Demonstration and characterization of dipeptide transport system activity in sheep omasal epithelium by expression of mRNA in Xenopus laevis oocytes.

Abstract

Research from this laboratory has recently demonstrated that the omasal epithelium of sheep is capable of absorbing dipeptides. In order to express proteins potentially responsible for the mediated absorption of small peptides, size-fractionated poly(A)+RNA (RNA) isolated from omasal epithelial tissue of sheep (average BW 67.5 kg) were injected into defolliculated Xenopus laevis oocytes. The ability of oocytes injected with RNA or water to absorb [14C]glycyl-L-sarcosine (Gly-Sar) from media (usually pH 5.5) was compared. After 4 d (P < .02) of culture, specific RNA fractions induced an increased (P < .02) rate of Gly-Sar absorption, as compared with water-injected oocytes. The dependency of Gly-Sar uptake on the presence of a pH gradient was evaluated at pH 5.0, 5.5, 6.0, 6.5, and 7.5. Inducible uptake increased (P < .001) in the presence of increasing proton concentrations, whereas endogenous uptake of Gly-Sar decreased (P < .001). At pH 5.5, induced Gly-Sar uptake was saturable (Kt = .4 mM), but endogenous uptake was not. The specificity of Gly-Sar absorption was studied by the co-incubation of .1 mM Gly-Sar with 5 mM levels of competing substrates (pH 5.5). Induced uptake was inhibited (P < .05) 44% by carnosine, 94% by methionylglycine, and 91% by glycylleucine, but not by glycine. Incubation of RNA with DNA oligomers that were complementary to the rabbit intestinal transporter completely inhibited (P < .05) induced Gly-Sar uptake. These results indicate that sheep omasal epithelial cells express messenger RNA that encode for proteins that are capable of H(+)-dependent dipeptide transport activity.