Endothelium-derived relaxing factor and coronary vasospasm.

The endothelium releases the powerful vasodilator and antiaggregatory substance, EDRF, both under basal conditions and upon stimulation by a wide variety of agents. Endothelial injury or dysfunction may play an important role in the spasmogenicity of the coronary artery, although other possible alterations related to atherosclerosis should also be considered. Among the possible stimuli, aggregating platelets are important as a source of vasoconstrictor substances. The endothelium may also produce the vasoactive substances EDHF and EDCF(s). Their pathophysiologic significance remains to be determined.link_to_subscribed_fulltex

Endothelium-derived endothelin-1

One year after the revelation by Dr. Furchgott in 1980 that the endothelium was obligatory for acetylcholine to relax isolated arteries, it was clearly shown that the endothelium could also promote contraction. In 1988, Dr. Yanagisawa’s group identified endothelin-1 (ET-1) as the first endothelium-derived contracting factor. The circulating levels of this short (21-amino acid) peptide were quickly determined in humans, and it was reported that, in most cardiovascular diseases, circulating levels of ET-1 were increased, and ET-1 was then tagged as “a bad guy.” The discovery of two receptor subtypes in 1990, ET(A) and ET(B), permitted optimization of the first dual ET-1 receptor antagonist in 1993 by Dr. Clozel’s team, who entered clinical development with bosentan, which was offered to patients with pulmonary arterial hypertension in 2001. The revelation of Dr. Furchgott opened a Pandora’s box with ET-1 as one of the actors. In this brief review, we will discuss the physiological and pathophysiological role of endothelium-derived ET-1 focusing on the regulation of the vascular tone, and as much as possible in humans. The coronary bed will be used as a running example in this review because it is the most susceptible to endothelial dysfunction, but references to the cerebral and renal circulation will also be made. Many of the cardiovascular complications associated with aging and cardiovascular risk factors are initially attributable, at least in part, to endothelial dysfunction, particularly dysregulation of the vascular function associated with an imbalance in the close interdependence of nitric oxide and ET-1

Ouabain inhibits endothelium-dependent relaxations to arachidonic acid in canine coronary arteries

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Oxygen-derived free radicals, endothelium, and responsiveness of vascular smooth muscle

Experiments were designed to determine the role of oxygen-derived free radicals in modulating contractions of vascular smooth muscle and endothelium-mediated relaxations to acetylcholine. The effects of generating or scavenging these radicals were studied in rings of canine coronary arteries suspended for isometric tension recording. Xanthine oxidase plus xanthine caused relaxations, which were greater in rings with endothelium than in rings without endothelium; the relaxations were not affected by superoxide dismutase or mannitol, but could be prevented by catalase. Xanthine oxidase plus xanthine depressed endothelium-mediated relaxations to acetylcholine; this effect was prevented by superoxide dismutase, but was not affected by catalase or mannitol. Exogenous hydrogen peroxide induced catalase-sensitive relaxations, which were depressed by the removal of the endothelium. Superoxide dismutase evoked catalase-sensitive relaxations only in rings with endothelium. Endothelium-mediated relaxations to acetylcholine were slightly depressed by superoxide dismutase or catalase alone; the combination of the two enzymes or mannitol caused a major shift to the right of the concentration-response curve to acetylcholine. In rings without endothelium, relaxations caused by sodium nitroprusside were not affected by the scavengers (alone or in combination) but were augmented by xanthine oxidase plus xanthine. These data suggest that 1) the endothelium-derived relaxing factor released by acetylcholine is not likely to be an oxygen-derived free radical; 2) hydrogen peroxide has a direct inhibitory action on coronary arterial smooth muscle and triggers endothelium-dependent relaxations; and 3) superoxide anions depress and hydroxyl radicals facilitate endothelium-dependent relaxations caused by activation of muscarinic receptors.link_to_subscribed_fulltex

