May 2008
Volume 49, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2008
Muscarinic Regulation of Epidermal Growth Factor Receptor in Mammalian Retinal Pigment Epithelial (RPE) Cells
Author Affiliations & Notes
  • V. A. Barathi
    Singapore Eye Research Institute, SERI/NUS, Singapore, Singapore
  • S. R. Weon
    Singapore Eye Research Institute, SERI/NUS, Singapore, Singapore
  • P. W. Y. Rebekah
    Singapore Eye Research Institute, SERI/NUS, Singapore, Singapore
  • R. W. Beuerman
    Singapore Eye Research Institute, SERI/NUS, Singapore, Singapore
    Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore
  • Footnotes
    Commercial Relationships  V.A. Barathi, None; S.R. Weon, None; P.W.Y. Rebekah, None; R.W. Beuerman, None.
  • Footnotes
    Support  NMRC 0985/2005, IBG
Investigative Ophthalmology & Visual Science May 2008, Vol.49, 3535. doi:https://doi.org/
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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. doi: https://doi.org/.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Methods: : 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.

Results: : 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.

Conclusions: : 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.

Keywords: retinal pigment epithelium • proliferation • receptors 
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