109 results on '"Bonanno JA"'
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2. Corneal Edema in Inducible Slc4a11 Knockout Is Initiated by Mitochondrial Superoxide Induced Src Kinase Activation.
- Author
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Ogando DG, Kim ET, Li S, and Bonanno JA
- Subjects
- Mice, Animals, src-Family Kinases metabolism, Superoxides metabolism, Antioxidants, Mice, Knockout, Lactates, Fluoresceins, Adenosine Triphosphatases metabolism, Anion Transport Proteins metabolism, Corneal Edema, Symporters
- Abstract
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 NH
3 -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.- Published
- 2023
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3. Nanoliposomes for Sensing Local Osmolarity of the Tear Film on the Corneal Surface.
- Author
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Thanuja MY, Ranganath SH, Bonanno JA, and Srinivas SP
- Subjects
- Humans, Tears, Osmolar Concentration, Cornea, Coloring Agents, Sucrose, Liposomes, Dry Eye Syndromes diagnosis, Dry Eye Syndromes drug therapy
- Abstract
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 (F
Green ), 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.- Published
- 2022
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4. Mitochondrial ROS in Slc4a11 KO Corneal Endothelial Cells Lead to ER Stress.
- Author
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Shyam R, Ogando DG, and Bonanno JA
- Abstract
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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Shyam, Ogando and Bonanno.)- Published
- 2022
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5. Rescue of the Congenital Hereditary Endothelial Dystrophy Mouse Model by Adeno-Associated Viruse-Mediated Slc4a11 Replacement.
- Author
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Shyam R, Ogando DG, Kim ET, Murugan S, Choi M, and Bonanno JA
- Abstract
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.- Published
- 2022
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6. The H + Transporter SLC4A11: Roles in Metabolism, Oxidative Stress and Mitochondrial Uncoupling.
- Author
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Bonanno JA, Shyam R, Choi M, and Ogando DG
- Subjects
- Animals, Anion Transport Proteins genetics, Gene Expression Regulation, Genetic Predisposition to Disease, Humans, Anion Transport Proteins metabolism, Mitochondria metabolism, Oxidative Stress genetics, Protons
- Abstract
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.- Published
- 2022
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7. RNA sequencing uncovers alterations in corneal endothelial metabolism, pump and barrier functions of Slc4a11 KO mice.
- Author
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Ogando DG and Bonanno JA
- Subjects
- Animals, Blotting, Western, Corneal Edema metabolism, Fluorescent Antibody Technique, Indirect, Glutamine metabolism, Glycolysis, Mice, Mice, Knockout, Oxidative Stress, Oxygen Consumption physiology, Real-Time Polymerase Chain Reaction, Sequence Analysis, RNA, Anion Transport Proteins genetics, Corneal Edema genetics, Endothelium, Corneal metabolism, Monocarboxylic Acid Transporters metabolism, Muscle Proteins metabolism, Sodium-Potassium-Exchanging ATPase metabolism, Symporters genetics, Symporters metabolism
- Abstract
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.)
- Published
- 2022
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8. The determination of thiocyanate in the blood plasma and holding water of Amphiprion clarkii after exposure to cyanide.
- Author
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Bonanno JA, Breen NE, Tlusty MF, Andrade L, and Rhyne AL
- Abstract
The illegal practice of cyanide fishing continues throughout the Indo-Pacific. To combat this destructive fishing method, a reliable test to detect whether a fish has been captured using cyanide (CN) is needed. We report on the toxicokinetics of acute, pulsed CN exposure and chronic thiocyanate (SCN) exposure, the major metabolite of CN, in the clownfish species, Amphiprion clarkii . Fish were pulse exposed to 50 ppm CN for 20 or 45 s or chronically exposed to 100 ppm SCN for 12 days and blood plasma levels of SCN were measured. SCN blood plasma levels reached a maximum concentration (301-468 ppb) 0.13-0.17 days after exposure to CN and had a 0.1 to 1.2 day half-life. The half-life of blood plasma SCN after chronic exposure to SCN was found to be 0.13 days. Interestingly, we observed that when a fish, with no previous CN or SCN exposure, was placed in holding water spiked to 20 ppb SCN, there was a steady decrease in the SCN concentration in the holding water until it could no longer be detected at 24 hrs. Under chronic exposure conditions (100 ppm, 12 days), trace levels of SCN (∼40 ppb) were detected in the holding water during depuration but decreased to below detection within the first 24 hrs. Our holding water experiments demonstrate that low levels of SCN in the holding water of A . clarkii will not persist, but rather will quickly and steadily decrease to below detection limits refuting several publications. After CN exposure, A. clarkii exhibits a classic two compartment model where SCN is eliminated from the blood plasma and is likely distributed throughout the body. Similar studies of other species must be examined to continue to develop our understanding of CN metabolism in marine fish before a reliable cyanide detection test can be developed., Competing Interests: Lawrence J. Andrade is an employee of Dominion Diagnostics., (©2021 Bonanno et al.)
- Published
- 2021
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9. Mitochondrial Targeting of the Ammonia-Sensitive Uncoupler SLC4A11 by the Chaperone-Mediated Carrier Pathway in Corneal Endothelium.
- Author
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Choi M and Bonanno JA
- Subjects
- Animals, Anion Transport Proteins metabolism, Cells, Cultured, Corneal Dystrophies, Hereditary metabolism, Corneal Dystrophies, Hereditary pathology, Endothelium, Corneal drug effects, Endothelium, Corneal pathology, Mice, Mice, Knockout, Mitochondria metabolism, Molecular Chaperones drug effects, Molecular Chaperones genetics, Molecular Chaperones metabolism, Protein Transport, Symporters metabolism, Ammonia pharmacology, Anion Transport Proteins genetics, Corneal Dystrophies, Hereditary genetics, Endothelium, Corneal metabolism, Mitochondria pathology, Proteomics methods, Symporters genetics
- Abstract
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.
- Published
- 2021
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10. Mitochondrial ROS Induced Lysosomal Dysfunction and Autophagy Impairment in an Animal Model of Congenital Hereditary Endothelial Dystrophy.
- Author
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Shyam R, Ogando DG, Choi M, Liton PB, and Bonanno JA
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- Animals, Anion Transport Proteins genetics, Basic Helix-Loop-Helix Leucine Zipper Transcription Factors metabolism, Blotting, Western, Cathepsin L metabolism, Cells, Cultured, Corneal Dystrophies, Hereditary genetics, Corneal Dystrophies, Hereditary pathology, Endothelium, Corneal drug effects, Endothelium, Corneal metabolism, Gene Expression Regulation, Immunohistochemistry, Injections, Intraperitoneal, Mice, Mice, Inbred C57BL, Mice, Knockout, Microscopy, Fluorescence, Organophosphorus Compounds pharmacology, Real-Time Polymerase Chain Reaction, Symporters genetics, Transfection, Ubiquinone analogs & derivatives, Ubiquinone pharmacology, Autophagy physiology, Corneal Dystrophies, Hereditary metabolism, Disease Models, Animal, Lysosomes metabolism, Mitochondria metabolism, Reactive Oxygen Species metabolism
- Abstract
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.
- Published
- 2021
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11. Inducible Slc4a11 Knockout Triggers Corneal Edema Through Perturbation of Corneal Endothelial Pump.
- Author
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Ogando DG, Shyam R, Kim ET, Wang YC, Liu CY, and Bonanno JA
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- Animals, Anion Transport Proteins metabolism, Endothelium, Corneal physiology, Mice, Oxidative Stress, Anion Transport Proteins genetics, Corneal Edema genetics, Corneal Edema metabolism, Corneal Edema physiopathology, Disease Models, Animal, Mice, Knockout genetics, Mice, Knockout metabolism, Symporters genetics
- Abstract
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.
