Journal qt NeurochemistryLippincott—Raven Publishers, Philadelphia© 1997 International Society for Neurochemistry

Nociceptin/Orphanin FQ Metabolism:Role of Aminopeptidase and Endopeptidase 24.15

Jose-Luis Montiel, Fabrice Cornille, Bernard P. Roques, and Florence Noble

Département de Pharmacochi,nie Moléculaire ci Structurale, U. 266 INSERM— URA D1500 CNRS,UFR des Sciences Pharmaceutiques et Biologiques, Faculté de Pharmacie, Paris, France

Abstract: The endogenous opioid receptor-like1 (ORL1)ligand, nociceptin/orphanin FQ (FGGFTGARKSAR-KLANQ), a heptadecapeptide structurally resembling dy-norphin A, has recently been identified. The wide distribu-tion of ORL1 mRNA and nociceptin/orphanin EQ precur-sor in the CNS, particularly in the limbic system regionsand in several areas known to be involved in pain percep-tion, suggests that nociceptin/orphanin FQ is potentiallyendowed with various central functions. In general, acti-vation and/or inactivation of regulatory peptides occurthrough the action of cell surface peptidases. The physio-logical mechanisms under which nociceptin/orphanin EQis metabolized should lead to a better understanding ofits physiological functions. Mouse brain cortical sliceswere incubated in medium containing the heptadecapep-tide in the presence or in the absence of peptidase inhibi-tors. The critical sites of enzymatic cleavage are Ph& —

G1y2, Ala7—Arg8, Ala~—Arg12,and Arg12—Lys13 bonds.

The major role played by metallopeptidases was con-firmed by the complete protection of metabolism in thepresence of EDTA. Aminopeptidase N and endopepti-dase 24.15 are the two main enzymes involved in noci-ceptin/orphanin FQ metabolism, whereas endopeptidase24.11 (involved in enkephalin [YGGEM(L)] catabolism)does notappear critically involved in nociceptin/orphaninFQ metabolism. The physiological relevance of amino-peptidase N and endopeptidase 24.15 in the heptadeca-peptide metabolism remains to be determined. KeyWords: Nociceptin/orphanin EQ—Metabolism—Amino-peptidase N— Endopeptidase 24.15—Brain slices— En-kephalin.J. Neurochem. 68, 354—361 (1997).

Opiates, as well as endogenous opioid peptides, ex-ert their physiological actions by interacting with threeclasses of opioid receptors, ~i, ~ and K. The recentcloning of these receptors has shown that opioid recep-tors belong to the G protein-coupled receptor super-family. The predicted amino acid sequences of thethree rodent opioid receptors are ‘-~65%identical withhighest similarity in the transmembrane spanning re-gions and intracellular loops (for review, see Reisine,1995). The opioid receptor-like

1 (ORL1) orphan re-ceptor, a new G protein-coupled receptor, was recently

identified on the basis of close homology with thepredicted amino acid sequence of opioid receptors(Bunzow et al., 1994; Chen et al., 1994; Fukuda et al.,1994; Mollereau et al., 1994; Wang et al., 1994; Wicket al., 1994; Lachowicz et al., 1995). The ORL~recep-tor is most closely related to opioid receptors not onlyon structural (sequence) but also on functionalgrounds. Thus, the ORL~receptors have been reportedto couple to K~channels and adenylyl cyclase (Meu-flier et al., 1995; Reinscheid et al., 1995; Zhang andYu, 1995; Vaughan and Christie, 1996).

It has been shown that ORLI does not bind any ofthe known opiate ligands with high affinity. It wastherefore proposed that the endogenous ligand for thisorphan receptor might be a novel opiate-related pep-tide. A heptadecapeptide (nociceptin/orphanin FQ;FGGFTGARKSARKLANQ) was identified and puri-fied from porcine and rat brain tissues as an endoge-nous ligand for ORL1 (Meunier et al., 1995;Reinscheid et al., 1995). All five of the major opioidpeptides characterized so far bear the amino-terminalsequence YGGF, whereas the newly isolated peptidebegins with the sequence FGGF and possesses furtherresemblance to the opioid peptide dynorphin A. Thewide distribution of ORLI mRNA and nociceptin/or-phanin FQ precursor in the CNS of the rodent (Bunzowet al., 1994; Chen et al., 1994; Fukuda et al., 1994;Mollereau et al., 1994; Houtani et al., 1996), particu-larly in the limbic system regions and in several areasknown to be involved in pain perception, including thehypothalamus, brainstem, and spinal cord dorsal horn,suggests that nociceptin/orphanin FQ is potentially en-dowed with various central functions.

