Functional characterization and cloning of amino acid transporter B 0,+ (ATB 0,+ ) in primary cultured rat pneumocytes

Cationic amino acid transport in primary cultured rat pneumocytes exhibiting characteristics of alveolar epithelial type I‐like cells are described. Asymmetry and activator ion dependency of 3 H‐ L ‐arginine uptake were characterized from the apical or basolateral fluid of pneumocytes grown on permeable support. Substrate specificity of transport was evaluated as a function of 3 H‐ L ‐arginine uptake inhibition in the presence of other amino acids. Transepithelial transport studies estimated 3 H‐ L ‐arginine flux in the apical‐to‐basolateral and basolateral‐to‐apical directions. Full length cDNA of rat amino acid transporter B 0,+ (rATB 0,+ ) was cloned and its relative expression level studied. Results indicate that uptake of 3 H‐ L ‐arginine from apical fluid is dependent on Na + and Cl − . Zwitterionic and cationic amino acids (excluding L ‐proline and anionic amino acids) inhibited uptake of 3 H‐ L ‐arginine from apical, but not basolateral incubation fluid. Apical‐to‐basolateral transepithelial flux of 3 H‐ L ‐arginine was 20× higher than basolateral‐to‐apical transport. Kinetic studies of 3 H‐ L ‐arginine uptake from apical fluid revealed maximal velocity (V max ) and Michaelis–Menten constants (K t ) of 33.32 ± 2.12 pmol/mg protein/15 min and 0.50 ± 0.11 mM, respectively, in a cooperative process having a coupling ratio of 1.18 ± 0.16 with Na + and 1.11 ± 0.13 with Cl − . Expression of rATB 0,+ mRNA was identified by RT‐PCR and Northern analysis. Corresponding cloned 3.2 kb rATB 0,+ cDNA sequence exhibits pronounced homology in deduced amino acid sequence to mouse (95% identity and 97% similarity) and human (89% identity and 95% similarity) ATB 0,+ homologues. We conclude that rat pneumocytes express ATB 0,+ , which may partly contribute towards recovering cationic and neutral amino acids from alveolar luminal fluid. J. Cell. Physiol. 214: 645–654, 2008. © 2007 Wiley‐Liss, Inc.