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

Abstract

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.

Nitric oxide facilitates NFAT-dependent transcription in mouse myotubes

“…[3][4][5][6]. Treatment of myotubes with thapsigargin (2 M) for 4 h to inhibit uptake of calcium by the sarcoplasmic reticulum also caused an increase in NFAT transcriptional activity, as previously reported (2). Similar to the calcium ionophore experiment, cotreatment with L-NAME inhibited this effect (Fig.…”

“…N G -nitro-L-arginine methyl ester (L-NAME), 1H- [1,2,4]oxadiazolo [4,3-a]quinoxalin-1-one (ODQ), 3-(5Ј-hydroxymethyl-2Јfuryl)-1-benzyl indazole (YC-1), diethylenetriamine-NONO (DETA-NO), methylamine hexamethylene methylamine-NONO (MAHMA-NO), and 3-(2-hydroxy-2-nitroso-1-propylhydrazino)-1-propanamine-NONO (PAPA-NO) were obtained from Cayman Chemical (Ann Arbor, MI). Lithium chloride (LiCl) was purchased from Acros Organics (Morris Plains, NJ).…”

Molecular Mechanisms and New Treatment Strategies for Non-Alcoholic Steatohepatitis (NASH)

“…Increasing SERCA2b levels reduce ER stress in the liver and increases glucose tolerance [69]. The SERCA inhibitor thapsigargin induces an increase in cytosolic calcium content with a decrease in ER calcium resulting in mitochondrial cytochrome C release and apoptosis of cultured primary rat hepatocytes [70]. …”

Hepatoprotective properties of extensively studied medicinal plant active constituents: Possible common mechanisms

“…In those articles, we looked for possible underlying common mechanisms of cytoprotection from molecular levels to whole animal studies of various structurally unrelated molecules. We found that several compounds experimentally showed some hepato-ameliorative properties and enumerated possible explanations (Canova et al, 2007;Cerny et al, 2009;Farghali & Masek, 1998;Farghali et al, 1991aFarghali et al, ,b, 1994Farghali et al, , 1996aFarghali et al, ,b, 1997Farghali et al, , 2000bFarghali et al, , 2002Farghali et al, , 2003Gasbarrini et al, 1992aGasbarrini et al, ,b, 1993Kmonickova et al, 2001;Lekic et al, 2011;Sakr et al, 1991a,b).…”