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Expression of Muscarinic and Adrenergic Receptors in Normal Human Conjunctival Epithelium

¹From the Institute of Applied Ophthalmobiology (IOBA), University of Valladolid, Valladolid, Spain; and ²Allergan Inc., Irvine, California.

PURPOSE. To study the presence of muscarinic and - and ß-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 _1A-, _1B-, _1D-, _2A-, _2B-, _2C-, ß₁-, ß₂-, and ß₃-adrenergic receptor subtypes were used. Different culture media were tested, including the addition of tumor necrosis factor (TNF)- and/or interferon (IFN)-. Normal human conjunctiva biopsy specimens and rat tissues were used in control experiments.

RESULTS. By immunofluorescence microscopy, all receptor subtypes, except the _2C-adrenergic receptor, were detected in control biopsy specimens. By flow cytometry, the M₂- and M₃-muscarinic receptors and _1A-, _1B-, _1D-, _2A-, _2B-, _2C-, ß₁-, and ß₃-adrenergic receptors were detected intracellularly and in cell membranes of the IOBA-NHC cells. M₁-muscarinic and ß₂-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 _1A-, _2A-, _2B-, ß₁- and ß₂-adrenergic receptor subtypes. Western blot analyses showed bands for all receptors. M₂-muscarinic and _1B- and _2B-adrenergic receptors expression was upregulated when cells were treated with the proinflammatory cytokines IFN and/or TNF.

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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