hum. a... in“? fivpugmm rm. .mmmasf gnaw... .één firuai 2mg. .3... ...mu% .2” . .. 1. ($.sz JHJIEP-V’de ;u..l.3v. 3......nbéian. i ‘.m.¥o. '.U...:».... I“? .nv “khaki“: I)... .MV..34 $3.11 51.1. 4... z 1:5... $5.4... 5...: This is to certify that the dissertation entitled Physiological Significance of the S-HTZB and 5-HT1B Receptors in Deoxycorticosterone Acetate-salt Hypertension presented by Amy Kissiah Lynn Banes has been accepted towards fulfillment of the requirements for Ph . D . degree in Pharmacology 8. Toxicology Skew CU L004? Major professor Date June 10, 2002 MS U is an Affirmative Action/Equal Opportunity Institution 0-12771 LIBRARY Michigan State University 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 7111111 5 20% , “VT 2 3 ZGM ‘L 5 1 ll. 0 4 6/01 cJClFle’DatODUOpBS-DJ 5 PHYSIOLOGICAL SIGNIFICANCE OF THE 5-HT23 AND 5- HT1B RECEPTORS IN DEOXYCORTICOSTERONE ACETATE-SALT HYPERTENSION BY Amy Kissiah Lynn Banes A DISSERTATION Submitted to Michigan State University In partial fulfillment of the requirements For the degree of DOCTOR OF PHILOSOPHY Department of Pharmacology and Toxicology 2002 ABSTRACT PHYSIOLOGICAL SIGNIFICANCE AND MECHANISMS OF THE UPREGULATION OF THE 5-HT2,3 AND 5-HT1B RECEPTORS IN DEOXYCORTICOSTERONE ACETATE-SALT HYPERTENSION BY Amy Kissiah Lynn Banes 5-HT is a neurotransmitter which possesses a plethora of actions in the body including vasoconstrictor and mitogenic actions in the vasculature. The specific aims of this project were to determine the mechanisms of enhanced sensitivity to 5-HT observed in DOCA-salt hypertension and the physiological relevance of the 5-HT receptor subtypes, which mediate contraction to 5-HT in the vasculature. I tested the hypothesis that during the condition of DOCA-salt hypertension there is a switch from the 5-HT2A receptor to the 5-HT1B and 5-HT28 receptors as the receptors which primarily mediate 5-HT-induced contraction in arteries from hypertensive rats. To address this hypothesis we characterized the 5-HT receptor subtypes found in vascular smooth muscle With Southern analysis. There was mRNA detected for 5-HT13, 5-HT,D, 5-HT,F, 5-HT2A, and 5-HT23 receptors. Contractility experiments with selective agonists of these receptors demonstrated no role for 5- HT1D and 5-HT1F receptors in arteries from norrnotensive sham and hypertensive DOCA-salt rats. 5-HT.8 and 5-HT23 receptor agonists elicited contraction only in arteries from DOCA-salt rats. Inhibition of both 5-HT23 and 5-HT1B receptors normalized the contractile response to 5-HT in arteries from DOCA-salt hypertensive rats compared to that observed in arteries from normotensive sham rats. This suggests that both the 5-HT1B and 5-HT28 receptors mediate the hyperresponsiveness to 5-HT observed in arteries from hypertensive rats. The functional upregulation of these receptors under conditions of DOCA- salt hypertension were supported by Western analysis. We found a significant increase in the level of 5-HT"3 and 5-HT2,3 receptor protein levels in the aorta and mesenteric arteries from DOCA-salt hypertensive rats. However, we found no increase in the level of 5-HT"3 and 5-HT2.3 receptor mRNA levels. This finding suggests that the receptor upregulation is not occurring at the transcriptional level. Additional studies revealed that aldosterone in vitro increased the 5-HT“3 and 5- HT2.3 receptor protein levels in a time and concentration-dependent manner. These effects were blocked by the mineralocorticoid receptor antagonist spironolactone. Further studies also demonstrated that prior to an increase in blood pressure, an increase in 5-HT2.3 receptor function can be observed after 24 hr of treatment with DOCA and salt. Additional data from the time course studies while suggesting that down stream effectors in the signaling cascade may be altered also support the idea that the level of 5-HT1B and 5-HT2E3 receptor proteins is not the critical change which allows for functional receptor coupling to contraction. These data suggest that the 5-HT2.3 and possibly the 5-HT"3 receptors may be important to the development and maintenance of hypertension. Dedication To my parents, James and Patricia, for teaching me to be grateful for the blessing that I have received and remembering what really matters. iv ACKNOWLEDGEMENTS I would first like to acknowledge my mentor, Dr. Stephanie Watts. My heartfelt thanks for the guidance and support, both personal and professional. Your patience and encouragement have helped me to grow and have set an example for me to follow. Thank you. I would like to thank the members of my thesis committee, Dr. Greg Fink, Dr. James Galligan, Dr. Donna Wang and Dr. Norbert Kaminski for your insight, time and guidance. To Dr. Sue Barman I also direct a thank you. She always made time in her busy schedule to listen, encourage and offer advice. Thank you, Sue. I would also like to thank the members of the Watts lab, both present and past, for their friendship and support. I would especially like to thank Carrie Northcott for her friendship and support. She has been a constant source of support and encouragement when I needed it the most. I will miss you Carrie. Finally, I want to express my sincere gratitude, appreciation and love to my family for their love and support, both personal and financial. I could not have done this without you. TABLE OF CONTENTS List of Tables .......................................................................................................... viii List of Figures .......................................................................................................... ix List of Abbreviations ............................................................................................... xiii I. Introduction A. Serotonin ......................................................................................... 1 1. Biosynthesis and storage ................................................................... 2 2. 5-HT Receptors ............................................................................... 2 a. 5-HT2A Receptor .......................................................................... 3 i. Physiology ...................................................................................... 3 ii. Location .......................................................................................... 4 iii. Regulation ...................................................................................... 4 iv. Pharmacology ................................................................................ 5 b. 5-HT2.3 Receptor ......................................................................... 8 i. Physiology ...................................................................................... 8 ii. Location ........................................................................................ 10 iii. Regulation .................................................................................... 11 iv. Pharmacology ............................................................................... 11 c. 5-HT,.3 and 5-HT1F Receptorsl8 i. Physiology ..................................................................................... 18 ii. Location ............. 18 iii. Regulation ..................................................................................... 19 iv. Pharmacology ............................................................................... 19 d. 5-HT1B Receptor ...................................................................................... 20 i. Physiology ..................................................................................... 20 ii. Location ......................................................................................... 21 iii. Regulation ..................................................................................... 22 iv. Pharmacology ............................................................................... 22 B. Hypertension ................................................................................ 23 1 .Hemodynamics ............................................................................... 26 2.Mineralocorticoids....................................................................— ..... 27 3.lncreased responsiveness .......................................................................... 31 C. Hypothesis........: ......................................................... ' ................................... 3 7 ll. Methods .............................................................................................................. 38 A.General Animal Methods ............................................................................... 38 1. Animals ..................................................................................................... 38 2. Mineralocorticoid Hypertension ................................................................ 38 3. Blood Pressure Measurement .................................................................. 38 vi B. Isolated Smooth Muscle Contractility Measurements in Aorta and Superior Mesenteric Arteries ............................................................................................. 39 C. Depolarization Protocol ...................................................................................... 40 D. Aldosterone Incubation Procedures ................................................................... 40 E. Tissue Homogenization for Protein Work .................................. 41 F. Western Analysis Procedures ........................................................................... 41 G. Real Time RT-PCR ............................................................................................ 42 H. Measurement of Plasma 5-HT ........................................................................... 43 I. Data Analysis and Statistics .............................................................................. 44 III. Results ............................................................................................................... 46 A. Hypothesis l ................................................................................................ 46 1. Profiling of 5-HT Receptors .................................................................... 46 2. Unmasking “Silent Receptors” ............................................................... 68 B. Hypothesis ll ............................................................................................... 78 1. Receptor Upregulation ........................................................................... 78 C. Hypothesis Ill ............................................................................................. 86 1. Mechanisms of Receptor Regulation ....................................................... 86 2 Aldosterone-incubation Time Course Studies ........................................... 86 3. Aldosterone-incubation Concentration response Curve Studies ............. 87 4. Aldosterone- and DOCA-incubation Studies with Spironolactone ........... 92 5. Aldosterone-incubation and Contraction Studies ..................................... 97 6. DOCA-salt Time Course Studies ............................................................. 97 7. Systolic Blood Pressure ......................................................................... 101 8. Contractile Studies on Day 1 ................................................................. 109 9. Contractile Studies on Day 3 ................................................................. 109 10. Contractile Studies on Day 5 ............................................................... 114 11. Contractile Studies on Day 7 ............................................................... 117 12. Protein Analysis Studies ...................................................................... 120 D. Hypothesis lV ............................................................................................ 126 1. Measurement of Free Plasma 5-HT Levels .......................................... 126 IV. Discussion ...................................................................................................... 129 A. Characterization of 5-HT receptors in Vascular Smooth Muscle ............ 130 B. 5-HT,B Receptors in Hypertension ........................................................... 131 C. 5-HT2.3 Receptors in Hypertension ........................................................... 132 D. Investigation of 5-HT Receptor “Unmasking” .......................................... 135 E. Upregulation of 5-HT18 and 5-HT2.3 Receptors in DOCA-salt Hypertension ............................................................................................ 138 F. Regulation of 5-HT1B and 5-HT23 Receptors ............................................. 138 G. Speculation .............................................................................................. 142 V. Conclusion ....................................................................................................... 148 VI. References ...................................................................................................... 152 vii Table 1. Table 2. LIST OF TABLES Binding affinities obtained from the literature for the 5"HT1D! S'HT1F! 5'HT2A, 5'HT28 and 5'HT1B receptors ...................................................................... 16 Systolic blood pressures, EC50 values for BW723086, CP93129 and 5-HT and the maximal contractile responses to BW723086, CP93129 and 5-HT .......... 108 viii Figure 1. Figure 2. Figure 3. Figure 4. Figure 5. Figure 6. Figure 7. Figure 8. Figure 9. Figure 10. Figure 11. Figure 12. LIST OF FIGURES Proposed signaling pathways for the 5-HT2A receptor .......................... 7 Proposed signaling pathways for the 5-HT23 receptor ........................ 14 Proposed signaling pathways for the 5-HT1B receptor ........................ 25 Effect of 5-HT in endothelium-denuded rat aorta and mesenteric arteries from normotensive Sham and hypertensive DOCA-salt rats .................................................................................................... 34 Results of RT-PCR analysis and northern blotting of rat thoracic aorta denuded of endothelium for 5-HT receptors ............................. 48 Effect of the 5-HT1') receptor agonist PNU0142633 in endothelium- denuded thoracic aorta from normotensive Sham and hypertensive DOCA-salt rats ................................................................................... 50 Effect of the 5-HT1,: receptor agonists BRL54443 and LY344864 in the endothelium-denuded thoracic aorta from normotensive Sham and hypertensive DOCA-salt rats ...................................................... 51 Effect of the 5-HT2A antagonist ketanserin on BRL54443-induced contraction in endothelium-denuded thoracic aorta from normotensive Sham and hypertensive DOCA-salt rats ..................... 54 Effect of the 5-HT2A receptor antagonist ketanserin on BRL54443- induced contraction in the endothelium-denuded superior mesenteric artery of normotensive Sham and hypertensive DOCA-salt rats ...... 56 Effect of 5-HT,.3 agonists in endothelium-denuded thoracic aorta from normotensive Sham and hypertensive DOCA-salt rats ..................... 60 Effect of the 5-HT,B antagonist GR55562 on 5-HT—induced contraction in the aorta and mesenteric arteries from normotensive Sham and hypertensive DOCA-salt rats ........................................... 63 Effect of the 5-HT,.3 receptor antagonist GR55562 on the 5-HT“3 receptor agonist CP93129-induced contraction in the mesenteric artery from normotensive Sham and hypertensive DOCA-salt rats .................................................................................................... 65 ix Figure 13. Figure 14. Figure 15. Figure 16. Figure 17. Figure 18. Figure 19. Figure 20. Figure 21. Figure 22. Effect of the 5-HT,.3 and 5-HT2B receptor antagonists GR55562 and LY272015 on the 5-HT-induced contraction in the mesenteric artery from normotensive Sham and hypertensive DOCA-salt rats .................................................................................. 67 Effect of 15 mM KCI depolarization on 5-HT"3 agonist-stimulated contraction in the rat aorta in the presence of ketanserin. Effect of 15mM KCI depolarization on sumatriptan-, CP93129, BW723C86 and RU24969-induced contraction in aorta from normal Sprague-Dawley rats ............................................................ 73 Effect of ketanserin on the 5-HT-induced contraction with 15 mM KCl in the mesenteric artery of normal Sprague-Dawley rats and hypertensive DOCA-salt rats ............................................................. 75 Effect of KCI (15 mM) on CP93129- and 5-HT-induced contraction and effects of the or, adrenergic agonist phenylephrine in the presence and absence of ketanserin in the mesenteric artery from normal Sprague-Dawley rats. .......................................................... 77 Measurement of 5-HT,B and 5-HT2,3 receptor density in the endothelium-denuded thoracic aorta from normotensive Sham and hypertensive DOCA-salt rats ...................................................... 81 Measurement of 5-HT,B and 5-HT2,3 receptor density in the endothelium-denuded mesenteric artery from normotensive Sham and hypertensive DOCA-salt rats ...................................................... 83 Real Time PCR measurements of 5-HT,B and 5-HT2.3 receptor mRNA in the endothelium-denuded thoracic aorta from normotensive Sham and hypertensive DOCA-salt rats ...................... 85 Measurement of 5-HT,.3 and 5-HT2,, receptor density to determine the effects of aldosterone (100 nM) for variable lengths of time (8,12,24 and 48 hrs) in the endothelium-denuded thoracic aorta from normal Sprague-Dawley rats ..................................................... 89 Measurement of 5-HT1B and 5-HT2,3 receptor density to determine the effects of aldosterone (1 nM — 100 nM) for 12 hrs in the endothelium-denuded thoracic aorta from normal Sprague-Dawley rats ..................................................................................................... 91 Measurement of 5-HT1B and 5-HT2.3 receptor density to determine the effects of incubation of Spironolactone on aldosterone-induced upregulation of the 5-HT“3 and 5-HT28 receptors in endothelium- denuded thoracic aorta from normal Sprague-Dawley rats ................ 95 X Figure 23. Figure 24. Figure 25. Figure 26. Figure 27. Figure 28. Figure 29. Figure 30. Figure 31. Figure 32. Figure 33. Measurement of 5-HT,,3 and 5-HT2,3 receptor density to determine the effects of incubation of spironolactone on DOCA-induced upregulation of 5—HT1B and 5-HT28 receptor proteins in endothelium- denuded thoracic aorta from normal Sprague-Dawley rats ................. 96 Effect of aldosterone (100 nM; 12 hr) incubation on 5-HT—, BW723C86- and CP93129-induced contraction in the endothelium-denuded thoracic aorta from normal Sprague-Dawley rats ....................................................................................................... 98 Measurement of 5-HT,,3 and 5-HT28 receptor density in the endothelium—denuded thoracic aorta from Sprague-Dawley rats incubated with aldosterone and contracted to 5-HT, BW723CB6 and CP93129 ........................................................................................... 100 Measurement of mass in Sham, DOCA-salt, High salt and DOCA-low salt on days 1,3,5 and 7 of treatment ............................. 103 Measurement of fluid consumption by day and treatment group ..... 105 Measurement of systolic blood pressures. ...................................... 106 Effect of 5-HT, BW723086 and CP93129 in endothelium-denuded thoracic aorta from Sham, DOCA-salt, High salt and DOCA-low salt rats on day one of treatment ............................................................ 110 Effect of 5-HT, BW723C86 and CP93129 in endothelium-denuded thoracic aorta from Sham, DOCA-salt, High salt and DOCA-low salt rats on day three of treatment .......................................................... 113 Effect of 5-HT, BW723086 and CP93129 in endothelium-denuded thoracic aorta from Sham, DOCA-salt, High salt and DOCA-low salt rats on day five of treatment ............................................................ 116 Effect of 5-HT, BW723086 and CP93129 in endothelium-denuded thoracic aorta from Sham, DOCA-salt, High salt and DOCA-low salt rats on day seven of treatment ........................................................ 119 Measurement of 5-HT,,3 receptor protein density in endothelium- denuded thoracic aortic homogenates from Sham, DOCA-salt, DOCA-low salt and High salt treated rats on days 1,3,5 and 7 of treatment ......................................................................................... 121 xi Figure 34. Figure 35. Figure 36. Measurement of 5-HT2,3 receptor protein density in endothelium- denuded thoracic aortic homogenates from Sham, DOCA-salt, DOCA-low salt and High salt treated rats on days 1,3,5 and 7 of treatment ......................................................................................... 123 Level of 5-HT in platelet poor and platelet rich fractions of plasma from normotensive Sham and hypertensive DOCA-salt rats ................................................................................................. 128 Speculation on signaling pathways which may be involved in 5-HT-induced contraction via the 5-HT2,3 and 5-HT,B receptors in arteries from DOCA-salt hypertensive rats .................................... 