Lee M R, Li L, Kitazawa T
Department of Physiology and Biophysics, Georgetown University Medical Center, Washington, D. C. 20007, USA.
J Biol Chem. 1997 Feb 21;272(8):5063-8. doi: 10.1074/jbc.272.8.5063.
Using permeabilized, arterial smooth muscle strips where membrane-associated pathways remain intact but intracellular Ca2+ stores are depleted, we investigated mechanism(s) for the Ca2+ desensitization of contractile force by cGMP. The nonhydrolyzable analog 8-bromo-cGMP, when applied to these strips with submaximal Ca2+ levels clamped, dramatically and reversibly reduced the steady state levels of phosphorylation at 20-kDa myosin light chain and contractile force, with a nanomolar concentration required to obtain 50% reduction. Supramaximal concentrations of 8-bromo-cGMP (10 microM), however, did not change the steady state relationship between phosphorylation and force. When light chain phosphatase activity was blocked at pCa 6.7, 10 microM 8-bromo-cGMP did not affect the rates of rise of light chain phosphorylation and contractile force. When light chain kinase activity was blocked, 10 microM 8-bromo-cGMP significantly accelerated light chain dephosphorylation and force relaxation from the maximal contraction steady state. The light chain phosphorylation time course of a pCa 6. 0-induced contraction in the presence of 8-bromo-cGMP exhibited kinetics that are predictable from a mathematical model in which only light chain phosphatase activity is increased. The results of this study strongly suggest that cGMP indirectly activates light chain phosphatase, the first proposed mechanism for cGMP-induced Ca2+ desensitization in vasodilatation.
我们使用通透处理的动脉平滑肌条，其中膜相关途径保持完整，但细胞内钙储存已耗尽，研究了环磷酸鸟苷（cGMP）使收缩力钙脱敏的机制。当将不可水解的类似物8-溴-cGMP应用于这些处于亚最大钙水平钳制状态的条带时，它显著且可逆地降低了20 kDa肌球蛋白轻链的磷酸化稳态水平和收缩力，获得50%降低所需的浓度为纳摩尔级。然而，超最大浓度的8-溴-cGMP（10 μM）并未改变磷酸化与力之间的稳态关系。当在pCa 6.7时轻链磷酸酶活性被阻断，10 μM 8-溴-cGMP不影响轻链磷酸化和收缩力的上升速率。当轻链激酶活性被阻断时，10 μM 8-溴-cGMP显著加速了轻链去磷酸化以及从最大收缩稳态的力松弛。在存在8-溴-cGMP的情况下，pCa 6.0诱导收缩的轻链磷酸化时间进程呈现出的动力学可从仅增加轻链磷酸酶活性的数学模型预测。本研究结果强烈表明，cGMP间接激活轻链磷酸酶，这是血管舒张中cGMP诱导钙脱敏的首个提出的机制。
