The Veterinary Journal 193 (2012) 228–233

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The Veterinary Journal

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A comparison of in vitro relaxant responses to ipratropium bromide,b-adrenoceptor agonists and theophylline in feline bronchial smooth muscle

Jérôme Leemans a,b,⇑, Nathalie Kirschvink b, Pascal Gustin a

a Section of Pharmacology, Pharmacotherapy and Toxicology, Department of Functional Sciences B41, Faculty of Veterinary Medicine, University of Liège, 4000 Liège, Belgiumb Veterinary Integrated Research Unit, Department of Veterinary Medicine, Faculty of Sciences, University of Namur, 5000 Namur, Belgium

a r t i c l e i n f o

Article history:Accepted 31 October 2011

Keywords:CatIsolated bronchial smooth muscleBronchodilatorsFunctional antagonism

1090-0233/$ - see front matter � 2011 Elsevier Ltd. Adoi:10.1016/j.tvjl.2011.10.026

⇑ Corresponding author at: Veterinary Integrated RVeterinary Medicine, Faculty of Sciences, UniversiBelgium. Tel.: +32 81 72 43 78.

E-mail address: jerome.leemans@gmail.com (J. Lee

a b s t r a c t

This study compares the potency and efficacy of different relaxant drugs including anticholinergic, b-adrenergic and methylxanthine agents on acetylcholine-contracted feline bronchi, and investigates theinfluence of the initial muscarinic-induced tone on bronchodilator response. Feline bronchi wereremoved from euthanased client-owned cats and were contracted with acetylcholine to cause either40% or 80% of the acetylcholine maximal contraction. The efficacy and potency of bronchodilating drugswere obtained from cumulative dose–response curves with efficacy (Emax) as the maximal relaxantresponse and potency (�logEC50) as the logarithm of the concentration of drug inducing 50% of maximalrelaxation.

Under low contractile tone (40%), all bronchodilators relaxed feline bronchi in a concentration-depen-dent manner with the following rank order of potency: formoterol > ipratropium bromide > feno-terol > isoprenaline > salbutamol P salmeterol > theophylline. Emax values ranged from 80% to 100%depending on the tested drug. Constriction of feline bronchi with high-dose acetylcholine (80%) causeda rightward and downward shift of the b2-mimetic dose–response curves. Significant decreases in�logEC50 and Emax values were reported for salbutamol, formoterol and salmeterol. This study providesevidence that existing classes of bronchodilators produce effective relaxation of acetylcholine-contractedfeline bronchi and that airway responsiveness to b2-stimulants is dependent on the magnitude of the ini-tial muscarinic-induced tone. The clinical relevance of these in vitro findings has yet to be explored inclinical trials.

� 2011 Elsevier Ltd. All rights reserved.

Introduction

Bronchodilators are first-line treatments for obstructive lung dis-eases in humans such as asthma and chronic obstructive pulmonarydisease (COPD) and are intended for use as relief medication andmaintenance therapy. Pharmacologically-mediated bronchodila-tion is primarily obtained with methylxanthines, anticholinergicagents and b-adrenergic agonists delivered either locally (as aero-sols) or systemically. Current knowledge on the use and effects ofbronchodilators in feline medicine is quite limited. Varying doseregimens and routes of administration have been empirically sug-gested (Padrid, 2006) and, except for investigations on theophyllineand its derivatives (McKiernan et al., 1983; Dye et al., 1989; Guen-ther-Yenke et al., 2007), no pharmacokinetic data are available.

ll rights reserved.

esearch Unit, Department ofty of Namur, 5000 Namur,

mans).

In terms of efficacy, neither prophylactic nor interventionaladministration of inhaled bronchodilators (b2-mimetics, anticho-linergics) have been proven to be effective in alleviating allergen-induced bronchospasm in cats with experimental asthma, whichis at odds with current and empirical-based recommendations thatprompt clinical use of bronchodilators in feline practice (Leemanset al., 2010, 2011).

