ORIGINAL ARTICLE

Parasympathetic neurotransmission in rabbit isolated bronchus ismodulated at prejunctional sites via endothelinB

receptor stimulation

K O. McKAY,1 P R. A. JOHNSON,1 J L. BLACK1 AND C L. ARMOUR2

Departments of 1Pharmacology and 2Pharmacy, The University of Sydney, New South Wales, Australia

Parasympathetic neurotransmission in rabbit isolated bronchus is modulated at prejunctionalsites via endothelinB receptor stimulationMcKAY KO, JOHNSON PRA, BLACK JL, ARMOUR CL. Respirology 2000; 5: 343–353Objective: The aim of this study was to investigate the mechanism involved in endothelin-inducedpotentiation of the response to parasympathetic nerve stimulation.Methodology: We used autoradiographic and functional studies in rabbit isolated bronchi.Results: Autoradiography revealed dense binding sites for radiolabelled endothelin-3 overbronchial parasympathetic ganglia. The contractile response of the bronchus to electrical field stimulation was significantly potentiated by endothelin-3, endothelin-1, sarafotoxin S6c and BQ-3020 to 326 ± 53%, 293 ± 63%, 514 ± 119% and 655 ± 178%, respectively, of control values. Theendothelin-3-induced potentiation of neurally evoked responses was not affected by the presenceof propranolol, phentolamine or hexamethonium. The potentiation was also unaltered by pretreat-ment with the endothelinA receptor antagonist BQ-123 (3 µmol/L), but was significantly reduced inthe presence of the combined endothelinA/endothelinB receptor antagonist PD 145065, indicatingthat the potentiation was mediated via endothelinB receptors. Confirmation of endothelinB receptorinvolvement in the neuropotentiation was obtained by demonstration of a significant ameliorationof the potentiation in the presence of the endothelinB receptor selective antagonist BQ-788, and afterendothelinB receptor desensitization by the endothelinB receptor selective agonist sarafotoxin S6b.Conclusions: These results suggest that the endothelin-induced potentiation of parasympatheticneural responses in the rabbit bronchus is mediated via endothelinB receptor activation.

Key words: autoradiography, electrical field stimulation, endothelinB receptor, neuromodulation,parasympathetic neurotransmission.

endothelin have been visualized autoradiographi-cally on bronchial and vascular smooth muscle.5 Thepresence of specific binding sites for endothelin-1and endothelin-2 on human bronchial parasympa-thetic ganglia has also been observed.6 These sitessuggested a possible neuromodulatory role forendothelin in the bronchi in addition to the estab-lished direct effects of endothelin on bronchial tone.Indeed, we previously noted that in segments ofrabbit isolated bronchus, endothelin-3 producedmarked potentiation of the response to parasym-pathetic nerve stimulation.7 Subsequent studies confirmed this neuromodulatory effect of theendothelins in human bronchus8 and in the trachealisof the mouse,9 rat10 and rabbit.11

The parasympathetic nervous system is theprimary neural network within the lung and stimula-tion of cholinergic nerves leads to a marked contrac-tion of airway tissue. The results of previous studies

Respirology (2000) 5, 343–353

INTRODUCTION

The endothelins1 have many properties, which maybe relevant to normal and aberrant airway structureand function. These include mitogenesis of airwaysmooth muscle,2 direct contraction of airway smoothmuscle,3 and an increased presence in bronchialbiopsies of asthmatics.4 Specific binding sites for

Correspondence: Dr Karen McKay, Children’s ChestResearch Centre, Department of Respiratory Medicine,Royal Alexandra Hospital for Children, PO Box 3515, Par-ramatta NSW 2124, Australia. Email: karenm8@nch.edu.au

Received 11 April 2000; accepted for publication 18July 2000.

suggest that parasympathetic neurotransmissionmay be modified at sites including the ganglion.12,13

Indeed, alpha and beta adrenoceptors have beendemonstrated on pulmonary parasympatheticganglia14 and it has been suggested that occupation ofthese receptors may modulate cholinergic neuro-transmission.15 Thus, the ganglion is a likely site atwhich modulation of airway parasympathetic neuraltransmission may occur and, therefore, neuropoten-tiation produced by endothelin in rabbit bronchusmay also occur at this site.

At least two types of endothelin receptor have beenidentified; the endothelinA receptor at which theendothelin-1 and endothelin-2 isoforms are equipo-tent and more potent than endothelin-3, and theendothelinB receptor at which all three isoforms haveequal potency.16 Selective agonists for the endothelinB

receptor have been identified and these include BQ-3020 and the peptide sarafotoxin S6c. These, and alimited number of selective endothelinA, endothelinB

and combined endothelinA/endothelinB receptorantagonists, allow identification of the receptorsubtype mediating specific effects. Previous studieshave shown that there are endothelinB receptors on parasympathetic neurons17 and that the endo-thelinB receptor is involved in neuromodulation inrabbit trachealis11 in addition to other organs andtissues.8,9,18

