Rubin R, Thomas A P, Hoek J B
Arch Biochem Biophys. 1987 Jul;256(1):29-38. doi: 10.1016/0003-9861(87)90422-x.
Guanine nucleotides are thought to mediate the interaction of the receptors for calcium-mobilizing hormones and phosphoinositide-specific phospholipase C. In the present study the characteristics of guanine nucleotide-dependent phospholipase C activation were studied in [3H]inositol-labeled permeabilized hepatocytes. The nonhydrolyzable GTP analogs guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) and guanyl-5'-yl imidodiphosphate stimulated the production of inositol phosphates by phospholipase C. The effect was concentration-dependent with half-maximal and maximal stimulation occurring with 0.6 and 10 microM GTP gamma S, respectively. The guanine nucleotide-induced stimulation of phosphoinositide breakdown was selective for phosphatidylinositol (4,5)-bisphosphate over phosphatidylinositol (4)-phosphate. The individual inositol phosphates formed after maximal GTP gamma S exposure were analyzed by high-performance liquid chromatography. Inositol 1,4,5-trisphosphate was rapidly produced, followed by the formation of inositol 1,3,4,5-tetrakisphosphate and inositol 1,3,4-trisphosphate. Ethanol is known to activate hormone-sensitive phospholipase C in intact rat hepatocytes. Ethanol (0.3 M) was ineffective in altering the characteristics of GTP gamma S-stimulated phospholipase C activation, in both digitonin-treated and sonicated hepatocytes. The metabolism of the various inositol phosphate isomers was unaffected by ethanol. The findings demonstrate the potential for the use of permeabilized hepatocytes in the analysis of phospholipase C activation by guanine nucleotides. Ethanol does not activate phospholipase C by altering this process.
鸟嘌呤核苷酸被认为可介导钙动员激素受体与磷酸肌醇特异性磷脂酶C之间的相互作用。在本研究中，我们在[3H]肌醇标记的通透化肝细胞中研究了鸟嘌呤核苷酸依赖性磷脂酶C激活的特性。不可水解的GTP类似物鸟苷5'-O-(3-硫代三磷酸)(GTPγS)和鸟苷-5'-基亚氨基二磷酸刺激磷脂酶C产生肌醇磷酸。该效应呈浓度依赖性，半最大刺激和最大刺激分别在0.6和10μM GTPγS时出现。鸟嘌呤核苷酸诱导的磷酸肌醇分解刺激对磷脂酰肌醇(4,5)-二磷酸比对磷脂酰肌醇(4)-磷酸具有选择性。在最大程度暴露GTPγS后形成的各个肌醇磷酸通过高效液相色谱进行分析。肌醇1,4,5-三磷酸迅速产生，随后是肌醇1,3,4,5-四磷酸和肌醇1,3,4-三磷酸的形成。已知乙醇可激活完整大鼠肝细胞中的激素敏感性磷脂酶C。在洋地黄皂苷处理和超声处理的肝细胞中，乙醇(0.3 M)均无法改变GTPγS刺激的磷脂酶C激活特性。乙醇不影响各种肌醇磷酸异构体的代谢。这些发现证明了通透化肝细胞在分析鸟嘌呤核苷酸激活磷脂酶C方面的应用潜力。乙醇不会通过改变这一过程来激活磷脂酶C。
