Corneal Edema in Inducible Slc4a11 Knockout Is Initiated by Mitochondrial Superoxide Induced Src Kinase Activation.

PURPOSE: Inducible Slc4a11 KO leads to corneal edema by disruption of the pump and barrier functions of the corneal endothelium (CE). The loss of Slc4a11 NH3-activated mitochondrial uncoupling leads to mitochondrial membrane potential hyperpolarization-induced oxidative stress. The goal of this study was to investigate the link between oxidative stress and the failure of pump and barrier functions and to test different approaches to revert the process. METHODS: Mice which were homozygous for Slc4a11 Flox and Estrogen receptor -Cre Recombinase fusion protein alleles at 8 weeks of age were fed Tamoxifen (Tm)-enriched chow (0.4 g/Kg) for 2 weeks, and controls were fed normal chow. During the initial 14 days, Slc4a11 expression, corneal thickness (CT), stromal [lactate], Na+-K+ ATPase activity, mitochondrial superoxide levels, expression of lactate transporters, and activity of key kinases were assessed. In addition, barrier function was assessed by fluorescein permeability, ZO-1 tight junction integrity, and cortical cytoskeleton F-actin morphology. RESULTS: Tm induced a rapid decay in Slc4a11 expression that was 84% complete at 7 days and 96% complete at 14 days of treatment. Superoxide levels increased significantly by day 7; CT and fluorescein permeability by day 14. Tight junction ZO-1 distribution and the cortical cytoskeleton were disrupted at day 14, concomitant with decreased expression of Cldn1, yet with increased tyrosine phosphorylation. Stromal lactate increased by 60%, Na+-K+ ATPase activity decreased by 40%, and expression of lactate transporters MCT2 and MCT4 significantly decreased, but MCT1 was unchanged at 14 days. Src kinase was activated, but not Rock, PKCα, JNK, or P38Mapk. Mitochondrial antioxidant Visomitin (SkQ1, mitochondrial targeted antioxidant) and Src kinase inhibitor eCF506 significantly slowed the increase in CT, with concomitant decreased stromal lactate retention, improved barrier function, reduced Src activation and Cldn1 phosphorylation, and rescued MCT2 and MCT4 expression. CONCLUSIONS: Slc4a11 KO-induced CE oxidative stress triggered increased Src kinase activity that resulted in perturbation of the pump components and barrier function of the CE.

Nanoliposomes for Sensing Local Osmolarity of the Tear Film on the Corneal Surface.

Purpose: Local hotspots of elevated tear hyperosmolarity (exceeding 900 mOsM) are predicted in dry eye disease (DED) but have not been measured. This study aims to develop, characterize, and evaluate the suitability of fluorescent nanoliposomes for noninvasive sensing of the local osmolarity of the tear film. Methods: Fluorescent nanoliposomes, loaded with calcein (susceptible to self-quenching; sensor dye) and sulforhodamine 101 (SR101; reference dye), were produced by the thin-film hydration method. Results: Dynamic light scattering measurements and Cryo-TEM showed that liposomes were negatively charged (-23.7 ± 1.5 mV), spherical (diameter = 117.9 ± 6.4 nm), and uniform in size (polydispersity index = 0.15 ± 0.02). These properties were unaffected by cold storage (4°C; 14 days), but dye leakage was significant after 3 days. Swelling and shrinkage of the liposomes by exposure to hypoosmotic and hyperosmotic media led to rapid dequenching and quenching of calcein fluorescence (FGreen), with no effect on SR101 fluorescence (FRed). The ratio FGreen/FRed decreased with increasing osmolarity and vice versa, obeying the Boyle van't Hoff relationship. When liposomes were dispersed in a gelatin film with dynamic radial sucrose gradients, local FGreen/FRed decreased with increasing hyperosmolarity as predicted. When instilled on the hydrophilic surface of contact lenses or ex vivo corneas, nanoliposomes dispersed evenly as thin films. Importantly, the measured FGreen/FRed declined continuously with evaporation and consequent increase in their osmolarities. Conclusions: The study provides proof of principle for noninvasive measurement of local tear film osmolarity based on osmosensitive fluorescent nanoliposomes. The strategy can potentially advance our understanding of the pathophysiology of DED.