- Published
- 2021
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12. Bicarbonate activates glycolysis and lactate production in corneal endothelial cells by increased pH i .
- Author
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Li S, Shyam R, Ogando DG, and Bonanno JA
- Subjects
- Animals, Biological Transport, Active, Cattle, Cells, Cultured, Endothelium, Corneal cytology, Endothelium, Corneal metabolism, Hydrogen-Ion Concentration, Models, Animal, Bicarbonates metabolism, Lactic Acid biosynthesis
- Abstract
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, pHi was greatest in BR>HEPES>BF. Increasing pHi with NH4 Cl 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., (Copyright © 2020 Elsevier Ltd. All rights reserved.)- Published
- 2020
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13. Corneal Endothelial Pump Coupling to Lactic Acid Efflux in the Rabbit and Mouse.
- Author
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Li S, Kim E, Ogando DG, and Bonanno JA
- Subjects
- Animals, Anion Transport Proteins metabolism, Biological Transport, Active physiology, Cornea metabolism, Corneal Edema physiopathology, Enzyme Inhibitors pharmacology, Female, Male, Mice, Knockout, Ouabain pharmacology, Rabbits, Sodium-Potassium-Exchanging ATPase antagonists & inhibitors, Symporters metabolism, Endothelium, Corneal metabolism, Lactic Acid metabolism
- Abstract
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.
- Published
- 2020
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14. Ammonia sensitive SLC4A11 mitochondrial uncoupling reduces glutamine induced oxidative stress.
- Author
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Ogando DG, Choi M, Shyam R, Li S, and Bonanno JA
- Subjects
- Ammonia pharmacology, Animals, Endothelial Cells, Endothelium, Corneal metabolism, Gene Knockout Techniques, Humans, Matrix Metalloproteinases metabolism, Mice, Mitochondria drug effects, Mitochondria genetics, Models, Biological, Oxidative Phosphorylation, Oxygen metabolism, SLC4A Proteins genetics, Ammonia metabolism, Glutamine metabolism, Mitochondria metabolism, Oxidative Stress drug effects, SLC4A Proteins metabolism
- Abstract
SLC4A11 is a NH
3 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., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)- Published
- 2019
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15. Evidence for a GPR18 Role in Chemotaxis, Proliferation, and the Course of Wound Closure in the Cornea.
- Author
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Murataeva N, Daily L, Taylor X, Dhopeshwarkar A, Hu SS, Miller S, McHugh D, Oehler O, Li S, Bonanno JA, Mackie K, and Straiker A
- Subjects
- Animals, Cattle, Cell Movement physiology, Cells, Cultured, Mice, Mice, Inbred C57BL, RNA, Messenger metabolism, Up-Regulation, Cell Proliferation physiology, Chemotaxis physiology, Corneal Injuries metabolism, Epithelium, Corneal metabolism, Receptors, G-Protein-Coupled physiology, Wound Healing physiology
- Abstract
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.
- Published
- 2019
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16. On the half-life of thiocyanate in the plasma of the marine fish Amphiprion ocellaris : implications for cyanide detection.
- Author
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Breen NE, Bonanno JA, Hunt S, Grossman J, Brown J, Nolte H, and Rhyne AL
- Abstract
The illegal practice of using cyanide (CN) as a stunning agent to collect fish for both the marine aquarium and live fish food trades has been used throughout the Indo-Pacific for over 50 years. CN fishing is destructive to all life forms within the coral reef ecosystems where it is used and is certainly one of many anthropogenic activities that have led to 95% of the reefs in the Indo-Pacific being labeled at risk for degradation and loss. A field-deployable test for detecting fish caught using CN would assist in combating the use of this destructive practice, however, no reliable and robust test exists. Further, there is little toxicokinetic data available on marine fish to support the development of such a test, yet such data is critical to establishing the concentration range and time scale over which such a test would be viable. This study presents the first direct measurement of the half-life of the metabolite thiocyanate (SCN) after pulsed exposure to CN in a marine fish. SCN was measured in the plasma of Amphiprion ocellaris after exposure to 50 ppm CN for three exposure times (20, 45, and 60 s) using HPLC-UV and a C30 column pre-treated with polyethylene glycol. Plasma SCN levels observed are dose-dependent, reflecting a longer time for conversion of CN to SCN as the dose of CN increases. SCN plasma levels reached a maximum concentration (1.2-2.3 ppm) 12-20 h after exposure to CN. The half-life for the elimination of SCN was 1.01 ± 0.26 days for 45 s exposure and 0.44 ± 0.15 days for 20 s exposure. Fish were also directly exposed to SCN (100 ppm for 11 days) and the observed half-life for SCN elimination was 0.35 ± 0.07 days. Plasma SCN levels did not return to control levels, even after 41 days when exposed to CN but did return to control levels after 48 days when exposed to SCN. The similar half-lives observed for CN and SCN exposure suggests that SCN exposure can be used as a proxy for measuring the rate of SCN elimination following CN exposure. In order for plasma SCN to be used as a marker for CN exposure, these results must be extended to other species and endogenous levels of SCN in wild caught fish must be established., Competing Interests: The authors declare that they have no competing interests.
- Published
- 2019
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17. R125H, W240S, C386R, and V507I SLC4A11 mutations associated with corneal endothelial dystrophy affect the transporter function but not trafficking in PS120 cells.
- Author
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Li S, Hundal KS, Chen X, Choi M, Ogando DG, Obukhov AG, and Bonanno JA
- Subjects
- Ammonium Chloride metabolism, Animals, Anion Transport Proteins genetics, Cell Line, Corneal Dystrophies, Hereditary metabolism, Cricetinae, Fibroblasts metabolism, Humans, Hydrogen-Ion Concentration, Mutagenesis, Site-Directed, Patch-Clamp Techniques, Transfection, Anion Transport Proteins metabolism, Antiporters genetics, Cell Membrane metabolism, Corneal Dystrophies, Hereditary genetics, Point Mutation, Protein Transport physiology
- Abstract
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 NH4 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 NH4 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 NH4 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.)- Published
- 2019
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18. Hypoxia and the Prolyl Hydroxylase Inhibitor FG-4592 Protect Corneal Endothelial Cells From Mechanical and Perioperative Surgical Stress.
- Author
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Bhadange Y, Lautert J, Li S, Lawando E, Kim ET, Soper MC, Price FW Jr, Price MO, and Bonanno JA
- Subjects
- Animals, Cell Line, Cell Survival, Cytoprotection, Descemet Stripping Endothelial Keratoplasty, Endothelium, Corneal pathology, Glycine pharmacology, Humans, Perioperative Care, Pilot Projects, Rabbits, Stress, Mechanical, Corneal Endothelial Cell Loss prevention & control, Endothelium, Corneal drug effects, Glycine analogs & derivatives, Hypoxia prevention & control, Ischemic Preconditioning, Isoquinolines pharmacology, Oxygen pharmacology, Prolyl-Hydroxylase Inhibitors pharmacology
- Abstract
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.
- Published
- 2018
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19. Conditionally Immortal Slc4a11-/- Mouse Corneal Endothelial Cell Line Recapitulates Disrupted Glutaminolysis Seen in Slc4a11-/- Mouse Model.
- Author
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Zhang W, Ogando DG, Kim ET, Choi MJ, Li H, Tenessen JM, and Bonanno JA
- Subjects
- Animals, Anion Transport Proteins metabolism, Antigens, Polyomavirus Transforming genetics, Blotting, Western, Cell Line, Cell Proliferation physiology, Corneal Dystrophies, Hereditary genetics, Corneal Dystrophies, Hereditary pathology, Disease Models, Animal, Endothelium, Corneal pathology, Female, Gas Chromatography-Mass Spectrometry, Ion Transport physiology, Male, Mice, Mice, Inbred C57BL, Mice, Knockout, Mice, Transgenic, Real-Time Polymerase Chain Reaction, Anion Transport Proteins genetics, Corneal Dystrophies, Hereditary metabolism, Endothelium, Corneal metabolism, Glutamine metabolism, Symporters genetics
- Abstract
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.
