Okajima T I, Pepperberg D R, Ripps H, Wiggert B, Chader G J
Lions of Illinois Eye Research Institute, Department of Ophthalmology, Chicago, IL.
Proc Natl Acad Sci U S A. 1990 Sep;87(17):6907-11. doi: 10.1073/pnas.87.17.6907.
Interphotoreceptor retinoid-binding protein (IRBP) has been hypothesized to function as an intercellular shuttle in the vertebrate eye, serving to transport retinoids between the retinal pigment epithelium (RPE) and photoreceptors in the process by which visual pigment is regenerated after photolysis. This hypothesis was tested in preparations utilizing the toad (Bufo marinus) eye and purified, initially ligand-free IRBP obtained from the bovine eye. Rod outer segments (ROS) or neural retinas were isolated and bleached, then incubated with native RPE (RPE-eyecup) in the presence or absence of IRBP. The amount of rhodopsin present after incubation was determined by spectrophotometric analysis and compared with that in control preparations receiving bovine serum albumin or Ringer's solution only. Supplementation with IRBP enhanced the formation of rhodopsin in both the ROS/RPE-eyecup and retina/RPE-eyecup preparations. Regeneration in ROS/RPE-eyecups receiving IRBP (1.8 nmol) increased in a roughly linear manner with the period of incubation (0-4 hr), at a rate of 0.44 nmol/hr. The extent of regeneration was graded with the quantities of IRBP and opsin introduced into the RPE-eyecup. With increasing amounts of IRBP (up to 5.2 nmol) or of initially available opsin (up to 15.6 nmol), the amount of rhodopsin formed (3-hr incubation) approached the same plateau value, about 2.5 nmol. Analysis of IRBP-supplemented Ringer's solution incubated in the RPE-eyecup showed 11-cis-retinal to be virtually the only retinoid withdrawn from the RPE. With large quantities of IRBP (3.2-9.2 nmol), the amount of 11-cis-retinal (2.7 +/- 0.5 nmol) withdrawn from the RPE during a 3-hr incubation was similar to the plateau value of rhodopsin formed in the ROS/RPE-eyecup. No 11-cis-retinal was observed in albumin-supplemented Ringer's solution (0.4-11.2 nmol of bovine serum albumin) or in Ringer's alone after similar incubation in the RPE-eyecup. The results suggest that an IRBP-mediated transfer of 11-cis-retinal from the RPE to the rods supports rhodopsin regeneration in vivo.
光感受器间类视黄醇结合蛋白（IRBP）被推测在脊椎动物眼中起到细胞间穿梭的作用，在视色素光解后再生的过程中，负责在视网膜色素上皮（RPE）和光感受器之间运输类视黄醇。利用蟾蜍（海蟾蜍）眼睛以及从牛眼中获取的最初无配体的纯化IRBP进行相关实验，对这一假说进行了验证。分离并漂白视杆外段（ROS）或神经视网膜，然后在有或没有IRBP存在的情况下，与天然RPE（RPE眼杯）一起孵育。孵育后通过分光光度分析测定视紫红质的含量，并与仅接受牛血清白蛋白或林格氏液的对照制剂中的含量进行比较。补充IRBP可增强ROS/RPE眼杯和视网膜/RPE眼杯制剂中视紫红质的形成。接受IRBP（1.8 nmol）的ROS/RPE眼杯中的再生随着孵育时间（0 - 4小时）大致呈线性增加，速率为0.44 nmol/小时。再生程度与引入RPE眼杯中的IRBP和视蛋白的量相关。随着IRBP量的增加（高达5.2 nmol）或初始可用视蛋白量的增加（高达15.6 nmol），形成的视紫红质量（3小时孵育）接近相同的平台值，约为2.5 nmol。对在RPE眼杯中孵育的补充IRBP的林格氏液进行分析表明，11 - 顺式视黄醛实际上是从RPE中提取的唯一类视黄醇。在大量IRBP（3.2 - 9.2 nmol）存在的情况下，3小时孵育期间从RPE中提取的11 - 顺式视黄醛量（2.7 +/- 0.5 nmol）与ROS/RPE眼杯中形成的视紫红质平台值相似。在RPE眼杯中进行类似孵育后，在补充白蛋白的林格氏液（0.4 - 11.2 nmol牛血清白蛋白）或仅林格氏液中未观察到11 - 顺式视黄醛。结果表明，IRBP介导的11 - 顺式视黄醛从RPE向视杆的转移支持了体内视紫红质的再生。
