J Mol Cell Cardiol 32, 1567–1574 (2000)

doi:10.1006/jmcc.2000.1192, available online at http://www.idealibrary.com on

Interactions of Dynorphin A-(1–13) andNociceptin with Cardiac D2 BindingSites: Inhibition of Ischemia-evokedRelease of Noradrenaline fromSynaptosomal–mitochondrial FractionsMichel Dumont and Simon LemaireDepartment of Molecular and Cellular Medicine, Faculty of Medicine, University of Ottawa, 451Smyth Road, Ottawa, Ontario, Canada K1H 8M5

(Received 21 May 1999, accepted in revised form 16 May 2000)

M. D S. L. Interactions of Dynorphin A-(1–13) and Nociceptin with Cardiac D2 BindingSites: Inhibition of Ischemia-evoked Release of Noradrenaline from Synaptosomal–mitochondrial Fractions.Journal of Molecular and Cellular Cardiology (2000) 32, 1567–1574. The effect of dynorphin A (Dyn A)-related peptides and nociceptin on the binding of the D2 receptor antagonist, [3H]raclopride, was examinedin membrane preparations of rat heart. Non-linear regression saturation binding analysis of [3H]raclopridebinding revealed the presence of a single high-affinity binding site with a Kd of 4.1 n and a Bmax of220 fmol/mg protein. The D2 stereospecificity of [3H]raclopride binding was demonstrated by competitionexperiments using two enantiomer pairs of antagonists. (+)-Butaclamol (IC50: 8.0 n) and (−)-sulpiride (IC50:112.3 n) were 27 000 and 24 times more potent than (−)-butaclamol (IC50: >100 �) and (+)–sulpiride(IC50: 2666 n), respectively. Nociceptin and Dyn A-(1–13) were also potent inhibitors of the binding of[3H]raclopride with shallow inhibition curves that fitted best with two sites model. Their order of potencyon the low affinity site [�-Neo-endorphin>nociceptin>Dyn A-(2–13)>Dyn A-(1–13)>Dyn B>Dyn A-(6–10)]correlated well with their ability to inhibit the binding of [3H]nociceptin (r=0.82). The indirect nature ofthe inhibitory effects of the peptides on the D2 receptor was demonstrated by their inability to inhibit[3H]raclopride binding to a membrane preparation (Sf9 cells transfected with the human D2long receptor) thatdoes not contain the ORL1 receptor and the lack of effect of raclopride (0.1 n–10 �) on both [3H]nociceptinand [3H]Dyn A-(1–13) binding. Isolated cardiac mitochondrial–synaptosomal fractions submitted to ischemicconditions (1 m iodoacetate +2 m NaCN, 5 min at 37°C) released 10.9% of their content in preloaded[3H]noradrenaline ([3H]NA). Dyn A-(1–13) (10 �), nociceptin (10 �) and the selective D2 receptor agonist,quinpirole (10 �) were potent blockers of the release of [3H]NA evoked by the ischemic conditions. Theinhibitory effect of Dyn A-(1–13), nociceptin and quinpirole were antagonized by the selective D2 receptorantagonist, raclopride (10 �); whereas naloxone, at a concentration (1 �) known to affect the ORL1

receptor, blocked the effects of the peptides but not those of quinpirole. The results demonstrate the presenceof D2 receptors in rat heart and suggest that Dyn A-(1–13) and nociceptin modulate ischemia-induced NArelease by a mechanism that involves the participation of both ORL1 and D2 receptors.

2000 Academic Press

K W: Dynorphin A-(1–13); Nociceptin; D2 receptors; Heart; Ischemia.

Please address all correspondence to: Simon Lemaire, Department of Molecular and Cellular Medicine, Faculty of Medicine, Universityof Ottawa, 451 Smyth Road, Ottawa, Ontario, Canada K1H 8M5. Tel: (613) 562-5800 Ext: 8350; Fax: (613) 562-5434; E-mail:slemaire@uottawa.ca

