Small airway hyperresponsiveness in COPD: relationship between structure and function in lung slices.

The direct relationship between pulmonary structural changes and airway hyperresponsiveness (AHR) in chronic obstructive pulmonary disease (COPD) is unclear. We investigated AHR in relation to airway and parenchymal structural changes in a guinea pig model of COPD and in COPD patients. Precision-cut lung slices (PCLS) were prepared from guinea pigs challenged with lipopolysaccharide or saline two times weekly for 12 wk. Peripheral PCLS were obtained from patients with mild to moderate COPD and non-COPD controls. AHR to methacholine was measured in large and small airways using video-assisted microscopy. Airway smooth muscle mass and alveolar airspace size were determined in the same slices. A mathematical model was used to identify potential changes in biomechanical properties underlying AHR. In guinea pigs, lipopolysaccharide increased the sensitivity of large (>150 µm) airways toward methacholine by 4.4-fold and the maximal constriction of small airways (<150 µm) by 1.5-fold. Similarly increased small airway responsiveness was found in COPD patients. In both lipopolysaccharide-challenged guinea pigs and patients, airway smooth muscle mass was unaltered, whereas increased alveolar airspace correlated with small airway hyperresponsiveness in guinea pigs. Fitting the parameters of the model indicated that COPD weakens matrix mechanical properties and enhances stiffness differences between the airway and the parenchyma, in both species. In conclusion, this study demonstrates small airway hyperresponsiveness in PCLS from COPD patients. These changes may be related to reduced parenchymal retraction forces and biomechanical changes in the airway wall. PCLS from lipopolysaccharide-exposed guinea pigs may be useful to study mechanisms of small airway hyperresponsiveness in COPD.

Prostaglandin E2 promotes MYCN non-amplified neuroblastoma cell survival via ß-catenin stabilization.

Amplification of MYCN is the most well-known prognostic marker of neuroblastoma risk classification, but still is only observed in 25% of cases. Recent evidence points to the cyclic adenosine monophosphate (cAMP) elevating ligand prostaglandin E2 (PGE2 ) and ß-catenin as two novel players in neuroblastoma. Here, we aimed to define the potential role of PGE2 and cAMP and its potential interplay with ß-catenin, both of which may converge on neuroblastoma cell behaviour. Gain and loss of ß-catenin function, PGE2 , the adenylyl cyclase activator forskolin and pharmacological inhibition of cyclooxygenase-2 (COX-2) were studied in two human neuroblastoma cell lines without MYCN amplification. Our findings show that PGE2 enhanced cell viability through the EP4 receptor and cAMP elevation, whereas COX-2 inhibitors attenuated cell viability. Interestingly, PGE2 and forskolin promoted glycogen synthase kinase 3ß inhibition, ß-catenin phosphorylation at the protein kinase A target residue ser675, ß-catenin nuclear translocation and TCF-dependent gene transcription. Ectopic expression of a degradation-resistant ß-catenin mutant enhances neuroblastoma cell viability and inhibition of ß-catenin with XAV939 prevented PGE2 -induced cell viability. Finally, we show increased ß-catenin expression in human high-risk neuroblastoma tissue without MYCN amplification. Our data indicate that PGE2 enhances neuroblastoma cell viability, a process which may involve cAMP-mediated ß-catenin stabilization, and suggest that this pathway is of relevance to high-risk neuroblastoma without MYCN amplification.

Negative modulation of mitochondrial calcium uniporter complex protects neurons against ferroptosis.

Ferroptosis is an iron- and reactive oxygen species (ROS)-dependent form of regulated cell death, that has been implicated in Alzheimer's disease and Parkinson's disease. Inhibition of cystine/glutamate antiporter could lead to mitochondrial fragmentation, mitochondrial calcium ([Ca2+]m) overload, increased mitochondrial ROS production, disruption of the mitochondrial membrane potential (ΔΨm), and ferroptotic cell death. The observation that mitochondrial dysfunction is a characteristic of ferroptosis makes preservation of mitochondrial function a potential therapeutic option for diseases associated with ferroptotic cell death. Mitochondrial calcium levels are controlled via the mitochondrial calcium uniporter (MCU), the main entry point of Ca2+ into the mitochondrial matrix. Therefore, we have hypothesized that negative modulation of MCU complex may confer protection against ferroptosis. Here we evaluated whether the known negative modulators of MCU complex, ruthenium red (RR), its derivative Ru265, mitoxantrone (MX), and MCU-i4 can prevent mitochondrial dysfunction and ferroptotic cell death. These compounds mediated protection in HT22 cells, in human dopaminergic neurons and mouse primary cortical neurons against ferroptotic cell death. Depletion of MICU1, a [Ca2+]m gatekeeper, demonstrated that MICU is protective against ferroptosis. Taken together, our results reveal that negative modulation of MCU complex represents a therapeutic option to prevent degenerative conditions, in which ferroptosis is central to the progression of these pathologies.

