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Purpose: SLC4A11, an electrogenic H+ transporter, is found in the plasma membrane and mitochondria of corneal endothelium. However, the underlying mechanism of SLC4A11 targeting to mitochondria is unknown., Methods: The presence of mitochondrial targeting sequences was examined using in silico mitochondrial proteomic analyses. Thiol crosslinked peptide binding to SLC4A11 was screened by untargeted liquid chromatography/tandem mass spectrometry (LC-MS/MS) analysis. Direct protein interactions between SLC4A11 and chaperones were examined using coimmunoprecipitation analysis and proximity ligation assay. Knockdown or pharmacologic inhibition of chaperones in human corneal endothelial cells (HCECs) or mouse corneal endothelial cells (MCECs), ex vivo kidney, or HA-SLC4A11-transfected fibroblasts was performed to investigate the functional consequences of interfering with mitochondrial SLC4A11 trafficking., Results: SLC4A11 does not contain canonical N-terminal mitochondrial targeting sequences. LC-MS/MS analysis showed that HSC70 and/or HSP90 are bound to HA-SLC4A11-transfected PS120 fibroblast whole-cell lysates or isolated mitochondria, suggesting trafficking through the chaperone-mediated carrier pathway. SLC4A11 and either HSP90 or HSC70 complexes are directly bound to the mitochondrial surface receptor, TOM70. Interference with this trafficking leads to dysfunctional mitochondrial glutamine catabolism and increased reactive oxygen species production. In addition, glutamine (Gln) use upregulated SLC4A11, HSP70, and HSP90 expression in whole-cell lysates or purified mitochondria of HCECs and HA-SLC4A11-transfected fibroblasts., Conclusions: HSP90 and HSC70 are critical in mediating mitochondrial SLC4A11 translocation in corneal endothelial cells and kidney. Gln promotes SLC4A11 import to the mitochondria, and the continuous oxidative stress derived from Gln catabolism induced HSP70 and HSP90, protecting cells against oxidative stress.

Purpose: The Slc4a11 knock out (KO) mouse model recapitulates the human disease phenotype associated with congenital hereditary endothelial dystrophy (CHED). Increased mitochondrial reactive oxygen species (ROS) in the Slc4a11 KO mouse model is a major cause of edema and endothelial cell loss. Here, we asked if autophagy was activated by ROS in the KO mice., Methods: Immortalized cell lines and mouse corneal endothelia were used to measure autophagy and lysosome associated protein expressions using Protein Simple Wes immunoassay. Autophagy and lysosome functions were examined in wild type (WT) and KO cells as well as animals treated with the mitochondrial ROS quencher MitoQ., Results: Even though autophagy activation was evident, autophagy flux was aberrant in Slc4a11 KO cells and corneal endothelium. Expression of lysosomal proteins and lysosomal mass were decreased along with reduced nuclear translocation of lysosomal master regulator, transcription factor EB (TFEB). MitoQ reversed aberrant lysosomal functions and TFEB nuclear localization in KO cells. MitoQ injections in KO animals reduced corneal edema and decreased the rate of endothelial cell loss., Conclusions: Mitochondrial ROS disrupts TFEB signaling causing lysosomal dysfunction with impairment of autophagy in Slc4a11 KO corneal endothelium. Our study is the first to identify the presence as well as cause of lysosomal dysfunction in an animal model of CHED, and to identify a potential therapeutic approach.

Purpose: The conventional Slc4a11 knockout (KO) shows significant corneal edema at eye opening, a fact that complicates the study of the initial events leading to edema. An inducible KO would provide opportunities to examine early events following loss of Slc4a11 activity., Methods: Slc4a11 Flox (SF) mice were crossed with mice expressing the estrogen receptor Cre Recombinase fusion protein and fed tamoxifen (Tm) for two weeks. Corneal thickness (CT) was measured by OCT. At eight weeks endpoint, oxidative damage, tight junction integrity, stromal lactate concentration, endothelial permeability, differentially expressed transporters, and junction proteins were determined. Separately, a keratocyte only inducible Slc4a11 KO was also examined., Results: At four weeks post-Tm induction Slc4a11 transcript levels were 2% of control. Corneal thickness increased gradually and was 50% greater than Wild Type (WT) after eight weeks with significantly altered endothelial morphology, increased nitrotyrosine staining, significantly higher stromal lactate, decreased expression of lactate transporters and Na-K ATPase activity, higher ATP, altered expression of tight and adherens junctions, and increased fluorescein permeability. No significant differences in CT were found between WT and keratocyte only Slc4a11 KO., Conclusions: The Slc4a11 inducible KO shows development of a similar phenotype as the conventional KO, thereby validating the model and providing a tool for further use in examining the sequence of cellular events by use of noninvasive in vivo physiological probes.

