(Circulation. 1999;99:2523-2529.)
© 1999 American Heart Association, Inc.
Clinical Investigation and Reports |
Calcium Antagonists Ameliorate Ischemia-Induced Endothelial Cell Permeability by Inhibiting Protein Kinase C
From the Franz Volhard Clinic and Max Delbrück Center for Molecular Medicine (A.H., C.L., C.M., M.M., R.B., F.C.L., H.H.), Medizinische Fakultät der Charité, Humboldt University of Berlin, and Physiologisches Institut (T.N.), Justus Liebig University, Giessen, Germany.
Correspondence to Hermann Haller, MD, Franz Volhard Clinic, Wiltberg Strasse 50, 13122 Berlin, Germany. E-mail haller{at}fvk-berlin.de
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Abstract |
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Background—Dihydropyridines block calcium channels; however, they also influence endothelial cells, which do not express calcium channels. We tested the hypothesis that nifedipine can prevent ischemia-induced endothelial permeability increases by inhibiting protein kinase C (PKC) in cultured porcine endothelial cells.
Methods and Results—Ischemia was induced by potassium
cyanide/deoxyglucose, and permeability was measured by albumin
flux. Ion channels were characterized by patch clamp.
[Ca2+]i was measured by fura 2. PKC activity
was measured by substrate phosphorylation after cell
fractionation. PKC isoforms were assessed by Western blot and confocal
microscopy. Nifedipine prevented the
ischemia-induced increase in permeability in a dose-dependent
manner. Ischemia increased [Ca2+]i,
which was not affected by nifedipine. Instead,
ischemia-induced PKC translocation was prevented by
nifedipine. Phorbol ester also increased
endothelial cell permeability, which was dose
dependently inhibited by nifedipine. The effects of
non–calcium-channel–binding dihydropyridine
derivatives were similar. Analysis of the PKC isoforms showed
that nifedipine prevented ischemia-induced
translocation of PKC-
and PKC-
. Specific inhibition of PKC
isoforms with antisense oligodeoxynucleotides demonstrated
a major role for PKC-.
Conclusions—Nifedipine exerts a direct effect on
endothelial cell permeability that is independent of
calcium channels. The inhibition of ischemia-induced
permeability by nifedipine seems to be mediated primarily
by PKC- inhibition. Anti-ischemic effects of
dihydropyridine calcium antagonists
could be due in part to their effects on endothelial
cell permeability.
Key Words: cells • ischemia • proteins • calcium
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Introduction |
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Calcium antagonists ameliorate ischemia in patients with chronic stable angina, unstable angina, variant angina, and silent ischemia and are used effectively in the treatment of hypertension.1 Calcium antagonists also prevent myocardial injury after ischemia and reperfusion.2 Calcium antagonists enhance vasodilatation, improve regional blood flow, and reduce ischemia-induced injury.3 Their actions have generally been attributed to specific L-type calcium channel blocking effects on vascular smooth muscle cells.4 However, calcium antagonists may also influence endothelial cells, which have not been shown to possess L-type calcium channels.5 Direct effects on endothelial cells could be important, because the endothelium plays a major role in ischemia-induced tissue injury. Adhesion molecule expression, NO release, and the maintenance of endothelial cell–layer permeability are disturbed with ischemia.6 Orth et al7 recently demonstrated that calcium antagonists inhibit the proliferation of mesangial cells, which also have no L-type calcium channels. Possible L-type calcium channel–independent mechanisms include inhibition of ATP utilization, blockade of K+ channels, inhibition of agonist-receptor interactions, inhibition of Na+/H+ exchange, and inhibition of hydrophobic compound traffic via multidrug resistance proteins.8 Block et al9 10 previously reported a calcium antagonist–mediated inhibition of protein kinase C (PKC) in vascular smooth muscle cells. Because PKC plays a central role in several endothelial cell functions, such as adhesion and permeability, we tested the hypothesis that the calcium antagonist nifedipine influences ischemia-induced permeability in endothelial cells via PKC inhibition.
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Methods |
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Phorbol ester TPA (phorbol 12-myristate 13-acetate), histone type III-S, DEAE cellulose, and all other materials, if not stated otherwise, were purchased from Sigma Chemical Co.
