Nitric Oxide Inhibits Neuronal Nitric Oxide Synthase by Interacting with ...

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DEAE-Sephacel(l.5 x 12 cm) pre-equilibrated with Buffer A. Following a wash with ..... ence of H,B plus a H,B-regenerating system, NO synthase ac- tivity increased by ... vert NO to nitrosonium (NO'), which could react with nucleo- philic groups ...

THE JOURNAL OF B I O ~ I C CHEMISTRY ~L 0 1994 by The American Society for Biochemistry and Molecular Biology, Ioc.

Vol. 269, No. 34, Issue of August 26, pp. 21644-21649, 1994 Printed i n U.S.A.

Nitric Oxide Inhibits Neuronal NitricOxide Synthase by Interacting with the Heme Prosthetic Group ROLE OF TETRAHYDROBIOPTERIN IN MODULATING THE INHIBITORY ACTION OF NITRIC OXIDE* (Received for publication, March 11, 1994, and in revised form, May 25, 1994)

Jeanette M. Griscavage, Jon M. Fukuto, Yumiko Komori, and Louis J. IgnarroS From the Department of Molecular Pharmacolom. UCLA School of Medicine, Center for the Health Sciences, Los Angeles,~C!alifornia~90024 "~

The objectiveof this study was toelucidate the mechanism by which nitric oxide (NO) inhibits NO synthase. Previous studies revealed that NO inhibits unpurified preparations of NO synthase. In the present study, the mechanism by which NO inhibits purified neuronal NO synthase from rat cerebellum was examined. The rate of L-citrulline formation from L-arginine was non-linear despite thepresence of excess substrate and cofactors and was further inhibited by 30%by 200 unitdm1 superoxide dismutase. In contrast, 30 p . oxyhemoglobin ~ increased NO synthase activity by 2-fold and made the reaction rate linear. Theseobservations were consistent with the hypothesis that enzymatically generated NO inhibits NO synthase activity. ExogenousNO (0.1-10 p ~ (but ) not NO,, nitrite, or nitrate)also inhibited NO synthase, and enzyme inhibition was not competitive with L-arginine. NO synthase inhibition by NO and other heme ligands supports the view that heme is involved in thecatalytic activity of NO synthase. Oxyhemoglobin prevented but could not reverse enzyme inhibition by NO. NOsynthase inhibition by NO wasmarkedlydiminished and reversed, however,by tetrahydrobiopterin (50 p . ~or ) a tetrahydrobiopterin-regenerating system, and the latter made the reaction rate linear. In contrast, NO synthase inhibition by NO was markedly enhanced by heme oxidants (LO p . methylene ~ blue; 3 JIM ferricyanide), and these oxidants directly inhibited NO synthase activity. These observations suggest that NO interacts with enzyme-bound ferric heme to inhibit NO synthase activity. In support of this view, NO inhibited enzyme activity in the absence of turnover, when the heme iron is in the ferric state,and this inhibition was reversed by tetrahydrobiopterin. Therefore, the oxidation state of heme iron appears to be one important determinant for the inhibitory action of NO, and tetrahydrobiopterin may increase NO synthase activity by diminishing the inhibitory action of NO.

that NO inhibits the activities of the constitutive isoforms of NO synthase fromrat cerebellum and bovine aortic endothelial cells, as well as the inducible isoform of NO synthase from activated rat alveolar macrophages (4-6). Other laboratories have confirmed these observations (7, 8). All of these initial experiments were performed, however, with unpurified preparations of NO synthase. Therefore, it was not possible to ascertain whether NO produces its inhibitory effect by a direct interactionwith NO synthaseorby an indirectmechanism involving enzymes or other constituents in the crudecytosolic preparations. The objective of the present study was to determine whether the inhibitory action of NO on NO synthase represents an indirect or direct effect on NO synthase and to elucidate the mechanism of action of NO. The approach that was taken was to study theeffects of exogenous NO, as well as enzymatically generated NO on purified preparationsof constitutive NO synthase from rat cerebellum (neuronal NO synthase). An inhibitory effect of NO on purified NO synthase would indicate that a direct interaction between NO and enzyme protein occurs, and subsequent experiments could be focused on the mechanism of this inhibitory action of NO. NO was found to elicit a marked inhibitory effect on purified preparations of NO synthase. Therefore, subsequent experiments were designed to deNO itself is theinhibitoryspeciesand terminewhether whether the mechanism of action of NO involves an interaction with enzyme-bound heme.

