This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Campos-Toimil, M. Articles by Takeda, K. Search for Related Content PubMed PubMed Citation Articles by Campos-Toimil, M. Articles by Takeda, K. Pubmed/NCBI databases Compound via MeSH Substance via MeSH Hazardous Substances DB CALCIUM COMPOUNDS CALCIUM, ELEMENTAL Related Collections Receptor pharmacology Endothelium/vascular type/nitric oxide Cell signalling/signal transduction Imaging

(Arteriosclerosis, Thrombosis, and Vascular Biology. 2000;20:e34.)
© 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

Manuel Campos-Toimil; Claire Lugnier; Marie-Thérèse Droy-Lefaix; Kenneth Takeda

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

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
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 (K_i=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

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
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 brain^{4 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 endothelium^{8 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 studies^{5 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

	Methods

Top Abstract Introduction Methods Results Discussion References

 
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 (K_i) 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.

View larger version (36K): [in this window] [in a new window]   Figure 6. Effects of EGb 761, rolipram, and db-cAMP on (a) 10 µmol/L histamine–, (b) 10 µmol/L ATP–, and (c) 5000 U/L thrombin–induced increases in [Ca2+]i in HUVECs bathed in normal and Ca2+-free external solutions, assessed by using the surface area under the Ca2+ response curves from individual cells. Each value is mean±SEM of at least 10 cells. *P<0.05 with respect to control values.

	Results

Top Abstract Introduction Methods Results Discussion References

 
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 K_i 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 K_m 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.

View larger version (29K): [in this window] [in a new window]   Figure 1. Inhibitory effects of EGb 761 on isolated vascular PDE isoforms. a, Dose-effect curves for PDE4 and PDE2 in their basal and activated states. Lineweaver-Burk representation of the inhibitory effects of EGb 761 against PDE4 (b), PDE5 (c), and activated PDE2 (d). Data are representative of 3 experiments.

View this table: [in this window] [in a new window]   Table 1. Effects of EGb 761 on the Hydrolytic Activity of Isolated Vascular Phosphodiesterases (PDEs)

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.

View larger version (24K): [in this window] [in a new window]   Figure 2. Histamine-induced [Ca2+]i rise in a single HUVEC loaded with fura-2. [Ca2+]i was measured with a digital image analysis system.

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).

View larger version (34K): [in this window] [in a new window]   Figure 3. a, Effects of 2-minute preincubation of HUVECs with different concentrations of EGb 761 on the maximal [Ca2+]i increase induced by histamine. Effects of 2-minute preincubation of HUVECs with EGb 761 (100 mg/L) on [Ca2+]i increases induced by histamine (b), ATP (c), and thrombin (d) in a 2 mmol/L Ca2+–containing external solution. Each curve represents mean±SEM of at least 10 individual cells. *P<0.05 with respect to control values.

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).

View larger version (34K): [in this window] [in a new window]   Figure 4. The selective PDE4 inhibitor rolipram (50 µmol/L) reduces increases in [Ca2+]i evoked by histamine (a), ATP (b), and thrombin (c) in a 2 mmol/L Ca2+–containing external solution. d, Inhibitory effect of 100 µmol/L db-cAMP on histamine-induced increase in [Ca2+]i. Each curve represents mean±SEM of at least 14 individual cells. *P<0.05 with respect to control values.

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).

View larger version (33K): [in this window] [in a new window]   Figure 5. Effects of 2-minute preincubation with EGb 761 (100 mg/L) on [Ca2+]i increases in HUVECs induced by histamine (a), ATP (b), and thrombin (c) in a Ca2+-free external solution. Inhibitory effects of 50 µmol/L rolipram (d) and 100 µmol/L db-cAMP (e) on histamine-induced increases in [Ca2+]i in a Ca2+-free external solution. Ca2+ entry provoked by application of 2 mmol/L Ca2+–containing external solution is inhibited by 100 mg/L EGb 761 (f) in thapsigargin-pretreated HUVECs bathed in a Ca2+-free external solution. Each curve represents mean±SEM of at least 11 individual cells. *P<0.05 with respect to control values.

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).

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
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 (K_i=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 761^{8 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.

