Relaxation Of Rabbit Aortic Rings Research Articles

The impairment of endothelium-dependent arterial relaxation by 7-ketocholesterol is associated with an early activation of protein kinase C.

Among components of oxidized low density lipoproteins, cholesterol derivatives oxidized in position 7 inhibit endothelium-dependent arterial relaxation by decreasing the release of the main endothelium-derived relaxing factor, nitric oxide (NO). The aim of the present study was to bring new insights into the molecular mechanism by which 7-ketocholesterol can block the endothelium-dependent arterial relaxation. Superoxide dismutase did not prevent the inhibitory effect of 7-ketocholesterol on endothelium-dependent relaxation, and consistent observations were made whether superoxide dismutase was conjugated or not to polyethylene glycol. In addition, neither glutathione supplementation, nor oxypurinol, i.e. a xanthine oxidase inhibitor could reverse the effect of 7-ketocholesterol, indicating that NO was not inactivated by superoxide anion. A direct alteration of the activity of the calcium-dependent NO synthase could also be ruled out, since identical relaxing effects of the calcium ionophore A23187 were observed whether arterial rings were treated or not with 7-ketocholesterol. 4 Whereas the above observations come in support of an early, inhibitory action of 7-ketocholesterol, the specific blockade of one given subtype of membrane receptors could be discarded, and similar inhibitions were observed when either muscarinic or purinergic receptors were stimulated. Finally, the blockade of protein kinase C activity by chelerythrine arose as the sole relevant tool in preventing the effect of 7-ketocholesterol on the endothelium-dependent relaxation of rabbit aortic rings. In addition, complementary studies on cultured bovine aortic endothelial cells came in direct support of the ability of 7-ketocholesterol to activate PKC. In conclusion, 7-ketocholesterol that is present in human hypercholesterolaemic plasma, in atherosclerotic arteries, and in many processed foods can block the release of NO by vascular endothelial cells through its ability to activate PKC.

Divergent effects of vitamin C on relaxations of rabbit aortic rings to acetylcholine and NO-donors.

1. Vitamin C may influence NO-dependent relaxation independently of effects on oxidant stress. 2. We investigated effects of vitamin C (0.1 -- 10 mmol l(-1)) on relaxation of pre-constricted rabbit aortic rings to acetylcholine (ACh), authentic NO and the NO-donors glyceryl trinitrate (GTN), nitroprusside (NP) and S-nitroso-N-acetyl-penicillamine (SNAP). DETCA (2 -- 6 mmol l(-1)), a cell permeable inhibitor of endogenous Cu-Zn superoxide dismutase (SOD) was used to increase intracellular superoxide anion (O(2)(-)). 3. Vitamin C reduced the response to ACh (71 +/- 7% inhibition of maximum relaxation at 10 mmol l(-1)) and inhibited relaxation to authentic NO. Vitamin C inhibited relaxation to GTN but potentiated relaxations to NP and SNAP, causing a parallel shift to a lower concentration range of the log dose-response curve by approximately one log unit at the highest dose. 4. Vitamin C increased the concentration of NO in bath solution (plus EDTA, 1.0 mmol l(-1)) following the addition of SNAP from 53 +/- 14 to 771 +/- 101 nmol l(-1) over the range 0.1-3.0 mmol l(-1). 5. DETCA inhibited relaxation to ACh (71 +/- 9% inhibition of maximum relaxation). This inhibition was abolished by a cell permeable SOD mimetic, but not by vitamin C. DETCA inhibited relaxation to SNAP but not that to NP nor to GTN. 6. Vitamin C inhibits endothelium-dependent relaxations of rabbit aortic rings to ACh and authentic NO and does not reverse impaired relaxation resulting from increased intracellular oxidant stress. Vitamin C potentiates relaxation to the NO-donors NP and SNAP by a mechanism that could involve release of NO from nitrosothiols.