147 xii AA ACE ADP AMPK Ang II bHLH BW723086 CAMP Ca2+ CGRP 5-CT c-fos CO DAG DOC DOCA-salt DMEM DMSO EDTA ET-1 List of Abbreviations Arachidonic Acid Angiotensin I converting enzyme Adenosine diPhosphate AMP-Activated Protein Kinase Angiotensin II Basic Helix Loop Helix 1-[5(2-thienylmethoxy)-1 H-3-indolyl]propan-2- amine hydrochloride Cyclic Adenosine monophosphate Calcium Calcitonin Gene Related Product 5-Carboxamidotryptamine Finkel-Biskis-Jinkins Osteosarcoma oncogene homologue Cardiac Output Diacylglycerol Deoxycorticosterone Deoxycorticosterone acetate-salt Dulbecco’s Modified Eagle Medium Dimethylsulfoxide Ethylenediamine Tetraacetic Acid Endothelin-l xiii eNOS ERK GAPDH G protein GRE GTP 5HIAA HPLC H202 5-HT lHC iNOS 5-NOT IPa JNK LNAME LNNA LY27201 5 LY344864 MAP Endothelial Nitric Oxide Synthase Extracellular Signal Regulated Kinase Gcheraldehyde-Phosphate Dehydrogenase GTP-dependent regulatory protein Glucocorticoid Response Element Guanosine triphosphate 5-hydroxyindole acetic acid High performance liquid chromatography Hydrogen Peroxide 5-hydroxytryptamine lmmunohistochemistry Inducible Nitric Oxide Synthase 5-Nonyloxytryptamine lnositol triphosphate c-Jun N-terminal Kinase NG-nitro-L-arginine methylester Nm-Nitro-L-Arginine 6- methyl-1 ,2,3,4-tetrahydro-1-[3,4- dimethoxyphenyl)methyl]-9H-pyrido[3,4- b]indole hydrochloride (R)-N-[3Dimethylamino-2,3,4,9-tetrahydro-1 H- carbazol-6-yl]-4-fluorobenzamine Mean Arterial Pressure xiv MAPK MRE MAPKK MUPP1 mRNA NAD(P)H Oxidase NO' 02. PCR PDGF PDZ domain PE Pl3-K PKB PKC PLA2 PLC PLD PSS RAS RG84 ROS Mitogen Activated Protein Kinase Mineralocorticoid Response Element Mitogen Activated Protein Kinase Kinase Multi-PDZ Domain Protein1 Messenger Ribonucleic Acid Nicotinamide Adenine Dinucleotides (Phosphate) Hydride Oxidase Nitric Oxide Superoxide Anion Polymerase Chain Reaction Platelet Derived Growth Factor PSD-95/Discs Large/ZO-l Phenylephrine Phosphatidylinositol 3— Kinase Protein Kinase B Protein Kinase C Phospholipase A2 Phospholipase C Phospholipase D Physiological Salt Solution Renin-Angiotensin System Regulator of G-Protein Signaling Protein 4 Reactive Oxygen Species XV SAPK SBP SIK SDS SH-3 SHR SNFl SOS SRF TBS TBS-T TCA TPR Stress Activated Protein Kinase Systolic Blood Pressure Salt-Inducible Kinase Sodium Dodecyl Sulfate Src Homology-3 Spontaneously Hypertensive Rat Sucrose-Nonfermenting 1 Protein Kinase Sodium Octyl Sulfate Serum Response Element Tris Buffered Saline Tris Buffered Saline + Tween Trichloroacetic Acid Total Peripheral Resistance xvi Introduction I. Serotonin Serotonin (5-hydroxytryptamine, 5-HT) was isolated from bovine serum by Rapport and colleagues in 1948 (Rapport et al, 1948). Since its discovery, 5-HT has been a molecule which has generated much interest and controversy in cardiovascular as well as behavioral and neurochemical research fields. 5-HT possesses a plethora of vascular effects including vasoconstriction (Hoyer, 1989), vasodilation (Ellis et al, 1995) and mitogenic actions (Launay et al, 1996). These vascular effects have led to the implication of 5-HT in diseases such as migraine (Fozard, 1995),-pulmonary hypertension (Keegan et al, 2001), atherosclerosis (lshida et al, 2001), mineralocorticoid-induced hypertension (Watts and Fink, 1999), Raynaud’s phenomenon (Coleiro et al, 2001, Igarashi et al, 2000) and as a contributor to liver injury from ischemia and reperfusion (Nakamura et al, 2001). 5-HT is involved in irritable bowel syndrome (Miwa et al, 2001), brain development (Witaker-Azmitia, 2001) and cardiac development (Nebigil and Maroteaux, 2001 ). Interestingly, 5-HT potentiates both agonist- induced contraction (Watts, 2000) and agonist-induced smooth muscle cell proliferation for other vasoactive substances such as endothelin (Watanabe et al, 2001). The wide variety of actions possessed by 5-HT necessitates an understanding of the role of 5-HT in normal and diseased state. Additionally, understanding the receptors that 5-HT interacts with to produce its myriad of effects in normal and diseased states is also necessary. Biosynthesis and storage 5-HT is an indolethylamine and is produced both centrally by serotonergic neurons and peripherally in the enterochromaffin cells of the gut. The synthesis of 5-HT begins with the uptake of the amino acid L-tryptophan. L-tryptophan is converted by tryptophan hydroxylase to 5-hydroxytryptophan. 5- hydroxytryptophan is then decarboxylated by aromatic amino acid decarboxylase to 5-HT. 5-HT is metabolized by monoamine oxidase to form 5- hydroxyindoleacetaldehyde. This is then converted by the enzyme aldehyde dehydrogenase to 5-hydroxyindole acetic acid (5HIAA). In the human body 5HIAA is the primary metabolite excreted (T yce, 1985). Both platelets and nerves store 5-HT and platelets serve as the primary source of 5-HT for the vasculature. 5-HT Receptors 5-HT acts at plasma membrane receptors to mediate its myriad of effects. 5-HT receptors have been divided into seven classes with subtypes in many of these classes. Most of the 5-HT receptors are classified as heptahelical, G- protein coupled receptors. The 5-HT3 class of receptors is the one exception as the members of the 5-HT3 class of receptors are ion channels. Among the known 5-HT receptors, there are five that have been suggested to mediate vascular smooth muscle contraction to 5-HT; the 5-HT 2A, 5-HT 23, 5-HT ,3, 5-HT1D and the 5-HT ,F receptors (Smith etal, 1999, Watts etal, 1996,Razzaque etal, 1999, VanDenBrink et al, 2000). 5-HT2A Receptor Physiology The 5-HT2A receptor has been characterized as a heptahelical G-protein coupled receptor (Roth ef al, 1995). Specifically, the 5-HT2A receptor couples to the Gq,11 family of G-proteins (Li et al, 1997). 5-HT2A receptors couple to the extracellular signal regulated kinase (ERK)/ mitogen activated protein kinase pathway (MAPK) (Grewal et al, 1999). The ERK/MAPK pathway is one of the three recognized mitogen activated protein kinase pathway (MAPK) pathways. , Of the three canonical MAPK pathways 5-HT, acting via the 5-HT2A receptor, appears to only activate the ERK/MAPK pathway. The c-Jun N-terminal kinase (JNK)/ stress activated protein kinase (SAPK) and p38 pathways are not activated by 5-HT via the 5-HT2A receptor (Banes et al, 2001). Activation of the ERK/MAPK pathway by the 5-HT2A receptor has been linked to generation of superoxide anion (02') and H202 (Lee et al, 2001, Greene et al, 2000). This receptor couples to phospholipase C (PLC), protein kinase C (PKC), src and L- type calcium channels (Pandey et al, 1995, Watts et al, 1994, Banes et al, 1999) (figure 1). Furthermore, internalization of this receptor is dependent on dynamin and independent of arrestins (Bhatnagar et al, 2001). It has been suggested that this internalization may be involved in receptor resensitization (Gray et al, 2001). The 5-HT2A receptor is the only 5-HT receptor that has been characterized so thoroughly with regards to internalization and its effects on signaling mechanisms. Location The 5-HT2A receptor mRNA has been located in vascular smooth muscle cells (Watts et al, 2001) and in regions of the brain and spinal cord (Cyr et al, 2000, Helton et al, 1994). Functionally the 5-HT2A has been suggested to be in platelet membranes (Kagaya et al, 1990), renal messangial cells (Greene et al, 2000) and bovine pulmonary artery smooth muscle cells (Lee et al, 2001). 5-HT2A receptors have been implicated in several tissues as mediators of contraction including: the rat aorta (Florian and Watts, 1998), rabbit mesenteric artery (Y ildiz and Tuncer, 1995), rat trachea and guinea pig aorta (Baez et al, 1994), human umbilical artery (Lovren etal, 1999), rat pulmonary artery (MacLean etal, 1996) and human coronary artery (Nilsson et al, 1999a). Under conditions of normal blood pressure the 5-HT2A receptor appears to be the predominate receptor which mediates contraction to 5-HT in the rat aorta and mesenteric arteries. Regulation Currently, there is very little known about the regulation of the 5-HT2A receptor. This receptor has been clonedand assigned to chromosome 13q14- q21 in the human (Sparkes et al, 1991) and the promoter for the human gene has been under investigation. Currently, sequence analysis of the 5-HT2A receptor promoter has revealed the presence of at least two promoter regions, a silencer, Sp1 sites, PEA3 sites, cyclic AMP response element (CRE) sequences and E-boxes (Zhu et al, 1995). However, the presence of these elements has not lead to any further characterization of the mechanisms by which expression of this gene is controlled or what substances may effect expression of this receptor. There have been several studies performed which involved receptor downregulation through agonist and antagonist exposure (Roth et al, 1990, Roth, 1999,Berry et al, 1996). Interestingly, changes in the levels of 5-HT2A receptor mRNA have been noted to be both independent and dependent on protein kinase C (PKC) activation (Anji et al, 2001, Wohlpart and Molinoff, 1998). The decreases in 5-HT2A receptor mRNA levels in the C6 glioma cell line implicate PKCa and/or PKCy activation in response to 5-HT stimulation (Anji et al, 2001). In P11 cells, an immortalized cells line, an increase in 5-HT2A receptor mRNA levels through PKC dependent and independent mechanisms has been demonstrated (Wohlpart and Molinoff, 1998). These increases appear to be due to an increased stability of the 5-HT2A receptor mRNA. Pharmacology Phan'nacologically, the 5-HT2A receptor is characterized by stimulation with the agonists 5-HT, or-methyl 5-HT and 1-(2,5-dimethoxy-4-iodophenyI)-2- aminopropane (DOl). Ketanserin is a widely utilized 5-HT2A antagonist. Another 5-HT2A antagonist, MDL100907, is considered the most selective 5-HT2A antagonist based upon binding affinity (Kehne et al, 1996). Figure 1. Diagram of the proposed signaling mechanisms utilized by the 5-HT2A receptor. Abbreviations: PLC=Phospholipase C; PKC2 Protein Kinase C, ERK=Extracellular signal regulated kinase; PLA2= Phospholipase A2; IP3= lnositol triphosphate; DAG: Diacylglycerol; 02': Superoxide; NAD(P)H oxidase: Nicotinamide adenine dinucleotides (phosphate) hydride; H202 = hydrogen peroxide; MAPKK: Mitogen activated protein kinase kinase; Srf= Serum response factor; SRE: Serum response element; c-fos: Finkel-Biskis-Jinkins osteosarcoma oncogene homologue. c365 :8 I - . \NONI $mSE£u oExoLoaam / o\V $6:me IIV cosombcoo > “0 v_v_n_<_2 ommExo IEVO + 325% 62.6 2.52 1 Bacomm 3.53 Al ._.I-m .8 _m__m£ouco .. m m T - w .m a w c. w M m m m I I 14 Table 1: Binding affinities obtained from the literature for the 5-HT1B receptor, 5- HT1D receptor, 5-HT1,: receptor, 5-HT2A receptor and 5-HT2,3 receptor. Values are reported as pKi = - log Ki [M]. 15 <2 em 3 no 2 2.: mmmmmmw 2m 3 m v E. Em at 5 2282962 8 mg a v a v s v a: $885 wumEomaEa. <2 a A <2 <2 S c 5 mm 580 <2 <2 <2 ms 3 a: mmmmv 5:22 <2 <2 we <2 No 5 <09.va m v mm as am I 3&3 8.3 35% ox mm 3 mg m6 E 9433mm NH <2 <2 ms me w 89 mmmvmnm me E as No E mwOmRZm 12923 Imfimmm oe am as 8d. 5 .:._-m mumEom< mgr—Am <~h:-m ”— 5.....m 05.1% m _.._.:-m ucsonEoo E: 2 521% 2an 22:2 9.62m 9399: are 16 References for Table 1: 1. Hoyer D, In: The peripheral actions of 5-hydroxytryptamine. Fozard, JA., ed. Oxford: Oxford University Press, 1989. 2. Wainscott D, Cohen ML, Schenck KW, Audia JE, Nissen JS, Baez M, Kursar JD, Lucaites VL, Nelson DL. Pharmacological characteristics of the newly cloned rat 5-hydroxytryptaminezp receptor. Mol. Pharmacol. 43: 419-426, 1993. 3. Bonhaus DW, Bach C, DeSouza A, Salazar FH, Matsuoka BD, Zuppan P, Chan HW, Eglen RM. The pharmacology and distribution of human 5-hydroxytryptamine23 (5- HT2B) receptor gene products: comparisonwith 5-HT2A and 5-HTZC receptors. Br. J. Pharmacol. 1 15: 622-628, 1995. 4. Kennett GA, Bright F, Trail B, Baxter GS, Blackburn TP. Tests of the 5- HT28 receptor agonist, BW723086, on three rat models of anxiety. Br. J. Pharmacol. 117: 1443-1448.1996. 5. Choi DS, Birraux G, Launay JM, Maroteaux L. The human serotonin 5-HT23 receptor: pharmacological link between 5-HT2 and 5-HT,D receptors. FEBS Lett. 352(3): 393- 399, 1 994. 6. Adham N, Romanienko P, Harlig P, Weinshank RL, Branchek T. The rat 5- hydroxytryptaminem receptor is the species homologue of the human 5- hydroxytryptaminem beta receptor. Mol. Pharmacol. 41: 1-7, 1992. 7. Brown AM, Avenell K, Young TJ, Ho M, Porter RA, Vimal M, Middlemiss DN. BRL54443, a potent agonist with selectivity for human cloned 5-HT,E and 5-HT,F receptors. abstract 233P, 1989. 8. Wainscott DB, Lucaites VL, Kursar JD, Baez M, Nelson DL. Pharrnacologic characterization of the human 5-hydroxytryptaminezg receptor: evidence for species differences. J. Pharrnaool. Exp. Ther. 276: 720-727, 1996. 9. Phebus LA, Johnson KW, Zgombick JM, Gilbert PJ, Van Belle K, Mancuso V, Nelson DL, Calligaro DO, Kiefer AD, Branchek TA, Flaugh ME. Characterization of LY344864 as a pharmacological tool to study 5-HT,F receptors: binding affinities, brain penetration and activity in the neurogenic dural inflammation model of migraine. Life Sci. 61(21): 2117-2126, 1997. 10.Personal communication with Robert McCall, Upjohn-Pharmicia,1999 11.Personal communication with Tocris .2000. 12.Cohen ML, Schenck KW, Mabry TE, Nelson DL, Audia JE. LY272015, a potent, selective and orally available 5-HT 29 receptor antagonist. J Ser. Res: 1-14, 1996. 13.Adham N, Kao HT, Checter LE, Bard J, Olsen M, Urquhart D, Durkin M, Hartig PR, Weinshank RL, Branchek TA. Cloning of another human serotonin receptor (5-HT,F): a fifth 5-HT1 receptor subtype coupled to the inhibition of adenylate cyclase. Proc. Natl. Acad. Sci. USA. 90(2): 408-412,1993. 14.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. Euro. J. Pharmacol. 372: 49-56,1999. l7 5-HT1D and 5-HT,F Receptors Physiology The 5-HT1D and 5-HT1F receptors are heptahelical G-protein coupled receptors, utilizing Gi as well as to Go in signal transduction (Adham et al, 1993, Wurch and Pauwel, 2000). Members of this. family of receptors couple to inhibition of adenylate cyclase. Until recently there has been much controversy as to the roles of the 5-HT,,3 (also known as the 5-HT,DB receptor) and 5-HT1D (also known as the 5-HT,D,, receptor) receptors due to a lack of specific pharmacological tools to discriminate between these two structurally similar receptors. Interestingly, 5-HT,D receptors are found to be co-localized by double immunohistochemical staining with substance P in the rat trigeminal ganglion neurons and with substance P, calcitonin gene-related peptide (CGRP) and nitric-oxide synthases in neurons in humans (Ma et al, 2001, Hou et al, 2001). These studies provide correlative evidence for the theory that anitmigraine drugs which activate 5-HT1D receptors may inhibit substance P and CGRP release. Location 5-HT,F receptor mRNA has been localized to the human middle meningeal arteries (Razzaque et al, 1999), human trigeminal ganglia (Bouchelet et al, 1996), human coronary arteries (Bouchelet et al, 2000) and the rabbit saphenous vein (Cohen and Schenck, 1999). Additionally, 5-HT1,: receptor mRNA has also been localized to the guinea pig trigeminal ganglion neurons where they mediate 5-HT stimulated inhibition of neurogenic dural inflammation (Johnson et al, 1997). 5-HT1F receptors decrease c-fos expression in the rat 18 trigeminal nucleus caudalis (Mitsikostas et al, 1999). The 5-HT1D receptor mRNA has been localized to human heart valve interstitial cells (Roy et al, 2000), porcine cerebral cortex (Bhalla et al, 2000) and as autoreceptors in the guinea pig mesencephalic raphe, hippocampus and frontal cortex (el Mansari and Blier, 1996). The 5-HT1D receptor has been localized by immunohistochemical staining in rat trigeminal ganglion neurons (Ma et al, 2001) and human trigeminal ganglion (Hou et al, 2001). Regulation There is a dearth of information about the regulation of the 5—HT1D and 5- HT1F receptors. Most current research has been directed to the functional and pharmacological characterization of these receptors. No analysis of the promoters has been reported. Additionally, there have been no studies reported looking at regulators of these receptors. Signaling mechanisms utilized by these receptors are also not clearly defined. Pharmacology The 5-HT,,= receptor is pharmacologically characterized by the agonists BRL54443 and (R)-N-[3Dimethylamino-2,3,4,9-tetrahydro-1H-carbazol-6-yl]-4- fluorobenzamide also known as LY344864 (Phebus et al, 1997). Currently, there are no 5-HT,F receptor antagonists available. The 5-HT,D receptor is characterized by the agonist PN0142633 (Gomez-Mancill et al, 2001). There are no selective 5-HT1D antagonists available. All of the 5-HT,D antagonists available (Le. GR127935) also have a high affinity for the 5-HT,B receptor. 19 5-HT1.3 Receptor Physiology The 5-HT,,3 receptor is a heptahelical G-protein coupled receptor which specifically utilizes Gi as well as to Go for signal transduction (Wurch and Pauwels, 2000). As a member of the 5-HT, class of receptors it is coupled to inhibition of adenylate cyclase. Additionally, 5-HT acting via 5-HT1B receptors has been linked to stimulation of DNA synthesis in fibroblasts (Seuwen et al, 1998). Similar to the 5-HT23 receptors in human coronary artery endothelial cells the 5- HT“; receptor is positively coupled to NO‘ production (Ishida et al, 1998). In the presence of elevated extracellular potassium concentrations, the 5-HT,B receptor activates phospholipase D (PLD) (Hinton et al, 1999). Additionally, in bovine endothelial cell cultures the 5-HT,,3 receptor has been linked to release of NO’ (McDuffie et al, 1999) as well as activation of the ERK/MAPK pathway (McDuffie et al, 2000) (figure 3). Furthermore, 5-HT1B receptors have been linked to activation of Akt/Protein kinase B in BE(2)-C neuroblastoma cells. This activation is inhibited by the regulator of G-protein signaling protein 4 (RGS4) (Lione et al, 2000). Stimulation of 5-HT1B receptors in native smooth muscle and primary cultures of rabbit renal artery smooth muscle resulted in activation of phosphatidylinositol 3- kinase (PIS-kinase) as well as the MAPK pathway (Hinton et al, 2000). In Chinese hamster ovary (CHO) cells activated 5-HT,B receptors stimulate p70 36 kinase (Pullarkat et al, 1998). p70 86 kinase participates in the phosphoinositide 20 3-kinase (Pl3-K) signaling pathway and in cell proliferation (Romanelli et al, 1999). Human 5-HT,,3 receptors transfected into CHO cells have also demonstrated synergy with Gq coupled receptors as sumatriptan synergistically enhanced P2U purinoceptor stimulated [3H] inositol phosphate accumulation through a pertussive toxin-sensitive mechanism (Dickenson and Hill, 1998). To date, contractile signaling pathways for this receptor has not been completely elucidated in vascular smooth muscle. Additionally, 5-HT,.3 receptors are “unmasked” in the tail artery from rats with normal blood pressure by a depolarizing stimuli (Craig and Martin, 1993). This “unmasking” of a silent 5-HT,B receptor has also been described for the rabbit ear artery (Movahedi and Purdy, 1997) and rabbit femoral artery (Chen et al, 2000). The mechanisms for this “unmasking” of silent receptors is not completely clear yet. However, it seems to entail the sensitization of the contractile myofilaments to calcium by the influx of calcium through voltage gated calcium channels activated by the depolarizing stimulus (Hill et al, 2000). Location The 5-HT,.3 receptor mRNA has been localized to: mouse striatum (Knobleman et al, 2000), rat trigeminal ganglion cells (Wotherspoon and Priestley, 2000), human coronary artery (Nilsson et al, 1999a), human cerebral artery (Nilsson at al, 1999b), human heart valve interstitial cells (Roy et al, 2000), human umbilical artery (Lovren et al, 1999) and human temporal artery (Verheggen et al, 1998). 21 Regulation Interestingly, 5-HT,B receptors form both homodimers as well as heterodimers with the 5-HT,D receptors (Lee et al, 2000, Xie et al, 1999). Furthermore, posttranscriptional modifications that have been described for the 5-HT,.3 receptor include phosphorylation and palmitoylation (Ng et al, 1993). The presence or absence of these posttranscriptional modifications and dimers, homo or hetero, may affect the function of the 5—HT1B receptors. While the human 5-HT,.3 gene has a naturally occurring Phe-124-Cys variant which changes the pharmacological properties, G-protein coupling and second messenger formation (Kiel at al, 2000) of this receptor very little else is known about the gene and promoter of this receptor. Currently, there is a dearth of information about regulators and mechanisms of regulation of transcription of this receptor. There is also no published information about a characterization of the promoter, internalization and the ability to of this receptor be resensitized and recycled to the membrane. It is also not clear why in some tissues the 5-HT,B receptor requires “unmasking” and in other tissues this “unmasking” is not necessary to observe a functional response. Pharmacology The 5-HT“3 receptor is characterized by the agonists: sumatriptan, CP93129, RU24969, CGS120668, 5-carboxamidotryptamine (5-CT) and 5- nonyloxytryptamine (5-NO'l). The antagonists that are used to characterize this receptor are: isamoltane, GR55562, GR127935 and 88216641. The binding data 22 reported for these compounds, with the exception of CP93129, were determined primarily at the human receptor. This is problematic as there is significant variability in this receptor subtype between species. Interestingly, this pharmacological variation between the rodent and human receptors is due to a single amino acid difference, a switch between a threonine at residue 355 in the human to an asparagine in the rodent 5-HT1B receptor (Oksenberg et al, 1992). ll. Hypertension The role of 5-HT in hypertension remains controversial. Much of this controversy stems, in part, from prior studies in which patients with hypertension were treated with the 5-HT2A antagonist ketanserin. These studies were inconclusive due to ketanserin’s ability to act as both a 5-HT2A receptor antagonist as well as an a, adrenergic receptor antagonist (Balasubramaniam et al, 1993, Vanhoutte, 1991, van Zwieten et al, 1992). Hypertension is defined by the Sixth Report of the Joint National Committee on Detection, Evaluation and Treatment of High Blood Pressure as an average systolic blood pressure >140 mmHg, an average diastolic blood pressure >90 mmHg or current antihypertensive therapy (JNC VI, 1997). This condition is a major risk factor for end-stage renal disease, myocardial infarction, peripheral vascular disease, stroke and congestive heart failure. There is substantial evidence to demonstrate that lowering blood pressure reduces cardiovascular related mortality. However, 23 Figure 3. Diagram of the proposed signaling pathways utilized by the 5-HT1B receptor. Abbreviations: PLC=Phospholipase C; PKC: Protein Kinase C, ERK=Extracellular signal regulated kinase; PLA2= Phospholipase A2; PLC=Phospholipase C; MAPKK: Mitogen activated protein kinase kinase; DAG: Diacylglycerol; eNOS: endothelial nitric oxide synthase; PKB =Protein kinase B; NO’: Nitric oxide; Regulator of G-protein signaling protein 4 = RG84; Srf= Serum response factor; SRE: Serum response element; c-fos: Finkel-Biskis-Jinkins osteosarcoma oncogene homologue. 