Poly(ADP-ribose) synthetase activation mediates increased permeability induced by peroxynitrite in Caco-2BBe cells

doi:10.1016/S0016-5085(98)70534-7

Gastroenterology

Volume 114, Issue 3, March 1998, Pages 510-518

https://doi.org/10.1016/S0016-5085(98)70534-7 Get rights and content

Abstract

Background & Aims: Peroxynitrite induces cytotoxicity by generating DNA single-strand breaks and activating poly(ADP-ribose) synthetase (PARS), a nuclear enzyme that consumes oxidized nicotinamide adenine dinucleotide (NAD⁺) and depletes cellular adenosine triphosphate (ATP). The aim of this study was to examine this mechanism of injury in an intestinal epithelial cell model after exposure to exogenous peroxynitrite (ONOO⁻) and nitric oxide (NO). Methods: Caco-2BBe cell monolayers exposed to donors of peroxynitrite (3-morpholino-sydnonimine [SIN-1], 3 mmol/L) or NO (S-nitroso-N-acetyl penicillamine [SNAP]; 3 mmol/L) were analyzed for DNA strand breaks, [NAD⁺], [ATP], and transepithelial flux of fluorescein sulfonic acid. Results: SIN-1 but not SNAP induced DNA single-strand breakage. Both SIN-1 and SNAP reduced [ATP], but only SIN-1 reduced [NAD⁺]. Inhibition of PARS activity by the PARS inhibitors 5-iodo-6-amino 1,2- benzopyrone or 3-aminobenzamide prevented the SIN-1–induced reduction in [NAD⁺] and [ATP] but had no effect on the SNAP-induced reduction in [ATP]. PARS inhibition reduced SIN-1–but not SNAP-induced hyperpermeability. Conclusions: Peroxynitrite but not NO increases transepithelial permeability by inducing DNA strand breaks that activate the PARS pathway and cause the depletion of intracellular energy stores. Inhibition of PARS activity may represent a novel strategy in ameliorating peroxynitrite-mediated epithelial injury during intestinal inflammation.

GASTROENTEROLOGY 1998;114:510-518

Section snippets

Cell culture

Primary cultures of human enterocytes are heterogeneous and have limited ex vivo survival. Therefore, transformed cells were used in the present studies. In selecting a model system for studying the mechanisms of oxidant injury in intestinal epithelia, we considered several principal criteria. First, because of variations in the effects of oxidant stress, both between species and among different cell types, the use of human cells of intestinal origin was preferred. Second, the model system

Results

Caco-2BBe cell monolayers were exposed to various concentrations of SIN-1 and SNAP to provide cells with a continuous exposure to ONOO⁻ and NO, respectively. SIN-1 releases approximately equimolar quantities of NO and O₂⁻, which immediately react to generate ONOO⁻ in situ.40, 41 Dose-response studies at 3 hours indicated that SIN-1, at 1 mmol/L, did not significantly affect cellular ATP. SIN-1 (3 mmol/L) induced an approximately 50% decrease in cellular ATP levels, whereas 5 mmol/L ATP caused

Discussion

This study shows that exposure to ONOO⁻, but not its precursor NO, damages cultured intestinal epithelial cells via the activation of the PARS pathway. Exposure of cells to ONOO⁻ induced DNA strand breaks and triggered an energy consuming pathway catalyzed by PARS. The consequent reduction of cellular stores of ATP and NAD⁺ was associated with the development of increased permeability of the epithelial monolayer. Although NO exposure also reduced cellular ATP, this effect was independent of

Acknowledgements

The authors thank Vivien Xue for assistance with the tissue culture studies.

