Expression of muscarinic and adrenergic receptors in normal human conjunctival epithelium.
OBJECTIVE
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)-, beta(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)-, alpha(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 IFN gamma and/or TNF alpha.


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.
Enríquez de Salamanca
Amalia
A
Institute of Applied Ophthalmobiology, University of Valladolid, Valladolid E-47005, Spain. amalia@ioba.med.uva.es
Siemasko
Karyn F
KF
Diebold
Yolanda
Y
Calonge
Margarita
M
Gao
Jianping
J
Juárez-Campo
Mónica
M
Stern
Michael E
ME
D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D003228:Conjunctiva / Q000187:drug effects / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004848:Epithelium / Q000187:drug effects / Q000378:metabolism; D005434:Flow Cytometry; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D007371:Interferon-gamma / Q000494:pharmacology; D008856:Microscopy, Fluorescence; D051381:Rats; D011942:Receptors, Adrenergic, alpha / Q000378:metabolism*; D011943:Receptors, Adrenergic, beta / Q000378:metabolism*; D011976:Receptors, Muscarinic / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D015854:Up-Regulation
D011942:Receptors, Adrenergic, alpha; D011943:Receptors, Adrenergic, beta; D011976:Receptors, Muscarinic; D014409:Tumor Necrosis Factor-alpha; D007371:Interferon-gamma