Expression and function of muscarinic receptor subtypes on human cornea and conjunctiva.
OBJECTIVE
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.
Liu
Shaohui
S
Singapore Eye Research Institute, National University of Singapore, Singapore.
Li
Jing
J
Tan
Donald T H
DT
Beuerman
Roger W
RW
D000818:Animals; D015153:Blotting, Western; D002217:Carbachol / Q000494:pharmacology; D049109:Cell Proliferation; D002478:Cells, Cultured; D018679:Cholinergic Agonists / Q000494:pharmacology; D003228:Conjunctiva / Q000166:cytology / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D016850:Limbus Corneae / Q000166:cytology / Q000378:metabolism*; D008251:Macaca; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D010766:Phosphorylation; D020033:Protein Isoforms / Q000145:classification / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D011976:Receptors, Muscarinic / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors
D018679:Cholinergic Agonists; D020033:Protein Isoforms; D012333:RNA, Messenger; D011976:Receptors, Muscarinic; D002217:Carbachol; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3