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Flavonoids are polyphenolic compounds that are widely present in food and Chinese medicine. The aim of the present study was to identify the flavonoids with anti-inflammatory effects in the airway; and to determine the role of anti-oxidant and cyclic adenosine monophosphate (cAMP) in the anti-inflammatory effect. Human bronchial epithelial BEAS-2B cells were exposed to bacterial endotoxin lipopolysaccharide (LPS) in the absence or presence of different flavonoids, which are categorized according to their chemical structures in seven subclasses [anthocyanidins, chalcones, flavanes, flavanones, flavones, flavonols, isoflavones]. Among the 17 flavonoids tested, only apigenin (flavones), luteolin (flavones), daidzein (isoflavones) and genistein (isoflavones) reduced LPS-induced release of inflammatory cytokines/chemokines interleukin (IL)-6, IL-8 and monocyte chemoattractant protein-1 in BEAS-2B cells. Quercetin caused further increase in LPS-induced IL-6 and IL-8 levels. It alone significantly increased nuclear factor-kappa B (NF-κB) p65 activity and the cellular oxidative stress marker malondialdehyde (MDA) level in BEAS-2B cells. By contrast, apigenin and genistein reduced LPS-induced increases in nuclear NF-κB activity and MDA level. Apigenin and genistein, but not quercetin, increased the cAMP level in BEAS-2B cells, and the cell-permeable cAMP analogue, 8-Br-cAMP, inhibited LPS-induced increase of IL-8 level. These findings suggest that the presence of C5-OH, C7-OH, C2=C3 and C4=O functional groups in the flavonoids is associated with greater anti-inflammatory effect, while that of C3-OH or glycosylation group at the A-ring greatly decreased the anti-inflammatory effect. The anti-inflammatory effect of these flavonoids may be related to their anti-oxidant properties, and partly to their ability in increasing cAMP level., (Copyright © 2019 Elsevier B.V. All rights reserved.)

A reduction in airway beta-adrenoceptor density has been reported in cystic fibrosis lung but the mechanism underlying this defect remains unclear. In this study, we have investigated whether the decrease in beta2-adrenoceptor associates with altered G protein-coupled receptor kinase (GRK) levels. We assessed GRK activity by rhodopsin phosphorylation, and beta2-adrenoceptor and GRK at the mRNA and protein levels by Northern and Western blotting in peripheral lung samples from normal donors and patients with cystic fibrosis. GRK activity was significantly increased in peripheral cystic fibrosis lung with parallel increases in GRK2/5 mRNAs and protein expression. Functionally, isoproterenol-stimulated adenylyl cyclase activity was also diminished by 65% in cystic fibrosis lung homogenates. These data suggest that the increase in GRK activity may be one of the mechanisms underlying alterations in the coupling between beta2-adrenoceptor and adenylyl cyclase via G-protein and may thus contribute to the downregulation of beta2-adrenoceptor in cystic fibrosis lung.

The responsiveness of the pulmonary circulation to acetylcholine changes in the newborn piglet. Therefore muscarinic receptors have been studied in the developing porcine lung from birth to adulthood using ligand binding, Northern blotting and in situ hybridisation. Maximal binding capacity of [N-methyl-3H] scopolamine and the affinity of the receptor in lung membranes increased between birth and 16 days (p < 0.05). Subtype affinity changed with age, but always M3, > M1 > M2. Northern blots of porcine muscarinic receptor subtypes showed m1, m2 and m3 mRNA present in lung membranes. m2 mRNA was present at all ages and decreased with age. m1 mRNA was absent at birth and m3 mRNA was absent at 3 days. Autoradiographic localisation showed ligand binding to the parenchyma and airway smooth muscle and nerves, there was no binding to intrapulmonary vessels. In situ hybridisation localised mRNA of all three subtypes to the smooth muscle cells of both vessels and airways. Changes in the receptor subtypes may explain the pharmacological changes during postnatal adaptation.