Calcium and activation of the release of endothelium-derived relaxing factor

Indirect and direct experimental evidence demonstrates that both the entry of extracellular calcium and the liberation of calcium from intracellular stores can contribute to an increase in free cytoplasmic calcium concentration in endothelial cells, which seems to be an essential step in the synthesis and/or release of endothelium-derived relaxing factor(s). A variety of Ca2+ transport mechanisms may be involved in the regulation of cytoplasmic calcium in endothelial cells. Ca2+ entry may occur via voltage-operated Ca2+ channels. If they do exist, these channels may have characteristics different from those in underlying vascular smooth muscle cells. Sustained activation of the release of EDRF by various receptor agonists (e.g., acetylcholine, adenine nucleotides, and bradykinin) is also dependent on Ca2+ entry, but it is insensitive to organic Ca2+ channel antagonists. These findings indicate that, when used clinically in various cardiovascular diseases, organic calcium channel antagonists are not expected to interfere with endothelium-dependent relaxation evoked by endogenous vasoactive substances (e.g., ADP, serotonin). Since amiloride and its analogues blocked endothelium-dependent relaxations in different arterial preparations, Na+ transport and Na+/Ca2+ exchange were suggested to play a role in calcium-dependent release of EDRF. The exact nature of Ca2+ transport mechanisms and also the calcium-sensitive cellular processes that lead to the synthesis/release of endothelium derived relaxing factor(s) remain to be determined. However, the available data suggest that calcium handling by the vascular smooth muscle and endothelial cells may be different, allowing potentially selective modulation of Ca2+ activtion in these two cell types.link_to_subscribed_fulltex

Potassium-free solution prevents the action but not the release of endothelium-derived relaxing factor

Exposure to a K+-free solution reversibly inhibited the acetylcholine-induced relaxation in perfused canine femoral artery segments with endothelium and in superfused bioassay femoral artery rings without endothelium. Infusion of 6.7 mM K+ into the K+-free perfusate downstream of the perfused artery restored the acetylcholine-induced relaxation in the bioassay ring, whereas no relaxation was observed in the perfused segment still exposed to the K+-free solution. These data demonstrate that the K+-free solution depresses the endothelium-dependent relaxation to acetylcholine in femoral arteries by preventing the action, but not the production, of endothelium-derived relaxing factor.link_to_subscribed_fulltex

Potassium-induced release of endothelium-derived relaxing factor from canine femoral arteries

Experiments were designed in a bioassay system to analyze the effect of elevated (from 5.9 mM to 7.5-45.9 mM) extracellular K+ concentration on the release of endothelium-derived relaxing factor. Segments of canine femoral artery with endothelium (donor segment) were mounted in an organ bath and perfused with modified Krebs-Ringer bicarbonate solution; the effluent from the donor segment was used to superfuse a canine coronary artery ring without endothelium (bioassay tissue). Elevation of perfusate K+ concentration by 1.6-15 mM by intraluminal infusion of potassium chloride upstream of the donor segment evoked further contractions of bioassay rings contracted with prostaglandin F(2α). In contrast, the bioassay rings progressively relaxed when increasing concentrations of potassium chloride (10-40 mM) were added extraluminally to the organ bath where the perfused segment was mounted. Extraluminal application of phenylephrine or prostaglandin F(2α) did not evoke relaxations in the bioassay ring. Removal of the endothelium from the donor segment or selective exposure of the segment (but not the bioassay ring) to Ca2+-deficient solution prevented the K+-induced relaxations. Treatment of the donor segment and the bioassay ring with inhibitors of known endogenous vasoactive substances (acetylcholine, norepinephrine, adenine nucleotides, and prostanoids) had no significant effect on the relaxation of the bioassay ring evoked by extraluminal application of potassium chloride. Simultaneous measurements of changes in isometric force in the donor segment and bioassay ring revealed that extraluminal elevation of K+ concentation relaxed the segments as well and that the relaxations could not be prevented by simultaneous intraluminal infusion of potassium chloride. Prolongation of the transit time between the donor segment and bioassay ring from 1 to 6 seconds depressed the relaxations of the bioassay tissue evoked by extraluminal elevation of K+ concentrations that could be prevented by superoxide dismutase (infused downstream of the donor segment). These findings indicate that K+ can stimulate the release of endothelium-derived relaxing factor from perfused canine femoral arteries but only when applied extraluminally. It is postulated that K+ triggers the release of a still unidentified mediator in the blood vessel wall that stimulates the release of the relaxing factor from the endothelial cells.link_to_subscribed_fulltex

Heterogeneity of endothelium-dependent responses to acetylcholine in canine femoral arteries and veins. Separation of the role played by endothelial and smooth muscle cells

