Isolated Rabbit Aorta Research Articles

Characterization of BIBS 39 and BIBS 222: two new nonpeptide angiotensin II receptor antagonists

Two new nonpeptide angiotensin II (AII) receptor antagonists, 4'-[(2-n-butyl-6-cyclohexylaminocarbonylamino-benzimidazole-1-yl)-methyl] biphenyl-2-carboxylic acid (BIBS 39) and 2-n-butyl-1-[4-(6-carboxy-2,5-dichlorobenzoylamino)-benzyl]-6-N-(methylaminocarbonyl)-n-pentylamino-benzimidazole (BIBS 222) were characterized in radioligand binding assays, and in vitro and in vivo experiments. BIBS 39 displaced [ 125I] AII from its specific binding sites with a K i value of 29 ± 7 nM for the AII subtype 1 (AT 1) receptor and a K i value of 480 ± 110 nM for the AII subtype 2 (AT 2) receptor. BIBS 222 showed a K i value of 20 ± 7 nM for the AT 1 subtype and a K i value of 730 ± 170 nM for the AT 2 subtype. Thus BIBS 39 was 17 times more selective for the AT 1 subtype and BIBS 222 37 times. Both compounds were specific for AII receptors as they did not show high affinity for other receptors. BIBS 39 shifted the AII concentration-contractile response curves in isolated rabbit aorta to the right in a parallel fashion. A pA 2 value of 8.14 ± 0.08 and a slope of 1.06 ± 0.07 were calculated. BIBS 222 caused nonparallel shifts to the right and reduced the maximal response induced by AII by about 25%. A K B value of 9.01 (±3.22).10 −8 M was determined. At 10 −5 M, neither compounds altered the contractile responses to noradrenaline and KCl. In pithed rats, BIBS 39 dose dependently shifted the dose-response curve of AII to the right without affecting the maximal response. BIBS 222 also caused parallel shifts to the right but a significant reduction of the maximal responses was observed at 3 and 10 mg/kg i.v. These results show that the benzimidazole derivatives BIBS 39 and BIBS 222 are potent and selective AII receptor antagonists. Substitution with a benzimidazole moiety results into a considerable loss of selectivity for the AT 1 receptor subtype compared with an imidazole moiety as, for instance, in DuP 753.

The endothelin receptor antagonist, BQ-123, inhibits angiotensin II-induced contractions in rabbit aorta

The purpose of this study was to examine the specificity of the cyclic pentapeptide ET A receptor antagonist BQ-123. BQ-123 competitively antagonized endothelin-1-induced contractions in rabbit aorta, increases in inositol phosphates in cultured rat vascular smooth muscle A10 cells, and binding of [ 125I]endothelin-1 to the cloned ET A receptor cDNA expressed in Cos 7 cells. In contrast, BQ-123 was a weak antagonist of [ 125I]endothelin-3 binding to rat cerebellar membranes and to membranes from Cos 7 cells transfected with the cloned ET B receptor cDNA. BQ-123 shifted concentration-response curves in isolated rabbit aorta elicited by angiotensin II, but did not bind to angiotensin II receptors nor affect angiotensin II-induced increases in inositol phosphates. BQ-123 also did not affect contractions induced by KCl or norepinephrine. These data suggest that endothelin may play a role in angiotensin II-induced contractions of rabbit aorta.

