Nitric oxide and blood vessels: physiological role and clinical implications

130

NITRIC OXIDE AND BLOOD CLINICAL IMPLICATIONS

VESSELS:

PHYSIOLOGICAL

ROLE

AND

ALISON CALVER, JOE COLLIER and PATRICK VALLANCE D e p a r t m e n t of Clinical Pharmacology, St G e o r g e ' s Hospital Medical School, C r a n m e r Terrace, L o n d o n S W l 7 ORE, U K

Introduction

Nitric oxide (NO) is an important mediator of the control of blood vessel tone, neurotransmission and certain aspects of host defence. Its actions, at least on blood vessels, are mimicked by the nitrovasodilator class of drugs, including glyceryi trinitrate and sodium nitroprusside, which act as an exogenous source of NO.1 Thus endogenous NO can be thought of as the body's 'endogenous nitrovasodilator'.2 The fascinating story of this very simple but potent compound will surely be of interest to students on biochemistry and pharmacology courses as well as to medical students. This article discusses the physiological role of NO within the vasculature and its clinical implications in disease. (For a review of neural NO, see Snyder and Bredt, Scientific American, May 1992, p 28.)

Synthesis of NO

NO is synthesised from the terminal guanidino nitrogen atom of the semi-essential amino acid L-arginine3 (Figure 1). L-citrulline is produced as a by-product. This process is catalysed by a stereospecific enzyme NO synthase; D-arginine is not a substrate for the enzyme. 4 The supply of L-arginine does not normally appear to be rate-limiting for the enzyme. 5 NO synthase incorporates molecular oxygen into both NO and citrulline. 6 Two broad groups of NO synthases have been isolated: a constitutive, Ca2+-calmodulin dependent enzyme, and an inducible, Ca2+-independent enzyme. 7 Both enzymes require NADPH as a co-factor and tetrahydrobiopterin enhances enzyme activity. The constitutive enzyme is present in endothelium, neural tissue and platelets. 7 A complementary DNA for brain NO synthase has now been cloned and reveals recognition sites for

GENERATOR

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H2N COOH L-arglnlne

Figure 1

H2N COOH N~hydroxyL-arglnlne

NO

f

x

/

Biosynthetic pathway for nitric oxide (NO). Molecular oxygen (02) is incorporated into both NO and L-citrulline via an intermediate l~-hydroxy-L-arginine, by NO synthase. N A D P H is an essential co-factor for this reaction and L-NMMA acts as an inhibitor of (at least) two steps in the pathway. X represents the putative carrier molecule for NO. (Reproduced from McCall T and Vallance P (1992) TIPS 13, 1-6)

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131 NADPH, FAD, flavin mononuleotide and calmodulin in addition to phosphorylation sites, indicating that the enzyme is regulated by many different factors. 8 The sequence of NO synthase has striking homology with that of cytochrome P-450 reductase, another electron transferase enzyme characterised by having binding sites for NADPH and two flavins8 (Figure 2). The inducible, Ca2+-independent NO synthase can be expressed in a variety of cells including endothelial cells, vascular smooth muscle cells and immune cells such as macrophages and neutrophils. 7 The precise conditions required for the induction of NO synthase in these cell types varies, but endotoxin, tumour necrosis factor and interleukin-1 have all been shown to induce the enzyme. 7 Expression of inducible NO synthase can be prevented by prior administration of glucocorticoids.9'1° A complementary DNA has now also been cloned for the inducible NO synthase from macrophages. 11 Comparison of the sequence of this inducible NO synthase with the brain NO synthase8 identifies shared binding sites for NADPH and flavins, and an additional conserved region near the N-terminus that may recognise L-arginine or contribute to the active site 11 (Figure 2).

Effects of NO

Nitric oxide is a mediator of cell-cell communication. Once formed in 'NO generator cells', NO diffuses to 'NO target cells' and binds to the haem moiety of guanylate cyclase causing a conformational change in the enzyme and enzyme activation, leading to a rise in intracellular cyclic GMP concentration. 12 The increase in cyclic GMP level is the major mechanism underlying many of the cardiovascular and neural effects of NO. Within the vasculature the rise in cyclic GMP induces a sequence of protein phosphorylation which ultimately causes a fall in intracellular Ca 2÷ and smooth muscle relaxation. NO also acts to regulate platelet function, preventing platelet aggregation 13 and adhesion to endothelial cells. TM Both central and peripheral neurones release NO which may act as a neurotransmitter. In the brain NO is released following stimulation of the NMDA glutamate receptor. 15 In the peripheral nervous system evidence is accumulating that NO is the neurotransmitter of non-cholinergic, non-adrenergic neurones that mediate smooth muscle relaxation. 16 The cytotoxic mechanisms of NO released from immune cells are less well understood, but NO synthesis is a mechanism of 'non-specific' immunity in macrophages 17 and leads to both cytostatic18'w and cytotoxic2° control of microorganisms. In evolutionary terms, NO is a highly preserved mediator being present in the horseshoe crab, Limulus, a species over 500 million years old. 21 Synthesis of NO from L-arginine is inhibited by certain analogues of L-arginine, such as NG-mono-methyl-L-arginine (L-NMMA), N-iminoethyl-L-ornithine (L-NIO) and /VaN H2 BRAIN NITRIC OXIDE SYNTHASE COOH NADPH

