Nonpigmented Ciliary Epithelial Cells Research Articles

Endothelin-like immunoreactivity in the aqueous humour and in conditioned medium from cultured ciliary epithelial cells.

Endothelin-like immunoreactivity was detected in human (15.6 +/- 2.7 pg/ml) and bovine (11.1 +/- 0.98 pg/ml) aqueous humour of the eye. These concentrations are 2-3 times higher than the corresponding plasma levels. Cultured human nonpigmented ciliary epithelial cells released endothelin-like immunoreactivity with a maximum of 2.1 +/- 0.32 pg/(cm2* 48 h). The release was stimulated by fetal calf serum, thrombin, carbachol and phorbol ester and blocked by cycloheximide. Immunocytochemistry showed cytoplasmic staining of cultured human nonpigmented ciliary epithelial cells for endothelin-1. Endothelin-1 was shown to induce contractions in isolated human ciliary muscle by isometric force measurements. Endothelin in the aqueous humour may play a role in the regulation of intraocular pressure.

Sodium channels in ocular epithelia.

Voltage-gated, tetrodotoxin(TTX)-blockable sodium channels are found in most excitable cells and are the primary contributors to action potentials generated by many of these cells. To date, there has only been one report of a non-cultured vertebrate epithelial cell type containing TTX-blockable Na+ channels: rabbit non-pigmented ciliary body epithelial cells [Cilluffo MC et al. (1991) Invest Opthalmol Vis Sci 32: 1619-1629], and three reports of cultured epithelial cells containing TTX-blockable Na+ channels: rabbit non-pigmented and pigmented ciliary body epithelium [Ciluffo MC et al. (1991) Invest Opthalmol Vis Sci 32: 1619-1629; Fain GL, Farahbakhsh (1989) J Physiol (Lond) 417: 83-103] and human lens epithelium [Cooper K et al. (1990) J Membr Biol 117: 285-298]. We report here the presence of sodium currents in two different non-cultured, freshly dissociated transporting epithelial cell types: the rabbit corneal endothelium and the frog lens epithelium. We also report the occurrence of sodium currents in six additional cultured ocular epithelial cell types from three different species. These currents have a current/voltage (I/V) relationship consistent with traditional voltage-gated Na+ currents, are quinidine- and TTX-blockable (of the low-affinity TTX-sensitive type), and disappear following bath substitution of Na+ with Cs+ or K+.

Formation and release of vesicles from the basal surfaces of rat eye non‐pigmented ciliary epithelial cells: A novel secretory mechanism?

When rat ciliary body is processed by high pressure freezing and freeze substitution, numerous membrane-bound vesicle profiles are seen in the vitreous associated with the pars plana and in the valleys between the ciliary processes. They consist of a homogeneously distributed fine granular matrix and varying numbers of ribosome-like structures. The mechanism by which these vesicles are secreted appears to follow an apocrine-type pattern, albeit at the basal cell surface. Matrix material accumulates between the basal plasma membrane of non-pigmented ciliary epithelial cells and a cortical layer of cytoskeletal components; the blebs thus formed protrude through a discontinuity in the basal lamina and, by a progressive narrowing of the neck region, are eventually pinched off, giving rise to free vesicles. Under conventional aqueous chemical fixation conditions, most of these vesicles are washed away or their contents solubilized and extracted, which accounts for their not having been identified hitherto as genuine morphological structures. They are nonetheless apparent, albeit in reduced numbers and mostly empty. Such vesicles are also observed in tissue processed according to several other chemical fixation techniques, namely, conventional fixation in the presence of the cationic dye ruthenium hexamine trichloride, simultaneous glutaraldehyde/osmium tetroxide fixation, and microwave fixation. In the latter instance, comparable vesicle preservation to that obtained by high pressure freezing/freeze substitution may be achieved if fixation is followed by cryoprotection, plunge freezing, and freeze substitution instead of conventional post-fixation and dehydration procedures.