Mode of relaxing action of FK336, a new antianginal agent, in rabbit aorta

1. 1. FK336 f(10 −6–10 -4M) inhibited contractile responses to norepinephrine (NE), KCl and Ca 2+ in isolated rabbit aortas. 2. 2. Relaxing effect of FK336 on KCl-response was inhibited by nitroglycerin (NG), but not by nifedipine or verapamil. 3. 3. FK336 inhibited residual NE response and a subsequent Ca 2+ response in Ca 2+-free medium. FK336 did not affect the inositol monophosphate level. 4. 4. Relaxing effect of FK336 on NE response was inhibited by methylene blue, NG, K +-channel inhibitors and acetylcholine (ACh), and potentiated by M&B 22,948 and theophylline. 8-Br cGMP and dibutyl cAMP had no effect. 5. 5. FK336 increased cGMP level in rat aorta. 6. 6. Potentiation of isoproterenol-relaxation by FK336 was inhibited by methylene blue. 7. 7. The inhibitory effect of ACh on FK336-relaxation was eliminated by endothelium removal, nordihydroquaiaretic acid and guinacrine, but not by indomethacin. These treatments themselves did not affect FK336-relaxation. 8. 8. The mode of vasorelaxing action of FK336 is discussed.

Characteristics of vasoinhibitory action of FK 453 (a pyrazolo-pyridine derivative), a new antihypertensive agent with diuretic action in isolated rabbit aorta.

The vasoinhibitory effect of FK 453 was examined in isolated rabbit aorta. FK 453 inhibited contractile responses to norepinephrine, angiotensin-I and KCl. Pretreatment of the tissue with FK 453 failed to affect the relaxing effect of verapamil on the KCl response and the inhibitory effect of prazosin on the phenylephrine response. FK 453 inhibited both the residual norepinephrine response and the subsequent Ca2+ response in a Ca(2+)-free medium containing EGTA and nifedipine. The inhibitory effect of a combined treatment with either FK 453 plus nitroglycerin or FK 453 plus theophylline, but not with FK 453 plus M & B 22,948 (2-o-propoxyphenyl-8-azapurine-6-one; May & Baker), was much greater than that of any single treatment. Pretreatment with FK 453 also potentiated relaxing effects of nitroglycerin and isoproterenol on the PGF2 alpha response. The effect of a combined treatment with FK 453 plus theophylline, but not with FK 453 plus M & B 22,948, was much greater than that of any single treatment. FK 453 also inhibited the activity of phosphodiesterase from canine aorta to convert cyclic [3H]-GMP and cyclic [3H]-AMP to 5'-GMP and 5'-AMP, respectively. These results suggest that the inhibitory action of FK 453 is not due to inhibition of voltage-operated Ca2+ channels or alpha-adrenoceptors, but due to increase in cyclic GMP level.

Additive and superadditive vasodilating combinations of diltiazem and glyceryl trinitrate in isolated rabbit aorta

AbstractThe combined use of two drugs with overtly similar action is a common therapeutic strategy. Combinations in doses that produce synergism are frequently sought. In this study, we examined the vasodilating action of diltiazem (DIL) and glyceryl trinitrate (GTN), tested in several fixed‐ratio combinations on an isolated preparation of the rabbit aorta. This pair of drugs, widely used clinically, exhibited significant superadditivity (synergism) in certain fixed‐ratio combinations tested, i.e., 30:1 and 100:1 of DIL:GTN (based on weight), and only simple additivity in other dose ratios, 1:15, 1:1, and 15:1. A demonstration of synergism requires that the total dose of the mixture needed to produce a level of effect be significantly less than the theoretically additive dose calculated; hence, statistical confidence limits of the additive value are needed. Testing of this nature has been limited for statistical reasons to drug combinations that produce parallel regressions of effect on log (dose). In this paper we illustrate the application of new statistical methodology that permits testing of drug combinations that produce nonparallel assays and report our experimental findings, which show that only certain fixed‐ratio combinations of DIL and GTN are superadditive (synergistic) in this test. Although vasodilation alone does not imply antianginal action, these findings may serve as a guide for further clinical testing.

P-125 - Characteristics of the vasorelaxing effect of KW-3196, a new vasodilator, in isolated rabbit aorta

P-125 - Characteristics of the vasorelaxing effect of KW-3196, a new vasodilator, in isolated rabbit aorta

In vitro and ex vivo Ca-antagonistic effect of 2-methoxyethyl(E)-3-phenyl-2-propen-1-yl(+/-)-1,4-dihydro-2,6-dimethyl- 4-(3- nitrophenyl)pyridine-3,5-dicarboxylate (FRC-8653), a new dihydropyridine derivative.

