(Hypertension. 2001;37:240.)
© 2001 American Heart Association, Inc.
Scientific Contributions |
Nifedipine Increases Endothelial Nitric Oxide Bioavailability by Antioxidative Mechanisms
From the Institute of Pharmacology (R.B., H.B., R.R., W.K.) and Clinic IV of Internal Medicine (G.E., T.A.M.), University of Cologne, Cologne, Germany.
Correspondence to Dr Reinhard Berkels, Institut fuer Pharmakologie, Gleueler Str 24, 50931 Koeln, Germany. E-mail Reinhard.Berkels{at}medizin.uni-koeln.de
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Abstract |
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Short-term treatment of the endothelium with dihydropyridine calcium antagonists resulted in an increased release in NO that is not due to a modulation of L-type calcium channels, because macrovascular endothelial cells do not express this channel. We investigated whether long-term (48 hours) treatment of porcine endothelial cell cultures with the dihydropyridine calcium antagonist nifedipine resulted in a similar enhanced NO liberation. Regarding to the underlying mechanism, we examined whether (1) nifedipine changed the mRNA and protein levels of the constitutive endothelial NO synthase (NOS) in endothelial cell cultures or (2) nifedipine exerts an NO protective effect via its antioxidative properties, as revealed in a cell culture model and with native endothelium from porcine coronary arteries. Nifedipine induced a significant time- and concentration-dependent increase (132±47%, 1 µmol/L, 40 minutes’ incubation) in the basal NO liberation (oxyhemoglobin assay). This increased NO release was not due to elevated NOS (type III) mRNA (Northern blot analysis) and protein (Western blot analysis) levels. However, nifedipine (both short- and long-term treatment) significantly reduced the basal and glucose (20 and 30 mmol/L)-stimulated formation of reactive oxygen species (lucigenin assay) of endothelial cell cultures and native cells. We conclude that the calcium antagonist nifedipine enhances the bioavailability of endothelial NO without significantly altering the NOS (type III) mRNA and protein expression, possibly via an antioxidative protection. This increased NO availability may cause part of the vasodilation and might contribute to the antithrombotic, antiproliferative, and antiatherosclerotic effects of dihydropyridine calcium antagonists.
Key Words: antioxidants • calcium antagonists • endothelium • nitric oxide • nitric oxide synthase • protein • nifedipine
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Introduction |
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Dihydropyridine (DHP)-type calcium antagonists are important drugs in the treatment of hypertension and coronary heart disease. They induce their specific pharmacological effects by binding to L-type calcium channels,1 2 which results in a reduced calcium influx with impaired electromechanical coupling both in vascular smooth muscle cells and in the heart.3 A few years ago, however, it was observed that removal of the endothelium or blockade of the guanylate cyclase of the vessel wall reduced the efficacy of the DHP-induced vasorelaxation,4 which indicated an endothelium-responsive cGMP-mediated process as part of the DHP action. Because macrovascular endothelial cells lack voltage-operated L-type calcium channels,5 6 the DHPs must exert these effects via other mechanisms. In this context, it is worth mentioning that DHPs may also exert antihrombotic7 8 and antiatherosclerotic9 10 effects in different experimental and clinical settings, of which the underlying signal transduction remains obscure.
With this background, it is tempting to speculate that NO, one of the most prominent endothelium-derived factors,11 12 which relaxes smooth muscle cells via the cGMP signal cascade,13 might be involved in these DHP actions. In fact, in various models, evidence has accumulated that DHPs stimulate the endothelial NO release,14 15 16 17 18 19 which may mediate or at least contribute to the above-mentioned calcium channel–independent effects.
