.- ..l. E, . . . .Afimksiwfigfi quirk... V , . .. . . . . . . 2 sow? LIBRARY Michig - ' Qtate University This is to certify that the thesis entitled Chronic 5-HT Causes a Long-Term Blood Pressure Fall in DOCA-Salt Hypertension: Role of Nitric Oxide presented by Jessica Lynn Diaz has been accepted towards fulfillment of the requirements for the Master of degree in Pharmacology and Toxicology Science 3mm w walla Major Professor’s Signature %/;%200}' Date MSU is an affirmative-action, equal-opportunity employer PLACE IN RETURN BOX to remove this checkout from your record. TO AVOID FINES return on or before date due. MAY BE RECALLED with earlier due date if requested. DATE DUE DATE DUE DATE DUE M27509 6/07 p:/ClRC/DateDue.indd-p.1 CHRONIC 5-HT CAUSES A LONG-TERM BLOOD PRESSURE FALL IN DOCA—SALT HYPERTENSION; ROLE OF NITRIC OXIDE By Jessica Lynn Diaz A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Pharmacology and Toxicology 2007 ABSTRACT CHRONIC 5-HT CAUSES A LONG-TERM BLOOD PRESSURE FALL IN DOCA- SALT HYPERTENSION; ROLE OF NITRIC OXIDE By Jessica Lynn Diaz We have shown that chronic serotonin (5-HT) causes a dramatic fall in mean arterial pressure (MAP) in both DOCA-salt hypertensive rats (deoxycorticosterone acetate) and Sham rats (Shamo). We hypothesized that 5- HT acts to promote the function of nitric oxide synthase (NOS) in vivo, and that this hypotensive effect would not be seen in a hypertensive model in which NOS is inhibited using L-NNA (Nw-nitro-L-arginine). 5-HT (25 uglkg/min) or Vehicle was administered to DOCA-salt and LNNA hypertensive rats and Shams. Within 24 hours, MAP in the DOCA 5-HT infused group fell 53 mmHg while in the Shamo rats, MAP fell less dramatically (-21 mmHg). This hypotensive response was not observed in the L-NNA 5-HT infused group, in which MAP remained unchanged. In contrast, ShamL 5-HT-infused rats experienced a similar drop in MAP to Shamo rats (-19 mmHg). Ganglionic blockade using hexamethonium given on Day 4 of 5-HT or vehicle infusion demonstrated marked sympatho- inhibition in the DOCA 5-HT infused rats (peak MAP fall of 40.3:5.9 mmHg), in contrast to DOCA Vehicle infused rats (peak fall MAP 90.61140 mmHg). This sympatho-inhibitory effect of 5-HT was abolished in LNNA hypertension in which there was no difference between 5-HT-infused (peak fall MAP 54.714] mmHg) and vehicle-infused rats (peak fall MAP 51.11129 mmHg). These data suggest that 5-HT inhibits the sympathetic nervous system in a NOS-dependent fashion. To my understanding and supportive family: Man'o, Janis, Marisa, Johanna, Marcus, and all our creatures great and small, who helped me through. ACKNOWLEDGEMENT I would like to thank Dr. Stephanie Watts, PhD, for your freely-given mentorship, guidance, and enormous support, both as a budding scientist in your lab and as a person. I do not know if I can everrepay your kindness and willingness to take me into your lab when I brought little more than a desire to try something new and off the beaten path of veterinary medicine. You opened your home and shared your beautiful family with me on several occasions, and for that, I thank you from the bottom of my heart. You have truly been a role model for me, and I will endeavor to carry everything you taught me with me as I continue on my life path, because it was so much more than science that I learned here along the way. I would also like to gratefully acknowledge my committee members, Dr. J.R. Haywood, and Dr. Greg Fink. I sincerely appreciate your tremendous time and effort spent on committee meetings, going over data with me, reviewing abstracts and drafts of this thesis, allowing my name to be associated with yours on this work, and most importantly for teaching me to remember the bigger picture. I would like to thank Dr. Andrew King for always dropping what you were doing to help me, with everything from telemetry surgery, statistics, being a sounding-board for my ideas, and finally for helping me understand the implications of my own data. I'm truly grateful. I need to thank members of the Watts lab, both past and present, as i could not have completed my project without your incredible help every single day. Dr. Wei Ni, Dr. Keshari Thakali, Dr. Elizabeth Linder, Dr. Theo Szasz, Robert Burnett, Nathan Tykocki, Jessie Priestly, Kevin Ogden, C.J. Bush, visiting summer scholar Merete "Mell" Ellekilde, and summer medical student R. Patrick Davis. Janice Thompson, I cannot thank you enough for everything I learned from you. Quite frankly, I don't know how students in other labs can possibly learn to do Western blots without the added benefit of your high energy and stellar sound effects! I would like to thank the current graduate students of the Department of Pharmacology and Toxicology for including me as part of the group! I enjoyed getting know you all both in and outside the classroom and laboratory. From my family, I have received nothing but love and unwavering support, which helped cultivate my internal spark and enabled me the freedom to try everything that I want to do, both as a person and in building my future career. Thank you. TABLE OF CONTENTS List of Tables ............................................................................... viii List of Figures .............................................................................. ix List of Abbreviations ..................................................................... xiii Introduction .............................................................................. 1 l. Discovery of 5-HT .................................................... 1 ll. 5-HT Receptors ........................................................ 2 a. Cardiac 5-HT receptors .......................................... 3 b. Renal 5-HT receptors ............................................ 4 c. Vascular 5-HT receptors ........................................ 4 III. Role of 5-HT in disease states ..................................... 5 IV. 5-HT in systemic hypertension ...................................... 7 V. Actions of acute infusions of 5-HT ................................ 9 VI. A gap in the knowledge ............................................... 10 Hypotheses ................................................................................. 12 Methods ...................................................................................... 12 l. Animal Use ............................................................... 12 ll. Euthanasia... ... .......................................................... 12 Ill. Animal Models ........................................................... 13 a. Mineralocorticoid Hypertension ................................ 13 b. L-NNA Hypertension ............................................... 14 c. DOCA plus L-NNA Hypertension ............................... 14 IV. Blood Pressure Measurements ...................................... 14 V. Osmotic Mini-pump implantation .................................... 15 VI. Tissue Basal 5-HT Level Measurement ........................... 17 VII. 5-HIAA and 5-HT Concentration Measurement from Whole Blood ........................................................................ 17 VIII. 5-HIAA and 5-HT Measurement from selected Vessels....... 18 IX. Protein Isolation ........................................................... 19 X. BCA Protein Assay ....................................................... 19 XI. Western Blotting .......................................................... 20 a. eNOS .................................................................... 20 b. PECAM-1 ................................................................ 20 c. a-actin .................................................................... 21 Xll. isolated Smooth Muscle Contractility Measurement ........... 