Effects of nonintermittent treatment of rabbits with pentaerythritol tetranitrate on vascular reactivity and superoxide production

Pentaerythritol tetranitrate is an organic nitrate ester that undergoes metabolization to pentaerythritol, pentaerythritol trinitrate, pentaerythritol dinitrate and pentaerythritol mononitrate. Recent data suggested that pentaerythritol tetranitrate is endowed with vasoprotective activities in experimental atherosclerosis. This study was undertaken to gain insight into the underlying mechanism. The basic mechanism of action of all pentaerythritol nitrates was evaluated by measuring liberation of nitric oxide (NO), stimulation of human soluble guanylate cyclase and vasorelaxation in rabbit aorta. A subsequent in vivo study in New Zealand White rabbits was performed to investigate the effects of a 4 months lasting nonintermittent oral treatment with 6 mg pentaerythritol tetranitrate kg −1 day −1 on vascular superoxide production, endothelium dependent vasorelaxation and vasorelaxation to pentaerythritol tetranitrate itself. The formation rates of NO from the pentaerythritol nitrates (100 μM, n=5) in presence of 5 mM cystein were (in nM min −1): 62.1±3.2 (pentaerythritol tetranitrate), 21.3±0.9 (pentaerythritol trinitrate), 6.4±0.6 (pentaerythritol dinitrate) and 3.2±0.4 (pentaerythritol mononitrate). Similarly, the pD 2 values (−log M) for half-maximal activation of soluble guanylate cyclase decreased from pentaerythritol tetranitrate (3.391±0.09, n=4) to pentaerythritol mononitrate (2.655±0.04, n=3) as did the p D 2 values (in −log M) for half-maximal relaxation of rabbit aortic rings ( n=7) from pentaerythritol tetranitrate (8.3±0.17) to pentaerythritol mononitrate (5.0±0.11). Significant correlations were found between the NO formation rates and the p D 2 values for enzyme stimulation ( r=0.98, P=0.002) and vasorelaxation ( r=0.90, P=0.049) suggesting that these effects of the pentaerythritol nitrates were mediated by NO. The results of the in vivo study showed that aging induces a significant increase of aortic superoxide production (median values, n=10) from 2.45 nM mg −1 min −1 (age 7 months) to 3.39 nM mg −1 min −1 (age 11 months, P<0.01) that was prevented by concurrent treatment with pentaerythritol tetranitrate (2.76 nM mg −1 min −1). In vitro vasorelaxation to pentaerythritol tetranitrate was identical in all groups indicating absence of nitrate tolerance. Endothelium-dependent vasorelaxation was also identical in all groups. These data suggest that oral treatment with pentaerythritol tetranitrate reduces vascular oxidant stress by an NO-dependent pathway, which may contribute to the vasoprotective activity of pentaerythritol tetranitrate in experimental atherosclerosis.

Dietary L-arginine normalizes endothelin-induced vascular contractions in cholesterol-fed rabbits.

The endothelium regulates vascular function by releasing the vasodilator autacoid nitric oxide (NO) and the vasoconstrictor peptide endothelin-1 (ET-1). Impaired activity of NO as well as excessive activity of ET-1 have been demonstrated in hypercholesterolemia and atherosclerosis. Because dietary L-arginine can restore NO function and improve abnormal endothelium-dependent relaxation in hypercholesterolemic rabbits, we examined the effects of dietary supplementation with L-arginine in cholesterol-fed rabbits on endothelium-dependent vascular relaxation and ET-1-induced vascular contraction, as well as the systemic synthesis of ET-1. Rabbits were initially fed a diet enriched with 1% cholesterol for 4 weeks, followed by 0.5% cholesterol alone or supplemented with 2% L-arginine in drinking water during the next 12 weeks. Cholesterol feeding impaired endothelium-dependent relaxation of rabbit aortic rings ex vivo and increased urinary immunoreactive ET-1 excretion, along with decreased urinary nitrate excretion, an index of NO production. L-Arginine partially restored endothelium-dependent relaxation in parallel to increased urinary nitrate excretion and decreased urinary immunoreactive ET-1 excretion. Selective inhibition of ET-A receptors with BQ123 partially restored endothelium-dependent relaxation in hypercholesterolemic rabbits but had no effect on arterial rings from rabbits supplemented with L-arginine or from control animals. The contractile vascular response of aortic rings to exogenous ET-1 was increased in rabbits fed a high-cholesterol diet; this enhanced contractility to ET-1 was completely reversed by L-arginine. These data suggest that L-arginine restores endothelial function and normalizes the synthesis and vasoconstrictor response to ET-1 in hypercholesterolemia.

Ceruloplasmin impairs endothelium-dependent relaxation of rabbit aorta.

