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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.

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.

Eight putative, low-molecular-weight peptides have been found in the media of bovine corneal tissue cocultures and in bovine aqueous fluid. By investigating amine fluid extracts from these sources by HPLC, possible amino acids, vitamins and urea were eliminated as the unknown peaks of interest. The molecular weights of these substances were equal to or less than 1,000 D. The peaks did not correspond to glutathione or bradykinin either which fall in this molecular weight range. As the cultures aged from 10 to 32 days, it was found that 5 of the 8 peptides varied in concentration. Five peptides increased in concentration to day 17 and slowly decreased afterwards. Efforts are not directed toward the identification of these peptides.

Bovine corneal endothelial cells showed a strong migratory response to specific simple sugars (D-glucose and sucrose, but not L-glucose, sorbitol, lactose, or D-galactose) at concentrations above 10 mM. Checkerboard analysis of the migratory responses in modified Boyden chambers indicated both chemotactic and chemokinetic effects. Serum starvation of the cultures increased the chemotaxis towards D-glucose and 2-deoxy-D-glucose, but not towards sucrose. Migration to sucrose and glucose was inhibited by chelation of extracellular calcium or by inhibition of Na+,K+ ATPase with ouabain. To date, this migratory response has been found only in corneal endothelial cells. Neither human melanoma cells, human breast carcinoma cells, bovine aortic endothelial cells, nor bovine microvascular endothelial cells migrated towards simple sugars, although all cell types migrated toward fibronectin in chemotaxis assays. After 16–19 passages in culture, bovine corneal endothelial cells retained their ability to migrate towards fibronectin, but lost their ability to migrate towards sugars. This loss of migratory response was accompanied by a sevenfold decrease in Na+,K+ ATPase activity. Although loss of Na+,K+ ATPase activity accompanied the loss of migratory response, pretreatment of cell cultures with 25 mM glucose did not stimulate, but rather lowered Na+,K+ ATPase activity in low or high passage cultures. © 1993 Wiley-Liss, Inc.

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.

An effort was made to determine whether the beta-blocking agents: timolol maleate and betaxolol hydrochloride could affect the activity of the plasma membrane, transport ATPases of corneal endothelial and epithelial cells grown in culture. It was found that both agents were able to inhibit the activity of Na+K+ATPase in endothelial cells in the concentration range from 10−12 to 10−4 M (47– 77% inhibition with timolol and 24-78% inhibition with betaxolol). In concentrations of 10−9 M or greater (for betaxolol) and 10−12 M or greater (for timolol) the inhibition was highly significant (p

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 corneal endothelium is a monolayer of cells in the posterior cornea that is responsible for maintaining a clear cornea. Corneal endothelial cells may be induced to divide, but it has been held that they do not divide in the normal cornea of an adult human. Some studies have suggested that a stem cell population for the corneal endothelium exists. This population could give rise to mature corneal endothelial cells and may reside either in the peripheral corneal endothelium or in the adjacent posterior limbus. This study was initiated to demonstrate the presence of such stem cells in the region of the posterior limbus and to show the response of these cells to corneal wounding.Unwounded and wounded corneas with their attached limbal sections were analyzed by immunofluorescence for the presence of nestin, telomerase, Oct-3/4, Pax-6, Wnt-1, and Sox-2. Alkaline phosphatase activity was observed with an enzyme-based reaction that produced a fluorescent product.In the unwounded cornea, stem cell markers nestin, alkaline phosphatase, and telomerase were found in the trabecular meshwork (TM) and in the transition zone between the TM and the corneal endothelial periphery (including Schwalbe's line). Telomerase was also present in the peripheral corneal endothelium. When wounded corneas and their attached limbii were tested, the same markers were found. However, after wounding, additional stem cell markers, Oct-3/4 (in the TM) and Wnt-1 (in both the TM and the transition zone), appeared. Moreover, the differentiation markers Pax-6 and Sox-2 were seen. Pax-6 and Sox-2 were also manifest in the peripheral endothelium post-wounding.Well documented specific stem cell markers were found in the TM and the transition zone of the human posterior limbus. Wounding of the corneas activated the production of two additional stem cell markers (Oct-3/4, Wnt-1) as well as two differentiation markers (Pax-6, Sox-2), the latter of which also appeared in the corneal endothelial periphery. It is suggested that stem cells reside in the posterior limbus and respond to corneal wounding to initiate an endothelial repair process. The stem cells may also contribute to a normal, slow replacement of corneal endothelial cells.

