An investigation of calcium-induced calcium-release (CICR) in cultured rat sensory neurones
In this study the mechanisms of Ca2+-induced-Ca2+-release, effects of membrane depolarizations and the actions of pharmacological intracellular Ca2+-modulators were examined in cultured rat dorsal root ganglion (DRG) neurones. The whole cell configuration of the patch clamp technique was used to record action potentials, action potential after-potentials and voltage-activated calcium currents, (ICa), calcium-activated chloride currents, (ICI(Ca)), and non-selective cation currents, (ICAN), under current and voltage clamp recording conditions, respectively. A sub population of DRG neurones expressed action potential after-depolarizations and ICI(Ca) tail currents which were due to activation of Ca2+-activated Cl- channels as a result of Ca2+ entry. ICAN was dominantly activated due to Ca2+ release from intracellular stores evoked by pharmacological Ca2+-releasing agents such as caffeine, ryanodine and dihydrosphingosine. Calcium-activated conductances were identified by estimating reversal potentials of the activated currents, using selective pharmacological blockers and extracellular ionic replacement studies. Calcium-dependence of activated currents was also examined by using high concentration of intracellular Ca2+ buffer, EGTA, to prevent elevation of intracellular Ca2+-levels and by rapidly buffering raised intracellular Ca2+ using intracellular 'caged Ca2+ chelator', diazo-2. The involvement of intracellular Ca2+- stores was examined by performing experiments in Ca2+-free extracellular recording medium and pharmacologically inhibiting release of Ca2+ from intracellular stores, using dantrolene. Ryanodine had complex actions on DRG neurones, which reflected its ability to mobilize Ca2+, deplete Ca2+ stores, and inhibit Ca2+ release channels. Ryanodine inhibited action potential after-depolarizations and ICI(Ca) tail currents by interacting with intracellular stores and preventing amplification of Ca2+ signalling by CICR. It was found that CICR observed under physiological conditions in rat DRG neurones involves intracellular Ca2+ stores which were sensitive to ryanodine. In addition to ryanodine sensitivity these intracellular Ca2+ stores could be mobilized by caffeine and dihydrosphingosine.