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-ß-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.

The interaction of l-cysteine/H2S pathway and muscarinic acetylcholine receptors (mAChRs) in mouse corpus cavernosum.

The aim of this study was to investigate the possible interaction of l-cysteine/H2S pathway and muscarinic acetylcholine receptors (mAChRs) in the mouse corpus cavernosum (CC). l-cysteine (endogenous H2S substrate; 10-6-10-3 M), sodium hydrogen sulfide (NaHS; exogenous H2S; 10-6-10-3 M) and acetylcholine (10-9-10-4 M) produced concentration-dependent relaxation in isolated mouse CC tissues. Relaxations to endogenous and exogenous H2S were reduced by non-selective mAChR antagonist atropine (5 × 10-5 M), selective M1 mAChR antagonist pirenzepine (5 × 10-5 M) and selective M3 mAChR antagonist 4-DAMP (10-7 M) but not by selective M2 mAChR antagonist AF-DX 116 (10-6 M). Also, acetylcholine-induced relaxations were reduced by atropine, pirenzepine, 4-DAMP and AF-DX 116, confirming the selective effects of mAChR antagonists. Furthermore, acetylcholine-induced relaxations were attenuated by cystathionine-gamma-lyase (CSE) inhibitor d,l-propargylglycine (PAG, 10-2 M) and cystathionine-ß-synthase inhibitor (CBS) aminooxyacetic acid (AOAA, 10-3 M). l-nitroarginine, nitric oxide synthase inhibitor, augmented the inhibitory effects of mAChR antagonists and H2S enzyme inhibitors on acetylcholine-induced relaxations. In addition, the existence and localization of CSE, CBS and 3-MST were demonstrated in mouse CC. Furthermore, tissue acetylcholine release was significantly increased by l-cysteine but not by exogenous H2S. The increase in acetylcholine level was completely inhibited by AOAA and PAG. These results suggest that M1 and M3 mAChRs contributes to relaxant effect mediated by endogenous H2S but at same time l-cysteine triggers acetylcholine release from cavernosal tissue. Also, the role of NO in the interaction of l-cysteine/H2S pathway and muscarinic acetylcholine receptors (mAChRs) could not be excluded.