Thapsigargin,a selective inhibitor of sarco-endoplasmic reticulum Ca2+-ATPases,modulates nitric oxide production and cell death of primary rat hepatocytes in culture

Increased cytosolic calcium ([Ca²⁺] _i ) and nitric oxide (NO) are suggested to be associated with apoptosis that is a main feature of many liver diseases and is characterized by biochemical and morphological features. We sought to investigate the events of increase in [Ca²⁺] _i and endoplasmic reticulum (ER) calcium depletion by thapsigargin (TG), a selective inhibitor of sarco-ER-Ca²⁺-ATPases, in relation to NO production and apoptotic and necrotic markers of cell death in primary rat hepatocyte culture. Cultured hepatocytes were treated with TG (1 and 5 μmol/L) for 0–24 or 24–48 h. NO production and inducible NO synthase (iNOS) expression were determined as nitrite levels and by iNOS-specific antibody, respectively. Hepatocyte apoptosis was estimated by caspase-3 activity, cytosolic cytochrome c content and DNA fragmentation, and morphologically using Annexin-V/propidium iodide staining. Hepatocyte viability and mitochondrial activity were evaluated by ALT leakage and MTT test. Increasing basal [Ca²⁺] _i by TG, NO production and apoptotic/necrotic parameters were altered in different ways, depending on TG concentration and incubation time. During 0–24 h, TG dose-dependently decreased iNOS-mediated spontaneous NO production and simultaneously enhanced hepatocyte apoptosis. In addition, TG 5 μmol/L produced secondary necrosis. During 24–48 h, TG dose-dependently enhanced basal NO production and rate of necrosis. TG 5 μmol/L further promoted mitochondrial damage as demonstrated by cytochrome c release. A selective iNOS inhibitor, aminoguanidine, suppressed TG-stimulated NO production and ALT leakage from hepatocytes after 24–48 h. Our data suggest that the extent of the [Ca²⁺] _i increase and the modulation of NO production due to TG treatment contribute to hepatocyte apoptotic and/or necrotic events.