© 2000 American Heart Association, Inc.
Vascular Biology |
Inhibition of Type 4 Phosphodiesterase by Rolipram and Ginkgo biloba Extract (EGb 761) Decreases Agonist-Induced Rises in Internal Calcium in Human Endothelial Cells
From Pharmacologie et Physico-chimie des Interactions Cellulaires et Moléculaires (M.C.-T., C.L., K.T.), UMR CNRS 7034, Faculté de Pharmacie, Université Louis Pasteur de Strasbourg, Illkirch, and IPSEN (M.-T.D.-L.), Paris, France. Dr Campos-Toimil is currently at the Department of Pharmacology, University of Cambridge, Cambridge, UK.
Correspondence to Dr K. Takeda, Pharmacologie et Physico-chimie des Interactions Cellulaires et Moléculaires, UMR CNRS 7034, Faculté de Pharmacie, BP 24, 67401 Illkirch, France. E-mail kt{at}aspirine.u-strasbg.fr
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Abstract—The effects of Gingko biloba extract EGb 761 on 5 isolated, vascular, cyclic nucleotide phosphodiesterase (PDE) isoforms were evaluated. EGb 761 preferentially inhibited PDE4 (IC50=25.1 mg/L), the isoform that is mainly present in endothelial cells, in a competitive manner (Ki=12.5 mg/L). Because changes in cyclic nucleotide levels may affect intracellular calcium ([Ca2+]i) levels in endothelial cells, we examined the effects of EGb 761 on both resting [Ca2+]i levels and agonist-induced rises in [Ca2+]i in single human umbilical vein endothelial cells (HUVECs) in culture. The effects of EGb 761 were compared with those of rolipram, a selective PDE4 inhibitor that increases cellular cAMP levels, and the cAMP analogue dibutyryl cAMP (db-cAMP). EGb 761 (20 and 100 mg/L), rolipram (50 µmol/L), and db-cAMP (100 µmol/L) significantly inhibited histamine-, ATP-, and thrombin-induced [Ca2+]i increases in HUVECs without modifying resting [Ca2+]i levels. Similar results were obtained by using a Ca2+-free bath solution. EGb 761 (100 mg/L), but not rolipram (50 µmol/L) or db-cAMP (100 µmol/L), also inhibited Ca2+ influx into cells having thapsigargin-depleted internal Ca2+ stores and bathed in a Ca2+-free external solution. Our results are consistent with an inhibition of PDE activity that causes a reduction of agonist-induced increases in [Ca2+]i in HUVECs, mainly by inhibition of Ca2+ mobilization from internal stores. It thus may be that the cardiovascular effects of EGb 761 involve inhibition of PDE4 activity and subsequent modification of Ca2+ signaling in endothelial cells.
Key Words: Gingko biloba • calcium • phosphodiesterases • rolipram • human umbilical vein endothelial cells
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Introduction |
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EGb 761, a highly standardized extract prepared from the leaves of Ginkgo biloba L, is widely used in human therapeutics to treat peripheral arterial occlusive disease1 and cerebral insufficiency in the elderly2 as well as related neurological disorders (for a review, see Reference 3 ). Experimental evidence has shown that EGb 761 may decrease functional alterations in the brain4 5 and sensorial tissues.6 7 In the cardiovascular system, the extract has been shown to have complex and multiple effects (for a review, see Reference 8 ). It increases peripheral9 and cerebral10 blood flow and microcirculation11 and reduces capillary permeability.12 EGb 761 also improves blood rheology13 and has antithrombotic effects14 similar to those of aspirin in an experimental model of thrombosis.15
A striking observation is that EGb 761 relaxes spasmodic blood vessels and contracts those that are abnormally dilated.8 16 17 Contractions elicited by EGb 761 appear to involve a release of catecholamines.16 The vasorelaxant effect of EGb 761 seems to depend on an intact endothelium8 16 and is mediated, at least in part, by the scavenging of free radicals,18 thereby protecting NO from oxidation. However, it is unclear whether EGb 761 interferes with the generation and release of endothelial vasoactive factors. One important mechanism of action underlying the vascular effects of EGb 761, in addition to its free radical–scavenging capability, may be its inhibition of cyclic nucleotide phosphodiesterase (PDE) activity, since an inhibitory effect of EGb 761 on cAMP PDE activity has been previously described.19
It is generally accepted that important cross-talk between cyclic nucleotide levels and intracellular Ca2+ ([Ca2+]i) levels occurs in various cell types, although these interactions are controversial, because contradictory results have been reported.20 21 It is also well established that many endothelium-dependent processes that contribute to the regulation of vascular tone and blood cell activation depend on [Ca2+]i in endothelial cells. Indeed, the synthesis and release of vasoactive factors such as NO (and consequently, cGMP), prostacyclin, endothelin, or von Willebrand factor and increases in endothelial permeability are dependent on agonist-induced rises in [Ca2+]i (for a review, see Reference 22 ). Thus, substances that influence endothelial cell [Ca2+]i homeostasis potentially influence the production of endothelial factors (notably NO, because of the Ca2+ sensitivity of constitutive NO synthase in the endothelium) and underlying vascular tone.
