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Spleen tyrosine kinase (SYK) is a key activator of signaling pathways downstream of multiple surface receptors implicated in asthma. SYK function has been extensively studied in mast cells downstream of the high-affinity IgE receptor, FcεR1. Preclinical studies have demonstrated a role for SYK in models of allergic inflammation, but a role in airway constriction has not been demonstrated. Here, we have used a potent and selective pharmacological inhibitor of SYK to determine the role of SYK in allergen-mediated inflammation and airway constriction in preclinical models. Attenuation of allergic airway responses was evaluated in a rat passive anaphylaxis model and rat and sheep inhaled allergen challenge models, as well as an ex vivo model of allergen-mediated airway constriction in rats and cynomolgus monkeys. Pharmacological inhibition of SYK dose-dependently blocked IgE-mediated tracheal plasma extravasation in rats. In a rat ovalbumin-sensitized airway challenge model, oral dosing with an SYK inhibitor led to a dose-dependent reduction in lung inflammatory cells. Ex vivo analysis of allergen-induced airway constriction in ovalbumin-sensitized brown Norway rats showed a complete attenuation with treatment of a SYK inhibitor, as well as a complete block of allergen-induced serotonin release. Similarly, allergen-mediated airway constriction was attenuated in ex vivo studies from nonhuman primate lungs. Intravenous administration of an SYK inhibitor attenuated both early- and late-phase allergen-induced increases in airway resistance in an Ascaris-sensitive sheep allergen challenge model. These data support a key role for SYK signaling in mediating allergic airway responses.

Accumulating evidence indicates protective actions of mineralocorticoid antagonists (MR antagonists) on cardiovascular pathology, which includes blunting vascular inflammation and myocardial fibrosis. We examined the anti-inflammatory and anti-fibrotic potential of MR antagonists in rodent respiratory models. In an ovalbumin allergic and challenged Brown Norway rat model, the total cell count in nasal lavage was 29,348±5451, which was blocked by spironolactone (0.3–60 mg/kg, p.o.) and eplerenone (0.3–30 mg/kg, p.o.). We also found that MR antagonists attenuated pulmonary inflammation in the Brown Norway rat. A series of experiments were conducted to determine the actions of MR blockade in acute/chronic lung injury models. (1) Ex vivo lung slice rat experiments found that eplerenone (0.01 and 10 µM) and spironolactone (10 µM) diminished lung hydroxyproline concentrations by 55±5, 122±9, and 83±8%. (2) In in vivo studies, MR antagonists attenuated the increases in bronchioalveolar lavage (BAL) neutrophils and macrophages caused by lung bleomycin exposure. In separate studies, bleomycin (4.0 U/kg, i.t.) increased lung levels of hydroxyproline by approximately 155%, which was blocked by spironolactone (10–60 mg/kg, p.o.). In a rat Lipopolysaccharide (LPS) model, spironolactone inhibited acute increases in BAL cytokines with moderate effects on neutrophils. Finally, we found that chronic LPS exposure significantly increased end expiratory lung and decreased lung elastance in the mouse. These functional effects of chronic LPS were improved by MR antagonists. Our results demonstrate that MR antagonists have significant pharmacological actions in the respiratory system.

Morphology, orientation and diffraction patterns of δ-Ni2Si precipitates in a Cu–Ni–Si alloy are investigated by TEM and HREM. Many disk-shaped nanosized δ-Ni2Si precipitates are uniformly distributed in Cu–1.5 wt%Ni–0.34 wt%Si alloy in an over-aging condition. The δ-Ni2Si precipitates preserve an orthogonal structure with a = 0.708 ± 0.005 nm, b = 0.504 ± 0.005 nm, and c = 0.364 ± 0.005 nm. The orientation relationships of six variants of δ-Ni2Si precipitates with Cu-matrix can be determined as (1 0 0)p||{0 0 1}Cu, [1 0 0]p||〈1 1 0〉Cu and [0 1 0]p||〈1 1 0〉Cu. Two typical diffraction pattern models under [0 0 1]Cu and [1 1 1]Cu zone axis are simulated according to diffraction effects of multiple variants, secondary diffraction and the size of particles, which coincide with the experimental results perfectly.

The deformation and aging behaviors of a hot rolled–quenched Cu–Cr–Zr–Mg–Si alloy were investigated by microhardness and electrical resistivity measurements, and transmission electron microscopy observations. The resistivity of aged alloy increases almost linearly with increasing of the secondary cold deformation reductions, and a faster and larger increase in resistivity of the peak-aged alloy is found during the deformation process due to the occurrence of resolution phenomenon. Quantitative analysis shows that about 0.0385 wt.% solutes are re-dissolved into the peak-aged alloy after 70% cold deformation. Results of the following aging treatment show that the deterioration caused by resolution phenomenon in electrical properties can be eliminated by the secondary aging treatment. A good combination of high hardness and low resistivity can be achieved in the Cu–Cr–Zr–Mg–Si alloy after suitable thermomechanical treatments.

Cu–Cr system alloy strips were manufactured by an online hot rolling–quenching (HR–Q) process and subsequent thermomechanical treatments. The microstructure and properties of the alloys were investigated by observations of optical microscopy and transmission electron microscopy, and measurements of microhardness and electrical conductivity. The results show that the HR–Q process and thermomechanical treatments are successfully developed to manufacture Cu–Cr system alloy strips with good combinations of strength, conductivity and softening resistance. Ordered fcc structure Cr precipitates, which are decomposed from the thermomechanical treated alloys, are considered to be precursors to the formation of equilibrium bcc Cr precipitates and responsible for the improvement of properties during near peak aging. Small additions of Zr, Mg and Si effectively improve the hardness and softening resistance of Cu–Cr alloy, and slightly reduce the electrical conductivity. The achievement of high strength and high electrical conductivity in the alloys is ascribed to the interactions of grain boundary strengthening, strain hardening and precipitation hardening.