- Published
- 2017
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20. Glutaminolysis is Essential for Energy Production and Ion Transport in Human Corneal Endothelium.
- Author
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Zhang W, Li H, Ogando DG, Li S, Feng M, Price FW Jr, Tennessen JM, and Bonanno JA
- Subjects
- Amino Acid Transport System X-AG genetics, Amino Acid Transport System X-AG metabolism, Animals, Carrier Proteins genetics, Carrier Proteins metabolism, Cell Line, Cells, Cultured, Citric Acid Cycle, Corneal Dystrophies, Hereditary genetics, Corneal Dystrophies, Hereditary metabolism, Corneal Dystrophies, Hereditary pathology, Epithelium, Corneal cytology, Epithelium, Corneal metabolism, Fuchs' Endothelial Dystrophy genetics, Fuchs' Endothelial Dystrophy metabolism, Fuchs' Endothelial Dystrophy pathology, Gene Expression, Humans, Ion Transport, Mice, Knockout, Microscopy, Fluorescence, Mutation, Rabbits, Reverse Transcriptase Polymerase Chain Reaction, SLC4A Proteins genetics, SLC4A Proteins metabolism, Adenosine Triphosphate metabolism, Endothelium, Corneal metabolism, Energy Metabolism, Glutamine metabolism
- Abstract
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 NH
3 :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., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)- Published
- 2017
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21. Fluid transport by the cornea endothelium is dependent on buffering lactic acid efflux.
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Li S, Kim E, and Bonanno JA
- Subjects
- Animals, Bicarbonates metabolism, Biological Transport, Active, Buffers, Carbonic Anhydrase Inhibitors pharmacology, Endothelium, Corneal drug effects, Female, HEPES chemistry, Hydrogen-Ion Concentration, In Vitro Techniques, Isotonic Solutions metabolism, Male, Models, Biological, Monocarboxylic Acid Transporters antagonists & inhibitors, Perfusion, Rabbits, Ringer's Lactate, Sodium-Potassium-Exchanging ATPase antagonists & inhibitors, Sodium-Potassium-Exchanging ATPase metabolism, Symporters antagonists & inhibitors, Time Factors, Endothelium, Corneal metabolism, Lactic Acid metabolism, Monocarboxylic Acid Transporters metabolism, Symporters metabolism
- Abstract
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 (β), whereas lactate efflux was directly related to β. 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., (Copyright © 2016 the American Physiological Society.)
- Published
- 2016
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22. Human SLC4A11 Is a Novel NH3/H+ Co-transporter.
- Author
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Zhang W, Ogando DG, Bonanno JA, and Obukhov AG
- Subjects
- Anion Transport Proteins chemistry, Anion Transport Proteins genetics, Antiporters chemistry, Antiporters genetics, Bicarbonates metabolism, Humans, Ion Transport, Protons, Symporters chemistry, Symporters genetics, Ammonia metabolism, Anion Transport Proteins metabolism, Antiporters metabolism, Hydrogen metabolism, Symporters metabolism
- Abstract
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., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)
- Published
- 2015
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23. Cannabinoid-induced chemotaxis in bovine corneal epithelial cells.
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Murataeva N, Li S, Oehler O, Miller S, Dhopeshwarkar A, Hu SS, Bonanno JA, Bradshaw H, Mackie K, McHugh D, and Straiker A
- Subjects
- Analysis of Variance, Animals, Benzoxazines pharmacology, Calcium Channel Blockers pharmacology, Cannabinoids metabolism, Cattle, Cell Proliferation drug effects, Cells, Cultured, Chemotaxis drug effects, Epithelial Cells drug effects, Epithelium, Corneal drug effects, Morpholines pharmacology, Naphthalenes pharmacology, RNA, Messenger analysis, Receptor, Cannabinoid, CB1 antagonists & inhibitors, Signal Transduction physiology, Wound Healing, Cannabinoids pharmacology, Chemotaxis physiology, Epithelial Cells physiology, Epithelium, Corneal cytology, Receptor, Cannabinoid, CB1 physiology
- Abstract
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.
- Published
- 2015
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24. Loss of ion transporters and increased unfolded protein response in Fuchs' dystrophy.
- Author
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Jalimarada SS, Ogando DG, and Bonanno JA
- Subjects
- Aged, Aged, 80 and over, Anion Transport Proteins genetics, Antiporters genetics, Case-Control Studies, Endothelium, Corneal metabolism, Gene Expression, Humans, Ion Transport genetics, Metabolic Networks and Pathways, Middle Aged, Mutation, Oxidative Stress, Protein Processing, Post-Translational, RNA, Messenger genetics, RNA, Messenger metabolism, Fuchs' Endothelial Dystrophy genetics, Fuchs' Endothelial Dystrophy metabolism, Ion Pumps genetics, Unfolded Protein Response genetics
- Abstract
Purpose: Fuchs' endothelial corneal dystrophy (FECD), which affects approximately 5% of the population over 40 in the U.S.A., is a major cause of corneal transplantation. FECD is associated with mutations of a variety of unrelated genes: SLC4A11, COL8A2, TCF8, and LOXHD1. The current pathological description of the dystrophy includes deficiency of corneal endothelium (CE) pump function and induction of the unfolded protein response (UPR). This study aims to determine the contribution of the two mechanisms by assessing the expression levels of (1) seven endothelial ion transporters known to regulate stromal hydration and (2) UPR related genes in a set of six CE samples obtained from FECD patients compared to that of normal controls., Methods: CE samples collected during FECD keratoplasty or from an eye bank (normal control) were transferred into an RNA stabilizing agent and refrigerated. Total RNA from each CE specimen was individually extracted. The expression levels of ion transporters and UPR genes were tested using quantitative real-time (RT) PCR and a UPR specific PCR array, respectively., Results: In normal CE, the comparative expression levels of ion transporters in decreasing order were SLC4A11, Na(+)/K(+) ATPase, pNBCe1, and NHE1, followed by the isoforms of monocarboxylate transporters (MCTs). In FECD samples, Na(+)/K(+) ATPase and MCTs 1 and 4 were significantly downregulated compared to normal controls (p<0.05). The PCR array tested 84 UPR related genes. Data analysis showed upregulation of 39 genes and downregulation of three genes, i.e., approximately 51% of the tested genes had their expression altered in FECD samples with a difference greater than ± twofold regulation. Thirteen of the altered genes showed significant changes (p<0.05). The PCR array results were validated by quantitative RT-PCR., Conclusions: FECD samples had evident UPR with significant changes in the expression of the protein processing pathway genes. The significant downregulation of ion transporters indicates simultaneous compromised CE pump function in Fuchs' dystrophy.
- Published
- 2014
25. CD147 required for corneal endothelial lactate transport.
- Author
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Li S, Nguyen TT, and Bonanno JA
- Subjects
- Animals, Anterior Chamber metabolism, Blotting, Western, Carbonic Anhydrase Inhibitors pharmacology, Corneal Stroma metabolism, Fluorescent Antibody Technique, Indirect, Gene Silencing physiology, Hydrogen-Ion Concentration, Ion Transport, RNA, Small Interfering genetics, Rabbits, Real-Time Polymerase Chain Reaction, Sulfonamides pharmacology, Thiazines pharmacology, Transfection, Basigin physiology, Endothelium, Corneal metabolism, Lactic Acid metabolism, Monocarboxylic Acid Transporters metabolism
- Abstract
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.)
- Published
- 2014
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26. Cornea and ocular surface disease: application of cutting-edge optometric research.
