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Vol. 286, Issue 2, 938-944, August 1998
Institute of Chemical Kinetics and Combustion, Novosibirsk 630090, Russia (S.D.), Institute of Applied Physiology, University Freiburg, Freiburg 79104, Germany (B.F., E.B.), and ISIS Pharma, Zwickau 08005, Germany (D.S.)
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Abstract |
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Anti-ischemic therapy with organic nitrates is complicated by
tolerance. Induction of tolerance is incompletely understood and likely
multifactorial. Recently, increased production of reactive oxygen
species (ROS) has been investigated, but it has not been clear if this
is a direct consequence of the organic nitrate on the vessel or an
in vivo adaptation to the drugs. To examine the possibility that nitrates could directly stimulate vascular ROS production, we compared the development of nitrate tolerance with the
formation of ROS induced by pentaerithrityltetranitrate (PETN) or
nitroglycerin (GTN) in vitro in porcine smooth muscle
cells, endothelial cells, washed ex vivo platelets and
whole blood. By examining cGMP formation, it was found that 24-hr
treatment with GTN but not PETN induced significant nitrate tolerance,
which was prevented by parallel treatment with Vit C. Incubation of vascular cells acutely with 0.5 mM GTN doubled the rate of ROS generation, whereas PETN had no such effect. The rate of ROS
(peroxynitrite and O2
) formation
detected by specific spin traps in tolerant smooth muscle cells,
treated for 24 hr with 0.01 mM GTN, was substantially higher (30.5 nM/min) than in control cells acutely treated with 0.5 mM GTN (25 nM/min). In contrast to PETN, GTN induces nitrate tolerance and also
increases the formation of ROS both in vascular cells and in whole
blood. ROS formation is minimally stimulated by PETN comparable to data
obtained in Vit C-suppressed GTN tolerance. ROS formation induced by
organic nitrates seems to be a key factor in the development of nitrate
tolerance.
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Introduction |
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Organic
nitrates (e.g., PETN and GTN) are used in
the therapy of a large variety of cardiovascular diseases in which
enhanced vasodilator responses of certain vascular sections are
beneficial, such as in myocardial ischemia. The organic nitrates do not
spontaneously release NO but must undergo a metabolic biotransformation
to NO (Mülsch et al., 1995
) before stimulating cGMP
production and vascular relaxation (Mellion et al., 1983).
Treatment with GTN and other organic nitrates is limited by the
development of nitrate tolerance, especially during nonintermittent
administration. Nitrate tolerance, a multifactorial phenomenon, is
characterized by neurohormonal counterregulation, enhanced responses to
vasoconstrictor agonists, as well as diminished responses to the
endothelium-derived relaxing factor (Laursen et al., 1996).
Despite many hypotheses and investigations, the mechanisms of nitrate
tolerance have not yet been fully perceived.
Recently, it was found that a GTN treatment of animals increased the
formation of superoxide (O2) in blood vessels
and that this seemed to inactivate NO (Münzel et al.,
1994, 1995); other studies indicated that 3-day GTN treatment resulted
in elevated vascular NADH-oxidase activity. The latter significantly
enhances the basal generation of O2 radicals
(Griendling et al., 1994; Münzel et al.,
1996) and promotes the inactivation of NO (Gryglewski et
al., 1986) and the formation of peroxynitrite (Huie and Padmaja,
1993). Superoxide radicals (O2) react
effectively with NO (k = 6.7×109
M
1 sec1) to
form peroxynitrite (Huie and Padmaja, 1993), which is a strong oxidant
(Pryor and Squadrito, 1995). The formation of peroxynitrite in
GTN-treated ex vivo platelets (Skatchkov et al.,
1996) and in human blood under GTN therapy was recently reported
(Skatchkov et al., 1997). Thiols are principal targets of
peroxynitrite in cells (Radi et al., 1991). Peroxynitrite
is capable of modifying enzyme activities, for example by irreversibly
oxidizing SH groups including those of sGC. The NO-mediated increase in
the activity of sGC depends on the state of SH groups (Braughler,
1983). Peroxynitrite causes vascular dysfunction in isolated hearts
(Villa et al., 1994). The action of GTN as an exogenous NO
donor and a promoter of cGMP-dependent vasodilation depends on the
specific balance between the concentrations of NO on one hand and the
rate of the GTN-induced simultaneous ROS-formation (superoxide radicals
and peroxynitrite) on the other hand.
