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1 Department of Pediatrics, Baylor College of Medicine, United States Department of Agricultural / Agricultural Research Service, Children's Nutrition Research Center, Houston, TX, USA; Department of Animal and Poultry Science, University of Guelph, Guelph, Ontario, Canada
2 Department of Animal Science, University of Kentucky, Lexington, KY, USA
3 Department of Pediatrics, Baylor College of Medicine, United States Department of Agricultural / Agricultural Research Service, Children's Nutrition Research Center, Houston, TX, USA
4 Department of Animal and Poultry Science, University of Guelph, Guelph, Ontario, Canada
* To whom correspondence should be addressed. E-mail: dburrin{at}bcm.tmc.edu.
Enteral L-glutamate is extensively utilized as an oxidative fuel by the gut mucosa in the neonate. To identify major uptake pathways and to understand the uptake regulation, we examined the transport kinetics and molecular identities of the apical membrane L-glutamate transporters in epithelial cells sequentially isolated along the small intestinal crypt-villus axis from milk protein-fed, 16-d-old pigs. The distended intestinal sac method was used to isolate 12 sequential cell fractions from the tip villus to the bottom crypt. Initial rates and kinetics of L- glutamate uptake were measured with L-[G-3H]glutamate by fast filtration in apical membrane vesicles prepared by Mg2+-precipitation and differential centrifugation with membrane potential clamped by SCN-. Initial L-glutamate uptake results suggested the presence of both the Bo and the X-AG transport systems, but the X-AG system was predominant for the uptake across the apical membrane. Kinetic data suggested that L-glutamate uptake through the X-AG system was associated with higher maximal transport activity, but lower transporter affinity in crypt cells than in villus cells. Molecular identity of the X-AG glutamate transporter, based on immunoblot and reverse transcription-polymerase chain reaction (RT-PCR) analysis, was primarily the defined excitatory amino acid carrier 1 (EAAC1). The expression of EAAC1 was increased with the cell differentiation and regulated at the transcription and translation levels from the crypt to the upper villus cells. In conclusion, efficiency and capacity of luminal L-glutamate uptake across the apical membrane are regulated by changing expression of the system X-AG transporter gene EAAC1 at the transcription and translation levels as well as the maximal uptake activity and transporter affinity along the intestinal crypt-villus axis in the neonate.
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