Proc. Nati. Acad. Sci. USAVol. 88, pp. 10860-10864, December 1991Biochemistry

Reversible conversion of nitroxyl anion to nitric oxide bysuperoxide dismutase

(endothelium-derived relaxing factor/catalase/3-morpholinosydnonimine hydrochloride)

MICHAEL E. MURPHY* AND HELMUT SIEStInstitut fur Physiologische Chemie I, Universitift Dusseldorf, Moorenstrasse 5, D-4000 Dusseldorf 1, Federal Republic of Germany

Communicated by Bruce N. Ames, August 16, 1991

ABSTRACT Superoxide dismutase (SOD) rapidly scav-enges superoxide (O°) and also prolongs the vasorelaxanteffects of nitric oxide (NO), thought to be the endothelium-derived relaxing factor. This prolongation has been ascribed toprevention ofthe reaction between °2- with NO. We report thatSOD supports a reversible reduction of NO to NO-. Whencyanamide and catalase were used to generate NO- in thepresence ofSOD, NO was measured by the conversion ofHbO2to MetHb. When SOD[Cu(I)] was exposed to NO anaerobi-cafly, NO- was trapped by MetHb forming nitrosylmyoglobin.When NO was generated by 3-morpholinosydnonimine hydro-chloride in the presence of SOD, NO- or a similar reductantwas formed, which reduced catalase compound II and pro-moted the formation of the catalase[Fe(H)J-NO complex. It is,therefore, conceivable that SOD may protect NO and endo-thelium-derived relaxing factor by a mechanism in addition toO° scavenging and that NO- may be a physiologically impor-tant form of endothelium-derived relaxing factor.

Nitric oxide is thought to be the endothelium-derived relaxingfactor (EDRF), a vasodilator produced from arginine. Thesimilar chemical properties ofNO and EDRF, including theirapparent reactivity with °2, support this proposal (1-4). Thereaction ofNO with O2 (reactions la and b) has been directlyobserved by using pulse-radiolysis (5), but evidence of thereaction of EDRF with °2 is indirect. Namely, superoxidedismutase (SOD) prolongs the effects of both EDRF andexogenous NO, whether or not there is a simultaneousaddition of compounds thought to generate O°. This consis-tent effect of SOD has been attributed (1-4) to its knownability to catalyze 0° dismutation (6).

NO + °2 4 ONOO- [la]

ONOO- NO0 [lb]

One report (5) suggests the rate constant for reaction lamay be much slower than diffusion limited (-56 x 106M-1 s-1 at 370C). Also, the intracellular concentration of °2is estimated to be quite low [e.g., in liver <66 pM (7)], andbasal release of O2 from cultured endothelial cells was toolow to be measured (8). Although all of these estimates aretentative, there is a lack of direct evidence that sufficient 02is released from endothelial cells to account for a significantportion ofNO destruction. Indeed, the reactions ofNO with02 (reactions 2 and 3) may be the major route of NOdecomposition, at least in vitro.

H202NO+O2-~2N02- NQ +NO + 2H [2]

H20N02+NO ±N2 3-2NO2 + 2H [3]

Analyses of the metabolites ofNO in vivo also fail to provethat reaction la is significant. Although peroxynitrite(ONOO-) can decompose to NO2 and 0OH in the absence ofSOD (9) or may convert to NO' in its presence (10), nitrate(NO-) is still a major product of reaction la (5, 9). Therefore,when reaction la is significant, SOD should not only protectNO but should also decrease NO production in favor ofNOand NO-. However, product analyses have failed to meet thelatter expectation (11, 12) and led us to examine reactionsinvolving SOD and NO in more detail.

METHODS3-Morpholinosydnonimine hydrochloride (SIN-1) was fromCassella AG (Frankfurt), CuZn-SOD (from bovine erythro-cytes), cyanamide, and hydroxylamine were from Sigma, andcatalase (from bovine liver) was from Boehringer Mannheim.HbO2 was prepared by reduction of MetHb (Aldrich) withdithionite, oxygenation, and purification on a Sephadex G-25column (in Krebs/Hepes buffer containing 140mM NaCl, 2.7mM KCl, 0.42 mM NaH2PO4, 1 mM MgCl2, 1.8 mM CaCI2,20 mM Hepes, adjusted to pH 7.4 at 37°C by adding NaOH)and measured at 415 nm (e = 131 mM-1-cm-1) (13). Met-myoglobin (MetMb) was prepared from myoglobin (Sigma)by oxidizing residual oxymyoglobin with equimolar SIN-1,followed by purification on a Sephadex G-25 column (in 0.1M KPO4, pH 7).NO- was trapped by MetMb under N2 (14) as follows.