Epac as a novel effector of airway smooth muscle relaxation.

Dysfunctional regulation of airway smooth muscle tone is a feature of obstructive airway diseases such as asthma and chronic obstructive pulmonary disease. Airway smooth muscle contraction is directly associated with changes in the phosphorylation of myosin light chain (MLC), which is increased by Rho and decreased by Rac. Although cyclic adenosine monophosphate (cAMP)-elevating agents are believed to relieve bronchoconstriction mainly via activation of protein kinase A (PKA), here we addressed the role of the novel cAMP-mediated exchange protein Epac in the regulation of airway smooth muscle tone. Isometric tension measurements showed that specific activation of Epac led to relaxation of guinea pig tracheal preparations pre-contracted with methacholine, independently of PKA. In airway smooth muscle cells, Epac activation reduced methacholine-induced MLC phosphorylation. Moreover, when Epac was stimulated, we observed a decreased methacholine-induced RhoA activation, measured by both stress fibre formation and pull-down assay whereas the same Epac activation prevented methacholine-induced Rac1 inhibition measured by pull-down assay. Epac-driven inhibition of both methacholine-induced muscle contraction by Toxin B-1470, and MLC phosphorylation by the Rac1-inhibitor NSC23766, were significantly attenuated, confirming the importance of Rac1 in Epac-mediated relaxation. Importantly, human airway smooth muscle tissue also expresses Epac, and Epac activation both relaxed pre-contracted human tracheal preparations and decreased MLC phosphorylation. Collectively, we show that activation of Epac relaxes airway smooth muscle by decreasing MLC phosphorylation by skewing the balance of RhoA/Rac1 activation towards Rac1. Therefore, activation of Epac may have therapeutical potential in the treatment of obstructive airway diseases.

Effects of (a Combination of) the Beta2-Adrenoceptor Agonist Indacaterol and the Muscarinic Receptor Antagonist Glycopyrrolate on Intrapulmonary Airway Constriction.

Expression of bronchodilatory ß2-adrenoceptors and bronchoconstrictive muscarinic M3-receptors alter with airway size. In COPD, (a combination of) ß2-agonists and muscarinic M3-antagonists (anticholinergics) are used as bronchodilators. We studied whether differential receptor expression in large and small airways affects the response to ß2-agonists and anticholinergics in COPD. Bronchoprotection by indacaterol (ß2-agonist) and glycopyrrolate (anticholinergic) against methacholine- and EFS-induced constrictions of large and small airways was measured in guinea pig and human lung slices using video-assisted microscopy. In guinea pig lung slices, glycopyrrolate (1, 3 and 10 nM) concentration-dependently protected against methacholine- and EFS-induced constrictions, with no differences between large and small intrapulmonary airways. Indacaterol (0.01, 0.1, 1 and 10 µM) also provided concentration-dependent protection, which was greater in large airways against methacholine and in small airways against EFS. Indacaterol (10 µM) and glycopyrrolate (10 nM) normalized small airway hyperresponsiveness in COPD lung slices. Synergy of low indacaterol (10 nM) and glycopyrrolate (1 nM) concentrations was greater in LPS-challenged guinea pigs (COPD model) compared to saline-challenged controls. In conclusion, glycopyrrolate similarly protects large and small airways, whereas the protective effect of indacaterol in the small, but not the large, airways depends on the contractile stimulus used. Moreover, findings in a guinea pig model indicate that the synergistic bronchoprotective effect of indacaterol and glycopyrrolate is enhanced in COPD.

Disruption of AKAP-PKA Interaction Induces Hypercontractility With Concomitant Increase in Proliferation Markers in Human Airway Smooth Muscle.

