Chitosan gels for buccal delivery of Schinus molle L. essential oil in dogs: characterization and antimicrobial activity in vitro.

Periodontal disease is considered the main oral cavity disorder in dogs. Essential oils have the potential for use in the prevention and treatment of oral diseases. The antimicrobial activity of Schinus molle L. essential oil (SMEO) has already been reported. Chitosan, a natural product with antimicrobial activity and good biocompatibility has potential in biodental applications. In this study, we evaluated the in vitro antimicrobial activity of SMEO against bacteria associated with periodontal disease in dogs, developed and evaluated the physicochemical properties of a novel chitosan-based buccal delivery system containing SMEO. SMEO showed antimicrobial activity against Gram positive and Gram negative bacteria associated with canine periodontitis, with MIC values of 750 µg.mL-1 for Staphylococcus spp. and Streptococcus spp, 1000 µg.mL-1 for Corynebacterium spp. and 1250 µg.mL-1 for Pseudomonas spp. All formulations evaluated presented adequate physicochemical properties, good stability, and pH values close to buccal pH (5.0-7.0). Chitosan gel loaded with SMEO showed potential as a SMEO delivery system, having the ideal physicochemical and rheological properties (pseudoplastic and apparent viscosities) required for application on buccal tissue. Thus, we can conclude that formulation has the potential to be used for buccal mucosa delivery in the prevention and treatment of periodontal disease in dogs.

In vitro efficacy of essential oils and extracts of Schinus molle L. against Ctenocephalides felis felis.

Extracts and essential oils from plants are important natural sources of pesticides. These compounds are considered an alternative to control ectoparasites of veterinary importance. Schinus molle, an endemic species of Brazil, produces a high level of essential oil and several other compounds. The aim of this work was to determinate the chemical composition of extracts and essential oils of S. molle and further to evaluate the activity against eggs and adults of Ctenocephalides felis felis, a predominant flea that infests dogs and cats in Brazil. In an in vitro assay, the non-polar (n-hexane) extract showed 100% efficacy (800 µg cm(-2); LD50 = 524·80 µg cm(-2)) at 24 and 48 h. Its major compound was lupenone (50·25%). Essential oils from fruits and leaves were evaluated, and had 100% efficacy against adult fleas at 800 µg cm(-2) (LD50 = 353·95 µg cm(-2)) and at 50 µg cm(-2) (LD50 = 12·02 µg cm(-2)), respectively. On the other hand, the essential oil from fruits and leaves was not active against flea eggs. This is the first study that reports the insecticidal effects of essential oils and extracts obtained from Schinus molle against Ctenocephalides felis felis.

Cytotoxicity of Extracts from Petiveria alliacea Leaves on Yeast.

Petiveria alliacea L. is a plant used in traditional medicine harboring pharmacological properties with anti-inflammatory, antinociceptive, hypoglycemiant and anesthetic activities. This study assessed the potential cytotoxic, genotoxic and mutagenic effects of ethanolic extract of P. alliacea on Saccharomyces cerevisiae strains. S. cerevisiae FF18733 (wild type) and CD138 (ogg1) strains were exposed to fractioned ethanolic extracts of P. alliacea in different concentrations. Three experimental assays were performed: cellular inactivation, mutagenesis (canavanine resistance system) and loss of mitochondrial function (petites colonies). The chemical analyses revealed a rich extract with phenolic compounds such as protocatechuic acid, cinnamic and catechin epicatechin. A decreased cell viability in wild-type and ogg1 strains was demonstrated. All fractions of the extract exerted a mutagenic effect on the ogg1 strain. Only ethyl acetate and n-butanol fractions increased the rate of petites colonies in the ogg1 strain, but not in the wild-type strain. The results indicate that fractions of mid-polarity of the ethanolic extract, at the studied concentrations, can induce mutagenicity mediated by oxidative lesions in the mitochondrial and genomic genomes of the ogg1-deficient S. cerevisiae strain. These findings indicate that the lesions caused by the fractions of P. alliacea ethanolic extract can be mediated by reactive oxygen species and can reach multiple molecular targets to exert their toxicity.

Comparative bioinformatic and structural analyses of pepsin and renin.

Pepsin, the archetypal pepsin-like aspartic protease, is irreversibly denatured when exposed to neutral pH conditions whereas renin, a structural homologue of pepsin, is fully stable and optimally active in the same conditions despite sharing highly similar enzyme architecture. To gain insight into the structural determinants of differential aspartic protease pH stability, the present study used comparative bioinformatic and structural analyses. In pepsin, an abundance of polar and aspartic acid residues were identified, a common trait with other acid-stable enzymes. Conversely, renin was shown to have increased levels of basic amino acids. In both pepsin and renin, the solvent exposure of these charged groups was high. Having similar overall acidic residue content, the solvent-exposed basic residues may allow for extensive salt bridge formation in renin, whereas in pepsin, these residues are protonated and serve to form stabilizing hydrogen bonds at low pH. Relative differences in structure and sequence in the turn and joint regions of the ß-barrel and ψ-loop in both the N- and C-terminal lobes were identified as regions of interest in defining divergent pH stability. Compared to the structural rigidity of renin, pepsin has more instability associated with the N-terminus, specifically the B/C connector. By contrast, renin exhibits greater C-terminal instability in turn and connector regions. Overall, flexibility differences in connector regions, and amino acid composition, particularly in turn and joint regions of the ß-barrel and ψ-loops, likely play defining roles in determining pH stability for renin and pepsin.

