The Effects Of Src Family Kinases On Na, K-ATPase In Formation Of Cortex Cataract In Vitro

BackgroundNa,K-ATPase is responsible for maintaining the correct concentrations of sodium and potassium in the lens cells.Na,K-ATPase activity in lens fibers and epithelium is subject to regulation as the result of protein tyrosine phosphorylation.Src-family kinases(SFK) are one of those most important tyrosine kinases.There is a convincing link between abnormal elevation of lens sodium and the opacification of the lens cortex that occurs in age-related cataract. Our previous studies demonstrated that inhibition of SFK activity prevented development of cataract in vitro,but the mechanism is not clear.It is valuable to understand the interactions between SFK and Na,K-ATPase in formation of cortical cataract.AimTo investigate the effects of PP1,specific inhibitor of SFK,on the expression of Na,K-ATPaseα1 and its activity in the cultured chicken embryonic lenses.MethodsE10 embryonic chicken lenses were cultured in medium199 with 10%FBS for 10 days.The lenses without treatment with PP1 were in control group,while the lenses treated with PP1 were in PP1 group.E10 lenses were used as normal control.In culture day 5 and day10,the lenses were picked for the following experiments.Lens cryosections were prepared for immunofluorence staining to detect Na,K-ATPaseα1 and c-Src.The lenses were micro dissected into three parts, lens epithelium(E),peripheral fibers(FP) and central fibers(FC),and then were used in Western Blot,RT-PCR and Na,K-ATPase activity assay to evaluate the expression of Na,K-ATPase and its activity.The anterior capsules of age-related cataract patients,obtained from cataract surgeries were collected for immunofluorence staining to detect Na,K-ATPaseα1.ResultsThe expression of Na,K-ATPaseα1 was detected in the lens epithelial cells, but its fluorence density was decreased with age.The cultured lenses developed opacification at the periphery cortex both in control and PP1-treated group,but the percentage of opacification area in cultured day 4,6,8 and 10 was lower in PP1-treated lenses than control lenses (P＜0.05).With immunofluorence staining and Western blot assay,Na,K-ATPaseα1 was strongly present on the cell membranes of lens epithelial cells,and became weaker in the lens fibers in E10 lenses.The activity of Na,K-ATPase presented the same pattern.In the cultured lenses of day5 and day 10 with the treatment of PP1,the expression of Na,K-ATPaseα1 was enhanced at both protein and mRNA levels,by Western blot and RT-PCR assay,but its pattern in lens tissues was not change.Unexpectedly,the activity of Na,K-ATPase was inhibited by PP1.ConclusionsNa,K-ATPaseα1 is mainly present in the epithelial cells and superficial fiber cells of E10 lenses.The distribution and specific activity of Na,K-ATPase is not uniform in the lenses,and they have the same pattern.Inhibition of SFK with PP1 prevents cortical cataract formation in vitro not by enhancing the activity of Na,K-ATPase,but by upregulating the expression of Na,K-ATPaseα1.Src/Na, K-ATPaseα1 complex may participate in the signaling pathways involving formation of cataract.