Biosynthesis and action of nitric oxide in mammalian cells

TIBS 22 - DECEMBER 1997

proteins in addition to t~anscdptio~ termination. It will also be interesting to understand why, out o~ a]~ the possible DNA-binding motifs, the myb DNA- binding mot~ was chosen in both fungi and mamma~s to serve in the terminator proteh~. We look forward to structural studies of pol I terminator proteins, b,~0und to their cognate DNA sequence, which might illuminate this problem.

RdeteBces 2 Reede;, R. H. and Lang, W. (1994} Mal.

Microbial 12, 11-15 2 Ju, Q. Morrow, B. E. and Warner, J. R. (1990)

MaL Cell. Bi l l tO, 5226-5234 3 Lang, W. and Reeder, R. H. (1993) MoL Cell.

BiaL 13, 649-658 4 Lang, W. et aL (19941 Ce# 79, 527-534 5 Evers, R. et aL (1995) EMBO J. 14, 1248-1256 6 [vers. R. and Grummt, L (1995) Proc. Natl.

Acad. Sci. O, S. A. 92, 5827-5831

7 Zhao, A., Guo, A., Liu, Z, and Pape, L. (1997) Nucleic Acids Res. 25, 904-910

8 Aasland, R., Stewart, A. E and Gibson, 1. i1996) Trends Biochem. ScJ. 21, 87-88

9 ~uhn, A., Bartsch, I. and Grummt, I. (1990} Nature 344, 559-562

10 Lang, W. and Reeder, R. H. (1995) Prac. Natl. Acad. Sci. U, S. A. 92, 9781-9785

11 Mason, S. W. Sander. E. E. and G~umrnL i (!997) EMBO J. 16, 163-172

12 Landick, R. (1997) Cell 88, 741-744 13 Labhart, P. (1995) Nucleic Acids Res, 23,

2252-2258 14 Jeong, S. W., Lang, W, and Reeder, R. H. (1995)

MoL Cell. Biol. 15, 5929-5936 15 Labhart, P. and Reeder, R. H, (1990) Nucleic

Acids Res. 18, 5271-5277 16 Pfleiderer, C,, Staid, A., Bartsch, I, and

Grummt, I, (1990) Nucleic Acids Res. 18, 4727-4736

17 Mason, S. W. Wallisch, M. and Grummt, I. J. Mal. Biol. (in press)

i 8 Jeang, S-W., Lang, W. H. and Reeder, R. H. (1996) J. BioL Cicero. 271, 16104-16110

19 Jacques, J-P. and Kolakafsky, D. (1991)

EV!EWS Genes Dev. 5, 707-713

20 Jacques, J-P., Hausmann, S. an~ ~%ia[~.afsxy, D. (1994) EMBO J. 13, 5496-5503

21 Chasman, D. Let as. (]990) Genes Dev 4, 503-53.4

22 Fedor, M. J., Lue, N, F. and ~arnb,~rg, R. 0 (1988) J, MaL Biol, 204, _109-127

23 Langst, G., 8lank. T. A. Becket, P. 9~ and Grummt, L (1997) ~MSO ./. 16, 760--168

24 McStay, B. and Reeder. R. H. (1990) Genes Dev 4, 1240-1252

25 Kulkens, T. et aL (1992) EMBO J. 11, 4665-4674

26 Lucchini, R. and Sago, J. M. (1995) Nature 374, 276-280

27 Johnson, S, P. and Warner, J. R (1989) Mol. CelL Biol. 9. 4986-4993

28 Sander, E. and Grurnmt, I. (1997) Nucleic Acids Res. 25, 1142-1147

29 Brewer, g. J. and Fan~:rnan, W, L {I98P~) Cell 55, 637-643

30 Gerber, J-K. et al. i !997) C0190, 559-557 31 Nudler, E., Mustaev, A., Lukhtanov, C. and

Goldfarb, A. (1997) CeJI89, 33-4~ 32 Grummt, L et ai. i'J.9~6) CoP .'15, 8~? ~46

Biosynthesis and action of nitrk oxide in m malian cells

Bernd Mayer and Benjamin Hernmens Nitric oxide (NO) can act as a vasorelaxant, a modulator of neurotransmission and a defence against pathogens. However, under certain conditions, NO can also have damaging effects to cells. Whether NO is useful or harmful depends on its chemical fate, and on the rate and location of its production. Here, we discuss progress in NO chemistry and the enzymology of NO synthases, and we will also attempt to explain its actions in the cardiovascular, nervous and immune systems.

