In terms of the abnormalities of photoreceptor cells in RCS rats, several changes were noted in the RCS ROS including opsin,
16 arrestin,
17 18 and ROS protein phosphorylation levels,
19 which may affect quenching of the phototransduction pathway. In our previous study in RCS rats compared with SD rats,
20 we performed proteome analysis and found significantly lower levels of expression of the mRNA of α-A crystalline and rhodopsin kinase (RK), which are thought to be involved in post-Golgi processing of opsin and Rho phosphorylation, respectively. In contrast, expression levels of other major proteins of ROS were almost comparable to those in 3-week-old SD rats according to SDS- PAGE analysis.
20 Therefore, we suggested that the kinetics of Rho phosphorylation and dephosphorylation are specifically affected in RCS rats. Thus, in 3-week-old RCS rats, it is reasonable to think that only the kinetics of Rho dephosphorylation is impaired, whereas the kinetics of Rho regeneration is comparable to wild-type rats in the present study. Our previous study involving the in vitro biochemical assay revealed that levels of Rho phosphorylation in 3-week-old RCS rats were slightly lower than those in 3-week-old SD rats, but, in contrast, dephosphorylation of phosphorylated Rho showed much slower kinetics in RCS than in SD rat ROS (3 weeks old). Furthermore, with our recent method of using specific antibodies to Rho phosphorylated at the
334Ser or
338Ser sites, which are known to be major phosphorylation sites in Rho in vivo,
11 21 we were able to evaluate the kinetics of dephosphorylation of phosphorylated photolyzed Rho in RCS and wild-type rats and SD and BN rats in vivo. This method applied during dark adaptation showed that dephosphorylation of
338Ser and
334Ser sites was completed within several hours (0.2–2 hours) in SD and BN rat retinas. However, those antibodies directed toward phosphorylated-
338Ser and -
334Ser sites were diminished within 4 to 7 days in RCS rat retinas. Therefore, we hypothesized that extremely prolonged survival of phosphorylated forms of Rho may contribute to persistent misregulation of phototransduction processes in retinal degeneration in RCS rat.
10 22 Our present results suggest that light-induced retinal degeneration may be caused by the same mechanism of RCS rat retinal degeneration as just described. since we found that light-induced stress also caused significant delay in the kinetics of Rho dephosphorylation. Furthermore, the retinal photoreceptor degenerative model of cancer-associated retinopathy (CAR), which is produced by intravitreous administration of anti-recoverin antibody to rats, showed significant high levels of Rho phosphorylation.
23 24 In addition, Rho mutants within the C terminus, in which
345Val and
347Pro are the most common sites of mutations causing autosomal dominant retinitis pigmentosa (adRP),
25 26 were also phosphorylated at significantly higher levels than in wild-type.
27 Therefore, prolonged survival of phosphorylated Rho by lower phosphatase activities or enhanced Rho kinase activities may be one of the common mechanisms responsible for most of the retinal photoreceptor degeneration. Although slow kinetics in Rho dephosphorylation was commonly observed in these retinal degenerations, the degree of kinetic change did not correspond to the severity of retinal degeneration. Additional unknown mechanisms must therefore be present to account for retinal degeneration.