	Acknowledgments

 
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.

	References

Top Abstract Introduction Methods Results Discussion References

 

1. Kleijnen J, Knipschild P. Ginkgo biloba. Lancet. 1992;340:1136–1139.
2. Kleijnen J, Knipschild P. Ginkgo biloba for cerebral insufficiency. Br J Clin Pharmacol. 1992;34:352–358.[Medline] [Order article via Infotrieve]
3. DeFeudis FV, ed. Ginkgo biloba Extract (EGb 761): Pharmacological Activities and Clinical Applications. Paris, France: Elsevier; 1991:1–181.
4. Barkats M, Venault P, Christen Y, Cohen-Salmon C. Effect of long-term treatment with EGb 761 on age-dependent structural changes in the hippocampi of three inbred mouse strains. Life Sci. 1995;56:213–222.[Medline] [Order article via Infotrieve]
5. Ahlemeyer B, Möwes A, Krieglstein J. Inhibition of serum deprivation- and staurosporine-induced neuronal apoptosis by Ginkgo biloba extract and some of its constituents. Eur J Pharmacol. 1999;367:423–430.[Medline] [Order article via Infotrieve]
6. Szabo ME, Droy-Lefaix MT, Doly M. EGb 761 and the recovery of ion imbalance in ischemic reperfused diabetic rat retina. Ophthalmic Res. 1995;27:102–109.[Medline] [Order article via Infotrieve]
7. Jastreboff PJ, Zhou S, Jastreboff MM, Kwapisz U, Gryczynska U. Attenuation of salicylate-induced tinnitus by Ginkgo biloba extract in rats. Audiol Neurootol. 1997;2:197–212.[Medline] [Order article via Infotrieve]
8. DeFeudis FV. Overview of the actions of Ginkgo biloba extract (EGb 761) on the cardiovascular system: from basic pharmacology to the clinic. In: Clostre F, DeFeudis FV, eds. Advances in Ginkgo biloba Extract Research, Volume 3: Cardiovascular Effects of Ginkgo biloba Extract (EGb 761). Paris, France: Elsevier; 1994:105–119.
9. Mouren X, Caillard P, Schwartz F. Study of the anti-ischemic action of EGb 761 in the treatment of peripheral arterial occlusive disease by TcPO2 determination. Angiology. 1994;45:413–417.
10. Krieglstein J, Beck T, Seibert A. Influence of an extract of Ginkgo biloba on cerebral blood flow and metabolism. Life Sci. 1986;39:2327–2334.[Medline] [Order article via Infotrieve]
11. Stucker O, Pons C, Duverger JP, Drieu K, D’Arbigny P. Effect of Ginkgo biloba extract (EGb 761) on the vasospastic response of mouse cutaneous arterioles to platelet activation. Int J Microcirc Clin Exp. 1997;17:61–66.[Medline] [Order article via Infotrieve]
12. Lagrue G, Behar A, Kazandjian M, Rahbar K. Idiopathic cyclic oedema: role of capillary hyperpermeability and its correction by Ginkgo biloba extract. Presse Med. 1986;15:1550–1553.
13. Witte S, Anadere I, Walitza E. Improvement of hemorheology with Ginkgo biloba extract: decreasing a cardiovascular risk factor. Fortschr Med. 1992;110:247–250.[Medline] [Order article via Infotrieve]
14. Kim YS, Pyo MK, Park KM, Park PH, Hahn BS, Wu SJ, Yun-Choi HS. Antiplatelet and antithrombotic effects of a combination of ticlopidine and Ginkgo biloba extract (EGb 761). Thromb Res. 1998;91:33–38.[Medline] [Order article via Infotrieve]
15. Belougne E, Aguejouf O, Imbault P, Azougagh-Oualane F, Doutremepuich F, Droy-Lefaix MT, Doutremepuich C. Experimental thrombosis model induced by laser beam: application of aspirin and an extract of Ginkgo biloba: EGb 761. Thromb Res. 1996;82:453–458.[Medline] [Order article via Infotrieve]
