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Anoxia and Na⁺/H⁺ exchange activity in rat hippocampal neurons Sheldon, Claire Alexis

Abstract

In the present study, the effects of anoxia on intracellular pH (pH[sub i]) and intracellular free sodium concentration ([Na⁺][sub i]) were examined in isolated rat hippocampal neurons loaded with H⁺- and/or Na⁺-sensitive fluorophores, and the contribution of changes in Na⁺/H⁺ exchange activity to the changes in pH[sub i] and [Na⁺], observed during and after anoxia were assessed. This assessment was aided by the development of a microspectrofluorimetric technique which permitted concurrent measurements of pH[sub i] and [Na⁺][sub i] in the same neuron. I found that, in hippocampal neurons, Na⁺/H⁺ exchange activity was reduced shortly following the onset of anoxia, possibly as a result of declining internal ATP levels, and did not contribute to the increases in pH[sub i] or [Na⁺][sub i] observed at this time. In contrast, Na⁺/H⁺ exchange activity was stimulated immediately after anoxia and contributed to acid extrusion and Na⁺ influx during this particularly vulnerable period. As a result, the reported neuroprotective actions of Na⁺/H⁺ exchange inhibitors are likely mediated in the immediate postanoxic period, consequent upon reductions in acid extrusion and/or internal Na⁺ loading. A Zn²⁺ - sensitive H⁺ efflux pathway, possibly a voltage-activated H⁺ conductance activated by membrane depolarization, also contributed to acid extrusion during and immediately after anoxia and may act to limit the potentially detrimental activation of Na⁺/H⁺ exchange activity observed after anoxia. The final series of experiments identified additional mechanisms that contribute to the changes in [Na⁺][sub i] evoked by anoxia in cultured postnatal rat hippocampal neurons. Na⁺ influx occurred through multiple pathways, the relative contributions of which differed not only during and after anoxia but also in neurons maintained in culture for different durations of time. Understanding the fundamental cellular mechanisms that contribute to anoxia-evoked changes in pH[sub i] and [Na⁺][sub i] in mammalian central neurons may uncover novel therapeutic strategies for the treatment of stroke.

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