References (66)

SJ Middleton et al.
Increased nitric oxide synthesis in ulcerative colitis
Lancet
(1993)
M. Guslandi
Nitric oxide in ulcerative colitis
Lancet
(1993)
M Mourelle et al.
Induction of nitric oxide synthase in colonic smooth muscle from patients with toxic megacolon
Gastroenterology
(1995)
VW. Yang
Eicosanoids and inflammatory bowel disease
Clin North Am
(1996)
DD Tran et al.
Enhanced systemic nitric oxide production in inflammatory bowel disease
Lancet
(1993)
II Singer et al.
Expression of inducible nitric oxide synthase and nitrotyrosine in colonic epithelium in inflammatory bowel disease
Gastroenterology
(1996)
MJS Miller et al.
Inducible nitric oxide synthase gene expression and peroxynitrite formation in guinea pig ileitis
Gastroenterology
(1995)
C Szabó et al.
Endogenous peroxynitrite is involved in the inhibition of cellular respiration in immuno-stimulated J774.2 macrophages
Biochem Biophys Res Commun
(1995)
C Szabó et al.
Peroxynitrite-mediated oxidation of dihydrorhodamine 123 occurs in early stages of endotoxic and hemorrhagtic shock and ischemia-reperfusion injury
FEBS Lett
(1995)
C. Szabó
DNA strand breakage and activation of poly-ADP ribosyltransferase: a cytotoxic pathway triggered by peroxynitrite
Free Radic Biol Med
(1996)

C. Szabó

DNA strand breakage and activation of poly-ADP ribosyltransferase: a cytotoxic pathway triggered by peroxynitrite

Free Radic Biol Med

(1996)

M Banasik et al.

Specific inhibitors of poly (ADP-ribose) synthetase and mono(ADP-ribosyl) transferase

J Biol Chem

(1992)

N Munfakh et al.

Protection of the hypertrophied myocardium by crystalloid cardioplegia

J Surg Res

(1991)

S Dimmeler et al.

Nitric oxide causes ADP-ribosylation and inhibition of glyceraldhyde-3-phosphate dehydrogenase

J Biol Chem

(1992)

PR Gardner et al.

Nitric oxide sensitivity of the aconitases

J Biol Chem

(1997)

L Castro et al.

Aconitase is readily inactivated by peroxynitrite, but not by its precursor, nitric oxide

J Biol Chem

(1994)

A Hausladen et al.

Superoxide and peroxynitrite inactivate aconitases, but nitric oxide does not

J Biol Chem

(1994)

RP. MacDermott

Alterations of the mucosal immune system in inflammatory bowel disease

J Gastroenterol

(1996)

H Wiseman et al.

Damage to DNA by reactive oxygen and nitrogen species: role in inflammatory disease and progression to cancer

Biochem J

(1996)

NK Boughton-Smith et al.

Nitric oxide synthase activity in ulcerative colitis and Crohn's disease

Lancet

(1993)

D Rachmilewitz et al.

Enhanced colonic nitric oxide generation and nitric oxide synthase activity in ulcerative colitis and Crohn's disease

Gut

(1995)

AL. Salzman

Nitric oxide in the gut

New Horizons

(1995)

MB Grisham et al.

Effects of nitric oxide synthase inhibition on the pathophysiology observed in a model of chronic granulomotous colitis

J Pharm Exp Ther

(1994)

MB Grisham et al.

Neutrophils, nitrogen oxides, and inflammatory bowel disease

Ann NY Acad Sci

(1992)

AL Salzman et al.

Induction and activity of nitric oxide synthase in cultured human intestinal epithelial monolayers

Am J Physiol

(1995)

SC Linn et al.

Transcriptional regulation of the human inducible nitric oxide synthase gene in an intestinal epithelial cell line

Am J Physiol

(1997)

AL Salzman et al.

Nitric oxide dilates tight junctions and depletes ATP in cultured Caco-2BBe intestinal epithelial monolayers

Am J Physiol

(1994)

C. Szabó

The role of peroxynitrite in the pathophysiology of shock, inflammation, and ischemia-reperfusion injury

Shock

(1996)

C Szabó et al.

Peroxynitrite-mediated activation of poly-ADP ribosyl synthetase contributes to the vascular failure in shock

B Zingarelli et al.