Palosuran inhibits binding to primate UT receptors in cell membranes but demonstrates differential activity in intact cells and vascular tissues.

BACKGROUND AND PURPOSE: The recent development of the UT ligand palosuran (1-[2-(4-benzyl-4-hydroxy-piperidin-1-yl)-ethyl]-3-(2-methyl-quinolin-4-yl)-urea sulphate salt) has led to the proposition that urotensin-II (U-II) plays a significant pathological role in acute and chronic renal injury in the rat. EXPERIMENTAL APPROACH: In the present study, the pharmacological properties of palosuran were investigated further using a series of radioligand binding and functional bioassays. KEY RESULTS: Palosuran functioned as a 'primate-selective' UT ligand in recombinant cell membranes (monkey and human UT K(i) values of 4 +/- 1 and 5 +/- 1 nM), lacking appreciable affinity at other mammalian UT isoforms (rodent and feline K(i) values >1 microM). Paradoxically, however, palosuran lost significant (10- to 54-fold) affinity for native and recombinant human UT when radioligand binding was performed in intact cells (K(i) values of 50 +/- 3 and 276 +/- 67 nM). In accordance, palosuran also exhibited diminished activity in hUT (human urotensin-II receptor)-CHO (Chinese hamster ovary) cells (IC50 323 +/- 67 nM) and isolated arteries (K(b)>10 microM in rat aorta; K(b)>8.5 microM in cat arteries; K(b)>1.6 microM in monkey arteries; K(b) 2.2 +/- 0.6 microM in hUT transgenic mouse aorta). Relative to recombinant binding K(i) values, palosuran was subjected to a 392- to 690-fold reduction in functional activity in monkey isolated arteries. Such phenomena were peculiar to palosuran and were not apparent with an alternative chemotype, SB-657510 (2-bromo-N-[4-chloro-3-((R)-1-methyl-pyrrolidin-3-yloxy)-phenyl]-4,5-dimethoxybenzenesulphonamide HCl). CONCLUSIONS AND IMPLICATIONS: Collectively, such findings suggest that caution should be taken when interpreting data generated using palosuran. The loss of UT affinity/activity observed in intact cells and tissues cf. membranes offers a potential explanation for the disappointing clinical efficacy reported with palosuran in diabetic nephropathy patients. As such, the (patho)physiological significance of U-II in diabetic renal dysfunction remains uncertain.

The novel endocannabinoid receptor GPR55 is activated by atypical cannabinoids but does not mediate their vasodilator effects.

BACKGROUND AND PURPOSE: Atypical cannabinoids are thought to cause vasodilatation through an as-yet unidentified 'CBx' receptor. Recent reports suggest GPR55 is an atypical cannabinoid receptor, making it a candidate for the vasodilator 'CBx' receptor. The purpose of the present study was to test the hypothesis that human recombinant GPR55 is activated by atypical cannabinoids and mediates vasodilator responses to these agents. EXPERIMENTAL APPROACH: Human recombinant GPR55 was expressed in HEK293T cells and specific GTPgammaS activity was monitored as an index of receptor activation. In GPR55-deficient and wild-type littermate control mice, in vivo blood pressure measurement and isolated resistance artery myography were used to determine GPR55 dependence of atypical cannabinoid-induced haemodynamic and vasodilator responses. KEY RESULTS: Atypical cannabinoids O-1602 and abnormal cannabidiol both stimulated GPR55-dependent GTPgammaS activity (EC50 approximately 2 nM), whereas the CB1 and CB2-selective agonist WIN 55,212-2 showed no effect in GPR55-expressing HEK293T cell membranes. Baseline mean arterial pressure and heart rate were not different between WT and GPR55 KO mice. The blood pressure-lowering response to abnormal cannabidiol was not different between WT and KO mice (WT 20+/-2%, KO 26+/-5% change from baseline), nor was the vasodilator response to abnormal cannabidiol in isolated mesenteric arteries (IC50 approximately 3 micro M for WT and KO). The abnormal cannabidiol vasodilator response was antagonized equivalently by O-1918 in both strains. CONCLUSIONS: These results demonstrate that while GPR55 is activated by atypical cannabinoids, it does not appear to mediate the vasodilator effects of these agents.

P2X1 stimulation promotes thrombin receptor-mediated platelet aggregation.

