit or stimulate adenylyl cyclase and by different agonist and antagonist binding profiles (Mark, and Olah, 2001; Stephen et al., 1994; Fredholm et al., 2003). We focused on adenosine A1 receptors (A1Rs) which couple to inhibition of norepinephrine release. A1Rs couple to the inhibitory G protein, Gi, leading to inhibition of adenylyl cyclase (Tabrizchi and Bedi, 2001; Linden, 2001; Ralevic and Burnstock, 1998). Activation of A1Rs in the nervous system inhibits Ca2+ currents resulting in suppression of neurotransmitter release (Ralevic and Burnstock, 1998). Sympathetic nerves supplying mesenteric arteries release norepinephrine, ATP and/or !nicotinamide adenine dinucleotide (!"NAD)(Burnstock, 2004; Smyth et al; 2004). Norepinephrine (NE), ATP and !"NAD can act on postjunctional !1 adrenergic receptors and P2X1 purinoceptors respectively to cause arterial smooth muscle contraction (Mutafova-Yambolieva et al., 2000; Smyth et al., 2004; Demel and Galligan, 2008). NE and ATP/!"NAD release is regulated by prejunctional !2-receptors (!2ARs) and A1Rs (Demel and Galligan, 2008; Illes et al., 1988; Ralevic, 2000; Rongen et al., 1996). The action of ATP is quickly terminated by enzymatic degradation to ADP, AMP, and adenosine as a final product (Todorov et al., 1997; 1999; Westfall et al., 2002). Adenosine binds to prejunctional A1Rs to inhibit NE and ATP/!"NAD release. Chronic treatment of rats with a non-selective adenosine receptor antagonist, 1,3-dipropyl-8sulphophenylxanthine (DPSPX), increased blood pressure and purinergic and adrenergic neurotransmission (Guinarães et al., 2003). This indicates that adenosine regulates blood pressure partly by acting at prejunctional adenosine receptors. Data from animal models and from human studies showed that increased sympathetic ! ""#! activation contributes to hypertension (Schlaich et al., 2004; Anderson et al., 1989). Previous work has shown that augmented sympathetic activity in DOCA-salt hypertensive rats is due partly to disruption of prejunctional !2AR function (deChamplain et al., 1987; Luo et al., 2004). However, it is not known if impairment of A1R function also occurs in hypertension. Thus, the purpose of this study was to determine if the disruption of prejunctional autoreceptors is specific to !2ARs or if there is a more general disruption of prejunctional autoreceptor function in hypertension. Furthermore, we tested the hypothesis that dysfunction of prejunctional A1Rs contributes to increased norepinephrine release from sympathetic nerves in DOCA-salt hypertensive rats. This model is a low rennin form of salt-sensitive hypertension that is driven by increased sympathetic nerve activity (Schenk and McNeil, 1992). Finally, there have been a number of studies of adenosine receptor function on periarterial sympathetic nerves but interactions of adenosine with sympathetic nerves supplying mesenteric veins in normotensive or hypertensive animals have not been studied. ! ""#! 3.3 Materials and Methods Preparation of DOCA-salt hypertensive rats Animals use protocols were approved by the Institutional Animal Care and Use Committee at Michigan State University. Adult male Sprague-Dawley rats (250-275g) were obtained from Charles River Laboratories (Portage, MI) and they were acclimated 2-3 days before entry into experimental protocols. Rat chow (Harlan/Teklad 8640 Rodent Diet) and tap water were provided ad libitum. Rats were housed in temperature and humidity controlled room with 12:12 hour dark-light cycle. The surgical procedures and drug treatment protocols for producing sham control and DOCA-salt hypertensive rats have been described in detail previously (Luo et al., 2004). After recovery from the surgical procedures, rats were housed under standard conditions for 4 weeks. DOCA-implanted rats received standard pelleted rat chow and drinking solution containing 1%NaCl+0.2%KCl in distilled water, while sham rats receive standard pelleted rat chow and tap water. Blood pressure was measured using tail-cuff plesmythography 3-5 days prior to experimentation. Rats with mean arterial pressure >150 mmHg were considered hypertensive. Tissue preparation for in vitro studies! 4 weeks after DOCA-salt or sham surgery, rats were euthanized with a lethal injection of pentobarbital (100 mg/kg i.p.). The mesentery was removed and transferred to a silicone elastomer-lined Petri dish with Krebs solution (mol/L): 117 NaCl, 4.7 KCl, 2.5 CaCl2, 1.2 MgCl2, 25 NaHCO3, 1.2 NaHPO4, and 11, glucose. Sections of tertiary mesenteric arteries (145-250!m outside diameter) and veins (190-390!m outside diameter) were dissected out and transferred to a recording chamber. The tissue was ! ""#! pinned flat with 50 !m-diameter stainless steel pins. Mesenteric arteries and veins were cleaned of adipose and connective tissue under a dissecting microscope. The chamber was mounted on the stage of an inverted microscope and tissues were superfused continuously at flow rate of 3 ml/min with warmed (36o C), oxygenated (95% O2, 5% CO2) Krebs solution. Tissues were allowed to equilibrate for 30 minutes before beginning experiments. Video images were obtained using a black-and-white video camera (Hitachi model KP-111; Yokohama, Japan) connected to the microscope and fed to a Picolo frame grabber board (Euresys Inc., Itasca, IL) mounted in a personal computer. Video images were analyzed using Diamtrak® edge tracking software (Adelaide, Australia). Diameter changes of 1 µm can be resolved. Drug-induced constrictions Drugs were applied using a 3-way stopcock system so that the superfusing Krebs solution could be changed to one containing a known drug concentration. Blood vessels were initially constricted with 3 consecutive applications of NE (10!M for arteries and 1!M for veins) at 15-minute intervals to verify viability and response stability. Responses greater than 20% of initial diameter in arteries and 30% in veins were considered acceptable and only those blood vessels were used for further 6 experiments. Adenosine (0.001-100!M) and N -cyclopentyl-adenosine (CPA, 0.0110!M) were added non-cumulatively. Successive concentrations were applied at 45minute intervals. Antagonists were applied for 30 minutes before testing the effect of agonists. Increasing concentrations of the A1R antagonist, 1,3-dipropl-8- cyplopentylxanthine (DPCPX) were applied cumulatively with a 15 minute incubation ! ""#! period for each concentration. Dipyridamole dose response curves were obtained nonaccumulatively with 15-minute wash between concentrations. Transmural stimulation of perivascular nerves Perivascular sympathetic nerves were stimulated using Ag/AgCl wire electrodes placed parallel to the length of the blood vessel. Parameters for nerve stimulation were: 30 stimuli (0.5ms duration) at 10Hz, 60-80V. Pilot experiments and previously published work (Park et al., 2007, Park et al., 2010) showed that these parameters produced a maximum constriction. Constrictions caused by electrical stimulation were blocked by the Na+ channel blocker, tetrodotoxin (0.3µM). Amperometric detection of norepinephrine (NE) Construction of carbon fiber microelectrodes was described in detail previously (Park et al., 2007). The microelectrode was positioned parallel to the blood vessel so that NE flux from nearby release site will be sensed by the electrode surface. The microelectrode was pressed gently against the vessel to enable the microelectrode to maintain contact with the vessel during stimulation-evoked constrictions. A platinum wire counter electrode and a commercial “no leak” Ag-AgCl (3M KCl, model EE009, Cypress System Inc., USA) reference electrode also were mounted in the chamber to complete the electrochemical cell. Continuous amperometric measurements were made with an Omni 90 analog potentiostat (Cypress Systems Inc.), a Minidigi analog-to-digital converter and a computer running Axoscope 9.0 (Axon Instruments, Union City, CA, USA). Data were obtained at a 100 Hz sampling rate. An applied potential of 600 mV was used to detect NE currents because this is the oxidation potential for NE at a masstransfer limited rate (Park et al., 2007). Currents were low pass filtered with at a time ! ""#! constant of 200 ms. Data were stored on the computer hard drive for further analysis. A bipolar focal stimulation electrode positioned along the surface of the vessel was used to excite perivascular nerves. The electrode was placed at a distance of 200 !m from the carbon fiber microelectrode to minimize the stimulus artifact in the current recording. Immunohistochemistry. MA and MV were excised, cleaned of excess fat and connective tissue. Vessels were perfused with saline to remove blood from the lumen. Tissues were cut into 1 cm lengths and fixed in Zamboni fixative overnight at 4 °C. The next day, the tissues were washed 3 times with 0.1 M PBS and then incubated in 4% goat serum in PBS for 1 h. Then, the vessels were incubated overnight with primary antibodies against particular adenosine receptor subtypes (Table 5) and tyrosine hydroxylase (TH) (mouse monoclonal anti-TH antibody, 1:200 in PBS, Millipore). To control for non-specific binding, control vessels were incubated without primary antibodies. After overnight incubation, unbound primary antibody was rinsed with 3 washes with PBS and the blood vessels were incubated with fluorescein isothiocyanate-conjugated goat anti-mouse IgG (1:40) and Cy3-conjugated goat anti-rabbit IgG (1:50, Jackson ImmunoResearch Laboratories, Inc.) to visualize TH and A1R staining respectively. After 1 hour incubation, unbound secondary antibody was rinsed away with 3 washes with PBS. Specimens were mounted on microscope slides in buffered glycerol (pH, 8.6) and were viewed under using a fluorescence microscope. Images are acquired and analyzed using MetaMorph software. ! ""#! Table 5. Sources of primary and secondary antibodies and working dilutions for adenosine receptor staining. ! Primary antibodies Antigen Source Santa Cruz Sigma Sigma Host Species Rabbit (polyclonal) Rabbit (polyclonal) Rabbit (polyclonal) Dilution 1:200 1:100 1:100 A2B R Santa Cruz Rabbit (polyclonal) 1:200 A3 R TH Sigma Rabbit (polyclonal) 1:100 Millipore Mouse (monoclonal) 1:100 Secondary antibodies Antigen Source Rabbit IgG Jackson immunolaboratory Host species Goat Dilution 1:400 Fluorophore Cy3 Mouse IgG Sheep 1:50 FITC A1R A2AR Jackson immunolaboratory Drugs. All drugs were obtained from Sigma Aldrich, USA. All drugs, except DPCPX, were dissolved in deionized water as a concentrated stock solution kept at 0oC. DPCPX and was made as stock solution in ethanol. Control experiments with the highest concentration of ethanol (0.01% vol/vol) did not affect neurogenic or agonist-induced blood vessel contraction (data not shown). Statistics. Data are reported as mean ± S.E., N values are the number of animals. Differences between groups were assessed using Student’s t-test. Differences in agonist concentration response curves were assessed first by 2-way ANOVA and Bonferoni’s test for multiple comparisions (GraphPad Prism 5.0, LaJolla, CA). ! "#$! Adenosine and CPA half maximal inhibitory concentrations (IC50) and maximum effect values (Emax) were determined from individual concentration response curves using a non-linear fitting routine with a logistic equation (Origin 8.0, Northampton, MA). Mean IC50 and Emax values were compared using Student’s t-test. IC50 values were expressed as the negative log of the drug concentration that causes 50% of the maximum inhibitory response (pIC50). P<0.05 was considered significant. ! "#"! 3.4 Results 3.4.1 Immunohistochemical localization of A1R, A2AR, A2BR, and A3R on sympathetic nerves supplying MA and MV in sham and DOCA-salt hypertension. Antibodies raised against A1Rs and tyrosine hydroxylase (TH), a marker for sympathetic nerves, were used to show that immunoreactivity for A1Rs and TH is coexpressed in sympathetic nerves innervating sham mesenteric arteries and veins. There was no obvious difference for A1R expression on TH-containing structure between sham and DOCA-salt arteries and veins (Figure 28). Additionally, immunostaining for the other three adenosine receptor subtypes was also performed by using their particular specific antibodies shown in table 5. Figure 29-30 shows co-expression of TH and A2AR or A2BR subtypes, respectively in arteries and veins of sham and DOCA-salt hypertensive rats. There was also no obvious difference in A2AR or A2BR expression on TH-containing fibers between sham and DOCA-salt arteries and veins. However, A3R are likely expressed by non-neuronal structures, most likely fibroblasts (Fig. 31 A, D, and G) and/or vascular smooth muscle cells (Fig. 31 C, F). ! "##! Figure 28. Localization of A1Rs on periarterial and perivenous sympathetic nerves of sham and DOCA hypertensive rats. Sympathetic nerves were labeled with an anti-TH antibody shown in green labeling. A1Rs were detected by anti-A1R antibody shown in red labeling. Overlay of photomicrographs shows the co-localization of TH and A1Rs (yellow labeling). The images were obtained by confocal microscopy. Bars indicate magnification. MA, mesenteric artery; MV, mesenteric vein. ! "#$! Figure 28. (Cont’d) ! "#$! Figure 29. Localization of A2ARs on periarterial and perivenous sympathetic nerves of sham and DOCA hypertensive rats. Sympathetic nerves were labeled with an anti-TH antibody shown in green labeling. A2ARs were detected by anti-A2AR antibody shown in red labeling. Overlay of photomicrographs shows the co-localization of TH and A2ARs (yellow labeling). The images were obtained by confocal microscopy. Bars indicate magnification. MA, mesenteric artery; MV, mesenteric vein. ! "#$! Figure 29. (Cont’d) ! ! ! "#$! Figure 30. Localization of A2BRs on periarterial and perivenous sympathetic nerves of sham and DOCA hypertensive rats. Sympathetic nerves were labeled with an anti-TH antibody shown in green labeling. A2BRs were detected by anti-A2BR antibody shown in red labeling. Overlay of photomicrographs shows the co-localization of TH and A2BRs (yellow labeling). The images were obtained by confocal microscopy. Bars indicate magnification. MA, mesenteric artery; MV, mesenteric vein. ! "#$! Figure 30. (Cont’d) ! "#$! Figure 31. A3Rs does not express on periarterial and perivenous sympathetic nerves. Sympathetic nerves were labeled with an anti-TH antibody shown in green. A3Rs were detected by anti-A3R antibody shown in red. Overlay of photomicrographs shows A3R staining outside the TH-containing nerves (A-G). A-F, Series of overlay images of A3R and TH-containing nerves from adventitia down to smooth muscle layer. Z indicates z-axis distance from adventitia. The images were obtained by confocal microscopy. Bars indicate magnification. MA, mesenteric artery; MV, mesenteric vein. ! "#$! 