Endothelial response to stress from exogenous Zn2+ resembles that of NO-mediated nitrosative stress, and is protected by MT-1 overexpression

Dean A. Wiseman, Sandra M. Wells, Jason Wilham, Maryann Hubbard, Jonathan E. Welker, Stephen Matthew Black

Research output: Contribution to journalArticle

56 Citations (Scopus)

Abstract

While nitric oxide (NO)-mediated biological interactions have been intensively studied, the underlying mechanisms of nitrosative stress with resulting pathology remain unclear. Previous studies have demonstrated that NO exposure increases free zinc ions (Zn2+) within cells. However, the resulting effects on endothelial cell survival have not been adequately resolved. Thus the purpose of this study was to investigate the role of altered zinc homeostasis on endothelial cell survival. Initially, we confirmed the previously observed significant increase in free Zn2+ with a subsequent induction of apoptosis in our pulmonary artery endothelial cells (PAECs) exposed to the NO donor N-[2-aminoethyl]-N-[2-hydroxy-2- nitrosohydrazino]-1,2-ethylenediamine. However, NO has many effects upon cell function and we wanted to specifically evaluate the effects mediated by zinc. To accomplish this we utilized the direct addition of zinc chloride (ZnCl 2) to PAEC. We observed that Zn2+-exposed PAECs exhibited a dose-dependent increase in superoxide (O2-.) generation that was localized to the mitochondria. Furthermore, we found Zn 2+-exposed PAECs exhibited a significant reduction in mitochondrial membrane potential, loss of cardiolipin from the inner leaflet, caspase activation, and significant increases in TdT-mediated dUTP nick end labeling-positive cells. Furthermore, using an adenoviral construct for the overexpression of the Zn2+-binding protein, metallothionein-1 (MT-1), we found either MT-1 overexpression or coincubation with a Zn 2+-selective chelator, N,N,N′,N′-tetrakis(2- pyridylmethyl)ethylene-diamide, in PAECs significantly protected the mitochondria from both NO and Zn2+-mediated disruption and induction of apoptosis and cell death. In summary, our results indicate that a loss of Zn2+ homeostasis produces mitochondrial dysfunction, increased oxidative stress, and apoptotic cell death. We propose that regulation of Zn2+ levels may represent a potential therapeutic target for disease associated with both nitrosative and oxidative stress.

Original languageEnglish (US)
JournalAmerican Journal of Physiology - Cell Physiology
Volume291
Issue number3
DOIs
StatePublished - Sep 13 2006

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Metallothionein
Nitric Oxide
Endothelial Cells
Pulmonary Artery
Zinc
ethylenediamine
Cell Survival
Mitochondria
Oxidative Stress
Homeostasis
Cell Death
Apoptosis
Diamide
Cardiolipins
Nitric Oxide Donors
Mitochondrial Membrane Potential
Chelating Agents
Caspases
Superoxides
Carrier Proteins

Keywords

  • Apoptosis
  • Mitochondrial dysfunction
  • Reactive nitrogen species

ASJC Scopus subject areas

  • Physiology
  • Cell Biology

Cite this

Endothelial response to stress from exogenous Zn2+ resembles that of NO-mediated nitrosative stress, and is protected by MT-1 overexpression. / Wiseman, Dean A.; Wells, Sandra M.; Wilham, Jason; Hubbard, Maryann; Welker, Jonathan E.; Black, Stephen Matthew.

In: American Journal of Physiology - Cell Physiology, Vol. 291, No. 3, 13.09.2006.

Research output: Contribution to journalArticle

Wiseman, Dean A. ; Wells, Sandra M. ; Wilham, Jason ; Hubbard, Maryann ; Welker, Jonathan E. ; Black, Stephen Matthew. / Endothelial response to stress from exogenous Zn2+ resembles that of NO-mediated nitrosative stress, and is protected by MT-1 overexpression. In: American Journal of Physiology - Cell Physiology. 2006 ; Vol. 291, No. 3.
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AB - While nitric oxide (NO)-mediated biological interactions have been intensively studied, the underlying mechanisms of nitrosative stress with resulting pathology remain unclear. Previous studies have demonstrated that NO exposure increases free zinc ions (Zn2+) within cells. However, the resulting effects on endothelial cell survival have not been adequately resolved. Thus the purpose of this study was to investigate the role of altered zinc homeostasis on endothelial cell survival. Initially, we confirmed the previously observed significant increase in free Zn2+ with a subsequent induction of apoptosis in our pulmonary artery endothelial cells (PAECs) exposed to the NO donor N-[2-aminoethyl]-N-[2-hydroxy-2- nitrosohydrazino]-1,2-ethylenediamine. However, NO has many effects upon cell function and we wanted to specifically evaluate the effects mediated by zinc. To accomplish this we utilized the direct addition of zinc chloride (ZnCl 2) to PAEC. We observed that Zn2+-exposed PAECs exhibited a dose-dependent increase in superoxide (O2-.) generation that was localized to the mitochondria. Furthermore, we found Zn 2+-exposed PAECs exhibited a significant reduction in mitochondrial membrane potential, loss of cardiolipin from the inner leaflet, caspase activation, and significant increases in TdT-mediated dUTP nick end labeling-positive cells. Furthermore, using an adenoviral construct for the overexpression of the Zn2+-binding protein, metallothionein-1 (MT-1), we found either MT-1 overexpression or coincubation with a Zn 2+-selective chelator, N,N,N′,N′-tetrakis(2- pyridylmethyl)ethylene-diamide, in PAECs significantly protected the mitochondria from both NO and Zn2+-mediated disruption and induction of apoptosis and cell death. In summary, our results indicate that a loss of Zn2+ homeostasis produces mitochondrial dysfunction, increased oxidative stress, and apoptotic cell death. We propose that regulation of Zn2+ levels may represent a potential therapeutic target for disease associated with both nitrosative and oxidative stress.

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