Characterization of endothelial cell amino acid transport systems involved in the actions of nitric oxide synthase inhibitors.
N omega-Substituted analogues of L-arginine have proven useful as specific inhibitors of nitric oxide formation in various biological systems. In the present study we describe the characteristics of amino acid transporters that mediate uptake of N omega-methyl-L-arginine (L-NMA) and N omega-nitro-L-arginine (L-NNA) into cultured porcine aortic endothelial cells. The transport of L-[14C]NMA showed biphasic kinetics, with Km values of 4 and 368 microM, and was inhibited by L-arginine, L-homoarginine, L-lysine, and L-ornithine but not by L-leucine or L-isoleucine. Similar transport kinetics (Km values of 6 and 609 microM) and substrate specificities were obtained for L-[3H]arginine uptake, indicating that L-arginine and L-NMA are transported by the same system. In contrast to L-arginine and L-NMA transport, uptake of L-[3H]NNA was monophasic (Km = 617 microM) and was inhibited by L-leucine and L-isoleucine but not by L-arginine, L-homoarginine, L-NMA, L-lysine, or L-ornithine. Uptake studies with L-[3H]leucine revealed that the transport of this amino acid occurred in a manner very similar to that of L-[3H]NNA transport, suggesting that the uptake of both compounds may be mediated by the same system. In additional experiments, we determined the effects of L-NMA and L-NNA on the A23187-induced accumulation of intracellular cGMP, to establish to what extent these transport systems are involved in the actions of nitric oxide synthase inhibitors. L-Lysine and L-ornithine, which both inhibited L-NMA uptake, increased the IC50 of L-NMA from 7.8 microM to 57 microM but did not reduce the inhibitory effects of L-NNA. In the presence of L-leucine or L-isoleucine, however, which both inhibited L-NNA uptake, the IC50 of L-NNA was increased from 1.2 microM to 37 microM but the inhibitory actions of L-NMA remained unaffected. These data demonstrate that the endothelial transport systems for L-arginine and L-leucine mediate the biological effects of L-NMA and L-NNA, respectively.
Schmidt
K
K
Institut für Pharmakologie und Toxikologie, Universität Graz, Austria.
Klatt
P
P
Mayer
B
B
D000594:Amino Acid Oxidoreductases / Q000037:antagonists & inhibitors*; D000596:Amino Acids / Q000378:metabolism*; D000818:Animals; D001120:Arginine / Q000031:analogs & derivatives* / Q000378:metabolism / Q000493:pharmacokinetics / Q000494:pharmacology; D001692:Biological Transport / Q000502:physiology; D002478:Cells, Cultured; D006152:Cyclic GMP / Q000378:metabolism; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D007930:Leucine / Q000378:metabolism; D019001:Nitric Oxide Synthase; D019335:Nitroarginine; D013552:Swine; D019323:omega-N-Methylarginine
D000596:Amino Acids; D019335:Nitroarginine; D019323:omega-N-Methylarginine; D001120:Arginine; D019001:Nitric Oxide Synthase; D000594:Amino Acid Oxidoreductases; D007930:Leucine; D006152:Cyclic GMP