J Mol Cell Cardiol 30, 2751–2760 (1998)Article No. mc980838

Characterization of the High Affinity[3H]Nociceptin Binding Site inMembrane Preparations of Rat Heart:Correlations with the Non-opioidDynorphin Binding SiteMichel Dumont and Simon LemaireDepartment of Cellular and Molecular Medicine, Faculty of Medicine, University of Ottawa,451 Smyth Road, Ottawa, Ontario, Canada

(Received 2 June 1998, accepted in revised form 24 September 1998)

M. D S. L. Characterization of the High Affinity [3H]Nociceptin Binding Site in MembranePreparations of Rat Heart: Correlations with the Non-opioid Dynorphin Binding Site. Journal of Molecular andCellular Cardiology (1998) 30, 2751–2760. The binding parameters of [3H]nociceptin were examined in membranepreparations of rat heart and compared with those of [3H]dynorphin A-(1-13) ([3H]Dyn A-(1-13)). Scatchardanalysis of [3H]nociceptin binding revealed the presence of two distinct sites: a high affinity (Kd: 583 n) lowcapacity (Bmax: 132 pmol/mg protein) site and a low affinity (Kd: 10 316 n) high capacity (1552 pmol/mgprotein) site. Dyn A and related peptides were potent competitors of the binding to the high affinity site with thefollowing rank order of potency: a-neo-endorphin>Dyn A-(2-13)=Dyn A-(3-13)>Dyn A-(5-13)>Dyn A-(1-13)>Dyn A>Dyn B>Dyn A-(6-10)>>Dyn A-(1-8). Nociceptin was 6.7 times less potent than Dyn A with a Ki of4.8 l as compared with 0.72l for Dyn A. The order of potency of the various peptides in inhibiting[3H]nociceptin binding correlated well (r=0.93) with their ability to compete with the binding of [3H]Dyn A-(1-13) (Dumont and Lemaire, 1993). In addition, the high affinity [3H]nociceptin and non-opioid [3H]Dyn A-(1-13)sites were both sensitive to NaCl (120 m) and the phospholipase C (PLC) inhibitors, U-73122 and neomycin(100l). The binding activities were less affected by the weak PLC inhibitor, U-73343, and no effect was observedwith the non-hydrolysable GTP analogs, Gpp(NH)p and GTP-c-S. Nociceptin (1–50l) was also shown to inhibitthe uptake of [3H]noradrenaline ( [3H]NA) by cardiac synaptosomal preparations. In spontaneously hypertensiverats (SHR), the potency of nociceptin in inhibiting [3H]NA uptake was increased by 1.6-fold as compared withWistar Kyoto (WKY) control rats and such effect was accompanied by comparable increased levels of cardiacORL1 mRNA and [3H]nociceptin high affinity sites. These changes correlated well with the previously observedincreased levels of non-opioid cardiac [3H]Dyn A-(1-13) sites in SHR (1.3 times as compared with WKY) andincreased potency of Dyn A-(1-13) in inhibiting [3H]NA uptake by cardiac synaptosomes in SHR (2.2-fold ascompared with WKY) (Dumont and Lemaire, 1995). The results demonstrate that in rat heart the characteristicsof the high affinity, low capacity [3H]nociceptin binding site are similar to those of the non-opioid Dyn bindingsite. The stimulation of this site by nociceptin, Dyn A or related peptides is more likely to produce a modulationof PLC activity and [3H]NA uptake and may participate to the pathophysiology of hypertension.

1998 Academic Press

K W: Nociceptin; Dynorphin; ORL1 receptor; Non-opioid receptor; G-protein; Phospholipase C; Heart;Hypertension.

Please address all correspondence to: Simon Lemaire. E-mail:slemaire@uottawa.ca

0022–2828/98/122751+10 $30.00/0 1998 Academic Press

M. Dumont and S. Lemaire2752

Dyn site (Dumont and Lemaire, 1993). In addition,Introductionspontaneously hypertensive rats (SHR) display anincreased Bmax with no change in membrane affinityNociceptin (Meunier et al., 1995; Meunier, 1997)

also known as orphanin FQ (Reinscheid et al., 1995) for both Dyn and high affinity nociceptin sites,suggesting that such site(s) may be involved in theis a 17 amino acid peptide possessing high affinity

for the G protein-coupled opioid-like receptor ORL1 pathophysiology of hypertension.(Mollereau et al., 1994; Wang et al., 1994).Although, the ORL1 receptor shares sequencehomology with previously described l, d and j Materials and Methodsopioid receptors (Bunzow et al., 1994; Chen et al.,1994; Fukuda et al., 1994; Mollereau et al., 1994;

