Acid/base transport in glial cells of Necturus optic nerve

Membrane properties of glial cells of Necturus maculosus optic nerve were studied using conventional microelectrodes and double-barreled, pH-sensitive microelectrodes. The effects of Ba$\sp{++}$, a known K$\sp+$-channel blocker, were studied with conventional microelectrodes. The addition of Ba$\sp{++}$ (2-5 mM) reversibly depolarized glial cells by 20 to 50 mV and reduced the sensitivity of the membrane to changes in bath (K$\sp+$). These effects were accompanied by an increase in the input resistance of the membrane, strongly suggesting that Ba$\sp{++}$ decreases the K$\sp+$ conductance of the glial cells. With the K$\sp+$ conductance decreased by Ba$\sp{++}$, the membrane response to HCO$\sb3\sp-$ was investigated. Addition of HCO$\sb3\sp-$ at constant pH caused a hyperpolarization which was Na$\sp+$-dependent, SITS (4-acetamido-4$\sp\prime$-isothiocyanato-stilbene-2,2$\sp\prime$-disulfonic acid)-sensitive, Cl$\sp-$-independent, and strophanthidin-insensitive. These results strongly suggest the presence in the glial membrane of an electrogenic Na$\sp+$/HCO$\sb3\sp-$ cotransporter that transports Na$\sp+$, HCO$\sb3\sp-$, and net negative charge in the same direction across the cell membrane. To determine the relation between intracellular pH (pH$\sb{\rm i}$) and acid/base transport mechanisms like electrogenic Na$\sp{+}$/HCO$\sb3\sp{-}$ cotransport, glial cells were studied with double-barreled, pH-sensitive microelectrodes. At a bath pH of 7.5, the mean initial pH$\sb{\rm i}$ was 7.32 (S.D. 0.03, n = 6) in HEPES-buffered Ringer\u27s solution and 7.39 (S.D. 0.1, n = 6) in HCO$\sb3\sp{-}$/CO$\sb2$-buffered solution. These values for pH$\sb{\rm i}$ are more than 1 pH unit alkaline to the pH$\sb{\rm i}$ predicted from a passive distribution of protons; thus, glial cells actively extrude acid. Acid extrusion mechanisms were determined by inducing acidifications from the steady-state pH$\sb{\rm i}$ and analyzing the ionic dependence and pharmacology of the pH$\sb{\rm i}$ recovery. Superfusion followed by withdrawal of 15 mM NH$\sb4\sp{+}$ induced an acidification of 0.1 to 0.3 pH unit after which the pH$\sb{\rm i}$ recovered toward the original steady-state over the next 7 to 16 min. In the absence of HCO$\sb3\sp{-}$/CO$\sb2$, the recovery from acidification was Na$\sp{+}$-dependent, and amiloride-sensitive. Recovery from acidification was stimulated by adding HCO$\sb3\sp{-}$/CO$\sb2$ at constant pH. In HCO$\sb3\sp{-}$/CO$\sb2$-buffered solution, the recovery was Na$\sp{+}$-dependent, SITS-sensitive, and associated with a membrane hyperpolarization. The data strongly suggest that the relatively alkaline pH$\sb{\rm i}$ of glial cells is due to the activity of at least two mechanisms, Na$\sp{+}$/H$\sp{+}$ exchange and electrogenic Na$\sp{+}$/HCO$\sb3\sp{-}$ cotransport