Chem. Res. Toxicol. 1990, 3, 71-76
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Effect of Cyanide on the Reaction of Nitroprusside with Hemoglobin: Relevance to Cyanide Interference with the Biological Activity of Nitroprusside' Dean E. Wilcox,t Harriet Kruszyna,t R o b e r t Kruszyna,$ and Roger P. Smith**' Department of Chemistry, Dartmouth College, Hanouer, New Hampshire 03755, and Department of Pharmacology and Toxicology, Dartmouth Medical School, Hanouer, New Hampshire 03756 Received July 3, 1989 The reaction of sodium nitroprusside (SNP) with deoxyhemoglobin (Hb) results in two distinct EPR-detectable species, the one-electron-reduced nitroprusside ion [ (CN),FeN0I3- and nitrosylhemoglobin (HbNO). In the presence of excess cyanide (CN-) only the signal for [(CN),FeN0I3- is observed. Thus, while free CN- does not interfere with H b reduction of SNP, it prevents transfer of the NO moiety to Hb. Electrolytic reduction of S N P under similar conditions, however, leads to [(CN)6FeN0]3-and a small amount of [ (CN),FeN0I2- resulting from loss of the CN- trans to the NO. Excess free CN- shifts the equilibrium between these two species toward [(CN),FeN0I3-, thereby reducing the concentration of [(CN),FeN0I2-. Thus, [(CN),FeN0I2- appears to be responsible for the transfer of NO to Hb. Consistent with this mechanism, both [(CN),FeN0I3- and [(CN),FeNOl2- are observed when S N P is added to erythrocyte lysates. Under these conditions HbNO is formed more rapidly due to the higher concentration of the latter species with the labile NO. This observation suggests that red blood cell constituents capable of binding CN- shift the equilibrium between the reduced S N P ions toward [(CN),FeN0l2-. In the reaction of reduced glutathione (GSH) with SNP, [ (CN),FeN0I3is formed as well as low concentrations of an EPR-detectable GSH-SNP adduct. Excess free CN- introduces a lag in the appearance of these signals, suggesting that GSH mediates SNP reduction by a different mechanism from that of Hb, although it too is inhibited by CN-. The CN- stabilization of [(CN),FeN0I3-, the reduced SNP species lacking a labile NO moiety, probably accounts for the ability of CN- to block or reverse the biological effects of SNP on aortic strips and human blood platelets. This chemical interaction appears to meet many of the criteria for competitive antagonism. By comparison, the vasodilator 3-morpholinosydnoneimine, which is a metabolite of molsidomine and which has an alkyl cyano and an alkyl nitroxide group, releases NO by an entirely different mechanism since free CN- has no effect on its biological activity.
Introduction Sodium nitroprusside (SNP),2Na2[(CN),FeN0], is employed as an emergency antihypertensive agent and in cases of severe cardiac failure. It and a variety of other compounds (sodium nitrite, sodium azide, hydroxylamine hydrochloride, glyceryl trinitrate, other alkyl nitrates, certain N-nitrosamines, etc.) are postulated (1)to induce vasodilation through a putative common intermediate, nitric oxide (NO), which activates guanylate cyclase and the pathways resulting in smooth muscle relaxation and the inhibition of aggregation and adhesion of platelets. Pertinent to this biological activity, the reaction of SNP, and other so-called nitrogenous vasodilators, with RBC under reducing conditions results (2) in various nitrosylated forms of hemoglobin. However, the aerobic reaction of SNP and HbOz has been shown (3) to result in the formation of MetHb and the complete decomposition of SNP. All 5 CN- equiv of SNP are released, and one of these is found complexed with the oxidized heme of MetHb to yield CNMetHb. If, however, excess free CNis present at the start of the reaction, the decomposition of SNP is inhibited. Although the Hb02 (or Hb) is oxidized, only the CN- trans to the NO ligand on SNP is
* Author to whom correspondence should be addressed. Dartmouth College.
