Anoxia-evoked intracellular pH and Ca2+ concentration changes in cultured postnatal rat hippocampal neurons

The ratiometric indicators 2',7'-bis-(2-carboxyethyl)-5-(and-6)-caroxyfluorescein and Fura-2 were employed to examine, respectively, intracellular pH (pH(i)) and calcium ([Ca2+](i)) changes evoked by anoxia in cultured postnatal rat hippocampal neurons at 37 degrees C. Under both HCO3-/CO2- and HEPES-buffered conditions, 3-, 5- or 10-min anoxia induced a triphasic change in pH(i) consisting of an initial fall in pHi, a subsequent rise in pH(i) in the continued absence of O-2 and, finally, a further rise in pH(i) upon the return to normoxia, which recovered towards preanoxic steady-state pH(i) values if the duration of the anoxic insult was less than or equal to 5 min. In parallel experiments performed an sister cultures, anoxia of 3, 5 or 10 min duration evoked rises in [Ca2+](i) which, in all cases, commenced after the start of the fall in pHi, reached a peak at or just following the return to normoxia and then declined towards preanoxic resting levels. Removal of external Ca2+ markedly attenuated increases in [Ca2+](i), but failed to affect the pH(i) changes evoked by 5 min anoxia. The latency from the start of anoxia to the start of the increase in pH(i) observed during anoxia was increased by perfusion with media containing either 2 mM Na+, 20 mM glucose or 1 mu M tetrodotoxin. Because each of these manoeuvres is known to delay the onset and/or attenuate the magnitude of anoxic depolarization, the results suggest that the rise in pH(i) observed during anoxia may be consequent upon membrane depolarization. This possibility was also suggested by the findings that Zn2+ and Cd2+, known blockers of voltage-dependent proton conductances, reduced the magnitude of the rise in pH(i) observed during anoxia. Under HCO3-/CO2-free conditions, reduction of external Na+ by substitution with N-methyl-D-glucamine (but not Li+) attenuated the magnitude of the postanoxic alkalinization, suggesting that increased Na+/H+ exchange activity contributes to the postanoxic rise in pH(i). In support, rates of pH(i) recovery from internal acid loads imposed following anoxia were increased compared to control values established prior to anoxia in the same neurons. In contrast, rates of pH(i) recovery from acid loads imposed during anoxia were reduced, suggesting the possibility that Na+/H+ exchange is inhibited during anoxia. We conclude that the steady-state pHi response of cultured rat hippocampal neurons to transient anoxia is independent of changes in [Ca2+](i) and is characterized by three phases which are determined, at least in part, by alterations in Na+/H+ exchange activity and, possibly, by a proton conductance which is activated during membrane depolarization. (C) 1999 IBRO. Published by Elsevier Science Ltd.