NMDA receptor characterization and subunit expression in rat cultured mesencephalic neurones

NMDA receptor characterization and subunit expression in rat cultured mesencephalic neurones NMDA‐induced changes in free intracellular Ca2+ concentration ((Ca2+)i) were determined in individual cultured rat mesencephalic neurones by the fura‐2 method. mRNA expression encoding NMDA receptor subunits (NR1, NR2A‐D) was examined by RT–PCR. NMDA (1–100 μM, plus 10 μM glycine) induced a concentration‐dependent increase in (Ca2+)i (EC50=5.7 μM). The effect of NMDA was virtually insensitive to tetrodotoxin (0.3 μM) and nitrendipine (1 μM), but dependent on extracellular Ca2+. 5,7‐Dichlorokynurenic acid (10 μM), a specific antagonist at the glycine binding site on the NMDA receptor, abolished the NMDA response. Memantine, an open‐channel blocker, and ifenprodil, a preferential non‐competitive NR1/NR2B receptor antagonist diminished the NMDA effect with an IC50 value of 0.17 and 1 μM, respectively. Ethanol at 50 and 100 mM caused about 25 and 45%‐inhibition, respectively. Agarose gel analysis of the PCR products followed by ethidium bromide fluorescence or CSPD chemiluminescence detection revealed an almost exclusive expression of the NR1 splice variants lacking exon (E) 5 and E22. The 3′ splice form without both E21 and E22 exceeded that containing E21 by approximately 4 fold. The relative amounts of NR2A, NR2B, NR2C corresponded to approximately 1:2:1. NR2D mRNA was also detectable. In conclusion, mesencephalic neurones bear ethanol‐sensitive NMDA receptors which might be involved in the development of ethanol dependence and withdrawal. The high affinity of NMDA to this receptor, its sensitivity to ifenprodil and memantine may suggest that the mesencephalic NMDA receptor comprises the NR1 splice variant lacking E5, NR2B, and NR2C, respectively. British Journal of Pharmacology (1999) 126, 121–130; doi:10.1038/sj.bjp.0702284 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png British Journal of Pharmacology Wiley

NMDA receptor characterization and subunit expression in rat cultured mesencephalic neurones

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Publisher
Wiley
Copyright
1999 Nature Publishing Group
ISSN
0007-1188
eISSN
1476-5381
DOI
10.1038/sj.bjp.0702284
pmid
10051128
Publisher site
See Article on Publisher Site

Abstract

NMDA‐induced changes in free intracellular Ca2+ concentration ((Ca2+)i) were determined in individual cultured rat mesencephalic neurones by the fura‐2 method. mRNA expression encoding NMDA receptor subunits (NR1, NR2A‐D) was examined by RT–PCR. NMDA (1–100 μM, plus 10 μM glycine) induced a concentration‐dependent increase in (Ca2+)i (EC50=5.7 μM). The effect of NMDA was virtually insensitive to tetrodotoxin (0.3 μM) and nitrendipine (1 μM), but dependent on extracellular Ca2+. 5,7‐Dichlorokynurenic acid (10 μM), a specific antagonist at the glycine binding site on the NMDA receptor, abolished the NMDA response. Memantine, an open‐channel blocker, and ifenprodil, a preferential non‐competitive NR1/NR2B receptor antagonist diminished the NMDA effect with an IC50 value of 0.17 and 1 μM, respectively. Ethanol at 50 and 100 mM caused about 25 and 45%‐inhibition, respectively. Agarose gel analysis of the PCR products followed by ethidium bromide fluorescence or CSPD chemiluminescence detection revealed an almost exclusive expression of the NR1 splice variants lacking exon (E) 5 and E22. The 3′ splice form without both E21 and E22 exceeded that containing E21 by approximately 4 fold. The relative amounts of NR2A, NR2B, NR2C corresponded to approximately 1:2:1. NR2D mRNA was also detectable. In conclusion, mesencephalic neurones bear ethanol‐sensitive NMDA receptors which might be involved in the development of ethanol dependence and withdrawal. The high affinity of NMDA to this receptor, its sensitivity to ifenprodil and memantine may suggest that the mesencephalic NMDA receptor comprises the NR1 splice variant lacking E5, NR2B, and NR2C, respectively. British Journal of Pharmacology (1999) 126, 121–130; doi:10.1038/sj.bjp.0702284

Journal

British Journal of PharmacologyWiley

Published: Jan 1, 1999

References

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