24 5266 __m0 mxmw III-vs COZOQLSCOO > / xans. e 95 > 3. $58.98; $6.26 68359.. 9 w + e a 8:525 we \ . oz / 2% z. 95 . a 3.. 36¢ $22 88 AI 2.6 538?. .oz Slim 826 + 558?. 1 o a mPPIé AllFIum =oO _m__m£oocm 25 the goals of antihypertensive therapy are no longer simply to lower the blood pressure. The goals now include preventing or reducing target organ damage as well as reducing concomitant risk factors (Messerli and Laragh, 2000). Hypertension is a complex and heterogeneous disease with two diagnostic categories, essential or primary hypertension and secondary hypertension. The diagnosis of secondary hypertension is applied when the . elevation in blood pressure has a definable cause. This is a condition often caused by a change in hormone secretion or renal function. With the appropriate treatment of the cause, secondary hypertension is generally curable. The diagnosis of essential or primary hypertension is applied when the cause of the increased pressure is undetermined. Due to the involvement of several physiological systems in the regulation of blood pressure, determining the cause is often impossible. The course of treatment is often a combination of lifestyle changes (diet, exercise, weight loss act.) as well as pharmacological intervention. Essential hypertension may be the result of multiple abnormalities in the systems which regulate blood pressure. These abnormalities may be genetic in origin or may be environmently related (Messerli and Laragh, 2000). Hemodynamics Blood pressure is generally thought of as being determined by two factors, cardiac output (CO) and total peripheral resistance (TPR). Blood pressure can be calculated by the following formula: MAP=TPR x CO. Total peripheral resistance can be calculated as the mean arterial pressure (MAP) divided by the cardiac 26 output: TPR=MAPICO (Messerli and Laragh, 2000). The cardiac output is determined by the stroke volume (SV) and the heart rate (HR): CO=SV x HR. While the sympathetic nervous system plays a large role in determining overall blood pressure the total peripheral resistance is mainly determined in the small ‘ muscular arteries. Vascular smooth muscle cells and endothelial cells are primarily responsible for maintenance of the vessel diameter. This contractile tissue regulates flow and resistance by changing the diameter of the vessels via contraction or relaxation. Therefore, factors which affect the vascular smooth muscle, either to cause relaxation or contraction, will have an effect on the blood pressure. Hypertrophy and hyperplasia of vascular smooth muscle also contributes to hemodynamic changes seen in hypertension. By encroaching on the lumen of the vessel the new growth creates a narrowing of the diameter and thus an increase in resistance. Mineralocorticoid hypertension Hypertension can be created experimentally and it is an established protocol to administer exogenous mineralocorticoids such as aldosterone or deoxycorticosterone (DOC) to create experimental models of hypertension. Mineralocorticoid excess, also known as primary aldosteronism, is a well-known form of secondary hypertension in humans. Aldosterone is produced by the enzyme aldosterone synthase in glomerulosa of the adrenal cortex as well as locally in the vasculature (Takeda et al, 1995). Aldosterone is a component of the renin-angiotensin system (RAS). Renin, an aspartyl protease, is produced by the 27 granular cells of the juxtaglomerular apparatus of the kidney. Renin’s substrate angiotensinogen is produced in the liver and circulates in the blood. Renin cleaves angiotensinogen to form angiotensin I. Angiotensin l circulates to the lung where it is enzymatically converted to angiotensin II by angiotensin converting enzyme (ACE). Angiotensin II has many effects, one of which is to act _ on the adrenal glomerulosa to cause an increase in aldosterone synthesis. Mineralocorticoids exert their effects by initially binding to mineralocorticoid receptors, a type of steroid hormone receptor. These are intracellular receptors, generally located in the cytosol. They are bound by heat shock proteins, which serve to anchor the receptors in the cytosol. After the hormone binds to the receptor, the receptor dissociates from the heat shock proteins and dimerizes with another steroid receptor. This dimer then translocates to the nucleus where it binds to steroid response elements in the DNA. Mineralocorticoids have been implicated in affecting the expression of the human sodium/potassium-ATPase B1 gene promoter (Derfoul et al, 1998), K-ras (Stockand et al, 1999), and the 5-HT1A receptor in the rat dentate gyrus (Meijer et al, 1994). Additionally, aldosterone induces methylation of ras in renal epithelial cells (Al-Baldawi at al, 2000). This suggests that aldosterone not only changes the level of protein expression but also the state of activation of those proteins. Important to the changes seen in hypertension, activation of renal mineralocorticoid receptors will lead to cellular changes which result in salt and water retention by the kidney. Ultimately there is a suppression of the renin— 28 angiotensin system due to the inhibitory effects of high sodium on renin secretion. Mineralocorticoids are also known to act in other tissues to modulate both gene expression as well as to exert nongenomic effects. In vascular smooth muscle, aldosterone increases levels of cAMP within one minute of exposure (Christ of al, 1999). Other nongenomic effects of aldosterone are rapid activation of the sodium/proton exchanger and the inositol 1,4,5-triphosphate/calcium pathway, both of which occur within one to two minutes (Wehling et al, 1992). An interesting substrain of rats, the Wistar and Wistar-Furth rats, are used to study mineralocorticoid hypertension. In this model the Wistar-Furth rats are insensitive to the hypertensive effects of DOCA-salt treatment (Kayes et al, 1996, Ullian et al, 1997, Sciotti and Gallant, 1987). The mechanisms for this are still under investigation. These animals do become hypertensive with renal artery stenosis (Sciotti and Gallant, 1987) and 5/6"1 nephrectomy (Fitzgibbon et al, 1999). Wistar-Furth rats appear to be resistant to end-organ damage associated with mineralocorticoids even though there is a measurable increase in circulating level of mineralocorticoids. (Sciotti and Gallant, 1987). In the Wistar-Furth rat model, there are increases in responsiveness, as measured by isometric contractile force, to both serotonin and the 5-HT2,3 agonist BW723086 (Watts and Harris, 1999, Bruner, 1992). Additionally, in the NG-nitro-L-arginine methyl ester (LNAME) model of hypertension where nitric oxide synthase is inhibited, the mineralocorticoid antagonist spironolactone has been reported to lower blood pressure (Mandarim-de-Lacerda et al, 2001). The thoracic aorta from another rat 29 model of nitric oxide synthase inhibition, the LNNA (Nw-Nitro-L-Arginine) hypertensive rats, demonstrate an increased contraction to BW723086 as well (Russell and Watts, 1999). The endogenous levels of aldosterone have not been reported in the LNAME or LNNA hypertensive rats. However, when considered together, these results suggest that increases in the level of mineralocorticoids as well as pressure may play regulatory roles in 5-HT28 receptor gene expression in hypertension. Additionally, the roles of an increase in pressure and elevated levels of salt in modulating gene expression and vascular reactivity can not be ignored. High salt treatment increases the expression in the adrenal gland of the salt- inducible kinase (SIK) which is a member of the myocardial sucrose- nonferrnenting 1 protein kinase (SNF1)/AMP-activated protein kinase (AMPK) family of serine/threonine protein kinases (Feldman et al, 2000). High salt has been implicated in vascular changes independent of an increase in pressure. These changes include alterations in the extracellular matrix in conduit arteries (Safar et al, 2000), membrane depolarization of both conduit and resistance arteries in salt loaded spontaneously hypertensive rats (SHR) (Fujii et al, 1999), impaired dilation of skeletal muscle resistance arteries (Weber and Lombard, 2000) and generation of reactive oxygen species (Lenda et al, 2000), specifically through the xanthine oxidase pathway (Swei et al, 1999). Increased vascular pressure is often modeled as increases in shear stress, mechanical stretch and mechanical strain. In endothelial cells exposed to 30 mechanical strain, there was a sequential activation of PKC and ERK activation (Cheng et al, 2001). Pulmonary artery endothelial cells subjected to increased sheer stress increased expression of endothlin-1 but decreased expression of the adrenomedullin receptor and urotensin lI (Dschietzig et al, 2001). Interestingly, elevated perfusion pressure increases expression of endothelin-1 and the endothelin (ET) B receptor in rabbit carotid arteries (Lauth et al, 2000). The mechanisms of changes in receptor expression by increased pressure has not yet been characterized. The effects of increased pressure on 5-HT receptor subtypes has never been examined. Increased responsiveness Arteries in many models of experimental hypertension and in other disease states display an increased responsiveness to 5-HT (T ompson and Webb, 1987, Ito etal, 1995, Miwa etal, 1994, Watts, 1998, Uematsu etal, 1987, Roson et al, 1990). Increased responsiveness is defined as an increase in potency, a decrease in threshold of activation and/or an increase in the maximal contraction elicited to an agonist. Increased responsiveness to 5-HT is pronounced in the DOCA-salt model of hypertension (figure 4). Mesenteric arteries, which control approximately 25 % of the cardiac output (Beme and Levy, 1997) and are therefore important contributors to total peripheral resistance, demonstrate a greater degree of hyperresponsiveness to 5-HT than does the thoracic aorta (figure 4). This hyperreactivity to 5-HT may be 31 physiologically relevant in that it may play a role in the increased total peripheral resistance observed in hypertension. Our laboratory and collaborators have published pharmacological data which suggest that the 5-HT-induced contraction in vascular smooth muscle in the DOCA—salt hypertensive and sham normotensive animals is mediated predominantly by the 5-HT 23 and 5-HT 2A receptors, respectively. (Watt and Fink, 1999, Watts, 1997, Watts and Webb, 1994). The contraction in arteries from normotensive sham rats is sensitive to inhibition by ketanserin. This suggests that this contraction is mediated by the 5-HT 2A receptor. This is not the case with the tissue from DOCA-salt treated animals. The 5-HT concentration response curve in the arteries from DOCA-salt hypertensive rats is not sensitive to inhibition by ketanserin at the lower concentrations. As the data in table 1 demonstrate, 5-HT2.3 and 5-HT1B receptors vary significantly in their pharmacology. Ketanserin, a 5-HT MC receptor antagonist, has a very low binding affinity for the 5-HT 23 receptor. Therefore, ketanserin will not be able to inhibit an effect mediated by the 5-HT2,3 receptor. The lack of inhibition of 5-HT- induced contraction by ketanserin in arteries from hypertensive DOCA-salt rats suggest that the 5HT2A receptor is not mediating this response. However, these data are consistent with the hypothesis that the ketanserin-insensitive 5-HT2,3 receptor does mediate the contraction to 5-HT in the arteries from DOCA-salt hypertensive rats. 32 Figure 4. Top: Effect of 5-HT in the endothelium-denuded thoracic aorta from normotensive sham and DOCA-salt hypertensive rats. Bottom: Effect of 5-HT in the endothelium-denuded superior mesenteric artery from normotensive sham and hypertensive DOCA-salt rats. Data are reported as a percentage of the initial phenylephrine (PE) 10'5 M contraction. Points represent the mean and the vertical bars represent the standard error of the mean for the number of experiments indicated in parentheses. * represents statistically significant difference (p<0.05) between the sham and DOCA-salt responses. 33 Aorta 200 1 50- _L 0 Cl) Percentage of PE (10'5 M) Contraction 01 o l +Sham (N=1 1) +DOCA-salt (N=13) -1o -9 -8 -7 -6 -5 -4 -3 Log 5-HT [M] 200 Mesenteric Artery +Sham (N=6) * C .2 +DOCA-salt * g (N=6) 8150- * o S I “P 3100- A “J I Cl. “5 1 a) v V .8 50— * ‘ C r q) I g 1 a, . 0.. 0‘ r I I 1 -1|0 -9 -8 -7 -6 -5 -4 -3 Log 5-HT [M] 34 Other pharmacological data which provide support for a change in the predominate receptor subtype which mediates contraction are the effects of the 5-HT2,3 receptor agonist BW723C86 and the 5-HT 28 antagonist LY272015. BW72SC86 does not contract an artery from a normotensive sham rat. However, BW723086 produces a significant contraction in the arteries from the DOCA-salt hypertensive animal (Watts and Fink, 1999, Watts and Harris, 1999). The 5-HT 2~2c antagonist ketanserin did not reduce the BW723086—induced contraction in the artery from the DOCA-salt treated animal (Watts and Harris, 1999). Moreover, the 5-HT 2., antagonist LY272015 produced a significant inhibition of the 5-HT 29 agonist BW723C86-induced contraction of the mesenteric artery from the DOCA-salt hypertensive rat (Watts and Harris, 1999). A physiological role for this receptor subtype switch is supported by the data which show that LY272015 reduced the blood pressure of only hypertensive DOCA-salt treated rats (Watts and Fink, 1999). This indicates that the 5-HT2.3 receptor is endogenously activated and plays a role in the maintenance of the elevated blood pressure in these rats. While these functional data suggest a change in the expression of the 5-HT2,3 receptor the levels of 5-HT2,3 receptor protein have never been measured. Surprisingly, there has been no thorough characterization of the 5-HT receptors, neither those linked to contraction nor those linked to growth pathways, in arteries from hypertensive rats. 5-HT has the potential to play a role in the pathology of hypertension both in the hypertrophy and hyperplasia 35 seen in arteries from hypertensive rats as well as in its capacity as a vasoconstrictor. Collectively, these pharmacological data all support the ideas that the 5- HT-induced contraction in a vessel from a normotensive sham rat is mediated by a 5-HT 2A receptor and that the contractions in the vessels from the DOCA-salt hypertensive rats are mediated primarily through 5-HT 23 receptors with the 5- HTZA receptor being activated at the higher concentration of 5-HT. This proposed change in the receptor subtypes mediating contraction to 5-HT in hypertension has both functional and physiological effects. Functionally, the arteries from DOCA-salt hypertensive rats are much more sensitive to 5-HT compared to the arteries from the normotensive sham rats (figure 4). An increased sensitivity to 5- HT means that it takes less endogenous 5-HT to activate the 5-HT 2,, receptor than the 5-HT2A receptor; potentially a small increase due to platelet dysfunction, or a thrombotic event could result in an increase in 5-HT sufficient to activate the 5-HT 23 receptor (Kamal et al, 1984, Baudouin-Legros et al, 1985, Guicheney et al, 1985, Carrascol etal, 1998). 36 Hypothesis l hypothesize that during normotensive conditions the 5-HT 2A receptor is the predominant 5-HT receptor which mediates contraction in vascular smooth muscle. Under conditions of increased vascular pressure and/or in the presence of DOCA there is a change in the receptor subtype(s) which mediate contraction in vascular smooth muscle, specifically a change from only the 5-HT 2A receptor to the 5-HT,.3 and 5-HT 2., receptors in addition to the 5-HT2A receptor. My specific aims were to determine the mechanism of enhanced serotonergic sensitivity in DOCA-salt hypertension and the physiological relevance of the 5-HT receptor subtypes, which mediate the contraction in the vasculature. To do this I tested the following four specific hypotheses: Subhypothesis £1; The 5-HT1D and the 5-HT1F receptors are not involved in the contraction of the aorta and superior mesenteric artery from normotensive and DOCA-salt hypertensive rats. 5-HT ,8 receptors are involved in the contraction of the aorta and superior mesenteric artery from DOCA-salt hypertensive rats. W There is an increase in the level of the smooth muscle 5-HT1B and 5-HT2B receptors in the vasculature of DOCA-salt hypertensive rats. Subhypothesrifl Elevated pressure and mineralocorticoid(s) treatment independently increase the levels of the 5-HT,,3 and 5-HT 28 receptor proteins. Subhypothesis #4: The level of free 5-HT circulating in the blood is increased in the DOCA-salt hypertensive rat. 37 Methods I. General Animal Methods: Animals Allanimal procedures were followed in accordance with the institutional guidelines of Michigan State University. Male Sprague-Dawley rats were purchased from Charles River (Portage, MI). Until surgery, animals were kept in clear plastic boxes with free access to standard rat chow (Teklad) and tap water. Mineralocorticoid Hygfiension: Male Sprague-Dawley rats (250-3009), under isoflurane (lsoFlo‘D, Abbott Laboratories, North Chicago, IL.) anesthesia, were uninephrectomized and deoxycorticosterone acetate (DOCA, 200mg/kg in silicone rubber) was implanted subcutaneously. Postoperatively, rats were given a solution of 1% NaCI and 0.2 % KCI for drinking. Normotensive sham rats were uninephrectomized, received no DOCA and drank normal tap water. Animals remained on this regimen for four weeks (unless otherwise specified) prior to use. Blood Pressure Measurement: Systolic blood pressure of rats was determined in the conscious state by the tail cuff method (pneumatic transducer). Briefly, the rat was placed in a plastic pail with wood chip bedding covering the bottom. This pail was placed on a heating pad and the rat was contained by placing a steel cage over it. A warming light was placed over the steel cage. The rat was warmed in this manner for 6 minutes. Warming the rat serves to vasodilate the tail artery which facilitates the 38 measurement of the blood pressure. The rat was then placed into a restraint and the blood pressure cuff was slipped onto the tail. The balloon transducer was placed onto the ventral side of the tail and secured with tape. The blood pressure was monitored until a stable pulse pressure was observed. The blood pressure was measured utilizing a sphygmomanometer in conjunction with the pulse transducer. Blood pressure measurements were taken three times to obtain an average systolic blood pressure. -- . :10 A. cl onr 'li men in . . a .12 2.: i-r W: Rats were anesthetized with pentobarbital (50 mg/kg, i.p.) and the tissues were dissected and removed. Tissues were placed in physiologic salt solution consisting of (in mM) NaCl, 103; KCI, 4.7; KHZPO“ 1.18; MgSO, ~7H20, 1.17; CaCl2 -2HzO, 1.6; NaHCOa, 14.9; dextrose, 5.5; and CaNaZEDTA, 0.03. The aorta and superior mesenteric artery were cleaned of fat and connective tissue, cut into helical strips, and mounted on stainless steel holders in tissue baths (50 ml) for isometric tension recordings using Mac Lab (Chart 3.4 software) and transducers. Strips were placed under optimum resting tension (1500 mg for aorta, 600 mg for superior mesenteric determined previously), and strips from normotensive and hypertensive rats were placed in the same bath, thereby controlling for experimental variations. Tissue baths were filled with warmed (37°C), aerated (95% 02, 5% C02), physiological salt solution (PSS). Endothelium was removed by gently rubbing the luminal face of the vessel with a 39 moistened cotton swab. Functional integrity of the endothelial cells was evaluated by testing endothelium-dependent relaxation of acetylcholine (1 nM) in strips contracted with phenylephrine (10 pM). Removal of the endothelium was to simplify evaluation of vascular smooth muscle cell response by removing a potential source of complicating modulators (i.e. nitric oxide). Cumulative concentration response cuwes to agonists were performed. Antagonists, inhibitors or vehicle were incubated with vessels for one hour prior to experimentation. WM Tissues were incubated for 1 hour with the 5-HT2A receptor antagonist ketanserin (10 nM). This concentration of ketanserin does not block 5-HT,B or 5-HT2,3 receptors. Tissues were then depolarized with 15 mM KCI. This contraction was allowed to plateau (15-20% of maximal PE 10 p M contraction) and then cumulative concentration response curves to agonists were performed. WWW Rats were anesthetized with pentobarbital (50 mg/kg, i.p.). The thoracic aorta from male Sprague-Dawley rats with normal blood pressure was removed and cleaned of fat and connective tissue, cut into helical strips, and out into four equal pieces. The aortic tissues were incubated under tissue culture conditions (37 °C, 5 % 002) in Dulbecco’s Modified Eagle Media (DMEM) (Gibco- BRL, Rockville, MD) supplemented with 10% fetal bovine serum (Hyclone, Logan, UT), 2% penicillin/streptomycin and 2% L-glutamine (Gibco-BRL). Aldosterone (Sigma- 40 RBI, St. Louis, MO) or vehicle was added to the media. The mineralocorticoid antagonist spironolactone (10pM, Sigma-RBI) was added thirty minutes prior to the addition of aldosterone. Tissues were removed from media and taken through the