Jan 9, 1995 - Meng, F., Xie, G.-X., Thompson, R. C., Mansour, A., Goldstein,. A., Watson, S. J. & Akil, H. (1993) Proc. Natl. Acad. Sci. USA 90,. 9954-9958. 14.
Proc. Natl. Acad. Sci. USA Vol. 92, pp. 7006-7010, July 1995
Pharmacology
K-Opioid receptor in humans: cDNA and genomic cloning, chromosomal assignment, functional expression, pharmacology, and expression pattern in the central nervous system FRJEDJERIC SIMONIN*, CLAIRE GAVE'RIAUX_RUFF*, KATIA BEFORT*, HANS MATTHES*, BEATRICE LANNESt, GABRIEL MICHELETTIt, MARIE-GENEVIEVE MArTEIt, GIS'ELE CHARRON§, BERTRAND BLOCH§, AND BRIGITTE KIEFFER*fl *Ecole Superieure de Biotechnologie, Parc d'innovation, Boulevard Sebastien Brandt, F-67400 Illkirch-Graffenstaden, France; tlnstitut de Physiologie, 11 rue Humann, F-67085 Strasbourg Cedex, France; tInstitut National de la Sante et de la Recherche Medicale U242, H6pital d'Enfants de la Timone, F-13385 Marseille Cedex 5, France; and §Equipe Postulante Centre National de la Recherche Scientifique 74, Laboratoire d'Histologie-Embryologie, Universite de Bordeaux II, 146 rue Leo Saignat, 33076 Bordeaux, France
Communicated by Avram Goldstein, Stanford, CA, March 17, 1995 (received for review January 9, 1995)
ABSTRACT Using the mouse 8-opioid receptor cDNA as a probe, we have isolated genomic clones encoding the human ,L- and ic-opioid receptor genes. Their organization appears similar to that of the human 8 receptor gene, with exon-intron boundaries located after putative transmembrane domains 1 and 4. The c gene was mapped at position qll-12 in human chromosome 8. A full-length cDNA encoding the human K-opioid receptor has been isolated. The cloned receptor expressed in COS cells presents a typical icl pharmacological profile and is negatively coupled to adenylate cyclase. The expression of K-opioid receptor mRNA in human brain, as estimated by reverse transcription-polymerase chain reaction, is consistent with the involvement of X-opioid receptors in pain perception, neuroendocrine physiology, affective behavior, and cognition. In situ hybridization studies performed on human fetal spinal cord demonstrate the presence of the transcript specifically in lamina II of the dorsal horn. Some divergences in structural, pharmacological, and anatomical properties are noted between the cloned human and rodent receptors.
(hKOR and hMOR, respectively) and present the molecular characterization and expression pattern of hKOR.11 tors
MATERIALS AND METHODS Genomic Cloning. Standard protocols were used (6) unless otherwise stated. A human genomic library was screened under stringent conditions with a randomly 32P-labeled 976-bp Pst I-Not I fragment containing most of the coding region of the mouse 8-opioid receptor cDNA (4), and several clones were isolated. Sac I fragments 6 and 1.7 kb in size (K-opioid receptor), as well as a 7.5-kb Sac I fragment (,u-opioid receptor), were obtained from three isolated clones, subcloned into pBluescript (Stratagene) and sequenced. Gene Mapping by in Situ Hybridization. Chromosome spread preparations from phytohemagglutinin-stimulated human lymphocytes were hybridized with a 3H-labeled probe
prepared from the 1.7-kb Sac I genomic hKOR fragment (including the last coding exon), exposed for 15 days, and developed as described (7). cDNA Cloning. A cDNA encoding hKOR was obtained by reverse transcription-polymerase chain reaction (RT-PCR). Total RNA (10 jig) was prepared (8) from human placenta, reverse transcribed, and amplified (9) with the forward primer 5' -GAGAGCTCGCGGCCGCGAGCTGCAGCGTCACCATG-3' and the reverse primer 5'-AGACCCAAGCTTGCCCGGGGACATCTCCACGACTAGTCA-3'. PCR products ranging from 1 to 1.3 kb were purified and a second amplification was performed with a new reverse primer, 5'-AGACCCAAGCTTGCCCGGGTCCACGACTAGTCATACTGG-3', and the same forward primer under the same conditions. The final product, 1.2 kb in size, was purified, made blunt-ended, and inserted into the