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

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 (pH_i) 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 pH_i are more than 1.2 pH units alkaline to the pH_i predicted from a passive distribution of protons; thus, these cells actively regulated pH_i. 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 pH_i. 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 pH_i of glial cells and also contributes to the ability of glial cells to buffer changes in pH in the neuronal microenvironment.