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Investigation of the subtype of U2-adrenoceptor mediatingprejunctional inhibition of cardioacceleration in the pithedrat heart

'Karen Smith, Katherine Gavin & 2James R. Docherty

Department of Physiology, Royal College of Surgeons in Ireland, 123 St. Stephen's Green, Dublin 2, Ireland

1 We have investigated the subtype of a2-adrenoceptor mediating prejunctional inhibition of cardioac-celeration in the pithed rat heart in comparison with M2-adrenoceptor ligand binding sites.2 In pithed rats, prejunctional M2-adrenoceptors were investigated in terms of the ability of M2-adrenoceptor antagonists to shift the inhibitory potency of the M2-adrenoceptor agonist, xylazine, againstthe tachycardia to a single electrical stimulus given via the pithing rod.3 Antagonist potency at prejunctional M2-adrenoceptors in pithed rat heart was correlated withantagonist affinity at M2-adrenoceptor ligand binding sites in membranes of rat kidney and subman-dibular gland labelled with [rH]-yohimbine.4 The correlation with the prejunctional M2-adrenoceptor in pithed rat heart was best for the M2D-adrenoceptor ligand binding site of rat submandibular gland (r = 0.98, n = 10, P <0.0001), as comparedto correlations with the M2A-adrenoceptor ligand binding site of human platelet (r = 0.90, n = 9,P <0.001), the x2B-adrenoceptor ligand binding site of rat kidney (r = 0.82, n = 10, P <0.01) and withpublished results for the x2c-adrenoceptor ligand binding site (r = 0.48, n = 6, NS).5 It is concluded that the functional prejunctional M2-adrenoceptor of pithed rat heart closely resemblesthe M2D-adrenoceptor ligand binding site of rat submandibular gland.

Keywords: Prejunctional a2-adrenoceptors; a2D-adrenoceptors; pithed rat heart

Introduction Methods

X2-Adrenoceptors have been subdivided into at least foursubtypes, a2A-, a2B-, OC2C- and a2D-adrenoceptors, based onligand binding and molecular cloning studies (Lorenz et al.,1990; Bylund, 1992), although the aX2D-adrenoceptor of ratsubmandibular gland may be a species homologue of thehuman M2A-adrenoceptor, reducing the number of genescoding for M2-adrenoceptors to three (Lanier et al., 1991;Harrison et al., 1991; Smith & Docherty, 1992), so that theterm oE2A/D-adrenoceptor could be used to describe thesehomologues. However, since the functional receptors differpharmacologically, the term a2A-adrenoceptor is used in thispaper to describe the ligand binding site in human plateletand M2D-adrenoceptor to describe the site on rat subman-dibular gland.We have previously investigated functional prejunctional

a2-adrenoceptors of adrenergic nerves in a number of tissuesand suggested that the prejunctional x2-adrenoceptors in ratvas deferens and rat submandibular gland resemble the M2D-adrenoceptor ligand binding site of rat submandibular gland(Smith & Docherty, 1992), whereas the functional prejunc-tional cK2-adrenoceptor of rat atrium differed (Smith et al.,1992).

In this study, we have examined the functional prejunc-tional M2-adrenoceptor in pithed rat heart in relation toligand binding sites in rat kidney (M2B-adrenoceptor: seeMichel et al., 1989; Smith & Docherty, 1992), and rat sub-mandibular gland (a2D-adrenoceptor: see Bylund, 1992). Forcomparison, ligand binding data for human platelet mem-branes (a2A-adrenoceptor: see Bylund, 1985; 1992) areincluded.

Male Wistar rats (250-350 g)College Dublin and employedoutlined below.

were obtained from Trinityin a number of studies, as

Pithed rat preparation

Rats were pithed by the method of Gillespie et al. (1970) andventilated with 100% 02 at a rate of 60 per min. Heart ratewas derived from carotid arterial pressure and drugs wereinjected into the jugular vein. The pithing rod was used as anelectrode positioned at Ti to stimulate the cardio-acceleratornerves with a single stimulus pulse (0.5 ms, supramaximalvoltage) every 2 min (see Docherty & Hyland, 1986).

