Poly(A)+ RNA from sheep omasal epithelium induces expression of a peptide transport protein(s) in Xenopus laevis oocytes.

Abstract

Animal and Poultry Sciences (ABSTRACT)In order to verify the research from this laboratory that sheep omasal epithelium contains mRNA encoding for a peptide transporter (s) and to determine di-to octapeptide transport capability, poly(A) + RNA isolated from sheep omasal epithelium was injected into Xenopus laevis oocytes. Poly(A) + RNA was functionally expressed in Xenopus oocytes 4 to 7 d post-injection. Peptide (5 di-, 10 tri-, 6 tetra-, 2 penta-, 1 hepta-, 1 septa-, 1 octapeptide) transport capability was measured by impaling oocytes with a microelectrode to monitor membrane potential (V m ). Oocytes were maintained in pH 5.5 buffer. Peptide transport was identified as being expressed when, in the presence of a buffered peptide substrate (1 mM), the oocyte membrane showed persistent depolarization (a more positive V m ). In the absence of peptide transport, the membrane became depolarized with the addition of buffered substrate, but rapidly repolarized to the resting potential. Peptide transport was expressed for some di-, tri-, and tetrapeptides.iii Measured depolarization ranged from 9.6 mV to 42.1 mV. Larger peptides were not transported by the oocytes. When transport expression was measured with the substrates in a pH 7.5 buffer, no transport occurred indicating that transport was dependent on a proton gradient. The data indicate that sheep omasal epithelium contains mRNA that code for a protein(s) capable of proton-dependent di-, tri-, and tetrapeptide transport. This provides further evidence that absorption of peptides from the ruminant stomach is possible. INTRODUCTIONIn recent years, the utilization of peptides as a source of amino acids and nitrogen has been recognized to be an important biological process in living species from microorganisms to animals and plants (Payne and Smith, 1994). Much information has been obtained regarding the ability of the gastrointestinal tract and tissues such as liver (Lochs et al., 1986), kidney (Loch et al., 1988), and skeletal muscle (Roth et al., 1988) to use peptides. Overall, little is known about the magnitude of peptide absorption and the metabolic significance of this phenomenon in ruminants. Results from a recent study indicated that peptides constitute about 65 to 78% of the blood plasma amino acid pool in ruminants, and that the forestomach may be the primary site of peptide absorption in ruminants (DiRienzo and Webb, 1995). Carnosine and methionylglycine were shown to be transferred intact across both ruminal and omasal epithelial tissue without hydrolysis when these tissues were mounted in parabiotic chambers (Matthews and Webb, 1995).However, the four-strata structure of all forestomach epithelia makes it difficult to purify and further characterize the transporter protein(s) using conventional procedures. With the success of expression cloning techniques, the identification and characterization of peptide transporter protein(s) in the ruminant forestomach becomes possible. Using expression system, poly(A) + RNA isolated from omasal epithelia were ...

Tissue distribution of a peptide transporter mRNA in sheep, dairy cows, pigs, and chickens.

“…Xenopus laevis oocytes injected with mRNA from sheep omasal epithelium provided further evidence that peptide transport occurs in the ruminant stomach (Matthews et al, 1996;Pan et al, 1997). Results from the present study indicate that the ruminal and omasal epithelium of sheep and dairy cows have a 2.8 kb mRNA that encodes for a peptide transport protein.…”

“…previous studies from our laboratory, a peptide transport protein(s) capable of transporting peptides up to four amino acids in length is present in sheep omasal epithelium (Matthews et al, 1996;Pan et al, 1997). Therefore, sheep omasal epithelium mRNA was used as the template for RT-PCR to develop a probe for northern blot analysis.…”

“…Studies from our laboratory have shown the existence of a peptide transporter(s) in the forestomach of sheep (Matthews et al, 1996;Pan et al, 1997). Translation of mRNA in Xenopus oocytes showed that the mRNA is present in sheep omasal epithelial tissue encoding for a peptide transporter(s) capable of transporting di-to tetrapeptides.…”

Transcellular Transport of a Highly Polar 3+ Net Charge Opioid Tetrapeptide

“…Whether PEPT1 or PEPT2 would transport tetrapeptides is less clear. Although the tetrapeptide TyrPro-Phe-Pro was not transported by PEPT2 (Tiruppathi et al, 1991), an H ϩ -coupled peptide transporter cloned from sheep omasal epithelium actively transported tetrapeptides (Pan et al, 1997). It therefore remains possible that [Dmt 1 ]DALDA may penetrate cell membranes via protoncoupled peptide transporters such as PEPT1 or PEPT2.…”

Molecular characterization and nutritional regulation of specificity protein 1 (Sp1) in grass carp (Ctenopharyngodon idella )

“…Previous studies have demonstrated proteins of the solute carrier (SLC) family play a crucial role in the transport of small peptides across the cell membrane. As a key member of the SLC superfamily, peptide transporter 1 (PepT1) was shown to be involved in transporting di‐/tripeptides across the brush‐border membranes of intestine epithelial cells (Cukrowicz & Duprez, ; Matthews, Wong, Bender, Bloomquist, & Webb, ; Pan, Wong, Bloomquist, & Webb, ). It has been reported that the expression of intestinal PepT1 is regulated by various factors, including cytokines, hormones and dietary conditions (Nielsen, Amstrup, Steffansen, Frokjaer, & Brodin, ; Pan, Terada, & Inui, ; Tanaka, Yamamoto, & Taketani, ; Terada & Inui, ).…”