Expression of muscarinic receptors in human and mouse sclera and their role in the regulation of scleral fibroblasts proliferation.
OBJECTIVE
To determine the expression of muscarinic receptor subtypes (mAChRs) in human and mouse scleral fibroblasts (SFs), to investigate the mechanism that mediate the role mAChRs play in cell proliferation, and to explore the underlying intracellular signaling pathways involved in mouse SFs with treatment of muscarinic agents.


METHODS
Reverse transcription polymerase chain reaction (RT-PCR) was used to detect mRNA expression of mAChRs in the human and mouse sclera. Western blot analysis and immunocytochemistry were used to detect proteins of mAChRs in the cultured SFs. An immunohistochemical study was used to further detect the presence of mAChR proteins in frozen scleral sections. BrdU (5-bromo-2-deoxyuridine ) cell proliferation assay was performed to measure DNA synthesis. Enzyme linked immunosorbent assay (ELISA) was used to measure in vitro kinase activity for epidermal growth factor receptor (EGF-R), fibroblast growth factor (FGF-2), transforming growth factor (TGF)-beta1, and extracellular signal-regulated kinase (ERK)1/2. Expressions of epidermal growth factor-receptor (EGF-R); protein kinase C (PKC); Proline-rich tyrosine kinase 2 (Pyk-2), v-raf murine sarcoma viral oncogene homolog B1 (B-Raf), Rat Sarcoma (Ras), c-Jun N-terminal kinases (JNK1/2), and ERK1/2 were detected by immunoblot.


RESULTS
mAChR for subtypes M(1)-M(5) were detected in both mouse and human SFs by protein, cellular, and mRNA analysis. EGF-R, PKC, Pyk-2, B-Raf, Ras, JNK1/2, and ERK1/2 were activated after treatment by agonists and antagonists, indicated by changes in phosphorylation of these proteins. Atropine abolished the carbachol-induced activation of SF cell proliferation in a concentration-dependent manner. Carbachol also activated p42/44 mitogen-activated protein kinase (MAPK) and Ras in a time-dependent manner. Muscarinic agents also modulated fibroblast growth factor expression in these cells.


CONCLUSIONS
This study confirms the presence and functional role of all five mAChRs in human and mouse SFs. These results show that proliferative responses of SFs to muscarinic receptor stimulation are mediated via the activation of the classical MEK-ERK-MAPK cascade.
Barathi
V A
VA
Singapore Eye Research Institute, Singapore.
Weon
S R
SR
Beuerman
R W
RW
D000704:Analysis of Variance; D000818:Animals; D002217:Carbachol / Q000494:pharmacology; D049109:Cell Proliferation; D018679:Cholinergic Agonists / Q000494:pharmacology; D066246:ErbB Receptors / Q000378:metabolism; D016222:Fibroblast Growth Factor 2 / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism*; D005786:Gene Expression Regulation; D006801:Humans; D007150:Immunohistochemistry; D051379:Mice; D008807:Mice, Inbred BALB C; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D011976:Receptors, Muscarinic / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012590:Sclera / Q000166:cytology* / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D018631:ras Proteins / Q000378:metabolism
D018679:Cholinergic Agonists; D011976:Receptors, Muscarinic; D016222:Fibroblast Growth Factor 2; D002217:Carbachol; C541485:EGFR protein, mouse; D066246:ErbB Receptors; D020928:Mitogen-Activated Protein Kinases; D018631:ras Proteins