Fernando K C, Gregory R B, Barritt G J
Department of Medical Biochemistry, School of Medicine, Faculty of Health Sciences, Flinders University, G.P.O. Box 2100, Adelaide, South Australia, 5001, Australia.
Biochem J. 1998 Mar 15;330 ( Pt 3)(Pt 3):1179-87. doi: 10.1042/bj3301179.
The roles of a trimeric GTP-binding regulatory protein, protein kinase A and mitochondria in the regulation of store-activated (thapsigargin-stimulated) Ca2+ inflow in freshly-isolated rat hepatocytes were investigated. Rates of Ca2+ inflow were estimated by measuring the increase in the fluorescence of intracellular fura-2 following the addition of extracellular Ca2+ (Ca2+o) to cells incubated in the absence of added Ca2+o. Guanosine 5'-[gamma-thio]-triphosphate (GTP[S]) and AlF4(-) inhibited the thapsigargin-stimulated Ca2+o-induced increase in cytoplasmic free Ca2+ concentration ([Ca2+]c) and this inhibition was prevented by the Rp diastereoisomer of adenosine 3',5'-(cyclic)phosphoro[thioate]. cAMP, forskolin and glucagon (half-maximal effect at 10 nM) mimicked inhibition of the thapsigargin-stimulated Ca2+o-induced increase in [Ca2+]c by GTP[S], but had little effect on thapsigargin-induced release of Ca2+ from intracellular stores. Azide and carbonyl cyanide p-trifluoromethoxyphenylhydrazone inhibited the thapsigargin-stimulated Ca2+o-induced increase in [Ca2+]c in the presence of increased cAMP (induced by glucagon). In contrast, Ruthenium Red markedly enhanced the thapsigargin-stimulated Ca2+o-induced increase in [Ca2+]c in both the presence and absence of increased cAMP (induced by forskolin and dibutyryl cAMP). It is concluded that, in hepatocytes, protein kinase A regulates the disposition of Ca2+, which enters the cytoplasmic space through store-activated Ca2+ channels, by directing some of this Ca2+ to the mitochondria. The idea that caution should be exercised in using observed values of Ca2+o-induced increase in [Ca2+]c as estimates of rates of agonist-stimulated Ca2+ inflow is briefly discussed.
研究了三聚体GTP结合调节蛋白、蛋白激酶A和线粒体在新鲜分离的大鼠肝细胞中对储存激活（毒胡萝卜素刺激）Ca2+内流的调节作用。通过测量在无细胞外Ca2+（Ca2+o）条件下孵育的细胞中加入细胞外Ca2+（Ca2+o）后细胞内fura-2荧光的增加来估计Ca2+内流速率。鸟苷5'-[γ-硫代]-三磷酸（GTP[S]）和AlF4（-）抑制毒胡萝卜素刺激的Ca2+o诱导的细胞质游离Ca2+浓度（[Ca2+]c）升高，腺苷3',5'-（环）磷[硫代]酸的Rp非对映异构体可阻止这种抑制作用。cAMP、福斯可林和胰高血糖素（10 nM时产生半数最大效应）模拟了GTP[S]对毒胡萝卜素刺激的Ca2+o诱导的[Ca2+]c升高的抑制作用，但对毒胡萝卜素诱导的细胞内储存Ca2+释放影响很小。叠氮化物和羰基氰对三氟甲氧基苯腙在cAMP升高（由胰高血糖素诱导）的情况下抑制毒胡萝卜素刺激的Ca2+o诱导的[Ca2+]c升高。相反，钌红在存在和不存在cAMP升高（由福斯可林和二丁酰cAMP诱导）的情况下均显著增强毒胡萝卜素刺激的Ca2+o诱导的[Ca2+]c升高。得出的结论是，在肝细胞中，蛋白激酶A通过将部分Ca2+导向线粒体来调节通过储存激活的Ca2+通道进入细胞质空间的Ca2+的分布。简要讨论了在使用观察到的Ca2+o诱导的[Ca2+]c升高值作为激动剂刺激的Ca2+内流速率估计值时应谨慎的观点。
