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BACKGROUND AND PURPOSE Inhaled corticosteroids, anticholinergics and β2-adrenoceptor agonists are frequently combined for treating chronic respiratory diseases. We examine the corticosteroid, budesonide, and novel NO-donating derivative, TPI 1020, against histamine- and methacholine-induced bronchoconstriction and whether they enhance the β2-adrenoceptor agonist formoterol or muscarinic antagonist tiotropium in conscious guinea pigs. EXPERIMENTAL APPROACH Dunkin-Hartley guinea pigs received inhaled histamine (3 mM) or methacholine (1.5 mM) and specific airway conductance (sGaw) was measured before and 15 or 75 min after treatment with budesonide, TPI 1020, tiotropium or formoterol alone or in combinations. KEY RESULTS Formoterol (0.7-10 µM) and budesonide (0.11-0.7 mM) inhibited histamine-induced bronchoconstriction and tiotropium (2-20 µM) inhibited methacholine-induced bronchoconstriction by up to 70.8 ± 16.6%, 34.9 ± 4.4% and 85.1 ± 14.3%, respectively. Formoterol (2.5 µM) or tiotropium (2 µM) alone exerted small non-significant bronchoprotection. However, when co-administered with TPI 1020 0.11 mM, which alone had no significant effect, there was significant inhibition of the bronchoconstriction (45.7 ± 12.2% and 79.7 ± 21.4%, respectively). Co-administering budesonide (0.11 mM) with tiotropium (2 µM), which alone had no effect, also significantly inhibited the methacholine bronchoconstriction (36.5 ± 13.0%), but there was no potentiation of formoterol against histamine. The NO scavenger, CPTIO, prevented the bronchoprotection by SNAPand TPI 1020. CONCLUSIONS AND IMPLICATIONS TPI 1020 potentiated the bronchoprotection by formoterol and tiotropium. Budesonide also enhanced the effects of tiotropium but not formoterol. Combination of TPI 1020 with a long-acting β2-adrenoceptor agonist or muscarinic receptor antagonist may therefore be a more potent therapeutic approach for treatment of chronic respiratory diseases. [ABSTRACT FROM AUTHOR]

Breast cancer is a global concern as a leading cause of death for women. Early and precise diagnosis can be vital in handling the disease efficiently. Breast cancer subtyping based on estrogen receptor (ER) status is crucial for determining prognosis and treatment. This study uses metabolomics data from plasma samples to detect metabolite biomarkers that could distinguish ER-positive from ER-negative breast cancers in a non-invasive manner. The dataset includes demographic information, ER status, and metabolite levels from 188 breast cancer patients and 73 healthy controls. Recursive Feature Elimination (RFE) with a Random Forest (RF) classifier identified an optimal subset of 30 features-29 biomarkers and age-that achieved the highest area under the curve (AUC). To address the class imbalance, Gaussian noise-based augmentation and Adaptive Synthetic Oversampling (ADASYN) were applied, ensuring balanced representation during training. Four machine learning (ML) algorithms-Random Forest, Support Vector Classifier (SVC), XGBoost, and Logistic Regression (LR)-were evaluated using grid search. The Random Forest classifier emerged as the top performer, achieving an AUC of 0.95 and an accuracy of 93%. These results suggest that ML has great promise for identifying specific metabolites linked to ER expression, paving the development of a novel analytical tool that can minimize current challenges in identifying ER status, and improve the precision of breast cancer subtyping.

Breast cancer remains a major public health concern, and early detection is crucial for improving survival rates. Metabolomics offers the potential to develop non-invasive screening and diagnostic tools based on metabolic biomarkers. However, the inherent complexity of metabolomic datasets and the high dimensionality of biomarkers complicates the identification of diagnostically relevant features, with multiple studies demonstrating limited consensus on the specific metabolites involved. Unlike previous studies that rely on singular feature selection techniques such as Partial Least Square (PLS) or LASSO regression, this research combines supervised and unsupervised machine learning methods with random sampling strategies, offering a more robust and interpretable approach to feature selection. This study aimed to identify a parsimonious and robust set of biomarkers for breast cancer diagnosis using metabolomics data. Plasma samples from 185 breast cancer patients and 53 controls (from the Cooperative Human Tissue Network, USA) were analyzed. This study also overcomes the common issue of dataset imbalance by using propensity score matching (PSM), which ensures reliable comparisons between cancer and control groups. We employed Univariate Naïve Bayes, L2-regularized Support Vector Classifier (SVC), Principal Component Analysis (PCA), and feature engineering techniques to refine and select the most informative features. Our best-performing feature set comprised 11 biomarkers, including 9 metabolites (SM(OH) C22:2, SM C18:0, C0, C3OH, C14:2OH, C16:2OH, LysoPC a C18:1, PC aa C36:0 and Asparagine), a metabolite ratio (Kynurenine-to-Tryptophan), and 1 demographic variable (Age), achieving an area under the ROC curve (AUC) of 98%. These results demonstrate the potential for a robust, cost-effective, and non-invasive breast cancer screening and diagnostic tool, offering significant clinical value for early detection and personalized patient management.

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. The murine-selective TAAR1 antagonist, EPPTB, had no effect on either β-PEA or RO5256390-induced vasoconstriction. The α 1 -adrenoceptor antagonist, prazosin, and the α 2 -adrenoceptor antagonist, yohimbine, induced a shift of the β-PEA concentration response curve too small to be ascribed to antagonism of α 1 -or α 2 -adrenoceptors, respectively. The α 2 -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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Parkinson's disease (PD) is a neurodegenerative and progressive disease that impacts the nerve cells in the brain and varies from person to person. The exact cause of PD is still unknown, and the diagnosis of PD does not include a specific objective test with certainty. Although deep learning has made great progress in medical neuroimaging analysis, these methods are very susceptible to biases present in neuroimaging datasets. An innovative decorrelated deep learning technique is introduced to mitigate class bias and scanner bias while simultaneously focusing on finding distinguishing characteristics in resting-state functional MRI (rs-fMRI) data, which assists in recognizing PD with good accuracy. The decorrelation function reduces the nonlinear correlation between features and bias in order to learn bias-invariant features. The publicly available Parkinson's Progression Markers Initiative (PPMI) dataset, referred to as a single-scanner imbalanced dataset in this study, was used to validate our method. The imbalanced dataset problem affects the performance of the deep learning framework by overfitting to the majority class. To resolve this problem, we propose a new decorrelated convolutional neural network (DcCNN) framework by applying decorrelation-based optimization to convolutional neural networks (CNNs). An analysis of evaluation metrics comparisons shows that integrating the decorrelation function boosts the performance of PD recognition by removing class bias. Specifically, our DcCNN models perform significantly better than existing traditional approaches to tackle the imbalance problem. Finally, the same framework can be extended to create scanner-invariant features without significantly impacting the performance of a model. The obtained dataset is a multiscanner dataset, which leads to scanner bias due to the differences in acquisition protocols and scanners. The multiscanner dataset is a combination of two publicly available datasets, namely, PPMI and FTLDNI-the frontotemporal lobar degeneration neuroimaging initiative (NIFD) dataset. The results of t-distributed stochastic neighbor embedding (t-SNE) and scanner classification accuracy of our proposed feature extraction-DcCNN (FE-DcCNN) model validated the effective removal of scanner bias. Our method achieves an average accuracy of 77.80% on a multiscanner dataset for differentiating PD from a healthy control, which is superior to the DcCNN model trained on a single-scanner imbalanced dataset.

A high percentage of asthma patients have symptoms that are not well controlled, despite effective drugs being available. One potential reason for this may be that poor inhaler technique limits the dose delivered to the lungs, thereby reducing the therapeutic efficacy. The aim of this study was to assess the prevalence of poor inhaler technique in an asthma patient population and to probe the impact of various demographic parameters on technique quality. This study was conducted at community pharmacies across Wales, UK. Patients diagnosed with asthma and 12 years or older were invited to participate. An aerosol inhalation monitor (AIM, Vitalograph ® ) was used to measure the quality of patient inhaler technique. A total of 295 AIM assessments were carried out. There were significant differences in the quality of inhaler technique across the different inhaler types ( p < 0.001, Chi squared). The best technique was associated with dry-powder inhalers (DPI devices, 58% of 72 having good technique), compared with pressurized metered-dose inhalers (pMDI) or pMDIs with a spacer device (18% of 174 and 47% of 49 AIM assessments, respectively). There were some significant associations between gender, age, and quality of inhaler technique, as determined with adjusted odds ratios. It seems that the majority of asthmatic patients were not using their inhalers appropriately. We recommend that healthcare professionals place more emphasis on assessing and correcting inhaler technique, as poor inhaler technique might be responsible for the observed lack of symptom control in the asthma patient population.

The role of the community pharmacist has evolved to include the provision of more clinical services for patients. Those people who have stable chronic conditions will be managed in community pharmacies. This qualitative study used semi-structured in-depth interviews to understand the potential of providing additional patient-centred care for patients with stable chronic conditions in community pharmacies and identify potential limitations of this approach. Participants were recruited from Welsh Government, Local Health Boards (LHBS), Community Pharmacy Wales (CPW) and the Royal Pharmaceutical Society Wales (RPSW). The interviews were audio-recorded, transcribed verbatim, and analysed thematically. Eight interviews were conducted. The identified themes were as follows: (1) inconsistency and bureaucracy in commissioning pharmacy services; (2) availability of funding and resources; (3) disagreement and uncertainty about the contribution of the community pharmacy sector; (4) continuity of patient medical information and fragmented care; (5) accessibility, capacity and facilities in community pharmacy; (6) pharmacy education and clinical expertise, and (7) patient acceptability. It was clear that the potential benefit of managing stable chronic diseases in community pharmacies was recognised; however, several limitations expressed by stakeholders of pharmacy services need to be considered prior to moving forward.