EXPERIMENTALPROCEDURES Chemicals and Materials-L-Arginine, L-citrulline, NADPH, NADP, NADH, flavin adenine dinucleotide, flavin mononucleotide, calmodulin, hemoglobin (human), superoxide dismutase (bovine erythrocytes), dihydropteridine reductase, potassium cyanide, dithiothreitol, triethanolamine, glycerol, methylene blue, potassium ferricyanide, phenylmethylsulfonyl fluoride, pepstatin A, leupeptin, EDTA,EGTA, and DEAESephacel were purchased from Sigma. 2',5'-ADP Sepharose 4B was obtained from Pharmacia Biotech Inc. Tetrahydrobiopterin was purDespite the current knowledge of cofactor and calcium rechased fromSchircks Laboratories (Jona, Switzerland). Solutions (2 mM quirements forNO synthase activity andthe basic oxygenation in 50 mM triethanolamine HCl, pH 7.4, containing 1 mM dithiothreitol) mechanisms involved the in conversion of L-arginine toNO plus were prepared fresh daily, and subsequent dilutions were made in di(99.5%), nitrogen dioxide L-citrulline (1-3), relatively little is known about other endog- thiothreitol-free buffer.Carbonmonoxide NO synthase activity in mam- (99.5%),and argon were obtained from Liquid Carbonic. NO gas (99%; enous factors that may modulate malian cells. Previous studies from this laboratory indicated Matheson) was purified further by passage through 5 N KOH in water to remove any NO, and then through a column of potassium hydroxide to remove any traces of water from the NO. The NO was collected in a * This work was supported by National Institutes of Health Grants glass bulb fitted with a rubber septum. Aliquots of 40 mlof gas were HL-40922, HL-35014and HL-43688,the Laubisch Fund for Cardiovas- bubbled through 1ml of distilled water contained in a small test tube cular Research, and the Tobacco-Related Disease Research Program. The costs of publication of this article were defrayed in part by the that had been vacuum evacuated for 20 min, followed by venting with payment of page charges. This article must therefore be hereby marked argon for 20 min. This procedure yields a nearly saturated solution of NO in water at 25 "C and 1 atm (approximately 1mM, as determined by "aduertisement"in accordance with 18 U.S.C. Section1734solelyto chemiluminescence).Serial dilutions were then made in oxygen-free indicate this fact. water with the aid of gas-tight syringes. S-Nitroso-N-acetylpenicilla$ To whom correspondence should be addressed.

