Takuwa Y, Kelley G, Takuwa N, Rasmussen H
Department of Cell Biology and Internal Medicine, Yale University School of Medicine, New Haven, CT 06510.
Mol Cell Endocrinol. 1988 Nov;60(1):71-86. doi: 10.1016/0303-7207(88)90121-9.
The protein phosphorylation changes associated with the contraction and relaxation of bovine carotid artery smooth muscle were studied using two-dimensional gel electrophoresis of labeled phosphoproteins. Muscle was stimulated with histamine, angiotensin II, 12-deoxyphorbol 13-isobutyrate (DPB) or high extracellular K+. Histamine induced a rapid and sustained contraction which was associated with an early (2 min) phosphorylation of 20 kDa myosin light chain (MLC) and two cytosolic proteins, Nos. 1 and 2, and with the late (60 min) phosphorylation of MLC, two isoelectric variants of desmin and ten other cytosolic proteins. Additionally, there was a decrease in the extent of phosphorylation of two cytosolic proteins, Nos. 9 and 10. Angiotensin II induced a rapid but transient contraction which was associated with the same early (2 min) phosphorylation changes, but with none of the late (60 min) changes. Elevation of the extracellular K+ concentration to 110 mM led to a sustained contraction which was associated with the phosphorylation of MLC and proteins Nos. 1 and 2 at both 2 and 60 min, but none of the other late phase phosphoproteins were seen. Addition of DPB, an activator of protein kinase C, induced a slowly developing but sustained contractile response which was associated with none of the early (5 min) phosphorylation changes. However, nearly all of late (60 min) protein phosphorylation changes were the same as those seen after histamine action. Addition of forskolin to either control or histamine-treated muscle led to an increase in the phosphorylation of three cytosolic proteins (Nos. 3, 8 and 13), and in the histamine-contracted muscle the dephosphorylation of MLC and proteins Nos. 4, 9, 10, 15 and 16. Similarly, forskolin induced a relaxation of DPB-treated muscle and the dephosphorylation of proteins Nos. 4, 9, 10, 15 and 16. These results suggest that there are two pathways by which histamine activates contraction: a Ca2+-calmodulin pathway which initiates the response, and a protein kinase C pathway which, along with the Ca2+-calmodulin pathway, sustains contraction.
利用标记磷蛋白的二维凝胶电泳技术，研究了与牛颈动脉平滑肌收缩和舒张相关的蛋白质磷酸化变化。用组胺、血管紧张素II、12 - 脱氧佛波醇13 - 异丁酸酯（DPB）或高细胞外钾离子刺激肌肉。组胺诱导快速且持续的收缩，这与20 kDa肌球蛋白轻链（MLC）以及两种胞质蛋白（第1号和第2号）的早期（2分钟）磷酸化有关，还与MLC、结蛋白的两种等电变体以及其他十种胞质蛋白的晚期（60分钟）磷酸化有关。此外，两种胞质蛋白（第9号和第10号）的磷酸化程度有所降低。血管紧张素II诱导快速但短暂的收缩，这与相同的早期（2分钟）磷酸化变化有关，但与晚期（60分钟）的变化无关。将细胞外钾离子浓度提高到110 mM会导致持续收缩，这与2分钟和60分钟时MLC以及第1号和第2号蛋白的磷酸化有关，但未观察到其他晚期磷蛋白的变化。添加蛋白激酶C的激活剂DPB会诱导缓慢发展但持续的收缩反应，这与早期（5分钟）的磷酸化变化无关。然而，几乎所有晚期（60分钟）的蛋白质磷酸化变化都与组胺作用后观察到的变化相同。向对照或组胺处理的肌肉中添加福斯可林会导致三种胞质蛋白（第3号、第8号和第13号）的磷酸化增加，并使组胺收缩的肌肉中MLC以及第4号、第9号、第10号、第15号和第16号蛋白发生去磷酸化。同样，福斯可林会使DPB处理的肌肉舒张，并使第4号、第9号、第10号、第15号和第16号蛋白去磷酸化。这些结果表明，组胺激活收缩有两条途径：一条是启动反应的Ca2 + -钙调蛋白途径，另一条是与Ca2 + -钙调蛋白途径一起维持收缩的蛋白激酶C途径。