The present study was conducted to examine whether some of the cardiovascular effects of EGb 761 might be explained by an interference of the extract with the [Ca2+]i handling in endothelial cells and whether this interference could be mediated by an inhibition of cyclic nucleotide PDE activity. We first characterized the effects of EGb 761 (at doses used in other in vitro studies5 19 ) on the hydrolytic activity of cyclic nucleotide PDE isoforms isolated from bovine aortic smooth muscle and endothelial cells, the latter of which contain mainly PDE2 and PDE4.23 We then investigated whether EGb 761 modifies resting [Ca2+]i levels and agonist-induced [Ca2+]i rises in single human umbilical vein endothelial cells (HUVECs) in culture. Specifically, we tested the actions of the extract on [Ca2+]i increases evoked by ATP, histamine, and thrombin. To correlate the PDE-inhibitory activity of EGb 761 with its possible actions on [Ca2+]i signals, we compared the effect of the extract to that of rolipram, a selective PDE4 inhibitor, which decreases ATP-induced [Ca2+]i rises in rat aortic smooth muscle cells,24 and to that of dibutyryl cAMP (db-cAMP), a membrane-permeable, nonhydrolyzable analogue of cAMP.
Our data provide insight into the cellular basis of the beneficial cardiovascular effects of EGb 761. The reduction of agonist-evoked rises in [Ca2+]i in endothelial cells could be implicated in the venotonic action or in the increases in microcirculation described for EGb 761, since this modulation of Ca2+ signaling can lead to a decrease in the liberation of endothelium-derived relaxing factors. Furthermore, the inhibitory effects of EGb 761 on cyclic nucleotide PDE activity may lead to different actions in other vascular layers containing different amounts of PDE isoforms and having a different pattern of interaction between cyclic nucleotides and [Ca2+]i levels. A preliminary account of some of these results has been published in abstract form.25
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Methods |
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Studies on the Hydrolytic Activity of PDE Isoforms
Cytosolic PDE isoforms (PDE1, PDE3, PDE4, and PDE5) were purified by anion-exchange chromatography from the medial layer of bovine aorta by a modification26 of a previously described method.27 Cytosolic PDE2 was isolated from cultured bovine aortic endothelial cells.23
PDE activities were measured by radioenzymatic assay28 at a substrate concentration of 1 µmol/L cAMP or cGMP in the presence of 15 000 counts per minute of [3H]cAMP or [3H]cGMP as a tracer, respectively. The buffer solution contained the following components (in mmol/L): 48 Tris HCl, 2 magnesium acetate, 1 g/L bovine serum albumin, and 1 EGTA, pH 7.5. PDE1 was assayed in the basal (in the presence of 1 mmol/L EGTA) or calmodulin-activated (with 10 µmol/L CaCl2 and 18 nmol/L calmodulin instead of 1 mmol/L EGTA) states by using [3H]cGMP as a substrate. PDE2 was assayed in the absence (basal state) or presence of 5 µmol/L cGMP (activated state) by using [3H]cAMP as a substrate. To prevent cross-contamination between isolated PDE3 and PDE4, studies of these isoforms with the use of [3H]cAMP as a substrate were always carried out in the presence of 10 µmol/L rolipram or 100 µmol/L cGMP, respectively. EGb 761 dose-effect curves on PDE activity were made with 6 concentrations of the extract.
