Garrison J C, Borland M K, Florio V A, Twible D A
J Biol Chem. 1979 Aug 10;254(15):7147-56.
Angiotensin II, catecholamines, and vasopressin are thought to stimulate hepatic glycogenolysis and gluconeogenesis via a cyclic AMP-independent mechanism that requires calcium ion. The present study explores the possibility that angiotensin II and vasopressin control the activity of regulatory enzymes in carbohydrate metabolism through Ca2+-dependent changes in their state of phosphorylation. Intact hepatocytes labeled with [32P]PO43- were stimulated with angiotensin II, glucagon, or vasopressin and 30 to 33 phosphorylated proteins resolved from the cytoplasmic fraction of the cell by electrophoresis in sodium dodecyl sulfate polyacrylamide slab gels. Treatment of the cells with angiotensin II or vasopressin increased the phosphorylation of 10 to 12 of these cytosolic proteins without causing measurable changes in cyclic AMP-dependent protein kinase activity. Glucagon stimulated the phosphorylation of the same set of 11 to 12 proteins through a marked increase in cyclic AMP-dependent protein kinase activity. The molecular weights of three of the protein bands whose phosphorylation was increased by these hormones correspond to the subunit molecular weights of phosphorylase (Mr = 93,000), glycogen synthase (Mr = 85,000), and pyruvate kinase (Mr = 61,000). Two of these phosphoprotein bands were positively identified as phosphorylase and pyruvate kinase by affinity chromatography and immunoprecipitation, respectively. Incubation of hepatocytes in a Ca2+-free medium completely abolished the effects of angiotensin II and vasopressin on protein phosphorylation but did not alter those of glucagon. Treatment of hepatocytes with angiotensin II, glucagon, or vasopressin stimulated phosphorylase activity by 250 to 260%, inhibited glycogen synthase activity by 50%, and inhibited pyruvate kinase activity by 30 to 35% (peptides) to 70% (glucagon). The effects of angiotensin II and vasopressin on the activity of all three enzymes were completely abolished if the cells were incubated in a Ca2+-free medium while those of glucagon were not altered. The results imply that angiotensin II, catecholamines, and vasopressin control hepatic carbohydrate metabolism through a Ca2+-requiring, cyclic AMP-independent pathway that leads to the phosphorylation of important regulatory enzymes.
血管紧张素II、儿茶酚胺和血管加压素被认为可通过一种不依赖环磷酸腺苷（cAMP）且需要钙离子的机制来刺激肝糖原分解和糖异生。本研究探讨了血管紧张素II和血管加压素是否通过其磷酸化状态的钙离子依赖性变化来控制碳水化合物代谢中调节酶活性的可能性。用[32P]PO43 -标记的完整肝细胞分别用血管紧张素II、胰高血糖素或血管加压素刺激，然后通过十二烷基硫酸钠聚丙烯酰胺平板凝胶电泳从细胞的细胞质部分分离出30至33种磷酸化蛋白。用血管紧张素II或血管加压素处理细胞可使其中10至12种胞质蛋白的磷酸化增加，而不会引起环磷酸腺苷依赖性蛋白激酶活性的可测量变化。胰高血糖素通过显著增加环磷酸腺苷依赖性蛋白激酶活性来刺激这同一组11至12种蛋白的磷酸化。这些激素使其磷酸化增加的三条蛋白带的分子量分别与磷酸化酶（Mr = 93,000）、糖原合酶（Mr = 85,000）和丙酮酸激酶（Mr = 61,000）的亚基分子量相对应。通过亲和层析和免疫沉淀分别确定其中两条磷蛋白带分别为磷酸化酶和丙酮酸激酶。在无钙培养基中孵育肝细胞完全消除了血管紧张素II和血管加压素对蛋白磷酸化的影响，但未改变胰高血糖素的作用。用血管紧张素II、胰高血糖素或血管加压素处理肝细胞可使磷酸化酶活性提高250%至260%，使糖原合酶活性抑制50%，使丙酮酸激酶活性抑制30%至35%（肽类）至70%（胰高血糖素）。如果在无钙培养基中孵育细胞，血管紧张素II和血管加压素对所有三种酶活性的影响会完全消除，而胰高血糖素的影响则不受改变。结果表明，血管紧张素II、儿茶酚胺和血管加压素通过一条需要钙离子、不依赖环磷酸腺苷的途径来控制肝脏碳水化合物代谢，该途径导致重要调节酶的磷酸化。
