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

It is now clear that oxygen-derived free radicals play an important part in several models of experimentally induced reperfusion injury. Although there are certainly multiple components to clinical ischemic and reperfusion injury, it appears likely that free-radical production may make a major contribution at certain stages in the progression of the injury. The primary source of superoxide in reperfused reoxygenated tissues appears to be the enzyme xanthine oxidase, released during ischemia by a calcium-triggered proteolytic attack on xanthine dehydrogenase. Reperfused tissues are protected in a variety of laboratory models by scavengers of superoxide radicals or hydroxyl radicals or by allopurinol or other inhibitors of xanthine oxidase. Dysfunction induced by free radicals may thus be a major component of ischemic diseases of the heart, bowel, liver, kidney, and brain.

Oxygen-derived free radicals in postischemic tissue injury.

Publisher Summary This chapter discusses the role of free radicals and catalytic metal ions in human disease. The importance of transition metal ions in mediating oxidant damage naturally leads to the question as to what forms of such ions might be available to catalyze radical reactions in vivo . The chapter discusses the metabolism of transition metals, such as iron and copper. It also discusses the chelation therapy that is an approach to site-specific antioxidant protection. The detection and measurement of lipid peroxidation is the evidence most frequently cited to support the involvement of free radical reactions in toxicology and in human disease. A wide range of techniques is available to measure the rate of this process, but none is applicable to all circumstances. The two most popular are the measurement of diene conjugation and the thiobarbituric acid (TBA) test, but they are both subject to pitfalls, especially when applied to human samples. The chapter also discusses the essential principles of the peroxidation process. When discussing lipid peroxidation, it is essential to use clear terminology for the sequence of events involved; an imprecise use of terms such as initiation has caused considerable confusion in the literature. In a completely peroxide-free lipid system, first chain initiation of a peroxidation sequence in a membrane or polyunsaturated fatty acid refers to the attack of any species that has sufficient reactivity to abstract a hydrogen atom from a methylene group.

Role of free radicals and catalytic metal ions in human disease: an overview.

The objective of this study was to determine whether endogenous nitric oxide (NO) inhibits leukocyte adhesion to vascular endothelium. This was accomplished by superfusing a cat mesenteric preparation with inhibitors of NO production, NG-monomethyl-L-arginine (L-NMMA) or NG-nitro-L-arginine methyl ester (L-NAME), and observing single (30-microns diameter) venules by intravital video microscopy. Thirty minutes into the superfusion period the number of adherent and emigrated leukocytes, the erythrocyte velocity, and the venular diameter were measured; venular blood flow and shear rate were calculated from the measured parameters. The contribution of the leukocyte adhesion glycoprotein CD11/CD18 was determined using the CD18-specific monoclonal antibody IB4. Both inhibitors of NO production increased leukocyte adherence more than 15-fold. Leukocyte emigration was also enhanced, whereas venular shear rate was reduced by nearly half. Antibody IB4 abolished the leukocyte adhesion induced by L-NMMA and L-NAME. Incubation of isolated cat neutrophils with L-NMMA, but not L-NAME, resulted in direct upregulation of CD11/CD18 as assessed by flow cytometry. Decrements in venular shear rate induced by partial occlusion of the superior mesenteric artery in untreated animals revealed that only a minor component of L-NAME-induced leukocyte adhesion was shear rate-dependent. The L-NAME-induced adhesion was inhibited by L-arginine but not D-arginine. These data suggest that endothelium-derived NO may be an important endogenous modulator of leukocyte adherence and that impairment of NO production results in a pattern of leukocyte adhesion and emigration that is characteristic of acute inflammation.

https://www.pnas.org/content/pnas/88/11/4651.full.pdf

Nitric oxide: an endogenous modulator of leukocyte adhesion.

Resistant starch (RS) is starch and products of its small intestinal digestion that enter the large bowel. It occurs for various reasons including chemical structure, cooking of food, chemical modification, and food mastication. Human colonic bacteria ferment RS and nonstarch polysaccharides (NSP; major components of dietary fiber) to short-chain fatty acids (SCFA), mainly acetate, propionate, and butyrate. SCFA stimulate colonic blood flow and fluid and electrolyte uptake. Butyrate is a preferred substrate for colonocytes and appears to promote a normal phenotype in these cells. Fermentation of some RS types favors butyrate production. Measurement of colonic fermentation in humans is difficult, and indirect measures (e.g., fecal samples) or animal models have been used. Of the latter, rodents appear to be of limited value, and pigs or dogs are preferable. RS is less effective than NSP in stool bulking, but epidemiological data suggest that it is more protective against colorectal cancer, possibly via butyrate. RS is a prebiotic, but knowledge of its other interactions with the microflora is limited. The contribution of RS to fermentation and colonic physiology seems to be greater than that of NSP. However, the lack of a generally accepted analytical procedure that accommodates the major influences on RS means this is yet to be established.

