Nitric oxide and neurons Solomon Johns
Hopkins
Medical
In the past 2 years oxide functions nervous in the
evidence
of the
Abnormal
the neurotoxicity
Current
such
hippocampus, secretion
USA
it may
as
long-term
oxide
play a role
receptors
in Neurobiology
in
potentiation depression
in
may be responsible that
and neurodegenerative
Introduction
and peripheral
that
and long-term
of nitric
by NMDA
of strokes
Opinion
Maryland,
in both the central suggests
plasticity
mediated
pathophysiology
Baltimore,
evidence has emerged to suggest that nitric
of synaptic
region
the cerebellum. for
powerful Recent
forms
CA1
Institution,
as a neurotransmitter
systems.
mediating
H. Snyder
results
in the
diseases.
1992, 2:323-327
The regulation of NOS by Ca2+ and calmodulin accounts for the ability of NO to mediate glutamate stimulation of cGMP via N-methyl-D-aspartate (NMDA) receptors. The extremely rapid activation of NOS by NMDA occurs as a result of the opening of neuronal membrane Ca2+ charnels The resulting influx of Ca2+ binds calmodulin to directly activate NOS.
Nitric oxide (NO) was first identified as a mediator of blood vessel relaxation and immune responses. Blood vessel relaxation in response to substances such as acetycholine, bradykinin and ATP does not involve actions upon receptors on smooth muscles of the blood vessel wall. Instead, endothelial cells are targeted, which in turn secrete an ‘endothelial-derived relaxing factor’ (EDRF) whose diffusion to adjacent smooth muscle cells initiates relaxation [ 11, The realization that NO is EDRF derived came in part from research showing that NO is the active metabolite responsible for blood vessel relaxation induced by nitroglycerin, and the organic nitrates used in the treatment of cardiac angina [2]. At about the same time other workers showed that NO is responsible for the tumoricidal and bactericidal actions of macrophages [3,4], Stimulation of macrophages by endotoxin activates their ‘killing’ activities coincident with a major increase in NO formation. Inhibition of NO synthesis by removal of arginine, the precursor of NO, or by inhibitors of NO synthase (NOS) enzymatic activity, blocks the bactericidal and tumoricidal actions of macrophages.
Although it is a most improbable neurotransmitter can didate (it is a gas which is not stored in synaptic vesicles or released by exocytosis, and which does not act at conventional membrane associated receptors), the bio logical roles of NO may be more prominent than most conventional transmitter molecules. Indeed, in several peripheral systems NO fulfrls all of the major criteria for a neurotransmitter. This review will discuss the evidence in support of this, Biosynthesis NO is formed directly from arginine by NOS, which exists in at least two distinct forms, NOS catalyzes the oxidation of one of the guanidine groups of arginine to give NO, along with the stoichiometric formation of citrulline. The macrophage enzyme is present at negligible levels in the basal state, but can be induced to extremely high levels when activated by y-interferon and lipopolysaccharide. This ‘inducible’ NOS has been purified as a dimer composed of two identical subunits of about 135 kD, which require FAD, FMN, and tetrahydrobiopterin as oxidative co-factors [Cl.
The first evidence showing the presence of NO in the brain came from observations of dissociated cultures of neonatal cerebellar cells, which release a factor whose actions on blood vessels are reminiscent of NO [5]. The biosynthetic enzyme for NO, NOS, which converts arginine into NO and citrulline, occurs in discrete neuronal populations in the brain and peripheral autonomic nervous system. Purified and molecularly cloned brainendothelial forms of NOS display multiple regulatory sites that include phosphorylation by three distinct enzymes, and oxidation mediated by multiple co-factors, including NADPH, flavin adenine dinucleotide (FAD), fhvin mononucleotide (FMN), and tetrahydrobiopterin.
Brain NOS, which is similar if not identical to the endothelial form, differs from the macrophage enzyme. It also requires FAD, FMN, NADPH, and tetrahydrobiopterin as co-factors. In addition, it is absolutely de-
Abbreviations CPR-mmcytochromeP-450 reductase; EDRF-endothelial-dewed FMN---flavin
mononucleotide;
LTD-long-term
NDP--NADPH-diaphorase;
depression;
LTP
NMDA-N-methyl-u-aspartate;
PKC--protein
Biology
potentiation;
N&nitric
kinase C; VIP-vasoactive
@ Current
relaxing factor; FAD-flavin
long-term
intestinal
NANC
adenrne dinucleotide; non-adrenergic
oxide; NOGnitric
non-cholinergrc;
oxide synthase;
polypeptide.
Ltd ISSN 0959-4388
323
324
Signaliing mechanisms
pendent upon Ca2+ and calmodulin [7]. Indeed, the brain enzyme initially resisted purification, because enzyme activity was lost due to the dissociation of calmodulin [7]. Purified brain NOS, a monomer of 150 kD, is a highly regulated enzyme, being phosphorylated by the Ca2+/phospholipid-dependent protein kinase, protein kinase C (PKC), and Ca2+/calmodulindependent protein kinase II [8]. With all three enzymes phosphorylation is stoichiometric and on serine, but the enzymes phosphotylate distinct peptides. In kidney cells stably transfected with NOS cDNA, activation of PKC with phorbol esters leads to a rapid phosphorylation of NOS and an associated 50% or greater decrease in NOS activity [S] This might provide a system for ‘cross-talk between the phosphoinositide and NO messenger systems. How Ca2+/calmodulin and cAMP-dependent phosphorylation of NOS inlluence activity is not yet clear. Molecular cloning of brain NOS has revealed a number of striking properties [9=-l. Well defined sites for the binding of FMN, FAD and NADPH are evident as well as consensus sequences for calmodulin binding and PKC phosphorylation. Of all known mammalian protein sequences, NOS shows close homology only with cytochrome P-450 reductase (CPR). The two proteins are most similar in the carboxyterminal portion, where NOS is almost 60% homologous with CPR over more than 600 amino acids. This similarity suggests that in phylogeny CPR may have provided the electron transport necessary for NOS activity even as a distinct protein. When the amino-terminal and carboxyterminal halves of NOS are expressed together, the mixture provides NOS catalytic activity suggesting that the carboxyterminal can indeed transfer electrons ‘in trams’ to the amino-terminal domain, something that CPR might be able to do as well (DS Bredt and SH Snyder, unpublished data). Molecularly cloned macrophage NOS shows substantial homology with brain NOS, particularly in the carboxy-terminal portion [lo]. A recently purified neutrophil NOS differs in some ways from both macrophage and brain enzymes in requiring Ca2+ but not calmodulin [ 111. Most forms of NOS that have been described are cytoplasmic, but in endothelium a particulate form has been discovered
[=I.