NITRIC OXIDE (NO) is a cardiovascular regulator of direct clinical importance, it is also used by the immune system as a weapon against pathogens, with atten- dant danger for host tissue if overpro- duced. NO also has a role in the central nervous system, where it is implicated in learning and memory, again with a poten- tial ior deleterious effects if its regulation is disturbed. The actions of NO are dependent on its chemical reactivity, so that fundamental research on its inor- ganic chemistry has immediate physiological relevance. Many insights into the

B. Mayer and B. Hemmens are at the Institut for Pharmakologie und Toxikologie, KarI-Franzens-Universit~t Graz, Universit~tsplatz 2, A-8010 Graz, Austria. Email: [email protected]

regulation of NO production have come from the study of the NO synthase enzymes, which use components familiar from other enzymes in new and uncon- ventional ways to achieve their unique catalytic chemistry ffig. 1). These factors add up to a rapidly expanding field that draws on an wide spectrum of disciplines.

In the following account, we have had to be selective and so have concentrated on the advances from the past few years. For more-detailed discussions, especially of the many essential, but older contri- butions that we cannot mention here, we recommend some existing reviews: on the chemistry of NO ], NO synthase enzymology 2 and its physiology in the cardiovascular system 3, brain 4 and immune system 5. There is also an NO homepage at http://www.apnet.com/no

Copyright © 1997, Elsevier Science Ltd. All rights reserved. 0968-0004/97/$17.00 PII: S0968-0004(97)01147-X

Chemistry of NO NO is not a classical imercellular

messenger, Le. one that is secreted by a transporter and which binds to a morn- brahe-bound receptor. Instead, it is freely diffusible and its biological effects are determined by its chemical reactivity. As a free radical, it reacts rapidly with species containing unpaired electrons, such a~ molecular oxygen, superoxide a~li~ and metals.

NO reacts with 0 2 in aqueous solution according to the ,}~ erall equation:

4NO + O~ = 4NOC + 4H +

An intermediate is formed (N203 or a related species) that can efficiently nitro- sate cellular thiols and amines. This could in principle represent an important path- way of NO cytotoxicity. However, the re-. action is second order with respect to NO (overall third-order rate constant 6.3 to 9.2 x 106 M -~- s -1) and is therefore negligibly slow at physiologically low, submicromolar NO concentrations. The formation of S-nitrosothiols observed in vivo 6 most likely occurs by other routes.

NO reacts with superoxJde anion (02-) at nearly diffusion-controlled rates (4.3 tO 6.7 × 109 M -1 S -I) to give peroxynitfite (ONOO-). The ONOO- anion is stable, but if it is protonated (pK a = 6.8) it de- composes rapidly (t|/2 = 1 s at pH7.4 and 37°C) to a potently cytotoxic intermediate with hydroxyl radical-like activity (ONOOH*) 7. However, in vivo this reaction is outcompeted by the reactions of the ONOO- anion with reduced glu- tathione (GSH) and CO 2, leading to GSH

477

REVIEWS H

(~) (~)1 _ O y NH2 HaN , , ~ N H 2 H2N . . ~ N OH /

/NH O2. / N H O2~ ~ N ( H +

~,, 1 ~ ~.. 0.5 NADPH"

L-Arginine NO-Hydroxy-L-arginine L-Citrulline

oN=O

Nitric oxide

Figure ~. The nitric oxide synthase reaction.

oxidation (k = 2.7 x 10 3 M -1 S-I; Ref. 8) and formation of nitrosoperoxycarbon- ate (0NO2CO2-) (k = 3 x l0 4 U -1 S-l; Re[. 9), respectively. The latter species is a potent nitrating agent and may cause the tyrosine nitration seen in some pathological conditions.

