Adrenergic and cholinergic receptors in isolated non-pigmented ciliary epithelial cells

Methods were developed to isolate non-pigmented ciliary epithelium from bovine eyes (BCBE) free from other cell types in the ciliary process. Once background binding due to pigment was minimized, it was possible to assay specific beta adrenergic receptors in these preparations. The BCBE beta adrenergic receptors showed appropriate stereospecificity and an order of binding affinities compatible with a beta 2 adrenergic selectivity. Interestingly, specific high affinity muscarinic cholinergic receptors were detected in both BCBE and primate non-pigmented ciliary epithelium. These muscarinic receptors showed an appropriate affinity and selectivity for agonists and antagonists. Further evaluations of the receptors and responses of non-pigmented ciliary epithelial cells may prove useful in understanding the effects of adrenergic and cholinergic agents on aqueous humor inflow.     

Isolation of non-pigmented epithelial cells from rabbit ciliary body

We describe a new technique for the separation and isolation of nonpigmented ciliary body epithelial cells from rabbit. Excised ciliary body is incubated in a medium buffered with EGTA to a free-Ca2+ concentration of 10(-8) M, and the nonpigmented cell layer is separated from the pigmented layer by microdissection under direct visualization. This technique yields intact cells which are greater than 99% nonpigmented. It can be used to produce preparations of nonpigmented cell plasma membrane and of viable whole cells, which may be useful for biochemistry, pharmacology, cell transport studies and tissue culture.     

Growth of non-pigmented ciliary epithelial cells in serum-free hormone-supplemented media

Bovine non-pigmented ciliary epithelial (NPE) cells are cultured following a dissecting procedure that preserves the original cellular polarity. Cell growth of primary cultures of NPE cells is supported by hormonally defined serum-free culture media and an artificial substrate with high binding affinity to attach cells in culture. Cell proliferation of primary cultures of bovine NPE cells was limited to 3-5 weeks. Nerve growth factor (NGF) was found to have a profound effect on the cellular junctions of growing NPE cells when added to the culture media. Nerve growth factor induced the formation abundant microfilament bundles associated with junctional complexes of undetermined nature between adjacent cells. Electron microscopy of NPE in culture shows a high intracellular transport activity with abundant coated pits and clathrin-coated vesicles, a complex network of microfilaments, microtubules and intermediate filaments associated with desmosomes.     

Regulatory volume decrease by cultured non-pigmented ciliary epithelial cells.

Cells (ODM C1-2/SV40) derived from human non-pigmented ciliary epithelial cells were studied by electronic cell sizing. The time course of the cell volume (vc) was monitored after suspending cells in paired experimental and control, isosmotic and hyposmotic solutions of identical ionic composition. Following anisosmotic cell swelling, the cells displayed the regulatory volume decrease (RVD) previously described. The RVD primarily reflects loss of cell KCl since: (1) the K(+)-channel blockers quinidine and Ba2+ both inhibit the RVD; and (2) replacement of external Cl- with gluconate or addition of the Cl- channel blocker NPPB also inhibits the RVD. Bicarbonate has previously been reported to speed the RVD. This action likely reflects pH dependence of the channels since: (1) increasing the external pH speeds the RVD, whether or not HCO3- is present; and (2) DIDS (a blocker of Cl- channels and of Cl-/HCO3- exchange) is an effective inhibitor of the RVD, even after blocking Cl-/HCO3- exchange by removing external HCO3-. The RVD could also be inhibited by reducing the availability of Ca2+, either by omitting Ca2+ from the external medium or by blocking mobilization of intracellular Ca2+ with TMB-8. Furthermore, the RVD was slowed and incomplete in the presence of the calcium/calmodulin blocker trifluoperazine. We conclude that anisosmotic swelling triggers a series of events, mediated at least in part by calcium/calmodulin, leading to the extrusion of KCl through parallel K+ and Cl- channels.     

