No detectable NO synthesis from L-arginine or N-G-hydroxy-L-arginine in fMLP-stimulated human blood neutrophils despite production of nitrite, nitrate, and citrulline from N-G-hydroxy-L-arginine

Nitric oxide (NO) is a well-documented effector molecule iu rodent phagocyt es but its synthesis in human neutrophils has been controversial. In this s tudy, NO production in human neutrophils activated by chemotactic peptide N -formyl-methionyl-leucyl-phenylalanine (fMLP) was measured in the presence of L-arginine (L-Arg) and N-G-hydrosy-L-arginine (OH-L-Arg), the precursor and intermediate amino acids in NO synthesis, respectively. Incubation of f MLP-activated neutrophils with OH-L-Arg resulted in a production of nitrite , nitrate, and citrulline that was greater than with unstimulated neutrophi ls but was not inhibited by the NOS inhibitors L-NMMA and L-NIO or the cyto chrome P-450 inhibitor troleandomycin and was not seen when OH-L-Arg was re placed with L-Arg, This nitrite, nitrate, and citrulline production was not associated with any detectable NO synthesis because no increases in cyclic GMP were observed in the presence of phosphodiesterase inhibitors and in t he presence or absence of superoxide dismutase, Moreover, no increases in t he formation of the reaction product of NO with superoxide, peroxynitrite, were observed on addition of either OH-L-Arg or L-Arg to activated neutroph ils, as assessed either by dihydrorhodamine oxidation or protein nitration, This suggests that, in spite of the production of nitrite, nitrate, and ci trulline, commonly used indicators of NO formation, normal human blood neut rophils, are not producing detectable amounts of either NO or peroxynitrite when stimulated with fMLP in the presence of OH-L-Arg.