2-(2,4-dihydroxyphenyl)-5-(E)-propenylbenzofuran promotes endothelial nitric oxide synthase activity in human endothelial cells.

Abstract

Endothelial nitric oxide synthase (eNOS) mediates important vaso-protective and immunomodulatory effects. Aim of this study was to examine whether lignan derivatives isolated from the roots of the anti-inflammatory medicinal plant Krameria lappacea influence eNOS activity and endothelial nitric oxide (NO) release. The study was performed using cultured human umbilical vein endothelial cells (HUVECs) and HUVEC-derived EA.hy926 cells. Among the eleven isolated compounds only 2-(2,4-dihydroxyphenyl)-5-(E)-propenylbenzofuran (DPPB) was able to increase eNOS enzyme activity. DPPB (1-10 μM) treatment for 24 h induced a significant and dose-dependent increase in eNOS activity as determined by the [(14)C]L-arginine/[(14)C]L-citrulline conversion assay. Immunoblotting studies further revealed a time-dependent DPPB-induced increase in eNOS-Ser(1177) and decrease in eNOS-Thr(495) phosphorylation, as well as increased AMPK phosphorylation at Thr(172), whereas Akt phosphorylation at Ser(473) was not affected. Si-RNA-mediated knockdown of AMPK and inhibition of CaMKKβ by STO 609, as well as intracellular Ca(2+) chelation by Bapta AM abolished the stimulating effect of DPPB on eNOS-Ser(1177) and AMPK-Thr(172) phosphorylation. Furthermore, we could show that DPPB increases intracellular Ca(2+) concentrations assessed with the fluorescent dye Fluo-3-AM. DPPB enhances eNOS activity and endothelial NO release by raising intracellular Ca(2+) levels and increases signaling through a CaMKKβ-AMPK dependent pathway.

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2-(2,4-dihydroxyphenyl)-5-(E)-propenylbenzofuran promotes endothelial
nitric oxide synthase activity in human endothelial cells
Angela Ladurnera, Atanas G. Atanasova, Elke H. Heissa, Lisa Baumgartnerb, Stefan Schwaigerb,
Judith M. Rollingerb, Hermann Stuppnerb, Verena M. Dirscha,*
aDepartment of Pharmacognosy, University of Vienna, Althanstrasse 14, 1090 Vienna, Austria
bInstitute of Pharmacy/Pharmacognosy and Center for Molecular Biosciences Innsbruck, University of Innsbruck, CCB-Center, Innrain 80/82, A-6020 Innsbruck, Austria
1. Introduction
The endothelium is responsible for the maintenance of vascular
homeostasis thereby regulating vascular tone, thrombosis, and
smooth muscle cell proliferation and migration [1,2]. In a diseased
state this balance is disturbed and the endothelium is in a
dysfunctional state. Endothelial dysfunction is a key event in the
development of atherosclerosis and can therefore be regarded as
an early marker for cardiovascular disease [3]. Hallmark of a
dysfunctional endothelium is an impaired action of the enzyme
endothelial nitric oxide synthase (eNOS) [4]. The reaction
catalyzed by eNOS leads to the conversion of molecular oxygen
and L-arginine to L-citrulline and nitric oxide (NO). NO is a major
vasoprotective mediator released by the endothelium, with
multiple effects including next to vasodilatation also inhibition
of platelet aggregation and leukocyte adhesion as well as control of
smooth muscle cell proliferation. This overall leads to an anti-
inflammatory and immunomodulatory action of NO [5–8].
Besides different cofactors (e.g. NADPH, FMN, FAD, BH4), adaptor
and regulatory proteins, eNOS requires Ca2+for sustained action.
When the intracellular Ca2+concentration ([Ca2+]i) reaches a certain
level, eNOS binds to the regulatory protein Ca2+/calmodulin (CaM),
thereby leading to an increased rate of electron transfer from NADPH
bound at the reductase domain of the enzyme to the heme center at
the N-terminal oxygenase domain, activating the enzyme [9].
Extracellular ligands of G-protein coupled receptors, such as
acetylcholine, bradykinin, adenosine, thrombin or histamine,
rapidly increase [Ca2+]i and activate eNOS via this mechanism
[10–12]. ENOS activation, however, can also be induced by stimuli
that act independently of a [Ca2+]iincrease like shear stress induced
by blood flow [13]. Regulation of eNOS further involves modulation
of eNOS protein expression and post-translational modification of
the protein such as phosphorylation [14]. Among the most
important kinases directly activating eNOS by phosphorylation
are Akt and AMP-activated protein kinase (AMPK) [15].