EcoRV site of plasmid pcDNA/Amp (Invitrogen). Binding Assays. COS-1 cells were transfected with purified plasmid, and P2 membranes were prepared (4). All binding assays were conducted as described (9). For saturation experiments, variable concentrations of [3H]U-69,593 (57 Ci/mmol, Amersham; 1 Ci = 37 GBq) and [3H]diprenorphine (30 Ci/mmol, Amersham) were used. For competition studies, binding sites were labeled with [3H]diprenorphine at 0.6 nM. All competing agents were purchased from Sigma, except bremazocine, norbinaltorphimine (nor-BNI), and naltrexone, which were obtained from Research Biochemicals. Binding assays using endogenous opioid peptides as competitors were
Opiates exert their strong analgesic action through three major opioid receptor types, named ,u, 8, and K, which belong to the superfamily of G-protein-coupled receptors. These receptors control a wide variety of physiological functions such as the autonomic, neuroendocrine, and immune systems, as well as mood and cognitive functions (for a review see ref. 1). Despite their common involvement in the body response to pain and stress, each receptor type displays a unique tissue distribution pattern (2) and a distinct pharmacological profile (3). K-Opioid receptors have a widespread distribution in the central nervous system and display highest affinity toward prodynorphinderived peptides. Rat and guinea pig animal models have been most intensively used in opioid research. Since significant differences in the anatomical distribution of opioid receptor binding sites have been observed across species, particularly concerning receptors of the K type (3), it is of utmost importance to study the endogenous K-opioid system specifically in humans. The molecular characterization of the human K-opioid receptor (hKOR) is therefore a necessary step to evaluate animal models and delineate the molecular basis of K-opioid function in humans. We (4) and others (5) have isolated a cDNA encoding a mouse 8-opioid receptor by expression cloning. We have used this DNA probe for the cloning of human opioid receptor genes. In this paper we report the analysis of genomic clones containing coding regions for human K- and ,u-opioid recep-
Abbreviations: hKOR, human K-opioid receptor; hMOR, human ,u-opioid receptor; hDOR, human 6-opioid receptor; nor-BNI, norbinaltorphimine; RT, reverse transcription. ITo whom reprint requests should be addressed. "The sequence of hKOR cDNA has been submitted to GenBank (accession no. U17298).
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
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Proc. Natl. Acad. Sci. USA 92 (1995)
Pharmacology: Simonin et al.
the adjacent 3' coding exon down to the termination codon, encoding aa 204-380. These exons appeared to be homologous to the second and third coding exons of the human 8-opioid receptor (hDOR) gene (9). None of the isolated clones contained the 5' coding region of the hKOR gene. Another genomic clone was found to contain coding regions of the hMOR as indicated by the high sequence similarity with the rat ,u-opioid receptor cDNA (18). This was later confirmed by sequence identity with the recently published hMOR cDNA (19). Sequence analysis of a 7.5-kb Sac I fragment subcloned from this hMOR genomic clone revealed two exons (encoding aa 96-212 and 213-386 of the receptor) with high similarity to the above-mentioned 8 and K exons. The C-terminal 12 aa of hMOR are not encoded by these exons and are presumably encoded by a 3' additional exon, as was shown in the case of the mouse gene (ref. 20; H.M., unpublished results). The two exons with homologous splice sites across the three human opioid receptor genes are represented in Fig. 1. Using a DNA fragment derived from the hKOR gene (see Materials and Methods), we mapped this gene to the 8q1-8q12 region in the human genome (data not shown). This finding is in accordance with a recently published gene mapping