Relaxation Of Rat Aortic Rings Research Articles

Nitric oxide synthase responsible for L-arginine-induced relaxation of rat aortic rings in vitro may be an inducible type.

1. Characteristics of L-arginine-induced non-endothelial nitric oxide (NO) formation in rat isolated thoracic aorta were investigated. 2. Relaxation to L-arginine in arterial rings devoid of endothelium developed about 2 h after the first challenge with L-arginine and reached a maximum after a further 4 h of incubation. 3. After the arteries had relaxed in response to L-arginine, guanosine 3':5'-cyclic monophosphate (cyclic GMP) production was stimulated. In fresh arteries that had not yet relaxed in response to L-arginine, L-arginine failed to elevate cyclic GMP levels. 4. Glucocorticoids, actinomycin D and polymyxin B prevented the development of L-arginine-induced relaxation and L-arginine-stimulated increase in cyclic GMP formation. 5. Once L-arginine-induced relaxation developed, these agents no longer suppressed the relaxation and increase in cyclic GMP formation to L-arginine. 6. From these results, it is suggested that in the isolated thoracic aorta of the rat, endotoxin in the medium triggers induction of a non-endothelial NO synthase during prolonged incubation, which accelerates production of NO from added L-arginine to cause relaxation of the arteries via cyclic GMP. Glucocorticoids and protein synthesis inhibitors may prevent induction of NO synthase. It is suggested that the NO synthase mediating the production of muscle-derived NO from L-arginine is an inducible type.

In Vitro Pharmacology of R 80122, a Novel Phosphodiesterase Inhibitor

The cardiac in vitro effects of R 80122, a novel phosphodiesterase (PDE) inhibitor, were investigated and compared with those of the reference compound milrinone and of the calcium-sensitizer adibendan. In guinea pig left atria, both milrinone and R 80122 increased contractile force; 10 microM milrinone was equieffective to 1 microM R 80122. The rate of spontaneously beating atria was not altered by R 80122 in the concentration range of 0.01-0.3 microM. Higher concentrations (1-10 microM) led to a statistically insignificant increase of 20%. Milrinone's effect on frequency was more pronounced and amounted to 21% at 10 microM and to 40% at 100 microM. Adibendan increased heart rate (HR) by 10% at a concentration of only 0.03 microM. This effect was not enhanced any further by increasing the concentration. In papillary muscle, the positive inotropic effects of both milrinone and R 80122 were inhibited by carbachol, indicating involvement of cyclic AMP. Further indications for a cyclic AMP-dependent action were obtained by induction of slow action potentials and synergism with isoprenaline. In electrophysiologic measurements, milrinone reduced action potential duration (APD) in a high concentration whereas R 80122 had no effect. Action potential changes elicited by a toxic concentration of ouabain were reduced by R 80122. Relaxation of rat aortic rings contracted by KCl and relaxation of guinea pig aortic rings contracted by norepinephrine (NE) was comparable for both milrinone and R 80122. R 80122 also caused relaxation of canine coronary arteries constricted with prostaglandin F2 alpha (PGF2 alpha) both with and without endothelium. NE-induced contractions in canine gastrosplenic arteries were not affected by R 80122. Cardiac contractility that had been impaired to various degrees by pentobarbital or by aging was restored to control values by both milrinone and R 80122. R 80122 enhanced cardiac contractility at lower concentrations than milrinone with no concomitant increase in frequency or shortening of the action potential, which may be advantageous for treatment of heart failure.

Analysis of the vasorelaxant action of jatrophone in the isolated aorta of the rat: influence of potassium channel blockers

