MECHANISMS OF 5-HYDROXYTRYPTAMINE-INDUCED HYPOTENSION By Robert Patrick Davis A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSPHY Pharmacology and Toxicology 2011 ABSTRACT MECHANISMS OF 5-HYDROXYTRYPTAMINE-INDUCED HYPOTENSION By Robert Patrick Davis Chronic serotonin (5-hydroxytryptamine, 5-HT) infusion results in a sustained fall in blood pressure in the sham and deoxycorticosterone acetate (DOCA)-salt rat. The exact mechanism(s) underlying a 5-HT-induced fall in blood pressure are not yet known. We hypothesize that 5-HT lowers blood pressure in a nitric oxide (NO)-dependent manner by reducing total peripheral resistance (TPR), either through a reduction in sympathetic nervous system (SNS) tone and/or direct stimulation of vascular 5-HT receptors. 5-HT (25 µg/kg/min; s.c.) or vehicle was infused to male Sprague Dawley or DOCA-salt hypertensive rats. Mean arterial pressure (MAP) was measured via radiotelemetry. Cardiac output (CO) was measured via aortic flow probes. 5-HT produced a significant reduction in MAP, increase in cardiac output (CO) and stroke volume (SV), and a significant reduction in total peripheral resistance (TPR). Additionally, 5-HT produced a sustained (one-month) fall in blood pressure in the DOCA-salt rat, but did not prevent the development of DOCA-salt hypertension. Isometric contractile force was measured in the sham and DOCA-salt superior mesenteric artery (SMA) to determine whether direct 5-HT receptor activtation was capable of relaxing the SMA. BW 723C86 (5-HT2B), CP 93129 (5-HT1B), and LP-44 (5-HT7) did not relax the SMA from sham or DOCA-salt rats vs. vehicle. Electrical field stimulation (EFS) was used to determine whether 5-HT was acting to inhibit neurogenic contraction in the isolated SMA. EFS-induced contraction was not significantly different in 5-HT incuabted vs. vehicle incuabted SMA under several conditions. Finally, several studies implicate the importance of the serotonin transporter (SERT) in mediating the effects of 5-HT. 5-HT produced a significantly greater fall in MAP in the SERT WT rat vs. the SERT KO rats suggesting a potentially important role for SERT in producing a 5-HT-induced fall in blood pressure. This research fills a gap in our understanding of how 5-HT functions in the cardiovascular system and validates our hypothesis that 5-HT lowers blood pressure through a reduction in total peripherl resistance. However, this research suggests that 5-HT is not acting through direct receptor mediated vascular relxation or through inhibition of NE release at the nerve terminal in the superior mesenteric artery to lower blood pressure.   ACKNOWLEDGEMENTS I wish to thank my advisor Dr. Stephanie W. Watts for her support throughout my graduate training. I also wish to thank my thesis committee, Dr. Anne Dorrance, Dr. Gregory Fink, and Dr. Elahé Crockett, for their support during my training. Finally, I wish to acknowledge all past and present members of the Watts laboratory, as well as those members of the department who have assisted and supported my training as a graduate student.   iv     TABLE OF CONTENTS List of Tables…………………………………………………………………………………..vii List of Figures…………………………………………………………………………………vii List of Abbreviations………………………………………………………...……………...xiii Chapter 1: Introduction……………………………………………………………………….1 Hypothesis…………………………………………………………………..………….12 Specific Aims…………………………………………………………………….……..12 Chapter 2: One-month serotonin infusion results in a prolonged fall in blood pressure in the male deoxycorticosterone acetate (DOCA)-salt rat…………………………….……..15 Introduction………………………………………………………………………...…..17 Methods……………………………………………………………….……….……….21 Results………………………………….………………………………………………25 Discussion………………………..……………………………….……..……...……..31 Chapter 3: 5-hydroxytryptamine (5-HT) does not reduce total peripheral resistance through direct receptor mediated vascular relaxation of the superior mesenteric artery…………………………………………………………………………..………………..44 Introduction…………………………………………………………………..……...…47 Methods…………………………………………………………………..…………….50 Results…………………………………………………………………………….……58 Discussion………………………………………………………………….…………..65 Chapter 4: 5-Hydroxytryptamine (5-HT) does not inhibit sympathetic neurogenic contraction or sympathetic pre-/post-ganglionic sympathetic nerve activity in the rat splanchnic circulation……………………………………………………..…………………..80 Introduction………………………………………………………………………..…...82 Methods………………………………………………………………………..……….84 Results….………………………………………………………………………………90 Discussion……………………………………………….……………………………..94 Chapter 5: Lack of the serotonin transporter (SERT) reduces the ability of 5hydroxytryptamine to lower blood pressure…………………...…………………………..110   Introduction……………………………………………………………………………112   v     Methods……………………………………………………………………………….114 Results…………………………..…………………………………….………………116 Discussion……………………………………………………...……………………..118 Discussion………………………………………………………………..….………………123 References…………………………………………………………………………………...137   vi     LIST OF TABLES Table 1: MAP and HR changes in male and female SERT WT and SERT KO rats…………………….………….…………………………126 Table 2: Free plasma 5-HT levels……………………………………….…………..141   vii     LIST OF FIGURES Figure 1.1: Schematic diagram of current hypotheses……………………………..….12 Figure 1.2: A. Effect of chronic 5-HT/ Vehicle administration on mean arterial blood pressure. B. Effect of chronic administration of 5-HT or Vehicle on heart rate (HR)…………………………………………………………………….…13 Figure 2.1: Plasma (platelet poor plasma; PPP) and platelet (platelet rich plasma; PRP) 5-HT content in established DOCA-salt hypertensive rats following 28-day infusion of vehicle or 5-HT…………………………………………...37 Figure 2.2: A. Effect of chronic 5-HT or vehicle infusion on mean arterial pressure (MAP) in established DOCA-salt hypertensive rats. B. Effect of chronic 5HT or vehicle infusion on heart rate (HR) in established DOCA-salt hypertensive rats…………………………………………………………...….38 Figure 2.3: Cumulative concentration response curve to A. phenylephrine (PE), B. 5HT, or C. acetylcholine (ACh) in the thoracic aorta of established DOCAsalt hypertensive rats…………………………………………………….……40 Figure 2.4: Plasma (platelet poor plasma; PPP) and platelet (platelet rich plasma; PRP) 5-HT content in Sprague Dawley rats following a one month infusion with either 5-HT or vehicle, followed by administration of a DOCA-salt pellet……………………………………………………………....42 Figure 2.5: Effect of chronic 5-HT or vehicle infusion on mean arterial pressure (MAP) in in Sprague Dawley rats following a one month infusion with either 5-HT or vehicle, followed by administration of a DOCA-salt pellet…………….43 Figure 2.6: Cumulative concentration response curve to A. phenylephrine (PE), B. 5HT, or C. acetylcholine (ACh) in the thoracic aorta of DOCA-salt rats treated with either 5-HT or vehicle during the development of DOCA-salt hypertension…………………………………………………………………….44 Figure 3.1: Effect of chronic 5-HT or vehicle infusion on A. mean arterial pressure (MAP), B. cardiac output (CO), C. cardiac index (CI), D. stroke volume, and E. total peripheral resistance (TPR)………………………………...…72 Figure 3.2: A. mRNA expression of 5-HT receptors in the superior mesenteric artery (SMA) of sham (n=4) and DOCA-salt (n=4) rats using standard real time PCR analysis. B. Cumulative concentration response curve to 5-HT generated from baseline, contracted with prostaglandin F2 alpha (PGF2α), C. and in the presence of ketanserin in the sham superior mesenteric resistance artery. D. Cumulative concentration response   viii     curve to 5-HT in SMA contracted with U46619 in sham (left) and DOCAsalt (right) SMA………………………………………………………………..74 Figure 3.3: A. Western blot analyses for identification of the 5-HT2B receptor in homogenates of SMA from sham (n=6) and DOCA-salt (n=6) rats. B. Immunohistochemical staining of the 5-HT2B receptor in SMA sections from sham and DOCA-salt rats. C. Cumulative concentration response curve to BW723C86 vs. vehicle (H20) in SMA contracted with U46619 in sham and DOCA-salt SMA………………………………………………….77 Figure 3.4: A. Western blot analyses for identification of the 5-HT7 receptor in homogenates of SMA from sham (n=6) and DOCA-salt (n=6) rats. B. Immunohistochemical staining of the 5-HT7 receptor in SMA sections from sham and DOCA-salt rats. C. Cumulative concentration response curve to LP-44 vs. vehicle (DMSO) in SMA contracted with U46619 in sham and DOCA-salt SMA………………………………………….………80 Figure 3.5: A. Western blot analyses for identification of the 5-HT1B receptor in homogenates of SMA from sham (n=6) and DOCA-salt (n=6) rats. B. Immunohistochemical staining of the 5-HT1B receptor in SMA sections from sham and DOCA-salt rats. C. Cumulative concentration response curve to CP 93129 vs. vehicle (H2O) in SMA contracted with U46619 in sham and DOCA-salt SMA………………………………………………….83 Figure 4.1: 5-HT-induced fall in mean arterial blood pressure of conscious animals receiving 5-HT chronically for one week (25 ug/kg/min); A. or B. anesethetized animals receiving 5-HT (25 ug/kg/min) over the course of a 30-60 minute infusion………………………………………………………..105 Figure 4.2: Effect of acute 5-HT infusion on preganglionic sympathetic nerve activity. A. Tracing of mean arterial pressure (MAP), preganglionic sympathetic nerve activity (SNA), and integrated (Integ. Spl) preganglionic nerve activity following intravenous 5-HT infusion in the SD rat. B. Time course of changes in preganglionic sympathetic nerve activity (SNA) based on integrated records in the SD rat…………………………………………….107 Figure 4.3: Effect of acute 5-HT infusion on postganglionic sympathetic nerve activity. A. Tracing of mean arterial pressure (MAP), postganglionic splanchnic sympathetic nerve activity (SNA), and integrated (Integ. Spl) postganglionic splanchnic nerve activity following intravenous 5-HT infusion in the SD rat. B. Time course of changes in postganglionic sympathetic nerve activity (SNA) based on integrated records in the SD rat………………………………………………………………………………109   ix     Figure 4.4: Vasoconstrictor response to EFS frequency response curves (0.2 Hz – 20 Hz) in superior mesenteric arteries from Sprague-Dawley rats incubated -8 with either vehicle (H2O) or 5-HT (10 M) for 5 min; A. or B. Vasoconstrictor response to EFS frequency response curves (0.2 Hz – 20 -8 Hz) in SMA (SD) incubated with either vehicle (H2O) or 5-HT (10 M) for 5 -6 min. in the presence of fluoxetine (10 Figure 4.5: M)…………………………….….111 Vasoconstrictor response to 10 Hz stimulus in superior mesenteric arteries -8 from Sprague-Dawley rats incubated with either vehicle (H2O), 5-HT (10 -5 -6 M), or UK 14304 (10 M) in the presence of fluoxetine and (10 M) and ketanserin (50 nM) for 30 min………………………………………………113 Figure 4.6: A. Vasoconstrictor response to 10 Hz stimulus in superior mesenteric arteries from Sprague-Dawley rats incubated with either vehicle (H2O), or -5 -6 CP 93129 (10 M) in the presence of fluoxetine and (10 M) and ketanserin (50 nM) for 30 min. B. Vasoconstrictor response to 10 Hz stimulus in superior mesenteric artery from either sham or DOCA-salt rats -8 incubated with either vehicle (H2O), or 5-HT (10 M) in the presence of -6 fluoxetine and (10 M) and ketanserin (50 nM) for 30 min……………...114 Figure 5.1: A. Effect of chronic 5-HT infusion on mean arterial pressure (MAP) in male SERT WT and SERT KO rats. B. Effect of chronic 5-HT infusion on mean arterial pressure (MAP) in female SERT WT and SERT KO rats……….127 Figure 6.1: Schematic diagram of current hypothesis. (TPR) represents total peripheral resistance. (SNS) represents sympathetic nervous system. (BP) represents blood pressure………………………………………...…142   x     LIST OF ABBREVIATIONS 5-HT ACh Acetylcholine BP Blood pressure bpm Beats per minute (heart rate) CNS Central nervous system DOCA Deoxycorticosterone acetate eNOS Endothelial nitric oxide synthase i.p. Intraperiotoneal L-NNA N-nitro-L-arginine MAO Monoamine oxidase MAP Mean arterial pressure NaCl Sodium chloride NO Nitric oxide NOS Nitric oxide synthase nNOS Neuronal nitric oxide synthase PPP Platelet-poor plasma PRP Platelet-rich plasma PE Phenylephrine PSS Physiological salt solution SERT   5-Hydroxytryptamine, serotonin Serotonin transporter xi     SEM SNS Sympathetic nervous system s.c. Subcutaneously SSRI Selective serotonin reuptake inhibitor TPR   Standard error of the mean Total peripheral resistance xii     CHAPTER 1 INTRODUCTION   1     5-hydroxytryptamine (5-HT, serotonin) is a vasoactive amine synthesized primarily in the enterochromaffin cells of the gastrointestinal tract and the raphe nucleus of the central nervous system (CNS) (Erspamer and Asero, 1952; Dahlstroem and Fuxe, 1964). 5-HT is synthesized from the dietary precursor tryptophan and the ratelimiting enzyme of 5-HT synthesis is tryptophan hydroxylase (TPH). TPH converts the essential amino acid tryptophan, to 5-hydroxytryptophan (5-HTP) (Walther et. al. 2003). 5-HTP is then acted upon by a nonspecific decarboxylase to form 5-HT. Two distinct isoforms of TPH exist: 1) tryptophan hydroxylase 1 (TPH1), expressed in the periphery, responsible for 5-HT synthesis primarily in the gut; and 2) tryptophan hydroxylase 2 (TPH2), expressed in the central nervous system (CNS), responsible for 5-HT synthesis within the neuron(s) in the CNS (Zhang et. al. 2004). The physiological actions of 5-HT are terminated by: 1) the serotonin transporter (SERT), which actively takes up free extracellular 5-HT, and 2) the mitochondrial enzyme monoamine oxidase (MAO), which inactivates 5-HT to form the metabolite 5hydroxyindole acetic acid (5-HIAA) (Marshall et. al. 2010). In the periphery, 5-HT is rapidly taken up by the platelet, which stores large amounts (millimolar) of 5-HT. The rapid uptake of 5-HT, its storage within the platelet, and metabolism by MAO results in comparatively low levels of free circulating 5-HT in the periphery (Figueras et al. 2005). The physiological actions of 5-HT are mediated by seven major receptor subtypes (5-HT1 – 5-HT 7). Each receptor subtype (with the exception of the 5-HT3 receptor) are heptahelical receptors, coupled to G proteins (Gs, Gi, Gq) with diverse effects. The 5-HT3 receptor is a ligand gated ion channel (Watts and Davis, 2011). 5-   2     HT is responsible for a variety of physiological functions in numerous major organ systems including: the cardiovascular, pulmonary, gastrointestinal, genitourinary, and central nervous system(s) (Berger et al. 2009). Our focus will be primarily on the actions of 5-HT in the cardiovascular system, paying particular attention to the effects of 5-HT on blood pressure. Blood Pressure Blood pressure is defined as the amount of pressure exerted against the arterial wall. Cardiac output (CO) is the amount of blood pumped by the heart (in particular the amount of blood ejected from the left ventricle) during systolic contraction. Total peripheral resistance (TPR) is the amount of force opposing the flow of blood through the circulatory system back to the heart. These opposing forces exert pressure on the arterial wall (recognized as arterial blood pressure) and changes to either CO or TPR result in changes in blood pressure. Thus, blood pressure can be calculated from the following equation: blood pressure (BP) = cardiac output (CO) x total peripheral resistance (TPR). Clinically, blood pressure is reported as the pressure exerted during systole (when the heart contracts) over the pressure exerted during diastole (when the heart is filling). Blood pressure < 120mmHg/80mmHg (millimeter mercury) is considered optimal in an adult (JNC VII; Chobanian et. al. 2003). Changes in blood pressure are achieved by a variety of major organs/systems, including (briefly), the heart, the peripheral vasculature, the kidney, and the sympathetic nervous system (SNS). For example, an increase in the amount of blood pumped from the heart, will result in a change in the force exerted against the arterial wall and a subsequent change in blood pressure. Alternatively, the kidney is capable of changing   3     total blood volume (through increased/decreased excretion of H2O, which will change the amount of blood returned to the heart. This change in blood volume will ultimately effect the amount of blood ejected from the left ventricle (CO) and thus will change the force exerted against the arterial wall (i.e. Δ in blood pressure). Finally, a change in SNS activity can change blood pressure at both the level of the heart and the peripheral vasculature. For example, the peripheral vasculature is extensively innervated by SNS fibers and changes in SNS activity, will result in a change in norepinephrine (NE; potent vasoconstrictor) release from the neuroeffector junction. An increase in NE release will increase the tone of the arterial wall, thereby increasing arterial resistance, and blood pressure. Thus, changes in blood pressure can be the result of a single or multiple changes/insults on the cardiovascular system. Hypertension Transient changes in blood pressure (i.e. physical activity, increase in blood pressure, or rest, decrease in blood pressure) are not considered clinically significant and exist primarily to ensure adequate delivery/exchange of nutrients/waste when metabolic demand is varied. However, chronic increases in blood pressure result in the clinical condition known as hypertension. Hypertension is defined as a systolic blood pressure > 140mmHg or a diastolic pressure > 90 mmHg (JNC VII; Chobanian et. al. 2003). Essential hypertension is a significant risk factor for common causes of morbidity and mortality, including stroke, myocardial infarction, heart failure, and kidney disease (Campese et. al. 2011). Unfortunately, the etiology of primary or essential hypertension remains largely unknown.   4     Historically, 5-HT has been described as a potential contributor to essential hypertension. This distinction is based on decades of research demonstrating that 5-HT is a potent constrictor of isolated blood vessels (Rapport et. al. 1948). Additionally, studies demonstrate elevated levels of free plasma 5-HT in both human and animal models of hypertension compared to normotensive controls (Brenner et. al. 2007; Fetkovska et. al. 1990). 5-HT receptor antagonists lower blood pressure in select animal models of hypertension (Watts and Fink, 1999). Arteries of hypertensive animals are hyperresponsive to the constrictor effects of 5-HT, and selective 5-HT reuptake inhibitors elevate blood pressure (Thompson and Webb, 1987; Amsterdam et. al. 1999). This evidence originally led investigators to postulate that elevated levels of free plasma 5-HT might be acting to constrict the vasculature, increasing vascular resistance, and blood pressure. 5-HT in the vasculature In the peripheral vasculature, the 5-HT1B, 5-HT2A, 5-HT2B, and 5-HT7 receptors are present and active (Bordoff et. al. 2002; Calama et. al. 2003; Saxena and Villalon, 1990). The literature describes 5-HT as both a constrictor and dilator of peripheral blood vessels. Under normotensive conditions in the rat, the 5-HT2A receptor is responsible for contraction of isolated blood vessels (Yildiz et. al. 1998). Under hypertensive conditions in the rat, the 5-HT1B, and 5-HT2B receptor(s) are linked to arterial hyperresponsiveness   5     to 5-HT (Banes and Watts, 2003; Banes and Watts, 2001). Thus, if 5-HT were acting primarily as a constrictor in the peripheral vasculature, this would result in an increase in vascular resistance and blood pressure. 5-HT may also function as a dilator of peripheral blood vessels. The 5-HT1B, 5HT2B, and 5-HT7 receptors have all been linked to dilation of peripheral blood vessels in the rat. The 5-HT1B/1D and 5-HT7 receptor(s) are located on vascular smooth muscle and described to mediate relaxation, but they are mechanistically not well-understood (Calama et. al. 2003; Saxena and Villalon, 1990). The pharmacological agonists CP 93129 (5-HT1B/1D receptor agonist) and LP-44 (5-HT7 receptor agonist) are selective agonists that aid in the investigation of these receptors. The 5-HT2B receptor is linked to falling blood pressure in patients placed on cardiopulmonary bypass (Bordoff et. al. 2002). Investigators of this study demonstrated that a bypass-induced (mechanical platelet disruption/5-HT release) fall in blood pressure was nitric oxide (NO) dependent. This finding suggested that activation of the 5-HT2B receptor might mediate NO release, thereby reducing total peripheral resistance, and lower blood pressure. However, the exact role of the rat 5-HT2B receptor remains controversial as this receptor is found to be upregulated in the smooth muscle of hypertensive rats, and acts as a potent constrictor of isolated blood vessels (Banes and Watts, 2003). The pharmacological agonist, BW 723C86 (5-HT2B receptor agonist) is another tool available for investigation of this particular 5-HT receptor.   6     5-HT in the SNS Preganglionic and postganglionic neurons carry/transmit signals from the central nervous system (CNS) to various organ systems (heart, adrenal gland, kidney, blood vessels) that are involved in regulating blood pressure. Preganglionic neurons originate within the CNS and spinal cord (the thoracolumbar region) traveling to sympathetic ganglia where they synapse with postganglionic neurons/nerve fibers. At the synapse, preganglionic neurons release acetylcholine (ACh), which activates nicotinic ACh receptors on the postganglionic neuron, depolarizing the cell, leading to signal propagation along the postganglionic neuron. Postganglionic nerve fibers travel to peripheral target tissues where they synapse and release norepinephrine (NE). NE then traverses the synaptic space and activates adrenergic receptors. These receptors are responsible for the effects of the SNS. For example, NE released along the peripheral vasculature will cause alpha-1 receptor activation and contract the artery and/or vein (depending on innervation and release). Therefore, an increase or decrease in NE release (at the neuroeffector junction) is capable of increasing or decreasing vessel tone, vascular resistance (total peripheral resistance; TPR), and blood pressure. Alternatively, changes in signal initiation/generation (represented by changes in preganglionic activity) or changes in signal propagation (i.e. inhibition of signal transfer at the ganglia, represented by changes in postganglionic activity) are capable of increasing or decreasing blood pressure. The 5-HT1B/1D receptor is expressed on the nerve terminal (neuroeffector junction) and activation of this receptor results in an inhibition of NE release (Molderings et. al. 1990). Thus, a reduction in NE release results in a reduction in peripheral vessel tone, a   7     fall in TPR, and a reduction in blood pressure. This suggests 5-HT may be capable of acting as a sympatholytic and capable of decreasing blood pressure. However, alterative reports in the literature suggest 5-HT can be actively taken up at the nerve terminal, resulting in NE displacement and NE release (Villalon and Centurion, 2007). This suggests 5-HT may be acting as an indirect sympathomimetic. Thus, 5-HT may be capable of increasing NE release, increasing TPR, and increasing blood pressure. This suggests 5-HT may be capable of acting as an indirect sympathomimetic and able to increase blood pressure. 5-HT and the serotonin transporter (SERT) SERT is a bidirectional transporter responsible for the uptake and release of 5-HT (Ni et. al. 2008). SERT is member of the sodium chloride-dependent transporter family (SLC6) and while SERT is well known for its uptake of 5-HT into platelets, it is also located on the neuron, the ganglia (rat superior cervical ganglia) (Nishimura M et. al. 1999), in the gastrointestinal system (Gershon and Tack, 2007), pulmonary system (Dodson et. al. 2004), immune system (Gordon and Barnes, 2003), and on the heart (Pavone et. al. 2007). Recently, SERT was also recognized on the systemic artery and vein (Ni et. al. 2008). Thus, both the artery and the vein also possess a transporter, which allows local control of intracellular and extracellular 5-HT concentrations. Moreover, SERT was recently identified on the blood brain barrier in the central nervous system (CNS) and thus suggests bidirectional transport of 5-HT across the blood brain barrier is possible (Nakatani et. al. 2008; Brust et. al. 2000; Wakayama et. al. 2002).   