Search

Purpose: To determine the refractive and corneal topographic recovery after the use of reverse-geometry contact lenses for overnight orthokeratology., Methods: Both eyes of 15 subjects were fitted with reverse-geometry contact lenses that were worn by the subjects for 1 month. Uncorrected visual acuity, refractive correction (sphere and spherical equivalent), corneal curvature, and corneal thickness were measured during this time and for 2 weeks after discontinuation of lens wear., Results: Ten subjects completed the investigation. Uncorrected visual acuity, refractive correction, and corneal curvature had changed significantly (P = 0.01) after 1 month of lens wear. By the end of 1 month, central corneal thickness was significantly thinner than the baseline value (P = 0.01), but it recovered fully after one night of no lens wear. Recovery of corneal curvature was complete 1 week after lens wear was discontinued. Refractive correction and binocular uncorrected visual acuity recovered fully after 2 weeks. Monocular uncorrected visual acuity remained significantly (P = 0.01) different from baseline acuity 2 weeks after lens discontinuation., Conclusions: Full effect of overnight orthokeratology in low myopes is achieved within 1 week of initiating use of reverse-geometry lenses. Recovery after short-term use of reverse-geometry lenses is rapid for corneal thickness and corneal curvature. Refractive correction and binocular uncorrected visual acuity recovered fully after 2 weeks. Monocular uncorrected visual acuity was the slowest to recover and had not achieved full recovery after 2 weeks.

Background: Bicarbonate activated Soluble Adenylyl Cyclase (sAC) is a unique cytoplasmic and nuclear signaling mechanism for the generation of cAMP. HCO3- activates sAC in bovine corneal endothelial cells (BCECs), increasing [cAMP] and stimulating PKA, leading to phosphorylation of the cystic fibrosis transmembrane-conductance regulator (CFTR) and increased apical Cl- permeability. Here, we examined whether HCO3- may also regulate the expression of sAC and thereby affect the production of cAMP upon activation by HCO3- and the stimulation of CFTR in BCECs., Results: RT-competitive PCR indicated that sAC mRNA expression in BCECs is dependent on [HCO3-] and incubation time in HCO3-. Immunoblots showed that 10 and 40 mM HCO3- increased sAC protein expression by 45% and 87%, respectively, relative to cells cultured in the absence of HCO3-. Furthermore, 40 mM HCO3- up-regulated sAC protein expression in Calu-3 cells by 93%. On the other hand, sAC expression in BCECs and Calu-3 cells was unaffected by changes in bath pH or osmolarity. Interestingly, BCECs pre-treated with10 microM adenosine or 10 microM forskolin, which increase cAMP levels, showed decreased sAC mRNA expression by 20% and 30%, respectively. Intracellular cAMP production by sAC paralleled the time and [HCO3-]-dependent expression of sAC. Bicarbonate-induced apical Cl- permeability increased by 78% (P < 0.01) in BCECs cultured in HCO3-. However for cells cultured in the absence of HCO3-, apical Cl- permeability increased by only 10.3% (P > 0.05)., Conclusion: HCO3- not only directly activates sAC, but also up-regulates the expression of sAC. These results suggest that active cellular uptake of HCO3- can contribute to the basal level of cellular cAMP in tissues that express sAC.

Light scattering is an empirical technique employed to measure rapid changes in cell volume. This study describes a new configuration for the method of light scattering and its corroboration by measurements of cell height (as a measure of cell volume). Corneal endothelial cells cultured on glass cover-slips were mounted in a perfusion chamber on the stage of an inverted microscope. A beam of light was focused on the cells from above the stage at an angle of 40 degrees to the plane of the stage. The scattered light intensity (SLI), captured by the objective and referred to as forward light scatter (FLS), increased and decreased in response to hyposmotic and hyperosmotic shocks, respectively. The rapid increase and decrease in SLI corresponded to cell swelling and shrinkage, respectively. Subsequently, SLI decreased and increased as expected for a regulatory volume decrease (RVD) and increase (RVI), respectively. These data are in agreement with measurements of cell height, demonstrating that the method of light scatter in FLS mode is useful for monitoring rapid changes in cell volume of cultured cells. Changes in SLI caused by gramicidin were consistent with cell volume changes induced by equilibration of NaCl and KCl concentrations across the cell membrane. Similarly, an additional decrease in SLI was recorded during RVD upon increasing K+ conductance by valinomycin. Decreasing K+ conductance of the cell membrane with Ba2+ changed the time course of SLI consistent with the effect of the K+ channel blocker on RVD. Bumetanide and dihydro-ouabain inhibited increases in SLI during RVI. In conclusion, FLS is a valid method for qualitative analysis of cell volume changes with a high time resolution.

Fluid secretion by the corneal endothelium is associated with the net flux of HCO(3)(-) from basolateral (stromal) to apical (anterior chamber) sides of the tissue. In this study we asked if Na(+)/HCO(3)(-) cotransporter (NBC-1) protein expression and functional activity are present in freshly isolated human corneal endothelium. Immunoblot analysis using a polyclonal antibody to NBC-1 showed a single band at approximately 130 kDa. Indirect immunofluorescence indicated that NBC-1 is expressed on the basolateral, but not apical side of human corneal endothelium. RT-PCR was used to determine whether the kidney or pancreatic isoform of NBC-1 is expressed. Using the specific primers for pNBC and kNBC isoforms, RT-PCR showed that only pNBC could be detected in human corneal endothelium. The product was cloned and confirmed by sequencing. Full-length NBC-1 was also cloned from human corneal endothelium. This clone (hcNBC) is 100% identical to the longer, more common form of NBC [pNBC; 1079 amino acids (aa); 122 kDa in human heart, pancreas and prostate]. To test for functional activity of NBC-1, freshly isolated endothelium was loaded with the pH sensitive fluorescent dye BCECF and HCO(3)(-) fluxes were measured. HCO(3)(-) fluxes were Na(+)-dependent, electrogenic and H(2)-DIDS sensitive. We conclude that the long isoform of the sodium bicarbonate cotransporter (pNBC-1) is expressed on the basolateral side of fresh human corneal endothelium (hcNBC). The shorter form, kNBC, could not be detected. As in bovine corneal endothelium, hcNBC is instrumental in loading HCO(3)(-) into endothelial cells from the basolateral membrane.

Purpose: To achieve an optimal fit with reverse geometry Contex OK lenses and to determine a time course for and the stability of visual and corneal changes in achieving maximal refractive, corneal curvature, and corneal thickness changes after overnight wear of OK B and D series lenses., Methods: This investigation was conducted under an Food and Drug Administration IDE G000059. Both eyes of 10 subjects were fitted with the lenses, and uncorrected visual acuity, refractive correction, contrast sensitivity, corneal curvature, and corneal thickness were measured at baseline and at 1 day, 1week, 1 month, and 3 months after lenses were worn. Except for baseline, data were collected at four different times during the day, immediately following lens removal and 4, 8, and 12 hours after lens removal., Results: The results from eight subjects showed that uncorrected visual acuity, refractive correction, contrast sensitivity, and corneal curvature all changed significantly (P=0.01) overnight. By the end of 1 week, all corneal and visual changes had reached a maximal level and remained fairly stable during the day. These changes were sustained at 3 months. The epithelial thickness data from four subjects showed that the corneal epithelial thickness was reduced by approximately 19 microm after 3 months of lens wear., Conclusions: Successful fitting of OK B and D series lenses requires a thorough understanding of the lens-cornea relationship. Full effect of overnight orthokeratology is achieved by the end of 1 week. The visual and corneal changes remain stable for all waking hours of the day and allow patients to enjoy excellent device-free vision (20/20).

cAMP-dependent activation of the cystic fibrosis transmembrane conductance regulator (CFTR) regulates fluid transport in many tissues. Secretion by the corneal endothelium is stimulated by cAMP and dependent on HCO(3)(-). We asked whether HCO(3)(-) can secondarily increase CFTR permeability in bovine corneal endothelial cells (BCEC) by activating soluble adenylyl cyclase (sAC). Immunofluorescence suggests that sAC is distributed throughout the cytoplasm. HCO(3)(-) (40 mM) increased cAMP concentration 42% in the presence of 50 microM rolipram (a phosphodiesterase 4 inhibitor), and a standard HCO(3)(-) Ringer solution (28.5 mM) increased apical Cl(-) permeability by 78% relative to HCO(3)(-)-free solution. The HCO(3)(-)-dependent increase in Cl(-) permeability was reduced 60% by 20 mM NaHSO(3) (a weak agonist of sAC). NaHSO(3) alone increased apical Cl(-) permeability by only 13%. The HCO(3)(-)-dependent increase in Cl(-) permeability was reduced 57% in the presence of 50 microM Rp-adenosine 3',5'-cyclic monophosphorothioate, and 86% by 50 microM 5-nitro-2-(3-phenylpropyl-amino)benzoic acid but unaffected by 200 microM apical H(2)DIDS. CFTR phosphorylation was increased 23, 150, and 32% by 20 mM HSO(3)(-), 28.5 mM HCO(3)(-), and 28.5 mM HCO(3)(-) + 20 mM HSO(3)(-), respectively. Activation of apical Cl(-) permeability by 5 microM genistein was increased synergistically by HCO(3)(-) over that due to genistein and HCO(3)(-) alone. We conclude that HCO(3)(-)-stimulated sAC is a form of autocrine signaling that contributes to baseline cAMP production, thereby affecting baseline CFTR activity in BCEC. This form of autocrine signaling may be important in tissues that express sAC and exhibit robust HCO(3)(-) influx (e.g., ocular ciliary epithelium, choroid plexus, and airway epithelium).

