Mechanism of a methylxanthine drug theophylline-induced Ca2+ signaling and cytotoxicity in AML12 mouse hepatocytes

Abstract

Theophylline is a methylxanthine drug used in therapy for respiratory diseases. However, the impact of theophylline on Ca²⁺ signaling has not been explored in liver cells. This study examined whether theophylline affected Ca²⁺ homeostasis and its related cytotoxicity in AML12 mouse hepatocytes. Cell viability was measured by the cell viability reagent (WST-1). Cytosolic Ca²⁺ concentration ([Ca²⁺]_i) was measured by the Ca²⁺-sensitive fluorescent dye fura-2. Theophylline (25-125 μM) induced [Ca²⁺]_i rises and cause cytotoxicity in AML12 cells. This cytotoxic response was reversed by chelation of cytosolic Ca²⁺ with BAPTA/AM. In Ca²⁺-free medium, treatment with the endoplasmic reticulum Ca²⁺ pump inhibitor thapsigargin abolished theophylline-induced [Ca²⁺]_i rises. Conversely, treatment with theophylline also abolished thapsigargin-induced [Ca²⁺]_i rises. However, inhibition of PLC failed to alter theophylline-evoked [Ca²⁺]_i rises. In Ca²⁺-containing medium, modulators of store-operated Ca²⁺ channels inhibited 30% of the [Ca²⁺]_i rises, whereas the PKC modulators had no effect. Furthermore, theophylline-induced Ca²⁺ influx was confirmed by Mn²⁺-induced quench of fura-2 fluorescence. Together, in AML12 cells, theophylline caused Ca²⁺-associated cytotoxicity and induced Ca²⁺ entry through PLC-independent Ca²⁺ release from the endoplasmic reticulum and PKC-insensitive store-operated Ca²⁺ channels. BAPTA-AM with its protective effects may be a potential compound for prevention of theophylline-induced cytotoxicity.

Keywords: AML12 mouse hepatocytes; Ca2+ signaling; cytotoxicity; endoplasmic reticulum; store-operated Ca2+ channels; theophylline.