Endothelium-denuded Rings Research Articles

Endothelium-dependent and NO-mediated desensitization to vasopressin in rat aorta.

1. The present study was performed to characterize the tachyphylaxis of rat aortae to vasopressin. Isometric tension generated by rat thoracic aorta sliced in 4 mm rings, was recorded. 2. Tension generated by intact rings increased with cumulative additions of vasopressin up to 10 nM (1.51 +/- 0.15 g). After this concentration, most rings lost their tension and relaxed to 1.09 +/- 0.17 g (P < 0.001) despite further addition of vasopressin. This tachyphylaxis was not observed in endothelium-denuded rings (from 2.87 +/- 0.12 g to 2.68 +/- 0.17 g). 3. Repeated administrations of supramaximal concentration (100 nM) of vasopressin confirmed an enhanced desensitization in intact rings, compared to endothelium-denuded rings. No desensitization to phenylephrine was observed in intact or in endothelium-denuded rings. 4. Dose-response curves to a V1 receptor agonist, [Phe2, Ile3, Orn8]-vasopressin, and to a V2 receptor agonist, [deamino-Cys1,D-Arg8]-vasopressin, were performed in intact preparations. An increase in tension, followed by a desensitization was observed with the V1 receptor agonist. In contrast, the V2 receptor agonist did not induce any response. 5. Pretreatment of intact aortic rings with the cyclo-oxygenase inhibitor, diclofenac (1 microM), did not prevent the desensitization to vasopressin. In contrast, NO synthase inhibition with NG-nitro-L-arginine (30 microM) resulted in an attenuated desensitization to vasopressin in intact rings (from 2.46 +/- 0.17 to 2.25 +/- 0.22 g, NS). 6. To confirm the involvement of NO, endothelium-denuded rings were pretreated with sodium nitroprusside (SNP). At a concentration of 10 nM, SNP induced a desensitization to vasopressin comparable with that observed in intact rings. 7. Pretreatment of endothelium-denuded rings with 8-bromo-cyclic GMP (100 microM) reduced maximum contraction to vasopressin without producing any desensitization. In contrast, guanylate cyclase inhibition with either LY 83,583 (10 microM) or methylene blue (10 microM) blocked completely the desensitization of intact rings to vasopressin. 8. The results suggest that the endothelium-dependent tachyphylaxis to vasopressin is due to rapid desensitization and is mediated by NO. However, it is unclear whether this effect of NO involves cyclic GMP.

Endothelial modulation of alpha 1-adrenoceptor contractile responses in the tail artery of spontaneously hypertensive rats.

1. Vascular contraction induced by phenylephrine was studied in tail artery rings from spontaneously hypertensive (SHR) and Wistar Kyoto rats (WKY) with particular focus on the role of endothelium. The influence of receptor reserve and the density of alpha 1-adrenoceptors on the possible differences observed were also analysed. 2. Phenylephrine (0.01-100 microM) induced concentration-dependent vasoconstrictions. The maximum response (alpha, P < 0.001) was greater but the pEC50 (P < 0.05) smaller in rings from SHR than from WKY rats irrespective of the presence or absence of endothelium. 3. Removal of endothelial cells resulted in a decrease of the maximum contraction with no modification in the pEC50 in arteries from both WKY and SHR. 4. The density of alpha 1-adrenoceptors (Bmax) and the dissociation constant (KD) were found to be the same for preparations from SHR and WKY rats in [3H]-prazosin binding experiments. 5. The apparent affinity (pKA) determined by the nested hyperbolic method and the operational model was similar in tail arteries from the two rat strains, irrespective of the presence or absence of endothelium. However, in endothelium-denuded rings, the pKA value was enhanced when compared with intact rings, in both SHR and WKY rats. 6. In rings from hypertensive rats, the operational parameter maximum possible effect (Em) was greater and the agonist efficacy (tau) was smaller than in rings from normotensive rats. When the endothelium was removed log tau and Em diminished in preparations from both rat strains. 7. In summary, the increased maximum responsiveness to phenylephrine in rings from SHR could be due to enhancement in Em. The log tau values indicate a deterioration in the transduction of the stimulus provided by the agonist in tail arteries from hypertensive animals. This study also suggests that the absence of endothelium modifies the alpha 1-adrenoceptor-mediated vasoconstriction probably by altering the transduction signalling mechanisms. The importance of analysing the degree of endothelium functionality when comparing results from different groups of rats is stated.