Asthma is still an incurable disease, and there is a recognized need for novel small-molecule therapies for people with asthma, especially those poorly controlled by current treatments. We previously demonstrated that calcium-sensing receptor (CaSR) negative allosteric modulators (NAMs), calcilytics, uniquely suppress both airway hyperresponsiveness (AHR) and inflammation in human cells and murine asthma surrogates. Here we assess the feasibility of repurposing four CaSR NAMs, which were originally developed for oral therapy for osteoporosis and previously tested in the clinic as a novel, single, and comprehensive topical antiasthma therapy. We address the hypotheses, using murine asthma surrogates, that topically delivered CaSR NAMs 1) abolish AHR; 2) are unlikely to cause unwanted systemic effects; 3) are suitable for topical application; and 4) inhibit airway inflammation to the same degree as the current standard of care, inhaled corticosteroids, and, furthermore, inhibit airway remodeling. All four CaSR NAMs inhibited poly-L-arginine-induced AHR in naïve mice and suppressed both AHR and airway inflammation in a murine surrogate of acute asthma, confirming class specificity. Repeated exposure to inhaled CaSR NAMs did not alter blood pressure, heart rate, or serum calcium concentrations. Optimal candidates for repurposing were identified based on anti-AHR/inflammatory activities, pharmacokinetics/pharmacodynamics, formulation, and micronization studies. Whereas both inhaled CaSR NAMs and inhaled corticosteroids reduced airways inflammation, only the former prevented goblet cell hyperplasia in a chronic asthma model. We conclude that inhaled CaSR NAMs are likely a single, safe, and effective topical therapy for human asthma, abolishing AHR, suppressing airways inflammation, and abrogating some features of airway remodeling. SIGNIFICANCE STATEMENT: Calcium-sensing receptor (CaSR) negative allosteric modulators (NAMs) reduce airway smooth muscle hyperresponsiveness, reverse airway inflammation as efficiently as topical corticosteroids, and suppress airway remodeling in asthma surrogates. CaSR NAMs, which were initially developed for oral therapy of osteoporosis proved inefficacious for this indication despite being safe and well tolerated. Here we show that structurally unrelated CaSR NAMs are suitable for inhaled delivery and represent a one-stop, steroid-free approach to asthma control and prophylaxis., (Copyright © 2020 by The American Society for Pharmacology and Experimental Therapeutics.)

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. This study aimed 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 sensitivity. Guinea pigs were sensitized to Ova and challenged with inhaled Ova alone or combined with LPS. Airway function was determined by measuring specific airway conductance via whole-body plethysmography. Airway hyper-responsiveness to histamine was determined before and 24 hours post-Ova challenge. Airway inflammation and underlying mechanisms were determined from bronchoalveolar lavage cell counts and lung tissue cytokines. Vehicle or dexamethasone was administered by once-daily i.p. 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 histamine bronchoconstriction, and further elevating airway inflammation. Intraperitoneal dexamethasone (20 mg/kg) significantly reduced the elongated EAR and airway 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 they are only partially sensitive to systemic, dexamethasone. This finding suggests that the route of corticosteroid administration may be important in determining corticosteroid sensitivity of asthmatic responses., (Copyright © 2017 by The Author(s).)

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 (sG aw ) 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 (sG aw ) was measured over 24h after OVA exposure. Airway hyperresponsiveness to inhaled histamine and inflammatory cells in bronchoalveolar lavage fluid were recorded 24h 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., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Purpose: Asthma is associated with reversible airway obstruction, leucocyte infiltration, airways hyperresponsiveness (AHR), and airways remodeling. Fluid accumulation causes pulmonary edema contributing to airways obstruction. We examined the temporal relationship between the late asthmatic response (LAR) following allergen challenge of sensitized guinea-pigs and pulmonary edema measured by magnetic resonance imaging (MRI)., Materials and Methods: Ovalbumin (OVA) sensitized 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 (sG(aw)) 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 edemic signals were quantified., Results: Ovalbumin caused early bronchoconstriction, followed at 7 h by an LAR and at 24 h AHR and leucocyte influx. The bright-intensity MRI edema signal, peaking at 7 h, was significantly (P < .05) greater after chronic (9.0 ± 0.7 × 10(3) mm(3)) than acute OVA (7.6 ± 0.2 × 10(3) mm(3)). Dexamethasone treatment before acute OVA abolished the AHR and LAR and significantly reduced eosinophils and the bright-intensity MRI edema from 9.1 ± 1.0 to 6.4 ± 0.3 × 10(3) mm(3)., Conclusion: We show a temporal relationship between edema and the LAR and their parallel reduction, along with eosinophils and AHR, by dexamethasone. This suggests a close causative association between pulmonary edema and impaired airways function.

Background and Purpose: Asthma exacerbations contribute to corticosteroid insensitivity. LPS is ubiquitous in the environment. It causes bronchoconstriction and airway inflammation and may therefore exacerbate allergen responses. This study examined whether LPS and ovalbumin co-administration could exacerbate the airway inflammatory and functional responses to ovalbumin in conscious guinea pigs and whether these exacerbated responses were insensitive to inhaled corticosteroid treatment with fluticasone propionate (FP)., Experimental Approach: Guinea pigs were sensitized and challenged with ovalbumin and airway function recorded as specific airway conductance by whole body plethysmography. Airway inflammation was measured from lung histology and bronchoalveolar lavage. Airway hyper-reactivity (AHR) to inhaled histamine was examined 24 h after ovalbumin. LPS was inhaled alone or 24 or 48 h before ovalbumin and combined with ovalbumin. FP (0.05-1 mg·mL(-1) ) or vehicle was nebulized for 15 min twice daily for 6 days before ovalbumin or LPS exposure., Key Results: Ovalbumin inhalation caused early (EAR) and late asthmatic response (LAR), airway hyper-reactivity to histamine and influx of inflammatory cells into the lungs. LPS 48 h before and co-administered with ovalbumin exacerbated the response with increased length of the EAR, prolonged response to histamine and elevated inflammatory cells. FP 0.5 and 1 mg·mL(-1) reduced the LAR, AHR and cell influx with ovalbumin alone, but was ineffective when guinea pigs were exposed to LPS before and with ovalbumin., Conclusions and Implications: LPS exposure exacerbates airway inflammatory and functional responses to allergen inhalation and decreases corticosteroid sensitivity. Its widespread presence in the environment could contribute to asthma exacerbations and corticosteroid insensitivity in humans., (© 2015 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of The British Pharmacological Society.)

Airway hyperresponsiveness and inflammation are fundamental hallmarks of allergic asthma that are accompanied by increases in certain polycations, such as eosinophil cationic protein. Levels of these cations in body fluids correlate with asthma severity. We show that polycations and elevated extracellular calcium activate the human recombinant and native calcium-sensing receptor (CaSR), leading to intracellular calcium mobilization, cyclic adenosine monophosphate breakdown, and p38 mitogen-activated protein kinase phosphorylation in airway smooth muscle (ASM) cells. These effects can be prevented by CaSR antagonists, termed calcilytics. Moreover, asthmatic patients and allergen-sensitized mice expressed more CaSR in ASMs than did their healthy counterparts. Indeed, polycations induced hyperreactivity in mouse bronchi, and this effect was prevented by calcilytics and absent in mice with CaSR ablation from ASM. Calcilytics also reduced airway hyperresponsiveness and inflammation in allergen-sensitized mice in vivo. These data show that a functional CaSR is up-regulated in asthmatic ASM and targeted by locally produced polycations to induce hyperresponsiveness and inflammation. Thus, calcilytics may represent effective asthma therapeutics., (Copyright © 2015, American Association for the Advancement of Science.)

Introduction: Inhalation of antigen in atopic asthma induces early (EAR) and late asthmatic responses (LARs), inflammatory cell infiltration and airways hyperresponsiveness (AHR). Previously, we have established a protocol of sensitisation and subsequent ovalbumin (Ova) inhalation challenge in guinea-pigs which induced these 4 features (Smith & Broadley, 2007). However, the responses of guinea-pigs to Ova challenge have recently declined, producing no LAR or AHR and diminished EAR and cells. By making cumulative modifications to the protocol, we sought to restore these features., Methods: Guinea-pigs were sensitised with Ova (i.p. 100 or 150 μg) on days 1 and 5 or days 1, 4 and 7 and challenged with nebulised Ova (100 or 300 μg/ml, 1h) on day 15. Airway function was measured in conscious guinea-pigs by whole-body plethysmography to record specific airway conductance (sGaw). Airway responsiveness to aerosolized histamine (0.3mM) was determined before and 24h after Ova challenge. Bronchoalveolar lavage was performed for total and differential inflammatory cell counts. Lung sections were stained for counting of eosinophils., Results: Lack of AHR and LAR with the original protocol was confirmed. Increasing the Ova challenge concentration from 100 to 300 μg/ml restored AHR and eosinophils and increased the peak of the EAR. Increasing the number of sensitisation injections from 2 to 3 did not alter the responses. Increasing the Ova sensitisation concentration from 100 to 150 μg significantly increased total cells, particularly eosinophils. A LAR was revealed and lymphocytes and eosinophils increased when either the Al(OH)3 concentration was increased or the duration between the final sensitisation injection and Ova challenge was extended from 15 to 21 days., Discussion: This study has shown that declining allergic responses to Ova in guinea-pigs could be restored by increasing the sensitisation and challenge conditions. It has also demonstrated an important dissociation between EAR, LAR, AHR and inflammation., (Copyright © 2014. Published by Elsevier Inc.)

