Heart 2001;85:342–350 which was subsequently identified as nitric GENERAL CARDIOLOGY oxide.23 L-arginine: nitric oxide pathway Endothelial function and nitric oxide: Endothelium derived nitric oxide is synthesised clinical relevance from the amino acid L-arginine by the endothelial isoform of nitric oxide synthase, 342 yielding L-citrulline as a byproduct.4 Nitric Patrick Vallance, Norman Chan oxide is labile with a short half life (< 4 seconds Centre for Clinical Pharmacology, University College London, in biological solutions). It is rapidly oxidised to London, UK nitrite and then nitrate by oxygenated haemo- globin before being excreted into the urine.4 Several co-factors are required for nitric oxide he vascular endothelium is a monolayer biosynthesis. These include nicotinamide of cells between the vessel lumen and adenine dinucleotide phosphate (NADPH), Tthe vascular smooth muscle cells. Far flavin mononucleotide, flavin adenine dinucle- from being inert, it is metabolically active and otide, tetrahydrobiopterin (BH4), and cal- produces a variety of vasoactive mediators. modulin. Once synthesised, the nitric oxide Among these mediators, endothelial derived diVuses across the endothelial cell membrane nitric oxide is essential in the maintenance of and enters the vascular smooth muscle cells vascular homeostasis, and defects in the where it activates guanylate cyclase, leading to L-arginine: nitric oxide pathway have been an increase in intracellular cyclic guanosine- implicated in a variety of cardiovascular 3’,5-monophosphate (cGMP) concentrations4 diseases. (fig 1). As a second messenger, cGMP mediates many of the biological eVects of nitric Historic perspectives oxide including the control of vascular tone and platelet function. In addition, nitric oxide has other molecular targets which include From EDRF to nitric oxide haem or other iron centred proteins, DNA, and In 1980, Furchgott and Zawadzki showed that thiols. These additional reactions may mediate the presence of vascular endothelial cells is changes in functions of certain key enzymes or essential for acetylcholine to induce relaxation ion channels. Nitric oxide also interacts with of isolated rabbit aorta.1 If the vascular enzymes of the respiratory chain including Correspondence to: endothelium was removed, the blood vessel complex I and II, and aconitase, and through Professor Patrick failed to relax in response to acetylcholine but these eVects alters tissue mitochondrial respi- Vallance, Centre of Clinical Pharmacology, still responded to glyceryl trinitrate. This ration. Interaction of nitric oxide with super- Gower Street Campus, endothelium dependent relaxation of vascular oxide anion can attenuate physiological re- 3rd Floor, Rayne Institute, smooth muscle to acetylcholine is mediated by sponses mediated by nitric oxide and produce 5 University Street, irreversible inhibitory eVects on mitochondrial London WC1E 6JJ, UK an endogenous mediator initially named en- 1 [email protected] dothelium derived relaxing factor (EDRF), function as a result of the formation of peroxynitrite (ONOO−), a powerful oxidant Endothelial cell Vascular smooth muscle cell species. Nitric oxide synthase isoforms Three isoforms of nitric oxide synthase (NOS) GTP cGMP O2 Citrulline have been identified: the endothelial isoform (eNOS), neuronal isoform (nNOS), and mac- L-arginineNO NO-haem GC rophage or inducible isoform (iNOS). All three NOS isoforms play distinct roles in the regula- NOS tion of vascular tone (fig 2). The genes encod- ing eNOS, nNOS, and iNOS are located on chromosome 7, 12, and 17, respectively.5 Figure 1. The L-arginine: nitric oxide pathway. NO, nitric oxide; NOS, nitric oxide Whereas eNOS and nNOS are normal con- synthase; GC, guanylate cyclase; cGMP, cyclic guanosine-3’,5-monophosphate; stituents of healthy cells, iNOS is not usually GTP, guanosine triphosphate expressed in vascular cells and its expression is seen mainly in conditions of infection or AB C inflammation. Biological effects of nitric oxide Nitric oxide and the vasculature Endothelium derived nitric oxide is a potent vasodilator in the vasculature, and the balance between nitric oxide and various endothelium Figure 2. (A) In most, if not all, vessels nitric oxide is synthesised within the derived vasoconstrictors and the sympathetic endothelium. (B) In certain vessels (for example, cerebral vessels) nitric oxide is nervous system maintains blood vessel tone. also synthesised by nerves in the adventitia (nitrogenic nerves). (C) After exposure to endotoxin or cytokines, iNOS is expressed throughout the vessel wall In addition, nitric oxide suppresses platelet and produces large amounts of nitric oxide. aggregation, leucocyte migration, and cellular www.heartjnl.com Education in