Blockade of nitric oxide-induced relaxation of rabbit aorta by cysteine and homocysteine.

AIM: To examine the inhibition by L-cysteine (Cys) and L-homocysteine (HoCys) of NO-induced relaxation of aorta. METHODS: The tension of rabbit aortic rings in oxygenated Krebs' solution was recorded isometrically. RESULTS: Pretreatment of endothelium-denuded rings with Cys or HoCys inhibited the NO-induced increase in cGMP. The inhibitory effects of Cys or HoCys on relaxation responses to subsequent additions of NO 75 nmol.L-1 gradually diminished with time, which was consistent with the loss of the sulfhydryl concentration of Cys and HoCys. Superoxide dismutase (SOD) 35 kU.L-1 attenuated the inhibition by Cys and HoCys of NO-induced relaxation. Neither boiled SOD nor catalase 100 kU.L-1 antagonized the inhibitory effects of Cys. Preaddition of SOD 35 kU.L-1 inhibited the reduction of cytochrome C by Cys. Increasing concentrations of SOD from 35 to 350 kU.L-1 intensified the cytochrome C reduction. Addition of xanthine 300 mumol.L-1 plus xanthine oxidase 1 U.L-1 to the mixture of cytochrome C 60 mumol.L-1 and Cys 100 mumol.L-1 produced an additional augmentation of SOD-inhibitable reduction of cytochrome C. The rate of the reduction of cytochrome C induced by HoCys 100 mumol.L-1 was much slower than with Cys. Addition of NO reduced the SH concentrations of both the supernatant of aortic homogenate and Cys in Krebs' solution. CONCLUSION: The inhibition by the SH compounds of NO is mediated partly by the superoxide generated by the auto-oxidation of these compounds, and partly by a direct reaction of SH groups with NO.

The 1996 Albert Lasker Medical Research Awards. The discovery of endothelium-derived relaxing factor and its importance in the identification of nitric oxide.

The discovery of endothelium-derived relaxing factor (EDRF) and its importance in the identification of nitric oxide (NO) originated with studies using rabbit aorta to examine drug-receptor interactions in vascular smooth muscle. Smooth muscle relaxation by acetylcholine and a number of other agonists was found to be dependent on the presence of endothelial cells, which, when stimulated by the agonist, released a diffusable, very labile, nonprostanoid substance, termed EDRF, that acted on vascular smooth muscle cells to activate relaxation. The characteristics of EDRF, when released from endothelial cells, were similar to the characteristics of NO. It is now established that EDRF, either as NO or some related nitrosyl substance, has a major role in a variety of important biological processes, including the regulation of vascular tone, local blood flow, and blood pressure, inhibition of platelet aggregation and adhesion, and involvement in postischemic reperfusion, memory function, and central nervous system degenerative diseases.

A research trail over half a century.

The author describes his major research activities from the time of his PhD thesis work (1937-1940) on properties of erythrocyte membranes to the present. His involvement in research on circulatory shock during World War II led to a continuing interest in the physiology and pharmacology of smooth muscle and cardiac muscle. From 1956 to 1978, his main areas of research were photorelaxation of blood vessels, factors influencing contractility of cardiac muscle, peripheral adrenergic mechanisms, and receptor theory. The major findings of his and his collaborators in these areas are described. He then recounts how an accidental finding in an experiment in 1978 on preparations of rabbit aorta eventually led to the discovery of endothelium dependent relaxation and the endothelium-derived relaxing factor (EDRF); and how additional findings led him to propose in 1986 that EDRF is nitric oxide.

Inhibition by sulfhydryl compounds of vascular relaxation induced by nitric oxide and endothelium-derived relaxing factor.

