Advances in Enzymology and Related Areas of Molecular Biology, Volume 65 Edited by Alton Meister Copyright © 1992 by John Wiley & Sons, Inc.

M A M M A L I A N NITRIC O X I D E SYNTHASES DENNIS J . STUEHR," Division of Hemutology-Oncology, Department of Medicine, and OWEN W. GRIFFITH, Department of Biochemistry, Cornell University Medical College, New York, New York CONTENTS

I. Introduction 11. Nitric Oxide Synthase Isoforms: Characterization and Tissue and Subcellular

Distribution 111. Purification and Properties A. Constitutive Nitric Oxide Synthase B. Inducible Nitric Oxide Synthase IV. Physiological and Pharmacological Regulation Regulation of Constitutive Nitric Oxide A. Calcium/Calmodulin-Mediated Synthase B. Induction and Expression of Inducible Nitric Oxide Synthase C. Inhibition by Arginine Analogs D. Inhibition by Cofactor Antagonists E. Pharmacological Control in Pathological States V. Studies of Mechanism VI. Conclusion and Perspective Acknowledgments References

I.

Introduction

Prior to 1981, nitrogen oxide biosynthesis through either an oxidative (nitrification) or reductive (denitrification) pathway was established only in microorganisms (1 -3 and refs. therein). Suggestive evidence for a mammalian pathway was reported, however, as early as 1916 with the finding that the urine of rats, pigs, and humans contained more nitrate than was present in their diets (4). In 1981 definitive diet studies using stable isotopes ("N) and germ-free animals were reported by Tannenbaum and co-workers (5, 6) and by Witter et al. (7) and established that nitrogen oxides were normal * Current address: Immunology Section, Research Institute, The Cleveland Clinic, Cleveland, Ohio.

287

288

DENNIS J. STUEHR AND OWEN W. GRIFFITH

and quantitatively significant mammalian metabolites. Subsequent experiments revealed that injection of sterile irritants into rats induced substantial increases in nitrate biosynthesis (8). This finding led in 1985 to the demonstration by Stuehr and Marletta (9) that activated macrophages stimulated in v i m with lipopolysaccharide express a nitrogen oxide synthase activity and produce nitrite and nitrate. Further studies indicated that macrophages form nitrite and nitrate by enzymatic oxidation of one of the two chemically equivalent guanidino nitrogens of L-arginine and that citrulline is a coproduct (10, 11). Hibbs and co-workers (10, 12) determined further that macrophage-mediated tumor cell cytostasis was L-arginine-dependent and reported that N"-methyl-L-arginine was a potent, reversible, and stereoselective inhibitor of both tumor cell cytostasis and nitritehitrate synthesis by macrophages. The chronology of these and related developments is outlined in Table 1. Contemporaneously with but independently of these immunological studies, other investigators were attempting to elucidate the observation by Furchgott and Zawadzki (18) that endothelial cells had an obligatory role in the acetylcholine-mediated relaxation of vascular smooth muscle. Although a variety of potential mediators were considered as endothelium-derived relaxing factor (EDRF) candidates, by 1986 both Furchgott (21) and Ignarro et a]. (34) had concluded that the free radical nitric oxide (NO.) was the primary endogenous vasodilator released by the vascular endothelium (reviewed in 16, 35, 36). Detection of NO. as the bioactive principle was made difficult by the rapidity with which it reacts with O2 in solution ( t l l z of several seconds) to form other nitrogen oxides including the free radical nitrogen dioxide (NO*), the nitrosating agents and ulnitrous anhydride (N203) and dinitrogen tetroxide (N204), timately, the stable products nitrite (NOT) and nitrate (NO;) (Scheme 1). Whereas the accumulation and quantitation of nitrite and nitrate in neutral or alkaline solutions allowed total nitrogen oxide formation to be conveniently determined, demonstration of the biological formation of NO- per se required the application of sensitive bioassay and chemoluminescent techniques (19, 20, 37). Soon afterward, macrophages were shown to generate NO- as a primary product (22-24), and L-arginine was found to be the precursor of endothelium-derived NO- (EDNO) and L-citrulline (26,27) (Table 1). These convergent lines of research thus established that

1988-1989

1987

1983 I985

1981

1916

Year

Mammals secrete more nitrate than they ingest (4) Rats and humans synthesize nitrate (5-7) Immunostimulants increase nitrate biosynthesis in rats (8) Immunostimulated mouse macrophages synthesize nitrite and nitrate (9). L-Arginine is converted to nitrite, nitrate, and L-citrulline by immunostimulated macrophages (10, 11) N"-Methyl-L-arginine inhibits nitrogen oxide synthesis in macrophages (12) Immunostimulated macrophages synthesize nitric oxide as a primary product (22-24)

Toxicolog ylImmunology

1990

1989

1988

1986-1987

1980

1977-1979

Year

Nitric oxide activates soluble guanylyl cyclase and this accounts for its vasodilatory properties (13-15, reviewed in 16, 17) Endothelium is obligatory for acetylcholineinduced vasorelaxation (18) Endothelium-derived relaxing factor is identified as nitric oxide (19-21, 34) Endothelium-derived nitric oxide and Lcitrulline are synthesized from L-arginine (25-27, 94) Endothelium-derived nitric oxide regulates normal blood pressure (28, 29) The profound hypotension seen following in vivo exposure to endotoxin or cytokines is due largely to nitric oxide and can be reversed by inhibitors of nitric oxide synthase (30-33)

Phy siology/Pharmacology

TABLE 1 Some Significant Findings in Mammalian Nitrogen Oxide Biosynthesis

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DENNIS J . STUEHR AND OWEN W. GRIFFITH

.N= 0 + .NO2 @ N203

Scheme I . nitrogen.

H20

* 2NO:+

2H@

Reaction of nitric oxide with O2 and H20to form higher oxides of

a common metabolic pathway synthesizing NO. from L-arginine was expressed under quite different circumstances in the vasculature and immune systems (Fig. 1). Following these early studies, nitrogen oxide biosynthesis in a variety of cells and tissues has been documented by many investigators, and the importance of NO. in mammalian physiology and pathophysiology is now evident (Table 1). The biological effects of endogenous NO. depend on the flux of NO. reaching a target cell. When generated in small amounts, many of the effects of NO. are mediated through the activation of soluble guanylyl cyclase (16, 17). Activation of guanylyl cyclase by NO. is involved in the control of vascular tone (19-21,38), platelet function (37, 39), renal function (40, 41), neural signaling (42-45), and possibly chemotaxis (46). Activity of a cytosolic ADP-ribosyltransferase that modifies a specific 39 kDa protein is also reported to be increased by physiological concentrations of NO. (47). When generated in larger amounts, NO. inhibits ribonucleotide reductase (48) and cellular DNA synthesis (23, 49), blocks oxidative phosphorylation by inactivating iron-sulfur centers in aconitase and complex

L-Arginine

L-Citrulline

Nitric oxide

Nitrite + Nitrate

Figure 1. Biosynthetic pathway for production of NO. in mammals.