3.4.2 Adenosine and CPA constricted mesenteric vein but not artery. Adenosine and, selective A1 adenosine receptor, CPA were tested for their constrictor effects in mesenteric arteries and veins. NE produced a sustained constriction of arteries while neither adenosine nor CPA constricted arteries (Fig. 32A, B). In sham arteries adenosine (10µM) and CPA (1µM) caused changes in diameter of only 0.4+0.3% (n=6) and 0.8+0.5% (n=8), respectively. Adenosine and CPA caused sustained constrictions of veins while NE produced a transient constriction of veins (Fig. 32C, D). In sham veins, adenosine and CPA caused decreases in diameter of 19+2% (n=5) and 28+3% (n=3), respectively. Similar data were obtained in DOCA-salt mesenteric arteries and veins. ! "#$! Figure 32. Representative traces of norepinephrine (NE) and adenosine induced-constrictions of sham mesenteric arteries and veins. A, NEinduced constriction of arteries was smaller than veins (C) in peak amplitude but the constriction was sustained throughout the period of NE application. Venous constriction showed rapid desensitization to NE (C). D, Adenosine constricted mesenteric veins but not arteries (B). Agonists were applied during the period indicated by the bar. Y-axis is vessel diameter (!m) ! ! "#"! 3.4.3 The prejunctional A1Rs inhibited neurogenic constriction of mesenteric arteries. The actions of adenosine and CPA on neurogenic constrictions in mesenteric veins were not studied because these agonists constricted veins directly. However, adenosine and CPA both caused concentration-dependent inhibition of neurogenic constriction in arteries (Fig. 33A). The concentration response curve for CPA was leftshift compared to that for adenosine. The pIC50 values for CPA and adenosine were 7.0±0.4 and 5.6±0.2, respectively (P<0.05). We next compared adenosine and CPA concentration-response curves for inhibition of neurogenic constrictions in arteries from sham and DOCA-salt hypertensive rats. Adenosine and CPA concentration response curves were both right shifted in DOCA-salt arteries compared to those obtained in sham arteries (Fig. 33B,C). The rightward shift was greater for adenosine compared to that for CPA. ! "#$! Figure 33. Adenosine and CPA concentration-response curves (CRCs) for inhibition of neurogenic constrictions of sham, but not DOCA-salt mesenteric arteries. A, Adenosine and CPA inhibited neurogenic constriction in sham arteries. The CPA curve was significantly left-shifted compared to the adenosine CRC (p<0.05). B-C, Adenosine and CPA-induced inhibition of neurogenic constriction were reduced in DOCA-salt arteries compared to sham arteries. Data are mean + S.E. Data were analyzed using a 2-way ANOVA and Bonferoni’s post-hoc test. *indicates significantly different from sham arteries. ! ! "##! 3.4.4 Adenosine and CPA did not affect mesenteric artery reactivity Control experiments were performed to determine whether the inhibition of neurogenic constriction produced by adenosine or CPA was due to pre- or postjunctional effects. We determined the influence of exogenous adenosine and CPA on two postjunctional receptors, the !1 adrenergic and the P2X1 receptors. The selective !1-adrenergic receptor agonist, phenylephrine, and the P2X1 receptor agonist, !,"-methylene ATP both caused concentration dependent constrictions of mesenteric arteries. Neither adenosine (1 µM) nor CPA (1 µM) altered phenylephrine or !,"methylene ATP-induced constrictions (Fig. 34A-D). This indicates that inhibition of the neurogenic constriction by adenosine and CPA was mediated prejunctionally. ! "#$! Figure 34. CPA and adenosine do not inhibit constrictions caused by phenylephrine (!1-adrenergic receptor agonist) and !,"-MeATP (P2X receptor agonists). Phenylephrine CRCs were not altered by adenosine (A) or CPA (B) in sham MA. !,"-MeATP CRCs were not altered by adenosine (C) or by CPA (D) in sham MA. Data are mean + S.E., n values represent the number of animals for each experiment. ! "#$! 3.4.5 A1Rs but not A2ARs mediated inhibition of NE release Neurogenic constriction was used as an indirect measure of NE release, which could be performed only in arteries because adenosine and CPA both constricted veins directly. In the next experiments, we made more direct measures of NE release using continuous amperometry with carbon-fiber microelectrode. This allowed measurement of NE release in real time near the surface of both arteries and veins. CPA was used in these studies as it is A1R selective and actions at other adenosine receptors would not complicate our data interpretation. In addition, adenosine could not be used in these studies as it interacted with the carbon fiber electrode making the electrode baseline current unstable. Short trains of stimulation evoked an oxidation current whose time course tracked the time course of the neurogenic constriction in mesenteric arteries (Fig. 35A,B). Besides, the data exhibit positive correlations between NE oxidation currents and neurogenic arterial contractions (Fig. 35C). ! "#$! Figure 35. Representative traces of CPA-mediated inhibition of NE oxidation currents and constrictions in sham MA. Electrically-evoke NE release (60 stimuli, 10 Hz frequency, 0.5 ms pulse duration, 60-80 V intensity) was detected by amperometry with a carbon fiber microelectrode. Peri-arterial sympathetic nerves were stimulated and oxidation currents (A) and constrictions (B) were inhibited by CPA. C, Positive correlation between NE oxidation current and neurogenic contraction. Duration of nerve stimulation (ns) is indicated by the bars. Imax indicates amplitude of NE oxidation current. n indicates the number of animals. ! R, relative correlation; s, second; "#$! SD, standard deviation.! CPA produced a concentration dependent inhibition of the oxidation current and the neurogenic constriction (Fig. 35A,B 36A). Similar data were obtained in mesenteric veins (where only oxidation current was monitored due to direct constrictor effect of CPA). Peak NE currents in sham veins were greater than sham arteries by 36% (13.7±1.5 vs. 7.5±0.9 pA, P<0.05)(Fig. 36A). Furthermore, DPCPX (0.1µM), a selective A1R antagonist, caused a rightward shift in the CPA concentration response curve in sham arteries and veins (Fig. 36C, 36D). The pIC50 values for CPA in sham arteries in the absence and presence of DPCPX were 6.6±0.2 and 4.7±0.2 respectively (P<0.05). The pIC50 values for CPA in sham veins in the absence and presence of DPCPX were 9.6±0.2 and 5.2±0.4 respectively (P<0.05). Amperometric measurement of NE release after brief electrical stimulation (60 stimuli at 10Hz) showed that the A2AR agonist CGS21680 in a range of concentrations selective for the A2AR (1-100nM) did not alter significantly NE oxidation currents (Fig. 36B). ! "#$! Figure 36. CPA, but not CGS21680 inhibit norepinephrine (NE) oxidation currents in sham mesenteric arteries and veins. A, Nerve stimulation was used to evoke NE oxidation currents, which were inhibited by CPA in sham arteries and veins. * Indicates that baseline oxidation currents were significantly larger in veins compared to arteries. B, Increasing concentrations of A2A adenosine receptor agonist CGS 21680 did not significantly change amplitude of NE oxidation currents in sham mesenteric arteries or veins. CPA concentration response curves for inhibition of NE oxidation currents in arteries were right shifted by the A1R antagonist DPCPX in arteries (C) and veins (D). Data are mean ± S.E. ! "#$! 3.4.6 Impaired function of prejunctional A1Rs in DOCA-salt mesenteric artery but not vein The data presented above indicate that adenosine and CPA act pre-junctionally to inhibit NE oxidation currents and neurogenic constrictions. The next study determined if this mechanism was impaired in DOCA-salt hypertension. It was found that adenosine- and CPA-induced inhibition of neurogenic constriction was reduced in DOCA-salt artery (Fig. 33B, C). CPA produced a concentration-dependent inhibition of NE oxidation currents in arteries and veins but DOCA-salt arteries were less sensitive to the inhibitory effects of CPA effect compared to sham arteries (Fig. 37A, P<0.05). In DOCA-salt arteries, the maximum inhibition of NE release produced by CPA (100 !M) was reduced and the pIC50 value was decreased compared to sham artery values (Table 6). The effects of CPA on NE oxidation currents were not different in sham and DOCA-salt veins (Fig. 37B, Table 6). ! "#$! Figure 37. Impaired A1R function in DOCA-salt mesenteric arteries but not veins. A, A 10 Hz stimulus train was used to evoke NE oxidation currents. CPA was then added in increasing concentrations. The CPA CRC was right shifted in DOCA-salt arteries compared to sham arteries. B, There were no differences in CPA CRCs between sham and DOCA-salt veins. Data are mean ± S.E. *Indicates significantly different from sham arteries. # baseline values. ! ! ! ! "#"! Indicates significantly different from Figure 37. Cont’d ! ! ! "#$! Table 6. Analysis of concentration response curves for the effects of the A1R agonist, CPA, on norepinephrine oxidation currents recorded from the surface of mesenteric arteries and veins from sham and DOCA-salt rats. pIC50 values and maximum inhibition were determined from nonlinear curve fits of concentration response curves from individual preparations. Mean values were then compared using Student’s t-test. N pIC50 % Max Inhibition Sham 9 7.6 ± 0.2 94 ± 2.0 DOCA-salt 9 6.4 ± 0.3* 64 ± 4.2* Mesenteric arteries Mesenteric veins Sham 5 8.5 ± 0.5 95 ± 3.6 DOCA-salt 6 8.6 ± 0.2 99 ± 1.4 * Indicates significantly different from sham values. ! ! "#$! 3.4.7 Role of endogenous adenosine in modulation of adrenergic transmission. We next determined the role of endogenous adenosine as a neuromodulator of sympathetic neurotransmission in mesenteric arteries and veins. DPCPX, a selective A1R antagonist, was used to block A1R autoinhibition. DPCPX caused an increase in NE current in concentration-dependent manner in arteries. Conversely, DPCPX caused a reduction of NE current in concentration-dependent manner in veins (Fig. 38A). Furthermore, dipyridamole, an adenosine transporter blocker, was used to produce an accumulation of endogenous adenosine providing a greater A1R activation. Dipyridamole produced a decrease in NE current in concentration-dependent manner is arteries, but not veins (Fig. 38B). DPCPX and dipyridamole did not change NE oxidation currents in DOCA-salt arteries (Fig. 38C-D). ! "##! Figure 38. Role of endogenous adenosine in adrenergic transmission of sham and DOCA-salt rats. Dose-response curves of DPCPX (A1R antagonist) and dipyridamole (adenosine transporter blocker) were performed under a 10 Hz stimulus train to evoke NE oxidation currents. A, DPCPX increased NE current in concentration-dependent manner in sham mesenteric arteries, nut not veins. B, dipyridamole caused a reduction of NE current in sham mesenteric arteries, but not veins. DPCPX (C) and dipyridamole (D) did not alter the amplitude of NE oxidation currents in DOCA-salt arteries. Data are mean ± S.E. # Indicates significantly different from baseline values. n values indicate the number of animals in the experiment. ! ! "#$! 3.5 Discussion 3.5.1 Adenosine A1, A2A, A2B, but not A3 receptors localize on periarterial and perivenous sympathetic nerves My data show that there are three subtypes of adenosine receptors (A1, A2A, and A2B, but not A3) located on the sympathetic nerve fibers supplying mesenteric arteries and veins. My research focuses on A1 and A2A subtypes as they are high affinity adenosine receptors (Olah et al., 1995). Thus, these two prejunctional receptors are more relevant to physiologic sympathetic neurotransmission where adenosine concentrations in the neurovascular junction are ranged in the nanomolar range. The low affinity A2BR may play a pivotal role under pathologic conditions where adenosine concentrations may reach the micromolar range (Ralevic et al., 1998). 3.5.2 Adenosine constricts mesenteric veins but not arteries via A1R activation Our data show that adenosine can modulate venous tone by directly constricting venous smooth muscle and by inhibiting NE release from perivenous sympathetic nerves. Adenosine does not constrict mesenteric arteries and acts only to inhibit NE release from periarterial sympathetic nerves. Adenosine-induced constriction of mesenteric veins was mimicked by the A1R agonist, CPA and adenosine produced a sustained constriction indicating that the A1R mechanism is desensitization resistant. It is not surprising that adenosine or CPA did not constrict mesenteric arteries as previous work has shown that adenosine dilates mesenteric arterioles (Mian et al., 1995) and this ! "#$! effect is mediated by A2R in the rat stomach (Nagata et al., 1996). In rabbit mesenteric arteries, adenosine induced vasodilation is mediated also by the A2AR subtype (de Brito et al., 2002). We did not detect a vasodilation in our studies because we did not preconstrict the blood vessels. A novel finding in our study is the A1R mediated constriction of mesenteric vein in light of previous studies showing that adenosine dilated rat mesenteric vein in situ (Mian et al., 1995). In these studies, the veins were blood perfused while our veins were maintained in a physiological buffer solution in vitro. Methodological differences might contribute to these differences in results. As the focus of our study was adenosine modulation of sympathetic neuroeffector transmission we did not pursue this issue. 