RELATIONSHIP BETWEEN FLUID TRANSPORT AND IN-SITU INHIBITION OF NA-K+ ADENOSINE-TRIPHOSPHATASE IN CORNEAL ENDOTHELIUM()

Purpose. To examine the relationship between the activity of the sodiu m pump of the corneal endothelium and corneal thickness. It was postul ated that because imbibition pressure of the stroma decreases as thick ness increases, a partially inhibited sodium pump would result in a ne w steady-state thickness of the cornea when reduced rates of fluid inf lux and efflux were equal. Measurements of physiologic behavior and bi ochemical activity were to be made in the same tissue and thus establi sh the relationship directly. Methods. Rabbit corneas were superfused with a bicarbonate Ringer solution containing different concentrations of ouabain. Exposure to ouabain was either continuous for 4 hours or for an initial 10 minutes followed by ouabain-free superfusion. Thickn ess was measured, and, after superfusion, endothelium was removed from the corneas, sonicated, and assayed for Na+-K+ adenosine triphosphata se (ATPase) activity without further addition of ouabain to the assay medium. Thickness was also measured during superfusion with suboptimal concentrations of Na+ or HCO3- and with brefeldin A, an inhibitor of protein trafficking. Results. Continuous exposure to ouabain caused co rneas to swell, but no new steady-state thickness was reached. At low concentrations, swelling rates increased with time, as did the extent of inhibition of the Na+-K+ ATPase. With only a 10-minute exposure to ouabain, swelling rates with 10(-4) M to 10(-5) M decreased with the d uration of ouabain-free superfusion. Similar swelling curves were obta ined by reductions in Na+ or HCO3- concentrations in the superfusion m edium, indicating that partial inhibition of the endothelial fluid tra nsport processes, whether via the Na+-K+ ATPase or by suboptimal ionic conditions, led toward a new equilibrium thickness of the cornea. How ever, when superfusion was continued for more than 4 hours, the cornea s exposed for 10 minutes to 3 X 10(-5) M or lower-concentration ouabai n showed increasing Na+-K+ ATPase activity and began to thin, indicati ng a recovery of fluid transport capability. This recovery was blocked by addition of brefeldin A during the ouabain-free superfusion. Concl usions. Inhibition of Na+-K+ ATPase by low concentrations of ouabain i ncreases with time. Temporary exposure to ouabain causes swelling at r ates that decline with time as ouabain dissociates from enzyme sites. This dissociation, together with the turnover of Na+-K+ ATPase in the plasma membrane, can lead to recovery of normal thickness in ouabain-e xposed corneas. Twenty percent of Na+-K+ ATPase in the endothelium is estimated to be intracellular, and about 20% of the activity can be in hibited without inducing swelling.