Characterization of glucose-induced in situ protein kinase C activity in cultured vascular smooth muscle cells.
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.
Williams
B
B
Department of Medicine, University of Colorado School of Medicine, Denver 80262.
Schrier
R W
RW
D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D000595:Amino Acid Sequence; D000704:Analysis of Variance; D000804:Angiotensin II / Q000494:pharmacology; D000818:Animals; D001011:Aorta / Q000201:enzymology*; D001127:Arginine Vasopressin / Q000494:pharmacology; D002463:Cell Membrane Permeability; D002478:Cells, Cultured; D004072:Digitonin / Q000494:pharmacology; D004075:Diglycerides / Q000494:pharmacology; D004789:Enzyme Activation; D005947:Glucose / Q000494:pharmacology*; D007546:Isoquinolines / Q000494:pharmacology; D007700:Kinetics; D008969:Molecular Sequence Data; D009131:Muscle, Smooth, Vascular / Q000201:enzymology*; D009842:Oligopeptides / Q000378:metabolism; D010766:Phosphorylation; D010879:Piperazines / Q000494:pharmacology; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D004075:Diglycerides; D007546:Isoquinolines; D009842:Oligopeptides; D010879:Piperazines; C044295:1,2-dioctanoylglycerol; D000804:Angiotensin II; D001127:Arginine Vasopressin; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D011493:Protein Kinase C; D005947:Glucose; D004072:Digitonin; D013755:Tetradecanoylphorbol Acetate