Purpose: To determine if the variability in contact lens-induced corneal swelling is associated with variability in corneal oxygen consumption (Q(C)) or corneal endothelial function., Methods: Corneal swelling was induced in 30 non-contact lens wearers by 2 hours of closed-eye contact lens wear, using thick (oxygen transmission [Dk/t] = 4.0 x 10-9) and thin (Dk/t = 12 x 10-9) hydrogel lenses of identical design. Following the induction of swelling, corneal thickness was continually measured by pachymetry until open-eye steady-state (OESS) thickness was achieved. The percentage of recovery per hour was calculated as a measure of endothelial function. Tear oxygen tension (PO(2)) beneath the hydrogels was measured in the open eye and after 5 minutes of eye closure to obtain estimates of Q(C). A change in corneal pH during eye closure while wearing the hydrogels was used as a measure of hypoxic acidosis. Associations between corneal swelling and endothelial cell density or corneal epithelial thickness were also tested., Results: There were modest but significant (P < 0.05) correlations between thick-lens corneal swelling and thick-lens closed-eye PO(2) (r = -0.36); thin lens corneal swelling and thin-lens closed-eye and open-eye PO(2) (r = -0.40 and -0.39, respectively). Corneal swelling also increases with increasing Q(C) (r = + 0.29 to + 0.33). Corneal swelling was associated with the decrease in pH during lens wear (r = + 0.30 and + 0.20 for thick and thin lenses, respectively). Thick- and thin-lens corneal swelling was significantly associated with percentage of recovery per hour (r = -0.40 and -0.34, respectively). Multiple regression analysis of corneal swelling with PO(2) and percentage of recovery per hour suggested an additive effect, however the effects were not significant. There was a modest inverse association between corneal swelling and OESS, however there were no associations between corneal swelling and endothelial cell density or epithelial thickness., Conclusions: The variability in contact lens-induced corneal swelling is associated with both corneal metabolic activity (Q(C) and pH) and endothelial function (percentage of recovery per hour). Our interpretation is that individuals with larger Q(C) produce more lactic acid (i.e., more swelling) whereas stronger endothelial function resists swelling. The modest correlations, however, suggest that other factors also are involved in explaining the phenomenon of corneal swelling.

Corneal transparency is dependent on regulation of the hydration of the corneal stroma. Water is driven into the cornea across the epithelial and endothelial cell layers by the stromal swelling pressure. This fluid leak into the cornea is counterbalanced by the corneal fluid pump, which is predominantly attributed to the ion and fluid transport capacity of the endothelial cell layer. Primary and secondary active transport mechanisms are responsible for generating a net ion flux from the stromal to anterior chamber side of the endothelium; however, the identity and location of all the components of this transport system are not known. The endothelial fluid pump is dependent on the presence of Cl(-) and HCO(3)(-), and can be slowed by carbonic anhydrase inhibitors. A number of anion transport mechanisms have been identified and characterized in the endothelium, including basolateral Na(+)/2HCO(3)(-) cotransport, Na(+)/K(+)/2Cl(-) cotransport, Cl(-)/HCO(3)(-) exchange, and apical anion channels permeable to both Cl(-) and HCO(3)(-). Furthermore, there is evidence for a carbonic anhydrase mediated CO(2)-diffusive mode of apical HCO(3)(-) flux. These findings are incorporated into a new model of transendothelial anion transport, which suggests that there are a number of alternate pathways for anion transport. There have been few studies on activation of signal transduction pathways that could stimulate endothelial fluid transport. Interestingly, recent studies show that multiple autocrine signaling pathways are in place that could be upregulated during physical stimulation and may be responsible for maintaining basal levels of fluid secretion.

Purpose: To examine if the variability in hypoxic corneal swelling in non-contact lens wearing and contact lens wearing groups can be explained by the variability in corneal metabolic activity or endothelial function., Methods: Corneal thickness was measured by Orbscan before and after 2 hr of closed-eye hydrogel lens wear. De-swelling rates (percentage recovery per hour) were determined by continued corneal thickness measurements until open-eye steady-state thickness was reached. Corneal oxygen consumption was estimated by measuring tear oxygen tension while wearing hydrogels of known oxygen transmissibility. The change in stromal pH because of hypoxia was measured by ratio fluorescence of stromal fluorescein., Results: Corneal swelling was inversely correlated with open- or closed-eye tear oxygen tension while wearing the test hydrogels. Corneal swelling was directly correlated with the change in stromal pH during hypoxic lens wear. Unexpectedly, corneal swelling was also inversely correlated with the percent recovery per hour. These parameters could not explain all the swelling variability. The contact lens wearing group showed significantly less swelling and change in stromal pH with the same lenses. However, percent recovery per hour and tear oxygen tension was the same., Conclusion: Corneal swelling is associated with corneal metabolic activity and endothelial function; however, other factors must also be involved. Contact lens wearers do not adapt by modifying oxygen consumption but probably by using glucose more efficiently.

Purpose: We sought to develop a direct, noninvasive measurement of tear oxygen tension behind soft contact lenses, and to use the tear oxygen tension data to determine if subjects with greater comeal oxygen consumption experience more comeal edema under hypoxic conditions. The relationship between tear oxygen tension and comeal thickness was also analyzed to determine if thicker comeas have higher metabolic rates., Methods: Tear oxygen tension measurement was based on the quenching of a phosphorescent dye by oxygen. By coating a soft contact lens with the dye and placing it in the eye, the rate of decay of phosphorescence could be measured and the tear oxygen tension calculated. Corneal oxygen consumption could then be calculated from the tear oxygen tension. Central corneal swelling was measured after 3 hours of hypoxia induced by contact lens wear under closed-eye conditions., Results: Seven right eyes of seven subjects were tested. Baseline central corneal thickness ranged from 517 to 616 microns. After 3 hours of hypoxia, central corneal swelling ranged from 24.5 to 74.3 microns (3.9% to 13.4%). Steady-state open-eye tear PO2 behind the lens varied between 42.7 and 67.1 torr Pearson correlation suggested possible inverse relationship between open-eye tear oxygen tension and central comeal swelling during hypoxia (r = -0.711, p = 0.0734)., Conclusions: Phosphorescent-based measurements provided a measure of tear oxygen tension in human beings. Low tear film oxygen tensions (high corneal metabolic demand) appeared to predict greater amounts of swelling in subjects' corneas. No relation was found between corneal thickness and tear oxygen tension, suggesting that epithelial and endothelial metabolic rates are more important than stromal thickness in determination of comeal oxygen demand.

Purpose: Fluid transport by the corneal endothelium is dependent on the presence of HCO(3)(-) and the activity of carbonic anhydrase (CA)-II and -IV, the cytoplasmic and membrane-bound CAs, respectively. This study was conducted to examine the inhibition of CA activity in cultured bovine corneal endothelial cells (BCECs) by dorzolamide, a topical CA inhibitor used in glaucoma therapy., Methods: BCECs were grown on glass coverslips and then perfused with HCO(3)(-)-free Ringer's. The inward flux of CO(2) was induced by exposure to CO(2)-HCO(3)(-) Ringer's and the opposing outward flux by returning to HCO(3)(-)-free Ringer's. Consequent transients in intracellular pH (pH(i)) were measured using the pH-sensitive fluorescent dye 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF). During the inward flux of CO(2,) the maximum rate of change of pH(i) was taken as a quantitative measure of the overall CA activity in BCECs., Results: Exposure to CO(2)-HCO(3)(-) Ringer's led to a transient decrease in pH(i) (component A), followed by a rapid increase to a new steady state (component B). However, when the CO(2)-HCO(3)(-) Ringer's was removed, the pH(i) increased transiently (component C) and then rapidly returned to the original pH(i) (component D). Component A, caused by an inward flux of CO(2) and its subsequent hydration by CA-II, was blocked by dorzolamide in a dose-dependent manner with an 50% inhibitory concentration (IC)(50) of 2.4 micro M (95% confidence interval: 0.5 -10.85 microM). However, the inhibition of the outward flux of CO(2), inward flux of HCO(3)(-), and outward flux of HCO(3)(-) (associated with components C, B, and D, respectively) was not dose dependent. Cells that were exposed to 500 nM of the drug for longer than 30 minutes did not show a significantly greater inhibition of any of the components. Dorzolamide and acetazolamide (500 microM) did not show additive inhibition of any of the components (P = 0.13; n = 6)., Conclusions: Dorzolamide significantly inhibits CA activity in BCECs at micromolar levels. Because these levels are encountered in the cornea and aqueous humor after topical administration, dorzolamide may compromise corneal hydration control, especially when the functional reserve of corneal endothelium is low. Dorzolamide does not appear to accumulate in the cells, because the inhibition of CA-II did not increase after prolonged exposure to the drug.

Purpose: Acidic organelles, including Golgi bodies and lysosomes, are known to operate as Ca(2+) storage sites in many cell types. This study demonstrates the presence of Ca(2+) stores in lysosomes of bovine corneal endothelial cells (BCECs) and examines their interaction with Ins(1,4,5)P(3)-sensitive Ca(2+) stores., Methods: Glycyl-L-phenylalanine-beta-naphthylamide (GPN) was used to release Ca(2+) from lysosomes by inducing their selective osmotic swelling. Ca(2+) released into the cytoplasm was measured with fura-2 or fura-PE3 fluorescent dyes. Fluorescence of acridine orange (AO), which selectively sequesters into acidic organelles, was used to establish swelling of lysosomes in response to GPN., Results: Exposure to GPN (100-200 microM) in cultured BCECs produced an increase in free cytosolic Ca(2+) ([Ca(2+)](i)) equivalent to approximately 79% of the peak response to uridine triphosphate (UTP), a P2Y agonist (n = 19). The endothelium of the freshly isolated cornea also produced [Ca(2+)](i) transients similar to those in cultured BCECs; however, the peak [Ca(2+)](i) increase was smaller ( approximately 43% of the peak response to UTP; n = 13). In cultured BCECs, the response to UTP was unaffected by pretreatment with GPN with extracellular calcium ([Ca(2+)](o)) at 0 and 1.2 mM (n = 10). Neither pretreatment with thapsigargin (5 microM) nor with U73122 (a phospholipase C inhibitor; 10 microM) blocked the peak GPN response (n = 6). Exposure to 20 microM monensin produced a [Ca(2+)](i) increase with [Ca(2+)](o) at 0 and 1.2 mM and also reduced the subsequent peak response to GPN (n = 6)., Conclusions: GPN-sensitive lysosomal Ca(2+) stores, distinct from Ins(1,4,5)P(3)-sensitive Ca(2+) stores, are found in both cultured cells and fresh tissue. These stores are susceptible to depletion by the loss of the pH gradient across lysosomes and P2 agonists. The latter occurs through mechanisms independent of phospholipase C (PLC) activation or Ins(1,4,5)P(3). The GPN stores also induce [Ca(2+)](o) influx in response to their depletion.

