Survey of regulatory roles of protein dephosphorylation process in cell motility and trans-membrane ion movements

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During the term of this project we carried out physiological and biochemical experiments chiefly in smooth and cardiac muscles. Part of the results have been published (see the reprint included in the pamphlet 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) phosporylation. 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 still phosphorylated even when contraction was completely inhibited by OA.These results indicate that there are several steps toward smooth muscle contract … More ion 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-carboxylicacid (9AC), aCl^- 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.(4) We have developed a new assay method for protein phosphatase inhibitors which utilizes firefly bioluminescence. The methods is especially suitable for quantitative analysis of inhibitors of PP2A. Less