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The discovery, early studies and identification of endothelium-derived relaxing factor (EDRF) as nitric oxide, are described.

The presence of air bubbles, and thus the unintentional removal of endothelial cells, may have prevented the discovery of endothelium-dependent vasodilation at an earlier date. In investigations with perfused rabbit central ear artery the infusion of acetylcholine, a known potent prejunctional inhibitor of adrenergic neurotransmission, did not inhibit vasoconstriction produced by infused norepinephrine. It was later determined that air bubbles in the perfusion line had mechanically removed the endothelial cells, and as a result no release of endothelium-derived relaxing factor (EDRF) could take place.

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.

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.

Furchgott’s epochal discovery in 1980 of endothelium-dependent relaxation of arteries by acetylcholine led to identification of a labile material released by endothelial cells first termed endothelium-derived relaxing factor and later shown to be nitric oxide (NO). NO is now recognized as a key mediator in multiple physiologic processes that are perturbed in diabetes. In induced diabetes in the rat, for example, the hypotensive response to acetylcholine infusion is sharply attenuated, perhaps due to the action of advanced glycosylated endproducts. Another illustration of impaired NO effect in diabetes is the finding that penile corpora cavernosa obtained from impotent diabetic men show total absence of relaxation on exposure to acetylcholine. While an exact role for NO in the pathogenesis of diabetic complications has yet to be defined, there is no doubt that it will. Therapeutic initiatives to restore normal NO equilibrium are rational objectives once the pathophysiology of diabetes is elucidated.

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.

It is by now well established that the synthesis of endothelium-derived relaxing factor (EDRF) in endothelial cells of systemic arteries depends on the oxidation of a guanidinium nitrogen of L-arginine to nitric oxide (NO) by the constitutive enzyme nitric oxide synthase, an oxygenase for which NADPH is a cosubstrate with L-arginine, and L-citrulline is a coproduct with NO.1The enzyme is dependent on Ca2Vcalmodulin for activation, and agonists that stimulate synthesis and release of EDRF from endothelial cells do so by acting on receptors that mediate an increase in the concentration of intracellular Caz+ ions.’ With the use of arginine analogs that competitively inhibit the enzyme, such asNGmonomethyl-L-arginine (L-NMMA), evidence has now been obtained that the endothelial cells of peripheral resistance vessels also generate EDRF via the nitric oxide synthase.1Findings with these arginine analog inhibitors also indicate that nitric oxide synthase is responsible for formation of EDRF in isolated canine cerebral arteries.2On cat cerebral arterioles observed under cranial windows, recent work with arginine analog inhibitors also indicates that the nitric oxide synthase is responsible for the synthesis of EDRF released in response to stimulation by topical acetylcholine (ACh).3

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.

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.

Despite its very potent vasodilating action in vivo, acetylcholine (ACh) does not always produce relaxation of isolated preparations of blood vessels in vitro. For example, in the helical strip of the rabbit descending thoracic aorta, the only reported response to ACh has been graded contractions, occurring at concentrations above 0.1 muM and mediated by muscarinic receptors. Recently, we observed that in a ring preparation from the rabbit thoracic aorta, ACh produced marked relaxation at concentrations lower than those required to produce contraction (confirming an earlier report by Jelliffe). In investigating this apparent discrepancy, we discovered that the loss of relaxation of ACh in the case of the strip was the result of unintentional rubbing of its intimal surface against foreign surfaces during its preparation. If care was taken to avoid rubbing of the intimal surface during preparation, the tissue, whether ring, transverse strip or helical strip, always exhibited relaxation to ACh, and the possibility was considered that rubbing of the intimal surface had removed endothelial cells. We demonstrate here that relaxation of isolated preparations of rabbit thoracic aorta and other blood vessels by ACh requires the presence of endothelial cells, and that ACh, acting on muscarinic receptors of these cells, stimulates release of a substance(s) that causes relaxation of the vascular smooth muscle. We propose that this may be one of the principal mechanisms for ACh-induced vasodilation in vivo. Preliminary reports on some aspects of the work have been reported elsewhere.

A brief review is first presented of findings during the past few years by the authors and by others on the nonprostaglandin endothelium-dependent relaxation of isolated arteries by a large number of vasoactive agents. Among these agents are acetylcholine (ACh); the calcium ionophore A23187; ATP and ADP; substance P; bradykinin (canine, human, and porcine arteries); histamine, acting via an H1-receptor (rat arteries); thrombin (canine arteries); serotonin (canine coronary artery); and norepinephrine, acting via an alpha2-receptor (canine coronary artery). The endothelium-derived relaxing factor (EDRF) released by ACh and other agents has not yet been identified. Our original hypothesis that arachidonic acid is the precursor of EDRF is not supported by the finding that other unsaturated fatty acids in addition to arachidonic acid, and even stearic acid, elicited nonprostaglandin endothelium-dependent relaxations. Methylene blue and hemoglobin (but not methemoglobin) rapidly inhibited relaxation of rabbit aorta by ACh or A23187, suggesting that our proposal that EDRF is a labile free radical may be correct. The endothelium-dependent relaxation by each of these agents was shown to be preceded by an endothelium-dependent increase in cyclic GMP in the smooth muscle--a finding consistent with the hypothesis that EDRF stimulates guanylate cyclase in the muscle, leading to an increase in cyclic GMP that somehow activates relaxation. Some questions relating to the potential physiological important of endothelium-dependent relaxations are discussed.

