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Transcellular transport of amino acids by epithelial tissues includes their import across apical membranes and sequential export through basolateral membranes. In the cell, the basolateral efflux step is driven by the amino acid concentration gradient and is strictly regulated by the cooperative action of various transporter proteins. The basolateral transporter LAT2-4F2hc (SLC7A8-SLC3A2) is an obligatory exchanger of neutral amino acids and does therefore not directly contribute to the net amino acid transport. Another basolaterally located protein, TAT1 (SLC16A10), transports aromatic amino acids by facilitated diffusion and we have previously shown in X. laevis oocytes that it can complement the vectorial transport of LAT2- 4F2hc by recycling exchange substrates1. Our hypothesis proposes that the interplay between exchangers and uniporters controls both the vectorial transport of amino acids and the cellular amino acid concentration. We aim at testing the cooperation of these two amino acid transporters (LAT2-4F2hc and TAT1) in a double knock out mouse model and in primary cell culture made from their proximal tubule cells. We are currently characterizing the newly generated LAT2-/-TAT1-/- double knock outs and comparing different metabolic parameters with those of the LAT2-/- and TAT1-/- single knock outs, and their wild type littermates2,3. Preliminary results show that double knock out mice under high protein diet excrete aromatic amino acids in the urine at a much higher level compared to LAT2-/- and even TAT1-/- mice. Moreover, other neutral amino acids that are substrates of LAT2 but not of TAT1 are hyperexcreted by the LAT2-/-TAT1-/- to a much higher extent than in both the single knock out models. This latter observation supports the hypothesis that the basolateral efflux of LAT2 substrates depends on the recycling activity of aromatic amino acids via TAT1. [ABSTRACT FROM AUTHOR]

The pancreas efficiently absorbs amino acids for the synthesis of enzymes, but also secrets free amino acids in the pancreatic juice which are re-absorbed by the small intestine. Under free protein diet, the release of amino acids on the pancreatic juice (PJ) may play important role on the homeostasis maintenance of the small intestine. From the 20 proteinogenic amino acids analyzed in the PJ, L-glutamate (Glu) was 4 fold more concentrated in PJ when compared to plasma levels. The concentrative mechanism could be due to an increase in the transport of Glu and the synthesis of Glu in the exocrine pancreas. Since there is a high expression of neutral amino acids and L-glutamine (Gln) transporters in the pancreas and they localize to the basolateral membrane of the acinar cells, we hypothesize Glu is synthesized in acinar cells and secreted to the pancreatic juice. We analyzed at mRNA level the expression of the 2 glutaminase isoforms (gls1 and gls2), glutamine synthase (Glul), alanine aminotransferase 1 and 2 (GPT1, GPT2) and aspartate aminotransferase 1 and 2 (GOT1 and GOT2) in pancreas. Our results showed that the two glutaminase isoforms (Gls1 and Gls2) are expressed in the pancreas, GLS2 is localized in the acinar cells, and that the levels of gls2 and of the cytoplasmatic GPT were elevated in animals receiving diet free of protein. These results suggest Glu may be synthesized in the exocrine pancreas from Gln and L-alanine and that the dietary protein content can modulate the expression of enzymes involved in the synthesis of Glu. The secretory mechanism of Glu to PJ could involve zymogen vesicles and transport via apical membrane. Our experiments showed that Glu and its precursors were not concentrating in zymogen granules vesicles (ZGV) but were found at high concentrations in the cytoplasmicfraction, suggesting the secretory mechanism does not involve exocytosis. Immunofluorescence and western blotting of isolated ZGV suggested that the sodium-dependent Glu transporter EAAT1 (Slc1a3) is present on the zymogen vesicles membrane. Additionally, immunofluorescence of pancreatic tissue showed that EAAT1 localizes proximal to the apical membrane of acinar cells. Our results support the hypothesis that EAAT1 is involved in the secretion of Glu during vesicular docking to the apical membrane of acinar cells. These preliminary exciting results suggest a new mechanism for Glu concentration and secretion on the pancreatic juice, as well a recycling of neutral amino acids (L-alanine, Gln) and Glu between the pancreas and intestine. Where applicable, the authors confirm that the experiments described here conform with The Physiological Society ethical requirements. [ABSTRACT FROM AUTHOR]