Purpose: Confirm that the corneal endothelial pump uses a lactate-coupled water efflux mechanism., Methods: Corneal thickness, lactate efflux, and stromal [lactate] were measured in de-epithelialized swollen and nonswollen ex vivo-mounted rabbit corneas perfused with bicarbonate-rich and bicarbonate-free Ringers, ouabain, or acetazolamide to determine if the relationships among these parameters were similar to previous data using intact corneas. The role of barrier function was tested by perfusion with calcium-free EGTA. Predictions of [lactate] in endothelial dystrophy were examined in the Slc4a11 knock out mouse., Results: De-epithelialized corneal swelling, lactate efflux, and stromal [lactate] in response to bicarbonate-free Ringers, ouabain, and acetazolamide perfusion had the same relationship as in intact corneas. The absolute amount of lactate efflux and stromal [lactate] in the de-epithelialized corneas was about half of intact corneas. De-epithelialized, swollen corneas deswelled fully with bicarbonate-rich, partially in the presence of acetazolamide, but continued to swell with bicarbonate-free or ouabain. The relationship among corneal thickness, lactate efflux, and [lactate] was the same as with nonswollen de-epithelialized corneas. In intact corneas swollen by perfusion with calcium-free EGTA, the relationship between swelling and lactate flux was the inverse of control corneas. The relationship between corneal swelling and [lactate] of intact corneas exposed to ouabain, but perfused with 7 mM lactate to simulate aqueous humor, was the same as without lactate. Corneal [lactate] in Slc4a11 knock out was twice that of wild type., Conclusions: The corneal endothelial pump works via a lactate efflux mechanism that requires an intact osmotic barrier.

Purpose: To establish conditionally immortal mouse corneal endothelial cell lines with genetically matched Slc4a11+/+ and Slc4a11-/- mice as a model for investigating pathology and therapies for SLC4A11 associated congenital hereditary endothelial dystrophy (CHED) and Fuchs' endothelial corneal dystrophy., Methods: We intercrossed H-2Kb-tsA58 mice (Immortomouse) expressing an IFN-γ dependent and temperature-sensitive mutant of the SV40 large T antigen (tsTAg) with Slc4a11+/+ and Slc4a11-/- C57BL/6 mice. The growth characteristics of the cell lines was assessed by doubling time. Ion transport activities (Na+/H+ exchange, bicarbonate, lactate, and Slc4a11 ammonia transport) were analyzed by intracellular pH measurement. The metabolic status of the cell lines was assessed by analyzing TCA cycle intermediates via gas chromatography mass spectrometry (GC-MS)., Results: The immortalized Slc4a11+/+ and Slc4a11-/- mouse corneal endothelial cells (MCECs) remained proliferative through passage 49 and maintained similar active ion transport activity. As expected, proliferation was temperature sensitive and IFN-γ dependent. Slc4a11-/- MCECs exhibited decreased proliferative capacity, reduced NH3:H+ transport, altered expression of glutaminolysis enzymes similar to the Slc4a11-/- mouse, and reduced proportion of TCA cycle intermediates derived from glutamine with compensatory increases in glucose flux compared with Slc4a11+/+ MCECs., Conclusions: This is the first report of the immortalization of MCECs. Ion transport of the immortalized endothelial cells remains active, except for NH3:H+ transporter activity in Slc4a11-/- MCECs. Furthermore, Slc4a11-/- MCECs recapitulate the glutaminolysis defects observed in Slc4a11-/- mouse corneal endothelium, providing an excellent tool to study the pathogenesis of SLC4A11 mutations associated with corneal endothelial dystrophies and to screen potential therapeutic agents.

