Bovine Corneal Endothelium Research Articles

Morfologia das células endoteliais corneanas bovinas obtidas com alizarina vermelha e microscopia óptica

Abstract The aim of this study was to determine the endothelial cell morphology in the different regions of healthy bovine corneal endothelium using optical microscopy. Twenty eyeballs from 10 male Brangus cattle, aged 24 months, were studied. The corneal endothelium was stained with the vital dye alizarin red and then examined with an optical microscope and photographed. Thirty endothelial cells from each corneal region were included in the analysis. Endothelial cell morphology was analysed in the central, superior, inferior, lateral and medial regions of the cornea. Comparisons between regions were performed using repeated measures analysis of variance (ANOVA). Differences were considered statistically significant at P < 0.05. Normal endothelial cells were mainly hexagonal (83.7%), pentagonal (7.45%) and heptagonal (8.8%), with a minimal number of cells of other shapes present. No statistical differences were observed in the endothelial cell morphology when comparing different regions of the cornea. Regarding endothelial cell morphology, there were no differences between the corneal regions.

Specular microscopy of the corneal endothelial cells of bovines: an ex vivo study.

The endothelium is the most posterior layer of the cornea and is essential for maintaining corneal transparency. Due to variations in corneal endothelial parameters among different species, knowledge of the normal parameters for each species is crucial. To evaluate the corneal endothelium of bovines using contact specular microscopy. Twenty eyeballs from 10 male Brangus (Bos taurus) aged 24 months were evaluated. Contact specular microscopy was performed on the central corneal area. The analyzed parameters were endothelial cell density (ECD) and endothelial cell morphology. The ECD in the central area was 1,277 cells/mm2. Regarding the morphology, mainly cells with six (74.3%), five (14.7%) and seven sides (10%) were found. There were no significant differences in ECD and morphology between left and right eyes. Contact specular microscopy facilitated the analysis and measurement of corneal endothelial parameters in bovines. The data obtained will serve as a reference for the analysis of bovine corneal endothelium.

A mathematical model of wound healing in bovine corneal endothelium

We developed a mathematical model to describe healing processes in bovine corneal endothelial (BCE) cells in culture, triggered by mechanical wounds with parallel edges. Previous findings from our laboratory show that, in these cases, BCE monolayers exhibit an approximately constant healing velocity. Also, that caspase-dependent apoptosis occurs, with the fraction of apoptotic cells increasing with the distance traveled by the healing edge. In addition, in this study we report the novel findings that, for wound scratch assays performed preserving the basal extracellular matrix: i) the healing cells increase their en face surface area in a characteristic fashion, and ii) the average length of the segments of the cell columns actively participating in the healing process increases linearly with time. These latter observations preclude the utilization of standard traveling wave formalisms to model wound healing in BCE cells. Instead, we developed and studied a simple phenomenological model based on a plausible formula for the spreading dynamics of the individual healing cells, that incorporates original evidence about the process in BCE cells. The model can be simulated to: i) obtain an approximately constant healing velocity; ii) reproduce the profile of the healing cell areas, and iii) obtain approximately linear time dependences of the mean cell area and average length of the front active segments per column. In view of its accuracy to account for the experimental observations, the model can also be acceptably employed to quantify the appearance of apoptotic cells during BCE wound healing. The strategy utilized here could offer a novel formal framework to represent modifications undergone by some epithelial cell lines during wound healing.

Bovine posterior limbus: an evaluation of an alternative source for corneal endothelial and trabecular meshwork stem/progenitor cells.

A growing body of evidence has revealed that stem-like cells in the posterior limbus of the eye between the corneal endothelium (CE) and trabecular meshwork (TM) may be able to rejuvenate these tissues in disease. However, these cells have not been clearly defined and we have named them PET cells (progenitor cells of the endothelium and trabeculum). A good and inexpensive animal model for PET cells is lacking, so we investigated bovine eyes as an effective large tissue source. We showed the presence of stem/progenitor cells in the bovine CE, transition zone, and TM in situ. Floating spheres cultured from the CE and TM showed similar stem cell marker expression patterns. Both the CE and TM spheres were bipotent and highly proliferative, but with limited secondary sphere-forming capability. They were highly prone to differentiate back into the cell type of their tissue of origin. It is speculated that the PET cells become more tissue-specific as they migrate away from their niche. Here, we showed that PET cells are present in the posterior limbus of bovine eyes and that they can be successfully cultured and expanded. PET cells represent an attractive target for developing new treatments to regenerate both the CE and TM, thereby reducing the requirement for donor tissue for corneal transplant and invasive treatments for glaucomatous patients.

