KINETIC MODELING OF THE NITRIC-OXIDE GRADIENT GENERATED IN-VITRO BY ADHERENT CELLS EXPRESSING INDUCIBLE NITRIC-OXIDE SYNTHASE

Inducible nitric oxide (NO) synthase produces a long-lasting NO flux w hich can exert cytotoxic effects on target cells. A prerequisite for t he understanding of the molecular basis of NO action is quantitative d ata on the availability of this small neutral radical molecule at both the spatial and temporal levels. The limits of NO availability depend on the respective rates of NO production, diffusion and autoxidation by molecular oxygen. Kinetic modelling of these processes has been per formed for a widely used experimental system consisting of a monolayer of adherent cells cultured in vitro for hours in unstirred culture me dium. It appears that: (i) the maximal NO concentration in the culture is in the immediate vicinity of the monolayer, where target cells wil l sediment; (ii) the steady-state NO concentration in this area is low er than 4 to 5 mu M; and (iii) measurements of nitrite/nitrate or citr ulline accumulation in the bulk cell medium culture during a given tim e period significantly underestimate (by a factor of up to 3 to 4) the true rate of NO synthesis at the level of the producer cell. This rat e can be, nevertheless, easily estimated from the rate of production o f the stable NO synthase products.