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Slc4a11 KO mice show significant corneal edema, altered endothelial morphology, and mitochondrial ROS at an early age without a decrease in endothelial cell density. We examined the differential gene expression profile between wild type (WT) and KO with the goal of finding pathways related to corneal endothelial metabolic, pump and barrier function that can explain the corneal edema. Freshly dissected Corneal Endothelium-Descemet's Membrane (CEDM) and cultured Mouse Corneal Endothelial Cells (MCEC) were obtained from WT and Slc4a11 KO mice. RNA sequencing Ingenuity Pathway Analysis (IPA) predicted activation, inhibition or differential regulation of several pathways. QPCR and Western analysis validated downregulation of Glycolytic enzymes, Mitochondrial complex components and Ion transporters. Functional testing revealed decreases in endothelial lactate production, Extracellular Acidification Rate (ECAR), glutaminolysis, and Oxygen Consumption Rate (OCR) of KO CEDM in the presence of Glutamine (Gln) that was not compensated by fatty acid oxidation. Stromal lactate was significantly elevated in KO along with decreased expression of MCT1 and MCT4 lactate transporters in endothelial cells. ATP levels were 2x higher in KO CEDM, concomitant with a 3-fold decrease in Na-K-ATPase activity and reduced basolateral membrane localization. Genes for cholesterol biosynthesis, glutathione metabolism and tight and adherens junctions were elevated. Alteration of tight junction structure and cortical cytoskeleton is evident in KO corneal endothelium with a significant increase in trans-endothelial fluorescein permeability. We conclude that Slc4a11 KO induces a coordinated decrease in glycolysis, glutaminolysis, lactate transport and Na-K-ATPase activity. These changes together with an altered barrier function cause an accumulation of stromal lactate in Slc4a11 KO mice leading to chronic corneal edema., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Recent studies have shown that lactate coupled water flux is the underlying mechanism of the corneal endothelial pump, which is highly dependent on the presence of bicarbonate. In this study we test the hypothesis that the increased intracellular pH (pH i ) caused by bicarbonate stimulates glycolytic activity and the production of lactate by endothelial cells. Primary cultures of bovine corneal endothelial cells (BCEC) were incubated in bicarbonate-free (BF) ringer, a high [HEPES] ringer, and bicarbonate-rich (BR) ringer all at pH 7.5. Lactate production and glucose consumption were greatest in BR>HEPES >BF. Similarly, pH i was greatest in BR>HEPES>BF. Increasing pH i with NH 4 Cl also increased lactate production in BF or BR, indicating that the increased lactate production in BR is not due to HCO 3 - itself. Glucose transport capacity, as measured by 2-N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)Amino-2-Deoxyglucose (2-NBDG) uptake was unaffected by the three incubation conditions. Using Laconic, a FRET sensor for lactate, we found that intracellular [lactate] increased immediately and transiently when cells were switched from BF to BR perfusion indicating increased lactate production with subsequent matching of efflux. Moreover, induction of acute lactate influx by perfusion pulses of 10 mM lactate increased intracellular [lactate] significantly faster in BF than in BR, consistent with higher lactate production and efflux in BR. In summary, our results indicate that glycolytic flux and lactate production increase in BR due to increased pH i , consistent with the well-known pH sensitivity of phosphofructokinase, the rate limiting enzyme in glycolysis., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