High-resolution electron microscopy was used to research the age-hardening behaviors in 6005A alloy and the microstructural characterizations of precipitates. It was found that β″, β′ and Q′ precipitates had 12 orientation variants, respectively, the smaller size and a high-density β′′ precipitates existed in alloy for a long time, which played a very important role in controlling an anti-overaged softening behavior in 6005A alloy. Further, by the crystallographic interface and morphology analysis, a main reason was that a 2-dimensions coherency strain-field not only had the high-density β′′ precipitates become the biggest obstacle of dislocations movement, but also made them transform into β′ and Q′ precipitates with more difficulty. Moreover, it was also found that β′ and Q′ precipitates had weaker relatively strain-fields, the larger size and a lower density, which were largely associated with the reduction in hardness that occurred upon overaging. Further, the strengthening of precipitates on alloys could be summarized as: β″ > Q′ > β′. Based on the structural information, the quantitative Moire fringes analytical technique was also used to verify the lattice parameters and orientation of precipitates.

In order to assess the antiinflammatory effects of mometasone furoate (CAS 83919-23-7, Sch 32088, MF) in an animal model of allergic airway disease, nasal inflammation (total cell count [TCC] in nasal lavage [NL]) and pulmonary inflammation (total and differential cell count) in bronchoalveolar lavage (BAL), forced vital capacity (FVC) and peak expiratory flow (PEF) were measured in ovalbumin (OVA)-sensitized and -challenged Brown Norway rat following pretreatment with MF or vehicle. Rats were sensitized twice over 14 days with OVA and placed into 1 of 4 protocols: Group 1: intranasal (i. n.) MF (0.001–100 µg/mL) or vehicle once daily for 3 days; Group 2: intratracheal (i. t.) MF (0.001 −0.3 mg/kg i. t.) or vehicle, one dose; Group 3: i. t. MF (0.1–1 mg/kg, i. t.) or vehicle, one dose; Group 4: nose-only inhalation (n. o. i.) of dry powder MF (estimated pulmonary deposition of MF, 1.4, 4.1, and 13.3 µg/kg) or vehicle, once daily for 3 days. Group 1 was challenged with i. n. OVA (1%) 2 h after last treatment dose. Groups 2–4 were challenged with aerosolized OVA (1%) 5 h after treatment. Assessments were performed 24 h post-challenge (Group 1: NL; Group 2: BAL; Group 3 and 4: FVC and PEF). Rats treated with MF demonstrated significant and dose-dependent improvements in nasal inflammation and lung function compared to those treated with vehicle; normalization of these markers to levels consistent with non-sensitized animals were noted at the highest MF doses. Improvements in lung function were similar with i. t. and n. o. i. administration. Pulmonary infiltration of total cells and eosinophils was significantly attenuated after one dose of i. t. MF (0.003–0.3 mg/kg). In this established model of allergic airway disease, MF significantly attenuated cellular infiltration in the upper and lower airways and normalized lung function following allergen provocation.

The microstructure and properties of a hot rolled-quenched Cu-Cr-Zr-Mg-Si alloy were investigated by transmission electron microscopy observations, micro-hardness and tensile strength testing, and electrical conductivity measurement. The results show that the hot rolling-quenching (HR-Q) process and subsequent thermomechanical treatments are successfully developed to manufacture Cu-Cr-Zr-Mg-Si alloy strips. Solution treatment can be finished during the HR-Q process, and good combinations of strength, conductivity, and softening resistance are obtained for the thermomechanically treated strips. Ordered fcc structure precipitates which exhibit a cube-on-cube orientation relationship with the matrix are responsible for the improvements of properties. The properties of hot rolled-quenched Cu-Cr-Zr-Mg-Si alloy strips can be further improved by two-step rolling and aging process. The improvements of properties are attributed to the interactions of precipitation strengthening and strain hardening.

Transition matrix, combine with high-resolution transmission electron microscopy (HRTEM) and stereographic projection, was used in this article to predict and characterize the orientations and electron diffraction patterns of β″ precipitates in Al-Mg-Si-(Cu) alloys. It was found that the β″ phases as the main strengthening precipitates had 12 variants with Al matrix, and the orientation relationships could be expressed as (010) β″//{100}Al, [001] β″//〈310〉Al, and [100] β″//〈230〉Al. Further, based on the C-centered monoclinic structure of β″ precipitate, a new diffraction patterns model under the [001]Al zone axis could be established, which was in good agreement with the experiment date. Further, it could be used to explain reasonably some issues (for example, the “cross-shaped” diffraction streaks phenomenon). Simultaneously, we also found that the β″ precipitates had only three different zone axes, [010] β″, [304] β″, and $$ \left[ {\bar{1}06} \right]_{{\beta^{\prime \prime } }} , $$ parallel to the [001]Al direction when they were precipitated from the Al matrix, and the β″ precipitate might deviate 1.6 deg from its original orientation because of the lattice relaxation.