0022–2828/00/081567+08 $35.00/0 2000 Academic Press

M. Dumont and S. Lemaire1568

with the cardiac D2 receptor and investigateIntroductionwhether such interaction could modulate the re-lease of [3H]NA from cardiac synaptosomes sub-The endogenous peptides dynorphin A (Dyn A) and

nociceptin produce various peripheral cardio- mitted to ischemic conditions. The results indicatethat Dyn A-(1–13) and nociceptin modulate thevascular effects. These peptides have been shown to

modulate blood pressure,1–5 cardiac enzymes,1,6,7 as cardiac D2 receptor and inhibit the ischemia-evokedrelease of [3H]NA by cardiac synaptosomes throughwell as noradrenaline (NA) release from sympathetic

nerve endings.1,8,9 In rat heart, the opioid peptide a mechanism that may involve both ORL1 and D2receptors.[3H]Dyn A-(1–13) was demonstrated to bind to a low

affinity, high capacity non-opioid site.10 The bindingactivity of [3H]Dyn A-(1–13) was insensitive to spe-cific ligands of � (U-50,488H; U-69,593) � ([D-Ala2, Materials and MethodsMePhe4, Glyol5]Enk) and � ([D-Ser2, Thr6]Leu-Enk)opioid receptors but displayed selectivity and struc-

Materialsture specificity for Dyn A and related peptides.10 Re-cently, nociceptin,11,12 a 17 amino acid peptide

[3H]Raclopride (78.4 Ci/mmol), [3H]NA (36.5 Ci/showing structural homology to Dyn A and pos-mmol) and [3H]nociceptin (157–172 Ci/mmol)sessing high affinity for a G protein-coupled opioidwere obtained from Amersham (England). [3H]Dynlike receptor, the ORL1 receptor,13 was shown to bindA-(1–13) (50 Ci/mmol) was purchased from Newto a site that displayed the same characteristics asEngland Nuclear (Boston, MA, USA). Iodoacetate,those of the cardiac non-opioid [3H]Dyn A-(1–13)nialamide, bacitracin, captopril, bestatin, thiorphanbinding site.14 It was suggested that Dyn A and no-and bovine serum albumin (BSA) were obtainedciceptin may bind to the same receptor in the heart,from Sigma Chemical Co. (St Louis, MO, USA).a G protein-coupled receptor.14

NaCN was purchased from BDH (London, Ontario,Dopamine (DA), the endogenous ligand for DACanada). (+)– and (−)-Butaclamol, (+)- and (−)-G protein-coupled receptors, produces peripheralsulpiride, raclopride, SCH 23390, quinpirole andcardiovascular effects similar to those of Dyn A-membranes of Sf9 cells transfected with the human(1–13) and nociceptin.15,16 DA receptors have beenD2long receptor were purchased from Research Bio-divided into two subgroups on the basis of receptorchemicals International (RBI, Natick, MA, USA).pharmacology, biochemical mechanisms of signalNaloxone was a product of Endo Laboratories (Mon-transduction and molecular biology.16 The two pre-treal, Quebec). Nociceptin, Dyn B and �-Neo-en-dominant DA receptors were classified as D1-likedorphin (�-Neo-End) were purchased from(D1 and D5) and D2-like (D2, D3 and D4).16 Ac-Peninsula Laboratories (Belmont, CA, USA). Dyntivation of both subgroups of DA receptors resultsA-(1–13), Dyn A-(2–13) and Dyn A-(6–10) werein a decrease in blood pressure due to a D2 receptor-synthesized in our laboratory as described by Le-mediated inhibition of sympathetic activity and amaire et al.26

D1 receptor-mediated arterial vasodilatation.15,17 Incardiac tissue, the pharmacological profile of thebinding of DA agonists and antagonists is consistentwith the presence of D2-like receptors.18–20 Recently, Membrane preparationa role for DA agonists has been proposed in thetreatment of cardiovascular diseases.17 Beneficial Membrane preparations of rat hearts were obtained

with a few modifications of the procedure alreadyeffects of DA agonists include reduction of ischemiaseverity due to coronary artery occlusion,21 decrease described.10 Briefly, the hearts of male Wistar (WR)

rats (Charles River, St Constant, Quebec, Canada)of the hemodynamic burden on the myocardiumin congestive heart failure15,22 and lowering of blood were removed from the animals after decapitation.

They were perfused through the aorta with 20 mlpressure in essential hypertension.23 An importantmechanism of action by which D2 receptor agonists of ice-cold solution of 0.32 sucrose containing

5 m Tris-HCl (pH 7.4; buffer A), minced, ho-may exert their beneficial cardiovascular effects isthe inhibition of NA release from sympathetic nerve mogenized using a Brinkman polytron (15 s at

setting 5) in 40 ml of buffer A and centrifuged atendings.15,17 The fact that Dyn A-(1–13), nociceptinand DA bind to a G protein-coupled receptor14,16 1000×g for 30 min at 4°C. The supernatant was

centrifuged at 26 000×g for 30 min at 4°C. Theand modulate noradrenergic neurotransmission14,