Role of MMP-9 in the breakdown of barrier integrity of the corneal endothelium in response to TNF-α

TNF-α induces loss of barrier integrity of the corneal endothelium through mechanisms involving the activation of p38 MAP kinase. This study has investigated the role of matrix metalloproteinase-9 (MMP-9), known to be activated by mechanisms downstream of p38 MAP kinase, on the breakdown of the barrier integrity. Experiments were performed with primary cultures of bovine corneal endothelium. Changes in the trans-endothelial electrical resistance (TER), a measure of barrier integrity, were measured by electric cell-substrate impedance sensing. The integrity of the apical junctional assembly was imaged by immunolocalization of ZO-1. MMP-9 activity in the conditioned medium of cells treated with TNF-α was visualized by gelatin zymography. Transcriptional activation of MMP-9 was assessed by real-time RT-PCR. Exposure to TNF-α led to significant disruption of ZO-1 and also caused a continuous decline in TER for more than 20 h. These effects were opposed by cycloheximide (protein synthesis inhibitor), GM-6001 (broad spectrum inhibitor of MMPs), minocycline (MMP-2 and MMP-9 inhibitor), and MMP-9 inhibitor I (selective MMP-9 inhibitor). Cycloheximide, GM-6001, and MMP-9 inhibitor I also attenuated the increase in permeability to FITC-dextran (10 kDa). In addition, TNF-α led to an increased MMP-9 activity in the conditioned medium as well as a nearly 20-fold increase in mRNA for MMP-9 but not for MMP-2. The functional activity and increase in mRNA levels of MMP-9 were blocked by SB-203580 (selective p38 MAP kinase inhibitor) and cycloheximide. In conclusion, transcriptional and translational activation of MMP-9, downstream of p38 MAP kinase signaling, is involved in the (TNF-α)-induced loss of corneal endothelial barrier integrity.

Dopaminrezeptoren des Hornhautepithels und -endothels

Dopamine is a major neurotransmitter and its two receptor subgroups, termed D1-like and D2-like receptors, are found both in the central and peripheral nervous systems. D1-like receptors signal through increases, D2-like receptors through decreases in cAMP production. Reports about the presence of dopamine receptors in the cornea are rare and inconsistant. The aim of this study was to examine if native bovine corneal epithelial and endothelial cells express dopamine receptors and whether these receptors belong to the D1-like or D2-like group. Dopamine receptors were studied using polyclonal antibodies. The cAMP concentration after receptor stimulation with dopamine was determined by means of an enzyme immunoassay. In bovine corneal epithelium and endothelium immunohistochemical staining was positive for D1-like receptors but not for D2-like receptors. Stimulation of corneal D1-like receptors with dopamine revealed a dose-dependent increase of the intracellular cAMP concentration which was blocked by SCH23 390 (a selective D1-like antagonist). Our data demonstrate that bovine corneal epithelium and endothelium express a functional D1-like receptor positively coupled to adenylyl cyclase and cAMP production. However, at the present time the physiological role of this receptor remains a matter of speculation.