In contrast to classic neurotransmitters, activation

Received June 17, 1996; revised manuscript received August 13.1996; accepted August 13, 1996.

Address correspondence and reprint requests to Dr. B. P. Roquesat Département de Pharmacochimie Moléculaire et Structurale, U.266 INSERM—URA D1500 CNRS, 4, Avenue de l’Observatoire,75270 Paris Cedex 06, France.

Abbreviations used: ACSF, artificial CSF; APN, aminopeptidaseN; NEP, neutral endopeptidase; ORLI, opioid receptor-like1 PC 18.[~HN-CH(CH2-CH,-S-CH1)-CH2-S-],.

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NOCICEPTIN/ORPHANIN FQ METABOLISM 355

and/or interruption of theresponses induced by regula-tory peptides are ensured by ectoenzymes that cleavethe peptide into active or inactive fragments. For in-stance, it has been shown that the antinociceptive andbehavioral responses to various physical or mentalstresses induced by interaction of the endogenous opi-oid peptides methionine (Met-) - and leucine (Leu-) -

enkephalins [YGGFL(M)] with the opioid receptorsare potentiated by protecting the peptides from enzy-matic inactivation (Roques and Fournié-Zaluski, 1986;Roques, 1988). Thus, the characterization of amino-peptidase N (APN) and neutral endopeptidase (NEP)and subsequently the design of selective inhibitorshave been, and will continue to be, of great value indetermining the role of enkephalins, and orally activedual inhibitors are now in preclinical trial as new anal-gesics (for review, see Roques et al., 1993). Similarly,identification of the physiological mechanisms underwhich nociceptin/orphanin FQ is metabolized shouldlead to a better understanding of its functions. For thispurpose, mouse brain slices were incubated in mediumcontaining nociceptin/orphanin FQ in the presence orabsence of various peptidase inhibitors. Fractions weresuccessively collected, and hydrolysis products of theheptadecapeptide obtained were identified and quanti-fied by HPLC. In addition, purified preparations ofpeptidases have been used to confirm the hydrolysissites.

EXPERIMENTAL PROCEDURES

ChemicalsAssembly of the protected peptide chains was carried out

using the stepwise solid-phase method of Merrifield (1963)on an Applied Biosystems model 431A automated peptidesynthesizer with Applied Biosystems small-scale Fmocchemistry. Peptides were cleaved from the resin, depro-tected, diethyl ether-precipitated, and washed in accordancewith Applied Biosystems guidelines. All the peptides werepurified by reverse-phase HPLC on a Vydac C~8column(250 x 10 mm) using acetonitrile (CHSCN) gradients in0.1% trifluoroacetic acid. [f FT3N-CH (CH2-CH2-S-CH3) -

CH2-S-]2 (PC 18) (Fournié-Zaluski et al., 1992) andretrothiorphan [HS-CH2-CH (CH2~)-NH-CO-CH2-COOH](Roques et al., 1983) were synthesized in the laboratory asdescribed previously. APN was purchased from BoehringerMannheim (Meylan, France). Purified endopeptidase 24.15was donated by Dr. F. Checler (UPR 411 CNRS, France).The selective inhibitor of endopeptidase 24.15 [Z-1t..01Phe~IJ-(PO2CH2)(L,D)Ala-Arg-Phel, the selective inhibitor of en-dopeptidase 24.16 Pro- (L) Phe’I’( PO2CH2) Gly-Pro], andthe mixed inhibitor of endopeptidase 24.15 and endopepti-dase 24.16 [~CH2-CH2kIt(PO2NH) -Gly-Pro-NieI werekindly provided by Dr. V. Dive (Commissariat a l’EnergieAtomique, Centre d’Etudes Nucléaires de Saclay, Saclay,France). Bestatin was from Roger Bellon (France). EDTAand puromycin were from Sigma Chemical (St. Quentin-Fallavier, France). Leu-enkephalin was from Bachem (Bu-bendorf, Switzerland).