16. Auguet M, Delaflotte S, Chabrier PE, Braquet P. Ginkgo biloba extract (EGb 761) and the regulation of vascular tone. In: Clostre F, DeFeudis FV, eds. Advances in Ginkgo biloba Extract Research, Volume 3: Cardiovascular Effects of Ginkgo biloba Extract (EGb 761). Paris, France: Elsevier; 1994:19–26.
17. Lee K, Ku J, Koh S, Kim K. Effects of methanol extract of Ginkgo biloba (EGb), its ethyl acetate fraction (EAF) and butanol fraction (BF) on the isolated aorta. Jpn J Pharmacol. 1992;58:377P. Abstract.
18. Maitra I, Marcocci L, Droy-Lefaix MT, Packer L. Peroxyl radical scavenging activity of Ginkgo biloba extract EGb 761. Biochem Pharmacol. 1995;49:1649–1655.[Medline] [Order article via Infotrieve]
19. Macovschi O, Prigent AF, Nemoz G, Pacheco H. Effects of an extract of Ginkgo biloba on the 3'-5'-cyclic AMP phosphodiesterase activity of the brain of normal and triethyltin-intoxicated rats. J Neurochem. 1987;49:107–114.[Medline] [Order article via Infotrieve]
20. Bolz SS, Pohl U. Indomethacin enhances endothelial NO release: evidence for a role of PGI₂ in the autocrine control of calcium-dependent autacoid production. Cardiovasc Res. 1997;36:437–444.[Abstract/Free Full Text]
21. Vischer UM, Wollheim CB. Purine nucleotides induce regulated secretion of von Willebrand factor: involvement of cytosolic Ca²⁺ and cyclic adenosine monophosphate-dependent signaling in endothelial exocytosis. Blood. 1998;91:118–127.[Abstract/Free Full Text]
22. Furchgott RF. The role of the endothelium in the responses of vascular smooth muscle to drugs. Annu Rev Pharmacol Toxciol. 1997;24:175–197.
23. Lugnier C, Schini VB. Characterization of cyclic nucleotide phosphodiesterases from cultured bovine aortic endothelial cells. Biochem Pharmacol. 1990;39:75–84.[Medline] [Order article via Infotrieve]
24. Eckly-Michel A, Martin V, Lugnier C. Involvement of cyclic nucleotide-dependent protein kinases in cyclic AMP-mediated vasorelaxation. Br J Pharmacol. 1997;122:158–164.[Medline] [Order article via Infotrieve]
25. Campos-Toimil M, Lugnier C, Takeda K. Agonist-induced increases in internal calcium in single human endothelial cells are inhibited by rolipram and extract from Ginkgo biloba (EGb 761). Pharmacol Toxicol. 1998;83(suppl 1):106. Abstract.
26. Komas N, Lugnier C, Stoclet JC. Endothelium-dependent and independent relaxation of the rat aorta by cyclic nucleotide phosphodiesterase inhibitors. Br J Pharmacol. 1991;104:495–503.[Medline] [Order article via Infotrieve]
27. Lugnier C, Schoeffter P, Le Bec A, Strouthou E, Stoclet JC. Selective inhibition of cyclic nucleotide phosphodiesterases of human, bovine and rat aorta. Biochem Pharmacol. 1986;35:1743–1751.[Medline] [Order article via Infotrieve]
28. Keravis TM, Wells JN, Hardman JG. Cyclic nucleotide phosphodiesterase activities from pig coronary arteries: lack of interconvertibility of major forms. Biochim Biophys Acta. 1980;613:116–129.[Medline] [Order article via Infotrieve]
29. Klein-Soyer C, Beretz A, Millon-Collard R, Abecassis J, Cazenave JP. A simple in vitro model of mechanical injury of confluent cultured endothelial cells to study quantitatively the repair process. Thromb Haemost. 1986;56:232–235.[Medline] [Order article via Infotrieve]
30. Lynch JW, Soares Lemos V, Bucher B, Stoclet JC, Takeda K. A pertussis toxin-insensitive calcium influx mediated by neuropeptide Y₂ receptors in a human neuroblastoma cell line. J Biol Chem. 1994;269:8226–8233.[Abstract/Free Full Text]
31. Grynkiewicz G, Poenie M, Tsien RY. A new generation of Ca²⁺ indicators with greatly improved fluorescence properties. J Biol Chem. 1985;260:3440–3450.[Abstract/Free Full Text]
