Molecular cloning and tissue distribution of the ovine somatostatin receptor subtype 5: osst5

Molecular cloning and tissue distribution of the ovine somatostatin receptor subtype 5: osst5

Domestic Animal Endocrinology 23 (2002) 397–410 Molecular cloning and tissue distribution of the ovine somatostatin receptor subtype 5: osst5 N. Debu...

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Domestic Animal Endocrinology 23 (2002) 397–410

Molecular cloning and tissue distribution of the ovine somatostatin receptor subtype 5: osst5 N. Debus a,∗ , A. Dutour a , F. Boudouresque b , V. Vuaroqueaux a,b , C. Oliver a , L’H. Ouafik b a

b

Laboratoire de Neuroendocrinologie Expérimentale, INSERM U501, IFR Jean Roche, Bvd P. Dramard, 13916 Marseille Cedex 20, France Cancérologie Expérimentale, IFR Jean Roche, Bvd P. Dramard, 13916 Marseille Cedex 20, France Received 19 January 2002; accepted 25 April 2002

Abstract The sheep is a valuable model to study growth hormone (GH) neuroregulation since its GH secretion pattern is close to that in humans and an integrated physiological approach is possible in this species. Somatostatin receptor subtype 5 (sst5) appears to be important in GH regulation but the ovine sst5 gene (osst5) has not yet been cloned. We report here the cloning of sst5 in that species. We screened a cDNA sheep library and isolated a 1.24 kb cDNA, which includes the whole coding region of osst5. The predicted protein consists of 367 amino acids exhibiting a putative seven transmembrane domain topology typical of G protein-coupled receptors. Nucleotide sequence comparisons with that of other species sst5 showed that osst5 displays 83.8, 81 and 79.7% homology with human, rat, and mice sst5, respectively. Southern blot analysis of ovine cortex DNA demonstrated that osst5 is encoded by a single gene. Osst5 transiently expressed in Chinese Hamster ovary (CHO) cells exhibit a high affinity for somatostatin-14. Reverse transcriptase-polymerase chain reaction (RT-PCR) studies demonstrated that osst5 mRNAs are present in pituitary, cortex, hypothalamus, hippocampus, colon and adrenal gland. The cloning of osst5 should provide a useful tool to study the mechanisms through which somatostatin inhibits hormone secretion in the sheep. © 2002 Elsevier Science Inc. All rights reserved.

∗ Corresponding author. Present address: UMR 868 Elevage des Ruminants en R´egions Chaudes, ENSA.M-INRA, 2 place P. Viala, 34060 Montpellier Cedex 1, France. Tel.: +33-4-99-61-29-60; fax: +33-4-67-54-56-94. E-mail address: [email protected] (N. Debus).

0739-7240/02/$ – see front matter © 2002 Elsevier Science Inc. All rights reserved. PII: S 0 7 3 9 - 7 2 4 0 ( 0 2 ) 0 0 1 7 7 - 7

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1. Introduction Most studies on the neuroregulation of growth hormone (GH) secretion and particularly on the involvement of somatostatin (SRIH), and its receptor subtype selective biological action have been performed in the male rat. There are experimental limitations in rodent models and growing interest in a more relevant animal model, such as the sheep, for the study of human GH neuroregulation. Indeed, GH secretion, in the male rat, is characterized by a strikingly regular ultradian rhythm, rather different from the irregular secretory bursts found in other species including humans and sheep. Furthermore the response to various physiological situations or secretagogues is very similar in humans and sheep: for example, endotoxin causes a decrease of GH secretion in rats and an increase in both humans and sheep [1,2]. In addition in sheep it is possible to collect large volume of hypophysial portal blood (HPB) and therefore to directly assess the secretion of neurohormones into HPB under physiological conditions, especially without the biases induced by anesthesia and surgical stress. This adds further interest to the study of this species. Somatostatin is a cyclic tetradecapeptide originally isolated from ovine hypothalamic extracts [3] which inhibits GH secretion as well as that of numerous other pituitary, pancreatic, and gastrointestinal hormones. SRIH is known to be an important multifunctional peptide regulating neurotransmission and inducing inhibition of various secretions and cell proliferation [4]. SRIH biological effects are mediated by high-affinity receptors [5]. Five distinct somatostatin receptor subtypes (sst1–5) have been identified in humans, and rodents, and form a family of G protein-coupled receptors [6–21]. In the pituitary, all five sst mRNAs are present in major pituitary cell types, sst5 mRNA being the most expressed especially in somatotrophs [22,23]. The somatotostatin receptor subtypes involved in hypothalamic and/or pituitary regulation of GH are only partially characterized. Using 17 somatostatin analogues, we observed major differences between rat and sheep analogue potencies to inhibit GH secretion in vitro and demonstrated that in sheep their potencies in inhibiting GH release in vitro were highly correlated with their binding affinities for sst2 and sst5 [24]. Similar results have been found in humans [24,25]. Human and murine sst5 cDNAs have been identified, but ovine sst5 (osst5) cDNA has not yet been cloned. To further define the role of sst5 in GH regulation in the powerful sheep model, we cloned and sequenced ovine sst5 cDNA from a sheep pituitary cDNA library and studied its tissue distribution.

