Cell Biology and Toxicology

, Volume 23, Issue 5, pp 337–354 | Cite as

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

Article

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.

Keywords

apoptosis intracellular free calcium necrosis nitric oxide rat hepatocytes thapsigargin 

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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© Springer Science + Business Media, Inc. 2007

Authors and Affiliations

  1. 1.Institute of Pharmacology, 1st Faculty of MedicineCharles UniversityPrague 2Czech Republic
  2. 2.Institute of Histology and Embryology, 1st Faculty of MedicineCharles UniversityPragueCzech Republic
  3. 3.Institute of Experimental MedicineThe Academy of Sciences of the Czech RepublicPragueCzech Republic

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