Contraction Of Rabbit Aortic Rings Research Articles

Two novel Bv8-like peptides from skin secretions of the toad Bombina maxima

Two novel bioactive peptides were purified from skin secretions of the toad Bombina maxima. The partial N-terminal sequences of these two peptides were determined by automated Edman degradation. This allowed the cloning of full-length cDNAs encoding these two peptides from a cDNA library prepared from the toad skin. The deduced complete amino acid sequences indicate that both peptides are composed of 77 amino acids. A fasta search in the databanks revealed that they exhibit 86–91% sequence identity with Bv8, a peptide originally isolated from skin secretions of Bombina variegata. They were thus named as Bv8-like peptide 1 (Bv8-LP1) and Bv8-like peptide 2 (Bv8-LP2), respectively. Sequence differences between Bv8-LP1 and 2 were due to six amino acid substitutions at positions 6, 11, 23, 24, 62 and 63. Bv8-LP1 and 2 differed from Bv8 with eleven and seven amino acid substitutions, respectively. Like Bv8, Bv8-LP1 and 2 possessed contractile activity on isolated guinea pig ileum. Additionally, they stimulated contraction of rabbit aortic rings in a dose-dependent manner at nanomolar concentrations.

Calcium antagonist properties of the bisbenzylisoquinoline alkaloid cycleanine

The alkaloid cycleanine ([12aR-(12aR,24aR)]-2,3,12a,13,14,15,24,24a-octa hydro-5,6,17,18- tetramethoxy-1,13-dimethyl-8, 11:20,23-dietheno-1H,12H [1,10]dioxacyclooctadecino[2,3,4-ij:11,12,13-i'j']diisoquinolin e) was extracted from the bulbs of Stephania glabra (Roxb) Miers and its effects on cardiac and smooth muscle preparations were studied and compared to those of nifedipine (1,4-dihydro-2, 6-dimethyl-4-(2-nitrophenyl)-3,5-pyridine dicarboxylic acid dimethylesther). Cycleanine inhibited the KCl-induced contraction of rabbit aortic rings with higher potency than nifedipine. IC50s for cycleanine and nifedipine were 0.8 and 7.10(-9) M respectively. Cycleanine had minor effects on the norepinephrine-induced contraction of rabbit aortic rings. Cycleanine and nifedipine also depressed the contraction of rat ventricular preparations but with lower potency (IC50 = 3 and 0.03.10(-6) M respectively). Action potential duration of rat right ventricular strips was decreased by both compounds. L-type Ca-current (ICaL) of single rat ventricular cardiomyocytes was inhibited by cycleanine in a voltage- and frequency-dependent manner. With a higher potency nifedipine inhibited ICaL in a tonic and almost frequency-independent manner. The results suggest that cycleanine can act as a potent vascular selective Ca-antagonist.

Antivasoconstrictor and Antiaggregatory Activities of Picotamide Unrelated to Thromboxane A2 Antagonism

Picotamide is a dual thromboxane (Tx) A2 receptor antagonist/Tx synthase inhibitor although some observations suggest an anti-vasoconstrictor effect independent of TxA2 inhibition/antagonism. The aim of our study was to assess whether picotamide antagonises vascular contractions induced by different vasoactive substances in vitro. Picotamide inhibited competitively the contraction of rabbit aortic rings induced by the TxA2 mimetic U46619 (pA2 = 3.59) but also the contractions induced by phenylephrine (pA2 = 3.93) and serotonin (5-HT) (pA2 = 5.81) although in a not competitive way. Picotamide did not inhibit potassium-induced contractions, thus excluding aspecific effects on vascular smooth muscle. Picotamide inhibited 5-HT-induced platelet aggregation in vitro with an IC50 (212 microM) similar to that found when other aggregating stimuli are used, but it did not affect shape change (IC50 > 1 mM) suggesting that the effects of picotamide can not be ascribed to 5-HT2-receptor antagonism; in the same experimental conditions neither a Tx-receptor antagonist (BM13.177) nor a dual Tx-receptor antagonist/synthase inhibitor (ridogrel) affected 5-HT-induced platelet responses. Our studies demonstrate that picotamide exerts antivasoconstrictor and platelet inhibitory effects unrelated to TxA2 antagonism. This activity may contribute to the anti-thrombotic/anti-ischaemic effects of the drug in vivo.