8     Pharmacological manipulation of SERT in the CNS is widely accepted as effective treatment for a variety of psychological disorders, most notably, depression. Antidepressant drugs (selective serotonin reuptake inhibitors; SSRI) target SERT, blocking the reuptake of 5-HT via SERT inhibition. These clinically efficacious drugs increase free 5-HT concentrations in the extracellular space and it is believed that this increase is responsible for the antidepressant effects of these drugs. (Keers and Aitchison, 2010). However, changes in blood pressure are noted side effects and therefore suggests changes in SERT function may be capable of modifying systemic blood pressure. Interestingly, 5-HT uptake via SERT has recently been demonstrated to enhance neuronal nitric oxide synthase (nNOS) activity in cells coexpressing both SERT and nNOS (Chanrion et. al. 2007). This finding suggests that 5-HT may be taken up by SERT, resulting in nNOS activation and the production of nitric oxide (NO), a powerful vasodilator. This evidence suggests 5-HT uptake via SERT may be capable of lowering blood pressure through activation of nNOS and NO production. The triphasic response 5-HT (delivered over seconds) results in a classic blood pressure response known as the triphasic response. 5-HT produces a fast depressor response, (mediated by an abrupt bradycardia, known as the Bezold-Jarisch reflex via the 5-HT3 receptor), followed by a prominent pressor response (mediated by arterial smooth muscle contraction via 5-HT2A receptor), and then a long lasting depressor response (likely mediated by activation of the 5-HT7 receptor producing smooth muscle relaxation).   9     This suggests 5-HT is capable of increasing and/or decreasing blood pressure when delivered acutely (over seconds). The triphasic response investigated changes in blood pressure when delivered over seconds to minutes. This is an important distinction, as it requires us to think differently about the mechanism(s) potentially involved in a chronic 5-HT-induced change in blood pressure. Acutely, changes in blood pressure are largely governed by changes mediated by the baroreceptors and baroreceptor reflex. The baroreceptors are stretch receptors located predominately at the carotid sinus and the aortic arch. If for example, blood pressure were to increase, the baroreceptor reflex would respond by slowing the heart, and removing sympathetic nervous system tone to the peripheral vasculature, resulting in a dilation of blood vessels and lower blood pressure. Chronic changes in blood pressure suggest an alternative mechanism, which may potentially be mediated by a change in vessel tone, or SNS activity. 5-HT infusion 5-HT elicits various cardiovascular responses. Thus, our laboratory designed the following experiment to investigate whether elevated levels of free plasma 5-HT would result in increased blood pressure, supporting active 5-HT-induced vasoconstriction or decreased blood pressure, supporting a completely different mechanism, in the sham and deoxycorticosterone acetate (DOCA)-salt rat. Despite significant literature to support the hypothesis that 5-HT may be contributing to essential hypertension (via peripheral artery contraction), the results of this experiment demonstrated that elevated levels of free plasma 5-HT were actually lowering   10     blood pressure in the sham and DOCA-salt rat (Diaz et. al. 2008) (Figure 2). This study unequivocally suggested that instead of constricting the peripheral vasculature, contributing to an increase in TPR, and blood pressure, a completely different mechanism is acting when 5-HT is delivered chronically. The exact mechanism(s) of a 5-HT-induced fall in blood pressure are not yet fully understood. However, Diaz et. al. demonstrated an absolute dependence on nitric oxide synthase (NOS) to produce a 5HT-induced fall in blood pressure. The following hypothesis was designed to investigate the mechanism underlying a 5-HT-induced fall in blood pressure.   11     Hypothesis 5-HT lowers blood pressure, in a nitric oxide dependent manner, by reducing total peripheral resistance, either through a reduction in sympathetic nervous system tone and/or direct stimulation of vascular 5-HT receptors Specific aim 1: Will test the hypothesis that 5-HT relaxes peripheral blood vessels through activation of peripheral vascular receptor(s). Specific aim 2: Will test the hypothesis that 5-HT lowers blood pressure through a reduction in SNS tone. Specific aim 3: Will test the hypothesis that 5-HT uptake via SERT is critical to enabling a 5-HT-induced fall in blood pressure. Figure 1.1 &'&%()*+,+-.)% !"#$% /01.8+40-.)% /0123405%)+65+2%.7+89% :;60<9%!"#$&=>$% $?>% $?>% /01.8+40-.)% /01.8+40-.)% @?% $?>% Figure 1.1. Schematic diagram of current hypotheses. (TPR) represents total peripheral resistance. (SNS) represents sympathetic nervous system. (BP) represents blood pressure.   12     Figure 1.2 Mean Arterial Pressure (mmHg) A. 200 5-HT/Vehicle 175 150 * 125 * 100 * 75 50 C1 C2 S1 * S2 * * * * * * * * * * S3 S4 S5 S6 DOCA Veh (n=7) DOCA 5-HT (n=7) Sham Veh (n=4) Sham 5-HT (n=4) S7 Time (Day) B. 450 5-HT/Vehicle Heart rate (bpm) * * 400 * * Sham 5-HT (n=4) Sham Veh (n=4) 350 DOCA 5-HT (n=7) DOCA Veh (n=7) 300 250 C1 C2 S1 S2 S3 S4 S5 S6 S7 Time (Day) Figure 1.2. A. Effect of chronic 5-HT/Vehicle administration on mean arterial blood pressure. Time (days) is represented on the x-axis. C represents control days. S   13     represents 5-HT administration. B. Effect of chronic administration of 5-HT or Vehicle on heart rate (HR). (n) represents the number of animals used. (*) Represents statistically significant difference (p<0.05) from control period C2. Vertical dotted line represents start of 5-HT/vehicle. Reference: Diaz et al. J Pharmacol Exp. Ther. 325:1 031-1038, 2008.   14     CHAPTER 2 One-month serotonin infusion results in a prolonged fall in blood pressure in the male deoxycorticosterone acetate (DOCA)-salt rat Robert Patrick Davis, Theodora Szasz, Hannah Garver, Robert Burnett, Nathan R. Tykocki and Stephanie W. Watts Department of Pharmacology and Toxicology, Michigan State University, East Lansing, Michigan                           Prepared as a manuscript for submission to American Journal of Physiology Heart and Circulatory Physiology   15     ABSTRACT Chronic serotonin (5-hydroxytryptamine, 5-HT) infusion results in a sustained fall in blood pressure in the deoxycorticosterone acetate (DOCA)-salt rat and is accompanied by improved ACh-induced arterial relaxation. These studies tested the hypothesis that a longer (one-month) and more clinically significant 5-HT infusion could cause a sustained fall in blood pressure in the DOCA-salt rat, and tested the new hypothesis that 5-HT could also attenuate the development of DOCA-salt hypertension. 5-HT or vehicle (Veh) were delivered via osmotic pump (25 µg/kg/min; sc) to established male DOCA-salt rats or Sprague Dawley rats prior to DOCA-salt administration for 28 days. Free plasma 5HT concentrations were significantly higher in 5-HT-infused DOCA-salt rats compared to Veh-infused DOCA-salt rats. On the final day of 5-HT infusion, mean arterial pressure (MAP; radiotelemetry) was significantly lower in 5-HT-infused (135 ± 4 mmHg) vs. Vehinfused (151 ± 7 mmHg) established DOCA-salt rats. However, 5-HT-infusion did not prevent the development of DOCA-salt hypertension (144 ± 7 mmHg) vs. Veh infusion (156 ± 6 mmHg). Isometric contractile force was measured in aortic strips of 5-HTinfused and Veh-infused DOCA-salt rats. Maximum relaxation to ACh was not significantly greater in the aorta of 5-HT-infused vs. Veh-infused established DOCA-salt rats, or in 5-HT vs. Veh-infused rats when 5-HT was delivered prior to the onset of DOCA-salt hypertension. This study demonstrates 5-HT is capable of lowering blood pressure in established DOCA-salt hypertensive rats over the course of one-month and suggests a potentially beneficial role for 5-HT in the cardiovascular system.   16     INTRODUCTION Hypertension is characterized by an elevation in arterial pressure, increased vascular resistance, cardiac hypertrophy, increased sympathetic nervous system tone, and altered renal function (Campese et. al. 2011). Despite the myriad of deleterious changes, the etiology of essential hypertension remains largely unknown, but is thought to be multifactorial, and include both genetic and environmental factors. Interestingly, several lines of evidence suggest an important association between 5-HT and hypertension. 5-HT is a vasoactive amine synthesized primarily in the enterochromaffin cells of the gastrointestinal tract, and the raphe nucleus in the central nervous system (Erspamer and Aerso, 1952; Rapport et. al. 1948; Dahlstrom and Fuxe, 1964). Studies demonstrate elevated levels of free plasma 5-HT in both human and animal models of hypertension compared to normotensive human and animal controls (Brenner et. al. 2007; Fetkovska et. al. 1990). 5-HT receptor antagonists lower blood pressure in select animal models of hypertension (Watts and Fink, 1999). The arteries of hypertensive animals are hyperresponsive to the constrictor effects of 5-HT, and selective 5-HT reuptake inhibitors elevate blood pressure (Thompson and Webb, 1987; Amsterdam et. al. 1999). Collectively, this evidence suggests that elevated levels of free plasma 5-HT might be acting to increase vascular resistance and blood pressure, thereby contributing to essential hypertension. Surprisingly, experimental efforts to elevate free plasma 5-HT actually lowered blood   17     pressure in the deoxycorticosterone acetate (DOCA)-salt and sham rat over the course of 7 days (Diaz et. al. 2008). This suggested a completely different mechanism, that 5HT might be acting to lower blood pressure in both human and animal models of hypertension. Thus, instead of contributing to essential hypertension, the elevated levels of 5-HT observed in hypertensive patients, may actually represent an adaptation to elevated blood pressure. The exact mechanism(s) of a 5-HT-induced fall in blood pressure are not yet fully understood, but were initially suggested in a study published in 2008 (Diaz et. al. 2008). Diaz et. al. demonstrated that inhibition of nitric oxide synthase (NOS) prevented a oneweek 5-HT-induced fall in blood pressure in the sham and DOCA-salt hypertensive rat. This study suggested an important role for NOS in enabling a 5-HT-induced fall in blood pressure. Additionally, Diaz et. al. demonstrated improved acetylcholine (ACh)-induced relaxation (a measure of endothelial cell health) in the aorta of 5-HT infused vs. vehicle infused DOCA-salt rats (10). This suggests a one-week 5-HT infusion may improve endothelial cell function in the DOCA-salt rat, potentially as part of the mechanism for a decrease in blood pressure. The DOCA-salt model is characterized by subcutaneous delivery of the mineralocorticoid deoxycorticosterone acetate (200 mg/kg/day) and ingestion of salt water (1% NaCl, 0.2% KCl) (Sun and Zhang, 2005; Pinto et. al. 1998). Additionally, renal mass is significantly reduced through unilateral nephrectomy. Collectively, this leads to neurohumoral activation and volume expansion in the DOCA-salt rat (Yemane   18     et. al. 2009). Further, the DOCA-salt model is considered a nitric oxide (NO)-depleted model of hypertension, due to characteristic endothelial cell dysfunction and a reduction in ACh-induced arterial relaxation (Voorde and Leusen, 1986; Cordellini et. al. 1988). Lastly, the DOCA-salt rat could also be considered a resistant model of hypertension due to the fact that after approximately 2 months of treatment with DOCA, removal of the pellet and return to normal H2O does not return blood pressure to pre-hypertensive levels (Beilin and Ziakas, 1972). The previously studied infusion protocol (7 days) is a relatively short clinical exposure given that most hypertensive patients require lifelong antihypertensive therapy. Thus, part of the present study investigates whether elevated levels of free plasma 5-HT were capable of lowering blood pressure for one-month in the established DOCA-salt hypertensive rat. In addition, this study takes a different tactic to investigate how 5-HT decreases blood pressure by determining if it can prevent the development of DOCAsalt hypertension. We hypothesized 5-HT could do so because of support from experiments done decades ago, which demonstrated that chronic treatment with L-5hydroxytryptophan (5-HTP) prevented the development of DOCA-salt hypertension in the rat (Fregly et. al. 1987). 5-HTP is the committed precursor to the synthesis of 5-HT and is synthesized by tryptophan hydroxylase from the dietary precursor tryptophan. Thus, we investigated whether chronically elevated free plasma 5-HT might also impact the development of DOCA-salt hypertension in the rat. This study has not been done before.   19     Finally, in both the reversal (delivery of 5-HT to established DOCA-salt rats) and prevention (delivery of 5-HT prior to the onset of DOCA-salt hypertension) studies, we sought to investigate whether endothelial cell health, measured by ACh-induced relaxation, was preserved and/or improved as has been observed with a shorter 1 week course of 5-HT.   20     METHODS Animal The Michigan State University Institutional Animal Care and Use Committee (IACUC) approved all protocols. Male Sprague-Dawley rats (250–300 g; Charles River Breeding Laboratories, Portage, MI) underwent one of 2 separate experimental protocols: 1) Male Sprague-Dawley rats (250–300 g) underwent left uninephrectomy, and a DOCA pellet was implanted s.c. (200 mg/kg). Water was supplemented with 1% NaCl and 0.2% KCl for the duration of the study. After 4 weeks these rats were considered established DOCA-salt hypertensive rats and were administered either 5-HT or vehicle via osmotic pump (s.c.) for one-month. 2) Male Sprague-Dawley rats (250–300 g) underwent left uninephrectomy, and were then administered either vehicle or 5-HT vehicle via osmotic pump (s.c.) for one-month. After 5 days of treatment, Sprague Dawley rats received a DOCA pellet (200 mg/kg) and their water was supplemented with 1% NaCl and 0.2% KCl for the remaining duration of the one-month infusion. Anesthesia and Analgesia All rats were anesthetized with isoflurane (2% in 100% O2) and ventilated mechanically. At the time of surgery, the incision site was treated with topical antibiotic containing analgesic to prevent irritation and infection. Incisions were closed with silk suture. Rats were treated with amoxicillin (150 mg/kg/i.m.) following surgery and 3 days thereafter. All rats were treated with rimadyl (5 mg/kg, s.c. for 2 days) for general analgesia. Surgical Methods: Blood pressure probe Radiotelemeters (DSI PhysioTel PA series transmitter model PA-C40) were implanted subcutaneously in the lower abdomen and catheters introduced into the left femoral   21     artery. Pressure sensing tips were advanced into the thoracic aorta. All rats were given 7 days to recover prior to any measure. Mean arterial pressure (MAP) and heart rate (HR) were recorded at 10-minute intervals (10 second recording) for the duration of the study. Surgical Methods: Alzet osmotic pump A small incision was made at the base of the neck. Blunt dissection was used to create a small subcutaneous pocket between the scapulae. The pump (Alzet Osmotic Pump, Model 2ML4, Duret Corporation, Cupertino, CA, 2.5 µL/hr 28 days) was inserted and the skin sutured closed. To each pump, a 5-HT creatinine complex (25 µg/kg/min) and 1% ascorbic acid, antioxidant (0.02 g/pump) or vehicle (1% ascorbic acid, antioxidant) was loaded. The solution was dissolved in 1 mol/L HCl, and a pH-balance (~7) was achieved with 4 mol/L NaOH. Plasma 5-HT measurements Blood collection and platelet poor plasma (PPP) / platelet rich plasma (PRP) preparation were conducted according to our previously published methods (Diaz et. al. 2008). Briefly, five milliliters (ml) of blood was collected from left cardiac ventricle using a heparin coated syringe. The blood was transferred into a EDTA anticoagulant vacutainer tube. Ten µmol/L pargyline and 10 µmol/L ascorbic acid were added. The EDTA tubes were spun at 160 x g for 30 minutes at 4°C for platelet-rich-plasma (PRP). Two ml of supernatant containing plasma and buffy coat were pipetted into EDTA-coated plastic tubes and mixed with a 1:1 dilution of 0.5 mol/L EDTA. Ten µmol/L pargyline and 10 µmol/L ascorbic acid were added. These tubes were centrifuged for 20 minutes at 1350 x g at 4°C for platelet-poor-plasma (PPP). To the remaining pellet (platelet layer), 1 ml of platelet buffer   22     (145 mmol/L NaCl, 5 mmol/L KCl, 1 mmol/L CaCl2, 1 mmol/L MgSO4 10 mmol/L Dglucose) and 1 µmol/L ADP was added. 10 µmol/L pargyline and 10 µmol/L ascorbic acid were added. These tubes were then vortexed and allowed to sit on ice for 15 minutes for platelets to become activated and degranulate. The tubes were then centrifuged at 730 x g for 10 minutes at 4°C. Ten percent trichloroacetic acid (TCA) was added to deproteinize both sets of samples and allowed to sit on ice for 10 minutes. These tubes were centrifuged at 4500 x g for 20 minutes at 4°C. Finally, the samples were ultracentrifuged at 280,000-x g for 2 hours. 5-HT concentrations were measured using high-performance liquid chromatography (HPLC) at 0.2 V and 0.6 ml/min flow rate. Isolated tissue bath Rats were anesthetized with pentobarbital (60 mg/kg i.p.) and the thoracic aorta was removed and placed in physiological salt solution (PSS) containing (mmol/L): NaCl 130; KCl 4.7; KH2PO4 1.8; MgSO4 * 7H2O 1.7; NaHCO3 14.8; dextrose 5.5; CaNa2EDTA 0.03, CaCl2 1.6 (pH 7.2). The endothelium-intact thoracic aorta was cleaned and cut into helical strips. It was mounted in a warmed (37°C), and aerated (95% O2, 5% CO2) tissue bath connected to Grass isometric transducer (FT03; Grass instruments, Quincy, MA, USA), which was connected to an ADInstruments PowerLab (ADInstruments, Colorado Springs, CO). Tissues were placed under optimal resting tension (1500 mg; previously determined) and allowed to equilibrate for 1 hour. The initial contraction was stimulated by -5 a maximal concentration (10 -10 response curves to PE (10   mol/L) of phenylephrine (PE). Cumulative concentration -5 to 10 -9 mol/L), and 5-HT (10 23   -5 to 10 mol/L) were   generated. The aorta was also half-maximally contracted to PE, followed by generation of -9 an acetylcholine (ACh) concentration response curve (10 -5 to 10 mol/L) to determine endothelial cell function. Each concentration response curves was performed in the aorta in the following order, PE, 5-HT, ACh. Potency of an agonist was expressed as = -log EC50 calculated by non-linear regression in GraphPad Prism (GraphPad Software Inc., San Diego, CA), where EC50 is the effective concentration of the agonist (mol/L) that induces 50% of the maximal response. Statistical analysis For in vivo data analysis, within-group differences were assessed by a one-way ANOVA with post hoc multiple comparisons using Student Newman-Keuls test (Graphpad Prism 5). An unpaired Students t-test was performed to measure point-to-point differences. In all cases, a p value of < 0.05 was considered significant. All results are presented as means ± SEM.   24     RESULTS 5-HT infusion in established DOCA-salt hypertensive rats In this series of experiments, Sprague Dawley rats underwent DOCA-salt surgery (uninephrectomy, DOCA pellet 200 mg/kg, and water supplemented with 1% NaCl and 0.2% KCl) and were given 4 weeks to become established DOCA-salt hypertensive rats. These rats then received either 5-HT (25 µg/kg/min) or vehicle (via osmotic pump) for one-month. Figure 1 shows the concentration of 5-HT, as measured by high performance liquid chromatography (HPLC), in platelet poor plasma (PPP; free 5-HT) and platelet rich th plasma (PRP; platelet bound 5-HT). Blood was collected on the 28 day of 5-HT or vehicle infusion in established DOCA-salt rats. PPP was higher in rats that received 5HT (282.9 ± 48.7 ng/ml) compared to those rats that received vehicle (87.9 ± 43.9 ng/ml) (p < 0.05). HPLC analysis revealed no differences between the 5-HT content contained within the osmotic pump and a freshly made 5-HT standard, suggesting a one-month 5-HT infusion did not degrade the 5-HT infused into established DOCA-salt rats. Figure 2A summarizes the effect of 5-HT or vehicle infusion on MAP in established DOCA-salt hypertensive rats. Following 5-HT infusion, mean arterial pressure (MAP) was significantly reduced (105 ± 5 mmHg) compared to rats receiving vehicle (138 ± 6 mmHg) at S2. Despite modest recovery in 5-HT-infused rats over the one-month study,   25     MAP never returned to control levels (152 ± 6 mmHg at C2 vs. 135 ± 4 mmHg at S27). Blood pressure did not change in rats receiving vehicle (142 ± 5 mmHg at C2 vs. 151 ± 7 mmHg at S27) (p > 0.05). Figure 2B summarizes the effect of 5-HT or vehicle infusion on HR in established DOCA-salt hypertensive rats. Immediately following 5-HT infusion (C4 post-pump), HR was significantly increased (426 ± 10 bpm) compared to rats receiving vehicle (357 ± 3 bpm). However, by the 3 rd day of 5-HT/vehicle infusion, HR had returned to normal in those rats receiving 5-HT despite significantly lower MAP compared to rats receiving vehicle. Isometric contractility in established DOCA-salt hypertensive rats infused with 5HT or vehicle Aortic strips were mounted in warmed and aerated tissue baths for measurement of isometric contractile force to determine whether a one month infusion of 5-HT or vehicle effected the response to: the α adrenergic receptor agonist, phenylephrine (PE), 5-HT, or ACh. Initial contraction was stimulated by a maximal concentration of PE (10-5 M) and the magnitude of this response was not statistically different (p > 0.05) in established DOCA-salt 5-HT-infused hypertensive rats (1165 ± 107.8 mg) compared to established DOCA-salt Veh-infused hypertensive rats (1415 ± 126.7 mg). These values were then used to normalize subsequent contractile responses. A concentration response curve to PE revealed no significant differences in potency between established DOCA-salt rats treated with 5-HT (-logEC50 = 7.45 ± 0.05)   26     compared to rats treated with vehicle (-logEC50 = 7.34 ± 0.1) (Figure 3A). Maximal responses to PE were not different in 5-HT-infused established DOCA-salt rats (143 ± 6.0% of initial PE-induced contraction) compared to Veh-infused established DOCA-salt rats (133 ± 14% of initial PE-induced contraction) (p > 0.05). Similarly, a concentration response curve to 5-HT revealed no significant differences in agonist potency between 5-HT-infused established DOCA-salt rats (-logEC50 = 5.96 ± 0.14) compared to Vehinfused established DOCA-salt rats (-logEC50 = 5.97 ± 0.09) (p >0.05) (Figure 3B). Maximal contraction to 5-HT was not different in 5-HT-infused established DOCA-salt rats (126 ± 20%) compared to Veh-infused DOCA-salt rats (140 ± 11%) (p > 0.05). Finally, an ACh-induced concentration response curve was performed. Data are reported as a percentage of the contraction elicited by half maximal (EC50) concentration of PE in 5-HT-infused established DOCA-salt rats (972 ± 130.2 mg) and Veh-infused established DOCA-salt rats (1083 ± 102.8 mg) (p > 0.05). 5-HT infusion did not improve aortic sensitivity to ACh in 5-HT-infused established DOCA-salt rats (logEC50 = 7.06 ± 0.17) compared to Veh-infused established DOCA-salt rats (-logEC50 = 6.76 ± 0.34) (p > 0.05) (Figure 3C). However, ACh-induced relaxation was slightly greater (although not statistically significant) in 5-HT-infused established DOCA-salt rats (33 ± 8.7% PE contraction remaining) vs. Veh-infused established DOCA-salt rats (58 ± 13.5% PE contraction remaining) (p > 0.05).   