Role of Protein Kinase C in Regulation of Bradykinin Receptor Activation in Endothelial Cells

Document Type

Article

Publication Date

12-1-1997

Description

Bradykinin (BK), a known inflammatory mediator, may induce alterations in the shape of endothelial cells via an intracellular pathway involving protein kinase C (PKC). Endogenous alkylglycerol (AG), a product produced by lipid metabolism, concomitantly with diacylglycerol (DAG), has been shown to reduce the translocation of PKC in various cell types; thus, this study investigates the potential inhibitory role of AG in the BK-induced translocation of PKC. Human endothelial cells (HUVEC) were incubated for 15 min and 3 hr in either control media or BK (1μM), 1-O-dodecyl-rac-glycerol (DDG, a synthetic AG, 30μM), phorbal myristale acetate (PMA, 0.1μM) or BK/DDG or PMA/DDG. Cytosolic and membrane fractions were collected via digitonin (0.5%) and Trilon X-100 (1%) extractions, respectively; then, after measurements of protein content (Pierce BCA), immunoblotting for specific isoforms. PKCα, PKCε, PKCζ, were completed by Western blot and imaging for band intensity was performed. In the control (untreated) HUVEC, PKC isoforms were found in both cytosol and membrane fractions with decreasing particulate levels of PKCε, PKCα, and PKCζ, respectively, while DDG reduced all isoform translocation. BK did not appear to induce translocation at either timepoint; however, with both DDG and BK present, translocation of PKCε and PKCα was increased. PMA induced translocation of PKCα and PKCε with no translocation of PKCζ; treatment with DDG and PMA did not alter the PMA-induced translocation. These results imply that DDG initiates a BK-induced translocation of the DAG-depended PKC isoforms. Therefore, the roles of transcellular signalling systems via DAG and PKC is complex, isoform specific, and can be amplified by DDG/AG.

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