-[32P]ATP was obtained from Amersham.
1,2-Diolein and phosphatidylserine were purchased
from Avanti Polar Lipids. CY3-conjugated anti-mouse IgG antibodies were
obtained from Dianova. The PKC inhibitor Goe 7624 was
obtained from Calbiochem. The fluorescent probe fura 2-AM was
purchased from Serva. All dihydropyridines were a
gift from Bayer, Leverkusen, Germany. Porcine aortic endothelial cells were isolated and cultured as previously described in detail.11 Experiments were performed with confluent monolayers, 4 days after they were seeded onto filters. The purity of these cultures was >98% endothelial cells. Permeability across the endothelial cell monolayer was studied in a 2-compartment system separated by a filter membrane.12 Both compartments contained modified Tyrode solution supplemented with 10% (vol/vol) FCS. The luminal compartment containing the monolayer had a volume of 2.5 mL, whereas the abluminal compartment had a volume of 10.5 mL. The fluid in the abluminal compartment was constantly stirred. Trypan blue–labeled albumin (60 µmol/L) was added to the luminal compartment. We continuously monitored the appearance of Trypan blue–labeled albumin in the abluminal compartment by pumping the liquid through a 2-wavelength photometer (model Specord S-10; Carl Zeiss; wavelength of 580 nmol/L for Trypan blue and 720 nm for the reference wavelength). The concentration of Trypan blue–labeled albumin in the luminal compartment was determined every 10 minutes of incubation. The albumin flux (F) across the monolayer was determined from the increase in albumin concentration ([A]2) in the abluminal compartment (volume V): F=d[A2]/dtxV, where t is time. Data were expressed as percentage of a defined control situation.
The term ischemia (from the Greek ischo, to keep back) refers to local anemia due to mechanical vascular obstruction. In our cell system, we actually performed energy depletion to mimic some of the features of ischemia in a cell culture system. Effects of energy depletion were studied in the presence of potassium cyanide (KCN; 1 mmol/L), an inhibitor of oxidative phosphorylation, and 2-deoxy-D-glucose (DG; 1 mmol/L), an inhibitor of glycolysis, as described previously.12 The effect of KCN/DG on endothelial cell permeability was fully reversible.
PKC activity was measured in cultured confluent cells after isolation
of a particulate fraction by previously described
techniques.13 Data presented in Figure 6
were calculated by subtraction of nonspecific activity. Western blot
analysis was performed as described elsewhere.13
The antisense methods were described in previous
publications.11 14 Phosphorothioate
oligodeoxyribonucleotides (ODNs) were purchased from TIB
Molbiol. For transfection, cells were incubated with lipofectin (10
µg/mL) and ODN (1 µmol/L) in the absence of FCS at 37°C for
4 hours, washed 2 times with medium, and then incubated with medium and
ODN (1 µmol/L) for another 4 hours. Afterward, the medium was
changed to 10% FCS for 24 hours before the start of the
experiments.
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For [Ca+2]i measurements, cells were sowed on coverslips and incubated at 37°C for 4 days.15 The calcium measurements were performed with a Spex fluorolog 2 spectrofluorometer, which was connected to a Nikon Diaphot 300 microscope and a variable-aperture photometer to isolate individual cells on the microscope stage (Spex Industries Inc). Cultured endothelial cells were loaded with fura 2-AM with 15 minutes' incubation in PBS containing 5 µmol/L fura 2-AM (added from a 5 mmol/L stock solution in DMSO). Ca2+ channel currents were recorded as previously described in the whole-cell configuration with a List patch-clamp amplifier (model EPC 7).16
Statistical Analysis
Statistical analysis was performed with the commercially
available statistics SPSS (SPSS Inc). Because the data comprise time
series, general linear modeling with repeated measures and
between-subject effects were calculated. In case of significant group
effects, data obtained at 30 minutes were compared between groups by
ANOVA with post hoc Scheffé tests to allow for multiple
testing.
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Results |
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Administration of KCN (1 mmol/L) and DG (1 mmol/L) resulted in a rapid decrease in ATP from 15±2 to 8±2.4 nmol/mg protein within 10 minutes (n=5; P
0.05; data not shown).
