Ocular Epithelium Research Articles

HCO(3)(-)-dependent soluble adenylyl cyclase activates cystic fibrosis transmembrane conductance regulator in corneal endothelium.

cAMP-dependent activation of the cystic fibrosis transmembrane conductance regulator (CFTR) regulates fluid transport in many tissues. Secretion by the corneal endothelium is stimulated by cAMP and dependent on HCO(3)(-). We asked whether HCO(3)(-) can secondarily increase CFTR permeability in bovine corneal endothelial cells (BCEC) by activating soluble adenylyl cyclase (sAC). Immunofluorescence suggests that sAC is distributed throughout the cytoplasm. HCO(3)(-) (40 mM) increased cAMP concentration 42% in the presence of 50 microM rolipram (a phosphodiesterase 4 inhibitor), and a standard HCO(3)(-) Ringer solution (28.5 mM) increased apical Cl(-) permeability by 78% relative to HCO(3)(-)-free solution. The HCO(3)(-)-dependent increase in Cl(-) permeability was reduced 60% by 20 mM NaHSO(3) (a weak agonist of sAC). NaHSO(3) alone increased apical Cl(-) permeability by only 13%. The HCO(3)(-)-dependent increase in Cl(-) permeability was reduced 57% in the presence of 50 microM Rp-adenosine 3',5'-cyclic monophosphorothioate, and 86% by 50 microM 5-nitro-2-(3-phenylpropyl-amino)benzoic acid but unaffected by 200 microM apical H(2)DIDS. CFTR phosphorylation was increased 23, 150, and 32% by 20 mM HSO(3)(-), 28.5 mM HCO(3)(-), and 28.5 mM HCO(3)(-) + 20 mM HSO(3)(-), respectively. Activation of apical Cl(-) permeability by 5 microM genistein was increased synergistically by HCO(3)(-) over that due to genistein and HCO(3)(-) alone. We conclude that HCO(3)(-)-stimulated sAC is a form of autocrine signaling that contributes to baseline cAMP production, thereby affecting baseline CFTR activity in BCEC. This form of autocrine signaling may be important in tissues that express sAC and exhibit robust HCO(3)(-) influx (e.g., ocular ciliary epithelium, choroid plexus, and airway epithelium).

Extensive hepatic uptake of Pz-peptide, a hydrophilic proline-containing pentapeptide, into isolated hepatocytes compared with colonocytes and Caco-2 cells

The objective of the present study was to investigate the uptake process of 4-Phenylazobenzoxycarbonyl-Pro-Leu-Gly-Pro-D-Arg (Pz-peptide), a hydrophilic and collagenase-labile pentapeptide, by isolated hepatocytes. For comparison, the uptake of Pz-peptide by Caco-2 cells and colonic cells, two known paracellular mutes of Pz-peptide, was also evaluated. A simple and sensitive reversed-phase HPLC assay method using UV detection has been developed. The coefficient of variation for all the criteria of validation were less than 15%. The method was, therefore, considered to be sutable for measuring the concentration of Pz-peptide in the biological cells. Pz-peptide was extensively uptaked into hepatocytes. The initial velocity of Pz-peptide uptake assessed from the initial slope of the curve was plotted as Eadie-Hofstee plots. The maximum velocity (Vmax) and the Michaelis constant (Km) were 0.190+/-0.020 nmol/min/10(6) cells and 12.1+/-3.23 microM, respectively. The permeability-surface area product (PS(influx)) was calculated to be 0.0157 ml/min/10(6) cells. Vmax and Km values for Caco-2 cells were calculated to be 6.22+/-0.930 pmol/min/10(6) cells and 82.8+/-8.37 microM, respectively, being comparable with those of colonocytes (6.04+/-1.03 pmol/min/10(6) cells and 87.8+/-13.2 microM, respectively). PS(influx) values for Caco-2 cells and colonocytes were calculated to be 0.0751 microl/min/10(6) cells and 0.0688 microl/min/10(6) cells, respectively. The more pronounced uptake of Pz-peptide by hepatocytes, when compared with Caco-2 cells and colonocytes, is probably due to its specific transporter. In conclusion, Pz-peptide, a paracellularly transported pentapeptide in the intestine and ocular epithelia, was uptaked into hepatocytes extensively. Although Pz-peptide is able to be uptaked into the Caco-2 cells and colonocytes, it is less pronounced when compared with hepatocytes. PS(influx) values of Caco-2 cells and colonocytes for unbound Pz-peptide under linear conditions were less than 0.4% when compared with that of hepatocytes.

