A simple cell line based in vitro test system for N-methyl-D-aspartate (NMDA) receptor ligands

The generation of cell lines stably expressing the functional recombinant N-methyl-D-aspartate (NMDA) receptors (NRs) and their use for ligand testing in a simple excitotoxicity model is described. The mouse fibroblast cell line L(tk-) was co-transfected stably with cDNAs encoding the human NR subunits, NR1-1a/NR2A or NR1-1a/NR2B, respectively. The NR expression and functionality in resulting clones have been verified by RT-PCR, Western blotting, immunocytochemistry and fluo-4 calcium imaging. Stimulation of NR expressing clones with L-glutamate and glycine resulted in necrosis of cultures within 1 h. Therefore, a lactate dehydrogenase-based excitotoxicity assay was used for the pharmacological characterisation. The two selected clones exhibited pharmacological properties corresponding to the distinct NR subunit assemblies. Both cell lines showed proton inhibition of cell death in the range of physiological pH. EC50-values for L-glutamate under saturated D-serine concentrations were 3.7 microM for L12-G10 (NR1-1a/NR2A) and 2.8 microM for L13-E6 (NR1-1a/NR2B), respectively. Competitive antagonists (RS)-APV and (RS)-CPP as well as glycine B site antagonist DCKA prevented L-glutamate/glycine-induced cell death. NR2B selective antagonists such as ifenprodil or haloperidol did only protect L13-E6 cells. Spermine (300 microM) triggered cell death selectively in the L13-E6 clone in a pH-dependent manner.