Nitric oxide produced by cytokine-activated pulmonary artery smooth muscle cells is cytotoxic to cocultured endothelium.
BACKGROUND
We recently demonstrated that rat pulmonary artery smooth muscle (RPASM) generates maximal nitric oxide (NO) when exposed to inflammatory cytokines, such as tumor necrosis factor (TNF)-alpha and interferon (IFN)-gamma. Our hypothesis is that NO produced by cytokine-stimulated RPASM has local cytotoxic effects on endothelium. Accordingly, we designed a pulmonary smooth muscle and endothelial coculture experiment in which the effects of NO on endothelium can be distinguished from the direct effects of cytokines.


METHODS
RPASM cells were incubated with a mixture of TNF-alpha (500 units/ml) and IFN-gamma (100 units/ml) for 24 hours. This cytokine mixture was then removed and the NO-producing smooth muscle cells were incubated in a coculture transwell system with rat pulmonary artery endothelial (RPAE) cells. Subsequent NO production (as measured by nitrite concentration in cell supernatants), and the number of viable attached endothelial cells were then measured at 48 hours.


RESULTS
RPASM continued to produce large amounts of NO, in the absence of further cytokine stimulation, after a 24-hour exposure to TNF-alpha and IFN-gamma. This RPASM-generated NO decreased the number of viable attached endothelial cells after 24 hour RPASM-RPAE coculture by 57%. The competitive stereospecific inhibitor of inducible NO synthase (iNOS), NG-monomethyl-L-arginine (NMA), returned the inducible NO production to basal levels and reversed the cytotoxic effects on endothelial cells. The number of viable attached endothelial cells returned to control levels.


CONCLUSIONS
The NO produced by cytokine-activated RPASM has local cytotoxic effects on RPAE in coculture. Such NO produced in the vasculature may be a factor in the origin of acute lung injury under conditions of trauma and sepsis.
Thomae
K R
KR
Department of Surgery, University of North Carolina at Chapel Hill, USA.
Joshi
P C
PC
Davies
P
P
Pitt
B R
BR
Billiar
T R
TR
Simmons
R L
RL
Nakayama
D K
DK
D000818:Animals; D002478:Cells, Cultured; D004730:Endothelium, Vascular; D007371:Interferon-gamma / Q000502:physiology*; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000378:metabolism*; D009569:Nitric Oxide / Q000096:biosynthesis*; D011651:Pulmonary Artery / Q000166:cytology / Q000378:metabolism*; D051381:Rats; D014409:Tumor Necrosis Factor-alpha / Q000502:physiology*
D014409:Tumor Necrosis Factor-alpha; D009569:Nitric Oxide; D007371:Interferon-gamma