Rabbit Aortic Strip Preparations Research Articles

Does alpha 1-acid glycoprotein act as a non-functional receptor for alpha 1-adrenergic antagonists?

The ability of a variety of alpha 1-acid glycoproteins (AAG) to affect the intrinsic activity of the alpha 1-adrenergic antagonist prazosin was studied in rabbit aortic strip preparations. From these studies, the activity of AAG appears to be linked to their ability to bind the antagonist. However, a capability to bind prazosin was not the only requirement for this effect. The removal of sialic acid and partial removal of the galactose and mannose residues by periodate oxidation of human AAG all but eliminated the ability of AAG to affect the intrinsic pharmacologic activity of prazosin, although the binding of prazosin was not significantly affected. The presence of bovine AAG, a protein that has a low ability to bind prazosin, reduced the effect of human AAG on prazosin activity. Based upon these results, we propose that AAG is able to bind in the vicinity of the alpha 1-adrenoceptors, therefore extending the binding region for antagonists in such a way as to decrease the ability of the antagonist to interact with the receptor. The carbohydrate side-chains are important for the binding of AAG in the region of the adrenoceptor.

Modulation of tachyphylaxis to angiotensin II in rabbit isolated aorta by the angiotensin AT1 receptor antagonist, losartan.

In rabbit aortic strip preparations, angiotensin II (AII) concentration-contractile response curves (0.3-100 nM) were highly reproducible. However, tachyphylaxis to the contractile response elicited by AII could be induced by repeated exposure to a supramaximal concentration (10 microM) of the peptide. In contrast, a correspondingly supramaximal concentration of the alpha 1-adrenoceptor agonist, phenylephrine (1 mM), did not cause the tissue to become tachyphylactic to phenylephrine. Furthermore, phenylephrine responses were unaffected in tissues previously made tachyphylactic to AII. When the non-peptide, competitive angiotensin AT1 receptor antagonist, losartan (300 nM), was administered before each supramaximal AII challenge, tachyphylaxis did not subsequently occur. Additionally, in tissues made tachyphylactic to AII, subsequent incubation with losartan (300 nM) reversed the AII tachyphylaxis. Thus, losartan may prevent the loss of contractility by preventing AII from interacting with its receptor in a manner which induces tachyphylaxis. However, since losartan can also completely reverse the loss of contractility, it appears capable of restoring the AT1 receptor to a state which allows subsequently administered AII to fully activate the contractile pathway.

Effects of peptidase inhibition on angiotensin receptor agonist and antagonist potency in rabbit isolated thoracic aorta.

1. Experiments were performed with peptidase inhibitors on rabbit aortic strip preparations, to determine whether endogenous peptidase activity can influence the potency estimates for angiotensin receptor agonists and antagonists in this tissue. 2. Angiotensin II (A II) and angiotensin III (A III) both induced concentration-related contractions of rabbit aortic strip preparations. A III was approximately 38 fold less potent than A II, and the gradient of the A III concentration-response curve (1.00 +/- 0.04) was significantly more shallow than that (1.76 +/- 0.05) of the A II curve. 3. Neither the aminopeptidase-A and -M inhibitor, amastatin, nor the aminopeptidase-B and -M inhibitor, bestatin, affected the potency of, or the maximum response to, A II. In contrast, the potency of A III was increased by both amastatin and bestatin. Amastatin had the most marked effect and at 10 microM caused approximately a 12 fold increase in the potency of A III (EC50 values, 102 nM and 8.6 nM in the absence and presence of amastatin, respectively), and also significantly steepened the gradient of the A III concentration-response curve. Amastatin did not affect the position or shape of the concentration-response curve to the alpha 1-adrenoceptor agonist, phenylephrine. Finally, the carboxypeptidase-N inhibitor, D-L-mercaptomethyl-3-guanidine-ethylpropanoic acid (MERGETPA) did not change the position or shape of the concentration-response curves to either A II or A III.4. In the presence of amastatin, the potency of the peptide angiotensin receptor antagonist, Ile7-A III (100nM-l microM ), was increased approximately 13 fold (pA2, with A II as the agonist, 7.0 +/- 0.1 and 8.1 +/- 0.1, in the absence and presence of amastatin, respectively). However, the potency of the nonpeptide angiotensin receptor antagonist, DuP 753 (30-300 nM), was little affected by amastatin (pA2, 8.2 +/- 0.1 and 8.1 +/- 0.1 in the absence and presence of amastatin, respectively).5. The results of this study suggest that endogenous aminopeptidase activity in the rabbit thoracic aorta can profoundly affect estimates of the potency of peptide angiotensin receptor agonists and antagonists.A suitable aminopeptidase inhibitor should therefore be included in studies, using this tissue, which aim to classify angiotensin receptor subtype(s) based on the rank order of peptide angiotensin receptor agonist and/or antagonist potencies.