Rescue of the Congenital Hereditary Endothelial Dystrophy Mouse Model by Adeno-Associated Viruse-Mediated Slc4a11 Replacement.

Purpose: Congenital hereditary endothelial dystrophy (CHED) is a rare condition that manifests at an early age showing corneal edema, increased oxidative stress, mitochondrial dysfunction, and eventually apoptosis of the endothelium due to loss of function of the membrane transport protein SLC4A11. This project tested whether replacing Slc4a11 into the Slc4a11 -/- CHED mouse model can reverse the disease-associated phenotypes. Design: Experimental study. Participants: Five-week-old or 11-week-old Slc4a11 -/- mice. Age- and gender-matched Slc4a11 +/+ animals were used as controls. A total of 124 animals (62 female, and 62 male) were used in this study. Fifty-three animals of the genotype Slc4a11 +/+ were used as age- and gender-matched noninjected controls. Seventy-one Slc4a11 -/- mice were administered anterior chamber injections of adeno-associated virus (AAV). Methods: Anterior chambers of young (5 weeks old) or older (11 weeks old) Slc4a11 -/- mice were injected once with adeno-associated virus serotype 9 (AAV9) mouse Slc4a11 or AAV9-Null vectors. Corneal thickness was measured using OCT. End point analysis included corneal endothelial cell density, mitochondrial oxidative stress, and corneal lactate concentration. Main Outcome Measures: Corneal thickness, endothelial cell loss, lactate levels, and mitochondrial oxidative stress. Results: In the young animals, AAV9-Slc4a11 reversed corneal edema, endothelial cell loss, mitochondrial oxidative stress, lactate transporter expression, and corneal lactate concentration to the levels observed in wild-type animals. In the older animals, gene replacement did not reverse the phenotype but prevented progression. Conclusions: Functional rescue of CHED phenotypes in the Slc4a11 -/- mouse is possible; however, early intervention is critical.

Mitochondrial ROS in Slc4a11 KO Corneal Endothelial Cells Lead to ER Stress.

Recent studies from Slc4a11 -/- mice have identified glutamine-induced mitochondrial dysfunction as a significant contributor toward oxidative stress, impaired lysosomal function, aberrant autophagy, and cell death in this Congenital Hereditary Endothelial Dystrophy (CHED) model. Because lysosomes are derived from endoplasmic reticulum (ER)-Golgi, we asked whether ER function is affected by mitochondrial ROS in Slc4a11 KO corneal endothelial cells. In mouse Slc4a11 -/- corneal endothelial tissue, we observed the presence of dilated ER and elevated expression of ER stress markers BIP and CHOP. Slc4a11 KO mouse corneal endothelial cells incubated with glutamine showed increased aggresome formation, BIP and GADD153, as well as reduced ER Ca2+ release as compared to WT. Induction of mitoROS by ETC inhibition also led to ER stress in WT cells. Treatment with the mitochondrial ROS quencher MitoQ, restored ER Ca2+ release and relieved ER stress markers in Slc4a11 KO cells in vitro. Systemic MitoQ also reduced BIP expression in Slc4a11 KO endothelium. We conclude that mitochondrial ROS can induce ER stress in corneal endothelial cells.

The H+ Transporter SLC4A11: Roles in Metabolism, Oxidative Stress and Mitochondrial Uncoupling.