- Author
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Robertson DM, Alexander LJ, Bonanno JA, Fleiszig SM, and McNamara N
- Subjects
- Biomedical Research trends, Humans, Optometry trends, Biomedical Research organization & administration, Conjunctival Diseases therapy, Corneal Diseases therapy, Dry Eye Syndromes therapy, Eye Infections therapy, Eyelid Diseases therapy, Optometry organization & administration
- Abstract
: Clinician-scientists bridge the gap between basic research and patient care. At the 2012 Annual Meeting, a symposium highlighting the application of cutting-edge optometric research within the anterior segment was held to present and discuss some of the recent basic scientific advances that will both shape and guide the development of future clinical care practices. This article summarizes this work, bringing together four experts, all clinician-scientists in the field of cornea and ocular surface. Collectively, this work provides new insights to clinicians and researchers alike, as well as brings forth a greater appreciation of the impact of ongoing optometric bench research in advancing clinical care.
- Published
- 2014
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27. SLC4A11 is an EIPA-sensitive Na(+) permeable pHi regulator.
- Author
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Ogando DG, Jalimarada SS, Zhang W, Vithana EN, and Bonanno JA
- Subjects
- Amiloride pharmacology, Amino Acid Sequence, Ammonium Chloride metabolism, Animals, Anion Transport Proteins genetics, Anion Transport Proteins metabolism, Antiporters genetics, Antiporters metabolism, CHO Cells, Cricetinae, Cricetulus, Dose-Response Relationship, Drug, HEK293 Cells, Humans, Hydrogen-Ion Concentration, Ion Transport, Molecular Sequence Data, Sodium Hydroxide metabolism, Time Factors, Transfection, Amiloride analogs & derivatives, Anion Transport Proteins antagonists & inhibitors, Antiporters antagonists & inhibitors, Cell Membrane Permeability drug effects, Epithelial Sodium Channel Blockers pharmacology, Sodium metabolism
- Abstract
Slc4a11, a member of the solute linked cotransporter 4 family that is comprised predominantly of bicarbonate transporters, was described as an electrogenic 2Na(+)-B(OH)4(-) (borate) cotransporter and a Na(+)-2OH(-) cotransporter. The goal of the current study was to confirm and/or clarify the function of SLC4A11. In HEK293 cells transfected with SLC4A11 we tested if SLC4A11 is a: 1) Na(+)-HCO3(-) cotransporter, 2) Na(+)-OH(-)(H(+)) transporter, and/or 3) Na(+)-B(OH)4(-) cotransporter. CO2/HCO3(-) perfusion yielded no significant differences in rate or extent of pHi changes or Na(+) flux in SLC4A11-transfected compared with control cells. Similarly, in CO2/HCO3(-), acidification on removal of Na(+) and alkalinization on Na(+) add back were not significantly different between control and transfected indicating that SLC4A11 does not have Na(+)-HCO3(-) cotransport activity. In the absence of CO2/HCO3(-), SLC4A11-transfected cells showed higher resting intracelllular Na(+) concentration ([Na(+)]i; 25 vs. 17 mM), increased NH4(+)-induced acidification and increased acid recovery rate (160%) after an NH4 pulse. Na(+) efflux and influx were faster (80%) following Na(+) removal and add back, respectively, indicative of Na(+)-OH(-)(H(+)) transport by SLC4A11. The increased alkalinization recovery was confirmed in NHE-deficient PS120 cells demonstrating that SLC4A11 is a bonafide Na(+)-OH(-)(H(+)) transporter and not an activator of NHEs. SLC4A11-mediated H(+) efflux is inhibited by 5-(N-ethyl-N-isopropyl) amiloride (EIPA; EC50: 0.1 μM). The presence of 10 mM borate did not alter dpHi/dt or ΔpH during a Na(+)-free pulse in SLC4A11-transfected cells. In summary our results show that SLC4A11 is not a bicarbonate or borate-linked transporter but has significant EIPA-sensitive Na(+)-OH(-)(H(+)) and NH4(+) permeability.
- Published
- 2013
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28. Ion transport function of SLC4A11 in corneal endothelium.
- Author
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Jalimarada SS, Ogando DG, Vithana EN, and Bonanno JA
- Subjects
- Animals, Basement Membrane metabolism, Biological Transport, Blotting, Western, Cattle, Cells, Cultured, Electrophoresis, Polyacrylamide Gel, Endothelium, Corneal cytology, Fluorescent Antibody Technique, Indirect, Gene Expression, Gene Silencing physiology, Genetic Vectors, Hydrogen-Ion Concentration, Ion Transport, RNA, Messenger metabolism, RNA, Small Interfering genetics, Real-Time Polymerase Chain Reaction, Sodium metabolism, Transfection, Anion Transport Proteins physiology, Antiporters physiology, Bicarbonates metabolism, Borates metabolism, Endothelium, Corneal metabolism, Hydroxides metabolism
- Abstract
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⁻.
- Published
- 2013
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29. Depletion of SLC4A11 causes cell death by apoptosis in an immortalized human corneal endothelial cell line.
- Author
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Liu J, Seet LF, Koh LW, Venkatraman A, Venkataraman D, Mohan RR, Praetorius J, Bonanno JA, Aung T, and Vithana EN
- Subjects
- Anion Transport Proteins biosynthesis, Antiporters biosynthesis, Apoptosis genetics, Blotting, Western, Cell Count, Cell Line, Cell Proliferation, Cell Survival, Endothelium, Corneal pathology, Gene Silencing, Humans, Immunohistochemistry, In Situ Nick-End Labeling, Mutation, RNA, Small Interfering biosynthesis, Reverse Transcriptase Polymerase Chain Reaction, Anion Transport Proteins genetics, Antiporters genetics, Cell Death genetics, Down-Regulation genetics, Endothelium, Corneal metabolism, Gene Expression Regulation, RNA, Messenger genetics, RNA, Small Interfering genetics
- Abstract
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.
- Published
- 2012
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30. Lactate-H⁺ transport is a significant component of the in vivo corneal endothelial pump.
- Author
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Nguyen TT and Bonanno JA
- Subjects
- Animals, Biological Transport, Active physiology, Blotting, Western, Endothelium, Corneal cytology, Hydrogen-Ion Concentration, Ion Transport, Rabbits, Endothelium, Corneal metabolism, Lactic Acid metabolism, Monocarboxylic Acid Transporters biosynthesis, Muscle Proteins biosynthesis, Symporters biosynthesis
- Abstract
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.
- Published
- 2012
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31. Molecular mechanisms underlying the corneal endothelial pump.
- Author
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Bonanno JA
- Subjects
- Animals, Aquaporins metabolism, Bicarbonates metabolism, Carbonic Anhydrases metabolism, Chlorides metabolism, Electroosmosis, Humans, Ion Transport, Models, Biological, Osmotic Pressure, Sodium-Potassium-Exchanging ATPase metabolism, Cornea blood supply, Endothelial Cells metabolism, Ion Pumps metabolism
- Abstract
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.)
- Published
- 2012
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32. Telomerase immortalization of human corneal endothelial cells yields functional hexagonal monolayers.