We have fabricated multilayer p–n hetero-junction structure La0.9Sr0.1MnO3/SrNb0.05Ti0.95O3 colossal magneto-resistivity thin films. The observations of transmission electron microscopy showed that the interfacial structure between La0.9Sr0.1MnO3 and SrNb0.05Ti0.95O3 was of great epitaxy and atomic level smooth. There was a superstructure within La0.9Sr0.1MnO3 thin films which was clarified in terms of the ordered partial substitution of La with Sr at center body of unit cell. Its chemical composition and distribution information investigated by electron energy loss spectrum and element mapping in the multilayer thin films were discussed.

The microstructure of precipitates in Cu–8.0Ni–1.8Si–0.15Mg (wt.%) alloy with super-high strength and high electrical conductivity was investigated by transmission electron microscope and selected-area electronic diffraction. The sequence of phase transformation in the alloy with aging time is that: spinodal decomposition + ordering(β-Ni3Si) → ordering(β-Ni3Si) + δ-Ni2Si → δ-Ni2Si + β-Ni3Si. And the relationship between the matrix and precipitates is that: ( 1 1 0 ) m / / ( 1 1 0 ) β / / ( 2 1 1 ¯ ) δ , [ 1 1 2 ¯ ] m / / [ 1 1 ¯ 2 ] β / / [ 3 2 4 ] δ . Spinodal decomposition, ordering (β-Ni3Si) and precipitates (β-Ni3Si + δ-Ni2Si) are attributed to the strengthening during aging, whose multiple interactions results in the variation of micro-hardness. After solution treatment at 970 °C for 6 h, cool rolled by 50%, and aged at 450 °C for 180 min, the Cu–8.0Ni–1.8Si–0.15Mg alloy has an average tensile strength of 1005 MPa, 0.2% proof strength of 768 MPa, elongation of 5.6% and an average electrical conductivity of 31.5.0% IACS. The morphology of the fracture surface of the alloy contains shallow dimple and quasi-cleavage pattern.

Background: Histamine and cysteinyl leukotrienes are pivotal mast cell mediators which contribute considerably and likely complementary to the symptoms of allergic rhinitis. Currently, we sought to explore the direct actions of histamine and leukotriene D4 (LTD4), a cysteinyl leukotriene, on porcine nasal arteries and veins. We also studied combined blocks of histamine and cysteinyl leukotrienes using loratadine and montelukast in an in vivo model of allergy-mediated nasal inflammation. Methods: For the evaluation of the action of histamine and LTD4 on arteries and veins, porcine nasal mucosa was isolated and cut into slices (100–300 µm thick). Real-time images of the nasal arteries and veins were recorded and vessel activities estimated by changes in cross-sectional area before and after the tested drugs. For the in vivo studies, the effect of loratadine and montelukast given alone and in combination was examined on upper airway inflammation in ovalbumin-sensitized and -challenged Brown Norway rats. Results: Both histamine (0.001–10 µmol/l) and LTD4 (0.001–10 µmol/l) produced a concentration-dependent increase in the lumen area of nasal mucosa arteries and veins. Histamine (0.01 µmol/l) alone produced a 24 and 12% increase in cross-sectional areas of arteries and veins, respectively. LTD4 (0.001 µmol/l) alone increased artery and vein dilation by about 17 and 9%, respectively. Combination treatment with histamine (0.01 µmol/l) and LTD4 (0.001 µmol/l) increased vessel dilation by 65% (arteries) and 26% (veins). In our in vivo Brown Norway rat studies, oral loratadine (0.01–10 mg/kg) and montelukast (0.01–10 mg/kg) significantly reduced antigen-induced total nasal inflammatory cell infiltration in a dose-dependent manner. The antiinflammatory dose-response curve of loratadine was shifted to the left when studied in combination with montelukast (0.01 mg/kg). Similarly, the dose-response characteristics of montelukast (0.01–10 mg/kg) was shifted in the presence of loratadine (0.01 mg/kg). Conclusion: Our studies support the position that histamine and cysteinyl leukotrienes may act collaboratively to elicit allergic nasal pathologies such as upper airway inflammation and nasal vessel dilation (which may translate into increased nasal mucosal engorgement). Furthermore, the current results are supportive of the hypothesis that combined treatment of allergic rhinitis with an H1 receptor antagonist and a CysLT1 receptor antagonist may have greater benefit than sole treatment with these agents alone.

The vascular bed in nasal mucosa of different species, including human, is highly vascularized and an extensive sinusoidal network of large capacitance vessels is present deep within the submucosa. When this network of venous sinusoids is engorged with blood, the swollen mucosa reduces the size of the airway lumen and congestion ensues. Nasal vasculature tone is strongly influenced by the sympathetic nervous system and the only drugs approved specifically to relieve vascular nasal obstruction are alpha-adrenoceptor sympathomimetic agents. Due to their vasoconstrictor action, the sympathomimetic decongestants oppose vasodilation, reducing nasal airway resistance and thus facilitating nose breathing. However, standard decongestants that are non-selective alpha-adrenoceptor agonists are associated with the potential for side-effect liabilities including hypertension, stroke, insomnia and nervousness. We propose than a selective alpha 2-adrenoceptor agonist, by acting preferentially on nasal venous capacitance vessels, will elicit decongestion with a reduced side-effect liability. In the present study, we evaluated the effects of the selective alpha 2-adrenoceptor agonist BHT-920 in a real-time tissue contractility assay using isolated pig nasal explants and in an in vivo cat model of congestion. The vasoconstrictor and decongestant effects of BHT-920 were compared to the non-selective alpha-adrenoceptor agonist epinephrine and the standard decongestant oxymetazoline. Our results showed that the alpha 2-adrenoceptor agonist BHT-920 preferentially contracts venous sinusoids confirming previous observations [Corboz MR, Varty LM, Rivelli MA, Mutter JC, Mingo G, McLeod R, et al. Effects of an alpha 2-adrenoceptor agonist in nasal mucosa. Arch Physiol Biochem 2003;11: 335-6, Corboz MR, Rivelli MA, Varty LM, Mutter J, Cartwright M, Rizzo CA, et al. Pharmacological characterization of postjunctional alpha-adrenoceptor in human nasal mucosa. Am J Rhinol 2005;19: 495-502] and displays decongestion without affecting blood pressure. Therefore, an alpha 2-adrenoceptor agonist, by causing constriction in the capacitance vessels of nasal mucosa, can produce nasal decongestion without the effects on blood pressure observed with the standard selective alpha 1-adrenoceptor and non-selective alpha-adrenoceptor sympathomimetic decongestants.