There is an increasing body of evidence that some natural
products contained in dietary sources or in specific medicinal
plants may positively influence endothelial NO production and
Biochemical Pharmacology 84 (2012) 804–812
A R T I C L E
I N F O
Article history:
Received 10 May 2012
Accepted 28 June 2012
Available online 6 July 2012
Keywords:
Endothelial nitric oxide synthase (eNOS)
Ca2+
Benzofuran derivative
AMPK
CaMKKb
A B S T R A C T
Endothelial nitric oxide synthase (eNOS) mediates important vaso-protective and immunomodulatory
effects. Aim of this study was to examine whether lignan derivatives isolated from the roots of the anti-
inflammatory medicinal plant Krameria lappacea influence eNOS activity and endothelial nitric oxide
(NO) release. The study was performed using cultured human umbilical vein endothelial cells (HUVECs)
and HUVEC-derived EA.hy926 cells. Among the eleven isolated compounds only 2-(2,4-dihydrox-
yphenyl)-5-(E)-propenylbenzofuran (DPPB) was able to increase eNOS enzyme activity.
DPPB (1–10 mM) treatment for 24 h induced a significant and dose-dependent increase in eNOS
activity as determined by the [14C]L-arginine/[14C]L-citrulline conversion assay. Immunoblotting studies
further revealed a time-dependent DPPB-induced increase in eNOS-Ser1177and decrease in eNOS-Thr495
phosphorylation, as well as increased AMPK phosphorylation at Thr172, whereas Akt phosphorylation at
Ser473was not affected. Si-RNA-mediated knockdown of AMPK and inhibition of CaMKKb by STO 609, as
well as intracellular Ca2+chelation by Bapta AM abolished the stimulating effect of DPPB on eNOS-
Ser1177and AMPK-Thr172phosphorylation. Furthermore, we could show that DPPB increases
intracellular Ca2+concentrations assessed with the fluorescent dye Fluo-3-AM. DPPB enhances eNOS
activity and endothelial NO release by raising intracellular Ca2+levels and increases signaling through a
CaMKKb–AMPK dependent pathway.
? 2012 Elsevier Inc. All rights reserved.
* Corresponding author. Tel.: +43 1 4277 55270; fax: +43 1 4277 55969.
E-mail addresses: angela.ladurner@univie.ac.at (A. Ladurner), atanas.atanaso-
v@univie.ac.at (A.G. Atanasov), elke.heiss@univie.ac.at (E.H. Heiss), l.baumgart-
ner@gmx.net
(L.
Baumgartner),
stefan.schwaiger@uibk.ac.at
judith.rollinger@uibk.ac.at (J.M. Rollinger), hermann.stuppner@uibk.ac.at
(H. Stuppner), verena.dirsch@univie.ac.at (V.M. Dirsch).
(S.
Schwaiger),
Contents lists available at SciVerse ScienceDirect
Biochemical Pharmacology
jo u rn al h om epag e: ww w.els evier.c o m/lo cat e/bio c hem p har m
0006-2952/$ – see front matter ? 2012 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.bcp.2012.06.029

Page 2

thus promote endothelial function [14]. The identification of such
compounds and better understanding of their molecular mecha-
nism of action provides valuable input for both the prevention and
the treatment of cardiovascular disease. Plant secondary metabo-
lites from the benzofuran class are known to exhibit a broad range
of bioactivities, including anti-inflammatory [16,17], cardio- and
vaso-protective [18,19], cytostatic [20], and antioxidant actions
[21,22]. However, an influence on eNOS activity was not described
so far.
The roots of Krameria lappacea (Dombey) Burdet et Simpson
(syn. K. triandra Ruiz et Pavon), which are a rich source for such
compounds, have been traditionally used in South America for the
treatment of different inflammation-related complaints, and have
been introduced in the European medicine since the 18th century
[23–25]. We have isolated eleven lignans (nine benzofuran lignans
and two epoxy lignans) including the benzofuran derivative 2-(2,4-
dihydroxyphenyl)-5-(E)-propenylbenzofuran (DPPB) from the
dried roots of K. lappacea. So far only one report shows the
identification of DPPB as the major active compound of a
standardized K. triandra root extract exhibiting a potent cytopro-
tective effect on different cell lines exposed to stress [23]. We
examine here a potential positive effect of the isolated lignan
derivatives on eNOS activity in endothelial cells. Furthermore, we
investigate the molecular mode of action of the most active
compound DPPB and characterize the upstream signaling path-
ways that lead to increased eNOS activity.
