New bronchodilators selected by molecular topology.

Molecular topology has been applied to find new lead compounds with bronchodilator activity. Among the selected compounds stands out 3-(1H-tetrazol-5yl)-9H-thioxanthene-9 -one-10,10-dioxide, anthrarobin, 9-oxo-9H-thioxantene-3-carboxylic-10,10-dioxide acid, acenocoumarol and griseofulvin, with a percentage of relaxation, at 0.1 mM, of 91, 92, 85, 69, and 74%, respectively. Theophylline shows a correspondent value of 77% (Emax = 100% at 1 mM).

Acute cardio-respiratory effects in rats of PS4α, an antineoplastic peptidoglycan from Mycobacterium vaccae

Abstract PS4α is a high molecular weight peptidoglycan extracted from Mycobacterium vaccae, which has demonstrated considerable antineoplastic activity in-vivo without apparent toxicity. Available for testing in only small quantities, a sensitive in-vivo method for measuring pulse and breathing rates in cannulated rats was applied to this compound at doses of 5, 50 and 500 μg kg−1. Various parameters (mean arterial pressure, maximum transpulmonary pressure, compliance, heart rate, minute volume, respiratory rate and tidal volume) were followed for up to 1 h and demonstrated no significant deviation in the baseline values obtained before injection. This compound at doses up to 500 μg kg−1 ha…

EFFECTS OF PHORBOL 12,13-DIACETATE AND ITS INFLUENCE ON SPASMOGENIC RESPONSES IN NORMAL AND SENSITIZED GUINEA-PIG TRACHEA

Abstract We have studied the effects of phorbol 12,13-diacetate (PDA) and its influence on a variety of spasmogenic responses in trachea isolated in from normal and sensitized guinea-pigs. Tracheal preparations were denuded of epithelium, treated with indomethacin (2·8 μm), and cooled to 20°C. In these experimental conditions, tracheal strips contracted to PDA (0·1 nm-1 μm). Contractions to PDA (1 μm) were greater in sensitized tissues. In normal trachea, contractions to PDA (0·1 μm) were depressed by H-7, 1-(5-isoquinolinyl-sulphonyl)-2-methylpiperazine, (50 μm), amiloride (10 μm), verapamil (10 μm) and Ca2+-free exposure. Similar effects were obtained in sensitized trachea except that PDA…

In vivo antioxidant treatment protects against bleomycin-induced lung damage in rats

1. This study examines the activity of the antioxidant N-acetylcysteine on bleomycin-induced pulmonary fibrosis in rats with emphasis on the early inflammatory phase. 2. Rats receiving N-acetylcysteine (300 mg kg(-1) day(-1), intraperitoneal) had less augmented lung wet weight, and lower levels of proteins, lactate dehydrogenase, neutrophil and macrophage counts in bronchoalveolar lavage fluid and lung myeloperoxidase activity with a betterment of histological score at 3 days postbleomycin. 3. A diminished lung GSH/GSSG ratio and augmented lipid hydroperoxides were observed 3 days postbleomycin. These changes were attenuated by N-acetylcysteine. Alveolar macrophages from bleomycin-exposed r…

Aclidinium inhibits human lung fibroblast to myofibroblast transition

Background Fibroblast to myofibroblast transition is believed to contribute to airway remodelling in lung diseases such as asthma and chronic obstructive pulmonary disease. This study examines the role of aclidinium, a new long-acting muscarinic antagonist, on human fibroblast to myofibroblast transition. Methods Human bronchial fibroblasts were stimulated with carbachol (10 −8 to 10 −5  M) or transforming growth factor-β1 (TGF-β1; 2 ng/ml) in the presence or absence of aclidinium (10 −9 to 10 −7  M) or different drug modulators for 48 h. Characterisation of myofibroblasts was performed by analysis of collagen type I and α-smooth muscle actin (α-SMA) mRNA and protein expression as well as α…

Nickel induces intracellular calcium mobilization and pathophysiological responses in human cultured airway epithelial cells.

Abstract Environmental exposure to nickel is associated to respiratory disorders and potential toxicity in the lung but molecular mechanisms remain incompletely explored. The extracellular Ca 2+ -sensing receptor (CaSR) is widely distributed and may be activated by divalent cations. In this study, we investigated the presence of CaSR in human cultured airway epithelial cells and its activation by nickel. Nickel transiently increased intracellular calcium (−log EC 50  = 4.67 ± 0.06) in A549 and human bronchial epithelial cells as measured by epifluorescence microscopy. Nickel (20 μM)-induced calcium responses were reduced after thapsigargin or ryanodine exposure but not by Ca 2+ -free medium…

Evaluation of the quality of publications on randomized clinical trials using the Consolidated Standards of Reporting Trials (CONSORT) statement guidelines in a Spanish tertiary hospital.

The main reason for conducting a clinical trial (CT) is to test the effect of a drug or medical procedure to improve treatment of a disease. CTs contribute most when they are rigorously conducted and the results are published adequately. The aim of this study is to assess, using the CONSORT statement guidelines, the quality of reporting of completed CTs conducted at a tertiary hospital to determine which sections of the articles should be improved. CTs published between 2002 and 2008 were identified by searching the MEDLINE and Cochrane Library. Forty of 127 completed CTs were published. There was a marked increase in the number of articles and the quality of the journals that published the…

Effects of low temperature and pharmacological interventions on the responses of the isolated guinea-pig trachea

Cooling the guinea-pig isolated trachea from 37°C to 20°C virtually abolished the response to CaCl2 (in K+-depolarized tissues) and depressed that to histamine (about 75% reduction), KCl and 5-hydroxytryptamine (around 50% inhibition) while the response to acetylcholine remained unaffected. A further cooling to 10°C was necessary to inhibit acetylcholine-induced contractions. Hyporesponsiveness to spasmogens by cooling was not associated with subsensitivity (increased EC50) except for 5-hydroxytryptamine. Contractile responses to KCl (50 mmol/l), histamine (1 mmol/l) and 5-hydroxytryptamine (0.1 mmol/l) in a Ca2+-free EGTA (0.1 mmol/l)-containing solution were inhibited by cooling to 20°C b…

Influence of low temperature on bronchodilatation induced by terbutaline administered by metered dose or dry powder inhalers in asthmatics.

Low temperatures may affect dose delivery efficacy and clinical effectiveness of medication aerosols. In this study we examine the effect of cold ambient temperature on the bronchodilatation produced by terbutaline delivered from a chlorofluorocarbon pressurized metered dose inhaler (pMDI) compared to a multi-dose dry powder inhaler (DPI). Fourteen stable asthmatics were studied on two consecutive days. On day 1, after measuring FEV1 at room temperature (22 degrees C), each patient was randomized to receive 500 microg of terbutaline delivered from pMDI or DPI stored for 24 h at 22 degrees C with FEV1 recorded 20 min post-dose; then, patients were placed in a chamber at -10 degrees C, and af…

Effects of bemiparin on airway responses to antigen in sensitized Brown-Norway rats.

Heparins have demonstrated activity in asthma. The effects of bemiparin, a low molecular weight heparin, were examined on antigen-induced responses in sensitized Brown-Norway rats. Inhaled bemiparin (1 mg/ml) reduced the acute bronchospasm produced by aerosol antigen, prevented airway hyperresponsiveness to 5-hydroxytryptamine postantigen exposure, and reduced the eosinophil count (from 0.205+/-0.062 to 0.054+/-0.016 x 10(6) cells/ml in antigen and antigen+bemiparin groups, respectively; P<0.05), eosinophil peroxidase activity, and proteins in the bronchoalveolar lavage fluid (BALF), as well as the transiently augmented mucin Muc5ac expression. Hyperresponsiveness to adenosine was not affec…

Stimulation of the ADRB3 Adrenergic Receptor Induces Relaxation of Human Placental Arteries: Influence of Preeclampsia1

Preeclampsia, which complicates 3-8% of pregnancies, is one of the leading causes of neonatal morbidity and mortality. Its pathophysiology remains unclear. The aim of the present study was to investigate the presence and the role of beta2- and beta2-adrenergic receptors (ADRB2 and ADRB3, respectively) in human placental arteries and to assess the influence of preeclampsia on ADRB responsiveness. SR 59119A, salbutamol, and isoproterenol (ADRB3, ADRB2, and nonselective ADRB agonists, respectively) induced a concentration-dependent relaxation of placental artery rings obtained from women with uncomplicated or preeclamptic pregnancies. SR 59119A-induced relaxation was unaffected by the blockade…

The spasmogenic effects of vanadate in human isolated bronchus

1. Inhalation of vanadium compounds, particularly vanadate, is a cause of occupational bronchial asthma. We have now studied the action of vanadate on human isolated bronchus. Vanadate (0.1 microM-3 mM) produced concentration-dependent, well-sustained contraction. Its -logEC50 was 3.74 +/- 0.05 (mean +/- s.e.mean) and its maximal effect was equivalent to 97.5 +/- 4.2% of the response to acetylcholine (ACh, 1 mM). 2. Vanadate (200 microM)-induced contraction of human bronchus was epithelium-independent and was not inhibited by indomethacin (2.8 microM), zileuton (10 microM), a mixture of atropine, mepyramine and phentolamine (each at 1 microM), or by mast cell degranulation with compound 48/…

Analgesic and thermic effects, and cerebrospinal fluid and plasma pharmacokinetics, of intracerebroventricularly administered morphine in normal and sensitized rats.

Abstract The relationship between asthma and opioids has barely been investigated. This study examines whether active sensitization of rats changes the analgesic and thermic effects of intracerebroventricular morphine or the pharmacokinetics of the drug. Morphine (5, 10 and 20 μg) was given intracerebroventricularly to sensitized (active immunization to ovalbumin and Al(OH)3 then airway challenge with ovalbumin after 12 days) and normal (i.e. non-sensitized) male Sprague-Dawley rats. The tail-flick latencies and changes in colon temperature were determined before morphine injection and at 30 min intervals for a period of 300 min afterwards. Results were expressed as the area under the time-…

Critical role of fractalkine (CX3CL1) in cigarette smoke-induced mononuclear cell adhesion to the arterial endothelium.

Background Cigarette smoking is an important risk factor for the development of cardiovascular disease, yet the pathways through which this may operate are poorly understood. Therefore, the mechanism underlying cigarette smoke (CS)-induced arterial endothelial dysfunction and the potential link with fractalkine/CX3CL1 upregulation were investigated. Methods and results Stimulation of human arterial umbilical endothelial cells (HUAECs) with pathophysiological concentrations of CS extract (1% CSE) increased CX3CL1 expression. Neutralisation of CX3CL1 activity under dynamic flow conditions significantly inhibited CSE-induced mononuclear cell adhesion to HUAECs (67%). The use of small interferi…

Roflumilast improves corticosteroid resistance COPD bronchial epithelial cells stimulated with toll like receptor 3 agonist

Chronic obstructive pulmonary disease (COPD) is characterised by chronic pulmonary inflammation punctuated by periods of viral exacerbations. Recent evidence suggests that the combination of roflumilast with corticosteroids may improve the compromised anti-inflammatory properties of corticosteroids in COPD. We analyzed differential and combination anti-inflammatory effects of dexamethasone and roflumilast N-oxide in human bronchial epithelial cells (HBECs) stimulated with viral toll like receptor (TLR) agonists. Lung tissue and HBECs were isolated from healthy (n = 15), smokers (n = 12) and smokers with COPD (15). TLR3 expression was measured in lung tissue and in HBECs. IL-8 secretion was …

Muscarinic M2 receptors in acetylcholine-isoproterenol functional antagonism in human isolated bronchus

The muscarinic functional antagonism of isoproterenol relaxation and the contribution of muscarinic M2 receptors were examined in human isolated bronchus. In intact tissues, acetylcholine (ACh) precontraction decreased isoproterenol potency and maximal relaxation (−log EC50 shift = −1.49 ± 0.16 and Emax inhibition for 100 μM ACh = 30%) more than the same levels of histamine contraction. The M2receptor-selective antagonist methoctramine (1 μM) reduced this antagonism in ACh- but not histamine-contracted tissues. Similar results were obtained for forskolin-induced relaxation. After selective inactivation of M3 receptors with 4-diphenylacetoxy- N-(2-chloroethyl)piperadine hydrochloric acid (3…

Effects of Mn2+ on the responses induced by different spasmogens in the oestrogen-primed rat uterus

Abstract We investigated the effect of Mn 2+ on the mechanical responses evoked by high K + (60 mM) or low Na + (25 mM) solutions, oxytocin and neurokinin A in the oestrogen-primed rat uterus. In a Ca 2+ -free, Mn 2+ (0.54 mM)-containing solution, high K + or low Na + solutions produced contractions of smaller amplitude than those observed in a normal Ca 2+ (0.54 mM) solution, which were abolished by nifedipine (1 μM). Oxytocin (1 μM) and neurokinin A (1 μM, in the presence of phosphoramidon 1 μM) evoked nifedipine-insensitive contractile responses similar to (oxytocin) or smaller (neurokinin A) in amplitude than those observed in Ca 2+ (0.54 mM)-containing solution. In strips loaded with C…

Rolipram inhibits leukocyte-endothelial cell interactionsin vivothrough P- and E-selectin downregulation

1. Rolipram, a selective phosphodiesterase (PDE) type 4 inhibitor, was used to characterize leukocyte recruitment mechanisms in models of acute and subacute inflammation. Intravital microscopy within the rat mesenteric microcirculation was employed. 2. Mesentery superfusion with PAF (0.1 microM) induced a significant increase in leukocyte rolling flux, adhesion and emigration at 60 min. Rolipram pretreatment, markedly inhibited these parameters by 100, 95 and 95% respectively. 3. Similar effects were observed when the mesentery was superfused with LPS (1 microg ml(-1)) for the same time period and these leukocyte parameters were nearly abrogated by rolipram pretreatment. 4. LPS exposure of …

Investigation into the role of phosphodiesterase IV in bronchorelaxation, including studies with human bronchus.

