Johan Zaagsma

Muscarinic receptor signaling in the pathophysiology of asthma and COPD

Anticholinergics are widely used for the treatment of COPD, and to a lesser extent for asthma. Primarily used as bronchodilators, they reverse the action of vagally derived acetylcholine on airway smooth muscle contraction. Recent novel studies suggest that the effects of anticholinergics likely extend far beyond inducing bronchodilation, as the novel anticholinergic drug tiotropium bromide can effectively inhibit accelerated decline of lung function in COPD patients. Vagal tone is increased in airway inflammation associated with asthma and COPD; this results from exaggerated acetylcholine release and enhanced expression of downstream signaling components in airway smooth muscle. Vagally derived acetylcholine also regulates mucus production in the airways. A number of recent research papers also indicate that acetylcholine, acting through muscarinic receptors, may in part regulate pathological changes associated with airway remodeling. Muscarinic receptor signalling regulates airway smooth muscle thickening and differentiation, both in vitro and in vivo. Furthermore, acetylcholine and its synthesizing enzyme, choline acetyl transferase (ChAT), are ubiquitously expressed throughout the airways. Most notably epithelial cells and inflammatory cells generate acetylcholine, and express functional muscarinic receptors. Interestingly, recent work indicates the expression and function of muscarinic receptors on neutrophils is increased in COPD. Considering the potential broad role for endogenous acetylcholine in airway biology, this review summarizes established and novel aspects of muscarinic receptor signaling in relation to the pathophysiology and treatment of asthma and COPD.

Muscarinic M3 receptors mediate contraction of human central and peripheral airway smooth muscle

The muscarinic receptor subtype involved in human airway smooth muscle contraction was characterised for the first time, using subtype-selective muscarinic antagonists. It was demonstrated that methacholine-induced contraction of central (trachea) and peripheral (small bronchi) airway smooth muscle preparations was antagonised by pirenzepine, AF-DX 116, 4-DAMP methobromide, hexahydrosiladifenidol, and methoctramine with pA2-values characteristic of M3 (smooth muscle/glandular) muscarinic receptors. Since these pA2-values demonstrate significant correlations with those found in bovine and guinea-pig tracheal smooth muscle contraction, it is concluded that these animal tissues provide a good model for the study of M3 subtype-selective muscarinic antagonists to be used as bronchodilators.

Arginase and asthma: novel insights into nitric oxide homeostasis and airway hyperresponsiveness

For many years it has been supposed that the production of an excess of nitric oxide (NO) by inducible NO synthase (iNOS) plays a major role in inflammatory diseases, including asthma. However, recent studies indicate that a deficiency of beneficial, bronchodilating constitutive NOS (cNOS)-derived NO is important in allergen-induced airway hyperresponsiveness. Although several mechanisms are proposed to explain the reduction of cNOS activity, reduced substrate availability, caused by a combination of increased arginase activity and decreased cellular uptake of L-arginine, appears to play a key role. Recent evidence also indicates that iNOS-induced pathophysiological effects involve substrate deficiency. Thus, at low concentrations of L-arginine iNOS produces both NO and superoxide anions, which results in the increased synthesis of the highly reactive, detrimental oxidant peroxynitrite. Based on these observations, we propose that a relative deficiency of NO caused by increased arginase activity and altered L-arginine homeostasis is a major factor in the pathology of asthma.

Inhibition of allergen-induced airway remodelling by tiotropium and budesonide: a comparison

Chronic inflammation in asthma and chronic obstructive pulmonary disease drives pathological structural remodelling of the airways. Using tiotropium bromide, acetylcholine was recently identified as playing a major regulatory role in airway smooth muscle remodelling in a guinea pig model of ongoing allergic asthma. The aim of the present study was to investigate other aspects of airway remodelling and to compare the effectiveness of tiotropium to the glucocorticosteroid budesonide. Ovalbumin-sensitised guinea pigs were challenged for 12 weeks with aerosolised ovalbumin. The ovalbumin induced airway smooth muscle thickening, hypercontractility of tracheal smooth muscle, increased pulmonary contractile protein (smooth-muscle myosin) abundance, mucous gland hypertrophy, an increase in mucin 5 subtypes A and C (MUC5AC)-positive goblet cell numbers and eosinophilia. It was reported previously that treatment with tiotropium inhibits airway smooth muscle thickening and contractile protein expression, and prevents tracheal hypercontractility. This study demonstrates that tiotropium also fully prevented allergen-induced mucous gland hypertrophy, and partially reduced the increase in MUC5AC-positive goblet cell numbers and eosinophil infiltration. Treatment with budesonide also prevented airway smooth muscle thickening, contractile protein expression, tracheal hypercontractility and mucous gland hypertrophy, and partially reduced MUC5AC-positive goblet cell numbers and eosinophilia. This study demonstrates that tiotropium and budesonide are similarly effective in inhibiting several aspects of airway remodelling, providing further evidence that the beneficial effects of tiotropium bromide might exceed those of bronchodilation.

