ATP-autocrine/paracrine release and its intracellular Ca^<2+> signals

ATP-自分泌/旁分泌释放及其胞内Ca^2信号

• 批准号: 13670107
• 负责人: KATSURAGI Takeshi
• 金额: $ 1.79万
• 依托单位: Fukuoka University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13670107/
• 关键词: Release mechanism of ATP; Intracellular Ca^<2+> signals; Cultured smooth muscle cells; Angiotensin II; AT_1-receptor; Inositol(1,4,5)trisphosphate; Caffeine; Ryanodine receptor; 初代培養結腸紐平滑筋細胞; ATPの放出機構; オートクリン; パラクリン物質; P2-受容体刺激; 平滑筋細胞; ryano

The studies were undertaken to determine what kind of intracellular Ca^<2+> signals is involved in the autocrine/paracrine release of ATP evoked by receptor stimulation from cultured smooth muscle cells (SMCs) from guinea-pigs. Angiotensin II (Ang II)(0.3-1 μM) elicited substantial release of ATP from cultured taenia coli SMCs, but not from a human fibroblast cell line. However, Ang II even at 10 μM failed to cause a leakage of lactate dehydrogenase (LDH) from the SMCs. The release of ATP by Ang II was suppressed by 10 μM SC52458, an AT_1 receptor antagonist, not by 10 μM PD123319, an AT_2 receptor antagonist. The evoked release of ATP was almost completely inhibited in the presence of 10 μM U-73122, a phospholipase C inhibitor, and 0.5 μM thapsigargin, a Ca^<2+>-ATPase inhibitor. Furthermore, the release was hampered by 50 μM BAPTA/AM, an intracellular Ca^<2+> chelator, but not by 0.1 μM nifedipine, a voltage gated Ca^<2+> channel inhibitor. The basal release of ATP was increased by B … More APTA/AM, but was reduced by U-73122. Ang II enhanced instantaneously inositol(1,4,5)trisphosphate (Ins(1,4,5)P_3) accumulation in the cells. The enhancing effect was perfectly antagonized by SC52458. These findings suggest that intracellular Ca^<2+> signals activated via stimulation of Ins(1,4,5)P_3 receptor are involved in the release of ATP evoked by Ang II. In Ca^<2+> free Krebs solution, caffeine at 3 mM instantaneously caused a remarkable release of ATP from cultured vas deferens SMCs, not from a human fibroblast cell line. The evoked release of ATP was markedly abolished by treatment with BAPTA/AM and thapsigargin. Furthermore, the release was completely inhibited by ryanodine and tetracaine, ryanodine receptor antagonists. From the study of [Ca^<2+>]i measurement with fluo 4, caffeine induced an elevation of [Ca^<2+>]i and the effect of caffeine was absolutely antagonized by tetracaine. In RT-PCR studies, the expression of mRNA from RyR-2, but not from RyR-1, was observed in vas deferens SMCs as well as ileal longitudinal SMCs from guinea-pigs. These findings suggest that the release of ATP induced by caffeine is triggered by the intracellular Ca^<2+> signal via ryanodine receptor (RyR-2) stimulation on endoplasmic/sarcoplasmic reticulum. Less

本研究旨在确定哪种胞内Ca^<2+>信号参与了培养豚鼠平滑肌细胞（SMCs）受体刺激引起的自分泌/旁分泌ATP释放。血管紧张素II (angii)（0.3-1 μM）诱导培养的大肠带绦虫SMCs大量释放ATP，但在人成纤维细胞系中没有。然而，即使在10 μM的浓度下，Ang II也未能引起SMCs中乳酸脱氢酶（LDH）的泄漏。AT_1受体拮抗剂10 μM SC52458能抑制Ang II对ATP的释放，而AT_2受体拮抗剂10 μM PD123319不能抑制Ang II对ATP的释放。10 μM U-73122（磷脂酶C抑制剂）和0.5 μM thapsigargin （Ca^<2+>-ATP酶抑制剂）对ATP的诱导释放几乎完全抑制。此外，50 μM BAPTA/AM（细胞内Ca^<2+>螯合剂）对释放有抑制作用，而0.1 μM硝苯地平（电压门控Ca^<2+>通道抑制剂）对释放无抑制作用。B…More APTA/AM增加了ATP的基础释放，而U-73122则降低了ATP的基础释放。Ang II增强了肌醇（1,4,5）三磷酸（Ins(1,4,5)P_3）在细胞中的瞬时积累。SC52458完全拮抗了这种增强作用。这些发现表明，通过刺激Ins(1,4,5)P_3受体激活的细胞内Ca^<2+>信号参与了Ang II诱导的ATP释放。在Ca^<2+> free Krebs溶液中，3 mM的咖啡因瞬间引起培养的输精管SMCs中ATP的显著释放，而不是从人成纤维细胞系中。经BAPTA/AM和thapsigarin处理后，ATP的诱发释放明显减少。此外，ryanodine和tetracaine， ryanodine受体拮抗剂完全抑制释放。从氟4测定[Ca^<2+>]i的研究来看，咖啡因诱导[Ca^<2+>]i升高，并且咖啡因的作用被丁卡因完全拮抗。在RT-PCR研究中，在豚鼠输精管SMCs和回肠纵向SMCs中观察到RyR-2而非RyR-1 mRNA的表达。这些发现表明，咖啡因诱导的ATP释放是通过刺激内质/肌浆网的ryanodine受体（RyR-2），通过胞内Ca^<2+>信号触发的。少

项目成果

Shinya Ueno: "Bidirectional modulation of P2X receptor-mediated response by divalent cations in rat dosal motor nucleus of the vagus neurons"J. Nurochem.. 78. 1009-1018 (2001)

Shinya Ueno：“大鼠迷走神经元背侧运动核中二价阳离子对 P2X 受体介导的反应的双向调节”J。

Katsuragi, T., Sato, C., Usune, S. and Ueno, S.: "ATP is released by ryanodine-receptor stimulation with caffeine from cultured smooth muscle cells"Mol. Pharmacol.. (in press).

Katsuragi, T.、Sato, C.、Usune, S. 和 Ueno, S.：“ATP 是通过咖啡因刺激培养的平滑肌细胞中的兰尼碱受体而释放的”Mol。

Katsuragi, T., Sato, C., Lou, G. and Honda, K.: "Inositol(1,4,5)trisphosphate signal triggers a receptor-mediated ATP release"Biochem. Biophys. Res. Commun.. 293. 686-690 (2002)

Katsuragi, T.、Sato, C.、Lou, G. 和 Honda, K.：“肌醇 (1,4,5) 三磷酸信号触发受体介导的 ATP 释放”Biochem。

Sono Fujiki: "Differences in the signalling transduction pathways involved in the P2-receptor mediated ATP release from the guinea pig vas deferens and ileum"Naunyn-Schmiedeb. Arch. Pharmacol.. (in press). (2002)

Sono Fujiki：“参与 P2 受体介导的豚鼠输精管和回肠 ATP 释放的信号转导途径的差异”Naunyn-Schmiedeb。

naoaki Harada: "Ischemia/reperfusion-induced increase in the hepatic level of prostacyclin is mainly mediated by activation of capsaicin-sensitive sensory neurons in rats"J. Lab. Clin. Med. (in press). (2002)

naoaki Harada：“缺血/再灌注引起的大鼠肝脏前列环素水平增加主要是通过激活大鼠辣椒素敏感的感觉神经元介导的”J.