localization Purification of brain NOS has permitted the generation of antibodies and an immunohistochemical localization [13,14**15]. The antiserum employed reacts with both brain and endothelial NOS, underscoring the biochemical similarities of the enzymes, but does not recognize macrophage NOS. Throughout the body NOS immunoreactivity occurs only in neurons and the endothelium of blood vessels. Within the brain NOS neurons display quite unique localizations. In some areas, such as the cerebellum, NOS occurs in all neurons of a given class, such as basket cells and granule cells, but not at all in other neurons, such as Purkinje cells. By contrast, in the cerebral cortex and hippocampus NOS occurs in only I%-2% of neurons, in scattered isolated cells that are medium to large aspiny neurons. While granule cells of the dentate gyms possess abundant NOS, no pyrami-
dal cells in the hippocampus are stained. In the corpus striatum NOS appears in scattered, medium to large as piny neurons in both the cell bodies and neuropil. Most brain regions contain some NOS cells, while prominent fiber staining NOS is most evident in the Islands of Callejae. The localizations of NOS do not fit with any known neurotransmitter but do co-localize with NADPH-diaphorase NDP staining occurs as a blue precipi(NDP) [l&,15]. tate with nitroblue tetrazolium in the presence of NADPH [16]. With few exceptions, in the brain and peripheral nervous systems, NDP and NOS localizations are essen tially identical both in rat and monkey. Kidney 293 cells transfected with NOS cDNA stain for NOS and NDP in proportions identical to those observed in neurons [15] indicating that NOS catalytic activity fully accounts for NDP staining. It has not been possible to characterize NDP as an enzyme protein by purification, because the diverse oxidative-reductive activities that can provide reduction of the dye are apparent only in multiple protein fractions. Hope et al. [ 17**], however, have recently shown that one fraction of NDP catalytic activity co-punfies with NOS activity. NDP neurons are selectively resistant to destruction in neurodegenerative diseases such as Huntington’s disease, following vascular stroke, or destruction of neural tissue by glutamate acting through NMDA receptors [ 181. How does NOS activity produce resistance to neurotoxicity? This is not known for certain, but NOS neurons also possess high levels of the manganese form of superoxide dismutase which is known to ‘detoxify’ NO (T Dawson, M Fotuhi, DS Bredt and SH Snyder, unpublished data). In the periphery, NOS occurs in association with NDP exclusively in neurons [15]. In the pituitary, prominent NOS staining fibers and terminals in the posterior lobe arise from NOS enriched cells in the supraoptic and paraventricular nuclei of the hypothalamus. Whether these regulate vasopressin and oxytocin secretion is unclear. In the adrenal gland, NOS occurs in discrete ganglion cells and fibers in the medulla, making close contact with catecholamine secreting chromalfin cells. Splanchnit nerve stimulation enhances catecholamine secretion from the adrenal medulla and augments blood flow. Nitroarginine, a potent inhibitor of NOS, blocks the increases in blood flow produced by nerve stimulation with no effect on catecholamine secretion (M Breslow, R Traystman, CD Ferris and SH Snyder, unpublished data). In the retina, a plexus of nerve fibers in the choroid stain for NOS, as do a limited number of amacrine cells in the inner nuclear layer, and occasional cells in the ganglion cell layer. The NOS neurons in the blood vessels of the choroid and limbus derive from cells in the sphenopalatine (pterygopalatine in the rat) ganglia, many of which also stain for vasoactive intestinal polypeptide (VIP) (R Stone, DS Bredt and SH Snyder, unpublished data). In blood vessels, NOS is concentrated in endothelial layers of large vessels such as the aorta, coronary arteries and cerebral arteries [ 131. The large cerebral blood vessels also display abundant NOS in nerve fibers within the adventitia, something not evident in most peripheral or distal cerebral vessels, These adventitial neurons also de-
Nitric oxide and neurons Snyder
rive from the sphenopalatine ganglion. Stimulating the adventitial nerve fibers elicits vascular relaxation, which through the use of NOS inhibitors, can be shown to be mediated via NO [ 19=]. Thus, NO satisfies a major criteria for a neurotransmitter in the innervation of cerebral arter ies. A localization of NOS to the adventitial innervation of blood vessels is also apparent in the penile corpora cavemosae of the rat (A Burnett, C Lowenstein, DS Bredt, T Chung, and SH Snyder, unpublished data). In rats, penile erections elicited by physiological nerve stimulation are reversed by as little as 5 mg kg-1 of the NOS inhibitor ni troarginine (A Burnett, C Lowenstein, DS Bredt, T Chung, and SH Snyder, unpublished data). Thus, NO may play a role in the physiology of penile erection,
like functions. The myenteric plexus mediates nonadrenergi-non-cholinergic (NANC) relaxation, a major component of peristalsis [29]. Classic biogenic amine transmitters do not account for NANC relaxation, and evidence for other candidates such as purines and neuropeptides has not been definitive. Myenteric plexus neurons do stain for NOS, however [13]. In numerous parts of the gastrointestinal system, including the lower esophageal sphincter [30], the stomach [31], and the large and small intestines [ 32-341, physiological NANC relaxation is blocked by NOS inhibitors. These effects are reversed by arginine and mimicked by nitroprusside. NOS inhibitors also hinder neurally mediated relaxation of penile corpora cavernosa [35-37,38-l and cerebral blood vessels [I’?].
