Reappraisal of the mechanism of cardiovascular responses to sympathomimetic amines in anaesthetised rats: dual α1-adrenoceptor and trace amine receptor mechanisms

Established dogma is that sympathomimetic amines, including β-phenylethylamine (PEA), increase blood pressure by releasing noradrenaline from sympathetic neurons. Recent evidence allowing longer contact with isolated immersed tissues indicates other mechanisms. The present study re-evaluates the mechanism of pressor responses to PEA in anaesthetised rats with longer exposure to infusions. Blood pressure and heart rate were monitored by cannulating a common carotid artery of anaesthetised male Sprague–Dawley rats. Drugs were administered by bolus doses or by 20-min infusions via a cannulated jugular vein. Increases in blood pressure by bolus doses of the α-adrenoceptor agonist, phenylephrine, were converted to depressor responses by prazosin and therefore α-adrenoceptor-mediated. Pressor responses to bolus doses of PEA were reduced. PEA infusions yielded four-phase responses: An initial increase in pressure (phase 1) blocked by prazosin was due to α-adrenoceptor vasoconstriction and a secondary fall in pressure (phase 2) due to vasodilatation by nitric oxide release. A later pressure increase (phase 3), further elevated after infusion stopped (phase 4), was not attenuated by prazosin and therefore non-adrenergic. This study showed for the first time that the sympathomimetic amine, β-phenylethylamine, increases blood pressure by two mechanisms. The established indirect sympathomimetic mechanism applies to bolus dose administration. However, with prolonged exposure to infusions, an additional slow-onset sustained non-adrenergic blood pressure increase occurs, most likely mediated via trace amine-associated receptors (TAAR-1). This response will dominate with prolonged exposures in clinical practice. These results prompt a re-evaluation of established dogma on the indirect sympathomimetic mechanisms of these amines

Trace amine-induced vasoconstriction of human mammary artery and saphenous vein

Sympathomimetic amines, including β-phenylethylamine (PEA), constrict animal blood vessels but their mechanism of action is not now thought to be through α-adrenoceptors and release of noradrenaline but via trace amine-associated receptors (TAARs). This information is not available for human blood vessels. Functional studies were therefore performed on human arteries and veins to establish whether they constrict to PEA and whether any constrictions are adrenoceptor-mediated. Isolated internal mammary artery or saphenous vein rings were set up in Kreb's-bicarbonate solution at 37 ± 0.5 °C gassed with O2:CO2 (95:5) under class 2 containment. Isometric contractions were measured and cumulative concentration-response curves for PEA or the α-adrenoceptor agonist, phenylephrine were established. PEA showed concentration-related contractions. The maximum was significantly greater in arteries (1.53 ± 0.31 g, n = 9) than veins (0.55 ± 0.18 g, n = 10), but not when plotted as % of KCl contractions. PEA showed slowly developing contractions plateauing at 17,3 ± 3.7 min in mammary artery. The reference α-adrenoceptor agonist, phenylephrine, exhibited more rapid onset (peak 5.0 ± 1.2 min) but non-sustained contractions. In saphenous veins, PEA (62.8 ± 10.7%) and phenylephrine (61.4 ± 9.7%, n = 4) displayed the same maximum, but phenylephrine was more potent. The α1-adrenoceptor antagonist, prazosin (1 μM), blocked phenylephrine contractions of mammary arteries but not PEA contractions in either vessel. PEA causes substantial vasoconstriction of human saphenous vein and mammary artery, which explains its vasopressor actions. This response, however, was not mediated via α1-adrenoceptors, but likely due to TAARs. The classification of PEA as a sympathomimetic amine on human blood vessels is therefore no longer valid and requires revision

Effects of nebulised magnesium sulphate on inflammation and function of the guinea-pig airway