Pharmacology of SC-52458, an Orally Active, Nonpeptide Angiotensin AT1 Receptor Antagonist

We describe the pharmacologic properties of SC-52458, 5-[(3,5-dibutyl-1H-1,2,4-triazol-1-yl)methyl]-2-[2-(1H-tetrazol - 5-ylphenyl)]pyridine, a novel nonpeptide angiotensin II (AII) receptor antagonist. SC-52458 was a potent inhibitor of [125I]AII binding to AT1 receptors in rat adrenal cortex and uterine smooth muscle membranes (IC50 values of 2.8 and 6.9 nM, respectively). Contraction of rabbit aortic rings by AII was antagonized by SC-52458 in a competitive and reversible manner (pA2 of 8.18). SC-52458 had no effect on the activity of angiotensin converting enzyme (ACE) or renin in vitro. In normotensive rats, administration of SC-52458, either intravenously (i.v.) or by gavage, inhibited the pressor response to AII. Daily treatment with SC-52458 at 20, 30, and 50 mg/kg by gavage for 4 days decreased blood pressure (BP) in conscious, spontaneously hypertensive rats (SHR). Further studies in renal-artery ligated rats and sodium-deficient dogs demonstrated that oral administration of SC-52458 decreased BP and that this activity was correlated with significant plasma levels of the compound. SC-52458 is an orally active, competitive AT1-receptor antagonist with antihypertensive properties.

Cromakalim Inhibits Multiple Mechanisms of Smooth Muscle Activation with Similar Stereoselectivity

Purified cromakalim trans enantiomers were tested for their ability to antagonize three specific mechanisms of smooth muscle activation, i.e., depolarization-induced Ca2+ entry through voltage-gated channels, agonist-induced Ca2+ entry, and agonist-induced Ca2+ release. Cromakalim effects were studied in rabbit aortic rings contracted by stimuli corresponding to the above mechanisms. First, aortic rings were contracted by increase in extracellular [K+] (to 27 mM), which causes partial membrane depolarization. Under these conditions, (-)-cromakalim exhibited an EC50 of 0.18 microM and a 150-fold higher relaxing potency than the (+)-enantiomer. Second, in aortic rings tonically contracted by 1 microM norepinephrine (NE) in the presence of 1 microM nifedipine, i.e., in rings contracted mainly owing to NE-stimulated Ca2+ entry through receptor-operated channels, (-)-cromakalim induced relaxation with an EC50 of 0.68 microM and exhibited a 191-fold higher potency than the (+)-enantiomer. Third, phasic, NE-induced contractions of rabbit aortic rings in the absence of extracellular Ca2+, i.e., contractions that reflect release of Ca2+ from intracellular stores, were antagonized with an EC50 of 0.29 microM and a 144-fold higher potency than the (+)-enantiomer. All effects of (-)-cromakalim were blocked by either completely depolarizing the vessels with high extracellular [K+] (40 mM) or by addition of the K+ channel blocker glibenclamide (10 microM). Cromakalim relaxed rabbit aorta independent of the mechanism underlying smooth muscle tone. Cromakalim effects were equivalent with respect to dose dependence, stereoselectivity, and sensitivity to extracellular [K+] and glibenclamide.(ABSTRACT TRUNCATED AT 250 WORDS)

Endothelin Release and Shift in Prostaglandin Balance Are Involved in the Modulation of Vascular Tone by Recombinant Erythropoietin

Hypertension is the main side effect developing in patients suffering from renal anemia who are treated with recombinant human erythropoietin (rhEPO). We investigated the effect of rhEPO on the vascular tone of rabbit aorta. rhEPO had no direct vasoconstrictor effect, but it enhanced norepinephrine (NE)-induced contractions of rabbit aortic rings. Relaxations to acetylcholine (ACh, 1 microM) were unaltered in the presence or absence of rhEPO, indicating that the endothelium-dependent NO pathway was not affected by rhEPO. In rings of human renal artery and rabbit aorta, rhEPO (200 U/ml) increased the synthesis of constrictor prostanoids. The cyclooxygenase inhibitors indomethacin and aspirin abolished the increase in prostanoid production. However, they did not completely suppress the rhEPO-induced enhancement of NE contractions in rabbit aorta. We further investigated the effect of rhEPO on prostanoid and endothelin-1 synthesis in cultured human endothelial cells. Endothelial cells from human umbilical veins (HUVEC) were isolated and cultured. After incubation with rhEPO, the formation of prostaglandin (PG) I2 (analyzed as its stable metabolite 6-keto-PGF1 alpha), PGF2 alpha, PGE2, thromboxane (Tx) B2, and of endothelin-1 (ET-1) was measured by radioimmunoassay (RIA). rhEPO (200 U/ml) increased the formation of PGF2 alpha and TxB2 and decreased the formation of PGI2 in HUVEC. The release of ET-1 was increased by nearly 90% in the presence of rhEPO (200 U/ml). We conclude that a shift in the balance of constrictor and relaxing prostanoids as well as an increased synthesis of ET-1 may contribute to the hypertensive side effect of rhEPO therapy. ET-1 may at least in part be responsible for the unexpectedly low inhibitory effect of indomethacin on rhEPO-enhanced contractions of rabbit aorta.