32. Follenius A, Gerard D. Fluorescence investigations of calmodulin hydrophobic sites. Biochem Biophys Res Commun. 1984;119:1154–1160.[Medline] [Order article via Infotrieve]
33. Bregestovski P, Bakhramov A, Danilov S, Moldobaeva A, Takeda K. Histamine-induced inward currents in cultured endothelial cells from human umbilical vein. Br J Pharmacol. 1988;95:429–436.[Medline] [Order article via Infotrieve]
34. Von der Weid PY, Serebryakov VN, Orallo F, Bergmann C, Snetkov VA, Takeda K. Effects of ATP on cultured smooth muscle cells from rat aorta. Br J Pharmacol. 1993;108:638–645.[Medline] [Order article via Infotrieve]
35. Oike M, Droogmans G, Nilius B. Amplitude modulation of Ca²⁺ signals induced by histamine in human endothelial cells. Biochim Biophys Acta. 1994;1222:287–291.[Medline] [Order article via Infotrieve]
36. Souness JE, Diocee BK, Martin W, Moodie SA. Pig aortic endothelial-cell cyclic nucleotide phosphodiesterases. Biochem J. 1990;266:127–132.[Medline] [Order article via Infotrieve]
37. Lanza F, Beretz A, Stierle A, Corre G, Cazenave JP. Cyclic nucleotide phosphodiesterase inhibitors prevent aggregation of human platelets by raising cyclic AMP and reducing cytoplasmic free calcium mobilization. Thromb Res. 1987;45:477–484.[Medline] [Order article via Infotrieve]
38. Moore TM, Chetham PM, Kelly JJ, Stevens T. Signal transduction and regulation of lung endothelial cell permeability: interaction between calcium and cAMP. Am J Physiol. 1998;275:L203–L222.[Abstract/Free Full Text]
39. Lückhoff A, Mülsch A, Busse R. cAMP attenuates autacoid release from endothelial cells: relation to internal calcium. Am J Physiol. 1990;258:H960–H966.[Abstract/Free Full Text]
40. Carson MR, Shasby SS, Shasby DM. Histamine and inositol phosphate accumulation in endothelium: cAMP and a G protein. Am J Physiol. 1989;257:L259–L264.[Abstract/Free Full Text]
41. Lawrie AM, Zolle O, Simpson AWM. Modulation of mitochondrial Ca²⁺ in ECV304 endothelial cells by agents which elevate cAMP. Cell Calcium. 1997;22:229–234.[Medline] [Order article via Infotrieve]
42. Buchan KW, Martin W. Modulation of agonist-induced calcium mobilisation in bovine aortic endothelial cells by phorbol myristate acetate and cyclic AMP but not cyclic GMP. Br J Pharmacol. 1991;104:361–366.[Medline] [Order article via Infotrieve]
43. Graier WF, Groschner K, Schmidt K, Kukovetz WR. Increases in endothelial cyclic AMP levels amplify agonist-induced formation of endothelium-derived relaxing factor (EDRF). Biochem J. 1992;288:345–349.
44. Graier WF, Kukovetz WR, Groschner K. Cyclic AMP enhances agonist-induced Ca²⁺ entry into endothelial cells by activation of potassium channels and membrane hyperpolarization. Biochem J. 1993;291:263–267.
45. Kessler T, Lugnier C. Rolipram increases cyclic GMP content in L-arginine-treated cultured bovine aortic endothelial cells. Eur J Pharmacol. 1995;290:163–167[Medline] [Order article via Infotrieve]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Campos-Toimil, M. Articles by Takeda, K. Search for Related Content PubMed PubMed Citation Articles by Campos-Toimil, M. Articles by Takeda, K. Pubmed/NCBI databases Compound via MeSH Substance via MeSH Hazardous Substances DB CALCIUM COMPOUNDS CALCIUM, ELEMENTAL Related Collections Receptor pharmacology Endothelium/vascular type/nitric oxide Cell signalling/signal transduction Imaging