Viral exacerbations of allergen-induced pulmonary inflammation in pre-clinical models reportedly reduce the efficacy of glucocorticoids to limit pulmonary inflammation and airways hyper-responsiveness to inhaled spasmogens. However, exacerbations of airway obstruction induced by allergen challenge have not yet been studied. hPIV-3 (human parainfluenza type 3 virus) inoculation of guinea-pigs increased inflammatory cell counts in BAL (bronchoalveolar lavage) fluid and caused hyper-responsiveness to inhaled histamine. Both responses were abolished by treatment with either dexamethasone (20 mg/kg of body weight, subcutaneous, once a day) or fluticasone propionate (a 0.5 mg/ml solution aerosolized and inhaled over 15 min, twice a day). In ovalbumin-sensitized guinea-pigs, allergen (ovalbumin) challenge caused two phases of airway obstruction [measured as changes in sGaw (specific airways conductance) using whole body plethysmography]: an immediate phase lasting between 4 and 6 h and a late phase at about 7 h. The late phase, airway hyper-responsiveness to histamine and inflammatory cell counts in BAL were all significantly reduced by either glucocorticoid. Inoculation of guinea-pigs sensitized to ovalbumin with hPIV-3 transformed the allergen-induced airway obstruction from two transient phases into a single sustained response lasting up to 12 h. This exacerbated airway obstruction and airway hyper-responsiveness to histamine were unaffected by treatment with either glucocorticoid whereas inflammatory cell counts in BAL were only partially inhibited. Virus- or allergen-induced pulmonary inflammation, individually, are glucocorticoid-sensitive, but in combination generate a phenotype where glucocorticoid efficacy is impaired. This suggests that during respiratory virus infection, glucocorticoids might be less effective in limiting pulmonary inflammation associated with asthma.

Trace amines including β-phenylethylamine (β-PEA) and amphetamines classically exert pharmacological actions via indirect sympathomimetic mechanisms. However, there is evidence for other mechanisms and this study explores the receptors mediating vasoconstriction in rat aorta. β-PEA, d-amphetamine, MDMA, cathinone and methylphenidate caused concentration-dependent contractions of rat isolated aortic rings which were unaffected by prazosin (1 μM), ICI-118,551 (1 μM), cocaine (10 μM) and pargyline (10 μM), to inhibit α1- and β2-adrenoceptors, neuronal transport and monoamine oxidase (MAO), respectively. Octopamine concentration-response curves, however, were shifted to the right. In the presence of the inhibitors, the rate of onset of octopamine contractions was slowed. Lineweaver-Burk analysis of the kinetics of the response generated different KM values for octopamine in the absence (2.35 × 10(-6)M) and presence (6.09 × 10(-5)M) of inhibitors, indicating mediation by different receptors. Tryptamine-induced vasoconstriction also resisted blockade by adrenergic inhibitors and the 5-HT1A, 1B, 1D and 5-HT2A receptor antagonists, methiothepin (50 nM) and ketanserin (30 nM), respectively. Trace amines and amphetamines therefore exert vasoconstriction independently of adrenoceptors, neuronal transport and 5-HT receptor activation. There was no evidence of tachyphylaxis or cross-tachyphylaxis of the vasoconstriction to these amines. Tyramine was a partial agonist and in its presence, β-PEA, d-amphetamine and octopamine were antagonised indicating that they all act through a common receptor for which tyramine serves as an antagonist. We conclude that the vasoconstriction is via TAAR-1, because of structural similarities between amines, ability to stimulate recombinant trace amine-associated receptor 1 (TAAR-1) and the presence of TAAR-1 in rat aorta., (© 2013 Elsevier B.V. All rights reserved.)

Tryptamine is an endogenous and dietary indoleamine-based trace amine implicated in cardiovascular pathologies, including hypertension, migraine and myocardial infarction. This study aimed at identifying the signalling pathways for the vasoconstrictor response to tryptamine in rat isolated perfused mesenteric arterial beds and co-released vasodilator modulators of tryptamine-mediated vasoconstriction. Tryptamine caused concentration-dependent vasoconstriction of the mesenteric bed, measured as increases in perfusion pressure. These were inhibited by the 5-HT(2A) receptor antagonist, ritanserin, indicating mediation via 5-HT(2A) receptors. The response was inhibited by the phospholipase C (PLC) and phospholipase A(2) (iPLA(2)) inhibitors, U-73122 and PACOCF(3), suggesting involvement of phospholipase pathways. Activation of these pathways by tryptamine releases cyclooxygenase (COX) products since indomethacin (non-selective inhibitor of COX-1/2) and nimesulide (selective COX-2 inhibitor) reduced the vasoconstriction. The most likely COX vasoconstrictor product was prostaglandin PGE(2) since the responses to tryptamine were reduced by AH-6809, a non-selective EP(1) receptor antagonist. Involvement of the Rho-kinase pathway in the tryptamine-evoked vasoconstriction was also indicated by its reduction by the Rho-kinase inhibitors, Y-27,632 and fasudil. The tryptamine vasoconstriction is modulated by the co-released endothelial vasodilator, nitric oxide. Thus, circulating tryptamine can regulate mesenteric blood flow through a cascade of signalling pathways secondary to stimulation of 5-HT(2A) receptors., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Pre-clinical evaluation of asthma therapies requires animal models of chronic airways inflammation, airway hyperresponsiveness (AHR) and lung remodelling that accurately predict drug effectiveness in human asthma. However, most animal models focus on acute allergen challenges where chronic inflammation and airway remodelling are absent. Chronic allergen challenge models have been developed in mice but few studies use guinea-pigs which may be more relevant to humans. We tested the hypothesis that a chronic rather than acute pulmonary inflammation model would best predict clinical outcome for asthma treatments. Guinea-pigs sensitized with ovalbumin (OVA) received single (acute) or nine OVA inhalation challenges at 48 h intervals (chronic). Airways function was recorded as specific airways conductance (sG(aw)) in conscious animals for 12 h after OVA challenge. AHR to inhaled histamine, inflammatory cell influx and lung histology were determined 24 h after the single or 9th OVA exposure. The inhaled corticosteroid, fluticasone propionate (FP), the phosphodiesterase 4 inhibitor, roflumilast, and the inducible nitric oxide synthase (iNOS) inhibitor, GW274150, orally, were administered 24 and 0.5 h before and 6 h after the single or final chronic OVA exposure. Both models displayed early (EAR) and late (LAR) asthmatic responses to OVA challenge, as falls in sG(aw), AHR, as increased histamine-induced bronchoconstriction, and inflammatory cell influx. Tissue remodelling, seen as increased collagen and goblet cell hyperplasia, occurred after multiple OVA challenge. Treatment with FP and roflumilast inhibited the LAR, cell influx and AHR in both models, and the remodelling in the chronic model. GW274150 also inhibited the LAR, AHR and eosinophil influx in the acute model, but not, together with the remodelling, in the chronic model. In the clinical setting, inhaled corticosteroids and phosphodiesterase 4 inhibitors are relatively effective against most features of asthma whereas the iNOS inhibitor GW274150 was ineffective. Thus, while there remain certain differences between our data and clinical effectiveness of these antiasthma drugs, a chronic pulmonary inflammation guinea-pig model does appear to be a better pre-clinical predictor of potential asthma therapeutics than an acute model., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Aim of the Study: The present study evaluates the Philippine medicinal plant Artemisia vulgaris for antagonistic activity at selected biogenic amine receptors on smooth muscle of the airways and gastrointestinal tract in order to explain its traditional use in asthma and hyperactive gut., Materials and Methods: The antagonistic activity of chloroform crude extract (AV-CHCl(3)) and methanol crude extract (AV-MeOH) of Artemisia vulgaris was studied against concentration-response curves for contractions of the guinea pig ileum and trachea to 5-hydroxytrptamine (5-HT(2) receptors), methacholine (M(3) muscarinic receptors), histamine (H(1) receptors) and β-phenylethylamine (trace amine-associated receptors, TAAR1)., Results and Discussion: The Artemisia vulgaris chloroform (AV-CHCl(3)) and methanol (AV-MeOH) extract showed histamine H1 antagonism in the ileum and trachea. Further analysis of AV-CHCl(3) isolated two major components, yomogin and 1,2,3,4-diepoxy-11(13)-eudesmen-12,8-olide. Yomogin, a sesquiterpene lactone, exhibited a novel histamine H1 receptor antagonism in the ileum., Conclusion: The presence of a specific, competitive histamine receptor antagonist and smooth muscle relaxant activity in Artemisia vulgaris extracts on the smooth muscle in ileum and trachea explains its traditional use in the treatment of asthma and hyperactive gut., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Inhaled bradykinin causes bronchoconstriction in asthmatic subjects but not nonasthmatics. To date, animal studies with inhaled bradykinin have been performed only in anesthetized guinea pigs and rats, where it causes bronchoconstriction through sensory nerve pathways. In the present study, airway function was recorded in conscious guinea pigs by whole-body plethysmography. Inhaled bradykinin (1 mM, 20 s) caused bronchoconstriction and influx of inflammatory cells to the lungs, but only when the enzymatic breakdown of bradykinin by angiotensin-converting enzyme and neutral endopeptidase was inhibited by captopril (1 mg/kg i.p.) and phosphoramidon (10 mM, 20-min inhalation), respectively. The bronchoconstriction and cell influx were antagonized by the B(2) kinin receptor antagonist 4-(S)-amino-5-(4-{4-[2,4-dichloro-3-(2,4-dimethyl-8-quinolyloxymethyl)phenylsulfonamido]-tetrahydro-2H-4-pyranylcarbonyl}piperazino)-5-oxopentyl](trimethyl)ammonium chloride hydrochloride (MEN16132) when given by inhalation (1 and 10 μM, 20 min) and are therefore mediated via B(2) kinin receptors. However, neither intraperitioneal MEN16132 nor the peptide B(2) antagonist icatibant, by inhalation, antagonized these bradykinin responses. Sensitization of guinea pigs with ovalbumin was not sufficient to induce airway hyperreactivity (AHR) to the bronchoconstriction by inhaled bradykinin. However, ovalbumin challenge of sensitized guinea pigs caused AHR to bradykinin and histamine. Infection of guinea pigs by nasal instillation of parainfluenza-3 virus produced AHR to inhaled histamine and lung influx of inflammatory cells. These responses were attenuated by the bradykinin B(2) receptor antagonist MEN16132 and H-(4-chloro)DPhe-2'(1-naphthylalanine)-(3-aminopropyl)guanidine (VA999024), an inhibitor of tissue kallikrein, the enzyme responsible for lung synthesis of bradykinin. These results suggest that bradykinin is involved in virus-induced inflammatory cell influx and AHR.

