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

Abstract

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.