Stable expression of varied levels of inducible nitric oxide synthase in primary cultures of endothelial cells

Nitric oxide (NO.), generated by nitric oxide synthase (NOS II) from immuno stimulated cells during infection, plays an important role in host immune d efense against microbial invasion. The impact of different rates of NO. pro duction on host cell function has not been defined. Herein, we describe the development of a method to express varied levels of murine NOS II in bovin e pulmonary artery endothelial cells. A retroviral vector (pMFGSNOS) encodi ng NOS II was used to transduce primary cultures of endothelial cells. Bovi ne endothelial cells were susceptible to this transduction and up to 18% of the cells expressed immunodetectable murine NOS II. The NOS II-transduced endothelial cells were cultured on the three-dimensional matrix, Gelfoam, f or 8-10 days. Stable expression of NOS II was assessed by measuring nitrite accumulation in media every 2 days. By day 10, endothelial cells on Gelfoa m were found to secrete NO. at a rate exceeding 1.0 muM/h/10(6) cells, conc omitant with an enhanced level of NOS II activity. Argininosuccinate synthe tase, a key enzyme in the metabolism of L-citrulline to L-arginine, increas ed as well, perhaps in response to dimunition of the intracellular arginine pool corresponding to the observed high output of NO.. In spite of the con tinuous flux of NO., endothelial cell viability was not effected. This syst em provides the opportunity to assess the impact of different levels of sus tained NO. production on endothelial cell physiology. (C) 2000 Academic Pre ss.