27     5-HT infusion prior to the development of DOCA-salt hypertension In this series of experiments, Sprague Dawley (SD) rats received either 5-HT (25 µg/kg/min) or vehicle (via osmotic pump) for 5 days and then underwent DOCA-salt surgery (uninephrectomy, DOCA pellet 200 mg/kg, and water supplemented with 1% NaCl and 0.2% KCl) to determine whether 5-HT was capable of inhibiting the development of DOCA-salt hypertension. Figure 4 shows the concentration of 5-HT in platelet poor plasma (PPP) and platelet rich plasma (PRP) in SD rats following a one month infusion with either 5-HT or vehicle and administration of a DOCA-salt pellet. PPP was significantly (p < 0.05) higher in rats that received 5-HT (177.1 ± 10.9 ng/ml) compared to those rats that received vehicle (13.3 ± 3.9 ng/ml). Figure 5 summarizes the effect of 5-HT or vehicle infusion on developing DOCA-salt hypertensive rats over the course of one-month. Initially, 5-HT infusion resulted in a significantly (p < 0.05) lower MAP compared to Veh-infused rats. Following the DOCAsalt pellet implant, 5-HT did not significantly impact the development of DOCA-salt hypertension in either 5-HT-infused rats or Veh-infused rats. The absolute magnitude of MAP reached in 5-HT-infused rats (144 ± 6.5 mmHg) was not significantly different compared with Veh-infused rats (156 ± 5.9 mmHg). Scheduled sampling was briefly stopped (S12) during one-month infusion; MAP was monitored for the remainder of the study (S13-S28).   28     Isometric contractility in DOCA-salt hypertensive rats infused with 5-HT or vehicle prior to DOCA-salt administration Aortic strips were mounted in warmed and aerated tissue baths for measurement of isometric contractile force to determine whether 5-HT infusion initiated prior to DOCAsalt administration would affect contractility. -5 maximal concentration of PE (10 Initial contraction was stimulated by a M) and the magnitude of this response was not statistically different (p > 0.05) in DOCA-salt 5-HT-infused hypertensive rats (1094 ± 106.8 mg) compared to DOCA-salt Veh-infused hypertensive rats (939 ± 121.6 mg). These values were then used to normalize subsequent contractile responses. A concentration response curve to PE revealed no significant differences in potency of PE between 5-HT-infused DOCA-salt rats (-logEC50 = 7.64 ± 0.1) compared to Vehinfused DOCA-salt rats (-logEC50 = 7.65 ± 0.2) (Figure 6A). Maximal responses to PE were also not different in 5-HT-infused DOCA-salt rats (123 ± 5.2% of initial PE-induced contraction) compared to Veh-infused DOCA-salt rats (146 ± 12.1% of initial PEinduced contraction) (p > 0.05). Similarly, a concentration response curve to 5-HT was performed. No significant differences existed in 5-HT potency between 5-HT-infused DOCA-salt rats (-logEC50 = 6.39 ± 0.11) compared to Veh-infused DOCA-salt rats (logEC50 = 6.32 ± 0.16) (p >0.05) (Figure 6B). Maximal responses to 5-HT were not different in 5-HT-infused DOCA-salt rats (145 ± 13% PE contraction remaining) compared to vehicle-infused DOCA-salt rats (178 ± 23% PE contraction remaining) (p > 0.05) when 5-HT was given prior to DOCA-salt administration.   29     Finally, an ACh-induced concentration response curve was performed. Data are reported as a percentage of the contraction elicited by half maximal (EC50) concentration of PE in 5-HT-infused DOCA-salt rats (748 ± 39.2 mg) and Veh-infused DOCA-salt rats (690 ± 65 mg) (p > 0.05). 5-HT infusion did not improve aortic sensitivity to ACh in 5-HT-infused DOCA-salt rats (EC50 = 7.09 ± 0.19) compared to Veh-infused established DOCA-salt rats (EC50 = 6.85 ± 0.24) (p > 0.05) (Figure 6C). However, AChinduced maximum relaxation was also slightly greater (although not statistically significant) in 5-HT-infused DOCA-salt rats (38 ± 9.2%) vs. Veh-infused DOCA-salt rats (59 ± 8.4%) (p > 0.05) when 5-HT infusion was initiated prior to DOCA-salt administration.   30     DISCUSSION In this study, we took 2 independent but related approaches towards examining the ability of chronically elevated free 5-HT to decrease blood pressure asking these questions: 1) Is 5-HT capable of decreasing blood pressure in the established DOCAsalt hypertensive rat over the course of one-month; and 2) Is 5-HT capable of inhibiting the development of DOCA-salt hypertension over one-month? Endpoints of this study included HPLC analysis to validate elevation of free plasma 5-HT levels, blood pressure measurement via radiotelemetry, and isometric contractile studies. 5-HT-induced hypotension in established DOCA-salt hypertensive rats 5-HT infusion led to a significant reduction in blood pressure over one-month and a statistically significant increase in free plasma 5-HT in established DOCA-salt hypertensive rats (282.9 ± 48.7 ng/ml) vs. vehicle (87.9 ± 43.9 ng/ml). For comparison, free plasma 5-HT levels in the normotensive sham rat were 2.7 ± 0.29 ng/ml in Vehinfused rats (7 days), and 47.1 ± 23 ng/ml in 5-HT-infused rats (7 days) (Diaz et. al. 2008). These values demonstrate an increase in free plasma 5-HT in our hypertensive animal model (the established DOCA-salt rat) compared to a normotensive animal control and a further increase in free plasma 5-HT with a one-mouth infusion. This study suggests elevated levels of free 5-HT (observed in both human and experimental models of hypertension) may be acting to lower blood pressure (rather than contributing to essential hypertension) and demonstrated 5-HT is capable of maintaining lower blood pressure(s) following 2 months of DOCA-salt treatment, a time   31     at which DOCA-salt hypertension becomes irreversible. Further this protocol also demonstrated that 5-HT infusion (delivered over one month to established DOCA-salt rats) does not improve ACh-induced relaxation (despite an observable trend toward improved ACh-induced relaxation). Moreover, the lack of a change in potency to the effects of PE and 5-HT demonstrated the potential of blood vessels to contract to normal stimuli (PE) and that those blood vessels do not lose there contractile response to 5-HT (desensitization) with either infusion protocol. In the established DOCA-salt rat, elevated blood pressure is characterized by hypervolaemia, high levels of mineralocorticoid, and high salt intake (Yermane et. al. 2009). Several factors contribute to the maintenance of established blood pressure and include: 1) enhanced sympathetic nerve activity, 2) increased vasopressin release, 3) increased endothelin expression, 4) attenuation of the baroreceptor reflex, 5) vascular remodeling, and 6) nitric oxide depletion due to endothelial cell damage/dysfunction (Schenk and McNeill, 1992; Crofton et. al. 1979; Lariviere et. al. 1993). Each of these factors represent a potential target by which 5-HT may be acting to lower blood pressure in the established DOCA-salt rat. The exact mechanism(s) by which 5-HT is acting to lower blood pressure in this established model have not been identified, but the above factors give some insights. For example, one possibility is that 5-HT might be acting to inhibit sympathetic nerve activity (SNA), thereby acting to reduce total peripheral resistance (TPR) and lower blood pressure. The 5-HT1B/1D receptor is located on the presynaptic nerve terminal and, when stimulated by 5-HT, results in an inhibition of norepinephrine (NE) release   32     (Molderings et. al. 1990; Göthert et. al. 1991; Shepard and Vanhoutte, 1985). Moreover, recent evidence suggests that 5-HT may be able to cross the blood brain barrier (Nakatani et. al. 2008). Thus, we cannot rule out the possibility that 5-HT might be acting in the central nervous system to lower blood pressure. Future studies will need to be undertaken to elucidate the exact mechanism of 5-HT-induced hypotension in the established DOCA-salt rat. 5-HT on the development of DOCA-salt hypertension 5-HT was significantly elevated at the end of the one-month study in 5-HT treated rats vs. vehicle treated rats. This measure validated our infusion protocol and suggested that any change or lack of change in the development of DOCA-salt hypertension was not due to insufficient delivery and/or degradation of 5-HT during this study. Despite elevated levels of free 5-HT, there was no significant difference in the development of DOCA-salt hypertension or absolute magnitude of blood pressure reached in 5-HT vs. vehicle treated rats. This was a surprising finding given that previous studies demonstrated that 5-HTP, the committed precursor to 5-HT, prevented the development of DOCA-salt hypertension in the rat (Fregly et. al. 1987). This suggests that 5-HT cannot prevent DOCA-salt hypertension despite, the previously studied effects of 5HTP. We further demonstrated that 5-HT infusion was not capable of improving AChinduced relaxation even if started prior to the development of DOCA-salt hypertension. Several lines of evidence suggest that enhanced sympathetic nerve activity (SNA) is crucial to the developmental phase of DOCA-salt hypertension (Yemane et. al. 2009;   33     Schenk and McNeill, 1992). While 5-HT was capable of lowering blood pressure in the established DOCA-salt rat, it was unable to prevent the development of DOCA-salt hypertension. Given the importance of enhanced SNA to the development of DOCA-salt hypertension, this finding might suggest another mechanism (rather then SNS inhibition) by which 5-HT lowers blood pressure. Further, while we do not dispute the importance of enhanced SNA in the established DOCA-salt model, an alternate mechanism may help explain why 5-HT was effective in lowering blood pressure in the established DOCA-salt rat but was unable to prevent the development of DOCA-salt hypertension. Thus, another possibility by which 5-HT may be acting to lower blood pressure is through direct action(s) on the peripheral vasculature. Indirect support for this hypothesis is provided by Diaz et. al. who demonstrated improved aortic ACh-induced relaxation in the DOCA-salt rat following 5-HT infusion and the absolute dependence on NOS for enabling a 5-HT-induced fall in blood pressure (Diaz et. al. 2008). This suggests that 5-HT might be acting on the endothelial cell to improve overall function and promote nitric oxide (NO) release. Alternatively, the 5-HT2B receptor is located on the endothelial cell and is capable of nitric oxide (NO) release (Ishida et. al. 1998). Thus, this receptor may be a potential target for 5-HT, promoting NO release, and a 5HT-induced fall in blood pressure. However, we cannot rule out the possibility that the improved aortic ACh-induced relaxation is due to lower blood pressure in 5-HT treated DOCA-salt rats compared with vehicle treated DOCA-salt rats. In other words, the improved aortic relaxation may be a consequence of lower blood pressure in DOCA-salt rats rather then 5-HT acting directly on the endothelial cell to improve NOS function and   34     NO release. In the present study, 5-HT did not improve aortic ACh-induced relaxation when infused over the course of one-month. Despite an observable trend toward an improved maximum relaxation to ACh in 5-HT treated rats compared to vehicle treated rats, this evidence suggests an alternate mechanism by which 5-HT lowers blood pressure. Finally, several clinical scenarios correlate well with our investigation of elevated levels of free 5-HT and changes to blood pressure. Cardiopulmonary bypass, hemodialysis (Borgdoff et. al. 2002), and anaphylactic shock (Meurer et. al. 1981) are all associated with release of 5-HT from either the platelet or enterochromaffin cell and are associated with significant falls in blood pressure. Pharmacological inhibition of SERT via treatment with an SSRI (selective serotonin reuptake inhibitor) increases extracellular concentrations of 5-HT and changes in blood pressure are noted as side effects of treatment with these drugs (Rodriguez et. al. 2001). Further, 5-HTP, the committed substrate to 5-HT synthesis, is taken as a sleep aide, potentially exposing patients to elevated levels of free 5-HT (Das et. al. 2004). Taken together, these data suggest, that there are several clinically relevant situations in which patients would have significantly elevated free plasma 5-HT levels. Understanding exactly how 5-HT affects cardiovascular function is critical to understanding and treating both current and future patients populations.   35     Perspectives and limitations One potential limitation of this study is the dose of DOCA-salt (200 mg/kg) used in our protocol to test the hypothesis of whether 5-HT was capable of preventing the development of DOCA-salt hypertension. Previously, a dose of 100 mg/kg of DOCA-salt was used to demonstrate 5-HTP (a committed precursor to 5-HT) was capable of preventing the development of DOCA-salt hypertension (Fregly et. al. 1987). The higher dose of 200 mg/kg of DOCA-salt (used throughout this study) may have masked the ability of 5-HT to inhibit DOCA-salt hypertension. Another potential limitation of this study is the length of our infusion protocol. Clinically, patients often require antihypertensive medications for a lifetime, thus a one-month infusion remains a relatively short exposure. Newly available infusion pumps may be able to extend this exposure even further (Abe et. al. 2009) In summary, this study reinforces the finding that chronic 5-HT-infusion lowers blood pressure in the established DOCA-salt hypertensive rat, and demonstrates that 5-HT is capable of lowering blood pressure over the course of one-month. 5-HT was unable to prevent the development of DOCA-salt hypertension, but this study suggests a potentially beneficial role for 5-HT in the cardiovascular system.   36                   Concentration [ng/ml] Figure 2.1                             8000 * 4000 (n=6) 500 * (n=5) 250 0 PPP PRP PPP Vehicle PRP 5-HT           Figure 2.1: Plasma (platelet poor plasma; PPP) and platelet (platelet rich plasma; PRP) 5-HT content in established DOCA-salt hypertensive rats following 28-day infusion of vehicle or 5-HT. Bars represent mean +/- SEM for the number of animals in parentheses. * Statistically significant difference (p < 0.05) vs. vehicle.   37     Figure 2.2 5-HT/Vehicle 200 150 * Vehicle (n=5) 5-HT (n=6) S9 S1 2 S1 5 S1 8 S2 1 S2 4 S2 7 S6 S3 um 4 (p os t-p C p) 100 2 Mean Arterial Pressure (mmHg) A C Time (Day) B Heart Rate (bpm) 500 5-HT/Vehicle 450 * * 400 Vehicle (n=5) 5-HT (n=6) * 350 300 S9 S1 2 S1 5 S1 8 S2 1 S2 4 S2 7 S3 p) um S6 Time (Day) C 4 (p os t-p C 2 250   38     Figure 2.2: A. Effect of chronic 5-HT or vehicle infusion on mean arterial pressure (MAP) in established DOCA-salt hypertensive rats. Data points indicate 24-hour averaged MAP +/- SEM for the number of animals in parentheses. The vertical line denotes 5-HT or vehicle osmotic pump implant. Time is represented by days on the x-axis. C represents control recordings. S represents 5-HT/vehicle infusions. B. Effect of chronic 5-HT or vehicle infusion on heart rate (HR) in established DOCA-salt hypertensive rats. Data points indicate 24-hour averaged HR (bpm) +/- SEM for the number of animals in parentheses. The vertical line denotes 5-HT or vehicle osmotic pump implant. Time is represented by days on the x-axis. C represents control recordings. S represents 5HT/vehicle infusions. * Statistically significant difference (p < 0.05) vs. vehicle.   39     Percentage Contraction (10-5 mol/L) PE A 175 140 105 70 Veh (n=7) 5-HT (n=8) 35 0 -10 -9 -8 -7 -6 -4 Percentage Contraction (10-5 mol/L) PE B 160 120 80 Veh (n=7) 5-HT (n=8) 40 0 -10 -9 -8 -7 -6 -5 -4 log 5-HT [mol/L] C 125 100 75 50 25 0 -10 Veh (n=7) 5-HT (n=8) -9 -8 -7 -6 log ACh [mol/L]   -5 log PE [mol/L] Percentage Contraction (EC50) PE Figure 2.3                                                                               40   -5 -4   Figure 2.3: Cumulative concentration response curve to A. phenylephrine (PE), B. 5-HT, or C. acetylcholine (ACh) in the thoracic aorta of established DOCA-salt hypertensive rats. Data points represent means +/- SEM for the number of animals in parentheses.   41     Concentration (ng/ml) Figure 2.4                 8000     6000     4000     2000     *     400 (n=9)     200   (n=9)     0   PPP PRP PPP PRP   Vehicle 5-HT                     Figure 2.4: Plasma (platelet poor plasma; PPP) and platelet (platelet rich plasma; PRP) 5-HT content in Sprague Dawley rats following a one month infusion with either 5-HT or vehicle, followed by administration of a DOCA-salt pellet. Bars represent mean +/- SEM for the number of animals in parentheses. * Statistically significant difference (p < 0.05) vs. vehicle.   42     Mean Arterial Pressure (mmHg) Figure 2.5       200           150           100           5-HT (25 ug/kg/min) DOCA Pellet (200 mg/kg) Vehicle (n=8) 5-HT (n=10) * C 5 pr C 1 e- C pu 3 m p S1 S3 S5 S7 S9 S1 1 S1 3 S1 5 S1 7 S1 9 S2 1 S2 3 S2 5 S2 7 50 Time (days) Figure 2.5: Effect of chronic 5-HT or vehicle infusion on mean arterial pressure (MAP) in in Sprague Dawley rats following a one month infusion with either 5-HT or vehicle, followed by administration of a DOCA-salt pellet. Data points indicate 24-hour averaged MAP +/- SEM for the number of animals in parentheses. The vertical line denotes 5-HT or vehicle osmotic pump implant and DOCA pellet implant. Time is represented by days on the x-axis. C represents control recordings. S represents 5-HT/vehicle infusions. * Statistically significant difference (p < 0.05) vs. vehicle.   43   B C   Percentage Contraction (10-5M) PE A Percentage Contraction (EC50) PE Figure 2.6 Percentage Contraction (10-5M) PE   160 120 80 40 0 Veh (n=8) 5HT (n=8) -10 -9 -8 -7 -6 -4 log PE [M] 250 200 150 100 50 0 -10 Veh (n=8) 5-HT (n=8) -9 -8 -7 -6 -5 -4 log 5-HT [M] 125 100 75 50 25 0 -10 Veh (n=8) 5-HT (n=8) -9 -8 -7 -6 log ACh [M]   -5 44   -5 -4   Figure 2.6: Cumulative concentration response curve to A. phenylephrine (PE), B. 5-HT, or C. acetylcholine (ACh) in the thoracic aorta of DOCA-salt rats treated with either 5-HT or vehicle during the development of DOCA-salt hypertension. Data points represent means +/- SEM for the number of animals in parentheses.   45     Chapter 3 5-hydroxytryptamine (5-HT) does not reduce total peripheral resistance through direct receptor mediated vascular relaxation of the superior mesenteric artery Robert Patrick Davis1, Jill Pattison1, Janice M. Thompson1, Ruslan Tiniakov2, Karie E. Scrogin2, and Stephanie W. Watts1 1 Department of Pharmacology and Toxicology, Michigan State University, East Lansing, MI 48824 2 Department of Pharmacology and Experimental Therapeutics, Loyola University Chicago, Stritch School of Medicine, Maywood, IL 60153 Submitted as a manuscript to Journal of Pharmacology and Experimental Therapeutics   46     ABSTRACT Serotonin (5-hydroxytryptamine; 5-HT) delivery over 1 week results in a sustained fall in blood pressure in the deoxycorticosterone acetate (DOCA)-salt and sham rat. We hypothesized that 5-HT lowers blood pressure through direct receptor-mediated vascular relaxation. In vivo, we show that 5-HT reduced mean arterial pressure (MAP), increased cardiac output (CO), stroke volume (SV), the cardiac index (CI), and reduced total peripheral resistance (TPR) in the normotensive rat when compared to rats receiving vehicle. Real-time RT-PCR demonstrated that mRNA transcripts for the 5HT2B, 5-HT1B, and 5-HT7 receptors were present in sham and DOCA-salt superior mesenteric artery (SMA). Immunohistochemistry and western blotting validated the presence of the 5-HT2B, 5-HT1B and 5-HT7 receptors in sham and DOCA-salt SMA. Isometric contractile force was measured in endothelial-intact sham mesenteric resistance arteries and SMA from sham and DOCA-salt rats. 5-HT did not cause relaxation in the ketanserin-incubated, U46619-contracted resistance artery and superior mesenteric arteries of sham and DOCA-salt rats. Maximum concentrations of BW-723C86 (5-HT2B agonist) and CP 93129 (5-HT1B agonist) did not relax the SMA from DOCA-salt (118 ± 6.7%; 180 ± 15% of half-maximal U46619-induced contraction) or sham (81 ± 7.1%; 88 ± 8.9%) rats vs. H2O vehicle. Further, maximum concentrations of LP-44 (5-HT7 agonist) did not relax the SMA from DOCA-salt (75 ± 4.7%) or sham (55 ± 6.4%) rats vs. vehicle. Thus, although these 5-HT receptors are present in the   47     SMA, their activation does not play a role in relaxation, and are likely not involved in a 5HT-induced fall in MAP.   48     INTRODUCTION 5-HT is a vasoactive amine synthesized in the enterochromaffin cells of the gastrointestinal tract and the raphe nucleus of the central nervous system (Erspamer and Asero, 1952; Dahlstroem and Fuxe, 1964). The physiological actions of 5-HT are mediated by 7 major receptor subtypes (5-HT1-5-HT7) (Watts and Davis, 2010). 5-HT is actively taken up by the serotonin transporter (SERT) and metabolized by monoamine oxidase (MAO) to its inactive metabolite 5-hydroxyindole acetic acid (5-HIAA). 5-HT is circulated throughout the peripheral vasculature by platelets, which actively take up and store (millimolar) concentrations of 5-HT. This results in comparatively low levels of circulating free plasma 5-HT under normotensive conditions (Figueras et. al. 2005). 5-HT was initially identified as a potent constrictor of isolated blood vessels in the rat (Rapport et al. 1948). The primary contractile receptor in the peripheral vasculature of the rat is the 5-HT2A receptor (Yildiz et. al. 1998). Activation of 5-HT receptors also mediates contraction of human blood vessels, including: the human coronary arteries (Nilsson et. al. 1999), human saphenous vein, internal mammary artery (Gul et. al. 2003), human cutaneous hand vein (Bodelsson et. al. 1992), and human pulmonary artery (Cortijo et. al. 1997). This led to an initial hypothesis that elevated levels of free plasma 5-HT, present in both human and experimental models of hypertension (Brenner et al. 2007; Fetkovska et al. 1990) were acting to constrict the peripheral vasculature, resulting in elevated blood   49     pressure. However, Diaz et. al. demonstrated that chronic 5-HT infusion produces a sustained fall in blood pressure in the deoxycorticosterone acetate (DOCA)-salt and sham rat (Diaz et. al. 2008). This suggested that elevated levels of free plasma 5-HT might be acting to lower blood pressure. The exact mechanism by which chronic 5-HT infusion lowers blood pressure is not yet known. However, Diaz et. al. also demonstrated that inhibition of nitric oxide synthase (NOS) prevented the chronic 5-HTinduced fall in blood pressure in the sham and DOCA-salt rat. This suggested an important role for NOS, and potentially nitric oxide (NO), in enabling a 5-HT-induced fall in blood pressure. The 5-HT2B, 5-HT1B, and 5-HT7 receptors have all been linked to dilation of peripheral arteries (Bordoff et. al. 2002; Calama et. al. 2003; Saxena and Villalon, 1990). Reports of falling blood pressure in patients placed on cardiopulmonary bypass led Bordoff et. al. to postulate that 5-HT released from mechanically disrupted platelets (a result of cardiopulmonary bypass) might be acting to lower blood pressure. Bordoff et. al. went on to demonstrate that treatment with either a 5-HT2B receptor antagonist or an inhibitor of NOS abolished a bypass-induced fall in blood pressure in the male normotensive Wistar rat (Bordoff et. al. 2002). This suggests 5-HT may be acting at the 5-HT2B receptor to stimulate NOS, and potentially release NO, thereby lowering blood pressure in the rat. Additionally, Calama et. al. demonstrated that intra-arterial administration of L-694, 247 (5-HT1B/1D receptor agonist) produced vasodilation in the hindquarter of the anesthetized rat (Calama et. al. 2003). This suggests 5-HT may be acting at the 5-   50     HT1B/1D receptor to lower blood pressure in the rat. Finally, the 5-HT7 receptor is associated with vascular smooth muscle relaxation, and a long-lasting depressor response to 5-HT in the rat (Saxena and Villalon, 1990). This suggests another potential site (the 5-HT7 receptor) at which 5-HT may be acting to lower blood pressure in the rat. Collectively, this evidence suggests that the 5-HT2B, 5-HT1B, or the 5-HT7 receptor(s) may represent potential site(s) of action by which 5-HT acts to lower blood pressure. Thus, we tested the hypothesis that 5-HT lowers blood pressure through direct receptormediated vascular relaxation. To test this hypothesis, we investigated whether 5-HT infusion was lowering blood pressure through a reduction in total peripheral resistance (TPR) or by reducing cardiac output (CO). We then utilized in vitro pharmacological techniques to demonstrate the presence of the 5-HT2B, 5-HT1B, and 5-HT7 receptors in the superior mesenteric artery (SMA) of the sham and DOCA-salt rat. Classical agonists selective for each receptor subtype were used, as well as 5-HT itself. The SMA is a model resistance artery and changes in the tone of this blood vessel reflect changes in blood pressure following 5HT infusion. Isolated mesenteric arteries were studied at the level of mRNA (real time PCR), protein (western blot and immunohistochemical analysis), and functionally (isolated contractile studies; conducted in the presence of ketanserin, to potentially unmask a relaxant receptor) to determine whether 5-HT caused direct relaxation of the isolated mesenteric artery.   