Endothelium-derived relaxing and contracting factors

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.     

The role of the endothelium in the development of functional hyperemia of the skeletal muscles]

Experiments on anesthesized dogs demonstrated that gastrocnemius muscle vessels working hyperemia substantially decreased after chemical destruction of endothelium by saponin, inhibition of endothelium-derived relaxing factor synthesis by gossypol and inhibition of quanylate cyclase by methylene blue. Reaction was not decreased after cyclooxygenase inhibition by indomethacin. The endothelium-derived relaxing factor predecessor--L-arginine essentially increased working hyperemia. We concluded that endothelium plays an important role in reaction of working hyperemia by endothelium-derived relaxing factor release.     

Adrenomedullin-induced dilation of human placental arteries is modulated by an endothelium-derived constricting factor

Adrenomedullin is synthesized and secreted by fetoplacental tissues. Given that the placenta lacks autonomic innervation, we proposed that adrenomedullin acts locally to control blood flow in the placental vasculature through a balance of dilatory and constrictive pathways. Placental stem villous arteries (200 microm) from normotensive human pregnancies were dissected and mounted on a wire myograph. The vessels were preconstricted with the thromboxane A(2) mimetic U46619 (EC(80) concentration), and exposed to cumulative concentrations of adrenomedullin (1 x 10(-9) to 3 x 10(-7) mol/L). Adrenomedullin caused concentration-dependent vasorelaxation which, in endothelium-intact vessels, was attenuated in the presence of the nitric oxide synthase inhibitor L-NMMA. This suggested that the vasodilation was mediated, at least in part, through nitric oxide. However, removal of the endothelium did not similarly alter the response. Nor did L-NMMA have any effect in endothelium-denuded vessels. We hypothesized that adrenomedullin must induce release of both endothelium-derived relaxing (nitric oxide) and constricting factors. When we blocked the two major pathways through which adrenomedullin is known to induce vasodilation, by incubating the vessels with L-NMMA (nitric oxide synthase inhibitor) and Rp-cAMPS (cAMP-dependent protein kinase inhibitor), adrenomedullin induced concentration-dependent vasoconstriction. This was not mediated through endothelin, since addition of the non-specific endothelin receptor antagonist PD142893 failed to alter the response to adrenomedullin. We conclude that, in addition to increasing endothelial nitric oxide biosynthesis in placental stem villous arteries, adrenomedullin induces release of an endothelium-derived constricting factor.     

Endothelium, arachidonic acid, and coronary vascular tone

Vasoactive mediators play an important role in the control of coronary vascular tone. Arachidonic acid (AA) metabolites and endothelium-derived vasoactive factors have been implicated in coronary vasoregulation. AA can be metabolized via three separate routes in blood vessels, mediated by cyclooxygenase, lipoxygenase, and cytochrome P-450-dependent monooxygenase enzymes. AA can evoke endothelium-dependent relaxations that are due in part to the formation of cytochrome P-450-dependent metabolites, inasmuch as drugs that modify cytochrome P-450 activity produce parallel changes in endothelium-dependent relaxations to AA. Moreover, some cytochrome P-450-derived metabolites formed biologically cause relaxations of isolated blood vessels. A cytochrome P-450-dependent pathway does not appear to contribute to endothelium-dependent relaxations induced by acetylcholine, which suggests that there may be a number of endothelium-derived relaxing factors (EDRFs). In addition, two endothelium-derived contractile factors have been described, including an unidentified cyclooxygenase metabolite of AA and a polypeptide isolated from cultured cells. As both prostaglandin I2 and acetylcholine-induced EDRF also inhibit platelet aggregation, endothelial injury and loss of these factors may predispose to vasospasm precipitated by release of platelet-derived mediators such as thromboxane A2 (TXA2) and 5-hydroxytryptamine. Unstable angina may be a clinical syndrome in which these events occur, which can be alleviated by inhibition of platelet activation and TXA2 formation with aspirin. Attenuation of endothelium-dependent relaxations can also occur without loss of endothelial cells. Neutrophil-endothelium interactions, precipitated by an ischemic episode, may initiate endothelial dysfunction and underlie the development of vasospasm in some conditions. Whether increased production of endothelium-derived contractile factors also occurs in vasospastic conditions remains to be determined.     

