Activities of transport enzymes located in the plasma membranes of corneal endothelial cells.

Plasma membranes from bovine corneal endothelial cells have been prepared for assay of ion transport enzymes (ATPase, EC 3.6.1.8). The membrane were judged to be highly purified by protein recovery, electron microscopy, and assay of enzyme markers. Sodium-potassium-stimulated ATPase was more than six times more active in the membranes (6.7 mumoles phosphate released/mg protein/30 min) than the whole homogenates (1.1 mumoles phosphate released). However, no increase in activity was seen with anionic ATPase of membranes (2.5 mumoles phosphate released with bicarbonate stimulation) vs. whole homogenates (3.0 mumoles phosphate released; insignificant difference). In contrast, the bicarbonate-stimulated ATPase activity of a mitochondrial preparation from the same cells was 10.6 mumoles phosphate released (p less than 0.001). It is suggested that anionic ATPase has no direct role in deturgescence as part of and endothelial cell "pump."

Glutathione in rabbit corneal endothelia: the effects of selected perfusion fluids.

Although the ameliorating effect of glutathione on corneal deturgescence is known, its chemical mechanism is not understood. An endeavor toward the latter was made by perfusing freshly excised rabbit corneas with selected perfusion fluids, measuring corneal thickness, and assaying the endothelial cells for reduced and oxidized glutathione after 2 and 5 hr of perfusion. Ringer's solution, containing either lactate or bicarbonate, caused significant decreases in both forms of glutathione after perfusion. The corneas increased in thickness considerably during these periods. When 5 mM glucose was added to bicarbonate-Ringer's solution, the corneas swelled about half as much as before. However, glutathione levels were as depressed as with simple Ringer's fluid. Adenosine (0.5 mM) in the presence of glucose (bicarbonate-Ringer's) caused a further swelling decrease so that the corneas were maintained at near normal thickness. The levels of glutathione were 84% of control values compared to 35% to 45% for Ringer's solutions (+/- glucose). The addition of glutathione to glucose (bicarbonate-Ringer's) caused intracellular glutathione levels to be higher than control values while allowing minimal tissue swelling. Glutathione in combination with adenosine, glucose, and bicarbonate produced the highest intracellular glutathione levels and a slight corneal deswelling. After oxidation of intracellular glutathione with t-butyl hydroperoxide in glucose (bicarbonate-Ringer's), endothelial cells were destroyed within 1 hr. The oxidant, however, may have had a direct effect upon the endothelial cell membranes.

Adenosine-stimulated production of sugar-phosphates in bovine corneal endothelium.

Exogenous adenosine promotes deturgescence of in vitro corneal preparations, but the biochemical mechanism underlying this effect has yet to be elucidated. This study sought to investigate the possibility that adenosine promotes deswelling by activating the pentose shunt in corneal endothelium. Homogenized extracts of bovine corneal endothelial tissue cultures were incubated with [32P]K2HPO4 in the presence or absence of 5 mM adenosine for periods up to 30 min. Radiolabelled sugar-phosphates were separated on polyethyleneimine-cellulose thin layers, autoradiographed, removed, and counted by liquid scintillation. At 30 min, radiolabelled ribose-1-phosphate (R-1-P), ribose-5-phosphate (R-5-P), and sedoheptulose-7-phosphate (S-7-P) were identified and found to be significantly higher than control (without adenosine) levels by factors of 2.7, 4.2, and 2.4, respectively. The increases occurred first for the ribose-phosphates and later for sedoheptulose-7-phosphate. These results suggest that exogenous adenosine activates the pentose shunt by contributing its ribose moiety as a substrate, which may ultimately provide cellular energy for the deturgescence mechanism.

Age as a function of ATPase activity in cultured corneal endothelial cells.