Neurotransmitter-like
In the above systems, the criteria for neurotransmitter sta tus that are satisfied by NO include the presence of its synthetic enzyme in relevant neurons, the ability of NO to mimic the effects of physiological nerve stimulation, and the prevention of the effects of nerve stimulation on blockade of NO formation. Yet NO is most unlike conventional transmitters, which are stored in synaptic vesicles and released by exocytosis. Immunohistochemistry at the electron microscopic level has established that in myenteric plexus nerve terminals NOS is not contained in synaptic vesicles nor associated with the plasma membrane, but is largely cytoplasmic (IJ Llewellyr-Smith, ZM Song, M Costa, DS Bredt, and SH Snyder, unpublished data). This fits with a notion of NO as a messenger synthesized on demand, which diffuses out of neurons rather than being released by exocytosis.
functions
In the brain NO mediates certain actions of glutamate involving NMDA receptors, Glutamate and NMDA increase cGMP levels about tenfold in cerebellar slices [20] and, with an identical concentration-response relationship, triple NOS levels in a matter of seconds [ 21,221. NOS inhibitors block both increased NOS activity and the augmentation of cGMP levels [ 21,221. The exact physiological role of the cGMP increases, which take place in Purkinje cells, is unclear. Stimulation of climbing fibers increases Purkinje cell cGMP levels and also evokes a release of NO [23**1. As Purkinje cells themselves do not possess NOS, the source of this NO is not clear. Possibilities include parallel fibers of granule cells and basket cells, both of which are abundant in NOS and also make contact with Purkinje cells. How the synaptic input of climbing fibers to Purkinje cells is transmitted to parallel fibers or basket cells has not been established, but some sort of retrograde message is a possibility. For instance, Purkmje cells stain markedly for adenosine, and parallel fiber terminals are selectively enriched in adenosine receptors [ 241. Climbing fiber stimulation in the cerebellum, together with parallel fiber stimulation, evokes long-term depres sion (LID) of parallel fiber-Purkinje cell transmission [25]. Nitroprusside, which generates NO, or SbromocGMP can substitute for stimulation of climbing fibers in eliciting LTD [ 23**]. Moreover, hemoglobin, which binds NO, and N-monomethylarginine, a NOS inhibitor, block LTD. Thus, in this model of synaptic plasticity NO is a reasonable candidate for a key mediator. Recent evidence favors a similar role for NO in the synaptic plasticity of long-term potentiation (LTP). Several laboratories have shown that NOS inhibitors block LTP in slice preparations of the CA1 region of the hiphas simpocampus [ 26=-280*]. Moreover, hemoglobin ilar effects, and direct injections of NOS inhibitors into hippocampal pyramidal cells also block LTP. In cultured hippocampal pyramidal cells, NO administration enhances spontaneous transmitter release. If NO is a retrograde mediator of hippocampal LTP, one would expect it to be generated in hippocampal pyramidal cells. These cells do not stain for NOS, however [ 151. NOS neurons in smooth muscle provide the most elegant systems in which to directly test neurotransmitter-
In several peripheral neuronal systems, including neural innervation of structures in the eye, cerebral vasculature, penile corpora cavernosa, and the myenteric plexus, NOS is colocalized with VIP. Could NO and VIP function as cotransmitters? No direct synergy has been demonstrated between the two. However, NANC relaxation of stomach muscle can be reduced about 30% by antibodies to VIP, while NOS inhibitors abolish all the remaining NANC relaxation [ 391. Besides serving as a neurotransmitter candidate and somehow providing protection from neurotoxicity, NO may itself induce neurotoxicity, particularly that resulting from glutamatergic stimulation of NMDA receptors. This may seem paradoxical as NMDA stimulates NO formation in NOS neurons, yet the neurons are remarkably resistant. This paradox could be resolved if the NOS neurons rem leased NO to kill adjacent neurons, In primary cerebral cortical neuronal cultures NMDA destroys up to 90% of neurons within 24 h [ 18]. In this system, NOS inhibitors prevent neurotoxicity, as does removal of arginine from the culture medium or addition of hemoglobin [40**]. Moreover, nitroprusside is neurotoxic to these cultures. As NOS only occurs in about 2% of cortical neurons, how could NOS neurons kill so many other cells? Staining of NMDA-treated cultures for NOS, and with trypan blue to label dead neurons, reveals that NOS neuronal profiles ramify extensively, with clumps of dead neurons generally lying along the path of NOS neuronal processes (V Dawson, T Dawson, G Uhl and SH Snyder, unpublished data).
325
326
Signalling mechanisms
NMDA-receptor stimulation has been implicated in the neurotoxicity of vascular strokes, so might NO play a role? In mice repeated 1 mg kg- l intraperitoneal doses of the NOS inhibitor nitroarginine markedly reduce the volume of infarcted brain tissue following ligations of the middle cerebral artery [41-•]. Neuronal protection occurs even when the drug is administered after arterial ligation, suggesting a potential therapeutic application in stroke patients. Strikingly, as much as 70% protection is provided by nitroarginine, while maximal protection by the NMDA antagonist m-801 is only about 5@60% [41-J.
GARTHWAITE
6.
STUEHRDJ, CHO HJ, KWON NS, WE&E MF, NATHAN CF: PuriIication and Characterization of the Cytokine-Induced Macrophage Nitric Oxide Synthase: an FAD- and FMNContaining Flavoprotein Proc Nat1 Acad Sci USA 1991, 88:7773-7777.
7.
BREDT DS, SNYDER SH: Isolation of Nitric Oxide Synthetase, a Cahnodulin-Requiring Enzyme. Prcu Nat1 Acad Sci [JSA 1990, 87:682-685.
8.