1. We have investigated the role of cyclic nucleotide phosphodiesterase IV (PDE IV) in the relaxation of human bronchus and guinea-pig trachea in vitro and in guinea-pigs in vivo. 2. Functional studies showed that the selective PDE IV inhibitors, rolipram and denbufylline, relaxed human and guinea-pig preparations in vitro. 3. Two clinically used xanthine non-selective PDE inhibitors, theophylline and pentoxifylline, were also effective in these preparations, but were much less potent than the selective agents used. 4. The rank order of potency for the four PDE inhibitors in both species was similar. 5. Biochemical studies indicated that PDE IV was the major PDE isoform present in the human…

Extracellular calcium-sensing receptor mediates human bronchial epithelial wound repair

The airway epithelium routinely undergoes damage that requires repair to restore epithelial barrier integrity. Cell migration followed by proliferation are necessary steps to achieve epithelial repair. Calcium-sensing receptor (CaSR) is implicated in cell migration and proliferation processes. Thus we hypothesized that CaSR mediates lung epithelial wound repair. We detected CaSR expression in human lung and in well-differentiated human bronchial epithelial cells (HBEC). To test the CaSR functionality, HBEC loaded with fura-2 were stimulated with extracellular Ca(2+) ([Ca(2+)](out)) which resulted in a concentration-dependent intracellular Ca(2+) ([Ca(2+)](i)) increase (potency approximately…

Rolipram inhibits airway microvascular leakage induced by platelet-activating factor, histamine and bradykinin in guinea-pigs.

Abstract Rolipram (0·1–1000 μg kg−1, i.v.) reduced the increase in microvascular permeability induced by platelet-activating factor (PAF; 50 ng kg−1, i.v.) at different sites of the guinea-pig airways. Rolipram (1–100μg kg−1, i.v.) inhibited histamine (30μg kg−1, i.v.)-and bradykinin (0·3 μg kg, i.v.)-induced airway microvascular leakage. These effects of rolipram were obtained at doses which inhibit histamine (7–20 μg kg−1 min−1)-induced bronchoconstriction (IC50 = 3 ± 1 μg kg, i.v.) without depressing arterial blood pressure in the guinea-pig. Aminophylline (50 mg kg−1) did not change the effect of PAF. The anti-exudative effect of rolipram is of potential therapeutic value in asthma.

Oral N-acetylcysteine attenuates the rat pulmonary inflammatory response to antigen.

Oxidative stress is involved in the pathophysiology of inflammatory airway diseases including asthma; therefore, antioxidants might be of clinical benefit in asthma treatment. In the present study, the effects of N-acetylcysteine on sensitised brown Norway rats were examined. N-Acetylcysteine (3 mmol kg body weight(-1) administered orally) was given daily for 1 week before challenge and various antigen-induced pulmonary responses were studied. Antigen exposure increased lipid peroxidation in bronchoalveolar lavage fluid (BALF) and oxidised glutathione levels in lung tissue 2 h after challenge. Lung nuclear transcription factor-KB-binding activity was increased 2 h after challenge, and BALF …

A critical role for TNFα in the selective attachment of mononuclear leukocytes to angiotensin-II-stimulated arterioles

Abstract Angiotensin II (Ang-II) exerts inflammatory activity and is involved in different cardiovascular disorders. This study has evaluated the involvement of tumor necrosis factor alpha (TNFα) in the leukocyte accumulation elicited by Ang-II. Ang-II (1 nM intraperitoneally in rats) induced TNFα release at 1 hour followed by neutrophil and mononuclear cell recruitment. The administration of an antirat TNFα antiserum had no effect on Ang-IIinduced neutrophil accumulation but inhibited the infiltration of mononuclear cells and reduced CC chemokine content in the peritoneal exudate. Pretreatment with either an anti-TNFα or an anti-IL-4 antiserum decreased Ang-II-induced arteriolar mononuclea…

Taurine chloramine inhibits functional responses of human eosinophils in vitro

10 páginas, 7 figuras, 1 tabla.

MUC1 intracellular bioactivation mediates lung fibrosis

BackgroundSerum KL6/mucin 1 (MUC1) has been identified as a potential biomarker in idiopathic pulmonary fibrosis (IPF), but the role of MUC1 intracellular bioactivation in IPF is unknown.ObjectiveTo characterise MUC1 intracellular bioactivation in IPF.Methods and resultsThe expression and phosphorylation of Thr41 and Tyr46 on the intracellular MUC1-cytoplasmic tail (CT) was increased in patients with IPF (n=22) compared with healthy subjects (n=21) and localised to fibroblasts and hyperplastic alveolar type II cells. Transforming growth factor (TGF)-β1 phosphorylated SMAD3 and thereby increased the phosphorylation of MUC1-CT Thr41 and Tyr46 in lung fibroblasts and alveolar type II cells, ac…

Olive Oil–Based Lipid Emulsion's Neutral Effects on Neutrophil Functions and Leukocyte–Endothelial Cell Interactions

Infection remains a drawback of parenteral nutrition (PN), probably related, among other factors, to immunosuppressive effects of its lipid component. Newer preparations may have lesser immunosuppressive impact. This study examines the effects of an olive oil-based lipid emulsion (long-chain triacylglycerols-monounsaturated fatty acids [LCT-MUFA]; ClinOleic) on various functions of human neutrophils in vitro and on rat leukocyte-endothelial cell interactions in vivo compared with LCT (Intralipid) and 50% LCT-50% medium-chain triacylglycerols (MCT; Lipofundin) mixture.Neutrophils isolated from healthy donors were incubated with concentrations (0.03-3 mmol/L) of lipid emulsions encompassing c…

CXCR2 blockade impairs angiotensin II-induced CC chemokine synthesis and mononuclear leukocyte infiltration.

Objective—Angiotensin II (Ang-II) and mononuclear leukocytes are involved in atherosclerosis. This study reports the inhibition of Ang-II–induced mononuclear cell recruitment by CXCR2 antagonism and the mechanisms involved.Methods and Results—Ang-II (1 nmol/L, i.p. in rats) induced CXC and CC chemokines, followed by neutrophil and mononuclear cell recruitment. Administration of the CXCR2 antagonist, SB-517785-M, inhibited the infiltration of both neutrophils (98%) and mononuclear cells (60%). SB-517785-M had no effect on the increase in CXC chemokine levels but reduced MCP-1, RANTES, and MIP-1α release by 66%, 63%, and 80%, respectively. Intravital microscopy showed that pretreatment with S…

Relaxation by Calcium Antagonists of Potassium-contracted Trachea from Normal and Sensitized Guinea-pigs: Influence of Epithelium and the Surface of Drug Entry

Abstract A technique by which drug access was restricted to either the mucosal or the adventitial surface of tracheal rings, isolated from normal (unsensitized) or sensitized guinea-pigs, was used to study the role of the epithelium in the relaxation produced by calcium antagonists (verapamil, nifedipine, cinnarizine and flunarizine) of K+-induced contraction. In trachea from normal guinea-pigs, the relaxation to verapamil for unrestricted or mucosal drug entry was reduced in the absence of epithelium, whereas the relaxation produced by nifedipine, cinnarizine or flunarizine was unchanged. In sensitized trachea, the relaxation elicited by the calcium antagonists tested was similar in intact…

LEUKOTRIENE RECEPTORS ON HUMAN PULMONARY VASCULAR ENDOTHELIUM

1. Cysteinyl-leukotrienes cause contractions and/or relaxations of human isolated pulmonary vascular preparations. Although, the localization and nature of the receptors through which these effects are mediated have not been fully characterized, some effects are indirect and not mediated via the well-described LT1 receptor. 2. In human pulmonary veins (HPV) with an intact endothelium, leukotriene D4 (LTD4) induced contraction above basal tone. This response was observed at lower concentrations of LTD4 in the presence of nitric oxide synthase inhibitor N omega-nitro-L-arginine (L-NOARG). Contractions (in the absence and presence of L-NOARG) were partially blocked by the LT1 antagonists (MK 5…

Aclidinium inhibits cholinergic and tobacco smoke-induced MUC5AC in human airways.

Mucus hypersecretion and mucin MUC5AC overexpression are pathological features of chronic obstructive pulmonary disease (COPD). This study examines the inhibitory effect of aclidinium, a new long-acting muscarinic antagonist, on MUC5AC expression in human airway epithelial cells. MUC5AC mRNA (RT-PCR) and protein expression (ELISA and immunohistochemistry) were studied in human bronchial tissue and differentiated human airway epithelial cells activated with carbachol (100 μM) or cigarette smoke extract in the absence or presence of aclidinium. Carbachol increased MUC5AC mRNA and protein expression in human bronchus and cultured epithelial cells. Aclidinium inhibited the carbachol-induced MUC…

Functional, biochemical and morphological studies on human bronchi after cryopreservation

1. Human isolated bronchi have been investigated as fresh tissue or after storage (7 and 30 days) at -196 degrees C in foetal calf serum containing 1.8 M dimethyl sulphoxide. 2. After cryopreservation, the maximal contractile response to acetylcholine (3 mM) was reduced (approximately 25%) but the difference did not reach significance statistically. Maximal responses to other spasmogens tested (histamine, [Nle10]NKA(4-10), bradykinin, leukotriene D4, U46619, and KCl) did not differ between unfrozen and frozen/thawed tissues. The sensitivity of cryopreserved tissues to the constrictor agents tested was similar to that of fresh tissues. 3. The accumulation of inositol phosphates produced by a…

Pharmacological activity of PF-904 in guinea pig in vivo, and on human bronchus and neutrophils in vitro.

The effects of PF-904 (4-amino-1-ethyl-6-methylpyrazino[2,3-c][1,2,6]thiadiazine 2,2-dioxide), a pyrazinothiadiazine derivative, were examined in guinea-pig airways in vivo, in human isolated bronchus and human polymorphonuclear leukocytes. PF-904 (12.5-200 mg/kg, intraduodenal) reduced bronchoconstriction in response to histamine, arachidonic acid, platelet-activating factor (PAF) and methacholine. PF-904 (50-200 mg/kg) prevented PAF-induced airways hyperreactivity and inhibited antigen-induced bronchoconstriction, airway microvascular leakage and eosinophil lung accumulation, but antigen-induced airways hyperresponsiveness was not reduced. PF-904 (1 microM-1 mM) produced complete inhibiti…

Angiotensin II Induces Neutrophil Accumulation In Vivo Through Generation and Release of CXC Chemokines

Background—Angiotensin II (Ang II) is implicated in the development of cardiac ischemic disorders in which prominent neutrophil accumulation occurs. Ang II can be generated intravascularly by the renin-angiotensin system or extravascularly by mast cell chymase. In this study, we characterized the ability of Ang II to induce neutrophil accumulation.Methods and Results—Intraperitoneal administration of Ang II (1 nmol/L) induced significant neutrophil recruitment within 4 hours (13.3±2.3×106neutrophils per rat versus 0.7±0.5×106in control animals), which disappeared by 24 hours. Maximal levels of CXC chemokines were detected 1 hour after Ang II injection (577±224 pmol/L cytokine-inducible neut…

MUC1 deficiency mediates corticosteroid resistance in chronic obstructive pulmonary disease.

