Fig. 5.

AdiNOS (adenoviral vectors containing the human iNOS gene)-induced iNOS overexpression in bPAEC and arginine uptake was necessary for AdiNOS-induced nitric oxide production and changes in cellular proliferation. To isolate effects of iNOS alone, without cytokine-induced effects on CAT or arginase, we studied bPAEC, wherein iNOS was overexpressed using AdiNOS. The adenoviral vector also contained the gene for green fluorescent protein. The transfection efficiency in the bPAEC was good, as can be seen in the two examples: A is phase contrast, and B is fluorescence. C: transfection of bPAEC with Adβ-gal did not result in detectable iNOS mRNA bands on RT-PCR, while transfection of bPAEC with AdiNOS resulted in easily detectable iNOS mRNA bands. When 10 mM l-leu was added to the media, there was no effect on the AdiNOS-induced iNOS mRNA expression. Transfection with AdiNOS resulted in readily detectable iNOS protein bands on Western blotting, and l-leu had no effect on AdiNOS-mediated iNOS protein expression. D: transfection of bPAEC with AdiNOS resulted in a significantly greater NO production than in Adβ-gal-transfected bPAEC. The addition of 10 mM l-leu (Leu) to the media significantly attenuated NO production in AdiNOS-transfected bPAEC. Values are means ± SE; n = 6 for each group. *AdiNOS different from Adβ-gal, P < 0.01. #AdiNOS + Leu different from AdiNOS, P < 0.01. E: transfection of bPAEC with AdiNOS reduced viable cell numbers compared with transfection with Adβ-gal. Inhibiting l-arginine uptake with l-leu returned cellular numbers in AdiNOS-transfected bPAEC to those seen in Adβ-gal-transfected bPAEC. The number of viable bPAEC were counted after a 48-h incubation. Values are means ± SE; n = 4–6 for each group. *AdiNOS viable cell number different from Adβ-gal viable cell number, P < 0.01. #AdiNOS + Leu different from AdiNOS alone, P < 0.01.