Interactions between NMDA receptors and mGlu5 receptors expressed in HEK293 cells

1 Ca2+ imaging was used to investigate interactions between responses induced by N-methyl-D-aspartate (NMDA; 15 muM) and (RS)-3,5-dihydroxyphenyl-glycine (DHPG; 30 muM) in human embryonic kidney (HEK) 293 cells, transiently transfected with rat recombinant NR1a, NR2A and mGlu(5a) cDNA. 2 Responses to NMDA were reversibly depressed by DHPG from 244 +/- 14 to 194 +/- 12% of baseline. Treatment with thapsigargin (1 muM, 10 min) prevented this effect. 3 After thapsigargin pretreatment, repeated applications of NMDA showed a gradual rundown in amplitude over a period of several hours, and were unaffected by DHPG. 4 Continuous perfusion with staurosporine (0.1 muM), 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. 5 The protein tyrosine kinase (PTK) inhibitors genistein (50 muM) and 3-(4-chlorophenyl)1-(1,1-dimethylethyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP2; 1 muM) inhibited the staurosporine- and DHPG-induced potentiation of NMDA responses. 6 The protein phosphatase (PTP) inhibitors orthovanadate (100 muM) and phenyl arsine oxide (PAO, 1 muM) facilitated the staurosporine-evoked potentiation of NMDA responses and occluded DHPG-induced potentiation. 7 In conclusion, complex interactions can be demonstrated between mGlu, 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, mGIU(5) receptor and PTK-dependent potentiation of NMDA receptors that can be augmented by either stimulating mGlu(5) receptors or by inhibiting PTPs.