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Schmedt T, Chen Y, Nguyen TT, Li S, Bonanno JA, and Jurkunas UV
- Subjects
- Biomarkers metabolism, Cell Differentiation, Cell Line, Cell Proliferation, Cell Shape, Cellular Senescence, Cyclin D metabolism, Cyclin-Dependent Kinase 4 metabolism, Endothelium, Corneal enzymology, Endothelium, Corneal metabolism, Humans, Ion Pumps metabolism, Ion Transport, Telomerase genetics, Transduction, Genetic, Tumor Suppressor Protein p53 metabolism, Endothelium, Corneal cytology, Telomerase metabolism
- Abstract
Human corneal endothelial cells (HCEnCs) form a monolayer of hexagonal cells whose main function is to maintain corneal clarity by regulating corneal hydration. HCEnCs are derived from neural crest and are arrested in the post-mitotic state. Thus cell loss due to aging or corneal endothelial disorders leads to corneal edema and blindness-the leading indication for corneal transplantation. Here we show the existence of morphologically distinct subpopulations of HCEnCs that are interspersed among primary cells and exhibit enhanced self-renewal competence and lack of phenotypic signs of cellular senescence. Colonies of these uniform and hexagonal HCEnCs (HCEnC-21) were selectively isolated and demonstrated high proliferative potential that was dependent on endogenous upregulation of telomerase and cyclin D/CDK4. Further transduction of HCEnC-21 with telomerase yielded a highly proliferative corneal endothelial cell line (HCEnT-21T) that was devoid of oncogenic transformation and retained critical corneal endothelial cell characteristics and functionality. This study will significantly impact the fields of corneal cell biology and regenerative medicine.
- Published
- 2012
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33. Oxygen-deficient metabolism and corneal edema.
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Leung BK, Bonanno JA, and Radke CJ
- Subjects
- Contact Lenses, Hydrophilic adverse effects, Corneal Edema etiology, Humans, Hypoxia etiology, Hypoxia metabolism, Ion Pumps metabolism, Models, Theoretical, Corneal Edema metabolism, Epithelium, Corneal metabolism, Oxygen metabolism
- Abstract
Wear of low-oxygen-transmissible soft contact lenses swells the cornea significantly, even during open eye. Although oxygen-deficient corneal edema is well-documented, a self-consistent quantitative prediction based on the underlying metabolic reactions is not available. We present a biochemical description of the human cornea that quantifies hypoxic swelling through the coupled transport of water, salt, and respiratory metabolites. Aerobic and anaerobic consumption of glucose, as well as acidosis and pH buffering, are incorporated in a seven-layer corneal model (anterior chamber, endothelium, stroma, epithelium, postlens tear film, contact lens, and prelens tear film). Corneal swelling is predicted from coupled transport of water, dissolved salts, and especially metabolites, along with membrane-transport resistances at the endothelium and epithelium. At the endothelium, the Na+/K+ - ATPase electrogenic channel actively transports bicarbonate ion from the stroma into the anterior chamber. As captured by the Kedem-Katchalsky membrane-transport formalism, the active bicarbonate-ion flux provides the driving force for corneal fluid pump-out needed to match the leak-in tendency of the stroma. Increased lactate-ion production during hypoxia osmotically lowers the pump-out rate requiring the stroma to swell to higher water content. Concentration profiles are predicted for glucose, water, oxygen, carbon dioxide, and hydronium, lactate, bicarbonate, sodium, and chloride ions, along with electrostatic potential and pressure profiles. Although the active bicarbonate-ion pump at the endothelium drives bicarbonate into the aqueous humor, we find a net flux of bicarbonate ion into the cornea that safeguards against acidosis. For the first time, we predict corneal swelling upon soft-contact-lens wear from fundamental biophysico-chemical principles. We also successfully predict that hypertonic tear alleviates contact-lens-induced edema., (Copyright © 2011 Elsevier Ltd. All rights reserved.)
- Published
- 2011
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34. Bicarbonate, NBCe1, NHE, and carbonic anhydrase activity enhance lactate-H+ transport in bovine corneal endothelium.
- Author
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Nguyen TT and Bonanno JA
- Subjects
- 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid pharmacology, Amiloride analogs & derivatives, Amiloride pharmacology, Animals, Blotting, Western, Carbonic Anhydrase Inhibitors pharmacology, Cattle, Cells, Cultured, Endothelium, Corneal drug effects, Fluorescent Antibody Technique, Indirect, Hydrogen-Ion Concentration, Niflumic Acid pharmacology, Real-Time Polymerase Chain Reaction, Bicarbonates metabolism, Carbonic Anhydrases metabolism, Endothelium, Corneal metabolism, Lactic Acid metabolism, Monocarboxylic Acid Transporters metabolism, Sodium-Bicarbonate Symporters metabolism, Sodium-Hydrogen Exchangers metabolism
- Abstract
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.
- Published
- 2011
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35. Soluble adenylyl cyclase mediates bicarbonate-dependent corneal endothelial cell protection.
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Li S, Allen KT, and Bonanno JA
- Subjects
- Adenylyl Cyclase Inhibitors, Adenylyl Cyclases genetics, Animals, Annexin A5 analysis, Apoptosis drug effects, Caspase 3 analysis, Cattle, Cells, Cultured, Cyclic AMP analysis, Cyclic AMP antagonists & inhibitors, Cyclic AMP metabolism, Cyclic AMP Response Element-Binding Protein metabolism, Cytochromes c metabolism, Epithelium, Corneal drug effects, Epithelium, Corneal physiopathology, Estradiol analogs & derivatives, Estradiol pharmacology, Isoquinolines pharmacology, Phosphodiesterase 4 Inhibitors pharmacology, Phosphorylation, Protein Kinase Inhibitors pharmacology, Proto-Oncogene Proteins c-bcl-2 analysis, RNA, Small Interfering genetics, Rolipram pharmacology, Staurosporine pharmacology, Sulfonamides pharmacology, Adenylyl Cyclases metabolism, Bicarbonates metabolism, Epithelium, Corneal enzymology
- Abstract
Cyclic AMP produced from membrane receptor complex bound adenylyl cyclases is protective in corneal endothelial cells (CEC). CEC also express soluble adenylyl cyclase (sAC), which is localized throughout the cytoplasm. When activated by HCO(3)(-), cAMP concentration ([cAMP]) increases by ∼50%. Here we ask if cAMP produced from sAC is also protective. We examined the effects of HCO(3)(-), pH, phosphodiesterase 4 inhibition by rolipram, sAC inhibition by 2HE (2-hydroxyestradiol), and sAC small interfering RNA (siRNA) knockdown on basal and staurosporine-mediated apoptosis. HCO(3)(-) (40 mM) or 50 μM rolipram raised [cAMP] to similar levels and protected endothelial cells by 50% relative to a HCO(3)(-)-free control, whereas 2HE, which decreased [cAMP] by 40%, and H89 (PKA inhibitor) doubled the apoptotic rate. sAC expression was reduced by two-thirds in the absence of HCO(3)(-) and was reduced to 15% of control by sAC siRNA. Protection by HCO(3)(-) was eliminated in siRNA-treated cells. Similarly, caspase-3 activity and cytochrome c release were reduced by HCO(3)(-) and enhanced by 2HE or siRNA. Analysis of percent annexin V+ cells as a function of [cAMP] revealed an inverse, nonlinear relation, suggesting a protective threshold [cAMP] of 10 pmol/mg protein. Relative levels of phosphorylated cAMP response element binding protein and phosphorylated Bcl-2 were decreased in CEC treated with 2HE or siRNA, suggesting that HCO(3)(-)-dependent endogenous sAC activity can mobilize antiapoptotic signal transduction. Overall, our data suggest a new role for sAC in endogenous cellular protection.
- Published
- 2011
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36. SOD2 contributes to anti-oxidative capacity in rabbit corneal endothelial cells.
