K+-INDUCED ALKALINIZATION IN MOUSE CEREBRAL ASTROCYTES MEDIATED BY REVERSAL OF ELECTROGENIC NA+-HCO3- COTRANSPORT

Raising extracellular K+ concentration ([K+](o)) induces an alkaline shift of intracellular pH (pH(i)) in astrocytes. The mechanism of this effect was examined using the fluorescent pH(i) indicator 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein in primary cultures of mouse cerebral astrocytes. Raising [K+](o) from 3 to 12 mM increased pH(i) by 0.28 pH units in 26 mM HCO3--buffered solution. In nominally HCO3--free solution (containing similar to 95 mu M HCO3-), the alkalinization fell to 0.21 pH units and further to 0.08 pH units on removal of atmospheric CO2, suggesting a process with high affinity for HCO3-. This effect was Na+ dependent, Cl- independent, and inhibited by 0.5 mM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, indicating the involvement of Na+-HCO3- cotransport. The relationship between pH(i) and log[K+](o) was found to be linear and to predict a stoichiometry of at least two HCO3- transported with each Na+. After removal of exogenous CO2/HCO3-, the direction of changes in pH(i) elicited by adding 1 mM HCO3- showed that net flux of HCO3- via the Na+-HCO3- cotransporter was outward at rest and was reversed by depolarization.