title: TNF-stimulated arginine transport by human vascular endothelium requires activation of protein kinase C.
abstract: OBJECTIVE
  The authors determined the endothelial arginine transport mechanism and the potential role of a tumor necrosis factor (TNF)-alpha-mediated signal transduction pathway involving protein kinase C (PKC) in regulating this transport in cultured endothelial cells.
  
  
  BACKGROUND
  The vascular endothelium metabolizes arginine to generate nitric oxide (NO), and an increase in NO production can be stimulated by several cytokines. The mechanism(s) responsible for the accelerated arginine transport are poorly understood.
  
  
  METHODS
  Arginine transport was assayed in confluent human umbilical vein endothelial cells in the presence of TNF +/- the PKC inhibitor chelerythrine chloride.
  
  
  RESULTS
  Carrier-mediated arginine transport was accomplished by two Na(+)-independent transporters, System y+ (80% of total transport) and System b0,+ (20% of transport). Tumor necrosis factor (0.1-2 ng/mL) increased System y(+)-mediated arginine transport in a time- and dose-dependent manner by augmenting System y+ transport maximal capacity (control Vmax = 1325 +/- 60 pmol/mg protein/minute vs. TNF Vmax = 3015 +/- 110 pmol/mg protein/minute, p < 0.01) without affecting transporter affinity (control Km = 30 +/- 1.4 microM vs. 34 +/- 1.3 microM arginine, p = NS). Stimulation was maximal at the 8-hour time point and was inhibited by both actinomycin D and cycloheximide. In addition, inhibition of PKC with chelerythrine abrogated the TNF-augmented arginine transport. Similarly, incubation of cells with the direct PKC activator TPA (phorbol ester 12-myristate 13-acetate) stimulated System y(+)-mediated arginine transport nearly fivefold, secondary to an increase in transporter Vmax (TPA Vmax = 5349 +/- 310 pmol/mg protein/minute, p < 0.001 vs. control), with no change in Km. This TPA-induced stimulation of arginine transport also was blocked by chelerythrine CI, actinomycin D, and cycloheximide. Incubation of TNF-stimulated cells with two NO synthase inhibitors did not reduce transport activity, suggesting that the arginine transporter and the NO synthase enzyme may, in part, be independently regulated.
authors.affiliation: Department of Surgery, Massachusetts General Hospital, Boston, USA.
authors.forename: M
authors.initials: M
authors.lastname: Pan
authors.forename: M
authors.initials: M
authors.lastname: Wasa
authors.forename: D S
authors.initials: DS
authors.lastname: Lind
authors.forename: J
authors.initials: J
authors.lastname: Gertler
authors.forename: W
authors.initials: W
authors.lastname: Abbott
authors.forename: W W
authors.initials: WW
authors.lastname: Souba
mesh_terms: D000470:Alkaloids; D000594:Amino Acid Oxidoreductases / Q000037:antagonists & inhibitors; D001120:Arginine / Q000378:metabolism*; D053119:Benzophenanthridines; D001692:Biological Transport / Q000502:physiology; D002148:Calmodulin-Binding Proteins / Q000037:antagonists & inhibitors; D002478:Cells, Cultured; D017868:Cyclic AMP-Dependent Protein Kinases / Q000502:physiology; D004730:Endothelium, Vascular / Q000378:metabolism*; D006801:Humans; D019001:Nitric Oxide Synthase; D010617:Phenanthridines / Q000494:pharmacology; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000494:pharmacology / Q000502:physiology*; D015290:Second Messenger Systems; D015398:Signal Transduction / Q000502:physiology*; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology / Q000502:physiology*; D014471:Umbilical Veins / Q000166:cytology
chemical_list: D000470:Alkaloids; D053119:Benzophenanthridines; D002148:Calmodulin-Binding Proteins; D010617:Phenanthridines; D014409:Tumor Necrosis Factor-alpha; D001120:Arginine; C016299:chelerythrine; D019001:Nitric Oxide Synthase; D000594:Amino Acid Oxidoreductases; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C