On rings of rabbit thoracic aorta precontracted with phenylephrine, L-cysteine (Cys) and dithiothreitol (DTT) (1-100 microM), but not glutathione (GSH), produced dose-dependent augmentation of contraction in endothelium-intact, but not in endothelium-denuded rings. The augmentation appeared to be due to inhibition of basally released endothelium-derived relaxing factor (EDRF), and was abolished by pretreatment with superoxide dismutase (SOD, 15 U/ml). At a high dose (1 mM), Cys and GSH produced transient, and DTT produced sustained endothelium-independent relaxation, not influenced by SOD. Cys and DTT (10 microM), but not GSH, produced a small but significant inhibition of acetylcholine-induced endothelium-dependent relaxation, and this inhibition was prevented by SOD. In a perfusion-bioassay system in which EDRF was released by acetylcholine from endothelium of a perfused segment of rabbit aorta, Cys and DTT (20 microM), but not GSH, infused into the perfusate between the segment and an endothelium-denuded bioassay ring, partially inhibited relaxation by the EDRF, but not when SOD was present. In organ chamber experiments, the large transient relaxation of endothelium-denuded rings produced by 75 nM nitric oxide (NO) was partially inhibited in a concentration-dependent manner by Cys, DTT and GSH (0.1-100 microM). Moderate relaxation by 15 nM NO was almost completely inhibited by each compound at 10 microM. The order of potency was Cys > DTT > GSH. Cystine, glutathione disulfide and alanine did not inhibit. Inhibition of NO-induced relaxation was largely attenuated by SOD.(ABSTRACT TRUNCATED AT 250 WORDS)

Introduction to EDRF research.

In response to acetylcholine, endothelial cells were shown to release a nonprostanoid factor, called endothelium-derived relaxing factor (EDRF), which caused relaxation of vascular smooth muscle. Since this discovery in 1980, many additional agents have been shown to stimulate release of EDRF from endothelium. Biological and chemical evidence has supported the proposal that EDRF is nitric oxide (NO), a potent vasodilator. Research on the synthesis, inhibition, and physiological roles of EDRF/NO has led to studies of this factor in vascular regulation and in various disease states, including hypertension, atherosclerosis, and diabetes.

Vasodilation induced by acetylcholine and by glyceryl trinitrate in rat aortic and mesenteric vasculature.

In endothelium-containing rings of rat aorta which had been precontracted with phenylephrine, addition of acetylcholine (ACh) (0.010-10 microM) resulted in concentration-dependent, graded relaxation through the release of endothelium-derived relaxing factor (EDRF). Hemoglobin (3 and 10 microM) and methylene blue (10 microM) both produced marked inhibition of this EDRF-mediated relaxation. In the perfused mesenteric arterial vasculature of the rat, ACh-induced vasodilation was also inhibited by hemoglobin and by methylene blue, although to a lesser extent than was ACh-induced relaxation of aortic rings by these two agents. These findings indicate that EDRF mediates in large part ACh-induced relaxation of resistance vessels in the mesenteric vascular bed as well as large arteries. The nitrovasodilator glyceryl trinitrate (GTN) caused endothelium-independent relaxation of aortic rings as well as vasodilation of mesenteric arterial vasculature. GTN-induced relaxation of aortic rings was antagonized by hemoglobin as well as methylene blue, but to a lesser extent than was ACh-induced relaxation. However, hemoglobin did not inhibit and methylene blue actually potentiated GTN-induced vasodilation in the perfused mesenteric vasculature. Possible explanations of these paradoxical results are discussed.

Responses of rabbit aorta to nitric oxide and superoxide generated by ultraviolet irradiation of solutions containing inorganic nitrite.

In a perfusion-superfusion system, an endothelium-free ring of rabbit thoracic aorta precontracted with phenylephrine was used to bioassay relaxing activity. As previously reported, when NaNO2 (1 mM) was added to the standard Krebs' solution (37 degrees C oxygenated, containing 30 microM EDTA), irradiation of the solution with long wavelength ultraviolet light, while it was perfusing through a glass tube proximal to the bioassay ring, resulted in a small degree of relaxation of the ring, and this relaxation was markedly potentiated when superoxide dismutase (SOD, 10 U/ml) was also present in the solution. The full potentiation by SOD of the relaxation produced by photoactivation of NO2- was matched by cytochrome c (30 microM), MnCl2 (30 microM) and CuCl2 (100 microM), all of which are scavengers of superoxide (O2-). No potentiation was produced by catalase (1,000 U/ml), ZnCl2 (100 microM) and boiled SOD. Also, CuCl2, when its concentration was no greater than that of the EDTA in the Krebs' solution, failed to potentiate the relaxation. These results are consistent with our earlier conclusion that the relaxant produced by irradiation of NO2- is NO and that it is normally inactivated rapidly by O2- also present in the irradiated perfusion fluid. When EDTA was omitted from the perfusing Krebs' solution, photoactivation of NO2- in the solution produced a very large relaxation of the bioassay ring, which was not enhanced by addition of SOD or other O2- scavengers.(ABSTRACT TRUNCATED AT 250 WORDS)

