Depression of endothelium-dependent relaxation in aorta from rats with Brugia pahangi lymphatic filariasis

A role for altered endothelial cell function is emerging in the pathogenesis of disease. We have previously demonstrated that Dirofilaria immitis, the canine heartworm, depresses endothelium-dependent responses and alters the mechanism of relaxation in the in vivo femoral artery of infected dogs. Exposure of rat aorta to the parasite or parasite-conditioned medium selectively depresses endothelium-dependent relaxation. D. immitis is closely related to the major human filarial pathogens. This study was designed to examine the effect of chronic infection with the filarial nematode Brugia pahangi on endothelium-mediated responses of the rat aorta in vitro. We tested the hypothesis that endothelium-dependent responses are depressed in the aorta from rats infected with B. pahangi. Rings of thoracic and abdominal aorta were suspended in muscle baths for measurement of isometric tension. Dose-response relations to norepinephrine, endothelium-dependent dilators (acetylcholine, histamine, and A23187), and nitroglycerin were done. In some experiments, inhibitors of cyclooxygenase (indomethacin and aspirin), guanylate cyclase (methylene blue), and nitric oxide formation (N-nitro-L-arginine methyl ester; L-NOARG) were used. No differences in vascular reactivity were detected in the thoracic aorta. In contrast, endothelium-dependent responses in abdominal aorta of Brugia-infected rats were significantly depressed when compared with control aorta from noninfected rats. Acetylcholine relaxation was further depressed by indomethacin and aspirin. After L-NOARG, acetylcholine relaxation in control abdominal aorta was completely abolished; however, in abdominal aorta of Brugia-infected rats, acetylcholine still caused relaxation. Methylene blue inhibited acetylcholine relaxation in both control and Brugia-infected abdominal aorta; however, relaxation in Brugia-infected aorta was significantly greater than control. This study demonstrates that endothelium-dependent relaxation can be altered by chronic experimental filarial infection in the absence of direct contact between the blood vessel and the parasite. The mechanism of relaxation in the Brugia-infected abdominal aorta appears to be altered when compared with control, suggesting that parasites are capable of modulating vascular reactivity by inducing changes in endothelial cell behavior. The mechanism may involve parasite-induced local inflammation or alterations in endothelial cell metabolism. Understanding how chronic experimental filarial infection alters vascular reactivity may enhance our understanding of the pathogenesis of human filariasis.     

Age and hypertension promote endothelium-dependent contractions to acetylcholine in the aorta of the rat

This study was undertaken to compare age-related changes in endothelium-dependent vascular responses in both hypertensive and normotensive rats. Aorta from normotensive Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR) aged 4-6 weeks (young), 3-6 months (adult), and 12-25 months (old) were examined for relaxation to acetylcholine, adenosine 5'-triphosphate (ATP), and sodium nitroprusside. Rubbed (endothelium denuded) aorta from all groups displayed neither relaxation nor contraction to acetylcholine. Maximal relaxation responses to acetylcholine were reduced progressively with increasing age in unrubbed aorta of both SHR and WKY rats. In addition, acetylcholine caused not only dose-dependent relaxations at lower concentrations but also increases in tension at higher concentrations in unrubbed aorta of old WKY rats as well as adult and old SHR. However, indomethacin completely inhibited the tension development. As a result, aorta treated with indomethacin demonstrated similar acetylcholine-induced, endothelium-dependent relaxations in all groups. The thromboxane A2 synthetase inhibitor (E)-7-phenyl-7-(3-pyridyl)-6-heptanoic acid (CV-4151) partially but significantly depressed the increases in tension in aorta of old WKY rats. The degrees of endothelium-dependent relaxations to ATP and endothelium-independent relaxations to sodium nitroprusside were almost similar in all groups. These findings suggest that the release of or vascular responsiveness to endothelium-derived relaxing factor in the aorta is well maintained through senescence in both strains and that, in the aorta of not only SHR but also old normotensive WKY rats, the endothelium releases contracting factors that may be thromboxane A2 and other vasoconstrictor prostanoids.     