2. Materials and methods 2.1. Isolation and sequencing of the osst5 cDNA A partial human sst2 (hsst2) cDNA (700 bp, 320–1020) [26] was radiolabeled with [␣-32 P] dCTP (ICN Pharmaceuticals, Orsay, France) using a random priming kit (Quick Prime, Amersham Pharmacia biotech, Orsay, France) and employed as a probe to screen a sheep pituitary cDNA ␭ZAPII library obtained from Stratagene. Recombinant plaques (1 × 106 in total) were plated at the density of 25,000 pfu/150 mm plate of Escherichia coli XL1-blue. After incubation for 12 h at 37◦ C, plates were lifted with nitrocellulose filters (Schleicher and Schuell,

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Ecquevilly, France). Membranes were prehybridized, hybridized, and washed under reduced stringency (2 × SSC, 0.1% SDS at room temperature for 25 min twice then 0.2 × SSC, 0.1% SDS at 50◦ C for 25 min twice) as previously described [27]. Filters were exposed to Kodak biomax film (Kodak, Rochester, NY, USA) for 2 days. A single positive clone was detected then plaque purified and subcloned into pBluscript (Stratagene, La Jolla, CA, USA) by phage rescue. DNA sequencing was performed using the dideoxy chain termination method of Sanger et al. [28], with a T7 sequencing kit purchased from Amersham Pharmacia biotech. Sequence analysis, comparison, and dendrogram were performed using MacVectorTM software (International Biotechnology Inc., New Haven, CT, USA) and Bizance, a French service for access to biomolecular sequence databases [29]. 2.2. Transfection of cells The sequence preceding the initiator MET in osst5 was modified to provide a consensus ribosome-binding site (Kozak) and a convenient restriction site (HindIII). The polymerase chain reaction was used to generate a cDNA fragment with the desired modifications. The oligonucleotide used to modify the sequence around the translational start site had the sequence 5 -CCAAGCTTGCCGCCACCATGGAGCCTCTGTTCCTGGCC-3 . The desired fragment was amplified from a pBluescript (Stratagene) plasmid containing osst5 cDNA by using this oligonucleotide and 5 -GGA TCC CAC TCA CAG CTT GCT GGT CTG-3 localized on 3 -UTR of osst5 cDNA containing a convenient restriction site (BamHI). Oligonucleotides were synthesized by Genset Oligos (Paris, France). The amplified fragment was digested with HindIII/BamHI (Gibco BRL, Life Technologies, Cergy Pontoise, France) and was inserted into the polylinker region of the pcDNA 3.1 (+) expression vector (Invitrogene, Groningen, The Netherlands). The nucleotide sequence was confirmed by DNA sequence analysis. Chinese Hamster ovary (CHO) cells were utilized for transient transfection with a plasmid prepared with Nucleobond AX Kit (Machery Nagel, Hoerdt, France). The CHO cells were transfected by the calcium phosphate method [30] and grown for 24–48 h in Dulbecco’s modified Eagle’s medium (Gibco BRL), then harvested for RNA extraction [31]. The transfection of osst5 was confirmed by the presence of osst5 mRNA in transfected but not control CHO cells, analyzed by Northern blot analysis of total RNA as previously described [26]. 2.3. Binding assay [125 I]-labeled Tyr0 -DTrp8 -SRIH 14 (specific activity: 2200 Ci/mmol) was prepared in our laboratory by lactoperoxydase iodination and purified by reverse phase HPLC [32]. Tyr0 DTrp8 -SRIH 14 was synthesized by Neosystem (Strasbourg, France). The iodinated peptide was diluted 1:2 in binding buffer, stored at 4◦ C and used within 3 days. Binding experiment was performed as previously described [33]. For binding experiments 106 cells/tube were used. Transiently transfected cells were washed twice with 5 mL of PBS followed by PBS containing 5 mM EDTA to detach the cells. Binding was performed for 30 min at 25◦ C in 20 mM HEPES, pH=7.4, 5 mM MgCl2 , 100 ␮g/mL bacitracin, 230 ␮M phenylmethylsulfonyl fluoride, 0.46 TIU/mL aprotinin, 0.1% bovine serum albumin (BSA, fraction V) in a total volume of 0.1 mL containing 0.25 nM [125 I]Tyr0 -DTrp8 -SRIH 14 without (total binding) or with