Cultured dog aortic endothelial cells release vasodilatory products in response to acetylcholine and adenosine

We tested the hypothesis that products released from contracting skeletal muscle could induce the release of vasodilatory products from vascular endothelium. Superfused endothelium-denuded rabbit aortic and dog femoral artery rings contracted with 1 microM phenylephrine delivered at 1-2 mL/min were subjected to 3-mL bolus challenges that had been briefly exposed to either a culture dish devoid of endothelial cells (control) or a dish containing dog aortic endothelial cells (experimental) immediately prior to their application. Challenges were alternated and included Krebs-Henseleit bicarbonate buffer (Krebs) pH 7.4 (vehicle); 1 microM acetylcholine; and the muscle metabolites 6 mM potassium chloride, 1 mM sodium phosphate monobasic, 10 microM adenosine, acidic Krebs pH 6.8, and 100 microM ammonium chloride. Only the products released from cultured endothelial cells after the addition of acetylcholine and adenosine resulted in a significant relaxation of vascular rings compared with control. Prior incubation of the cultured endothelial cells with 10 microM 8-phenyltheophylline eliminated the relaxation induced by adenosine, and all relaxations were eliminated by prior incubation of the cells with either 10 microM N omega-nitro-L-arginine or a combination of 10 microM indomethacin and 10 microM nordihydroguaiaretic acid. This indicates that at least one metabolite released from contracting skeletal muscle could induce the release of vasodilatory products from the endothelium, which would act as a local amplifier of functional hyperemia.

Endothelin-1 Does Not Mediate the Endothelium-Dependent Hypoxic Contractions of Small Pulmonary Arteries in Rats

Various pulmonary artery preparations in vitro demonstrate sustained endothelium-dependent contractions upon hypoxia. To determine whether endothelin-1 could mediate this phenomenon, we examined the effect of bosentan, a new antagonist of both the ETA and ETB subtypes of the endothelin receptor. Small (300 pm) pulmonary arteries from rats were mounted on a myograph, precontracted with prostaglandin F2 alpha and exposed to hypoxia (PO2, 10 to 15 mm Hg, measured on-line) for 45 min. Endothelium-intact control rings exhibited a biphasic response, with a transient initial vasoconstriction (phase 1) followed by a second slowly developing sustained contraction (phase 2). Expressed in percent of the maximal response to 80 mmol/L KCl, the amplitudes of phase 1 (peak tension) and 2 (tension after 45 min of hypoxia) averaged 37 +/- 12% and 17 +/- 14%, respectively (n = 11). In endothelium-denuded rings, phase 1 persisted while the amplitude of phase 2 was reduced to 2 +/- 12% (p < 0.05, n = 8), showing the endothelium dependence of this contraction. Neither phase was significantly decreased in rings treated with 10(-5) mmol/L bosentan (38 +/- 15% and 17 +/- 12%, respectively, n = 6). The PO2 threshold for onset of hypoxic contraction was not significantly different among these three groups and averaged 32 +/- 24 mm Hg. In a separate experiment, we assessed the inhibitory effect of 10(-5) mol/L bosentan on the response to 10(-8) mol/L endothelin-I. Rings treated for 45 min with 10(-8) mol/L endothelin-1 alone exhibited a maximal contraction of 75 +/- 27% (n = 6). This was reduced to 4 +/- 17% (p < 0.01, n = 6) in rings treated with both 10(-8) mol/L endothelin-1 and 10(-5) mol/L bosentan. We conclude that complete blockade of all endothelin receptor subtypes has no effect on either endothelium-dependent or -independent hypoxic contractions in this preparation. This suggests that endothelial factors other than endothelin-I mediate the acute hypoxic contractions of small pulmonary arteries in the rat.

Hypoxia impairs nitrovasodilator-induced pulmonary vasodilation: role of Na-K-ATPase activity.

To elucidate the effect of hypoxia on nitrovasodilator-induced pulmonary vasodilation, we studied canine pulmonary arterial rings under isometric conditions in vitro. Exposure to hypoxia inhibited the relaxant responses of KCl-contracted tissues to sodium nitroprusside (SNP), so that the maximal relaxation (Emax) and the negative logarithm of molar concentration required to produce 50% relaxation (pD2) were decreased from 92 +/- 7 to 62 +/- 5% and from 5.8 +/- 0.2 to 4.7 +/- 0.3, respectively (means +/- SE, P < 0.01 for each). This effect was likewise observed when 8-bromoguanosine-3',5'-cyclic monophosphate was used as a relaxant. The impairment of SNP-induced relaxation of endothelium-denuded rings under hypoxia was abolished by ouabain or K(+)-free solution. Incubation with SNPincreased intracellular cGMP contents in a dose dependent manner, an effect that was not altered by hypoxia. SNP also increased ouabain-sensitive 86Rb uptake, and this effect was inhibited by hypoxia. These results suggest that hypoxia reduces nitrovasodilator-induced relaxation of pulmonary artery, probably through an inhibition of cGMP-dependent sarcolemmal Na-K-adenosine triphosphatase activity.

Endothelins contract guinea-pig pulmonary artery and enhance its adrenergic response via ET(A) receptors.