This study evaluated the effects of ceruloplasmin, the copper-containing blue oxidase of vertebrate plasma, on the relaxation of rabbit aortic rings after endothelial release of nitric oxide (NO). Ceruloplasmin at physiological, i.e., micromolar, concentrations inhibited relaxation of rabbit aorta induced by endothelium-dependent agonists like acetylcholine or ADP, whereas it was ineffective toward vasodilation due to direct stimulation of smooth muscle cells by nitroglycerin. The effect was reversible and specific for native, fully metalated ceruloplasmin, since relaxation was not impaired by the heat-treated or metal-depleted derivatives. A trapping mechanism, involving a direct interaction of NO or other NO-containing species (like nitrosothiols and iron-dinitrosyls) with the copper sites and/or with the free thiol of ceruloplasmin, could be safely excluded on the basis of spectroscopic and chemical analyses of the protein exposed to authentic NO, nitrosothiols, or iron-dinitrosyls. The data presented in this paper constitute the first evidence of impairment of the endothelium-dependent vasodilatation by a plasma protein and may shed some light on the still uncertain physiological role of ceruloplasmin.

The soluble guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) inhibits relaxation of rabbit aortic rings induced by carbon monoxide, nitric oxide, and glyceryl trinitrate

Carbon monoxide (CO), a vasodilator, has been implicated as an activator of soluble guanylyl cyclase (sGC) to effect smooth muscle relaxation; however, this idea has not received universal support. The purpose of this study was to examine the effects of the sGC inhibitor 1H-[1,2,4]oxadiazolo[4,3-alpha]quinoxalin-1-one (ODQ) on relaxation of rabbit aortic rings (RARs) induced by CO. Administration of 10 microM ODQ completely abolished relaxation of RARs by CO (30 microM), whereas only a partial attenuation of NO-induced relaxation was achieved by the same concentration of ODQ. The results of this study suggest that CO-mediated relaxation of RARs is mediated by sGC and indicate that ODQ may serve as a useful tool in the investigation of the actions of CO. Furthermore, these observations support the idea that ODQ is less potent in inhibiting relaxations by NO, thereby implicating a component of NO-induced relaxation that is independent of sGC/cGMP.

Evans' blue dye abolishes endothelium-dependent relaxation of rabbit aortic rings

Dear Editors, Anitschkow [1] observed that areas of rabbit aortic wall downstream of branch points are prone to diet-induced disease and also show an enhanced uptake of circulating trypan blue dye. Numerous subsequent studies using trypan blue or, more commonly, its isomer Evans’ blue dye (EBD) have confirmed for a range of species, ages and diets that these dyes accumulate preferentially downstream of branch points. Studies employing other tracers, however, have only found this pattern in immature vessels [2–5]; in mature vessels, elevated transport occurs upstream of branches [6,7]. We have recently demonstrated that the adult pattern of uptake reverts to the juvenile one in mature rabbit aortas perfused with an inhibitor of NO synthesis [8]. Consequently, the lack of an age-related reversal in the pattern of EBD uptake near branches could be explained if the dye, like methylene blue, inhibits the release or action of NO. To investigate this possibility, we have examined the effect of EBD on NO-mediated relaxation of aortic rings. Three female New Zealand White (NZW) rabbits aged 99–100 days (2.72–2.75 kg) were heparinised (1000 USP units, Sigma) and anaesthetised with pentobarbital (240 mg Sagatal, Rhone Merieux) via the marginal ear vein. In each animal, the abdomen was opened along the midline, the phrenic arteries were clamped, the diaphragm was cut away and the sternum was split along its length. Descending thoracic aortas were gently excised and placed in Holman’s buffer (120 mM NaCl, 5 mM KCl, 2.5 mM CaCl2, 1.3 mM NaH2PO4, 25 mM NaHCO3, 10 mM sucrose, 11.1 mM D-glucose; pH 7.4) equilibrated with 95% O2 and 5% CO2. At least 6 rings 2–3 mm wide were cut from each vessel, two being de-endothelialised by abrasion with a matchstick. The aortic rings were suspended on isometric force transducers (Harvard Apparatus) in Student tissue water baths (Harvard Apparatus) containing 50 ml of the same buffer at 37°C. They were tensioned to 2 g for 1 h before being constricted by addition of 1 mM phenylephrine (L-phenylephrine hydrochloride, Sigma). This dose was used because a preliminary series of experiments [9] using other NZW rabbits had shown that it gives approximately 50% of the maximum phenylephrine-induced constriction and therefore allows the demonstration of further constriction as well as relaxation. Representative chart recorder traces for the constriction and for the changes described below are shown in Fig. 1. After 5 min, by which time a steady force was being generated, 10 mM indomethacin (Calbiochem-Novabiochem) was added to avoid effects mediated by cyclo-oxygenase products in subsequent interventions. After a further 5 min, 1 mM acetylcholine (Sigma) was added to stimulate the release of endotheliumderived relaxing factor. The preliminary experiments [9] had shown this to be the lowest concentration giving maximal relaxation of rings preconstricted with 1 mM * Corresponding author. Tel.: +44 1734 875123 ext. 7053; fax: +44 1734 310180; e-mail P.D.Weinberg@reading.ac.uk

