ATP induces dephosphorylation of myosin light chain in endothelial cells

In cultured porcine aortic endothelial monolayers, the effect of ATP on myo sin light chain (MLC) phosphorylation, which controls the endothelial contr actile machinery, was studied. ATP (10 mu M) reduced MLC phosphorylation bu t increased cytosolic Ca2+ concentration ([Ca2+](i)). Inhibition of the ATP -evoked [Ca2+](i) rise by xestospongin C (10 mu M), an inhibitor of the ino sitol trisphosphate-dependent Ca2+ release from endoplasmic reticulum, did not affect the ATP-induced dephosphorylation of MLC. MLC dephosphorylation was prevented in the presence of calyculin A (10 nM), an inhibitor of prote in phosphatases PP-1 and PP-2A. Thus ATP activates MLC dephosphorylation in a Ca2+-independent manner. In the presence of calyculin A, MLC phosphoryla tion was incremented after addition of ATP, an effect that could be abolish ed when cells were loaded with the Ca2+ chelator 1,2-bis(2-aminophenoxy)eth ane-N,N,N',N'-tetraacetic acid acetoxymethyl ester (10 mu M). Thus ATP also activates a Ca2+-dependent kinase acting on MLC. In summary, ATP simultane ously stimulates a functional antagonism toward both phosphorylation and de phosphorylation of MLC in which the dephosphorylation prevails. In endothel ial cells, ATP is the first physiological mediator identified to activate M LC dephosphorylation by a Ca2+-independent mechanism.