1. This study has pharmacologically characterized endothelin (ET) receptor subtype(s) mediating contraction and enhancement of adrenergic contraction in guinea-pig pulmonary artery. Isometric tension of the isolated endothelium-denuded ring preparations was measured in the presence of indomethacin (10(-5) mol/L) and N(G)-nitro-L-arginine methyl ester (L-NAME; 3 x 10(4) mol/L) to exclude a mechanism via endothelium, cyclo-oxygenase-generated eicosanoids and nitric oxide. 2. In the additional presence of tetrodotoxin (TTX; 3 x 10(-7) mol/L), ET-1 (10(-11)-10(-7) mol/L) concentration-dependently contracted the preparations. The rank order of potency to contract the preparations among ET receptor agonists was ET-1, sarafotoxin (STX)6b > ET-3 > IRL 1620, STX 6c. BQ-123 (7 x 10(-7)-7 x 10(-6) mol/L) concentrations-dependently shifted the concentration-contraction curve for ET-1 to the right in a parallel manner. Pretreatment with STX 6c (3 x 10(-7) mol/L for 30 min) did not significantly desensitize contractions to ET-1, ET-3 or IRL 1620 (P > 0.05; t-test, 10 d.f). 3. ET-1 (10(-10)-10(-9) mol/L) and STX 6b (10(-9)-10(-8) mol/L) significantly enhanced the electrical field stimulation-induced contraction in a BQ-123-sensitive manner (P < 0.05: t-test, 24-38 d.f), while ET-3 (10(-11)-10(-8) mol/L) and STX 6c (10(-11)-10(-7) mol/L) did not affect contractions. ET-1 (10(-11) mol/L) significantly enhanced contractions to exogenous noradrenaline in the presence of TTX (3 x 10(-7) mol/L) (P < 0.05; t-test, 16 d.f.). 4. These data indicate that the BQ-123-sensitive ET(A) receptor mediates both contraction and enhancement of adrenergic contractions in the guinea-pig pulmonary artery.

Halothane and Isoflurane Inhibit Vasodilation Due to Constitutive but Not Inducible Nitric Oxide Synthase

Inhalational anesthetics inhibit the nitric oxide-guanylyl cyclase signaling pathway, but the site of this inhibition is not yet clear. This study was designed to test the hypothesis that receptor activation or downstream signaling events leading to nitric oxide synthase activation are important sites for this inhibition by comparing the effect of anesthetics on vasodilation caused by the calcium-dependent constitutive endothelial nitric oxide synthase versus the calcium-independent inducible nitric oxide synthase. Endothelium-intact or -denuded rat thoracic aorta rings preincubated with or without lipopolysaccharide were mounted for isometric tension measurement, constricted with phenylephrine, then relaxed with methacholine in the presence or absence of halothane (1-3%) or isoflurane (1-3%). The cyclic guanosine 3,5-monophosphate content in the endothelium-denuded rings preincubated with or without lipopolysaccharide in the presence or absence of 3% halothane or 3% isoflurane was quantified by radioimmunoassay. The activity of partially purified inducible nitric oxide synthase from activated mouse macrophage was assayed in the presence or absence of halothane (1-4%) or isoflurane (1-5%) by the conversion of 3H-L-arginine to 3H-L-citrulline. Halothane and isoflurane inhibited methacholine-stimulated, nitric oxide-mediated vasorelaxation in endothelium-intact aortic rings. Neither halothane nor isoflurane affected the vasorelaxation caused by basal endothelial nitric oxide synthase or inducible nitric oxide synthase activity. Neither anesthetic altered the cyclic guanosine 3,5-monophosphate increase caused by inducible nitric oxide synthase in the lipopolysaccharide-treated rings. The results demonstrated that halothane and isoflurane inhibit only receptor/calcium-activated nitric oxide synthase action and that direct inhibition of nitric oxide synthase, soluble guanylyl cyclase, or an interaction with nitric oxide are not responsible for anesthetic inhibition of endothelium-dependent vasorelaxation.

GR127935 is a potent antagonist of the 5-HT 1-like receptor mediating contraction in the canine coronary artery

The effects of the recently developed 5-HT id receptor antagonist, GR127935 ( N-[4-methoxy-3-(4-methyl-1-piperazinyl)phenyl]-2′-methyl-4′-(5-methyl-1,2,4-oxadiazol-3-yl)[1,1,-biphenyl]-4-carboxamide), and those of the preferential human 5-HT 1Da receptor antagonist, ketanserin, on the isometric contraction induced by 5-hydroxytryptamine (5-HT) and sumatriptan in endothelium-denuded ring segments of canine coronary artery were analyzed. Sumatriptan mimicked 5-HT with lower potency but similar efficacy. GR127935 (1, 3 and 10 nM) concentration dependently antagonized the contractions elicited by both agonists; only the 5-HT maximum was reduced. Ketanserin and mianserin (both at 1 μM) were inactive. These data strongly suggest that a 5-HT id receptor mediates contraction in the dog coronary artery. The possibility that this 5-HT id receptor resembles the cloned human 5-HT idβ subtype is discussed.

