Cavernosal Smooth Muscle Contraction Research Articles

Involvement of RhoA/Rho-kinase in l-cysteine/H2S pathway-induced inhibition of agonist-mediated corpus cavernosal smooth muscle contraction

Rho-kinase activity is a key regulator in the maintenance of corporal vasoconstriction and penile detumescense. Also, importance of l-cysteine/H2S pathway in erectile tissue has been shown; however it is currently unknown the role RhoA/Rho-kinase pathway in H2S-induced inhibition in cavernosal tissue. We investigated the role of RhoA/Rho-kinase pathway in the inhibitory effect of l-cysteine and NaHS, as endogenous and exogenous H2S, respectively, on phenylephrine-induced contractions of mouse cavernosal strips. Phenylephrine, α1 receptor agonist, (10 nM-100 μM) induced a concentration-dependent contraction in CC. l-cysteine (endogenous H2S substrate; 10 mM) and exogenous H2S (NaHS; 1 mM) significantly inhibited the contractile response to phenylephrine (P < 0.05). Inhibition of CSE and CBS enzymes by PAG (10 mM) and AOAA (1 mM), respectively, significantly reversed the inhibitory effects of l-cysteine on phenylephrine-induced contraction (P < 0.05). Y-27632 (1 μM), a specific Rho-kinase inhibitor, significantly augmented the inhibitory effect of l-cysteine and NaHS on phenylephrine-induced contraction, and this inhibition was reversed by PAG and AOAA (P < 0.05). In addition, the formation of H2S was increased by approximately 1.8 fold over basal values after incubation of tissue homogenates with l-cysteine. Y-27632 significantly increased both basal and l-cysteine-induced H2S formation and this augmentation diminished by PAG and AOAA (P < 0.05). Furthermore, the pMYPT-1 expression was significantly decreased by l-cysteine, NaHS or Y-27632 alone. Also, pMYPT-1 expression was completely abolished by the l-cysteine/NaHS plus Y-27632 combination, and this inhibition was reversed by PAG and AOAA (P < 0.05). These results suggest that there is an interaction between Rho-kinase and H2S pathways. Rho-kinase may be, at least in part, inhibits CSE/CBS enzymes in mouse corpus cavernosal tissue; however, it is not excluded the other kinases such as PKC and Zip-kinase.

The effect of sub-chronic systemic ethanol treatment on corpus cavernosal smooth muscle contraction: the contribution of RhoA/Rho-kinase.

The aim of this study was to evaluate whether the sub-chronic systemic ethanol exposure has direct effect on cavernosal smooth muscle contractions induced by KCl (depolarizing) and phenylephrine (α1-receptor agonist), and the possible involvement of RhoA/Rho-kinase pathway. Sub-chronic systemic ethanol was applied to mice with inhalation route for 14 days. The blood levels in ethanol-treated mice averaged 121.2 ± 9.1 mg/dl. KCl (80 mM) and phenylephrine (10 nM-100 μM) induced sustained contractions in corpus corporal strips from sham-treated mice. Sub-chronic ethanol treatment reduced the contractions to KCl. However, phenylephrine-induced contractions were not affected by ethanol treatment. Rho-kinase inhibitor fasudil (50 μM) and Y-27632 (50 μM) inhibited contractions to KCl and phenylephrine in sham-treated mice. Ethanol treatment increased the inhibitory effect of Rho-kinase inhibitors on contractions to phenylephrine. The relaxations induced by fasudil (100 μM) and Y-27632 (500 μM) did not change in ethanol treatment group. In ethanol-treated group, the expression of RhoA decreased compared to sham-treated group. Also, ROCK1 expression was reduced by ethanol but not statically significant to sham-treated group; however, the expression of ROCK2 increased in ethanol group. From these findings, it seems that phenylephrine and KCl-induced contractions depends on RhoA/Rho-kinase-mediated Ca(2+) sensitization. Also, these results suggest that the ethanol treatment decreased the expression of RhoA, and the inhibitory effect of ethanol on KCl-induced contractions may be due to, at least in part, the inhibition of a RhoA/Rho-kinase in mouse corpus cavernosum.

Excessive Penile Norepinephrine Level Underlies Impaired Erectile Function in Adenosine A1 Receptor Deficient Mice

