PROTEIN-KINASE-C ACTIVITY AND ITS RELATIONSHIP TO MYOINOSITOL UPTAKE DURING HYPERGLYCEMIC CONDITIONS IN CULTURED BOVINE LENS EPITHELIAL-CELLS

Incubation of cultured bovine lens epithelial cells (BLECs) in minimal essential medium (MEM) containing An mM galactose for 20 hr results i n an attenuation of H-3-myo-inositol (H-3-MI) concentrating ability. D ecreased 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 betw een PKC activity, myo-inositol transport and hyperglycemic conditions. PKC activities in the cytosol and particulate fractions of bovine len s epithelial cells in culture were quantitated using a mixed micelle a ssay following DEAE-cellulose (DE52) and Sephadex G-25 chromatography. Protein kinase C activity was assessed as Ca2+ and phospholipid-depen dent Ac-myelin basic protein substrate peptide phosphorylation and con firmed using a PKC pseudosubstrate inhibitor peptide (PKC 19-36). Tota l PKC activity was similar in galactose-incubated cells (871 +/- 64 pm ol/mg total protein/min) and control cells (881 +/- 8 pmol/mg total pr otein/min) after 20 hr. In unstimulated cells, approximately 90% of th e total cellular PKC activity was recovered in the cytosolic fraction. Enzyme translocation was induced with the tumor promoting phorbol est er. phorbol 12-myristate 13-acetate (PMA), resulting in a 6-fold incre ase in membrane-associated PKC activity. A similar PMA-induced translo cation was observed in BLECs incubated with 40 mM galactose MEM-mainta ined cells briefly treated with PMA or the non-phobol PKC activators, SC-1 0 and mezerein, displayed a rate of H-3-MI uptake similar to the untreated control cells. Treatment of galactose-incubated cells with a ny of the three PKC activators failed to alter the galactose-attenuate d H-3-MI accumulation. These observations demonstrated the presence of a Ca2+ and phospholipid-dependent PKC activity in BLECs that appeared to function equally well in acute hyperglycemic conditions (20 hr) as in cells maintained in physiological medium. Moreover, these studies indicated that MI transport was not regulated directly by PKC and the galactose-induced attenuation in H-3-MI-concentrating capability was n ot a consequence of a derangement in PKC activity under these incubati on conditions.