35-MM K+-STIMULATED CA-45(2+) UPTAKE IN CEREBELLAR GRANULE CELL-CULTURES MAINLY RESULTS FROM NMDA RECEPTOR ACTIVATION

In primary cultures of cerebellar granule cells, the Ca2+ influx resulting from K+ depolarization (35 mM) was equal to one-third of that observed with 100 muM N-methyl-D-aspartate (NMDA) and was reduced in a major part (90%) by NMDA receptor antagonists. The rank order of potency of these competitive and non-competitive NMDA receptor antagonists was very close to their affinity for the NMDA and phencyclidine sites respectively. Granular cell depolarization with 35 mM K+ also induced a large increase in the extracellular glutamate concentration. Repeated washes of the culture wells, addition of glutamate pyruvate transaminase (+2 mM pyruvate), or pretreatment of the cells with tetanus toxin resulted in a parallel reduction of the extracellular glutamate concentration and Ca-45(2+) uptake measured after a 35 mM K+ stimulation. Dihydropyridine (BAY K-8644) stimulated the release of glutamate in a nifedipine-sensitive manner in the presence of 15 mM K+. However, nifedipine (1 muM), which decreased by 60% the K+-induced Ca-45(2+) uptake, did not reduce the 35 mM K+-evoked glutamate release. Taken together, these results demonstrated that in cerebellar granule cell cultures, 90% of the 35 mM K+-stimulated Ca-45(2+) influx resulted from the release of glutamate and the consecutive activation of NMDA receptors. Activation of these glutamate receptors then allows Ca2+ influx to occur through L-type voltage-operated Ca2+ channels.