obtained by fluorescence in situ hybridization using another genomic fragment of the hKOR gene as a probe (17). Primary Structure of hKOR. To achieve functional expression of hKOR, we isolated a full-length cDNA from human placenta, which has been reported to be a rich source for K receptors (21). We devised a RT-PCR strategy for the specific amplification of hKOR cDNA; we used human sequence information from our gene analysis for the design of two 3' reverse primers; the 5' forward primer was based on the published mouse sequence (10). A cDNA was amplified and the deduced protein sequence is shown in Fig. 2. To confirm that no mutation had been introduced into the cDNA clone by the PCR amplication, we compared the 3' nucleotide sequence (corresponding to aa 87-380 in Fig. 2) with the previously determined genomic sequence and found 100% identity. The 5' nucleotide sequence (aa 1-86) was confirmed by comparing sequences of PCR products obtained from three independent experiments. Comparison of the human nucleotide sequence with those of previously cloned mouse, rat, and guinea pig K-opioid receptors indicated 87% homology with all three rodent sequences. The deduced hKOR amino acid sequence is 94% identical to both the mouse and rat sequences, and 91% identical to the guinea pig sequence. The high sequence similarity indicates that the cDNA indeed represents the human counterpart of the rodent K receptor. Functional Expression of hKOR and Pharmacological Properties. We transiently expressed hKOR in COS-1 cells, using the mammalian expression vector pcDNA/Amp (Bm., 6 ± 2 pmol/mg of membrane protein). No specific opioid binding was observed on control mock-transfected cells. In saturation experiments (data not shown), both the selective K
conducted in the absence and in the presence of a mixture of protease inhibitors (leupeptin, pepstatin, aprotinin, antipain, and chymostatin, each at 2.5 jug/ml). Nonspecific binding was determined in the presence of 1 ,uM naloxone (Sigma). Adenylate Cyclase Inhibition. COS-1 cells (1.5 x 106 per 14-cm dish) were trypsinized 24 hr after transfection, seeded in 12-well plates (1.2 x 104 cells per well), and grown for 48 hr. Incubation of the cells with 3-isobutyl-1-methylxanthine (Sigma), forskolin (Sigma), and opioid ligands was followed by measurement of intracellular cAMP (9). RT-PCR Detection of Human Transcripts in Brain. Three human brains were taken from 50- to 70-year-old normal Caucasian subjects 5-20 hr postmortem, according to guidelines from French law (loi Caillavet). Structures of interest were dissected out, total RNA was prepared, and cDNA was synthesized from the RNA (9). For the specific amplification and detection of the hKOR transcript, the following oligonucleotides were used: forward primer, 5'-GAGAGCTCGCGGCCGCGTCTACTTGATGAATTCCTGG-3' (A); reverse primer, 5 '-GGAAGCTTGAATTCCTGCTAGTGCTCTGCCGCTC-3' (B); and detection probe, 5'-GCACCTCCCACAGCACAGCTGC-3' (C). One-tenth of the cDNA was amplified (see ref. 9) with primers A and B. PCR products were analyzed by Southern blot hybridization with 32P-labeled probe C. Blots were exposed to x-ray film for 1.5 hr. In Situ Hybridization. Human fetal spinal cords were obtained at autopsy after therapeutic or spontaneous abortion at the Centre Hospitalier Regional de Bordeaux (France) in accordance with guidelines and recommendations of the Comite National d'Ethique. Spinal cords were dissected, cut into 5-mm fragments, fixed by immersion for 24 hr in 1% paraformaldehyde, frozen in isopentane (-45°C), and stored at -80°C until sectioning. [a-[35S]thio]UTP-labeled antisense and sense RNA probes were synthesized by in vitro transcription of a 0.8-kb fragment of hKOR cDNA spanning from transmembrane domain 2 to the stop codon. In situ hybridization was performed on 15-,um-thick sections of human spinal cord (as will be described elsewhere), with 3 months of exposure time prior to development.