* Dartmouth Medical School. 0893-228x/90/2703-0071$02.50/0
labilized as evidenced ( 4 ) by its exchange with 13CN- in solution. This effect of cyanide on the reactivity of SNP may form the basis for CN- reversal or inhibition of the effects of SNP in various biological systems (5). The interaction between these two compounds exhibits many of the characteristics of competitive antagonism. The antagonist (CN-) has no action of its own in the concentrations used, the effects of both the antagonist and agonist (SNP) are reversible, the antagonist is selective in affecting some but not all of a group of similar agonists, and the antagonist produces parallel shifts to the right in log dose-response curves for the agonist (6, 7). But true competitive antagonism can occur only if the agonist and antagonist act at the same drug receptor site. It has long been assumed, however, that SNP is a directly acting agonist, meaning that it does not act through a drug receptor. Instead, it is presumed to act directly on the biochemical pathway that is responsible for smooth muscle relaxation or the inhibition of platelet aggregation. A direct chemical reA preliminary report on these findings was made at the 28th Annual Meeting of the Society of Toxicology, Atlanta, GA (27). Abbreviations: SNP, sodium nitroprusside; Hb, deoxyhemoglobin; Hb02, oxyhemoglobin; MetHb, methemoglobin; HbNO, nitrosylated hemoglobin; RBC, red blood cells; SIN-1,3-morpholinosydnone imine; IEF, isoelectric focusing; EPR, electron paramagnetic resonance; NMR, nuclear magnetic resonance; GSH, reduced glutathione; GSSG, oxidized glutathione. 0 1990 American Chemical Society
72 Chem. Res. Toxicol., Vol. 3, No. 1, 1990 & 0-N-T-Y n
onN-p-5H
u
Wilcox et al.
N+C=NH
-H+
‘0’
YO
CN
1’:
A
2.000
SIN - 1A
SIY - I
H-
F i g u r e 1. Structure of SIN-1 and its pH-dependent tautomer, SIN-1A.
100 G
action between an agonist and an antagonist also superficially resembles competitive antagonism, but CN- does not react directly with SNP. Thus, the biological interaction between CN- and SNP apparently involves neither a receptor nor direct chemical reaction. To characterize further the mechanism by which SNP exerts its biological activity, we have investigated the effect of CN- on the reaction between SNP and Hb. This reaction in the absence of CN- results in the decomposition of SNP and the formation of HbNO. These and similar data for the effect of CN- on the reaction of SNP with GSH and with lysates have allowed us to describe what may be a unique type of drug-xenobiotic interaction between SNP and CN-. For comparison, we have also examined a metabolite of molsidomine, SIN-1 (Figure l),a vasodilator that contains an alkyl cyano group and that spontaneously releases NO (8).
”.- I
Experimental Section Washed human RBC were added to an equal volume of distilled water and frozen and thawed twice to produce complete hemolysis. In some experiments these lysates were used directly, whereas in others the lysates were centrifuged a t 25000g for 30 min a t 5 “C and the supernatant was removed for further purifcation. The supernatant was then subjected to preparative IEF. The HbOz fraction was eluted and reduced in volume in a Minicon concentrator. This preparation was shown to be free of GSH and GSSG (4),and demonstrated to be pure by analytical IEF. The HbOz concentrate was diluted with 0.1 M phosphate buffer, p H 7.4, to 1.3 m M in heme(I1) and purged with 02-free Nz in a tonometer a t 20 “C. Either NaCN in buffer or plain buffer was added by injection through a rubber septum in a side arm of the tonometer followed by injection of SNP. The tonometer was again purged with Nz for 1min before resealing. Samples were removed anaerobically via the septum a t periodic intervals and frozen immediately in liquid Nz for E P R analysis. Solutions of GSH (3 mM in buffer) were also reacted with S N P according to the same anaerobic protocol. The experiments with S N P and suspensions of human blood platelets, aggregated with either epinephrine or ADP, were as previously described (6). The experiments using SIN-1 and rabbit aortic strips were also as previously described (5). The electrolytic reduction of S N P (1.6 mM in 0.1 M phosphate buffer, pH 7.4) was carried out with vigorous N2 purging by using Pt electrodes a t 25 V and 100 mA; samples were periodically removed under anaerobic conditions and frozen in liquid nitrogen for E P R analysis. The EPR spectra were obtained on a Bruker ESP300 X-band EPR spectrometer operating a t 20-mW microwave power, 100-kHz modulation frequency, and 5-G modulation amplitude. Samples were frozen a t 77 K in a Wilmad EPR Dewar. The EPR spectra were analyzed by subtracting the signals of the individual reduced S N P species obtained from the electrolysis experiment. Power saturation analysis of the S = l / z E P R signals from HbNO and [ (CN)5FeN0]3-indicated there is negligible saturation of these signals using the above spectrometer settings. This allowed quantitative comparison of the concentrations of the EPR-detectable species by double integration of their EPR signal and comparison to the doubly integrated signal from samples of known HbNO concentration as previously described (2) using the Bruker E S P software.