3.5.3 A1Rs couple to inhibition of NE release Adenosine, a degradation product of ATP metabolism in the neuroeffector junction (Tabrizchi et al., 2001; Todorov et al., 1997; Westfall et al., 2002) modulates sympathetic neurotransmission in the mesenteric circulation (Diniz et al., 2004; Donoso et al., 2006; Illes et al., 1988; Ralevic, 1995), in the rat tail artery (Bucher et al., 1992; Diniz et al., 2004), rat caudal arteries (Shinozuka et al., 1988), and in human forearm blood vessels (Rongen et al., 1996). Adenosine-induced inhibition of sympathetic neuroeffector transmission is mediated by prejunctional A1Rs (Diniz et al., 2004; Ralevic, 1995; Ralevic, 2000) and/or A2ARs (Donoso et al., 2006). These previous studies showed that A2ARs could either couple to increased or decreased NE release ! "#$! from perivascular sympathetic nerves (Diniz et al., 2004; Donoso et al., 2006). They used long trains of electrical stimulation and NE was measured in the bath solution collected by overflow and high performance liquid chromatography. We used amperometry to measure NE release in real time near a few release sites on a single blood vessel with brief trains of electrical stimulation that more closely mimic sympathetic nerve activity in vivo. Under these conditions, we found that the A2AR agonist CGS21680 in a range of concentrations selective for the A2AR (1-100nM) did not alter significantly NE oxidation currents. Our immunohistochemical data show that A1Rs are localized to perivascular sympathetic nerves supplying rat mesenteric arteries and veins. Sympathetic nerves were identified by localizing tyrosine hydroxylase (TH) immunoreactivity in nerve fibers. TH is the rate-limiting enzyme in NE synthesis. The immunohistochemical data are consistent with our conclusion that A1Rs modulate NE release in the mesenteric circulation. This differs from the conclusion of Donoso and co-workers (Donoso et al., 2006) who concluded that A1Rs do not couple to inhibition of NE release in the rat mesentery. This conclusion is based partly on RT-PCR studies, which did not detect A1R transcripts in the mesentery. However, mRNA encoding the A1R protein would be found in highest concentrations in the nerve cell body of sympathetic neurons in prevertebral ganglia. It is not surprising that they failed to detect the A1R mRNA in the mesentery where sympathetic nerve terminals are located. Immunohistochemical methods detect the A1R protein, which was found in periarterial and perivenous nerve ! "#$! fibers. These methodological differences would account in part for differences in conclusions about the role of the A1R in perivascular sympathetic nerves. The result also demonstrates that there are TH negative nerve fibers expressing A1R immunoreactivity. It is possible that these A1Rs are localized to periarterial primary afferent nerve fibers (Burnstock et al., 1996). We did not measure mRNA or protein levels of prejunctional A1R in mesenteric blood vessels as A1Rs are expressed by sympathetic nerves fibers, and by vascular smooth muscle in mesenteric veins. Hence, measurements of A1R expression in whole blood vessel wall protein extracts would not distinguish the amount of A1Rs in the nerve fibers vs. smooth muscle or other cell types such as fibroblasts (Gines et al., 2000) in the blood vessel wall. As adenosine and CPA constricted the vein we could not study neurogenic venous constrictions in the presence of these agonists. However, we were able to study A1R modulation of arterial constrictions. Neurogenic constriction also permits comparisons between adenosine and CPA for inhibition of this response. Adenosine could not be used in our amperometry studies as it interacted with the microelectrodes causing an unstable baseline. Neurogenic constrictions of rat mesenteric arteries are mediated by NE acting at !1-adrenergic receptors and ATP acting at P2X1 receptors (Dunn et al., 1999). We confirmed that A1R stimulation does not directly affect arterial smooth muscle reactivity by showing that constrictions caused by phenylephrine or the P2X1 receptor agonist, !,"-methylene ATP, were unaffected by adenosine and CPA. However, adenosine and CPA produced concentration dependent decreases in nerve! "#$! mediated constrictions supporting the existing data that A1R activation couples to inhibition of neurotransmitter release. Our data show that inhibition of neurogenic constriction by adenosine is more sensitive to impairment in DOCA-salt hypertension compared to CPA. This may be due to different potency profile of adenosine analogues acting at A1Rs. Additionally, adenosine as a non-selective AR agonist may also act at other AR subtypes that couple to inhibition of neurotransmitter release. A previous study revealed that NE overflow of periarterial nerves was decreased by activation of A3 adenosine receptors (A3Rs) (Donoso et al., 2006). It is possible that A3R function is also impaired in DOCA-salt hypertension. Further studies are needed to clarify this issue. Measurement of neurogenic constrictions is an indirect measure of neurotransmitter release and it does not discriminate between effects mediated by NE or ATP release. Continuous amperometry with carbon fiber microelectrodes detects NE release from sympathetic nerves (Dunn et al., 1999; Park et al., 2007). We used amperometry to directly assess adenosine receptor modulation of NE release from sympathetic nerves. Our amperometry data confirm previous work showing that A1Rs mediate prejunctional inhibition of sympathetic neuroeffector transmission in the mesentery but we expand on these findings in two ways. Firstly, previous work used 3 increases in vascular perfusion pressures, arterial contractions or overflow of H-NE following long trains of electrical stimulation to assess A1R-mediated modulation of NE release. We used microelectrodes to measure NE release from a small number of ! "#$! nerve fibers on the blood vessel surface. This technique allows measurements in real time and also permits use of short trains of stimulation that more closely mimic sympathetic nerve activity occurring in vivo. Secondly, we show that endogenous adenosine can inhibit NE release. This conclusion is based on the finding that the A1R selective antagonist, DPCPX, increased NE oxidation currents while the adenosine uptake inhibitor, dipyridamole, decreased NE oxidation currents in mesenteric arteries from normotensive rats. It is important to note that enhancement of NE release by endogenously released adenosine could be due only to blockade of inhibitory A1Rs or to a combination of inhibition of A1Rs which reveals an action of adenosine at facilitatory A2A or A2B receptors on sympathetic nerve terminals (Diniz et al., 2004; Fresco et al., 2002). Additional studies are needed to address this issue. However, previous work has shown that prejunctional A2ARs could couple to increased or decreased NE release from perivascular sympathetic nerves (Diniz et al., 2004; Donoso et al., 2006). These investigators used long trains of electrical stimulation and NE was measured in the bath solution collected by overflow and high performance liquid chromatography. We used amperometry to measure NE release in real time near a few release sites on a single blood vessel with brief trains of electrical stimulation that more closely mimic sympathetic nerve activity in vivo. Under these conditions, we found that the A2AR agonist CGS21680 in a range of concentrations selective for the A2AR did not alter significantly NE oxidation currents. The function of ! "#"! facilitatory A2A receptors may be most prominent when A1Rs are blocked. Additional studies are needed to address these differences in results and their interpretation. 3.5.4 Impaired prejunctional A1R function in DOCA-salt MA Sympathetic nerves regulate blood pressure and increased sympathetic nerve activity contributes to increased blood pressure in DOCA-salt hypertension (deChamplain et al., 1987). Increased sympathetic vasoconstrictor tone in DOCA-salt hypertension is not due to vascular hyperreactivity to NE or ATP released by sympathetic nerves (Luo et al., 2003) as might occur in angiotensin-induced hypertension(Griendling et al., 1997). One possibility is that augmented sympathetic tone is due to loss of inhibitory !2-AR function on sympathetic nerve terminals (deChamplain et al., 1987; Demel et al., 2008; Luo et al., 2004). Prior to this study, it was not clear if A1R function is also impaired in DOCA-salt hypertension. Here, we assessed whether dysfunction of the prejunctional autoreceptors is specific to the !2-AR or if there is a more general disruption of prejunctional mechanisms controlling neurotransmitter release from periarterial sympathetic nerves. We found that the CPA concentration response curve for inhibition of neurogenic constrictions of mesenteric arteries and for inhibition of NE release from periarterial nerves are both right-shifted in DOCA-salt hypertension suggesting reduced A1R function. Impaired A1R function is not specific for the exogenously applied agonist, but it also affects the inhibitory actions of endogenously released adenosine. ! This conclusion is based on data showing that "#$! increases in NE oxidation currents caused by DPCPX (A1R antagonist) and reductions in NE oxidation currents caused by dipyridamole (adenosine uptake inhibitor) were also reduced in DOCA-salt arteries. We also found that concentration response curves for inhibition of neurogenic constrictions by exogenous adenosine and CPA were right shifted in MA from DOCA-salt hypertensive rats. These data are similar to those obtained using !2-AR agonists to inhibit neurogenic constrictions (Luo et al., 2004; Park et al., 2010). These results also indicate that prejunctional !2-ARs are not selectively affected in DOCA-salt hypertension, but there is a more general disruption of prejunctional mechanisms controlling neurotransmitter release form periarterial sympathetic nerves. Indeed, there is evidence that !2-ARs and A1Rs interact to modulate NE released from sympathetic nerves supplying the rat tail artery (Bucher et al., 1992). It is also important to note that A1R modulation of NE release from perivenous sympathetic nerves was not altered in DOCA-salt hypertension. Similar results have been obtained when !2-AR function was studied in perivenous sympathetic nerves from DOCA-salt hypertensive rats (Park et al., 2010). The mechanisms responsible for decreased function of prejunctional autoreceptors in arteries are unclear. It is possible that arterial autoreceptor impairment is due to receptor downregulation. Western blot analysis or radioligand binding studies would be useful for these measurements. However, neuronal protein in the arterial wall is a small fraction of total protein present. Furthermore, A1Rs are both expressed by nerve fibers and other cell types (smooth muscle, fibroblasts (Bruns, 1979; Gines et al., 2000)) in the arterial wall. ! "#$! These limitations would make it difficult to determine if there are neuronal specific changes in receptor expression in arteries in DOCA-salt hypertension. 3.6 Perspective. Our data support previous findings that A1Rs localized to periarterial nerves couple to inhibition of NE release. We also show that the disruption of autoreceptor function on periarterial sympathetic nerves is not specific for the !2-adrenergic receptors as A1 autoreceptors on periarterial nerves of DOCA-salt hypertensive rats are also impaired. We also show for the first time that A1Rs also couple to inhibition of NE release in perivenous sympathetic nerves. An important difference between mesenteric arteries and veins is that A1R and !2-AR function is disrupted in periarterial but not perivenous sympathetic nerves in DOCA-salt hypertension. !2-AR function is at least partly restored in DOCA-salt hypertensive rats that had been treated with apocynin an - NADPH oxidase inhibitor (Demel et al., 2010). NADPH oxidase produces O2 in the vasculature(Szasz et al., 2007) and this enzyme is also localized to perivascular - sympathetic nerves (Cao et al., 2009). Increased O2 levels in arteries is associated - with higher arterial pressures and elevated O2 could disrupt signaling mechanisms linking !2-AR and A1R to inhibition of NE release. Venous pressure is not increased in - DOCA-salt hypertension (Ralevic, 2000) so levels of O2 would not be elevated in veins ! "#$! and this might spare autoreceptor function. There are also likely to be differences in arteries and veins in the enzyme systems that produce and degrade reactive oxygen species (Szasz et al., 2007). This difference could also contribute to maintained autoreceptor function in perivenous sympathetic nerves in DOCA-salt hypertension. ! "##! BIBLIOGRAPHY ! "#$! BIBLIOGRAPHY Bucher B, Corriu C, Stoclet JC (1992). Prejunctional opioid p-receptors and adenosine A1-receptors on the sympathetic nerve endings of the rat tail artery interact with the alpha 2-adrenoceptors. N-S Arch Pharmacol 345: 37-43. Burnstock G, Wood JN (1996). Purinergic receptors: their role in nociception and primary afferent neurotransmission. Curr Opin Neurobiol 6(4): 526-532. Cao X, Demel SL, Quinn MT, Galligan JJ, Kreulen D (2009). Localization of NADPH oxidase in sympathetic and sensory ganglion neurons and perivascular nerve fibers. Auton Neurosci 151(2): 90-97. de Brito MTV, Canto A, Correia JHD, Cunha RA, Marques MC (2002). Adenosine A2A receptors in portal hypertension: their role in the abnormal response to adenosine of the cranial mesenteric artery in rabbits. Brit J Pharmacol 135: 1324-1330. deChamplain J, Bouvier M, Drolet G (1987). Abnormal regulation of the sympathoadrenal system in deoxycorticosterone acetate - salt hypertensive rats. Can J oPhysiol Pharm 65(8): 1605-1614. Demel SL, Dong H, Swain GM, Wang X, Kreulen DL, Galligan JJ (2010). Antioxidant treatment restores prejunctional regulation of purinergic transmission in mesenteric arteries of doca-salt hypertensive rats. Neuroscience 168(2): 335-345. Demel SL, Galligan JJ (2008). Impaired purinergic neurotransmission to mesenteric arteries in DOCA-salt hypertensive rats. Hypertension 52(2): 322-329. Diniz C, Fresco P, Leal S, Goncalves J (2004). Adenosine receptors involved in modulation of noradrenaline release in isolated rat tail artery. Eur J Pharmacol 504: 1725. Donoso MV, Aedo F, Huidobro-Toro JP (2006). The role of adenosine A2A and A3 receptors on the differential modulation of norepinephrine and neuropeptide Y release from peripheral sympathetic nerve terminals. J Neurochem 96: 1680-1695. Dunn WR, Brock JA, Hardy TA (1999). Electrochemical and electrophysiological characterization of neurotransmitter release from sympathetic nerves supplying rat mesenteric arteries. Brit J Pharmacol 128: 174-180. Fresco P, Diniz C, Queiroz G, GoncËalves J (2002). Release inhibitory receptors activation favours the A2A-adenosine receptor-mediated facilitation of noradrenaline release in isolated rat tail artery. Brit J Pharmacol 136: 230-236. ! "#$! Gines S, Hillion Jl, Torvinen M, Le Crom Sp, ! VC, Canela EI, et al. (2000). Dopamine D1 and adenosine A1 receptors form functionally interacting heteromeric complexes. Proc Natl Acad Sci 97: 8606-8611. Griendling KK, Ushio-Fukai M, Lassègue B, Alexander RW (1997). Angiotensin II Signaling in Vascular Smooth Muscle: New