protein isolation procedure listed below. V. Tissue Homogenization for Protein Work: Aorta and superior mesenteric arteries from sham and DOCA-salt treated rats were removed, cleaned, denuded of endothelium and cut into helical strips. The tissue was frozen in liquid nitrogen, pulverized in a liquid nitrogen-cooled mortar and pestle and solubilized in a lysis buffer (0.5 mol/L Tris HCI [pH 6.8], 10% SDS, 10% glycerol) with protease inhibitors (0.5 mmol/L PMSF, 10 pg/ml aprotinin and 10 pg/ml leupeptin). Homogenates were centrifuged (11,000 x g for 10 minutes, 4°C) and supernatant total protein measured (BCA, Sigma). flzflestemflgn'mg; Tissue homogenate supernatant (4:1 in denaturing sample buffer, boiled for 5 minutes), was separated on SDS-polyacrylamide gels (10% or 12 %) and transferred to lmmobilon—P membrane. Membranes were blocked [4-6 hours in Tris-buffer saline + Tween-20 (0.1%; TBS-T) containing 4% chick egg ovalbulmin and 0.025% sodium azide] and probed overnight (4°C) with primary antibody. The following antibodies were used: mouse anti-serotonin 2B receptor (5-HT28R) monoclonal antibody (0.5 ug/ml, PharMingen, San Diego, CA) and guinea pig anti-serotonin 1B receptor (5-HT,B) polyclonal antibody (1 :1000, Chemicon, Temecula, CA). Blots were washed three times with TBS-T (30 minutes, 5 41 minutes, 5 minutes) and once with TBS (5 minutes). An antimouse horseradish peroxidase linked secondary antibody (1 :10,000, Amersham Laboratories, Arlington Heights, IL) or an antiguinea pig horseradish peroxidase linked secondary antibody (1 :10,000, Chemicon) was added for one hour and incubated with blots at 4°C. Blots were washed using the described regimen. Enhanced chemiluminescence was performed using standard reagents (Amersham Laboratories). Each blot was washed and redeveloped using an or-smooth muscle actin antibody (1:400, Oncogene Research Products, Boston, MA; anti- mouse secondary antibody, 1:5,000, Amersham Laboratories). Equal lane loading of protein was ensured by comparing or-smooth muscle actin densitometry. V I. Ti e RT-P R' Total RNA was isolated from tissues using standard procedures and TRI reagent (Molecular Research Center). Concentration/purity/integrity of RNA was ascertained spectrophotometrically (AZSOIAZBO) and by running a qualitative 1% agarose gel to visualize all samples with ethidium bromide (188/288 check). Samples were diluted to 50 ng/ul RNA and taken through reverse transcription on a Perkin Elmer GeneAmp 5700 for Real-time PCR in nuclease-free buffer containing appropriate reagents and Reverse Transcriptase (45 °C, 30 min.). Samples from sham and DOCA-salt tissues were run at the same time and run with samples that did not contain reverse transcriptase. After reverse transcription, the appropriate primer [synthesized by the Biochemistry 42 department at MSU]; 5-HT1B forward primer = 5’-CACTAGGCCAGGTGGTCTGC- 3’, reverse 3’-GCAGCGAAATCGAGATGGAG; 5-HT23 forward primer = 5’- ACAGAAAGGCGAATGGCTTC-3’, reverse = 5’-CGGCAGTCTGC'ITCA1TTCC - 3’; annealing temperature of 60°C for 60 seconds, 35 cycles] and SYBR Green master mix was added for PCR. Samples were normalized to a GAPDH signal (GAPDH primers purchased from PE Applied Biosystems). WWI: Blood samples from both sham and DOCA-salt hypertensive rats were collected into 5 ml syringes coated with heparin (2000 units/mL) and anticoagulant solution [consisting of (in mM) citric acid, 13; sodium citrate, 12.6; D-glucose, 11; and 150 pl of 4% EDTA per tube] from the hepatic vein. After blood collection, the contents of the syringes was carefully mixed with anticoagulant solution (.1mL/mL of blood). This mixture was centrifuged for ten minutes at 160 x g and 4°C to obtain platelet rich plasma. Plasma (2 ml) was removed and a 1:1 dilution was performed with EDTA (0.4 M) and these samples were centrifuged again for 20 minutes at 1350 x g and 4°C to obtain platelet poor plasma. The remaining platelet rich plasma was resuspended in 1 ml of platelet buffer (pH =7.4) consisting of [(in mM) NaCl, 145; KCI, 5; CaCIz, 1; MgSO,, 1; D-glucose, 10], adenosine diphosphate (ADP) (1.0 uM/L) was added and then samples were vortexed and allowed to remain on ice for 15 minutes. The samples were deproteinized with trichloroacetic acid (0.5 M) for thirty minutes and then centrifuged at 4500 x g for twenty minutes at 4°C. The supernatant was centrifuged again at 100,000 x g at 4°C for two hours to remove 43 all proteins. The monoamine oxidase inhibitor parglyine (10 pM) and ascorbic acid (10 uM) were added to the blood after it was initially placed into anticoagulant solution and was added again after each centrifugation step as well as to the supernatant after deproteinization. The concentration of trichloroacetic acid used was chosen after a concentration response curve experiment was performed with one ng standards of 5-HT to ensure that no degradation occurred. Measurements were made using high performance liquid chromatography (HPLC). The chromatography column was packed with 6 pm spherical c18 bonded to silica gel particle. (Waters, Division of Millipore, Milford, MA). A precolumn Nova Pack 018 (Waters) was used. The mobile phase used was: EDTA 0.1 M, 0.03% sodium octyl sulfate (SOS), 0.05M NaPO, and 15% methanol. IX, Data Analysis and Statistics: Data are presented as means i standard error of the mean for the number of animals in parentheses. Contraction was reported as a percentage of response to maximum contraction of phenylephrine (10 nM). EC5o values (agonist concentration necessary to produce a half-maximal response) were determined using non-linear regression analysis and was reported as the mean of the negative logarithm (-log) of the E050 value (M). When comparing two groups, the appropriate Student’s t test was used. ANOVA followed by a Student- Newman-Kuels post hoc test was performed when comparing three or more groups. In all cases, a p value less than or equal to 0.05 was considered statistically significant. Band density was quantified using the program NIH Image. HPLC data was normalized for the plasma volume of the samples. Results from the Real-Time PCR experiments were reported as values normalized by GAPDH or as ACT (threshold values) 45 Results Hypothesis l: . Profiling of 5-HT Receptors Which 5-HT receptors comprise the complete complement of 5-HT receptors in the vasculature of the normotensive and hypertensive rats is currently undetermined. We have data from RT-PCR in endothelium-denuded rat aorta from a normotensive rat (figure 5) which examines which 5-HT receptor mRNA are expressed in aortic smooth muscle cells. While all of the known 5-HT receptors were not probed for in this experiment, those probed for are receptors linked to contraction or reported to modulate vascular tone in other vascular beds. These data show the presence of the following receptor’s mRNA: 5-HT2A, 5-HT28, 5-HT,D_ 5-HT,B, 5-HT,F and 5-HT7 receptors. The mRNA for the 5-HT1E (not shown) receptor was not detected. These findings suggest that in addition to the 5-HT2A and 5-HT2E3 receptors, 5-HT ,8. 5-HT1D and 5-HT1F receptors may also be involved in mediating 5-HT-induced vascular smooth muscle contraction because their message is present. Of the five receptors for which mRNA message was detected, four have been suggested to be involved in mediating vascular smooth muscle cell contraction to 5-HT (Smith et al, 1999, Watts of al, 1996,Razzaque et al, 1999, VanDenBrink et al, 2000). These contractile receptors are the 5-HT,B, 5-HT2A, 5-HT2B and 5-HT,F receptors. The 5-HT7 receptor is known to couple to activation of G, and thereby mediate vascular relaxation (Adham etal, 1998, Cushing etal, 1996). 46 Figure 5: Results of RT-PCR analysis and Southern blotting of rat thoracic aorta denuded of endothelium for 5-HT receptors. Data shown for the 5-HT2A, 5-HT23, 5-HT15, 5-HT,F, 5-HT1D and 5-HT7 receptors. Data for the 5-HT1': receptor not shown. Experiments were preformed at Eli Lily, Indianapolis, IN in collaboration with Dr. Mel Baez. Abbreviations: +RT = Reverse Transcriptase present; - RT= Reverse Transcriptase absent. 47 RT-PCR of 5-HT Receptors in Rat Aorta 18 1D 1F +RT -RT '+RT -RT +RT -RT 2A 2B 7 +RT _-RT +RT, -RT +RT :RT 48 Based on these data, we next moved to contractile experiments to determine which of the receptors for which message was detected were involved in mediating 5-HT-induced contraction. The first receptor addressed was the 5-HT1D receptor. We performed contractile experiments utilizing the selective 5-HT1D agonist PNU014633 (10‘9 to 10‘5 M) in the aorta from both normotensive sham and hypertensive DOCA-salt rats (figure 6). The average systolic blood pressure for the sham rats utilized in the following studies was 123 _-r_- 2 mm Hg. The average systolic blood pressure for the DOCA-salt rats used in the following studies was 194 i 10 mm Hg. There was no contraction observed in the aorta from either the normotensive sham or the hypertensive DOCA-salt rats. Based on the contractile data we conclude that the 5-HT1D receptor does not mediate contraction in rat aortic vascular smooth muscle either under conditions of normal or high blood pressure. To address the involvement, if any, of the 5-HT,F receptor in mediating 5- HT-induced contraction we performed contractile experiments using the 5-HT1F agonists BRL54443 and LY344864 in aorta from both normotensive sham (figure 7,top) and hypertensive DOCA-salt rats (figure 7,bottom). LY344864 (10‘9 to 10"" M) did not elicit a contraction in the aorta from either the normotensive sham or the hypertensive DOCA-salt rats. BRL54443 (10'9 to 10'5 M) did cause a contraction in the aorta and the mesenteric artery from both the normotensive sham (figure 8, 49 Aorta 200 +Sham(N=4) 5 + DOCA-salt (N=4) '8 g 150- 0 ’2" In '52 I 100- D. “5 (D Cl :9. E g 50‘ (D D. o-_——a—I—q—a—H—m+¢—I——.—— -1o -9 -8 -7 -6 -5 -4 -3 Log PNU0142633 [M] Figure 6. Effect of the 5-HT1D receptor agonist PNU0142633 in endothelium- denuded thoracic aorta from normotensive sham and hypertensive DOCA-salt rats. Data are reported as a percentage of the initial phenylephrine (PE) 10'5 M contraction. Points represent the mean and the vertical bars represent the standard error of the mean for the number of experiments indicated in parentheses. 50 Figure 7. Top: Effect of the 5-HT1F receptor agonists BRL54443 and LY344864 in endothelium-denuded thoracic aorta from normotensive sham rats. Bottom: Effect of the 5-HT1F receptor agonists BRL54443 and LY344864 inendothelium-denuded thoracic aorta from hypertensive DOCA-salt rats. Data are reported as a percentage of the initial phenylephrine (PE) 10'5 M contraction. Points represent the mean and the vertical bars represent the standard error of the mean for the number of experiments indicated in parentheses. 51 Percentage of PE (10'5 M) Contraction Percentage of PE (10‘5 M) Contraction Sham Aorta 200 —-¥—BRL54443 N=4) +5-HT N=11 +LY 864( =6) 150- 100- f 0_ -1 . -1o -9 -8 -7 s -5 -3 Log Agonist [M] 200 DOCA-salt Aorta —V-—BRL54443 (N=4) +5-HT (N=13) +LY344864(N=6) 150- 100- j 50- / /' / 0' _ ‘1 -1o -9 -8 -7 s -5 -3 Log Agonist [M] 52 Figure 8. Top: Effect of the 5-HT2A receptor antagonist ketanserin (10 nM) on the 5-HT1F receptor agonist BRL54443-induced contraction in endothelium-denuded thoracic aorta from normotensive sham rats. Bottom: Effect of the 5-HT2A receptor antagonist ketanserin (10 nM) on the 5-HT1': receptor agonist BRL54443-induced contraction in endothelium-denuded thoracic aorta from hypertensive DOCA-salt rats. Data are reported as a percentage of the initial phenylephrine (PE) 10'5 M contraction. Points represent the mean and the vertical bars represent the standard error of the mean for the number of experiments indicated in parentheses. * represents statistically significant difference (p<0.05) between the vehicle-incubated and ketanserin-incubated responses. 53 Sham Aorta 200 + Vehicle (N=4) + Ketanserin (10 nM; N=4) 150- 100‘ Percentage of PE (10'5 M) Contraction -10 -9 -8 -7 -6 -5 -4 Log BRL54443 [M] 200 DOCA-salt Aorta E + Vehicle (N=4) ‘5? + Ketanserin (10 nM; N=4) E 8 150 — 3 l0 '0 f; too-R a. “5 Q) g E 50— Q) 8 G) a a 0 I I I I -10 -9 -8 -7 -6 -5 -4 -3 Log BRL54443 [M] 54 Figure 9. Top: Effect of the 5-HT2A receptor antagonist ketanserin (10 nM) on the 5-HT1': receptor agonist BRL54443-induced contraction in endothelium-denuded superior mesenteric artery from normotensive sham rats. Bottom: Effect of the 5-HT2A receptor antagonist ketanserin (10 nM) on the 5-HT,F receptor agonist BRL54443-induced contraction in endothelium-denUded superior mesenteric artery from hypertensive DOCA-salt rats. Data are reported as a percentage of the initial phenylephrine (PE) 10'5 M contraction. Points represent the mean and the vertical bars represent the standard error of the mean for the number of experiments indicated in parentheses. * represents statistically significant difference (p<0.05) between the vehicle-incubated and ketanserin- incubated responses. 55 Sham Mesenteric Artery 2 00 +Vehicle (N=4) + Ketanserin (10 nM; N=4) Percentage of PE (10'5 M) Contraction S R o I I . '10 '9 -8 -7 .6 -5 -4 _3 Log BRL54443 [M] DOCA-salt c 200 Mesenteric Artery % +Vehicle (N=4) g + Ketanserin (10 nM; N=4) C 8 1501 E “9 £3,100 UJ D. “5 g, 50- :9 C (D e g 0- f l -10 -9 -8 -7 J5 -5 .4 -3 Log BRL54443 [M] 56 top, figure 9,top) as well as the hypertensive DOCA-salt rats (figure 8, bottom, figure 9,bottom). However, the contraction elicited by BRL54443 in the aorta and mesenteric artery from the sham rat appears to be due to activation of the 5-HT 2A receptor as it is almost completely inhibited in the presence of the 5-HT2A receptor antagonist ketanserin (figure 8, top, figure 9, top). In the superior mesenteric arteries from DOCA-salt treated rats ketanserin has no effect on the BRL54443- induced contraction (figure 9, bottom). This is a markedly different response from that seen in the thoracic aorta, where ketanserin inhibited BRL54443-induced contraction (-log ECso value (M) 6.42 i 0.04 and 5.26 i 0.11, vehicle- and ketanserin-treated, respectively) (figure 8, bottom). These data suggest that BRL54443 is acting through the 5-HT2A receptor in the aorta from sham and DOCA-salt hypertensive rats as well as in the mesenteric artery from the sham rats. Furthermore, these data suggest that the BRL54443-induced contraction in the superior mesenteric arteries of the DOCA-salt hypertensive rats is not mediated predominately by the 5-HT2A receptor but rather either by the 5-HT2,3 receptor or the 5-HT,.3 receptor. BRL54443 has affinity for both of these receptors (table 1). Further contractile studies with the 5--HT2.3 antagonist LY272015 and the 5-HT,,3 antagonist GR55562 would determine which of these two possible receptors is mediating the BRL54443-induced contraction in the arteries from hypertensive DOCA-salt rats. However, based on the data, using multiple 5-HT1F agonists, it is appropriate to rule out the involvement of the 5-HT,F receptor in S7 mediating contraction in arteries from normotensive and DOCA-salt hypertensive rats. Lastly, we have contractile data demonstrating that the 5-HT,,3 receptor agonists sumatriptan (10'9 to 10'5 M), RU24969 (10'9 to10'5 M) and the rodent selective 5-HT1B agonist CP93129 (10'9t010'5 M) have no effect in arteries from normotensive rats (figure 10, top). The presence of the 5-HT1B antagonist GR55562 (100 nM) produced no effect on the 5-HT—induced contraction in the aorta or superior mesenteric artery from normotensive sham rats (figure 11, top left, bottom left). This suggests that under conditions of normal blood pressure, the 5-HT ,3 receptor does not mediate contraction in vascular smooth muscle. However, these 5-HT1B receptor agonists all produced a contraction in the aorta from hypertensive DOCA-salt rats (figure 10,bottom). This contraction was only a partial contraction compared to that elicited by 5-HT. The 5--HT,B receptor antagonist GR55562 (100 nM) did produce a small, but significant inhibition of the 5-HT-induced contraction in both the thoracic aorta (-Iog EC50 values [M] 6.26 :t 0.08 and 5.88 i 0.04, vehicle- and GR55562-treated, respectively) and superior mesenteric artery (-log EC,o values [M] 6.90 5; 0.10 and 6.29 i 0.13, vehicle- and GR55562-treated, respectively) from DOCA-salt hypertensive rats (figure 11, top right, bottom right). The affinity of GR55562 is currently unknown at the 5-HT2,, receptor. Therefore, to determine the selectivity of GR55562 and the resultant response seen, we tested GR55562 (100 nM) against the 5-HT,B rodent receptor selective agonist CP93129. In the mesenteric artery of 58 normotensive sham rats CP93129 (10'9 to 10’5 M) and the combined treatment of GR55562 and CP93129 had no effect. In the mesenteric artery of hypertensive DOCA-salt rats, GR55562 (100 nM) produced a small but significant inhibition of the CP93129-induced contraction (-log E050 values [M] 6.73 i 0.07 and 5.68 i 0.05, vehicle- and GR55562-treated, respectively) (figure 12, bottom). Interestingly, when the 5-HT,B antagonist GR55562 (100 nM) and the 5-HT2,, receptor antagonist LY272015 (10 nM) are incubated with the mesenteric artery from hypertensive DOCA-salt rats simultaneously the 5-HT-induced contraction is shifted rightward (-Iog ECso values [M] 6.79 1; 0.08 and 5.81 i 0.07, dual vehicle- and GR55562 and LY272015 combined treatment, respectively) (figure 13, bottom). This response is normalized to the response seen from arteries from sham rats leftward (-log EC50 values [M] 5.99 i 0.03 and 5.81 1: 0.07, sham dual vehicle- and DOCA-salt GR55562 and LY272015 combined, treatment, respectively) (figure 13, bottom). Neither the 5-HT1B receptor antagonist GR55562 nor the 5-HT23 receptor antagonist LY272015 had any significant effect on the 5-HT-induced contraction in the superior mesenteric arteries from the normotensive rats (figure 13, top). The variability of the response seen in the tissues from the sham rats is due to the different concentrations of dimethyl sulfoxide (DMSO) used as the vehicles for the antagonists. The one vehicle contains either 25 pl or 50 pl of DMSO. The two or dual vehicle conditions contain 75 pl of DMSO. These data suggest that 5-HT,B receptors mediate, at 59 Figure 10. Top: Effect of the 5-HT1B receptor agonists in endothelium- denuded thoracic aorta from normotensive sham rats. Bottom: Effect of the 5-HT1B receptor agonists in endothelium-denuded thoracic aorta from hypertensive DOCA-salt rats. Data are reported as a percentage of the initial phenylephrine (PE) 10'5 M contraction. Points represent the mean and the vertical bars represent the standard error of the mean for the number of experiments indicated in parentheses. 6O Percentage of PE (10"5 M) Contraction Percentage of PE (10'5 M) Contraction Sham Aorta + 5-HT (N=11) + RU24969 (N=9) + Sumatriptan (N=4) 150 _. 4' CP93129 (N=7) 200 100— 50—- o— . -1o -9 -8 -7 -6 -5 -4 -3 Log Agonist [M] DOCA-salt Aorta 200 +5-HT(N=13) + RU24969 (N=9) + Sumatriptan (N=4) 100- 50" I ’155’ ‘65" 4- a I 0" “' I I I I ~10 -9 -8 -7 -6 -'5 -4 -3 Log Agonist [M] 61 Figure 11. Top: Effect of the 5-HT1B receptor antagonist GR55562 (100 nM) on 5-HT-induced contraction in the endothelium-denuded thoracic aorta from normotensive sham (left) and hypertensive DOCA-salt rats (right). Bottom: Effect of 5-HT,,3 receptor antagonist GR55562 (100 nM) on 5-HT-induced contraction in endothelium-denuded superior mesenteric artery from normotensive sham (left) and hypertensive DOCA-salt rats (right). Data are reported as a percentage of the initial phenylephrine (PE) 10‘5 M contraction. Points represent the mean and the vertical bars represent the standard error of the mean for the number of experiments indicated in parentheses. * represents statistically significant difference (p<0.05) between the vehicle-incubated and GR55562-incubated responses. 62 Percentage of PE (10'5 M) Contraction Percentage of PE (10'5 M) Contraction Sham Aorta DOCA-salt Aorta 200 200 . +Vehicle (N=4) Hehlcle (N=4) ‘+GR55562 (100 nM; N=4) +G855562 (100 nM; N=4) 150- 150: 100- 1004 50— 50- 0.. I 1 1 0- I I I I I -10 -9 -8 -7 -6 -5 -4 -3 -10 -9 -8 -7 -6 -5 -4 -3 Log 5-HT [M] Log 5-HT [M] Sham DOCA-salt 200 Mesenteric Artery 200 Mesenteric Artery +Vehicle (N=4) -I—Vehicle (N=4) -‘-— GR55562 (100 nM;N=4) +G855562 (100 nM;N=4) 1509 150- roo~ 100 - 50— 50- 0‘ I I I I O- ‘ -1o -9 -s -7 -6 -5 -4 -3 -10 -9 -3 37 4'5 -é «'1 -3 Log 5-HT [M] Log 5-HT [M] 63 Figure 12. Top: Effect of the 5-HT,,3 receptor antagonist GR55562 (100 nM) on the 5-HT1,, receptor agonist CP93129-induced contraction in endothelium-denuded superior mesenteric artery from normotensive sham rats. Bottom: Effect of the 5-HT,.3 receptor antagonist GR55562 (100 nM) on the 5-8HT1B receptor agonist CP93129-induced contraction in endothelium-denuded superior mesenteric artery from hypertensive DOCA-salt rats. Data are reported as a percentage of the initial phenylephrine (PE) 10‘5 M contraction. Points represent the mean and the vertical bars represent the standard error of the mean for the number of experiments indicated in parentheses. * represents statistically significant difference (p<0.05) between the vehicle-incubated and GR55562-incubated responses. Sham Mesenteric Artery _L O O + Vehicle (N=4) + GR55562 (100 nM;N=5) \l ‘1‘ Percentage of PE (10‘5 M) Contraction N 01 01 O l I O-fi‘W— -1o -9 -8 -7 -6 -5 -4 -3 Log CP93129 [M DOCA-salt : Mesenteric Artery .9100 . *5 +Vehrcle(N=4) g + GR55562 (100 nM;N=5) C 8 75- E LO '2 E: D. “6 (D U) 9 C (D e (D o_ I -10 -9 -8 -7 -6 -5 -4 -3 Log CP93129 [M 65 Figure 13. Top: Effect of the 5-HT1B receptor antagonist GR55562 (100 nM) and the 5-HT2,3 receptor antagonist LY272015 (10 nM) on 5-HT- induced contraction in endothelium-denuded superior mesenteric artery from normotensive sham rats. Bottom: Effect of the 5-HT1B receptor antagonist GR55562 (100 nM) and 5-HT28 receptor antagonist LY272015 (10 nM) on the 5-HT-induced contraction in endothelium-denuded superior mesenteric artery from hypertensive DOCA-salt rats. Data are reported as a percentage of the initial phenylephrine (PE) 10‘5 M contraction. Points represent the mean and the vertical bars represent the standard error of the mean for the number of experiments indicated in parentheses. * represents statistically significant different (p<0.05) from the DOCA-salt vehicle-incubated response. + represents statistically significant difference (p<0.05) from the combined vehicle (2 Vehicle) response. 