The role of rhoA/rho-kinase and PKC in the inhibitory effect of L-cysteine/H2S pathway on the carbachol-mediated contraction of mouse bladder smooth muscle

We investigated the role of RhoA/Rho-kinase (ROCK) and PKC in the inhibitory effect of L-cysteine/hydrogen sulfide (H2S) pathway on the carbachol-mediated contraction of mouse bladder smooth muscle. Carbachol (10(-8)-10(-4) M) induced a concentration-dependent contraction in bladder tissues. L-cysteine (H2S precursor; 10(-2) M) and exogenous H2S (NaHS; 10(-3) M) reduced the contractions evoked by carbachol by 49 and 53%, respectively, relative to control. The inhibitory effect of L-cysteine on contractions to carbachol was reversed by 10(-2) M PAG ( 40%) and 10(-3) M AOAA ( 55%), cystathionine-gamma-lyase (CSE) and cystathionine-beta-synthase (CBS) inhibitor, respectively. Y-27632 (10(-6) M) and GF 109203X (10(-6) M), a specific ROCK and PKC inhibitor, respectively, reduced contractions evoked by carbachol ( 18 and 24% respectively), and the inhibitory effect of Y-27632 and GF 109203X on contractions was reversed by PAG (29 and 19%, respectively) but not by AOAA. Also, Y-27632 and GF 109203X reduced the inhibitory responses of L-cysteine on the carbachol-induced contractions (38 and 52% respectively), and PAG abolished the inhibitory effect of L-cysteine on the contractions in the presence of Y-27632 (38%). Also, the protein expressions of CSE, CBS, and 3-MST enzymes responsible for endogenous H2S synthesis were detected by Western blot method. H2S level was increased by L-cysteine, Y-27632, and GF 109203X (from 0.12 +/- 0.02 to 0.47 +/- 0.13, 0.26 +/- 0.03, and 0.23 +/- 0.06 nmol/mg respectively), and this augmentation in H2S level decreased with PAG (0.17 +/- 0.02, 0.15 +/- 0.03, and 0.07 +/- 0.04 nmol/mg respectively). Furthermore, L-cysteine and NaHS reduced carbachol-induced ROCK-1, pMYPT1, and pMLC20 levels. Inhibitory effects of L-cysteine on ROCK-1, pMYPT1, and pMLC20 levels, but not of NaHS, were reversed by PAG. These results suggest that there is an interaction between L-cysteine/H2S and RhoA/ROCK pathway via inhibition of ROCK-1, pMYPT1, and pMLC20, and the inhibition of RhoA/ROCK and/or PKC signal pathway may be mediated by the CSE-generated H2S in mouse bladder.

Automated summary

This study investigated the role of RhoA/Rho-kinase (ROCK) and PKC in the inhibitory effect of L-cysteine/hydrogen sulfide (H2S) pathway on carbachol-induced contraction in mouse bladder smooth muscle. L-cysteine and H2S reduced the contractions, and ROCK and PKC inhibitors also decreased the contractions, suggesting an interaction between L-cysteine/H2S and the RhoA/ROCK pathway. The inhibitory effect of L-cysteine on ROCK-1, pMYPT1, and pMLC20 levels was reversed by PAG, indicating that CSE-generated H2S mediates the inhibition.