Trace amines including tyramine and β-phenylethylamine (β-PEA) increase blood pressure and cause vasoconstriction which is attributed to indirect sympathomimetic actions. However, there is evidence that they may also have non-sympathomimetic mechanisms. This study examined whether β-PEA causes vasoconstriction of rat aorta by a sympathomimetic action or through the recently described trace-amine-associated receptors (TAAR). Concentration-response curves (CRCs) for β-PEA were constructed either cumulatively or non-cumulatively in rat isolated aortic rings. TAAR-1 and TAAR-4 protein expression was determined in rat aorta by Western blotting and TAAR-1 mRNA by reverse transcriptase polymerase chain reaction (RT-PCR). β-PEA caused concentration-related constriction of rat aorta. The contractions were unaffected by endothelium removal or the nitric oxide synthase inhibitor, N(ω)-nitro-L-arginine methyl ester (L-NAME, 100 μM) or the cyclooxygenase inhibitor, indomethacin (10 μM). Non-cumulative CRCs showed greater contractions and sensitivity to β-PEA than cumulative. The α(1)-adrenoceptor antagonist, prazosin, failed to inhibit either curve. The β-adrenoceptor antagonist, propranolol, the adrenergic neuronal transport inhibitor, cocaine, and the monoamine oxidase inhibitor, pargyline, also failed to alter the CRC. In the combined presence of prazosin, cocaine, pargyline, and the selective β(2)-adrenoceptor antagonist, ICI-118,551, the trace amine contractile potency order was tryptamine > β-PEA > octopamine > D: -amphetamine > tyramine. Western blotting and RT-PCR revealed the presence of TAAR-1 in rat aorta, but TAAR-4 was poorly expressed. Vasoconstriction of rat aorta by β-PEA appears not to be an indirect sympathomimetic action. The presence of TAAR-1 suggests that vasoconstriction may be via these receptors; however, the potency order differed from that reported for transfected cells expressing rat TAAR-1.

Inhaled corticosteroids are regularly co-administered with beta(2)-adrenoceptor agonists. This study evaluates in conscious guinea-pigs the bronchodilator effect, alone or combined with salbutamol, of TPI 1020, a novel anti-inflammatory corticosteroid and nitric oxide (NO) donor derived from budesonide. Guinea-pigs received inhaled histamine (3 mM) and specific airway conductance (sG(aw)) measured. Responses to histamine were measured before and on the next day 15 min after a 15 min inhalation of vehicle, salbutamol, TPI 1020, budesonide, the NO-donor, S-nitroso-N-acetylpenicillamine (SNAP), or combinations of these drugs. Salbutamol and TPI 1020 caused concentration-dependent bronchodilatation measured as inhibition of histamine-induced bronchoconstriction. TPI 1020-induced bronchodilatation was blocked by the guanylyl cyclise inhibitor, ODQ, indicating cGMP-dependence through released NO. While salbutamol at 80 microM did not exert significant bronchodilatation, significant inhibitions were observed when co-administered with TPI 1020, 0.11 and 0.33 mM. The combined effects of TPI 1020 and salbutamol lasted significantly longer than either drug alone. Inhaled budesonide was a weak bronchodilator and when co-administered with salbutamol there was enhanced bronchodilatation. Addition of the NO-donor, SNAP (0.1 mM), to the budesonide/salbutamol combination, also improved the inhibition of histamine-induced bronchoconstriction. This study has shown that TPI 1020 potentiates the bronchodilator activity of salbutamol, and their combination lasted longer than either drug administered individually. Both the corticosteroid and NO-releasing activities of TPI 1020 appear to be required for the potentiation of salbutamol. Combination of TPI 1020 with a beta(2)-adrenoceptor agonist may therefore be useful against acute bronchoconstriction episodes in asthma, and may offer an opportunity for reducing doses of inhaled beta(2)-adrenoceptor agonists., (Copyright (c) 2010 Elsevier B.V. All rights reserved.)

The mechanisms leading to airway hyper-responsiveness (AHR) in asthma are still not fully understood. AHR could be produced by hypersensitivity of the airway smooth muscle or hyperreactivity of the airways. This study was conducted to ascertain whether AHR in a murine model of asthma is produced by changes at the level of the airway smooth muscle. Airway smooth muscle responses were characterised in vitro in isolated trachea spirals from naive mice and from an acute ovalbumin (OVA) challenge model of allergic asthma. AHR was investigated in vivo in conscious, freely moving mice. Inflammatory cell influx into the lungs and antibody responses to the antigen were also measured. In vitro study of tracheal airway smooth muscle from naïve mice demonstrated concentration-related contractions to methacholine and 5-HT, but no responses to histamine or adenosine or its stable analogue, 5'-N-ethyl-carboxamidoadenosine. The contractions to 5-HT were inhibited by ketanserin and alosetron indicating involvement of 5-HT(2A) and 5-HT(3) receptors, respectively. In an acute model of allergic asthma, OVA-treated mice were shown to be atopic by inflammatory cell influx to the lungs after OVA challenge, increases in total IgE and OVA-specific IgG levels and contractions to OVA in isolated trachea. In the asthmatic model, AHR to methacholine was demonstrated in conscious, freely moving mice in vivo and in isolated trachea in vitro 24 and 72h after OVA challenge. No AHR in vitro was seen for 5-HT, histamine or adenosine. These results suggest that, in our mouse model of asthma, changes occur at the level of the muscarinic receptor transduction pathway of coupling to airway smooth muscle contraction. These changes are maintained when tissues are removed from the inflammatory environment and for at least 3 days., (Copyright (c) 2010 Elsevier Ltd. All rights reserved.)

Trace amines, including tyramine and beta-phenylethylamine (beta-PEA), are constituents of many foods including chocolate, cheeses and wines and are generated by so-called 'friendly' bacteria such as Lactobacillus, Lactococcus and Enterococcus species, which are found in probiotics. We therefore examined whether these dietary amines could exert pharmacological effects on the gut and its vasculature. In the present study we examined the effects of tyramine and beta-PEA on the contractile activity of guinea-pig and rat ileum and upon the isolated mesenteric vasculature and other blood vessels. Traditionally, these amines are regarded as sympathomimetic amines, exerting effects through the release of noradrenaline from sympathetic nerve endings, which should relax the gut. A secondary aim was therefore to confirm this mechanism of action. However, contractile effects were observed in the gut and these were independent of noradrenaline, acetylcholine, histamine and serotonin receptors. They were therefore probably due to the recently described trace amine-associated receptors. These amines relaxed the mesenteric vasculature. In contrast, the aorta and coronary arteries were constricted, a response that was also independent of a sympathomimetic action. From these results, we propose that after ingestion, trace amines could stimulate the gut and improve intestinal blood flow. Restriction of blood flow elsewhere diverts blood to the gut to aid digestion. Thus, trace amines in the diet may promote the digestive process through stimulation of the gut and improved gastrointestinal circulation.

The effects of ischaemia and reoxygenation on cardiac contractile function can be abrogated by ischaemic preconditioning (IPC). We tested whether beta-adrenoceptor agonists could mimic IPC and whether IPC was dependent on beta-adrenoceptor activation in rat-isolated cardiac tissues. Paced left atria and right ventricular strips were set-up in Krebs solution and isometric developed tension recorded. Ischaemia was simulated by replacing with hypoxic glucose-free Krebs solution for 30 min. IPC and isoprenaline (10(-7) M) preconditioning for 10 min were examined. Developed tension post-reoxygenation was expressed as a percentage of the pre-ischaemic baseline. Recovery at 15 min was significantly increased by IPC in atria (47 +/- 4.0% vs. 29.3 +/- 1.7%, p < 0.05) and ventricles (39.0 +/- 5.2% vs. 22.4 +/- 2.8%, p < 0.05). At 60 min, isoprenaline-treated atria recovery (75.8 +/- 16.6%) was significantly (p < 0.05) greater than controls (47.9 +/- 2.3%). Propranolol (10(-6) M) abolished both effects. Therefore, both IPC and beta-adrenoceptor agonist-induced improvement of contractile recovery was propranolol-sensitive and beta-adrenoceptor-mediated.

