Jans D A, Resink T J, Hemmings B A
Friedrich Miescher-Institut, Basel, Switzerland.
Arch Biochem Biophys. 1991 Mar;285(2):377-81. doi: 10.1016/0003-9861(91)90376-t.
A novel "cAMP-resistant" variant of LLC-PK1 renal epithelial cells which is impaired in in vivo down-regulation of response following hormonal stimulation of adenylate cyclase (AC) is described. Compared to parental cells, the BIB27 mutant exhibited markedly higher in vivo activation of cAMP-dependent protein kinase (cAMP-PK) in response to the hormones salmon calcitonin (SCT) or [Arg8]-vasopressin (AVP) or the AC activator forskolin. The activation of cAMP-PK subsequent to agonist stimulation also persisted much longer in the mutant than in LLC-PK1 cells, although the cAMP-PK of BIB27 cells was normal in terms of both absolute levels and regulation by cAMP in vitro. Intracellular cAMP accumulation was also much higher in BIB27 than in LLC-PK1 cells following agonist stimulation. Production of cAMP could be detected in BIB27 cells even 12 h after treatment with AVP or SCT, whereas cAMP production in LLC-PK1 had returned to basal within 1 and 8 h, respectively. High levels of free cAMP-PK catalytic (C) subunit in BIB27 persisted even 12 h after hormone addition, meaning that the higher cAMP production in BIB27 did not result in the normal down-regulation of cAMP-PK C subunit levels. In vitro AC activity in BIB27 cell homogenates could be stimulated by hormones or receptor-independent agonists, but to a lesser extent than in LLC-PK1 cell homogenates. The SCT and AVP concentrations promoting half-maximal AC activation in BIB27 cells were about 10- and 3-fold higher than parental, respectively. BIB27 accordingly appeared to possess a mutation in AC responsible for the impairment of both in vitro response to agonists and the normal in vivo down-regulation processes following hormonal stimulation.
本文描述了一种新型的LLC-PK1肾上皮细胞“抗cAMP”变体，该变体在激素刺激腺苷酸环化酶（AC）后体内反应的下调过程中受损。与亲代细胞相比，BIB27突变体对鲑鱼降钙素（SCT）、[Arg8]-血管加压素（AVP）或AC激活剂福斯高林等激素的反应，在体内表现出明显更高的cAMP依赖性蛋白激酶（cAMP-PK）激活。尽管BIB27细胞的cAMP-PK在体外的绝对水平和cAMP调节方面均正常，但在激动剂刺激后，BIB27突变体中cAMP-PK的激活持续时间也比LLC-PK1细胞长得多。激动剂刺激后，BIB27细胞内的cAMP积累也比LLC-PK1细胞高得多。在用AVP或SCT处理12小时后，仍可在BIB27细胞中检测到cAMP的产生，而LLC-PK1细胞中的cAMP产生分别在1小时和8小时内恢复到基础水平。即使在添加激素12小时后，BIB27中高水平的游离cAMP-PK催化（C）亚基仍然存在，这意味着BIB27中较高的cAMP产生并未导致cAMP-PK C亚基水平的正常下调。BIB27细胞匀浆中的体外AC活性可被激素或不依赖受体的激动剂刺激，但程度低于LLC-PK1细胞匀浆。在BIB27细胞中促进AC激活达到半数最大效应的SCT和AVP浓度分别比亲代细胞高约10倍和3倍。因此，BIB27似乎在AC中存在一个突变，该突变导致其对激动剂的体外反应受损以及激素刺激后体内正常下调过程受损。
