Nofziger Charity, Kalsi Kameljit, West T Aaron, Baines Deborah, Blazer-Yost Bonnie L
Department of Biology, Indiana University, Indianapolis, USA.
Cell Physiol Biochem. 2008;22(5-6):487-96. doi: 10.1159/000185505. Epub 2008 Dec 9.
AMP-activated protein kinase (AMPK) is a regulatory kinase coupling cellular metabolism with ion transport. Madin-Darby Canine Kidney-Clone 7 (MDCK-C7) cells possess characteristics of the renal principal cell type, express the cystic fibrosis transmembrane regulator and the epithelial Na(+) channel, and display NPPB and amiloride-sensitive transepithelial transport when stimulated with [Arg(8)]-vasopressin. [Arg(8)]-vasopressin binding to its receptor on the basolateral membrane of MDCK-C7 results in cAMP production, activation of cAMP-dependent protein kinase A (PKA), and increases in Cl(-) and Na(+) transport. Ussing-style electrophysiology showed that the PKA inhibitor, H89, blocked Cl(-) and Na(+) transport. Unexpectedly, [Arg(8)]-vasopressin stimulation resulted in the dephosphorylation of pAMPK(thr172). H89 did not prevent this, suggesting that the dephosphorylation is independent of PKA. 24 hour, but not 15 minute, incubation with the AMPK activator, AICAR, also blocked [Arg(8)]-vasopressin-stimulated currents. Contrary to previous studies, immunoblotting revealed that AICAR did not increase abundance of the active, phosphorylated form of AMPK (pAMPK(thr172)); although, AICAR treatment significantly blocked [Arg(8)]-vasopressin -stimulated cAMP production. [Arg(8)]-vasopressin still caused pAMPK(thr172) dephosphorylation in the presence of AICAR, suggesting that this effect is also independent of cAMP. In summary, these data suggest [Arg(8)]-vasopressin regulates AMPK phosphorylation and that AICAR inhibits ion transport independently of AMPK in MDCK-C7 cells.
AMP激活的蛋白激酶（AMPK）是一种将细胞代谢与离子转运相偶联的调节性激酶。犬肾Madin-Darby克隆7（MDCK-C7）细胞具有肾主细胞类型的特征，表达囊性纤维化跨膜调节因子和上皮钠通道，并在受到[精氨酸（8）] - 血管加压素刺激时表现出对NPPB和阿米洛利敏感的跨上皮转运。[精氨酸（8）] - 血管加压素与其在MDCK-C7细胞基底外侧膜上的受体结合会导致cAMP产生、cAMP依赖性蛋白激酶A（PKA）激活以及Cl（-）和Na（+）转运增加。Ussing式电生理学表明，PKA抑制剂H89可阻断Cl（-）和Na（+）转运。出乎意料的是，[精氨酸（8）] - 血管加压素刺激导致pAMPK（thr172）去磷酸化。H89并不能阻止这种情况，这表明去磷酸化独立于PKA。用AMPK激活剂AICAR孵育24小时而非15分钟，也会阻断[精氨酸（8）] - 血管加压素刺激的电流。与先前的研究相反，免疫印迹显示AICAR不会增加活性磷酸化形式的AMPK（pAMPK（thr172））的丰度；尽管如此，AICAR处理可显著阻断[精氨酸（8）] - 血管加压素刺激的cAMP产生。在存在AICAR的情况下，[精氨酸（8）] - 血管加压素仍会导致pAMPK（thr172）去磷酸化，这表明这种作用也独立于cAMP。总之，这些数据表明[精氨酸（8）] - 血管加压素调节AMPK磷酸化，并且AICAR在MDCK-C7细胞中独立于AMPK抑制离子转运。