In vitro responsiveness of feline airways to different classes ofrelaxant drugs is poorly documented. Previous works showed thatb-adrenoceptor agonists (i.e. isoprenaline, fenoterol, salbutamoland terbutaline) caused a dose-dependent relaxation of basal orcarbachol-induced tone in feline airway preparations (Lulichet al., 1976; O’Donnell and Wanstall, 1983). However, to the bestof our knowledge, no in vitro studies have specifically addressedthe effects of long-acting b2-agonists (salmeterol, formoterol) orhave examined on a comparative basis the ability of feline bronchito respond to currently prescribed bronchodilators.

The in vitro potency and efficacy of b2-agonists are not onlydependent on the intrinsic characteristics of the drugs but alsoon the distribution of b-adrenoceptor subtypes in the tissue (b1-,

J. Leemans et al. / The Veterinary Journal 193 (2012) 228–233 229

b2- or mixed b1/b2 population) (Lulich et al., 1976), the magnitudeof the induced contractile tone (Lemoine and Overlack, 1992; Lin-den et al., 1993) and the nature of the contractile stimuli (Torphy,1984). In dogs, guinea-pigs and humans, pre-contracting airwaytissues with increasing concentrations of muscarinic agonistscauses a rightward shift of b-stimulant dose–response curves alongwith a marked decline in maximal relaxing response (Torphy et al.,1983; Lemoine and Overlack, 1992; Linden et al., 1993; Nalineet al., 1994; Molimard et al., 1998). More interestingly, differencesin activity among b-adrenergic agonists are more likely to be evi-denced as the muscarinic tone increases.

Formoterol is a nearly full agonist of b2-adrenoceptors whilesalbutamol and salmeterol act as partial agonists. Under strongmuscarinic stimulation, this partial agonism becomes functionallyrelevant and much higher concentrations of b2-mimetics arenecessary to induce relaxation (Lemoine and Overlack, 1992;Molimard et al., 1998). A review of the literature has indicatedthat such functional antagonism between cholinergic and b-adrenergic pathways has never been demonstrated in feline airwaypreparations.

The aims of the present study were (1) to investigate the in vitroresponsiveness of feline bronchial smooth muscle to differentrelaxant drugs, including a range of b-adrenoceptor agonists (iso-prenaline, fenoterol, salbutamol, salmeterol, formoterol), an anti-cholinergic agent (ipratropium bromide) and a methylxanthinederivative (theophylline); and (2) to assess whether sensitivity offeline airways to b-adrenoceptor agonists is influenced by the initialmuscarinic-induced tone. We hypothesised that all bronchodilatorswould relax feline bronchi in a concentration-dependent mannerand that pre-treating tissues with high concentrations of musca-rinic agonists would drastically increase the dose of b-stimulantrequired to induce relaxation.

Materials and methods

Tissue collection and preparation of bronchial segments

Bronchial tissues were removed from client-owned cats, which were euthan-ased for reasons other than respiratory disease at local veterinary clinics. Immedi-ately after euthanasia, the cats were transported to the laboratory, the chest openedand the lungs carefully excised and placed in Krebs–Henseleit (KH) solution (NaCl118.0; KCl 5.4; CaCl2 2.5; KH2PO4 1.2; MgSO4 1.2; NaHCO3 25.0; glucose11.7 mmol/L) aerated with 95% O2 and 5% CO2. The cats were of both sexes, varyingage (range 0.5–10 years) and were free from respiratory disease at necropsy. Thisstudy was approved by the local Ethical Committee for Animal Experiments ofthe University of Liège (Protocol number 74).

Segmental bronchi with an inner diameter of 1–3 mm were dissected from cau-dal lobes of the lungs. Upon removal of fat and adhering connective tissues, 2–4segments of the same bronchus (�5 mm in length) were prepared and suspendedunder an initial tension of 2.0 g in water-jacketed organ baths containing oxygen-ated KH solution at 37 �C. The tissues were washed every 15 min with fresh KHsolution which gave a final preload of 0.5–1 g after a 60-min equilibration period.Before subsequent experiments, feline bronchi were maximally contracted withacetylcholine 3 � 10�3 mol/L to confirm their viability and response to spasmogenicstimuli. Afterwards, preparations were washed and allowed to rest until basal tonewas re-established (�90 min). Changes in tension were recorded by isometric forcetransducers connected to an amplifier and a data acquisition system (IOX 1.587,EMKA Technologies).