The well-known reluctance of human corneal endothelial cells (HCECs) to divide has continually intrigued investigators. Related to this, the discovery of an increased endothelial cell population in the periphery of the human cornea has prompted an investigation for evidence of the existence of stem-like cells in the endothelial periphery. Showing that stem cells or transient amplifying cells may exist in the periphery might explain the origin of HCECs and indicate a source for these cells in wound repair. In addition, these cells might be of value in culturing or as a source for the synthesis of artificial corneas.Human corneas with attached scleral rims were obtained from eye banks and were assayed for telomerase activity and BrdU (bromodeoxyridine) incorporation to determine, respectively, the presence of a stem-like cell marker and replicative activity. In the case of telomerase activity, the tissues were divided into central, intermediate and peripheral areas by the use of trephines. BrdU staining (using alkaline phosphatase bound secondary antibody) was performed on whole corneas plus scleral rims exposed to BrdU antibodies on the endothelial side whereas BrdU fluorescence (using fluorescein bound secondary antibody) was obtained from transverse sections of the these tissues by the same procedure. Some corneas were wounded to determine whether the wounded areas stimulated BrdU (by staining or fluorescence) followed by the synthesis of transforming growth factor beta (TGFbeta). The latter was determined by quantitative ELISA. Rabbit corneas were also assayed for BrdU incorporation to compare their evidence of cell division with that of humans.After dividing corneas into central, intermediate, and peripheral sections, the dissected endothelial tissues exhibited positive telomerase activity in the peripheral and intermediate sections. No activity was observed in the central endothelial tissues or the limbus between the trabecular meshwork and Schwalbe's line. BrdU staining with alkaline phosphatase was occasionally observed in the wounded area's human corneal endothelial cells after wounding. When BrdU fluorescence assays were made on corneal transverse sections with fluorescein, fluorescence occurred in an area just at and adjacent to the trabecular meshwork, but was not seen at the corneal endothelium. After wounding, BrdU fluorescence extended into the corneal endothelium. TGF-beta levels were increased in fluids bathing the endothelium following wounding, but the increases lagged behind the wounding event.It is suggested stem-like cells may be sequestered in a niche at the junctional region where the corneal endothelial cells and the trabecular meshwork come together. These putative stem cells may supply new cells for both the corneal endothelium and the trabeculae. Evidence suggests that cells from this area migrate (perhaps as transient amplifying cells) to the endothelial periphery and, perhaps, to wounded areas of the corneal endothelium when needed. The migration may not be constant and may be age dependent.

To determine the protein and mRNA expression of p53, p63, and p73 in central and peripheral human corneal endothelial cells. Since these proteins are known to be involved in the regulation of cell division, this study seeks information about their influence in regulating cell proliferation in the human corneal endothelium.Human donor corneas were separated into central and peripheral sections. The endothelial tissue from these samples was dissected and samples were analyzed for mRNA transcription of p53, transactivating p63 (TAp63), delta N p63 (DeltaNp63), transactivating p73 (TAp73), and delta N p73 (DeltaNp73) via the reverse transcriptase-polymerase chain reaction (RT-PCR). Additional samples were analyzed for p53, p63, and p73 protein expression via SDS-PAGE, western blotting, and immunodetection. Frozen corneal sections were immunostained for p53 and analyzed via fluorescence microscopy.p53 and TAp63 mRNA and protein expression were detected in central and peripheral human corneal endothelium. p53 and TAp63 protein expression were greater in central than in peripheral tissue. DeltaNp63 and all isoforms of p73 were not detected in either central or peripheral corneal endothelium.p53 is expressed in both peripheral and central human corneal endothelium, although it is more highly expressed in the central endothelium. Similarly, TAp63 is more highly expressed in central rather than in peripheral endothelium. This suggests that the peripheral endothelium may have more potential for cell division than the central endothelium. DeltaNp63, a stem cell marker, was not detected in the corneal endothelium. Neither the TAp73 nor the DeltaNp73 isoforms were detected in either central or peripheral human corneal endothelium.

