FIG. 1.

Induction of IFN-γ-mediated IDO mRNA expression and measurement of IDO activity in ASMC with the effects of the inhibitor 1-MT. (A) ASMC were treated with increasing concentrations of IFN-γ for 48 h and pulse-labeled with [³H]tryptophan for 4 h. IDO activity was expressed as the percentage of pulse-labeled [³H]tryptophan converted to metabolites, determined by paper chromatography. (Inset) RT-PCR analysis of IDO mRNA expression in ASMC treated with 500 U of IFN-γ per ml or left untreated. Lane 1, molecular weight standard; lane 2, 12-h IFN-γ treatment; lane 3, 12 h, untreated; lane 4, 24-h IFN-γ treatment; lane 5, 24 h, untreated; lane 6, negative control (distilled H₂O); lane 7, positive control (IDO cDNA cloned into pUC19 as described in the text). (B) ASMC monolayers were pretreated for 1 h with 1-MT, followed by IFN-γ (25 U/ml) stimulation for 48 h. Monolayers were pulse-labeled with [³H]tryptophan for 4 h, and IDO activity was measured by [³H]tryptophan catabolism using paper chromatography and calculated as the percentage converted to metabolite fractions.