BREDT DS, FE~UUS CD, SNYDERSH: Nitric Oxide Synthase Reg-
ulatory Protein Protein Binding
Conclusions Rapidly accumulating evidence indicates that NO is a neuronal messenger, fulfilling many criteria of a neurotransmitter. As it is such an atypical transmitter, one must either revise classical definitions or introduce a new category for messenger molecules such as NO. One other candidate for such a novel class is carbon monoxide. This gas is formed by two types of heme oxygenase: type I, which occurs primarily in peripheral tissues and is inducible; and type II, which is not inducible but occurs in high concentrations in the brain, strikingly reminiscent of the two major forms of NOS [42]. Using in situ hybridization my colleagues and I have demonstrated selective localizations for type II heme oxygenase in discrete neuronal populations in the hippocampus and cerebellum (D Hirsch, A Verma, C Glatt, and SH Snyder, unpublished data). Whatever the exact function of NO in the nervous system, it certainly has major physiological roles and may well be a valuable target for the design of important new therapeutic agents.
Supported by USPHS grams DA-00266, MH-18501, Contract DA~27190 7408, Research Scientist Award DA-00074, and a gift from BristolMeyers Squibb.
and recommended
Papers of particular interest, published view, have been highlighted as: . of special interest .. of outstanding interest 1.
2.
reading
within the annual period of rep
BREDT DS, HWANG PH, GLATI C, ILWENSTEIN
10.
ILXXNSTEIN CJ, GLUT CS, BREDT DS, SUER SH: Cloned and Expressed Macrophage Nitric Oxide Synthase Contrasts with Brain Enzyme. Proc Nat1 Acad Sci USA 1992, m press.
11.
Y~I Y, HAT-TORI R, KOSUGA K, EU.AWA H, HIKI K, KAWAI C: Cahnodulin-Independent Nitric Oxide Synthase from
C, REED RR, SH: Cloned and Expressed Nitric Oxide Synthase Structurally Resembles Cytochrome P-450 Reductase. Nature 1991, 351:714-718. The hrst molecular cloning of a NOS, revealing recognition sites on the enzyme for three oxidative cofactors (NADPH, FAD, FMN), calmodulin and a consensus PKA phosphorylation sequence. Its sequence resem~ blance to cytochrome P~450 reductase suggests a neural role for P-450 systems.
F, MITRAL CK, A~UVOLDWP,
SWER
Rat Polymorphonuclear 266:12544-12547.
NeutrophiIs.
J Biol
Chem
1991,
12.
FOKTERMANNU, SCHMIDT HHHW. POLLOCKJS, SHENC H, MITCHELL JA, WARNERTD, NAKANE M, M~IRAD F: Isoforms of Nitric Oxide Synthase. Rio&em Pharmacoll991,42:184~1857.
13.
BKEIX DS, HWANGPM, SNYDERSH: Localization of Nitric Oxide Synthase Indicating a Neural Role for Nitric Oxide. Nature 1990, 3471768-770.
14. ..
DAWSON TM, BKED~‘DS, FOTUHI M, HWANC PM, SNYDER SH:
15.
BREDT DS, GIA~
Nitric Oxide Synthase and Neuronal NADPH Diaphorase are Identical in Brain and Peripheral Tissues. Proc Nat1 Acad Sci C&4 1991, S&7797-7801. Co-localizations of NDP and NOS are demonstrated in all neuronal sites throughout the brain and periphery. Proportions of NOS and NDP staining are identical in cultured cells transfected with NOS and in neurons, proving that NOS catalytic activity fully accounts for all neuronal NDP staining. CE, HWANG PM, FOTfJflI M, DAWSON TM, SH: Nitric Oxide Synthase Protein and mRNA are Discreetly Localized in Neuronal Populations of the Mammalian CNS Together with NADPH Diaphorase. Neu~ ran 1991, 7:615-624. SNYDER
MONCADA S, PALMER RMJ, Hlt;cs EA: Nitric Oxide: Physiology, Pathophysiology and Pharmacology. Pharmacol Rez’ 1991, 43:10!+142. Mrrf~~
Sites: Phosphorylation by Cyclic AMP Dependent Kinase, Protein KInase C, and CaIciumKaImoduIin Kinase; Identification of Flavin and Cahnodulin Sites. J Biol Cbem 1992, in press.
9. ..
Acknowledgements
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BON C, STLJTZMANN JIM, DOBLE A, BUNCHARDJ-C: Possible Involvement of Nitric Oxide in Long-Term Potentiation. Eur J Pharmacol 1991, 199:37?381. LTP in the CA1 region of the hippocampus is prevented by low concen~ trations of nitroarginine, and the effects are stereospecifically reversed by L-arginine. Sodium nitroprusside, which generates NO, is found to elicit LTP.
RAJFERJ, ARONSONWJ, BUSH PA, DOREY FJ, IGNARROLJ: Nitric Oxide as a Mediator of the Corpus Cavemosum in Response to Nonadrenergic Noncholinergic Transmission. New Eng J Med 1992, 326:9c94. In corpora cavernosae of human penises from impotent males, inhibitors of NOS block nerve-evoked relaxation, consistent with a role for NO in penile erection, There is no comparison with tissue from non-impotent subjects, precluding any conclusions about causes of impotence.
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O’DELL TJ, HAWKINS RD, KANDEL ER, ARANCIO 0: Tests of the Roles of Two Diffusible Substances in Long-Term Potentiation: Evidence for Nitric Oxide as a Possible Early Retrograde Messenger. Proc Nat1 Acad Sci USA 1991, 88:11285-11289. The authors lind that LTP in hippocampal CA1 is reversed by higher concentrations of nitroarginine than those in [ 26**] and also by Nmethylarginine. Both inhibitors are active when added to the bath or injected with pyramidal cells. Hemoglobin was also found to block LTP.
