Identification of a Na+-dependent cationic and neutral amino acid transporter, B(0,+), in human and rabbit cornea.
The purpose of this study was to identify and functionally characterize an active transport system for L-arginine on rabbit corneal epithelium and human cornea and study its interaction with the amino acid ester prodrugs of acyclovir (Anand, B. S.; Mitra, A. K. Pharm. Res. 2002, 19, 1194-1202). Transport characteristics of [3H]-L-arginine across freshly excised rabbit corneas were determined at various concentrations, in the absence of sodium and chloride ions. Inhibition studies were conducted in the presence of other amino acids, ouabain, and amino acid ester prodrugs of acyclovir (glycine-ACV, phenylalanine-ACV and gamma-glutamate-ACV). Reverse transcription-polymerase chain reaction (RT-PCR) for amino acid transporter B(0,+) was carried out on total RNA isolated from rabbit cornea, rabbit corneal epithelium, and human cornea. Transport of L-Arg across rabbit cornea was saturable (Km = 306 +/- 72 microM and Vmax = 0.12 +/- 0.01 nmol min(-1) cm(-2)) and was Na+, Cl-, and energy dependent. Transport was inhibited by neutral and cationic amino acids and a B(0,+) system specific inhibitor, BCH (Sloan, J. L.; Mager, S. J. Biol. Chem. 1999, 274, 23740-23745), but not by anionic amino acids. Amino acid prodrugs of ACV (Glu-ACV and Phe-ACV) also inhibited transport of [3H]-L-Arg across rabbit cornea. Amino acid transporter B(0,+) was identified by RT-PCR and its identity confirmed by subcloning and sequencing in rabbit cornea, rabbit corneal epithelium, and human cornea. A Na+-, Cl(-)-, and energy-dependent carrier for L-Arg, B(0,+), was identified on rabbit corneal epithelium and human cornea. Glu-ACV and Phe-ACV appear to be substrates for this transporter. The presence of such transporters on the corneal epithelium may provide new opportunities for transporter-targeted prodrug design for enhanced corneal absorption.
Jain-Vakkalagadda
Blisse
B
Division of Pharmaceutical Sciences, School of Pharmacy, University of Missouri-Kansas City, 5005 Rockhill Road, Kansas City, Missouri 64110-2499, USA.
Pal
Dhananjay
D
Gunda
Sriram
S
Nashed
Yasser
Y
Ganapathy
Vadivel
V
Mitra
Ashim K
AK
D000212:Acyclovir / Q000031:analogs & derivatives / Q000138:chemical synthesis / Q000494:pharmacology; D026905:Amino Acid Transport Systems; D026921:Amino Acid Transport Systems, Neutral / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D000596:Amino Acids / Q000494:pharmacology; D000818:Animals; D001120:Arginine / Q000187:drug effects / Q000378:metabolism / Q000493:pharmacokinetics; D001692:Biological Transport / Q000187:drug effects; D002712:Chlorides / Q000494:pharmacology; D003315:Cornea / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D004355:Drug Stability; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008297:Male; D015394:Molecular Structure; D010042:Ouabain / Q000494:pharmacology; D011355:Prodrugs / Q000737:chemistry / Q000494:pharmacology; D011817:Rabbits; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017421:Sequence Analysis; D012964:Sodium / Q000494:pharmacology*; D013379:Substrate Specificity; D013997:Time Factors
D026905:Amino Acid Transport Systems; D026921:Amino Acid Transport Systems, Neutral; D000596:Amino Acids; D002712:Chlorides; D011355:Prodrugs; C503995:SLC6A14 protein, human; D010042:Ouabain; D001120:Arginine; D012964:Sodium; D000212:Acyclovir