Concepts. Hypertension 29: 366-370. Illes P, Jackisch R, Regenold JT (1988). Presynaptic p1-purinoceptors in jejunal branches of the rabbit mesenteric artery and their possible function. J Physiol 397: 1329. Luo M, Fink GD, Lookingland KJ, Morris JA, Galligan JJ (2004). Impaired function of alpha2-adrenergic autoreceptors on sympathetic nerves associated with mesenteric arteries and veins in DOCA-salt hypertension. Am J Physiol 286: H1558-H1564. Luo M, Hess MC, Fink GD, Olson LK, Rogers J, Kreulen DL, et al. (2003). Differential alterations in sympathetic neurotransmission in mesenteric arteries and veins in DOCAsalt hypertensive rats. Auton Neurosci 104: 47-57. Mian R, Marshall JM (1995). The role of adenosine in mediating vasodilatation in mesenteric circulation of the rat in acute and chronic hypoxia. J Physiol 489(1): 225234. Nagata H, Sekizuka E, Morishita T, Tatemichi M, Kurokawa T, Mizuki A, et al. (1996). Adenosine A2-receptor mediates ethanol-induced arteriolar dilation in rat stomach. Am J Physiol 271: G1028-G1033. Olah ME, Stiles GL (1995). Adenosine receptor subtypes: characterization and therapeutic regulation. Annu Rev Phamloeol Toxicol 35: 581-606. Park J, Galligan JJ, Fink GD, Swain GM (2010). Alterations in sympathetic neuroeffector transmission to mesenteric arteries but not veins in DOCA-salt hypertension. Auton Neurosci 152(1-2): 11-20. Park J, Galligan JJ, Fink GD, Swain GM (2007). Differences in sympathetic neuroeffector transmission to rat mesenteric arteries and veins as probed by in vitro continuous amperometry and video imaging. J Physiol 584(3): 819-834. Ralevic V (1995). Modulation by nicotinamide adenine dinucleotide of sympathetic and sensory-motor neurotransmission via P1-purinoceptors in the rat mesenteric arterial bed. Brit J Pharmacol 114: 1541-1548. Ralevic V (2000). Sympathoinhibition by adenosine A1 receptors, but not P2 receptors, in the hamster mesenteric arterial bed. Eur J Pharmacol 387: 287-293. ! "#$! Ralevic V, Burnstock G (1998). Receptors for purines and pyrimidines. Pharmacol Rev 50(3): 413-492. Rongen GA, Lenders JWM, Lambrou J, Willemsen JJ, Belle HV, Thien T, et al. (1996). Presynaptic inhibition of norepinephrine release from sympathetic nerve endings by endogenous adenosine. Hypertension 27: 933-938. Shinozuka K, Bjur RA, Westfall DP (1988). Characterization of prejunctional purinoceptors on adrenergic nerves of the rat caudal artery. N-S Arch Pharmacol 338: 221-227. Szasz T, Thakali K, Fink GD, Watts SW (2007). A comparison of arteries and veins in oxidative stress: producers, destroyers, function, and disease. Exp Biol Med 232: 27-37. Tabrizchi R, Bedi S (2001). Pharmacology of adenosine receptors in the vasculature. Pharmacol Therapeut 91: 133-147. Todorov LD, Mihaylova-Todorova S, Westfall TD, Sneddon P, Kennedy C, Bjur RA, et al. (1997). Neuronal release of soluble nucleotidases and their role in neurotransmitter inactivation. Nature 387: 76-79. Westfall DP, Todorov LD, Mihaylova-Todorova ST (2002). ATP as a cotransmitter in sympathetic nerves and its inactivation by releasable enzymes. J Pharmacol Exp Ther 33(2): 439-444. ! ! "#$! CHAPTER 4 DOCA-SALT HYPERTENSION FAILED TO ENHANCE SYMPATHETIC NE CLEARANCE THROUGH PREJUNCTIONAL A2A ADENOSINE RECEPTOR ACTIVATION ! "#$! 4.1 Abstract Norepinephrine (NE) and ATP are released from perivascular sympathetic nerves to control arterial resistance and venous capacitance. NE is cleared from the neuroeffector junction via the NE transporter (NET). ATP is hydrolyzed to produce adenosine, which activates prejunctional A1-adenosine receptors (A1Rs) to inhibit NE release. A2A-adenosine receptors (A2ARs) are also located on perivascular nerves and these receptors facilitate sympathetic transmission. In the central nervous system A2ARs also stimulate neurotransmitter re-uptake. This effect has not been investigated in peripheral nerves. We tested the hypothesis that A2ARs enhance NE clearance at the sympathetic neuroeffector junction by increasing NET activity, and this response is impaired in DOCA-salt hypertensive rats. Electrically evoked NE release was measured in vitro in real time using amperometry. Drug effects on the kinetics of NE oxidation currents were measured. Activation of A2ARs by CGS21680 (1-10nM, A2AR agonist) enhanced NE uptake via NET in control mesenteric veins, but not arteries, an A2AR antagonist (SCH58261 400nM), and NET blocker (cocaine 10µM) prevented this effect. Forskolin (1 µM, adenylyl cyclase activator) mimicked CGS21680 effects and H-89 (1 µM, protein kinase A inhibitor) blocked CGS21680-mediated effects. In DOCA-salt veins, CGS21680 failed to promote NE clearance, but forskolin restored the response. Impaired A2AR function to promote NE clearance in perivascular veins accounts for ! "#"! increased NE availability in mesenteric veins of DOCA-salt hypertension. DOCA-salt hypertension impairs this pathway by inhibiting A2AR activation of adenylyl cyclase. ! "#$! 4.2 Introduction Hypertension (HT), a major risk factor for stroke, cardiac and renal injury and HT affects 1 billion people worldwide (Chobanian et al., 2003). HT is associated with elevated sympathetic nerve activity and circulating catecholamines (Bouvier et al., 1986; deChamplain et al., 1976). Mesenteric arteries and veins are highly innervated by sympathetic nerves which supply the adventitia-media border of arteries and tunica media of veins (Birch et al., 2008). Periarterial sympathetic nerves control vascular resistance by releasing NE and ATP as co-transmitters (Astrand et al., 1989; Bobalova et al., 2001b). Once released, NE and ATP act at the postjunctional !1-adernergic and P2X1 receptors respectively to cause constriction. The actions of NE and ATP are terminated by two mechanisms. Firstly, NE is quickly taken up into nerve terminals via the high affinity NE transporter (NET) while ATP is degraded by extracellular 5’ectonucleotidase enzyme into ADP, AMP and adenosine as a final breakdown product. Secondly, NE and adenosine activate prejunctional !2-adrenergic and A1adenosine autoreceptors, respectively to stop further transmitter release. Deoxycorticosterone acetate (DOCA)-salt hypertension is a salt sensitive, renin independent model of hypertension (Schenk et al., 1992). Periarterial and perivenous sympathetic nerves in DOCA-salt hypertensive rats release more NE (Luo et al., 2004) causing increased vascular tone. Increased NE release in DOCA-salt mesenteric arteries is partly due to impaired sympathetic autoreceptor function (deChamplain, 1990; Luo et al., 2004). However, autoreceptor function is preserved in mesenteric veins (Park et al., 2010). Luo and co-workers also reported downregulation of the ! "#$! postjunctional !1-adrenoceptor resulting in decreased sensitivity to NE and phenylephrine (an !1-receptor agonist) in mesenteric veins of DOCA-salt hypertensive rats (Luo et al., 2003). Since the number of adrenergic receptors is in part regulated by catecholamines (Michel et al., 1990), it is possible that the receptor downregulation is caused by elevated NE availability (deChamplain et al., 1976). It is unclear what drives elevated NE availability in DOCA-salt veins. Adenosine acts on four different G-protein coupled receptor subtypes: A1, A2A, A2B, and A3-adenosine receptors. A1 and A2A subtypes are high affinity receptors that desensitize quickly (Olah et al., 1995). In the nervous system, activation of A2ARs requires higher adenosine concentrations of adenosine compared to those required to activate A1R (Ralevic et al., 1998). Low affinity A2BRs require sustained, high concentrations of adenosine for activation suggesting that A2BRs are activated only under pathophysiological conditions where adenosine levels are high (ischemia for example) (Eckle et al., 2008; Koeppen et al., 2011; Ralevic et al., 1998). A2ARs are also located on sympathetic nerves supplying mesenteric arteries and 2+ veins. Neuronal A1Rs couple to Ca channel inhibition (Ralevic et al., 1998) resulting in inhibition of transmitter release. A2ARs couple to the stimulator G-protein Gs to 2+ activate adenylyl cyclase and Ca channels leading to facilitation of neurotransmitter release. Recent studies have revealed a novel mechanism of A2AR in modulating ! "#$! neurotransmission where A2ARs modulate GABA and glutamate uptake via and action on GABA and glutamate transporters, respectively (Cristóvão-Ferreira et al., 2011; 2009; Gonzalez et al., 2006), (Matos et al., 2012). However, A2AR-modulated transporter function has not been investigated in peripheral nerves. Thus, the first aim of this study was to test the hypothesis that prejunctional A2ARs couple to enhanced NET function to remove NE from the sympathetic neuroeffector junction in mesenteric blood vessels. The second aim was to determine whether this mechanism was impaired in DOCA-salt hypertensive rats. 4.3 Materials and Methods 4.3.1 Animal. All animal use protocols were approved by the Institutional Animal Care and Use Committee at Michigan State University. Male Sprague-Dawley rats (250275g) were obtained from Charles River Laboratories (Portage, MI). Upon arrival, rats were placed in temperature and humidity controlled room with 12:12 hour light-dark cycle. They were provided with Rat chow (Harlan/Teklad 8640 Rodent Diet) and tap water were provided ad libitum. The animals were acclimated 2-3 days before entry into experimental protocols. 4.3.2 DOCA-salt hypertensive rats. The surgical procedures and drug treatment protocols for producing sham control and DOCA-salt hypertensive rats have been described in detail previously (Luo et al., 2003). After surgery, rats were housed under standard conditions for 4 weeks for recovery. DOCA-implanted rats received standard ! "#$! pelleted rat chow and drinking solution containing 1%NaCl+0.2%KCl in distilled water. Sham rats received standard pelleted rat chow and distilled water. Blood pressure was measured using tail-cuff plesmythography 3-5 days prior to experimentation. Rats with mean arterial pressure >150 mmHg were considered hypertensive. 4.3.3 Tissue preparation for in vitro studies.! 4-weeks post surgery, blood pressure had risen substantially in DOCA-salt but not in sham control rats. At this point, DOCAsalt or sham rats were sacrificed with a lethal dose of pentobarbital intra peritoneal injection (100 mg/kg). The whole mesentery transferred into cold Krebs solution (mM): 117 NaCl, 4.7 KCl, 2.5 CaCl2, 1.2 MgCl2, 25 NaHCO3, 1.2 NaHPO4, and 11, glucose. A section of tertiary mesenteric arteries (145-250 !m outside diameter) and veins (190390 !m outside diameter) was dissected out and transferred to a recording chamber. The tissue was pinned flat with 50 !m-diameter stainless steel pins. Adipose and connective tissue was gently removed under dissecting microscope. The tissue was opened as a small window to allow stimulating and recording electrodes to be positioned on the vessel. The recording chamber was then mounted on the stage of an inverted microscope and tissue was superfused continuously (2.5 ml/min) with warmed (36oC), oxygenated (95% O2, 5% CO2) Krebs solution. Tissues were allowed to equilibrate for 30 minutes before beginning experiments. During this time the blood vessels relaxed to stable resting diameter. Video images were obtained using a blackand-white video camera (Hitachi model KP-111; Yokohama, Japan) connected to the microscope and fed to a Picolo frame grabber board (Euresys Inc., Itasca, IL) mounted ! "##! in a personal computer. Video images were analyzed using Diamtrak® edge tracking software (Adelaide, Australia). Diameter changes of 1 µm can be resolved. ! 4.3.4 Amperometric detection of norepinephrine (NE) release. A bipolar stimulating electrode made from silver wire was inserted into a 2-barrel glass capillary filled with Krebs solution. The electrode was mounted on a holder and positioned on the vessel surface using a micromanipulator. Sympathetic nerves were stimulated using trains of electrical stimulation (10 Hz, 0.5 ms pulse duration 6 s train duration, 60-80V). Released NE was detected as an oxidation current using amperometry with carbon fiber microelectrodes. Construction and preparation of carbon fiber microelectrodes was described previously (Park et al., 2007). The microelectrode was positioned horizontally and pressed gently against the blood vessel surface so that NE flux from nearby release sites could be detected. The carbon fiber microelectrode was placed at a distance of 200!m from the stimulating electrode to minimize the stimulus artifact during the current recording. A commercial “no leak” Ag-AgCl reference electrode (3M KCl, model EE009, Cypress System Inc., USA) and a platinum wire counter electrode were also mounted in the chamber to complete the electrochemical cell. Continuous amperometric measurements were made with an Omni 90 analog potentiostat (Cypress Systems Inc.), a Minidigi analog-to-digital converter and a computer running Axoscope 9.0 (Axon Instruments, Union City, CA, USA). Data were obtained at a 100 Hz sampling rate. An applied potential of 0.6 volts was used to detect NE currents because this is the selective oxidation potential for NE at mass-transfer limited rate (Park et al., 2007). ! "#$! Currents were low pass filtered with at a time constant of 200 ms. Data were stored on the computer hard drive for further analysis. 4.3.5 Drug Application. Stock solutions of drugs were diluted to working concentrations with Krebs solution on the day of experiment. The vessels were initially performed 3 consecutive trains without drug to verify viability and response stability. Drugs were applied using a 3-way stopcock system and it took 3 minutes for drugs to reach the chamber. To obtain concentration response curves, agonists were applied 15 minutes before nerve stimulation with 45-minute wash between each concentration. Antagonists were applied for 30 minutes before agonist application. 