Induction of nitric oxide synthase in human vascular smooth muscle: interactions between proinflammatory cytokines.
OBJECTIVE
We have attempted to demonstrate the induction of inducible nitric oxide synthase in human vascular tissue and define the capacity of different cytokines to induce this enzyme.


METHODS
Segments of human arteries were stimulated with lipopolysaccharide (10 micrograms/ml), interleukin-1 beta (5 U/ml), tumor necrosis factor-alpha (10 U/ml), and interferon-gamma (200 U/ml). Cytokines were either used alone or in certain combinations, as well as in the presence of L-NG-monomethyl-arginine (100 mumol/l) or cycloheximide (1 mumol/l). Induction was assessed by measurement of mRNA expression, immunocytochemical localisation of the expressed protein, nitric oxide synthase activity and levels of nitrite, a product of nitric oxide formation.


RESULTS
PCR analysis showed the presence of mRNA for iNOS in stimulated samples which could be inhibited by cycloheximide. There was positive staining with an antibody against human iNOS in the media of stimulated vessel segments. Stimulated segments were also shown to contain Ca(2+)-independent nitric oxide synthase activity. The cytokines and lipopolysaccharide together gave a significant rise in levels of nitrite in the medium after 36 and 48 h, which was inhibited by L-NG-monomethyl-arginine and cycloheximide. Only interferon-gamma incubated alone was capable of increasing nitrite levels. This effect was enhanced by co-incubation with either interleukin-1 beta, tumor necrosis factor-alpha or lipopolysaccharide.


CONCLUSIONS
We have shown that increased production of nitrite by human vascular tissue in response to cytokines is associated with induction of iNOS as shown at the molecular and protein levels, and further supported by the presence of increased Ca(2+)-independent nitric oxide synthase activity following cytokine stimulation.
Chester
A H
AH
Department of Cardiothoracic Surgery, Imperial College of Science, Technology and Medicine, National Heart and Lung Institute, Middlesex, UK.
Borland
J A
JA
Buttery
L D
LD
Mitchell
J A
JA
Cunningham
D A
DA
Hafizi
S
S
Hoare
G S
GS
Springall
D R
DR
Polak
J M
JM
Yacoub
M H
MH
D003513:Cycloheximide / Q000494:pharmacology; D016207:Cytokines / Q000378:metabolism*; D004357:Drug Synergism; D004790:Enzyme Induction; D006801:Humans; D007150:Immunohistochemistry; D066298:In Vitro Techniques; D007371:Interferon-gamma / Q000494:pharmacology; D007375:Interleukin-1 / Q000494:pharmacology; D008070:Lipopolysaccharides / Q000494:pharmacology; D009131:Muscle, Smooth, Vascular / Q000737:chemistry / Q000201:enzymology*; D019001:Nitric Oxide Synthase / Q000032:analysis / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II; D009573:Nitrites / Q000032:analysis; D016133:Polymerase Chain Reaction; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D012333:RNA, Messenger / Q000032:analysis; D013268:Stimulation, Chemical; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D019323:omega-N-Methylarginine / Q000494:pharmacology
D016207:Cytokines; D007375:Interleukin-1; D008070:Lipopolysaccharides; D009573:Nitrites; D011500:Protein Synthesis Inhibitors; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; D019323:omega-N-Methylarginine; D007371:Interferon-gamma; D003513:Cycloheximide; C496319:NOS2 protein, human; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II