Inhibitory ryanodine prevents ryanodine receptor-mediated Ca2+ release without affecting endoplasmic reticulum Ca2+ content in primary hippocampal neurons

Abstract

Ryanodine is a cell permeant plant alkaloid that binds selectively and with high affinity to ryanodine receptor (RyR) Ca2þ release channels. Sub-micromolar ryanodine concentrations activate RyR channels while micromolar concentrations are inhibitory. Several reports indicate that neuronal synaptic plasticity, learning and memory require RyR-mediated Ca2þ-release, which is essential for muscle contraction. The use of micromolar (inhibitory) ryanodine represents a common strategy to suppress RyR activity in neuronal cells: however, micromolar ryanodine promotes RyR-mediated Ca2þ release and endoplasmic reticulum Ca2þ depletion in muscle cells. Information is lacking in this regard in neuronal cells; hence, we examined here if addition of inhibitory ryanodine elicited Ca2þ release in primary hippocampal neurons, and if prolonged incubation of primary hippocampal cultures with inhibitory ryanodine affected neuronal ER calcium content. Our results indicate that inhibitory ryanodine does not cause Ca2þ release from the ER in primary hippocampal neurons, even though ryanodine diffusion should produce initially low intracellular concentrations, within the RyR activation range. Moreover, neurons treated for 1 h with inhibitory ryanodine had comparable Ca2þ levels as control neurons. These combined findings imply that prolonged incubation with inhibitory ryanodine, which effectively abolishes RyR-mediated Ca2þ release, preserves ER Ca2þ levels and thus constitutes a sound strategy to suppress neuronal RyR function.