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

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

Research output: Contribution to journalArticle

18 Citations (Scopus)

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
Externally publishedYes

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Conjunctiva
Muscarinic Receptors
Cornea
Epithelial Cells
Mitogen-Activated Protein Kinase 1
Carbachol
Real-Time Polymerase Chain Reaction
Fibroblasts
Western Blotting
Cell Proliferation
Bromodeoxyuridine
Cultured Cells
Muscarinic M1 Receptors
Pirenzepine
Muscarinic Agonists
Polymerase Chain Reaction
Muscarinic Antagonists
Atropine
Epidermal Growth Factor
Immunohistochemistry

ASJC Scopus subject areas

  • Ophthalmology
  • Sensory Systems
  • Cellular and Molecular Neuroscience

Cite this

Expression and function of muscarinic receptor subtypes on human cornea and conjunctiva. / Liu, Shaohui; Li, Jing; Tan, Donald T.H.; Beuerman, Roger W.

In: Investigative Ophthalmology and Visual Science, Vol. 48, No. 7, 01.07.2007, p. 2987-2996.

Research output: Contribution to journalArticle

Liu, Shaohui ; Li, Jing ; Tan, Donald T.H. ; Beuerman, Roger W. / Expression and function of muscarinic receptor subtypes on human cornea and conjunctiva. In: Investigative Ophthalmology and Visual Science. 2007 ; Vol. 48, No. 7. pp. 2987-2996.
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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.",
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N2 - 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.

AB - 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.

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