Inhibition of swelling-activated Cl- currents by functional anti-ClC-3 antibody in native bovine non-pigmented ciliary epithelial cells

PURPOSE: To determine the potential role of ClC-3, a PKC-inhibitable Cl(-) channel, in mediating the swelling-activated Cl(-) current (I(Cl,swell)) of native bovine nonpigmented ciliary epithelial (NPE) cells. METHODS: Native bovine NPE cells were freshly harvested by enzymatic digestion. Whole-cell currents were recorded by patch-clamp measurements either in the presence or absence of a functional anti-ClC-3 antibody. RESULTS: Baseline whole-cell currents were small under isotonic conditions. Hypotonic cell swelling stimulated outwardly rectifying I(Cl,swell), which was reversibly inhibited by the Cl(-) channel blockers, phloretin (300 microM) or 5-nitro-2-(phenylpropylamino)-benzoate (NPPB, 100 microM). Intracellular dialysis with anti-ClC-3 C(670-687) antibody did not affect baseline currents, but significantly delayed and inhibited hypotonic stimulation of I(Cl,swell). Preabsorption of the antibody with its antigen prevented the inhibition of I(Cl,swell) by antibody. In addition, intracellular dialysis with control Ex(133-148) antibody did not affect the I(Cl,swell). Moreover, activation of PKC by pretreatment with 100 nM phorbol 12,13-dibutyrate (PDBu) significantly inhibited the initial stimulation of I(Cl,swell), but had no effects on the steady state currents. CONCLUSIONS: The results suggest that endogenous ClC-3 is involved in mediating I(Cl,swell) of native bovine NPE cells. The delayed stimulation of I(Cl,swell) by PDBu may reflect upregulation of swelling-activated Cl(-) channels of different subtypes, especially when the function of ClC-3 is blocked. This information will be useful in understanding the mechanisms controlling aqueous humor formation and thereby intraocular pressure.     

Phenylarsine oxide inhibits phosphate uptake in human ciliary non-pigmented epithelial cells.

Phenylarsine oxide (PAO), a sulfhydryl modifying reagent and a widely used inhibitor for tyrosine phosphatases and endocytosis, was tested on the level of phosphorylation in human nonpigmented ciliary epithelial ocular (HNPE) cells. Pretreatment with (PAO, 10 microM) for 30 min followed by incubation with 32Pi to stimulate endogenous phosphorylation surprisingly resulted in a total reduction in 32Pi labeled proteins. PAO (10-50 microM) dose-dependently inhibited both sodium-dependent and -independent phosphate uptake in cells. p-Hydroxymercuribenzoate (pHMB, 10 microM), another sulfhydryl modifying reagent failed to mimic PAO effects. However, metabolic inhibitors (iodoacetamide (0.1 mM) and 2,4-dinitrophenol (DNP, 0.5 mM) also mimicked PAO effects, suggesting that the inhibition of ATP production may be responsible for attenuation of both phosphate uptake mechanisms. However, sodium-dependent phosphate uptake in isolated plasma membrane vesicles pretreated with PAO was also significantly lower than control vesicles treated with dimethlysulfoxide (DMSO), suggesting that PAO may be directly targeting a component of the sodium-dependent cotransporter. It is suggested that PAO is a novel inhibitor of phosphate uptake in HNPE cells that acts indirectly by inhibiting ATP production and directly by inhibiting the Na-dependent cotransporter.     

Cell signaling in bovine ciliary epithelial organ culture.

The ciliary epithelium secretes aqueous humor, an intraocular fluid whose production is regulated in part by transmembrane signaling pathways including those mediated by G protein-coupled receptors. Many drugs, such as beta-adrenergic receptor (AR) antagonists and alpha2-AR agonists, are used to lower intraocular pressure by presumably decreasing fluid transport across this epithelium. Hence, our purpose was to establish a ciliary epithelial organ culture system suitable for the study of cell signaling pathways. A trypsin-mediated dissection method was established to isolate bovine ciliary epithelial sheets. These sheets were cultured in a 5% CO2 incubator. The quality was assessed by light microscopy, by protein analysis, and by the evaluation of epinephrine-mediated phosphoinositide turnover. The cultured epithelial explants were viable as evidenced by minimal trypan blue staining. The explants were composed primarily of nonpigmented cells and some pigmented cells, but no other ciliary body tissues were present on histology. Membrane preparations showed proteins with a distribution from 31 to 116 kDa. Epinephrine caused a dose-dependent increase in [3H]inositol phosphates (InsPs) accumulation with a maximal increase of two- to three-fold over basal levels. This epinephrine-mediated increase was inhibited by prazosin. We established an organ culture system of isolated bovine ciliary epithelium suitable for the study of transmembrane signaling pathways.     