With the ability to switch between proliferative and contractile phenotype, airway smooth muscle (ASM) cells can contribute to the progression of airway diseases such as asthma and chronic obstructive pulmonary disease (COPD), in which airway obstruction is associated with ASM hypertrophy and hypercontractility. A-kinase anchoring proteins (AKAPs) have emerged as important regulatory molecules in various tissues, including ASM cells. AKAPs can anchor the regulatory subunits of protein kinase A (PKA), and guide cellular localization via various targeting domains. Here we investigated whether disruption of the AKAP-PKA interaction, by the cell permeable peptide stearated (st)-Ht31, alters human ASM proliferation and contractility. Treatment of human ASM with st-Ht31 enhanced the expression of protein markers associated with cell proliferation in both cultured cells and intact tissue, although this was not accompanied by an increase in cell viability or cell-cycle progression, suggesting that disruption of AKAP-PKA interaction on its own is not sufficient to drive ASM cell proliferation. Strikingly, st-Ht31 enhanced contractile force generation in human ASM tissue with concomitant upregulation of the contractile protein α-sm-actin. This upregulation of α-sm-actin was independent of mRNA stability, transcription or translation, but was dependent on proteasome function, as the proteasome inhibitor MG-132 prevented the st-Ht31 effect. Collectively, the AKAP-PKA interaction appears to regulate markers of the multi-functional capabilities of ASM, and this alter the physiological function, such as contractility, suggesting potential to contribute to the pathophysiology of airway diseases.

PKA and Epac cooperate to augment bradykinin-induced interleukin-8 release from human airway smooth muscle cells.

BACKGROUND: Airway smooth muscle contributes to the pathogenesis of pulmonary diseases by secreting inflammatory mediators such as interleukin-8 (IL-8). IL-8 production is in part regulated via activation of Gq-and Gs-coupled receptors. Here we study the role of the cyclic AMP (cAMP) effectors protein kinase A (PKA) and exchange proteins directly activated by cAMP (Epac1 and Epac2) in the bradykinin-induced IL-8 release from a human airway smooth muscle cell line and the underlying molecular mechanisms of this response. METHODS: IL-8 release was assessed via ELISA under basal condition and after stimulation with bradykinin alone or in combination with fenoterol, the Epac activators 8-pCPT-2'-O-Me-cAMP and Sp-8-pCPT-2'-O-Me-cAMPS, the PKA activator 6-Bnz-cAMP and the cGMP analog 8-pCPT-2'-O-Me-cGMP. Where indicated, cells were pre-incubated with the pharmacological inhibitors Clostridium difficile toxin B-1470 (GTPases), U0126 (extracellular signal-regulated kinases ERK1/2) and Rp-8-CPT-cAMPS (PKA). The specificity of the cyclic nucleotide analogs was confirmed by measuring phosphorylation of the PKA substrate vasodilator-stimulated phosphoprotein. GTP-loading of Rap1 and Rap2 was evaluated via pull-down technique. Expression of Rap1, Rap2, Epac1 and Epac2 was assessed via western blot. Downregulation of Epac protein expression was achieved by siRNA. Unpaired or paired two-tailed Student's t test was used. RESULTS: The beta2-agonist fenoterol augmented release of IL-8 by bradykinin. The PKA activator 6-Bnz-cAMP and the Epac activator 8-pCPT-2'-O-Me-cAMP significantly increased bradykinin-induced IL-8 release. The hydrolysis-resistant Epac activator Sp-8-pCPT-2'-O-Me-cAMPS mimicked the effects of 8-pCPT-2'-O-Me-cAMP, whereas the negative control 8-pCPT-2'-O-Me-cGMP did not. Fenoterol, forskolin and 6-Bnz-cAMP induced VASP phosphorylation, which was diminished by the PKA inhibitor Rp-8-CPT-cAMPS. 6-Bnz-cAMP and 8-pCPT-2'-O-Me-cAMP induced GTP-loading of Rap1, but not of Rap2. Treatment of the cells with toxin B-1470 and U0126 significantly reduced bradykinin-induced IL-8 release alone or in combination with the activators of PKA and Epac. Interestingly, inhibition of PKA by Rp-8-CPT-cAMPS and silencing of Epac1 and Epac2 expression by specific siRNAs largely decreased activation of Rap1 and the augmentation of bradykinin-induced IL-8 release by both PKA and Epac. CONCLUSION: Collectively, our data suggest that PKA, Epac1 and Epac2 act in concert to modulate inflammatory properties of airway smooth muscle via signaling to the Ras-like GTPase Rap1 and to ERK1/2.