51     METHODS Animals The Michigan State University and Loyola University Chicago, Stritch School of Medicine Institutional Animal Care and Use Committees (IACUC) approved all protocols. Male Sprague Dawley rats (225-250 g, Charles River Laboratories) were used. Hemodynamic studies (measurement of mean arterial pressure (MAP), cardiac output (CO), stroke volume (SV), and calculations of cardiac index (CI), and total peripheral resistance) were completed in conscious freely moving Sprague Dawley rats. Isolated tissue experiments (RT-PCR, western blot, immunohistochemistry, and isolated contractile experiments) were performed in the SMA from sham and DOCA-salt rats. DOCA-salt hypertension Sprague-Dawley rats underwent left uninephrectomy, and a DOCA pellet was implanted subcutaneously (200 mg/kg). Sham rats underwent left uninephrectomy, but were not implanted with DOCA pellet. Rats were given standard rat chow ad libitum. DOCA-salt water was supplemented with 1% NaCl and 0.2% KCl for the duration of the study. Sham rats received tap water for the duration of the study. Hypertension was established after 4 weeks of initiation of DOCA. Sham systolic blood pressure was < 140 mmHg, while DOCA-salt systolic blood pressure was > 140 mmHg measured by tail cuff method. Anesthesia and Analgesia All rats were anesthetized with isoflurane (2% in 100% O2) and ventilated mechanically. At the time of surgery, the incision site was treated with topical antibiotic containing   52     analgesic to prevent irritation and infection. Incisions were closed with silk suture. Rats were treated with amoxicillin (150 mg/kg/i.m.) following surgery and 3 days thereafter. All rats were treated with rimadyl (5 mg/kg, s.c. for 2 days) for general analgesia. Surgical Methods: Alzet osmotic pump A small incision was made at the base of the neck. Blunt dissection was used to create a small subcutaneous pocket between the scapulae. The pump (Alzet Osmotic Pump, Model 2ML4, Duret Corporation, Cupertino, CA, 2.5 µL/hr 28 days) was inserted and the skin sutured closed. To each pump, a 5-HT creatinine complex (25 µg/kg/min) and 1% ascorbic acid, antioxidant (0.02 g/pump) or vehicle (1% ascorbic acid, antioxidant) was loaded. The solution was dissolved in 1 mol/L HCl, and a pH-balance (~7) was achieved with 4 mol/L NaOH. Surgical Methods: Ascending aortic flow probe Rats were given an injection of antisialogogue, glycopyrrolate (4 mg/kg, s.c.), then anesthetized with isoflurane, intubated and mechanically ventilated with room air. Through an incision in the third intercostal space, an ultrasonic transit-time flow probe (model 3SB; Transonic Systems Inc., Ithaca, NY) was placed around the ascending aorta. The thoracic incision was closed in layers, and the lungs were reinflated with negative pressure. The probe cable was tunneled s.c. and externalized at the nape of the neck using silastic cuff.   53     Surgical Methods: Blood pressure probe During the same surgery, radiotelemetric transmitters (PhysioTel C50-PXT series, Data Science International, St. Paul, MN, USA) for blood pressure and ECG recording were implanted subcutaneously. Through the groin incision, left femoral artery was catheterized and the tip of catheter was advanced into the abdominal aorta. All rats were allowed at least 10 days for recuperation prior to beginning data collection. Mean arterial pressure (MAP), heart rate (HR), and aortic blood flow (i.e. CO) were recorded daily, between 10:00 am and 2:00 pm. Following the connection to recording instruments, animals were allowed at least 60 min for habituation, after which MAP and HR were recorded continuously for 1 hour. Total peripheral resistance (TPR) (mmHg/ml/min/kg) and cardiac Index (CI) are reported as normalized parameters. CI = CO / (9.1 x g 0.66 0.66 ), where g = body mass in grams, and (9.1 x g ) = body surface area 2 in cm ). Tissue preparation Rats were anesthetized with pentobarbital (60 mg/kg i.p.) and the superior mesenteric artery or the mesenteric resistance artery was removed and placed in physiological salt solution (PSS) containing (mM): NaCl 130; KCl 4.7; KH2PO4 1.8; MgSO4 * 7H2O 1.7; NaHCO3 14.8; dextrose 5.5; CaNa2EDTA 0.03, CaCl2 1.6 (pH 7.2) Real-Time RT-PCR Superior mesenteric artery was removed and placed in sterile water, then cleaned of fat and blood. Total RNA was isolated using the MELT Total Nucleic Acid Isolation System   54     (Ambion, Austin, TX) and reverse transcribed with Superscript II reverse transcriptase (Invitrogen, Carlsbad, CA). Standard real-time RT-PCR was done using a GeneAMP 7500 Real-Time PCR machine (Applied Biosystems, Carlsbad, CA) and SYBR Green PCR Master Mix (Applied Biosystems). Rat primers were purchased from SABiosciences (Frederick, MD): 5-HT1A (RefSeq Accession #: NM_012585.1; 191 bp amplicon), 5-HT1B (RefSeq Accession #: NM_022225.1; 103 bp amplicon), 5-HT1D (RefSeq Accession #: NM_012852.1; 173 bp amplicon), 5-HT2A (RefSeq Accession #: NM_017254.1; 191 bp amplicon), 5-HT2B (RefSeq Accession #: NM_017250.1; 140 bp amplicon), 5-HT3A (RefSeq Accession #: NM_024394.2; 179 bp amplicon), 5-HT4 (RefSeq Accession #: NM_01285.31; 83 bp amplicon), 5-HT5A (RefSeq Accession #: NM_013148.1; 154 bp amplicon), 5-HT6 (RefSeq Accession #: NM_024365.1; 186 bp amplicon), 5-HT7 (RefSeq Accession #; NM_022938.2; 99 bp amplicon), and calibrator control (beta-2 microglobulin) (RefSeq Accession #: NM_012512, 128 bp amplicon). PCR conditions were: 95oC for 10 minutes followed by 40 cycles of (95oC, 15 sec; 60oC, 60 sec). A standard dissociation curve was run following the above cycle conditions. Western Blot Analysis Superior mesenteric arteries were cleaned of fat and blood, placed into liquid nitrogen and ground to a powder. Ice-cold homogenation buffer (125 mM Tris (pH 6.8), 4% SDS, 20% glycerol, 0.5 mM phenylmethylsulfonyl fluoride, 1 mM orthovanadate, 10 ug/ml aprotinin, 10 ug/ml leupeptin) was added and the homogenates were vortexed briefly   55     and sonicated for 1 minute (5x each). Samples were centrifuged at 11,000 rpm for 10 minutes at 4°C to pellet debris. Supernatant was collected and placed in clean microtubes, then held at -80°C until protein concentrations could be determined. Protein concentration was determined with the BCA protein kit (Sigma, catalog #BCA1), following standard procedure. Western analysis of rat superior mesenteric artery homogenates (50 µg) was done and proteins were transferred to PVDF (5-HT1B and 5HT7) or nitrocellulose (5-HT2B). Blots were then incubated overnight at 4°C with 5-HT1B (1 ug/mL; Abcam, Cambridge, MA; Catalog # ab13896), 5-HT2B (1:1000; BD Pharmingen, San Diego, CA; Catalog # 556334), or 5-HT7 (1:1000; Abcam, Cambridge, MA; Catalog # ab13898). Following 5-HT receptor antibody incubation, blots were reprobed with smooth muscle α-actin (1:2000; EMD Chemicals/Calbiochem, Gibbstown, NJ) to ensure equal protein loading. All blots were developed using species-specific HRP-conjugated secondary antibodies and ECL reagents (Amersham/GE Healthcare Life Sciences, Piscataway, NJ). Immunohistochemistry Slides containing 8 µm sections of paraffin-embedded rat superior mesenteric artery were dewaxed in Histochoice Clearing Agent (2X, 3 minutes each; Amresco, Solon, OH), followed by isopropanol (4X, 3 minutes each) and distilled water (2X, 3 minutes each). Slides were placed in Antigen Unmasking Solution (Vector Laboratories, Burlingame, CA) and microwaved on high (2X, 30 seconds each), then cooled and dried. Samples were incubated with 0.3% hydrogen peroxide in phosphate-buffered saline (PBS) for 30 minutes to block endogenous peroxidases, followed by blocking for   56     nonspecific binding with 1.5% blocking serum in PBS for 30 minutes. Slides were incubated overnight in a humidified chamber at 4oC with 5-HT1B, 5-HT2B or 5-HT7 antibody (5 µg/mL) (same antibodies used for western blots) in 1.5% blocking serum. Slides were washed in PBS (3X) and incubated with a peroxidase-conjugated secondary antibody in 1.5% blocking serum for 30 minutes, followed by 30 minutes with Vectastain Elite ABC Reagent (Vector Laboratories). 3,3-diaminobenzidine/H2O2 was applied until staining appeared (1-4 minutes), stopped with PBS washing, and counterstained with hematoxylin (Vector Laboratories, Burlingame, CA). Slides were air dried and coverslipped and pictures were taken using an inverted Nikon microscope with a digital camera (Nikon, Tokyo, Japan). Images were captured using MMI Cellcut software (MMI Inc., Rockledge, FL). Isolated tissue bath Endothelium-intact superior mesenteric arteries (SMA) were cleaned and cut into helical strips for measurement of isometric contractile force. SMA strips were mounted in warmed (37°C) and aerated (95% O2, 5% CO2) tissue baths (30 mL) on Grass isometric transducers (FT03; Grass instruments, Quincy, MA, USA), which were connected to an ADInstruments PowerLab (ADInstruments, Colorado Springs, CO). Tissues were placed under optimal resting tension (600 mg; previously determined) and allowed to equilibrate for 1 hour before an initial challenge with a maximal concentration of phenylephrine (10 -5 M). After the initial challenge, tissues were washed until toned returned to baseline. Then, -7 a half-maximal concentration of phenylephrine (10   57   M) was added to the bath, followed   -6 by acetylcholine (ACh; 10 M) to determine the integrity of the endothelial layer. Tissues that relaxed >50% to ACh were considered endothelium-intact. Tissues were washed until they returned to baseline. Strips were incubated with ketanserin (50 nM; 5-HT2A antagonist) for 15 minutes to prevent activation of the 5-HT2A contractile receptor. Tissues were then contracted half-maximally to U46619 (thromboxane A2 agonist) (~ 30 minute incubation with ketanserin). Cumulative concentration-response curves were generated for each of the following agonists: 5-HT vs. vehicle (H2O), BW-723C86 (5HT2B agonist) vs. vehicle (H2O), CP 93129 (5-HT1B agonist) vs. vehicle (H2O), and LP44 (5-HT7 agonist) vs. vehicle (DMSO). All data were captured in the program Chart. Wire Myograph Under a stereomicroscope with a calibrating eyepiece and in cold PSS, two tungsten wires were inserted through the lumen of a cleaned mesenteric resistance artery (~200250 µm diameter). One wire was attached to an isometric force transducer that can detect forces from 0.002 – 10 grams, the other to a micrometer-attached support. Force generation was recorded on a Grass Model 7D polygraph. The dual chamber, kept at 37o C and perfused via a peristaltic pump with warmed and oxygenated PSS, allowed mounting of two parallel vessels. A passive tension of ~400 mg (appropriate for generating optimal force in a resistance artery) was applied. Vessels were allowed to -5 equilibrate for 1 hr prior to intial challenge with PE (10 M). After initial washout, experiments were performed as described above for isolated tissue bath.   58     Statistical analysis For in vivo data analysis, between group differences were assessed by a two-way ANOVA with repeated measures followed by post hoc testing using Bonferroni’s procedure (GraphPad Prism 5). For isometric contractile studies, relaxation is reported as a percentage of initial contraction to a half-maximal concentration of U46619 (thromboxane A2 agonist). Repeated measures two-way ANOVA followed by the Bonferroni post hoc test was used to compare concentration response curves. An unpaired students t-test was used to compare differences in maximal response(s) between agonist vs. vehicle. In all cases, p < 0.05 was considered significant. All results are presented as the mean ± SEM.   59     RESULTS Effect of 5-HT on CO and TPR There was no between-group difference in blood pressure prior to beginning of 5-HT or vehicle infusion (C1-C2). Continuous 5-HT infusion resulted in a significant fall in MAP on days S1 and S2 (106 ± 5 and 106 ± 3 mmHg) compared to vehicle-infused group (120 ± 2 and 116 ± 3 mmHg) (Figure 1A). Starting on day S3, MAP tended to normalize yet remained significantly below baseline level. Heart rate was significantly elevated 1 day after the start of 5-HT infusion (447 ± 6 bpm vs. 404 ± 9 bpm in vehicle group, P<0.01) but then returned to baseline level. Infusion of 5-HT produced a significant increase in cardiac output (CO; 353 ± 24.8 ml/min/kg; S1), stroke volume (SV; 0.25 ± 2 0.01 ml; S1) and cardiac index (CI; 0.28 ± 0.2 ml/min/cm ; S1) compared to vehicle 2 infusion (236 ± 4.1 ml/min/kg; 0.18 ± 0.01 ml; 0.18 ± 0.01 ml/min/cm , respectively; S1). All these parameters remained significantly elevated throughout the experiment (Figure 1). Rats receiving 5-HT developed a significant reduction in total peripheral resistance (TPR; 0.31 ± 0.03 mmHg/ml/min/kg; S1) compared to vehicle-treated animals (0.51 ± 0.01 mmHg/ml/min/kg). This effect persisted throughout the remainder of the study. Real Time RT-PCR 5-HT receptor transcripts were quantified using real time RT-PCR (2 -delta Ct x1000) in the SMA of sham and DOCA-salt rats using beta-2 microglobulin as a calibrator (Figure 2A). The 5-HT2B receptor was expressed at high levels in the SMA of both the sham   60     and DOCA-salt rat (4.46 ± 0.44; 10.41 ± 2.32 respectively). mRNA transcripts for the 5HT1B (0.83 ± 0.24; 0.81 ± 0.11), and 5-HT7 (0.78 ± 0.62; 0.21 ± 0.04) were expressed at significantly lower levels vs. the 5-HT2B receptor, in the SMA of sham and DOCA-salt rats (p < 0.05). Effect of 5-HT on arterial tone Endothelial-intact mesenteric resistance vessels (200 µm diameter) were mounted in a -9 wire myograph chamber for measurement of isometric contractile force. 5-HT (10 M 10 -5 M) contracted the isolated mesenteric resistance vessel from baseline and when 9 contracted with prostaglandin F2 alpha (PGF2α) (Figure 2B). 5-HT (10- M - 10 -5 M) also contracted the isolated mesenteric resistance vessel when contracted with PGF2α and incubated with ketanserin (100 nM; 5-HT2A receptor antagonist) (Figure 2C). These -6 same vessels relaxed to ACh (10 M) and forskolin (1 µM). Relaxation to 5-HT was not observed under any condition. Endothelium-intact helical SMA strips from sham and DOCA-salt rats were mounted in an isolated tissue bath for measurement of isometric contractile force. Tissues were contracted half-maximally to U46619 and were incubated with ketanserin to block the contractile effects of the 5-HT2A receptor (these conditions apply to all remaining -9 -5 experiments). 5-HT (10 M – 10 M) did not relax the SMA from sham or DOCA-salt   61     rats compared to vehicle (Figure 2D). Maximum concentrations of 5-HT (10 -5 M) contracted the sham SMA (125 ± 9.1% of half-maximal U46619-induced contraction) compared to vehicle (78 ± 6.1% of half-maximal U46619-induced contraction). Similarly, a maximum concentration of 5-HT (10 -5 M) also contracted the DOCA-salt SMA (144 ± 16.2% of half-maximal U46619-induced contraction) compared to vehicle (83 ± 9% of half-maximal U46619-induced contraction). At the end of each experiment, sodium nitroprusside (SNP) was added to each bath to demonstrate the SMA was able to relax under experimental conditions set forth (SNP was also used in all remaining experiments to demonstrate relaxation under various conditions). These findings suggest that 5-HT alone does not relax the SMA in either the sham or DOCA-salt rat. 5-HT2B receptor Homogenates of SMA from sham and DOCA-salt rats were processed for western blot analysis using antibodies that recognized the 5-HT2B receptor (amino acids 1-58) (Figure 3A). The 5-HT2B receptor antibody recognized protein (~ 45-55 kDa) in lanes loaded with SMA from sham and DOCA-salt homogenates. A band was present in rat stomach fundus (control), thus validating use of this antibody. α-actin was similarly expressed in all SMA homogenates loaded. Immunohistochemical detection of the 5-HT2B receptor was performed on sections from the SMA of the sham and DOCA-salt rats (Figure 3B). Staining was observed as black/brown precipitate and appeared to be present in the endothelial, smooth muscle,   62     and adventitial layers of the SMA from sham and DOCA-salt rats. Staining was not observed in those tissues that were not incubated with primary antibody. Black/brown staining was observed in the rat brain (control), thus validating use of this antibody. No staining was observed in rat brain sections that were not incubated with primary antibody. Collectively, western analysis and IHC staining provided qualitative evidence that the 5-HT2B receptor was present in the SMA of the sham and DOCA-salt rat. The 5-HT2B receptor is located on the endothelial cell of the SMA and is thus a potential target for a 5-HT-induced, nitric oxide dependent, fall in blood pressure. SMA strips that -6 relaxed > 50% to ACh (10 M) were considered endothelial cell-intact and this test -9 preceded each concentration response curve. BW-723C86 (10 M – 10 -5 M; 5-HT2B receptor agonist) did not relax the helical SMA strip from either sham or DOCA-salt rats -5 compared to vehicle (Figure 3C). Maximum concentrations of BW-723C86 (10 M) did not relax the sham SMA (81 ± 7.1% of half-maximal U46619-induced contraction) compared to vehicle (41 ± 8.3 % of half-maximal U46619-induced contraction). -5 Additionally, maximum concentrations of BW-723C86 (10 M) contracted the DOCA- salt SMA (118 ± 6.7 of half-maximal U46619-induced contraction) compared to vehicle (60 ± 5.5%of half-maximal U46619-induced contraction). This evidence suggests that 5HT is not acting at the 5-HT2B receptor in the SMA of either the sham or DOCA rat to lower blood pressure.   63     5-HT7 receptor The 5-HT7 receptor antibody (amino acids 13-28) recognized protein (~ 50 kDa) in homogenates of sham and DOCA-salt SMA (Figure 4A). A strong band was also present in rat brain lysate homogenate (control), thus validating use of this antibody. αactin was similarly expressed in all SMA homogenates loaded and α-actin was not present in rat brain lysate homogenate. Immunohistochemical detection of the 5-HT7 receptor was similarly performed in sections of SMA from sham and DOCA-salt rats (Figure 4B). Negligible staining was observed as black/brown precipitate in the adventitia of SMA from sham and DOCA-salt rats. In our positive control (rat brain) we observed staining. 5-HT7 receptor proteins was identified by western blot with greater confidence, suggesting this antibody may not perform as well in IHC analysis, but that the 5-HT7 receptor is present in the SMA of both the sham and DOCA-salt rat. We next determined if the 5-HT7 receptor was functionally active by investigating -9 relaxation to LP-44, a 5-HT7 receptor agonist. LP-44 (10 -5 M – 10 M) did not relax the SMA from sham or DOCA-salt rats compared to vehicle (Figure 4C). Maximum concentrations of LP-44 (10 -5 M) did not relax the SMA from sham rats (55 ± 6.4% of half-maximal U46619-induced contraction) compared to vehicle (51 ± 7.8 % of halfmaximal U46619-induced contraction). Similarly, maximum concentrations of LP-44 (10   64   -   5 M) did not relax the SMA from DOCA-salt rats (75 ± 4.7 of half-maximal U46619- induced contraction) compared to vehicle (73 ± 7.8%of half-maximal U46619-induced contraction). Collectively, this evidence suggests that 5-HT is not likely acting at the 5HT7 receptor to lower blood pressure. 5-HT1B receptor Homogenates of SMA from sham and DOCA-salt rats were processed for western blot analysis using antibodies that recognized the 5-HT1B receptor (amino acids 8-26). The 5-HT1B receptor antibody recognized multiple proteins in the SMA of sham and DOCAsalt rats (Figure 5A). A band was present in rat brain lysate homogenate (control), which was consistent with one of the bands observed in the SMA from sham and DOCA-salt rats (arrow). α-actin was similarly expressed in all SMA homogenates loaded, and was expressed at low levels in rat brain lysate homogenate. Immunohistochemical detection of the 5-HT1B receptor was also performed in SMA of sham and DOCA-salt rats (Figure 5B). Staining was observed in the media and adventitia, as black/brown precipitate, in sections of SMA from sham and DOCA-salt rats. Robust staining was observed in rat brain sections, thereby validating use of the 5HT1B antibody. Taken together, western blot analysis and IHC staining provided evidence that the 5-HT1B receptor was present in the SMA of sham and DOCA-salt rats.   65     Finally, the same functional protocol was undertaken in endothelium-intact helical SMA strips from sham and DOCA-salt rats to determine if activation of the 5-HT1B receptor -9 was capable of relaxing the SMA. CP 93129 (10 -5 M – 10 M; 5-HT1B receptor agonist) did not relax the SMA of sham or DOCA-salt rats compared to vehicle (Figure 5C). A -5 maximum concentration of CP 93129 (10 M) did not relax the SMA from sham rats (88 ± 8.9% of half-maximal U46619-induced contraction) compared to vehicle (41 ± 8.3 % of half-maximal U46619-induced contraction). In contrast, a maximum concentration of -5 CP 93129 (10 M) contracted the SMA from DOCA-salt rats (180 ± 15 of half-maximal U46619-induced contraction) compared to vehicle (60 ± 5.5% of half-maximal U46619induced contraction). This suggests that 5-HT is not acting at the 5-HT1B receptor to lower blood pressure.   66     DISCUSSION The main objective(s) of this study were to: 1) identify whether 5-HT was lowering blood pressure through a reduction in total peripheral resistance (TPR) or cardiac output (CO), 2) identify the 5-HT2B, 5-HT7, and 5-HT1B receptors in the superior mesenteric artery (SMA) of sham and DOCA-salt rats via PCR, western, and IHC analysis, 3) determine whether activation of these receptors were capable of relaxing the SMA of sham or DOCA-salt rats. 5-HT reduces total peripheral resistance in sham and DOCA-salt rats 5-HT infusion produced a significant reduction in MAP and TPR, supporting our hypothesis that 5-HT might be acting to lower blood pressure through direct receptor mediated vascular relaxation. This is the first time the effects of 5-HT on TPR in the conscious animal have been reported. Further, our finding that 5-HT infusion increases CO is consistent with previous studies (Diaz et. al. 2008), which demonstrated that 5-HT infusion reduces MAP, and elevates heart rate (HR) in sham and DOCA-salt rats. Defining these important hemodynamic parameters (CO, CI, and TPR) allowed us to pursue a careful investigation of the vasculature, the site of TPR, as the primary site of action for a 5-HT-induced fall in blood pressure. 