Glucagon stimulation of hepatic Na(+)-pump activity and alpha-subunit phosphorylation in rat hepatocytes.
Glucagon stimulation of hepatic Na(+)-pump activity and alpha-subunit phosphorylation in rat hepatocytes.
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胰高血糖素刺激大鼠肝细胞中的肝钠泵活性和α亚基磷酸化。
DOI：
10.1042/bj3130983
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发表时间：
1996
期刊：
影响因子：
--
通讯作者：
Hazen,SA
中科院分区：
文献类型：
--
作者：
Lynch,CJ;McCall,KM;Ng,YC;Hazen,SA
在这项研究中，研究了Na+流入、花生四烯酸介体和α-亚基磷酸化在肝Na+/K+-ATP酶对胰高血糖素的刺激反应中的可能作用。新鲜分离的大鼠肝细胞中哇巴因敏感的86Rb+摄取的胰高血糖素刺激在激素添加后不到1分钟内达到最大水平，并且在浓度为2.4(±1.3)×10-10M时为最大水平的一半(EC50)。该响应的 K+ 依赖性分析表明，K+ 对表观 Vmax. 有影响，但表观 K0.5 没有显着变化。与莫能菌素不同，胰高血糖素对Na+/K+-ATP酶介导的转运活性的刺激与22Na+流入的增加不相关。这表明胰高血糖素对Na+/K+-ATP酶的刺激并非继发于Na+流入的增加。花生四烯酸介体在这种效应中的作用似乎也不太可能，因为无论是基础的还是胰高血糖素刺激的哇巴因敏感的86Rb+摄取都不受环加氧酶、脂氧合酶、细胞色素P-450或磷脂酶A2抑制剂的超最大浓度的显着影响。为了研究蛋白激酶介导的磷酸化在刺激哇巴因敏感的86Rb+摄取中的可能作用，用[32P]Pi对肝细胞进行代谢放射性标记。胰高血糖素刺激 32P 掺入 95 kDa 磷蛋白中，该磷蛋白在二维凝胶电泳中与 Na+/K+-ATP 酶 α 亚基免疫反应性共迁移。使用抗（大鼠肾 Na+/K+-ATP 酶）血清，可以从去污剂溶解的肝细胞颗粒部分中免疫沉淀 α 亚基。当肝细胞用[32P]Pi进行代谢放射性标记时，免疫沉淀的α亚基含有32P。胰高血糖素使 32 P 掺入免疫沉淀亚基的量增加了 197±21% (n= 6)。针对 α 亚基中的氨基酸序列制备的兔抗肽血清（“抗 LEAVE”血清）也观察到了类似的结果。胰高血糖素刺激的α-亚基磷酸化(1×10-10M)的EC50与哇巴因敏感的86Rb+摄取的胰高血糖素刺激的EC50非常接近。总之，胰高血糖素对肝脏 Na+/K+-ATP 酶介导的转运活性的刺激并不是继发于 Na+ 内流的增加或花生四烯酸介质水平的变化。这些数据支持了以下假设：胰高血糖素对肝细胞中 Na+ 泵活性的刺激可能与蛋白激酶介导的 α 亚基磷酸化状态的变化有关。
In this study the possible role of Na+influx, arachidonate mediators and α-subunit phosphorylation in the stimulatory response of hepatic Na+/K+-ATPase to glucagon was examined. Glucagon stimulation of ouabain-sensitive86Rb+uptake in freshly isolated rat hepatocytes reached maximal levels in less than 1 min after hormone addition and was half-maximal (EC50) at a concentration of 2.4(±1.3)×10-10M. Analysis of the K+-dependence of this response indicates an effect on the apparentVmax.for K+with no significant change in the apparentK0.5. Unlike monensin, glucagon stimulation of Na+/K+-ATPase-mediated transport activity was not associated with an increase in22Na+influx. This indicates that the stimulation of Na+/K+-ATPase by glucagon is not secondary to an increase in Na+influx. A role for arachidonate mediators in this effect also appears unlikely because neither basal nor glucagon-stimulated ouabain-sensitive86Rb+uptake was significantly affected by supramaximal concentrations of cyclo-oxygenase, lipoxygenase, cytochromeP-450 or phospholipase A2inhibitors. To study the possible role of protein kinase-mediated phosphorylation in the stimulation of ouabain-sensitive86Rb+uptake, hepatocytes were metabolically radiolabelled with [32P]Pi. Glucagon stimulated incorporation of32P into a 95 kDa phosphoprotein that co-migrates with Na+/K+-ATPase α-subunit immunoreactivity in two-dimensional gel electrophoresis. The α-subunit could be immunoprecipitated from detergent-solubilized particulate fractions of hepatocytes using an anti-(rat kidney Na+/K+-ATPase) serum. When hepatocytes were metabolically radiolabelled with [32P]Pi, the immunoprecipitated α-subunit contained32P. Glucagon increased the incorporation of32P into the immunoprecipitated subunit by 197±21% (n= 6). Similar results were observed with a rabbit anti-peptide serum (‘anti-LEAVE’ serum) prepared against an amino acid sequence in the α-subunit. The EC50for glucagon-stimulated phosphorylation of the α-subunit (1×10-10M) was very close to that for glucagon stimulation of ouabain-sensitive86Rb+uptake. In conclusion, it appears that glucagon stimulation of hepatic Na+/K+-ATPase-mediated transport activity is not secondary to increases in Na+influx or changes in the levels of an arachidonate mediator. The data provide support for the hypothesis that glucagon stimulation of Na+-pump activity in hepatocytes may be related to protein kinase-mediated changes in the phosphorylation state of the α-subunit.