Penile erection is a complex neurovascular physiological event controlled by multiple factors and signaling pathways. A considerable amount of evidence indicates that adenosine plays a significant role in cavernosal smooth muscle relaxation. However, the specific role of adenosine and its receptors in erectile physiology and pathology is not fully understood. To determine the role of the adenosine A1 receptor (ADORA1) in penile erection. Adenosine A1 receptor deficient (Adora1-/-) mice and aged-matched wild-type (WT) mice were utilized. We evaluated the in vivo erectile function by measuring the intracavernosal pressure (ICP) in response to cavernous nerve stimulation (CNS). Enzyme-linked immunosorbent assay was used to measure the norepinephrine (NE) plasma concentration in the corpus cavernosum and systemic circulation. We also evaluated the myosin light chain phosphorylation (p-MLC) in penile tissue pre- and post-CNS. The main outcome measurement of this research was the evaluation of in vivo erectile response to CNS by measuring the ICP in Adora1-/- mice and WT mice and to identify the localization and specific neuron types of ADORA1 expression by dual immunostaining and immunofluorescence co-localization. In vivo, both the ratio of CNS-induced Maximum ICP to mean arterial pressure and CNS-induced slope in Adora1-/- mice were significantly lower than WT mice. At the cellular level in penile tissue, we determined that ADORA1 was highly abundant in neuronal cells. During penile erection, Adora1-/- mice exhibited a higher level of NE plasma concentration in the penis than WT mice. And WT mice had a significantly greater reduction in p-MLC compared to Adora1-/- mice. Our results show that ADORA1 is enriched on neuron cells where it functions to control NE release. Activation of this receptor during penile erection results in reduced NE release and reduced cavernosal smooth muscle contraction, therefore facilitating penile erection.

Oral alpha adrenoceptor blockade as a treatment of erectile dysfunction.

The sympathetic nervous system, via release of noradrenaline (NA) and stimulation of alpha adrenoceptors (ARs), is considered to be the prime determinant of cavernosal smooth muscle contraction and detumescence. A relative predominance of NA-induced contraction over nitric oxide-mediated relaxation may contribute to erectile dysfunction (ED). Therefore alpha AR antagonism seems an attractive way of treating ED, but so far the therapeutic success of oral treatment has been limited. Modest activity has been documented for the alpha2 AR antagonist, yohimbine, which is believed to act in the central nervous system. Phentolamine, mainly blocking alpha1 and alpha2 ARs peripherally, has been shown to have beneficial effects, but the efficacy compared to other alternatives, e.g., sildenafil, has not been established. To improve oral treatment of ED with alpha AR antagonists, new drugs are required. However, little is known about central noradrenergic mechanisms involved in erection, or which alpha AR subtypes in the penile erectile tissues are the most important for mediation of contraction. It is still unclear what profile (alpha1 vs alpha2 ARs; selectivity for alpha1 and/or alpha2 AR subtypes) is the most advantageous for an alpha AR antagonist in the treatment of ED.

RELATIVE ROLES OF CYCLOOXYGENASE AND NITRIC OXIDE SYNTHASE PATHWAYS IN ISCHEMIA-INDUCED INCREASED CONTRACTION OF CAVERNOSAL SMOOTH MUSCLE

Purpose To study the mechanism of chronic ischemia-induced increased cavernosal smooth muscle contraction in an animal model of vasculogenic erectile dysfunction. Materials and Methods New Zealand White rabbits were divided into control (n = 6, fed with a regular diet), hypercholesterolemic (n = 9, fed with a diet containing 0.5% cholesterol) and chronic cavernosal ischemia (CCI, n = 10, underwent balloon de-endothelialization of iliac arteries and received a diet containing 0.5% cholesterol) groups. After 16 weeks, the relationship between iliac artery blood flow and cavernosal smooth muscle contraction was studied. The roles of cyclooxygenase and nitric oxide (NO) pathways in chronic ischemia-induced increased smooth muscle contraction were also examined. Results Iliac artery blood flow in the CCI group was significantly reduced compared with the control and hypercholesterolemic groups. Hypercholesterolemia alone did not affect cavernosal smooth muscle contraction. Atherosclerosis-induced chronic cavernosal arterial insufficiency did not affect contraction to norepinephrine while causing a significant increase in electrical field stimulation-induced neurogenic contraction. Inhibition of the cyclooxygenase pathway by indomethacin decreased electrical field stimulation-induced contraction in all animals but failed to normalize the differences between CCI and control groups. In the presence of indomethacin, L-arginine decreased electrical field stimulation-induced contraction in the control and hypercholesterolemic groups but not in the CCI group. In the presence of indomethacin, treatment with nitric oxide synthase (NOS) inhibitor, N G-monomethyl-L-arginine (L-NMMA), increased electrical field stimulation-induced contraction in all groups. This effect of L-NMMA on smooth muscle contraction was significantly greater in the control and hypercholesterolemic groups compared with the CCI group. After tissue treatment with L-NMMA, the magnitude of contraction in cavernosal tissue from control and hypercholesterolemic groups was similar to those observed in the CCI group. Conclusions Mechanism of chronic ischemia-induced increased cavernosal smooth muscle contraction involves increased output of constrictor eicosanoids and impairment of the inhibitory influence of NO pathway in cavernosal tissue.

Effect of angiotensin II on corpus cavernosum smooth muscle in relation to nitric oxide environment: in vitro studies in canines.

