NO: Recent Developments

by Kirk Maxey, M.D. and Jeff Johnson, Ph.D.

Nitric oxide (NO) is a free radical gas that is involved in the regulation of a vast number of biological processes. The importance of NO in biomedical research was highlighted in 1998 when the Nobel Prize in Physiology & Medicine was awarded to Robert Furchgott, Louis Ignarro and Ferid Murad for their pioneering work in this field. Their initial efforts to understand the role of NO in the nervous system, cardiovascular and immune systems expanded to numerous other biomedical disciplines including apoptosis, inflammation, kidney function, diabetes, oxidative stress and aging, to name only a few. Research in this field continues to be highly active. This article seeks to provide a succinct review of the basic fundamentals of NO biochemistry and physiology, as well as highlight some recent developments and the complementary research tools available from Cayman Chemical for work in this field.

Formation of Nitric Oxide

Biosynthetic NO is derived from the amino acid arginine in an oxidative reaction that consumes molecular oxygen and reducing equivalents in the form of NADPH. The products of the reaction are NO, NADP⁺ and citrulline. Because NO cannot be stored and released as needed, the regulated synthesis of NO in biological systems is essential. The enzyme responsible for NO synthesis is Nitric Oxide Synthase (NOS), a remarkable enzyme requiring FAD, FMN, Heme, Ca²⁺, calmodulin and 6(R)-tetra-hydro-L-biopterin (BH₄) as cofactors. Three NOS iso-enzymes have been characterized and the salient features are summarized in Figures 1 and 2. Structurally, all NOS isozymes consist of a carboxy-terminal reductase domain which binds the flavin cofactors. A Ca²⁺/calmodulin binding domain lies in the center followed by an oxygenase domain, with electronic absorption properties similar to P450 enzymes, where binding of Heme, O₂, BH₄ and arginine substrate all take place (see schematic in Figure 2). The crystal structures of the oxygenase domains of iNOS and eNOS have been published.^1,2,3 However, the three-dimensional structure of the reductase domain remains unknown, as does the overall conformation of the homodimer.

Three distinct NOS enzymes, each the product of a unique gene, have been identified and characterized. The names given to these 3 NOS isoforms are based primarily on the tissue or cell source or the order in which each isoform was first characterized. The neuronal form (nNOS, NOS-1, Type I) is a Ca²⁺-dependent enzyme found in neuronal tissue and skeletal muscle. Four splice variants of full length nNOS (nNOSα), have more recently been identified (nNOSβ, nNOSγ, nNOSμ, and nNOS-2). A second isoform of NOS (iNOS, NOS-2, Type II) is inducible in a variety of cells and tissues in response to cytokine or endotoxin activation. iNOS binds Ca²⁺/calmodulin so tightly that at normal physiologic levels its activity is functionally Ca²⁺-independent. A third form, first found in vascular endothelial cells (eNOS, NOS-3, Type III), is also Ca²⁺ dependent, but differs from the neuronal form by its smaller size compared to nNOSα. eNOS is myristoylated and palmitoylated at the N-terminus, modifications which are required for localization to the plasmalemmal caveolae of endothelial cells. The human enzymes exhibit approximately 51-57% homology at the amino acid level. nNOS and eNOS were initially characterized as constitutive enzymes, but it is now clear that all three enzymes can be induced, albeit to different levels and by different stimuli.

Nitric Oxide Function

Cellular signaling with nitric oxide involves the highly regulated synthesis of NO by NOS, diffusion of NO into an adjacent cell, and activation of the soluble isoform of guanylate cyclase (sGC) leading to the synthesis of the second messenger cGMP. The mechanism of activation of sGC involves NO binding to a pentacoordinate ferrous heme that appears to be uniquely tuned to interact with NO. When NO is synthesized in the vascular endothelium by eNOS it causes relaxation of the adjacent smooth muscle. This relaxation is mediated by the transient increase in smooth muscle cellular cGMP, which presumably activates a cGMP-dependent protein kinase. A similar mode of action has been proposed for cell to cell signaling between neurons and smooth muscle. NO produced by nNOS in non-adrenergic, non-cholinergic (NANC) neurons of the autonomic nervous system relaxes adjacent smooth muscle via the same cGMP-dependent mechanism. NANC nerves play important roles in producing relaxation of smooth muscle in the cerebral circulation and the gastrointestinal, urogenital, and respiratory tracts. Dysregulated NO production in these nerves is associated with migraine headache, hypertrophic pyloric stenosis and male impotence.

