title: Intracellular L-arginine concentration does not determine NO production in endothelial cells: implications on the "L-arginine paradox".
abstract: We examined the relative contributory roles of extracellular vs. intracellular L-arginine (ARG) toward cellular activation of endothelial nitric oxide synthase (eNOS) in human endothelial cells. EA.hy926 human endothelial cells were incubated with different concentrations of (15)N(4)-ARG, ARG, or L-arginine ethyl ester (ARG-EE) for 2h. To modulate ARG transport, siRNA for ARG transporter (CAT-1) vs. sham siRNA were transfected into cells. ARG transport activity was assessed by cellular fluxes of ARG, (15)N(4)-ARG, dimethylarginines, and L-citrulline by an LC-MS/MS assay. eNOS activity was determined by nitrite/nitrate accumulation, either via a fluorometric assay or by(15)N-nitrite or estimated (15)N(3)-citrulline concentrations when (15)N(4)-ARG was used to challenge the cells. We found that ARG-EE incubation increased cellular ARG concentration but no increase in nitrite/nitrate was observed, while ARG incubation increased both cellular ARG concentration and nitrite accumulation. Cellular nitrite/nitrate production did not correlate with cellular total ARG concentration. Reduced (15)N(4)-ARG cellular uptake in CAT-1 siRNA transfected cells vs. control was accompanied by reduced eNOS activity, as determined by (15)N-nitrite, total nitrite and (15)N(3)-citrulline formation. Our data suggest that extracellular ARG, not intracellular ARG, is the major determinant of NO production in endothelial cells. It is likely that once transported inside the cell, ARG can no longer gain access to the membrane-bound eNOS. These observations indicate that the "L-arginine paradox" should not consider intracellular ARG concentration as a reference point.
authors: Department of Pharmaceutical Sciences, University at Buffalo, The State University of New York, Buffalo, NY 14260, USA.
authors: Soyoung
authors: S
authors: Shin
authors: Srinidi
authors: S
authors: Mohan
authors: Ho-Leung
authors: HL
authors: Fung
mesh_terms: D001120:Arginine / Q000032:analysis / Q000378:metabolism* / Q000494:pharmacology; D027201:Cationic Amino Acid Transporter 1 / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D004730:Endothelium, Vascular / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D009569:Nitric Oxide / Q000032:analysis / Q000096:biosynthesis*; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics; D014162:Transfection
chemical_list: D027201:Cationic Amino Acid Transporter 1; D034741:RNA, Small Interfering; D009569:Nitric Oxide; D001120:Arginine; C496314:NOS3 protein, human; D052250:Nitric Oxide Synthase Type III