Proton-driven dipeptide uptake in primary cultured rabbit conjunctival epithelial cells.
OBJECTIVE
To characterize proton-driven carrier-mediated dipeptide uptake in primary cultured conjunctival epithelial cells of the pigmented rabbit using beta-alanyl-L-histidine (L-carnosine) as a model dipeptide substrate.


METHODS
Uptake of tritiated L-carnosine was monitored using conjunctival epithelial cells on days 6 through 8 in culture on a filter support. The structural features of dileucine stereoisomers and cephalexin contributing to interaction with the dipeptide transporter were evaluated by computer modeling and inhibition of tritiated L-carnosine uptake.


RESULTS
Uptake of L-carnosine by primary cultured conjunctival epithelial cells in the presence of an inwardly directed proton gradient showed directional asymmetry (favoring apical uptake by a factor of five), temperature dependence, and saturability correlated with substrate concentration, with a Michaelis-Menten constant (Km) of 0.3 +/- 0.03 mM and a maximum uptake rate (Vmax) of 22.0 +/- 1.0 picomoles per milligram protein per minute. L-Carnosine uptake was optimal at pH 6.0 and was reduced by 60% and 35%, respectively, by 50 microM p-trifluoromethoxyphenylhydrazone (a proton ionophore) and by acid preloading with 50 mM NH4Cl. The constituent amino acids did not inhibit L-carnosine uptake. L-Carnosine uptake was inhibited, however, from 50% to 80% by other dipeptides and structurally similar drugs such as bestatin, beta-lactam antibiotics, and angiotensin-converting enzyme inhibitors. The LL, LD, or DL forms of the dipeptide Leu-Leu inhibited tritiated L-carnosine uptake by approximately 60%, 40%, and 70%, respectively. By contrast, the DD form did not inhibit uptake. Results from computer modeling suggest that an appropriate dipeptide N-terminal to C-terminal distance and a favorable orientation of the side chains may be important for substrate interaction with the conjunctival dipeptide transporter.


CONCLUSIONS
Uptake of the dipeptide L-carnosine in primary cultured pigmented rabbit conjunctival epithelial cells is probably mediated by a proton-driven dipeptide transporter. This transporter may be used for optimizing the uptake of structurally similar peptidomimetic drugs.
Basu
S K
SK
Department of Pharmaceutical Sciences, School of Pharmacy, University of Southern California, Los Angeles 90033, USA.
Haworth
I S
IS
Bolger
M B
MB
Lee
V H
VH
D000643:Ammonium Chloride / Q000494:pharmacology; D000818:Animals; D001426:Bacterial Proteins / Q000378:metabolism*; D001692:Biological Transport / Q000187:drug effects; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone / Q000494:pharmacology; D002336:Carnosine / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism*; D002478:Cells, Cultured; D002506:Cephalexin / Q000378:metabolism; D003198:Computer Simulation; D003228:Conjunctiva / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D004151:Dipeptides / Q000378:metabolism; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007476:Ionophores / Q000494:pharmacology; D007930:Leucine / Q000031:analogs & derivatives / Q000378:metabolism; D008297:Male; D026901:Membrane Transport Proteins; D011522:Protons; D011817:Rabbits; D013237:Stereoisomerism
D001426:Bacterial Proteins; D002352:Carrier Proteins; D004151:Dipeptides; D007476:Ionophores; D026901:Membrane Transport Proteins; D011522:Protons; D000643:Ammonium Chloride; C086845:dtpT protein, Lactococcus lactis; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; D002336:Carnosine; D007930:Leucine; C012211:ubenimex; D002506:Cephalexin