POLY(ADP-RIBOSE) SYNTHETASE ACTIVATION MEDIATES INCREASED PERMEABILITY INDUCED BY PEROXYNITRITE IN CACO-2BBE CELLS

Background & Aims: Peroxynitrite induces cytotoxicity by generating DN A single-strand breaks and activating poly(ADP-ribose) synthetase (PAR S), a nuclear enzyme that consumes oxidized nicotinamide adenine dinuc leotide (NAD(+)) and depletes cellular adenosine triphosphate (ATP). T he aim of this study was to examine this mechanism of injury in an int estinal epithelial cell model after exposure to exogenous peroxynitrit e (ONOO-) and nitric oxide (NO). Methods: Caco-2BBe cell monolayers ex posed 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 on ly SIN-1 reduced [NAD(+)]. Inhibition of PARS activity by the PARS inh ibitors 5-iodo-6-amino 1,2-benzopyrone or 3-aminobenzamide prevented t he 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 no t NO increases transepithelial permeability by inducing DNA strand bre aks that activate the PARS pathway and cause the depletion of intracel lular energy stores. Inhibition of PARS activity may represent a novel strategy in ameliorating peroxynitrite-mediated epithelial injury dur ing intestinal inflammation.