title: Protein kinase C activity and its relationship to myo-inositol uptake during hyperglycemic conditions in cultured bovine lens epithelial cells.
abstract: 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)
authors.affiliation: Department of Anatomy and Cell Biology, Texas College of Osteopathic Medicine/University of North Texas, Fort Worth 76107.
authors.forename: P R
authors.initials: PR
authors.lastname: Cammarata
authors.forename: W
authors.initials: W
authors.lastname: Fan
authors.forename: Y
authors.initials: Y
authors.lastname: Jin
authors.forename: T
authors.initials: T
authors.lastname: Yorio
mesh_terms: 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
chemical_list: D003470:Culture Media; D007294:Inositol; D011493:Protein Kinase C; D005947:Glucose; D013755:Tetradecanoylphorbol Acetate; D005690:Galactose