NO binds, at diffusion-controlled rates, to the haem group of soluble guanylate cyclase (sfiC), which is the major target for the physiological effects of NO. This stimulates cGMP formation by several hundredfold at nanomolar concentrations of NO, This function of sGC as an NO sensor results from a special environ- ment of the Hls-Iigated haem that, unlike haemoglobin or myogiobin, can form a pentacoordinate ferrous nitrosyl com- plex even under aerobic conditions I°.

Staraler and c'~workers u propose a physiologk'al role k,r S-nitroso- haemoglobin. They ?'esented some -vl- donee for a cycle revolving S-nitrosation of haemoglobin in the lung and release d S-nitrosoglutathione from erythrocytes in the tissues.

NO also reacts with several other metalloproteins. For example, in cells that can synthesize the inducible NO synthase isoenzyme, NO binding converts the iron-sulphur enzyme cis-aconitase to an mRNA-binding form that regulates cellular iron metabolism :2.

BiosynthesLs of NO All known NO synthases constitute a

family of three Isoenzymes that represent distinct gene products: the constitutive neuronal (nNOS) and endothelial (eNOS) forms and the inducible 0NOS) macro- phage form. The human NOS genes are located on chromosomes 12 (nNOS: 150 kb, 29 exons), 7 (eNOS: 21-22 kb, 26 exons), and 17 0NOS: 37 kb, 26 exons) 13.

As may be expected from the com- plexicity and structural diversity of the NOS genes, there z.e several reports on

478

alternatively spliced variants. For in- stance, two catalytically active splice forms of nNOS (nNOS {3 and ~/) have been identified in mice with targeted deletions of oxen 2. These splice forms might be important in certain brain regions and probably account for the residual nNOS activity in nNOS-knockout mice ~4.

Each NOS isoform has the same lay- out of catalytic domains: a C-terminal reductase with one binding site each for FAD, FMN and NADPH, and an N-terminal oxygenase section. The oxygenase domain contains bound haem and the binding site for the cofactor tetrahy- drobiopterin (H4biopterin). H4biopterin Is essential for the coupling of NADPH- dependent 0 z activation to NO synthesis. Calmodulin (CAM) binds just at the N-terminal side of the reductase. Each isoenzyme has a different N4erminal extension, which is not essential for catalysis and probably functions in the intracellular localisation of the enzyme. Fig. 2 summarises various structural fea- tures that have been identified.

H4biopterin~ependent cyto©hrome P-450 The oxygenase of NOS contains a haem

group with a thiolate iigand resulting in typical cytochrome PA50-1ike spectral properties. However, the oxygenase of NOS has no convincing sequence simi- larity with other P450 enzymes, and the recent crystal structure revealed a fold unlike any other known haemcprotein, being composed Largely of [~-sheet 5°.

The first unique feature of the NOS oxygenase is its haem-dependent dimefis- atinniS (see Fig. 3). Native NOS is a homo- dimer. The intersubunit interface is contained within the oxygenase region. Without bound haem, NOS was found to be monomeric. This monomeric form had no NOS activity, but did have full cytochrome-c reductase activity, normal ffavin content and a normal circular


dichroism spectrum. It could bind neither L-arg~nine nor H4biopterin, and incubation with haemin resulted in dimer formation.

Another unique feature of the NOS o~genase is that it is the only "known H4biopterin-dependent P450. It also contrasts with the other H4biopterin- dependent enzymes, in which H4hioptedn is oxidised to the dihydro form, which must dissociate in order to be reduced by a separate reductase enzyme ~6. in NOS, H4biopterin does not undergo net oxidation during the enzyme reaction. The cofactor affects both the conformation and the activity of NOS; much effort is currently being directed to teasing apart the relative impo~'tance of each type of contribution to the overall [unction ol the enzyme.

