AN ESSENTIAL ROLE OF MYOSIN LIGHT-CHAIN KINASE IN THE REGULATION OF AGONIST-STIMULATED AND FLUID FLOW-STIMULATED CA2-CELLS( INFLUX IN ENDOTHELIAL)

Cytosolic Ca2+ ([Ca2+](i)) plays an important role in endothelial cell signaling. Although it has been suggested that the influx of Ca2+ can be triggered by depletion of intracellular Ca2+ stores, the mechanism (or mechanisms) underlying this phenomenon needs further elaboration. In the present study, involvement of myosin light-chain kinase (MLCK) in the regulation of Ca2+ signaling was investigated in agonist-and f luid flow-stimulated endothelial cells loaded with Ca2+-sensitive dyes . Bradykinin (BK) and thapsigargin caused an increase in [Ca2+](i) fol lowed by a sustained rise due to Ca2+ influx from extracellular space and shifted total myosin light-chain (MLC) from the unphosphorylated t o the diphosphorylated form. ML-9 (100 mu M), an inhibitor of MLCK, ab olished Ca2+ influx and prevented MLC diphosphorylation in BK-and thap sigargin-treated cells, but did not affect Ca2+ mobilization from inte rnal stores. Fluid flow stimulation (shear stress=5 dynes/cm(2)) incre ased [Ca2+](i) and enhanced MLC phosphorylation. ML-9 also inhibited C a2+ response and MLC phosphorylation in fluid flow-stimulated cells. T he Ca2+ influx in response to BK was linearly correlated with the diph osphorylation of MLC in ML-9 treated cells. Effects of ML-5 and ML-7, analogs of ML-9, to inhibit Ca2+ influx paralleled their potencies to inhibit MLCK activity. These findings demonstrate that MLCK plays an e ssential role in regulating the plasmalemmal Ca2+ influx in agonist-an d fluid flow-stimulated endothelial cells. This study is the first to report the close relationship between Ca2+ influx and MLC diphosphoryl ation.