Neurokinin A is not only a potent bronchoconstrictor, but also has immuno-modulatory effects in animals and man, mediated via tachykinin NK2 receptors. We have examined the effect of the glucocorticoid, dexamethasone, on tachykinin NK2 receptor mRNA and the number of tachykinin NK2 receptors in bovine tracheal smooth muscle in vitro by Northern blot analysis using a human tachykinin NK2 receptor cDNA probe and receptor binding assay using [3H]SR48968 [(S)-N-methyl-N[4-acetylamino-4-phenylpiperidino-2(3,4-dichlorophenyl) butyl]benzamide]. Tachykinin NK2 receptor mRNA showed a time-dependent suppression (62% reduction after 6 h at 10(-7) M of dexamethasone), as well as a concentration-dependent suppression after the incubation with dexamethasone (IC50 = 1.3 x 10(-8) M). This suppression was abolished by the glucocorticoid receptor antagonist, mifepristone (RU38486), indicating that dexamethasone acts via the glucocorticoid receptor. It was also abolished by the protein synthesis inhibitor, cycloheximide (10 microg/ml), indicating that new protein synthesis is required on this suppression. Using the RNA polymerase inhibitor actinomycin D (5 microg/ml), we showed that the stability of tachykinin NK2 receptor mRNA was not affected by dexamethasone (t1/2 = 5 h). Nuclear run-on assays revealed a 51% reduction in the rate of tachykinin NK2 receptor gene transcription after treatment with dexamethasone for 6 h. Radioligand binding assay using an selective tachykinin NK2 receptor antagonist, [3H]SR48968 showed a significant decrease in the number of receptor binding sites after 16 h (Bmax = 262 +/- 23 versus 213 +/- 13 fmol/mg protein for vehicle and dexamethasone treatment respectively, P < 0.05), with no significant change at the earlier time points. These results suggest that glucocorticoids act on glucocorticoid receptors to decrease tachykinin NK2 receptor expression by decreasing the rate of tachykinin NK2 receptor gene transcription.

beta-Adrenoceptor agonists induce the down-regulation of beta 2-adrenoceptors and mRNA expression in animal lung. The down-regulation of beta 2-adrenoceptors may limit the therapeutic efficacy of beta 2-adrenoceptor-mediated bronchodilators in obstructive airways disease. We examined the effects of three selective beta 2-adrenoceptor agonists, salbutamol, salmeterol and formoterol on beta 2-adrenoceptor binding and mRNA levels in human lung in vitro. Human lung was obtained from cardiac transplantation donors. Peripheral lung was chopped and incubated with three selective beta 2-adrenoceptor agonist for 3 h or 24 h at three different concentrations (0.1, 1 and 10 microM). The affinity and density of beta 2-adrenoceptors was determined by [125I]iodocyanopindolol equilibrium binding in a lung membrane preparation in the presence of 0.1 microM CGP 20712 A (1-{2-[(3-carbamoyl-4-hydroxy)phenoxy] ethylamino}-3-[4-(1-methyl-4-trifluoromethyl-2-imidazolyl) phenoxy]-propan-2-ol), a selective beta 1-adrenoceptor antagonist. Although treatment with salbutamol for 3 h did not change beta 2-adrenoceptor density, both salmeterol and formoterol reduced beta 2-adrenoceptor density, and exposure to each agonist for 24 h reduced beta 2-adrenoceptor density at all concentrations. Treatment with 10 microM salmeterol increased the equilibrium dissociation constant (Kd), and also shifted the competition curves of (-)-isoprenaline to the left, beta 2-Adrenoceptor mRNA, measured by Northern blot analysis using a human beta 2-adrenoceptor cDNA probe, was reduced after exposure to all beta 2-adrenoceptor agonists at 3 h. Our data provide evidence for down-regulation of beta 2-adrenoceptor protein and mRNA after selective beta 2-adrenoceptor agonist treatment in human lung.

The cellular localisation and distribution of mRNAs encoding beta-adrenoceptor subtypes in human lung were studied by in situ hybridisation and Northern blot analysis. The 851-bp SmaI/PvuII fragment of human beta 1-adrenoceptor cDNA, the 439-bp SmaI fragment of human beta 2-adrenoceptor cDNA and the 975-bp SmaI fragment of human beta 3-adrenoceptor cDNA bound to single mRNA species of approximately 3.2 kb, 2.2 kb and 2.3 kb in size, respectively. Human lung and heart and rabbit lung expressed both beta 1- and beta 2-adrenoceptor mRNAs with no detectable level of beta 3-adrenoceptor mRNA, while rabbit perirenal adipose tissue expressed beta 1-, beta 2- and beta 3-adrenoceptor mRNAs. Cultured human airway epithelial cells and airway smooth muscle cells expressed only beta 2-adrenoceptor mRNA. In situ hybridisation in human lung, using 35S-labelled antisense RNA probes revealed a high level of expression of beta 1- and beta 2-adrenoceptor mRNAs in the pulmonary blood vessels, high level of expression of beta 2-adrenoceptor mRNA in the alveolar walls with less expression of beta 1-adrenoceptor mRNA. There was a moderate expression of beta 2-adrenoceptor but not beta 1-adrenoceptor mRNA in airway epithelium and smooth muscle of peripheral airways and no detectable beta 3-adrenoceptor mRNA in any lung structures.