Identification of endothelin converting enzyme-1 in human non-pigmented ciliary epithelial cells.

Endothelins (ETs), potent vasoactive peptides, are present in many ocular tissues including the ciliary epithelium where active ET-1 is produced from the precursor Big ET-1 by a membrane-bound metalloprotease, endothelin-converting enzyme (ECE). Although the role of ocular ET's are uncertain, they are elevated in the aqueous humor of normal as well as glaucomatous eyes and have been shown to lower the intraocular pressure for prolonged periods of time. In the current study, an endothelin-converting enzyme-1 (ECE-1) has been identified and its activity has been studied in SV-40 transformed human non-pigmented ciliary epithelial (HNPE) cells. Western blotting using polyclonal antibodies against ECE-1, detected a 124 kDa protein in the plasma membrane but not in the cytosol. Further characterization of the enzymatic activity of ECE-1 (conversion of Big ET-1 to ET-1) using the plasma membrane fraction of HNPE cells was performed by a novel assay involving(125)I-Big ET-1 (substrate; 80 fmoles mg(-1)protein) and polyclonal antibodies specific for Big ET-1. Mean ECE-1 activity (expressed as fmoles(125)I-ET-1 produced mg protein(-1)time(-1)) increased linearly with time and was similar to that observed in rat lung tissue. ECE-1 activity was enhanced with increasing concentrations of substrate ((125)I-Big ET-1; 30-200 fmoles mg protein(-1)180 min(-1)) as well as with increasing concentrations of protein (5-20 microgram proteins at the substrate concentration of 80 fmoles mg protein(-1)180 min(-1)). Treatments with CGS-26303 (100 micrometer), an inhibitor of ECE-1 and thiorphan (2 mM; a metalloprotease inhibitor), significantly decreased ECE-1 activity. Furthermore, both, acidification of the reaction buffer (from pH 7.4 to 6.4) and the addition of a metal chelator, EGTA (2 mM) decreased ECE-1 activity by nearly 60%. These results suggest that the ECE-1 localized in HNPE cells, is a neutral pH-sensitive metalloprotease which is similar in its activity to that observed in lung tissue and is essential for the production of ET-1 in HNPE cells. The physiological importance of the unusual proteolytic processing by ECE-1 in ocular tissue may reflect on how ET regulates intraocular pressure.     

Alterations of intracellular calcium in human non-pigmented ciliary epithelial cells of the eye

The ciliary epithelium of the eye is thought to be responsible for the active production of aqueous humor. The role of hormones and neuropeptides in mediating this process is unclear. Alterations in intracellular calcium in response to several hormones were assayed by fluorometric detection utilizing Quin2 in human SV-40 transformed non-pigmented ciliary epithelial cells grown in monolayer culture. A dose-dependent increase in intracellular calcium was found for the following drugs, which are given with their respective EC50 values: carbachol (15.7 +/- 4 microM), ATP (1.67 +/- 0.4 microM), arginine vasopressin (52 +/- 14 nM), bradykinin (2.4 +/- 0.7 nM), histamine (0.7 +/- 0.1 microM), and angiotensin II (6.4 +/- 1 nM). The following increases in calcium levels above typical resting levels of 45.9 +/- 4.6 nM were observed: 30% (0.1 microM angiotensin II), 50% (0.1 microM bradykinin, 2 microM arginine vasopressin), 100% (10 microM histamine), and 150% (1 mM carbachol, 10 microM ATP). Dopamine, KCl, phorbol esters, propranolol, epinephrine, and norepinephrine failed to increase intracellular calcium levels. The mobilization of intracellular calcium was unaffected by changes in the pH of the extracellular medium (over the pH range of 7.6 to 6.9) induced by glacial acetic, sulfuric or hydrochloric acids. Phosphatidic acid, however, did cause an elevation in intracellular calcium and is consistent with its putative role as an ionophore in other non-excitable exocrine tissues. These studies suggest a role for the hormonally induced mobilization of intracellular calcium which may underlie the secretion of aqueous humor by these cells.     

