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(Investigative Ophthalmology and Visual Science. 2007;48:2987-2996.)
© 2007 by The Association for Research in Vision and Ophthalmology, Inc.
DOI:  10.1167/iovs.06-0880
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Expression and Function of Muscarinic Receptor Subtypes on Human Cornea and Conjunctiva

Shaohui Liu,1,2 Jing Li,1,2,3 Donald T. H. Tan,1,3,4 and Roger W. Beuerman1,3

1From the Singapore Eye Research Institute and the 3Department of Ophthalmology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore; and the 4Singapore National Eye Center, Singapore.

PURPOSE. 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.

METHODS. 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.

RESULTS. 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.

CONCLUSIONS. 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.






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