Chronic mild acidosis specifically reduces functional expression of N-methyl-D-aspartate receptors and increases long-term survival in primary cultures of cerebellar granule cells.
Previous studies suggest that chronic depolarization by addition of 25 mM KCl or N-methyl-D-aspartate to primary cultures of cerebellar granule cells promotes expression of the N-methyl-D-aspartate subtype of glutamate receptor, as determined by electrophysiological responsiveness and susceptibility to excitotoxicity. Recent studies have demonstrated that acute mild acidosis reduces N-methyl-D-aspartate receptor channel activity by a non-competitive action of H+ on an extracellular site of the receptor channel complex. Since the level of N-methyl-D-aspartate receptor expression in granule cell cultures is activity-dependent, we examined whether chronic mildly acidotic culture conditions would selectively diminish the level of N-methyl-D-aspartate responsiveness in granule cells, in effect producing a functional level of expression more comparable to that observed in vivo. To test this, cerebellar granule cells from eight-day neonatal rats were grown in an HCO3-buffered medium containing elevated K+ (25 mM KCl) either under standard conditions (95% air/5% CO2, pH 7.4), or under chronic mildly acidotic conditions (90% air/10% CO2, estimated pH of 7.1). Glutamate receptor subtype expression was subsequently assessed using standard neurotoxicity assays, a quantitative immunoblotting assay for N-methyl-D-aspartate receptors and whole cell patch clamp recordings. Cells grown in the 10% CO2 environment exhibited a significant reduction in susceptibility to L-glutamate neurotoxicity (at least 10-fold), but not kainate-induced neurotoxicity, relative to cells grown in 5% CO2. In both culture conditions, L-glutamate- and kainate-induced toxicity were mediated by activation of N-methyl-D-aspartate and non-N-methyl-D-aspartate receptors, respectively, as determined by the sensitivity of agonist-induced toxicity to specific receptor antagonists. Using polyclonal antibodies generated against a peptide sequence recognizing five of eight splice variants in the common "R1" subunit of N-methyl-D-aspartate receptors, a 31% reduction in the amount of immunoreactive protein was observed in membrane preparations from cells grown in 10% CO2, relative to the amount detected in cells grown in 5% CO2. Moreover, perfusion of cells with glutamate (50 microM) in a nominally Mg(2+)-free solution containing glycine (2 microM) elicited N-methyl-D-aspartate antagonist-sensitive inward currents in proportionately fewer cells cultured in 10% CO2, relative to cells cultured in 5% CO2. Long-term survival was also significantly enhanced in cells exposed chronically to mild acidotic culture conditions, relative to cells grown under standard pH conditions (22 days, 10% CO2 vs 16 days, 5% CO2).(ABSTRACT TRUNCATED AT 400 WORDS)
Leahy
J C
JC
Department of Pharmacology, SUNY Health Science Center at Syracuse 13210.
Chen
Q
Q
Vallano
M L
ML
D000138:Acidosis / Q000378:metabolism*; D000818:Animals; D002470:Cell Survival; D002478:Cells, Cultured; D002531:Cerebellum / Q000473:pathology*; D002908:Chronic Disease; D004594:Electrophysiology; D018698:Glutamic Acid / Q000506:poisoning; D007608:Kainic Acid / Q000506:poisoning; D016202:N-Methylaspartate / Q000378:metabolism; D009474:Neurons / Q000187:drug effects / Q000502:physiology; D009498:Neurotoxins / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D016194:Receptors, N-Methyl-D-Aspartate / Q000187:drug effects / Q000378:metabolism*; D013997:Time Factors
D009498:Neurotoxins; D016194:Receptors, N-Methyl-D-Aspartate; D018698:Glutamic Acid; D016202:N-Methylaspartate; D007608:Kainic Acid