Magnesium sulphate is a potential treatment for acute severe asthma. However, the mechanisms and dose-response relationships are poorly understood. The first objective of this study was to examine whether inhaled magnesium sulphate exerts bronchodilator activity measured as bronchoprotection against histamine-induced bronchoconstriction in conscious guinea-pigs alone and combined with salbutamol. Secondly, we examined whether inhaled magnesium sulphate inhibits airways inflammation and function in models of neutrophilic and eosinophilic lung inflammation induced, respectively, by inhaled lipopolysaccharide or the inhaled antigen, ovalbumin (OVA). Airway function was measured in conscious guinea-pigs as specific airway conductance (sGaw) by whole-body plethysmography. Anti-inflammatory activity was measured against lung inflammatory cell influx induced by OVA inhalation in OVA-sensitised animals or by lipopolysaccharide (LPS) exposure of non-sensitised animals. Airway function (sGaw) was measured over 24 h after OVA exposure. Airway hyperresponsiveness to inhaled histamine and inflammatory cells in bronchoalveolar lavage fluid were recorded 24 h after OVA or LPS challenge. Histamine-induced bronchoconstriction was inhibited by inhaled magnesium sulphate or salbutamol alone and in combination, they produced synergistic bronchoprotection. LPS-induced neutrophil influx was inhibited by 6 days pretreatment with magnesium sulphate. Early and late asthmatic responses in OVA sensitised and challenged animals were attenuated by magnesium sulphate. Lung inflammatory cells were increased by OVA, macrophages being significantly reduced by magnesium sulphate. Nebulised magnesium sulphate protects against histamine-induced bronchoconstriction in conscious guinea-pigs and exerts anti-inflammatory activity against pulmonary inflammation induced by allergen (OVA) or LPS. These properties of magnesium sulphate explain its beneficial actions in acute asthma

Is there a role for biogenic amine receptors in mediating β-phenylethylamine and RO5256390-induced vascular contraction?

Background: Substantial evidence indicates trace amines can induce vasoconstriction independently of noradrenaline release. However, the mechanism underlying noradrenaline-independent vasoconstrictor responses to trace amines has not yet been established. This study evaluates the role of trace amine-associated receptor 1 (TAAR1) and other biogenic amine receptors in mediating β-phenylethylamine and the TAAR-1 selective agonist RO5256390-induced vasoconstriction. Methods: Vasoconstrictor responses to β-PEA and the TAAR1-selective agonist, RO5256390 were assessed in vitro in endothelium-denuded aortic rings and third-order mesenteric arteries of male Sprague Dawley rats. Results: β-PEA and RO5256390 induced concentration-dependent vasoconstriction of aortic rings but not third-order mesenteric arteries. Vasoconstrictor responses in aortic rings were insensitive to antagonists of 5-HT and dopamine. The murine-selective TAAR1 antagonist, EPPTB, had no effect on either β-PEA or RO5256390-induced vasoconstriction. The α1-adrenoceptor antagonist, prazosin, and the α2-adrenoceptor antagonist, yohimbine, induced a shift of the β-PEA concentration response curve too small to be ascribed to antagonism of α1- or α2-adrenoceptors, respectively. The α2-adrenoceptor antagonist atipamezole had no effect on β-PEA or RO5256390-induced vasoconstriction. Conclusion: Vasoconstrictor responses to trace amines are not mediated by classical biogenic amine neurotransmitter receptors. Insensitivity of β-PEA vasoconstrictor responses to EPPTB, may be explained by its low affinity for rat rather than murine TAAR1. Therefore, TAAR1 remains the most likely candidate receptor mediating vasoconstrictor responses to trace amines and that prazosin and yohimbine have low affinity for TAAR1

A stereoselective synthesis of a 3,4,5-substituted piperidine of interest as a selective muscarinic (M1) receptor agonist

