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Dana Garcia, Vicente Carlos Quintanilla, Satish Cheepala, Floyd Weckerly, John D Schuetz; ATP-binding Cassette Protein C4 (Abcc4) in the Retina of Zebrafish: Role in Regulating Melanosome Aggregation in the Retinal Pigment Epithelium. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):2319.
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© ARVO (1962-2015); The Authors (2016-present)
Elevated cyclic AMP (cAMP) in photoreceptors is associated with dark adaptation in the vertebrate retina, and cAMP elicits dark-adaptive melanosome aggregation in the retinal pigment epithelium (RPE) of fishes. We hypothesized that zebrafish ATP-binding cassette protein C4 (Abcc4) exports cAMP and as such could mediate signal exchange via cAMP transmitted from the neural retina to the RPE.
Zebrafish Abcc4 (ZfAbcc4) was expressed in Saos-2 cells. Cells were tested by enzyme immunoassay for their ability to export cAMP as compared to Saos-2 cells transfected with an empty vector. To determine whether nucleotide export was inhibited by sildenafil, HEK293 cells expressing ZfAbcc4 were challenged to export [3H]-PMEA as a proxy for cAMP in the absence or presence of the Abcc4 inhibitor MK571 or sildenafil. Localization of Abcc4 in light- and dark-adapted zebrafish retina was determined immunohistochemically. As a functional assay, sildenafil citrate was injected intraocularly into light-adapted fish which were dark-adapted for 2 hours, after which the position of melanosomes (pigment index; PI) was evaluated in retinal sections. Sodium citrate or DMSO was injected intraocularly as counterion and carrier controls.
Saos-2 cells expressing Abcc4 exported 140 pmol cAMP/105 cells compared to 55 pmol cAMP/105 cells transfected with empty vector. Abcc4’s transport activity in HEK293 cells was inhibited (p<0.05) by both MK571 and sildenafil, causing cells to retain 10- and 5-times more PMEA, respectively, than untreated cells. In zebrafish retina, Abcc4 localized to photoreceptor, RPE and outer plexiform layer with labeling more intense in dark-adapted than light-adapted fish. Dark-adapted, uninjected fish fully aggregated melanosomes (PI = 0.42 ± 0.02); sildenafil treatment completely blocked aggregation (PI = 0.78 ± 0.02; p<<<0.0001). Estimation of the 95% confidence interval for the differences in the PI between injected and contralateral eyes indicated a strong treatment effect for sildenafil, a small effect for DMSO, and no effect for Na citrate injection.
These results support a model in which Abcc4 exports cAMP from photoreceptors into the subretinal space from which it can be imported into the RPE, where it induces melanosomes aggregation.
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