Relationship between NO synthesis, arginine transport, and intracellular arginine levels in vascular smooth muscle cells.
The present study was designed to evaluate the relevance of arginine transport in nitric oxide (NO) synthesis in vascular smooth muscle cells. For this purpose, NO synthesis and arginine transport (system B0,+ and y+) were evaluated in cells treated with IL-1beta or angiotensin II (Ang II). In addition, the effects of 5 mM lysine and glutamine, competitive inhibitors of systems y+ and B0,+ respectively, were examined. L-arginine transport was estimated with 3H-labelled arginine and NO was determined with the Griess reagent. These studies were done in control conditions, arginine-starved cells, and in cells incubated in media containing 10 mM arginine. Our data indicate that induction of NO biosynthesis by IL-1beta depends on external arginine when cells are arginine-depleted for 24 hours. The concentration of arginine producing half maximal activation of NO synthesis in arginine-depleted cells ([arginine]i < 10 microM) was 41.1 +/- 18 microM. By contrast, in normal culture conditions, NO synthesis occurred independently of arginine transport. Neither 5 mM lysine or glutamine which abolished arginine transport through systems y+ and B0,+, respectively, reduced nitrite release in cells incubated in normal media. This suggests that the relevance of arginine uptake to NO synthesis depends on the status of intracellular arginine pools. Intracellular arginine concentrations were not affected by the stimulation of NO production using IL-1beta or its inhibition using Ang II, but were markedly reduced by arginine starvation for 48h. Aspartate levels were also reduced by arginine-depletion, but were not affected in cells incubated with 10 mM arginine. By contrast, glutamate levels were reduced in arginine-starved cells and were increased in cells incubated in arginine-supplemented medium. Ornithine levels were markedly increased by arginine supplementation. Altogether, these findings indicate that NO synthesis is normally independent of membrane transport. However in arginine-depleted cells, membrane transport is essential for NO synthesis. It is concluded that arginine transport is required for the long-term maintenance of intracellular arginine pools.
Escobales
N
N
Department of Physiology, University of Puerto Rico, School of Medicine, San Juan 00936-5067, USA. nescobal@prtc.net
Rivera-Correa
M
M
Altieri
P I
PI
Rodriguez
J F
JF
D000804:Angiotensin II / Q000494:pharmacology; D000818:Animals; D001120:Arginine / Q000172:deficiency / Q000378:metabolism*; D001224:Aspartic Acid / Q000378:metabolism; D001692:Biological Transport; D002478:Cells, Cultured; D005260:Female; D018698:Glutamic Acid / Q000378:metabolism; D005973:Glutamine / Q000378:metabolism; D007375:Interleukin-1 / Q000494:pharmacology; D008239:Lysine / Q000378:metabolism; D009131:Muscle, Smooth, Vascular / Q000378:metabolism*; D009569:Nitric Oxide / Q000096:biosynthesis* / Q000378:metabolism; D009573:Nitrites / Q000378:metabolism; D009952:Ornithine / Q000378:metabolism; D051381:Rats; D020318:Rats, Long-Evans
D007375:Interleukin-1; D009573:Nitrites; D005973:Glutamine; D000804:Angiotensin II; D001224:Aspartic Acid; D009569:Nitric Oxide; D018698:Glutamic Acid; D001120:Arginine; D009952:Ornithine; D008239:Lysine