Purpose: Cannabinoid CB1 receptors are found in abundance in the vertebrate eye, with most tissue types expressing this receptor. However, the function of CB1 receptors in corneal epithelial cells (CECs) is poorly understood. Interestingly, the corneas of CB1 knockout mice heal more slowly after injury via a mechanism proposed to involve protein kinase B (Akt) activation, chemokinesis, and cell proliferation. The current study examined the role of cannabinoids in CEC migration in greater detail., Methods: We determined the role of CB1 receptors in corneal healing. We examined the consequences of their activation on migration and proliferation in bovine CECs (bCECs). We additionally examined the mRNA profile of cannabinoid-related genes and CB1 protein expression as well as CB1 signaling in bovine CECs., Results: We now report that activation of CB1 with physiologically relevant concentrations of the synthetic agonist WIN55212-2 (WIN) induces bCEC migration via chemotaxis, an effect fully blocked by the CB1 receptor antagonist SR141716. The endogenous agonist 2-arachidonoylglycerol (2-AG) also enhances migration. Separately, mRNA for most cannabinoid-related proteins are present in bovine corneal epithelium and cultured bCECs. Notably absent are CB2 receptors and the 2-AG synthesizing enzyme diglycerol lipase-α (DAGLα). The signaling profile of CB1 activation is complex, with inactivation of mitogen-activated protein kinase (MAPK). Lastly, CB1 activation does not induce bCEC proliferation, but may instead antagonize EGF-induced proliferation., Conclusions: In summary, we find that CB1-based signaling machinery is present in bovine cornea and that activation of this system induces chemotaxis.

Purpose: CD147/basigin is a chaperone for lactate:H(+) cotransporters (monocarboxylate transporters) MCT1 and MCT4. We tested the hypothesis that MCT1 and -4 in corneal endothelium contribute to lactate efflux from stroma to anterior chamber and that silencing CD147 expression would cause corneal edema., Methods: CD147 was silenced via small interfering ribonucleic acid (siRNA) transfection of rabbit corneas ex vivo and anterior chamber lenti-small hairpin RNA (shRNA) pseudovirus in vivo. CD147 and MCT expression was examined by Western blot, RT-PCR, and immunofluorescence. Functional effects were examined by measuring lactate-induced cell acidification, corneal lactate efflux, [lactate], central cornea thickness (CCT), and Azopt (a carbonic anhydrase inhibitor) sensitivity., Results: In ex vivo corneas, 100 nM CD147 siRNA reduced CD147, MCT1, and MCT4 expression by 85%, 79%, and 73%, respectively, while MCT2 expression was unaffected. CD147 siRNA decreased lactate efflux from 3.9 ± 0.81 to 1.5 ± 0.37 nmol/min, increased corneal [lactate] from 19.28 ± 7.15 to 56.73 ± 8.97 nmol/mg, acidified endothelial cells (pHi = 6.83 ± 0.07 vs. 7.19 ± 0.09 in control), and slowed basolateral lactate-induced acidification from 0.0034 ± 0.0005 to 0.0012 ± 0.0005 pH/s, whereas apical acidification was unchanged. In vivo, CD147 shRNA increased CCT by 28.1 ± 0.9 μm at 28 days; Azopt increased CCT to 24.4 ± 3.12 vs. 12.0 ± 0.48 μm in control, and corneal [lactate] was 47.63 ± 6.29 nmol/mg in shCD147 corneas and 17.82 ± 4.93 nmol/mg in paired controls., Conclusions: CD147 is required for the expression of MCT1 and MCT4 in the corneal endothelium. Silencing CD147 slows lactate efflux, resulting in stromal lactate accumulation and corneal edema, consistent with lactate efflux as a significant component of the corneal endothelial pump., (Copyright 2014 The Association for Research in Vision and Ophthalmology, Inc.)

