Bovine corneal endothelium in vitro

MacCallum, Donald K.; Lillie, John H.; Scaletta, Lawrence J.; Occhino, Joseph C.; Frederick, William C.; Ledbetter, Steven

doi:10.1016/0014-4827(82)90313-5

Cited by 73 publications

(18 citation statements)

References 28 publications

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“…Primary cultures of bovine corneal endothelial cells were established by a method similar to the one of MacCallum et al [31]. Eyes from 3-to 4-years-old steers were obtained from a local abbattoir.…”

Section: Cell Culturesmentioning

confidence: 99%

Effect of bicarbonate, pH, methazolamide and stibenes on the intracellular potentials of cultured bovine corneal endothelial cells

et al. 1984

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Micropuncture of cultured bovine corneal endothelial cells led to registrations stable for hours. Intracellular potentials were mainly in the range of -40 to -55 mV, average 46.3 +/- 0.6 mV (SEM). Changes of extracellular [HCO-3] led to voltage transients, their amplitude depending logarithmically on [HCO-3] with a mean slope of 37.3 +/- 8.8 (SD) mV. After removal of bicarbonate/CO2, a steady-state depolarization was seen. This steady-state depolarization, but not the voltage transients, could be reduced by 1 mM Ba++. After removal of bicarbonate, the voltage response to changes of extracellular potassium was reduced. Alteration of pHi induced by permeable buffers (butyrate, glycodiazine and ammonium) also resulted in voltage transients, internal acidification being correlated with a hyperpolarization, and internal alkalinization with a depolarization. Also changes of external pH caused voltage responses, alkalinization causing a hyperpolarization, acidification a depolarization. Methazolamide, an inhibitor of carbonic anhydrase, as well as stilbenes (SITS or DIDS) caused a reduction of the voltage response to HCO-3 and pH. Their effects were additive. It is suggested that corneal endothelial cells possess one or two electrogenic transporters for HCO-3 or related species, one of which is inhibitable by stilbenes.

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Section: Cell Culturesmentioning

confidence: 99%

Effect of bicarbonate, pH, methazolamide and stibenes on the intracellular potentials of cultured bovine corneal endothelial cells

et al. 1984

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“…Bovine corneal endothelial cells (BCEC) were cultured to confluence onto 25-mm round coverslips, 13-mm Anodisc filters, or T-25 flasks as previously described (7,24). Briefly, primary cultures from fresh cow eyes were established in T-25 flasks with 3 ml of Dulbecco's modified Eagle's medium (DMEM), 10% bovine calf serum, and antibiotic (100 U/ml penicillin, 100 U/ml streptomycin, and 0.25 g/ml Fungizone), gassed with 5% CO2-95% air at 37°C, and fed every 2-3 days.…”

Section: Methodsmentioning

confidence: 99%

Role of NBC1 in apical and basolateral HCO₃⁻ permeabilities and transendothelial HCO₃⁻ fluxes in bovine corneal endothelium

