Inability of rat alveolar macrophages to recycle L-citrulline to L-arginine despite induction of argininosuccinate synthetase mRNA and protein, and inhibition of nitric oxide synthesis by exogenous L-citrulline.
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.
Hammermann
R
R
Institut für Pharmakologie und Toxikologie der Universität Bonn, Germany.
Bliesener
N
N
Mössner
J
J
Klasen
S
S
Wiesinger
H
H
Wessler
I
I
Racké
K
K
D000818:Animals; D001120:Arginine / Q000096:biosynthesis*; D001123:Argininosuccinate Lyase / Q000235:genetics* / Q000378:metabolism; D015153:Blotting, Western; D002478:Cells, Cultured; D002956:Citrulline / Q000378:metabolism* / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D005260:Female; D066298:In Vitro Techniques; D008070:Lipopolysaccharides / Q000494:pharmacology; D016676:Macrophages, Alveolar / Q000378:metabolism*; D008297:Male; D009569:Nitric Oxide / Q000096:biosynthesis*; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats
D008070:Lipopolysaccharides; D012333:RNA, Messenger; D002956:Citrulline; D009569:Nitric Oxide; D001120:Arginine; D001123:Argininosuccinate Lyase