P2X1 receptors are ATP-gated channel demonstrated to be involved in multiple platelet responses, although in vitro analysis has been complicated by the effects of rapid desensitization. To further investigate potential roles of P2X1 receptors in platelet activation, the current study employed methods which maximally preserved P2X1 functionality. In preliminary in vivo studies, P2X1-deficiency reduced thrombus formation following the laser-induced, but not FeCl3-induced injury. Given the multiple potential mechanisms involved in thrombus formation in vivo, including tissue-factor/thrombin generation pathways, subsequent studies were designed to investigate the effects of P2X1 inhibition or stimulation on platelet activation in vitro; specifically, the interaction of P2X1 with thrombin receptor stimulation. Aggregation initiated by low/threshold levels of a protease-activated receptor (PAR)4 agonist was reduced in P2X1-deficient murine platelets, and inhibition of P2X1 in wild-type platelets similarly reduced PAR4-mediated aggregation. In human platelets, aggregation to low/threshold stimulation of PAR1 was inhibited with the P2X1 antagonist MRS2159. In addition, P2X1 stimulation primed human platelet responses, such that subsequent sub-threshold PAR1 responses were converted into significant aggregation. Selective ADP receptor inhibitors attenuated P2X1-mediated priming, suggesting that the synergy between P2X1 and sub-threshold PAR1 stimulation was in part because of enhanced granular release of ADP. Overall, the present study defines a novel interaction between platelet P2X1 and thrombin receptors, with P2X1 functioning to amplify aggregation responses at low levels of thrombin receptor stimulation.

Human urotensin-II, the most potent mammalian vasoconstrictor identified to date, as a therapeutic target for the management of cardiovascular disease.

The novel cyclic undecapeptide human urotensin-II (hU-II) and its high-affinity G-protein-coupled receptor, GPR14, are both expressed within the human cardiovasculature (vascular smooth muscle, endothelium, myocardium, coronary atheroma, etc.) and may, therefore, contribute to the (patho)physiological regulation of cardiovascular homeostasis in humans. Indeed, hU-II is an efficacious, sustained spasmogen of mammalian isolated blood vessels including those from rats, rabbits, dogs, pigs, non-human primates and humans (where it is one to two orders of magnitude more potent than endothelin(ET)-1). In vivo, hU-II markedly alters systemic hemodynamics in the anesthetized primate (increase cardiac contractility [dP/dt], increase stroke volume, decrease total peripheral resistance) ultimately resulting in fatal cardiovascular collapse. As such, the development of selective hU-II receptor antagonists may be of utility in the management of cardiovascular disorders characterized by aberrant vasoconstriction, myocardial dysfunction and/or cardiac remodeling (e.g., myocardial infarction, congestive heart failure).

Selective ETA receptor antagonism with BQ-123 is insufficient to inhibit angioplasty induced neointima formation in the rat.

OBJECTIVE: The aim was to assess whether or not the endothelin ETA receptor selective antagonist BQ-123 could inhibit neointima formation in vivo following balloon angioplasty. METHODS: The effect of either acute administration of BQ-123 (0.1 mg.kg-1.min-1 intra-arterial infusion for 1 h before and 1 h after angioplasty) or chronic administration (bolus intraperitoneal injection, 2.5 mg.kg-1 twice daily; continuous intraperitoneal infusion, 0.8 and 8 mg.kg-1.d-1) on neointima formation was examined in rats which had undergone left common carotid artery balloon angioplasty. RESULTS: Neither acute intra-arterial infusion nor either mode of chronic intraperitoneal administration of BQ-123 had a significant effect on the degree of neointima formation observed following balloon angioplasty. CONCLUSIONS: Neither acute nor chronic ETA receptor blockade is sufficient to inhibit angioplasty induced neointima formation in the rat. Since it was previously shown that the ETA/B antagonist SB 209670 was effective in this model, while the ETA selective antagonist BQ-123 is now found to be ineffective, the data implicate the ETB receptor subtype in the pathogenesis of neointima formation.

Antihypertensive actions of the novel nonpeptide endothelin receptor antagonist SB 209670.

Indirect evidence has implicated endothelin-1 in the pathogenesis of hypertension. In the present study we examined such a role directly with SB 209670, a novel nonpeptide endothelin receptor antagonist. The antihypertensive and hemodynamic effects of SB 209670 were examined in conscious, unrestrained spontaneously hypertensive (SHR), normotensive Wistar-Kyoto (WKY), and renin-hypertensive rats. Sustained intravenous infusion of SB 209670 (10 micrograms.kg-1.min-1 for 6 hours) produced a significant, reversible reduction in mean arterial pressure in SHR but not in WKY rats. The antihypertensive response to 10 micrograms.kg-1.min-1 SB 209670 (approximately 25 mm Hg reduction in blood pressure) was associated with bradycardia (16% decrease in heart rate) but only a minimal reduction (3%) in cardiac output, because stroke volume was evaluated (by 15%). Therefore, the antihypertensive effect of SB 209670 resulted from a decrease (13%) in total peripheral resistance. A sustained antihypertensive effect could also be observed after intraduodenal administration of SB 209670 (3 mg/kg) in conscious SHR (reduction of approximately 35 mm Hg 5 hours after administration). SB 209670 (3 mg/kg intravenous bolus) did not alter the pressor response or tachycardia observed in pithed SHR after stimulation of thoracolumbar sympathetic outflow. SB 209670 was also antihypertensive in renin-hypertensive rats, lowering blood pressure to an extent similar to that observed in SHR. Thus, the data presented provide evidence to support a role for endothelin-1 in the pathophysiology of two animal models of hypertension.

