Cardiovascular Research 47 (2000) 602–608
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Inhibition of purified soluble guanylyl cyclase by L-ascorbic acid
a,
b ,1
a
a
Astrid Schrammel *, Doris Koesling , Kurt Schmidt , Bernd Mayer
a
¨ Pharmakologie und Toxikologie, Karl-Franzens-Universitat
¨ Graz, Universitatsplatz
¨
Institut f ur
2, 8010 Graz, Austria
b
¨ Pharmakologie, Freie Universitat
¨ Berlin, Thielallee 69 -73, D-14195 Berlin, Germany
Institut f ur
Received 17 November 1999; accepted 11 January 2000
Abstract
Objective: L-Ascorbic acid has been described to exert multiple beneficial effects in cardiovascular disorders associated with impaired
nitric oxide (NO) / cGMP signalling. The aim of the present study was to investigate the effect of vitamin C on the most prominent
physiological target of endogenous and exogenous NO, i.e. soluble guanylyl cyclase (sGC). Methods: To address this issue we used a
highly purified enzyme preparation from bovine lung (from the slaughterhouse). Enzymic activity was measured by a standard assay
based on the conversion of [a- 32 P]GTP to [ 32 P]cGMP and the subsequent quantification of the radiolabelled product. NO was quantified
using a commercially available Clark-type electrode. Results: Stimulation of sGC by the NO donor 2,2-diethyl-1-nitroso-oxyhydrazine
was inhibited by ascorbate with an IC 50 of |2 mM. Maximal enzyme inhibition (|70%) was observed at 0.1–1 mM vitamin C.
Stimulation of sGC by the NO-independent activator protoporphyrin-IX was also inhibited with similar potency. The effect of ascorbate
on sGC was largely antagonised by reduced glutathione (1 mM) and the specific iron chelator diethylenetriaminepentaacetic acid (0.1
mM). Electrochemical experiments revealed that NO is potently scavenged by vitamin C. Consumption of NO by ascorbate was prevented
by reduced glutathione (1 mM), diethylenetriaminepentaacetic acid (0.1 mM) and superoxide dismutase (500 units / ml) whereas up to
5000 units / ml superoxide dismutase failed to restore sGC activity. Conclusions: Our results suggest that physiological concentrations of
L-ascorbic acid diminish cGMP accumulation via both scavenging of NO and direct inhibition of sGC.  2000 Elsevier Science B.V. All
rights reserved.
Keywords: Nitric oxide; Oxygen consumption; Second messengers; Smooth muscle; Vasoconstriction / dilatation
1. Introduction
Stimulation of soluble guanylyl cyclase (GTP pyrophosphate lyase (cyclising) EC 4.6.1.2; sGC) by L-arginine-derived nitric oxide (NO) and consequent formation
of the second messenger cGMP represents a widespread
signal transduction mechanism that is involved in a variety
of biological processes [1,2]. In the cardiovascular system,
accumulation of cGMP critically contributes to the regulation of vascular smooth muscle tone as well as platelet
aggregation and adhesion. Impaired NO / cGMP signalling
is implicated in diverse pathologies such as diabetes,
hypertension, coronary artery disease, hypercholesterolemia and chronic heart failure [3–7].
*Corresponding author.
E-mail address: [email protected] (A. Schrammel).
1
¨ Pharmakologie, Ruhr Universitat
¨ Bochum,
Present address: Institut fur
MA N1 / 39, D-44780 Bochum, Germany.
NO-sensitive sGC is a heterodimer composed of an aand a b-subunit with an overall molecular mass of 150
kDa [8]. The enzyme contains stoichiometric amounts of
protoporphyrin-IX-type heme bound to the N-terminal
portion of the b-subunit [9]. High-affinity binding of NO
to the prosthetic heme group results in the formation of a
ferrous nitrosyl heme complex which triggers a change in
protein conformation and consequent enzyme activation
[10].
