gy 550 (2006) 155–161www.elsevier.com/locate/ejphar

European Journal of Pharmacolo

A Rho-kinase inhibitor, Y-27632, reduces cholinergiccontraction but not neurotransmitter release

Lynette Fernandes a,⁎, Angela D'Aprile a, Glenn Self a, Melanie McGuire a,Tracey Sew a, Peter Henry a, Roy Goldie b

a Western Australian Institute for Medical Research and Pharmacology Unit, School of Medicine and Pharmacology,University of Western Australia, Perth, Australia

b Faculty of Health Sciences, Flinders University, Adelaide, Australia

Received 4 August 2005; received in revised form 1 November 2005; accepted 11 August 2006Available online 24 August 2006

Abstract

This study examined the effects of the selective Rho-kinase inhibitor Y-27632 [(+)-(R)-trans-4-(1-aminoethyl)-(4-pyridyl)cyclohexanecarbox-amide dihydrochloride]) on cholinergic nerve-mediated contraction and neurotransmitter release in murine and guinea-pig isolated trachealpreparations. In tracheal preparations obtained from both species, Y-27632 shifted carbachol concentration–effect curves to the right and reducedthe maximal contractile response. Repeated electrical field stimulation (EFS) evoked transient, consistent and reproducible contractions in murineand guinea-pig tracheal preparations. Y-27632 inhibited these cholinergic nerve-mediated contractions in a concentration-dependent manner. EFS(0.1–30 Hz) elicited frequency-dependent cholinergic nerve-mediated contractile responses. In murine tracheal preparations, Y-27632 (3 μM and10 μM) shifted frequency–response curves to EFS to the right by 5.5 and 13.0 fold respectively and markedly reduced the maximal contractileresponse. In murine and guinea-pig tracheal preparations loaded with [3H]-choline, Y-27632 (10 μM) significantly increased the EFS-inducedoutflow of radioactivity from airway cholinergic nerves by 27% and 54% respectively. Thus, Y-27632 inhibited both carbachol-induced andcholinergic nerve-mediated contractile responses. Conversely, Y-27632 increased neurotransmitter release from airway cholinergic nerves.However, since antagonism of acetylcholine-induced contraction by Y-27632 overwhelmed the increased neurotransmitter release, the overalleffect of this Rho-kinase inhibitor was to inhibit cholinergic nerve-mediated contraction.© 2006 Elsevier B.V. All rights reserved.

Keywords: Rho-kinase; Trachea; Smooth muscle contraction; Cholinergic nerve; Acetylcholine release

1. Introduction

It has been suggested that cholinergic nerve function may beupregulated in airway diseases such as asthma and chronicobstructive pulmonary disease (COPD) (Jacoby and Fryer, 2001;Jartti, 2001). Anti-cholinergic bronchodilators such as ipratro-pium and tiotropium are effective bronchodilators in themanagement of both asthma and COPD (Barnes, 2004; Rodrigoand Rodrigo, 2002; Westby et al., 2004). Indeed, some asthmaticpatients show a greater bronchodilator response to ipratropium ascompared with salbutamol (Chhabra and Pandey, 2002),suggesting increased cholinergic bronchoconstriction in thissub-group of individuals. Some common triggers of asthma

⁎ Corresponding author. Tel.: +61 8 9346 4517; fax: +61 8 9346 3469.E-mail address: Lynette.Fernandes@uwa.edu.au (L. Fernandes).

0014-2999/$ - see front matter © 2006 Elsevier B.V. All rights reserved.doi:10.1016/j.ejphar.2006.08.018

exacerbations include respiratory tract viral infection, inhalationof allergens or exposure to irritant air pollutants. These triggershave been shown to increase cholinergic nerve function in theairways and may explain the bronchodilator effect of anti-cholinergic agents in acute asthma (Jacoby and Fryer, 2001).Furthermore, since cholinergic tone is the main reversiblecomponent of COPD, anti-cholinergic drugs play an importantrole in the treatment of bronchial obstruction in COPD (Barnes,2004).

