Characterization of monocarboxylate uptake and immunohistochemical demonstration of monocarboxylate transporters in cultured rabbit corneal epithelial cells.

OBJECTIVES: This study aimed to characterize the mechanisms of monocarboxylate uptake by cultured rabbit corneal epithelial cells (RCECs) using l- and d-lactic acids as model substrates. METHODS: l-/d-Lactic acid uptake was evaluated by measuring the accumulation in confluent RCECs. Also, we demonstrated the distribution of monocarboxylate transporters (MCTs) in RCECs by immunohistochemistry. KEY FINDINGS: The accumulation of (14) C-labelled l- and d-lactic acids was dependent on time, pH and temperature. The Arrhenius plots of the uptake were biphasic. The initial uptake of (14) C-labelled l-lactic acid exhibited concentration dependence and was greater than that of the d-isomer. The initial uptake of (14) C-labelled l- and d-lactic acids involved saturable and nonsaturable processes; the saturable process exhibited higher affinity for l-lactic acid than for the d-isomer. l-/d-lactic acid uptake was inhibited by chiral monocarboxylate in a stereoselective manner. The uptake of (14) C-labelled l- and d-lactic acids was sensitive to metabolic inhibitors and other monocarboxylates. MCT expression in RCECs was confirmed immunohistochemically. In particular, MCT2 expression was detected in RCECs, whereas MCT1, MCT4 and MCT5 expression was detected in the surface layer. CONCLUSION: These results indicate that the carrier-mediated transport system specific for monocarboxylates elicits lactic acid uptake in RCECs. Therefore, the transcorneal permeation of drugs with a monocarboxylic moiety may be dependent on the activity of a specific pH-dependent transporter as well as passive diffusion according to the pH-partition theory.

Diquafosol elicits increases in net Cl- transport through P2Y2 receptor stimulation in rabbit conjunctiva.

The purpose of the present study was to understand the mechanisms of action of diquafosol, a stable derivative of uridine 5'-triphosphate, on Cl(-) transport across the isolated rabbit conjunctiva. Rabbit conjunctivas were isolated and mounted in a modified Ussing chamber. Under short-circuit conditions, the effects were determined of mucosal (tear) side diquafosol application on the short-circuit current (Isc). Diquafosol rapidly and dose-dependently increased the Isc at concentrations ranging from 0.1 to 968 microM when added to the mucosal side of the conjunctiva. In the absence of the serosal Cl(-), the Isc induced by 10 microM diquafosol was substantially reduced. On the contrary, in the absence of mucosal side Na(+), the diquafosol-induced increases in Isc were unchanged. Following 45-min preincubation, the P2Y(2) antagonist suramin inhibited the diquafosol-induced increases in the Isc whereas the P2Y(1) antagonist pyridoxal-phosphate-6-azophenyl-2'4'-disulfonic acid had no effect. These studies suggest that diquafosol stimulates net Cl(-) secretion from the serosal to the mucosal side via stimulation of P2Y(2) receptors in the rabbit conjunctiva.