Retinomotor movements include elongation and contraction of rod and cone photoreceptors, and mass migration of melanin-containing pigment granules (melanosomes) of the retinal pigment epithelium (RPE) within the eyes of fish, frogs, and other lower vertebrates. Eyes of these animals do not contain dilatable pupils; therefore the repositioning of the rods and cones and a moveable curtain of pigment granules serve to modulate light intensity within the eye. RPE from sunfish (Lepomis spp.) can be isolated from the eye and dissociated into single cells, allowing in vitro studies of the cytoskeletal and regulatory mechanisms of organelle movement. Pigment granule aggregation from distal tips of apical projections into the cell body can be triggered by the application of underivatized cAMP, and dispersion is effected by cAMP washout in the presence of dopamine. While the phenomenon of cAMP-dependent pigment granule aggregation in isolated RPE was described many years ago, whether cAMP acts through the canonical cAMP-PKA pathway to stimulate motility has never been demonstrated. Here, we show that pharmacological inhibition of PKA blocks pigment granule aggregation, and microinjection of protein kinase A catalytic subunit triggers pigment granule aggregation. Treatment with a cAMP agonist that activates the Rap GEF, Epac (Effector protein activated by cAMP), had no effect on pigment granule position. Taken together, these results confirm that cAMP activates RPE pigment granule motility by the canonical cAMP-PKA pathway. Isolated RPE cells labeled with antibodies against PKA RIIα and against PKA-phosphorylated serine/threonine amino acids show diffuse, punctate labeling throughout the RPE cell body and apical projections. Immunoblotting of RPE lysates using the anti-PKA substrate antibody demonstrated seven prominent bands; two bands in particular at 27 and 64 kD showed increased levels of phosphorylation in the presence of cAMP, indicating their phosphorylation could contribute to the pigment granule aggregation mechanism.

中文翻译：

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蛋白激酶 A 调节鱼类视网膜色素上皮细胞中色素颗粒运动，Lepomis spp。

视网膜运动包括视杆和视锥细胞的伸长和收缩，以及鱼类、青蛙和其他低等脊椎动物眼内视网膜色素上皮 (RPE) 的含黑色素色素颗粒（黑素体）的大规模迁移。这些动物的眼睛不包含可扩张的瞳孔；因此，视杆和视锥细胞的重新定位以及可移动的色素颗粒帘用于调节眼睛内的光强度。翻车鱼的 RPE (莱波米斯spp.) 可以从眼睛中分离出来并解离成单个细胞，从而使体外细胞器运动的细胞骨架和调控机制的研究。使用未衍生化的 cAMP 可以触发从顶端突起的远端尖端进入细胞体的色素颗粒聚集，并且在多巴胺存在下通过 cAMP 冲洗来实现分散。虽然多年前描述了孤立的 RPE 中 cAMP 依赖性色素颗粒聚集的现象，但 cAMP 是否通过典型的 cAMP-PKA 途径刺激运动性从未得到证实。在这里，我们显示 PKA 的药理抑制作用会阻止色素颗粒聚集，而显微注射蛋白激酶 A 催化亚基会触发色素颗粒聚集。用激活 Rap GEF、Epac（由 cAMP 激活的效应蛋白）的 cAMP 激动剂处理对色素颗粒位置没有影响。综合起来，这些结果证实 cAMP 通过经典 cAMP-PKA 途径激活 RPE 色素颗粒运动。用针对 PKA RIIα 和针对 PKA 磷酸化丝氨酸/苏氨酸氨基酸的抗体标记的分离的 RPE 细胞在整个 RPE 细胞体和顶端突出处显示出弥散的点状标记。使用抗 PKA 底物抗体对 RPE 裂解物进行免疫印迹显示了七个显着条带；特别是在 27 和 64 kD 的两条带在 cAMP 存在下显示磷酸化水平增加，表明它们的磷酸化可能有助于色素颗粒聚集机制。整个 RPE 细胞体和顶端突起的点状标记。使用抗 PKA 底物抗体对 RPE 裂解物进行免疫印迹显示了七个显着条带；特别是在 27 和 64 kD 的两条带在 cAMP 存在下显示磷酸化水平增加，表明它们的磷酸化可能有助于色素颗粒聚集机制。整个 RPE 细胞体和顶端突起的点状标记。使用抗 PKA 底物抗体对 RPE 裂解物进行免疫印迹显示了七个显着条带；特别是在 27 和 64 kD 的两条带在 cAMP 存在下显示磷酸化水平增加，表明它们的磷酸化可能有助于色素颗粒聚集机制。