Rho/rho-kinase signalling in chronically alcohol-fed mice.

BACKGROUND/AIM: The effects of chronic ethanol consumption on male sexual function are debateable, and its effects on the Rho/Rho-kinase pathway are unknown. Therefore, we investigated smooth muscle reactivity and Rho/Rho-kinase signalling in the corpus cavernosum of ethanol-fed mice. MATERIALS AND METHODS: Ethanol was added to drinking water at 5% concentration in the first 2 days with 5% increment every subsequent 2 days, up to a final concentration of 20% for 45 days. Corpora cavernosa were isolated and a cumulative dose-response curve to phenylephrine was obtained. Acetylcholine (10-6 M), electrical field stimulation (EFS, 40 V, 8-16 Hz) and Y-27632 (10-6-10-5 M)-induced relaxations were compared in the control and ethanol groups. RESULTS: Blood ethanol levels in the control and ethanol-treated mice were 1.5 ± 0.3 mg/dL and 37.4 ± 4.1 mg/dL (P < 0.001), respectively. Phenylephrine-induced contractile responses were potentiated in the ethanol group, with pD2 values of 4.92 ± 0.18 and 5.71 ± 0.21 (P < 0.01) in the corpus cavernosum obtained from control and alcohol-fed mice, respectively. The relaxant responses to acetylcholine and EFS, but not Y-27632, were significantly augmented in the corpus cavernosum obtained from ethanol-treated mice. However, expression and activation of Rho-kinase remained unchanged in the alcohol group. CONCLUSION: Ethanol drinking may increase sensitivity to both vasoconstrictors and vasodilators in the mouse corpus cavernosum without any alteration in Rho/Rho-kinase signalling.

Nitric oxide does not downregulate Rho-kinase (ROCK-2) expression in rat coronary endothelial cells.

Rho kinase (ROCK) and nitric oxide (NO) are important targets in cardiovascular diseases. Therefore, we investigated the possible influence of NO on Rho kinase (ROCK-2 isoform) expressions in cultured rat coronary microvascular endothelial cells. The cells were isolated from Wistar rats on a Langendorff system, and were incubated overnight (approximately 16 h) with an NO generator, A-23187 (10 to 10 M), NO donors, such as sodium nitroprusside (10 to 10 M), glyceryl trinitrate (10 to 10 M), 2,2'-(hydroxynitrosohydrazono)bis-ethanimine (10 to 10 M), and NaNO2 (10 to 10 M) or a nitric oxide synthase (NOS) inhibitor, N-nitro-L-arginine methylester (2 x 10 M), or two ROCK inhibitors, (+)-(R)-trans-4-(1-aminoethyl)- N-(4-pyridyl) cyclohexanecarboxamide dihydrochloride monohydrate (Y-27632, 10 M) and fasudil (10 M) in the absence or presence of thrombin (4 U/mL). ROCK-2 and endothelial NOS (eNOS) expressions were detected by Western blotting. Moreover, nitrite/nitrate levels were detected by Griess method in the presence of the ROCK inhibitors. The NO donors and the NO generator had no significant effects on ROCK-2 expression. Y-27632 and fasudil did not alter eNOS expression and NO production. Nitrite/nitrate levels were 4.4 +/- 0.32 microM in control and 4.0 +/- 0.93 microM and in Y-27632 group. These results demonstrate that prolong NO donation could not suppress the expression of ROCK-2 protein, and the ROCK inhibitor did not change e-NOS expression and NO production in the cultured rat coronary microvascular endothelial cells.

Involvement of rho kinase in the ouabain-induced contractions of the rat renal arteries.

Agonist and depolarization-induced vascular smooth muscle contractions include the activation of rho/rho kinase pathway. However, there are no reports addressing the question whether this pathway is involved in ouabain-induced vascular smooth muscle contractions. Therefore, in this study, the possible participation of the rho/rho kinase pathway in ouabain-induced contractions was evaluated in rat renal arteries. Effects of rho kinase inhibitors (fasudil and Y-27632) on ouabain-induced contractions, and phosphorylation of myosin binding subunits (MYPT/MBS85) of myosin phosphatase were determined using isolated tissue and Western blot experiments, respectively. Fasudil and Y-27632 inhibited ouabain-induced contractions in a concentration-dependent manner. The phosphorylation of MYPT was not altered by ouabain. However, ouabain significantly increased MBS85 phosphorylation of myosin phosphatase. The phosphorylation of both subunits of myosin phosphatase was inhibited by Y-27632. These results indicate that activation of rho kinase and the subsequent phosphorylation of MBS85 are involved in ouabain-induced contraction of rat renal arteries. This mechanism may be important in essential hypertension with elevated endogenous ouabain levels.

Rho-kinase (ROCK-1 and ROCK-2) upregulation in oleic acid-induced lung injury and its restoration by Y-27632.

