DOI
https://doi.org/10.25772/P4AW-KM98
Defense Date
2013
Document Type
Thesis
Degree Name
Master of Science
Department
Physiology
First Advisor
Srinivasa Karnam
Abstract
Inhibitory neurotransmitters, chiefly nitric oxide and vasoactive intestinal peptide, cause MLC20 dephosphorylation and muscle relaxation via inhibition of myosin light chain (MLC) kinase and activation of MLC phosphatase. Hydrogen sulfide (H2S) produced as a byproduct by luminal sulfate-reducing commensal bacteria or as an endogenous signaling molecule synthesized from L-cysteine via cystathionine-γ-lyase (CSE) and cystathionine-β-synthase (CBS) regulates muscle contraction. However, the role of H2S in the regulation of MLC phosphatase activity and MLC20 phosphorylation is not known. The aim of the present study was to examine the expression of CSE and CBS in smooth muscle cells and to elucidate the molecular mechanism of H2S-induced muscle relaxation. Expression of CSE and CBS was determined by RT-PCR and western blot in muscle cells. The effect of H2S on CCh-stimulated RhoA/Rho kinase pathway and muscle contraction was examined using an endogenous activator of CSE (L-cysteine) and an exogenous H2S donor (NaHS). Isometric contraction in isolated muscle strips and scanning micrometry in isolated muscle cells was measured. Rho kinase activity was measured by immunokinase assay. Expression of CSE, but not CBS was detected in smooth muscle cells of stomach and colon from mouse, rabbit and human. Carbachol-induced contraction in muscle strips and in freshly dispersed muscle cells was inhibited by L-cysteine and NaHS in a concentration-dependent manner (1 to 100 mM). Glibenclamide, an inhibitor of KATP channels and a known target of H2S, had no effect on the inhibition of contraction by H2S. L-cysteine (10 mM) or NaHS (1 mM) inhibited carbachol-induced Rho kinase activity, and the inhibition by L-cysteine was blocked in cells transfected with CSE-specific siRNA. We conclude that both endogenous and exogenous H2S induce muscle relaxation, and the mechanism is inhibition of RhoA/Rho kinase activity and stimulation of MLCP activity leading to MLC20 dephosphorylation.
Rights
© The Author
Is Part Of
VCU University Archives
Is Part Of
VCU Theses and Dissertations
Date of Submission
May 2013