Molecular Psychiatry (1997) 2, 300–310 1997 Stockton Press All rights reserved 1359–4184/97 $12.00 PROGRESS Nitric oxide in health and disease of the nervous system H-Y Yun1,2, VL Dawson1,3,4 and TM Dawson1,3 Departments of 1Neurology; 3Neuroscience; 4Physiology, Johns Hopkins University School of Medicine, Baltimore, MD, USA Nitric oxide (NO) is a widespread and multifunctional biological messenger molecule. It mediates vasodilation of blood vessels, host defence against infectious agents and tumors, and neurotransmission of the central and peripheral nervous systems. In the nervous system, NO is generated by three nitric oxide synthase (NOS) isoforms (neuronal, endothelial and immunologic NOS). Endothelial NOS and neuronal NOS are constitutively expressed and acti- vated by elevated intracellular calcium, whereas immunologic NOS is inducible with new RNA and protein synthesis upon immune stimulation. Neuronal NOS can be transcriptionally induced under conditions such as neuronal development and injury. NO may play a role not only in physiologic neuronal functions such as neurotransmitter release, neural development, regeneration, synaptic plasticity and regulation of gene expression but also in a variety of neurological disorders in which excessive production of NO leads to neural injury. Keywords: nitric oxide synthase; endothelium-derived relaxing factor; neurotransmission; neurotoxic- ity; neurological diseases Nitric oxide is probably the smallest and most versatile NO synthases isoforms and regulation of NO bioactive molecule identified. Convergence of multi- generation disciplinary efforts in the field of immunology, cardio- vascular pharmacology, chemistry, toxicology and neu- NO is formed by the enzymatic conversion of the guan- robiology led to the revolutionary novel concept of NO idino nitrogen of l-arginine by NO synthase (NOS). as an unorthodox messenger.1–3 The discovery of NO Three NOS isoforms, neuronal NOS (nNOS; type I), as a messenger molecule in the nervous system4 also immunologic NOS (iNOS; type II) and endothelial NOS revised conventional concepts of neurotransmitters. (eNOS; type III) have been isolated and cloned5–8 NO is freely diffusible across membranes and is not (Table 1). All three isoforms are expressed in the cen- stored in synaptic vesicles and released by exocytosis tral nervous system (CNS). NO can also be generated upon membrane depolarization. NO seems to be ter- nonenzymatically by the direct reduction of nitrite to minated primarily by reaction with its targets. Thus, NO under acidic and highly reduced conditions that control of NO synthesis, by NOS, is the primary way may occur under ischemic conditions.9–10 nNOS and to regulate NO actions. Rapid progress is being made eNOS are calcium/calmodulin-dependent and under in understanding the regulation of NOS activity and most conditions are constitutively expressed. However, the cellular and molecular targets of NO under physiol- recent studies indicate that both enzymes can be ogic and pathologic conditions. Some of the emerging induced (ie new protein synthesis) under conditions roles of NO in the nervous system include regulation or of stress, injury and differentiation.11 iNOS is calcium- control of neuronal morphogenesis, short-term or long- independent and its expression is primarily regulated term synaptic plasticity, regulation of gene expression at the level of transcription. Interestingly, in the and modification of sexual and aggressive behavior. absence of the substrate, l-arginine, nNOS is able to − Deregulated formation of NO from all three NOS iso- produce superoxide anion (O2· ) and hydrogen per- forms plays a role in neural injury in a host of neuro- oxide.12,13 Under conditions of reduced arginine con- logical disorders. centrations substantial amounts of superoxide may be 14 − generated by NOS. NOS generation of O2· and NO in arginine-depleted cells can lead to peroxynitrite- mediated cellular injury.15 Since NO is freely diffusible, highly reactive, and synthesized on demand without being stored, its gener- Correspondence: Dr TM Dawson, Department of Neurology, Johns ation is the only way by which it is regulated. Thus, Hopkins University School of Medicine, 600 N Wolfe St there is a multiplicity of regulatory sites on the Pathology 2-210, Baltimore, MD 21287, USA. E-mail: enzymes that synthesize NO. Consensus sites for phos- ted dawsonKqmail.bs.jhu.edu 2Present address; Department of Biochemistry, Chung-Ang Uni- phorylation are evident in all NOS isoforms. Although versity College of Medicine, Seoul, Korea there are some discrepancies, it appears likely that Received and accepted 27 February 1997 nNOS catalytic activity is decreased by phosphoryl- Nitric oxide in the nervous system H-Y Yun et al 301 Table 