Allergic asthma is a chronic disease of the airways, with superimposed acute inflammatory episodes which correspond to exacerbations of asthma. Two novel models of allergic asthma have been developed in mice receiving the same allergen sensitisation, but with acute or chronic allergen exposures, the latter to mimic the human situation more closely. Ovalbumin-sensitised mice were challenged by ovalbumin inhalation twice on the same day for the acute model, and 18 times over a period of 6 weeks for the chronic model. Lung function was monitored in conscious, unrestrained mice immediately after the last challenge for up to 12 h. Airway responsiveness to inhaled methacholine and serum antibody levels were determined 24 h after challenge. Bronchoalveolar inflammatory cell recruitment was determined at 2 or 24 h. Acute and chronically treated mice had similar early and late asthmatic responses peaking at 2 h and 7-8 h, respectively. IgE and IgG antibody levels, compared with naïve mice, and eosinophil infiltration, compared with naïve and saline challenge, were elevated. Airway hyperresponsiveness to methacholine was observed 24 h after challenge in both models. The acute model had higher levels of eosinophilia, whereas the chronic model showed hyperresponsiveness to lower doses of methacholine and had higher levels of total IgE and ovalbumin-specific IgG antibodies. Both novel murine models of allergic asthma bear a close resemblance to human asthma, each offering particular advantages for studying the mechanisms underlying asthma and for evaluating existing and novel therapeutic agents.

Adrenaline (epinephrine) has been used for cardiopulmonary resuscitation (CPR) since 1896. The rationale behind its use is thought to be its alpha-adrenoceptor-mediated peripheral vasoconstriction, causing residual blood flow to be diverted to coronary and cerebral circulations. This protects these tissues from ischaemic damage and increases the likelihood of restoration of spontaneous circulation. Clinical trials have not demonstrated any benefit of adrenaline over placebo as an agent for resuscitation. Adrenaline has deleterious effects in the setting of resuscitation, predictable from its promiscuous pharmacological profile. This article discusses the relevant pharmacology of adrenaline in the context of CPR. Experimental and clinical evidences for the use of adrenaline and alternative vasopressor agents in resuscitation are given, and the properties of an ideal vasopressor are discussed.

Although the endocannabinoids 2-arachidonoylglycerol (2-AG) and anandamide share a similar pharmacology, 2-AG reportedly limits myocardial ischaemia-reperfusion injury whereas anandamide does not. We therefore investigated whether or not anandamide reduces infarct size and which, if any, of the known cannabinoid-signalling pathways are involved. Rat isolated perfused hearts were subjected to global, no-flow ischaemia (30 min) and reperfusion (1 h). Agonists were present from 5 min before ischaemia until the end of reperfusion. Antagonists, where used, were present throughout the protocol. Recovery of left ventricular developed pressure and coronary flow was incomplete in control hearts and not significantly affected by any drug treatment. In vehicle-treated hearts, 26+/-3% (n=13) of the left ventricle was infarcted at the end of reperfusion. Infarction of the left ventricle was significantly reduced after 1 microM anandamide (10+/-1%, n=7) or 1 microM methanandamide (12+/-4%, n=6) but not 1 microM HU210. Neither ACPA (1 microM; CB1 receptor agonist) nor JWH133 (1 microM; CB2 receptor agonist), individually or combined significantly affected infarct size. Anandamide (1 microM) did not reduce infarct size in the presence of the CB1 receptor antagonist rimonabant (SR141716A, 1 microM) or the CB2 receptor antagonist, SR144528 (1 microM). Despite sensitivity to CB1 and CB2 receptor antagonists, the infarct-limiting action of anandamide was not mimicked by agonists selective for CB1 or CB2 receptors suggesting the involvement of a novel cannabinoid site of action.

Structural and functional maturation of a number of fetal organs and physiological systems occurs in the immediate period prior to term, in association with the prepartum increase in fetal plasma cortisol concentration. At present, little is known about how myocardial sensitivity to adrenergic and muscarinic cholinergic stimulation changes as the fetus approaches term, nor the role of the prepartum increase in plasma cortisol concentration in mediating these changes. This study used a novel Langendorff, biventricular, ovine fetal heart preparation to investigate the effects of advancing gestation and cortisol treatment on myocardial sensitivity to adrenergic (isoprenaline) and muscarinic cholinergic (carbachol) stimulation. It was hypothesized that cortisol infusion would fully mimic developmental changes in myocardial responsiveness to adrenergic and cholinergic stimulation. Sixteen Welsh Mountain sheep fetuses were surgically prepared under general anaesthesia with vascular catheters. At 125 +/- 1 days gestational age (dGA; term, 145 dGA) fetuses were infused with saline vehicle (n= 7; Premature Control) or with cortisol (n= 4; 2-3 mg kg(-1) d(-1)i.v.; Premature Cortisol) for 5 days. The Term Control group (n= 5) comprised fetuses that were surgically prepared at 130 dGA and infused with vehicle for 5 days prior to delivery (n= 2), or that received no surgery (n= 3). Under terminal anaesthesia, Premature Control and Premature Cortisol fetuses were delivered at 130 dGA and Term Control fetuses between 135 and 143 dGA. Following exsanguination under anaesthesia, fetal hearts were mounted in the Langendorff preparation, allowing measurement of left ventricular (LV) developed pressure and right ventricular (RV) developed pressure, heart rate (HR), coronary perfusion pressure and perfusate distribution to the myocardium. Cortisol infusion elevated fetal plasma cortisol concentrations to values similar to those measured close to term (45.0 +/- 7.1 ng ml(-1)). Advancing gestational age, but not cortisol treatment, enhanced fetal LV developed pressure, RV developed pressure and HR responses to carbachol (P < 0.05). Advancing gestational age, but not cortisol treatment, suppressed fetal LV developed pressure, RV developed pressure and HR responses to isoprenaline (P < 0.05). Maximum doses of either carbachol or isoprenaline had no effect on coronary perfusate distribution. Changes in myocardial responsiveness to adrenergic and muscarinic cholinergic stimulation with advancing gestation provide mechanisms that contribute to the maturation of the cardiovascular system as the ovine fetus approaches term. These changes in myocardial responsiveness are not solely dependent on preparturient elevations in fetal plasma cortisol concentration.

Mitogen-activated protein kinases (MAPKs) have been implicated during ischemia-reperfusion (IR) and angiotensin II (AngII) type 2 receptor (AT2R) blockade has been shown to induce cardioprotection involving protein kinase Cepsilon (PKCepsilon) signaling after IR. We examined whether the 3 major MAPKs, p38, c-Jun NH2-terminal kinase (JNK-1 and JNK-2), and extracellular signal regulated kinases (ERK-1 and ERK-2) are activated after IR and whether treatment with the AT2R antagonist PD123,319 (PD) alters their expression. Isolated rat hearts were randomized to control (aerobic perfusion, 80 min), IR (no drug; 50 min of perfusion, 30 min global ischemia and 30 min reperfusion; working mode), and IR + PD (0.3 micromol/l) and left ventricular (LV) work was measured. We measured LV tissue content of p38, p-p38, p-JNK-1 (54 kDa), p-JNK-2 (46 kDa), p-ERK-1 (44 kDa), p-ERK-2 (42 kDa) and PKCepsilon proteins by immunoblotting and cGMP by enzyme immunoassay. IR resulted in significant LV dysfunction, increase in p-p38 and p-JNK-1/-2, no change in p-ERK-1/-2 or PKCepsilon, and decrease in cGMP. PD improved LV recovery after IR, induced a slight increase in p-p38 (p < 0.01 vs. control), normalized p-JNK-1, did not change p-ERK-1/-2, and increased PKCepsilon and cGMP. The overall results suggest that p38 and JNK might play a significant role in acute IR injury and the cardioprotective effect of AT2R blockade independent of ERK. The activation of p38 and JNKs during IR may be linked, in part, to AT2R stimulation.

Cannabinoids include not only plant-derived compounds (of which delta9-tetrahydrocannabinol is the primary psychoactive ingredient of cannabis), but also synthetic agents and endogenous substances termed endocannabinoids which include anandamide (2-arachidonoylethanolamide) and 2-arachidonoylglycerol. Cannabinoids act on specific, G-protein-coupled, receptors which are currently divided into two types, CB1 and CB2. Relatively selective agonists and antagonists for these receptors have been developed, although one agent (SR141716A) widely used as an antagonist at CB1 receptors has non-cannabinoid receptor-mediated effects at concentrations which are often used to define the presence of the CB1 receptor. Both cannabinoid receptors are primarily coupled to Gi/o proteins and act to inhibit adenylyl cyclase. Stimulation of CB1 receptors also modulates the activity of K+ and Ca2+ channels and of protein kinase pathways including protein kinase B (Akt) which might mediate effects on apoptosis. CB, receptors may activate the extracellular signal-regulated kinase cascade through ceramide signalling. Cannabinoid actions on the cardiovascular system have been widely interpreted as being mediated by CB1 receptors although there are a growing number of observations, particularly in isolated heart and blood vessel preparations, that suggest that other cannabinoid receptors may exist. Interestingly, the currently identified cannabinoid receptors appear to be related to a wider family of lipid receptor, those for the lysophospholipids, which are also linked to Gi/o protein signalling. Anandamide also activates vanilloid VR1 receptors on sensory nerves and releases the vasoactive peptide, calcitonin gene-related peptide (CGRP), which brings about vasodilatation through its action on CGRP receptors. Current evidence suggests that endocannabinoids have important protective roles in pathophysiological conditions such as shock and myocardial infarction. Therefore, their cardiovascular effects and the receptors mediating them are the subject of increasing investigative interest.