CuZn-SOD (100 1, 20 mg/ml in 50 mM KPO4/1 mM EDTA)was reduced with NaBH4 (100 ul, 50mM in 0.1M NaBO3, pH10) for 3 min (15). Acetic acid (50 Al, 0.7 M) was added tobring the pH to -5 and eliminate excess NaBH4. After H2formation was complete, K2HPO4 (100 ,ul, -0.4 M) wasadded to bring the pH to 7.0. MetMb (50 ,ul, 0.5 mM) wasadded, and NO gas (Merck, >99%, for synthesis) wasintroduced over the mixture (=0.2 cm3.s-) with gentlemixing for 20 sec, followed by N2 for 1 min to removeunbound NO. The mixture was brought to 0.8 ml withdeoxygenated KPO4, and the spectrum was read immedi-ately. Authentic nitrosylmyoglobin (MbNO) was prepared byreducing MetMb to myoglobin with a slight excess of dithio-nite, followed by exposure to NO and N2 as above. Bothtrapped and authentic MbNO were unstable when exposed toair, reverting to MetMb within 30 min.

Copper-free SOD (apo-SOD) was prepared by chelationwith diethyldithiocarbamate, centrifugation, and dialysis(16). Native SOD was handled similarly, except that nodiethyldithiocarbamate was included. Reconstituted SOD

Abbreviations: SOD, superoxide dismutase; apo-SOD, copper-freeSOD; CAT-NO, catalase-nitric oxide complex; EDRF, endotheli-um-derived relaxing factor; MbNO, nitrosylmyoglobin; MetMb,metmyoglobin; SIN-1, 3-morpholinosydnonimine hydrochloride.*Present address: Department of Pharmacology, College of Medi-cine, University of Vermont, Burlington, VT 05405.tTo whom reprint requests should be addressed.

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(apo-SOD plus Cu) was prepared by mixing apo-SOD with 10AM CUSO4 and then dialyzing to remove excess copper.Native and apo-SOD were handled similarly, except that noCuSO4 was added.The rate of 2- generation by xanthine oxidase (Boehringer

Mannheim) was determined by the rate of SOD-inhibitablereduction of 20 FM ferricytochrome c (17) using E550 = 21mM-'cm-l. The rate of H202 generation by glucose oxidase(Boehringer Mannheim) was determined colorimetrically(18). The Krebs/Hepes buffer was supplemented with 1 mMxanthine and 5 mM glucose in experiments using xanthineoxidase or glucose oxidase. The rate ofNO release by SIN-1was determined by the loss of HbO2 (578-592 nm; a band ofHbO2 vs. isosbestic point with MetHb) using Ae = 11.2mM-1cm-1 (13). One unit of SOD activity was defined asyielding half-maximal inhibition of 20 JLM cytochrome creduction by xanthine plus xanthine oxidase in the presenceof 10 ,ug of catalase (6, 17). Dual-wavelength absorbancemeasurements and spectra were made using a Sigma ZWSIIspectrophotometer (Biochem, Munich).

RESULTS AND DISCUSSIONSOD Requirement for NO Formation from NO-. Hydrox-

ylamine is converted to NO by catalase in the presence of asource of H202 (19). When HbO2 is added to such a mixture,it is converted to MetHb due to a diffusion-limited stoichio-metric oxidation of HbO2 by NO, yielding MetHb and NO3(reaction 4) (20). This conversion can be followed via the lossof the a-absorption band of HbO2 at 578 nm. NO generationby a H202/hydroxylamine/catalase mixture does not requireSOD and is not influenced by its presence (Fig. 1A and Table1).