Drugs

The following drugs were used: acetylcholine chloride, isoprenaline hydrochlo-ride, fenoterol hydrobromide, ipratropium bromide monohydrate (Sigma); salbuta-mol (Cambrex); salmeterol base, formoterol fumarate (gifts from Professor C.Advenier). Theophylline was used as an injectable solution (Euphyllin, ALTANAPharma). All working solutions were prepared before use, kept on ice and protectedfrom light. Formoterol was dissolved in distilled water in the presence of dimethyl-sulfoxide (DMSO; maximal amount added to baths, 0.002% [v/v]) and subsequentlydiluted in KH solution. Salmeterol was dissolved in distilled water in the presenceof HCl 0.01 mol/L and DMSO (maximal amount added to baths, 0.011% [v/v]) and

thereafter diluted in KH solution. Others drugs were dissolved in distilled waterand diluted in KH solution. Final concentration of drug vehicles in the organ bathdid not affect smooth muscle tone (data not shown).

Experimental procedures

Relaxant responses to bronchodilators were evaluated at two different levels ofacetylcholine-induced pre-contraction. Separate preliminary experiments werethus conducted to determine the concentrations of acetylcholine eliciting 40%(EC40) and 80% (EC80) of its own maximal contraction. Both provocative concentra-tions were calculated by linear interpolation of acetylcholine dose–response curves(10�9–3 � 10�3 mol/L). These calculations were based on three subjects (n = 3),each providing 6–8 preparations for acetylcholine evaluation.

Following the resting period, bronchial segments were contracted with acetyl-choline to cause either 40% (1.6 � 10�5 mol/L) or 80% (1.2 � 10�4 mol/L) of the acetyl-choline maximal contraction. Once a stable contractile tone was achieved,cumulative dose–response curves to isoprenaline (10�10–3 � 10�6 mol/L), salbuta-mol (10�9–10�5 mol/L), fenoterol (10�10–3 � 10�7 mol/L), formoterol (10�11–3 �10�8 mol/L), salmeterol (10�10–10�5 mol/L), ipratropium bromide (10�10–3� 10�7 mol/L) and theophylline (10�6–3 � 10�3 mol/L) were constructed. The con-centration of relaxant drugs was increased in half log steps except for salmeterolwhich was added to baths in full log increments. After the maximal effect of eachcompound was obtained, the remaining active tension was abolished by theophylline3 � 10�3 mol/L which produces a maximal relaxation. Only one preparation of thesame subject was used for each specific drug evaluation, e.g. ‘drug x – low or high in-duced tone’. Thus, n equals the number of studied subjects.

Expression and statistical analysis of the results

As detailed elsewhere (Naline et al., 1994), the dose–response curves to relaxantdrugs were obtained by plotting the negative logarithm (base 10) of the molar con-centration of drug versus the corresponding relaxant response, expressed as per-centage of the action of theophylline 3 � 10�3 mol/L. Agonist efficacy was definedas the maximal relaxant response (Emax) and potency as the negative logarithm(base 10) of the molar concentration of drug causing 50% of the maximal relaxationinduced by theophylline 3 � 10�3 mol/L (�logEC50). The intrinsic activity (IA) ofb2-agonists was expressed as the ratio between Emax of the studied drug and Emax

of isoprenaline (reference compound).Results are expressed as means ± SEM (standard error of the mean). Data were

analysed by a generalised linear model to assess the effects of drugs, induced mus-carinic tone and drug by tone interaction. Post-hoc pairwise comparisons were per-formed using a least square mean difference test. All variables supported the nullhypothesis stating that the data fit a normal distribution. Level of significancewas set at P < 0.05.