Cell Culture
Fragments of human umbilical veins were opened under sterile
conditions, and their endothelial cells (HUVECs) were
gently isolated by using a scalpel blade and then cultured as described
previously.29 The culture medium was medium 199/RPMI-1640
(1/1, vol/vol) containing 10 mmol/L HEPES, 2 mmol/L
L-glutamine, antibiotics (105 U/L
penicillin and 100 mg/L streptomycin), 0.25 mg/L amphotericin B, and
20% human serum (vol/vol). HUVECs were frozen in
LN2 at the second passage and used in experiments
from the third to the fifth passage. We verified that this number of
passages did not alter cell responses. The cells were subcultured in
75-cm2 flasks and, for experiments, in 35-mm
Petri dishes into which a 20-mm-diameter hole had been cut in the base
and replaced by a thin (0.07-mm) glass coverslip. All plates were first
treated with polylysine (0.5 g/L), sterilized by UV light, and then
incubated with type I collagen (0.06 g/L) from rat tail at 4°C
overnight. Cells were seeded at low density
(2x103 cells/cm2) and kept
in culture (37°C, 5% CO2 in air) for 2 to 4
days before the experiments.
Measurement of [Ca2+]i
[Ca2+]i levels
were determined as described previously.30 In brief, cells
(plated on Petri dishes as described above) were incubated for 30
minutes at 37°C in a normal bath solution (composition in
mmol/L: NaCl 140, KCl 5, CaCl2 2,
MgCl2 2, HEPES 10, and glucose 11, pH 7.4)
containing 5 µmol/L fura 2-acetoxymethyl ester (fura
2-AM), washed 3 times with a normal bath solution, and then
allowed to rest for 30 minutes in the incubator. Cells were placed on
an inverted microscope (Nikon Diaphot) and continuously superfused at
0.5 mL/min with either a normal bath solution or a
Ca2+-free solution (no added
CaCl2 and 1 mmol/L EGTA). Measurements were
made at room temperature on isolated cells or small groups of dispersed
cells viewed with a 40x oil-immersion objective (u.v.-Fluor, numerical
aperture 1.3; Nikon) with the use of a digital imaging system. Fura 2
was excited alternately at 340±10 and 380±10 nm with a 100-W mercury
lamp (HBO, Osram), with emitted fluorescence at 510±20 nm
measured by an intensified CCD camera (Darkstar-800, Photonic
Sciences).
Ratiometric Ca2+ images were generated at 2-second intervals by using 4 averaged images at each wavelength. Images were digitally stored and analyzed with software from IMSTAR. For each cell, [Ca2+]i was averaged from pixels within manually outlined cell areas. The [Ca2+]i was calculated, after background subtraction, as reported elsewhere,30 31 by using in situ–determined values for the limiting ratios of saturating (10 mmol/L Ca2+ in the bath) and minimal (0 mmol/L Ca2+ and 10 mmol/L EGTA in the bath) [Ca2+]i both in the presence of 10 µmol/L ionomycin. Drugs were locally applied by pressure ejection from fine-tipped glass pipettes (2-µm internal diameter) placed 200 to 300 µm from the target cells. In preliminary experiments, strong desensitization for repeated agonist application to individual cells was found; thus, different cells were used as controls and treated cells. Puffer application of external solution alone was without effect.
Drugs and Chemicals
Cell culture media (medium 199 and RPMI-1640), HEPES,
L-glutamine, penicillin, streptomycin, amphotericin B, and
trypsin-EDTA solution were from Gibco; endotoxin-free human
serum was from Etablissement de Transfusion Sanguine (Strasbourg,
France). The serum was obtained from a pool of 13 to 15 healthy donors
negative for hepatitis B virus and HIV, and it was
complement-inactivated at 56°C for 30 minutes.
ATP, histamine dihydrochloride, db-cAMP, poly-L-lysine
hydrobromide, and thapsigargin were from Sigma Chemical Co; ionomycin
free acid from Calbiochem; fura 2-AM from Molecular Probes;
and [3H]cAMP and
[3H]cGMP from Amersham. Human 
-thrombin
(3x106 U/L) was a kind gift of Dr J.-M.