MaterialsWang et al., 1994), it has poor affinity for opioidreceptor ligands (Meunier et al., 1995; Reinscheid

[3H]Nociceptin (157–172 Ci/mmol) and [3H]no-et al., 1995; Pan et al., 1996). The ORL1 receptorradrenaline ([3H]NA, 36 Ci/mmol) were obtainedis present in the central nervous system (CNS;from Amersham (England). [3H]Dyn A-(1-13)Bunzow et al., 1994; Fukuda et al., 1994; Mollereau(50 Ci/mmol) was purchased from New Englandet al., 1994; Wang et al., 1994) as well as inNuclear (Boston, MA, USA). Desipramine, ni-peripheral organs such as the intestine, vas deferens,soxetine, ouabain, 4-aminopyridine, 5′guanyly-liver and spleen (Wang et al., 1994). In the CNS,limidodiphosphate (GppNHp), neomycin, baci-ORL1 transcripts are localized in brain regionstracin, captopril, bestatin, thiorphan, bovine serumknown to regulate blood pressure (Bunzow et al.,albumin (BSA) and polyethylenimine (PEI) were1994; Fukuda et al., 1994; Mollereau et al., 1994;obtained from Sigma Chemical Co. (St Louis, MO,Wang et al., 1994), suggesting that the ORL1 re-USA). Guanosine-5′-o-(3-thiotriphosphate)tetra-ceptor may have a role to play in the regulation oflithium (GTP-c-S), 1-[6[[(17b)-3-Methoxyestra-1,cardiovascular functions. In this regard, nociceptin3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-was recently shown to produce various peripheraldione (U-73122) and 1-[6[[(17b)-3-Methoxyestra-cardiovascular effects that include vasorelaxation of1,3,5(10)-trien-17-yl]amino]hexyl]-2,5-pyrrolidin-precontracted arterial rings (Gumusel et al., 1997),edione (U-73343) were purchased from Researchdepressor responses in anaesthetized rats (Cham-Biochemicals International (Natick, MA, USA).pion and Kadowitz, 1997; Giuliani et al., 1997)Trans-(+)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolid-and inhibitory modulation of transmitter release ininyl) cyclohexyl benzene-acetamide methane-guinea-pig left atria (Guiliani and Maggi, 1997).sulfonate hydrate (U-50,488H) were donated byWe have previously demonstrated that Dyn A isthe Upjohn Company (Kalamazoo, MI, USA). No-a potent modulator of catecholamine uptake inciceptin, Dyn A, Dyn B, Leu-enkephalin (Leu-Enk),isolated cardiac synaptosomes (Dumont and Le-a-Neo-endorphin (a-Neo-End), [D-Ser2, Thr6]Leu-maire, 1995), an effect that is more likely mediatedEnk (DSLET), [D-Ala2, MePhe4, Gly-ol5]by an interaction of the peptide with a non-opioidEnk (DAGO), neuromedin B (NeB), substance Preceptor (Dumont and Lemaire, 1993). The ex-(SP) were purchased from Peninsula Laboratoriesistence of a structural homology between Dyn A(Belmont, CA, USA). Dyn A-(1-13), Dyn A-(2-13),and nociceptin (Meunier et al., 1995; Reinscheid etDyn A-(3-13), Dyn A-(5-13), Dyn A-(1-8) and Dynal., 1995) and the fact that both the ORL1 receptorA-(6-10) were synthesized in our laboratory asand the cardiac non-opioid Dyn binding site aredescribed by Lemaire et al. (1986). All primers forsensitive to Dyn A but not to l, d and j opioidthe polymerase chain reaction (PCR) were syn-receptor ligands (Dumont and Lemaire, 1993; Ad-thesized and HPLC-purified at the Biotechnologyapa and Toll, 1997; Mathis et al., 1997), led us toResearch Institute (University of Ottawa, Ottawa,hypothesize that Dyn A and nociceptin may produceOntario, Canada).their cardiovascular effects through an interaction

with a common site. In the present study, we havecharacterized the binding sites for [3H]nociceptin inmembrane preparations of rat heart and verified Membrane preparationthe possible interaction of Dyn A and related pep-tides with these sites. The results indicate that the Membrane preparations of rat hearts were obtained

with a few modifications of the procedure alreadybinding characteristics of the high affinity, lowcapacity [3H]nociceptin site correlate well with described (Dumont and Lemaire, 1993). Briefly, the

hearts of male Wistar (WR), Wistar Kyoto (WKY)those of the previously described cardiac non-opioid