Background Lung inflammation in COPD is poorly controlled by inhaled corticosteroids (ICS). Strategies to improve ICS efficacy or the search of biomarkers who may select those patients candidates to receive ICS in COPD are needed. Recent data indicate that MUC1 cytoplasmic tail (CT) membrane mucin can mediate corticosteroid efficacy in chronic rhinosinusitis. The objective of this work was to analyze the previously unexplored role of MUC1 on corticosteroid efficacy in COPD in vitro and in vivo models. Methods MUC1-CT expression was measured by real time PCR, western blot, immunohistochemistry and immunofluorescence. The inflammatory mediators IL-8, MMP9, GM-CSF and MIP3α were measured by EL…

Effects of taurine on pulmonary responses to antigen in sensitized Brown-Norway rats.

Oxidative stress appears relevant to asthma. Therefore, the effects of the antioxidant taurine (oral, 1 and 3 mmol x kg(-1) x day(-1) for 7 days before challenge) were examined on antigen-induced responses in sensitized Brown-Norway rats. Taurine did not reduce the bronchospasm produced by aerosol antigen but prevented airway hyperreactivity to 5-hydroxytryptamine (5-HT) at 24 h after antigen exposure, and reduced the eosinophils (from 0.178+/-0.038x10(6) to 0.044+/-0.014x10(6)* and 0.048+/-0.013x10(6)* cells ml(-1) in antigen and antigen+taurine 1 or 3 mmol x kg(-1), respectively; *P0.05 vs. antigen), lipid hydroperoxides, and Evans blue dye extravasation in bronchoalveolar lavage fluid. T…

Non-neuronal cholinergic system contributes to corticosteroid resistance in chronic obstructive pulmonary disease patients

Background: Inhaled corticosteroid (ICS) with long-acting beta-2 agonists is a well-documented combination therapy for chronic obstructive pulmonary disease (COPD) based on its additive anti-inflammatory properties. By contrast, the recommendation of ICS in combination with long-acting muscarinic antagonist (LAMA) is not evidence-based. In this study, neutrophils obtained from COPD patients were used to compare the anti-inflammatory effects of aclidinium bromide (a long-acting muscarinic antagonist) with corticosteroids and their potential additive effect. Methods: Human sputum and blood neutrophils were isolated from healthy individuals ( n = 37), patients with stable COPD ( n = 52) and th…

Effects of sensitization on vasoactive intestinal polypeptide-induced relaxation and its concentration and binding in guinea-pig airways.

We investigated the relaxant effect of vasoactive intestinal polypeptide (VIP) in trachea and lung parenchyma from normal and sensitized guinea-pigs. A technique by which drug access was restricted to either the mucosal or the adventitial surface of tracheal rings was used. In intact trachea, concentration-response curves for VIP entering from the mucosal surface (pD2 = 6.61 +/- 0.06) were displaced to the right compared with those for adventitial entry (pD2 = 6.78 +/- 0.04). Epithelium removal produced a leftward shift (approximately 2.8-fold) in the mucosal VIP concentration-response curve. Sensitization did not alter the responsiveness (maximal effect) or sensitivity (pD2 values) of trac…

Effects of dantrolene on the responses to methylxanthines in the isolated guinea-pig trachea.

The effect of dantrolene on the responses to methylxanthines (caffeine and theophylline) and a beta-adrenoceptor agonist (salbutamol) was studied in isolated guinea-pig trachea. Caffeine and theophylline (1 microM-10 mM) and salbutamol (1 nM-10 microM) produced concentration-dependent relaxation of spontaneous and stimulated (acetylcholine 1 mM) tone. Responses to high concentrations (10 mM) of caffeine and theophylline added to tracheal strips either unstimulated (spontaneous tone) or indomethacin (2.8 microM)-treated were reversed to contractions in the presence of dantrolene (3 microM-0.3 mM). This effect was not observed for salbutamol or when relaxant responses to the agonists were gen…

The human near-term myometrialβ3-adrenoceptor but not theβ2-adrenoceptor is resistant to desensitisation after sustained agonist stimulation

1. In order to compare the beta(2)- and beta(3)-adrenoceptor (beta-AR) desensitisation process in human near-term myometrium, we examined the influence of a pretreatment of myometrial strips with either a beta(2)- or a beta(3)-AR agonist (salbutamol or SR 59119A, respectively, both at 10 microm, for 5 and 15 h) on the relaxation and the cyclic adenosine monophosphate (cAMP) production induced by these agonists. 2. To assess some of the mechanisms potentially implicated in the beta-AR desensitisation process, we studied the influence of such treatment on the number of beta(2)- and beta(3)-AR binding sites, the beta(2)- and beta(3)-AR transcripts expression and the phosphodiesterase 4 (PDE4) …

ROLIPRAM INHIBITS PAF-INDUCED AIRWAY MICROVASCULAR LEAKAGE IN GUINEA-PIG - A COMPARISON WITH MILRINONE AND THEOPHYLLINE

The effects of 3 phosphodiesterase (PDE) inhibitors, rolipram (PDE IV), milrinone (PDE III) and theophylline (non-selective) on PAF (50 ng kg-1; iv)-induced airway vascular leakage have been evaluated in guinea-pigs. Rolipram (3-300 micrograms kg-1; iv) reduced the increase in permeability induced by PAF at all airway levels whereas milrinone (10-1000 micrograms kg-1; iv) and theophylline (30 mg kg-1; iv) were without effects. The anti-leakage activity of rolipram may be of therapeutic value in asthma.

Oral N-acetylcysteine reduces bleomycin-induced lung damage and mucin Muc5ac expression in rats

Oxidative stress is involved in the pathogenesis of pulmonary fibrosis, therefore antioxidants may be of therapeutic value. Clinical work indicates that N ‐acetylcysteine (NAC) may be beneficial in this disease. The activity of this antioxidant was examined on bleomycin-induced lung damage, mucus secretory cells hyperplasia and mucin Muc5ac gene expression in rats. NAC (3 mmol·kg−1·day−1) or saline was given orally to Sprague-Dawley rats for 1 week prior to a single intratracheal instillation of bleomycin (2.5 U·kg−1) and for 14 days postinstillation. NAC decreased collagen deposition in bleomycin-exposed rats (hydroxyproline content was 4,257±323 and 3,200±192 µg·lung−1 in vehicle- and NAC…

Binding of [3H][D-Ala2, MePhe4, Gly-ol5] Enkephalin, [3H][D-Pen2, D-Pen5]Enkephalin, and [3H]U-69,593 to Airway and Pulmonary Tissues of Normal and Sensitized Rats

Abstract Bhargava, H. N., V. M. Villar, J. Cortijo and E. J. Morcillo. Binding of [3H][D-Ala2, MePhe4, Gly-ol5]enkephalin, [3H][D-Pen2, D-Pen5]enkephalin, and [3H]U-69,593 to airway and pulmonary tissues of normal and sensitized rats. Peptides 18(10) 1603–1608, 1997.—The role of endogenous opioid peptides in the regulation of bronchomotor tone, as well as in the pathophysiology of asthma is uncertain. We have studied the binding of highly selective [3H]labeled ligands of μ-([D-Ala2, MePhe4, Gly-ol5]enkephalin; DAMGO), δ ([D-Pen2, D-Pen5]enkephalin; DPDPE), and κ-(U-69,593) opioid receptors to membranes of trachea, main bronchus, lung parenchyma and pulmonary artery obtained from normal (uns…

Phosphodiesterase 4 inhibition decreases MUC5AC expression induced by epidermal growth factor in human airway epithelial cells

Background: A common pathological feature of chronic inflammatory airway diseases such as asthma and chronic obstructive pulmonary disease (COPD) is mucus hypersecretion. MUC5AC is the predominant mucin gene expressed in healthy airways and is increased in asthmatic and COPD patients. Recent clinical trials indicate that phosphodiesterase type 4 (PDE4) inhibitors may have therapeutic value for COPD and asthma. However, their direct effects on mucin expression have been scarcely investigated. Methods: MUC5AC mRNA and protein expression were examined in cultured human airway epithelial cells (A549) and in human isolated bronchial tissue stimulated with epidermal growth factor (EGF; 25 ng/ml).…

The effects of phorbol 12,13-diacetate on responses of guinea-pig isolated trachea to methylxanthines, isoprenaline and ryanodine

1. Using guinea-pig isolated trachea, we have studied how phorbol 12,13-diacetate (PDA) modulates mechanical responses of the tissue to methylxanthines, isoprenaline and ryanodine. 2. Caffeine (10 microM-5 mM), theophylline (10 microM-5 mM) and isoprenaline (1 nM-1 microM), each inhibited the spontaneous tone of the trachea. Pretreatment with PDA (0.1-10 microM) converted relaxant responses to high concentrations of the methylxanthines into contractions. PDA produced no equivalent effect against isoprenaline. Pretreatment with verapamil (1 or 10 microM), nifedipine (0.1 microM) or incubation with Ca(2+)-free, EGTA (0.1 mM)-containing physiological salt solution (PSS) suppressed the contract…

Menopause and ovariectomy cause a low grade of systemic inflammation that may be prevented by chronic treatment with low doses of estrogen or losartan.

Abstract The incidence of cardiovascular diseases in premenopausal women is lower than in men or postmenopausal women. This study reports the discovery of a low grade of systemic inflammation, including monocyte adhesion to arterial endothelium, elicited by menopause or estrogen depletion. Chronic treatment with low dose of 17-β-estradiol or inhibition of the renin-angiotensin system reduced this inflammation. Using an in vitro flow chamber system with human arterial and venous endothelial cells, we found that leukocytes from healthy postmenopausal women were more adhesive to the arterial endothelium than those from premenopausal women regardless of the stimulus used on endothelial cells. I…

Angiotensin II-Induced Mononuclear Leukocyte Interactions with Arteriolar and Venular Endothelium Are Mediated by the Release of Different CC Chemokines

Abstract Angiotensin II (Ang-II) is associated with atherogenesis and arterial subendothelial mononuclear leukocyte infiltration. We have demonstrated that Ang-II causes the initial attachment of mononuclear cells to the arteriolar endothelium. We now report on the contribution of CC chemokines to this response. Intraperitoneal administration of 1 nM Ang-II induced MCP-1, RANTES, and MIP-1α generation, maximal at 4 h, followed by mononuclear leukocyte recruitment at 8 and 24 h. Using intravital microscopy within the rat mesenteric microcirculation 4 h after exposure to 1 nM Ang-II, arteriolar mononuclear cell adhesion was 80–90% inhibited by pretreatment with Met-RANTES, a CCR1 and CCR5 ant…

Erythromycin exertsin vivoanti-inflammatory activity downregulating cell adhesion molecule expression

1. Macrolides have long been used as anti-bacterial agents; however, there is some evidence that may exert anti-inflammatory activity. Therefore, erythromycin was used to characterize the mechanisms involved in their in vivo anti-inflammatory activity. 2. Erythromycin pretreatment (30 mg kg(-1) day(-1) for 1 week) reduced the lipopolysaccharide (LPS; intratracheal, 0.4 mg kg(-1))-induced increase in neutrophil count and elastase activity in the bronchoalveolar lavage fluid (BALF) and lung tissue myeloperoxidase activity, but failed to decrease tumor necrosis factor-alpha and macrophage-inflammatory protein-2 augmented levels in BALF. Erythromycin pretreatment also prevented lung P-selectin,…

Effects of Ca2+ channel antagonists in guinea-pig normal and skinned gall bladder.

CaCl2 (0.01-50 mM, in K(+)-depolarized tissues), KCl (0.1-100 mM) and acetylcholine (1 nM-10 mM) produced concentration-dependent contractions of guinea-pig isolated gall bladder. Nifedipine (1-100 microM), verapamil (1-100 microM), diltiazem (1-100 microM), cinnarizine (1-100 microM), and flunarizine (1-100 microM) each produced a concentration-related inhibition of the log concentration-effect curve for CaCl2. The rank order of potencies of these antagonists, measured as the IC50 against Ca2+ (50 mM)-induced contraction of depolarized gall bladder, was diltiazem (0.25 microM)or = verapamil (0.8 microM) approximately nifedipine (1.2 microM)cinnarizine (25 microM) approximately flunarizine …

DIFFERENT ABILITY OF TRIFLUOPERAZINE TO INHIBIT AGONIST-INDUCED CONTRACTION OF LUNG PARENCHYMA STRIPS FROM CONTROL AND SENSITIZED GUINEA-PIGS

Abstract There is increasing interest in the therapeutic potential of calcium antagonists in asthma. Among them the use of calmodulin antagonists deserves consideration. In the present work the effect of trifluoperazine on contractions generated by different mechanisms (CaCl2, KCl, acetylcholine, histamine and 5-hydroxytryptamine) in lung parenchyma strip isolated from control and actively sensitized guinea-pigs has been studied. Trifluoperazine produced both in unsensitized and sensitized lung strips, a concentration-dependent, right, downward displacement of the concentration-response curves to the agonists used, although the sensitization procedure resulted in a potentiation in the abili…

In vitro anti-inflammatory effects of AZD8999, a novel bifunctional muscarinic acetylcholine receptor antagonist /β2-adrenoceptor agonist (MABA) compound in neutrophils from COPD patients.