Up to now, however, all of these findings were obtained only after acute exposure to DHPs (lasting minutes to hours). Because patients usually take calcium antagonists for a longer period of time, we investigated whether long-term treatment of endothelial cell cultures with nifedipine may alter the basal endothelial NO release as well as the expression of the constitutive endothelial NO synthase (ecNOS) mRNA and protein. Furthermore, because NO is rapidly deactivated by reactive oxygen species (ROS)20 and the DHPs may act as scavengers as known from different in vitro models,21 22 23 we also determined the antioxidative potency of nifedipine in endothelial cell cultures as well as in native cells to reveal a potential NO-protection effect as an underlying mechanism of the increased NO bioavailability.
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Methods |
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Cell Culture of Porcine Aortic Endothelial Cells
Porcine aortas were obtained fresh from a local abattoir. Immediately after removal, the vessels were stored in sterile PBS (4°C) with penicillin (200 mg/L) and streptomycin (200 mg/L) (Sigma Chemical Co). The endothelial cells were isolated enzymatically (1 mg/mL dispase; Boehringer Mannheim) and cultured in Medium 199 (GIBCO) with 10% FCS (GIBCO), 100 mg/L penicillin, 100 mg/L streptomycin, and HEPES. Cells were grown in an incubator (Heraeus) at 37°C in a 5% CO2 atmosphere and saturated humidity. The culture medium was replaced every day to compensate for decomposition of the substances during treatment. On confluency (2 to 4 days), the cells were detached through treatment with trypsin (0.05% and 0.02% EDTA; Sigma Chemical Co), washed once, and seeded into 25-cm2 flasks and again to confluence. Only passage 2 was used for the experiments. The purity of the porcine aortic endothelial cell (PAEC) culture was tested by uptake of Dil-labeled acetylated LDL, factor VIII immunostaining, and anti–smooth muscle actin immunostaining (Sigma Chemical Co). To avoid decomposition of the light-sensitive drug, nifedipine was added under yellow light, and the cultures were kept in the dark. Nifedipine was dissolved in DMSO (stock solution 10 mmol/L) and subsequently diluted in medium, resulting in final concentrations of 0.1 and 1.0 µmol/L and a maximum DMSO concentration of 0.01%, which did not cause any solvent effects on the parameters tested.
Measurement of Antioxidative Effects
The release of ROS from endothelial
cells was measured in cultured endothelial cell
suspensions and in native endothelial cells with a
lucigenin chemiluminescence assay. Briefly, coronary arteries
of female pigs were prepared immediately after excision of the hearts
and stored in Tyrode’s solution saturated with oxygen. During the
removal of connective tissue, special care was taken to preserve the
endothelium. The arteries were cut into rings of 0.3 cm
(wet weight 2 to 3 g), and the rings opened with a longitudinal
cut. The pieces were incubated in 450 µL HEPES-buffered saline
solution (in mmol/L) (HEPES 5, NaCl 140, KCl 5,
CaCl2 2, MgCl2 1, and
glucose 5, adjusted to pH 7.4) with or without nifedipine
or superoxide dismutase (SOD; 70 U/mL; Sigma Chemical Co) and with
different glucose concentrations (5, 10, or 30 mmol/L) for 20
minutes at 37°C. Basal ROS formation of native cells and cell
cultures was measured at 5 mmol/L glucose. Hyperglycemia (30
mmol/L) was used to induce an increase in the ROS release from native
endothelial
cells.24 25 26 27
After 20 minutes, lucigenin (0.25 mmol/L) was added (final volume
500 µL), and the emitted chemiluminescence was immediately
recorded for 1 minute with a commercially available counter
(Berthold Biolumat LB 9500). The respective backgrounds were
subtracted. The solvent (DMSO) did not interfere with the
measurements.
