Function and metabolism
Yvelines, France). The calcium ionophore A23187 was from Calbiochem
(Meudon, France). Unlabelled and [ 3 H]-alkyl-Gro were obtained as
described previously (Hichami et al., 1997). PAEC were isolated from
porcine aorta of Large Writh-Pietrain (6 month-old, 100 kg) treated
with trypsin for one minute to detach endothelial cells and cultured
in M199 medium supplemented with 1.6% antibiotics-antimycotics,
0.6% Fungizone® and 20% FBS.
Incorporation of [ 3 H]-alkyl-Gro into membrane PL
PAEC were incubated (5.10
5 cells/60 mm diameter Petri dish) with
[3H]-alkyl-Gro (10 - 5 M, 92.5 mCi/mmol) during 6, 12, 24 and 48 hours.
The culture medium was removed, the cells were washed with phosphate buffer saline, and the total lipicls were extracted according to
Bligh & Dyer (1959). The membrane PL were separated by thin-layer
chromatography (TLC) on silica gel plate (chloroform: methanol: acetic acid, 35: 14 :2.7, v/v, v). Radioactive material vvas visualized on a
radiochromatogram scanner (Bioscan, Washington, IJSA) and the PL
classes were identified by their retention factor. The zones on silica gel
plates corresponding to radioactive phospholipids were scrapped off, and
radioactivity was measured in a liquid scintillation counter (Packard,
Rungis, France).
Permeability studies
Primary cultures of PAEC were performed on microporous membrane
support at the density of 0.5. 10 6 cells per insert, and rhe permeability
of the cell monolayer was evaluated at cell confluence: the inserts were
placed into beakers at a maintained temperature of 37°C. This experimental system, according to Patterson et al. (1992), afforded the possibility of measuring the permeability of dye-labelled albumin through
the endothelial monolayer. Luminal and abluminal chambers both
contained the same concentration of 0.5% albumin. The experiments
started when Evans blue dye-labelled albumin was added at the luminal
side of the endothelial monolayer which had been incubated at 37°C
during 24 h before the experiments with or without 10 -5 M alkyl-Gro.
Then, at regular intervals, samples of abluminal medium were collected
and Evans blue concentration was measured by spectrophotometry at
620 nonometres. Clearance rates, V
1
were calculated according to the
following formula: V 1 = A
ab
x V
ab
x (A 1 ) -1 , where A
ab
is the abluminal
sample absorbance, V ab is the abluminal volume (in pl), and A 1 îs the
absorbance of undiluted luminal medium at t= 0. At t 30 mn, permeability was stimulated by PMA (2.10 -7 M) or A23187 (10 - 6 M).
l-O-[ 3 H]-Alkyl-2-acyl-sn-glycerol production
PAEC were incubated with [ 3 H]-alkyl-Gro (10 -5 M, 370 m Ci/mmol)
during 24 hours. The culture medium was then removed and the cells
washed with phosphate buffer saline, then maintained for 60 min in
medium without alkyl-Gro. PAEC were then incubated in the presence
or absence of PMA (2.10 - 7 M) or A23187 (10 -6 M) during rwo minutes.
149
Yvelines, France). The calcium ionophore A23187 was from Calbiochem
(Meudon, France). Unlabelled and [ 3 H]-alkyl-Gro were obtained as
described previously (Hichami et al., 1997). PAEC were isolated from
porcine aorta of Large Writh-Pietrain (6 month-old, 100 kg) treated
with trypsin for one minute to detach endothelial cells and cultured
in M199 medium supplemented with 1.6% antibiotics-antimycotics,
0.6% Fungizone® and 20% FBS.
Incorporation of [ 3 H]-alkyl-Gro into membrane PL
PAEC were incubated (5.10
5 cells/60 mm diameter Petri dish) with
[3H]-alkyl-Gro (10 - 5 M, 92.5 mCi/mmol) during 6, 12, 24 and 48 hours.
The culture medium was removed, the cells were washed with phosphate buffer saline, and the total lipicls were extracted according to
Bligh & Dyer (1959). The membrane PL were separated by thin-layer
chromatography (TLC) on silica gel plate (chloroform: methanol: acetic acid, 35: 14 :2.7, v/v, v). Radioactive material vvas visualized on a
radiochromatogram scanner (Bioscan, Washington, IJSA) and the PL
classes were identified by their retention factor. The zones on silica gel
plates corresponding to radioactive phospholipids were scrapped off, and
radioactivity was measured in a liquid scintillation counter (Packard,
Rungis, France).
Permeability studies
Primary cultures of PAEC were performed on microporous membrane
support at the density of 0.5. 10 6 cells per insert, and rhe permeability
of the cell monolayer was evaluated at cell confluence: the inserts were
placed into beakers at a maintained temperature of 37°C. This experimental system, according to Patterson et al. (1992), afforded the possibility of measuring the permeability of dye-labelled albumin through
the endothelial monolayer. Luminal and abluminal chambers both
contained the same concentration of 0.5% albumin. The experiments
started when Evans blue dye-labelled albumin was added at the luminal
side of the endothelial monolayer which had been incubated at 37°C
during 24 h before the experiments with or without 10 -5 M alkyl-Gro.
Then, at regular intervals, samples of abluminal medium were collected
and Evans blue concentration was measured by spectrophotometry at
620 nonometres. Clearance rates, V
1
were calculated according to the
following formula: V 1 = A
ab
x V
ab
x (A 1 ) -1 , where A
ab
is the abluminal
sample absorbance, V ab is the abluminal volume (in pl), and A 1 îs the
absorbance of undiluted luminal medium at t= 0. At t 30 mn, permeability was stimulated by PMA (2.10 -7 M) or A23187 (10 - 6 M).
l-O-[ 3 H]-Alkyl-2-acyl-sn-glycerol production
PAEC were incubated with [ 3 H]-alkyl-Gro (10 -5 M, 370 m Ci/mmol)
during 24 hours. The culture medium was then removed and the cells
washed with phosphate buffer saline, then maintained for 60 min in
medium without alkyl-Gro. PAEC were then incubated in the presence
or absence of PMA (2.10 - 7 M) or A23187 (10 -6 M) during rwo minutes.
149