Metabotropic glutamate receptor pharmacology and signalling in model and neuronal cell systems

In rat cerebellar granule cells measurement of phosphoinositide turnover and cAMP formation revealed that mGluR effects on these two parameters were developmentally regulated. Inhibitory effects on cAMP formation and phosphoinositide turnover increased in parallel followed by a decline to lower values coinciding with an increased sensitivity of the neurons to NMDA and L-glutamate excitotoxicity. Phosphoinositide responses mediated by group I mGluRs present on cerebellar granule neurons (thought previously to be predominantly mediated by mGluR1 in these cells) were insensitive to pertussis toxin (PTx). Consequently, the effect of PTx on mGluR1-mediated phosphoinositide signalling in BHK cells was the subject of further investigation in the present work.;In the present study, NMDA concentration-dependently induced neuronal cell death assessed using the [3H]-ouabain binding technique. However, mGluR-mediated second messenger responses could no longer be demonstrated in mature neurons that had become sensitive to glutamate excitotoxicity. Thus, in contrast to hippocampal and cerebral cortical cultures, rat cerebellar granule cells appear to be unsuitable for investigations of this type. However, validation studies using the [3H]-ouabain binding method indicated that this technique reliably predicts the extent of neuronal cell death in cerebellar granule cells.;Pre-treatment of NHK-mGluR1 cells with PTx led to a dramatic increase in the phosphoinositide turnover in the absence of added agonist. Although only very low levels of L-glutamate could be detected in medium taken from control and PTx-treated cell monolayers, the PTx-elicited effect on basal phosphoinositide turnover was reversed by pre-incubation in the presence of glutamic-pyruvic transaminase (GPT) and pyruvate, suggesting that an increased sensitivity to endogenous glutamate was responsible for the activation of phospholipase C (PLC) following PTx treatment. Consistent with this hypothesis, in the presence of GPR/pyruvate, maximal phosphoinositide responses to quisqualate were augmented and agonist potency increased in PTx-treated compared to control cells.