66 Percentage of PE (10'5 M) Contraction Percentage of PE (1 0‘5 M) Contraction Sham Mesenteric Artery 100] O- -U— Vehicle (N=4) -'— GR55562 (100 nM; N=4) + LY272015 (10 nM; N=6) -I- 2 Vehicle (N=4) + GR55562 (100 nM) and LY272015 (10 nM; N=4) -10 ~94: -7 e -5 4 Log 5-HT [M] DOCA-salt Mesenteric Artery 100.. -10-9 43 -7 e -5 4 Log 5-HT [M] 67 —El— Vehicle (N=6) + GR55562 (100 nM; N=4) + LY272015 (10 nM; N=6) + 2 Vehicle (N=3) + GR55562 (100 nM) and LY272015 (10 nM; N=4) + Sham 2 Vehicle (N=4) least partially, the 5-HT-induced contraction in both the thoracic aorta and superior mesenteric artery of DOCA-salt hypertensive rats. Unmasking “Silent Receptors” It has been reported in some forms of hypertension that vascular smooth muscle exhibits a slightly depolarized resting membrane potential (Kwan and Grover, 1983). This may be due to defects in the handling of calcium or a loss of potassium channel function (Pamnami at al, 1985, Herrnsmeyer, 1993, Kwan, 1985, Borges et al, 1999, Yuan at al, 1998). It has also been reported that the 5- HT“3 receptor may require a depolarizing stimulus in order to become activated (Craig and Martin, 1993, Movahedi and Purdy, 1997). There is almost unanimous agreement among investigators that in order to be enabled, the 5-HT18 receptor must be activated following a stimulus which either activates or increases the probability of opening of L-type calcium channels. This 5-HT,B-mediated contraction is dependent on the influx of external calcium (Hill et al, 2000). These “silent receptors” have been found in several different arteries of normotensive subjects including human coronary artery, human pulmonary arteries, rabbit ear artery and tail artery of the rat (Nilsson et al, 1999, Morecroft et al, 1999, Movahedi and Purdy, 1997, Craig and Martin, 1993). In order to determine if the 5-HT1B receptors in the rat thoracic aorta and mesenteric artery requires a depolarizing stimulus, we performed isolated tissue bath experiments on normal Sprague-Dawley rat thoracic aorta in the presence of KCI (15 mM) and the 5-HT2A receptor antagonist ketanserin (10 nM). KCI was 68 chosen as a primary stimulus due to the fact that it activates L-type calcium channels and thereby acts as the depolarizing stimulus; this is similar to the published experimental protocols investigating “silent receptors”. The 5-HT2,,,2¢ receptor antagonist ketanserin (10 nM) was used to inhibit the contribution of the 5-HT2A receptor to the 5-HT-induced contraction. For comparison values the responses of arteries from hypertensive DOCA-salt rats and sham rats with normal blood pressures have been included. These data demonstrate that acute depolarization alone was not sufficient to result in an increase in the maximal contraction to 5-HT in the aorta or mesenteric artery from the normal Sprague- Dawley rats (figure 14, top, figure 15, bottom). The observed leftward shift in potency (-log EC,o value [M] 6.31 :I: 0.05 and 5.87 _-_I-_ 0.05, with KCI and without KCI, respectively in aorta) (-Iog EC50 value [M]6.55 i 0.08 and 5.91 i 0.03, with KCI and without KCI, respectively in mesenteric artery) may be due to activation of “silent" 5-HT2A receptors. “Silent” 5-HT2A receptor have been previously noted in the rabbit ear artery (Smith et al, 1999). This notion is support by the data obtained in the presence of ketanserin. In the presence of KCI and ketanserin the 5-HT-induced contraction is rightward shifted (-Iog EC50 value [M] 6.55 :1: 0.08 and 5.24 i 0.03, with KCI and without ketanserin and with KCI and with ketanserin, respectively in mesenteric artery) (figure 15, top). This response is very similar to that seen in the thoracic aorta where the enhanced contraction to 5-HT is also sensitive to inhibition by ketanserin (-log EC50 value [M] 6.31 i 0.05 and 5.24 i 0.03, with KCI and without 69 ketanserin and with KCI and with ketanserin, respectively) (figure 14, top). Ketanserin’s ability to inhibit the 5-HT-induced contraction suggests that this is a 5-HT2A mediated response. Additionally, the 5-HT—induced response in arteries from hypertensive DOCA-salt rats is insensitive to inhibition by ketanserin (30 nM) (figure 15, bottom). The lack of inhibition by ketanserin indicates that the 5- HT-induced contraction is not mediated by the 5-HT2A receptor. Therefore, even in the presence of a depolarizing stimulus, the arteries from rats with normal blood pressure do not mimic the response seen in arteries taken from hypertensive DOCA-salt rats (figure 14, top, figure 16, top). To determine if this enhanced response was selective for 5-HT we repeated these studies in the mesenteric artery with the or, adrenergic receptor agonist phenylephrine. This appears to be a selective enhancement of the 5-HT response as there was no change in the phenylephrine-induced contraction in the presence of the KCI (15 mM) (figure 16, bottom). The concentration of ketanserin is also selective for the 5-HT2A receptor, not the or, receptor, at the concentration used as the phenylephrine-induced contraction was unaffected in the presence of ketanserin (figure 16, bottom). Additional acute depolarization data was obtained by performing contractile studies using the 5-HT1B agonists RU24969, CP93129 and sumatriptan as well as the 5-HT2,3 agonist BW723086. Even in the presence of the depolarizing stimulus, the 5-HT2E3 receptor agonist BW723C86 and the 5-HT,B receptor agonist sumatriptan did not elicit a contraction in the thoracic aorta from 70 a Sprague-Dawley rat with normal blood pressure (figure 14, bottom). The 5-HT1E3 agonist RU24969 only caused a very small contraction at the very highest concentrations (figure 14, bottom). CP93129 also did not cause a contraction in the mesenteric artery in the presence of the KCI (15 mM) (figure 16, top). These additional data do not support the role of “silent 5-HT,,3 receptors” in the rat thoracic aorta or mesenteric artery. 71 Figure 14.Top: Effect of KCI (15 mM) depolarization on 5-HT-induced contraction in the presence and absence of the 5-HT,W2C receptor antagonist ketanserin (10 nM) in the thoracic aorta from normal Sprague- Dawley rats. Sham and DOCA-salt responses were obtained in the absence of KCI (15 mM). Bottom: Effect of KCI (15 mM) depolarization on the 5-HT2,3 receptor agonist BW723086-induced response and the 5-HT,B receptor agonists sumatriptan, CP93129 and RU24969-induced responses in the‘thoracic aorta from normal Sprague-Dawley rats. Responses obtained the thoracic aorta from hypertensive DOCA-salt rats were obtained in the absence of KCI. Data are reported as a percentage of the initial phenylephrine (PE) 10'5 M contraction. Points represent the mean and the vertical bars represent the standard error of the mean for the number of experiments indicated in parentheses. * represents statistically significant difference (p<0.05) between the response obtained in the sham aorta. Abbreviations: w/= with; w/o= without. 72 Percentage of PE (10'5 M) Contraction Percentage of PE (10‘5 M) Contraction Aorta 200 150- -I—DOCA- salt (N: 13) + Sham (N=11) +With KCI & Ketanserin (N=4) +(Nithn 199144 without Ketanserin 100- 501 0- ‘ I I -10 -9 -8 -7 -6 -5 -4 -3 Log 5-HT [M] Aorta 20° Wi h K I 1mM + DOCA-salt Sumatri tan (N=4) + DOCA-salt RU249 9N 150. + DOCA-saltCP93129 N =5 + DOCA-salt BW723CB (N=4) 100- With KCI 15 mM 4:— Sumatriptan( =4) -5 4 Log Agonist [M] 73 + RU24969 (N=7) 4— CP93129 (N=4) A— BW723C86<(N=4) Figure 15. Top: Effect of KCI (15 mM) in the presence and absence of the 5-HT2N20 receptor antagonist ketanserin (10 nM) on the 5-HT-induced contraction in mesenteric artery from rats with normal blood pressure. Bottom: Effect of the 5-HT2A,2C receptor antagonist ketanserin (30 nM) on the 5-HT—induced contraction in the endothelium-denuded superior mesenteric artery from hypertensive DOCA-salt rats. Data are reported as a percentage of the initial phenylephrine (PE) 10'5 M contraction. Points represent the mean and the vertical bars represent the standard error of the mean for the number of experiments indicated in parentheses. * represents statistically significant difference (p<0.05) between the response obtained in the sham artery. Abbreviations: W: with; w/o= without. 74 Sham Mesenteric Artery 300 + w/o KCI & Ketanserin (N=8) + w/ KCI&w/o Ketanserin (N=4) + w/ KCI & Ketanserin (N=4) Ketanserin =10 nM 200 “ 100— Percentage of PE (10'5 M) Contraction -10 -9 -s -7 -6 -5 -4 Log 5-HT [M] DOCA-salt Mesenteric Artery 300 + Vehicle (N=3) —I— Ketanserin (30 nM;N=3) 200 " 100‘ Percentage of PE (10'5 M) Contraction -1O -9 -8 -7 -6 -5 -4 Log 5-HT [M] 75 Figure 16. Top: Effect KCI (15 mM) on the 5-HT,B receptor agonist CP93129- and 5-HT-induced contraction in the endothelium-denuded mesenteric artery from rats with normal blood pressure. Responses obtained the thoracic aorta from hypertensive DOCA-salt rats were obtained in the absence of KCI. Bottom: Effect of KCI (15 mM) depolarization on the a, adrenergic receptor agonist phenylephrine- induced contraction in the presence and absence of the 5-HTMC receptor antagonist ketanserin (10 nM) in the mesenteric artery of normal Sprague- Dawley rats. Data are reported as a percentage of the initial phenylephrine (PE) 10-5 M contraction. Points represent the mean and the vertical bars represent the standard error of the mean for the number of experiments indicated in parentheses. * represents statistically significant difference (p<0.05) between the response obtained in the sham artery. Abbreviations: w/= with; w/o= without. 76 Mesenteric Artery no ketanserin treatment * + a-HT & MKIS-l-4) 150 - + 8P931N 29 & =50 J—r-s f —o—fih -)HT ShaNm w/o KCI +?- -HT )DOCA w/o KCI N=8) O-Io -9 -8 7 -6 -5 -4 -3 Log Agonist [M] Percentage of PE (10'5 M) Contraction 8 o l C ":9; Mesenteric Artery g 200 + w/KCI&Ket(N=4) 8 + w/KCI &w/o Ket (N=4) E 150' -O- w/o KCI&Ket(N=4) l0 '0 C 100- LIJ D. “6 _ a, 50 U) :9. C 8 0'- I I I I g -10 -9 -8 -7 -6 -5 -4 -3 Log Phenylephrine [M] 77 Hypothesis ll Receptor Protein Levels All of the pharmacological data from functional studies implicates the 5- HT2,3 and 5-HT1B receptors in the hyperresponsiveness to 5-HT observed in arteries from hypertensive DOCA-salt rats. These experiments do not address the mechanism by which the hyperresponsiveness to 5-HT occurs. Therefore, we proposed the hypothesis that the 5--HT2,3 and 5-HT,,3 receptor protein levels were increased under the conditions of DOCA-salt hypertension. The increased levels of receptor proteins would, at least partially, explain why there was no contraction to 5-HT2,3 receptor agonist and 5-HT,,3 receptor agonists under conditions of normal blood pressure but why there was contraction to these agonists under conditions of hypertension. We speculated that under conditions of normal blood pressure there were not enough 5-HT2,3 and 5-HT1B receptors to couple efficiently with either the G-proteins and/or effector molecules to mediate 5-HT-induced contraction. To address this hypothesis we utilized Western analysis and antibodies to 5-HT,,3 and 5-HT2,3 receptors to determine if the change in contractility to 5-HT1,, receptor and 5-HT2,3 receptor agonists was due to an increase in the levels of the receptor protein. 5-HT,,3 and 5-HT2,3 receptor proteins were both increased approximately 2-fold in aorta from Sprague-Dawley DOCA-salt hypertensive rats as compared to controls (figure 17). These data are similar to those obtained from the mesenteric artery where 5-HT1B and 5-HT2,3 receptor proteins were both 78 increased approximately 2-fold in the mesenteric artery from Sprague-Dawley DOCA-salt hypertensive rats as compared to controls (figure 18). These data suggest that an increase in the level of receptor protein may be at least partially responsible for the changes observed to 5-HT1B receptor and 5-HT2,3 receptor agonists. Interestingly, when the mRNA for the 5-HT2,, receptor in the thoracic aorta from normotensive sham and hypertensive DOCA-salt was examined by Real Time PCR there was no difference in the levels of mRNA (figure 19, bottom). The CT values were 23.82 i 0.35 for the sham and 24.02 i 0.35 for the DOCA-salt (ACT values of 6.93 _-l_- 0.65 and 7.71 i 0.62, sham and DOCA-salt, respectively). The mRNA for the 5-HT,,3 receptor in the thoracic aorta from sham rats and hypertensive DOCA-salt rats also showed no difference in levels of mRNA (figure 19, top). The CT values were 24.53 i 0.40 for the sham and 23.39 i 0.27 for the DOCA-salt (ACT values of 7.01 :I: 0.31 and 6.92 :I: 0.76, sham and DOCA-salt, respectively). The CT values for GAPDH were similar for the sham and DOCA- salt (CT values 16.89 i 0.62 and 16.311 _-l_- 0.40, sham and DOCA-salt, respectively). These data, while demonstrating an increase in the level of 5- HT“, and 5-HT28 receptor proteins, suggest that the increase is not due to an increase in transcription. This would suggest post-transcriptional or translational modifications and/or a decrease in the degradation of these receptors. However, as we did not perform any RNAse protection assays and did not investigate the 79 stability and rate of degradation of the 5-HT,B and 5-HT2,3 receptor mRNA we can not conclusively rule out a transcriptional regulation of these receptors. 80 Figure 17. Top: Measurement of 5-HT1B receptor protein levels in thoracic aorta from normotensive sham and hypertensive DOCA-salt rats. Bottom: Measurement of 5-HT2,3 receptor protein levels in thoracic aorta from normotensive sham and hypertensive DOCA-salt rats. Units are reported as arbitrary densitometry units. Blots are representative of four experiments. Total protein loaded in each lane was 25 pg/pL. Vertical bars represent the mean and the standard error of the mean for the number of experiments indicated in parentheses. * represents statistically significant difference (p<0.05) between the response obtained in the sham aorta 81 Thoracic Aorta 5-HT1 B Receptor 7500 (N=4) 5000‘ 2500'“ Arbitrary Densitometry Units DOCA-salt V‘s'ha’m” DOCA-saltSham DOCA-salt 5'HTZB Receptor 7500 (N=4) £3 * 'E '_l— D E 50004 Q) E .8 U) C (D o 2‘ 93 2§ <2: DOCA-salt Sham DOCA-salt Sham DOCA-salt 82 Figure 18. Top: Measurement of 5-HT1B receptor protein levels in the superior mesenteric artery from normotensive sham and hypertensive DOCA-salt rats. Bottom: Measurement of 5-HT2,3 receptor protein levels in the superior mesenteric artery from normotensive sham and hypertensive DOCA-salt rats. Units are reported as arbitrary densitometry units. Blots are representative of four experiments. Total protein loaded in each lane was 25 pg/pL. Vertical bars represent the mean and the standard error of the mean for the number of experiments indicated in parentheses. * represents statistically significant difference (p<0.05) between the response obtained in the sham artery 83 Mesenteric Artery 5-HT1 B Receptor 1 0,000 7500 ‘ 5000 '- Arbitrary Densitometry Units 2500 " (N=7) * T Sham DOCA-salt Sham DOCA-salt 5‘HTZB Receptor 10,00Cf 7500-J 5000'— 2500 " Arbitrary Densitometry Units (N=7) * T Sham DOCA-salt Sham DOCA-salt 84 m- 3‘." mgr-w Figure 19. Top: Real Time PCR measurement of 5-HT,,3 receptor mRNA in the thoracic aorta from normotensive sham and hypertensive DOCA- salt rats 28 days after initial surgery. Bottom: Real Time PCR measurement of 5-HT2,3 receptor mRNA in the thoracic aorta from normotensive sham and hypertensive DOCA-salt rats. Units are reported as ACT values, where CT values for 5-HT,,3 and 5-HT2E3 receptor mRNA are corrected for GAPDH mRNA expression. Vertical bars represent the mean and the standard error of the mean for the number of experiments indicated in parentheses. 85 _\ O 5-HT13Receptor ACT Value T 9’ 9° N l O (N=4) __I_ 1O Sham DOCA-salt 5'HT2B Receptor ACT Value 1‘ ‘i’ °° (N=4) __[__ Sham DOCA-salt 86 Hypothesis lll Mechanisms of Receptor Regulation There is little information currently available about the regulation of the 5- HT 23 and 5-HT1B receptors. Previous studies in our laboratory with Wistar and Wistar-Furth rats (Watts and Harris, 1999) have suggested a role for both DOCA treatment and increased vascular pressure as variables which may regulate the expression of the 5-HT 2,, receptor. However, separation of these two factors is difficult to achieve in vivo. Due to the commonality of mineralocorticoids in the model of DOCA-salt hypertension and Wistar-Furth rats treated with DOCA and salt, we speculated that mineralocorticoids would have the ability to upregulate 5- HTz,3 and 5-HT1B receptors. Previous studies have demonstrated aldosterone’s ability to increase expression of the Na-K-ATPase in vascular smooth muscle cells (Oguchi et al, 1993). Investigation of the promoters for the rat genes for the 5-HT1B receptor and the 5-HT2.3 receptor revealed that they contain mineralocorticoid response elements (MRE)(Foguet et al, 1993, Hamblin et al, 1992). Therefore, we tested the hypothesis that aldosterone-incubation would cause an increase in the levels of 5-HT,,3 receptor and 5-HT2,3 receptor proteins, independent of an increase in blood pressure. Aldosterone-Incubation Time Course Studies To test the effects of mineralocorticoid treatment in the absence of an increase in blood pressure, we performed aortic incubation studies using aldosterone. Endothelium-denuded thoracic aorta from male Sprague-Dawley 87 rats with normal blood pressure was incubated under tissue culture conditions with aldosterone. Incubation with aldosterone (100 nM) for 8 and 12 hours resulted in a significant increase (approximately 2-fold above vehicle treated) in both 5-HT,,3 receptor (figure 20, top) and 5-HT2,3 receptor (figure 20, bottom) protein levels. Incubation for 24 and 48 hours with aldosterone (100 nM) did not result in a significant increase in either 5-HT,.a or 5-HT28 receptor protein levels above vehicle. Unexpectedly, aldosterone-incubation at 24 and 48 hour time points did not result in an increase in 5-HT,B or 5-HT28 receptor proteins (figure 20, top and bottom). These data suggest that while aldosterone causes an acute increase in the level of the receptor proteins, a second cellular signal is necessary to maintain the increased levels of receptor proteins. A co-incubation pilot study with vasopressin and aldosterone (data not shown) demonstrated that treatment with vasopressin alone or in combination with aldosterone had no effect on 5-HT2,3 and 5-HT,,3 receptor protein levels. This suggests that vasopressin is not the maintenance signal for continued increase in the level of the 5-HT23 and 5-HT1B receptor proteins. Aldosterone-incubation Concentration Response Curve Studies Additionally, tissues were incubated for 12 hours with varying concentrations of aldosterone (1 nM - 100 nM). The concentrations of aldosterone which resulted in a statistically significant increase in 5-HT,.3 receptor protein levels above that of the vehicle were 30, 50 and 100 nM (figure 21, top). 5-HT2,3 receptor protein levels were statistically increased by the 10, 30, 50 and 88 Figure 20. Top: Measurement of 5-HT1B receptor protein levels to determine the effects of Aldosterone (Aldo, 100 nM) incubation for variable lengths of time (8, 12, 24 and 48 hours) in thoracic aorta from Sprague- Dawley rats with normal blood pressure. Bottom: Measurement of 5-HT2,3 receptor protein levels to determine the effects of Aldosterone (Aldo, 100 nM) incubation for variable lengths of time (8, 12, 24 and 48 hours) in thoracic aorta from Sprague-Dawley rats with normal blood pressure. Blots are representative of seven experiments. Units are reported as arbitrary densitometry units. Blots are representative of four experiments. Total protein loaded in each lane was 50 pg/pL. Vertical bars represent the mean and the standard error of the mean for the number of experiments indicated in parentheses. * represents statistically significant difference (p<0.05) between the response obtained in the vehicle- incubated artery. 89 1 5000 5-HT1 B Receptor SE (N=7) ,, E‘ | c» 10000—- E * .8; _ 2 T 8 5000— a S Li :8 o— “ VehmliAldo VenIeIeAIdo vghmmo Veme “do 8 hr 12 hr 24 hr 48 hr VehicIeAldo Vmo VehrcleAldngmglgAlgo 8 hr 12 hr 24 hr 48 hr 5'HTZB Receptor 1 5000 .9 g (N=7) E‘ . ‘1’ 10000— E * III; E T 8 5000- a e 1:5 I < 0‘v VehiclaeFAldo VehIcILAldo VehicliAldo VehicleAldo 12 hr 24 hr 48 hr VehiclLAIdo VehicleAldo \LehlcleAldo VehiclaAldo 8 hr 12 hr hr 48 hr 56 kDa 90 Figure 21. Top: Measurement of 5-HT,,3 receptor protein levels to determine the effects of Aldosterone (Aldo, 1 nM to 100 nM) incubation for 12 hours in thoracic aorta from Sprague-Dawley rats with normal blood pressure. Bottom: Measurement of 5-HT2,3 receptor protein levels to determine the effects of Aldosterone (Aldo, 1 nM to 100 nM) incubation for 12 hours in thoracic aorta from Sprague-Dawley rats with normal blood pressure. V indicates vehicle treatment. Blots are representative of four to ten experiments. Units are reported as arbitrary densitometry units. Total protein loaded in each lane was 50 pg/pL. Vertical bars represent the mean and the standard error of the mean for the number of experiments indicated in parentheses. * represents statistically significant difference (p<0.05) between the response obtained in the vehicle-incubated artery. 91 5-HT1 B Receptor a) g15000 (M440) * * V e \‘ <1E’Ioooo— 2 '1— 173 c T a) D 5000‘ 2* S \ 2:: o - A < . (>0 1 5 10 30 50 100 \‘0‘0‘ Aldosterone [nM] V 1 5 10 so 9 50 100 Aldosterone [nM] 5'HTZB Receptor (I) 'E15000 (N=4-10) :3 2‘ * 4—0 * 09 10000- "I" E “4:44: 0 [3:45 (D “5*A“A 5 sooo— 122:2: D .32., I :Z:I:I:I 2‘ :I:I:I CU .»:«:«: E 0 , :«.4~:« % .