Marked regional differences in response to stimu-lation have been reported for the lung. Neurallyinduced relaxant responses in ferret trachea wereshown to be solely adrenergic in nature, while in bronchi from the same animals, the relaxationoccurred via a combination of adrenergic andinhibitory NANC stimulation.19 Moreover, potentia-tion of cholinergic responsiveness by thromboxane A2

has been demonstrated in rabbit bronchi but nottrachea,20 and substance P-induced inhibition ofresponsiveness is dependent upon the region of therabbit airway tree under examination, with a greatereffect recorded in more proximal airways.21 In thepresent investigations, we have therefore attemptedto localize the site and, in particular, the receptor, at which endothelin-3 induced neuromodulationoccurs within the bronchi of the rabbit lung. We haveused autoradiography to detect binding sites forendothelin on bronchial ganglia and have used spe-cific agonists and antagonists in functional studies toidentify the mechanism through which this neuro-modulation is mediated.

METHODS

Mature New Zealand White rabbits (weight range3.0–3.8 kg) were killed by a blow to the head or exsan-guination, methods approved by the Animal EthicalReview Committee of the University of Sydney, NewSouth Wales, Australia. The entire cardiopulmonarysystem from the larynx downwards was removed enbloc from the thoracic cavity and immediately placedin ice-cold Krebs’–Henseleit solution which had beensaturated with carbogen gas (5% CO2 in oxygen). Theheart was removed, and the trachea and intrapul-monary main bronchi were dissected from the sur-

rounding parenchymal tissue. A segment of trachea(measuring 1 cm in length) immediately proximal tothe bifurcation was excised as were 4–5 mm long ringsof the intrapulmonary main bronchus from the lowerlobe of each lung.

Autoradiographic studies

Five airway segments (one tracheal and four bron-chial) from each animal were frozen in Tissue TekOCT embedding medium over liquid nitrogen andstored at –70°C until required. Between 30 and 60 sec-tions were cut from each of the five segments, andairways from three rabbits were used. All slides wereplaced in a –20°C freezer until use in autoradio-graphic studies incorporating previously publishedmethodology6 and 25 pmol/L [125I]-endothelin-3 inthe presence of 10 µmol/L phosphoramidon, with theaddition of 0.1 µmol/L endothelin-3 to the incubationmixture to assess ‘non-specific binding’.

Functional studies

Segments of intralobar main bronchi were paired(one segment from approximately the identical posi-tion in the right and left lung) and mounted onperspex rods (equipped with stainless steel elec-trodes) under 1 g of preload in glass tissue baths con-taining Krebs’–Henseleit solution maintained at 37°Cand bubbled with carbogen gas. This preload hasbeen shown to be optimal for rabbit bronchi.22

After calibration, the tissues were washed at 15 minintervals for 1 h. When a stable baseline had beenobtained, the tissues were exposed to either acetyl-choline (1 mmol/L, termed the reference response) orelectrical field stimulation according to the followingprotocols. Antagonists were in contact with the tissuefor 30 (cumulative concentration–response curveexperiments) or 32 (electrical field stimulation exper-iments) min before subsequent experimentation inall series using antagonists.

Effect of endothelin-3 on the frequency–response relationship

In these experiments, four bronchial rings from eachanimal were used, two from the left and two from thecorresponding position in the right lung. When stablebaseline tone was attained after the 1 h equilibrationperiod, frequency–response curves were performedin each tissue using Grass SD9 stimulators with thevoltage set at 20 V and the stimulus duration at 0.6 msec for each tissue. The frequency settings wereincreased incrementally from 0.25 to 64 Hz, the stim-ulus was applied for 20 s at 4 min intervals and the frequency of stimulation was doubled every 4 minuntil 64 Hz was reached. These settings resulted incontractile responses which were equal to 4–10%Tmax(Ach) at 1 Hz. Two frequency–response curveswere elicited in each tissue; 20 min was allowedbetween each curve. After the first frequency–response curve (baseline curve), either endothelin-3

344 KO McKay et al.

(1 nmol/L, 10 nmol/L or 100 nmol/L) or diluent wereadded to the baths before the second frequency–response curve was elicited in each tissue. Atropine (1 µmol/L) was added to the baths at the conclusionof the experiment and the absence of an evokedresponse to any frequency confirmed stimulation ofparasympathetic nerves.

Effect of endothelin receptor agonists onparasympathetic neurotransmission

In this series of experiments, duplicate pairs of tissues(two from the left and two from the right lung) fromeach animal were field stimulated with constant fre-quency and voltage. The settings for the voltage andfrequency delivered by the stimulators were altereduntil a submaximal contractile response with a mag-nitude in the range 200–600 mg of generated forcewas attained. In all cases, the frequency settingrequired for such a response was between 2 and 12 Hz, the voltage setting between 8 and 30 V, theduration of stimulation was 0.6 msec, and the fieldwas applied for 20 s every 4 min. When three consec-utive responses of equivalent magnitude (differingfrom their mean by less than 10%) were obtained ineach tissue, endothelin-1 (1 pmol/L) was added toone tissue from each pair and diluent to the other.Eight responses (over 32 min) were elicited betweenthe addition of each increasing concentration ofendothelin-1 (1 pmol/L to 300 nmol/L at half-log unit increments). In further series of experiments,this procedure was repeated with either endothelin-3, sarafotoxin S6c or BQ-3020 instead of endothelin-1. Again, atropine (1 µmol/L) was added to the bathsat the conclusion of each experiment to confirm stimulation of parasympathetic nerves.

