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Accumulating evidence has indicated the involvement of glutamatergic neurotransmission in the pathophysiology of excitotoxicity and in the mechanism of action of antidepressants. We have previously shown that tricyclic desipramine and the selective serotonin reuptake inhibitor fluoxetine inhibit NMDA receptors (NMDARs) in the clinically relevant, low micromolar concentration range. As the different subtypes of NMDARs are markedly different in their physiological and pathological functions, our aim was to investigate whether the effect of antidepressants is subtype-specific. Using whole-cell patch-clamp recordings in rat cortical cell cultures, we studied the age-dependence of inhibition of NMDA-induced currents after treatment with desipramine and fluoxetine, as the expression profile of the NMDAR subtypes changes as a function of days in vitro. We also investigated the inhibitory effect of these antidepressants on NMDA-induced currents in HEK 293 cell lines that stably expressed rat recombinant NMDARs with GluN1a/GluN2A or GluN1a/GluN2B subunit compositions. The inhibitory effect of desipramine was not age-dependent, whereas fluoxetine displayed a continuously decreasing inhibitory profile, which was similar to the GluN1/GluN2B subtype-selective antagonist ifenprodil. In HEK 293 cells, desipramine equally inhibited NMDA currents in both cell lines, whereas fluoxetine showed an inhibitory effect only in cells that expressed the GluN1/GluN2B subtype. Our data show that fluoxetine is a selective inhibitor of GluN2B-containing NMDARs, whereas desipramine inhibits both GluN1/GluN2A and GluN1/GluN2B subtypes. As the clinical efficacy of these drugs is very similar, the putative NMDAR-associated therapeutic effect of antidepressants may be mediated only via inhibition of the GluN2B-containing subtype. The manifestation of the GluN1/GluN2B-selectivity of fluoxetine suggests the neuroprotective potential for this drug in both acute and chronic neurodegenerative disorders., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

In this study, we have established a non-neuronal cell line stably and inducibly expressing recombinant NMDA receptors (NRs) composed of rat NR1a/NR2A subunits. EcR-293 cells were transfected with rat NR1a and NR2A cDNAs using the inducible mammalian expression vector pIND. Cell colonies resistant for the selecting agents were picked and tested for NR2A mRNA as well as protein expression using quantitative RT-PCR and flow cytometry based immunocytochemistry. Clonal cells expressing functional NMDA receptors were identified by measuring NMDA-evoked ion currents, and NMDA-induced increase in cytosolic free calcium concentration in whole-cell patch-clamp and fluorimetric calcium measurements, respectively. One clone named D5/H3, which exhibited the highest response to NMDA, was chosen to examine inducibility of the expression and for pharmacological profiling of recombinant NR1a/NR2A NMDA receptors. To check inducibility, NR2A subunit expression in D5/H3 cells treated with the inducing agent muristerone A (MuA) was compared with that in non-induced cells. Both NR2A mRNA and protein expression was several folds higher in cells treated with the inducing agent. As part of the pharmacological characterization, we examined the activation of the expressed NR1a/NR2A receptors as a function of increasing concentration of NMDA. NMDA-evoked concentration-dependent increases in cytosolic [Ca2+] with an EC50 value of 41 +/- 1 microM. In addition, whereas the NMDA response was concentration-dependently inhibited by the channel blocker MK-801 (IC50 = 58 +/- 6 nM), NR2B subunit selective NMDA receptor antagonists were ineffective. Thus, this cell line, which stably and inducibly expresses recombinant NR1a/NR2A NMDA receptors, can be a useful tool for testing NMDA receptor antagonists and studying their subunit selectivity., ((c) 2005 Elsevier Ltd. All rights reserved.)