Effects of arginase isoforms on NO Production by nNOS

Abstract

Both arginase isoforms (AI and AII) regulate high-level NO production by the inducible NOS, but whether the arginase isoforms also regulate low-level NO production by neuronal NOS (nNOS) is not known. In this study, 293 cells that stably overexpress nNOS gene (293nNOS cells) were transfected with rat AI (pEGFP-AI) or AII (pcDNA-AII) plasmids, and nitrite production was measured with or without supplemental L-arginine. Transfection with pEGFP-AI increased AI expression and activity 10-fold and decreased intracellular l-arginine by 50%. Nitrite production was inhibited by >80% when no l-arginine was supplemented but not when 1 mM L-arginine was present. The inhibition was reversed by an arginase inhibitor, N(omega)-hydroxy-L-arginine. Transfection with pcDNA-AII increased AII expression and activity but had little effect on nitrite production even if no l-arginine was added. These results suggest that, in 293nNOS cells, AI was more effective in regulating NO production by nNOS, most likely by competing for L-arginine.

FIG. 1.

Western blot analysis of AI from cells transfected with pEGFP-AI expression plasmid: lanes 1–4, pEGFP-AI; lanes 5–7, pEGFP-C3. Each lane represents different experiments. Protein concentration for each lane: 20 μg.

FIG. 2.

Fluorescent microscopic pictures of 293nNOS cells transfected with pEGFP-C3 (A) and pEGFP-AI (B). Arrowheads indicate cells expressing GFP-AI. Magnification: 60×.

FIG. 3.

Western blot analysis of AII from cells transfected with pcDNA-AII expression plasmid: lane 1, pcDNA-3C; lane 2, pcDNA-AII. Protein concentration for each lane: 20 μg.

FIG. 4.

Intracellular arginine, citrulline, and ornithine in cells 16 h after incubation with control, pEGFP-AI, and pcDNA-AII plasmids. *P < 0.05 vs control plasmids.

FIG. 5.

Nitrite production by 293nNOS cells incubated with (A) pEGFP-AI (n = 6) and (B) pcDNA-AII (n = 6) for 16 h and exposed to 0 or 1 mM l-arginine in the medium for 2 h. *P < 0.05 vs pEGFP-C3.

FIG. 6.

Nitrite production by 293nNOS cells incubated with control, pEGFP-AI, and pcDNA-AII plasmids for 16 h and exposed to 0 mM l-arginine in the medium for 2 h. Some cells were treated with 1 mM N^ω-hydroxy-l-arginine added at the beginning of the 2-h incubation. Nitrite production by 293 cells incubated with control, pEGFP-AI, and pcDNA-AII plasmids for 16 h and exposed to 1 mM l-arginine in the medium for 2 h is also shown for comparison. *P < 0.05 vs 0 mM l-arginine without N^ω-hydroxy-l-arginine; N = 5 each for experiments with N^ω-hydroxy-l-arginine.