MAMMALIAN NITRIC OXIDE SYNTHASES

291

I and I1 of the mitochondria1 electron transport chain (49-54), and decreases protein synthesis by a unknown mechanism (55). Large fluxes of NO. are cytostatic and potentially cytotoxic, but in the absence of cell death, the inhibitions identified are reversible with time. The relative importance of reactivation and new enzyme synthesis in restoring cell function is not yet established. Mouse macrophage-derived NO. has been shown to block the growth of tumor cells and a variety of bacterial and fungal pathogens (56-60). Because macrophages generate sufficient NO. to cause significant nitrosation of amines, endogenous NO. formation has also been implicated in carcinogenesis (61, 62). The present review focuses mainly on the enzymology and control of L-arginine-dependent NO. synthesis. Other aspects of this subject including the pharmacology (16, 35, 36, 63), immunology (64, 65), and toxicology (66-68) of NOhave been reviewed elsewhere. 11. Nitric Oxide Synthase Isoforms: Characterization and Tissue

and Subcellular Distribution

All mammalian species examined to date (rats, mice, ferrets, and humans) exhibit a basal level of endogenous nitrate biosynthesis (69). Immunostimulated nitrate synthesis has also been observed in rats and mice (8, 70) and possibly in humans (68, 71). On the basis of substrate and inhibitor specificities and antigenic reactivity it has become apparent that basal and immunostimulated nitrate formation are due to the activities of distinct constitutive and cytokine-inducible nitric oxide synthases, respectively. Some characteristics of the constitutive and inducible isoforms are summarized in Table 2; these and other properties of the synthases are discussed in detail in subsequent sections. The several mammalian tissues and cells known to express nitric oxide synthase activity are listed in Table 3. Although most studies have focused on rat tissues and cells, the species distribution of nitric oxide synthase is undoubtably broad. Wider testing for both the constitutive and inducible activities will be possible as isoform-specific molecular probes and antibodies become available. To date only endothelial cells [and, possibly, macrophages/monocytes (87)] have been shown to express both constitutive (88, 111, 112) and inducible (32, 33) nitric oxide synthase, but it is likely that this list

292

DENNIS 1. STUEHR AND OWEN W. GRIFFITH

TABLE 2 Nitric Oxide Synthases: Characteristics of Enzyme Isoforms Nitric Oxide Synthase Characteristic

Inducible

Constitutive

CELLS

Prototypic Other examples

Macrophages Hepatocytes Tumor cells

Cerebellar cells Endothelid cells Platelets

No No Yes

Yes Yes Yes Yes

DEPENDENT ON

Calcium Calmodulin Tetrahydrobiopterin FAD FMN

Yes

Yes

Unknown

Partially No NAA = NMA > NNA

Completely" Yes NAA = NNA > N M A Yes

INHIBITED BY

EGTA Trifluoperazine N"-substituted argininesh Dipheny leneiodonium

Yes

See text. Comparative potencies of major inhibitors of this class: NMA, N"-methyl-Larginine; NAA, N"-amino-L-arginine; NNA. Nu-nitro-L-arginine. "

will increase as more tissues are tested after agonist stimulation as well as cytokine induction. It is noted that the extent to which constitutive and inducible nitric oxide synthases are expressed simultaneously even in endothelial cells remains an open question; it has not yet been possible to demonstrate agonist-stimulated NO- synthesis by the constitutive enzyme in endothelial cells after cytokinemediated induction of the other synthase isoform. Recently, human monocyte-derived macrophages have been reported to express an induced nitric oxide synthase in culture (114). This finding contrasts with previous studies in which NO. synthesis could not be induced in cultured human macrophages by cytokine treatment (115, 116). Although the effects of specific culture conditions on enzyme yield are poorly defined, induction of NO. synthesis in human cells may depend on their being infected with an intracellular parasite (i.e., Mycobacterium avium) during treatment with cytokines (1 14). More work is needed to determine if induction

MAMMALIAN NITRIC OXIDE SYNTHASES

293

TABLE 3 Tissues and Cells That Express Nitric Oxide Synthase Source

Synthase lsoform

Species (Ref.)

TISSUES

Artery and vein

Constitutive

Mesenteric vasculature Corpus cavernosurn Heart Brain Liver Adrenals Lung Spleen

Constitutive Constitutive Constitutive Constitutive Inducible Constitutive Inducible Inducible

Cow, rabbit, rat, pig, human, dog, mouse, guinea pig (reviewed in 167, 35, 36, 72) Rat (73) Rabbit (74) Rabbit, guinea pig (72, 75, 76) Pig, rat (77-80) Rat (81-82) Rat (83) Rat (82) Rat (84)

Inducible Constitutive Constitutive Inducible Inducible Constitutive Constitutive Constitutive Inducible Constitutive Inducible Inducible Constitutive Constitutive

Mouse, rat (22-24, 81, 85, 86) Human (87) Cow, pig (27, 88, 90-97) Mouse, pig (28, 33, 98) Rat (99, 100) Rat, human (87, 101-104) Human (39) Rat (105) c o w (40) Rat (106) Mouse (107) Mouse (108, 109) Rat (110) Pig (41)

CELLS

Macrophages Endothelial cells Hepatocytes Neutrophils Platelets Astrocytes Mesangial cells Mast cells Fibroblasts Adenocarcinoma Neuroblastoma Epithelial

of nitric oxide synthase in human macrophages is more tightly regulated than in rodent macrophages. The tissue distribution of constitutive nitric oxide synthase has been characterized in rat brain and other tissues using polyclonal antibodies raised against the rat cerebellar enzyme (1 17). There was a striking localization of nitric oxide synthase in rat cerebellum (glomeruli, granule cells, and basket cells) and olfactory bulb, along with the islands of Callejea, diagonal bands of Broca, and the mammillary nuclei. Other sites included neuronal projections into the posterior lobe of the pituitary and adrenal medulla, and several autonomic

