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

• Published in: Naunyn-Schmiedeberg's archives of pharmacology Vol. 396; no. 9; pp. 2023 - 2038
• Main Authors: Dalkir, Fatma Tugce, Aydinoglu, Fatma, Ogulener, Nuran
• Format: Journal Article
• Language: English
• Published: Berlin/Heidelberg Springer Berlin Heidelberg 01.09.2023; Springer Nature B.V
• Subjects: Biomedical and Life Sciences; Biomedicine; Bladder; Carbachol; Cysteine; Hydrogen sulfide; Kinases; Muscle contraction; Neurosciences; Pharmacology/Toxicology; Protein kinase C; Rho-associated kinase; RhoA protein; Smooth muscle; Hydrogen sulfide; Rho-kinase; L-cysteine; Protein kinase C; Urinary bladder; Calcium sensitization
• ISSN: 0028-1298; 1432-1912
• DOI: 10.1007/s00210-023-02440-6

We investigated the role of RhoA/Rho-kinase (ROCK) and PKC in the inhibitory effect of L-cysteine/hydrogen sulfide (H 2 S) 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 (H 2 S precursor; 10 −2  M) and exogenous H 2 S (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-β-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 H 2 S synthesis were detected by Western blot method. H 2 S 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 H 2 S 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/H 2 S 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 H 2 S in mouse bladder.