Stimulation of muscarinic acetylcholine G-protein-coupledreceptors is now known to be linked to a signal transductionpathway involving RhoA and its downstream modulator Rho-kinase. Activation of the RhoA/Rho-kinase pathway by agonistssuch as acetylcholine can promote phosphorylation of myosinlight chain kinase and increase [Ca2+]i, resulting in contractionof airway smooth muscle.

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The Rho-kinase inhibitor Y-27632 [(+)-(R)-trans-4-(1-ami-noethyl)-(4-pyridyl)cyclohexanecarboxamide dihydrochloride]has been shown to induce relaxation in airway preparationsobtained from various animal species as well as human airways.Indeed, Y-27632 has been shown to reverse airway tone caused bya range of spasmogens in vitro (Burdyga et al., 2003; Chiba et al.,2001; Gosens et al., 2004; Nakahara et al., 2000; Setoguchi et al.,2001; Yoshii et al., 1999) and in vivo (Hashimoto et al., 2002a,b;Iizuka et al., 2000). The Rho-kinase inhibitor Y-27632 has alsobeen shown to inhibit cholinergic, noradrenergic and purinergicnerve-mediated contractile responses in isolated smooth musclepreparations. For example, Y-27632 reversed electrical fieldstimulation (EFS)-induced nerve-mediated contractions in mouse(Büyükafşar and Levent, 2003), rabbit and human penile corpuscavernosum tissue (Rees et al., 2001), in rat and human prostaticsmooth muscle (Rees et al., 2003) as well as in mouse vasdeferens (Büyükafşar and Levent, 2003). In addition, Y-27632attenuated electrically-evoked contractions in rat urinary bladder(Wibberley et al., 2003) and sheep ureter (Levent and Büyükafşar,2004). Consistent with a direct inhibitory influence on cholinergicnerve function, Y-27632 has been shown to cause both decreasedneurotransmitter release from cholinergic nerves in the mousegastric fundus (Büyükafşar and Levent, 2003) and relaxation ofthese preparations. These data suggest that Y-27632 acts pre-synaptically to reduce excitatory neurotransmitter release. How-ever, Y-27632 may also act post-synaptically to directly reducesmooth muscle tone. In the airways, the effect of Y-27632 tomodulate cholinergic nerve-mediated contraction as well asneurotransmitter release has not been examined.

The Rho-kinase inhibitor Y-27632 has proven bronchodilator(Hashimoto et al., 2002a,b; Iizuka et al., 2000) and anti-inflam-matory (Henry et al., 2005) activity. The ability ofY-27632 to alsoreduce neurotransmitter release from airway cholinergic nerveswould further enhance the therapeutic potential of Rho-kinaseinhibitors in the treatment of asthma and COPD, since this anti-spasmogenic, pre-junctional action would be matched by its wellrecognised post-synaptic inhibitory action against acetylcholine-induced contraction. Accordingly, the present study investigatesthe effect of the Rho-kinase inhibitor Y-27632 on cholinergicnerve-mediated contraction in murine and guinea-pig isolatedtrachea and evaluates the influence of Y-27632 on acetylcholinerelease from airway cholinergic nerves.

2. Methods

2.1. Tissue preparation

All experimental protocols were approved by the AnimalEthics Committee, University of Western Australia and werecarried out in accordance with the National Health and MedicalResearch Council's Australian code of practice for the care anduse of animals for scientific purposes. Mice (CBA/CaH, 5–10 weeks old) and guinea-pigs (SR/C Tricolour, 200–300 g)were sacrificed with Nembutal (sodium pentobarbitone,300 mg/kg i.p.) and exsanguinated via the renal vein. Thetrachea was dissected free of associated tissues and placed inKrebs Bicarbonate Solution (KBS; composition in mM: NaCl

117, KCl 5.36, NaHCO3 25, KH2PO4 1.03, MgSO4·7H2O 0.57,CaCl2·2H2O 2.5 and glucose 11.1).

