Octanol, a gap junction uncoupling agent, changes intracellular [H+] in rat astrocytes

Abstract

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.