Histamine stimulation of inositol phosphate metabolism in cultured human non-pigmented ciliary epithelial cells

Treatment of cultured human non-pigmented ciliary epithelial cells with 100 microM histamine for 30 minutes resulted in a 3-5 fold increase in intracellular inositol phosphates. The stimulation by histamine was dose-dependent, with a half-maximal concentration of 3 microM and a maximal concentration of 100 microM. In response to histamine, inositol monophosphate increased approximately linearly for 30 min in the presence of 10 mM LiCl2, while inositol bisphosphate and inositol trisphosphate showed rapid rises complete within a few minutes. Treatment of cells with the H1 antagonist diphenhydramine resulted in a complete inhibition of the histamine effect at 1 microM, with a half-maximal inhibition at 56 nM, whereas cimetidine, an H2 antagonist, had little effect at any concentration tested. Schild analysis of the diphenhydramine/histamine receptor interaction gave an apparent dissociation constant of 7.1 nM. The data suggest that human non-pigmented ciliary epithelial cells possess H1 histamine receptors.     

Bovine non-pigmented and pigmented ciliary epithelial cells in culture: comparison of catalase, superoxide dismutase and glutathione peroxidase activities

Non-pigmented epithelial (NPE) cells and pigmented epithelial (PE) cells were dissociated from bovine ciliary processes by brief digestion with pronase and grown in a culture medium containing high fetal bovine serum for at least 25 generations. Both types of cells grown to confluence showed the presence of intermediate junctions with associated tonofilaments. PE cells were distinguished from NPE cells by pigmentation during the early passages. Gamma-glutamyl transpeptidase activity was associated almost exclusively with NPE cells and proved to be a useful enzymatic marker to distinguish NPE from PE. A comparison was made between NPE and PE cells as to the levels of enzymes involved in the detoxification of active oxygen species. Catalase, Se-dependent glutathione peroxidase and superoxide dismutase activities were significantly higher in NPE than in PE cells. The results suggest that NPE cells play the major role in detoxification of active oxygen species during aqueous humor formation.     

Volume regulation of cultured, transformed, non-pigmented epithelial cells from human ciliary body

Electronic cell sizing has been used to measure the volume of cells suspended in isosmotic and in hyposmotic solutions of identical ionic composition. Without inhibitors, the cells displayed a regulatory volume decrease (RVD) following anisosmotic cell swelling with a time constant (tau) of 6.3 +/- 0.9 min (mean +/- S.E.). The RVD was markedly impaired by substituting gluconate for external Cl-, and tau was prolonged by: (i) reducing the chemical gradient favoring K+ loss (by elevating the external [K+] and blocking the Na. K-exchange pump), (ii) blocking the K+ channels with Ba2+, (iii) blocking Cl- channels and Cl-/HCO3 = exchange with DIDS, and (iv) removing external HCO3-. Withdrawing HCO3- may have altered the RVD either directly by inhibiting a Cl-/HCO3- antiport, or indirectly by affecting intracellular pH. The regulatory volume response of ODM Cl-2/SV40 cells is in several respects qualitatively similar to that of non-pigmented epithelial cells of the intact ciliary body. These common characteristics suggest that the cultured cells can serve as a useful model for studying solute and fluid transport across the human ciliary epithelium. The basis for the RVD is likely to be activation of separate K+ and Cl- channels, with or without the parallel operation of coupled K+/H+ and Cl-/HCO3- antiports.     

Separation of bovine pigmented ciliary epithelial cells by density gradient and further characterization in culture

Dissociated pigmented epithelial (PE) cells from ciliary processes (CP) of bovine eyes were separated by isopycnic centrifugation in metrizamide gradients and further cultivated. The level of cellular contamination during separation of PE by non-pigmented epithelial (NPE) cells was estimated to be 5-10% and by non-epithelial cells (i.e. endothelial, fibroblasts and blood cells) less than 1%. Bovine PE cells were found to have different serum requirements than NPE to grow in vitro. PE cells were actively stimulated to proliferate in the presence of fetal calf serum for a few generations. Short-term culture of NPE was only obtained in media containing a low concentration of L-tyrosine and no serum. Two morphological markers, namely desmosomes and intermediate-sized filaments (IF) of the vimentin type, were found by immunolabeling to be coexpressed in cultured PE and were used as criteria to define the epithelial identity of these cells. The present study was unable to detect any tyrosinase activity in cultured PE cells.     