Purpose: Mutations in SLC4A11, a member of the SLC4 superfamily of bicarbonate transporters, give rise to corneal endothelial cell dystrophies. SLC4A11 is a putative Na⁺ borate and Na⁺:OH⁻ transporter. Therefore we ask whether SLC4A11 in corneal endothelium transports borate (B[OH]₄⁻), bicarbonate (HCO3⁻), or hydroxyl (OH⁻) anions coupled to Na⁺., Methods: SLC4A11 expression in cultured primary bovine corneal endothelial cells (BCECs) was determined by semiquantitative PCR, SDS-PAGE/Western blotting, and immunofluorescence staining. Ion transport function was examined by measuring intracellular pH (pHi) or Na⁺ ([Na⁺](i)) in response to Ringer solutions with/without B(OH)₄⁻ or HCO₃⁻ after overexpressing or small interfering RNA (siRNA) silencing of SLC4A11., Results: SLC4A11 is localized to the basolateral membrane in BCEC. B(OH)₄⁻ (2.5-10 mM) in bicarbonate-free Ringer induced a rapid small acidification (0.01 pH unit) followed by alkalinization (0.05-0.1 pH unit), consistent with diffusion of boric acid into the cell followed by B(OH)₄⁻. However, the rate of B(OH)₄⁻-induced pHi change was unaffected by overexpression of SLC4A11. B(OH)₄⁻ did not induce significant changes in resting [Na⁺(i)] or the amplitude and rate of acidification caused by Na⁺ removal. siRNA-mediated knockdown of SLC4A11 (∼70%) did not alter pHi responses to CO₂/HCO₃⁻-rich Ringer, Na⁺-free induced acidification, or the rate of Na⁺ influx in the presence of bicarbonate. However, in the absence of bicarbonate, siSLC4A11 knockdown significantly decreased the rate (43%) and amplitude (48%) of acidification due to Na⁺ removal and recovery (53%) upon add-back. Additionally, the rate of acid recovery following NH₄⁺ prepulse was decreased significantly (27%) by SLC4A11 silencing., Conclusions: In corneal endothelium, SLC4A11 displays robust Na⁺-coupled OH⁻ transport, but does not transport B(OH)₄⁻ or HCO₃⁻.

Purpose: To investigate the effects of SLC4A11 gene depletion in human corneal endothelial cells., Methods: To achieve stable downregulation of SLC4A11 gene expression in immortalized human corneal endothelial cells (HCECs), short-hairpin RNA (shRNA) targeted against SLC4A11 was used. Cell growth and viability were determined using the real-time cell analyzer and trypan blue staining respectively. Apoptosis was investigated by Annexin V and TUNEL assays. Alterations in apoptotic gene expression following SLC4A11 silencing were determined using the RT(2)Profiler PCR array for human apoptosis while activation of the apoptotic pathway was ascertained by western analysis., Results: SLC4A11 silencing in HCECs could be achieved by stable expression of shRNA targeted against SLC4A11. SLC4A11 knockdown suppressed HCEC growth and reduced HCEC viability compared to the control. This reduction in cell growth is associated with increased apoptosis in SLC4A11-silenced cells., Conclusions: Our data suggest that the reduction of cell number with time in SLC4A11-depleted HCECs is due to an increase in cell death by apoptosis. This suggests that SLC4A11 is necessary for cell survival and may explain the pathologic corneal endothelial cell loss in endotheliopathies due to SLC4A11 mutations.