Muscarinic M2 receptors in bovine tracheal smooth muscle: discrepancies between binding and function

Previous work showing that AF-DX 116, a cardioselective muscarinic antagonist in functional experiments, does not discriminate between muscarinic receptors in bovine cardiac and tracheal membranes has been extended. In addition to AF-DX 116 we used the muscarinic antagonists, atropine, pirenzepine, 4-DAMP methobromide, gallamine, hexahydrosiladifenidol and methoctramine, in radioligand binding experiments on bovine cardiac left ventricular and tracheal smooth muscle membranes. The functional antagonism of the methacholine-induced contraction of bovine tracheal smooth muscle strips was also evaluated. An excellent correlation was found for all compounds between the binding affinities for muscarinic receptors in cardiac and tracheal smooth muscle membranes; moreover, the affinities found in cardiac membranes correspond with the pA2 values reported for atrial preparations of rat and guinea pig. However, significant and occasionally marked discrepancies were found between binding and functional affinities of these muscarinic antagonists on bovine tracheal smooth muscle.

Increased arginase activity underlies allergen‐induced deficiency of cNOS‐derived nitric oxide and airway hyperresponsiveness

A deficiency of constitutive nitric oxide synthase (cNOS)‐derived nitric oxide (NO), due to reduced availability of L‐arginine, importantly contributes to allergen‐induced airway hyperresponsiveness (AHR) after the early asthmatic reaction (EAR). Since cNOS and arginase use L‐arginine as a common substrate, we hypothesized that increased arginase activity is involved in the allergen‐induced NO deficiency and AHR. Using a guinea‐pig model of allergic asthma, we addressed this hypothesis by examining the effects of the specific arginase inhibitor Nω‐hydroxy‐nor‐L‐arginine (nor‐NOHA) on the responsiveness to methacholine of isolated perfused tracheae from unchallenged control animals and from animals 6 h after ovalbumin challenge. Arginase activity in these preparations was investigated by measuring the conversion of L‐[14C]arginine to [14C]urea. Airways from allergen‐challenged animals showed a 2 fold (P<0.001) increase in responsiveness to intraluminal (IL) administration of methacholine compared to controls. A similar hyperresponsiveness (1.8 fold, P<0.01) was observed in control airways incubated with the NOS inhibitor Nω‐nitro‐L‐arginine methyl ester (L‐NAME, 0.1 mM, IL), while L‐NAME had no further effect on the airways from challenged animals. Remarkably, 5 μM nor‐NOHA (IL) normalized the hyperresponsiveness of challenged airways to basal control (P<0.001), and this effect was fully reversed again by 0.1 mM L‐NAME (P<0.05). Moreover, arginase activity in homogenates of the hyperresponsive airways was 3.5 fold (P<0.001) enhanced compared to controls. The results indicate that enhanced arginase activity contributes to allergen‐induced deficiency of cNOS‐derived NO and AHR after the EAR, presumably by competition with cNOS for the common substrate, L‐arginine. This is the first demonstration that arginase is involved in the pathophysiology of asthma.

Detrimental Effects of β-Blockers in COPD: A Concern for Nonselective β-Blockers

Introduction β-Blockers are known to worsen FEV 1 and airway hyperresponsiveness (AHR) in patients with asthma. Both characteristics determine the outcome of COPD, a disease with frequent cardiac comorbidity requiring β-blocker treatment Objective To determine the effects of β-blockers on AHR (provocative concentration of methacholine causing a 20% fall in FEV 1 [PC 20 ]), FEV 1 , and response to formoterol in patients with COPD Design A double-blind, placebo-controlled, randomized, cross-over study Setting An ambulatory, hospital outpatient clinic of pulmonary diseases Patients Patients with mild-to-moderate irreversible COPD and AHR Intervention Fifteen patients received propranolol (80 mg), metoprolol (100 mg), celiprolol (200 mg), or placebo for 4 days, followed by a washout period ≥ 3 days. On day 4 of treatment, FEV 1 and PC 20 were assessed. Immediately hereafter, formoterol (12 μg) was administered and FEV 1 was measured for up to 30 min Results PC 20 was significantly lower (p 1 deteriorated only after propranolol treatment (2.08 ± 0.31 L) [mean ± SD] compared with placebo (2.24 ± 0.37 L). The fast bronchodilating effect of formoterol was hampered by propranolol (mean increase in FEV 1 at 3 min, 6.7 ± 8.9%) but was unaffected by the other β-blockers (16.9 ± 9.8%, 22 ± 11.6%, and 16.9 ± 9.0% for placebo, metoprolol, and celiprolol, respectively) Conclusions Pulmonary effects did not occur by celiprolol. Only propranolol reduced FEV 1 and the bronchodilating effect of formoterol. Both metoprolol and propranolol increased AHR. Thus, different classes of β-blockers have different pulmonary effects. The anticipated beneficial cardiovascular effects of a β-blocker must be weighted against the putative detrimental pulmonary effects, ie , effect on FEV 1 , AHR, and response to additional β 2 -agonists

