digitonin and 1-2-dioctanoylglycerol

The activation of protein kinase C was investigated in digitonin-permeabilized human neuroblastoma SH-SY5Y cells by measuring the phosphorylation of the specific protein kinase C substrate myelin basic protein4-14. The phosphorylation was inhibited by the protein kinase C inhibitory peptide PKC19-36 and was associated to a translocation of the enzyme to the membrane fractions of the SH-SY5Y cells. 1,2-Dioctanoyl-sn-glycerol had no effect on protein kinase C activity unless the calcium concentration was raised to concentrations found in stimulated cells (above 100 nM). Calcium in the absence of other activators did not stimulate protein kinase C. Phorbol 12-myristate 13-acetate was not dependent on calcium for the activation or the translocation of protein kinase C. The induced activation was sustained for 10 min, and thereafter only a small net phosphorylation of the substrate could be detected. Calcium or dioctanoylglycerol, when applied alone, only caused a minor translocation, whereas in combination a marked translocation was observed. Arachidonic acid (10 microM) enhanced protein kinase C activity in the presence of submaximal concentrations of calcium and dioctanoylglycerol. Quinacrine and p-bromophenacyl bromide did not inhibit calcium- and dioctanoylglycerol-induced protein kinase C activity at concentrations which are considered to be sufficient for phospholipase A2 inhibition.

Topics: Acetophenones; Amino Acid Sequence; Arachidonic Acids; Calcium; Cell Membrane; Cell Membrane Permeability; Digitonin; Diglycerides; Dose-Response Relationship, Drug; Enzyme Activation; Humans; Molecular Sequence Data; Myelin Basic Protein; Neuroblastoma; Phospholipases A; Phospholipases A2; Phosphorus; Phosphorus Radioisotopes; Phosphorylation; Protein Kinase C; Quinacrine; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

The VSMC is an important target for the injurious effects of hyperglycemia in vivo. PKC plays a key role in the regulation of VSMC contraction and growth. This study examines whether elevated extracellular glucose concentrations (10-30 mM [180-540 mg/dl]) activate PKC in cultured rat VSMCs in vitro. A new, rapid, and highly specific assay was used to determine in situ PKC activity in digitonin-permeabilized VSMCs. PKC activity in VSMCs responded rapidly to variations in extracellular glucose concentrations. PKC was activated significantly within 10 min of exposure to D-glucose (20 mM) versus glucose (5 mM). Moreover, with continued exposure to D-glucose (20 mM), PKC activation was sustained for up to 48 h. Reducing D-glucose concentrations to 5 mM restored PKC activity to control values within 1 h. PKC activation was also glucose-concentration dependent. A threshold of only 15 mM (270 mg/dl) was required to significantly and maximally activate PKC in VSMC. PKC was not activated in the presence of osmotic control media that contained either elevated mannitol or L-glucose concentrations. In marked contrast to the sustained PKC activation induced by D-glucose in VSMCs, the normal physiological PKC response to the pressor hormones, AII and AVP, was short-lived and returned to base line within minutes. Sustained PKC activation in the presence of elevated D-glucose concentrations in vitro could disturb the normal physiological regulation of VSMC function and growth and thereby may contribute to the apparent vasotoxicity of hyperglycemia in vivo.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Amino Acid Sequence; Analysis of Variance; Angiotensin II; Animals; Aorta; Arginine Vasopressin; Cell Membrane Permeability; Cells, Cultured; Digitonin; Diglycerides; Enzyme Activation; Glucose; Isoquinolines; Kinetics; Molecular Sequence Data; Muscle, Smooth, Vascular; Oligopeptides; Phosphorylation; Piperazines; Protein Kinase C; Rats; Rats, Sprague-Dawley; Tetradecanoylphorbol Acetate