- Author
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Liu C, Ogando D, and Bonanno JA
- Subjects
- Animals, Apoptosis radiation effects, Blotting, Western, Cells, Cultured, Cornea cytology, Cornea radiation effects, Endothelial Cells cytology, Endothelial Cells radiation effects, Endothelium, Corneal cytology, Endothelium, Corneal radiation effects, Gene Expression, Gene Silencing, Membrane Potential, Mitochondrial radiation effects, Mitochondria genetics, Mitochondria radiation effects, Oxidative Stress, RNA, Small Interfering genetics, RNA, Small Interfering metabolism, Rabbits, Reactive Oxygen Species metabolism, Reverse Transcriptase Polymerase Chain Reaction, Superoxide Dismutase genetics, Ultraviolet Rays, Antioxidants metabolism, Cornea enzymology, Endothelial Cells enzymology, Endothelium, Corneal enzymology, Mitochondria enzymology, Superoxide Dismutase metabolism
- Abstract
Purpose: Corneal endothelial cells are rich in mitochondria, a potential source of reactive oxygen species (ROS). ROS have been implicated in endothelial cell loss during aging or in endothelial dystrophies. In this study we examined the anti-oxidative role of mitochondrial superoxide dismutase (SOD2) in corneal endothelial cells., Methods: SOD2 expression was examined by RT-PCR and western blot analysis in fresh rabbit corneal endothelium (RCE) and cell cultures. SOD2 activity, total reactive oxygen species (ROS), mitochondrial ROS, mitochondrial membrane potential (MMP), and apoptotic levels were examined in untreated, SOD2 siRNA and viral vector shRNA treated RCE cells. Scrambled siRNA and shRNA sequence targeting non-mammalian genes were used as controls., Results: SOD2 is expressed in both fresh and cultured rabbit corneal endothelium. SOD2 expression was reduced by ~80%-90% in cultured RCE using either siRNA or shRNA approaches. SOD2 activity was decreased by ~70%-80% for both approaches. Total cell ROS was significantly increased in shSOD2 lentivirus treated cells (9%±6%) relative to control transduction (0.4%±0.1%). MitoSOX™ staining for mitochondrial ROS in siSOD2 treated RCE cells was dramatically increased. Two minutes of UV irradiation increased total ROS levels by 15%, whereas in shSOD2 treated cells UV induced ROS was increased 29%±5% (p<0.05). MMP was reduced in shSOD2 viral treated cells by 66%±3%, significantly greater than in control transduced cells (15%±8%, p<0.05). Apoptosis increased by 1.5 fold in shSOD2 virus treated samples compared with scrambled virus and untreated cells., Conclusions: SOD2 is expressed in both fresh and cultured rabbit corneal endothelium. siRNA and shRNA approaches are able to efficiently knockdown SOD2 expression and reduce enzyme activity in RCE cells. Decreased SOD2 activity causes elevated ROS production, mitochondrial membrane potential loss and early cell apoptosis. These results indicate that SOD2 is a significant anti-oxidative enzyme in RCE cells.
- Published
- 2011
37. Knockdown of NBCe1 in vivo compromises the corneal endothelial pump.
- Author
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Liu C, Cheng Q, Nguyen T, and Bonanno JA
- Subjects
- Animals, Bicarbonates metabolism, Biological Transport, Active drug effects, Blotting, Western, Carbonic Anhydrase Inhibitors pharmacology, Carbonic Anhydrases physiology, Gene Knockdown Techniques, Genetic Vectors, Green Fluorescent Proteins genetics, Green Fluorescent Proteins metabolism, Immunodeficiency Virus, Feline genetics, Intraocular Pressure, Microscopy, Fluorescence, RNA Interference, Rabbits, Reverse Transcriptase Polymerase Chain Reaction, Sulfonamides pharmacology, Thiazines pharmacology, Transfection, Endothelium, Corneal metabolism, Gene Silencing physiology, Membrane Transport Proteins physiology, Sodium-Bicarbonate Symporters physiology
- Abstract
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.
- Published
- 2010
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38. A hierarchy of endothelial colony-forming cell activity displayed by bovine corneal endothelial cells.
- Author
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Huang L, Harkenrider M, Thompson M, Zeng P, Tanaka H, Gilley D, Ingram DA, Bonanno JA, and Yoder MC
- Subjects
- Animals, Cattle, Cell Culture Techniques, Cell Proliferation, Cells, Cultured, Colony-Forming Units Assay, Coronary Vessels, Endothelium, Corneal metabolism, Endothelium, Vascular metabolism, Flow Cytometry, Gene Expression, Immunophenotyping, Leukocyte Common Antigens metabolism, Lipoproteins, LDL metabolism, Reverse Transcriptase Polymerase Chain Reaction, Stem Cells metabolism, Telomerase metabolism, Endothelium, Corneal cytology, Endothelium, Vascular cytology, Stem Cells cytology
- Abstract
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.
- Published
- 2010
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39. Hypoxia reduces TGFbeta1-induced corneal keratocyte myofibroblast transformation.
- Author
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Xing D and Bonanno JA
- Subjects
- Animals, Cell Differentiation drug effects, Cell Hypoxia drug effects, Cell Line, Transformed, Enzyme Activation drug effects, Extracellular Signal-Regulated MAP Kinases metabolism, Fibroblasts enzymology, Phosphoproteins metabolism, Phosphorylation drug effects, Protein Binding drug effects, Rabbits, Smad3 Protein metabolism, rhoA GTP-Binding Protein metabolism, Cornea cytology, Fibroblasts cytology, Fibroblasts drug effects, Transforming Growth Factor beta1 pharmacology
- Abstract
Purpose: The purpose of this study was to determine whether transient hypoxia had an effect on transforming growth factor beta1 (TGFbeta1)-induced rabbit corneal keratocyte myofibroblast transformation., Methods: Primary isolated rabbit corneal keratocytes were cultured in a serum-free medium. The effect of transient hypoxia treatment (1% oxygen, 4 h/day) on TGFbeta1 (5 ng/ml)-induced alpha-smooth muscle actin (alpha-SM actin) expression was examined by immunofluorescence, flow cytometry, and immunocytochemistry 72 h after treatment. We found that hypoxia treatment significantly reduced the myofibroblast phenotype and alpha-SM actin expression that was induced by TGFbeta1. To explore the possible mechanism for this effect, we screened for the effects of hypoxia on several early TGFbeta-dependent signaling events including activated pSmad3, CREB (cAMP response element binding) binding protein (CBP), MAPKs (Mitogen-activated protein kinase), and RhoA by co-immunoprecipitation and western blotting., Results: Hypoxia alone increased alpha-SM actin expression and the association of pSmad3 to CBP, but it did not induce the myofibroblast phenotype. The levels of pERK (the extracellular signal-regulated protein kinase) and pSmad3 or the extent of the interaction between pSmad3 and CBP induced by TGFbeta1 were not affected by hypoxia whereas the activation of RhoA induced by TGFbeta1 was significantly reduced., Conclusions: We conclude that hypoxia can inhibit TGFbeta1-induced corneal myofibroblast transformation and alpha-SM actin expression. Our data show that this inhibition does not occur by altering Smads or MAPK signaling but possibly by reducing the early activation of RhoA.
