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

在血管平滑肌中，一些激动剂即使在没有细胞外Ca^<2+>的情况下也能诱导持续收缩，而不增加细胞内游离Ca^<2+>水平（[Ca^<2+>]i）。为了评估Ca2+非依赖性收缩机制，我们研究了各种药物对Ca^<2+>缓冲液中完整血管平滑肌（大鼠胸主动脉条）的影响，重点关注[Ca^<2+>]i，肌球蛋白轻链（MLC）和MLC-磷酸酶活性。内皮素-1 （ET-1）、12-肉豆酸酯- 13-乙酸酯（PMA）、12-二丁酸酯（PDB）和钙动蛋白-a可产生Ca^<2+>-缺失大鼠胸主动脉条的持续收缩，该条带是在Ca^<2+>无缓冲液中反复应用去甲肾上腺素或咖啡因制备的。在不含Ca^<2+>的缓冲液中孵育10分钟后，去甲肾上腺素诱导了典型的相性收缩和[Ca^<2+>]i的短暂增加，这是用fura 2测量的。另一方面，PMA和PDB诱导Ca^<2+> [Ca^<2+>]i中Ca^<2+> free buffer持续收缩。pdb诱导的收缩与20kda MLC的磷酸化有关。对20-kDa MLC进行二维磷酸肽图谱分析，结果表明，90%的多肽来源于MLC激酶催化反应，10%的多肽来源于蛋白激酶c的磷酸化反应。该酶由58-kDa调控亚基和38- kDa催化亚基组成。虽然ET-1和PDB对纯化的磷酸酶活性没有明显影响，但PDB对完整主动脉条的mlc -磷酸酶活性有抑制作用。这些结果提示血管平滑肌中mlc -磷酸酶的调控可能在Ca^<2+>-不依赖性收缩中起重要作用。

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.