The soluble guanylyl cyclase inhibitor 1&lt;I&gt;H&lt;/I&gt;-[1,2,4]oxadiazolo[4,3-&lt;I&gt;a&lt;/I&gt;]quinoxalin-1-one (ODQ) inhibits relaxation of rabbit aortic rings induced by carbon monoxide, nitric oxide, and glyceryl trinitrate

The soluble guanylyl cyclase inhibitor 1&lt;I&gt;H&lt;/I&gt;-[1,2,4]oxadiazolo[4,3-&lt;I&gt;a&lt;/I&gt;]quinoxalin-1-one (ODQ) inhibits relaxation of rabbit aortic rings induced by carbon monoxide, nitric oxide, and glyceryl trinitrate

Effect of different oxygen tensions on the nitric oxide-induced relaxation of rabbit aortic rings

Effect of different oxygen tensions on the nitric oxide-induced relaxation of rabbit aortic rings

Methylmethacrylate monomer produces direct relaxation of vascular smooth muscle in vitro

Methylmethacrylate bone cement is associated with severe hypotensive reactions during surgery and anesthesia. The purpose of this in vitro study was to determine if methylmethacrylate monomer could produce hypotension by acting directly on vascular smooth muscle. Segments of human saphenous vein or rabbit thoracic aorta were cut into rings. The rings were mounted in isolated tissue chambers in order to measure isometric tension development. Methylmethacrylate monomer (methylmethacrylic acid ester) produced direct relaxation of venous or aortic rings preconstricted with either potassium ion or noradrenaline. The relaxation was concentration-dependent, occurring at concentrations from 10(-3) to 10(-1) M. The relaxation of rabbit aortic rings (preconstricted with noradrenaline) was unaffected by pre-treatment with atropine, propranolol, cimetidine, indomethacin, or methylene blue. Endothelial stripping with Triton X-100, sufficient to completely abolish acetylcholine-induced relaxation, also had little effect on methylmethacrylate-induced relaxation. Methylmethacrylate produced direct relaxation of rabbit aortic rings constricted with either potassium or noradrenaline in calcium-deficient media, and inhibited subsequent calcium-induced constriction. These results suggest that methylmethacrylate monomer may interfere with intracellular and extracellular calcium mobilization and excitation/contraction coupling in vascular smooth muscle. The direct relaxation of venous and arterial smooth muscle produced by methylmethacrylate monomer may contribute in part to the hypotension that can occur when acrylic bone cement is employed during orthopedic procedures.

Effect of temperature on glyceryl trinitrate induced relaxation of rabbit aorta

It has previously been shown that the vasodilatory response to glyceryl trinitrate (GTN) was decreased during hypothermic cardiopulmonary bypass. The purpose of these experiments was to determine the effect of temperature on GTN-induced relaxation and on GTN biotransformation in rabbit aorta. It was determined that the EC50 of GTN on rabbit aortic rings (RARs) was increased significantly from 1.8 x 10(-8) M at 37 degrees C to 3.4 x 10(-8) M at 27 degrees C (p < 0.05). The production of NO by rabbit aortic strips (RASs) was significantly less at 27 degrees C compared with 37 degrees C after 80 min, being 9.62 x 10(-11) +/- 13.2 x 10(-11) mol NO/g wet wt. RASs compared with 5.71 x 10(-10) +/- 9.43 x 10(-11) mol NO/g wet wt. RASs, respectively (p < 0.05), after 80 min incubation. There was no difference in the amount of glyceryl-1,2-dinitrate (1,2-GDN) produced from GTN at the two temperatures. The ED20 for NO-induced relaxation of RARs increased from 3.46 x 10(-10) +/- 2.24 x 10(-10) mol at 37 degrees C to 1.01 x 10(-9) +/- 4.51 x 10(-10) mol at 27 degrees C (p < 0.05). These data indicate that the biotransformation of GTN and the release of NO were impaired by hypothermia, and that this, as well as a decrease in the tissue response to NO at 27 degrees C, explains the decrease in GTN activity at reduced body temperatures.

Lipoproteins as factors in vessel tone and reactivity modulation.

