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Anoxia-evoked changes in intracellular pH in acutely dissociated adult rat hippocampal neurons Sheldon, Claire Alexis

Abstract

The ratiometric fluorophore, 2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) was utilized to measure pHi in acutely dissociated adult rat hippocampal CA1 neurons in response to periods of anoxia of 3, 5 and 10 min duration. At 37°C, in the presence or absence of HC03-, the pHi response to anoxia was characterized by three phases: i) during the anoxic period, there was an intracellular acidification; ii) in the continued absence of oxygen, there was a rise in pHi toward pre-anoxic pHi values; and iii) in the immediate post-anoxic period, there was an intracellular alkalinization above pre-anoxic pHi values. These changes were not secondary to anoxia-evoked changes in intracellular free Ca2+ concentration. Next, the mechanisms underlying the anoxia-evoked changes in pHi were examined. In addition to supporting the established roles of an increased rate of ATP hydrolysis and intracellular lactate accumulation, evidence is presented that a decreased activity of the Na+/H+ exchanger contributed to the production of the intracellular acidification observed during an anoxic period. The rise in pHi observed in the continued absence of oxygen was attributed, at least in part, to activation of a Zn2+-sensitive, voltage-dependent proton conductance. Finally, an increased activity of the Na+/H+ exchanger contributed to the production of the post-anoxic alkalinization. Reductions in extracellular pH and ambient temperature were found to attenuate the post-anoxic activation of Na+/H+ exchange, as was an inhibitor of PKA. The pHi changes characterized in the present study may determine, at least in part, neuronal viability following an anoxic insult. [Scientific formulae used in this abstract could not be reproduced.]

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