294

DENNIS J. STUEHR AND OWEN W. GRlFFlTH

nerves that contract smooth muscle. The antibodies also localized nitric oxide synthase in the brain capillary endothelium, consistent with the view that a single constitutive synthase isoform accounts for the activity of the neurons and vasculature. Conversely, the antibodies did not react with the inducible macrophage nitric oxide synthase, a finding indicating that this isoform is distinct from the constitutive enzyme. By direct assay of cytosolic fractions of rat brain, activity was highest in cerebellum and decreased in the order hypothalamus = midbrain > striatum = hippocampus = cerebral cortex > medulla oblongata (45). Early studies with brain, endothelial cell, and macrophage lysates showed that nitric oxide synthase was localized exclusively in the soluble fraction (22, 77, 88, 89, 111). More recently, investigators at Abbott Laboratories (112, 113) have reported that a major portion (SO%) of bovine endothelial cell nitric oxide synthase activity is in the particulate fraction of cell homogenates. Activity could be removed from the membranes by the detergent CHAPS but not by KCl washes. Since the particulate fraction used in the studies would be expected to contain plasma membranes, microsomes, and, possibly, intracellular organelles, the actual subcellular localization of the activity remains to be determined. The particulate and soluble activities displayed identical dependence on NADPH, Ca" , and calmodulin, and both were inhibited by Nu-nitro-L-arginineand Numethyl-L-arginine. The authors concluded that the particulate enzyme represents a distinct third isoform and postulated that it may have a different physiological role than the soluble activity. Additional kinetic, immunological and sequencing studies with purified particulate and cytosolic activities are expected to determine if the particulate enzyme is an anchored form of the previously characterized soluble, constitutive isoform or if it is, in fact, a distinct species. It may be noted that reassessment of the subcellular distribution of mouse peritoneal macrophage nitric oxide synthase indicated that only 10-15% of the total activity remained in the washed pellet following sonication, and there was no indication that the pelleted activity was distinct from the bulk of the activity isolated in the cytosol(l18). Nonetheless, the finding of a particulate endothelial enzyme suggests that the membraneous fractions of other cell types should be examined thoroughly with due regard for the pos-

295

MAMMALIAN NITRIC OXIDE SYNTHASES

sibility that such fractions may require supplementation with various cofactors (see below) in order to show activity. 111. Purification and Properties A. CONSTITUTIVE NITRIC OXIDE SYNTHASE

A soluble, constitutive nitric oxide synthase has been purified to homogeneity from rat (77, 78) and porcine cerebellum (79). The various chromatographic procedures applied, purification factors obtained, specific activities, and overall yields are summarized in Table 4. In all cases, enzyme activity proved to depend on Ca2+ and a Ca2+-binding protein indistinguishable from and replacable by calmodulin (77, 91). Discovery of this requirement and the inclusion of Ca2+/calmodulin in assay mixtures used with purified fractions was key to the successful isolation of the enzyme (77). In an effort to increase yields and stability, several investigators have also used buffers containing proteinase inhibitors (77, 78) and thiols (43, 77TABLE 4 Purification of Brain Nitric Oxide Synthases

Source Rat cerebellum" Rat cerebellumb

Pig cerebellum'

Purification Steps Chromatography on DEAE and then 2',5'-ADP Sepharose Chromatography on 2',5'-ADP Sepharose and then calmodulin agarose Ammonium sulfate precipitation and then 2',5'-ADP Sepharose chromatography

Fold Purifiedd

Specific Activity" (nmol pdt. x min-' x m g - ' )

6000

960

30

8925

107

19

4500

730

5

Yield (%)

Data from refs. 77," 78," and 79.' Fold purification and specific activity determined by assaying the conversion of L-[I4C]arginine to L-[''C]citruIIine.

296

DENNIS J . STUEHR AND OWEN W. GRIFFITH

79). Nevertheless, the purified enzyme is unstable, and various additives have been identified to mitigate this problem partially (7779). Physical and kinetic properties determined with nitric oxide synthases purified from rat and porcine cerebellum are summarized in Table 5. Also shown are more limited data obtained with the unpurified nitric oxide synthase activities of rat synaptosomal cytosol or rat neuroblastoma N1E-115 cell cytosol. Based on velocity sedimentation (Mr = 279,000) and SDS-PAGE (Mr = 155,000) data, soluble, constitutive rat cerebellar nitric oxide synthase appears to be a homodimer in its native state (78). Hydrophobic interactions between the enzyme and some gel matrices (78) or partial proteolysis may account for some lower estimations of the native molecular weight ( M , = 200,000) and to the suggestion that the enzyme is a monomer (e.g., 77). All studies with constitutive cytosolic enzyme purified from brain indicate that exogenous NADPH, thiol, L-arginine, Caz , and calmodulin are required for NO- synthesis; the requirement for each additive is absolute (77-79). Although stimulation by (6R)-5,6,7,8tetrahydro-L-biopterin (H4-biopterin) was not observed in initial studies with the constitutive enzyme, Ca2+/calmodulin-dependent +

TABLE 5

Reported Physical and Kinetic Parameters of Brain Nitric Oxide Synthases

Parameter M,(X low3) native Denatured Pl Specific activity" App. K,,, (L-Arg, pM) APP. Ki "MA, pMY ("A, pM) EC5o Ca2+ (pM) EC5o Calmodulin (nM)

Purified Rat CerebellaP

Crude Rat Synaptosomal or Neuroblastoma Cytosolh

200, 279

150, 155 6.1 960, 107 1.5, 2.2 1.4 0.4 0.20, 0.35 10, 3.5

Purified Porcine Cerebellar" 200

160 730 8.4, 1.2 0.7, 1.0 0.4, 0.3

and 79." Data obtained from refs. 77 and 78," 80 and 1 nmoI L-citruhe per min per mg. NMA,N"-methyl-L-arginine;NNA,N"-nitro-L-arginine.

0.4 70

MAMMALIAN NITRIC OXIDE SYNTHASES

297

nitric oxide synthase purified from porcine brain has recently been shown by Mayer et al. to be stimulated three- to fivefold by H4an oxidized form of the cofactor, biopterin; 7,8-dihydro-~-biopterin, was inactive (79). Although a requirement for H4-biopterin had previously been shown for the inducible isoform of nitric oxide synthase ( 1 19, 120, see below), the demonstration that H4-biopterin is a cofactor for a constitutive enzyme is of considerable significance in establishing a uniformity of chemical mechanism among the various synthase isoforms. Additional characterization of the pig brain enzyme showed it to be a flavoprotein containing approximately one molecule of FAD, one molecule of FMN (both bound noncovalently), and 1 nonheme iron per 160 kDa monomer (121). In addition, pterin analysis showed that a portion of the purified enzyme (3-9 mol %) contained noncovalently bound biopterin (90%as H4-biopterin). Redox cycling of this bound H4-biopterin could provide an explanation of the lack of complete dependence on exogenous H4-biopterin observed with the constitutive pig brain enzyme (79). The discovery of stoichiometrically bound nonheme iron is important and consistent with the occurrence of such iron in other H4-biopterin-dependent enzymes such as phenylalanine hydroxylase and tyrosine hydroxylase ( 1 22, 123). Although it cannot be assured that the iron is redox active or catalytically essential until further studies are reported for the cerebellar enzyme, the precedent for iron involvement in an oxygenand H4-biopterin-dependent hydroxylation reaction in NO. synthesis is compelling (123, 124). An unusual nitric oxide synthase was recently purified 520-fold from rat inflammatory neutrophils collected from the peritoneal cavity following injection of a sterile irritant (oyster glycogen) (125). Gel filtration and electrophoretic evidence suggested the enzyme was a monomer of 150 kDa. The purified synthase exhibited a V,,, of 485 nmol citrullinelmin per milligram of protein and had a pZ of 5.6. The enzyme displayed no calmodulin dependence and was not affected by several inhibitors of calcium-binding proteins; its activity was enhanced only 20-30% by Ca2' (125, 126). FAD and H4-biopterin enhanced the activity (125) and an uncharacterized factor from neutrophil cytosol reportedly stabilized the activity, which was otherwise very unstable (127). From the data available, the neutrophi1 enzyme appears to have characteristics of both the constitutive