HbO2 + NO -- MetHb + NO0 [4]

On the other hand, cyanamide is converted by H202 pluscatalase toHNO (21), which deprotonates to NO- [pKa = 4.7(22)]. When HbO2 is added to such a H202/cyanamide/catalase mixture, it is converted to MetHb in the presence ofSOD but not in its absence (Fig. 1B). The loss of HbO2 alsofails to occur when either H202, catalase, or cyanamide aremissing, whereas a source of °- has no effect on this loss(Table 1). We hypothesize that SOD accepts an electron fromNO-, converting it to NO (reaction 5a and Fig. 2). Alterna-tive explanations are not excluded, however, such as areaction of NO- with 02 to form ONOO-, the conversion ofONOO- to NO+ by SOD, followed by the oxidation ofHbO2to MetHb by NO2 -

hydroxylamineCATHbO2 GI

A; 2

.005AU

1 min

SOD

cyanamideCATHbO2 W.

B ;-. -= -SOD

+ SOD

FIG. 1. Effect of SOD on conversion of HbO2 to MetHb by NO-or NO--generating systems. Reaction mixtures contained catalase(200 Ag/ml), glucose (5 mM), and HbO2 (2 I&M). NO was generatedfrom 20 ,M hydroxylamine (A). NO- was generated from 1 mMcyanamide (B). Glucose oxidase (G.O.) was added to generate H202at 17 nM s-s. Some samples contained SOD (100 pg/ml). Loss ofHbO2 was measured at 578-592 nm.

Table 1. Effect of SOD on rate of HbO2 loss in the presence of aNO-- or NO-generating system

Reaction mixture*

Catalase + glucose oxidase+ hydroxylamine + SOD- SOD- catalase- glucose oxidase

Catalase + glucose oxidase+ cyanamide + SOD- SOD- catalase or- cyanamide or

Rate of HbO2 conversionto MetHb,t nM-s-'

5.3 + 0.15.3 + 0.20.9 + 0.10.36 ± 0.01

4.6 ± 0.50.3 ± 0.1

- glucose oxidase <0.1+ xanthine oxidase 4.6 ± 0.1

*Conditions were as for Fig. 3. Xanthine oxidase (plus 1 mMxanthine) generated O2 at 7 nM-s-1.tValues are the means (±SEMs) from three experiments.

5aNO- + SOD[Cu(II)] ;± NO + SOD[Cu(I)]

5b[5]

Reaction 5a is analogous to the normal reaction of SOD, inwhich it accepts an electron from O2 (reaction 6a).

6a0j + SOD[Cu(II)] ±- 02 + SOD[Cu(I)]

6b[6]

The copper atom ofCuZn-SOD has a formal potential (E°')of +0.42 V vs. NHE (normal hydrogen electrode) at pH 7.4(23). Only limited data are available on the redox potential ofNO/NO-, but a midpoint potential of -+0.315 V vs. SCE(saturated calomel electrode) in 4 M H2SO4 can be derivedfrom one report (24). Adjusted to reflectNHE and pH 7.4, E°'of NO/NO- should be near +0.255 V; thus reaction 5a isthermodynamically feasible.

Reaction 5b, the reduction of NO to NO-, appears ther-modynamically unfavorable. However, a proton donationfrom SOD itself may facilitate a reduction of NO to HNO(reaction 7), a modified version ofreaction 5b. This reductionwould be analogous to the other normal reaction ofSOD withO2 (reaction 8), which yields H202. Reaction 5b may also becompared with reaction 6b, which occurs, although unfavor-able (25). SOD[Cu(I)] reportedly transfers an electron toother small molecules, such as H202 (26). And although onestudy reported no stable binding between NO and CuZn-SOD(27), this result does not preclude an electron transfer to NO.

NO + SOD[Cu(I)]-H+ ± HNO + SOD[Cu(II)] [7]

°2 + SOD[Cu(I)]-H+ + H+ ± H202 + SOD[Cu(II)] [8]

SOD Requirement for NO- Formation from NO. Evidencethat SOD can reduce NO to NO- was obtained by trappingNO- with MetMb, yielding MbNO (reaction 9) (14). When

SIN-1

Hydroxylamif/e

Cyanamide V 02 _

NO SOD H2O2NO- SOD(H20FIG. 2. Reaction pathways involving SOD and NO. Numbers in

parentheses refer to reactions discussed in text. Reaction 5 is thenonclassical reaction, whereas reactions 6 and 8 are the classicaldismutation pathway.

