DIVALENT-CATION ENTRY IN CULTURED RAT CEREBELLAR GRANULE CELLS MEASURED USING MN2-2 FLUORESCENCE( QUENCH OF FURA)

In this study the rate of Mn2+ quench of fura-2 fluorescence evoked by glutamatergic and cholinergic agonists, depolarization and Ca2+ store modulators was measured in cultured cerebellar granule cells, in orde r to study their effects on Ca2+ entry in isolation from effects on Ca 2+ store release. The rate of fluorescence quench by 0.1 mM Mn2+ was m arkedly increased by 25 mM K+-evoked depolarization or by 200 mu M N-m ethyl-D-aspartate (NMDA), with a significantly greater increase occurr ing during the rapid-onset peak phase compared to the plateau phase of the K+- or NMDA-evoked [Ca2+](i) response. The stimulatory effect of NMDA on Mn2+ quench was abolished by dizocilpine (10 mu M), but nitren dipine (2 mu M), while decreasing the rate of basal quench, did not af fect NMDA-stimulated Mn2+ entry. This suggests that nitrendipine may n ot act on NMDA channels in granule cells, at least under these conditi ons, and that voltage-operated Ca2+ channels are involved in control q uench whereas the NMDA-evoked quench is dependent on entry through the receptor channel. The t(1/2) of quench was unaffected by alpha-amino- hydroxyisoxazole propionic acid (200 mu M) and carbamyl choline (1 mM) . Neither thapsigargin (10 mu M) nor dantrolene (30 mu M) significantl y affected the rate of quench under control or NMDA- or K+-stimulated conditions, which confirms that the previously reported inhibitory eff ects on [Ca2+](i) elevations evoked by these agents are due to actions on Ca2+ stores. However, thapsigargin elevated [Ca2+](i) in the prese nce of normal [Ca2+](0) but not in nominally Ca2+-free medium, indicat ing that it evokes Ca2+ entry in cerebellar granule cells, probably su bsequent to store depletion, which appears to be either too small to b e detected by Mn2+ quench or to occur via Mn2+-impermeant channels.