Pharmacological characterisation of the mechanisms underlying the relaxant effect of adrenomedullin in the rat carotid artery.

Abstract

We investigated the mechanisms underlying the relaxant effect of adrenomedullin (AM) in the rat carotid artery and verified the expression of AM system components in this tissue. The carotid artery was isolated from male Wistar rats and immunohistochemical, Western immunoblotting, real-time polymerase chain reaction and functional assays were conducted. Protein and mRNA expression of AM, calcitonin receptor-like receptor (CRLR) and receptor activity-modifying proteins (RAMP)1, 2, 3 were detected in carotid segments from male Wistar rats. Immunohistochemical assays showed that AM and CRLR receptors are expressed in the endothelium and smooth muscle cells. Functional assays showed that AM concentration dependently relaxed carotid rings with intact endothelium. Endothelial removal reduced, but not abolished, the relaxation induced by AM. AM22-52 (selective antagonist for AM receptors) and calcitonin gene-related peptide (CGRP)8-37 (selective CGRP receptor antagonist) reduced AM-induced relaxation in endothelium-intact rings. Pre-incubation of endothelium-intact rings with N-nitro-L-arginine methyl ester, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one or Rp-8-Bromo-?-phenyl-1,N2-ethenoguanosine 3',5'cyclic monophosphorothioate reduced AM-induced relaxation. Inhibition of cyclooxygenase-1 and protein kinase A (PKA) reduced AM-induced relaxation. The relaxation induced by AM was attenuated by the K(+) channel blockers apamin and glibenclamide. AM increased nitrate levels and 6-keto-prostaglandin F1α (stable product of prostacyclin) in the rat carotid. In endothelium-denuded rings, AM22-52 , glibenclamide and PKA inhibition by H89 reduced AM-induced relaxation. The novelty of this work is that it first demonstrated functionally that AM-induced relaxation is mediated by AM and CGRP receptors located on the endothelium and AM receptors located on smooth muscle of rat carotid arteries. AM-induced relaxation involves the nitric oxide-cGMP pathway, a vasodilator prostanoid, the opening of K(+) channels and the activation of PKA.