NH 2 MACROPHAGE NITRIC OXIDE SYNTHASE

COOH NH2

NADPH

CYTOCHROME P-450 REDUCTASE TMD

Figure 2

COOH

A schematic model showing the spatial relationships of the co-factor recognition sites within brain and macrophage NO synthase and cytochrome P-450 reductase. The predicted sites for calmodulin binding (CAM) and protein kinase A phosphorylation (P) within the brain NO synthase sequence and the transmembrane domain (TMD) in the cytochrome P-450 reductase sequence are noted, the conseved N-terminal region in the brain and macrophage enzymes is shaded. (Redrawn from Bredt D S et al (1991) Nature 351, 714-718, and Lyons R C et al (1992) J Biol Chem 267, 6370-6374)

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132 nitro-L-arginine methyl ester (L-NAME), which act as stereospecific competitive inhibitors of NO synthase5 (Figure 3). These analogues are powerful tools for examining the physiological role of the L-arginine:NO pathway in the cardiovascular system of animals and humans.

HN

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Figure 3

NO and the Control of Normal Vascular Tone

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Structural formulae of L-arginine, NC-monomethyl-L-arginine (L-NMMA), N-iminoethylL-ornithine (L-NIO), NC-nitro-L-arginine methyl ester (L-NAME) and asymmetric dimethylarginine (A D MA ) In 1980, Furchgott and Zawadzki discovered that the vasodilator actions of acetylcholine depended upon the release from an intact endothelium of a substance they called endothelium-derived relaxing factor (EDRF). 22 Acetylcholine stimulates the release of EDRF which acts on the smooth muscle to cause blood vessel relaxation. It is now known that EDRF is NO or possibly NO attached to a carrier molecule. 23 In vitro studies using animal5'24 and human 25 blood vessels have shown that inhibition of NO synthesis using L-NMMA causes an endothelium-dependent vasoconstriction which can be reversed by giving L-arginine. In some vessels it therefore seems that NO is released continuously. Removal of this constant basal NO-mediated vasodilator tone by LNMMA results in vasoconstriction. This is also seen in vivo, where systemic inhibition of NO synthesis using intravenous L-NMMA in rabbits, 24 guinea-pigs26 and rats 27 causes a rise in blood pressure, which can be reversed with excess L-arginine infusion. Ex-vivo studies on aortic segments from L-NMMA treated rabbits show a reduced release of NO from these tissues, confirming that the response in vivo is due to diminished N O . 24 These results suggest that NO contributes to the regulation of resting vascular tone and blood pressure. This also appears to be true in humans: local infusion of L-NMMA into the brachial artery of healthy volunteers causes about a 40% fall in forearm blood flow, implying that human forearm resistance vessels in vivo are in a constant state of NOmediated vasodilatation. 2s However, there is an important difference between arteries and veins in terms of NO release at least in humans in vivo. Whereas local infusion of LNMMA in healthy volunteers causes vasoconstriction in arterioles, in veins it does not, indicating that there is no basal release of NO in this vascular bed, in healthy volunteers. 29 In addition to basal NO formation, the synthesis of NO from L-arginine is stimulated by a variety of hormones and autacoids, including acetylcholine, bradykinin and substance P, and by physical stimuli such as increases in flow and shear stress within the vessel wall. 7 By analogy with other physiological regulators it has been speculated that an endogenous inhibitor of NO synthase must exist. L-NMMA and other guanidinosubstituted arginine analogues have provided useful tools to study the L-arginine:NO pathway in vivo. It is now clear that certain methylated arginines, including L-NMMA, occur naturally in animals and humans. One such compound, asymmetric dimethylarginine (ADMA; Figure 3), circulates in human plasma and is found in micromolar

BIOCHEMICAL EDUCATION 20(3) 1992

133 concentrations in human urine. 3° ADMA inhibits in vitro preparations of NO synthase, increases vascular tone in vitro, elevates systemic blood pressure in guinea pigs, and reduces forearm blood flow in healthy volunteers. 3° All these actions are attenuated by Larginine, confirming that ADMA is a competitive inhibitor of the enzyme. 3° It is possible that ADMA, or indeed L-NMMA could act as endogenous inhibitors of the L-arginine:NO pathway. 3°,31

Clinical Implications

Hypertension, diabetes and hyperlipidaemia all have similar long term complications, in particular the incidence of occlusive vascular disease is high. Within the circulation, in addition to its actions on blood vessels, NO also inhibits platelet aggregation 13 and adhesion) 4 Consequently, if the L-arginine:NO pathway were to be generally impaired in these diseases, this would tend to tip the balance within the vessel towards vasoconstriction and thrombosis, providing a common mechanism whereby these different pathologies might result in similar long term complications.