2. Methods
2.1. Chemicals and cell culture reagents
Dulbecco’s modified Eagle’s medium (DMEM) without phenol
red containing 4.5 g/L glucose, endothelial growth medium EBMTM,
EGMTMSingleQuots, glutamine, amphotericin B, benzylpenicillin
and streptomycin were purchased from Lonza (Verviers, Belgium),
HAT supplement (100 mM hypoxanthine, 0.4 mM aminopterin and
16 mM thymidine) from Biochrom (Berlin, Germany), and trypsin
from Cambrex (Verviers, Belgium). Fetal bovine serum (FBS) was
obtained from Gibco via Invitrogen (Paisley, UK). A23187 and 4,5-
diaminofluorescein (DAF-2) were bought from Alexis Biochemicals
(Lausen, Switzerland) and [14C]L-arginine (346 mCi/mmol) from
New England Nuclear (Bosten, MA, USA). Bapta AM was purchased
from Tocris (Bristol, UK) and Fluo-3-AM was bought from
Invitrogen (Paisley, UK). Antibodies were obtained from the
following companies: eNOS from BD (Becton, Dickinson and
Company; Franklin Lakes, NJ, USA), phospho-eNOS-Ser1177, phos-
pho-eNOS-Thr495, phospho-AMPK-Thr172, AMPK, phospho-Akt-
Ser473, Akt, and horseradish peroxidase-conjugated goat anti-
rabbit secondary antibody from Cell Signaling (Bosten, MA, USA),
anti-tubulin from Santa Cruz (CA, USA), anti-actin from MP
Biomedicals (Solon, OH, USA), and horseradish peroxidase-
conjugated goat anti-mouse secondary antibody from Upstate
(Millipore, Vienna, Austria). All other chemicals were bought from
Sigma–Aldrich (Vienna, Austria). TLC plates were bought from
Machery-Nagel (Markus Bruckner Analysentechnik, Linz, Austria).
2.2. Origin of tested compounds
Compounds 1–11 were isolated and identified in a previous
study [26] as: 5-(3-hydroxypropyl)-2-(2-methoxy-4-hydroxyphe-
nyl)benzofuran (1), (?)-larreatricin (2), meso-3,30-didemethox-
ynectandrin B (3), (2S,3S)-2,3-dihydro-3-hydroxymethyl-2-(4-
hydroxyphenyl)-5-(E)-propenylbenzofuran (4), 2-(2-hydroxy-4-
methoxyphenyl)-5-(3-hydroxypropyl)benzofuran
dihydroxyphenyl)-5-(E)-propenylbenzofuran (DPPB; 6), (+)-con-
ocarpan (7), 2-(4-hydroxyphenyl)-5-(E)-propenylbenzofuran (8),
(5),
2-(2,4-
rataniaphenol III (9), rataniaphenol I (10), and rataniaphenol II
(11). The purity of all isolated compounds was ?96% (determined
by HPLC). The used plant material (Ratanhiae radix, dried ground
roots of Krameria lappacea (Dombey) Burdet et Simpson (syn. K.
triandra Ruiz et Pavon; Krameriaceae); 500 g; KL 6269) was
purchased from Mag. pharm. Kottas-Heldenberg & Sohn (Vienna,
Austria) and complied with the European Pharmacopoeia. A
voucher specimen (KL 6269) is deposited at the Institute of
Pharmacy/Pharmacognosy, University of Innsbruck (Austria).
2.3. Cell culture
The human endothelial cell line EA.hy926 (kindly provided by
Dr. C.-J.S. Edgell, University of North Carolina, Chapel Hill, NC, USA)
[28] was grown in DMEM without phenol red supplemented with
2 mM glutamine, 100 U/mL benzylpenicillin, 100 mg/mL strepto-
mycin, HAT supplement, and 10% FBS until passage 26. For
experiments, cells were seeded in six-well plates at a density of
5 ? 105cells/well and treated with test compounds at confluence,
after approximately 72 h. HUVECs were obtained from Lonza and
cultivated in EBMTMgrowth medium supplemented with 10% FBS,
EGMTMSingleQuots, 100 U/mL benzylpenicillin, 100 mg/mL strep-
tomycin, and 1% amphotericin until passage five. For experiments
cells were seeded in gelatine-coated six-well plates at a density of
1 ? 105cells/well. DPPB and other tested compounds of K.
lappacea, STO 609, compound C, and Bapta AM were dissolved
in dimethyl sulfoxide (DMSO) and stored at ?80 8C. Final DMSO
concentrations did not exceed 0.1%. Control cells were always
treated with an equal volume of solvent.
