June 2017
Volume 58, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2017
The role of cAMP and protein kinase A (PKA) in regulation of pigment granule aggregation in RPE of sunfish, Lepomis spp.
Author Affiliations & Notes
  • Christina King-Smith
    Biology, Saint Joseph's University, Philadelphia, Pennsylvania, United States
  • Joseph Quinlan
    Biology, Saint Joseph's University, Philadelphia, Pennsylvania, United States
  • Nicole Fischer
    Biology, Saint Joseph's University, Philadelphia, Pennsylvania, United States
  • Elizabeth Del Rio
    Biology, Saint Joseph's University, Philadelphia, Pennsylvania, United States
  • Melissa Messalti
    Biology, Saint Joseph's University, Philadelphia, Pennsylvania, United States
  • Footnotes
    Commercial Relationships   Christina King-Smith, None; Joseph Quinlan, None; Nicole Fischer, None; Elizabeth Del Rio, None; Melissa Messalti, None
  • Footnotes
    Support  None
Investigative Ophthalmology & Visual Science June 2017, Vol.58, 1038. doi:
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      Christina King-Smith, Joseph Quinlan, Nicole Fischer, Elizabeth Del Rio, Melissa Messalti; The role of cAMP and protein kinase A (PKA) in regulation of pigment granule aggregation in RPE of sunfish, Lepomis spp.. Invest. Ophthalmol. Vis. Sci. 2017;58(8):1038.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : Retinomotor movements in eyes of fish and other lower invertebrates include long-distance migration of RPE pigment granules in response to light or dark. Since eyes of these species do not contain dilateable pupils, aggregation and dispersion of pigment granules within long RPE apical projections serves to control light flux to photoreceptors. In dissociated, cultured RPE cells, pigment granule aggregation can be triggered by cAMP. We sought to confirm that cAMP stimulation of aggregation occurs through activation of protein kinase A (PKA), and characterized targets of PKA phosphorylation in RPE to better understand the regulation of pigment granule motility.

Methods : Isolated RPE cells from sunfish (Lepomis spp.) were microinjected with PKA catalytic subunit and the position of pigment granules (aggregated vs. dispersed) was compared to un-injected cells. To identify PKA targets, RPE tissue was treated with phosphatase inhibitors and cAMP or dopamine (the chemical trigger for pigment granule dispersion). PKA-phosphorylated proteins were identified by immunoblotting and immunoprecipitation using an anti-Ser/Thr PKA phosphorylated substrate antibody.

Results : Injection of PKA catalytic subunit triggered pigment granule aggregation (N=6 cells) while pigment granules in uninjected cells remained dispersed, demonstrating that cAMP activation of aggregation occurs via PKA activity. Immunoblotting revealed numerous PKA-phosphorylated proteins in cAMP-treated samples; the most abundant were at 112, 82, and 48 kD, in contrast to dopamine-treated RPE cells, which revealed no PKA phosphoproteins via immunoblotting.

Conclusions : While cAMP has been shown to play other roles in cells besides activation of PKA, in fish RPE cells, cAMP elicits pigment granule aggregation via activation of PKA. Previous work has shown that cAMP-triggered pigment granule aggregation involves Rho kinase and non-muscle myosin II. Potential candidates for PKA-phosphorylated targets include proteins associated with the activation of myosin II.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.

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