Purpose: To confirm the expression of monocarboxylate transporters (MCT) 1, 2, and 4 in rabbit CE and to test the hypothesis that cellular buffering contributed by HCO₃⁻, NBCe1, and carbonic anhydrase (CA) activity facilitates lactate-H⁺ efflux thereby controlling corneal hydration in vivo., Methods: MCT1-4 expression of rabbit endothelium was examined by Western blotting and immunofluorescence staining. Lactate-induced acidification (LIA) was measured in perfused CE in the presence and absence of HCO₃⁻ and acetazolamide (ACTZ) using tissue treated with siRNA specific to MCT1, 2, and 4. Corneal thickness and lactate concentration were measured in New Zealand White rabbits treated with the topical CA inhibitor Azopt, and from eyes that were injected intracamerally with ouabain, disodium 4,4'-diisothiocyanatostilbene-2,2'-disulfonate (DIDS), and shRNA specific to the 1Na⁺:2HCO₃⁻ cotransporter NBCe1., Results: MCT1 and MCT4 are localized to the lateral membrane, while MCT2 is apical. Cell pH measurements showed LIA in response to 40 mM lactate in bicarbonate free (BF) Ringer's that was inhibited by niflumic acid and by MCT siRNA knockdown, and significantly reduced in the presence of HCO₃⁻. Lactate-dependent proton flux in vitro was not significantly greater in the presence of HCO₃⁻ or reduced by ACTZ. However, when active transport, NBCe1, or CA activity was disrupted in vivo, corneal edema ensued and was associated with significant corneal lactate accumulation., Conclusions: MCT1, 2, and 4 are expressed in rabbit CE on both the apical and basolateral surfaces and function to transport lactate-H⁺. Lactate-H⁺ flux is facilitated by active transport, HCO₃⁻ transport and CA activity, disruption of which causes corneal edema in vivo and indicates that facilitation of lactate efflux is a component of the endothelial pump.

Purpose: To identify and localize the monocarboxylate transporters (MCTs) expressed in bovine corneal endothelial cells (BCEC) and to test the hypothesis that buffering contributed by HCO(3)(-), sodium bicarbonate cotransporter (NBCe1), sodium hydrogen exchanger (NHE), and carbonic anhydrase (CA) activity facilitates lactate flux., Methods: MCT1-4 expression was screened by RT-PCR, Western blot analysis, and immunofluorescence. Endogenous lactate efflux and/or pH(i) were measured in BCEC in HCO(3)(-)-free or HCO(3)(-)-rich Ringer, with and without niflumic acid (MCT inhibitor), acetazolamide (ACTZ, a CA inhibitor), 5-(N-Ethyl-N-isopropyl)amiloride (EIPA) (Na(+)/H(+) exchange blocker), disodium 4,4'-diisothiocyanatostilbene-2,2'-disulfonate (DIDS; anion transport inhibitor), or with NBCe1-specific small interfering (si) RNA-treated cells., Results: MCT1, 2, and 4 are expressed in BCEC. MCT1 was localized to the lateral membrane, MCT2 was lateral and apical, while MCT4 was apical. pH(i) measurements showed significant lactate-induced cell acidification (LIA) in response to 20-second pulses of lactate. Incubation with niflumic acid significantly reduced the rate of pHi change (dpH(i)/dt) and lactate-induced cell acidification. EIPA inhibited alkalinization after lactate removal. Lactate-dependent proton flux was significantly greater in the presence of HCO(3)(-) but was reduced by ACTZ. Efflux of endogenously produced lactate was significantly faster in the presence of HCO(3)(-), was greater on the apical surface, was reduced on the apical side by ACTZ, as well as on the apical and basolateral side by NBCe1-specific siRNA, DIDS, or EIPA., Conclusions: MCT1, 2, and 4 are expressed in BCEC on both the apical and basolateral membrane (BL) surfaces consistent with niflumic acid-sensitive lactate-H(+) transport. Lactate dependent proton flux can activate Na(+)/H(+) exchange and be facilitated by maximizing intracellular buffering capacity through the presence of HCO(3)(-), HCO(3)(-) transport, NHE and CA activity.

