Properties of ryanodine receptors in cultured cerebellar granule neurons: effects of hexachlorocyclohexane isomers and calcium.
The binding of [3H]ryanodine was determined in microsomal membrane preparations obtained from cultured cerebellar granule cells. A KD of 1 nM and a Bmax of 64 fmol/mg protein were calculated from saturation experiments. This binding was calcium dependent and maximum values were obtained at 100-300 microM Ca+2. Caffeine increased [3H]ryanodine binding only at Ca+2 concentrations lower than optimum. The binding of [3H]ryanodine was inhibited by ruthenium red, procaine and the delta-isomer of hexachlorocyclohexane (delta-HCH). Dantrolene, a ryanodine receptor antagonist in skeletal sarcoplasmic reticulum, and the pesticide gamma-HCH (lindane) had no effect on [3H]ryanodine binding. The obtained binding parameters, the Ca+2 dependence and the effects of the agents tested agree with previous reports using brain microsomal membranes, further indicating a neuronal localization of [3H]ryanodine binding sites. When the interaction between dantrolene and gamma- and delta-HCH was tested, no changes were detected on the effects of HCH isomers on [3H]ryanodine binding. Dantrolene, which inhibits Ca+2 release from sarcoplasmic reticulum and from unidentified internal Ca+2 stores in neurons, also inhibits the intracellular Ca+2 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 Pharmacol, in press). Thus, the results obtained in this work verify the presence of different intracellular sites of action for the two HCH isomers: the ryanodine Ca+2 channel for delta-HCH and an unidentified dantrolene-sensitive Ca+2 channel for the gamma-HCH isomer.
Rosa
R
R
Department of Pharmacology, Instituto de Investigaciones Biomédicas de Barcelona, Consejo Superior de Investigaciones Científicas, Spain.
Sanfeliu
C
C
Rodríguez-Farré
E
E
Frandsen
A
A
Schousboe
A
A
Suñol
C
C
D000779:Anesthetics, Local / Q000494:pharmacology; D000818:Animals; D001667:Binding, Competitive / Q000502:physiology; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology*; D015220:Calcium Channels / Q000378:metabolism*; D002148:Calmodulin-Binding Proteins / Q000502:physiology; D002273:Carcinogens / Q000737:chemistry / Q000494:pharmacology*; D002478:Cells, Cultured / Q000737:chemistry / Q000187:drug effects; D002492:Central Nervous System Depressants / Q000737:chemistry / Q000494:pharmacology*; D002531:Cerebellum / Q000166:cytology; D004396:Coloring Agents / Q000494:pharmacology; D003620:Dantrolene / Q000494:pharmacology; D001556:Hexachlorocyclohexane / Q000737:chemistry / Q000494:pharmacology*; D006706:Homeostasis / Q000187:drug effects; D007536:Isomerism; D008861:Microsomes / Q000737:chemistry / Q000378:metabolism; D009124:Muscle Proteins / Q000378:metabolism*; D009125:Muscle Relaxants, Central / Q000494:pharmacology; D009474:Neurons / Q000737:chemistry* / Q000166:cytology / Q000187:drug effects; D011343:Procaine / Q000494:pharmacology; D051381:Rats; D012430:Ruthenium Red / Q000494:pharmacology; D012433:Ryanodine / Q000378:metabolism / Q000494:pharmacology; D019837:Ryanodine Receptor Calcium Release Channel; D014316:Tritium
D000779:Anesthetics, Local; D015220:Calcium Channels; D002148:Calmodulin-Binding Proteins; D002273:Carcinogens; D002492:Central Nervous System Depressants; D004396:Coloring Agents; D009124:Muscle Proteins; D009125:Muscle Relaxants, Central; D019837:Ryanodine Receptor Calcium Release Channel; D014316:Tritium; D012430:Ruthenium Red; D012433:Ryanodine; C086916:delta-hexachlorocyclohexane; D011343:Procaine; D001556:Hexachlorocyclohexane; D003620:Dantrolene; D002118:Calcium