The discovery of nitric oxide and its role in vascular biology

Abstract

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.

Figure 1

Bioassay system used to detect the release of EDRF from endothelial cells. Porcine aortic endothelial cells were grown in culture on microcarrier beads (approximately 70 μm), which were packed into a modified chromatographic column. Inset shows an electron micrograph of a bead covered in endothelial cells. The beads were perfused with Krebs buffer at 37°C and the perfusate was allowed to flow over the bioassay tissues (four rabbit aortae denuded of endothelium). The time taken for the superfusate to reach the first tissue was 1 s and the gap between each subsequent tissue was 3 s. From Gryglewski et al. (1986a), with permission.

Figure 2

Detection of exogenous and endogenous NO. (a) Bioassay. The rabbit aorta was relaxed in a concentration-dependent manner by EDRF released from the endothelial cells by bradykinin (BK, TC) and by NO (OT), as in Figure 1. (b) Chemiluminescence. EDRF was released by bradykinin from a replicate column of the cells used in the bioassay. The amounts of both EDRF (endogenously-produced NO) and of exogenously-applied authentic NO which relaxed the bioassay tissue were also detectable by chemiluminescence. From Palmer et al. (1987), with permission.

Figure 3

Inhibition of platelet aggregation by EDRF and NO. Control (C) aggregation of human platelet-rich plasma was induced by collagen (Coll). Pretreatment for 1 min with supernatant from endothelial cells stimulated with bradykinin at different concentrations (1–10 nM, C.S, left panel) or with authentic NO (right panel) prevented collagen-induced aggregation in a concentration-dependent manner.

Figure 4

Effect of diabetes and L-NAME on nitrergic nerves and erectile function in rats. (A) Immunostaining for nNOS in the corpus cavernosum of a control rat (a), in an 8-week-diabetic rat (b, note that the nitrergic nerves became very sparse) and in an 8-week-diabetic rat treated with L-NAME (c, note that the structure and number of the nitrergic nerve fibres were preserved during diabetes). Tyrosine hydroxylase immunostaining in a control rat (d), an 8-week-diabetic rat (e, note that the morphology and density of noradrenergic nerves were unchanged) and an 8-week-diabetic rat treated with L-NAME (f). Scale bar is 33–43 μM. (B) Typical changes in intracavernous pressure (ICP) in response to stimulation in a control rat (upper trace), a 12 week diabetic rat (middle trace: note that the increase in ICP could not be maintained during the stimulation period) and a 12 week diabetic rat treated with L-NAME (lower trace). L-NAME was withdrawn 72 h before the experiments. The vertical scale corresponds to 100 cm H₂O ICP. The horizontal scale corresponds to 2 min. From Cellek et al. (1999), with permission.

Figure 5

The effect of L-NMMA on vascular tone and blood pressure. (a) The effect of N^G-monomethyl L-arginine (L-NMMA) and its inactive isomer (D-NMMA) on the basal tone of pre-contracted rabbit aortic rings, with and without endothelium. (b) Long-lasting effect of L-NMMA on blood pressure in the anaesthetised rabbit. The lower part of the trace shows the reversal of this effect by L-arginine. The heart rate is also shown. From (a) Rees et al. (1989a); (b) Rees et al. (1989b), with permission.

Figure 6

Time course of activation of left ventricular NO synthase and changes in plasma NO_x⁻ (NO₂⁻ plus NO₃⁻) concentration after i.p. injection of endotoxin (LPS) or pyrogen-free saline in rats. (a) Ca²⁺-dependent (○) and Ca²⁺-independent (•) NO synthase activity in the left ventricular wall after treatment with LPS. C represents control values 6 h after injection of pyrogen-free saline. (b) Level of NO_x⁻ in plasma at time rats were killed after treatment with LPS. C Represents control values 6 h after injection of pyrogen-free saline. Reprinted from Schulz et al. (1992), with permission.