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. 2000 Feb;46(2):156–162. doi: 10.1136/gut.46.2.156

Exogenous nitric oxide inhibits apoptosis in guinea pig gastric mucous cells

C Potter 1, P Hanson 1
PMCID: PMC1727833  PMID: 10644307

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 µM N-hexanoyl-D-sphingosine (C6-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.


Keywords: nitric oxide; gastric mucosa; stomach; apoptosis

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Figure 1  .

Figure 1  

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Effect of 1 mM S-nitroso-N-acetyl-penicillamine (SNAP; shaded bars) on apoptosis in guinea pig gastric mucosal cells in the presence and absence of 25 µM C6-ceramide. Apoptosis was detected by staining of nuclei with Hoechst 33258 in A and by nick end labelling in B, with data presented as mean (SEM) of six and four separate cell preparations, respectively. Data were analysed by analysis of variance followed by a Newman-Keuls multiple comparison test. *p<0.05, ***p<0.001 for the effect of SNAP in comparison with the same treatment without SNAP.

Figure 2  .

Figure 2  

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Nuclear staining with Hoechst 33258. (A) Cells treated with 1 mM S-nitroso-N-acetyl-penicillamine that were attached to the culture plate. One of the few apoptotic cells is labelled A. (B) Cells that had detached spontaneously under control conditions.

Figure 3  .

Figure 3  

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Fragmentation pattern of low molecular weight DNA as revealed by electrophoresis on agarose. Cells were treated with 25 µM C6-ceramide in the presence (lanes 2 and 4) and absence (lanes 1 and 3) of 1 mM S-nitroso- N-acetyl-penicillamine. Extracts from cells attached to the culture plate are in lanes 1 and 2 and cells that had detached are in lanes 3 and 4. Markers are in lane 5. 

Figure 4  .

Figure 4  

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Effect of the concentration of S-nitroso-N-acetyl-penicillamine (SNAP) on apoptosis in cells attached (A) and detached (B) that had been treated with 25 µM C6-ceramide. Apoptosis was quantified by staining of nuclei with Hoechst dye. Data are the mean (SEM) of four separate cultures and have been analysed by analysis of variance followed by Dunnett's test. *p<0.05, **p<0.01 for comparison with the result obtained in the absence of SNAP.

Figure 5  .

Figure 5  

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Effect of 1 mM S-nitroso-N-acetyl-penicillamine (SNAP; shaded bars) on caspase 3 like activity in extracts of guinea pig gastric mucosal cells incubated for 24 hours in the presence and absence of 25 µM C6-ceramide. Data are presented as mean (SEM) of five separate cell preparations. Data were analysed by two factor analysis of variance, to remove the effect of variation in caspase activity between experiments, followed by a Newman-Keuls multiple comparison test. **p<0.01, ***p<0.001 for the effect of SNAP in comparison with the same treatment without SNAP.

Figure 6  .

Figure 6  

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Effect of 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (carboxy-PTIO) on apoptosis (Hoechst staining). (A) Data are mean (SEM) for cells which had detached from the plate in three separate experiments with the effect of 1 mM SNAP (shaded bars) determined by paired t test; *p<0.05, **p<0.01. (B) Data are for attached cells in the presence (shaded bars) and absence (clear bars) of 25 µM C6-ceramide and are mean (SEM) of four separate cell preparations; *p<0.05, **p<0.01 for the difference from the appropriate control by a Newman-Keuls test.

Figure 7  .

Figure 7  

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Effect of ODQ (1H-1,2,4-oxadiazole-4,3-a-quinoxaline-1-one) on the action of 1 mM S-nitroso-N-acetyl- penicillamine (SNAP, shaded bars) on apoptosis (Hoechst 33258 staining), cyclic GMP content, and on detachment of cells from the culture plate. The attached cells in panels A and C had been treated with 25 µM C6-ceramide to induce apoptosis. Results in B are for detached cells. Data in A, B, C, and D are mean (SEM) of five, four, three, and three cell preparations, respectively, and were subjected to analysis of variance followed by a Newman-Keuls multiple comparison test. *p<0.05, **p<0.01,***p<0.001 for the effect of SNAP; †p<0.05 for the effect of ODQ in the presence of SNAP.

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