Protein kinase C activity and its relationship to myo-inositol uptake during hyperglycemic conditions in cultured bovine lens epithelial cells.
Incubation of cultured bovine lens epithelial cells (BLECs) in minimal essential medium (MEM) containing 40 mM galactose for 20 hr results in an attenuation of 3H-myo-inositol (3H-MI) concentrating ability. Decreased MI uptake could negatively impact on normal phosphoinositide turnover and diacylglycerol production, and presumably, protein kinase C (PKC) activation. The present report examines the relationship between PKC activity, myo-inositol transport and hyperglycemic conditions. PKC activities in the cytosol and particulate fractions of bovine lens epithelial cells in culture were quantitated using a mixed micelle assay following DEAE-cellulose (DE52) and Sephadex G-25 chromatography. Protein kinase C activity was assessed as Ca2+ and phospholipid-dependent Ac-myelin basic protein substrate peptide phosphorylation and confirmed using a PKC pseudosubstrate inhibitor peptide (PKC 19-36). Total PKC activity was similar in galactose-incubated cells (871 +/- 64 pmol/mg total protein/min) and control cells (881 +/- 8 pmol/mg total protein/min) after 20 hr. In unstimulated cells, approximately 90% of the total cellular PKC activity was recovered in the cytosolic fraction. Enzyme translocation was induced with the tumor promoting phorbol ester, phorbol 12-myristate 13-acetate (PMA), resulting in a 6-fold increase in membrane-associated PKC activity. A similar PMA-induced translocation was observed in BLECs incubated with 40 mM galactose MEM-maintained cells briefly treated with PMA or the non-phorbol PKC activators, SC-10 and mezerein, displayed a rate of 3H-MI uptake similar to the untreated control cells.(ABSTRACT TRUNCATED AT 250 WORDS)
Cammarata
P R
PR
Department of Anatomy and Cell Biology, Texas College of Osteopathic Medicine/University of North Texas, Fort Worth 76107.
Fan
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W
Jin
Y
Y
Yorio
T
T
D000818:Animals; D001692:Biological Transport; D002417:Cattle; D002478:Cells, Cultured; D003470:Culture Media; D004848:Epithelium / Q000201:enzymology; D005690:Galactose / Q000378:metabolism*; D005947:Glucose / Q000378:metabolism; D007294:Inositol / Q000378:metabolism*; D007908:Lens, Crystalline / Q000201:enzymology*; D011493:Protein Kinase C / Q000302:isolation & purification / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D003470:Culture Media; D007294:Inositol; D011493:Protein Kinase C; D005947:Glucose; D013755:Tetradecanoylphorbol Acetate; D005690:Galactose