Endotoxin-induced nitric oxide production in pulmonary artery endothelial cells is regulated by cytokines.
BACKGROUND
L-Arginine is the sole precursor of nitric oxide (NO). Bacterial lipopolysaccharide (endotoxin) (LPS) stimulates carrier-mediated L-arginine transport in porcine pulmonary artery endothelial cells (PAECs) through an autocrine pathway that involves interleukin-1 alpha (IL-1 alpha) and tumor necrosis factor alpha (TNF-alpha).


OBJECTIVE
To determine if Escherichia coli LPS stimulates NO synthesis in PAECs and, if so, if LPS stimulation of NO production is also mediated by autocrine secretion of IL-1 alpha and TNF-alpha.


METHODS
Monolayers of PAECs were incubated with various concentrations of LPS, recombinant human TNF-alpha, or IL-1 alpha, and total nitrate-nitrite accumulation was measured at different time points with the Greiss reagent following cadmium reduction. Release of TNF-alpha and IL-1 alpha release by LPS-stimulated PAECs were measured using the WEHI (for TNF-alpha) and A375.S2 (for IL-1 alpha) bioassays. The PAECs were then incubated with saline solution or LPS in the presence or absence of either a polyclonal antibody to human TNF or IL-1 receptor antagonist, and nitrate-nitrite accumulation was measured at 48 hours.


RESULTS
Production of NO by PAECs was increased 230% by LPS (1 microgram/mL), 350% by TNF-alpha (1000 U/mL), and 240% by IL-1 alpha (1000 U/mL) (P < .05 vs control). The LPS-stimulated NO production was inhibited by IL-1 receptor antagonist (100 micrograms/mL) or antibody to TNF (10 micrograms/mL) to control levels (P < .05 vs LPS; difference vs saline solution was not significant). The LPS-stimulated TNF-alpha secretion by PAECs and TNF-alpha activity were maximal at 6 hours (400 +/- 42 pg/mL). The IL-1 alpha activity was not detectable in LPS-stimulated PAECs by the A375.S2 bioassay.


CONCLUSIONS
Endotoxin, TNF-alpha, and IL-1 alpha stimulated NO synthesis in PAECs. Endotoxin-stimulated NO synthesis through an autocrine pathway involving the cytokines TNF-alpha and IL-1 alpha.
Cendan
J C
JC
Department of Surgery, University of Florida, Gainesville.
Moldawer
L L
LL
Souba
W W
WW
Copeland
E M
EM
Lind
D S
DS
D000818:Animals; D001120:Arginine / Q000031:analogs & derivatives / Q000276:immunology; D004730:Endothelium, Vascular / Q000276:immunology / Q000378:metabolism*; D004926:Escherichia coli; D007375:Interleukin-1 / Q000276:immunology / Q000378:metabolism*; D008070:Lipopolysaccharides / Q000276:immunology*; D009569:Nitric Oxide / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000276:immunology; D011651:Pulmonary Artery / Q000166:cytology*; D013552:Swine; D013997:Time Factors; D014409:Tumor Necrosis Factor-alpha / Q000276:immunology / Q000378:metabolism*; D019323:omega-N-Methylarginine
D007375:Interleukin-1; D008070:Lipopolysaccharides; D014409:Tumor Necrosis Factor-alpha; D019323:omega-N-Methylarginine; D009569:Nitric Oxide; D001120:Arginine