Further studies of electrogenic Na+/HCO cotransport in glial cells of necturus optic nerve: Regulation of pHi

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Abstract
In the presence of Ba++, an increase in the bath HCO at constant CO2 (i.e., Variable bath pH) produced a hyperpolarization. The hyperpolarizing effect of adding HCO3−/CO2 at constant bath pH was not significantly affected by the presence of 50 μmol/l strophanthidin. In the absence of Ba++, addition of HCO3−/CO2 at constant bath pH produced a Na+-dependent hyperpolarization. Therefore, CO2 movements, electrogenic Na+/K+ pump activity and changes in Ba++ binding do not contribute significantly to the hyperpolarization induced by HCO3−. These results along with the results of previous studies (Astion et al: J Gen Physiol 93:731, 1989) strongly suggest that the hyperpolarization induced by the addition of HCO3− is due to an electrogenic Na+/HCO3− cotransporter, which transports Na+, HCO3− (or its equivalent), and net negative charge across the glial membrane. To study the role of electrogenic Na+/HCO cotransport in the regulation of pHi in glial cells, we used intracellular double-barreled, pH-sensitive microelectrodes. At a bath pH of 7.5, the mean initial intracellular pH (pHi) was 7.32 (SD 0.03, n = 6) in HEPES-buffered Ringer's solution and 7.39 (SD 0.1, n = 6) in HCO3−/CO2 buffered solution. These values for pHi are more than 1.2 pH units alkaline to the pHi predicted from a passive distribution of protons; thus, these cells actively regulated pHi. Superfusion and with-drawal of 15 mmol/l NH4+ induced an acidification of 0.2 to 0.3 pH units, which recovered toward the original steady-steady-state pHi. Recovery from acidification was stimulated by adding HCO3−/CO2 at constant pH. In HCO3−/CO2-buffered solutions, the recovery was Na+-dependent, inhibited by 4-acetamido-4′-isothiocyanato-stilbene-2,2′-disulfonic acid (SITS), and associated with a hyperpolarization of the membrane. Thus it appears that the electrogenic Na+/HCO3− cotransporter helps maintain the relatively alkaline pHi of glial cells and also contributes to the ability of glial cells to buffer changes in pH in the neuronal microenvironment.