ACh-induced rebound stimulation of L-type Ca2+ current in guinea-pig ventricular myocytes, mediated by Gβγ-dependent activation of adenylyl cyclase
ACh-induced rebound stimulation of L-type Ca2+ current in guinea-pig ventricular myocytes, mediated by Gβγ-dependent activation of adenylyl cyclase
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DOI：
10.1111/j.1469-7793.2001.00677.x
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发表时间：
2001-11-01
影响因子：
5.5
通讯作者：
Harvey, RD
中科院分区：
文献类型：
--
作者：
Belevych, AE;Sims, C;Harvey, RD
1.采用全细胞膜片钳技术研究了M受体对豚鼠心室肌细胞L-型Ca ~（2+）电流cAMP依赖性调节的影响.毒蕈碱激动剂ACh抑制β-肾上腺素能激动剂异丙肾上腺素（Iso）刺激的Ca 2+电流，ACh洗脱显示刺激反应，表现为Ca 2+电流振幅的短暂反弹增加。乙酰胆碱诱导的刺激作用，没有观察到在Iso.3的情况下。在H-2组胺受体活化和霍乱毒素处理的情况下也观察到ACh诱导的反弹刺激，其与β-肾上腺素能受体活化一样以刺激性G蛋白（G（s））依赖性方式增强腺苷酸环化酶（AC）活性。ACh诱导的反跳刺激在forskolin的存在下没有观察到，forskolin以G（s）非依赖性的方式增强AC活性。百日咳毒素（PTX）治疗阻断ACh的刺激和抑制作用。用QEHA（一种结合游离G蛋白β γ亚基的肽）进行细胞内透析，选择性地拮抗刺激作用，留下增强的抑制作用。通过豚鼠心室肌细胞膜的Western印迹分析，获得了AC 4表达的证据，AC 4是AC的一种亚型，可以被G β γ刺激，但仅在G α存在的情况下。这些结果表明，毒蕈碱受体刺激促进和抑制cAMP依赖性调节的Ca 2+电流，净反应是这两个行动之间的平衡。我们认为，刺激作用是由于直接激活AC 4的PTX敏感的G蛋白的β-γ亚基。
1. The effects that muscarinic receptor stimulation have on the cAMP-dependent regulation of L-type Ca2+ currents were studied in isolated guinea-pig ventricular myocytes using the whole-cell configuration of the patch-clamp technique.2. The muscarinic agonist ACh inhibited the Ca2+ current stimulated by the beta -adrenergic agonist isoprenaline (Iso), and washout of ACh revealed a stimulatory response that appeared as a transient rebound increase in the amplitude of the Ca2+ current. The ACh-induced stimulatory effect was not observed in the absence of Iso.3. ACh-induced rebound stimulation was also observed in the presence of H-2 histamine receptor activation and cholera toxin treatment, which like beta -adrenergic receptor activation enhance adenylyl cyclase (AC) activity in a stimulatory G protein (G(s))-dependent manner. ACh-induced rebound stimulation was not observed in the presence of forskolin, which enhances AC activity in a G(s)-independent manner.4. Pertussis toxin (PTX) treatment blocked both the stimulatory and inhibitory effects of ACh. Intracellular dialysis with QEHA, a peptide that binds free G protein,beta gamma subunits, selectively antagonized the stimulatory effect, leaving an enhanced inhibitory effect.5. Evidence for the expression of AC4, an isoform of AC that can be stimulated by G beta gamma but only in the presence of G alpha (s), was obtained by Western blot analysis of guinea-pig ventricular myocyte membrane preparations.6. These results suggest that muscarinic receptor stimulation facilitates as wen as inhibits cAMP-dependent regulation of the Ca2+ current and that the net response is a balance between these two actions. We suggest that the stimulatory effect is due to a direct activation of AC4 by the beta gamma subunits of a PTX-sensitive G protein.