Impaired endothelium-dependent relaxations in rabbits subjected to aortic coarctation hypertension

Rabbits were rendered hypertensive by suprarenal coarctation of the abdominal aorta. Seven days later, endothelium-dependent and endothelium-independent vascular relaxations were examined in vascular rings taken from hypertensive (thoracic aorta, carotid artery) and normotensive (abdominal aorta) regions. Relaxation of phenylephrine-contracted rings in response to endothelium-dependent agonists (acetylcholine, A23187) was impaired, compared with that in sham-operated and intact controls, in regions exposed to the elevated blood pressure (i.e., above the coarctation). Responses to acetylcholine and A23187 in the abdominal aorta, below the coarctation, were not altered. The diminished endothelium-dependent responses in the thoracic aorta were not affected by pretreatment with the cyclooxygenase inhibitor indomethacin. In contrast to acetylcholine and A23187, responses to the endothelium-independent agonist nitroprusside were not attenuated in vessels from hypertensive regions, indicating that the defect occurred in the endothelium. The EC50 for acetylcholine-induced relaxations of thoracic aorta correlated significantly with mean arterial pressure above the coarctation, indicating that the extent to which endothelium-dependent relaxation is impaired is in proportion to the degree of blood pressure elevation. This study suggests that the diminished relaxations by endothelium-dependent agonists is a local response to the elevation of blood pressure and is not due to a circulating factor.     

Endothelium-dependent relaxation of rat aorta and main pulmonary artery by the phytoestrogens genistein and daidzein

OBJECTIVE: The dietary phytoestrogens genistein and daidzein have been shown to relax agonist-preconstricted arteries in vitro; the mechanisms of relaxation remain incompletely understood. This study aimed to determine whether the relaxation of phenylephrine (PE)-constricted rat aorta and main pulmonary artery by genistein and daidzein was endothelium-dependent. METHODS: Effects of endothelial-denudation, and pretreatment with with 100 microM L-N(G)-nitroarginine methyl ester (L-NAME) and/or 10 microM indomethacin on relaxation of PE (1 microM)-preconstricted contractures by genistein (1-100 microM) and daidzein (3-100 microM) were assessed by measuring isometric force development by rat arterial rings. The effect of L-NAME on relaxation to 17beta-estradiol (10 microM) was also measured in aorta. RESULTS AND CONCLUSIONS: Genistein and daidzein caused concentration-dependent relaxation of aorta rings preconstricted with PE (1 microM). The IC50 values were 5.7 microM (n=8, 95% confidence limits 4.3-7.7 microM) and 36.7 microM (n=12, 95% confidence limits 25.7-44.1 microM), respectively. Removal of the endothelium and pretreatment with L-NAME (100 microM) significantly inhibited relaxation at 3, 10 and 30 microM genistein and 10 and 30 microM daidzein. The contracture evoked in rat aorta by depolarization with 75 mM K+ solution was similarly relaxed by genistein in a partially endothelium-dependent manner. 17Beta-estradiol (10 microM) caused a 48.7+/-5.0% (n=11) relaxation of the PE contracture, which was significantly reduced to 25.1+/-5.3% (n=7) by L-NAME. Relaxations brought about by 17beta-estradiol, genistein, and daidzein were not significantly affected by the genomic estrogen receptor antagonist ICI 182,780 (10 microM). Similar endothelium-dependent effects of genistein were observed in the main pulmonary artery. The results show that the relaxation of these rat arteries by concentrations of genistein and daidzein which overlap those present in human plasma after ingestion of soybean-containing meals is largely endothelium dependent.     

Antigen-induced release of airway epithelium-derived inhibitory factor

The ability of guinea pig trachea to produce a vasoactive epithelium-derived inhibitory factor (EpDIF) in response to mast cell-derived mediators was assessed in a coaxial bioassay system. The mast cell degranulating agent compound 48/80 (10 micrograms/ml) and histamine caused reductions in phenylephrine-induced tone in endothelium-denuded rat aorta preparations mounted coaxially within epithelium-intact guinea pig tracheal tube tissue. Relaxation responses to histamine and to compound 48/80 (10 micrograms/ml) were markedly reduced in the presence of mepyramine (50 microM) or when the epithelium was removed from coaxially mounted guinea pig trachea, indicating that they were mediated via the release of EpDIF. Coaxial bioassay assemblies were also prepared using EpDIF donor tracheal tissue obtained from guinea pigs actively sensitized to ovalbumin. Subsequent challenge with ovalbumin (10(-7) to 10(-1) mg/ml) produced concentration-dependent relaxation mediated by EpDIF. Ovalbumin-induced relaxation responses were not inhibited in the presence of either mepyramine (20 and 100 microM) or SKF 104353-Z2 (10 microM) alone but were significantly reduced when both mepyramine (20 microM) and SKF 104353-Z2 (10 microM) were present. Antigen-induced relaxation was apparently mediated by EpDIF in response to mast cell-derived histamine and leukotrienes. Rat tracheal airway smooth muscle did not relax in response to EpDIF, suggesting selectivity of action on vascular smooth muscle. Vasoactive EpDIF may play a role in protecting against antigen-induced bronchoconstriction by regulating bronchial circulation flow.     