Results A series of experiments was undertaken to investigate the reactions of SNP with Hb, GSH, and erythrocyte lysates and the effect of CN- on these reactions. All reac-
E
0.3
W
E 0.2
.-
& 0.1 0.0 0
50
100
150 200
250
300
Time (min) F i g u r e 2. (A) 77 K E P R spectra taken a t various times during the reaction of 1.6 mM S N P with 1.3 mM H b heme: (a) 4 min (intensity X 8); (b) 18 min; (c) 60 min; (d) 120 min; and (e) 300 min (intensity X (a’) 77 K EPR spectrum of [(CN)5FeN0]3obtained by anaerobic electrolysis (25 V and 100 mA for 90 min) of a 0.1 M phosphate pH 7.4 solution, 1.6 mM in SNP. (B) Plot of the concentration of EPR-detectable species for this reaction in the absence of excess CN- [total signal intensity (0with boldface line) and intensity of the [(CN),FeNO]” EPR signal (0 with lightface line)] and in the presence of 1.3 mM NaCN [total signal intensity ( A with boldface line) and intensity of the [(CN)SFeN0]3-EPR signal (A with lightface line)] as a function of reaction time.
tions, except where indicated, were performed under N2 and were monitored by EPR spectroscopy, which has the capability of detecting and quantifying reduced forms of SNP, GSH radicals, and various forms of HbNO. Further, in contrast to other studies (9,lO) of these reactions, aliquots were removed at various times and frozen in liquid nitrogen to stop the reaction. The EPR spectra taken on these frozen aqueous samples allowed us to follow the concentrations of the various paramagnetic species as the reaction proceeded. Reaction of SNP with Hb. Figure 2A shows the 77 K EPR spectra taken at various times during the reaction of 1.6 mM SNP with Hb (1.3 mM in heme). Early in the reaction the EPR features of a reduced SNP species with g, = 2.003, a,(14N) = 30 G, and gll = 1.930 are observed; these spectral parameters are confirmed by comparing (Figure 2A) this weak signal to one obtained during SNP electrolysis (see below). By quantitative comparison to reported (11) 77 K EPR spectra of reduced SNP species, this signal is due t o the ion [(CN)5FeN0]*. Thus, electron transfer between SNP and Hb leads to this one-electron-
Chem. Res. Toxicol., Vol. 3, No. 1, 1990 73
Reduced Nitroprusside and Excess Cyanide I
1
A
g = 2.000
I
9 = 2.000
100 G
io0 G
H -
c i c
Figure 3. 77 K EPR signal (-) from an aliquot of an aqueous buffered solution of SNP after 6 min of electrolysis at 25 V and 100 mA under a N2 atmosphere. The EPR signal resulting from subtraction of the spectrum of [(CN),FeN0I3- (Figure 2A, spectrum a') is also indicated (- - -).