Notwithstanding extensive efforts, the mechanism of capacitative calcium entry (CCE) remains unclear. Two seemingly opposed theories have been proposed: secretion-like coupling (Patterson, R. L., van Rossum, D. B., and Gill, D. L. (1999) Cell 98, 487-499) and the calcium influx factor (CIF) (Randriamampita, C., and Tsien, R. Y. (1993) Nature 364, 809-814). In the current study, a combinatorial approach was taken to investigate the mechanism of CCE in corneal endothelial cells. Induction of cytochrome P-450s by beta-naphthoflavone (BN) enhanced CCE measured by Sr(2+) entry after store depletion. 5,6-Epoxyeicosatrienoic acid (5,6-EET), a proposed CIF generated by cytochrome P-450s (Rzigalinski, B. A., Willoughby, K. A., Hoffman, S. W., Falck, J. R., and Ellis, E. F. (1999) J. Biol. Chem. 274, 175-182), induced Ca(2+) entry. Both BN-enhanced CCE and the 5,6-EET-induced Ca(2+) entry were inhibited by the CCE blocker 2-aminoethoxydiphenyl borate, indicating a role for cytochrome P-450s in CCE. Treatment with calyculin A (CalyA), which causes condensation of cortical cytoskeleton, inhibited CCE. The actin polymerization inhibitor cytochalasin D partially reversed the inhibition of CCE by CalyA, suggesting a secretion-like coupling mechanism for CCE. However, CalyA could not inhibit CCE in BN-treated cells, and 5,6-EET caused a partial activation of CCE in CalyA-treated cells. These results further support the notion that cytochrome P-450 metabolites may be CIFs. The vesicular transport inhibitor brefeldin A inhibited CCE in both vehicle- and BN-treated cells. Surprisingly, Sr(2+) entry in the absence of store depletion was enhanced in BN-treated cells, which was also inhibited by 2-aminoethoxydiphenyl borate. An integrative model suggests that both CIF from cytochrome P-450 metabolism and secretion-like coupling mechanisms play roles in CCE in corneal endothelial cells.

HCO-dependent fluid secretion by the corneal endothelium controls corneal hydration and maintains corneal transparency. Recently, it has been shown that mRNA for the cystic fibrosis transmembrane conductance regulator (CFTR) is expressed in the corneal endothelium; however, protein expression, functional localization, and a possible role in HCO transport have not been reported. Immunoblotting for CFTR showed a single band at approximately 170 kDa for both freshly isolated and primary cultures of bovine corneal endothelial cells. Indirect immunofluorescence confocal microscopy indicated that CFTR locates to the apical membrane. Relative changes in apical and basolateral chloride permeability were estimated by measuring the rate of fluorescence quenching of the halide-sensitive indicator 6-methoxy-N-ethylquinolinium iodide during Cl(-) influx in the absence and presence of forskolin (FSK). Apical and basolateral Cl(-) permeability increased 10- and 3-fold, respectively, in the presence of 50 microM FSK. FSK-activated apical chloride permeability was unaffected by H(2)DIDs (250 microM); however, 5-nitro-2-(3-phenylpropyl-amino)benzoic acid (NPPB; 50 microM) and glibenclamide (100 microM ) inhibited activated Cl(-) fluxes by 45% and 30%, respectively. FSK-activated basolateral Cl(-) permeability was insensitive to NPPB, glibenclamide, or furosemide but was inhibited 80% by H(2)DIDS. HCO permeability was estimated by measuring changes in intracellular pH in response to quickly lowering bath [HCO]. FSK (50 microM) increased apical HCO permeability by twofold, which was inhibited 42% by NPPB and 65% by glibenclamide. Basolateral HCO permeability was unaffected by FSK. Genistein (50 microM) significantly increased apical HCO and Cl(minus sign) permeability by 1.8- and 16-fold, respectively. When 50 microM genistein was combined with 50 microM FSK, there was no further increase in Cl(-) permeability; however, HCO permeability was reduced to the control level. In summary, we conclude that CFTR is present in the apical membrane of bovine corneal endothelium and could contribute to transendothelial Cl(-) and HCO transport. Furthermore, there is a cAMP-activated Cl(-) pathway on the basolateral membrane that is not CFTR.

Purpose: In this study, the involvement of signaling pathways in the regulation of HCO(3)(-) permeability across the apical membrane of the corneal endothelium was examined., Methods: Cultured bovine corneal endothelial cells (CBCECs) were grown to confluence on permeable membranes. Apical and basolateral sides were perfused with a HCO(3)(-)-rich Cl(-)-free Ringer's solution (28.5 mM; pH 7.5). Relative changes in apical HCO(3)(-) permeability were assayed by pulsing the apical perfusion bath with a low-HCO(3)(-) Cl(-)-free Ringer's solution (2.85 mM; pH 6.5), in the presence or absence of agonists or inhibitors, and comparing the rates of change in intracellular pH (pH(i)), as measured with a pH-sensitive dye. Ca(2+)-activated signaling was measured with the Ca(2+)-sensitive dye Fura-2. Qualitative changes in membrane potential (E(m)) were measured with a voltage-sensitive dye. RT-PCR using calcium-activated chloride channel (CLCA)-specific primers was used to examine the expression of CLCA in the corneal endothelium., Results: The adenoceptor agonist adenosine (20 M) enhanced HCO(3)(-) permeability by a factor of 2. Forskolin (40 microM) exerted a 6.3-fold increase of HCO(3)(-) permeability, which was inhibited by the Cl(-) channel blockers, glibenclamide (50 microM) and niflumic acid (100 microM). Adenosine triphosphate (ATP) and ATPgammaS, P(2) receptor agonists that increased intracellular Ca(2+) in corneal endothelium, enhanced HCO(3)(-) permeability by 87% and 79%, respectively. ATPgammaS induced depolarization of the E(m), consistent with anion channel activation, rather than activation of Ca(2+)-dependent K(+) channels, which could secondarily increase extrusion of anions by E(m) hyperpolarization. Cyclopiazonic acid (CPA), an endoplasmic reticulum (ER) Ca(2+)-pump inhibitor that increased [Ca(2+)](i), also enhanced HCO(3)(-) permeability by 95%. Both the calmodulin kinase II (CaMKII) inhibitor KN-62 and the PKC inhibitor bisindolylmaleimide I (BIMI), decreased HCO(3)(-) permeability induced by ATPgammaS. The PKC activator PMA also increased HCO(3)(-) permeability by a factor of 1.8. RT-PCR using CLCA-specific primers showed the expression of CLCA1 in both fresh and cultured BCECs., Conclusions: Activation of adenoceptors and purinoceptors enhances HCO(3)(-) permeability across the apical membrane of the cultured corneal endothelium. Multiple signaling pathways (PKA, PKC, and Ca(2+)/CaMKII) contribute to the HCO(3)(-) transport in cultured corneal endothelium. Both cAMP and Ca(2+)-activated Cl(-) channels (possibly CLCA) may be involved in HCO(3)(-) transport.

Purpose: To devise a procedure for direct estimation of corneal oxygen consumption in human subjects., Methods: Tear oxygen tension (PO2) was measured at the posterior surface of two standard hydrogel contact lenses (38% water, 0.2 and 0.06 mm thick, oxygen transmissibility [Dk/t] = 4.2 and 14 x 10(-9) cm x mL O2/mL x sec x torr) and one newly available hydrogel-silicone polymer lens (Dk/t = 99 x 10(-9)). The oxygen-sensitive dye, Pd-meso-tetra (4-carboxyphenyl) porphine, bound to bovine serum albumin, was incubated with the lenses overnight. The lenses, coated with the protein-dye complex, were placed on four subjects' eyes, and tear PO2 was measured in the open eye and after 5 minutes of eye closure, using a time-domain phosphorescence measurement system. Given the tear PO2, lens Dk/t, and corneal thickness, oxygen consumption (Q(C), in mL O2/cm(3) x sec) could be calculated from established oxygen diffusion models., Results: Protein-dye complex bound to the lens surface enabled reporting of tear PO2 for long periods. As expected, estimated tear PO2 was higher in subjects wearing lenses with higher Dk/t: mean open-eye PO2 = 30.6 +/- 3.1 and 8.1 +/- 1.3 torr for the thin and thick hydrogel lenses, respectively, and 97.6 +/- 22.9 torr for the hydrogel-silicone lens. After 5 minutes of eye closure, tear PO2 was significantly reduced and reached a new steady state in approximately 20 seconds after eye opening. Fitting a single exponential model to the data and extrapolating to t = 0 provided an estimate of PO2 under the closed lid for the thin hydrogel (PO2 = 7 +/- 2.3 torr) and the hydrogel-silicone lens (PO2 = 22.6 +/- 4 torr). After 5 minutes of eye closure with the thick hydrogel lens, tear PO2 remained constant for approximately 10 seconds after eye opening (mean PO2 = 3.9 +/- 0.7) before increasing to a new steady state. This delay could be accounted for by the time needed for oxygen to diffuse to the posterior surface of the lens. Calculated Q(C) ranged from 2.2 x 10(-4) to 3.7 x 10(-6) mL O2/cm(3) x sec) at the highest and lowest PO2s, respectively, and is comparable to previous in vitro and in vivo estimates., Conclusions: Tear PO2 behind hydrogel lenses can be measured in human subjects using the phosphorescence of the porphyrin-protein complex bound to the lens surface. The method is simple, fast, reliable, and noninvasive, allowing quick and direct estimates of Q(C). In addition to contact lens wear, this method should be useful for examining the effects of disease, surgery, or topical drugs on the corneal oxygen consumption rate.

Contact lens wear can cause a number of physiological changes in the cornea. Two areas of interest in my laboratory have been contact lens effects on the endothelium and, more recently, the role of metabolic activity in predicting corneal swelling. The first part of this review focuses on the function of the corneal endothelium, the nature of its fluid pump, and the effects of contact lens-induced hypoxia and corneal pH changes on corneal endothelial function. In the second part, the etiology of hypoxia-induced corneal swelling is reviewed in relation to new studies on the causes of intersubject corneal swelling variability. The results indicate that corneal swelling is influenced by both corneal metabolic activity and endothelial function.

Purpose: Corneal endothelial fluid transport is dependent on HCO(3)(-) and CO(2) fluxes. CO(2) permeability (P:CO(2)) measurements in an oocyte expression system and in reconstituted proteoliposomes have suggested that the water channel AQP1 can transport CO(2). An AQP1 knockout mouse model, however, showed no evidence for CO(2) transport through AQP1 in erythrocytes or lung. Because HCO(3)(-) and CO(2) fluxes are essential to endothelial function, the current study was conducted to determine whether AQP1 expression levels in confluent cultures of bovine corneal endothelial cells (BCECs) affects membrane PCO(2)., Methods: BCEC endogenous AQP1 expression was reduced by antisense oligonucleotide (AO) transfection or adenoviral antisense-AQP1 (AV) infection. AQP1 was overexpressed by adenoviral sense-AQP1 (SV) infection, which directs expression of recombinant AQP1., Results: Expression of AQP1 and osmotic water permeability (control P(f) = 0.046 +/- 0.005 cm/sec) were reduced 45% and 36.5%, respectively, by AO transfection and reduced 67% and 49%, respectively, by AV infection. SV infection induced a more than threefold overexpression of AQP1 but showed only a 37% increase in P(f). Adenoviral empty virus (EV) infection did not change AQP1 expression or P(f). PCO(2) was determined by measuring the rate of intracellular pH decrease after exposure to CO(2)/HCO(3)(-)-rich solutions, as measured by the pH-sensitive fluorescent dye 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF). Apparent PCO(2) of BCEC (0.0036 +/- 0.00023 cm/sec) was not different among control, oligonucleotide-transfected, and adenoviral-infected cells. P(f) could also be reduced more than 50% by 3 to 5 minutes' exposure of control cells to 0.5 mM p-chloromercuriphenylsulfonic acid (pCMBS), but this had no effect on rates of intracellular pH decrease., Conclusions: AQP1 does not contribute to PCO(2) in corneal endothelial cells.

