Endothelium-derived nitric oxide relaxes nonvascular smooth muscle

A bioassay cascade superfusion procedure was used to compare and contrast the actions of arterial and venous endothelium-derived relaxing factor (EDRF) with authentic nitric oxide (NO) on several nonvascular smooth muscle preparations. EDRF was released from human umbilical vein or bovine pulmonary artery by A23187 and allowed to superfuse two nonvascular and one vascular precontracted smooth muscle strips arranged in a cascade. NO or S-nitroso-N-acetylpenicillamine was delivered by superfusion. Both EDRF and NO relaxed bovine trachea, although artery was 10 times more sensitive than trachea to either relaxant. Similarly, rabbit taenia coli and rat fundus relaxed in response to high concentrations of NO or large amounts of EDRF released from umbilical vein. Vascular and nonvascular relaxant responses to both EDRF and NO were inhibited by oxyhemoglobin, methylene blue or superoxide, and were enhanced by superoxide dismutase. Perfusion of pulmonary artery or umbilical vein with A23187 resulted in contraction of guinea pig ileum and relaxation of pulmonary artery, whereas NO relaxed both preparations. Oxyhemoglobin enhanced the contractile and abolished the relaxant responses. Thus, ileum is more sensitive to endothelium-derived contracting factor(s) than to EDRF. NO raised cyclic GMP levels in all smooth muscle preparations, but a greater fold increase was observed in artery than in nonvascular smooth muscle. EDRF released from human umbilical vein was identified chemically as NO or a nitroso compound, as was done previously for EDRF from bovine pulmonary artery and vein. These observations support the view that one EDRF from artery and vein is NO or a labile nitroso compound.     

Role of endothelium-formed nitric oxide on vascular responses

1. Endothelial cells of blood vessels generate factors which can modulate underlying smooth muscle tone, inducing vasorelaxation, (endothelium-derived relaxing factor, EDRF, and endothelium-derived hyperpolarizing factor) and/or vasoconstriction (endothelium-derived contracting factors, EDCFs, including the peptide endothelin). 2. EDRF is nitric oxide (NO) or a RNO compound from which this oxide is released. Its half-life is very short (6-50 sec), and it produces rapid vasodilations and inhibits platelet aggregation. 3. NO is formed from the terminal guanidino of L-arginine, but not of D-arginine. NO effects and NO formation are inhibited by NG-monomethyl-L-arginine (L-NMMA), but not by D-NMMA. These inhibitory effects are blocked by L-arginine. 4. Removal of endothelium or pathological situations that can induce endothelial dysfunction (atherosclerosis, diabetes, hypertension or subarachnoid hemorrhage) cause increases on the vascular contractility elicited by agonists (noradrenaline, serotonin, EDCFs, etc.). These findings suggest that EDRF produces a physiological inhibitory modulation of vascular smooth muscle tone and its alteration produces or facilitates the development of diseases such as hypertension or coronary and cerebral vasospasm.     

Influence of haemoglobin and erythrocytes on the effects of EDRF, a smooth muscle inhibitory factor, and nitric oxide on vascular and non-vascular smooth muscle.

1. The relaxant action of endothelium-derived relaxing factor (EDRF), the smooth muscle inhibitory factor (IF) isolated from the bovine retractor penis (BRP), nitric oxide (NO) and sodium nitroprusside (NaNP) on four vascular and non-vascular smooth muscle preparations has been examined. Their sensitivity to EDRF, the IF and NO was the same, suggesting all might be NO. Sodium nitroprusside produced complete relaxation of the rat anococcygeus at low doses, suggesting an action additional to the intracellular release of NO. 2. Haemoglobin added to solutions of EDRF, activated IF or NO completely removed their relaxant properties, consistent with all three acting by virtue of NO. 3. Suspensions of red blood cells with a haemoglobin concentration equivalent to to that used in the previous experiments were as effective as haemoglobin in abolishing the relaxant effect of EDRF or NO but were ineffective against the activated IF. 4. The similarity in sensitivity of a series of smooth muscles and the binding by haemoglobin are consistent with NO being the active principle of both EDRF and the acid activated IF. The abolition of the effect of EDRF by red blood cells (RBCs) is further confirmation for this hypothesis, but the ineffectiveness of RBCs against acid-activated IF suggests that either the latter is not NO or that it is bound in a way which makes it unable to diffuse through cell membranes.     

