Characteristics of large neutral amino acid‐induced release of preloaded L‐glutamine from rat cerebral capillaries in vitro: Effects of ammonia, hepatic encephalopathy, and γ‐glutamyl transpeptidase inhibitors

Abstract

The release of newly loaded L-[14C]glutamine (L-Gln) from rat cerebral cortical capillaries was stimulated by L-transport system substrates: tryptophan (TRY), leucine (leu), and nonlabeled L-Gln, respectively, by 32, 50, and 40% above the basal release resulting from superfusion with standard Krebs-Henseliet buffer. However, no stimulation was observed upon treatment with D-Gln or L-glutamate (L-Glu), which are not the L-system substrates, or with ammonium chloride. The stimulatory effect of TRY was temperature dependent but sodium independent, and was abolished in the presence of a sulfhydryl reagent N-ethylmaleimide (NEM). The results support the view that the L-Gln-stimulated uptake of large neutral amino acids (LNAA) across the blood-brain barrier involves the L-system mediated Gln-LNAA exchange. The TRY-stimulated Gln release was enhanced in vitro by simultaneous addition of ammonium chloride, and in capillaries derived from rats with acute hepatic encephalopathy (HE). These results confirm the role of Gln-LNAA exchange in the excessive accumulation of LNAA in brain observed in a variety of hyperammonemic conditions. Superfusion of L-Gln-loaded capillaries in a buffer containing gamma-glutamyl transpeptidase (GGT) inhibitors, serine borate (SB) or 6-diazo-5-oxo-L-norleucine (DON), increased the basal L-Gln release and made it irresponsive to subsequent treatment with TRY. However, the basal release was also increased by superfusion with serine alone or Leu, and this treatment abolished the subsequent effect of TRY as well. Moreover, DON stimulated L-Gln release from capillaries superfused in a standard way, and the effects of DON and TRY were additive. Hence, in the present conditions, SB and DON acted as L-system substrates rather than as GGT inhibitors. Taken together, the results do not support the concept that GGT mediates the Gln-LNAA exchange.

Alterations of Blood Brain Barrier Function in Hyperammonemia: An Overview

“…In this way, enhanced activation of GGT could contribute to raised Try and other LNAA levels as observed in rats with TAA-induced ALF (Hilgier et al 1990). The above speculations were confirmed in a follow up study from the author’s laboratory showing that GGT affects the L system-mediated amino acid exchange (Hilgier et al 1992). …”

“…This inference has been proven directly in studies in which increased tryptophan (Try) uptake in exchange with Gln via the L-transport system was recorded in cerebral capillary microvessels isolated from PCA rats (Cangiano et al 1983). Vice versa, release of newly loaded Gln from the capillaries was promoted by the Try and leucine (Leu), and the effect was more pronounced when the capillaries were isolated from TAA rats or following their incubation with ammonia than in control preparations (Hilgier et al 1992). In the in situ setting, Rigotti et al (1985) showed that treatment of PCA rats with an inhibitor of Gln synthesis, methionine sulfoximine (MSO), reduced the increased accumulation of the AAA in the brain in a manner correlated with increased ammonia accumulation.…”

Glutamine‐induced free radical production in cultured astrocytes

“…DON completely inhibited free radical production in cultured astrocytes exposed to glutamine, suggesting that ammonia, resulting from glutamine hydrolysis, is responsible for the generation of free radicals. DON has also been reported to inhibit ␥-glutamyl transpeptidase (Inoue et al, 1977), to increase glutamine release (Hilgier et al, 1992), to inhibit Na ϩ -dependent glutamine transport into cells (Taylor et al, 1992), and to block the transport of glutamine into mitochondria (Goldstein, 1975). These effects of DON could result in decreased amounts of mitochondrial glu- Fig.…”

Inhibition of glutamine synthetase during ammonia exposure in rainbow trout indicates a high reserve capacity to prevent brain ammonia toxicity

“…In mammals, it has been shown that the increase in brain glutamine concentrations during an ammonia challenge results in the uptake of the large neutral amino acids (LNAAs) tryptophan, phenylalanine and tyrosine across the blood-brain barrier (Cangiano et al, 1983;Cardelli-Cangiano et al, 1984;Rigotti et al, 1985;Gorgievski-Hrisoho et al, 1986;Hilgier et al, 1992) (for a review, see Bachmann, 2002). The driving force for the increase in brain LNAAs are amino acid exchangers with 1:1 stoichiometries that transfer glutamine out of the brain at the same time as LNAAs are transported in.…”