Purpose: Chloride and bicarbonate are necessary for maintenance of fluid transport by the corneal endothelium, however there is little information on the identity of anion transport proteins that could serve as anion efflux mechanisms in endothelial cells. Therefore, we ask whether mRNA for the anion transport related proteins, CFTR, CLC-2, ClC-3, ClC-5 and AE2, are expressed in human, bovine or rabbit corneal endothelium., Methods: RT-PCR was performed for CFTR, CLC-2, ClC-3, ClC-5 and AE2 using total RNA from fresh human, bovine and rabbit corneal endothelium as well as cultured bovine corneal endothelial cells (CBCEC). Specificity of PCR products was confirmed by sequencing., Results: RT-PCR analysis gave positive bands at the predicted size for CLC-3 and CLC-5 from fresh human, rabbit and bovine as well as CBCEC. However, for CLC-2, no band was apparent around the predicted size from fresh and cultured corneal endothelium. A band at the predicted size was obtained for CFTR from fresh human, rabbit and bovine endothelium, as well as from CBCEC. RT-PCR analysis for AE2 produced specific bands from fresh human, rabbit and bovine corneal endothelium, but no positive band was obtained from CBCEC. Sequencing analysis further confirmed the identities of CLC-3, CLC-5, CFTR and AE2 in corneal endothelium., Conclusions: CFTR, CLC-3 and ClC-5 are expressed in fresh and cultured corneal endothelial cells. However, consistent with previous immunoblots studies, AE2 is only expressed in fresh corneal endothelium. These results have implications for modeling possible apical anion efflux mechanisms in corneal endothelium.

Functional studies support the presence of the Na(+)-HCO(3)(-) cotransporter (NBC) in corneal endothelium and possibly corneal epithelium; however, molecular identification and membrane localization have not been reported. To test whether NBC is expressed in bovine cornea, Western blotting was performed, which showed a single band at approximately 130 kDa for freshly isolated and cultured endothelial cells, but no band for epithelium. Two isoforms of NBC have recently been cloned in kidney (kNBC) and pancreas (pNBC). RT-PCR was run using cultured and fresh bovine corneal endothelial and fresh corneal epithelial total RNA and specific primers for kNBC and pNBC. RT-PCR analysis for pNBC was positive in endothelium and weak in epithelium. The RT-PCR product was subcloned and confirmed as pNBC by sequencing. No specific bands for kNBC were obtained from corneal cells. Indirect immunofluorescence and confocal microscopy indicated that NBC locates predominantly to the basolateral membrane in corneal endothelial cells. Furthermore, Na(+)-dependent HCO(3)(-) fluxes and HCO(3)(-)-dependent cotransport with Na(+) were elicited only from the basolateral side of corneal endothelial cells. Therefore, we conclude that pNBC is present in the basolateral membrane of both fresh and cultured bovine corneal endothelium and weakly expressed in the corneal epithelium.

Purpose: To examine whether Na(+)-K(+)-2Cl(-) cotransport has the potential to contribute to corneal endothelial ion and fluid transport in cultured and fresh bovine corneal endothelial cells., Methods: Cl- and Na+ sensitive fluorescent dyes were used to measure furosemide-dependent ion fluxes in cultured and fresh endothelial cells. Immunoblot analysis and immunofluorescence were used to determine expression and location of the Na(+)-K(+)-2Cl(-)cotransporter (NKCC1)., Results: Application of furosemide (50-100 microM) reduced Cl- and Na+ influx in approximately 50% of trials using cultured cells and only 10% of trials with fresh cells; however, in all cases pretreatment with furosemide slowed Cl- efflux when cells were bathed in Cl(-)-free Ringer's. Double-sided perfusion of cultured cells indicated that furosemide-sensitive Cl- fluxes were located on the basolateral side. Immunoblot analysis revealed 174-kDa bands in both fresh and cultured cells, but the bands were denser in fresh endothelial cells. Immunofluorescence showed distinct lateral membrane staining in addition to significant amounts of perinuclear staining., Conclusions: The Na(+)-K(+)-2Cl(-) cotransporter is present in both fresh and cultured bovine corneal endothelium, and the expression is apparently higher in the fresh cells. The cotransporter is present on the lateral membrane consistent with a role in loading endothelial cells with Cl-, thereby possibly contributing to a transendothelial Cl- flux. However, in the resting cell, net flux through the transporter is often not apparent.

Purpose: (1) To update Fatt's mathematical model of the distribution of oxygen tension (pO2) across the cornea and contact lens (CL) to include the recent finding that corneal oxygen consumption increases with the acidification that occurs with CL wear. (2) To estimate the minimum transmissibility (CL Dk/t) to avoid epithelial anoxia or to avoid stromal anoxia., Methods: A five-layer static and one-dimensional mathematical model of oxygen diffusion through the cornea based on Fatt's models was used. The relationships between acidosis and increased QO2, and acidosis and CL Dk/t were used to estimate corneal QO2 for a given CL Dk/t., Results: (1) Revised model predictions are in agreement with direct tear pO2 measurements beneath CLs in the rabbit. (2) For the human eye, the minimum CL Dk/t for oxygen delivery to the basal epithelial cells was determined to be 23 for the open eye and 89 for the closed eye. To prevent anoxia throughout the entire corneal thickness the Dk/t requirements are 35 for the open eye and 125 for the closed eye., Conclusions: (1) Model predictions of the oxygen distribution beneath contact lenses are significantly lower than previous models that did not include the effect of acidosis on corneal QO2. (2) Minimum Dk/t values that allow oxygen delivery to the basal epithelium are in agreement with the Dk/t needed to avoid corneal edema.

Corneal endothelial function is dependent on HCO3- transport. However, the relative HCO3- permeabilities of the apical and basolateral membranes are unknown. Using changes in intracellular pH secondary to removing CO2-HCO3- (at constant pH) or removing HCO3- alone (at constant CO2) from apical or basolateral compartments, we determined the relative apical and basolateral HCO3- permeabilities and their dependencies on Na+ and Cl-. Removal of CO2-HCO3- from the apical side caused a steady-state alkalinization (+0.08 pH units), and removal from the basolateral side caused an acidification (-0.05 pH units). Removal of HCO3- at constant CO(2) indicated that the basolateral HCO3- fluxes were about three to four times the apical fluxes. Reducing perfusate Na+ concentration to 10 mM had no effect on apical flux but slowed basolateral HCO3- flux by one-half. In the absence of Cl-, there was an apparent increase in apical HCO3- flux under constant-pH conditions; however, no net change could be measured under constant-CO2 conditions. Basolateral flux was slowed approximately 30% in the absence of Cl-, but the net flux was unchanged. The steady-state alkalinization after removal of CO2-HCO3- apically suggests that CO2 diffusion may contribute to apical HCO3- flux through the action of a membrane-associated carbonic anhydrase. Indeed, apical CO2 fluxes were inhibited by the extracellular carbonic anhydrase inhibitor benzolamide and partially restored by exogenous carbonic anhydrase. The presence of membrane-bound carbonic anhydrase (CAIV) was confirmed by immunoblotting. We conclude that the Na+-dependent basolateral HCO3- permeability is consistent with Na+-nHCO3- cotransport. Changes in HCO3- flux in the absence of Cl- are most likely due to Na+-nHCO3- cotransport-induced membrane potential changes that cannot be dissipated. Apical HCO3- permeability is relatively low, but may be augmented by CO2 diffusion in conjunction with a CAIV.

Swelling induced enhancement of anion permeability was investigated using the halide-sensitive fluorescent dye SPQ in cultured bovine corneal endothelial cells (BCEC). Rates of anion influx were quantified in terms of the rate of change of SPQ fluorescence during exposure to short duration pulses of Cl-, I-or NO3-while the cells were being perfused with I-, NO3-or Cl-Ringer, respectively. Since SPQ fluorescence is quenched to different extents by these anions, their influx or efflux causes significant changes in fluorescence. The ratio of the maximum rate of change of fluorescence during the pulse period under hyposmotic conditions to that under isosmotic conditions, referred to as the enhancement ratio (ER), was calculated as a measure of the increase in anion permeability. When cells were perfused with NO3-Ringer, exposure to I-pulses yielded an ER=9.0+/-2.6 for 110+/-5 mosmhyposmotic shock. This was higher than with Cl-/I-(6.4+/-0.7) or NO3-/Cl-(3.2+/-0.8) anion-pairs for the same level of shocks. In all cases, the enhancement occurred within approximately 100 seconds after swelling but decreased with continued progress of regulatory volume decrease (RVD). ER returned to approximately 1 within 4 minutes after returning to isosmotic conditions. The membrane potential (Em) depolarized immediately after hyposmotic shock. When cells were depolarized prior to the shocks by high [K+], changes in Emwere relatively small. ER, for the NO3-/I-anion-pair, was significantly reduced by DIDS (100% at 500 microm), NPPB ( approximately 80% at 100 microm) and tamoxifen (approximately 85% at 12 microm). Tamoxifen and NPPB also inhibited swelling induced depolarization. Increasing cationic conductance with Gramicidin D at approximately 2 minutes following hyposmotic shock induced NPPB-inhibitable secondary swelling or accelerated RVD under normal or low Na+conditions, respectively. These results demonstrate that BCEC express swelling activated Cl-channels, which facilitate RVD by enhancing anionic permeability and also by providing a favorable electrical gradient for K+efflux., (Copyright 1999 Academic Press.)

Purpose: To determine whether corneal acidosis, which occurs during contact lens wear, alters corneal O2 consumption (QO2) and if so, whether increased ion transport activity could contribute to altered QO2 during acidosis., Methods: PO2 was measured, using the phosphorescence quenching of Pd-meso-tetra-(4-carboxyphenyl) porphine, in an airtight chamber that held a trephined rabbit cornea. The rate of change in chamber PO2 was used as a measure of QO2. QO2 was measured at pH 7.5 and then at either pH 6.7, 7.1, or 7.3. Measurements of QO2 at pHs 7.5 and 6.7 were repeated in the presence of 0.5 mM amiloride and 0.5 mM ouabain., Results: When pH was changed from 7.5 to 6.7, 7.1, or 7.3, O2 consumption increased by a factor of 1.80+/-0.11 (+/-SE), 1.65+/-0.12, and 1.44+/-0.06, respectively. Amiloride (0.5 mM) and ouabain (0.5 mM) inhibited 50% and 65%, respectively, of the increase in QO2 at pH 6.7., Conclusions: Corneal acidosis leads to increased QO2 in a dose-dependent manner. The increased QO2 is in part secondary to the activation of pH regulatory mechanisms, including Na+/H+ exchange, which then stimulates Na+/ K+-ATPase activity. These findings indicate that contact lens-induced acidosis can exacerbate corneal hypoxia and related complications.

