HIGH GLUCOSE-CONCENTRATIONS INCREASE ENDOTHELIAL-CELL PERMEABILITY VIA ACTIVATION OF PROTEIN-KINASE C-ALPHA

Endothelial cell permeability is impaired in diabetes mellitus and may be increased by high extracellular glucose concentrations. High gluco se activates protein kinase C (PKC), a family of kinases vital to intr acellular signaling. We tested the hypothesis that high glucose concen tration activates PKC in endothelial cells and leads to an increase in endothelial cell permeability via distinct PKC isoforms, Porcine aort ic endothelial cells were used, and the PKC isoforms alpha, delta, eps ilon, zeta, and theta were identified in these cells. Glucose caused a rapid dose-dependent increase in endothelial cell permeability, with an EC50 of 17.5 mmol/L. Phorbol 12-myristate 13-acetate (TPA) induced an increase in permeability very similar to that elicited by glucose. The effect of glucose and TPA was totally reversed by preincubating th e cells with the PKC inhibitors staurosporine (10(-8) mol/L) and Goe 6 976 (10(-8) mol/L). Downregulation of PKC by preincubation with TPA fo r 24 hours also abolished the effect of glucose and TPA on endothelial cell permeability. High glucose (20 mmol/L) caused an increase in PKC activity at 2, 10, and 30 minutes. Cell fractionation and Western blo t analysis showed a glucose-induced translocation of PKC alpha and PKC epsilon. Confocal microscopy confirmed the translocation and showed a n association of PKC alpha and PKC epsilon with nuclear structures and the cell membrane. Specific antisense oligodesoxynucleotides (ODNs) a gainst PKC alpha reduced the expression of the isoform, abolished the effects of glucose on endothelial cell permeability completely, and re duced the TPA effect significantly. In contrast, specific antisense OD Ns against PKC epsilon had no effect on glucose-induced permeability a nd only a minor effect on the TPA-induced increase in permeability. We conclude that an increase in extracellular glucose leads to a rapid d ose-dependent increase in endothelial cell permeability via the activa tion of PKC and that this effect is mediated by the PKC isoform alpha.