Abstract
Purpose:
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.
Methods:
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.
Results:
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.
Conclusions:
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.