Expression and function of muscarinic receptor subtypes on human cornea and conjunctiva
Expression and function of muscarinic receptor subtypes on human cornea and conjunctiva
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DOI：
10.1167/iovs.06-0880
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发表时间：
2007-07-01
影响因子：
4.4
通讯作者：
Beuerman, Roger W.
中科院分区：
文献类型：
--
作者：
Liu, Shaohui;Li, Jing;Beuerman, Roger W.
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.