In the brain nNOS is coupled to N-methyl-D-aspartate (NMDA) receptors through a scaffolding mechanism involving the PDZ domains of both proteins as well as PSD-95 and PSD-93. Proposed roles of NO in the brain are less clear but include long-term potentiation and behavior. In support of the latter, nNOS knockout mice are aggressive and exhibit inappropriate sexual behavior.⁶

The immune system has harnessed the toxic properties of NO to kill invading organisms, pathogens and tumor cells. The elevated concentrations of NO generated by iNOS in immune cells interferes with iron homeostasis and results in disruption of biologically important heme and non-heme iron-containing proteins such as cytochrome P450⁷, catalase, aconitase and ribonucleotide reductase.^8,9 NO also reacts rapidly with superoxide (O₂^-) to form peroxynitrite, a potent oxidant with the potential to disrupt protein structures through the nitration of protein tyrosine residues. In support of this role, nitrotyrosine has been identified both immunohistochemically and analytically in the debris of tissue damaged by inflammation and is useful as an endpoint marker of oxidative stress. In support of the role of NO in cell death, iNOS knockout mice have reduced defenses against microorganisms and against lymphoma tumor proliferation.¹⁰

Therapeutic Potential

Although high concentrations of NO can be beneficial as an anti-bacterial and anti-tumor agent, an excess of NO or related nitrogen reactive species, such as peroxynitrite, can be detrimental or even fatal. Inhibition of NOS, in particular isozyme selective inhibition, has great potential therapeutic value. This is especially evident in endotoxic shock where levels of NO vastly exceed normal physiologic levels.¹² The clinically observed extreme hypotension in shock has been linked to elevated levels of NO and has been reversed by NOS inhibition in both animal models of shock and in humans. In the brain, over-stimulation of NMDA receptors and the resulting activation of neuronal NOS results in massive release of NO. The predominant mechanism of NO mediated toxicity appears to be via its reaction with superoxide to make peroxynitrite. Through this mechanism, NO mediates the post-ischemic damage of stroke and may also play a major role in the pathophysiology of neurodegenerative diseases such as Parkinson’s disease, Huntington’s disease, ALS and Alzheimer’s disease.¹¹

Inhibitors selective for the neuronal NOS, such as N^ω-propyl-L Arginine, may find a role in the treatment of stroke, as nNOS selective inhibitors are neuroprotective in animal models of focal ischemia.¹¹ Research on selective NOS inhibition has focused on iNOS that is associated with inflammation or shock. For instance, 1400W is a highly selective iNOS inhibitor which is effective in preventing endotoxin-induced vascular leak, as well as reducing lesion size associated with inflammation following cerebral ischemia.^13,14 Despite its effectiveness, acute toxicity at high doses prevents its safe therapeutic use in humans. New inhibitors such as GW273629 and GW274150 may have potential for the treatment of a variety of inflammatory disorders such as rheumatoid arthritis and ulcerative colitis, in addition to the acute inflammatory indications such as shock in which iNOS is implicated.

Interestingly, a relative deficiency of NO also seems evident in a number of clinical conditions. One of the most popular drugs in recent history uses an indirect approach to solve a problem consistent with NO deficiency. Sildenafil (Viagra) treats erectile dysfunction by enhancing the action of endogenous NO in relaxing the corpora cavernosum smooth muscle. Inhibition of cGMP-specific phospho-diesterase type 5 by sildenafil effectively prolongs NO-mediated activation of sGC by preventing hydrolytic breakdown of its second messenger, cGMP.

Nitric oxide donors offer the best means to resolve clinical problems related to low NO levels. By far the most common example is oral nitroglycerin, which acts to augment NO in the coronary arteries and increases blood flow to ischemic myocardium. The diazenium-diolate class of NO donors (NONOates; see Table II) offers the potential to fine-tune the NO delivery in a variety of ways including incorporation of the drug on immobilized supports, formulation in aerosols for treatment of pulmonary hypertension and synthesis of prodrugs such as V-PYRRO/NO.^15,16 NO-NSAIDs incorporate an NO-releasing moiety with classical Non-Steroidal Anti-Inflammatory Drugs. NO released from these compounds counteracts the toxic effect of NSAIDs on the gastrointestinal tract and also aids in wound healing.^17,18 Exciting data is emerging for the use of NO donors (in particular DETA NONOate) to induce neurogenesis and restore brain function following stroke (see inset article by Dr. Michael Chopp).

