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.

/pdf/nitric-oxide-and-peroxynitrite-in-health-and-disease-3uuuqgfdcz.pdf

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.

Nitric oxide (NO), the smallest signalling molecule known, is produced by three isoforms of NO synthase (NOS; EC 1.14.13.39). They all utilize l-arginine and molecular oxygen as substrates and require the cofactors reduced nicotinamide-adenine-dinucleotide phosphate (NADPH), flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN), and (6R-)5,6,7,8-tetrahydrobiopterin (BH(4)). All NOS bind calmodulin and contain haem. Neuronal NOS (nNOS, NOS I) is constitutively expressed in central and peripheral neurons and some other cell types. Its functions include synaptic plasticity in the central nervous system (CNS), central regulation of blood pressure, smooth muscle relaxation, and vasodilatation via peripheral nitrergic nerves. Nitrergic nerves are of particular importance in the relaxation of corpus cavernosum and penile erection. Phosphodiesterase 5 inhibitors (sildenafil, vardenafil, and tadalafil) require at least a residual nNOS activity for their action. Inducible NOS (NOS II) can be expressed in many cell types in response to lipopolysaccharide, cytokines, or other agents. Inducible NOS generates large amounts of NO that have cytostatic effects on parasitic target cells. Inducible NOS contributes to the pathophysiology of inflammatory diseases and septic shock. Endothelial NOS (eNOS, NOS III) is mostly expressed in endothelial cells. It keeps blood vessels dilated, controls blood pressure, and has numerous other vasoprotective and anti-atherosclerotic effects. Many cardiovascular risk factors lead to oxidative stress, eNOS uncoupling, and endothelial dysfunction in the vasculature. Pharmacologically, vascular oxidative stress can be reduced and eNOS functionality restored with renin- and angiotensin-converting enzyme-inhibitors, with angiotensin receptor blockers, and with statins.

/pdf/nitric-oxide-synthases-regulation-and-function-50kgzzd2xv.pdf

Nitric oxide synthases: regulation and function.

This review focuses on the role of oxidative processes in atherosclerosis and its resultant cardiovascular events. There is now a consensus that atherosclerosis represents a state of heightened oxidative stress characterized by lipid and protein oxidation in the vascular wall. The oxidative modification hypothesis of atherosclerosis predicts that low-density lipoprotein (LDL) oxidation is an early event in atherosclerosis and that oxidized LDL contributes to atherogenesis. In support of this hypothesis, oxidized LDL can support foam cell formation in vitro, the lipid in human lesions is substantially oxidized, there is evidence for the presence of oxidized LDL in vivo, oxidized LDL has a number of potentially proatherogenic activities, and several structurally unrelated antioxidants inhibit atherosclerosis in animals. An emerging consensus also underscores the importance in vascular disease of oxidative events in addition to LDL oxidation. These include the production of reactive oxygen and nitrogen species by vascular cells, as well as oxidative modifications contributing to important clinical manifestations of coronary artery disease such as endothelial dysfunction and plaque disruption. Despite these abundant data however, fundamental problems remain with implicating oxidative modification as a (requisite) pathophysiologically important cause for atherosclerosis. These include the poor performance of antioxidant strategies in limiting either atherosclerosis or cardiovascular events from atherosclerosis, and observations in animals that suggest dissociation between atherosclerosis and lipoprotein oxidation. Indeed, it remains to be established that oxidative events are a cause rather than an injurious response to atherogenesis. In this context, inflammation needs to be considered as a primary process of atherosclerosis, and oxidative stress as a secondary event. To address this issue, we have proposed an "oxidative response to inflammation" model as a means of reconciling the response-to-injury and oxidative modification hypotheses of atherosclerosis.

Role of Oxidative Modifications in Atherosclerosis

Background Primary pulmonary hypertension is a progressive disease for which no treatment has been shown in a prospective, randomized trial to improve survival. Methods We conducted a 12-week prospective, randomized, multicenter open trial comparing the effects of the continuous intravenous infusion of epoprostenol (formerly called prostacyclin) plus conventional therapy with those of conventional therapy alone in 81 patients with severe primary pulmonary hypertension (New York Heart Association functional class III or IV). Results Exercise capacity was improved in the 41 patients treated with epoprostenol (median distance walked in six minutes, 362 m at 12 weeks vs. 315 m at base line), but it decreased in the 40 patients treated with conventional therapy alone (204 m at 12 weeks vs. 270 m at base line; P<0.002 for the comparison of the treatment groups). Indexes of the quality of life were improved only in the epoprostenol group (P<0.01). Hemodynamics improved at 12 weeks in the epoprostenol-treated pat...

https://www.nejm.org/doi/pdf/10.1056/NEJM199602013340504

A Comparison of Continuous Intravenous Epoprostenol (Prostacyclin) with Conventional Therapy for Primary Pulmonary Hypertension

Mechanism of vascular smooth muscle relaxation by organic nitrates, nitrites, nitroprusside and nitric oxide: evidence for the involvement of S-nitrosothiols as active intermediates.