BQ-123, a selective endothelin subtype A-receptor antagonist, lowers blood pressure in different rat models of hypertension.

OBJECTIVE: To investigate the role of endothelin-1 in the pathogenesis of hypertension directly by using the selective endothelin subtype A-receptor antagonist BQ-123. METHODS: The antihypertensive and hemodynamic effects of sustained BQ-123 administration were examined in conscious, unrestrained spontaneously hypertensive rats (SHR), normotensive Wistar-Kyoto (WKY) rats and renin hypertensive rats. RESULTS: Sustained infusions of BQ-123 (0.16-164 nmol/kg per min, intravenously, for 6 h) produced dose-dependent reductions in mean arterial pressure in SHR, the maximal reduction being obtained with a dose of 16 nmol/kg per min. This reversible response was evident up to 14 h after the cessation of antagonist infusion. The antihypertensive response to a maximal dose of BQ-123 was associated with bradycardia, but only a minimal reduction in cardiac output (since the stroke volume was elevated) in the SHR. Therefore, the antihypertensive effect of BQ-123 resulted from a decrease in total peripheral resistance. In contrast, in WKY rats the infusion of the high dose (164 nmol/kg per min, intravenously for 6 h) produced a small but significant reduction in mean arterial pressure. BQ-123 did not alter the pressor response or tachycardia observed in pithed SHR following stimulation of the thoracolumbar sympathetic outflow. BQ-123 was also antihypertensive in renin hypertensive rats, lowering the blood pressure to an extent similar to that observed in SHR. CONCLUSION: The data presented indicate a role for endothelin in the pathophysiology of hypertension.

Endothelium-dependent mesenteric vasorelaxant effects and systemic actions of endothelin (16-21) and other endothelin-related peptides in the rat.

1. The rat isolated superior mesenteric bed, perfused with Krebs-Henseleit solution containing 10 microM indomethacin and precontracted with 100 microM methoxamine, was used to study the vasorelaxation produced by some fragments of endothelin-1, by two alanyl-substituted analogues, and by human and porcine proendothelins. The systemic cardiovascular effects of some of these peptides were also studied in anaesthetized rats. 2. Endothelin (16-21) was an endothelium-dependent vasodilator about 10 times less potent than acetylcholine and its amide was about 500 times less potent than the free acid. Human proendothelin-1 and porcine proendothelin-1 also caused endothelium-dependent relaxations but neither [Ala3,11]endothelin-1 nor [Ala1,3,11,15]endothelin-1 produced significant reductions in the methoxamine-induced tone. Sodium nitroprusside was approximately 200 times less potent than acetylcholine in the presence of the endothelium and was the only vasorelaxant to be active after destruction of the endothelium by perfusion with 0.3% CHAPS; in the absence of the endothelium it was 3.7 times more potent as a vasodilator than in its presence. 3. Porcine proendothelin-1 had weak contractile activity in the isolated mesenteric bed but porcine endothelin (22-39), endothelin (16-21) and endothelin (16-21) amide did not have vasoconstrictor actions. 4. Endothelin-3, [Ala3,11]endothelin-1, [Ala1,3,11,15]endothelin-1 and endothelin (16-21) all had systemic blood pressure effects in the anaesthetized rat. Although endothelin (16-21) did not cause vasoconstriction in vitro, it increased mean arterial pressure in vivo but was at least 100 times less potent than endothelin-3. Despite causing no vasorelaxation in vitro, [Ala1,3,11,15]endothelin-1 and [Ala3,11]endothelin-1 induced short-lived systemic depressor responses which were followed by pressor responses. Endothelin-3 was more potent as a systemic depressor agent than as a pressor agonist and, as a vasodepressor agent, it was 3-4 times more potent than either of the alanyl-substituted analogues. Endothelin-3, [Ala3,11]endothelin-1 and [Ala1,3,11,15]endothelin-1 were equipotent vasopressor agents. Porcine endothelin (22-39) had no systemic blood pressure effects. 5. This study shows directly that the presence of both disulphide bonds is required in the endothelins for them to be able to cause endothelium-dependent relaxation in the mesenteric vascular bed and that this response is mediated by different receptors from those causing contraction of the vascular smooth muscle in the mesentery. Vasorelaxation caused by endothelin (16-21) and its amide suggests that there is a receptor on the endothelium similar to one reported in the guinea-pig trachea. Finally, the ability of endothelin peptides to cause systemic vasodepressor responses is not related to their ability to cause endothelium-dependent relaxation, at least in the mesenteric circulation, and, for endothelin (16-21), its systemic pressor response is not likely to be the result of vasoconstriction in the mesenteric bed.

Endothelium-dependent vascular activities of endothelin-like peptides in the isolated superior mesenteric arterial bed of the rat.

