Department of Physiology, University of Tübingen, Gmelinstrasse 5, D72076 Tübingen, Germany.
Biochem Biophys Res Commun. 2010 Aug 6;398(4):677-82. doi: 10.1016/j.bbrc.2010.06.135. Epub 2010 Jul 4.
AMP-activated protein kinase (AMPK) is activated upon energy depletion and serves to restore energy balance by stimulating energy production and limiting energy utilization. Specifically, it enhances cellular glucose uptake by stimulating GLUT and SGLT1 and glucose utilization by stimulating glycolysis. During O(2) deficiency glycolytic degradation of glucose leads to formation of lactate and H(+), thus imposing an acid load to the energy-deficient cell. Cellular acidification inhibits glycolysis and thus impedes glucose utilization. Maintenance of glycolysis thus requires cellular H(+) export. The present study explored whether AMPK influences Na(+)/H(+) exchanger (NHE) activity and/or Na(+)-independent acid extrusion. NHE1 expression was determined by RT-PCR and Western blotting. Cytosolic pH (pH(i)) was estimated utilizing BCECF fluorescence and Na(+)/H(+) exchanger activity from the Na(+)-dependent re-alkalinization (DeltapH(i)) after an ammonium pulse. As a result, human embryonic kidney (HEK) cells express NHE1. The pH(i) and DeltapH(i) in those cells were significantly increased by treatment with AMPK stimulator AICAR (1mM) and significantly decreased by AMPK inhibitor compound C (10 microM). The effect of AICAR on pH(i) and DeltapH(i) was blunted in the presence of the Na(+)/H(+) exchanger inhibitor cariporide (10microM), but not by the H(+) ATPase inhibitor bafilomycin (10nM). AICAR significantly enhanced lactate formation, an effect significantly blunted in the presence of cariporide. These observations disclose a novel function of AMPK, i.e. regulation of cytosolic pH.
AMP 激活的蛋白激酶（AMPK）在能量耗竭时被激活，通过刺激能量产生和限制能量利用来恢复能量平衡。具体而言，它通过刺激 GLUT 和 SGLT1 增强细胞葡萄糖摄取，并通过刺激糖酵解增强葡萄糖利用。在缺氧时，葡萄糖的糖酵解降解导致乳酸和 H+的形成，从而给能量不足的细胞带来酸负荷。细胞酸化抑制糖酵解，从而阻碍葡萄糖利用。因此，维持糖酵解需要细胞 H+外排。本研究探讨了 AMPK 是否影响 Na+/H+交换器（NHE）的活性和/或 Na+非依赖性酸外排。通过 RT-PCR 和 Western blot 测定 NHE1 的表达。利用 BCECF 荧光测定细胞质 pH（pH(i)），并通过铵脉冲后 Na+依赖性再碱化（DeltapH(i)）测定 Na+/H+交换器活性。结果表明，人胚肾（HEK）细胞表达 NHE1。用 AMPK 激动剂 AICAR（1mM）处理可显著增加 pH(i)和 DeltapH(i)，而用 AMPK 抑制剂化合物 C（10μM）处理则显著降低 pH(i)和 DeltapH(i)。在 Na+/H+交换器抑制剂 cariporide（10μM）存在的情况下，AICAR 对 pH(i)和 DeltapH(i)的作用减弱，但 H+ATP 酶抑制剂 bafilomycin（10nM）则无此作用。AICAR 显著增强乳酸形成，在 cariporide 存在的情况下，该作用显著减弱。这些观察结果揭示了 AMPK 的一个新功能，即调节细胞质 pH。