21 Xlll. Data Analysis and Statistics ........................................... 22 Results ...................................................................................... 24 Hypothesis 1 ..................................................................... 24 vi Validation of model: plasma 5-HT measurements ....................... 24 Validation of model: peripheral vascular tissue 5-HT measurements ...................................................................... 25 DOCA-salt hypertension: Effect of 5-HT on telemetric measurements ...................................................................... 28 Osmotic pump function and 5-HT viability after 7 days of infusion ................................................................................ 28 5-HT HCI infusion .................................................................. 30 Effect of 5-HT on DOCA-salt water intake ................................. 30 Effect of hexamethonium ........................................................ 50 Isolated tissue contractility ...................................................... 50 Role of NO ........................................................................... 52 L-N NA hypertension: plasma 5-HT measurements ..................... 52 L-N NA hypertension: Effect of 5-HT of telemetric measurements ...................................................................... 53 L-N NA hypertension: Effect of hexamethonium ........................ 55 Role of NOS inhibition in DOCA ............................................. 73 DOCA+L-NNA: plasma 5-HT measurements ............................. 74 DOCA+L-NNA: Effect of 5-HT on telemetric measurements ......... 75 DOCA+L-NNA: Effect of hexamethonium ................................... 76 DOCA+L-NNA: Isolated tissue contractility ................................. 77 Discussion ................................................................................... 92 Plasma 5-HT levels .............................................................. 93 Involvement of 5-HT receptors ................................................ 95 Effect of 5-HT on MAP ........................................................... 97 Conclusion .................................................................................. 100 Speculation/Future Study ............................................................ 101 References .................................................................................. 103 Bibliography ................................................................................ 108 vii LIST OF TABLES Table 1. 5-HT quantification comparison of PPP in the Shamo, DOCA, ShamL, L-NNA, ShamD+L-NNA, and DOCA+L-NNA .............. 67 viii LIST OF FIGURES Figure 1. Blood pressure tracing of acute iv infusion of 5-HT .............. 11 Figure 2. Top: Chromatogram showing separation of 10 ng standards using HPLC. Bottom: Detection of basal levels of 5-HIAA and 5-HT in thoracic aorta .................................................................. 33 Figure 3. Quantification and comparison of 5-HT in the platelet-poor plasma (PPP) and platelet-rich plasma (PRP) in Shamo rats receiving Vehicle or 5-HT infusion at Day 7 of infusion... .. 34 Figure 4. Quantification and comparison of 5-HT in the platelet-poor plasma (PPP) and platelet-rich plasma (PRP) in DOCA rats receiving Vehicle or 5-HT infusion at Day 7 of infusion ........... 35 Figure 5. Quantification and comparison of 5-HIAA and 5-HT in aorta, vena cava, carotid, jugular vein, and superior mesenteric artery harvested from ShamD rats receiving Vehicle or 5-HT infusion at Day 7 of infusion .............................................. 37 Figure 6. Quantification and comparison of 5-HIAA and 5-HT in aorta, vena cava, carotid, jugular vein, and superior mesenteric artery harvested from DOCA rats receiving Vehicle or 5-HT infusion at Day 7 of infusion .............................................. 39 Figure 7. Top: Twenty-four hour-average blood pressure measurement in ShamD and DOCA rats receiving Vehicle or 5-HT infusion for 7 days. Middle: Twenty-four hour-average heart rate measurement in ShamD and DOCA rats receiving Vehicle or 5-HT infusion for 7 days. Bottom: Twenty-four hour-average activity level measurement in ShamD and DOCA rats receiving Vehicle or 5-HT infusion for 7 days ............................................... 41 Figure 8. Top: Quantification of remaining Vehicle or 5-HT pump fluid volumes after 7 days of infusion. Middle: HPLC detection and quantification of 5-HT from the remaining pump fluid. Bottom: Determination of the viability of the 5-HT from the remaining pump fluid at the end of 7 days of in vivo infusion ix Figure 9. Figure 10. Figure 11. Figure 12. Figure 13. Figure 14. Figure 15. Figure 16. Figure 17. Figure 18. in an isolated tissue bath .................................................. Top: Chromatogram of remaining Vehicle pump fluid after 7 days of infusion. Bottom: Chromatogram of remaining 5-HT pump fluid diluted 1:105 after 7 days of infusion ............................................ Twenty-four hour-average blood pressure measurement in ShamD and DOCA rats receiving 5-HT creatine sulfate or 5-HT HCI infusion for 7 days .................................................... Top: Grouped averages of the ShamD and DOCA fluid consumption measurements over the 7 day duration of the Vehicle or 5-HT infusion. Bottom: Twenty-four hour-average blood pressure measurement in DOCA rats receiving Vehicle or 5-HT infusion for 7 days and the effect of restricting salt-water intake on the MAP of DOCA Vehicle rats ................................................ Peak response to hexamethonium administered on Day 4 of Vehicle or 5-HT infusion in ShamD and DOCA rats ................ Effect of 7-day Vehicle or 5-HT infusion on cumulative response curves to PE, ACh, and 5-HT in aorta from ShamD rats .............................................................. Effect of 7-day Vehicle or 5-HT infusion on cumulative response curves to PE, ACh, and 5-HT in superior mesenteric artery from ShamD rats ..................................................... Effect of 7-day Vehicle or 5-HT infusion on cumulative response curves to PE, ACh, and 5—HT in aorta from DOCA rats ............................................................................... Effect of 7-day Vehicle or 5-HT infusion on cumulative response curves to PE, ACh, and 5-HT in superior mesenteric artery from DOCA rats ...................................................... Western blot analysis examining eNOS expression in aorta from DOCA Vehicle or 5-HT-infused rats .............................. Quantification and comparison of 5-HT in the platelet-poor plasma (PPP) and platelet-rich plasma (PRP) in ShamL rats 43 45 46 48 49 57 59 61 63 64 Figure 19. Figure 20. Figure 21. Figure 22. Figure 23. Figure 24. Figure 25. Figure 26. Figure 27. receiving Vehicle or 5-HT infusion at Day 7 of infusion ............ 65 Quantification and comparison of 5-HT in the platelet-poor plasma (PPP) and platelet-rich plasma (PRP) in L-NNA rats receiving Vehicle or 5-HT infusion at Day 7 of infusion ............ 66 Top: Twenty-four hour-average blood pressure measurement in ShamL and L-NNA rats receiving Vehicle or 5-HT infusion for 7 days. Middle: Twenty-four hour-average heart rate measurement in ShamL and L-NNA rats receiving Vehicle or 5-HT infusion for 7 days. Bottom: Twenty-four hour-average activity level measurement in ShamL and L-NNA rats receiving Vehicle or 5-HT infusion for 7 days ....................................................................... 69 Twenty-four hour-average blood pressure measurement comparing L-NNA and DOCA rats receiving Vehicle or 5-HT infusion for 7 days ........................................................... 