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Tunde Molnar, Amber Frye, Peter Barabas, Daniel Ryskamp, David Krizaj; The molecular mechanisms of store-operated calcium entry in Müller glia. Invest. Ophthalmol. Vis. Sci. 2013;54(15):6344.
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© ARVO (1962-2015); The Authors (2016-present)
Müller cells play a key function in retinal volume regulation, metabolism, glutamate recycling and pathology. Here we characterize a fundamental Ca2+ signaling pathway in Müller astroglia, determine its molecular mechanism and investigate its contribution to Müller gliosis induced by ocular hypertension.
Optical imaging from fura-2 -loaded cells and whole-cell patch-clamp recording were performed in dissociated mouse Müller cells isolated from wild type and Trpc1/Trpc3 (TRPC1/3-/-) double knockout mice. Store-operated calcium signals were evoked by prolonged depletion of endoplasmic reticulum (ER) Ca2+ stores in Ca2+-free saline supplemented with cyclopiazonic acid (CPA). Intraocular pressure (IOP) was elevated for 7 and 14 days by injection of microbeads into the anterior chamber of wild type and TRPC1/3-/- mice. Müller cell reactivity was assessed with an anti-GFAP antibody.
Depletion of ER Ca2+ stores induced marked store-operated calcium entry (SOCE) (568 ± 159 nM over the baseline of 232 ± 36 nM; N=17 cells) which was manifested as [Ca2+]i overshoots following re-exposure of cells to the inward-directed calcium gradient. Induction of SOCE was associated with intracellular Ca2+ waves that propagated from the endfoot to the apical regions of Müller cells. Both SOCE and Ca2+ wave propagation were suppressed by a mix of SOC channel inhibitors 2-APB, SKF96365 and Gd3+. Likewise, depletion of ER stores induced a prominent inward Ca2+/Na+ current (~ 50 pA) in the presence of TEA and Cs+. The depletion-evoked current was antagonized by 2-APB, SKF96365 and Gd3+. The amplitude of SOCE signals was significantly diminished in mice lacking TRPC1 and 3 isoforms (299 ± 94 nM; N=14 cells; P<0.01). GFAP immunoreactivity in the ocular hypertension model was markedly stronger in TRPC1/3-/- mice compared to wild type animals.
We found that SOCE mediates significant Ca2+ influx in mouse Müller cells mainly through TRPC1 and/or TRPC3 channels. The enhanced expression of GFAP in glaucomatous TRPC1/3 -/- retinas indicates that calcium signals associated with TRPC1 and TRPC3 channels modulate the induction and/or maintenance of Müller gliosis in the diseased retina.
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