Participation of pigment epithelium in ocular immune privilege. 3. Epithelia cultured from iris, ciliary body, and retina suppress T-cell activation by partially non-overlapping mechanisms

Purpose : The ocular microenvironment is immunosuppressive and anti-inflammatory. Since various ocular pigmented epithelia contribute to this microenvironment, we studied the relative capacities of pigment epithelial (PE) cells cultured from the iris, ciliary body, and retina of mouse eyes to suppress T-cell activation in vitro. Methods : Pigment epithelium was cultured from iris, ciliary body, and retina for 14 days, then assayed for the capacity, directly or across transwell membranes, to suppress mixed lymphocyte reactions and anti-CD3 stimulation of T cells. Potential molecules responsible for suppression were examined by attempting to block suppression with appropriate reagents, and by using mice with pertinent mutant or disrupted genes. Results : We found that PE cells from all three ocular tissue sources profoundly suppressed T-cell activation in vitro. While iris PE suppressed poorly when separated from T cells by a transwell membrane (implying that cell contact is necessary), retina PE suppressed fully even in the presence of such a membrane (implying that soluble factors were responsible). Ciliary body PE used both soluble factors as well as cell contact to achieve suppression. Suppression could not be ascribed to TGFß, IFN?, TNFa, CD48, or ICAM-1, or to interactions between CD40 and CD154, or CD95 and CD95 ligand. Galectin-1, a galactoside-binding protein, was found to be expressed on all cultured PE cells, but only retinal pigment epithelium (RPE) from galectin-1 KO mice showed reduced capacity to inhibit T-cell activation. Conclusions : Cultured pigment epithelia from iris, ciliary body, and retina comparably suppress T-cell activation in vitro, but by partially different mechanisms. Although RPE cells suppress in part through expression of galectin-1, the molecular mediators of suppression by iris and ciliary body PE remain to be identified.

Uveal melanocytes, ocular pigment epithelium, and Müller cells in culture: in vitro toxicology.

Uveal melanocytes and the ocular pigment epithelium are located in the middle and inner layers of the eye. Müller cells (a type of glial cell) are located in the neural retina. Melanocytes, retinal pigment epithelium (RPE), and Müller cells do not participate directly in the detection or transfer of visual information, but they have various functions that support the neural retina and are essential for the maintenance of vision. Methods for the isolation and cultivation of melanocytes, RPE, and Müller cells have been established by us and other investigators. These cultured cells can be used as in vitro model systems for studying the toxicology of visible light, ultraviolet (UV) radiation, drugs, and other potentially toxic agents. Toxic effects on these cells may give rise to altered retinal function and result in impaired vision. Both melanocytes and pigment epithelium contain melanin, which has the ability to bind organic amines and metal ions. This results in the accumulation of these substances in the eye. Melanin may protect cells from chemical stress by binding toxic chemicals; but in chronic exposure, increased and lengthy binding may cause damage to these cells. Two different types of melanin are found in the eye: eumelanin and pheomelanin, which may have photoprotective and phototoxic effects, respectively. Pigment epithelium contains mainly eumelanin, whereas melanocytes contain both eumelanin and pheomelanin. Melanin is an antioxidant and with age, the antioxidant properties may diminish to the point that it may even become a prooxidant. There are also other functions of pigment epithelium and uveal melanocytes not related to melanin and there are also several functions of Muller cells that play a role in the toxicological aspects of the eye. Cultured uveal melanocytes, pigment epithelial cells, and Müller cells can be used to study the toxicology of these cells in vitro.

Differential regulation of gene expression of neurotensin and prohormone convertases PC1 and PC2 in the bovine ocular ciliary epithelium: possible implications on neurotensin processing

Prohormone convertases PC1 and PC2 are enzymes involved in the intracellular processing of pro-neurotensin/neuromedin N (pro-NT/NN) through the regulated secretory pathway. In this study, we present evidence of the differential gene expression of pro-NT/NN, pro-PC1 and pro-PC2 in two cell lines established from the neuroendocrine ocular ciliary epithelium. Dexamethasone and forskolin were found to synergistically up-regulate NT/NN mRNA expression in both cell types. The pigmented cells released NT, and this release was enhanced by agents that induced its biosynthesis. In contrast, nonpigmented cells exhibited a significantly reduced neurotensin secretion in response to inducers, leading to an accumulation of the peptide. PC1 and PC2 mRNA expression was induced in a cell-specific manner by the same agents that enhanced pro-NT/NN biosynthesis. These results demonstrate cell-specific processing of pro-NT/NN by the ciliary epithelium.

