Effects of a myosin light chain kinase inhibitor, wortmannin, on cytoplasmic Ca2+ levels, myosin light chain phosphorylation and force in vascular smooth muscle.
Biochemical studies have shown that wortmannin is an inhibitor of myosin light chain (MLC) kinase (Nakanishi et al. (1992) J. Biol. Chem. 267: 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 microM) decreased MLC phosphorylation 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 Ca2+ levels ([Ca2+]i). In contrast, wortmannin only partially inhibited the sustained contractions induced by phenylephrine (1 microM) and prostaglandin F2 alpha (PGF2 alpha, 10 microM) without a change in the [Ca2+]i. On the other hand, wortmannin (1 or 10 microM) reduced the increase in MLC phosphorylation induced by phenylephrine and PGF2 alpha 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 in MLC phosphorylation. Wortmannin completely inhibited the increase in MLC phosphorylation and contraction induced by caffeine without affecting the increase in [Ca2+]i. In the absence of external Ca2+, phenylephrine induced a small transient increase in [Ca2+]i, MLC phosphorylation and generation of force. This was followed by a small sustained contraction without an increase in [Ca2+]i and MLC phosphorylation. Wortmannin (1 microM) inhibited the transient phase of the contraction and the increase in MLC phosphorylation without affecting the transient increase in [Ca2+]i nor the sustained contraction. Wortmannin inhibited the Ca2(+)-induced contraction in permeabilized rat mesenteric artery, although it did not inhibit the Ca(2+)-independent, ATP-induced contraction in the thiophosphorylated muscle. These results suggest that wortmannin inhibits MLC phosphorylation due to an increase in the entry of Ca2+ or through the release of Ca2+ from the sarcoplasmic reticulum. The results also suggest that the activation of receptors by norepinephrine and PGF2 alpha induces a contraction via a MLC phosphorylation-independent pathway or through a pathway which is dependent on the resting level of MLC phosphorylation. We conclude that wortmannin is a useful tool in studies of the physiological role of MLC kinase.
Takayama
M
M
Department of Veterinary Pharmacology, Graduate School of Agriculture and Life Sciences, University of Tokyo, Japan.
Ozaki
H
H
Karaki
H
H
D000730:Androstadienes / Q000494:pharmacology*; D000818:Animals; D002110:Caffeine / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D066298:In Vitro Techniques; D008297:Male; D009131:Muscle, Smooth, Vascular / Q000187:drug effects* / Q000502:physiology; D018994:Myosin Light Chains / Q000378:metabolism*; D009219:Myosin-Light-Chain Kinase / Q000037:antagonists & inhibitors*; D010656:Phenylephrine / Q000494:pharmacology; D010766:Phosphorylation; D051381:Rats; D017208:Rats, Wistar; D014661:Vasoconstriction / Q000187:drug effects*; D000077191:Wortmannin
D000730:Androstadienes; D004791:Enzyme Inhibitors; D018994:Myosin Light Chains; D010656:Phenylephrine; D002110:Caffeine; D009219:Myosin-Light-Chain Kinase; D002118:Calcium; D000077191:Wortmannin