THE RELATIONSHIP BETWEEN L-ARGININE-DEPENDENT NITRIC-OXIDE SYNTHESIS,NITRITE RELEASE AND DINITROSYL-IRON COMPLEX-FORMATION BY ACTIVATED MACROPHAGES

We identified the source of the nitrogen included into nitric oxide (N O) and studied the relationship between formation of NO, intracellular dinitrosyl ferrous iron complex (DNIC) and release of nitrite by muri ne bone-marrow-derived macrophages stimulated with E. coli lipopolysac charide (LPS). NO was trapped in the cell membrane by iron-diethyldith iocarbamate complex (FeDETC) and was detected as a paramagnetic NOFe(D ETC)2 complex by electron paramagnetic resonance (EPR) spectroscopy. M acrophages stimulated for 7 h up to 48 h with LPS and then incubated f or 2 h with DETC exhibited an anisotropic EPR signal of axial symmetry with g-factor values g(perpendicular-to) = 2.035, g(parallel-to) = 2. 02 and a triplet hyperfine structure (hfs) at g(perpendicular-to) char acteristic for NOFe(DETC)2. In cells incubated with [N-15(G)]L-arginin e instead of [N-14(G)]L-arginine the EPR signal of [N-15]OFe(DETC)2 wa s detected with a doublet hfs at g(perpendicular-to), indicating that NO was generated exclusively from the terminal guanidino-nitrogen of e xtracellular L-arginine. The ratio of NO formation and of nitrite rele ase changed with time of exposure to LPS, nitrite exceeding NO at earl y stages of macrophage activation, and NO exceeding nitrite at later s tages. DNIC with thiolate ligands (0.5 nmol/10(7) Cells) was observed in stimulated macrophages not loaded with DETC. Furthermore, DNIC rele ased from macrophages was trapped in the extracellular medium by bovin e serum albumin (BSA) (1 nmol/107 cells per 2 h) by formation of a par amagnetic DNIC with BSA. DNIC release not only provides a route for ir on loss from activated macrophages, but may also play a role in the cy totoxic and microbiostatic activity of macrophages.