Li¹,

Sun²,

Bonanno³

2005

American Journal of Physiology-Cell Physiology

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Ϫ transport properties of the corneal endothelium. Recent work (13) suggested the presence of an apical 1Na ϩ -3HCO 3 Ϫ cotransporter (NBC1) in addition to a basolateral 1Na ϩ -2HCO 3 Ϫ cotransporter. We examined whether the NBC1 cotransporter contributes significantly to basolateral or apical HCO 3 Ϫ permeability and whether the cotransporter participates in transendothelial net HCO 3 Ϫ 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 Ϫ permeabilities were determined by measuring the rate of pHi change when HCO 3 Ϫ was removed from the bath under constant pH or constant CO2 conditions. Using either protocol, we found that cultures treated with NBC1 siRNA had sixfold lower basolateral HCO 3 Ϫ permeability than untreated or siCONTROL siRNA-treated cells. Apical HCO 3 Ϫ permeability was unaffected by NBC1 siRNA treatment. Net non-steady-state HCO 3 Ϫ flux was 0.707 Ϯ 0.009 mM ⅐ min Ϫ1 ⅐ cm 2 in the basolateral-to-apical direction and increased to 1.74 Ϯ 0.15 when cells were stimulated with 2 M 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 Ϫ flux to 0.236 Ϯ 0.002. NBC1 siRNA treatment or 100 M ouabain also eliminated steady-state HCO 3 Ϫ flux, as measured by apical compartment alkalinization. Collectively, reduced basolateral HCO 3 Ϫ permeability, basolateral-to-apical fluxes, and net HCO 3 Ϫ flux as a result of reduced expression of NBC1 indicate that NBC1 plays a key role in transendothelial HCO 3 Ϫ flux and is functional only at the basolateral membrane. corneal endothelium; sodium bicarbonate cotransporter; small interfering RNA; bicarbonate transport THE ION AND FLUID TRANSPORT PROPERTIES of the corneal endothelium are responsible for maintaining the hydration and transparency of the cornea. Corneal endothelial fluid transport requires the presence of HCO 3 Ϫ (15, 18, 29) and Cl Ϫ (44), is sensitive to carbonic anhydrase inhibitors (19,22,29), and is completely eliminated by ouabain, a Na ϩ -K ϩ -ATPase inhibitor. Net stroma to anterior chamber HCO 3 Ϫ flux is responsible for the measured short-circuit current and the small, negative transendothelial potential (19), suggesting that HCO 3 Ϫ is the primary secreted anion. Although significant progress has been made in identifying and locating plasma membrane transporters in the corneal endothelium, the contribution of the transporters to net HCO 3 Ϫ transport is largely unknown. In this study we have examined the role of the sodium bicarbonate cotransporter (NBC1) in transendothelial HCO 3 Ϫ transport. Previous studies have shown that the uptake of HCO 3 Ϫ across the basolateral membrane of corneal endothelial cells occurs by a potent Na ϩ -dependent, Cl Ϫ -independent, DIDSsensitive, and electrogenic Na ϩ -2HCO 3 Ϫ cotransporter (5, 8, 21...

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“…BCECs were cultured to confluence onto 25 mm round coverslips, 13 mm anodiscs, or T-25 flasks as previously described (Bonanno and Giasson, 1992;MacCallum et al, 1982). Briefly, primary cultures from fresh cow eyes were established in T-25 flasks with 3 ml of Dulbecco's modified Eagle's medium, 10% bovine calf serum, and antibiotics (penicillin 100 u/ml), gassed with 5% CO 2 -95% air at 37 °C and fed every 2-3 days.…”

Section: Cell Culturementioning

confidence: 99%

Molecular expression and functional involvement of the bovine calcium-activated chloride channel 1 (bCLCA1) in apical HCO3− permeability of bovine corneal endothelium

Zhang

Xie

et al. 2006

Experimental Eye Research

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Corneal endothelium secretes from basolateral (stroma) to apical (anterior chamber) compartments. Apical 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 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 permeability. In situ hybridization confirmed prominent bCLCA1-specific mRNA expression in CBCEC. bCLCA1 antiserum detected the heterologously expressed bCLCA1 in HEK293 cells and a 90 kDa 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 [ ] by application of ATPγS or cyclopiazonic acid (CPA) increased apical permeability in siControl transfected CBCEC, while having no effect on apical permeability in bCLCA1 specific siRNA transfected cells. Baseline 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 permeability, but not resting permeability, across the corneal endothelium.

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Bovine corneal endothelium in vitro

Cited by 73 publications

References 28 publications

Effect of bicarbonate, pH, methazolamide and stibenes on the intracellular potentials of cultured bovine corneal endothelial cells

Effect of bicarbonate, pH, methazolamide and stibenes on the intracellular potentials of cultured bovine corneal endothelial cells

Role of NBC1 in apical and basolateral HCO₃⁻ permeabilities and transendothelial HCO₃⁻ fluxes in bovine corneal endothelium

Molecular expression and functional involvement of the bovine calcium-activated chloride channel 1 (bCLCA1) in apical HCO3− permeability of bovine corneal endothelium

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Bovine corneal endothelium in vitro

Cited by 73 publications

References 28 publications

Effect of bicarbonate, pH, methazolamide and stibenes on the intracellular potentials of cultured bovine corneal endothelial cells

Effect of bicarbonate, pH, methazolamide and stibenes on the intracellular potentials of cultured bovine corneal endothelial cells

Role of NBC1 in apical and basolateral HCO3− permeabilities and transendothelial HCO3− fluxes in bovine corneal endothelium

Molecular expression and functional involvement of the bovine calcium-activated chloride channel 1 (bCLCA1) in apical HCO3− permeability of bovine corneal endothelium

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Role of NBC1 in apical and basolateral HCO₃⁻ permeabilities and transendothelial HCO₃⁻ fluxes in bovine corneal endothelium