Purpose: To determine the apical versus basolateral polarity of the putative anion exchanger in cultured bovine corneal endothelial cells (BCECs) and to examine the influence of Cl--dependent membrane potential (Em) changes on HCO3- transport., Methods: BCECs grown on permeable supports were used for independent perfusion of apical and basolateral surfaces. Intracellular pH (pHi) was measured using the fluorescent dye BCECF. Relative changes in Em were measured using the fluorescent dye bis-oxonol. Western blot analysis was used to detect immunoreactivity against the anion exchanger (AE1 or AE2)., Results: Cl- removal from apical and basolateral surfaces produced cellular alkalinization (apical side, 0.07 pH units; basolateral side, 0.06 pH units; both sides, 0.20 pH units). Application of 100 microM H2-4,4'-diisothiocyanatodihydrostilbene-2,2'-disulfonic acid (DIDS), an anion exchange inhibitor, on the apical side produced an alkalinization (0.02 pH units) followed by acidification (-0.05 pH units), whereas basolateral H2DIDS caused a substantial acidification (-0.16 pH units). In the absence of Na+, Cl- removal from the apical side caused a transient alkalinization (0.03 pH units) followed by a return to baseline; Cl- removal from the basolateral side caused a small (-0.03) acidification. In Na+-free Ringer, apical H2DIDS produced a transient alkalinization (0.02 pH units), whereas basolateral exposure had no effect. 5-Nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), N-phenylanthranilic acid (DPC), and niflumic acid (50-200 microM), known Cl- channel blockers, produced cellular acidification in control Ringer. Niflumic acid hyperpolarized Em and inhibited depolarization after Cl- removal. Western blot analysis failed to detect AE2 expression in cultured BCECs. However, fresh BCECs produced a trace response., Conclusions: Physiological activity of an apical anion exchanger is weak in cultured BCECs. Cultured BCECs have significant Cl- conductance. Thus, cellular alkalinization after Cl- removal is caused primarily by depolarization of Em, which drives HCO3- influx through the basolateral electrogenic Na+:nHCO3- cotransporter. In contrast with cultured BCECs, AE2 may be present in fresh cells.

Purpose: To characterize Ca2+ mobilization by P2 receptors in the bovine corneal endothelial cells (BCEC)., Methods: Changes in intracellular Ca2+ ([Ca2+]i) were measured by fluorescence imaging of cultured and fresh BCEC cells loaded with the Ca2+-sensitive dye Fura-PE3. Relative rates of Ca2+ influx were measured employing Mn2+ as a surrogate for Ca2+., Results: Exposure of cultured cells to uridine 5'-triphosphate (UTP), 2-methyl-thio ATP (msATP) and ATP caused biphasic changes in [Ca2+]i consisting of a peak followed by a plateau phase. Based on the peak responses to 100 microM agonist, the magnitude of UTP responses were similar to that of ATP but greater than that of msATP or ADP. UTP and msATP stimulated Mn2+ influx following [Ca2+]i peak similar to that observed in response to cyclopiazonic acid (CPA), an inhibitor of ER Ca2+-ATPase. Under Ca2+-free conditions, peak responses were similar to those in the presence of external Ca2+, but reduced when the cells were pre-exposed to CPA. Reactive Blue-2 (RB2), inhibited msATP responses by 60.4 +/- 18.8% but UTP responses by only 10.6 +/- 9.5%. Repeated exposures to UTP or msATP reduced [Ca2+]i mobilization indicating homologous desensitization. Response to UTP was not affected by a prior exposure to msATP. However, response to msATP was reduced by a prior exposure to UTP indicating mixed heterologous desensitization. Fresh cells responded to UTP (50 microM) with temporal characteristics of [Ca2+]i mobilization similar to that of cultured cells., Conclusion: BCEC express P2 receptors belonging to the P2Y subfamily. The emptying of the IP3-sensitive stores, leading to the initial peak in [Ca2+]i response, subsequently caused capacitative Ca2+ influx leading to the onset of the plateau phase. A significant homologous desensitization to UTP and msATP, selective heterologous desensitization between UTP and msATP, and selective inhibition by RB2 indicate the coexistence of multiple P2Y receptors.

This study describes a quantitative analysis of the enhancement in anion permeability through swelling-activated Cl- channels, using the halide-sensitive fluorescent dye 6-methoxy-N-(3-sulfopropyl)quinolinium (SPQ). Cultured bovine corneal endothelial monolayers perfused with NO3- Ringer's were exposed to I- pulses under isosmotic and, subsequently, hyposmotic conditions. Changes in SPQ fluorescence due to I- influx were significantly faster under hyposmotic than under isosmotic conditions. Plasma membrane potential (Em) was -58 and -32 mV under isosmotic and hyposmotic conditions, respectively. An expression for the ratio of I- permeability under hyposmotic condition to that under isosmotic condition (termed enhancement ratio or ER) was derived by combining the Stern-Volmer equation (for modeling SPQ fluorescence quenching by I-) and the Goldman flux equation (for modeling the electrodiffusive unidirectional I- influx). The fluorescence values and slopes at the inflection points of the SPQ fluorescence profile during I- influx, together with Em under isosmotic and hyposmotic conditions, were used to calculate ER. Based on this approach, endothelial cells were shown to express swelling-activated Cl- channels with ER = 4.9 when the hyposmotic shock was 110 +/- 10 mosM. These results illustrate the application of the SPQ-based method for quantitative characterization of swelling-activated Cl- channels in monolayers.

Background: Fluorophotometry can be used to quantify changes in epithelial permeability (Pdc), corneal pH, and tear exchange (T95; time to deplete 95% of a fluorescent dye from beneath a contact lens) associated with contact lens wear. Using fluorometric procedures, we present previously reported data in order to review the effects of contact lens wear on Pdc and pH. We also introduce a new method for measuring tear exchange beneath a soft contact lens and present preliminary data., Methods: Pdc was assessed on 32 subjects after 1 h of closed-eye soft contact lens wear. Stromal pH was assessed on 21 subjects wearing lenses with a range of different oxygen transmissibilities (Dk/L). T95 was assessed on 7 subjects who wore disposable lenses. Pdc estimates were derived by measuring the rate at which topically applied fluorescein crossed the epithelial barrier from the tears; corneal pH was quantified by alternately exciting a fluorescein-loaded cornea with two wavelengths of blue light and calculating the fluorescence intensity ratio (490/450 nm), which is pH-sensitive; and the T95 was estimated by applying 2 microliters of FITC-Dextran to the posterior surface of a soft contact lens and the monitoring the exponential decay of dye under the lens., Results: On average, 1 h of closed-eye contact lens wear caused a 41% increase in Pdc compared to the control eye. Corneal pH varied directly with a decrease in Dk/L. On average the T95 under contact lenses (mean = 29 min) was slow compared to normal rates with no lens (approximately 5 min)., Conclusions: Fluorophotometry can be used to quantify some interesting effects associated with contact lens wear, and the use of these techniques may provide new information about the impact of contact lens wear on corneal structure and function.

Purpose: Tear oxygen tension beneath contact lenses, measured in a previous study, was found to be lower than mathematical model predictions. In this study, the authors developed a phosphorescence-based technique for measuring corneal O2 consumption (QO2) to determine whether errors in previous determinations of QO2 could explain the discrepancy between measured and predicted tear oxygen tension., Methods: Corneal oxygen consumption is measured using the phosphorescence quenching of Pd-meso-tetra-(4-carboxyphenyl)porphine by oxygen. Oxygen tension of a pH 7.5 Ringer's solution is measured in an airtight chamber that holds an excised rabbit cornea, and QO2 is calculated from the decreased PO2 in the stirred chamber solution. QO2 rates are measured for the whole trephined rabbit cornea with the epithelium, the endothelium, or both, removed, which makes possible the estimation of the consumption rates of the epithelium, stroma, and endothelium., Results: Control experiments indicated that the cornea QO2 was constant for 3 hours and that exposure to 2,4-dinitrophenol increased QO2. Mean QO2 rates (microliters of O2/cm2 per hour) were: whole cornea, QO2 = 7.53; epithelium, QO2 = 3.73; stroma, QO2 = 2.97; and endothelium, QO2 = 0.86., Conclusions: Phosphorescence-based measurements can be used to determine the QO2 of the component layers of the cornea. Estimates of QO2 in this study were similar to previous measurements, indicating that the discrepancy of tear PO2 measures and model predictions are not a result of errors in the QO2 measurements at pH 7.5.

Purpose: Hypoxic corneal swelling in human subjects is reduced by CO2 induced corneal acidosis. We investigated whether this effect could be explained by a reduction in lactate production under acidic conditions., Methods: Lactate production from isolated bovine corneas was assayed under normoxic and hypoxic conditions at normal pH (7.5) and low pH (7.05). Low pH was achieved using a CO2/HCO3- Ringer or a HEPES buffered Ringer., Results: Normoxic corneas produced significantly less (approximately 25%) lactate under all acidic conditions. In contrast, hypoxic corneas produced the same or slightly less lactate under acidic conditions (weighted average of all conditions = 6% less lactate)., Conclusions: From these results, we concluded that pH can have a substantial effect on lactate production, but its influence on glycolytic activity during hypoxia is suppressed and may be negligible.

