Hammermann R, Bliesener N, Mössner J, Klasen S, Wiesinger H, Wessler I, Racké K
Institut für Pharmakologie und Toxikologie der Universität Bonn, Germany.
Naunyn Schmiedebergs Arch Pharmacol. 1998 Dec;358(6):601-7. doi: 10.1007/pl00005300.
In the present study it was tested whether rat alveolar macrophages (AMphi) convert L-citrulline to L-arginine to maintain nitric oxide (NO) synthesis under conditions of limited availability of L-arginine. Rat AMphi (0.5 x 10(6) cells/well, cultured for 20 h in the absence or presence of 1 microg/ml lipopolysaccharides, LPS), were incubated for 6 h in amino acid-free Krebs solution and nitrite accumulation was determined as a measure of NO synthesis. After culture in the absence of LPS, nitrite in the incubation media was at the detection limit, independent of the addition of L-arginine or L-citrulline. AMphi, cultured in the presence of LPS, produced about 4 nmol per 10(6) cells and 6 h nitrite, and L-arginine enhanced nitrite accumulation in a concentration-dependent manner, maximally about threefold (EC50: 55 microM). In LPS-treated AMphi L-citrulline (up to 10 mM) failed to enhance nitrite accumulation, but rather inhibited it by about 50% in the presence of 100 microM L-arginine, i.e. when NO synthesis was enhanced. L-Arginine in the culture medium was 3H-labelled and its metabolism analysed by HPLC. In medium of AMphi exposed to LPS [3H]-L-arginine was reduced by about 60% after a 20-h culture period and this was almost balanced by an almost equal increase in [3H]-L-citrulline and [3H]-L-ornithine, i.e. L-arginine was markedly consumed. When [14C]-L-citrulline was added to the culture medium of AMphi, no significant formation of [14C]-L-arginine could be detected. On the other hand, argininosuccinate synthetase mRNA (by RT-PCR) and protein (by Western blot) was marginally detectable in control AMphi, but clearly induced after exposure to LPS. Finally, L-citrulline was shown to inhibit L-arginine uptake in a concentration dependent manner, by about 50% at 10 mM. In conclusion, although the expression of argininosuccinate synthetase in rat AMphi can be induced by LPS, AMphi appear not to be able to recycle significant amounts of L-citrulline to L-arginine to maintain sustained NO synthesis. On the contrary, at high concentrations L-citrulline can reduce NO synthesis, and this effect appears to be caused by inhibitory effects on L-arginine uptake.
在本研究中，检测了大鼠肺泡巨噬细胞（AMphi）是否能在L-精氨酸供应有限的条件下将L-瓜氨酸转化为L-精氨酸以维持一氧化氮（NO）的合成。将大鼠AMphi（0.5×10⁶个细胞/孔，在不存在或存在1μg/ml脂多糖（LPS）的情况下培养20小时）在无氨基酸的Krebs溶液中孵育6小时，并测定亚硝酸盐积累量作为NO合成的指标。在不存在LPS的情况下培养后，孵育培养基中的亚硝酸盐处于检测限，与添加L-精氨酸或L-瓜氨酸无关。在存在LPS的情况下培养的AMphi，每10⁶个细胞在6小时内产生约4nmol亚硝酸盐，L-精氨酸以浓度依赖的方式增强亚硝酸盐积累，最大约为三倍（EC50：55μM）。在LPS处理的AMphi中，L-瓜氨酸（高达10mM）未能增强亚硝酸盐积累，反而在存在100μM L-精氨酸时（即当NO合成增强时）抑制亚硝酸盐积累约50%。培养基中的L-精氨酸用³H标记，并通过HPLC分析其代谢。在暴露于LPS的AMphi培养基中，经过20小时的培养期后，[³H]-L-精氨酸减少了约60%，而[³H]-L-瓜氨酸和[³H]-L-鸟氨酸几乎等量增加，几乎与之平衡，即L-精氨酸被大量消耗。当将[¹⁴C]-L-瓜氨酸添加到AMphi的培养基中时，未检测到[¹⁴C]-L-精氨酸的显著形成。另一方面，在对照AMphi中可微量检测到精氨琥珀酸合成酶mRNA（通过RT-PCR）和蛋白质（通过Western印迹），但在暴露于LPS后明显诱导。最后，L-瓜氨酸以浓度依赖的方式抑制L-精氨酸摄取，在10mM时抑制约50%。总之，尽管大鼠AMphi中精氨琥珀酸合成酶的表达可被LPS诱导，但AMphi似乎无法将大量L-瓜氨酸再循环为L-精氨酸以维持持续的NO合成。相反，在高浓度下，L-瓜氨酸可降低NO合成，这种效应似乎是由对L-精氨酸摄取的抑制作用引起的。
