Potter C L, Hanson P J
Pharmaceutical Sciences Institute, Aston University, Aston Triangle, Birmingham B4 7ET, UK.
Gut. 2000 Feb;46(2):156-62. doi: 10.1136/gut.46.2.156.
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.
To determine whether exogenous NO, derived from an NO donor, might promote or counteract apoptosis in gastric mucous epithelial cells.
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.
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.
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.
一氧化氮（NO）合酶活性增加和细胞凋亡增强是幽门螺杆菌感染胃黏膜的特征，并且有人提出了因果关系。然而，尽管NO可促进细胞凋亡，但其作用因细胞类型而异。
确定来自NO供体的外源性NO是否可能促进或抵消胃黏液上皮细胞中的细胞凋亡。
将豚鼠胃黏膜细胞的原代培养物暴露于NO供体S-亚硝基-N-乙酰青霉胺（SNAP）24小时。通过用Hoechst 33258进行核染色、DNA原位缺口末端标记以及细胞提取物中DNA“梯带”的存在来检测细胞凋亡。分别通过免疫测定和荧光测定法测定环鸟苷酸（cGMP）含量和半胱天冬酶活性。
1 mM的SNAP并未改变培养板上表现出细胞凋亡特征的小部分细胞（3%-6%）。然而，当用25 μM N-己酰基-D-鞘氨醇（C6-神经酰胺）增强或通过使细胞从培养板上脱离时，SNAP产生了对细胞凋亡和半胱天冬酶3样活性的抑制作用。鸟苷酸环化酶抑制剂1H-1,2,4-恶二唑-4,3-a-喹喔啉-1-酮（ODQ）可阻止SNAP对cGMP的刺激，但不能阻止NO供体的抗凋亡作用。cGMP类似物8-溴-cGMP和8-（4-氯苯硫基）鸟苷-3',5'-环一磷酸并未显著抑制黏膜细胞中的细胞凋亡。
外源性NO通过不涉及cGMP升高的机制抑制豚鼠胃黏液细胞中的细胞凋亡。因此，如果在幽门螺杆菌感染期间由NO合酶产生NO，则可能抵消该病原体的促凋亡作用。
