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 20x 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.
Uchiyama
Tomomi
T
First Department of Biochemistry, Kyushu University of Health and Welfare, Nobeoka, Miyazaki, Japan.
Fujita
Takuya
T
Gukasyan
Hovhannes J
HJ
Kim
Kwang-Jin
KJ
Borok
Zea
Z
Crandall
Edward D
ED
Lee
Vincent H L
VH
D000595:Amino Acid Sequence; D026962:Amino Acid Transport System ASC / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D000818:Animals; D001120:Arginine / Q000378:metabolism; D001692:Biological Transport; D002478:Cells, Cultured; D002712:Chlorides; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004847:Epithelial Cells / Q000378:metabolism; D005786:Gene Expression Regulation; D057927:Hydrophobic and Hydrophilic Interactions; D007700:Kinetics; D008297:Male; D008969:Molecular Sequence Data; D050479:Neurotransmitter Transport Proteins; D011650:Pulmonary Alveoli / Q000166:cytology* / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D012964:Sodium; D013379:Substrate Specificity; D013997:Time Factors; D014316:Tritium / Q000378:metabolism
D026962:Amino Acid Transport System ASC; D002712:Chlorides; D018076:DNA, Complementary; D050479:Neurotransmitter Transport Proteins; D012333:RNA, Messenger; C515774:Slc6a14 protein, rat; D014316:Tritium; D001120:Arginine; D012964:Sodium