2.2. Carbachol-mediated contractions

Murine and guinea-pig tracheal ring preparations were sus-pended between 2 stainless steel hooks in 2 ml organ bathscontaining KBS and connected to an isometric force transducer.The resting tension of tracheal preparations was adjusted to 200–350 mg for murine and 600–700 mg for guinea-pig trachealpreparations. KBS was aerated with 5% CO2 in oxygen andmaintained at 37 °C. KBS contained indomethacin (3 μM) sincecyclooxygenase products including prostaglandins have beenshown to influence airway tone. Tracheal preparations wereequilibrated for 30 min during which time the bathing mediumwas replaced. The tension in murine and guinea-pig trachealpreparations was readjusted as required to 200–350 mg or 600–700 mg, respectively. Tracheal rings were then exposed tocumulative concentrations of carbachol (0.2 and 10 μM) toconfirm tissue viability. This process was repeated to establish themaximum contractile response to carbachol at 10 μM (Cmax).Isometric contractile responses were measured using an FTO3force-displacement transducer (Grass Instruments, Quincy, MA)linked to a pre-amplifier and a computer-based data acquisitionsystem.

Concentration–effect curves to carbachol (0.03–30 μM)were constructed in the absence and presence of Y-27632 (0.3–10 μM). In the latter experiments, tracheal preparations wereequilibrated with Y-27632 for 15 min prior to the constructionof each carbachol concentration–effect curve. Y-27632 failed toalter baseline tone in either murine or guinea-pig trachealpreparations, suggesting the absence of inherent tone in thepresence of indomethacin. Changes in carbachol-induced tonein the absence and presence of Y-27632 were measured as apercentage of the response to 10 μM carbachol.

2.3. Cholinergic nerve-mediated contractions

Murine tracheal preparations were suspended in organ bathsbetween two parallel platinum electrodes. Electrical fieldstimulation (EFS) was delivered by a Grass S44 stimulatorconnected to a Med-Lab Stimu-Splitter II (Med-Lab Instruments,Loveland, Co) and an automated timing device. Trachealpreparations were exposed to indomethacin (3 μM), propranolol(1 μM) and Nw-nitro-L-arginine methyl ester (L-NAME,100 μM). Cyclooxygenase products have been shown to bereleased from airway tissue following EFS and have the potentialto modulate cholinergic nerve-mediated contractions and neuro-transmitter release (Fernandes et al., 1994; Spicuzza et al., 1998.Propranolol and L-NAME were used to inhibit adrenergic andinhibitory non-adrenergic non-cholinergic nerve-mediatedresponses respectively. The EFS parameters used were: 0.1–30 Hz, 11–15 V, 150–200 mA, 0.5 ms pulse duration, 10 s trainwith 3 min intervals between successive stimulations. Thesestimulation parameters have been shown to selectively activateairway cholinergic nerves (Ellis and Undem, 1990). EFS-inducedcontractions were abolished in the presence of either atropine

Table 1Carbachol pD2 and Emax values in the absence and presence of Y-27632

Carbachol pD2 Carbachol Emax (%)

Murine tracheaCarbachol alone 6.7±0.2 105±8+0.3 μM Y-27632 6.8±0.1 102±7+1 μM Y-27632 6.7±0.1 110±4+3 μM Y-27632 6.1±0.1a 93±4+10 μM Y-27632 5.5±0.1a 71±4a

Guinea-pig tracheaCarbachol alone 7.0±0.1 105±1+0.3 μM Y-27632 7.0±0.1 101±1+1 μM Y-27632 6.8±0.1 99±2+3 μM Y-27632 6.7±0.1a 99±2+10 μM Y-27632 6.5±0.1a 93±2a

Values represent mean±S.E.M. (n=7–10). Statistical differences were calcu-lated using one-way ANOVA followed by Bonferroni t-test. aPb0.05 comparedwith carbachol alone.

Fig. 1. (A) Representative trace of cholinergic nerve-mediated contractileresponses induced by repeated EFS in murine tracheal rings in the absence(Time control) and presence of Y-27632. The EFS parameters used were 3 Hz, 11–15 V, 150–200 mA, 0.5 ms pulse duration, 10 s train with 3 min intervals betweensuccessive stimulations. Cholinergic nerve-mediated contractile responses inducedby repeated EFS in (B) murine and (C) guinea-pig tracheal rings in the absence(open bars) and presence (closed bars) of Y-27632. At 0 μM Y-27632, the closedbar represents the initial contractile responses obtained prior to addition of theRho-kinase inhibitor. EFS-induced contractile responses obtained in the presenceof Y-27632 were calculated as a percentage of this initial contractile response.Data represents mean±S.E.M. (n=6–8). Statistical differences were calculatedusing unpaired t-test. *Pb0.05 compared with corresponding data obtained in theabsence of Y-27632.

