Analysis of molecular mechanism in Ca^<2+>-independent vasocontraction

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In vascular smooth muscle, some agonists induce sustained contraction even in the absence of extracellular Ca^<2+> without increases in intracellular free Ca^<2+> level ([Ca^<2+>]i). To assess the Ca2+-independent contractile mechanism(s), we examined the effects of various agents on intact vascular smooth muscle (rat thoracic aorta strips) in Ca^<2+>-free buffer, focusing on [Ca^<2+>]i, myosin light chain (MLC) and MLC-phosphatase activity. Endothelin-1 (ET-1), phorbol 12-myristate 13-acetate (PMA), phorbol 12,13-dibutyrate (PDB) and caliculin-A produced sustained contractions of Ca^<2+>-depleted rat thoracic aorta strips, prepared by repeated applications of norepinephrine or caffeine in Ca^<2+>-free buffer. After 10 min incubation in Ca^<2+>-free buffer, norepinephrine induced a typical phasic contraction and a transient increase in [Ca^<2+>]i, which measured with fura 2. On the other hand, PMA and PDB induced sustained contraction in Ca^<2+>-free buffer in [Ca^<2+>]i. The PDB-induced contraction was associated with the phosphorylation of 20-kDa MLC. Two-dimentional phosphopeptide mapping of 20-kDa MLC revealed that about nine-tenths of the phophopeptides was derived from MLC kinase-catalyzed reaction and about one-tenth was due to phosphorylation by protein kinase C. MLC-phosphatase was purified from chicken gizzard. The enzyme consisted of 58-kDa regulatory subunits and 38- kDa catalytic subunit. Although ET-1 and PDB did not show any significant effects on the purified phosphatase activity, MLC-phosphatase activity of intact aorta strip was inhibited by the treatment with PDB. These results suggest that the regulation of MLC-phosphatase in vascular smooth muscle may play important roles in the Ca^<2+>-independent contraction.