Nitric oxide and the vascular endothelium.

The vascular endothelium synthesises the vasodilator and anti-aggregatory mediator nitric oxide (NO) from L-arginine. This action is catalysed by the action of NO synthases, of which two forms are present in the endothelium. Endothelial (e)NOS is highly regulated, constitutively active and generates NO in response to shear stress and other physiological stimuli. Inducible (i)NOS is expressed in response to immunological stimuli, is transcriptionally regulated and, once activated, generates large amounts of NO that contribute to pathological conditions. The physiological actions of NO include the regulation of vascular tone and blood pressure, prevention of platelet aggregation and inhibition of vascular smooth muscle proliferation. Many of these actions are a result of the activation by NO of the soluble guanylate cyclase and consequent generation of cyclic guanosine monophosphate (cGMP). An additional target of NO is the cytochrome c oxidase, the terminal enzyme in the electron transport chain, which is inhibited by NO in a manner that is reversible and competitive with oxygen. The consequent reduction of cytochrome c oxidase leads to the release of superoxide anion. This may be an NO-regulated cell signalling system which, under certain circumstances, may lead to the formation of the powerful oxidant species, peroxynitrite, that is associated with a variety of vascular diseases.

The discovery of nitric oxide and its role in vascular biology.

Nitric oxide (NO) is a relative newcomer to pharmacology, as the paper which initiated the field was published only 25 years ago. Nevertheless its impact is such that to date more than 31,000 papers have been published with NO in the title and more than 65,000 refer to it in some way. The identification of NO with endothelium-derived relaxing factor and the discovery of its synthesis from L-arginine led to the realisation that the L-arginine: NO pathway is widespread and plays a variety of physiological roles. These include the maintenance of vascular tone, neurotransmitter function in both the central and peripheral nervous systems, and mediation of cellular defence. In addition, NO interacts with mitochondrial systems to regulate cell respiration and to augment the generation of reactive oxygen species, thus triggering mechanisms of cell survival or death. This review will focus on the role of NO in the cardiovascular system where, in addition to maintaining a vasodilator tone, it inhibits platelet aggregation and adhesion and modulates smooth muscle cell proliferation. NO has been implicated in a number of cardiovascular diseases and virtually every risk factor for these appears to be associated with a reduction in endothelial generation of NO. Reduced basal NO synthesis or action leads to vasoconstriction, elevated blood pressure and thrombus formation. By contrast, overproduction of NO leads to vasodilatation, hypotension, vascular leakage, and disruption of cell metabolism. Appropriate pharmacological or molecular biological manipulation of the generation of NO will doubtless prove beneficial in such conditions.

Subcellular localization of nitric oxide synthase in the cerebral ventricular system, subfornical organ, area postrema, and blood vessels of the rat brain.

The distribution of neuronal nitric oxide synthase (nNOS) has been studied in the more rostral portion of the lateral ventricle, subfornical organ, area postrema and blood vessels of the rat central nervous system. nNOS was located by means of a specific polyclonal antibody, by using light and electron microscopy. Light microscopy showed immunoreactive varicose nerve fibers and terminal boutons-like structures in the lateral ventricle, positioned in supra- and subependimal areas. The spatial relationships between immunoreactive neuronal processes and the wall of the intracerebral blood vessels were studied. Electron microscopy showed numerous nerve fibers in the wall of the lateral ventricle; many were nNos-immunoreactive and established very close contact with ependymal cells. Immunoreactive neurons and processes were found in the subependymal plate of the ventricular wall, the subfornical organ, the area postrema, and the circularis nucleus of the hypothalamus. In these last three areas, the immunoreactive neurons were found close to the perivascular space of fenestrated and nonfenestrated blood vessels. The nNOS immunoreactivity was localized to the endoplasmic reticulum, cisterns, ribosomes, neurotubules, and in the inner part of the external membrane. In the terminal boutons, the reaction product was found surrounding the vesicle membranes. This distribution showed nNOS as a predominantly membrane-bound protein. The nitrergic nerve fibers present in the wall of the ventricular system might regulate metabolic functions as well as neurotransmission in the subfornical organ, area postrema and circularis nucleus of the hypothalamus.

Molecular mechanisms and therapeutic strategies related to nitric oxide.

The formation of nitric oxide (NO) from L-arginine is now recognized as a ubiquitous biochemical pathway involved in the regulation of the cardiovascular, central, and peripheral nervous systems, as well as in other homeostatic mechanisms. The L-arginine:NO pathway comprises a substrate, L-arginine, a family of enzymes, the NO synthases, and at least one physiological effector system, the soluble guanylate cyclase. NO also inhibits enzymes in target cells and can interact with oxygen-derived radicals to produce other toxic substances. Thus, NO also plays a role in immunological host defense and in the pathophysiology of certain clinical conditions. Several steps in the L-arginine:NO pathway are amenable to manipulation. Some substances will change the concentration and/or actions of NO with consequences that, in certain cases, may be therapeutic. In addition, other agents themselves generate NO and thus mimic the actions of the endogenous mediator. This brief overview will discuss some possible interventions in the pathway and the potential benefits as well as undesirable side effects that might arise from them.