The water-soluble antioxidant L-ascorbic acid has been
reported to exert beneficial effects on several cardiovascular diseases. Thus, endothelial dysfunction in the course of
essential hypertension was improved by supplementation
of vitamin C [11]. Others described the preventive effect of
ascorbate on the development of tolerance during longterm administration of organic nitrates [12]. Recently a
stimulation of NO biosynthesis by L-ascorbic acid has been
Time for primary review 22 days.
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PII: S0008-6363( 00 )00019-5
A. Schrammel et al. / Cardiovascular Research 47 (2000) 602 – 608
observed in human endothelial cells [13] and the antioxidant was shown to sensitise isolated coronary arteries
towards NO-induced vasodilation [14].
However, besides its multiple antioxidant properties,
vitamin C was shown to become prooxidative under
certain conditions [15]. The autoxidation of ascorbic acid
is catalysed by trace metals such as copper and iron and
involves two successive one-electron oxidation steps,
yielding the ascorbyl radical and dehydroascorbate, respectively (Scheme 1). Ascorbate is regenerated from dehydroascorbate by diverse enzymatic [16–18] or non-enzymatic
[19] mechanisms. At physiological conditions dehydroascorbate is unstable and hydrolyses to give 2,3-diketogulonic acid, a reaction recently found to be triggered
603
by bicarbonate [20]. The open-chained compound 2,3diketogulonic acid undergoes further fragmentation to
yield a variety of products with five or less carbons [21].
Metal-catalysed autoxidation of ascorbate may lead to
the formation of reactive oxygen species including
superoxide (O 2
2 ), hydrogen peroxide (H 2 O 2 ), and hydroxyl radicals (OH ? ) [22]. Additionally, the accumulation
of reactive aldehydes [23], capable of damaging proteins
via Maillard chemistry [24], was observed following
ascorbate autoxidation. Indeed, it has been suggested that
‘‘ascorbylation’’ of proteins may contribute to the pathophysiology associated with certain diseases, including
diabetes, cataract and renal failure [25–27]. The present
study was designed to investigate the effect of L-ascorbic
acid on sGC using highly purified enzyme preparations
from bovine lung. Our results suggest that cGMP accumulation is inhibited in vitro by physiologically relevant
concentrations of L-ascorbic acid [28] via two different
mechanisms.
2. Methods
2.1. Materials
sGC was purified from bovine lung as described [29].
2,2-Diethyl-1-nitroso-oxyhydrazine sodium salt (DEA /
NO) was purchased from Alexis (Lausen, Switzerland).
[a- 32 P]GTP (.3000 Ci / mmol) was from Humos Diagnostika (Vienna, Austria). L-Ascorbic acid, dehydroascorbic
acid, protoporphyrin-IX, superoxide dismutase (SOD),
catalase and all other chemicals were purchased from
Sigma (Vienna, Austria). All solutions were prepared in
Nanopure water (Barnstead ultrafiltered type I, resistance5
18 MV / cm). Stock solutions of ascorbic acid and dehydroascorbic acid (10 mM each) were prepared in a 0.1 M
sodium acetate buffer, pH 5.0 and kept on ice to minimise
autoxidation. Further dilutions were made in a 50 mM
K 2 HPO 4 –KH 2 PO 4 buffer, pH 7.4 immediately before use.
2.2. Determination of sGC activity
Scheme 1. Simplified mechanism of ascorbate autoxidation.
Purified sGC (50–100 ng; maximal activity (vmax ) | 15–
18 mmol /(mg?min)) was incubated at 378C for 10 min in a
total volume of 0.1 ml of a 50 mM K 2 HPO 4 –KH 2 PO 4
buffer pH 7.4 containing 0.5 mM [a- 32 P]GTP (200 000–
300 000 cpm), 3 mM MgCl 2 and 1 mM cGMP. L-Ascorbic
acid, dehydroascorbic acid, reduced glutathione (GSH),
chelators and other additives were present as indicated.