15,17,21,24,25 prompted us to verify the possible inter- pellets were resuspended in buffer A, incubated at37°C for 30 min and centrifuged at the same speed.action of nociceptin, Dyn A and related peptides

Dyn A-(1–13), Nociceptin and Cardiac D2 Receptors 1569

The resulting pellets were resuspended in 0.3 ociceptin binding were performed as already de-scribed.10,14KCl, stirred at 4°C for 60 min and centrifuged at

26 000×g for 30 min at 4°C. They were washedonce with buffer A and centrifuged as describedabove. The final pellets were resuspended in buffer

Preparation of cardiac synaptosomal–mitochondrialA at a concentration of 1mg protein/ml and frozen

fractionsat −80°C. Proteins were measured by the methodof Lowry et al.27 using BSA as the standard.

Cardiac synaptosomal–mitochondrial fractions ofWR rats were prepared as already described.29 Theanimals were decapitated and their hearts were

Binding assaysremoved, perfused through the aorta with 20 mlof ice-cold 0.32 sucrose containing 1 m EGTA

Binding assays were performed with modifications(pH 7.5), minced and transferred to an oxygenated

of a previously described method.28 Briefly, [3H]ra-HEPES-buffered Krebs–Ringer solution (KRH in m:

clopride binding was carried out at room tem-HEPES 50, NaCl 144, MgCl2 1.2, CaCl2 1.2, KCl 5,

perature for 60 min in 1 ml of buffer A containingglucose 10, ascorbic acid 1, and nialamide

30 � bestatin, 25 � bacitracin, 10 � captopril,0.0124 m, pH 7.4) containing 12 U of collagenase

0.3 � thiorphan and 100 �g of membrane protein.(Worthington Biochemicals, NJ, USA) per mg of

Binding experiments were terminated by filtrationtissue. This suspension was then incubated at 37°C

under reduced pressure through 934-AH Whatmanfor 40 min. Heart tissue was collected by low-speed

filters. The filters were washed four times withcentrifugation (120×g), washed with KRH, and

3 ml aliquots of ice-cold buffer A, placed in 10 mlrecentrifuged at the same speed. The tissue was

Ecolume (ICN, Biochemicals Inc., Mississauga, On-homogenized in 10 vol of ice-cold 0.32 sucrose

tario, Canada) and counted in a Wallac Scintillationwith a glass Teflon homogenizer with a clearance

counter (WinSpectral 1414). Specific binding ofof 0.25 mm. The homogenate was centrifuged at

[3H]raclopride was defined as the difference between650×g for 10 min at 4°C. The resulting super-

the total radiolabel bound and that bound in thenatant was centrifuged at 21 000×g for 20 min at

presence of 10 � (+)-butaclamol and represented4°C. The pellets were resuspended in oxygenated

40% at a concentration of 5 n. The equilibriumice-cold KRH at a concentration of 1.5 mg protein/

dissociation constant (Kd ) and the maximum bind-ml. Proteins were measured by the method of Lowry

ing capacity (Bmax) were obtained from non-linearet al.,27 using BSA as the standard.

regression saturation binding analysis using theprogram GraphPad PRISM (GraphPad SoftwareInc., San Diego, CA, USA). Competition experimentswere performed with 5 n of [3H]raclopride in the [3H]NA uptake and releasepresence of cold ligands (0.1 n–10 �). The con-centration that produces a 50% inhibition of [3H]ra- The cardiac synaptosomal–mitochondrial fraction

(1.5 mg protein/ml) was loaded with 300 nclopride binding (IC50) was derived using the non-linear regression analysis program GraphPad [3H]NA by a 1 h incubation at 37°C in KRH. At

the end of the incubation period, the preparationPRISM. For one binding site analysis the followingequation was used: Y= Bottom+(Top–Bottom)/ was put on ice, diluted with ice-cold KRH, and

collected by centrifugation at 900×g for 10 min1+10x-LogIC50 where top and bottom are the Y valuesat the top and bottom plateau of the curve. For at 4°C. The pellet was washed with KRH and

recentrifuged at the same speed. The final pellettwo binding sites analysis the following equationwas used: Y=Bottom+(Top–Bottom) [Fraction1/ was resuspended in KRH. Ischemic conditions were

obtained by incubating the synaptosomal–1+10x-LogIC501+1–Fraction1/1+10x-LogIC502 where Yis specific binding and X is the logarithm of the mitochondrial fraction in the presence of 1 m