The purpose of the present study was to determine time course relationship between cyclic GMP accumulation and relaxation in bovine coronary artery and evaluate the effects of recently identified inhibitors, methylene blue and methemoglobin, on these relationships. Arterial strips were suspended in specially mounted tissue baths which permitted continuous recording of isometric tension until rapid freeze-clamping for subsequent determination of cyclic GMP levels by radioimmunoassay. Relaxation and cyclic GMP levels were measured in submaximally contracted strips at zero time (untreated) or 5-sec to 5-min intervals after exposure to 0.5 microliter of nitric oxide, 1 microM glyceryl trinitrate, 1 microM sodium nitroprusside of 1 mM sodium nitrite in the absence or presence of 10 mM methylene blue or 1 microM methemoglobin. Cyclic GMP accumulation preceded onset of relaxation elicited by nitric oxide and glyceryl trinitrate and temporally correlated with relaxation induced by sodium nitroprusside and sodium nitrite. Methylene blue simultaneously inhibited cyclic GMP accumulation and relaxation induced by all four relaxants. In contrast to methylene blue, methemoglobin abolished cyclic GMP accumulation and relaxation elicited by nitric oxide without altering responses to glyceryl trinitrate, sodium nitroprusside and sodium nitrite. These findings are consistent with and strongly support an involvement of cyclic GMP formation in vascular smooth muscle relaxation elicited by nitrogen oxide-containing vasodilators.

Relationship between cyclic guanosine 3':5'-monophosphate formation and relaxation of coronary arterial smooth muscle by glyceryl trinitrate, nitroprusside, nitrite and nitric oxide: effects of methylene blue and methemoglobin.

The objective of this study was to ascertain whether "endothelium-derived relaxing factor" (EDRF) released from bovine intrapulmonary artery and vein is capable of directly activating soluble guanylate cyclase, thereby accounting for elevated vascular levels of cyclic GMP during EDRF release. Isolated arterial and venous rings, after equilibration and depolarization in bath chambers, were transferred to reaction tubes and incubated with soluble guanylate cyclase that had been purified to homogeneity from bovine lung. Addition of test agents to either bath chambers or enzyme reaction mixtures enabled the determination of their sites of action. Arterial and venous rings caused an endothelium-dependent 2- to 3-fold enzyme activation that was inhibited by methylene blue. Endothelium-dependent enzyme activation in artery but not vein was enhanced several-fold by acetylcholine in an atropine-sensitive manner. Bradykinin, which relaxes both artery and vein when endothelium is intact, activated guanylate cyclase upon addition of endothelium-intact rings to enzyme reaction mixtures. Vasoactive intestinal peptide, which causes endothelium-dependent relaxation of artery but not vein, also activated guanylate cyclase in the presence of endothelium-intact artery but not vein. Arachidonic acid activated the enzyme directly as well as through EDRF release from artery but not vein. Atrial peptides, prostacyclin, isoproterenol and nitroglycerin were inactive. Methylene blue was a powerful inhibitor of EDRF-elicited activation of guanylate cyclase but was without effect when rings were merely pretreated with methylene blue in bath chambers with no further addition to enzyme reaction mixtures. Thus, methylene blue did not interfere with the formation, release or chemical stability of EDRF, but rather inhibited its influence on guanylate cyclase. No agent was found to inhibit EDRF generation or release.(ABSTRACT TRUNCATED AT 250 WORDS)

Activation of purified soluble guanylate cyclase by endothelium-derived relaxing factor from intrapulmonary artery and vein: stimulation by acetylcholine, bradykinin and arachidonic acid.

L-Arginine Inhibits Balloon Catheter-Induced Intimal Hyperplasia

Recent studies have suggested that cyclic GMP accumulation in platelets mediates the antiaggregatory effects of certain nitrogen oxide-containing agents such as sodium nitroprusside, nitric oxide, nitrosoguanidines, and related agents. The vasodilator effect of these agents may involve the formation of S-nitrosothiol intermediates which relax vascular smooth muscle, elevate tissue levels of cyclic GMP, and activate guanylate cyclase. The purpose of this study was to investigate the effects of various synthetic S-nitrosothiols on human platelet aggregation. The S-nitroso derivatives of N-acetylpenicillamine, cysteine, and beta-D-thioglucose inhibited human platelet aggregation in a concentration-dependent fashion when ADP, collagen, U46619, or sodium arachidonate was employed as the aggregating agent. The antiaggregatory effects of the S-nitrosothiols were associated with a rapid and marked increase in intracellular platelet cyclic GMP levels, whereas cyclic AMP levels remained unchanged. Additionally, S-nitrosothiols disaggregated platelets which had been aggregated while concomitantly elevating platelet cyclic GMP levels. Moreover, guanylate cyclase, partially purified from the soluble fraction of human platelets, was markedly activated by S-nitrosothiols in a heme-dependent manner. Methemoglobin, a hemoprotein with a high affinity for nitric oxide, partially reversed the antiaggregatory effects, attenuated the accumulation of cyclic GMP, and inhibited the activation of guanylate cyclase by S-nitrosothiols. These data are consistent with the hypothesis that S-nitrosothiols could serve as active intermediates in the inhibitory action of sodium nitroprusside, nitric oxide, and related nitrogen oxides on platelet aggregation.

Inhibition of human platelet aggregation by S-nitrosothiols. Heme-dependent activation of soluble guanylate cyclase and stimulation of cyclic GMP accumulation.

P. J. Kadowitz

Papers

Mechanism of vascular smooth muscle relaxation by organic nitrates, nitrites, nitroprusside and nitric oxide: evidence for the involvement of S-nitrosothiols as active intermediates.

Relationship between cyclic guanosine 3':5'-monophosphate formation and relaxation of coronary arterial smooth muscle by glyceryl trinitrate, nitroprusside, nitrite and nitric oxide: effects of methylene blue and methemoglobin.

Activation of purified soluble guanylate cyclase by endothelium-derived relaxing factor from intrapulmonary artery and vein: stimulation by acetylcholine, bradykinin and arachidonic acid.

L-Arginine Inhibits Balloon Catheter-Induced Intimal Hyperplasia

Inhibition of human platelet aggregation by S-nitrosothiols. Heme-dependent activation of soluble guanylate cyclase and stimulation of cyclic GMP accumulation.