Reactions were started by adding tenfold concentrated
stock solutions of DEA / NO, protoporphyrin-IX or vehicle
to the assay mixture and transfer of the samples from 48C
to 378C. Incubations were terminated by ZnCO 3 precipitation, and [ 32 P]cGMP was isolated by column chromatography as described previously [30]. To determine basal
604
A. Schrammel et al. / Cardiovascular Research 47 (2000) 602 – 608
activity (without activators), a higher enzyme concentration
of 0.2 mg / 0.1 ml was used and the reaction time was
extended to 20 min. Experiments with DEA / NO were
performed with a maximal stimulatory concentration of 1
mM [31,32]. Results were corrected for enzyme-deficient
blanks and recovery of cGMP. The concentration–response
curves were fitted to the Hill equation.
2.3. Electrochemical detection of NO
NO was quantified with a Clark-type electrode (Iso-NO;
World Precision Instruments, Berlin, Germany) connected
to an Apple Macintosh computer by an analog-to-digital
converter (Mac Lab; World Precision Instruments; New
Haven, CT, USA). The electrode was calibrated daily by
addition of sodium nitrite to a helium-gassed solution of
0.1 M KI in 0.1 M H 2 SO 4 . The electrode exhibited a linear
response to up to 10 mM NO with an average slope of 0.6
nM NO / pA output current. Experiments with DEA / NO
were performed in water-jacketed open plastic vials containing L-ascorbic acid, dehydroascorbic acid and other
additives as specified in a total volume of 1 ml. The
reaction mixtures used were the same as in the sGC assay
described above, except that [a- 32 P]GTP was omitted.
Reactions were started by adding 100-fold concentrated
stock solutions of DEA / NO to the samples. In another
series of experiments aliquots of saturated NO solutions
(|2 mM) were injected through a septum into 1.8 ml glass
vials completely filled with the sGC assay mixture. All
experiments were performed under constant stirring at
378C and data were recorded with a sampling rate of 0.5
Hz. NO was quantified from the peak concentrations using
the CHART software program for Apple Macintosh. The
concentration–response curves were fitted to the Hill
equation. Data represent mean values6standard error of
three experiments.
3. Results
L-Ascorbic acid was found to inhibit stimulation of sGC
by the NO donor DEA / NO (1 mM) with an IC 50 of
2.460.4 mM (Fig. 1A). Maximal enzyme inhibition
(|70%) was observed at 0.1–1 mM vitamin C. To
investigate whether sGC inhibition by ascorbate is limited
to the NO-stimulated enzyme, we used protoporphyrin-IX
(10 mM) as NO-independent activator of sGC. As shown
in Fig. 1B, the protoporphyrin-IX-stimulated enzyme was
similarly sensitive to ascorbate with |65% inhibition
observed at 1 mM vitamin C. The IC 50 value was in the
low micromolar range (not shown). Stimulation of sGC by
protoporphyrin-IX yielded about 20–30% of the maximally achievable enzymic activity (not shown). Ascorbate did
not appreciably affect basal cGMP formation: less than
20% inhibition was observed at 1 mM of the vitamin (Fig.
1B).
Fig. 1. Effect of L-ascorbic acid on stimulation of soluble guanylyl
cyclase. (A) Purified sGC (0.05 mg) was stimulated with DEA / NO (1
mM) and assayed for activity in the presence of increasing concentrations
of ascorbate as described under Methods. Data represent mean
values6standard errors of three experiments with duplicate determination. (B) Purified sGC was stimulated with DEA / NO (1 mM) or
protoporphyrin-IX (10 mM) or was assayed under basal conditions.
Formation of cGMP was measured in the absence and presence of
L-ascorbic acid (1 mM). Data are presented as percent of the respective
controls and represent mean values6standard errors of 18 (DEA / NO),
eight (protoporphyrin-IX) or three (basal conditions) experiments with
duplicate determination.