Cough is an important defensive pulmonary reflex that removes irritants, fluids, or foreign materials from the airways. However, when cough is exceptionally intense or when it is chronic and/or nonproductive it may require pharmacologic suppression. For many patients, antitussive therapies consist of OTC products with inconsequential efficacies. On the other hand, the prescription antitussive market is dominated by older opioid drugs such as codeine. Unfortunately, "codeine-like" drugs suppress cough at equivalent doses that also often produce significant ancillary liabilities such as GI constipation, sedation, and respiratory depression. Thus, the discovery of a novel and effective antitussive drug with an improved side effect profile relative to codeine would fulfill an unmet clinical need in the treatment of cough. Afferent pulmonary nerves are endowed with a multitude of potential receptor targets, including TRPV1, that could act to attenuate cough. The evidence linking TRPV1 to cough is convincing. TRPV1 receptors are found on sensory respiratory nerves that are important in the generation of the cough reflex. Isolated pulmonary vagal afferent nerves are responsive to TRPV1 stimulation. In vivo, TRPV1 agonists such as capsaicin elicit cough when aerosolized and delivered to the lungs. Pertinent to the debate on the potential use of TRPV1 antagonist as antitussive agents are the observations that airway afferent nerves become hypersensitive in diseased and inflamed lungs. For example, the sensitivity of capsaicin-induced cough responses following upper respiratory tract infection and in airway inflammatory diseases such as asthma and COPD is increased relative to that of control responses. Indeed, we have demonstrated that TRPV1 antagonism can attenuate antigen-induced cough in the allergic guinea pig. However, it remains to be determined if the emerging pharmacologic profile of TRPV1 antagonists will translate into a novel human antitussive drug. Current efforts in clinical validation of TRPV1 antagonists revolve around various pain indications; therefore, clinical evaluation of TRPV1 antagonists as antitussive agents will have to await those outcomes.

Asthma presents as a heterogeneous syndrome characterized by airway obstruction, inflammation and hyper-reactivity (AHR). Spleen tyrosine kinase (Syk) mediates allergen-induced mast cell degranulation, a central component of allergen-induced inflammation and AHR. However, the role of Syk in IgE-mediated constriction of human small airways remains unknown. In this study, we addressed whether selective inhibition of Syk attenuates IgE-mediated constriction and mast cell mediator release in human small airways.Human precision cut lung slices (hPCLS) ex vivo derived from non-asthmatic donors were incubated overnight with human IgE, dexamethasone, montelukast, antihistamines or a selective Syk inhibitor (SYKi). High-affinity IgE receptor (FcεRI) activation by anti-IgE cross-linking was performed, and constriction and mediator release measured. Airway constriction was normalized to that induced by maximal carbachol stimulation. Syk expression (determined by qPCR and immunoblot) was also evaluated in human primary airway smooth muscle (HASM) cells to determine whether Syk directly modulates HASM function.While dexamethasone had little effect on FcεR-mediated contraction, montelukast or antihistamines partially attenuated the response. SYKi abolished anti-IgE-mediated contraction and suppressed the release of mast cell or basophil mediators from the IgE-treated hPCLS. In contrast, SYKi had little effect on the non-allergic contraction induced by carbachol. Syk mRNA and protein were undetectable in HASM cells.A selective Syk inhibitor, but not corticosteroids, abolished FcεR-mediated contraction in human small airways ex vivo. The mechanism involved FcεRI receptor activation on mast cells or basophils that degranulate causing airway constriction, rather than direct actions on HASM.

It has been suggested that the rat is relatively more susceptible to toxicity induced by inhibitors for type 4 cAMP-specific phosphodiesterase (PDE4). In this study designed to elucidate possible biochemical basis for the higher susceptibility, we compared PDE4 expression levels and their functional relevance among rats, monkeys and humans. In several toxicologically relevant tissues and blood leukocytes, the mRNA expression levels of PDEs 4A, 4B, 4C and 4D were significantly higher in rats than in humans. We confirmed that higher PDE4 expression levels were correlated with a higher enzyme activity level in rat leukocytes. The PDE4 enzyme activity level of leukocytes in monkeys fell between that of rats and humans. Functionally, the potencies of the PDE4 inhibitors rolipram, SB 207499 and SCH 351591 in inhibiting tumor necrosis factor production from leukocytes were in the following order: rat > monkey > human. In addition, rolipram was about 10-fold more potent in rats than in humans in inhibiting phenylephrine-induced contraction of renal artery. These inhibitors were confirmed to be highly selective for PDE4 in comparison to all other PDE families, and to inhibit rat and human PDE4s with identical potencies. Taken together, these results suggest that the higher susceptibility of rats to PDE4 inhibitor-induced toxicity might be due to their higher expression levels of PDE4, and that PDE4 inhibitors may be safer in humans than in monkeys and, particularly, rats.