5-HT does not directly relax the mesenteric arterial vasculature 5-HT alone was not capable of relaxing the mesenteric resistance vessel or SMA and thus, suggests 5-HT is not likely acting at the level of the SMA to mediate direct receptor mediated vascular relaxation and lower blood pressure. However, the literature   67     suggests several 5-HT receptors (5-HT2B, 5-HT7, and 5-HT1B) are capable of peripheral artery dilation (Bordoff et. al. 2002; Calama et. al. 2003; Saxena and Villalon, 1990). Therefore, we sought to determine whether these receptors were present in the SMA of the sham or DOCA-salt rat and whether activation of these receptors, in the presence of ketanserin (5-HT2A receptor antagonist), might unmask a potential relaxant role for 5-HT in the SMA. Moreover, the literature also suggests that the 5-HT2B receptor is located on the endothelial cell and is capable of nitric oxide (NO) release (Ishida et. al. 1998). Given the absolute dependence of a 5-HT-induced fall in blood pressure on nitric oxide synthase (NOS) (Diaz et al. 2008), the 5-HT2B receptor was a leading candidate in our investigation. However, a significant body of evidence suggests an alternate role for the 5-HT2B receptor, which may depend on the location within the vasculature (endothelial cell; relaxant or smooth muscle; contractile) and/or underlying pathology (presence or absence of hypertension; contractile) (Ishida et. al. 1998; Banes and Watts, 2003). In the aorta of the DOCA-salt rat, the functional 5-HT2B receptor is up regulated and mediates contraction of the aorta vs. the sham rat (Banes and Watts, 2003). Despite instances in which this receptor has been described as contractile, we determined whether this receptor was present in the SMA of the sham or DOCA-salt rat and whether activation of this receptor in endothelial intact SMA strips might elicit a relaxant response.   68     The 5-HT2B, 5-HT7, and 5-HT1B receptor transcripts were identified via PCR analysis, thus validating our interest in these receptors as potential targets mediating a fall in resistance, and a 5-HT-induced fall in blood pressure. Prominent expression of the 5HT2B receptor transcript compared to the 5-HT7 and 5-HT1B transcripts also validated our interest in this receptor as a potential mediator of a 5-HT-induced fall in blood pressure. Western blot and IHC analysis identified the 5-HT2B, 5-HT7, and 5-HT1B receptors in the SMA of sham and DOCA-salt rats. The 5-HT7 receptor antibody recognized protein (via western blot) in the SMA of the sham and DOCA-salt rat, but the same antibody did not elicit a strong immunohistochemical stain in sections of SMA from the sham or DOCA-salt rat. Despite repeated attempts with this antibody, and attempts with a different antibody (ImmunoStar 5-HT7 receptor antibody; catalog # 24430), we accepted a limited ability to detect the 5-HT7 receptor via IHC analysis. Fortunately, western blot analysis revealed strong bands in the SMA of both sham and DOCA-salt rats, giving us confidence that the 5-HT7 receptor protein was present in the SMA of the sham and DOCA-salt rat. Detection of the 5-HT2B, 5-HT7, and 5-HT1B receptors allowed us to proceed with functional investigation. Despite their presence and literature to support our hypothesis (5-HT lowers blood pressure through receptor mediated vascular relaxation), no significant relaxation was observed in the SMA of the sham or DOCA-salt rat. Instead,   69     activation of the 5-HT2B receptor (via BW723C86) and 5-HT1B (via CP 93129) caused contraction of the SMA of the DOCA-salt rat. These responses are consistent with the contractile responses seen in previous studies in the DOCA-salt aorta (Banes and Watts, 2003; Banes and Watts, 2001). Activation of the 5-HT7 receptor did not elicit a contractile response, but it also did not relax the SMA of the sham or DOCA-salt rat. Therefore, we must conclude that the 5-HT2B, 5-HT7, and 5-HT1B are not involved in relaxing the SMA and it is unlikely that activation of these receptors in the mesentery is responsible for mediating a 5-HT-induced fall in blood pressure. Potential mechanism(s) underlying a 5-HT-induced fall in blood pressure Several other possibilities might explain a 5-HT-induced fall in blood pressure. 5-HT may be acting in a different vascular bed to lower total peripheral resistance (TPR) and blood pressure. Other vascular beds 5-HT might be acting in to lower blood pressure include: the skeletal muscle vascular bed (Proctor and Parker, 2006), or the cutaneous vascular bed (Kellogg et. al. 2008). Changes in resistance in either of these vascular beds might be capable of eliciting a 5-HT-induced fall in blood pressure. Another possibility is that 5-HT is acting to inhibit sympathetic nerve activity (SNA), thereby reducing vascular resistance and lowering blood pressure. The 5-HT1B/1D receptor is located on the presynaptic nerve terminal and when stimulated by 5-HT, results in an inhibition of norepinephrine (NE) release (Molderings et. al. 1990). Alternatively, 5-HT may be acting centrally to inhibit SNA, reducing total peripheral resistance and eliciting a 5-HT-induced fall in blood pressure. Recent studies have   70     demonstrated that 5-HT is capable of crossing the blood brain barrier via the serotonin transporter (SERT) (Nakatani et. al. 2008), though this remains controversial. Thus, we cannot rule out the possibility that 5-HT may be acting centrally to lower blood pressure. Perspectives and Limitations 5-HT infusion produced a persistent cyanosis in both the tip of the tail and the left hind limb (instrumented with an arterial catheter) of the sham and DOCA-salt rats. This observation raises the possibility that 5-HT might be acting to constrict the vasculature in the tail and hind limb despite lowering mean arterial pressure in these rats. However, it might also be possible that these beds are hypo-perfused, concurrent with the fall in mean arterial pressure, producing the observed cyanosis. This observation suggests further study is necessary to address exactly how 5-HT is functioning in the cardiovascular system. A limitation of this study is the use of only a single specific 5-HT receptor agonist to investigate each 5-HT receptor (5-HT2B, 5-HT1B, 5-HT7) of interest. In summary, this study demonstrates the presence of the 5-HT2B, 5-HT7, and 5-HT1B receptors in the SMA of sham and DOCA-salt rats, but activation of these receptors does not participate in relaxation of the SMA and therefore these receptors are not likely involved in a 5-HT-induced fall in blood pressure.     71   C. D. E. 140 120 100 80 Vehicle (n=5) 5-HT (n=6) * * C1 S1 S3 S5 S7 Time (days) CO (mL/min/kg) B. Stroke Volume (ml) Cardiac Index (ml/min/cm2) A. TPRI (mmHg/ml/min/kg) Figure 3.1   MAP (mmHg)   400 * 300 * * * * * * Vehicle (n=5) 5-HT (n=6) 200 100 C1 S1 S3 S5 S7 Time (days) 0.35 0.30 0.25 0.20 0.15 * * * * * * * C1 S1 S3 S5 S7 Time (days) 0.3 * 0.2 0.1 * * Vehicle (n=5) 5-HT (n=6) C1 S1 S3 S5 S7 Time (days) 0.6 0.5 0.4 0.3 0.2 * * * * * * * C1 S1 S3 S5 S7 Time (days)   Vehicle (n=5) 5-HT (n=6) 72   Vehicle (n=5) 5-HT (n=6)   Figure 3.1: Effect of chronic 5-HT or vehicle infusion on A) mean arterial pressure (MAP), B) cardiac output (CO), C) cardiac index (CI), D) stroke volume, and E) total 0.66 peripheral resistance (TPR). CI = CO/ (9.1 x g 0.66 and (9.1 x g ), where g – body mass in grams, 2 ) – body surface area in cm . Data points are 1-hour average +/- SEM. Number of animals per group is shown in parentheses. The vertical line denotes 5-HT or vehicle osmotic pump implant. Time is represented by days on the x-axis. C1 and C2 control recordings. S1 – S7 – days since beginning of 5-HT/vehicle infusion. * P < 0.05 vs. vehicle.   73     Figure 3.2 A. 2^(-deltaCT) x 1000 15 10 5 Sham (n=4) DOCA (n=4) 2 1 T 7 6 H T 5- H 5A 5- 4 H T T 5- H 3A 5- T H 5- H T 2B 2A T 5- H 5- T 1D 1B H T 5- H 5- 5- H T 1A 0 (20 µM) Contraction C. Percentage PGF2! B. Percentage PE (10-5 M) or PGF2! (20 µM) Contracted 5-HT receptor subtypes 300 200 100 Baseline (n=4) PGF2!-Contracted (n=11) 0 -10-9 -8 -7 -6 -5 -4 log 5-HT [M] 400 Ketanserin (100 nM) (n=6) 300 200 100 0 -10-9 -8 -7 -6 -5 -4 log 5-HT [M]   74     Figure 3.2 (cont’d) Percentage Contraction (EC50) U46619 D. Sham 175 150 125 Sham 100 75 50 25 0 -10 Vehicle (n=6) 5-HT (n=7) -9 -8 -7 -6 -5 -4 Percentage Contraction (EC50) U46619 log 5-HT [M] 175 150 125 DOCA 100 75 50 25 0 -10 Vehicle (n=9) 5-HT (n=9) -9 -8 -7 -6 -5 -4 log 5-HT [M] Figure 3.2: A. mRNA expression of 5-HT receptors in the superior mesenteric artery (SMA) of sham (n=4) and DOCA-salt (n=4) rats using standard real time PCR analysis. B. Cumulative concentration response curve to 5-HT generated from baseline, contracted with prostaglandin F2 alpha (PGF2α), C. and in the presence of ketanserin   75     in the sham superior mesenteric resistance artery. D. Cumulative concentration response curve to 5-HT in SMA contracted with U46619 in sham (left) and DOCA-salt (right) SMA. Data points represent mean ± SEM for the number of animals in parentheses. * P < 0.05 vs. sham 5-HT1B and 5-HT7 receptors. † P < 0.05 vs. DOCA-salt 5-HT1B and 5-HT7 receptors.   76       Figure 3.3   A. 5-HT2B Sham                   52 kDa       !-actin         B. Sham   Stomach Fundus DOCA Control A E M E S M A A E M M E A 77   No Primary                                               DOCA Primary       Figure 3.3 (cont’d) C. Percentage Contraction (EC50) U46619             Sham                           DOCA                     150 125 100 75 50 25 0 -10 Vehicle (n=6) BW723C86 (n=5) -9 -8 -7 -6 -5 -4 Percentage Contraction (EC50) U46619 log BW723C86 [M] 150 125 100 75 50 25 0 -10 Vehicle (n=4) BW723C86 (n=4) -9 -8 -7 -6 -5 -4 log BW723C86 [M]   Figure 3.3: A. Western blot analyses for identification of the 5-HT2B receptor in homogenates of SMA from sham (n=6) and DOCA-salt (n=6) rats. B. Immunohistochemical staining of the 5-HT2B receptor in SMA sections from sham and DOCA-salt rats. No primary sections were exposed only to secondary antibody. Images were taken at 20X objective. E = endothelium, M = media, A = adventitia, S = staining in   78     rat stomach fundus, L = lumen. C. Cumulative concentration response curve to BW723C86 vs. vehicle (H20) in SMA contracted with U46619 in sham and DOCA-salt SMA. Data points represent mean ± SEM for the number of animals in parentheses.   79     Figure 3.4   5-HT7 Sham         50 kDa       !- Actin       B. Sham                       DOCA DOCA A Control S A A A   80   Brain Lysate Primary     No Primary   A.   Figure 3.4 (cont’d) Percentage Contraction (EC50) U46619 C.   150 125 Sham 100 75 50 25 0 -10 Veh (n=6) LP-44 (n=9) -9 -8 -7 -6 -5 -4 Percentage Contraction (EC50) U46619 log LP-44 [M] 150 125 100 75 DOCA 50 25 0 -10 Veh (n=6) LP-44 (n=6) -9 -8 -7 -6 -5 -4 log LP-44 [M] Figure 3.4: A. Western blot analyses for identification of the 5-HT7 receptor in homogenates of SMA from sham (n=6) and DOCA-salt (n=6) rats. B. Immunohistochemical staining of the 5-HT7 receptor in SMA sections from sham and DOCA-salt rats. No primary sections were exposed only to secondary antibody. Images   81     were taken at 20X objective. A = adventitia, S = staining in rat brain lysate, L = lumen. C. Cumulative concentration response curve to LP-44 vs. vehicle (DMSO) in SMA contracted with U46619 in sham and DOCA-salt SMA. Data points represent mean ± SEM for the number of animals in parentheses.   82     Figure 3.5 Ly sa te n ai Br DO CA am Sh am Sh DO CA am Sh DO CA 5-HT1B A. - !-actin B. Sham DOCA A Control A M S M L L A M A M L   L 83     Figure 3.5 (cont’d) Percentage Contraction (EC50) U46619 C. 150 125 100 Sham 75 50 25 0 -10 Veh (n=5) CP 93129 (n=5) -9 -8 -7 -6 -5 -4 Percentage Contraction (EC50) U46619 log CP 93129 [M] 150 125 100 DOCA 75 50 25 0 -10 Veh (n=5) CP 93129 (n=5) -9 -8 -7 -6 -5 -4 log CP 93129 [M] Figure 3.5: A. Western blot analyses for identification of the 5-HT1B receptor in homogenates of SMA from sham (n=6) and DOCA-salt (n=6) rats. B. Immunohistochemical staining of the 5-HT1B receptor in SMA sections from sham and DOCA-salt rats. No primary sections were exposed only to secondary antibody. Images   84     were taken at 20X objective. M = media, A = adventitia, S = staining in rat brain lysate, L = lumen. C. Cumulative concentration response curve to CP 93129 vs. vehicle (H2O) in SMA contracted with U46619 in sham and DOCA-salt SMA. Data points represent mean ± SEM for the number of animals in parentheses.   85     CHAPTER 4 5-Hydroxytryptamine (5-HT) does not inhibit sympathetic neurogenic contraction or sympathetic pre-/post-ganglionic sympathetic nerve activity in the rat splanchnic circulation 1 1 1,2 Robert Patrick Davis , Susan M. Barman , Hakan S. Orer Stephanie W. Watts 1 3 , Shaun F. Morrison and 1 Department of Pharmacology and Toxicology, Michigan State University, East Lansing, Michigan 2 Department of Pharmacology, Faculty of Medicine, Hacettepe University, Ankara, Turkey 3 Oregon Health Sciences University, Beaverton Oregon Prepared for submission to American Journal of Physiology Regulatory, Integrative and Comparative Physiology   86     ABSTRACT Serotonin (5-hydroxytryptamine; 5-HT) infusion for 7 days results in a sustained fall in blood pressure (BP) in rodents, and this is accompanied by a robust fall in total peripheral resistance (TPR). Because TPR is significantly maintained by sympathetic nervous system tone, we hypothesized that 5-HT would 1) reduce sympathetic nerve activity (SNA); and/or 2) inhibit sympathetic neuroeffector function. The model was the splanchnic circulation of the rat. In urethane-anesthetized, paralyzed, and artificially respired Sprague-Dawley rats, mean BP was significantly (p<0.05) reduced from 101 ± 4 to 63 ± 3 mm Hg during slow infusion of 5-HT (25 µg/kg/hr, iv). Pre- and postganglionic splanchnic SNA was unaffected during 5-HT infusion. In superior mesenteric arterial rings mounted in isolated tissue bath for electrical field stimulation, -9 5-HT (10 – 10 -5 M; 5 min) did not inhibit neurogenic contraction compared to vehicle (99 ± 11% at 20 Hz; 96 ± 16% at 20 Hz; respectively). Further, 5-HT did not inhibit neurogenic contraction in the presence of fluoxetine (reuptake inhibitor), ketanserin (5HT2A antagonist), and a longer incubation with 5-HT (30 min) vs. vehicle in arteries from normal, sham and DOCA-salt hypertensive rats. The 5-HT1B receptor agonist CP 93129 (5-HT1B agonist) was also ineffective. This study demonstrates that 5-HT does not interact with the peripheral sympathetic nervous system (neuroeffector junction, pre and postganglionic SNA) to remove sympathetic tone from the splanchnic circulation, and suggests an alternate mechanism(s) or vascular bed is mediating a 5-HT-induced fall in blood pressure.   87     INTRODUCTION 5-hydroxytryptamine (5-HT, serotonin) is synthesized in the enterochromaffin cells of the gastrointestinal tract and the raphe nucleus of the central nervous system (CNS) and is well documented as a potent constrictor of isolated blood vessels (Berger et al, 2009; Doggrell, 2003; Erspamer et al, 1952; Kaumann and Levy, 2006; Page and McCubbin 1953a, b; Ramage and Villalon, 2008; Rapport et al, 1948; Watts 2005, 2009). Historically, 5-HT has been considered a pathogenic factor or potential contributor to the development of essential hypertension due in particular to the knowledge that free plasma 5-HT levels are elevated in both human and experimental models of hypertension compared to normotensive controls (Brenner et al., 2007; Diaz et al., 2008). If 5-HT is indeed a vasoconstrictor in vivo, elevations in free plasma 5-HT would logically promote vasoconstriction, elevation of total peripheral resistance (TPR) and elevation in blood pressure. However, when we experimentally elevate free plasma 5-HT levels in the rodent, mean arterial blood pressure is lowered. Administration of 5HT has typically been performed through Alzet osmotic pumps, and is done chronically over one week. This is true in multiple strains of rats (Sprague Dawley, Wistar; Diaz et al., 2008), and male and female (Davis et al., 2011a). In the mineralocorticoid- dependent, hypertensive deoxycorticosterone acetate (DOCA) salt rat, 5-HT nearly normalized arterial blood pressure. More recently, we have demonstrated that 5-HT causes a robust fall in total peripheral resistance (Davis et. al., 2011b). This important piece of information suggests that the vasculature, or control of the vasculature, is the site of action for 5-HT to reduce blood pressure.   88     Since the sympathetic nervous system is essential for maintaining normal levels of total peripheral resistance, removal of sympathetic tone is a means by which blood pressure could be reduced. 5-HT has an interesting history with respect to sympathetic tone. Several studies suggest that 5-HT inhibits norepinephrine (NE) release at the sympathetic neuroeffector junction (Gothert et al., 1991). The 5-HT1B/1D receptor has been implicated in presynaptic inhibition and inhibition of NE release (Molderings et al, 1990). This evidence suggests 5-HT may be acting to reduce sympathetic outflow at the neuroeffector junction, thereby reducing vascular resistance, and potentially lowering blood pressure. Central actions of 5-HT include both increases and decreases in SNA (Barnes and Sharp, 1999; Ramage and Villalon, 2008). Thus, if systemically administered 5-HT can cross the blood-brain barrier, a reduction in sympathetic nerve activity (SNA) could contribute to its vasodepressor response. There are serotonergic receptors on sympathetic ganglia (Hertzler, 1961; Jones et al., 1995; Meehan and Kreulen, 1991; Pickering et al., 1994; Ramage and Villalon, 2008; Sheng-Rong et al, 1999; Watkins and Newberry, 1996), so this is another potential target for the site of action of 5-HT to reduce BP. The current study was designed to study the role of sympathoinhibition in mediating the depressor response to a slow intravenous infusion of 5-HT in anesthetized rats. We hypothesized that 5-HT would 1) reduce sympathetic nerve activity (SNA); and/or 2) inhibit sympathetic neuroeffector function.   89     METHODS Animal The Michigan State University Institutional Animal Care and Use Committee (IACUC) approved all protocols. Male Sprague Dawley rats (225-465 g, Charles River Laboratories) were used in this study. DOCA-salt hypertensive and normotensive sham rats were also used. Methods for studies involving SNA recordings Anesthesia Sprague Dawley rats used for recordings of SNA were anesthetized with urethane (1 - 1.1 g/kg ip) and paralyzed with gallamine triethiodide (initial dose of 20 mg/kg iv; supplemental doses administered as needed to maintain paralysis) following induction with isoflurane. The trachea was cannulated for maintenance of artificial respiration using positive pressure ventilation (Harvard Apparatus Inspira ASV ventilator). Rectal temperature was maintained near 37°C with the aid of a heat lamp. Catheterization Arterial and venous catheters were introduced into the femoral artery and femoral vein. The arterial catheter was connected to a pressure transducer to monitor changes in blood pressure. Venous catheter(s) was used to delivered drug to the anesthetized rat. Nerve recording   90     Potentials were recorded monophasically from the cut central ends of the preganglionic or postganglionic sympathetic sphlanchic nerve placed on platinum bipolar electrodes. The capacity-coupled preamplifier bandpass was set at 1 – 1,000 Hz. Preganglionic or postganglionic sympathetic nerve activity (SNA) was then rectified and integrated (1-volt reset). This signal was then quantified in 1 or 10-min data blocks at baseline and during 5-HT infusion. Hexamethonium (10 mg/kg, iv) was administered at the end of each experiment to verify that recordings were from preganglionic or postganglionic branches of the splanchnic nerve. Methods for studies involving infusion of 5-HT in conscious animals Anesthesia All rats were anesthetized with isoflurane (2% in 100% O2) and ventilated mechanically. At the time of surgery, the incision site was treated with topical antibiotic containing analgesic to prevent irritation and infection. Incisions were closed with silk suture. Rats were treated with amoxicillin (150 mg/kg/i.m.) following surgery and 3 days thereafter. All rats were treated with rimadyl (5 mg/kg, s.c. for 2 days) for general analgesia. Blood pressure probe Radiotelemeters (DSI PhysioTel PA series transmitter model PA-C40) were implanted subcutaneously in the lower abdomen and catheters introduced into the left femoral artery. Pressure sensing tips were advanced into the thoracic aorta. All rats were given 7   91     days to recover prior to any measure. Mean arterial pressure (MAP) and heart rate (HR) were recorded at 10-minute intervals (10 second recording) for the duration of the study. Alzet osmotic pump A small incision was made at the base of the neck. Blunt dissection was used to create a small subcutaneous pocket between the scapulae. The pump (Alzet Osmotic Pump, Model 2ML1, Duret Corporation, Cupertino, CA, 10 µL/hr 7 days) was inserted and the skin sutured closed. To each pump, a 5-HT creatinine complex (25 µg/kg/min) and 1% ascorbic acid, antioxidant (0.02 g/pump) or vehicle (1% ascorbic acid, antioxidant) was loaded. The solution was dissolved in 1 mol/L HCl, and a pH-balance (~7) was achieved with 4 mol/L NaOH. Methods for electrical field stimulation DOCA-salt hypertension Under isoflurane anesthesia, Sprague-Dawley rats underwent left uninephrectomy, and a deoxycorticosterone acetate (DOCA) pellet was implanted subcutaneously (200 mg/kg). Sham rats also underwent left uninephrectomy, but were not implanted with a DOCA pellet. Rats were given standard rat chow ad libitum. DOCA-salt water was supplemented with 1% NaCl and 0.2% KCl for the duration of the study. Hypertension was established after 4 weeks of initiation of DOCA. Sham rats received tap water for the duration of the study. Sham systolic blood pressure was < 140 mmHg, while DOCAsalt systolic blood pressure was > 140 mmHg measured by tail cuff method.   