Recent evidence indicates that AZD8999 (LAS190792), a novel muscarinic acetylcholine receptor antagonist and β2-adrenoceptor agonist (MABA) in development for chronic respiratory diseases, induces potent and sustained relaxant effects in human bronchi by adressing both muscarinic acetylcholine receptors and β2-adrenoceptor. However, the anti-inflammatory effects of the AZD8999 monotherapy or in combination with corticosteroids are unknown. This study investigates the anti-inflammatory effects of AZD8999 in monotherapy and combined with fluticasone propionate in neutrophils from healthy and chronic obstructive pulmonary disease (COPD) patients. Peripheral blood neutrophils from healthy and C…

Vascular effects of sildenafil in patients with pulmonary fibrosis and pulmonary hypertension: an ex vivo/in vitro study

Sildenafil improves the 6-min walking distance in patients with idiopathic pulmonary fibrosis (IPF) and right-sided ventricular systolic dysfunction.We analysed the previously unexplored role of sildenafil on vasoconstriction and remodelling of pulmonary arteries from patients with IPF and pulmonary hypertension (PH) ex vivo. Pulmonary arteries from 18 donors without lung disease, nine IPF, eight PH+IPF and four PH patients were isolated to measure vasodilator and anti-contractile effects of sildenafil in isometric organ bath. Ventilation/perfusion was explored in an animal model of bleomycin lung fibrosis.Sildenafil relaxed serotonin (5-HT) pre-contracted pulmonary arteries in healthy dono…

Trans- but not cis-resveratrol impairs angiotensin-II-mediated vascular inflammation through inhibition of NF-κB activation and peroxisome proliferator-activated receptor-gamma upregulation.

Abstract Angiotensin II (Ang-II) displays inflammatory activity and is implicated in several cardiovascular disorders. This study evaluates the effect of cis- and trans (t)-resveratrol (RESV) in two in vivo models of vascular inflammation and identifies the cardioprotective mechanisms that underlie them. In vivo, Ang-II–induced arteriolar leukocyte adhesion was inhibited by 71% by t-RESV (2.1 mg/kg, i.v.), but was not affected by cis-RESV. Because estrogens influence the rennin-angiotensin system, chronic treatment with t-RESV (15 mg/kg/day, orally) inhibited ovariectomy-induced arteriolar leukocyte adhesion by 81%, partly through a reduction of cell adhesion molecule (CAM) expression and c…

JAK2 mediates lung fibrosis, pulmonary vascular remodelling and hypertension in idiopathic pulmonary fibrosis: an experimental study

BackgroundPulmonary hypertension (PH) is a common disorder in patients with idiopathic pulmonary fibrosis (IPF) and portends a poor prognosis. Recent studies using vasodilators approved for PH have failed in improving IPF mainly due to ventilation (V)/perfusion (Q) mismatching and oxygen desaturation. Janus kinase type 2 (JAK2) is a non-receptor tyrosine kinase activated by a broad spectrum of profibrotic and vasoactive mediators, but its role in PH associated to PH is unknown.ObjectiveThe study of JAK2 as potential target to treat PH in IPF.Methods and resultsJAK2 expression was increased in pulmonary arteries (PAs) from IPF (n=10; 1.93-fold; P=0.0011) and IPF+PH (n=9; 2.65-fold; P&lt;0.00…

Effects of Calcium Antagonists on Rat Normal and Skinned Fundus

Abstract Calcium chloride (CaCl2) (0·1−25 Mm, in K+-depolarized tissue), KCl (10−112 Mm) and acetylcholine (1 × 10−9 m−1 Mm) produced concentration-dependent contractions of rat isolated fundus. Verapamil (0·01−100 μm), cinnarizine (1−100 μm), trifluoperazine (10−500 μm) and dantrolene (50−250 μm) each produced a concentration-related rightward and downward shift of the log concentration-effect curve for CaCl2. The rank order of potencies of these antagonists, measured as the IC50 against Ca2+ (25 Mm)-induced contraction of depolarized fundus, was verapamil (2.5 μm) &amp;gt; cinnarizine (8·7 μm) &amp;gt; trifluoperazine (85·1 μm) &amp;gt; dantrolene (&amp;gt; 250 μm). Cinnarizine (0·5 Mm) a…

In vitro tracheal hyperresponsiveness to muscarinic receptor stimulation by carbachol in a rat model of bleomycin-induced pulmonary fibrosis

Summary 1 Bleomycin-induced lung injury is widely used as an experimental model to investigate the pathophysiology of pulmonary fibrosis but the alterations in the pharmacological responsiveness of airways isolated from bleomycin-exposed animals has been scarcely investigated. The aim of this study was to examine the in vitro tracheal responses to muscarinic receptor stimulation with carbachol in a rat bleomycin model. 2 Concentration–response curves to carbachol (10 nm to 0.1 mm) were obtained in tracheal rings isolated from Sprague–Dawley rats 14 days after endotracheal bleomycin or saline. The intracellular calcium signal in response to carbachol (10 μm) was measured by epifluorescence m…

Effects of alkylxanthines on contractility of diaphragm fibres isolated from normal and sensitized guinea-pigs.

Abstract This study investigates the effects of alkylxanthines on twitch tension generated by electrical stimulation (supramaximal pulses, 0·2 ms duration, 1 Hz) of diaphragm muscle fibres isolated from normal and actively-sensitized guinea-pigs. Caffeine, theophylline and theobromine increased, in a concentration-dependent manner (50–500 μm), twitch tension in normal and sensitized diaphragm. Caffeine (500 μm) enhanced contractility to a greater extent than theophylline or theobromine. Twitch potentiation by caffeine (500 μm) was significantly greater in sensitized diaphragm. Verapamil (0·1–100 μm) did not alter twitch contractions in the absence or presence of alkylxanthines in normal or …

Aclidinium Partially Prevents Human Lung Fibroblast Activation In Vitro

MUC4 is overexpressed in idiopathic pulmonary fibrosis and collaborates with transforming growth factor β inducing fibrotic responses.

Several mucins are implicated in idiopathic pulmonary fibrosis (IPF); however, there is no evidence regarding the role of MUC4 in the development of IPF. Here we demonstrated that MUC4 was overexpressed in IPF patients (n = 22) compared with healthy subjects (n = 21) and located in pulmonary arteries, bronchial epithelial cells, fibroblasts, and hyperplastic alveolar type II cells. Decreased expression of MUC4 using siRNA–MUC4 inhibited the mesenchymal/myofibroblast transformations of alveolar type II A549 cells and lung fibroblasts, as well as cell senescence and fibroblast proliferation induced by TGF-β1. The induction of the overexpression of MUC4 increased the effects of TGF-β1 on mesen…

Evaluation of Mucociliary Clearance by Three Dimension Micro-CT-SPECT in Guinea Pig: Role of Bitter Taste Agonists

Different image techniques have been used to analyze mucociliary clearance (MCC) in humans, but current small animal MCC analysis using in vivo imaging has not been well defined. Bitter taste receptor (T2R) agonists increase ciliary beat frequency (CBF) and cause bronchodilation but their effects in vivo are not well understood. This work analyzes in vivo nasal and bronchial MCC in guinea pig animals using three dimension (3D) microCT-SPECT images and evaluates the effect of T2R agonists. Intranasal macroaggreggates of albumin-Technetium 99 metastable (MAA-Tc99m) and lung nebulized Tc99m albumin nanocolloids were used to analyze the effect of T2R agonists on nasal and bronchial MCC respecti…

Cigarette smoke exposure up-regulates endothelin receptor B in human pulmonary artery endothelial cells: molecular and functional consequences

BACKGROUND AND PURPOSE Pulmonary arteries from smokers and chronic obstructive pulmonary disease patients show abnormal endothelium-dependent vascular reactivity. We studied the effect of cigarette smoke extract (CSE) on endothelin receptor B (ETB) expression in human pulmonary artery endothelial cells (HPAECs) and its role in endothelial dysfunction. EXPERIMENTAL APPROACH ETB receptor expression was measured by real time RT-PCR, Western blot and immunofluorescence. Cell contraction, intracellular Ca2+, F/G-actin, RhoA activity, myosin light chain phosphorylation, ET, NO, thromboxane (Tx)A2 and reactive oxygen species (ROS) were measured by traction microscopy, fluorescence microscopy, phal…

Cigarette smoke-induced pulmonary endothelial dysfunction is partially suppressed by sildenafil.

Abstract Cigarette smoke mediated oxidative stress and endothelial dysfunction are important processes in the pathogenesis of several lung disorders. In this study we evaluated the effect of PDE5 inhibition on pulmonary artery endothelial dysfunction induced by cigarette smoke in vitro . Human pulmonary artery endothelial cells (HPAEC) were incubated in the absence or presence of PDE5 inhibitor sildenafil (10 nM–1 μM), PKG agonist 8-Br-cGMP (1 mM), or the antioxidants dyphenyleneiodonium (DPI 1 μM) and N -acetylcysteine (NAC 1 mM) for 30 min. Then, cigarette smoke extract (CSE) was added for 24 h. CSE (2.5–10%)-induced ROS generation was suppressed by DPI, and partially reversed by sildenaf…

Cigarette Smoke-Induced Fibroblast Activation Is Attenuated By Aclidinium In Vitro

β-Adrenoceptor stimulation up-regulates phosphodiesterase 4 activity and reduces prostaglandin E 2 -inhibitory effects in human neutrophils

Human neutrophils were treated for 4 h with a combination of salbutamol (1 µM), a β2-adrenoceptor agonist, and rolipram (30 µM), a selective phosphodiesterase 4 inhibitor, to investigate whether this treatment produces up-regulation of phosphodiesterase activity with functional consequences. Anion-exchange chromatography coupled with the use of selective activators and inhibitors demonstrated that a phosphodiesterase activity with characteristics of the isoenzyme type 4 was increased in drug-treated cells. Kinetic analysis showed a ~1.5-fold increase in V max without alteration of K m values. The augmented phosphodiesterase activity in drug-treated cells was abolished by actinomycin D. Cycl…

Mucin 1 deficiency mediates corticosteroid insensitivity in asthma

BACKGROUND: The loss of corticosteroid efficacy is an important issue in severe asthma management and may lead to poor asthma control and deterioration of airflow. Recent data indicate that Mucin 1 (MUC1) membrane mucin can mediate corticosteroid efficacy in chronic rhinosinusitis, but the role of MUC1 in uncontrolled severe asthma is unknown. The objective was to analyze the previously unexplored role of MUC1 on corticosteroid efficacy in asthma. METHODS: Mucin 1 expression was evaluated by real-time PCR in human bronchial epithelial cells (HBEC) and blood neutrophils from uncontrolled severe asthma (n=27), controlled mild asthma (n=16), and healthy subjects (n=13). IL-8, MMP9, and GM-CSF …

Arterial and Venous Endothelia Display Differential Functional Fractalkine (CX 3 CL1) Expression by Angiotensin-II

Objective— Angiotensin-II (Ang-II) promotes the interaction of mononuclear cells with arterioles and neutrophils with postcapillary venules. To investigate the mechanisms underlying this dissimilar response, the involvement of fractalkine (CX 3 CL1) was explored. Methods and Results— Enhanced CX 3 CL1 expression was detected in both cremasteric arterioles and postcapillary venules 24 hours after Ang-II intrascrotal injection. Arteriolar leukocyte adhesion was the unique parameter significantly reduced (83%) in animals lacking CX 3 CL1 receptor (CX 3 CR1). Human umbilical arterial and venous endothelial cell stimulation with 1 μmol/L Ang-II increased CX 3 CL1 expression, yet neutralization …

CLONIDINE, A PARTIAL AGONIST OF THE HISTAMINERGIC H2 RECEPTORS?

Changes inα-tocopherol and retinol levels during cardiopulmonary bypass correlate with maximal arterial partial pressure of oxygen

Cardiopulmonary bypass (CPB) is associated with oxidative stress. This study examined antioxidant levels in adults undergoing CPB surgery and their correlation with clinical variables. Arterial blood samples were obtained from 27 patients undergoing CPB. The time-course variation of vitamin C (spectrofluorimetry), alpha-tocopherol and retinol (HPLC) levels were determined. Plasma vitamin C rose initially but gradually decayed during reperfusion until 60% reduction of baseline values post-surgery. alpha-Tocopherol and retinol were reduced along CPB with post-operative values approximately 25% lower than baseline. No significant changes were found for selenium and glutathione peroxidase. PaO(…

Retinoid X receptor agonists impair arterial mononuclear cell recruitment through peroxisome proliferator-activated receptor-γ activation.

