Interphotoreceptor retinoid-binding protein promotes rhodopsin regeneration in toad photoreceptors

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IRBP-supplemented Ringer's solution incubated in the RPE-eyecup showed 11-cis-retinal to be virtually the only retinoid withdrawn from the RPE. With large quantities of IRBP (3.2-9.2 nmol), the amount of 11-cis-retinal (2.7 +/- 0.5 nmol) withdrawn from the RPE during a 3-hr incubation was similar to the plateau value of rhodopsin formed in the ROS/RPE-eyecup.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.