Receptor-mediated Ca2+ signalling in cultured rat cerebellar granule cells.

Neurones possess many pathways which can mediate Ca2+ entry, and two mechanisms, involving inositol 1,4,5-trisphosphate (InsP3) and ryanodine receptors, by which Ca2+ in intracellular stores can be released. Cultured cerebellar granule cells were utilized in this study as a model neuronal system for investigating the relationships between entry and store release, and between the different mechanisms of Ca2+ release. Ca2+ entry mechanisms activated by K+-depolarization and N-methyl-D- aspartate (NMDA) were studied using epifluorescence microscopy with the Ca2+-binding dye fura 2, for [Ca2+]i and Mn2+ quench studies. The voltage-operated Ca2+ channels involved in the K+-evoked response were characterized, while the NMDA response was found to be mediated by the receptor channel and to display considerable variability between cell groups. Both agents were found to evoke significant Ca2+ release from stores, a phenomenon apparently dependent on Ca2+ entry. Interactions between responses evoked by the metabotropic glutamate receptor agonist 1-aminocyclopentane-1S,3R-dicarboxylic acid (ACPD) and NMDA were studied and found to be complex, including a variable ACPD-evoked inhibition of NMDA [Ca2+]i responses which was apparently mediated via protein kinase C activation. Carbachol (CCh) acting on muscarinic M3 receptors evoked a marked elevation of total inositol phosphates, InsP3 mass and [Ca2+]i. The [Ca2+]i response was inhibited by Ca2+ store-modulating agents. Both CCh- and NMDA-evoked [Ca2+]i responses appeared to involve activation of ryanodine receptors, the muscarinic activation of ryanodine receptors probably occurring secondary to Ca2+ release via InsP3 receptors. An apparent pharmacological differentiation of the primary stores involved in the two responses suggested that InsP3 receptor-expressing stores may be physically different from ryanodine receptor-expressing stores in these neurones. CCh, like ACPD, downregulated expression of InsP3 receptors during chronic stimulation, as measured by Western blotting, leading to subsequently decreased responses to both CCh and ACPD, but was apparently without effect on directly ryanodine receptor-mediated responses. After chronic CCh pre-treatment, NMDA responses were unchanged and so may not require significant activation of InsP3 receptors. Thus several aspects of the complexity of neuronal Ca2+ signalling have been elucidated in this study, notably the activation of Ca2+ release by Ca2+ entry, the activation of both ryanodine and InsP3 receptors by muscarinic stimulation, and the possible expression of at least two different Ca2+ stores. These findings may have important implications for our understanding of how neuronal signalling occurs in vivo.