Hypertension

Most patients with high blood pressure have a raised peripheral resistance and reduced NO synthesis could mediate part of this increased resistance. There is evidence that the Larginine:NO:guanylate cyclase pathway is abnormal in human hypertension. Stimulated NO-induced vasodilatation is diminished in patients with essential hypertension as the forearm blood flow response to acetylcholine is blunted in this group of pateints.32'33 Basal NO-mediated vasodilatation, which determines resting vascular tone, is also reduced in these patients as their forearm blood flow response to L-NMMA is reduced. 34 Furthermore the diminution in the L-NMMA response is related to the blood pressure, such that the higher the blood pressure the greater the abnormality in basal NO-mediated vasodilatation. 34 The mechanisms of reduced NO synthesis are not yet clear, but one possibility would be the presence of increased levels of an inhibitor of NO synthase. Recently, it has been demonstrated that plasma concentrations of ADMA, an endogenous inhibitor of NO synthase, are greatly increased in patients with chronic renal failure and may be sufficient to raise vascular resistance. 3° Thus increases in plasma ADMA may contribute to the hypertension that occurs in patients with chronic renal failure irrespective of the original cause of renal impairment.

Diabetes mellitus

Endothelial abnormalities are recognised features of diabetes and these could contribute to the development of the vascular complications seen in this condition. Studies in vitro using blood vessels from animals with genetic, 35 or drug-induced, 36 diabetes have shown impaired endothelium-dependent relaxation. Blood vessels taken from human subjects with insulin dependent diabetes have diminished responses to acetylcholine, 37 while in vivo the response in the forearm arteriolar bed to L-NMMA is reduced. 38 These findings suggest that basal NO production or the effect of NO is impaired. The precise abnormality in the NO system in insulin dependent diabetes is not yet clear and requires further study, some, 38 but not all, 39 studies suggest that the vascular smooth muscle may be less sensitive to NO in patients with insulin dependent diabetes.

Hyperlipidaemia

Studies in animal models of hyperlipidaemia have demonstrated that blood vessels from these animals do not relax normally in response to pharmacological stimuli that release N O . 40 In studies in hyperlipidaemic patients the forearm vasodilator response to acetylcholine is blunted, as is the response to sodium nitroprusside, suggesting that the abnormality in NO-mediated vasodilatation lies at the level of the vascular smooth muscle.41 While in normal tissues the supply of L-arginine substrate does not appear to be rate-limiting for NO synthase, the endothelial dysfunction seen in cholesterol fed rabbits 4° and the coronary circulation of hyperlipidaemic patients 42 can be reversed by providing excess L-arginine.

Septic shock

Excessive production of NO within the vasculature may also lead to disease. Septic shock is associated with hypotension and a reduced response of blood vessels to vasoconstrictors such as noradrenaline. In animals, endotoxin and cytokines produce expression of the inducible NO synthase in endothelial cells 1° and the vascular smooth muscle. 9 Consequently, large amounts of NO are synthesised throughout the vessel wall and this might be

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134 responsible for the hypotension associated with septic shock. 9 In animal models of septic shock low doses of L-NMMA restore blood pressure 43"44 and the vascular response to vasoconstrictors. 9 L-NMMA has now been used in four patients with septic shock, in all cases it restored blood pressure and vascular responsiveness to noradrenaline. 45'46 Further evaluation of its role in the treatment of septic shock is required, and awaits the results of a randomised controlled trial of NO synthase inhibitors.

Conclusions

Nitric oxide is now firmly established as a mediator involved in the control of vascular tone, platelet function, non-specific immune responses and neurotransmission. Possible derangement of the NO system has been identified in a variety of clinical conditions. The diminished NO mediated vasodilator tone in hypertension, diabetes and hyperlipidaemia may partly explain the development of similar long term vascular complications in these disorders, while increased production of NO seems to mediate the hypotension of septic shock. Although the supply of L-arginine is not rate limiting under normal circumstances, it may become so in disease states. In the same way, although circulating levels of the endogenous inhibitor ADMA are unlikely to affect resting vascular tone or immune modulation under normal conditions, concentrations of ADMA may rise significantly in disease and may reach functional levels. Therapies based on the L-arginine:NO pathway are now emerging. The next step will be to manipulate the NO systems in different cell types selectively in order to find new therapeutic options.

References

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