REGULATION OF CORNEAL ENDOTHELIAL BARRIER FUNCTION BY ADENOSINE, CYCLIC-AMP, AND PROTEIN-KINASES

PURPOSE. TO determine which processes or factors that regulate corneal hydration are responsible for the hydration-modulating effects of ade nosine. Influx of fluid to the stroma and efflux to the aqueous humor are governed, respectively, by the imbibition pressure of the stromal matrix and the transendothelial ionic gradients determined by the perm eability and active transport characteristics of this monolayer. The f ocus of this study was to assess the effects of adenosine on these end othelial parameters. METHODS. Isolated corneas freshly dissected from rabbit eyes were used throughout. Active ion transport was assessed by measurement of Rb-86(+) uptake by the endothelial cells of intact cor neas incubated for 30 minutes in 25 mM HCO3--Ringer with agents promot ing corneal deturgescence or corneal swelling. Intracellular and extra cellular fluid in the scraped endothelial cell mass was estimated from simultaneous counts of H-3-mannitol and C-14-urea, allowing calculati on of tissue-to-medium (T-M) ratios of Rb-86(+) in cell water. Permeab ility of the endothelium was determined by measuring the efflux into t he superfusate of 5-carboxyfluorescein (CE) applied to the stroma of d eepithelialized corneas superfused at the endothelial surface with the same media described for Rb-86(+) uptake. Thickness of these corneas and of others fixed for scanning electron microscopy was monitored wit h a specular microscope. RESULTS. In the control medium, 25 mM HCO3--R inger, Rb-86(+) was accumulated to yield a T-M ratio of 6.21. Neither adenosine nor other agents that increase cyclic adenosine monophosphat e (cAMP)-that is, forskolin and dibutyryl cAMP-changed this value to a significant extent. Bumetanide had no effect, but ouabain caused a de crease in T-M to 1.30, a 79% inhibition. Elimination of Nat or HCO; al so caused marked decreases in uptake. Permeability to CF in control me dium was 3.40 x 10(-4) cm/min. A decrease of more than 20% (P < 0.05) was seen in the presence of adenosine and cAMP promoters and also with the protein kinase inhibitor H-8, whereas phorbol myristate acetate c aused an increase to 4.50 x 10(-4) cm/min (P < 0.01). Ouabain caused n o change, but blocked the effects of adenosine. Reducing the Ca2+ conc entration of the superfusing medium caused time-dependent increases in permeability to 4.57 at 15 to 45 minutes and 12.5 at SO to 110 minute s. At the earlier time, this increase in permeability could be prevent ed by the addition of adenosine or H-8. Elimination of Na+ or HCO3- io ns from the medium caused a small decrease in permeability and, like o uabain, blocked the effect of adenosine. Changes in thickness of corne as were consistent, in most cases, with the observed alterations in Rb -86(+) uptake or permeability to CF. Scanning electron microscopy show ed contraction and rounding of endothelial cells in low Ca2+ medium, w ith stretching of intercellular borders, features that were largely el iminated when adenosine was also present. CONCLUSIONS. Adenosine, thro ugh increasing cAMP, decreases permeability of the corneal endothelium . This effect, rather than a change in the active transport (fluid pum p) mechanism, is responsible for the promotion of deturgescence and ma intenance of lower steady state thickness of corneas exposed to adenos ine. The mechanism may involve the phosphorylation state of cytoskelet al proteins and seems to be dependent on an undisturbed environment of monovalent ions.