Intermediate-filament expression in ocular tissue.

Intermediate-filament proteins (IFPs) occur in the intracellular cytoskeleton of eukaryotic cells, and their expression in diverse tissues is related both to embryology as well as to differentiation. Although the available information concerning their functional properties in vivo is still incomplete, antibodies against individual IFPs are commonly used in immunohistochemical procedures as markers for differentiation, and these antibodies are of outstanding value in the routine histopathological evaluation of tumor specimens. This review presents a compilation of the currently available data concerning IFP expression in normal and diseased ocular tissues. Representatives of every known class of IFP have been detected in normal ocular tissues. The external epithelia exhibit complex expression patterns of cytokeratin (CK) polypeptides, with CK3 and CK12 being specific markers of the corneal epithelium. Recent research has revealed that single mutant CK polypeptides may play a role in the pathogenesis of corneal dystrophies. The internal ocular epithelia reveal simple but specific patterns of IFP expression, these comprising simple-epithelial CKs and/or the mesenchymal IFP, vimentin. The IFP complement of the neuronal structures of the eye embraces several distinct IFP classes and reflects the diversity of the cell types present at these sites. With respect to ocular tumors, the IFP profile of melanomas might be correlated with metastatic potential. In conclusion, IFP analysis may be able to cast light on the pathogenesis of ocular diseases, as well as being a valuable adjunct in ophthalmopathological diagnosis.

Molecular mechanisms of water transport in the eye

The four major sites for ocular water transport, the corneal epithelium and endothelium, the ciliary epithelium, and the retinal pigment epithelium, are reviewed. The cornea has an inherent tendency to swell, which is counteracted by its two surface cell layers, the corneal epithelium and endothelium. The bilayered ciliary epithelium secretes the aqueous humor into the posterior chamber, and the retinal pigment epithelium transports water from the retinal to the choroidal site. For each epithelium, ion transport mechanisms are associated with fluid transport, but the exact molecular coupling sites between ion and water transport remain undefined. In the retinal pigment epithelium, a H+-lactate cotransporter transports water. This protein could be the site of coupling between salt and water in this epithelium. The distribution of aquaporins does not suggest a role for these proteins in a general model for water transport in ocular epithelia. Some water-transporting membranes contain aquaporins, others do not. The ultrastructure is also variable among the cell layers and cannot be fitted into a general model. On the other hand, the direction of cotransport in symporters complies with the direction of fluid transport in both the corneal epi- and endothelium, as well as the ciliary epithelium and retinal pigment epithelium.

Molecular Evidence That Human Ocular Ciliary Epithelium Expresses Components Involved in Phototransduction

Here we report the expression, in the human ocular ciliary epithelium and in a human nonpigmented (NPE) ciliary epithelial cell line, of genes usually restricted to cone and rod photoreceptor cells of the retina. By RT-PCR and DNA sequencing we identified the expression of rhodopsin and components linked to its deactivation, including rhodopsin kinase, recoverin, and visual arrestin. We also detected the expression of transducin (T-α), phosphodiesterase (PDE-α), and cGMP-gated channel α-subunits. Cultured NPE cells responded to treatment with phorbol ester by enhancing the expression of rhodopsin mRNA three- to fourfold. Indirect immunofluorescence of the intact ciliary epithelium with monoclonal antibodies (MAbs) against rhodopsin, rhodopsin kinase, and visual arrestin revealed labeling preferentially restricted to the NPE cells. Furthermore, Western blot analysis of whole lysates from the pars plicata region of the human ciliary epithelium with MAbs demonstrated immunochemical cross-reactivity with proteins of molecular mass similar to rhodopsin (36 kDa), rhodopsin kinase (64 to 66 kDa), and arrestin (48–52 kDa) from the human retina. These results provide the first molecular evidence that components of a non-visual phototransduction pathway are expressed in the human ocular NPE ciliary epithelium, which may be linked to circadian entrainment tasks.

Otx genes are required for tissue specification in the developing eye.