21644

21645

Nitric Oxide Inhibition of Nitric Oxide Synthase mine (SNAP)' was prepared, stored, and used as described previously (9). Oxyhemoglobin was prepared from hemoglobin as described previously (10). DowexAGBOW-X8 (H'form; 100-200 mesh), Dowex AG 1-X8 (acetate form; 100-200 mesh), and Tris base (electrophoresis grade) were purchased from Bio-Rad. Sodiumnitrite and sodium nitrate were obtained from Fisher Chemical Co. Aquasol-2 was purchased from Du Pont NEN. Chemicals used for gel electrophoresis were obtained from Bio-Rad. Isolation and Purification of NO Synthase-Rat cerebellum was used as the source ofNO synthase. Cerebella were excised from approximately 75 rat brains (Pel-Freez Biologicals) and rinsed, and homogenates (3635% w/v) were prepared in 50 m~ triethanolamine HCl, pH 7.4, containing 0.1 m~ EDTA, 0.1 mM EGTA, 2 mM dithiothreitol, 10% (w/v) glycerol, 1 mM phenylmethylsulfonyl fluoride, 1 p~ pepstatin A, and 2 p~ leupeptin (BufferA) at 0-4 "C with the aid of a tissue grinder fitted with a ground glass pestle. Homogenates were centrifuged at 100,000x g for 60 min at 4 OC, and the supernatant was subjected to further purification by a modification of methods reported previously (11).Briefly, the supernatant was applied (2 mumin) to a column of DEAE-Sephacel(l.5 x 12 cm) pre-equilibrated with Buffer A. Following a wash with five column volumesof Buffer A, NO synthase activity was eluted with 150 ml of a lineargradient of 0-400 m~ NaCl in Buffer A (2 mumin), employing a GradiFrac system from Pharmacia. Fractions containing in excess of 60% of total NO synthase activity were pooled and applied (0.5 mumin) to a column of 2',5'-ADP Sepharose 4B (1x 2.5 cm) pre-equilibrated with 50 mM triethanolamine HC1, pH 7.4, containing 2 mM dithiothreitol, 30% (w/v) glycerol, 4 p~ FAD, and 4 PM H,B (Buffer B). The column was washed (0.5 mumin) successively with 10 column volumesof Buffer B, 12 column volumesof Buffer B containing 0.6 M NaCl, and 12 column volumes of Buffer B containing 1.1 mM NADP. NO synthase was eluted by passage of seven column volumesof Buffer B containing 30 m~ NADPH through the column (0.5 mumin). A 3-fold higher yield of NO synthase was achieved by using 30 mM instead of 10 mM NADPH to elute the enzyme. Small aliquots (25-50 pl) of NO synthase were stored at -75 "C for up to 4 weeks without appreciable loss of enzymatic activity. Enzyme aliquots were diluted in ice-cold 50 m~ triethanolamine HC1, pH 7.4, just prior to use such that the final concentration of NADPH in enzyme reaction mixtures was 100 p~ or less. Gel Electrophoresis-The homogeneity of enzyme fractions was determined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (Mini-PROTEAN I1 Electrophoresis Cell, Bio-Rad)essentially according to Laemmli (12). T h e 7.5% separating gel contained 0.375 M Tris-HC1, pH 8.8, and 0.1% (w/v) SDS, whereas the 4% stacking gel contained 0.125 M Tris-HC1, pH 6.8, and 0.1% SDS.The electrode buffer was 0.025 M Tris-HC1, pH 8.3, containing 0.192 M glycine and 0.1% SDS. Enzyme fractions were diluted 4-fold, heated at 95 "C for 5 min in 0.0625 M Tris-HC1, pH 6.8,containing 10%(w/v) glycerol,2% (w/v) SDS, 5% (v/v) 2-mercaptoethanol, and 0.001% (w/v) bromphenol blue, and applied to the stacking gel. Approximately 100 ng of sample protein were applied to each lane. The gels wererun for approximately 45 min at 60 mA/gel at constant voltage (200 V). Gels werestained using the procedure outlined in the Bio-RadSilver Stain Kit, employing high range silver stain standards. A single band of protein was observed, with a molecular mass of approximately 150 kDa. The two-column purification procedure yielded NO synthase preparations that were about 95% pure with specific activities ranging from 450 to 650 nmol L-citrulline/min/mg protein, as reported by other laboratories (11, 13, 14). Protein Determination-Protein concentrations in cerebellum supernatant fractions were determined by the Bradford CoomassieBrilliant Blue method as described by Bio-Rad. Bovine serum albumin was used as the standard. Assay of NO Synthase Activity-NO synthase activity was determined by monitoring the formation of L-citrulline from L-arginineby a modification of methods described previously (11, 15). Enzymatic reactions were conducted at 37 "C in 50 mM triethanolamine HC1, pH 7.4, containing 50 p~ L-arginine, approximately 200,000 dpm of ~-[2,3,4,53HlarginineHCl(77 Cilmmol; Amersham), 100 p~ NADPH, 10 PMFAD, 2 mM CaCl,, 1 pg of calmodulin, 10 p~ H,B, 0.1-0.5 pg of enzyme protein, and other test agents as indicated, in afinal incubation volume of 100 pl. Substrate and cofactor concentrations were modified in certain experiments as indicated. The ~-[2,3,4,5-~H]arginine HCl was purified by anionic exchange chromatography on columns ofDowexAG 1-X8, OH- form (prepared from the acetate form), to remove traces of

I

10

e

Control

o 20USOD

0

2.5

5

7.5

10

Time (min) FIG.1. Time course of L-citrulline formationby NO synthase and the influence of superoxide dismutase and oxyhemoglobin. Standard enzyme incubations were conducted as described under "Experimental Procedures," with the indicated concentrations of superoxide dismutase (SOD) and oxyhemoglobin (HbO,) added toreaction mixtures just prior t o initiation of reactions by addition of 0.11 pg of NO synthase. Values represent the mean S.E. of duplicate determinations from five separate experiments. [3H]citrulline.Enzymatic reactions were initiated at 37 "C by addition ofNO synthase and were terminated after 10 min (except for timecourse experiments) by addition of 2 ml of ice-cold 20 mM sodium acetate, pH 5.5, containing 1 m~ L-citrulline, 2 m~ EDTA, and 0.2 mM EGTA (Stop Buffer). Samples were applied to columns (1cm diameter) containing 1ml of Dowex AG50W-X8, Na' form (prepared from the H' form), pre-equilibrated with Stop Buffer. Theeluate (2 ml) was collected into a liquid scintillation vial. Columns were eluted with 2 ml of water vial and collected into another vial. Aquasol-2(10ml) was added to each and counted in a Beckman LS 3801 liquid scintillation spectrometer. L-Citrulline was recoveredin the first 4ml of Dowex column eluate to the extent of 96%, and data were correctedto account forsuch recovery. RESULTS

