THROMBIN INHIBITS MYOSIN LIGHT-CHAIN DEPHOSPHORYLATION IN ENDOTHELIAL-CELLS

Histamine and thrombin increase myosin light-chain kinase-mediated pho sphorylation of myosin light chain (MLC) in human umbilical vein endot helial cells (HUVEC). The increase in MLC phosphorylation caused by th rombin persists longer (330 min) than the increase caused by histamine (<5 min), although both increase cell calcium similarly. We hypothesi zed that some of the longer duration of the increase in MLC phosphoryl ation caused by thrombin was because of inhibition of myosin dephospho rylation by thrombin. Calyculin A, an inhibitor of type 1 and 2A prote in phosphatases, caused a time-dependent increase in MLC phosphorylati on in unstimulated HUVEC. As thrombin-stimulated phosphorylation appro ached its peak at 15 min, calyculin A caused progressively less of an increase in MLC phosphorylation in thrombin-stimulated HUVEC, and no i ncrease at the peak of thrombin stimulation. In HUVEC in which cell ca lcium was maintained at 600 nM, thrombin increased MLC phosphorylation above the level caused by increased calcium alone at a time coincidin g with the peak of thrombin stimulation. However, when phosphatase act ivity was already inhibited with calyculin A, thrombin did not further increase MLC phosphorylation in cells in which calcium was maintained at 600 nM calcium. Thrombin increases MLC phosphorylation in HUVEC no t only by increasing cell calcium but also by inhibiting calyculin A-s ensitive dephosphorylation of MLC.