Results

Evidence for the validity of experimental conditions

Two different levels of acetylcholine-induced pre-contractionwere elicited by adding acetylcholine at concentrations of1.6 � 10�5 mol/L (EC40) and 1.2 � 10�4 mol/L (EC80). Both provoca-tive concentrations produced a sustained contraction correspond-ing to 35.01 ± 2.48% (EC40) and 70.91 ± 3.46% (EC80) of the initialmaximal contraction to acetylcholine (3 � 10�3 mol/L), which rea-sonably reflects expected changes in muscular tone.

Maximum time for completing dose–response curves to anyrelaxant drugs was less than 90 min. The spontaneous decreaseof tension occurring within 90 min after acetylcholine-inducedpre-contraction had plateaued equalled 11.25 ± 7.31% (n = 5) and14.73 ± 5.33% (n = 5) for low and high contractile tones, respec-tively. These values significantly differed from Emax values com-puted for studied bronchodilators (P < 0.05).

Potency of relaxant agonists

Under low contractile tone (EC40), all bronchodilators relaxedfeline bronchi in a concentration-dependent manner with thefollowing rank order of potency: formoterol (EC50 value 3.2-fold(x) lower than the next most potent agent) > ipratropium bromide(5.9x) > fenoterol (2.3x) > isoprenaline (2.2x) > salbutamol (1.3x)P salmeterol (3802x) > theophylline (Table 1). Dose–responsecurves were displaced to the right as the acetylcholine concentration

Table 1Characterisation of potency, efficacy and intrinsic activity of some relaxant agonists under two different levels of acetylcholine-induced tone in isolated feline bronchi.

EC40 EC80

n �logEC50 Emax (%) IA n �logEC50 Emax (%) IA

Isoprenaline 6 7.96 ± 0.14 96.20 ± 1.52 1 6 7.78 ± 0.21 88.63 ± 3.96 1Salbutamol 6 7.62 ± 0.21 91.0 ± 2.13 0.95 6 6.80 ± 0.13§ 73.16 ± 4.18§,� 0.83Fenoterol 4 8.33 ± 0.08 98.30 ± 0.12 1.02 4 7.84 ± 0.16 89.11 ± 3.04 1.01Formoterol 7 9.60 ± 0.16 93.59 ± 3.29 0.97 7 9.06 ± 0.20§ 82.55 ± 5.52§ 0.93Salmeterol 6 7.51 ± 0.22 83.62 ± 4.50� 0.87 6 – 48.26 ± 6.45§,� 0.54Ipratropium bromide 5 9.10 ± 0.10 99.94 ± 0.14 – 5 8.64 ± 0.11 99.30 ± 0.44 –Theophylline 6 3.93 ± 0.15 100 – 7 3.68 ± 0.22 100 –

Bronchial segments were contracted with two concentrations of acetylcholine (EC40 = 1.6 � 10�5 mol/L and EC80 = 1.2 � 10�4 mol/L), producing 40% and 80% of the acetyl-choline maximal contraction, respectively. �logEC50 (potency), negative logarithm (base 10) of the molar concentration of drug causing 50% of the maximal relaxationinduced by theophylline 3 � 10�3 mol/L; Emax (efficacy), maximal relaxant response expressed as a percentage of the action of theophylline 3 � 10�3 mol/L; IA (intrinsicactivity), ratio between Emax of a b2-agonist and Emax of isoprenaline (reference compound). �logEC50 and Emax values are shown as mean ± SEM.§ Significantly different from low-level contractile tone (EC40), P < 0.05.� Significantly different from isoprenaline, P < 0.05.