Localization of the site of parasympatheticneuromodulation by endothelin-3

Sets of four bronchial rings from each animal wereused in these series, and only one antagonist wasused in each experimental series. As above, whenthree consecutive equivalent contractile responses tofield stimulation at constant frequency and voltagewere obtained, the antagonist under investigationwas added to two baths and an equivalent volume ofdiluent to the remaining two baths. After eight stim-ulations 1 nmol/L endothelin-3 was added to onebath containing diluent and one containing antago-nist, while the other baths received equal volumes ofdiluent. Eight responses to stimulation (over 32 min)were elicited between the addition of each increasingconcentration of endothelin-3 (1–100 nmol/L at half-log unit increments). The antagonists studiedwere the beta adrenoceptor antagonist propranolol (1 µmol/L), the alpha adrenoceptor antagonist phen-tolamine (1 µmol/L) and the ganglion blocker hexa-methonium (1 µmol/L). Again, atropine (1 µmol/L)was added to the baths at the conclusion of eachexperiment to confirm stimulation of parasympa-thetic nerves.

Localization of neuromodulation to a specificendothelin receptor type

To further localize the site of modulation and to char-acterize the endothelin receptor type involved in neuromodulation, a number of specific endothelinantagonists, and the effect of endothelinB receptordesensitization, on the endothelin-induced neuropo-tentiation were examined. Preliminary experimentswere performed to characterize the effectiveness ofvarious endothelin antagonists in affecting directcontraction of rabbit bronchial smooth muscle inresponse to endothelin. In multiple series of experi-ments, cumulative concentration–response curves toendothelin-1 (1 pmol/L–300 nmol/L at half-log unitincrements) were conducted in the presence andabsence of the combined endothelinA/endothelinB

receptor antagonist PD 145065 (at either 0.3 µmol/L,3 µmol/L or 30 µmol/L), the endothelinA-selectivereceptor antagonists PD 147953 (at either 0.3 µmol/L,3 µmol/L or 30 µmol/L) and BQ-123 (at either 0.3, 1 or 3 µmol/L), the endothelinB-selective receptor anta-gonist BQ-788 (at either 0.1 µmol/L, 1 µmol/L or 10 µmol/L), and a combination of endothelinA andendothelinB antagonists all of which were applied 30 min prior to commencing the endothelin-1 cumu-lative concentration–response curves. Endothelin-3was not used for these experiments as we have been unable to generate sufficient direct contraction(greater than 20% of the reference response to acetylcholine) in response to endothelin-3 in order to produce contractile cumulative concentration–response curves in this tissue.23

The effect of the antagonists on the neuropotenti-ation induced by endothelin-3 was examined in sep-arate series of experiments using four tissues fromeach animal. When stable responses to electrical fieldstimulation at constant frequency and voltage were obtained as described above, the tissues fromeach animal were treated in the following manner.Diluent for the antagonist was added to one pair of tissues, while the remaining pair received theantagonist under investigation. After eight stimula-tions 1 nmol/L endothelin-3 was added to one bathcontaining diluent and one containing antagonist,while the other baths received equal volumes ofdiluent. Eight responses to stimulation (over 32 min)were elicited between the addition of each increasingconcentration of endothelin-3 (1–100 nmol/L at logunit increments). The antagonists studied were BQ-123 at 3 µmol/L, PD 145065 at 30 µmol/L and BQ-788at 10 µmol/L. In addition, the effect of the combina-tion of BQ-123 (3 µmol/L) and BQ-788 (10 µmol/L) onthe potentiation induced by endothelin-3 was exam-ined. At the conclusion of the experiment, atropine (1 µmol/L) was added to the tissues to confirmparasympathetic nerve stimulation had occurred.

In a separate and final series, after stimulationparameters required to produce a response between200 and 600 mg of generated force were determinedas above, endothelinB receptors in one tissue fromeach tissue pair were desensitized by exposure to 300 nmol/L sarafotoxin S6c. The remaining tissuefrom each pair was treated with diluent and served as a control. When the contractile response to

Endothelin and airway neuromodulation 345

sarafotoxin S6c had reached a plateau and the tonehad spontaneously returned to baseline, the tissueswere washed three times (at 10 min intervals). Thetissues were then repeatedly field stimulated as aboveusing the same stimulation parameters, and whenstable responses were obtained, increasing concen-trations of sarafotoxin S6c were added to each tissueat 32 min intervals.