Development of Tolerance to Inhibitory Effect of Ethanol on Endothelium-dependent Vascular Relaxation in Ethanol-fed Rats

Rats were maintained on liquid diets containing ethanol (35% of total calories) or an equicaloric volume of sucrose instead of ethanol for 10 wk. Vascular strips of isolated rat aortas were mounted in organ chambers to record isometric tension. Ethanol in vitro inhibited the endothelium-dependent relaxation responses to acetylcholine and ATP in both pair-fed control and ethanol-fed rats. The inhibitory effect of ethanol was greater in the pair-fed rats. In addition, the magnitudes of these relaxation responses in the absence of ethanol in vitro in pair-fed rats were similar to those in the presence of ethanol in ethanol-fed rats. In the absence of ethanol in vitro, the relaxations in response to acetylcholine and ATP in the ethanol-fed rats were greater than in the pair-fed rats. These results suggest that chronic ethanol consumption can induce tolerance to ethanol-induced inhibition of endothelium-dependent relaxation responses to acetylcholine and ATP, and that the relaxations can become adapted to the presence of plasma levels of ethanol, which may inhibit the relaxation in vivo. The augmented relaxation in the ethanol-fed rats may result from the mechanism causing tolerance to the inhibitory effect of ethanol.     

Gender difference in rat aorta vasodilation after acute exposure to high glucose: involvement of protein kinase C beta and superoxide but not of Rho kinase

OBJECTIVES: Several reports suggest that acute hyperglycemia affects male and female vascular beds differently. However, little is known about the interactions between hyperglycemia and gender in the vasculature. The objectives of our study were to investigate if there is a gender-based difference in the relaxation response of rat aorta after acute exposure to high glucose concentration, and the potential role of protein kinase C-beta (PKCbeta), superoxide, and Rho kinase in the gender-specific effect of acute high glucose on the relaxation response. METHODS: Endothelium-dependent dilator responses to acetylcholine (ACh, 10(-8) to 10(-5) M) were obtained before and after 3 h treatment with Krebs' solution containing high glucose (46 mM) in aortic rings pre-contracted with phenylephrine (2 microM) taken from female and male Sprague-Dawley rats. Similar experiments were generated in the presence of 1 microM LY379196, a selective PKCbeta inhibitor, 25 microM MnTMPyP, a superoxide dismutase mimetic, or 1 microM Fasudil, a Rho kinase inhibitor. Furthermore, protein expression of PKCbeta isoforms was measured by Western blotting. RESULTS: We demonstrated that a 3 h incubation with elevated level of glucose impairs ACh responses only in the female rat aortic rings. Inhibition of PKCbeta or superoxide production but not Rho kinase prevents the high glucose-induced impairment of endothelium-dependent relaxation of female rat aorta. In addition, PKCbeta2 expression is significantly higher in the female rat aorta than that in male rat aorta. CONCLUSION: These results suggest that the gender difference in the impairment of endothelium-dependent vasodilation after acute exposure to high glucose in rat aorta is possibly due to differences in PKCbeta2 expression.     

Endothelin-1-induced constriction in the coronary resistance vessels and abdominal aorta of the guinea pig

The purpose of this study was to examine contractile properties of endothelin-1, a newly discovered vasoactive peptide, in guinea pig coronary resistance vessels and abdominal aorta. Changes in perfusion pressure after injections of endothelin-1 were measured using a constant-flow modified Langendorff preparation. The ED10 values of coronary perfusion pressure were about 100-fold less for endothelin-1 than for prostaglandin F2 alpha. After the endothelium was damaged by exposure to free radicals, maximal coronary constriction in response to endothelin-1 (10(-9) moles) was not altered, whereas dilator responses to low doses of endothelin-1 were converted to constrictor responses. Removal of the endothelium from aortic rings significantly increased responsiveness to endothelin-1 and the maximal response to the peptide. In calcium-free medium, endothelin-1 induced small increases both in perfusion pressure in coronary vessels and in tension in the aorta. Reintroduction of calcium in the coronary and aortic preparations produced a rapid increase in perfusion pressure and tension, respectively. Further, endothelin-1-induced coronary constriction was inhibited 59% +/- 7% by nifedipine (10(-7) moles). We conclude that endothelin-1 is a more potent constrictor than prostaglandin F2 alpha in the coronary vasculature. Endothelin-1-induced constriction in the coronary vasculature of the guinea pig is not mediated through an endogenous constricting factor released from the endothelium or a constrictor prostaglandin. Further, endothelin-1-induced dilation in the coronary vasculature and attenuation of endothelin-1-induced contraction in the abdominal aorta of the guinea pig are mediated through the release of a factor from the endothelium.(ABSTRACT TRUNCATED AT 250 WORDS)     

Endothelium-dependent relaxation and L-arginine metabolism in genetic hypertension.

