Abstract
Purpose: :
The cornea is very glycolytic and produces a significant amount of lactic acid that needs to be removed in order to maintain corneal transparency. The purpose of this study is to determine the presence of monocarboxylate transporters (MCTs) in the rabbit corneal endothelium (CE) and their role in transporting lactic acid. Regulation of lactate:H+ transport will also be examined.
Methods: :
Total RNA from fresh rabbit CE was screened for the expression of MCT 1-4 mRNA by RT-PCR. Western blotting (WB) was used to detect expression of MCT 1-4 protein and immunofluorescent staining (IF) was done to localize the proteins. Physiological experiments were performed using fresh rabbit CE mounted in a double-sided perfusion chamber. Cells were loaded with the intracellular pH sensitive fluorescent dye 2’,7’-bis(2-carboxyethl)-5(6)-carboxyfluorescein acetoxy-methylester (BCECF-am). Change in the rate of lactate-induced acidification (LIA) was measured in the presence of bicarbonate free (BF) and bicarbonate rich (BR) medium and in 100µM niflumic acid (non-specific MCT inhibitor), 100 µM acetazolamide ( a carbonic anhydrase (CA) inhibitor) and 10 µM EIPA ( Na+/H+ exchange blocker).
Results: :
RT-PCR and WB indicate MCT 1, 2, and 4 are expressed in the rabbit CE. IF staining showed MCT 1 predominately expressed on the BL membrane and MCT 2 & 4 on the apical and BL membranes. BCECF fluorescence measurements showed a significant decrease in pHi following 1 min of 20mM of lactate exposure to either the basal and apical surface in tissues perfused with ringer solution containing no bicarbonate . This acidification was followed by a pHi increase over baseline (overshoot). In the presence of bicarbonate, there was no decrease in pHi (apical surface) or very small decrease in pHi (BL surface) with lactate exposure. Five-minute incubation with niflumic acid on the BL surface in BF Ringer reduced the rate (from .25 to .10 pH units/min) and amount of LIA (from .075 pH unit reduction to .035). Acetazolamide did not change the rate of LIA in tissues perfused with no bicarbonate. However, in the presence of bicarbonate, acetazolamide produced a small increase in LIA on the BL surface (from .047 pH units/min to .057 pH units/min). Lastly, introduction of EIPA eliminated the overshoot seen after removing lactate.
Conclusions: :
MCTs 1, 2, and 4 are expressed in rabbit CE. IF and physiology confirm the presence of MCTs on both the apical and BL membranes. Bicarbonate in conjunction with CA activity significantly buffers the H+ cotransported with lactate producing little or no LIA. Na+/H+ exchange also participates in ameliorating a lactic acid load.
Keywords: pump/barrier function • cornea: endothelium • pH