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



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.


apoptosis intracellular free calcium necrosis nitric oxide rat hepatocytes thapsigargin 





alanine aminotransferase


adenosine triphosphate


endoplasmic reticulum


endoplasmic reticulum calcium


intracellular free calcium


cytochrome c


mitochondrial permeability transition


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


nuclear factor-kappa B


nitric oxide


nitric oxide synthase


endothelial NOS


inducible NOS


mitochondrial NOS


neuronal NOS




reactive oxygen species


sarco-endoplasmic reticulum Ca2+-ATPases




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  1. Al-Abed Y, Bucala R. Efficient scavenging of fatty acid oxidation products by aminoguanidine. Chem Res Toxicol. 1997;10(8):875–9.PubMedCrossRefGoogle Scholar
  2. Bras M, Queenan B, Susin SA. Programmed cell death via mitochondria: different modes of dying. Biochemistry (Mosc). 2005;70(2):231–9.CrossRefGoogle Scholar
  3. Bustamante J, Di Libero E, Fernandez-Cobo M, Monti N, Cadenas E, Boveris A. Kinetic analysis of thapsigargin-induced thymocyte apoptosis. Free Radic Biol Med. 2004;37(9):1490–8.PubMedCrossRefGoogle Scholar
  4. Cardozo AK, Ortis F, Storling J, et al. Cytokines downregulate the sarcoendoplasmic reticulum pump Ca2+ ATPase 2b and deplete endoplasmic reticulum Ca2+, leading to induction of endoplasmic reticulum stress in pancreatic beta-cells. Diabetes. 2005;54(2):452–61.PubMedCrossRefGoogle Scholar
  5. Chae HJ, Kim HR, Xu C, et al. BI-1 regulates an apoptosis pathway linked to endoplasmic reticulum stress. Mol Cell. 2004;15(3):355–66.PubMedCrossRefGoogle Scholar
  6. Chen BC, Hsieh SL, Lin WW. Involvement of protein kinases in the potentiation of lipopolysaccharide-induced inflammatory mediator formation by thapsigargin in peritoneal macrophages. J Leukoc Biol. 2001;69(2):280–8.PubMedGoogle Scholar
  7. Chen T, Zamora R, Zuckerbraun B, Billiar TR. Role of nitric oxide in liver injury. Curr Mol Med. 2003;3(6):519–26.PubMedCrossRefGoogle Scholar
  8. Chen YJ, Hsu KW, Tsai JN, Hung CH, Kuo TC, Chen YL. Involvement of protein kinase C in the inhibition of lipopolysaccharide-induced nitric oxide production by thapsigargin in RAW 264.7 macrophages. Int J Biochem Cell Biol. 2005;37(12):2574–85.PubMedCrossRefGoogle Scholar
  9. Chung HT, Pae HO, Choi BM, Billiar TR, Kim YM. Nitric oxide as a bioregulator of apoptosis. Biochem Biophys Res Commun. 2001;282(5):1075–9.PubMedCrossRefGoogle Scholar
  10. Dilworth C, Bigot-Lasserre D, Bars R. Spontaneous nitric oxide in hepatocyte monolayers and inhibition of compound-induced apoptosis. Toxicol In Vitro. 2001;15(6):623–30.PubMedCrossRefGoogle Scholar
  11. Farghali H, Kamenikova L, Martinek J, Lincova D, Hynie S. Preparation of functionally active immobilized and perfused mammalian cells: an example of the hepatocyte bioreactor. Physiol Res. 1994;43(2):121–5.PubMedGoogle Scholar
  12. Farghali H, Canova N, Gaier N, et al. Inhibition of endotoxemia-induced nitric oxide synthase expression by cyclosporin A enhances hepatocyte injury in rats: amelioration by NO donors. Int Immunopharmacol. 2002;2(1):117–27.PubMedCrossRefGoogle Scholar