Preparation and incubation of slices from mousebrain cortex

Male CD~mice (weighing 25—30 g) were obtained fromCharles River France (Saint-Aubin-Lès-Elbeuf, France).The animals were killed, and the brains were rapidly re-moved and washed in ice-cold artificial CSF (ACSF) thatwas pregassed with 95% 02 and 5% CO2 (0.6 mM MgSO4,1.25 mM NaH2PO4, 2 mM CaCL. 5 mM KCI, 124 mMNaC1, II mM glucose, and 26 mM NaHCO~.pH 7.4). Thebrains were cut into slices (400 pm) using a Mcllwain tissuechopper from +3.20 mm to —0.80 mm, which refers tomillimeters rostral to the bregma according to the atlas ofPaxinos and Watson (1986). The slices were rapidly placedin gassed ACSF, and the cortex region was punched. Thesepunches (2 mm in diameter) were washed three times eachfor 10 mm in gassed ACSF.

In polypropylene tubes (final volume, 100 pl), sixpunches (-~10 ~igof total protein) were preincubated for 20mm in ACSF at 37°Cin the absence or in the presence ofinhibitors (bestatin. 100 pM; PC 18, 10 pM; endopeptidase24.15 inhibitor, 10 pM: mixed endopeptidase 24.15/endo-peptidase 24.16 inhibitor, 10 pM; retrothiorphan, 10 pM;EDTA, 100 pM). Nociceptin/orphanin FQ was finally added(final concentration, 100 pM) and incubated for 60 mm at37°C.Enzymatic reactions were terminated by addition of50 ~.elof 0.2 M HC1. The mixture was immediately cooledto 4°Cand centrifuged (48 g, 15 mm at 4°C).Controls wereperformed under the same conditions by heating the tissueat 80°Cfor 10 mm before incubation.

In the time course study, the same procedure was fol-lowed. Nociceptin/orphanin FQ or Leu-enkephalin wasadded (final concentration, 50 pM) and incubated for 10,20, 40, or 60 mm at 37°C.

Assay for enzymatic activitiesNociceptin/orphanin FQ (100 pM) was preincubated for

20 mm at 37°Cin the absence or in the presence of specificinhibitors (PC 18, 10pM; endopeptidase 24.15 inhibitor, 10pM; mixed endopeptidase 24.15 /endopeptidase 24.16 inhib-itor, 10 1.eM) and incubated (60 mm at 37°C)with 45 or 1.3pg of APN or endopeptidase 24.15, respectively, in a finalvolume of 100 j.el of 50 mM Tris-HCI, pH 7.4. Reactionswere stopped by addition of 50 pl of 0.2 M HCI. Blankswere obtained by heating the enzymatic preparation (80°C,10 mm) before the beginning of incubation, and controlscorrespond to samples without inhibitors. For determinationof kinetic parameters of the cleavage of nociceptin by APNand endopeptidase 24.15, we used increasing concentrationsof nociceptin in the range 40—800 pM. Values were obtainedusing ENZFITTER Biosoftware.

HPLC analysisSamples (100 pl) were automatically injected (model

SIL-IOA; Shimadzu, Kyoto, Japan) on a Vydac-C5 column(250 X 4.6 mm; particle size, 5 pm). The samples wereanalyzed using an isocratic gradient of 0% acetonitrile and0.35% trifluoroacetic acid (pH 2.0) versus 0.5% trifluoro-acetic acid (pH 2.0) for 5 mm and then a linear gradientrising from 0 to 30% acetonitrile in 50 mm at a flow rateof 0.8 mI/mm. The cleavage products formed during theincubations were detected at 214 nm. The major metaboliteswere identified and quantified by comparison with syntheticmarkers or by sequencing (Applied Biosystems model 473Asequencer) of manually collected fragments.

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356 J.-L. MONTIEL ET AL.

drolyzed the heptadecapeptide mainly at the Phe’ —

Gly2, A1a7—Arg8, Ala”—Arg12, and Arg’2—Lys13bonds. The appearance of the amino acid Phe stronglysuggested involvement of aminopeptidase activities inthe metabolism of nociceptin/orphanin FQ. This wasconfirmed by addition of bestatin (final concentration,100 ,uM) to the incubation medium, which stronglydecreased but did not abolish the formation of Phe(Fig. 1D) and reduced the percentage of nociceptin/orphanin FQ degradation (Fig. IC; 22% vs. 38% inthe absence of bestatin).

