Bicarbonate, NBCe1, NHE, and carbonic anhydrase activity enhance lactate-H+ transport in bovine corneal endothelium.

Corneal transparency is a cumulative result of avascularity, regular arrangement of stromal collagen fibrils, maintenance of hydration by the corneal endothelium, and uniform refractive index within the epithelium aided by the relative lack of mitochondria. As a consequence of the paucity of epithelial mitochondria, the cornea is highly glycolytic. Eighty-five percent of glucose consumed by the cornea is converted to lactate,1 which results in a substantial [lactate] gradient between the cornea and aqueous humor.2 Accumulation of lactate, for example induced by hypoxia, creates an osmotic load that leads to corneal edema.2 Because the outer layers of corneal epithelium are impermeable to lactate,2 lactate efflux must occur posteriorly across the corneal endothelium and into the anterior chamber. Failure to remove lactate, even under normoxic conditions, will lead to corneal edema and loss of corneal transparency, indicating that lactate needs to be efficiently removed from the cornea. Lactic acid has a pKa of 3.86, so at physiological pH, 99.96% is the anion lactate− so cellular transport will need to be facilitated. Lactate produced in the epithelium is transported into the stroma via a lactate-H+ cotransport mechanism.3 Lactate efflux across the corneal endothelium could be transcellular or paracellular; however, because most of the surface area is cellular, it would be most efficient if stroma to aqueous efflux were transcellular. Indeed, facilitated cotransport of lactate-H+ has been demonstrated in the corneal endothelium.4 Using rabbit corneas, Giasson and Bonanno4 found that stromal lactate acidifies endothelial cells suggesting an H+ coupled mechanism. Since then, the monocarboxylate transporter family (MCT, SLC16) has emerged as the molecular mechanism for facilitated lactate-H+ cotransport. MCTs are responsible for transport of lactate, pyruvate, and ketone bodies across the plasma membrane in many tissues.5 Of the fourteen members of the MCT family, only the first four members (MCT1–4) have been demonstrated experimentally to facilitate the proton-linked transport of lactate.6,7 MCT1 is the most widely distributed isoform and is expressed in most mammalian tissues. MCT2 can be found in the liver, kidney, brain, and sperm tail. Although MCT1 and MCT2 can be found coexpressed in the same tissue, their locations within the tissue can be different, suggesting that each has a unique functional role. MCT3 has been found exclusively in the basolateral membrane (BL) of the retinal pigment epithelium (RPE) and choroid plexus epithelium.8 Lastly, MCT4 can be found in glycolytic tissues such as white skeletal muscle fibers, astrocytes, white blood cells, and chondrocytes. Chidlow et al.9 have examined the distribution of the MCT1–4 isoforms in the anterior structures of the rat eye, including the cornea. They found all four MCT mRNAs present in the rat cornea. Immunohistochemistry data however, showed immunoreactivity only to MCT1 and MCT2 in the corneal endothelium. MCT dependent lactate-H+ flux is facilitated by bicarbonate transporters and carbonic anhydrase (CA) activity in various cells and tissues.10–16 Becker et al.13 reported that lactate transport activity by MCT1 is increased two-fold when coexpressed with the sodium bicarbonate cotransporter (NBC) in Xenopus oocytes. They also reported that MCT1 and MCT4 transport activity is increased by interaction with carbonic anhydrase II (CAII).10,14,15 Similarly, CA activity has been found to facilitate lactic acid transport in rat skeletal muscle fibers11 as well as in neurons and astrocytes.12 These studies suggest that HCO3− in conjunction with CA increases the effective buffering capacity and thus attenuates the dissipation of H+ gradient across the cell membrane hence increasing MCT activity.13–15 Buffering capacity has a marked influence on the change in pHi and is itself enhanced by the activity of many acid and/or base transporters in a cell. Because of the presence of HCO3− transporters, several CAs, and lactate-H+ cotransporters, we postulate that a similar facilitation mechanism occurs in the corneal endothelium. Interestingly, Giasson and Bonanno4 found that basolateral lactate influx in the rabbit corneal endothelium is partially sodium dependent suggesting that the basolateral sodium bicarbonate cotransporter (NBCe1) could act to augment lactate-H+ flux. There is also evidence that Na+/H+ exchange (NHE1, SLC9) exists in the corneal endothelium and is active when pHi is reduced.17 Lactate-induced acidification could activate NHE1, which may also be involved in regulating the transport of lactate-H+ across the corneal endothelium. In this study, we have attempted to examine the role of buffering capacity in the transport of lactic acid in bovine corneal endothelium. We first sought to identify the MCT isoforms that facilitate lactate/H+ transport in cultured bovine CE and to determine their membrane localization. We then examined the effect of HCO3− and CA activity on buffering capacity and lactate-induced cell acidification (LIA). Direct lactate flux was examined by measuring endogenous lactate appearance in the apical and BL domains while inhibiting NBCe1, NHE1, and CA activity.