Analysis of results

All responses were measured isometrically usingGrass FT03 transducers (Grass, Quincy, MA, USA)coupled to either a Grass polygraph or MacLab computerized analogue to digital recording device(ADI, Sydney, NSW, Australia). In the frequency–response studies, the response obtained at each fre-quency in the presence or absence of endothelin-3,was expressed as a percentage of the maximalresponse obtained in the baseline frequency–response curve obtained in the same tissue. Curvesrelating percentage response at each frequency weredrawn for each tissue and the frequency at which halfthe maximal response was attained (EF50) in eachcurve was derived. An overall mean curve was drawnfor each concentration of endothelin-3 (and one for control tissues treated with diluent alone), and ageometric mean EF50 value with 95% confidencelimits calculated for each intervention. Differences in magnitude of response and EF50 between treatedand control tissues were compared using Statview for Macintosh software by using analysis of variancefollowed by Fisher’s PLSD (least significant differ-ence) post-hoc test at 0.05 level of significance().

In all other electrical field stimulation experiments,the mean of the three pretreatment equivalentresponses was recorded and designated the ‘initialresponse’ and all subsequent responses were ex-pressed as a percentage of this initial response. Thegreatest response to field stimulation in each 32 minperiod following the addition of endothelin (orendothelin receptor agonist) or diluent to each tissuewas recorded and expressed as a percentage of theinitial response. Where duplicate tissues from ananimal were used, the values from the duplicatetissues were then averaged to give a single value forstatistical analysis. Responses for each experimentalcondition (i.e. in the presence or absence of antago-nists, with and without receptor desensitization) fromeach animal were combined with those from allanimals used within an experimental series and themean response (with standard error of the mean) forthe treatment calculated. The mean response for eachtreatment was compared with the correspondingmean control value using A N O VA and Fisher’s PLSDpost-hoc test.

In experiments analyzing the efficacy of endothe-lin antagonists on the concentration–response rela-tionships to endothelin-1, mean curves generated inthe presence and absence of an antagonist were alsocompared by A N O VA followed by Fisher’s PLSD post-hoc test. The responses to each concentration of

endothelin-1 were first expressed as a percentage ofthe reference response to acetylcholine and values forduplicate tissues were averaged prior to calculation ofan overall mean for each treatment. The potency ofendothelin in the presence and absence of the anta-gonists was assessed by recording the log of the concentration of endothelin-1 at which 30% of thereference response to acetylcholine was obtained (– log EC30(ACh)). Mean values within each experimen-tal series were compared using A N O VA followed byFisher’s PLSD post-hoc test.

Compounds used

Krebs’–Henseleit solution (composition in mmol/L:NaCl 118.4, KCl 4.7, CaCl2.2H2O 2.5, MgSO4.7H2O 1.2,KH2PO4 1.2, NaHCO3 25.0 and D-glucose 11.1) was pre-pared using laboratory grade reagents. Atropine sulfate in sodium chloride was obtained from Astra Pharmaceuticals (Sydney, NSW, Australia) andpropranolol hydrochloride was from ICI Pharma-ceuticals (Sydney, NSW, Australia). BQ-788 was purchased from Peptides International (Louisville, KY, USA). PD 145065 (Ac-((R)-2-(10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5-yl)Gly-L-Leu-L-Asp-L-Ile-L-Ile-L-Trp.Na)) and PD 147953 (N-[(hexahydro-1H-azepin-1-yl) carbonyl]) L-Leu-(1-Me)D-Trp-(3-(2-pyridinyl))D-Ala.Na) were gifts from Parke-DavisWarner Lambert (Ann Arbor, MI, USA). Tissue Tek is aproduct of Miles Laboratories (Elkhart, IN, USA).Copper sulfate and potassium ferricyanide were fromAjax Chemicals (Sydney, NSW, Australia). Acetyl-choline, acetylthiocholine iodide, bacitracin, bovineserum albumin, hexamethonium bromide, methylgreen pyronin liquid stain, phosphoramidon (N-(a-rhamnopyranosyloxyphosphinyl)-L-leucyl-L-trypto-phan) ammonium salt, phentolamine and Tris-HClwere from Sigma (St Louis, MO, USA). Sodium acetate,sodium citrate, dimethylsulfoxide, haematoxylin andDePeX (Gurr) mounting medium were purchasedfrom BDH (Sydney, NSW, Australia). Radiolabelledendothelin-3 (specific activity 2000 Ci/mmol) wasfrom Amersham (Amersham, UK) and was reconsti-tuted in distilled water. Endothelin-1, endothelin-3,BQ-123, BQ-3020 and sarafotoxin S6c were purchasedfrom Auspep (Melbourne, Victoria, Australia).

Stock solutions, which were stored at –70°C, wereprepared by dissolving endothelin-1 and endothelin-3 in 0.1 mol/L acetic acid, BQ-3020 and BQ-123 in a mixture of 12% dimethylsulfoxide and NH3

(0.2 mol/L), and all other compounds in distilledwater. All agents were diluted in Krebs’–Henseleitsolution (which served as the diluent in controltissues) on the day of experimentation and were kepton ice for the duration of the experiment.

RESULTS

Autoradiographic studies

Under light field illumination, in sections in whichstaining of acetylcholinesterase had been performed,

346 KO McKay et al.

parasympathetic ganglia were visualized as collec-tions of brown-stained nerve cell bodies (Fig. 1a) andin those which had been stained with methyl-greenpyronin, the cell bodies of the ganglia had a pink/bluecolouring (Fig. 1b). Ganglia were identified in airwaysfrom all three rabbits but not in every airway segmenttaken from each animal. When darkfield illuminationwas used, binding sites appeared as uniformly sizedilluminated silver grains. Dense binding sites for [125I]-endothelin-3 were apparent over the ganglia in bothtracheal and bronchial sections (Fig. 1d). ‘Non-spe-cific’ binding was present (Fig. 1c) but was noticeablymore sparse than in adjacent sections demonstrating‘total binding’.