Biochemistry: Murphy and Sies

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10862 Biochemistry: Murphy and Sies

0.4

0.3

< 0.2-

0.1 -

II \ MbNO

Native SOD / \

-SOD or -NO \

450 500 550Wavelength (nm)

600

FIG. 3. Absorbance spectra ofMetMb and MbNO. Reactants andprocedures are described in Methods. SOD Cu(I), a mixture ofreduced CuZn-SOD and MetMb exposed to NO; (-SOD or -NO), thesame procedure but with SOD or NO omitted; Native SOD, nonre-duced SOD was used (NaBH4 and acetic acid were premixed beforeaddition to SOD); MbNO, authentic nitrosylmyoglobin. The refer-ence wavelength was 640 nm, and 0.1 M KPO4 buffer, pH 7, was theblank.

CuZn-SOD was reduced to SOD[Cu(I)] and mixed withMetMb, MbNO formed upon addition ofNO (Fig. 3). MbNOfailed to form when either SOD or NO was omitted. Whennative SOD [mostly Cu(II)] was used, only a small amount ofMbNO was formed. Thus, SOD[Cu(I)] appears to generateNO- from NO.

Mb[Fe(III)] + NO-+ Mb[Fe(II)]-NO [91

A series of experiments involving catalase and SIN-1 alsosuggests that SOD can reduce NO to NO-. SIN-1 producesboth NO and O2 during its thermal decomposition (28),

Hbf2 zH2

0.01AU

\ 1 minA B 1m

CATSIN-1 SIN-i14_ 4 _

HqO,Catalase

Fe(lIl)

CAT-NOFe(IlI)-NO

z 'Z C- Compound IH2 02 Fe(V) or Fe(IV)-O

Compound IIFe(IV) or Fe(Ill)-O

FIG. 5. Reaction pathways involving catalase and NO. auto,autodecomposition.

continually providing reducing equivalents that could allowSOD to convert NO to NO- (Fig. 2). The rate of NO releasefrom SIN-1, as determined by the loss of HbO2 (Fig. 4A) isunaffected by SOD (data not shown) or catalase (Fig. 4B).When SOD and SIN-1 were added together with catalase

(in any order), an absorption at 578 nm developed, eventhough HbO2 was absent (Fig. 4C, first half). Virtually noabsorbance occurred in the absence of SOD, and none in theabsence of catalase (data not shown). The absorbing com-pound was identified as the catalase[Fe(III)]-nitric oxidecomplex (CAT-NO, Fig. 5) by its characteristic a and Pbandsat 580 nm and 540 nm (ref. 29, Fig. 6). The identity ofCAT-NO was confirmed by its ability to act as an immediateand stoichiometric source of NO upon addition of eitherexcess (Fig. 4C, middle of trace) or limiting (data not shown)HbO2. The spectrum of HbO2 was distinguishable fromCAT-NO (Fig. 6).When xanthine oxidase was added to catalase, the absorp-

tion at 578 nm rose (Fig. 4D, first half). This absorbingcompound was identified as catalase compound II by itsspectrum (refs. 19, 30, Fig. 6) and its 02-dependent, SOD-inhibitable formation (ref. 30, data not shown). The concen-tration of compound II increased even further when bothxanthine oxidase and SIN-1 were present with catalase (Fig.4D, middle of trace). The subsequent addition of SOD in thepresence ofSIN-1 caused a rapid disappearance ofcompoundH and then an equally rapid formation ofCAT-NO (Fig. 4D).Three traits of the loss of compound II indicate that it is dueto a reaction with a reductant forming from a product ofSIN-1: (i) the loss is rapid, although compound II is usuallystable and unaffected by SOD in the absence of SIN-1 (refs.

0.03

CSIN-1

CAT /SOD

4

0.01

AU

1 min

0.02

00.01

SOD4

D SIN AUCAT.CAT 4 10 min4

FIG. 4. Absorbance changes at 578-592 nm induced by combi-nations of SIN-1, SOD, catalase, HbO2, and xanthine oxidase. SIN-1(200 PM), HbO2 (4 ,uM), catalase (CAT; 200 pg/ml), CuZn-SOD (100pg/ml), and xanthine oxidase (X.O., to generate 0- at 20 nM s 1)were added at the points indicated to a total of 1 ml of Krebs/Hepesbuffer plus 1 mM xanthine, pH 7.4, at 37°C. Each trace representsone recording out of at least three to four replicates. AU, absorbanceunits.