Hypotonic stimulation induces airway constriction in normal and asthmatic airways. However, the osmolarity sensor in the airway has not been characterized. TRPV4 (also known as VR-OAC, VRL-2, TRP12, OTRPC4), an osmotic-sensitive cation channel in the transient receptor potential (TRP) channel family, was recently cloned. In the present study, we show that TRPV4 mRNA was expressed in cultured human airway smooth muscle cells as analyzed by RT-PCR. Hypotonic stimulation induced Ca2+influx in human airway smooth muscle cells in an osmolarity-dependent manner, consistent with the reported biological activity of TRPV4 in transfected cells. In cultured muscle cells, 4α-phorbol 12,13-didecanoate (4-αPDD), a TRPV4 ligand, increased intracellular Ca2+level only when Ca2+was present in the extracellular solution. The 4-αPDD-induced Ca2+response was inhibited by ruthenium red (1 μM), a known TRPV4 inhibitor, but not by capsazepine (1 μM), a TRPV1 antagonist, indicating that 4-αPDD-induced Ca2+response is mediated by TRPV4. Verapamil (10 μM), an L-type voltage-gated Ca2+channel inhibitor, had no effect on the 4-αPDD-induced Ca2+response, excluding the involvement of L-type Ca2+channels. Furthermore, hypotonic stimulation elicited smooth muscle contraction through a mechanism dependent on membrane Ca2+channels in both isolated human and guinea pig airways. Hypotonicity-induced airway contraction was not inhibited by the L-type Ca2+channel inhibitor nifedipine (1 μM) or by the TRPV1 inhibitor capsazepine (1 μM). We conclude that functional TRPV4 is expressed in human airway smooth muscle cells and may act as an osmolarity sensor in the airway.

We have previously shown that N/OFQ, the endogenous peptide ligand for the ‘opioid-like’ NOP receptor, inhibits cough in guinea pigs and cats. In the present study we sought to continue our characterization of the cough-suppressant effects of NOP stimulation by profiling the pulmonary and antitussive effects of a novel non-peptide NOP agonist, Ro-64-6198, in guinea pigs. In receptor-binding assays, we confirmed that Ro-64-6198 selectively binds to NOP receptors over other opioid receptors. The Ki values for Ro-64-6198 at NOP, MOP, KOP and DOP receptors was 0.3, 36, 214 and 3,787 nmol/l, respectively. In GTPγS-binding assays, Ro-64-6198 displayed >900-fold functional selectivity at NOP relative to MOP receptors. We evaluated the effects of Ro-64-6198 (3 and 10 µmol/l) in isolated guinea pig nodose ganglia cells on the increases in intracellular Ca2+ concentration evoked by capsaicin stimulation (1 × 10–8–1 × 10–6 mol/l). Similar to previously reported data with N/OFQ, Ro-64-6198 (3 and 10 µmol/l) significantly attenuated Ca2+ responses in nodose ganglia cells produced by exposure to capsaicin. The effect of Ro-64-6198 (3 µmol/l) on capsaicin-induced intracellular Ca2+ responses was blocked by the NOP antagonist, J113397 (3 µmol/l). In guinea pig in vivo studies, aerosolized capsaicin (10–300 µmol/l) produced a dose-dependent increase in cough number. Ro-64-6198 given i.p. significantly inhibited cough due to capsaicin (300 µmol/l) exposure. In a duration study we found that the maximum antitussive effect (42 ± 8% inhibition) of Ro-64-6198 (3 mg/kg) was observed at 1 h after i.p. administration. Also at 1 h after administration, Ro-64-6198 (0.003–3.0 mg/kg, i.p.) produced a dose-dependent inhibition of cough. The antitussive effect of Ro-64-6198 (3 mg/kg, i.p.) was blocked by J113397 (12 mg/kg, i.p.) but not by the classical opioid antagonist naltrexone (10 mg/kg, i.p.). Although the antitussive action of Ro-64-6198 may be mediated by a central and/or a peripheral site of action, we hypothesize that selective oral NOP agonists that do not penetrate the blood-brain barrier may provide a novel approach for the treatment of cough. Moreover, because these drugs do not interact at MOP receptors, they may be devoid of codeine-like side effects such as respiratory depression, sedation, constipation or proclivities for addictive liabilities.

1. Endogenous neuronal lipid mediator anandamide, which can be synthesized in the lung, is a ligand of both cannabinoid (CB) and vanilloid receptors (VR). The tussigenic effect of anandamide has not been studied. The current study was designed to test the direct tussigenic effect of anandamide in conscious guinea-pigs, and its effect on VR1 receptor function in isolated primary guinea-pig nodose ganglia neurons. 2. Anandamide (0.3-3 mg.ml(-1)), when given by aerosol, induced cough in conscious guinea-pigs in a concentration dependent manner. When guinea-pigs were pretreated with capsazepine, a VR1 antagonist, the anandamide-induced cough was significantly inhibited. Pretreatment with CB1 (SR 141716A) and CB2 (SR 144528) antagonists had no effect on anandamide-induced cough. These results indicate that anandamide-induced cough is mediated through the activation of VR1 receptors. 3. Anandamide (10-100 micro M) increased intracellular Ca(2+) concentration estimated by Fluo-4 fluorescence change in isolated guinea-pig nodose ganglia cells. The anandamide-induced Ca(2+) response was inhibited by two different VR1 antagonists: capsazepine (1 micro M) and iodo-resiniferatoxin (I-RTX, 0.1 micro M), indicating that anandamide-induced Ca(2+) response was through VR1 channel activation. In contrast, the CB1 (SR 141716A, 1 micro M) and CB2 (SR 144528, 0.1 micro M) receptor antagonists had no effect on Ca(2+) response to anandamide. 4. In conclusion, these results provide evidence that anandamide activates native vanilloid receptors in isolated guinea-pig nodose ganglia cells and induces cough through activation of VR1 receptors.