Vascular action of bradykinin is mediated by B2-subtype receptors in the canine lingual artery.

The mechanisms underlying the responses of the canine lingual artery to bradykinin (BK) have been examined. In endothelium-intact lingual artery ring preparations contracted to a stable plateau tension by the addition of norepinephrine (NE), BK produced B2-receptor antagonist [D-Phe7]-BK-sensitive and endothelium-dependent relaxations. Theobserved effect was significantly inhibited by nitric oxide (NO) inactivator hemoglobin, methylene blue, a good inhibitor of soluble guanylate cyclase, and by NG-monomethyl-L-arginine, a NO synthase inhibitor, but not by B1-receptor antagonist des-Arg9-[Leu8]-BK, suggesting endothelial production of the vasodilator agent NO by B2-receptors activation. In endotheliumdenuded rings in the absence of active tension caused by NE, BK increased muscle tension and cytosolic Ca2+ level; these were inhibited by [D-Phe7]-BK. The concentration-dependent contraction produced by BK of endothelium-denuded rings was also attenuated by [D-Phe7]-BK; protein kinase C inhibitor H-7 and phospholipase C inhibitor neomycin B reduced the BK-induced contraction. Indomethacin was without effect. Hence, it can be concluded that BK can cause relaxation and contraction in the canine lingual artery via B2-receptors activation. The relaxation response to BK might be mediated by endothelial production of No.BK has the potential to be a vasoconstrictor by exerting an effect on vascular smooth muscle cells, but not on endothelial cells, via B2-receptors activation. It is also postulated that the contractile response to BK may be due to the production of second messengers derived from the action of phospholipase C, but not due to contractile prostanoids, when B2-receptors are activated.

Effects of Acute Acetaldehyde, Chronic Ethanol, and Pargyline Treatment on Agonist Responses of the Rat Aorta

The purpose of this investigation was to determine the mechanism(s) underlying the vasorelaxant effects of acute versus chronic acetaldehyde (ACA) exposure, in particular, the role of intact endothelium on contractile responses of rat aortic ring segments to potassium chloride (KCl) or norepinephrine (NE). The acute effects of ACA were assessed in preparations from normal animals maintained on a standard rat chow (non-ethanol-ingesting). The monamine oxidase inhibitor, pargyline, elevates plasma ACA levels by decreasing acetaldehyde dehydrogenase activity. Accordingly, preparations from pargyline-treated ethanol-ingesting animals (PE) were used to assess the effects of chronic (12 weeks) ACA and results were compared to those of pargyline-treated non-ethanol-ingesting (P) rats fed a standard liquid diet. In normal and P preparations, maximal inotropic response to KCl and NE were greater in endothelium-denuded than in endothelium-intact preparations. However, in aortic rings obtained from PE rats, the maximal inotropic response to KCl was depressed only in endothelium-denuded rings and that to NE was nearly identical in endothelium-denuded compared to endothelium-intact preparations. Acute ACA (30 mM) exposure significantly reduced both NE- and KCl-induced contractile responses in muscles from all groups. The magnitude of the vasorelaxant effect of this [ACA] on NE-induced responses was endothelium-independent and was similar between groups. However, the vasorelaxant effect of ACA (30 mM) on KCl-induced contractile responses was significantly attenuated in muscles from PE animals with greater inhibition occurring in endothelium-denuded preparations. These results suggest that chronic acetaldehyde exposure leads to an impairment in the inotropic response to membrane depolarization in endothelium-denuded preparations resulting in depressed responsiveness. In addition, the acute vasorelaxant effect of ACA on KCl-induced contractures is significantly attenuated in preparations chronically exposed to ACA which suggests a possible development of tolerance.

Lack of endotoxin-induced hyporesponsiveness to U46619 in isolated neonatal porcine pulmonary but not mesenteric arteries.

