Conditions to Study Nitric Oxide Generation by Polymorphonuclear Cells from an Inflammatory Exudate in Rats

Superoxide and nitric oxide release by leukocytes has been usually performed after exposure to a particular stimulus. We measured the generation of superoxide and nitric oxide by cells isolated from an inflammatory exudate of rats in either the absence or the presence of a variety of stimuli. Nonstimulated leukocytes generated superoxide radical (1.2 nmol·106cells−1) and nitric oxide (3.8 nmol·106cells−1) after 2 h incubation. When cells were incubated with lipopolysaccharides, opsonized zymosan or phorbol 12-myristate 13-acetate, superoxide level increased while nitric oxide decreased. Phorbol 12-myristate 13-acetate (100 ng/ml) induced a decrease of 0.88 nmol·106cells−1compared with nonstimulated cells, and incubation withN-iminoethyl-L-ornithine increased superoxide production by 0.81 nmol·106cells−1. These results provide clear evidence that cells from an inflammatory exudate which are already triggered are able to generate a considerable amount of nitric oxide and in less proportion superoxide, that the measure of nitric oxide must be performed without a further stimulus, and that both molecules react in an equimolar proportions to give peroxynitrite anion.