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J. A. Murphy, M. L. Archibald, W. H. Baldridge, B. C. Chauhan; The Role of Endothelin B in Endothelin-1 Induced Rat Optic Nerve Head Astrocyte Proliferation and Ca2+ Signaling. Invest. Ophthalmol. Vis. Sci. 2010;51(13):2099.
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© ARVO (1962-2015); The Authors (2016-present)
The goal of this research was to characterize the influence of endothelin-1 (ET-1) on rat optic nerve head astrocyte (ONHA) proliferation and Ca2+ signaling in cells lacking functional endothelin B (ETB) receptors.
Optic nerve head astrocytes (ONHAs) were isolated from adult wild type (WT) and transgenic spotted lethal (TSL) rats which lack functional ETB receptors. Astrocyte specificity was determined immunohistochemically with positive labeling for glial fibrillary acidic protein (GFAP), and negative labeling for A2B5 (a marker for Type II astrocytes located outside the optic nerve head) and myelin basic protein (MBP). The mitogenic effects of ET-1 at concentrations of 10-7, or 10-9 M or vehicle were investigated for 48 or 72 hrs in TSL and WT ONHAs. To investigate intracellular calcium ([Ca2+]i) in ONHAs, cells were loaded with fura-2 AM calcium indicator dye.
The specificity of the cultured cells as ONHAs was confirmed with positive labeling for GFAP, and negative labeling for A2B5 and MBP. In TSL cells, ET-1 induced proliferation was significantly blunted at 48 hrs (by 37% at 10-7 M, p = 0.072; and by 33% at 10-9 M, p = 0.039) and 72 hrs (by 117% at 10-7 M, p = 0.004; and by 100% at 10-9 M) compared with WT cells. There was, however, significant proliferation of TSL cells with ET-1 compared to vehicle compared with WT cells; at 48 hrs (by 36% at 10-7 M, p<0.001; and by 25% at 10-9 M, p = 0.002) and at 72 hrs (by 30% at 10-7 M, p = 0.002; and by 30% at 10-9 M, p = 0.003). Increases in ONHA [Ca2+]i following ET-1 exposure were significantly greater in TSL cells (by 20% at 10-7 M, p=0.001; and by 48% at 10-8 M, p=0.001). ET-1 at 10-9 M did not alter [Ca2+]i.
These findings indicate that (1) ET-1 induces proliferation of ONHAs which lack functional ETB receptors, (2) that this proliferation is reduced compared to cells which possess both ETB and ETA receptors, and (3) that absence of functional ETB receptors increases ET-1 induced [Ca2+]i.
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