4.3.6 Immunohistochemistry of mesenteric blood vessels. The whole mesentery was removed and transferred to a clear silicone bottom plate containing ice-cold phosphate buffered saline (PBS, 0.1 M). The tissues were pinned flat and cleaned of excess fat and connective tissue. Blood was flushed from the lumen by cold 0.9% saline perfusion with 27G needle into the superior mesenteric artery and vein. Tissues were fixed in ice-cold Zamboni fixative for 2 h and were washed 3x with PBS. Then, the tissues were incubated in 4% goat+sheep serum in PBS-0.1%Tritron-X at room temperature for 1 h. The vessels were cut into 1-cm length and incubated with primary antibodies against A2ARs (rabbit polyclonal, 1:100 dilution in PBS, Sigma) and tyrosine hydroxylase (TH) (mouse monoclonal anti-TH antibody, 1:100 in PBS, Millipore) overnight at 4°C. To control for non-specific binding, control vessels were incubated without primary antibodies. After overnight incubation, unbound primary antibody was ! "#$! rinsed 3x with PBS and the blood vessels were incubated with fluorescein 3 isothiocyanate (FITC)-conjugated sheep anti-mouse IgG (1:50) and Cy -conjugated goat anti-rabbit IgG (1:400, Jackson ImmunoResearch Laboratories, Inc.) for 1 h at room temperature. Then, unbound secondary antibodies were rinsed 3x with PBS and 1x with deionized water to reduce PBS-produced autofluorescence. Specimens were mounted on microscope slides with anti-fade media (Prolong-Gold, Invitrogen) and viewed under a confocal microscopy (Ziess Pascal model). 4.3.7 Drugs. All drugs except cocaine and guanethidine were obtained from Sigma Aldrich, USA. Cocaine was acquired from Mallinckrodt, Missouri, USA. Guanethidine (CIBA Pharmaceutical, New Jersey) was a generous gift from Dr. Susan Barman. Forskolin, CGS21680 and SCH58261 were made as stock solutions in DMSO and kept at 0°C. The highest concentration of DMSO present in working solutions (0.001% vol/vol) did not affect vascular or sympathetic nerve function. Cocaine, guanethidine, and H-89 were dissolved in deionized water as concentrated stock solutions and kept at 0°C. All drug stocks were freshly diluted with Krebs buffer on the day of experiment. 4.3.8 Statistical Analysis. All data are reported as mean ± S.E., n values indicate the number of rats. Differences between groups were assessed using Student’s t-test. Differences in agonist concentration response curves were assessed first by a 2 way ANOVA and Bonferoni’s test for multiple comparisions (GraphPad Prism 5.0, LaJolla, CA). Agonist IC50 (half maximal inhibitory drug concentration) and maximum effect values were determined from individual concentration response curves using a non- ! "#$! linear fitting routine and a logistic equation (Origin 8.0, Northampton, MA). IC50 and maximum effect values where then compared using Student’s t-test. IC50 values are expressed as the negative log of the drug concentration that causes 50% of the maximum inhibitory response (pIC50). P<0.05 was considered statistically significant. Kinetic of NE clearance is represented by !-ratio, which is calculated by area under the curve (AUC) of the pre-peak current (pre-Imax) divided by AUC of post-Imax (Fig. 33A). The lower !-ratio indicates the faster NE clearance. ! "#$! 4.4 Results We first present the result of experiments that show expression of A2AR on sympathetic nerves supplying mesenteric arteries and veins from sham rats. The second section of the results describes A2AR function in enhancing NE clearance of sham rats. The third section we report the results of experiments that examine the disruption of A2AR in DOCA-salt hypertension. Finally, we present results of studies that involve downstream mechanism of A2AR activation and illustrates impaired Gsstimulated adenylase cyclase (AC) step of DOCA-salt hypertension causing failure to enhance NE clearance during A2AR activation. 4.4.1 A2ARs are localized to sympathetic nerves supplying mesenteric arteries and veins. Tyrosine hydroxylase (TH), the rate-limiting enzyme in for the NE biosynthetic pathway, and A2ARs antibodies were used to identify perivascular sympathetic nerves and A2ARs. A2ARs were co-localized with TH in peri-arterial and peri-venous nerves (Fig. 39). ! "#"! Figure 39. Localization of A2A adenosine receptors (A2ARs) on sympathetic nerves supplying mesenteric artery and vein of sham rats. Mouse monoclonal TH (1:100) and rabbit polyclonal A2AR (1:100) antibodies were used to identify sympathetic nerves and A2AR, respectively. Images of whole mount preparations were acquired using confocal microscopy. TH is a protein marker for sympathetic nerves. Arteries (top panel) and veins (bottom panel) of sham rats contain TH (green) and A2AR s (red). Merged images showed co-localization of TH and A2AR (yellow). Scale bar=20µm. TH=tyrosine hydroxylase. ! ! "#$! 4.4.2 CGS21680 (selective A2AR agonist) enhanced NE clearance in mesenteric veins, but not arteries of normotensive rats. This experiment was designed to determine how A2AR activation modulated kinetics of NE release. The basal amplitude of NE oxidation currents in sham mesenteric veins was slightly higher than in sham arteries (15.6±2.2pA, n=10 vs 12.46±1.58pA, n=8, p>0.05). CGS21680 (1- 100nM) did not change peak oxidation currents in arteries and reduced this current in veins only at the highest concentration (Fig. 40B). Basal current half-life of mesenteric veins was longer than in arteries (16.9±7.4 s, n=10 vs 2.8±0.4 s, P < 0.05). The !-ratio is a measure of NE clearance rate. The lower !-ratio describes the faster NE reuptake. Basal !-ratios in mesenteric veins were significantly higher than in arteries (6.1 ± 1.2, n=9 vs 1.0 ± 0.1, n=7, P < 0.05) (Fig. 40C). CGS21680 caused a concentration dependent (1 – 100 nM) reduction in the !!ratio in veins, but not arteries (Fig. 40C). ! "#$! Figure 40. CGS21680 enhanced NE clearance in mesenteric vein, but not artery of normotensive rats. Selective agonist CGS21680 was used to activate A2AR. Concentration-response curves of CGS21680 (1-100nM) were performed and kinetics of NE oxidation currents was analyzed. A, each oxidation current was calculated for peak amplitude (Imax) and ! ratio. ! represents how fast NE was removed. The lower ! ratio indicates the faster NE clearance. Basal NE clearance in perivenous nerves was dramatically slow compared to periarterial nerves since !-ratio in the absence of agonist was much greater in mesenteric veins (C). CGS21680 (1-10nM) did not alter peak current, but high concentration of CGS21680 slightly decreased peak current due to non-specific affinity toward A1 adenosine receptors (B). CGS21680 also caused a reduction of ! ratio in concentration-dependent manner (C). High concentration These CGS-mediated effects were specific to mesenteric vein, but not artery. Data are mean±S.E., n=number of rats, Data were performed repeated one-way ANOVA with Bonferroni multiple comparison post test. * indicates different from control (p<0.05). ! ! "#$! Figure 40. (cont’d) ! ! "#$! 4.4.3 Cocaine blocked CGS-induced effects. The data above suggest that activation of A2AR increased NE clearance in veins. In the next experiment a NET blocker, cocaine, was used to determine if NE transport was involved in A2AR-enhanced NE clearance. CGS21680 (10 nM) itself caused a reduction in !-ratio (Fig. 41A), and current half-life (Fig. 41B) without affecting the peak amplitude of NE oxidation current (Fig. 41C). The CGS-induced effects were prevented by cocaine (10 µM)(Fig. 41A-B). ! "#$! Figure 41. NE transporters was involved in A2AR-enhanced NE clearance in sham mesenteric veins. NE transporters (NETs) were blocked by cocaine to examine if A2AR enhanced NE clearance via NET. Series of paired experiments were performed. CGS21680 (10nM) enhanced NE clearance as it decreased ! ratio (A) and current half-life (B). After 45 minute-wash, cocaine was incubated for 30 minutes before the agonist. Cocaine fully prevented CGS21680-induced effects (A-B). Neither cocaine nor CGS21680 affect peak amplitude of NE oxidation current(C). Data are mean±S.E., n=10. Data were analyzed by repeated one-way ANOVA with Bonferroni multiple comparison post test. # indicates different between groups (P<0.05). ! ! "##! 4.4.4 SCH58261 (selective A2AR antagonist) antagonized CGS-induced effects. SCH58261 was used to examine if enhancement of NE clearance was mediated by A2AR activation. CGS21680 enhanced NE clearance (Fig. 42A,B) and this effect was blocked by SCH58261 (Fig. 42C,D). Figure 42. CGS21680 effects were antagonized by SCH58261. To confirm that A2A activation enhances NE clearance via NET, a selective A2AR antagonist SCH58261 (400nM) was used to block CGS-mediated effect. A-B, control experiments illustrated that CGS21680 promoted NE clearance and CGS-induced effect was fully antagonized by a selectiveA2AR antagonist, SCH58261 (400nM, C, D). SCH58261 itself did not significantly affect basal NE clearance (C, D). Data are mean±S.E., n=6-15. Data were analyzed by ordinary one-way ANOVA with Bonferroni multiple comparison post test. * indicates different between groups (P<0.05). ! ! "#$! Figure 42. (cont’d) ! "#$! 4.4.5 NE clearance is impaired in DOCA-salt hypertension We next tested if A2AR-induced enhancement of NE clearance is impaired in DOCA-salt hypertension. Only DOCA-salt veins were studied because CGS21680 did not alter NE clearance in arteries. The basal !-ratio and current half-life were higher in DOCA-salt compared to sham veins (Fig.43 A, B). DOCA-salt veins were less responsive to CGS21680 (10 nM) compared to sham veins (Fig 43A). CGS21680 decreased the !-ratio in DOCA-salt and sham veins by 13.06±4.9% and 49.05 ± 7.9%, (P < 0.05, Fig. 43A)) respectively. CGS21680 (10 nM) also shortened oxidation current half-life in DOCA-salt and sham veins by 10 ± 11%, and 40±7.2(P<0.05), respectively (Fig. 43B). The data above demonstrate that NE clearance in DOCA-salt veins is slower than in sham vein. The next experiment was performed to determine if impaired NET function contributes to inefficient NE clearance of DOCA-salt veins. Guanethidine, a substrate for NET was used to assess NET function. Guanethidine is taken up into sympathetic nerve terminals and then blocks NE release resulting in a reduction of NE oxidation current. Basal NE release in DOCA-salt veins was higher then in sham veins (9.1±2.1, n=4 vs 15.8±1.7pA, n=8, Fig. 43C). The concentration response curves for guanethidine-induced inhibition of NE oxidation currents were similar in sham and DOCA-salt veins (Fig. 43C). Maximal inhibition in sham and DOCA-salt veins were 100± 0.0%, n=4 and 95.4±3.3%, n=8, P > 0.05) respectively. The pIC50 values for guanethidine were similar in sham and DOCA-salt veins (7.4±0.0, n=4 and 7.7±0.2, n=8 Why is there a different “n” value here compared to the max inhibition n=4) respectively P>0.05). ! "#$! Figure 43. CGS21680 revealed impaired prejunctional A2AR function of DOCA-salts mesenteric veins. A, Basal ! ratio of DOCA-salt mesenteric vein is higher than in control veins and DOCA-salt vein is relatively resisted to CGS21680-induced effect compared to control. B, CGS21680 shortened half-life of NE oxidation current in control vein, but not DOCA-salt vein. C, Guanethidine was used to determine NE transporter function. Guanethidine suppressed NE release in control and DOCA-salt veins with similar potency and efficacy, indicating that NET function is preserved in DOCA-salt vein. * indicates different between treatments of control rat (P<0.05). # salt and control rats(P<0.05). ! "#"! indicates different between DOCA- 4.4.6 Forskolin restored NE clearance of DOCA-salt hypertension Since A2AR is a Gs-coupled receptor and the next experiment was designed to determine if the A2AR-enhanced NE clearance used the adenylate cyclase/PKA/cAMP pathway. Forskolin (adenylase cyclase activator) decreased the !-ratio and NE oxidation current half-life in control veins. Forskolin also restored clearance in DOCAsalt veins (Fig. 44A,B). CGS21680 induced increases in NE clearance were blocked by H89, a PKA inhibitor (Fig. 44C,D). ! "#$! Figure 44. Forskolin restored NE clearance mechanism of DOCA-salt mesenteric veins. PKA activator (forskolin) and inhib itor (H89) were used to reveal downstream mechanism of A2AR activation. Forskolin mimicked CGSinduced effects in control veins in lowering !-ratio and NE current half-life (A, B) and CGS-induced effects were prevented by H89 (C, D). Forskolin restored NE clearance mechanism in DOCA-salt veins suggesting that A2AR disruption occurs at Gs-adenylase cyclase coupling step. * indicates different between treatments (P<0.05). ! "#$! 4.5 Discussion 4.5.1 A2AR expression on sympathetic nerves supplying mesenteric arteries and veins. Our results show that sympathetic nerves more densely supply mesenteric arteries compared to veins as shown previously (Birch et al., 2008; Park et al., 2007). We also show that A2AR immunoreactivity co-localization with TH a marker for sympathetic nerves in arteries and veins. We also detected some non-neuronal A2AR immunoreactivity. This is not surprising as A2AR couple to vasodilation in mesenteric artery (Hiley et al., 1995) and coronary artery (Belardinelli et al., 1998; Glover et al., 1996), pial artery(Shin et al., 2000) and renal artery (Hansen et al., 2003; Rump et al., 1987). Hence, the non-neuronal A2AR immunoreactivity is likely to be arterial smooth muscle cells. Conversely, there was far less non-neuronal A2AR immunoreactivity in mesenteric veins. We have shown previously that adenosine (Sangsiri et al., 2012) constricts mesenteric veins, but not arteries suggesting that A1R is more prominent in controlling venomotor tone directly. 4.5.2 Analysis of NE oxidation current kinetics. The rise and fall of NE oxidation currents reflect NE release and clearance respectively. During the rising phase, a short train of focal stimulation causes NE release, which accumulates near the electrode producing the peak current (Imax). The rising phase consists of 2 components. Phase1 is rapid and is due to NE release from ! "#$! multiple varicosities. Phase2 is slower and is influenced by exocytosis and the activation of !2-adrenergeic and A1-adenosine autoreceptors as NE accumulates in the junction causing a decline in the release rate (Park et al., 2010). The decay phase is controlled largely by neuronal reuptake via NET, and diffusion away from release sites and the microelectrode. Diffusion is constant across different experimental conditions due to the constant solution flow rate and fixed microelectrode position. Thus, the current decay under different conditions reflects changes in NE clearance. Changes in the !-ratio could be due to changes in the rise and/or decay of the NE oxidation. However, none of the drugs used in this study altered the rising phase of NE current in mesenteric veins. Thus, changes in the !