298

DENNIS J. STUEHR AND OWEN W. GRIFFITH

cerebellar enzyme (Mr, pZ near 6, instability) and the inducible macrophage enzyme (calmodulin independence, partial FAD dependence, fold purification to homogeneity). Further study is required to understand how this enzyme is related to the other isoforms. A soluble, constitutive activity forming citrulline from L-arginine and inhibitable by Nu-methyl-L-argininehas also been identified and partially characterized in homogenates of porcine vascular endothelial cells (88). Although formation of nitrogen oxides could not be shown (88), activation of soluble guanylyl cyclase by the reaction product was later demonstrated (90, 91, 128). More recently, Gross (129) has lysed bovine aortic endothelial cells in the presence of both thiols and proteinase inhibitors; activity in the resulting cytosol is higher than reported in other studies (88) and NOT formation is easily demonstrated. Purification of the physiologically important endothelial enzyme to homogeneity has, however, not yet been reported. The brain and endothelial nitric oxide synthases bind L-arginine with high affinity. In unfractionated pig endothelial cell cytosol the ECso for L-arginine-dependent activation of guanylyl cyclase is 6 pM (90).With nitric oxide synthase purified from rat cerebellum the K , for L-arginine is about 2 pM (77,78); a value of 6 pM was determined with rat forebrain homogenates (43). In the latter studies, L-homoarginine was shown to be an alternative substrate reacting with a K , of about 170 p M and a V,,, 57% of that seen with L-arginine (43). A number of L-arginine containing compounds including esters, amides, and dipeptides have also been identified as substrates in crude systems, but it is likely that they are hydrolyzed to L-arginine in situ. Although Na-benzoyl-L-arginineethyl ester has vasorelaxant activity (1301, it is not a substrate of nitric oxide synthase and its effects have been shown not to be mediated by NO. (131). Thus other than L-homoarginine, the substrate specificity of constitutive nitric oxide synthase appears to be quite strict; a number of the analogs shown not to be substrates are listed in Table 6. Inhibition by arginine analogs is discussed in Section 1V.C. B. INDUCIBLE NITRIC OXIDE SYN'I'HASE

A soluble nitric oxide synthase activity has been purified to homogeneity from immunostimulated rat macrophages (222) and from

299

MAMMALIAN NITRIC OXIDE SYNTHASES

TABLE 6 Substrate Specificity of Nitric Oxide Synthase Isoforms Substrate

Constitutive Isoform"

Induced Isoform"

++ +

++

L- Arginine

L-Hornoarginine N"-hydroxy-L-arginine L-a-Amino-P-guanidinopropionate L - a - Amino-y-guanidinobutyrate D,L-a-Methylarginine L-Canavanine Agmdtine Argininic acid o-Arginine L-Ornithine Ammonium chloride Hydroxylamine

++ ++

ND ND

ND ND

+ ND ND -

ND ND

++

' , Activity as a substrate is comparable to L-arginine; + , less active than L-arginine; - , not active as a substrate; ND, not determined. Data obtained from refs. 36, 132, and 133. an interferon-y and lipopolysaccharide-inducedmacrophage cell line (Table 7) (134). In the presence of L-arginine,NADPH, H4-biopterin, FAD and thiol, the purified enzymes had specific activities of 944 and 1300 nmol NO. per min per milligram protein, respectively. The activity of the purified enzymes was not enhanced significantly by Ca2+ and/or calmodulin, and several inhibitors of Ca2+ binding proTABLE 7 Purification of Cytokine-induced Macrophage Nitric Oxide Synthase" Protein

Fraction

(rng)

Total Activityb

Specific Activity'

Yield (%)

Purification Factor

~

Lysate supernatant Mono Q anion exchange FPLC 2',5'-ADP Sepharose TSK G3000

198 7.6

0.27 0.04

487 141

50 42.4

2.5 21.3 197 1060

100

1

29

9

10.2 8.7

83 426

" RAW 264.7 cells were harvested after 10-12 hr incubation with interferon-y and lipopolysaccharide. Values shown are averages of three purifications, each starting with about 5 x lo9 cells. nrnol NoTimin. ' nrnol NOT/min per rng protein (NOi- was not measured).

300

DENNIS J. STUEHR AND OWEN W. GRIFFITH

teins had no effect. On SDS-PAGE, the mouse macrophage nitric oxide synthase exhibited three closely spaced bands between 125 and 135 kDa. These bands were not present in identically purified preparations from noninduced macrophages, suggesting that the enzyme was newly synthesized in response to cytokine treatment. Gel filtration studies indicated that the induced mouse macrophage enzyme is catalytically active as a dimer of -250 kDa, but could be dissociated into inactive monomers of 130 kDa in the absence of L-arginine, H4-biopterin, and FAD. The enzyme contained 1 FAD and 0.5 FMN bound noncovalently per 130 kDa subunit (i.e., 2 FAD and 1 FMN per functional dimer) (1 34). Analysis of enzyme-bound transition metals or He-biopterin has not been carried out with the induced macrophage nitric oxide synthase. A summary of these results is given in Table 8. Although the flavin content of the induced macrophage nitric oxide synthase differed quantitatively from that of the constitutive pig cerebellar enzyme [ 1 FAD and 1 FMN per monomer (121)], it is not yet clear if this represents an actual difference in the number of flavin binding domains or if it merely reflects differences in cofactor retention during purification. In either case, it is remarkable that the induced and constitutive nitric oxide synthases contain both FAD and FMN. The only other mammalian protein known to contain both flavins is NADPH-cytochrome P-450 oxidoreductase (cytochrome C reductase) (135). Of note, this enzyme and the rat cerebellar nitric oxide synthase are reported to share sequence homology in their flavin binding domains (136). Implications of these findings

-

TABLE 8 Physical and Kinetic Characteristics of Cytokine-induced Macrophage Nitric Oxide Synthase Native M P Denatured M,b VI¶tZtX

K, , L-arginine K,, NADPH FAD content FMN

250 kDa 125-1 35 kDa 1300 nmol NO'lmin per mg protein 2.8 pM 0.3 pM I. 1 per I30 kDa subunit 0.55 per 130 kDa subunit

" Native M , was estimated by gel filtration on TSK G3000 SW and G4000 SW columns. ' Denatured M , was estimated by SDS-PAGE.