Gamma-interferon induces differential expression of HLA-DR, -DP and -DQ in human ciliary epithelial cells

The antigen-specific activation of T-helper lymphocytes is dependent on the presentation of antigen in context with the gene products of the major histocompatibility complex class II (MHC II). Aberrant expression of MHC II on the ciliary epithelium has been observed in uveitic eyes which may enable these cells to specifically interact with lymphocytes and may play a role in ocular autoimmunity. Human MHC II consists of three subclasses termed HLA-DR, -DP and -DQ, which seem to be differentially regulated and may have different functions. The present study was initiated to investigate the dynamics of the differential MHC II expression on cultured human non-pigmented ciliary epithelial cells (NPE cells) in response to gamma-interferon (gamma-IFN) by means of immunohistochemistry. NPE cells grown in control tissue-culture medium did not express MHC class II. HLA-DR and -DP could be induced by incubation with 100 mu/ml gamma-IFN for 3 days. HLA-DQ was expressed only weakly and at higher doses of gamma-IFN (greater than or equal to 500 mu/ml) and longer incubation periods (greater than or equal to 5 days). After removal of the gamma-IFN stimulus, all three MHC II subclasses persisted for several days. The differential expression of HLA-DR and -DP as compared with HLA-DQ in response to gamma-IFN in the ciliary epithelium is similar to observations in other non-lymphoid ocular cells but appears to be different from the regulation of MHC II expression on lymphoid cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Transepithelial resistance of ciliary epithelial cells in culture: Functional modification by protamine and extracellular calcium

1. l. Bovine pigmented and human non-pigmented ciliary epithelial cells were cultured on porous filter supports to obtain measurements of transepithelial electrical parameters. 2. 2. The non-pigmented cells showed maximal transepithelial resistance of 15–30 Ωcm 2 from the third to seventh day in culture. 3. 3. The pigmented ciliary cells reached maximal resistances of 9–20 Ωcm 2 after the fourth day in culture. 4. 4. The transepithelial resistances of the cultured epithelia were functionally increased by protamine. This effect could be reversed by heparin. 5. 5. We conclude that the range of resistances in cultured ciliary epithelial cells is the same as in whole ciliary preparations. Thus, cultured ciliary epithelial cells can be used for studies on transepithelial transport.

Histamine H1receptor occupancy triggers inositol phosphates and intracellular calcium mobilization in human non-pigmented ciliary epithelial cells

Agonists and antagonists of histamine were used to characterize the stimulation of inositol phosphates formation and elevation of intracellular Ca2+ by histamine in cultured non-pigmented epithelial (NPE) cells from human ciliary body. Agonists specific for the H1 histamine receptor subtype were 20- to 200-fold more potent than the H2-specific agonists tested, and 5-16% as potent as histamine in inositol phosphates stimulation. An H1 antagonist was 10,000-fold more potent than an H2 antagonist in blocking histamine stimulation of inositol phosphates. H1 agonists also mimicked and H1 antagonists inhibited the elevation of intracellular Ca2+ by histamine. The first phase of the Ca2+ response to histamine was largely independent of extracellular Ca2+ while the second phase required extracellular Ca2+. Dose-response curves for histamine elevation of intracellular Ca2+ (EC50 = 10 microM, maximum at 100 microM) and inositol phosphates (EC50 = 2 microM, maximum at 100 microM) were similar. These data support the characterization of the NPE histamine receptor as an H1 receptor linked to elevation of inositol phosphates and intracellular Ca2+.