Cough is an important defensive pulmonary reflex that removes irritants, fluids or foreign materials from the airways. However, often cough is non-productive and requires suppression. Opioid mu receptor agonists, such as codeine are commonly used as antitussive agents and are among the most widely administered drugs in the world. Codeine suppresses the responsiveness of one or more components of the central reflex pathway for cough and is an efficacious antitussive drug for cough due to diverse aetiologies. However, opioids produce side effects that include sedation, addiction potential and constipation. Therefore, novel cough suppressant therapies should maintain or improve upon the antitussive efficacy profile of opioids. Moreover, these novel therapies should have a safety profile significantly better than current antitussive therapies. Presently, we discuss preclinical findings showing that activation of the 'opioid-like' receptor (NOP(1)) inhibits cough in the guinea pig and cat.

Nociceptin/orphanin FQ (N/OFQ), an endogenous opioid-like orphan receptor (NOP receptor, previously termed ORL1 receptor) agonist, has been found to inhibit capsaicin-induced bronchoconstriction in isolated guinea-pig lungs and in vivo. The underlying mechanisms are not clear. In the present studies, we tested the effect of N/OFQ on VR1 channel function in isolated guinea-pig nodose ganglia cells. Capsaicin increased intracellular Ca(2+) concentration in these cells through activation of vanilloid receptors. Capsaicin-induced Ca(2+) responses were attenuated by pretreatment of nodose neurons with N/OFQ (1 microM). N/OFQ inhibitory effect on the Ca(2+) response in nodose ganglia cells was antagonized by tertiapin (0.5 microM), an inhibitor of inward-rectifier K(+) channels, but not by verapamil, a voltage gated Ca(2+) channel blocker, indicating that an inward-rectifier K(+) channel is involved in N/OFQ inhibitory effect. In isolated guinea-pig bronchus, N/OFQ (1 microM) inhibited capsaicin-induced airway contraction. Tertiapin (0.5 microM) abolished the N/OFQ inhibition of capsaicin-induced bronchial contraction. Capsaicin (10 microg) increased pulmonary inflation pressure in the isolated perfused guinea-pig lungs. This response was significantly attenuated by pretreatment with N/OFQ (1 microM). Tertiapin also abolished the N/OFQ inhibitory effect on capsaicin-induced bronchoconstriction in perfused lungs. Capsaicin increased the release of substance P and neurokinin A from isolated lungs. N/OFQ (1 microM) blocked the capsaicin-induced tachykinin release. These results indicate that N/OFQ-induced hyperpolarization of tachykinin containing airway sensory nerves, through an inward-rectifier K(+) channel activation, accounts for the inhibition of capsaicin-evoked broncoconstriction.

Molecular dynamics (MDs) simulations were used to explore the thermal stability of Au nanoparticles (NPs) with decahedral, cuboctahedral, icosahedral and Marks NPs. According to the calculated cohesive energy and melting temperature, the Marks NPs have a higher cohesive energy and melting temperature compared to these other shapes. The Lindemann index, radial distribution function, deformation parameters, mean square displacement and self-diffusivity have been used to characterize the structure variation during heating. This work may inspire researchers to prepare Marks NPs and apply them in different fields.

Injury to lung epithelial cells has a role in multiple lung diseases. We previously identified mitsugumin 53 (MG53) as a component of the cell membrane repair machinery in striated muscle cells. Here we show that MG53 also has a physiological role in the lung and may be used as a treatment in animal models of acute lung injury. Mice lacking MG53 show increased susceptibility to ischemia-reperfusion and over-ventilation induced injury to the lung when compared with wild type mice. Extracellular application of recombinant human MG53 (rhMG53) protein protects cultured lung epithelial cells against anoxia/reoxygenation-induced injuries. Intravenous delivery or inhalation of rhMG53 reduces symptoms in rodent models of acute lung injury and emphysema. Repetitive administration of rhMG53 improves pulmonary structure associated with chronic lung injury in mice. Our data indicate a physiological function for MG53 in the lung and suggest that targeting membrane repair may be an effective means for treatment or prevention of lung diseases.

Cystic fibrosis transmembrane conductance regulator (CFTR) chloride channels are regulated tightly by protein kinases and phosphatases. The regulatory domain of CFTR has about 20 potential sites for phosphorylation by protein kinases A (PKA) and C (PKC). The reason for this large number of sites is not known, however their conservation from fish to humans implies that they play important roles in vivo. PKA is an important activator, and its stimulation of CFTR is enhanced by PKC via mechanisms which are not fully understood. The physiological stimuli of CFTR are not known for some epithelia, and it appears likely that other serine/threonine and even tyrosine kinases also regulate CFTR in particular tissues. Phosphatases that deactivate CFTR have yet to be identified definitively at the molecular level, however CFTR is regulated by a membrane-bound form of protein phosphatase-2C (PP2C) in several cell types. Patch-clamp studies of channel rundown, co-immunoprecipitation, chemical cross-linking studies, and pull-down assays all indicate that CFTR and PP2C are closely associated within a stable regulatory complex. Understanding the regulation of CFTR by PP2C is a priority due to its potential as a target for pharmacotherapies in the treatment of cystic fibrosis.