This study characterizes the effects of L-arginine and NG-monomethyl L-arginine on dilator responsiveness of vascular tissue from Wistar-Kyoto rats and stroke-prone spontaneously hypertensive rats. Rings of abdominal aorta were suspended in tissue baths for measurement of isometric force. After contraction induced by phenylephrine, cumulative addition of acetylcholine, L-arginine, or A23187 to the muscle bath caused a similar relaxation of aortic rings in both animal groups. To test the hypothesis that arginine metabolism is altered in hypertension, aortic rings were incubated with NG-monomethyl L-arginine. NG-monomethyl L-arginine (10-300 microM) did not affect contractile responses to phenylephrine (10(-10) to 10(-4) M) in either animal group (EC50, 10(-7) M). Exposure of aortic rings to NG-monomethyl L-arginine resulted in a greater inhibition of relaxation response to acetylcholine (10(-10) to 10(-6) M) in hypertensive animals. NG-monomethyl L-arginine (300 microM) caused complete inhibition of relaxation to acetylcholine in the hypertension group. Incubation with L-arginine (10-100 microM) overcame the inhibition of acetylcholine-induced relaxation produced by NG-monomethyl L-arginine in both groups. Exposure of aortic ring segments to NG-monomethyl L-arginine attenuated relaxation responses to A23187 (10(-10) to 3 x 10(-6) M) in both groups. L-Arginine-induced reversal of the inhibitory effect of NG-monomethyl L-arginine on the relaxation responses to A23187 was similar between groups.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of cytochrome P-450 inhibitors on endothelium-dependent relaxation in rabbit aorta

Endothelium-dependent relaxation in rabbit aorta was inhibited by exposure to two cytochrome P-450 inhibitors, metyrapone and SKF-525A. Aortic rings were contracted to a stable plateau by addition of phenylephrine (10(-7) M). Relaxation was elicited by the cumulative addition of methacholine (3 X 10(-8) - 3 X 10(-6) M) or A23187 (10(-8) - 10(-6) M). Exposure to metyrapone (500 microM) or SKF-525A (10 micrograms/ml) was found to inhibit relaxation in response to concentrations of methacholine exceeding 10(-7) M. Maximal relaxation was inhibited 73% by metyrapone. Relaxation stimulated by concentrations of A23187 exceeding 10(-7) M was also found to be inhibited by both metyrapone and SKF-525A exposure. Maximum A23187-induced relaxation (55% of the phenylephrine contractile response) was inhibited 40% by metyrapone and 55% by SKF-525A. Arachidonic acid (10-100 microM) also elicited endothelium-dependent relaxation in rings pretreated with indomethacin (10 micrograms/ml) and contracted with phenylephrine. This relaxation response was abolished by exposure to metyrapone or SKF-525A. These results suggest that cytochrome P-450 may be involved in endothelium-dependent relaxation responses, perhaps by metabolizing arachidonic acid to active products.     

Diminished endothelium-derived relaxing factor activity in an experimental model of chronic heart failure

Abnormalities in vasomotor tone, including enhanced vasoconstriction at rest and diminished vasodilation in response to various stimuli, develop as a consequence of chronic heart failure. This study was undertaken to evaluate whether a specific local mechanism, namely endothelium-derived relaxing factor (EDRF) activity, might be impaired in an experimental model of chronic heart failure. Segments of thoracic aorta (TA) and pulmonary artery (PA) were isolated from a group of rats that had hemodynamic evidence of heart failure 10 weeks after ligation of the left coronary artery (n = 25) and from a group of sham-operated control rats (n = 18). Both endothelium-dependent and endothelium-independent vascular responses were assessed by exposing arterial segments to increasing concentrations of agonists. All studies were performed in the presence of 10 microM indomethacin to avoid the influence of vasoactive prostanoids. The dose-response curve for EDRF-mediated relaxation to acetylcholine was shifted rightward in rats with heart failure, and the concentrations of acetylcholine required to achieve 50% maximal relaxation (EC50) were increased compared with those of control rats in both TA and PA segments. Additionally, the dose-response curve for relaxation to ADP was shifted rightward with significantly increased EC50 in PA segments from rats with heart failure. In contrast, EDRF-mediated relaxation to the calcium ionophore A23187 was similar in the groups. Endothelium-independent relaxation to nitroglycerin was slightly increased in TA but not PA segments in the heart-failure group. Basal EDRF activity, as assessed by the increase in force after exposure to hemoglobin, was diminished in PA segments from rats with heart failure.(ABSTRACT TRUNCATED AT 250 WORDS)     

