INTRACELLULAR PH REGULATION IN SINGLE CULTURED ASTROCYTES FROM RAT FOREBRAIN
INTRACELLULAR PH REGULATION IN SINGLE CULTURED ASTROCYTES FROM RAT FOREBRAIN
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DOI：
10.1002/glia.440080404
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发表时间：
1993-08-01
期刊：
影响因子：
6.2
通讯作者：
BORON, WF
中科院分区：
文献类型：
--
作者：
BOYARSKY, G;RANSOM, B;BORON, WF
我们使用荧光pH敏感染料2 '，7'-双（羧乙基）-5，6-羧基荧光素（BCECF）监测从新生大鼠前脑培养的单个星形胶质细胞的细胞内pH（pH（i））。当在37 ℃下暴露于用HEPES缓冲至pH 7.40的标称无CO2/HCO 3培养基时，细胞的平均pH（i）为6.89。切换到用5% CO2和25 mM HCO缓冲至pH 7.40的培养基，导致稳态pH（i）平均增加0.35，表明存在HCO 3依赖性酸挤出机制。在持续的碱化之前，有时会出现短暂的小酸化。在星形胶质细胞暴露于名义上无HCO 3（HEPES缓冲）溶液的实验中，20 mM细胞外NH 4+的应用和撤回导致pH（i）下降到远低于初始值的值。pH（i）自发地从该酸负荷恢复，稳定在比施加NH 4+之前的值高约0.1的值。在对沐浴在HEPES缓冲溶液中的细胞进行的其他实验中，去除细胞外Na+会导致pH（i）迅速下降0.5。返回Na+导致pH（i）迅速升高，表明存在Na+依赖性/HCO 3非依赖性酸挤出机制;返回Na+后的最终pH（i）比初始值高出约0.08。通过返回Na+引起的pH（i）恢复基本上不受50 μ M乙基异丙基氨氯吡嗪（EIPA）的影响，但被50 μ M 4，4 '-二异硫氰基芪-2，2'-二磺酸盐（DIDS）加速。将[K+]o从5 mM增加到25 mM导致pH（i）在标称无CO2/HCO 3-的溶液中可逆地增加约0.2，在含CO2/HCO 3-的溶液中可逆地增加约0.1，尽管在CO2/HCO 3-存在下初始pH（i）高约0.17。这些结果表明存在去极化诱导的碱化。我们的研究结果表明，在培养的哺乳动物星形胶质细胞中存在HCO 3依赖性和非依赖性酸碱转运系统，并表明星形胶质细胞pH（i）对膜电压或[K+]o本身的变化敏感。(C)1993 Wiley-Liss，Inc.
We used the fluorescent pH-sensitive dye 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF) to monitor intracellular pH (pH(i)) in single astrocytes cultured from the forebrain of neonatal rats. When exposed to a nominally CO2/HCO3--free medium buffered to pH 7.40 with HEPES at 37-degrees-C, the cells had a mean pH(i) of 6.89. Switching to a medium buffered to pH 7.40 with 5% CO2 and 25 mM HCO:, caused the steady-state pH(i) to increase by an average of 0.35, suggesting the presence of a HCO3--dependent acid-extrusion mechanism. The sustained alkalinization was sometimes preceded by a small transient acidification. In experiments in which astrocytes were exposed to nominally HCO3--free (HEPES-buffered) solutions, the application and withdrawal of 20 mM extracellular NH4+ caused pH(i) to fall to a value substantially below the initial one. pH(i) spontaneously recovered from this acid load, stabilizing at a value approximately 0.1 higher than the one prevailing before the application of NH4+. In other experiments conducted on cells bathed in HEPES-buffered solutions, removing extracellular Na+ caused pH(i) to decrease rapidly by 0.5. Returning the Na+ caused pH(i) to increase rapidly, indicating the presence of an Na+-dependent/HCO3--independent acid-extrusion mechanism; the final pH(i) after returning Na+ was approximately 0.08 higher than the initial value. This pH(i) recovery elicited by returning Na+ was not substantially affected by 50 muM ethylisopropylamiloride (EIPA), but was speeded up by 50 muM 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS). Increasing [K+]o from 5 to 25 mM caused pH(i) to increase reversibly by approximately 0.2 in nominally CO2/HCO3--free solutions, and by approximately 0.1 in CO2/HCO3--containing solutions, although the initial pH(i) was approximately 0.17 higher in the presence of CO2/HCO3-. These results suggest the presence of a depolarization-induced alkalinization. Our results suggest the presence of both HCO3- dependent and -independent acid-base transport systems in cultured mammalian astrocytes, and indicate that astrocyte pH(i) is sensitive to changes in either membrane voltage or [K+]o per se. (C) 1993 Wiley-Liss, Inc.