As cells grown in tissue culture age, they typically become altered when compared to their in vivo counterparts. Bovine corneal endothelial cells were grown in culture for periods up to 60 days (primary = 10 days; secondary = 50 days). The plasma membrane enzymes: Na+K+ ATPase and Mg2+ ATPase were assayed for specific activity at selected intervals throughout the growth periods. In primary cultures, it was found that Na+K+ ATPase was quite low at 5 days (0.05 units), but increased fourfold at 10 days (0.22 units). By contrast, Mg2+ ATPase changed little over the same period. Tissue cultures grown secondarily had Na+K+ ATPase activity fall 0.3 units (0.52 to 0.22) for 35 days after a single trypsinization. After five trypsinizations over a 50-day period, the activity fell 0.23 units (0.33 to 0.10). The alterations in Mg2+ ATPase activity were more complex in secondary cultures. In the instance in which a single trypsinization was used, the activity fell 0.15 units at day 20, rose 0.12 units (above the starting value) at day 25, then returned to the initial level by day 35. When multiple trypsinizations were used, the activity fell 0.06 units by day 30, then remained stable for the duration. The data may be indicative of mechanisms such as receptor alteration, feedback inhibition and genetic instability when these cells are grown in culture.

Glucose oxidation in the chick cornea: effect of diamide on the pentose shunt.

Chick embryo corneas (stages 38 and 45) have been used to study variations in pentose shunt activity following the use of a glutathione-specific oxidizing agent, diamide, and a sulfydryl blocking agent, N-ethylmaleimide (NEM). Shunt activity was measured by the ratio of radiolabeled carbon 1 (14C-1) of glucose to radiolabeled carbon 6 (14C-6) of glucose derived as expired 14CO2. Diamide and NEM were both found to increase pentose shunt activity relative to glycolysis, although by different means. Diamide appeared to exert its effect by oxidizing glutathione and creating a demand for higher shunt activity to facilitate glutathione reduction by NADPH. Both C-1 and C-6 oxidation were increased, but C-1 oxidation was increased to a much greater extent. In contrast, NEM decreased both C-1 and C-6 oxidation, with C-6 preferentially affected. Thus NEM appears to preferentially inhibit the enzymatic machinery of the glycolytic-tricarboxylic acid cycle pathway and acts as an effective metabolic stress on the cornea. Our data suggest that the pentose shunt in the cornea may serve as an important alternative pathway under conditions of metabolic stress for glucose utilization and the production of energy (ATP) in the corneal cells.

Relationship of telomeres and p53 in aging bovine corneal endothelial cell cultures.

PURPOSE: To demonstrate a relationship between telomere lengths and levels of p53 in cultured bovine corneal endothelial cells (CECs) during aging. METHODS: Bovine CECs were grown and aged as long-term cultures. Telomere lengths were determined directly on gels with 32P probes after treatment of isolated DNA with RsaI and HinfI. Protein p53 was determined using an enzyme-linked immunosorbent sandwich assay. Cellular aging and the development of replicative senescence were monitored by the appearance of senescent morphology and the beta-galactosidase assay. RESULTS: Bovine CEC telomeres lost 4 kb (from 12.8 to 8.8 kb) over 1 year (89 population doublings [PDs]). The p53 levels in bovine CECs were initially small (approximately 60 pg/million cells), but rose 3.5-fold by culture age of 260 days (64 PDs). On initiation, cultured bovine CECs did not stain for the senescent marker beta-galactosidase. However, these cells stained at 89 PDs and senescent morphology was observed in the cultures at 64 PDs. CONCLUSIONS: The data indicate an inverse relationship between telomere lengths (decreasing) and levels of p53 (increasing) in bovine CECs during aging. These properties may influence the ability of these cells to divide as they enter into replicative senescence.

How might 12 (R) HETE cause the inhibition of Na,K-ATPase?