Cayman Chemical provides novel and useful research tools for NO research. Lists containing properties of NO-donors and NOS inhibitors supplied by Cayman Chemical are provided in Tables I and II. In addition Cayman Chemical offers assay kits to measure NOS activity and quantification of Nitrate/Nitrite. Other products for NO and NOS research are featured in this issue.

Nitric Oxide and Neurogenesis

by Michael Chopp, Ph.D. and Ruilan Zhang, M.D.
Henry Ford Health Sciences Center, Detroit, Michigan, USA

Just when we think that we have a full understanding of this pleiotropic molecule, another fascinating application and function of Nitric Oxide (NO) arises. In this brief essay, I will describe and outline our findings that NO donor molecules reduce neurological deficits and evoke neurogenesis in ischemic brain, and this neurogenesis may contribute to functional benefit found after cerebral injury such as stroke. The logic for initiating research for the effects of NO on brain plasticity arises from observations that NO plays a prime role in the development of the embryonic brain.¹⁹ Nitric oxide synthase is greatly increased in the embryonic brain and NO may facilitate production of new brain cells.^19,20 NO is associated with long-term potentiation and activates the NMDA receptor.²¹ Armed with data from the role of NO as a facilitator of neuronal proliferation in the developing brain and an idea that restoration of function after cerebral injury may be associated with similar events that enhance long term potentiation, we tested the hypothesis that administration of an NO donor to rats induces neurogenesis and reduces neurological and functional deficits after stroke.²²

The only FDA approved treatment of stroke, recombinant tissue plasminogen activator, must be initiated within three hours of stroke onset. However, with restorative treatment, rapid early intervention may not be needed and clinically it may be reasonable to wait before initiating treatment until there is a clear indication of the extent and degree of functional deficit. Thus, in our investigations of restorative therapy with nitric oxide donors, we time onset of treatment at not less than one day after stroke. Therefore, in the study to be described, nitric oxide donors were first administered 24 hours after onset of stroke.

Male Wistar rats were subjected to occlusion of the right middle cerebral artery by means of an embolus, a model of stroke very similar to embolic stroke in humans. The rats were treated with (Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl) aminio] diazen-1-ium-1,2-diolate (DETA/NONOate) and behavioral, functional, and physiological measurements were performed over time to 42 days after stroke. Compared to saline treated rats, rats treated with an NO donor showed a significant reduction in functional and physiological deficits measured using a rotarod test, a somatosensory test, and body weight. To test the underlying mechanisms associated with the improved functional performance with treatment, rats were injected with bromodeoxyuridine (BrdU) a thymidine analog which provides a measurement of newly formed DNA. Rats were injected (IP) with BrdU for 14 days, and were sacrificed at either 14 or 42 days after stroke onset. Significant increases of newly formed cells were detected in the subventricular zone, the olfactory bulb, and the dentate gyrus in ipsilateral and contralateral hemispheres of NO donor treated rats compared to shamącontrol rats subjected to embolic stroke. More than 90% of the cells generated within the dentate gyrus were identified as neurons or precursor neurons, as evaluated with double immunohistochemical staining methods. Cells formed in the subventricular and the olfactory bulb exhibited neuronal, astrocytic, and developmental phenotype. Animals treated with NO donors as expected also showed a significant increase in cerebral levels of cyclic GMP, suggesting that the mechanisms of action promoting neurogenesis may be coupled to the effects of cyclic GMP. Parallel studies with similar results were performed with sodium nitroprusside dihydrate (SNP) as the NO donor and NO donors were also shown to evoke neurogenesis in both young and aged normal non-ischemic rats.

These data provide provocative evidence that neural injury, aging, and possibly neurodegenerative disease can be treated pharmacologically to induce remodeling of the brain and enhancement of function. In the data presented here, administration of an NO donor evoked neurogenesis and functional repair. NO regulates vascular and neural activities and is required for proper function of many organs. NO may also play an important role in neurogenesis and recovery from neural injury and possibly from neurodegenerative disease.

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