-ratio represent changes in NE clearance by NET. 4.5.3 Activation of A2A receptors enhances NE clearance in perivenous, but not periarterial sympathetic plexus. Recent studies have reported a novel mechanism of A2AR in modulating GABA and glutamate uptake via and action on GABA and glutamate transporters, respectively (Cristóvão-Ferreira et al., 2011; 2009; Gonzalez et al., 2006), (Matos et al., 2012). In the present study, we have revealed a novelty of A2ARs modulation of NE transporters in peripheral nervous system. Activation of the prejunctional A2AR using CGS2168 enhances NE clearance. This effect was blocked by cocaine and the A2AR antagonist, SCH58261. This suggests that enhancement of NE transport is mediated by A2AR activation and that NET is ! "#$! required. This mechanism may considered as a cross regulation of release of clearance by co-released neurotransmitters (NE and ATP) via their respective autoreceptors. A2AR enhanced NE clearance is more prominent in perivenous compared to periarterial sympathetic nerves. Artery-vein differences in the arrangement of sympathetic nerves could partly explain this difference in clearance. The sympathetic nerve plexus is denser in mesenteric arteries compared to veins. Periarterial nerves could clear NE more efficiently than perivenous nerves because NET expressed by neighboring or varicosities near release sites could contribute to NE clearance. This is analogous to “lateral inhibition” mediated by !2AR autoreceptors (Brock, 1995). Lateral inhibition does not occur in perivenous nerves since their varicosities are too far from one another to detect NE released from adjacent varicosities or nerve fibers. This would explain the slow basal NE clearance in mesenteric veins. Secondly, the periarterial plexus resides at the adventitial and media border while the perivenous plexus spreads deeper into muscle layer (Birch et al., 2008). Thus, neurogenic venous contraction occurs more rapidly than arterial contraction because of the closer proximity of varicosities to the muscle cells based on nerve geography (Park et al., 2007). Third, veins are more sensitive to NE-mediated contraction than arteries (Pérez-Rivera et al., 2004). Therefore, small increases in NE availability would constrict veins and reduce capacitance. Finally, prolonged !1-adrenergic receptor activation causes desensitization in mesenteric veins, but not arteries (Pérez-Rivera et al., 2004; Xu et al., 2007), indicating a need for regulation of NE availability at the neuroeffector junction. Thus, although veins do not make efficient use of the A2AR autoreceptor to regulate NE ! "#$! availability they may use regulation of NE clearance to buffer junctional NE. This mechanism may permit veins to re One may question about the source of adenosine that modulates basal perivenous sympathetic transmission. Is it from neuronal or non-neuronal sites? An !1adrenergic receptor blocker, prazosin, completely blocked neurogenic constriction in veins, but only partly reduces arterial contraction (Park et al., 2007). In addition, purine receptor blockers do not affect neurogenic constrictions in veins while markedly inhibiting arterial constrictions (Park et al., 2007). These results support the idea that NE is the major transmitter released from perivenous sympathetic nerves. It has been argued that although P2X1 receptor agonist, !, !-methylene ATP failed to contract mesenteric vein, ATP and UTP, an agonist for P2Y2/P2Y4 receptors could induce venous contraction, and this effect were prevented by sumarin a P2Y2 receptor antagonist (Galligan et al., 2001). Moreover, Bobalova et al detected electrically-evoked ATP release from canine and guinea-pig mesenteric veins. It should be noted that basal neurogenic ATP release was much lower than basal NE release, particularly in canine mesenteric veins (approximately 10 times lower than NE)(Bobalova et al., 2001a; Bobalova et al., 2001b). Thus, it is possible that ATP is co-released with NE in perivenous nerves and the small amount of ATP released may provide the adenosine needed to modulate adrenergic transmission. Postjunctional P2Y receptors may only be activated when high concentrations of ATP are achieved such as might occur during cell injury (Yin et al., 2007). ! "#$! Since activation of A2AR requires brief, but high (micromolar) concentrations of adenosine (Olah et al., 1995), enhanced NE clearance by A2AR action may serve as a rescue mechanism to shut down sympathetic nerve activity to reduce oxygen consumption. Adenosine is known as a key regulator of vasomotor tone during hypoxia (Tabrizchi et al., 2001). Unlike previous studies showing that A2AR facilitate NE release in the rat tail artery (Fresco et al., 2004), we did not see facilitation of NE release through A2AR activation in mesenteric arteries or veins. It is possible facilitation of NE release could be masked by A1R-coupled NE release inhibition since we did not preincubate the tissue with A1R antagonist. One should keep in mind that although CGS21680 is considered as selective A2AR agonist, it is likely to distinguish A2AR well from A2BR but not from the other subtypes. CGS21680 is 170-fold selective for A2A over A1R in rat and its selectivity is far lower in humans (Klotz, 2000). A more selective A2AR agonist is needed. 4.5.4 Impaired A2AR function in DOCA-salt hypertension occurs at Gs-protein coupling step. We studied A2AR function in DOCA-salt mesenteric veins because CGS21680 did not alter arterial NE clearance in sham rats. In DOCA-salt veins, CGS21680 failed to enhance NE clearance suggesting that the prejunctional A2AR function is impaired in ! "##! DOCA-salt hypertensive veins. A2AR dysfunction probably occurs at the Gs-stimulated adenylase cyclase step because forskolin could restore NE clearance in DOCA-salt mesenteric veins. Previous work showed impaired NET function in DOCA-salt arteries, but not veins (Dong, 2009), which is consistent with our data showing that NET function is not altered in DOCA-salt veins. Western blot analysis revealed overexpression of NET in mesenteric arteries, veins, and superior cervical and stellate sympathetic ganglia in DOCA-salt rats (Luo et al., 2003). This suggests that NET overexpression in DOCA-salt arteries may occur to compensate for impaired NET function. Upregulation of NET in DOCA-salts veins may result from impaired prejunctional A2AR stimulation of NET activity. Overall, A2AR dysfunction accounts for impaired NE clearance DOCA-salt veins and increased NE availability to act at postjunctional receptors. The present study is the first to show disruption of prejunctional receptors on perivenous sympathetic nerves. Relatively high sensitivity to NE-induced constriction plus elevated NE level due to slow clearance mechanism in DOCA-salt mesenteric veins could contribute to increased venomotor tone and a decrease in vascular capacitance (Fink, 2009). ! "#$! BIBLIOGRAPHY ! "#$! BIBLIOGRAPHY Astrand P, Stjärne L (1989). ATP as a sympathetic co-transmitter in rat vasomotor nerves--further evidence that individual release sites respond to nerve impulses by intermittent release of single quanta. Acta Physiol Scand 136(3): 355-365. Belardinelli L, Shryock JC, Snowdy S, Zhang Y, Monopoli A, Lozza G, et al. (1998). The A2a adenosine receptor mediates coronary vasodilation. The Journal of Pharmacology and Experimental Therapeutics 284: 1066-1073. Birch DJ, Turmaine M, Boulos PB, Burnstock G (2008). Sympathetic innervation of human mesenteric artery and vein. J Vasc Res 45: 323-332. Bobalova J, Mutafova-Yambolieva VN (2001a). Co-release of endogenous atp and noradrenaline from guinea-pig mesenteric veins exceeds co-release from mesenteric arteries. Clinical and Experimental Pharmacology and Physiology 28: 397-401. Bobalova J, Mutafova-Yambolieva VN (2001b). Presynaptic 2-adrenoceptor-mediated modulation of adenosine 5¢ triphosphate and noradrenaline corelease: differences in canine mesenteric artery and vein. Journal of Autonomic Pharmacology 21: 47-55. Bouvier M, deChamplain J (1986). Increased basal and reactive plasma norepinephrine and epinephrine levels in awake DOCA-salt hypertensive rats. Journal of the Autonomic Nervous System 15: 191-195. Brock JA (1995). Chapter 5. Modulation of neurotransmitter release by autoreceptors. In: Neurotransmitter release and its modulation, Powis DA, Bunn SJ (eds). Cambridge: Cambridge University Press. Chobanian AV, Bakris GL, Black HR, Cushman WC, Green LA, Izzo-Jr JL, et al. (2003). Seventh report of the joint national committee on prevention, detection, evaluation, and treatment of high blood pressure. Hypertension 42: 1206-1252. Cristóvão-Ferreira S, Navarro G, Brugarolas M, Perez-Capote K, Vaz SH, Fattorini G, et al. (2011). Modulation of GABA Transport by Adenosine A1R–A2AR Heteromers, Which Are Coupled to Both Gs- and Gi/o-Proteins. The Journal of Neuroscience 31(44): 15629-15639. Cristóvão-Ferreira S, Vaz S, Ribeiro J, AM S (2009). Adenosine A2A receptors enhance GABA transport into nerve terminals by restraining PKC inhibition of GAT-1. J Neurochem 109(2): 336-347. ! "#"! deChamplain J (1990). Pre- and postsynaptic adrenergic dysfunctions in hypertension. J Hypertens 8(7): S77-85. deChamplain J, Farley L, Cousineau D, Ameringenj M-RV (1976). Circulating catecholamine levels in human and experimental hypertension. Circulation Research 38: 109-114. Dong H Disruption of norepinephrine release and clearance mechanisms in sympathetic nerves associated with mesenteric arteries but not veins in salt-sensitive hypertension. Ph.D Dissertation, Michigan State University, East-Lansing, 2009. Eckle T, Faigle M, Grenz A, Laucher S, Thompson LF, Eltzschig HK (2008). A2B adenosine receptor dampens hypoxia-induced vascular leak. Blood 111: 2024-2035. Fink GD (2009). Sympathetic activity, vascular capacitance, and long-term regulation of arterial pressure. Hypertension 53: 307-312. Fresco P, Diniz C, Goncalves J (2004). Facilitation of noradrenaline release by activation of adenosine A2A receptors triggers both phospholipase C and adenylate cyclase pathways in rat tail artery. Cardiovascular Research(63): 739-746. Galligan JJ, Hess MC, Miller SB, Fink GD (2001). Differential localization of p2 receptor subtypes in mesenteric arteries and veins of normotensive and hypertensive rats. J Pharmacol Exp Ther 296(2): 478-485. Glover DK, Ruiz M, Y J, Yang, Koplan BA, Allen TR, et al. (1996). Pharmacological stress thallium scintigraphy with 2 cyclohexylmethylidenehydrazinoadenosine (WRC0470), a novel, short-acting adenosine A2A receptor agonist. Circulation 94: 1726-1732. Gonzalez B, Paz F, Floran L, Aceves J, Erlij D, Floran B (2006). Adenosine A2A receptor stimulation decreases GAT-1-mediated GABA uptake in the globus pallidus of the rat. Neuropharmacology 51: 154-159. Hansen PB, Schnermann J (2003). Vasoconstrictor and vasodilator effects of adenosine in the kidney. Am J Physiol Renal Physiol 285: F590-F599. Hiley CR, Botril FE, Warnock J, Richardson PJ (1995). Effects of pH on responses to adenosine, CGS 21680,carbachol and nitroprusside in the isolated perfused superior mesenteric arterial bed of the rat. British Joumal of Phamacology 116: 2641-2646. Klotz K-N (2000). Adenosine receptors and their ligands. Naunyn-Schmiedeberg’s Arch Pharmacol 362: 382-391. Koeppen M, Eckle T, Eltzschig HK (2011). Interplay of hypoxia and A2B adenosine receptors in tissue protection. Advances in Pharmacology 61: 145-186. ! "#$! Luo M, Fink GD, Lookingland KJ, Morris JA, Galligan JJ (2004). Impaired function of α2adrenergic autoreceptors on sympathetic nerves associated with mesenteric arteries and veins in DOCA-salt hypertension. Am J Physiol Heart Circ Physiol 286: H15581564. Luo M, Hess MC, Fink GD, Olson LK, Rogers J, Kreulen DL, et al. (2003). Differential alterations in sympathetic neurotransmission in mesenteric arteries and veins in DOCAsalt hypertensive rats. Autonomic Neuroscience: Basic and Clinical 104: 47-57. Matos M, Augusto E, Santos-Rodrigues AD, Schwarzschild MA, Chen J-F, Cunha RA, et al. (2012). Adenosine a2a receptors modulate glutamate uptake in cultured astrocytes and gliosomes. Glia 60: 702-716. Michel MC, Brodde O-E, Insel PA (1990). Peripheral adrenergic receptors in hypertension. Hypertension 16: 107-120. Olah ME, Stiles GL (1995). Adenosine receptor subtypes: characterization and therapeutic regulation. Annu Rev Phamloeol Toxicol 35: 581-606. Park J, Galligan JJ, Fink GD, Swain GM (2010). Alterations in sympathetic neuroeffector transmission to mesenteric arteries but not veins in DOCA-salt hypertension. Auton Neurosci 152(1-2): 11-20. Park J, Galligan JJ, Fink GD, Swain GM (2007). Differences in sympathetic neuroeffector transmission to rat mesenteric arteries and veins as probed by in vitro continuous amperometry and video imaging. J Physiol 584(3): 819-834. Pérez-Rivera AA, Fink GD, Galligan JJ (2004). Increased reactivity of murine mesenteric veins to adrenergic agonists: functional evidence supporting increased α1adrenoceptor reserve in veins compared with arteries. J Pharmacol Exp Ther 308(1): 350-357. Ralevic V, Burnstock G (1998). Receptors for purines and pyrimidines. Pharmacological Reviews 50(3): 413-492. Rump L, Jabbari-T J, von Kügelgen I, Oberhauser V (1987). Adenosine mediates nitricoxide-independent renal vasodilation by activation of A2A receptors. J Hypertens 12(2): 1987-1993. Sangsiri S, Dong H, Swain GM, Galligan JJ, Xu H (2012). DOCA-salt hypertension impairs perivascular sympathetic A1 adenosine receptor function. East-Lansing. Schenk J, McNeill JH (1992). The pathogenesis of DOCA-salt hypertension. Journal of Pharmacological and Toxicological Methods 27: 161-170. ! "#$! Shin HK, Park SN, Hong KW (2000). Implication of adenosine A2A receptors in hypotension-induced vasodilation and cerebral blood flow autoregulation in rat pial arteries. Life Sciences 67(12): 1435-1445. Tabrizchi R, Bedi S (2001). Pharmacology of adenosine receptors in the vasculature. Pharmacology & Therapeutics 91: 133-147. Xu H, Fink GD, Galligan JJ (2007). Increased sympathetic venoconstriction and reactivity to norepinephrine in mesenteric veins in anesthetized DOCA-salt hypertensive rats. Am J Physiol Heart Circ Physiol 293(1): H160-H168. Yin J, Xu K, Zhang J, Kumar A, Yu F-SX (2007). Wound-induced ATP release and EGF receptor activation in epithelial cells. Journal of Cell Science 120: 815-825. ! ! "#$! CHAPTER 5 GENERAL DISCUSSION, PERSPECTIVES AND FUTURE DIRECTIONS ! "#$! 