Masuo M, Reardon S, Ikebe M, Kitazawa T
Department of Physiology, University of Virginia School of Medicine, Charlottesville 22908.
J Gen Physiol. 1994 Aug;104(2):265-86. doi: 10.1085/jgp.104.2.265.
Mechanisms of Ca2+ sensitization of both myosin light chain (MLC) phosphorylation and force development by protein kinase C (PKC) were studied in permeabilized tonic smooth muscle obtained from the rabbit femoral artery. For comparison, the Ca2+ sensitizing effect of guanosine 5'-O-(gamma-thiotriphosphate) (GTP gamma S) was examined, which had been previously shown to inhibit MLC phosphatase in phasic vascular smooth muscle. We now report that PKC activators (phorbol esters, short chain synthetic diacylglycerols and a diacylglycerol kinase inhibitor) and GTP gamma S significantly increase both MLC phosphorylation and force development at constant [Ca2+]. Major phosphorylation site occurring in the presence of phorbol-12,13-dibutyrate (PDBu) or GTP gamma S at constant [Ca2+] is the same serine residue (Ser-19) as that phosphorylated by MLC kinase in response to increased Ca2+ concentrations. In an ATP- and Ca(2+)-free solution containing 1-(5-chloronaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine (ML-9), to avoid the kinase activity, both PDBu and GTP gamma S significantly decreased the rate of MLC dephosphorylation to half its control value. However, PDBu inhibited the relaxation rate more than did GTP gamma S. In the presence of microcystin-LR to inhibit the phosphatase activity, neither PDBu nor GTP gamma S affected MLC phosphorylation and force development. These results indicate that PKC, like activation of GTP binding protein, increases Ca2+ sensitivity of both MLC phosphorylation and force production through inhibition of MLC phosphatase.
在从兔股动脉获取的透化的紧张性平滑肌中，研究了蛋白激酶C（PKC）对肌球蛋白轻链（MLC）磷酸化和力产生的Ca2+致敏机制。为作比较，检测了5'-O-(γ-硫代三磷酸)鸟苷（GTPγS）的Ca2+致敏作用，先前已表明其可抑制相性血管平滑肌中的MLC磷酸酶。我们现在报告，PKC激活剂（佛波酯、短链合成二酰基甘油和二酰基甘油激酶抑制剂）和GTPγS在恒定[Ca2+]时显著增加MLC磷酸化和力产生。在恒定[Ca2+]下，佛波醇-12,13-二丁酸酯（PDBu）或GTPγS存在时发生的主要磷酸化位点与MLC激酶在Ca2+浓度增加时磷酸化的丝氨酸残基（Ser-19）相同。在含有1-(5-氯萘-1-磺酰基)-1H-六氢-1,4-二氮杂䓬（ML-9）的无ATP和Ca(2+)溶液中，为避免激酶活性，PDBu和GTPγS均显著降低MLC去磷酸化速率至其对照值的一半。然而，PDBu比GTPγS更能抑制松弛速率。在存在微囊藻毒素-LR以抑制磷酸酶活性的情况下，PDBu和GTPγS均不影响MLC磷酸化和力产生。这些结果表明，PKC与GTP结合蛋白的激活一样，通过抑制MLC磷酸酶增加MLC磷酸化和力产生的Ca2+敏感性。
