Ca²⁺ signaling as a mechanism of haloperidol-induced cytotoxicity in human astrocytes and assessing the protective role of a Ca²⁺ chelator

Abstract

Haloperidol, a typical antipsychotic medication, has been shown to possess various biological effects in different brain models. However, the impact of haloperidol on Ca²⁺ signaling in astrocytes is elusive. This study explored the effect of haloperidol on cytosolic free Ca²⁺ levels ([Ca²⁺]_i) and viability, and established these two connections in Gibco® Human Astrocytes (GHAs) and DI TNC1 rat astrocytes. Haloperidol (5–20 μM) caused [Ca²⁺]_i rises in a concentration-dependent manner in GHAs but not in DI TNC1 cells. Furthermore, removal of extracellular Ca²⁺ reduced haloperidol’s effect by approximately 30% in GHAs. Haloperidol (20–40 μM) evoked concentration-dependent cytotoxicity in GHAs and DI TNC1 cells. However, chelating cytosolic Ca²⁺ with the Ca²⁺ chelator BAPTA/AM significantly reversed haloperidol’s cytotoxicity only in GHAs. In GHAs, haloperidol-induced Ca²⁺ entry was inhibited by store-operated Ca²⁺ modulators (2-APB and SKF96365) and the protein kinase C (PKC) inhibitor GF109203X. This Ca²⁺ entry induced by haloperidol was confirmed by Mn²⁺ entry-induced quench of fura-2 fluorescence. In Ca²⁺-free medium, treatment with the endoplasmic reticulum Ca²⁺ pump inhibitor 2,5-di-tert-butylhydroquinone (BHQ) abolished haloperidol-induced [Ca²⁺]_i rises. Conversely, treatment with haloperidol inhibited 45% of BHQ-evoked [Ca²⁺]_i rises. Moreover, haloperidol-induced Ca²⁺ release from the endoplasmic reticulum was abolished by inhibition of phospholipase C (PLC) by U73122. Together, in GHAs but not in DI TNC1 cells, haloperidol caused Ca²⁺-associated cell death, induced Ca²⁺ entry via PKC-sensitive store-operated Ca²⁺ channels, and evoked PLC-dependent Ca²⁺ release from the endoplasmic reticulum. The protective effect of Ca²⁺ chelating on haloperidol-induced cytotoxicity in human astrocytes was also demonstrated.