PURPOSE: 12 (R) hydroxy 5,8,10,14-eicosatetraenoic acid [12 (R) HETE] is a potent inhibitor of Na,K-ATPase. This study was an attempt to determine how the eicosanoid might inhibit the enzyme by using molecular modeling. METHODS: Models were generated using the program HyperChem 2.0 for Windows. Models of 12 (R) HETE, 12 (S) HETE (the "S" isomer of 12 (R) HETE), and 8 (R) hydroxy-hexadecatrienoic acid [8 (R) HHDTrE, a catabolic isomer of 12 (R) HETE] were formed and docked with phosphatidyl choline and the H3-H4 peptide of the alpha-subunit of Na,K-ATPase. In addition, models of 12 (R) HETE, and related compounds, were formed and complexed with calcium, and then docked with phosphatidyl choline. The energies of stabilization were calculated for each optimal docking. RESULTS: Optimal steric fitting and calculated energies of stabilization indicated that 12 (R) HETE and 8 (R) HHDTrE had the best fits when bound to the fatty acid portions of phosphatidyl choline. However, when Ca-HETE complexes were modeled, it was found that they formed even more stable complexes when bound to phosphatidyl choline. Calculated energies of 12 (S) HETE, whether complexed to calcium or not, were less favorable than the other HETE compounds. CONCLUSIONS: The results of the study indicate that plasma membrane lipids rather than Na,K-ATPase itself are more likely to be bound by 12 (R) HETE and its related compounds. Moreover, it was found that the calcium complexes of 12 (R) HETE and 8 (R) HHDTrE are even more likely to dock with plasma membrane lipids. This suggests that such complexes may be able to transport calcium into the cell and make it available for the inhibition of Na, K-ATPase at the enzyme's sodium binding site.

Tritiated thymidine experiments in the cat: a description of techniques and experiments to define the time-course of radioactive thymidine availability.

Tritiated thymidine [(3H]thymidine) autoradiographic techniques have been used to define birthdates for cells in a variety of animals. In an effort to include [3H]thymidine experiments in our ongoing studies of visual system development, we have used an intrauterine injection procedure that affords [3H]thymidine labeling of dividing nerve cells in the cat. This report contains a detailed description of the injection procedures used, as well as the results of experiments undertaken to define the period of time during which the exogenous [3H]thymidine introduced by such injections remains available for uptake.

Effects of mannitol on cultured corneal endothelial cell Na,K-ATPase activity.

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.

Alteration of ATPase activity and duplex DNA in corneal cells grown in high glucose media.

An investigation was made on the possible effects of high levels (450 mg/dl) of glucose on the activities of Na,K-ATPase [E.C. (Enzyme Commission) 3.6.1.37] and Mg-ATPase (E.C. 3.6.1.4) in plasma membrane preparations of bovine corneal endothelial cells grown in tissue culture. The activities of these enzymes were compared with the activities of the same enzymes from cells grown in low or "fasting" levels (90 mg/dl) of glucose. All activities were assayed from cells that were secondary cultures (15-25 days after trypsinization). The results indicated a 76% decrease in activity for Na,K-ATPase (0.04 units in high glucose vs. 0.17 units in low glucose). The activity for Mg-ATPase also decreased by 33% (0.24 units in high glucose vs. 0.36 units in low glucose). A t-test for significance indicated that the loss of activity in both enzymes was highly significant (p < 0.001) in the high glucose media. Assays for ATPase activity of plasma membranes were also made directly in high glucose after removal of the membranes from cells grown in low-glucose media. However, those membrane ATPases showed no significant decrease in activity. Tests for DNA damage of cells grown in the presence of high levels of glucose indicated a 15.5% change (decrease) in the amount of double-stranded DNA remaining after alkali treatment. This change was highly significant also (p < 0.001). These data suggest that the diabetic state may negatively affect membrane-bound ATPases of the corneal endothelium and further point to the possibility of an altered synthetic rate of ATPase polypeptides as a result of DNA damage.

Sorption of sodium hydroxide by type I collagen and bovine corneas.

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.

The effects of glutathione and adenosine on plasma membrane ATPases of the corneal endothelium. An hypothesis on the stimulatory mechanism of perfused glutathione upon deturgescence.

Reduced glutathione (0.3 mM) stimulates the activity of sodium-potassium activated ATPase (Na+K+ATPase) by 54% in plasma membranes prepared from bovine corneal endothelial cells. Oxidized glutathione, however, has no effect on Na+K+ATPase activity in the same tissue, although it does inhibit magnesium activated ATPase (Mg++ATPase) by approximately 30%. Adenosine neither stimulates nor inhibits either Na+K+ATPase or Mg++ATPase in these plasma membranes. It is postulated that the stimulatory effect of glutathione on deturgescence stems from the direct reaction of the reduced form of the tripeptide on sulfhydryl groups located on plasma membranes of corneal endothelial cells. It is highly probable that these sulfhydryl groups are part of the Na+k+ATPase complex itself.

