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W. W. Kothmann, S. C. Massey, J. O'Brien; Dopamine D1-Receptor Mediated Modulation of Connexin36 Phosphorylation in AII Amacrine Cells. Invest. Ophthalmol. Vis. Sci. 2008;49(13):1515.
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© ARVO (1962-2015); The Authors (2016-present)
Connexin36 (Cx36)-mediated coupling of AII amacrine cells is required for functionality of the primary rod pathway in the retina. Tracer coupling of AII amacrine cells is modulated by background light intensity and dopamine signaling. Dopamine reduces tracer coupling of AII amacrine cells in a D1 receptor-mediated manner. One hypothesized mechanism for this reduced coupling is phosphorylation at regulatory serine residues on Cx36, although changes in the phosphorylation state of Cx36 have not been directly measured. We have thus tested the ability of dopaminergic agents to alter Cx36 phosphorylation in AII amacrine cells.
Adjacent pieces of superior retina (approx. 0.25 cm2) still attached to the sclera were collected from the enucleated eyes of adult New Zealand rabbits. Each piece was incubated for 20 minutes under normal room lights in oxygenated Ames medium (at 35°C) containing: no additive (control), D1 agonist, D1 antagonist, or D1 agonist plus protein kinase A (PKA) inhibitor. After subsequent fixation the pieces of retina were isolated from the sclera, immunolabeled with antibodies against Cx36, phosphoSer293-Cx36, and calretinin, and imaged on a confocal microscope using identical settings. Phosphorylation at individual gap junction plaques was measured as the ratio of phosphoSer293 labeling to Cx36 labeling.
Phosphorylation of Cx36 gap junctions on AII amacrine cells in control pieces was highly variable. Incubation with the D1 agonist SKF38393 (100 µM) significantly reduced the mean phosphorylation of Cx36 at Ser293, while application of the D1 antagonist SCH23390 (100 µM) significantly increased phosphorylation at Ser293. The effect of SKF38393 (100 µM) on Cx36 phosphorylation was partially blocked by the PKA inhibitor Rp-8-cpt-cAMPS (20 µM).
The dopamine D1 receptor pathway modulates Cx36 phosphorylation in AII amacrine cells. This pathway classically activates PKA, and we have demonstrated that PKA partially mediates the effects of D1 agonist application. It is thus paradoxical that application of a D1 agonist leads to reduced phosphorylation of Cx36 at Ser293. We predict that the changes in the phosphorylation state of Cx36 in AII amacrine cells are associated with changes in the coupling state of these cells.
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