Endothelial function and nitric oxide: clinical relevance.

The vascular endothelium is a monolayer of cells between the vessel lumen and the vascular smooth muscle cells. Far from being inert, it is metabolically active and produces a variety of vasoactive mediators. Among these mediators, endothelial derived nitric oxide is essential in the maintenance of vascular homeostasis, and defects in the L-arginine: nitric oxide pathway have been implicated in a variety of cardiovascular diseases.

Historic perspectives

From EDRF to nitric oxide

In 1980, Furchgott and Zawadzki showed that the presence of vascular endothelial cells is essential for acetyicholine to induce relaxation of isolated rabbit aorta. [1] If the vascular endothelium was removed, the blood vessel failed to relax in response to acerylcholine but still responded to glyceryl trinitrate. This endothelium dependent relaxation of vascular smooth muscle to acetyicholine is mediated by an endogenous mediator initially named endothelium derived relaxing factor (EDRF), [1] which was subsequently identified as nitric oxide. [2 3]

L-arginine: nitric oxide pathway

Endothelium derived nitric oxide is synthesised from the amino acid L-arginine by the endothelial isoform of nitric oxide synthase, yielding L-citrulline as a byproduct. [4] Nitric oxide is labile with a short half life ([less than] 4 seconds in biological solutions). It is rapidly oxidised to nitrite and then nitrate by oxygenated haemoglobin before being excreted into the urine. [4] Several co-factors are required for nitric oxide biosynthesis. These include nicotinamide adenine dinucleotide phosphate (NADPH), flavin mononucleotide, flavin adenine dinucleotide, tetrahydrobiopterin ([BH.sub.4]), and calmodulin. Once synthesised, the nitric oxide diffuses across the endothelial cell membrane and enters the vascular smooth muscle cells where it activates guanylate cyclase, leading to an increase in intracellular cyclic guanosine 3', 5-monophosphate (cGMP) concentrations [4] (fig 1). As a second messenger, cGMP mediates many of the biological effects of nitric oxide including the control of vascular tone and pl atelet function. In addition, nitric oxide has other molecular targets which include haem or other iron centred proteins, DNA, and thiols. These additional reactions may mediate changes in functions of certain key enzymes or ion channels. Nitric oxide also interacts with enzymes of the respiratory chain including complex I and II, and aconitase, and through these effects alters tissue mitochondrial respiration. Interaction of nitric oxide with superoxide anion can attenuate physiological responses mediated by nitric oxide and produce irreversible inhibitory effects on mitochondrial function as a result of the formation of peroxynitrite (ONOO), a powerful oxidant species.

Nitric oxide synthase isoforms

Three isoforms of nitric oxide synthase (NOS) have been identified: the endothelial isoform (eNOS), neuronal isoform (nNOS), and macrophage or inducible isoform (iNOS). All three NOS isoforms play distinct roles in the regulation of vascular tone (fig 2). The genes encoding eNOS, nNOS, and iNOS are located on chromosome 7, 12, and 17, respectively. [5] Whereas eNOS and nNOS are normal constituents of healthy cells, iNOS is not usually expressed in vascular cells and its expression is seen mainly in conditions of infection or 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 derived vasoconstrictors and the sympathetic nervous system maintains blood vessel tone. In addition, nitric oxide suppresses platelet aggregation, leucocyte migration, and cellular adhesion to the endothelium, and attenuates vascular smooth muscle cell proliferation and migration. Furthermore, nitric oxide can inhibit activation and expression of certain adhesion molecules, and influence production of superoxide anion. Loss of endothelium derived nitric oxide would be expected to promote a vascular phenotype more prone to atherogenesis, a concept supported by studies in experimental animals. [6]

Nitric oxide release from the vascular endothelium

There is a continuous basal synthesis of nitric oxide from the vascular endothelium to maintain resting vascular tone. A number of chemical and physical stimuli may activate eNOS and lead to increased nitric oxide production.

Basal nitric oxide release

The synthesis of nitric oxide in vascular endothelial cells in culture or intact vascular tissue can be inhibited by [N.sup.G] monomethyl-L-arginine (L-NMMA), an analogue of L-arginine in which one of the guanidino nitrogen atoms is methylated. This inhibitory effect of L-NMMA is readily reversed by L-arginine. [7] L-NMMA and similar substrate based inhibitors have been used to examine the role of nitric oxide in various vascular beds in vitro and in both human and animal models.

In rings of rabbit aorta, L-NMMA causes significant endothelium dependent contraction. Intravenous infusion of L-NMMA into experimental animals induces a dose related increase in blood pressure which is reversed by intravenous administration of L-arginine, and in the human forearm vasculature infusion of L-NMMA into the brachial artery causes substantial dose dependent vasoconstriction. Thus continuous generation of nitric oxide is crucial in maintaining peripheral vasodilatation in humans [7] (fig 3). This basal nitric oxide mediated dilatation has been seen in every other arterial bed studied including cerebral, pulmonary, renal, and coronary vasculature. In contrast, in the venous system inhibitors of NOS do not lead to an increase in basal tone in a variety of venous preparations from animals or humans, [8] suggesting that basal nitric oxide production does not have a major role in the maintenance of the resting tone in most veins. In conduit vessels, there is some basal nitric oxide mediated dilatation b ut it appears to be less than that seen in resistance vessels.

Agonist stimulated nitric oxide release

Many chemical substances such as acetylcholine, bradykinin, serotonin, and substance P are able to induce endothelium dependent vasodilatation. In rings of rabbit aorta, endothelium dependent relaxation induced by acetylcholine, calcium ionophore (A23187) or substance P is inhibited by L-NMMA. This provides in vitro evidence that vasorelaxation induced by endothelium dependent agonists is nitric oxide mediated. L-NMMA also attenuates the hypotensive effect of acetylcholine in vivo in animals. However, the blockade is far from complete and there is now growing evidence for additional mechanisms underlying endothelium dependent responses to acetylcholine, particularly in resistance vessels. Similarly in humans, L-NMMA inhibits agonist stimulated relaxation in resistance, [7] 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 derived …