Peroxynitrite-mediated DNA strand breakage activates poly-ADP ribosyl synthetase and causes cellular energy depletion in macrophages stimulated with bacterial lipopolysaccharide

J Immunol

(1996)

C Szabó et al.

DNA strand breakage, activation of poly-ADP ribosyl synthetase, and cellular energy depletion are involved in the cytotoxicity in macrophages and smooth muscle cells exposed to peroxynitrite

Proc Natl Acad Sci USA

(1996)

C Szabó et al.

Role of poly-ADP ribosyltransferase activation in the nitric oxide– and peroxynitrite-induced vascular failure

Circ Res

(1996)

C Szabó et al.

Activation of poly (ADP-ribose) synthetase is involved in the endothelial dysfunction in response to peroxynitrite and endotoxin shock

Crit Care Med

(1996)

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^☆: Address requests for reprints to: Andrew L. Salzman, M.D., Division of Critical Care Medicine, Children's Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, Ohio 45229. e-mail: [email protected]; fax: (513) 636-4267.

^☆☆: Supported in part by grant 9650090N from the American Heart Association (to A.L.S.), by Octamer, Inc. (Mill Valley, California) (to C.S.), and by grant T32BK07727 from the National Institutes of Health (to M.K.).

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Alimentary TractPoly(ADP-ribose) synthetase activation mediates increased permeability induced by peroxynitrite in Caco-2BBe cells☆,☆☆

Abstract

Section snippets

Cell culture

Results

Discussion

Acknowledgements

Lancet

Lancet

Gastroenterology

Clin North Am

Lancet

Gastroenterology

Gastroenterology

Biochem Biophys Res Commun

FEBS Lett

Free Radic Biol Med

Free Radic Biol Med

J Biol Chem

J Surg Res

J Biol Chem

J Biol Chem

J Biol Chem

J Biol Chem

Alterations of the mucosal immune system in inflammatory bowel disease

J Gastroenterol

Damage to DNA by reactive oxygen and nitrogen species: role in inflammatory disease and progression to cancer

Biochem J

Nitric oxide synthase activity in ulcerative colitis and Crohn's disease

Lancet

Enhanced colonic nitric oxide generation and nitric oxide synthase activity in ulcerative colitis and Crohn's disease

Gut

Nitric oxide in the gut

New Horizons

Effects of nitric oxide synthase inhibition on the pathophysiology observed in a model of chronic granulomotous colitis

J Pharm Exp Ther

Neutrophils, nitrogen oxides, and inflammatory bowel disease

Ann NY Acad Sci

Induction and activity of nitric oxide synthase in cultured human intestinal epithelial monolayers

Am J Physiol

Transcriptional regulation of the human inducible nitric oxide synthase gene in an intestinal epithelial cell line

Am J Physiol

Nitric oxide dilates tight junctions and depletes ATP in cultured Caco-2BBe intestinal epithelial monolayers

Am J Physiol

The role of peroxynitrite in the pathophysiology of shock, inflammation, and ischemia-reperfusion injury

Shock

Peroxynitrite-mediated activation of poly-ADP ribosyl synthetase contributes to the vascular failure in shock

Peroxynitrite-mediated DNA strand breakage activates poly-ADP ribosyl synthetase and causes cellular energy depletion in macrophages stimulated with bacterial lipopolysaccharide

J Immunol

DNA strand breakage, activation of poly-ADP ribosyl synthetase, and cellular energy depletion are involved in the cytotoxicity in macrophages and smooth muscle cells exposed to peroxynitrite

Proc Natl Acad Sci USA

Role of poly-ADP ribosyltransferase activation in the nitric oxide– and peroxynitrite-induced vascular failure

Circ Res

Activation of poly (ADP-ribose) synthetase is involved in the endothelial dysfunction in response to peroxynitrite and endotoxin shock

Crit Care Med

Alimentary Tract
Poly(ADP-ribose) synthetase activation mediates increased permeability induced by peroxynitrite in Caco-2BBe cells☆,☆☆