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