On the other hand, after 40—50 mm of incubation,a plateau was obtained with the three main peptidemetabolites: FGGFTGA, RKLANK, and KLANQ(Fig. 1B). This could be explained by a further degra-dation by arninopeptidases of these primary formedmetabolites. Indeed, in the presence of bestatin in theincubation medium, levels of FGGFTGA and RKL-ANK time-dependently increased (Fig. 1 D).

FIG. 1. Time course of nociceptin/orphanin FQ metabolism inthe cortical slice assay. Heptadecapeptide (100 pM) was incu-bated, and metabolites were analyzed as described in Experi-mental Procedures. A: Nociceptin degradation (control). B: Ap-pearance of main metabolites. F1, retention time (r.t.) = 19.2mm; G2—017, r.t. = 35 mm; F1—A7, r.t. = 42 mm; F1—A~,r.t.= 41 mm; R12—Q17, r.t. = 23 mm; K13—Q17, r.t. = 21 mm; noci-ceptin, r.t. = 40 mm. C: Nociceptin degradation in the presenceof bestatin (100 pM). D: Appearance of main metabolites in thepresence of bestatin. Data in A and C are mean ± SEM (bars)percentages of degradation (n = 5); data in B and D are mean±SEM (bars) values of relative absorbance with respect to thecomplete peptide (n = 5). O.D., optical density.

Data analysisData are mean ±SEM values from three (assays for enzy-

matic activities) or five independent experiments.

RESULTS

Metabolism of nociceptin/orphanin FQ in thecortical slice assay

As shown in Fig. IA, the metabolism of nociceptin/orphanin FQ was time dependent, and different peptidefragments progressively appeared (Fig. 1B; only themain fragments have been illustrated). The nociceptin/orphanin FQ metabolites were identified on the basisof their coelution with standard peptides and/or sub-jected to sequencing. Thus, peptidase activities hy-

Effects of selective endopeptidase inhibitors onnociceptin/orphanin FQ metabolism in thecortical slice assay

As shown in Table 1, addition of EDTA in the incu-bation medium completely protected hydrolysis ofnociceptin/orphanin FQ. This result demonstrated thecrucial role played by metallopeptidases in the hepta-decapeptide metabolism, and these enzymes were thencharacterized by using selective endopeptidase inhibi-tors.

Involvement of aminopeptidases in nociceptin/or-phanin FQ metabolism was clearly demonstrated withthe aminopeptidase inhibitor bestatin. Indeed, the pres-ence of this inhibitor in the incubation medium reducedthe heptadecapeptide metabolism (35% protection; Ta-ble 1) and strongly decreased the appearance of themetabolite Phe (76% reduction; Table 2). Similarly,the presence of a selective APN inhibitor, PC 18, inthe incubation medium prevented enzymatic degrada-tion of nociceptin/orphanin FQ (29% protection; Ta-ble 1), essentially by protection of the Phe’ —G1y2bond, illustrated by the important reduction (72%; Ta-ble 2) of Phe appearance. Concomitantly the amountsof some of the other metabolites were increased by 5—55% (Table 2).

Three of the main metabolites generated by nocicep-tin/orphanin FQ metabolism are FGGFTGA, RKL-ANQ, and KLANQ, which could result from endopep-tidase activities. These enzymes were characterized bymeasuring the effects of selective endopeptidase inhib-itors on the heptadecapeptide metabolism. Retrothior-phan, a highly selective NEP inhibitor, produced onlya small change in nociceptin/orphanin FQ metabolism(9% protection; Table 1), whereas the selective endo-peptidase 24.15 and the mixed endopeptidase 24.15/endopeptidase 24.16 inhibitors efficiently reduced theheptadecapeptide metabolism (63 and 56%, respec-tively). With regard to the production of the peptidemetabolites, the selective endopeptidase 24.15 and the

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NOCICEPTIN/ORPHANIN FQ METABOLISM 357

TABLE 1. Effects of inhibitors in nociceptin/orphaninFQ ,netabolism

Inhibition(%)

Control 0Retrothiorphan 10 ±3.1Bestatin 35 ±2.1PCI8 29 ±4.2Endopeptidase 24.15 inhibitor 63 ±4.5Mixed endopeptidase 24.15/24.16 inhibitor 56 ± 1.8Bestatin + endopeptidase 24.15 inhibitor 70 ±4.3Bestatin + mixed endopeptidase 24.15/24.16 inhibitor 77 ±2.7PC 18 + mixed endopeptidase 24.15/25.16 inhibitor 73 ±4.2EDTA 95 ±2.6