70 Peak response to hexamethonium administered on Day 4 of Vehicle or 5-HT infusion in ShamL and L-NNA rats ................ 71 Effect of 7-day Vehicle or 5-HT infusion on cumulative response curves to PE, ACh, and 5-HT in aorta from ShamL rats ............................................................................... 80 Effect of 7-day Vehicle or 5-HT infusion on cumulative response curves to PE, ACh, and 5-HT in aorta from L-NNA rats ............................................................................... 82 Quantification and comparison of 5-HT in the platelet-poor plasma (PPP) and platelet-rich plasma (PRP) in ShamD + L-N NA rats receiving Vehicle or 5-HT infusion at Day 7 of infusion .......................................................................... 83 Quantification and comparison of 5-HT in the platelet-poor plasma (PPP) and platelet-rich plasma (PRP) in DOCA + L-NNA rats receiving Vehicle or 5-HT infusion at Day 7 of infusion .......................................................................... 84 Top: Twenty-four hour-average blood pressure measurement in ShamD +L-NNA and DOCA+L-NNA rats receiving Vehicle xi Figure 28. Figure 29. Figure 30. or 5-HT infusion for 7 days. Middle: Twenty-four hour-average heart rate measurement in ShamD +L-NNA and DOCA+L-NNA rats receiving Vehicle Bottom: Twenty-four hour-average activity level measurement in ShamD +L-NNA and DOCA+L-NNA rats receiving Vehicle or 5-HT infusion for 7 days ............................................... 86 Peak response to hexamethonium administered on Day 4 of Vehicle or 5-HT infusion in ShamD +L-NNA and DOCA+L—NNA rats ............................................................................... 87 Effect of 7-day Vehicle or 5—HT infusion on cumulative response curves to PE, ACh, and 5-HT in aorta from ShamD + L-N NA rats ..................................................................... 89 Effect of 7-day Vehicle or 5-HT infusion on cumulative response curves to PE, ACh, and 5-HT in aorta from DOCA+ L-NNA rats ..................................................................... 91 xii LIST OF ABBREVIATIONS 5-HIAA 5-Hydroxyindoacetic acid 5-HT 5-Hydroxytryptamine, serotonin ACh Acetylcholine BCA Bicinchoninic Acid BP Blood pressure bpm Beats per minute (heart rate) BSA Bovine Serum Albumin CNS Central Nervous System DOCA Deoxycorticosterone acetate eNOS Endothelial nitric oxide synthase i.p. lntraperiotoneally KCI Potassium chloride L-NNA Nw-nitro-L-arginine MAO Monoamine oxidase MAP Mean arterial pressure NaCl Sodium chloride NO Nitric oxide NOS Nitric oxide synthase PCPA Para-chlorophenyalanine PPP Platelet-poor plasma xiii PRP Platelet-rich plasma PE Phenylephrine PECAM-1 Platelet/endothelial cell adhesion molecule—1 PSS Physiological salt solution SERT Serotonin transporter SEM Standard error of the mean SHR Spontaneously hypertensive rats SNS Sympathetic Nervous System s.q. Subcutaneously SSRls Selective Serotonin Reuptake Inhibitors TBS Tris Buffered Saline TBS-T Tris Buffered Saline + Tween TPR Total peripheral resistance xiv Introduction Discovery of 5-HT Serotonin or 5-HT (5-hydroxytryptamine) is a diverse physiological agent, acting as a neurotransmitter in the central nervous system, a vasoactive agent capable of causing smooth muscle contraction in the cardiovascular system, a regulator of gastrointestinal peristalsis, and as an aggregator of platelets. 5-HT was discovered independently in two laboratories in the mid-20th century. Rapport, Green, and Page discovered a powerful endogenous vasoconstricting substance at low concentrations in clotted blood which they called serotonin: "sero—" for being isolated from serum and "-tonin" for its vasoconstrictor activity (Rapport et al, 1948). Erspamer reported an indole compound obtained from the enterochromaffin cells of the intestine that could cause smooth muscle contraction in isolated tissues, which he called enteramine. In 1949, the chemical structure of serotonin was determined to be 5-hydroxytryptamine. In the 1950s, enteramine and serotonin were found to be the same compound. The vast majority (95%) of the 5-HT produced in the body is made in the enterochromaffin cells of the gut. A small percentage is produced in the neuroendocrine cells of the lung. Another site of 5-HT production is the central nervous system, largely kept distinct from peripheral 5-HT by the blood-brain- barrier (BBB) (Maurer-Spurej, 2005). 5-HT is produced by a series of enzymatic reactions, beginning with the dietary essential amino acid tryptophan (cote et al., 2004). As a polar molecule, 5-HT itself cannot cross the BBB in physiologic conditions, in contrast to tryptophan, which is able to transverse the BBB (O'Kane and Hawkins, 2003). Tryptophan hydroxylase (TPH) is the rate-limiting enzyme in 5-HT production from tryptophan, and 2 isoforrns exist: TPH1 (peripheral) and TPH2 (central) (Coté et al., 2004). The action of 5-HT is terminated after its degradation to an inactive metabolite, 5-hydroxyindole acetic acid (5-HIAA), via the action of mitochondrial monoamine oxidase (C6té et al., 2004). While investigations are ongoing, currently there is no published evidence for the enzymatic machinery for 5-HT handling and production being present in peripheral arteries or veins. But it has been reported that there exists a functional serotonin transporter (SERT) on peripheral arteries, whereby smooth muscle cells are able to take up 5-HT from the extracellular to intracellular space (Ni et al., 2004). Thus, the current theory is that 5-HT gets transported from the site of production in the intestine to the 5-HT receptors and/or SERT located on the cellular membranes of the peripheral vasculature via the circulating platelets (Ni et al., 2004). Platelets, as anucleate cells, lack the machinery to produce 5- HT themselves, but are able to take up 5-HT via SERT localized to the platelet cell membrane, and have the ability to store 5-HT in their dense granules (Ni et al., 2004; Maurer-Spurej, 2005). Platelet storage may be a protective mechanism to keep freely circulating 5-HT levels low in the plasma: 0.1 ng/ml measured by HPLC, while platelet-stored 5-HT levels have been measured at 8841202 ng/109 platelets (Maurer—Spurej, 2005). 5-HT receptors To date, there are currently seven families of 5-HT receptors (5-HT1-7), some of which have subfamilies. 5-HT3 receptors are ligand-gated ion channels, while the other families are transmembrane-spanning G-protein-coupled receptors. 5-HT exerts its effects in the cardiovascular system either directly on cardiovascular 5-HT receptors or indirectly via receptors in the central nervous system. The 5-HT receptors present in the tissues of the cardiovascular system include 5-HT1B, 5-HT2 family, 5-HT3, 5-HT4, and 5-HT7 receptors. CidLac 5-HT receptors: 5-HT affects the heart in a myriad of ways, due to the existence of numerous 5-HT receptors on the various tissue types. There are 5-HT receptors on the cells of the sino-atrial node, the pacemaker of the heart, which influence heart rate. Tachycardia results from activation of the 5-HT4 receptors (human), 5-HT23 receptors (rat), or 5-HT7 receptors (cat) (COté et al., 2004). Increased circulating 5-HT can cause sinus tachycardia and atrial fibrillation (Cbté et al., 2004). In contrast, bradycardia can also potentially be induced by activation of the 5-HT3 receptors located on vagal nerve endings innervating the sino-atrial node (Coté et al., 2004). Cardiac contractility in the myocardial cells is mediated primarily by 5-HT23 receptors (C6té et al., 2004). Expression of 5-Hng receptors is vitally important to heart development. Lack of the 5-HT23 receptors in knock- out mice results in mid-gestational lethality due to defects in the myocardial architecture, including markedly decreased ventricular wall thickness, and a gross absence of ventricular myocardial trabeculation (Nebigil et al., 2000). Renal 5-HT receptors_: There are species differences in the receptors mediating effects of 5-HT in the renal artery. In many species, the 5-HT2A receptor is responsible for contraction to 5-HT. Mr 5—HT receptors: In the peripheral vasculature, 5-HT1B, 5-HT2A, 5-HT23, 5-HT.;, and 5-HT7 receptors are present (C6té et al., 2004; Hoyer et al., 2002; Watts, 2005). 