The purpose of this study was to determine whether heterogeneity in endothelium-dependent responses to acetylcholine between canine blood vessels of different anatomical origin reflects variations in endothelial function or in responsiveness of vascular smooth muscle cells. Experiments were conducted in a bioassay system, where segments of femoral artery or vein with endothelium were perfused intraluminally and the perfusate used to superfuse rings of femoral arteries or veins without endothelium. Indomethacin was present in all experiments to prevent the synthesis of prostanoids. The blood vessels were contracted by phenylephrine. Measurement of wall tension in both the perfused segment and bioassay ring allowed simultaneous detection of endothelium-derived relaxing factor(s) released abluminally (segment) and intraluminally (ring). Intraluminal infusion of acetylcholine (ACh) induced relaxations in the perfused artery but not in vein segments. During arterial superfusion ACh induced relaxation in femoral arterial rings but contraction in venous rings. After treatment with atropine the arterial perfusate evoked relaxations in venous rings. Infusion of ACh through the femoral vein evoked only moderate relaxations in arterial rings. These data demonstrate that depressed endothelium-dependent relaxation to ACh in femoral veins compared to femoral arteries is due to a masking effect of the direct stimulating action of ACh and decreased release of the same mediator or the release of a different relaxing factor from venous endothelium.link_to_subscribed_fulltex

Nature of endothelium-derived relaxing factor: Are there two relaxing mediators?

The role of arachidonic acid in forming endothelium-derived relaxing factor remains controversial. This controversy may be explained if more than one factor exists. To test this hypothesis, the effects of various inhibitors of arachidonic acid metabolism were studied. The perfusate from canine femoral arteries with endothelium was bioassayed with coronary artery rings without endothelium. Treatment of the perfused segment (but not the bioassay ring) with inhibitors of phospholipase A2 (quinacrine) or cytochrome P450 (metyrapone) had no effect on the basal relaxing activity of the effluent; treatment with the inhibitor of lipoxygenase, nordihydroguaiaretic acid, significantly depressed it. With increasing concentrations of acetylcholine, a biphasic concentration-relaxation curve was obtained; the enzyme inhibitors depressed or prevented the first phase but affect the second phase. Infusion of arachidonic acid or soybean lipoxidase directly on the bioassay ring did not cause relaxation; together, they evoked concentration-dependent relaxations. These data suggest that acetylcholine can trigger the release of two chemically different relaxing mediators from the endothelium of the canine femoral artery. One factor may be a product of lipoxygenase (or epoxigenase). The second factor is not a metabolite of arachidonic acid and may be released under basal conditions. The existence of two (or more) chemically different endothelium-derived mediators may help to explain the controversial data regarding the nature of the factor(s).link_to_subscribed_fulltex

Ouabain inhibits endothelium-dependent relaxations to arachidonic acid in canine coronary arteries

Experiments were designed to analyze the effects of ouabain on the actions of exogenous arachidonic acid on endothelial and vascular smooth muscle cells. Rings or strips were prepared from left circumflex canine coronary arteries and suspended for isometric tension recording in organ chambers filled with oxygenerated modified Krebs-Ringer-bicarbonate solution. During contractions evoked by prostaglandin F(2α), arachidonic acid caused relaxations both in the presence and the absence of endothelium. However, removal of the endothelium reduced its inhibitory action. Indomethacin prevented the relaxations in rings without endothelium, but did not affect the response to high doses (10-6 to 10-5 M) of arachidonic acid in preparations with endothelium. The inhibitor of lipoxygenase, nordihydroguaiaretic acid, had no effect on the inhibitory responses to arachidonic acid in rings with or without endothelium. Ouabain abolished both the endothelium-dependent and the direct relaxations to arachidonic acid. Endothelium-dependent relaxations in response to oleic acid, elaidic acid, adenosine diphosphate and thrombin were not affected by ouabain. In the presence of indomethacin, coronary artery strips without endothelium were relaxed by arachidonic acid only when layered (intimal surface against intimal surface) with a longitudinal strip with endothelium. In layered preparations, treatment of the intact longitudinal strip with ouabain before layering prevented the relaxation, whereas pretreatment of the strip without endothelium had no effect. These experiments demonstrate that: 1) arachidonic acid stimulates the release of a relaxing factor from the endothelial cells of canine coronary arteries which differs from prostaglandins; 2) ouabain prevents this effect by acting on endothelial cells; and 3) the selective effect of ouabain on endothelium-dependent relaxations induced by arachidonic acid indicates that it either stimulates the release of a factor different from that produced by other endothelium-dependent relaxing substances and/or causes the release of the same relaxing mediator by different means.link_to_subscribed_fulltex