- Published
- 2009
40. Hypoxia preconditioning protection of corneal stromal cells requires HIF1alpha but not VEGF.
- Author
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Xing D and Bonanno JA
- Subjects
- Animals, Apoptosis, Cadmium Chloride pharmacology, Cattle, Cells, Cultured, Corneal Stroma metabolism, Gene Expression Regulation drug effects, Gene Knockdown Techniques, Hypoxia-Inducible Factor 1, alpha Subunit antagonists & inhibitors, Hypoxia-Inducible Factor 1, alpha Subunit genetics, In Situ Nick-End Labeling, RNA Interference, RNA, Small Interfering, Stress, Physiological, Ultraviolet Rays, Cell Hypoxia, Corneal Stroma cytology, Hypoxia-Inducible Factor 1, alpha Subunit metabolism, Vascular Endothelial Growth Factor A metabolism
- Abstract
Purpose: Hypoxia preconditioning protects corneal stromal cells from stress-induced death. This study determined whether the transcription factor HIF-1alpha (Hypoxia Inducible Factor) is responsible and whether this is promulgated by VEGF (Vascular Endothelial Growth Factor)., Methods: Cultured bovine stromal cells were preconditioned with hypoxia in the presence of cadmium chloride, a chemical inhibitor of HIF-1alpha, and HIF-1alpha siRNA to test if HIF-1alpha activity is needed for hypoxia preconditioning protection from UV-irradiation induced cell death. TUNEL assay was used to detect cell apoptosis after UV-irradiation. RT-PCR and western blot were used to detect the presence of HIF-1alpha and VEGF in transcriptional and translational levels., Results: During hypoxia (0.5% O2), 5 muM cadmium chloride completely inhibited HIF-1alpha expression and reversed the protection by hypoxia preconditioning. HIF-1alpha siRNA (15 nM) reduced HIF-1alpha expression by 90% and produced a complete loss of protection provided by hypoxia preconditioning. Since VEGF is induced by hypoxia, can be HIF-1alpha dependent, and is often protective, we examined the changes in transcription of VEGF and its receptors after 4 h of hypoxia preconditioning. VEGF and its receptors Flt-1 and Flk-1 are up-regulated after hypoxia preconditioning. However, the transcription and translation of VEGF were paradoxically increased by siHIF-1alpha, suggesting that VEGF expression in stromal cells is not down-stream of HIF-1alpha., Conclusions: These findings demonstrate that hypoxia preconditioning protection in corneal stromal cells requires HIF-1alpha, but that VEGF is not a component of the protection.
- Published
- 2009
41. Effect of cAMP on TGFbeta1-induced corneal keratocyte-myofibroblast transformation.
- Author
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Xing D and Bonanno JA
- Subjects
- Actins metabolism, Animals, CREB-Binding Protein metabolism, Cell Count, Cell Differentiation drug effects, Cells, Cultured, Colforsin pharmacology, Cornea metabolism, Culture Media, Serum-Free, Fibroblasts cytology, Fibroblasts metabolism, Flow Cytometry, Fluorescent Antibody Technique, Indirect, Mitogen-Activated Protein Kinases metabolism, Phosphorylation, Rabbits, Smad3 Protein metabolism, rhoA GTP-Binding Protein metabolism, Cornea cytology, Cyclic AMP pharmacology, Transforming Growth Factor beta1 pharmacology
- Abstract
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.
- Published
- 2009
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42. Dependence of cAMP meditated increases in Cl- and HCO(3)- permeability on CFTR in bovine corneal endothelial cells.
- Author
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Li J, Allen KT, Sun XC, Cui M, and Bonanno JA
- Subjects
- Animals, Cattle, Cell Membrane Permeability drug effects, Cell Membrane Permeability physiology, Cells, Cultured, Colforsin pharmacology, Cystic Fibrosis Transmembrane Conductance Regulator genetics, Endothelial Cells drug effects, Endothelial Cells metabolism, Endothelium, Corneal cytology, Endothelium, Corneal drug effects, RNA Interference, RNA, Small Interfering genetics, Transfection, Bicarbonates metabolism, Chlorides metabolism, Cyclic AMP physiology, Cystic Fibrosis Transmembrane Conductance Regulator physiology, Endothelium, Corneal metabolism
- Abstract
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.
- Published
- 2008
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43. Role of carbonic anhydrase IV in corneal endothelial HCO3- transport.
- Author
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Sun XC, Li J, Cui M, and Bonanno JA
- Subjects
- Animals, Basement Membrane metabolism, Benzolamide pharmacology, Biological Transport, Carbon Dioxide metabolism, Carbonic Anhydrase IV antagonists & inhibitors, Carbonic Anhydrase Inhibitors pharmacology, Cattle, Cell Membrane Permeability, Cells, Cultured, Endothelium, Corneal cytology, Fluorescent Antibody Technique, Indirect, Hydrogen-Ion Concentration, Immunoblotting, Intracellular Membranes, Microscopy, Confocal, RNA, Small Interfering genetics, Reverse Transcriptase Polymerase Chain Reaction, Sodium-Bicarbonate Symporters metabolism, Transfection, Bicarbonates metabolism, Carbonic Anhydrase IV physiology, Endothelium, Corneal enzymology
- Abstract
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.
- Published
- 2008
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44. Molecular expression and functional involvement of the bovine calcium-activated chloride channel 1 (bCLCA1) in apical HCO3- permeability of bovine corneal endothelium.
- Author
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Zhang Y, Li J, Xie Q, and Bonanno JA
- Subjects
- Animals, Antibodies immunology, Calcium metabolism, Cattle, Cell Membrane Permeability physiology, Cells, Cultured, Chloride Channels metabolism, Endothelial Cells metabolism, Fluorescent Antibody Technique methods, In Situ Hybridization methods, Microscopy, Confocal methods, RNA, Messenger analysis, RNA, Small Interfering metabolism, Bicarbonates metabolism, Chloride Channels immunology, Endothelium, Corneal metabolism
- Abstract
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.
- Published
- 2006
- Full Text
- View/download PDF
45. Hypoxia preconditioning protects corneal stromal cells against induced apoptosis.
- Author
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Xing D, Sun X, Li J, Cui M, Tan-Allen K, and Bonanno JA
- Subjects
- Animals, Apoptosis radiation effects, Blotting, Western, Cattle, Cells, Cultured, Cornea radiation effects, Culture Media, Conditioned, Fas Ligand Protein, Fibroblasts pathology, Fibroblasts radiation effects, Hypoxia-Inducible Factor 1, alpha Subunit physiology, In Situ Nick-End Labeling, Membrane Glycoproteins physiology, Stromal Cells radiation effects, Tumor Necrosis Factors physiology, Ultraviolet Rays, fas Receptor physiology, Apoptosis physiology, Cell Hypoxia physiology, Cornea pathology, Ischemic Preconditioning, Stromal Cells pathology
- Abstract
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.
- Published
- 2006
- Full Text
- View/download PDF
46. TRPC4 knockdown suppresses epidermal growth factor-induced store-operated channel activation and growth in human corneal epithelial cells.
- Author
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Yang H, Mergler S, Sun X, Wang Z, Lu L, Bonanno JA, Pleyer U, and Reinach PS
- Subjects
- Blotting, Western, Calcium metabolism, Cell Proliferation, Cells, Cultured, Cornea metabolism, Egtazic Acid chemistry, Electrophysiology, Humans, Immunoblotting, Microscopy, Fluorescence, Nickel pharmacology, Patch-Clamp Techniques, RNA, Messenger metabolism, RNA, Small Interfering metabolism, Reverse Transcriptase Polymerase Chain Reaction, TRPC Cation Channels, Thymidine metabolism, Time Factors, Transfection, Cornea cytology, Epidermal Growth Factor metabolism, Epithelial Cells cytology, Ion Channels genetics
- Abstract
Epidermal growth factor (EGF) in corneal epithelial cells stimulates proliferation by inducing capacitative calcium entry (CCE). However, neither the identity nor the mechanism of activation of the plasma membrane influx pathway that mediates CCE is known. Accordingly, we determined, in human corneal epithelial cells, whether or not (i) CCE is dependent upon stimulation of storeoperated channel (SOC) activity, (ii) the canonical transient receptor potential (TRP) protein isoform TRPC4 is a component of such channels, and (iii) suppression of TRPC4 protein expression decreases EGF-induced stimulation of SOC activity and proliferation. The whole cell patch-clamp technique was used to monitor TRPC4-mediated stimulation of SOC activity following intracellular calcium store depletion and induction of CCE. TRPC4 small interfering RNA transfection suppressed TRPC4 protein expression. Reverse transcription-PCR and Western blot analysis were used to assess knockdown efficiency of mRNA and protein expression. [(3)H]Thymidine incorporation was used to evaluate EGF-in-duced mitogenesis. Ca(2+) transients were measured by single-cell fluorescence imaging. TRPC4 knockdown decreased mRNA and protein expression by 89 and 87%, respectively. In these cells, EGF-induced SOC activation elicited by intracellular calcium store depletion was obviated; 2) EGF-induced CCE fell by 76%; 3) EGF-induced stimulation of SOC activity was eliminated; and 4) EGF-induced increases in proliferation fell by 54%. Thus, TRPC4 is a component of SOC in human corneal epithelial cells whose activation by EGF is requisite for an optimum mitogenic response to this growth factor.
