PH regulation after acid load in primary cultures of mouse astrocytes

Abstract

Intracellular pH (pH i) recovery in primary cultures of mouse astrocytes after acid-loading was studied with the ion transport inhibitors (amiloride, SITS, acetazolamide, ouabain and bumetanide), and by reducing the concentration of Na + or Cl − in HCO 3-free HEPES-buffered (HEPES) and in HCO − 3/CO 2 Hanks' balanced salt solution (HBSS). The pH i of astrocytes exposed to 15 mM NH 4Cl decreased abruptly and began to recover slowly after 5 min. Exposure of the cells to NH 4Cl for 2 min and reincubation in HEPES HBSS decreased pH i further within 1–2 min after removal of NH 4Cl; pH i then recovered toward the control value. Cultures exposed to HCO 3/CO 2 HBSS (10 mM/2%) showed changes in pH i in the opposite direction. These responses are unique to astrocytes and differ from those occurring in most other cells. Recovery of pH i after NH 4Cl prepulse was markedly inhibited in low-Na + and in amiloride-containing HEPES HBSS. Ouabain also reduced pH i recovery rate; however, SITS, acetazolamide and bumetanide did not. Therefore, Na +-H + exchange is the major process for pH i recovery from acidification in HCO 3-free solution. In HCO − 3/CO 2 HBSS pH i recovery was markedly inhibited by SITS and acetazolamide, but not by amiloride, ouabain, or bumetanide. The inhibitory effect of SITS on pH i recovery was enhanced in low-Na + HBSS. These results indicate that both Na + and HCO 3 are directly related to pH i recovery in HCO − 3/CO 2 solution after acid-load. Low-Cl HEPES HBSS and low-Cl HCO − 3/CO 2 HBSS media did not alter pH recovery rate. Thus, pH i recovery after acid-load is not Cl −-dependent, and therefore, does not involve a Na +-dependent Cl −-HCO 3 exchange process. It appears that mouse astrocytes possess 3 acid-regulating systems: Na +-H + exchange, Na +-HCO − 3 co-transport and Na +-independent Cl −-HCO − 3 exchange.