Abstract Mononuclear cell migration into the vascular subendothelium constitutes an early event of the atherogenic process. Because the effect of retinoid X receptor (RXR)α on arterial mononuclear leukocyte recruitment is poorly understood, this study investigated whether RXR agonists can affect this response and the underlying mechanisms involved. Decreased RXRα expression was detected after 4 h stimulation of human umbilical arterial endothelial cells with TNF-α. Interestingly, under physiological flow conditions, TNF-α–induced endothelial adhesion of human mononuclear cells was concentration-dependently inhibited by preincubation of the human umbilical arterial endothelial cells with RXR…

Effect of serotonin and calcium in normal and sensitized guinea pig isolated trachea

Tracheal strips from normal and actively sensitized guinea pigs were studied to determine the responses to serotonin (5-hydroxytryptamine, 5-HT ; 1 nM - 0.1 mM) and ouabain (0.1 μM - 0.1 mM), and the effects of increasing the extracellular calcium (Ca o ) concentration on tonic contractions elicited by 5-HT. Sensitized trachea exhibited an increased responsiveness and sensitivity to 5-HT and ouabain. Increases in Ca o to achieve final concentrations of 5, 10 and 20 mM caused concentration-related relaxations of normal and sensitized tissues contracted to a similar plateau level with 5-HT. Inhibition of the Na + /K + -ATPase by ouabain (10 μM) reversed the effects of Ca o from relaxation to …

MUC4 impairs the anti-inflammatory effects of corticosteroids in patients with chronic rhinosinusitis with nasal polyps

Background Current evidence suggests that membrane-tethered mucins could mediate corticosteroid efficacy, interacting with glucocorticoid receptor (GR) in patients with chronic rhinosinusitis with nasal polyps (CRSwNP). Mucin 4 (MUC4)–tethered mucin is expressed in nasal polyp (NP) epithelial cells and upregulated under inflammatory conditions. Moreover, MUC4β has the capacity to interact with other intracellular proteins. We hypothesized that MUC4 modulates corticosteroid efficacy of patients with CRSwNP. Objective We sought to analyze the role of MUC4 in corticosteroid effectiveness in different cohorts of patients with CRSwNP and elucidate the possible mechanisms involved. Methods Eighty…

Effects of selective phosphodiesterase inhibitors on platelet-activating factor- and antigen-induced airway hyperreactivity, eosinophil accumulation, and microvascular leakage in guinea pigs.

There is currently interest in the potential use of selective inhibitors of cyclic nucleotide phosphodiesterases (PDE) in the treatment of asthma. In this study we examined the effects of three selective PDE inhibitors, milrinone (PDE III), rolipram (PDE IV) and zaprinast (PDE V), on the broncoconstriction produced by antigen and histamine, the airway hyperreactivity and microvascular leakage after aerosol exposure to platelet-activating factor (PAF) and antigen, and the antigen-induced eosinophil infiltration in guinea-pig lung. Inhaled rolipram (0.01-10 mg ml-1) inhibited dose dependently the bronchospasm produced by aerosol antigen (5 mg ml-1) an anaesthetised, ventilated guinea-pigs. Ro…

Roflumilast N-Oxide Partially Prevents The Loss Of Ciliated Human Airway Epithelial Cells Secondary To Incubation With Cigarette Smoke Extracts In Vitro

LATE-BREAKING ABSTRACT: MUC1 downregulation induces corticosteroid resistance in asthma'sin vitroandin vivomodels

Background: Corticosteroids are in the first-line of Asthma treatment. However a subset of asthmatic patients is insensitive to anti-inflammatory effects of corticosteroids. Our previous data demonstrated that MUC1 downregulation correlates corticosteroid resistance in Nasal Polyposis and COPD. We hypothesized that MUC1 is also involved in corticosteroid resistance in Asthma. Objective: To analyze the role of MUC1 in the corticosteroid effectiveness in Asthma9s in vitro and in vivo models. Methods: 20 patients with steroid resistant Asthma were recruited. The expression of MUC1 was determined by real-time PCR in inflammatory cells from peripheral blood. HMVEC-L and U937 cells knockdown with…

Oxidative stress-induced glucocorticoid resistance is prevented by dual PDE3/PDE4 inhibition in human alveolar macrophages

Summary Background Oxidative stress is present in airway diseases such as severe asthma or Chronic Obstructive Pulmonary Disease and contributes to the low response to glucocorticoids through the down-regulation of histone deacetylase (HDAC) activity. Objective To study the effects of the phosphodiesterase (PDE)-3 and 4 inhibitors and their combination vs. glucocorticoids in a model of lipopolysaccharide (LPS)-induced cytokine release in alveolar macrophages under oxidative stress conditions. Methods Differentiated U937 or human alveolar macrophages were stimulated with H2O2 (10–1000 μm) or cigarette smoke extract (CSE, 0–15%) for 4 h before LPS (0.5 μg/mL, 24 h) addition. In other experime…

PDE4 inhibitors as new anti-inflammatory drugs: effects on cell trafficking and cell adhesion molecules expression.

Phosphodiesterase 4 (PDE4) is a major cyclic AMP-hydrolyzing enzyme in inflammatory and immunomodulatory cells. The wide range of inflammatory mechanisms under control by PDE4 points to this isoenzyme as an attractive target for new anti-inflammatory drugs. Selective inhibitors of PDE4 have demonstrated a broad spectrum of anti-inflammatory activities including the inhibition of cellular trafficking and microvascular leakage, cytokine and chemokine release from inflammatory cells, reactive oxygen species production, and cell adhesion molecule expression in a variety of in vitro and in vivo experimental models. The initially detected side effects, mainly nausea and emesis, appear at least pa…

Role of adenylate kinase type 7 expression on cilia motility: possible link in primary ciliary dyskinesia.

Background Adenylate kinase 7 (AK7) mediates the reaction 2ADP ↔ ATP + AMP, providing energy for the beating of cilia. A study recently showed that AK7 expression may be correlated with the primary ciliary dyskinesia (PCD) phenotype in mice. In this study, we characterized AK7 expression in vitro in an air–liquid interface (ALI) model and in middle nasal turbinate biopsy specimens from a cohort of patients with PCD to elucidate whether AK7 expression is correlated with ciliary malfunction. Methods AK7 expression was measured by real-time reverse-transcription polymerase chain reaction and Western blotting. In vitro differentiated nasal human epithelial cell siRNA experiments were performed …

Sphingosine-1-phosphate increases human alveolar epithelial IL-8 secretion, proliferation and neutrophil chemotaxis

Sphingosine-1-phosphate (S1P) has been presented recently as a pro-inflammatory agent in the airway epithelium since S1P levels are increased in bronchoalveolar lavage fluid of human asthmatics. However, the effects of S1P over the alveolar epithelium and neutrophil interactions are poorly understood. Here, we show that S1P increased interleukin 8 (IL-8) gene expression and protein secretion and proliferation in alveolar epithelial cells A549 at physiological concentrations (1 microM). At the same time, S1P increased intracellular Ca2+ concentration (potency 17.91 microM, measured by epifluorescence microscopy), phospholipase D (PLD) activity (measured by chemiluminiscence method) and extra…

Roflumilast N-oxide combined with PI3Kδ inhibitor improves the phenotype of early-onset COPD

Background: Severe, early-onset COPD (Chronic Obstructive Pulmonary Disease) is characterized by a rapid decline in lung function at an early age with neutrophil over-activation. Roflumilast is approved as treatment for moderate and severe COPD at risk of exacerbation as add on therapy. Recent evidence indicates that the combination of PDE4 and PI3Kδ inhibitors show synergic anti-inflammatory properties. Objectives: To explore the effects from adding a selective PI3Kδ inhibitor to roflumilast N-oxide (RNO) in neutrophils isolated from peripheral blood of severe, early-onset COPD patients in in vitro models. Methods: Neutrophils were isolated from peripheral blood of 20 severe, early-onset C…

Direct effect of cigarette smoke on human pulmonary artery tension.

The effect of chronic cigarette smoke on pulmonary artery (PA) tension has been studied extensively; nevertheless, the direct effect of cigarette smoke is poorly understood. We investigated the direct effect of cigarette smoke extract (CSE) on PA tension in non-smokers, smokers, and COPD patients in vitro. PA samples from 35 patients who underwent lung resection were examined by measuring isometric tension in response to increasing serotonin concentrations. CSE dose dependently inhibited the response to serotonin in smokers and COPD patients, and to a lesser extent in non-smokers. CSE-induced relaxation was similarly inhibited by the nonspecific nitric oxide synthase (NOS) inhibitor l-NOARG…

Arachidonic acid relaxes human pulmonary arteries through K+ channels and nitric oxide pathways.

We aimed to investigate the role of K(+) channels and nitric oxide (NO) on the relaxant effects of arachidonic acid in the human intralobar pulmonary arteries. Arachidonic acid produced a concentration-dependent relaxation (E(max)=93+/-3% of maximal relaxation induced by papaverine 0.1 mM;-log EC(30)=7.03+/-0.09) that was antagonized by the cyclooxygenase inhibitor indomethacin (1 microM), by the combination of cyclooxygenase blockade and cytochrome P450 (CYP) blockade with 17-octadecynoic acid (17-ODYA, 10 microM), by the combination of cyclooxygenase inhibition and NO synthase (NOS) inhibition with N(omega)-nitro-l-arginine (l-NOARG, 100 microM), by the simultaneous inhibition of CYP and …

Roflumilast, a phosphodiesterase 4 inhibitor, alleviates bleomycin-induced lung injury

Mandarin translation of abstract Background and purpose:  The effects of a phosphodiesterase 4 (PDE4) inhibitor, roflumilast, on bleomycin-induced lung injury were explored in ‘preventive’ and ‘therapeutic’ protocols and compared with glucocorticoids. Experimental approach:  Roflumilast (1 and 5 mg·kg−1·d−1, p.o.) or dexamethasone (2.5 mg·kg−1·d−1, p.o.) was given to C57Bl/6J mice from day 1 to 14 (preventive) or day 7 to 21 (therapeutic) after intratracheal bleomycin (3.75 U·kg−1). In Wistar rats, roflumilast (1 mg·kg−1·d−1, p.o.) was compared with methylprednisolone (10 mg·kg−1·d−1, p.o.) from day 1 to 21 (preventive) or from day 10 to 21 (therapeutic), following intratracheal instillatio…

Bronchodilator and anti-inflammatory activities of glaucine:In vitrostudies in human airway smooth muscle and polymorphonuclear leukocytes

Selective phosphodiesterase 4 (PDE4) inhibitors are of potential interest in the treatment of asthma. We examined the effects of the alkaloid S-(+)-glaucine, a PDE4 inhibitor, on human isolated bronchus and granulocyte function. Glaucine selectively inhibited PDE4 from human bronchus and polymorphonuclear leukocytes (PMN) in a non-competitive manner (Ki=3.4 μM). Glaucine displaced [3H]-rolipram from its high-affinity binding sites in rat brain cortex membranes (IC50∼100 μM). Glaucine inhibited the spontaneous and histamine-induced tone in human isolated bronchus (pD2∼4.5). Glaucine (10 μM) did not potentiate the isoprenaline-induced relaxation but augmented cyclic AMP accumulation by isopre…

Biotransformation in vitro of the 22R and 22S epimers of budesonide by human liver, bronchus, colonic mucosa and skin.

The pharmacological effects of glucocorticoids are greatly influenced by their pharmacokinetic properties. In the present report, the in vitro biotransformation of the 22R and 22S epimers of the topical steroid budesonide was studied in the S-9 fraction of human liver, bronchus, skin and colonic mucosa. The disappearance of unchanged epimers of budesonide was measured during 90 min of incubation by high performance liquid chromatography. The rate of disappearance was high in human liver while little biotransformation occurred in bronchial tissue and colonic mucosa, and none was detected in the skin. A marked decay of the initial concentration of unchanged budesonide epimers was noticed afte…

Bafetinib inhibits functional responses of human eosinophils in vitro

Eosinophils play a prominent role in the process of allergic inflammation. Non-receptor associated Lyn tyrosine kinases generate key initial signals in eosinophils. Bafetinib, a specific Abl/Lyn tyrosine kinase inhibitor has shown a potent antiproliferative activity in leukemic cells, but its effects on eosinophils have not been reported. Therefore, we studied the effects of bafetinib on functional and mechanistic responses of isolated human eosinophils. Bafetinib was more potent than non-specific tyrosin kinase comparators genistein and tyrphostin inhibiting superoxide anion triggered by N-formyl-Met-Leu-Phe (fMLF; 100 nM) (−log IC50=7.25±0.04 M; 6.1±0.04 M; and 6.55±0.03 M, respectively).…

Calcium dependence of the contraction produced by endothelin (ET-1) in isolated guinea-pig trachea.