In extracavernosal vascular tissue, smooth muscle tone is modulated by a balance between angiotensin II (Ang II) and nitric oxide (NO). We hypothesized that these substances also play an important role in regulating cavernosal smooth muscle contractility. We therefore studied the in vitro effects of an Ang II receptor antagonist and a nitric oxide synthase (NOS) inhibitor on the contractile effects of Ang II, phenylephrine, and electrical field stimulation in canine corpus cavernosum. Ang II caused a dose-dependent contraction of cavernosal smooth muscle which was inhibited by the Ang II receptor antagonist and augmented by NOS inhibition. Contractions induced by phenylephrine or electrical stimulation were diminished by the Ang II receptor antagonist, and augmented by NOS inhibition. We conclude that Ang II plays an important role in modulating cavernosal smooth muscle tone, and that the contractile effect of Ang II on cavernosal tissue is modulated by the local NO environment.

Tissue Angiotensin II as a Modulator of Erectile Function. I. Angiotensin Peptide Content, Secretion and Effects in the Corpus Cavernosum

Purpose Although Angiotensin II (Ang II) is a major modulator of regional blood flow in the extracavernosal segments of the vascular bed, its role in erectile function is unknown. The corpus cavernosum penis is a modified vascular tissue that contains endothelial and smooth muscle cells. In other segments of the vascular bed, these cell types produce Ang II. Therefore, we explored the presence and function of an Ang II producing paracrine system in the corpus cavernosum. Methods The angiotensin content of the human corpus cavernosum was measured by radio-immunoassay. The distribution pattern of Ang II containing cells within the corpus cavernosum was assessed by an immunohistochemical technique, and the rate of its secretion was determined by superfusion. The effects of Ang II and its antagonist, losartan, on intracavernosal pressure were determined under in vivo conditions, in anesthetized dogs. Results Human corpus cavernosum contained 1178 +/− 223 (SEM) fmol Ang II, 528 +/− 171 fmol Ang I, 475 +/− 67 fmol des-asp-Ang I, and 1897 +/− 371 fmol des-asp-Ang II/gm. tissue (n = 4). Ang II was found mainly in endothelial cells lining blood vessels and smooth muscle bundles within the corpus cavernosum. Superfused cavernosal tissue secreted immuno-reactive Ang II (Ang II ir) at a rate of 57 +/− 36.5 fmol Ang II ir/gm. tissue/minute (n = 10). The amount of Ang II released per gram of tissue in an hour was 3-fold greater than the Ang II content/gm. tissue, suggesting a local production of Ang II. Papaverine and prostaglandin E 1 suppressed Ang II secretion significantly (p <0.001, p = 0.013). The responsiveness to inhibition was a function of the initial rate of Ang II secretion. Tissue samples with a high rate of secretion were less responsive to the inhibitors than tissue that secreted small amounts of Ang II (n = 6). In anesthetized dogs, intra-cavernosal injection of Ang II terminated spontaneous erections, while losartan increased the intracavernosal pressure in a dose dependent manner up to the mean arterial pressure (n = 4). Conclusions The corpus cavernosum produces and secretes physiologically relevant amounts of Ang II. The rate of Ang II secretion can be modulated by pharmacologic agents that regulate cytosolic calcium levels and are used clinically to treat erectile dysfunction. Intracavernosal injection of Ang II causes contraction of cavernosal smooth muscle and terminates spontaneous erection in anesthetized dog, while administration of an Ang II receptor antagonist results in smooth muscle relaxation and thus erection.

Characterization of Cyclic Nucleotide and Inositol 1, 4, 5-Trisphosphate-Sensitive Calcium-Exchange Activity of Smooth Muscle Cells Cultured from the Human Corpora Cavernosa1

Smooth muscle-mediated expansion and contraction of the vascular sinusoids of the corpora cavernosa may modulate male erectile function. To elucidate the biochemical events that control erection by promoting or inhibiting contraction of cavernosal smooth muscle, tissue from a potent man was grown in cell culture. The cells grew as noncontractile cultures, but had the following smooth muscle cell properties: These cells expressed desmin, the muscle cell-specific intermediate filament protein. They accumulated 45Ca2+ from the medium, which was released by exposure to the ionophore A23187, to cyclic nucleotides (cyclic guanosine 5'-monophosphate [GMP] much greater than cyclic adenosine 3',5'-monophosphate [AMP]), and to the phosphodiesterase inhibitor, papaverine; and; they accumulated Ca2+ in an ATP-dependent manner when the cultured cells were permeabilized by digitonin extraction. ATP-dependent Ca2+ uptake was inhibited approximately 80% by ruthenium red and simulated by cyclic GMP much greater than cyclic AMP. Inositol 1,4,5-trisphosphate (IP3), which is thought to mediate the release of Ca2+ by the smooth muscle cell sarcoplasmic reticulum in vivo, released approximately 0.85 pmol Ca2+/million cells from the digitonin-extracted cells. IP3-dependent release occurred in the presence of ruthenium red and was not affected by cyclic GMP or cyclic AMP. These results indicate that smooth muscle from this human source can be grown successfully in cell culture and that the biochemical pathways that regulate tension in vivo may be perpetuated in vitro. Moreover, some of the clinical responses to drugs administered in situ for erectile dysfunction (e.g. papaverine) may be the result of altered cavernosal smooth muscle cell Ca2+ exchange and may be mediated by cyclic GMP.