Expression of muscarinic and adrenergic receptors in normal human conjunctival epithelium

PURPOSE. To study the presence of muscarinic and alpha- and beta-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 M-1-, M-2-, and M-3-muscarinic and to alpha(1A)-, alpha(1B)-, alpha(1D)-, alpha(2A)-, alpha(2B)-, alpha(2C)-, alpha(1)-, beta(2)-, and beta(3)-adrenergic receptor subtypes were used. Different culture media were tested, including the addition of tumor necrosis factor (TNF)-alpha and/or interferon (IFN)-gamma. Normal human conjunctiva biopsy specimens and rat tissues were used in control experiments. RESULTS. By immunofluorescence microscopy, all receptor subtypes, except the alpha(2C)-adrenergic receptor, were detected in control biopsy specimens. By flow cytometry, the M-2- and M-3-muscarinic receptors and alpha(1A)-, alpha(1B)-, alpha(1D)-, alpha(2A)-, alpha(2B)-, alpha(2C)-, beta(1)-, and beta(3)-adrenergic receptors were detected intracellularly and in cell membranes of the IOBA-NHC cells. M-1-muscarinic and beta(2)-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 M-2 and M-3-muscarinic and alpha(1A)-, alpha(2A)-, beta(2B)-, beta(1)- and beta(2)-adrenergic receptor subtypes. Western blot analyses showed bands for all receptors. M-2-muscarinic and alpha(1B)- and alpha(2B)-adrenergic receptors expression was upregulated when cells were treated with the proinflammatory cytokines IFNgamma and/or TNFalpha. 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.