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

Coca‐Prados, Miguel; Kondo, Kazuyoshi

doi:10.1016/0014-4835(85)90142-3

Cited by 26 publications

(7 citation statements)

References 18 publications

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“…8-SVHCE can also be grown as a polarized epithelial monolayer on permeable substrates such as Millipore filters (M.C.-P., unpublished results), and the formation of desmosomes by 8-SVHCE in culture (Fig. LA) can be confirmed by indirect immunofluorescence using antibodies recognizing desmosome plaques, as previously described for bovine CE (9). The successful preservation of cellular identity and function of the 8-SVHCE cells reported here is encouraging in the pursuit to establish differentiated CE cells in culture.…”

Section: Resultsmentioning

confidence: 99%

“…In addition, several attempts have been made to grow CE in tissue culture media after its dissociation and separation (9) from ciliary processes. Some of the differentiated characteristics ofhuman CE cells, such as their structural polarity and epithelial cell type, are retained in primary tissue cultures and have been studied by morphological and immunological criteria (9,10). Activation of adenylate cyclase in human CE cells by catecholamines results in increased intracellular cAMP and significant stimulation of protein phosphorylation, particularly of vimentin (a major cytoskeletal component) (11).…”

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confidence: 99%

“…Immunofluorescence Microscopy. 8-SVHCE cells were grown on glass coverslips, fixed, and stained by indirect immunofluorescence (9). The primary reagent was undiluted hybridoma medium containing a monoclonal antibody directed against desmosomal proteins from bovine muzzle epidermis and designated MmDGI-1 (17), which was kindly provided by M. S. Steinberg and C. Blanck (Princeton University).…”

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confidence: 99%

“…Indirect immunofluorescence was carried out by incubation for 30 min at 370C with fluorescein isothiocyanateconjugated goat anti-mouse IgG (Miles). The coverslips were mounted and photographed as described (9).…”

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Transformation of human ciliary epithelial cells by simian virus 40: induction of cell proliferation and retention of beta 2-adrenergic receptors.

Coca‐Prados¹,

Wax²

1986

Proc. Natl. Acad. Sci. U.S.A.

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Ciliary epithelial cells derived from human eye were successfully propagated through many generations after transformation with simian virus 40. The cell clone 8-SVHCE was isolated and characterized by immunoprecipitation and pharmacological studies that demonstrated the presence of several functional properties observed in the parent cells of this tissue. Immunoprecipitation revealed the presence of large tumor (T) antigen, and Southern blot analysis showed the incorporation of viral DNA into high molecular weight ciliary epithelial cell DNA. Studies of catecholamine-stimulated cellular cAMP production and of isoproterenol-dependent protein phosphorylation of vimentin in 8-SVHCE indicated the functional conservation of 6-adrenergic receptor-mediated processes that are thought to be important in the regulation of aqueous humor production by the ciliary epithelium in vivo.Ciliary epithelium (CE) in the mammalian eye is thought to play an essential role in the formation of aqueous humor. The composition of aqueous humor is largely dependent on the mechanism of active transport of plasma proteins and electrolytes in these cells. The rate of formation of aqueous humor is modulated by the sympathetic (adrenergic) system (1). The stroma in the ciliary processes is innervated by adrenergic fibers whose terminations remain close to the CE (2). Two structural features of these cells in vivo are (i) a structural polarity where the apical domain of a nonpigmented epithelial (NPE) cell faces the apical domain of a pigmented epithelial (PE) cell and (ii) a physiological, enzyme-based active transport system, Na+,K+-ATPase, located in the basolateral domains in both cell types. The relationship of the structural and physiological polarity in the CE to the regulation of ion transport (i.e., fluid flow from the stroma to the posterior chamber) is unclear.Previous studies (3-5) using crude membrane preparations from whole ciliary processes have indicated the presence of 8-adrenergic receptors in the ciliary epithelium of eyes from a variety of mammalian species. It is possible, however, that vascular endothelial cells from microvessels in the stroma of ciliary processes may have contributed to the stimulation of adenylate cyclase by 83-adrenergic receptors that was observed in these preparations. Attempts have been made to dissociate the CE cells from the stroma tissue to measure the properties and characteristics of the P-adrenergic receptor/ adenylate cyclase system in a mixed population of NPE and PE cells (6), after their separation in a density gradient (7), and in isolated NPE cells (8). In these studies, p-adrenergic receptors were found in both cell types. In addition, several attempts have been made to grow CE in tissue culture media after its dissociation and separation (9) from ciliary processes. Some of the differentiated characteristics ofhuman CE cells, such as their structural polarity and epithelial cell type, are retained in primary tissue cultures and have been studied by morphological and immunological...

