Molecular identification of high-affinity glutamate transporters in sheep and cattle forestomach, intestine, liver, kidney, and pancreas

Glutamate metabolism is essential to support many facets of metabolism. The objective of this study was to determine the tissue distribution of glutam ate transporters known to support the tissue metabolism of glutamate. The e xpression of proteins capable of high-affinity glutamate transport (system X-AG(-)) by epithelia isolated from the rumen, omasum, duodenum, jejunum, i leum, cecum, and colon and homogenates of liver, kidney, and pancreatic tis sues from wethers (n = 4; BW = 28.4 +/- 8.4 kg) and steers (n = 3; BW = 426 +/- 32.3 kg) fed forage-based diets was evaluated by immunoblot analysis. Proteins EAAC1 (62, 93 kDa) and GLT-1 (142, 188, > 202 kDa) were expressed by every tissue examined. In contrast, GLAST1 (140 kDa) was expressed only by the pancreas, and EAAT4 (67 kDa) was detected only in sheep brain. To co rroborate protein expression data, the presence and size of transporter mRN A in ileal, liver, and pancreatic homogenates were evaluated by Northern an alysis. GLAST1 mRNA (2.4, 4.3 kb) was detected only in the pancreas, wherea s EAAC1 (2.2, 2.8 kb) and GLT-1 (12.1 kb) mRNA transcripts were detected in all three tissues. The expression of EAAT4 and GLT-1 mRNA was confirmed by reverse transcriptase-polymerase chain reaction analyses. Sequencing of th e resulting partial-length ovine GLT-1 cDNA revealed 100% identity with the rat homolog. Overall, these data demonstrate that sheep and cattle share t he same pattern of system X-AG(-) transporter expression, which differed am ong tissues and transporter isoforms. Accordingly, these data provide the f undamental knowledge to initiate research that determines whether the expre ssion of high-affinity glutamate transporters by ruminants is sensitive to ontogenic and(or) dietary regulation.