RESULTS Isolation of Exons Coding for hKOR and hMOR Genes. We screened a human genomic library with a probe derived from the mouse 8-opioid receptor cDNA (4). Sequence analysis of two overlapping clones indicated the presence of K-opioid receptor coding regions, as suggested by their strong homology with the recently described rodent K-opioid receptor nucleotide sequences (10-16). A 6-kb Sac I fragment derived from the first clone contained one exon encompassing putative transmembrane regions 2-4 of the receptor (aa 87-203). This exon had been identified previously by other authors (17). A 1.7-kb Sac I fragment derived from the second clone contained
hDOR
-L TTCTAAG1GTAC TYr78
hKOR
hMOR
CTTTTAGIATAC
ZXON
2
-CQG
G |GTGAG
192 Arg
202 Arg
CTCCTAGIATAC Tyr96
211 Arg
CGGCAAG
\
GACTCAGIATGGT
ZXON 3
Gly 194
CGGAA&GIGTAAG
Tyr87
7007
CCTCCAG ACGTC Val 205
GTGAG
\CTTCTAGI TTCC Ser214
FIG. 1. Conserved positions of splice sites within the human opioid receptor gene family. Partial genomic organization of hDOR, hKOR, and hMOR genes is presented. hDOR genomic organization is taken from Simonin et al. (9). The two exons encoding putative transmembrane regions 2-4 (exon 2) and transmembrane region 5 to the stop codon (exon 3) are shown in bold. The sequences of exon-intron boundaries are indicated together with the adjacent homologous codons for the three genes. Amino acid residues are numbered with respect to their position in the deduced protein sequences of hDOR (9), hKOR (Fig. 2), and hMOR (19) cDNAs. Consensus donor/acceptor sites, AG/GT, are found at all the splice sites.
7008 hKOR mKOR rKOR
gpKOR hKOR mKOR rKOR
Proc. Natl. Acad. Sci. USA 92 (1995)
Pharmacology: Simonin et al. * 1* MDSPIQIFRG EPGPTCAPSA CLPPNSSAWF PGWAEPDSNG -E -------- D-----S -----L----S-- -N5---S----E-------- ---------- --L----S-- -N---S----GRRR-GPAQ PASELP-RN- --L--G---L -------G--
50 SAGSEDAQLE -V ----Q---
-V----Q-----PQ-E---
I 51 100 PAHISPAIPV IITAVYSVVF VVGLVGNSLV MFVIIRYTKM KTATNIYIF S--
gpKOR hKOR
mKOR rKOR gpKOR
II 101 LALADALVTT TMPFQSTVYL -------------------
------A--------A---
III 150 MNSWPFGDVL CKIVISIDYY NMFTSIFTLT
_______ ______
-------------------
151
hKOR
mKOR rKOR
gpKOR hKOR
mKOR rKOR
gpKOR
MMSVDRYIAV _ ________ __________ __________
IV CHPVKALDFR
TPLKAKIINI CIWLLSSSVG ISAIVLGGTK -----
----------
----------
A-------- A----
----------
----------
----
----------
----------
1+
VREDVDVIEC SLQFPDDDYS WWDLFMKICV ---------- ------- E-- ------------------- ------- E-- ---------------I--- ---------- ----------
251 # hKOR mKOR rKOR
gpKOR
#VI K-----K------
----------
----------
---
----------
----------
---
----------
----------
----------
hKOR
mKOR
----------
----------
-------
----V--
----------
----------------
gpKOR
__________
----------
I-----
V 250 FIFAFVIPVL TTTVCYTLMI - V-------- ---------- V-------- ---------- V-------- ----------
.LRLKSVRLLS GSREKDRNLR RITRLVLVVV AVFVVCWTPI
301 STSHSTAALS SYYFCIALGY
rKOR
200
II ----II ------- II -----
300
HJIFILVEALG
---
----------
---
-------------------
350
VII
TNSSEELNIYAF-LDENFKRC FRDFCFPLKM V-V--
hKOR
351 # #380 RMERQSTSRV RNTVQDPAYL RDIDGMNKPV
mKOR
------- N--
--------
SM --VG -----
rKOR
------- N--
--------
gpKOR
----------
---------
SM --VG ----M -NV--V ----
----------------------------
I--------I-------I-
-------
FIG. 2. Deduced protein sequence of hKOR and comparison with the mouse, rat, and guinea pig sequences. In rodent sequences, only residues which are different from the human sequence are represented. Putative transmembrane domains are underlined (I-VII). Consensus N-linked glycosylation sites (*) and potential phosphorylation sites (#) are indicated. Exon-intron boundaries are shown by arrows.