Immunocytochemical study of phenobarbital-and 3-methylcholanthrene-inducible cytochrome P450 isozymes in primary cultures of porcine ciliary epithelium

We found in the previous study that the induction of 7-pentoxyresorufin O-dealkylase and 7-ethoxyresorufin O-dealkylase activities by phenobarbital and 3-methylcholanthrene, respectively, is more pronounced in porcine ciliary nonpigmented epithelial cells than in pigmented epithelial cells. In order to determine whether cytochrome P450 isozymes that mediate the O-dealkylase activities are also induced in nonpigmented cells under the conditions, primary cultures of porcine ciliary processes were treated with phenobarbital and 3-methylcholanthrene and the expression and localization of cytochrome P450 isozymes induced by these compounds were investigated by immunocytochemical methods using antibodies against the individual P450 isozymes. Intense labeling of nonpigmented epithelial cells was observed when ciliary processes treated with phenobarbital were reacted with anti-P450 (phenobarbital) antibody and when the processes treated with 3-methylcholanthrene were incubated with anti-P450 (methylcholanthrene) antibody. The labeling patterns supported the conclusion that the O-dealkylase activities and P450 isozymes specific for these activities are co-induced in and localized to the endoplasmic reticulum. This study is the first report presenting direct evidence of cytochrome P450 induction in primary cultures of ocular tissues and demonstrates the usefulness of porcine ciliary epithelial cells for studying the induction of ocular drug metabolizing enzymes.

Glutathione-dependent detoxification of peroxide in bovine ciliary body

Perfusion of the bovine eye with a buffer solution containing t-butyl hydroperoxide and the glutathione reductase inhibitor nitrofurantoin caused significant decreases in reduced glutathione level in ciliary body and iris. The result was interpreted to suggest that the organic hydroperoxide was decomposed by the glutathione peroxidase-reductase system. The glutathione reductase reaction requires NADPH. Since the level of NADPH is maintained by the hexose monophosphate shunt in many tissues, we investigated whether this is also the case with bovine uveal tissues. CO 2 formation from [1- 14C]glucose but not from [6- 14C]glucose was markedly stimulated by t-butyl hydroperoxide and was inhibited by the glutathione reductase inhibitor 1,3-bis(2-chloroethyl)-1-nitrosourea, thus supporting the importance of the hexose monophosphate shunt for hydroperoxide decomposition through the glutathione peroxidase-reductase system. The peroxidase-reductase activity was found both in non-pigmented and pigmented ciliary epithelial cells in culture. Purification studies isolated two forms of glutathione reductase [GR I (140 kDa) with subunit Mr of 70 kDa and GR II (> 670 kDa) with subunit Mr of 45 kDa] and a novel glutathione peroxidase (112 kDa with subunit Mr of 29 kDa). The peroxidase is active both with H 2O 2 and organic hydroperoxides, does not contain selenium and shows no glutathione S-transferase activity.

Pars Plicata in Equine Recurrent Uveitis

Pars Plicata in Equine Recurrent Uveitis

G protein complement of SV40-transformed ciliary epithelial cells

Guanine nucleotide-binding proteins (G proteins) are a family of receptor-coupled signal-transducing proteins that regulate a variety of second-messenger systems and ion channels. The complement of G proteins in SV40-transformed pigmented and nonpigmented ciliary epithelial cells was determined by Western blot analysis utilizing peptide and holoprotein derived antisera to known G protein alpha and beta subunits and cholera toxin catalyzed ADP-ribosylation. The complement of alpha subunits found in both SV40-transformed NPE and PE cells includes Gs alpha and all three members of the Gi alpha family. Neither cell type contains Go alpha or Gz alpha. Both cell lines contain beta 35 and beta 36. Future studies will examine the functional involvement of these G proteins in the regulation of aqueous humor stimulus-secretion coupling.

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 μ m), ATP (1·67±0·4 μ m), arginine vasopressin (52±14 n m), bradykinin (2·4±0·7 n m), histamine (0·7±0·1 μ m), and angiotensin II (6·4±1 n m). The following increases in calcium levels above typical resting levels of 45·9±4·6 n m were observed: 30% (0·1 μ m angiotensin II). 50% (0·1 μ m bradykinin, 2 μ m arginine vasopressin), 100% (10 μ m histamine), and 150% (1 m m carbachol, 10 μ m 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.

Heterogeneity of the ciliary epithelium of the rat eye as revealed by spot 35 protein (a Purkinje cell specific protein)-like immunoreactivity.