Mechanical Interferometry Imaging for Creep Modeling of the Cornea

A novel nanoindentation technique was used to biomechanically characterize each of three main layers of the cornea by using Hertzian viscoelastic formulation of creep, the deformation resulting from sustained-force application. The nanoindentation method known as mechanical interferometry imaging (MII) with <1-nm displacement precision was used to observe indentation of bovine corneal epithelium, endothelium, and stroma by a spherical ferrous probe in a calibrated magnetic field. For each specimen, creep testing was performed using two different forces for 200 seconds. Measurements for single force were used to build a quantitative Hertzian model that was then used to predict creep behavior for another imposed force. For all three layers, displacement measurements were highly repeatable and were well predicted by Hertzian models. Although short- and long-term stiffnesses of the endothelium were highest of the three layers at 339.2 and 20.2 kPa, respectively, both stromal stiffnesses were lowest at 100.4 and 3.6 kPa, respectively. Stiffnesses for the epithelium were intermediate at 264.6 and 12.2 kPa, respectively. Precise, repeatable measurements of corneal creep behavior can be conveniently obtained using MII at mechanical scale as small as one cell thickness. When interpreted in analytical context of Hertzian viscoelasticity, MII technique proved to be a powerful tool for biomechanical characterization of time-dependent biomechanics of corneal regions.

Polyamidoamine dendrimer hydrogel for enhanced delivery of antiglaucoma drugs

Dendrimer hydrogel (DH), made from ultraviolet-cured polyamidoamine dendrimer G3.0 tethered with three polyethylene glycol (PEG, 12,000 Da)–acrylate chains (8.1% w/v) in pH 7.4 phosphate buffered saline (PBS), was studied for the delivery of brimonidine (0.1% w/v) and timolol maleate (0.5% w/v), two antiglaucoma drugs. DH was found to be mucoadhesive to mucin particles and nontoxic to human corneal epithelial cells. DH increased the PBS solubility of brimonidine by 77.6% and sustained the in vitro release of both drugs over 56–72 hours. As compared to eye drop formulations (PBS-drug solutions), DH brought about substantially higher human corneal epithelial cells uptake and significantly increased bovine corneal transport for both drugs. DH increased timolol maleate uptake in bovine corneal epithelium, stroma, and endothelium by 0.4- to 4.6-fold. This work demonstrated that DH can enhance the delivery of antiglaucoma drugs in multiple aspects and represents a novel platform for ocular drug delivery. From the Clinical EditorDendrimer hydrogel was studied as agent for simultaneous delivery of two anti-glaucoma drugs, one hydrophobic and one hydrophilic. Superiority over standard PBS-based formulation was clearly demonstrated for both drugs. The work may be a novel platform for ocular drug delivery.

Spiroplasma found in the eyes of scrapie affected sheep

Scrapie, a transmissible spongiform encephalopathy (TSE) occurring naturally in sheep, characteristically shows a severe retinopathy that is well developed in the terminal phases of the disease. In this study, we set out to demonstrate similar retinal changes in our ruminant spiroplasmosis TSE model. The eyes from deer, sheep, and goats that were inoculated intracranially with the laboratory strain of spiroplasma (suckling mouse cataract [SMCA] strain of Spiroplasma mirum) or with Spiroplasma sp. isolated from the brains affected with scrapie or with chronic wasting disease were examined by light microscopy for pathologic changes and by immunocytochemistry for distribution of spiroplasma antigen. The eyes were also obtained from a research flock of sheep with terminal scrapie, from which the intraocular tissues were submitted aseptically for culture assay in M1D broth or as explants on bovine corneal endothelia (BCE). The eyes from the spiroplasmosis ruminant models showed retinopathy remarkably similar to eye lesions seen in sheep with scrapie. The spiroplasma antigen accrued in the ruminant model eye tissues, particularly in the retina, the vitreous humor, and the corneal endothelia. A Spiroplasma sp. grew out of the scrapie-affected eyes both in the M1D broth and in the BCE cultures but did not expand. These new spiroplasma isolates differed immunologically from SMCA. These data showed a clear association of spiroplasma with scrapie suggesting that these bacteria have a role in the pathogenesis of TSE and that the eye should be a research focus for future studies of TSE.

