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Down-regulation of G-protein-mediated Ca2+ sensitization in smooth muscle
M C Gong1, H Fujihara, L A Walker
1Department of Molecular Physiology and Biological Physics, University of Virginia Health Sciences Center, Charlottesville 22906-0011, USA.
Abstract:
Prolonged treatment with guanosine 5'-[gamma-thio]triphosphate (GTP gamma S; 5-16 h, 50 microM) of smooth muscle permeabilized with Staphylococcus aureus alpha-toxin down-regulated (abolished) the acute Ca2+ sensitization of force by GTP gamma S, AIF-4, phenylephrine, and endothelin, but not the response to phorbol dibutyrate or a phosphatase inhibitor, tautomycin. Down-regulation also abolished the GTP gamma S-induced increase in myosin light chain phosphorylation at constant [Ca2+] and was associated with extensive translocation of p21rhoA to the particulate fraction, prevented its immunoprecipitation, and inhibited its ADP ribosylation without affecting the immunodetectable content of G-proteins (p21rhoA, p21ras, G alpha q/11, G alpha i3, and G beta) or protein kinase C (types alpha, beta 1, beta 2, delta, epsilon, eta, theta, and zeta). We conclude that the loss of GTP gamma S- and agonist-induced Ca2+ sensitization through prolonged treatment with GTP gamma S is not due to a decrease in the total content of either trimeric (G alpha q/11, G alpha i3, and G beta) or monomeric (p21rhoA and p21ras) G-protein or protein kinase C but may be related to a structural change of p21rhoA and/or to down-regulation of its (yet to be identified) effector.
Insights
Prolonged guanosine 5'-[gamma-thio]triphosphate (GTP gamma S) treatment desensitizes smooth muscle force. This desensitization is linked to p21rhoA changes, not G-protein or kinase levels.
Area of Science:
- Biochemistry
- Cellular Biology
- Physiology
Background:
- Smooth muscle contraction is regulated by calcium (Ca2+) sensitization.
- G-proteins, including p21rhoA, play a role in this sensitization.
- The effects of prolonged guanosine 5'-[gamma-thio]triphosphate (GTP gamma S) on these pathways are not fully understood.
Purpose of the Study:
- To investigate the long-term effects of GTP gamma S on smooth muscle Ca2+ sensitization.
- To determine the role of G-proteins and protein kinase C in GTP gamma S-induced desensitization.
- To explore potential structural changes in p21rhoA.
Main Methods:
- Permeabilized smooth muscle cells were treated with GTP gamma S for extended periods (5-16 hours).
- Force, myosin light chain phosphorylation, and G-protein/kinase C levels were measured.
- p21rhoA translocation and ADP ribosylation were assessed.
Main Results:
- Prolonged GTP gamma S treatment abolished Ca2+ sensitization induced by various agonists.
- This desensitization correlated with p21rhoA translocation and inhibited ADP ribosylation.
- No significant changes were observed in the total content of G-proteins or protein kinase C.
Conclusions:
- Long-term GTP gamma S exposure leads to desensitization of smooth muscle force.
- The mechanism involves alterations in p21rhoA, possibly structural, rather than changes in protein levels.
- Further research is needed to identify the specific effector of p21rhoA involved.