The mechanism underlying the relaxant response of rat aortic rings to the diterpene jatrophone was investigated. Jatrophone (3 and 10 μM) did not affect acetylcholine-induced endothelium-dependent relaxations, but caused concentration-dependent inhibition of noradrenaline (NA)-induced concentrations in unrubbed, and to a lesser extent, in denuded rings. Jatrophone (30 μM) fully prevented responses to angiotensin II and NA, while responses to KCl (up to 220 mM) were unaffected. In depolarizing medium (KCl 40 mM), jatrophone (3–30 μM) anatagonized Ca 2+-induced contractions in a concentration-dependent and noncompetitive manner, while verapamil (10–100 nM) caused a concentration-dependent, rightward displacement and depression of the Ca 2+ concentration-response curve. Jatrophone (1 to 300 μM) concentration dependently relaxed rat aortic rings precontraction with either NA (1 μM) of KCl (80 mM), yielding EC 50 s of 11 and 24 μM, respectively. These relaxant responses to jatrophone were unaffected by glibenclamide (1 μM), but the concentration-response curve was displaced to the right (2- to 8-fold) by other K + channel blockers such as tetraethylammonium (10 and 30 mM), 4-aminopyridine (3 and 10 mM) or procaine (1 and 3 mM). These results indicate that jatrophone relaxes the rat aorta, at least in part, by activating K + channels distinct from the ATP-sensitive subtype. Since jatrophone, like verapamil, relaxed preparations contracted with KCl and inhibited Ca 2+-induced contractions in depolarized preparations, this diterpene may also block Ca 2+ influx through voltage-sensitive channels. However, additional actions of jatrophone on receptor-operated Ca 2+ channels causing Ca 2+ efflux and/or release cannot be fully ruled out.

Endothelium-dependent and independent relaxation of the rat aorta by cyclic nucleotide phosphodiesterase inhibitors.

1. The effects of selective inhibitors of adenosine 3':5'-cyclic monophosphate (cyclic AMP) and guanosine 3':5'-cyclic monophosphate (cyclic GMP) phosphodiesterases (PDEs) were investigated on PDEs isolated from the rat aorta and on relaxation of noradrenaline (1 microM) precontracted rat aortic rings, with and without functional endothelium. 2. Four PDE forms were isolated by DEAE-sephacel chromatography from endothelium-denuded rat aorta: a calmodulin-activated PDE (PDE I) which hydrolyzed preferentially cyclic GMP, two cyclic AMP PDEs (PDE III and PDE IV) and one cyclic GMP-specific PDE (PDE V). The latter was selectively and potently inhibited by zaprinast. The two cyclic AMP PDEs were discriminated by specific inhibitors: one was inhibited by cyclic GMP (PDE III) and by new cardiotonic agents (milrinone, CI 930, LY 195115 and SK&F 94120); the other was inhibited by denbufylline and rolipram (PDE IV). None of these drugs significantly inhibited PDE I. 3. The PDE III inhibitors caused endothelium-independent relaxations of rat aortic rings with the following EC50 values (microM concentration producing 50% relaxation): LY 195115: 3.4, milrinone: 5.7, CI 930; 7.8, SK&F 94120: 14.7. Neither NG-monomethyl-L-arginine (L-NMMA, 300 microM), an inhibitor of the L-arginine-NO pathway, nor L-arginine (1 mM) modified the effect of PDE III inhibitors. However, methylene blue (10 microM) an inhibitor of soluble guanylate cyclase abolished relaxation induced by PDE III inhibitors except in the case of compound CI 930. 4. The specific PDE IV and PDE V inhibitors both produced endothelium-dependent relaxations which were inhibited by L-NMMA and by methylene blue (10 microM). In the presence of L-NMMA, relaxation was restored by subsequent addition of L-arginine. 5. The relaxant effects of denbufylline and rolipram were studied in the presence of drugs stimulating either adenylate cyclase (forskolin and isoprenaline) or soluble guanylate cyclase (sodium nitroprusside, SNP), or inhibiting PDE III (milrinone). In endothelium-denuded rings, a relaxing effect of both denbufylline and rolipram was found in the presence of milrinone (EC5o values 1.7 and 12 microM, respectively) or SNP (EC50 values 12.3 and 124 microM, respectively), but not in the presence of forskolin or isoprenaline. However in the presence of functional endothelium, relaxations produced by PDE IV inhibitors were significantly potentiated by forskolin, isoprenaline, milrinone and SNP (respective EC50 values for denbufylline: 2, 2, 0.4 and 0.7 microM and for rolipram: 7, 13, 7 and 1.2 microM). 6. These results indicate that the relaxant effects of inhibitors of the cyclic AMP-specific PDE IV are markedly enhanced by cyclic GMP elevating agents and by the PDE III inhibitor milrinone. They support the hypothesis that cyclic GMP enhances cyclic AMP-mediated relaxation, possibly through the inhibition of the cyclic GMP-inhibited PDE III.

