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      Expression of muscarinic receptors in human and mouse sclera and their role in the regulation of scleral fibroblasts proliferation

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      1 , 1 , 1 , 2 ,
      Molecular Vision
      Molecular Vision

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          Abstract

          Purpose

          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)-β1, 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.

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          Most cited references51

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          International Union of Pharmacology. XVII. Classification of muscarinic acetylcholine receptors.

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            Muscarinic receptors: their distribution and function in body systems, and the implications for treating overactive bladder.

            1. The effectiveness of antimuscarinic agents in the treatment of the overactive bladder (OAB) syndrome is thought to arise through blockade of bladder muscarinic receptors located on detrusor smooth muscle cells, as well as on nondetrusor structures. 2. Muscarinic M3 receptors are primarily responsible for detrusor contraction. Limited evidence exists to suggest that M2 receptors may have a role in mediating indirect contractions and/or inhibition of detrusor relaxation. In addition, there is evidence that muscarinic receptors located in the urothelium/suburothelium and on afferent nerves may contribute to the pathophysiology of OAB. Blockade of these receptors may also contribute to the clinical efficacy of antimuscarinic agents. 3. Although the role of muscarinic receptors in the bladder, other than M3 receptors, remains unclear, their role in other body systems is becoming increasingly well established, with emerging evidence supporting a wide range of diverse functions. Blockade of these functions by muscarinic receptor antagonists can lead to similarly diverse adverse effects associated with antimuscarinic treatment, with the range of effects observed varying according to the different receptor subtypes affected. 4. This review explores the evolving understanding of muscarinic receptor functions throughout the body, with particular focus on the bladder, gastrointestinal tract, eye, heart, brain and salivary glands, and the implications for drugs used to treat OAB. The key factors that might determine the ideal antimuscarinic drug for treatment of OAB are also discussed. Further research is needed to show whether the M3 selective receptor antagonists have any advantage over less selective drugs, in leading to fewer adverse events.
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              Epidermal growth factor receptor (EGFR) and EGFR mutations, function and possible role in clinical trials.

              The epidermal growth factor receptor (EGFR) is a growth factor receptor that induces cell differentiation and proliferation upon activation through the binding of one of its ligands. The receptor is located at the cell surface, where the binding of a ligand activates a tyrosine kinase in the intracellular region of the receptor. This tyrosine kinase phosphorylates a number of intracellular substrates that activates pathways leading to cell growth, DNA synthesis and the expression of oncogenes such as fos and jun. EGFR is thought to be involved the development of cancer, as the EGFR gene is often amplified, and/or mutated in cancer cells. In this review we will focus on: (I) the structure and function of EGFR, (II) implications of receptor/ligand coexpression and EGFR mutations or overexpression, (III) its effect on cancer cells, (IV) the development of the malignant phenotype and (V) the clinical aspects of therapeutic targeting of EGFR.
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                Author and article information

                Journal
                Mol Vis
                MV
                Molecular Vision
                Molecular Vision
                1090-0535
                2009
                30 June 2009
                : 15
                : 1277-1293
                Affiliations
                [1 ]Singapore Eye Research Institute, Singapore
                [2 ]Department of Ophthalmology, Yong Loo Lin School of Medicine, National University of Singapore, National University Hospital, Singapore
                Author notes

                The first two authors contributed equally to this work.

                Correspondence to: Professor Beuerman, PhD, Singapore Eye Research Institute, 11 Third Hospital Avenue, Singapore, 168751; Phone: (+65) 6322 4544; FAX: (+65) 6322 4599; email: rwbeuer@ 123456mac.com
                Article
                136 2008MOLVIS0397
                10.1017/S0952523809990198
                2704914
                19578554
                0eb285e2-c764-4194-b866-10d29102254c
                Copyright @ 2009

                This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

                History
                : 09 December 2008
                : 23 June 2009
                Categories
                Research Article
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                Beuerman

                Vision sciences
                Vision sciences

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