Lipoprotein fractions purified from fresh canine plasma--very low density lipoprotein (VLDL), low density lipoprotein (LDL) and high density lipoprotein (HDL)--produce relaxation of isolated ring segments of coronary arteries of canine (porcine and human) and rabbit aorta precontracted with high potassium, 30 mM, or phenylephrine, 10(-6) M, respectively. Lipoproteins do not possess high species specificity and produce relaxation of arteries of other species which differ quantitatively. There was no marked endothelium-dependent relaxation induced by animal lipoproteins although human LDL produced endothelium-dependent relaxation induced by animal lipoproteins although human LDL produced endothelium-dependent relaxation of rabbit aortic rings. Canine LDL and HDL decreased endothelium-dependent relaxation of rabbit aortic rings produced by acetylcholine (10(-9)-10(-6) M), which was more pronounced when the LDL and HDL were present in the organ bath. In this case, LDL and HDL reduced the rate of development and amplitude of contraction produced by phenylephrine, 10(-6) M. We suggest that lipoprotein effects may result from blockade of some steps in electromechanical coupling or from inhibition of voltage- and receptor-operated Ca-channels of vessel smooth muscle cell membrane.

L-arginine is the physiological precursor for the formation of nitric oxide in endothelium-dependent relaxation

The formation of nitric oxide (NO) from L-arginine by vascular endothelial cells and its relationship to endothelium-dependent relaxation of vascular rings was studied. The release of NO, measured by bioassay or chemiluminescence, from porcine aortic endothelial cells stimulated with bradykinin was enhanced by infusions of L-, but not D-arginine. The release of 15NO, determined by high resolution mass spectrometry, from L-guanidino 15N (99%) arginine was also observed, indicating that NO is formed from the terminal guanidino nitrogen atom(s) of L-arginine. L-NG-monomethyl arginine (L-NMMA), but not D-NMMA, inhibited both the generation of NO by endothelial cells in culture and the endothelium-dependent relaxation of rabbit aortic rings. Both these effects were reversed by L-arginine. These data indicate that L-arginine is the physiological precursor for the formation of NO which mediates endothelium-dependent relaxation.

Effect of SKF 525A on the release of nitric oxide and prostacyclin from endothelial cells

The effect of SKF 525A on the endothelium-dependent relaxation of rabbit aortic rings and on the release of nitric oxide and prostacyclin from porcine aortic endothelial cells in culture was examined. SKF 525A (10–30 μM) inhibited acetylcholine- but not the calcium ionophore A23187-induced endothelium-dependent relaxation without affecting the release of endothelium-derived relaxing factor from endothelial cells stimulated with bradykinin. Higher concentrations of SKF 525A (30–200 μM) caused transient endothelium-dependent relaxation of rabbit aortic rings and the release of nitric oxide and prostacyclin from the endothelial cells without inducing release of lactate dehydrogenase. The mechanism whereby SKF 525A releases nitric oxide and prostacyclin from endothelial cells requires further investigation.

Effect of 5-isosorbide mononitrate on isosorbide dinitrate-induced relaxation of rabbit aortic rings.

The ability of isosorbide dinitrate (ISDN) and its two metabolites, 5-isosorbide mononitrate (5-ISMN) and 2-isosorbide mononitrate (2-ISMN), to relax phenylephrine-contracted rabbit aortic rings was compared. The three organic nitrates demonstrated similar efficacy. ISDN was found to be the most potent (median effective dose (ED50); 1.5 X 10(-7) +/- 1.1 X 10(-7) M), followed by 2-ISMN (ED50, 1.8 X 10(-6) +/- 9 X 10(-7) M) and 5-ISMN (ED50, 8.2 X 10(-6) +/- 3.6 X 10(-6) M). The log dose-response curve of ISDN in rabbit aortic rings was constructed in the absence and presence of three fixed concentrations of 5-ISMN (5 X 10(-6), 10(-5), and 3 X 10(-5) M). No shift in the ISDN dose-response curve at high ISDN concentrations was noted in the presence of 5-ISMN. Using the isobolographic method with fixed ISDN/5-ISMN ratio mixtures, no evidence for an antagonistic effect of 5-ISMN on ISDN-induced vasodilation was obtained. Analysis of the fixed ISDN/5-ISMN ratio mixture responses by the median-effect plot showed no antagonistic effect. It is concluded that in rabbit aortic rings 5-ISMN, the major metabolite of ISDN, is not an antagonist of ISDN at a "nitrate receptor," and no support is provided for the hypothesis that the accumulation in plasma of metabolites (e.g., 5-ISMN) with longer half-lives than the parent drug explains tolerance to organic nitrates.