The characteristics of calcium antagonism and vascular effect of 2-methoxyethyl(E)-3-phenyl-2-propen-1-yl(+/-)-1,4-dihydro-2,6-dime thyl-4-(3- nitrophenyl)pyridine-3,5-dicarboxylate (FRC-8653) were investigated. FRC-8653 inhibited an increase in intracellular free calcium concentration during membrane depolarization in PC12 cells. FRC-8653 also inhibited the specific binding of 3H-nitrendipine to cardiac membranes, in a similar manner to nifedipine and nicardipine. FRC-8653 inhibited KCl- and CaCl-induced contractions in isolated rabbit aorta, but failed to affect norepinephrine-induced contraction. The vasorelaxing effect of FRC-8653 in rabbit aorta developed more slowly than those of nifedipine and nicardipine. In ex vivo experiment, the inhibitory effect of orally administered FRC-8653 against KCl-contraction in rat aorta lasted longer than that of nifedipine. These findings suggest that FRC-8653 dilates blood vessels by blocking calcium influx via dihydropyridine-sensitive, voltage-dependent calcium channels and that the vascular effects are slow in development and long in duration.

O-517 - Adrenergic nature of contractile response of Isolated rabbit aorta to electrical field stimulation

O-517 - Adrenergic nature of contractile response of Isolated rabbit aorta to electrical field stimulation

Inactivation of Endothelin by Polymorphonuclear Leukocyte-Derived Lytic Enzymes

AbstractCultured bovine aortic endothelial cells (BAEC) released endothelin-1 (ET-1) in the culture medium in a time-dependent fashion. Coincubation of fMLP-activated human polymorphonuclear leukocytes (PMN) with BAEC caused a fast (maximal activity was reached within 15 minutes) and cell number-dependent disappearance of ET-1 from the medium. This effect was direct to ET-1, because it was also present when PMN were incubated with the synthetic peptide in the absence of BAEC. PMN-dependent disappearance of ET-1 was associated with loss of constrictor activity on isolated rabbit aorta. PMN-released products were responsible for ET-1 degrading activity, because supernatants of activated PMN were equally effective as the intact cells. Resting PMN, in the same time frame, were uneffective. Eglin C, a potent blocker of PMN-derived elastase and cathepsin G, reversed the ET-1 inhibitory activity of fMLP-stimulated PMN and of their supernatant. Direct addition of elastase and cathepsin G to synthetic ET-1 destroyed its immunoreactivity and this effect was blocked by eglin C. High-performance liquid chromatography (HPLC) analysis supported the hypothesis that ET-1 degradation by PMN was due to enzymatic proteolysis. These data provide evidence that activated PMN are able to degrade ET-1 through the release of proteases. Because physiologic concentrations of PMN can destroy high amounts (up to 100 nmol/L) of ET-1 within a few minutes, we propose that this mechanism of ET-1 inactivation has biologic relevance.

Inactivation of endothelin by polymorphonuclear leukocyte-derived lytic enzymes

Cultured bovine aortic endothelial cells (BAEC) released endothelin-1 (ET-1) in the culture medium in a time-dependent fashion. Coincubation of fMLP-activated human polymorphonuclear leukocytes (PMN) with BAEC caused a fast (maximal activity was reached within 15 minutes) and cell number-dependent disappearance of ET-1 from the medium. This effect was direct to ET-1, because it was also present when PMN were incubated with the synthetic peptide in the absence of BAEC. PMN-dependent disappearance of ET-1 was associated with loss of constrictor activity on isolated rabbit aorta. PMN-released products were responsible for ET- 1 degrading activity, because supernatants of activated PMN were equally effective as the intact cells. Resting PMN, in the same time frame, were uneffective. Eglin C, a potent blocker of PMN-derived elastase and cathepsin G, reversed the ET-1 inhibitory activity of fMLP- stimulated PMN and of their supernatant. Direct addition of elastase and cathepsin G to synthetic ET-1 destroyed its immunoreactivity and this effect was blocked by eglin C. High-performance liquid chromatography (HPLC) analysis supported the hypothesis that ET-1 degradation by PMN was due to enzymatic proteolysis. These data provide evidence that activated PMN are able to degrade ET-1 through the release of proteases. Because physiologic concentrations of PMN can destroy high amounts (up to 100 nmol/L) of ET-1 within a few minutes, we propose that this mechanism of ET-1 inactivation has biologic relevance.