Conditions were as follows: control, without inhibitor; retrothior-phan (10 pM); bestatin (100 pM); PC 118 (10 pM); endopeptidase24.15 inhibitor(l0 pM); mixed endopeptidase 24.15/24.16 inhibitor(10 pM); bestatin (100 pM) + endopeptidase 24.15 inhibitor (10pM); bestatin (100 pM) + mixed endopeptidase 24.15/24.16 inhibi-tor (10 pM); PC 18 (10 pM) + mixed endopeptidase 24.15/24.16inhibitor (10 pM); and EDTA (100 pM). Data are mean ±SEMvalues (n = 5).

mixed endopeptidase 24.15 /endopeptidase 24.16 in-hibitors in the incubation medium reduced the produc-tion of FFGGFTGA (71 and 100%, respectively),RKLANQ (68 and 100%, respectively), and KLANQ(93 and 100%, respectively). A reduction of Phe ap-pearance was also observed in the presence of theseendopeptidase inhibitors (56 and 41% with endopepti-dase 24.15 and mixed inhibitors, respectively; Table2). A highly selective endopeptidase 24.16 inhibitorwas also tested and was unable to inhibit the heptadeca-peptide hydrolysis (data not shown).

In the presence of aminopeptidase (bestatin or PC18) and endopeptidase (endopeptidase 24.15 or mixedendopeptidase 24.15 /endopeptidase 24.16) inhibitors,the reductions of nociceptin/orphanin FQ metabolismand of the generated peptide metabolites were higherthan that observed with aminopeptidase or endopepti-dase inhibitors used alone (Tables 1 and 2).

Nociceptin/orphanin FQ degradation by purifiedenzyme

Using purified APN and endopeptidase 24.15, a pat-tern of nociceptin/orphanin FQ metabolites similar tothat observed in the presence of brain slices was ob-tained. As shown in Fig. 2A, the heptadecapeptide wasrapidly and time-dependently metabolized by APN,with appearance of the amino acid Phe and the peptidefragment Gly2—Gln 7~The presence of PC 18 (10 ~.tM)or bestatin (100 ~tM) in the incubation medium abol-ished completely the generation of these metabolites(Fig. 2B).

Similarly, in the presence of purified endopeptidase24.15, nociceptin/orphanin FQ was time-dependentlymetabolized, with appearance of the peptide fragmentscorresponding to the cleavage of A1a7—Arg8, Alatt—Argt2, and Arg’2—Lyst3 bonds (Fig. 3A). Generation

of these fragments was completely abolished by theselective endopeptidase 24.15 inhibitor (Fig. 3B). Asshown in Table 3, the kinetic parameters of degradationof nociceptin by APN were K~,= 1,260 ±300 pM,Keat = 1.6 ±0.2 mint, and Kcat/Km = 1.27, whereasthose corresponding to the cleavage of nociceptin/or-phanin FQ by endopeptidase 24.15 were Km = 97 ±8.0pM, K~

0= 1,575 ±100 min~, and Keat/Km = 1.6)< l0~.

Comparative study of nociceptin/orphanin FQand Leu-enkephalin degradation in the corticalslice assay

As shown in Fig. 4, the specific rates of degradationof nociceptin/orphanin FQ and Leu-enkephalin were6.2 and 18 nmol/min/mg of protein, respectively.

Also, the results obtained show that the heptadeca-peptide had a very low inhibitory potency toward pureNEP and APN (both involved in enkephalin metabo-lism), exhibiting K~values of 1,700 and 9 pM, respec-tively.

DISCUSSION

In the present study, the respective roles of amino-peptidase and endopeptidase 24.15 in nociceptin/or-

FIG. 2. In vitro nociceptmn/orphanmn EQ (100 pM) hydrolysis bypurified APN, as described in Experimental Procedures. A: Timecourse of metabolite appearance. B: Inhibition of metabolite ap-pearance. Blank (B), heat-inactivated APN; control (C), withoutinhibitor; bestatin (Best), 100 pM; PC 18, 10 pM. Insets: Timecourse degradation (A) or selective inhibition of degradation (B)of the heptadecapeptide. Data are mean ± SEM (bars) values(n = 3). O.D., optical density.