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 µM), 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.

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.

Bradykinin elicits relaxation of isolated transverse rings of canine coronary, celiac, superior mesenteric, renal, splenic, pulmonary, gastric, and femoral arteries. After endothelial cells of the vessel wall are removed by rubbing of the intimal surface, canine arteries fail to relax upon addition of bradykinin. The endothelium-dependent relaxation of canine arteries remains intact after treatment with cyclooxygenase inhibitors (indomethacin and flurbiprofen), and this argues against mediation by prostaglandins. When they are stimulated with bradykinin, endothelial cells of canine arteries appear to release a substance mediating vascular smooth muscle relaxation. In contrast, preparations of arteries of cats (superior mesenteric) and rabbits (superior mesenteric and celiac) may be rubbed on the intimal surface without a consistent loss of sensitivity to the relaxing effects of bradykinin. In addition, relaxation of the cat and rabbit arteries is completely blocked by cyclooxygenase inhibitors. Preliminary studies indicate that bradykinin relaxes human arteries in an endothelium-dependent manner and that this effect is not mediated by prostaglandins. We have previously reported that arteries of all species tested require the presence of endothelial cells for relaxation in response to acetylcholine and we have also demonstrated, using the rabbit aorta, that this effect is mediated by the release of an uncharacterized substance from these cells that relaxes vascular smooth muscle. We conclude that bradykinin relaxes canine and human arteries via a similar mechanism but that it relaxes cat and rabbit arteries by stimulating release of prostaglandins from as yet undefined cell types.

1. The electrophoretic mobility of unhemolyzed human red cells has been determined as a function of ionic strength at approximately constant pH in isotonic mixtures of glucose solution and saline-phosphate buffer solution. 2. Above an ionic strength of about 0.02 the cells behave as particles with a smooth surface of large radius of curvature. Below an ionic strength of about 0.02, changes of the surface occur, probably involving a decrease of charge density and perhaps connected with injury of the surface. 3. The mobility as a function of pH at an ionic strength of 0.172 has been determined for human red cells, for the lipid extract of the cells, and for the stroma protein of the cells. The isoelectric points of cells, lipid, and protein have been found to be about 1.7, 2.6, and 4.7 respectively. 4. The pH-mobility data lead to the conclusion that a red cell surface is composed largely of lipid and dominated by strong acid groups, possibly the phosphoric acid groups of cephalin molecules.

1. Experimental evidence has been presented to show that the sphering of mammalian red cells in saline suspension between new slide and cover-glass is a result of these two changes: (1) the p H of the suspension is raised over 9.2 by the alkaline effect of the glass; (2) the glass adsorbs out from the suspension a protein, which, if present, prevents sphering until a p H of about 11.3 is reached, but if absent allows sphering around a p H of 9.2. 2. Anti-sphering factor is contained by both serum and cells. It cannot be adequately removed from cells by washing them with saline, but it can be removed from them by absorption on clean glass surfaces. 3. Changes of cell shape occurring between slide and cover-glass may be duplicated in the bulk of a suspension. 4. The p H and amount of anti-sphering factor present also influence the intermediate crenated shapes between the disk and sphere shapes. 5. Substances from the skin which counteract the activity of the anti-sphering factor may be introduced into a suspension by shaking it against the palm of the hand. Oleic or palmitic acids in a suspension also counteracts the activity of the anti-sphering factor present. 6. Watery ghosts show a peculiar temporary sphering between new slide and cover-glass. 7. The disk-sphere transformation in the absence of anti-sphering factor is reversible. In repeated reversing of the transformation the biconcavities and particular crenations of any one cell always reappear on the same parts of the surface of that cell.

1. Experimental evidence has been presented showing that crystalbumin (the carbohydrate-poor fraction of serum albumin) is the factor which prevents mammalian red blood cells from becoming spherical at pH values over 9.2. 2. The amount of crystalbumin taken up from a solution of it by red cells previously freed of it is of the order of 800 mg. per 100 c.c. of red cells. If this amount is all taken up at the red cell surface, it would form a layer only a few molecules thick. 3. The electrophoretic mobility in phosphate buffer of pH 7.4 is the same for cells containing crystalbumin, cells free of crystalbumin, cells with anti-sphering activity counteracted by fatty acid, and ghosts which have been temporarily sphered by a rise in pH. The mobilities in a saline-glycine buffer solution of pH 10.1 for the first three classes of cells just mentioned are also the same. The mobility of cells sphered with lecithin in a saline-phosphate buffer solution is the same as that for untreated discoidal cells.

