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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.

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.

Last month, 70 members of the U.S. Congress, including Henry Hyde, Chairman of the House Judiciary Committee, and J. C. Watts Jr. Republican Conference Chairman, signed a letter urging the federal government to ban all research on stem cells obtained from human embryos and fetuses. The letter calls upon the U.S. Department of Health and Human Services (DHHS) to reverse National Institutes of Health (NIH) Director Harold Varmus's decision to allow funding of pluripotent stem cell research. The lawmakers object “in the strongest possible terms” to Varmus's decision, as well as to the memorandum issued in January by DHHS General Counsel Harriet Rabb, which served as the legal basis for Varmus's position. In their letter, the members of Congress state, “Any NIH action to initiate funding of such research would violate both the letter and spirit of the federal law banning federal support for research in which human embryos are harmed or destroyed.” Federal laws and regulations, they claim, have protected human embryos and fetuses “from harmful experimentation at the hands of the Federal government” for more than two decades. “This area of law has provided a bulwark against government's misuse and exploitation of human beings in the name of medical progress. It would he a travesty for this Administration to attempt to unravel this accepted ethical standard.”

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 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.

Several years ago we accidentally discovered that relaxation of isolated prepa­ rations of arteries (rings, transverse strips, or helical strips) by acetylcholine (ACh) was strictly dependent on the presence of endothelial cells on the intimal surface of the preparations (1-3). This discovery helped resolve the paradox that ACh, a potent vasodilator of arteries in vivo, often produced no relaxation or even contraction of isolated preparations of arteries in vitro (4, 5). Apparent­ ly those isolated preparations that had failed to relax had had their endothelial cells unintentionally rubbed off during the course of their preparation for experiments. This discovery also led to the finding that a number of other agents, including some but not all of the most potent known endogenous vasodilators, also require endothelial cells to produce relaxation in isolated arteries. A number of laboratories are now engaged in research on various aspects of the endothelium-dependent relaxation of blood vessels by various agents. This review attempts to bring together the more important findings in this new area of research. A fuller description of early experimental results in this area obtained by the author and others is available in another recent review (6). The reader is also referred to that review for a discussion of a postulated role for endothelial cells in mediating or facilitating contractions of some blood vessels under special 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.

Pharmacological evidence is presented supporting the theory that the increase in contractile strength of isolated cardiac muscle under suprathreshold stimulation is due to the release of an adrenergic mediator (norepinephrine). However, release of this material does not account for changes in contractile strength associated with changes in frequency of stimulation at threshold levels.