Syntheses of (1RS,2SR,6SR)-2-alkoxymethyl-, 2-hetaryl-, and 2-(hetarylmethyl)-7-arylmethyl-4,7-diaza-9-oxabicyclo[4.3.0]nonan-8-ones, of interest as potential muscarinic M1 receptor agonists, are described. A key step in the synthesis of (1RS,2SR,6SR)-7-benzyl-6-cyclobutyl-2-methoxymethyl-4,7-diaza-9-oxabicyclo[4.3.0]nonan-8-one, was the addition of isopropenylmagnesium bromide to 2-benzyloxycarbonylamino-3-tert-butyldimethylsilyloxy-2-cyclobutylpropanal. This gave the 4-tert-butyldimethylsilyloxymethyl-4-cyclobutyl-5-isopropenyloxazolidinone with the 5-isopropenyl and 4-tert-butyldimethylsilyloxymethyl groups cis-disposed about the five-membered ring by chelation controlled addition and in situ cyclisation. This reaction was useful for a range of organometallic reagents. The hydroboration–oxidation of (4SR,5RS)-3-benzyl-4-(tert-butyldimethylsilyloxymethyl)-4-cyclobutyl-5-(1-methoxyprop-2-en-2-yl)-1,3-oxazolidin-2-one gave (4SR,5RS)-3-benzyl-4-(tert-butyldimethylsilyloxymethyl)-4-cyclobutyl-5-[(SR)-1-hydroxy-3-methoxyprop-2-yl]-1,3-oxazolidin-2-one stereoselectively. 4,7-Diaza-9-oxabicyclo[4.3.0]nonan-8-ones with substituents at C2 that could facilitate C2 deprotonation were unstable with respect to oxazolidinone ring-opening and this restricted both the synthetic approach and choice of 2-heteroaryl substituent. The bicyclic system with a 2-furyl substituent at C2 was therefore identified as an important target. The addition of 1-lithio-1-(2-furyl)ethene to 2-benzyloxycarbonylamino-3-tert-butyldimethylsilyloxy-2-cyclobutylpropanal gave (4SR,5RS)-4-tert-butyldimethylsilyloxymethyl-4-cyclobutyl-5-[1-(2-furyl)ethenyl]-1,3-oxazolidinone after chelation controlled addition and in situ cyclisation. Following oxazolidinone N-benzylation, hydroboration at 35 °C, since hydroboration at 0 °C was unexpectedly selective for the undesired isomer, followed by oxidation gave a mixture of side-chain epimeric alcohols that were separated after SEM-protection and selective desilylation. Conversion of the neopentylic alcohols into the corresponding primary amines by reductive amination, was followed by N-nosylation, removal of the SEM-groups and cyclisation using a Mitsunobu reaction. Denosylation then gave the 2-furyloxazolidinonyl-fused piperidines, the (1RS,2SR,6SR)-epimer showing an allosteric agonistic effect on M1 receptors. Further studies resulted in the synthesis of other 2-substituted 4,7-diaza-9-oxabicyclo[4.3.0]nonan-8-ones and an analogous tetrahydropyra

Route of administration affects corticosteroid sensitivity of a combined ovalbumin and lipopolysaccharide model of asthma exacerbation in guinea-pigs

Lipopolysaccharide (LPS) contributes to asthma exacerbations and development of inhaled corticosteroid insensitivity. Complete resistance to systemic corticosteroids is rare and most patients lie on a continuum of steroid responsiveness. The objective of this study was to examine the sensitivity of combined ovalbumin- (Ova) and LPS-induced functional and inflammatory responses to inhaled and systemic corticosteroid in conscious guinea-pigs, to test the hypothesis that the route of administration affects its sensitivity. Guinea-pigs were sensitised to Ova and challenged with inhaled Ova alone or combined with LPS. Airways function was determined by measuring specific airways conductance via whole-body plethysmography. Airways hyperresponsiveness to histamine was determined pre- and 24h post-Ova challenge. Airways inflammation and underlying mechanisms were determined from bronchoalveolar lavage cell counts and lung tissue cytokines. Vehicle or dexamethasone was administered by once-daily intraperitoneal injection (5, 10 or 20 mg/kg) or twice-daily inhalation (4 or 20 mg/ml) for 6 days before Ova challenge or Ova with LPS. LPS exacerbated Ova-induced responses, elongating early asthmatic responses (EAR), prolonging bronchoconstriction by histamine and further elevating airways inflammation. Intraperitoneal dexamethasone (20 mg/kg) significantly reduced the elongated EAR and airways inflammation but not the increased bronchoconstriction to histamine. In contrast, inhaled dexamethasone (20 mg/ml), which inhibited responses to Ova alone, did not significantly reduce functional and inflammatory responses to combined Ova and LPS. Combined Ova and LPS-induced functional and inflammatory responses are insensitive to inhaled but only partially sensitive to systemic dexamethasone. These results suggest that the route of corticosteroid administration may be important in determining the sensitivity of asthmatic responses to these agents