SKF-525A Does Not Inhibit Release of Endothelium-Derived Relaxing Factor from Rat Thoracic Aorta and Dog Mesenteric and Femoral Artery

SKF-525A (proadifen) inhibits endothelium-dependent relaxations induced by acetylcholine, arachidonic acid and the calcium ionophore A23187. This suggests that SKF-525A is an inhibitor of endothelium-derived relaxing factor (EDRF) and that EDRF may be a product of arachidonic acid metabolism formed via a cytochrome P-450-dependent pathway or that EDRF release is dependent on cytochrome P-450. We tested this postulate using both isolated rings of rat thoracic aorta and dog mesenteric and femoral artery and the perfusion-superfusion bioassay. Rings of rat thoracic aorta and dog mesenteric and femoral artery with intact endothelium were precontracted with an EC50 concentration of norepinephrine (0.1 nmol/l) or U46619 (0.05 mumol/l) and the relaxation to acetylcholine (ACh), bradykinin, adenosine triphosphate (ATP) or nitroglycerin (GTN) were obtained before, 30 min after addition of, and 30 min after washout of SKF-525A (50 mumol/l). SKF-525A inhibited ACh-induced endothelium-dependent relaxation of rat aortic rings and endothelium-dependent relaxation of the dog mesenteric and femoral artery produced by ACh and ATP, but did not affect relaxation to bradykinin or GTN. The inhibitory effect on SKF-525A on ACh and ATP-induced relaxation was partially reversed upon its washout from the muscle chamber. Pretreatment of the blood vessels with ibuprofen (1 mumol/l) did not attenuate SKF-525A-mediated inhibition of the relaxations to any agonist. Selective exposure of dog femoral artery (donor) to SKF-525A (50 mumol/l) for 60 min did not affect the relaxation responses of endothelium-rubbed coronary artery (bioassay tissue) to basal EDRF nor to the effluent from donor tissues stimulated with ACh (10-1,000 pmol), ATP (1-100 nmol) or bradykinin (3-100 pmol). The results show that SKF-525A exhibited a reversible inhibition of endothelium-dependent relaxation by a smooth muscle mechanism unrelated to the generation of EDRF from endothelium.

Endothelium-Derived Relaxing Factor and Muscle-Derived Relaxing Factor in Rat Aorta

Summary: The aim of the present study was to characterize the toxicity of cyclosporine, a widely used immunosuppressant, toward vascular endothelial and smooth muscle cells. Rat aortic rings in classical organ baths and perfused rabbit thoracic aortas were exposed to cyclosporine or solvent in vitro. Cyclosporine reduced the maximum endothelium-dependent relaxation to acetylcholine and the ionophore A23187, but not to SIN-1, a metabolite of molsidomine that spontaneously releases nitric oxide (NO). l-Arginine, which is the precursor for the synthesis of NO from endothelial cells, relaxed rat aortic rings with or without endothelium that had been incubated for 8 h in physiological solution under O2 and CO2 at 37°C. Nitroarginine, a potent inhibitor of NO synthesis from l-arginine, and methylene blue, an inhibitor of guanylate cyclase, inhibited this relaxation in both types of rings, whereas cyclosporine did not. From these data, we conclude that the cells of the vascular wall synthetize not only an endothelium-derived relaxing factor (EDRF), but also a media-derived relaxing factor (MDRF), the nature of which is probably close to NO. Cyclosporine does not affect the synthesis of these two factors, but inhibits the endothelial cell mechanisms responsible for the stimulated EDRF release that follows muscarinic receptor activation and intracellular rise in calcium.

