Volume 114, Issue 3, Pages 510-518 (March 1998) Poly(ADP-ribose) synthetase activation mediates increased permeability induced by peroxynitrite in Caco-2BBe cells  Michelle Kennedy*, Alvin G. Denenberg‡, Csaba Szabó‡, Andrew L. Salzman‡  Gastroenterology  Volume 114, Issue 3, Pages 510-518 (March 1998) DOI: 10.1016/S0016-5085(98)70534-7 Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 1 (A) DNA single-strand break time course in Caco-2BBe cells exposed to peroxynitrite. Caco-2BBe cell monolayers (n = 7) were exposed to the peroxynitrite donor SIN-1 (3 mmol/L) for 1, 2, and 4 hours. The extent of DNA strand breaks was assessed indirectly by measuring the quantity of double-stranded DNA using the alkaline unwinding assay. #P < 0.05, *P < 0.001 vs. control. (B) DNA single-strand breakage in Caco-2BBe cells exposed to NO and peroxynitrite. Caco-2BBe cell monolayers (n = 10–12) were exposed for 4 hours to the peroxynitrite donor SIN-1 (3 mmol/L) and the NO donor SNAP (3 mmol/L). The extent of DNA strand breakage in cell extracts was assessed indirectly by measuring the quantity of double-stranded DNA using the alkaline unwinding assay. *P < 0.05 vs. control. Gastroenterology 1998 114, 510-518DOI: (10.1016/S0016-5085(98)70534-7) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 2 Time course of ATP depletion in Caco-2BBe cells exposed to (A) SIN-1 or (B) SNAP. Caco-2BBe cell monolayers (n = 6–9) were exposed to 3 mmol/L SIN-1 or SNAP for 0, 1, 3, and 6 hours. ATP was determined by HPLC. *P < 0.001 vs. control. Gastroenterology 1998 114, 510-518DOI: (10.1016/S0016-5085(98)70534-7) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 3 Concentration of NAD+ in Caco-2BBe cells exposed to (A) the peroxynitrite generator SIN-1 or (B) the NO generator SNAP. Caco-2BBe cell monolayers (n = 6–9) were exposed for 2 hours to 3 mmol/L SIN-1 or SNAP in the absence or presence of the PARS inhibitors 3-AB (3 mmol/L) and INH2BP (100 μmol/L). [NAD+] was determined by HPLC. *P < 0.05 vs. control. #P < 0.05 vs. cells exposed to SIN-1 or SNAP. Gastroenterology 1998 114, 510-518DOI: (10.1016/S0016-5085(98)70534-7) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 4 Concentration of ATP in Caco-2BBe cells exposed to (A) the peroxynitrite generator SIN-1 or (B) the NO generator SNAP. Caco-2BBe cell monolayers (n = 8–9) were exposed for 2 hours to 3 mmol/L SIN-1 or SNAP in the absence or presence of the PARS inhibitors 3-AB (3 mmol/L) and INH2BP (100 μmol/L). ATP was determined by HPLC. *P < 0.05 vs. control. #P < 0.05 vs. cells exposed to SIN-1 or SNAP. Gastroenterology 1998 114, 510-518DOI: (10.1016/S0016-5085(98)70534-7) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 5 (A) Permeability and (B) ATP content of Caco-2BBe cell monolayers exposed to the peroxynitrite generator SIN-1. Caco-2BBe cell monolayers (n = 12) were exposed for 24 hours to 3 mmol/L SIN-1 at an extracellular pH of 7.0 or 7.4. At pH 7.0, one group of cells was treated with the PARS inhibitor 3-AB (1 mmol/L). Permeability was determined by the transepithelial passage of FS acid (492 kilodaltons) and expressed as the apical to basolateral flux of FS acid as a percent of control. ATP concentration was determined by HPLC. *P < 0.05 vs. control. #P < 0.05 indicates a significant effect of 3-AB compared with the response in cells exposed to SIN-1 in the absence of the PARS inhibitor. Groups (left to right) are as follows: control pH 7.4, SIN-1 pH 7.4, control 7.0, SIN-1 pH 7.0, 3-AB, and SIN-1 pH 7.0 + 3-AB. Gastroenterology 1998 114, 510-518DOI: (10.1016/S0016-5085(98)70534-7) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 6 (A) Permeability and (B) ATP content of Caco-2BBe cell monolayers exposed to the NO generator SNAP. Caco-2BBe cell monolayers (n = 12) were exposed for 24 hours to 3 mmol/L SNAP at an extracellular pH of 7.0 or 7.4. At pH 7.0, one group of cells was treated with the PARS inhibitor 3-AB (1 mmol/L). Permeability was determined by the transepithelial passage of FS acid (492 kilodaltons) and expressed as the apical to basolateral flux of FS acid as a percent of control. ATP concentration was determined by HPLC. *P < 0.05 vs. control. #P < 0.05 indicates a significant effect of 3-AB compared with the response in cells exposed to SNAP in the absence of the PARS inhibitor. Groups (left to right) are as follows: control pH 7.4, SNAP pH 7.4, control 7.0, SNAP pH 7.0, 3-AB, and SNAP pH 7.0 + 3-AB. Gastroenterology 1998 114, 510-518DOI: (10.1016/S0016-5085(98)70534-7) Copyright © 1998 American Gastroenterological Association Terms and Conditions