SLC4A11 mutations are associated with Fuchs' endothelial corneal dystrophy (FECD), congenital hereditary endothelial dystrophy (CHED) and Harboyan syndrome (endothelial dystrophy with auditory deficiency). Mice with genetically ablated Slc4a11 recapitulate CHED, exhibiting significant corneal edema and altered endothelial morphology. We recently demonstrated that SLC4A11 functions as an NH 3 sensitive, electrogenic H + transporter. Here, we investigated the properties of five clinically relevant SLC4A11 mutants: R125H, W240S, C386R, V507I and N693A, relative to wild type, expressed in a PS120 fibroblast cell line. The effect of these mutations on the NH 4 Cl-dependent transporter activity was investigated by intracellular pH and electrophysiology measurements. Relative to plasma membrane expression of NaK ATPase, there were no significant differences in plasma membrane SLC4A11 expression among each mutant and wild type. All mutants revealed a marked decrease in acidification in response to NH 4 Cl when compared to wild type, indicating a decreased H + permeability in mutants. All mutants exhibited significantly reduced H + currents at negative holding potentials as compared to wild type. Uniquely, the C386R and W240S mutants exhibited a different inward current profile upon NH 4 Cl challenges, suggesting an altered transport mode. Thus, our data suggest that these SLC4A11 mutants, rather than having impaired protein trafficking, show altered H + flux properties., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The corneal endothelium is responsible for maintaining the hydration of the cornea. This is through a "Pump-Leak" mechanism where the active transport properties of the endothelium represent the "Pump" and the stromal swelling pressure represents the "Leak". For the "Pump", Na(+), K(+) ATPase activity and the presence of HCO(3)(-), Cl(-), and carbonic anhydrase activity are required. Several basolateral (stromal side) anion transporters, apical (facing the aqueous humor) ion channels and water channels have been identified that could support a model for ion secretion as the basis for the endothelial pump, however evidence of sustained anion fluxes, osmotic gradients or the need for water channels is lacking. This has prompted consideration of other models, such as Electro-osmosis, and consideration of metabolite flux as components of the endothelial pump. Although the conditions under which the "Pump" is supported are known, a complete model of the endothelial "Pump" has yet to emerge., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The cystic fibrosis transmembrane conductance regulator (CFTR) is present on the apical membrane of corneal endothelial cells. Increasing intracellular [cAMP] with forskolin stimulates an NPPB and glibenclamide-inhibitable apical Cl(-) and HCO(3)(-) permeability [Sun, X.C., Bonanno, J.A., 2002. Expression, localization, and functional evaluation of CFTR in bovine corneal endothelial cells. Am. J. Physiol. Cell Physiol. 282, C673-C683]. To definitively determine that the increased permeability is dependent on CFTR, we used an siRNA knockdown approach. Apical Cl(-) and HCO(3)(-) permeability and steady-state HCO(3)(-) flux were measured in the presence or absence of forskolin using cultured bovine corneal endothelial cells that were transfected with CFTR siRNA or a scrambled sequence control. CFTR protein expression was reduced by approximately 80% in CFTR siRNA treated cultures. Forskolin (10 microM) increased apical chloride permeability by 7-fold, which was reduced to control level in siRNA treated cells. CFTR siRNA treatment had no effect on baseline apical chloride permeability. Apical HCO(3)(-) permeability was increased 2-fold by 10 microM forskolin, which was reduced to control level in siRNA treated cultures. Similarly, there was no effect on baseline apical HCO(3)(-) permeability by knocking down CFTR expression. The steady-state apical-basolateral pH gradient (DeltapH) at 4h in control cultures was increased approximately 2.5-fold by forskolin. In CFTR siRNA treated cells, the baseline DeltapH was similar to control, however forskolin did not have a significant effect. We conclude that forskolin induced increases in apical HCO(3)(-) permeability in bovine corneal endothelium requires CFTR. However, CFTR does not have a major role in determining baseline apical chloride or HCO(3)(-) permeability.

Corneal endothelium secretes HCO(3)(-) from basolateral (stroma) to apical (anterior chamber) compartments. Apical HCO(3)(-) permeability can be enhanced by increasing [Ca(2+)](i). We hypothesized that the bovine calcium-activated chloride channel 1 (bCLCA1), shown previously by PCR screening to be expressed in corneal endothelium, is involved in Ca(2+) activated apical HCO(3)(-) permeability. bCLCA1 expression in cultured bovine corneal endothelial cells (CBCEC) was examined by in situ hybridization analysis, immunoblotting, immunofluorescence and confocal microscopy. Rabbit polyclonal antibodies were generated using a 14 aa polypeptide (417-430) from the predicted sequence of bCLCA1. The small interference RNA (siRNA) knock down technique was used to evaluate the functional involvement of bCLCA1 in apical HCO(3)(-) permeability. In situ hybridization confirmed prominent bCLCA1-specific mRNA expression in CBCEC. bCLCA1 antiserum detected the heterologously expressed bCLCA1 in HEK293 cells and a 90kDa band in CBCEC, which was absent when using the pre-immune serum or antigen absorption of serum. Immunofluoresence staining with anti-bCLCA1 antibody and confocal microscopy indicates an apical membrane location in CBCEC. In CBCEC transfected with bCLCA1 specific siRNA, bCLCA1 expression was reduced by 80%, while transfection with siControl scrambled sequence had no effect. Increasing [Ca(i)(2+)] by application of ATPgammaS or cyclopiazonic acid (CPA) increased apical HCO(3)(-) permeability in siControl transfected CBCEC, while having no effect on apical HCO(3)(-) permeability in bCLCA1 specific siRNA transfected cells. Baseline HCO(3)(-) permeability, however, was not different between controls and siRNA treated cells. We conclude that the calcium-activated chloride channel (bCLCA1) is expressed in bovine corneal endothelial cells and can contribute to Ca(2+) dependent apical HCO(3)(-) permeability, but not resting permeability, across the corneal endothelium.

The purpose of this study, was to determine whether hypoxia preconditioning can protect corneal stromal cells from UV stress and cytokine mediated apoptosis. Two models were implemented. First, primary cultured bovine corneal fibroblasts were preconditioned with 0.5-1.5% O2 for 4 hr and stressed with UV-irradiation or stimulation of Fas receptor. Second, bovine eyes were preconditioned with 0.5% O2 for 4 hr and stressed by epithelial scraping to induce anterior keratocyte apoptosis. Cell fate was analyzed at 4 hr after stress using quantitative TUNEL or condensed nuclei assays. Cell apoptotic rates in hypoxia preconditioned groups were significantly lower (50-80%) than that of normoxia control groups. Hypoxia prevented the degradation of the transcription factor HIF-1alpha. CoCl2 (100-200 microM), a chemical inducer of HIF-1alpha, also produced strong protection against UV and Fas induced apoptosis. Moreover, hypoxia preconditioned media protected cells against UV-induced apoptosis. These findings demonstrate that hypoxia preconditioning has a generalized protective effect against stromal fibroblast and keratocyte apoptosis and suggest that HIF-1alpha mediated expression and secretion of protective factors is involved.

