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J. L. Flanagan, S. Xu, P. A. Simmons, J. G. I. Vehige, Jr., M. D. Willcox, Q. Garrett; OCTN2 Mediates Active Transport of L-Carnitine in Human Corneal and Conjunctival Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2010;51(13):2364.
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Imbalance of L-Carnitine (β-hydroxy-gamma-trimethylaminobutyrate) levels has been implicated in ocular surface damage of dry eye syndrome. Previously we demonstrated expression and localization of carnitine/organic cation transporters, OCTN1 and OCTN2, in human corneal and conjunctival epithelia. The present study aimed to characterize L-carnitine transport in cultured human limbal corneal (HCLE) and conjunctival epithelial (HCjE) cells.
Time-course, Na+ dependence, kinetics, and pH- dependence of L-carnitine transport were investigated using L-[3H]carnitine uptake in HCLE and HCjE cells. The effect of D-carnitine, acetyl-L-carnitine, γ-butyrobetaine, tetraethylammonium (TEA), 2-amino-2-norbornane carboxylic acid (BCH) and the blocking effect of OCTN1 and/or OCTN2 specific antibodies on the uptake of L-[3H]carnitine by cells were determined.
L-[3H]carnitine uptake in both HCLE and HCjE cells increased in a linear, time-dependent manner, beyond 90 min. Choline replacement of Na+ in the uptake buffer inhibited by 90% (p = 0.001).Uptake was saturable and linear (1-24 µM). A low and high affinity transport system was observed for both cell lines. Differences in Km and Vmax for L-carnitine transport were significant: Km 33.9 ± 12.7 vs. 18.9 ± 5.3 (high) and 216.7 ± 44.4 vs.172.3 ± 37.1 (low); Vmax 1.1 ± 0.3 vs. 0.5± 0.1 (high) and 3.5 ± 0.3 and 1.99 ± 0.2 (low) for HCLE and HCjE respectively. L-carnitine, D-carnitine, acetyl-L-Carnitine and γ-butyrobetaine inhibited the uptake of L-[3H]carnitine by HCLE and HCjE cells. L-carnitine was showed stronger inhibition than D-carnitine and acetyl-L-carnitine (p < 0.01); gamma-butyrobetaine was equal to L-carnitine. There was no difference between D-carnitine and acetyl-L-carnitine. Inhibition was concentration dependent. L-[3H]carnitine uptake was highest pH 5.5. Anti-OCTN2 antibodies blocked transport.
L-carnitine enters HCLE and HCjE cells by active carrier transport which is time-, Na+-, and pH- dependent. OCTN2 appears to play a dominant role in this process.
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