2.4. [14C]L-arginine/[14C]L-citrulline conversion assay
The enzymatic reaction catalyzed by eNOS converts the amino
acid arginine into citrulline and NO. [14C]L-citrulline production
can thus serve as a surrogate marker of NO production. The assay
was performed as previously described [29]. Briefly, endothelial
cells were equilibrated in HEPES buffer (HEPES 10 mM, NaCl
145 mM, KCl 5 mM, MgSO4
CaCl2? 2H2O 1.5 mM, pH 7.4) for 10 min at 37 8C. Then
0.32 mM [14C]L-arginine (346 mCi/mmol) and 1 mM of the calcium
ionophore A23187 were added. The reaction was stopped by lysing
cells followed by extraction with ethanol and ethanol/water. The
extracts were dried under vacuum (SPD 1010 SpeedVac, Thermo
Savant, Thermo Scientific, Langenselbold, Germany) and resolved
in water/methanol (1:1). After separation of [14C]L-arginine from
[14C]L-citrulline by thin layer chromatography (Polygram SIL N-HR,
Machery-Nagel, Austria) in the solvent system water:chloroform:-
methanol:ammonium hydroxide 25% (2:1:9:4, v/v/v/v) [14C]L-
citrulline was quantified by autoradiography in a phosphoimager
(BAS-1800II, Fujifilm, Du ¨sseldorf, Germany). AIDA software
(raytest, Langenzersdorf, Austria) was used for densitometric
analysis.
2 mM, a-D(+)-Glucose 10 mM,
2.5. Quantification of NO release by 4,5-diaminofluorescein-2 (DAF-2)
Quantification of NO released from endothelial cells was
performed using 4,5-diaminofluorescein-2 (DAF-2), a NO-sensitive
fluorescent probe. EA.hy926 cells were seeded in 96-well plates at
a density of 2.5 ? 104cells/well and were treated with test
compounds at confluence after approximately 72 h. Cells were
washed two times with PBS+ (137 mM NaCl, 2.68 mM KCl, 8.1 mM
Na2HPO4, 1.47 mM KH2PO4, 0.5 mM MgCl2? 6H2O, 0.68 mM
CaCl2? 2H2O) containing 100 mM arginine and equilibrated
10 min in this buffer. Then A23187 was added to a final
concentration of 1 mM and DAF-2 to a final concentration of
0.1 mM and the cells were incubated for 1 h at 37 8C. Addition of
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L-NAME to a final concentration of 200 mM allowed correction for
non-NO-specific fluorescence. The supernatant was transferred to
a black 96-well plate and fluorescence was measured in a plate
reader (Genios Pro, Tecan, Gro ¨dig, Austria) with an excitation
wavelength of 485 nm and an emission wavelength of 520 nm.
Fluorescence values were normalized to viable cells as determined
by resazurin conversion method [30]. For this, the cells were
incubated with 0.1 mg/mL resazurin in PBS for 30 min before
measuring the fluorescence in a plate reader (Genios Pro, Tecan,
Gro ¨dig, Austria) at an excitation wavelength of 535 nm and an
emission wavelength of 590 nm.
2.6. Gel electrophoresis and immunoblot analysis
Preparation of cell extracts, SDS-PAGE, immunoblot analysis,
and densitometric evaluations were performed as described
previously [31]. For detection of multiple proteins with similar
molecular weights in one sample, two or more identical
membranes were processed in parallel.
2.7. siRNA mediated knockdown of AMPKa
HUVECs were seeded in six-well plates at a density of 0.3 ? 106
cells/well and transfected one day later with 33 pmol AMPKa
siRNA (Santa Cruz, CA, USA) and scrambled control (Invitrogen,
Paisley, UK), respectively, using the OptiMEM/Oligofectamine
system (Invitrogen, Paisley, UK). 72 h after transfection the cells
were used for experiments. Successful knockdown of the target
proteins was confirmed by Western blot analysis.
2.8. Determination of intracellular Ca2+concentration
The intracellular calcium concentration ([Ca2+]i) was measured
using the intracellular calcium indicator Fluo-3-AM. EA.hy926 cells
were seeded at a density of 0.5 ? 106cells/well in a 6-well plate and
treated with test compounds at confluence, approximately after
72 h. Cells were loaded with the fluorescent dye Fluo-3-AM (2 mM)
for 45 min at 37 8C. Then [Ca2+]iwas measured in a flow cytometer.
2.9. Statistical methods
Statistical analysis was done using GraphPad Prism software
version 4.03 (GraphPad Software Inc., La Jolla, CA, USA). To
determine statistical significance Student’s t test or one- or two-
way analysis of variance (ANOVA) were performed. P values < 0.05
were considered significant (*). Figures with bar graphs represent
means ? SEM of at least three independent experiments.
