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Cardiovascular Research 2000 47(3):602-608; doi:10.1016/S0008-6363(00)00019-5
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Copyright © 2000, European Society of Cardiology

Inhibition of purified soluble guanylyl cyclase by L-ascorbic acid

Astrid Schrammela,*, Doris Koesling1,b, Kurt Schmidta and Bernd Mayera

aInstitut für Pharmakologie und Toxikologie, Karl-Franzens-Universität Graz, Universitätsplatz 2, 8010 Graz, Austria
bInstitut für Pharmakologie, Freie Universität Berlin, Thielallee 69-73, D-14195 Berlin, Germany

* Corresponding author astrid.schrammel{at}kfunigraz.ac.at

Received 17 November 1999; accepted 11 January 2000


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
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 [{alpha}-32P]GTP to [32P]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 IC50 of ~2 µM. 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.

KEYWORDS Nitric oxide; Oxygen consumption; Second messengers; Smooth muscle; Vasoconstriction/dilatation


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
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].

NO-sensitive sGC is a heterodimer composed of an {alpha}- and a β-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 β-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 long-term administration of organic nitrates [12]. Recently a stimulation of NO biosynthesis by L-ascorbic acid has been 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 by bicarbonate [20]. The open-chained compound 2,3-diketogulonic acid undergoes further fragmentation to yield a variety of products with five or less carbons [21].


Figure 1
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  		Scheme 1 Simplified mechanism of ascorbate autoxidation.

 
Metal-catalysed autoxidation of ascorbate may lead to the formation of reactive oxygen species including superoxide (O2–), hydrogen peroxide (H2O2), 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
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
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). [{alpha}-32P]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, resistance=18 M{Omega}/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 K2HPO4–KH2PO4 buffer, pH 7.4 immediately before use.

2.2 Determination of sGC activity
Purified sGC (50–100 ng; maximal activity (vmax)~15–18 µmol/(mg·min)) was incubated at 37°C for 10 min in a total volume of 0.1 ml of a 50 mM K2HPO4–KH2PO4 buffer pH 7.4 containing 0.5 mM [{alpha}-32P]GTP (200 000–300 000 cpm), 3 mM MgCl2 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 4°C to 37°C. Incubations were terminated by ZnCO3 precipitation, and [32P]cGMP was isolated by column chromatography as described previously [30]. To determine basal activity (without activators), a higher enzyme concentration of 0.2 µg/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 µM [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 H2SO4. The electrode exhibited a linear response to up to 10 µM 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 [{alpha}-32P]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 37°C 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 values±standard error of three experiments.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
L-Ascorbic acid was found to inhibit stimulation of sGC by the NO donor DEA/NO (1 µM) with an IC50 of 2.4±0.4 µM (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 µM) 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 IC50 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).


Figure 1
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  		Fig. 1 Effect of L-ascorbic acid on stimulation of soluble guanylyl cyclase. (A) Purified sGC (0.05 µg) was stimulated with DEA/NO (1 µM) and assayed for activity in the presence of increasing concentrations of ascorbate as described under Methods. Data represent mean values±standard errors of three experiments with duplicate determination. (B) Purified sGC was stimulated with DEA/NO (1 µM) or protoporphyrin-IX (10 µM) 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 values±standard 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 µM) 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 µM) instead of DEA/NO and obtained similar results (Fig. 2B). In another series of experiments increasing concentrations of ascorbate (0.5 µM –1 mM) were added to the assay mixture prior to injection of DEA/NO (1 µM) and the release of NO was quantified as described. As shown in Fig. 2C, vitamin C decreased the peak concentrations of NO with an IC50 of ~2 µM. In the presence of the highest ascorbate concentration tested (1 mM) we measured 0.02 µM of NO.


Figure 2
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  		Fig. 2 Interference of ascorbate with NO autoxidation. (A) Release of NO from DEA/NO (1 µM) was measured with a Clark-type electrode as described under Methods. Reactions were started by the addition of DEA/NO (1 µM) 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 µM. 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 µM) was quantified under control conditions and in the presence of increasing concentrations of L-ascorbic acid (0.5 µM–1 mM). Peak concentrations of NO were plotted against the ascorbate concentration. Data represent mean values±standard errors of three experiments.

 
It is well established that ascorbic acid becomes oxidised in the presence of trace metals, thereby generating superoxide (O2–), hydroperoxide (H2O2) and hydroxyl radicals (OH•). To investigate whether NO reacts with an 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.


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  		Table 1 Effects of scavengers and chelators on ascorbate-mediated NO consumption and sGC inhibition; n=3

 
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 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).


Figure 3
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  		Fig. 3 Effect of dehydroascorbic acid on sGC activity. Purified sGC (0.1 µg) was stimulated with DEA/NO (1 µM) or protoporphyrin-IX (10 µM) 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 values±standard 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).

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
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 O2– 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 (O2–, 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 O2– 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 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 protoporphyrin-IX-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.

Time for primary review 22 days.


   Acknowledgements
 
This work was supported by grants 13211-MED, 13586-MED, 13013-MED (to B.M.) and 12191-MED (to K.S.) of the Fonds zur Förderung 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.


   Notes
 
1 Present address: Institut für Pharmakologie, Ruhr Universität Bochum, MA N1/39, D-44780 Bochum, Germany. Back


   References
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 

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