5- HT1B receptors are located in the CNS, but can also mediate contraction in rat caudal arteries and are expressed on cerebral arteries (Hoyer et al., 2002). The 5-HT2A receptor is widely distributed both centrally and peripherally and mediates contraction in a number of tissues containing smooth muscle (Hoyer et al., 2002). The 5-HT23 receptor is present on smooth muscle cells (Kaumann and Levy, 2006; Hoyer et al., 2002) and is upregulated in arteries from DOCA-salt hypertensive rats. The 5-HT23 receptor becomes the 5-HT receptor predominately responsible for vascular smooth muscle contraction in hypertensive rats, as opposed to the 5-HT2A receptor in normotensive rats (Watts et al., 1996; Banes and Watts, 2002; Banes and Watts, 2003). In some tissues, activation of the 5-HT23 receptor results in endothelium- and nitric oxide- dependent vasorelaxation, including the pig pulmonary arteries (Jahnichen et al., 2005) and rat jugular vein (Ellis et al., 1995). In addition to localization to the heart, 5-HT4 receptors are expressed in both pig and human coronary artery smooth muscle cells, and weakly expressed in human endothelial cells (Ullmer et al., 1995). The 5-HT4 receptor is positively coupled to adenylate cyclase, but its definitive function has yet to be fully uncovered (Hoyer et al., 2002; Ullmer et al., 1995). Finally, 5-HT7 receptors are expressed extensively in the vasculature and are responsible for mediating endothelium-independent vasorelaxation (Terron and Martinez-Garcia, 2007; De Vries, 1999). While 5-HT is a vasoconstrictor on its own, its most probable physiologic contribution to blood pressure regulation is by modulation of other vasoactive agents and hormones. Subcontractile concentrations of 5-HT have the ability to potentiate smooth muscle contraction of isolated tissues to norepinephrine (NE), angiotensin II (Angll), and the potent vasoconstrictor endothelin-1 (ET-1) (Watts, 2000.) Role of 5-HT in disease states Because of the physiological diversity of 5-HT receptors and function in the body, dysregulation of 5-HT production, metabolism, or cellular responses are capable of affecting a number of physiological systems. 5-HT has been implicated in a number of gastrointestinal, neurological, hemostatic, as well as cardiovascular diseases. 5-HT in the brain influences a number of behaviors, including compulsion, aggression, anxiety, depression, sleep, cognition, sensory perception, motor activity, appetite, temperature regulation, nociception, sexual behavior and hormone secretion (Goodman and Gilman, 2001). Peripherally, 5- HT plays a role in other disease conditions including migraine headaches and gastrointestinal disorders, (Goodman and Gilman, 2001) in addition to vascular diseases such as thrombosis and atherosclerosis (Vikenes et al., 1999; Ishida et al., 2001). In the cardiovascular system, 5-HT is a vascular smooth muscle cell mitogen, associated with cellular proliferation and vascular remodeling, which is a hallmark of systemic hypertension. Hypersensitivity to 5-HT has been observed in cardiovascular diseases, including atherosclerosis (Ishida et al., 2001) and systemic hypertension (Watts et al., 1995; Watts, 1998; Banes and Watts, 2001) Excessive levels of circulating 5-HT have been associated with heart valvular pathology. Gustafsson et al. observed that 3 months of daily subcutaneous injection of 5-HT creatinine sulfate complex (50 mglkg for the first 3 days and 20 mglkg throughout the duration of the study) resulted in pathology of the aortic and/or pulmonic heart valves (Gustafsson, 2005). In this particular study, no measurements of blood pressure were made with 5-HT administration. There is a strong connection between increased levels of 5-HT and increased SERT function in patients with pulmonary hypertension (Egermayer et al., 1999). Neuroendocrine cells lining the lung secrete vasoactive mediators, including 5-HT, in response to pulmonary insult, like airway hypoxia and hypercapnia (Egermayer et al., 1999). In fact, 5-HT is identified as the most powerful pulmonary vasoconstrictor known to date (Egermayer et al., 1999). Anorexogenic fluramines act on SERT as 5-HT releasers, thereby increasing 5- HT in circulation (Chapman and Vlfideman, 2006; Egermayer et al., 1999), and use of these drugs has been associated with development of primary pulmonary hypertension (Chapman and Wideman, 2006). SERT plays a central role in the pathogenesis of pulmonary-artery smooth muscle cell proliferation, by bringing 5- HT intracellularly to exert physiological effects, ultimately leading to pulmonary hypertension (Guignabert et al., 2006; Nemecek et al., 1986). Patients with platelet-storage pool diseases, in which the platelets have defects in the ability to store substances intracellularly, which results in increased 5-HT levels in plasma, have an increased incidence of developing pulmonary hypertension (Egermayer et al, 1999; Maurer-Spurej, 2005). As a corollary, the use of SERT-inhibitors, like fluoxetine (Prozac®), is protective against pulmonary hypertension (Maclean et al., 2004). Maurer-Spurej et al. were the first to show that the use of fluoxetine inhibits the release of 5-HT from platelets during aggregation, preventing elevations in plasma 5-HT, which is perhaps the mechanism of this protection (2004). While the association between 5-HT and pulmonary hypertension is established currently, the link between 5-HT and systemic hypertension is less so, and more controversial. 5-HT in systemic hypertension The use of SERT-inhibitors, namely fluoxetine, causes increases in blood pressure in both rodents (Lazartigues et al., 2000) and humans (Amsterdam et al,1999) Freely circulating levels of 5-HT in the plasma (platelet poor plasma, PPP) are reported to be increased in hypertensive patients and experimental models of hypertension (Maurer-Spurej, 2005; Brenner et al., 2007), most likely the result of impaired SERT kinetics and reduced ability of platelets to take up and store 5-HT (Fetkovska et al., 1990; Brenner et al., 2007). Brenner et al. specifically showed that PPP 5-HT levels of hypertensive patients are elevated while the platelet- stored 5-HT (platelet rich plasma, PRP) levels are diminished. They also showed decreased expression of SERT protein on the cellular surface of platelets from hypertensive patients in addition to a decreased rate of uptake (Vmax) (Brenner et aL,2007) A strong association between elevated plasma levels of 5-HT and angiographically-confirmed coronary artery disease has been observed in human patients (Vikenes et al., 1999). Platelets are known to become activated and aggregate at sites of endothelial injury or atherosclerosis. Tremendous concentrations of 5-HT are capable of being locally released from platelet granules upon activation and contribute to the decreasing coronary vessel lumen size by vasoconstriction. Additionally, as a smooth muscle cell mitogen, cellular proliferation exacerbates the disease (Vikenes, et al., 1999). An important hallmark of human hypertension and experimental models of hypertension, such as the mineralocorticoid model of hypertension, the DOCA- salt model (deoxycorticosterone