Cardiac Nociceptin Binding Sites 2753

or spontaneously hypertensive (SHR) rats (Charles curve fitting computer program BDATA (EMF Soft-ware Inc., Knoxville, TN, USA). Competition ex-River, St Constant, Quebec, Canada), as indicated,

were removed from the animals after decapitation. periments were performed with 0.75 n of[3H]nociceptin in the presence of 6–9 concentra-They were perfused through the aorta with 20 ml

of ice-cold solution of 0.32 sucrose containing tions of cold ligands (10−8–10−4 ). The con-centration that produces a 50% inhibition of50 m Tris-HCl (pH 7.4; buffer A), minced, ho-

mogenized using a Brinkman polytron (15 s at [3H]nociceptin binding (IC50) was derived using thenon-iterative least square computer programsetting 5) in 40 ml of buffer A and centrifuged at

1000×g for 30 min at 4°C. The supernatant was CDATA (EMF Software Inc.). The inhibition constant(Ki) was calculated according to the equation: Ki=centrifuged at 26 000×g for 30 min at 4°C. The

pellets were resuspended in buffer A, incubated at IC50/(1+[L]/Kd) were [L] is the concentration of[3H]nociceptin and Kd its equilibrium dissociation37°C for 30 min and centrifuged at the same speed.

The resulting pellets were resuspended in 0.3 KCl constant (Cheng and Prusoff, 1973). [3H]Dyn A-(1-13) binding was performed as already described(Lee et al., 1982), stirred at 4°C for 60 min and

centrifuged at 26 000×g for 30 min at 4°C. They (Dumont and Lemaire, 1993).were washed once with buffer A and centrifugedas described above. The final pellets were re-suspended in buffer A at a concentration of 1 mg Preparation of cardiac synaptosomal-mitochondrial

fractionsprotein/ml and frozen at −80°C. Proteins weremeasured by the method of Lowry et al. (1951)

Cardiac synaptosomal-mitochondrial fractions ofusing BSA as the standard.WKY and SHR were prepared as already described(Aloyo et al., 1991). The animals were decapitatedand their hearts were removed, perfused throughBinding assaysthe aorta with 20 ml of ice-cold 0.32 sucrosecontaining 1 m EGTA (pH 7.5), minced and trans-Binding assays were performed with modifications

of a previously described method (Butour et al., ferred to an oxygenated HEPES-buffered Krebs-Ringer solution (KRH in m: HEPES 50, NaCl 144,1997). Briefly, [3H]nociceptin binding was carried

out at 4°C for 120 min in 0.5 ml of buffer A con- MgCl2 1.2, CaCl2 1.2, KCl 5, glucose 10, ascorbicacid 1, and nialamide 0.0124 m, pH 7.4) con-taining 0.1% BSA, 30 l bestatin, 25 l ba-

citracin, 10 l captopril, 0.3 l thiorphan and taining 12 U of collagenase (Worthington Bio-chemicals, NJ, USA) per mg of tissue. This50 lg of membrane protein. The binding ex-

periments were terminated by filtration under re- suspension was then incubated at 37°C for 40 min.Heart tissue was collected by low-speed cent-duced pressure through 934-AH Whatman filters

pretreated with 0.33% PEI in buffer A. The filters rifugation (120×g), washed with KRH, and re-centrifuged at the same speed. The tissue waswere washed four times with 3 ml aliquots of ice-

cold 10 m Tris-HCl (pH: 7.4), placed in 10 ml homogenized in 10 vol of ice-cold 0.32 sucrosewith a glass Teflon homogenizer with a clearanceEcolume (ICN, Biomedicals Inc., Mississauga, On-

tario, Canada) and counted in a Wallac Scintillation of 0.25 mm. The homogenate was centrifuged at650×g for 10 min at 4°C. The resulting super-counter (WinSpectral 1414). Specific binding of

