Submitted on May 22, 2003
Accepted on March 23, 2004
Expression of apical membrane L-glutamate transporters in neonatal porcine epithelial cells along the small intestinal crypt-villus axis
Ming Z. Fan1, James C. Matthews2, Nadege M. P. Etienne2, Barbara Stoll3, Dale Lackeyram4, and Douglas G. Burrin3*
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.
