Role of protein phosphorylation in Ca2+ regulated intracellular messenger systems

We investigated the effects of a newly synthesized compound 1-(5-chloronaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine (ML-9), a myosin light chain kinase (MLC-kinase) inhibitor, on contractile responses and on phosphorylation of 20 KDa myosin light chain (LC20) in collagen induced human platelet, and the intact and skinned vascular smooth muscle cells. ML-9 were found to bind at or near ATP-binding site of MLC-kinase molecule and inhibit the enzyme activity in competitive fashion with respect to ATP, with a Ki value of 3.8 microM in the presence or absence of Ca2+-calmodulin. ML-9 had no or little effect on any other enzymes tested in vitro over similar concentration range to inhibit MLC-kinase. ML-9 delayed the time course of LC20 phosphorylation, sequentially led to a delay in aggregation and serotonin secretion in a dose-dependent fashion in collagen-induced human platelet. ML-9 (10 microM) produced a shift to the right and down in dose-response curves of rabbit vascular strips to all agonist tested. ML-9 also suppressed the Ca2+-induced contraction of saponin-treated skinned fibers. We then examined the relationship between ML-9-induced inhibition of isometric tension development after addition of 50 mM KCl and the extent of LC20 phosphorylation in vascular smooth muscle. ML-9 not only inhibited the maximal extent of LC20 phosphorylation but also delayed the time course of the phosphorylation, in a dose-dependent manner. The initial rate of isometric tension development correlated to the extent of phosphorylation of the LC20. Thus, ML-9 should prove to be a specific and potent MLC-kinase inhibitor for investigating the physiological role of myosin light chain phosphorylation by MLC-kinase, in vivo and in vitro.