Freyssinet (INSERM U143, Institut de Hematologie, Université
Louis Pasteur Strasbourg, France); rolipram was a kind gift from
Schering AG (Berlin, Germany). EGb 761 was from IPSEN, and
calmodulin was purified as previously
described.32 All other reagents were of analytical grade
(Merck).
For calcium-imaging experiments, all drugs were dissolved in deionized water, except fura 2-AM, ionomycin, and rolipram, which were dissolved in dimethyl sulfoxide (DMSO). The final concentration of DMSO never exceeded 0.2%. Control groups received treatment with the solvent alone. For studies on PDE activity, all compounds were dissolved in DMSO, and the final concentration of DMSO in the assay (1%) did not affect PDE activity.
Data Analysis
Results are expressed as mean±SEM. Significant differences
between means (P<0.05) were determined by Student’s
t test for unpaired data. The area under the
Ca2+ curves from individual cells
(Figure 
6)
was determined by the trapezoid rule
(Prism 2 software, Graphpad). The IC50 values of
EGb 761 against the different PDE isoforms were calculated by nonlinear
regression. Apparent inhibitory constant
(Ki) values for EGb 761 were determined by
using substrate concentrations ranging from 0.1 to 20 µmol/L.
Lineweaver-Burk plots were analyzed by least-squares linear
regression analysis.
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Effects of EGb 761 on Vascular Cyclic Nucleotide PDE Isoforms
EGb 761 preferentially inhibited PDE4 with an IC50 value of 25 mg/L (with a 95% confidence interval of [10.0]) as seen in Figure 1a
and, to a lesser extent,
PDE5 (IC50=47.3 [25.2] mg/L; the
Table ), with negligible effects on PDE1 and PDE2 in
their basal states (IC50 >200 mg/L; the
Table ). However, EGb 761 significantly inhibited the
Ca2+-calmodulin–activated
state of PDE1, indicating a possible interaction with
calmodulin (the Table). Moreover, PDE2 was more
sensitive to EGb 761 in its cGMP-stimulated state (Figure 1a).
Kinetic studies showed that EGb 761 competitively inhibited PDE4, PDE5,
and activated PDE2, with Ki values
of 12.5±1.1 mg/L (Figure 1b), 14.0±1.6 mg/L (Figure 1c ), and 62.7±5.9 mg/L (Figure 1d), respectively. The
apparent Km values for PDE4, PDE5, and
activated PDE2 were 0.89±0.07 µmol/L cAMP,
0.44±0.03 µmol/L cGMP, and 14.28±0.12 µmol/L cAMP,
respectively.
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Effects of EGb 761, Rolipram, and db-cAMP on Basal
[Ca2+]i Levels
The mean basal
[Ca2+]i level in HUVECs
was 79.9±2.3 nmol/L (n=72) and was unchanged over the experimental
time course. Resting
[Ca2+]i levels were not
significantly affected by a 2-minute preincubation with EGb 761 (1 to
100 mg/L) rolipram (10 and 50 µmol/L), or db-cAMP (100
µmol/L).
Effects of EGb 761, Rolipram, and db-cAMP on Agonist-Induced
[Ca2+]i Rises
A 60-second application of histamine (10 µmol/L; Figure 2)
or ATP (10 µmol/L) caused a biphasic increase in
[Ca2+]i, which returned
to near the resting level within 120 to 140 seconds. A similar
application of thrombin (5000 U/L) caused a biphasic increase in
[Ca2+]i, which returned
to near the resting level within 180 to 200 seconds. The percentage of
responsive cells in a given culture did not vary greatly and was, on
average, 65% to 70% for ATP and 90% to 95% for histamine and
thrombin when responses from many preparations were considered.
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As illustrated in Figures 3b through 3d, a 2-minute
preincubation of cells with 100 mg/L EGb 761 significantly reduced
agonist-induced [Ca2+]i
increases evoked by 10 µmol/L histamine, 10 µmol/L ATP,
and 5000 U/L thrombin compared with untreated cells. This effect was
also observed for 20 mg/L EGb 761 (Figure 6
). Lower doses of
EGb 761 were without effect, as shown in the concentration-effect curve
made with 6 different concentrations of the extract (Figure 3a).
