Intracellular distribution and regulatory function of protein phosphatases

本项目的目的是研究可逆性蛋白磷酸化在细胞运动和离子转运调节中的作用。在本项目期间，我们主要在平滑肌和心肌中进行了生理和生化实验，总结如下。部分研究结果已经发表（见所附重印本）。(1)冈田酸（OA）是蛋白磷酸酶1和2A（PP 1和PP 2A）的有效抑制剂，其对PP 2A的亲和力高于对PP 1的亲和力。在具有完整质膜的平滑肌制备物中，OA可逆地抑制收缩和肌球蛋白轻链（MLC）磷酸化。另一种PP抑制剂，互变霉素，表现出更高的亲和力PP 1比PP 2A是已知的增强收缩和肌球蛋白轻链磷酸化。在本实验中，我们观察到OA强烈抑制互变霉素的这种收缩作用。加入OA也降低了MLC的磷酸化水平，但约10%的MLC磷酸化水平降低。 关于我们 这些结果表明，平滑肌收缩是由蛋白磷酸化抑制的几个步骤。(2)在离体牛眼睫状体平滑肌细胞上，用全细胞钳技术研究了卡巴胆碱对细胞膜电位和电流的影响。我们已经证明了一个非选择性阳离子通道的存在，它是由属于M3亚型的毒蕈碱受体激活。这一通道接纳Ca^<2+>，可能至少部分地作为持续收缩所需的Ca^<2+>进入通道。(3)在离体豚鼠心室肌细胞上，我们观察了Cl^-通道抑制剂蒽-9-羧酸（9AC）对CFTR通道的影响。我们已经证明，异丙肾上腺素或毛喉素对CFTR的激活作用在9AC存在下可逆地增强和延长。我们还表明，9AC抑制一部分细胞内的对硝基苯基磷酸酶活性，这对已知的磷酸酶抑制剂，如OA，酒石酸或溴四咪唑不敏感。少

The aim of this project has been to investigate the roles of reversible protein phosphorylation in regulation of cell motility and ion transport. During the term of this project we carried out physiological and biochemical experiments chiefly in smooth and cardiac muscles, as summarized below. Part of the results obtained have been published (see the reprints attached).(1) Okadaic acid (OA) is a potent inhibitor of protein phosphatase 1 and 2A (PP1 and PP2A) having a higher affinity to PP2A than to PP1. In smooth muscle preparations with intact plasma membrane, OA reversibly inhibits contraction and myosin light-chain (MLC) phosphorylation. Another PP inhibitor, tautomycin, which exhibits a higher affinity PP1 than PP2A is known to enhance contraction and myosin light chain phosphorylation. In the present experiments we have observed that OA strongly inhibits this contractile effect of tautomycin. The MLC phosphorylation was also decreased by addition of OA but about 10 % of MLC were s … More till phosphorylated even when contraction was completely inhibited by OA.These results indicate that there are several steps toward smooth muscle contraction which is suppressed by protein phosphorylation.(2) In smooth muscle cells isolated from bovine ciliary body we have studied effects of carbachol on the membrane potential and current using the whole-cell clamp technique. We have demonstrated the existence of a non-selective cation channel which is activated by muscarinic receptors belonging to the M3 subtype. This channel, which admits Ca^<2+>, may serve at least partly as a Ca^<2+> entry required for sustained contraction.(3) In isolated myocyte of guinea-pig ventricle, we have examined the effect of anthracene-9-carboxylic acid (9AC), a Cl^- channel inhibitor, on the CFTR channel. We have shown that the activating effect of isoprenaline or forskolin on the CFTR is reversibly enhanced and prolonged in the presence of 9AC.We have also shown that 9AC inhibits a fraction of intracellular p-nitrophenyl phosphatase activity, which is insensitive to known phosphatase inhibitors, such as OA, tartaric acid or bromotetramisole. Less