The Journal of Neurophysiology Vol. 87 No. 5 May 2002, pp. 2209-2224
Copyright ©2002 by the American Physiological Society

Intracellular pH Response to Anoxia in Acutely Dissociated Adult Rat Hippocampal CA1 Neurons

Departments of Anatomy and Physiology, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada

Sheldon, Claire and John Church. Intracellular pH Response to Anoxia in Acutely Dissociated Adult Rat Hippocampal CA1 Neurons. J. Neurophysiol. 87: 2209-2224, 2002. 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/CO₂- or HEPES-buffered media (pH 7.35) at 37°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 O₂, 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 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 µM 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 pH_i recovery from internal acid loads imposed immediately after anoxia were increased, and the application of Zn²⁺ 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°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 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.