Two main structural correlates of H4biopterin activation have been observed. The first is that it can confer on the dimer a surprising resistance to dissociation by SDS 17. The second is that it binds to the enzyme only when the haem is in a high-spin state ~. As the high-spin/ low-spin transition in P450s genera|ly reflects the absence or presence of a sixth ligand to the haem iron, this would be compatib|e with a structure in which H4biopterin binds so close to the haem that it competes sterically with the sixth ligand. L-Arginine also binds only to the high-spin form, suggesting that the chemistry of NO formation occurs in a confined space near the haem.

H41fie~efln ~ i n g and ~roxynll~ite synthesis As already mentioned, H4biopterin is

essential for coupling NADPH-dependent 0 2 activation to NO synthesis IG. in its absence, the haem centre can release 02-. When considering how this effect is achieved, it may be such that the lack of net oxidation of H4bioptedn by no means rules out the possibility that it is tran- siently oxidised during the enzyme turn- over. The analogue 4-amino-H4biopterin, which cannot undergo the H4/H 2 redox cycle, binds to NOS with almost the same affinity as H4biopterin does, but efficiently antagonises the activation by H4biopterin. At least for iNOS, 4-amino- H4biopterin appears to cause both the low-to-high spin transition and the stabilisation of the dimer zg. This hints strongly that the conformational effects of H4biopterin alone might not be suffi- cient to activate the enzyme, and that the cofactor may be a direct chemical par- ticipant at some point in the reaction.

The binding of H4biopterin to NOS shows strong negative cooperativity between the two binding sites of the

TIBS 2 2 - DECEMBER 1997

homodime~, which are located one on each ol the two subunits ~g. The first mob ecu]e of H4biopterin binds with a dissociation constant in the low nanomolar range, while the second dissociation constant is about three orders of magnitude ~arge~. This explains Che observation that the purified enzyme usually contains one molecule of H4biopterin per dimer.

The H4biopteri~4ree and H4biopterin- containing subunits appea~ to be cata- lyticaRy independent. This seems likely to have important physiological impli- cations. Over a wide range of H4biopterin concentrations, one site per dimer will be occupied, so that the overall rate of NO synthesis could be fairly insensitive to fh~ctuMions in H~biopterin concentration. However, the activity of the H4biopterin- free subunit should be noted, in light of its ability to catalyse O 2- production. This activity is low in eNOS and iNOS, but is much higher in nNOS 2°. O 2- reacts rapidly with NO to form peroxynitrite, which is cytotoxic.

The possibility that, under certain conditions, nNOS could work elfectively as a peroxyaitrite synthase might account for the apparent conh'ibution of this isoenzyme to brain ischaemia-reperfusion injury. Unfortunately, we cannot say whether the enzyme is really exposed in rive to H4biopterin concentrations that would allow the production of pero~,- nitrite, mainly because the average H4biopterin concentration for a tissue may be quite different from the lucal concentration in the cells that contain NOS. 1"he best indication timt H4bi~pterin may become limiting for NO biosynthesis in rive is the restoration o| endothelium- dependent vasorelaxation by exogenous H4biopterin in tissues affected by a variety of cardiovascular disease states2~; whether similar effects are relevant in brain is an open question.

Isoemyme-spedfic regulation by calmodulin NOS is str ict ly dependent on cal-

modulin (CAM), which activates the enzyme by facilitating electron transfer within the reductase. An important difference between the isoenzymes is that, while in eNOS and nNOS, CaM binding and activation respond to physiological changes in Ca 2+, iNnS already binds CaM and is fully active at the lowest Ca 2+ concentrations encountered in rive.

An obvious difference between the sequences of iNnS and the other two isoenzymes is the presence in eNOS and nNOS of a 40-residue insert within the FMN-binding domain (residues 594-638 in human eNOS and 830-870 in rat nNOS).

