Induction of cortical cataracts in cultured mouse lenses with H-89, an inhibitor of protein kinase A.
OBJECTIVE
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.
Calvin
Harold I
HI
Department of Biochemistry, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark, NJ, USA. calvinhl@umdnj.edu
Wu
Kaili
K
Li
Wenjie
W
Guo
Li
L
Banerjee
Urmi
U
Fu
S-C Joseph
SC
D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine / Q000494:pharmacology; D000818:Animals; D000831:Animals, Newborn; D019328:Buthionine Sulfoximine / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D002386:Cataract / Q000139:chemically induced* / Q000378:metabolism / Q000473:pathology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors*; D015180:Electrophoresis, Gel, Two-Dimensional; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005260:Female; D007546:Isoquinolines / Q000494:pharmacology*; D007904:Lens Cortex, Crystalline / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D008297:Male; D051379:Mice; D009924:Organ Culture Techniques; D010766:Phosphorylation; D011188:Potassium / Q000378:metabolism; D011247:Pregnancy; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D012964:Sodium / Q000378:metabolism; D013054:Spectrophotometry, Atomic; D013449:Sulfonamides; D038202:alpha-Crystallin A Chain / Q000378:metabolism
D004791:Enzyme Inhibitors; D007546:Isoquinolines; D013449:Sulfonamides; D038202:alpha-Crystallin A Chain; D019328:Buthionine Sulfoximine; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D012964:Sodium; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; C063509:N-(2-(4-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide; D011188:Potassium; D002118:Calcium