230 J. Leemans et al. / The Veterinary Journal 193 (2012) 228–233

was increased from 1.6�10�5 mol/L (EC40) to 1.2�10�4 mol/L(EC80) (Figs. 1A–F). At the high level of muscarinic contraction(EC80), salmeterol potency was not determined since its relaxant ef-fects failed to reach 50% of the relaxation induced by theophylline3 � 10�3 mol/L, thus precluding meaningful analysis. The �logEC50

of isoprenaline, salbutamol, fenoterol, formoterol, ipratropium bro-mide and theophylline were reduced by 0.18, 0.82, 0.49, 0.54, 0.46and 0.25 log units, respectively. This indicates that the concentra-tions of each agonist need to be increased 1.5, 6.6, 3.1, 3.5, 2.9 and1.8 times, respectively, to obtain the same relaxing effect. This lossof potency was significant for salbutamol (P = 0.002) and formoterol(P = 0.021). The rank order of potency remained broadly the same asthat observed for the low-level stimulation: formoterol (EC50 value2.6-fold lower than the next most potent agent) > ipratropium bro-mide (6.3x) > fenoterol (1.1x) P isoprenaline (9.5x) > salbutamol(1318x) > theophylline (Table 1).

Efficacy and intrinsic activity

Efficacy of theophylline, which was the reference drug used in thisbronchodilator assay, was arbitrarily set at 100%. Under low level ofmuscarinic contraction (EC40), the rank order of efficacy was: the-ophylline = ipratropium bromide P fenoterol P isoprenaline Pformoterol P salbutamol > salmeterol. All b2-adrenergic agonistswere less active as the acetylcholine-induced tone increased (Figs.1A–E). Significant differences in Emax were found between isoprena-line and salbutamol (P = 0.010) or salmeterol (P < 0.001) under highcontractile tone (EC80) (Table 1). Pearson’s correlation analysis dem-onstrated that mean Emax values for b2-mimetics under low contrac-tile tone were significantly and negatively correlated with meanlosses in efficacy after high muscarinic stimulation (Fig. 2;r = �0.966, P = 0.007). These data were further substantiated by ace-tylcholine-mediated changes in IA. At the high level of precontrac-tion (EC80), the IA of b2-mimetics was reduced to varying degrees:highly for salmeterol and salbutamol, moderately for formoterol, un-changed for fenoterol (Table 1).

Discussion

Experimental models of feline asthma provide evidence thatbronchoconstriction strongly contributes to pulmonary dysfunc-tion (Padrid et al., 1995; Kirschvink et al., 2007). As the numberof cats being diagnosed with so-called ‘feline asthma’ has increasedover the last two decades (Ranivand and Otto, 2008), there is anemerging need to establish accurately that bronchodilators maybe useful in feline practice for prophylactic and/or symptomatictreatment of airflow limitation. In this respect, in vitro preliminaryscreening of bronchodilators is a critical step towards demonstrat-ing drug efficacy. By conducting cumulative dose–response exper-

iments in a tissue chamber system, we found that anticholinergic,b-adrenergic and methylxanthine agents produced effective relax-ation of acetylcholine-contracted feline bronchi. Furthermore, andconsistent with our working hypothesis, feline airway responsive-ness to b-adrenoceptor agonists was dependent on the magnitudeof the initial muscarinic-induced tone.

Under high contractile tone (EC80), the �logEC50 values for the-ophylline and ipratropium bromide were 3.68 ± 0.22 and 8.64 ±0.11 respectively. Our results roughly confirm previous estimatesfor theophylline (3.64–3.90) and ipratropium bromide (9.92) ob-tained on guinea-pig and human airways precontracted with mus-carinic agonists (at a dose producing 70–85% of maximalcontraction) (Diamond and O’Donnell, 1981; Karlsson and Persson,1981; Finney et al., 1985). To the authors’ knowledge, theophyllineis the only active bronchodilator whose pharmacokinetic proper-ties have been specifically addressed in cats. Most recommendeddosages were calculated assuming a therapeutic concentrationrange of 10–20 lg/mL – i.e. those usually considered in humanbeings (McKiernan et al., 1983; Guenther-Yenke et al., 2007). Thistheoretical therapeutic range (4.29–3.99 in logarithmic molar con-centrations) agrees well with the �logEC50 value (3.93) reportedhere for theophylline after low muscarinic stimulation (EC40).