Dextronatrin: A potent atrial natriuretic peptide analog; Vascular and natriuretic effects

AbstractA shortened 23 amino acid analog (dextronatrin) of rat ANP(1–28) in which, in position 6 next to Cys, there is a D‐Tyr residue, has been compared to rat ANP(1–28) for its diuretic and electrolyte‐excreting properties in male normotensive rats. Both compounds elicited an equally large but short‐lasting diuresis. The natriuresis was also short‐lasting as were the changes in Cl− and K+ excretion.In a rabbit aortic strip preparation dextronatrin antagonized the vasoconstricting effects of noradrenaline but to a lesser extent than rat ANP(1–28).

Effect of lipid feeding on endothelium dependent relaxation in rabbit aortic preparations.

The effect of a cholesterol enriched diet on concentration responses to 5-hydroxytryptamine, ergometrine, and acetylcholine and on endothelium dependent relaxation was investigated in rabbit aortic strip preparations. Sensitivity to ergometrine was slightly reduced after both two and 10 weeks of cholesterol feeding. Sensitivity to 5-hydroxytryptamine was slightly reduced after two but not after 10 weeks of cholesterol feeding. Sensitivity to acetylcholine was not altered at two or 10 weeks in the absence of endothelium, but the endothelium dependent lowering of the constrictor concentration-response was almost abolished after 10 weeks of cholesterol feeding. Likewise the endothelium dependent relaxant response of preconstricted preparations to acetylcholine was almost abolished after 10 weeks of cholesterol feeding. Thus this model of hyperlipidaemia leads after 10 weeks to loss of endothelium dependent relaxation.

Haptoglobin-haemoglobin complex in human plasma inhibits endothelium dependent relaxation: evidence that endothelium derived relaxing factor acts as a local autocoid

Endothelium dependent relaxation of isometrically mounted rabbit aortic strip preparations was rapidly inhibited by human plasma at dilutions down to 1:1000. Gel filtration and ion exchange chromatography were used to demonstrate that this inhibitory activity was present in fractions containing haptoglobin. Purified haptoglobin itself possessed no inhibitory action against endothelium dependent relaxation, but the haptoglobin-haemoglobin complex did, consistent with the documented ability of haemoglobin to inhibit this phenomenon. The concentration of haemoglobin normally bound to haptoglobin is sufficient to account for the inhibitory properties of human plasma. This suggests that endothelium derived relaxing factor exerts no downstream intravascular effect in vivo and thus that its physiological dilator role is that of a local autocoid acting on subjacent smooth muscle.

Production of endothelium derived relaxant factor is dependent on oxidative phosphorylation and extracellular calcium.

The production (synthesis or release or both) of endothelium derived relaxant factor was studied in rabbit aortic strip preparations and an aortic-coronary artery bioassay system. Production of endothelium derived relaxant factor was rapidly inhibited by agents that inhibit mitochondrial electron transport or F1-ATPase, or which uncouple oxidative phosphorylation, but was only slowly impaired by inhibition of glycolysis. It was dependent also on the presence of extracellular calcium with a rapid on-off response time. This study shows that production of endothelium derived relaxant factor appears to be dependent on both oxidative phosphorylation and extracellular calcium.

Evidence for the presence of histamine in Gaboon viper ( Bitis gabonica) venom

A. K. Tilmisany, A. A. Mustafa, A. Abdel Aziz and O. H. Osman. Evidence for the presence of histamine in Gaboon viper ( Bitis gabonica) venom. Toxicon 24, 1159 – 1161, 1986. — Venom from B. gabonica produced a dose-dependent contraction of both isolated guinea-pig ileum and rabbit aortic strip preparations. The venom-induced contraction was not antagonised by pretreatment with atropine, phentolamine, methysergide or indomethacin, however, it was blocked by the prior addition of chlorpheniramine or cyproheptadine. Upon exhaustive dialysis of the venom, the dialysate contracted both preparations, while the dialysed venom had no effect. Paper and thin-layer chromatography of the venom showed a spot with an R f value corresponding to authentic histamine. Biological assay of the crude venom on the guinea-pig ileum showed that it contained the equivalent of 2 – 10 μg of histamine per mg dry weight of crude venom. pA 2 values for chlorpheniramine using either the venom or histamine as agonists were not significantly different.

