Bimodal modulation of store-operated Ca²⁺ channels by clozapine in astrocytes

Clozapine (Clz) and olanzapine (Olz) are second generation (atypical) antipsychotics, used widely for treating schizophrenia and bipolar disorder. These drugs share multiple sites of actions, however their mechanisms remain incompletely understood. Here, we analyzed the effects of these drugs on primary cultures of rat cortical astrocytes and C6 glioma cells using fura-2-based Ca²⁺ imaging. C6 cells, but not cortical astrocytes, express the serotonin 2A receptor subtype, which couples to phospholipase C. Clz (1μM) significantly blocked serotonin-induced Ca²⁺ transients in C6 cells, consistent with known antagonistic actions of Clz. Interestingly, at higher concentrations (>10μM), Clz but not Olz increased intracellular Ca²⁺ concentrations in both cortical astrocytes and C6 cells. This Clz-induced Ca²⁺ increase was concentration-dependent and completely blocked by removal of extracellular Ca²⁺ using ethylene glycol tetraacetic acid (EGTA). Furthermore, 2-aminoethyl diphenylborinate or SKF-96365, blockers for store-operated Ca²⁺ channels, significantly inhibited the Clz-induced Ca²⁺ increase. Therefore, we analyzed the effects of Clz and Olz during Ca²⁺ re-entry through store-operated Ca²⁺ channels, which was maximized following depletion of internal Ca²⁺ stores by thapsigargin and EGTA. The results demonstrated that Clz decreased Ca²⁺ re-entry through store-operated Ca²⁺ channels in cortical astrocytes and C6 cells whereas Olz failed to modulate the Ca²⁺ re-entry. These results suggest Clz-specific bimodal actions via store-operated Ca²⁺ channels in astrocytic cells. Since intracellular Ca²⁺ homeostasis in astrocytes is an important determinant for neighboring synaptic signal transmission, our results may explain Clz-specific adverse effects or differential actions between Clz and Olz reported in the treatment of psychiatric disorders.