Purpose: To evaluate the role of the sodium bicarbonate cotransporter (NBCe1) as a component of the corneal endothelial pump in the in vivo rabbit eye., Methods: Lentiviruses with NBCe1 shRNA and GFP expression cassettes were injected intracamerally. Knockdown efficacy was determined 1 week to 4 weeks later by immunofluorescence, Western blot analysis, and PCR. Functional effects were monitored by corneal thickness (CT) and brinzolamide sensitivity., Results: Within 24 hours there was a modest anterior chamber inflammation that resolved within 48 hours. At 4 × 10(6) IFU, more than 95% of the corneal endothelial surface showed GFP fluorescence above background within 7 days. At 14 to 21 days, signs of anterior chamber inflammation reemerged, and endothelial cell GFP fluorescence disappeared within 40 days after injection. The second phase of inflammation could be avoided by using GFP-less viruses. There was no significant difference in CT between scrambled sequence and NBCe1 shRNA-injected eyes over 3 weeks. Two drops of 1% brinzolamide produced 7.85% ± 3.3% corneal swelling within 5 hours of topical instillation. However, in corneas showing more than 25% NBCe1 knockdown (30 of 42 rabbits; 59% ± 15% knockdown), corneal swelling was significantly higher (10.1% ± 2.9%) relative to control eyes., Conclusions: FIV-based lentiviral vectors can transfect CE with shRNA in rabbits. The response to GFP is consistent, with previous studies showing the production of anti-GFP antibodies. Partial knockdown of NBCe1 did not affect baseline CT, which is consistent with the corneal endothelium having a substantial functional reserve. Provocative testing using, brinzolamide, however, revealed an underlying deficiency, confirming the importance of NBCe1 bicarbonate transport and demonstrating the concerted action between NBCe1 and carbonic anhydrases.

Purpose: To test the hypothesis that the robust expansion of bovine corneal endothelial cells (BCECs) in vitro is due to the presence of individual endothelial cells with various levels of proliferative potential., Methods: BCECs and bovine vascular endothelial cells (ECs) derived from aorta, coronary artery, and pulmonary artery were cultivated in optimized medium. These cell populations were confirmed by morphologic features, functional assays, and gene expression profiles. Moreover, ECs were plated in a single-cell clonogenic assay to evaluate colony-forming ability., Results: Both corneal and vascular ECs were confirmed to be pure populations of endothelium uncontaminated with hematopoietic cells. A complete hierarchy of endothelial colony-forming cells (ECFCs) was identified in BCECs by a single-cell clonogenic assay. The distribution of the various types of ECFCs was similar to the control ECs removed from the systemic vessels., Conclusions: Cultured BCECs display clonal proliferative properties similar to those of vascular ECs.

Purpose: TGFbeta is the major mediator to induce myofibroblast differentiation in the corneal wound-healing process. Elevated cAMP can reduce TGFbeta-induced fibrosis in other tissues. This study was conducted to determine whether elevated cAMP can inhibit TGFbeta1-induced rabbit corneal keratocyte-myofibroblast transformation., Methods: Primary isolated rabbit corneal keratocytes were cultured in serum-free medium. The effects of the adenylate cyclase agonist forskolin (FSK; 2 microM) on TGFbeta1 (5 ng/mL)-induced alpha-smooth muscle actin (alpha-SMA) expression was examined by immunofluorescence, flow cytometry, and immunochemistry 72 hours after treatment. The effects of TGFbeta+FSK on activated pSmad3, CREB binding protein (CBP), MAPKs, and RhoA were determined by coimmunoprecipitation and Western blot., Results: FSK significantly reduced the myofibroblast phenotype and alpha-SMA expression induced by TGFbeta1 in rabbit corneal keratocytes. TGFbeta1 increased the phosphorylation of ERK and Smad3. TGFbeta1-induced alpha-SMA expression was reduced by MEK inhibition (U0126); however, the levels of pERK, pSmad3, or the extent of the interaction between pSmad3 and CBP induced by TGFbeta1 were not affected by FSK. TGFbeta1 also activated RhoA and ROCK (Y27632) inhibition reduced alpha-SMA expression. Activation of RhoA was significantly reduced by FSK., Conclusions: Raising cAMP by FSK treatment inhibits the TGFbeta1-induced corneal myofibroblast transformation and alpha-SMA expression and thereby provides a promising method to control corneal fibrosis. The data suggest that cAMP-dependent inhibition does not occur by altering Smads or MAPK signaling, but possibly by reducing the activation of RhoA.

