Interactions between NMDA receptors and mGlu5 receptors expressed in HEK293 cells

Ca2+ imaging was used to investigate interactions between responses induced by N-methyl-D-aspartate (NMDA; 15 μM) and (RS)-3,5-dihydroxyphenyl-glycine (DHPG; 30 μM) in human embryonic kidney (HEK) 293 cells, transiently transfected with rat recombinant NR1a, NR2A and mGlu5a cDNA. Responses to NMDA were reversibly depressed by DHPG from 244±14 to 194±12% of baseline. Treatment with thapsigargin (1 μM, 10 min) prevented this effect. After thapsigargin pretreatment, repeated applications of NMDA showed a gradual rundown in amplitude over a period of several hours, and were unaffected by DHPG. Continuous perfusion with staurosporine (0.1 μM), after thapsigargin pretreatment, converted the run-down to a small increase in NMDA responses to 123±6 % of baseline. DHPG induced a further and sustained potentiation of NMDA responses to 174±12% of the initial baseline. The protein tyrosine kinase (PTK) inhibitors genistein (50 μM) and 3-(4-chlorophenyl)1-(1,1-dimethylethyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP2; 1 μM) inhibited the staurosporine- and DHPG-induced potentiation of NMDA responses. The protein phosphatase (PTP) inhibitors orthovanadate (100 μM) and phenyl arsine oxide (PAO, 1 μM) facilitated the staurosporine-evoked potentiation of NMDA responses and occluded DHPG-induced potentiation. In conclusion, complex interactions can be demonstrated between mGlu5 and NMDA receptors expressed in HEK293 cells. There is a negative inhibitory influence of Ca2+ release and PKC activation. Inhibition of these processes reveals a tonic, mGlu5 receptor and PTK-dependent potentiation of NMDA receptors that can be augmented by either stimulating mGlu5 receptors or by inhibiting PTPs. British Journal of Pharmacology (2004) 142, 991–1001. doi:10.1038/sj.bjp.0705861