acetate), the L-NNA model (N-w-L-arginine; an inhibitor of nitric oxide synthase), and the spontaneously hypertensive rat (SHR) ‘ is a hyperresponsiveness or increased sensitivity of isolated vessels to 5-HT, demonstrated by a left-ward shift of the cumulative dose response curve as compared to normotensive sham tissues (Watts, 1998; Russell et al., 2002). This increase in sensitivity begins as early as Day 5 in the DOCA-salt rat (Watts, 1998). Mineralocorticoid hypertension (i.e. rat DOCA-salt experimental model) is dependent on 5-HT23 receptor expression, specifically causing an increase in the 5-HT23 receptor activation, as 5-HT2;; receptor antagonism decreases blood pressure (Banes and Watts, 2003; Watts and Fink, 1999). The DOCA-salt model of hypertension is not the only model in which 5-HT receptor function is altered. In the L-NNA hypertensive model, the 5-HT23 receptor expression level and function were increased compared to shams (Russell et al., 2002). Actions of acute infusions of 5-HT The response to exogenous 5-HT acutely varies across species, dose ranges, and time of infusion, and the effect is complex. A triphasic response in blood pressure within minutes was observed in the conscious state in dogs and cattle. This triphasic response is shown in Figure 1 and included 1) an initial transient fall in arterial pressure with concurrent bradycardia, then 2) a pressor response, and finally 3) a prolonged depressor response (Page, 1952; Page and McCubbin, 1953; Comroe et al., 1953; Rudolph and Paul, 1956; Emerson, 1968; Linden et al., 1999). In anesthetized cats and rabbits, an intravenous 5-HT infusion caused bradycardia and hypotension (Page and McCubbin, 1953; Comroe et al., 1953). Blood pressure increases result after intravenous adminstration of 5-HT to conscious sheep (Nelson et al., 1987). Much is unknown to date as to what 5-HT receptors may be responsible for the triphasic response to acute exogenous 5-HT. However, Terrén helped shed some light on the complex response by using selective agonist and antagonists against the cloned 5- HT7 receptor to implicate that receptor in the long-lasting hypotensive response to 5-HT (1997). A gap in the knowledge 5-HT is clearly important in the embryological development of a normal cardiovascular system and plays a role in a myriad of adult body systems, including the CNS, gastrointestinal, and cardiovascular systems. Of particular interest are the effects of 5-HT in the peripheral vasculature and in the cardiovascular system as a whole. The contribution of 5-HT to the regulation of systemic blood pressure is controversial and highly disputed. Chronic exogenous 5-HT administration with concomitant blood pressure measurement has not been published in the literature to date. Elucidating the role of 5-HT in the regulation of systemic blood pressure and the mechanism of action is the focus of this study. 10 130' 120‘ 110‘ 100‘ 9.. l Systolic Pressure (mmHg) ‘7 ontrol 1 2 3 Time (min) \I o C' Figure 1 Figure 1. Effect of 100 pg intravenous 5-HT administered to a conscious dog (arrow indicates time of administration). Systolic blood pressure is shown on the y-axis and time in minutes on the x-axis (adapted from Page, 1952). 11 Hypotheses: Hypothesis 1: Chronic 5-HT infusion will lead to increased blood pressure and arterial 5-HT content in normotensive rats; these responses will be enhanced in DOCA-salt hypertensive rats. Our results led to Hypothesis 2. Hypothesis 2: Chronic 5-HT acts to promote the function of nitric oxide synthase (NOS) in vivo, and that this effect will not be seen in a model of LNNA hypertension where NOS is inhibited. Methods: I. Animal Use All animal procedures were followed in accordance with the institutional guidelines of Michigan State University. Normal male Sprague-Dawley rats (225- 250 g) were purchased from Charles River (Portage, MI) for DOCA and ShamD or Harlan Industries, Inc. (Indianapolis, IN) for L-NNA administration. Rats were kept in clear plastic boxes maintained in dedicated animal rooms kept at 2112°C and 50110% humidity under 12:12 hour light/dark cycle with standard rat chow (Teklad®) and tap water provided ad Iibitum, except where stated. ll. Euthanasia: 12 Rats were deeply anesthetized with pentobarbital (60 mglkg i.p.) prior to inducing bilateral pneumothorax by opening the thoracic cavity and severing aortic arch. Ill. Animal Models: Mineralocorticoid Hypertension: Male Sprague—Dawley rats (250-300 9; Charles River, Portage, MI) were anesthetized with isoflurane (lsoFlo®) in preparation for left uninephrectomy. The left flank region and upper cervical dorsal region were clipped free of fur and the skin cleaned with povidone iodine solution. Animals were placed in right lateral recumbency, and an incision made perpendicular to the spine and caudal to the last costa. The left kidney was identified, gently exteriorized, and excised after ligation of the left renal artery. A two-layer closure was made, using 6.0 non- absorbable nylon suture to close the abdominal muscle layer, and 4.0 non- absorbable nylon suture to close the skin incision. The animal was then re- positioned to sternal recumbency and a skin incision made on the nape of the neck approximately 1 centimeter caudal to the ears. A Silastic® (Dow Corning, Midland, MI) implant impregnated with DOCA (150 or 200 mglkg) was placed subcutaneously. The skin was closed with 6.0 non-absorbable nylon suture Post-operatively, the rats were returned to their home cages and examined daily for evidence of redness, swelling, or discharge at the incision sites. Rats were given a solution of 1% NaCl and 0.2% KCI to drink. Sham rats also received a uninephrectomy as described, but received no DOCA implant and drank normal 13 tap water. Animals were fed standard rat chow and had free access to food and their respective water. The animals remained on this regimen for five weeks. Normotensive sham rats (ShamD) were used as a comparator to DOCA hypertensive rats. At the same time, Shamo rats underwent uninephrectomy surgery as described for DOCA, but received no DOCA pellet. ShamD rats were given tap water to drink for the duration of the experiment. L-NNA Hypertension: Male Sprague-Dawley rats (250-300 g; Harlan, Indianapolis, IN, USA) were given tap water mixed with Nw-nitro-L-arginine (L-NNA, 0.5 g/L) (Sigma- Aldrich Chemicals, St. Louis, MO, USA). The animals were on this regimen for 17 days. Normotensive sham rats (ShamL) were used as a comparator to DOCA hypertensive rats. ShamL rats were given tap water to drink for the duration of the experiment. DOCA plus LNNA Hypertension: Male Sprague-Dawley rats (Charles River, Portage, MI, USA) underwent uninephrectomy and DOCA pellet implant as described above. On Day 25 post- DOCA surgery, rats were given 1% NaCl, 0.2% KCI, and 0.01 g/L LNNA to drink for an additional 10 days. IV. Blood Pressure Measurements: 14 Under isoflurane anesthesia, radiotelemeter devices (Data Sciences International, MN, USA) with attached catheters with pressure-sensing tips were implanted subcutaneously through a 1-1.5 cm incision in the left inguinal area. The left femoral artery was gently separated from the femoral nerve and vein, and gentle tension applied with silk suture to occlude blood flow temporarily. After splashing 1% lidocaine to the vessel to prevent vasospasm, catheters were introduced into the femoral artery 3-5 mm distal to the level of the peritoneal wall, and the pressure-sensing tip was advanced approximately 5 cm to the abdominal aorta. The catheter was ligated in place and the subcutaneous and skin layers were closed with 6.0 and 4.0 non-absorbing nylon suture, respectively. The rats were allowed 3-4 days to recover post-operatively in their home cages with daily monitoring for