Endothelium-dependent and -independent vasodilation involving cyclic GMP: relaxation induced by nitric oxide, carbon monoxide and light.

The characteristics of carbon monoxide (CO)-induced, endothelium-independent relaxation of rabbit aorta were compared with those of nitric oxide (NO)-induced and light-induced relaxation and endothelium-dependent relaxation mediated by endothelium-dependent relaxing factor (EDRF). CO was less than one thousandth as potent as NO as a relaxant. Various findings, including an increase in cyclic GMP associated with CO-induced relaxation, led to the conclusion that CO - like NO, EDRF and light - produces relaxation as a result of its stimulation of guanylate cyclase. LY 83583, which generates superoxide, was a potent, fast-acting inhibitor of acetylcholine-induced endothelium-dependent relaxation and NO-induced relaxation, and a fairly potent, moderately fast-acting inhibitor of photorelaxation, but only a very weak inhibitor of CO-induced relaxation. The ability of LY 83583 as well as hemoglobin to inhibit photorelaxation is consistent with the hypothesis that on radiation a photo-induced relaxing factor is formed which can stimulate guanylate cyclase and which can be inactivated by superoxide and by hemoglobin.

Endothelium-derived relaxing and contracting factors.

Endothelium-dependent relaxation of blood vessels is produced by a large number of agents (e.g., acetylcholine, ATP and ADP, substance P, bradykinin, histamine, thrombin, serotonin). With some agents, relaxation may be limited to certain species and/or blood vessels. Relaxation results from release of a very labile non-prostanoid endothelium-derived relaxing factor (EDRF) or factors. EDRF stimulates guanylate cyclase of the vascular smooth muscle, with the resulting increase in cyclic GMP activating relaxation. EDRF is rapidly inactivated by hemoglobin and superoxide. There is strong evidence that EDRF from many blood vessels and from cultured endothelial cells is nitric oxide (NO) and that its precursor is L-arginine. There is evidence for other relaxing factors, including an endothelium-derived hyperpolarizing factor in some vessels. Flow-induced shear stress also stimulates EDRF release. Endothelium-dependent relaxation occurs in resistance vessels as well as in larger arteries, and is generally more pronounced in arteries than veins. EDRF also inhibits platelet aggregation and adhesion to the blood vessel wall. Endothelium-derived contracting factors appear to be responsible for endothelium-dependent contractions produced by arachidonic acid and hypoxia in isolated systemic vessels and by certain agents and by rapid stretch in isolated cerebral vessels. In all such experiments, the endothelium-derived contracting factor appears to be some product or by-product of cyclooxygenase activity. Recently, endothelial cells in culture have been found to synthesize a peptide, endothelin, which is an extremely potent vasoconstrictor. The possible physiological roles and pathophysiological significance of endothelium-derived relaxing and contracting factors are briefly discussed.

Interactions of light and sodium nitrite in producing relaxation of rabbit aorta.