Influence of Superoxide Dismutase and Oxyhemoglobin on NO Synthase Activity and on the Inhibitory Action of NO-The rate of L-citrulline formation was non-linear during a 10-min incubation period at 37 "C (Fig. 1). This was not attributed to substrate/cofactor depletion or enzyme instability as reaction rates remained non-linear after increasing the concentrations of L-arginine,NADPH, FAD, and calmodulin 5-fold (not shown)

or adding a 100-fold excess of bovine serum albumin to reaction mixtures. In order to determine whether superoxide anion, a product of the enzymatic reaction (13, 16, which could also be 171, was responsible for the non-linear reaction rates, superoxdiside dismutase was added to reaction mixtures. Superoxide mutase, however, inhibitedNO synthase activity and made the rate of L-citrulline formation more non-linear(Fig. 1). To determine indirectly if enzymatically formed NO, which could be increased in the presence of superoxide dismutase, might be responsible for inhibitingNO synthase, oxyhemoglobin (a good scavenger of NO) was added to reaction mixtures. Oxyhemoglobin increased NO synthase activity and made the r a t e of L-citrulline formation nearly linear (Fig.1). In order to determine whether the NO reaction product was responsible for inhibitingNO synthase activity, exogenousNO was tested. NO elicited a potent, concentration-dependent inhibitory effect on NO synthase (Table I). SNAP, an NO donor compound (91, also elicited a concentration-dependent (1-100 PM) inhibition of NO synthase (discussed below). NO is relatively unstable and undergoes oxidation to nitrite and, in the presence of certain catalysts,nitrate (18). NO, is a likely intermediate in these oxidation reactions. Additionof 10 p~ NO,, 1 mM nitrite, or 1 mM nitrate to reaction mixtures failedto alter The abbreviations used are: SNAP, S-nitroso-N-acetylpenicillamine; NO synthase activity (Table I). Thus, NO itself is the likely H,B, (6R)-5,6,7,8-tetrahydro-~-biopterin. inhibitory species. The effects of superoxide dismutaseand oxy~

'

~~

Nitric Oxide Inhibitionof Nitric Oxide Synthase

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TABLE I Influence ofNO, superoxide dismutase, oxyhemoglobin, nitrite, nitrate, and NO, on L-citrulline formationby NO synthase Test agents“

NO synthase activity

+ 20 units of SODh

Control

+ 30 PM HbO,C

nmol L-citrullinelrninimg protein

None 0.1 p~ NO 1 VM NO 10 p~ NO 1 mM NaNO, 1 mM NaNO, 10 I.IM NO,

496 -c 359 38d 361197 .c 25 237 5 18 81 f 11 487 f 32 521 f 43 483 -c 36

f834 27

2

64

16 707 f 55

0.1 0.2 0.3 0.4 0.5 a Standard enzyme incubations were conducted for 10 min as described under “Experimental Procedures.” SOD andHbO, were added 1/L-Arginine ( W ” ) to reaction mixturesjust prior to, whereas test agents were added just after, initiation of reactions by addition of 0.15 pg of NO synthase. FIG.2. Effects of superoxide dismutase and SNAP on the K,,, for Superoxide dismutase. L-arginine and the V,, of NO synthase. Standard enzyme incubae Oxyhemoglobin. tions were conducted for 10 min as described under “Experimental Mean -c S.E. of duplicate determinationsfrom three tofive separate Procedures,” withthe indicated concentrationsof L-arginine, SNAP, and experiments. superoxide dismutase (SOD). Superoxide dismutase was added to reaction mixtures just prior to, whereas SNAP was added just after, hemoglobin on the inhibitory action of added NO (Table I) were initiation of reactions with 0.12 pg of NO synthase. Data points represent mean valuesof duplicate determinationsfrom one of five separate similar to their effects on enzymatically formed NO (Fig. 1). A experiments.