Purpose: Carbonic anhydrase activity has a central role in corneal endothelial function. The authors examined the role of carbonic anhydrase IV (CAIV) in facilitating CO(2) flux, HCO(3)(-) permeability, and HCO(3)(-) flux across the apical membrane., Methods: Primary cultures of bovine corneal endothelial cells were established on membrane-permeable filters. Apical CAIV was inhibited by benzolamide or siRNA knockdown of CAIV. Apical CO(2) fluxes and HCO(3)(-) permeability were determined by measuring pH(i) changes in response to altering the CO(2) or HCO(3)(-) gradient across the apical membrane. Basolateral to apical (B-to-A) HCO(3)(-) flux was determined by measuring the pH of a weakly buffered apical bath in the presence of basolateral bicarbonate-rich Ringer solution. In addition, the effects of benzolamide and CAIV knockdown on steady state DeltapH (apical-basolateral compartment pH) after 4-hour incubation in DMEM were measured., Results: CAIV expression was confirmed, and CAIV was localized exclusively to the apical membrane by confocal microscopy. Both 10 microM benzolamide and CAIV siRNA reduced apparent apical CO(2) flux by approximately 20%; however, they had no effect on HCO(3)(-) permeability or HCO(3)(-) flux. The steady state apical-basolateral pH gradient at 4 hours was reduced by 0.12 and 0.09 pH units in benzolamide- and siRNA-treated cells, respectively, inconsistent with a net cell-to-apical compartment CO(2) flux., Conclusions: CAIV does not facilitate steady state cell-to-apical CO(2) flux, apical HCO(3)(-) permeability, or B-to-A HCO(3)(-) flux. Steady state pH changes, however, suggest that CAIV may have a role in buffering the apical surface.

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.

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.

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: 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: 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: 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: 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: 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)

The effects of tear-film hypoxia and contact lens wear on human in vivo stromal pH was tested using a non-invasive fluorometric technique. Hypoxia was produced by exposing the normal open eye to 100% nitrogen gas passed through tight-fitting goggles. Stromal pH dropped from 7.53 +/- 0.02 to 7.34 +/- 0.03 (n = 12, +/- SD) within 90 min of nitrogen gas exposure, t1/2 = 20 min. After removing the goggles, stromal pH returned to baseline in 35 min, t1/2 = 10 min. Wearing a thick hydrogel contact lens which caused a tear PO2 less than or equal to 2 mm Hg with the eyes open, reduced stromal pH from 7.55 +/- 0.02 to 7.15 +/- 0.04 (n = 12, +/- SD) in 80 min, t1/2 = 9.5 min. After removing the lens, baseline pH was reached in 40 min, t1/2 = 4.5 min. The stromal pH differences between hypoxia (N2 only) and contact lens wear were not due to differences in tear temperature between the two procedures (contact lens wear 32 +/- 1.5 degrees C, goggles 33 +/- 1.0 degrees C). However exposing the eye to 95% nitrogen-5% carbon dioxide reduced stromal pH to 7.16 +/- 0.05 (n = 7, +/- SD) in 80 min, t1/2 = 8 min, which was similar to that produced during contact lens wear. These experiments show that contact lens wear causes corneal acidosis by: (1) the production of protons from hypoxic metabolism, and (2) the accumulation of carbon dioxide behind the lens due to low lens CO2 transmissibility.

The pH sensitive fluorescent properties of fluorescein were utilized to noninvasively measure human in vivo stromal pH in the normal open eye and following eye closure. Stromal pH after either 20 or 90 min of eye closure was 7.39 +/- 0.01 (n = 12, +/- SEM) and returned to 7.54 within 10-15 min of opening the eye, t1/2 = 3 min. Eye closure was simulated by exposing the eye to a gas mixture of 7.1% O2, 6.7% CO2, balance N2, which was passed through tight-fitting goggles. This gas mixture resulted in a steady-state stromal pH of 7.29 +/- 0.02 within 10-15 min, t1/2 = 2.2 min. The time course of the return of stromal pH to open eye levels after removal of the goggles, t1/2 = 2.3 min, was similar to that after eye opening. The extent of the pH change however, was 0.1 pH units greater with the test gas. Exposure of the eyes to 5% CO2 (CO2 concentration of blood) and balance air, produced a stromal pH of 7.38 + 0.01 (n = 6, +/- SEM), which closely matches that following eye closure suggesting that conjunctival [CO2] is 5% and is the major component controlling stromal pH when the eyes are closed.