Characterization of the metabotropic glutamate receptors mediating phospholipase C activation and calcium release in cerebellar granule cells: Calcium-dependence of the phospholipase C response

In this study we have determined the metabotropic glutamate receptors (mGluRs) involved in the glutamate activation of phospholipase C (PLC) and Ca2+ mobilization in cerebellar granule cells at 9 days in vitro; and studied the Ca2+ modulation of the PLC response. Both PLC activation and Ca2+ signalling were found to be mediated exclusively by the mGluR1 subtype, although both group I mGluRs, mGluR1α(and mGluR5, could be detected in cell extracts. Exposure of cells to medium devoid of Ca2+ for various times before agonist stimulation reduced the PLC response, which was quickly recovered following the re-exposure of cells to Ca2+-containing medium. The extent of the glutamate response correlated well with changes in the cytosolic Ca2+ concentration. On the other hand, loading of the intracellular Ca2+ stores by a transient depolarization followed by washing in nondepolarizing buffer, allowed glutamate to release stored Ca2+ in the majority of cells and enhanced glutamate activation of PLC. Under such conditions, the absence of extracellular Ca2+ during stimulation and the chelation of cytosolic Ca2+ with BAPTA/AM inhibited both glutamate-elicited Ca2+ response and PLC activation. Overall, these results indicate that the mGluR-mediated activation of PLC depends on the presence of extracellular Ca2+ and can be modulated by moderate changes of cytosolic Ca2+. Furthermore, ryanodine reduced PLC stimulation by glutamate in predepolarized cells but not in control cells, suggesting that ryanodine receptors could play a role in the potentiation of the mGluR-mediated activation of PLC by Ca2+ release in predepolarized cells.