A relaxing factor released by phospholipase A2 in the absence of endothelium

Summary Phospholipase A₂ (PLA₂) produced slow dose dependent relaxation in intact and endothelium-deprived precontracted rabbit aortic strips. In endothelium-deprived preparations, relaxation induced by PLA₂ is inhibited by hemoglobin, methylene blue and parabromophenacylbromide (PBPB), and is potentiated by superoxide dismutase (SOD). Indomethacin has no effect. Relaxation is accompanied by a rise in c-GMP. Phospholipase C causes a significant increase in tension, while Phospholipase D has no effects. In intact aortic strips PLA₂ causes a biphasic response with no elevation in c-GMP. The results indicate several common features of the PLA₂ released factor with endothelium-derived relaxing factor (EDRF). However PLA₂ induced relaxation is not dependent on endothelial cells. Apparently in addition to nitric oxide which may be the endothelium-derived relaxing factor, a second smooth muscle relaxing factor exists which is initiated by PLA₂ and is independent of endothelium. The production of the PLA₂ produced relaxation is dependent on its specific hydrolytic activity. We call this relaxing factor the phospholipid-derived relaxing factor (PDRF).     

Role of the endothelium in the development of reactive hyperemia

The experiments on anesthetized dogs demonstrated that reaction of the femoral vessels reactive hyperemia essentially decreased after chemical inhibition of endothelium by saponin, inhibition of lipoxygenase by quercetin and guanylate cyclase by methylene blue. Reaction was increased after cyclooxygenase inhibition by indomethacin. We concluded that the endothelium plays an important role in reaction of reactive hyperemia by endothelium-derived relaxing factor release.     

The role of lysolecithin in the relaxation of vascular smooth muscle

The effect of lysolecithin (lysophosphatidylcholine) on the relaxation of rabbit aortic strip closely resembled that produced by acetylcholine (ACh) which releases the endothelium-derived relaxing factor (EDRF). Relaxation induced by lysolecithin depended on the presence of endothelium and was inhibited by hemoglobin and methylene blue. It appeared to be mediated by the second messenger, c-GMP. Lysolecithin induced relaxation was slower but more persistent than that resulting from the endothelium-derived relaxing factor (EDRF) produced by acetylcholine (ACh). Like lysolecithin, Triton X-100, a non-ionic detergent, also preferentially relaxed aortic strips with intact endothelium. The results demonstrate the importance of phospholipids derived from cell membranes in vascular smooth muscle relaxation. Endothelium-derived relaxing factors appear as a group of heterogeneous substances.     

Endothelial cell-dependent phosphorylation of a platelet protein mediated by cAMP- and cGMP-elevating factors.

We reported previously that a 46/50-kDa membrane-associated vasodilator-stimulated phosphoprotein (VASP) is phosphorylated in intact human platelets in response to both cGMP- and cAMP-elevating vasodilator drugs and presented evidence that this is mediated by cGMP- and cAMP-dependent protein kinases, respectively. VASP was recently purified and an antibody against it was developed which detects a phosphorylation-induced mobility change of VASP in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Halbrügge, M., Friedrich, C., Eigenthaler, M., Schanzenb?cher, P., and Walter, U. (1990) J. Biol. Chem. 265, 3088-3093). We have now used these methods for the quantitative analysis of VASP phosphorylation during coincubations of human endothelial cells and human platelets. Endothelial cell-derived factors caused the rapid, stoichiometric, and reversible phosphorylation of platelet VASP during these coincubations. Other experiments indicated that the endothelium-derived factors which stimulate VASP phosphorylation are prostacyclin and endothelium-derived relaxing factor whose effects are mediated by cAMP/cAMP-dependent protein kinase and cGMP/cGMP-dependent protein kinase, respectively. The results suggest that VASP phosphorylation is an important component of the inhibitory effects of prostacyclin and endothelium-derived relaxing factor on platelet activation and that VASP phosphorylation is a useful biochemical marker for the interaction of endothelial cells and platelets.     