\\ _. E , (>5 1 5 10 30 50 100 < \‘Q‘p‘ Aldosterone [nM] v 1 5 10 30 50 100 Aldosterone [nM] 92 100 nM concentrations of aldosterone (figure 21, bottom). Aldosterone- and DOCA-incubation Studies with Spironolactone Furthermore, aldosterone-stimulated increase of 5-HT,B and 5-HT2,, receptor protein levels was inhibited by the mineralocorticoid receptor antagonist spironolactone (10 pM)(figure 22 top and bottom, respectively). These data indicate that aldosterone acted via a mineralocorticoid receptor to cause the increase in the 5-HT,,3 and 5-HT2,3 receptor protein levels was inhibited by the mineralocorticoid receptor antagonist spironolactone. Taken together, these data support the hypothesis that aldosterone can increase the 5-HT1B and 5-HT2,a receptor protein levels, independent of an increase in pressure. To examine whether the increase in the levels of the 5-HT13 and 5-HT2,3 receptor proteins was selective to aldosterone we used deoxycorticosterone acetate (DOCA) as another mineralocorticoid receptor agonist. Thoracic aorta from male Sprague-Dawley rats was incubated with DOCA (100 nM) for twelve hours in the presence and absence of the mineralocorticoid receptor antagonist spironolactone (10 pM). DOCA incubation increased the level of the 5-HT2,3 receptor protein approximately 2-fold (figure 23, top) and 1-fold (figure 23, bottom) for the 5-HT1B and 5-HT28 receptors, respectively. This increase in the level of receptor proteins was inhibited by the presence of the mineralocorticoid antagonist spironolactone. These data suggest that both mineralocorticoid receptor agonists, aldosterone and DOCA, increase the level of the 5-HT2,3 and 5-HT,B receptor proteins through the mineralocorticoid receptor. 93 Figure 22. Measurement of 5-HT,B receptor protein levels (top) and 5- HT2,3 receptor protein levels (bottom) to determine the effects of incubation of spironolactone (SP, 10 pM) on Aldosterone-induced (Aldo, 100 nM) increase in the level of 5-HT,B and 5-HT2,3 receptor proteins in thoracic aorta from Sprague-Dawley rats with normal blood pressure. DMSO indicates dimethyl sulfoxide treatment. ETOH indicates ethanol treatment. Blots are representative of seven experiments. Units are reported as arbitrary densitometry units. Total protein loaded in each lane was 50 pg/pL. Vertical bars represent the mean and the standard error of the mean for the number of experiments indicated in parentheses. * represents statistically significant difference (p<0.05) between the response obtained in the DMSO/ETOH-incubated artery. + represents statistically significant difference (p<0.05) between the response obtained in the Aldo/SP-incubated artery. 94 5-HT1 B Receptor 15000 (N=7) * + T 10000- 5000- ~ R ///, k 0‘ DMSO DMSO Aldo Aldo ETOH SP ETOH SP Arbitrary Densitometry Units 44 kDa Wsfimm. 21.“. . DMSO DMSO Aldo Aldo ETOH SP ETOH SP .‘2 5-HT23 Receptor .5 * + (D E 10000- 'I .9 '27) 6 D 5000- i i / \ £- 0_ /// .\\ 9 DMSO DMSO Aldo - Aldo <1: ETOH SP ETOH SP DMSO DMSO Aldo Aldo ETOH SP ETOH SP 95 Figure 23. Measurement of 5-HT1,; receptor (top) and 5-HT29 receptor (bottom) protein levels to determine the effects of incubation of spironolactone (SP, 10 pM) on deoxycorticosterone acetate-induced (DOCA, 100 nM) increases in the level of 5-HT,,3 receptor and 5-HT28 receptor proteins in thoracic aorta from Sprague-Dawley rats with normal blood pressure. PPG indicates propylene glycol treatment. ETOH indicates ethanol treatment. Blots are representative of four experiments. Units are reported as arbitrary densitometry units. Total protein loaded in each lane was 50 pg/pL. Vertical bars represent the mean and the standard error of the mean for the number of experiments indicated in parentheses. * represents statistically significant difference (p<0.05) between the response obtained in the PPG/ETOH-incubated artery. + represents statistically significant difference (p<0.05) between the response obtained in the DOCA/SP-incubated artery. 96 Arbitrary Densitometry Units Arbitrary Densitometry Units 10000 5-HT1 B Receptor (N=4) 7500— 5000— T 2500- ‘ PPG/ PPG/SP DOCA/ DOCA/SP ETOH ETOH PPG/.7 PPG/SP DOCA/ ooCAéP ETOH ETOH 5-HT Rece tor 10000 23 p (N=4) 7500- * + 5000— T 2500— PPG/ PPG/SP DOCA/ DOCA/SP ETOH ETOH ‘” . . 1‘ PPG/ PPG/SP DOCA/ DOCA/SP- ETOH ETOH 97 Aldosterone-incubation and Contraction Studies To determine if an acute increase in the level of 5-HT1B and 5-HT2,3 receptor proteins was sufficient to cause an enhanced contractile response to 5- HT we incubated endothelium-denuded thoracic aorta from male Sprague- Dawley rats with aldosterone (100 nM) for 12 hours and then placed the tissues into an isolated tissue bath for contractile studies. The aldosterone-incubated tissues did not have an enhanced response to 5-HT (figure 24). lmportantly, Western analysis of the tissues after removal from the bath demonstrated a significant increase in the levels of 5-HT2B and 5-HT1B receptor proteins (figure 25). These data suggest that an acute increase in the level of the receptor proteins is not sufficient to enable the hyperresponsiveness to 5-HT seen in arteries from hypertensive DOCA-salt rats. Alternatively, these data also suggest that other factors, such as the function of down stream signaling molecules, such as Rho-kinase (Weber and Webb, 2001) or the coupling of the receptor to the G- protein may also be chronically altered in the arteries from the hypertensive DOCA-salt rats by factors other than aldosterone. DOCA-salt Time Course Studies In order to determine the separate effects, in viva, of mineralocorticoids, pressure and salt we performed a time course study. On day zero, male Sprague-Dawley rats were weighed and their blood pressure measured. All rats received an uninephrectomy and the rats in the DOCA-salt 98 Aorta 200 + 5-HT (N=4) +CP93129 (N=4) Aldosterone incubation + 5-HT (N=4) + BW723086 (N=4) —0— CP93129 (N=4) 100T 50- Percentage of PE (10'5 M) Contraction Log Agonist [M] Figure 24. Effect of aldosterone (100 nM, 12 hr) incubation on 5-HT-, the 5-HT28 receptor agonist BW723C86- and the 5-HT1B receptor agonist CP93129-induced contraction in the endothelium-denuded rat thoracic aorta. Data are reported as a percentage of the initial phenylephrine (PE) 10‘5 M contraction. Points represent the mean and the vertical bars represent the standard error of the mean for the number of experiments indicated in parentheses. 99 Figure 25. Top: Measurement of 5-H'l'1B receptor protein levels in thoracic aorta from Sprague-Dawley rats incubated with aldosterone (100 nM; 12 hr) after contraction to S-HT, the 5-HT2E3 receptor agonist BW723086 and the 5-HT,B receptor agonist CP93129. Bottom: Measurement of 5-HT2.3 receptor protein levels in thoracic aorta from Sprague-Dawley rats incubated with aldosterone (100 nM; 12 hr) after contraction to 5-HT, the 5-HT2,3 receptor agonist BW723086 and the 5-HT1B receptor agonist CP93129. Blots are representative of four experiments. Units are reported as arbitrary densitometry units. Total protein loaded in each lane was 50 ug/uL. Vertical bars represent the mean and the standard error of the mean for the number of experiments indicated in parentheses. * represents statistically significant difference (p<0.05) from the response obtained in the Vehicle-incubated artery. lOO 5-HT1 B Receptor ~v' .V’r 2‘ *5 15000 E * .9 "Z’ a) £2 10000- D '5. 2C) g 5000- 1 £3 < 0 Vehicle- Aldosterone- Incubated Incubated (N=4) (N=4) Aldo — Aldo Vehicle Vehicle 5'HTZB Receptor 2‘ 15000 *5 * E .9 '7) 10000-1 c 03 £2 2; .T_ g 5000- E < 0 Vehicle- Aldosterone- lncubated Incubated (N=4) (N=4) ,4 ‘ , . .. I . .. .' M ' 'r , “. 99.x. ‘ ‘_ , i)? , ~ ‘ m t ' ' V . 3' Vehicle Vehicle Aldo Aldo 101 and DOCA-low salt groups received implants. Sham and DOCA-low salt rats were given normal tap water to drink. The DOCA-salt and high salt rats received high salt water (1% NaCl and 0.2% KCI) to drink. On days 1,3,5 and 7 after starting the protocol the rats were weighed and the systolic blood pressure was measured. Only the sham rats on day seven of the protocol showed a significant gain in body weight (figure 26, top right). None of the rats in the high salt, DOCA- salt and DOCA-low salt groups gained significant body weight during the protocol. By day three the DOCA-salt rats were consuming significantly more fluid than the sham, high salt and DOCA-low salt rats (figure 27, top right). The DOCA-salt rats continued to increase their fluid consumption through day seven. Additionally, the rats placed on the high salt water alone increased intake significantly by day five and remained elevated through day seven (figure 27, bottom left). The sham and DOCA-low salt rats did not vary significantly in their fluid consumption during the study. Systolic Blood Pressure The systolic blood pressure of the sham rats remained relatively constant over the seven days of treatment (figure 28). The DOCA-salt rats showed a significant increase in blood pressure by day three (average systolic blood pressure (SBP) 124 i- 7.6 mm Hg and 110 i 2.6 mm Hg, DOCA-salt and sham, respectively) (figure 27). The systolic blood pressure of the DOCA-salt rats reached hypertensive levels by day five (table 2). High salt rats had increased blood pressures by day seven but did not reach hypertensive levels (table 2). By 102 “auxin—fr Figure 26. Top left: Measurement of mass (9) of Sham rats on day s 1-7. Top right: Measurement of mass (9) of DOCA-salt rats on days 1-7. Bottom left: Measurement of mass (9) of DOCA-low salt rats on days 1-7. Bottom right: Measurement of mass (9) of High salt rats on days 1-7. Vertical bars represent the mean and the standard error of the mean for the number of experiments indicated in parentheses. * represents statistically significant difference (p<0.05) between the response obtained in the sham. 103 H5859... so 50 6-... uzv :3 262000 EoEfimfi so >8 n m -m P o 2: 8m can as uzv o ow “396.000 (5) ssew Became... so 23 :8 :9... EoEEE... “_o 30 52m 00.. 8 N (6) ssew O O ('0 00¢ oov 104 Figure 27. Measurement of fluid consumption by day and treatment group. Sham and DOCA-low salt rats received normal tap water to drink. High salt and DOCA-salt rats received salt water containing 1 % NaCl and 0.2 % KCI to drink. Vertical bars represent the mean and the standard error of the mean for the number of experiments indicated in parentheses. * represents statistically significant difference (p<0.05) between the response obtained from day one of treatment. 105 9.90 Sesame... gum 2mm 26— Ewcm -mo 9.0.0 5558:. tam 2mm 30— zmm Emnm -<00n_ -mo menl lnnk l and ldard .590 “$58.... gmm «3m 26. «Rm 0 52m .500 .58 so... - e V\ 8 .... \ Ir .. on H m. m. 2: m \ . 2: w W , w c c MI W 3 8m ) - 8m ) w w. m>mo 2.20 Eo5m9... =90. gum 26. =8 o 52¢ .500 .50 8.... r o , . I on .. on m H. mm 02 m. l 9: I p m. 9: M - 9.: H ... .2. m 08 mm m 8m w. A z. - Bu ( 0:?" com com F .30 l the 3565. 106 175 + Sham (N=7) ’5: + DOCA-salt (N=7) I + DOCA-low salt (N=4) E + High salt (N=4-6) .5. 93 150- 1 c?» (I) * (D .. L— a all. ‘0 ..‘i * O .2 CD 125 1 .9 _ ._ :<:> 4+ w - a ’II/ -_ ‘(7 * * 100 I I I l l I 0 1 2 3 4 5 6 7 8 Day of Treatment Figure 28. Measurement of systolic blood pressures. Data are reported in mm Hg. Points represent the mean and the vertical bars represent the standard error of the mean for the number of experiments indicated in parentheses. * represents statistically significant difference (p<0.05) between the response obtained from the sham response on each day . 107 Table 2. Systolic blood pressures (SBP), EC50 values for BW723C86, CP93129 and 5-HT and the maximal contractile responses to BW723C86, CP93129 and 5-HT represented as a percentage of phenylephrine (10 pM); PE) for sham, high salt, DOCA-low salt and DOCA-salt rats. Values represent the means i standard error of the mean for each group. The number of animals in each group is indicated in parentheses. * represents statistical difference from response obtained in the shams. a. 108 :0 H 0.00 0.0 H 0.00 0.0 H 0.0: 00.0 H 00.0 0.0 H 000 .0. 0.00 00.: 0 0 0. 0 H 0.0: 00.0 H 00.0 0.0 H 000 .0. ..00 0.2.500 0 0 H 0.00 0.0 H 0.00 0.2 H 0.000 00.0 H 00.0 0.0 H 000 .0. 0.00-500 0 0 0.0 H 0.00 00.0 H 00.0 0.0 H 0: .0. 0.9.0 0 000 0.0 H 0.00 0.0 H 0.00 0. I H 0.000 00.0 + 00.0 0.0 H 000 .0. 0.00 00.: 0 0 0.0 H 0.0: 00.0 H 00.0 0.0 H 000 .0. 0.00 2.2-0.000 0d + 9mm m. E H mém 0.0 H mdow 0.0.0 H mad 3. H 0.3 3. =00-00 0 0 0.0 H 0.000 00.0 H 8.0 0.0 H 000 .0. 0.00 00.: 0 0.0. H 0.00 0.0 H 0.000 00.0 H 00.0 0.0 H 000 .0. 0.00 30.0000 c We H mdm 9w H 0.000 mod H 090 0.0. H o: E =00-00 omcommom L uncommon. L ... EE amw mm ..mmao 0003an .......m 3508:. .50 ..2 E: .0E_x0_2 .9585. 2...; SUN mo. . 109 da the H9 san (Sor arte BVV7 conh reSpc 29,u: 0f the that \ Contra Contra eXPOSI becom Tecepu Contra There is day seven the DOCA-low salt rats had significantly lower blood pressure than their sham, DOCA-salt and high salt counterparts (average SBP 114 i 2.3 mm Hg, 142 i 8.4 mm Hg, 130 i 9.3 mm Hg and 101 i 2.1 mm Hg, sham, DOCA- salt, high salt and DOCA-low salt, respectively). Contractile Studies on Day 1 Although there was no increase in systolic blood pressure on day one the arteries from the DOCA-salt rats contracted to the 5-HT2,3 receptor agonist BW723086 (maximal contraction 52.5 % i 10.5 of phenylephrine (PE) 10'5 M contraction). The arteries from DOCA-low salt, high salt and sham rats did not respond to either BW723C86 or to the 5-HT“3 receptor agonist CP93129 (figure 29, top and middle). There was no enhanced contraction to 5-HT observed in any of the arteries from the treatment groups (figure 29, bottom). These data suggest that while the 5-HT2,3 receptor is functional on day one, as indicated by contraction to BW723086, this is not sufficient to result in an enhanced contraction to 5-HT. It is interesting to note that after twenty-four hours of exposure to elevated mineralocorticoid and salt levels the 5-HT2.3 receptor becomes functionally linked to contraction. The change that enables the 5-HT23 receptor to mediate contraction occurs in the absence of an increase in pressure. Contractile Studies on Day 3 On day three of the time course the 5-HT2.3 agonist BW723C86 elicits a contraction in the arteries from the DOCA-salt and high salt rats (figure 30, top). There is also a significant contraction observed to the 5-HT1B agonist CP93129 in 110 Figure 29. Top: Effect of the 5-HT28 receptor agonist BW723086 in endothelium-denuded thoracic aorta from high salt, DOCA-salt, DOCA-low salt and sham rats on day one of treatment. Middle: Effect of the 5-HT"3 receptor agonist CP93129 in endothelium-denuded thoracic aorta from high salt, DOCA-salt, DOCA-low salt and sham rats on day one of treatment. Bottom: Effect of 5-HT in endothelium-denuded thoracic aorta from high salt, DOCA-salt, DOCA-low salt and sham rats on day one of treatment. Data are reported as a percentage of the initial phenylephrine (PE) 10'5 M contraction. Points represent the mean and the vertical bars represent the standard error of the mean for the number of experiments indicated in parentheses. * represents statistically significant difference (p<0.05) between the response obtained in the sham artery. 111 Percentage of PE (10'5 M) Contraction Percentage of PE (10'5 M) Contraction Percentage of PE (10"5 M) Contraction Day 1 20° +th salt (N=4) —o— D 100- 50" 0— -1o -9 -8 -7 J—I—DOCA-salt SN: ) 150 -'— Sham (N=6 CA-Iow salt N=4) 1 £2.9 . -6 -5 -4 -3 Log BW723086 [M] 200 150‘ 100‘ 50q +Hi h salt (N=4) +0 CA-low salt (N=4) +DOCA-salt (N=6) +Sham (N=6 -1o -9 -8 -7 -6 -5 -4 -3 Log CP93129 [M] 200 150‘ 100‘ 50‘ ‘+ DOCA-salt +High salt (N=6) +DOCA-low salt (N=4) +DOCA-salt (N=5) —¥—Sham N=5) +Sham 8 day (N=9) . a 0 23 day (N=1 1) 0 -9 -8 -7 -6 -5 -4 -3 Log 5-HT [M] 112 Figure 30. Top: Effect of the 5-HT28 receptor agonist BW723086 in endothelium-denuded thoracic aorta from high salt, DOCA-salt, DOCA-low salt and sham rats on day three of treatment. Middle: Effect of the 5-HT“3 receptor agonist CP93129 in endothelium-denuded thoracic aorta from high salt, DOCA-salt, DOCA-low salt and sham rats on day three of treatment. Bottom: Effect of 5-HT in endothelium-denuded thoracic aorta from high salt, DOCA-salt, DOCA-low salt and sham rats on day three of treatment. Data are reported as a percentage of the initial phenylephrine (PE) 10‘5 M contraction. Points represent the mean and the vertical bars represent the standard error of the mean for the number of experiments indicated in parentheses. * represents statistically significant difference (p<0.05) between the response obtained in the sham artery. 113 Percentage of PE (10'5 M) Contraction Percentage of PE (10'5 M) Contraction Percentage of PE (10'5 M) Contraction Day 3 +lbligh salt (N=4) CA- low salt (N=4) —I-DOCA-salt N=6 150+ +Sham (N__ _6§ ) 200 Log BW723C86 [M] «Ir—High salt (N=4) —O-D CA-low salt (N=4) +DOCA-salt (N=6) 1-4—Sham (N=6 :39 Log CP93129 [M] 200 ::HDi h salt (N=4) CA-low sal—éN=4) -l-DOCA-salt (N- 150 :‘F-Sham N=5) +Sham 8 lday (N=9) +DOCA-salt 28 day (N=11) 100- Log 5-HT [M] 114 the arteries from the DOCA-salt rats (figure 30, middle). The arteries from the DOCA-low salt and sham rats do not show a significant contractile response to BW723C86 or CP93129 (figure 30, top and middle). Contraction to 5-HT is enhanced is arteries from high salt rats (figure 30, bottom). The arteries from the DOCA-salt rats showed no change in the maximal contraction to 5-HT but demonstrated enhanced 5-HT potency (-log E050 [M] values 6.025 :9; 0.08, 5.740 i 0.05, 6.017 i 0.03, DOCA-salt 3 day, Sham, DOCA-salt 28 day, respectively). Unexpectedly, the arteries from DOCA-low salt rats show an increased maximal response to 5-HT (maximal contraction 141.2 -_1- 11.8 % PE 10‘5 M contraction). Since there was no response to the 5-HT,B agonist CP93129 and very little response to the 5-HT2.3 agonist BW723C86 this enhanced contraction to 5-HT is surprising. However, this enhancement of 5-HT-induced contraction in arteries from DOCA-low salt rats only occurs on day three. Contractile Studies on Day 5 Experiments performed on day five of the time course demonstrate a similar profile of results. Arteries from the DOCA-salt and" high salt rats contracted to both the 5-HT2.3 agonist BW72SC86 (maximal contraction 54.3 i 11.9 % and 46.3 i 6.6 % of PE 10'5 M contraction, DOCA-salt and high salt, respectively) and the 5-HT,B agonist CP93129 (maximal contraction 35.0 :1: 8.6% and 27.8 1: 7.4 % of PE 10'5 M contraction, DOCA-salt and high salt, respectively) (figure 31, top and middle). Arteries from high salt rats also displayed an increased maximal contraction (maximal contraction 148.5 x 11.4 % and 106.2 i 115 Figure 31. Top: Effect of the 5-HT2E3 receptor agonist BW723C86 in endothelium-denuded thoracic aorta from high salt, DOCA-salt, DOCA- low salt and sham rats on day five of treatment. Middle: Effect of the 5- HT1B receptor agonist CP93129 in endothelium-denuded thoracic aorta from high salt, DOCA-salt, DOCA-low salt and sham rats on day five of treatment. Bottom: Effect of 5-HT in endothelium-denuded thoracic aorta from high salt, DOCA-salt, DOCA-low salt and sham rats on day five of treatment. Data are reported as a percentage of the initial phenylephrine (PE) 10’5 M contraction. Points represent the mean and the vertical bars represent the standard error of the mean for the number of experiments indicated in parentheses. * represents statistically significant difference (p<0.05) between the response obtained in the sham artery. 116 Percentage of PE (10'5 M) Contraction Day 5 200 100- t-A—High salt (N=4) +DOCA- low salt N=4) +DOCA-salt (N=) 1504 +Sham (N=6 -1 0 -9 -8 -7 -6 -5 -4 Log BW723086 [M] zoo‘trnfgm salt(N (N=4) CA-low salt N=4) c -I-DOCA-salt g: .g 150_+Sham (N=6N O "6 9 8. E S 0 1000 C A 8 2 5“.) o. o 1: 501 * 0-. . -1o -9 e -7 -6 -5 -4 -3 Log CP93129 [M] 200 :HDigh salt (N=4) c CA- low salt N=5N=4) w 0 +DOCA—saltN * o. “3 150-""Sham m=5N . .. g -D-Sham 8d y(N=9) ' g E +DOCA-salt + g 8 100- 28day(N=11) E A o 2 2m ° '0 °' 1: 50~ Log 5-HT [M] 117 8.7 % of PE 10'5 M contraction, high salt and sham, respectively), a decrease in the threshold of activation of contraction and an increase in potency, and as compared to sham arteries (-log EC50 value [M] 6.06 i 0.05 and 5.60 i 0.07 high salt and sham, respectively) (figure 31, bottom). The arteries from DOCA- salt rats also show a decrease in the threshold of activation of contraction (figure 31, bottom). Contractile Studies on Day 7 On day seven of the time course, arteries from DOCA-salt rats contracted to both BW723086 (maximal contraction 57.4 g]; 5.9 % of PE 10‘5 M contraction) and CP93129 (maximal contraction 63.8 :t 7.8 % of PE 10'5 M contraction) (figure 32, top and middle). Arteries from high salt rats also contracted to both BW723086 (maximal contraction 24.5 i 9.4 % of PE 10‘5 M contraction) and CP93129 (maximal contraction 19.6 :t 8.1 % of PE 10‘5 M contraction) (figure 31, top and middle). Interestingly, arteries from high salt rats have been exposed to both increased levels of salt as well as an increased pressure. However, these arteries do not contract to the same magnitude of contraction in the presence of BW723086 and CP93129 as those that are taken from DOCA-salt rats on day seven (table 2). These data suggest that the combination of increased levels of salt and mineralocorticoids as well as the increased pressure are all important regulators of the changes which enable the 5-HT2E3 and 5-HT,.3 receptors to participate in contraction. Additionally, arteries from DOCA-salt and high salt rats both show hyperresponsiveness to 5-HT (figure 32, bottom). The characteristic 118 Figure 32. Top: Effect of the 5-HT2E3 receptor agonist BW723C86 in endothelium-denuded thoracic aorta from high salt, DOCA-salt, DOCA- low salt and sham rats on day seven of treatment. Middle: Effect of the 5- HT“, receptor agonist CP93129 in endothelium-denuded thoracic aorta from high salt, DOCA-salt, DOCA-low salt and sham rats on day seven of treatment. Bottom: Effect of 5-HT in endothelium-denuded thoracic aorta from high salt, DOCA-salt, DOCA-low salt and sham rats on day seven of treatment. Data are reported as a percentage of the initial phenylephrine (PE) 10'5 M contraction. Points represent the mean and the vertical bars represent the standard error of the mean for the number of experiments indicated in parentheses. * represents statistically significant difference (p<0.05) between the response obtained in the sham artery. 