Background: The stress-responsive mitogen-activated protein kinases (MAPKs) (p38-MAPK, c-Jun NH2-terminal kinase [JNK-1 and JNK-2], and extracellular signal regulated kinases [ERK-1 and ERK-2]) might be involved in angiotensin II (AII)-induced ischemia-reperfusion injury. Cardioprotection induced by AII type 1 (AT1) and type 2 (AT2) receptor blockade during ischemia-reperfusion is associated with protein kinase Cepsilon (PKCepsilon), nitric oxide, and cyclic guanosine monophosphate (cGMP) signaling. Our aim was to assess the effect of selective AT1 and AT2 receptor blockade with losartan and PD123,319, respectively, on MAPK expression after ischemia-reperfusion in isolated working rat hearts., Methods: Groups of six hearts were subjected to global ischemia (30 minutes) followed by reperfusion (30 minutes) and exposed to no drug/no ischemia-reperfusion (control), ischemia-reperfusion/no drug, and ischemia-reperfusion with losartan (1 microM), or PD123,319 (0.3 microM) and additional groups. AT1/AT2 receptor expression, MAPKs, PKCepsilon, and cGMP, and changes in mechanical function were measured. Western blotting was done on left ventricular tissue for AT1/AT2, p38/phosphorylated-p38 (p-p38), phosphorylated (p)-JNK-1/-2, phosphorylated (p)-ERK-1/-2, and PKCepsilon proteins; Northern blots for AT1/AT2 mRNA; and enzyme immunoassay for cGMP., Results: Compared with controls, ischemia-reperfusion induced significant left ventricular dysfunction, decreased AT2 protein and mRNA, increased p-p38 and p-JNK-1/-2, did not change p-ERK-1/-2 or PKCepsilon, and decreased cGMP. PD123,319 improved left ventricular recovery after ischemia-reperfusion, increased AT2 protein and mRNA, mildly increased p-p38, normalized p-JNK-1, did not change p-ERK-1/-2, and increased PKCepsilon and cGMP. Losartan did not change p-p38, increased p-JNK-1, and did not change pERK-1/-2, PKCepsilon, or cGMP., Conclusions: The overall results suggest that the activation of p38-MAPK and JNK might be linked to AII signaling and play a significant role in acute ischemia-reperfusion injury as well as in the cardioprotective effect of AT2 receptor blockade.

We assessed ANG II type 1 (AT(1)) and type 2 (AT(2)) receptor (R) expression and functional recovery after ischemia-reperfusion with or without AT(1)R/AT(2)R blockade in isolated working rat hearts. Groups of six hearts were subjected to global ischemia (30 min) followed by reperfusion (30 min) and exposed to no drug and no ischemia-reperfusion (control), ischemia-reperfusion and no drug, and ischemia-reperfusion with losartan (an AT(1)R antagonist; 1 micromol/l), PD-123319 (an AT(2)R antagonist; 0.3 micromol/l), N(6)-cyclohexyladenosine (CHA, a cardioprotective adenosine A(1) receptor agonist; 0.5 micromol/l as positive control), enalaprilat (an ANG-converting enzyme inhibitor; 1 micromol/l), PD-123319 + losartan, ANG II (1 nmol/l), or ANG II + losartan. Compared with controls, ischemia-reperfusion decreased AT(2)R protein (Western immunoblots) and mRNA (Northern immunoblots, RT-PCR) and impaired functional recovery. PD-123319 increased AT(2)R protein and mRNA and improved functional recovery. Losartan increased AT(1)R mRNA (but not AT(1)R/AT(2)R protein) and impaired recovery. Other groups (except CHA) did not improve recovery. The results suggest that, in isolated working hearts, AT(2)R plays a significant role in ischemia-reperfusion and AT(2)R blockade induces increased AT(2)R protein and cardioprotection.

Objective: To evaluate oral misoprostol use before office endometrial biopsy., Methods: Forty-two nonpregnant women aged 35-77 years were randomized to a prospective, double-blind study to receive either 400 microg oral misoprostol or placebo 3 hours before office endometrial biopsy. Misoprostol effects were assessed by 1) cervical resistance, 2) ease of performing the endometrial biopsy, 3) success rate of obtaining an endometrial biopsy, 4) pain intensity associated with the endometrial biopsy, and 5) adverse clinical side effects., Results: Patients in the misoprostol group experienced significantly (P <.01) more pain associated with the endometrial biopsy. The observed power to detect this difference in misoprostol-placebo comparison using the Wilcoxon rank sum test at 0.05 level of significance is 89%. In addition, significantly (P <.05) more patients had the adverse side effect of uterine cramping at 1.5 hours after medication ingestion in the misoprostol group. The observed power to detect this difference is 98%. There were no differences between the misoprostol and placebo groups in cervical resistance, ease of performing the biopsy, success rate for obtaining an endometrial biopsy, or adverse side effects at 3 hours post medication ingestion., Conclusion: Oral misoprostol 400 microg caused more uterine cramping and pain in nonpregnant women undergoing office endometrial biopsy when given 3 hours before biopsy attempt. No other cervical effects were noted.

1. Cannabinoids are known to cause coronary vasodilatation and reduce left ventricular developed pressure (LVDP) in isolated hearts although the identity of the receptor(s) mediating these responses is unknown. Our objective was to pharmacologically characterize cannabinoid receptors mediating cardiac responses to the endocannabinoid, anandamide. 2. Dose-response curves for coronary perfusion pressure (CPP) and LVDP were constructed to anandamide, R-(+)-methanandamide, palmitoylethanolamide (PEA) and JWH015 in isolated Langendorff-perfused rat hearts. Anandamide dose-response curves were also constructed in the presence of antagonists selective for CB(1), CB(2) or VR(1) receptors. 3. Anandamide and methanadamide significantly reduced CPP and LVDP but the selective CB(2) receptor agonists, PEA and JWH015 had no significant effect, compared with equivalent vehicle doses. 4. Single bolus additions of the selective CB(1)-receptor agonist, ACEA (5 nmol), decreased LVDP and CPP. When combined with JWH015 (5 nmol) these responses were not augmented. 5. Anandamide-mediated reductions in CPP were significantly blocked by the selective CB(1) receptor antagonists SR 141716A (1 microM) and AM251 (1 microM) and the selective CB(2) receptor antagonist SR 144528 (1 microM) but not by another selective CB(2) receptor antagonist AM630 (10 microM) nor the vanilloid VR(1) receptor antagonist capsazepine (10 microM). 6. SR 141716A, AM281 and SR 144528 significantly blocked negative inotropic responses to anandamide that were not significantly affected by AM251, AM630 and capsazepine. 7. One or more novel sites mediate negative inotropic and coronary vasodilatatory responses to anandamide. These sites can be distinguished from classical CB(1) and CB(2) receptors, as responses are sensitive to both SR 141716A and SR 144528.

1. The cannabinoid arachidonyl ethanolamide (anandamide) caused concentration-dependent relaxation of 5-HT-precontracted, myograph-mounted, segments of rat left anterior descending coronary artery. 2. This relaxation was endothelium-independent, unaffected by the fatty acid amide hydrolase inhibitor, arachidonyl trifluoromethyl ketone (10 microM), and mimicked by the non-hydrolysable anandamide derivative, methanandamide. 3. Relaxations to anandamide were attenuated by the cannabinoid receptor antagonist, SR 141716A (3 microM), but unaffected by AM 251 (1 microM) and AM 630 (1 microM), more selective antagonists of cannabinoid CB(1) and CB(2) receptors respectively. Palmitoylethanolamide, a selective CB(2) receptor agonist, did not relax precontracted coronary arteries. 4. Anandamide relaxations were not affected by inhibition of sensory nerve transmission with capsaicin (10 microM) or blockade of vanilloid VR1 receptors with capsazepine (5 microM). Nevertheless capsaicin relaxed coronary arteries in a concentration-dependent and capsazepine-sensitive manner, confirming functional sensory nerves were present. In contrast, capsazepine and capsaicin did inhibit anandamide relaxations in methoxamine-precontracted rat small mesenteric arteries. 5. Relaxations to anandamide were inhibited by TEA (1 mM) or iberiotoxin (50 nM), blockers of large conductance, Ca(2+)-activated K(+) channels (BK(Ca)). Gap junction inhibition with 18alpha-glycyrrhetinic acid (100 microM) did not affect anandamide relaxations. 6. This study shows anandamide relaxes the rat coronary artery by a novel mechanism. Anandamide-induced relaxations do not involve the endothelium, degradation into active metabolites, or activation of cannabinoid CB(1) or CB(2) receptors, but may involve activation of BK(Ca). Vanilloid receptor activation also has no role in the effects of anandamide in coronary arteries, even though functional sensory nerves are present.