kinetic analysis revealed that NO (tested as SNAP) inhibited NO synthase by mechanisms that are not competitive with L-arginine when L-arginine concentrations were in the rangeof 2-20 m (Fig. 2). Similarly, superoxide dismutase decreased the V,, without altering theK,,, for L-arginine. In view of the relatively shorthalf-life of NO in thepresence of oxygen, the marked inhibitory effect of a one-time addition of NO to enzyme reaction mixtures suggested thatNO may cause inhibition by an irreversible mechanism. Oxyhemoglobin completely prevented the inhibitoryeffect of NO when added prior to NO, but the additionof oxyhemoglobin to reaction mixtures at 15 s after NO failed t o reverse the inhibitory action of NO (Fig. 3). Although these findings suggest thatNO inhibits NO synthase irreversibly, there areconditions under which enzyme inhibition is reversible, and this is discussed below. Comparison of the Effects of NO, Carbon Monoxide 6‘01, and Cyanide Ion on NO Synthase Activity-The three heme ligands CO (about 1 mM), and cyanide ion (10 mM KCN) each NO (1 p ~ ) , inhibited NO synthase activity by 4 5 4 5 % (not shown). NO was much more potent than either CO or cyanide, and this is consistent with the relative bindingaffinities of these ligands for heme iron-containing proteins and theview that NO interacts with the heme iron inNO synthase. At the highestconcentrations obtainable, 100 p~ NO, 80% CO, and 100 mM KCN inhibited NO synthase activity by 80-100%. Reversal of the Inhibitory Action of NO on NO Synthase by [email protected] a n d Thiols-In view of the possibility that NO inhibits NO synthase by forming a nitrosyl complex with the heme iron in NO synthase, chemical agents known t o modify the oxidation state of heme iron by reduction were tested. Dithiothreitol (5 mM), 5 mM glutathione, and 10mM cysteine partially prevented the inhibitoryaction of 10 PM NO (shift from 20% to about35% of control; not shown). Much lower concentrations of H,B (0.05 mM), however, markedly prevented the inhibitoryaction of 10 p~ NO (shift from 20% t o 70% of control). The influence of an elevated concentrationof H,B was compared with thatof other cofactors including NADPH, FAD, and calmodulin on NO synthaseinhibition byNO. Increasingtheconcentrations of NADPH (from 0.1 to 1 mM),FAD (from 0.1 to 0.5 mM), or calmodulin (from l to 5 pg) failed to modify the inhibitory action of NO (not shown). Thus, the inhibitoryeffect of NO was independent of the concentrationsof NADPH, FAD, or calmodulin. Addition of a H,B-regenerating system (100 p~ NADH plus 2.3 unitdm1 dihydropteridine reductase) toreaction mixtures containing only 10 p~ H,B nearly doubled product formation and

800

0 -NO

600

400

200

0

I

b CTRL NO

A

B

p 3 0 r”HbOpl

FIG,3. Influence of timeof addition of oxyhemoglobin on modification by NO of L-citrulline formation by NO synthase. Standard enzyme incubations were conducted for 10 min as described under “Experimental Procedures,” with the indicated concentrations of NO and oxyhemoglobin (HbO,). NO was added to reaction mixtures just after initiation of reactions by addition of 0.125 pg of NO synthase. Oxyhemoglobin was added to reaction mixtures either just prior to addition of NO synthase (A) or 15 s after addition of NO ( B ) . CTRL signifies control enzymatic activity; -NO signifies absenceof added NO. Values represent the mean -c S.E. of duplicate determinationsfrom four separate experiments.

abolished the inhibitory actionof NO (Fig. 4).Moreover, addition of this H,B-regenerating systemto reaction mixtures caused the rate of product formation to become nearly linear over 10 min of incubation a t 37 “C (not shown). In the absence of a regenerating system, the addition of 100 VM H,B produced similar results.Addition of 100 1”H,B to reaction mixtures a t 30 s after addition of NO caused a 75430% reversal of the inhibitory effect of NO on NO synthase activity, whereas 5 mM dithiothreitol in the presence of 10 PM H,B caused only 20-25% reversal (not shown). Enhancement of the InhibitoryAction of NO on NO Synthase by Oxidizing Agents-Low concentrations of methylene blue and ferricyanide ion directly inhibited NO synthase in a concentration-dependent manner (Fig. 5). Methylene blue (10 1”) and ferricyanide (3 p ~ enhanced ) the inhibitoryeffect of 1 pm NO from 38 to56%and from 38 to87%, respectively. The higher concentrations of oxidants increased the inhibitoryeffect of 1 p~ NO to 100%. The inhibitory effects of the oxidants on NO synthase activity were unaltered in the presence of 2 mM di-