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DAWSONVL, DAWSONTM, LONDON ED, BREDT DS, SNYDERSH: Nitric Oxide Mediates Glutamate Neurotoxicity in Primary Cortical Culture. Proc Nat1 Acad Sci USA 1991, 88:636ti371. In primary cerebral cultures of fetal rats NMDA neurotoxicity is prevented by NOS inhibitors or removal of arginine from the medium. Sodium nitroprusside is also neurotoxic. These results show that NO mediates NMDA neurotoxicity, which is thought to be involved in stroke damage and is blocked by NMDA antagonists. 40. ..
NOWICKIJP, Duv.~ D, POIGNET H, SCATON B: Nitric-Oxide Mediates Neuronal Death After Focal Cerebral Ischemia in the Mouse. Eur ,I Pharmacol 1991, 204:33+340. A direct test of the implications of [40**] that NO is involved in stroke damage. Low doses of nitroarginine provide better protection than NMDA antagonists against tissue destruction following middle cerebral artey ligation. 41. ..
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SH Snyder, Departments of Neuroscience, Pharmacology and Molecular Sciences, Psychiatry and Behavioral Sciences, Johns Hopkins Medical Institution, 725 North Wolfe Street, Baltimore, Malyland 21205, LJSA.
327
Hopkins
Medical
In the past 2 years oxide functions nervous in the
evidence
of the
Abnormal
the neurotoxicity
Current
such
hippocampus, secretion
USA
it may
as
long-term
oxide
play a role
receptors
in Neurobiology
in
potentiation depression
in
may be responsible that
and neurodegenerative
Introduction
and peripheral
that
and long-term
of nitric
by NMDA
of strokes
Opinion
Maryland,
in both the central suggests
plasticity
mediated
pathophysiology
Baltimore,
evidence has emerged to suggest that nitric
of synaptic
region
the cerebellum. for
powerful Recent
forms
CA1
Institution,
as a neurotransmitter
systems.
mediating
H. Snyder
results
in the
diseases.
1992, 2:323-327
The regulation of NOS by Ca2+ and calmodulin accounts for the ability of NO to mediate glutamate stimulation of cGMP via N-methyl-D-aspartate (NMDA) receptors. The extremely rapid activation of NOS by NMDA occurs as a result of the opening of neuronal membrane Ca2+ charnels The resulting influx of Ca2+ binds calmodulin to directly activate NOS.
Nitric oxide (NO) was first identified as a mediator of blood vessel relaxation and immune responses. Blood vessel relaxation in response to substances such as acetycholine, bradykinin and ATP does not involve actions upon receptors on smooth muscles of the blood vessel wall. Instead, endothelial cells are targeted, which in turn secrete an ‘endothelial-derived relaxing factor’ (EDRF) whose diffusion to adjacent smooth muscle cells initiates relaxation [ 11, The realization that NO is EDRF derived came in part from research showing that NO is the active metabolite responsible for blood vessel relaxation induced by nitroglycerin, and the organic nitrates used in the treatment of cardiac angina [2]. At about the same time other workers showed that NO is responsible for the tumoricidal and bactericidal actions of macrophages [3,4], Stimulation of macrophages by endotoxin activates their ‘killing’ activities coincident with a major increase in NO formation. Inhibition of NO synthesis by removal of arginine, the precursor of NO, or by inhibitors of NO synthase (NOS) enzymatic activity, blocks the bactericidal and tumoricidal actions of macrophages.
Although it is a most improbable neurotransmitter can didate (it is a gas which is not stored in synaptic vesicles or released by exocytosis, and which does not act at conventional membrane associated receptors), the bio logical roles of NO may be more prominent than most conventional transmitter molecules. Indeed, in several peripheral systems NO fulfrls all of the major criteria for a neurotransmitter. This review will discuss the evidence in support of this, Biosynthesis NO is formed directly from arginine by NOS, which exists in at least two distinct forms, NOS catalyzes the oxidation of one of the guanidine groups of arginine to give NO, along with the stoichiometric formation of citrulline. The macrophage enzyme is present at negligible levels in the basal state, but can be induced to extremely high levels when activated by y-interferon and lipopolysaccharide. This ‘inducible’ NOS has been purified as a dimer composed of two identical subunits of about 135 kD, which require FAD, FMN, and tetrahydrobiopterin as oxidative co-factors [Cl.
The first evidence showing the presence of NO in the brain came from observations of dissociated cultures of neonatal cerebellar cells, which release a factor whose actions on blood vessels are reminiscent of NO [5]. The biosynthetic enzyme for NO, NOS, which converts arginine into NO and citrulline, occurs in discrete neuronal populations in the brain and peripheral autonomic nervous system. Purified and molecularly cloned brainendothelial forms of NOS display multiple regulatory sites that include phosphorylation by three distinct enzymes, and oxidation mediated by multiple co-factors, including NADPH, flavin adenine dinucleotide (FAD), fhvin mononucleotide (FMN), and tetrahydrobiopterin.
Brain NOS, which is similar if not identical to the endothelial form, differs from the macrophage enzyme. It also requires FAD, FMN, NADPH, and tetrahydrobiopterin as co-factors. In addition, it is absolutely de-
Abbreviations CPR-mmcytochromeP-450 reductase; EDRF-endothelial-dewed FMN---flavin
mononucleotide;
LTD-long-term
NDP--NADPH-diaphorase;
depression;
LTP
NMDA-N-methyl-u-aspartate;
PKC--protein
Biology
potentiation;
N&nitric
kinase C; VIP-vasoactive
@ Current
relaxing factor; FAD-flavin
long-term
intestinal
NANC
adenrne dinucleotide; non-adrenergic
oxide; NOGnitric
non-cholinergrc;
oxide synthase;
polypeptide.