Identification and partial purification of a thiol endothelin-converting enzyme from porcine aortic endothelial cells.

Endothelin is a potent peptide vasoconstrictor. The final step in the processing of endothelin has been postulated to be the cleavage of the Trp21-Val22 peptide bond in proendothelin by a putative endothelin-converting enzyme. A soluble extract of primary porcine aortic endothelial cells was found to contain an enzyme activity that converted proendothelin-1 (proET-1) to an endothelin-1 (ET-1)-like peptide as determined by the rabbit aortic ring contraction assay. This enzyme was partially purified by DE52 ion-exchange chromatography. Incubation of proET-1 with the partially purified enzyme generated a product which had a retention time on HPLC identical to that of authentic ET-1. Further analysis of the product showed that it caused contraction of rabbit aortic rings, had a molecular weight identical to ET-1 as measured by fast atom bombardment mass spectrometry, and competed for [125I]ET-1 binding in an RIA using specific antibodies which recognize the carboxy terminal tryptophan of ET-1. The enzyme activity could be inhibited by thiol protease inhibitors such as Z-phe-pheCHN2 and p-hydroxymercuribenzoate, but not by serine- or metalloprotease inhibitors. The optimal pH for the enzymatic activity was between 7.0 and 7.5, and no activity was detected at pH 4.0. These results demonstrate that this thiol protease is a potential endothelin-converting enzyme.

A Vascular Smooth Muscle Relaxant Effect of Rosmarinus officinalis

AbstractThe effects of the volatile oil of Rosmarinus officinalis leaves on the vascular smooth muscle of rabbit were tested in vitro using isolated aortic segments (rings). The volatile oil of R. officinalis leaves inhibited the contractions of rabbit aortic rings induced by norepinephrine (NE) stimulation in Ca2+-containing solution. Also, the volatile oil inhibited the contractions of rabbit aortic rings induced by high potassium (K+) solutions. This inhibition was dose-dependent and reversible. Furthermore, the volatile oil inhibited the contractions of rabbit aortic rings induced by NE stimulation in Ca2+-free solution. These data suggest that the volatile oil of R. officinalis leaves has a direct vascular smooth muscle relaxant effect.

Nonpeptidic angiotensin II antagonists: synthesis and in vitro activity of a series of novel naphthalene and tetrahydronaphthalene derivatives

Starting from the structure of the novel nonpeptidic angiotensin II antagonist DuP 753, a series of more rigid analogues was prepared by replacing the biphenyl part of DuP 753 with a naphthalene ring. Five different regioisomers (compounds 6a-e) were synthesized, and receptor binding in rat smooth muscle cell preparations as well as inhibition of angiotensin II induced contraction of rabbit aortic rings was measured and the order of potency was compared with predictions made on the basis of a molecular modeling study. In good agreement with the predictions, the 2,6-substituted regioisomer 6d and its analogue 7 (isomeric at the imidazole substituent) were found to be most potent, but were still weaker than DuP 753. Tetrahydronaphthalene derivatives with and without an additional methyl group in the alpha-position to the acidic function and with this same 2,6-substitution pattern (compounds listed in Table III) were then prepared with the expectation of getting a further increase in potency. Whereas the carboxylic acid derivatives 13a,b showed activity in the expected potency range, surprisingly no further potency increase was observed after replacement of the carboxylic acid function by a tetrazole (compounds 18a,b). These results may indicate that the compounds do not bind to the AT1 receptor in the same way as DuP 753.

