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

血管痉挛的分子机制：平滑肌细胞Ca^2 的细胞内信号网络。

• 批准号: 13470149
• 负责人: KANAIDE Hideo
• 金额: $ 8万
• 依托单位: KYUSHU UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2003
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-13470149/
• 关键词: vascular smooth muscle cells; Ca^<2+> signaling; Ca^<2+> sensitization; vasospasm; thrombin; subarachnoid hemorrhage; myosin phosphorylation; myosin phosphatase; Protease-actvated receptors; トロンビン受容体; 細胞質[Ca^<++>]i; ミオシン軽鎖燐酸化; 細胞質Ca^<2+>濃度; T

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.

血管痉挛的分子机制，特别是平滑肌细胞Ca^<2+>-致敏的细胞内信号网络被确定。得到以下结果：(1)在无Ca^<2+>、无Ca^<2+>的培养基中，用1 mM的ATPγS处理α-毒素渗透环，可增强Ca^<2+>浓度增加引起的后续收缩，增强Ca^<2+>的敏感性，而rho激酶抑制剂Y-27632仅能部分抑制这一作用。我们发现除了Rho激酶外，这种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+>-敏感性方面具有重要作用。(2)肌球蛋白磷酸酶在肌丝Ca^<2+>的增强中起关键作用。磷酸酶活性主要由110kDa调控亚基MYPT1调控。为了确定MYPT1在完整平滑肌中参与肌球蛋白磷酸酶调控的区域，我们将MYPT1片段作为融合蛋白与HIV Tat蛋白的细胞穿透肽（Tat -MYPT1，一种包含Tat肽和MYPT1区域的构建体）引入猪冠状动脉条带，并检测其对收缩性的影响。研究发现，区域1-296对这种增强作用至关重要，而区域297-374则起补充作用。(3)采用兔双腔出血模型，探讨凝血酶及其受体PAR1在蛛网膜下腔出血过度收缩发生中的作用。蛛网膜下注射自体血液诱导离体基底动脉对凝血酶的超收缩反应。这种高反应性可能是由于PAR1脱敏功能的上调和受损。出血引起的凝血酶活化在诱导高反应性中起重要作用。因此，凝血酶和PAR1可作为SAH治疗的新靶点。

项目成果

Koga M: "The long-term deficiency of estrogen enhances the contractile response without affecting the Ca^<2+>-sensitivity of the contractile apparatus in arterial smooth muscle of the female rabbit."J Soc Gynecol Invest. (in press).

Koga M：“雌激素的长期缺乏增强了收缩反应，但不影响雌性兔子动脉平滑肌中收缩装置的Ca^2-敏感性。”J Soc Gynecol Invest。

Ihara E, Derkach DN, Hirano K, Nishimura J, Nawata H, Kanaide H: "Ca^<2+> influx in the endothelial cells is required for the bradykinin-induced endotheliurn-dependent contraction in the porcine interlobar renal artery."J Physiol. 534. 701-711 (2001)

Ihara E、Derkach DN、Hirano K、Nishimura J、Nawata H、Kanaide H：“猪间肾动脉中缓激肽诱导的内皮依赖性收缩需要 Ca^2 流入内皮细胞。”J Physiol

Maeda Y, Hirano K, Nishimura J, Sasaki T, Kanaide H: "Rho-kinase inhibitor inhibits both myosin phosphorylation-dependent and-independent enhancement of myofilament Ca^<2+> sensitivity in bovine middle cerebral artery."Br J Pharmacol. 140. 871-880 (2003)

Maeda Y、Hirano K、Nishimura J、Sasaki T、Kanaide H：“Rho 激酶抑制剂抑制牛大脑中动脉中肌球蛋白磷酸化依赖和独立的肌丝 Ca^2 敏感性增强。”Br J Pharmacol。

Hirano K., Hirano M., Zeng Ying, Nishimura J., Kanaide H.: "Cloning and functional expression of a degradation-resistant novel isoform of p27^<Kip1>"Biochem J.. 353. 51-57 (2001)

Hirano K.、Hirano M.、Zeng Ying、Nishimura J.、Kanaide H.：“p27^<Kip1>的抗降解新亚型的克隆和功能表达”Biochem J.. 353. 51-57 (2001)

Ise S: "Theophylline attenuates Ca^<2+> sensitivity and modulates BK channels in porcine tracheal smooth muscle."Br J Pharmacol. 140. 939-947 (2003)

Ise S：“茶碱可减弱猪气管平滑肌中的 Ca^2 敏感性并调节 BK 通道。”Br J Pharmacol。