Angiotensin-converting enzyme inhibitors reveal non-NO-, non-prostacycline-mediated endothelium-dependent relaxation in internal thoracic artery of hypertensive patients

BACKGROUND: We have shown that treatment of hypertension with ACE inhibitors (ACE-I) enhances relaxation to acetylcholine in human internal thoracic artery (ITA) above this in nonhypertensive patients receiving no ACE-I. Present study assesses the endothelium-dependent responses mediated by neither NO nor prostacyclin in human ITA. METHODS: We compared isolated ITA rings from hypertensive patients treated with ACE-I (ACE-I group) with those from normotensive patients on no ACE-I (control group). Relaxation to acetylcholine was assessed before and after inhibition of NO synthase and cyclooxygenase with L-NMMA and indomethacin, respectively. RESULTS: The maximal relaxation in ACE-I group was 79+/-3.3% and was depressed by incubation with L-NMMA and indomethacin to 41+/-2.7% (p<0.001); pD(2)=7.7+/-0.1 vs. 7.4+/-0.8 (p=0.265). The maximal relaxation to acetylcholine was lower in the control group: 65+/-3.3% (p=0.01); pD(2)=7.5+/-0.1 (p=0.07). Incubation with L-NMMA and indomethacin produced contraction to acetylcholine with a maximum of 43+/-7% (p<0.001); pD(2)=5.3+/-0.3 (p<0.001). The area under the concentration-response curve for acetylcholine-induced relaxation in ACE-I group equaled [arbitrary units] 596+/-71 and after incubation with L-NMMA and indomethacin 281+/-40 (p=0.002). Estimated LNMMA- and indomethacin-resistant relaxation, absent in control group, accounted for 47+/-4% of relaxation to acetylcholine in ACE-I group. Estimated NO- and prostacyclin-mediated relaxation was higher in control group than ACE-I group: 628+/-74 vs. 315+/-47 (p=0.009). CONCLUSIONS: The results suggest that therapy with ACE-I improves endothelial function of hypertensive patients mainly by enhancing the endothelium-derived hyperpolarizing factor (EDHF) (and not NO)-mediated responses. It seems that it reveals measurable non-NO- non-PGI-mediated endothelium-dependent relaxation otherwise absent in conduit arteries.     

Endothelium-dependent relaxation and hyperpolarization in aorta from control and renal hypertensive rats.

Endothelium-dependent relaxations are depressed in hypertension. In this study we investigated the possible involvement of endothelium-dependent smooth muscle hyperpolarization in this phenomenon. In isolated aortic segments from control rats, acetylcholine (10(-8)-10(-5) M) elicits relaxations after precontraction with norepinephrine (10(-7) M), and acetylcholine or carbachol (10(-5) M) induce smooth muscle hyperpolarization (10.6 +/- 0.9 mV). Both effects disappear after removal of the endothelium and are depressed by tetraethylammonium (3 x 10(-3) M), a rather nonspecific blocker of K+ channels, but not by glibenclamide (10(-5) M), a potent blocker of the ATP-regulated K+ channels, which has a marked effect on the relaxation induced by BRL 38227. The relaxation effect of acetylcholine is impaired in norepinephrine-contracted preparations from hypertensive rats but is not further depressed by tetraethylammonium. In aorta from hypertensive rats, hyperpolarization induced by carbachol was significantly reduced to a mean of only 21.8% of the values obtained in preparations from normotensive rats. From the relaxation-hyperpolarization relation obtained with BRL 38227 (opening K+ channels), it is derived that the endothelium-dependent hyperpolarization (approximately 10 mV) contributes for at least 20-30% of the maximal relaxation effect of acetylcholine on rat aorta. It is concluded that the diminished endothelium-dependent hyperpolarization may contribute to the depression of the endothelium-dependent relaxation in hypertension.     

