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S. Narayan, T. Yorio; Endothelin-thrombin Interactions at the Blood Retinal Barrier . Invest. Ophthalmol. Vis. Sci. 2003;44(13):337.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: The potent vasoactive peptide-endothelin-1 (ET-1) is expressed in the mammalian retina. It is also known that deregulated ET-1 secretion along with other proinflammatory mediators promote acute changes in the ocular blood flow accompanied by an alteration in axonal transport in the optic nerve. The primary focus of this study is to delineate the regulatory aspects of ET-1 secretion with its relation to thrombin in the retinal pigment epithelium. Methods: Enucleated eyes from adult male wistar rats and brown norway rats were used to confirm the expression of ET-1 in the retina by light microscopy and electron microscopy. Human retinal pigment epithelial cells (ARPE-19) were used to study thrombin-mediated regulation of ET-1. Confluent cells (3-4 weeks in culture) were treated with thrombin, at both physiological (~ 3 nM) and pathological concentrations (5-10 nM) for various time points and immunoreactive ET-1 was measured by light microscopy and radioimmunoassay. The expression of the tight-junction associated protein ZO-1, in treated and untreated cells was measured by indirect immunofluorescence and immunoblot analysis. Functional studies to assess thrombin-induced mobilization of intracellular Ca2+ ([Ca2+]i) by its action on protease-activated-receptors (PAR-1/4) were done in real-time by fura-2 imaging. PAR-1 selective agonist, SFLLR (thrombin-receptor activating peptide, TRAP-6) and hirudin (a direct thrombin antagonist) were also included in the study. TNF-α, a known inducer of ET-1 secretion was used as positive control in all our experiments. Results: The mammalian retinal pigment epithelium expresses ET-1, as measured in whole retinas as well as in isolated cell culture systems. Thrombin, both at lower and higher concentrations greatly enhanced [Ca2+]i levels in ARPE-19 cells, an effect mimicked by SFLLR and blocked by hirudin suggesting PAR-1 activation. Thrombin and TNF-α at higher concentrations resulted in disruption of the tight-junction barrier in a time-dependent manner with concomitant increase in ET-1 secretion. Conclusions: The RPE may act as a source of ET-1 at the region of the blood retinal barrier. Thrombin and TNF-α may disrupt the tight-junction barrier and promote a feed-forward signal to enhance the release of ET-1. Elevated ET-1 levels may decrease ocular blood flow and axonal transport in the optic nerve in a manner consistent with optic neuropathies.
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