In rings of rabbit aorta mounted in muscle chambers, endothelial-independent reversible relaxation produced by irradiation with long wavelength was potentiated in the presence of 1 mM NaNO2. Photorelaxation in the presence of NaNO2, but not in its absence, was further potentiated by superoxide dismutase. The interaction of light and NaNO2 in producing relaxations was studied in a perfusion-superfusion system in which Krebs' solution (37 degrees C, 95% O2-5% CO2) with or without added NaNO2 was perfused (2 ml/min) through a glass tube (1.25 mm, inside diameter) on its way to superfusing an endothelium-free aortic ring a few seconds downstream. UV irradiation either of the solution flowing through the tube or of the aortic ring could be made separately. The ring, mounted for tension recording, was precontracted to a similar level of tone in the absence or presence of NaNO2 by l-phenylephrine HCl. Photorelaxation on direct irradiation of the ring was potentiated markedly by 1 mM NaNO2, was not significantly potentiated further by 10 U/ml of superoxide dismutase, and was inhibited markedly by Hb (1-10 microM) but not by MetHb (10 microM). Irradiation of the perfusing solution produced no relaxation of the aortic ring downstream in the absence of NaNO2, and only small relaxation at 1 mM NaNO2. When superoxide dismutase was present along with NaNO2, irradiation of the perfusing solution (15-120 sec) produced marked relaxation, with magnitude increasing with concentration of NaNO2 (1 microM-1 mM). This relaxation was inhibited by Hb (complete at 0.3-1 microM) but only partly by MetHb (10 microM).(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence for endothelium-dependent vasodilation of resistance vessels by acetylcholine.

In the perfused mesenteric arterial vasculature of the rabbit, vasodilation by acetylcholine (ACh) was almost completely blocked after a 15-min perfusion of the vasculature with 0.2% collagenase, an enzyme capable of removing endothelial cells. In the perfused mesenteric arterial vasculature of the rat, vasodilation by ACh was markedly, though not completely, inhibited by hemoglobin (10 microM), an agent which can inactivate endothelium-derived relaxing factor (EDRF). These results suggest that a major component of vasodilation of mesenteric resistance vessels in rabbit and rat by ACh is mediated by EDRF.

Depression of contractile responses in rat aorta by spontaneously released endothelium-derived relaxing factor.

Removal of endothelial cells on rings of rat aorta increased the sensitivity to the selective alpha-1 adrenoceptor agonist phenylephrine, to the nonselective alpha adrenoceptor agonist norepinephrine and to the selective alpha-2 adrenoceptor agonist clonidine. In the case of the first two, which are strong agonists for the alpha-1 adrenoceptor-mediating contraction, removal of endothelium increased sensitivity 4- and 6-fold at the EC30 level, but produced little or no increase in maximum. In the case of clonidine, a partial agonist for the alpha-1 adrenoceptor, which gave only about 15% of the maximum given by phenylephrine on endothelium-containing rings, removal of the endothelium not only shifted the curve to the left but also increased the maximum to about 50% of that given by phenylephrine. The depression of sensitivity to these agonists in rings with endothelium appeared to be due to the vasodepressor action of endothelium-derived relaxing factor (EDRF), as hemoglobin, a specific blocking agent of EDRF, abolished this depression. It is unlikely that the endothelium-dependent depression was due to stimulation of release of EDRF, because clonidine did not produce endothelium-dependent relaxation in precontracted rings even when its contractile action was blocked by the alpha-1 adrenoceptor antagonist prazosin. Further evidence against alpha adrenoceptor agents stimulating release of EDRF was that neither phenylephrine nor clonidine induced a rise in cyclic GMP in aortic rings, whereas acetylcholine, which does release EDRF, caused a large rise in cyclic GMP content. The possibility that the muscle cells of intact rat aortic rings were under the tonic influence of released EDRF was supported by the finding that, in the absence of any contractile agent, hemoglobin induced a fall in the basal level of cyclic GMP in endothelium-containing rings. Also consistent with EDRF being released spontaneously was the finding that contraction induced by 5-hydroxytryptamine, like that by alpha-adrenergic agonists, was also depressed in endothelium-containing rings of aorta. When the efficacy of phenylephrine as an alpha-1 agonist was reduced to about the initial efficacy of clonidine by irreversible inactivation of a very large fraction of alpha-1 adrenoceptors of the smooth muscle cells by pretreatment with dibenamine, the concentration-contraction curves for phenylephrine for both endothelium-containing rings and for endothelium-denuded rings now became very similar to the corresponding curves obtained for clonidine before receptor inactivation.(ABSTRACT TRUNCATED AT 400 WORDS)