Lu S C, Kuhlenkamp J, Garcia-Ruiz C, Kaplowitz N
Department of Medicine, University of Southern California School of Medicine, Los Angeles 90033.
J Clin Invest. 1991 Jul;88(1):260-9. doi: 10.1172/JCI115286.
Our present work characterized the role of hormone-mediated signal transduction pathways in regulating hepatic reduced glutathione (GSH) synthesis. Cholera toxin, dibutyryl cAMP (DBcAMP), and glucagon inhibited GSH synthesis in cultured hepatocytes by 25-43%. Cellular cAMP levels exhibited a lower threshold for stimulation of the GSH efflux than inhibition of its synthesis. The effect of DBcAMP was independent of the type of sulfur amino acid precursor and cellular ATP levels and unassociated with increased GSH mixed disulfide formation or altered GSH/oxidized glutathione ratio. In liver cytosols, addition of DBcAMP and cAMP-dependent protein kinase (A-kinase) inhibited GSH synthesis from substrates (cysteine, ATP, glutamate, and glycine) by approximately 20% which was prevented by the A-kinase inhibitor. However, if only substrates of the second step in GSH synthesis were used (gamma-glutamylcysteine, glycine, and ATP), DBcAMP and A-kinase exerted no inhibitory effect. Phenylephrine, vasopressin, and phorbol ester also inhibited GSH synthesis in cultured cells by approximately 20%, and depleted cell GSH independent of the type of sulfur amino acid precursor. Cellular cysteine level was unchanged despite the significant fall in GSH after glucagon or phenylephrine treatment. Pretreatment with either staurosporine, C-kinase inhibitor, or calmidazolium, a calmodulin inhibitor, partially prevented but, together, completely prevented the inhibitory effect of phenylephrine. The same combination had no effect on the inhibitory effect of glucagon. The effects of hormones were confirmed in both the intact perfused liver and after in vivo administration. Thus, two classes of hormones acting through distinct signal transduction pathways may down-regulate hepatic GSH synthesis by phosphorylation of gamma-glutamylcysteine synthetase.
我们目前的工作确定了激素介导的信号转导途径在调节肝脏还原型谷胱甘肽（GSH）合成中的作用。霍乱毒素、二丁酰环磷腺苷（DBcAMP）和胰高血糖素可使培养的肝细胞中GSH合成减少25%-43%。细胞内环磷腺苷水平对GSH外流的刺激阈值低于对其合成的抑制阈值。DBcAMP的作用与硫氨基酸前体类型和细胞三磷酸腺苷水平无关，且与GSH混合二硫化物形成增加或GSH/氧化型谷胱甘肽比值改变无关。在肝脏胞质溶胶中，添加DBcAMP和环磷腺苷依赖性蛋白激酶（A激酶）可使底物（半胱氨酸、三磷酸腺苷、谷氨酸和甘氨酸）合成GSH的量减少约20%，而A激酶抑制剂可阻止这种减少。然而，如果仅使用GSH合成第二步的底物（γ-谷氨酰半胱氨酸、甘氨酸和三磷酸腺苷），DBcAMP和A激酶则无抑制作用。去氧肾上腺素、血管加压素和佛波酯也可使培养细胞中的GSH合成减少约20%，并使细胞内GSH耗竭，且与硫氨基酸前体类型无关。尽管胰高血糖素或去氧肾上腺素处理后GSH显著下降，但细胞内半胱氨酸水平未改变。用星形孢菌素（一种C激酶抑制剂）或钙调蛋白抑制剂卡咪唑预处理可部分阻止但联合使用则完全阻止去氧肾上腺素的抑制作用。相同的联合处理对胰高血糖素的抑制作用无影响。在完整的灌注肝脏和体内给药后均证实了激素的作用。因此，两类通过不同信号转导途径起作用的激素可能通过γ-谷氨酰半胱氨酸合成酶的磷酸化来下调肝脏GSH的合成。