Human bronchial cyclic nucleotide phosphodiesterase isoenzymes: biochemical and pharmacological analysis using selective inhibitors.

1 The aims of the present study were to characterize the cyclic nucleotide phosphodiesterase (PDE) isoenzyme activities present in human bronchi and to examine the ability of selective isoenzyme inhibitors to relax histamine and methacholine precontracted preparations of human bronchi. 2 Three separations of pooled human bronchial tissue samples were performed. Ion‐exchange chromatography showed that the soluble fraction of human bronchial preparations contains PDE I, II, III, IV and V isoenzyme activities. Multiple forms of PDE I and PDE IV were observed and PDE IV was the main cyclic AMP hydrolytic activity. 3 3‐Isobutyl‐1‐methylxanthine (IBMX) non‐selectively inhibited all separated isoenzyme activities. Zaprinast selectively inhibited PDE V, but also effectively inhibited one of the two PDE I isoforms identified. The PDE IV selective inhibitors rolipram and RO‐201724, inhibited the PDE IV activities as did the dual PDE III/IV inhibitor, Org 30029. Org 9935, a PDE III selective inhibitor, potently attenuated part of the PDE IV activity peak in one of three separations performed, indicating that some PDE III activity may co‐elute with PDE IV under the experimental conditions employed. 4 PDE IV‐selective (rolipram), PDE III‐selective (Org 9935) and dual PDE III/IV (Org 30029) inhibitors were effective relaxants of human bronchial smooth muscle. The PDE V/PDE I inhibitor, zaprinast was relatively ineffective. 5 The present study demonstrates in human bronchi, as in animal airways smooth muscle, that inhibitors of PDE III, PDEIV and dual PDE III/IV have potentially useful bronchodilator activity and are worthy of further consideration as anti‐asthma drugs.

Characterization of the muscarinic receptor subtype involved in phosphoinositide metabolism in bovine tracheal smooth muscle

1 The muscarinic receptor subtype involved in the methacholine‐induced enhancement of phosphoinositide metabolism in bovine tracheal smooth muscle was identified by using the M2‐selective antagonist AF‐DX 116 and the M3‐selective antagonist 4‐diphenylacetoxy‐N‐methylpiperidine (4‐DAMP) methobromide, in addition to the M1‐selective antagonist pirenzepine, in a classical Schild analysis. 2 All the antagonists shifted the methacholine dose‐response curve to the right in a parallel and concentration‐dependent fashion, yielding Schild plots with slopes not significantly different from unity. The pA2 values (6.94, 6.32 and 8.54 for pirenzepine, AF‐DX 116 and 4‐DAMP methobromide respectively) indicate that it is the M3 (smooth muscle/glandular), but not the M2 (cardiac) muscarinic receptor subtype, present in this tissue, that mediates phosphoinositide turnover, in accordance with our previous contractile studies. 3 The results provide additional evidence for the involvement of phosphoinositide turnover in the pharmacomechanical coupling between muscarinic receptor stimulation and contraction in (bovine tracheal) smooth muscle.

Airway hyperresponsiveness in asthma: lessons from in vitro model systems and animal models

Airway hyperresponsiveness (AHR) is a hallmark clinical symptom of asthma. At least two components of AHR have been identified: 1) baseline AHR, which is persistent and presumably caused by airway remodelling due to chronic recurrent airway inflammation; and 2) acute and variable AHR, which is associated with an episodic increase in airway inflammation due to environmental factors such as allergen exposure. Despite intensive research, the mechanisms underlying acute and chronic AHR are poorly understood. Owing to the complex variety of interactive processes that may be involved, in vitro model systems and animal models are indispensable to the unravelling of these mechanisms at the cellular and molecular level. The present paper focuses on a number of translational studies addressing the emerging central role of the airway smooth muscle cell, as a multicompetent cell involved in acute airway constriction as well as structural changes in the airways, in the pathophysiology of airway hyperresponsiveness.