5.1 Result Summary Sympathetic nervous system hyperactivity is associated with salt-sensitive hypertension. However, the mechanism of sympathetic nerve overactivity is not fully understood. From the autoreceptor perspective, it is possible that local regulations of neurotransmitter release and clearance by prejunctional receptors at sympathetic nerve varicosities are compromised (deChamplain et al., 1987; Dong, 2009). This leads to an increase in sympathetic transmitter release and gradual removal of transmitters from the neuroeffector junction causing sustained vascular contraction, thereby an increase in arterial peripheral resistance and a rise in blood pressure. degradation product These present studies focus on role of an ATP adenosine as a neuromodulator in sympathetic neurotransmission of mesenteric circulation. In these studies, DOCA-salt hypertensive rats were used, as this model is a low renin form of salt-sensitive hypertension with an increase in sympathetic nerve activity. Video camera and continuous amperometry with carbon fiber microelectrode were used to detect vasoconstriction and real-time NE release from sympathetic nerve varicosities in response to brief trains of electrical nerve stimulation. The overall finding of these present studies is that DOCA-salt hypertension is associated with disruption of prejunctional adenosine receptor function. Decreased activity of prejunctional adenosine A1 receptors coupled to inhibition of NE release leads to increased NE release from sympathetic varicosities. Additionally, dysfunction of prejunctional adenosine A2A receptors linked to increasing NE transporter activity results in increased NE availability at the neuroeffector junction to act on the postjunctional ! "#$! receptors. These findings indicate a fine-tuning cross-regulation between purinergic and adrenergic neurotransmission and reveal a local dysregulation of sympathetic transmission at the neuroeffector junction, which occurs independently from the central nervous system. The key findings of these present studies include: 5.1.1. Prejunctional adenosine A1 receptors (A1Rs) inhibits NE release from perivascular sympathetic nerves and impaired A1R function contributes to increased NE release in DOCA-salt hypertension. This finding is consistent with previous published studies reporting that A1Rs couple to inhibition of NE release from sympathetic nerves supplying several vascular beds including rat tail arteries (Diniz et al., 2004) , rat caudal arteries (Shinozuka et al., 1988), and human forearm vessels (Rongen et al., 1996). In the present study, it is found that activation of the prejunctional A1Rs located on periarterial and perivenous sympathetic nerves inhibits NE release from sympathetic nerve varicosities supplying mesenteric arteries and veins. The function of the prejunctional A1Rs is predominant in periarterial nerves under normal physiologic condition. Previous work has shown that the prejunctional !2-adrenergic autoreceptor function is impaired in DOCA-salt hypertension (deChamplain et al., 1987). My data show that this effect is not specific for the !2-adrenergic autoreceptor as A1 autoreceptor function is also impaired. Similar to the prejunctional !2-adrenergic ! "#$! receptors, A1R function is impaired specifically in periarterial, but the receptor function is preserved in perivenous sympathetic nerves. 5.1.2. Adenosine A2A receptor (A2AR) stimulation enhances sympathetic NE clearance and this response is impaired in DOCA-salt hypertension. Herein, the novel mechanism of A2ARs in modulating NE uptake via NE transporter in peripheral nervous system is revealed. A2ARs are part of a local mechanism to regulate NE clearance from the neurovascular junction. Unlike A1R function, which is predominant in periarterial nerves, the A2AR activation enhances NE clearance occurring prominently in perivenous nerves. This mechanism provides such a precise regulation of NE availability to perivenous sympathetic neuroeffector junction, where low efficiency of NE clearance occurs. This mechanism also permits rapid removal of !1-adrenergic receptor desensitization in response to NE providing efficient dynamics of venomotor tone to maintain normal blood pressure during postural hypotension. Prejunctional A2AR function is impaired in DOCA-salt hypertension and impairment probably occurs at the Gs-protein coupling step as forskolin (an adenylyl cyclase activator) restored NE clearance in DOCA-salt veins. This work is the first showing the disruption of prejunctional receptors on perivenous sympathetic nerves. ! "#$! 5.2 General Discussion 5.2.1 Why perivascular sympathetic nerves contain and release more than one transmitter? Firstly, co-stored transmitters may be a simple way to balance ions within synaptic vesicles. The major transmitter NE with positively charged amine might be neutralized by negatively charged ATP and/or NPY molecules. Secondly, the pattern of sympathetic nerve firing can vary. In some circumstances, the physiological function of an organ might require short burst of high frequency that release certain transmitters, while during other periods it might require a slow tonic release of another transmitter. For instance, sympathetic nerves innervating the vas deferens contain NE, ATP, and NPY as cotransmitters. Under low frequency nerve stimulation, NE and ATP, but not NPY are released. NPY is likely to be released during intense stimulation (Kennedy et al., 1997) to produce strong contraction and enhance action of NE (Ekblad et al., 1984). Thirdly, release of multiple transmitters may provide synergism and flexibility of postjunctional responses. For example, NE and ATP are co-released by perivascular sympathetic nerves onto the postjunctional !1 adrenergic and P2 receptors, respectively to synergistically stimulate contraction. In arteries, stimulation of the postjunctional P2X receptors by ATP elicits rapid arterial contraction following by sustained contraction mediated by the action of NE on the metabotropic !1 adrenoceptors. Moreover, NE action on the !1 adrenoceptors is likely to desensitize quickly in mesenteric veins. Conversely, adenosine, an ATP degradation product, provides sustained venous contraction ! "##! through activation of A1 adenosine receptors (see chapter3). This indicates a rescue mechanism from receptor desensitization by using multiple transmitters to activate different effector pathways. Moreover, while neuronal ATP acts on P2 receptors, NE-induced activation of !1 adrenoceptors also causes the release of ATP from vascular smooth muscle cells to potentiate the contraction indicating flexibility to produce neurogenic contraction (Billaud et al., 2011). Lastly, cotransmitters modulate release of one another to provide precise control of neurotransmission. For example, NE provides the autoinhibition to inhibit further NE release, NE also inhibits ATP release (Demel et al., 2008a). Likewise, adenosine, an ATP metabolite, interacts with the prejunctional A1 adenosine receptor to inhibit NE (Sangsiri et al., 2010) and ATP release (Demel et al., 2008a). Prejunctional adenosine receptors may serve as a spare mechanism to control major transmitter release under pathologic conditions (hypertension, for example). One should keep in mind that the co-transmitters released by perivascular sympathetic nerves are not fixed and could be altered by aging and pathologic diseases (Burnstock, 1990). Periarterial sympathetic nerves in DOCAsalt hypertension have decreased purinergic transmission due to ATP depletion (Demel et al., 2008b). Conversely, adrenergic transmission is likely to be enhanced, as tyrosine hydroxylase is upregulated (Grobecker et al., 1976) and increased plasma NE is detected (Bouvier et al., 1986). Furthermore, NE and NPY bioavailability is increased, but purinergic transmission is decreased in ! "##! perivascular sympathetic nerves of spontaneously hypertensive rats (SHR) (Burnstock, 1990) . 5.2.2 Is G-protein coupling a key pathological target? As my work suggests the impaired function of the presynaptic A1R, and A2AR, likely to occurs at the receptor-G-protein coupling step. This work supports the hypothesis that failure of the receptor-G-protein interaction may account for the impaired negative modulation of NE release provided by the prejunctional receptors on perivascular sympathetic nerves. Autoinhibitory feedback is a general mechanism for control of neurotransmitter release throughout the nervous system. The basic mechanism 2+ of !2-AR inhibition is based on reduced opening probability of Ca 2+ the inhibitory "# subunit of GO-protein leading to decreased Ca channel by currents and inhibition of exocytosis. Lipscombe and co workers showed that NE-induced !22+ AR activation strongly inhibited the N-type, but not L-type Ca channels in frog sympathetic ganglia (Lipscombe et al., 1989). Likewise, Yawo and Chuhma showed that adenosine inhibited $-conotoxin-sensitive Ca 2+ channels (Yawo et 2+ al., 1993) suggesting that the N-type Ca channels are the target for adenosine receptor-mediated autoinhibition as $-conotoxin is a selective inhibitor of the Ntype Ca ! 2+ channels. It should be noted that the concentration of $-conotoxin in "#$! their study was 10 µM which if far above the concentration that is selective for N2+ type channels (Yawo et al., 1993). Demel showed that N-type Ca channel function is preserved in DOCA-salt hypertensive rats (Demel, 2008). Thus, it is logical to imply that autoinhibitory feedback failure of the !2-ARs and A1Rs found in DOCA-salt arteries probably occurs somewhere upstream the activation of 2+ Ca channels which could be either extracellularly at agonist-receptor binding step or intracellularly at the receptor-G protein interaction step. Fardoun and coworkers have reported that reactive oxygen species (ROS) interfere with the coupling of dopamine D1 receptors and the target G-protein by causing an increase in the G protein receptor kinase (GRK) level in old Fischer 344 rats (Fardoun et al., 2006). GPCRs are substrates for GRK phosphorylation, which promotes receptor downregulation via a "-arrestin-dependent pathway. It is possible that changes in GRK level or activity would alter the activity of the GPCRs and their coupling activity with G-proteins. Growing evidence suggests that increased GRK level is involved with impaired receptor-G protein coupling resulting in reduced adenylyl cyclase activation (Gros et al., 1997). GRK2 is highly expressed in immune cells and its expression is affected by oxidative stress (Lombardi et al., 2002). The role of GRK2 in phosphorylation of "adrenergic receptors, which link to Gs/ adenylyl cyclase/ cAMP pathway under normal and hypertensive conditions has been studied widely. However, the study of the GRK mediated phosphorylation of the !2-adrenergic, A1, and A2A adenosine receptors in hypertension has not been undertaken. Fortunately, the ! "#"! process of A1R desensitization has been studied intensively and it has been shown that GRK2 is also responsible for A1R desensitization, where the GRK2 phosphorylation site is located in the third intracellular loop (Nie et al., 1997) on serine and threonine residues (Jajoo et al., 2010) and at the threonine residue on the C-terminus (Palmer et al., 1996). A2AR/Gs couple receptor desensitization is also mediated by GRK2 phosphorylation, and tumor necrosis factor (TNF)-! prevented A2AR desensitization and this TNF-! induced-effect occurs similarly in "-adrenergic receptors. TNF-! prevents receptor desensitization by blocking translocation of GRK2 to the phosphorylation site of the receptor (Khoa et al., 2006). This phenomenon may account for the selective impairment of the function of prejunctional Gi-, but not Gs-coupled receptors in DOCA-salt arteries. Interestingly, GRK4 is associated with salt-sensitive hypertension in humans and in animal models (Gros et al., 1997; Gros et al., 2000; Zeng et al., 2008). Increased levels of GRK lead to hyperphosphorylation of the GPCR allowing "arrestin binding, which in turn prevents the G-protein interaction with the receptor. Differential increases of GRK protein subtypes may account for differential disruption of receptor-G protein coupling as observed in arteries versus veins. Moreover, it has been reported that ROS induced a rapid transient 2+ increase of intracellular Ca 2+ ([Ca ]in) in various cell types including smooth 2+ muscle cells. The rapid increased [Ca ]in is caused by ER store leakage as ! "#$! ROS inhibit the ATP-dependent Ca2+ pump (SERCA) on the ER membrane (Thannickal et al., 2000). Thus, the cells could not maintain a low concentration 2+ 2+ (10-100 nM) of free [Ca ]in. The rapid increase of [Ca ]in is followed by a slow 2+ increased [Ca ]in mostly derived from extracellular site (Thannickal et al., 2000). This mechanism could partly account for a slightly depolarized resting membrane potential of DOCA-salt arterial smooth muscle cells compared to sham (Demel, 2008) . ! "#$! 5.3 Therapeutic significance by modulation of perivascular sympathetic transmission These results suggest that sympathetic nerve overactivity in salt-sensitive hypertension is partly independent from the central control. The physiological relevance of perivascular sympathetic transmission has been studied by several groups (Brock et al., 1997; Bucher et al., 1992; Donoso et al., 1997; Ekblad et al., 1984; Nilsson et al., 1985; Ralevic, 1995; Shinozuka et al., 1988; Stjarne, 1989; Westfall et al., 1995). However, the pharmacological differences between arteries and veins, and the possible pre-and postjunctional interactions between drugs and the different neurotransmitter system at arterial vs venous nerve varicosities have not been widely studied. For example, one drug may exert pharmacological action differently by periarterial versus perivenous nerves. In this case, cocaine (NE transporter blocker) and CGS21680 (A2AR agonist) provide a good illustration. Cocaine Alternately, how can a drug that prevents one co-transmitter’s postjunctional effect also abolish the contraction effect mediated by the other cotransmitters? Specifically, why would the pharmacological action of prazosin significantly lower blood pressure in vivo, while PPAD (P2X1 receptor antagonist) does not (Demel, 2008)? Prazosin strongly inhibits vascular contraction by two major mechanisms. First, prazosin blocks the !1 adrenergic receptors (!1AR) so that NE is no longer produce vascular contraction. Second, the postjunctional !1AR blockade prevents a large amount of ATP release from vascular smooth ! "#$! muscle cells through pannexin1 channels. Conversely, PPAD failed to lower blood pressure in vivo as PPAD only blocks the P2X receptors, but the cotransmitter NE still activates !1AR and produce arterial contraction. Moreover, how would a drug exert multiple effects on the same transmitter release system? Specifically, low concentrations of dipyridamole (adenosine transporter blocker) cause a decreased synaptic concentration of NE. Hence, the NE-dependent postjunctional effects would be decreased. Alternatively, high concentrations of dipyridamole produce an increased concentration of NE in the neurovascular junction, which would increase NE-dependent postjunctional effects including the non-neuronal release of ATP. The evidence above indicates that differential nerve organization between arteries and veins, and the complex interaction between cotransmitters activating multiple pre- and postjunctional mechanisms certainly make pharmacological investigation more intricate. This complexity provides wider opportunities for more precise modification of the neurotransmission process, which could ultimately constitute a potential therapeutic advantage. As the alterations in the prejunctional adenosine receptor function in DOCA-salt hypertension occurs, drugs selective for presynaptic adenosine receptors may have some usefulness in the treatment of salt-sensitive hypertension. Currently, it is unclear whether the prejunctional adenosine receptor dysfunction is involved in human salt-sensitive hypertension ! "#$! 