Evidence for generation of a large amount of nitric oxide-like vascular smooth muscle relaxant by cholesterol-rich neutrophils

To determine the effect of cholesterol incorporation on the ability of neutrophils to generate superoxide radicals and nitric oxide-like vasorelaxant material, isolated human neutrophils were incubated with cholesterol-rich liposomes, which increased total cholesterol content by 141% and esterified cholesterol content by 523%. Cholesterol loading resulted in 5 to 7 fold increase in cytosolic calcium in resting as well as in PMA or f-MLP-stimulated cells, but a marked (P less than 0.01) reduction in both PMA- and f-MLP-stimulated superoxide radical generation by these cells. Nitric oxide-like activity measured as relaxation of rat aortic rings was more pronounced (P less than 0.02) in cholesterol-rich than in cholesterol-poor cells. The greater relaxation of aortic rings in response to cholesterol-rich neutrophils was observed in rings with or without intact endothelium, and was potentiated by superoxide dismutase and inhibited by oxyhemoglobin as well as L-NMMA, thus suggesting that the vasorelaxant material was nitric oxide. The greater generation of nitric oxide by cholesterol-rich neutrophils occurs perhaps in response to increased cytosolic calcium.

Cytochrome P450-Dependent Arachidonic Acid Metabolites, 19− and 20-Hydroxyeicosatetraenoic Acids, Enhance Sodium-Potassium ATPase Activity in Vascular Smooth Muscle

Several cytochrome P450-dependent arachidonic acid metabolites have been shown to affect Na+,K(+)-ATPase activity. In the present study, we tested the effect of omega- and omega - 1-hydroxylated products, i.e., 19- and 20-hydroxyeicosatetraenoic acids (19- and 20-HETE), on the K-induced relaxation in rat aortic rings. 19-HETE and 20-HETE increased the magnitude of the potassium-induced relaxation in a dose-dependent fashion (10(-7)-10(-5) M). The inhibitory effect of ouabain on the potassium-induced relaxation was reversed by both 19- and 20-HETE. In addition, indomethacin fully inhibited the stimulatory effect of 19- and 20-HETE on relaxation induced by potassium. Vascular ouabain-sensitive 86Rb uptake was also increased by 19- and 20-HETE. These observations suggest that 19- and 20-HETE stimulate vascular Na+,K(+)-ATPase via their conversion by cyclooxygenase to prostaglandin-like material.

Desmopressin is a Potent Vasorelaxant of Aorta and Pulmonary Artery Isolated from Rabbit and Rat

Hypotension related to the intraoperative use of desmopressin acetate to improve platelet function following cardiopulmonary bypass has recently been reported. To investigate the direct vascular actions of this drug as a potential mechanism of its induced hypotension, cumulative, dose-dependent (3.7 X 10(-10) to 1.2 X 10(-7) M) effects of desmopressin were studied in isolated phenylephrine precontracted rings of rat and rabbit thoracic aorta and rabbit pulmonary artery. Desmopressin was a potent vasodilator of all vessel types studied with significant (P less than 0.05) vasodilation beginning at 7.5 X 10(-9) M. Vascular relaxation of all vessels was greater when the vascular endothelium was intact (P less than 0.05). Indomethacin potentiated (P less than 0.05) vascular relaxation in rat and rabbit aortic rings and partially inhibited (P less than 0.05) relaxation in rabbit pulmonary artery rings. Selective antagonists of vasopressin V1 (d(CH2)5-Tyr(Me)AVP, 1 X 10(-6) M) and V2 (d(CH2)5[D-Ile2,Ala-NH2(9)] AVP, 1 X 10(-6) M) receptors and of histamine H1 (diphenhydramine, 1 X 10(-5) M) and H2 (cimetidine 1 X 10(-5) M) receptors had no effect on desmopressin-induced relaxation of rat aortic rings. Chlorobutanol, the diluent in which desmopressin is supplied, was devoid of vascular effects. To study the effects of desmopressin on vascular cyclic GMP and cyclic AMP concentrations, a cultured bovine aortic smooth muscle--rat vascular smooth muscle coculture model was employed. Desmopressin (1 X 10(-7) and 1 X 10(-8) M) did not significantly alter control values of either cyclic nucleotide.(ABSTRACT TRUNCATED AT 250 WORDS)

The interaction of parafluorohexahydrosiladiphenidol at muscarinic receptors in vitro

