Identification and Localization of Pkcisoforms in Human Endothelial Cells: Age Related Differences and Activation by Bradykinin

PKC has been linked to functional and morphological changes in endothelial cells which could be involved in their response to inflammation. PKC α, ε, and ζ isotbrms have been shown to be the most prominent in Human Umbilical Vein Endothelial Cells (HUVEC). We hypothesize that: 1) high and low passed cells would have the same isoform distribution, and 2) bradykinin (luM) and PMA (100nM) activate PKC isoforms in high (-20th) and low (4-5th) passed HUVEC. The cells were incubated for 1 and 15 min with either bradykinin or PMA and then scraped, sonicated and fractionated. PKC in the cytosolic and membranebound fractions was assayed by Western blot. These experiments revealed that: • In the control samples, a and Çisoforms were present in cytosolic but not membrane-bound fractions, whereas e was present only in the membranebound fraction. • Bradykinin did not cause a change in a or Çisoform distribution, but the amount of e was attenuated in the membrane bound fraction at 15 min. • PMA activated the DAG-dependent a and e isoforms but not the DAG-independent Çisoform. • All the above results were consistent for both high and low passed cells. This study suggests that bradykinin inhibits a Ca-independent, DAG-dependent PKC isoform (e), potentially by activating an inhibitory G protein. Further, HUVEC clearly display a non-uniform basal distribution of PKC α, ε and ζ in HUVEC