Despite strong in vitro and in vivo evidence for using short-act-ing (salbutamol) and long-acting b2-agonists (salmeterol, formo-terol) in the management of obstructive pulmonary diseases inhumans, little is known on their relative efficacy as relaxing agentsin feline airway preparations. In the present study, the rank orderof potency for b-mimetics under low contractile tone (EC40) wasformoterol > fenoterol > isoprenaline > salbutamol P salmeterol andwas broadly in accordance with those established in other species– i.e. on isolated guinea-pig trachea precontracted with carbachol(Lemoine and Overlack, 1992; Linden et al., 1993) and on isolatedhuman bronchi at resting tone (Naline et al., 1994, 2007). Our re-sults together with the reports cited above also indicated that for-moterol was the most potent b2-stimulant with EC50 valuesapproximately 10 to 100-fold higher than those recorded for salbu-tamol and salmeterol. Thus, it may be argued that feline bronchialsmooth muscle shows the same sensitivity to b2-adrenoceptor ago-nists as other airway preparations in which b2-adrenoceptors pre-dominate over the b1-subtype. Studies using pharmacologicalagonists and antagonists of b-adrenoceptors have revealed thatrelaxation of feline airways is mediated by a mixed population ofb-adrenoceptors, predominantly of the b1-subtype in trachea whilebeing of the b2-subtype in parenchymal lung strips (Lulich et al.,1976; O’Donnell and Wanstall, 1983). Even if any conclusions onb-adrenergic receptor subtypes would require further pharmaco-logical and radioligand binding studies, our results highly suggesta predominantly b2-adrenoceptor population in the feline bron-chial smooth muscle.

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Fig. 1. (A–F) Mean cumulative log10 dose–response curves to: (A) isoprenaline; (B) salbutamol; (C) fenoterol; (D) formoterol; (E) salmeterol; and (F) ipratropium bromide onisolated feline bronchi. Bronchial segments were contracted with acetylcholine to cause either 40% (EC40 = 1.6 � 10�5 mol/L; filled symbols) or 80% (EC80 = 1.2 � 10�4 mol/L;open symbols) of the acetylcholine maximal contraction. Values are means with SEM and are presented as a percentage of the action of theophylline 3 � 10�3 mol/L.Horizontal dotted lines represent 50% of the maximal response to theophylline 3 � 10�3 mol/L.

J. Leemans et al. / The Veterinary Journal 193 (2012) 228–233 231

As the concentration of acetylcholine was increased (EC80), theIA was highly reduced for salmeterol and salbutamol, moderatelyfor formoterol, and unchanged for fenoterol (Table 1). The fact thatthe IA of b2-agonists was reduced to different extents after highmuscarinic contraction (EC80) indicates that some of these ago-nists had greater efficacy at b2-adrenoceptors than others. More-over, it was found that the lower the efficacy under lowmuscarinic tone, the more partial was the agonist under high con-tractile tone. On this basis, it may be stated that fenoterol and for-moterol act as full (or nearly full) agonists of b2-adrenoceptors,salbutamol and salmeterol as partial agonists, the latter displaying

more partial agonistic activity than any of the other b2-mimetics.These in vitro findings raise concerns about how partial agonisticproperties of b2-agonists impact their clinical efficacy. Studies per-formed on healthy cats have shown that salmeterol at a dose elic-iting maximal response produces weaker bronchoprotectiveeffects against muscarinic stimuli in comparison to salbutamol(Leemans et al., 2009). Our results are consistent with those gen-erated in humans and strengthen the current body of evidencethat salmeterol is a partial agonist of b2-adrenoceptors in vitroas well as in vivo (Naline et al., 1994; Molimard et al., 1998;Palmqvist et al., 1999).

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Fig. 2. Relationship between mean Emax values for b2-agonists under low acetyl-choline-induced tone (EC40) and mean losses in efficacy after high muscarinicstimulation. Coefficient of correlation (r) and P are indicated.