Comparison of the survival of endothelium-derived relaxing factor and nitric oxide within the isolated perfused mesenteric arterial bed of the rat.

1. The survival of the endothelium-derived relaxing factor (EDRF) released by acetylcholine (ACh) in the rat isolated perfused mesenteric vascular bed was compared to that of exogenously applied nitric oxide (NO) by direct bioassay of the effluent from the mesenteric bed on rabbit aortic strips (RbAs). 2. NO (0.1-10 nmol) produced dose-related vasodilatations in the mesenteric vascular bed and also survived in the effluent in concentrations sufficient to provoke relaxations of the RbAs. ACh (1.7 pmol-300 nmol) caused dose-related vasodilatations in the mesenteric bed but no EDRF was detected in the effluent. This was true even when ACh provoked much larger vasodilatations in the mesenteric bed than those to doses of NO that survived passage through the bed. 3. Removal of the endothelial cells in the mesenteric vascular bed abolished vasodilatations in response to ACh but did not significantly affect those to NO. Survival of NO through the mesenteric bed was not increased. 4. Superoxide dismutase (SOD, 10 u ml-1) did not significantly affect the vasodilator effects of either ACh or NO. It did increase the survival of NO through the mesenteric vascular bed but native EDRF was still not detected in the effluent. 5. The absence of bioassayable EDRF in the effluent following ACh-induced vasodilatations might be explained by the volume into which EDRF is released abluminally being much smaller than that into which it is released luminally.     

Comparison of relaxation responses of vascular and non-vascular smooth muscle to endothelium-derived relaxing factor (EDRF), acidified sodium nitrite (NO) and sodium nitroprusside

Summary Smooth muscle relaxant activity of endothelium-derived relaxing factor (EDRF) released from columns of cultured bovine endothelial cells by bradykinin (0>1 — 3 nmol/l) was measured in four non-vascular preparations: guinea-pig taenia caeci, guinea-pig trachea, rat stomach (fundus) and rat anococcygeus. Each preparation was contracted to a steady level of force with a variety of agonists such that they relaxed optimally to sodium nitroprusside (SNP). The EDRF-induced relaxations in each preparation were compared with those obtained in de-endothelialized ring preparations of greyhound coronary artery by means of paired bioassays run in parallel. EDRF released from the endothelial cell columns caused 80–100% relaxation of the coronary artery, 40–80% in the guinea-pig taenia caeci, 50–70% in the rat anococcygeus, 5–8% in the guinea-pig trachea and was undetectable in the rat stomach strip. By comparison, SNP caused maximal relaxation in all tissues compared with the coronary artery.In separate organ bath experiments the sensitivity to nitric oxide (NO: generated by adding acidified solutions of NaNO₂) and SNP was compared in each preparation. SNP caused maximal relaxation in all tissues with the following order of potency: dog coronary artery > guinea-pig trachea > guinea-pig taenia = rat anococcygeus > rat stomach strip. In contrast, the concentration of acidified NaNO₂ (NO, 300 nmol/l) that caused 96 ± 4% relaxation in the dog coronary artery caused 84 ± 7% and 48 ± 1 % relaxation in the taenia and anococcygeus respectively. No response attributable to NO was detected in either the trachea or rat stomach strip.In conclusion, EDRF relaxed both vascular and certain non-vascular smooth muscles with the same order of potency and magnitude as NO generated from acidified solutions of NaNO₂. This correlation provides further evidence that NO and EDRF are similar, if not identical. Send offprint requests to T. M. Cocks at the above address     

Species and vascular bed responses of veins and arteries to endothelial-derived relaxing factor in dog and pig

