Pharmacological characterization of the relaxant effect induced by adrenomedullin in rat cavernosal smooth muscle

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.

The Effects of Vasopressin and Desmopressin on the Contractile and Relaxation Responses of Rabbit Cavernosal Smooth Muscle / 대한비뇨기과학회지

PURPOSE: This study aimed to investigate the effects of vasopressin and desmopressin on the contractile and relaxative responses of rabbit cavernosal smooth muscle. MATERIALS AND METHODS: Isometric tension studies were conducted to investigate the effects of vasopressin(10(-14)-10(-8)M) and desmopressin(10(-14)- 10(-8)M) on the contraction and relaxation responses of rabbits cavernous muscle strips in an organ bath. The effects of pretreatment with phenylephrine(10(-5)M), L-NAME(10(-5)M) and indomethacin(10(-5)M) on the contraction and relaxation responses of the vasopressin and desmopressin were also investigated. The statistics were analyzed by Student's t-test and ANOVA. RESULTS: Vasopressin contracted the strips in a dose-dependent manner, while desmopressin did not. The phenylephrine-induced contraction was dose-dependently increased by vasopressin, but it was dose-dependently relaxed by desmopressin. L-NAME pre-treatment did not block the relaxation response, but indomethacin pre-treatment did. Vasopressin- induced contraction occurred the via V(1) receptor, while desmopressin- induced relaxation occurred via the V(2) receptor. CONCLUSIONS: Vasopressin, in pathophysiological circumstances, would worsen erectile dysfunction. On the contrary, desmopressin, which may induce an endothelium-dependent relaxation of the cavernous smooth muscles, would be good for erectile function.

Effect of Glucocorticoid on Rabbit Corpus Cavernosum Smooth Muscle / 대한비뇨기과학회지

PURPOSE: Glucocorticoid contribute to the pathogenesis of hypertension through intracellular signals that stimulate vascular smooth muscle contraction. However, some in vitro and in vivo studies have shown that glucocorticoid has the potential role of vasorelaxation. Therefore, we tried to investigate the effect of short acting glucocorticoid (hydrocortisone; HCS) on the isolated rabbit cavernosal smooth muscle for evaluation of the possibility of using this material as a pharmacoerecting agent. MATERIALS AND METHODS: Strips of rabbit corpus cavernosum were mounted in organ chambers. On the precontracted muscle strips with phenylephrine(PHE; 5x10-6M), HCS was treated with increasing concentration from 10microgram/mL. The relaxing activity of ACh(10-7M), phentolamine(10-8M), papaverine(10-8M), verapamil(10-6M), PGE1(10-2M), SNAP(5x10-5M) were observed with the preparation of HCS. Depolarization by KCl were observed with HCS to investigate the relationship of HCS effects to K+. RESULTS: Pretreatment of muscle strips with low dose of HCS caused concentration-related increase of a PHE induced contraction. On muscle strips submaximally precontracted with PHE, HCS(10microgram/mL to 100microgram/mL) showed no relaxations. Pretreatment of muscle strips with 10microgram/mL dose of HCS caused a potentiation of a relaxation effects of ACh, papaverine, verapamil, SNAP, PGE1 and 50microgram/mL dose of HCS caused a potentiation of a relaxation effects of ACh, phentolamine. However, 100microgram/mL dose of HCS did not produce changes of these potentiating responses to relaxation. Also, HCS did not influenced the depolarization with any concentrations of KCl. CONCLUSIONS: HCS has the potentiating effect of both the PHE-induced contraction, and the relaxation of the ACh, phentolamine, papaverine, verapamil, SNAP, PGE1 at 10-50microgram/mL concentration on the cavernosal smooth muscle.

The Effect of Testosterone on Nitric Oxide Synthase in the Penis of the Rabbit / 대한비뇨기과학회지

