Molecular mechanisms of vasospasm : Intracellular signaling network underlying the Ca^<2+> of smooth muscle cells.

血管痉挛的分子机制，特别是平滑肌细胞Ca^2+敏感化的细胞内信号网络，已经确定。获得了以下结果。（1）在无Ca^<2+>、无ATP的培养液中，用1 mM ATPγS处理α-毒素透化的血管环，可增强随后由Ca^<2+>浓度增加引起的收缩，并增强Ca^<2+>敏感性，而Rho激酶抑制剂Y-27632仅部分抑制这种作用。我们发现这种不同于Rho激酶的新激酶在调节血管平滑肌肌球蛋白磷酸酶活性和肌丝Ca^2+敏感性中具有重要作用。（2）肌球蛋白磷酸酶在增强肌丝Ca^<2+>中起关键作用。磷酸酶活性主要由110 kDa的调节亚基MYPT 1调节。为了确定MYPT 1参与调节完整平滑肌中肌球蛋白磷酸酶的区域，我们将MYPT 1片段作为与HIV达特蛋白的细胞穿透肽的融合蛋白（TAT-MYPT 1，包含Tat的构建体）引入猪冠状动脉条中。达特肽和MYPT 1区域），并检查它们对收缩性的影响。我们发现，区域1-296是必不可少的，而区域297-374起着补充作用。(3)A在兔双出血模型中研究了凝血酶及其受体PAR 1在蛛网膜下腔出血中过度收缩发展中的作用。蛛网膜下腔注射自体血可引起离体基底动脉对凝血酶的过度收缩反应。高反应性可能是由于PAR 1的上调和脱敏作用受损所致。出血引起的凝血酶激活在高反应性的诱导中起重要作用。因此，凝血酶和PAR 1被认为是有用的作为一个新的治疗靶点在SAH的管理。

Molecular mechanisms of vasospasm, especially the intracellular signaling network working for the Ca^<2+>-sensitization of smooth muscle cells, were determined. Following results were obtained. (1)Treatment of α-toxin-permeabilized rings with 1 mM ATPγS in the Ca^<2+>-free, ATP-free media enhanced the subsequent contraction induced by increment of Ca^<2+> concentration, and potentiated the Ca^<2+>-sensitivity, which was only partly inhibited by Rho-kinase inhibitor, Y-27632. We found a fundamental role of this new kinase other than Rho kinase in the regulation of the myosin phosphatase activity and myofilament Ca^<2+>-sensitivity in vascular smooth muscle. (2)Myosin phosphatase plays a critical role in potentiating the myofilament Ca^<2+>. The phosphatase activity is primary regulated by the 110kDa regulatory subunit, MYPT1. To determine the region of MYPT1 involved in regulation of myosin phosphatase in intact smooth muscle, we introduced MYPT1 fragments into the strips of porcine coronary artery as fusion protein with a cell penetrating peptide of HIV Tat protein(TAT-MYPT1, a construct containing Tat peptide and regions of MYPT1), and examined their effect on the contractility. We found that the region 1-296 is essential for this augmentation, while region 297-374 plays a supplemental role. (3)A role of thrombin and its receptor PAR1 in development of hypercontractility in subarachnoid hemorrhage was investigated in a rabbit double hemorrhage model. The subarachnoidal injection of autologous blood induced hyper-contractile response of the isolated basilar artery toward thrombin. The hyper-responsiveness was suggested to be due to the up-regulation and impairment of the desensitization of PAR1. The activation of thrombin due to hemorrhage was suggested to play an important role in induction of the hyper-responsiveness. Thrombin and PAR1 were thus suggested to be useful as a new therapeutic target in the management of SAH.