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 immunostimulated cells during infection, plays an important role in host immune defense against microbial invasion. The impact of different rates of NO* production 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 bovine pulmonary artery endothelial cells. A retroviral vector (pMFGSNOS) encoding NOS II was used to transduce primary cultures of endothelial cells. Bovine 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, for 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 Gelfoam were found to secrete NO* at a rate exceeding 1.0 microM/h/10(6) cells, concomitant with an enhanced level of NOS II activity. Argininosuccinate synthetase, a key enzyme in the metabolism of l-citrulline to l-arginine, increased as well, perhaps in response to dimunition of the intracellular arginine pool corresponding to the observed high output of NO*. In spite of the continuous flux of NO*, endothelial cell viability was not effected. This system provides the opportunity to assess the impact of different levels of sustained NO* production on endothelial cell physiology.
Zhang
B
B
Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy, Baltimore, Maryland 21201, USA.
Cao
G L
GL
Domachowske
J
J
Jackson
M J
MJ
Porasuphatana
S
S
Rosen
G M
GM
D000818:Animals; D001124:Argininosuccinate Synthase / Q000378:metabolism; D002417:Cattle; D002470:Cell Survival; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000166:cytology / Q000201:enzymology*; D015870:Gene Expression; D051379:Mice; D009569:Nitric Oxide / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000235:genetics*; D052247:Nitric Oxide Synthase Type II; D012190:Retroviridae / Q000235:genetics; D014161:Transduction, Genetic
D009569:Nitric Oxide; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse; D001124:Argininosuccinate Synthase