Intracellular pH response to anoxia in acutely dissociated adult rat hippocampal CA1 neurons

The effects of anoxia on intracellular pH (pH(i)) were examined in acutely isolated adult rat hippocampal CA1 neurons loaded with the H+-sensitive fluorophore, 2', 7'- bis-(2-carboxyethyl)-5-( and-6)-carboxyfluorescein. During perfusion with HCO3-/CO2- or HEPES-buffered media (pH 7.35) at 37degrees C, 5- or 10-min anoxic insults were typified by an intracellular acidification on the induction of anoxia, a subsequent rise in pHi in the continued absence of O-2, and a further internal alkalinization on the return to normoxia. The steady-state pH(i) changes were not consequent on changes in [Ca2+](i) and, examined in the presence of HCO3-, were not significantly affected by (DIDS). In the absence of HCO3-, the magnitude of the postanoxic alkalinization was attenuated when external Na+ was reduced by substitution with N-methyl-D-glucamine (NMDG(+)), but not Li+, suggesting that increased Na+/H+ exchange activity contributes to this phase of the pH(i) response. In contrast, 100-500 muM Zn2+, a known blocker of H+-conductive pathways, reduced the magnitudes of the internal alkalinizations that occurred both during and following anoxia. The effects of NMDG(+)-substituted medium and Zn2+ to reduce the increase in pH(i) that occurred after anoxia were additive. Consistent with the steady-state pH(i) changes, rates of pHi recovery from internal acid loads imposed immediately after anoxia were increased, and the application of Zn2+ and/ or perfusion with NMDG(+)-substituted medium slowed pHi recovery. Reducing extracellular pH from 7.35 to 6.60, or reducing ambient temperature from 37degrees C to room temperature, also attenuated the increases in steady-state pH(i) observed during and after anoxia and reduced rates of pHi recovery from acid loads imposed in the immediate postanoxic period. Finally, inhibition of the cAMP/protein kinase A second-messenger system reduced the magnitude of the rise in pHi after anoxia in a manner that was dependent on external Na+; conversely, activation of the system with isoproterenol increased the postanoxic alkalinization, an effect that was attenuated by pretreatment with propranolol, Rp-cAMPS, or when NMDG(+) (but not Li-)(+) was employed as an external Na+ substitute. The results suggest that a Zn2+-sensitive acid efflux mechanism, possibly a H+-conductive pathway activated by membrane depolarization, contributes to the internal alkalinization observed during anoxia in adult rat CA1 neurons. The rise in pH(i) after anoxia reflects acid extrusion via the H+-conductive pathway and also Na+/H+ exchange, activation of the latter being mediated, at least in part, through a cAMP-dependent signaling pathway.