Accordingly, these experiments were performed with suspensions of cultured porcine endothelial cells. Briefly, confluent monolayers (passage 2) were detached with trypsin (0.05% and 0.02% EDTA; Sigma Chemical Co) and resuspended in HEPES buffer. The cells were counted, and 50 000 cells were used for each measurement (no stirring). According to our experiments with native vessels, the cell suspensions were treated (short term) with nifedipine (20 minutes before the measurement). Then, 20 mmol/L glucose (coadministration with nifedipine) was used to induce an enhanced ROS release. In addition, we treated cells with nifedipine in different concentrations for all of passage 2 (48 hours). These cells were detached, and measurements similar to those with untreated cells were performed. In contrast to the experiments with untreated cells, during the incubation with glucose, no nifedipine was present. The respective backgrounds were subtracted. The solvent (DMSO) did not interfere with the measurements.
Nonradioactive Northern Blot
Analysis
Before isolation of the mRNA from confluent PAEC
monolayers, the cells were treated with nifedipine
(untreated cells as control) for the entire passage (48 hours). Total
cellular mRNA was extracted from endothelial cells
according to a modification of a mRNA isolation kit (Boehringer
Mannheim). Briefly, endothelial cells were solubilized
in lysis buffer consisting of 5 mol/L guanidinium thiocyanate, 0.7
mol/L Tris-HCl, pH 8.0, 1.3 mol/L LiCl, 65 mmol/L EDTA, 0.25%
dithiothreitol, and 0.13% laurylsarcosine. Poly(A) mRNA was
subsequently incubated with biotin-labeled
oligo(dT)20 probe and magnetic streptavidin
particles at 37°C for 5 minutes. Streptavidin-mRNA complex was
resuspended in buffer (10 mmol/L Tris buffer, 0.2 mol/L LiCl,
1 mmol/L EDTA, pH 7.5), repetitively washed, and heat dissociated
(65°C, solvent H2O) before being magnetically
separated.
RNA was then size-fractionated in a 1.2% agarose gel electrophoresis before being transferred and immobilized on a nylon membrane (Boehringer Mannheim). Hybridization was performed as RNA-RNA hybrids because of superior strength and higher affinity. Membranes were hybridized simultaneously with a linearized 1.5-kb fragment of digoxigenin-UTP–labeled NOS RNA and with digoxigenin-UTP–labeled ß-actin RNA (Boehringer Mannheim) for verification of equal amounts of RNA. Digoxigenin-UTP labeling had been performed through in vitro transcription of the 1.5-kb human NOS cDNA insert with T7 RNA polymerase. The ecNOS-specific probe was kindly donated by Kenneth Bloch (Department of Anesthesia, Massachusetts General Hospital, Boston, Mass). Sequencing of 200 bp of the probe confirmed the identity of the ecNOS. RNA-RNA hybrids were visualized with chemiluminescence after binding of anti-digoxigenin alkaline phosphatase conjugates with CDP-Star and were exposed to Lumi-Film x-ray film (Boehringer Mannheim). The amount of mRNA expression was quantified through densitometry with National Institutes of Health Image software on an Apple Macintosh computer equipped with a Microtech scanner and corrected for ß-actin expression.
Cell Lysis and Immunoblot
Analysis
Treated and untreated confluent PAECs were washed
with ice-cold PBS and immediately solubilized in 500 µL lysis buffer
(150 mmol/L NaCl, 50 mmol/L HEPES, 1% SDS, and 1 tablet/50
mL complete protease inhibitor cocktail; Boehringer
Mannheim). The amount of protein was determined with a Bio-Rad protein
kit before equal amounts of protein were used in 1 blot. Cell lysate
protein was size-fractionated on an 8% SDS gel and blotted onto a PVDF
(Bio-Rad) membrane with wet blotting. These immunoblots
were then blocked with nonfat milk powder (2%, 30 minutes), incubated
with the primary monoclonal mouse anti–endothelial
cell NOS antibody (1: 3000, 1 hour), and washed in buffer (TBS-T)
before being incubated with the secondary horseradish
peroxidase–conjugated anti-mouse IgG antibody (1: 3000, 1 hour)
(Affinity Research). Protein-antibody conjugates were visualized with
chemiluminescence reagent (Boehringer Mannheim), which uses
horseradish peroxidase to oxidize luminol, and exposed to Kodak medical
x-ray film for 0.5, 1, 1.5, and 2 minutes. The protein amount was
quantified through densitometry with National Institutes of Health
Image software on an Apple Macintosh computer equipped with a Microtech
scanner.