  13. Farghali H, Canova N, Kucera T, Martinek J, Masek K. Nitric oxide synthase inhibitors modulate lipopolysaccharide-induced hepatocyte injury: dissociation between in vivo and in vitro effects. Int Immunopharmacol. 2003;3(12):1627–38.PubMedCrossRefGoogle Scholar
  14. Feng XQ, You Y, Xiao J, Zou P. Thapsigargin-induced apoptosis of K562 cells and its mechanism. Zhongguo Shi Yan Xue Ye Xue Za Zhi. 2006;14(1):25–30.PubMedGoogle Scholar
  15. Fouad D, Siendones E, Costan G, Muntane J. Role of NF-kappaB activation and nitric oxide expression during PGE protection against d-galactosamine-induced cell death in cultured rat hepatocytes. Liver Int. 2004;24(3):227–36.PubMedCrossRefGoogle Scholar
  16. Frankfurt OS, Krishan A. Identification of apoptotic cells by formamide-induced DNA denaturation in condensed chromatin. J Histochem Cytochem. 2001;49(3):369–78.PubMedGoogle Scholar
  17. Fukuda K, Kojiro M, Chiu JF. Demonstration of extensive chromatin cleavage in transplanted Morris hepatoma 7777 tissue: apoptosis or necrosis? Am J Pathol. 1993;142(3):935–46.PubMedGoogle Scholar
  18. Furuya Y, Lundmo P, Short AD, Gill DL, Isaacs JT. The role of calcium, pH, and cell proliferation in the programmed (apoptotic) death of androgen-independent prostatic cancer cells induced by thapsigargin. Cancer Res. 1994;54:6167–75.PubMedGoogle Scholar
  19. Geller DA, Nussler AK, Di Silvio M, et al. Cytokines, endotoxin, and glucocorticoids regulate the expression of inducible nitric oxide synthase in hepatocytes. Proc Natl Acad Sci USA. 1993;90(2):522–6.PubMedCrossRefGoogle Scholar
  20. Ghafourifar P, Cadenas E. Mitochondrial nitric oxide synthase. Trends Pharmacol Sci. 2005;26(4):190–5.PubMedCrossRefGoogle Scholar
  21. Grynkiewicz G, Ponie M, Tsien RY. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem. 1985;260:3440–50.PubMedGoogle Scholar
  22. Guinzberg R, Uribe S, Diaz-Cruz A, Hernandez Cruz A, Pina E. In rat hepatocytes, different adenosine receptor subtypes use different secondary messengers to increase the rate of ureagenesis. Life Sci. 2006;79(4):382–90.PubMedCrossRefGoogle Scholar
  23. Gumpricht E, Dahl R, Yerushalmi B, Devereaux MW, Sokol RJ. Nitric oxide ameliorates hydrophobic bile acid-induced apoptosis in isolated rat hepatocytes by non-mitochondrial pathways. J Biol Chem. 2002;277(28):25823–30.PubMedCrossRefGoogle Scholar
  24. Haynes V, Elfering S, Traaseth N, Giulivi C. Mitochondrial nitric-oxide synthase: enzyme expression, characterization, and regulation. J Bioenerg Biomembr. 2004;36(4):341–6.PubMedCrossRefGoogle Scholar
  25. He Q, Lee DI, Rong R, Yu M, et al. Endoplasmic reticulum calcium pool depletion-induced apoptosis is coupled with activation of the death receptor 5 pathway. Oncogene. 2002;21(17):2623–33.PubMedCrossRefGoogle Scholar
  26. Hu P, Han Z, Couvillon AD, Kaufman RJ, Exton JH. Autocrine tumor necrosis factor alpha links endoplasmic reticulum stress to the membrane death receptor pathway through IRE1alpha-mediated NF-kappaB activation and down-regulation of TRAF2 expression. Mol Cell Biol. 2006;26(8):3071–84.PubMedCrossRefGoogle Scholar
  27. Humez S, Legrand G, Vanden-Abeele F, et al. Role of endoplasmic reticulum calcium content in prostate cancer cell growth regulation by IGF and TNFalpha. J Cell Physiol. 2004;201(2):201–13.PubMedCrossRefGoogle Scholar