Cyclic nucleotides in anatomical subdivisions of the bovine lens.

Adenosine 3', 5'-cyclic monophosphate (cAMP) and guanosine 3', 5'-cyclic monophosphate (cGMP) have been measured in whole and anatomically subdivided areas of bovine lenses. The highest values of cAMP and cGMP (2.843 pmoles/mg protein and 0.0139 pmoles/mg protein respectively) were found in the epithelium. In contrast, the lowest values of cAMP and cGMP (.007 pmoles/mg protein and .001 pmoles/mg protein respectively) were detected in the central nucleus. Values for the cyclic nucleotides in the whole tissue more closely resembled those of the whole nucleus.

Null effect of adenosine on cyclic nucleotides of the corneal endothelium: possible implications for adenosine-stimulated corneal deturgescence.

Adenosine has been reported by several investigators to stimulate corneal deturgescence, but the actual biochemical mechanism of this effect remains unknown. Effects observed include increased magnitude and rate of corneal thinning, increased endothelial fluid transport rate, support of adenosine triphosphate (ATP) levels, stimulation of magnesium activated ATPase (Mg++ATPase) and sodium-potassium activated ATPase (Na+K+ ATPase) activities in endothelium whole cell preparations, but a lack of stimulation of Mg++ATPase and Na+K+ATPase activities in endothelial plasma membrane preparations. This study examined the possibility that adenosine alters the corneal endothelial levels of two biochemical effectors: adenosine 3',5'-cyclic monophosphate (cAMP) and guanosine 3',5'-cyclic monophosphate (cGMP). Bovine corneal endothelium was studied as fresh tissue and after growth in tissue culture. The samples were processed both at room temperature and in liquid nitrogen. Incubation of fresh and cultured endothelia in media containing adenosine was found to have no effect on the cAMP and cGMP levels regardless of the processing method. The data suggest that cyclic nucleotides do not mediate adenosine-stimulated corneal deturgescence.

Sodium and potassium saturation kinetics of Na+K+-ATPase in plasma membranes from corneal endothelium: fresh tissue vs. tissue culture.

The maximal velocity (Vmax) and the apparent dissociation constant (K0.5) of Na+K+-ATPase have each been estimated with respect to sodium and potassium ion activation. These estimations were made from the enzymatic activity of plasma membrane preparations derived from bovine corneal endothelial cells. The determinations were made on cells obtained from fresh tissue and from secondary tissue cultures. Two methods were used to obtain the estimates: the first used a combination of Eadie-Hofstee and Hill plots; the second used Eisenthal-Cornish-Bowden plots. The Vmax for sodium was 5.58-5.60 mumol Pi/mg protein/30 min for fresh tissue vs. 2.00-1.80 mumoles Pi/mg protein/30 min for tissue cultures. The corresponding K0.5 values were 62-57 mM (fresh tissue) vs. 7.9-6.7 mM (tissue culture). Vmax for potassium was 4.28-4.00 mumoles Pi/mg protein/30 min for fresh tissue vs. 1.37-1.34 mumoles Pi/mg protein/30 min for tissue cultures. The corresponding K0.5 values were 3.3-3.1 mM (fresh tissue) and 1.7-1.7 mM (tissue culture). The results indicate a lowered activity and change in affinity of the enzyme for the two ions in tissue cultures compared to fresh tissues. The detergent Lubrol W-X increased activity in both tissue sources. Sonication had no significant effect on the activity. Variations in pH (7-9) indicated that the highest activity was obtained at pH 7.8 for the enzyme in tissue culture while activity was highest at pH 8.0 for the enzyme in fresh tissues. These differences in kinetic activity suggest a response to changes in the ion requirements of these cells due to their environment, developmental stage or some other parameter.

Common cryopreservation media deplete corneal endothelial cell plasma membrane Na+,K+ ATPase activity.