Measurement of NO
To determine the NO release of cultured cells, we
used the oxyhemoglobin
assay.28 29 30
This technique measures the conversion of oxyhemoglobin to
met-hemoglobin by NO. Briefly, PAECs were cultured in
60-cm2 Petri dishes and treated with
nifedipine during the entire passage (untreated cells as a
control). The cells were washed 3 times with HEPES-buffered saline
solution. A freshly prepared oxyhemoglobin solution (1 µmol/L) in
HEPES buffer was added (2 mL) to the dishes and directly measured. The
cells were kept at 37°C during the measurement. The spectra were
measured with a single-wavelength spectrophotometer (DU 7500; Beckman
Instruments). To access the autoxidation of the oxyhemoglobin, a dish
without cells was identically prepared for comparison. The
oxyhemoglobin was measured every 10 minutes for 40 minutes. The
autoxidation spectra were subtracted to obtain difference spectra, and
the increasing met-hemoglobin formation was measured at 402 nm compared
with the isosbestic point at 411 nm.
Preparation of Oxyhemoglobin
Oxyhemoglobin was prepared by dissolving rabbit
hemoglobin (containing <50% met-hemoglobin) in phosphate buffer.
During stirring, the mixture was exposed to oxygen (95% plus 5%
CO2) for 20 minutes. Then, a molar excess of
sodium dithionite was added and gassed for 15 minutes. The resulting
oxyhemoglobin was purified through gel chromatography
to remove the sodium dithionite. The purity was controlled with
spectroscopy, and the concentration was determined with the
Lambert-Beer law.
Statistics
All data are expressed as mean±SEM for n
experiments. The results were analyzed by the
nonparametric Wilcoxon test for matched pairs and
the Mann-Whitney test for nonmatched pairs. A value of
P<0.05 was considered
statistically significant.
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Results |
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NO Measurement
The treatment of PAECs with nifedipine for 48 hours (throughout the entire passage) resulted in an increased basal NO release of these monolayers (cells from the same animal). During the measurement, nifedipine was not present in the supernatant. At 20 minutes after integration of the NO liberation through the formation of met-hemoglobin, a significant concentration-dependent increase in the NO release by nifedipine (0.1 and 1 µmol/L) could be demonstrated, in contrast to untreated cells (n=6) (Figure 1), which persisted for the entire course of the experiment. Preincubation with NNA (100 µmol/L) resulted in a complete inhibition of the NO formation of the cells (n=3) (Figure 1).
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Indicates P<0.05 from control group.
Northern Blot Analysis
The treatment of endothelial cells with
nifedipine did not result in a significant alteration in
the NOS mRNA expression
(Figure 2), although at a concentration of 1 µmol/L, a
slight increase could be shown (n=4)
(Figure 2).
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Western Blot Analysis
The Western blot analysis revealed the same
behavior as the Northern blot analysis. Nifedipine
at a concentration of 0.1 µmol/L did not alter the NOS protein
expression (n=4), whereas at the higher concentration (1 µmol/L), a
slight but nonsignificant increase was seen (n=4)
(Figure 2).
An antibody against the inducible NOS (type II NOS) (Western blot) showed no results (data not shown).
Antioxidative Properties of
Nifedipine
Nifedipine (1 µmol/L) induced a
significant decrease in the basal ROS release (lucigenin assay) of
endothelial cell cultures (n=6)
(Figure 3) and of native cells of porcine coronary
arteries (-85±21% at 1 µmol/L, n=4). This effect could be
achieved by both short-term (20 minutes) and long-term (48 hours)
incubation of the cells with nifedipine (1 µmol/L) (n=6)
(Figure 3).
