Endogenously Produced Nitric Oxide Increases Tumor Necrosis Factor-a Production in Transfected Human U937 Cells

Various functions of human phagocytes are modulated by exposing intact cells to calcium ionophore using the cGMP reporter cell assay (P ! .0001). After differentiation with nitric oxide (NO). We transfected the human U937 monoblastoid cell line with an expression vector containing human phorbol-12-myristate-13-acetate (PMA), iNOS transfectants produced more tumor necrosis factor-a (TNF-a) (124.9Ô 25.4 endothelial NO synthase (eNOS) or murine inducible NOS (iNOS) cDNA to study the regulatory role of NO without the pg/5 Ì 10 cells per 24 hours) than did empty-vector transfected cells (21.9 Ô 1.9 pg/5 Ì 10 cells per 24 hours; P ! nonspecific effects associated with exogenous NO sources. Western blot confirmed expression of eNOS or iNOS in re.02). This effect was inhibited by 500 mmol/L L-NMA (54.4 Ô 3.1 pg/5 Ì 10 cells per 24 hours; P ! .05). However, in the spectively transfected cells, but not in naive or empty-vector transfected cells. Transfectants expressing iNOS, a calciumpresence of high concentrations of lipopolysaccharide (1 mg/ mL), which further increased NO production in iNOS transindependent enzyme, but not eNOS, a calcium-dependent enzyme, spontaneously produced NO (P Ú .001). The NO fected cells (P! .044), TNF-a production was similar comparing PMA-differentiated iNOS and empty-vector transfectants release from iNOS-transfected cells, as measured by nitrite and nitrate accumulation and by cyclic guanosine mono(12.2 Ô 0.8 and 13.1 Ô 1.7 ng/5 Ì 10 cells per 24 hours, respectively; P ! .5). The results show that under certain phosphate (cGMP) increases in rat reporter cells, was inhibitable (P Ú .01 for both) with N-methyl-L-arginine (L-NMA), conditions endogenously produced NO can upregulate TNFa production in human phagocytes. a NOS inhibitor. The eNOS transfectants were shown to contain functional enzyme by the conversion of L-arginine This is a US government work. There are no restrictions on its use. to L-citrulline in fractionated cells (P ! .0001) and by