Department of Medicine, Renal Electrolyte Division, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15263, USA.
Am J Physiol Renal Physiol. 2010 May;298(5):F1162-9. doi: 10.1152/ajprenal.00645.2009. Epub 2010 Feb 10.
The vacuolar H(+)-ATPase (V-ATPase) in type A kidney intercalated cells is a major contributor to acid excretion in the collecting duct. The mechanisms of V-ATPase-trafficking regulation in kidney intercalated cells have not been well-characterized. In developmentally related epididymal clear cells, we showed previously that PKA, acting downstream of soluble adenylyl cyclase (sAC), induces V-ATPase apical membrane accumulation. These PKA-mediated effects were inhibited by activators of the metabolic sensor AMP-activated kinase (AMPK) in clear cells. Here, we examined the regulation of V-ATPase subcellular localization in intercalated cells by PKA and AMPK in rat kidney tissue slices ex vivo. Immunofluorescence labeling of kidney slices revealed that the PKA activator N(6)-monobutyryl cAMP (6-MB-cAMP) induced V-ATPase apical membrane accumulation in collecting duct intercalated cells, whereas the V-ATPase had a more cytosolic distribution when incubated in Ringer buffer alone for 30 min. V-ATPase accumulated at the apical membrane in intercalated cells in kidney slices incubated in Ringer buffer for 75 min, an effect that was prevented by treatment with PKA inhibitor (mPKI). The V-ATPase distribution was cytosolic in intercalated cells treated with the carbonic anhydrase inhibitor acetazolamide or the sAC inhibitor KH7, effects that were overridden by 6-MB-cAMP. Preincubation of kidney slices with an AMPK activator blocked V-ATPase apical membrane accumulation induced by 6-MB-cAMP, suggesting that AMPK antagonizes cAMP/PKA effects on V-ATPase distribution. Taken together, our results suggest that in intercalated cells V-ATPase subcellular localization and therefore its activity may be coupled to acid-base status via PKA, and metabolic status via AMPK.
空泡质子泵 (V-ATPase) 在 A 型肾闰细胞中是集合管酸排泄的主要贡献者。V-ATPase 在肾闰细胞中的运输调节机制尚未得到很好的描述。在发育相关的附睾透明细胞中，我们之前表明，PKA 作为可溶性腺苷酸环化酶 (sAC) 的下游，诱导 V-ATPase 顶端膜积累。在透明细胞中，代谢传感器 AMP 激活的蛋白激酶 (AMPK) 的激活剂抑制了这些 PKA 介导的效应。在这里，我们在离体大鼠肾组织切片中研究了 PKA 和 AMPK 对闰细胞中 V-ATPase 亚细胞定位的调节。肾切片的免疫荧光标记显示，PKA 激活剂 N(6)-单丁酰环腺苷酸 (6-MB-cAMP) 诱导集合管闰细胞的 V-ATPase 顶端膜积累，而当在 Ringer 缓冲液中孵育 30 分钟时，V-ATPase 则分布在细胞质中。在 Ringer 缓冲液中孵育 75 分钟的肾切片中，V-ATPase 聚集在闰细胞的顶端膜上，而用 PKA 抑制剂 (mPKI) 处理则阻止了这一作用。在闰细胞中用碳酸酐酶抑制剂乙酰唑胺或 sAC 抑制剂 KH7 处理，V-ATPase 分布在细胞质中，而这一作用被 6-MB-cAMP 所逆转。在 AMPK 激活剂预处理肾切片后，阻断了 6-MB-cAMP 诱导的 V-ATPase 顶端膜积累，这表明 AMPK 拮抗 cAMP/PKA 对 V-ATPase 分布的作用。总之，我们的结果表明，在闰细胞中，V-ATPase 的亚细胞定位及其活性可能通过 PKA 与酸碱状态相关，通过 AMPK 与代谢状态相关。