agonist [3H]U69,593 and the nonselective opioid antagonist [3H]diprenorphine displayed high affinity and saturable specific binding at a single class of sites with Kd values of 1.49 ± 0.01 nM and 0.77 ± 0.12 nM, respectively. The pharmacological profile of the cloned human receptor was determined by measuring [3H]diprenorphine displacement with a set of competitors (Table 1). No significant change in Ki values was observed when the endogenous opioid peptides were assayed in the presence or absence of protease inhibitors, ruling out possible peptide degradation during competition experiments. Prodynorphin-derived opioid peptides (dynorphin A, dynorphin A-(1-8), dynorphin B, a-neoendorphin, and B3-neoendorphin) and the K-selective ligands U50,488 and nor-BNI competed efficiently with [3H]diprenorphine binding. The nonselective opioid ligands bremazocine, naltrexone, levorphanol and naloxone also displayed high affinity for hKOR. In contrast, the receptor interacted weakly with the ,u ligand [D-Ala2,MePhe4,Gly-ol5]enkephalin (DAGO) or the 8 ligand [D-Ala2,D-Leu5]enkephalin (DADLE). Similarly, the proopiomelanocortin- and preproenkephalin-derived opioid peptides (3-endorphin and [Leu5]- and [Met5]enkephalin) exhibited poor affinity for hKOR. We conclude that the cloned human receptor presents the typical binding characteristics of K-opioid receptor. Further, the strong potency of arylacetamides (U69,593 and U50,488) and nor-BNI in binding at the receptor defines hKOR as a Kl subtype (22).
Table 1. Pharmacological profile of cloned hKOR expressed in COS cells Kd or Ki, nM Ligand Ligand Ki, nM Saturation ,u/8-Selective 0.77 DAGO > 1000 [3H]Diprenorphine 1.49 DADLE >1000 [3H]U69,593
Competition K-Selective Dynorphin A Dynorphin A-(1-8) Dynorphin B a-Neoendorphin
13-Neoendorphin
5.50 9.14 8.80 5.23 13.5
502 ,3-Endorphin [Leu5]Enkephalin >1000 [Met5]Enkephalin >1000 Nonselective Bremazocine Naltrexone
Levorphanol
0.54 4.07 20.4
15.5 Naloxone 26.8 U50,488 1.15 nor-BNI Saturation isotherms were done in triplicate with [3H]diprenorphine and [3H]U69,593. Competition experiments using [3H]diprenorphine were repeated two or three times in duplicate for each compound. Kd and Ki values were determined with the EBDA/LIGAND program (G. A. McPherson, Biosoft, U.K.) DAGO, [D-Ala2,MePhe4,Gly-o15]enkephalin; DADLE, [D-Ala2,D-Leu5]enkephalin.
We examined functional coupling of the cloned receptor transiently expressed in COS cells (data not shown). The addition of 500 nM U50,488 (a nonpeptide selective agonist), bremazocine (a nonpeptide nonselective agonist), or dynorphin A (a K-preferring peptide agonist) induced inhibition of the forskolin-stimulated production of cAMP by 45 ± 2% independent of the agonist used. This inhibitory effect was reversed by the antagonist naloxone. Our results indicate that hKOR expressed in COS cells is negatively coupled to adenylate cyclase and that signal transduction occurs following activation of the receptor by three different types of agonist molecules. Expression Pattern of hKOR in Human Brain. Total RNA was prepared from various human brain structures, as well as from placenta, which we used as an external control. Regardless of the postmortem period, high molecular weight mRNAs and intact rRNAs were obtained, indicating the absence of RNA degradation (data not shown). RT-PCR was used to detect the hKOR transcript. The design of K-specific oligonucleotide primers was based on hKOR sequences found in regions which are highly divergent between opioid receptor subtypes (first extracellular loop and C-terminal region), excluding any possible cross-hybridization to 8 and j, receptor cDNAs. RT-PCR produced a DNA fragment of the expected size (754 bp), and this fragment further hybridized to a third 32P-labeled K-specific oligonucleotide, confirming the selective detection of the K-opioid receptor transcript. Each brain region was tested from at least two individuals and a consistent expression pattern was found (Fig. 3). The reproducible signal intensity over several brain samples suggests that RT-PCR provides a reasonable estimation of relative hKOR expression levels across investigated brain areas. The hKOR transcript was detected in every region tested, with the exception of the internal globus pallidus, where no mRNA was detected. In the telencephalon, the signal was particularly strong in most regions, including the various neocortical areas, the olfactory bulb, the amygdala, and the basal ganglia. A weaker signal was observed in the external globus pallidus and hippocampus. In the diencephalon, the transcript was abundant in both posterior and anterior thalamus and clearly present in the hypothalamus and mamillary bodies. The transcript was detected with moderate intensity in all midbrain and brainstem regions investigated, with a predominant signal in the ventral tegmental area and locus coeruleus. Finally, hKOR was also found in the cerebellar cortex, in the pituitary gland, and in the spinal cord at cervical, dorsal, and lombar levels.