Nitric Oxide Inhibition

Control

of Nitric Oxide Synthase

+ H4B Regenerating System

FIG.4. Influence of a tetrahydrobiopterin-regeneratingsystem on the inhibitory actionof NO on L-citrulline formation by NO synthase.Standard enzyme incubations were conducted for 10 min as described under “Experimental Procedures,” with the following modifications. Controlreactions contained standard concentrations (10 w) of tetrahydrobiopterin (H,B). Reaction mixtures with the H4B-regenerating system contained 10 p~ H,B, 100 p~ NADH, and 0.23 units of dihydropteridine reductase. NO, at the indicated concentrations, was added to reaction mixtures just after initiation of reactions by addition of 0.17 pg of NO synthase. -NO signifies absence of added NO. Values represent the mean S.E. of duplicate determinations from four separate experiments.

600

1

21647

+HdB - G B

rHqB-RS +H4B

-++30 sec 4 Time of L-Arginine / NADPH Addition After NO Synthase

+O

sec

FIG.6. Influence of enzymatic turnover on the inhibitory action of NO on L-citrulline formation by NO synthase. Standard enzyme incubations wereconductedfor 10 min as described under “Experimental Procedures,” with the following modifications. ~-Arginine plus NADPH were added together to reaction mixtures just prior to addition of 0.22 pg of NO synthase (0 s ) or at 30 s after addition of NO synthase, as indicated. NO was always added immediately after addition of NO synthase. -NO signifies absence of added NO; +H4B signifies addition of 10 V M H4Bprior to NO synthase; +H4B-RSsignifies addition of 10 p~ H,B, 100 V M NADH, and 0.23 units of dlhydropteridine reductase just prior to L-arginine plus NADPH. Values represent the mean 5 S.E. of triplicate determinations from five separate experiments.

0-NO

NADPH addition was made. Whenpresent, H,B was added just prior to enzyme, whereas the H,B-regenerating system was added justprior to L-arginineNADPH (approximately 30 s after NO addition). As NO is chemically labile in the reaction mixtures, it is likely that any effect of NO on NO synthase would have been elicited within several seconds after addition. NO (10 VM) inhibited NO synthase activity by the same magnitude (75% inhibition) whether it was added t o enzyme-containing reaction mixtures after, or 30 s before, addition of Larginine plus NADPH. Therefore, the inhibitory action of NO wasindependent of enzymaticturnover. In the absence of CTRL A B C A B C added H,B, NO synthase activity decreased by about 40% and Meth. Blue FeCN enzyme inhibition by NO increased t o over 90%. In the presFIG.5. Influence of methylene blue and potassium ferricya- ence of H,B plus a H,B-regenerating system, NO synthase acnide on the inhibitory action of NO on L-citrulline formation by NO synthase.Standard enzyme incubations were conducted for 10 min tivity increased by 33-75% over enzyme activity observed in as described under “Experimental Procedures,” with the indicated con- the presence of H,B alone. Moreover, addition of the H,B-recentrations of NO, methylene blue (Meth. Blue), and potassium fem- generating system t o reaction mixtures approximately 30 s ) cyanide (FeCN).Methylene blue (A, 10 PM;B , 30 PM; C , 100 p ~ and after addition of NO largely reversed the enzyme inhibition by FeCN (A, 3 p ~B;, 10 p;C, 30 p ~were ) added toreaction mixtures just prior to, whereas NO was added just after, initiation of reactions by NO, as well as the apparent instability of NO synthase incuaddition of 0.26 pg of NO synthase. CTRL signifies control reactions; bated for 30 s in theabsence of added L-arginine and NADPH. -NO signifies absence of added NO. Values represent the mean % S.E. of duplicate determinations from three to five separate experiments.

