Komatsu M, McDermott A M, Gillison S L, Sharp G W
Department of Pharmacology, Cornell University College of Veterinary Medicine, Ithaca, New York 14853-6401, USA.
Endocrinology. 1995 May;136(5):1857-63. doi: 10.1210/endo.136.5.7720630.
Male Sprague-Dawley rats were injected ip with 1 microgram pertussis toxin (PTX)/100 g BW. The rats were killed 24, 48, and 72 h after injection, and their pancreases were removed. At each time point, insulin secretion by isolated islets was measured under basal and glucose-stimulated conditions and in the absence and presence of norepinephrine. cAMP levels were measured under basal and forskolin-stimulated conditions in the absence and presence of norepinephrine. PTX-induced ADP ribosylation of Gi/Go proteins in vivo was monitored by ADP ribosylation in vitro using PTX and 32P-labeled NAD and also by Western blotting. At 24 h, 1 microM norepinephrine inhibited glucose-stimulated insulin secretion by 92% in the control islets, but by only 53% in the PTX-treated islets; at 48 h, norepinephrine still inhibited secretion (by 40%) in the PTX-treated islets; at 72 h, the inhibitory effect of norepinephrine was abolished. Therefore, contrary to recent suggestions, all of the effect of norepinephrine to inhibit insulin release is PTX sensitive. The effects of PTX on the ability of norepinephrine to lower cAMP levels were similar to those observed for the inhibition of insulin release. PTX partially blocked the effect of norepinephrine to lower cAMP levels at 24 and 48 h, and the block was complete after 72 h. The extent of the in vivo ADP ribosylation of the Gi/Go proteins, monitored at each time point by in vitro [32P]ADP-ribosylation and Western blotting, demonstrated a profound ADP ribosylation at 48 and 72 h. As detected by Western blotting, the rates of ADP ribosylation by PTX and the onset of decreased expression varied among the different G-proteins. G alpha o was virtually eliminated after only 24 h of PTX treatment. G alpha i2 was markedly affected by 48 h; G alpha i3 was little affected until 72 h.
将1微克百日咳毒素（PTX）/100克体重腹腔注射给雄性Sprague-Dawley大鼠。在注射后24、48和72小时处死大鼠，并取出其胰腺。在每个时间点，在基础和葡萄糖刺激条件下以及在有无去甲肾上腺素的情况下测量分离胰岛的胰岛素分泌。在基础和福斯高林刺激条件下以及在有无去甲肾上腺素的情况下测量cAMP水平。通过使用PTX和32P标记的NAD进行体外ADP核糖基化以及蛋白质印迹法监测体内PTX诱导的Gi/Go蛋白的ADP核糖基化。在24小时时，1微摩尔去甲肾上腺素在对照胰岛中使葡萄糖刺激的胰岛素分泌抑制92%，但在PTX处理的胰岛中仅抑制53%；在48小时时，去甲肾上腺素在PTX处理的胰岛中仍抑制分泌（40%）；在72小时时，去甲肾上腺素的抑制作用消失。因此，与最近的观点相反，去甲肾上腺素抑制胰岛素释放的所有作用对PTX敏感。PTX对去甲肾上腺素降低cAMP水平能力的影响与观察到的对胰岛素释放抑制的影响相似。PTX在24和48小时部分阻断了去甲肾上腺素降低cAMP水平的作用，72小时后阻断作用完全。在每个时间点通过体外[32P]ADP核糖基化和蛋白质印迹法监测的Gi/Go蛋白的体内ADP核糖基化程度在48和72小时显示出显著的ADP核糖基化。通过蛋白质印迹法检测，PTX的ADP核糖基化速率和表达降低的起始时间在不同的G蛋白之间有所不同。仅PTX处理24小时后，Gαo几乎完全消失。48小时时Gαi二明显受影响；直到72小时Gαi三几乎没有受到影响。