It is still very difficult to quantify the formation of superoxide
radicals and of peroxynitrite in biological systems. Recently, it was
shown (Vásquez-Vivar et al., 1997) that the
application of lucigenin-chemiluminescence for peroxynitrite
quantification is unexpectedly associated with the presence of
artifacts, for example with an additional lucigenin-induced formation
of superoxide radicals. However, without an amplification of
chemiluminescence (e.g., with lucigenin) the GTN-induced
formation of peroxynitrite and
O2 in vascular cells remains
below the detection limit. Therefore, in our experiments, we used ESR
spectroscopy with spin trapping techniques.
To clarify the mechanisms involved in the development of nitrate
tolerance and to elaborate and demonstrate a more effective and less
detrimental vasodilator, it is important to compare different organic
nitrates with regard to the development of nitrate tolerance and the
concomitant formation of ROS (superoxide radical and of peroxynitrite).
It has been reported that GTN treatment causes nitrate tolerance both
in vitro and in vivo (Feelisch and Kelm, 1991;
Tsutamoto et al., 1994). Fink and Bassenge (1997) reported that a nonintermittent PETN administration in dogs did not cause tolerance. As mentioned above, GTN induces ROS formation in vascular cells. However, no quantitative data are available hitherto quantifying the formation of superoxide radicals and peroxynitrite as a consequence of PETN and GTN metabolism. Consequently, we studied the effects of
PETN and GTN on ROS formation in vascular cells and in blood.
It is known that treatment with organic nitrates induces vasodilation
by increasing cGMP formation (Feelisch and Kelm, 1991). In
nitrate-tolerant organs, the administration of organic nitrates does
not cause vasodilation because the contents and release of cGMP cannot
be enhanced (Tsutamoto et al., 1994). Therefore, we used the
nitrate-induced effects on cGMP contents as a marker of the development
of nitrate tolerance in vitro.
The aim of our study was to compare the development of nitrate tolerance in vitro with the concomitant formation of ROS (superoxide radicals and peroxynitrite) induced by PETN and GTN in suspensions of cultured SMC and EC in washed ex vivo platelets and in whole blood.
Four partly newly elaborated spin traps were used in our ESR experiments. To differentiate between the formation of superoxide radicals and peroxynitrite, the spin traps DMPO and TMIO were used. The rates of ROS formation were determined using TEMPONE-H and CP-H.
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Materials and Methods |
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Abstract
Introduction
Materials & Methods
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Discussion
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Processing of cells.
EC and SMC were washed from porcine
aortas as described by Hecker et al. (1994) and grown in
culture (cat. no. 31095-029; GIBCO BRL Life Technologies, Eggenstein,
Germany) as described previously (Campbell and Campbell, 1987). The
purity of the EC and SMC cultures were checked as described by Hecker
et al. (1994). Cell viability was checked by fluorescence
microscopy and by measuring the increase in the cGMP induced by
incubation with the NO donor sodium nitroprusside (2 µM, 3 min).
Washed ex vivo platelets were obtained from trained,
conscious dogs by venopuncture. Platelets were washed from plasma and
resuspended in 50 mM phosphate buffer (PBS), pH 7.4, as in Bassenge and
Fink (1996). EC (4000 cells/µl) and SMC (2500 cells/µl) obtained
from cultures (fifth passage), as well as washed platelets
(100,000/µl), were incubated with 0.5 mM GTN or PETN in PBS for 15 min at 20°C in the presence of cysteine (20 µM) as a cofactor of
nonenzymatic systems involved in GTN metabolism (Weber et
al., 1996). To induce tolerance to GTN or PETN both SMC and EC
were incubated in culture with GTN, GTN with Vit-C (20 µg/ml) or with
PETN for 24 hr at 37°C (initial concentration of nitrates was 10 µM) (Salvemini et al., 1993). The state of platelet
function was analyzed by measuring the thrombin-induced increase in
intracellular Ca++ using
Ca++-dependent fluorescence of FURA 2 dye as
described by Bassenge and Fink (1996). The protein concentrations were
determined using the Lowry method. Blood was drawn from the carotid
artery of dogs into a citric acid solution (6:1 v/v) (Bassenge and
Fink, 1996).
Spin trapping experiments.