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Because in different pathological situations, such as diabetes and atherosclerosis, a marked increase in the ROS concentration has been found,24 31 32 we induced an increased ROS release in native vessels and in cell suspensions by increasing the glucose concentrations (30 or 20 mmol/L glucose). In cell suspensions, the effect was less pronounced, and at 20 mmol/L glucose, a maximal effect was elicited (data not shown). Mannitol was used as an osmotic control (30 or 20 mmol/L), and it did not induce an ROS release of the endothelial cells (n=6, data not shown).
The glucose-induced ROS release of native
endothelium from porcine coronary arteries as
well as from suspended endothelial cultures was
significantly reduced by nifedipine (short-term treatment,
20 minutes) in a concentration-dependent manner
(Figures 4 and 5) (n=6 to 8). This short-term effect could be
validated with suspensions of nifedipine-treated (48 hours,
no nifedipine present during measurement)
endothelial cells
(Figure 5). In cells that had been pretreated with
nifedipine (0.1 and 1 µmol/L), the glucose-induced ROS
release was significantly reduced (n=6)
(Figure 5). Furthermore, 
50% of the ROS release could be
abolished by incubation with SOD (70 U/mL)
(Figures 4 and 5), indicating that superoxide anions or the
product of superoxide anions and NO forms a substantial part of the
ROS. Denudation of the endothelium from the artery
segments completely abolished (-87±10%; n=4) the ROS release,
indicating that the endothelium is the source of the
ROS.
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Discussion |
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It is known that various DHP calcium antagonists enhance the NO release from endothelial cells.14 15 16 17 18 19 Using an NO-sensitive electrode, we previously demonstrated that nifedipine in the micromolar and submicromolar concentration ranges stimulates the NO release from the native endothelium.33 34 These findings were obtained after acute exposure to DHP calcium antagonists. Therefore, we investigated whether (1) a long-term treatment (48 hours, entire passage 2) of porcine endothelial cell cultures with nifedipine resulted in a similarly altered NO release and (2) an increased formation and/or an impaired inactivation of NO might be involved by measuring the ecNOS (NOS isoform III) mRNA and protein expression and the antioxidative potency of nifedipine, respectively.
In contrast to the short-term measurements during long-term treatment, nifedipine was not present in the buffer during the antioxidative and NO experiments.
We could demonstrate that treatment with nifedipine (concentration and time dependently) significantly increased the NO liberation of endothelial cells. The NO release could be completely blocked by N-nitro-L-arginine, an NOS inhibitor. This is consistent with findings of Ding and Vaziri,19 who showed by measuring nitrite and nitrate (the degradation products of NO) that the treatment of endothelial cells with the DHP calcium antagonist felodipine (0.1 to 10 µmol/L, 24 hours) increased the endothelial NO release from rat aortic endothelial cells. To investigate whether this increased NO release is due to a modulation of the expression of the ecNOS gene, we performed Northern and Western blotting. We found slightly but not significantly increased mRNA and protein levels after treatment with nifedipine. This stands in contrast to the study of Ding and Vaziri,19 who found a significant rise in the ecNOS protein mass after felodipine treatment. This may be due to differences in the cell culture model or to the use of a different calcium antagonist. They used rat endothelial cells in passage 7, whereas we studied porcine endothelial cells in passage 2, which may be closer to the physiological situation because the cell physiology may change during advanced passages. Moreover, others could show that the activity of the ecNOS enzyme is not altered by nifedipine,35 which could be confirmed with the use of another DHP calcium antagonist (pranidipine).36
The maximum concentration (1 µmol/L) used in our study is
higher than the plasma concentrations of nifedipine in vivo
(0.2 µmol/L),35 but it
is not an unusual phenomenon that ex vivo higher concentrations of
calcium antagonists are required to achieve pharmacological
effects. Furthermore, in vivo the lipophilic nifedipine
accumulates in membranous structures, resulting in markedly higher
local concentrations.37
Nifedipine at the concentrations used in our study did not
significantly alter the cell proliferation of PAECs, so there were no
differences in cell numbers that could explain the different NO release
(data not shown).