Since little is known about the contribution to function of the N-terminal cytoplasmic loops (CL1, residues 139-194; CL2, residues 242-307) of cystic fibrosis transmembrane conductance regulator (CFTR), all nine point mutations identified in CLs 1 and 2 from patients with cystic fibrosis were reconstructed in the expression vector pcDNA3-CFTR and expressed transiently in COS-1 and HEK-293 cells and stably in Chinese hamster ovary (CHO) cells. Four amino acid substitutions retarded production of mature, fully glycosylated CFTR, suggesting that misprocessing of the channel causes the disease symptoms in the affected patients. Protein maturation could not be promoted by cell culture conditions of reduced temperature (26 degrees C). When properly processed mutants were evaluated for functional defects by the iodide efflux method, the G178R- and E193K-CFTR-expressing cell lines showed impaired anion translocation activities. Patch-clamp studies of single channels revealed that E193K variants had a significantly decreased open probability, which resulted from an increase in the mean closed time of the channels. This contrasted with a previous study of disease-associated point mutations in CL3 that mainly affected the mean open time. None of the maturation-competent CL 1 and 2 mutants had altered conductance. Thus, the N-terminal CLs appear not to contribute to the anion translocation pathway of CFTR; rather, mutations in CL1 can impede transition to the open state. Interestingly, the ability of the non-hydrolyzable ATP analogue adenylyl imidodiphosphate (AMP-PNP) to lock the channel into open bursts was abolished by the I148T and G178R amino acid substitutions.

Protein kinase A (PKA) stimulates Cl secretion by activating the cystic fibrosis transmembrane conductance regulator (CFTR), a tightly regulated Cl- channel in the apical membrane of many secretory epithelia. The CFTR channel is also modulated by protein kinase C (PKC), but the regulatory mechanisms are poorly understood. Here we present evidence that PKA-mediated phosphorylation alone is not a sufficient stimulus to open the CFTR chloride channel in the presence of MgATP; constitutive PKC phosphorylation is essential for acute activation of CFTR by PKA. When patches were excised from transfected Chinese hamster ovary cells, CFTR responses to PKA became progressively smaller with time and eventually disappeared. This decline in PKA responsiveness did not occur in the presence of exogenous PKC and was reversed by the addition of PKC to channels that had become refractory to PKA. PKC enhanced PKA stimulation of open probability without increasing the number of functional channels. Short-term pretreatment of cells with the PKC inhibitor chelerythrine (1 microM) reduced the channel activity that could be elicited by forskolin in cell-attached patches. Moreover, in whole cell patches, acute stimulation of CFTR currents by chlorophenylthio-cAMP was abolished by two chemically unrelated PKC inhibitors, although an abrupt, partial activation was observed after a delay of15 min. Modulation by PKC was most pronounced when basal PKC phosphorylation was reduced by briefly preincubating cells with chelerythrine. Constitutive PKC phosphorylation in unstimulated cells permits the maximum elevation of open probability by PKA to reach a level that is approximately 60% of that attained during in vitro exposure to both kinases. Differences in basal PKC activity may contribute to the variable cAMP responsiveness of CFTR channels in different cell types.

The aim of the current studies was to investigate the possibility that a decreased relaxant response to nitric oxide (NO) might contribute to strain-related differences in airway responsiveness in the rat. Isolated tracheal rings from hyperresponsive. Fisher rats were confirmed to be more responsive to carbachol [mean effective concentration (EC50) = 2.45 x 10(-7) M] than those from Lewis (EC50 = 3.60 x 10(-7) M, P < 0.03) and ACI (EC50 = 3.85 x 10(-7) M, P < 0.01) rats. Sodium nitroprusside (SNP), a NO donor, caused relaxation of the carbachol (10(-6) M) contracted tracheal rings, but the half-maximal inhibition concentration (IC50) SNP in Fisher rats (5.60 x 10(-6) M) was significantly higher than in Lewis (1.34 x 10(-6) M, P < 0.001) and ACI rats (1.13 x 10(-6) M, P < 0.0005). The inhibitory effect of SNP on airway responsiveness to inhaled methacholine (MCh) in vivo was also less pronounced for Fisher than Lewis rats. SNP induced an accumulation of guanosine 3',5'-cyclic monophosphate (cGMP) in cultured tracheal smooth muscle cells (TSM). Fisher TSM produced less cGMP on exposure to SNP compared with TSM from ACI (P < 0.01) and Lewis (P < 0.0001) rats. A decreased guanylyl cyclase activity may account for the impaired relaxant effect of SNP in Fisher rats.

The introduction of A ring pyrazole modification to the hydrocortisone C-21 heteroaryl thioethers generated compounds with excellent transrepression potency (IL-8 inhibition) compared to their hydrocortisone analogs. However, the transcriptional transactivation activity of these compounds were considerably higher than the corresponding hydrocortisone analogs. Among all the compounds evaluated, a quinoxaline thioether modification demonstrated the best overall in vitro separation.

The prednisolone C-21 heteroaryl thioethers have been synthesized and evaluated in cell based transrepression and transactivation assays. Most of the compounds demonstrated weak transactivational activity in both human and rat tyrosineaminotransferase functional assay while keeping potent anti-inflammatory activity. The benzimidazole thioether 7 exhibited comparable anti-inflammatory activity and improved safety profile compared to the classical oral steroid prednisolone.

