First published online 18 March 2003
doi: 10.1242/jcs.00378
Permeabilization in a cerebral endothelial barrier model by pertussis toxin involves the PKC effector pathway and is abolished by elevated levels of cAMP
Kerstin E. Brückener1,
Ali el Bayâ1,
Hans-Joachim Galla2 and
M. Alexander Schmidt1,*
1 Institut für Infektiologie — Zentrum für Molekularbiologie der
Entzündung (ZMBE), Universitätsklinikum Münster,
Von-Esmarch-Str. 56, 48149 Münster, Germany
2 Institut für Biochemie, Westfälische Wilhelms-Universität
Münster, Von-Esmarch-Str. 56, 48149 Münster, Germany
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Fig. 1. In vitro ADP-ribosylation of available  -Gi subunits
extracted from BCEC, various Plexus-chorioideus-derived epithelial
cell lines (ESP, SP-R and SCP), and isolated porcine Plexus
chorioideus epithelial cells (lower panels) after preincubation with PT
in culture. Cells at 80% confluency were incubated with 200 ng/ml PT for up to
5 hours and the solubilized membrane proteins were used as substrate in an in
vitro ADP-ribosylation assay with 140 ng activated PT.
32P-ADP-ribose labeled target proteins were measured using a
Phosphoimager. All assays were performed in four independent experiments. The
standard deviations (error bars, n=4) are indicated. The values have
been normalized to the amount of protein employed in the assay. The signals
obtained in solubilized cells without prior incubation with PT has been set to
100% for each cell type. The residual ADP-ribosylation was measured after
pre-incubation with PT for the indicated time. Prior incubation with Brefeldin
A (1 µg/ml) for 1 hour inhibits the PT-mediated ADP-ribosylation as shown
for BCECs (top right).
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Fig. 2. Transendothelial resistance (TER) and permeability for horse-radish
peroxidase (HRP) of BCEC monolayers. TER was measured using a custom-made
epithelial tissue voltohmmeter designed for Transwell filter inserts. The
transendothelial transport of HRP to the lower compartment was assessed
spectrophotometrically. OD values were representative of the traversed HRP and
thus for the level of relative permeability. The standard deviations (error
bars, n=4) are indicated.
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Fig. 3. Time course of PT-enhanced permeability and reduced TER. BCEC monolayers
were exposed to medium or medium containing 200 ng/ml PT for the times
indicated. TER and permeability for HRP was measured as described in the
legend to Fig. 1. The standard
deviations (error bars, n=6) are indicated.
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Fig. 4. Measurements of intracellular cAMP levels after treatment with PT, CT or
forskolin. BCEC monolayers were incubated with PT (low: 200 ng/ml, high: 1
µg/ml), cholera toxin (CT; 1 µg/ml) or forskolin (50 µM) for 2 hours.
Intracellular cAMP levels were determined using a cAMP enzyme immunoassay
system (Amersham Pharmacia Biotech, Braunschweig, Germany). The values shown
represent the means of analysis performed in triplicate. The standard
deviations (error bars, n=3) are indicated.
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Fig. 5. Influence of elevated cAMP levels on barrier permeability. BCEC monolayers
were incubated as indicated with medium or with medium containing PT (200
ng/ml) and/or CT (1 µg/ml) and/or forskolin (50 µM) for 4 hours. The
transendothelial HRP transport was assessed by measuring HRP concentrations
spectrophotometrically in the lower compartment. OD values were taken as a
measure for the traversed HRP indicating the relative permeability. The
standard deviations (error bars, n=6) are indicated.
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Fig. 6. H-7 and staurosporine enhance the PT-mediated permeabilization of BCECs.
BCEC monolayers were exposed to medium, medium containing the drug H-7 (50
µM), staurosporine (50 nM), or PT (200 ng/ml) alone or medium with H-7 in
combination with PT for 4 hours (+1 hour for the permeability assay). The
transendothelial HRP transport was assessed by measuring HRP concentration
spectrophotometrically in the lower compartment. OD values were representative
of the traversed HRP as a measure of relative permeability. The standard
deviations (error bars, n=6) are indicated. (A) Time-course of
relative permeability upon incubation with PT and PKC inhibitors H-7 or
staurosporine. (B) Effect of PKC inhibitors H-7 and staurosporine on the PT
induced permeability after 4 hours of incubation.
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Fig. 7. PKC-activating cellular drugs dioctanoyl-sn-glycerol and PMA reduce the
PT-mediated permeabilization of endothelial monolayers. BCEC monolayers were
exposed to medium, medium containing the drugs dioctanoyl-sn-glycerol (100
µg/ml) or PMA (500 nM), alone or in combination with PT (200 ng/ml) for 4
hours (+1 hour for the permeability assay). The transendothelial HRP transport
was assessed by measuring HRP concentration spectrophotometrically in the
lower compartment. OD values indicated the traversed HRP as a measure of
relative permeability. The standard deviations (error bars, n=6) are
indicated.
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Fig. 8. Effect of the PI3 kinase inhibitors wortmannin and LY294002 on PT-mediated
permeabilization of BCEC monolayers. BCEC monolayers were exposed to medium,
medium containing wortmannin (100 nM), LY294002 (10 µM) or PT (200 ng/ml)
alone or medium with wortmannin or LY294002 in combination with PT for 4 hours
(+1 hour for the permeability assay). The transendothelial HRP transport was
assessed by measuring HRP concentration spectrophotometrically in the lower
compartment. OD values indicated the traversed HRP as a measure of relative
permeability. The standard deviations (error bars, n=6) are
indicated.
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© The Company of Biologists Ltd 2003
