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 HCO/CO(2)- or
HEPES-buffered media (pH 7.35) at 37 degrees C, 5- or 10-min anoxic insults were typified by an intracellular acidification on the induction of
anoxia, a subsequent rise in pH(i) 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 [Ca(2+)](i) and, examined in the presence of HCO, were not significantly affected by (
DIDS). In the absence of HCO, 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 microM Zn(2+), 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 Zn(2+) to reduce the increase in pH(i) that occurred after
anoxia were additive. Consistent with the steady-state pH(i) changes, rates of pH(i) recovery from internal
acid loads imposed immediately after
anoxia were increased, and the application of Zn(2+) and/or perfusion with NMDG(+)-substituted medium slowed pH(i) recovery. Reducing extracellular pH from 7.35 to 6.60, or reducing ambient temperature from 37 degrees C to room temperature, also attenuated the increases in steady-state pH(i) observed during and after
anoxia and reduced rates of pH(i) 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 pH(i) 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 Zn(2+)-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.