Aprotinin impairs endothelium-dependent relaxation in rat aorta and inhibits nitric oxide release from rat coronary endothelial cells

OBJECTIVE: Aprotinin, a non-specific serine protease inhibitor, reduces postoperative bleeding after coronary artery surgery. The mechanism of action for this 'blood-sparing' effect of aprotinin is only partially clarified. We therefore aimed to investigate the effect of aprotinin on the release of nitric oxide (NO), a vasodilator and antiaggregant factor, from rat coronary microvascular endothelial cells and on the NO-mediated endothelium-dependent relaxation of rat thoracic aorta. METHODS: Endothelium-intact and endothelium-denuded thoracic aortic rings from Wistar rats (250-300 g) were suspended in organ chambers. Contractile and relaxant responses in the absence and presence of aprotinin (125, 250 and 500 KIU/ml) were recorded via a mechanotransducer. Coronary microvascular endothelial cells (CMEC) were isolated on a Langendorff system by collagenase perfusion of the hearts from the same rats. Calcium ionophore- (1 microM) induced release of NO from confluent cells was determined spectrophotometrically by measuring its stable metabolites, nitrite and nitrate, via Griess reaction. RESULTS: Aprotinin selectively enhanced phenylephrine-induced contractions in endothelium-intact rat thoracic aortic rings, but not in the endothelium-denuded rings. The use of a nitric oxide synthesis inhibitor Nomega-nitro-L-arginine methyl ester (100 microM) on endothelium-intact rings produced a similar increase in phenylephrine-induced contractions. KCl-induced contractions remained unaltered. Aprotinin inhibited acetylcholine-, calcium ionophore- and L-arginine-induced endothelium-dependent relaxations, but not sodium nitroprusside-induced endothelium-independent relaxation. Aprotinin had no significant effect on basal nitrite-nitrate release from CMEC, while it inhibited calcium ionophore-induced total nitrite accumulation in the supernatants. CONCLUSION: Aprotinin selectively impairs endothelium-dependent relaxation as well as basal NO availability in rat thoracic aortic rings and inhibits NO release from rat CMEC. This effect of the drug may contribute to its 'blood-sparing' action and may also account for the increase in perioperative restenosis risk observed in clinical practice during aprotinin therapy.     

Arachidonic acid elicits endothelium-dependent release from the rabbit aorta of a constrictor prostanoid resembling prostaglandin endoperoxides

This study was designed to investigate the mediator(s) of endothelium-dependent arterial constrictor responses evoked by arachidonic acid in vitro. A segment of descending rabbit thoracic aorta was isolated and perfused (1-2 ml/min) with oxygenated Krebs' bicarbonate buffer. Changes in the vascular smooth muscle-contracting activity of the aortic effluent were detected by superfusion bioassay using either strips of rabbit aorta or rings of dog saphenous vein, both denuded of endothelium and exposed to indomethacin (10 microM). Arachidonic acid (5-50 micrograms) injected into the inflow of the perfused aorta caused a dose-related increase in the vascular smooth muscle-contracting activity of the aortic effluent, whereas arachidonic acid added directly into the aortic effluent did not. The arachidonic acid-induced elevation of vascular smooth muscle-contracting activity in the aortic effluent was not apparent when indomethacin (10 microM) was added to the aortic inflow to inhibit cyclooxygenase, when the endothelium of the perfused aorta was removed by rubbing, or when the thromboxane A2/prostaglandin H2 receptors of the vascular tissues used for bioassay were blocked with an antagonist (1 microM SQ29548), and was unaffected when an inhibitor of thromboxane synthase (10 microM CGS 13080) was added to the aortic inflow. This effect of arachidonic acid was accompanied by release of prostaglandin H2 (measured as prostaglandin F2 alpha after reduction with SnCl2) in amounts sufficient to elicit contraction of the vascular tissues used for bioassay and was attenuated when a reducing agent (2 mM FeCl2) that converts prostaglandin H2 to 12-heptadecatrienoic acid was added to the aortic effluent. Collectively, these observations suggest that arachidonic acid stimulates endothelium-dependent release from the perfused aorta of a prostanoid that contracts vascular smooth muscle via interaction with thromboxane A2/prostaglandin H2 receptors. The study also suggests that the prostanoid responsible for the vascular smooth muscle-contracting activity of the aortic effluent is a prostaglandin endoperoxide(s) rather than thromboxane A2.     

