Regular Article
Nitric Oxide and Peroxynitrite-Dependent Aconitase Inactivation and Iron-Regulatory Protein-1 Activation in Mammalian Fibroblasts

https://doi.org/10.1006/abbi.1998.0898Get rights and content

Abstract

The reaction of reactive oxygen and nitrogen species with the [4Fe–4S]2+cluster of mitochondrial (m-) and cytosolic (c-) aconitases leads to loss of catalytic activity and, in the case of the c-aconitase, triggers total cluster disruption to yield the iron-regulatory protein-1 (IRP-1). Herein we have studied the relative contribution and interplay of reactive oxygen species (O [equation] and H2O2), nitric oxide (NO), and peroxynitrite in the modulation of m- and c-aconitase and IRP-1 activities in V79-M8 mammalian fibroblasts, identifying key variables that control the various reactivities at the cellular level. Extracellular production of H2O2led to inactivation of both m- and c-aconitase and IRP-1 activation, while extracellular [equation] had no effect. However, increased intracellular production of [equation] caused a loss in m- and c-aconitase activity and IRP-1 activation. Nitric oxide released from NOC-12 had a more complex effect on aconitase and IRP-1 activities. Mitochondrial aconitase was more sensitive than c-aconitase toNO-mediated inactivation and minimal activation of IRP-1 was observed during a 30-min exposure to theNO donor. The action ofNO was down- or upregulated by the presence of extra- or intracelular [equation], respectively. Extracellular [equation] decreased theNO-mediated inactivation of aconitases, due to the preferential extracellular decomposition and the lower diffusivity of peroxynitrite compared toNO. On the other hand,NO exposure concomitant with enhanced intracellular [equation] fluxes lead to intracellular peroxynitrite formation as evidenced by Western blot analysis of nitrated proteins, which increased the effects observed withNO alone. Peroxynitrite-mediated aconitase inactivation, IRP-1 activation, and cellular protein nitration were more pronounced in cells with low GSH content such as V79-M8 glutathione-depleted cells as well as in pGSOD4 cells which contain 32% of the GSH of the parental strain. Mechanistically, our results imply that the differential actions of the studied reactive species toward cellular aconitases depend on at least three critical factors: (i) their reaction rates with aconitases, (ii) the cellular compartment where they are formed, and (iii) the intracellular status of glutathione.

References (72)

  • O. Melefors et al.

    Blood Rev.

    (1993)
  • Y. Yu et al.

    J. Biol. Chem.

    (1992)
  • E.W. Mullner et al.

    Cell

    (1988)
  • P.H. Brown et al.

    J. Biol. Chem.

    (1989)
  • C.R. Bhasker et al.

    J. Biol. Chem.

    (1993)
  • L. Zheng et al.

    Arch. Biochem. Biophys.

    (1992)
  • D.H. Flint et al.

    J. Biol. Chem.

    (1993)
  • P.R. Gardner et al.

    J. Biol. Chem.

    (1991)
  • E.A.L. Martins et al.

    Arch. Biochem. Biophys.

    (1995)
  • L. Castro et al.

    J. Biol. Chem.

    (1994)
  • A. Hausladen et al.

    J. Biol. Cel.

    (1994)
  • M.C. Kennedy et al.

    J. Biol. Chem.

    (1997)
  • K. Keyer et al.

    J. Biol. Chem.

    (1997)
  • A. Denicola et al.

    Arch. Biochem. Biophys.

    (1996)
  • R. Radi et al.

    J. Chem. Biol.

    (1991)
  • L. Castro et al.

    Arch. Biochem. Biophys.

    (1996)
  • R.M. Uppu et al.

    Arch. Biochem. Biophys.

    (1996)
  • A. Gow et al.

    Arch. Biochem. Biophys.

    (1996)
  • A. Denicola et al.

    Arch. Biochem. Biophys.

    (1996)
  • M.M. Bradford

    Anal. Biochem.

    (1976)
  • C.C. Winterbourn
  • I.A. Rose et al.

    J. Biol. Chem.

    (1967)
  • F. Tietze

    Anal. Biochem.

    (1969)
  • F.R. Gadelha et al.

    Arch. Biochem. Biophys.

    (1997)
  • O.W. Griffith et al.

    J. Biol. Chem.

    (1979)
  • P.R. Gardner et al.

    J. Biol. Chem.

    (1992)
  • P.R. Gardner et al.

    J. Biol. Chem.

    (1995)
  • C. Bouton et al.

    J. Biol. Chem.

    (1996)
  • C. Bouton et al.

    J. Biol. Chem.

    (1997)
  • G.R. Upchurch et al.

    Adv. Pharmacol.

    (1995)
  • A. Denicola et al.

    Arch. Biochem. Biophys.

    (1993)
  • L. Zhu et al.

    Arch. Biochem. Biophys.

    (1992)
  • P.R. Gardner et al.

    Arch. Biochem. Biophys.

    (1993)
  • A.H. Robbins et al.

    Proc. Natl. Acad. Sci. USA

    (1989)
  • H.H. Emptage et al.

    J. Biol. Chem.

    (1983)
  • Cited by (93)

    • Fluoroacetate

      2020, Handbook of Toxicology of Chemical Warfare Agents
    • The effect of nitric oxide on mitochondrial respiration

      2019, Nitric Oxide - Biology and Chemistry
    • Mitochondria and Nitric Oxide

      2017, Nitric Oxide: Biology and Pathobiology: Third Edition
    • Peroxynitrite Formation and Detection in Living Cells

      2017, Nitric Oxide: Biology and Pathobiology: Third Edition
    View all citing articles on Scopus

    Packer, L.

    1

    To whom correspondence should be addressed at Departmento de Bioquı́mica, Facultad de Medicina, Universidad de la República, Av. Gral. Flores 2125, 11800 Montevideo, Uruguay. Fax: (5982) 9249563. E-mail:[email protected].

    View full text