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increasing amounts (10−10 –10−6 M) of SRIH 14 (Bachem, Voisins-le-Bretonneux, France). Specific binding was calculated as the difference between total and residual binding in the presence of 10−6 M unlabeled SRIH 14. This saturable component represented between 40 and 50% of the total binding. The incubation was stopped by diluting the mixture with 5 mL ice cold 50 mM Tris–HCl buffer containing 1% BSA. Separation of free and bound radioligand was obtained by rapid filtration in vacuo through Whatman GF/C glass-filter circles (2.5 cm in diameter) (Merk Eurolab, Strasbourg, France), presoaked in 50 mM Tris–HCl containing 0.3% polyethyleneimine and 0.1% BSA. Filters were washed with 10 mL ice cold buffer. Filters were recovered in polystyrene tubes and counted in a Cobra II (Packard Bioscience, Rungis, France) (99% efficiency for 125 I). In the absence of cell preparations less than 1% of the radioactive material was retained on the filter. All assays were performed in triplicate. 2.4. Analysis of osst5 mRNA distribution by reverse transcriptase-polymerase chain reaction (RT-PCR) Total RNA was prepared from normal sheep tissues using the acid guanidinium thiocyanate phenol/chloroform extraction method [31]. Since somatostatin receptor genes do not have introns and in order to avoid genomic DNA contamination, 10 ␮g of total RNA were treated with 10 U RNase Free DNase I-RQI (Promega, Lyon, France) for 2 h at 37◦ C [34]. Total RNA was reverse transcribed into cDNA using M-MLV reverse transcriptase, as described by the manufacturer (Gibco BRL); briefly, each sample was separated in one positive reaction (5 ␮g total RNA in presence of reverse transcriptase) and one negative reaction (absence of reverse transcriptase) to check for DNA contamination. Amplification of cDNA was performed in a 50 ␮L reaction volume with the manufacturer’s buffer in the presence of 1 ␮M of each primer (Genset Oligos), 200 ␮M of each dNTP (Promega), and 2.5 U of ExpandTM long template PCR system polymerase (Boerhinger, Mannheim, Germany). Optimal temperature and cycling conditions were 94◦ C during 5 min for denaturation, followed by 40 cycles of annealing at 62◦ C for 35 s, elongation at 68◦ C for 50 s, and denaturation at 94◦ C for 30 s. Reactions were performed using a Biometra Trio-thermoblock cycler (Biometra, Gröttingen, Germany). Sens primer sequence was AAGAAGCCCAGCACGGA; antisens primer sequence was GCCAAGATGAAGACGGT; PCR product size was 400 bp. Samples were fractionated on agarose gels in 89 mM Tris, 89 mM boric acid, 2.5 mM EDTA, pH 8, and visualized by staining with ethidium bromide. RT-PCR was performed on tissues which were collected from three different animals. 2.5. Southern blot analysis Genomic DNA from ovine cortex was isolated using standard techniques [35]. DNA (10 ␮g) was digested with restriction enzymes BamHI, EcoRI, HindIII, XbaI, and SalI (Gibco BRL) and fractionated on a 0.7% agarose gel, transferred to a Hybond-N+ membrane (Amersham Pharmacia biotech) using an alkali blotting procedure, immobilized, and hybridized with ovine sst5 cDNA probe (1239 bp). The membrane was exposed to Biomax film (Kodak). The southern blot analysis has been performed on three different ovine genomic DNA samples.

