Localization of the peptide transporter PEPT2 in the lung: implications for pulmonary oligopeptide uptake

Abstract

Pulmonary delivery of peptidomimetic antibiotics is frequently used for local drug therapy in pulmonary infections. Identification of transport pathways into airway epithelia can lead to new strategies of therapy. Here we describe the distribution of the beta-lactam-transporting high-affinity proton-coupled peptide transporter PEPT2 in mammalian lungs. Using reverse transcriptase-polymerase chain reaction and Northern blot analysis, PEPT2-mRNA was detected in lung extracts. The expression of PEPT2-mRNA and protein was localized to alveolar type II pneumocytes, bronchial epithelium, and endothelium of small arteries of rat lung by nonisotopic in situ hybridization and immunohistochemistry. In addition, transport studies using murine whole-organ preparations revealed transporter-mediated uptake of a fluorophore-conjugated dipeptide derivative into bronchial epithelial cells and type II pneumocytes. This transport was competitively inhibited by cephalosporins and dipeptides that are reported as PEPT2-carried substrates. Cell specificity of the PEPT2-mediated uptake pattern was confirmed by double labeling with Lycopersicon esculentum lectin. Together these data suggest that PEPT2 is the molecular basis for the transport of peptides and peptidomimetics in pulmonary epithelial cells. In conclusion PEPT2 may be an interesting target for pulmonary delivery of peptides and peptidomimetics.

Figure 1.

Substrates of ex vivo uptake studies. The fluorophore-conjugated dipeptide d-Ala-Lys-AMCA, unlabeled glycyl-(l)-glutamine, and unlabeled cefadroxil served as substrates for the in situ uptake studies.

Figure 2.

Detection of PEPT2-mRNA in rat lung by RT-PCR. Five μg of total RNA from rat lung and kidney were subjected to RT-PCR using primer pairs specific for PEPT2, PEPT1, or GAPDH. The RT-PCR products were separated by agarose gel electrophoresis and visualized with ethidium bromide.

Figure 3.

Detection of PEPT2-mRNA in rat lung by Northern blot analysis. Samples of total RNA (10 μg) from rat lung and kidney were separated by agarose gel electrophoresis, blotted, and hybridized with a specific PEPT2 antisense probe.

Figure 4.

Localization of PEPT2-mRNA and PEPT2-like immunoreactivity in the rat lung. Localization of PEPT2-like immunoreactivity in type II pneumocytes (a) and bronchial epithelium (b) of rat lung. Sections incubated with the PEPT2-antibody in the presence of the antigen peptide do not exhibit specific immunoreactivity (c). Distribution of PEPT2-mRNA in rat lung was detected by nonradioactive in situ hybridization. Cryostat sections (8 μm) of rat lung were hybridized with digoxigenin-labeled antisense (d and e) or sense (f) PEPT2-cRNA probe. Hybridization signals were obtained in bronchial epithelium (e) and type II pneumocytes (d). Scale bars: 22.5 μm (a), 45 μm (b), 34 μm (c), 16 μm (d), and 30 μm (e and f).

Figure 5.

Localization of PEPT2-like immunoreactivity in the murine lung. Immunofluorescence localization of PEPT2-like immunoreactivity in the bronchial epithelium (a and c) and type II pneumocytes (b) of murine lung. d: A control section stained for PEPT2-like immunoreactivity in the presence of the antigen peptide. Scale bars: 80 μm (a), 16 μm (b), 10 μm (c), and 14 μm (d).

Figure 6.

Uptake studies in murine whole-organ preparations. d-Ala-Lys-AMCA uptake was restricted to bronchial epithelial cells (arrows in a). There was no visible uptake as a result of adding 1 mmol/L of either cefadroxil (d) or Gly-(l)-Gln (c) to the d-Ala-Lys-AMCA solution. In the alveolar space, fluorescence accumulation was detected in type II pneumocytes (e, arrows in f) but signal was absent in type I cells (f). Inhibition studies with cefadroxil (g) or Gly-(l)-Gln (h) lead to a reduction of the signal in the type II pneumocytes. Scale bars: 60 μm (a, d, and e), 12 μm (f), 22 μm (b and c), 18 μm (g and h).

Figure 7.

Combined ex vivo uptake and immunohistochemistry studies. d-Ala-Lys-AMCA fluorescence was not present in type I pneumocytes of the peripheral lung. Double labeling of the same section with LEA (b), a specific marker for type I cells, revealed the mutually exclusive presence. The uptake of d-Ala-Lys-AMCA (a) was restricted to type II pneumocytes (arrow) that are flanked by type I cell membranes (arrowheads) that displayed LEA-activity (b). Scale bar: 15 μm.