Solute-linked cotransporter, SLC4A11, a member of the bicarbonate transporter family, is an electrogenic H+ transporter activated by NH3 and alkaline pH. Although SLC4A11 does not transport bicarbonate, it shares many properties with other members of the SLC4 family. SLC4A11 mutations can lead to corneal endothelial dystrophy and hearing deficits that are recapitulated in SLC4A11 knock-out mice. SLC4A11, at the inner mitochondrial membrane, facilitates glutamine catabolism and suppresses the production of mitochondrial superoxide by providing ammonia-sensitive H+ uncoupling that reduces glutamine-driven mitochondrial membrane potential hyperpolarization. Mitochondrial oxidative stress in SLC4A11 KO also triggers dysfunctional autophagy and lysosomes, as well as ER stress. SLC4A11 expression is induced by oxidative stress through the transcription factor NRF2, the master regulator of antioxidant genes. Outside of the corneal endothelium, SLC4A11's function has been demonstrated in cochlear fibrocytes, salivary glands, and kidneys, but is largely unexplored overall. Increased SLC4A11 expression is a component of some "glutamine-addicted" cancers, and is possibly linked to cells and tissues that rely on glutamine catabolism.

RNA sequencing uncovers alterations in corneal endothelial metabolism, pump and barrier functions of Slc4a11 KO mice.

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.

Mitochondrial Targeting of the Ammonia-Sensitive Uncoupler SLC4A11 by the Chaperone-Mediated Carrier Pathway in Corneal Endothelium.

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.

Mitochondrial ROS Induced Lysosomal Dysfunction and Autophagy Impairment in an Animal Model of Congenital Hereditary Endothelial Dystrophy.

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.

Inducible Slc4a11 Knockout Triggers Corneal Edema Through Perturbation of Corneal Endothelial Pump.

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.

Bicarbonate activates glycolysis and lactate production in corneal endothelial cells by increased pHi.

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 (pHi) 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, pHi was greatest in BR>HEPES>BF. Increasing pHi with NH4Cl also increased lactate production in BF or BR, indicating that the increased lactate production in BR is not due to HCO3- 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 pHi, consistent with the well-known pH sensitivity of phosphofructokinase, the rate limiting enzyme in glycolysis.

Corneal Endothelial Pump Coupling to Lactic Acid Efflux in the Rabbit and Mouse.

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.

Ammonia sensitive SLC4A11 mitochondrial uncoupling reduces glutamine induced oxidative stress.

SLC4A11 is a NH3 sensitive membrane transporter with H+ channel-like properties that facilitates Glutamine catabolism in Human and Mouse corneal endothelium (CE). Loss of SLC4A11 activity induces oxidative stress and cell death, resulting in Congenital Hereditary Endothelial Dystrophy (CHED) with corneal edema and vision loss. However, the mechanism by which SLC4A11 prevents ROS production and protects CE is unknown. Here we demonstrate that SLC4A11 is localized to the inner mitochondrial membrane of CE and SLC4A11 transfected PS120 fibroblasts, where it acts as an NH3-sensitive mitochondrial uncoupler that enhances glutamine-dependent oxygen consumption, electron transport chain activity, and ATP levels by suppressing damaging Reactive Oxygen Species (ROS) production. In the presence of glutamine, Slc4a11-/- (KO) mouse CE generate significantly greater mitochondrial superoxide, a greater proportion of damaged depolarized mitochondria, and more apoptotic cells than WT. KO CE can be rescued by MitoQ, reducing NH3 production by GLS1 inhibition or dimethyl αKetoglutarate supplementation, or by BAM15 mitochondrial uncoupling. Slc4a11 KO mouse corneal edema can be partially reversed by αKetoglutarate eye drops. Moreover, we demonstrate that this role for SLC4A11 is not specific to CE cells, as SLC4A11 knockdown in glutamine-addicted colon carcinoma cells reduced glutamine catabolism, increased ROS production, and inhibited cell proliferation. Overall, our studies reveal a unique metabolic mechanism that reduces mitochondrial oxidative stress while promoting glutamine catabolism.

Evidence for a GPR18 Role in Chemotaxis, Proliferation, and the Course of Wound Closure in the Cornea.