Ischemia increased permeability within minutes, reaching a
maximum at 30 minutes (148±9% of control values; n=7;
P0.01) and remaining stable thereafter (Figure 1). Afterward,
endothelial cell permeability declined to a plateau
phase. The ischemia-induced increase in permeability was
totally reversible after the KCN/DG-containing medium was removed (data
not shown). Figure 2 shows the
effect of nifedipine on the time course of
ischemia-induced increases in permeability.
Nifedipine decreased both the initial peak and the plateau
phase of ischemia-induced permeability. The ameliorating effect
of nifedipine was observed at a concentration of
10-10 mol/L (n=7; P0.01 compared
with KCN/DG). We observed a nonspecific cation current in the
endothelial cells. Characteristics of
L-type calcium channels were not detected (n=7;
data not shown). We then analyzed whether calcium influx could
occur by other mechanisms. Cadmium (5x10-5
mol/L) was used as a nonspecific inhibitor of calcium flux
(Figure 3). Cadmium had no effect on
basal permeability and did not influence the ischemia-induced
alteration (n=5). In addition, cadmium did not interfere with the
effects of nifedipine on ischemia-induced
permeability changes.
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Possible effects of nifedipine on intracellular free
calcium concentrations in endothelial cells were
investigated in the next set of experiments. A
representative experiment is shown in Figure 4. Incubation of
endothelial cells with KCN/DG led to a rapid and
sustained increase in intracellular free calcium concentration from
143±29 to 472±35 nmol/L (n=8; P0.01). Concomitant
incubation with nifedipine did not influence the rapid
increase, nor did incubation with nifedipine alter the
sustained elevation in intracellular free calcium concentration during
ischemia (472±35 versus 458±39 nmol/L; n=8; P=NS).
After removal of KCN/DG from the medium, intracellular free calcium
concentrations returned to baseline (data not shown).
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Figure 5 shows the effect of different
PKC inhibitors on endothelial cell
permeability after 30 minutes of exposure to KCN/DG.
Endothelial cell incubation with
staurosporine (10-8 mol/L) 30
minutes before KCN/DG exposure reduced ischemia-induced
permeability to almost basal values (n=6; P<0.05). The PKC
inhibitor Goe 7624 (10-8 mol/L) also
reduced ischemia-induced endothelial
permeability (n=6; P<0.05). These findings indicate that
the effects of KCN/DG are mediated by PKC. This assumption was
supported by downregulation of PKC. Preincubation with TPA (100 nmol/L)
for 24 hours, a time period that downregulates the phorbol
ester–sensitive PKC isoforms, abolished the effects of KCN/DG (n=7;
P<0.05). The observed effects of known PKC
inhibitors were similar to the effects achieved with
nifedipine (10-8 mol/L; n=6; Figure 5).
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Figure 6
shows the effects of
nifedipine on TPA-induced endothelial cell
permeability. TPA (10-8 mol/L) produced a rapid
increase in endothelial cell permeability.
Preincubation with nifedipine (30 minutes) led to a
dose-dependent inhibition of the phorbol ester–induced effect. At a
concentration of 10-7 mol/L,
nifedipine reduced the TPA-induced increase in permeability
almost completely. Comparison of nifedipine with the
well-characterized PKC inhibitor staurosporine
showed a similar efficacy of the calcium antagonist in
restoring phorbol ester–induced permeability. In addition to phorbol
ester stimulation, we used glucose as a stimulus of PKC
activation.11 Glucose (20 mmol/L) induced a rapid
increase in endothelial cell permeability (168±19% of
basal values; n=8). This effect was almost completely blocked by
preincubation with nifedipine (10-8
mol/L; 117±24%; P0.05 versus control; n=5) (data not
shown).
Figure 7 shows the effect of
nifedipine on PKC activity in the particulate fraction
after cell fractionation. KCN/DG induced a 4-fold increase in
membrane-bound (particulate) PKC activity at 10 minutes (n=6;
P0.05). Preincubation with nifedipine
(10-8 mol/L) reduced PKC activity in the
membrane fraction significantly (n=6; P0.05). We next
investigated whether nifedipine specifically inhibits the
translocation of specific PKC isoforms in endothelial
cells. In Figure 8, the results of
translocation experiments for PKC-, -
, -
, and - are shown.