Inhibition of platelet aggregation by S-nitroso-cysteine via cGMP-independent mechanisms: evidence of inhibition of thromboxane A2 synthesis in human blood platelets

S-Nitroso-cysteine (SNC), a putative endothelium-derived relaxing factor, potently inhibited collagen- and arachidonic acid-induced platelet aggregation (IC₅₀=100 nM) and thromboxane A₂ (TxA₂) synthesis of human blood platelets. ODQ, a selective inhibitor of the soluble guanylyl cyclase, inhibited SNC-induced formation of cGMP but did not reverse inhibition by SNC of collagen- and arachidonic acid-induced platelet aggregation. Combination of ODQ with SQ-29548, a specific platelet TxA₂ receptor antagonist, did not modify the antiaggregatory action of SNC. Our study shows that SNC inhibits platelet aggregation by cGMP-independent mechanisms that may involve inhibition of TxA₂ synthesis in human platelets.     

Endothelin-3 stimulates production of endothelium-derived nitric oxide via phosphoinositide breakdown.

Cultured bovine endothelial cells (EC) have specific receptors for endothelin (ET)-3 functionally coupled to phosphoinositide breakdown. We studied whether ET-3 stimulates synthesis of nitric oxide (NO), an endothelium-derived relaxing factor that activates soluble guanylate cyclase in EC, and whether the ET-3-induced NO formation involves G-proteins. ET-3 dose-dependently stimulated production of intracellular cGMP in EC, of which effects were abolished by pretreatment with NG-monomethyl L-arginine, an inhibitor of NO synthesis, and methylene blue, an inhibitor of soluble guanylate cyclase. The stimulatory effects of ET-3 on cGMP production, inositol trisphosphate formation and increase in cytosolic free Ca2+ concentration were similarly blocked by pretreatment with pertussis toxin (PTX). These data suggest that ET-3 induces synthesis of NO mediated by phosphoinositide breakdown via PTX-sensitive G-protein in EC.     

Endothelium-derived relaxing factor (nitric oxide) has protective actions in the stomach

The role that nitric oxide, an endothelium-derived relaxing factor, may play in the regulation of gastric mucosal defence was investigated by assessing the potential protective actions of this factor against the damage caused by ethanol in an ex vivo chamber preparation of the rat stomach. Topical application of glyceryl trinitrate and sodium nitroprusside, which have been shown to release nitric oxide, markedly reduced the area of 70% ethanol-induced hemorrhagic damage. Topical application of a 0.01% solution of authentic nitric oxide also significantly reduced the severity of mucosal damage. Pretreatment with indomethacin precluded the involvement of endogenous prostaglandins in the protective effects of these agents. The protective effects of NO were transient, since a delay of 5 minutes between NO administration and ethanol administration resulted in a complete loss of the protective activity. The protection against ethanol afforded by 10 micrograms/ml nitroprusside could be completely reversed by intravenous infusion of either 1% methylene blue or 1 mM hemoglobin, both of which inhibit vasodilation induced by nitric oxide. Intravenous infusion of 1% methylene blue significantly increased the susceptibility of the mucosa to damage induced by topical 20% ethanol. These results indicate that ethanol-induced gastric damage can be significantly reduced by nitric oxide. The mechanisms underlying the protective actions of nitric oxide are unclear, but may be related to its vasodilator or anti-aggregatory properties.     

Contractile and relaxant effects of jellyfish toxin on the vascular and intestinal tissues

A toxic component (pCrTX) of jellyfish Carybdea rastonii (10(-8)-10(-6)g/ml) caused a contraction in both rat aorta and guinea-pig taenia coli which was partially inhibited by indomethacin or aspirin. pCrTX (10(-7)-10(-6)g/ml) relaxed the norepinephrine-induced contraction in rat aorta which was inhibited by removing endothelium or by adding methylene blue. These results suggest that a portion of the pCrTX-induced contraction is due to release of prostaglandin(s) and that the pCrTX-induced relaxation is due to release of an endothelium-derived relaxing factor.     