iodoacetate and 2 m NaCN to inhibit the glycoliticconcentration of the unlabeled ligand. Fraction1 isthe fraction of the receptors that have an affinity enzyme, glyceraldehyde phosphate dehydrogenase,

and the oxidative phosphorylation enzyme, cyto-described by Log IC50.1 The remainder of the re-ceptors have an affinity described by Log IC50.2 chrome c oxidase, respectively.30 [3H]NA release

assays were performed in 1 ml of KRH. Tubes con-[3H]Raclopride binding on membranes from Sf9cells transfected with the human D2long receptor taining 800 �l of KRH, iodoacetate, NaCN and the

various test drugs were prewarmed at 37°C forwere performed using the recommended assay con-ditions from RBI. [3H]Dyn A-(1–13) and [3H] 5 min. The release experiment was started by the

M. Dumont and S. Lemaire1570

Dyn A-(1–13) and nociceptin may bind to the sameG protein-coupled receptor.14 In order to verify ifthis receptor may be related to the D2 G protein-coupled receptor we have examined the effects ofincreasing concentrations of Dyn A related peptidesand nociceptin on [3H]raclopride binding. No-ciceptin and Dyn A-(1–13) were potent inhibitorsof the binding of [3H]raclopride but had shallowinhibition curves (low Hill coefficient, Table 1). Theorder of potency of nociceptin and various Dyn Arelated peptides in inhibiting [3H]raclopride bindingto the low affinity site was as follows: �-Neo-End>nociceptin>Dyn A-(2–13)>Dyn A-(1–13)>DynFigure 1 Saturation curve and Scatchard plot (inset) ofB>Dyn A-(6–10) (Table 1). This order of potencyspecific [3H]raclopride binding to rat heart membranes.

Binding experiments were performed as described under correlated well with their previously reported ability‘‘Materials and Methods’’. The results represent the to inhibit the binding of [3H]nociceptin to rat heartmean±.. of three separate experiments conducted in membranes (r=0.82 as compared with 0.49 forduplicate.

the high affinity site; Fig. 2).14

In order to verify if the interaction with the D2receptor was direct or indirect, we have examinedaddition of 200 �l of the [3H]NA-loaded syn-the inhibitory potency of Dyn A related peptidesaptosomal–mitochondrial fraction (300 �g of pro-(10 �) and nociceptin (10 �) on [3H]raclopridetein). The mixture was vortexed and incubated forbinding in membranes from Sf9 cells transfected5 min at 37°C. Release experiments were ter-with the human D2long receptor (no ORL1 receptorminated by placing the samples on ice for 10 minpresent). The results show that Dyn A related pep-followed by centrifugation at 900×g for 10 min.tides and nociceptin display no or little inhibitoryAliquots of the supernatants were collected, placedeffect on [3H]raclopride binding (Table 1). In ad-in 10 ml Ecolume and counted in a Wallac scin-dition, raclopride (0.1 n–10 �) did not affect thetillation counter. The basal release was obtained bybinding of [3H]Dyn A-(1–13) and [3H]nociceptin tosubtracting the background of counts in the absencethe cardiac membrane preparation, suggesting thatof drug at 4°C from the counts in the medium afterthe inhibitory effects of Dyn A related peptides andincubation at 37°C. Net [3H]NA release in thenociceptin on the D2 receptor may be indirect.presence of ischemic conditions was obtained after

In order to verify if the interaction of Dyn A-subtraction of both background and basal release(1–13) and nociceptin with the cardiac D2 receptorand expressed as a percentage of the synaptosomalcould induce the same effects as D2 ligands oncontent of [3H]NA (before the release experiment).cardiovascular functions, Dyn A-(1–13), nociceptinand the D2 receptor agonist, quinpirole, were testedfor their ability to modulate the release of [3H]NAevoked by ischemic conditions in isolated cardiacResultssynaptosomal–mitochondrial fractions. Incubationof the cardiac synaptosomal–mitochondrial frac-Saturation binding experiments with [3H]raclopride

at a concentration range of 1–20 n revealed the tions with 1 m iodoacetate+2 m NaCN (isch-emic conditions) at 37°C for 5 min caused apresence of a single high affinity binding site in

rat heart. Nonlinear regression saturation binding 10.9±1.0% release of their content in preloaded[3H]NA. Dyn A-(1–13) (10 �), nociceptin (10 �)analysis gave a Kd of 4.1±1.1 n and a Bmax of