PKC- (Figure 8A) showed a single band at 82 kDa and was
mostly located in the cytosolic fraction under resting conditions.
Endothelial cell exposure to KCN/DG resulted in a
translocation of PKC- from the cytosolic fraction to the membrane or
particulate fraction. Preincubation with nifedipine
prevented the ischemia-induced translocation. Densitometric
analysis of the translocation experiments (n=3;
P0.05 compared with control and nifedipine) is
also shown in Figure 8. In Figure 8B, the effects of
KCN/DG on PKC- are shown. Ischemia had no significant effect
on the translocation of PKC-. In Figure 8C, the effects of
ischemia on PKC- are shown. Ischemia had no effect
on the translocation of PKC-. PKC- (Figure 8D) showed a
double band in both the cytosol and the particulate fractions. KCN/DG
induced an increase in the upper band in the particulate fraction and a
concomitant decrease in the cytosolic fraction. This effect was
completely prevented by nifedipine (n=3; P0.05
compared with control and nifedipine).
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To analyze which PKC isoform is responsible for the increase in
ischemia-induced permeability, we used antisense ODN to
specifically suppress expression of the respective PKC isoform (Figure 9; n=4). Endothelial
cells were incubated with lipofectin (10 µg/mL) and antisense ODN,
sense ODN, or reversed ODN against PKC-, -, or - before
exposure to KCN/DG. Antisense ODN for PKC- almost completely
inhibited the increase in ischemia-induced
endothelial cell permeability. In contrast to the
effects of antisense against PKC-, antisense ODN against PKC- and
- did not reduce ischemia-induced permeability
significantly.
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Finally, we examined whether the effects of nifedipine are
stereoselective with a dihydropyridine
nifedipine enantiomer (BAY K005552). In addition, a
dihydropyridine compound that does not bind to
L-type calcium channels (BAY R001223) was investigated.
These compounds are similar in structure but have no effect on the
L-type calcium channel (personal communication from Bayer
Inc, 1998). The drugs (Figure 10) were similar in efficacy to
nifedipine in terms of blocking ischemia-induced
endothelial cell–layer permeability increases (n=4;
P0.05 compared with KCN/DG alone).
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Discussion |
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Ischemia increased endothelial cell permeability, which was associated with an increase in [Ca2+]i and PKC activation. Nifedipine inhibited this effect dose dependently without changes in [Ca2+]i homeostasis but with inhibition of PKC. PKC-
and - inhibition may be responsible
for the ameliorating effect of nifedipine on
ischemia-induced endothelial cell permeability.
Our findings suggest that PKC is the molecular target that influences
ischemia-induced increases in endothelial cell
permeability. Lynch et al17 were the first to observe that
PKC activation is an important signal transduction pathway to increase
macromolecular transport across endothelial monolayers.
Several reports subsequently supported this finding in
vitro.18 PKC activation plays a role in the increased
endothelial cell permeability induced by
bradykinin,19 hydrogen peroxides,20
thrombin,18 and endothelins.21 Thus, in
addition to an increase in
[Ca2+]i, the activation
of PKC appears to be a major determinant of an increase in
endothelial cell permeability.22 Increased
PKC activity may be subsequent to or actually induced by the increase
in [Ca2+]i. On the other
hand, increased endothelial cell permeability after
direct activation of PKC via phorbol ester suggests a mechanism
independent of changes in
[Ca2+]i. However, the
exact mechanism of PKC action within endothelial cells
is not clear. Exposure to phorbol ester leads to
phosphorylation and redistribution of the cytoskeletal
proteins caldesmon and vimentin, in concert with agonist-mediated
endothelial cell contraction and resultant barrier
dysfunction.23 This observation indicates that PKC
activation increases endothelial cell permeability by
an interaction with cytoskeletal proteins. This hypothesis is supported
by recent observations in epithelial cells, in which PKC-dependent
actin reorganization led to modulation of intercellular
permeability.24
We observed an effect of KCN/DG on the PKC isoforms and but not
and . Translocation of PKC- after ischemia has also
been observed by others. Wang and coworkers25 demonstrated
that PKC- is responsible for the opening of ion channels with
ischemia. Others26 have found a PKC-
translocation in ischemic myocardium and myocardial
cells. We13 14 showed previously that PKC- plays an
important role in the regulation of endothelial cell
permeability. Our results with KCN/DG support a role for this PKC
isoform in mediating endothelial cell permeability