DISCUSSION

thiothreitol (not shown). Influence of Enzymatic lhrnover on Znhibition of NO Synthase by NO-The objective of this experiment was t o ascertain whether NO could inhibit NO synthase activity in theabsence of substrate and electron donor. Under such conditions the heme iron in NO synthase is oxidized, whereas it undergoes reduction duringcatalysisafter addition of L-arginine plus NADPH. Two types of experiments were conducted for Fig. 6. In the first type, a standard enzyme incubation was conducted where L-arginineNADPH and either H,B or the H,B-regenerating systemwere added t o reaction mixtures justprior to NO synthase. NO was added immediately after enzyme addition. This is representedin Fig. 6 as 0 s, signifying thatthe L-arginineNADPH addition was made just prior to enzyme addition. In the second type, NO was added t o reaction mixtures immediately after NO synthase and reaction mixtures were preincubated for 30 s, after which time the L-arginine/

The present data that 100 nM t o 10 p~ NO inhibited purified neuronal NO synthase extend earlier findings made with unpurified enzyme (4) and indicate that the inhibitory action of NO is probably attributed to a direct interaction between NO and enzyme protein. The inhibitory action of NO on NO synthase appears to be due t o NO itself rather thanto a chemically related species. In order to address the question of whether enzymatically generated NO also inhibits NO synthase activity, we examined the effects of compounds that either prolong or diminish the biological half-life of NO (19). Superoxide dismutase inhibited, whereas oxyhemoglobin enhanced, NO synthase activity. Since superoxide dismutase presumably raises, whereas oxyhemoglobin lowers, the NO concentration, these data were taken as indirect evidence that enzymatically generated NO inhibits NO synthase activity. The mechanism of action of NO as an inhibitorof NO synthase is attributed toa directinteraction between NO and some functional group on NO synthase, such asheme. NO synthase

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Nitric Oxide Inhibition of Nitric Oxide Synthase

is a hemoprotein (14,21-23), and NO has a high binding a E n ity for the heme in other hemoproteins such ashemoglobin and myoglobin (24, 25). NO also binds t o heme iron in cytochrome P-450 (26-28) and guanylate cyclase (29). Since NO synthasebound heme presumably binds and activates molecular oxygen to catalyze the oxidation of L-arginine to NO plus L-citrulline, any interference with this process by heme ligands(21-23, 30) would inhibit product formation. Indeed, NO interferes with substrate oxidation by binding to theheme of cytochrome P450 (311, and cyclooxygenase and lipoxygenase (32). An interaction between NO and enzyme-bound heme, rather than theL-arginine binding site, would be consistent with the kinetic data showing that NO does not compete with L-arginine. Other heme ligands including cyanide ion and CO inhibited NO synthase activity, as reported previously (14, 21-23, 30). NO, however, was much more potent than cyanide ion or CO. Imidazole and phenylimidazoles also inhibited NO synthase by interacting with heme iron (33). The inhibition of NO synthase by NO supports the view that heme ligands interfere with the enzymatic oxidation of L-arginine (14). The oxidizing agents methylene blue and ferricyanide ion inhibited NO synthase activity and markedly enhanced the inhibitory action of NO. The possibility that oxidants may convert NO to nitrosonium (NO'), which could react with nucleophilic groups such as thiols on NO synthase to inhibit enzymatic activity, was ruled out on the basis of the findings that the inhibitory influence of oxidants on NO synthase was not altered in thepresence of 2 mM dithiothreitol. A more reasonable explanation relates to thefact that theseoxidizing agents are known to oxidize heme iron in hemoproteins (34-371, including NO synthase (38).It is possible that the inhibition of NO synthase by oxidants is mediated by enzymatically generated NO. If NO inhibits NO synthase by forming a nitrosylheme complex, one possible explanation is that NO binds preferentially to oxidized heme iron in NO synthase. Although the reduced heme iron in hemoglobin and myoglobin has a higher binding affinity for NO than does oxidized heme iron (35, 391, the heme environment inNO synthase, like that incytochrome P450, is different from that inhemoglobin or myoglobin (14,221 and this difference may favor binding of NO to oxidized heme iron. The observations that NO inhibited NO synthase activity independently of enzymatic turnover strongly suggests that NO binds to oxidized heme iron in NO synthase, as spectral studies have shown that the heme iron in NO synthase in the absence of substrate is in the oxidized state (14,21-23,30). NO may form a more chemically stable nitrosyl complex with oxidized than reduced heme iron inNO synthase, and thiswould suggest that NO inhibits NO synthase activity by interfering with the function of the heme prosthetic group. Additional experiments, however, are necessary to test this hypothesis. In contrast to the enhancing influence of oxidizing agents, reducing agents not only prevented but also reversed the inhibitory action of NO on NO synthase. H,B was much more potent and effective than sulfhydryl reducing agents. This action differed from that elicited by oxyhemoglobin, which prevented but did not reverse the inhibitory effect of NO. A direct chemical interaction between NO and H,B is unlikely, as a direct chemical reaction between NO and H4B could not be detected inexperiments where NO loss was monitored by chemiluminescence detection.' One possible explanationfor these observations is that the reducing agents favor the maintenance of the oxidation state of enzyme-bound heme iron in the Fez+ state, and such an effect diminishes the inhibitory effect of NO on NO synthase. Assuming that NO binds preferJ. M. Fukuto, unpublished data.