1. The vasoconstrictor activities of endothelin-2, endothelin-3, sarafotoxin S6b, human proendothelin1-38 and mouse vasoactive intestinal contractor (VIC) were studied in the isolated Krebs-Henseleit perfused mesenteric arterial bed of the rat in the presence and absence of the endothelium. The vasoconstrictor properties of endothelin-1 were studied in control preparations and in preparations treated with methylene blue or N omega-nitro-L-arginine methyl ester (NAME). Finally, the direct vasodilator properties of endothelin-2, endothelin-3 and sarafotoxin S6b were studied in preparations preconstricted with methoxamine. 2. In the presence of an intact endothelium, all of the peptides caused dose-dependent increases in perfusion pressure and sarafotoxin S6b was a full agonist relative to the other peptides studied (maximum increase in perfusion pressure, Rmax = 106 +/- 11 mmHg). Endothelin-1, endothelin-2 and VIC were more potent vasoconstrictors (ED50 93.0 +/- 40.0, 90.8 +/- 20.5 and 106 +/- 63 pmol, respectively) than endothelin-3 and sarafotoxin S6b, which were found to be equipotent (ED50 values 411 +/- 195 and 345 +/- 86 pmol, respectively). A full dose-response relationship could not be constructed for proendothelin, but the highest dose used (4 nmol) increased the perfusion pressure by 15.4 +/- 1.6 mmHg. 3. Destruction of the endothelium with the zwitterionic detergent 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulphonate (CHAPS) significantly enhanced the pressor activity of all 5 peptides. The Rmax for sarafotoxin S6b was not significantly altered by removal of the endothelium but its potency was significantly increased (ED50 = 115 +/- 15 pmol). Although their R,,, values were significantly increased, endothelin-2 and VIC were still partial agonists relative to sarafotoxin S6b in CHAPSpretreated preparations; their potencies were unchanged (ED5o values 118 + 53 and 416 + 196pmol, respectively). Removal of the endothelium significantly reduced the potency of endothelin-3 (ED5o, 6.3 + 2.2 nmol) but this peptide then exhibited full agonist activity (R..x = 106 + 14 mmHg). After endothelial cell destruction, the pressor responses to proendothelin were increased; 4 nmol gave a response of 38.8 + 5.5 mmHg. 4. Exposure of preparations to either 100 microM NAME (R,,,X = 42.6 + 2.4mmHg and EDSo = 57.5 + 13.7 pmol) or 10 microM methylene blue (R,,,. = 36.0 + 5.1 mmHg and ED50 = 81.5 + 26.1 pmol) significantly enhanced the maximum pressor responses to endothelin-l (control: R.,=X = 22.5 + 2.6 mmHg; ED5o = 93.0 + 40.Opmol). The values in the presence of NAME or methylene blue were not significantly different from those found previously for endothelin-1 after removal of the endothelium with CHAPS. 5. Endothelin-2, endothelin-3 and sarafotoxin S6b all caused vasorelaxation in preparations which had been precontracted with 100 microM methoxamine. This action was endothelium-dependent as it was abolished by perfusing the mesentery with CHAPS. Endothelin-3 and sarafotoxin S6b caused relaxation at much lower doses than were needed with endothelin-1 and endothelin-2. 6. The endothelium significantly modulates the vasoconstrictor activity of all the endothelin-like peptides studied, including the precursor peptide proendothelin (which was the least potent of the peptides). This modulation is likely to be due to the release of endothelium-derived relaxing factor, since similar results to destruction of the endothelium were obtained when endothelin-l was investigated in the presence of either methylene blue or NAME (an inhibitor of nitric oxide formation) in the perfusion fluid. The vasodilator effects of the peptides were also endothelium-dependent. There was a different order of potency for vasoconstriction and vasodilatation supporting the suggestion that there are sub-types of receptor for the endothelin-like peptides in the vasculature; one type on the vascular smooth muscle and a second type on the endothelium.

Endothelial modulation and changes in endothelin pressor activity during hypoxia in the rat isolated perfused superior mesenteric arterial bed.