- Published
- 2005
- Full Text
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47. Expression and functional evaluation of transient receptor potential channel 4 in bovine corneal endothelial cells.
- Author
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Xie Q, Zhang Y, Cai Sun X, Zhai C, and Bonanno JA
- Subjects
- Animals, Blotting, Western methods, Calcium metabolism, Calcium Channels metabolism, Cattle, Cells, Cultured, Chromatography, Affinity, Ion Channels genetics, Ion Channels immunology, RNA, Small Interfering genetics, Reverse Transcriptase Polymerase Chain Reaction methods, TRPC Cation Channels, Transfection, Calcium Signaling physiology, Endothelium, Corneal metabolism, Ion Channels metabolism
- Abstract
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.
- Published
- 2005
- Full Text
- View/download PDF
48. Molecular cloning and characterization of a human AIF-like gene with ability to induce apoptosis.
- Author
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Xie Q, Lin T, Zhang Y, Zheng J, and Bonanno JA
- Subjects
- Amino Acid Sequence, Apoptosis physiology, Apoptosis Inducing Factor, Cloning, Molecular, Female, Flavoproteins chemistry, Flavoproteins physiology, Gene Expression, Humans, Male, Membrane Potentials, Membrane Proteins chemistry, Membrane Proteins physiology, Mitochondria metabolism, Molecular Sequence Data, Pregnancy, Protein Structure, Tertiary, RNA, Messenger genetics, RNA, Messenger metabolism, Recombinant Proteins chemistry, Recombinant Proteins genetics, Recombinant Proteins immunology, Reverse Transcriptase Polymerase Chain Reaction, Sequence Homology, Amino Acid, Tissue Distribution, Transfection, Apoptosis genetics, Flavoproteins genetics, Membrane Proteins genetics
- Abstract
In this study, we cloned and characterized a human gene homologous to the apoptosis-inducing factor (AIF), which is named AIF-like (AIFL). Human AIFL has 598 amino acids, with a characteristic Rieske domain and a pyridine nucleotide-disulfide oxidoreductase domain (Pyr_redox). AIFL shares 35% homology with AIF, mainly in the Pyr_redox domain. Reverse transcriptase-PCR analysis showed the expression of AIFL mRNA in all tissues tested, i.e. brain, colon, heart, kidney, liver, lung, muscle, ovary, pancreas, placenta, small intestine, and testis. We developed antibodies against human AIFL using fusion proteins as antigens. The antibodies specifically recognized the antigen and heterologously expressed AIFL proteins. The expression of AIFL proteins in human tissues was also ubiquitous, demonstrated by immunohistochemistry in tissue array slides. Subcellular fractionation and immunofluorescence staining studies revealed that AIFL is predominantly localized to the mitochondria. Similar to AIF, overexpression of AIFL induced apoptosis, as shown by increased cytoplasmic nucleosomes and subdiploid cell populations in AIFL-transfected cells. The segment 1-190 containing the Rieske domain induced apoptosis, whereas the segment containing the Pyr_redox domain did not contribute to the pro-apoptotic function. The mitochondrial membrane potential of cells transfected with AIFL was significantly more depolarized than that of the control. AIFL transfection-induced cytochrome c release and cleavage of caspase 3. Furthermore, the pan-caspase inhibitor Z-VAD-fmk inhibited AIFL induced apoptosis. In summary, AIFL induces apoptosis in a caspase-dependent manner when heterologously expressed.
- Published
- 2005
- Full Text
- View/download PDF
49. Characterization of adenosine receptors in bovine corneal endothelium.
- Author
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Tan-Allen KY, Sun XC, and Bonanno JA
- Subjects
- Adenosine pharmacology, Animals, Blotting, Western, Calcium analysis, Cattle, Cell Membrane Permeability, Cells, Cultured, Chlorides metabolism, Cyclic AMP analysis, Endothelium, Corneal metabolism, Flavins pharmacology, Fluorescent Antibody Technique, Indirect, Membrane Potentials, Purinergic P2 Receptor Antagonists, RNA, Messenger analysis, Receptors, Purinergic genetics, Receptors, Purinergic P1 analysis, Receptors, Purinergic P1 genetics, Receptors, Purinergic P2 analysis, Receptors, Purinergic P2 genetics, Reverse Transcriptase Polymerase Chain Reaction, Endothelium, Corneal chemistry, Receptors, Purinergic analysis
- Abstract
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).
- Published
- 2005
- Full Text
- View/download PDF
50. Role of NBC1 in apical and basolateral HCO3- permeabilities and transendothelial HCO3- fluxes in bovine corneal endothelium.
- Author
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Li J, Sun XC, and Bonanno JA
- Subjects
- Animals, Cattle, Cells, Cultured, Colforsin pharmacology, Cornea metabolism, Endothelial Cells cytology, Endothelial Cells drug effects, Enzyme Inhibitors pharmacology, Humans, Ouabain pharmacology, Permeability, RNA, Small Interfering genetics, RNA, Small Interfering metabolism, Sodium-Bicarbonate Symporters antagonists & inhibitors, Sodium-Bicarbonate Symporters genetics, Bicarbonates metabolism, Cell Polarity, Cornea anatomy & histology, Endothelial Cells metabolism, Sodium-Bicarbonate Symporters metabolism
- Abstract
Corneal transparency and hydration control are dependent on HCO(3)(-) transport properties of the corneal endothelium. Recent work (13) suggested the presence of an apical 1Na(+)-3HCO(3)(-) cotransporter (NBC1) in addition to a basolateral 1Na(+)-2HCO(3)(-) cotransporter. We examined whether the NBC1 cotransporter contributes significantly to basolateral or apical HCO(3)(-) permeability and whether the cotransporter participates in transendothelial net HCO(3)(-) flux in cultured bovine corneal endothelium. NBC1 protein expression was reduced using small interfering RNA (siRNA). Immunoblot analysis showed that 5-15 nM siRNA decreased NBC1 expression by 80-95%, 4 days posttransfection. Apical and basolateral HCO(3)(-) permeabilities were determined by measuring the rate of pH(i) change when HCO(3)(-) was removed from the bath under constant pH or constant CO(2) conditions. Using either protocol, we found that cultures treated with NBC1 siRNA had sixfold lower basolateral HCO(3)(-) permeability than untreated or siCONTROL siRNA-treated cells. Apical HCO(3)(-) permeability was unaffected by NBC1 siRNA treatment. Net non-steady-state HCO(3)(-) flux was 0.707 +/- 0.009 mM.min(-1).cm(2) in the basolateral-to-apical direction and increased to 1.74 +/- 0.15 when cells were stimulated with 2 muM forskolin. Treatment with 5 nM siRNA decreased basolateral-to-apical flux by 67%, whereas apical-to-basolateral flux was unaffected, significantly decreasing net HCO(3)(-) flux to 0.236 +/- 0.002. NBC1 siRNA treatment or 100 muM ouabain also eliminated steady-state HCO(3)(-) flux, as measured by apical compartment alkalinization. Collectively, reduced basolateral HCO(3)(-) permeability, basolateral-to-apical fluxes, and net HCO(3)(-) flux as a result of reduced expression of NBC1 indicate that NBC1 plays a key role in transendothelial HCO(3)(-) flux and is functional only at the basolateral membrane.
- Published
- 2005
- Full Text
- View/download PDF
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