REVIEWS Conserved tsoenzyme-specific

N-termina! sequence

Oxygenase

CaM-binding site

Reductase

NH 2 _£_

H4bioptedn-binding cysteine -- Haem axia~ migand cysteine - -

Arg-binding glutamate - -

f Phosphate ~ " I

FMN domain I

L .. IsoMIoxazine J

('Pyrophosphate I Isoallox~zine

Ribose

DPH domain Adenine

COO -

< - - Fatty acy~ation sites (eNOS}

<,--- PDZ domain (nNOS)

Ca2+-independent CaM binding (iNOS)

CaM inhibitor sequence "¢'-'- (eNOS and nNOS)

Figure 2 Functional elements m the nitric o×ide (NO) synthase sequence. For references to earlier work on the identification of the haem-binding cysteine (Cys415 in nNOS, Cys184 in eNOS and Cys194 in iNOS), [[he PDZ domain of nlqOS and fatty acylatien sites in eNOS, see Ref, 52, Consensus sequences for calmodulin (CaM)-binding and putative contact sites for the isoalloxazine ring of FMN, and for FAD and NADPN were first recognized in nNOS and are conserved between all NOS isoforms ~2. The CaM-inhibRory sequence was identified by J. C. Salerno and colleagues 22. Site-directed mutagenesis studies revealed elements important for H4biopterin binding 53.54 and a glutamate residue involved in L-arginine binding tn eNOS s~ and to the oxygenase domain of iNOS 55.

This was linked to the function of C',uM by the finding that synthetic pepfides with this same sequence competed with CaM binding 22. in other CaM-dependent enzymes, it has often been found that CaM must displace an inhibitory loop of the enzyme from the vicinity of its binding site, and a similar role was proposed for this insert in NOS.

More light was shed on this question by an elegant study in which the CaM- binding sequences were exchanged between the different isoenzymes 23. A chimera of eNOS, containing the CaM- binding sequence from iNnS, bound CaM independently of Cat+; however the enzyme still required Ca a÷ to become active.

Combining these f~ndings it would seem that the Caa+-independent binding

of CaM to NOS is conferred by the specific CaM-binding sequence of iNnS, but that in the isoenzymes containing the inhibitory insert, onTy the Ca2÷-bound form of (:aM can displace the insert and activate the enzyme. ;n iNnS the acti. vation is presumably Ca2+-independent because the inhibitory insert is absent.

PhygoPogy of NO Catdiova~cu[a¢ sptem. Cardiovascular

NO, which regulates blood pressure and platelet function, originates mainly, but not exclusively, from endothelial cells: cardiomyocytes, for example, also syn- tuesize eNOS 24. Treatment with NOS inhibitors 25, and more recently, disruption of the gene encoding eNOS 26'27, both significantly increased the blood pressure

M9

REVIEWS

Cytochrome-c reductase

+ haem

FAD FMN CaM haem J

NADPH oxidase

+ BH4

f ham CaM FMN FAD 1

I FAD FMN CaM l ~ h u m J

Peroxynitrite synthase

+ BH4

f ~mem

IFAD FMN CaM ~ ~ ~

NO synthase

Rgm 3 Subunit assembly of neuronal nitric oxide (NO) syno thase. Dimerization of NO synthase depends on binding of the prosthetic haem group ts, and binding of H4blopterln (BH 4) stabilizes the dlmer zT. Negative co-

ts operativtty of H4biopterln binding underlies the isolation of an enzyme with one H4blopterln per dlmer, which Is likely to have peroxynitrtte synthase activity, Only In the presence of saturating concentrations of H4blopterin does the enzyme become a pure NO synthase.

in laboratory animals. These results indicate that tonic endothelial NO synthesis is essential for maintenance of normal blood pressure. Endothelial NO release, and therefore the regulation of eNOS, is relevant to hypertension, athero- sclerosis and heart failure: these disease states all involve impaired endothelium- dependent relaxation of blood vessels. eNOS is regulated by both chemical and mechanical factors that can alter the enzyme's production, activity and sub- cellular localization ~.

