Vanhoutte, Paul M.

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.— F urchgott , R. F.; V anhoutte , P. M. Endothelium‐derived relaxing and contracting factors. FASEB J. 3: 2007‐2018; 1989.

Chronic treatment with estrogens enhances some endothelium-dependent relaxations. Whether or not progesterone would exert a similar effect is unknown. Experiments were designed to determine the effect of chronic treatment with progesterone on endothelium-dependent responses. Adult female dogs were ovariectomized and pellets containing carrier substance, estrogen, progesterone, or estrogen plus progesterone were implanted subcutaneously. After 14-21 days coronary arteries were removed, cut into rings, and suspended for the measurement of isometric force in organ chambers in the presence of indomethacin. Endothelium-dependent relaxations to ADP, bradykinin, or the calcium ionophore did not differ among groups. However, relaxations to acetylcholine and to the alpha 2-adrenergic agonist BHT-920 were greater in the estrogen-treated group than in the estrogen plus progesterone-treated group. In rings without endothelium, relaxations to nitric oxide and isoproterenol did not differ among groups. However, relaxations of the smooth muscle to ADP were greater in the progesterone-treated group than in the progesterone plus estrogen group. These results suggest that progesterone alone minimally affects endothelium-dependent responses. However, progesterone seems to antagonize the stimulatory effects of estrogen on two endothelium-dependent responses that are associated with pertussis toxin-sensitive guanine nucleotide regulatory proteins and the production of nitric oxide. These studies suggest that a specific receptor/second messenger system can be modulated by female reproductive steroid hormones.

Experiments were designed to determine the effects of chronic estrogen treatment on endothelium-dependent responses to arachidonic acid in the aorta of the rabbit. Ovariectomized rabbits were treated with either placebo or 17 beta-estradiol for 14 days. The aortas were removed, and rings cut from each blood vessel were suspended for measurement of isometric force. In rings contracted with a submaximal concentration of norepinephrine, endothelium-dependent contractions to arachidonic acid were augmented by estrogen treatment. Prostaglandin F2 alpha, E2, the thromboxane analogue U 46619, and prostacyclin contracted aortic smooth muscle. Only the contractions to prostacyclin were augmented by estrogen treatment. Estrogen treatment enhanced the contractions to norepinephrine in aortas with, but not in those without, endothelium; these contractions were not affected by inhibition of uptake of the neurotransmitter. Indomethacin inhibited contractions to norepinephrine in rings with endothelium from estrogen-treated rabbits. These results indicate that chronic treatment with estrogens can affect arterial reactivity to norepinephrine through an endothelium-dependent mechanism that may involve the metabolism of arachidonic acid by cyclooxygenase. The altered sensitivity of the smooth muscle to prostacyclin may contribute in part to the augmented contractions to arachidonic acid with estrogen treatment.

This study was designed to investigate whether the respiratory epithelium can modulate the tone of the underlying smooth muscle in response to decreases in partial pressure of O2 (PO2). Canine bronchial segments with or without epithelium (diameter, 4-6 mm; length, 50-60 mm) were mounted in organ chambers and perfused intraluminally with modified Krebs-Ringer bicarbonate solution [temperature, 37 degrees C; PO2 varying from 600 (control) to 40 mmHg; PCO2, 36 mmHg]. Isometric tension was recorded by means of stirrups passed through the wall of the central part of the bronchial segment. During contractions to carbachol, the tissues with epithelium showed epithelium-dependent relaxations when the PO2 was decreased. The level of relaxation was dependent on the PO2. The epithelium-dependent relaxation could not be blocked by the following agents: indomethacin, methylene blue, propranolol, or tetrodotoxin (antagonists or blockers of cyclooxygenase, guanylate cyclase, beta-adrenoceptors, and sodium channels, respectively). The epithelium-dependent relaxation was not accompanied by the release of an assayable relaxing factor in the bronchial lumen. The experiments suggest that 1) lowering the PO2 induces the epithelium to release a relaxing factor(s), which is neither a product of cyclooxygenase nor endothelium-derived relaxing factor; 2) a local reflex mechanism is not involved in the phenomenon; and 3) the relaxing factor(s) either is not released into the bronchial lumen or, if it is, is catalyzed rapidly in the lumen on release.

Exercise is associated with release of catecholamines and vasoactive intestinal polypeptides. Recurrent exposure to catecholamines modifies the sensitivity of adrenoceptors. To test the hypothesis that exercise training may affect the sensitivity of the epicardial coronary arteries, we performed studies on isolated coronary arteries from male dogs capable of running on a treadmill. The animals were separated randomly into two groups: sedentary and exercise training. After 11 wk, rings of left circumflex and left anterior descending coronary arteries were studied in vitro. Contractions to alpha 1-adrenergic agonists (norepinephrine and phenylephrine) were not affected by exercise training. During contractions with prostaglandin F2 alpha, endothelium-dependent relaxations to alpha 2-adrenergic agonists (norepinephrine and UK 14304) were not reduced significantly by exercise training. The concentration-relaxation curves to beta-adrenergic agonists (norepinephrine, isoproterenol, and epinephrine) were shifted to the right after training. The concentration-response curves to vasoactive intestinal polypeptide, but not that to substance P, were shifted to the right in rings with endothelium from exercise-trained animals. These findings demonstrate a decrease in responsiveness of canine vascular smooth muscle to beta-adrenergic agonists and to vasoactive intestinal polypeptide after exercise training.