[3H]nociceptin was defined as the difference between natant was centrifuged at 21 000×g for 20 min at4°C. The pellets were resuspended in oxygenatedthe total radiolabel bound and that bound in the

presence of 10 l Dyn A-(1-13). Saturation ex- ice-cold KRH at a concentration of 1.5 mg protein/ml. Proteins were measured by the method of Lowryperiments with [3H]nociceptin (0.25 to 10 000 n)

were performed by adding [3H]nociceptin to a con- et al., (1951) using BSA as the standard.centration up to 1 n; greater concentrations wereprepared by mixing 1 n of [3H]nociceptin withunlabeled nociceptin to obtain final concentrations Uptake studiesranging from 2.5 to 10 000 n. The results areexpressed after correction by multiplication of the Uptake assays of [3H]NA into cardiac synaptosomal-

mitochondrial fractions were performed in 1 ml ofradiolabeled bound with the dilution factor(1+[unlabeled nociceptin]/[labeled nociceptin]). KRH containing 0.5% BSA, 30 l bestatin, 25 l

bacitracin, 10 l captopril and 0.3 l thiorphanThe equilibrium dissociation constant (Kd) and themaximum binding capacity (Bmax) were obtained (buffer B). Tubes containing 600 ll of buffer B,

100 ll of 0.1 l [3H]NA in buffer B and 100 ll offrom Scatchard plot analysis using the iterative

M. Dumont and S. Lemaire2754

non-labeled nociceptin (10−7–10−4 ) in buffer Bwere prewarmed at 37°C for 5 min. The uptakereaction was initiated by addition of 200 ll of thesynaptosomal-mitochondrial fractions (300 lg ofprotein). The mixture was vortexed and incubatedfor 10 min at 37°C. Uptake experiments were ter-minated by placing the samples on ice for 10 min,followed by filtration under reduced pressurethrough 934-AH Whatman filters. Filters werewashed with 4×3-ml aliquots of ice-cold KRH,placed in 10 ml Ecolume (ICN) and counted in aWallac Scintillation counter (WinSpectral 1414).

Figure 1 Association time course of specific [3H]n-Uptake was measured by the difference in countsociceptin binding to rat heart membrane preparationsobtained without and with 1 l nisoxetine. The for the indicated times at 4° and 25°C. Binding ex-

concentration of nociceptin that produces 50% in- periments were performed as described under “Materialshibition of [3H]NA uptake (IC50) was obtained with and Methods” and each point represents the mean±..

of three experiments conducted in duplicate.the non-linear regression curve-fitting programGraphPad PRISM (GraphPad Software, Inc, SanDiego CA, USA).

cDNA will produce a 521 and 249 bp band for theORL1 receptor and GADPH primers, respectively.

RNA isolation PCR was performed in 25 ll of Tris-Cl buffer (10 m,pH 8.3) containing 2 m MgCl2, 50 m KCl,

Hearts of 8 week-old male WKY and age-matched 200 l of each dNTP, 0.15 l of each primers,SHR rats were removed from the animals after 0.625 U of TaqGold polymerase (Perkin-Elmer) anddecapitation. They were perfused through the aorta 1 ll of the RT reaction. A PCR 2400 instrumentwith 20 ml of ice-cold solution of 0.32 in buffer (Perkin-Elmer) was used with the following pro-A, frozen in liquid nitrogen and stored at −80°C gram: 12 min at 94°C, 30 cycles of amplificationuntil use. Total RNA was extracted using TriZol (1 min at 94°C, 1 min at 55°C, 1 min at 72°C),reagent (Gibco-BRL, Burlington, Ontario, Canada). followed by 7 min at 72°C and soaking at 4°C.

Reaction products were electrophoresed on an agar-ose gel (1.5%) and stained with SyBrGreen (Mo-

Reverse transcription and polymerase chain reaction lecular probes, Eugene, OR, USA). The gels were(RT-PCR) scanned with a chemifluorescence imaging system

(Storm 860, Molecular Dynamics, Sunnyvale, CA,RT was performed with 2 lg of total RNA that was USA). Quantification of the bands were obtainedreverse transcribed using 2.5 U of Moloney Murine using the ImageQuaNT computer program (Mo-Leukemia Virus reverse transcriptase (M-MLV RT; lecular Dynamics). The relative abundance of thePerkin-Elmer, Applied Biosystems Division, Forest ORL1 receptor was normalized to the internal con-City, CA, USA) in 10 m Tris-Cl pH 8.3, 50 m trol, GADPH.KCl, 5 m MgCl2, 1 m each dNTP and 0.125 l