L-NG-monomethyl arginine inhibits the vasodilating effects of low dose of endothelin-3 on rat mesenteric arteries

We have reported that low doses of endothelin-3 (ET-3) elicited continuous vasodilation of rat mesenteric arteries, which is possibly related to endothelium-derived relaxing factor (EDRF). In order to clarify whether or not the vasodilating effects of ET-3 are associated with EDRF, we examined the effects of L-NG-monomethyl arginine (L-NMMA), an analog of L-arginine, on low-dose ET-3 induced vasodilation of rat mesente-Hc arteries. Infusion of 50 microM L-NMMA inhibited the vasodilation induced by 10(-13) M ET-3 and rather elicited an increase in perfusion pressure, which itself was decreased by infusion of 150 microM L-arginine. In the presence of 50 microM L-NMMA, 10(-13) M ET-3 did not elicit any vasodilation of the mesenteric arteries preconstricted with NE, in which 150 microM L-arginine, but not D-arginine, caused considerable vasodilation. These data suggest that the vasodilating effects of low doses of ET-3 are associated with EDRF as an endothelium-derived nitric oxide.     

Methylene blue inhibits angiogenesis in chick chorioallontoic membrane through a nitric oxide-independent mechanism

Angiogenesis is the process of generating new blood vessels from preexisting vessels and is considered essential in many pathological conditions. The purpose of the present study was to evaluate the effect of methylene blue in chick chorioallantoic membrane angiogenesis model in vivo. In this well characterized model, methylene blue inhibited angiogenesis in a concentration-dependent manner. In addition, when methylene blue was combined with sodium nitroprusside, a spontaneous generator of nitric oxide, an inhibition of angiogenesis was evident which was comparable with that observed by the application of methylene blue alone. Sodium nitroprusside, alone, caused a significant inhibition in basal angiogenesis. These results provide evidence that methylene blue inhibits angiogenesis independently of nitric oxide pathway and suggest that methylene blue may be useful for treating angiogenesis-dependent human diseases.     

Vascular effects of oxygen-derived free radicals

This review attempts to summarize the available data regarding the vascular actions of free oxygen radicals. Studies on blood vessels in situ and in vitro demonstrate that free oxygen radicals can evoke both vasodilation and vasoconstriction. Free oxygen radicals can modulate the tone of vascular smooth muscle by acting directly on the smooth muscle cells, and also via indirect mechanisms by changes in the production or biological activity of vasoactive mediators. The individual oxygen radicals may have different (sometimes opposite) vascular effects. Superoxide anion inactivates endothelium-derived relaxing factor and the adrenergic neurotransmitter norepinephrine. Hydrogen peroxide and the hydroxyl radical evoke vasodilation by acting directly on vascular smooth muscle and also by stimulating the synthesis/release of endothelium-derived relaxing factor. In acute arterial hypertension or experimental brain injury oxygen radicals are important mediators of vascular damage. Production of oxygen-derived free radicals by activated neutrophils may be responsible for vasodilation and increased permeability of capillary membrane during the acute inflammatory process. Free oxygen radicals also play an important role in reperfusion injury of various organs, and vascular actions of the free radicals may contribute to the damage of parenchymal tissues.     

Alterations in relaxation to lactate and H(2)O(2) in human placental vessels from gestational diabetic pregnancies

We determined whether alterations in the mechanism of relaxation to H(2)O(2) potentially contribute to the enhanced prostaglandin-mediated contractile response to H(2)O(2) and posthypoxic reoxygenation seen in human placental vessels of pregnancies with gestational diabetes mellitus (GDM). Isolated placental arteries and veins from GDM and uncomplicated full-term pregnancies were precontracted with prostaglandin F(2alpha) (PO(2) 35-38 Torr) and then exposed to lactate (1-10 mM), arachidonic acid (0.01-10 microM), nitroglycerin (1 nM-1 microM), forskolin (0.01-10 microM), or H(2)O(2) (1 microM-1 mM + 10 microM indomethacin). The rates of tissue H(2)O(2) metabolism by catalase and nitrite production were measured. The relaxation to lactate was reduced in GDM placental arteries and veins by 54-85 and 66-80%, and the relaxation to H(2)O(2) was inhibited by 80-94% in GDM placental veins compared with vessels from uncomplicated full-term pregnancies. H(2)O(2) caused only minimal relaxation of placental arteries. Responses to other relaxing agents were not altered in the GDM placental vessels. Diabetic vessels showed rates of nitrite production that were increased by 113-195% and rates of H(2)O(2) metabolism by catalase that were decreased by 44-61%. The loss of relaxation to H(2)O(2) and lactate (mediated via H(2)O(2)), perhaps as a result of the inhibition of catalase by nitric oxide, may explain the previously reported enhancement of prostaglandin-mediated contractile responses to H(2)O(2) and posthypoxic reoxygenation seen in GDM placental vessels.     