redness, swelling and/or discharge from the incision sites, and then 3-4 days of baseline measurements were made at a sampling schedule of 10 seconds every 10 minutes. Mean arterial pressure, pulse pressure, heart rate, and rat activity levels were recorded at the same sampling schedule throughout the duration of the experiments. V. Osmotic Mini-pump Implantation: One week after radiotelemeter placement and under isolfurane anesthesia, osmotic pumps with a release rate of 10.0 ul/hour and release duration of 7 days (Alzet Osmotic Pumps, Model 2ML1, Durect Corporation, Cupertino, CA, USA) were implanted subcutaneously between the scapulae. Two experimental groups were used: 1) Control group (vehicle pump), 2) 5-HT 15 (25 pg serotonin creatinine sulfate complex/kg/min) (Sigma-Aldrich Chemicals, St. Louis, MO, USA). On the day of pump implantation, the serotonin creatinine sulfate complex and 1% (w/v) ascorbic acid (as an antioxidant) was fully dissolved in 1N HCI using sonication and vortex, and then pH-balanced to between 6-7 with 4 N NaOH, and finally balanced with distilled water. The solution was then passed through a sterile Millex®-GS syringe driven filter 0.22 uM filter (Millipore, Carrigtwohill, Co. Cork, Ireland) to finally load the osmotic pumps with a 25-gauge blunt-tipped needle provided by Alzet with the osmotic pumps. The solution for the vehicle pumps contained 1% ascorbic acid, a proportional volume of 1N HCI as used for the 5-HT pumps, and pH-balanced to between 6-7 with 4N NaOH, and finally balanced with distilled water. Vehicle pumps were loaded as described with a new syringe, syringe filter, and needle. The pumps were loaded at room temperature immediately before surgical implantation. Since the pumps were not used in combination with a catheter, nor was it imperative that steady state of 5-HT infusion to be reached immediately, it was not essential to prime them prior to use (i.e. load the pumps and then place the tubes in sterile isotonic saline at 37°C for at least 4-6 hours prior to surgical implantation). Subsequently, a continuous rate of infusion was not reached for several hours after surgical implantation in vivo. In addition to serotonin creatinine sulfate complex, an alternate form of 5- HT, 5-HT HCI, was used in one small experiment to verify that the response to 5- HT was due to 5-HT and not the creatinine sufate salt (Sigma-Aldrich Chemicals, 16 St. Louis, MO, USA). 5-HT HCl was dissolved in water with 1% ascorbic acid to load the osmotic pumps. At the end of selected experimental protocols, remaining fluid from the osmotic pumps was retrieved, volume recorded, and finally 5-HT concentration was quantified using HPLC to look for evidence of pump function and potential degradation of 5-HT after 7 days within the pump in vivo. VI. Tissue Basal 5-HT Level Measurement: Thoracic aortae, vena cavae, superior mesenteric arteries, jugular veins, and carotid arteries were removed from pentobarbitaI-anesthetized normtotensive and hypertensive rats, cleaned of fat, connective tissue, and blood, and placed in 75 uL of 0.05 mM sodium phosphate and 0.03 mM citric acid buffer (pH 2.5) containing 15% methanol. Samples were frozen at -80°C at least four hours prior to HPLC quantification. Vll. 5-HIAA and 5-HT Concentration Measurement from Whole Blood: In anesthetized rats, 5 ml blood was collected from left cardiac ventricle using a heparinized (1000 U/L) 5 ml syringe and 22 gauge needle. The blood was gently transferred into a 7.0 ml EDTA anticoagulant vacutainer tube. Ten uM pargyline and 10 (1M ascorbic acid were added. The EDTA tubes were spun at 160 x g (1000 RPM) for 30 minutes at 4°C for platelet-rich-plasma (PRP). Two ml of supernatant containing plasma and buffy coat layer was gently pipetted into EDTA-coated plastic tubes and mixed gently with a 1:1 dilution of 0.5 M EDTA. 17 Ten uM pargyline and 10 (1M ascorbic acid were added. These tubes were centrifuged for 20 minutes at 1350 x g at 4°C for PPP. To the remaining pellet (platelet layer), 1 ml of platelet buffer and 1 uM ADP was added for PRP. Ten uM pargyline and 10 uM ascorbic acid were added. These tubes were vortexed and allowed to sit on ice for 15 minutes for platelets to become activated and degranulate. Then the tubes were centrifuged at 730 x g for 10 minutes at 4°C. Ten percent (T CA) 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. The samples were ultracentrifuged at 280,000 x g for 2 hours. The supernatant was collected, placed into new tubes and the samples were ready for quantification analysis. 5-HT and 5-HIAA concentrations were measured using electrochemical detection high-performance liquid chromatography (HPLC) at 0.4 V and 1.25 ml/min flow rate. VIII. 5-HIAA and 5-HT Measurement from selected vessels: Samples were thawed, sonicated for 3 seconds and centrifuged for 1 minute (10,000 x g). Supernatant was collected and transferred to new tubes. Tissue pellets were dissolved in 1.0 M NaOH and assayed for protein. The concentration of 5-HIAA and 5-HT in tissue supernatants was determined by isocratic High Performance Liquid Chromatography (HPLC) coupled with electrochemical detection. Twenty microliters of tissue supernatant was injected onto a C18 reverse phase analytical column (ESA, Bedfore, MA, USA). This column was coupled to a coulometric electrode conditional cell in series with dual 18 electrode analytical cells (ESA, Bedfore, MA, USA) at 0.4 V and 1.25 mI/min flow rate. 5-HIAA and 5-HT content was determined by comparing peak height in samples with a standard curve and from standards run the same day. Values are reported as a concentration normalized to vessel protein content. IX. Protein Isolation: Rat thoracic aortae, vena cavae, superior mesenteric arteries, jugular veins, and carotid arteries were removed from the rat and placed in PSS and cleaned as described above. Arteries were snap-frozen and pulverized in a liquid nitrogen-cooled mortar and pestle and solubilized in lysis buffer [0.5 M Tris HCI (pH 6.8), 10% SDS, 10% glycerol] with protease inhibitors [0.5 mM phenylmethylsulfonyl fluoride, 10 ugluL aprotinin/10 ug/ml leupeptin, and 0.1 M orthovanadate]. Homogenates were centrifuged (11,000 g for 10 min, 4°C). The Bicinchoninic Acid (BCA) method for protein measurement was used (Sigma, St. Louis, MO). Samples were stored at -80°C until use. X. BCA Protein Assay: The bovine serum albumin (BSA) protein standard, consisting of a known concentration of BSA, was utilized to make the standard curve to which the protein samples were compared. The working reagent was made by mixing BCA with copper ll sulfate (50:1). To determine the protein concentrations of samples, 5 (IL protein from each sample, 95 uL H20 and 2 ml working reagent were mixed and incubated in the absence of carbon dioxide for 30 minutes at 37°C. The 19 samples were analyzed on a spectrophotometer at an absorbance of 562 nm and the protein concentration determined by plotting these values on the standard curve performed on the same day. XI. Western Blotting: eNOS Tissue homogenates (4:1 in denaturing sample buffer, boiled for 5 min) were separated on SDS-polyacrylamide gels and transferred to lmmobilin-FL membrane. Membranes were blocked for 3 hours (Odyssey Blocking Buffer, Ll- COR Biosciences, Nebraska, USA) at 4°C. Blots were probed overnight with mouse lgG anti-eNOS/NOS Type III primary antibody (BD Transduction Laboratories) at a dilution of 1:1000 at 4°C, rinsed in TBS-Tween (TBS-T, pH 7.6) (20 mM Tris, 137 mM sodium chloride and 0.1% Tween-20), and incubated with fluorescent anti-mouse 800 secondary antibody for 1 hour in the dark at 4°C following with TBS-T washes in the dark. Blots were directly detected on the LI- COR Odyssey Infrared Fluorescent Imaging System (Ll-COR