Patterning of the vertebrate eye appears to be controlled by the mutual regulation and the progressive restriction of the expression domains of a number of genes initially co-expressed within the eye anlage. Previous data suggest that both Otx1 and Otx2 might contribute to the establishment of the different eye territories. Here, we have analysed the ocular phenotype of mice carrying different functional copies of Otx1 and Otx2 and we show that these genes are required in a dose-dependent manner for the normal development of the eye. Thus, all Otx1(-/-); Otx2(+/-) and 30% of Otx1(+/-); Otx2(+/-) genotypes presented consistent and profound ocular malformation, including lens, pigment epithelium, neural retina and optic stalk defects. During embryonic development, optic vesicle infolding was severely altered and the expression of pigment epithelium-specific genes, such as Mitf or tyrosinase, was lost. Lack of pigment epithelium specification was associated with an expansion of the prospective neural retina and optic stalk territories, as determined by the expression of Pax6, Six3 and Pax2. Later in development the presumptive pigment epithelium region acquired features of mature neural retina, including the generation of Islet1-positive neurones. Furthermore, in Otx1(-/-); Otx2(+/-) mice neural retina cell proliferation, cell differentiation and apoptotic cell death were also severely affected. Based on these findings we propose a model in which Otx gene products are required for the determination and differentiation of the pigment epithelium, co-operating with other eye patterning genes in the determination of the specialised tissues that will constitute the mature vertebrate eye.

Ectopic gland induction by lens-specific expression of keratinocyte growth factor (FGF-7) in transgenic mice

During mammalian embryogenesis, epithelial-mesenchymal interactions play a determining role in normal tissue patterning and development. Keratinocyte growth factor (KGF), a member of the fibroblast growth factor (FGF) family, is a mesenchymally-derived mitogen for epithelial cells. As the KGF receptor is expressed by epithelial cells of numerous tissues and KGF is produced in adjacent stromal cells, KGF is thought to play a role in mediating epithelial cell behaviour. To further investigate the role of this molecule in the development of ocular epithelia we employed transgenic mice engineered to overexpress human KGF in the eye. The most striking phenotypic development was the hyperproliferation of embryonic corneal epithelial cells and their subsequent differentiation into functional lacrimal gland-like tissues. This indicates that stimulation of the KGF receptor early in development, in surface ectoderm normally destined to form corneal epithelium, is sufficient to alter the fate of these cells. Furthermore, this suggests that the correct spatial and temporal expression of FGFs plays a critical role in normal lacrimal gland induction. These transgenic mice provide a valuable model system to study the mechanisms underlying cell fate decisions during ocular morphogenesis.

Surfactant Protein C Promoter-Driven Expression of T1-Alpha Induces Lung Inflammation

T1-alpha (TIA) is a cell membrane protein expressed with high specificity by the alveolar (primarily type I cell), choroid plexus, and ciliary (ocular) epithelia in the rat. In the lung, TIA undergoes upregulation prior to birth as well as in several models of lung injury. The unusual pattern of basal expression and upregulation with physiologically critical events supports an important function for TIA. Analysis of predicted protein structure suggests that TIA is a type Ia membrane glycoprotein, but no homologies or motifs provide clues to function.

Location and clonal analysis of stem cells and their differentiated progeny in the human ocular surface.

We have analyzed the proliferative and differentiation potential of human ocular keratinocytes. Holoclones, meroclones, and paraclones, previously identified in skin, constitute also the proliferative compartment of the ocular epithelium. Ocular holoclones have the expected properties of stem cells, while transient amplifying cells have variable proliferative potential. Corneal stem cells are segregated in the limbus, while conjunctival stem cells are uniformly distributed in bulbar and forniceal conjunctiva. Conjunctival keratinocytes and goblet cells derive from a common bipotent progenitor. Goblet cells were found in cultures of transient amplifying cells, suggesting that commitment for goblet cell differentiation can occur late in the life of a single conjunctival clone. We found that conjunctival keratinocytes with high proliferative capacity give rise to goblet cells at least twice in their life and, more importantly, at rather precise times of their life history, namely at 45-50 cell doublings and at approximately 15 cell doublings before senescence. Thus, the decision of conjunctival keratinocytes to differentiate into goblet cells appears to be dependent upon an intrinsic "cell doubling clock. " These data open new perspectives in the surgical treatment of severe defects of the anterior ocular surface with autologous cultured conjunctival epithelium.

