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
D. Ross et Wl. Joyner, 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, Endothelium, 5(4), 1997, pp. 321-332
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.