Impaired membrane translocation of thrombin stimulated PKC epsilon by high glucose.
Hyperglycaemia is known to cause endothelial dysfunction and to promote diabetic angiopathy. Therefore, this study was designed to evaluate the effect of long term incubation (16 +/- 1 days) in 30 mM vs 5 mM glucose on ligand induced translocation of protein kinase C (PKC) in paired cultures of individual isolates of human umbilical vein endothelial cells (HUVECs). Cells were stimulated with increasing concentrations of thrombin (0.01, 0.1, 1, 10 and 100 nM) for 30 seconds in the presence of 5 mM and 30 mM glucose, respectively, and analyzed by immunoblotting for PKC-isoforms alpha and epsilon. Stimulation by thrombin of confluent cultures displayed a concentration dependent rise in membrane bound PKC alpha and epsilon. Translocation of PKC alpha by thrombin remained unaffected by high versus normal ambient glucose, whereas translocation of PKC epsilon in cells grown in 30mM glucose was reduced at maximal thrombin concentrations (area under the curve, AUC: 90.4 +/- 7% of control cells; p < 0.008; n = 6) versus control cultures kept in 5mM glucose. In the identical isolates translocation of PKC epsilon was not reduced by 30 mM mannitol used as osmotic control. No change was induced by long term incubation of resting cells with 30 mM vs 5 mM glucose as to total and membrane bound PKC alpha or PKC epsilon. The obtained data suggest modulation by 30 mM glucose of ligand induced PKC translocation in an isoform specific manner, whereas subcellular distribution of PKC isoforms in the absence of thrombin stimulation remains unaffected by 30 mM glucose.
Reining
G
G
Department of Internal Medicine III, University of Vienna, Austria.
Baumgartner-Parzer
S
S
Waldhäusl
W
W
D001692:Biological Transport; D002462:Cell Membrane / Q000378:metabolism*; D002478:Cells, Cultured; D003600:Cytosol / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004730:Endothelium, Vascular / Q000187:drug effects / Q000378:metabolism*; D005947:Glucose / Q000494:pharmacology*; D006801:Humans; D015151:Immunoblotting; D007527:Isoenzymes / Q000378:metabolism*; D011493:Protein Kinase C / Q000378:metabolism*; D013268:Stimulation, Chemical; D013917:Thrombin / Q000494:pharmacology*; D013997:Time Factors; D014471:Umbilical Veins
D007527:Isoenzymes; D011493:Protein Kinase C; D013917:Thrombin; D005947:Glucose