Functional Expression of Components of the Natriuretic Peptide System in Human Ocular Nonpigmented Ciliary Epithelial Cells

The expression of the natriuretic peptide system in the human ocular ciliary epithelium (CE) and in cultured nonpigmented (NPE) ciliary epithelial cells was examined. By RT-PCR and DNA sequencing, we demonstrated that the CE and NPE cells express mRNA for (i) ANP; (ii) BNP; (iii) NPR-A, NPR-B, and NPR-C receptors; and (iv) the neutral endopeptidase 24.11. Radioimmunoassay results indicate that BNP is secreted by cultured NPE cells at much higher levels than ANP. NPR-A and NPR-B receptors elicited a cGMP response to ANP, BNP, and CNP, in a rank order of potency (CNP ⪢ ANP ≥ BNP), indicative that the NPR-B receptor is predominant in NPE cells. A71915, an inhibitor of NPR-A activity, attenuated (65–75%) cGMP response to ANP and BNP, but not to CNP. C-ANP4–23elicited an inhibitory effect (30–37%) on basal levels of cAMP in NPE cells and on forskolin NPE-treated cells, indicative that the NPR-C receptor is functional in these cells. PMA induced, in NPE cells, a long-term downregulation (75–85%) of NPR-C receptor mRNA, but not of NPR-A or NPR-B receptor mRNA, suggesting a differential regulation of NPR-C receptor mRNA via activation of PKC. Collectively, our data provide molecular evidence that all the components of the natriuretic peptide system with the exception of CNP are coexpressed in the ocular NPE ciliary epithelial cells, where they may function as local autocrine/paracrine modulators to influence eye pressure.

Epithelial Hyperproliferation and Transglutaminase 1 Gene Expression in Stevens-Johnson Syndrome Conjunctiva

In Stevens-Johnson syndrome, pathological keratinization of the ordinarily nonkeratinized corneal and conjunctival mucosal epithelia results in severe visual loss. We examined conjunctiva covering cornea in five eyes in the chronic cicatricial phase of Stevens-Johnson syndrome. Normal conjunctiva from five age-matched individuals was studied also. The number of epithelial cells in Stevens-Johnson syndrome conjunctiva that were immunoreactive with a monoclonal antibody, Ki-67, to a nuclear antigen found only in proliferating cells was greater than normal (93.8+/-19.8 cells above 100 basal cells versus 12.8+/-0.5 cells above 100 basal cells; P = 0.009). In addition, although clinical inflammation was mild, massive lymphocytic infiltration was seen in the substantia propria of conjunctiva covering cornea. In situ hybridization documented transglutaminase 1 (keratinocyte transglutaminase) mRNA in suprabasal cells of the abnormally thickened conjunctival epithelium in all Stevens-Johnson syndrome patients. In contrast, no message was detected in normal conjunctival or corneal epithelia. Transglutaminase 1 is expressed during the terminal differentiation of keratinocytes where it helps synthesize cornified cell envelopes. We speculate that in Stevens-Johnson syndrome, epithelial hyperproliferation, and transglutaminase 1 gene expression lead to the pathological keratinization of ocular surface mucosal epithelia.

Immunolocalization of transcription factor AP1 in human ocular surface epithelia

Purpose. The present study examined whether normal human ocular surfcae epithelia express AP1 components. Changes in expression patterns of these components in a case of ocular surface epithelial dysplasia was also evaluated before and after topical mitomycin C treatment. Methods. Specimens of normal corneas (n = 2) and conjunctiva (n = 4) were obtained from 4 patients during cataract surgery or post mortem, while specimens of dysplastic epithelial tissue from the limbus were obtained from one patient. Specimens were immunohistochemically studied using antibodies against components of AP1. Results. The normal corneal epithelium showed no staining with antibodies against c-Fos, Fra-2, FosB, c-Jun or JunB, whereas the limbal and bulbar conjunctival epithelia were positive for c-Fos, Fra-2, and c-Jun. Anti-FosB and -JunB antibodies reacted weakly with the conjunctival epithelium. JunD was absent in normal corneal and conjunctival epithelia. The dsyplastic epithelium showed positive labelling for c-Fos, Fra-2, c-Jun, and JunD throughout its thickness. Fra-1 was present in all specimens of epithelia examined. The dysplastic epithelium treated with mitomycin C was not labeled by anti-c-Fos or -Fra-2 antibody. Conclusion. Individual AP1 components show specific expression patterns in normal ocular surface epithelia and a case of dysplastic epithelium before and after topical MMC treatment, implying that these factors may play important roles in modulating epithelial cell function, e.g., proliferation and differentiation.