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(0.1 μM) or tetrodotoxin (3 μM), demonstrating that contractileresponses were both cholinergic and neural in nature.

Tracheal preparations were repeatedly stimulated (3 Hz) inthe absence or presence of Y-27632 (0.3–10 μM). EFS-inducedcontractile responses obtained in the presence of Y-27632 werecalculated as a percentage of the initial contractile responseobtained in the absence of the Rho-kinase inhibitor. In otherexperiments, some preparations were equilibrated with Y-27632(1–10 μM) for 15 min prior to the construction of frequency–response curves to EFS (0.1–30 Hz). Thus, frequency–responsecurves to EFS were constructed in the absence and presence ofY-27632. EFS-induced contractile responses were assessed as apercentage of the contractile response induced at 30 Hz in theabsence of Y-27632 (Emax). The frequency of EFS causing acontractile response equivalent to 20% Emax (EF20) wasdetermined. The magnitude of cholinergic nerve-mediatedcontractile responses induced at 30 Hz were also determined.

2.4. Neurotransmitter release

Wessler et al. (1994) have shown that EFS-induced efflux ofradioactivity derived from [3H]-choline is higher in epithelium-denuded compared with epithelium-intact tracheal preparations.Thus, epithelium-denuded tracheal preparations from either 5mice or 1 guinea-pig were used in each experiment. Epitheliumdenudation was confirmed by histological examination. Trache-al tube preparations were mounted onto a wire platinumelectrode and loaded into an acrylic flow chamber. A secondplatinum electrode was embedded into the wall of the chamber.Tracheal preparations were superfused (∼2ml/min) with KBS at37 °C and aerated with 5% CO2 in O2, containing indomethacin(3 μM), propranolol (1 μM) and L-NAME (100 μM). Trachealpreparations were allowed to equilibrate for 20 min before beingelectrically stimulated (5–8 V, 150–200 mA, 0.5 ms pulseduration, 1 Hz) for 10 min. This period of EFS was used topromote acetylcholine turnover in cholinergic nerves. EFS(10 Hz) was then applied for 60 min, during which 3 ml of KBScontaining [3H]-choline (30 μM) was continuously superfusedover the tracheal tissue. This loaded cholinergic nerves with

[3H]-choline for conversion to [3H]-acetylcholine. This loadingperiod was followed by a 90 min washout period where trachealpreparations were superfused with KBS containing the cholineuptake inhibitor hemicholinium-3 (10 μM) and the M2

muscarinic acetylcholine antagonist gallamine (100 μM) tomaximise neurotransmitter release from cholinergic nerves. Theentire tracheal superfusate was collected over the next 90 minperiod. Thus, 30 aliquots of superfusate were collected over3 min periods. The release of radioactivity (dpm/g/3 min) fromtracheal preparations was also measured during two×2 minperiods (S1 and S2) of EFS (3 Hz) between the 42nd and 44thmin and 69th and 71st min of the collection period. In someexperiments, tracheal preparations were exposed to Y-27632(10 μM) or the neurotoxin tetrodotoxin (3 μM) for 15 min priorto S2 (between the 54th and 71st min). Liquid scintillation

Fig. 2. Cholinergic nerve-mediated frequency–response curves to EFS wereconstructed in murine tracheal preparations in the (○) absence and presence of(▴) 1 μM, (♦) 3 μM and (▾) 10 μM Y-27632. EFS-induced contractileresponses were assessed as a percentage of the contractile response induced at30 Hz in the absence of Y-27632. Data represents mean±S.E.M. (n=7–8).

Table 2Cholinergic nerve-mediated frequency–response curves to EFS wereconstructed in murine tracheal preparations in the absence and presence of Y-27632 to determine mean EF20 and Emax values

EF20 Emax (% Control at 30 Hz)

Murine tracheaControl 0.2±0.0 96±1+1 μM Y-27632 0.4±0.1 79±4+3 μM Y-27632 1.1±0.2a 52±6a

+10 μM Y-27632 2.6±0.7a 48±4a

EF20 is defined as the effective frequency causing 20% of the maximum EFS-induced contraction (30 Hz, Control preparations). Values represent mean±S.E.M.(n=7–8). Statistical differences were calculated using one-way ANOVA followedby Bonferroni t-test. aPb0.05 compared with contractile responses obtained in theabsence of Y-27632.