301

MAMMALIAN NITRIC OXIDE SYNTHASES

with respect to the mechanism of NO. synthesis are discussed in Section V. In contrast to the constitutive enzyme isolated from brain, macrophage nitric oxide synthase exhibits good stability in both purified and crude form. During catalysis at 37"C, activity of the purified mouse macrophage enzyme decreased only 44% after 6 hr (134), and specific activity in a 3 hr assay remained constant over a wide range of enzyme concentrations (50-1300 ng/ml). As illustrated in Fig. 2, crude macrophage enzyme is stable over a pH range of 6.0-8.5 but exhibits a narrow range of maximal activity between pH 7.8 and 8.0 ( I 33). Identical results have been obtained with partially purified macrophage enzyme assayed in a completely defined reaction mixture (137). The cofactor requirements for the inducible isoform of nitric oxide synthase are complex. Whereas NO- production (assayed as NO; and NOT formation) by unfractionated macrophage cytosol required only L-arginine and NADPH (22, 89), dilution of the homogenate or partial purification of the enzyme revealed a requirement for addiI

n

82

W

TI Q)

@-a

loo

0-0

75

-

IM 50 0

-

0

3

TI

en

z

I (v 0 +-

z

1

I

I

I

Activity Stability

25-

0

/

3

4

5

6

PH

7

a

9

10

Figure 2 . pH-Activity and pH-stability of crude macrophage nitric oxide synthase. From (133).

302

DENNIS J. STUEHR AND OWEN W.

GRIFFITH

tional cofactors (89). Studies with a desalted macrophage cytosol (119) or with nitric oxide synthase partially purified by chromatography on 2’,5’-ADP Sepharose (120) demonstrated a requirement for nanomolar concentrations of H4-biopterin. Only the R stereoisomer was active suggesting that the enzyme has a specific H4-biopterin binding site (1 19). The concentration of H4-biopterin measured in unfractionated macrophage cytosol was sufficient to account for the H4-biopterin-independent activity of those fractions and was consistent with the measured ECso values. The ability of several other reduced pteridines to activate nitric oxide synthase was also examined (Table 9) (120). As shown, only H4-biopterin was an effective cofactor when present at 0.5 pM, but 6-methyl-H4-biopterinand H4neopterin could partially substitute for H4-biopterin when studies were carried out with 50 p M pteridine. Interestingly, NO- synthesis by desalted macrophage cytosol was >95% H4-biopterin-dependent in assays carried out over 18 hr (119) but was only 60% H4-biopterindependent in a 3 hr assay (120). This result suggests that H4-biopterin dissociates from the synthase only slowly. It is notable that H4-biopterin enhanced the activity of partially purified macrophage nitric oxide synthase only when the reaction mixture was supplemented with cytosol from noninduced macrophages. Investigation of this dependence established that the cytosol from noninduced cells contained a NADPH-dependent dihydropteridine reductase activity able to recycle H4-biopterin during NO. TABLE 9 Activity of Various H4-Pteridines in Enhancing NO‘ Synthesis Concentration Pteridine

( I N

0.5

50

Data from ref. 120.

None H4-Biopterin HrNeopterin 6-(CH~)-Hd-Biopterin 6,7-(CH&-H4-Biopterin H4-Biopterin H4-Neopterin 6-(CH&H4-Biopterin 6,7-(CH&-H4-Biopterin

“02 1 (PM)

58.1 ? 130.5 ? 53.2 -L 55.3 ? 53.4 ? 146.9 2 77.0 2 82.1 k 67.0 5

1.2 1.7 1.2 1.9 0.1 3.1

3.3 2.9 4.2

MAMMALIAN NITRIC OXIDE SYNTHASES

303

synthesis (120) (Fig. 3). Cytosol from noninduced cells also provided FAD and glutathione, cofactors subsequently shown to significantly stimulate partially purified nitric oxide synthase (138). These studies allowed the definition of a complete assay system consisting of Larginine, NADPH, Hcbiopterin, FAD, and thiol. Because H4-biopterin can be regenerated nonenzymatically from its oxidized form (quinonoid H2-biopterin) by thiols such as dithiothreitol, glutathione, or cysteine (139, 140), addition of dihydropteridine reductase was not required, In a 3 hr assay under defined conditions, partially purified nitric oxide synthase showed a complete dependence on Larginine and NADPH, and a strong but incomplete dependence on thiol, FAD, and H4-biopterin when they were individually omitted NADPH

Dihydrofolate Reductoso

2~-

HpBiopterin

I1

NAw+

Dih dropkridine

\

H4Ei;terin

Q-H$iopterin

+

L-Citruilins

L-Argininc

Figure 3. Proposed scheme for cycling of H4biopterin during NO. synthesis in macrophage cytosol. Hdbiopterin can be regenerated in two ways: dihydropteridine reductase catalyzes the reduction of quinonoid Hzbiopterin (Q-Hzbiopterin), which is the presumed product of nitric oxide synthase; dihydrofolate reductase reduces H*biopterin, which arises spontaneously from Q-H2biopterin. Methotrexate (MTX) inhibits dihydrofolate reductase (213), while Nu-methyl-L-arginine (NMA) inhibits NO synthase (10). Reproduced from (120).

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DENNIS J . STUEHR AND OWEN W. GRIFFITH

from the reaction mixture (Table 10). A partial dependence on exogenous FMN has also recently been observed (134). Essentially no NO. was formed in reaction mixtures containing L-arginine and NADPH but not containing H4-biopterin, FAD, or thiol (138). AIthough total activity depended on the assay conditions, reaction stoichiometry was unchanged; L-citrulline and NO; /NO: formation were in all cases equivalent. More detailed kinetic studies established that the partial dependence on FAD and FMN was constant throughout a 3 hr assay period but that almost all of the H4-biopterinor thiol-independent NO. synthesis occurred in the initial 30-60 min of the reaction period. After 60 min, dependence on thiol or H4biopterin was essentially complete (134, 138). This result, obtained with either partially or completely purified enzyme, again suggests that H4-biopterin dissociates from the enzyme slowly during purification and catalytic turnover. The requirement for reduced pyridine nucleotide has also been examined for the inducible isoform. Partially purified macrophage nitric oxide synthase exhibited a strict requirement for NADPH; NADP+, NADH, and NAD’ were inactive (89). Although crude, desalted macrophage cytosol supports NO- synthesis in the presence of substoichiometric amounts of NADPH (1 19), such synthesis was TABLE 10 Cofactor Requirements of a Partially Purified Induced Macrophage Nitric Oxide Synthase ~

Cofactor Omitted None NADPH H,-Biopterin FAD Thiol”

Saturating Concentration

ECso

Relative NO;/NOC Synthesis when Indicated Cofactor is Omitted“ (5%) 100

ND 30 nM 0.2 pM 0.8 m M

5

ND

2100 nM 2 2 JLM

15-35

35-55 20-55

2 2 mM

~

~

~~

Data are from refs. I18 and 138. Similar results were obtained with the purified enzyme (134). ND = not determined. a In a 3 hr end point assay. Similar results were obtained using glutathione, dithiothreitol, or cysteine. When thiol was omitted, assays were carried out in the presence of dihydropteridine reductase to regenerate H4-biopterin.

