Resting distribution and stimulated translocation of protein kinase C isoforms alpha, epsilon and zeta in response to bradykinin and TNF in human endothelial cells.
Protein kinase C (PKC) has been linked to functional and morphological changes in endothelial cells involved in increased microvessel permeability. Bradykinin and TNF are potent inflammatory mediators which translocate PKC from the cytosol to the membrane of various cell types, including endothelial cells. The PKC isoforms alpha, epsilon and zeta have been demonstrated as the most prominent in human umbilical vein endothelial cells (HUVEC). We propose that bradykinin and TNF cause increased microvascular permeability via a PKC-dependent endothelial cell signalling pathway. HUVEC were incubated at 37 degrees C and 5% CO2 for 1 min, 15 min and 3 h with either bradykinin (1 microM) or TNF (100 U/ml). PMA incubation served as a positive control (100 nM, 15 min). Cytosolic and membrane-bound extracts were obtained by incubation in digitonin (0.5%) and Triton X100 (1%). PKC isoforms were assayed by Western blot and membrane fractions calculated. These experiments revealed that: HUVEC clearly displayed a non-uniform basal membrane fraction distribution of PKC isoforms, with zeta (35.4%) greater than epsilon (30.6%) and both much greater than alpha (8.6%); Bradykinin caused significant translocation of PKC alpha with 15 min and 3 h of treatment but not 1 min; TNF caused dramatic translocation of PKC alpha at 1 min treatment which subsided at 15 min and 3 h but remained significantly elevated; and PMA caused dramatic translocation of alpha and epsilon but not zeta. Treatments of bradykinin and TNF that translocated PKC also showed cytoskeletal rearrangement of rhodamine-phalloidin stained actin, causing it to become more prevalent near cell membranes and concentrated at focal points between cells. These results suggest that PKC alpha may contribute to long term low grade increases in microvessel permeability in response to bradykinin, and that PKC alpha could be involved in both transient and sustained microvessel permeability changes induced by TNF. Also, cytoskeletal actin organization appears to be a downstream pathway in the activation process, possibly leading to alteration in endothelial cell shape and contact points.
Ross
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Department of Physiology, James H. Quillen College of Medicine, East Tennessee State University, Johnson City 37614, USA.
Joyner
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D001920:Bradykinin / Q000494:pharmacology*; D002451:Cell Compartmentation; D002462:Cell Membrane / Q000201:enzymology; D002478:Cells, Cultured; D003599:Cytoskeleton / Q000201:enzymology; D004730:Endothelium, Vascular / Q000187:drug effects* / Q000201:enzymology; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism*; D051571:Protein Kinase C-alpha; D051744:Protein Kinase C-epsilon; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*; D014471:Umbilical Veins; D000099221:Protein Kinase C zeta
D001920:Bradykinin; D007527:Isoenzymes; D008565:Membrane Proteins; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; D051744:Protein Kinase C-epsilon; D013755:Tetradecanoylphorbol Acetate; D014409:Tumor Necrosis Factor-alpha; D000099221:Protein Kinase C zeta; C496628:PRKCA protein, human; C497255:PRKCE protein, human