Activation of phospholipase C (PLC) upon inhibition of mGluR5. (A) Intracellular Ca2+ concentrations were determined by measuring the fluorescence intensity of the fluorochrome Fluo-4 AM. BV-2 cells were pretreated for 1 hour with 5 μM U-73122 prior to the addition of 100 μM MPEP. Relative changes in the Ca2+ concentrations detected by Fluo-4 AM were analyzed after 15 minutes. Results are expressed as ΔF/F (in %), with F being the baseline fluorescence and ΔF the variation of fluorescence. Data represent the mean ± SD. (B) Cells were incubated for 24 hours with 100 μM MPEP in the presence or absence of 5 μM of the phospholipase C inhibitor U-73122, followed by determination of mRNA expression of CHOP, GRP78 and GRP95 by quantitative PCR. Results were normalized to GAPDH mRNA control. Results represent the mean ± SD from three independent experiments, each performed in triplicate. (C), BV-2 cells were incubated with 100 μM MPEP for 2 hours with or without pretreatment for 1 hour with 5 μM U-73122, followed by determination of PLC activity. PLC activity (mU/μg total protein) is given as the mean ± SD from three independent experiments. (D) BV-2 cells were incubated with 100 μM MPEP in the absence or presence of 5 μM PLC inhibitor U-73122 or 1 μM Ca2+ chelator BAPTA-AM, followed by the determination of GRP78 protein expression by immunoblotting. (E) Densitometric analysis of GRP78 band normalized against β-actin. **P <0.01; ***P <0.001. CHOP, C/EBP homologous protein; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; MPEP, 2-methyl-6-(phenylethynyl)-pyridine.