Transport of L-carnitine in human corneal and conjunctival epithelial cells

Abstract

Purpose: 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 examine the characteristics of L-carnitine transporters in cultured human limbal corneal (HCLE) and conjunctival epithelial (HCjE) cells.

Methods: Time-course, Na(+)-dependence, kinetics, energy- and pH- dependence of L-carnitine transport were investigated by monitoring L-[(3)H]carnitine uptake into HCLE and HCjE cells. To determine the specificity of action, competition and inhibition studies were performed.

Results: The uptake of L-carnitine into HCLE and HCjE cells was saturable and time-dependent. An Eadie-Hofstee plot showed two distinct components: a high- and a low- affinity carnitine transport system in HCLE and/or HCjE cells. L-carnitine transport was significantly inhibited by the metabolic inhibitors (sodium azide, dinitrophenol, iodoacetic acid). The L-carnitine analogs (D-carnitine, acetyl-L-carnitine and γ-butyrobetaine), tetraethylammonium (TEA), 2-amino-2-norbornane carboxylic acid (BCH), strongly inhibited uptake of L-[(3)H]carnitine. Uptake of L-[(3)H]carnitine also required the presence of Na(+) in the external medium and the uptake activity was maximal at pH 5.5. The anti-OCTN2 antibody blocked L-carnitine uptake in both HCLE and HCjE cells whereas the anti-OCTN1 antibody did not significantly block L-carnitine uptake.

Conclusions: L-carnitine is transported into HCLE and HCjE cells by an active carrier mediated transport system that is time-, Na(+)-, energy- and pH- dependent. The carnitine/organic cation transporter OCTN2 appears to play a dominant role in this process.

Figure 1

Time course and Na⁺-dependence of L-carnitine uptake. The uptake of 12nM L-[³H]carnitine by HCLE (A) or HCjE (B) cells was measured at pH 7.4 and 37 °C in the presence or absence of Na⁺. For Na⁺-free buffer, NaCl was replaced by an equimolar concentration of choline. Values are the mean±SD (n=4).

Figure 2

Concentration dependence and the kinetic characteristics of L-carnitine uptake.   A: Concentration dependence of carnitine uptake by HCLE or HCjE cells; B and C:  the kinetic characteristics of L-[³H]carnitine uptake with the Eadie–Hofstee plot indicating dependence of the uptake rate (v) on uptake rate/carnitine concentration (v/s) for HCLE (B) and HCjE (C) cells, respectively. HCLE or HCjE cells were pre-incubated for 60 min with uptake buffer, then different concentrations of L-carnitine were added to the incubation medium and the uptake was measured for 30 min at pH 7.4 and 37 °C in the presence of Na⁺. The concentration of L-[³H]carnitine was kept constant at 24 nM and the concentration of L-carnitine was varied (range 1-480 μM) by addition of unlabeled L-carnitine. Values are the mean±SD (n=4).

Figure 3

Effect of the pH of the medium on the uptake of L-[³H]carnitine by HCLE or HCjE cells at 37 °C. HCLE or HCjE cells were pre-incubated for 60 min in uptake buffer of different pH values at 37 °C; L-[³H]carnitine (24 nM) was then added and incubation was continued for 30 min. Each value is the mean±SD of results from three experiments. #p<0.005 for HCLE and 0.002 for HCjE cells respectively compared with medium at pH 7.4. *p=0.002 for HCLE and, p=0.007 for HCjE, respectively compared with medium at pH 7.4.

Figure 4

Relative expression of OCTN1, OCTN2, and ATB^0,+ in human ocular epithelial cells. Representative image of semi-quantitative RT PCR -amplified human OCTN1, OCTN2, ATB^0,+, and ACTB products. In the image, Lanes 1,3,5,7 show HCLE product and Lanes 2,4,6,8 show HCjE product: Lanes 1–2 OCNT1; Lanes 3–4 OCTN2; Lanes 5–6 ATB^0,+; and Lanes 7–8 ACTB.

Figure 5

Blocking effect of OCTN1 and/or OCTN2 antibody on the uptake of L-[³H]carnitine by HCLE or HCjE cells at 37 °C. HCLE or HCjE cells were pre-incubated for 60 min in uptake buffer containing OCTN1 (1:500) and/or OCTN2 (1:500) antibody at 37 °C; L-[³H]carnitine (24 nM) was then added and incubation was continued for 30 min. Each value is the mean±SD of results from three experiments. *p<0.01 compared with the control group.