Under normal physiologh.. ' -'mditions, eNOS is regulated by Intracellui~_ ~.a z÷ and is activated by Ca ~+ agunists, suC: as acetylchollne or bradykinln. Vascular NO synthesis is also sensitive to blood flow. Endothelial cells respond to haemo- dynamic shear stress with an acute NO release, but without an increase of

480

bulk cytosolic Ca'-'L The effect might represent a Ca 2+- independent activation of eNOS, but new sensors are allowing the detection of Io- calised Ca z+ increases near the plasma membrane, which could also account for the activation 29. Tyrosine phos- phorylation of eNOS itseIP ° or an eNOS-associated protein 3~ has also been suggested as an activation mechanism.

eNOS can be myristoylated and/or palmitoylated near its N-terminus 28. These events appear to be regulated by cAMP a2 and seem likely to govern the targeting of the enzyme to the plasma membrane, the Golgi apparatus and caveolae, and its transio- cation between these sites. eNOS binds to caveolin-1 and -3 in endothelial cells and cardiomyocytes, respectively, and caveolin-derived synthetic peptides inhibit eNOS activity in vitro aS. These observations are new and their exact contribution to NO release is not yet clear, but it is reasonable to expect a coherent account in the near future.

In addition to these acute effects on enzyme activity, several physiological factors appear to regulate the steady- state eNOS mRNA levels. The most important among these factors are hypoxia 34 and laminar shear stress 3~, both of which upregulate eNOS pro-

duction and lead to increased NO release. Central nervous system. NO is produced

by neurons in many parts of the brain. It can both link local blood flow to neuronal activity and modulate neurotrans- mitter release 4.

In postsynaptic terminals, binding of glutamate to N-methyl-D-aspartate- (NMDA)-type receptors leads to Ca 2+ influx, which would activate nNOS. nNOS binds via its N-terminal PDZ domain to certain postsynaptic density proteins (PSD-g5, PSD-93) in the brain 3°, as well as to the dystrophin-associated protein ~-syntrophin in skeletal muscle.

Peptides binding with high affinity to the PDZ domain of nNOS were found to potently and selectively uncouple NOS activity from NMDA receptor stimulation, suggesting that the interaction with postsynaptic proteins targets nNOS to the


NMDA receptors, creating the link be= tween postsynaptic glutamate binding and NO synthesis sT.

The function of NO in normal physiology of the brain is controversial. It has been proposed to play a role in fang-term potentiation (LTP) of excitatory neurotransmission in the hippocampus, a phenomenon thought to be involved in learning and memory formation. LTP in- volves glutamatergic neurotransmission, and NOS inhibitors both inhibited presynaptic glutamate release and reduced LTP 3s. The involvement of NO has been disputed, because the effects of NOS inhibitors were found to depend on experimental conditions 39, CA| hippo- campal neurons have no nNOS, and nNOS-knockout mice had normal LTP 4°. A possible resolution of the contoversy has been suggested by the findings that the relevant neurons contain eNOS, and that LTP was seriously impaired in mice lacking the gone for eNOS 4~,42.

Induction of LTP by NO appears to depend on cGMP formation, because the sGC inhibitor, 1H-[ 1,2,4]oxadiazolo[4,3-a] quinoxalin-l-one (ODQ), caused a signifi- cant reduction of LTP 43. cGMP can trigger Ca2+-influx through cyclic nucleotide- gated ion channels, causing release of glutamate from presynaptic terminals. NO is supposed to act as a retrograde messenger completing the feedback loop responsible for sustaining LTP.

The role of NO in ischaemia-reperfusion injury of the brain has also been the subject of debate. Both protective and harmful eifect~ of NO have been proposed depending on the stage of the ischaemic process and on the cellular source of NO ~. The development of selective phar- macological tools and the specific disruption of genes coding for NOS isoenzymes have begun to illuminate this area. Mice lacking the nNOS gene were found to be less susceptible to experimental stroke than control animaiP 5, whereas infarct size was significantly increased in animals without eNOS 4s. Thus, the positive effect of endothelial-derived NO on blood flow appears to protect the brain from excitotoxicity, while neuronal NO contributes to neurodegeneration. This last effect might be explained by the ability of nNOS to synthesize superoxide and]or peroxynitrite (see above).