The vascular endothelium is a source of a mediator of vascular smooth muscle (VSM) relaxation known as endothelial-derived relaxing factor (EDRF). Endothelial-dependent relaxation is dependent on the type of vessel under study (artery vs. vein) and on the vascular site from which the blood of vessels are derived. EDRF is released by bradykinin, adenosine, and adenosine triphosphate, substance P, serotonin, arachidonic acid, thrombin, and other relaxant substances. EDRF appears to be a labile substance with a six-second half-life that contains a labile ketone or carbonyl moiety in its active site. EDRF appears to be selectively inhibited by ouabain, pyruvate, and metabolic inhibitors and inactivated by potassium and sodium borohydride. EDRF appears to act by stimulating an increase in cyclic GMP in the smooth muscle cells of porcine tissues and possibly by other mechanisms in canine tissues. Canine veins possess receptors for porcine aortic EDRF but may not make EDRF itself or may have a different endothelial-derived substance than canine arteries. Little is known concerning the factors that release or affect the action of EDRF, its mechanism of action or its chemical composition, especially in the pulmonary vasculature. The differences between release of EDRF from artery and vein and the responses to, and composition of, EDRF in vein and artery remain an area for further research.     

EDRF generation and release from perfused bovine pulmonary artery and vein

A bioassay cascade superfusion procedure was utilized to study the release and effects of endothelium-derived relaxing factor (EDRF) from isolated bovine pulmonary artery and vein. Three precontracted, endothelium-denuded strips of pulmonary artery or vein mounted in series served as the detector of EDRF. Perfusion of artery with acetylcholine, bradykinin or A23187, in the presence of indomethacin, caused the release of EDRF (t1/2 = 3-5 s) which relaxed superfused strips of both artery and vein. Similarly, perfusion of vein with bradykinin or A23187, but not acetylcholine, caused the release of EDRF (t1/2 = 3-5 s) which relaxed superfused strips of both vein and artery. EDRF-elicited vascular smooth muscle relaxation was associated with cyclic GMP accumulation in the superfused strips. EDRF activity was increased and its duration prolonged by superoxide dismutase or low oxygen tension, whereas oxyhemoglobin and methylene blue were markedly inhibitory. The similar patterns of release, instability, and properties of EDRF from intact artery and vein suggest that arterial and venous EDRF are closely similar.     

Tracheal epithelium releases a vascular smooth muscle relaxant factor: demonstration by bioassay

Acetylcholine caused no relaxation of a rubbed strip from rabbit aorta (RASR) which was precontracted with phenylephrine. When the same strip was taken into epithelium-intact guinea-pig trachea, acetylcholine produced a slow-developing relaxation which was antagonized by atropine but not by propranolol, indomethacin, theophylline or hydroquinone. RASR was not relaxed by acetylcholine when it was taken from epithelium-rubbed trachea. The results indicate that acetylcholine releases vascular smooth muscle relaxant factor from tracheal epithelium.     

Discrimination by the NO-trapping agent, carboxy-PTIO, between NO and the nitrergic transmitter but not between NO and EDRF.

1. The effects of carboxy-PTIO, a scavenger of free radical nitric oxide (NO), were studied on endothelium-dependent relaxations of rat aorta and nitrergic nerve stimulation-induced relaxations of anococcygeus muscle and gastric fundus strips to test the hypothesis that endothelium-derived relaxing factor (EDRF) and the transmitter released by nitrergic nerves is free radical NO. 2. Carboxy-PTIO (10-300 microM) produced concentration-dependent reductions of relaxations elicited by exogenous NO, and relaxations mediated by EDRF released by acetylcholine and ATP in rings of rat aorta. The inhibitory effect of carboxy-PTIO was removed by washing the tissues. 3. In the rat anococcygeus muscle, carboxy-PTIO (10-300 microM) produced concentration-dependent reductions of relaxations to exogenous NO; however, in concentrations up to 2000 microM it did not reduce relaxations elicited by nitrergic nerve stimulation (1-2 Hz), in fact, concentrations of 300 microM or more slightly enhanced them. 4. In rat gastric fundus strips, carboxy-PTIO (100 and 300 microM) reduced relaxations to exogenous NO, but relaxations elicited by stimulation of the nitrergic component of non-adrenergic, non-cholinergic nerves were not affected. 5. These results suggest that EDRF is free radical NO and may be designated EDNO, but the transmitter released from nitrergic nerves does not appear to be identical to EDNO and may not be free radical NO.     