Functional studies

Effect of endothelin-3 on the frequency–response relationship

There was no significant difference between themaximal amplitude or the geometric mean EF50

values in successive frequency–response curves inthe control tissues. Addition of endothelin-3 to theorgan baths did, however, significantly alter the frequency–response relationship. Endothelin-3 at 100 nmol/L caused a significant decrease in the geo-metric mean EF50 value with a leftward shift in the

frequency–response curve to electrical field stimula-tion at constant voltage (Fig. 2). In the presence ofendothelin-3 (at 100 nmol/L), the geometric meanEF50 value was 3.5 Hz (95% confidence limits: 2.8, 4.5 Hz) and this was significantly lower than incontrol tissues where the value was 7.3 Hz (95% con-fidence limits: 4.4, 12.2 Hz, A N O VA and Fisher’s PSLDat 0.05 level of significance, n = 5). The presence of 1 nmol/L or 10 nmol/L endothelin-3 did not signifi-cantly alter the geometric mean EF50 values in tissuesfrom five rabbits.

Endothelin-3 at 100 nmol/L also significantlyincreased the amplitude of the response to electricalfield stimulation (Fig. 2). For example, at 1 Hz, the response was 6.6 ± 1.3% of the maximal response in control tissues and 46.0 ± 5.2% in the presence of 100 nmol/L endothelin-3 (significantly differentusing A N O VA and Fisher’s PLSD at 0.05 level of signi-ficance, n = 5). The amplitude of the response to elec-trical field stimulation was not affected by either 1 nmol/L or 10 nmol/L endothelin-3 at any frequencystudied.

Effect of endothelin receptor agonists onparasympathetic neurotransmission

In addition to confirming our previously reportedpotentiation of the response to electrical field stimu-

Endothelin and airway neuromodulation 347

a b

c d

Figure 1 Photomicrographs of para-sympathetic ganglia in sections ofrabbit bronchus. The upper panels arelight field photomicrographs identify-ing ganglia by staining for (a) acetyl-cholinesterase and with (b) methyl-green pyronin. The lower panels are dark field photomicrographs of sections incubated in [125I]-endothelin-3 in (c) the presence and(d) the absence of excess unlabelledendothelin-3. sm, Smooth muscle; g,ganglionic cell body.

lation in the presence of endothelin-3,7 we observeda marked enhancement of the response in the pres-ence of endothelin-1, sarafotoxin S6c and BQ-3020(Fig. 3). In the case of endothelin-1 (Fig. 3a), this significant potentiation occurred over the concentra-tion range 0.01–0.3 µmol/L and was maximal at 0.1 µmol/L where the magnitude of the response was293 ± 63% of the response prior to the addition ofendothelin-1 (P < 0.05 and Fisher’s PLSD post-hoc test, n = 4). The potentiation induced by theendothelinB receptor-specific agonist sarafotoxin S6coccurred between 1 and 30 nmol/L with the highestconcentration producing a response to nerve stimu-lation which was 747 ± 334% of the baseline response(P < 0.05 and Fisher’s PLSD post-hoc test, n = 4;Fig. 3c). In addition, BQ-3020, another endothelinB

receptor-selective agonist, also induced a markedpotentiation of the response to field stimulation, inthis case to 655 ± 178% of the baseline response at 3 µmol/L (P < 0.05 and Fisher’s PLSD post-hoctest, n = 4; Fig. 3d). All of these agonists also caused anincrease in the basal tone of the tissues but there wasno temporal or quantitative association between theincrease in tone and the maximal potentiation of theresponse to electrical field stimulation. All neurallymediated responses were abolished in the presence ofatropine confirming parasympathetic stimulation.

Localization of the site of parasympatheticneuromodulation by endothelin

To localize the site of endothelin-induced poten-tiation, the effects of various antagonists on the

neuropotentiation were assessed. Hexamethonium, anicotinic receptor antagonist and parasympatheticganglionic transmission blocker, had no effect on the response to electrical field stimulation and didnot alter the potentiation induced by endothelin-3 (response to electrical field stimulation with 100 µmol/L endothelin-3 was 259 ± 64% of the refer-ence response in the presence of 1 µmol/L hexam-ethonium and 271 ± 33% in control tissues; n = 5, P >0.05). The beta-adrenergic antagonist propranolol,and the alpha receptor antagonist phentolamine,similarly had no effect on the basal tension or on theresponse to field stimulation in the presence orabsence of endothelin-3 at any of the concentrationsexamined (response to electrical field stimulationwith 100 nmol/L endothelin-3 was 295 ± 127% [pro-pranolol 1 µmol/L] and 294 ± 36% [control], and 284 ± 52% [phentolamine 10 µmol/L] and 414 ± 65%[control]; all n = 5, P > 0.05).