Organ selective angiotensin antagonists: Sarcosyl 1-cysteinyl(s-methyl) 8-angiotensin I

An angiotensin antagonist, Sarcosyl 1-Cysteinyl(S-Methyl) 8-angiotensin I [Sar 1-Cys(Me) 8-ANG I] was synthesized and its pharmacological properties evaluated in vivo (rat blood pressure assay) and in vitro (rabbit aortic strip, guinea-pig ileum and rat uterus assay). It was found to be an extremely potent angiotensin II (ANG II) antagonist in the rat pressor assay (dose ratio for ANG II of 1300 during infusion of 5.0 μg/kg/min Sar 1-Cys-(Me) 8-ANG I) and a moderately effective antagonist in guinea-pig ileum (pA 2 ≅ 8.2). Moderate antagonism was also seen in the rabbit aortic strip preparation (pA 2 ≅ 8.1) while the analog was inactive in the rat uterus assay. In each of the preparations where antagonist activity was observed there was evidence of non-competitive antagonism. Most striking was the inability of extremely high doses (up to 125 μg ANG II/kg) of ANG II to overcome the Sar 1-Cys(Me) 8-ANG I blockade. In both the rat pressor and guinea-pig ileum assays the Sar 1-Cys(Me) 8-ANG I antagonism is completely abolished in the presence of the converting enzyme inhibitor SQ14225 (Captopril®-Squibb). Organ selectivity of this analog is discussed in terms of the inherent activity of the active principle (i.e. the Sar 1-Cys(Me) 8-angiotensin II [Sar 1-Cys(Me) 8-ANG II] released by the action of converting enzyme) and the availability of converting enzyme in each bioassay.

Changes of adrenergic reaction pattern in experimental diabetes mellitus.

Changes of the adrenergic reaction pattern in experimental diabetes have been studied in rabbit aortic strip preparations. Strips taken from alloxan-treated diabetic rabbits showed increased noradrenaline (NA) sensitivity when compared to control preparations. A similar increase in NA sensitivity was noted in strips from prednisolone-treated, non-diabetic animals. Whereas in the control and prednisolone-treated groups, NA contractions could be blocked only by alphaand not by beta-adrenergic blocking agents, in diabetes both phentolamine, 0.5 |ig/ml, and propranolol, 0.5-2 M-g/ml, produced a significant parallel shift to the right of the NA dose-response curve. In control preparations isoproterenol (ISO) exerted only a contraction at high concentrations, which could be blocked by alpha-blocking agents. In diabetes sensitivity to this effect increased, and it became susceptible to beta-blocking agents. Sensitivity to phenylephrine remained the same in all 3 groups. After cold storage at + 5 C for 24 hr the NA sensitivity of strips from diabetic animals remained increased, whereas the inhibitory action of propranolol disappeared. It is concluded that separable quantitative (supersensitivity) and qualitative (vasoconstriction sensitive also to beta antagonists) changes in adrenergic reactions develop in experimental diabetes. Possible mechanisms for the 2 phenomena are discussed. {Endocrinology 93: 752, 1973) E H A N C E D responsiveness of the peripheral blood vessels to endogenous catecholamines in diabetes is a well-known phenomenon and it may account for the appearance of vascular disease in diabetic patients (1 ) . Since similar functional changes in the vascular reaction pattern precede by far and may underlie morphologic changes of the vessels, it is important to know whether increased catecholamine sensitivity is the only change in the functional state of the vessels or whether qualitative changes in the adrenergic receptor mechanism also appear in di-

Substrate requirements for angiotensin I conversion in vivo and in vitro.