of oligo(dt)16. The primers used for the rat ORL1

receptor (GenBank accession number: U05239)were: forward primer, 5′-GTCATCCTCAGGCA- ResultsCACCAA-3′ (bases 214–233 of the coding region);reverse primer, 5′-AAGCAGACGGACACCACGAA- Cardiac membrane preparations from male WR rats

were used for the characterization of [3H]nociceptin3′ (bases 716–735 of the coding region). The in-ternal control used in the present study was the rat binding. The specific binding of [3H]nociceptin was

time and temperature dependent (Fig. 1). The bind-glyceraldehyde-phosphate dehydrogenase (GADPH;GenBank accession number: M17701). Sequences ing of [3H]nociceptin was maximal after 10 min

at 25°C, while that performed at 4°C reached aof the primers for this internal control were: forwardprimer, 5′-TGATGACATCAAGAAGGTGGTGAAG-3′ maximum at 30 min and remained maximal for at

least 120 min. Since the maximal binding observed(bases 758–782 of the coding region); reverseprimer, 5′-TCCTTGGAGGCCATGTAGGCCAT (bases at 25°C represented only 85% of that obtained at

4°C, all subsequent experiments were performed at974–997 of the coding region). Amplification of

Cardiac Nociceptin Binding Sites 2755

Figure 2 Saturation curve and Scatchard plot (inset) ofspecific [3H]nociceptin binding to rat heart membranes.Membrane preparations were incubated in presence ofthe indicated concentrations of the peptide. Binding ex-periments were performed as described under “Materialsand Methods” and the results are expressed as themean±.. of three experiments conducted in duplicate.

4°C for 120 min. Under these conditions the specificbinding represented 85% of total binding.

Saturation binding experiments with a con-centration range of 0.25 to 10 000 n [3H]n-ociceptin revealed the presence of two distinct sites

Figure 3 Inhibition of [3H]nociceptin (A) and [3H]Dyn(Fig. 2): a high affinity (Kd; 583±94 n), low A-(1-13) (B) binding by NaCl and the non-hydrolyzablecapacity (Bmax; 132±21 pmol/mg protein) site and GTP analogs, Gpp(NH)p and GTP-c-S. Binding ex-a low affinity (Kd; 10 316±3148 n), high capacity periments were performed as described under “Materials

and Methods” and each value represents the mean±..(Bmax; 1552±252 pmol/mg protein) site.of four experiments conducted in duplicate. StatisticalThe relative potency of Dyn A and related peptidessignificance was determined by using one-way analysisin inhibiting the binding of [3H]nociceptin to the of variance followed by Newman-Keuls post hoc test.

high affinity site (e.g. in the presence of a low ∗ P<0.05 as compared to control. ∗∗ P<0.05 as comparedconcentration [0.75 n] of [3H]nociceptin) was de- to NaCl alone.termined. Dyn A inhibited the binding of [3H]nociceptin with a Ki of 0.72 l. Dyn A-(1-13) anda-Neo-End were also potent inhibitors of [3H] exhibited little or no inhibition of [3H]nociceptin

binding. Other compounds possessing little or nonociceptin binding with Ki of 0.55 and 0.25 l,respectively. Dyn B displayed moderate affinity with inhibitory effects included the Na+K+ ATPase in-

hibitor, ouabain, the noradrenaline uptake blockers,a Ki of 13.2 l. High concentrations (100 l) ofDyn A-(1-8) showed little inhibition. The non-opioid desipramine and nisoxetine and the peptide ne-

uromedin B. Substance P displayed a low ability tofragments, Dyn A-(2-13), Dyn A-(3-13) and DynA-(5-13), were potent inhibitors of [3H]nociceptin compete with [3H]nociceptin binding with a Ki of

32.5 l.with Ki of 0.43, 0.44 and 0.49 l, respectively.The smallest active fragment tested was Dyn A-(6- In order to verify if [3H]nociceptin and [3H]Dyn

A-(1-13) bind to a G protein-coupled receptor, we10) with a Ki of 18.8 l. The order of potency ofDyn A and related peptides (including nociceptin) have determined the effect of NaCl and non-hydro-

lysable GTP analogs, Gpp(NH)p and GTP-c-S (Fig.in inhibiting [3H]nociceptin binding was as follows:a-Neo-End>Dyn A-(2-13)=Dyn A-(3-13)>Dyn A- 3). [3H]nociceptin and [3H]Dyn A-(1-13) binding

were both sensitive to NaCl (120 m). Gpp(NH)p(5-13)>Dyn A-(1-13)>Dyn A>nociceptin>DynB>Dyn A-(6-10)>>Dyn A-(1-8). This order of po- and GTP-c-S (100 l) did not affect the binding

activities per se, but GTP-c-S (100 l) slightly po-tency correlated well with that observed for theinhibition of [3H]Dyn A-(1-13) binding to rat heart tentiated the inhibitory effect of NaCl on [3H]Dyn

