The discovery that mammalian cells have the ability to synthesize the free radical nitric oxide (NO) has stimulated an extraordinary impetus for scientific research in all the fields of biology and medicine. Since its early description as an endothelial-derived relaxing factor, NO has emerged as a fundamental signaling device regulating virtually every critical cellular function, as well as a potent mediator of cellular damage in a wide range of conditions. Recent evidence indicates that most of the cytotoxicity attributed to NO is rather due to peroxynitrite, produced from the diffusion-controlled reaction between NO and another free radical, the superoxide anion. Peroxynitrite interacts with lipids, DNA, and proteins via direct oxidative reactions or via indirect, radical-mediated mechanisms. These reactions trigger cellular responses ranging from subtle modulations of cell signaling to overwhelming oxidative injury, committing cells to necrosis or apoptosis. In vivo, peroxynitrite generation represents a crucial pathogenic mechanism in conditions such as stroke, myocardial infarction, chronic heart failure, diabetes, circulatory shock, chronic inflammatory diseases, cancer, and neurodegenerative disorders. Hence, novel pharmacological strategies aimed at removing peroxynitrite might represent powerful therapeutic tools in the future. Evidence supporting these novel roles of NO and peroxynitrite is presented in detail in this review.

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Nitric Oxide and Peroxynitrite in Health and Disease

Evolution has resorted to nitric oxide (NO), a tiny, reactive radical gas, to mediate both servoregulatory and cytotoxic functions. This article reviews how different forms of nitric oxide synthase help confer specificity and diversity on the effects of this remarkable signaling molecule.

https://faseb.onlinelibrary.wiley.com/doi/pdf/10.1096/fasebj.6.12.1381691

Nitric oxide as a secretory product of mammalian cells.

This review concentrates on advances in nitric oxide synthase (NOS) structure, function and inhibition made in the last seven years, during which time substantial advances have been made in our understanding of this enzyme family. There is now information on the enzyme structure at all levels from primary (amino acid sequence) to quaternary (dimerization, association with other proteins) structure. The crystal structures of the oxygenase domains of inducible NOS (iNOS) and vascular endothelial NOS (eNOS) allow us to interpret other information in the context of this important part of the enzyme, with its binding sites for iron protoporphyrin IX (haem), biopterin, L-arginine, and the many inhibitors which interact with them. The exact nature of the NOS reaction, its mechanism and its products continue to be sources of controversy. The role of the biopterin cofactor is now becoming clearer, with emerging data implicating one-electron redox cycling as well as the multiple allosteric effects on enzyme activity. Regulation of the NOSs has been described at all levels from gene transcription to covalent modification and allosteric regulation of the enzyme itself. A wide range of NOS inhibitors have been discussed, interacting with the enzyme in diverse ways in terms of site and mechanism of inhibition, time-dependence and selectivity for individual isoforms, although there are many pitfalls and misunderstandings of these aspects. Highly selective inhibitors of iNOS versus eNOS and neuronal NOS have been identified and some of these have potential in the treatment of a range of inflammatory and other conditions in which iNOS has been implicated.

/pdf/nitric-oxide-synthases-structure-function-and-inhibition-1t31plqilr.pdf

Nitric oxide synthases: structure, function and inhibition

Over the past decade, the Nomenclature Committee on Cell Death (NCCD) has formulated guidelines for the definition and interpretation of cell death from morphological, biochemical, and functional perspectives. Since the field continues to expand and novel mechanisms that orchestrate multiple cell death pathways are unveiled, we propose an updated classification of cell death subroutines focusing on mechanistic and essential (as opposed to correlative and dispensable) aspects of the process. As we provide molecularly oriented definitions of terms including intrinsic apoptosis, extrinsic apoptosis, mitochondrial permeability transition (MPT)-driven necrosis, necroptosis, ferroptosis, pyroptosis, parthanatos, entotic cell death, NETotic cell death, lysosome-dependent cell death, autophagy-dependent cell death, immunogenic cell death, cellular senescence, and mitotic catastrophe, we discuss the utility of neologisms that refer to highly specialized instances of these processes. The mission of the NCCD is to provide a widely accepted nomenclature on cell death in support of the continued development of the field.

https://biblio.ugent.be/publication/8555786/file/8555788.pdf

Molecular mechanisms of cell death: recommendations of the Nomenclature Committee on Cell Death 2018.