1. The antagonistic actions of parafluorohexahydrosiladiphenidol (pFHHSiD) at muscarinic receptors has been studied in cardiac muscle, smooth muscle and cell culture preparations. In this paper, the classification scheme of Doods et al. (1987) is employed. This scheme is based upon differential affinities of muscarinic antagonists. pFHHSiD exhibited high pA2 values at M3 receptors mediating contractions of guinea-pig ileum and oesophageal muscularis mucosae (7.8 and 8.2 respectively) whereas low values were determined at M2 receptors mediating negative inotropic responses in guinea-pig atria (6.0). Intermediate pA2 values were determined at M1 receptors mediating contractions of the canine femoral and saphenous veins. 2. The pA2 values of pFHHSiD at receptors mediating endothelial-dependent relaxation of rat aortic rings, rabbit jugular vein and canine femoral artery (7.6-7.9) were similar to those determined on the ileum. However, the pA2 values of pFHHSiD at receptors mediating contractions of the guinea-pig trachea (7.1), which has been previously shown to possess M3 receptors, were different from those determined in the ileum. 3. The similarity in pA2 values of pFHHSiD between the M3 receptors in guinea-pig ileum and the receptors mediating endothelial-dependent relaxations provide further evidence for the role of M3 receptors in this vascular response. Taken together, pA2 values for pFHHSiD range from 7.1 to 8.2, depending upon the M3 preparation used. The selectivity of the compound therefore for the M3 versus the M2 muscarinic receptor ranged from 13 to 163 fold. 4. At muscarinic receptors mediating stimulation of phosphatidylinositol hydrolysis, pFHHSiD paradoxically displayed a high affinity for the M1 receptor in the SH-SY5Y cell line (pA2 = 7.9) as well as for the M3 receptor in the human astrocytoma (1321 NI cell line (pA2 = 7.6). The value at the M1 receptor in SH-SY5Y cells was greater than was observed at M1 receptors mediating contractions of both the canine saphenous and femoral veins (7.1). 5. pFHHSiD, therefore, clearly delineated M3 from M2 muscarinic receptors, whilst the separation between M1 and M3 receptors was variable. The reason for the anomalous affinity estimates in some functional studies remains unclear. These data indicate that the pA2 values for pFHHSiD appear to be tissue-dependent since the M3 selectivity varies according to the preparations studied. As a result the utility of pFHHSiD in muscarinic receptor classification is limited.

Possible Mechanism of Benzodiazepine-Induced Relaxation of Vascular Smooth Muscle

This study investigated the mechanism of benzodiazepine-induced relaxation of vascular smooth muscle. The ability of several benzodiazepine and isoquinolinecarboxamide compounds, including a pair of enantiomers, to inhibit [3H]Ro5-4864 binding to the peripheral-type benzodiazepine binding site in rat aortic smooth muscle was compared with their relative ability to induce relaxation of rat aortic rings. The binding was performed in a membrane fraction obtained from a pellet centrifuged at 11,400 g and enriched with high-affinity [3H]Ro5-4864 binding. The rank order of potency (Ki) for inhibition of [3H]Ro5-4864 binding to isolated membranes was: (-)PK 14067 (6.4 +/- 0.7 nM) = PK 11195 (6.6 +/- 0.8 nM) greater than Ro5-4864 (17.6 +/- 2.1 nM) greater than diazepam (600 +/- 180 nM) = (+)PK 14068 (530 +/- 70 nM) greater than clonazepam (14,300 +/- 2,100 nM). However, micromolar concentrations of these agents were required to induce relaxation of rat aortic rings contracted with KCl and/or norepinephrine (NE). Moreover, the relaxations induced by these agents were not stereoselective. The rank order of potency (IC50) for relaxation of KCl-induced contracted muscle was: Ro5-4864 (6.6 +/- 0.3 microM) = PK 11195 (6.7 +/- 0.9 microM) = (-)PK 14067 (11.6 +/- 0.7 microM) = (+)PK 14068 (7.6 +/- 1.1 microM) greater than diazepam (47.4 +/- 5.3 microM) = clonazepam (47.5 +/- 5.7 microM). Further investigation of the mechanism of benzodiazepine-induced relaxation showed that (-)PK 14067 and (+)PK 14068 inhibited CaCl2-induced contractions. The benzodiazepines relaxed muscle contracted with KCl to a greater magnitude than those contracted with NE or prostaglandin F2 alpha (PGF2 alpha).(ABSTRACT TRUNCATED AT 250 WORDS)