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cocktail (Star Scint™, Packard, Netherlands) was added to eachaliquot and assayed for radioactivity using a liquid scintillationcounter (TriCarb 1500, Packard Instrument Co. Inc., USA).

The release of radioactivity derived from [3H]-choline wasplotted against time and a line of regression extrapolated usingvalues obtained between the 3rd–42nd and 69th–90th min.These lines of regression were used to provide an estimate ofbasal radioactivity outflow from tracheal preparations. EFS-induced radioactivity outflow was assessed as the differencebetween that released at S1 and S2 and the relevant basal outflowof radioactivity.

2.5. Drugs and chemicals

Nembutal (sodium pentobarbitone) was obtained fromBoehringer Ingelheim (Ingelheim, Germany). Atropine, carba-chol, gallamine, hemicholinium-3, indomethacin, L-NAME,propranolol and tetrodotoxin were obtained from Sigma (St.Louis, MO, USA). Y-27632 was obtained from Tocris (Avon-mouth, UK). [3H]-choline was obtained from AmershamBiosciences (Buckinghamshire, UK; specific activity 81.0 Ci/mmol). All other chemicals were of analytical grade andobtained from standard commercial sources.

2.6. Statistical analysis

Results are expressed as mean±S.E.M. unless otherwisestated. The n value represents the number of animals used. Anexception to this are neurotransmitter release experiments usingmurine tracheal preparations where the n value represents thenumber of experiments conducted. EC50, EF20 and S2:S1 ratioswere normalised by transformation to pD2, log EF20, and − log10(S2:S1) values respectively for statistical analysis. In repeated EFSstudies, an unpaired t-test was used to determine the effect ofY-27632 compared with the corresponding Control. Differencesinmean data obtainedwere determined using one-way analysis ofvariance (ANOVA) followed by a post hoc Dunnett's orBonferroni t-test where appropriate (Wallenstein et al., 1980). AP value of less than 0.05 was considered statistically significant.

3. Results

3.1. Y-27632 inhibits carbachol- and cholinergicnerve-mediated contractions

Lower concentrations of the Rho-kinase inhibitor Y-27632(0.3 μMand 1 μM) failed to alter carbachol potency or maximumcontractile responses in murine or guinea-pig isolated trachealpreparations. Inmurine trachea, 3μMand 10μMY-27632 shiftedcarbachol concentration–effect curves to the right by 3.5 and 16.2fold respectively (Table 1). At 10μM, the highest concentration ofY-27632 used, carbachol Emax values were reduced by 34%(Table 1). Similar results were obtained in guinea-pig trachea.Here, 3 μMand 10μMY-27632 shifted carbachol concentration–effect curves to the right by 2 and 3 fold respectively. Y-27632(10 μM) also reduced carbachol Emax values by 12% (Table 1).

Repeated EFS (3 Hz) evoked transient, consistent andreproducible contractile responses in murine and guinea-pigisolated tracheal preparations. The Rho-kinase inhibitor Y-27632reversed cholinergic nerve-mediated contractions in a concentra-tion-dependent manner. In murine tracheal preparations, 1 μM,3 μM and 10 μM Y-27632 suppressed cholinergic contractileresponses by 52%, 61% and 70% respectively (Fig. 1B). Inguinea-pig tracheal preparations, these concentrations of Y-27632reduced cholinergic contractions by 44%, 66% and 80%respectively (Fig. 1C).

