PURPOSE: To compare the effects of two serine-threonine protein kinase inhibitors in a mouse lens culture system previously designed to investigate cortical cataracts caused by L-buthionine sulfoximine (BSO), inhibitor of GSH biosynthesis. METHODS: Cataract development in HL-1 medium was evaluated visually or by measurement of lens Na+/K+ ratio through atomic absorption. Protein changes were evaluated by 32P-labeling, 2D-gel electrophoresis, phosphorimaging and mass spectrometry. Results. H-7 (50 microM), inhibitor of protein kinase A (PKA) and protein kinase C (PKC), did not cause cataracts, but inhibited BSO cataract development. By contrast, 25 microM H-89, selective inhibitor of PKA, caused large annular cortical cataracts and 100-fold elevation of Na+/K+ within 30 hr in day 10 lenses, in either the presence or absence of BSO. H-89 cataracts were also seen in day 12 and day 21 lenses. 32P-labeling of day 12 lenses pretreated with H-89 displayed more than 80% decrease in phosphorylation of alphaA crystallin, a known substrate of PKA, in the insoluble protein fraction. 2D-gel electrophoresis of day 12 H-89 cataract lens fractions revealed limited degradation of alpha and beta crystallins, degradation of cytoskeletal proteins, and elevated lens Ca2+ (>4 nmol/mg wet wt.), suggesting Ca2+-activated proteolysis. Conclusions. High Na+/K+ cataracts are induced by H-89, selective inhibitor of PKA, but not by H-7, an inhibitor of both PKA and PKC that impeded BSO-induced Na+/K+ elevation and cataract. These results suggest contrasting effects of PKA and PKC on lens cation transport and cortical cataract development.
PURPOSE: To compare the effects of two serine-threonine protein kinase inhibitors in a mouse lens culture system previously designed to investigate cortical cataracts caused by L-buthionine sulfoximine (BSO), inhibitor of GSH biosynthesis. METHODS:Cataract development in HL-1 medium was evaluated visually or by measurement of lens Na+/K+ ratio through atomic absorption. Protein changes were evaluated by 32P-labeling, 2D-gel electrophoresis, phosphorimaging and mass spectrometry. Results. H-7 (50 microM), inhibitor of protein kinase A (PKA) and protein kinase C (PKC), did not cause cataracts, but inhibited BSOcataract development. By contrast, 25 microM H-89, selective inhibitor of PKA, caused large annular cortical cataracts and 100-fold elevation of Na+/K+ within 30 hr in day 10 lenses, in either the presence or absence of BSO. H-89cataracts were also seen in day 12 and day 21 lenses. 32P-labeling of day 12 lenses pretreated with H-89 displayed more than 80% decrease in phosphorylation of alphaA crystallin, a known substrate of PKA, in the insoluble protein fraction. 2D-gel electrophoresis of day 12 H-89cataract lens fractions revealed limited degradation of alpha and beta crystallins, degradation of cytoskeletal proteins, and elevated lens Ca2+ (>4 nmol/mg wet wt.), suggesting Ca2+-activated proteolysis. Conclusions. High Na+/K+ cataracts are induced by H-89, selective inhibitor of PKA, but not by H-7, an inhibitor of both PKA and PKC that impeded BSO-induced Na+/K+ elevation and cataract. These results suggest contrasting effects of PKA and PKC on lens cation transport and cortical cataract development.