Ion coupled fluid transport by the corneal endothelium is stimulated by adenosine through a cAMP dependent mechanism. This study examines if anion conductance is enhanced by cAMP and, hence by adenosine. Cl- fluxes, measured by changes in fluorescence of the Cl- sensitive dye SPQ, following removal or re-addition of Cl- Ringer, could be accelerated by 20 microM forskolin or 10 microM adenosine. The cAMP cocktail (20 microM forskolin + 100 microM IBMX + 100 microM cpt-cAMP) had no effect on resting [Cl-]i. However, when Cl- influx was inhibited by 100 microM furosemide, net Cl- efflux was observed in response to the cAMP cocktail. Exposure to the cAMP cocktail alone depolarized the resting membrane potential. Conversely, the cAMP cocktail caused a relative hyperpolarization in cells which had been previously depolarized beyond the equilibrium potential for Cl- (ECl-), by application of 1 microM Gramicidin D. cAMP dependent changes in membrane potential could be inhibited by 50 microM NPPB, but not by 200 microM DPC, 100 microM H2DIDS or 50 microM glibenclamide. Taken together, these results are consistent with NPPB-sensitive, cAMP activated Cl- channels. To examine if these channels are permeable to HCO3-, changes in pHi in response to the cAMP cocktail were measured in acidified and depolarized cells in the absence of Na+. The cAMP cocktail caused an increase in pHi only when HCO3- was present, consistent with HCO3- influx. In control HCO3- Ringer, the cAMP cocktail caused a transient decrease in pHi, which could not be accounted for by inhibition of Na+:nHCO3- cotransport or stimulation of Cl-/HCO3- exchange. These results are consistent with conductive HCO3- efflux through cAMP activated channels. We conclude that cultured bovine corneal endothelial cells possess cAMP activated anion channels.

The intracellular fluorescence of 6-methoxy-N-(3-sulfopropyl)-quinolinium (SPQ), a Cl(-) -sensitive fluorescent dye, is quenched by intracellular organic anions and proteins of unknown identity. The concentration of these intracellular quenchers (ICQs), however, is dependent on cell volume. In the absence of Cl-, changes in the observed SPQ fluorescence may therefore reflect changes in cell volume. This concept has been applied to determine relative changes in cell volume of cultured corneal endothelium in response to anisosmotic shocks, using NO3- as the Cl- substituent. SPQ fluorescence increased with decreasing osmolarity and vice versa. A 20 mosM hypertonic shock was needed to detect a change in SPQ fluorescence with a signal-to-noise ratio of >25. Assuming dynamic quenching by ICQs, we applied an extension of the Stern-Volmer equation to develop a simple relationship between the measured SPQ fluorescence and relative changes in cell volume. For large hyposmotic shocks, regulatory volume decrease (RVD) was observed. The rate of RVD could be enhanced by exposure to 0.5 microM gramicidin in low-Na+ Ringer solution (i.e., K+-NO3- efflux), indicating that K+ conductance is rate limiting for RVD. These results demonstrate the principle of using fluorescence quenching to measure changes in cell volume in real time. Because SPQ is sensitive to Cl-, its usefulness as a quenching probe is limited. However, a structure-activity study can be expected to yield useful Cl(-)-insensitive analogs.

Purpose: Previous studies have shown that contact lens oxygen transmissibility correlates with binding of Pseudomonas aeruginosa to the rabbit cornea after overnight lens wear. Studies of human lens wear stratified by oxygen transmissibility will be required to validate these animal results. In humans, bacterial binding to shed cells obtained through corneal irrigation cytology may provide an indirect measure of in vitro binding. The purpose of this study was to establish the relationship between binding to shed cells and to the residual corneal surface in an animal model of lens wear prior to initiation of human studies., Methods: The test contact lenses used were: rigid lens A (Dk/L = 10 x 10(-9) [cm/ sec][mL O2/mL mmHg]); rigid lens B (Dk/L = 97); soft lens A (Dk/L = 9); soft lens B (Dk/L = 20); and, soft lens C (Dk/L = 39). There were six rabbits in each group, except for the soft lens C group, which had seven rabbits. After overnight lens wear, the corneal surface was irrigated with a corneal irrigation chamber to collect surface cells before exposure to a bacterial suspension (1 x 10(7) CFU/mL) for 30 minutes. The number of bacteria adherent to the residual corneal surface was then assessed by CFU determination. Cells collected from the corneal surface (9 mL) were incubated with 1 mL bacterial suspension containing 10(8) (CFU/mL) for 30 minutes. The number of bacteria adherent to shed cells was assessed by staining with acridine orange and direct counting by epifluorescence microscopy., Results: The differences in the number of bacteria adhering to shed epithelial cells between the treated and the control eyes were 2.90 +/- 1.20 and 0.23 +/- 0.41 for rigid lenses A and B, respectively, and 5.97 +/- 1.54, 3.67 +/- 2.32, and 0.90 +/- 1.45 (bacterial/cell) for soft lenses A, B, and C, respectively. Overnight contact lens wear induced a significant increase in bacterial binding to shed corneal epithelial cells for rigid lens A and for soft lenses A and B. There were significant differences among lens groups (P = 0.00017, ANOVA), with significant differences between rigid lenses A and B, soft lenses A and C, and soft lenses B and C. The binding of bacteria to shed cells was significantly correlated with the binding of bacteria to the residual corneal surface, both confirming and extending previous results (R = 0.78, P < 0.001)., Conclusion: These results demonstrate a positive correlation between P. aeruginosa adherence to shed corneal cells and to the residual corneal surface in the rabbit eye following contact lens wear. In light of the results from prior animal studies, examination of the behavior of P. aeruginosa binding to exfoliated cells appears to be a promising and valid method for future assessment of similar lens-induced increases in bacterial binding in prospective human clinical studies.

Purpose: To develop a noninvasive measurement of tear oxygen tension and to determine tear oxygen tension and calculated oxygen flux rates during wear of contact lenses of varying oxygen transmissibility (Dk/L)., Methods: The basis of the tear oxygen tension (PO2) measurement is phosphorescence quenching of Pd-meso-tetra-(4-carboxyphenyl) porphine by oxygen. A fiber-optic bundle, positioned approximately 2 mm from a cuvette or from the corneal surface, delivers an excitation flash (< 4 microseconds, 539 +/- 23 nm) and collects the phosphorescence emission (> 645 nm) of the dye at 1 MHz for 2 ms. Porphine (500 microM) + 1% albumin is instilled directly into the tears of a sedated rabbit, and phosphorescence is measured before and after contact lens insertion., Results: Phosphorescence intensity decay lifetimes are related inversely to PO2 and follow the Stern-Volmer relationship. Calibration control experiments revealed a temperature effect on lifetime of 1% per degree, dye concentration independence over a 100-fold dilution, and no significant pH effect between 6.8 and 7.9. Phosphorescence lifetimes indicated that tear PO2 beneath contact lenses reached steady state within 3 to 8 minutes. Mean steady state tear PO2 (mm Hg +/- SE; N > 7) beneath contact lenses was: polymethylmethacrylate (Dk/L = 0), tear PO2 = 0.58 +/- 0.22; Oxyflow f30 (Dk/L = 15.3), tear PO2 = 27.7 +/- 3.4; Fluorex 700 Dk/L = 30), tear PO2 = 52.5 +/- 3.8; Oxyflow 151 (Dk/L = 51.9), tear PO2 = 78.7 +/- 23.9. Steady state tear PO2 beneath an Oxyflow f30 after application of topical anesthetic and 10 minutes of lens wear was 41 +/- 1.2 mm Hg, significantly higher than without anesthetic., Conclusions: Phosphorescence-based measurements allow the direct, sensitive, and noninvasive assessment of oxygen availability beneath a contact lens. Oxygen levels beneath permeable contact lenses found by direct measurement are significantly lower than what mathematical models predict. The measured values, however, are close to PO2 estimates using the equivalent oxygen percentage (EOP) technique. Tear PO2 measurements may be used to examine metabolic disturbances as shown by reduced oxygen consumption rates after application of topical anesthetic.

Purpose: Changes in pH in the corneal epithelium, stroma, endothelium and aqueous humor following contact lens wear were evaluated., Methods: Intracellular pH, stromal pH, and aqueous humor pH were measured either in vivo or in vitro in humans and in rabbits using pH-sensitive fluorescent dyes. Fluorescence was measured before and during lens wear using a slit lamp fluorimeter (in vivo experiments) or a microscope fluorimeter (in vitro experiments)., Results: The pH of corneal epithelium, stoma epithelium and aqueous humor decreased significantly with lenses up to Dk/L = 100. Hypoxia had large effects on the corneal epithelium and stroma, but had no effect on the endothelium or aqueous. Conversely, carbon dioxide accumulation caused significant acidosis in all compartments measured., Conclusions: Corneal acidosis during contact lens wear is significant under a variety of wearing conditions and, thus, must be considered when evaluating the morphological or functional changes that occur following long-term contact lens wear.

Corneal acidosis has been shown to reduce corneal hydration control (CHC) as measured by the rate, expressed as the percent recovery per hour (PRPH), at which the thickness of the cornea decreases exponentially after an increased hydration load. Since the effect of pH on corneal function is of scientific interest and may have clinical implications, we explored the relationship between pH and PRPH in greater detail by examining the effect of different stromal pH levels on corneal hydration control. Corneal edema was induced using a 90-min exposure to wearing a hypoxic contact lens (CL). Following removal of the CL, random assignment over four eye-test combinations of either 0, 3, 5, and 7% CO2 were made while pH and corneal thickness were monitored using slit lamp fluorophometry and optical pachometry to measure corneal pH and corneal thickness, respectively. From these measurements we determined the pH-dose/PRPH relationship. The average stromal pH +/- 1SD resulting from exposure to either the 0, 3, 5, and 7% CO2, was 7.65 +/- 0.11, 7.30 +/- 0.09, 7.15 +/- 0.08 and 7.04 +/- 0.07 (p < 0.001), respectively. Analysis based on a quadratic model of the dose-response relationship between PRPH and corneal pH indicates that PRPH is relatively unchanged for pH in the physiological range (pH = 7.40-7.65) and then decreases notably below the physiological range.

Carbonic anhydrase inhibitors are known to inhibit fluid transport by the corneal endothelium. This phenomenon could be due to a combination of effects involving disruption of intracellular pH regulation, reduced gradients for diffusion of CO2, substrate limitation to HCO3- transport systems or direct inhibition of membrane HCO3- transport. We examined the effects of the carbonic anhydrase inhibitor, Acetazolamide (ACTZ), on intracellular pH (pHi) and HCO3- transport in cultured bovine corneal endothelium. The pHi was measured utilizing the pH sensitive fluorescent dye, BCECF. Na+:HCO3- cotransport and Cl-/HCO3- exchange activities were studied by measuring the HCO3(-)-dependent flux of Na+ and Cl-, respectively. Na+ and Cl- fluxes were measured using the ion-sensitive dyes SBFI and SPQ, respectively. Application of 100 or 500 microM ACTZ to cells perfused under HCO3(-)-rich conditions, significantly reduced steady-state pHi by 0.06 +/- 0.01 (n = 14, P < 0.05). ACTZ also eliminated rapid pHi transients due to CO2 diffusion, significantly slowed the initial rate of pHi changes (50 +/- 10% of control, n = 7, P < 0.05) secondary to Na+:HCO3- cotransport or Cl-/HCO3- exchange (37 +/- 1% of control, P < 0.05, n = 7). However, the flux of the cotransported ions, Na+ and Cl-, and the steady-state levels of these ions were not affected by ACTZ. We conclude that the drop in steady-state pHi, the elimination of CO2 induced pHi transients and the slowed pHi changes secondary to HCO3- transport were due to inhibition of cytosolic carbonic anhydrase by ACTZ, i.e. slowing the equilibrium among CO2, HCO3- and H+ and not due to limitation of substrate availability or direct inhibition of the membrane transporters.

