Novel role of the Ca2+-ATPase in NMDA-induced intracellular acidification

The mechanism involved in N-methyl-D-glucamine (NMDA)-induced Ca2+-dependen t intracellular acidosis is not clear. In this study, we investigated in de tail several possible mechanisms using cultured rat cerebellar granule cell s and microfluorometry [fura 2-AM or 2',7'-bis(2-carboxyethyl)-5(6)-carboxy fluorescein-AM]. When 100 mu M NMDA or 40 mM KCl was added, a marked increa se in the intracellular Ca2+ concentration ([Ca2+](i)) and a decrease in th e intracellular pH were seen. Acidosis was completely prevented by the use of Ca2+-free medium or 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-AM, suggesting that it resulted from an influx of extracellular Ca2+. The following four mechanisms that could conceivably have been involved wer e excluded: 1) Ca2+ displacement of intracellular H+ from common binding si tes; 2) activation of an acid loader or inhibition of acid extruders; 3) ov erproduction of CO2 or lactate; and 4) collapse of the mitochondrial membra ne potential due to Ca2+ uptake, resulting in inhibition of cytosolic H+ up take. However, NMDA/KCl-induced acidosis was largely prevented by glycolyti c inhibitors (iodoacetate or deoxyglucose in glucose-free medium) or by inh ibitors of the Ca2+-ATPase (i.e., Ca2+/H+ exchanger), including La3+, ortho vanadate, eosin B, or an extracellular pH of 8.5. Our results therefore sug gest that Ca2+-ATPase is involved in NMDA-induced intracellular acidosis in granule cells. We also provide new evidence that NMDA-evoked intracellular acidosis probably serves as a negative feedback signal, probably with the acidification itself inhibiting the NMDA-induced [Ca2+](i) increase.