Adrenergic stimulation of bovine non-pigmented ciliary epithelial cells raises cAMP but has no effect on K+ or Cl- currents.

Freshly isolated bovine nonpigmented ciliary epithelial cells were studied using the whole-cell voltage-clamp and permeabilized patch techniques. In a study of 148 cells three classes of cell were found; those containing inward currents alone, 31%; those containing outward currents alone, 37% and those containing both inward and outward currents, 32%. The outward currents exhibited slow, delayed activation, were blocked by tetraethylammonium (TEA+) but were not effected by changes in [Cai] suggesting they are K(+)-currents similar to IK(V), the delayed rectifier. The inward currents were reduced by TEA+ and blocked by Cs+ suggesting they are inward rectifier K(+)-currents. Intracellular cAMP levels were increased by isoprenaline with a Ka of 20 nM. However, the resting membrane potential recorded from whole ciliary processes in intracellular microelectrode studies was not effected by adrenergic stimulation, neither were the leak current, the outward current nor the sustained inward current significantly effected by isoprenaline, noradrenaline or dibutyryl-cAMP in whole-cell and permeabilized patch clamp studies.     

Dexamethasone regulates endothelin-1 and endothelin receptors in human non-pigmented ciliary epithelial (HNPE) cells

Endothelin-1 (ET-1) lowers intraocular pressure (IOP) in animal models by regulating aqueous humour dynamics through both inflow and outflow mechanisms. Moreover, ET's concentration is elevated in glaucoma patients and in animal models of glaucoma. Glucocorticoid therapy often can lead to increase IOP in susceptible individuals including patients with primary open angle glaucoma (POAG). In this study, we examined the effects of dexamethasone (Dex), a frequently used anti-inflammatory glucocorticoid, on the synthesis and release of endothelin-1 and on the expression of endothelin receptors in human non-pigmented ciliary epithelial (HNPE) cells, an established source for ET-1 in the anterior chamber. As measured by ET-1 immunoreactivity, ET-1 was concentration-dependently increased following 24hr Dex treatment, with a maximum concentration (100 nM) causing a threefold increase of ET-1 release. Western blot analysis of HNPE cells showed the expression of endothelin receptor A (ET(A)) and endothelin receptor B (ET(B)) with approximate molecular weights of 40 kDa. Dex treatment decreased ET(A) receptor expression at all Dex doses, but up-regulated ET(B) receptors with 10nM Dex having the greatest effect. Quantitative PCR demonstrated that Dex also increased the mRNA of pre-pro-ET-1 (ppET-1) and ET(B) but decreased the mRNA of ET(A). RU486, a glucocorticoid receptor antagonist, was able to block Dex's actions on ET release and ET(B) receptor expression, but did not block its action on ET(A) receptor expression. Endothelin receptors were minimally expressed in HNPE cells as determined in binding experiments (B(max): ET(A) 17, ET(B) 25 fmolmg(-1) membrane protein). However Dex treatment stimulated a dramatic increase in ET(B) receptor density while decreasing ET(A) receptors (B(max): ET(A) 11, ET(B) 116 fmolmg(-1) membrane protein). The regulation of endothelin and its receptors could be a novel mechanism associated with glucocorticoid's effects on intraocular pressure. The increase in ET-1 and disproportionate regulation in ET receptor expression by Dex could promote dysregulation in ET's mechanism on both inflow and outflow, thus affecting aqueous humour dynamics in the anterior chamber of the eye.     

Volume regulation of non-pigmented cells from ciliary epithelium

We describe a new method for investigating ion and water transport in ciliary epithelium. A single ciliary process from the rabbit is isolated, placed in a chamber, and rapidly perfused with physiological Ringer. If this process is then viewed at its edge with a light microscope using Hoffman modulation contrast optics, it is possible to record the image of a single layer of non-pigmented epithelial cells. When these cells are exposed to hypotonic Ringer, they swell and then reduce their volume by extruding salt and water. We have used the rate of swelling to calculate the hydraulic conductivity of the non-pigmented cells. These measurements show that the aqueous humor could be secreted by the generation of a modest osmotic gradient across the non-pigmented cell basal membrane. We have also used this preparation to investigate the mechanism of the decrease in cell volume after swelling in hypotonic medium. This volume regulatory decrease can occur at a rate as large as the rate of normal fluid transport during the secretion of the aqueous. It appears to be caused in part by active Na extrusion, since it is partially inhibited by exposure to ouabain. In addition, there appears also to be a contribution to the regulatory response from a swelling-induced K efflux, since the rate of volume regulation can be altered by perfusion with Ba2+ and by changing the extracellular K concentration.     