119 Percenatge of PE (10'5 M) Contraction Percenatge of PE (10'5 M) Contraction Percenatge of PE (10'5 M) Contraction 200+ 100- 200 150- -rJ-Sham 28 day (N=9) 1000 Day 7 ih salt( (N=6) CA- low sal N=4) ... DOCA-salt (N ) 150- . Sh am (N=6 Log BW723086 [Ml i—High salt (N=6) +D CA- low sal_éN=4) +DOCA-salt 1501 +Sham (N=6 Log CP93129 [M] +High silt (N=6) +oooA- low salt lN=4) -l-DOCA-salt N- ) +Sham (N=5 * +DOCA-salt 28 day (N=11) Log 5-HT [M] 120 increased maximal contraction (maximal contraction 130.3 i 19.5 °/o and 119.5 i 5.1 % of PE 10'5 M contraction, DOCA-salt and high salt, respectively), the decreased threshold for contraction and the increased potency (- log EC50 value [M] 6.41 i 0.09, 6.13 .t 0.03 and 5.70 i: 0.07, DOCA-salt, high salt and sham, respectively). Protein Analysis Studies There is no increase in 5-HT1B receptor protein levels at any of the treatment groups at any time point (figure 33), although there is a trend towards increasing levels of protein in the aortic homogenates from the DOCA-salt rats. This was an unexpected finding as the arteries from both the DOCA-salt and high salt rats contract to the 5-HT,B agonist CP93129 starting on day three of the time course. These data suggest that although the receptor protein levels were increased by 28 days of DOCA-salt treatment, increased receptor protein levels are not required to enable this receptor to participate in contraction. Furthermore, the 5-HT2B receptor protein levels are significantly increased by day 3 of DOCA-salt treatment (figure 34). The 5-HT2E3 receptor protein levels are not increased in any of the other treatment groups at any time point. Interestingly, the level of receptor protein is not significantly increased on day 1, even though there is a significant contraction to the 5-HT2,3 receptor agonist BW723C86. There is also no significant increase in the 5-HT2.3 receptor levels in the aortic homogenates from the high salt rats in which contraction to BW723C86 does 121 Figure 33. Top: Measurement of 5-HT,B receptor protein levels in aortic homogenates from sham, DOCA-low salt, high salt and DOCA-salt rats on days 1, 3, 5,and 7. Bottom: Representative Western blot probed for the 5- HT1B receptor protein. Total protein loaded in each lane was 50 ug/uL. Blots are representative of 4-6 experiments. Units are reported as arbitrary densitometry units. Vertical bars represent the mean and the standard error of the mean for the number of experiments indicated in parentheses. * represents statistically significant difference (p<0.05) between the response obtained in the corresponding sham. Abbreviations: S= Sham; DS= DOCA-salt; DLS= DOCA-low salt; HS: High salt. 122 .3958 n >8 m >8 m >8 _ >8 >88 add do d an ad mu m an flu mu m ..wfllqalmdlml «.358 > >8 m >8 m >8 F >8 >88 an ad ma w an «.6 mu m an win mu m wnlmdlj w_w>w._ £99m .ofiooom m _.._.I-m suun Mtewotgsueq MBJllQJV 123 Figure 34. Top: Measurement of 5-HT2.3 receptor protein levels in aortic homogenates from sham, DOCA-low salt, high salt and DOCA-salt rats on days 1, 3, 5,and 7. Bottom: Representative Western blot probed for the 5- HT"3 receptor protein. Total protein loaded in each lane was 50 ug/uL. Blots are representative of 4-6 experiments. Units are reported as arbitrary densitometry units. Vertical bars represent the mean and the standard error of the mean for the number of experiments indicated in parentheses. * represents statistically significant difference (p<0.05) between the response obtained in the corresponding sham. Abbreviations: S= Sham; DS= DOCA-salt; DLS= DOCA-low salt; HS: High salt. 124 =8-8 m >8 m >8 F >8 >8 mm madam mfilfldj mulfldlwdllw Illfi. till? it .....Ili uni... l. £3-58 > >8 m >8 m >8 _ >8 >8 mm mnlmdllmqllw mnlfldlmdlw mflddlmdllw m_m>m.. 5991 .2882”. ._._._-m stlun Mtewousuea MelthJv 125 occur. These data would suggest that increased levels of the 5-HT2,3 receptor protein are not essential for contraction to occur. Additionally, these data suggest that an increase in pressure is also not required for the 5-HT2.3 receptor to become functionally coupled to contraction. Collectively, these data support the hypothesis that mineralocorticoids can upregulate the 5-HT2B and 5-HT,B receptors, independent of an increase in blood pressure. These data also demonstrate that increased levels of salt and pressure are also important regulators of these receptors. Additionally, an increase in the level of receptor protein is not required to enable these receptors to mediate contraction. These data also suggest that changes in the signaling cascades and receptor/effector coupling are potentially involved in this enhanced contractility to 5-HT and agonists of the 5-HT2,3 and 5-HT1B receptors under conditions of DOCA-salt hypertension. 126 Hypothesis IV Measurement of Free Plasma 5-HT Levels To test the hypothesis that the level of free 5-HT circulating in the blood is increased in the DOCA-salt model of hypertension, we measured the levels of free 5-HT. The normal physiological levels of 5-HT are reported to be between 5 and 120 nM in both human models and rat models of hypertension (Marasini et al, 1985, Zhao et al, 1999, Biondoet al, 1986). The increase in free 5-HT may occur for several reasons. There may be a decrease in platelet uptake (Kamal et al, 1984) of 5-HT or pulmonary endothelial damage results in a decrease in 5-HT clearance. Platelets from hypertensive subjects tend to aggregate which would result in 5-HT release. This increase in aggregation may be attributable to an increase in calcium and a decrease in NO' levels (Camilletti et al, 2001, Nityanand et al, 1993). There are also reports in the literature that the platelets from hypertensive subjects are defective in either storage and/or uptake mechanisms of 5-HT (Nityanand et al, 1990, Fetkovska et al, 1990). This decreased platelet 5-HT levels may be due to defects in the 5-HT transporter. There is presently no one mechanism that has been clearly elucidated to account for this increase seen in hypertension. However, even a small increase in free 5-HT could be sufficient to produce endogenous activation of the 5-HT2,3 receptor in the vasculature of hypertensive subjects. We have preliminary data which suggest a trend towards an increase in the levels of free 5-HT in the platelet poor plasma fraction from the DOCA-salt 127 hypertensive rats (figure 35). There is a corresponding decrease in the level of 5- HT in the platelet rich plasma fraction from the DOCA-salt hypertensive rats (figure 35). These data are in agreement with previously published human studies which demonstrate an increase in the levels of 5-HT in the plasma. These data also suggest one possible mechanism for the endogenous activation of 5-HT23 receptors reported in the DOCA-salt model of hypertension (Watts and Fink, 1999). Another possible mechanism for the endogenous activation of 5-HT2,3 receptors in DOCA-salt hypertension is that the increase in the level of the receptor protein and _!' change in the functional coupling of the 5-HT2.3 receptors to mediate contraction is sufficient to result in activation of these receptors, which then participate in the maintenance of the increased blood pressure. 128 400 6 o 2 .Q 300 2 o .c 3 “5 200 E \ c» 5 I— 100 I. no 0 Sham Sham DOCA DOCA PRP PPP PRP PPP Figure 35. Level of 5-HT in the platelet poor and platelet rich fractions of plasma from normotensive sham and hypertensive DOCA-salt rats. Vertical bars represent the mean value and the standard error of the mean of the number of animals indicated in parenthesis. PPP represents platelet poor plasma. PFlP represents platelet rich plasma. 129 Discussion Since the isolation of 5-HT by Rapport and colleagues in 1948, a role for 5-HT in hypertension has been suggested. While the specific role which 5-HT plays in hypertension remains a controversial topic, there is no argument that 5- HT is both a vasoconstrictor and a mitogen. The ability of 5-HT to be involved in both of these processes may be important to 5-HT’s role in hypertension. It has been established that enhanced agonist-induced contraction as well as hypertrophy and hyperplasia of vascular smooth muscle are characteristic of arteries from hypertensive subjects in both human and experimental models. Vascular remodeling can be seen as a compensatory mechanism by which the arteries attempt to cope with the increased pressure. lntriguingly, this remodeling may involve the dedifferentiation of vascular smooth muscle cells, migration of smooth muscle cells, replication and differentiation into a contractile phenotype. This situation may create an opportunity to observe receptors involved in growth and migration that may not be expressed under the normal contractile phenotype. Furthermore, while there is controversy about the specific actions of 5-HT in many diseases, including depression, hypertension, pre-eclampsia, pulmonary hypertension, migraine and psychiatric disorders, the potential targeting of therapies to 5-HT receptors and transporters are currently being explored and utilized clinically. Therefore, understanding the 5-HT receptor subtypes and mechanisms which 5-HT utilizes in these pathological states is important to 130 achieving maximal therapeutic benefits as well as avoiding unwanted and potentially harmful side-effects This project was undertaken to characterize the 5-HT receptor subtypes utilized to mediate contractile hyperresponsiveness observed in arteries from hypertensive DOCA-salt rats and to investigate the mechanisms by which the expression and function of these receptors are changed under the conditions of DOCA-salt hypertension. Characterization of 5-HT Receptors in Vascular Smooth Muscle At the start of this project we set out to characterize which 5-HT receptors mediate 5-HT-induced contraction in vascular smooth muscle. Based on data from contractile experiments, we concluded that there was no contractile role for the 5-HT,D and 5-HT,F receptors in aorta from normotensive and hypertensive DOCA-salt rats. We were also unable to observe a contraction to the 5-HT"3 receptor agonists in aorta from normotensive rats. These findings for the 5-HT1D and 5-HT1F receptors are in agreement with findings by other investigators which demonstrate no role in smooth muscle contraction for the 5-HT,D and 5-HT1F receptors (Razzaque et al, 1999, Cohen and Schenck, 1999). Furthermore, in vascular smooth muscle cultured from aorta removed from rats with normal blood pressure, only the 5-HT2A receptor was able to activate the ERK/MAPK pathway (Watts et al, 2001). Agonists of the 5-HT23, 5-HT,.3 5-HT1D and 5-HT,F receptors showed no increase in the level of ERK/MAPK activation. These data further 131 support the idea that while the mRNA for these receptors are present, there does not appear to be an activatable receptor protein. These combined data support the conclusion that the 5-HT,D, 5-HT1F and 5-HT,,3 receptors do not participate in 5-HT-induced contraction under conditions of normal blood pressure. 5-HT“, Receptors in Hypertension Initially, we hypothesized that the 5-HT,B receptor mediated contraction ! under conditions of DOCA-salt hypertension. Our experiments using 5—HT, the 5- F HT“, antagonist GR55562, the 5-HT,,3 receptor agonists RU24969, sumatriptan I and the rodent selective 5-HT,B receptor agonist CP93129 support the conclusion that the 5-HT“3 receptor does mediate at least a portion of the 5-HT- and CP93129-induced contraction under conditions of DOCA-salt hypertension. These findings are in agreement with those of MacLean and colleagues in pulmonary hypertension. (MacLean and Morecroft, 2001, MacLean et al, 1996, Keegan et al, 2001). These studies found that arteries taken from control rats with normal blood pressure in the pulmonary arteries did not respond to agonists of the 5-HT,.3 receptor. Additionally, the 5-HT,.3 receptor antagonist GR555562 had no effect on 5-HT-induced contraction in arteries from the control rats. However, 5-HT-induced contraction in arteries from the control rats was sensitive to inhibition by the 5-HT,.,_,,,2c receptor antagonist ketanserin. Interestingly, in vivo administration of the 5-HT1W, receptor antagonist GR127935 (3 mg/kg/day) attenuated the increased right ventricular pressure, right ventricular hypertrophy 132 and pulmonary vascular remodeling in the hypertensive rats (Keegan et al, 2001). These data suggest that both the 5-HT2A and 5-HT,,3 receptors are involved in pulmonary hypertension. There have been no experiments which have examined the effects of in vivo administration of a 5-HT"3 antagonist on mean arterial blood pressure in the DOCA-salt model of hypertension. Additionally, in atherosclerotic coronary arteries from rabbits and rabbit carotid arteries which undergo collar placement, there is also a hypersensitivity to 5-HT and an increased contractile response to 5-HT,B receptor agonists (lshida et at, 2001, Geerts et al, 2000). Collectively, these data all suggest that under ' conditions of vascular diseases, such as hypertension and atherosclerosis, there is a hypersensitivity to 5-HT and a functional change in the 5-HT,.3 receptor which contributes to the development and/or maintenance of the diseased state. 54”,, Receptors in Hypertension Data implicating the 5-HT2,3 receptor in DOCA-salt hypertension has existed for several years. A change in the 5-HT receptor which primarily mediates contraction to 5-HT under the conditions of DOCA-salt hypertension has been suggested largely on the basis of functional data. These data include the use of the 5-HTMC receptor antagonist ketanserin (Watts, 1998). The mesenteric arteries from the hypertensive DOCA-salt rats by day 7 no longer respond to the 5-HTM receptor antagonist ketanserin with a rightward shift in the 5-HT concentration response curve (Watts, 1998). These data support the 133 ‘I-n'uumjhw— idea of the 5-HT2.3 receptor mediating 5-HT-induced contraction in arteries from hypertensive rats when one considers the low affinity of ketanserin for the 5-HT23 receptor (table 1). The 5-HT2,3 receptor is not sensitive to inhibition by ketanserin and any 5-HT-induced contraction mediated by this receptor would, therefore, not be inhibited. Further evidence for involvement of the 5-HT2,, receptor in hypertension comes from another study by Watts and colleagues in which they demonstrate that ketanserin did not shift the 5-HT-induced contraction in mesenteric arteries from 28 day hypertensive DOCA-salt rats (Watts of al, 1996). Additionally, the study by Watts and colleagues also demonstrated that in the rat stomach fundus the 5-HT2.3 receptor is phenoxybenzamine-insensitive. The 5- HT.)A receptor is sensitive to alkylation by phenoxybenzamine as the 5-HT2A receptor mediated contraction in the thoracic aorta from normotensive sham rats was inhibited in the presence of phenoxybenzamine (300 nM). The 5-HT- induced contraction in the arteries from the hypertensive DOCA-salt rats was relatively insensitive to the treatment with phenoxybenzamine (Watts 9! el, 1996). The idea of the 5-HT2,3 receptor involvement is also supported by the findings that the 5-HT2B receptor antagonist LY272015 inhibited 5-HT-induced contraction in the aorta from the DOCA-salt hypertensive rats but not the sham rats (Watts, 1997). Further evidence for the involvement of the 5-HT2.3 receptor comes from the use of the 5-HT2.3 receptor agonist BW723C86. Watts and Harris showed that BW723086 elicited a contraction in the mesenteric arteries from DOCA-salt rats but not in their corresponding sham counterparts (Watts and 134 ‘— hum-..- n. Harris, 1999). These data suggest that the predominant receptor which mediates 5-HT-induced contraction in the mesenteric artery from DOCA-salt hypertensive rats is the 5-HT28 receptor. The physiological importance of this receptor is emphasized by the finding that administration of the 5-HT2.,-, receptor antagonist LY272015, in vivo, decreased the blood pressure of hypertensive DOCA-salt rats (Watts and Fink, 1999). However, chronic administration of LY272015 to determine whether 5- HT?B receptors are required for development of the increased blood pressure as well as smooth muscle cell hypertrophy and hyperplasia has not yet been done in any model of hypertension. Additional support for the importance of 5-HT29 receptors in the cardiovascular system comes from 5-HT2,3 receptor knockout mice. 5-HT2B receptor knockout mice show severe cardiovascular abnormalities (Choi et al, 1997, Nebigil et al, 2001b, Nebigil et al, 2000b). However, the use of 5-HT2,3 receptor knockout mice to study the importance of this receptor in the development of hypertension has also not yet been done. Since 5-HT2.3 receptors are required for morphogenesis, cell migration and cell cycle progression, one can speculate that 5-HT2.a receptors may be important to the vascular changes observed in arteries during the development of hypertension. However, immunohistochemical studies to examine location of the 5-HT2.3 receptors in arteries from hypertensive rats have not been performed yet. Furthermore, localization of the 5-HT18 and 5-HT23 receptors within the smooth muscle cells, membrane or cytosol. has not been done. These localization studies would 135 address the idea that 5-HT,,3 and 5-HT28 receptors may not be activated in smooth muscle cells from normotensive rats because they are stored in the cytosol and therefore not able to be activated by an agonist. Additionally, no studies have been done, to date, which examine the involvement of the 5-HT28 receptors in pulmonary hypertension although the 5- HT28 receptor mRNA has been localized to pulmonary smooth muscle cells with immunohistochemistry (Choi and Maroteaux, 1996). Furthermore, the 5-HT2,, receptor has been implicated in the LNNA model of hypertension as well. (Russell and Watts, 1999). These findings suggest that the 5-HT2.3 receptor may be involved in many models of hypertension and could be a potential therapeutic target for the treatment of established essential hypertension. Investigation of 5-HT Receptor “Unmasking” Currently, the mechanisms of receptor “unmasking” are not clearly understood. Additionally, there have been no studies which address the question of why the 5-HT"3 receptor requires “unmasking” in some but not all arteries. Furthermore, there are no studies to date which address the phenomenon of “unmasking” with regards to the 5-l-lT2,3 receptor. However, the work by Smith and colleagues in the rabbit ear artery would suggest that both the 5-HT,B and 5- HT” receptors may function as “silent receptors” (Smith et al, 1999). This may be due to a lack of coupling to second messenger systems under non- depolarized conditions. They suggest that the depolarization caused by elevated 136 K+ may induce an influx of extracellular calcium which in turn enhances the coupling to the second messenger pathways which mediate vasoconstriction (Smith et al, 1999). It is not known if the depolarization-dependent “unmasking” is a phenomenon selective for 5-HT,.3 and 5-HT2A receptors or if other 5-HT receptors might also be “unmasked” by this mechanism. Based on this information about “unmasking”, we hypothesized that membrane depolarization alone would enable an artery from a normotensive sham rat to respond in the same manner to 5-HT and 5--HT,.3 receptor agonists as an artery from a hypertensive DOCA-salt rat. However contrary to this hypothesis, we were unable to observe a contraction even in the presence of KCI (15 mM) to the rodent selective 5-HT"3 receptor agonist CP93129 in arteries from normotensive rats. Therefore, we conclude that these data do not support a role for silent 5—HT1B receptors in the mesenteric arteries from normotensive rats. Additionally, we were also unable to observe a contraction to the 5-HT2B receptor agonist BW723C86 and the 5-HT“3 receptor agonists RU24969 and sumatriptan in the rat thoracic aorta from normotensive rats in the presence of KCI (15 mM). The data presented, herein demonstrate that acute depolarization alone was not sufficient to enhance arterial responses to agonists of the 5-HT1B and 5-HT2,3 receptors. interestingly, another artery from the rat, specifically the rat tail artery, has been described as requiring “unmasking” to observe a response to 5-HT,.a receptor agonists (Craig and Martin, 1993). The difference which enables the tail 137 artery 5-HT,B receptor to be “unmasked” and respond to CP93129 while the aorta and mesenteric artery do not is yet known. However, this difference may involve the ability of the 5-HT,B receptor to activate mechanisms which lead to increases in intracellular calcium levels. The role of calcium in 5-HT1B receptor —mediated contraction appears to vary between species and vessel. In the rabbit ear artery 5-HT,.3 receptor- induced contraction, following “unmasking” with preconstriction with the or1 adrenergic receptor agonist phenylephrine, requires extracellular calcium and activation of L-type calcium channels (Movahedi and Purdy, 1997). However, in i the rabbit saphenous vein contraction to 5-HT“3 receptor agonists is insensitive to the L-type calcium channel blocker nifedipine (Razzaque et al, 1995). Additionally, in vascular smooth muscle cells from the bovine basilar artery, 5- l-lT1B receptor stimulation induces release of calcium from intracellular stores. In contrast, stimulation of the rabbit mesenteric artery and the rabbit renal artery fails to result in any release of intracellular calcium and appears to be dependent on L-type voltage gated calcium channels (Seager et al, 1994, Hill et al, 2000). It is interesting to note that the 5-HT2.3 receptor has been linked to activation of L-type calcium channels for contraction in the rat stomach fundus (Cox and Cohen, 1995). The signaling pathway used by the 5-HT,,3 and 5-HT2E3 receptors to activate the L-type calcium channels and