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首页> 外文期刊>American Journal of Physiology >Alkaline pH- and cAMP-induced V-ATPase membrane accumulation is mediated by protein kinase A in epididymal clear cells
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Alkaline pH- and cAMP-induced V-ATPase membrane accumulation is mediated by protein kinase A in epididymal clear cells

机译：附睾透明细胞中蛋白激酶A介导碱性pH和cAMP诱导的V-ATPase膜积累

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First published December 26, 2007; doi:10.1152/ajpcell.00537.2007.-In the epididymis, low luminal bicarbonate and acidic pH maintain sperm quiescent during maturation and storage. The vacuolar H+-ATPase (V-ATPase) in epididymal clear cells plays a major role in luminal acidification. We have shown previously that cAMP, luminal alkaline pH, and activation of the bicarbonate-regulated soluble adenylyl cyclase (sAC) induce V-ATPase apical accumulation in these cells, thereby stimulating proton secretion into the epididymal lumen. Here we examined whether protein kinase A (PKA) is involved in this response. Confocal immunofluorescence labeling on rat epididymis perfused in vivo showed that at luminal acidic pH (6.5), V-ATPase was distributed between short apical microvilli and subapical endo-somes. The specific PKA activator A-monobutyryl-S'-S'-cyclic monophosphate (6-MB-cAMP, 1 mM) induced elongation of apical microvilli and accumulation of V-ATPase in these structures. The PKA inhibitor myristoylated-PKI(mPKI, 10 pM) inhibited the apical accumulation of V-ATPase induced by 6-MB-cAMP. Perfusion at pH 6.5 with 8-(4-chIorophenylthio)-2-0-methyl-cAMP (8CPT-2-O-Me-cAMP; 10 mu), an activator of the exchange protein activated by cAMP (Epac), did not induce V-ATPase apical accumulation. When applied at a higher concentration (100 pM), 8CPT-2-O-Me-cAMP induced V-ATPase apical accumulation, but this effect was completely inhibited by mPKI, suggesting crossover effects on the PKA pathway with this compound at high concentrations. Importantly, the physiologically relevant alkaline pH-induced apical V-ATPase accumulation was completely inhibited by pretreatment with raPKI. We conclude that direct stimulation of PKA activity by cAMP is necessary and sufficient for the alkaline pH-induced accumulation of V-ATPase in clear cell apical microvilli.
机译：首次发布于2007年12月26日; doi：10.1152 / ajpcell.00537.2007.-在附睾中，低碳酸氢根和酸性pH可使精子在成熟和储存过程中保持静止。附睾透明细胞中的液泡H + -ATPase（V-ATPase）在管腔酸化中起主要作用。先前我们已经表明，cAMP，管腔碱性pH值和碳酸氢盐调节的可溶性腺苷酸环化酶（sAC）的激活在这些细胞中诱导V-ATPase顶端积累，从而刺激质子分泌到附睾腔中。在这里，我们检查了蛋白激酶A（PKA）是否参与此反应。体内灌注大鼠附睾的共聚焦免疫荧光标记显示，在管腔酸性pH（6.5）下，V-ATPase分布在短根尖微绒毛和根尖内体之间。特定的PKA活化剂A-单丁酰-S'-S'-环一磷酸酯（6-MB-cAMP，1 mM）诱导这些结构中根尖微绒毛伸长和V-ATPase积累。 PKA抑制剂myristoylated-PKI（mPKI，10 pM）抑制了6-MB-cAMP诱导的V-ATPase的顶端积累。在pH 6.5时，未注入由cAMP（Epac）激活的交换蛋白激活剂8-（4-氯苯硫基）-2-0-甲基-cAMP（8CPT-2-O-Me-cAMP; 10 mu）灌注诱导V-ATPase顶端积累。当以较高浓度（100 pM）施用时，8CPT-2-O-Me-cAMP会诱导V-ATPase顶端积累，但这种作用已被mPKI完全抑制，表明该化合物在高浓度下对PKA途径有交叉效应。重要的是，用raPKI预处理可完全抑制生理相关的碱性pH诱导的顶端V-ATPase积累。我们得出结论，通过cAMP直接刺激PKA活性对于在透明细胞根尖微绒毛中碱性pH诱导的V-ATPase积累是必要和充分的。

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