Angiogenesis is an important step in the development of ocular onchocercaisis. In previous studies, it has been demonstrated that Onchocerca volvulus homologues of the Ancylostoma secreted protein family have pronounced angiogenic activity. The overall goal of the current study was to determine if this angiogenic effect is exerted through a direct or indirect mechanism. These studies focused on one member of this family, OvASP-2, as this protein is expressed in microfilaria, the stage of the parasite that causes ocular onchocercaisis. Clones encoding truncated and full length open reading frames were expressed as fusion proteins with Escherichia coli maltose binding protein (MBP), and angiogenic activity was compared in vitro and in vivo with MBP alone. Truncated constructs expressing only the first 105 amino acids of OvASP-2 were as active as the full length protein in inducing new blood vessel formation. The full length fusion protein did not stimulate proliferation or production of vascular endothelial growth factor in vascular endothelial cells in vitro, indicating that OvASP-2 does not directly stimulate angiogenesis. Sequence analysis demonstrated that the gene encoding OvASP-2 contained five introns. Sequence comparisons of the genomic loci from West African blinding and non-blinding strains of O. volvulus revealed that some polymorphism existed among the various isolates tested. However, none of these polymorphisms could be used to differentiate the parasite strains, suggesting that qualitative variation in OvASP-2 could not explain the difference in ocular pathogenic potential of the two parasite strains.

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.

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+ AT Pase 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.

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.

Liposomes have been prepared with a phospholipid ratio and a cholesterol content similar to that of the outer segments of rod photoreceptor cells. Studies were made of the appearance, electrophoretic mobility and sodium leakage rates of the liposomes, with and without the inclusion of protein (albumin, myoglobin, ribonuclease, lysozyme, proteolipid or opsin). The findings indicate that although the appearance and properties of these liposomes closely resemble those of other liposomes reported in the literature, the liposomes become markedly changed in appearance, electrophoretic mobility and sodium permeability by the addition of opsin. These data emphasize the hydrophobic nature of the opsin-lipid interaction and suggest an ability of opsin to alter the ion leakage properties of a rod outer segment membrane.

Cytoplasmic glutathione peroxidase (GPx) activity has been found to be consistently greater in bovine corneal endothelium compared to bovine corneal epithelium and whole lens. Freezing reduced the activity in all tissues examined. Triton X-100 increased GPx activity in the presence of sodium azide, but had no significant effects in its absence.

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.

Quantities of adenosine 3′,5′-cyclic monophosphate were measured in rabbit corneal endothelial cells taken from fresh tissue and tissue culture. Higher levels of both nucleotides were observed when the cells were initially frozen in liquid nitrogen compared with those that were not. Adenosine 3′,5′-cyclic monophosphate was 14 times lower in the primary cultures compared to the fresh tissues while subcultures were about two-thirds that of the fresh tissue amount. Guanosine 3′,5′-cyclic monophosphate was four times higher in the primary cultures compared to the fresh tissue while subcultures were about two-thirds that of the fresh tissue amount. It is emphasized that the ratios of adenosine 3′,5′-cyclic monophosphate to guanosine 3′,5′-cyclic monophosphate were similarly high in both fresh tissue and subcultured cells approaching confluency, but quite low in primary outgrowths.

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.

Cluck neural retinal cells are relatively undifferentiated in appearance for several weeks in culture. Cells with long, thin processes are subsequently observed as well as foci of small, pigmented cells and large semi-transparent masses of cells or other material. Retinal cells thus appear to be able to differentiate into several divergent cell types in vitro. Chick pigment epithelial (PE) cells rapidly differentiate in culture, forming microvilli, pigment granules and other specialized subcellular organelles. 5-Bromodeoxyuridine greatly inhibits cellular differentiation of cultured PE cells as assessed by decreased visible pigmentation and by colonial disorganization. In contrast, several prostaglandins and dibutyryl cyclic-AMP plus isobutylmethylxanthine increase pigmentation and induce a more mature type of cell morphology and colonial development. By electron microscopy, increased numbers of pigment granules are apparent as well as an increase in the length of microvilli. Prostaglandins also increase glutathione levels in the cells. Prostaglandins and cyclic-AMP thus promote the general state of differentiation of PE cells in culture.