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

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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 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.

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Abbreviations

AG:

aminoguanidine

ALT:

alanine aminotransferase

ATP:

adenosine triphosphate

ER:

endoplasmic reticulum

[Ca2+]ER :

endoplasmic reticulum calcium

[Ca2+] i :

intracellular free calcium

cyt.c :

cytochrome c

MPT:

mitochondrial permeability transition

MTT:

3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide

NF-κB:

nuclear factor-kappa B

NO:

nitric oxide

NOS:

nitric oxide synthase

eNOS:

endothelial NOS

iNOS:

inducible NOS

mtNOS:

mitochondrial NOS

nNOS:

neuronal NOS

p-NA:

p-nitroaniline

ROS:

reactive oxygen species

SERCAs:

sarco-endoplasmic reticulum Ca2+-ATPases

TG:

thapsigargin

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Authors and Affiliations

  1. Institute of Pharmacology, 1st Faculty of Medicine, Charles University, Albertov 4, 128 00, Prague 2, Czech Republic

    N. Kutinová Canová & H. Farghali

  2. Institute of Histology and Embryology, 1st Faculty of Medicine, Charles University, Prague, Czech Republic

    J. Martínek

  3. Institute of Experimental Medicine, The Academy of Sciences of the Czech Republic, Prague, Czech Republic

    E. Kmoníčková & Z. Zídek

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  1. N. Kutinová Canová
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  2. E. Kmoníčková
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  3. J. Martínek
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  5. H. Farghali
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Correspondence to N. Kutinová Canová or H. Farghali.

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Canová, N.K., Kmoníčková, E., Martínek, J. et al. Thapsigargin, a selective inhibitor of sarco-endoplasmic reticulum Ca2+-ATPases, modulates nitric oxide production and cell death of primary rat hepatocytes in culture. Cell Biol Toxicol 23, 337–354 (2007). https://doi.org/10.1007/s10565-007-0185-6

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