In vitro culture of human fetal corneal endothelial cells

To explore and optimize a proper culture system for human fetal corneal endothelial cells (hFCECs), including the methods of primary culture, passage and cryopreservation. Fresh fetal corneas were explanted to propagate primary corneal endothelial cells. The cells were cultured in DMEM/F-12 supplemented with 10% fetal bovine serum (FBS) in the absence or presence of the extracts from bovine corneal endothelium cells (bCECs), and the cells were identified with immunocytochemical staining. The passage and cryopreservation of hFCECs were optimized according to previous reports on adult corneal endothelial cells. Using the explant culture method, hFCECs migrated successfully within 3 days and assumed polygonal-shaped corneal endothelial morphology. The optimizing methods were 0.125% trypsin + 0.02% EDTA for passage and 10% DMSO + 90% FBS for cryopreservation. Recovered hFCECs from cryopreservation remained typical morphology and immunological markers of corneal endothelial cells, including positive staining of NSE, Nestin, Ki67 and ZO-1, and negative staining of CK3/12, which demonstrated that they retained the characterizations of corneal endothelial cells and proliferative capacity. Moreover, the extracts from bCECs can promote the proliferative capacity of hFCECs significantly, while maintaining their typical endothelial morphology. The culture conditions of human fetal corneal endothelial cells were firstly optimized, including the primary culture, passage and cryopreservation. Meanwhile, we confirmed that the extracts from bovine corneal endothelium promoted the proliferative capacity while maintaining the morphology of hFCECs in vitro.

Lovastatin Inhibits the Thrombin-Induced Loss of Barrier Integrity in Bovine Corneal Endothelium

Increased actomyosin contraction of the dense band of actin cytoskeleton at the apical junctional complex (perijunctional actomyosin ring, PAMR) breaks down the barrier integrity of corneal endothelium. This study has investigated the efficacy of statins, which inhibit activation of RhoA, in opposing the thrombin-induced loss of barrier integrity of monolayers of cultured bovine corneal endothelium. Myosin light chain (MLC) phosphorylation, a biochemical measure of actomyosin contraction, was assayed by urea-glycerol gel electrophoresis, followed by western blot analysis. The locus of MLC phosphorylation and changes in the organization of the PAMR were visualized by immunostaining. Phosphorylation of MYPT1, a regulatory subunit of myosin light-chain phosphatase (MLCP), was assessed by Western blot analysis to determine down-regulation of RhoA. The barrier integrity was assessed in terms of trans-endothelial electrical resistance (TER), and further confirmed by determining permeability to FITC dextran (10 kDa) and distribution of ZO-1, a marker of tight junctional assembly. Lovastatin, a prototype of lipophilic statins, induced MLC dephosphorylation under basal conditions. It opposed increase in phosphorylation of MLC and MYPT1 in response to thrombin and nocodazole, agents known to activate RhoA in the endothelium. Pretreatment with the statin opposed the thrombin- and nocodazole-induced disruption of the PAMR and the thrombin-induced decline in TER. Lovastatin also opposed the thrombin- and nocodazole-induced increase in permeability to FITC dextran and redistribution of ZO-1. However, upon supplementation with GGPP (geranylgeranyl pyrophosphate), lovastatin failed to oppose the effects of thrombin and nocodazole on the PAMR, ppMLC, and ZO-1 distribution. Lovastatin attenuates RhoA activation in the corneal endothelium presumably by reducing its isoprenylation. This underlies the suppression of the thrombin-induced loss in barrier integrity of the corneal endothelium.

Dependence of cAMP meditated increases in Cl− and HCO3− permeability on CFTR in bovine corneal endothelial cells

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 HCO3− 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 HCO3− permeability and steady-state HCO3− 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 ∼80% in CFTR siRNA treated cultures. Forskolin (10μM) 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 HCO3− permeability was increased 2-fold by 10μM forskolin, which was reduced to control level in siRNA treated cultures. Similarly, there was no effect on baseline apical HCO3− permeability by knocking down CFTR expression. The steady-state apical-basolateral pH gradient (ΔpH) at 4h in control cultures was increased ∼2.5-fold by forskolin. In CFTR siRNA treated cells, the baseline ΔpH was similar to control, however forskolin did not have a significant effect. We conclude that forskolin induced increases in apical HCO3− permeability in bovine corneal endothelium requires CFTR. However, CFTR does not have a major role in determining baseline apical chloride or HCO3− permeability.