Stereoselective and nonstereoselective inhibition exhibited by the enantiomers of verapamil

The interaction of the isomers of verapamil with sites on the calcium channel and alpha 1-adrenergic receptor has been examined. The inhibitory potency of these enantiomers differ with respect to the agonist. KCl- or clonidine-induced contractions of rabbit aortic rings were inhibited in a stereoselective manner by the enantiomers of verapamil with the (-)-isomer being more potent than the (+)-isomer. Similarly, (-)-verapamil was also more potent at displacing (-)-[N-methyl-3H]desmethoxyverapamil than was the (+)-isomer. In contrast, the inhibition of norepinephrine- or phenylephrine-induced aortic contractions was not stereoselective. Differences in enantiomer potency were also observed in vivo. The ability of clonidine to increase blood pressure in the anesthetized rat was blocked in a stereoselective manner by the verapamil enantiomers, while inhibition of the pressor actions of phenylephrine was not. In summary, for agents that rely heavily on calcium channel function (KCl, clonidine), stereoselective inhibition was observed. Stereoselective inhibition was not observed against high efficacy alpha 1-agonists. This difference in stereochemistry argues that verapamil does not act at the same site when inhibiting clonidine or KCl action when compared with norepinephrine or phenylephrine.

Elevation of cyclic AMP by prostacyclin is accompanied by relaxation of bovine coronary arteries and contraction of rabbit aortic rings

The role of cyclic AMP (cAMP) in the control of vascular smooth muscle tension was examined by comparing the effects of prostacyclin (PGI 2) on tension and cAMP levels in helical strips of bovine coronary arteries and in rabbit aortic rings, both denuded of endothelium. In bovine coronary arteries, PGI 2 elevated cAMP levels and relaxed the muscles. The PGI 2-induced cAMP elevation preceded the relaxation and both parameters were altered in a dose-dependent manner by increasing concentrations of PGI 2 (0.3, 3 and 30 μM). These results are consistent with a role for cAMP as a mediator of vascular smooth muscle relaxation. Cyclic AMP levels were also elevated by PGI 2 in a concentration- and time-dependent manner in rabbit aortic rings. However, in direct contrast to the results in the bovine coronary arteries, PGI 2-induced elevation of cAMP in the aortic rings was accompanied by contraction rather than relaxation. Isoproterenol, a drug which is generally believed to relax smooth muscles by virtue of its ability to increase tissue levels of cAMP, relaxed PGI 2-contracted aortic rings with no further elevation of cAMP beyond that caused by the PGI 2 alone. These results demonstrate that cAMP elevation and relaxation of vascular smooth muscle are not always well correlated. It is possible that some form of functional compartmentalization of cAMP or cAMP-dependent protein kinase exists in these tissues.

The facilitative actions of bay K 8644 on norepinephrine and KCL-induced contractures of rabbit aortic rings

The effect of the calcium channel agonist BAY K 8644 on the ability of KCl and norepinephrine to induce contractions of rabbit aortic rings has been examined in Krebs-Henseleit buffer containing either 4.0 or 6.8 mM potassium. BAY K 8644 (10 −8 to 10 −6 M) alone induced slowly developing aortic contractures which were 10 (at 4.0 mM potassium) or 20 (at 6.8 mM potassium) percent of the maximum obtainable with norepinephrine. These contractions were not observed in every experiment, but were more likely to occur at 6.8 mM (71% at 10 −6 M BAY K 8644) when compared to 4.0 mM (31% at 10 −6 M BAY K 8644) potassium buffer. BAY K 8644, in either potassium buffer, induced a statistically significant shift to the left in the norepinephrine dose-response curve. The norepinephrine dose-response curve was significantly curvilinear in the presence of 3 × 10 −8 M BAY K 8644 (6.8 mM potassium) and 10 −6 M BAY K 8644 (4.0 mM potassium). Similarly, BAY K 8644 induced sinistral shifts in the KCl dose-response curve with a curvilinear function observed at 3 × 10 −7 M BAY K 8644. These data show that BAY K 8644 is capable of inducing aortic contractures at potassium concentrations significantly lower than previously reported. Furthermore, BAY K 8644 facilitates opening of calcium channels by either potassium or norepinephrine. In contrast to others, our data indicates that BAY K 8644 can effect calcium channels activated by norepinephrine. Finally, our data suggest that the alpha and dihydropyridine receptors are capable of interacting and that occupation of one receptor can affect the action of a compound binding to the other receptor.