1. The isolated superior mesenteric arterial bed of the rat, perfused with Krebs-Henseleit solution containing 10 microM indomethacin, was used to study the effects of reducing dissolved O2 tension on the pressor responses to endothelin-1, endothelin-3 and sarafotoxin S6b. The modulation of these responses by the endothelium was investigated by removing the intima with the detergent CHAPS and, for endothelin-1, by inhibiting nitric oxide production with N omega-nitro-L-arginine methyl ester (L-NAME). Comparison was made with the effects of lowering O2 tension on the pressor responses to noradrenaline and 5-hydroxytryptamine. 2. Lowering the perfusate O2 tension from 551 +/- 2 mmHg to 14.0 +/- 0.5 mmHg did not change the ED50 for endothelin-1 but its maximal responses (Rmax) were increased by 2.1 and 2.7 fold, respectively, in the presence and absence of endothelium. The Rmax values for endothelin-3 were also greater in hypoxia either in the presence (by 2.3 fold) or absence of the endothelium (by 1.6 times) but those for sarafotoxin S6b were only enhanced significantly by hypoxia in the absence of the intima. hypoxia reduced the potencies of endothelin-3 and sarafotoxin S6b whether or not endothelium was present. 3. Endothelial destruction, whether in hypoxic or oxygenated conditions, increased the Rmax values for endothelin-1 and endothelin-3; at both O2 tensions those for endothelin-3 increased more than those for endothelin-1. The ED50 for endothelin-1 was unchanged by destroying the endothelium but endothelin-3 was less potent in the absence of an endothelium than in its presence. Removal of the endothelium did not change the R.ax for sarafotoxin S6b but increased its potency in both hypoxic and oxygenated tissues. 4. In hypoxia, and in the presence of both the endothelium and 100 microM L-NAME, the Rmax for endothelin-1 was 1.6 times greater than that in hypoxia in the absence of L-NAME. Co-infusion of 100 microM L-arginine, but not of 100 mircoM D-arginine, with 100 microM L-NAME reversed this effect. The presence of L-NAME decreased the potency of endothelin-1. 5. Destroying the endothelium did not affect the Rmax for noradrenaline in either oxygenated conditions or hypoxia. Changing 02 tension when the endothelium was intact had no effect on the Rmax but it was 11% greater in oxygenated, than in hypoxic, endothelium denuded preparations. Endothelial destruction decreased the potency of noradrenaline in hypoxia but increased it in oxygenated tissues. In hypoxia, L-NAME had no effect on the ED50 relative to control preparations with endothelium but the Rmax was 30% greater. 6. 5-Hydroxytryptamine gave very small pressor responses in the presence of endothelium in both oxygenated and hypoxic tissues but the Rmax was 1.7 times greater in hypoxia. L-NAME increased the R,,x by 9.8 times in oxygenated preparations and 6.3 fold in hypoxia. The ED5o values were the same in all conditions. 7. It is concluded that, although hypoxia generally increased the R.. for the endothelin/sarafotoxin peptides, the changes could not be explained by a simple increase in receptor number since hypoxia decreased the potency of endothelin-3 and sarafotoxin S6b. Thus alterations in receptor binding or activation properties, or both, also occurred. The changes associated with hypoxia were not common to all vasoconstrictor agonists since, in the absence of endothelial function, hypoxia did not affect the Rmax values for either noradrenaline or 5-hydroxytryptamine. Also, the pressor responses to the peptides and both the amines can be modulated by the endothelium in hypoxia as well as in oxygenated conditions.

Endothelin-1 does not mediate hypoxic vasoconstriction in canine isolated blood vessels: effect of BQ-123.

1. The role of endothelin-1 in mediating the phenomenon of hypoxic vasoconstriction was examined in canine, isolated pulmonary, circumflex coronary and femoral arterial rings. 2. In tissues with an intact endothelium, the exogenous application of endothelin-1 (0.1-300 nM) caused concentration-dependent increases in canine, isolated pulmonary artery tone. Endothelin-3 (1-300 nM) was approximately 30 fold less potent than endothelin-1 as a vasoconstrictor in this tissue. In contrast, the selective ETB-receptor agonist, sarafotoxin S6c (0.01-1 microM), failed to elicit vasoconstriction in this tissue. Thus, endothelin isopeptide-induced vasoconstriction of the canine isolated pulmonary artery is mediated exclusively by the ETA-receptor subtype. 3. The concentration-dependent increases in isometric tension induced by endothelin-1 (0.1-300 nM) were antagonized by the ETA-selective antagonist, BQ-123 (10 microM); this concentration of antagonist caused a shift to the right in the concentration-response curve for endothelin-1 of approximately two orders of magnitude. This concentration of BQ-123 did not unmask any ETB-receptor-mediated vasoconstriction since sarafotoxin S6c (0.01-1 microM) still failed to elicit contraction in the presence of this concentration of BQ-123. 4. The hypoxia-induced vasoconstriction of canine, isolated pulmonary, circumflex coronary and femoral arterial rings was unaffected by pretreatment with the endothelin receptor antagonist, BQ-123 (10 microM), a concentration shown previously to antagonize the contractile actions of exogenously applied endothelin-1 in the isolated pulmonary artery. 5. These results are the first to provide direct evidence showing that the endothelium-dependent vasoconstriction observed during acute periods of hypoxia in vitro is not mediated by an endothelin-related isopeptide.

Vascular activities of endothelin-1 and some alanyl substituted analogues in resistance beds of the rat.