MAMMALIAN NITRIC OXIDE S Y N l H A S E S

305

accounted for by dithiothreitol-dependent regeneration of N ADPH from NADP' in the crude system (119, 138). When cysteine was used in place of dithiothreitol, maximal NO- synthesis by partially purified enzyme required a fourfold molar excess of NADPH (138). Much of the NADPH oxidation in the presence of cysteine was found, however, to be arginine-independent, suggesting that NADPH-dependent formation of reduced oxygen species (e.g., superoxide or hydrogen peroxide) was occurring and, in the presence of arginine, might be contributing to the enzymatic formation of NO.. That this was not the case was demonstrated by showing that hydrogen peroxide could not replace NADPH and that catalase or superoxide dismutase did not decrease NO. synthesis relative to controls (1 18). It was also established that NADPH did not function solely to regenerate H4-biopterin. Thus, although a dihydropteridine reductase activity was present in unpurified cytosol (120), this activity was removed during purification of the enzyme (138, 141). Similarly, NADPH was shown to be required even in the presence of excess H4-biopterin ( 1 19). The data strongly support a direct role of NADPH in NO. synthesis. Studies with highly purified macrophage nitric oxide synthase showed that 70-95% of the observed NADPH oxidation is L-arginine-dependent in complete assay mixtures (134, 141). Direct comparison of L-arginine-dependent NADPH oxidation and nitrogen oxide formation indicated that 1.5 mol of NADPH is oxidized per mol of NOT/NO; formed in assay mixtures where H4-biopterin could be recycled by thiol but not by NADPH. Conditions that prevented or decreased NO. synthesis (adding N"-methyl-L-arginine, replacing L-arginine with D-arginine, or omission of added FAD) affected substrate-specific NADPH oxidation proportionately (Table 11) (118). These results support the accuracy of the NADPH/NO. stoichiometry indicated above and serve to elucidate the mechanism by which nitric oxide synthase carries out the five-electron oxidation of arginine (see Section V). The activity of crude macrophage homogenates is reported to be stimulated by Mg2+ (22). In addition, NO. synthesis by macrophage cytosol was shown to be completely inhibited by micromolar concentrations of Cu2+ and Zn2* and to be partially inhibited by divalent metal ion chelating agents (Table 12) (138). Although the iron chelators TIRON and 1,lO-phenanthroline did not inhibit NO. syn-

306

DENNIS J . STUEHR AND OWEN W . GRIFFITH

TABLE 11 NO Synthesis and Substrate-specific NADPH Oxidation under Various Circumstances Substrate (1 mM) L-Arginine

Added NMA" -

4-

D-Arginine

None (I

-

FAD"

NO; plus NOT (Rel. %)

+ + + +

Substrate-specific NADPH Oxidation (Rel. %)

~

I00 5

40 0 0

100 15

36 0 0

NMA, Nu-methyl-L-arginine at 0.5 mM; FAD at 4 (LM.

thesis completely, mechanistic considerations (Section V) suggest that it will be useful to carry out more extensive chelation studies with the purified synthases. The substrate specificity of induced nitric oxide synthase with respect to L-arginine analogs appears to be qualitatively similar to TABLE 12 Effect of Some Divalent Metal Ions and Chelating Agents on NO' Synthesis by Macrophage Cytosol NO;' plus NO< Synthesis (Rel. %) At 10pM

At 100 pM

METAL ION

None Fez CaZ +

cu2+ +

Zn2

+

100

91 2 3 106 2 3 87 2 3 68 ? 3

98 2 2

105 f 3 12 2 1 8 * 1

At I mM CHELATOR

None EDTA EGTA DTPA TIRON 1,IO-Phenanthroline

100

29 ? 1 64 t 2 55 ? 3 85 r+ 1 64 2 2

None of the metal ions or chelating agents interfered with detection of NOT and NOT.

MAMMALIAN NITRlC OXIDE SYNTHASES

307

that of the constitutive enzyme, but a few quantitative differences have been identified. With enzyme purified from mouse macrophages, L-arginine reacts with a K , of 2.3-2.8 pM,a value comparable to that seen with the constitutive enzyme (134,141). L-Homoarginine, which is a relatively poor substrate of the constitutive enzyme, is a moderately good substrate of the inducible enzyme, reacting with a V,,, approximately equal to that of L-arginine and a K,,, of 4 0 pM (118). N"-Hydroxy-L-arginine, a putative intermediate in the conversion of L-arginine to citrulline and NO.(Section V), is an excellent substrate; it reacts with a K,,, of 6.6 FM and a V,,, nearly twice that of L-arginine (141). The only other L-arginine analog known to react is a-methyl-DL-arginine,a substrate of the induced isoform of endothelial cells; its activity has not yet been kinetically characterized (118). The ability or inability of several arginine analogs to act as substrates is summarized in Table 6. It is notable that D-arginine, as well as the lower homologs of L-arginine, and analogs lacking the a-amino or carboxyl functions of L-arginine (argininic acid and agmatine, respectively) do not react. Studies with L-arginine-containingdipeptides, L-arginineamide, L-arginine methyl ester, and L-arginine hydroxamate showed that none were substrates of macrophage nitric oxide synthase when arginase was added to the assay mixtures to remove free arginine (118). Taken together, the data suggest a very restricted L-arginine binding site with a high degree of specificity for an ionizable carboxyl and amino group in the proper configuration. In this respect the enzyme differs from mammalian arginine deiminases, which deiminate carboxyl- and/or amino-substituted arginine derivatives but not free L-arginine (142144). Arginine deiminases do not appear to play a role in the conversion of arginine to NO.. Consistent with this conclusion, preparations of macrophage nitric oxide synthase do not convert ammonia or hydroxylamine to NO. (Table 6).