PURPOSE: We previously showed that cannabinoid-related GPR18 receptors are present in the murine corneal epithelium, but their function remains unknown. The related CB1 receptors regulate corneal healing, possibly via chemotaxis. We therefore examined a potential role for GPR18 in corneal epithelial chemotaxis and wound healing. METHODS: We examined GPR18 messenger RNA (mRNA) and protein expression in the cornea. We additionally examined GPR18 action in cultured bovine corneal epithelial cells (bCECs) using Boyden and tracking assays, as well as proliferation and signaling. Finally, we examined wound closure in murine corneal explants. RESULTS: GPR18 mRNA was upregulated with injury in the mouse cornea. GPR18 protein was present in basal epithelial cells of the mouse and cow and redistributed to the wound site upon injury. GPR18 ligand N-arachidonoylglycine induced bCEC chemotaxis. The endocannabinoid arachidonoylethanolamine also induced chemotaxis via fatty acid amide hydrolase-mediated metabolism to N-arachidonoylglycine. GPR18 receptor activation additionally induced bCEC proliferation. In an explant model, the GPR18 antagonist O-1918 slowed corneal epithelial cell migration and the rate of corneal wound closure. CONCLUSIONS: Corneal GPR18 activation induced both chemotaxis and proliferation in corneal epithelial cells in vitro and impacted wound healing. GPR18 may contribute to the maintenance of corneal integrity.

R125H, W240S, C386R, and V507I SLC4A11 mutations associated with corneal endothelial dystrophy affect the transporter function but not trafficking in PS120 cells.

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 NH3 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 NH4Cl-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 NH4Cl 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 NH4Cl 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.

Hypoxia and the Prolyl Hydroxylase Inhibitor FG-4592 Protect Corneal Endothelial Cells From Mechanical and Perioperative Surgical Stress.

PURPOSE: To determine whether hypoxia preconditioning can protect corneal endothelial cells from mechanical stress and perioperative procedures mimicking Descemet stripping automated endothelial keratoplasty (DSAEK). METHODS: Preconditioning was delivered by 2 hours of 0.5% oxygen incubation in a hypoxia chamber or by exposure to the prolyl hydroxylase inhibitor FG-4592, which prevents hypoxia-inducible factor-1 alpha degradation. Damage to whole corneas was produced by brief sonication. To mimic use with DSAEK, FG-4592-preconditioned and control donor corneas were dissected with a microkeratome, and the posterior donor button was pulled through a transplant insertion device (Busin glide). The area of endothelial damage was determined by trypan blue staining. RESULTS: In all cases, hypoxia preconditioning or incubation with FG-4592 protected corneal endothelial cells from death by mechanical stress. Hypoxia-preconditioned human and rabbit corneas showed 19% and 29% less cell loss, respectively, relative to controls, which were both significant at P < 0.05. FG-4592 preconditioning reduced endothelial cell loss associated with preparation and insertion of DSAEK grafts by 23% relative to the control (P < 0.01). CONCLUSIONS: These results support the hypothesis that preconditioning by hypoxia or exposure to FG-4592 improves corneal endothelial cell survival and may also provide protection during surgical trauma.

Conditionally Immortal Slc4a11-/- Mouse Corneal Endothelial Cell Line Recapitulates Disrupted Glutaminolysis Seen in Slc4a11-/- Mouse Model.

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.

Glutaminolysis is Essential for Energy Production and Ion Transport in Human Corneal Endothelium.

Corneal endothelium (CE) is among the most metabolically active tissues in the body. This elevated metabolic rate helps the CE maintain corneal transparency by its ion and fluid transport properties, which when disrupted, leads to visual impairment. Here we demonstrate that glutamine catabolism (glutaminolysis) through TCA cycle generates a large fraction of the ATP needed to maintain CE function, and this glutaminolysis is severely disrupted in cells deficient in NH3:H+ cotransporter Solute Carrier Family 4 Member 11 (SLC4A11). Considering SLC4A11 mutations leads to corneal endothelial dystrophy and sensorineural deafness, our results indicate that SLC4A11-associated developmental and degenerative disorders result from altered glutamine catabolism. Overall, our results describe an important metabolic mechanism that provides CE cells with the energy required to maintain high level transport activity, reveal a direct link between glutamine metabolism and developmental and degenerative neuronal diseases, and suggest an approach for protecting the CE during ophthalmic surgeries.