Endothelium-Derived Nitric Oxide: Pharmacology and Relationship to the Actions of Organic Nitrate Esters

Vascular smooth muscle relaxation elicited by various endogenous substances results from their interaction with vascular endothelial cells to trigger the formation of endothelium-derived relaxing factor (EDRF). EDRF from pulmonary and peripheral arteries and veins and from cultured and freshly harvested aortic endothelial cells has been identified pharmacologically and chemically as nitric oxide (NO) or a labile nitroso compound. Endothelium-derived NO (EDNO) and authentic NO activate the cytoplasmic form of guanylate cyclase by heme-dependent mechanisms and thereby stimulate intra-cellular cyclic GMP accumulation in cells including vascular smooth muscle and platelets. Cyclic GMP functions as a second messenger to cause vascular smooth muscle relaxation and inhibition of platelet aggregation and adhesion to vascular endothelial surfaces. EDNO is synthesized from L-arginine and perhaps arginine-containing peptides by an unidentified calcium-requiring process coupled to the occupation of extracellular endothelial receptors. The biological actions of EDNO are terminated by spontaneous oxidation to NO₂ ^– and NO₃ ^–. The biological half-life of the very lipophilic EDNO is only 3–5 sec and this allows EDNO to function locally as an autacoid. Nitroglycerin and other organic nitrate esters elicit endothelium-independent relaxation after entering vascular smooth muscle cells and undergoing denitration and formation of NO. The pharmacological actions of nitroglycerin are therefore essentially the same as those of EDNO, and the endogenous NO receptor is the heme group bound to soluble guanylate cyclase. EDNO may serve a biological role to modulate local blood flow and platelet function.     

Human red blood cells inhibit endothelium-derived relaxing factor (EDRF) activity

We have used a bioassay-cascade system to investigate the inhibitory effects of human red blood cells on EDRF activity. The vascular smooth muscle relaxant effect of EDRF released from an endothelium-intact pig coronary artery by the calcium ionophore A23187 was inhibited by washed red cells. This inhibition of EDRF activity by red cells was similar to that expected from their haemoglobin content. This study provides further evidence that in vivo EDRF acts as a local autocoid.     

Increased vasodilator response to acetylcholine of renal blood vessels from diabetic rats

The vasodilator effect of acetylcholine (ACh) and nitroprusside and the vasoconstrictor effect of noradrenaline was assessed in the perfused kidney of streptozocin diabetic rats. Compared with control animals injected with acidified saline, the renal vasoconstrictor effect of noradrenaline was increased in diabetic rats both in terms of the dose required to produce 50% of the maximal effect (EC50) and in the maximal response achieved. The renal vasodilator effect of ACh (but not nitroprusside) was similarly enhanced in diabetic animals. The effect of ACh (but not nitroprusside) in the perfused kidney of both control and diabetic rats was reduced or abolished by mepacrine (10 microM), metyrapone (10 microM) or methylene blue (100 microM) suggesting that ACh exhibits vasodilator activity in the rat kidney by virtue of releasing endothelium derived relaxing factor (EDRF). These results are in contrast to previous published reports demonstrating reduced biosynthesis of EDRF in the aorta of diabetic rats. The mechanism which underlies the increased renal vascular response to ACh is not known. However, increased endothelial cell turnover or cholinoceptor number, elevated activity of enzyme(s) which synthesis EDRF or hyperresponsiveness of vascular smooth muscle to released EDRF should all be considered.     

Vascular smooth muscle sensitivity to endothelium-derived relaxing factor is different in different arteries.

1. The relaxation responses of pre-constricted pig coronary artery (PCA) and rabbit aorta (RA) without endothelium, to endothelium-derived relaxing factor (EDRF) released from either a PCA or RA with intact endothelium have been studied by use of a bioassay cascade system. Effects of EDRF have been compared with sodium nitroprusside (NaNP) and 8-bromo-cyclic GMP. 2. The time course of changes in cyclic GMP levels in response to EDRF in PCA and RA have also been studied. 3. EDRF (released from a PCA or RA) caused significantly greater relaxation in the PCA than the RA, whether 5-hydroxytryptamine or high extracellular potassium was used as the constrictor agonist. 4. These differences in sensitivity to EDRF were paralleled by NaNP but not 8-bromo-cyclic GMP. 5. Cyclic GMP levels peaked earlier in the RA (30s) than in the PCA (180s) but the peak levels were significantly greater in the PCA (2.45 fold) than the RA (1.48 fold). 6. These data show that the previously described differences in EDRF activity between different artery types can be explained in part by differences in the responsiveness of the smooth muscle to EDRF.     