1. In order to test the hypothesis that angiotensin II exacerbates myocardial ischaemia-reperfusion (IR) injury, we examined the effects of graded angiotension II concentrations of angiotensin II on IR injury in both working and non-working (Langendorff) isolated rat hearts. 2. Non-working hearts were subjected to 30 min aerobic perfusion (baseline) then 25 min of global, no-flow ischaemia followed by 30 min of reperfusion either in the absence (control, n=7) or presence of 1 (n=6) or 10 nM (n=5) angiotensin II). Recoveries of LV developed pressure and coronary flow after 30 min reperfusion in control hearts (58+/-9 and 40+/-8% of baseline levels, respectively) were no different from hearts treated with 1 or 10 nM angiotensin II. Infarct size (determined at the end of reperfusion by triphenyltetrazolium chloride staining) was reduced by angiotensin II in a concentration-dependent manner (from a control value of 27+/-3 to 18+/-4% and 9+/-3% of the LV, respectively). 3. Working hearts were subjected to 50 min pre-ischaemic (pre-I) aerobic perfusion then 30 min of global, no-flow ischaemia followed by 30 min of reperfusion either in the absence (control, n=14) or presence of 1 (n=8), 10 (n=7) or 100 nM (n=7) angiotensin II). In controls, post-ischaemic (post-I) left ventricular (LV) work and efficiency of oxygen consumption were depressed (43+/-9 and 42+/-10% of pre-I levels, respectively). The presence of angiotensin II throughout IR had no effect on LV work compared with control. 4. Thus, angiotensin II reduces infarct size in a concentration-dependent manner but has no effect on contractile stunning associated with IR in isolated rat hearts.

This study investigated the role of beta-adrenoceptors in the cardioprotective and metabolic actions of adenosine A(1) receptor stimulation. Isolated paced (300 beats min(-1)) working rat hearts were perfused with Krebs-Henseleit solution containing 1.2 mM palmitate. Left ventricular minute work (LV work), O(2) consumption and rates of glycolysis and glucose oxidation were measured during reperfusion (30 min) following global ischaemia (30 min) as well as during aerobic conditions. Relative to untreated hearts, N(6)-cyclohexyladenosine (CHA, 0.5 microM) improved post-ischaemic LV work (158%) and reduced glycolysis and proton production (53 and 42%, respectively). CHA+propranolol (1 microM) had similar beneficial effects, while propranolol alone did not affect post-ischaemic LV work or glucose metabolism. Isoprenaline (10 nM) impaired post-ischaemic function and after 25 min ischaemia recovery was comparable with 30 min ischaemia in untreated hearts (41 and 53%, respectively). Relative to isoprenaline alone, CHA+isoprenaline improved recovery of LV work (181%) and reduced glycolysis and proton production (64 and 60%, respectively). In aerobic hearts, CHA, propranolol or CHA+propranolol had no effect on LV work or glucose oxidation. Glycolysis was inhibited by CHA, propranolol and CHA+propranolol (50, 53 and 52%, respectively). Isoprenaline-induced increases in heart rate, glycolysis and proton production were attenuated by CHA (85, 57 and 53%, respectively). The cardioprotective efficacy of CHA was unaffected by antagonism or activation of beta-adrenoceptors. Thus, the mechanism of protection by adenosine A(1) receptor activation does not involve functional antagonism of beta-adrenoceptors.

Background: Whether cardioprotection induced by the angiotensin II (AngII) type 2 receptor (AT(2)R) antagonist PD123,319 (PD) after ischemia-reperfusion (IR) is influenced by the concentration of PD, presence of AngII, timing of exposure, or inhibition of proton production from glucose metabolism is not known., Methods and Results: We examined these factors in isolated working rat hearts subjected to IR injury, no treatment (control), or treatment with N(6)-cyclohexyl adenosine (CHA, 0.5 micromol/L), an adenosine A(1) receptor agonist that induces cardioprotection by decreasing protons ("positive" control). Compared with control, 1 micromol/L PD present throughout IR improved recovery of left ventricular work (73 +/- 5 vs. 40 +/- 8%) to the level with CHA (82 +/- 5%), but 0.1 micromol/L PD did not (58 +/- 6 vs. 40 +/- 8%). AngII (1 nmol/L) did not effect postischemic recovery associated with 1 micromol/L PD (73 +/- 7%) but improved that associated with 0.1 micromol/L PD (86 +/- 3%). PD (1 micromol/L), present solely during reperfusion, enhanced postischemic left ventricular recovery to 72 +/- 5%. Also, PD (1 micromol/L) did not affect glycolytic rates or proton production in nonischemic or IR hearts., Conclusion: PD-induced cardioprotection is 1) PD concentration-dependent, 2) AngII-sensitive, 3) mediated during reperfusion, and 4) independent of proton production, suggesting that reduction in IR injury and indirect AT(1)R stimulation might be involved.

The relaxant effect of adenosine and 5'-(N-ethylcarboxamido)adenosine (NECA) against alpha-adrenoceptor-mediated contractile tone in guinea-pig isolated aortic rings has been examined to determine if this A2B-receptor-mediated relaxation was dependent upon the contracting agent, and whether the contractions were dependent upon intracellular or extracellular calcium. Relaxation responses were consistently greater for aortic rings pre-contracted with phenylephrine (3x10(-6) M) than for rings pre-contracted with noradrenaline (3x10(-6) M). Maximum inhibition by NECA was significantly greater for phenylephrine-contracted aortae than for noradrenaline-contracted (81.9+/-2.8% compared with 25.0+/-1.5%). These differences persisted in the presence of beta- and alpha2-adrenoceptor blockade and could not, therefore, be attributed to stimulation of these receptors by noradrenaline. The ratio of the contractions obtained before and in the presence of adenosine or NECA was compared with the control ratio obtained before and after vehicle. Experiments were performed both in the presence of normal calcium levels and under calcium-free conditions. In normal-calcium medium, NECA inhibited phenylephrine-induced contractions (test ratio, 76.7+/-3.9%; control ratio, 133.1+/-9.8%) to a greater extent than noradrenaline-induced contractions (108.4+/-4.1 and 123.4+/-4.9%); adenosine similarly inhibited phenylephrine-induced contractions more than those induced by noradrenaline. Under calcium-free conditions, adenosine (36.7+/-11.9 and 110.7+/-26.6%) and NECA (55.2+/-9.1 and 87.1+/-14.9%) were only effective against phenylephrine-induced contractions. This suggests that activation of the A2B-receptor by these agonists inhibited intracellular mobilization of calcium for phenylephrine-induced contractions only. The effects on extracellular calcium influx were examined for phenylephrine- and noradrenaline-induced contractions in normal-calcium medium but in the presence of ryanodine to prevent intracellular calcium mobilization. NECA inhibited phenylephrine-induced contractions (77.3+/-12.4 and 111.4+/-9.3%), presumably by interfering with influx of calcium through receptor-operated calcium channels. In contrast, NECA failed to reduce noradrenaline-induced contractions (121.5+/-10.7 and 122.4+/-11.6%), suggesting that the effect on noradrenaline is predominantly via interaction with intracellular calcium. Adenosine was consistently a more effective relaxant than NECA, possibly because of an additional intracellular component of the response. We conclude that adenosine receptor agonists inhibit phenylephrine-induced contractions of guinea-pig aorta more selectively than noradrenaline-induced contractions. A2B-receptor stimulation might reveal a fundamental difference between the modes of contraction elicited by these two alpha-adrenoceptor agonists.

The vasorelaxant actions of adenosine and its analogue, 5'-(N-ethylcarboxamido)-adenosine (NECA), were investigated in guinea-pig isolated aortic rings by addition to the tissue prior to induction of a contraction by the alpha1-adrenoceptor agonist phenylephrine (PE, 3x10(-6) M). The effect was calculated from the ratio (C2/C1) of the contraction to PE before (C1) and in the presence of adenosine or NECA (C2). This was compared with a control ratio obtained at the same time in which no vasorelaxant was present during C2. Experiments were performed in either "normal" or "Ca2+ -free" bathing medium. Both adenosine and NECA caused inhibition of contractions in "normal" and "Ca2+ -free" conditions, the latter indicating that the vasorelaxant action was due in part to inhibition of intracellular Ca2+ mobilization. To determine whether inhibition of influx of extracellular Ca2+ is a target for the vasorelaxation, contractions to PE were obtained in "normal" Ca2+ and in the presence of ryanodine (10(-5) M), which prevents the release of intracellular Ca2+. These contractions were inhibited by NECA indicating that stimulation of A2-receptors by NECA interferes with the influx of Ca2+ via the opening of receptor-operated Ca2+ channels (ROCs). This study has demonstrated that cell surface A2-receptor stimulation in the guinea-pig aorta inhibits phenylephrine-induced contractions by interfering with both the release of intracellular Ca2+ and the influx of extracellular Ca2+, presumably via ROCs.

Adenosine has previously been shown to stimulate K(+)-efflux and to block L-type calcium channels in atrial myocytes. The aim of the present study was to evaluate the contribution of K(+)-channels in the development of the negative inotropic and chronotropic responses to adenosine agonists in guinea-pig left and right atria, respectively. Tetraethylammonium (TEA) potentiated the negative inotropic and chronotropic responses to R-(-)-N6-(2-phenyl-isopropyl)-adenosine (R-PIA), seen as leftward shifts of the concentration-response curves. Glibenclamide had no effect on the negative inotropic response to R-PIA but increased the rate of onset of the negative chronotropic response in right atria. 4-Aminopyridine (4-AP, 10 mM), potentiated the left atrial inotropic responses to R-PIA, seen as a leftward shift of the concentration-response curve, but slowed the speed of onset of the response to a single concentration (10(-6) M) of R-PIA. This reduction in speed of onset of the response can explain the differences in effects of 4-AP on concentration-response curves reported here and previously. In the right atria, 4-AP (10 mM) inhibited the negative chronotropic responses to R-PIA, seen as a rightward shift of the concentration-response curve and reduction of the maximum response. 4-AP also slowed the onset of the right atrial rate response to R-PIA. These results support the theory that K(+)-efflux plays only a minor role in the negative inotropic responses of guinea-pig left atria to R-PIA. This apparently controls the speed of onset of the response. The negative chronotropic response of guinea-pig right atria to R-PIA appears to be much more dependent upon K(+)-efflux than for the negative inotropic response of the left atria.