DesArg 9D-Arg[Hyp 3,Thi 5,D-Tic 7,Oic 8]bradykinin (desArg 10-[Hoe140]) is a bradykinin B 1 receptor antagonist

DesArg 9-D-Arg[Hyp 3,Thi 5,D-Tic 7,Oic 8]BK is a potent and stable B 1 bradykinin (BK) receptor antagonist which was one order of magnitude more potent (IC 50 1.2 × 10 −8 M) in the isolated rabbit aorta than the known selective B 1 BK receptor antagonist, desArg 9-[Leu 8]BK (IC 50 1.1 × 10 −7 M). DesArg 9-D-Arg[Hyp 3,Thi 5,D-Tic 7,Oic 8]BK is the desArg 10 derivative of Hoe 140, a new, potent, stable, selective and long-acting B 2 BK receptor antagonist. In B 2 organ preparation it was three orders of magnitude less potent than Hoe140. Since it is potent and stable it could contribute to the investigation of B 1 BK receptor function.

Antihypertensive and cardiovascular effects of the new calcium antagonist YH334

Antihypertensive effect of YH334 was examined in various experimental hypertension rat models and the systemic and regional hemodynamic profiles of the compound were investigated in conscious spontaneously hypertensive rats (SHR). The antihypertensive potency of YH334 is found to be more than 10 times stronger than that of nitrendipine in the all hypertensive models. The effective doses to lower the initial blood pressure by 20% (ED20) of YH334 were 1.4 mg/kg in normotensive rats (NR), 0.7 mg/kg in SHR, 0.1 mg/kg in DOCA salt hypertensive rats (DHR) and 0.4 mg/kg in renal hypertensive rats (RHR), and the ED20 values of nitrendipine were 15.8 mg/kg in NR, 7.1 mg/kg in SHR, 1.7 mg/kg in DHR and 4.8 mg/kg in RHR. The primary hemodynamic effect of YH334 was characterized by increasing CI and SVI and reducing TPRI of which hemodynamic profile is similar to that of nitrendipine. Both compounds seem to produce potent antihypertensive effects by lowering peripheral resistance in the skeletal muscles. In the organ bath study using isolated rabbit aorta, YH334 was found to be a potent voltage dependent calcium channels blocker without significant inhibitory effect on the receptor operated calcium channels like the most of other dihydropyridine type calcium antagonists. Furthermore, YH334 showed acute diuretic and natriuretic effects in conscious SHR, which may render the unnecessary restriction of sodium in the diet of those patients on long term hypertension therapy. This effect would provide an additional benefit to its potent antihypertensive activity.

2-Nicotinamidoethyl acetate (SG-209) is a potassium channel opener: structure activity relationship among nicorandil derivatives.