Heart Basal nitric oxide release The L-arginine: nitric oxide pathway The synthesis of nitric oxide in vascular endothelial cells in culture or intact vascular x Nitric oxide is synthesised from L-arginine tissue can be inhibited by NG monomethyl-L- to yield citrulline arginine (L-NMMA), an analogue of L-arginine in which one of the guanidino Co-factors required for nitric synthesis: x nitrogen atoms is methylated. This inhibitory – nicotinamide adenine dinucleotide eVect of L-NMMA is readily reversed by 343 phosphate (NADPH) 7 – flavin mononucleotide L-arginine. L-NMMA and similar substrate – flavin adenine dinucleotide based inhibitors have been used to examine the role of nitric oxide in various vascular beds in – tetrahydrobiopterin (BH4) – calmodulin vitro and in both human and animal models. In rings of rabbit aorta, L-NMMA causes x Enzymes responsible for nitric oxide significant endothelium dependent contrac- synthesis: tion. Intravenous infusion of L-NMMA into – endothelial nitric oxide synthase experimental animals induces a dose related (expressed in normal cells) (eNOS) increase in blood pressure which is reversed by – neuronal nitric oxide synthase intravenous administration of L-arginine, and (expressed in normal cells) (nNOS) in the human forearm vasculature infusion of – inducible nitric oxide synthase L-NMMA into the brachial artery causes sub- (expressed during infection/ stantial dose dependent vasoconstriction. Thus inflammation) (iNOS) continuous generation of nitric oxide is crucial in maintaining peripheral vasodilatation in x Nitric oxide activates guanylate cyclase in 7 vascular smooth muscle cells to synthesise humans (fig 3). This basal nitric oxide cyclic guanosine-3’,5-monophosphate mediated dilatation has been seen in every (cGMP) which causes many of its other arterial bed studied including cerebral, biological eVects pulmonary, renal, and coronary vasculature. In contrast, in the venous system inhibitors of NOS do not lead to an increase in basal tone in adhesion to the endothelium, and attenuates a variety of venous preparations from animals 8 vascular smooth muscle cell proliferation and or humans, suggesting that basal nitric oxide migration. Furthermore, nitric oxide can in- production does not have a major role in the hibit activation and expression of certain adhe- maintenance of the resting tone in most veins. sion molecules, and influence production of In conduit vessels, there is some basal nitric superoxide anion. Loss of endothelium derived oxide mediated dilatation but it appears to be nitric oxide would be expected to promote a less than that seen in resistance vessels. vascular phenotype more prone to atherogen- esis, a concept supported by studies in experi- Agonist stimulated nitric oxide release mental animals.6 Many chemical substances such as acetylcho- line, bradykinin, serotonin, and substance P are Nitric oxide release from the vascular able to induce endothelium dependent va- endothelium sodilatation. In rings of rabbit aorta, endothe- There is a continuous basal synthesis of nitric lium dependent relaxation induced by acetyl- oxide from the vascular endothelium to main- choline, calcium ionophore (A23187) or tain resting vascular tone. A number of chemi- substance P is inhibited by L-NMMA. This cal and physical stimuli may activate eNOS and provides in vitro evidence that vasorelaxation lead to increased nitric oxide production. induced by endothelium dependent agonists is nitric oxide mediated. L-NMMA also attenu- ates the hypotensive eVect of acetylcholine in vivo in animals. However, the blockade is far 5 from complete and there is now growing evidence for additional mechanisms underlying endothelium dependent responses to acetyl- choline, particularly in resistance vessels. Simi- larly in humans, L-NMMA inhibits agonist 7 3 stimulated relaxation in resistance, conduit, and venous vessels in vivo. However, the degree of inhibition to agonist dependent dilatation varies between vascular beds, and mechanisms (for example, prostaglandins and endothelium Forearm blood flow Forearm blood derived hyperpolarising factors) other than that (ml/100 ml forearm/min) 1 L-NMMA mediated by nitric oxide appear to be involved. 124 (µmol/min) Physical forces and nitric oxide release Haemodynamic shear stress exerted by the 010 30 50 70 viscous drag of flowing blood is an important physiological stimulus in the regulation Minutes after cannulation of nitric oxide release from the endothelium. Figure 3. Inhibition of endogenous nitric oxide by L-NMMA produces a dose The mechanisms of shear stress induced dependent reduction in forearm blood flow. nitric oxide release is complex, involving www.heartjnl.com Education in Heart (1) extremely rapid initiation via ion channel supported