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358 J. -L. MONTIEL ET AL.

TABLE 2. Effects of specific inhibitors on the generation of metabolitesduring nociceptin/orphanin EQ hydrolysis

Metabolite

F FGGFTGA RKLANQ KLANQ

ControlRetrothiorphanBestatinPC 18Inh 24.15Inh mxBestatin + Inh 24.15Bestatin + lnh mxPC18+InhmxEDTA

10095.0 ±1.024.1 ±1.228.0 ±8.045.6 ±3.458.4 ±4.55.8 ±4.24.7 ±4.0

11.6±3.10

10088.4 ±5.0

137.0 ±14.0105.6 ±7.028.6 ±7.2

018.0 ±3.6

000

10084.2 ±

155.8 ±152.0 ±32.1 ±

020.0 ±

000

3.818.1)18.06.0

8.0

10089.2 ±3.093.3 ±10.090.5 ±8.0

6.8 ±3.000000

Conditions were as follows: retrothiorphan (100 pM); bestatin (100 pM); PC 18 (10 pM); Inh 24.15,endopeptidase 24.15 inhibitor (10 pM); Inh mx, mixed endopeptidase 24.15/endopeptidase 24.16 inhibitor(10 pM). Data are mean ±SEM percentages for metabolite appearance compared with the control(n = 5).

phanin FQ metabolism have been demonstrated usingbrain cortical slices. This preparation is physiologicallymore relevant than homogenates in which the anatomi-cal organization of the tissue is destroyed and that arelikely to contain intracellular peptidases as contami-

FIG. 3. In vitro nociceptmn/orphanin EQ (100 pM) hydrolysis byendopeptidase 24.15, as described in Experimental Procedures.A: Time course study. B: Inhibition study. Blank (B), heat-inacti-vated endopeptidase 24.15; control (C), without inhibitor; endo-peptidase 24.15 inhibitor (1-24.15), 10 pM; mixed endopepti-dase 24.15/24.16 inhibitor (l-mx), 10 pM. Insets: Time coursedegradation (A) or selective inhibition of degradation (B) of theheptadecapeptide. Data are mean ±SEM (bars) values (n = 3).O.D., optical density.

nants. Moreover, in situ hybridization studies haveshown that both ORL1 receptor (Fukuda et al., 1994;Mollereau et al., 1994) and nociceptin/orphanin FQprecursor (Houtani et al., 1996) are densely present inthe cortex (parietal and frontal), which is also rich indifferent peptidases (Waksman et al., 1986; Lucius etal., 1995; Waters et al., 1995).

Incubation of nociceptin/orphanin FQ with braincortical slices led to the generation of numerous frag-ments that were identified on the basis of their sequenc-ing and/or coelution with standard peptides. Four criti-cal sites of enzymatic cleavage have been character-ized, Phe

1—G1y2, Ala7—Arg5, Ala”—Arg’2, andArgt2—Lys’3, suggesting a complex metabolism,which has also been observed with other neuropep-tides, such as dynorphin A (Young et al., 1987; Safaviand Hersh, 1995) or neurotensin (Checler et al., 1988).

The main role played by metallopeptidases in thenociceptin/orphanin FQ metabolism was demonstratedby the almost complete protection of the heptadecapep-tide in the presence of EDTA. APN is a zinc-containingproteolytic ectoenzyme (Kenny et al., 1987) recently

FIG. 4. Comparative degradation of Leu-enkephalin (50 pM)and nociceptin/orphanin EQ (50 pM) in the cortical slice assay,as described in Experimental Procedures. Data are mean ±SEM(bars) values (n = 3).

