Expression and Function of Muscarinic Receptor Subtypes on Human Cornea and Conjunctiva

• Published in: Investigative ophthalmology & visual science 2007-07, Vol.48 (7), p.2987-2996
• Main Authors: Liu, Shaohui, Li, Jing, Tan, Donald T. H, Beuerman, Roger W
• Format: Article
• Language: English
• Subjects: Animals; Biological and medical sciences; Blotting, Western; Carbachol - pharmacology; Cell Proliferation; Cells, Cultured; Cholinergic Agonists - pharmacology; Conjunctiva - cytology; Conjunctiva - metabolism; Dose-Response Relationship, Drug; Enzyme Activation; Epithelial Cells - drug effects; Epithelial Cells - metabolism; Eye and associated structures. Visual pathways and centers. Vision; Fibroblasts - drug effects; Fibroblasts - metabolism; Fundamental and applied biological sciences. Psychology; Humans; Immunohistochemistry; Limbus Corneae - cytology; Limbus Corneae - metabolism; Macaca; Mitogen-Activated Protein Kinase 1 - metabolism; Mitogen-Activated Protein Kinase 3 - metabolism; Phosphorylation; Protein Isoforms - classification; Protein Isoforms - genetics; Protein Isoforms - metabolism; Receptors, Muscarinic - classification; Receptors, Muscarinic - genetics; Receptors, Muscarinic - metabolism; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger - metabolism; Time Factors; Vertebrates: nervous system and sense organs
• ISSN: 0146-0404; 1552-5783; 1552-5783
• DOI: 10.1167/iovs.06-0880

To investigate the cellular distribution of the muscarinic receptor (MR) subtypes m1-m5 on the ocular surface and to determine their function in cell growth. Human limbal and conjunctival epithelial cells and conjunctival fibroblasts were isolated and cultured. RT-PCR, real-time PCR, immunostaining and Western blot analyses for m1-m5 were performed on cultured cells and tissues and a human conjunctival epithelial cell line (IOBA-NHC). Cell proliferation and p42/44 mitogen-activated protein (MAP) kinase (MAPK) activation in response to MR agonists and antagonists were analyzed by bromodeoxyuridine [BrdU] incorporation and Western blot analysis, respectively. RT-PCR revealed the presence of m1-m5 transcripts in cultured limbal and conjunctival epithelial cells and conjunctival fibroblasts. Relative quantitative real-time PCR showed that the m1 transcript level in conjunctival cells was higher than that in limbal cells; m2, m3, and m4 expression levels were higher in conjunctival fibroblasts than in epithelial cells. Absolute quantitative real-time PCR showed that the m5 mRNA level in the three cell types was higher than those of m1-m4. Immunohistochemistry and Western blot analysis confirmed the presence of m1-m5 proteins in the cultured cells and in tissues. Carbachol increased the incorporation of BrdU into conjunctival epithelial cells in a dose-dependent manner, which was totally inhibited by atropine, but only partially inhibited by pirenzepine, AF-DX116, and 4-DAMP. Carbachol also activated p42/44 MAPK in a time-dependent manner. Preincubation with U0126 abolished carbachol-induced p42/44 MAPK activation and cell proliferation. All five MR subtypes were found on corneal and conjunctival cells. The MRs have a role in epithelial cell proliferation through the phosphorylation of p42/44 MAPK in a time-dependent fashion similar to EGF.