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V. A. Barathi, S. R. Weon, P. W. Y. Rebekah, R. W. Beuerman; Muscarinic Regulation of Epidermal Growth Factor Receptor in Mammalian Retinal Pigment Epithelial (RPE) Cells. Invest. Ophthalmol. Vis. Sci. 2008;49(13):3535.
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Primary cells were harvested by trypsinization from non-human primate, B6 mouse and human donors. In addition to that we used the ARPE-19 cell line as a positive control. Cultured RPE cells were used at passage 2-4. Luminescent ELISA assays for proliferation were carried out with atropine, pirenzipine and carbachol at 0, 0.1, 1, 10, 100 µM concentrations. The muscarinic toxins of MT-1 and MT-7 were used at 0, 0.005, 0.05, 0.5, 5 µM. RPE primary cells were established using immunostaining (anti-cytokeratin KG 8.13 antibody) and specific features were studied by transmission electron microscopy (TEM). Presence of mAChRs and EGF-R were investigated using immunocytochemistry and immunoblotting.
Early stage outgrowth of RPE explants took approximately 1 week. RPE primary cells were identified via anti-cytokeratin KG 8.13, which is specific for RPE cells and cultures of 90% confluency were processed for TEM that confirmed the specific features of RPE cells. Western blots confirmed the presence of all five mAChR sub types and EGF-R on RPE cells. M1 specific antagonists of pirenzipine and MT-7 inhibited proliferation of RPE cells in a dose dependent manner (p < 0.05, n = 6, ANOVA). Atropine, a pan muscarinic antagonist also inhibited proliferation of RPE cells in a dose dependent manner (p < 0.05, n = 6, ANOVA). In contrast, the muscarinic receptor agonist carbachol and MT-1 increased cell proliferation in a dose dependent manner (p < 0.05, n = 6, ANOVA). Atropine and pirenzipine completely blocked carbachol stimulated proliferation. Carbachol activated EGF-R within minutes of application peaking at 30 minutes.
Our results confirm all 5 subtypes of mAChRs are expressed in these mammalian RPE cells. Muscarinic receptors participate in the control of cell proliferation in monkey, mouse and human RPE cells through EGF-R activation.
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