Poly(A)⁺ RNA from the mucosa of rat jejunum induces novel Na⁺-dependent and Na⁺-independent leucine transport activities in in oocytes of Xenopus laevis

Summary

Complementary DNA clones have been isolated recently from rat (D2) and rabbit kidney (rBAT) which induce increased Na⁺—independent Leu and Lys transport activities (System b^0,+) when expressed in oocytes of Xenopus laevis. These cDNAs encode type II membrane glycoproteins which show significant homology to the heavy chain of the human and mouse 4F2 surface antigen (4F2hc). Injection of human 4F2hc cRNA into oocytes also results in induction of Leu/Lys transport activity, but with differing cation requirements for the two amino acids (Na⁺-dependent for Leu, Na⁺-independent for Lys: system y⁺L). System y ⁺ L is a newly discovered zwitterionic/cationic amino acid transporter first described in human erythrocytes. Here we have examined the characteristics of Leu transport in Xenopus oocytes microinjected with mRNA from the mucosa of rat jejunum. L-Leu uptake during 10 min (02 mm₁, 20d`C) reached 20 pmol/oocyte compared with endogenous uptake by water-injected oocytes of typically 3–4 pmol/oocyte. The expressed transport activity was 80% Na⁺-dependent. The Na⁺-dependent component of the expressed flux was saturable (K_mapp 020 mm) and inhibited by Lys, but not by Ala or Phe. The minor Na⁺-independent component of expressed Leu transport activity was also saturable (K_mapp 010 mm). Amino acid inhibition studies resolved this flux into two main components, one of which was inhibited by Lys, Ala and Phe and another which was only inhibited by Lys. There was a small residual component of Na ⁺-independent Leu transport which was insensitive to inhibition by Lys. Experiments utilizing polymerase chain reaction (PCR) demonstrated the presence of both D2 and 4F2hc message in rat jejunum. Hybrid-depletion of jejunal mRNA with an antisense oligonucleotide complementary to D2 had no effect on the expression of Na ⁺—linked Leu transport activity, but reduced the smaller Na ⁺—independent component of Leu transport by 40%, suggesting only a minor role of D2 in the expression of rat intestinal Leu transport activity. Although the properties of Na⁺-dependent Leu transport were, with the exception of a lack of inhibition by Ala and Phe, consistent with erythrocyte y ⁺ L, hybrid-depletion of jejunal mRNA with an antisense oligonucleotide complementary to 4F2hc had no detectable effect on the expressed transport activity. We conclude, therefore, that mRNA from rat Jejunum encodes novel Na ⁺—dependent and Na ⁺—independent transport activities unrelated to the D2/4F2hc glycoproteins. In preparation for the expressin cloning of these proteins, we have determined that maximal Leu transport activity is encoded by an mRNA size fraction of 1·5-2-2·5 kb (peak 2·0 kb).

Key Words:

• Leu
• amino acid transport
• rat jejunum
• D2 glycoprotein
• 4F2hc glycoprotein