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. 2001 Mar 1;531(Pt 2):417-23.
doi: 10.1111/j.1469-7793.2001.0417i.x.

The role of L-tryptophan transport in L-tryptophan degradation by indoleamine 2,3-dioxygenase in human placental explants

Y Kudo  1 , C A Boyd
Affiliations

The role of L-tryptophan transport in L-tryptophan degradation by indoleamine 2,3-dioxygenase in human placental explants

Y Kudo et al. J Physiol. .

Abstract

The physiological importance of L-tryptophan transport for placental indoleamine 2,3-dioxygenase-mediated degradation of L-tryptophan has been studied using human placental chorionic villous explants. L-Tryptophan influx into villous explants is supported exclusively by transport system L and is substantially inhibited by the L-system-specific substrate 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH) and also by 1-methyl-tryptophan which is also an inhibitor of indoleamine 2,3-dioxygenase. L-Tryptophan influx is enhanced 2.3-fold following in vitro culture of the villous explant. Interferon-gamma, which increases villous explant indoleamine 2,3-dioxygenase expression, has no effect on L-tryptophan influx. In explants both BCH and 1-methyl-tryptophan inhibit indoleamine 2,3-dioxygenase-mediated L-tryptophan degradation. This also applies when L-tryptophan degradation has been stimulated by interferon-gamma. These findings show transport of L-tryptophan into the trophoblast to be a rate-limiting step for indoleamine 2,3-dioxygenase-mediated L-tryptophan degradation and therefore for the normal physiology of mammalian pregnancy.

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Figures

Figure 1
Figure 1. Time course of l-tryptophan influx into placental explants
Radioactive l-tryptophan content of explants of villous tissue was measured as a function of time over the period indicated. Medium contained 2 μml-[3H]tryptophan (2 μCi ml−1 or 74 KBq ml−1) in the presence or absence (choline) of sodium as described in Methods. •, sodium; ○, choline. Data represent the means ±s.d. of three separate experiments with triplicate assays from three different placentae.
Figure 2
Figure 2. Effect of BCH and 1-methyl-tryptophan and l-tryptophan on radioactive l-tryptophan influx into placental explants
l-Tryptophan influx over a 30 s period was measured in medium containing 2 μml-[3H]tryptophan (2 μCi ml−1 or 74 KBq ml−1) with indicated concentrations of unlabelled l-tryptophan (A) and BCH (BCH) or 1-methyl-tryptophan (B) in the absence (choline) of sodium as described in Methods. Data show carrier-mediated influx rate defined by subtracting the diffusional component from the total influx. The diffusional component was determined by measuring the influx of l-[3H]tryptophan in the presence of 20 mm unlabelled l-tryptophan. The lines were fitted using least-square regression analysis. Each point represents the mean ±s.d. of one representative of three separate experiments from three different placentae with five replicate assays. •, l-tryptophan (A) or BCH (B); ○, 1-methyl-tryptophan.
Figure 3
Figure 3. Effect of in vitro culture on l-tryptophan influx in placental explants
l-Tryptophan flux into either fresh or cultured (with or without 1000 units ml−1 interferon-γ (IFN)) explant of villous tissue over a 30 s period was measured in medium containing 2 μml-[3H]tryptophan (2 μCi ml−1 or 74 KBq ml−1) with or without unlabelled amino acid (2 mm for l-tryptophan, BCH or 1-methyl-tryptophan, final concentration) in the presence or absence (choline) of sodium as described in Methods. Data show carrier-mediated influx defined by subtracting the diffusional component from the total influx (in either the presence or absence of sodium). The diffusional component was determined by measuring the influx of l-[3H]tryptophan in the presence of 20 mm unlabelled l-tryptophan. Data represent the means ±s.d. of three separate experiments with triplicate assays from three different placentae. The mediated BCH-sensitive fluxes in fresh and in cultured tissue with or without interferon-γ are, respectively, 8.56 ± 1.58, 19.98 ± 2.20 and 19.64 ± 1.85 pmol (mg protein)−1 (30 s)−1. The rate following culture is significantly greater than for fresh samples (P value was less than 0.01 in both control and interferon-γ group).
Figure 4
Figure 4. Effect of 1-methyl-tryptophan on l-tryptophan catabolism by indoleamine 2,3-dioxygenase in placental explants
Villous explants were cultured with 1000 units ml−1 interferon-γ and/or 2 mm 1-methyl-tryptophan or vehicle (control) for the time indicated. Concentrations of l-tryptophan (A) and l-kynurenine (B) in the conditioned medium were analysed by HPLC and indoleamine 2,3-dioxygenase activity (C) in the tissue extract was determined colorimetrically as described in Methods. •, control; ○, interferon-γ; ▾, 1-methyl-tryptophan; ▿, interferon-γ and 1-methyl-tryptophan. Data represent the means ±s.d. of three separate experiments with triplicate assays from three different placentae.
Figure 5
Figure 5. Effect of BCH on l-tryptophan catabolism by indoleamine 2,3-dioxygenase in placental explants
Villous explants were cultured with 1000 units ml−1 interferon-γ and/or 2 mm BCH or vehicle (control) for the time indicated. Concentrations of l-tryptophan (A) and l-kynurenine (B) in the conditioned medium were analysed by HPLC and indoleamine 2,3-dioxygenase activity (C) in the tissue extract was determined colorimetrically as described in Methods. •, control; ○, interferon-γ; ▾, BCH; ▿, interferon-γ and BCH. Data represent the means ±s.d. of three separate experiments with triplicate assays from three different placentae.
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