Nitric oxide (NO), apparently identical to endothelium-derived relaxing factor in blood vessels, is also formed by cytotoxic macrophages, in adrenal gland and in brain tissue, where it mediates the stimulation by glutamate of cyclic GMP formation in the cerebellum Stimulation of intestinal or anococcygeal nerves liberates NO, and the resultant muscle relaxation is blocked by arginine derivatives that inhibit NO synthesis It is, however, unclear whether in brain or intestine, NO released following nerve stimulation is formed in neurons, glia, fibroblasts, muscle or blood cells, all of which occur in proximity to neurons and so could account for effects of nerve stimulation on cGMP and muscle tone We have now localized NO synthase protein immunohistochemically in the rat using antisera to the purified enzyme We demonstrate NO synthase in the brain to be exclusively associated with discrete neuronal populations NO synthase is also concentrated in the neural innervation of the posterior pituitary, in autonomic nerve fibres in the retina, in cell bodies and nerve fibres in the myenteric plexus of the intestine, in adrenal medulla, and in vascular endothelial cells These prominent neural localizations provide the first conclusive evidence for a strong association of NO with neurons

Localization of nitric oxide synthase indicating a neural role for nitric oxide

Nitric oxide mediates vascular relaxing effects of endothelial cells, cytotoxic actions of macrophages and neu- trophils, and influences of excitatory amino acids on cerebellar cyclic GMP. Its enzymatic formation from arginine by a soluble enzyme associated with stoichiometric production of citruline requires NADPH and Ca2 . We show that nitric oxide synthetase activity requires calmodulin. Utilizing a 2',5'-ADP affmity col- umn eluted with NADPH, we have purified nitric oxide synthetase 6000-fold to homogeneity from rat cerebellum. The purified enzyme migrates as a single 150-kDa band on SDS/PAGE, and the native enzyme appears to be a monomer. Endothelium-derived relaxing factor, a labile substance formed by endothelial cells, which mediates vasodilation, has been shown to be identical to nitric oxide (NO) (1-3). In addition to relaxing blood vessels, NO has multiple messen- ger functions as it has been demonstrated in macrophages (4) and in brain tissue (5-7). NO appears responsible for the cytotoxic effects of macrophages and neutrophils (8). We have obtained direct evidence for NO as a messenger for the influences of the excitatory amino acid glutamate on cGMP in the cerebellum (7). We showed a striking enhancement by glutamate and other excitatory amino acids of the conversion of arginine to NO and the associated formation of citrulline. Moreover we observed that Nw-monomethyl-L-arginine (MeArg), an inhibitor of the enzymatic conversion of arginine to NO, inhibits glutamate-elicited cGMP formation, an influ- ence selectively reversed by excess arginine. Evidence that NO mediates functions of tissues as diverse as the brain, endothelium, and blood cells suggests a wide- spread role for NO as a messenger molecule. Localizing NO formation at a cellular level throughout the body would be greatly facilitated by immunohistochemical identification of NO synthetase, the NO-forming enzyme. The mechanism of conversion of arginine to NO, presently unclear (9), would be greatly clarified by purification of NO synthetase. Charac- terization of this enzyme has been hampered by the complex assays required to assay the enzyme by monitoring NO formation directly. We have shown that NO formation is accompanied by the stoichiometric conversion of arginine to citrulline, permitting a simple, sensitive, and specific enzyme assay measuring the transformation of (3H)arginine to (3H)citrulline (7). Utilizing this assay in the present study, we have purified NO synthetase to homogeneity. We demon- strate that NO synthetase is a calmodulin-requiring enzyme, explaining numerous reports of a crucial role for calcium in endothelium-dependent smooth muscle relaxation.