The cells were resuspended in 50 mM PBS, pH 7.4, containing 0.2 mM DTPA and 0.9% NaCl. Probes for ESR
measurements were analyzed in quartz capillaries of an internal
diameter of 1 mm. To inhibit the formation of hydroxyl radicals from
H2O2 catalyzed by traces of
transition metals potentially present in the buffer, DTPA was added to
the cell suspensions (final concentration, 0.2 mM) (Rosen and Freeman,
1984). The absence of paramagnetic impurities was checked in stock
solutions of spin traps by ESR spectroscopy. ESR measurements were
performed at room temperature using an EMX-A ESR spectrometer (Bruker,
Karlsruhe, Germany). The ESR settings were the following: field center,
3474 G; field sweep, 60 G; microwave frequency, 9.72 GHz; microwave
power, 20 mW; magnetic field modulation, 100 kHz; modulation amplitude,
2.0 G; conversion time, 655 msec; detector time constant, 1024 msec;
magnetic field sweep time, 671 sec. The ESR spectra were recorded 5 min
after equilibration of the samples in the ESR cavity.
Determination of cGMP contents.
The cGMP contents were
assayed in whole cells using radioimmunoassay (Biotrend, Cologne,
Germany) as described by Bassenge and Fink (1996). The probes were
prepared for the determination of cGMP formation induced by an acute
addition of the nitrate (100 µM), according to the following
protocol: cell suspensions were incubated for 1 min with different
nitrate compounds and the process then stopped by the addition of
trichloracetic acid (final concentration, 5%) after the probes were
frozen in liquid nitrogen. To determine the basal level of cGMP in the
control cells, after an incubation with nitrates in culture (24 hr),
the probes were tested parallel to probes that had been acutely treated with nitrates (in control cells, saline was added instead of the solution-containing nitrates).
Preparation of CP-H and TEMPONE-H spin trap stock solutions. CP-H and TEMPONE-H were dissolved in oxygen-free (nitrogen bubbled) 0.1 M sodium phosphate buffer, pH 7.4, in the presence of 0.9% NaCl and 1 mM DTPA. DTPA was used to decrease the self-oxidation of hydroxylamines catalyzed by traces of transition metal ions. The concentration of CP-H and TEMPONE-H in the stock solutions amounted to 10 mM. Before the experiments, stock solutions were kept frozen or in a cool airtight place.
Superoxide radical determination.
Superoxide radical
generation in platelets, SMC and EC was determined using the spin trap
DMPO (Rosen and Freeman, 1984) in a concentration of 0.1 M quantifying
the DMSO-resistant DMPO-OH spin adduct (Dikalov et al.,
1997a). DMSO (0.1% final concentration in probes) was used as a
peroxynitrite scavenger. DMPO-OH formation in cell suspensions was
measured using the amplitude of the second low-field component of the
ESR spectra of the DMPO-OH spin-adduct.
Peroxynitrite determination.
Peroxynitrite formation was
determined by the spin traps DMPO and TMIO. Using DMPO (0.1 M), the
peroxynitrite formation in cell suspensions was quantified by the
DMSO-inhibited DMPO-OH spin adduct (final concentration DMSO, 0.1%).
Peroxynitrite also was determined by TMIO (0.2 M) by monitoring the ESR
signal of the TMIO-OH spin-adduct (Dikalov et al., 1996)
with a spectrum corresponding to the hyperfine interaction constants
aN = 14.3 G and aH = 16.3 G. Solutions of known concentrations of peroxynitrite were used to
obtain the corresponding calibration curve. For this purpose, small
aliquots of peroxynitrite (pH 13) were mixed with 0.2 M TMIO dissolved
in 50 mM phosphate buffer (pH 7.4) containing 0.2 mM DTPA.
Hydroxyl radical formation in cell suspensions.
The
contribution of hydroxyl radicals formed from
H2O2 to the formation of
DMPO-OH and TMIO-OH spin adducts was tested using two methods: (1) by
measuring the intensity of the ESR spectra of the spin adduct
DMPO-CH°3 in the presence of DMSO (a
methyl radical CH°3 is formed during the
reaction of hydroxyl radicals with DMSO, which in turn forms the
DMPO-CH3° adduct from DMPO; Rosen and Rauckman,
1984); and (2) by the detection of a TMIO-OH spin adduct in the model
system containing H2O2 and
DTPA. It was found that the presence of 0.2 mM DTPA effectively
inhibited the formation of the DMPO-CH°3 spin
adduct. Moreover, no ESR signal was observed in the samples containing
50 µM H2O2, 0.2 mM DTPA and 0.2 M TMIO in PBS (pH 7.4). Therefore, the presence of 0.2 mM DTPA
effectively inhibited the iron catalyzed formation of hydroxyl
radicals. Dismutation of superoxide radicals and the consequent
formation of H2O2 did not
contribute to the formation of TMIO-OH in the presence of DTPA. Thus,
the contribution of hydroxyl radicals to the formation of TMIO-OH and
DMPO-OH spin adducts was negligible under our experimental conditions.