The effects of endotoxin from Escherichia coli on the vasoconstrictor responses to noradrenaline (10 nM-100 microM) and the thromboxane A2 analog U46619 (100 pM-1 microM) were evaluated on isolated pulmonary and mesenteric arteries from neonatal piglets. Incubation for 20 h with endotoxin (1 microgram ml-1) induced a decrease in the contractile responses to noradrenaline in both arteries (p < 0.05) which was inhibited by NG-nitro-L-arginine-methyl ester (L-NAME, 100 microM). Endotoxin-treated mesenteric arteries also showed a reduction of the maximal contractions induced by U46619 (p < 0.05) and this effect was inhibited by L-NAME. In contrast, the contractile responses to U46619 were similar in control and endotoxin-treated pulmonary arteries. In endothelium-denuded pulmonary rings, endotoxin was also unable to modify the contractile responses to U46619. In pulmonary rings, the contractions induced by U46619 (100 nM) were much less sensitive to sodium nitroprusside, 8-bromo-cyclic GMP or dipyridamole than those induced by 10 microM noradrenaline. In conclusion, endotoxin-treated pulmonary arteries exhibited decreased responses to noradrenaline due to enhanced nitric oxide release but not to the thromboxane A2 analog U46619. This lack of hyporesponsiveness to U46619 in pulmonary arteries may be attributed to a relative insensitivity to nitric oxide. The absence of pulmonary hyporesponsiveness to U46619 may explain why pulmonary hypertension occurs in septic shock despite Ca(2+)-independent nitric oxide synthase induction in the lung.

Distribution of Endothelin-1-Receptor Subtypes in Rat Portal Vein

Endothelin-1 (ET-1), a potent vasoactive peptide, was first isolated from cultured porcine endothelial cells. Subsequent studies revealed the existence of two additional related peptides, ET-2 and ET-3, and at least two distinct ET-receptor subtypes, ETA (selective for ET-1) and ETB (nonselective for ET isopeptides). These isopeptides and receptors are widely distributed in many tissues and are involved in numerous biological responses. The aim of this study was to identify the eventual distribution of the two distinct endothelin-receptor subtypes in isolated endothelium-denuded rat portal vein rings (PVRs) and strips (PVSs). BQ-123 (0.6, 1, and 6 microM) and PD-145065 (0.06, 0.1, 0.6, and 6 microM) were used to differentiate the subtypes because they are selective antagonists for ETA and nonselective antagonists for ETA-ETB receptors, respectively. To characterize the ET receptors further, sarafotoxin-S6c (a selective ETB-receptor agonist) and IRL-1038 (a selective ETB-receptor antagonist) were used. In PVRs, cumulative additions of ET-1 (0.1-100 nM) caused graded and slow contractions and potentiated spontaneous rhythmic contractions. The EC50 values and maximal response to 100 nM of ET-1 were 2.72 nM and 0.75 g, respectively (n = 7). PVSs showed ET-1 EC50 values very similar to those of PVRs, but Emax values to 100 nM of ET-1 were significantly lower (Emax = 0.33 g; n = 7). Moreover, ET-1 clearly increased the amplitude and frequency of spontaneous contractions in both types of specimens, although these were greater in the PVSs. Thirty-minute incubation with the selective ETA-receptor antagonist BQ-123 blunted ET-1-induced effects in PVS specimens but only weakly antagonized ET-1-induced contractions in PVRs. In contrast, the nonselective ETA-ETB-receptor antagonist PD-145065 significantly shifted the ET-1 concentration-response curve to the right in PVRs and partially inhibited ET-1 effects in PVSs. Moreover, sarafotoxin-S6c (0.1-100 nM) contracted PVRs and PVSs in a similar manner to ET-1; its effects were antagonized by IRL-1038 only at the PVR level. The differences observed in PVR and PVS specimens in response to agonists and antagonists of ET confirmed the great heterogeneity of endothelin-sarafotoxin receptors. In our experimental models, functionally ETB-like (or non-ETA) receptors seem mostly to mediate vasoconstriction.

Short exposure to endotoxin increases the constriction induced by angiotensin II in rat aorta.

The contraction elicited by angiotensin II (ANG II) was studied by using standard isometric tension techniques in aortic rings exposed for 1 h to 1 or 10 micrograms/ml Escherichia coli lipopolysaccharide endotoxin (LPS). This contraction was 18 and 71% greater for the two doses of LPS, respectively, than in unexposed control rings. In endothelium-denuded rings, the LPS-induced increase in contraction in response to ANG II was completely abolished. Because the contraction induced by ANG II is modulated by the simultaneous release of prostaglandins, we tested the hypothesis that LPS interferes with this modulation. We found that the LPS-induced increase in contraction to ANG II was inhibited in the presence of the cyclooxygenase inhibitor indomethacin (10(-5) M) or the prostaglandin H2/thromboxane A2-receptor antagonist SQ-29548 (2 x 10(-7) M). Conversely, the LPS-induced increase in contraction in response to ANG II was not inhibited by the presence of dexamethasone (10(-6) M), which inhibits new protein synthesis. In addition, there was no loss of vasodilator response to the endothelium-dependent receptor agonist acetylcholine (10(-8)-10(-4) M) or in the constrictor responses to norepinephrine (10(-9)-10(-5) M) and KCl (20-100 mM). We conclude that short exposure to LPS produces a specific increase in the constrictor response to ANG II via mechanisms mediated by prostaglandin H2/thromboxane A2. This effect could be a LPS-induced shift in favor of constrictor prostanoids in the balance of dilator/constrictor prostanoids, the release of which is associated with stimulation by ANG II.