The mechanism of the vasodilating action of 2-nicotinamidoethyl acetate (SG-209), a derivative of nicorandil, was examined in the isolated rabbit aorta. Comparison was made using 2-nicotinamidoethyl alcohol (SG-86) and 2-nicotinamidoethyl nitrate (nicorandil; SG-75) to reveal any structure-activity relationships. SG-209 and nicorandil caused concentration-dependent relaxation in preparations precontracted with phenylephrine (10(-7) mol/l), while SG-86 produced a relaxation only at very high concentrations. The pD2 values (-log[EC50]) of SG-209 and nicorandil were 3.59 +/- 0.07 and 5.95 +/- 0.10, respectively. The vasorelaxant activity of nicorandil was associated with significant increases in cyclic GMP content, while that of SG-209 was not. Methylene blue (10(-5) mol/l) attenuated the relaxant effect of nicorandil, but had no effect on that of SG-209. Furthermore, the relaxant effect of nicorandil was not affected by glibenclamide (10(-5) mol/l), whilst the relaxant effect of SG-209 was abolished by this compound. In the presence of methylene blue (10(-5) mol/l), however, glibenclamide (10(-5) mol/l) attenuated the relaxant effect of higher concentrations of nicorandil (greater than or equal to 10(-5) mol/l). These results indicate that the relaxant effect of SG-209 is mostly if not exclusively due to the activation of potassium channels, while this action contributes to the vasodilating action of nicorandil only at higher concentrations.

Inhibitory effect of 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8) in vascular smooth muscle

The inhibitory effects of 8-(N,N-diethylarnmo)octyl-3,4,5-trirnethoxybenzoate (TMB-8) on vascular smooth muscle contraction and cytosolic Ca 2+ level ([Ca 2+] i) were examined using isolated rabbit aorta loaded with a fluorescent Ca 2+ indicator, fura-2. TMB-8 (100 μM) decreased the high K +-induced increase in muscle tension, and [Ca 2+] i and 45Ca 2+ influx to their respective resting levels. TMB-8 (100 μM) almost completely inhibited the increase in [Ca 2+] i and 45Ca 2+ influx due to norepinephrine although muscle tension was only partially decreased. A higher concentration of TMB-8 (300 μM) inhibited the remaining portion of the contraction without additional decrease in [Ca 2+] i. The inhibitory effect of TMB-8 on high K +-induced contraction, but not on the norepinephrine-induced contraction, was antagonized by the increase in external Ca 2+ concentrations or by the Ca 2+ channel activators, CGP 28,392 and by Bay K8644. In Ca 2+-free solution, norepinephrine-induced transient increases in [Ca 2+] i and muscle tension and 100 μM TMB-8 inhibited these changes. The caffeine-induced transient increases in [Ca 2+] i and muscle tension were also inhibited by TMB-8 at concentrations higher than those needed to inhibit the norepinephrine-induced transient changes. In permeabilized smooth muscle, TMB-8 (300 μM) did not inhibit the Ca 2+-induced contraction. These results suggest that TMB-8 inhibits vascular smooth muscle contractility by inhibiting Ca 2+ influx, Ca 2+ release and Ca 2+ sensitization of contractile elements.

Vascular Smooth Muscle Relaxation by α1-Adrenoceptor Blocking Action of Denopamine in Isolated Rabbit Aorta

We investigated the mechanism of vascular relaxation by denopamine (Deno), an oral positive inotropic agent that has selective beta 1-adrenergic action. Deno relaxed, dose-dependently (0.1-30 microM), ring segments of rabbit aorta, which were partially precontracted with 1 microM phenylephrine (Phe) or norepinephrine (NE), but did not relax those precontracted with 5 microM prostaglandin F2 alpha or 40 mM K+. The relaxation was not significantly inhibited by pretreatment with 10 microM propranolol or metoprolol. Deno produced parallel shifts in concentration-response curves to Phe, but this was not true for clonidine. The Schild plot analysis resulted in a linear regression of a slope of 1.075 +/- 0.063, which was not significantly different from unity, and the pA2 value of Deno against Phe was 5.57 +/- 0.02. The specific binding of [3H]prazosin to a rabbit aorta membrane preparation was displaced in a concentration-dependent manner by the simultaneous addition of Deno. The slope of a Hill plot was not significantly different from unity (1.102 +/- 0.147). The pK1 value for Deno calculated from the displacement curve was 5.29 +/- 0.17, which was not significantly different from the pA2 value of Deno. In conclusion, vascular smooth muscle relaxation by Deno was mediated by the blocking effect of alpha 1-adrenoceptors. Thus, these findings suggest that Deno may be effective in the treatment of congestive heart failure because it elicits a positive inotropic effect by beta 1-adrenergic action and vasodilation by alpha 1-adrenergic blocking action.

