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 micromol/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 micromol/L produced secondary necrosis. During 24-48 h, TG dose-dependently enhanced basal NO production and rate of necrosis. TG 5 micromol/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.
Canová
N Kutinová
NK
Institute of Pharmacology, 1st Faculty of Medicine, Charles University, Albertov 4, Prague 2, Czech Republic. ncano@lf1.cuni.cz
Kmonícková
E
E
Martínek
J
J
Zídek
Z
Z
Farghali
H
H
D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D002118:Calcium / Q000502:physiology; D053148:Caspase 3 / Q000378:metabolism; D016923:Cell Death / Q000187:drug effects; D002467:Cell Nucleus / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D006146:Guanidines / Q000494:pharmacology; D022781:Hepatocytes / Q000187:drug effects / Q000502:physiology*; D008928:Mitochondria / Q000187:drug effects / Q000502:physiology; D009569:Nitric Oxide / Q000096:biosynthesis*; D052247:Nitric Oxide Synthase Type II / Q000037:antagonists & inhibitors / Q000378:metabolism; D051381:Rats; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D019284:Thapsigargin / Q000494:pharmacology*
D006146:Guanidines; D009569:Nitric Oxide; D019284:Thapsigargin; D052247:Nitric Oxide Synthase Type II; C496318:Nos2 protein, rat; D053148:Caspase 3; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; C004479:pimagedine; D002118:Calcium