Localization of neuromodulation to a specificendothelin receptor type

Neither endothelinA receptor selective antagonist,BQ-123 nor PD 147953, had an effect on the cumula-tive concentration–response curve to endothelin-1 atany of the three concentrations studied (Table 1) indi-cating an absence of involvement of endothelinA

receptors in direct contraction of rabbit bronchus toendothelin-1. Conversely, the combined endothelinA

and endothelinB receptor antagonist PD 145065 sig-nificantly reduced both the potency and efficacy ofendothelin-1 in rabbit isolated bronchus (Table 1).This effect was only apparent at 30 µmol/L PD145065, the highest concentration of the antagoniststudied and the antagonist was non-competitive atthis concentration as the maximal response toendothelin-1 was 35 ± 5% of the reference response toacetylcholine in treated tissues and 74 ± 6% in thecontrol tissues (P < 0.05, and Fisher’s PLSDpost-hoc test, n = 4). BQ-788 at 10 µmol/L was a com-petitive antagonist for endothelin-1 in rabbit bronchi.The lower concentrations of BQ-788 studied (0.1 and1 µmol/L) had no effect on the endothelin-1 cumula-tive concentration–response curve (Table 1).

Blockade of endothelinA receptors with BQ-123 hadno effect on the neuropotentiation induced byendothelin-3 (Table 2). Endothelin-3-induced poten-tiation was, however, significantly ameliorated intissues in which endothelinB receptors had beenblocked by BQ-788 at 10 µmol/L (P < 0.05 andFisher’s PLSD post-hoc test, n = 6) (Table 2). Abolitionof the potentiation was also seen in tissues treatedwith the combined endothelinA/endothelinB receptorantagonist PD 145065 and also in tissues treated witha combination of antagonists, specifically, BQ-123and BQ-788. There was no difference in resultsobtained with endothelinB receptor blockade aloneand combined endothelinA/endothelinB receptorblockade.

Desensitization of the endothelinB receptor byprior exposure to sarafotoxin significantly altered thesubsequent potentiation of electrical field stimula-tion by each concentration of sarafotoxin S6c (0.1–

348 KO McKay et al.

Figure 2 The effect of endothelin-3 on the frequency–response relationship in rabbit isolated bronchus. Themean responses to increasing frequency of stimulation in the presence (�, 10 nmol/L and �, 100 nmol/L) andabsence (�) of endothelin-3 are expressed as a percentageof the maximal response in the ‘baseline curve’. The meanresponses from five animals are shown and the vertical barsindicate SEM. Where no bars are shown, the size of the SEMis smaller than the symbol representing the mean value.*Significantly different from response in absence ofendothelin-3 (P < 0.05 and Fisher’s PLSD post-hoctest, n = 5).

100 nmol/L, P < 0.05 and Fisher’s PLSD post-hoc test, n = 4). For example, in the presence of 1 nmol/L sarafotoxin S6c, the mean maximalresponse to parasympathetic nerve stimulation was882.5 ± 271.4% of the initial response while in desen-sitized tissues this potentiation was reduced to 179.5 ± 29.6% of the initial response (P < 0.05 and Fisher’s PLSD post-hoc test, n = 4).

DISCUSSION

The primary objective of the present study was to elu-cidate the mechanism for endothelin-3-induced neuropotentiation within rabbit bronchus. In ourprevious report, we showed that the endothelin-3-induced potentiation of the response to parasympa-thetic nerve stimulation was not a result of en-hanced post-junctional responsiveness to acetyl-choline,7 therefore in the present study we examinedall possible prejunctional neuromodulatory sites, aswell as the endothelin receptor subtype/s involved. Inthe present report we demonstrated autoradi-ographic binding sites for endothelin-3 on airwayparasympathetic ganglia and that endothelin-3 notonly augmented the magnitude of the response tonerve stimulation, but it also decreased the frequencyrequired to produce half the maximal contraction(the EF50). It is unlikely that the effect of endothelin-3was a result of sympathetic influences on parasym-

pathetic neurotransmission as both alpha- and beta-antagonists had no effect on the neuromodulation.The endothelin-induced neuropotentiation, there-fore, likely occurs at prejunctional sites, and theseresults with specific endothelin agonists, antagonistsand receptor desensitization, suggest that the recep-tor involved is likely to be the endothelinB receptor.

There are specific binding sites for endothelin-1and endothelin-2 on cholinergic nerves5 and ganglia6

in human bronchi, and endothelin-like immunoreac-tivity has been detected in the vagus nerve.24 In thepresent study, we showed that there are also bindingsites for iodinated endothelin-3 on rabbit airwayganglia. These ganglia form part of the parasympa-thetic nervous system as the cell bodies within thesestructures stained positively for acetylcholinesterase.Airway ganglia process neural signals and haveinhibitory25 and excitatory26 input. Endothelin hasbeen shown to modulate synaptic transmission infeline colonic27 and rabbit pelvic parasympatheticganglia28 suggesting that the endothelin receptorsdetected on the ganglia may be involved in neuro-modulation in the bronchi.

