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Amalia Enríquez de Salamanca, Karyn F. Siemasko, Yolanda Diebold, Margarita Calonge, Jianping Gao, Mónica Juárez-Campo, Michael E. Stern; Expression of Muscarinic and Adrenergic Receptors in Normal Human Conjunctival Epithelium. Invest. Ophthalmol. Vis. Sci. 2005;46(2):504-513. doi: 10.1167/iovs.04-0665.
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purpose. To study the presence of muscarinic and α- and β-adrenergic receptors in a normal human conjunctival epithelial (IOBA-NHC) cell line.
methods. Neurotransmitter receptors were determined in IOBA-NHC cells by flow cytometry, immunofluorescence, and Western blot analysis. Antibodies to M1-, M2-, and M3-muscarinic and to α1A-, α1B-, α1D-, α2A-, α2B-, α2C-, β1-, β2-, and β3-adrenergic receptor subtypes were used. Different culture media were tested, including the addition of tumor necrosis factor (TNF)-α and/or interferon (IFN)-γ. Normal human conjunctiva biopsy specimens and rat tissues were used in control experiments.
results. By immunofluorescence microscopy, all receptor subtypes, except the α2C-adrenergic receptor, were detected in control biopsy specimens. By flow cytometry, the M2- and M3-muscarinic receptors and α1A-, α1B-, α1D-, α2A-, α2B-, α2C-, β1-, and β3-adrenergic receptors were detected intracellularly and in cell membranes of the IOBA-NHC cells. M1-muscarinic and β2-adrenergic receptors were detected only intracellularly, but were mobilized to the cell membrane when cholera toxin and hydrocortisone were omitted from the culture medium. Confocal microscopy detected the M2 and M3-muscarinic and α1A-, α2A-, α2B-, β1- and β2-adrenergic receptor subtypes. Western blot analyses showed bands for all receptors. M2-muscarinic and α1B- and α2B-adrenergic receptors expression was upregulated when cells were treated with the proinflammatory cytokines IFNγ and/or TNFα.
conclusions. The IOBA-NHC cell line maintained expression of the neurotransmitter receptors expressed in normal human conjunctival epithelium. A proinflammatory medium upregulated expression of some receptors. Although the functional state of these receptors is unknown, these findings justify further use of the IOBA-NHC cell line to study the neural component of conjunctival inflammation.
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