Regulation of photoreceptor gap junction phosphorylation by adenosine in zebrafish retina

Abstract

Electrical coupling of photoreceptors through gap junctions suppresses voltage noise, routes rod signals into cone pathways, expands the dynamic range of rod photoreceptors in high scotopic and mesopic illumination, and improves detection of contrast and small stimuli. In essentially all vertebrates, connexin 35/36 (gene homologs Cx36 in mammals, Cx35 in other vertebrates) is the major gap junction protein observed in photoreceptors, mediating rod-cone, cone-cone, and possibly rod-rod communication. Photoreceptor coupling is dynamically controlled by the day/night cycle and light/dark adaptation, and is directly correlated with phosphorylation of Cx35/36 at two sites, serine110 and serine 276/293 (homologous sites in teleost fish and mammals, respectively). Activity of protein kinase A (PKA) plays a key role during this process. Previous studies have shown that activation of dopamine D4 receptors on photoreceptors inhibits adenylyl cyclase, down-regulates cAMP and PKA activity, and leads to photoreceptor uncoupling, imposing the daytime/light condition. In this study, we explored the role of adenosine, a nighttime signal with a high extracellular concentration at night and a low concentration in the day, in regulating photoreceptor coupling by examining photoreceptor Cx35 phosphorylation in zebrafish retina. Adenosine enhanced photoreceptor Cx35 phosphorylation in daytime, but with a complex dose-response curve. Selective pharmacological manipulations revealed that adenosine A2a receptors provide a potent positive drive to phosphorylate photoreceptor Cx35 under the influence of endogenous adenosine at night. A2a receptors can be activated in the daytime as well by micromolar exogenous adenosine. However, the higher affinity adenosine A1 receptors are also present and have an antagonistic though less potent effect. Thus, the nighttime/darkness signal adenosine provides a net positive drive on Cx35 phosphorylation at night, working in opposition to dopamine to regulate photoreceptor coupling via a push-pull mechanism. However, the lower concentration of adenosine present in the daytime actually reinforces the dopamine signal through action on the A1 receptor.

Figure 1

Effects of adenosine on the phosphorylation level of OPL gap junctions in daytime, light-adapted retina. A) Confocal micrograph of immunostaining for total Cx35 (red) and phospho-S276 Cx35 (green) in the OPL of light-adapted daytime zebrafish retina in an eyecup preparation. Phosphorylated Cx35 (shown alone in A′) is very low. Scale bar in B applies to all images in A and B. B) 25 minute incubation with 10 μM adenosine increased Cx35 phosphorylation (phospho-S276 Cx35 shown alone in B′). C) Dose-response relationship of Cx35 phosphorylation level with adenosine concentration. Curves fit to the data are a third order polynomial model (solid line) and a fourth order polynomial model (dashed line). Data are means ± SE of median values for 4–7 animals. * P<0.05; ** P<0.01; *** P<0.001 compared to 0 added adenosine. D) Results table of orthogonal polynomial contrast following one-way ANOVA to assess the best fit of multiphasic models to the adenosine dose-response data. The orthogonal polynomial contrast tested the 4 possible orders of polynomial trends including linear, quadratic, cubic, and quartic terms; each contrast has 1 degree of freedom (DF) and they are orthogonal. The F value is obtained by taking the mean square for the contrast (DF=1) divided by the mean square for error (DF=21). The cubic polynomial fit (solid line in Fig1C) provided a significant improvement in fit over a quadratic model, while no significant improvement resulted from the quartic fit (dashed line in Fig 1C).

Figure 2

Contributions of A2 receptors to regulation of Cx35 phosphorylation. A) Dose-response relationship of Cx35 phosphorylation level for incubation of adenosine A2a receptor agonist CGS 21680. Data are means ± SE of median values for 2–7 animals. B) Comparison of Cx35 phosphorylation level for incubation of non-drug control, 10 μM adenosine, 10 μM adenosine plus 100 nM SCH 442416 (A2a receptor antagonist), and 10 μM adenosine plus 5 nM MRS 1706 (A2b receptor antagonist). Data are means ± SE of median values for 3–5 animals.

Figure 3

Contribution of A1 receptor to regulation of Cx35 phosphorylation. A) Dose-response relationship of Cx35 phosphorylation level for incubation of adenosine receptor agonist NECA alone (solid curve) and in the presence of 20 nM A1 receptor antagonist DPCPX (dashed curve). Asterisks mark significance compared to no drug while plus signs mark significance comparing NECA with NECA+DPCPX in two-way ANOVA followed by Bonferroni post-hoc tests. Data are means ± SE of median values for 2–7 animals. B) Comparison of Cx35 phosphorylation levels for incubation of no drug control, 20 nM DPCPX, and 10 μM CGS 21680. Data are means ± SE of median values for 3–5 animals.

Figure 4

Influence of adenosine receptors on the phosphorylation level of OPL gap junctions in nighttime, dark-adapted retina. A) Confocal micrograph of immunostaining for total Cx35 (red) and phospho-S276 Cx35 (green) in the OPL of nighttime, dark-adapted zebrafish retina in an eyecup preparation. Phosphorylated Cx35 (shown alone in A′) is much higher than in daytime. B) 25 minute incubation with 1 μM A2a receptor antagonist CSC reduced Cx35 phosphorylation (phospho-S276 Cx35 shown alone in B′). Scale bar in B′ applies to all images in A and B. C) Dose-response relationship of Cx35 phosphorylation level for incubation of CSC. D) Dose-response relationship of Cx35 phosphorylation level for incubation of less selective adenosine receptor antagonist DMPX. Median values for 2 animals are shown.