Abstract
Purpose :
Previous work from the Fischbarg lab showed that cultured bovine corneal endothelium (BCEC) was capable of fluid transport. Subsequently, we showed that bicarbonate buffered ringers enhances apical compared to basolateral lactate efflux from BCEC, consistent with the model of a lactate-efflux dependent endothelial pump. Here we asked if bicarbonate or highly buffered bicarbonate-free ringers enhanced lactate production and could explain the increased lactate efflux in these cells.
Methods :
Primary cultures of BCEC were produced from fresh cow eyes. Lactate production and glucose consumption were measured in bicarbonate-free (BF), High-HEPES bicarbonate-free (HEPES), and bicarbonate-rich (BR) ringers all at pH 7.5. Cells were transfected with Laconic, a FRET based lactate sensor to measure relative changes in intracellular [lactate]. BCECF was used to measure pHi. Glut transporter activity was measured by NBDG uptake.
Results :
Lactate production by corneal endothelial cells was linear over two hours and was 24±1.8 in BR, 15.9±1.8 in HEPES, and 8.6±1.3 nMoles/hr in BF per 2x105 cells (n=6). When laconic transfected cells were perfused with BF, switching to BR caused an immediate increase in intracellular [lactate] (Laconic signal) followed by a gradual reduction back to the starting level, indicating a burst of lactate production that was shortly matched by increased efflux. Lactate pulses (10 mM) in the perfusate caused sharp increases of 14.3±3.2% in Laconic signal (lactate influx) in BF, 4.5±2.3% in HEPES, and 2.4±0.4% in BR (n=6) consistent with higher lactate production and efflux in BR. Glut transporter activity was not significantly different in the three ringers. Intracellular pHi was 7.04±.10 , 7.11±.13, and 7.23±.11 in BF, HEPES, and BR, respectively.
Conclusions :
These data suggest that glycolysis is activated (and therefore lactate production) by raising pHi most likely due to the known pH sensitivity of phosphofructokinase. Glycolytic intermediate analysis is needed to determine if phosphofructokinase is responsible.
This is a 2020 ARVO Annual Meeting abstract.