Adventures in vascular biology: a tale of two mediators

Abstract

I would like to thank the Royal Society for inviting me to deliver the Croonian Lecture. In so doing, the Society is adding my name to a list of very distinguished scientists who, since 1738, have preceded me in this task. This is, indeed, a great honour. For most of my research career my main interest has been the understanding of the normal functioning of the blood vessel wall and the way this is affected in pathology. During this time, our knowledge of these subjects has grown to such an extent that many people now believe that the conquering of vascular disease is a real possibility in the foreseeable future. My lecture concerns the discovery of two substances, prostacyclin and nitric oxide. I would like to describe the moments of insight and some of the critical experiments that contributed significantly to the uncovering of their roles in vascular biology. The process was often adventurous, hence the title of this lecture. It is the excitement of the adventure that I would like to convey in the text that follows.

Figure 1

Metabolic pathway of arachidonic acid as it was known in (a) 1971, (b) 1975 and (c) 1976.

Figure 2

Aggregation of indomethacin-treated human platelet-rich plasma induced by prostaglandin endoperoxide (PGG₂) and by thromboxane A₂ (TXA₂). TXA₂ is far more potent than PGG₂.

Figure 3

Differential bioassay of rabbit aorta-contracting substances. PGG₂ (200 ng) was added to 500 μl of Tris buffer at 0 °C and a 50 μl sample (equivalent to 20 ng) was tested on the bioassay tissues. Immediately after testing, horse platelet microsomes were added to the PGG₂ solution and 50 μl was tested 2 min later. The responses to a high dose of PGG₂ and to PGE₂ are also shown for comparison. Reprinted, with permission, from Needleman et al. (1976).

Figure 4

Conversion of PGG₂ by aortic microsomes. In a bioassay using (a) rabbit aortic strip and (b) rat colon, PGE₂ and PGF_2α contracted rat colon, whereas PGG₂ contracted rabbit aorta. The spontaneous disappearance of PGG₂ following incubation for the times indicated resulted in the appearance of PGE- and PGF-like activity. In the presence of aortic microsomes (AM), however, the contractile activity of PGG₂ disappeared within 0.5 min but no PGE- or PGF-like activity was formed, even after 20 min. This did not occur when the AM were boiled. Reprinted, with permission, from Moncada et al. (1976a).

Figure 5

Differential bioassay of prostacyclin (PGX) and PGE₂. The tissues were arranged in a cascade, one above the other, and PGX (10 ng ml⁻¹) and PGE₂ (2 ng ml⁻¹) were infused over the tissues for 1 min. PGX, but not PGE₂, caused a relaxation of the bovine coronary artery.

Figure 6

Comparison of the anti-aggregatory potencies of prostacyclin (PGX) and PGE₁. PGX was obtained by incubation of 100 ng PGH₂ with 500 μg of aortic microsomes in 100 μl of 0.05 M Tris buffer for 2 min at 22 °C and then stored on ice. PGX and PGE₁ were added to human platelet-rich plasma 1 min before the platelets were aggregated with arachidonic acid (AA, 1 mM). In this experiment, PGX is at least 25 times more potent as an anti-aggregating agent than PGE₁. Reprinted, with permission, from Moncada et al. (1976a).

Figure 7

Inhibition of platelet aggregation by prostacyclin (PGX) generated from rings of mesenteric artery from indomethacin-treated and control rabbits. Cut rings of artery (6 mg) from a control rabbit were incubated in 1 ml Tris buffer for 3 min. An aliquot (20 μl) of the supernatant from the incubation mixture added to the platelet-rich plasma (PRP) inhibited the aggregation induced by adenosine diphosphate (ADP; 10 mM). Addition of 6 mg of the rings themselves also prevented platelet aggregation. Supernatant from rings of mesenteric artery (6 mg), removed from an indomethacin-treated rabbit and incubated in Tris buffer as above, did not inhibit platelet aggregation. However, when the rings were added to the PRP, ADP-induced aggregation was prevented. Thus, the rings were able to use endoperoxides generated by the platelet to synthesize PGX. Reprinted from Bunting et al. (1976). Copyright (1976) with permission from Elsevier.