To investigate whether L-ascorbic acid interferes with
NO autoxidation, we performed electrochemical experiments using a Clark-type NO-sensitive electrode. As
shown in Fig. 2A, NO released from DEA / NO (1 mM)
was potently scavenged by ascorbate. To exclude that the
observed effect arises from a specific reaction of ascorbate
with the NO donor, we repeated the experiments with NO
solutions (|4 mM) instead of DEA / NO and obtained
similar results (Fig. 2B). In another series of experiments
increasing concentrations of ascorbate (0.5 mM –1 mM)
were added to the assay mixture prior to injection of
DEA / NO (1 mM) and the release of NO was quantified as
described. As shown in Fig. 2C, vitamin C decreased the
A. Schrammel et al. / Cardiovascular Research 47 (2000) 602 – 608
Fig. 2. Interference of ascorbate with NO autoxidation. (A) Release of
NO from DEA / NO (1 mM) was measured with a Clark-type electrode as
described under Methods. Reactions were started by the addition of
DEA / NO (1 mM) to the reaction mixture. At the indicated time point
L-ascorbic acid (1 mM) was injected into the reaction mixture. An
original trace representative of three experiments is presented. (B)
Aliquots of saturated NO solutions were injected through a septum into
1.8 ml glass vials to give |4 mM. At the indicated time point L-ascorbic
acid (1 mM) was added to the reaction mixture. An original trace
representative of two experiments is presented. (C) NO released from
DEA / NO (1 mM) was quantified under control conditions and in the
presence of increasing concentrations of L-ascorbic acid (0.5 mM–1 mM).
Peak concentrations of NO were plotted against the ascorbate concentration. Data represent mean values6standard errors of three experiments.
peak concentrations of NO with an IC 50 of |2 mM. In the
presence of the highest ascorbate concentration tested (1
mM) we measured 0.02 mM of NO.
It is well established that ascorbic acid becomes oxidised in the presence of trace metals, thereby generating
superoxide (O 2
2 ), hydroperoxide (H 2 O 2 ) and hydroxyl
radicals (OH ? ). To investigate whether NO reacts with an
605
oxygen species arising from metal-driven ascorbate autoxidation, we performed electrochemical experiments in the
presence of SOD, catalase, the combination of both and the
hydroxyl radical scavenger mannitol. As shown in Table 1,
SOD (500 units / ml) and its combination with catalase
(500 units / ml) largely prevented ascorbate-mediated NO
consumption, whereas catalase (500 units / ml) or mannitol
(1 mM) were ineffective. Additionally we tested the
specific iron chelator diethylenetriaminepentaacetic acid
(DTPA), the Cu(I)-selective chelator neocuproine and the
reductant glutathione for their ability to prevent NO
scavenging by ascorbate. As shown in Table 1, DTPA (0.1
mM) and glutathione (1 mM) were found to strongly
antagonise the effect of ascorbate, whereas neocuproine
was relatively ineffective.
We next examined whether these effects of scavengers
and chelators on ascorbate / NO chemistry were relevant for
sGC activity. As summarised in Table 1, the specific iron
chelator DTPA (0.1 mM) and glutathione (1 mM) almost
completely reversed ascorbate-mediated sGC inhibition,
whereas catalase (500 units / ml), the hydroxyl radical
scavenger mannitol (1 mM) and the Cu(I)-selective
chelator neocuproine (0.1 mM) had no appreciable effect.
Surprisingly, we found that SOD (#5000 units / ml) did
not restore sGC activity. Similar results were obtained in
experiments using Mn-SOD instead of the Cu,Zn-containing enzyme (not shown). Ascorbate-mediated sGC
inhibition was only partially overcome by a combination of
SOD and catalase (500 units / ml; each).
