Expression and function of muscarinic receptor subtypes on human cornea and conjunctiva

Shaohui Liu, Jing Li, Donald T.H. Tan, Roger W. Beuerman

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19 Scopus citations

Abstract

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 ml 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. Pre-incubation 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.

Original languageEnglish (US)
Pages (from-to)2987-2996
Number of pages10
JournalInvestigative Ophthalmology and Visual Science
Volume48
Issue number7
DOIs
StatePublished - Jul 1 2007

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ASJC Scopus subject areas

  • Ophthalmology
  • Sensory Systems
  • Cellular and Molecular Neuroscience

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