Overexpression of Endothelin A and B Receptors Enhances Calcium Mobilization in Ocular Astrocytes and Ciliary Epithelial Cells

Purpose: Endothelin-1 (ET-1), a vasoactive peptide, binds ETA receptor and ETB receptor to exert its role in multiple cellular processes. A growing body of evidence suggests that elevated levels of ET-1 and activation of its receptors contributes to neurodegeneration in glaucoma, where reactive astrocytes are found to induce damage of retinal ganglion cells. Overexpression of c-Jun, a transcription factor, has been shown to increase levels of ETB receptor, suggesting that the expression of ETB receptor is regulated by c-Jun. This study attempts to determine if overexpression of ET-1 receptors affect calcium influx in response to ET-1 treatment. Methods: Primary astrocytes were isolated from retina and optic nerve of rat pups postnatal 4-7 days. Calcium imaging using Fura-2-AM fluorescent dye was used to determine calcium influx following treatment of ET-1, in the presence and absence of BQ610 (ETA selective antagonist), or BQ788 (ETB selective antagonist) or no treatment (control). ETA, ETB and c-Jun were also overexpressed in Human Non-Pigmented Epithelial (HNPE) cells using DNA transfection and calcium mobilization was measured. Results: Overexpression of ETA or ETB in HNPE cells significantly increased [Ca2+]i levels compared to control following ET-1 treatment at p2+]i in primary astrocytes. Treatment with either BQ610 or BQ788 in primary astrocytes significantly (p2+]i levels compared to control following ET-1 treatment. Conclusion: This study demonstrated that ETA and ETB can mediate calcium influx in HPNE cells and primary astrocytes. ETA receptor stimulation produced a similar calcium influx in HPNE cells as ETB receptor activation, suggesting that both receptors may be involved in [Ca2+]i signaling. The increase in calcium can result in activation of cell death pathways that may explain the ET-1 neurodegenerative actions.