Dose-response curves to the M2-adrenoceptor agonists,xylazine, clonidine, UK 14,304 and oxymetazoline were con-structed from the effects of increasing doses (10 fold in-crements) administered at 5 min intervals. Prejunctionaleffects of agonists were assessed as the inhibition of thecardio-acceleration to a single stimulus. Antagonist drugs, orvehicle, were administered intravenously 10 min before start-ing the agonist dose-response curve. The potency of xylazinewas expressed as an ID50 (dose causing 50% inhibition of thecardio-acceleration to a single stimulus), and the effects ofantagonists were assessed from the difference between agonistpotency in the individual antagonist experiment and themean agonist potency in vehicle experiments. Shifts inagonist potency were expressed as log(DR - 1), where DR isthe xylazine dose ratio (difference in potency of xylazinebetween vehicle experiments and experiments in the presenceof antagonist), and mean values (± s.e.mean) were obtainedfrom the effects of the antagonist in n experiments.

Antagonist dose administered was expressed in mol kg-' toallow direct comparison between agents of widely differingmolecular weights. Antagonist potency was expressed as anapparent pA2 (- log mol kg-') from the calculation: - logantagonist dose (mol kg-') + log(DR - 1). Expression of

' Present address: Institute of Physiology, University of Glasgow,Glasgow G12 8QQ.2 Author for correspondence.

BrRish Journal of Pharmacology (1995) 115, 316-320

K. Smith et al Cardiac prejunctional o2-adrenoceptors

antagonist potency in this form allowed direct comparisonwith ligand binding data.

In some experiments, cardioaccelerator responses to iso-prenaline (1-1Ongkg-') were examined before and follow-ing exposure to xylazine (I mg kg-') or vehicle, to assesspostjunctional actions of xylazine, if any.

Radioligand binding studies

Preparation of human platelet and rat kidney membraneswas carried out as described in Connaughton & Docherty(1990), and preparation of submandibular gland membraneswas as described for rat kidney. The resultant pellets wereused immediately or stored at -20'C for later use. Pelletswere reconstituted in 2.5ml (platelet), 5 volumes (subman-dibular) or 10 volumes (kidney) of incubation buffer.

In saturation experiments, aliquots of membrane suspen-sion were incubated with various concentrations of [3H]-yohimbine (specific activity: 81 Ci mmol -', Amersham) at370C (human platelet: 0.3-20 nM; incubation buffer: Tris-HC1 50 mM, MgCI2 8 mM, EGTA 5 mM, pH 7.4 at 370C) orat 25°C (rat kidney: 0.5-30 nM; rat submandibular gland:2.0-40 nM; incubation buffer: Tris-HCl 50 mM, EDTA5 mM, pH 7.4 at 250C). In competition studies, [3H]-yohimbine (5 or 10 nM) was incubated with competingligands in concentrations from 0.1 nM to 1 mM in 0.5 log unitincrements for 30 min. Non-specific binding was determinedin the presence of phentolamine (10 pM). Specific binding of[3H]-yohimbine was 70-90% of total binding. Assays wereterminated by washing with ice-cold incubation buffer, fol-lowed by rapid vacuum filtration through Whatman GF/Cfilters, using a Brandel Cell Harvester. Radioactivity retainedon filters was determined by liquid scintillation spectroscopy.The inhibition constant (Ki) for inhibition of radiolabelled

ligand binding was determined from the formula:

Ki = IC50/(1 + [3H]/kD)where IC50 is the concentration of competing ligand thatinhibits radioligand specific binding by 50%, kD is the dis-sociation constant for the radioligand (human platelet:5.50 nM, 95% confidence limits 3.80-7.94 nM, n = 6; ratkidney: 8.71 nM, 95% confidence limits 7.41-10.2 nM, n = 7;rat submandibular gland: 23.4 nM, 95% confidence limits18.2-30.2 nM, n = 4), and 3H is the concentration of tritiatedyohimbine employed (5 nM: platelet and kidney; 10 nM: sub-mandibular gland). Bma,, values obtained in saturationexperiments were 115±10 (n=6), 94±13 (n=7) and146 ± 19 (n = 4) fmol mg-' protein in human platelet, ratkidney and rat submandibular gland, respectively.