220±19 fmol/mg of protein (Fig. 1). The stereo- and quinpirole (10 �) significantly inhibited therelease of [3H]NA evoked by the ischemic conditionsspecificity of [3H]raclopride binding to cardiac D2

receptors was verified using two enantiomer pairs (Fig. 3). Their inhibitory effects were blocked by theselective D2 receptor antagonist raclopride (10 �;of antagonists. (+)-Butaclamol (IC50: 8.0±0.3 n)

was at least 27 000 times more potent than (−)- P<0.05; Fig. 3) but not by the D1 receptor an-tagonist, SCH 23390 (1 �; Fig. 3). The opioidbutaclamol (IC50>100 �) in competing with

[3H]raclopride binding, whereas (−)-sulpiride (IC50: receptor antagonist, naloxone, at a concentrationknown to block opioid receptors (0.2 �; the af-112.3±5.5 n) was 24 times more potent than

(+)-sulpiride (IC50: 2666±257 n). finities of naloxone for �, � and k receptors are1.78, 27.0 and 17.2 n, respectively31) did notRecently, our laboratory has demonstrated that

Dyn A-(1–13), Nociceptin and Cardiac D2 Receptors 1571

Table 1 Inhibition by nociceptin and Dyn A-related peptides of [3H]raclopride binding tomembrane preparations of rat heart and Sf9 cells transfected with the human D2long receptor

[3H]raclopride bindingRat heart Sf9 cells

IC50H IC50L Hill slope IC50

Peptides (n) (�) (�)

Nociceptin 9.1±2.7 0.43±0.19 0.65±0.05 NA

Dyn A related peptidesDyn A-(1–13) 24.2±7.1 3.3±1.4 0.53±0.05 >10Dyn A-(2–13) 484±178 0.83±0.08 NADyn A-(6–10) 87.1±15.4 24.6±11.1 0.44±0.08 NADyn B 75.9±8.7 10.6±4.9 0.53±0.07 >10�-Neo-End 43±2 1.29±0.09 NA

Values represent the means±.. of three separate experiments each conducted in duplicate. [3H]Ra-clopride binding was performed as described under ‘‘Materials and Methods’’.

affect the inhibitory effects of Dyn A-(1–13) and skeletal muscle, vas deferens, spleen and heart.14,

32 Fine binding analysis indicated that the ORL1nociceptin (Table 2). However, a higher con-centration of naloxone (1 �) produced a significant receptor was linked to a G protein.33 This site

binds well to nociceptin but also displays bindingreversal of the inhibitory effects of the peptidesbut not that of quinpirole. Neither raclopride, SCH sensitivity to Dyn A related peptides.34,35 The facts

that Dyn A-(1–13), nociceptin and D2 agonists23390 nor naloxone, at the concentrations used,affected the release of [3H]NA evoked by ischemic bind to a G protein-coupled receptor14,16 and inhibit

NA release in the heart9,15,17,24,25 prompted us toconditions (Fig. 3 and Table 2).verify the possible interaction (direct or indirect) ofDyn A related peptides and nociceptin with cardiacD2 receptors. Our results show that Dyn A-relatedpeptides and nociceptin are potent inhibitors ofDiscussion[3H]raclopride-binding. The order of potency of

The present study demonstrates the presence of a these peptides [�-Neo–End>nociceptin>Dyn A-(2–single high affinity [3H]raclopride binding site in 13)>Dyn A-(1–13)>Dyn B>Dyn A-(6–10)] in in-rat heart with characteristics of stereospecific D2 hibiting the low affinity [3H]raclopride binding sitereceptors.18–20 The affinity (Kd: 4.1 n) and number corresponds to their ability to compete with theof binding sites (Bmax: 220 fmol/mg of protein) of binding of [3H]nociceptin to cardiac membranes.14

[3H]raclopride binding to rat cardiac membranes However, the inability of these peptides (10 �) tocorrelate well with those reported for [3H]DA in inhibit [3H]raclopride binding to membranes of Sf9guinea-pig heart and [3H]spiperone in rat and cells, a membrane preparation that does not containhuman heart (Kd: from 0.4 to 11.8 n; Bmax: from the ORL1 receptor (data not shown), combined with40 to 267 fmol/mg of protein).18–20 The variations the lack of effect of raclopride on both [3H]Dynin the binding characteristics between these studies A-(1–13) and [3H]nociceptin binding suggest anmay be explained by the use of different DA receptor indirect interaction of the peptides with the cardiactritiated ligands (antagonist v agonist), tissue pre- D2 receptor. Together, these results suggest thatparations (membranes v frozen sections) or species Dyn A related peptides and nociceptin indirectly(rat v guinea-pig and human) used for sources of modulate cardiac D2 receptors through an inter-cardiac D2 receptors. action with the ORL1 receptor which may cause a

