Mileo A M, Palma E, Polenzani L, Limatola C, Grassi F, Eusebi F
Laboratory of Biophysics, Experimental Research Center, Rome, Italy.
J Neurosci Res. 1995 Jul 1;41(4):443-51. doi: 10.1002/jnr.490410403.
The modulation of acetylcholine-activated current (IACh) by protein kinase C (PKC) was studied in Xenopus laevis oocytes microinjected with either mRNA extracted from C2C12 myotubes (C2C12 mRNA) or RNAs encoding murine alpha beta gamma delta subunits of the nicotinic ACh receptor (nAChR). Voltage-clamped oocytes were treated for 90 sec with 12-O-tetradecanoylphorbol-13-acetate (TPA, 300 nM), a potent PKC activator. Transient increase in the amplitude and acceleration in the decay of IACh were invariably observed within minutes of TPA application, and were independent of extracellular Ca2+ concentration. Both parameters recovered to control within 20-30 min; then a slight depression of IACh developed. By this time, an initial PKC down regulation was observed. At the peak of TPA-induced potentiation, dose-response relations suggested an increased binding affinity of nAChR for the neurotransmitter. 4 alpha-phorbol 12,13-didecanoate (300 nM), a biologically inactive analogue of TPA, did not affect IACh, while staurosporine (5-10 microM), a potent inhibitor of PKC activity, suppressed the action of TPA on IACh. In oocytes co-injected with C2C12 mRNA and with rat brain mRNA, IACh was potentiated by 5-hydroxy-tryptamine (10 microM), whose receptors are coupled to phosphoinositide hydrolysis. The nAChR-channel activity in cell-attached patches increased when TPA was applied to the oocytes. In 50% of the oocytes examined, a sustained depression of the single channel activity followed. We conclude that in Xenopus oocytes an endogenous PKC system regulates the function of embryonic-type muscle nAChRs.
在非洲爪蟾卵母细胞中研究了蛋白激酶C（PKC）对乙酰胆碱激活电流（IACh）的调节作用，这些卵母细胞被显微注射了从C2C12肌管中提取的mRNA（C2C12 mRNA）或编码烟碱型乙酰胆碱受体（nAChR）的小鼠αβγδ亚基的RNA。用强效PKC激活剂12 - O - 十四酰佛波醇 - 13 - 乙酸酯（TPA，300 nM）对电压钳制的卵母细胞处理90秒。在应用TPA后的几分钟内，总是观察到IACh的幅度瞬时增加以及衰减加速，且与细胞外Ca2+浓度无关。两个参数在20 - 30分钟内恢复到对照水平；然后IACh出现轻微下降。此时，观察到PKC最初的下调。在TPA诱导的增强作用达到峰值时，剂量反应关系表明nAChR对神经递质的结合亲和力增加。4α - 佛波醇12,13 - 二癸酸酯（300 nM），一种TPA的生物无活性类似物，不影响IACh，而星形孢菌素（5 - 10 μM），一种强效的PKC活性抑制剂，抑制了TPA对IACh的作用。在共注射C2C12 mRNA和大鼠脑mRNA的卵母细胞中，IACh被5 - 羟色胺（10 μM）增强，其受体与磷酸肌醇水解偶联。当将TPA应用于卵母细胞时，细胞贴附片膜中的nAChR通道活性增加。在所检查的50%的卵母细胞中，随后出现单通道活性的持续下降。我们得出结论，在非洲爪蟾卵母细胞中，内源性PKC系统调节胚胎型肌肉nAChRs的功能。
