Salvemini D, Pistelli A, Mollace V, Anggård E, Vane J
William Harvey Research Institute, St Bartholomew's Hospital Medical College, London, U.K.
Biochem Pharmacol. 1992 Jul 7;44(1):17-24. doi: 10.1016/0006-2952(92)90032-e.
The metabolism of glyceryl trinitrate (GTN) to nitric oxide (NO) was studied in the mouse macrophage cell line J774 and in the human monocytic cell line U937 in the absence or presence of Escherichia coli lipopolysaccharide (LPS). Two bioassay systems were used: inhibition of platelet aggregation and measurement of cGMP after stimulation by NO of guanylate cyclase in J774 cells. In addition, NO produced from GTN by cells or by cellular fractions was measured as nitrite (NO2-) one of its breakdown products. J774 cells (1.25 x 10(5) cells) treated with indomethacin (10 microM) enhanced the platelet inhibitory activity of GTN (22-352 microM) but not that of sodium nitroprusside (4 microM). This effect was abrogated by co-incubation with oxyhaemoglobin (oxyHb, 10 microM) indicating release of NO from GTN. U937 cells (up to 60 x 10(5)) did not metabolize GTN to NO. LPS (0.5 micrograms/mL for 18 hr) enhanced at least 2-fold the capacity of J774 cells but not that of U937 cells to form NO from GTN and this enhancement was attenuated when cycloheximide (10 micrograms/mL) was incubated together with LPS. In the absence of LPS stimulation, cycloheximide had no effect. Furthermore, when incubated with GTN (200 microM), J774 cells treated with LPS released more NO from GTN as indicated by a 3-fold greater increase in their level of cGMP which was prevented by oxyHb (10 microM). Incubation of J774 cells with GTN (75-600 microM) for 30 min led to a concentration-dependent increase in NO2- which was substantially reduced when the cells were boiled. The microsomal fraction was more potent than the cytosol in producing NO2- from GTN (1.2-2.4 mM). Release of NO2- from GTN by J774 cells was not affected by treating the cells with the NO synthase inhibitor, NG-monomethyl-L-arginine (MeArg, 300 microM). In J774 cells made tolerant to GTN, potentiation of the anti-platelet effects of GTN (11-352 microM) and release of NO2- from GTN was reduced. Thus, J774 cells but not U937 cells convert GTN to NO. This enzymic pathway (present mainly in the microsomal fraction of the J774 cells) is induced by LPS and is not regulated by endogenous NO released from L-Arg by the enzyme NO synthase. Furthermore, when compared to normal cells, tolerant J774 cells metabolize GTN to NO less effectively as assessed by a reduced capacity to potentiate the anti-platelet effect of GTN and to release NO2-.(ABSTRACT TRUNCATED AT 400 WORDS)
在存在或不存在大肠杆菌脂多糖（LPS）的情况下，研究了甘油三硝酸酯（GTN）在小鼠巨噬细胞系J774和人单核细胞系U937中向一氧化氮（NO）的代谢。使用了两种生物测定系统：抑制血小板聚集以及在J774细胞中由NO刺激鸟苷酸环化酶后测量cGMP。此外，将细胞或细胞组分从GTN产生的NO作为其分解产物之一的亚硝酸盐（NO2-）进行测量。用吲哚美辛（10μM）处理的J774细胞（1.25×10⁵个细胞）增强了GTN（22 - 352μM）的血小板抑制活性，但未增强硝普钠（4μM）的活性。与氧合血红蛋白（oxyHb，10μM）共同孵育可消除此效应，表明GTN释放出NO。U937细胞（高达60×10⁵个）未将GTN代谢为NO。LPS（0.5μg/mL，处理18小时）使J774细胞从GTN形成NO的能力增强至少2倍，但未增强U937细胞的此能力，并且当与环己酰亚胺（10μg/mL）与LPS一起孵育时，这种增强作用减弱。在不存在LPS刺激的情况下，环己酰亚胺无作用。此外，当与GTN（200μM）孵育时，用LPS处理的J774细胞从GTN释放出更多的NO，这通过其cGMP水平增加3倍得以表明，而这被oxyHb（10μM）阻止。将J774细胞与GTN（75 - 600μM）孵育30分钟导致NO2-浓度依赖性增加，当细胞煮沸时该增加显著降低。微粒体部分比胞质溶胶从GTN产生NO2-（1.2 - 2.4 mM）的能力更强。J774细胞从GTN释放NO2-不受用NO合酶抑制剂NG-单甲基-L-精氨酸（MeArg，300μM）处理细胞的影响。在对GTN产生耐受性的J774细胞中，GTN（11 - 352μM）的抗血小板作用增强以及从GTN释放NO2-均降低。因此，J7
