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G. Adamus, S. Shiraga, S. Webb, R. Duvoisin; Anti–Recoverin Antibody Induce Intracellular Calcium Increase . Invest. Ophthalmol. Vis. Sci. 2004;45(13):3439.
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Purpose: Anti–recoverin antibodies that are found in patients with CAR syndrome penetrate retinal cells and induce apoptosis via a mitochondrial pathway involving the release of cytochrome c and proteins of bcl–2 family. The goal of this study was to investigate whether the antibody entry caused a change in intracellular Ca2+ and whether blocking of Ca2+ release in retinal cells treated with anti–recoverin antibody will prevent the activation of apoptotic mechanism. Methods: The concentration of free intracellular Ca2+ was measured using the Ca2+ sensitive fluorescent dye Fura–2AM in living E1A.NR3 retinal cells (gift from Dr. Gail Seigel) treated with anti–recoverin MAb Rec–1. Bcl–xL from antibody–treated and untreated cells was measured by Western blotting and cytosolic cytochrome c was assessed using an ELISA. Results: The exposure of retinal cells to MAb Rec–1 in vitro caused a significant increase in intracellular Ca2+, while non–specific antibodies did not have such effect. Cotreatment with antibody and nifedipine, a calcium channel blocker depressed the Ca2+ increase and significantly decreased bcl–xL expression after 20 min before slowly returning to baseline conditions. Nifedipine alone did not cause a decrease in bcl–xL expression. Retinal cells treated with Rec–1 and nifedipine showed also a decrease in cytosolic cytochrome c compared to those treated with anti–recoverin antibody alone. Normal antibody or nifedipine alone did not induce cytochrome c release. Conclusions: Intracellular Ca2+ is increased in cells treated with anti–recoverin antibody, which results in the activation of the mitochondrial apoptotic pathway. Our results suggest that antibody entry contributes to pathogenicity of CAR syndrome.
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