Second M3 muscarinic receptor binding site contributes to bronchoprotection by tiotropium.

BACKGROUND AND PURPOSE: The bronchodilator tiotropium binds not only to its main binding site on the M3 muscarinic receptor but also to an allosteric site. Here, we have investigated the functional relevance of this allosteric binding and the potential contribution of this behaviour to interactions with long-acting ß-adrenoceptor agonists, as combination therapy with anticholinergic agents and ß-adrenoceptor agonists improves lung function in chronic obstructive pulmonary disease. EXPERIMENTAL APPROACH: ACh, tiotropium, and atropine binding to M3 receptors were modelled using molecular dynamics simulations. Contractions of bovine and human tracheal smooth muscle strips were studied. KEY RESULTS: Molecular dynamics simulation revealed extracellular vestibule binding of tiotropium, and not atropine, to M3 receptors as a secondary low affinity binding site, preventing ACh entry into the orthosteric binding pocket. This resulted in a low (allosteric binding) and high (orthosteric binding) functional affinity of tiotropium in protecting against methacholine-induced contractions of airway smooth muscle, which was not observed for atropine and glycopyrrolate. Moreover, antagonism by tiotropium was insurmountable in nature. This behaviour facilitated functional interactions of tiotropium with the ß-agonist olodaterol, which synergistically enhanced bronchoprotective effects of tiotropium. This was not seen for glycopyrrolate and olodaterol or indacaterol but was mimicked by the interaction of tiotropium and forskolin, indicating no direct ß-adrenoceptor-M3 receptor crosstalk in this effect. CONCLUSIONS AND IMPLICATIONS: We propose that tiotropium has two binding sites at the M3 receptor that prevent ACh action, which, together with slow dissociation kinetics, may contribute to insurmountable antagonism and enhanced functional interactions with ß-adrenoceptor agonists.

Epac: effectors and biological functions.

Epac1 (also known as cAMP-GEF-I) and Epac2 (also known as cAMP-GEF-II) are cyclic AMP-activated guanine nucleotide exchange factors for Ras-like GTPases. Since their discovery about 10 years ago, it is now accepted that Epac proteins are novel cAMP sensors that regulate several pivotal cellular processes, including calcium handling, cell proliferation, cell survival, cell differentiation, cell polarization, cell-cell adhesion events, gene transcription, secretion, ion transport, and neuronal signaling. Recent studies even indicated that Epac proteins might play a role in the regulation of inflammation and the development of cardiac hypertrophy. Meanwhile, a plethora of diverse effectors of Epac proteins have been assigned, such as Ras and Rho GTPases, phospholiase C-epsilon, phospholipase D, mitogen-activated protein kinases, protein kinase B/Akt, ion channels, secretory-granule associated proteins and regulators of the actin-microtubule network, the latter probably involved in the spatiotemporal dynamics of Epac-related signaling. This review highlights multi-faceted effectors and diverse biological functions driven by Epac proteins that might explain certain controversial signaling properties of cAMP.

Potentiation of beta-adrenoceptor function in bovine tracheal smooth muscle by inhibition of protein kinase C.

To examine the role of contractile agonist-induced activation of protein kinase C (PKC) in functional antagonism of airway smooth muscle contraction by beta-adrenoceptor agonists, we examined the effects of the specific PKC-inhibitor GF 109203X (2-[1-(3-dimethylaminopropyl)-1H-indol-3-yl]-3-(1H-indol-3-yl) maleimide) on isoprenaline-induced relaxation of bovine tracheal smooth muscle contracted by various concentrations of methacholine and histamine. In the absence of GF 109203X, the potency of isoprenaline (pD(2)) was gradually reduced at increasing methacholine- and histamine-induced smooth muscle tones, but the maximal relaxation (E(max)) was decreased only at higher concentrations of methacholine. In the presence of GF 109203X, pD(2) values were significantly increased for both methacholine- and histamine-induced contractions. Moreover, isoprenaline E(max) values in the presence of high concentrations of methacholine were also increased. Although both methacholine- and histamine-induced contractions were slightly reduced by GF 109203X, the changes in isoprenaline pD(2) could only partially be explained by reduced contractile tone. In contrast to isoprenaline, forskolin-induced relaxations were not affected by GF 109203X. The results indicate that PKC activation contributes to the reduced beta-adrenergic responsiveness induced by methacholine and histamine, which may involve uncoupling of the beta-adrenoceptor from the effector system. Since many mediators and neurotransmitters in allergic airway inflammation can activate PKC, this cross talk may be important in the reduced bronchodilator response of patients with severe asthma.