Rat heart membrane preparations were pre- D2 agonist-like alteration of the conformation ofviously shown to contain a low affinity high cap- the D2 site. In this regard, a receptor–receptoracity non–opioid receptor for [3H]Dyn A-(1–13).10 interaction with D2 receptor has already beenThe Dyn-specific receptor was subsequently shown shown for the adenosine A2 receptor in the neo-to possess the same characteristics as the ORL1 striatum.36

receptor for the endogenous peptide nociceptin.11, In recent years, DA agonists, including DA itself,12,14 The ORL1 receptor was initially detected in the have been proposed as an alternative approach

in the treatment of cardiovascular diseases.17 Thebrain,13 but also shown to be present in the intestine,

M. Dumont and S. Lemaire1572

Figure 2 Correlation between the relative potency ofvarious compounds in inhibiting the binding of [3H]nociceptin and [3H]raclopride to the high affinity site (A)and low affinity site (B) to membrane preparations of ratheart. The values were derived from Table 1 and Dumontand Lemaire14. The correlation coefficients are (r)=0.49for the high affinity site and (r)=0.82 for the low affinitysite. The competition curve of [3H]nociceptin by no-ciceptin itself had a Hill slope less than unity (0.51)suggesting receptor heterogeneity.14 Analysis of this com-petition curve using non-linear regression parameters oftwo binding sites showed that the affinities of nociceptinfor the high and low affinity ORL1 binding sites are11.5 n and 6 �, respectively. The value of 11.5 n Figure 3 Inhibitory effects of Dyn A-(1–13) (A), no-was taken to do the correlations. ciceptin (B) and quinpirole (C) on the release of [3H]NA

evoked by ischemic conditions from synaptosomal–mitochondrial fractions and reversal by the D2 receptorantagonist, raclopride. [3H]NA release experiments were

benefits of D2 agonists were demonstrated by the performed as described under ‘‘Materials and Methods’’.The results represent the mean±.. of five separatereduction of ischemia severity due to coronaryexperiments conducted in duplicate. ∗P<0.05 as com-artery occlusion,21 decrease of the hemodynamicpared with control, Student’s t-test. ∗∗P<0.05 as com-burden on the myocardium in congestive heart pared with either Dyn A-(1–13), nociceptin or quinpirole

failure15,22 and blood pressure in essential hyper- alone, Student’s t-test.tension.23 One common mechanism by which DAand DA receptor agonists exert their beneficialcardiovascular effects is the presynaptic D2 re- are significantly reversed by the D2 selective an-

tagonist, raclopride, but they are not affected byceptor-mediated inhibition of NA release from sym-pathetic nerve endings.15,17 Herein, the results the D1 antagonist, SCH 23390. Interestingly, the

opioid antagonist, naloxone (1 �), antagonized theindicate that Dyn A-(1–13) and nociceptin are aspotent as quinpirole, a D2 agonist, in inhibiting the inhibitory effect of Dyn A-(1–13) and nociceptin

but not that of quinpirole. The high concentrationrelease of [3H]NA from cardiac synaptosomal–mitochondrial fractions subjected to ischemic con- of naloxone necessary to inhibit the effects of the

peptides and the poor affinity of nociceptin for opioidditions. The inhibitory effects of these compounds

Dyn A-(1–13), Nociceptin and Cardiac D2 Receptors 1573

Table 2 Effects of Dyn A-(1–13), nociceptin and quinpirole on the releaseof [3H]NA evoked by ischemic conditions from synaptosomal–mitochondrialfractions in the absence and presence of the opioid antagonist, naloxone

Control Dyn A-(1–13) Nociceptin Quinpirole(10 �) (10 �) (10 �)

Naloxone (�)0 12.7±1.2 6.1±0.6∗ 7.6±0.5∗ 6.7±1.5∗0.2 12.5±1.2 7.8±1.5 9.0±1.0 7.5±0.91.0 10.8±2.0 10.9±0.7∗∗ 11.3±0.6∗∗ 7.5±1.4

[3H]NA release experiments were performed as described under ‘‘Materials and Methods’’.The results represent the mean±.. of five separate experiments conducted in duplicate.Statistical significance was determined using one way ANOVA followed by the Newman

Keuls post-hoc test.∗P<0.05 as compared with control.∗∗P<0.05 as compared to Dyn A-(1–13) and nociceptin alone.

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