This study describes the effects of three cryopreservation media on the specific activity of corneal endothelial plasma membrane Na+,K+ ATPase activity, a transporter required for the fluid pump in the cornea. Bovine corneal endothelial cell cultures were used as a model system for these studies. Cryopreserved primary cells were thawed and passaged once to increase cell number. The specific activity plasma membrane Na+,K+ ATPase activity was subsequently measured on 4-6 replicate cultures. One freeze/thaw cycle depleted the Na+,K+ ATPase specific activity of corneal endothelial cell cultures by approximately 90%, as compared to cells of equivalent passage which had not been cryopreserved. Cell morphology of the cryopreserved cultures was indistinguishable from that of control cultures. In other experiments, first passage cultures which had not been subjected to cryopreservation were incubated with a dimethyl sulfoxide-, glycerol-, or propane diol-based freezing medium and Na+,K+ ATPase was measured on plasma membranes subsequently isolated from the cultures. Incubation of cells with cryopreservation media in the absence of the freezing process also depleted Na+,K+ ATPase by approximately 90%. Radiolabeled ouabain was used to measure Na+,K+ ATPase sites on cell cultures pretreated with dimethyl sulfoxide-based freezing media. A 4 h treatment with DMSO-based freezing medium had no effect on ouabain binding; treatment for 18 h reduced binding by only 50%. Thus, the method used to assess pump function (determination of Na+,K+ ATPase specific activity versus ouabain binding) may provide conflicting data concerning the level of pump function cultured cells. The cryoprotectants present in many common media used to freeze tissue culture cells appear to inhibit corneal endothelial Na+,K+ ATPase. Since the fluid pump of corneal endothelial cells is coupled to Na+,K+ ATPase activity, care must be taken to insure that pump function is not impaired during cryopreservation of cell cultures.

The inhibition of sodium, potassium-stimulated ATPase and corneal swelling: the role played by polyols.

BACKGROUND: Sodium, potassium-stimulated adenosine triphosphatase (Na,K-ATPase; E. C. 3.6.1.37) is considered to be the principal corneal enzyme responsible for deturgescence. Some metabolites are known to inhibit Na,K-ATPase, including glucose and galactose. In diabetes, some cells take up high levels of glucose and are converted to the polyhydroxy alcohol: sorbitol. In galactosemia, a similar uptake of galactose may cause the intracellular formation of the polyhydroxy alcohol: galactatol. This study tested the possibility that polyhydroxy alcohols (polyols), metabolized from carbohydrates, might decrease Na,K-ATPase activity. METHODS: Cultured corneal endothelial cells were separately exposed to 20 mM of the polyols: sorbitol, galactitol, and xylitol. Media containing 5 mM glucose served as the control. Plasma membrane vesicles, containing the Na,K-ATPase, were isolated and tested for the enzyme's activity. RESULTS: We found that the enzyme, after exposure to polyols, had only 15%-20% of its normal activity when assayed with the control. CONCLUSIONS: Since corneal endothelial cells can generate polyols in diabetes mellitus, this result may explain why corneas are able to swell in individuals with poorly controlled diabetes.

The inhibition of Na, K-ATPase, and Mg-ATPase by timolol maleate in cultured non-pigmented epithelial cells of the ciliary body.

Bovine, non-pigmented, ciliary body epithelial cells were isolated and grown in culture to determine whether timolol maleate might affect the activity of their plasma membrane ATPases. The possible effects were tested in drug concentrations in a range of 5 x 10(-19) to 5 x 10(-5) M over an incubation period of 30 min at 37 degrees. Assays of specific activity showed that the drug significantly (p less than .001 for most concentrations) inhibited both Na,K-ATPase and Mg-ATPase. However, the inhibition was partially reversed in concentrations greater than 10(-6) M for Na,K-ATPase and 10(-5) M for Mg-ATPase. The latter enzyme also indicated a second partial reversal in activity at concentrations between 10(-12) and 10(-9) M. These reversals in activity suggest that more than one binding site is involved in the inhibition of both enzymes. Since Na,K-ATPase in non-pigmented, ciliary body cells is responsible for the generation of aqueous fluid and the intraocular pressure (IOP), this inhibition demonstrates a possible mechanism for the pharmacological action of timolol maleate in lowering IOP.