Effects of U46619 on contractions to 5-HT, sumatriptan and methysergide in canine coronary artery and saphenous vein in vitro.

1. The aim of this study was to investigate the mechanism of enhanced reactivity to 5'-hydroxytryptamine (5-HT) and sumatriptan previously observed in human isolated coronary arteries when active force was raised with the thromboxane A2-mimetic, U46619. 2. Ring segments of dog isolated coronary artery and saphenous vein were suspended in organ baths and cumulative concentration-contraction curves to 5-HT, sumatriptan and methysergide were constructed in the absence and presence of low concentrations of U46619. 3. In both endothelium-intact and endothelium-denuded rings of coronary artery, precontraction with U46619 to low (< 10% Fmax; the contraction to a maximum depolarizing 125 mM KCl Krebs solution; KPSS) levels of active force had no effect on either the maximum contraction or sensitivity (pEC50) to 5-HT, sumatriptan and methysergide. 4. Ketanserin (1 microM) had no effect on contractions to sumatriptan and methysergide in endothelium-denuded coronary artery rings, but reduced the maximum contraction to 5-HT by approximately 90% to a value (5% Fmax) similar to that for sumatriptan and methylsergide. Under these conditions, U46619 precontraction had no effect on either pEC50 or maximum for 5-HT, sumatriptan or methysergide. 5. In rings of saphenous vein with endothelium and treated with ketanserin (1 microM), 5-HT and sumatriptan caused equal maximum responses of 65% Fmax which were approximately double that of methysergide (32% Fmax). The maximum responses and sensitivity to 5-HT, sumatriptan, methysergide and noradrenaline were unaffected by precontraction with U46619. 6. Pretreatment of the saphenous vein with sodium nitroprusside (SNP; 10 microM) caused a small sustained relaxation and significantly depressed the maximal contraction to 5-HT without affecting sensitivity and abolished the contraction curve to sumatriptan and methysergide. When the relaxation response to SNP was reversed with U46619 (1-4 nM), the contraction curves to 5-HT, sumatriptan and methysergide were similar to those obtained prior to relaxation with SNP. In contrast, the same treatment with SNP had little affect on the contraction curve to noradrenaline.7 In conclusion, the pattern of U46619-enhanced reactivity of 5-HT, sumatriptan and methysergide in SNP-treated dog saphenous vein, highlights the importance of functional antagonism when assessing reactivity to contractile agonists in isolated blood vessels.

The role of increased nitric oxide in the vascular hyporeactivity to noradrenaline in long-term portal vein ligated rats

To test the possible role of nitric oxide production in long-term portal vein ligation in the rat, where the hyperdynamic circulation was reported to be absent, in vivo experiments on isolated thoracic aortic rings from partial portal vein ligated or sham-operated rats were performed, 6 months postoperatively. The concentration-response curves to noradrenaline ofboth intact and endothelium-denuded rings from portal hypertensive rats were significantly shifted to the right as compared to those from sham-operated animals. In intact rings, addition of N G-nitro-L-arginine, a specific inhibitor of nitric oxide synthase, resulted in a significant shift of the curves to the left in sham-operated and portal vein ligated rats. In endothelium-denuded rings, addition of N G-nitro-L-arginine resulted in a significant shift of the curves to the left in portal vein ligated but not in sham-operated animals. After blockade of the nitric oxide biosynthesis with N G-nitro-L-arginine, the negative logarithm of the concentration of nonadrenaline causing half-maximal response did not significantly differ any more between portal vein ligated and sham-operated rats; in endothelium-denuded rings hyporeactivity to noradrenaline persisted in portal vein ligated rats. Only in the intact rings did N G-nitro-L-arginine significantly increase the maximal contractions. No differences were demonstrated in endothelium-dependent relaxations to acetylcholine between sham-operated and portal hypertensive animals. From these results, it can be concluded that in vitroaortic hyporeactivity to noradrenaline is still present in long-term portal vein ligated rats, and that it results at least partially from activation of the L-arginine: nitric oxide pathway in the aortic vascular wall.

A comparison of the contractile effects of 5-hydroxytryptamine, sumatriptan and MK-462 on human coronary artery in vitro.

