PAF-induced structural and biochemical alterations of endothelial actin
pools. A, phalloidin staining of endothelial actin in control
conditions and after PAF-R activation. Actin staining of growth
hormone-starved monolayers (control) showed the presence of a well
organized peripheral dense band (PDB) and the existence of a few
stress fibers (SF) scattered in the center of the cell. When
confluent monolayers of HUVECs were stimulated with PAF for 1 min, 5 min, 15
min, and 30 min (e), we observed the formation of large cellular
folds in the first 1 min (b, arrows), the dilation of IEJs
(asterisk in b-e), the dissolution of PDB (c-e),
and the formation of SF in the first 5 min (b and c). These
alterations were less numerous at the later time points (d and
e). The mentioned changes of endothelial cell shape and of
endothelial actin pools are PAF-R-dependent, since the treatment of the
monolayer with BN 52078 prevented them (e). Bar, 30 μm in
a, b, e, and f and 25 μm in c and d
(n = 8). B, high resolution field emission scanning EM of
PAF-induced alterations in endothelial cells. a, the IEJ between two
resting ECs appears as a continuous line without interruptions or
irregularities. The figure also illustrates the presence of plasmalemmal
openings of endothelial vesicular carriers on both cells
(arrowheads). Bar, 0.1 μm (n = 6). b,
IEJ gaps formed between endothelial cells that are filled with filopodia and
lamellipodia (asterisk). The wrinkles present on the surface of one
of the endothelial cells (arrow) represent plasma membrane folds over
the newly formed SF inside the cell. Bar, 1 μm (n = 6).
c, within 5 min of PAF exposure, there is a marked increase in
membrane projections (especially filopodia), and the newly formed filopodia
are also found on the surface of PAF-treated cells (arrowheads).
Bar, 5 μm (n = 6). d, by 15 min after PAF
exposure, membrane projections remained abundant, whereas the dimensions
(width and length) of IEJs gaps increased. Bar, 2 μm (n =
6). e and f, the number and length of the filopodia were
maximal by 30 min. Bar, 5 μm for e and 10 μm for
f (n = 6 in both cases). C and D, PAF-R
activation induced the shift of G- to F-actin. C, different pools of
actin from confluent monolayers of HUVECs were analyzed by immunoblotting with
an anti-actin polyclonal Ab. The panel illustrates changes in G- and
F-actin pools in control and PAF-treated (10-10 m)
monolayers. Note the changes in G- and F-actin from basal conditions
(control) to more F-actin in the first 5 min and the reversal of the
ratio (with much more G-actin) at 15 and 30 min. The last lanes show
that lyso-PAF (10-6 m for 30 min) did not change the
ratio of different pools of actin (n = 8). D, the blot shown
illustrates the absence of actin pool changes induced by PAF challenge
following pretreatment of monolayers with the PAF-R antagonist BN 52078
(n = 6). Statistical significance was as follows: *,
p < 0.01; **, p < 0.05; ‡,
p < 0.001, by comparison with controls (Student's t
test).