Fluid transport by the cornea endothelium is dependent on buffering lactic acid efflux.

Maintenance of corneal hydration is dependent on the active transport properties of the corneal endothelium. We tested the hypothesis that lactic acid efflux, facilitated by buffering, is a component of the endothelial fluid pump. Rabbit corneas were perfused with bicarbonate-rich (BR) or bicarbonate-free (BF) Ringer of varying buffering power, while corneal thickness was measured. Perfusate was collected and analyzed for lactate efflux. In BF with no added HEPES, the maximal corneal swelling rate was 30.0 ± 4.1 µm/h compared with 5.2 ± 0.9 µm/h in BR. Corneal swelling decreased directly with [HEPES], such that with 60 mM HEPES corneas swelled at 7.5 ± 1.6 µm/h. Perfusate [lactate] increased directly with [HEPES]. Similarly, reducing the [HCO3 (-)] increased corneal swelling and decreased lactate efflux. Corneal swelling was inversely related to Ringer buffering power (ß), whereas lactate efflux was directly related to ß. Ouabain (100 µM) produced maximal swelling and reduction in lactate efflux, whereas carbonic anhydrase inhibition and an monocarboxylic acid transporter 1 inhibitor produced intermediate swelling and decreases in lactate efflux. Conversely, 10 µM adenosine reduced the swelling rate to 4.2 ± 0.8 µm/h and increased lactate efflux by 25%. We found a strong inverse relation between corneal swelling and lactate efflux (r = 0.98, P < 0.0001). Introducing lactate in the Ringer transiently increased corneal thickness, reaching a steady state (0 ± 0.6 µm/h) within 90 min. We conclude that corneal endothelial function does not have an absolute requirement for bicarbonate; rather it requires a perfusing solution with high buffering power. This facilitates lactic acid efflux, which is directly linked to water efflux, indicating that lactate flux is a component of the corneal endothelial pump.

Cannabinoid-induced chemotaxis in bovine corneal epithelial cells.

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.

Human SLC4A11 Is a Novel NH3/H+ Co-transporter.

SLC4A11 has been proposed to be an electrogenic membrane transporter, permeable to Na(+), H(+) (OH(-)), bicarbonate, borate, and NH4 (+). Recent studies indicate, however, that neither bicarbonate or borate is a substrate. Here, we examined potential NH4 (+), Na(+), and H(+) contributions to electrogenic ion transport through SLC4A11 stably expressed in Na(+)/H(+) exchanger-deficient PS120 fibroblasts. Inward currents observed during exposure to NH4Cl were determined by the [NH3]o, not [NH4 (+)]o, and current amplitudes varied with the [H(+)] gradient. These currents were relatively unaffected by removal of Na(+), K(+), or Cl(-) from the bath but could be reduced by inclusion of NH4Cl in the pipette solution. Bath pH changes alone did not generate significant currents through SLC4A11, except immediately following exposure to NH4Cl. Reversal potential shifts in response to changing [NH3]o and pHo suggested an NH3/H(+)-coupled transport mode for SLC4A11. Proton flux through SLC4A11 in the absence of ammonia was relatively small, suggesting that ammonia transport is of more physiological relevance. Methylammonia produced currents similar to NH3 but with reduced amplitude. Estimated stoichiometry of SLC4A11 transport was 1:2 (NH3/H(+)). NH3-dependent currents were insensitive to 10 µM ethyl-isopropyl amiloride or 100 µM 4,4'- diisothiocyanatostilbene-2,2'-disulfonic acid. We propose that SLC4A11 is an NH3/2H(+) co-transporter exhibiting unique characteristics.