3. Results
3.1. DPPB is the only compound isolated from K. lappacea that
stimulates endothelial NO release and increases eNOS enzyme activity
Eleven lignan derivatives (Fig. 1) were isolated from the dried
roots of K. lappacea [26] and investigated for their ability to alter
NO release from endothelial cells. Treatment of endothelial
EA.hy926 cells [28] with compounds 1, 5, and 9 showed a
significantly decreased release in endothelial NO whereas only
compound 6 increased it (Fig. 2A). The other compounds had no
significant effect. We also determined possible toxic effects of the
isolated compound on endothelial EA.hy926 cells by a LDH release
and resazurin conversion assay. None of the compounds were toxic
(data not shown). Since eNOS activity is the major determinant of
the endothelial NO production, we further determined eNOS
enzyme activity by quantifying the conversion of [14C]L-arginine to
[14C]L-citrulline. We tested compounds 6, 7, 8 and 11 in this assay,
as they were the most promising compounds regarding the results
from the endothelial NO release measurement, although only
compound 6 showed a statistically significant effect (Fig. 2B).
Interestingly only compound 6 (DPPB, 2-(2,4-dihydroxyphenyl)-5-
(E)-propenylbenzofuran) was able to significantly increase eNOS
enzyme activity. Therefore DPPB was further investigated.
Treatment of endothelial EA.hy926 cells with 10 mM DPPB for
24 h resulted in a more than 2-fold increase in NO availability in
comparison to the solvent control (Fig. 2C). Mevastatin, which is
known to potently upregulate eNOS expression and thereby NO
release, was used as a positive control [32,33]. Additionally, DPPB
treatment resulted in a dose-dependent increase of eNOS activity
in EA.hy926 cells, reaching 1.2-fold activation at a concentration of
10 mM (Fig. 2D). As a positive control for the [14C]L-arginine/[14C]L-
citrulline conversion assay we used ascorbic acid, which is known
to stabilize the eNOS cofactor tetrahydrobiopterin and therefore
increases eNOS activity [34,35]. To verify the activity of DPPB also
in primary HUVECs, cells were treated with 10 mM DPPB and eNOS
enzyme activity was measured using the [14C]L-arginine/[14C]L-
citrulline conversion assay, resulting in a very similar activity of
DPPB (Fig. 2E).
3.2. DPPB changes the eNOS phosphorylation pattern
Modulation of eNOS activity is linked to changes in the
phosphorylation state of the enzyme [15]. Enhanced phosphory-
lation at eNOS-Ser1177, and concomitantly decreased phosphory-
lation at eNOS-Thr495, is known to stimulate eNOS activity [15].
Treatment of EA.hy926 cells and HUVECs with DPPB led to an
increase in eNOS-Ser1177phosphorylation and a decrease in eNOS-
Thr495phosphorylation in a time-dependent manner (Fig. 3A and
B). DPPB had its maximal effect after 24 h of incubation. Therefore
we decided to continue with this condition. On the contrary, total
eNOS protein level (Fig. 3A and B) remained unchanged, overall
suggesting a direct stimulatory effect of DPPB on eNOS enzyme
Fig. 1. Chemical structures of investigated lignan derivatives of K. lappacea: 5-(3-
hydroxypropyl)-2-(2-methoxy-4-hydroxyphenyl)benzofuran (1), (?)-larreatricin
(2),
meso-3,30-didemethoxynectandrin
hydroxymethyl-2-(4-hydroxyphenyl)-5-(E)-propenylbenzofuran (4), 2-(2-hydroxy-
4-methoxyphenyl)-5-(3-hydroxypropyl)benzofuran (5), 2-(2,4-dihydroxyphenyl)-5-
(E)-propenylbenzofuran (DPPB; 6), (+)-conocarpan (7), 2-(4-hydroxyphenyl)-5-(E)-
propenylbenzofuran (8), rataniaphenol III (9), rataniaphenol I (10), and rataniaphenol
II (11).
B
(3),
(2S,3S)-2,3-dihydro-3-
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activity. Additionally we checked for changes in neuronal and
inducible NOS expression upon treatment with DPPB, but no
differences were detectable (data not shown).
3.3. DPPB does not affect Akt but increases the phosphorylation of
AMPK-Thr172
Among the most prominent upstream determinants of eNOS
activity, known to directly catalyze the phosphorylation of eNOS-
Ser1177and thereby stimulate endothelial NO production, are the
kinases Akt and AMPK [36,37]. Akt activation was estimated by
analyzing the phosphorylation of the Akt-Ser473residue [37]. The
activation of AMPK was monitored by determining the phosphor-
ylation of the AMPK-Thr172residue [38]. Treatment with DPPB did
neither alter Akt phosphorylation in EA.hy926 cells nor in HUVECs
(Fig. 3C and D), indicating no changes in Akt activity. However,
upon DPPB treatment the phosphorylation of AMPK at Thr172was
increased in both cell types (Fig. 3C and D), indicating an increased
AMPK activity.