1 NOS isoforms Type 1 nNOS Type II iNOS Type III eNOS Ca2+ dependency Activity depends on elevated Activity is independent of Activity depends on elevated Ca2+ elevated Ca2+ Ca2+ First identified in Neurons Macrophages Endothelial cells Expression Constitutive Inducible Constitutive Inducible under appropriate Inducible under appropriate conditions conditions Putative AP-2, TEF-1/MCBF IFN-g, TNF-a, NF-kB AP-1, AP-2, NF-1 transcription CREB/ATF/cFos A-activator-binding Heavy metal acute phase regulatory sites NRF-1, Ets, NF-1 response, Shear stress and NF-kB sterol regulatory elements Subcellular Soluble or membrane-bound Soluble or membrane-bound Largely membrane-bound localization Sarcoplasmic membrane localization in skeletal muscle Phenotypes of null Pyloric stenosis Increased susceptibility to Deficient acetylcholine mutant mice Resistant to vascular stroke Listeria and Leishmania vasodilation Resistant to MPTP neurotoxicity infection and lymphoma cell Elevated mean blood pressure Excessive sexual and aggressive proliferation l-nitroarginine-induced behavior Resistant to endotoxin hypotension Urinary bladder hypertrophy hypotension and carrageenan Increased infarct volume and loss of urethral and inflammation following MCA occlusion bladder muscle relaxation Reduced infarct volume by NO Normal hippocampal LTP and inhibitor in MCA occlusion cerebellar LTD Reduced hippocampal LTP in nNOS and eNOS double knockouts ation by protein kinase A (PKA), protein kinase C tochemistry has localized nNOS to the Golgi apparatus, (PKC), cyclic GMP-dependent protein kinase (PKG) endoplasmic reticulum, spines, dendritic shafts and and calcium-calmodulin protein kinase (CAM-k).16,17 axon terminals.24,25 In addition, nNOS and the NMDA- Calcineurin, a calcium-calmodulin dependent protein receptor subunits are colocalized in nNOS neurons.26 phosphatase, is able to dephosphorylate NOS and sub- The presence of a PDZ domain (DHR domain or GLGF sequently increase its catalytic activity.18 The immuno- repeat) in nNOS27 and its role in the membrane associ- suppressant and calcineurin inhibitor, FK506 ation of nNOS in skeletal muscle28 may provide insight (Tacrolimus), protects cultured cortical neurons into regulation of nNOS. PDZ domain interactions may against glutamate neurotoxicity by maintaining the mediate binding of nNOS to synaptic junctions through phosphorylated, catalytically inactive state of nNOS.18 interactions with postsynaptic density-95 protein NO formation is also tightly regulated by controlling (PSD-95) and PSD-93.29 Skeletal muscle membrane the levels of various necessary cofactors within the from patients with Duschene Muscular Dystrophy cell.19 Recent studies identified a novel protein, PIN, which lack dystrophin also lack nNOS.28,30 nNOS is which interacts with and potently inhibits the activity also significantly reduced in the skeletal muscle from of nNOS.20 PIN destabilizes the nNOS dimer, a confor- mdx mice, which do not express full-length dystro- mation necessary for activity. The highly conserved phin. nNOS mRNA, protein and enzymatic activity are sequence of PIN suggests it plays a role in regulating all markedly reduced in mdx mouse skeletal muscle,31 numerous NO-related biological processes. Regulation suggesting a role of NO in maintaining integrity and of iNOS primarily occurs at the level of gene transcrip- functional regulation of skeletal muscle membranes. tion and analysis of the promoter region indicates that iNOS has a complex pattern of regulation.21,22 iNOS Biochemistry of NO expression is induced in response to cytokines, lipo- polysaccharide (LPS) and a host of other agents.19 In NO is considered a highly reactive free radical due to the nervous system, NO generated by nNOS in glial its short half-life (3–6 s) in comparison to traditional cells negatively regulates iNOS expression through neuronal messenger molecules. NO, once produced, inhibition of NFkB.23 does not persist in its native form for very long, in that NOS may also be regulated by changes in its intra- it is either rapidly oxidized, reduced, or complexed to cellular localization. Electron microscopic immunohis- various biomolecules (Table 2). The biological outcome Nitric oxide in the nervous system H-Y Yun et al 302 Table 2 Cellular targets of NO Molecules Actions Cellular consequences Guanylyl cyclase Activation Increase cGMP and possible neuroprotection Ras Activation Activation of extracellular signal-activated protein kinase and possible neuroprotection NADPH-ubiquinone oxidoreductase Inhibition Decreased oxidative phosphorylation NADH-succinate oxidoreductase Inhibition Decreased oxidative phosphorylation cis-Acotinase Inhibition Decreased glycolysis Cytochrome oxidase Inhibition Decreased oxidative phosphorylation Creatine