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Mannitol was introduced into the media of bovine corneal endothelial cells grown in culture. This was accomplished in order to compare its influence on Na,K-ATPase activity with that of high levels of glucose that inhibit the enzyme. The study was conducted with the intent of showing possible adverse osmolar effects on enzyme activity. Mannitol was found to inhibit NA,K-ATPase when compared with mannitol-free medium (11 U vs. 202 U). However, this inhibition was significantly greater than that produced by high levels of glucose. When mannitol was introduced directly into the assay for the plasma membrane-extracted enzyme, the inhibition was as severe (14 U) as for that placed in the culture medium. By way of comparison, glucose introduced into the enzyme assay caused no significant inhibition (189 U). The mannitol concentration used was 20 mM and in the media there was an osmotic pressure of 347 mOsmol/kg. The high glucose concentration was 25 mM and the osmotic pressure in the media was 341 mOsmol/kg. These osmotic pressures were compared with that of the control medium (with 5 mM glucose), which generated a pressure of 308 mOsmol/kg. None of these values were judged sufficiently high to rupture cell plasma membranes or alter cell morphology as seen by vital staining and phase contrast microscopy. In addition, it was found that mannitol had no effect on cellular DNA, whereas a previous study showed that high glucose caused an increased unwinding of duplex DNA. This study suggests that mannitol inhibits endothelial cell Na,K-ATPase by a different mechanism than that of glucose. It further points out that osmotic effects may not be involved with either mechanism.

There are no quantitative studies on the uptake of alkali into corneal tissues. To study this phenomenon, both type I collagen and bovine corneas were incubated in sodium hydroxide (NaOH) under varying conditions for periods up to 27.5 h. The sorption (absorption or adsorption) of the alkali to protein and tissue was measured as the quantity of NaOH no longer available for titration to neutrality with hydrochloric acid. Sorption was found to be dependent on the concentration of NaOH (0.01-1 N) but independent of the incubation temperature (4-35 degrees C). In whole cornea, sorption of 1 N NaOH began immediately and increased with time up to 6 h. After 6 h, sorption decreased, together with the observed degradation and solubilization of the tissue. Stripping of the corneal endothelium alone or of the endothelium and epithelium increased sorption in a similar manner when compared to whole corneas for periods up to 4 h. These observations are compatible with ionic and nonionic bonding of hydroxide ions to collagen (including that of the cornea) and the subsequent release of hydroxide ions during hydrolysis of the protein itself. Indirect evidence also suggests the inclusion of quantities of unbound hydroxide ions in hydrated gels of glycosaminoglycans. It is proposed that in a chemical burn of the cornea, alkali is both stored in the tissue (by sorption) and reacted with it (by hydrolysis), without any net consumption of alkali taking place.