Antihypertensive activity of himbacine in anesthetized cats

AbstractHimbacine is a plant alkaloid isolated from Galbulimima baccata. Intravenous administration of himbacine produced a fall in blood pressure and heart rate in anesthetized normotensive cats. These effects were similar in areflexic and normal cats, suggesting direct peripheral action with no central component involvement. Presence of himbacine did not alter the preganglionic stimulation of cat nictitating membrane, ruling out the possibility of ganglion blockade. In isolated rabbit aorta, himbacine inhibited K+‐induced contraction, an effect similar to that of papaverine. These data indicate that antihypertensive and bradycardiac actions of himbacine are mediated through its direct depressant action on myocardium and blood vessels, and that the compound is relatively safe with i.p. LD50 in mice of 230 mg/kg.

A potent vasoconstrictor in the rat submandibular gland.

We detected a novel vasoconstrictor in an arginine esterase fraction separated from fractions containing tonin and other esterases that were obtained from a rat submandibular gland extract. When tested on isolated rabbit aorta rings, the substance caused dose-related contractions that were slow in onset, long-lasting, and difficult to reverse by rinsing. The substance acts directly on vascular smooth muscle, since preincubation with plasma or intact endothelium is not required. The fact that the constrictor was destroyed by heat and incubation with pronase suggests that it is a protein. Molecular sieving indicates an estimated molecular weight of 24,000 Da. It has a neutral isoelectric point that is higher than the pI of tonin, from which it can be separated by anion exchange chromatography. A small amount of the vasoconstrictor was obtained by gel filtration and eluted from isoelectric focusing polyacrylamide gels. The purified substance showed a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It was a potent vasoconstrictor; an estimated concentration of 2.5 nM induced contraction of isolated rabbit aorta rings ranging from 15% to 40% of the maximum contraction obtained by 60 mM KCl. Contraction was completely blocked by 1 mM (p-amidinophenyl)methanesulfonyl fluoride, a serine protease inhibitor. Contractile activity was not affected by hirudin, a thrombin inhibitor, but was completely inhibited by soybean trypsin inhibitor and blunted by aprotinin; thus it may be a trypsin-like serine protease. Purified vasoconstrictor preparation showed hydrolyzing activity on Pro-Phe-Arg-methyl-coumarin amide, a kallikrein substrate. We conclude that a novel vasoconstrictor serine protease is present in the rat submandibular gland.

Synthesis and biological activity of N-terminus modified [Ile8] angiotensin II analogues

Semi-peptidic analogues of the angiotensin II (AII) antagonist [Ile8] AII have been prepared by the solid phase method and, for some steps, by synthesis in solution. In the modified analogues 4–15, the N-terminal di- or tripeptidic fragments (Asp-Arg or Asp-Arg-Val) in [Ile8]AII was replaced by non-peptidic moieties featuring amino or guanidino groups as substituent of simple alkyl spacers of various lengths. The compounds synthesized have been tested for their ability to produce an AII-like contraction and/or to inhibit AII-induced contractions in isolated rabbit aortae. All of them were found devoid of agonistic activity and most retained significant antagonistic activity in this assay. Analogue 5 antagonized AII-induced blood pressure effect in the anesthetized rat when infused at 30 μg/kg/min. The structure-activity relationships for these compounds are discussed.

An endothelium-dependent contraction induced by acetylcholiine in isolated rabbit aorta

An endothelium-dependent contraction induced by acetylcholiine in isolated rabbit aorta

Study of the role of EDRF(s) after prolonged administration of sodium chloride on the response of isolated rabbit aorta

Study of the role of EDRF(s) after prolonged administration of sodium chloride on the response of isolated rabbit aorta