We previously found that activation of purinergic receptors mobilizes Ca2+ and enhances bicarbonate transport in bovine corneal endothelial cells (BCEC). Since transient receptor potential channel 4 (TRPC) has been reported to be a candidate for capacitative calcium entry (CCE) and receptor operated calcium entry (ROC), we examined the expression of TRPC4 and evaluated the potential involvement of TRPC4 in CCE or ROC in BCEC. The C-terminus of TRPC4 was fused into the glutathione S-transferase (GST) expression vector. The fusion protein GST-TRPC4c was induced in bacteria and purified by affinity chromatography. An antibody was raised in rabbit by using the purified GST-TRPC4c antigen. In Western blotting, the TRPC4 antibody recognized the fusion protein while the pre-immune IgG did not. The TRPC4 antibody recognized a band at around 80 kD for membrane proteins from both the fresh and cultured BCEC. The pre-immune IgG could not detect bands at the same size. Incubation with the TRPC4c antigen abolished the 80 kD band. Immunofluorescence using the TRPC4 antibody stained both fresh and cultured BCEC, while pre-immune IgG did not. RNAi knocked down the expression of TRPC4 in cultured BCEC. Ca2+ entry induced by the purinergic receptor agonist ATP, was increased in TRPC4-siRNA transfected cells compared with the scrambled siRNA control, while Ca2+ entry induced by store depletion through blocking the endoplasmic reticulum Ca2+ pump, did not differ between the siRNA and scrambled siRNA-treated cells. Taken together, these results show that TRPC4 protein is expressed in the bovine corneal endothelial cells and may be a negative regulator in ROC stimulated by purinergic activation, but not by store depletion itself.

Previous studies indicated that adenosine can increase [cAMP](i) and stimulate fluid transport by corneal endothelium. The purpose of this study was to determine which adenosine receptor subtype(s) are expressed and to examine their functional roles in modulating [cAMP](i), [Ca(2+)](i) and effects on Cl(-) permeability in corneal endothelium. We screened bovine corneal endothelium (BCE) for adenosine receptor subtypes by RT-PCR and immunoblotting, and examined the effects of pharmacological agents on adenosine stimulated Cl(-) transport, [cAMP](i) and [Ca(2+)](i). RT-PCR indicated the presence of A(1) and A(2b) adenosine receptors, while A(2a) and A(3) were negative. Western blot (WB) confirmed the presence of A(2b) ( approximately 50 kDa) and A(1) ( approximately 40 kDa) in fresh and cultured BCE. Ten micromolar adenosine increased [cAMP](i) by 2.7-fold over control and this was inhibited 66% by 10 microm alloxazine, a specific A(2b) blocker. A(1) activation with 1 micromN(6)-CPA (a specific A(1) agonist) or 100 nm adenosine decreased [cAMP](i) by 23 and 6%, respectively. Adenosine had no effect on [Ca(2+)](i) mobilization. Indirect immunofluorescence localized A(2b) receptors to the lateral membrane and A(1) to the apical surface in cultured BCE. Adenosine significantly increased apical Cl(-) permeability by 2.2 times and this effect was nearly abolished by DMPX (10 microm), a general A(2) blocker. Adenosine-induced membrane depolarization was also inhibited by 33% (n=6) in the presence of alloxazine. Bovine corneal endothelium expresses functional A(1) and A(2b) adenosine receptors. A(1), preferentially activated at <1 microm adenosine, acts to decrease [cAMP](i) and A(2b), activated at >1 microm adenosine, increase [cAMP](i).

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.

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.)

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.

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.

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)

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.

Intracellular pH (pHi) was measured in basal corneal epithelial cells from fresh corneal explants using the pH sensitive fluorescent dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). The overlying superficial and wing cells were removed by mechanical scraping to expose basal cells attached to their basal lamina. Tissue pieces with attached, dye-loaded basal cells were mounted in a microscope-stage-perfusion chamber which allowed rapid changes of Ringer's bathing solutions while measuring BCECF fluorescence. In NaCl-Ringer's (bicarbonate free). pHo 7.40, resting cell pHi was 7.34 +/- 0.03 (+/- S.E.M., n = 31). Buffering capacity measured by NH4Cl treatment was 31 mM pH at pHi 7.34 and increased with decreasing pHi. Recovery from 20 mM NH4Cl-induced acid loads was dependent on the presence of Na and inhibited by 1 mM amiloride. Adding amiloride to resting cells caused a slow, reversible acidification (0.04 pH units min-1). These results indicate the presence of Na:H exchange, its role in responding to acid loads and in maintaining resting cell pHi. Activation of Na:H by Nao showed simple saturation kinetics, with Km = 44 mM. Net proton efflux via Na:H exchange increased with decreasing pHi and was enhanced by depleting cells of Nai, suggesting roles for both pHi and Nai in control of Na:H activation.