0

III

FIG. 6. Difference absorbance spectra of CAT-NO and com-pound II (vs. catalase) and HbO2 (vs. MetHb). Conditions were asfor Fig. 4. The blank spectrum for CAT-NO was recorded by using100 pg of SOD per ml and 160 pg of catalase per ml before additionof 200 AM SIN-1 (maximal absorption after -5 min). The blankspectrum for HbO2 vs. MetHb was recorded by using 4 ,uM HbO2before addition of200 ,uM SIN-1 (conversion to MetHb was completeafter -5 min). The blank spectrum for compound II was recorded byusing 160 pg of catalase per ml before addition of xanthine oxidaseto generate °- at 20 nM's-1 (maximal absorption after -30 min). Thereference wavelength was 640 nm, and no intermediate spectra wereseen (data not shown). AU, absorbance units.

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_- 400-

cmur

rl_ 200-

_<

0.00 0.05 0.101/Amount of SOD (pg/ml) -1

FIG. 7. Concentration dependence of SOD towards formation ofCAT-NO. Conditions were as for Fig. 4 using 100 pM SIN-1 and fourdifferent catalase (CAT) concentrations. Each point represents themean for two to five determinations. Lines were calculated simul-taneously using all data points and the assumption of a singlex-intercept point. AU, absorbance units.

30, 31, and data not shown); (ii) the loss is roughly linear, notfirst order, as would be expected were compound II decreas-ing only because it was no longer being formed; and (iii) therate of loss corresponds to that of SIN-1 breakdown (com-pare HbO2 loss in Fig. 4A to compound II loss in Fig. 4D).Proposed Scheme for CAT-NO Formation (Fig. 5). Catalase

normally cycles between the native and compound I [Fe(V)]states as each reacts with H202 (29-31). Compound I reactswith °- or may autodecompose to yield compound II {ferrylcatalase [Fe(IV)]} (30, 31). O2- reduces compound II to nativecatalase but only at a slow rate (30). NO-, generated fromreaction Sb, could act as a 1-e- reductant converting com-pound I to II and then compound II to native catalase (viareactions analogous to those involving 0j). Ultimately,CAT-NO accumulates because catalase is maintained in thenative [Fe(III)] form.

Additional observations lend support to this scheme. First,the ability of SOD-type compounds to promote CAT-NOformation apparently did not parallel their classic SOD ac-tivity. Fe-SOD, Mn-SOD, and free copper all possess classicSOD activity when used in place of CuZn-SOD, and all alsopromote the formation of CAT-NO (Table 2). Importantly,though, the ratio of these two activities differs, suggestingthat a single reaction does not account for both activities.Care had been taken to keep the two assay conditionsidentical in an attempt to avoid artifactual ionic effects.

Further, when supporting CAT-NO formation, SOD ap-peared to act more like a substrate than an enzyme, giving

xG

E0C-0 EZC

U) CO_n in

CO

O <

Or_

0.015

0.010 -

0.005 -

0.000 -

control= ApoSOD t C. ++

AcoSOD

Table 2. Comparison of SOD activity and promotion of CAT-NOformation by various types of SOD and copper ions

SOD activity*

Amount forAmount for half-maximal1 unit of CAT-NO

Compound SOD activity (A) formation (B) Ratio B/AtCuZn-SOD 0.21 + 0.03 pg/ml 43 ± 2 j.g/ml 205 ± 13Mn-SOD 0.26 ± 0.01 pg/ml 44 ± 6 ug/ml 167 ± 17Fe-SOD 0.36 + 0.09 pLg/ml 38 ± 8 pg/ml 104 ± 14*CuS04 0.18 ± 0.02 AM 5.1 ± 0.3 pM 29 ± 2t*Units of SOD activity were determined graphically (6, 17), and theamount of SOD or copper required for half-maximal formation ofCAT-NO was determined by the x intercept of double reciprocalplots as in Fig. 7. At least four values were used for each graph, andthe experiment was repeated three times.

tRatio was determined for individual experiments and then averaged.tSignificantly different from CuZn-SOD (P < 0.05 by analysis ofvariance with Scheffe's post hoc test).

straight lines on a double reciprocal plot (Fig. 7). However,CAT-NO formation depended on catalytically active SODbecause copper removal from the enzyme eliminated theactivity, which was restored by reconstituting the enzymewith copper (Fig. 8). Formation of CAT-NO linearly de-pended on the catalase concentration (data not shown).