IV. Physiological and Pharmacological Regulation A. CALCIUM/CALMODULIN-MEDIATED REGULATION OF CONSTITUTIVE NITRIC OXIDE SYNTHASE

As noted, the soluble, constitutive isoform of nitric oxide synthase from either rat (77, 78) or pig (79) brain is activated by Ca2+/ calmodulin; essentially no activity is observed in the absence of

308

DENNlS J . STUEHR AND OWEN W. GRIFFITH

Ca2+/calmodulin.Synthesis of NO. by N1E-115 neuroblastoma cell cytosol is also stimulated by Ca2+ and fully inhibited by calmodulin antagonists (145) (Table 13). Similarly, early studies by Singer and Peach showed that release of EDRF by endothelial cells was Ca2 dependent (146). Subsequent studies with endothelial cell cytosol have shown NO- synthesis to be inhibited by calmodulin antagonists (91) and to be Ca2+-dependentif endogenous Ca2+is either removed (90, 91) or chelated (88, 90). However, in contrast to studies with the brain enzyme, endothelial cell cytosol is reported to catalyze Ca2+-independent NO. synthesis even when free Ca2+ levels are reduced below 10 nM (91, 128). Because levels of Ca2+below about 80 nM reportedly do not stimulate NO. synthesis, it is presently unclear whether NO. synthesis at very low Ca2' represents a Ca2+/ calmodulin-independent activity of the endothelial soluble or particulate, constitutive enzymes or whether it is due to the activity of a distinct enzyme. In this regard it should be noted that the activities of soluble and particulate bovine endothelial nitric oxide synthases, even when partially purified by chromatography on 2' S'-ADP-Sepharose, were stimulated only 2.4-fold and 4.3-fold, respectively, by addition of calmodulin; activity in the absence of exogenous calmodulin was thus significant (1 12). Although these results suggest Ca2+/calmodulin-independent NO. synthesis is due to the known constitutive enzymes, affinity chromatography may not have removed all of any inducible nitric oxide synthase present. Unequiv+ -

TABLE 13 Calcium Binding-protein Inhibitors Isoform Inhibited Compound

Structural Name

Inducible

~

Trifluoperazine w-5 W-13

Calcineurin Melittin Calmidazolium

N-(6-aminohexyl)-lnaphthalenesulfonamide

N-(4-Aminobutyl)-5-chloro-2naphthalenesulfonamide

Based on data from refs. 77, 78, 90,91, and 112.

Constitutive ~

No No

Yes Yes

No

Yes Yes Yes Yes

MAMMALIAN NITRlC OXIDE SYNTHASES

3 09

ocal separation of the inducible isoform is important because Kilbourn and Belloni (32,33)have shown that endothelial cells produce the inducible nitric oxide synthase in response to cytokines and/or endotoxin. Because endotoxin is a common contaminant of buffers and cell culture media, it is possible that low levels of inducible isoform were present in the endothelial cytosols examined to date. Further studies using isoform specific antibodies, substrates, or inhibitors (Section 1V.C) are required to determine if significant Ca2+ / calmodulin-independent NO. synthesis by a constitutive nitric oxide synthase isoform occurs. Several studies have established that constitutive brain nitric oxide synthase is regulated by changes in Ca2' concentration in the physiological range. Thus the purified rat cerebellar enzyme is activated by Ca2+ with an ECso of 200 nM (77) or 350 nM (78). For the pig enzyme, the measured ECso value is 400 nM (79), and the enzyme is essentially inactive at Ca2 concentration below about 80 nM (78). Similar values for Ca2+ activation were determined in less purified preparations from brain and in NlE-115 neuroblastoma cells (78, 145). Because the resting free Ca2+ concentration in synaptosomes is about 80 nM (147), any significant agonist-mediated increase in intracellular Ca2 is expected to increase the activity of neuronal nitric oxide synthase (43). Similar conclusions apply to Ca2+/calmodulin-dependent endothelial cell NO. synthesis. Both the cytosolic and particulate fractions of bovine aortic endothelial cells exhibited stimulation by Ca2+ with an ECso value of about 50 nM (145). Studies with pig endothelial cytosol indicated an ECWof 60 nM (90) or 300 nM (128, 141). Considering that the free Ca2+ concentration of resting endothelial cells is 30-40 nM (90,148), and that Ca2+ increases to 160-700 nM (88, 90, 148) following stimulation by agonists, significant Ca2+-dependent activation of nitric oxide synthase seems assured. A wide variety of agonists have been shown or postulated to increase intracellular Ca2+ levels with consequent activation of constitutive nitric oxide synthase. In neurons it is believed that binding of glycine and glutamate to N-methybaspartate (NMDA) receptors allows Ca2+ to enter the cells (149). Kainate may also stimulate significant Ca2+ entry (149). In endothelial cells, a variety of vasodilatory compounds including bradykinin, adenosine, acetylcholine, leukotriene D4, histamine, dopamine, and the calcium iono+

+

3 10

DENNIS J . STUEHR AND OWEN W. GRIFFITH

phore A23187 have been shown to increase NO. synthesis (72, 150). All are believed to act via an increase in intracellular Ca2+ levels, but which, if any, of these agonists acts in vivo is not yet established. Recent studies suggest that endogenous NO. synthesis is required for vasomotor nerve-induced vasodilatation (15 l ) , but the mechanism and mediators of this stimulation remain to be elucidated. Studies with perfused vascular segments subjected to pustile flow suggest that mechanical stretching of the vasculature during normal blood flow may also stimulate NO. synthesis (152). Although additional studies are required to identify the true stimulus, it is quite clear that vasoactive amounts of NO. are produced in vivo, since administration of nitric oxide synthase inhibitors to guinea pigs (29), rabbits (28), and dogs (153) causes a marked (up to 60% in guinea pigs) increase in blood pressure. Although several investigators have considered the possibility that constitutive nitric oxide synthase might be regulated in part by the availability of L-arginine, studies to date suggest that this is rarely the case. While the Km of arginine is only a few p M , cultured endothelial cells maintain intracellular L-arginine concentrations of 0.1 to I mM, do not express arginase activity, and can probably regenerate some L-arginine from the L-citrulline formed during NO. synthesis (92, 164, 165). In vitro studies with vascular rings indicate that NO. synthesis is not diminished until after several hr in argininefree buffers (16, 164). Nitric oxide-mediated vasorelaxation as occurs in normal blood pressure homeostasis is thus not expected to be acutely dependent on exogenous L-arginine. Consistent with this view, intravenous administration of arginase to normal animals causes no change in blood pressure despite the fact that plasma arginine is reduced from 100-200 p M (rats) or 40-50 pM (guinea pigs) to very low levels ( < I p M ) (166). Similarly, intravenous administration of L-arginine has been shown not to affect normal blood pressure in several species including rat, guinea pig, dog and man (30, 31, 166). Interestingly, L-arginine administration was found to increase the duration and magnitude of the hypotensive response to acetylcholine, a finding suggesting that nonphysiological overstimulation of constitutive nitric oxide synthase can exhaust intracelM a r substrate and force a dependence on extracellular arginine (219).