1. MK-462 (N,N-dimethyl-2-[5-(1,2,4-triazol-1-ylmethyl)-1H- indol-3-yl]ethylamine) is a novel selective 5-HT1D-receptor agonist which in clinical trials has been shown to be an effective antimigraine agent. As angiographic studies have shown that sumatriptan (an established 5-HT1D-receptor agonist) can cause coronary artery vasoconstriction in patients, we compared the effects of MK-462 with those of 5-HT and those of sumatriptan, on isolated segments of human coronary artery in vitro. 2. Coronary arteries were obtained from explanted hearts from patients (n = 22, 2 females, 20 males, aged 21-60 years) undergoing cardiac transplantation. Endothelium-denuded ring segments of coronary artery, 2mm long were mounted in organbaths for isometric tension recording. For each arterial ring segment, a cumulative concentration-effect curve to either 5-HT, sumatriptan or MK-462 was determined. After maximal response to each agonist had been obtained, ketanserin (a 5-HT2 receptor antagonist) 0.6 microM was added to the tissue bath, followed by methiotepin (0.6 microM) and the reduction in tension produced by the addition of each antagonist was determined. 3. Out of 22 coronary arteries studied, only 10 showed any response (contraction) to 5-HT. Not all arteries which responded to 5-HT contracted in response to both sumatriptan and MK-462 (one ring from each artery being exposed to a single agonist in each case).(ABSTRACT TRUNCATED AT 250 WORDS)

Differential sensitivity of basal and acetylcholine-stimulated activity of nitric oxide to destruction by superoxide anion in rat aorta.

1. In this study we compared the ability of superoxide anion to destroy the relaxant activity of basal and acetylcholine (ACh)-stimulated activity of NO in isolated rings of rat aorta. 2. Superoxide dismutase (SOD, 1-300 u ml-1) induced a concentration-dependent relaxation of phenylephrine (PE)-induced tone in endothelium-containing rings which was blocked by NG-nitro-L-arginine (L-NOARG, 30 microM), but had no effect on endothelium-denuded rings. It was likely therefore that the relaxant action of SOD resulted from protection of basally produced NO from destruction by superoxide anion, generated either within the tissue or in the oxygenated Krebs solution. 3. In contrast, a concentration of SOD (50 u ml-1) which produced almost maximal enhancement of basal NO activity, had no effect on ACh (10 nM-3 microM)-induced relaxation. 4. In the presence of catalase (3000 u ml-1) to prevent the actions of hydrogen peroxide, superoxide anion generation using hypoxanthine (HX, 0.1 mM)/xanthine oxidase (XO, 16 mu ml-1) produced an augmentation of PE-induced tone in endothelium-containing but not endothelium-denuded rings. This was likely to have resulted from removal of the tonic vasodilator action of basally-produced NO by superoxide anion, since it was blocked in tissues treated with SOD (250 u ml-1), NG-monomethyl-L-arginine (L-NMMA, 30 microM) or L-NOARG (30 microM). Pyrogallol (0.1 mM) had a similar action to HX/XO, but produced an additional augmentation of tone by an endothelium-independent mechanism. 5. In contrast to their ability to destroy almost completely the basal activity of NO, HX (0.1 mM)/XO(16 mu ml-1) and pyrogallol (0.1 mM) had no effect on ACh-induced relaxation at any concentration. An increase in the concentration of HX to 1 mM or pyrogallol to 0.3 mM did, however, lead to a profound decrease in the magnitude and time course of ACh-induced relaxation at all concentrations.6. Treatment with diethyldithiocarbamate (DETCA, 0.1 mM, 1 h) to inhibit endogenous Cu-Zn SOD,augmented PE-induced tone in endothelium-containing rings and abolished the ability of HX (0.1 mM)/XO (16 mu ml-1) and L-NMMA (30 microM) to augment tone. It was likely that DETCA had led to the destruction of basal NO activity by increasing superoxide anion levels since its actions were reversed by exogenous SOD (10-300 upsilon ml-1).7. In contrast to its ability to destroy basal activity of NO completely, DETCA (0.1 mM) produced only a slight blockade of ACh-induced relaxation. However, if these tissues were subsequently treated with concentrations of HX (0.1 mM)/XO (16 mu ml-1) or pyrogallol (0.1 mM), which had no effect by themselves on ACh-induced relaxation, a profound blockade was seen and this was reversed completely with SOD (250 u ml-1).8. The data suggest that basal activity of NO is more sensitive to inactivation by superoxide anion than ACh-stimulated activity and this probably results from differential protection by endogenous Cu-ZnSOD. It is possible therefore that endogenous SOD lowers superoxide anion levels to such an extent that only small amounts of NO, such as those produced under basal conditions, are destroyed. Following generation of superoxide anion with HX/XO or pyrogallol, or inhibition of Cu-Zn SOD with DETCA,levels of the free radical will increase such that greater amounts of NO, such as those produced following stimulation with ACh, will then be destroyed.