A-(1-13) binding (10 m; Fig. 3), consistent withmembranes (r=0.93; Dumont and Lemaire, 1993).High concentrations (100 l) of j (U-50-488H), the possible involvement of a G protein. On the

other hand, the specific PLC inhibitor, U-73122d (DSLET) and l (DAGO) opioid receptor ligands

M. Dumont and S. Lemaire2756

Figure 4 Inhibition of [3H]nociceptin (A) and [3H]Dyn A-(1-13) (B) binding by U-73122, U-73343 and neomycin.Binding experiments were performed as described under“Materials and Methods” and each value represents themean±.. of four experiments conducted in duplicate.Statistical significance was determined by using one-wayanalysis of variance followed by Newman-Keuls post hoctest. ∗ P<0.05 as compared to control. ∗∗ P<0.05 ascompared to U-73122.

Figure 5 Representative RT-PCR analysis (A) and ORL1

receptor mRNA levels (B) in hearts of 8-week-old WKY(100 l), inhibited the binding of [3H]nociceptinand SHR rats. PCR amplification gave a 521 and 249 bpand [3H]Dyn A-(1-13) (Fig. 4). U-73343 (100 l), band for the ORL1 receptor and GADPH primers, re-

a weak or inactive PLC inhibitor, was significantly spectively. The mRNA levels were quantified by den-less potent than U-73122 (35.7±5.4% of control sitometric scanning and normalized to the internal

control GADPH. A value of 1 was ascribed to the ORL1as compared with 9.9±1.2% for [3H]nociceptinmRNA levels in the hearts of WKY rats. RT-PCR wasbinding and 40.0±6.3% of control as comparedperformed as described under “Materials and Methods”with 1.5±1.1% for [3H]Dyn A-(1-13) binding, re- and each value represents the mean±.. of four different

spectively; Fig. 4). In addition, neomycin (100 l), RNA preparations. Statistical significance was determineda potent PLC inhibitor, completely abolished the by using Student’s t-test. ∗ P<0.05 as compared to WKY

rats.two binding activities (Fig. 4).Using RT-PCR, the ORL1 receptor mRNA was

detected in hearts of 8 week-old WKY and age-matched SHR rats [Fig. 5(A)]. The ORL1 receptormRNA was 1.42-fold more abundant in SHR than Fig. 2 and Table 2). However, in hearts of 8 week-

old SHR rats, the Bmax value of the high affinity,in age-matched WKY rats [Fig. 5(B)]. To determinewhether the increase in the mRNA levels for the low capacity [3H]nociceptin binding site was in-

creased by 64% as compared to age-matched WKYORL1 receptor was parallelled by a change in [3H]nociceptin binding activity, we measured the sat- rats (from 122±17 to 200±12 pmol/mg protein;

P<0.05, Student’s t-test) whereas no change in theuration binding parameters of [3H]nociceptin in 8week-old WKY and age-matched SHR rats (Table 2). Kd values was observed (664±100 n for WKY

as compared to 519±83 n for SHR rats). NoScatchard analysis revealed no significant change inthe binding parameters of [3H]nociceptin in the significant difference in Kd and Bmax values was

observed for the low affinity, high capacityheart of the two control groups (WR and WKY;

Cardiac Nociceptin Binding Sites 2757

Table 1 Relative potency of various compounds in in-hibiting the binding of [3H]nociceptin to membrane pre-parations of rat heart

Compounds Ki1

(l)

Nociceptin 4.8±1.3Dyn A related peptides

Dyn A-(1-13) 0.55±0.28Dyn A-(2-13) 0.43±0.11Dyn A-(3-13) 0.44±0.09Dyn A-(5-13) 0.49±0.09Dyn A 0.72±0.18

Figure 6 Effect of nociceptin on [3H]NA uptake intoDyn A-(6-10) 18.8±2.5cardiac synaptosomal-mitochondrial fractions isolatedDyn A-(1-8) >100from 8-week-old WKY and SHR rats. Uptake assays wereDyn B 13.2±4.2performed as described under “Materials and Methods”a-Neo-End 0.25±0.04and each value represents the mean (.. <15%).