Proteinkinase C Isoformen in Hornhautepithel und -endothel

Protein kinase C (PKC) plays a key role in cell metabolism. Three subgroups and 12 isoforms have been isolated so far, catalysing specific functions in cell metabolism. The demonstration of PKC subtypes in corneal tissue has been inconsistent. The aim of this study was to verify the expression of several PKC subgroups and isoforms in human and bovine corneal epithelial and endothelial cells. PKC subgroups and isoforms were studied using polyclonal antibodies. Antibodies to PKC-alpha, -delta, -epsilon and -zeta, representing all three PKC subgroups, bound in human and bovine corneal epithelium and endothelium. No binding was found for antibodies to PKC-beta2. For the first time the presence of all three PKC subgroups was demonstrated in human and bovine corneal epithelial and endothelial cells. Further studies are needed to show the role of these subgroups in cellular functions such as cell proliferation and differentiation.

Retinal Pigment Epithelium Rescues Vascular Endothelium from Retinoic Acid-Induced Apoptosis

To determine whether retinoids are capable of inducing vascular endothelial cell apoptosis and whether the presence of an intact RPE monolayer can block retinoid-induced vascular endothelial cell death. Confluent fetal bovine aortic endothelial (FBAE) cells were incubated with various concentrations of all-trans or 9-cis retinoic acid (an analogue of 11-cis retinoic acid). Apoptosis rates were determined at 24 hours, and the effect of inhibition of protein synthesis and activation of protein kinase C on apoptosis was investigated by supplying culture medium with 0.1 mg/mL cycloheximide and 10 nM phorbol myristate acetate. To investigate the impact of RPE on retinoid-induced apoptosis, confluent FBAE cells were cultured with a confluent layer of RPE in inserts where retinoids were added to the upper compartment. A confluent bovine corneal endothelium monolayer was used as the control. The permeabilities of the RPE and bovine corneal endothelium monolayers to fluorescein (20 microg/mL) and 9-cis retinoic acid (3 x 10(-4) M) were also determined. 9-cis Retinoic acid induced higher rates of apoptosis in FBAE cells than did all-trans retinoic acid and the control (P = 0.004). This effect was dose-dependent, with an ED(50) of 1.4 microM (r = 0.99, P = 0.004). Cycloheximide did not inhibit 9-cis retinoic acid-induced apoptosis, but phorbol myristate acetate significantly decreased the apoptosis rate (P = 0.005). The presence of a confluent RPE monolayer reduced the 9-cis retinoic acid-induced apoptosis rate (P = 0.002), but the presence of a bovine corneal endothelial monolayer did not (P > 0.05). Both cell types established a similar diffusion barrier against fluorescein and 9-cis retinoic acid. 9-cis Retinoic acid is an important mediator of vascular endothelial apoptosis. A confluent monolayer of RPE can prevent endothelial cell apoptosis, and this effect is not due simply to establishment of a diffusion barrier by the RPE.

Muscarinic cholinoceptor-stimulated phosphatidyl inositol pathway in corneal epithelial and endothelial cells

Muscarinic cholinoceptors are distributed widely in both the central and peripheral nervous system. The presence of muscarinic cholinoceptors in corneal tissue is well established. Previous reports have shown that corneal muscarinic cholinoceptors are of the m2 or m4 subtype. However, recent studies have indicated the presence of the m5 muscarinic cholinoceptor subtype in human corneal epithelium and endothelium. The aim of the study was to confirm the presence of the m5 cholinoceptor subtype in bovine corneal epithelium and endothelium and the activation of phosphatidyl inositol pathway by its stimulation. Muscarinic m5 cholinoceptor sites, phosphatidyl inositol 4,5-biphosphate, inositol 1,4,5-triphosphate and protein kinase C, were studied using immunocytochemistry and immunofluorescence. Activation of protein kinase C after stimulation of the m5 muscarinic cholinoceptor subtype was measured using the HTS protein kinase C assay kit. Immunocytochemistry/immunofluorescence revealed the presence of the m5 muscarinic cholinoceptor subtype, phosphatidyl inositol 4,5-biphosphate and protein kinase C in bovine corneal epithelial and endothelial cells. In bovine corneal epithelium and endothelium, protein kinase C activity was stimulated by acetylcholine in a dose-dependent manner (P<0.0001). Our findings indicate that acetylcholine-induced stimulation of muscarinic m5 cholinoceptors activates the phosphatidyl inositol pathway in corneal epithelial and endothelial cells, resulting in increased protein kinase C activity. Further work will be needed to clear the physiologic role of this signaling pathway in corneal epithelium and endothelium.

