Faculty of Medicine and Health Sciences Heymans Institute of Pharmacology Promotor: Prof. Dr. R. A. Lefebvre Gastrointestinal effects of soluble guanylate cyclase activation by NO-independent compounds and by NO delivery via nitrite Sarah M. R. Cosyns 2014 Thesis submitted as partial fulfilment of the requirements for the degree of Doctor in Biomedical Sciences The studies described in this thesis were supported by grant BOF10/GOA/024 from the Special Investigation Fund of Ghent University, grant G.0021.09N from the Fund of Scientific Research Flanders and by COST action BM1005 (European Network on Gasotransmitters). List of abbreviations 8-Br-cGMP 8-bromoguanosine 3’, 5’ cyclic monophosphate AAC area above the curve ADHF acute decompensated heart failure ALT alanine transaminase ANOVA analysis of variance AST aspartate transaminase ataciguat 5-chloro-2-[[(5-chloro-2-thienyl)sulfonyl]amino]-N-[4-(4-morpholinyl sulfonyl)phenyl]benzamide (=HMR1766) ATP adenosine triphosphate AU arbitrary units AUC area under the curve BAY 41-2272 3-(4-amino-5-cyclopropylpyrimidin-2-yl)-1-(2-fluorobenzyl)-1H- pyrazolo[3,4-b] pyridine BK channel large conductance Ca 2+ -activated K + channel cGK cGMP-dependent protein kinase (= PKG) cGMP cyclic guanosine 3’-5’-monophosphate CGRP calcitonin gene-related peptide cinaciguat 4-[((4-carboxybutyl)-(2-[(4-phenethylbenzyl)oxy]phenethyl)amino) methyl]benzoic acid (= BAY 58-2667) CNS central nervous system CO carbon monoxide CORM-3 carbon monoxide-releasing molecule-3 COX-2 cyclo-oxygenase-2 CRF corticotrophin-releasing factor DAMPs damage associated molecular patterns DETA-NO diethylenetriamine NONOate DGME diethylene glycol monoethyl ether DMSO dimethylsulfoxide EC 50 half maximal effective concentration EDTA ethylene diaminetetraacetic acid EFS electrical field stimulation EGTA ethylene glycol tetraacetic acid EIA enzyme immunoassay ELISA enzyme-linked immunosorbent assay Emax maximum effect eNOS endothelial nitric oxide synthase (= NOS3) ENS enteric nervous system FD-70 fluorescein-labeled dextran 70 kDa FDA Food and Drug Administration GALT gut-associated lymphoid tissue GC geometric centre GMC giant migrating contractions GTP guanosine-5’-triphosphate HNE 4-hydroxy-2-non-enal H2S hydrogen sulfide ICAM-1 intercellular adhesion molecule-1 ICC interstitial cell of Cajal IFNγ interferon gamma IL-6 interleukin-6 IL-12 interleukin-12 IM intestinal manipulation iNOS inducible nitric oxide synthase (= NOS2) i.p. intraperitoneally I/R ischemia/reperfusion i.v. intravenously KATP channel ATP-sensitive potassium channel Lo optimal load L-012 8-amino-5-chloro-7-phenylpyrido[3,4-d]pyridazine-1,4(2H,3H)dione LES lower esophageal sphincter L-NAME Nω-nitro-L-arginine methyl ester MCP-1 monocyte-chemoattractant protein-1 MDA malondialdehyde MMC migrating motor complex MPO myeloperoxidase MRS2500 (1 R*,2 S*)-4-[2-iodo-6-(methylamino)-9H-purin-9-yl]-2-(phosphonooxy) bicycle[3.1.0]hexane-1-methanol dihydrogen phosphate ester NANC non-adrenergic non-cholinergic NF-κB nuclear factor-κB nNOS neuronal nitric oxide synthase (= NOS1) NO nitric oxide NOS nitric oxide synthase NSAIDS nonsteroidal anti-inflammatory drugs ODQ 1H[1,2,4,]oxadiazolo[4,3-a]quinoxalin-1-one PACAP pituitary adenylate cyclase-activating polypeptide PBS phosphate buffered saline PDE phosphodiesterase PGF 2α prostaglandin F2α PKG protein kinase G (= cGK) PMSF phenylmethylsulfonyl fluoride POI postoperative ileus PTIO 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide ROS reactive oxygen species sGC soluble guanylate cyclase S.E.M. standard error of the mean SK channel small conductance Ca 2+ -activated K + channel SNAP S-nitroso-N-acetyl-DL-penicillamine TCA trichloroacetic acid TNFα tumor necrosis factor alpha UES upper esophageal sphincter VIP vasoactive intestinal polypeptide WT wild-type YC-1 3-(5’-hydroxymethyl-2’-furyl)-1-benzylindazole Table of contents List of abbreviations……………….…………………………………………..………….………………………..……. 5 Table of contents……………………..…………………………………………..…………………………………..……. 9 Chapter I Literature survey………………………………………..…………………………………..……. 17 I.1 The gastrointestinal tract…………………………………………..………………………………………. 19 I.1.1 Anatomy and functions.……………………………………………………………………………. 19 I.1.2 Gastrointestinal motility……………………………………….…………………………………... 22 I.1.3 Neuronal control of gastrointestinal motility.……….………………………………..…. 24 I.1.4 Nitrergic neurotransmission…..…………………………………………………………………. 28 I.2 Soluble guanylate cyclase……….…………………………………....…...…………….………………. 30 I.2.1 Structure………………………………..……………………………………………………………….… 30 I.2.2 sGC in oxidative stress conditions.…………………………………………………….…….... 32 I.2.3 Role of sGC in gastrointestinal motility……..…………………………………………….... 33 I.3 NO pharmacotherapy and alternatives.……………………….……………………………..