MAMMALIAN NITRIC OXIDE SYNTHASES

311

B. INDUCTION AND EXPRESSION OF INDUCIBLE NITRIC OXIDE SYNTHASE

As noted, bacterial products or cytokines (alone or in combination) induce expression of a distinct isoform of nitric oxide synthase; expression typically begins 4-12 hr after exposure to the inducing agent(s) (70). In mouse macrophages, expression of the synthase appears to require protein synthesis during the induction period because cycloheximide prevented expression of enzyme activity only during that time period (154). Once expressed, the nitric oxide synthase activity of mouse macrophages remained constant in the presence of cycloheximide for at least 8 hr (154), and the activity may be stable for as long as 24-48 hr (70). Although it is unknown if cells synthesize the enzyme de novo in response to inducing agents or merely modify an existing protein, purification studies with the macrophage enzyme are consistent with de novo synthesis (134). Interestingly, certain cytokines can also prevent the induction of nitric oxide synthase in cultured cells (159, and anti-inflammatory drugs such as dexamethasone and hydrocortisone have been shown to prevent induction of the enzyme in rodent macrophages, lung, and liver (156, 157). Cytokine induction and suppression have been reviewed (64, 65, 155); the findings with respect to nitric oxide synthase are summarized in Fig. 4. There are presently no known mechanisms by which cells directly regulate inducible nitric oxide synthase following its expression. In contrast to the constitutive isoform of brain and endothelial cells, intracellular Ca+ + concentrations were found not to play a role in the expression or regulation of the inducible macrophage activity (158). Similarly, antagonists of calcium binding proteins do not block NO. synthesis in macrophages or macrophage lysates (134, 159). In some cases, however, cytokine-induced NO. synthesis may be indirectly controlled by the limited availability of L-arginine. Thus, macrophage arginase and nitric oxide synthase are induced by similar cytokines (57, 85, 160-162), and depletion of extracellular Larginine by macrophage arginase was shown to limit NO- synthesis in experimental wounds in rats (163). In endotoxic rats, where the acute hypotension is attributed to vigorous, unregulated NO. synthesis by induced enzyme (31, Section IV E), administration of arginase causes a modest increase in blood pressure (166). Thus, in

DENNIS J. STUEHR AND OWEN W. GRIFFITH

312 INDUCTION 0-12 hours

-

Inhibitors: Cytokines (MDF. TGF-Pi -Pa -P3)

-- Dexarnethasone Hydrocortisone Cycbheximide I

EXPRESSION 12-72 hours

-- N%ethyl-L-Arginine Diphenyleneiodoniurn

*N=O + L-Citrulline

Figure 4. Regulation of nitric oxide synthase induction and expression. Induction can be blocked by the macrophage deactivating factor (MDF) and tumor growth factors 8-1, 8-2, and 8-3 (155), or by the other drugs as illustrated. Once nitric oxide synthase activity is expressed, it can be inhibited reversibly by Nu-substituted Larginine analogs or irreversibly by diphenyleneiodonium.

endotoxemia and presumably in septic shock, NO. synthesis appears to be dependent at least in part on plasma arginine. Under some circumstances, NO. synthesis may also be controlled by H4-biopterin availability. In human and mouse cells, exposure to cytokines increased expression of GTP cyclohydrolase (167-171). In some cases, cytokines also induced 6-pyruvoyl-H4-biopterin synthase and sepiapterin reductase activities, and intracellular H4-biopterin concentrations were found to be increased (Fig. 5). In mouse fibroblasts, increased H4-biopterin synthesis correlated with expression of an inducible nitric oxide synthase activity (107). Inhibition of fibroblast GTP cyclohydrolase prevented H4-biopterin synthesis and greatly diminished the capacity of the cells to generate NO.. Adding exogenous sepiapterin eliminated the dependence on GTP cyclohydrolase and restored both HAiopterin concentrations and NO. production. Because human monocytes and macrophages do not express significant levels of 6-pyruvoyl-H4-biopterin synthase (167), they synthesize large quantities of H2-neopterinfollowing cytokine stimulation (Fig. 5 ) (167, 172). This metabolic defect is ap-

MAMMALIAN NITRIC OXIDE SYNTHASES

313

OH

" O H HO Z C d OH

I

GTP GTP qdohydrolase'

0

0

HO

OH

C-C-CH, HPN

B.pyruvoyl-H,Pterln

H,Biopterln

Figure 5. Biosynthesis of H4biopterin. Cytokines have been reported to increase expression of the enzymes marked with an asterisk. Human macrophages are deficient in 6-pyruvoyLH4pterin synthase, and therefore produce H2neopterin. Adapted from (107, 167-172, 176, 177, 214, 215).

parently restricted to human and monkey macrophages (167) and suggests that H4-biopterin availability may limit NO. synthesis in human macrophages. On the other hand, H4-biopterin synthesis is induced by cytokines in human T-lymphocytes (169), and it is possible that such cells might serve as a source of H4-biopterin for macrophages at inflammatory sites.

DENNIS J. STUEHR AND OWEN W. GRlFFlTH

314

C. 1NHlBITION BY ARGINlNE ANALOGS

Based on early studies showing that N"-methyl-~-arginine blocked macrophage-mediated L-arginine-dependent tumor cell cytostasis (10, 12), a variety of N"-substituted-L-arginine derivatives have been investigated as potential inhibitors of nitric oxide synthase (Table 14). When partially purified macrophage enzyme was assayed in the presence of 1 mM L-arginine as substrate, N"-amino-L-arginine, N"-ethyl-L-arginine, and Nu-methyl-L-arginine were the most effective inhibitors with EDso values of about 40 p M , 100 pM, and 200 FM, respectively. Nu-Nitro-L-arginine and L-canavanine were less effective with EDso values of about 1 mM, whereas the N"propyl and N"-butyl derivatives exhibited EDso values of 10 mM or greater (118, 173). Similar rank-order potency was observed with TABLE 14 Inhibition of Nitric Oxide Synthase by Nw-Substituted Arginines Extent of Inhibition Observed Arginine Analog Nu-Methyl N"',N"'-Dimethyl N",N"-Dimethyl N"-Ethyl N"-Propyl N"-Butyl N"-Nitro N"-Amino L -Can av an in e N'-Iminoethyl-L-ornithine N"-Amino-L-homoarginine

Structure

Inducible

Constitutive

CH3NH(C=NH)NH-R CH~NH(C=NCHI)NH-R (CHj)zN(C=NH)NH-R CzHsNH(C=NH)NH-R C3H,NH(C=NH)NH-R C4HsNH(C=NH)NH-R (NOz)NH(C=NH)NH-R NHzNH(C=NH)NH-R CH3NH(C=NH)NH--(T-R' CH3(C=NH)NH-R NHzNH(C=NH)NH-R"

++

++

K = -CH2CH2CH2CH(NH2)COOH. R' =