(Hypertension. 2000;36:291.)
© 2000 American Heart Association, Inc.
Scientific Contributions |
From the Evans Memorial Department of Medicine and Whitaker Cardiovascular Institute, Boston University School of Medicine, Boston, Mass.
Correspondence to Dr John F. Keaney, Jr, Whitaker Cardiovascular Institute, Boston University School of Medicine, 80 E Concord St, Room W507, Boston, MA 02118. E-mail jkeaney{at}bu.edu
| Abstract |
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Key Words: antioxidants oxidation-reduction blood vessels nitric oxide S-nitrosoglutathione relaxation
| Introduction |
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Under aerobic conditions, NO can combine with O210 or reduced thiols11 to form S-nitrosothiols, which possess bioactivity similar to authentic NO.12 13 In the intracellular space, the abundance of glutathione (GSH; 1 to 5 mmol/L14 ) renders the formation of S-nitrosoglutathione (GSNO) kinetically feasible,11 15 16 and GSNO formation has been implicated in cellular functions such as neutrophil oxidant production17 and signal transduction.18 The precise metabolic fate of GSNO and its implications for NO bioactivity, however, remain unclear.
Recent evidence suggests that S-nitrosothiol metabolism is sensitive to the local reducing environment. Reduced transition metal ions such as Cu+ catalyze the decomposition of S-nitrosothiols more effectively than do their oxidized forms (eg, Cu2+).16 19 20 Both ascorbic acid21 and GSH22 accelerate the decomposition of GSNO in vitro and may modulate the release of NO from S-nitrosothiols.23 Enhanced release of NO from S-nitrosothiols has been shown to augment the hypotensive response to these agents.24 However, the effect of ascorbic acid and GSH on the NO-like bioactivity of GSNO is not clear and serves as the purpose of the present study.
| Methods |
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(PGF2
), and all other compounds were purchased
from Sigma Chemical Co. Physiological salt solution (PSS) contained 118.3 mmol/L NaCl, 4.7 mmol/L KCl, 2.5 mmol/L CaCl2, 1.2 mmol/L MgSO4, 1.2 mmol/L KH2PO4, 25 mmol/L NaHCO3, and 11.1 mmol/L glucose. All solutions were prepared with double-distilled water and treated with Chelex before use. All glassware was acid-washed followed by thorough flushing with Chelex-treated water. Solutions of NO and GSNO were synthesized and quantified immediately before use as described.12 25 26
NO Bioassay
The NO-like bioactivity of GSNO was assessed in a PSS buffer
system with vascular relaxation as a bioassay with segments of Hartley
guinea pig thoracic aorta. Animals (450 to 500 g) were killed with
CO2, and aortic ring segments (5 mm) without
endothelium were prepared and suspended in organ
chambers as described.27 Organ chambers were acid-washed
followed by copious flushing with Chelex-treated water to minimize
contamination with metal ions. With this treatment, the buffer metal
ion content (as assessed by ascorbate oxidation28 ) was
reduced from 6±2 to <0.01 µmol/L (n=3). Animal studies were
approved by the Boston University Medical Campus Institutional Animal
Care and Use Committee.
Quantification of GSNO Degradation
Time-dependent changes in GSNO concentration were monitored
continuously at 332 nm (
=750 mol/L-1 ·
cm-1) over a period of 100 minutes with the use
of a Varian Cary 3 dual-beam spectrophotometer at 37°C. The rate of
GSNO decay as a function of time (d[GSNO]/dt)
was determined by the initial slope of the decay curve over the first
10 minutes of incubation.
| Results |
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Ascorbic acid and GSH had more uniform effects on the duration of GSNO-mediated arterial relaxation. Contracted arterial segments exposed to GSNO (0.1 µmol/L) demonstrated stable relaxation that was shortened considerably by 300 µmol/L ascorbic acid or GSNO (Figure 2A). Quantitatively, the half-time of GSNO (0.1 µmol/L) arterial relaxation (ie, the time required to restore 50% of the tension reduction produced by a vasodilator) exceeded 120 minutes (Figure 2B) and decreased to 22.5±3.5 minutes and 36.3±4.3 minutes in the presence of ascorbic acid and GSH, respectively (both P<0.05 versus control by 1-way ANOVA; n=6). Thus, both ascorbic acid and GSH appear to accelerate GSNO decomposition manifested as a shorter duration of arterial relaxation.
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Transition Metal Ions and Ascorbic AcidInduced GSNO
Decomposition
The decomposition of GSNO has been linked to reduced
transition metal ions,29 and ascorbic acid is known to
reduce the valence state of transition metal ions in
solution.30 In the presence of DTPA, a strong metal
chelator, we found that ascorbic acid had no effect on the duration of
GSNO-mediated arterial relaxation (Figure 3). Specific chelation of Cu(I) with
100 µmol/L bathocuproine sulfonate31 also abrogated
the effect of ascorbic acid, whereas the iron-specific chelator
deferoxamine (100 µmol/L) was ineffective (Figure 3).
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To examine the kinetics of GSNO decomposition, we followed GSNO decay as the change in absorbance at 332 nm. As shown in Figure 4, GSNO decay was significantly enhanced by ascorbic acid, and this effect was inhibited by chelation of transition metals with DTPA (Figure 4). The effect of ascorbic acid was specific for Cu(I), because it was inhibited by bathocuproine and not affected by deferoxamine (Figure 4). Individually, copper and ascorbic acid enhanced GSNO degradation, and their combination was additive (Figure 4). Thus, ascorbic acid enhances GSNO decomposition in a copper-dependent manner.
