Inositol 1,4,5-triphosphate-stimulated calcium release from permeabilized cerebellar granule cells.
1. Muscarinic cholinoceptor stimulation of phosphoinositide hydrolysis in rat cultured cerebellar granule cells results in a rapid, transient accumulation of inositol 1,4,5-trisphosphate (Ins(1,4,5)P3), which has been implicated in the release of non-mitochondrial intracellular Ca2+ stores. In the present study, the release of Ca2+ from intracellular stores and the Ins(1,4,5)P3 receptor responsible for this process have been investigated. 2. Monolayers of saponin-permeabilized granule cells accumulate 45Ca2+ in an ATP-dependent manner and the sequestered 45Ca2+ can be concentration-dependently released by Ins(1,4,5)P3 by a stereospecific and heparin-sensitive mechanism. The EC50 for Ins(1,4,5)P3-stimulated 45Ca2+ release was 80 +/- 3 nM. 3. Radioligand binding studies performed on a crude granule cell membrane fraction indicated the presence of an apparently homogeneous population of stereo-specific Ins(1,4,5)P3 receptors (KD 54.7 +/- 2.0 nM; Bmax 1.37 +/- 0.29 pmol mg-1 protein). 4. This study provides evidence for Ins(1,4,5)P3-sensitive intracellular Ca2+ stores in primary cultures of cerebellar granule cells and suggest that these cells provide an excellent model neuronal system in which to study the relative functional roles of Ca2+ release from intracellular stores and Ca(2+)-entry in neuronal Ca2+ homeostasis.
Whitham
E M
EM
Department of Pharmacology and Therapeutics, University of Leicester.
Challiss
R A
RA
Nahorski
S R
SR
D000818:Animals; D001667:Binding, Competitive / Q000187:drug effects; D002118:Calcium / Q000378:metabolism*; D002121:Calcium Channel Blockers / Q000494:pharmacology; D002132:Calcium Radioisotopes; D002463:Cell Membrane Permeability / Q000187:drug effects; D002531:Cerebellum / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D003594:Cytoplasmic Granules / Q000187:drug effects / Q000378:metabolism; D006706:Homeostasis / Q000187:drug effects; D066298:In Vitro Techniques; D015544:Inositol 1,4,5-Trisphosphate / Q000494:pharmacology*; D007700:Kinetics; D011869:Radioligand Assay; D051381:Rats; D012503:Saponins / Q000494:pharmacology
D002121:Calcium Channel Blockers; D002132:Calcium Radioisotopes; D012503:Saponins; D015544:Inositol 1,4,5-Trisphosphate; D002118:Calcium