entially to oxidized heme iron, H,B may elicit its effect by causing a reduction of the heme iron and facilitating thedissociation of NO from the heme complex. This view is consistent with evidence that NO forms a relatively labile nitrosyl-Fez+ heme complex with cytochrome P450 (271, in contrast to the stable nitrosyl-Fez+complex formed with hemoglobin (39).This difference has been attributed to a difference in the proximal axial ligand between heme iron and the protein. A histidine ligand is involved in the binding of heme iron to hemoglobin and myoglobin, whereas a thiolate ligand is involved in cytochrome P450 (40) and NO synthase (14, 22). Inhibition of NO synthase by NO may not be irreversible in the presence of elevated levels of H,B, and theconcentration of H4Bmay determine the magnitude of inhibition. Indeed, these observations suggest that H,B may modulate the formation of NO by controlling the negative feedback effect of NO on NO synthase. This is consistent with previous findings that H,B may not functionas a stoichiometric reactant butmay prevent the progressive loss of enzyme activity that occurs during incubation (41). The possibility that endogenously generated NO can interfere withthe function of endogenous hemoproteinsis supported by recent observations that NO generated by cytokine-activated macrophages in vivo in rats results in NO-mediated impairment of hepatic cytochrome P450 function (42).Thus, it is plausible that endogenously generated NO can inhibit NO synthase activity. Findings similar to those reported in the present study were made with unpurified NO synthase derived from bovine aortic endothelial cells (5), whereas inducible NO synthase from cytokine-activated rat alveolar macrophages was more resistant to inhibition by NO (6).Additional studies with isolated arterial rings and perfused vascular endothelial cells revealed that exogenous NO markedly inhibits endotheliumdependent formation of NO (5). Thus, it appears thatNO can functioning cells. It remains tobe inhibit NO synthase in intact determined whether neuronal NO synthase in intactcells can be inhibited by NO. Acknowledgments-We gratefully acknowledge the excellent techniea1 assistance provided by Norma E. Rogers, Debra A. Schmitz, and Nicole S. Arabolos. REFERENCES 1. Forstermann, U., Gorsky, L. D., Pollock, J. S., Ishii, K., Schmidt, H. H. H. W., Heller, M., and Murad, F. (1990)Mol. Pharmacol. 38, 7-13 2. Nathan, C . (1992)FASEB J. 6, 3051-3064 3. Bredt, D. S., Hwang, P. M., Glatt, C. E., Lowenstein, C., Reed, R. R., and Snyder, S. H. (1992)Nature 361, 714-718 4. Rogers, N. E., and Ignarro, L. J. (1992)Biochem. Biophys. Res. Commun. 189, 242-249 5. Buea G. M.. Griscavage. J. M., Rogers, N. E., and Ignarro, L. J. (1993)Circ. Res. 7 3 , 8 0 ~ 1 26. Griscavage, J. M., Rogers, N. E., Sherman, M. P., and Ignarro, L. J. (1993)J . Immunol. 151,6329-6337 7. Assreuy, J., Cunha, 1. Q., Liew, F. Y., and Moncada, S. (1993)BE J. Pharmacol. 108.8334337 , 8. Rengasamy, A,, and Johns, R. A. 11993)Mol. Pharmacol. 44, 124-128 9. Ignarro, L. J., Lippton, H., Edwards, J. C., Barieos, W. H., Hyman, A. L., Kadowitz, P. J., and Gruetter, C . A. (1981)J. Pharmacol. Exp. The% 218, 739-749 10. Ignarro, L. J., Byrns, R. E., Buga, G. M., and Wood, K. S.(1987)Circ. Res. 61, 866879 11. Bredt, D. S., and Snyder, S. H. (1990)Proc. Nutl.Acad. Sci. U.S . A . 87, ~~~

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