Endothelin (ET-1, 1 pM to 0.1 microM) produced a concentration-dependent contraction of isolated guinea-pig trachea. BAY K 8644 (1 microM) did not significantly alter the concentration-response curve for ET-1. Incubation with nicardipine (10 microM) partly inhibited responses to low concentrations (10 pM to 1 nM) of ET-1 while verapamil (10 microM) and diltiazem (10 microM) were ineffective. La3+ (10 microM) and Cd2+ (10 microM) preferentially depressed the responses evoked by high concentrations (30 nM-0.1 microM) of ET-1 without affecting the responses evoked by low concentrations of the peptide. Incubation in Ca2(+)-free (with EDTA, 1 mM) medium resulted in suppression of the responses e…

Contributions of ACE and mast cell chymase to endogenous angiotensin II generation and leucocyte recruitment in vivo

Aims In vitro studies suggest that mast cell chymase (MCP) is more important than angiotensin-converting enzyme (ACE) for generating angiotensin II (Ang II) within the cardiovascular system. We investigated in vivo the relative contributions of ACE and MCP to leucocyte recruitment induced by endogenously generated Ang II. Methods and results Exposure of the murine cremasteric microcirculation of C57BL/6 mice to Ang I (100 nM for 4 h) induced leucocyte–endothelium interactions. Either losartan (an Ang II receptor-1 antagonist, AT1) or enalapril (an ACE inhibitor), but not chymostatin (a chymase inhibitor), inhibited Ang I-induced responses. Mast cell degranulation with compound 48/80 (CMP48/…

Functional, biochemical and molecular biological evidence for a possible β3-adrenoceptor in human near-term myometrium

The possible existence of a β3-adrenoceptor (β3-AR) in human near-term myometrium was investigated by in vitro functional and biochemical studies and analysis of mRNA expression. SR 59119A and SR 59104A and CGP 12177 (two selective agonists and a partial agonist, respectively, of the β3-AR), salbutamol and terbutaline (β2-AR agonists) each produced a concentration-dependent relaxation of the myometrial spontaneous contractions. There were no differences in pD2 values for the relaxing potencies of terbutaline, salbutamol, CGP 12177 and SR 59119A. The rank order for their relaxing efficacies was SR 59119A>SR 59104A>terbutaline∼salbutamol∼CGP 12177 (Emax=52±7%, 42±12% and ∼ 30% respectively). …

Selective Inhibition of Phosphodiesterases 4A, B, C and D Isoforms in Chronic Respiratory Diseases: Current and Future Evidences

Chronic respiratory diseases affect millions of people every day. According to the World Health Organization estimates, ~235 million people suffer from asthma, ~64 million suffer from chronic obstructive pulmonary disease (COPD), and millions more suffer from allergic rhinitis around the world. In recent last years, the first phosphodiesterase 4 (PDE4) inhibitor, roflumilast, was approved as a treatment to reduce the risk of exacerbations in stable and severe COPD associated with chronic bronchitis and a history of exacerbations. PDE4 exists as four subtypes (A, B, C, and D) each with a capacity to degrade cAMP, a second messenger involved in inflammatory responses. PDE4 inhibitors inhibit …

Effectiveness of oral N -acetylcysteine in a rat experimental model of asthma

Oxidative stress appears to be relevant to asthma pathogenesis. Therefore, the effectiveness of the antioxidant N -acetylcysteine was examined on antigen-induced pulmonary responses in sensitized Brown-Norway rats. N -acetylcysteine (oral, 1 mmol kg(-1)per day for 7 days before challenge) did not reduce the immediate bronchospasm that followed aerosol antigen exposure but prevented airway hyperreactivity to 5-hydroxytryptamine at 24 h after antigen challenge, and reduced the eosinophils (from 0.178 +/- 0.038 in the absence to 0.064 +/- 0.020 x10(6)cells ml(-1)in the presence of N -acetylcysteine;P< 0.05), and Evans blue dye extravasation in bronchoalveolar lavage fluid. Taurine levels in br…

Piclamilast inhibits the pro-apoptotic and anti-proliferative responses of A549 cells exposed to H(2)O(2) via mechanisms involving AP-1 activation.

Reactive oxygen species (ROS) are involved in the pathogenesis of many inflammatory diseases such as chronic obstructive pulmonary disease (COPD). They can alter the expression of genes involved in cellular damage by activating transcription factors, including the NF-κB and the activator protein 1 (AP-1). Phosphodiesterase type 4 (PDE4) inhibitors have anti-inflammatory and antioxidant effects, as described in in vivo and in vitro COPD models. This study analysed the effects of piclamilast, a selective PDE4 inhibitor, on modulating the global gene expression profile in A549 cells exposed to H(2)O(2).Changes in gene expression were analysed using high-density Affymetrix microarrays and valid…

Effect of boldine, secoboldine, and boldine methine on angiotensin II-induced neutrophil recruitment in vivo.

AbstractAngiotensin-II (Ang-II) has inflammatory activity and is involved in different diseases associated with the cardiovascular system. This study has evaluated the effect of boldine (B), and two phenanthrene alkaloids semisynthesized by us, secoboldine (SB) and boldine methine (BM), on Ang-II-induced neutrophil recruitment. Intraperitoneal administration of 1 nM Ang-II induced significant neutrophil accumulation, which was maximal at 4–8 h. BM inhibited neutrophil infiltration into the peritoneal cavity at 4 h and 8 h by 73% and 77%, respectively, SB at 8 h by 55%, and B had no effect on this response. Although BM inhibited the release of cytokine-inducible neutrophil chemoattractant/ke…

Histamine up-regulates phosphodiesterase 4 activity and reduces prostaglandin E2-inhibitory effects in human neutrophils.

Objective: To investigate whether histamine produces up-regulation of phosphodiesterase (PDE) activity with functional consequences in human peripheral blood neutrophils.¶Methods: PDE activity was studied by a radioisotopic method following anion-exchange chromatography. Reverse transcriptase-polymerase chain reaction (RT-PCR) was used for detection of mRNA transcripts of PDE4 subtypes. Cyclic AMP (cAMP) levels were measured by enzyme-immunoassay, and superoxide generation by cytochrome c reduction.¶Treatment: Neutrophils were incubated for 4 h with histamine (1 μM).¶Results: PDE4 was the only isoenzyme activity increased in treated neutrophils. Kinetic analysis showed a ∼1.5-fold increase …

Roflumilast Prevents the Metabolic Effects of Bleomycin-Induced Fibrosis in a Murine Model

Fibrotic remodeling is a process common to chronic lung diseases such as chronic obstructive pulmonary disease (COPD), pulmonary fibrosis, acute respiratory distress syndrome and asthma. Based on preclinical studies phosphodiesterase 4 (PDE4) inhibitors may exhibit beneficial anti-inflammatory and anti-remodeling properties for the treatment of these respiratory disorders. Effects of PDE4 inhibitors on changes in the lung metabolome in models of pulmonary fibrotic remodeling have not yet been explored. This work studies the effects of the PDE4 inhibitor roflumilast on changes in the lung metabolome in the common murine model of bleomycin-induced lung fibrosis by nuclear magnetic resonance (…

A small molecule, orally active, α 4 β 1 /α 4 β 7 dual antagonist reduces leukocyte infiltration and airway hyper-responsiveness in an experimental model of allergic asthma in Brown Norway rats

α4β1 and α4β7 integrins are preferentially expressed on eosinophils and mononuclear leukocytes and play critical roles in their recruitment to inflammatory sites. We investigated the effects of TR14035, a small molecule, α4β1/α4β7 dual antagonist, in a rat model of allergic asthma. Actively sensitized rats were challenged with aerosol antigen or saline on day 21, and the responses evaluated 24 and 48-h later. TR14035 (3 mg kg−1, p.o.) was given 1-h before and 4-h after antigen or saline challenge. Airway hyper-responsiveness to intravenous 5-hydroxytryptamine was suppressed in TR14035-treated rats. Eosinophil, mononuclear cell and neutrophil counts, and eosinophil peroxidase and protein con…

Is the beta3-adrenoceptor (ADRB3) a potential target for uterorelaxant drugs?

International audience; The management of premature birth still remains unsatisfactory. Since the relative lack of efficiency and/or safety of current tocolytic agents have been highlighted, it is necessary to develop new uterorelaxant drugs deprived of important maternal and foetal side effects. Our work reported in this review focuses on a potential new target for tocolytic drugs, the beta3-adrenoceptor (ADRB3). This third type of ADRB is shown to be present and functional in human myometrium. We demonstrated that ADRB3 agonists are able to inhibit in-vitro spontaneous contractions of myometrial strips, via a cyclic AMP-mediated pathway. Furthermore, we established that ADRB3 is the predo…

EFFECTS OF VANADATE ON RESPONSES OF GUINEA-PIG ISOLATED TRACHEA TO SPASMOGENS

Abstract The effects of vanadate on the contractility of the guinea-pig isolated trachea was examined. Vanadate (0·1 Mm) produced a sustained contraction that was abolished in Ca2+-free EGTA (0·1 Mm)-containing physiological salt solution but was resistant to verapamil (1 μm). Vanadate (0·1 Mm) depressed tracheal responses to CaCl2 (in Ca2+-free depolarizing solution), KCl, acetylcholine, histamine and 5-hydroxytryptamine. For vanadate (10 μm), the inhibition of spasmogenic responses only reached statistical significance for histamine and 5-hydroxytryptamine. Caffeine (1 Mm)-induced spasm (trachea at 20°C in the presence of indomethacin (2·8 μm)) was not affected by vanadate (10 μm-0·1 Mm).…

The JAK2 pathway is activated in idiopathic pulmonary fibrosis

Background: Idiopathic pulmonary fibrosis (IPF) is the most rapidly progressive and fatal fibrotic disorder, with no curative therapies. The signal transducer and activator of transcription 3 (STAT3) protein is activated in lung fibroblasts and alveolar type II cells (ATII), thereby contributing to lung fibrosis in IPF. Although activation of Janus kinase 2 (JAK2) has been implicated in proliferative disorders, its role in IPF is unknown. The aim of this study was to analyze JAK2 activation in IPF, and to determine whether JAK2/STAT3 inhibition is a potential therapeutic strategy for this disease. Methods and results: JAK2/p-JAK2 and STAT3/pSTAT3 expression was evaluated using quantitative …

The preclinical pharmacology of roflumilast--a selective, oral phosphodiesterase 4 inhibitor in development for chronic obstructive pulmonary disease

After more than two decades of research into phosphodiesterase 4 (PDE4) inhibitors, roflumilast (3-cyclopropylmethoxy-4-difluoromethoxy-N-[3,5-di-chloropyrid-4-yl]-benzamide) may become the first agent in this class to be approved for patient treatment worldwide. Within the PDE family of 11 known isoenzymes, roflumilast is selective for PDE4, showing balanced selectivity for subtypes A-D, and is of high subnanomolar potency. The active principle of roflumilast in man is its dichloropyridyl N-oxide metabolite, which has similar potency as a PDE4 inhibitor as the parent compound. The long half-life and high potency of this metabolite allows for once-daily, oral administration of a single, 500…

Role of endothelium in the responses to noradrenaline in normal and sensitized guinea-pig aorta

1. The pharmacological reactivity of sensitized blood vessels has been less studied than that of the airways. Aorta rings were obtained from normal (non-sensitized) and actively sensitized guinea-pigs, and prepared for isometric recording of tension changes. 2. Noradrenaline (10 nM-0.1 mM), histamine (0.1 microM-0.1 mM) and KCl (10-100 mM) produced concentration-related contractions of normal tissues. Removal of endothelium resulted in a marked leftward and upward shift of the concentration-response curve for noradrenaline but it did not alter histamine- or KCl-induced responses. Pretreatment with L-NG-nitroarginine (L-NOARG, 30 microM), haemoglobin (5 microM) or ibuprofen (10 microM) enhan…

Functional Profile Of Aclidinium Bromide In Isolated Human Bronchi And Left Atria

Roflumilast inhibits leukocyte-endothelial cell interactions, expression of adhesion molecules and microvascular permeability

Background and purpose: The present study addressed the effects of the investigational PDE4 inhibitor roflumilast on leukocyte-endothelial cell interactions and endothelial permeability in vivo and in vitro. Experimental approach: In vivo, intravital video-microscopy was used to determine effects of roflumilast p.o. on leukocyte-endothelial cell interactions and microvascular permeability in rat mesenteric venules. In vitro, the effects of roflumilast N-oxide, the active metabolite of roflumilast in humans, and other PDE4 inhibitors on neutrophil adhesion to tumour necrosis factor α (TNFα)-activated human umbilical vein endothelial cells (HUVEC), E-selectin expression and thrombin-induced e…

Contraction of human airways by oxidative stress

We examined the in vitro effects of tert-butylhydroperoxide (tBu-OOH) in human bronchial muscle. tert-Butylhydroperoxide produced concentration-dependent contractions of bronchial rings (maximum effect was 56.5 +/- 9.6% of contraction by 1 mM acetylcholine; effective concentration 50% was approximately 100 microM). tert-Butylhydroperoxide (0.5 mM)-induced contraction was enhanced by epithelial removal but abolished by indomethacin (cyclooxygenase inhibitor) and zileuton (lipoxygenase inhibitor). tert-Butylhydroperoxide produced a transient rise in intracellular calcium in human cultured airway smooth muscle cells (HCASMC). The bronchial reactivity to acetylcholine and histamine was not alte…

Mucus and MUC in asthma.