Effects of resveratrol on vascular tone and endothelial function of human saphenous vein and internal mammary artery

BACKGROUND: The polyphenolic compound resveratrol presented in red wine has potent cardiovascular effect in animal. Here, we investigated the ability of resveratrol to relax human coronary bypass grafts, saphenous vein and internal mammary artery and also its effect on their endothelial reactivity. METHODS: Vascular rings were obtained from 38 male patients undergoing coronary artery bypass operation. The relaxant effects of resveratrol (10-70 microM) and acetylcholine (10(-8)-10(-4) M) were examined on precontracted saphenous vein and internal mammary artery rings. RESULTS: Resveratrol, at concentration of 70 microM caused relaxations of 34.2+/-5.7% in saphenous vein and 35.2+/-5.4% in internal mammary artery. Endothelium removal and l-NOARG (nitric oxide synthase inhibitor, 10(-4) M) pretreatment almost completely inhibited the relaxation to resveratrol in internal mammary artery but partially in saphenous vein rings. Indomethacin (cyclooxygenase inhibitor, 10(-5) M) slightly, but not significantly enhanced the relaxation to resveratrol in both vessels. The endothelium-dependent relaxations to acetylcholine were significantly improved in the presence of resveratrol of 20 microM in both grafts (E(max): 33.8+/-3.7% versus 46.8+/-4% in saphenous vein n=9; p<0.05; 54. 4+/-5.3% versus 69.3+/-5.4% in internal mammary artery, n=8, p<0.05). The relaxations to acetylcholine were fully eliminated by combination of resveratrol with l-NOARG (10(-4) M) in both vessels. CONCLUSIONS: Resveratrol produced mainly endothelium-dependent and nitric oxide-mediated vasodilation in human internal mammary artery but partially in saphenous vein rings and improved their endothelial reactivity. This may have a therapeutic potential in cardiovascular diseases.     

Arachidonic acid- and acetylcholine-induced relaxations of rabbit aorta.

The present study investigated the role of arachidonic acid and acetylcholine in mediating endothelium-dependent relaxations of rabbit aorta. Isolated thoracic aortic rings were precontracted with a submaximal concentration of norepinephrine, and the effect of various agents on arachidonic acid- and acetylcholine-induced relaxations was examined. Arachidonic acid elicited a concentration-related relaxation that was potentiated by the cyclooxygenase inhibitor indomethacin. Treatment with the lipoxygenase inhibitor nordihydroguaiaretic acid completely blocked but the cytochrome P450 inhibitor metyrapone had no effect on arachidonic acid-induced relaxation. NG-Monomethyl-L-arginine and nitro-L-arginine, compounds that inhibit the nitric oxide-like endothelium-derived relaxing factor, had little or no effect on arachidonic acid-induced relaxations. In contrast, nordihydroguaiaretic acid, metyrapone, NG-monomethyl-L-arginine, and nitro-L-arginine all attenuated the relaxation to acetylcholine; however, indomethacin had no effect on acetylcholine-induced relaxations. Arachidonic acid and acetylcholine had no effect on denuded rabbit aorta. Incubation of rabbit aorta with [14C]arachidonic acid resulted in the synthesis of major radioactive metabolites that comigrated with the prostaglandins and hydroxyeicosatetraenoic acids. Indomethacin selectively inhibited prostaglandin formation, nordihydroguaiaretic acid attenuated both prostaglandins and hydroxyeicosatetraenoic acids, and metyrapone blocked the epoxyeicosatrienoic acids. Additionally, acetylcholine elicited a twofold increase in tissue cyclic guanosine monophosphate content in contrast to a 59% reduction in cyclic guanosine monophosphate content observed with arachidonic acid. Therefore, these data suggest that in rabbit aorta, arachidonic acid-induced relaxations are mediated by an endothelium-dependent factor (or factors) that differs from the factor (or factors) released by acetylcholine. These results support the existence of multiple endothelium-derived relaxing factors.     

Aortic response to relaxing agents in Watanabe heritable hyperlipidemic (WHHL) rabbits of different age.

