INTERACTIONS BETWEEN CA2+ MOBILIZING MECHANISMS IN CULTURED RAT CEREBELLAR GRANULE CELLS

1. The interactions between IP3 receptor-mediated and Ca2+-induced Ca2+ release were investigated in cerebellar granule cell bodies, using the techniques of microfluorimetry and image analysis. 2. The IP3-sensitive Ca2+ release mechanism was activated using acetylcholine (ACh) and the selective metabotropic glutamate receptor agonist 1-aminocyclo-pentane-1S,3R-dicarboxylic acid (ACPD). Caffeine was used to activate, and ryanodine to inhibit, the Ca2+-induced Ca2+ release process. Thapsigargin was used to deplete intracellular Ca2+ stores. 3. Transient applications of caffeine (5-50 mm), ACPD (50-500 muM) and ACh (0.05-1 muM) mobilized intracellular Ca2+ ([Ca2+]i). Ca2+ mobilizing responses to 50 mM caffeine and 1 muM ACh increased with time in culture until day 4. However, beyond this period the responsiveness of cells to caffeine, but not to ACh, declined markedly. 4. Responses induced by ACPD and ACh were inhibited in the presence of caffeine at concentrations below those which mobilized Ca2+ (1-5 mM). This effect was not due to Ca2+ pool depletion, elevation of cAMP or inhibition of phosphodiesterases. 5. Prior challenge with ACh or ACPD inhibited Ca2+ mobilization induced by caffeine (50 mM). Transient exposure to caffeine inhibited subsequent responses to ACh through a mechanism which involved store depletion. 6. Thapsigargin (0.1-1 muM) inhibited, to a similar extent, Ca2+ mobilization induced by caffeine, ACPD and ACh. 7. Ryanodine (10 muM) antagonized Ca2+ mobilization induced by caffeine, ACh and ACPD. However, the ability of ryanodine to block inositol 1,4,5-trisphosphate-linked agonist responses varied considerably between cells. The sensitivity of ACh-induced responses to ryanodine correlated with the sensitivity of the cells to caffeine. 8. The possible explanations for the pronounced interactions between IP3 receptor-mediated and Ca2+-induced Ca2+ release processes in cerebellar granule cells are discussed.