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

本研究旨在探讨受体刺激引起的豚鼠血管平滑肌细胞(SMC)自分泌/旁分泌三磷酸腺苷(ATP)的自分泌/旁分泌信号与细胞内钙离子信号的关系。血管紧张素II(Ang II)(0.3-1μM)可诱导培养的结肠带绦虫SMC大量释放三磷酸腺苷，但不能诱导人成纤维细胞释放。然而，即使在10μM时，Ang II也不能引起SMC的乳酸脱氢酶漏出。AT1受体拮抗剂10μM SC52458可抑制血管紧张素Ⅱ释放三磷酸腺苷，而AT2受体拮抗剂10μM PD123319不能抑制血管紧张素Ⅱ的作用。10μM U-73122(磷脂酶C抑制剂)和0.5μM thapsigargin(Ca^&lt；2+&gt；-ATPase抑制剂)几乎完全抑制ATP的释放。50μM BAPTA/AM是细胞内钙离子的螯合剂，但不能被电压门控钙通道阻滞剂0.1μM硝苯地平抑制。B-…可增加三磷酸腺苷的基础释放更多的APTA/AM，但减少了U-73122。Ang II可促进细胞内三磷酸肌醇(Ins(1，4，5)P3)的瞬时蓄积。SC52458可完全拮抗这种增强作用。这些结果提示，Ins(1，4，5)P3受体激活的细胞内钙信号参与了血管紧张素Ⅱ诱导的ATP释放。在无钙Krebs溶液中，咖啡因浓度为3 mM时，可引起培养的输精管平滑肌细胞产生显著的ATP释放，而不是人成纤维细胞。BAPTA/AM和thapsigargin可明显抑制ATP的释放。此外，兰尼定和丁卡因这两种兰尼定受体拮抗剂可完全抑制该药物的释放。用Fluo 4测定[Ca~(2+)]i的研究表明，咖啡因可引起[Ca~(2+)]i升高，丁卡因可完全拮抗咖啡因的这种作用。在RT-PCR研究中，RyR-2的mRNA在豚鼠输精管和回肠纵行SMC中表达，而RyR-1则不表达。这些结果表明，咖啡因诱导的ATP释放是通过刺激内质/肌质网上的兰尼定受体(RyR-2)来触发细胞内Ca~(2+)和Gt~(2+)信号的。较少

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