Rate of transport of l-arginine is independent of the expression of inducible nitric oxide synthase in HEK 293 cells.
Expression of inducible nitric oxide synthase (iNOS) is generally accompanied by a parallel upregulation in l-arginine transport which is dependent, at least in part, on the synthesis of new carrier proteins. It is not clear however whether the induction of iNOS and its subsequent utilisation of l-arginine for NO synthesis contribute to the enhancement in l-arginine transport rates observed following induction of cells with pro-inflammatory mediators. To address this issue, we have transfected an iNOS construct in a pEGFP-N1 vector into HEK-293 cells and investigated the effects this has on l-arginine transport. The expression of iNOS through transfection resulted in the production of significant quantities of NO as detected by the standard Griess assay. Under these conditions, the transport of l-arginine was found to be unaltered, with rate of uptake being comparable in both transfected and non-transfected cells. Characterisation of the transporter(s) involved with uptake of l-arginine revealed features characteristic of the classical cationic amino acid transport system y(+). Further analysis of the expression profile of the cationic amino acid transporter (CAT) involved revealed the presence of transcripts for CAT-1 and CAT-2B. These data demonstrate that iNOS activity does not drive or enhance l-arginine transport despite the fact that HEK-293 cells transport l-arginine via the CATs, including CAT-2B which is thought to be critical for supply of substrate to iNOS.
Cui
Zhaoqiang
Z
Department of Biosciences, University of Hertfordshire, College Lane, Herts AL10 9AB, UK.
Tuladhar
Raj
R
Hart
Stephen L
SL
Marber
Michael S
MS
Pearson
Jeremy D
JD
Baydoun
Anwar R
AR
D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine / Q000378:metabolism*; D001692:Biological Transport / Q000502:physiology; D027201:Cationic Amino Acid Transporter 1 / Q000235:genetics / Q000502:physiology; D027202:Cationic Amino Acid Transporter 2 / Q000235:genetics / Q000502:physiology; D002460:Cell Line; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000032:analysis; D020869:Gene Expression Profiling; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007668:Kidney / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D008969:Molecular Sequence Data; D009569:Nitric Oxide / Q000096:biosynthesis; D019001:Nitric Oxide Synthase / Q000235:genetics* / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II; D009842:Oligopeptides / Q000494:pharmacology; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D012964:Sodium / Q000494:pharmacology
D027201:Cationic Amino Acid Transporter 1; D027202:Cationic Amino Acid Transporter 2; D018076:DNA, Complementary; D009842:Oligopeptides; D012333:RNA, Messenger; D049452:Green Fluorescent Proteins; D009569:Nitric Oxide; D001120:Arginine; D012964:Sodium; C496319:NOS2 protein, human; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496318:Nos2 protein, rat