……… 34 I.3.1 Classic NO pharmacotherapy and its limitations.…………………………………….... 34 I.3.2 sGC stimulators…………………………….……………………………………….………..…...…… 35 I.3.3 sGC activators....…………………………………………………………………………………..…... 37 I.3.4 NO pharmacotherapy for gastrointestinal disorders……………….………..…….… 40 I.3.5 Nitrite as a source of NO.………………………………………………………………………….. 41 I.4 Postoperative ileus……………………………….…………………….…………………………….………. 45 I.4.1 Pathogenesis…...……………………………………………………………………………..………... 46 I.4.2 Management of POI………….……………………………………...……………………….……... 52 I.5 References………………………………………………….…………………………………………………….. 57 Chapter II Aims…………………………………………………….………………………………………………... 71 II.1 References…………………………………………………….………………………………………………….. 76 Chapter III Heme deficiency of soluble guanylate cyclase induces gastroparesis…..….. 81 III.1 Abstract……………………………………………………….…………………………………………..……….. 83 III.2 Introduction………………………………………………………………....…...…………….……..……….. 85 III.3 Materials and methods……………………………………………….……………………………………… 85 III.3.1 Ethical approval..………………………………………………………………………………………. 85 III.3.2 Animals………………………………….…………………………………………………………..……… 85 III.3.3 Muscle tension experiments………………………………….………………….…………...… 86 III.3.3.1 Tissue preparation………….……………………………………….…….……….…… 86 III.3.3.2 Isometric tension recording………………………………………..….………...… 86 III.3.3.3 Protocol in fundic, jejunal and colonic strips……….………….…..……… 87 III.3.3.4 Protocol in pyloric rings…………………..................….…………….………… 88 III.3.3.5 Data analysis…………………………………..………….…………………….………… 90 III.3.4 Gastric emptying………………………………....……………………………..………….………… 91 III.3.5 Transit and small intestinal contractility…….….………..............……………..……… 91 III.3.5.1 Intestinal transit (fluorescein-labelled dextran method)...….….…... 91 III.3.5.2 Small intestinal contractility………….…………...……………..….…….……… 92 III.3.5.3 Whole gut transit time (carmine method)…..……………………….……… 92 III.3.5.4 Distal colonic transit……………………….…………………………….………..…… 93 III.3.6 Histology……………….……………………………………………………………………………..…… 93 III.3.7 sGC enzyme activity……………….………………………………………………..……………..… 93 III.3.8 Drugs used……………………………….…………………………………………………………..…… 94 III.3.9 Statistics……………………………………………………….………………….…………………..…… 94 III.4 Results…………………….……………………………………………………..……………………………..….. 95 III.4.1 General observations and histology……………………………………………..….….….… 95 III.4.2 sGC enzyme activity……………….………………………………………………….….…….....… 97 III.4.3 Muscle tension experiments…………………………………….……….……..………….…… 97 III.4.3.1 Tissue weight..………………………………………….……..……………….………… 97 III.4.3.2 Contractile responses to carbachol and PGF 2α ………………….….……… 97 III.4.3.3 Fundus.………………………….………………………………………………….……….. 98 III.4.3.4 Pyloric rings……………….………………………………………………………..…... 101 III.4.3.5 Jejunum………………………………………………………….….………………..…… 103 III.4.3.6 Colon…………………………………………………………………….……………..…… 105 III.4.4 Gastric emptying, small intestinal transit and whole gut transit time…….… 106 III.5 Discussion………………………..……...………………………………………………….………………….. 108 III.6 References…………………………………...…………………………………………………………………. 112 Chapter IV Mechanism of relaxation and interaction with nitric oxide of the soluble guanylate cyclase stimulator BAY 41-2272 in mouse gastric fundus and colon……….……. 117 IV.1 Abstract………………………………………………………………………….……………………………….. 119 IV.2 Introduction………………………………………………….………………………………………..…….…. 120 IV.3 Materials and methods…………………………………………….……………………….….…………. 121 IV.3.1 Animals…………………………………………………............................……………….…..… 121 IV.3.2 Muscle tension experiments…………………………..…………….………………………… 121 IV.3.2.1 Tissue preparation and isometric tension recording…………………. 121 IV.3.2.2 Protocols……………………..…………………………………………………………… 122 IV.3.2.3 Functional data analysis………………………….……………………..…….…… 124 IV.3.3 cGMP analysis………………………………………………….……………………………………… 125 IV.3.4 Drugs used………..………………………………………………………..…..……………………… 125 IV.3.5 Statistics…………………..……………………………………………………………………………… 126 IV.4 Results……………………….…………………………………………………………………………..……….. 127 IV.4.1 Role of sGC-cGMP in the relaxant effect of BAY 41-2272 in gastric fundus 125 and colon……………………………………………………………………..………………………..…………… 127 IV.4.1.1 Influence of ODQ and L-NAME on BAY 41-2272-induced 124 relaxations……………………………………………………………………………..……………….. 127 IV.4.1.2 Influence of phosphodiesterase-5 inhibition on BAY 41-2272- 125 induced relaxations…………..………….…………………..……………………………………. 129 IV.4.1.3 cGMP analysis……….………………………………………………..……..………… 129 IV.4.2 Interaction with endogenous and exogenous NO……………....................…… 130 IV.4.2.1 Influence of ODQ and L-NAME on EFS and exogenous NO………... 130 IV.4.2.2 Interaction of BAY 41-2272 with EFS and exogenous NO………….. 131 IV.4.2.3 Influence of phosphodiesterase-5 inhibition on EFS and 125 exogenous NO……………………………………………………………..………………………..…