Wollheim C B, Ullrich S, Meda P, Vallar L
Institut de Biochimie Clinique, University of Geneva, Switzerland.
Biosci Rep. 1987 May;7(5):443-54. doi: 10.1007/BF01362507.
The regulation of insulin secretion from RINm5F cells exposed to high voltage discharge has been investigated. Electron microscopy revealed that the overall structure of the cells was preserved after permeabilization. In this preparation insulin release was stimulated by Ca2+ (EC50 = 2.4 microM). The stable GTP analogue GTP gamma S enhanced secretion both at intermediate (nano- to micromolar) and vanishingly low (less than 10 pM) Ca2+ concentrations. At optimal Ca2+ (10 microM) the effect of GTP gamma S was greatly reduced. We investigated whether the secretory response to GTP analogues was mediated by any of three enzyme systems regulated by GTP-binding proteins, i.e. generation of cyclic AMP by adenylate cyclase, of diacylglycerol by phospholipase C and of arachidonic acid by phospholipase A2. The involvement of these messenger systems could be excluded as (i) cyclic AMP only had minor, Ca2+ dependent effects, (ii) phospholipase C was not activated in the absence of Ca2+ and insulin secretion due to the phorbol ester TPA displayed a different Ca2+ dependency, (iii) arachidonic acid did not elicit Ca2+ independent insulin secretion. These results, taken together with the finding that insulin secretion due to Ca2+ or TPA is attenuated by the inhibitory guanine nucleotide GDP beta S, suggest the existence of a regulatory site in exocytosis which is sensitive to guanine nucleotides.
对暴露于高压放电的RINm5F细胞胰岛素分泌的调节进行了研究。电子显微镜显示，通透处理后细胞的整体结构得以保留。在此制备物中，胰岛素释放受到Ca2+刺激（EC50 = 2.4微摩尔）。稳定的GTP类似物GTPγS在中等（纳摩尔至微摩尔）和极低（小于10皮摩尔）Ca2+浓度下均增强分泌。在最佳Ca2+（10微摩尔）时，GTPγS的作用大大降低。我们研究了对GTP类似物的分泌反应是否由受GTP结合蛋白调节的三种酶系统中的任何一种介导，即腺苷酸环化酶产生环磷酸腺苷、磷脂酶C产生二酰基甘油以及磷脂酶A2产生花生四烯酸。这些信使系统的参与可以被排除，因为（i）环磷酸腺苷仅具有微小的、依赖Ca2+的作用，（ii）在没有Ca2+的情况下磷脂酶C未被激活，并且由于佛波酯TPA引起的胰岛素分泌表现出不同的Ca2+依赖性，（iii）花生四烯酸不会引发不依赖Ca2+的胰岛素分泌。这些结果，连同Ca2+或TPA引起的胰岛素分泌被抑制性鸟嘌呤核苷酸GDPβS减弱这一发现，表明在胞吐作用中存在一个对鸟嘌呤核苷酸敏感的调节位点。