tl reduced SNP species. As the reaction proceeds, the broad signal, seen even within the first 5 min and characteristic of HbNO, rapidly grows with eventual loss of the [(CN),FeN0I3- signal after 2 h. The shape of the HbNO signal suggests that initially the valency hybrid species, (NOa2+P3+)2, is formed but that eventually the fully reduced and nitrosylated Hb tetramer (Figure 2A, spectrum e) is obtained (2). An analogous reaction between SNP and MetHb exhibited no EPR signals, indicating that the observed paramagnetic species result from a redox reaction between the reduced heme iron and SNP. If the same reaction is run with the addition of 1.3 mM NaCN, the total signal intensity is greatly reduced, amounting after 5 h to only 15% of that in the absence of CN-. Figure 2B shows the relative total signal intensity and the intensity of the [(CN),FeNO]* signal for the reaction in the presence and absence of CN-. The concentration of [(CN)5FeN0]3-is observed to be similar in magnitude whether CN- is present or not; however, in the presence of CN-, only after 2 h does the HbNO signal become the major EPR-detectable species. Thus, although cyanide does not inhibit reduction of SNP by Hb, it suppresses the nitrosylation of Hb by SNP. Electrolysis of SNP. To investigate the EPR-detectable species resulting from reduction of SNP under our experimental conditions, an aqueous buffered solution of SNP, vigorously purged with N2, was subjected to electrolysis. Figure 3 shows the 77 K EPR signal of this solution after 6 min of electrolysis. The dominant signal is that of [(CN),FeN0I3-; however, weaker features are observed at a slightly lower magnetic field. Longer periods of electrolysis lead eventually to the loss of the weaker features and a clean EPR spectrum of [(CN),FeN0I3-. Subtraction of the [(CN),FeN0I3- signal from the spectrum after 6 min of electrolysis results in another EPR signal (Figure 3) with g, = 2.038, a,(14N) = 14.5 G, gll = 2.008, and aI1('*N)= 16 G. This signal is quantitatively very similar to that observed (12) for [(CN),FeNOl2- resulting from loss of the CN- trans to the NO. Thus, reduction of SNP in aqueous pH 7.4 solution results in small amounts of [ (CN)4FeN0]2-,as well as [(CN),FeN0I3-. Reaction of SNP with GSH. Since one of the major biological reductants present in erythrocytes is GSH, we have investigated the reaction of SNP with GSH and the effect of CN- on this reaction. Figure 4A shows the 77 K EPR spectra taken at various times during the reaction of 1.3 mM SNP with 3.1 mM GSH. Initially, there is only
0
20
40
60
80
Time (min)
100
120
Fi,ure 4. (A)77 K EPR spectra taken at various times during the reaction of 1.3 mM SNP with 3.1 mM GSH: (a) 4 min (intensity X 8); (b) 8 min (intensity X 8);(c) 18 min; (d) 60 min; (e) 120 min. (B) Plot of the concentration of EPR-detectable species for this reaction in the absence of excess CN- [totalsignal intensity (0with boldface line), intensity of the [(CN)$eNO]" EPR signal (0with lightface line), and intensity of the g = 2.026 signal (-@-)I and in the presence of 1.3 mM NaCN [totalsignal intensity (A with boldface line), intensity of the [(CN),FeNO]" EPR signal (Awith lightface line), and intensity of the g = 2.026 signal (--A-)] as a function of reaction time.
a weak EPR signal at g = 2.026, which persists throughout the course of the reaction. The other EPR signal that appears is that of [(CN),FeN0I3-, which is the major species after 20 min. The g = 2.026 feature does not correspond to an expected GSH radical species, and we associate it with a paramagnetic GSH adduct of SNP. The concentration of this species relative to [ (CN)6FeN0]3after 1h is noted to be similar to that of [ (CN)4FeNO]2during the first several minutes of the electrolysis experiment (Figure 3). Numerous studies (13-15) have investigated the reaction of SNP with thiols, and a 520-nm absorption band associated with SNP-thiol adducts has been used (16) as a quantitative test for GSH. To determine if there is a correlation between the g = 2.026 species we observed in the anaerobic SNP reaction with GSH and any paramagnetic species resulting from the aerobic reaction, we used EPR to follow the aerobic reaction of SNP with GSH at pH 7.4. We observed (data not shown) only a weak EPR signal at g = 2.026 which is very similar to the signal seen in the anaerobic reaction; the time dependence of the intensity of this signal, however, does not correlate with the intensity of the 520-nm absorption band. Since the EPR signal of [(CN)$eNO]* is not observed, the reduced SNP ion appears to be more susceptible to oxidation than the
74
Chem. Res. Toxicol., Vol. 3, No. 1, 1990
Wilcox et al.
