June 2015
Volume 56, Issue 7
Free
ARVO Annual Meeting Abstract  |   June 2015
ATP-binding Cassette Protein C4 (Abcc4) in the Retina of Zebrafish: Role in Regulating Melanosome Aggregation in the Retinal Pigment Epithelium
Author Affiliations & Notes
  • Dana Garcia
    Biology, Texas State University, San Marcos, TX
  • Vicente Carlos Quintanilla
    Biology, Texas State University, San Marcos, TX
  • Satish Cheepala
    Pharmaceutical Sciences, St. Jude Children's Research Hospital, Memphis, TN
  • Floyd Weckerly
    Biology, Texas State University, San Marcos, TX
  • John D Schuetz
    Pharmaceutical Sciences, St. Jude Children's Research Hospital, Memphis, TN
  • Footnotes
    Commercial Relationships Dana Garcia, None; Vicente Quintanilla, None; Satish Cheepala, None; Floyd Weckerly, None; John Schuetz, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2015, Vol.56, 2319. doi:
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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)

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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.

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