Nitric oxide suppression of cellular proliferation depends on cationic amino acid transporter activity in cytokine-stimulated pulmonary endothelial cells.
Inducible nitric oxide (NO) synthase (iNOS) is a stress response protein upregulated in inflammatory conditions, and NO may suppress cellular proliferation. We hypothesized that preventing L-arginine (L-arg) uptake in endothelial cells would prevent lipopolysaccharide/tumor necrosis factor-α (LPS/TNF)-induced, NO-mediated suppression of cellular proliferation. Bovine pulmonary arterial endothelial cells (bPAEC) were treated with LPS/TNF or vehicle (control), and either 10 mM L-leucine [L-leu; a competitive inhibitor of L-arg uptake by the cationic amino acid transporter (CAT)] or its vehicle. In parallel experiments, iNOS or arginase II were overexpressed in bPAEC using an adenoviral vector (AdiNOS or AdArgII, respectively). LPS/TNF treatment increased the expression of iNOS, arginase II, CAT-1, and CAT-2 mRNA in bPAEC, resulting in greater NO and urea production than in control bPAEC, which was prevented by L-leu. LPS/TNF treatment resulted in fewer viable cells than in controls, and LPS/TNF-stimulated bPAEC treated with L-leu had more viable cells than LPS/TNF treatment alone. LPS/TNF treatment resulted in cleaved caspase-3 and cleaved poly(ADP-ribose) polymerase expression, which was attenuated by L-leu. AdiNOS reduced viable cell number, and treatment of AdiNOS transfected bPAEC with L-leu preserved cell number. AdArgII increased viable cell number, and treatment of AdArgII transfected bPAEC with L-leu prevented the increase in cell number. These data demonstrate that iNOS expression in pulmonary endothelial cells leads to decreased cellular proliferation, which can be attenuated by preventing cellular L-arg uptake. We speculate that CAT activity may represent a novel therapeutic target in inflammatory lung diseases characterized by NO overproduction.
Department of Pediatrics, The Ohio State University, Columbus, Ohio, USA.
Louis G
LG
Chicoine
Louis G
LG
Chicione
Michael R
MR
Stenger
Hongmei
H
Cui
Andrea
A
Calvert
Rebecca J
RJ
Evans
B Keith
BK
English
Yusen
Y
Liu
Leif D
LD
Nelin
D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D001119:Arginase / Q000378:metabolism; D001120:Arginine / Q000378:metabolism; D027201:Cationic Amino Acid Transporter 1 / Q000235:genetics / Q000378:metabolism*; D027202:Cationic Amino Acid Transporter 2 / Q000235:genetics / Q000378:metabolism*; D002417:Cattle; D002452:Cell Count; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D042783:Endothelial Cells / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D008070:Lipopolysaccharides / Q000494:pharmacology; D008168:Lung / Q000166:cytology*; D009569:Nitric Oxide / Q000096:biosynthesis / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*; D014508:Urea / Q000378:metabolism
D027201:Cationic Amino Acid Transporter 1; D027202:Cationic Amino Acid Transporter 2; D008070:Lipopolysaccharides; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; D009569:Nitric Oxide; D014508:Urea; D001120:Arginine; D052247:Nitric Oxide Synthase Type II; D001119:Arginase
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