Novel role of the Ca(2+)-ATPase in NMDA-induced intracellular acidification

Am J Physiol. 1999 Oct;277(4):C717-27. doi: 10.1152/ajpcell.1999.277.4.C717.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acidosis / chemically induced
  • Acids / metabolism*
  • Animals
  • Binding Sites
  • Calcium / metabolism
  • Calcium-Transporting ATPases / physiology*
  • Carbon Dioxide / metabolism
  • Hydrogen / metabolism
  • Hydrogen-Ion Concentration / drug effects
  • Intracellular Membranes / metabolism*
  • Mitochondria / metabolism
  • N-Methylaspartate / pharmacology*
  • Neurons / metabolism
  • Osmolar Concentration
  • Potassium Chloride / pharmacology
  • Rats
  • Rats, Wistar

Substances

  • Acids
  • Carbon Dioxide
  • N-Methylaspartate
  • Potassium Chloride
  • Hydrogen
  • Calcium-Transporting ATPases
  • Calcium