Inducible L-arginine/nitric oxide pathway in human internal mammary artery and saphenous vein

To characterize the L-arginine/nitric oxide (NO) pathway in human vascular smooth muscle (VSM), contractile responses of isolated internal mammary arteries (IMA) and saphenous veins (SV) were observed after induction of NO synthase by interleukin-1 beta (IL-1 beta) or by lipopolysaccharide (LPS). In IL-1 beta-treated endothelium-denuded rings, contractile responses to phenylephrine were reduced in SV rings only. Maximum phenylephrine-induced contraction was depressed by approximately 50%. This was not modified by the presence of indomethacin, NG-nitro-L-arginine methyl ester (L-NAME), or methylene blue (MeB). In LPS-treated vessels, contractile responses were depressed in both SV and IMA rings (40%), and this was not affected by indomethacin. In SV, L-NAME, NG-monomethyl-L-arginine, or MeB did not affect the inhibitory effect of LPS, whereas the effect was reversed in IMA by these inhibitors. In LPS-treated IMA, but not in SV, exogenous L-arginine evoked significant vasodilation (20%). We conclude that VSM of the human IMA possesses an L-arginine/NO pathway inducible by LPS. In SV, LPS or IL-1 beta treatment inhibits contraction by an unidentified system that is not dependent on NO synthase or on guanylate cyclase activities.

Calcitonin gene-related peptide mediated neurogenic vasorelaxation in the isolated canine lingual artery.

The nature of neurogenic relaxation was investigated in ring preparations of canine lingual artery. In all experiments, the preparations were previously treated with guanethidine (5 x 10(-6) M) to block neurogenic constrictor responses. In the presence of norepinephrine (10(-5) M) to induce tone, electrical stimulation (10 V, 4 to 16 Hz, for 45 sec) produced relaxation of the rings in an endothelium-independent fashion. The relaxant response in endothelium-denuded rings was not changed by propranolol (10(-5) M), and atropine (10(-5) M) did not affect the relaxation elicited by electrical stimulation in endothelium-intact rings. NG-monomethyl-L-arginine (10(-4) M) or NG-nitro-L-arginine methyl ester (10(-4) M), a nitric oxide (NO) synthase inhibitor, had no effect on the electrical stimulation-induced relaxation of endothelium-denuded rings. Human calcitonin gene-related peptide (CGRP)-(8 - 37) (2 x 10(-8) M), a CGRP1-receptor antagonist, inhibited neurogenic relaxation of endothelium-denuded rings; substance P (10(-6) M) failed to mimic the observed effect of electrical stimulation. The demonstrated effect of electrical stimulation was inhibited by glibenclamide (10(-5) M), but not tetraethylammonium (2 x 10(-4) M); glibenclamide abolished the relaxation in response to exogenous CGRP or the ATP-sensitive K+ channel opener cromakalim (10(-6) M) in endothelium-denuded rings. Moreover, tetrodotoxin (3.13 x 10(-6) M) inhibited the relaxation of endothelium-denuded rings induced by electrical stimulation. The relaxation was selectively inhibited when endogenous CGRP had been depleted from perivascular nerves by capsaicin (10(-6) M). These results suggest that CGRP, but not NO, released from non-adrenergic non-cholinergic nerves by electrical stimulation produces relaxation of canine lingual artery that is mediated by activation of CGRP1 receptors.

Role of nitric oxide in hyporeactivity to noradrenaline of isolated aortic rings in portal hypertensive rats

To test the hypothesis that induction of nitric oxide synthase causes systemic vascular hyporesponsiveness to vasopressors in portal hypertension, we performed in vitro experiments on isolated thoracic aortic rings from partial portal vein ligated or sham operated rats at 3 weeks postoperatively. The concentration-response curves to noradrenaline of intact and endothelium-denuded aortic rings from portal hypertensive rats were significantly shifted to the right as compared to those from sham operated animals. Maximal contractions did not significantly differ. Addition of N G-nitro- l-arginine, a specific inhibitor of nitric oxide synthase, shifted the curves to the left in both sham operated and portal hypertensive rats, so that in intact rings, the concentrations of noradrenaline producing half-maximal response did not significantly differ any more between sham operated and portal vein ligated rats. In endothelium-denuded rings, a hyporeactivity to noradrenaline persisted in portal vein ligated rats. Furthermore, N G-nitro- l-arginine induced an additional significant increase in the maximal response to noradrenaline in sham operated as compared to portal hypertensive rats. The endothelium-dependent relaxations to acetylcholine were attenuated in portal hypertensive rats as compared to sham operated animals. From these results, it can be concluded that increased nitric oxide production in the vascular wall of thoracic aorta of portal hypertensive rats is involved in their hyporesponsiveness to noradrenaline. Our findings in endothelium-denuded rings indicate the involvement of the inducible nitric oxide synthase in the smooth muscle layer. Involvement of an inducible nitric oxide synthase in the endothelium cannot be excluded. The endothelial constitutive nitric oxide synthase, however, seems to be suppressed in portal vein ligated rats.