ENDOTHELIUM-DERIVED RELAXING FACTOR RELEASED FROM CULTURED CELLS: DIFFERENTIATION FROM NITRIC OXIDE

1. Endothelium-derived relaxing factor (EDRF) is an extremely labile mediator thought to be identical to nitric oxide (NO). 2. A cascade superfusion technique was used to bioassay EDRF released from bovine aortic endothelial cells grown to confluence on microcarrier beads. 3. Bradykinin (1-100 nmol/l), infused through a 1 cm column of endothelial cells on microcarriers, released an EDRF-like substance that caused relaxations of de-endothelialized strips of rabbit aorta (precontracted with phenylephrine). These relaxations diminished on successive tissues in the cascade, when compared with those produced by glyceryl trinitrate as a stable standard. 4. Haemoglobin (1 mumol/l), infused directly over the bioassay tissues, abolished bradykinin-induced relaxations and these were restored within 5 min after removal of haemoglobin. The infusion did not affect the relaxations produced by glyceryl trinitrate in this system. 5. Methylene blue (20 mumol/l) inhibited bradykinin-induced relaxations when infused over the rabbit aortae, and reduced those relaxations produced by glyceryl trinitrate. The effects of bradykinin, but not glyceryl trinitrate, were partially restored after removing methylene blue. 6. These data are consistent with the known effects of these compounds on the activity of NO, and on EDRF in isolated blood vessels. 7. The activity of EDRF (released by bradykinin) was compared directly with NO on strips of guinea-pig trachea (de-epithelialized) interposed in cascade between two rabbit aortae; all strips were precontracted with histamine and phenylephrine. 8. A submaximal dose of NO that matched the relaxation produced by EDRF on the uppermost aorta, caused relaxation of the trachea, but EDRF had no effect on this tissue. In addition, the NO-induced relaxation of the lower aorta was greater than that produced by EDRF. 9. These data indicate that EDRF does not have identical biological activity to NO. EDRF could contain an NO moiety attached to a carrier molecule that is bound and stabilized in tracheal tissue.     

The pharmacology of endothelium-dependent vascular reactions

The review deals with the critical analysis of the recent publications showing an important role of the endothelium in the mechanism of vasodilation caused by endogenous agents (acetylcholine, bradykinin, substance P, ATP, histamine, thrombin) and pharmacological agents (clonidine, hydralazine, mellitin, calcium ionophore A 23187). The mechanism of the endothelium-dependent vasodilatation is based on the release of the endothelium-derived relaxant factor (EDRF). In 1987-1988 it was shown that in some cases EDRF is NO. The experimental evidence suggests that EDRF (NO) may directly activate guanylate cyclase that results in vascular smooth muscle relaxation due to cAMP accumulation. The possible physiological and pathophysiological significance of the endothelium-dependent vascular responses is discussed.     

Inhibition by nitric oxide and nitric oxide-producing vasodilators of DNA synthesis in vascular smooth muscle cells

Effects of nitric oxide (NO) and NO-producing vasodilators such as glyceryl trinitrate and sodium nitroprusside were tested on DNA synthesis in the clonal rat aortic smooth muscle cells, RACS-1. DNA synthesis was estimated by [3H]thymidine incorporation to DNA. NO and NO-producing vasodilators inhibited the DNA synthesis that was induced by 10% fetal calf serum. NO and NO-producing vasodilators also inhibited the basal level of DNA synthesis that occurred possibly as a result of autocrine mechanisms. NO-producing vasodilators also inhibited the fetal calf serum-induced proliferation of cells. Sodium nitroprusside inhibited the endothelin-mediated DNA synthesis. In another mesenchymal cell line, Chinese hamster fibroblast V79 cells, NO and NO-producing vasodilators failed to inhibit DNA synthesis, excluding the possibility of general cell toxicity. An exposure to NO and NO-producing vasodilators resulted in an increase of cyclic GMP (cGMP) content in the RACS-1 cells. A cGMP analog, 8-bromo-cGMP, inhibited DNA synthesis in the RACS-1 cells. These results suggest that EDRF/nitric oxide released from endothelium possibly contributes to inhibition of the DNA synthesis in vascular smooth muscle cells.     