92     Tissue preparation Rats were anesthetized with pentobarbital (60 mg/kg i.p.) and the superior mesenteric artery was removed and placed in physiological salt solution (PSS) containing (mM): NaCl 130; KCl 4.7; KH2PO4 1.8; MgSO4 * 7H2O 1.7; NaHCO3 14.8; dextrose 5.5; CaNa2EDTA 0.03, CaCl2 1.6 (pH 7.2). Sections of the artery were cut into rings for measurement of isometric contractile force, but the surrounding adventitial fat and connective tissue was not removed. Isolated tissue bath Two parallel stainless steel hooks were introduced into the lumen of the artery. One segment was fixed within the warmed (37°C), and aerated (95% O2, 5% CO2) tissue bath (30 mL). The other was connected to a Grass isometric force transducer (FT03; Grass instruments, Quincy, MA, USA), which was connected to an ADInstruments PowerLab (ADInstruments, Colorado Springs, CO). Tissues were placed under optimal resting tension (1200 mg; previously determined) and allowed to equilibrate for 1 hour before an -5 initial challenge with a maximal concentration of phenylephrine (10 M). After the initial PE challenge, tissues were washed until toned returned to baseline. Electrical field stimulation Superior mesenteric artery segments were mounted between two platinum electrodes (positioned within the tissue bath) connected to a Grass Instruments stimulator (S88; Quincy, MA) and electrical stimulus was delivered according to the following protocol: 30 stimuli, stimulus duration 0.5 ms, frequency 0.2 – 20 Hz, voltage 120 V). For each   93     stimulus, isometric contractile force was measured in an isolated tissue bath as outlined above. An initial 20 Hz maximal stimulus was delivered to each vascular segment. All subsequent data (means ± SEM) were expressed as a percentage of the initial 20 Hz maximal contraction. To validate the neural origin of the EFS-induced contractile response, the nerve impulse propagation blocker, tetrodotoxin (TTX; 300 nM) was added to the tissue bath after a brief wash out period following the initial 20 Hz stimulus. A second 20 Hz stimulus was delivered to validate contraction was abolished in the presence of TTX. In preliminary experiments, we validated this EFS-induced contraction was sympathetically mediated as the α1 adrenergic receptor antagonist prazosin (50 nM) abolished the 20 Hz-induced contraction. The following experimental conditions were used: 1) arterial segments alone, 2) segments incubated with the -6 serotonin transport reuptake inhibitor fluoxetine (10 M), 3) segments incubated with fluoxetine (50 nM) and the 5-HT2A/2C receptor antagonist ketanserin (50 nM; 15 minutes). EFS-induced contractile studies were conducted in the presence or absence of 5-HT (10 -8 -5 M), Vehicle (H2O), or CP 93129 (5-HT1B agonist; 10 M). Data analysis: For SNA data analysis, group differences were assessed by a two-way mixed design ANOVA and post-hoc testing at each time point was performed using Bonferroni’s procedure to correct for multiple comparisons (GraphPad Prism 5). In all cases, a p-value of <0.05 was considered significant. A paired t-test was used to compare the level of blood pressure changes during control periods versus at the nadir during 5-HT infusion. An unpaired students t-test was used to compare differences in maximal EFS   94     response(s) between groups. In all cases, p < 0.05 was considered significant. All results are presented as the means ± SEM.   95     RESULTS The effect of 5-HT (chronic and acutely administered) on blood pressure Normal male Sprague-Dawley rats were our initial model. In Figure 1A, we demonstrate the fall in blood pressure caused by 5-HT (25 ug/kg/min, Alzet pump) after one week in conscious rats. Blood pressure was measured through radiotelemetry. Figure 1B shows the effects of a slow administration of 5-HT (25 ug/kg/min, iv) over the course of 10 min to 1 hour in anesthetized male Sprague Dawley rats. In both experiments, 5-HT caused a fall in blood pressure. The similarity in qualitative and quantitative response allows for the statement that the mechanisms we observe in the acute situation may apply to that of the chronic situation, our ultimate concern. Having established this, we moved to two separate experimental protocols to investigate the ability of 5-HT to modify sympathetic nervous system tone. The effect of 5-HT on SNA in the splanchnic bed One mechanism by which 5-HT could reduce blood pressure is by removal of sympathetic tone at the pre- and/or post-ganglionic level. In urethane-anesthetized rats, we investigated both of these possibilities by recording from the splanchnic sympathetic nerve. Figures 2A and 3A show representative examples of the effect of 5-HT on preganglionic and postganglionic SNA, respectively. While 5-HT caused a robust fall in blood pressure in both experiments (top tracing in panel A), there was no change in integrated SNA (panel B of each figure). Removal of 5-HT infusion (figure 3A, right arrow) resulted in a rebound in blood pressure that, in some cases was accompanied by   96     an increase in SNA. In some experiments, the alpha adrenergic agonist phenylephrine (PE) was used to elicit a reflex-induced inhibition of. PE robustly inhibited preganglionic activity, suggesting that we would be able to detect a 5-HT-induced inhibition of SNA if it occurred. Effect of 5-HT on sympathetic synaptic transmission in mesenteric artery Another potential site of action for 5-HT to remove sympathetic tone from the vasculature is on the presynaptic terminal of the sympathetic nerve that innervates the mesenteric vasculature. In initial trials using a short incubation, 5-HT (5 minutes) did not inhibit neurogenic contraction following EFS frequency response curve (0.2 – 20Hz) vs. vehicle (H2O) (99 ± 11% at 20 Hz; 96 ± 16% at 20 Hz; respectively, reported as percentage of initial 20 Hz contraction) in the superior mesenteric artery of the Sprague Dawley rat (Figure 4A). 5-HT (5 minutes) did not inhibit neurogenic contraction in the -6 presence of fluoxetine (10 M; 5-HT reuptake inhibitor) vs. vehicle (H2O) (140 ± 26% at 20 Hz; 98 ± 6% at 20 Hz; respectively) in tissues from the Sprague-Dawley rat (Figure 4B). Fluoxetine was added to minimize uptake of 5-HT and thereby maximize the opportunity of 5-HT to interact with a presynaptic receptor. The addition of fluoxetine appeared to potentiate the neurogenic contractile response (140 ± 26% at 20 Hz, in the presence of fluoxetine vs. 99 ± 11% at 20 Hz, the absence of fluoxetine). However, this observation was not statistically significant. To block the effects of 5-HT on postsynaptic 5-HT2A receptors that are contractile, we next added the 5-HT2A/2C receptor antagonist ketanserin (50 nM) to the buffer. This   97     concentration of ketanserin is sufficient to block these 5-HT receptors but not the alpha adrenergic receptors that mediate EFS-induced contraction. 5-HT (10 -8 M) did not inhibit neurogenic contraction in the presence fluoxetine, ketanserin (50 nM; 5-HT2A/2C antagonist) and following a longer incubation with 5-HT (30 minutes) vs. vehicle (44 ± 11% at 10 Hz; 53 ± 7% at 10 Hz; respectively) in the artery from the Sprague-Dawley rat (Figure 5). We considered this protocol the ideal situation in which to observe 5-HTinduced inhibition of EFS-induced contraction. This was supported by a positive control, -5 use of the α2 agonist UK 14304 (10 M; 30 minutes) to virtually abolish neurogenic contraction vs. vehicle in the superior mesenteric artery of the Sprague-Dawley rat (4 ± 1% at 10 Hz; 50 ± 7% at 10 Hz; respectively; Figure 5). In an attempt to more selectively activate the 5-HT1B receptor, the lead candidate for presynaptic inhibition, we tested the 5-HT1B agonist CP 93129, and it did not inhibit neurogenic contraction vs. vehicle (42 ± 9% at 10 Hz vs 40 ± 4% at 10 Hz) in superior mesenteric artery of the Sprague-Dawley rat in the presence of fluoxetine, ketanserin, and following a 30 minute incubation period (Figure 6A). -6 Finally, we took this same protocol [fluoxetine, 10 M; 5-HT reuptake inhibitor; ketanserin 50 nM; 5-HT2A antagonist; 30-minute incubation period that showed robust inhibition with UK14304) and applied this to superior mesenteric arteries from sham and DOCA-salt hypertensive rats. Under these conditions, 5-HT did not inhibit neurogenic contraction in the superior mesenteric artery of the sham rat vs. vehicle (44 ± 11% at 10   98     Hz; 53 ± 7% at 10 Hz; respectively) or the DOCA-salt rat vs. vehicle (66 ± 7% at 10 Hz; 70 ± 4% at 10 Hz) (Figure 6B).   99     DISCUSSION This study took two different approaches to determining whether 5-HT caused the withdrawal of sympathetic tone, peripherally, from the splanchnic vasculature. Overall, our studies were negative and suggest that a reduction in SNA, at least to the splanchnic bed, does not contribute to the depressor response that accompanies a slow intravenous infusion of 5-HT. 5-HT and SNA We initiated this series of experiments because of previous findings. Diaz et al (2008) demonstrated that the blood pressure fall induced by hexamethonium, a ganglionic blocker, was significantly reduced in rats infused 5-HT vs those infused vehicle. This suggests, but does not prove, that 5-HT may be inhibiting or causing withdrawal of sympathetic tone, such that there is less to be reduced by hexamethonium. We appreciate an alternative conclusion, namely that the effect of hexamethonium may be smaller because blood pressure has already been reduced by 5-HT. Nonetheless, there was also sufficient literature supports that 5-HT may have effects on SNA and/or directly at the ganglion. In the rat, sympathetic preganglionic neurons in the supper thoracic spinal cord responded to 5-HT with largely excitation through what appeared to be a 5HT1-like receptor (Lewis and Coote, 1990). Hertzler (1961) demonstrated that 5-HT increased the amplitude of responses of the rat stellate ganglia to preganglionic stimulation, suggesting that 5-HT facilitates transmission, and this was reaffirmed by Pickering et al (1994). Sheng-Rong et al (1991) similarly demonstrated depolarizations of the guinea pig celiac and inferior mesenteric ganglion (1999). In guinea pig inferior   100     ganglion, 5-HT was shown to have both excitatory and inhibitory effects on sympathetic transmission (Meehan and Kreulen 1991), while in superior cervical ganglion 5-HT caused a depolarization that promoted transmission (Watkins and Newberry 1996). Jones et al showed 5-HT had a different effect in the cat, where 5-HT1D receptors mediate inhibition of sympathetic ganglionic transmission (1995). Unlike these multiple studies, we did not observe a change in either pre- or post-ganglionic splanchnic SNA during a slow infusion of 5-HT. These nerves were clearly sensitive to stimuli that change blood pressure (e.g. PE) and could invoke the appropriate physiological response. EFS-induced contraction in the superior mesenteric artery was also not reduced by 5-HT, and a number of conditions were tried so as to maximize the opportunity and chances of 5-HT being able to interact with a presynaptic receptor and indirectly cause relaxation through removal of sympathetic activity. While we readily observed robust inhibition of EFS-induced contraction by the α2 adrenergic agonist, UK14304, this was not observed, in any situation, with 5-HT. Multiple groups have demonstrated the ability of 5-HT, through multiple 5-HT receptors, to cause presynaptic inhibition of sympathetic nerves in blood vessels (Gothert et al., 1991; Kubo and Su, 1983; Molderings et al, 1990), and presynaptic inhibition was recently reviewed by Feuerstein (2008). Some of this early work was done in human saphenous veins, but Kubo and Su directly studied the mesentery. They indirectly suggested that 5-HT can inhibit presynaptic receptors because the ability of 5-HT to potentiate EFS-induced contraction was less than that of potentiating norepinephrine-induced contraction. Thus, it is unclear if 5-HT caused presynaptic inhibition of the sympathetic nerve innervating the mesentery.   101   The 5-   HT1B/1D receptor has been most heavily implicated in presynaptic inhibition, hence our use of CP93129. However, both 5-HT and CP93129 were unable to inhibit EFS-induced contraction in the superior mesenteric artery. Mechanism of 5-HT to decrease blood pressure? Other groups have demonstrated the acute and long lasting fall in BP in the rat administered 5-HT and/or a 5-HT-induced vasodilator response that one could envision participates in a fall in BP (Calama et al, 2003, 2005; Centurion et al, 2004, DeVries et al, 1999; Garcia et al, 2006; Hoffman et al., 1990; Terron, 1997; Terron et al, 2007). Our studies suggest that if 5-HT does inhibit the sympathetic nervous system, it is not at the level of the splanchnic circulation. We cannot rule out the possibility that this particular bed may be unimportant for the fall in BP caused by 5-HT, and that 5-HT at other sites may have a profound effect on BP. We have not, for example, taken a global measure of SNA such as norepinephrine overflow (Esler et al., 1989). Such a measure might also not be helpful if 5-HT is functioning discretely to influence a particular bed. What beds might those be? The cutaneous circulation is one. When administered to the rodent, 5HT causes a significant elevation in the temperature of the tail, indicative of heat shedding through vasodilation (Lin et al., 1983). Another is the skeletal muscle, which receives a significant amount (40%) of cardiac output and thus could account for large change in blood pressure. Alsip et al demonstrated the ability of 5-HT to dilate the arteriole of the rat skeletal muscle, supporting this possibility (1992).   102     Another possibility that is more controversial is that 5-HT moves into the central nervous system to reduce sympathetic tone. This is controversial because most state that 5-HT is unable to cross the blood brain barrier; this contrasts with its precursor, 5hydroxytryptophan, which freely moves across the blood brain barrier and, like 5-HT, can cause a fall in blood pressure (Baron et al., 1991; Fregly et al, 1987; Itskovitz et al., 1989). However, older studies support that 5-HT administration intravenously results in higher levels of 5-HT and its primarily metabolite, 5-hydroxyindole acetic acid (5-HIAA), in the brain (Bulat and Supek, 1968). Expression of the serotonin transporter on the capillary endothelial cells that comprise the barrier has been observed (Brust et al, 2000, Wakayama et al, 2002), making it possible for 5-HT to move in and out of the CNS, and Nakatani et al (2008) recently described the ability of 5-HT to pass from the CNS to the periphery. Westergaard demonstrated nearly 30 years ago that 5-HT itself can modify blood brain barrier function (1978). Activation of the nucleus tractus solitarious is described to inhibit sympathetic activity in rats (Comet et al., 2007), and this would be consistent with our observations. We recently published that the fall in blood pressure to chronically (one week) administered 5-HT was reduced by half in male rats that lack the serotonin transporter (SERT knock out or KO; Davis et al, 2011) compared to their wild type control (SERT WT). One explanation for this is that SERT, on the blood brain barrier, is important for 5-HT penetrating the CNS, and thus its genetic loss results in a reduced fall in blood pressure. There is an enormous amount of literature that goes against this idea, but it must be pursued.   103     In summary, this study suggests 5-HT is not acting to lower blood pressure through inhibition of NE release at the neuroeffector junction or through inhibition of pre- or postganglionic sympathetic neurons innervating the mesentery.   104     Figure 4.1 %&'()*+#,)-./*()# !"# MAP (mmHg) 150 0)123# 100 * 50 C on 5- H tro l T 0 $"# !+.45#,)-./*()# MAP (mmHg) 150 0)163# 100 * 50 C on 5- H tro l T 0 Figure 4.1: 5-HT-induced fall in mean arterial blood pressure of conscious animals receiving 5-HT chronically for one week (5-HT 25 ug/kg/min; A) or anesethetized animals receiving 5-HT (25 ug/kg/min) over the course of a 30-60 minute infusion (B). Bars represent mean arterial pressure (MAP) in normotensive Sprague Dawley rats before (control) and following 5-HT (reported at nadir of the MAP depressor response) for the   105     number of animals in parentheses. * statistically significant difference (p<0.05) vs. appropriate control.   106     107   60 50 40 30 m m m m m m m in in in in in in in 9&:#$"%:#;+78'+ !"#$%&$'+78'+ 3,!4+55+6$+ ,-+ 20 10 0 Integ Spl, % of Control   Figure 4.2 !"#$%&$'()&(*+ .-+ 150 /&012+ 100 50 0   Figure 4.2: Effect of acute 5-HT infusion on preganglionic sympathetic nerve activity. A: Tracing of mean arterial pressure (MAP), preganglionic sympathetic nerve activity (SNA), and integrated (Integ. Spl) preganglionic nerve activity following intravenous 5-HT infusion in the SD rat. Arrow marks the point at which 5-HT was delivered. B: Time course of changes in preganglionic sympathetic nerve activity (SNA) based on integrated records in the SD rat. Bars represent integrated preganglionic sympathetic nerve activity (means+SEM) at time(s) indicated following the start of intravenous 5-HT infusion for the number of animals in parentheses.   108     109   9 8 7 6 5 4 m m m m m m m m m m in in in in in in in in in in 9'$:%;&$:<+78(+ !"#$%&'%(+78(+ 3,!4+55+6%+ ,-+ 3 2 1 0 Integ Postgang Spl, % of Control   Figure 4.3 !"#$%&'%()"')*+ .-+ 150 /'012+ 100 50 0   Figure 4.3: Effect of acute 5-HT infusion on postganglionic sympathetic nerve activity. A. Tracing of mean arterial pressure (MAP), postganglionic splanchnic sympathetic nerve activity (SNA), and integrated (Integ. Spl) postganglionic splanchnic nerve activity following intravenous 5-HT infusion in the SD rat. Arrow marks the point at which 5-HT was delivered and when 5-HT infusion was stopped. B: Time course of changes in postganglionic sympathetic nerve activity (SNA) based on integrated records in the SD rat. Bars represent integrated postganglionic sympathetic nerve activity (means+SEM) at time(s) indicated following the start of intravenous 5-HT infusion for number of animals indicated in parentheses.   110     Frequency (Hz) 111   20 .0 .0 H H H H H H H z z z z z z z 20 .0 .0 5. 0 2. 0 1. 0 0. 5 0. 2 10 B. 10 5. 0 2. 0 1. 0 0. 5 0. 2 Percentage Contraction 20 Hz Test H H H H H H H z z z z z z z Percentage Contraction 20 Hz Test   Figure 4.4   A. 180 150 120 90 60 30 Veh (n=4) 5-HT (n=5) 0 Frequency (Hz) 180 150 120 90 60 30 Veh (n=4) 5-HT (n=5) 0   Figure 4.4: A. Vasoconstrictor response to EFS frequency response curves (0.2 Hz – 20 Hz) in superior mesenteric arteries from Sprague-Dawley rats incubated with either -8 vehicle (H2O) or 5-HT (10 M) for 5 min. B. Vasoconstrictor response to EFS frequency response curves (0.2 Hz – 20 Hz) in SMA (SD) incubated with either vehicle (H2O) or 5-8 HT (10 -6 M) for 5 min. in the presence of fluoxetine (10 M). Results (means ± SEM) are expressed as a percentage of the initial 20 Hz maximal contraction for number of animals in parentheses.   112     100 80 60 Vehicle (n=6) 5-HT (10-8 M) (n=5) Vehicle (n=4) UK 14304 (10-5 M) (n=4) 40 20 * 4 30 le 14 K U Ve h ic T H 5- ic le 0 Ve h Percentage Contraction 20 Hz test Figure 4.5 10 Hz test Figure 4.5: Vasoconstrictor response to 10 Hz stimulus in superior mesenteric arteries -8 from Sprague-Dawley rats incubated with either vehicle (H2O), 5-HT (10 -5 14304 (10 -6 M) in the presence of fluoxetine and (10 M), or UK M) and ketanserin (50 nM) for 30 min. Results (means ± SEM) are expressed as a percentage of the initial 20 Hz maximal contraction for number of animals in parentheses. * statistically significant difference (p < 0.05) vs. vehicle.   113     Figure 4.6 100 80 60 40 Vehicle (n=3) 20 CP 93129 (10-5 M) (n=5) le 93 12 9 0 C P Ve h ic Percentage Contraction 20 hz test A. 10 Hz test 100 80 60 Sham Vehicle (n=6) Sham 5-HT (10-8 M) (n=5) 40 DOCA Vehicle (n=4) DOCA 5-HT (10-8 M) (n=4) 20 T H le D O C A 5- ic Ve h A C O D Sh am Sh am ic 5H le T 0 Ve h Percentage Contraction 20 Hz test B. 10 Hz test   114     Figure 4.6: A. Vasoconstrictor response to 10 Hz stimulus in superior mesenteric arteries -5 from Sprague-Dawley rats incubated with either vehicle (H2O), or CP 93129 (10 -6 the presence of fluoxetine and (10 M) in M) and ketanserin (50 nM) for 30 min. Results (means ± SEM) are expressed as a percentage of the initial 20 Hz maximal contraction. B. Vasoconstrictor response to 10 Hz stimulus in superior mesenteric artery from either -8 sham or DOCA-salt rats incubated with either vehicle (H2O), or 5-HT (10 -6 presence of fluoxetine and (10 M) in the M) and ketanserin (50 nM) for 30 min. Results (means ± SEM) are expressed as a percentage of the initial 20 Hz maximal contraction for number of animals indicated in parentheses.                     115     Chapter 5 Lack of the serotonin transporter (SERT) reduces the ability of 5hydroxytryptamine to lower blood pressure. 1 2 1 Robert Patrick Davis , A. Elizabeth Linder , and Stephanie W. Watts 1 Department of Pharmacology and Toxicology, Michigan State University, East Lansing, MI, 48823 USA; 2 Department of Pharmacology, Universidade Federal de Santa Catarina, Florianopolis, SC, 88040-970, Brazil. Published in Naunyn-Schmiedeberg's Archives of Pharmacology: 383(5): 543. 2011   116     ABSTRACT Serotonin (5-hydroxytryptamine; 5-HT) is a potent constrictor of isolated blood vessels. However, recent studies demonstrate that chronic 5-HT infusion results in a prolonged fall in blood pressure in the rat. This finding highlights the need for further study of 5-HT in the cardiovascular system. We tested the hypothesis that a functional serotonin transporter (SERT) is critical to enabling a 5-HT-induced fall in blood pressure. Experiments were performed in male and female rats to determine whether gender significantly affected the ability of 5-HT to lower blood pressure and to determine whether SERT dependence was different in male vs. female rats. 5-HT (25 µg/kg/min; s.c.) was infused for 7 days to male and female, SERT wildtype (WT), and SERT knockout (KO) rats. Mean arterial pressure (MAP) and heart rate (HR) were monitored via radiotelemetry. 5-HT produced a significantly greater fall in MAP (at the nadir) in the male SERT WT rat (-20 ± 1 mmHg) compared to the male SERT KO rat (-10 ± 2 mmHg). Similarly, 5-HT also produced a significantly greater fall in MAP (at the nadir) in the female SERT WT rat (-19 ± 1 mmHg) compared to the female SERT KO rat (-15 ± 0.4 mmHg). While the lack of a functional SERT protein did not prevent a 5-HT-induced fall in blood pressure, it did reduce the ability of 5-HT to lower blood pressure in the male and female SERT rat, suggesting a potentially important role for SERT in producing a 5-HT-induced fall in blood pressure.   117     INTRODUCTION 5-hydroxytryptamine (5-HT) is a vasoactive amine synthesized in the enterochromaffin cells of the gastrointestinal tract and the raphe nucleus in the central nervous system (Erspamer and Asero 1952; Dahlstroem and Fuxe 1964). It is a potent constrictor of isolated blood vessels (Rapport et al. 1948) and its actions are terminated by the enzymatic activity of monoamine oxidase. Elevated levels of free plasma 5-HT are present in both human and experimental models of hypertension (Brenner et al. 2007; Fetkovska et al. 1990). Initially, this led investigators to postulate that 5-HT might be acting to constrict the peripheral vasculature, resulting in elevated blood pressure. However, chronic 5-HT infusion produced a prolonged fall in blood pressure in the DOCA-salt and sham rat (Diaz et al. 2008). This suggested elevated free plasma 5-HT might be acting to lower blood pressure in both human and animal models of hypertension. Thus, 5-HT might represent an adaptation to elevated pressure, rather than a contributing factor to essential hypertension. Interestingly, Diaz et. al. demonstrated that inhibition of nitric oxide synthase (NOS) prevented a 5-HT-induced fall in blood pressure, suggesting an important role for NOS and likely nitric oxide (NO) in eliciting a 5-HT-induced fall in blood pressure. However, the exact mechanism underlying a 5-HT-induced fall in blood pressure is not yet fully understood. Several studies implicate the importance of the serotonin transporter (SERT) as mechanistically important in mediating the effects of 5-HT. Chanrion et. al.   118     demonstrated 5-HT uptake via SERT enhanced neuronal nitric oxide synthase (nNOS) activity in cells coexpressing both SERT and nNOS (Chanrion et. al. 2007). This led Chanrion et. al. to speculate that 5-HT uptake via SERT leads to an allosteric change in nNOS, which increases its affinity for calmoudulin, resulting in activation. This suggests elevated levels of free 5-HT may be taken up via SERT leading to nNOS activation, NO production, and lower blood pressure. Moreover, reports also suggest that 5-HT can cross the blood brain barrier (BBB) via SERT (Nakatani et al. 2008). Thus, elevated levels of peripheral 5-HT might potentially cross the BBB and act centrally to activate nNOS via SERT uptake. Collectively, this evidence suggests that SERT may play an important role in eliciting a 5-HT-induced fall in blood pressure. We tested the hypothesis that SERT function and 5-HT uptake is essential to enabling a 5-HT-induced fall in blood pressure. To test this hypothesis, we utilized the SERT KO rat created by Edwin Cuppen [ethylnitrosurea-induced mutagenesis on a Wistar rat background] (Homberg et al. 2007). Previous studies validated the absence of a functional SERT and the presence of a truncated protein in the male SERT KO rat through immunohistochemical (IHC) staining and western blot analysis (Linder et al. 2008). Finally, reports suggest SERT may function differently in males vs. females (Maron et. al. 2010). Thus, we investigated chronic 5-HT infusion in male and female rats to determine whether gender significantly affected the ability of 5-HT to lower blood pressure.   