The possible contribution of Rho/Rho-kinase signalling in oleic acid (100 mg kg-1, i.v., for 4 h)-induced lung injury was investigated in rats. Furthermore, the possible protective effect of the administration of a Rho-kinase inhibitor, (+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl) cyclohexanecarboxamide dihydrochloride monohydrate (Y-27632, 0.5-5 mg kg-1, i.v., 15 min before the administration of oleic acid), was also examined. Western blot analysis as well as histopathological examination revealed that Rho-kinase (ROCK-1 and ROCK-2) was upregulated in lungs obtained from oleic acid-administrated rats. In addition, the markers of oxidative and nitrosative stress, i.e., malondialdehyde, myeloperoxidase, 3-nitro-L-tyrosine and nitrite/nitrate, in serum and lung tissue were also increased in the injury group. Treatment of rats with 5 mg kg-1 Y-27632 reversed the oleic acid-induced lung damage, which was demonstrated by histopathological assessment and confirmed in Western blot experiments: ROCK-blots were more intense in the oleic acid group than in control and Y-27632 treatment reversed ROCK upregulation. In addition, malondialdehyde, myeloperoxidase, 3-nitro-L-tyrosine and nitrite/nitrate were also normalized after the administration of Y-27632 (0.5 mg kg-1 and 5 mg kg-1). These findings suggest that ROCK-1 and ROCK-2 are involved in oleic acid-induced lung damage in rats, and that inhibition of this enzyme by Y-27632 may have a protective effect against such damage. Consequently, Rho kinase inhibitors may be potential therapeutic agents in the treatment of acute respiratory distress syndrome (ARDS).

Effects of HA-1077 and Y-27632, two rho-kinase inhibitors, in the human umbilical artery.

A role for the small G protein rho and rho-kinase has been shown in smooth muscle contraction regarding Ca++ sensitivity. However, there are no data in the literature assessing how this system operates in human umbilical arteries (HUA). Therefore, we evaluated the effects of HA-1077 and Y-27632, two rho-kinase inhibitors, on agonist-(5-hydroxytryptamine [5-HT]) and depolarization-induced (KCl) contractions of HUA. HA-1077 and Y-27632 inhibited 5-HT-induced contractile responses at 10-4M concentration but not at 10(-5) M. HA-1077 at 10(-4) M also significantly attenuated contractions induced by 20 mM KCl. In addition, HUA precontracted with 5-HT relaxed concentration dependently in response to HA-1077 and Y-27632. When precontracted with KCl, HUA also relaxed dose-dependently in response to HA-1077, but the maximal relaxation was significantly smaller than the response obtained when precontracted with 5-HT. To determine possible involvement of rho-kinase on agonist-induced intracellular calcium-mediated contractions, tissues were precontracted with 5-HT in Ca++-free Krebs solution before cumulative addition of HA-1077 or Y-27632 (10(-7) to 10(-4) M). Both rho-kinase inhibitors relaxed HUA completely. Maximum relaxations of HUA to HA-1077 and Y-27632 were significantly larger than the responses seen in normal Krebs solution and were obtained with lower concentrations of the drugs considered to be more specific for rho-kinase inhibition. However, preincubation of HUA with HA-1077 or Y-27632 (10(-5) M for both) did not affect the 5-HT-induced contractions in this medium. Finally, immunoblot experiments revealed the expression of rho-kinase isoform rockII protein in HUA. These results indicate that rhoA/rho-kinase pathway can contribute to agonist-induced contractions of HUA. However, this effect appears to be limited to intracellular calcium-induced contractions and may be more important in sustaining contractions rather than the initial phase of force development.

Upregulation of Rho-kinase (ROCK-2) expression and enhanced contraction to endothelin-1 in the mesenteric artery from lipopolysaccharide-treated rats.

Effects of bacterial lipopolysaccharide (Escherichia coli serotype, 055:B5, 20 mg kg(-1), i.p., for 6 h) and a Rho-kinase inhibitor, (+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl) cyclohexanecarboxamide dihydrochloride monohydrate, Y-27632 (10(-9)-10(-5) M) were investigated on the contractile responses of the rat mesenteric artery to phenylephrine (10(-9)-3 x 10(-5) M), angiotensin-2 (10(-10)-10(-6) M) and endothelin-1 (10(-10)-10(-7) M). Moreover, alteration in the level of Rho-kinase (ROCK-2) expression was examined in the superior mesenteric artery obtained from saline- and lipopolysaccharide-treated rats by Western blotting. Endotoxemic rat mesenteric rings exhibited no different contractions to phenylephrine and angiotensin-2 but augmented contractile activity to endothelin-1. In the mesenteric artery obtained from the endotoxemic rats, acetylcholine-induced vasorelaxation did not differ; pD2 value for acetylcholine was 7.85+/-0.12 in the endotoxemic rings; however, it was 7.81+/-0.15 in the control rings (P>0.05). Y-27632 induced relaxation, which was the same in the control arteries as in endotoxemic ones when contracting agent was phenylephrine. However, when endothelin-1 was used to precontract the rings, Y-27632 produced enhanced relaxation in endotoxemic vessels. pD2 values for Y-27632 were, respectively, 7.69+/-0.12 and 8.20+/-0.10 in control and endotoxemic rings precontracted by endothelin-1 (10(-8) M) (P<0.01). Moreover, Y-27632 (10(-5) M) suppressed the contraction induced by angiotensin-2 (10(-10)-10(-6) M). Western blot analysis revealed that Rho-kinase was upregulated significantly in the mesenteric artery obtained from the rats treated with LPS for 6 h. In addition, serum NO2-/NO3- level, which was detected by Griess method, was 10.0+/-1.4 microM in endotoxemic rats; however, it was 6.6+/-0.5 microM in control (P<0.05). Taken together, these results show that the expression of the contractile protein Rho-kinase could be upregulated in endotoxemic mesenteric artery and this upregulation may be coincided with an enhanced contraction to endothelin-1 but not phenylephrine and angiotensin-2.