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NOCICEPTIN/ORPHANIN FQ METABOLISM 359

TABLE 3. Cleavage rates of nociceptin/orphanin FQby APN and endopeptidase 24.15

Nociceptin

APN(min’) 1.6 ±0.2

K,,, (pM) 1,260 ±300K=~/Km 1.27

Endopeptidase 24.15K

0~,(min’) 1,575 ±100K~,(pM) 97 ±8.0

1.6 x 10°

Data are mean ±SEM values (n = 3).

cloned (Olsen et al., 1988; Watt and Yip, 1989). Inthe brain, APN has been shown to be involved in thedegradation of the opioid peptides, enkephalins(Waksman et al., 1985; Turner et al., 1987), dynorphin(Safavi and Hersh, 1995), and cholecystokinin octa-peptide (Deschodt-Lanckman and Bui, 1981; Durieuxet al., 1986). The membrane-bound APN has a broadspecificity (McDonald and Barrett, 1986), althoughhydrophobic residues, preferentially aromatic, in theNH2-terminal position are more rapidly removed. Inaddition, the S~’and S2’ subsites of APN also seemto prefer hydrophobic residues (Hernandez et al., 1988;Xie et al., 1989). This accounts for the somewhat slowhydrolysis of the Phe

1—G1y2 bond of nociceptin/or-phanin FQ. The involvement of APN was unambigu-ously confirmed by using the selective APN inhibitorPC 18 or bestatin, which induced a strong reductionof the Phe metabolite generation. According to thesefindings, purified APN has been shown to hydrolyzein vitro the heptadecapeptide at the Ph&—G1y2 bond.Minor participation of other membrane-bound amino-peptidase activities belonging to the group of zinc-metallopeptidases, present in large quantities in brain(Kenny et al., 1987), could also have occurred in thenociceptin/orphanin FQ metabolism. Indeed, the enzy-matic cleavage of the Phe t_G1y2 bond in the corticalslice preparation was not fully prevented by PC 18or bestatin. Moreover, bestatin and puromycin, whichinteract with several aminopeptidases (Umezawa et al.,1976), appeared more efficient than PC 18 (Fournié-Zaluski et al., 1992).

During hydrolysis of nociceptin/orphanin FQ, rapidcleavages occurred at the Ala7 —Argt, Ala — Arg 2

and Arg ‘2—Lys 3 bonds, which were not abolished inthe presence of aminopeptidase inhibitors, suggestingthe involvement of endopeptidase activities. Moreover,the formation of some of these metabolites was en-hanced after blockade of aminopeptidases. This con-comitant increase of endopeptidase activity in the pres-ence of aminopeptidase inhibitors has been previouslyreported with enkephalins (Waksman et al., 1985).

The sequence of endopeptidase 24.15 is characteris-tic of a metallopeptidase in that it contains the typical

consensus sequence HExxH (Pierotti et al., 1990; Ka-wabata et al., 1993; Habgood et al., 1994), which hasbeenfound in numerous other zinc endopeptidases (forreview, see Roques et al., 1993). Several studies sug-gest that endopeptidase 24.15 is a cytosolic enzymewith a weakly hydrophobic domain, which could ex-plain the presence of a minor membrane-associatedcomponent (10—20% of total activity) (Orlowski etal., 1983; Acker et al., 1987). This could account forthe involvement of this peptidase in the invivo metabo-lism of several neuropeptides (Kest et al., 1991; Ment-lein and Dahms, 1994; Vincent et al., 1995). Thus,Molineaux et al. (1990) have observed a reduction inneurotensin degradation after intraventricular injectionof an endopeptidase 24.15 selective blocker. The re-sults obtained in the present study suggest the involve-ment of this endopeptidase in the nociceptin/orphaninFQ metabolism. Indeed, in the presence of the selectiveendopeptidase 24.15 inhibitor in the incubation me-dium, the hydrolysis of the heptadecapeptide and thegeneration of the different peptide metabolites werestrongly decreased. Complete abolition of the Ala7—Argt, Alalt_Argt2, and Arg12—Lys’3 cleavages wasobserved in the presence of the mixed endopeptidase24.15/endopeptidase 24.16 inhibitor. In contrast, a se-lective endopeptidase 24.16 inhibitor did not modifythe nociceptin/orphanin FQ metabolism (data notshown), confirming the preferential involvement ofthe endopeptidase 24.15 activity in hydrolysis of theheptadecapeptide. This result is in good agreementwith the reported good affinity of endopeptidase 24.15for substrates with Arg or Lys in the P

2’ position (Jira-cek et al., 1995). Moreover, the expected metaboliteswere obtained by incubation of nociceptin in the pres-ence of pure endopeptidase 24.15, and the kinetic con-stants derived from these experiments showed that noc-iceptin is a very good substrate for this enzyme (Kcat/Km = 16,000). The difference observed between theselective endopeptidase 24.15 and the mixed endopep-tidase 24.15 /endopeptidase 24.16 inhibitors could bedue to the slow binding reaction of the former inhibitorto the peptidase (Jiracek et al., 1995; Vincent et al.,1995), requiring several hours of preincubation, whichis impossible with fresh brain slices. Taken togetherthese findings suggest that nociceptin/orphanin FQ isvery likely metabolized by endopeptidase 24.15 of thecell surface, although one cannot exclude an intracellu-lar cleavage of the peptide after internalization.

