Corneal Endothelial Pump Is Dependent on Buffering Lactic Acid Efflux

Abstract

Purpose : To answer two questions: Is HCO₃^- necessary for the corneal endothelial pump and 2) is Lactate and water efflux facilitated by HCO₃^- or other buffers?

Methods : Rabbit corneas were mounted in vitro and superfused on the endothelial surface with Glutathione Bicarbonate Ringer (control GBR, 28.5 mM HCO₃^-/5% CO₂, pH7.5) for 90’ and then switched to Ringer of varying [HCO₃^-] (5, 10, 18, 28.5 and 44 mM) or HCO₃^- free Ringer (BF), buffered with 0, 10, 25, 40, or 60 mM HEPES, all with 1mM phosphate, 37 deg C. Perfusate was collected over 30 minute intervals and assayed for [lactate]. Corneal thickness (CT) was measured every 15 min by OCT imaging. At the end of each experiment (5 hours), the lactate in the cornea was extracted. The effects of inhibitors of Na-K-ATPase, carbonic anhydrase, and Moncarboxylate Transporters as well as the endothelial pump agonist Adenosine, on CT and lactate flux were also examined.

Results : Standard GBR perfusion resulted in 5±0.36 µm/hr increase in corneal thickness. Reducing the [HCO₃^-] to 18, 10 and 5mM led to maximum corneal swelling rates of: 8.3±0.8; 13.7±1.26; and 20±1.1µm/hr, respectively, whereas increasing bicarbonate to 44mM was not significantly different from control. Reducing [HCO₃^-] to zero led to 30±2.1µm/hr of swelling. However, supplementing bicarbonate-free solutions with HEPES buffer, 10, 25, 40, and 60mM reduced swelling rates to: 22±1.1µm/hr; 13±1.1µm/hr; 10±1.1µm/hr; and 7.5±1.1µm/hr, respectively. Lactate efflux (90-300min) was greatest in GBR (28.5 & 44 mM HCO₃^-, 1350±50 nmols) and lowest in bicarb-free with 0 HEPES (750±39 nmols). Lactate retention in the cornea after 5 hours of perfusion was lowest in GBR (14±2.5nmols/mg protein) and highest in bicarb-free with 0 HEPES (22±35nmols/mg protein). Inhibitors of Na-K-ATPase, carbonic anhydrase, and Moncarboxylate Transporters produced swelling rates of 32±1.1, 10±2, & 11.5±1.3µm/hr, respectively, with concomitant decreases in lactate flux and increases in lactate retention. Conversely, relative to GBR, Adenosine decreased the swelling rate by 14%, increased lactate efflux by 25% and reduced lactate retention by 5%.

Conclusions : Maintenance of corneal hydration was directly related to perfusing solution-buffering capacity with or without bicarbonate. Bicarbonate and other buffers act by facilitating lactic acid efflux, which is linked to corneal hydration control.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.