1 (J'2000
lo9 (DR-I): I / I Iog[CN']
+
5 78
3
109
-6
B
0.41
0
I
50
100 150 200 250 300
Time (min) Figure 5. (A) 77 K EPR spectra taken at various times during the reaction of 1.6 mM SNP with lysates which are 1.3 mM in hemoglobin heme and 0.2 mM in GSH: (a) 4 min; (b) 13 min; (c) 18 min; (d) 60 min; (e) 120 min. (B) Plot of the total concentration of EPR-detectable species for this reaction in the absence of excess CN- (A with boldface line) and in the presence of 1.3 mM NaCN (A with lightface line) and the total concentration of EPR-detectablespecies for the reaction of 1.6 mM SNP with 1.3 mM Hb in the absence of excess CN- (0with boldface line) and in the presence of 1.3mM NaCN (0with lightface line) as a function of time.
paramagnetic species giving rise to the g = 2.026 signal. Similar experiments at pH 8.0, however, exhibited the EPR signal of [(CN)@eNO]* and a different weak paramagnetic signal. The EPR-detectable products of this aerobic reaction and their stability appear to be quite pH dependent. The effect of 1.3 mM NaCN on the total EPR signal intensity and the concentration of the EPR-detectable species in the anaerobic reaction of SNP and GSH is shown in Figure 4B. Excess cyanide dramatically retards the appearance of all EPR-detectable species. However, by 1 h the signals of [(CN)5FeN0]3-and the GSH adduct are clearly observed and their concentrations appear to be comparable. Thus, the major effect of cyanide is to introduce a lag period in the reaction between SNP and GSH. Reaction of S N P with Erythrocyte Lysates. For comparison between the reaction of SNP and purified Hb and the reactions of SNP in intact RBC, EPR was used to monitor the reaction between SNP and lysates under anaerobic conditions. Figure 5A shows the 77 K EPR spectra taken a t various times during the reaction of 1.6 mM SNP with 1.3 mM lysates, which contain 0.2 mM GSH quantified by HPLC ( 4 ) . Early in the reaction the EPR signals of both [(CN),FeN0I2- and [ (CN)5FeN0]3are observed; however, both signals diminish within 1 h,
(KcM-1
I
-5
log [CN-]
-4
-3
Figure 6. Schild plot for the inhibition of human platelet aggregation by SNP and the antagonism of that effect by CN- (6). The value derived for the "dissociation constant" for CN- from the "receptor" WBS 6.9 X lo4 M. The slope in such a plot should equal 1.0, and the value found here was 1.11. The correlation coefficient ( r ) was 0.73 with p = 0.097 (17).
to be replaced by the signal due to HbNO. Figure 5B shows the total EPR signal intensity for this reaction in the presence and absence of 1.3 m M NaCN. For comparison, the same data for the SNP reaction with purified Hb are also shown. In addition to the signal due to [(CN),FeNO]*-, which appears during the early portion of this reaction, the other unique feature of the reaction of SNP and lysates is the more rapid formation of HbNO than is observed with purified Hb. In the presence of 1.3 mM CN-, the reaction of SNP with lysates is indistinguishable from that with purified Hb; only the [(CN)6FeN0]3-EPR signal appears during the early portion of the reaction, and very little HbNO is formed. CN- Effect on S N P and SIN-1 Pharmacology. Data obtained on the inhibition of human blood platelet aggregation (6) with SNP and its antagonism by CN- can be fitted to a Schild plot (Figure 6), indicating that the dose ratio is a linear function of the antagonist concentration (17). Such a result is consistent with a mechanism of competitive antagonism between SNP and CN-. In other experiments, rabbit aortic strips, contracted by 1X to 1 X lo+ M norepinephrine (5),were relaxed to approximately an equal extent by either 1 X lo+ M SNP or 1 X lo4 M SIN-1. The relaxation induced by SNP was partially reversed by (1-2) X lo-, M CN-, whereas CNconcentrations as high as 3 X lo-, had no effect on the relaxation produced by SIN-1 (data not shown). Discussion It has been shown (2) that SNP will nitrosylate Hb under anaerobic in vitro conditions, and there is reason to believe that this chemical reaction is relevant to the vasodilatory action of SNP. However, this reaction also results ( 3 ) in the decomposition of SNP and the release of 5 equiv of CN-. Since MetHb is formed in the reaction, electron transfer from Hb to SNP is inferred. However, little else is known about the mechanism of the reaction. This study was undertaken to gain further insight into the biologically relevant NO-transfer reaction and the effect of cyanide on this chemical transformation. One group has used ambient temperature solution EPR to suggest (9,18) that reduction of SNP by Hb results in [(CN),FeN0I2-.