Proc. Natl. Acad. Sci. USA 92 (1995)
Pharmacology: Simonin et al. HUMAN BRAIN STRUCTURES
hKOR
_
TELENCEPHA LON Cortex frontalis
.l.itranscript I
Cortex temporalis
A few scattered neurons present in the dorsal and ventral gray matter are also labeled. Appropriate controls, including the use of a sense probe, demonstrated the specificity of the reaction (data not shown). The presence of the transcript in fetal spinal cord indicates expression of the K-opioid receptor during development of the nervous system in humans.
Cortex parietal
I
Cortex occipitalis
Hippocampus Corpus amygdaloideum Bulbus olfactorius Nucleus caudatus
DISCUSSION +++
.1 ++
Nucleus accumbens
I
Putamen
Globus pallidus, pars lateralis
+++
Globus pallidus, pars medialis +++
)IENCEPHA LON
S:
Nuclei anteriores thalami
I.
Nuclei posteriores thalami Hypothalamus
+++
+++
SI
Corpus mamillare
+++
MESENCEPHALON Substantia nigra
+++
Area tegmentalis ventralis
+++
Colliculus inferior
+++
Colliculus superior PONS, MEDULLA OBLONGATA Locus coeruleus
7009
++ ++
In this study we describe the isolation of genomic clones encoding hKOR and hMOR genes. For both genes, exonintron boundaries are found after putative transmembrane domains 1 and 4 of the deduced protein sequence. We have reported a similar organization in the hDOR gene (9). Comparison of genomic sequences indicates that these exon-intron boundaries are located at almost identical positions in the nucleotide sequence, defining homologous exons for the three opioid receptor genes. This result strongly suggests that the opioid receptor gene family might have originated from duplication of a single ancestral gene. Human genomic sequence analysis allowed us to isolate a hKOR cDNA from placenta by use of RT-PCR. Recently Mansson et al. (23) reported the isolation of a hKOR cDNA from the same source. The two sequences are identical with the exception of one conservative difference (Glu -- Asp) at position 2 in the deduced protein sequence. We investigated the pharmacological profile of hKOR expressed in COS cells and compared it with the profile of the rodent receptor. Although a good correlation is generally found, some significant differences (more than 1 order of magnitude) are observed between the human pharmacology presented in this study and the rodent pharmacology described elsewhere. In particular, some prodynorphin products (dynorphin A, dynorphin B, and 3-neoendorphin) bind more efficiently to the cloned rat receptor (13), and some alkaloid compounds (diprenorphine, U50,488, and nor-BNI) display highest affinity to the cloned mouse receptor (24). In such a comparison, variability might originate from the various experimental systems used by the different investigators or from real variations in binding potency of the tested ligands. More remarkable is the observation that all the prodynorphin products tested in this study displayed equivalent affinity for the human receptor
Nucleus olivaris
I
Cortex cerebelli
Hypophysis Medulla spinalis - Cervical level - Dorsal level - Lombar level Placenta
I I
f:c
..
*:
I.