Molecular identification and coexpression of galanin and GalR-1 galanin receptor in the human ocular ciliary epithelium: differential modulation of their expression by the activation of alpha2- and beta2-adrenergic receptors in cultured ciliary epithelial cells.

Here we report the coexpression of the neuropeptide galanin and GalR-1 galanin receptors in the human ciliary epithelium, a bilayer of neuroepithelial cells [nonpigmented (NPE) and pigmented (PE)] with neuroendocrine functions, and in a cell line (ODM-2) derived from the NPE cells. Stimulation of ODM-2 cells with phorbol ester [phorbol 12-myristate 13-acetate (PMA)] or forskolin resulted in an up-regulation (two- to threefold) of galanin mRNA expression. Procaterol, a selective beta2-adrenergic agonist, and the catecholamine isoproterenol exerted a long-term down-regulation on galanin mRNA expression when added alone or in combination with PMA or forskolin. These actions exerted by procaterol or isoproterenol were abolished in the presence of ICI 118,551, a selective beta2-adrenergic antagonist. A radioimmunoassay for galanin peptide indicated that galanin or a galanin-like product is present in the human aqueous humor fluid and is accumulated with time in the culture medium of ODM-2 cells. It is interesting that norepinephrine, which exhibited no effect on galanin mRNA expression, induced a down-regulation in the level of galanin or galanin-like product accumulated in the medium of cultured ODM-2 cells to levels even lower than those induced by beta2-adrenergic agonists. This effect is best explained by the concomitant up-regulation (four- to fivefold) of GalR-1 galanin receptor transcripts induced through the activation of alpha2-adrenergic receptors. These findings support the view that pathways elicited by the activation of alpha2- and beta2-adrenergic receptors influence the expression of galanin and GalR-1 galanin receptors in ciliary epithelial cells.

Novel paracrine signaling mechanism in the ocular ciliary epithelium.

The ciliary body contains an epithelial bilayer consisting of an outer pigmented cell layer (PE) and an inner nonpigmented cell layer (NPE) responsible for aqueous humor secretion. Secretion may be mediated in part by cytosolic Ca2+ concentration ([Ca2+]i), but whether or how the two layers could coordinate their Ca2+ signals to regulate secretion is unclear. To investigate interactions between PE and NPE, we examined [Ca2+]i signaling in isolated intact ciliary epithelial bilayers using confocal microscopy. Phenylephrine selectively increased [Ca2+]i in PE and acetylcholine increased [Ca2+]i in NPE, but epinephrine increased [Ca2+]i in both layers. This increase spread from PE to NPE, and [Ca2+]i signaling across the bilayer remained coordinated during [Ca2+]i oscillations. All epinephrine-induced [Ca2+]i signaling was blocked by the alpha1-adrenergic antagonist prazosin, whereas signaling in the NPE but not PE was blocked by the beta-adrenergic antagonist propranolol, the gap junction blockers octanol and 18alpha-glycyrrhetinic acid, or the A kinase inhibitor Rp diastereomer of adenosine 3',5'-cyclic monophosphothioate. The beta-adrenergic agonist isoproterenol failed to increase Ca2+ by itself, but isoproterenol plus phenylephrine-induced [Ca2+]i signals across the bilayer similar to those induced by epinephrine. Finally, isoproterenol increased cell-to-cell spread of lucifer yellow via gap junctions, whereas cell-to-cell spread of [Ca2+]i signals could be induced by photorelease of caged inositol 1,4,5-trisphosphate. Thus, calcium signals are coordinated in the epithelial bilayer so that adrenergic stimulation can increase [Ca2+]i in NPE, but only if NPE are primed by activation of endogenous adenylyl cyclase, whereupon they receive stimulation from adjacent PE via gap junctions. This novel interplay between endocrine and paracrine pathways may coordinate [Ca2+]i signaling across the ciliary epithelial bilayer.