119     METHODS Animal Use Male and female SERT knockout (SERT-KO), and Wistar-based wild type (WT) rats (Charles River) were used in these studies. The SERT-KO and WT rats were bred at ® Michigan State University under a breeding license obtained from genOway . All animal use procedures were performed in accordance with the Institutional Animal Care and Use Committee at Michigan State University. Experimental protocol Animals were anesthetized with isoflurane, and implanted with radiotelemetery devices (Data Sciences International, St. Paul, MN USA: PA-C40 introduced via the femoral artery). Rats were allowed 4 days to recover and 3 days of baseline measurements ® were made. An Alzet osmotic pump (Model 2ML1, Duret Corporation, Cupertino, CA; 10µL/hr 7 days) containing 5-HT (25 µg/kg/min) was inserted subcutaneously at the base of the neck. Mean arterial pressure (MAP) and heart rate (HR) were monitored for nd the duration of the study. In table 1, we report MAP and HR on the 2 control day as well as at the nadir. Hemodynamic measurements were sampled for 10 seconds every 10 minutes for the duration of the experiment. Data are reported as 24-hour averages. Blood was collected at the end of the study and fractionated into platelet poor plasma to validate elevated free plasma 5-HT levels chromatography.   120   using high performance liquid   Plasma 5-HT collection Blood collection and platelet poor plasma (PPP) preparation were conducted according to our previously published methods (Diaz et al. 2008). Briefly, five milliliters (ml) of blood was collected from left cardiac ventricle using a heparin coated syringe. The blood was transferred into an EDTA anticoagulant vacutainer tube. The tubes were spun at 160 x g for 30 minutes for platelet-rich-plasma (PRP). The supernatant containing plasma and buffy coat were pipetted into EDTA-coated plastic tubes. These tubes were centrifuged at 1350 x g for 20 minutes for platelet-poor-plasma (PPP). To the remaining pellet (platelet layer), 1 ml of platelet buffer and 1 µmol/L ADP was added. 10 µmol/L pargyline and 10 µmol/L ascorbic acid were added. The tubes were centrifuged at 730 x g for 10 minutes. Ten percent trichloroacetic acid (TCA) was added to deproteinize both sets of samples. All tubes were centrifuged at 4500 x g for 20 minutes. Finally, samples were ultracentrifuged at 280,000 x g for 2 hours. 5-HT concentrations were measured using HPLC at 0.4 V and 0.9 ml/min flow rate. Data analysis For in vivo data analysis, within-group differences were assessed by a one-way ANOVA with post hoc comparisons using Student Newman-Keuls test (GraphPad Prism 5). An unpaired Students t-test was performed to measure point-to-point differences. In all cases, a p value of < 0.05 was considered significant. All results are presented as means ± SEM.   121     RESULTS In the male rat, chronic 5-HT infusion significantly (p < 0.05) elevated free plasma 5-HT levels in the SERT WT rat (61 ± 16.2 ng/ml) compared to baseline measures (8.6 ± 1.3 ng/ml). Similarly, chronic 5-HT infusion also elevated free plasma 5-HT levels in the SERT KO rat (37.3 ± 24.2 ng/ml) compared to baseline measures (1.1 ± 0.5 ng/ml). At baseline, free plasma 5-HT levels were significantly higher in the SERT WT (8.6 ± 1.3 ng/ml) compared to the SERT KO rat (1.1 ± 0.5 ng/ml) rat (p < 0.05). In the female rat, chronic 5-HT infusion also significantly (p < 0.05) elevated free plasma 5-HT levels in the SERT WT (62 ± 8.4 ng/ml) compared to baseline measures (3.9 ± 0.6 ng/ml). Similarly, chronic 5-HT infusion also elevated free plasma 5-HT levels in the SERT KO rat (23 ± 7.3 ng/ml) compared to baseline (1.9 ± 1.0 ng/ml). At baseline, free plasma 5-HT levels were not significantly different in the SERT WT (3.9 ± 0.6 ng/ml) compared to the SERT KO (1.9 ± 1.0 ng/ml) rat. Table 1 summarizes changes in MAP and HR. MAP fell rapidly in all models reaching a nadir after 24-48 hours (Figure 1). HR was elevated (at the nadir) in all models following 5-HT infusion but not statistically different between groups. Both MAP and HR trended toward baseline (C2) throughout the remainder of the study. In the male rat, 5-HT produced a significantly greater fall in MAP in the SERT WT rat (20 ± 1 mmHg) compared to the SERT KO rat (-10 ± 2 mmHg) (Figure 1A). MAP remained significantly lower in the SERT WT rat compared to the SERT KO rat during   122     the 2 nd rd and 3 day of 5-HT infusion. MAP in the SERT WT also never returned to baseline (C2) at the end of the 7 day infusion whereas it returned to baseline in the SERT KO. In the female rat, 5-HT produced a significantly greater fall in MAP in the SERT WT (-19 ± 1 mmHg) compared to the SERT KO (-15 ± 0.4 mmHg) rat (Figure 1B). Baseline (C2) MAP was greater in the SERT WT rat (112 ± 1.3 mmHg) compared to the SERT KO rat (106 ± 2.0 mmHg). MAP in both the SERT WT and SERT KO returned to baseline (C2) by the end of the study. There was no significant difference in the fall in MAP between the male and female SERT WT or the male and female SERT KO rats.   123     DISCUSSION This study investigated whether a functional SERT protein was essential to enabling a 5-HT-induced fall in blood pressure. The absence of a functional SERT protein significantly reduced the magnitude of the 5-HT-induced fall in blood pressure in the SERT KO rat. This suggests an important role for 5-HT uptake in mediating a 5-HTinduced fall in blood pressure in the Wistar rat. The mechanisms by which SERT participates in a 5-HT-induced fall in blood pressure are numerous. However, the most compelling evidence for SERT involvement in a 5HT-induced fall in blood pressure is provided by Chanrion et. al. who suggests 5-HT uptake via SERT may activate nNOS and stimulate NO release. Interestingly, both arteries and veins also possess SERT and actively take up 5-HT (Ni et al. 2004; Linder et al. 2008). This suggests elevated free 5-HT may be taken up into the vasculature via SERT and lead to activation of endothelial nitric oxide synthase (eNOS), and release of NO. The present study also sought to determine whether gender affected the ability of 5-HT to lower blood pressure. No statistical differences in MAP were noted between the male SERT WT vs. the female SERT WT or the male SERT KO vs. the female SERT KO at the nadir. The absence of a functional SERT significantly reduced the ability of 5-HT to lower blood pressure in both male and female Wistar rats. Therefore we conclude gender does not significantly impact a 5-HT-induced fall in blood pressure. While the absence of a functional SERT significantly reduced the ability of 5-HT to lower   124     blood pressure, it did not completely abolish a 5-HT-induced fall in blood pressure. Therefore, this raises questions about whether 5-HT might be taken up by another transporter. Alternatively, another mechanism might exist, besides 5-HT uptake, which contributes to a 5-HT-induced fall in blood pressure. Further, one might expect free 5-HT levels to be higher in 5-HT infused SERT KO rats compared to 5-HT infused SERT WT rats due to the absence of a functional SERT to take up exogenously delivered 5-HT. However, following 5-HT infusion, we observed higher levels of free 5-HT in the SERT WT compared to the SERT KO rat. This raises questions about where exogenously delivered 5-HT might be deposited or whether 5HT may be rapidly excreted in the SERT KO rat compared to the SERT WT rat. Finally, we acknowledge that chronic 5-HT infusion will likely result in down regulation of SERT. Thus, we speculate that if 5-HT uptake via SERT is critical to enabling a 5-HTinduced fall in blood pressure, then down regulation of SERT may correlate with the observed trend in MAP (Figure 1) back towards baseline in 5-HT infused SERT WT and SERT KO rats. In summary, this study suggests an important role for 5-HT uptake in enabling a 5-HTinduced fall in blood pressure in the Wistar rat.   125     Table 1: MAP and HR changes in male and female SERT WT and SERT KO rats. Data are mean ± SEM. * P < 0.05 compared with SERT KO. Control BP (mmHg) HR (bpm) 106 ± 1 348 ± 6 101 ± 2 355 ± 7 113 ± 1 366 ± 3 106 ± 2 387 ± 4 Nadir BP (mmHg) HR (bpm) 85 ± 1 422 ± 6 90 ± 2 412 ± 7 93 ± 2 440 ±4 91 ± 2 456 ± 2 Change BP (mmHg) HR (bpm) 21 ± 1.0 * 74 ± 5 11 ± 2.0 57 ± 8 19 ± 1.0 * 75 ± 5 15 ± 0.4 69 ± 5 Model Male SERT WT rat (n=8) Male SERT KO rat (n=7) Female SERT WT rat (n=8) Female SERT KO rat (n=7) Male SERT WT rat (n=8) Male SERT KO rat (n=7) Female SERT WT rat (n=8) Female SERT KO rat (n=7) Male SERT WT rat (n=8) Male SERT KO rat (n=7) Female SERT WT rat (n=8) Female SERT KO rat (n=7)   126   Figure 1   Figure 5.1 Male 140 130 120 110 100 S3 S2 S1 3 C C C 2 70 S7 Male SERT WT (n = 8) Male SERT KO (n = 7) S6 * * S5 80 * † S4 90 1 Mean Arterial Pressure (mmHg) A Time (Day) B 140 130 120 # 110 100 90 80 Female SERT WT (n = 8) Female SERT KO (n = 7) Time (Day)   127   S7 S6 S5 S4 S3 S2 S1 3 C C C 2 70 1 Mean Arterial Pressure (mmHg) Female   Figure 5.1: A. Effect of chronic 5-HT infusion on mean arterial pressure (MAP) in male SERT WT and SERT KO rats. B. Effect of chronic 5-HT infusion on mean arterial pressure (MAP) in female SERT WT and SERT KO rats. Data points indicate 24-hour averaged MAP +/- SEM for the number of animals in parentheses. The vertical line denotes 5-HT or vehicle osmotic pump implant. Time is represented by days on the xaxis. C represents control recordings. S represents 5-HT infusion. significant * statistically (P < 0.05) difference compared with male SERT KO rat. † statistically significant (P < 0.05) difference compared to control (C2) in male SERT WT rat. # statistically significant (P < 0.05) difference compared to control (C2) in female SERT KO rat (C2).   128     DISCUSSION These studies investigated whether 5-HT lowers blood pressure, in a nitric-oxide dependent manner, by reducing total peripheral resistance, either through a reduction in sympathetic nervous system tone and/or direct stimulation of vascular 5-HT receptors. While the results of these studies were largely negative, significant progress has been made toward elucidating the mechanism underlying a 5-HT-induced fall in blood pressure. Several important findings revealed that: 1. 5-HT lowers blood pressure by reducing peripheral vascular resistance, 2. 5-HT is capable of lowering blood pressure over the course of one-month, 3. 5-HT is not acting in the mesenteric vasculature to lower blood pressure (either via direct receptor mediated dilation or by reducing sympathetic nervous system tone), and 4. SERT plays an important role in enabling a 5HT-induced fall in blood pressure. Despite these important findings, significant questions remain regarding the mechanism underlying a 5-HT-induced fall in blood pressure. 5-HT reduces peripheral vascular resistance The first major finding of this study was that 5-HT significantly reduces peripheral vascular resistance in the Sprague Dawley rat. This finding resolved the question of exactly how 5-HT is acting to lower blood pressure given that changes in blood pressure are governed by either changes in vascular resistance or changes in cardiac output. However, this study did not reveal exactly where 5-HT is acting to reduce peripheral vascular resistance? Our data strongly suggests against changes in the mesenteric   129     vasculature. Thus, understanding and elucidating the exact mechanism of 5-HT-induced hypotension will depend upon identifying exactly where 5-HT is acting. With this in mind, we can speculate about which vascular bed 5-HT may be acting on based indirectly on the distribution of cardiac output in the rat. We hypothesize that changes in vascular resistance in those beds receiving the greatest portion of cardiac output are most likely to exert significant changes on mean arterial pressure following 5-HT infusion. Thus, skeletal muscle, which receives 25-35% of total cardiac output, (depending on conditions) is a reasonable vascular bed to consider when investigating where 5-HT is acting to reduce peripheral resistance and lower blood pressure (Delp et. al. 1991). This contrasts with the mesenteric vasculature, and gastrointestinal tract, which receives approximately 14% of cardiac output and was extensively studied throughout this series of experiments (Delp et. al. 1991). Therefore, it may be reasonable for us to postulate that the changes in mean arterial pressure following 5-HT infusion are due to changes in vascular resistance in skeletal muscle due to receipt of a greater portion of cardiac output (thus potentially being capable of a greater change in blood pressure). However, evidence in the literature may suggest against this particular hypothesis. Investigators injected 5-HT (0.1 µmol/L and 1000 µmol/L) into the masseter muscle of 12 healthy patients and measured changes in blood flow using laser Doppler technique (Emberg et. al. 2006). These investigators revealed that 5-HT had no effect on local blood flow at either concentration in the microcirculation of the masseter   130     muscle. The results of this study suggest that 5-HT is not capable of reducing vascular resistance in skeletal muscle, as evidenced by the fact that there was no change in local blood flow. An increase in blood flow in the masseter muscle would have been consistent with the hypothesis that 5-HT is reducing vascular resistance in skeletal muscle. Moreover, investigators also demonstrated that treatment with a mixed 5HT1B/5-HT2A receptor antagonist improved perfusion in the lower limb following arterial occlusion and ischemia in the obese Zucker rat (Janiak et. al. 2002). This study suggests that 5-HT is acting as a constrictor in the collateral vasculature of the lower limb and that receptor blockade improves perfusion in these vessels following arterial occlusion. Thus, this study suggests against 5-HT acting to dilate skeletal muscle vasculature or collateral blood vessels and raises the question about whether another vascular bed may mediate a 5-HT-induced fall in blood pressure. The skin or cutaneous vasculature receives approximately 6% of cardiac output and thus may be another potential place 5-HT is acting to reduce peripheral resistance and lower blood pressure. Support for this hypothesis is provided by the clinical carcinoid syndrome, characterized by increased release of 5-HT, arising from a carcinoid tumor (Jackson et. al. 2009). Skin flushing, tachycardia, and changes in blood pressure are symptoms associated with carcinoid syndrome. Skin flushing can be interpreted as an increase in skin blood flow and therefore it is not unreasonable to hypothesize given the clinical symptoms of the carcinoid syndrome that 5-HT may act at the level of the cutaneous vascular to dilate these vessels causing a reduction in vascular resistance and a reduction in blood pressure.   131     However, inconsistent with this hypothesis is the observation of marked cyanosis in the lower limb of those rats receiving 5-HT (lower limb instrumented with radiotelemeter). This observation suggests 5-HT infusion decreases perfusion of the lower limb either through direct constriction of the peripheral/cutaneous vasculature or potentially hypo-perfusion of the lower limb due to lower mean arterial pressure. If the cutaneous vasculature were dilated then there would be an increase in perfusion and the skin surface (paw) would appear pink. The literature also suggests that changes in human cutaneous vascular tone are capable of altering blood pressure and that these changes are linked to neuronal nitric oxide synthase (nNOS) (Kellogg et. al. 2008). This suggests that 5-HT may be capable of reducing cutaneous vascular resistance through an nNOS mediated action(s). This hypothesis would be consistent with the finding by Diaz et. al. that a 5-HT-induced fall in blood pressure is dependent on nitric oxide synthase (NOS) (Diaz et. al. 2008). Collectively, both the skeletal muscle vasculature and the cutaneous vasculature are reasonable and appropriate choices to pursue further investigation toward understanding the exact mechanism underlying a 5-HT-induced fall in blood pressure. 5-HT is capable of lowering blood pressure over the course of one-month The second major finding of this study was that 5-HT was capable of reducing blood pressure in the DOCA-salt rat over the course of one-month but was unable to prevent the development of DOCA-salt hypertension. The original study that led to the discovery that 5-HT was capable of causing a pronounced fall in blood pressure (Diaz   132     et. al. 2008) was designed to address the actions of 5-HT on blood pressure, given the clinical observation that hypertensive patients presented with elevated levels of free plasma 5-HT compared to their normotensive counterparts. However, as has been previously discussed, the original study was a relatively short clinical exposure given that the human patients are likely exposed to elevated levels of free 5-HT for many months to years (rather than days). The finding that 5-HT is able to maintain lower blood pressures in the DOCA-salt rat over the course of one-month significantly strengthens the case that elevated levels of free plasma 5-HT are likely acting to lower blood pressure rather then increase or contribute to essential hypertension in human patients. Despite a significant advance in our understanding of what 5-HT is capable of in the cardiovascular system, a longer 5-HT infusion and/or varied 5-HT infusions would result in better understanding and the design of a more clinically relevant study. We ® were fortunate to work with the iPrecio micro infusion pump, a programmable infusion pump that allowed for a longer infusion protocol (moving beyond the previously studied ® one-month infusion via the Alzet osmotic infusion pump) and variable administration of 5-HT (start, stop, and change the dose of 5-HT delivered over the designated infusion period). We were able to establish that 5-HT is capable of repeated falls in blood ® pressure in the sham and DOCA-salt rats when the iPrecio micro infusion pump(s) were paired with animals instrumented with radiotelemetry devices. Moreover, we also undertook a dose response study, which demonstrated that 5-HT is capable of reducing   133     blood pressure in the Sprague Dawley rat at lower doses then had been previous delivered. ® In the future, the iPrecio micro infusion pump could be employed to study the long-term (> one-month) effects of 5-HT on blood pressure. This device could be employed to study a 3-month infusion of 5-HT in the normotensive and hypertensive rat (when infused continuously at 3.0 µL/hr). Thus, this device will be important in future efforts to elucidate the exact mechanism underlying a 5-HT-induced fall in blood pressure. If 5-HT is not acting at the mesenteric vasculature then where? The third major finding of this study was that 5-HT is not likely acting in the mesenteric vasculature (either through direct receptor mediated vascular relaxation or through inhibition of sympathetic nervous system tone) to lower blood pressure. Previous reports in the literature contradict our findings and suggest that direct vascular relaxation of an isolated mesenteric preparation is possible in the presence of ketanserin (Mclennan and Taylor, 1984). Thus, we are forced to ask the question about what differs between our investigation and the studies conducted by Mclennan and Taylor? One possibility is that we investigated the effects of 5-HT in a helical strip (mounted in an isolated tissue bath) as opposed to the perfused mesenteric vascular bed. The act of cutting the vessel into a helical strip may have disrupted the architecture of the superior mesenteric artery and resulted in a vessel that was unable to relax under the conditions we set forth. In the future, we may need to investigate an alternate   134     technique (rather then the helical strip) to investigate whether 5-HT is acting through direct receptor mediated relaxation to lower blood pressure. Unfortunately, our present data does not allow us to determine the specific mechanism underlying a 5-HT-induced fall in blood pressure, nor does it provide us with significant insight to determine whether 5-HT is acting through a direct receptor mediated vascular relaxation or through sympathoinhibition. Thus, the question regarding how 5-HT is acting to reduce peripheral vascular resistance remains, as does the question regarding the exact vascular bed in which 5-HT is acting to reduce peripheral vascular resistance and blood pressure (this possibility has been discussed above). While significant effort has been dedicated throughout this series of experiments to the possibility that 5-HT may have been acting in the periphery to mediate a 5-HTinduced fall in blood pressure, the possibility that 5-HT may be able to cross the blood brain barrier (BBB) and act centrally to mediate a 5-HT-induced fall in blood pressure has largely been ignored. In our laboratory, the crucial experiment that has not yet been undertaken to determine whether 5-HT (delivered subcutaneously) is capable of crossing the blood brain barrier involves radiolabeling exogenously delivered 5-HT to determine whether it is capable traversing the blood brain barrier. Despite the absence of this critical experiment, investigators demonstrated that intravenous delivery of 5-HT is capable of elevating 5-HT and 5-HIAA levels within the CNS (Bulat and Supek, 1968). Further, the presence of SERT on the endothelial cells   135     lining the blood brain barrier suggests 5-HT is capable of entering the CNS through a direct transport mediated carrier mechanism (Brust et. al. 2000) (Wakaymam et. al. 2008). Collectively, these studies suggest that a centrally mediated 5-HT-induced fall in blood pressure is possible and should be explored. However, we also discovered that 5-HT does not inhibit preganglionic splanchnic sympathetic nerve activity, and this result suggests against a centrally mediated effect given that changes in centrally generated nerve activity are reflected in changes in preganglionic nerve activity. This does not rule out the possibility that 5-HT acts centrally, but without a definite vascular bed to target, identifying another preganglionic nerve to record from will unlikely yield positive results. Thus, it will be crucial to identify whether 5-HT is capable of gaining access to the CNS and which vascular bed 5-HT is targeting to mediate a 5-HT-induced fall in blood pressure. Then important work can begin on elucidating the exact mechanism underlying a 5-HT-induced fall in blood pressure. SERT plays an important role in a 5-HT-induced fall in blood pressure The final major finding of this study was that SERT plays an important role in enabling a 5-HT-induced fall in blood pressure. These results suggested that 5-HT uptake is an important component to enabling a 5-HT-induced fall in blood pressure. However, the absence of SERT (SERT KO rats) did not completely abolish a 5-HTinduced fall in blood pressure and raises the question if 5-HT uptake is crucial to the mechanism underlying a 5-HT-induced fall in blood pressure, how is 5-HT taken up in   136     the absence of SERT? One possibility is that 5-HT is taken up by another monoamine transporter: the dopamine transporter (DAT) and/or the norepinephrine transporter (NET) (Zhou et. al. 2002; Suarez-Roca and Cubeddu, 2002). Uptake via either of these transporters would allow a 5-HT-induced fall in blood pressure in the absence of SERT. The importance of SERT in enabling a 5-HT-induced fall in blood pressure, also indirectly supports the hypothesis that elevated levels of free plasma 5-HT are antihypertensive. Our laboratory has previously demonstrated that SERT is dysfunctional in the aorta of the DOCA-salt and L-NNA hypertensive rat (Ni et. al. 2006). Thus, impaired 5-HT uptake via a dysfunctional SERT protein may contribute to elevated blood pressure in these models of hypertension given that 5-HT uptake via SERT lowers blood pressure. However, this study only investigated SERT function in the aorta, and thus we have not yet determined SERT dysfunction globally. Therefore, if SERT enables transport of 5-HT across the blood brain barrier, we cannot state that SERT is impaired in the DOCA-salt and/or L-NNA hypertensive rat without further study of SERT function across the blood brain barrier. The fact that 5-HT infusion lowered blood pressure in the DOCA-salt rat (when delivered over 7 days) (Diaz et. al. 2008) suggests that 5-HT is able to lower blood pressure despite a dysfunctional SERT protein. Thus, it may be possible that excess levels are taken up by alternative monoamine transporters (NET and/or DAT) enabling a nitric oxide-mediated fall in total peripheral resistance and a significant fall in blood pressure.   