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Afobazole Modulates Neuronal Response to Ischemia and Acidosis via Activation of σ-1 Receptors

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.