Asthma is characterized by chronic airway inflammation and a mucus hypersecretory phenotype comprising excess mucus secretion, goblet cell hyperplasia and submucosal gland hypertrophy. This augmented mucus secretion has been relatively undervalued in asthma compared with airway inflammation. However, mucus plugging contributes to airflow limitation and airway hyperresponsiveness, and to morbidity and mortality in asthma. We review recent contributions to this field and therapeutic avenues to control mucus hypersecretion.A distinct mucus hypersecretory phenotype may present in asthma. Overexpression of MUC5AC, MUC5B and MUC2 have been described in asthma secretions, but identification of def…

Protective effect of N-acetylcysteine on ischaemia-induced myocardial damage in canine heart.

The glutathione redox pathway is an important antioxidant system in the myocardium. N-Acetylcysteine is a low molecular weight glutathione precursor that has been used clinically to replenish glutathione stores. The present study was aimed at evaluating the protective effect of N-acetylcysteine on myocardial damage resulting from permanent coronary occlusion (without reperfusion) in anaesthetized dogs. N-Acetylcysteine (150 mg kg−1 i.v.) administered 2 min before occlusion rerduced infarct size in dogs subjected to 24 h ischemia. The infarct size as a percentage of the area at risk was 86.8 ± 3.6% (n = 11) in control (salinetreated) dogs and 68.2 ± 2.4% (n = 7; P < 0.05 vs control) in N-ace…

Effect of Glutathione on Canine Myocardial Ischaemia Without Reperfusion

Abstract The present study was to evaluate the effect of exogenous glutathione on myocardial damage resulting from permanent (no reperfusion) coronary ligation (3 or 6 h) in anaesthetized dogs. Haemodynamics, infarct size and myocardial glutathione content were determined. Erythrocyte superoxide dismutase (SOD) activity was also determined in coronary venous blood samples. Glutathione was administered by the intraperitoneal route, 100 mg kg−1 as initial dose given 5 min before coronary ligation, and successive doses of 25 mg kg−1 every 40 min throughout the study period. Saline-treated dogs showed myocardial infarction, a decrease in myocardial glutathione content, and a transient increase …

Diaphragmatic paralysis following minor cervical trauma.

Two asthmatic patients developed unilateral diaphragmatic paralysis from phrenic nerve injury, in one case following cervical chiropractic manipulation and in the other after a motorcycle accident. Both presented with increased dyspnea and orthopnea. Diagnosis, severity, and level of the lesion were established by neurophysiological methods, which are preferred to chest radiography and diaphragmatic ultrasonography. In spite of only partial electrophysiological recovery of the nerve, both patients were asymptomatic 1 year later.

Inhibitory effects of N-acetylcysteine on the functional responses of human eosinophils in vitro

Background Oxidative stress appears to be relevant in the pathogenesis of inflammation in allergic diseases like bronchial asthma. Eosinophils are oxidant-sensitive cells considered as key effectors in allergic inflammation. Objective The aim of this work was to study the effects of the clinically used antioxidant N-acetyl-L-cysteine (NAC) on the functional responses of human-isolated eosinophils. Methods Human eosinophils were purified from the blood of healthy donors by a magnetic bead separation system. The effects of NAC were investigated on the generation of reactive oxygen species (chemiluminescence and flow cytometry), Ca2+ signal (fluorimetry), intracellular glutathione (GSH; flow c…

Attenuation by oralN-acetylcysteine of bleomycin-induced lung injury in rats

Antioxidant therapy may be useful in diseases with impaired oxidant-antioxidant balance such as pulmonary fibrosis. This study examines the effect ofN-acetylcysteine (NAC) on bleomycin-induced lung fibrosis in rats.NAC (3 mmol·kg−1; oral) was given daily from 1 week prior to a single intratracheal instillation of bleomycin (2.5 U·kg−1) or saline, until 14 days postinstillation.NAC partially decreased the augmented collagen deposition in bleomycin-exposed rats (hydroxyproline content was 4,354±386 and 3,416±326 µg·lung−1in vehicle-treated and NAC-treated rats, respectively; p&lt;0.05). The histological assessment using a semiquantitative score showed less collagen deposition and inflammatory…

Effect of two phenanthrene alkaloids on angiotensin II-induced leukocyte-endothelial cell interactionsin vivo

The present study has evaluated the effect of two phenanthrene alkaloids, uvariopsine and stephenanthrine, on angiotensin II (Ang-II)-induced leukocyte–endothelial cell interactions in vivo and the mechanisms involved in their activity. Intravital microscopy within the rat mesenteric microcirculation was used. A 60 min superfusion with 1 nM Ang-II induced a significant increase in the leukocyte–endothelial cell interactions that were completely inhibited by 1 μM uvariopsine cosuperfusion. A lower dose of 0.1 μM significantly reduced Ang-II-induced leukocyte adhesion by 75%. When Ang-II was cosuperfused with 1 and 0.1 μM stephenanthrine, Ang-II-induced leukocyte responses were significantly …

Oxidative stress and pulmonary inflammation: pharmacological intervention with antioxidants.

Reactive oxygen and nitrogen species are generated by several inflammatory and structural cells of the airways. These oxidant species may have important effects on different lung cells as regulators of signal transduction, activators of key transcription factors, and modulators of gene expression and apoptosis. Thus, an increased oxidative stress accompanied by reduced endogenous antioxidant defences may have a role in the pathogenesis of a number of inflammatory pulmonary diseases including asthma. Although antioxidant drugs could play a useful role in the therapy of inflammatory lung diseases, their clinical impact is relatively modest at present. Rigorous clinical investigation with the …

Effects of ouabain on human bronchial muscle in vitro

The effects of ouabain, an inhibitor of the plasmalemmal Na(+)/K(+)-ATPase activity, were examined in human isolated bronchus. Ouabain produced concentration-dependent contraction with -logEC(50)=7.16+/-0.11 and maximal effect of 67+/-4% of the response to acetylcholine (1 mM). Ouabain (10 microM)-induced contraction was epithelium-independent and was not depressed by inhibitors of cyclooxygenase and lipoxygenase, antagonists of muscarinic, histamine H(1)-receptors and alpha-adrenoceptors, or neuronal Na(+) channel blockade. The inhibition of ouabain contraction in tissues bathed in K(+)-free medium, and the inhibition by ouabain of the K(+)-induced relaxation confirm that the contractile a…

Inhibitory effects of N-acetylcysteine on superoxide anion generation in human polymorphonuclear leukocytes.

Abstract It has been suggested that reactive oxygen species released by activated polymorphonuclear leukocytes (PMN) in man is one mechanism of tissue injury. Therapeutic action aimed at increasing antioxidant defence mechanisms is still a clinical challenge. This study examines the activity of N-acetylcysteine, a known antioxidant, in the protection of PMN exposed in-vitro to the chemoattractant peptide fMet-Leu-Phe (FMLP), the protein kinase C activator phorbol myristate acetate or the lipid peroxidation promoter t-butyl hydroperoxide. FMLP (3–300 nm) and phorbol myristate acetate (160 pm–160 nm) induced concentration-related superoxide anion generation. Pre-treatment with N-acetylcystein…

Relaxation of the isolated human internal anal sphincter by sildenafil.

Abstract Background Hypertonicity of the internal anal sphincter (IAS) appears to be involved in the pathogenesis of anal fissure. The relaxant effects of sildenafil, a selective phosphodiesterase 5 (PDE5) inhibitor, on isolated human IAS were investigated. Methods The efficacy (maximal effect, Emax) and potency (−log IC50, where IC50 is half-maximal inhibitory concentration) of the PDE5 inhibitors, sildenafil and zaprinast, and of nitric oxide donors, sodium nitroprusside and glyceryl trinitrate, as relaxants of histamine (0·1 mmol/l)-induced tone were examined in IAS strips under isometric contraction. The presence of PDE5 isoenzymes and changes in intracellular calcium and cyclic nucleot…

Hawthorn extract inhibits human isolated neutrophil functions.

Hawthorn extract is a popular herbal medicine given as adjunctive treatment for chronic heart failure. In contrast to the cardiac properties of hawthorn extract, its anti-inflammatory effect has been scarcely investigated. This study examines the effects of a dry extract of leaves and flowers of Crataegus laevigata on various functional outputs of human neutrophils in vitro. Incubation of human neutrophils obtained from peripheral blood of healthy donors with C. laevigata extract (0.75-250 microg/ml) inhibited N-formyl-Met-Leu-Phe (FMLP)-induced superoxide anion generation, elastase release and chemotactic migration with potency values of 43.6, 21.9, and 31.6 microg/ml, respectively. By con…

Aclidinium inhibits cigarette smoke-induced lung fibroblast-to-myofibroblast transition.

Cigarette smoking contributes to lung remodelling in chronic obstructive pulmonary disease (COPD). As part of this remodelling, peribronchiolar fibrosis is observed in the small airways of COPD patients and contributes to airway obstruction. Fibroblast-to-myofibroblast transition is a key step in peribronchiolar fibrosis formation. This in vitro study examined the effect of cigarette smoke on bronchial fibroblast-to-myofibroblast transition, and whether aclidinium bromide inhibits this process. Human bronchial fibroblasts were incubated with aclidinium bromide (10 −9 –10 −7 M) and exposed to cigarette smoke extract. Collagen type I and α-smooth muscle actin (α-SMA) expression were measured …

MUC1-CT mediates corticosteroid responses in COPD

Background: Corticosteroid resistance is an acquired condition in chronic obstructive pulmonary disease (COPD) patients and a challenge to develop new anti-inflammatory therapies. In previous reports we showed that cytoplasmic tail of the membrain tethered mucin 1 (MUC1-CT) interacts with glucocorticoid receptor (GR) mediating corticosteroid anti-inflammatory efficacy. Objectives: To analyze the role of MUC1-CT as a key marker of corticosteroid efficacy in COPD Methods: The expression of MUC1-CT and the anti-inflammatory role of dexamethasone were evaluated in neutrophils and bronchial epithelial cells from healthy and COPD patients. Anti-inflammatory effects of dexamethasone and glucocorti…

The relaxant effects of cromakalim (BRL 34915) on human isolated airway smooth muscle

Cromakalim (BRL 34915) is a potassium channel opener with therapeutic potential as a bronchodilator in asthma. Cromakalim (0.1–30 μmol/l) inhibited the spontaneous tone of human isolated bronchi n a concentration-related manner being nearly as effective as isoprenaline or theophylline. The order of relaxant potencies (expressed as -log10 IC50 mol/l; mean ±SEM) was isoprenaline (7.29 ± 0.27; n = 8) > cromakalim (5.89 ± 0.12; n = 7) > theophylline (4.07 ±0.13; n = 10). In human bronchi where tone had been raised by addition of histamine (0.1 mmol/l), acetylcholine (0.1 mmol/l) or leukotriene D4 (LTD4, 0.1 μmol/l), the relaxant effect of cromakalim was substantially reduced. Cromakalim suppres…

Aclidinium, a New Long-Acting Antimuscarinic, Inhibits Cholinergic and Cigarette Smoke-Induced Up Regulation of Mucin MUC5AC Expression in Human Airway Epithelial Cells

Effects of inhaled glaucine on pulmonary responses to antigen in sensitized guinea pigs.

The alkaloid (S)-(+)-1,2,9,10-tetramethoxyaporphine (glaucine) is a phosphodiesterase 4 inhibitor with bronchodilator and anti-inflammatory activity in vitro. In this study, we examined the in vivo effects of glaucine on an animal model of asthma. In ovalbumin sensitized guinea pigs, inhaled glaucine (10 mg ml(-1), 3 min) inhibited the acute bronchoconstriction produced by aerosol antigen (antigen response was 256+/-42 and 95+/-14 cm H(2)O l(-1) s(-1) in control and glaucine-treated animals, respectively; P<0.05). Pretreatment with glaucine (10 mg ml(-1), 10 min inhalation, 30 min pre- and 3 h post-antigen exposure) markedly reduced airway hyperreactivity to histamine, eosinophil lung accum…

N-acetyl-L-cysteine (NAC) inhibit mucin synthesis and pro-inflammatory mediators in alveolar type II epithelial cells infected with influenza virus A and B and with respiratory syncytial virus (RSV).