Figure 8

Bioassay system used to detect the release of EDRF from endothelial cells. Porcine aortic endothelial cells were grown in culture on microcarrier beads (approx. 70 μm), which were packed into a modified chromatographic column. Inset shows an electronmicrograph 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. Reprinted, with permission, from Gryglewski et al. (1986a).

Figure 9

Relaxation of rabbit aortae by EDRF and NO. A column packed with endothelial cells cultured on microcarrier beads (as described in figure 8) was perfused with Krebs buffer. The effluent was used to perfuse three spiral strips of rabbit aorta denuded of endothelium (RbA) in a cascade. The tissues were pre-contracted submaximally. Glyceryl trinitrate (GTN) over the tissues (OT) was used to standardize the sensitivity of the tissues. EDRF was released from the cells by an infusion (1 min) through the column (TC) of bradykinin (Bk). Nitric oxide (NO) was administered OT as a 1 min infusion. Reprinted, with permission, from Palmer et al. (1987).

Figure 10

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 9. (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. Reprinted, with permission, from Palmer et al. (1987).

Figure 11

Anti-aggregatory action of EDRF (NO) and its potentiation by prostacyclin. (a) Sub-threshold concentrations of NO (0.1 μM) and (b) amounts of EDRF released from 0.5 ml of endothelial cells, treated with indomethacin and stimulated with bradykinin, are both potentiated by a sub-threshold concentration of prostacyclin (PGI₂, 0.1 nM). The inhibition of aggregation induced by a combination of these sub-threshold concentrations of prostacyclin and NO was reversed by haemoglobin (Hb, 100 nM). Aggregation was induced by collagen (Coll); C represents control aggregation. Reprinted, with permission, from Radomski et al. (1987b).

Figure 12

The l-arginine : nitric oxide pathway.

Figure 13

The effect of l-NMMA on vascular tone, blood pressure and blood flow. (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 anaesthetized rabbit. The lower trace shows the reversal of this effect by l-arginine. The heart rate is also shown. (c) Effect of l-NMMA on blood flow in the brachial artery in five healthy human subjects. Reprinted, with permission, from (a) Rees et al. (1989b), (b) Rees et al. (1989a) and (c) Vallance et al. (1989). Copyright (1989) with permission from Elsevier.

Figure 14

Schematic of two types of hypertension. In the normal situation, vasoconstrictor influences from outside the blood vessel (grey arrows) are counterbalanced by basal production of nitric oxide (black arrows) by endothelial NO synthase. Hypertension can occur in a situation in which vasoconstrictor activity is increased and more NO is generated in an attempt to compensate (Type A). In Type B, reduced synthesis of NO would result in hypertension even in the presence of normal amounts of vasoconstrictor activity. Modified and reprinted, with permission, from Rees et al. (2000).

Figure 15

Effect of pretreatment with the NO synthase inhibitor l-NAME (N^G-nitro-l-arginine methyl ester) on oxygen consumption by endothelial cells. The dotted line shows oxygen consumption by control cells, which were not treated with l-NAME. The solid trace shows that cells pretreated for 20 min with l-NAME respire at a constant rate at all oxygen concentrations. Modified and reprinted, with permission, from Clementi et al. (1999). Copyright (1999) National Academy of Sciences, USA.

Figure 16

Effect of nitric oxide on the redox state of the electron transport chain (ETC). (a,b) The change in RE (reducing equivalents; a measure of the reduction state of the ETC cytochromes) at different oxygen concentrations, in the presence and absence of l-NMMA. (a) The results in RAW 246.7 (murine monocytic) cells and (b) the results in HUVEC (human vascular endothelial cells). (c,d) The effect of NO on the production of superoxide anion (O₂⁻). Fluorescence, measured by flow cytometry, indicates intracellular O₂⁻ production from intact cells incubated with DHE in the presence (shaded) or absence (clear) of l-NMMA at 21% oxygen (c) and 3% oxygen (d). (e) A Western blot of nuclear extracts of cells incubated at 21 and 3% oxygen detecting NFκB in the absence or presence of l-NMMA. Reprinted, with permission, from Palacios-Callender et al. (2004). Copyright (2004) National Academy of Sciences, USA.