Statistical evaluation

In functional studies, values are expressed as agonist ID50(dose producing 50% inhibition of the stimulation-evokedtachycardia), antagonist log (DR - 1) (where DR is thexylazine dose-ratio between potency in the presence ofantagonist and presence of vehicle), and antagonist pA2.Antagonist pA2 was calculated as the sum of antagonist dose(- log mol kg-') and log(DR - 1). Results are expressed asmean and 95% confidence limits or mean ± s.e. of mean.Effects of antagonist on xylazine ID50 were compared witheffects of vehicle by Student's t test for unpaired data andAnalysis of Variance. Correlation analysis was carried out onan Apple Macintosh using the Statworks and Cricketgraphprogrammes (Cricket software, Inc.).

Drugs

The following drugs were employed: abanoquil hydrochloride(gift: Pfizer, Sandwich, U.K.); ARC 239 (2-(2,4-(o-meth-oxyphenyl)-piperazin-1-yl)-ethyl4,4 dimethyl-l,3-(2H,4H)-iso-quinolindine chloride; gift: Karl Thomae, Biberach, Ger-many); BDF 8933 (4-fluoro-2-(imidazolin-2-ylamino)-isi-

indoline maleate, gift: Beiersdorf, Hamburg, Germany),benozathian hydrochloride (Research Biochemicals, Natick,U.S.A.); chlorpromazine hydrochloride (Sigma, Poole, U.K.),HV 723 (a-ethyl-3,4,5-trimethoxy-a-(3-((2-(2-methoxyphen-oxy)ethyl)-amino)-propyl)-benzene acetonitrile fumarate; gift:Hokurika, Seiyaku, Katsuyama, U.K.); idazoxan hydroch-loride (Research Biochemicals); isoprenaline hydrochloride(Sigma); oxymetazoline hydrochloride (Sigma); phentolaminehydrochloride (Sigma); prazosin hydrochloride (gift: Pfizer);spiroxatrine hydrochloride (gift: Janssen, Ireland); UK14,304 (5-bromo-6-(2-imidazolin-2-yl amino)quinoxaline; gift:Pfizer); WB 4101 (2-(2',6'-dimethoxyphenoxyethyl)amino-methyl- 1,4-benzodioxan; Research Biochemicals); xylazinehydrochloride (gift: Bayer, Ireland); yohimbine hydrochloride(Sigma).

Results

Pithed rat preparation

In pithed rat, resting blood pressure was 70.8 ± 1.2/36.0 ± 0.7 mmHg (n = 81), and resting heart rate was256 ± 3 beats min-'. Single pulse stimulation produced a car-dioacceleration of 23.4 ± 0.8 beats min-' (n = 81). Mostantagonist drugs caused a transient inhibition of thestimulation-evoked cardioacceleration but the response hadrecovered by 10 min (just before beginning the xylazine dose-response curve), except in the case of BDF 8933 (0.1 mgkg-') which significantly reduced the stimulation-evokedtachycardia to 65.1 ± 8.4% (n = 5) (vehicle: 96.6 ± 2.8%,n = 9, P <0.01). Chlorpromazine significantly increased thestimulated-evoked tachycardia to 239 ± 26% (n = 6) (P<0.001), and the response following chlorpromazine was pro-longed in a manner consistent with blockade of nor-adrenaline re-uptake (see Tuck et al., 1972).

In vehicle experiments, xylazine, clonidine, UK 14,304 andoxymetazoline inhibited the stimulation-evoked tachycardiato a single stimulus with ID50 values of 3.80jgkg-' (95%confidence limits: 3.0-4.8 jig kg-') (n = 22), 0.67 jig kg-'(0.37-0.98) (n=6), 0.57ttgkg-1 (0.39-0.81) (n=3) and0.47tjgkg-1 (0.12-1.74) (n=5), respectively. These valuesare 15.1, 2.51, 1.29 and 1.58 nmolkg-', respectively. Invehicle experiments, in which saline replaced the test agonist,the response to a single stimulus was not significantly altered(control: 24.4 ± 2.6 beats min-'; response following 5 salineinjections over approximately 20 min: 25.9 ± 2.0 beats min-';n = 8).