3.4. Increased eNOS-Ser1177phosphorylation upon DPPB treatment is
dependent on AMPK and CaMKKb
To check if the AMPK activity is indeed required for the DPPB-
induced eNOS-Ser1177activation we utilized compound C, an
inhibitor of AMPK. Application of compound C (10 mM) blocked
DPPB-mediated phosphorylation at AMPK-Thr172as well as at
eNOS-Ser1177in EA.hy926 cells (Fig. 4A), underlining the causal link
between the AMPK activation and the observed increase in eNOS
phosphorylation. This was further confirmed by downregulation of
AMPK levels by transfection with a specific siRNA. When HUVECs
were transfected with this siRNA, treatment with DPPB failed to
elicit enhanced eNOS-Ser1177phosphorylation (Fig. 4B). Interesting-
ly treatment of endothelial cells with compound C alone led to a
basally elevated eNOS phosphorylation at Ser1177and AMPK
phosphorylation at Thr172. In addition, AMPK siRNA transfection
also led to an elevated level of eNOS phosphorylation at Ser1177. This
is probably due to off-target effects or compensatory mechanisms
triggered by these treatments.
To strengthen the connection between AMPK and eNOS activity
upon DPPB treatment we further assessed the effect of DPPB on
eNOS enzyme activity by measuring conversion of [14C]L-arginine
to [14C]L-citrulline in the presence of compound C. Incubation of
EA.hy926 cells with 10 mM compound C completely blocked the
effect of DPPB on eNOS enzyme activity (Fig. 4C). This result
confirms a contribution of AMPK on the DPPB-induced increase in
eNOS activity.
To further study how AMPK is activated upon DPPB treatment,
we investigated a potential involvement of CaMKKb, an upstream
kinase of AMPK [38,39]. Upon incubation of EA.hy926 cells with
10 mM STO 609, an inhibitor of CaMKKb, DPPB failed to increase
AMPK-Thr172and eNOS-Ser1177phosphorylation, suggesting an
important role of CaMKKb for the DPPB-induced activation of
eNOS (Fig. 4D).
Fig. 2. DPPB increases eNOS activity and endothelial NO release. (A) EA.hy926 cells were treated with 10 mM of the indicated compound or solvent vehicle for 24 h.
Endothelial NO release of eleven lignan derivatives was measured by incubation with the NO-sensitive fluorescent probe diaminofluorescein-2. Fluorescence values were
normalized to the number of viable cells as determined by resazurin staining (*p < 0.05; **p < 0.01) (mean ? SEM, n = 3). (B) EA.hy926 cells were treated with 10 mM of the
indicated compound or solvent vehicle for 24 h. Then a [14C]L-arginine/[14C]L-citrulline conversion assay was performed as described in Section 2. [14C]L-citrulline production was
normalized to the untreated control (**p < 0.01) (mean ? SEM, n = 4). (C) EA.hy926 cells were incubated with 10 mM DPPB for 24 h. Mevastatin (Mev, 1 mM) was used as positive
control. NO release was quantified as in (A) (*p < 0.01) (mean ? SEM, n = 3). (D) EA.hy926 cells were treated with the indicated concentration of DPPB for 24 h. Then a [14C]L-
arginine/[14C]L-citrulline conversion assay was performed as in (B). Ascorbic acid (Asc, 100 mM) served as a positive control. [14C]L-citrulline production was normalized to the
untreated control (**p < 0.01; ***p < 0.001; n.s., not significant) (mean ? SEM, n = 3). (E) HUVECs were incubated with 10 mM of DPPB for 24 h, and eNOS activity was determined as
in (B) (**p < 0.01) (mean ? SEM, n = 3).
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3.5. DPPB enhances intracellular Ca2+levels
CaMKKb and eNOS are both enzymes that are regulated
by [Ca2+]i [12,40]. Therefore we were interested whether
the increase in
eNOS activity and NO production upon
treatment
For determination of [Ca2+]i we used the fluorescent probe
Fluo-3-AM. Incubation of EA.hy926 cells with different con-
centrations of DPPB showed a dose-dependent increase in [Ca2+]i
(Fig. 5A).
with
DPPB
is
due
to
an
increase
in
[Ca2+]i.
Fig. 3. Changes in phosphorylation status of different proteins upon DPPB treatment. (A), (C) EA.hy926 cells or (B), (D) HUVECs were treated with 10 mM of DPPB or solvent
vehicle for the indicated time points or for 24 h in figure (B) and (D) and subjected to Western blot and subsequent densitometric analysis for the detection and quantification
of (phospho-) eNOS levels, (phospho-) Akt levels or (phospho-) AMPK levels, respectively. One representative blot is shown. Band intensities are normalized to tubulin or actin
and expressed as fold activation in comparison to the untreated control (***p < 0.001; **p < 0.01; *p < 0.05; n.s., not significant) (mean ? SEM, n = 3).
