C L Potter1, P J Hanson. 1. Pharmaceutical Sciences Institute, Aston University, Aston Triangle, Birmingham B4 7ET, UK.
Abstract
BACKGROUND: Increased nitric oxide (NO) synthase activity and enhanced apoptosis are features of gastric mucosa infected with Helicobacter pylori and a causative relation has been suggested. However, although NO can promote apoptosis, its actions vary with cell type. AIMS: To determine whether exogenous NO, derived from an NO donor, might promote or counteract apoptosis in gastric mucous epithelial cells. METHODS: Primary cultures of guinea pig gastric mucosal cells were exposed to the NO donor S-nitroso-N-acetyl-penicillamine (SNAP) for 24 hours. Apoptosis was detected from nuclear staining with Hoechst 33258, in situ nick end labelling of DNA, and the presence of DNA "ladders" in cell extracts. Cyclic GMP content and caspase activity were determined by immunoassay and fluorimetric assay respectively. RESULTS: SNAP 1 mM did not alter the small proportion of cells on the culture plate (3-6%) which exhibited features of apoptosis. However, SNAP produced an inhibition of apoptosis, and of caspase 3 like activity, when enhanced by 25 microM N-hexanoyl-D-sphingosine (C(6)-ceramide), or by detachment of cells from the culture plate. The guanylate cyclase inhibitor, 1H-1, 2, 4-oxadiazole-4, 3-a-quinoxaline-1-one (ODQ), prevented the stimulation of cyclic GMP by SNAP, but not the anti- apoptotic effects of the NO donor. The cyclic GMP analogues 8-bromo-cyclic GMP and 8-(4-chlorophenylthio) guanosine-3',5'- cyclic monophosphate did not significantly inhibit apoptosis in the mucosal cells. CONCLUSIONS: Exogenous NO inhibited apoptosis in guinea pig gastric mucous cells by a mechanism which did not involve elevation of cyclic GMP. NO, if produced from NO synthase during infection with H pylori, may therefore counter the proapoptotic effects of this pathogen.
BACKGROUND: Increased nitric oxide (NO) synthase activity and enhanced apoptosis are features of gastric mucosa infected with Helicobacter pylori and a causative relation has been suggested. However, although NO can promote apoptosis, its actions vary with cell type. AIMS: To determine whether exogenous NO, derived from an NO donor, might promote or counteract apoptosis in gastric mucous epithelial cells. METHODS: Primary cultures of guinea pig gastric mucosal cells were exposed to the NO donorS-nitroso-N-acetyl-penicillamine (SNAP) for 24 hours. Apoptosis was detected from nuclear staining with Hoechst 33258, in situ nick end labelling of DNA, and the presence of DNA "ladders" in cell extracts. Cyclic GMP content and caspase activity were determined by immunoassay and fluorimetric assay respectively. RESULTS:SNAP 1 mM did not alter the small proportion of cells on the culture plate (3-6%) which exhibited features of apoptosis. However, SNAP produced an inhibition of apoptosis, and of caspase 3 like activity, when enhanced by 25 microM N-hexanoyl-D-sphingosine (C(6)-ceramide), or by detachment of cells from the culture plate. The guanylate cyclase inhibitor, 1H-1, 2, 4-oxadiazole-4, 3-a-quinoxaline-1-one (ODQ), prevented the stimulation of cyclic GMP by SNAP, but not the anti- apoptotic effects of the NO donor. The cyclic GMP analogues 8-bromo-cyclic GMP and 8-(4-chlorophenylthio) guanosine-3',5'- cyclic monophosphate did not significantly inhibit apoptosis in the mucosal cells. CONCLUSIONS: Exogenous NO inhibited apoptosis in guinea pig gastric mucous cells by a mechanism which did not involve elevation of cyclic GMP. NO, if produced from NO synthase during infection with H pylori, may therefore counter the proapoptotic effects of this pathogen.
Authors: Juan M Herrerías; José M Esteban; Antonio M Caballero-Plasencia; Manuel Valenzuela-Barranco; Virginia Motilva; Catalina Alarcón; José Martín; Juan M Herrerías; Pilar Esteban Journal: Dig Dis Sci Date: 2003-05 Impact factor: 3.199