Role of endothelium in the contractions induced by norepinephrine and clonidine in rat aorta

The inhibitory effects of endothelium-derived relaxing factor (EDRF) on the contractions induced by norepinephrine and clonidine in rat aorta were examined. Carbachol induced a relaxation of norepinephrine-induced contraction in rat aorta with endothelium. Removal of endothelium inhibited the carbachol-induced relaxation and increased the magnitude of norepinephrine-induced contraction. Quinacrine, a phospholipase A2 inhibitor, methylene blue, a guanylate cyclase inhibitor and tetraethylammonium, a potassium permeability inhibitor, inhibited carbachol-induced relaxation and augmented the magnitude of norepinephrine-induced contraction only when endothelium was present. Clonidine induced a contraction when endothelium was removed or muscle was treated with methylene blue. The contractions induced by norepinephrine and clonidine were equally sensitive to prazosin and equally less sensitive to yohimbine. Clonidine inhibited the norepinephrine-induced contraction, whereas it potentiated the angiotensin 11- or 12 mM K-induced contractions in the aorta with endothelium. The inhibitory effect of clonidine on the norepinephrine-induced contraction was reduced by endothelium-removal and by methylene blue but not by yohimbine. These results suggest that norepinephrine has a strong direct stimulating action and clonidine has a weak one on vascular smooth muscle cells possibly mediated by alpha 1-adrenoceptors, and their contractile effects are inhibited by the spontaneously released EDRF.     

The effects of diethylcarbamazine citrate on smooth muscle.

The effect of the anthelmintic drug diethylcarbamazine citrate (DECC) was examined on the guinea-pig isolated ileum, rabbit duodenum, chick oesophagus, rat portal vein and pig coronary artery. DECC contracted all the gastrointestinal smooth muscle preparations. The contractions were antagonized by hexamethonium and atropine but they were not affected by mepyramine or methysergide in concentrations that abolished, or markedly reduced, responses to histamine and 5-hydroxytryptamine. DECC inhibited the responses of the guinea-pig ileum to other spasmogens, namely, acetylcholine, histamine and nicotine. Physostigmine markedly potentiated the responses of the chick oesophagus and the rabbit duodenum to DECC. DECC relaxed the potassium chloride-induced contractions of the pig coronary artery strips; these relaxations were not modified by propranolol or calcium chloride. There was no evidence that DECC released histamine from skin or muscle.     

The effects of diethylcarbamazine citrate on smooth muscle

The effect of the anthelmintic drug diethylcarbamazine citrate (DECC) was examined on the guinea-pig isolated ileum, rabbit duodenum, chick oesophagus, rat portal vein and pig coronary artery. DECC contracted all the gastrointestinal smooth muscle preparations. The contractions were antagonized by hexamethonium and atropine but they were not affected by mepyramine or methysergide in concentrations that abolished, or markedly reduced, responses to histamine and 5-hydroxytryptamine. DECC inhibited the responses of the guinea-pig ileum to other spasmogens, namely, acetylcholine, histamine and nicotine. Physostigmine markedly potentiated the responses of the chick oesophagus and the rabbit duodenum to DECC. DECC relaxed the potassium chloride-induced contractions of the pig coronary artery strips; these relaxations were not modified by propranolol or calcium chloride. There was no evidence that DECC released histamine from skin or muscle.     

The anti-aggregating properties of vascular endothelium: interactions between prostacyclin and nitric oxide

1. The interactions between endothelium-derived nitric oxide (NO) and prostacyclin as inhibitors of platelet aggregation were examined. 2. Porcine aortic endothelial cells treated with indomethacin and stimulated with bradykinin (10-100 nM) released NO in quantities sufficient to account for the inhibition of platelet aggregation attributed to endothelium-derived relaxing factor (EDRF). 3. In the absence of indomethacin, stimulation of the cells with bradykinin (1-3 nM) released small amounts of prostacyclin and EDRF which synergistically inhibited platelet aggregation. 4. EDRF and authentic NO also caused disaggregation of platelets aggregated either with collagen or with U46619. 5. A reciprocal potentiation of both the anti- and the dis-aggregating activity was also observed between low concentrations of prostacyclin and authentic NO or EDRF released from endothelial cells. 6. It is likely that interactions between prostacyclin and NO released by the endothelium play a role in the homeostatic regulation of platelet-vessel wall interactions.