Contact lens wear causes significant epithelial and stromal acidosis. In this study, we tested whether lens wear can cause endothelial acidosis as well. Rabbit corneas were isolated and perfused in vitro. The endothelial intracellular pH (pHi) was measured with a pH sensitive fluorescent probe (BCECF). Three conditions were examined: 1) Polymethylmethacrylate (PMMA) and rigid gas-permeable (RGP) contact lens wear using a range of oxygen transmissibility (Dk/L) from 0 to 121, 2) epithelial hypoxia produced by exposure to oligomycin/sodium azide solution or epithelial perfusion with 100% N2 equilibrated Ringer's solution, and 3) epithelial exposure to Ringer's equilibrated with 5% CO2, balance air. PMMA and RGP contact lens wear acidified endothelial cells by 0.23 +/- 0.01 (n = 23) and 0.11 +/- 0.01 pH units (n = 23), respectively, within twenty min of lens insertion. Epithelial hypoxia, induced by sodium azide and oligomycin, reversibly acidified the endothelium by 0.04 +/- 0.01 pH units (n = 4). However, epithelial hypoxia induced by perfusion with 100% N2 equilibrated Ringer's did not have a significant effect on endothelial pHi. Introduction of 5% CO2 to the epithelium, acidified the endothelium by 0.15 +/- 0.02 pH units (n = 7) within 10 min. We conclude that contact lens wear can significantly acidify corneal endothelial cells. The endothelial pHi change is caused almost exclusively by a build up of CO2 behind the lens; hypoxia having very little contribution. As expected, RGP contact lenses induced less endothelial acidosis than PMMA controls.

Proton coupled lactate transport across the rabbit corneal endothelium was studied using a pH-sensitive intracellular fluorescent probe. Functional indications that lactate transport is carrier-mediated and coupled to H+ at the apical endothelium (aqueous humor facing) that were found were: (1) proton influx was a saturable function of lactate concentration; (2) L-lactate produced a faster maximal H+ influx and had a higher affinity for the transporter (Vmax = 1.6 mM min-1, apparent K1/2 = 31 mM) than its optical isomer, D-Lactate (Vmax = 0.9 mM min-1, apparent K1/2 = 59 mM); (3) the lactate-induced acidification was inhibitable by apical mersalyl acid. These results are consistent with the presence of an apical lac-:H+ cotransporter. On basolateral addition of lactate to a de-epithelialized cornea, the endothelial cells transiently acidified by 0.05 units, but then alkalinized by 0.07, 0.02 units over baseline. The basolateral lactate-induced acidification was inhibited by 75% by mersalyl acid, while the net alkalinization observed under control conditions was unaffected. The alkalinization was Na(+)-dependent yet amiloride-insensitive. Alkalinization on addition of basolateral lactate could also be inhibited by apical mersalyl. These results suggest that the acidification represents lactate influx through basolateral Lac-:H+ cotransport while the net alkalinization is due to basolateral Na(+)-dependent lactate uptake and lactate efflux through apical Lac-:H+ cotransport. These facilitated transport mechanisms could provide for the efficient removal of lactate from the highly glycolytic cornea.

Fluid transport by the corneal endothelium is supported in air equilibrated, nominally HCO3- free Ringer's, yet it is inhibited by carbonic anhydrase inhibitors indicating dependence on the presence of CO2. It is possible that the CO2/HCO3- contained in air and/or the CO2 derived from oxidative metabolism is sufficient to support HCO3- transport. This study investigates whether endothelial HCO3- transport mechanisms, Na+:HCO3- cotransport and Cl-/HCO3- exchange, are active in the nominal absence of HCO3-. In cultured bovine corneal endothelial cells the rate of intracellular pH (pHi) recovery following acid loading in air-equilibrated, nominally HCO(3-)-free solutions was slowed 75% by 0.5 mM amiloride (inhibitor of Na+/H+ exchange), yet 60% of the acid load was still pumped out. This residual acid efflux (or base influx) was N(+)-dependent, slowed 60 +/- 15% by equilibration of solutions with 100% N2 (to minimize CO2 availability) and totally blocked by pretreatment with 100 microM DIDS, an inhibitor of Na+:HCO3- cotransport. Resting cells were acidified 0.19 +/- 0.01 pH units by addition of 0.5 mM H2DIDS when perfused with air-equilibrated solutions, but only 0.03 +/- 0.02 when perfused with 100% N2 equilibrated solutions. Application of 500 microM acetazolamide (a carbonic anhydrase inhibitor) also acidified resting cells, 0.11 +/- 0.01 pH units, but the acidification was eliminated when cells were perfused with 100% N2-equilibrated solutions, indicating the presence of carbonic anhydrase activity under nominally HCO(3-)-free conditions. Cl-/HCO3- exchange activity was eliminated by 100% N2 perfusion and slowed 29% by acetazolamide.(ABSTRACT TRUNCATED AT 250 WORDS)

Purpose: Based on contact lens-induced stromal acidification of the cornea, it has been suggested that the corneal epithelial and endothelial cells also become acidotic during contact lens wear. This alleged acidification may have a role in altered cell appearance and metabolism during contact lens wear. This study investigated the effects of anoxia, carbon dioxide retention, and contact lens gas transmissibility on the epithelial and aqueous humor pH in living rabbits., Methods: Epithelial intracellular pH (pHi) and aqueous humor pH were fluorophotometrically measured with a pH sensitive-dye (BCECF) during contact lens wear or exposure to various gas mixtures., Results: Polymethylmethacrylate (PMMA) lens wear acidified epithelial cells by preventing CO2 efflux and by inducing hypoxia. Increasing lens oxygen transmissibility decreased epithelial acidification. After initiation of rigid, gas-permeable (RGP) lens wear or CO2-air exposure, pHi dropped transiently and then recovered partially. This recovery of pHi was not observed during anoxia, whether induced by PMMA lens wear or exposure to 100% N2. The aqueous humor also acidified during PMMA lens wear, a phenomenon not observed during RGP lens wear. Changes in aqueous pH were smaller, slower, and delayed when compared to their epithelial counterparts., Conclusions: Hypoxic contact lens wear acidifies the corneal epithelium and aqueous humor. The aqueous humor pH change indicates a probable endothelial acidification during hypoxic contact lens wear; the pH changes are caused by two separate and additive effects, CO2 retention and hypoxic acidosis. Increases in the oxygen transmissibility of the lens decrease the cellular acidosis, which might minimize cellular complications arising from contact lens wear. We estimate that a lens with an oxygen transmissibility (Dk/L) of 300 x 10(-11) (cm/sec)(ml O2/ml x mm Hg) is needed to prevent epithelial pHi changes in the open eye. In contrast, lenses with Dk/L as low as 18 x 10(-9) (cm/sec)(ml O2/ml x mm Hg) can prevent aqueous humor pH changes.

Purpose: Studies have shown that stromal acidosis reduces the rate of corneal thickness recovery after induced edema, providing the first human in vivo evidence that corneal pH can influence corneal hydration control. This finding raises the question of the possible effect that pH may have on induced corneal swelling. To explore this question, the corneal swelling response to hypoxia was measured while stromal pH was controlled., Methods: Corneal edema and stromal acidosis was induced in ten subjects by passing a mixture of nitrogen and carbon dioxide gas across the eyes through tight-fitting goggles. One eye of each subject received 100% N2, whereas the contralateral eye received a mixture of 95% N2 and 5% CO2. Exposures of 95% N2 + 5% CO2 lower pH on average to 7.16 versus 7.34 for 100% N2 alone. Before and after 2.5 hours of gas exposure, central corneal thickness (CCT) was measured., Results: Eyes exposed to the lower pH environment (eg, N2 + CO2) developed less change in CCT compared to the eyes receiving N2 alone. Overall increase in CCT was 29.9 +/- 5.3 microns for eyes exposed to the 95% N2 + 5% CO2 gas mixture, versus 37.1 +/- 4.8 microns for 100% N2 eyes (P < 0.0001)., Conclusions: The corneal swelling response to hypoxia can be reduced by lowering stromal pH. Because changes in corneal pH alone have not been found to alter steady-state CCT, it is proposed that pH exerts its effect only under non-steady-state conditions (ie, corneal swelling and deswelling). This suggests that acidosis may produce changes in the rate of lactate metabolism or alter endothelial hydraulic conductivity.

A detailed comparison of intracellular pH (pHi) regulatory mechanisms was made between fresh (FBCE) and cultured (CBCE) bovine corneal endothelium to: (1) identify the ion transport mechanisms that could directly or indirectly affect transendothelial HCO3- transport; and (2) determine if cultured cells could serve as a model for studying transendothelial bicarbonate transport. We used the pH-sensitive fluorescent probe BCECF-AM to measure pHi. FBCE and CBCE readily incorporated the dye and showed pHi calibration curves that were not significantly different with respect to pK (7.39 for FBCE and 7.35 for CBCE). Resting pHi in bicarbonate free Ringer's (pH 7.5) was significantly lower in cultured cells (7.17 +/- .02, n = 50) than in fresh cells (7.30 +/- .02, n = 54). Steady-state pHi was reduced by addition of 0.5 mmol/l amiloride, a Na+/H+ exchange blocker (-.16 pH U for FBCE, -.18 for CBCE) or removal of Na+ (-.47 pH U for FBCE, -.51 for CBCE). Recovery from an (NH4)2SO4-induced acid load was blocked by Na+ removal, and the rate of recovery was inhibited 74% and 79% in the presence of amiloride for FBCE and CBCE, respectively. The dependence of proton efflux on Na+0 showed simple saturating kinetics (apparent Km = 30 and 31 mmol/l for FBCE and CBCE, respectively), consistent with the presence of Na+/H+ exchange in FBCE and CBCE. Na+/H+ exchange activity, as measured by amiloride-sensitive acid recovery, was inversely proportional to pHi. The activity in FBCE was about twice that in CBCE. Furthermore, the zero flux point for Na+/H+ exchange was at least 0.1 pH U higher in FBCE. Changing from bicarbonate-free Ringer's to bicarbonate Ringer's (5% CO2/28 mmol/l HCO3-, pH 7.5) induced a rapid and short acidification followed by an alkalinization .09 and .18 pH U above the starting pHi for FBCE (final pHi 7.37) and CBCE (final pHi 7.33), respectively. This transition was unaffected by amiloride. Similarly, amiloride had no effect on resting pHi in bicarbonate Ringer's for FBCE or CBCE, indicating that Na+/H+ exchange does not contribute to the maintenance of the steady-state resting pHi in bicarbonate Ringer's. Although most of the characteristics of Na+/H+ exchange for FBCE and CBCE were similar, the differences in overall activity and the low levels of activity in resting cells must be considered when using CBCE to model ion coupled fluid transport in BCE.

