Rho kinase is involved in Ca²⁺ entry of rat penile small arteries

Abstract

Tonic physiological activity of RhoA/Rho kinase contributes to the maintenance of penile flaccidity through its involvement in the Ca 2ϩ sensitization of erectile tissue smooth muscle. The present study hypothesized that Rho kinase is also involved in the modulation of Ca 2ϩ entry induced by ␣1-adrenoceptor stimulation of penile arteries. Rat penile arteries were mounted in microvascular myographs for simultaneous measurements of intracellular Ca 2ϩ ([Ca 2ϩ ]i) and force. The Rho-kinase inhibitor Y-27632 markedly reduced norepinephrine-mediated electrically induced contractions and the increases in both [Ca 2ϩ ]i and tension elicited by the ␣1-adrenoceptor agonist phenylephrine (Phe). In contrast, the protein kinase C (PKC) inhibitor Ro-31-8220 reduced tension without altering the Phe-induced increase in [Ca 2ϩ ]i. In the presence of nifedipine, Y-27632 still inhibited the non-L-type Ca 2ϩ signal and blunted Phe contraction. Y-27632 did not impair the capacitative Ca 2ϩ entry evoked by store depletion with cyclopiazonic acid but largely reduced the Ba 2ϩ influx stimulated by Phe in fura-2 AM-loaded arteries. The addition of Y-27632 to arteries depolarized with high KCl markedly reduced tension without changing [Ca 2ϩ ]i. In ␣-toxin-permeabilized penile arteries stimulated with threshold Ca 2ϩ concentrations, Y-27632 inhibited the sensitization induced by either guanosine 5Ј-O-(3-thiotriphosphate) (GTP␥S) or Phe in the presence of GTP␥S. However, Y-27632 failed to alter contractions induced by a maximal concentration of free Ca 2ϩ . These results suggest that Rho kinase, besides its contribution to the Ca 2ϩ sensitization of the contractile proteins, is also involved in the regulation of Ca 2ϩ entry through a nonselective cation channel activated by ␣1-adenoceptor stimulation in rat penile arteries. penile arteries; calcium entry; nonselective cation channels; calcium sensitization PENILE ERECTION OCCURS when nitric oxide (NO) released from nerves and endothelium upon sexual stimulation relaxes smooth muscle of the corpus cavernosum (CC) and penile arteries, leading to blood filling of the sinuses and restriction of venous outflow (2, 25). During the flaccid state, erectile tissue is contracted by the release of neural and local factors, such as norepinephrine, neuropeptide Y, endothelin-1, and prostanoids that increase smooth muscle cytosolic Ca 2ϩ ([Ca 2ϩ ] i ) and/or Ca 2ϩ sensitization through activation G protein-coupled receptors (2,20,25). Erectile dysfunction (ED), considered as a sign of early endothelial dysfunction and cardiovascular disease, is three times more prevalent in men with Types 1 and 2 diabetes than in men without diabetes (3, 33). About 50% of these patients exhibit suboptimal responses to oral phophodiestarase 5 inhibitors, such as sildenafil (33), that enhance the NOmediated vasodilatation leading to penile erection. As an alternative, inhibition of the Rho/Rho-kinase signaling pathway has been proposed as a potential molecular target for the development of novel therapies for ED si...

Impaired Ca2+ handling in penile arteries from prediabetic Zucker rats: involvement of Rho kinase

“…In the present study, inhibition of RhoK markedly reduced the contractions induced by both stimulation of adrenergic nerves and selective activation of the ␣ 1 -adrenergic receptor with PE, with inhibition of PE responses by Y-27632 being greater in penile arteries from OZRs compared with LZRs. At the concentration used (10 M), the effects of Y-27632 on the contraction and Ca 2ϩ signal of rat arteries have been previously shown not to be mimicked or prevented by selective inhibition of PKC (20,41), which suggests that the effects of the RhoK inhibitor are mediated through its interaction with Rho-dependent kinase. The present findings therefore suggest that a larger component of the ␣ 1 -adrenergic vasoconstriction is mediated through the activation of RhoK in prediabetic arteries, which is in agreement with results reported by Naik et al (26) in skeletal muscle arterioles from OZRs and consistent with the more potent erectile responses to intracavernous injection of RhoK inhibitors in type 1 diabetic rats compared with controls (7) and with the ability of these compounds to improve the voltage-dependent erectile responses to stimulation of the cavernous nerves in prediabetic OZRs (46).…”

“…Our previous studies on the Ca 2ϩ signaling mechanisms of the ␣ 1 -adrenergic vasoconstriction in penile arteries have demonstrated that the RhoA/RhoK pathway plays a main role not only in the Ca 2ϩ sensitization mechanisms but also in the regulation of Ca 2ϩ entry through a transient receptor potential cation channel (TRPC) member (41). While pretreatment with Y-27632 had a dual inhibitory/enhancing effect on the increases in [Ca 2ϩ ] i induced by submaximal and maximal concentrations of PE in penile arteries from LZRs, the RhoK inhibitor did not alter the reduced PE-mediated Ca 2ϩ mobilization of arteries from OZRs, thus initially ruling out a role for RhoK in the regulation of Ca 2ϩ fluxes of prediabetic arteries.…”

“…Frequencyresponse curves to EFS were obtained at baseline tension over a range of 1-32 Hz by delivering 20-s pulse trains (0.3-ms pulse duration) at intervals of 5 min (34). To test the influence of RhoK inhibition on the contractions elicited by electrical stimulation of perivascular nerves in the arterial wall, frequency-response curves were performed in the absence and presence of a submaximal concentration of Y-27632 (10 M) (20,41). These experiments were carried out in the presence of the NO synthase (NOS) inhibitor N G -nitro-L-arginine (L-NNA; 100 M) to avoid the effects of neural-released NO.…”

“…Thus, RhoK regulates the contraction of erectile tissue in response to endothelin-1 and ␣ 1 -adrenergic agonists (24,41,45), and enhanced vasoconstriction of corporal tissue to these agonists has been demonstrated to be associated with augmented expression of RhoK in the prediabetic obese Zucker rat (OZR) (45,46). Furthermore, the beneficial effect of RhoK inhibition on the impaired erectile responses to nerve stimulation has been shown in models of both type 1 (7) and type 2 (46) diabetes-associated ED.…”

Opposing roles of smooth muscle BK channels and ryanodine receptors in the regulation of nerve-evoked constriction of mesenteric resistance arteries

“…Neurally released ATP can be monitored optically as a local elevation of Ca 2ϩ in smooth muscle, referred to as a junctional Ca 2ϩ transient (jCaT) (21). NE acts via ␣ 1 -adrenergic receptors to promote constriction through multiple mechanisms, including the induction of inositol 1,4,5-trisphosphate receptor-mediated Ca 2ϩ waves, membrane depolarization, and increased Ca 2ϩ -sensitivity (19,25,33,34). Resistance-sized mesenteric arteries constrict to both pressure and sympathetic nerve stimulation and play a key role in blood pressure regulation (8,10,18,24).…”