during ischemia. Other investigators have also observed an
effect of ischemia on the PKC isoform . However, we did not
observe an effect on the ischemia-induced increase in
endothelial cell permeability by downregulation of
PKC-. Because we used a different model and induced
metabolic rather perfusion-related ischemia, it is
possible that model-dependent characteristics play a role in these
discrepant findings. Our finding with respect to the PKC isoform
during ischemia is in accordance with the recent observation of
Mizukami et al.27
Block and coworkers10 showed that 3 calcium antagonists (nifedipine, verapamil, and diltiazem) all inhibited the action of recombinant platelet-derived growth factor in vascular smooth muscle cells and reduced PKC activation. They also showed that calcium antagonists modulate expression of HMG-CoA reductase and LDL-receptor genes stimulated by platelet-derived growth factor. These calcium antagonist–mediated effects on gene expression were observed at pharmacological concentrations that were 1 to 2 orders of magnitude lower than those required for inhibition of depolarization-induced opening of voltage-sensitive L-type calcium channels.28 Similar observations have been made by other groups.7 8 9 However, in the above-mentioned studies, the effects of calcium antagonists were investigated in cell types that possess L-type calcium channels, such as vascular smooth muscle cells. Recently, Orth et al7 investigated the effects of calcium antagonists in mesangial cells and could not associate the observed inhibitory effects on cell proliferation with changes in intracellular calcium. Our data demonstrate that the inhibitory effect of calcium channel blockers on PKC is not dependent on L-type calcium channels or on changes in intracellular calcium regulation. PKC inhibition by calcium antagonists is conceivably responsible for the effects of these compounds in other cell types that do not express L-type calcium channels.29 30 31
A direct interaction between nifedipine and PKC isoforms has not been observed in in vitro investigations. However, the inhibitory effect of nifedipine on TPA-induced permeability would favor such an explanation. Our findings suggest that nifedipine blocks the proximal part of the PKC activation pathway. Because [Ca2+]i was not influenced by nifedipine in the present study, we would rather speculate that nifedipine interferes with the release of phospholipids from the plasma membrane or other intracellular lipid pools. Possible candidates are diacylglycerol, ceramide, or other products of the phosphoinositide cycle.32 An increased production of these compounds in ischemia has been shown.33 Decreased generation of these compounds would then lead to a decrease in PKC activation. Another possible mechanism is the inhibition of PKC binding to intracellular sites and cell membranes.34
Our results may explain previous experimental studies in which calcium
antagonists were useful in protecting from ischemic
injury in various tissues. Translocation of PKC isoforms in
ischemic cardiomyocytes has been shown by Yoshida
et al.35 Translocation of protein kinase C isoforms has
been observed in both ischemic neuronal tissue and blood
vessels.34 A role for PKC- activation is also suggested
by the observation that this isoform opens ion channels in
ischemic tissue.36 The finding that calcium
antagonists influence PKC activity during ischemia
could be important, because PKC inhibition has been found to be
beneficial in protecting the heart, lung, and brain from
ischemia.37 38 The link between ischemia
and increased endothelial cell–layer permeability is
also relevant. For instance, the prevention of ischemia-induced
permeability increases could be useful therapeutically.
In summary, we observed that the dihydropyridine
calcium antagonist nifedipine prevented the
ischemia-induced increase in endothelial cell
permeability. We ruled out the possibility that this was dependent on
L-type calcium channels or was mediated by changes in
[Ca2+]i regulation. Instead, we observed PKC
inhibition in endothelial cells by
nifedipine, specifically, the inhibition of PKC-.
Although the exact molecular mechanism of this effect is unclear, our
findings provide a rationale for the observed vascular effects of
calcium antagonists after ischemia and may also
explain the effects of calcium antagonists in other cell
types that do not express L-type calcium channels.
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Acknowledgments |
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This project was supported by a grant from the Deutsche Forschungsgemeinschaft. Dr Maasch was supported by an educational grant from Bayer Inc, Leverkusen, Germany.
Received May 22, 1998; revision received February 16, 1999; accepted February 23, 1999.
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