Biosciences, Nebraska, USA). PECAM-1 Tissue homogenates (4:1 in denaturing sample buffer, boiled for 5 min) were separated on SDS-polyacrylamide gels and transferred to lmmobilin-FL membrane. Membranes were blocked for 3 hours (Odyssey Blocking Buffer, Ll- COR Biosciences, Nebraska, USA) at 4°C. Blots were probed overnight with mouse lgG anti-PECAM-1 (D-11) primary antibody (Santa Cruz Biotechnology, 20 Inc.) at a dilution of 1:200 at 4°C, rinsed in TBS-Tween (TBS-T, pH 7.6) (20 mM Tris, 137 mM sodium chloride and 0.1% Tween-20), with a final rinse in TBS (20 mM Tris and 137 mM sodium chloride), and incubated with horseradish perioxidase-associated anti-mouse secondary antibody for 1 hour at 4°C following with TBS-T washes. Blots were incubated with ECL‘3 reagents to visualize the bands. .01a_ctin Blots were probed for 2 hours with mouse lgG anti-a-actin primary antibody (Santa Cruz Biotechnology, Inc.) at a dilution of 125000 at 4°C, rinsed in TBS-Tween (TBS-T, pH 7.6) (20 mM Tris, 137 mM sodium chloride and 0.1% Tween-20), with a final rinse in TBS (20 mM Tris and 137 mM sodium chloride), and incubated with horseradish perioxidase-associated anti-mouse secondary antibody for 1 hour at 4°C following with TBS-T washes. Blots were incubated with ECL® reagents to visualize the bands. XII. Isolated Smooth Muscle Contractility Measurement: The thoracic aorta and superior mesenteric artery was removed from pentobarbital-anesthetized rats and submerged in PSS. The aorta was cleaned of fat, connective tissue, and blood and cut into helical strips. Aortic strips were then mounted onto stainless steel rods with silk suture and placed into 50 ml tissue baths for isometric tension recordings using a force transducer and Chart® software program (ADlnstruments, Colorado Springs, CO, USA). Strips were placed under previously-determined optimum resting tension (1,500 mg for rat 21 aorta, 600 mg for rat superior mesenteric artery) and allowed to equilibrate for one hour with frequent washes before exposure to pharmacological compounds. To the best of my ability, in experiments testing tissues from hypertensive rats, one aortic strip isolated from a normotensive control and one aortic strip from a hypertensive rat, or one aortic strip from a rat receiving vehicle and one strip from a rat receiving 5-HT were placed in the same bath, thereby controlling for potential experimental variations. Tissue baths contained warmed (37°C), aerated (95% 02/C02) PSS. Administration of an initial concentration of 10 uM PE was used to test arterial smooth muscle strip viability; for rat aorta, the strips must contract to a minimum of 600 mg to be considered viable. For rat superior mesenteric artery, the strips must contract to a minimum of 200 mg to be considered viable. Cumulative concentration curves to selected agonists were performed. XIII. Data Analysis and Statistics: For blood pressure data analysis, within group differences were assessed by a one-way repeated measures ANOVA with post-hoc multiple comparisons using Dunnett’s procedure (GraphPad lnstat 3). Between 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 4). In all cases, a p-value of <0.05 was considered significant. All results are presented as mean 1 SEM. When comparing two groups, 22 the appropriate Student's t—test was used. In all cases, a p value less than or equal to 0.05 is considered statistically significant. 5-HIAA and 5-HT concentrations from vascular tissue were quantified using a standard curve, and also using standards run the same day, and reported as a concentration normalized to protein content. Isometric contractions are reported as force (milligrams) or as a percentage of response to maximum contraction to PE. ACh response curves are reported as a percentage of response to half-maximal contraction to PE. Band density quantitation in Western analyses was performed using NIH Image (v.1.61). For each sample, the densities of the tested bands on Western blotting are normalized to the density of the corresponding actin band. 23 Results: Hypothesis 1: Chronic 5-HT infusion will lead to increased blood pressure and arterial 5-HT content in normotensive rats; these responses will be enhanced in DOCA-salt hypertensive rats. Validation of model: plasma 5-HT measurrnents We wanted to prove that 5-HT concentrations in the circulating plasma as well as the peripheral blood vessels increase with osmotic pump implantation loaded with a 5-HT creatinine sulfate solution in both normotensive sham rats and hypertensive rats. To prove that 5-HT was released from the osmotic pump and reached the systemic circulation, we used HPLC analysis to detect and 5- HIAA and 5-HT content first in the plasma, and then in selected vessels. Figure 2 shows a standard chromatogram of a representative of all standard-mix tracings from HPLC (top) and then a representative chromatogram of basal 5-HT and 5-HIAA in rat aorta (bottom). To prove that 5-HT was released from the osmotic pump reaching the systemic circulation and remained viable throughout the duration of the infusion, whole blood was collected at the time of rat sacrifice after 7 days of 5-HT or vehicle infusion. We used HPLC to quantify 5-HT in both platelet-poor and platelet-rich plasma from uninephrectomized normotensive sham rats, designated Shamo, shown in Figure 3. 5-HT concentrations are increased in free circulating plasma (platelet-poor plasma, PPP) in the ShamD 5-HT-infused group, 24 47.11232 nglml plasma, as compared to the Shamo Vehicle-infused group, 2710.3 nglml plasma, an increase of 17.4 fold. The 5-HT contained within the granules of platelets (platelet-rich plasma, PRP) increased in the ShamD 5-HT- infused group, 257.7140.2 nglml plasma, as compared to the Shamo Vehicle- infused group, 31 .919.7 nglml plasma, an increase of 8 fold. Figure 4 shows the increased 5-HT levels in both components of plasma in DOCA rats after 7 days of 5-HT as compared to Vehicle infusion. 5-HT concentrations are increased in PPP in the DOCA 5-HT-infused group, 114.5121.1 nglml plasma, as compared to the DOCA Vehicle-infused group, 24.9150 nglml plasma, a 4.5 fold increase. The PRP 5-HT concentration increased in the DOCA 5-HT-infused group, 591312125 nglml plasma, as compared to the DOCA Vehicle-infused group, 137913535 nglml plasma, a 4.2 fold increase. Plasma measurements of 5-HT using HPLC indicated that the 5- HT administered via osmotic pump was in fact released and reached the systemic circulation. Validation of model: peripheral gscuLar tissue 5-HT measurements The quantification of basal 5-HIAA and 5-HT content levels in selected ShamD rat vessels harvested on Day 7 of Vehicle or 5-HT infusion are reported in Figure 5. As arterial smooth muscle cells have a functional SERT, we expected an increase in basal 5-HIAA and 5-HT content in those vessels harvested from 5- HT infused ShamD rats. As 5-HT is rapidly metabolized by the action of monoamine oxidase, and we have not inhibited the metabolism of 5-HT in this 25 experiment, we cannot forget to examine the metabolite 5-HIAA. In aorta, we observed an increase in 5-HIAA content as well as 5-HT content in the 5-HT infused Shamo (5-HIAA: 1.8108 nglmg protein; 5-HT: 0.510.4 nglmg protein, n=4) as compared to ShamD Vehicle-infused (5-HIAA: 0.410.2 nglmg protein; 5- HT: 0.3102 nglmg protein, n=4). In carotid artery, we observed an increase in 5- HIAA content as well as 5-HT content in the 5-HT infused ShamD (5-HIAA: 3.1108 nglmg protein; 5-HT: 4.811.7 nglmg protein, n=4) as compared to Shamo Vehicle-infused (5-HIAA: 1.010.2 nglmg protein; 5-HT: 0.410.1 nglmg protein, n=4). In the superior mesenteric artery, we observed an increase in 5-HIAA content as well as 5-HT content in the 5-HT infused Shamo (5-HIAA: 4.712.1 nglmg protein; 5-HT: 3610.4 nglmg protein, n=4) as compared to Shamo Vehicle-infused (5-HIAA: 0.5103 nglmg protein; 5-HT: 0910.8 nglmg protein, n=4). In veins, we observe a new phenomenon; the 5-HT measured in veins does not seem to be as rapidly metabolized to 5-HIAA as it is in arteries. Thus, we can measure higher 5-HT content in veins as compared to arteries, even under basal conditions, and less 5-HIAA content in veins as compared to arteries. In the vena cava, we observed an increase in 5-HIAA content as well as 5-HT content in the 5—HT infused Shamo (5-HIAA: 3.5105 nglmg protein; 5-HT: 27.0199 nglmg protein, n=4) as compared to Shamp Vehicle-infused (5-HIAA: 0.610.1 nglmg protein; 5-HT: 13910.4 nglmg protein, n=4). Similarly, in the jugular vein, we observed an increase in 5-HIAA content as well as 5-HT content in the 5-HT infused ShamD (5-HIAA: 3.1109 nglmg protein; 5-HT: 39.11116 26 nglmg protein, n=4) as compared to Shamo Vehicle-infused (5-HIAA: 0.410.1 nglmg protein; 5-HT: 8913.1 nglmg protein, n=4). 