Molecular expression and functional involvement of the bovine calcium-activated chloride channel 1 (bCLCA1) in apical HCO 3− permeability of bovine corneal endothelium

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 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 [Ca 2+ i] by application of ATPγS 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.

Human Corneal Endothelial Cell Precursors Isolated by Sphere-Forming Assay

To isolate precursors of human corneal endothelial cells (HCECs) in vitro. HCECs were subjected to a sphere-forming assay in which spheres floated in serum-free medium containing growth factors. To promote differentiation, the isolated sphere colonies were plated in dishes coated with poly-L-lysine (PLL)/laminin or fetal bovine endothelium extracellular matrix. Marker expression of neural and mesenchymal cells was examined in the sphere colonies and their progenies by immunocytochemistry and/or reverse transcription-polymerase chain reaction (RT-PCR). Adherent differentiated cells from the sphere colonies were evaluated morphologically and functionally. HCECs formed primary and secondary spherical colonies, as shown by sphere-forming assay in vitro. The colonies expressed nestin, beta3-tublin, glial fibrillary acidic protein, and alpha-smooth muscle actin on immunocytochemistry. The progeny, proliferating on extracellular matrix derived from bovine corneal endothelium, but not on PLL/laminin-coated and noncoated dishes, expressed nestin and beta3-tublin. These markers were confirmed by RT-PCR. Adherent differentiated cells from the sphere colonies had an HCEC-like hexagonal shape and satisfactory transport activity that is essential in HCECs. These findings indicate that the HCEC contains precursor cells with a propensity to differentiate into HCECs and that these cells can also produce neuronal and mesenchymal cell proteins.

Characterization of adenosine receptors in bovine corneal endothelium

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 (∼50 kDa) and A 1 (∼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 μ m alloxazine, a specific A 2b blocker. A 1 activation with 1 μ m N 6-CPA (a specific A 1 agonist) or 100 n m 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 μ m), 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 μ m adenosine, acts to decrease [cAMP] i and A 2b, activated at >1 μ m adenosine, increase [cAMP] i.

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

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.

Effect of membrane potential depolarization on the organization of the actin cytoskeleton of eye epithelia. The role of adherens junctions

We have previously determined that the depolarization of the plasma membrane potential of confluent bovine corneal endothelial cells in culture, provokes a characteristic reorganization of the actin cytoskeleton. The purposes of the present work are to investigate whether similar responses are exhibited by other epithelia, irrespectively of their specific functions and embryonic origin, and to test the hypothesis that the cytoskeletal reorganization induced by membrane depolarization requires of a well-organized circumferential actin disposition in order to take place. For this, we have performed studies on three different cultured epithelia of the eye: bovine corneal endothelium, bovine retinal pigment epithelium and cellular lines of murine lens epithelium. For all of these cells, we explored the effects of plasma membrane depolarization, achieved via the incorporation of gramicidin D to the bathing media or by the replacement of extracellular sodium chloride by potassium gluconate, on the cadherin and actin distribution. The membrane potential changes were monitored by fluorescence microscopy using oxonol V; fluorescent probes were also used for F-actin and cadherin. Detergent extraction and Western blot analysis were employed to reveal the relative amount of cadherin attached to the cytoskeleton. The main findings of this study are that different confluent cultured epithelia exhibiting a well-defined circumferential pattern of actin distribution respond to plasma membrane depolarization by similar modifications in the actin cystoskeleton to those reported for bovine corneal endothelial cells. On the other hand, epithelia that do not exhibit such actin pattern in confluence, as well as non-confluent monolayers, do not display noticeable actin modifications in response to the depolarizing procedures. While in the former cells, cadherin is predominantly located at the lateral membrane domain, the cells that do not respond to membrane depolarization mainly display their cadherin in the intracellular compartment. We suggest that the typical peripheral disposition of actin, associated to well-established epithelial-type adherens junctions (i.e. zonula adherens), is a pre-requisite for the cytoskeletal organizational modifications exhibited by epithelial cells in response to the depolarization of the plasma membrane potential.