Proton-driven dipeptide uptake in primary cultured rabbit conjunctival epithelial cells

Abstract

Purpose: 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.