Opioid receptor ligandsU-50488H >100DSLET NA

DiscussionDAGO NAOther receptor ligands

The present study demonstrates the presence ofSubstance P 34.5±8.0Neuromedin B >100 two distinct [3H]nociceptin binding sites in cardiacOuabain NA membranes: a high affinity site and a low affinityDesipramine >100 site representing 7.8 and 92.2% of total specificNisoxetine >100

binding, respectively. Previous studies conducted in1 Values represent the means±.. of three experiments each mouse brain (Mathis et al., 1997), Chinese hamster

conducted in duplicate. [3H]nociceptin binding was performed ovary (CHO; Adapa and Toll, 1997) and humanas described under “Materials and Methods”. NA, non active.

embryonic kidney (HEK) 293 cells (Ardati et al.,1997) also demonstrated receptor heterogeneity fornociceptin. However, these authors have reporteddifferent binding characteristics for both the high[3H]nociceptin binding site between the two strains

of animals (Table 2). affinity (Kd: 0.004–0.38 v 583 n; Bmax: 0.03–1.6v 132 pmol/mg protein) and low affinity (Kd: 0.23–In order to verify if the presence of nociceptin

receptors in the heart was linked to some physio- 8.3 v 10 316 n: Bmax: 0.23–1.8 v 1552 pmol/mgprotein) [3H]nociceptin binding sites. These dif-logical and/or pathophysiological activity, no-

ciceptin was tested for its ability to modulate the ferences may be explained by the use of differentmembrane preparations, binding conditions anduptake of [3H]NA by cardiac synaptosomal-mito-

chondrial preparations from 8 week-old WKY (con- sources of ORL1 sites.On the other hand, the Kd and Bmax values of thetrol) and SHR rats. Nociceptin potently inhibited

[3H]NA uptake by cardiac synaptosomal-mito- high affinity, low capacity [3H]nociceptin bindingsite in membranes of rat heart correspond to thosechondrial fractions, its potency being 1.6 times

greater in SHR than in WKY (IC50 of 7.3±1.3 v of the non-opioid Dyn binding site in the samecardiac membrane preparation (Kd; 285 n: Bmax;11.7±1.1 l; P<0.05, Student’s t-test, Fig. 6).

Table 2 Binding parameters of [3H]nociceptin binding to rat heart membranes of 8-week-old WKY and SHR rats

High affinity Low affinity

Kd Bmax Kd Bmax

(n) (pmol/mg protein) (n) (pmol/mg protein)

WKY 664±100 122±17 9204±451 1623±163SHR 519±83 200±12∗ 7572±1974 1634±254

Values represent the means±.. of three experiments each conducted in duplicate. [3H]nociceptin binding was performed asdescribed under “Materials and Methods”. Statistical significance was determined by using Student’s t-test. ∗ P<0.05 as compared toWKY rats.

M. Dumont and S. Lemaire2758

215 pmol/mg protein; Dumont and Lemaire, 1993). ([Ca2+]i) and activate protein kinase C (PKC) (Co-ckcroft, 1987). More recently, the stimulation of theIn addition, the potency of Dyn A and related

peptides in inhibiting the binding of [3H]nociceptin ORL1 receptor by nociceptin was shown to activatePKC via the PLC/Ca2+ pathway (Lou et al., 1997).correlates well with their potency in inhibiting

[3H]Dyn A-(1-13) binding (r=0.93; Dumont and In SHR rats, a model of hypertension and myo-cardial hypertrophy, we have observed that theLemaire, 1993). The non-opioid peptides Dyn A-

