AMP-activated protein kinase inhibits alkaline pH- and PKA-induced apical vacuolar H+-ATPase accumulation in epididymal clear cells
AMP-activated protein kinase inhibits alkaline pH- and PKA-induced apical vacuolar H+-ATPase accumulation in epididymal clear cells
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DOI：
10.1152/ajpcell.00004.2009
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发表时间：
2009-04-01
影响因子：
5.5
通讯作者：
Pastor-Soler, Nuria M.
中科院分区：
文献类型：
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作者：
Hallows, Kenneth R.;Alzamora, Rodrigo;Pastor-Soler, Nuria M.
Hallows KR、Alzamora R、Li H、Gong F、Smolak C、Neumann D、Pastor-Soler NM。 AMP 激活的蛋白激酶抑制碱性 pH 和 PKA 诱导的附睾透明细胞中顶端液泡 H+-ATP 酶的积累。 Am J Physiol Cell Physiol 296：C672-C681，2009。首次发表于 2009 年 2 月 11 日； doi:10.1152/ajpcell.00004.2009.-酸性管腔 pH 值和低 [HCO3-] 使精子在附睾成熟过程中保持静止。透明细胞中的液泡 H+-ATP 酶（V-ATP 酶）是附睾管腔酸化的主要贡献者。我们之前已经证明，蛋白激酶 A (PKA) 在碱性管腔 pH 值或 HCO3- 刺激的可溶性腺苷酸环化酶下游发挥作用，诱导透明细胞中 V-ATP 酶顶膜积聚。在这里，我们检查了代谢传感器 AMP 激活蛋白激酶 (AMPK) 是否调节 PKA 诱导的 V-ATP 酶顶膜积累。大鼠和非人灵长类动物附睾的免疫荧光标记揭示了上皮细胞中的特异性 AMPK 表达。大鼠附睾的免疫荧光标记表明，体内灌注 AMPK 激活剂 5-氨基咪唑-4-甲酰胺-1-β-D-呋喃核苷 (AICAR) 或 A-769662 会诱导 V-ATP 酶重新分布到根尖下囊泡中，即使在管腔碱性 (pH 7.8) 缓冲液存在的情况下，与 对照组不加药物灌注。此外，用 AICAR 预灌注可阻断 N-6-单丁酰-cAMP (6-MB-cAMP) 诱导的 PKA 介导的 V-ATP 酶易位至透明细胞顶膜。纯化的 PKA 和 AMPK 均在体外磷酸化 V-ATPase A 亚基。在 HEK-293 细胞中，A 亚基体内的 [P-32] 正磷酸盐标记在 PKA 刺激后增加，并在 RNA 干扰介导的 AMPK 敲低后减少。最后，A 亚基的 PKA 依赖性体内磷酸化程度随着 AMPK 敲低而增加。总之，我们的研究结果表明，AMPK 抑制附睾透明细胞中 PKA 介导的 V-ATP 酶顶端积累，两种激酶在体外和体内直接磷酸化 V-ATP 酶 A 亚基，并且 AMPK 抑制该亚基的 PKA 依赖性磷酸化。 V-ATP 酶活性可通过 PKA 与酸碱状态感应相结合，并通过 AMPK 与代谢状态相结合。
Hallows KR, Alzamora R, Li H, Gong F, Smolak C, Neumann D, Pastor-Soler NM. AMP-activated protein kinase inhibits alkaline pH- and PKA-induced apical vacuolar H+-ATPase accumulation in epididymal clear cells. Am J Physiol Cell Physiol 296: C672-C681, 2009. First published February 11, 2009; doi:10.1152/ajpcell.00004.2009.-Acidic luminal pH and low [HCO3-] maintain sperm quiescent during maturation in the epididymis. The vacuolar H+-ATPase (V-ATPase) in clear cells is a major contributor to epididymal luminal acidification. We have shown previously that protein kinase A (PKA), acting downstream of soluble adenylyl cyclase stimulation by alkaline luminal pH or HCO3-, induces V-ATPase apical membrane accumulation in clear cells. Here we examined whether the metabolic sensor AMP-activated protein kinase (AMPK) regulates this PKA-induced V-ATPase apical membrane accumulation. Immunofluorescence labeling of rat and non-human primate epididymides revealed specific AMPK expression in epithelial cells. Immunofluorescence labeling of rat epididymis showed that perfusion in vivo with the AMPK activators 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR) or A-769662 induced a redistribution of the V-ATPase into subapical vesicles, even in the presence of a luminal alkaline (pH 7.8) buffer compared with that of controls perfused without drug. Moreover, preperfusion with AICAR blocked the PKA-mediated V-ATPase translocation to clear cell apical membranes induced by N-6-monobutyryl-cAMP (6-MB-cAMP). Purified PKA and AMPK both phosphorylated V-ATPase A subunit in vitro. In HEK-293 cells [P-32] orthophosphate in vivo labeling of the A subunit increased following PKA stimulation and decreased following RNA interference-mediated knockdown of AMPK. Finally, the extent of PKA-dependent in vivo phosphorylation of the A subunit increased with AMPK knockdown. In summary, our findings suggest that AMPK inhibits PKA-mediated V-ATPase apical accumulation in epididymal clear cells, that both kinases directly phosphorylate the V-ATPase A subunit in vitro and in vivo, and that AMPK inhibits PKA-dependent phosphorylation of this subunit. V-ATPase activity may be coupled to the sensing of acid-base status via PKA and to metabolic status via AMPK.