High-affinity uptake of L-kynurenine by a Na+-independent transporter of neutral amino acids in astrocytes

Abstract

L-Kynurenine (KYN), an intermediary product in the kynurenine pathway of tryptophan metabolism, is the common precursor from which are formed both quinolinic acid, a potent endogenous "excitotoxin," and kynurenic acid, a nonselective antagonist of excitotoxins. The present work examines 3H-KYN transport in primary astrocyte cultures derived from the cerebra of newborn mice. Influx and efflux of 3H-KYN were attributable almost entirely to carrier-mediated transport. The tritium recovered in uptake experiments was identifiable as 3H-KYN, indicating a low rate of KYN metabolism during incubations up to 30 min. KYN uptake decreased in the presence of extracellular Na+, at least in part because KYN efflux was accelerated. Marked trans stimulation of KYN efflux by extracellular KYN provided evidence of the exchanging nature of the carrier. Saturation curves for the initial velocity of KYN uptake conformed to a 1-component saturable system with Km of 32 microM and Vmax of 2.1 nmol mg-1 protein min-1. KYN was notably concentrated by the astrocytes, with an estimated steady-state distribution ratio of 180-fold for 1 microM KYN. Analog inhibition studies showed that the KYN transporter exhibited a clear preference for large neutral amino acids; leucine, tryptophan, and phenylalanine were recognized with relatively higher affinity than KYN. In summary, KYN is concentratively transported into astrocytes by a Na+-independent exchanger with high affinity for branched-chain and aromatic neutral amino acids. The substrate specificity and high affinity of this transport system resemble the properties of neutral amino acid transport across the blood-brain barrier in the rat and human.

Upregulation of tryptophanyl-tRNA synthethase adapts human cancer cells to nutritional stress caused by tryptophan degradation

“…To obtain an overview of the fate of Trp under conditions of Trp deficiency, we integrated gene expression data of U87-MG glioblastoma cells under Trp sufficiency or deprivation, into an extended version of a previously published mathematical model of Trp metabolism that is based on existing kinetic data for the enzymatic conversions and transporters 33 . In line with the previously described upregulation of amino acid transporters in response to Trp deprivation 34,35 , the model predicted increased flux of Trp into the cells (Figure 6C). Kyn is transported by some of the same antiporters as Trp.…”

“…By applying computational modelling of Trp usage in different pathways, we confirmed the upregulation of Trp transporters 35 , which resulted in enhanced influx of Trp into the cells but also increased efflux of Kyn out of the cells (Figure 6C). However, most strikingly, Trp shortage channeled Trp towards WARS (Figure 6C).…”

“…Diverse mechanisms including the suppression of mTOR signalling and the induction of autophagy have been reported to adapt cells to Trp starvation 45,46 . Furthermore, activation of GCN2-peIF2α-ATF4 signaling by Trp starvation was shown to mediate the upregulation of Trp transporters 35 , suggesting that the GCN2-peIF2α-ATF4 axis coordinates multiple adaptations to Trp starvation. Indeed, GCN2 enabled cells to slowly proliferate despite Trp depletion and to rapidly start proliferating upon Trp replenishment (Figure 5).…”

“…Although several studies have shown that amino acid transporters are upregulated in response to Trp deprivation to increase Trp import 34,35,47,49 , it is unclear whether this intracellular increase in Trp leads to further degradation by Trp catabolizing enzymes or can be used for protein synthesis. By applying computational modelling of Trp usage in different pathways, we confirmed the upregulation of Trp transporters 35 , which resulted in enhanced influx of Trp into the cells but also increased efflux of Kyn out of the cells (Figure 6C).…”

Breastfeeding and the gut-brain axis: is there a role for melatonin?

“…An array of different TRYCATs can be produced, including immune and neuroregulatory TRYCATs, such as the excitotoxic quinolinic acid and the more protective kynurenic acid. Peripheral increases in such TRYCATs lead to increased levels of somatization (20) as well as increasing the availability for kynurenine and kynurenic acid to be taken up over the blood-brain barrier, in turn altering neuroregulation centrally (21). Such increases in IDO, as well as the stress hormone cortisol-induced tryptophan 2,3-dioxygenase (TDO), not only increase peripheral and central TRYCATs, but, by decreasing serotonin availability, also decrease the activity of the melatonergic pathway, which requires serotonin as a precursor (22).…”