title: Novel role of the Ca(2+)-ATPase in NMDA-induced intracellular acidification.
abstract: The mechanism involved in N-methyl-D-glucamine (NMDA)-induced Ca(2+)-dependent intracellular acidosis is not clear. In this study, we investigated in detail several possible mechanisms using cultured rat cerebellar granule cells and microfluorometry [fura 2-AM or 2', 7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein-AM]. When 100 microM NMDA or 40 mM KCl was added, a marked increase in the intracellular Ca(2+) concentration ([Ca(2+)](i)) and a decrease in the intracellular pH were seen. Acidosis was completely prevented by the use of Ca(2+)-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 Ca(2+). The following four mechanisms that could conceivably have been involved were excluded: 1) Ca(2+) displacement of intracellular H(+) from common binding sites; 2) activation of an acid loader or inhibition of acid extruders; 3) overproduction of CO(2) or lactate; and 4) collapse of the mitochondrial membrane potential due to Ca(2+) uptake, resulting in inhibition of cytosolic H(+) uptake. However, NMDA/KCl-induced acidosis was largely prevented by glycolytic inhibitors (iodoacetate or deoxyglucose in glucose-free medium) or by inhibitors of the Ca(2+)-ATPase (i.e., Ca(2+)/H(+) exchanger), including La(3+), orthovanadate, eosin B, or an extracellular pH of 8.5. Our results therefore suggest that Ca(2+)-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 [Ca(2+)](i) increase.
authors.affiliation: Department of Physiology, College of Medicine, National Taiwan University Hospital, Taipei, Taiwan. mlw@ha.mc.ntu.edu.tw
authors.forename: M L
authors.initials: ML
authors.lastname: Wu
authors.forename: J H
authors.initials: JH
authors.lastname: Chen
authors.forename: W H
authors.initials: WH
authors.lastname: Chen
authors.forename: Y J
authors.initials: YJ
authors.lastname: Chen
authors.forename: K C
authors.initials: KC
authors.lastname: Chu
mesh_terms: D000138:Acidosis / Q000139:chemically induced; D000143:Acids / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000502:physiology*; D002245:Carbon Dioxide / Q000378:metabolism; D006859:Hydrogen / Q000378:metabolism; D006863:Hydrogen-Ion Concentration / Q000187:drug effects; D007425:Intracellular Membranes / Q000378:metabolism*; D008928:Mitochondria / Q000378:metabolism; D016202:N-Methylaspartate / Q000494:pharmacology*; D009474:Neurons / Q000378:metabolism; D009994:Osmolar Concentration; D011189:Potassium Chloride / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar
chemical_list: D000143:Acids; D002245:Carbon Dioxide; D016202:N-Methylaspartate; D011189:Potassium Chloride; D006859:Hydrogen; D000252:Calcium-Transporting ATPases; D002118:Calcium