The substrate requirements for angiotensin I-converting enzyme were studied in vivo in the dog lung and in vitro in plasma, using angiotensin I (AI), 5- D -Ile-AI, 7- D -Pro-AI, and 8-D-Phe-AI which had been synthesized by the solid-phase technique. All peptides were inactive in the rabbit aortic strip preparation. None of the D-amino acid-substituted peptides gave a pressor response in the anesthetized dog; none showed significant immunologic cross-reactivity with anti-AI serum or antiangiotensin II serum. Each peptide was labeled with 125 I, and the monoiodinated species was isolated. The iodinated peptides were incubated with diluted dog plasma or injected into the right ventricle of intact anesthetized dogs. In vitro, 125 I-AI, 125 I-5-D-IIe-AI, and 125 I-7-D-Pro-AI were converted to angiotensin II (AII). 8-D-Phe-AI was not converted. In vivo, 15 seconds after injecting 125 I-AI, 125 I-5-D-IIe-AI, or 125 I-7-D-Pro-AI into the right ventricle, 125 I-AJI accounted for 70, 60, and 45%, respectively, of the radioactive material in aortic blood. These results and our previous observations on the importance of the C-terminal sequence of AI for conversion indicate that the enzymatic binding sites for AI-converting enzyme in vivo and in vitro extend from position 10 to position 8 but not to position 7. D-amino acid substitution at positions 7 and 5 abolishes biologic and immunologic activity without interfering with conversion.

Mechanism of pulmonary conversion of angiotensin I to angiotensin II in the dog.

The mechanism of conversion of angiotensin I to angiotensin II was studied in vivo in the pulmonary circulation of the intact anesthetized dog and in vitro in plasma by using L-Leu-anglotensin I, D-Leu-angiotensin I, and des-Leu-angiotensin I which had been synthesized by the solid-phase technique. None of the peptides were active in the rabbit aortic strip preparation; D-Leu-angiotensin I and des-Leu-angiotensin I gave less than 5% of the pressor response of L-Leu-angiotensin I in the pentolinium-treated rat. D-Leu-angiotensin I and des-Leu-angiotensin I were not converted to angiotensin II when incubated with diluted human or dog plasma or partially purified converting enzyme from dog plasma. In a single pulmonary circulation time, following injection of a 20-nmole bolus of L-Leu-angiotensin I into the dog right ventricle, 56% of the material recovered had been converted to angiotensin II, as measured by radioimmunoassay. Conversion was accompanied by a rise in mean blood pressure of 30 mm Hg. After injection of equimolar amounts of D-Leu-angiotensin I and des-Leu-angiotensin I, no generation of angiotensin II and no pressor response occurred. These observations indicate that pulmonary conversion in vivo and plasma conversion in vitro occur via a dipepridylcarboxypeptidase and that a D-amino acid at the C-terminus prevents conversion.

Effects of isoxazolium cations on some isolated muscle preparations.

1. The quaternary ammonium compound, N,t-butyl-5-methyl isoxazolium perchlorate (BIP), an anionic group reagent, initially causes contractions of the rat phrenic-nerve diaphragm, guinea-pig ileum and rabbit aortic strip preparations in vitro.2. In addition, the drug produces an irreversible block of indirectly elicited twitch responses in the diaphragm and of contractions induced by acetylcholine, methylfurmethide, dimethyl-phenylpiperazinium, 5-hydroxytryptamine (5-HT), histamine, angiotensin and pilocarpine in the ileum, while direct electrical stimulation of the diaphragm and contractions of the ileum to Ba and K ions are relatively unaffected.3. BIP is also an irreversible inhibitor of acetylcholinesterase but not of butyrylcholinesterase.4. On rabbit aortic strip preparations, responses to histamine, noradrenaline and 5-HT were differentially sensitive to irreversible blockade by BIP.5. Diphenhydramine, used in conditions which gave complete protection of the histamine response to irreversible block by dibenamine, did not protect against the blocking action of BIP but increased the blockade.6. These results suggest that BIP reacts covalently with anionic groups which mediate receptor initiated stimuli. The isoxazolium group may be useful in conferring irreversible properties by its substitution in drug molecules for the pyrrole or pyrrolidine group.

Potentiation of smooth muscle contraction by adrenal steroids.

The rabbit aortic strip preparation of Furchgott was used to test the effect of adrenal steroids upon the muscle contraction produced by 0.1 to 0.2 µ g. of levarterenol. The steroids tested were corticosterone, hydrocortisone, aldosterone acetate, androsterone, and dehydroisoandrosterone. Amounts of 1 to 300 µ g. were added to the 20-ml. muscle bath. Potentiation was produced by each steroid; the percentage increase of amplitude of contraction was from 36.1 to 74.2 percent. Corticosterone produced consistent potentiation in amounts as small as 5 µ g.; 200 µ g. of aldosterone acetate was required to produce potentiation. Maximum potentiation was produced by hydrocortisone. The observed potentiation may be due to an effect of adrenal steroids upon transcellular sodium and potassium gradients.