Determination of rates of ROS formation.
The determination
of the rate of ROS formation by the DMPO spin trap is limited by the
half-life period of the DMPO-OH spin adduct and usually the
steady-state concentration of the DMPO-OH spin adduct is measured.
Using CP-H or TEMPONE-H as spin traps (Dikalov et al.,
1997a), the rate of ROS formation can be determined monitoring the
time-dependent accumulation of the corresponding stable nitroxyl
radicals TEMPONE or CP using ESR (Dikalov et al., 1997a). The rates of ROS formation (superoxide radical and
peroxynitrite) in vitro were determined by the analysis of
the oxidation of hydroxylamines CP-H (1 mM) and TEMPONE-H (1 mM) to
nitroxyl radicals CP and TEMPONE, respectively. The rates of
CP and TEMPONE formation were measured by the kinetics of nitroxyl
radical generation, which were obtained by monitoring the amplitude of
the low field component of the ESR spectrum. Experimental
concentrations of CP and TEMPONE in vitro were determined
using the dependence of the amplitude of the ESR spectrum on the
concentration of CP and TEMPONE obtained from Sigma (Deisenhofen,
Germany).
; Evans
et al., 1982). Nitroxyl radicals, formed in the reaction of
TEMPONE-H or CP-H with ROS, can be reduced by ascorbate to ESR-silent
hydroxylamines. The CP radical is much more resistant to the reaction
with ascorbate than TEMPONE nitroxyl radicals (Dikalov et
al., 1997a). Therefore, we used CP-H to avoid an underestimation
of the ROS formation both in platelets and in whole blood.
During GTN and PETN metabolism in vascular cells, it is possible that
superoxide radicals and peroxynitrite could be formed (Münzel
et al., 1996; Dikalov et al., 1997c; Skatchkov
et al., 1997). TEMPONE-H reacts with both these
ROS to form the nitroxyl radical TEMPONE. The formation of superoxide
radicals can be tested using SOD as competitive reagent. SOD competes
with TEMPONE-H for superoxide radicals, decreasing the TEMPONE
formation. The formation of ROS was measured as TEMPONE generation in
cultured SMC (2500 cells/µl) after the addition of 0.5 mM
PETN, 0.5 mM GTN, and 0.5 mM GTN plus 1000 units/ml SOD, after 24-hr
incubation with GTN.
The rate of ROS formation in suspensions of washed ex vivo
platelets (100,000 cells/µl) was measured as the rate of CP formation with the addition of 0.5 mM PETN, 0.5 mM GTN or 1 mM hydralazine plus
0.5 mM GTN.
The calibrations were obtained using xanthine/xanthine-oxidase as a
source of O2 radicals. All measurements were
performed in 50 mM PBS in the presence of 0.9% NaCl and 0.2 mM DTPA at
pH 7.4 at 20°C.
Statistics. All values are expressed as mean ± S.D. Statistical significance was determined by Student's t test for paired data. Two groups of data were considered to be significantly different at P < .05.
Chemicals and drugs. PETN was obtained from ISIS Pharma (Zwickau, Germany). GTN was from Pohl Boskamp (Hohenlockstedt, Germany). Hydralazine (antihypertensive drug) was from Ciba-Geigy (Basel, Switzerland). The spin traps TMIO, TEMPONE-H and CP-H were from Alexis Corporation (San Diego, CA). The spin trap DMPO, nitroxyl radicals TEMPONE and CP, DMSO, cysteine, glutathione, DTPA, bovine erythrocyte SOD, catalase and xanthine were obtained from Sigma. Xanthine oxidase was supplied by Fluka (Neu-Ulm, Germany). Peroxynitrite was obtained from Alexis. All other chemicals were of analytical grade and purchased from Merck (Darmstadt, Germany).