The purpose of this study was to characterize the adrenergic and cholinergic responsiveness of isolated rings of canine bronchial arteries. Electrical stimulation of the vessels produced frequency-dependent contractions that were inhibited by phenoxybenzamine, tetrodotoxin, and phentolamine but not by atropine. Norepinephrine caused concentration-dependent contractions of the rings; the concentration-response curve to norepinephrine was shifted to the right by phentolamine and prazosin, whereas rauwolscine had no effect. Isoproterenol, norepinephrine, tyramine, and electrical stimulation (in the presence of phentolamine) failed to elicit relaxation in rings contracted with prostaglandin F2 alpha. Contracted rings relaxed to acetylcholine in an endothelium- and concentration-dependent manner. At concentrations up to 10(-4) M, acetylcholine did not evoke contractions in these tissues. Endothelium-dependent relaxation to acetylcholine was inhibited by atropine and 4-diphenyl-acetoxy-N-methyl piperidine methiodine but not by pirenzepine. These results suggest that alpha 1-adrenoceptors mediate contraction to norepinephrine, and M3-muscarinic receptors mediate endothelium-dependent relaxation to acetylcholine in the canine bronchial artery. Moreover, the smooth muscle of these vessels does not respond directly to beta-adrenergic- or cholinergic-receptor stimulation.

Experiments were designed to compare the reactivity of canine basilar arteries to endothelium-derived relaxing factor (EDRF) and nitric oxide. Preparations with endothelium activated by bradykinin were used to study relaxations induced with EDRF, whereas the inhibitory effect of nitric oxide was studied in preparations without endothelium. All experiments were performed in the presence of indomethacin. EDRF- and nitric oxide-induced relaxations were significantly augmented in the presence of superoxide dismutase plus catalase but were reduced in the presence of methylene blue, LY 83583, and hemoglobin. M & B 22984 did not affect relaxations to either EDRF or nitric oxide. These results indicate that in the canine basilar artery EDRF is not an oxygen-derived free radical. The similar responsiveness of the basilar artery to EDRF and nitric oxide is consistent with the proposal that in the canine basilar artery nitric oxide is the factor.

Experiments were designed to analyze the effects of endothelium-derived relaxing factor(s) (EDRF; released basally or on stimulation with acetylcholine) and nitric oxide (NO) on smooth muscle of coronary arteries of different diameter. During contractions of the bioassay ring evoked with prostaglandin F2 alpha, the relaxations caused by basal EDRF were greater in the distal than in the proximal coronary arteries, whereas there was no difference in response to the EDRF released by acetylcholine. During direct superfusion, NO caused similar relaxations in proximal and distal coronary artery rings. Optimal tension, prostaglandin F2 alpha-induced contractions, and relaxations caused by sodium nitroprusside were comparable in both preparations. In rings of proximal and distal coronary artery studied in organ chambers, acetylcholine caused comparable endothelium-dependent, whereas sodium nitroprusside and NO cause comparable endothelium-independent relaxations. These experiments indicate a difference in response of different-sized coronary arteries to basally released EDRF and suggest that the basally released factor differs from NO.

The calcium ionophore A23187 causes endothelium-dependent contractions in canine basilar arteries. Removal of the endothelium, or treatment with indomethacin or superoxide dismutase (SOD), prevented the endothelium-dependent excitatory effect of the calcium ionophore. Catalase and deferoxamine were without effect. Superoxide anion generated by xanthine plus xanthine oxidase in the presence of catalase caused contractions of the vascular smooth muscle, which were abolished by SOD or heat inactivation of xanthine oxidase. The A23187-induced production of prostaglandins F2 alpha and E2 and thromboxane B2 was abolished by the removal of endothelium and by treatment with indomethacin but was not affected by the presence of SOD plus catalase. These observations are consistent with the hypothesis that superoxide anion, rather than prostaglandins generated by hydroperoxidase activity of cyclooxygenase, is an endothelium-derived contracting factor in canine cerebral arteries.

In canine systemic veins, in contrast to what is observed in mammalian systemic arteries, endothelium-dependent relaxations to the calcium ionophore A23187 are not diminished by the inhibitor of soluble guanylate cyclase, methylene blue. Therefore, experiments were designed to determine whether these relaxations in the veins are associated with the accumulation of guanosine 3',5'-cyclic monophosphate (cGMP). Rings of canine femoral arteries and veins with and without endothelium were suspended for the measurement of isometric force in organ chambers; cGMP was measured by radioimmunoassay. In arteries and veins contracted with norepinephrine, the tissue content of cGMP was greater in rings with than without endothelium. This difference was decreased by methylene blue (10(-5) M). A23187 (3 X 10(-7) M, for 1 min) increased the accumulation of cGMP, which was temporally related with the onset of relaxation in tissues with endothelium. Methylene blue inhibited the accumulation of cGMP in both blood vessels but inhibited the relaxations only in the arteries. In rings without endothelium, sodium nitroprusside (3 X 10(-7) and 10(-5) M) initiated increases in cGMP, which followed the onset of relaxation. Neither response to sodium nitroprusside was reduced by methylene blue. These results suggest that in canine femoral arteries and veins, relaxation of the smooth muscle to sodium nitroprusside are mediated by a mechanism distinct from changes in cGMP. Likewise, in canine systemic veins, endothelium-derived factor(s) released in response to A23187 also can initiate relaxation of the smooth muscle by a mechanism distinct from changes in cGMP.