Simultaneous recordings were made of action potential ( AP) and contractile strength ( CS) of electrically driven isolated left atria of guinea pigs at 25–27 C under various experimental conditions which alter CS. Reduction of CS produced by decreasing frequency from 1/sec to 0.1/sec, or by spontaneous failure, was associated with only small changes, if any, in AP configuration. The increased CS produced by strophanthin-K or high calcium was associated with shortening of the AP plateau duration; treatment with ryanodine increased AP plateau duration, and decreased CS. The first beat following a 3- to 10-sec rest interval (postrest ( PR) beat) exhibited greater CS, and an AP with shorter plateau and more pronounced "tailing off" than steady-state controls. With high calcium or strophanthin present, PR beats and steady-state beats were nearly identical in both CS and AP. After ryanodine, PR beats were markedly reduced in CS and had prolonged AP plateaus; these changes could be reversed by high calcium or strophanthin. A hypothesis is introduced which accounts for changes in CS under these conditions.

Sympathetic nerve stimulation and intraluminal norepinephrine infusion for more than 15 seconds produced a biphasic response in the isolated rabbit ear artery perfused with Krebs solution. This response consisted of an initial rapid constriction (phase A), which was followed by partial relaxation, and a final slowly developing constriction (phase B), which lasted for the duration of nerve stimulation or norepinephrine administration. Raising the potassium concentration of the Krebs solution to 12mM decreased the relaxation time between the two constrictor phases in response to norepinephrine; lowering the potassium concentration to 1.2 mM increased the relaxation time and decreased the degree of constriction of both phases. Biphasic vasoconstrictor responses were also elicited by the intraluminal infusion of phenylephrine, histamine, serotonin, or 35 m M potassium. When calcium was absent from the perfusing solution or when manganous sulfate (1m M ) was present, norepinephrine produced only a fast phase A constriction, with no subsequent slow phase B constriction. However, after treatment of the artery with ryanodine, the phase A constriction in response to norepinephrine was markedly inhibited, but the phase B constriction was not. We concluded that the fast phase A constriction depends on the release of calcium from an intracellular pool and that the slow phase B constriction depends on the influx of extracellular calcium.

Measurements of the electrical mobility of washed rabbit red cells and of ghosts produced by hypotonic solutions, freezing-and-thawing, chloroform, and saponin were made in the Abramson horizontal microelectrophoresis cell. These different forms of lysis, which corresponds to a variety of degrees of injury to the red cell, are unaccompanied by any change in electrical mobility. These observations are discussed from the standpoint of the possible structure of the cell membrane and the action of lysins upon it.

Relaxation of preparations of mammalian arteries by acetylcholine (ACh) requires the presence of endothelial cells (Furchgott & Zawadzki, 1980; Furchgott et al., 1981). It has been demonstrated that ACh stimulates these cells to produce and release a factor that acts on the smooth muscle cells to cause relaxation (Furchgott & Zawadzki, 1980; Furchgott, 1983). This endothelium-derived relaxing factor (EDRF) has not yet been identified. Indirect evidence suggests that it may be a free radical (Furchgott et al., 1981) or a very labile aldehyde or ketone (Griffith et al., 1984). Relaxation of arteries by some other vasodilators, including the calcium ionophore A23187, also depends on the release of EDRF (for reviews, see Furchgott, 1983, 1984). In contrast, some vasodilators, such as glyceryl trinitrate, do not require endothelial cells to produce relaxation, but act directly on the smooth muscle cells.

In 1980, Furchgott and Zawadzki reported that the relaxation of the smooth muscle of in vitro preparations of rabbit aorta and other arteries induced by acetylcholine and other agonists for muscarinic receptors was dependent on the presence of endothelial cells in the preparations. After complete removal of the endothelial cells, either mechanically or enzymatically, acetylcholine no longer induced any relaxation, but in most arteries, actually caused some contraction at higher concentrations (Figure 1). In the case of isolated rings or strips of rabbit thoracic aorta, the muscarinic receptor mediating relaxation appeared to be the same subtype (M2) as the receptor on the smooth muscle cells mediating contraction (Furchgott et al., 1981; Furchgott, 1986).

