Uncoupling of force and myosin light chain phosphorylation produced by slow stretch in vascular smooth muscle.

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Relationship between force and myosin light chain (MLC) phosphorylation in slow stretch-induced contraction in canine basilar artery was investigated. In the presence of tetraethyl-ammonium (5 mM), slow stretch at a rate of 1 mm/sec up to 1.5 times initial muscle length during a stimulus period of 15 min produced an increase in multiple phosphorylated MLC species (at least mono-, di- and tri-phosphorylated species) without any apparent contraction, indicating uncoupling of force and MLC phosphorylation. Slow stretch also increased translocation of protein kinase C (PKC)-α and PKC-δ from the cytosol to the membrane fraction. The MLC phosphorylation was inhibited by nicardipine (a 1,4-dihydropyridine Ca^<2+> channel blocker), ML-9 (an inhibitor of myosin light chain kinase) or calphostin C (a cPKC/nPKC inhibitor) to about 50% of that in drug-untreated artery. Y-27632 ( a Rho-kinase inhibitor) abolished the MLC phosphorylation. In contrast, rottlerin (5 μM, a putative inhibitor of PKC-δ) had no apparent effect on MLC phosphorylation. The translocation of PKC-α was inhibited by only calphostin C, and that of PKC-δ was attenuated by calphostin C, Y-27632 or rottlerin. Okadaic acid (an inhibitor of phosphatase) inhibited 80 mM KCl-induced contraction, but it increased multiple phosphorylated MLC species.Considering that the translocation of PKC-α and δ is important for their activation, the present results suggest that Rho/Rho-kinase activity and PKC other than PKC-δ are involved in MLC phosphorylation without contraction.