We describe the pharmacological and pharmacokinetic profiles of SCH 486757, a nociceptin/orphanin FQ peptide (NOP) receptor agonist that has recently entered human clinical trials for cough. SCH 486757 selectively binds human NOP receptor (Ki = 4.6 ± 0.61 nM) over classical opioid receptors. In a guinea pig capsaicin cough model, SCH 486757 (0.01–1 mg/kg) suppressed cough at 2, 4, and 6 h post oral administration with a maximum efficacy occurring at 4 h equivalent to codeine, hydrocodone, dextromethorphan and baclofen. The antitussive effects of SCH 486757 (3.0 mg/kg, p.o.) was blocked by the NOP receptor antagonist J113397 (12 mg/kg, i.p.) but not by naltrexone (10 mg/kg, p.o.). SCH 486757 does not produce tolerance to its antitussive activity after a 5-day BID dosing regimen. After acute and chronic dosing paradigms, SCH 486757 (1 mg/kg) inhibited capsaicin-evoked coughing by 46 ±9% and 40 ± 11%, respectively. In a feline mechanically-evoked cough model, SCH 486757 produces a maximum inhibition of cough and expiratory abdominal electromyogram amplitude of 59 and 61%, respectively. SCH 486757 did not significantly affect inspiratory electromyogram amplitude. We examined the abuse potential of SCH 486757 (10 mg/kg, p.o.) in a rat conditioned place preference procedure which is sensitive to classical drugs of abuse, such as amphetamine and morphine. SCH 486757 was without effect in this model. Finally, SCH 486757 displays a good oral pharmacokinetic profile in the guinea pig, rat and dog. We conclude that SCH 486757 has a favorable antitussive profile in preclinical animal models.

Nasal congestion, one of the major disease features of rhinitis, is induced by the filling of venous sinusoids causing mucosal engorgement with resultant obstruction of nasal airflow. The only available drugs that directly target the underlying vascular features driving nasal obstruction are the sympathomimetic alpha-adrenoceptor agonists due to their vasoconstrictor action. However, standard decongestants are nonselective alpha-adrenoceptor agonists, which have the potential for side-effects liabilities such as hypertension, stroke, insomnia and nervousness. In the present study, the effects of nonsubtype selective alpha(2)-adrenoceptor agonists BHT-920 and PGE-6201204 were evaluated in several isolated nasal mucosa contractile bioassays including dog, pig and monkey, and in a real-time tissue contractility assay using isolated pig nasal explants for BHT-920. The decongestant activity of PGE-6201204 was evaluated in vivo in a cat model of experimental congestion. Our results showed that alpha(2)-adrenoceptor agonists (1) contract nasal mucosa of different species, (2) exert a preferential vasoconstrictor effect on the capacitance vessels (veins and sinusoids), and (3) elicit decongestion. In conclusion, a selective alpha(2)-adrenoceptor agonist causing constriction preferentially in the large venous sinusoids and veins of nasal mucosa and producing nasal decongestion is expected to show efficacy in the treatment of nasal congestion without the characteristic arterio-constrictor action of the standard nonselective sympathomimetic decongestants.

The TRPV1 channel is mainly expressed in sensory nerves. Activation of the channel induces neuropeptide release from central and peripheral sensory nerve terminals, resulting in the sensation of pain, neurogenic inflammation, smooth muscle contraction and cough. The TRPV1 channel can be activated by vanilloids such as capsaicin, as well as endogenous stimulators including H(+), heat, lipoxygenase products and anandamide. TRPV1 channel function is upregulated by several endogenous mediators present in inflammatory conditions, which decreases the threshold for activation of the channel. Under these conditions, TRPV1 can be activated by physiological body temperature, slight acidification or lower concentration of TRPV1 agonists. There is evidence that TRPV1 plays a role in the development of pathophysiological changes and symptoms in several diseases. In this review, we discuss TRPV1 channel activation and regulation in normal and diseased conditions, the role of TRPV1 in pain, cough, asthma and urinary incontinence, and the potential use of TRPV1 antagonists as a novel therapy for these diseases.

Nitric oxide (NO) is known to be synthesized from l-arginine in a reaction catalyzed by NO synthase. Liver cytochrome P-450 enzymes also catalyze the oxidative cleavage of CN bonds of compounds containing a -C(NH2)NOH function, producing NO in vitro. The present study was designed to investigate whether there was evidence of a similar pathway for the production of NO in tracheal smooth muscle cells. Formamidoxime (10−2to 10−4M), a compound containing -C(NH2)NOH, relaxed carbachol-contracted tracheal rings and increased intracellular cGMP in cultured tracheal smooth muscle cells, whereasl-arginine had no such effect. NO was detectable in the medium containing cultured tracheal smooth muscle cells when incubated with formamidoxime. Ethoxyresorufin (10−7to 10−4M), an alternate cytochrome P-450 substrate, inhibited formamidoxime-induced cGMP accumulation as well as tracheal ring relaxation in cultured tracheal smooth muscle cells. The NO synthase inhibitors Nω-nitro-l-arginine (10−3M) and NG-monomethyl-l-arginine (10−3M) had no effect on formamidoxime-induced cGMP accumulation. These results suggest that NO can be synthesized from formamidoxime in tracheal smooth muscle cells, presumably by a reaction catalyzed by cytochrome P-450.