Nociceptin/orphanin FQ was not completely pro-

FIG. 5. Principal nociceptmn/orphanin FQ cleavage sites. APN,aminopeptidase activity; EP, endopeptidase activity.

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360 J.-L. MONTIEL ET AL.

tected (70—77% protection) from its metabolism bythe association of APN and endopeptidase 24.15 (orthe mixed endopeptidase 24.15/endopeptidase 24.16)inhibitors. This absence of complete protection sug-gests the participation of other peptidases, like car-boxypeptidases (Kenny et al., 1987) or the recentlydescribed Arg-specific dibasic cleaving enzyme (Csu-hai et al., 1995), which may play a minor role in theheptadecapeptide metabolism. Moreover, enkephalinand nociceptin/orphanin FQ have a high homology ofamino acid sequence, YGGF- and FGGF-, respec-tively. It is well established that NEP 24.11 cleavesthe G1y3—Phe4 bond of enkephalins (Malfroy et al..1978), suggesting a possible role for NEP in the hydro-lysis of nociceptin/orphanin FQ. Nevertheless, the re-sults obtained in the presence of retrothiorphan, ahighly selective NEP inhibitor (Roques et al., 1983),show that this enzyme is not involved in the heptadeca-peptide metabolism, whereas in the same conditionsa rapid NEP cleavage activity on Leu-enkephalin isobserved. Accordingly, pure NEP (200 ng) was unableto cleave significantly nociceptin (100 p~M)after a 90-mm incubation at 37°C(data not shown). NEP has abroad selectivity and can cleave various short linearor cyclic peptides, as well as polypeptides of intermedi-ate or long lengths (for review, see Roques et al.,1993). However, NEP shows little activity toward opi-oid peptides such as /3-endorphin or dynorphin despitethe fact that they contain the N-terminal sequence ofMet- or Leu-enkephalin (Turner et al., 1987; Roqueset al., 1993). The efficiency of NEP in cleaving theGly3—Phe4 bond is sensitive to the length of the aminoacid sequence added at the COOH terminus (Turneret al., 1987). Thus, the efficient metabolism of Leu-enkephalin by NEP could be due to a favorable ionicinteraction between the free COOH-terminal carboxylgroup and a well-positioned, positively charged aminoacid in the active site of NEP (Fournié-Zaluski et al.,1979; Beaumont et al., 1992). The fact that dynorphinand nociceptin/orphanin FQ are poor substrates ofNEP could be due to an unfavorable ionic interactionof the basic amino acids present in the vicinity ofthe F(Y)GGF sequences and/or to a conformationallyrelated hindered access of the enzyme-sensitive bondsto the active site.

In conclusion, the results obtained in this study byusing brain cortical slices show that nociceptin/or-phanin FQ is preferentially metabolized by the con-comitant action of APN and endopeptidase 24.15 (Fig.5). Moreover, its metabolism is different from that ofenkephalins. The extended 17-amino-acid nociceptin/orphanin FQ is hydrolyzed relatively slowly comparedwith the pentapeptide enkephalin. The conformationof the larger forms of peptides appears to render themmore resistant to enzymatic degradation (Churchill etal., 1987; Barnes et al., 1995; Safavi and Hersh, 1995).This observation suggests that nociceptin/uipLnin FQmay have long-lasting modulatory effects, which re-main to be determined.

Acknowledgment: We wish to acknowledge gratefullyDrs. V. Dive and F. Checler for providing us with purifiedendopeptidase 24.15, selective inhibitors of endopeptidase24.15 and endopeptidase 24.16, and mixed endopeptidase24.15/endopeptidase 24.16 inhibitor. We wish to acknowl-edge E. Ruffet for his technical assistance and P. Cone, C.Lenoir, and Prof. M. C. Fournié-Zaluski for the synthesis ofnociceptin/orphanin FQ, retrothiorphan, and PC 18.

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