Short-Chain Fatty Acids and Human Colonic Function: Roles of Resistant Starch and Nonstarch Polysaccharides

https://www.gastrojournal.org/article/0016-5085(81)90648-X/pdf

Superoxide radicals in feline intestinal ischemia

https://www.gastrojournal.org/article/0016-5085(82)90115-9/pdf

Ischemic injury in the cat small intestine: Role of superoxide radicals

Xanthine oxidase inhibitors attenuate ischemia-induced vascular permeability changes in the cat intestine

Recent evidence in vivo indicates that spon- taneously hypertensive rats (SHR) exhibit an increase in oxyradical production in and around microvascular endothe- lium. This study is aimed to examine whether xanthine oxidase plays a role in overproduction of oxidants and thereby may contribute to hypertensive states as a consequence of the increasing microvascular tone. The xanthine oxidase activity in SHR was inhibited by dietary supplement of tungsten (0.7 gykg) that depletes molybdenum as a cofactor for the enzyme activity as well as by administration of (2)BOF4272 ((2)-8- (3-methoxy-4-phenylsulfinylphenyl)pyrazolo(1,5-a)-1,3,5- triazine-4-monohydrate), a synthetic inhibitor of the enzyme. The characteristic elevation of mean arterial pressure in SHR was normalized by the tungsten diet, whereas Wistar Koto (WKY) rats displayed no significant alteration in the pres- sure. Multifunctional intravital videomicroscopy in mesentery microvessels with hydroethidine, an oxidant-sensitive f luoro- probe, showed that SHR endothelium exhibited overproduc- tion of oxyradicals that coincided with the elevated arteriolar tone as compared with WKY rats. The tungsten diet signifi- cantly repressed these changes toward the levels observed in WKY rats. The activity of oxyradical-producing form of xanthine oxidase in the mesenteric tissue of SHR was '3-fold greater than that of WKY rats, and pretreatment with the tungsten diet eliminated detectable levels of the enzyme activity. The inhibitory effects of the tungsten diet on the increasing blood pressure and arteriolar tone in SHR were also reproducible by administration of (2)BOF4272. These results suggest that xanthine oxidase accounts for a putative source of oxyradical generation that is associated with an increasing arteriolar tone in this form of hypertension. The progressive elevation of arterial blood pressure in the spontaneously hypertensive rat (SHR) is associated with an upward displacement in vascular resistance, which is especially striking in the small arterial or arteriolar segments of the microcirculation. Previous investigations focused on the mi- crocirculation of hypertensives have reported morphological changes in the wall of arterioles (1) as well as changes in the overall makeup of the arteriolar network (2-4), which could account for the elevation in peripheral resistance. An above- normal level of arteriolar tone can be demonstrated in the microcirculation of SHR down to the smallest precapillary ramifications of the terminal arterioles in different tissues (5-8). Several hypothesis have been advanced to account for the high blood pressure in SHR. Among the factors that have been proposed to account for the increase in arteriolar resistance are smooth muscle medial hypertrophy (9), vessel rarefaction (2), an elevation of tone in resistance arteries (10), an in- creased sympathetic activity (11), differences in catecholamine transmitter disposition and sensitivity of smooth muscle cells (12, 13), and differences in central vascular regulation (14). We previously reported that the level of microvascular oxidative stress in SHR was significantly above that displayed in its Wistar Kyoto (WKY) normotensive control strain. The en- hanced level in SHR showed a linear correlation with the increase in arteriolar tone (15). Superfusion of a synthetic xanthine oxidase (XO) inhibitor ((2)BOF4272, (2)-8-(3-

Xanthine oxidase activity associated with arterial blood pressure in spontaneously hypertensive rats (microcirculationyxanthine dehydrogenaseyendotheliumytungsten)

https://www.gastrojournal.org/article/0016-5085(85)90214-8/pdf

D. Neil Granger

Papers

Superoxide radicals in feline intestinal ischemia

Ischemic injury in the cat small intestine: Role of superoxide radicals

Xanthine oxidase inhibitors attenuate ischemia-induced vascular permeability changes in the cat intestine

Xanthine oxidase activity associated with arterial blood pressure in spontaneously hypertensive rats (microcirculationyxanthine dehydrogenaseyendotheliumytungsten)

“Forward” and “backward” flow mechanisms of portal hypertension: Relative contributions in the rat model of portal vein stenosis