The ability of acetylcholine and other muscarinic agonists to produce marked vasodilatation in various vascular beds in vivo was well established many years ago. In view of the potent vasodilating activity of muscarinic agonists in vivo, it was to be expected that these agents would produce relaxation of isolated blood vessels in vitro. More than 13 years ago it was demonstrated in a number of laboratories that acetylcholine and other muscarinic agonists could indeed produce relaxation (dilatation) of isolated perfused or superfused blood vessels contracted by stimulation of perivascular adrenergic nerves (e.g., Rand and Varma, 1970; Hume et al., 1972; Steinsland et al., 1973; Vanhoutte, 1974, 1977). This relaxation was attributed to an action of muscarinic agonists on the muscarinic “inhibitory receptor” of adrenergic nerve endings (Loffelholz and Muscholl, 1969), resulting in inhibition of release of norepinephrine from these terminals. This prejunctional inhibitory action of muscarinic agonists could explain in part the vasodilatation that they produced in innervated vascular beds in vivo; but it could not explain the ability of these agents to still cause marked dilatation in denervated vascular beds. In view of such vasodilatation, it was to be expected that muscarinic agonists would produce relaxation of spontaneous or drug-induced contractions of isolated preparations of blood vessels.

Relaxation of selected isolated arteries by acetylcholine, bradykinin, and certain other vasodilators is mediated by a factor released from endothelial cells. Relaxation by the factor (chemical identity still unknown) is accompanied by an increase of cyclic GMP. The finding that hemoglobin inhibits both endothelium-dependent relaxation and the increase in cyclic GMP may have pathophysiologic relevance.

This paper reviews studies performed by the authors and by others on the levels of high energy phosphate compounds—adenosine triphosphate (ATP), adenosine diphosphate (ADP), and creatine phosphate (CP)—of cardiac muscle under various conditions influencing contractile force. Interference with energy metabolism decreases both contractile force and high energy phosphates, especially CP. Certain types of experimental "failure" are associated with decreases in high energy phosphates; however, other types of "failure" occur without significant decreases in these phosphates. In addition, marked decreases or increases in contractile force, independent of significant changes in high energy phosphates, can be produced by drugs, by changes in heart rate, or by alterations in extracellular concentrations of cations. A decrease in force when levels of high energy phosphate are normal may be attributed to a deficiency in the utilization of these energy stores for mechanical work. The nature of this deficiency has been analyzed in isolated cat papillary muscles by simultaneously determining the activity oxygen consumption and contractile force per beat. In the case of decreases in contractile force due either to reduced heart rate or to spontaneous heart failure, the deficiency may be attributed almost completely to a loss in efficiency in the conversion of chemical energy in the high energy phosphates to mechanical energy (work). Cardiac glycosides, in restoring contractile force, do so by restoring the efficiency of this conversion. In the case of decreases in contractile force resulting from lowered extracellular Ca ++ , the deficiency may be attributed partly to a loss of efficiency in this conversion and partly to a reduction in amount of high energy phosphate utilized per beat.

Publisher Summary This chapter presents a study examining antagonism of propranolol to isoproterenol in guinea-pig trachea. In this study, four helically-cut strips from the trachea of a reserpine-pretreated guinea pig were mounted in Krebs solution at 37°. Each concentration–response curve for relaxation by isoproterenol (ISO) was obtained with cumulative additions of the drug after the smooth muscle had been brought to a steady level of contraction with carbachol. Each experimental strip was first tested with ISO in the absence, and then successively in the presence of one or more concentrations of propranolol (PPL). The exposure time to each PPL concentration was close to 75 min. Concentrations of ISO used for calculating dose ratios were those required to produce one-half maximal relaxation. In all experiments, one strip received no PPL and served as a control. To block extraneuronal uptake of ISO, some strips were pretreated with 3 μg/ml of dibenamine for 40 min. The results provide evidence for the conclusion that the pharmacologic antagonism between agonists and antagonists acting on adrenergic receptors is satisfactorily explained by the classical receptor theory for competitive antagonism.

A wide variety of tissues undergo a change of functional state on exposure to noradrenaline or adrenaline. Those molecular constituents of the effector cells of a tissue with which molecules of these catecholamines must first interact in order to produce a change of state — or response — of the tissue, are the so-called adrenoceptors (also commonly called adrenergic receptors). For convenience, we refer to noradrenaline, adrenaline and other agents which produce responses in tissues by interacting with adrenoceptors, as adrenergic agonists. An agent which specifically inhibits a response produced by an adrenergic agonist is referred to as adrenergic blocking agent or adrenergic antagonist.

SMOOTH muscle of isolated mammalian arteries in a state of active tonic contraction will relax when exposed to light1–3. Other types of mammalian smooth muscles are insensitive to light or much less sensitive than vascular smooth muscle. When other types of smooth muscle are treated with nitrite ion (an ion which increases the photosensitivity of rabbit aortic strips1), they, too, become very sensitive to light. This report deals with the effects of ultraviolet and visible radiation on non-vascular smooth muscle, especially after treatment with NaNO2.