5-HIAA and 5-HT values from selected DOCA rat vessels harvested on Day 7 of Vehicle or 5-HT infusion are shown in Figure 6. We again observed a difference in 5-HT uptake in veins versus arteries. In aorta, we observed an increase in 5-HIAA content as well as 5-HT content in the 5-HT infused DOCA (5-HIAA: 1.410.1 nglmg protein; 5-HT: 4.211.1 nglmg protein, n=4) as compared to DOCA Vehicle-infused (5-HIAA: 0.110.1 nglmg protein; 5-HT: 0610.1 nglmg protein, n=4). In carotid artery, we observed an increase in 5-HIAA content as well as 5-HT content in the 5-HT infused DOCA (5-HIAA: 2.5109 nglmg protein; 5-HT: 6.7128 nglmg protein, n=4) as compared to DOCA Vehicle-infused (5- HIAA: 0.210.1 nglmg protein; 5—HT: 1810.4 nglmg protein, n=4). In the superior mesenteric artery, we observed an increase in 5-HIAA content as well as 5-HT content in the 5—HT infused DOCA (5-HIAA: 2110.3 nglmg protein; 5-HT: 4.9124 nglmg protein, n=4) as compared to DOCA Vehicle-infused (5-HIAA: 0.410.1 nglmg protein; 5-HT: 1.110.3 nglmg protein, n=4). Again, in veins, we observe the 5-HT measured in veins does not seem to be as rapidly metabolized to 5- HIAA as it is in arteries. In the vena cava, we observed an increase in 5-HIAA content as well as 5-HT content in the 5-HT infused DOCA (5-HIAA: 2710.6 nglmg protein; 5-HT: 31.7111.4 nglmg protein, n=4) as compared to DOCA Vehicle-infused (5-HIAA: 0.210.1 nglmg protein; 5-HT: 10.6109 nglmg protein, n=4). Similarly, in the jugular vein, we observed an increase in 5-HIAA content as well as 5-HT content in the 5-HT infused DOCA (5-HIAA: 3610.7 nglmg 27 protein; 5-HT: 184.4147.2 nglmg protein, n=4) as compared to DOCA Vehicle- infused (5-HIAA: 0.210.1 nglmg protein; 5-HT: 6510.5 nglmg protein, n=4). This increase in 5-HT and 5-HIAA content measured in tissues harvested from rats receiving 5-HT via infusion indicates that the 5-HT is transported somehow and does get to the peripheral vasculature, where SERT on the smooth muscle cells transports the 5-HT intracellularly. It is an interesting observation to see a difference in distribution of 5-HT and metabolite content between arteries and veins, as it appears that most of the 5-HT is rapidly metabolized in arteries, but seems to be preserved as 5-HT in the veins. DOCA-silt hvpenensionzgfied of 5-HT on telemetric measurements: Figure 7 shows the 24-hour averaged MAP of both Shamo and DOCA rats with 2 days of control baseline measurements and 7 days of 5-HT or Vehicle infusion. MAP started to fall within six hours of 5-HT release from the osmotic pumps, and reached a nadir at Day 2 of 5-HT infusion in both normotensive ShamD and hypertensive DOCA rats. The DOCA rats infused with 5-HT, had a resting baseline MAP of 166.5 1 7.6 mmHg during the control period, and experienced a maximal MAP fall of -53.7 mmHg at Day 2, with a MAP of 112.8 1 2.8 mmHg. The normotensive ShamD rats infused with 5-HT had a resting baseline MAP of 100.8 1 2.4 mmHg, and experienced a maximal MAP fall of - 21.6 mmHg at Day 2, with a MAP of 79.2 1 1.5 mmHg. Osmotic pump function and 5—HT via_bilitv after 7 dgy_s of infusion: 28 We measured the volume of the fluid remaining in the mini-osmotic pump after 7 days of infusion in the DOCA and the ShamD rats. Each mini-pump starting volume was 2 ml, and as the top graph in Figure 8 shows, there was no statistical difference in the remaining volume between the four groups, indicating the pumps released virtually equal amounts of either 5-HT or Vehicle at the end of 7 days of infusion. We measured the concentration of the 5-HT from the fluid remaining in the osmotic pumps at the end of 7 days of in vivo infusion, and the quantification is shown in the middle graph on Figure 8. The Vehicle pump samples contained 0 nglml 5-HT both prior to infusion and after pump removal from the rat as confirmed by HPLC (n=12). A representative chromatogram from a sample retrieved from a Vehicle pump is shown in the top of Figure 9. The HPLC quantification of the 5-HT fluid sampled before implant was 20.7 jug/ml. Post-explant from the rat after 7 days of infusion, a sample of fluid from each of the 5-HT pumps was quantified using HPLC and contained an average of 17810.6 ug/ml (n=18). The 5-HT pump fluid was so concentrated that the sample had to be diluted down 1:106 to be amenable to detection on HPLC. A representative chromatogram from a diluted sample retrieved from a 5-HT pump is shown in the bottom of Figure 9. We also tested the vasoactivity of the 5-HT in the remaining fluid from the osmotic pump after 7 days of use in a rat. An aliquot of this fluid (5 ul of 17.4 pg/ml) was added to an isometric contractility tissue bath to challenge a ring of aorta. The bottom graph in Figure 8 shows the response of the aortic ring. A 29 robust tissue contraction of over 6000 mg was observed, indicating the sustained vasoactivity of 5-HT even after 7 days at body temperature. 5-HT HCI infusion: We repeated this 5-HT infusion with another formulation of 5-HT, 5-HT hydrochloride, in a small experiment to show that this effect on blood pressure is in fact due to 5-HT itself and was not attributable to the creatinine sulfate complex conjugated to the 5-HT molecule. The averaged 24-hour MAP of 5-HT HCI infusion in DOCA rats is plotted with DOCA Vehicle and DOCA 5-HT (serotonin creatinine sulfate complex) and is shown in Figure 10. This indicates that the profound decrease in MAP to 5-HT remains the same, regardless of the formulation of the compound. As a result of this experiment, we utilized the 5-HT creatinine sulfate complex formulation in all of the subsequent experiments. Effect of 5-HT on DOCA-salt water intafl We observed that the DOCA rats receiving 5-HT greatly reduced their salt water consumption. We hypothesized that the blood pressure fall may result, at least in part, from a decreased salt intake as compared to DOCA Vehicle rats, whose salt-water intake did not change. The top graph in Figure 11 shows a grouped 7-day average of fluid consumption in both ShamD and DOCA rats. It is important to point out that the ShamD rats are consuming tap water, and the DOCA rats are consuming water supplemented with 1% NaCI and 0.2% KCI. There was no statistical difference in the ShamD rats when 5-HT is administered, 30 but the salt-water consumption by the DOCA rats receiving 5-HT was decreased to nearly half the consumption of DOCA Vehicle rats. We did one experiment when we restricted the salt-water consumption of the DOCA Vehicle rats based on how much the DOCA 5-HT rats drank. The 24-hour—grouped MAP of the DOCA Vehicles was not significantly altered (a 7 mmHg drop in MAP) when their salt-water consumption was restricted to about half of what they would have consumed ad Iibitum. 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