Stereoselectivity at the Calcium Channel

The stereoisomers of the new dihydropyridine derivative 202-791 [isopropyl 4-(2,1,3-benzoxadiazol-4-yl)-1,4-dihydro-2,6-dimethyl-5-nitro-3 -pyridinecarboxylate] were synthesized separately and tested on isolated rabbit aortic rings for effects on depolarization-induced contraction and depolarization-stimulated uptake of 45Ca2+. The racemic mixture enhanced contraction of rabbit aortic rings at low levels of depolarization but inhibited contraction and 45Ca2+ uptake at high levels of depolarization. The IC50 values were 2.0 X 10(-7) and 1.7 X 10(-7) M, respectively. The R enantiomer inhibited contraction and 45Ca2+ uptake with IC50 values of 3.2 X 10(-8) and 4.3 X 10(-8) M, respectively. This compound showed no stimulant activity. By contrast, the S enantiomer of 202-791 shifted the concentration-response curve for depolarization-induced contraction in an almost parallel fashion to the left, thus enhancing contraction. The EC50 value for this effect at a KCl concentration of 16 mM was 1.8 X 10(-7) M. This compound enhanced 45Ca2+ uptake concentration dependently at all levels of depolarization tested. Thus, the stereoisomers of a dihydropyridine derivative may behave as a calcium entry blocker or a calcium entry enhancer on vascular smooth muscle, depending only on the stereochemistry. If asymmetric compounds elicit effects suggesting a dualistic action at the calcium channel, then the stereoisomers should be prepared.

Assessing calcium antagonism on vascular smooth muscle: A comparison of three methods

Three methods relying on depolarization-induced contraction of rabbit aortic rings were compared. The effects of hyperosmolarity induced by adding KCI to the bath and of a lowered NaCI concentration due to an exchange of NaCI for KCl were assessed. A dihydropyridine derivative (PY 108-068) and verapamil were used as antagonists. For the first method, 50.3 mM KCI was either added to the bath to reach a final concentration of 55 mM, or KCl was exchanged for NaCI to keep the bath isoosmotic. We found a noncompetitive antagonism and the results obtained from cumulative concentration-response curves appeared to be based on the slow tonic contraction. For the second method, the rings were suspended in a calcium-free Krebs-Henseleit (KH) or TRIS bath solution containing also 55 mM of KCl and calcium was added as an agonist. We found a competitive inhibition of the calcium effects. The slope of the Schild Plot was, however, −1.3 for PY 108-068 and −0.5 for verapamil. Very high concentrations of calcium had relaxant effects and thus distorted the results. For the third method, the rings were contracted by KCl and then relaxed by adding the calcium antagonist to the bath. Isoosmotic solution and solution made hyperosmotic with sucrose were also used. Hyperosmolarity by itself contributed to the tension development. In hyperosmolar solution, the calcium antagonists appeared to be less active. Such bath conditions can therefore considerably bias the results. The first and the second method, in combination with experiments using receptor stimulating agents such as noradrenaline, appear to be most suitable for investigating calcium antagonists on blood vessels.

PY 108-068, a new, potent, and selective inhibitor of calcium-induced contraction of rabbit aortic rings.

PY 108-068 (PY) is a new benzoxadiazolyle dihydropyridine derivative. It potently antagonizes calcium-induced contractions of rabbit aorta in depolarizing solution (PD'2:8.8). This antagonism is selective, since no relevant antagonistic effects against noradrenaline (NA)-, serotonin (5-HT)-, and angiotensin II (AII)-induced contractions are found at the highest concentration of PY (10(-5) M). Verapamil (V), examined for comparison, was less selective with respect to its antagonism: pA2 value against calcium-induced contractions in depolarizing solution: 7.6, pA2 against serotonin-induced contractions: 6.9. In calcium-free Krebs--Henseleit solution tachyphylaxis occurred after three to four administrations of NA, 5-HT, or AII. When calcium was added in the presence of one of these agonists, the slow phase of concentration, which depends on extracellular calcium, developed. This slow phase of contraction was not inhibited to a relevant extent by a 10(-5) M concentration of PY. Verapamil inhibited the tonic contraction induced by serotonin (pD'2: 5.5) but not to a relevant extent the contractions induced by the two other agonists. PY therefore, inhibits calcium-induced contractions of rabbit aorta in depolarizing solution very selectively, and shows hardly any effects on receptor-operated channels.