1. The effects of endothelin-1 and of three analogues in which alanyl residues had been substituted in place of cysteinyl residues were studied in the rat, isolated, Krebs-Henseleit-perfused mesenteric bed and the in situ, blood-perfused, mesenteric and hindquarters circulations of the rat. The effects on the vascular responses to these peptides of removing the endothelium by detergent perfusion or of cyclo-oxygenase inhibition by indomethacin were also determined. 2. In all three preparations, endothelin-1 was a potent vasoconstrictor (ED50 values ranged from 40 to 400 pmol) although it was rather less potent in the hindquarters than in the mesentery. Also, the maximum response was very much smaller in the isolated mesentery (24.7 +/- 2.1 mmHg) than in the in situ mesentery (81.8 +/- 2.6 mmHg) or hindquarters (107 +/- 10 mmHg). 3. Removal of the endothelium by perfusion with detergent significantly enhanced the potency of endothelin as a vasoconstrictor in the in situ messentery, but reduced the maximum response obtained, whereas removal of the endothelium in vitro significantly increased the maximum response without changing the ED50. The presence or absence of indomethacin had no significant effects in the blood-perfused hindquarters preparation or the isolated mesentery but, after administration of 5 mg kg-1 indomethacin to the in situ mesenteric preparation, the maximal response to endothelin-1 was enhanced. 4. When the preparations were preconstricted with alpha 1-adrenoceptor agonists, endothelin-1 had modest vasodilator effects. These vasodilator effects were abolished when the endothelium was destroyed by detergent perfusion. 5. Both [Ala3,11]endothelin-1 and [Ala1,15]endothelin-1 were also vasoconstrictor agents in the mesenteric preparations but they were less potent than endothelin-1 itself; [Ala3,11]endothelin-1 was intermediate in potency between endothelin-1 and [Ala1,15]endothelin-1. In the in situ preparation these analogues gave similar maximal responses to the parent peptide but, in vitro, they gave maximal responses that were much greater than that of endothelin-1 and which were similar to those found with all 3 peptides in the in situ mesentery. Destruction of the endothelium in vitro had no effect on the responses to these 2 analogues and the log dose-response curve for [Ala1,15]endothelin-1 in the isolated mesentery was biphasic. 6. A third analogue possessing no disulphide bridges [( Ala1,3,11,15]endothelin-1) was a partial agonist in the in situ preparations but had no vasoconstrictor effect in the in vitro mesentery. It had no vasorelaxant effect in the hindquarters preparation but it enhanced the responses to endothelin-l when the 2 peptides were administered together in all 3 preparations. 7. It is concluded that it is not essential for the endothelin family of peptides to possess 2 disulphide bridges for them to be vasoconstrictor agents. However, only endothelin-1, of the 4. peptides studied, showed either endothelium-dependent vasorelaxant activity or modulation by the endothelium of its pressor effects. This, together with some differences in the vasoconstrictor log dose-response curves to the peptides and the results of co-administration of [Ala' 3'11"15]endothelin-1 and endothelin-1, suggests that there may be more than one receptor type mediating vascular responses to the peptides studied.

Human urotensin-II is a potent spasmogen of primate airway smooth muscle.

The contractile profile of human urotensin-II (hU-II) was examined in primate airway and pulmonary vascular tissues. hU-II contracted tissues from different airway regions with similar potencies (pD(2)s from 8.6 to 9.2). However, there were regional differences in the efficacy of hU-II, with a progressive increase in the maximum contraction from trachea to smaller airway regions (from 9 to 41% of the contraction to 10 microM carbachol). hU-II potently contracted pulmonary artery tissues from different regions with similar potencies and efficacies: pD(2)s=8.7 to 9.3 and maximal contractions=79 to 86% of 60 mM KCl. hU-II potently contracted pulmonary vein preparations taken proximal to the atria, but had no effect in tissues from distal to the atria. This is the first report describing the contractile activity of hU-II in airways and suggests that the potential pathophysiological role of this peptide in lung diseases warrants investigation.

Human urotensin-II is an endothelium-dependent vasodilator in rat small arteries.

The possible role of the endothelium in modulating responses to human urotensin-II (U-II) was investigated using isolated segments of rat thoracic aorta, small mesenteric artery, left anterior descending coronary artery and basilar artery. Human U-II was a potent vasoconstrictor of endothelium-intact isolated rat thoracic aorta (EC(50)=3.5+/-1.1 nM, R(max)=103+/-10% of control contraction induced by 60 mM KCl and 1 microM noradrenaline). However the contractile response was not significantly altered by removal of the endothelium or inhibition of nitric oxide synthesis with L-NAME (100 microM). Human U-II did not cause relaxation of noradrenaline-precontracted, endothelium-intact rat aortae. Human U-II contracted endothelium-intact rat isolated left anterior descending coronary arteries (EC(50)=1.3+/-0.8 nM, R(max)=20.1+/-4.9% of control contraction induced by 10 microM 5-HT). The contractile response was significantly enhanced by removal of the endothelium (R(max)=55.4+/-16.1%). Moreover, human U-II caused concentration-dependent relaxation of 5-HT-precontracted arteries, which was abolished by L-NAME or removal of the endothelium. No contractile effects of human U-II were found in rat small mesenteric arteries. However the peptide caused potent, concentration- and endothelium-dependent relaxations of methoxamine-precontracted vessels. The relaxant responses were potentiated by L-NAME (300 microM) but abolished in the additional presence of 25 mM KCl (which inhibits the actions of endothelium-derived hyperpolarizing factor). The present study is the first to show that human U-II is a potent endothelium-dependent vasodilator in some rat resistance vessels, and acts through release of EDHF as well as nitric oxide. Our findings have also highlighted clear anatomical differences in the responses of different vascular beds to human U-II which are likely to be important in determining the overall cardiovascular activity of this peptide.