Finally, although CAT-NO formation was reversed andinhibited by a source of °- (xanthine plus xanthine oxidase,Fig. 8), it was inhibited equally well by a source of H202(glucose plus glucose oxidase, Fig. 8) and, transiently, byH202 itself (data not shown). Extra SOD never allowed theformation of CAT-NO to recover, but the addition of extraSIN-1 reversed the inhibition (Fig. 8). Apparently, a highratio of H202 versus NO production rates inhibits formationofCAT-NO, and O2- does not prevent formation ofCAT-NOdirectly but rather inhibits by being a source of H202. Thus,the results indicate that °2 dismutation is not the mechanismby which SOD promotes CAT-NO formation.

Implications Toward SOD Effects on EDRF. NO- could bean intermediate form of EDRF. NO-synthesizing enzymesmight actually convert arginine to NO- (a 4-e- oxidation).SOD could support the final 1-e- oxidation to NO, eliminat-ing the need for such a step in the reaction sequence of NOsynthase (32). In this regard it is interesting that endogenousSOD is reportedly necessary for EDRF generation but not forNO- release by endothelial cells (33) and that assays used to

t xanthine oxidase

+ SIN-1

+SOD

+ glucose oxidase

+SOD

- SIN-i* t

FIG. 8. Copper requirement of SOD in the formation of CAT-NO and inhibition of CAT-NO accumulation by sources of °- and H202.Conditions were as for Fig. 4 (200 ,g of catalase per ml, 100 Ag ofSOD per ml, SIN-1 to generate NO at 14 nM-so1), with additions as indicated.apo-SOD had <0.5% of native enzyme activity, whereas apo-SOD plus Cu had "80%o activity. The xanthine oxidase generated °O at 38 nM s1.The glucose oxidase generated H202 at 34 nM-so1. Where indicated, additional SOD at 272 pg/ml (+SOD) or sufficient SIN-1 to generate NOat 62 nM s-1 (+SIN-1) was added. Each value is the mean (±SEM) of at least three experiments. AU, absorbance units.*Data differ significantly from control.tData differ significantly from the addition of the xanthine oxidase or glucose oxidase alone (P < 0.05 by analysis of variance with Scheffe'spost hoc test).

Biochemistry: Murphy and Sies

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1 pg CAT

150 pg CAT

200 pg CAT

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measure NO synthase activity sometimes include SOD (34).Furthermore, nitrosothiols, which are potent vasorelaxants(35) and, perhaps, intermediate forms of EDRF (36), mayrelease NO-, rather than NO itself, as they decompose in thepresence of other thiols (37).One previous study addressed the question of whether

NO- could be EDRF by perfusing aortic rings with cyana-mide (38). The rings did not relax, but the production ofneither NO, HNO, nor NO- was measured in this perfusionmodel. Whether NO- itself could activate guanylate cyclase,an important target of EDRF action, might depend on theoxidation state of its bound heme. By analogy to the trappingof NO- by MetMb (Fig. 3), when the heme of guanylatecyclase is in the Fe(III) state within the cell, NO- might binddirectly to yield the Fe(II)heme-NO complex, which is theactivated form (39). On the other hand, guanylate cyclase isalso active with the heme iron in higher oxidation statesanalogous to compound 11 (39), and NO- might convert theseto the inactive Fe(III) state.Our results in no way suggest that reaction 1 is irrelevant

in situations where a high local concentration of O°2 occurs,such as the immediate vicinity of activated macrophages andneutrophils (40). However, the occurrence of reaction 5opens the possibility that SOD may also prolong NO andEDRF by a second mechanism in addition to °2 dismuta-tion-namely, a reversible conversion ofNO to NO-, but thisidea cannot be further appraised before basic information isobtained on the stability and action of NO- in vivo.

Finally, SOD is claimed to suppress ischemia/reperfusioninjury and other pathologies (41) and is part of the evidencesuggesting an excessive production of °2 in these cases.Because vascular tone is a factor in reperfusion injury, it wasfurther suggested that the ability ofSOD to protect EDRF (byeliminating O ) is an important component of this protection(3, 9). However, ifSOD could potentiate the effects ofEDRFby an additional mechanism, then no source of excessive °2production need be proposed to explain the beneficial effectsof SOD.

We thank the National Foundation for Cancer Research, Be-thesda, and the Jung-Stiftung fur Wissenschaft und Forschung,Hamburg, for support.

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