Heterogeneity in ornithine cytotoxicity of bovine retinal pigment epithelial cells in primary culture

Gyrate atrophy of the choroid and retina is a chorioretinal degeneration caused by hyperornithinemia and a deficiency of ornithine-delta-aminotransferase (OAT). We recently showed that ornithine exhibits cytotoxicity to human retinal pigment epithelial (RPE) cell lines treated with the OAT inhibitor, 5-fluoromethylornithine (5-FMOrn), and suggested that this system may be an in vitro model of gyrate atrophy. In the present study, in order to apply this system to primary cultured RPE cells, we freshly prepared RPE cells from bovine eyes and studied the effect of ornithine on cell damage. Two phenotypes, epithelioid and fusiform, which coexisted in the primary culture and epithelioid phenotype cells, but not fusiform ones, were severely damaged and partially detached from the substrate by 10 m m ornithine and 0.5 m m 5-FMOrn. Neither ornithine nor 5-FMOrn alone exhibited such cytotoxicity to both phenotypes of RPE cells. Proline significantly prevented the ornithine-induced cytotoxicity. Epithelioid and fusiform phenotypes isolated from the primary culture showed different distribution of actin filaments. A combination of ornithine and 5-FMOrn time-dependently inhibited [(3)H]thymidine incorporation in the epithelioid, but not fusiform, cells. Proline prevented the inhibition of [(3)H]thymidine incorporation by ornithine in 5-FMOrn-treated epithelioid cells. Furthermore, l -azetidine-2-carboxylic acid, a collagen synthesis inhibitor, reduced [(3)H]thymidine incorporation in epithelioid, but not fusiform, cells, which was reversed by proline. These results demonstrate that the epithelioid phenotype of bovine RPE cells becomes susceptible to ornithine following inactivation of OAT. The phenotypic cells and its prevention by proline may provide insight into biochemical triggers that induce gyrate atrophy.     

Adenylyl cyclase activity mediated by beta-adrenoceptors in immortalized human trabecular meshwork and non-pigmented ciliary epithelial cells.

Non-pigmented ciliary epithelial (NPE) and trabecular meshwork (TM) cells are important in maintaining normal aqueous humor dynamics through the inflow and outflow routes, respectively. The current studies were undertaken to evaluate the ability of several beta-adrenergic receptor agonists to stimulate various antagonists to inhibit cAMP production in cultured immortalized human TM and NPE cells using an automated enzyme immunoassay. Isoproterenol was the most potent agonist in both the NPE and TM cells. The rank order of potency of agonists in NPE and TM cells, respectively, was: isoproterenol [EC50 = 37 and 66 nM] > epinephrine [EC50 = 112 and 526 nM] > albuterol [EC50 = 426 and 785 nM] > norepinephrine [EC50 = 3 and > 10 microM] > phenylephrine [EC50 > 10 microM for both] = dopamine [EC50 > 10 microM for both](n = 3-19). The isoproterenol-induced cAMP production was inhibited by various antagonists with the following rank order of potency in NPE and TM cells, respectively: propranolol [Ki = 0.2 and 0.3 nM] = ICI-118551 [Ki = 0.5 and 0.4 nM] > levobunolol [Ki = 1.1 and 2.1 nM] > levobetaxolol [Ki = 13 and 14 nM] = racemic betaxolol [Ki = 43 and 19 nM] > dextrobetaxolol [Ki = 2,705 and 1,980 nM] > atenolol [Ki > 4,000 nM for both] (n = 3-7). These detailed pharmacological studies using a variety of agonists and antagonists further supported the presence of beta2-adrenergic receptors in immortalized human NPE and TM cells.