EFS (0.1–30 Hz) elicited frequency-dependent cholinergicnerve-mediated contractions in murine isolated trachea (Fig. 2).EFS-evoked contractile responses were smaller in magnitudecompared with those induced by carbachol. For example, inguinea-pig tracheal preparations, contractile responses obtainedat 30 Hzwere equivalent to approximately 50% of the contractileresponse obtained to 10 μM carbachol. Since higher concentra-tions of Y-27632 caused marked reductions in EFS-inducedcontractile responses, EF20 was selected to examine the effect ofthis Rho-kinase inhibitor on frequency–response curves to EFS.Y-27632 inhibited cholinergic nerve-mediated contractions in aconcentration-dependent manner across the entire frequencyrange. Whereas the lowest concentration of Y-27632 (1 μM)failed to alter either EF20 or Emax values, higher concentrationsof Y-27632 (3 μM and 10 μM) shifted the frequency–responsecurve to the right by 5.5 and 13 fold respectively and markedly

Table 3S2:S1 ratios and mean − log10(S2:S1 ratio) obtained following EFS of murine andguinea-pig tracheal preparations pre-loaded with [3H]-choline

S2:S1 ratio − log10(S2:S1 ratio)

Murine tracheaControl 0.35 0.46±0.03+10 μM Y-27632 0.44 0.35±0.01a

+3 μM tetrodotoxin 0.09 1.40±0.64a

Guinea-pig tracheaControl 0.51 0.31±0.04+10 μM Y-27632 0.78 0.12±0.05a

+3 μM tetrodotoxin 0.05 1.34±0.12a

S2:S1 ratios were calculated and transformed to − log10(S2:S1 ratio) for statisticalanalysis. Values represent mean±S.E.M. (n=3–7). Statistical differencesbetween − log10(S2:S1 ratio) were calculated using one-way ANOVA followedby Dunnett's t-test. aPb0.05 compared with respective Control.

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reduced the maximum contractile response (Table 2 and Fig. 2).Similar responses were also obtained in guinea-pig isolatedtracheal preparations (data not shown).

3.2. Y-27632 augments neurotransmitter release from airwaycholinergic nerves

The basal outflow of radioactivity (derived from [3H]-choline)from murine and guinea-pig tracheal preparations was consistentthroughout the collection period in the absence and presence ofeither Y-27632 (10 μM) or tetrodotoxin (3 μM). In Controlexperiments, the outflow of radioactivity increasedmarkedlywithEFS at S1 and S2. As expected, radioactivity outflow at S2was lessthan that at S1, producing S2:S1 ratios of less than 1. S2:S1 ratioswere calculated for each experiment conducted in the absence orpresence of either tetrodotoxin (3 μM) or Y-27632 (10 μM).These S2:S1 ratios were then normalised by transformation to− log10(S2:S1) values for statistical analysis. In the presence oftetrodotoxin (3 μM), the release of radioactivity associated withS2 was virtually abolished (Table 3). This confirmed that therelease of radioactivity associated with EFS was neuronal inorigin. When − log10(S2:S1) values obtained in the absence andpresence of Y-27632 (10 μM) were compared, the Rho-kinaseinhibitor was found to have significantly increased EFS-inducedrelease of radioactivity from murine and guinea-pig isolatedtracheal preparations by 27% and 54% respectively (Table 3).

4. Discussion

The current study confirms that Y-27632 reverses carbachol-induced airway smooth muscle tone in vitro (Yoshii et al., 1999).Y-27632 (3 and 10 μM) shifted carbachol concentration–effectcurves to the right by between 2 and 16 fold. At the highestconcentration used in this study (10 μM), Y-27632 also reducedthe maximal contractile response to carbachol. The inhibitoryeffect of Y-27632 on carbachol-induced contractile responsesappeared to be greater in murine compared with guinea-pigisolated trachea, suggesting a small species difference. Thus,3 μM and 10 μM Y-27632 shifted carbachol concentration–effect curves to the right by 3.5 and 16.2 fold respectively in

murine trachea and 2 and 3 fold respectively in guinea-pigtrachea. These data support previous reports indicating that themuscarinic acetylcholine receptor signal transduction pathway islinked to Rho-kinase (Ding and Murray, 2005; Schmidt et al.,1999).

