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 inf lammatory cytokines, such as tumor necrosis factor (TNF)-alpha and int erferon (IFN)-gamma. Our hypothesis is that NO produced by cytokine-st imulated RPASM has local cytotoxic effects on endothelium. Accordingly , we designed a pulmonary smooth muscle and endothelial coculture expe riment in which the effects of NO on endothelium can be distinguished from the direct effects of cytokines. Methods. RPASM cells were incuba ted with a mixture of TNF-alpha (500 units/ml) and IFN-gamma (100 unit s/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 supernatan ts), and the number of viable attached endothelial cells were then mea sured at 48 hours. Results. RPASM continued to produce large amounts o f NO, in the absence of further cytokine stimulation after a 24-hour e xposure to TNF-alpha and IFN-gamma. This RPASM-generated NO decreased the number of viable attached endothelial cells after 24 hour RPASM-RP AE coculture by 57%. The competitive stereospecific inhibitor of induc ible NO synthase (iNOS), N-G-monomethyl-L-arginine (NMA), returned the inducible NO production to basal levels and reversed the cytotoxic ef fects on endothelial cells. The number of viable attached endothelial cells returned to control levels. Conclusions. The NO produced by cyto kine-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 king injury under conditions of trauma and sepsis.