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

Document Type

Article

Publication Date

12-1-1996

Description

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.

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