Agonist binding to the beta 2-adrenoceptors and its mapping were studied using the newly developed radioligand [3H]formoterol. The results of [3H]formoterol saturation binding and formoterol inhibition of [3H]formoterol binding were consistent with binding to a single class of receptors (Kd = 1.34 +/- 0.15 nM, Bmax = 154.9 +/- 8.0 fmol/mg protein in guinea pig lung membranes, n = 8; Kd = 1.05 +/- 0.17 nM, Bmax = 67.8 +/- 8.1 fmol/mg protein in human lung membranes, n = 5) and competition assays with other agonists and antagonists disclosed only a single class of site. The nonhydrolyzable GTP analogue GTP gamma S caused a reduction in both Kd and Bmax, indicating that the receptors labelled by [3H]formoterol are coupled to a guanine nucleotide binding regulatory protein. Receptor mapping of [3H]formoterol binding sites shows that beta 2-adrenoceptors were widely distributed in both guinea pig and human lung, with dense labelling over airway epithelium and uniformly over alveolar walls, and sparse labelling of airway and vascular smooth muscle. In addition, submucosal glands were also sparsely labelled in human bronchus. The distribution of beta 2-adrenoceptors was similar to the pattern previously described with non-selective radiolabelled antagonists in the presence of selective beta 1-adrenoceptor antagonists.

Platelet-activating factor (PAF) is a potent inflammatory mediator and it actions are mediated via specific cell surface receptors which are coupled to G-proteins. PAF stimulates several functions in monocytes and may modulate the expression of its own receptor. To investigate the possible modulation of PAF receptor mRNA expression Northern blot analysis of total RNA from human monocytes was performed using the cDNA of human leukocyte PAF receptor as a probe. Following the addition of 100 nM PAF, there was a 2.0-fold increase in PAF receptor mRNA at 60 minutes after the stimulation, which was inhibited by pretreatment with the PAF receptor antagonist WEB 2086. This increase returned to control level at 120 and 180 min. The increase of PAF receptor mRNA was statistically significant for 10 nM to 1 microM of PAF, while 100 nM of lysoPAF did not increase PAF receptor mRNA levels. These results suggest that PAF receptor expression can be regulated by PAF itself at the transcriptional level.

The down-regulation by isoprenaline of beta 1 and beta 2-adrenoceptors in rat lung was investigated at the receptor protein and messenger RNA level. Rats were treated with either isoprenaline or vehicle for 2 h, 1 day and 7 days. Isoprenaline treatment resulted in significant decreases of both beta 1- and beta 2-adrenoceptor density after 1 day with maximal decreases of 65 +/- 7 and 65 +/- 5% for beta 1- and beta 2-adrenoceptors, respectively, at 7 days. The administration of isoprenaline had no effect on binding affinities of either beta 1 or beta 2-adrenoceptors. beta 1-Adrenoceptor mRNA was significantly decreased by 58 +/- 10, 73 +/- 4 and 51 +/- 11% at 2 h, 1 day and 7 days, respectively, after isoprenaline treatment. However, the beta 2-adrenoceptor mRNA was not changed at 2 h after treatment and was significantly decreased by 35 +/- 11 and 45 +/- 12% at 1 day and 7 days, respectively after treatment. This time course of beta 2-adrenoceptor mRNA correlated well with that of the transcription factor cyclic AMP response element binding protein-like DNA binding activity, determined by gel shift assay. These findings indicate the existence of distinct mechanisms for down-regulation of beta 1- and beta 2-adrenoceptors and suggest the involvement of cyclic AMP response element binding protein in the down-regulation of beta 2-adrenoceptors in rat lung.