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Results |
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Formation of ROS in suspensions of SMC, EC and platelets. The formation of the DMPO-OH spin adduct in suspensions of SMC is depicted in figure 1. An addition of GTN (0.5 mM) to SMC suspensions enhanced the DMPO-OH formation from 96 ± 10 to 180 ± 26 nM/mg protein (fig. 1), thus the 15-min incubation with GTN resulted in a significantly enhanced ROS formation in suspensions of SMC (fig. 1). Such an enhancement was not observed when PETN was used. The addition of 0.1% DMSO to the SMC suspension with 0.5 mM GTN reduced DMPO-OH formation from 180 ± 26 to 118 ± 12 nM/mg protein. SOD addition (1000 units/ml) to SMC suspensions inhibited the DMPO-OH formation to 58 ± 6 nM/mg protein.
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Spin trapping of peroxynitrite using TMIO in suspensions of SMC, EC
and platelets.
The spin trap TMIO does not trap superoxide
radicals but reacts with peroxynitrite producing the TMIO-OH spin
adduct (Dikalov et al., 1996). In our experiments, the spin
trap TMIO was used for the determination of peroxynitrite formation
during GTN and PETN metabolism (fig. 2).
It was found that an acute addition of 0.5 mM GTN to the suspensions of
vascular cells led to the formation of significant amounts of the
TMIO-OH spin adduct. In contrast to GTN, the contents of the TMIO-OH
spin adduct in suspensions of PETN-treated vascular cells did not
differ statistically from the control.
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Determination of the rate of ROS formation in SMC, EC, platelets and whole blood. The addition of 0.5 mM GTN to suspensions of SMC increased the rate of ROS formation by 83 ± 8% (from 13.6 in control to 25 nM/min/mg protein). The presence of extracellular SOD (1000 units/ml) effectively inhibited the rate of TEMPONE formation (from 25 to 12 nM/min/mg protein). Acute treatment of SMC with PETN did not result in a significant increase in ROS formation (fig. 3).
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;
Evans et al., 1982). Therefore, for the experiments with
blood, we used the spin trap CP-H, which is more resistant to ascorbate than TEMPONE-H (Dikalov et al., 1997a). The formation of ROS
in blood was measured as the rate of CP formation after the addition of
0.5 mM PETN, 0.5 mM GTN and hydralazine (1 mM) plus GTN (0.5 mM) as
depicted in figure 5A. It was found that
the addition of 0.5 mM GTN augmented the formation of ROS up to
182 ± 7% (from 17 ± 1 to 31 ± 2 nmol/min). In
contrast to GTN, the rate of ROS formation in blood with PETN was only
slightly increased (115 ± 8%) (fig. 5A).
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) to platelets before GTN treatment reduced the
rate of CP formation from 365% to 250%. However, a preincubation with
hydralazine in a concentration up to 10 mM did not decrease the
GTN-induced ROS formation to the basal level or to the level obtained
by the addition of SOD to GTN-treated platelets (data not shown).
It is interesting that in our experiments the addition of hydralazine
(1 mM) to the blood before GTN treatment reduced the formation of CP
during GTN treatment from 182 ± 7% to 132 ± 6% (fig. 5A).
This effect of hydralazine can be attributed to the inactivation of
NADH-oxidases of blood cells in the same way as in washed ex
vivo platelets and in isolated EC (Münzel et al., 1996).
Determination of cGMP contents. To compare the development of nitrate tolerance induced by PETN and GTN in vascular cells in vitro, we measured the formation of cGMP during acute administration and at the end of a 24-hr treatment with these nitrates. The formation of cGMP after acute treatment with nitrates was determined. It was found that the induction of cGMP formation in EC (similar to SMC) after a 1-min treatment with PETN or with GTN (concentrations varied from 10 to 500 µM) was almost the same (fig. 6, left columns). There was no significant difference between the PETN- or GTN-induced cGMP formation after a 24-hr treatment of EC cultures. The 24-hr treatment of EC with GTN showed clearly the development of tolerance, as neither GTN nor PETN stimulate the release of cGMP in EC (center columns). In contrast to GTN-treated cells, the formation of cGMP was stimulated both by GTN and PETN in EC treated for 24 hr with PETN (fig. 6, right columns). An incubation of EC with GTN plus an antioxidant (Vit C) significantly improved cGMP release from 0.8 pM/mg protein at control to 12 pM/mg protein after additional GTN/Vit-C treatment.
