Functional characterization and cloning of amino acid transporter B0,+ (ATB0,+) in primary cultured rat pneumocytes
✍ Scribed by Tomomi Uchiyama; Takuya Fujita; Hovhannes J. Gukasyan; Kwang-Jin Kim; Zea Borok; Edward D. Crandall; Vincent H.L. Lee
- Publisher
- John Wiley and Sons
- Year
- 2007
- Tongue
- English
- Weight
- 371 KB
- Volume
- 214
- Category
- Article
- ISSN
- 0021-9541
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✦ Synopsis
Abstract
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.