Purchase this article with an account.
Xu Wang, Balwantray C. Chauhan, William H. Baldridge; Endothelin-1 Induced Increase of Intracellular Calcium Concentration in Optic Nerve Astrocytes via Endothelin Receptors. Invest. Ophthalmol. Vis. Sci. 2012;53(14):2796.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Endothelin (ET)-1 is a vasoactive peptide implicated in varied ocular pathologies. Our previous work has demonstrated that application of ET-1 to optic nerve causes blockade of axonal transport and retinal ganglion cell (RGC) loss. As ET-1 has been shown to elevate intracellular calcium concentration ([Ca2+]i) in cultured optic nerve astrocytes, it is possible that ET-1-induced calcium dynamics in astrocytes lead to axonal transport dysfunction and RGC loss. The aim of the current study was to determine if ET-1 affects [Ca2+]i of optic nerve astrocytes in situ by using calcium-imaging techniques.
Pieces of isolated rat optic nerves were incubated in fluo-8 AM. Labelled nerves were placed in a recording chamber and superfused with Ames media. Cells loaded with fluo-8 were imaged with fluorescence microscopy and cooled CCD camera under computer control. Time-lapse images were obtained before, during and after ET-1 (10-11 - 10-5 M) application. Fluorescence changes (ΔF/F) were quantified by determining the change of fluorescence in cells compared to baseline. Involvement of specific ET receptors was assessed by testing the effect of ET-1 on [Ca2+]i in the presence of ETA (BQ610, 10 µM) and/or ETB (BQ788, 10 µM) antagonists. To identify the cell types, nerves were fixed and processed for GFAP (astrocytes marker) immunohistochemistry after calcium-imaging.
ET-1 produced dose-dependent increases of fluo-8 fluorescence (indicative of [Ca2+]i) of optic nerve astrocytes at ≥ 10-10 M with responses (%ΔF/F) ranging from 7.9% ± 4.0% (10-10 M, mean ± SD) to 31.7% ± 10.2% (10-5 M). The effect of 10-7 M ET-1 on optic nerve astrocytes [Ca2+]i was not affected by BQ610 or BQ788 alone (10 µM). However, when BQ610 and BQ788 were combined, the increase of astrocyte [Ca2+]i in response to 10-7 M ET-1 was reduced by 95.5%. The morphology of Fluo-8 labelled cells was consistent with GFAP immunostaining.
Our results indicate that application of ET-1 increases [Ca2+]i in optic nerve astrocytes in situ. The increase is mediated by both ETA and ETB receptors. These results suggest that optic nerve astrocytes likely plays a role in ocular pathology where a concomitant increase of ET-1 is observed via an increase in intracellular calcium.
This PDF is available to Subscribers Only