  28. Ilan E, Tirosh O, Madar Z. Triacylglycerol-mediated oxidative stress inhibits nitric oxide production in rat isolated hepatocytes. J Nutr. 2005;135(9):2090–5.PubMedGoogle Scholar
  29. Isaacs JT. New strategies for the medical treatment of prostate cancer. BJU Int. 2005;96(Suppl 2):35–40.Google Scholar
  30. Izumi T, Yamaguchi M. Overexpression of regucalcin suppresses cell death in cloned rat hepatoma H4-II-E cells induced by tumor necrosis factor-alpha or thapsigargin. J Cell Biochem. 2004;92(2):296–306.PubMedCrossRefGoogle Scholar
  31. Janssen S, Rosen DM, Ricklis RM, et al. Pharmacokinetics, biodistribution, and antitumor efficacy of a human glandular kallikrein 2 (hK2)-activated thapsigargin prodrug. Prostate. 2006;66(4):358–68.PubMedCrossRefGoogle Scholar
  32. Ji C, Kaplowitz N. ER stress: can the liver cope? J Hepatol. 2006;45(2):321–33.PubMedCrossRefGoogle Scholar
  33. Jordan ML, Rominski B, Jaquins-Gerstl A, Geller D, Hoffman RA. Regulation of inducible nitric oxide production by intracellular calcium. Surgery. 1995;118(2):138–45; discussion 145–6.Google Scholar
  34. Jun CD, Yoon HJ, Park YC, et al. Synergistic cooperation between thapsigargin and phorbol ester for induction of nitric oxide synthesis in murine peritoneal macrophages. Free Radic Biol Med. 1996;20(6):769–76.PubMedCrossRefGoogle Scholar
  35. Kaplowitz N. Mechanisms of liver cell injury. J Hepatol. 2000;32(1 Suppl):39–47.Google Scholar
  36. Kaplowitz N. Drug-induced liver injury. Clin Infect Dis. 2004;38(Suppl 2):S44–8.Google Scholar
  37. Kashiwagura T, Erecinska M, Wilson DF. pH dependence of hormonal regulation of gluconeogenesis and urea synthesis from glutamine in suspensions of hepatocytes. J Biol Chem. 1985;260(1):407–14.PubMedGoogle Scholar
  38. Kiemer AK, Vollmar AM. Elevation of intracellular calcium levels contributes to the inhibition of nitric oxide production by atrial natriuretic peptide. Immunol Cell Biol. 2001;79(1):11–7.PubMedCrossRefGoogle Scholar
  39. Kim JS, He L, Lemasters JJ. Mitochondrial permeability transition: a common pathway to necrosis and apoptosis. Biochem Biophys Res Commun. 2003;304(3):463–70.PubMedCrossRefGoogle Scholar
  40. Kim PK, Zamora R, Petrosko P, Billiar TR. The regulatory role of nitric oxide in apoptosis. Int Immunopharmacol. 2001;1(8):1421–41.PubMedCrossRefGoogle Scholar
  41. Kim YM, Talanian RV, Billiar TR. Nitric oxide inhibits apoptosis by preventing increases in caspase-3-like activity via two distinct mechanisms. J Biol Chem. 1997;272(49):31138–48.PubMedCrossRefGoogle Scholar
  42. Kishikawa H, Sakamoto A, Ogawa R. Nitric oxide suppresses hepatocyte apoptosis induced by free radicals. Biomed Res (Tokyo). 2001;22(2):83–9.Google Scholar
  43. Kmonickova E, Kutinova Canova N, Farghali H, Holy A, Zidek Z. Modulator of intracellular Ca(2+), thapsigargin, interferes with in vitro secretion of cytokines and nitric oxide. Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2005;149(2):321–4.PubMedGoogle Scholar
  44. Korge P, Weiss JN. Thapsigargin directly induces the mitochondrial permeability transition. Eur J Biochem. 1999;265(1):273–80.PubMedCrossRefGoogle Scholar
  45. Kumar A, Takada Y, Boriek AM, Aggarwal BB. Nuclear factor-kappaB: its role in health and disease. J Mol Med. 2004;82(7):434–48.PubMedCrossRefGoogle Scholar