Airway smooth muscle tone is controlled predominantly bythe parasympathetic nerve pathway. It has been suggested thatupregulation of this nerve pathway may contribute to increasedairway tone in asthma and COPD (Jacoby and Fryer, 2001; Jartti,2001). Indeed, anti-cholinergic bronchodilator drugs such asipratropium and tiotropium are useful in the management ofdisease in these patients (Barnes, 2004; Rodrigo and Rodrigo,2002; Westby et al., 2004). The Rho-kinase inhibitor Y-27632,in a concentration-dependent manner, inhibited EFS-evokedcontractile responses elicited by repeated stimulation in murineand guinea-pig isolated tracheal preparations. Furthermore, intracheal preparations obtained from both species, Y-27632shifted frequency–response curves to EFS to the right. Thisinhibitory effect on EFS-induced contractions was apparentacross the entire frequency range. This is in accord with previousstudies where Y-27632 inhibited nerve-mediated contractileresponses following stimulation of cholinergic, adrenergic andpurinergic nerves (Büyükafşar et al., 2003; Büyükafşar and Ün,2003; Levent and Büyükafşar, 2004; Rees et al., 2001, 2003;Wibberley et al., 2003).

The effect of Y-27632 on EFS-induced release of radioactivityfrom airway cholinergic nerves was investigated, since inhibitoryaction at either or both a pre- or post-junctional site could explainreduced response to EFS. Surprisingly, Y-27632 was found tosignificantly increase EFS-induced release of radioactivity fromcholinergic nerves in bothmurine and guinea-pig trachea. This is incontrast to the results of the study by Büyükafşar and Levent(2003), who demonstrated that Y-27632 caused relaxation anddecreased neurotransmitter release from cholinergic nerves ingastrointestinal smooth muscle. However, these authors (Büyü-kafşar and Levent, 2003) used a 10 times higher concentration ofY-27632 compared with the current study, raising the possibility ofa non-specific action in reducing neurotransmitter release.Importantly, each gastric fundal strip preparation was stimulatedonly once in neurotransmitter release studies (Büyükafşar andLevent, 2003). The use of two periods of EFS in the present studyprovided a more robust approach to examining the effect of agentssuch as Y-27632 on neurotransmitter release from cholinergicnerves. In the present study, Y-27632 increased the outflow ofradioactivity from guinea-pig tracheal preparations by 54%,compared with 27% in murine trachea. While these increases inoutflow of radioactivity are significant, the dominant effect ofY-27632 on airway smooth muscle tone remains inhibitory. Inter-estingly, β2-adrenoceptor bronchodilators, which have a domi-nant post-junctional relaxant effect, have also been shown toincrease neurotransmitter release from airway cholinergic nerves(Belvisi et al., 1996; De Haas et al., 1999; Zhang et al., 1995,1996a,b). The possible mechanisms by which Y-27632 increasesneurotransmitter release from airway cholinergic nerves areunclear and warrant further investigation. Y-27632 is a potent andspecific inhibitor of Rho-kinase and is unlikely to have non-specific actions at the concentrations used in the current study

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(Ihizaki et al., 2000). The effect of a range of concentrations of Y-27632 on neurotransmitter release could be investigated.However, detecting small changes in neurotransmitter releasewith low concentrations of Y-27632 may be a limiting factor. TheM2 muscarinic acetylcholine antagonist gallamine was used in allneurotransmitter release experiments to maximise neurotransmit-ter release from airway cholinergic nerves, ruling out any effect ofY-27632 on altering the function of this receptor. Finally, it ispossible that Y-27632 alters secretion-coupling mechanismslinked to neurotransmitter release from airway cholinergic nerves.This last option seems the most likely and warrants furtherinvestigation.

The data obtained in this study suggest that the inhibitory effectof Y-27632 on cholinergic nerve-mediated contraction is due toantagonism of acetylcholine-induced contraction. Furthermore,the inhibitory effect of Y-27632 on airway smooth muscleovercomes the increase in neurotransmitter release caused by thisRho-kinase inhibitor. That Y-27632, like β2-adrenoceptoragonists (Belvisi et al., 1996; De Haas et al., 1999; Zhang et al.,1995, 1996a,b), increases acetylcholine release from airwaycholinergic nerves should not rule out the potential for Rho-kinaseinhibitors in the treatment of asthma and COPD. Indeed, theoverall effects of Y-27632 in the airways are bronchodilator andanti-inflammatory (Henry et al., 2005).

Acknowledgements

This work was supported by grants from the WesternAustralian Institute for Medical Research as well as the Athelstanand Amy Saw Bequest.

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