新型合成三肽抑制巨噬细胞精氨酸转运

A new synthetic tripeptide inhibits L-arginine transport in macrophages

目的观察新型合成三肽[Arg(NO3)-Lys(OCH3)-Arg(NO3)]对巨噬细胞株(RAW264．7)左旋-精氨酸／一氧化氮 (L-Arg／NO)途径的影响。方法培养的巨噬细胞(培养液中含L-Arg 0．5 mmol／L)加入脂多糖(LPS,1μg/L)随机分为3组 (每组n=6),分别加入双蒸水、三肽(1×10-4mol／L)、NG甲基-L-精氨酸(L-NMMA,1×10-4mol／L),对照组只含L-Arg (n=6),作用24 h后,检测亚硝酸盐(NO2-)、3H-L-Arg转运量;培养的巨噬细胞[培养液中含L-Arg(0-2 mmol／L)]加入 LPS(1 μg／L)24 h后,检测NO2-;培养的巨噬细胞(培养液中含L-Arg 0．5mmol／L)加入LPS(1μg/L)后分别加三肽[ (0-10)×10-4 mol／L]24 h后,检测NO2-、3H-L-Arg转运量。结果 LPS刺激细胞产生NO的量和L-Arg率转运分别为非刺激组的50倍和2．7倍,三肽(1×10-4mol／L)即可明显降低NO的生成及抑制L-Arg的转运(分别降低71％、67％),较 L-NMMA(1×10-4 mol／L)作用要强(P<0．05);NO的生成依赖于细胞外L-Arg,并成浓度依赖性,其米氏常数(Km):(0．162± 0．015) mmol／L、最大转运速率(Vmax):(91．2±2．3)μmol／(24 h·106cells);三肽成浓度依赖性的降低细胞L-Arg转运和 NO的生成,半数有效抑制剂量(EC50)分别为0．21×10-4mol／L、1．27×104mol／L。结论 LPS作用于巨噬细胞引起L-Arg转运增加:三肽通过抑制细胞L-Arg转运及与L-Arg竞争性结合一氧化氮合酶作用位点,影响NO的生成。

Objective To observe the effect of a new synthetic tripeptide [Arg（NO3）- Lys（OCH3）- Arg（NO3）] on L-arginine/NO pathway in the macrophage cell strain RAW246.7. Methods The cultured macrophages exposed to lipopolysaccharide （LPS, 1μg/L） treatment were randomly divided into 3 groups （n=6） and treated with distilled water, 1×10^-4 mol/L tripeptide and 1×10^-4 mol/L L-arginine, NG-monomethyl-L-arginine （L-NMMA） for 24 h, respectively. The macrophages were incubated for 24 h with LPS （1μg/L） and in the presence of different concentrations of L-arginine （0 to 2 mmol/L）, or in normal culture medium （containing 0.5 mmol/L L-arginine） for 24 h with LPS （1μg/L） and in the presence of tripeptide of 0 to 10×10^-4 mol/L. The changes of [^3H]-L-arginine transport and NO production from the macrophages were measured. Result NO release from macrophages incubated in the LPS-containing culture medium was 50 folds, and [^3H]-L-arginine uptake 2.7 folds that in cells in normal culture medium. Tfipeptide （1×10^-4 mol/L） inhibited [^3H]-L-arginine transport and NO production by 67% and 71% respectively. The effect oftripeptide was stronger than L-NMMA （P〈0.05）. Extracelluar L-arginine caused a concentration-dependent increase in nitrite production, which reached the maximum at concentrations above 0.5 mmol/L Km for nitrite production of 0.162±0.015 mmol/L andVmax of 91.2±2.3μmol/（24h. 106cells）. L-arginine transport and NO production were inhibited by tripeptide, but their dose-dependent pattern of changes was different with EC50 of 0.21×10^-4 mol/L and 1.27×10^-4 mol/L, respectively. Conclusions Activation ofmacrophages with LPS induces nitrite accumulation in the culture medium, which is dependent on the presence of extracelluar L-arginine. The tripeptide induces dysfunction of L-arginine/NO pathway in macrophages, potently inhibits L-arginine transport and competitively combine the active sites of nitric oxide synthase.