1. Optimization of myocardial energy substrate metabolism improves the recovery of mechanical function of the post-ischaemic heart. This study investigated the role of K(ATP)-channels in the regulation of the metabolic and mechanical function of the aerobic and post-ischaemic heart by measuring the effects of the selective K(ATP)-channel activator, cromakalim, and the effects of the K(ATP)-channel antagonist, glibenclamide, in rat fatty acid perfused, working hearts in vitro. The role of K(ATP) channels in the cardioprotective actions of the adenosine A1-receptor agonist, N6-cyclohexyladenosine (CHA) was also investigated. 2. Myocardial glucose metabolism, mechanical function and efficiency were measured simultaneously in hearts perfused with modified Krebs-Henseleit solution containing 2.5 mM Ca2+, 11 mM glucose, 1.2 mM palmitate and 100 mu l(-1) insulin, and paced at 300 beats min(-1). Rates of glycolysis and glucose oxidation were measured from the quantitative production of 3H20 and 14CO2, respectively, from [5-3H/ U-14C]-glucose. 3. In hearts perfused under aerobic conditions, cromakalim (10 microM), CHA (0.5 microM) or glibenclamide (30 microM) had no effect on mechanical function. Cromakalim did not affect glycolysis or glucose oxidation, whereas glibenclamide significantly increased rates of glycolysis and proton production. CHA significantly reduced rates of glycolysis and proton production but had no effect on glucose oxidation. Glibenclamide did not alter CHA-induced inhibition of glycolysis and proton production. 4. In hearts reperfused for 30 min following 30 min of ischaemia, left ventricular minute work (LV work) recovered to 24% of aerobic baseline values. Cromakalim (10 microM), administered 5 min before ischaemia, had no significant effect on mechanical recovery or glucose metabolism. CHA (0.5 microM) significantly increased the recovery of LV work to 67% of aerobic baseline values and also significantly inhibited rates of glycolysis and proton production. Glibenclamide (30 microM) significantly depressed the recovery of mechanical function to < 1% of aerobic baseline values and stimulated glycolysis and proton production. 5. Despite the deleterious actions of glibenclamide per se in post-ischaemic hearts, the beneficial effects of CHA (0.5 microM) on the recovery of mechanical function and proton production were not affected by glibenclamide. 6. The data indicate that the cardioprotective mechanism of adenosine A1-receptor stimulation does not involve the activation of K(ATP)-channels. Furthermore, in rat fatty acid perfused, working hearts, stimulation of K(ATP)-channels is not cardioprotective and has no significant effects on myocardial glucose metabolism.

To determine whether intrinsic angiotensin II (ANG II) type 1 receptor (AT1-R) stimulation modulates recovery of postischemic mechanical function, we studied the effects of selective AT1-R blockade with losartan on proton production from glucose metabolism and recovery of function in isolated working rat hearts perfused with Krebs-Henseleit buffer containing palmitate, glucose, and insulin. Aerobic perfusion (50 min) was followed by global, no-flow ischemia (30 min) and reperfusion (30 min) in the presence (n = 10) or absence (n = 14) of losartan (1 mumol/l) or the cardioprotective adenosine A1 receptor agonist N6-cyclohexyladenosine (CHA, 0.5 mumol/l, n = 11). During reperfusion in untreated hearts (controls), left ventricular (LV) minute work partially recovered to 38% of aerobic baseline, whereas proton production increased to 155%. Compared with controls, CHA improved recovery of LV work to 79% and reduced proton production to 44%. Losartan depressed recovery of LV work to 0% without altering proton production. However, exogenous ANG II (1-100 nmol/l) in combination with losartan restored recovery of LV work during reperfusion in a concentration-dependent manner, suggesting that postischemic recovery of function depends on intrinsic AT1-R stimulation.

Objectives: To assess the effect of the angiotensin II type 1 receptor (AT1-R) antagonist L-158,809 on acute infarct expansion and left ventricular (LV) function during acute anterior myocardial infarction., Methods: Dogs were randomized to receive intravenous L-158,809 (0.1 mg/kg bolus and 0.6 microgram/kg/min infusion) or vehicle beginning 1 h after permanent left anterior descending coronary artery ligation and continued for 48 h. In vivo LV remodelling and function (quantitative echocardiography) and hemodynamics over 48 h, and postmortem remodelling after 48 h were measured., Results: L-158,809 produced 90% to 100% inhibition of the angiotensin II pressor response during the infusions. With respect to percentage changes over the 48 h in vivo, compared with vehicle controls, L-158,809 decreased mean arterial pressure (-20 +/- 4 versus -9 +/- 2%, P = 0.03) and left atrial pressure (-38 +/- 5 versus 25 +/- 6%, P < 0.0001) but did not change heart rate. These unloading effects were associated with a smaller percentage increase in infarct expansion index (-5 +/- 7% versus 27 +/- 2%, P = 0.001) and LV diastolic volume (11 +/- 11% versus 52 +/- 6%, P = 0.008), less shape deformation, fewer apical aneurysms (0 versus 100%, P = 0.0003), better global ejection fraction (49 +/- 2% versus 39 +/- 2%, P = 0.005), less ST segment elevation and fewer Q waves. Also compared with vehicle controls, with L-158,809 postmortem infarct size (19.8 +/- 2.4% versus 50.4 +/- 4.7% of risk region, P = 0.0002) and expansion index (2.06 +/- 0.09 versus 2.76 +/- 0.18, P = 0.006) were less and thinning ratio greater (0.92 +/- 0.02 versus 0.60 +/- 0.05, P = 0.0001)., Conclusions: The novel AT1-R antagonist L-158,809 produces significant AT1-R blockade, reduces LV loading, and effectively limits acute infarct expansion and early LV remodelling during canine myocardial infarction.

To determine whether changes in density (rho) of infarct and noninfarct zones during healing and remodeling after myocardial infarction influence estimates of left ventricular mass and detection of temporal changes by imaging, we measured weights (g) and volumes (mL) of infarct, noninfarct, and mixed tissue in hearts removed 1 to 42 days after anterior infarction in three groups of dogs: nonreperfused infarction treated with placebo or captopril, or infarcts reperfused after 2 h. In vivo mass was calculated from in vivo diastolic myocardial volumes (echocardiograms) and an assumed density of 1.05 g/mL or actual values derived from tissue weights and volumes. Over the 42 days, actual density deviated more from the assumed value of 1.05 in infarct than noninfarct zones, and the overall density was higher for reperfused than nonreperfused ventricles (1.09 vs. 1.06 g/mL, p < 0.01). Correction for density improved the correlation between absolute in vivo and postmortem mass slightly but not the detection of relative changes in mass in control, captopril, or reperfusion groups. These findings suggest that (i) densities of infarct and noninfarct zones differ and change during healing, especially after reperfusion, and (ii) correction for density provides more accurate estimates of volume-derived mass in reperfused hearts.

The P1 purinoceptor subtype mediating the negative inotropic responses of guinea-pig left atria and the negative chronotropic responses of beating right atria were characterized. Guinea-pig isolated paced left atria (2Hz, 5ms, threshold voltage+50%) and spontaneously beating right atria were set up in Krebs-bicarbonate solution and isometric tension and rate of contraction, respectively, were recorded. Concentration-response curves for the reduction of tension and rate, respectively, by adenosine receptor agonists, N6-cyclopentyladenosine (CPA), the R- and S- stereoisomers of N6-(2-phenylisopropyl) adenosine (R-PIA and S-PIA), 5'-(N-carboxamido) adenosine (NECA) and 2-p-((carboxyethyl)-phenethylamino)-5'-(N-carboxamido) adenosine (CGS21680) were obtained. The orders of potency on the left atria (CPA = NECA > R-PIA > S-PIA > CGS21680) and right atria (CPA = R-PIA > S-PIA > CGS21680) were consistent with the responses being mediated via A1 receptors. Antagonism of the responses to CPA or R-PIA by 8-cyclo-1,3-dimethylxanthine (CPT) was examined by a full Schild analysis. Concentration-response curves for CPA or R-PIA were obtained in the absence or presence of five or six concentrations (10(-7)-10(-5) or 3 x 10(-5)M) of CPT. The shift in the concentration-response by CPT was expressed as the concentration-ratio (CR) and plotted as -log(CR-1) against log molar concentration of CPT (Schild plot). pA2 values were calculated from the intercept on the concentration axis and by application of the equation; pA2 = log(antagonist concentration) -log (CR-1). The Schild plots had unity slopes indicating competitive antagonism and the pA2 values derived therefrom indicated that the responses were mediated via A1-receptor. Closer inspection of the Schild plots, however, showed that at the higher concentrations of CPT there was a limit to the displacement of the concentration-response curves of the left and right atria to CPA and of the left atria to R-PIA. There were also significant differences in the apparent pA2 values calculated from the equation, when different concentrations of antagonist were examined. These results indicated that at higher concentrations of agonist there may be a component of the response that is resistant to antagonism by CPT. Whether this is related to the proposal that cardiac responses are mediated via A3 receptors is discussed.