Induction of cortical cataracts in cultured mouse lenses with H-89, an inhibitor of protein kinase A

Abstract

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 32 P-labeling, 2D-gel electrophoresis, phosphorimaging and mass spectrometry. Results. H-7 (50µM), inhibitor of protein kinase A (PKA) and protein kinase C (PKC), did not cause cataracts, but inhibited BSO cataract development. By contrast, 25µM 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. 32 P-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 Ca 2+ (>4nmol/mg wet wt.), suggesting Ca 2+ -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.