Regulation of neuronal cell functions by GTP-binding protein, ARF.

加入KCl或ATP可刺激PC12细胞钙内流和去甲肾上腺素(NA)释放。佛波醇肉豆蔻酸醋酸酯对蛋白激酶C的激活和刺激剂增加cAMP含量增加了KCl-和ATP刺激的NA释放。百日咳毒素处理的PC12细胞经KCL和ATP刺激后的NA释放量与对照组相同。霍乱毒素(CTX)对PC12细胞有刺激和抑制作用，且呈时间依赖性。1-2 h后，CTX可促进KCl-和ATP刺激的NA释放。24 h后，尽管PC12细胞内cAMP水平较高，但CTX显著抑制NA的释放。接下来，我们研究了CTX对KCl和ATP刺激的PC12细胞膜蛋白ADP重糖基化作用。CTX催化膜上Gs的α亚基45 kDa蛋白的[32-P]ADP-核糖化。氯化钾和三磷酸腺苷刺激的细胞膜上45 kDa的ADP-核糖化量是对照(未刺激)细胞的两倍。免疫印迹分析显示，KCl2和ATP刺激的细胞中α亚基的蛋白含量与对照细胞相同。在氯化钾和三磷酸腺苷刺激的细胞膜上，CTX的反式-ADP核糖基化的量也增加。这些结果表明，在氯化钾和三磷酸腺苷刺激的细胞中，作为环磷酰胺催化的二磷酸腺苷核糖化的激活蛋白的二磷酸腺苷再糖化因子(ARF)增加。

Addition of KCl or ATP stimulated Ca influx and noradrenaline (NA) release in PC12 cells. The activation of protein kinase C by phorbol myristate acetate and increases of cAMP content by stimulants enhanced KCl- and ATP-stimulated NA releases. KCl- and ATP-stimulated NA releases from pertussis toxin-pretreated PC12 cells were same compared with those from control cells. Pretreatment with cholera toxin (CTX) showed stimulatory and inhibitory effects in PC12 cells, depending on pretreatment-time. After 1-2 h, KCl- and ATP-stimulated NA releases were enhanced by CTX.After 24 h, the NA releases were inhibited significantly by CTX,although the level of cAMP in PC12 cells was high.Next we investigated the ADP-rebosylation of membrane proteins by CTX from KCl- and ATP-stimulated PC12 cells. CTX catalyzed [32-P] ADP-ribosylation of 45 kDa protein, alpha subunit of Gs, in membranes. The amounts of ADP-ribosylation of 45 kDa in membranes from KCl- and ATP-stimulated cells increased two-fold, compared with that in the control (non-stimulated) cells. The protein contents of alpha subunit in KCl- and ATP-stimulated cells were same as that in control cells, by immunoblotting analysis. The amount of atuto-ADP-ribosylation by CTX also increased in membranes from KCl- and ATP-stimulated cells. These data suggest that ADP-rebosylation factor (ARF), which was an activator protein of CTX-catalyzed ADP-ribosylation, increased in KCl- and ATP-stimulated cells.