Proc. Natl. Acad. Sci. USA Vol. 83, pp. 1523-1527, March 1986 Neurobiology Autoradiographic comparison of the distribution of the neutral endopeptidase "enkephalinase" and of ja and 6 opioid receptors in rat brain (enkephailnase inhibitor/enkephalin) GILLES WAKSMAN*, EDITH HAMELt, MARIE-CLAUDE FOURNI&-ZALUSKI*, AND BERNARD P. ROQUES*t *DJpartement de Chimie Organique, U 266 Institut National de la Santd et de la Recherche Mddicale, UA 498 Centre National de la Recherche Scientifique, UER des Sciences Pharmaceutiques et Biologiques, 4, avenue de l'Observatoire, 75006 Paris, France; and tCerebral Circulation and Metabolism Group, Laboratoire d'Etudes et de Recherches Synthdlabo, 92220 Bagneux, France Communicated by Jean-Pierre Changeux, October 7, 1985 ABSTRACT The neutral endopeptidase EC 3.4.24.11, also inhibitors. Although an operational re-uptake mechanism designated enkephalinase, has been visualized by in vitro cannot be definitively excluded, it is generally accepted that autoradiography using the tritiated inhibitor [3H]-N-[(2RS)-3- extracellular hydrolysis of enkephalins by the endopeptidase hydroxyaminocarbonyl-2-benzyl-1-oxopropyl]glycine, EC 3.4.24.11 represents a major mechanism for terminating ([3H]HACBO-Gly). Specific binding of [3H]HACBO-Gly (Kd = the enkephalinergic signal (8-10). However, the ability of 0.4 ± 0.05 nM) corresponding to 85% of the total binding to purified enkephalinase to degrade a variety of neuropeptides brain slices was inhibited by 1 IAM thiorphan, a selective in vitro, including substance P, cholecystokinin-(26-33) [sul- inhibitor of enkephalinase, but remained unchanged in the fated cholecystokinin (CCK)-octapeptide], and neurotensin presence of captopril, a selective inhibitor of angiotensin- (reviewed in ref. 11), raises questions concerning the selec- converting enzyme. Very high levels of [3H]HACBO-Gly bind- ing were found in the choroid plexus and the substantia nigra. tive involvement of the neutral endopeptidase in enkephalin High levels were present in the caudate putamen, globus metabolism in vivo. A plausible hypothesis is that the pallidus, nucleus accumbens, olfactory tubercle, and in the specificity of enkephalinase action in vivo is determined by substantia gelatinosa of the spinal cord. Moderate densities the selective association of the enzyme with enkephalinergic were found in parts of the amygdala, the periaqueductal gray neuronal systems. matter, the interpeduncular nucleus, and the molecular layer To test this hypothesis and to obtain a better understanding of the cerebellum. The distribution of enkephalinase was of enkephalinergic function, we have compared the regional compared to that of ,u and 6 opioid receptors, selectively distributions of opioid receptors and enkephalinase in rat labeled with [3H]Tyr-D-Ala-Gly-MePhe-glycinol and [3H]Tyr- brain by autoradiography. Selective labeling of A and 8 D-Thr-Gly-Phe-Leu-Thr, respectively. In the caudate puta- receptor types (12-14) was achieved by use ofthe appropriate men, [3H]HACBO-Gly binding overlapped the clustered IA sites radiolabeled probes, [3H]DAMGE and [3H]DTLET, respec- but appeared more closely related to the diffusely distributed tively (15, 16). The enkephalinase was selectively visualized 6 sites. High levels of enkephalinase and ,u opioid binding sites with the recently described (17) potent and tritiated inhibitor were present at the level of the periaqueductal gray matter and [3H]-N-[(2RS)-3-hydroxyaminocarbonyl-2-benzyl-1-oxopro- in the substantia gelatinosa of the spinal cord, regions where pyliglycine ([3H]HACBO-Gly). The autoradiographic analy- only sparse 6 opioid receptors could be detected. The associ- sis of the neutral endopeptidase's distribution in many brain ation of enkephalinase with 6 and ,u opioid receptors in these regions is consistent with a selective role of enkephalinase in areas is consistent with the observed role of the enzyme in the termination of the enkephalinergic signal. regulating the effects of opioid peptides in striatal dopamine release and analgesia, respectively. Except for the choroid plexus and the cerebellum, the close similarity observed in MATERIALS AND METHODS numerous rat brain areas between the distribution of Chemicals. enkephalinase and that of it and/or 6 opioid binding sites could [3,5-3H]Tyr-D-Ala-Gly-MePhe-glycinol account for most of the pharmacological effects elicited by ([3H]DAMGE, 60 Ci/mmol; 1 Ci = 37 GBq) was purchased enkephalinase inhibitors. from Amersham. [3 ,5-3H]Tyr-D-Thr-Gly-Phe-Leu-Thr ([3H]DTLET, 45 Ci/mmol) (16) and [3H]HACBO-Gly were The endogenous opioid peptide enkephalins are chiefly and synthesized in our laboratory and tritiated by reduction, with rapidly metabolized in the brain (1) by both an aminopepti- 3H2, of the benzylidene precursor (17) at the Commissariat a dase activity (2, 3) and enkephalinase (4), a membrane-bound l'Energie Atomique (Saclay, France). Thiorphan was from enzyme identical to the neutral metalloendopeptidase EC our laboratory (6). Levorphanol was a gift