Immune system. NO synthesis is part of the inflammatory response against pathogens, such as bacteria, viruses and tumor cells. When bone marrow-derived macrophages are activated by cytokines or endotoxin, the gone encoding iNOS is induced, resulting in synthesis of new

TIBS 22 - DECEMBER 1997 REVIEWS iNOS protein and a sustained production of NO. The induction of iNOS is regulated at the transcriptional level by endotoxin and cytokines either activating [interleukin l OL-!), interferono~ 0FN-7), turnout necrosis factor-~ (TNF-~)] or inhib- iting [IL-4, -8 and -10, tumour growth factor-~, platelet-derived growth ~actors (PDGFs), insulin- like growth factor-1 0GF-I), etc.] gone transcription. The signalling cascades leading to iNOS production are cell-type specific, but appear to involve activation of tyrosine kinases and NF-KB ~3.

iNOS can be produced by other cells besides macrophages, includ- ing heart muscle, astroglia, liver and smooth muscle cells. Although iNOS induction is more ditficult to achieve in human than in routine cell lines, the most recent evidence confirms that it does indeed occur in infection and in- flammation in human systems 47A8. Induced NO synthesis in the vascular wall causes potentially lethal hypoten- sion in severe inflammatory conditions like septic shock or multi-organ failure. Endotoxic shock was less-often fatal in iNOS-knockout mice 49.

The large NO concentrations produced by iNOS raise the question of how the host cells protect themselves against NO. Pre-stimulating mudne macrophages with low levels of NO or repetitive cytokine/endotoxin treatment resulted in resistance against NO-mediated apop- tosis, presumably through increased inactivation of NO by superoxide S°. However, NO resistance is not confined to the host. In Escherichia coil, NO activates the redox-sensitive transcriptional regulator SoxR, triggering an oxidative stress response that defends the bacteria against activated macrophages St. Future studies on these host and pathogen mechanisms may illuminate pathologi- cally important issues, such as bact,'" ial resistance, host-versus-graft disease and control of tumour growth.

A©knowiedgements The salary of B. H. and the experimen-

tal work in the authors' laboratory were financed by grants from the Fonds zur FOrderung der Wissenschaftlichen Forschung in Austria.

References 1 Fukuto, J. M. (1995) Adv. Pharmacol. 34, 1-15 2 Stuehr, D. J. (1997) Annu. Rev. PharmacoL

ToxicoL 37, 339-359 3 Loscalzo, J. and Welch, G. (1995) Prog.

Cardiovasc. Dis. 38, 87-104

4 Garthwaite, J. and Boulton, C. L. (1995) Annu. Rev. Physiol. 57,683-706

5 Xie, Q. W. and Nathan, C. (1994) J. Leukocyte Biol. 56, 576-582

6 Gaston, B. et aL (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 10957-10961

7 Beckman, J. S. and Koppenol, W. H. (1996) Am. J. Physiol. Cell Physiol. 40, C1424-C1437

8 Quijana, C. et at. (1997) Biochem. J. 322, 167-173

9 Lymar, S. V. and Hurst, J. K. (1995) .L Am. Chem. Sac. 117, 8867-8868

10 Stone, J. R., Sands, R. H., Dunham, W. R. and Marietta, M, A. (1995} Biochem. Biophys. Res. Commun. 207,572-577

I 1 Jia, L., Bonaventura, C., Bonaventura, J. and Stamler, J. S. (1996) Nature 380, 221-226

12 Hentze, M. W. and Kuhn, L, C. (1996) Proc. Natl. Acad. ScL U. S. A. 93, 8175-8182

13 F6rstermann, U. and Kleinert, H. (1995) Naunyn-Schmiedeberg's Arch. Pharmacot. 352,351-364

14 Eliasson, M. J. L., Blackshaw, S., Schell, M. J. and Snyder, S. H. (1997) Proc. Natl. Acad. ScL U. S. A. 94, 3396-3401