5.4 Future directions 5.4.1 Measurement of ATP release by excitatory junction potential (EJP) recording As ATP is co-released with NE from perivascular sympathetic varicosities, it is important to further investigate how purinergic transmission is regulated. In arteries, ATP acts on the P2X1 receptors on smooth muscle cells. The P2X1 receptors are ion channels permeable to cations. The influx of cations in response to P2X1 stimulation causes membrane depolarization and vascular contraction. To record EJPs one applies submaximal electrical nerve stimulation to avoid contraction and loss of the microelectrode impalement during vascular contraction. EJP recordings have some limitations. The EJP is not a direct measurement of ATP release, but it measures membrane depolarization-induced by ATP action on the P2X ion channels. In addition, EJPs do not occur in mesenteric veins, as the majority of the postjunctional ATP receptors in the veins are the P2Y G-protein coupled receptors. Demel has revealed decreased ATP bioavailability and EJP rundown in DOCA-salt arteries. She also proposed that impaired purinergic transmission of DOCA-salt rats is not due to !2-adrenergic and A1-adenosine autoreceptors as exogenous NE, UK14304 (!2 agonist), and CPA (A1R agonist) produce EJP inhibition equally in both sham and DOCA-salt arteries (Demel et al., 2008a). It is possible that the neuronal ATP releaseinduced EJP results might be masked by the non-neuronal ATP release. Kügelgen and Starke proposed that approximately one third of stimulation-evoke ! "#$! ATP release is from sympathetic nerves, and the other two third of released ATP is from smooth muscle cells (guinea pig vas deferens), where ATP is released by neurogenic NE-stimulated !1 adrenoceptors (Kügelgen et al., 1991). As mentioned earlier, pannexin1 channels are crucial in facilitating intracellular ATP release in response to the postjunctional !1D receptor activation (see chapter 1). NE is also co-released during the nerve stimulation and acts on !1D receptors. Thus, ATP from neuronal and non-neuronal sources could produce the EJPs. Sympathetic NE: ATP release ratio is approximately 7:1 indicating that neuronal ATP release is quite small compared to NE (Lagercrantz, 1976). PPAD (P2X1 antagonist), dramatically inhibit neurogenic contraction in mesenteric artery indicating that ATP is a major vasoconstrictor in arteries. Thus, it is possible that ATP-induced contraction is mainly from non-neuronal site. The large amount of non-neuronal ATP may mask the neuronal ATP-mediated EJP and conceal the difference between DOCA-salt and sham responses to NE, and CPA in modulating purinergic neurotransmission. However, this could be argued that the time course of non-neuronal ATP release may be too slow to produce EJP and the direct effect of pannexin channel blockade on EJP amplitude has not been evaluated. 5.4.2 Measurement of endogenous adenosine To provide insight into how adenosine modulates sympathetic neurotransmission, direct measurement of adenosine concentration is needed. Recently, amperometry with an enzymatic-based biosensor has been used to ! "#$! measure real-time ATP release and its degradation product (adenosine) in several brain regions. Unfortunately, my attempt to measure adenosine concentration from sympathetic nerves supplying mesenteric arteries by using a commercial adenosine biosensor (Sarissa Biomedical™, Conventry, UK) failed. The failure to detect adenosine by using the commercial biosensors might related to the small amount of ATP release from the peripheral sympathetic nerves compared to the amount of ATP released in the CNS (Frenguelli et al., 2003; Gourine et al., 2008; Llaudet et al., 2005). Thus, small amounts of the ATP degradation product, adenosine, from a mesenteric artery may be below the sensitivity threshold of the biosensor. It should be noted that the amplitude of adenosine concentration detected in the brain slices during hypoxia was high up to 30 µM (Frenguelli et al., 2003). Further development of an adenosine biosensor with high sensitivity is needed. Cechova and Venton have introduced fast-scan cyclic voltametry with carbon fiber microelectrodes to detect endogenous adenosine and dopamine simultaneously from rat cuadate-putamen. As adenosine and dopamine have clearly different oxidation potentials (1.5 vs 0.6 Volts, respectively (Cechova et al., 2010)), it permits one to detect twotransmitters released simultaneously (Cechova et al., 2008). This study broadens the opportunities to detect adenosine or even simultaneous multi-detection of NE and ATP and/or adenosine by using amperometry or fast scans cyclic voltametry. Combination of amperometry with carbon fiber electrodes at the potential 1.5 Volts could permit adenosine to be detectable as an oxidation current. Additionally, fast scan cyclic voltametry with potential ranging for NE and ATP/or ! "#$! adenosine oxidation would allow one to measure multiple transmitters simultaneously. However, my major concern would be about the small proportion of ATP/adenosine release compared to NE (see chapter1) might be below the detection threshold. ! "#$! BIBLIOGRAPHY ! "##! BIBLIOGRAPHY Billaud M$! Lohman AW$! Straub AC$! Looft-Wilson R$! Johnstone SR$! Araj CA!"#$"%&'! %2011&'!Pannexin1 regulates 1-adrenergic receptor− mediated vasoconstriction'! Circulation Research!109!!80-85'! ! Bouvier M$! de Champlain J! %1986&'! Increased basal and reactive plasma norepinephrine and epinephrine levels in awake DOCA-salt hypertensive rats'! J Auton Nerv Syst!15!!191-195'! ! Brock JA$! Bridgewater M$! Cunnane TC! %1997&'! b-Adrenoceptor mediated facilitation of noradrenaline and adenosine 5'-triphosphate release from sympathetic nerves supplying the rat tail artery'! British Journal of Pharmacology! 120!!769-776'! ! Bucher B$! Corriu C$! Stoclet JC! %1992&'! Prejunctional opioid p-receptors and adenosine A1-receptors on the sympathetic nerve endings of the rat tail artery interact with the alpha 2-adrenoceptors'!N-S Arch Pharmacol!345!!37-43'! ! Burnstock G! %1990&'! Changes in expression of autonomic nerves in aging and disease'!Journal of the Autonomic Nervous System!30!!S25-S34'! ! Cechova S$!Elsobky AM$!Venton BJ!%2010&'!A1 receptors self-regulate adenosine release in the striatum: evidence of autoreceptor characteristics'! Neuroscience! 171!!1006-1015'! ! Cechova S$! Venton BJ! %2008&'! Transient adenosine efflux in the rat caudate– putamen'!Journal of Neurochemistry!105!!1253-1263'! ! deChamplain J$! Bouvier M$! Drolet G! %1987&'! Abnormal regulation of the sympathoadrenal system in deoxycorticosterone acetate - salt hypertensive rats'! Can J oPhysiol Pharm!65%8&!!1605-1614'! ! Demel SL! Impaired purinergic neurotransmission in mesenteric arteries in saltsensitive hypertension'! Doctoral of philosophy$! Michigan State University$! EastLansing$!2008'! ! Demel SL$! Galligan JJ! %2008a&'! Impaired Purinergic Neurotransmission to Mesenteric Arteries in Deoxycorticosterone Acetate-Salt Hypertensive Rats.! Hypertension!52%2&!!322-329'! ! ! "#"! Demel SL%! Galligan JJ! &2008b'(! Impaired purinergic neurotransmission to mesenteric arteries in DOCA-salt hypertensive rats(!Hypertension!52&2'!!322-329(! ! Diniz C%! Fresco P%! Leal S%! Goncalves J! &2004'(! Adenosine receptors involved in modulation of noradrenaline release in isolated rat tail artery(! Eur J Pharmacol! 504!!17-25(! ! Dong H! Disruption of norepinephrine release and clearance mechanisms in sympathetic nerves associated with mesenteric arteries but not veins in saltsensitive hypertension(! Ph.D! Dissertation%! Michigan State University%! EastLansing%!2009(! ! Donoso MV%! Brown N%! Carrasco C%! Cortes V%! Fournier A%! Huidobro-Toro JP! &1997'(! Stimulation of the sympathetic perimesenteric arterial nerves releases neuropeptide Y potentiating the vasomotor activity of noradrenaline: involvement of neuropeptide Y-Y1 receptors(!Journal of Neurochemistry!69!!1048-1059(! ! Ekblad E%!Edvinsson L%!Wahlestedt C%!Uddman R%!Hakanson R%!Sundler F!&1984'(! Neuropeptide Y co-exists and co-operates with noradrenaline in perivascular nerve fibers(!Regulatory Peptides!8!!225-235(! ! Fardoun RZ%! Asghar M%! Lokhandwala M! &2006'(! Role of oxidative stress in defective renal dopamine D1 receptor-G protein coupling and function in old Fischer 344 rats(!Am J Physiol Renal Physiol 291!!F945-F951(! ! Frenguelli BG%! Llaudet E%! Dale N! &2003'(! High-resolution real-time recording with microelectrode biosensors reveals novel aspects of adenosine release during hypoxia in rat hippocampal slices(!Journal of Neurochemistry!86!!1506–1515(! ! Gourine AV%! Dale N%! Korsak A%! Llaudet E%! Tian F%! Huckstepp R!" #$" %&'! &2008'(! Release of ATP and glutamate in the nucleus tractus solitarii mediate pulmonary stretch receptor (Breuer–Hering) reflex pathway(!J Physiol!586!!3963-3978(! ! Grobecker H%! Saavedra J%! Roizen M%! Weise V%! Kopin I%! Axelrod J! &1976'(! Peripheral and central catecholaminergic neurons in genetic and experimental hypertension in rats(!Clin Sci Mol Med Suppl!3!!337s-380s(! ! Gros R%! Benovic JL%! Tan CM%! Feldman RD! &1997'(! G-Protein–coupled receptor kinase activity is increased in hypertension(!J Clin Invest!99!!2087-2093(! ! Gros R%! Chorazyczewski J%! Meek MD%! Benovic JL%! Ferguson SSG%! Feldman RD! &2000'(! G-protein−coupled receptor kinase activity in hypertension : increased vascular and lymphocyte G-protein receptor kinase-2 protein expression(! Hypertension!35!!38-42(! ! ! "#$! Jajoo S%!Mukherjea D%!Kumar S%!Sheth S%!Kaur T%!Rybak LP!"#$"%&'!&2010'(!Role of -arrestin1/ERK MAP kinase pathway in regulating adenosine A 1 receptor desensitization and recovery(!Am J Physiol Cell Physiol!298!!C56-C65(! ! Kennedy B%! Shen GH%! Ziegler MG! &1997'(! Neuropeptide Y-mediated pressor responses following high-frequency stimulation of the rat sympathetic nervous system(! The Journal of Pharmacology and Experimental Therapeutics! 281!! 291296(! ! Khoa ND%! Postow M%! Danielsson J%! Cronstein BN! &2006'(! Tumor necrosis factorprevents desensitization of G s- coupled receptors by regulating GRK2 association with the plasma membrane(!Mol Pharmacol!69!!1311-1319(! ! Kügelgen I%! Starke K! &1991'(! Release of noradrenaline and ATP by electrical stimulation and nicotine in guinea-pig vas deferens(! Naunyn-Schmiedeberg's Archives Of Pharmacology!344!!419-429(! ! Lagercrantz H! &1976'(! On the composition and function of large dense cored vesicles in sympathetic nerves(!Neuroscience!1!!81-92(! ! Lipscombe D%! Kongsamut S%! Tsien RW! &1989'(! )*+,)-Adrenergic inhibition of sympathetic neurotransmitter release mediated by modulation of N-type calciumchannel gating(!Nature!340!!639-642(! ! Llaudet E%! Hatz S%! Droniou M%! Dale N! &2005'(! Microelectrode biosensor for realtime measurement of atp in biological tissue(!Anal Chem!77!!3267-3273(! ! Lombardi MS%!Kavelaars A%!Penela P%!Scholtens EJ%!Roccio M%!Schmidt RE!"#$"%&'! &2002'(! Oxidative Stress Decreases G Protein-Coupled Receptor Kinase 2 in Lymphocytes via a Calpain-Dependent Mechanism(!Molecular Pharmacology!62!! 379-388(! ! Nie Z%!Mei Y%!Ramkumar V!&1997'(!Short term desensitization of the A1 adenosine receptors in DDT1MF-2 cells(!Molecular Pharmacology!52!!456-464(! ! Nilsson H%!Ljung B%!Sjöblom N%!Wallin BG!&1985'(!The influence of the sympathetic impulse pattern on contractile responses of rat mesenteric arteries and vein(!Acta Physiologica Scandinavica!123!!303-309(! ! Palmer TM%! Benovic JL%! Stiles GL! &1996'(! Molecular basis for subtype-specific desensitization of inhibitory adenosine receptors(! The Journal of Biological Chemistry!271!!15272-15278(! ! ! "#$! Ralevic V! %1995&'! Modulation by nicotinamide adenine dinucleotide of sympathetic and sensory-motor neurotransmission via P1-purinoceptors in the rat mesenteric arterial bed'!Brit J Pharmacol!114!!1541-1548'! ! Rongen GA(! Lenders JWM(! Lambrou J(! Willemsen JJ(! Belle HV(! Thien T!" #$" %&'! %1996&'! Presynaptic inhibition of norepinephrine release from sympathetic nerve endings by endogenous adenosine'!Hypertension!27!!933-938'! ! Sangsiri S(!Galligan JJ!%2010&'!Differential inhibition of norepinephrine release by A1 adenosine receptors and alpha 2-adreno- ceptors in periarterial sympathetic nerves from normotensive and DOCA-salt hypertensive rats'! Hypertension! 556%4&!!e106'! ! Shinozuka K(! Bjur RA(! Westfall DP! %1988&'! Characterization of prejunctional purinoceptors on adrenergic nerves of the rat caudal artery'!N-S Arch Pharmacol! 338!!221-227'! ! Stjarne L! %1989&'! Basic mechanisms and local modulation of nerve impulseinduced secretion of neurotransmitters from individual sympathetic nerve varicosities'!Rev Physiol Biochem Pharmacol!112!!1-137'! ! Thannickal VJ(!Fanburg BL!%2000&'!Reactive oxygen species in cell signaling'!Am J Physiol Lung Cell Mol Physiol!279!!L1005-L1028'! ! Westfall TC(! Yang CL(! Curfman-Falvey M! %1995&'! Neuropeptide-Y-ATP interactions at vascular sympathetic neuroeffector junction'! J Cardiovasc Pharm! 26!!682-687'! ! Yawo H(! Chuhma N! %1993&'! Preferential inhibition of o-conotoxin-sensitive presynaptic Ca2+ channels by adenosine autoreceptors'!Nature!365!!256-258'! ! Zeng C(! Villar VAM(! Eisner GM(! Williams SM(! Felder RA(! Jose PA! %2008&'! G protein−coupled receptor kinase 4 : role in blood pressure regulation'! Hypertension!51!!1449-1455'! ! ! "#$!