PROPERTIES OF RYANODINE RECEPTORS IN CULTURED CEREBELLAR GRANULE NEURONS - EFFECTS OF HEXACHLOROCYCLOHEXANE ISOMERS AND CALCIUM

The binding of [H-3]ryanodine was determined in microsomal membrane pr eparations obtained from cultured cerebellar granule cells, A K-D of 1 nM and a B-max of 64 fmol/mg protein were calculated from saturation experiments, This binding was calcium dependent and maximum values wer e obtained at 100-300 mu M Ca++, Caffeine increased [H-3]ryanodine bin ding only at Ca++ concentrations lower than optimum, The binding of [H -3]ryanodine was inhibited by rutheniun red, procaine and the delta-is omer of hexachlorocyclohexane (delta-HCH). Dantrolene, a ryanodine rec eptor antagonist in skeletal sarcoplasmic reticulum, and the pesticide gamma-HCH (lindane) had no effect on [H-3]ryanodine binding, The obta ined binding parameters, the Ca++ dependence and the effects of the ag ents tested agree with previous reports using brain microsomal membran es, further indicating a neuronal localization of [H-3]ryanodine bindi ng sites, When the interaction between dantrolene and gamma- and delta -HCH was tested, no changes were detected on the effects of HCH isomer s on [H-3]ryanodine binding. Dantrolene, which inhibits Ca++ release f rom sarcoplasmic reticulum and from unidentified internal Ca++ stores in neurons, also inhibits the intracellular Ca++ mobilization induced by gamma-HCH but only marginally that induced by delta-HCH in the same preparation of cerebellar granule cells (Rosa et al.; Toxicol Appl Ph armacol, in press), Thus, the results obtained in this work verify the presence of different intracellular sites of action for the two HCH i somers: the ryanodine Ca++ channel for delta-HCH and an unidentified d antrolene-sensitive Ca++ channel for the gamma-HCH isomer. (C) 1997 Wi ley-Liss, Inc.