Responses to xylazine obtained in vehicle experiments werecompared with responses to xylazine obtained in experimentsin the presence of antagonist, taking as examples ARC 239(5 mg kg-'), HV 723 (2 mg kg-'), yohimbine (1 mg kg-') andBDF 8933 (0.1 mg kg-') (Figure 1). Antagonist dosesemployed and apparent pA2 values obtained are shown inTable 1. All antagonists in the doses employed significantlyaltered the potency of xylazine (Student's t test, P <0.05 orless; Analysis of Variance, P<0.0001) (Table 1).

Xylazine (1 mg kg-') did not significantly reduce the car-dioacceleration produced by isoprenaline. In vehicle experi-ments, the tachycardia to isoprenaline (3 ng kg-') wasreduced from 36.6 ± 8.1 beats min-' to 31.6 ± 6.8 beatsmin-' (n = 5), whereas xylazine (1 mg kg-') reduced thetachycardia from 32.0 ± 7.6 beats min' to 27.8 ± 7.6 beatsmin-' (n = 4) (no significant differences).

Radioligand binding studies

K, values for the inhibition by ligands of [3H]-yohimbinebinding to human platelet, rat kidney and rat submandibulargland membranes were obtained (see Table 2). Hill slopes forall ligands in all 3 tissues were close to unity, so that it wasassumed that a single homogeneous population of ligandbinding sites was present in all 3 tissues.

317

K. Smith et al Cardiac prejunctional a2-adrenoceptors

C

cJ04-)0

10 000

Xylazine concentration (jg kg-1)

Figure 1 Inhibition by the a2-adrenoceptor agonists, xylazine, of thetachycardia evoked by a single stimulus in the pithed rat preparationin the presence of antagonist or vehicle. Symbols: vehicle (0), ARC239 (5mg kg-') (0), HV 723 (2mg kg-') (0), yohimbine (1 mgkg-') (U), BDF 8933 (0.1 mg kg-') (A). Values (expressed as % ofcontrol responses) are mean ± s.e. of mean from at least 4 experi-ments.

-N

0)

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cL

0Uc

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9

8-

7.

Correlation between ligand binding sites and thefunctional prejunctional cx2-adrenoceptor in pithed ratheart

Correlations between a2B- and a2D-adrenoceptor ligand bind-ing sites and the functional site in the pithed rat heart areshown in Figures 2 and 3. The correlation of the functionalprejunctional M2-adrenoceptor of pithed rat heart was betterwith the M2D-adrenoceptor ligand binding site of rat subman-dibular gland (r = 0.98, n = 10, P<0.0001) (Figure 3) thanwith the CX2B-adrenoceptor ligand binding site of rat kidney(r = 0.82, n = 10, P<0.01) (Figure 2) or with the a2A-adrenoceptor ligand binding site of human platelet (r = 0.90,n = 9, P< 0.001) (Figure 4).The correlation between published ligand binding data for

the M2C-adrenoceptor ligand binding sites and functional sitein pithed rat heart was also examined. The correlationbetween the M2C-site (human a2-C4 gene expressed in COS-7cells: Bylund et al., 1992) and the functional receptor of thepithed rat heart was r = 0.48 (non-significant; n = 6 antagon-ists, excluding abanoquil, benoxathian, HV 723 and BDF8933) (Figure 5).

0

.Table 2 Affinities (pKl, -logM) of test agents at theM2-adrenoceptor ligand binding sites human platelet (M2A),rat kidney (M2B) and rat submandibular gland (a2D)

'90

6 7 8 9

Kidney pKi (-log M)

Figure 2 Correlation between antagonist Ki (- log M) obtained ata2B-adrenoceptor ligand binding sites in rat kidney membranes andthe antagonist pA2 obtained against xylazine in the pithed rat heart.r=0.82, n=10, P<0.01.

Test agent

ChlorpromazineWB 4101BDF 8933PrazosinARC 239YohimbinePhentolamineBenoxathianAbanoquilHV 723

Platelet Kidney Submand.