Isolation of nitric oxide synthetase, a calmodulin-requiring enzyme (endothelium-derived relaxing factor/arginine/cGMP)

The beta-adrenergic receptor kinase (beta ARK) phosphorylates the agonist-occupied beta-adrenergic receptor to promote rapid receptor uncoupling from Gs, thereby attenuating adenylyl cyclase activity. Beta ARK-mediated receptor desensitization may reflect a general molecular mechanism operative on many G-protein-coupled receptor systems and, particularly, synaptic neurotransmitter receptors. Two distinct cDNAs encoding beta ARK isozymes were isolated from rat brain and sequenced. The regional and cellular distributions of these two gene products, termed beta ARK1 and beta ARK2, were determined in brain by in situ hybridization and by immunohistochemistry at the light and electron microscopic levels. The beta ARK isozymes were found to be expressed primarily in neurons distributed throughout the CNS. Ultrastructurally, beta ARK1 and beta ARK2 immunoreactivities were present both in association with postsynaptic densities and, presynaptically, with axon terminals. The beta ARK isozymes have a regional and subcellular distribution consistent with a general role in the desensitization of synaptic receptors.

https://www.jneurosci.org/content/jneuro/12/10/4045.full.pdf

The G-protein-coupled receptor kinases beta ARK1 and beta ARK2 are widely distributed at synapses in rat brain.

We show that calmodulin-dependent phosphodiesterase (CAM-PDE) is selectively expressed in mature olfactory receptor neurons within the olfactory mucosa. Immunocytochemical staining reveals neuronal immunoreactivity that is most pronounced within cilia, dendritic knobs, and axon bundles. Neither sustentacular cells nor basal cells display immunoreactivity. The extent of loss of neuronal immunoreactivity following bulbectomy resembles loss of the neuronal population. High- affinity CAM-PDE activity in olfactory cilia is fivefold greater than in brain, when assayed at low micromolar cAMP. This activity is depleted in turbinates following bulbectomy. Olfactory mucosal PDE activity is composed of a minimum of two major forms. In the absence of Ca(2+), rolipram-sensitive PDE comprises 65% of total activity. Following stimulation by Ca2+, CAM-PDE activity is elevated sixfold to become the predominant form, thereby increasing total activity 300%, with half-maximal effect at 1 microM Ca2+. We propose that Ca2+ stimulation of CAM-PDE may be necessary for termination of olfactory signals.

https://www.jneurosci.org/content/jneuro/12/3/915.full.pdf

Calcium/calmodulin-activated phosphodiesterase expressed in olfactory receptor neurons

We have localized CDRK and DRK1, two novel K+ channels of the Shab subfamily by immunohistochemistry. The two channels are closely related in structure with about 90% amino acid identity in the N-terminal and middle portions and 60% identity in the C-terminal region. We observe striking differences in cellular localizations of the two channels. DRK1 tends to localize to cell bodies and proximal dendrites discretely, while CDRK is diffusely present in cell bodies and is also found on fibers in specific brain areas. In the cerebral cortex DRK1 is localized to pyramidal cells, whereas CDRK occurs in small cells, presumably interneurons. These localizations may reflect specialized delayed rectifier functions and targeting properties manifested differentially by K+ channel subfamily members.

https://www.jneurosci.org/content/jneuro/13/4/1569.full.pdf

Contrasting immunohistochemical localizations in rat brain of two novel K+ channels of the Shab subfamily

Nitric oxide acts as a neural messenger in both the central and peripheral nervous systems Mice with targeted disruption of the   neuronal isoform of nitric oxide synthase nNOS yr y are extremely aggressive relative to wild-type WT mice Male nNOS yr y mice exhibit an increase in the number and duration of aggressive encounters compared to WT animals when tested in a variety of paradigms used to test rodent aggression This inappropriate aggressive behavior has only been observed in male nNOS yr y mice; nNOS yr y females, like female WT mice, exhibit little or no aggression The present study sought to test the dependence of increased aggressive behavior in nNOS yr y males on testosterone Intact nNOS yr y males exhibited elevated levels of aggression relative to intact WT males Castration reduced aggression in both WT and nNOS yr y males to equivalent low levels Testosterone replacement restored aggression to precastration levels in both genotypes These data provide evidence that increased aggressive behavior of nNOS yr y mice, like aggression in WT mice, is testosterone-dependent q 1997 Elsevier Science BV

Solomon H. Snyder

Papers

Isolation of nitric oxide synthetase, a calmodulin-requiring enzyme (endothelium-derived relaxing factor/arginine/cGMP)

The G-protein-coupled receptor kinases beta ARK1 and beta ARK2 are widely distributed at synapses in rat brain.

Calcium/calmodulin-activated phosphodiesterase expressed in olfactory receptor neurons

Contrasting immunohistochemical localizations in rat brain of two novel K+ channels of the Shab subfamily

Research report Aggressive behavior in male mice lacking the gene for neuronal nitric oxide synthase requires testosterone