Stable expression of human inducible nitric oxide synthase in V79 Chinese hamster cells

Abstract

A recombinant expression vector containing the full-length cDNA for human inducible nitric oxide (NO) synthase was constructed for constitutive expression in V79 Chinese hamster cells. Expression was followed by Western analyses using three different NO synthase antisera. Activity remained stable during 4 months of continued cultivation. Activities were 25 pmol min −1 mg −1 cytosolic protein with l-arginine and 47 pmol min −1 mg −1 cytosolic protein with N G-hydroxy- l-arginine as substrates. Activity was concentration-dependently inhibited by inhibitors such as N G-methyl- l-arginine, N G-nitro- l-arginine, N G-nitro- l-arginine methyl ester, aminoguanidine, and S-methyl-isothiourea. The rank order of inhibitor potencies was different from published results obtained with rodent inducible NOS. Parental V79 cells do not express and cannot be induced for NO synthase activity. Therefore, the genetically engineered V79 cell line is defined for the cDNA-encoded human inducible NO synthase. The new cell line may serve as a useful tool to study human inducible NO synthase.