Arginase:A novel key enzyme in the pathophysiology of allergic asthma

Allergisch astma is een chronische luchtwegaandoening, die onder meer gekenmerkt wordt door allergeen-geïnduceerde vroege en late bronchusobstructieve reacties en ontwikkeling van overgevoeligheid van de luchtwegen voor luchtwegvernauwende prikkels na deze reacties. In dit proefschrift werd met behulp van geïsoleerde luchtwegpreparaten aangetoond dat luchtweghyperreactiviteit na de vroege astmatische reactie veroorzaakt wordt door een tekort aan zowel neuronaal als non-neuronaal bronchusverwijdend stikstofmonoxide (NO), als gevolg van een verlaagde beschikbaarheid van het substraat L-arginine voor constitutieve NO-synthases (cNOS). Ontdekt werd dat deze substraatdeficientie in hoge mate wordt veroorzaakt door een toegenomen activiteit van het enzym arginase in de luchtwegwand, dat L-arginine omzet in L-ornithine en ureum. Daarnaast bleek het tekort aan non-neuronaal NO mede veroorzaakt te kunnen worden door een verhoogde afgifte van polykationen door ontstekingscellen, waarschijnlijk door inhibitie van kationische aminozuurtransporters die zorgen voor de L-arginine-opname in de NO-producerende cellen. Toegenomen arginase-activiteit en polykationen bleken eveneens ten grondslag te liggen aan de luchtweghyperreactiviteit na de late astmatische reactie. Tijdens de late reactie wordt induceerbaar NOS (iNOS) tot expressie gebracht, dat bij lage argininebeschikbaarheid zowel NO als superoxide anionen produceert. Beide stoffen reageren tot de uiterst reactieve stikstofverbinding peroxynitriet dat de reactiviteit van de luchtwegen verhoogt. Naast substraatcompetitie met NOS draagt toegenomen arginase-activiteit tevens bij aan de hyperreactiviteit door de productie van L-ornithine, dat eveneens het cellulaire argininetransport kan remmen. Verhoging van de argininebeschikbaarheid door specifieke arginaseremmers bleek de reactiviteit van de hyperreactieve luchtwegen te normaliseren. Arginaseremmers openen daarmee een geheel nieuw perspectief in de behandeling van allergisch astma

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An alternative in treatment of obese asthma

Multiple facets of cAMP signalling and physiological impact:cAMP compartmentalization in the lung

Therapies involving elevation of the endogenous suppressor cyclic AMP (cAMP) are currently used in the treatment of several chronic inflammatory disorders, including chronic obstructive pulmonary disease (COPD). Characteristics of COPD are airway obstruction, airway inflammation and airway remodelling, processes encompassed by increased airway smooth muscle mass, epithelial changes, goblet cell and submucosal gland hyperplasia. In addition to inflammatory cells, airway smooth muscle cells and (myo)fibroblasts, epithelial cells underpin a variety of key responses in the airways such as inflammatory cytokine release, airway remodelling, mucus hypersecretion and airway barrier function. Cigarette smoke, being next to environmental pollution the main cause of COPD, is believed to cause epithelial hyperpermeability by disrupting the barrier function. Here we will focus on the most recent progress on compartmentalized signalling by cAMP. In addition to G protein-coupled receptors, adenylyl cyclases, cAMP-specific phospho-diesterases (PDEs) maintain compartmentalized cAMP signalling. Intriguingly, spatially discrete cAMP-sensing signalling complexes seem also to involve distinct members of the A-kinase anchoring (AKAP) superfamily and IQ motif containing GTPase activating protein (IQGAPs). In this review, we will highlight the interaction between cAMP and the epithelial barrier to retain proper lung function and to alleviate COPD symptoms and focus on the possible molecular mechanisms involved in this process. Future studies should include the development of cAMP-sensing multiprotein complex specific disruptors and/or stabilizers to orchestrate cellular functions. Compartmentalized cAMP signalling regulates important cellular processes in the lung and may serve as a therapeutic target

β3-Adrenoceptor-mediated relaxation of rat and human urinary bladder:roles of BKCa channels and Rho kinase

Previous studies suggest that the large-conductance Ca2+-activated K+ (BKCa) channel and Rho-kinase play major roles in the control of urinary bladder tone. Here, we investigated their involvement in beta-adrenoceptor (AR)-mediated relaxation of rat and human bladder. Concentration-response curves of isoprenaline and mirabegron-induced bladder relaxation were generated against passive tension and KCl- and carbachol-induced tone, in the absence or presence of the BKCa channel inhibitor iberiotoxin (100 nM) or the Rho-kinase inhibitor Y27,632 (1 mu M). Myosin light chain (MLC) phosphorylation was studied by Western blot. In rat, iberiotoxin only slightly altered isoprenaline- and mirabegron-induced relaxation against KCl-induced tone but attenuated relaxation by both agonists against carbachol-induced tone. Y27,632 enhanced isoprenaline- or mirabegron-induced relaxation only against carbachol-induced tone. In humans, iberiotoxin slightly enhanced relaxation by both agonists against carbachol-induced pre-contraction. Y27,632 did not change isoprenaline-induced relaxation but enhanced that by mirabegron. Under passive tension, MLC phosphorylation was markedly reduced by both beta-AR agonists, an effect insensitive to Y27,632. In the presence of carbachol, both beta-AR agonists increased MLC phosphorylation, an effect reduced by Y27,632 only in the presence of 1 mu M carbachol. These results indicate that the extent of BKCa channel and Rho-kinase involvement in relaxation induced by beta-AR agonists depends on pre contractile stimulus and species

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 β2-adrenoceptors and bronchoconstrictive muscarinic M3-receptors alter with airway size. In COPD, (a combination of) β2-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 β2-agonists and anticholinergics in COPD. Bronchoprotection by indacaterol (β2-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