Differences in nitric oxide synthase activity in a macrophage-like cell line, RAW264.7 cells, treated with lipopolysaccharide (LPS) in the presence or absence of interferon-c (IFN-c): possible heterogeneity of iNOS activity

Nitric oxide synthase (NOS) activities were compared in a macrophage-like cell line, RAW 264.7 cells, treated with bacterial lipopolysaccharide (LPS) alone or with LPS and inter-feron- 7 (IFN- 7). An about 5-6-fold higher amount of NO2 " originating from the nitric oxide radical ( • NO) was produced in the culture supernatant of macrophages treated with LPS + IFN- 7 than with LPS alone, depending on both the time of incubation and the dose of LPS. However, the difference in the amounts of iNOS protein in these macrophage extracts was much greater than that in the amount of released NO2". To estimate the NOS activity after induction of NOS at 37'C for 8 h, we examined both intact cells and cell extracts. The cells were washed and re-incubated in Hank's balanced salt solution (HBSS) containing 1 mM L-arginine and 100 pM carboxy-PTIO, which enabled us to detect trace amounts of-NO by means of rapid reaction of carboxy PTIO with »NO to form-NO2, which was finally converted to NO2~. The results showed an about 7-fold difference between the macrophages pretreated with LPS alone and those with LPS+IFN- 7. Using the extracts, the NOS activity was assayed with L-[C/-14C]arginine as a substrate for NOS in vitro, and the results again revealed an about 7-fold difference between the two types of cell extracts. A kinetic stud