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3.6. Chelation of intracellular Ca2+abolishes the positive effect of
DPPB on eNOS-Ser1177and AMPK-Thr172phosphorylation
The data obtained so far suggested that an increase in [Ca2+]i
may be the reason for the increased eNOS activity upon DPPB
treatment. To obtain proof for this assumption we tested whether
incubation of EA.hy926 cells with the Ca2+chelator Bapta AM
(30 mM) could abolish the activating effects induced by DPPB.
Chelation of intracellular Ca2+indeed abrogated the stimulating
effect of DPPB on eNOS-Ser1177and AMPK-Thr172phosphorylation
(Fig. 5B), similar to the effects previously observed with compound
C and STO 609 (Fig. 4A and B). Again, treatment of the cells with
Bapta AM led to a basal increase in AMPK phosphorylation at
Thr172, which, however, could not be further increased upon
treatment with DPPB. All these results regarding basal activation of
different proteins after treatment with pharmacological inhibitors
strengthens our belief that there is a delicate balance between the
upstream kinases of eNOS-Ser1177(Akt, CaMKKb, AMPK) and that
interference with one of these kinases is likely to trigger
compensatory actions by the others.
Fig. 4. eNOS phosphorylation is enhanced after DPPB administration in an AMPK- and CaMKKb- dependent manner. (A) EA.hy926 cells were pretreated with 10 mM
compound C and incubated with 10 mM DPPB for 24 h as indicated. Western blot and subsequent densitometric analyses were performed to detect and quantify (phospho-)
AMPK and (phospho-) eNOS protein levels. One representative blot is shown. Band intensities are normalized to tubulin and expressed as fold difference to the vehicle treated
control (**p < 0.01; n.s., not significant) (mean ? SEM, n = 3). (B) HUVEC were transfected with AMPKa siRNA or scrambled control prior to treatment with 10 mM DPPB for 1 h as
indicated. Western blot and subsequent densitometric analyses were performed to detect and quantify (phospho-) eNOS and AMPK protein. One representative blot is shown. Band
intensities are normalized to actin and expressed as fold untreated control (*p < 0.05; n.s., not significant) (mean ? SEM, n = 3). (C) EA.hy926 cells were pretreated with 10 mM
compound C and incubated with 10 mM DPPB for 24 h as indicated. Then a [14C]L-arginine/[14C]L-citrulline conversion assay was performed as described in Section 2. [14C]L-
citrulline production was normalized to the untreated control (***p < 0.001; n.s., not significant) (mean ? SEM, n = 3). (D) EA.hy926 cells were pretreated with 10 mM STO 609 and
incubated with 10 mM DPPB for 24 h as indicated. Western blot and subsequent densitometric analyses were performed to detect and quantify (phospho-) AMPK and (phospho-)
eNOS protein levels. One representative blot is shown. Band intensities are normalized to tubulin and expressed as fold untreated control (**p < 0.01; n.s., not significant)
(mean ? SEM, n = 3).
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All data therefore point to a strong causal link between the
increase in [Ca2+]i and the observed DPPB-induced increase of
eNOS activity.
4. Discussion
In this study we show that the benzofuran derivative DPPB
enhances eNOS activity leading to an increased NO availability in
cultured human endothelial cells. The observed effect is associated
with an increase in [Ca2+]ileading to the activation of eNOS via a
signal transduction pathway involving Ca2+/CaM, CaMKKb, and
AMPK (Fig. 6).
Secondary metabolites from the benzofuran class are known to
exhibit a broad range of bioactivities [16–18,20,21], however, an
influence on eNOS activity was not described so far. Interestingly,
out of several benzofuran derivatives isolated from K. lappacea
[26], DPPB is the only compound which promotes eNOS activity in
human endothelial cells.
DPPB increases endothelial NO release by a mechanism
involving increased [Ca2+]i. eNOS activation via an increase of
[Ca2+]iis a well-established mechanism since eNOS can be directly
activated by protein–protein interaction with the intracellular Ca2+
receptor CaM enabling the electron transfer from NADPH at the
reductase domain of the enzyme to the heme center at the
oxygenase domain [12]. In addition, CaM is a known mediator of
Ca2+signaling directly binding and regulating a number of
different target proteins, thereby affecting many different cellular
functions [41]. A well-established target of CaM is CaMKKb, which
further translates the intracellular Ca2+increase into phosphor-
ylation of downstream target proteins, including AMPK phos-
phorylation at the threonine residue 172 [38,39]. Although the
AMPK pathway is mainly considered as an important regulator of
metabolism, it is also crucial for the maintenance of endothelial
function and redox balance [42]. AMPK exerts anti-atherosclerotic
effects by influencing various signaling cascades resulting in
improved NO bioavailability, attenuated free radical generation
Fig. 5. The effect of DPPB is due to increase of intracellular Ca2+levels. (A) EA.hy926
cells were incubated with the indicated concentrations of DPPB for 24 h.