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The effects of EGb 761 against agonist-induced increases in
[Ca2+]i were mimicked by
a 2-minute preincubation of cells with 50 µmol/L rolipram, a
selective PDE4 inhibitor (Figures 4a through IVc
and VI ), but not by 10 µmol/L rolipram (not shown).
Similarly, a 2-minute preincubation with 100 µmol/L db-cAMP also
significantly reduced the
[Ca2+]i rise induced by
histamine (Figures 4d and 6a).
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Effects of EGb 761, Rolipram, and db-cAMP on Agonist-Induced
[Ca2+]i Rises in Ca2+-Free
Solution
In a Ca2+-free external solution, ATP
(10 µmol/L), histamine (10 µmol/L), or thrombin (5000
U/L) caused an initial
[Ca2+]i elevation similar
to that observed in the presence of 2 mmol/L
Ca2+, but with a much reduced second plateau
phase (Figures 5e through Ve). Again, a 2-minute preincubation
with EGb 761 (20 or 100 mg/L) under these conditions significantly
reduced both peak Ca2+ responses and the areas
under the curves to the 3 agonists (Figures 5a through 5c
and 6). In a similar fashion, rolipram (50 µmol/L,
Figure 5d) and db-cAMP (100 µmol/L, Figure 5e)
also reduced histamine-induced
[Ca2+]i signals from
cells bathed in a Ca2+-free external solution
(also see Figure 6).
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Effect of EGb 761 on the Ca2+ Influx Evoked by
Depletion of [Ca2+]i Stores
In the absence of extracellular Ca2+ and
after treatment of cells with 5 µmol/L thapsigargin for 10
minutes, 10 µmol/L histamine–, 10 µmol/L ATP–, and 5000
U/L thrombin–induced
[Ca2+]i mobilization was
completely abolished (not shown). Under these conditions, a 60-second
application of 2 mmol/L Ca2+–containing
external solution induced an increase in
[Ca2+]i (Figure
5f), which was observed in only 25% of the cells tested. This
Ca2+ entry was significantly reduced by a
2-minute preincubation with 100 mg/L EGb 761 (Figure 5f),
whereas lower doses of EGb 761 (1 or 20 mg/L), rolipram (10 or 50
µmol/L), or db-cAMP (100 µmol/L) were all without effect (not
shown).
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Our results show that a short (2-minute) preincubation of HUVECs with EGb 761 significantly decreases ATP-, histamine-, and thrombin-induced [Ca2+]i rises without modifying resting [Ca2+]i levels, in both the presence and absence of external Ca2+. Agonist-induced biphasic increases in [Ca2+]i in endothelial cells result from an initial transient elevation via an IP3-mediated release of Ca2+ from internal stores, followed by a slow, sustained plateau phase due to a Ca2+ influx via capacitative Ca2+ entry and/or receptor-operated, voltage-independent, nonspecific cationic channels.33 34 In the current investigation, agonist-induced [Ca2+]i rises in HUVECs were largely accounted for by mobilization from thapsigargin-sensitive internal stores and, in a minor way, from Ca2+ influx, because removal of extracellular Ca2+ did not affect the maximal [Ca2+]i achieved and the measured capacitative entry of Ca2+ was relatively weak. Similar findings in HUVECs have been previously reported.21 35
An inhibitory effect of EGb 761 on cAMP PDE activity in crude cytosolic and particulate fractions of rat brain was previously described.19 Among the vascular PDE isoforms tested herein, we found that EGb 761 preferentially inhibited PDE4 in a competitive manner (Ki=12.5±1.1 mg/L). Because vascular endothelial cells contain mostly PDE2 and PDE4,23 36 the inhibitory effects of EGb 761 on the agonist-induced [Ca2+]i mobilization currently observed might have been associated with PDE inhibition and subsequently increased cAMP levels. The mechanism involved may be similar to that proposed for human platelets after inhibition of PDE337 or for smooth muscle cells after inhibition of PDE4.24 This hypothesis is supported by the following considerations: (1) the cAMP-elevating agents rolipram and db-cAMP also inhibit agonist-induced [Ca2+]i rises in HUVECs (see below); (2) EGb 761 inhibits increases in both [Ca2+]i and PDE activity at the same range of concentrations; and (3) EGb 7618 16 and PDE4 inhibitors26 are endothelium-dependent relaxing agents.