  46. Lemaire C, Andreau K, Souvannavong V, Adam A. Inhibition of caspase activity induces a switch from apoptosis to necrosis. FEBS Lett. 1998;425(2):266–70.PubMedCrossRefGoogle Scholar
  47. Li J, Billiar TR. Nitric Oxide. IV. Determinants of nitric oxide protection and toxicity in liver. Am J Physiol. 1999;276(5 Pt 1):G1069–73.Google Scholar
  48. Li J, Holbrook NJ. Elevated gadd153/chop expression and enhanced c-Jun N-terminal protein kinase activation sensitizes aged cells to ER stress. Exp Gerontol. 2004;39(5):735–44.PubMedCrossRefGoogle Scholar
  49. Li J, Lee B, Lee AS. Endoplasmic reticulum stress-induced apoptosis: multiple pathways and activation of p53-up-regulated modulator of apoptosis (PUMA) and NOXA by p53. J Biol Chem. 2006;281(11):7260–70.PubMedCrossRefGoogle Scholar
  50. Lin XS, Denmeade SR, Cisek L, Isaacs JT. Mechanism and role of growth arrest in programmed (apoptotic) death of prostatic cancer cells induced by thapsigargin. Prostate. 1997;33(3):201–7.PubMedCrossRefGoogle Scholar
  51. Liu HF, Xie Q, Jiang S, et al. Inhibition of mouse hepatocyte apoptosis by anti-caspase-12 small interfering RNA. Zhonghua Gan Zang Bing Za Zhi. 2005;13(12):923–6.PubMedGoogle Scholar
  52. Liu J, Waalkes MP. Nitric oxide and chemically induced hepatotoxicity: beneficial effects of the liver-selective nitric oxide donor, V-PYRRO/NO. Toxicology. 2005;208(2):289–97.PubMedCrossRefGoogle Scholar
  53. Malhi H, Gores GJ, Lemasters JJ. Apoptosis and necrosis in the liver: a tale of two deaths? Hepatology. 2006;43(2 Suppl 1):S31–44.Google Scholar
  54. McNaughton L, Puttagunta L, Martinez-Cuesta MA, et al. Distribution of nitric oxide synthase in normal and cirrhotic human liver. Proc Natl Acad Sci USA. 2002;99(26):17161–6.PubMedCrossRefGoogle Scholar
  55. Meijer AJ, Lamers WH, Chamuleau RA. Nitrogen metabolism and ornithine cycle function. Physiol Rev. 1990;70(3):701–44.PubMedGoogle Scholar
  56. Narayanan B, Islam MN, Bartelt D, Ochs RS. A direct mass-action mechanism explains capacitative calcium entry in Jurkat and skeletal L6 muscle cells. J Biol Chem. 2003;278(45):44188–96.PubMedCrossRefGoogle Scholar
  57. Nissim I, Luhovyy B, Horyn O, Daikhin Y, Nissim I, Yudkoff M. The role of mitochondrially bound arginase in the regulation of urea synthesis: studies with [U-15N4]arginine, isolated mitochondria, and perfused rat liver. J Biol Chem. 2005;280(18):17715–24.PubMedCrossRefGoogle Scholar
  58. Ockner RK. Apoptosis and liver diseases: recent concepts of mechanism and significance. J Gastroenterol Hepatol. 2001;16(3):248–60.PubMedCrossRefGoogle Scholar
  59. Orrenius S, Zhivotovsky B, Nicotera P. Regulation of cell death: the calcium–apoptosis link. Nat Rev Mol Cell Biol. 2003;4(7):552–65.PubMedCrossRefGoogle Scholar
  60. Park YC, Jun CD, Kang HS, Kim HD, Kim HM, Chung HT. Role of intracellular calcium as a priming signal for the induction of nitric oxide synthesis in murine peritoneal macrophages. Immunology. 1996;87(2):296–302.PubMedCrossRefGoogle Scholar
  61. Rodriguez-Lopez AM, Martinez-Salgado C, Eleno N, Arevalo M, Lopez-Novoa JM. Nitric oxide is involved in apoptosis induced by thapsigargin in rat mesangial cells. Cell Physiol Biochem. 1999;9(6):285–96.PubMedCrossRefGoogle Scholar