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Section: Resultsmentioning

confidence: 99%

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confidence: 99%

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confidence: 99%

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confidence: 99%

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Transformation of human ciliary epithelial cells by simian virus 40: induction of cell proliferation and retention of beta 2-adrenergic receptors.

Coca‐Prados¹,

Wax²

1986

Proc. Natl. Acad. Sci. U.S.A.

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“…Tritiated prazosin (3H-PRZ, Ci/mmol), rauwolscine (3H-RAU, 70-90 Ci/mmol) and myo-inositol (3H-INS, [10][11][12][13][14][15][16][17][18][19][20] Ci/mmol) were purchased from New England Nuclear Corporation (Boston, MA).…”

Section: Methodsmentioning

confidence: 99%

Alpha₁-Adrenoceptors in the Albino Rabbit Ciliary Process

Mallorga¹,

Buisson²,

Sugrue³

1988

Journal of Ocular Pharmacology and Therapeutics

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H-Prazosin and^H-rauwolscine binding sites were identified in a membrane suspension prepared from albino rabbit iris + ciliary body. Scatchard analysis of saturation binding experiments demonstrated that both %-prazosin and %-rauwolscine bind to a single population of binding sites with Krj values of 0.87 nM and 5.33 nM, respectively. Bmax values of 65.7 and 198 fmol/mg protein were obtained for^H -prazosin and 3jj-rauwolscine, respectively. Displacement studies by several adrenergic agonists and antagonists indicated that^H -prazosin and^H-rauwolscine labelled a^-and o^-adrenoceptors, respectively, in the iris + ciliary body. Epinephrine, norepinephrine and phenylephrine were able to stimulate the synthesis of 3H-inosit.0l. phosphates in ciliary processes labelled with 3H-inositol, with EC50 values of 2.4, 12 and 10 yM, respectively. The corresponding maximum stimulations of basal activity were 433, 430 and 283%, respectively. Phenylephrine behaved like a partial agonist in this assay. The norepinephrine response could be potently antagonized by prazosin (K^= 27 nM), with rauwolscine being 285-fold less potent. An epithelial cell suspension was prepared from the ciliary process. Stimulation of phosphatidylinositol turnover by norepinephrine (0.1 mM) was observed, and this could be blocked by prazosin (10 \M), thus, indicating the presence of oci-adrenoceptors, coupled to phosphatidylinositol turnover, in epithelial cells of the rabbit ciliary process.

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Dual effect of ciliary body cells on T lymphocyte proliferation

et al. 1990

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The interaction between organ-resident cells from the anterior uvea of the eye and T helper (Th) cells was investigated. Cells from Lewis rat ciliary body processes (CB cells), grown in tissue culture using an explant technique, could be induced to express major histocompatibility complex class II (Ia) antigens by incubation with rat interferon-gamma. Ia+ CB cells only poorly functioned as antigen-presenting cells (APC) for a syngeneic, uveitogenic Th cell line specific for the retinal soluble antigen (SAg). Moreover, if added to an Ag-driven lymphocyte proliferation assay in the presence of conventional APC, the rat CB cells had an inhibiting effect on Th proliferation. This inhibitory activity was not species specific, since similar effects were observed with bovine and human ciliary epithelial cells. The suppressive activity of CB cells was composed of a soluble factor, as well as a membrane-associated inhibitor. The soluble activity did not appear to be related to transforming growth factor-beta (TGF-beta), since no reversal of inhibition by a neutralizing antibody to TGF-beta was found. Part of the soluble inhibitory activity could be reversed by indomethacin treatment. The membrane-associated component was trypsin sensitive, suggesting a protein molecule. After abrogation of the inhibitory capacity by trypsin treatment and fixation by glutaraldehyde, CB cells effectively presented SAg to Th cells. These data suggest that CB cells are capable of mediating both Ag presentation and inhibition of Th cell proliferation.

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

Cited by 26 publications

References 18 publications

Transformation of human ciliary epithelial cells by simian virus 40: induction of cell proliferation and retention of beta 2-adrenergic receptors.

Transformation of human ciliary epithelial cells by simian virus 40: induction of cell proliferation and retention of beta 2-adrenergic receptors.

Alpha₁-Adrenoceptors in the Albino Rabbit Ciliary Process

Dual effect of ciliary body cells on T lymphocyte proliferation

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

Cited by 26 publications

References 18 publications

Transformation of human ciliary epithelial cells by simian virus 40: induction of cell proliferation and retention of beta 2-adrenergic receptors.

Transformation of human ciliary epithelial cells by simian virus 40: induction of cell proliferation and retention of beta 2-adrenergic receptors.

Alpha1-Adrenoceptors in the Albino Rabbit Ciliary Process

Dual effect of ciliary body cells on T lymphocyte proliferation

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Alpha₁-Adrenoceptors in the Albino Rabbit Ciliary Process