intracellular calcium stores has not yet been elucidated. However, if these two receptors simultaneously couple to activation of different mechanisms which would increase intracellular 138 calcium levels in vascular smooth muscle cells it might, in part, explain their combined contribution to the hyperresponsiveness to 5-HT observed in arteries from hypertensive DOCA-salt rats and the lack of effect in vessels from normotensive rats. Increased Levels of 5-l-I‘l'“3 and 5-HT,. Receptor Proteins In DOCA-salt Hypertension Based on the contractile data which suggested a functional change in the 5-HT,.3 and 5-HT28 receptors, we speculated that an increase in the expression of these receptors in vascular smooth muscle which would provide an explanation for the physiological and pharmacological data which indicate a change in the complement of contractile serotonergic receptors in vascular smooth muscle under conditions of hypertension. These molecular data demonstrate an increase in the levels of 5-HT,.3 and 5-HT23 receptor proteins under conditions of established DOCA-salt hypertension in the thoracic aorta as well as the mesenteric artery. These are the first studies which have demonstrated on increased level of 5-HT2,3 and 5-HT“3 receptor proteins in a diseased state. Regulation of 5-HT,, and 5-HT“ Receptors With the knowledge of an increased level of 5-HT“3 and 5-HT2,3 receptor protein levels we next proposed to determine what factors might contribute to this increase in receptor protein levels. There are currently no studies which have 139 investigated the regulation of either of these receptors at the transcriptional and translational levels. Analysis of the rodent 5-HT“3 promoter revealed that there are multiple mineralocorticoid response elements (MRE’s) located in the promoter (Hamblin et al, 1992). Additional analysis also revealed that the rodent S-HTZB receptor promoter also contains two MRE’s (Foguet et al, 1992). This is relevant to the regulation of these receptors in a disease state such as in the DOCA-salt model, where hypertension is induced by the use of mineralocorticoids, as well as other models of hypertension where elevated levels of aldosterone may influence expression of this receptor. As previously mentioned, aldosterone has been implicated in increasing the expression of the K-ras (Stockand et al, 1999). Aldosterone also decreases the mRNA expression of c-Myc, c-Jun, c-Fos by post-transcriptional mechanisms while increasing Fra-2 mRNA by a transcriptional mechanism in epithelial cells (Verry et al, 2000). This is interesting to note because it suggests that aldosterone may act directly at a promoter via the MRE as well as indirectly through its actions on transcription factors. Additionally, aldosterone induces methylation of ras in renal epithelial cells (Al-Baldawi et al, 2000). This suggests that aldosterone not only changes the level of protein expression but also the state of activation of proteins involved in signaling cascades. While the signaling pathways used by the 5-HT,B and 5- HT,"3 receptors to cause contraction are currently unknown, both of these receptors have been shown to utilize the ERK/MAPK pathway, of which ras is a 140 member, for mitogenesis. Whether aldosterone is actingdirectly at the MRE’s or through its actions on other transcription factors to cause the upregulation of the 5-HT1B and 5-HT2E3 receptors in vivo is unclear as there is no increase in the level of 5-HT1B and 5-HT2,, receptor mRNA. However, the lack of an increase in mRNA is not conclusive as we did not measure the rate of mRNA degradation. The 5- HT"3 receptor promoter also contains eight basic helix loop helix (bHLH) sites, one glucocorticoid response element (GRE), several SP-1 sites as well as several AP-1 sites. The 5-HT23 receptor promoter contains ten bHLH sites, SP-1 sites and multiple AP-1 sites. Transcription factors from the bHLH family include a wide variety of proteins such as the aryl hydrocarbon receptor nuclear transporter (ARN'l) (Huffman et al, 2001), Myc (Miethe et al, 2001) and MyoD (Chen et al, 2001) among many others. Animals have been shown to contain members of 36 subfamilies of bHLH transcription factors, of the 44 subfamilies which have been defined (Ledent and Vervoot, 2001). Interestingly, another transcription factor, the SP-l transcription factor, is a target for NO‘ in vascular smooth muscle cells (Sellak et al, 2002). NC‘ was shown to inhibit the binding of SP-1 to the cGMP-dependent protein kinase (PKG) promoter (Sellak et al, 2002). Thus, in conditions such as hypertension where the increase in reactive oxygen species may affect the bioavailability of NO’, transcriptional regulation of genes by NO' may be affected contributing to the changes observed in this disease. Collectively, this information suggests that characterization of these promoters and the transcriptional mechanisms of 141 “Altamira-run regulation of these receptors is an undertaking which will be both time and labor intensive. It is also unknown at this point if aldosterone affects the members of the signaling cascades used by the 5-HT,3 and S-HT28 receptors to cause contraction. This is an intriguing possibility since the ability of the 5-HT1.3 and 5- HT2,3 receptors to mediate contraction does not appear to be dependent on the level of the receptor protein. We initially speculated that upregulation of 5-HT1B and 5-HT28 receptor proteins was, at least partially, responsible for the increased ‘_ I" .rv-a-v contractility to 5-HT seen in the arteries from hypertensive rats. Our data would 5' argue that an increase in the level of receptor protein is not sufficient to allow these receptors to participate in contraction. Additionally, the data obtained in the DOCA time course studies in vivo demonstrate that contraction can occur in the absence of an increase in the receptor protein. The mechanism by which the protein levels of the 5-HT1B and 5-HT2,3 receptors are increased may not to involve an increase in mRNA levels as these were unchanged in the Real Time PCR experiments from the rat thoracic aorta. However, a study by Watts and colleagues demonstrated an 2-fold increase in 5- HTZ,3 receptor mRNA in the mesenteric artery from hypertensive DOCA-salt rats (Watts et al, 1996). While the protein levels are increased in both the aorta and mesenteric artery (°/o increase from sham 218% and 171%, aorta and mesenteric artery, respectively), these data do not reflect the changes observed in the levels- of 5-HT2.3 receptor mRNA. Interestingly, in the mesenteric artery one observes a 142 greater enhancement of the 5-HT-induced contraction than that observed in the aorta (figure 4). However, these data do not address the issues of mRNA degradation. Therefore, based on the data presented herein we can not completely rule out transcriptional regulation of the 5-HT,B and 5-HT2.3 receptors. Furthermore, these findings further support the idea that hyperresponsiveness to 5-HT involves the 5--HT2B and 5-HT1B receptors through mechanisms involving more than just an increase in the level of receptor protein. There appear to be changes in the signaling mechanisms and/or changes in the receptor-effector coupling mechanisms. Speculation These data suggest that in addition to the upregulation of 5-HT,.3 and 5-HT23 receptors, other changes must be occurring in the vascular smooth muscle to enable the contractile responses observed. Additional second messengers which may be involved in 5-HT23 and 5-HT,B receptor signaling as second messengers in vascular smooth muscle but have not yet been thoroughly examined include reactive oxygen species, the Rho-Rho-kinase pathway and MUPP1 (figure 34). The use of reactive oxygen species as a signaling mechanism by 5-HT2,3 and 5-HT,.3 receptors has not yet been investigated. Interestingly, H202 mediates calcium-dependent contraction in canine basilar arteries by a mechanism which involves PKC and Pl-3 kinase (Yang et al, 1999). The 5-HT2., receptor is known to couple to PKC activation in the rat stomach fundus to cause contraction (Cox 143 and Cohen, 1995). Therefore, the involvement of H202 in the contractile signaling pathways used by the 5-HT23 receptor should be investigated. Furthermore, the 5-HT2B receptor and the 5-HT2A receptor use many of the same signaling pathways. 5-HT2A receptors are known to use H202 and 02‘ (Lee et al, 2001, Greene et al, 2000). If activation of the 5-HT28 or 5-HT,B receptors leads to an increased generation of reactive oxygen species the results may be an increase in contraction as well as a decrease in the available NC“ with a reduction in vasorelaxation. Additionally, since the involvement of reactive oxygen species has been implicated in many models of hypertension (Zalba et al, 2001, Makino et al, 2002, Gao and Lee, 2001), this merits further investigation. Another second messenger which should be examined further in regards to 5- HT-induced contractile signaling is the Rho-Rho—kinase pathway. The involvement of the Rho-Rho-kinase pathway in calcium sensitization induced by agonist stimulation (Hirata et al, 1992) has been suggested and provides another area which requires further study. While the mechanisms of calcium sensitization induced by agonist stimulation are not clearly understood this may be involved in the hyperresponsiveness to 5-HT observed in vessels from hypertensive rats. Interestingly, in mesenteric arteries from hypertensive DOCA-salt rats contracted with 5-HT, there is an enhanced relaxation to the Rho-kinase inhibitor Y-27632 (Weber and Webb, 2001). The results from this study suggest that the enhanced 5-HT-induced contraction may be due to involvement of the Rho-Rho-kinase pathway. Additionally, these data also suggest an increased level of Rho-Rho- 144 kinase activity. Therefore, examining the ability of 5-HT2,3 and 5-HT1B receptors to couple to this pathway maybe of importance in disease states, such as hypertension, where there is an upregulation and change in the functional coupling of the 5-HT2B and 5-HT1B receptors. Lastly, the PDZ-dependent signaling mechanisms used by the 5-HT2.3 receptor also merits further study. To date, 5-HT2B receptors have been shown to activate nitric oxide sYnthases via its group IPDZ motif located at the C-terminus ', (Manivet et al, 2000). This receptor-mediated stimulation of iNOS requires the 5- i HTZB receptor to be coupled to the Gor13 (Manivet et al, 2000). The loss of a vasodilator, specifically NC’ from endothelial cells potentially, through a 5-HT28 receptor, in arteries from hypertensive rats may contribute to the enhanced arterial tone. Interestingly, the MUPP1 protein, a PDZ binding protein which has 13 PDZ domains, interacts with the 5-HT28 receptor (Bécamel et al, 2001). The presence of MUPP1, which may serve as a scaffolding protein, in the vasculature of hypertensive DOCA-salt rats has not been examined at this time. The MUPP1 protein appears to be involved in clustering of 5-HT2c receptors at the cell membrane (Bécamel et al, 2001). This clustering of receptors may be required for dimerization of receptors as well as proper interaction with effector molecules. While no studies have yet been performed to examine if 5-l:lT2E3 receptors form dimers, it has been established that 5-HT1B receptors can form both hetero and homodimers. Dimerization affects the interaction of the receptor with G-proteins, 145 agonists and members of the signaling pathways. Therefore, the involvement of MUPP1 and the ability of the 5--HT2,3 receptor to form dimers should be studied. Furthermore, the class III Mint-1 protein, a PDZ domain containing scaffolding protein, has been linked to N-type Ca2+ channels (Maximov ef al, 1999). The interaction of L-type Ca2+ channels and PDZ-domain containing proteins has not yet been investigated. However, it has been shown that both the 5-HT2E3 and the 5-HT,.3 receptors can couple to L-type calcium channels. The exact mechanisms of this interaction have not yet been elucidated. Additionally, in the family of i membrane-associated guanylate kinases (MAGUKs), of which CASK is a " member, PDZ-domains are found together with src homology (SH-3) domains (Li et al, 2002). Many adaptor molecules have SH-3 and SH-2 domains which facilitate protein-protein interactions. Proteins with PDZ-domains have been shown to interact with PLC and PKC (Bahner et al, 2002, Huber ef al, 1998). Additionally, the interactions of scaffolding proteins with the 5-HT“3 receptor have also not yet been examined. However, this interaction between 5-HT receptors and their mechanisms of coupling to their signaling pathways may be an interesting avenue of future research. Furthermore, changes in calcium channel density and/or function may also participate in the enhanced 5-HT—induced contraction and BW723086- and CP93129-induced contractions observed in arteries from DOCA-salt hypertensive rats. In mesenteric arteries from hypertensive DOCA-salt rats L- type calcium channel expression was increased as compared to sham rats 146 (Molero et al, 2001). Changes in function of the calcium channels have also been suggested in several models of hypertension (Hermsmeyer, 1993, Matsuda et al, 1997, Cox and Lozinskaya, 1995). These changes in calcium channel density and function may contribute to the changes in the functional coupling of the 5- HT1B and 5-HT2B receptors. The combination of changes in calcium channel function and/or number combined with changes in the signaling pathways, such as Rho-Rho-kinase and calcium sensitization induced by agonist stimulation, , may be important to the hyperresponsiveness to 5-HT observed in hypertension. Physiologically, the changes in the levels of the 5-HT2,3 and 5-HT,B receptors r and their functional coupling may contribute to either the development and/ or maintenance of hypertension. The ability of the 5-HT28 receptor antagonist LY272015 to lower the blood pressure of DOCA-salt hypertensive rats suggests endogenous activation of this receptor to maintain the increased blood pressure (Watts and Fink, 1999). While no 5-HT,.3 receptor antagonists have been studied in vivo in the DOCA-salt model of hypertension, they have been used to study pulmonary hypertension. 5-HT has been implicated as a causative agent in pulmonary hypertension (Egermayer et al, 1999, Kereveur et al, 2000, MacLean et al, 2000, Miyata et al, 2000). The administration of 5-HT1!, receptor antagonists attenuate the pulmonary hypertension (Miyata et al, 2000). The common finding between these two models of hypertension, pulmonary and DOCA-salt-induced, is an increase in the level of free 5-HT in the plasma. These findings suggest that increases in plasma free 5-HT may be a marker for future 5-HT based 147 treatments. These studies suggest that 5-HT,,3 and 5-HT2,3 receptors are involved in mediating the arterial hyperresponsiveness to 5-HT observed in hypertension. This involvement appears to be due to changes independent of the level of receptor proteins as the functional response of contraction can be separated from an increase in the level of 5-HT28 and 5-HT,.3 receptor proteins. Currently, there is no precedent for receptors to be present but not activatable under conditions of normal blood pressure but which are involved as mediators of contraction and the development of a pathological condition such as hypertension. “5%; 148 Figure 36. Speculations on changes in signaling pathways which may be involved in 5-HT-induced contraction via the 5-HT28 and 5-HT“3 receptors in arteries from DOCA-salt hypertensive rats. Abbreviations: ERK: Extracellular signal regulated kinase; PKC: Protein Kinase C; MUPP1: multi-PDZ domain protein 1; PLC: Phospholipase C; DAG: Diacylglycerol; lP3= lnosital triphosphate; ROS: Reactive oxygen species; MAPKK: Mitogen activated protein kinase kinase; PLD: Phospholipase D; Pl3-K= Phosphoinosital 3 kinase. 149 L1... 882 mm own. ... In" Ohm lll' mm.— racella 1y be otors l 7 6 xmm +Nmo +Nmo a 3 m ivaled 150 Conclusion The main goals of this thesis were to investigate the mechanisms of the increase in the level of the 5-HT,,,3 and 5-HT,,3 receptor proteins in DOCA-salt hypertension and the physiological significance of these changes. The 5-HT2.3 receptor has been implicated as a possible mediator of the maintained increase in pressure observed in DOCA-salt hypertension. Additionally, hyperresponsiveness to 5-HT observed in arteries from hypertensive rats is another a physiological phenomenon which we investigated to determine if the 5- HT,"3 and 5-HT,.3 receptors were involved in the mechanisms of i‘ hyperresponsiveness. 5-HT is a known mitogen and vasoconstrictor. With the use of 5-HT receptors and transporters as dmg targets, it is necessary to understand how 5- HT mediates its actions. Therefore, we initially characterized the 5-HT receptors which are involved in 5-HT-induced contraction under conditions of both normal blood pressure as well as hypertension. From the studies presented herein we conclude that only the 5-HT2A receptor mediates 5-HT-induced contraction in arteries from rats with normal blood pressure. We also conclude that the 5-HT28, 5-HT1B and 5-HT2A receptors are involved in 5-HT—induced contraction under conditions of DOCA-salt hypertension. We were unable to detect any involvement of the 5-HT,D and 5-HT,F receptors under either conditions. We also conclude that the 5-HT,B and 5-HT2.3 receptors in the thoracic aorta of 151 normotensive rats can not be “unmasked” to participate in the 5-HT-induced contraction. We initially speculated that increases in the level of 5-HT,.3 and 5-HT2E3 receptor proteins were, at least partially, responsible for the increased contractility to 5-HT seen in the arteries from hypertensive rats. However, the results of our aldosterone incubation studies and the DOCA-salt time course studies do not support this idea. These data support the argument that an increase in the level of 5-HT“3 and 5-HT2.3 receptor proteins are not necessary to observe an increased contractile response to agonists of these receptors. However, there is an increased contractile response to the 5-HT2.5 receptor agonist BW723086 prior to an increase in blood pressure in the DOCA and salt treated rats. Therefore, while the 5--HT2.3 and 5--HT,.3 receptor protein levels are increased after four weeks of DOCA-salt treatment, the precise role of these receptors in the development of hypertension and possibly vascular remodeling are yet to be elucidated. The studies presented here also support the idea that mineralocorticoids in conjunction with increases in pressure and salt may be important mediators of the changes to both the level of 5-HT28 and 5--HT1B receptor protein levels and the signaling mechanisms utilized by these receptors. Aldosterone incubation, in vitro, produced an increase in the level of the 5-HT"3 and 5-HT2.3 receptor protein levels. The in vivo time course experiments, however, did not show any increase in the level of the 5-HT1B receptor protein levels. Additionally, only the DOCA-salt 152 rats showed an increase in the level of the 5-HT2,3 receptor protein. These data suggest that in vivo regulation mechanisms of these receptors maybe more complicated than in vitro experiment suggested as elevated levels of ‘ mineralocorticoids alone in vivo was not sufficient to increase the levels either the 5-HT,.a or the 5-HT2,3 receptor proteins. These data suggest that in vivo mineralocorticoids may not be acting directly at the transcriptional level. However, as we have not measured mRNA degradation or done an RNAse I protection assay, we can not rule out the possibility of transcriptional regulation. The functional changes resulting from a change from a 5-HT 2A receptor to L” the addition of the 5-HT2.3 and 5-HT,.3 receptors have significant physiological ramification. 5-HT has a 300 fold higher affinity for the 5-HT 2., receptor than for the 5-HT2A receptor. When comparing the 5-HT23, 5-HT,.3 and 5-HT2A receptors, 5-HT has the greatest affinity for the 5-HT2.3 receptor followed by the 5-HT,.3 receptor (table 1). This increased sensitivity to 5-HT in hypertension coupled to the possibility of an increase in free 5-HT, as observed in the HPLC experiments, and increased level of receptor proteins creates a situation where 5-l-lT may be acting endogenously to mediate an increase in total peripheral resistance. This increased 5-HT receptor activation maycontribute either to the development or maintenance of hypertension. The finding that the 5-HT2.3 receptor antagonist LY272015 can act as an antihypertensive agent in weeks three and four of DOCA-salt hypertension suggests that this may be an important receptor to consider in the treatment of established hypertension. While 5-HT"3 receptor 153 antagonists have not been tested in the DOCA-salt model of hypertension, they are effective in the treatment of pulmonary hypertension. If the 5--HT,,3 receptor antagonists prove effective in lowering blood pressure of the DOCA-salt hypertensive rats, this could create another potential target for therapy. This project is also significant in that the determination of serotonergic receptors which mediate vascular contraction will allow for targeting of drug therapy for migraine with fewer systemic side effects. One of the major side effects reported for migraine treatment with lmitrex", also known as sumatriptan, is chest pain associated with vasoconstriction of 5-HT“3 receptors (Ottervanger et al, 1998). With the increasing interest in utilizing 5-HT receptors as targets for treatment of neurological disorders, such as depression, determining the serotonergic receptors which mediate contraction to 5-HT in the vasculature of normotensive and hypertensive subjects is imperative. Understanding of the regulation of 5-HT receptors, their signaling pathways and functions in disease states may contribute to advances in these fields of therapeutic research. 154 33./“3 References Adham N, Kao HT, Checter LE, Bard J, Olsen M, Urquhart D, Durkin M, Hartig PR, Weinshank RL, Branchek TA. 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