64% of chronic obstructive pulmonary disease (COPD) exacerbations are caused by respiratory infections including influenza (strains A and B) and respiratory syncytial virus (RSV). They affect the airway epithelium increasing inflammatory and apoptosis events through mechanisms involving ROS generation, and induce the release of mucins from epithelial cells that are involved in the deterioration of the patient's health during the course of the disease. The antioxidant NAC has proved useful in the management of COPD reducing symptoms, exacerbations and accelerated lung function decline. It has been shown to inhibit influenza virus replication and to diminish the release of inflammatory and ap…

Taurine and the Lung

Taurine (TAU) is the most abundant free amino acid in many tissues and in particular in proinflammatory cells like polymorphonuclear leukocytes and tissues exposed to elevated levels of oxidants1. Furthermore, orally administered TAU has been reported to reduce lung oxidant damage from exposure to ozone, nitrogen dioxide, paraquat, amiodarone and bleomycin in animal models2–5.

Halothane inhibits endothelium-dependent relaxation elicited by acetylcholine in human isolated pulmonary arteries.

This study examined whether a clinically relevant concentration of the volatile anaesthetic halothane modifies the endothelium-dependent relaxation produced by acetylcholine (3 nM-10 microM), histamine (1 pM-0.1 microM) and anti-human immunoglobulin E (1:1000) in human isolated pulmonary arteries submaximally precontracted with noradrenaline. An inhibitor of nitric oxide formation, N(G)-nitro-L-arginine (100 microM), attenuated acetylcholine-induced relaxation but failed to inhibit histamine- and anti-human immunoglobulin E-induced relaxation. Indomethacin (2.8 microM, a cyclooxygenase inhibitor) preferentially reduced the relaxation to histamine and anti-human IgE. Halothane (2%) significa…

Electrophysiologic evaluation of phrenic nerve and diaphragm function after coronary bypass surgery: Prospective study of diabetes and other risk factors

Objective Phrenic neuropathy after coronary artery bypass grafting has been related to various risk factors with conflicting results. The aim of this study was to assess the incidence, characteristics, and clinical consequences of phrenic neuropathy and the influence of diabetes and other risk factors. Methods We conducted an observational, prospective study of parallel groups including 94 consecutive patients subjected to coronary artery bypass grafting, half of them with diabetes and associated polyneuropathy . Electrophysiologic study of phrenic nerve conduction as the reference method, chest radiography, diaphragm ultrasound, and functional respiratory tests were performed 24 to 48 hour…

The In Vitro Pharmacological Profile Of LAS100977 - A Potent, Selective And Long-acting Beta-2 Receptor Agonist

Activation of PPARβ/δ inhibits leukocyte recruitment, cell adhesion molecule expression, and chemokine release

Abstract Activation of the nuclear receptor PPARb/d inhibits acute inflammatory responses in vitro with human primary cells and in vivo by targeting the endothelial cell-leukocyte interaction. The infiltration of PMNs into tissues is a prominent feature in inflammation. The mechanism underlying PMN recruitment depends on the release of chemotactic mediators and CAM expression on endothelial cells. The nuclear receptor PPARβ/δ is widely expressed in many tissues, including the vascular endothelium; however, its role in acute inflammation remains unclear. Using intravital microscopy in the mouse cremasteric microcirculation, we have shown that activation of PPARβ/δ by its selective ligand GW5…

Massive triiodothyronine intoxication: Efficacy of hemoperfusion?

A case of massive accidental triiodothyronine intoxication (1000-fold the usual therapeutic dose, for 8 days) is reported with important disturbances of cardiovascular and central nervous systems that required intensive care support. Serum free triiodothyronine levels were 4789 pmol L-1 on admittance (normal values, 3.5-6.5 pmol L-1). In the absence of a specific treatment, hemoperfusions were performed but failed to accelerate significantly the decay of blood levels of free triiodothyronine (apparent half-life 25.9 hours; 95% confidence interval: 19.8-37.4 hours). The patient, a young woman, made a satisfactory recovery, in spite of important clinical complications.

Effect of dantrolene sodium in isolated guinea-pig trachea.

The effect of dantrolene sodium (3 microM-0.3 mM) on the spontaneous tone and responses to various contractile agonists was studied in isolated guinea-pig trachea. Dantrolene produced a concentration-related inhibition of the spontaneous tracheal tone, reaching a value of 94.8 +/- 4.8% of the relaxation induced by caffeine 10 mM. Removal of the epithelium did not affect the dantrolene-induced relaxation. Dantrolene did not alter the concentration-response curve for KCl and produced only small displacements of the concentration-response curves for CaCl2, acetylcholine and histamine, without affecting their maximal effects. Dantrolene dose relatedly inhibited the contraction induced by caffei…

Effects of beta-adrenoceptor drug stimulation on various models of gastric ulcer in rats.

Abstract 1. Experiments were designed to evaluate the effect of the pharmacological activation of beta-adrenoceptors on various models of gastric ulcer in the rat. 2. Pretreatment with the beta-adrenoceptor stimulant drugs, isoprenaline or salbutamol, significantly inhibited stress-induced gastric ulcers. This anti-ulcer effect was abolished by propranolol but not by atenolol, suggesting that beta 2-adrenoceptors mediate this response. 3. In the pylorus-ligation model, salbutamol inhibited lesion formation and reduced the intragastric content of hydrogen ions, histamine and pepsin although the latter was only affected with the higher dose of salbutamol. 4. Salbutamol also prevented the ulce…

EFFECTS OF CROMAKALIM (BRL-34915) IN TRACHEA ISOLATED FROM ACTIVELY SENSITIZED GUINEA-PIGS

Abstract The effects of cromakalim were examined in tracheal strips isolated from normal (unsensitized) guinea-pigs and from animals actively sensitized to bovine serum albumin. Sensitized tracheae exhibited hyper-responsiveness to KCl, acetylcholine and histamine. In normal and sensitized tracheae, cromakalim (0·01–10 μm) produced a concentration-related suppression of spontaneous tone. The ability of cromakalim to relax tracheal strips was reduced when tone was raised by KCl (25 Mm), acetylcholine (0·1 Mm) or histamine (0·1 Mm) and lost against KCl (120 Mm)-induced spasm. Procaine (5 Mm) abolished the relaxant effect of cromakalim whilst tetraethylammonium (8 Mm) was without effect. These…

Effects of fenspiride on human bronchial cyclic nucleotide phosphodiesterase isoenzymes: functional and biochemical study.

We have investigated the role of human bronchial cyclic nucleotide phosphodiesterases in the effects of fenspiride, a drug endowed with bronchodilator and anti-inflammatory properties. Functional studies on human isolated bronchi showed that fenspiride (10(-6)-3 x 10(-3) M, 30 min) induced a shift to the left of the concentration-response curves for isoprenaline and sodium nitroprusside with -logEC50 values of 4.1+/-0.1 (n = 7) and 3.5+/-0.2 (n = 8), respectively. Biochemical studies were carried out on three human bronchi in which separation of cyclic nucleotide phosphodiesterase isoenzymes was performed by ion exchange chromatography followed by determination of phosphodiesterase activity…

Characterization of 5-HT receptors on human pulmonary artery and vein: functional and binding studies

1. This study aimed to investigate the 5-hydroxytryptamine (5-HT) receptors mediating contraction of ring preparations isolated from human pulmonary arteries and veins. In functional studies, the responses to 5-HT, sumatriptan, ergotamine, serotonin-O-carboxymethyl-glycyl-tyrosinamide (SCMGT), alpha-methyl 5-HT (alpha-Me) and 2-methyl 5-HT (2-Me) were studied with WAY100635, GR127935, ritanserin, zacopride and SB204070 as antagonists. 2. All agonists produced concentration-dependent contractions of human pulmonary artery and vein preparations. The order of potency (-log ECS0 values) was ergotamine (6.88) > 5-HT (6.41) > or = SCMGT (6.20) = sumatriptan (6.19) > or = alpha-Me (6.04) in the ar…

Relaxation by β3-adrenoceptor agonists of the isolated human internal anal sphincter

In this study, responses of beta(3)-adrenoceptor agonists were examined on human isolated internal anal sphincter (IAS) in order to explore their relaxant effects on hypertonicity of IAS.The relaxant efficacy (E(max)) and potency (-logIC(50)) of BRL37344 and SR58611A, beta(3)-adrenoceptor agonists, were examined in contracted IAS muscle strips. The presence of beta(3)-adrenoceptors, and changes in intracellular calcium and cyclic nucleotide levels in IAS muscle were tested by Western blotting, epifluorescence microscopy and enzyme immunoassay, respectively.BRL37344 and SR58611A relaxed contracted IAS muscle (E(max)=27+/-3% and 35+/-3%; -logIC(50)=6.26+/-0.24 and 4.87+/-0.13; respectively). …

Bronchodilator and anti-inflammatory activities of SCA40: studies in human isolated bronchus, human eosinophils, and in the guinea-pig in vivo.

There is currently interest in the use of inhibitors of cyclic nucleotide phosphodiesterases (PDE) as potential anti-asthma agents. In this study we examined the effects of SCA40 (6-bromo-8-methylaminoimidazol-[1,2-a] pyrazine-2-carbonitrile), a preferential inhibitor of PDE 3 also endowed with PDE 4 and 5 inhibitory activities, on isolated bronchus and eosinophil functions and in an animal model of asthma. SCA40 (1 nM-0.1 mM) produced concentration-dependent inhibition of spontaneous and stimulated tone of human isolated bronchus and reached a maximal relaxation similar to that of theophylline (3 mM). The potency (-log EC50 values) of SCA40 against spontaneous tone (6.52 +/- 0.10) was grea…

Effects of SCA40 on human isolated bronchus and human polymorphonuclear leukocytes: comparison with rolipram, SKF94120 and levcromakalim

1. SCA40 (0.1 nM-0.1 mM) produced concentration-dependent suppression of the spontaneous tone of human isolated bronchus (-log EC50 = 6.85 +/- 0.09; n = 10) and reached a maximal relaxation similar to that of theophylline (3 mM). The potency (-log EC50 values) of SCA40 compared to other relaxants was rolipram (7.44 +/- 0.12; n = 9) > SCA40 > or = levcromakalim (6.49 +/- 0.04; n = 6) > SKF94120 (5.87 +/- 0.10; n = 9). 2. When tested against the activity of the isoenzymes of cyclic nucleotide phosphodiesterase (PDE) isolated from human bronchus, SCA40 proved highly potent against PDE III (-log IC50 = 6.47 +/- 0.16; n = 4). It was markedly less potent against PDE IV (4.82 +/- 0.18; n = 4) and …

Pharmacological and biochemical study on the effects of selective phosphodiesterase inhibitors on human term myometrium

This study was aimed at evaluating the in vitro effects of phosphodiesterase inhibitors and beta2-adrenoceptor agonists on spontaneous contractions of human term myometrium. Rolipram, RP 73401 (3-cyclopentyloxy-N-(3,5(-dichloro-4-pyridil)-4-methoxybenzamide) and Ro 20-1724 (1-4-(3-butoxy-4-methoxybenzyl)-2-imidozolidinone) (phosphodiesterase 4 inhibitors) inhibited spontaneous myometrial contractions (Emax approximately 100%; pD2 of 6.80+/-0.28, 6.84+/-0.32 and 6.31+/-0.03, respectively). Salbutamol and formoterol were less effective (Emax=40+/-6% and 35+/-12%, respectively) than phosphodiesterase 4 inhibitors to reduce myometrial contractility. Inhibitors of phosphodiesterase 3 (milrinone …

Effects of phorbol 12,13-diacetate on human isolated bronchus

Protein kinase C appears to be involved in the regulation of airway contractility. Phorbol 12,13-diacetate (PDA; 0.01-10 microM), a protein kinase C activator, produced a transient relaxation followed by a sustained contraction of human isolated bronchus. Different protein kinase C inhibitors (calphostin C, staurosporine and 1-(5-isoquinolinesulfonyl)-2-methylpiperazine) (H-7), nifedipine (NIF; 1 microM) or incubation with Ca(2+)-free medium, inhibited the spasmogenic response to phorbol, while ouabain (10 microM) suppressed only the initial relaxation. These results indicate that the initial relaxation, in response to PDA, is related to the activation of Na(+)/K(+)-ATPase, while the ensuin…