Ltd ISSN 0959-4388
323
324
Signaliing mechanisms
pendent upon Ca2+ and calmodulin [7]. Indeed, the brain enzyme initially resisted purification, because enzyme activity was lost due to the dissociation of calmodulin [7]. Purified brain NOS, a monomer of 150 kD, is a highly regulated enzyme, being phosphorylated by the Ca2+/phospholipid-dependent protein kinase, protein kinase C (PKC), and Ca2+/calmodulindependent protein kinase II [8]. With all three enzymes phosphorylation is stoichiometric and on serine, but the enzymes phosphotylate distinct peptides. In kidney cells stably transfected with NOS cDNA, activation of PKC with phorbol esters leads to a rapid phosphorylation of NOS and an associated 50% or greater decrease in NOS activity [S] This might provide a system for ‘cross-talk between the phosphoinositide and NO messenger systems. How Ca2+/calmodulin and cAMP-dependent phosphorylation of NOS inlluence activity is not yet clear. Molecular cloning of brain NOS has revealed a number of striking properties [9=-l. Well defined sites for the binding of FMN, FAD and NADPH are evident as well as consensus sequences for calmodulin binding and PKC phosphorylation. Of all known mammalian protein sequences, NOS shows close homology only with cytochrome P-450 reductase (CPR). The two proteins are most similar in the carboxyterminal portion, where NOS is almost 60% homologous with CPR over more than 600 amino acids. This similarity suggests that in phylogeny CPR may have provided the electron transport necessary for NOS activity even as a distinct protein. When the amino-terminal and carboxyterminal halves of NOS are expressed together, the mixture provides NOS catalytic activity suggesting that the carboxyterminal can indeed transfer electrons ‘in trams’ to the amino-terminal domain, something that CPR might be able to do as well (DS Bredt and SH Snyder, unpublished data). Molecularly cloned macrophage NOS shows substantial homology with brain NOS, particularly in the carboxy-terminal portion [lo]. A recently purified neutrophil NOS differs in some ways from both macrophage and brain enzymes in requiring Ca2+ but not calmodulin [ 111. Most forms of NOS that have been described are cytoplasmic, but in endothelium a particulate form has been discovered
[=I.
localization Purification of brain NOS has permitted the generation of antibodies and an immunohistochemical localization [13,14**15]. The antiserum employed reacts with both brain and endothelial NOS, underscoring the biochemical similarities of the enzymes, but does not recognize macrophage NOS. Throughout the body NOS immunoreactivity occurs only in neurons and the endothelium of blood vessels. Within the brain NOS neurons display quite unique localizations. In some areas, such as the cerebellum, NOS occurs in all neurons of a given class, such as basket cells and granule cells, but not at all in other neurons, such as Purkinje cells. By contrast, in the cerebral cortex and hippocampus NOS occurs in only I%-2% of neurons, in scattered isolated cells that are medium to large aspiny neurons. While granule cells of the dentate gyms possess abundant NOS, no pyrami-
dal cells in the hippocampus are stained. In the corpus striatum NOS appears in scattered, medium to large as piny neurons in both the cell bodies and neuropil. Most brain regions contain some NOS cells, while prominent fiber staining NOS is most evident in the Islands of Callejae. The localizations of NOS do not fit with any known neurotransmitter but do co-localize with NADPH-diaphorase NDP staining occurs as a blue precipi(NDP) [l&,15]. tate with nitroblue tetrazolium in the presence of NADPH [16]. With few exceptions, in the brain and peripheral nervous systems, NDP and NOS localizations are essen tially identical both in rat and monkey. Kidney 293 cells transfected with NOS cDNA stain for NOS and NDP in proportions identical to those observed in neurons [15] indicating that NOS catalytic activity fully accounts for NDP staining. It has not been possible to characterize NDP as an enzyme protein by purification, because the diverse oxidative-reductive activities that can provide reduction of the dye are apparent only in multiple protein fractions. Hope et al. [ 17**], however, have recently shown that one fraction of NDP catalytic activity co-punfies with NOS activity. NDP neurons are selectively resistant to destruction in neurodegenerative diseases such as Huntington’s disease, following vascular stroke, or destruction of neural tissue by glutamate acting through NMDA receptors [ 181. How does NOS activity produce resistance to neurotoxicity? This is not known for certain, but NOS neurons also possess high levels of the manganese form of superoxide dismutase which is known to ‘detoxify’ NO (T Dawson, M Fotuhi, DS Bredt and SH Snyder, unpublished data). In the periphery, NOS occurs in association with NDP exclusively in neurons [15]. In the pituitary, prominent NOS staining fibers and terminals in the posterior lobe arise from NOS enriched cells in the supraoptic and paraventricular nuclei of the hypothalamus. Whether these regulate vasopressin and oxytocin secretion is unclear. In the adrenal gland, NOS occurs in discrete ganglion cells and fibers in the medulla, making close contact with catecholamine secreting chromalfin cells. Splanchnit nerve stimulation enhances catecholamine secretion from the adrenal medulla and augments blood flow. Nitroarginine, a potent inhibitor of NOS, blocks the increases in blood flow produced by nerve stimulation with no effect on catecholamine secretion (M Breslow, R Traystman, CD Ferris and SH Snyder, unpublished data). In the retina, a plexus of nerve fibers in the choroid stain for NOS, as do a limited number of amacrine cells in the inner nuclear layer, and occasional cells in the ganglion cell layer. The NOS neurons in the blood vessels of the choroid and limbus derive from cells in the sphenopalatine (pterygopalatine in the rat) ganglia, many of which also stain for vasoactive intestinal polypeptide (VIP) (R Stone, DS Bredt and SH Snyder, unpublished data). In blood vessels, NOS is concentrated in endothelial layers of large vessels such as the aorta, coronary arteries and cerebral arteries [ 131. The large cerebral blood vessels also display abundant NOS in nerve fibers within the adventitia, something not evident in most peripheral or distal cerebral vessels, These adventitial neurons also de-
Nitric oxide and neurons Snyder
rive from the sphenopalatine ganglion. Stimulating the adventitial nerve fibers elicits vascular relaxation, which through the use of NOS inhibitors, can be shown to be mediated via NO [ 19=]. Thus, NO satisfies a major criteria for a neurotransmitter in the innervation of cerebral arter ies. A localization of NOS to the adventitial innervation of blood vessels is also apparent in the penile corpora cavemosae of the rat (A Burnett, C Lowenstein, DS Bredt, T Chung, and SH Snyder, unpublished data). In rats, penile erections elicited by physiological nerve stimulation are reversed by as little as 5 mg kg-1 of the NOS inhibitor ni troarginine (A Burnett, C Lowenstein, DS Bredt, T Chung, and SH Snyder, unpublished data). Thus, NO may play a role in the physiology of penile erection,
like functions. The myenteric plexus mediates nonadrenergi-non-cholinergic (NANC) relaxation, a major component of peristalsis [29]. Classic biogenic amine transmitters do not account for NANC relaxation, and evidence for other candidates such as purines and neuropeptides has not been definitive. Myenteric plexus neurons do stain for NOS, however [13]. In numerous parts of the gastrointestinal system, including the lower esophageal sphincter [30], the stomach [31], and the large and small intestines [ 32-341, physiological NANC relaxation is blocked by NOS inhibitors. These effects are reversed by arginine and mimicked by nitroprusside. NOS inhibitors also hinder neurally mediated relaxation of penile corpora cavernosa [35-37,38-l and cerebral blood vessels [I’?].
