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Functions of Sensory Trp Channels in Vascular Responses to Chemical and Thermal Stimuli

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Functions of Sensory Trp Channels in Vascular Responses to Chemical and Thermal Stimuli KU Leuven Biomedical Sciences Group Faculty of Medicine Department of Cellular and Molecular Medicine FUNCTIONS OF SENSORY TRP CHANNELS IN VASCULAR RESPONSES TO CHEMICAL AND THERMAL STIMULI Lucía ALONSO CARBAJO Jury: Dissertation presented in Promoter: Prof. Karel Talavera Pérez partial fulfilment of the Co-promoter: Prof. María Teresa Pérez García requirements for the Chair: Prof. Rudi Vennekens degree of Doctor in Jury members: Prof. Peter Vangheluwe Biomedical Sciences Prof. Félix Viana de la Iglesia June 2019 PROGRAMA DE DOCTORADO EN INVESTIGACIÓN BIOMÉDICA TESIS DOCTORAL: FUNCTIONS OF SENSORY TRP CHANNELS IN VASCULAR RESPONSES TO CHEMICAL AND THERMAL STIMULI Presentada por Lucía Alonso Carbajo para optar al grado de Doctor/a por la Universidad de Valladolid Dirigida por: Prof. Karel Talavera Pérez Prof. María Teresa Pérez García A mis padres y a mi marido, Alejandro Table of Contents List of abbreviations ......................................................................................................................i Chapter 1: Introduction ................................................................................................................ 1 1.1 The resistance arteries ........................................................................................................... 3 1.1.1 The vascular wall ............................................................................................................ 3 1.2 Perivascular nerves ................................................................................................................. 5 1.2.1 The sympathetic nervous system ................................................................................... 5 1.2.2 The sensory nervous system .......................................................................................... 8 1.3 The vascular tone ................................................................................................................. 11 1.4 Transient Receptor Potential channels ................................................................................. 13 1.5 Sensory TRP channels in the vascular system ....................................................................... 15 1.5.1 TRPA1 ........................................................................................................................... 15 1.5.2 TRPV1 ........................................................................................................................... 16 1.5.3 TRPM3 .......................................................................................................................... 17 1.5.4 TRPM8 .......................................................................................................................... 19 1.5.5 Other sensory TRP channels ......................................................................................... 20 1.6 Modulation of vascular function by thermal stimuli: role of sensory TRP channels ............. 21 Chapter 2: Hypothesis and objectives ........................................................................................ 25 Chapter 3: Materials and methods ............................................................................................. 29 3.1 Animals ................................................................................................................................. 31 3.2 Vascular smooth muscle cells isolation ................................................................................. 31 3.2.1 Surgery ......................................................................................................................... 31 3.2.2 Enzymatic digestion ..................................................................................................... 31 3.3 Sympathetic ganglia isolation ............................................................................................... 32 3.4 RNA extraction and cDNA synthesis ...................................................................................... 32 3.5 Quantitative and real-time polymerase chain reaction ........................................................ 32 3.6 Immunofluorescence microscopy ........................................................................................ 33 3.7 Pressure myography experiments ........................................................................................ 34 3.8 Intracellular Ca2+ fluorescence imaging ................................................................................ 36 3.9 Patch-clamp electrophysiology ............................................................................................ 36 3.10 Reagents ............................................................................................................................. 37 3.11 Statistical analysis ............................................................................................................... 37 Chapter 4: Activation of the cation channel TRPM3 in perivascular nerves induces vasodilation of resistance arteries .................................................................................................................. 39 4.1 Expression pattern of TRPM family in mouse mesenteric arteries ....................................... 41 4.2 Localization of TRPM3 in perivascular nerves of mesenteric arteries ................................... 42 4.3 TRPM3 activation induces vasodilation mainly via stimulation of CGRP receptors .............. 48 4.4 CGRP release induced vasodilation via activation of K+ channels ......................................... 52 4.5 Sympathetic nerves are not implicated in TRPM3-mediated vasodilation ........................... 55 Chapter 5: Role of TRPA1 and TRPM8channels in vascular responses to cold ........................... 59 5.1TRPA1 and TRPM8 channels are involved in intrinsic vascular responses to cold ................. 61 5.2 Implication of the perivascular innervation in intrinsic vascular responses to cold............. 66 Chapter 6: Discussion, general conclusions and future perspectives ......................................... 71 6.1 Discussion............................................................................................................................. 73 6.1.1 Activation of the cation channel TRPM3 in perivascular nerves induces vasodilation of resistance arteries...................................................................................................... 73 6.1.2 Role of TRPA1 and TRPM8channels in vascular responses to cold ............................... 77 6.2 General discussions .............................................................................................................. 82 6.3 Future perspectives .............................................................................................................. 83 6.3.1 Role of TRPM3 in hypertension .................................................................................... 83 6.3.2 Cold shock response in ´in vivo´ models: role of TRPA1 and TRPM8 channels ………..... 85 Chapter 7: References ................................................................................................................ 87 Chapter 8: Abstract .................................................................................................................. 103 8.1 Abstract .............................................................................................................................. 105 8.2 Resumen ............................................................................................................................ 106 Acknowledgements, personal contribution and conflict of interest statements ...................... 107 Chapter 9: List of publications and curriculum vitae ................................................................. 109 9.1 List of publications .............................................................................................................. 111 9.2 Curriculum Vitae ................................................................................................................. 113 Abbreviations List of abbreviations AC Adenylate cyclase ACh Acetylcholine AITC Allyl isothiocyanate α-SMA a-smooth muscle actin AngII Angiotensin II ATP Adenosine triphosphate BAPTA 1,2-bis(o-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid ACTB b-actin β-gal b-galactosidase 2+ + BKCa Ca -dependent K channels BPH Blood pressure high BPN Blood pressure normal BSA Bovine serum albumin cAMP Cyclic adenosine monophosphate CAT Choline acetyltransferase CGRP Calcitonin gene-related peptide CHO Chinese hamster ovary CLR Calcitonin-like receptor CNS Central nervous system CO Cardiac output Ct Threshold cycle DAG Diacylglycerol DHEA Dehydorepiandrosterone DIDS 4,4'-Diisothiocyano-2,2'-stilbenedisulfonic acid DMSO Dimethyl sulfoxide DRG Dorsal root ganglion DTE 1,4-Dithioerythritol EC Endothelial cells EC50 Half maximal concentration EGTA Ethylene glycol-bis(2-aminoethylether)-N,N,N’,N’-tetraacetic acid GAPDH Glyceraldehyde 3-phosphate dehydrogenase GPCR G protein-coupled receptors HEK293T Human embryonic kidney cells 293 SV40 T-antigen HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid HPLC High-performance liquid chromatography iNOS Inducible nitric oxide synthase + KATP ATP-dependent K channels + KIR Inward rectifier K channels KO Knockout Kv Voltage-dependent K+ channels L-DOPA Levodopa MLCK Myosin light chain kinase MAPK Mitogen-activated protein kinases NA Noradrenaline
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