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 F1α (6-keto-PGF1α; 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. AM22-52, a selective antagonist for AM receptors, reduced the relaxation induced by AM. Conversely, CGRP8-37, a selective antagonist for calcitonin gene-related peptide receptors, did not affect AM-induced relaxation. Preincubation of CSM strips with NG-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 (Ca2+-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.
Highlights
Immunohistochemical assays demonstrated that AM and calcitonin receptor-like receptor (CRLR) were expressed in rat cavernosal smooth muscle (CSM)
The initiation and maintenance of penile erection is caused by relaxation of the blood vessels in the cavernosal smooth muscle (CSM), which results in an increased blood flow into the trabecular spaces of the corpora cavernosa [1]
The results obtained by quantitative real-time polymerase chain reaction (qRT-PCR) showed that rat CSM expressed mRNA of pre-pro-AM, CRLR, and RAMP1, -2, and -3 (Figure 1B)
Summary
The initiation and maintenance of penile erection is caused by relaxation of the blood vessels in the cavernosal smooth muscle (CSM), which results in an increased blood flow into the trabecular spaces of the corpora cavernosa [1]. The control of CSM tone is mainly mediated by the adrenergic, cholinergic, and nonadrenergic, noncholinergic (NANC) systems [2]. Noradrenaline, released from sympathetic nerves, induces contraction of penile CSM while parasympathetic innervations mediate smooth muscle relaxation in the trabecular network and cavernosal arterial venous bed [3]. Nitric oxide (NO) released from NANC nerve endings and from the vascular endothelium is considered the most important mediator of CSM relaxation [4]. Release of endothelium-derived NO is regulated by several factors, including vasodilator substances such as adrenomedullin (AM), which has Received February 12, 2014.
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