Protein kinase A and the exchange protein directly activated by cAMP (Epac) modulate phenotype plasticity in human airway smooth muscle.

BACKGROUND AND PURPOSE: Platelet-derived growth factor (PDGF) modulates the airway smooth muscle (ASM) 'contractile' phenotype to a more 'proliferative' phenotype, resulting in increased proliferation and reduced contractility. Such phenotypic modulation may contribute to airway remodelling in asthma. We have previously shown that the cAMP effector molecules, protein kinase A (PKA) and the exchange protein directly activated by cAMP (Epac) inhibited PDGF-induced phenotypic modulation in bovine ASM. Here, we investigated these mechanisms in human ASM strips and cells. EXPERIMENTAL APPROACH: ASM strips were incubated with PDGF in the absence or presence of the activators of Epac (8-pCPT-2'-O-Me-cAMP) or of PKA (6-Bnz-cAMP) for 4 days. Strips were mounted for isometric contraction experiments or analysed for the expression of contractile markers. Cell proliferation was measured and proliferative markers were analysed under similar conditions. KEY RESULTS: Activation of Epac and PKA prevented PDGF-induced ASM strip hypocontractility, and restored the expression of smooth muscle actin, myosin and calponin, which had been markedly diminished by PDGF. Epac and PKA activation inhibited the PDGF-induced ASM cell proliferation and G(1)/S phase transition and the expression and phosphorylation of cell cycle regulators. CONCLUSIONS AND IMPLICATIONS: Epac and PKA maintain a normally contractile ASM phenotype in a mitogenic environment, suggesting that specific activators of Epac and PKA may be beneficial in the treatment of airway remodelling in asthma.

(Endo)cannabinoids mediate different Ca2+ entry mechanisms in human bronchial epithelial cells.

In human bronchial epithelial (16HBE14o(-)) cells, CB(1) and CB(2) cannabinoid receptors are present, and their activation by the endocannabinoid virodhamine and the synthetic non-selective receptor agonist CP55,940 inhibits adenylyl cyclase and cellular interleukin-8 release. Here, we analyzed changes in intracellular calcium ([Ca2+](i)) evoked by Delta(9)-tetrahydrocannabinol (Delta(9)-THC), CP55,940, and virodhamine in 16HBE14o(-) cells. Delta(9)-THC induced [Ca2+](i) increase and a large transient [Ca2+](i) mobilization, the latter probably reflecting store-depletion-driven capacitative Ca2+ entry (CCE). In contrast, CP55,940 induced a rather moderate Ca2+ influx and a sustained [Ca2+](i) mobilization. CP55,940-induced Ca2+ influx was inhibited by Ni2+, indicating CCE, possibly mediated by transient receptor potential channel TRPC1, the mRNA of which is expressed in 16HBE14o(-) cells. CP55,940-induced calcium alterations were mimicked by virodhamine concentrations below 30 microM. Interestingly, higher virodhamine induced an additional Ca2+ entry, insensitive to Ni2+, but sensitive to the TRPV1 antagonist capsazepine, the TRPV1-TRPV4 inhibitor ruthenium red, and the non-CCE (NCCE) inhibitors La3+ and Gd3+. Such pharmacological profile is supported by the presence of TRPV1, TRPV4, and TRPC6 mRNAs as well as TRPV1 and TRPC6 proteins in 16HBE14o(-) cells. Cannabinoid receptor antagonists increased virodhamine-induced Ca2+ entry. Virodhamine also enhanced arachidonic acid release, which was insensitive to cannabinoid receptor antagonism, but sensitive to the phospholipase A(2) inhibitor quinacrine, and to capsazepine. Arachidonic acid induced [Ca2+](i) increase similar to virodhamine. Collectively, these observations suggest that [Ca2+](i) alterations induced by Delta(9)-THC, CP55,940 and by low concentrations of virodhamine involve mobilization and subsequent CCE mechanisms, whereas such responses by high virodhamine concentrations involve NCCE pathways.