Effect of tryptophan on the early tri-iodothyronine uptake in mouse thymocytes

Department of Experimental Medicine and Pathology, University 'La Sapienza' of Rome, Italy.

OBJECTIVE: We have studied the effect of tryptophan on cellular [(125)I]tri-iodothyronine (T3) uptake by mouse thymocytes. MATERIALS AND METHODS: Mouse thymocytes (20 x 10(6 )cells/ml) were suspended in Krebs-Ringer solution buffered by Tris-HCl and incubation (23 degrees C at pH7.45+/-0.6), in the presence or absence of 1mM tryptophan, was started by adding 25 pM [(125)I]T3. At the end of incubation, samples were cooled in ice, centrifuged over a 30% sucrose cushion and the cell-associated radioactivity was measured in the pellet. RESULTS: Tryptophan reduced both the total and the saturable fraction of [(125)I]T3 uptake by 44% (P=0.0009) and 60% (P=0.0006) respectively, following 1 min of incubation. This effect was specific and dose-dependent, being maximal at 5mM concentration (-82%). In contrast, the pre-exposure of cells to tryptophan for up to 2h had no effect on the subsequent uptake of [(125)I]T3, in the absence of tryptophan. The effect of D-tryptophan on saturable T3 uptake was not different from that obtained using the L-stereoisomer. Tryptophan reduced the V(max) of the initial rate of saturable [(125)I]T3 uptake by two-thirds without affecting the apparent K(m) (2.2 nM) of the process, thus indicating the non-competitive nature of the inhibition. In sodium-free medium the saturable [(125)I]T3 uptake was reduced by 43%. The inhibitory effect of tryptophan on [(125)I]T3 uptake was exerted in both the presence and the absence of sodium. In fact, the inhibitory effect of tryptophan on T3 transport was greater and significantly different (P=0.0046) from that obtained by sodium depletion alone. CONCLUSIONS: Tryptophan interferes with both the sodium-dependent and -independent components of [(125)I]T3 uptake by a dose-dependent, non-competitive mechanism which operates in cis-modality at the plasma membrane level of mouse thymocytes.

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