From our data, we cannot conclude that nifedipine directly interferes with the expression of ecNOS mRNA and protein. On the other hand, it might be possible that nifedipine alters the signal transduction responsible for NOS activation. For example, Zhang and coworkers16 38 showed that amlodipine induced an increased nitrite (degradation product of NO) production in microvessels and macrovessels of dog hearts (acute addition of the DHP). This increase could be abolished with Hoe 140, a kinin receptor blocker, indicating that a kinin-mediated mechanism plays an important role. In contrast to our results, these authors did not find an enhanced nitrite production by nifedipine. This may be explained by species differences or the different preparation, especially of the microvessels (sequential-dissection and homogenization). Moreover, the measurement of nitrite by the Griess assay may be less sensitive than measurement by the oxyhemoglobin assay.39 Very recently, Kitakaze et al40 described increased cardiac NO levels by nifedipine in an open chest ischemia-reperfusion canine model.
In addition, there are reports (acute treatment of endothelial cells with DHP) that kinases,41 as well as an increased Ca2+ influx,33 may play a role in the DHP-induced NO release.
It is known that NO is rapidly deactivated by
ROS,20 and because there are
different findings that DHPs exert antioxidative
properties,21 23 42 43 44
we reinvestigated this aspect in both native
endothelial cells from porcine coronary
arteries and suspended endothelial cells by using a
lucigenin assay.45 The
previous studies usually used in vitro assays with enzymes
generating superoxide
anions,22 23
investigated the oxidation of LDL in
vitro46 or ex
vivo,47 or investigated the
lipid peroxidation of sarcolemmal membrane
preparations.21 42
In these reports, high micromolar (10 µmol/L) concentrations were
always needed to induce an antioxidative effect. In contrast, we used
native cells or cell cultures, which are more
physiological and offer the possibility that
nifedipine accumulates in membranes, thus reaching higher
concentrations.37 In
addition, we stimulated the ROS release of the
endothelial cells because this is a pathological
situation that often occurs in persons with diabetes and
atherosclerosis. We used acute hyperglycemia to
induce an ROS
increase.25 26 27 31
The basal and glucose-induced ROS releases of
endothelial cell suspensions and native cells were
significantly reduced by short-term treatment with
nifedipine, and long-term treatment (48 hours) of
endothelial cell cultures showed an even more
pronounced effect of nifedipine. Using the lucigenin assay,
we could not determine which ROS radicals are diminished by
nifedipine, but a large part of the ROS seems to be either
superoxide anions (inhibition by the SOD) or products that result
from a reaction of superoxide anions with NO, such as peroxynitrite. It
seems that at least part of the increased NO release by
nifedipine is due to a protection from ROS, which
deactivates NO. One could argue that the ROS level may also be
reduced due to a reaction with NO (increased by
nifedipine), but the studies conducted with
nifedipine in cell-free
systems22 23
confirm the antioxidative properties of the nifedipine
molecule per se.
In summary, we showed that the treatment of endothelial cells with nifedipine increased NO release that is not due to an altered expression of ecNOS mRNA and protein. It instead seems that there is an enhanced availability of NO by an antioxidative protection. This will not rule out that other mechanisms, such as kinin-mediated actions,16 38 play an important role, too, or act synergistically. The increased NO availability may contribute to beneficial effects of DHP calcium antagonists that were found by others, such as antiaggregatory,7 8 antiproliferative,48 49 and antiatherosclerotic9 10 49 effects.
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Acknowledgments |
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This work was supported by the Köln-Fortune Program of the Medical Faculty of the University of Cologne.
Received July 18, 2000; first decision July 31, 2000; accepted August 14, 2000.
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