Serum and aortic tissue cholesterol levels in parallel with aortic relaxation to endothelium-dependent and independent drugs were determined in Watanabe heritable hyperlipidemic (WHHL) rabbits in comparison with New Zealand (N.Z.) normocholesterolemic rabbits, aged 4-14 months. Serum cholesterol was elevated (626 +/- 99 mg/100 ml) in 4-6-month-old WHHL rabbits and significantly lower in 12-14-month-old animals (344 +/- 51 mg/100 ml). Cholesterol infiltration in thoracic aorta was high in young WHHL compared with N.Z. rabbits (0.88 +/- 0.3 mg/100 mg fresh tissue vs. 0.08 +/- 0.003 mg/100 mg, respectively) and it did not vary with age. In N.Z. rabbits, serum and aortic cholesterol levels were low from 4 to 14 months of age. The aortic relaxation to acetylcholine (0.03-3 microM) on EC50 noradrenaline precontracted rings was similar in 4-6-month-old WHHL and N.Z. rabbits of the same age. In WHHL rabbits, the relaxation to acetylcholine was significantly reduced in 7-11- (-35% at maximum) and in 12-14-month-old rabbits (-40% at maximum). In N.Z. rabbits the response to acetylcholine was not modified in the 3 age groups. The relaxation to ATP (30 microM to 3 mM) was reduced by age both in N.Z. and in WHHL rabbits, but in 12-14-month-old WHHL rabbits the maximal relaxing response was significantly more elevated than in age-matched N.Z. rabbits (50.1 +/- 2.5% vs. 35.1 +/- 3.2%, respectively). The aortic relaxation to NaNO2 (10 microM to 3 mM) was reduced by age both in N.Z. and in WHHL rabbits.(ABSTRACT TRUNCATED AT 250 WORDS)     

Arginine restores cholinergic relaxation of hypercholesterolemic rabbit thoracic aorta

BACKGROUND. Reduced synthesis of endothelium-derived relaxing factor (EDRF) may explain impaired endothelium-dependent vasodilation in hypercholesterolemia. Accordingly, we designed studies to determine if endothelium-dependent relaxation in hypercholesterolemic rabbits may be restored by supplying L-arginine, the precursor of EDRF. METHODS AND RESULTS. Normal or hypercholesterolemic rabbits received intravenous L-arginine (10 mg/kg/min) or vehicle for 70 minutes. Subsequently, animals were killed, thoracic aortas were harvested, and vascular rings were studied in vitro. Rings were contracted by norepinephrine and relaxed by acetylcholine chloride or sodium nitroprusside. Vasorelaxation was quantified by determining the maximal response (expressed as percent relaxation of the contraction) and the ED50 (dose of drug inducing 50% relaxation; expressed as -log M). In vessels from hypercholesterolemic animals receiving vehicle, there was a fivefold rightward shift in sensitivity to acetylcholine compared with normal animals (p = 0.05, n = 5 in each group). In vessels from hypercholesterolemic animals, L-arginine augmented the maximal response to acetylcholine (83 +/- 16% versus 60 +/- 15%, p = 0.04 versus vehicle) and increased the sensitivity to acetylcholine (ED50 value: 6.7 +/- 0.2 versus 6.2 +/- 0.2, p less than 0.05 versus vehicle). Arginine did not affect maximal and EC50 responses to acetylcholine in vessels from normal animals. Arginine did not potentiate endothelium-independent responses in either group. CONCLUSIONS. We conclude that the endothelium-dependent relaxation is normalized in hypercholesterolemic rabbit thoracic aorta by in vivo exposure to L-arginine, the precursor for EDRF.     

Direct evidence against a role of ATP as the nonadrenergic, noncholinergic inhibitory neurotransmitter in guinea pig tenia coli.

Electrical field stimulation of the isolated guinea pig tenia coli in the presence of a muscarinic receptor antagonist (atropine) and an adrenergic neuron blocker (guanethidine) produces relaxation. A large amount of indirect evidence has suggested that the neurotransmitter that is released from these nonadrenergic, noncholinergic inhibitory neurons is ATP or a related nucleotide, and the nerves have been termed "purinergic." A photoaffinity analog of ATP, arylazido aminopropionyl ATP, which produces a specific pharmacological antagonism of P2 purinergic receptors in isolated guinea pig vas deferens and urinary bladder, was utilized in the present study to evaluate directly whether ATP is the nonadrenergic, noncholinergic inhibitory neurotransmitter in tenia coli. By blocking postjunctional P2 receptors, arylazido aminopropionyl ATP produced a pronounced antagonism of relaxations induced by exogenously added ATP. Responses produced by ADP, AMP, and adenosine also were antagonized by arylazido aminopropionyl ATP, but to a lesser extent. Inhibitory responses to isoproterenol were not antagonized. Under these conditions of established, specific P2-receptor blockade of responses to exogenously added ATP, relaxations induced by field stimulation of intrinsic inhibitory nerves in the presence of atropine (1 microM) and guanethidine (1 microM) were not antagonized. Though these results provide no indication of the actual substance involved, they suggest strongly that the nonadrenergic, noncholinergic inhibitory neurotransmitter in the guinea pig tenia coli is not ATP.