| 2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE) is a unique endogenous ligand for the arylhydrocarbon receptor (AhR). The circulating and inactive form of ITE is 2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylic acid (ITC). We determined endogenous levels of ITC in a variety of cell lines, as well as in various tissues collected from the rat. Four cell lines IEC-6 (rat small intestine epithelial), MCF7 (human breast adenocarcinoma), CV-1 (green African monkey kidney cells), and SH-SY5Y (human neuroblastoma), and three rat tissues (kidney, lung, and intestine) were found to have detectable ITC. Based upon the structure of the ligand, we hypothesized the amino acids tryptophan and cysteine may be the ligand precursors. Rat kidney tissue homogenate was found to have the highest levels of ITC, which significantly increased with the addition of 2 x 10-3 M tryptophan or 2 x 10-3 M cysteine. We optimized the conditions for the incubation of kidney homogenate with tryptophan, and found that maximal ITC is produced at one hour with 2 x 10-3 M tryptophan. Incorporation of the added amino acids was proven with [3H] Trp and [ 35S] Cys, resulting in radiolabeled ITC. Incubation of metabolites of L-tryptophan and D-tryptophan, tryptamine and indole-3-pyruvate with rat kidney homogenate, were also found to increase ITC biosynthesis. Most significant was indole-3-pyruvic acid, which supported a 30 to 40-fold increase of ITC production over that of L-tryptophan. We have determined that tryptophan and cysteine are precursors to ITC, and that metabolites of tryptophan, especially indole-3-pyruvate, appear to be the preferred precursors of the indole portion of ITC. |