The comparison of intracellular pH (pHi) regulation between fresh (FBCE) and cultured (CBCE) bovine corneal endothelium was extended to HCO3- transport mechanisms. Upon introduction of CO2/HCO3- Ringer's solution, there was a small, sharp acidification followed by an alkalinization .09 and .18 pH U above the HCO3- free resting pHi for FBCE and CBCE, respectively. This increase in pHi totally depended upon the presence of Na+, independent of Cl- and blocked by the anion transport inhibitor, H2DIDS (0.5 mmol/l). Recovery from an (NH4)2SO4-induced acid load also was blocked by Na+ removal and inhibited 62% and 84% by 0.5 mmol/l H2DIDS for FBCE and CBCE, respectively. These results indicate the presence of a Na+ dependent HCO3- transporter. Additions of 22 mmol/l K+ or 5 mmol/l Ba2+ led to substantial H2DIDS-inhibitable base influx in HCO3- Ringer's, which was significantly reduced in the absence of HCO3- for FBCE and CBCE, consistent with the presence of electrogenic Na+:nHCO3- cotransport. Using the Na+ sensitive intracellular dye, SBFI, we confirmed that the addition of HCO3- resulted in a H2DIDS-sensitive Na+ influx. The mean steady-state [Na+i] = 14 +/- 3 mmol/l in bicarbonate-free Ringer's and 31.5 +/- 2 mmol/l in CO2/HCO3-. HCO3- induced Na+ influx was reduced 74% by 0.5 mmol/l H2DIDS. Removal of Cl- from bicarbonate Ringer's alkalinized FBCE and CBCE by .12 +/- .01 and .21 +/- .03, respectively. The increased pHi was completely blocked by 0.5 mmol/l H2DIDS. Similar alkalinizations were seen when Cl- was removed from air equilibrated bicarbonate-free Ringer's. However, because of the lowered buffering capacity in the absence of HCO3-, the flux was reduced by 80%. Cl- free alkalinizations were eliminated by equilibrating the bicarbonate-free Ringer's with 100% nitrogen gas, indicating that residual CO2 can act as a substrate. Bicarbonate efflux on readdition of Cl- was Na+ independent and pHi sensitive (inactivated by low pHi), and showed simple saturating kinetics with respect to bath Cl, K1/2 = 22 and 16 mmol/l for FBCE and CBCE, respectively. These data are consistent with the presence of Cl-/HCO3- exchange in FBCE and CBCE. Application of 0.5 mmol/l H2DIDS to resting cells in HCO3- Ringer's significantly reduced pHi by .23 +/- .03 and .18 +/- .02 in FBCE and CBCE, respectively, indicating net HCO3- influx in resting cells and suggesting that the stoichiometry of the Na+:nHCO3- cotransporter favors Na+ and HCO3- uptake, ie, n < or = 2.

An estimate of overall corneal hydration control can be obtained by measuring the rate of thickness recovery following induced corneal swelling; it is expressed as the percent recovery per hour (PRPH). This recovery is nearly, but not exactly, exponential, because there appears to be an initial slower recovery phase lasting about 30-40 minutes. This 30-40 minute period of slower recovery corresponds to the time when corneal pH is reduced secondary to the contact lens-induced swelling, suggesting the possibility that stromal acidosis may retard the corneal deswelling process. In this study, we explored the effects of corneal acidosis on hydration control by monitoring corneal recovery under normal and reduced pH conditions. Corneal pH was controlled by having subjects were goggles and exposing their eyes to air (normal pH) or a gas mixture providing 21% O2 and 7% CO2 (low pH). Relative corneal pH levels were monitored by measuring fluorescence intensity (FI) ratios, which showed that the average (+/- standard deviation) FI ratio was significantly lower under 7% CO2 (0.838 +/- .024) vs air (0.985 +/- .025; P = 0.0001), corresponding to approximate pH values of 7.25 vs 7.50. Under these reduced pH conditions, open-eye steady-state (OESS) corneal thickness was not substantially affected. For 10 subjects, mean (+/- SD) corneal thickness decreased 0.93 +/- 3.7 microns vs 1.10 +/- 4.50 microns after exposures to 60 minutes of 7% CO2 and 40 minutes of air (P greater than or equal to 0.45), respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Intracellular pH (pHi) can affect many cell functions including metabolic activity, protein synthesis, and cell growth rates. For the corneal epithelium these processes are important for maintenance of its barrier function and ion transport properties. This brief report reviews some of the recent findings from my laboratory, which describe basic ion transport mechanisms, e.g., Na+/H+ exchange and lactate-H+ cotransport, useful for regulating pHi in rabbit corneal epithelium. The effects of acid challenges, 5% CO2 (simulated eye closure), and hypoxia (simulated contact lens wear) on pHi are also examined. The 5% CO2 drops pHi by approximately 0.2 units with little or no recovery, indicating that steady-state pHi in vivo is reduced during eye closure. By contrast, hypoxia in vitro slowly raises pHi, approximately 0.1 units, partially due to the stimulation of lactate-H+ efflux.

Sections of whole ciliary body dissected from Dutch belted rabbits were incubated with the cell entrappable pH probe BCECEF-AM. This led to a highly specific localization of epifluorescence emission at the exposed, non-pigmented cell layer (npe) of the dual layered epithelium that covers this organ. The BCECF-loaded tissue sections were superfused in a flow-through chamber and the intracellular pH (pHi) of small groups (10-20) of cells was derived from the ratio of the emission intensities derived from excitations at 490 and 440 nm. In CO2/HCO3- Ringer's, npe pHi = 7.09 +/- 0.11. Replacement of CO2/HCO3- by Hepes increased pHi by 0.22 +/- 0.02, indicating alkali secretory activity under the bicarbonate-rich conditions. Replacement of Cl- by gluconate elicited a rapid, 0.6-U increase in pHi. This effect exhibited little dependence on Na+ and was inhibited by 0.5 mM dihydro-4,4'-diisothiocyanatostilbene -2,2'-disulfonate (H2DIDS). These results indicate the presence of an electroneutral Cl-/base exchange activity. Elevation of [K-] (by partial replacement of Na+) also elicited increases in pHi. In Cl(-)-free media pHi reached 7.8-8.0, a condition under which intracellular [HCO3-] is at least twice as high as its extracellular value. This effect did not occur in the absence of Na+. The Na(+)-dependent high [K+]-induced pHi increase was inhibited by H2DIDS. The effects of Ba2+ on pHi, alone and in combination with high [K+], as well as that of full K+ removal, suggested that the link between high [K+] and pHi increase was mainly due to the effect of cell depolarization on an electronegative Na+ dependent HCO3- transporter. Under normal physiological conditions, the two acid/base transport systems are the main determinants of npe pHi.

Rabbit corneal epithelial cells, loaded with the pH-sensitive fluorescent dye 2',7'-bis(2-carboxy-ethyl)-5(6)-carboxyfluorescein, show a profound acidification (pH 7.33 to 6.75) in HCO3(-)-free Ringer solution when exposed to the Na(+)-H+ exchange inhibitor amiloride. This indicates that the cells are under a constant acid load that is being countered by Na(+)-H+ exchange. Amiloride-induced acidification was affected neither by incubation in Cl(-)-free Ringer solution nor by hypoxia, indicating that the potential acid loaders, Cl(-)-HCO3- (or OH-) exchange or glycolytic metabolism, are not contributing to the acidification. The possibility of a H+ influx dependent on the outward K+ gradient was tested. Perfusion with a high-K+ Ringer solution (77 mM) caused Na(+)- and Cl(-)-independent alkalinizations. Membrane depolarization by gramicidin, Ba2+, Cl(-)-free Ringer solution, or ouabain all produced small (less than 0.1 pH units) acidifications, inconsistent with contribution by a membrane potential driven passive H+ influx. In Na(+)-free Ringer solution, intracellular pH (pHi) of 6.4-6.6, addition of nigericin (a 1K(+)-1H+ ionophore) produced no significant change in pHi, indicating that [K+]i/[K+]o = [H+]i/[H+]o. Both amiloride-induced acidifications and high-K(+)-induced alkalinizations were significantly stimulated by the presence of 1 mM ZnSO4 and unaffected by H2-DIDS (0.5 mM, an anion transport blocker) or 100 microM SCH28080 (K(+)-H(+)-ATPase blocker). In the absence of a demonstrable H+ conductance, it is concluded that amiloride-induced acidification and K(+)-induced pHi changes are via a carrier-mediated K(+)-H+ exchanger. In addition to pHi regulation, K(+)-H+ exchange may play a role in cell volume control.(ABSTRACT TRUNCATED AT 250 WORDS)

The presence of the membrane transport mechanism, lactate-H+ cotransport, was tested in explants of rabbit corneal epithelium. Basal corneal epithelial cells were loaded with the pH sensitive fluorescent dye BCECF. Intracellular pH (pHi) was measured by rationing the fluorescence emission output following excitation at 490 and 440 nm. Perfusion of explants in lactate-containing Ringer's, pH 7.40, produced a reversible decrease in pHi. The lactate induced proton influx (mM/min) followed saturating kinetics, Km = 10.7 mM lactate, Vmax = 10.2 mM/min. Proton influx following addition of 10 mM lactate was inhibited 36, 60 and 47% by pre-perfusion in 1 mM CHC (cyano-hydroxycinammic acid), 500 microM H2DIDS (4,4'-diisothiocyanato-dihydrostilbene-2,2'-disulfonic acid) and 1 mM LAIE (lactic acid isobutylester), respectively. These inhibitors of lactate-H+ cotransport were reversible. Mersalyl acid (500 microM) inhibited proton flux from 10 mM lactate addition by nearly 100%, but was irreversible. Stimulation of lactate production by perfusion in N2 equilibrated Ringer's (hypoxia) or the addition of 1 mM NaCN led to a slow alkalinization (0.1 pH unit in 10 min). Pre-perfusion with the reversible inhibitors slowed the hypoxic alkalinization by approximately 40%. It is concluded that lactate-H+ cotransport is present in the corneal epithelium and that it contributes to pHi regulation during hypoxia.