Pharmacological evidence for the presence of three distinct functional endothelin receptor subtypes in the rabbit lateral saphenous vein.

1. Contraction of the rabbit isolated saphenous vein is mediated by a heterogeneous endothelin (ET) receptor population. This study has characterized these receptor subtypes by use of several pharmacologically distinct ET receptor agonists and antagonists. 2. ET-1, ET-3, sarafotoxin S6c (STXc) and [Ala3,11]ET-1 produced biphasic, concentration-dependent contractions of the saphenous vein, responses which were best fitted by a two-site model comprised of a high (pM) and a low (nM) affinity site. In contrast, IRL 1620 only recognized one of these sites. ET(16-21) was devoid of contractile activity. ET-1, ET-3 and STXc were equipotent at the high affinity site (pD2s of 12.0 +/- 0.2, 12.2 +/- 0.2 and 12.3 +/- 0.3) indicating that this site had the characteristics of an ETB receptor. In contrast, the low affinity site had the functional characteristics of an ETc receptor since the pD2s for ET-3 (10.2 +/- 0.3) and STXc (10.6 +/- 0.3) were significantly greater than that for ET-1 (9.1 +/- 0.1). These contractile responses were insensitive to BQ-123, confirming that ETA receptors were not involved in mediating this effect. 3. SB 209670 differentially antagonized the high affinity phases of the isopeptide concentration-response curves in a fashion dependent on the competing agonist: relative to the KB obtained against STXc (0.15 nM). SB 209670 was 10 fold less potent when ET-1 was used as the competing agonist. This differential effect was not evident at the low affinity site (KB = 38 nM). SB 209670 produced parallel, concentration-dependent rightward shifts in the concentration-response curve to STXc Ro 47-0203 was approximately 1 to 2 orders of magnitude less potent than SB 209670 at inhibiting the high affinity component of the concentration-response curve to STXc, whereas BQ-788 and Ro 46-2005 were approximately 3 orders of magnitude less potent than SB 209670. In addition to RES-701 and BQ-123, the high affinity site was insensitive to PD 142893 suggesting that it may represent an ETB2 receptor. Ro 47-0203 and SB 209670 were equipotent at inhibiting the low affinity component of the STXc concentration-response curve. Although Ro 46-2005, BQ-788, PD 142893 and RES-701 produced significant antagonism at the low affinity site, they were at least ten fold less potent than SB 209670. 4. ET-1, ET-3 and STXc produced endothelium-dependent vasorelaxation in the precontracted saphenous vein. Antagonist IC50s were approximated as being: SB 209670, 3 nM; BQ-788 and RES 701,300 nM; Ro 46-2005 and PD 142893, 3 microM; BQ-123, > 10 .M, consistent with vasorelaxation being mediated by an ETB1 receptor.5. In summary, three pharmacologically distinct ET receptor subtypes have been identified in the rabbits aphenous vein. Two contractile receptors are present on the vascular smooth muscle, a high affinity site with the characteristics of an ETB2 receptor and a distinct lower affinity site with the characteristics of an ETc receptor. In addition, an ETBI receptor is present on the endothelium which mediates the vasodilator actions of this peptide family.

In vivo pharmacological characterization of the non-peptide endothelin receptor antagonist SB 209670.

1. The aim of the present study was to assess the ability of SB 209670, a high affinity non-peptide endothelin receptor antagonist (0.4 and 18 nM Kis at human cloned ETA and ETB receptors, respectively), to inhibit the haemodynamic actions of endothelin-1 in vivo. 2. Systemic administration of (+/-)-SB 209670, given either as a bolus i.v. injection or as a continuous i.v. infusion, did not alter basal haemodynamic parameters in the anaesthetized rat. 3. Infusion of (+/-)-SB 209670 (10 micrograms kg-1 min-1) selectively inhibited the depressor and carotid vasodilator response to exogenous endothelin-1: 100 micrograms kg-1 min-1 was required to inhibit significantly the biphasic haemodynamic actions of endothelin-1. The haemodynamic actions of angiotensin II and calcitonin gene-related peptide were unaltered by 100 micrograms kg-1 min-1 (+/-)-SB 209670. 4. Bolus i.v. administration of (+/-)-SB 209670 (1 mg kg-1) selectively inhibited the depressor and carotid vasodilator actions of endothelin-1: 10 mg kg-1 (+/-)-SB 209670 was required to inhibit the secondary vasoconstrictor actions of endothelin-1. 5. (+/-)-SB 209670 (10 mg kg-1) was also effective at antagonizing the pressor actions of endothelin-1 in the conscious rat for up to 3 h after intraduodenal administration thereby demonstrating that the antagonist was bioavailable upon enteric administration. This dose of (+/-)-SB 209670 did not alter basal haemodynamic parameters in the conscious rat. 6. Thus, ( +/- )-SB 209670 is an effective endothelin receptor antagonist in vivo. Using the doses defined in this study, SB 209670 may, therefore, serve as a useful tool for understanding the role of endogenous endothelin-I in the control of cardiovascular function under both physiological and pathophysiological conditions.