RESTING DISTRIBUTION AND STIMULATED TRANSLOCATION OF PROTEIN-KINASE-CISOFORM-ALPHA, ISOFORM-EPSILON AND ISOFORM-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 permea bility. Bradykinin and TNF are potent inflammatory mediators which tra nslocate PKC from the cytosol to the membrane of various cell types, i ncluding endothelial cells. The PKC isoforms a, alpha, epsilon and zet a have been demonstrated as the most prominent in human umbilical vein endothelial cells (HUVEC). We propose that bradykinin and TNF cause i ncreased microvascular permeability via a PKC-dependent endothelial ce ll signalling pathway. HUVEC were incubated at 37 degrees C and 5% CO2 for 1 min, 15 min and 3 h with either bradykinin (1 mu M) 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 d igitonin (0.5%) and Triton X100 (1%). PKC isoforms were assayed by Wes tern blot and membrane fractions calculated. These experiments reveale d 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 sign ificant translocation of PKC alpha with 15 min and 3 h of treatment bu t not 1 min; TNF caused dramatic translocation of PKC alpha at 1 min t reatment which subsided at 15 min and 3 h but remained significantly e levated; and PMA caused dramatic translocation of alpha and epsilon bu t not zeta. Treatments of bradykinin and TNF that translocated PKC als o showed cytoskeletal rearrangement of rhodamine-phalloidin stained ac tin, causing it to become more prevalent near cell membranes and conce ntrated at focal points between cells. These results suggest that PKC alpha may contribute to long term low grade increases in microvessel p ermeability in response to bradykinin, and that PKC alpha could be inv olved 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 alt eration in endothelial cell shape and contact points.