Neurotransmitter-like
In the above systems, the criteria for neurotransmitter sta tus that are satisfied by NO include the presence of its synthetic enzyme in relevant neurons, the ability of NO to mimic the effects of physiological nerve stimulation, and the prevention of the effects of nerve stimulation on blockade of NO formation. Yet NO is most unlike conventional transmitters, which are stored in synaptic vesicles and released by exocytosis. Immunohistochemistry at the electron microscopic level has established that in myenteric plexus nerve terminals NOS is not contained in synaptic vesicles nor associated with the plasma membrane, but is largely cytoplasmic (IJ Llewellyr-Smith, ZM Song, M Costa, DS Bredt, and SH Snyder, unpublished data). This fits with a notion of NO as a messenger synthesized on demand, which diffuses out of neurons rather than being released by exocytosis.
functions
In the brain NO mediates certain actions of glutamate involving NMDA receptors, Glutamate and NMDA increase cGMP levels about tenfold in cerebellar slices [20] and, with an identical concentration-response relationship, triple NOS levels in a matter of seconds [ 21,221. NOS inhibitors block both increased NOS activity and the augmentation of cGMP levels [ 21,221. The exact physiological role of the cGMP increases, which take place in Purkinje cells, is unclear. Stimulation of climbing fibers increases Purkinje cell cGMP levels and also evokes a release of NO [23**1. As Purkinje cells themselves do not possess NOS, the source of this NO is not clear. Possibilities include parallel fibers of granule cells and basket cells, both of which are abundant in NOS and also make contact with Purkinje cells. How the synaptic input of climbing fibers to Purkinje cells is transmitted to parallel fibers or basket cells has not been established, but some sort of retrograde message is a possibility. For instance, Purkmje cells stain markedly for adenosine, and parallel fiber terminals are selectively enriched in adenosine receptors [ 241. Climbing fiber stimulation in the cerebellum, together with parallel fiber stimulation, evokes long-term depres sion (LID) of parallel fiber-Purkinje cell transmission [25]. Nitroprusside, which generates NO, or SbromocGMP can substitute for stimulation of climbing fibers in eliciting LTD [ 23**]. Moreover, hemoglobin, which binds NO, and N-monomethylarginine, a NOS inhibitor, block LTD. Thus, in this model of synaptic plasticity NO is a reasonable candidate for a key mediator. Recent evidence favors a similar role for NO in the synaptic plasticity of long-term potentiation (LTP). Several laboratories have shown that NOS inhibitors block LTP in slice preparations of the CA1 region of the hiphas simpocampus [ 26=-280*]. Moreover, hemoglobin ilar effects, and direct injections of NOS inhibitors into hippocampal pyramidal cells also block LTP. In cultured hippocampal pyramidal cells, NO administration enhances spontaneous transmitter release. If NO is a retrograde mediator of hippocampal LTP, one would expect it to be generated in hippocampal pyramidal cells. These cells do not stain for NOS, however [ 151. NOS neurons in smooth muscle provide the most elegant systems in which to directly test neurotransmitter-
In several peripheral neuronal systems, including neural innervation of structures in the eye, cerebral vasculature, penile corpora cavernosa, and the myenteric plexus, NOS is colocalized with VIP. Could NO and VIP function as cotransmitters? No direct synergy has been demonstrated between the two. However, NANC relaxation of stomach muscle can be reduced about 30% by antibodies to VIP, while NOS inhibitors abolish all the remaining NANC relaxation [ 391. Besides serving as a neurotransmitter candidate and somehow providing protection from neurotoxicity, NO may itself induce neurotoxicity, particularly that resulting from glutamatergic stimulation of NMDA receptors. This may seem paradoxical as NMDA stimulates NO formation in NOS neurons, yet the neurons are remarkably resistant. This paradox could be resolved if the NOS neurons rem leased NO to kill adjacent neurons, In primary cerebral cortical neuronal cultures NMDA destroys up to 90% of neurons within 24 h [ 18]. In this system, NOS inhibitors prevent neurotoxicity, as does removal of arginine from the culture medium or addition of hemoglobin [40**]. Moreover, nitroprusside is neurotoxic to these cultures. As NOS only occurs in about 2% of cortical neurons, how could NOS neurons kill so many other cells? Staining of NMDA-treated cultures for NOS, and with trypan blue to label dead neurons, reveals that NOS neuronal profiles ramify extensively, with clumps of dead neurons generally lying along the path of NOS neuronal processes (V Dawson, T Dawson, G Uhl and SH Snyder, unpublished data).