6.48 ± 0.057.08 ± 0.228.72 ± 0.065.62 ± 0.125.45 ± 0.028.04±0.107.61 ± 0.126.85 ± 0.05

6.58 ± 0.10

6.29 ± 0.036.94 ± 0.038.91 ± 0.147.12 ± 0.047.06 ± 0.167.93 ± 0.027.24 ± 0.125.82 ± 0.157.18 ± 0.147.13 ± 0.20

5.26 ± 0.076.58 ± 0.108.81 ± 0.206.24 ± 0.125.54 ± 0.137.34 ± 0.067.31 ± 0.175.81 ± 0.055.98 ± 0.116.24 ± 0.06

Values are mean and s.e.mean from at least 3 experiments.

Table 1 Effects of vehicle and antagonists on the prejunctional potency of xylazine in the pithed rat preparation

Test agent

Vehicle(n = 22)Yohimbine (I mg kg-')(n = 4)Prazosin (I mg kg-')(n = 4)BDF 8933 (0.1 mg kg-')(n = 5)Phentolamine (1 mg kg-')(n = 5)HV 723 (2 mg kg-')(n = 4)Chlorpromazine (5 mg kg-')(n = 3)WB 4101 (2 mg kg-')(n = 3)Benoxathian (5 mg kg-')(n = 3)ARC 239 (Smgkg-')(n = 7)Abanoquil (2 mg kg-')(n = 3)

Xylazine ID50(llg kg-')

3.80(3.0-4.8)

302(240-380)

8.91(6.61-12.0)

589(389-891)

417(363-479)

60.2(40.7-89.1)

15.1(12.6-18.2)

58.9(40.7-85.1)

37.2(21.4-64.6)

12.9(9.55-17.4)

43.6(27.5-69.2)

log(DR - 1)

1.89 ± 0.08

0.24± 0.11

2.19± 0.18

2.04 ± 0.06

1.17 ± 0.18

0.47 ± 0.11

1.16 ± 0.17

0.94 ± 0.27

0.43 ± 0.15

1.02 ± 0.06

- log antagonist pA2(mol kg-')

7.48

5.86

8.71

7.62

6.66

5.32

6.44

5.84

5.38

6.01

Values are xylazine ID50 (dose producing 50% inhibition of the stimulation-evoked tachycardia) and 95% confidence limits, antagonistlog (DR - 1) and s.e. of mean, and antagonist pA2. Antagonist pA2 was calculated as the sum of - log antagonist dose (mol kg-') andlog(DR - 1), and so has the same s.e. of mean as the log (DR - 1) column.

318

K. Smith et al Cardiac prejunctional m2-adrenoceptors

9.

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0~

8-

7

6-

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-

0E

0.

Calic

0'._

C

0~6 7 8Submandibular pK (-log M)

Figure 3 Correlation between antagonist K, (- log M) obtained ata2D-adrenoceptor ligand binding sites in rat submandibular glandmembranes and the antagonist pA2 obtained against xylazine in thepithed rat heart. r = 0.98, n = 10, P<0.0001.

9,

-W0 8

< 7co

0.C4

< 76.2

0 5s

0

0

0

5 6 7

Platelet pK (-log M)

Figure 4 Correlation between antagonist K, (-a2A-adrenoceptor ligand binding sites in humanand the antagonist pA2 obtained against xylazirheart. r=0.90, n=9, P<0.001.

Discussion

The major finding of this study is that the fitional M2-adrenoceptor of pithed rat heartthe M2D-adrenoceptor ligand binding site in r

gland. This and other points of interestThe main objective of this study was tc

subtype of M2-adrenoceptor found prejurpithed rat heart. Using the pithed rat prepation by the M2-adrenoceptor agonist xylazin(dia evoked by a single stimulus given via tbuseful model of prejunctional a2-adrenocepantagonist potency can be assessed in ternagonist potency (see Docherty & McG&antagonist drugs were employed in the Iwhich three (ARC 239, chlorpromazine,marked selectivity between subtypes (> 10 Iaffinity), and doses were chosen which pr4agonist potency. The correlation between ti

junctional receptor in the pithed rat hearbinding site of submandibular gland was sand so significant (P<0.0001) that it is virthese are the same receptor. The correlafunctional prejunctional receptor in the pit]the ligand binding site of rat kidney cornpoor (r = 0.82), ruling out identity with the

7

6

.

