Regulation of N-methyl-D-aspartate receptor expression and N-methyl-D-aspartate-induced cellular response during chronic hypoxia in differentiated rat PC12 cells.
The purpose of the present study was to examine the effect of chronic hypoxia on N-methyl-D-aspartate-mediated cellular responses in differentiated PC12 cells. PC12 cells were differentiated by treatment with nerve growth factor. Patch-clamp analysis in differentiated PC12 cells showed that extracellularly applied N-methyl-D-aspartate induced an inward current that was abolished by the presence of the N-methyl-D-aspartate receptor antagonist MK-801. Results from Ca(2+) imaging experiments showed that N-methyl-D-aspartate induced an elevation in intracellular free Ca(2+) which was also abolished by MK-801. We also examined the effect of hypoxia on the N-methyl-D-aspartate-induced current in nerve growth factor-treated cells. We found that the N-methyl-D-aspartate-induced inward current and the N-methyl-D-aspartate-induced elevation in intracellular free Ca(2+) were markedly attenuated by chronic hypoxia. We next examined the possibility that the reduced N-methyl-D-aspartate responsiveness was due to down-regulation of N-methyl-D-aspartate receptor levels. Northern blot and immunoblot analyses showed that both messenger RNA and protein levels for N-methyl-D-aspartate receptor subunit 1 were markedly decreased during hypoxia. However, the messenger RNA for N-methyl-D-aspartate receptor subunit 2C was increased, whereas the protein level for subunit 2C did not change. Our results indicate that differentiated PC12 cells express functional N-methyl-D-aspartate receptors and that chronic exposure to hypoxia attenuates the N-methyl-D-aspartate-induced Ca(2+) accumulation in these cells via down-regulation of N-methyl-D-aspartate receptor subunit 1. This mechanism may play an important role in protecting PC12 cells against hypoxic stress.
Kobayashi
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Department of Molecular and Cellular Physiology, University of Cincinnati Medical Center, Cincinnati, OH 45267-0576, USA.
Millhorn
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D000818:Animals; D015214:Antigens, Differentiation, Myelomonocytic / Q000494:pharmacology*; D002454:Cell Differentiation; D002470:Cell Survival; D002908:Chronic Disease; D000860:Hypoxia / Q000378:metabolism* / Q000473:pathology* / Q000503:physiopathology; D020932:Nerve Growth Factor / Q000494:pharmacology; D016716:PC12 Cells / Q000187:drug effects / Q000378:metabolism / Q000473:pathology / Q000502:physiology; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D016194:Receptors, N-Methyl-D-Aspartate / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000494:pharmacology*
D015214:Antigens, Differentiation, Myelomonocytic; D020932:Nerve Growth Factor; D012333:RNA, Messenger; D016194:Receptors, N-Methyl-D-Aspartate; D014157:Transcription Factors; C075373:MNDA protein, human