(2-13), Dyn A-(3-13) and Dyn A-(5-13) were potent levels of ORL1 receptor mRNA, the number (Bmax)of high affinity [3H]nociceptin binding sites as wellinhibitors of both binding activities, demonstrating

the non-opioid nature of the two sites. Sensitivity as the inhibitory potency of nociceptin on [3H]NAuptake are increased as compared to WKY rats.of [3H]nociceptin binding to Dyn A and related

peptides was also observed in mouse brain (Mathis These increases correlate with those observed for[3H]Dyn A-(1-13) binding and the inhibitory po-et al., 1997) and CHO cells (Adapa and Toll, 1997),

but to a somewhat lower degree than that observed tency of Dyn A-(1-13) on [3H]NA uptake in SHRrats (Dumont and Lemaire, 1995). The possible rolein the cardiac membrane preparations. Therefore,

the binding of [3H]nociceptin to the high affinity of nociceptin, Dyn A and related peptides in thedevelopment of hypertension and accompanyingsite was more sensitive to Dyn A and some related

peptides than to nociceptin itself, suggesting that, cardiac hypertrophy in SHR remains to be es-tablished but such involvement would more likelyin an in vivo situation, this site may be mainly

stimulated by endogenous Dyn A and related pep- be through an increase in their activities. DynA was previously suggested to participate to thetides (Dumont et al., 1990).

Several studies demonstrate that the ORL1 re- pathophysiology of the hypertensive state in SHRthrough an enhanced inhibitory effect of the peptideceptor is influenced by GTP, GTP analogs and NaCl

(Adapa and Toll, 1997; Ardati et al., 1997; Butour on the uptake of NA by sympathetic nerve terminals(Dumont and Lemaire, 1997). The present data alsoet al., 1997; Makman and Dvorkin, 1997; Makman

et al., 1997). In rat cardiac membranes, the binding indicate that nociceptin displays similar inhibitoryeffects on the uptake of [3H]NA and that such effectsactivities of [3H]nociceptin and [3H]Dyn A-(1-13)

were sensitive to NaCl, but not to the non-hydro- are enhanced in SHR. Thus, the development ofhypertension in SHR may involve the participationlysable GTP analogs Gpp(NH)p and GTP-c-S. How-

ever, the combination of GTP-c-S (100 l) with of both nociceptin and Dyn A.The high levels of high affinity [3H]nociceptinNaCl (10 m) slightly enhanced the inhibitory effect

of NaCl alone, suggesting the possible involvement and/or [3H]Dyn A-(1-13) binding sites in the heartof SHR may lead to an increase in the stimulationof a G protein. The relative insensitivity of these

binding activities to GTP analogs may be explained of PLC and entry of Ca2+ into cardiomyocytes. Anociceptin and/or Dyn A modulation of PLC activityby the occurrence of G protein-linked receptor with

no or little sensitivity to GTP and analogs. In this in SHR is supported by the observation that vascularsmooth muscle cells of SHR possess high levels ofregard, the G protein a subunit, Gq, a protein linked

to the stimulation of PLC, was shown to be relatively PLC activity (Vehara et al., 1988) and that cardiacPKC activity (Makita and Yashuda, 1990), DAGinsensitive to GTP analogs (Pang and Sternweiss,

1990; Smrcka et al., 1991; Taylor et al., 1991; levels (Kondo et al., 1990) and myocardial cells[Ca2+]i (Andrawis et al., 1988) are increased inJhon et al., 1993; Smrcka and Sternweiss, 1993).

Interestingly, [3H]nociceptin and [3H]Dyn A-(1-13) SHR as compared with WKY rats.In conclusion, rat hearts possess a high affinity,bindings were inhibited by the PLC inhibitors, U-

73122 and neomycin. These results suggest that low capacity [3H]nociceptin binding site that displaythe same characteristics as those of the non-opioidnociceptin, Dyn A and related peptides may bind to

the same site in the rat heart and evoke the activation Dyn binding site, suggesting that the two peptidesmay bind to the same receptor. The high sensitivityof PLC involving a Gq type of G protein.

PLC is known to play an important role in the of these two sites to NaCl and PLC inhibitors indicatethat cardiac dynorphin and nociceptin may bindcontrol of myocardial function. The myocardial

inotropic and hypertrophic effects of angiotensin II, to a Gq type of G protein linked to the activationof PLC. The increased number of sites for Dyn Aendothelin and a1-adrenergic ligands are coupled to

a Gq protein-mediated activation of PLC (Shubeita et and nociceptin in cardiac SHR membranes and theenhanced inhibitory effects of the peptides on theal., 1990; Sadoshima and Izumo, 1993a, b; LaMorte

et al., 1994; Brodde et al., 1995). Stimulation of uptake of [3H]NA by cardiac synaptosomal-mito-chondrial preparations from SHR suggest that thePLC produces inositol triphosphate and diacylglycerol

(DAG) which in turn elevate intracellular Ca2+ two endogenous peptides, via an overstimulation

Cardiac Nociceptin Binding Sites 2759

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