Reduced Nitroprusside and Excess Cyanide
Chem. Res. Toxicol., Vol. 3, No. 1, 1990 75
Scheme I Our results, obtained by using anaerobic techniques and frozen solution EPR, indicate that a more complicated GS- + [(CN)5FeN0]2mechanism is involved. [(CN)5FeN(SG)O]3[(CN),FeN(SG)0l2- + CNThe 77 K EPR signal of HbNO is clearly distinguishable from other signals observed. However, spectroscopic differences between the fully nitrosylated Hb tetramer and the two valency hybrids, Le., ( N O ~ t ~ + for l ~(+~)t~~ + N O f l ~ + ) ~ , ’12GSSG + [(CN)5FeN0]3- 5 [(CN)4FeN0]2-+ ‘/,GSSG all of which contribute to the observed spectrum (19), with [(CN)5FeN0]3-or [(CN),FeN0I2-. Others have also prevent the subtraction and quantitation (except by difnoted (13) an additional weak EPR signal in the reaction ference methods) of the HbNO EPR signal. of thiols with SNP. We find that the g = 2.026 species and The EPR spectral properties of reduced forms of SNP [ (CN)5FeN0]3-are susceptible to aerobic oxidation; howhave been the subject of numerous studies since their first ever, higher pH conditions alter the EPR signal of this detection (20). For purposes of this study it is important paramagnetic thiolate adduct and stabilize [(CN),FeNO]* to consider the 77 K EPR signals found in frozen aqueous toward oxidation. solution. The two EPR signals of reduced SNP that we The effect of CN- on the GSH reaction with SNP is observe were first chemically and spectroscopically anamanifested as a lag period in the production of EPR-delyzed by Van Voorst and Hemmerich (20, who showed tectable species. Scheme I, where glutathione is reprethat these signals interconverted as a function of pH and sented by GS-, explains this phenomenon. If the primary suggested that they arise from [(CN),FeN0I3- and pathway for formation of [ (CN),FeN0I3- is through the [ (CN),FeNOHI2-. Subsequent work (22) with the crysintermediate [(CN),FeN(SG)OI2-,then CN- should inhibit tallographically characterized (12) complexes of its formation. Relevant to this point, it has been estimated [ (CN),FeN0I2-and studies (23)using nonaqueous solvents, (25)that -90% of the thiolate-SNP adduct exhibiting the however, indicated that the two signals were due to 520-nm absorption decays through a pathway involving the [(CN),FeNO]* and [(CN),FeN0I2-,the latter arising from species [ (CN),FeN(SR)0I2-. Binding of thiolate, presumloss of the CN- trans to NO. This assignment of the ably to the electrophylic N, appears to enhance the lability EPR-detectable species has been put on firm ground of the trans-CN-. The end of the lag phase indicates the through a study (11) of the radiation-induced electron loss of excess CN-, possibly through the reaction (25) beaddition to SNP in glasses at 77 K and analysis of the tween GSSG and CN-: effect of different glasses and annealing on the EPR signals of these reduced SNP species. GSSG + CN- GS- + GSCN (3) Our data for the reaction of SNP with Hb indicate fast (Figure 2) that reduction of SNP accompanies the nitroGSCN cyclic products (4) sylation of Hb. The lack of redox reactivity between SNP Small amounts of GSH, produced even in the presence of and MetHb demonstrates that the reduced heme Fe(I1) cyanide, would be involved in this reaction; upon conis the one-electron reductant of SNP. 13CNMR data have sumption of excess CN-, the reaction between GSH and been presented (9) as evidence for an SNP-Hb adduct, SNP would proceed as in the absence of CN-. which may be the precursor complex for inner-sphere The SNP reaction with erythrocyte lysates, which conelectron transfer. The effect of excess cyanide on this tain both GSH and Hb, is important for modeling SNP reaction indicates that while CN- does not prevent the chemistry in the intact red blood cell. The reaction is one-electron reduction of SNP to [(CN)5FeN0]3-by Hb, similar to that with purified Hb and results in the forit does interfere with the transfer of NO to reduced Hb. mation of HbNO. However, the more rapid formation of The concentration of [ (CN),FeN0I3- in aqueous solution HbNO, especially at the start of the reaction, and the depends on the equilibria: observation of the EPR signal from [ (CN)4FeN0]2-suggest [(CN)5FeN0]3-+ [(CN),FeN0I2- + CN(1) that the equilibrium between [ (CN),FeN0I3- and [(CN),FeNOl2-is shifted more toward [(CN)4FeN0]2-,the H+ + CN- + HCN (2) species responsible for nitrosylation of Hb. While a lower pH could cause this shift (eq 2), the use of highly buffered It has been estimated (24),on the basis of optical measolutions in these experiments suggests that there is some surements, that the equilibrium constant for eq l is 6.8 constituent in lysates that is binding the labile trans-CN-. (f1.9) X lo-, in the pH range 6.9-7.8. We have shown by Addition of excess CN-, which is comparable to the case electrolysis of SNP that under our experimental conditions when CN- accumulates from decomposition of SNP, ovthe amount of [(CN),FeN0I2- present in solution is deercomes this CN- “sink”, and the reaction with lysates now tectable by EPR. This is not inconsistent with the small quantitatively resembles that with purified Hb, Le., there equilibrium constant since the electrolysis solution was is little nitrosylation of the Hb. Lysates contain many purged with N2 which would lead to loss of HCN and a substances capable of binding CN- such as heme enzymes shift of the eq 1equilibrium to the right. Since the effect (catalase), GSSG, NADH, etc., and other evidence exists of additional cyanide would be to decrease the concento support the concept of a CN- “sink” in various tissues tration of [ (CN),FeN0I2- in solution, CN- inhibition of the formation of HbNO suggests that [(CN)4FeN0]2-is the (26). These experiments have indicated that it is reduced SNP species responsible for the nitrosylation of [(CN),FeNOl2-, the reduced S N P species that has lost the Hb. CN- trans to the NO, which has a labile NO moiety and The anaerobic reaction of SNP with GSH confirms is responsible for nitrosylation of Hb and other biological (Figure 4) that this physiologically important reducing constituents. The species resulting from loss of NO from agent also reacts with SNP to give primarily [(CN),FeN0J2-is suggested to further decompose to give [(CN),FeNOl3-. We find no evidence for [(CN)4FeN0]2the additional 4 equiv of CN- observed in the aerobic in contrast to a report (10) that this reduced SNP ion is reaction of S N P and Hb. CN- specifically inhibits the SNP formed in the reaction of GSH and SNP. The weak signal nitrosylation of Hb by lowering the concentration of at g = 2.026 observed throughout the reaction arises from a different paramagnetic species, possibly a GSH adduct [(CN),FeN0J2- through shifting the equilibrum (eq 1)
1
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-
-
76 Chem. Res. Toxicol., Vol. 3, No. 1, 1990
between it and [(CN),FeN0I3- toward the latter species. This explanation of the chemical mechanism translates directly to the phenomenon observed when CN- reverses the biological effects of SNP in human blood platelets or on aortic strips. Although, as shown here, this interaction satisfies many of the formal criteria for competitive antagonism, such cannot be the case because no drug receptor for these species is involved in this system. Nor can the phenomenon be ascribed to a direct chemical inactivation of SNP by CN-, which would also have kinetic features like those of competitive antagonism, because CN- does not react with SNP. Instead, excess CN- stabilizes a reduction product of SNP, [(CN)5FeN0]3-,which does not release NO, the biologically active moiety. For comparison with SNP, which possesses NO and CN- bound to a metal ion, none of these factors play a role in the biological activity of SIN-1 or its pH-dependent tautomer (Figure l), SIN-lA, where the organic cyano group does not affect its release of NO.
Acknowledgment. This work was supported in part by Grant HL 14127 from the National Heart, Lung and Blood Institute (R.P.S.). The Bruker ESP-300 spectrometer was purchased with funding from the NSF (Grant CHE-8701406). We thank Hoechst-Roussel Pharmaceuticals, Inc., for gifts of molsidomine and SIN-1. We are grateful for the critical suggestions of Dr. E. Lucile Smith, Department of Biochemistry, Dartmouth Medical School.
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