from Hoffman-La 3.4.24.11 (5). The physiological relevance of brain enkepha- Roche Laboratories (Basel, Switzerland). linase is supported by the naxolone-reversible decrease in the Binding Procedures. Male Sprague-Dawley rats (150-200 g) were decapitated, and their brains, including the upper responsiveness to nociceptive stimuli - elicited by intracerebral administration of highly potent enkephalinase segments of the cervical spinal cord, were rapidly removed inhibitors such as thiorphan {N-[(2RS)-2-mercaptomethyl-1- and frozen in isopentane at -45°C. Coronal sections (20 ,tm oxo-3-phenylpropyl]glycine} (6) or kelatorphan {N-[(2R)-3- thick) were cut on a cryostat at -17°C, thaw-mounted onto hydroxyaminocarbonyl-2-benzyl-1-oxopropyl]-L-alanine} gelatin-coated slides, and stored at -80'C until used. All (7), a compound belonging to a new series of bidentate Abbreviations: CCK, cholecystokinin; DAMGE, [D-Ala2,N-methyl- Phe4,glycinol5]enkephalin; DTLET, Tyr-D-Thr-Gly-Phe-Leu-Thr The publication costs of this article were defrayed in part by page charge ([D-Thr2,Leu5]enkephalin-Thr6); HACBO-Gly, N-[(2RS)-3-hydroxy- payment. This article must therefore be hereby marked "advertisement" aminocarbonyl-2-benzyl-1-oxopropyl]glycine. in accordance with 18 U.S.C. §1734 solely to indicate this fact. tTo whom reprint requests should be addressed. 1523 Downloaded by guest on September 27, 2021 1524 Neurobiology: Waksman et al. Proc. Natl. Acad. Sci. USA 83 (1986) sections were warmed to room temperature just prior to As shown in Fig. LA, binding of [3H]HACBO-Gly to rat incubation for binding as described below. brain tissue sections was highly specific; the nonspecific For selective labeling of g opioid binding sites, the sections binding determined in the presence of 1 ,uM thiorphan, a were incubated with 4 nM [3H]DAMGE for 30 min at room highly potent inhibitor of enkephalinase, was 12-15% oftotal temperature in 50 mM Tris HCl (pH 7.4) as described (15). binding (Fig. 1B). In contrast, the potent and selective The 8 opioid sites were selectively labeled with 3 nM inhibitor of angiotensin-converting enzyme, captopril (19), [3H]DTLET (18). At the end of the incubation, the sections was unable to modify the specific binding of [3H]HACBO- were washed twice for 10 min in fresh buffer at 0-40C, Gly (Fig. 1C). followed by a rapid rinse in ice-cold distilled water, and then Moreover, HACBO-Gly was shown to be a selective dried under a stream of cold air. For nonspecific binding, the inhibitor of enkephalinase with a Ki of approximately 0.4 nM sections were incubated as above with either [3H]DAMGE or but greater than 30 nM for the dipeptidyl aminopeptidase and [3H]DTLET but in the presence of 10 1LM levorphanol. In 1 uM for other brain metallopeptidases, such as aminopep- both cases, nonspecific binding accounted for <10%1 of total tidase M and angiotensin-converting enzyme (17). binding in all brain regions studied. Autoradiographic Localization of [H]HACBO-Gly Binding The enkephalinase was labeled with 3 nM [3H]HACBO- Sites in Rat Brain. [3H]HACBO-Gly binding sites were found Gly for 60 min at room temperature in 50 mM Tris HCl (pH to be discretely distributed in rat brain (Fig. 1A and Table 1), 7.4). At the end of the incubation, the sections were washed with the highest concentrations in the choroid plexus, in fresh ice-cold buffer for 1 min followed by two 5-min substantia nigra, caudate putamen, globus pallidus, olfactory rinses, rapidly washed in ice-cold distilled water, and imme- tubercle, nucleus accumbens, and the substantia gelatinosa diately dried. Nonspecific binding was determined in sections of the spinal cord. A second group of structures containing incubated as above but in the presence of 1 AM thiorphan and moderate levels of [3H]HACBO-Gly binding included parts accounted for <15% of the total binding. of the amygdala, the interpeduncular nucleus, the molecular Autoradiography. After being carefully dried, all labeled layer of the cerebellum, the periaqueductal gray matter, and sections were mounted with eight calibrated tritium stan- the hippocampus. dards and closely apposed to sheets of tritium-sensitive Interestingly, an accumulation of silver grains, intermin- Ultrofilm (LKB, Fisher) inside x-ray cassettes. The films gled with streaks of nonlabeled zones corresponding to the were exposed for 8-12 weeks at 4°C and then developed in presence of white matter tracts, was observed in the region Kodak LX-24 developer for 1.5 min at 18°C, fixed in Kodak connecting the substantia nigra and the caudate putamen L-4 fixative for 5 min, washed for 30 min under running tap (Fig. LA); this labeling was particularly dense in the area of water, and dried. the entopeduncular nucleus. Such a pattern of labeling has Quantitation of Binding Sites by Densitometry. All films been reported at the level of the nigrostriatal pathway for were analyzed by spot densitometry using a Quantimet 720 angiotensin-converting enzyme visualized with [3Hicaptopril (Cambridge Instrument Ltd., Cambridge, England).