Theparambil Shefeeq M, Naoshin Zinnia, Thyssen Anne, Deitmer Joachim W
Department of General Zoology, Faculty of Biology, University of Kaiserslautern, Kaiserslautern, Germany.
J Physiol. 2015 Aug 15;593(16):3533-47. doi: 10.1113/JP270086. Epub 2015 Jun 25.
The regulation of H(+) i from cytosolic alkalosis has generally been attributed to the activity of Cl(-) -coupled acid loaders/base extruders in most cell types, including brain cells. The present study demonstrates that outwardly-directed sodium bicarbonate cotransport via electrogenic sodium bicarbonate cotransporter 1 (NBCe1) mediates the major fraction of H(+) i regulation from cytosolic alkalosis in mouse cortical astrocytes. Cl(-) -coupled acid-loading transporters play only a minor role in the regulation of H(+) i from alkalosis in mouse cortical astrocytes. NBCe1-mediated H(+) i regulation from alkalosis was dominant, with the support of intracellular carbonic anhydrase II, even when the intra- and extracellular [HCO3 (-) ] was very low (<1mM), as in nominally CO2 /HCO3 (-) free condition. A reversed NBCe1 in astrocytes may also be significant for stabilizing extracellular pH in brain tissue.
Recovery of intracellular pH from cytosolic alkalosis has been attributed primarily to Cl(-) coupled acid loaders/base extruders such as Cl(-) /HCO3 (-) or Cl(-) /OH(-) exchangers. We have studied this process in cortical astrocytes from wild-type and transgenic mouse models with gene deletion for the electrogenic sodium bicarbonate cotransporter 1 (NBCe1) and for carbonic anhydrase (CA) isoform II. An acute cytosolic alkalosis was induced by the removal of either CO2 /HCO3 (-) or butyric acid, and the subsequent acid loading was analysed by monitoring changes in cytosolic H(+) or Na(+) using ion-sensitive fluorescent dyes. We have identified that NBCe1 reverses during alkalosis and contributes more than 70% to the rate of recovery from alkalosis by extruding Na(+) and HCO3 (-) . After CA inhibition or in CAII-knockout (KO) cells, the rate of recovery was reduced by 40%, and even by 70% in the nominal absence of CO2 /HCO3 (-) . Increasing the extracellular K(+) concentration modulated the rate of acid loading in wild-type cells, but not in NBCe1-KO cells. Removing chloride had only a minor effect on the recovery from alkalosis. Reversal of NBCe1 by reducing pH/[HCO3 (-) ] was demonstrated in astrocytes and in Xenopus oocytes, in which human NBCe1 was heterologously expressed. The results obtained suggest that reversed NBCe1, supported by CAII activity, plays a major role in acid-loading cortical astrocytes to support recovery from cytosolic alkalosis.
在包括脑细胞在内的大多数细胞类型中，胞质碱中毒时H⁺i的调节通常归因于Cl⁻偶联的酸加载器/碱排出器的活性。本研究表明，通过电中性碳酸氢钠共转运体1（NBCe1）向外的碳酸氢钠共转运介导了小鼠皮质星形胶质细胞中胞质碱中毒时H⁺i调节的主要部分。Cl⁻偶联的酸加载转运体在小鼠皮质星形胶质细胞碱中毒时H⁺i的调节中仅起次要作用。即使在细胞内和细胞外[HCO₃⁻]非常低（<1mM）的情况下，如在名义上无CO₂/HCO₃⁻的条件下，在细胞内碳酸酐酶II的支持下，NBCe1介导的碱中毒时H⁺i调节也是占主导的。星形胶质细胞中反向的NBCe1对于稳定脑组织细胞外pH可能也很重要。
胞质碱中毒后细胞内pH的恢复主要归因于Cl⁻偶联的酸加载器/碱排出器，如Cl⁻/HCO₃⁻或Cl⁻/OH⁻交换体。我们在野生型和转基因小鼠模型的皮质星形胶质细胞中研究了这一过程，这些模型分别缺失了电中性碳酸氢钠共转运体1（NBCe1）和碳酸酐酶（CA）同工型II。通过去除CO₂/HCO₃⁻或丁酸诱导急性胞质碱中毒，并使用离子敏感荧光染料监测胞质H⁺或Na⁺的变化来分析随后的酸加载情况。我们发现，碱中毒时NBCe1会反向，通过排出Na⁺和HCO₃⁻，其对碱中毒恢复速率的贡献超过70%。在CA抑制后或在CAII基因敲除（KO）细胞中，恢复速率降低了40%，在名义上无CO₂/HCO₃⁻的情况下甚至降低了70%。增加细胞外K⁺浓度可调节野生型细胞中的酸加载速率，但对NBCe1-KO细胞无此作用。去除氯离子对碱中毒的恢复只有轻微影响。在星形胶质细胞和异源表达人NBCe1的非洲爪蟾卵母细胞中，通过降低pH/[HCO₃⁻]证明了NBCe1的反向。所得结果表明，在CAII活性的支持下，反向的NBCe1在酸加载皮质星形胶质细胞以支持胞质碱中毒恢复中起主要作用。
