Inhibition of muscarinic-stimulated polyphosphoinositide hydrolysis and Ca2+ mobilization in cat iris sphincter smooth muscle cells by cAMP-elevating agents
Inhibition of muscarinic-stimulated polyphosphoinositide hydrolysis and Ca2+ mobilization in cat iris sphincter smooth muscle cells by cAMP-elevating agents
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DOI：
10.1016/s0898-6568(97)00018-1
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发表时间：
1997-09-01
影响因子：
4.8
通讯作者：
AbdelLatif, AA
中科院分区：
文献类型：
--
作者：
Ding, KH;Husain, S;AbdelLatif, AA
在 SV-40 转化的猫虹膜括约肌平滑肌 (SV-CISM-2) 细胞中研究了卡巴胆碱 (CCh) 对肌醇 1,4,5-三磷酸 (IP3) 产生和细胞内钙 ([Ca2+](i)) 动员的影响，以及 cAMP 升高剂对它们的调节。 CCh 产生 IP3 产量的时间和剂量依赖性增加； t(1/2)和EC50值分别为68秒和0.5μM。毒蕈碱激动剂引起 [Ca2+](i) 短暂增加，并在 77 秒内达到最大值，并以浓度依赖性方式增加 [Ca2+](i) 动员，EC50 为 1.4 μM。 Thapsigargin（一种 Ca2+-泵抑制剂）导致 [Ca2+](i) 快速增加，随后添加 CCh 没有效果。 CCh 诱导的 IP3 产生和 CCh: 诱导的 [Ca2+](i) 动员均被 4-DAMP（一种 M-3 毒蕈碱受体拮抗剂）比哌仑西平（一种 M-1 受体拮抗剂）更有效地拮抗，表明这两种反应都是通过 M3 受体亚型介导的。用磷脂酶 C (PLC) 抑制剂 U73122 处理细胞，导致 CCh 刺激的 IP3 产生和 [Ca2+]i 动员均出现浓度依赖性下降。这些数据表明这些平滑肌细胞中 IP3 产生增强与 [Ca2+](i) 动员之间存在密切相关性，并表明 CCh 刺激的 [Ca2+](i) 增加可能是通过 IP3 产生增加来介导的。异丙肾上腺素 (ISO) 以浓度依赖性方式抑制 CCh 诱导的 IP3 产生 (IC50 = 80 nM) 和 [Ca2+](i) 动员 (IC50 = 0.17 μM)。从 SV-CISM-2 细胞分离的微粒体级分含有磷脂酶 C (PLC)，其受到 CCh (10 μM) 和 GTP gamma S (0.1 μM) 的刺激。用 ISO 或毛喉素（各 5 μM）预处理细胞，产生膜级分，其中 CCh 刺激的 PLC 活性显着减弱。此外，当从 SV-CISM-2 细胞中分离的微粒体部分被蛋白激酶 A (PKA) 磷酸化时，CCh 和 GTP γ S 刺激的 IP3 产生显着受到抑制。从这些研究中可以得出结论，在 SV-CISM-2 细胞中，M-3 毒蕈碱受体的激活会刺激 PLC 介导的 PIP2 水解，产生动员 [Ca2+](i) 的 IP3。此外，cAMP 的升高可能通过涉及 PLC、G 蛋白、IP3 受体和/或 IP3 代谢酶的 PKA 依赖性磷酸化的机制抑制 IP3 产生和 [Ca2+](i) 动员。 (C) 1991 爱思唯尔科学公司。
The effects of carbachol (CCh) on inositol 1,4,5-trisphosphate (IP3) production and intracellular calcium ([Ca2+](i)) mobilization, and their regulation by cAMP-elevating agents were investigated in SV-40 transformed cat iris sphincter smooth muscle (SV-CISM-2) cells. CCh produced time-and dose-dependent increases in IP3 production; the t(1/2) and EC50 values were 68 s and 0.5 mu M, respectively. The muscarinic agonist provoked a transient increase in [Ca2+](i) which reached maximum within 77 s, and increased [Ca2+](i) mobilization in a concentration-dependent manner with an EC50 of 1.4 mu M. Thapsigargin, a Ca2+-pump inhibitor, caused a rapid rise in [Ca2+](i) and subsequent addition of CCh was without effect. Both CCh-induced IP3 production and CCh: induced [Ca2+](i) mobilization were more potently antagonized by 4-DAMP, an M-3 muscarinic receptor antagonist, than by pirenzepine, an M-1 receptor antagonist, suggesting that both responses are mediated through the M3 receptor subtype. Treatment of the cells with U73122, a phospholipase C (PLC) inhibitor, resulted in a concentration-dependent-decrease in both CCh-stimulated IP3 production and [Ca2+]i mobilization. These data indicate close correlation between enhanced IP3 production and [Ca2+](i) mobilization in these smooth muscle cells and suggest that the CCh-stimulated increase in [Ca2+](i) could be mediated through increased IP3 production. Isoproterenol (ISO) inhibited CCh-induced IP3 production (IC50 = 80 nM) and [Ca2+](i) mobilisation (IC50 = 0.17 mu M) in a concentration-dependent manner. Microsomal fractions isolated from SV-CISM-2 cells contained phospholipase C (PLC) which was stimulated by CCh (10 mu M) and GTP gamma S (0.1 mu M) Pretreatment of the cells with ISO or forskolin, 5 mu M each, produced membrane fractions in which CCh-stimulated PLC activity was significantly attenuated. Furthermore, when microsomal fractions isolated from SV-CISM-2 cells were phosphorylated with Protein kinase A (PKA), the CCh- and GTP gamma S-stimulated IP3 production were significantly inhibited. It can be concluded from these studies that in SV-CISM-2 cells, activation of M-3 muscarinic receptors results in stimulation of PLC-mediated PIP2 hydrolysis, generating IP3 which mobilizes [Ca2+](i). Furthermore, elevation of cAMP may inhibit IP3 production and [Ca2+](i) mobilization through mechanisms involving PKA dependent phosphorylation of PLC, G-proteins, IP3 receptor and/or IP3 metabolizing enzymes. (C) 1991 Elsevier Science Inc.