However, it should be noted that the effect of elevating cAMP on [Ca2+]i in endothelial cells is presently somewhat controversial (for a review, see Reference 38 ). It was reported that a 2-minute preincubation of HUVECs with forskolin (an adenylyl cyclase stimulator) reduced histamine-induced [Ca2+]i increases without affecting resting levels.20 Similarly, ATP-induced [Ca2+]i rises in bovine aortic endothelial cells were inhibited by 100 µmol/L db-cAMP.39 On the other hand, in HUVECs, nonselective PDE blockade by isobutyl methylxanthine failed to alter ATP-evoked [Ca2+]i responses,21 in accord with the lack of effect of theophylline (another nonselective PDE inhibitor), forskolin, and 8-bromo-cAMP on histamine-induced [Ca2+]i increases.40 Interestingly, protocols aimed at elevating cAMP levels significantly reduce ATP-evoked rises in mitochondrial Ca2+, but not [Ca2+]i, in ECV304 cells, a HUVEC-derived cell line.41 Nevertheless, a potentiating effect on agonist-induced [Ca2+]i mobilization was reported in bovine aortic endothelial cells, but only when the cAMP-elevating agents were added during the plateau phase of thrombin-, bradykinin-, or ATP-induced [Ca2+]i responses.42 Contrary to our results, it has been reported that cAMP-elevating agents increase bradykinin-induced NO formation by enhancing the agonist-induced rise in [Ca2+]i in pig aortic endothelial cells,43 probably by facilitating the activation of Ca2+-sensitive K+ channels and thereby promoting membrane hyperpolarization.44 The different cardiovascular effects provoked by EGb 761 in vivo may be explained in part by these sorts of differences for the cross-talk between cAMP and [Ca2+]i at different regions of the vascular tree.
We found that the effects of rolipram and db-cAMP on agonist-induced [Ca2+]i increases were generally similar to those of EGb 761. This supports the hypothesis that elevated cAMP levels interfere with Ca2+ mobilization and that the inhibitory effects of rolipram and EGb 761 on agonist-induced [Ca2+]i increases are both linked to an inhibition of PDE4, 1 of the major PDE isoforms present in vascular endothelial cells.23 36
These discrepancies observed for endothelial [Ca2+]i regulation by cAMP might possibly be explained by differences in the degree of cAMP accumulation and functional compartmentalization of the cAMP cascade, thereby allowing different physiological responses to various cAMP-increasing agents, as proposed for vascular smooth muscle.24 Indeed, the high cAMP hydrolytic activity of PDE2 can overcome global increases in cAMP due to local inhibition of PDE4.23 Thus, standard assays of cAMP do not reveal local increases in cAMP induced by PDE4 inhibition specifically implicated in physiological responses.36 45 At least 1 effect of EGb 761 (at 100 mg/L, close to the IC50 against PDE2) in reducing the Ca2+ influx evoked by the depletion of [Ca2+]i stores was not shared by rolipram or db-cAMP. This suggests that inhibition of other PDE isoforms, such as activated PDE2,23 36 may also be involved in the modulation of regulating [Ca2+]i homeostasis by EGb 761 in endothelial cells.
In conclusion, EGb 761 significantly reduced agonist-induced [Ca2+]i increases in HUVECs, most probably via inhibition of Ca2+ mobilization from internal stores. Given the measured inhibitory action of EGb 761 on PDE4 activity and the similar effects of rolipram and db-cAMP, this effect likely involves an elevation of cAMP levels. Our data indicate that the complex cardiovascular effects of EGb 761 involve, at least in part, an inhibition of PDE4 activity and a subsequent modification of calcium signaling in endothelial cells.
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Acknowledgments |
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M. Campos-Toimil was a recipient of a Fellowship from the Formación de Personal Investigador program of the Ministerio de Educación y Cultura, Spain. We thank M. Fischer and A. Le Bec for skillful technical assistance, G. Haan-Archipoff for help in setting up the HUVEC cultures, and J.-M. Freyssinet and Schering AG for providing thrombin and rolipram, respectively.
Received July 27, 1999; accepted February 14, 2000.
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