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[摘要]：CPI-17 is a unique phosphoprotein that specifically inhibits myosin light chain phosphatase in smooth muscle and plays an essential role in agonist-induced contraction. To elucidate the in situ mechanism for G protein-mediated Ca2+-sensitization of CPI-17 phosphorylation, alpha-toxin-permeabilized arterial smooth muscle strips were used to monitor both force development and CPI-17 phosphorylation in response to GTP gamma S with varying Ca2+ concentrations. CPI-17 phosphorylation increased at unphysiologically high Ca2+ levels of pCa <= 6. GTP gamma S markedly enhanced the Ca2+ sensitivity of CPI-17 steady-state phosphorylation but had no enhancing effect under Ca2+-free conditions, while the potent PKC activator PDBu increased CPI-17 phosphorylation regardless of Ca2+ concentration. CPI-17 phosphorylation induced by pCa 4.5 alone was markedly inhibited by the presence of PKC inhibitor but not ROCK inhibitor. In the presence of calyculin A, a potent PP1/PP2A phosphatase inhibitor, CPI-17 phosphorylation increased with time even under Ca2+-free conditions. Furthermore, as Ca2+ concentration increased, so did CPI-17 phosphorylation rate. GTP gamma S markedly enhanced the rate of phosphorylation of CPI-17 at a given Ca2+. In the absence of calyculin A, either steady-state phosphorylation of CPI-17 under Ca2+-free conditions in the presence of GTP gamma S or at pCa 6.7 in the absence of GTP gamma S was negligible, suggesting a high intrinsic CPI-17 phosphatase activity. In conclusion, cooperative increases in Ca2+ and G protein activation are required for a significant activation of total kinases that phosphorylate CPI-17, which together overcome CPI-17 phosphatase activity and effectively increase the Ca2+ sensitivity of CPI-17 phosphorylation and smooth muscle contraction. (C) 2010 Elsevier Inc. All rights reserved. |
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