We next investigated which redox state of ascorbate is
responsible for sGC inhibition by comparing the effects of
ascorbic acid (1 mM) with that of dehydroascorbic acid (1
mM) on the DEA / NO- and protoporphyrin-IX-stimulated
enzyme (Fig. 3). We found that dehydroascorbic acid
caused a marked decrease in enzyme activity with about
75% and 73% inhibition observed with the DEA / NO- and
the protoporphyrin-IX-stimulated sGC, respectively. Enzyme inhibition by dehydroascorbate was partially reversible by glutathione (1 mM) in the case of DEA / NO and
Table 1
Effects of scavengers and chelators on ascorbate-mediated NO consumption and sGC inhibition; n53
Sample a
NO max
Specific activity
(% of control)
DEA / NO (1 mM)
1AA (1 mM)
1SOD (500 u / ml)
1catalase (500 u / ml)
1SOD1catalase (500 u / ml each)
1mannitol (1 mM)
1DTPA (0.1 mM)
1neocuproine (0.1 mM)
1GSH (1 mM)
0.5560.05
0.0260.00
0.2960.00
0.0360.02
0.1860.02
0.0260.00
0.1960.06
0.0260.00
0.1560.01
;100
38.462.9
30.367.6
46.7612.3
62.4610.5
40.666.9
95.3620.8
45.360.8
81.964.4
a
DEA / NO, 2,2-diethyl-1-nitroso-oxyhydrazine; AA, L-ascorbic acid;
SOD, superoxide dismutase; DTPA, diethylenetriaminepentaacetic acid;
GSH, reduced glutathione.
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A. Schrammel et al. / Cardiovascular Research 47 (2000) 602 – 608
Fig. 3. Effect of dehydroascorbic acid on sGC activity. Purified sGC (0.1
mg) was stimulated with DEA / NO (1 mM) or protoporphyrin-IX (10
mM) and assayed for cGMP formation under control conditions and in the
presence of ascorbate (AA) or dehydroascorbate (DHA; 1 mM each).
Experiments were performed with and without glutathione (GSH; 1 mM).
Data are expressed as percent of control and represent values6standard
errors of three experiments with duplicate determination. Student’s
unpaired t-test was used to evaluate the statistical significance of the
effects of GSH (* P,0.05; ** P,0.01).
fully reversible if the enzyme was stimulated with
protoporphyrin-IX. Electrochemical experiments revealed,
that dehydroascorbic acid (1 mM) was comparably potent
in scavenging NO (not shown). Finally, we tested the two
major decomposition products oxalic acid and threonic
acid for their ability to mimic the effect of ascorbate.
Neither compound significantly affected sGC activity (not
shown).
4. Discussion
The objective of the present study was to characterise
the effect of ascorbic acid on sGC using a highly purified
enzyme preparation from bovine lung. Our results demonstrate that physiological concentrations of vitamin C
potently inhibit both NO-dependent and -independent
enzyme activation, whereas the basal activity of the
enzyme is not affected.
There are several mechanisms which may account for
the observed effect. It seems likely that O 2
2 generated in
the course of ascorbate autoxidation rapidly scavenges NO
to form peroxynitrite [33]. Since peroxynitrite per se does
not activate sGC [31], this mechanism would explain
ascorbate-mediated inhibition of the DEA / NO-stimulated
enzyme as well as the protective effect of GSH. In
accordance with our results obtained with the NO-sensitive
electrode, a recent study demonstrated that accumulation
of NO in the superfusate of isolated coronary arteries is
potently diminished in the presence of ascorbate [14].
However, there must be an additional NO-independent
mechanism of inhibition, since ascorbic acid showed a
similar effect on protoporphyrin-IX-stimulated sGC. Moreover, SOD prevented the consumption of NO, but failed to
restore sGC activity (Table 1).
It seems conceivable that reactive oxygen species arising
from metal-driven ascorbate autoxidation (O 2
2 , H2O2,
OH ? ) cause oxidative damage to sGC since a variety of
proteins were found susceptible to oxidative modification
[34]. In support of this hypothesis, glutathione and the iron
chelator DTPA were found to prevent ascorbate-mediated
enzyme inhibition. However, neither SOD nor catalase or
the hydroxyl radical scavenger mannitol restored enzyme
activity. In addition, dehydroascorbic acid proved similarly
effective in decreasing sGC activity, making such a
mechanism rather unlikely.
