Abstract

Afobazole is an anxiolytic medication that has been previously shown to be neuroprotective both in vitro and in vivo. However, the mechanism(s) by which afobazole can enhance neuronal survival remain poorly understood. Experiments were carried out to determine whether afobazole can decrease intracellular calcium overload associated with ischemia and acidosis and whether the effects of afobazole are mediated via interaction of the compound with σ receptors. Fluorometric Ca²⁺ imaging was used to resolve how application of afobazole affects intracellular Ca²⁺ handling in cortical neurons. Application of afobazole significantly depressed, in a concentration-dependent and reversible manner, the intracellular Ca²⁺ overload resulting from in vitro ischemia and acidosis. The IC₅₀ for afobazole inhibition of ischemia-evoked intracellular Ca²⁺ overload was considerably less than that for the inhibition of [Ca²⁺]_i increases induced by acidosis. However, afobazole maximally inhibited only 70% of the ischemia-evoked intracellular Ca²⁺ overload but effectively abolished intracellular Ca²⁺ increases produced by acidosis. The effects of afobazole on ischemia- and acidosis-induced intracellular Ca²⁺ dysregulation were inhibited by preincubating the neurons in the irreversible, pan-selective σ-receptor antagonist, metaphit. Moreover, the effects of afobazole on intracellular Ca²⁺ increases triggered by acidosis and ischemia were blocked by the selective σ-1-receptor antagonists, BD 1063 and BD 1047, respectively. Experiments examining the effects of afobazole on neuronal survival in response to ischemia showed that afobazole was neuroprotective. Taken together, these data suggest that afobazole regulates intracellular Ca²⁺ overload during ischemia and acidosis via activation of σ-1 receptors. This mechanism is probably responsible for afobazole-mediated neuroprotection.