Induction of L-arginine transport is inhibited by atrial natriuretic peptide: a peptide hormone as a novel regulator of inducible nitric-oxide synthase substrate availability.
BACKGROUND
The inducible nitric-oxide synthase (iNOS) synthesizes NO from L-arginine. Availability of L-arginine is maintained by a lipopolysaccharide (LPS)-induced induction of the CAT-2B amino acids transporter. Recently, we could show that the cardiovascular hormone atrial natriuretic peptide (ANP) inhibits the induction of iNOS in LPS-stimulated macrophages via its guanylate cyclase-coupled A-receptor.


OBJECTIVE
To investigate whether ANP exerts an effect on LPS-induced L-arginine uptake.


METHODS
Murine bone marrow derived macrophages were activated with LPS (1 microg/ml, 20 h) in the presence or absence of ANP or C-type natriuretic peptide (CNP). L-Arginine transport was determined by measuring the uptake of L-[(3)H]arginine. L-[(3)H]Arginine influx was also determined in LPS-activated cells in the presence of N(G)-monomethyl-L-arginine (L-NMMA), competitor amino acids, or ANP. Nitrite accumulation was determined in supernatants of LPS-activated cells cultured in the presence or absence of L-ornithine.


RESULTS
ANP dose dependently (10(-8)-10(-6)M) inhibited LPS-induced L-[(3)H]arginine uptake when added simultaneously with LPS, whereas it showed no effect when added simultaneously with L-[(3)H]arginine. The effect was abrogated by the A-receptor antagonist HS-142-1 (10 microg/ml). CNP (10(-6) M) did not influence L-arginine transport. Competitor amino acids (10(-2) M) inhibited L-[(3)H]arginine uptake. An excess of unlabeled L-arginine (10(-2) M) as well as its analog L-NMMA (10(-3) M) also reduced L-[(3)H]arginine influx. L-Arginine uptake was critical for production of NO because L-ornithine significantly decreased LPS-induced nitrite accumulation.


CONCLUSIONS
This work demonstrates that ANP inhibits LPS-induced L-arginine uptake via its guanylate cyclase-coupled A-receptor. Besides its influence on the induction of iNOS, this effect may represent an important and unique mechanism by which ANP regulates NO production in macrophages.
Kiemer
A K
AK
Department of Pharmacy, Center of Drug Research, University of Munich, Munich, Germany. alexandra.kiemer@cup.uni-muenchen.de
Vollmar
A M
AM
D000818:Animals; D001120:Arginine / Q000378:metabolism*; D009320:Atrial Natriuretic Factor / Q000494:pharmacology*; D001667:Binding, Competitive; D001692:Biological Transport / Q000187:drug effects; D002478:Cells, Cultured; D004347:Drug Interactions; D006162:Guanylate Cyclase / Q000378:metabolism; D006728:Hormones / Q000494:pharmacology; D008070:Lipopolysaccharides / Q000494:pharmacology; D051379:Mice; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II; D017461:Receptors, Atrial Natriuretic Factor / Q000378:metabolism; D013379:Substrate Specificity; D014316:Tritium
D006728:Hormones; D008070:Lipopolysaccharides; D014316:Tritium; D009320:Atrial Natriuretic Factor; D001120:Arginine; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse; D006162:Guanylate Cyclase; D017461:Receptors, Atrial Natriuretic Factor; C091715:atrial natriuretic factor receptor A