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Discussion |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Using a new, well validated technique for the detection of
specific compounds in ROS formation, we could convincingly demonstrate a generation of both O2 and peroxynitrite in
various types of vascular cells during acute exposure to organic
nitrates, especially after induction of tolerance after a 24-hr
continuous exposure to organic nitrates. We found such formation in
both SMC in EC in which tolerance was elicited before nitrate exposure
and, surprisingly, even in platelets of animals. By using a combination
of different nonspecific and several new specific spin traps, we could
differentiate exactly between peroxynitrite and
O2 generation in various vascular cells. In
addition, we found that different organic nitrates were associated with
substantially different rates of ROS generation in all three vascular
cell types tested, e.g., PETN produced substantially less ROS than GTN
(only one fourth of the amount). Unexpectedly, our in vitro
data closely resembled that of earlier in vivo data obtained
not only by us (Fink and Bassenge, 1997) but also by others, especially
with regard to the O2-mediated generation of
atheromatosis (Kojda et al., 1995).
As recent studies have shown, the development of tolerance to organic
nitrates is a multifactorial phenomenon. Apart from the formation of
ROS such as O2 and
ONOO, which have recently been shown to induce
tolerance (Münzel et al., 1995; Dikalov et
al., 1998), other changes have been recognized such as the
reduction in the concentration of low molecular thiols (Needleman and
Johnson, 1973) and the inhibition of sGC and the resulting decrease in
cGMP formation and vasorelaxation (Schröder et al.,
1988).
Previously, it was reported that treatment of vascular cells with GTN
induces formation of ROS (Münzel et al., 1996). These data were obtained using lucigenin-chemiluminescence, which was recently criticized, because of substantial artifacts caused by lucigenin-mediated additional formation of superoxide radicals (Vásquez-Vivar et al., 1997). To avoid this problem,
we therefore studied the nitrate-induced formation of ROS by ESR
spectroscopy with spin trapping techniques, which are not limited by
lucigenin-associated artifacts. The other advantage of using special
spin traps such as TMIO (Dikalov et al., 1996), TEMPONE-H
(Dikalov et al., 1997b) or CP-H (Dikalov et al.,
1997a) lies in the fact that we were able to analyze not only the
O2 release during exposition to nitrates but
also the formation of ONOO, which is known to
inhibit various enzymes containing SH-groups such as sGC. In addition,
by using the ESR technique, we were capable of measuring the rate of
ROS release in cell suspensions and in blood in the presence of
antioxidants and/or reductants. The data obtained confirmed the fact
that GTN induces the formation of ROS both in isolated vascular cells
and in whole blood. Moreover, it was shown in our study that GTN also
induced ROS formation in whole blood treated with hydralazine
[inhibitor of NAD(P)H-oxidoreductases] (Münzel et
al., 1996), which only partially decreased the GTN-induced formation of ROS.
In addition to GTN we also studied PETN, an organic nitrate, the new
interesting aspects of which were recently discussed by Hess et
al. (1997). This study showed on one hand an NO· release at
a redox potential of +3 and on the other hand that a particular PETN
metabolite (pentaerithrityldinitrate) could simultaneously act as a
reductant. When we analyzed the effects of PETN on ROS formation in
vascular cells and in whole blood, we could show for the first time
that PETN only slightly increased ROS formation in vascular cells and
in whole blood (not more than 20%). This could potentially be
attributed to the superimposed action of this particular
PETN-metabolite. Moreover, in contrast to PETN, the treatment of the
vascular cells with GTN caused a 200% increase in ROS formation
compared with nitrate-free control cell suspensions. Thus, it is
reflected in our data that PETN treatment does not lead to the
development of nitrate tolerance in vascular cells (and the effect of
PETN on ROS formation is negligible), in contrast to GTN, which induced
both ROS formation and substantial development of nitrate tolerance.
This was shown as a decrease in stimulated cGMP release. Therefore, it
seems rather likely that ROS formation is closely associated with the development of nitrate tolerance in vitro. This was also
convincingly shown in newly developed assays using more specific spin
traps, in addition to DMSO and SOD in different vascular cell
preparations. Therefore, we assume that the enhanced in
vitro formation of superoxide radicals and of peroxynitrite during
long-term treatment with GTN could also play a key role in the
development of nitrate tolerance under in vivo conditions.