15 Klatt, P. et aL (1996) J. Biol. Chem. 271. 7336-7342

16 Mayer, B. and Wemer, E. R. (1995) Naunyn-Schmiedeberg's Arch. Pharmacol. 351, 453-463

17 Klatt, P. et at. (1995) EMBO J. 14, 3687-3695 18 Gorren, A. C. F. et eL (1996) Biochemistry35,

16735-16745 19 Mayer. B. et aL (1997) Biochemistry 36,

8422-8427 20 List, 8. M. etaL (1997) 8iochem. J. 323,

159-165 21 Kinoshita, H., Tsutsui, M., Milstien, S. and

Katusic, Z. S. (1997) Prog. Neurobiol. 52, 295-302

22 Gross, S. S. etaL (1997) Jap. 1 PharmacoL 75 (Suppl. I), 6P (Abst 13)

23 Venema, R. C., Sayegh, H. S.. Kent, J. D. and Harrison, D. G. (1996) J. BioL Chem. 271, 6435-6440

24 Balligand, J. L. et al. (1995) J. Biol. Chem. 270, 14582-14586

25 Rees, D. D., Palmer, R. M. and Moncada, S. {1989) Proc. Natl. Acad. Sci. U. $. A. 86, 3375-3378

26 Huang, P. L. et aL (1995) Nature 377,239-242 27 Shesely, E. G. et at. (1996) Proc. Natl. Acad.

Sci. U. S. A. 93, 13176-13181 28 Sase, K. and Michel, T. (1997) Trends

Cardiovasc. Meal. 7, 26-37

29 GraJer, W. F. et at. J. PhysroL !in press} 30 Garcia-Cardena, G. et aL (1996} J. Biol.

Chem. 271, 27237-27240 31 Venema, V. J., Marrero, M. 8. and

Venema, R. C. (1996) Biochem. Biophys. Res. Commun. 226, 703-710

32 8elhassen, t, oral. (1997) 1. Biol. Chem. 272, 11198-11204

33 Ju, H., Zou, R., Venema, V. J. and Venema, R. C. (1997) J. Biol. Chem. 272, 18522-18525

34 &-net, U, A. et at, (1996) J. BiaL Chem. 271, 15069-15073

35 Topper, J. N., Cai, J. X., Falb, D. and Gimbrene, M. A. (1996) Proc. Natl. Acad. ScL U. S, A. 93, 10417-10422

36 Brenman, J. E. et aL (1996) Ce1184, 757-767

37 Bredt, D. S., Christoferson, K. S, and Brahman, J. E. (1997) Jap, J. Pharmacol. 75 (Suppl, [), 7P (Abst 20)

38 Malenka, R. C. (1994) Cell 78, 535-538 39 Williams, J. H. et al. (1993) Neuron 11,

877-884 40 Odell, T. J. et al. (1994) Science 265,

542-546 41 Son, H, et eL (1996) Ce1187,1015-1023

42 Wilson, R. I. et at. (1997) Nature 386, 338 43 Boulton, C. L., Southam, E. and Garthwaite, J,

(1995) Neuroscience 69, 699-703 44 ladecola, C. (1997) Trends Neurosci. 20,

132-139 45 Huang, Z, H. et aL (1994) Science 265,

1883-1885 46 Huang, Z. H. et aL (1996) J. Cereb. Blood Flow

Metab. 16, 981-987 47 Wheeler, M. A. et aL (1997) 1. Clin. Invest. 99,

110-116 48 Moilanen, E. et aL (1997) Am. J. PathoL 150,

881-887 49 MacMicking, J. D. et at. (1995) Cell 81,

641-650 50 Brine, B. et al. (1997) J. Biol. Chem. 272,

7253-7258 51 Nunoshiba, T. et at. (1993) Proc. Natl. Acad.

ScL U. S. A. 90, 9993-9997 52 Hemmens, 8. and Mayer, B. (1997) in Methods

in Molecular Biolo~/ (Vol. 100. Nitric Oxide Protocols) (Titheradge, M. A., ed.), pp. 1-32, Humane Press

53 Chen, P. F., Tsai, A, L, and Wu, K. K. (1995) Biochem. Biophys, Res. Commun, 215, 1119-1129

54 Ghosh, D. K., Wu, C., Mayer, B. and Stuehr, D. J. (1997) Biochemistry 36, 10609-10619

55 Gachhui, R. et aL (1997) Biochemistry 36, 5097-5103

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