PMID- 17447015 OWN - NLM STAT- MEDLINE DCOM- 20080111 LR - 20131121 IS - 0742-2091 (Print) IS - 0742-2091 (Linking) VI - 23 IP - 5 DP - 2007 Sep TI - Thapsigargin, a selective inhibitor of sarco-endoplasmic reticulum Ca2+ -ATPases, modulates nitric oxide production and cell death of primary rat hepatocytes in culture. PG - 337-54 AB - 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. FAU - Canova, N Kutinova AU - Canova NK AD - Institute of Pharmacology, 1st Faculty of Medicine, Charles University, Albertov 4, Prague 2, Czech Republic. ncano@lf1.cuni.cz FAU - Kmonickova, E AU - Kmonickova E FAU - Martinek, J AU - Martinek J FAU - Zidek, Z AU - Zidek Z FAU - Farghali, H AU - Farghali H LA - eng PT - Journal Article PT - Research Support, Non-U.S. Gov't DEP - 20070419 PL - Switzerland TA - Cell Biol Toxicol JT - Cell biology and toxicology JID - 8506639 RN - 0 (Guanidines) RN - 31C4KY9ESH (Nitric Oxide) RN - 67526-95-8 (Thapsigargin) RN - EC 1.14.13.39 (Nitric Oxide Synthase Type II) RN - EC 1.14.13.39 (Nos2 protein, rat) RN - EC 3.4.22.- (Caspase 3) RN - EC 3.6.3.8 (Sarcoplasmic Reticulum Calcium-Transporting ATPases) RN - SCQ4EZQ113 (pimagedine) RN - SY7Q814VUP (Calcium) SB - IM MH - Animals MH - Apoptosis/drug effects/*physiology MH - Calcium/physiology MH - Caspase 3/metabolism MH - Cell Death/drug effects MH - Cell Nucleus/drug effects/physiology MH - Cells, Cultured MH - Guanidines/pharmacology MH - Hepatocytes/drug effects/*physiology MH - Mitochondria/drug effects/physiology MH - Nitric Oxide/*biosynthesis MH - Nitric Oxide Synthase Type II/antagonists & inhibitors/metabolism MH - Rats MH - Sarcoplasmic Reticulum Calcium-Transporting ATPases/antagonists & inhibitors/*metabolism MH - Thapsigargin/*pharmacology EDAT- 2007/04/21 09:00 MHDA- 2008/01/12 09:00 CRDT- 2007/04/21 09:00 PHST- 2006/10/02 00:00 [received] PHST- 2007/01/21 00:00 [accepted] PHST- 2007/04/21 09:00 [pubmed] PHST- 2008/01/12 09:00 [medline] PHST- 2007/04/21 09:00 [entrez] AID - 10.1007/s10565-007-0185-6 [doi] PST - ppublish SO - Cell Biol Toxicol. 2007 Sep;23(5):337-54. doi: 10.1007/s10565-007-0185-6. Epub 2007 Apr 19.