Taking into account that the formation of hydrogen peroxide results
from the dismutation of superoxide radicals, one cannot exclude a
generation of hydroxyl radicals in our samples. However, the formation
of hydroxyl radicals from hydrogen peroxide is a transition
metal-catalyzed reaction, which can be suppressed by adding a chelating
agent such as DTPA (Butler and Halliwell, 1982). In fact, in our
probes, a formation of the TMIO-OH spin adduct was not detected in the
presence of 0.2 mM DTPA and 50 µM
H2O2, whereas a bolus
addition of peroxynitrite (50 µM) to 0.2 M TMIO in the presence of
0.2 mM DTPA in PBS (pH 7.4) led to the formation of a strong ESR signal
of the TMIO-OH spin-adduct. Thus, 0.2 mM DTPA effectively inhibited the
iron-catalyzed reaction of hydroxyl radical formation from
H2O2 but did not affect the
formation of the TMIO-OH spin-adducts by peroxynitrite.
The detailed molecular mechanism of the effect of ROS formation on the
development of nitrate tolerance is still not precisely known. However,
the enhanced formation of superoxide radicals and of peroxynitrite in
GTN-treated cells in vitro could lead to a pronounced
oxidative damage of the cells. Such oxidative damage can contribute to
the development of nitrate tolerance via an inactivation of
certain enzymes involved in the NO-induced stimulation of cGMP
formation and contents (particularly by damaging the SH-groups of
soluble guanylyl cyclase; Braughler, 1983), which were substantially
diminished in our cell suspensions incubated with GTN for 24 hr. This
was in contrast to PETN-incubated cells as well as to
non-nitrate-exposed cells and to cells which were incubated with GTN
along with the antioxidant Vit-C.
Such an additional supplementation with antioxidants could thus be
important not only under our in vitro conditions (because of
this excessive formation of ROS during therapy with organic nitrates)
but also under in vivo conditions in which similar
mechanisms were recently demonstrated (Bassenge and Fink, 1996; Fink
et al., 1998).
Conclusions.
GTN metabolism in vascular cells and in blood is
associated with a drastically enhanced formation of ROS (superoxide
radical and peroxynitrite) for which hydralazine-inhibited enzymatic
systems may be partially responsible. A 24-hr treatment of the vascular cells with 10 µM GTN leads to nitrate tolerance, whereas PETN has no
affect on the formation of cGMP. PETN causes a negligible rise in the
formation of ROS and does not induce nitrate tolerance, a finding that
we have also seen under in vivo conditions (Fink and
Bassenge, 1997).
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Footnotes |
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Accepted for publication April 10, 1998.
Received for publication November 17, 1997.
1 This study was supported by the German Heart Foundation (Deutsche Herzstiftung, Frankfurt/Main, Germany) and through a scholarship from the German Academic Exchange Service (Deutscher Akademischer Austausch- dienst, Bonn, Germany) (S.D.).
Send reprint requests to: Prof. Dr. Eberhard Bassenge, Institute of Applied Physiology, Hermann-Herder-Str. 7, D-79104 Freiburg, Germany. E-mail: angphys{at}ruf.uni-freiburg.de
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Abbreviations |
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CP, 3-carboxy-proxyl;
CP-H, 1-hydroxy-3-carboxy-pyrrolidine;
DMPO, 5,5-dimethyl-1-pyrroline-N-oxide;
DMPO-OH, 2-hydroxy-5,5-dimethylpyrrolidine-N-oxyl;
ESR, electron spin resonance;
EC, endothelial cells;
NO, nitric oxide;
GTN, nitroglycerin;
O2, superoxide radical;
PETN, pentaerithrityltetranitrate;
ROS, reactive oxygen species;
SMC, smooth
muscle cells;
SOD, erythrocyte superoxide dismutase;
TMIO, 3,5,5-trimethyl-imidazole-1-oxide;
TMIO-OH, 5-hydroxy-2,2,4,-trimethyl-3-imidazoline-1-oxyl;
TEMPONE-H, 1-hydroxy-2,2,6,6-tetramethyl-4-oxo-piperidine hydrochloride;
TEMPONE, 2,2,6,6-tetramethyl-4-oxo-piperidinoxyl;
Vit C, vitamin C.
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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-); after the addition of 0.5 mM GTN (-
-), 0.5 mM GTN + 1000 units/ml SOD (-
-) or 0.5 mM PETN (-
-); and in cells after 24 hr
of treatment with 0.1 mM GTN and resuspended in buffer-free from GTN
(-
-). Data are mean ± S.D.; n = 12; * P < 0.05 vs. PETN.
