Intracellular pH regulation in cultured astrocytes from rat hippocampus .1. Role of HCO3-
Intracellular pH regulation in cultured astrocytes from rat hippocampus .1. Role of HCO3-
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DOI：
10.1085/jgp.110.4.453
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发表时间：
1997-10-01
影响因子：
3.8
通讯作者：
Boron, WF
中科院分区：
文献类型：
--
作者：
Bevensee, MO;Weed, RA;Boron, WF
用2‘，7’-双羧乙基-5，6-羧基荧光素(BCECF)对大鼠海马区传代培养的单个星形胶质细胞进行pH(I)监测，研究细胞内pH(I)的调节。在pH(I)为7.0时，本征缓冲力(β(1))为10.5 mm(pH单位)(-1)，并随pH(I)呈线性下降；与数据的最佳拟合线斜率为-10.0 mm(pH单位)(-2)。在没有HCO3-的情况下，pH(I)的恢复主要是由Na-H交换器介导的，因为在pH(I)6.05时，阿米洛利抑制了88%的恢复，乙基异丙基阿米洛利(EIPA)抑制了79%的恢复。酸挤压的乙基异丙基阿米洛利敏感组分与pH(I)呈线性关系。用Li+取代镀液中的Na+，可抑制68%(pH(I)6.23)的出酸。由于Na+驱动的HCO3-转运体，从不含CO2/HCO3-的镀液切换到含CO2/HCO3-的浴液会导致平均稳态pH(I)从6.82增加到6.90。400亩M，4，4‘-二异硫氰基二苯乙烯-2，2’-二磺酸(DIDS)可抑制HCO3引起的pH(I)升高75%(pH(I)6.85)，400亩M，4，4‘-二异硫氰基二苯乙烯-2，2’-二磺酸(SITS)可抑制星形胶质细胞65%(pH(I)6.55~6.75)。用N-甲基-D-葡聚糖胺(NMDG(+))取代外加Na+可阻断CO2/HCO3-诱导的pH(I)升高。在HCO3-存在下，Na+驱动的HCO3-转运蛋白有助于pH(I)从酸负荷中恢复。例如，HCO3-使酸挤出速率与pH(I)的关系图在碱性方向上移动了0.15-0.3个pH单位。此外，在阿米洛利抑制Na-H交换的情况下，HCO3-使酸排出量增加3.8倍(pH(I)6.20)。当星形胶质细胞在以Li+为主要阳离子的阿米洛利中酸负载时，HCO3-不能引起pH(I)的显著升高。因此，在HCO3-转运体上，Li+似乎不能很好地替代NaI。我们得出结论，阿米洛利敏感的Na-H交换器和Na+驱动的HCO3-转运体是星形胶质细胞的主要酸排出器。
We studied the regulation of intracellular pH (pH(i)) in single cultured astrocytes passaged once from the hippocampus of the rat, using the dye 2',7'-biscarboxyethyl-5,6-carboxyfluorescein (BCECF) to monitor pH(i). Intrinsic buffering power (beta(1)) was 10.5 mM (pH unit)(-1) at pH(i) 7.0, and decreased linearly with pH(i); the best-fit line to the data had a slope of -10.0 mM (pH unit)(-2). In the absence of HCO3-, pH(i) recovery from an acid load was mediated predominantly by a Na-H exchanger because the recovery was inhibited 88% by amiloride and 79% by ethylisopropylamiloride (EIPA) at pH(i) 6.05. The ethylisopropylamiloride-sensitive component of acid extrusion fell linearly with pH(i). Acid extrusion was inhibited 68% (pH(i) 6.23) by substituting Li+ for Na+ in the bath solution. Switching from a CO2/HCO3--free to a CO2/HCO3--containing bath solution caused mean steady state pH(i) to increase from 6.82 to 6.90, due to a Na+-driven HCO3-transporter. The HCO3--induced pH(i) increase was unaffected by amiloride, but was inhibited 75% (pH(i) 6.85) by 400 mu M 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), and 65% (pH(i) 6.55-6.75) by pretreating astrocytes for up to similar to 6.3 h with 400 mu M 4-acetamide-4'-isothiocy anatostilbene-2,2'-disulfonic acid (SITS). The CO2/HCO3--induced pH(i) increase was blocked when external Na+ was replaced with N-methyl-D-glucammonium (NMDG(+)). In the presence of HCO3-, the Na+-driven HCO3- transporter contributed to the pH(i) recovery from an acid load. For example, HCO3- shifted the plot of acid-extrusion rate vs. pH(i) by 0.15-0.3 pH units in the alkaline direction. Also, with Na-H exchange inhibited by amiloride, HCO3- increased acid extrusion 3.8-fold (pH(i) 6.20). When astrocytes were acid loaded in amiloride, with Li+ as the major cation, HCO3- failed to elicit a substantial increase in pH(i). Thus, Li+ does not appear to substitute well for Nai on the HCO3- transporter. We conclude that an amiloride-sensitive Na-H exchanger and a Na+-driven HCO3- transporter are the predominant acid extruders in astrocytes.