The observation that sGC was comparably sensitive to
reduced and oxidised ascorbate implies either that enzyme
inhibition is not very selective in terms of redox state and
structure, or that a common metabolite, generated in the
course of ascorbate autoxidation downstream of dehydroascorbate, is the actual inhibitory compound. The protective effects of glutathione and DTPA are in agreement with
the latter hypothesis because both compounds prevent
ascorbate autoxidation. In the case of DTPA, the effect is
presumably due to its metal-chelating properties, whereas
for glutathione a direct redox effect might be important
[19].
Some degradation products of dehydroascorbate have
been considered to glycate amino groups of proteins,
subsequently leading to irreversible modifications including fragmentation or formation of protein cross-links
[24,35]. Although we did not detect any fragmentation of
sGC in the presence of ascorbate or dehydroascorbate (1
mM each) upon gel electrophoresis of the protein (not
shown), we cannot rule out that specific ‘‘ascorbylation’’
of critical amino acid residues of sGC might account for
the observed loss of enzyme activity. Further studies using
more sophisticated techniques would be required to settle
this issue.
In summary, our data suggest that ascorbic acid mediates inhibition of sGC via two different mechanisms. On
the one hand, NO is scavenged by O 2
2 generated in the
course of ascorbate autoxidation, a reaction that is accelerated by trace metals present in the reaction mixture. On the
other hand, a second, NO-independent mechanism appears
to operate which might involve a reactive product of
ascorbate or dehydroascorbate degradation.
Previous studies on the effects of vitamin C on sGC
yielded ambiguous results as the antioxidant was found to
potentiate [36] or to inhibit [37] enzyme activity. The
discrepancies may be due to the choice of different NO
donors and / or different redox properties of the crude
enzyme preparations used. A more recent study described
opposite effects of ascorbate and dehydroascorbate on
NO-induced vasorelaxation, as the reduced form was
found to sensitise and the oxidised form was found to
desensitise isolated coronary arteries against exogenous
A. Schrammel et al. / Cardiovascular Research 47 (2000) 602 – 608
NO [14]. From these observations it has been suggested
that the redox state of the heme iron of sGC might be
inversely regulated by ascorbate and dehydroascorbate.
These effects are in apparent contrast to our results
obtained with the purified enzyme and we have no
satisfactory explanation for this discrepancy to date. However, from our results obtained with the protoporphyrinIX-stimulated sGC, we can definitively exclude the heme
iron as the critical target in our system, since activation of
protoporphyrin-IX occurs independently of the heme.
The biological implications of these findings remain to
be established. The effect of ascorbic acid on NO / cGMP
signal transduction will critically depend on the vascular
GSH status and the efficiency of metal sequestration.
Under physiological conditions the levels of GSH and
ascorbate are in the millimolar range [38] and metals occur
primarily in non-catalytic protein-bound forms. Therefore,
negative effects of ascorbate, such as increases in blood
pressure are not expected to occur at normal cellular GSH
levels, in accordance with previous reports demonstrating a
decrease rather than an increase in blood pressure upon
supplementation of vitamin C [39,40]. However, intracellular GSH pools may be depleted in situations of oxidative
stress [41] and metals may be released from their stores.
Indeed, enhanced plasma levels of copper have been
reported in patients suffering from diabetes [42] and a
pronounced mobilisation of copper and iron was observed
following myocardial ischemia [43]. In addition, accumulation of iron in the brain due to impaired systemic iron
metabolism has been associated with Parkinson’s disease
[44]. Thus, the effect of ascorbate and / or dehydroascorbate on vascular NO / cGMP signalling may become significant under such conditions and contribute to the
severity of distinct pathologies.
[5]
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
[14]
[15]
[16]
[17]
[18]
Acknowledgements
[19]
This work was supported by grants 13211-MED, 13586MED, 13013-MED (to B.M.) and 12191-MED (to K.S.) of
¨
the Fonds zur Forderung
der Wissenschaftlichen Forschung
in Austria and by the Deutsche Forschungsgemeinschaft
(D.K.). We thank Dr. A.C.F. Gorren for helpful discussion
and Dr. B. Hemmens for critical reading of the manuscript.
[20]
[21]
[22]
[23]
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