By means of immunoreactivity for spot 35 protein, a novel cerebellar Purkinje cell-specific protein, the regional heterogeneity among non-pigmented ciliary epithelial cells of rats was demonstrated with reference to the antero-posterior and crest-valley directions of individual ciliary epithelial folds in immature and mature eyes. The functional significance of the occurrence of the spot 35 immunoreactivity in the posterior portion of the ciliary epithelium is briefly discussed in relation to the formation of the aqueous humor.

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.

Rhegmatogenous retinal detachment in Labrador retrievers. II. Proliferative vitreoretinopathy.

We observed proliferative vitreoretinopathy in Labrador retrievers with spontaneous rhegmatogenous retinal detachment. Cellular membranes and their associated connective tissue matrix were prominent on the surfaces of the detached formed vitreous and on the retina. Membrane contraction folded the underlying vitreous and retina and exerted traction on the structures to which they attached. Proliferation occurred in the retinal pigment epithelium under the detached retina and in the nonpigmented ciliary epithelium when it became detached. Retinal pigment epithelial cells, nonpigmented ciliary epithelial cells, macrophages, and glial cells appeared to contribute to the membranes. Our findings suggested a sequence that began with vitreous abnormalities and ended with fibrocellular membranes on the surfaces of the vitreous and the totally detached retina. Because a similar sequence can develop in humans, these dogs may be useful in future studies of proliferative vitreoretinopathy.

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.

Effects of pilocarpine and paracentesis on occluding junctions between the nonpigmented ciliary epithelial cells

The occluding junctions between the nonpigmented ciliary epithelial cells in the Rhesus monkey have been studied with the electron microscope using horseradish peroxidase as a tracer and the freeze-fracture technique. These junctions can be divided into two distinct classes: one type is made of parallel strands, while the other type consists of a net-like meshwork of junctional strands. The occluding junctions in the pars planar and the valley of the ciliary process are mainly of the parallel type which is composed of between three and 15 strands. After pilocarpine instillation, the straight and parallel strands in these portions became wavy but remained intact. This finding suggests that during accomodation the blood-aqueous barrier is maintained by changing the shape of the occulding junctions so as to resist the contraction of the ciliary muscle. The second type of junctional strands, made up of from five to 18 ridges and grooves, is mainly seen in the crest of the ciliary process. Following repeated paracentesis, every ciliary process of almost all the experimental monkeys developed cysts in the ciliary processes but only at their most anterior portion. The cyst walls were ruptured at several locations. The cysts, the intercellular spaces and the posterior chamber contained fibrin. In freeze-fractured replicas of these specimens, the occluding junctions at the anteriormost portion of the pars plicata were normally composed of from one to five strands of the parallel type, and which were often fragmented after paracentesis. This observation indicates that the occluding junctions in this portion of the ciliary process may be responsible for the leakage of the serum protein into the posterior chamber that is observed after paracentesis.

Cilia in the ciliary epithelium.

Cilia in the pigmented and nonpigmented ciliary epithelial cells were studied by electron microscopy in the normal Rhesus monkey eye. In addition cilia formation was studied following paracentesis as well as injection of hemoglobin into the vitreous cavity. In all eyes studied, cilia had a 9 + 0 microtubule pattern; however, some cilia had aberrant arrangement of microtubules. Cilia increased in number and showed elongation in the treated eyes, these newly-formed cilia occasionally extending towards the cell interior opposite to the normal orientation. The results suggested that the formation of ciia can be provoked by a variety of experimental stimuli either with or without drugs.

Levels of Glycolytic Enzyme Activity in the Ciliary Epithelium Prepared from Bovine Eyes

Activities of TIM, GAPDH, PGK, PGM, EN, PK, and LDH were tested in extracts of nonpigmented and pigmented ciliary epithelial cells isolated from the ciliary processes of bovine eyes. Correlated to cell count, the activity levels of TIM, GAPDH, PGK, PGM, EN, and LDH were found to be significantly higher in the nonpigmented cell type.