325
326
Signalling mechanisms
NMDA-receptor stimulation has been implicated in the neurotoxicity of vascular strokes, so might NO play a role? In mice repeated 1 mg kg- l intraperitoneal doses of the NOS inhibitor nitroarginine markedly reduce the volume of infarcted brain tissue following ligations of the middle cerebral artery [41-•]. Neuronal protection occurs even when the drug is administered after arterial ligation, suggesting a potential therapeutic application in stroke patients. Strikingly, as much as 70% protection is provided by nitroarginine, while maximal protection by the NMDA antagonist m-801 is only about 5@60% [41-J.
GARTHWAITE
6.
STUEHRDJ, CHO HJ, KWON NS, WE&E MF, NATHAN CF: PuriIication and Characterization of the Cytokine-Induced Macrophage Nitric Oxide Synthase: an FAD- and FMNContaining Flavoprotein Proc Nat1 Acad Sci USA 1991, 88:7773-7777.
7.
BREDT DS, SNYDER SH: Isolation of Nitric Oxide Synthetase, a Cahnodulin-Requiring Enzyme. Prcu Nat1 Acad Sci [JSA 1990, 87:682-685.
8.
BREDT DS, FE~UUS CD, SNYDERSH: Nitric Oxide Synthase Reg-
ulatory Protein Protein Binding
Conclusions Rapidly accumulating evidence indicates that NO is a neuronal messenger, fulfilling many criteria of a neurotransmitter. As it is such an atypical transmitter, one must either revise classical definitions or introduce a new category for messenger molecules such as NO. One other candidate for such a novel class is carbon monoxide. This gas is formed by two types of heme oxygenase: type I, which occurs primarily in peripheral tissues and is inducible; and type II, which is not inducible but occurs in high concentrations in the brain, strikingly reminiscent of the two major forms of NOS [42]. Using in situ hybridization my colleagues and I have demonstrated selective localizations for type II heme oxygenase in discrete neuronal populations in the hippocampus and cerebellum (D Hirsch, A Verma, C Glatt, and SH Snyder, unpublished data). Whatever the exact function of NO in the nervous system, it certainly has major physiological roles and may well be a valuable target for the design of important new therapeutic agents.
Supported by USPHS grams DA-00266, MH-18501, Contract DA~27190 7408, Research Scientist Award DA-00074, and a gift from BristolMeyers Squibb.
and recommended
Papers of particular interest, published view, have been highlighted as: . of special interest .. of outstanding interest 1.
2.
reading
within the annual period of rep
BREDT DS, HWANG PH, GLATI C, ILWENSTEIN
10.
ILXXNSTEIN CJ, GLUT CS, BREDT DS, SUER SH: Cloned and Expressed Macrophage Nitric Oxide Synthase Contrasts with Brain Enzyme. Proc Nat1 Acad Sci USA 1992, m press.
11.
Y~I Y, HAT-TORI R, KOSUGA K, EU.AWA H, HIKI K, KAWAI C: Cahnodulin-Independent Nitric Oxide Synthase from
C, REED RR, SH: Cloned and Expressed Nitric Oxide Synthase Structurally Resembles Cytochrome P-450 Reductase. Nature 1991, 351:714-718. The hrst molecular cloning of a NOS, revealing recognition sites on the enzyme for three oxidative cofactors (NADPH, FAD, FMN), calmodulin and a consensus PKA phosphorylation sequence. Its sequence resem~ blance to cytochrome P~450 reductase suggests a neural role for P-450 systems.
F, MITRAL CK, A~UVOLDWP,
SWER
Rat Polymorphonuclear 266:12544-12547.
NeutrophiIs.
J Biol
Chem
1991,
12.
FOKTERMANNU, SCHMIDT HHHW. POLLOCKJS, SHENC H, MITCHELL JA, WARNERTD, NAKANE M, M~IRAD F: Isoforms of Nitric Oxide Synthase. Rio&em Pharmacoll991,42:184~1857.
13.
BKEIX DS, HWANGPM, SNYDERSH: Localization of Nitric Oxide Synthase Indicating a Neural Role for Nitric Oxide. Nature 1990, 3471768-770.
14. ..
DAWSON TM, BKED~‘DS, FOTUHI M, HWANC PM, SNYDER SH:
15.
BREDT DS, GIA~
Nitric Oxide Synthase and Neuronal NADPH Diaphorase are Identical in Brain and Peripheral Tissues. Proc Nat1 Acad Sci C&4 1991, S&7797-7801. Co-localizations of NDP and NOS are demonstrated in all neuronal sites throughout the brain and periphery. Proportions of NOS and NDP staining are identical in cultured cells transfected with NOS and in neurons, proving that NOS catalytic activity fully accounts for all neuronal NDP staining. CE, HWANG PM, FOTfJflI M, DAWSON TM, SH: Nitric Oxide Synthase Protein and mRNA are Discreetly Localized in Neuronal Populations of the Mammalian CNS Together with NADPH Diaphorase. Neu~ ran 1991, 7:615-624. SNYDER
MONCADA S, PALMER RMJ, Hlt;cs EA: Nitric Oxide: Physiology, Pathophysiology and Pharmacology. Pharmacol Rez’ 1991, 43:10!+142. Mrrf~~
Sites: Phosphorylation by Cyclic AMP Dependent Kinase, Protein KInase C, and CaIciumKaImoduIin Kinase; Identification of Flavin and Cahnodulin Sites. J Biol Cbem 1992, in press.
9. ..
Acknowledgements
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RAJFERJ, ARONSONWJ, BUSH PA, DOREY FJ, IGNARROLJ: Nitric Oxide as a Mediator of the Corpus Cavemosum in Response to Nonadrenergic Noncholinergic Transmission. New Eng J Med 1992, 326:9c94. In corpora cavernosae of human penises from impotent males, inhibitors of NOS block nerve-evoked relaxation, consistent with a role for NO in penile erection, There is no comparison with tissue from non-impotent subjects, precluding any conclusions about causes of impotence.
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SH Snyder, Departments of Neuroscience, Pharmacology and Molecular Sciences, Psychiatry and Behavioral Sciences, Johns Hopkins Medical Institution, 725 North Wolfe Street, Baltimore, Malyland 21205, LJSA.
327
