Discrepant intracellular pH changes following intracellular Ca²⁺ increases induced by glutamate and Ca²⁺ ionophores in rat hippocampal neurons

We investigated changes in intracellular pH (pHi) in relation to intracellular Ca²⁺ concentration ([Ca²⁺]i) in primary cultured hippocampal neurons treated with glutamate. [Ca²⁺]i and phi were imaged with fluorescent dyes and confocal microscopy. Exposure to 1 mM glutamate for 10 min increased [Ca²⁺]i and evoked acidosis. These changes persisted for at least 60 min, even after removal of glutamate. The increase in [Ca²⁺]i and the acidosis were not observed in Ca²⁺-free solution and were attenuated in the presence of MK-801, an NMDA receptor antagonist. We also found that the increase in [Ca²⁺]i and acidosis could be induced by addition of Ca²⁺ to the extracellular solution in the cells pretreated with glutamate in Ca²⁺-free solution, even if glutamate did not exist in the extracellular solution. On the other hand, ionomycin and Br-A23187, calcium ionophores, increased [Ca²⁺]i to almost the same level as glutamate and increased phi. Extracellular Ca²⁺ was also indispensable for the increase in [Ca²⁺]i and the alkalosis. These results suggest the followings: 1) intracellular acidosis by glutamate is dependent on the presence of extracellular Ca²⁺; 2) the acidosis does not result from only the increase in [Ca²⁺]i; and 3) glutamate induces the irreversible disorder of regulatory mechanisms of [Ca²⁺]i not only by Ca²⁺-dependent process, but also by Ca²⁺-independent process.