PURPOSE: Androgen was thought to be linked to sexual activity in man but its peripheral action have not been identified precisely. Recently, androgens dependent nature of nitric oxide synthase(NOS), the enzyme system producing nitric oxide(NO) as the principal mediator in penile erection, has been studied. The present study was designed to determine if NO within the penis is regulated by androgen. MATERIALS AND METHODS: Effect of testosterone on the penile NO was investigated from three groups of rabbit; control(a sham operation), castration group (four sub-groups: 1, 2, 3 and 4 weeks after castration) and testosterone replacement group (four sub-groups: testosterone propionate(10mg/day) was given subcutaneously from 1, 2, 3 and 4 weeks after castration for 7 days respectively). Strips of rabbit corpus cavernosum were isolated and mounted in organ chambers. The relaxation effects were assessed with electrical field stimulation(EFS) in the precontracted cavernosal muscle strips with phenylephrine. Penile NOS activity was measured by the rate of 14C-L-arginine-citrulline conversion. Penile tissue was subsequently stained for the presence of NOS, using a nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) histochemistry. RESULTS: EFS evoked a relaxation of rabbit corpus cavernosum. A gradual decrease in the EFS evoked relaxation was recorded, proportional to the duration of being castrated, which was restored to the level of control by testosterone replacement. After castration, penile NOS activity was significantly reduced to 51.3%, 43.7%, 37.5%, and 32.7% of the level of control at 1, 2, 3, 4weeks. This decrease in penile NOS activity after castration was also restored by testosterone replacement. Diffusely scattered delicate nerve fibers showing blue color reaction after NADPH-d histochemical staining were reduced in castrated cavernosum in comparison with control. CONCLUSIONS: Based on presented data we conclude that testosterone plays a direct role in penile erection by regulating the activity of NOS within the penis.

Effect of Testosterone on the Relaxation of Rabbit Corpus Cavernosum / 대한비뇨기과학회지

Androgen was thought to be linked to sexual activity in man but it did not identify the peripheral action and its mechanism of androgen. The present study was designed to determine the role of testosterone in the relaxation of the corpus cavernosum. Effect of testosterone on the relaxation of rabbit corpus cavernosum was investigated from three groups of rabbit; two groups were castrated and the third underwent a sham operation (control). After castration, one group, received subcutaneous injection of testosterone (testosterone propionate 10 mg/day). Strips of rabbit corpus cavernosum were isolated and mounted in 10 ml organ chambers. Isometric tension measurement of acetylcholine (Ach: 10(-5)M) and papaverine (10(-5)M) in the precontracted cavernosal muscle strips with phenylephrine (5 x 10(-6)M) were performed in all groups; control, castration group (four sub-groups: 1, 2, 4 and 8 weeks after castration) and testosterone replacement group (four sub-groups: testosterone was given from 1, 2, 4 and 8 weeks after castration for 14 days respectively). The relaxation response of corpus cavernosal muscle to acetylcholine and papaverine was significantly decreased in castrated group and restored up to the level of control after testosterone replacement. The maximum inhibitory effect of castration on the relaxation of corpus cavernosal muscle began to be shown at 4 weeks after castration. Based on these results, we conclude that testosterone regulates the tone of cavernosal smooth muscle through the pathway including cholinergic endothelium mediated relaxation.

The Action Mechanism of Relaxation Effect of Atropine on the Isolated Rabbit Corpus Cavernosum / 대한비뇨기과학회지

Atropine is the classic antimuscarinic anticholinergic drug that bas been used to block cholinergic neurotransmission in basic research and received recent interest clinically in the intracavernous pharmacotherapy of erectile dysfunction. It has been suggested that at low dose(0.00000001M), atropine blocks muscarinic receptors, thereby reducing both cholinergic of the adrenergic and cholinergic excitation of the NANC neuroeffector systems, on the other hand, at large pharmacological dose(0.001M),induces the release of EDRF which recently has been identified as nitric oxide(NO) or NO like substance. Therefore, we tried to confirm the action of atropine in the cavernosal tissue and define its mechanism. Strip of rabbit corpus cavernosum were isolated and mounted in 10 ml organ chambers to measure isometric tension. Muscle strips submaximally precontracted with phenylephrine(5x0.000001M) and treated with increasing concentrations of atropine(0.00000000001M to 0.00lM) showed tension increase upto 0.00000001M of atropine, and thereafter, relaxed concentration dependently(0.0000001M: 43.7%, 0. 000001M: 63.0%,0.00001M:86.2%,0.0001M:93.6%,0.001M:100%). Relaxations to atropine(5x0.000001M) were not inhibited even partially by endothelial disruption or by pretreatment with methylene blue or pyrogallol. Pretreatment of muscle strips with atropine(5x0.00000lM) caused concentration- related inhibition of a phenylephrine induced contraction, and in calcium-free high potassium depolarizing solution, decreased basal tension as well as inhibited contraction induced by CaC12. However, atropine did not produce reduction of responses to depolarizing medium(20, 40, 80mM KCl). With these results we can confirm the relaxation effect of atropine at a larger dose(>0.0000001M)on the cavernosal smooth muscle and suggest that its action is mediated by increasing intracellular calcium sequestration,not by hyperpolarization or EDRF.