Pappas C A, Rioult M G, Ransom B R
Department of Neurology, Yale University School of Medicine New Haven, Connecticut 06510, USA.
Glia. 1996 Jan;16(1):7-15. doi: 10.1002/(SICI)1098-1136(199601)16:1<7::AID-GLIA2>3.0.CO;2-2.
Octanol rapidly closes gap junction channels but its mechanism of action is not known. Because intracellular [H+], pHi, also affects the conductance of gap junctions, we studied octanol's effects on pHi in cultured rat astrocytes, which are highly coupled cells. Octanol (1 mM) caused an acid shift in the pHi of 90% of rat hippocampal astrocytes which averaged -0.19 +/- 0.09 pH units in magnitude. In 58% of the cells tested, a biphasic change in pHi was seen; octanol produced an initial acidification lasting approximately 10 min that was followed by a persistent alkalinization. The related gap junction uncoupling agent, heptanol, had similar effects on pHi. Octanol-induced changes in pHi were similar in nominally HCO(3-)-free and HCO(3-)-containing solutions, although the rate of initial acidification was significantly greater in the presence of HCO3-. The initial acidification was inhibited in the presence of the stilbene DIDS, an inhibitor of Na+/HCO3- cotransport, indicating that octanol caused acidification by blocking this powerful acid extruder. The alkalinization was inhibited by amiloride which blocks the Na+/H+ exchanger (NHE), an acid extruder, suggesting that the alkaline shift induced by octanol was caused by stimulation of NHE. As expected, octanol's effects on astrocytic pHi were prevented by removal of external Na+, which blocks both Na+/HCO3- cotransport and NHE. Octanol had only small effects on intracellular Ca2+ (Ca2+i) in astrocytes. Hepatocytes which, like astrocytes, are strongly coupled to one another, showed no change in pHi with octanol application. Fluorescence recovery after photobleaching (FRAP) was used to study the effect of changes in astrocyte pHi on degree of coupling in hippocampal astrocytes. Coupling was decreased by intracellular acid shifts approximately -0.2 pH units in size. Octanol's effects on astrocyte pHi were complex but a prompt initial acidification was nearly always seen and could contribute to the uncoupling action of this drug in astrocytes. Because octanol uncouples hepatocytes without changing their pHi, this compound clearly can influence gap junctional conductance independent of changes in pHi.
辛醇能迅速关闭缝隙连接通道，但其作用机制尚不清楚。由于细胞内[H⁺]，即细胞内pH值（pHi），也会影响缝隙连接的电导，我们研究了辛醇对培养的大鼠星形胶质细胞pHi的影响，这些细胞是高度耦合的细胞。辛醇（1 mM）使90%的大鼠海马星形胶质细胞的pHi发生酸向偏移，其幅度平均为-0.19±0.09 pH单位。在58%的测试细胞中，观察到pHi的双相变化；辛醇产生了持续约10分钟的初始酸化，随后是持续的碱化。相关的缝隙连接解偶联剂庚醇对pHi有类似的影响。在名义上不含HCO₃⁻和含HCO₃⁻的溶液中，辛醇诱导的pHi变化相似，尽管在有HCO₃⁻存在时初始酸化速率明显更高。在存在芪类药物DIDS（一种Na⁺/HCO₃⁻共转运抑制剂）的情况下，初始酸化受到抑制，这表明辛醇通过阻断这种强大的酸排出器导致酸化。碱化受到氨氯地平的抑制，氨氯地平可阻断Na⁺/H⁺交换体（NHE），一种酸排出器，这表明辛醇诱导的碱向偏移是由NHE的刺激引起的。正如预期的那样，去除细胞外Na⁺可防止辛醇对星形胶质细胞pHi的影响，细胞外Na⁺会阻断Na⁺/HCO₃⁻共转运和NHE。辛醇对星形胶质细胞内的Ca²⁺（Ca²⁺i）只有很小的影响。肝细胞与星形胶质细胞一样，彼此之间紧密耦合，在应用辛醇后pHi没有变化。采用光漂白后荧光恢复（FRAP）技术研究星形胶质细胞pHi变化对海马星形胶质细胞耦合程度的影响。细胞内酸向偏移约-0.2 pH单位时，耦合程度降低。辛醇对星形胶质细胞pHi的影响很复杂，但几乎总是能看到迅速的初始酸化，这可能有助于该药物在星形胶质细胞中的解偶联作用。由于辛醇能使肝细胞解偶联而不改变其pHi，这种化合物显然可以独立于pHi的变化而影响缝隙连接的电导。