High affinity [3H]bradykinin (BK) receptor binding sites have been identified in human and guinea pig lung sections by in vitro autoradiography. [3H]BK was incubated with tissue sections for 120 min at 25 degrees C and non-specific binding determined by incubating adjacent serial sections in the presence of unlabelled BK. In saturation experiments with guinea pig lung sections, a single class of high affinity binding sites was identified with an apparent dissociation constant (Kd) of 0.5 +/- 0.1 nM and a maximal binding capacity (Bmax) of 35.2 +/- 2.9 fmol/mg protein (n = 5). The binding of [3H]BK was inhibited by unlabelled BK and NPC 349 (a specific B2 antagonist) at IC50 of 2.7 +/- 0.4 and 87 +/- 9 nM (n = 3), respectively. In contrast, no inhibition was found at 1 microM for a variety of vasoactive peptides such substance P, calcitonin gene-related peptide, vasoactive intestinal peptide and des-Arg9-[Leu8]BK (a specific B1-antagonist). Autoradiography revealed that BK receptors were widely distributed in human and guinea pig lung, with dense labelling over bronchial and pulmonary blood vessels of all sizes and in the lamina propria immediately subjacent to the basal epithelial cell layer in large airways. Airway smooth muscle was sparsely labelled in large airways, but greater labelling in smaller airways. There was also detectable labelling over submucosal glands and nerve fibres in human intrapulmonary bronchi and over alveolar walls in both species. The high density of BK receptors on bronchial and pulmonary blood vessels indicate that BK may play an important role in the regulation of airway and pulmonary blood flow, as well as airway epithelial regulation.

We have used in situ hybridization to study the localization of mRNA encoding the beta 2-adrenoceptor in tissue sections of the human and rat lung and compared this with the distribution of beta 2-receptor binding sites using receptor autoradiography. To localize beta 2-receptor mRNA, a [32P]labeled antisense RNA probe (riboprobe) was generated from human or rat beta 2-receptor cDNA. A similar distribution of beta 2-receptor mRNA was identified in both species. The highest intensity of beta 2-receptor mRNA was detected in smooth muscle of small airways, airway epithelium and pulmonary blood vessels. Lower intensity of beta 2-receptor mRNA was identified in smooth muscle of large airways, and alveolar epithelium (presumably type I and type II pneumocytes). No significant hybridization signal was detected in interstitial tissue. The specificity of the hybridization signal was confirmed with a sense probe (having identical sequence to the mRNA) and preincubation with RNase A, and by Northern blot analysis which revealed a single band of mRNA of 2.2 kb. There was a correspondence between mRNA localization and the distribution of beta 2-receptors visualized by [125I]iodocyanopindolol autoradiographically in the presence of CGP 20712 (a beta 1-selective antagonist). However, alveolar walls that showed a high beta 2-receptor density had relatively low levels of mRNA. This cellular heterogeneity may reflect differences in RNA stability or transcription rate in different lung cells. This approach opens up new options in the investigation of the regulation of pulmonary beta 2-receptor gene expression in health and disease.

Muscarinic receptor subtypes in human and guinea pig lung membranes were characterised using selective muscarinic antagonists. Competition experiments were carried out against [3H](-)-quinuclidinyl benzilate binding at 25 degrees C in Tris-HCl buffer; non-specific binding was determined in the presence of 1 microM atropine. Of all the antagonists examined, only the M1-selective antagonist pirenzepine exhibited a heterogeneous binding profile (nH less than 1.0), best described by two-binding sites of high and low affinity. Binding of [3H]pirenzepine confirmed the presence of a high proportion of high affinity (M1) receptors (60% of total) in human peripheral lung. The high potency of M3-selective antagonists 4-diphenylacetoxy-N-methyl-piperidine methiodide (4-DAMP) and hexahydrosiladifenidol suggested the presence of M3 receptors, but the low potency of AF-DX 116 and methoctramine indicated that there was no significant population of M2 receptors present. The existence of muscarinic receptor subtypes in lung may have important clinical implication but their cellular localisation remains to be determined.

Binding studies in several species have demonstrated a high proportion of M1 muscarinic receptors in the lung but their localization is uncertain. Using [3H]quinuclidinyl benzylate we have confirmed that binding sites with high affinity for pirenzepine account for 50% of muscarinic receptors in the rat lung. Our functional studies using the muscarinic antagonists 4-diphenylacetoxy-N-methylpiperidine (4-DAMP), methoctramine and pirenzepine have demonstrated that the muscarinic receptor on the rat pulmonary artery endothelium which mediates vasodilation is of the M3 subtype and cannot account for the high proportion of M1 receptors identified in lung homogenates.