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Transition Metal Ions and GSH-Induced GSNO Decomposition
Arterial relaxation to GSNO (0.1 µmol/L) was
significantly shortened by GSH (Figures 5A and 5B), but this effect was only
partially inhibited by DTPA (Figure 5A) and not materially
altered in the presence of copper or deferoxamine (Figure 5B). Consistent with this observation, GSNO decay was
significantly enhanced by GSH (Figure 6).
However, the effect of GSH on GSNO decay was not significantly
inhibited by chelation of transition metals with DTPA (Figure 6). In fact, GSH actually inhibited the action of copper to
enhance GSNO decomposition (Figure 6). Thus, GSH accelerates
GSNO decay through a mechanism that appears largely independent of
transition metals.
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The availability of redox-active copper in vivo is controversial.32 We sought to determine whether a physiologically relevant source of copper (eg, copper-zinc superoxide dismutase [Cu-Zn SOD]) would support the effects of ascorbic acid on GSNO-mediated arterial relaxation. As shown in Figure 7, Cu-Zn SOD enhanced the effect of ascorbic acid on GSNO-mediated arterial relaxation, and this effect was inhibited by DTPA. In contrast, Cu-Zn SOD had no effect on the ability of GSH to shorten the duration of GSNO-mediated arterial relaxation (Figure 7).
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| Discussion |
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The role of ascorbic acid in GSNO metabolism has been examined previously. Kashiba-Iwatsuki and colleagues21 found by electron spin resonance that extracellular levels of ascorbic acid (50 to 100 µmol/L) stimulated the decomposition of GSNO in vitro in association with ascorbyl radical production and that 2 moles of GSNO were consumed per mole of ascorbic acid. The data presented here extend these observations by linking the enhanced degradation of GSNO to an increased NO flux enhancing the extent (Figure 1) of GSNO-mediated arterial relaxation and reducing its duration of action (Figure 2). In contrast to prior studies,21 33 we found that the effect of ascorbic acid on GSNO-mediated arterial relaxation was dependent on the presence of copper (Figures 3 and 4). One possible explanation for this discrepancy might relate to tissue sources of metal ions or our bicarbonate buffer system compared with the phosphate system of Kashiba-Iwatsuki and colleagues.
Catalytic quantities of both copper and iron20 are known to accelerate the decomposition of S-nitrosothiols. Specifically, the reduced forms (eg, Cu+ and Fe2+) of these metal ions are responsible for the catalysis of S-nitrosothiol decomposition.16 19 20 Because both ascorbic acid and GSH reduce copper and iron,30 it is reasonable to speculate that both agents enhance GSNO bioactivity through transition metal reduction. Although our data support such speculation for ascorbic acid, the effect of GSH on GSNO decomposition and its NO-like bioactivity was not materially altered by metal ion chelation with DTPA (Figure 5). This observation is readily explained by observations that GSH forms a redox-inactive complex with copper34 35 36 at molar ratios of GSH:Cu exceeding 2.19 37 Given that our experiments were performed with a GSH concentration of 300 µmol/L (Figures 5 and 6) and that the contaminating metal ion concentration was <0.01 µmol/L, our conditions favored GSH-Cu(I) complex formation,37 thus explaining the lack of copper dependence. Although Fe(II) supports the decomposition of GSNO,19 20 it is unlikely that GSH-mediated GSNO decomposition involved iron, because DTPA and deferoxamine had no effect on this process (Figure 6).
There are data to suggest that GSH-mediated GSNO decomposition proceeds in the absence of transition metal ions. Hogg and coworkers23 used a metal ionfree system and found that GSNO formed in the presence of excess GSH was unstable, suggesting a direct reaction between GSH and GSNO. The proposed product of this reaction was peroxynitrite, a compound with limited vasodilating bioactivity compared with authentic NO.38 39 With this scenario, one would predict that GSH-mediated GSNO decomposition would not lead to enhanced NO-like bioactivity, consistent with the results reported here (Figure 1B). Thus, metal ionindependent reaction of GSH with GSNO can account for the reduced GSNO-mediated arterial relaxation in the presence of millimolar concentrations of GSH.
One important issue to consider is the relevance of our findings for S-nitrosothiol action and metabolism in vivo. Considerable evidence indicates that S-nitrosothiols are formed in vivo, and protein S-nitrosylation has been implicated in the modulation of energy metabolism,17 signal transduction,40 apoptosis,41 and even blood flow.42 For such events to be reversible, the cellular environment must provide some mechanism(s) to restore nitrosothiols back to their reduced form. Singh and colleagues16 have proposed that GSNO serves as a sink for functional protein NO+ moieties, leading to the "repair" of S-nitrosylated proteins, and that GSH-mediated GSNO decomposition facilitates this repair mechanism. Our data and those of others19 39 indicate that ascorbic acid may also facilitate GSNO decomposition, perhaps through copper-containing enzymes such as SOD (Figure 7),43 and enhance the NO-like bioactivity of GSNO (Figure 1). Prior reports that ascorbic acid enhances NO bioactivity44 and reduces blood pressure45 have prompted speculation on the role of GSNO decomposition in these observations.
In summary, the data presented here indicate that both ascorbic acid and GSH enhance the decomposition of GSNO. In the case of ascorbic acid, accelerated GSNO decomposition is metal ion dependent and associated with a modest increase in bioactivity (eg, vasodilation) that is reduced in duration. With GSH, however, accelerated GSNO decomposition is not metal ion dependent and does not seem to result in enhanced bioactivity. Instead, the main effect is a reduction in the duration of GSNO bioactivity. These data suggest that the reducing environment within the cell has important implications for S-nitrosothiol bioactivity.
| Acknowledgments |
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Received October 20, 1999; first decision November 12, 1999; accepted February 25, 2000.
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