Hexamethonium had no effect upon the responseto electrical field stimulation in the presence orabsence of endothelin-3. This finding suggests that inthis preparation, field stimulation did not lead to theactivation of preganglionic nerves and that the poten-tiation induced by endothelin-3 occurs postganglion-ically. It must be acknowledged, however, that the

Endothelin and airway neuromodulation 349

Figure 3 The effect of (a)endothelin-1, (b) endothe-lin-3, and the endothelinB

receptor selective agonists(c) sarafotoxin S6c and (d)BQ-3020 on the response toelectrical field stimulation inrabbit isolated bronchus.The responses are expressedas a percentage of the meancontrol responses (unfilledbars) obtained in tissuesprior to the addition of theagonists to the baths (filledbars). The capped verticallines indicate SEM values.*Significantly different fromresponse in control tissues (P< 0.05 and Fisher’sPLSD post-hoc test, n = 5).

350 KO McKay et al.

Table 1 Effect of endothelin antagonists on magnitude of the response** and the potency† of endothelin-1 in rabbit isolated bronchus

PD 147953 (n = 4)Control 0.3 mmol/L 3 mmol/L 30 mmol/L

Tmax(%ACh) 64 ± 7 57 ± 10 49 ± 2 48 ± 11- log EC30(ACh) 7.62 ± 0.17 7.51 ± 0.19 7.47 ± 0.22 7.30 ± 0.37

BQ-123 (n = 5)Control 0.3 mmol/L 1 mmol/L 3 mmol/L

Tmax(%ACh) 62 ± 5 76 ± 10 74 ± 11 68 ± 7- log EC30(ACh) 8.17 ± 0.08 8.23 ± 0.14 7.99 ± 0.17 8.38 ± 0.18

PD 145065 (n = 4)Control 0.3 mmol/L 3 mmol/L 30 mmol/L

Tmax(%ACh) 71 ± 8 73 ± 2 72 ± 11 35 ± 5*- log EC30(ACh) 7.66 ± 0.08 7.71 ± 0.17 7.69 ± 0.13 6.64 ± 0.09*

BQ-788 (n = 6)Control 0.1 mmol/L 1 mmol/L 10 mmol/L

Tmax(%ACh) 64 ± 4 69 ± 9 60 ± 6 62 ± 4- log EC30(ACh) 8.18 ± 0.07 8.11 ± 0.11 8.05 ± 0.05 7.76 ± 0.14*

BQ-123 (3 mmol/L) + BQ-788 (10 mmol/L) (n = 6)Control BQ-123 alone BQ-788 alone BQ-123 + BQ-788

Tmax(%ACh) 63 ± 4 70 ± 9 62 ± 4 36 ± 7*- log EC30(ACh) 8.16 ± 0.08 8.31 ± 0.16 7.75 ± 0.14* 6.87 ± 0.15*

** The maximum response is expressed as a percentage of the reference response to acetylcholine (Tmax(%Ach)) and is shownwith SEM values.

† Potency is expressed as the negative log of the EC30(ACh); the concentration of endothelin-1 required to produce a responseequal to 30% of the reference response to acetylcholine, and is shown with standard error of the mean values.

* Significantly different from the corresponding control value (P < 0.05 and Fisher’s PLSD post-hoc test).

Table 2 Effect of endothelinA and endothelinB receptor antagonism on the potentiation of the response† to electrical fieldstimulation by endothelin-3 in rabbit isolated bronchus

Endothelin-3 (nmol/L)1 nmol/L 10 nmol/L 100 nmol/L

BQ-123 (n = 6) 0 186 ± 48 283 ± 59 373 ± 953 mmol/L 144 ± 23 235 ± 43 246 ± 53

PD 145065 (n = 8) 0 — 310 ± 82 —30 mmol/L — 106 ± 10* —

BQ-788 (n = 6) 0 186 ± 48 283 ± 59 373 ± 9510 mmol/L 105 ± 5* 94 ± 4* 129 ± 20*

BQ-123 + BQ-788 0 186 ± 48 283 ± 59 373 ± 95(n = 6) 1 mmol/L 115 ± 11* 100 ± 11* 146 ± 18*

† The maximum response to electrical field stimulation in the presence of each concentration of endothelin-3 is expressedas a percentage of the initial response to electrical field stimulation (prior to the addition of endothelin-3) and is shown withSEM values.

* Significantly different from the corresponding mean value in paired control tissues (P < 0.05 and Fisher’s PLSDpost-hoc test).

preparation used in the present study is not optimalfor the study of preganglionic stimulation and it is nottherefore possible to exclude a functional role for theganglionic endothelin-3 binding sites visualized byautoradiography in the present study.

It has been suggested that sympathetic nerves maymodulate cholinergic neurotransmission.15 Modula-tion of neurotransmission through parasympatheticganglia by beta2- and alpha-adrenergic agonists hasbeen demonstrated in both ferret29 and rat30 tracheain vitro. This modulation was shown to be due to aninhibition of acetylcholine release from nerve termi-nals. In 1991, Kushiku et al. reported inhibition ofsympathetic ganglionic transmission by endothelin-3.31 It is therefore conceivable that inhibition of sympathetic neurotransmission by endothelin-3could have been responsible for decreased modu-latory activity of the sympathetic nervous system,thereby resulting in our observed enhanced cho-linergic responsiveness. This is clearly not the mechanism involved here, as neither phentolaminenor propranolol had a significant effect on theenhanced response to neural stimulation induced byendothelin-3.