Synthesis of calcitonin gene-related peptide (CGRP) by rat arterial endothelial cells

We investigated the protein and mRNA expression of calcitonin gene-related peptide (CGRP) in endothelial cells of the rat thoracic aorta and femoral artery. Light microscopic immunocytochemistry revealed that immunoreactivity for CGRP was preferentially located in the endothelium of both vessels. Immunoelectron microscopy showed that CGRP-immunoreactive gold particles were preferentially localized on cisterns of the rough endoplasmic reticulum and on the Weibel-Palade (WP) bodies in the endothelial cells. Prepro CGRP mRNA signals were also detected on the endothelium. Our results are the first to demonstrate that endothelial cells of both elastic and large muscular arteries synthesize CGRP and store it, in part, in WP bodies, implying that CGRP may act as an endothelium-derived relaxing factor in these vessels.     

Age-related endothelial dysfunction with respect to nitric oxide, endothelium-derived hyperpolarizing factor and cyclooxygenase products

Vascular aging is associated with both structural and functional changes that can take place at the level of the endothelium, vascular smooth muscle cells and the extracellular matrix of blood vessels. With regard to the endothelium, reduced vasodilatation in response to agonists occurs in large conduit arteries as well as in resistance arteries with aging. Reviews concerning the different hypotheses that may account for this endothelial dysfunction have pointed out alterations in the equilibrium between endothelium-derived relaxing and constricting factors. Thus, a decreased vasorelaxation due to nitric oxide and, in some arteries, endothelium-derived hyperpolarizing factor as well as an increased vasoconstriction mediated by cyclooxygenase products such as thromboxane A2 are likely to occur in age-induced impairment of endothelial vasodilatation. Furthermore, enhanced oxidative stress plays a critical role in the deleterious effect of aging on the endothelium by means of nitric oxide breakdown due to reactive oxygen species. The relative contribution of the above phenomenon in age-related endothelial dysfunction is highly dependent on the species and type of vascular bed.     

Secretory functions of the vascular endothelium.

The endothelial cells which line the blood vessels as a monolayer exert a remarkable control over the vascular system. Indeed, the endothelium can be regarded as a highly active metabolic and endocrine organ in its own right. On the hand, vasoactive substances such as serotonin and bradykinin are inactivated and on the other the cells can enzymatically produce the vasoconstrictor, angiotensin II and secrete endothelin-1 ((ET-1). Perhaps more importantly, the cells also produce two unstable vasodilator substances, which potently inhibit platelet clumping: prostacyclin and endothelium-derived relaxing factor (EDRF) which has been identified as nitric oxide (NO; 1). Both substances seem well designated as local hormones, released to influence adjacent cells. The endothelial cell, therefore, exerts control over the cardiovascular system by elaborating dilator substances as well as vasconstrictors.     

The arterial communication between the gastrocnemius muscle heads: a fresh cadaveric study and clinical implications

The purpose of this investigation was to describe the anatomy of the communicating (anastomotic) vessels between the gastrocnemius muscle heads and to record the extent of their supply potential. Ensuing clinical implications are discussed. Fourteen fresh cadaveric gastrocnemius muscles were examined. Detailed dissections of the communicating vessels were facilitated after injections of methylene blue or cadaveric blood solutions through the medial, lateral, or both sural arteries. The extent of the arterial cross-supply between the muscles' heads through these vessels was determined in-eight specimens after methylene blue perfusions through the lateral sural arteries, while one specimen was examined after injection of methylene blue and yellow ink through the lateral and medial sural arteries, respectively. Communicating vessels were detected in all 14 specimens. A mean number of 5.8 vascular bundles and single vessels was found. The bundles consisted of arterioles and, as all indications suggested, of concomitant venules as well. Regarding arterial cross-supply, it was clearly evident that each head could be vascularized solely from the contralateral one, mostly through these bundles. However, even if only a part of the bundles was preserved intact, vasculature was not affected.