232 J. Leemans et al. / The Veterinary Journal 193 (2012) 228–233

Constriction of feline airways with high-dose acetylcholinecaused a rightward shift of the b2-mimetic dose–response curveswith depression of the maximal relaxation. The relaxant effectsof salbutamol, formoterol and salmeterol were significantly re-duced both in potency and efficacy. These findings are the first re-ported evidence for a functional antagonism between non-selective muscarinic agonists (i.e. acetylcholine) and b-adrenergicagonists in feline bronchi and are in agreement with earlier exper-imental investigations on canine, guinea-pig and human airways(Torphy et al., 1983; Lemoine and Overlack, 1992; Linden et al.,1993; Naline et al., 1994; Molimard et al., 1998). The functionalantagonism between cholinergic and adrenergic pathways islinked to co-existence in airway smooth muscle of post-junctionalmuscarinic M2 and M3 receptors. M3 receptors mediate direct con-traction of smooth muscle. M2 receptors are not capable of elicitinga direct contractile response by themselves, but interact withinhibitory G proteins (Gi) to block adenylate cyclase and Ca2+-acti-vated K+ channels, thereby counteracting b-adrenoceptor-inducedbronchodilation (Coulson and Fryer, 2003; Ehlert, 2003).

Another finding of the present study was the inability ofhigh-dose acetylcholine (EC80) substantially to affect isoprena-line-induced relaxation. Pertussis toxin treatment, which uncou-ples M2-receptors from Gi proteins, enhances the relaxant effectsof forskolin (cAMP stimulating relaxant agent), but not those ofisoprenaline, on muscarinic-induced contractions of guinea-pigtrachea (Ostrom and Ehlert, 1999). Using both genetic and pharma-cological approaches, Matsui et al. (2003) have shown thatforskolin exhibits higher inhibitory effects against muscarinic-in-duced contractions in trachea from M2-receptor knockout miceas compared to wild-type mice, while isoprenaline did not. The factthat relaxant effects of isoprenaline are partly mediated by cAMP-and M2-receptor independent pathways may be responsible fordifferences in the magnitude of functional antagonism betweenisoprenaline and other b2-mimetics.

The functional antagonism between muscarinic and b2-adreno-ceptor agonists is difficult to translate in clinical terms. Salbutamoland ipratropium bromide were recently tested for preventive ef-fects against carbachol-induced bronchospasm in healthy cats(Leemans et al., 2009). Combining them enhanced their efficacyin a synergistic fashion, which presumably results from the non-selective blockade of muscarinic receptors by ipratropium bro-mide. As a non-selective anticholinergic, ipratropium bromideantagonises the contractile response of M3-receptors and mostlikely preserves salbutamol from the inhibitory regulation of M2-receptors. Although preliminary, these results support the proposalthat combination therapy would be more effective in the treatmentof airway disease with enhanced cholinergic tone compared to sin-gle drug treatment. This may be of critical importance in feline

practice as cats with idiopathic naturally acquired asthma arethought to have increased parasympathetic tone (Mitchell et al.,1997).

Conclusions

The existing classes of bronchodilators (anticholinergics,b-adrenergic agonists and methylxanthines) are effective in reduc-ing muscarinic-induced tone in isolated feline bronchi. This makescat airways well suited to respond to currently available bronchod-ilators and should prompt further therapeutic trials in cats withexperimentally-induced or naturally-occurring asthma. Increasingcholinergic tone antagonises the relaxant response to mostb2-stimulants. The underlying mechanism as well as the clinicalrelevance of this functional antagonism in cats still needs to beelucidated.

Conflict of interest statement

None of the authors of this paper has a financial or personalrelationship with other people or organisations that could inappro-priately influence or bias the content of the paper.

Acknowledgements

The authors acknowledge F. Delvaux and I. Habsch for theirtechnical assistance, Dr. L. Massart for his statistical support,Dr. E. Naline for his precious advice and Professor C. Advenier forthe generous gift of formoterol and salmeterol. Jérôme Leemanswas supported by FRIA, Belgium. This study was funded by a Grantfrom the Région Wallonne DGTRE, Belgium.

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