  62. Ruemmele FM, Dionne S, Levy E, Seidman EG. TNFalpha-induced IEC-6 cell apoptosis requires activation of ICE caspases whereas complete inhibition of the caspase cascade leads to necrotic cell death. Biochem Biophys Res Commun. 1999;260(1):159–66.PubMedCrossRefGoogle Scholar
  63. Saito Y, Nishio K, Ogawa Y, et al. Turning point in apoptosis/necrosis induced by hydrogen peroxide. Free Radic Res. 2006;40(6):619–30.PubMedCrossRefGoogle Scholar
  64. Shangari N, O'Brien PJ. The cytotoxic mechanism of glyoxal involves oxidative stress. Biochem Pharmacol. 2004;68(7):1433–42.PubMedCrossRefGoogle Scholar
  65. Schuchmann M, Galle PR. Apoptosis in liver disease. Eur J Gastroenterol Hepatol. 2001;13(7):785–90.PubMedCrossRefGoogle Scholar
  66. Sohn J, Khaoustov VI, Xie Q, Chung CC, Krishnan B, Yoffe B. The effect of ursodeoxycholic acid on the survivin in thapsigargin-induced apoptosis. Cancer Lett. 2003;191(1):83–92.PubMedCrossRefGoogle Scholar
  67. Srivastava RK, Sollott SJ, Khan L, Hansford R, Lakatta EG, Longo DL. Bcl-2 and Bcl-X(L) block thapsigargin-induced nitric oxide generation, c-Jun NH(2)-terminal kinase activity, and apoptosis. Mol Cell Biol. 1999;19(8):5659–74.PubMedGoogle Scholar
  68. Thastrup O, Cullen PJ, Drobak BK, Hanley MR, Dawson AP. Thapsigargin, a tumor promoter, discharges intracellular Ca2+ stores by specific inhibition of the endoplasmic reticulum Ca2+-ATPase. Proc Natl Acad Sci USA. 1990;87(7):2466–70.PubMedCrossRefGoogle Scholar
  69. Treiman M, Caspersen C, Christensen SB. A tool coming of age: thapsigargin as an inhibitor of sarco-endoplasmic reticulum Ca2+-ATPases. Trends Pharmacol Sci. 1998;19(4):131–5.PubMedCrossRefGoogle Scholar
  70. Ueda T, Takeyama Y, Hori Y, Takase K, Goshima M, Kuroda Y. Pancreatitis-associated ascitic fluid increases intracellular Ca(2+) concentration on hepatocytes. J Surg Res. 2000;93(1):171–6.PubMedCrossRefGoogle Scholar
  71. Wang H, Gao X, Fukumoto S, Tademoto S, Sato K, Hirai K. Post-isolation inducible nitric oxide synthase gene expression due to collagenase buffer perfusion and characterization of the gene regulation in primary cultured murine hepatocytes. J Biochem (Tokyo). 1998;124(5):892–9.Google Scholar
  72. Waring P, Beaver J. Cyclosporin A rescues thymocytes from apoptosis induced by very low concentrations of thapsigargin: effects on mitochondrial function. Exp Cell Res. 1996;227(2):264–76.PubMedCrossRefGoogle Scholar
  73. Xie Q, Khaoustov VI, Chung CC, et al. Effect of tauroursodeoxycholic acid on endoplasmic reticulum stress-induced caspase-12 activation. Hepatology. 2002;36(3):592–601.PubMedCrossRefGoogle Scholar
  74. Xu W, Liu L, Charles IG, Moncada S. Nitric oxide induces coupling of mitochondrial signalling with the endoplasmic reticulum stress response. Nat Cell Biol. 2004;6(11):1129–34.PubMedCrossRefGoogle Scholar
  75. Yamaguchi H, Bhalla K, Wang HG. Bax plays a pivotal role in thapsigargin-induced apoptosis of human colon cancer HCT116 cells by controlling Smac/Diablo and Omi/HtrA2 release from mitochondria. Cancer Res. 2003;63(7):1483–9.PubMedGoogle Scholar

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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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