Muscarinic receptors of the M2 subtype are locatedon postganglionic parasympathetic nerves and whenoccupied, provide inhibitory feedback to cholinergicinnervation.32 Decreased activity at these receptorswould therefore result in an increased cholinergicresponse and may have therefore explained ourobservations. Although present on the nerves inguinea-pig,33 canine34 and human35 airways, M2

receptors are absent from the rabbit lung36 and it istherefore most unlikely that the increased respon-siveness induced by endothelin is due to alterationsin M2 muscarinic receptor functioning in this tissue.

Endothelin-1, sarafotoxin S6c and BQ-3020 as wellas endothelin-3 potentiated the response to electricalfield stimulation. In all cases, the potentiation wasnot correlated with increases in basal smooth muscletone and, indeed, in the case of endothelin-3, waspresent in the absence of contraction to endothelin-3. This is consistent with our previous observationthat there was no relationship between increases inrabbit airway smooth muscle tone and parasympa-thetic neuropotentiation to prostaglandins.37 Theparasympathetic potentiation we have observed inthe presence of peptides of the endothelin family wasnot therefore related to the direct contractile effect ofthese agents on smooth muscle.

Endothelin-3 caused a leftward shift in the fre-quency–response curve to field stimulation as well asan augmentation of the magnitude of the response toelectrical field stimulation. A leftward shift in the fre-quency–response curve may indicate a depolariza-tion of the membrane of the nerve ending and anincreased probability of neurotransmitter release atlower stimulation frequencies. Such a mechanismwas suggested by Yamawaki et al.38 to explainincreased cholinergic responsiveness, in the presenceof the vasoactive peptide angiotensin II, in rabbitbronchus. Henry and Goldie have also observed agreater relative enhancement by the endothelinB

agonist sarafotoxin S6c at lower stimulation frequen-

cies in mouse airways.9 This suggests that such aneffect occurs across species. It is therefore likely,based upon the results in the Henry and Goldie study,that endothelin-3 binds to endothelin receptors onpostganglionic nerve endings, causing a partial depo-larization of nerve endings and when the tissue is stimulated, more acetylcholine is released, result-ing in a significantly greater contractile response. This proposed mechanism is supported by the reportof endothelin-induced facilitation of parasympa-thetic cholinergic neurotransmission as a result ofincreased neurotransmitter release in rat trachealis.10

The finding that the endothelinB receptor selectiveagonists sarafotoxin S6c and BQ-3020 caused thegreatest potentiation of the response to electrical fieldstimulation in the present study suggested that theneuromodulatory effects of endothelin in this tissuewere mediated by an endothelinB receptor. This was supported by the observation that the mixed endothelinA/endothelinB antagonist PD145065 markedly reduced the potentiation producedby endothelin-3, while this did not occur withendothelinA selective antagonists. Moreover, theendothelinB selective receptor antagonist BQ-788attenuated the potentiation induced by endothelin-3,further supporting the contention that an endothe-linB receptor was involved in this response. Our suc-cessful reduction of the neuropotentiation bydesensitization of the endothelinB receptors by priorexposure of the tissue to the endothelinB receptorselective agonist sarafotoxin S6c, was further evi-dence of the involvement of this receptor type in theneuromodulation. This contention is supported byother reports of potentiation of parasympatheticneural responses in human bronchus8 and impliesthat endothelinB receptors modulate cholinergic neu-rotransmission in the rabbit bronchus as well as therabbit trachea11 and that there are no regional differ-ences in endothelin-induced cholinergic neuromod-ulation in the rabbit lung.

We have therefore shown that endothelin-1, BQ-3020, sarafotoxin S6c as well as endothelin-3 signifi-cantly increase the response to parasympatheticnerve stimulation in rabbit bronchi. This effect is pre-junctional in nature, and the lack of effect of sympa-thetic receptor antagonists and the reported lack of inhibitory M2 muscarinic receptors in this speciesindicate that the potentiation is not mediated viaeither of these mechanisms. The persistence ofpotentiation in the presence of pure endothelinA

antagonists coupled with amelioration of this effectin the presence of a mixed endothelinA/endothelinB

antagonist PD 145065, and the endothelinB antago-nist BQ-788, and after endothelinB receptor desensi-tization, indicate that this neuropotentiation ismediated via activity at endothelinB receptors.

ACKNOWLEDGEMENTS

We acknowledge the technical assistance of DonnaCarey in carrying out a number of the functionalexperiments. Professor Black and Dr Johnson aresupported by the National Health and Medical

Endothelin and airway neuromodulation 351

Research Council of Australia. Dr McKay was the Aus-tralian Lung Foundation/Astra Fellow in RespiratoryMedicine and a Rolf Edgar Lake Fellow in the Facultyof Medicine at the University of Sydney at the time ofthis study.

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