Aquaporins in complex tissues: distribution of aquaporins 1–5 in human and rat eye

Multiple physiological fluid movements are involved in vision. Here we define the cellular and subcellular sites of aquaporin (AQP) water transport proteins in human and rat eyes by immunoblotting, high-resolution immunocytochemistry, and immunoelectron microscopy. AQP3 is abundant in bulbar conjunctival epithelium and glands but is only weakly present in corneal epithelium. In contrast, AQP5 is prominent in corneal epithelium and apical membranes of lacrimal acini. AQP1 is heavily expressed in scleral fibroblasts, corneal endothelium and keratocytes, and endothelium covering the trabecular meshwork and Schlemm's canal. Although AQP1 is plentiful in ciliary nonpigmented epithelium, it is not present in ciliary pigmented epithelium. Posterior and anterior epithelium of the iris and anterior lens epithelium also contain significant amounts of AQP1, but AQP0 (major intrinsic protein of the lens) is expressed in lens fiber cells. Retinal Müller cells and astrocytes exhibit notable concentrations of AQP4, whereas neurons and retinal pigment epithelium do not display aquaporin immunolabeling. These studies demonstrate selective expression of AQP1, AQP3, AQP4, and AQP5 in distinct ocular epithelia, predicting specific roles for each in the complex network through which water movements occur in the eye.

Adrenergic-mediated connexin43 phosphorylation in the ocular ciliary epithelium

PURPOSE. Gap junctions provide metabolic cooperativity between the nonpigmented and pigmented cells of the ciliary epithelium. Connexin43 is the major protein of these junctions. To learn whether the phosphorylation state of this gap junction is sensitive to adrenergic mediators, we exposed isolated intact ciliary epithelia to agonists of the G-protein receptor-coupled system and analyzed the phosphorylation state of connexin43 by western blot. METHODS. The double layer of intact ciliary epithelia was isolated and exposed to isoproterenol, forskolin, and the tumor promoter 12-o-tetradecanoylphorbol-13-acetate (TPA). The phosphorylation state of connexin43 was analyzed by western blot, using a monoclonal antibody that recognized both the phosphorylated and dephosphorylated connexin43. An upward shift in electrophoretic mobility confirmed the presence of a phosphate group. RESULTS. Connexin43 phosphorylation was rapidly induced by each of these agonists. One µM isoproterenol and 5 µM forskolin induced phosphorylation of connexin43, as did 16 nanomolar TPA. The effect of isoproterenol was partially blocked by 1 µM timolol. Addition of a phosphatase after forskolin treatment reversed the effect of forskolin. Control explant tissue not treated with these agents exhibited a slower but definite phosphorylation of connexin43. CONCLUSIONS. Phosphorylation of ciliary epithelial connexin43 is sensitive to modulators of the cAMP system as well as an agent that activates PKC. Isolated intact ciliary epithelia phosphorylate connexin43 by endogenous mechanisms, most likely as a protective response to stress.

Glycoconjugate expression during early mouse oculogenesis.

The carbohydrate side-chain of glycoconjugates can show a developmentally regulated expression pattern. In order to analyse these changes during the development of the eye, 13 lectins were used to reveal glycoconjugates histochemically in 8.5- to 14-day-old mouse embryos. During this period, eyes develop from the most immature vesiculation of the neural plate neuroepithelium into a primitive stage with all structures present, such as pigment epithelium, not yet differentiated neuroretina and lens. A striking diversity of carbohydrate side-chain expression was observed in the preocular somatoectoderm and neural plate of 8.5-day-old embryos, as indicated by the binding of nine different lectins. Binding sites at the apical poles of neuroepithelium of five of these lectins (PNA, LCA, SBA, LPA and GSA-II) disappeared completely during further development. The binding sites of four other lectins, WGA, MPA, Con A and BPA, remained expressed during the course of development, being indicative for the carbohydrate side-chains beta-GlcNAc(1-4)Gluc, alpha-Gal(1-3)GalNAc, alpha-D-Man/alpha-D-Gluc and alpha-GalNAc. In contrast, binding sites for GSA-I, RCA-I (alpha-D-Gal), UEA-I (alpha-L-Fuc) and DBA (alpha-GalNAc(1-3)GalNAc) were not present at any developmental stage. The time point of gross changes of lectin binding sites correlates well with the period of neural tube formation. During later development from neuroectoderm to the ocular pigment epithelium, a sharp reduction in all lectin binding sites at the apical cell poles, except for WGA and MPA, was observed. WGA binding sites were present until embryonic day 10, while those for MPA were present until day 9. At the basal cell poles of the pigment epithelium, all lectin binding sites except for WGA were lost after embryonic day 11.5. These results indicate that there are sophisticated kinetics of glycoconjugate expression during the course of early embryonic development of ectoderm into its descendent tissues.