EFFECTS OF A MYOSIN LIGHT-CHAIN KINASE INHIBITOR, WORTMANNIN, ON CYTOPLASMIC CA2-CHAIN PHOSPHORYLATION AND FORCE IN VASCULAR SMOOTH-MUSCLE(LEVELS, MYOSIN LIGHT)

Biochemical studies have shown that wortmannin is an inhibitor of myos in light chain (MLC) kinase (Nakanishi et al. (1992) J. Biol. Chem. 26 7: 2157-2163). To investigate the role of MLC kinase in smooth muscle contractions, we examined the effects of wortmannin on isolated smooth muscles of the rat aorta. Wortmannin (1 mu M) decreased MLC phosphory lation and the amplitude of contractions induced by high K+ (72.7 mM) to a level seen at rest. This occurred without a change in cytosolic C a2+ levels ([Ca2+](i)). In contrast, wortmannin only partially inhibit ed the sustained contractions induced by phenylephrine (1 mu M) and pr ostaglandin F-2 alpha (PGF(2 alpha), 10 mu M) without a change in the [Ca2+](i). On the other hand, wortmannin (1 or 10 mu M) reduced the in crease in MLC phosphorylation induced by phenylephrine and PGF,, to a level seen at rest. In the absence of external Ca2+, caffeine (20 mM) induced a transient increase in [Ca2+](i) and force with an increase i n MLC phosphorylation. Wortmannin completely inhibited the increase in MLC phosphorylation and contraction induced by caffeine without affec ting the increase in [Ca2+](i). In the absence of external Ca2+, pheny lephrine induced a small transient increase in [Ca2+](i), MLC phosphor ylation and generation of force. This was followed by a small sustaine d contraction without an increase in [Ca2+](i) and MLC phosphorylation . Wortmannin (1 CIM) inhibited the transient phase of the contraction and the increase in MLC phosphorylation without affecting the transien t increase in [Ca2+](i) nor the sustained contraction. Wortmannin inhi bited the Ca2+-induced contraction in permeabilized rat mesenteric art ery, although it did not inhibit the Ca2+-independent, ATP-induced con traction in the thiophosphorylated muscle. These results suggest that wortmannin inhibits MLC phosphorylation due to an increase in the entr y of Ca2+ or through the release of Ca2+ from the sarcoplasmic reticul um. The results also suggest that the activation of receptors by norep inephrine and PGF(2 alpha) induces a contraction via a MLC phosphoryla tion-independent pathway or through a pathway which is dependent on th e resting level of MLC phosphorylation. We conclude that wortmannin is a useful tool in studies of the physiological role of MLC kinase.