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

Octanol rapidly closes gap junction channels but its mechanism of action is not known. Because intracellular [H⁺], pH_i, also affects the conductance of gap junctions, we studied octanol's effects on pH_i in cultured rat astrocytes, which are highly coupled cells. Octanol (1 mM) caused an acid shift in the pH_i 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 pH_i was seen; octanol produced an initial acidification lasting ∼10 min that was followed by a persistent alkalinization. The related gap junction uncoupling agent, heptanol, had similar effects on pH_i. Octanol-induced changes in pH_i were similar in nominally HCO₃⁻-free and HCO₃⁻-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 pH_i 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 pH_i with octanol application. Fluorescence recovery after photobleaching (FRAP) was used to study the effect of changes in astrocyte pH_i on degree of coupling in hippocampal astrocytes. Coupling was decreased by intracellular acid shifts ∼−0.2 pH units in size. Octanol's effects on astrocyte pH_i 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 pH_i, this compound clearly can influence gap junctional conductance independent of changes in pH_i. © 1996 Wiley-Liss, Inc.