S
FIG. 3. Distribution of the hKOR transcript in human brain. Total RNA was prepared from a set of dissected human brain structures and from placenta. RT-PCR using two human K-specific oligonucleotide primers was employed to amplify selectively a region spanning transmembrane domains 2-7 of the hKOR transcript (see Materials and Methods). A representative autoradiogram for a set of brain structures from one subject is presented. Signal intensity: + + + +, very strong; + + +, strong; + +, moderate; +, low; -, undetectable.
Localization of the hKOR Transcript Within Spinal Cord. In situ hybridization using the hKOR probe demonstrated the presence of reactive neurons in the spinal cord of human fetus and neonate. Preliminary data indicated similar results with adult spinal cord. The pattern obtained from a 16-week fetus is presented in Fig. 4. Heavy labeling is seen in packed neurons in the dorsal horn of the gray matter, specifically in lamina II.
o ^ fw,*~~~
96
FIG. 4. Detection of the hKOR mRNA by in situ hybridization in the spinal cord of a 16-week human fetus. (A) Section of the spinal cord stained with toluidine blue. The ependymal canal is indicated by a c, the small arrow shows the dorsal horn, and the star shows the ventral horn of the spinal gray matter. (B) An adjacent section hybridized with the 35S-radiolabeled cRNA hKOR probe. A darkfield autoradiogram is presented. A heavy signal is present in the dorsal horn corresponding to reactive neurons in lamina II (arrow). (C and D) Details of B in the vicinity of the ependyma (C) and at the level of lamina II (D). Three labeled neurons (double arrows) are shown in the periependymal area. Numerous labeled packed neurons are visible in lamina II.
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Pharmacology: Simonin et al.
(5.23-13.5 nM; Table 1), whereas a high variability has been reported for the cloned guinea pig receptor (0.58-256 nM; ref. 16). This latter difference might be related to the strong N-terminal structural divergence between the two receptors (see Fig. 3). Species specificity should be evaluated and a strict comparison between the pharmacology of human and rodent cloned K receptors will be required in the development of new therapeutic compounds. The regional distribution of the hKOR transcript in human brain found in this study is in good agreement with many anatomical reports which describe a widespread distribution of K-opioid receptor throughout forebrain, midbrain, and brainstem structures in various mammalian species at both the protein level (for a review see ref. 2) and the mRNA level (13, 16, 25). Interestingly, these studies underline species differences among rodents in the distribution of K-opioid receptor. Our study indicates that such differences also exist between human and rodents. In particular, we have detected a high level of hKOR transcript in cerebellum. An abundant expression in this region has also been described in guinea pig (16), whereas it is completely absent in rat (13). In addition, we have found a surprisingly weak signal in hippocampus and substantia nigra, whereas in rodents, a high level of K-opioid receptor mRNA has been detected in both areas. These results underline the possibility of functional differences in K-opioid physiology between human and rodents. In the spinal cord we found abundant levels of hKOR almost exclusively expressed in lamina II of the dorsal horn. This region is the site for receipt and processing of primary afferent nociceptive information. Numerous pharmacological assays in vivo have demonstrated K agonist-induced analgesia at the spinal level (26) and anatomical studies in rats (2) and humans (27) have shown that K binding sites are predominantly localized in lamina II. The colocalization of receptor mRNA and binding sites in this area suggests that K-opioid receptors are found in interneurons of lamina II and further confirms a possible role of hKOR in spinal analgesia in humans. In conclusion, this study suggests some divergences between the human and the rodent receptors which might have functional implications. Further careful study of the human receptor at the molecular level will therefore be important for the understanding of K-opioid physiology. We are grateful to J. M. Garnier for providing the genomic libraries and to Dr. N. Foulkes for careful reading of the manuscript. We especially thank Prof. P. Chambon and Prof. J. F. Lefevre for supporting our work. This work was supported by the Universite Louis Pasteur, the Centre National de la Recherche Scientifique, the Institut National de la Sant6 et de la Recherche Medicale, the Association pour la Recherche sur le Cancer, Rhone-Poulenc Rorer (France), and Fabriques de Tabac Reunies (Switzerland). 1. Olson, G. A., Olson, R. D. & Kastin, A. J. (1993) Peptides 14, 1339-1378.
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(1995)
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