title: Pharmacological characterization of the relaxant effect induced by adrenomedullin in rat cavernosal smooth muscle.
abstract: The aim of the present study was to determine the mechanisms underlying the relaxant effect of adrenomedullin (AM) in rat cavernosal smooth muscle (CSM) and the expression of AM system components in this tissue. Functional assays using standard muscle bath procedures were performed in CSM isolated from male Wistar rats. Protein and mRNA levels of pre-pro-AM, calcitonin receptor-like receptor (CRLR), and Subtypes 1, 2 and 3 of the receptor activity-modifying protein (RAMP) family were assessed by Western immunoblotting and quantitative real-time polymerase chain reaction, respectively. Nitrate and 6-keto-prostaglandin F(1α) (6-keto-PGF(1α); a stable product of prostacyclin) levels were determined using commercially available kits. Protein and mRNA of AM, CRLR, and RAMP 1, -2, and -3 were detected in rat CSM. Immunohistochemical assays demonstrated that AM and CRLR were expressed in rat CSM. AM relaxed CSM strips in a concentration-dependent manner. AM(22-52), a selective antagonist for AM receptors, reduced the relaxation induced by AM. Conversely, CGRP(8-37), a selective antagonist for calcitonin gene-related peptide receptors, did not affect AM-induced relaxation. Preincubation of CSM strips with N(G)-nitro-L-arginine-methyl-ester (L-NAME, nitric oxide synthase inhibitor), 1H-(1,2,4)oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, quanylyl cyclase inhibitor), Rp-8-Br-PET-cGMPS (cGMP-dependent protein kinase inhibitor), SC560 [5-(4-chlorophenyl)-1-(4-methoxyphenyl)-3-trifluoromethyl pyrazole, selective cyclooxygenase-1 inhibitor], and 4-aminopyridine (voltage-dependent K(+) channel blocker) reduced AM-induced relaxation. On the other hand, 7-nitroindazole (selective neuronal nitric oxide synthase inhibitor), wortmannin (phosphatidylinositol 3-kinase inhibitor), H89 (protein kinase A inhibitor), SQ22536 [9-(tetrahydro-2-furanyl)-9H-purin-6-amine, adenylate cyclase inhibitor], glibenclamide (selective blocker of ATP-sensitive K(+) channels), and apamin (Ca(2+)-activated channel blocker) did not affect AM-induced relaxation. AM increased nitrate levels and 6-keto-PGF1α in rat CSM. The major new contribution of this research is that it demonstrated expression of AM and its receptor in rat CSM. Moreover, we provided evidence that AM-induced relaxation in this tissue is mediated by AM receptors by a mechanism that involves the nitric oxide-cGMP pathway, a vasodilator prostanoid, and the opening of voltage-dependent K(+) channels.
authors.affiliation: Programa de Pós-Graduação em Farmacologia, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP, Brasil.
authors.forename: L N
authors.initials: LN
authors.lastname: Leite
authors.affiliation: Programa de Pós-Graduação em Farmacologia, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP, Brasil.
authors.forename: N A
authors.initials: NA
authors.lastname: Gonzaga
authors.affiliation: Departamento de Cirurgia e Anatomia, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP, Brasil.
authors.forename: D P C
authors.initials: DP
authors.lastname: Tirapelli
authors.affiliation: Departamento de Cirurgia e Anatomia, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP, Brasil.
authors.forename: L F
authors.initials: LF
authors.lastname: Tirapelli
authors.affiliation: Laboratório de Farmacologia, Departamento de Enfermagem Psiquiátrica e Ciências Humanas, Escola de Enfermagem de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP, Brasil.
authors.forename: C R
authors.initials: CR
authors.lastname: Tirapelli
mesh_terms: D015761:4-Aminopyridine / Q000494:pharmacology; D015121:6-Ketoprostaglandin F1 alpha / Q000032:analysis; D053607:Adrenomedullin / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D000818:Animals; D015153:Blotting, Western; D058286:Calcitonin Receptor-Like Protein / Q000032:analysis* / Q000037:antagonists & inhibitors; D017869:Cyclic GMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors; D016861:Cyclooxygenase Inhibitors / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007150:Immunohistochemistry; D007191:Indazoles / Q000494:pharmacology; D008297:Male; D009126:Muscle Relaxation; D009130:Muscle, Smooth / Q000187:drug effects* / Q000378:metabolism; D009569:Nitric Oxide / Q000031:analogs & derivatives / Q000032:analysis; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors; D010276:Parasympatholytics / Q000494:pharmacology*; D010413:Penis / Q000187:drug effects* / Q000378:metabolism; D024642:Potassium Channels, Voltage-Gated / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D017208:Rats, Wistar; D060888:Real-Time Polymerase Chain Reaction; D058262:Receptor Activity-Modifying Protein 1 / Q000235:genetics / Q000378:metabolism; D058263:Receptor Activity-Modifying Protein 2 / Q000235:genetics / Q000378:metabolism; D058264:Receptor Activity-Modifying Protein 3 / Q000378:metabolism; D018015:Receptors, Calcitonin Gene-Related Peptide / Q000378:metabolism; D014665:Vasodilator Agents / Q000494:pharmacology*
chemical_list: D058286:Calcitonin Receptor-Like Protein; D016861:Cyclooxygenase Inhibitors; D004791:Enzyme Inhibitors; D007191:Indazoles; D010276:Parasympatholytics; D024642:Potassium Channels, Voltage-Gated; D012333:RNA, Messenger; C545508:Ramp1 protein, rat; C545509:Ramp2 protein, rat; C545510:Ramp3 protein, rat; D058262:Receptor Activity-Modifying Protein 1; D058263:Receptor Activity-Modifying Protein 2; D058264:Receptor Activity-Modifying Protein 3; D018015:Receptors, Calcitonin Gene-Related Peptide; D014665:Vasodilator Agents; D053607:Adrenomedullin; D009569:Nitric Oxide; D015121:6-Ketoprostaglandin F1 alpha; D015761:4-Aminopyridine; D019001:Nitric Oxide Synthase; D017869:Cyclic GMP-Dependent Protein Kinases; C080122:7-nitroindazole