Prolonged survival of the phosphorylated form of rhodopsin during dark adaptation of Royal College Surgeons rat

Abstract

Abstract: : Purpose:Our previous study revealed significant low levels of expression of rhodopsin kinase in Royal College of Surgeons (RCS) rat (Invest Ophthalmol Vis Sci. 1999; 40:2788–2794.), suggesting that dysfunction of rhodopsin (Rho) phosphorylation may be related to the RCS photoreceptor degeneration. The purpose of the present study is to study Rho phosphorylation and dephosphorylation in RCS rat retina. Methods: Specific antibodies toward major Rho phosphorylation sites in vivo, 334Ser or 338Ser (J Biol Chem. 1995; 270:14259–14262.) were prepared by immunization of authentic phosphorylated peptides, phospho–Rho334 peptide (DDEApSATASK) or phospho–Rho338 peptide (CEASATApSKT), in rabbit. Using these antibodies, immunohistochemical study was performed in conjunction with Rho phosphorylation and dephosphorylation assays. Results: ELISA and Western blot analysis revealed that the raised antibodies exclusively recognized either the phosphorylated 334Ser or 338Ser site. In immunofluorescence labeling, both antibodies recognized photoreceptor outer segments from light adapted retinas from Sprague Dawley (SD), Brown Norway (BN) and RCS rat. During dark adaptation, immunoreactivities toward p–338Ser and p–334Ser sites were diminished within several hours (0.2–2 hr) in SD and BN rat retinas. However, in contrast, those toward p–338Ser and p–334Ser sites were diminished within 4 to 7 days in RCS rat retinas. In vitro studies demonstrated decreased levels of both Rho phosphorylation and dephosphorylation reactions in RCS retinas. However, the dephosphorylation reaction was much more greatly affected than the phosphorylation reaction. Conclusions:Extremely prolonged survival of phosphorylated forms of Rho may contribute to persistent misregulation of phototransduction processes in retinal degeneration in RCS rat.

Keywords: photoreceptors • retinal development