Pizzonia J H, Ransom B R, Pappas C A
Department of Surgery (Neurosurgery), Yale University School of Medicine, New Haven, Connecticut 06510, USA.
J Neurosci Res. 1996 Apr 15;44(2):191-8. doi: 10.1002/(SICI)1097-4547(19960415)44:2<191::AID-JNR12>3.0.CO;2-9.
Astrocytes actively maintain their intracellular pH (pHi) more alkaline than expected by passive distribution of H+. Acid extruding transporters such as the amiloride-sensitive Na+/H+ exchanger (NHE) are necessary for pH regulation. Currently, four mammalian NHEs (NHE1-NHE4) have been cloned, with a fifth (NHE5) partially cloned. We attempted to determine which isoform(s) of NHE was present in cultured hippocampal astrocytes using amiloride sensitivity and immunospecificity as criteria. In the absence of HCO3-, amiloride blocked pHi recovery after an acid load with an IC50 of approximately 3.18 microM, similar to values reported for the amiloride-sensitive isoforms NHE1 and NHE2. Immunoblotting with a highly specific antibody for NHE1 identified a 100 kDa protein, indicating the presence of NHE1 in whole brain, hippocampus, and cultured hippocampal astrocytes. Further probing for an additional amiloride-sensitive NHE failed to detect evidence of the presence of NHE4. Surprisingly, application of the potent analog of amiloride, ethylisopropylamiloride (EIPA), caused a reversible alkalinization of pHi, suggesting the presence of an additional acid/base transport mechanism that is EIPA-sensitive.
星形胶质细胞通过主动调节维持其细胞内pH值（pHi），使其比H⁺被动分布时的预期值更偏碱性。诸如氨氯地平敏感的Na⁺/H⁺交换体（NHE）等酸外排转运体对于pH调节是必需的。目前，已克隆出四种哺乳动物NHE（NHE1 - NHE4），第五种（NHE5）已部分克隆。我们试图以氨氯地平敏感性和免疫特异性为标准，确定培养的海马星形胶质细胞中存在哪种NHE亚型。在没有HCO₃⁻的情况下，氨氯地平在酸负荷后阻断pHi恢复，IC₅₀约为3.18微摩尔，这与报道的氨氯地平敏感亚型NHE1和NHE2的值相似。用针对NHE1的高度特异性抗体进行免疫印迹鉴定出一种100 kDa的蛋白质，表明NHE1存在于全脑、海马和培养的海马星形胶质细胞中。进一步检测另一种氨氯地平敏感的NHE未能检测到NHE4存在的证据。令人惊讶的是，应用氨氯地平的强效类似物乙基异丙基氨氯地平（EIPA）导致pHi可逆性碱化，这表明存在一种对EIPA敏感的额外的酸/碱转运机制。