Intracellular Ca2+was quantified in a flow cytometer after incubation with the
fluorescent dye Fluo-3-AM (**p < 0.01; *p < 0.05) (mean ? SEM, n = 3). (B) EA.hy926
cells were treated with 10 mM DPPB for 24 h and with 30 mM Bapta AM for 2 h as
indicated. Western blot and subsequent densitometric analyses were performed to
detect and quantify (phospho-) eNOS and (phospho-) AMPK protein levels. One
representative blot is shown. Band intensities are normalize to actin and expressed as
fold untreated control (**p < 0.01, n.s., not significant) (mean ? SEM, n = 6).
Fig. 6. Scheme of the proposed mechanism by which DPPB increases eNOS activity.
Administration of DPPB results in an increase in intracellular Ca2+and enhanced
Ca2+/CaM signaling thereby activating CaMKKb, AMPK and finally eNOS, ultimately
resulting in enhanced NO production. The inhibitors used in the study (Bapta AM,
STO 609, AMPKa siRNA and Compound C) and their place of action is also indicated.
A. Ladurner et al. / Biochemical Pharmacology 84 (2012) 804–812
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and activation of angiogenic factors [39]. Furthermore, activated
AMPK is able to reduce the activation of NF-kB in endothelial cells
[43]. In addition to its beneficial effect on endothelial function,
AMPK inhibits the proliferation of vascular smooth muscle cells
thus supporting anti-atherosclerotic mechanisms [44]. Activated
AMPK enhances eNOS enzyme activity by phosphorylation at the
eNOS-Ser1177residue [15].
Upon DPPB treatment we observed a strong increase of AMPK-
Thr172phosphorylation concomitant with increased eNOS-Ser1177
phosphorylation (Fig. 3) suggesting that eNOS is not only activated
via a direct protein–protein interaction with Ca2+/CaM, but also via
an additional regulatory loop involving Ca2+/CaM-dependent
CaMKKb activation, AMPK activation, and finally increased
eNOS-Ser1177phosphorylation (Fig. 6). The causal connection of
these regulatory steps was indeed confirmed by the application of
CaMKKb and AMPK inhibitors (STO 609 and compound C,
respectively) as well as by knockdown of AMPK (Fig. 4). These
approaches were able to block the DPPB-induced increase of eNOS-
Ser1177phosphorylation, an accepted posttranslational modifica-
tion activating eNOS [15]. Accompanying the increase in eNOS-
Ser1177phosphorylation, we observed a decreased phosphoryla-
tion of eNOS-Thr495after treatment with DPPB (Fig. 3). The eNOS-
Thr495residue is located in the CaM-binding domain of the enzyme
and decreased phosphorylation of Thr495is known to correlate
with a stronger CaM-eNOS interaction due to interference of the
phosphorylated residue with CaM binding to the eNOS CaM-
binding domain [45,46].
It is difficult to attribute specific effects to AMPK activation as
there are several reports showing crosstalk between the Akt and
the AMPK pathway [39]. However, our data showed no influence of
DPPB on Akt, also a major regulator of eNOS-Ser1177phosphor-
ylation. Similar results have been shown for the regulation of eNOS
by some agonists of G protein-coupled receptors such as
bradykinin or histamine, which are independent on signaling
from the PI3K/Akt pathway, but are known to utilize an increase in
[Ca2+]ifor their signal transduction in endothelial cells [46,47].
Taken together our results reveal that the benzofuran derivative
DPPB, isolated from the medicinal plant K. lappacea, increases
eNOS activity and NO availability in cultured endothelial cells. This
action is mediated by a mechanism involving elevation of the
intracellular Ca2+levels and increased signaling via a pathway
involving Ca2+/CaM, CaMKKb, and AMPK. Furthermore, our study
outlines DPPB as an interesting natural product that might have
significant contribution in mediating the bioactivity of K. lappacea.
Clearly further studies are necessary to better estimate whether
DPPB has a good potential to be used as a pharmaceutical or health-
promoting food supplement additive.
Conflict of interest
None.
Funding
This work was supported by grants from the Austrian Science
Fund (FWF) [NFN S10703-B03 and S10703-B13 and S10704-B03
and S10704-B13].
Acknowledgements
We gratefully acknowledge the excellent technical assistance
of Daniel Schachner, Judith Benedics, and Hortenzia Beres. We
would also like to thank Dr. Hartmut Kleinert (Mainz, Germany)
for the human iNOS control cell lysates and the respective iNOS
antibody.
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