The discovery of nitric oxide as the endogenous nitrovasodilator.

Endothelium-derived relaxing factor (EDRF) is a labile humoral agent released by vascular endothelium that mediates the relaxation induced by some vasodilators, including acetylcholine and bradykinin. EDRF also inhibits platelet aggregation, induces disaggregation of aggregated platelets, and inhibits platelet adhesion to vascular endothelium. These actions of EDRF are mediated through stimulation of the soluble guanylate cyclase and the consequent elevation of cyclic guanosine 3',5'-monophosphate. EDRF has been identified as nitric oxide (NO). The pharmacology of NO and EDRF is indistinguishable; furthermore, sufficient NO is released from endothelial cells to account for the biological activities of EDRF. Organic nitrates exert their vasodilator activity following conversion to NO in vascular smooth muscle cells. Thus, NO may be considered the endogenous nitrovasodilator. NO is synthesized by vascular endothelium from the terminal guanido nitrogen atom(s) of the amino acid L-arginine. This indicates the existence of an enzymic pathway in which L-arginine is the endogenous precursor for the synthesis of NO. The discovery of the release of NO by vascular endothelial cells, the biosynthetic pathway leading to its generation, and its interaction with other vasoactive substances opens up new avenues for research into the physiology and pathophysiology of the vessel wall.

Prostaglandins in the pathogenesis and prevention of vascular disease.

Metabolism of arachidonic acid gives rise to a number of products with potent, and sometimes opposing, biological actions. Prostacyclin, the main product of arachidonic acid in vascular tissue, is a vasodilator and inhibitor of platelet aggregation whereas thromboxane A2, produced by the platelet, is a vasoconstrictor and inducer of platelet aggregation. Generation of these products may be modified in certain diseases, such as atherosclerosis and diabetes, so that prostacyclin production is reduced and thromboxane A2 production increased, resulting in a pro-thrombotic condition. Synthesis of arachidonic acid metabolites may be manipulated using drugs such as aspirin or imidazole analogues which selectively inhibit different enzymes in the metabolic pathway. Such drugs have proved beneficial in the treatment of some vascular disorders. Clinical use of prostacyclin has shown it to be effective in the treatment of peripheral vascular disease, Raynaud's Syndrome and pulmonary hypertension. Stable analogues of prostacyclin are being developed which may lead to a separation of the vasodilator and anti-platelet actions of prostacyclin.

Arachidonate metabolism in blood cells and the vessel wall.

Arachidonic acid (AA) is metabolized by the cyclo-oxygenase and the lipoxygenase pathways to give a number of products, some of which have potent and sometimes opposing biological activities. Different cell types produce different metabolites, so that the chief AA metabolite produced by the platelet is the pro-aggregatory thromboxane A2 (TXA2), whereas that produced by the vascular endothelium is the anti-aggregatory prostacyclin. White blood cells, on the other hand, are the chief source of the leukotrienes, which are implicated in the inflammatory process. Generation of these products may be modified in certain pathological conditions, such as atherosclerosis and diabetes, where prostacyclin synthesis is reduced and TXA2 synthesis increased, resulting in a pro-thrombotic state. Synthesis of AA metabolites may be inhibited, either totally or selectively, using drugs which inhibit different enzymes in the metabolic pathway. These drugs may be beneficial in the treatment of thrombotic disorders and inflammation. AA metabolism may also be modified by dietary substitution with eicosapentaenoic acid, a fatty acid present in fish oils.

Effects of intravenous infusion of prostacyclin (PGI2) in man.

Prostacyclin infused intravenously in human volunteers induces ex vivo inhibition of platelet aggregation, tachycardia and hypotension. The inhibition of platelet aggregation is obtained with slightly lower doses than those which exhibit cardiovascular effects. The cardiovascular effects disappeared within a few minutes after discontinuing the infusion of prostacyclin but the platelet effects were longer lasting. Prostacyclin did not have any effect on platelet count, platelet factor 3, accelerated partial thromboplastin time, prothrombin time, euglobulin clot lysis time, fibrinogen degradation products, blood glucose concentration or urine sodium potassium ratio.

Inflammatory effects of prostacyclin (PGI2) and 6-oxo-PGF1alpha in the rat paw.

In the rat paw prostacyclin was 5--10 times less potent than PGE2 in causing oedema, and 5 times less potent in potentiating carrageenin-induced oedema, which it did in a dose-related manner. Prostacyclin was 5 times more potent than PGE2 in producing hyperalgesia and as potent as PGE2 in restoring carrageenin-induced hyperalgesia. The effects on oedema were longer lasting than those on hyperalgesia. 6-oxo-PGF1alpha was 500 times less potent than PGE2 in causing oedema by itself and in potentiating carrageenin-induced oedema. It had no hyperalgesic activity in this test.