137     However, in the L-NNA hypertensive rat, 5-HT did not lower blood pressure. Thus, while a dysfunctional SERT protein did not prevent a 5-HT-induced fall in blood pressure in the DOCA-salt rat, these results suggest that in the absence of nitric oxide synthase (NOS) and NO production that a 5-HT-induced fall in blood pressure is not possible with or without a functional SERT. Collectively these data suggest and important association between SERT and NOS. 5-HT and nitric oxide (NO) Our laboratory has previously demonstrated an absolute dependence on NOS and likely NO production in enabling a 5-HT-induced fall in blood pressure (Diaz et. al. 2008). Additionally, we also demonstrated that SERT is an important component in enabling a 5-HT-induced fall in blood pressure. Thus, the hypothesis put forth regarding SERT and nNOS will need to be explored further given the direct connection between 5HT uptake via SERT and nNOS activation (Chanrion et. al. 2007). This hypothesis suggests that 5-HT uptake via SERT leads to an allosteric change in nNOS, increasing its affinity for calmoudulin, resulting in its activation, and likely production of NO. This would likely result in a reduction in vascular resistance, and the observed 5-HT-induced fall in blood pressure. However, another possibility exists. Investigators demonstrated that pretreatment with L-NAME (10 mg/kg) completely abolished a 5-HT1A receptor mediated prejunctional inhibition of an electrically stimulated pressor response in the diabetic   138     pithed rat (Garcia et. al. 2006). This study suggests an important association between 5-HT and nitric oxide (NO) in mediating a prejunctional inhibitory effect in the diabetic pithed rat. Thus, it may still be possible that 5-HT is acting through a prejunctional inhibitory mechanism and that this inhibition is dependent on NO synthesis, despite the results obtained in the superior mesenteric artery following EFS. This study raises questions about what differs between our study (which concluded 5-HT does not inhibit a prejunctional pressor response in the superior mesenteric artery) vs. the study conducted by Garcia et. al. One possibility is that the technique of pithing and/or stimulation of the entire sympathetic outflow allowed these investigators to uncover a 5-HT mediated prejunctional inhibition, which we were unable to see in our isolated superior mesenteric artery preparation. Alternatively, these studies utilized a diabetic rat model. Thus, the neurovascular complications of diabetes may have contributed to alternate findings compared to our study. Free plasma 5-HT levels Lastly, a table has been constructed comparing free plasma 5-HT levels in various animal models infused with 5-HT (Table 1). In the original 7-day study, free plasma 5-HT in the sham rat was greater following 5-HT infusion, compared to plasma 5-HT in the DOCA-salt following vehicle infusion (Diaz et. al. 2008). This comparison strongly suggests against the original hypothesis that elevated levels of free 5-HT are acting to elevate blood pressure as elevated levels in the sham rat did not raise blood pressure but rather lowered blood pressure. Moreover, this allows us to hypothesize   139     that elevated levels of free plasma 5-HT may serve as a brake to increasing blood pressure rather than a driver of elevated pressures.   140     Table 2: Free plasma 5-HT levels Model DOCA-salt rat (one month infusion in established rats) DOCA-salt rat (one month infusion prior to initiating DOCA-salt hypertension) 141   177.1 ± 10.9 5-HT (ng/ml) 61 ± 16.2 37 ± 24.2 62 ± 8.4 23 ± 7.3 Veh (ng/ml) 3 ± 0.3 25 ± 5.1 Sham (7 days infusion) DOCA-salt (7 day infusion in established rats) 13.3 ± 3.9 Baseline (ng/ml) 8 ± 1.3 1 ± 0.5 4 ± 0.6 2 ± 1.0 Male SERT WT rat (7 day infusion) Male SERT KO rat (7 day infusion) Female SERT WT rat (7 day infusion) Female SERT KO rat (7 day infusion)   Platelet Poor Plasma (PPP) Veh (ng/ml) 5-HT (ng/ml) 87.9 ± 43.9 282.9 ± 48.7 5-HT (ng/ml) 47 ± 23.2 138 ± 35.4   Conclusions The present studies provide us with several important findings that will aide in unraveling the exact mechanism of a 5-HT-induced fall in blood pressure. These studies demonstrated 5-HT is capable of reducing peripheral vascular resistance (TPR). In the future, it will be critical to identify the exact vascular bed in which resistance falls. This will enable future studies to determine whether 5-HT is acting through direct receptor mediated vascular relaxation or through a sympathoinhibitory mechanism (Figure 1). Additionally, this work defined an important role for SERT in enabling a 5-HT-induced fall in blood pressure and thus understanding how SERT and NOS interact will be another critical component to understanding the exact mechanism of a 5-HT-induced fall in blood pressure. Figure 6.1 ,/,%012030451%!"#$ /6%7+80)(+8% ')(29):%% !"#$% &'()*+%!"#$,-.$% $>.% ?>% ;)<580=)451%!"#$/6% 7+80)(+8%')(29):%% Figure 6.1: Schematic diagram of current hypothesis. (TPR) represents total peripheral resistance. (SNS) represents sympathetic nervous system. (BP) represents blood pressure   142     REFERENCES   143     REFERENCES Abe C, Tashiro T, Tanaka K, Ogihara R, Morita H. A novel type of implantable and programmable infusion pump for small laboratory animals. J Pharmacol Toxicol Methods. 59(1): 7-12. 2009. Alsip NL, Harris PD. Serotonin-induced dilatation of small arterioles is not mediated via endothelium-derived relaxing factor in skeletal muscle. Eur J Pharmacol 229: 117-124. 1992. Amsterdam JD, Garcia-Espana F, Fawcett J, Quitkin FM, Reimherr FW, Rosenbaum JF, Beasley C. Blood pressure changes during short-term fluoxetine treatment. J Clin Psychopharm. 19: 9-14. 1999. Barnes NM, Sharp T. A review of central 5-HT receptors and their function. Neuropharmacol 38: 1083-1152, 1999. Banes AK, Watts SW. Enhanced contraction to 5-hydroxytryptamine is not due to “unmasking” of 5-hydroxytrptamine(1b) receptors in the mesenteric artery of the deoxycorticosterone acetate-salt rat. Hypertension. 38(4): 891-895. 2001. Banes AK, Watts SW. Arterial expression of 5-HT2B and 5-HT1B receptors during development of DOCA-salt hypertension. BMC Pharmacol. 3: 1-15. 2003. Baron A, Riesselmann A, Fregly MJ. Reduction in the elevated blood pressure of Dahl Salt sensitive rats treated chronically with L-5-hydroxytryptophan. Pharmacology 42: 15-22. 1991. Beilin LJ, Ziakas G. Vascular Reactivity in Post-Deoxycorticosterone Hypertension in Rats and its Relation to ‘Irreversible’ Hypertension in Man. Clinical Science. 42: 579590. 1972. Berger M, Gray JA, Roth BL. The expanded biology of serotonin. Ann Rev Med 60:355366, 2009. Bodelsson M, Tornebrandt K, Bertilsson IL, Arneklo-Nobin B. Heterogeneity of contractile 5-HT receptors in human hand veins. Eur J Pharmacol. 219 (3): 455-460. 1992. Borgdorff P, Fekkes D, Tangelder GJ. Hypotension caused by extracorpeal circulation: serotonin from pump-activated platelets triggers nitric oxide release. Circulation 106: 2588-2593. 2002. Brenner B, Harney JT, Ahmed BA, Jeffus BC, Unal R, Mehta JL, Kilic F. Plasma serotonin levels and the platelet serotonin transporter. J Neurochem 102: 206-215, 2007.   144     Brust P, Friederich A, Krizbai IA, Bergmann R, Roux F, Ganapathy V, Johannsen B. Functional expression of the serotonin transporter in immortalized rat brain microvessel endothelial cells. J Neurochem 74: 1241-1248. 2000. Bulat M, Supek Z. Passage of 5-hydroxytryptamine through the blood brain barrier, its metabolism in the brain and elimination of 5-hydroxyindoleacetic acid from the brain tissue. J Neurochem 15: 383-389. 1968. Calama F, Garcia M, Jarque MJ, Moran A, Martin ML, San Roman L. 5hydroxytryptamine-induced vasodilator responses in the hindquarters of the anesthetized rat, involve beta2-adrenoreceptors. J Pharm Pharmacol 55(10): 1371-1378. 2003. Calama E, Ortiz de Urbina AV, Moran A, Martin ML, San Roman L. Effect of 5hydroxytryptamine on neurogenic vasoconstriction in the isolated, autoperfused hindquarters of the rat. Clin Exp Pharmacol Physiol 32: 894-900. 2005. Campese VM, Ku E, Park J. Sympathetic renal innervation and resistant hypertension. Int J Hypertens. 2011: 814354. 2010. Centurion D, Glusa E, Sanchez-Lopez A, Valdivia LF, Saxena PR, Villalon CM. 5-HT7, but not 5-HT2B, receptors mediate hypotension in vagosympathetctomized rats. Eur J Pharmacol. 502: 239-242. 2004. Chanrion B, Mannoury la Cour C, Bertaso F, Lerner-Natoli M, Freissmuth M, Millan M J, Brockaert J, Martin P. Physical interaction between the serotonin transporter and neuronal nitric oxide synthase underlies reciprocal modulation of their activity. PNAS 104: 8119-8124. 2007. Chobanian AV, Barkis GL, Black HR, Cushman WC, GreenLA, Izzo JL, Jones DW, Materson BJ, Oparil S, Wright JT, Roccella EJ. The Seventh Report of the Joint National Committee on Prevention, Detection, Evaluation, and Treatment of High Blood Pressure. Hypertension 42: 1206-1252. 2003. Cordellini S, Nigro D, Carvalho MH, Fortes ZB, Scivoletto R. Reactivity of macro- and microvessels of DOCA-salt hypertensive rats: role of the endothelial cell. Braz J Med Biol Res. 21(4): 845-9. 1988. Cortijo J, Martí-Cabrera M, Bernabeu E, Domènech T, Bou J, Fernández AG, Beleta J, Palacios JM, Morcillo EJ. Characterization of 5-HT receptors on human pulmonary artery and vein: functional and binding studies. Br J Pharmacol. 122: 1455-1463. 1997. Crofton JT, Share L, Shade RE, Lee-Kwon WJ, Manning M, Sawyer WH. The importance of vasopressin in the development and maintenance of DOCA-salt hypertension in the rat. Hypertension. 1: 31-38. 1979.   145     Dahlstroem A, Fuxe K. Evidence for the existence of monoamine-containing neurons in the central nervous system. 1. Demonstration of monoamines in the cell bodies of brain stem neurons. Acta Physiol Scand. 232(Suppl): 1–55. 1964. Das YT, Bagchi M, Bagchi D, Preuss HG. Safety of 5-hydroxy-L-tryptophan. Tox. Lett. 150: 111-122. 2004. Davis RP, Watts SW. Lack of the serotonin transporter (SERT) reduces the ability of 5hydroxytryptamine to lower blood pressure. Naunyn-Schmiedeberg’s Archiv Pharmacol 383: 543. 2001. De Vries P, De Visser PA, Heiligers JPC, Villalón CM, Saxena PR. Changes in systemic and regional haemodynamics during 5-HT7 receptor mediated depressor responses in rats. Naunyn-Schmiedeberg’s Archiv Pharmacol 359: 331-338. 1999. Delp MD, Manning RO, Bruckner JV, Armstrong RB. Distribution of cardiac output during diurnal changes of activity in rats, Am J Physiol. 261: H1487-1493. 1991. Diaz J, Ni W, Thompson J, King A, Fink G, Watts SW. 5-hydroxytryptamine lowers blood pressure in normotensive and hypertensive rats. J Pharmacol Exp Ther. 325: 1031-1038. 2008. Dodson AM, Anderson GM, Rhoden KJ. Serotonin uptake and metabolism by cultured guinea pig airway smooth muscle cells. Pulm Pharmacol Ther. 17: 19-25. 2004. Doggrell SA. The role of 5-HT on the cardiovascular and renal systems and the clinical potential of 5-HT modulation. Expert Opin Investig Drugs 12: 805-823. 2003. Emberg M, Hedenberg-Magnusson B, Kurita H, Kopp S. Effects of local serotonin administration on pain and microcirculation in the human masseter muscle. J Orofac Pain. 20(3): 241-248. 2006. Esler M, Jennings G, Lamberg G. Noraderenaline release and the pathophysiology of primary human hypertension. Am J Hypertens 2: 140S-146S. 1989. Erspamer V, Asero B. Identification of enteramine, the specific hormone of the enterochromaffin cell system, as 5-hydroxytryptamine. Nature. 169: 800-801. 1952. Fetkovska N, Pletscher A, Ferracin F, Amstein R, Buhler FR. Impaired uptake of 5hydroxytryptamine in platelet in essential hypertension: clinical relevance. Cardiovasc Drugs Ther. 4: 105-109. 1990 Feuerstein TJ. Presynaptic receptors for dopamine, histamine and serotonin. Handbook Exper Pharmacol 184: 289-338. 2008   146     Figueras J, Domingo E, Cortadellas J, Padilla F, Dorado D, Segura R, Garland R, Soler J Comparison of plasma serotonin levels in patients with variant angina pectoris versus healed myocardial infarction. Am J Cardiol. 96: 204-207. 2005. Fregly MJ, Lockley OE, Summers C. Chronic treatment with L-5-hydroxytryptophan prevents the development of DOCA-salt-induced hypertension in rats. J Hypertens. 5(5): 621-8. 1987. Garcia M, Moran A, Martin ML, Barthelmebs M, San Roma L. The nitric oxide synthesis/pathway mediates the inhibitory serotoninergic response of the pressor effect elicited by sympathetic stimulation in diabetic pithed rats. Eur J Pharmacol 537: 126143. 2006. Gershon MD, Tack J. The serotonin signaling system: from basic understanding to drug development for functional GI disorders. Gastroenterology. 132: 397-414. 2007. Gordon JR, Barnes NM. Lymphocytes transport serotonin and dopamine: agony or ecstasy. Trends Immunol. 24: 438-443. 2003. Göthert M, Molderings GJ, Fink K, Schlicker E. Heterogeneity of presynaptic serotonin receptors on sympathetic neurones in blood vessels. Blood Vessels. 28(1-3): 11-8. 1991. Gul H, Yildiz O, Simsek A, Balkan, Ersoz N, Cetiner S, Isimer A, Sen D. Pharmacologic Characterization of Contractile Serotonergic Receptors in Human Isolated Mesenteric Artery. J Cardiovasc Pharmacol. 41 (2): 307-315. 2003. Hertzler EC. 5-hydroxytryptamine and transmission in sympathetic ganglia. Pharmacol 17: 406-413. 1961. Br J Hoffman P, Carlsson S, Skarphedinsson JO, Thoren P. Role of different serotonergic receptors in the long-lasting blood pressure depression following muscle stimulation in the spontaneously hypertensive rat. Acta Physiol Scand 139: 305-310, 1990. Homberg JR, Olivier JDA, Smits BMG, Mul JD, Mudde J, Verheul, Nieuwenhuizen OFM, Cools AR, Ronken E, Cremers T, Schoffelmeer ANM, Ellenbroek BA, Cuppen E. Characterization of the serotonin transporter knockout rat: A selective change in the functioning of the serotonergic system. Neuroscience. 146: 1662-1676. 2007. Ishida T, Kawashima S, Hirata K, Yokoyama M. Nitric Oxide is produced via 5-HT1B and 5-HT2B receptor activation in human coronary artery endothelial cells. Kobe J Med Sci. 2: 51-63. 1998. Itskovitz HD, Werber JL, Sheridan AM, Brewer TF, Stier CT. 5-hydroxytrptophan and carbidopa in spontaneously hypertensive rats. J Hypertens 7: 311-315. 1989.   147     Jackson LN, Chen LA, Larson SD, Silva SR, Rychahou PG, Boor PJ. Li J, Defreitas G, Stafford WL, Townsend CM Jr, Evers BM. Development and characterization of a novel in vivo model of carcinoid syndrome. Clin Cancer Res. 15(8): 2747-2755. 2009. Janiak P, Lainee P, Grataloup Y, Luyt CE, Bidourd JP, Michel JB, O’Connor SE, Herbert JM. Serotonin receptor blockade improves distal perfusion after lower limb ischemia in the fatty Zucker rat. Cardiovasc Res. 56(2): 293-302. 2002. Jones JFX, Martin GR, Ramage AG. Evidence that 5-HT1D receptors mediate inhibition of sympathetic ganglionic transmission in anaesthetized cats. Br J Pharmacol 116:1715-1717, 1995. Kaumann AJ, Levy FO. 5-Hydroxytryptamine receptors in the human cardiovascular system. Pharmacol Ther 111: 674-706, 2006. Keers R, Aitchison KJ. Gender differences in antidepressant drug response. Int Rev Pyschiatry. 22(5): 485-500. 2010. Kellogg DL, Zhao JL, Wu Y. Neuronal nitric oxide synthase control mechanisms in the cutaneous vasculature of humans in vivo. J Physiol. 586(3): 847-857. 2008. Kubo T, Su C. Effect of serotonin and some other neurohumoral agents on adrenergic neurotransmission in spontaneously hypertensive rat vasculature. Clin Exp Hypertens A. 5: 1501-1510, 1983. Lariviere R, Day R, Schriffrin EL. Increased expression of endothelin-1 gene in blood vessel of deoxycorticosterone acetate-salt hypertensive rats. Hypertension. 21: 916920. 1993. Lewis DI, Coote JH. The influence of 5-hydroxytryptamine agonists and antagonists on identified sympathetic preganglionic neurons in the rat, in vivo. Br J Pharmacol 99: 667672. 1990. Lin MT, Wu JJ, Tsay BL Serotonergic mechanism in the hypothalamus mediated thermoregulatory responses in rats. Naunyn-Schmiedeberg’s Arch Pharmacol 322: 271278, 1983. Linder AE, Diaz J, Ni W, Szasz T, Burnett R, Watts SW. Vascular reactivity, 5-HT uptake, and blood pressure in the serotonin transporter knockout rat. Am J Physiol Heart Circ Physiol. 294: H1745-H1752. 2008. Linder AE, Ni W, Szasz T, Burnett R, Diaz J, Geddes TJ, Kuhn DM, Watts SW. A serotonergic system in veins: Serotonin transporter-independent uptake. J Pharmacol. Exp. Ther. 325: 714-22. 2008.   148     Maron E, Toru I, Hirvonen J, Tuominen L, Lumme V, Vasar V, Shlik J, Nutt DJ, Helin S, Nagren K, Tiihonen J, Hietala J. Gender differences in brain serotonin transporter availability in panic disorder. J Psychopharmacol. 0(0): 1-8. 2010. Marshall AM, Nommsen-Rivers LA, Hernandez LL, Dewey KG, Chantry CJ, Gregerson KA, Horseman ND. Serotonin transport and metabolism in the mammary gland modulates secretory activation and involution. J Clin Endocrinol Metab. 95(2): 837-846. 2010. Mclennan PL, Taylor DA. Antagonism by ketanserin of 5-HT-induced vasoconstriction unmasks a 5-HT-induced vasodilation. Eur J Pharmacol. 104: 313-318. 1984. Meehan AG, Kreulen DL. Electrophysiological studies on the interaction of 5hydroxytryptamine with sympathetic transmission in the guinea pig inferior mesenteric artery and ganglion. J Pharmacol Exp Ther 256:82-87, 1991. Meuer S, Ecker U, Hadding U, Bitter-Suermann D. Platelet serotonin release by C3a and C5a: two independent pathways of activation. J. Immunol. 126: 1506-1509. 1981. Molderings GJ, Werner K, Likungu J, Göthert M. Inhibition of noradrenaline release from the sympathetic nerves of the human saphenous vein via presynaptic 5-HT receptors similar to the 5-HT 1D subtype. Naunyn Schmiedebergs Arch Pharmacol. 324(4): 371-7. 1990. Nakatani Y, Sato-Suzuki I, Tsujino N, Nakasato A, Seki Y, Fumoto M, Arita H. Augmented brain 5-HT crosses the blood-brain barrier through the 5-HT transporter in rat. Eur J Neurosci. 29(9): 2466-72. 2008. Ni W, Geddes TJ, Priestley JR, Szasz T, Kuhn DM, Watts SW. The existence of a local 5-hydroxytrypatminergic system in peripheral arteries. Br J Pharmacol. 154(3): 663-674. 2008. Ni W, Lookingland K, Watts SW. Arterial 5-Hydroxytryptamine transporter function is impaired in deoxycorticosterone acetate and N-omega-nitro-L-arginine but not spontaneously hypertensive rats. Hypertension. 48(1): 134-140. 2006. Ni W, Thompson JM, Northcott CA, Lookingland K, Watts SW. The serotonin transporter is present and functional in peripheral arterial smooth muscle. J Cardiovasc. Pharmacol. 43:770-81. 2004. Nilsson T, Longmore J, Shaw D, Pantev E, Bard JA, Branchek T, Edvinsson L. Characterization of 5-HT receptors in human coronary arteries by molecular and pharmacological techniques. Eur J Pharmacol. 24 (372): 49-56. 1999.   149     Nishimura M, Sato K, Shimada S, Tohymama M. Expression of norepinephrine and serotonin transporter mRNAs in the rat superior cervical ganglion. Brain Res Mol Brain Res. 67(1): 82-86. 1999. Page IH, McCubbin JW. The variable arterial response to serotonin in laboratory animals and man. Circ Res 1:354-362. 1953a. Page IH, McCubbin JW. Modification of vascular responses to serotonin by drugs. Am J Physiol 174:436-440, 1953b. Pavone LM, Mithbaokar P, Mstellon V, Avalllone L, Gaspar P, Maharajan V, Baldini A. Fate map of serontonin transporter-expression cells in developing mouse heart. Genesis. 45: 689-695. 2007. Pickering AE, Spanswick D, Logan SD. 5-hydroxytryptamine evokes depolarizations and membrane potential oscillations in rat sympathetic preganglionic neurons. J Physiol 480: 109-121. 1994. Pinto YM, Paul M, Ganten D. Lessons from rat models of hypertension: from Goldblatt to genetic engineering. Cardiovas Res. 39: 77-88. 1998. Proctor DN, Parker BA. Vasodilation and vascular control in contracting muscle of the aging human. Microcirculation. 13: 315-327. 2006. Ramage AG, Villalon CM: 5-Hydroxytryptamine and cardiovascular regulation. Trends Pharmacol Sci 29: 472-481. 2008. Rapport MM, Green AA, Page IH. Serum vasoconstrictor (serotonin) IV. Isolation and characterization. J Biol Chem 176: 1243-1251. 1948. Rodriguez de la Torre B, Dreher J, Malevany I, Bagli M, Kolbinger M, Omran H, Lüderitz B, Rao ML. Serum levels and cardiovascular effects of tricyclic antidepressants and selective serotonin reuptake inhibitors in depressed patients. Ther Drug Monit. 4: 435440. 2001. Saxena PR, Villalon CM. Cardiovascular effects of serotonin agonists and antagonists. J Cardiovasc Pharmacol. 15: S17-S34. 1990. Schenk J, and McNeill JH. The Pathogenesis of DOCA-Salt Hypertension. J Pharmacol Toxicol Methods. 3: 161-170. 1992. Sheng-Rong C, Lie-Cheng W, De-Hu K, Zhen-Xin H, Ru-Chun M. Substance P and 5hydroxytryptamine-mediated depolarization in sympathetic ganglion neurons. Acta Physiological Sinica 51: 585-587. 1999.   150     Shephard JT, Vanhoutte PM. Local modulation of adrenergic neurotransmission in blood vessels. J Cardiovasc Pharmacol. 7 Suppl 3: S167-78. 1985. Suarez-Roca H, Cubeddu LX The selective serotonin reuptake inhibitor citalopram induces the storage of serotonin in catecholaminergic terminal. J Pharmacol Exp Ther. 302: 174-179. 2002. Sun Z, Zhang Z. Historic perspectives and recent advances in major animal models of hypertension. Acta Pharmacologica Sinica. 26(3): 295-301. 2005. Terrón JA. Role of 5-HT7 receptors in the long-lasting hypotensive response induced by 5-hydroxytryptamine in the rat. Br J Pharmacol 121: 563-571, 1997. Terron JA, Sanchez-Maldonado C, Martinez-Garcia E. Pharmacological evidence that 5-HT1B/1D receptors mediate hypotension in anesthetized rats. Eur J Pharmacol 576:132-135, 2007. Thompson LP, Webb C. Vascular responsiveness to serotonin metabolites in mineralocorticoid hypertension. Hypertension. 9: 277-281. 1987. Villalon CM, Centruion D. Cardiovascular responses produced by 5-hydroxytriptamine: a pharmacological update on the receptors/mechanisms involved and therapeutic implications. Naunyn Schmiedebergs Arch Pharmacol. 376: 45-63. 2007. Voorde JV, Leusen I. Endothelium-dependent and independent relaxation of aortic rings from hypertensive rats. Am J Physiol. 250: H711-7. 1986. Wakayama K, Ohtsuki S, Takanaga H, Hosoya K, Terasaki T. Localization of norepinephrine and serotonin transporter in mouse brain capillary endothelial cells. Neurosci Res 44: 173-180. 2002. Walther DJ, Peter JU, Bashammakh S, Hortnagl H, Voits M, Fink H, Bader M. Synthesis of serotonin by a second tryptophan hydroxylase isoform. Science. 299: 76. 2003. Watkins CJ, Newberry NR. Multiple 5-HT receptors in the guinea pig superior cervical ganglion. Br J Pharmacol 177: 21-28, 1996. Watts SW. 5-HT in systemic hypertension: foe, friend or fantasy? Clin Sci 108: 399-412. 2005. Watts SW. The love of a lifetime: 5-HT in the cardiovascular system. Am J Physiol Regul Integr Comp Physiol 296: R252-R256. 2009. Watts SW, Davis RP. 5-hydroxytryptamine receptors in systemic hypertension: an arterial focus. Cardiovas Ther. 29(1): 54-67. 2011.   151     Watts SW, Fink G. 5-HT2B-receptor antagonist LY-272015 is antihypertensive in DOCAsalt-hypertensive rats. Am J Physiol Heart Circ Physiol. 276: 944-952. 1999. Westergaard E. The effect of serotonin on the blood brain barrier to proteins. J Neural Transm Suppl 14: 9-15. 1978. Yemane H, Busauskas M, Burris SK, Knuepfer MM. Neurohumoral mechanisms in deoxycortictosterone acetate (DOCA)-salt hypertension in rats. Exp physiol. 95.1: 5155. 2009. Yildiz O, Smith JR, Purdy RE. Serotonin and Vasoconstrictor Synergism. Life Sci. 62(19): 1723-1732. 1998. Zhang X, Beaulieu JM, Sotnikova TD, Gainetdinov RR, Caron MG. Trytophan hydroxylase-2 controls brain serotonin synthesis. Science. 305: 217. 2004. Zhou FC, Lesch KP, Murphy DL. Serotonin uptake into neurons via dopamine transporters: a compensatory alternative. Brain Res. 942: 109-119. 2002.   152