The synthesis of a series of adenosine A3 receptor agonists

A series of 1′-(6-aminopurin-9-yl)-1′-deoxy-N-methyl-β-D-ribofuranuronamides that were characterised by 2-dialkylamino-7-methyloxazolo[4,5-b]pyridin-5-ylmethyl substituents on N6 of interest for screening as selective adenosine A3 receptor agonists, have been synthesised. This work involved the synthesis of 2-dialkylamino-5-aminomethyl-7-methyloxazolo[4,5-b]pyridines and analogues that were coupled with the known 1′-(6-chloropurin-9-yl)-1′-deoxy-N-methyl-β-D-ribofuranuronamide. The oxazolo[4,5-b]pyridines were synthesized by regioselective functionalisation of 2,4-dimethylpyridine N-oxides. The regioselectivities of these reactions were found to depend upon the nature of the heterocycle with 2-dimethylamino-5,7-dimethyloxazolo[4,5-b]pyridine-N-oxide undergoing regioselective functionalisation at the 7-methyl group on reaction with trifluoroacetic anhydride in contrast to the reaction of 4,6-dimethyl-3-hydroxypyridine-N-oxide with acetic anhydride that resulted in functionalisation of the 6-methyl group. To optimise selectivity for the A3 receptor, 5-aminomethyl-7-bromo-2-dimethylamino-4-[(3-methylisoxazol-5-yl)methoxy]benzo[d]oxazole was synthesised and coupled with the 1′-(6-chloropurin-9-yl)-1′-deoxy-N-methyl-β-D-ribofuranuronamide. The products were active as selective adenosine A3 agonists

Paracetamol reduces influenza-induced immunopathology in a mouse model of infection without compromising virus clearance or the generation of protective immunity

Background: Seasonal influenza A infection affects a significant cohort of the global population annually, resulting in considerable morbidity and mortality. Therapeutic strategies are of limited efficacy, and during a pandemic outbreak would only be available to a minority of the global population. Over-the-counter medicines are routinely taken by individuals suffering from influenza, but few studies have been conducted to determine their effectiveness in reducing pulmonary immunopathology or the influence they exert upon the generation of protective immunity. Methods: A mouse model of influenza infection was utilised to assess the efficacy of paracetamol (acetaminophen) in reducing influenza-induced pathology and to examine whether paracetamol affects generation of protective immunity. Results: Administration (intraperitoneal) of paracetamol significantly decreased the infiltration of inflammatory cells into the airway spaces, reduced pulmonary immunopathology associated with acute infection and improved the overall lung function of mice, without adversely affecting the induction of virus-specific adaptive responses. Mice treated with paracetamol exhibited an ability to resist a second infection with heterologous virus comparable with that of untreated mice. Conclusions: Our results demonstrate that paracetamol dramatically reduces the morbidity associated with influenza but does not compromise the development of adaptive immune responses. Overall, these data support the utility of paracetamol for reducing the clinical symptoms associated with influenza virus infection

Pulmonary oedema measured by MRI correlates with late-phase response to allergen challenge

Purpose: Asthma is associated with reversible airway obstruction, leucocyte infiltration, airways hyperresponsiveness (AHR) and airways remodelling. Fluid accumulation causes pulmonary oedema contributing to airways obstruction. We examined the temporal relationship between the late asthmatic response (LAR) following allergen challenge of sensitised guinea-pigs and pulmonary oedema measured by magnetic resonance imaging (MRI). Materials and Methods: Ovalbumin (OVA) sensitised guinea-pigs received either a single OVA inhalation (acute) or nine OVA inhalations at 48 h intervals (chronic). Airways obstruction was measured as specific airways conductance (sGaw) by whole body plethysmography. AHR to inhaled histamine and bronchoalveolar lavage for leucocyte counts were measured 24 h after a single or the final chronic ovalbumin challenges. MRI was performed at intervals after OVA challenge and high intensity oedemic signals quantified. Results: Ovalbumin caused early bronchoconstriction, followed at 7 h by a LAR and at 24 h AHR and leucocyte influx. The bright intensity MRI oedema signal, peaking at 7 h, was significantly (P<0.05) greater after chronic (9.0±0.7x103 mm3) than acute OVA (7.6±0.2x103 mm3). Dexamethasone treatment before acute OVA abolished the AHR and LAR and significantly reduced eosinophils and the bright intensity MRI oedema from 9.1±1.0 to 6.4±0.3x103 mm3. Conclusion: We show a temporal relationship between oedema and the LAR and their parallel reduction, along with eosinophils and AHR, by dexamethasone. This suggests a close causative association between pulmonary oedema and impaired airways function