7 8 9 10

a2C p/( (-log M)

Figure 5 Correlation between antagonist Ki (- log M) obtained ata2c-adrenoceptor ligand binding sites (human M2-C4 gene expressedin COS-7 cells: Bylund et al.,1992) and the antagonist pA2 obtainedagainst xylazine in the pithed rat heart. r = 0.48, n = 6, NS.

subtype. The correlation with the published data for theId x2c-adrenoceptor ligand binding site (Bylund et al., 1992) was

also relatively poor. Hence the functional prejunctional a2-adrenoceptor of pithed rat heart resembles the functionalprejunctional a2-adrenoceptors of rat vas deferens and sub-mandibular gland in that they all appear to resemble closelythe M2D-adrenoceptor ligand binding site of rat subman-dibular gland (Smith et al., 1992; Smith & Docherty, 1992).

Relative potencies of a series of agonists were also inves-tigated in the pithed rat heart. Clonidine, UK 14,304 andoxymetazoline were approximately equipotent but 10 times

8 9 more potent then xylazine. This order of potency is similar tothat found for these agonists at the functional prejunctionalM2-adrenoceptor of rat vas deferens, which may be of the

logM) obtained at x2D-adrenoceptor subtype (Smith & Docherty, 1992).platelet membranes Other authors have examined subtypes of functional pre-ie in the pithed rat junctional M2-adrenoceptors. Functional prejunctional a2-

adrenoceptors in rat submandibular gland (Limberger et al.,1992), rat cerebral cortex (Trendelenberg et al., 1993) and ratkidney (Bohmann et al., 1993) resemble the M2D-adrenoceptorligand binding site, whereas those in rabbit cerebral cortex(Trendelenberg et al., 1993) and human cerebral cortex(Raiteri et al., 1992) resemble the a2A-adrenoceptor. Since the

unctional prejunc- 'x2A-adrenoceptor and X2D-adrenoceptor may be speciesclosely resembles homologues (see Introduction), this would suggest that the,at submandibular a2A/D-adrenoceptor may be the predominant prejunctional a2-vill be discussed. adrenoceptor.v characterize the However, the functional prejunctional M2-adrenoceptor innctionally in the rat isolated atrium does not closely resemble the a2D-ration, the inhibi- adrenoceptor (Smith et al., 1992; Limberger et al., 1992), ande of the tachycar- this has led to the suggestion that the receptor may resembleie pithing rod is a the %2B-adrenoceptor ligand binding site of rat kidney cortex)tor function, and (Smith et al., 1992). Indeed, for the 10 antagonists employedns of the shift in in the current study, the correlation between prejunctionalrath, 1980). Ten potency in rat atrium (authors, unpublished results, seepresent study, of Smith et al., 1992) with the kidney M2B-adrenoceptor ligandprazosin) showed binding site (r = 0.94) was better than with the rat subman-fold differences in dibular M2D-adrenoceptor ligand binding site (r = 0.89). Thisoduced a shift in leads to an apparent anomaly that the prejunctional receptorhe functional pre- activated by xylazine in the pithed rat heart closely resemblesrt and the ligand the rat submandibular a2D-adrenoceptor ligand binding site,o close (r = 0.98) whereas the prejunctional receptor activated by noradrenalinetually certain that in rat isolated atrium does not. This may mean that thetion between the receptor activated by xylazine to inhibit tachycardia differshed rat heart and from the receptor activated by noradrenaline to inhibittex was relatively release of transmitter in atria, suggesting two subtypes ofI2B-adrenoceptor prejunctional receptor in atria, although studies in vitro may

319

'.- -- A

320 K. Smith et al Cardiac prejunctional k-adrenoceptors

not examine exclusively nerves involved in control of heartrate. We are currently investigating this problem but have noanswer at present.

In conclusion, the prejunctional a2-adrenoceptor in pithedrat heart mediating the cardioinhibitory actions of xylazine

closely resembles the M2D-adrenoceptor ligand binding site ofrat submandibular gland.

Supported by the Health Research Board (Ireland) and RCSI.Generous gifts of drugs are gratefully acknowledged.

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(Received December 5, 1994Revised January 21, 1995

Accepted January 30, 1995)