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3. Results In order to perform sst5 cloning in sheep, we used the hsst2 open reading frame fragment as a probe to screen a sheep pituitary cDNA library under reduced stringency. A clone of 1239 bp was isolated and sequenced. The cloned cDNA contained a single open reading frame, the sequence of which predicts a protein product of 367 amino acids with an estimated molecular mass of 40,266 Da, and was designated osst5 (Fig. 1). Comparison of the deduced amino acid sequence with those of the members of the hsst family reveal 44, 47, 48, 52, 44, and 78% sequence identity to hsst1, hsst2A, hsst2B, hsst3, hsst4, and hsst5, respectively, indicating that we have isolated the ovine sst5 receptor (Fig. 2). Osst5 shows 83.8% nucleotide sequence identity to the hsst5 sequence and 81 and 79.7% with mouse sst5 (msst5) (the sequence published by Baumeister et al. [19] was used for the comparison) and rat sst5 (rsst5) sequences, respectively. Comparison of the amino acid sequence with that of the already cloned sst5 reveals great similarity with human, rat and mouse sst5 with 78, 76, and 77% sequence identity, respectively (Fig. 3). The dendrogram shows close homology to human, rat, and mouse sst5 sequences, with a closer homology between osst5 and hsst5 than between osst5 and rsst5 or msst5 (Fig. 4). Computer analysis of the hydropathic profile of the osst5 protein sequence showed the presence of seven hydrophobic domains separated by stretches of hydrophilic amino acids, which are typical of G protein-coupled receptors [36]. Sequences of sst5 proteins show greatest similarity in the region corresponding to the putative membrane-spanning domain and diverge the most at their amino and carboxyl termini. The osst5 displays the structural motifs common to the superfamily of G protein-coupled receptors, including other ssts, such as the DRY sequence in the second intracellular loop (IC2), an aspartic acid residue (Asp119 ) in the third transmembrane domain (TM3), and cysteine residues in the first (EX1) (Cys112 ) and second (EX2) (Cys187 ) extracellular loop, which are likely to form a disulfide bridge [36–38]. Moreover, we observed a consensus sequence in IC3 identifying a potential site for G protein coupling [39] located in the 22-residue region of Lys224 –Arg245 , and made up of two basic residues at the amino-terminal site (KLK) and the RKVTR motif at the carboxyl-terminal end of the loop as previously reported by Panetta et al. [17]. Furthermore, within TM7, we identified the sequence YANSCANPXLY. This YANSCANPXLY sequence is a signature sequence for the somatostatin receptor family is not present in other seven transmembrane domain receptors. In sst1–4 the X amino acid of the YANSCANPXLY motif corresponds to Ile. In human, rat, mouse, and now in sheep sst5, Ile is substituted by another amino acid. This substitution is specific to the somatostatin receptor subtype 5. In addition, rat, mouse, and human sst5 differ from sst1 to 4 by their lack of a glutamate residue adjacent to the conserved LAXAD motif in TM2. Osst5 also lacks this glutamate residue. Sst1–5 can be separated into two subgroups according to their EX3 length, sst1 and 4 having EX3 regions that are four amino acids shorter than those seen in sst2, 3, and 5. Osst5 shows the same EX3 length as hsst5 and rsst5. Using MacVectorTM Software and Bizance, we found that osst5 has two potential Nglycosylation sites within the putative extracellular region of the protein (at Asn14 in the amino-terminal segment and at Asn188 in EX2), two consensus sequences for phosphorylation by cAMP-dependant protein kinase A (at Ser243 and at Thr248 in IC3), and nine sites for protein

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Fig. 1. Nucleotide and deduced amino acid sequence of osst5 cDNA and protein. Amino acids are annotated by the single-letter amino acid code. The putative transmembrane domains are underlined. Potential N-glycosylation (䉲), cAMP-dependent protein kinase A (䊐), protein kinase C (䊏), and palmitoylation (䉱) sites are indicated on the sequence. The sequence has been submitted to the EMBL Data Bank with accession number AJ441116.

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Fig. 2. Alignment of osst5 with the six human somatostatin receptor subtypes. The different amino acid sequences are aligned using the single-letter amino acid code. Amino acid residues identical or similar within the seven sequences are indicated by asterisks (∗ ) or points (:), respectively. Gaps (indicated by dashes) in the sequences have been introduced to maximize alignment. The putative transmembrane domains are indicated in the sequences. Amino acid residues specifics to G protein-coupled receptors, somatostatin receptors or somatostatin receptor subtype 5 are highlighted.

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Fig. 3. Alignment of osst5 with hsst5, msst5, and rsst5. The different amino acid sequences are aligned using the single-letter amino acid code. Amino acid residues identical or similar within the five sequences are indicated by asterisks (∗ ) or points (:), respectively. Gaps (indicated by dashes) in the sequences have been introduced to maximize alignment. The putative transmenbrane domains are indicated in the sequences.

Fig. 4. Sequence relationship of cloned receptors depicted in a dendrogram. The amino acid sequences of the various cloned ssts were compared for homology using the bizance program [29] and plotted in a dendrogram. Sequence relationships are indicated reciprocally by the length of the lines. The amino acid sequences were taken from GenBank.

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Fig. 5. Southern blot analysis of sst5 in the ovine genome. Ten micrograms of genomic DNA isolated from ovine cortex were restriction cut with HindIII, BamHI, XbaI, SalI or EcoRI and fractionated on a 0.7% agarose-TBE gel before transfer onto a nitrocellulose membrane. The membrane was subsequently hybridized osst5 cDNA probe (1239 bp). The autoradiograph showed only one band per lane.

kinase C phosphorylation (at Ser21 in the amino-terminal segment, at Ser147 in IC2, at Ser238 , Thr239 , and Ser243 in IC3, and at Ser315 , Ser343 , Ser355 , and Thr364 in the carboxyl-terminal segment). Moreover, the intracellular COOH-terminal domain of osst5 is serine–threonine-rich and could serve as a substrate for serine/threonine protein kinases. Osst5 has a cysteine residue (Cys321 ) surrounded by leucine-rich sequences in the carboxyl-terminal region, which may be palmitoylated as been reported for the ␤-adrenergic receptor [40]. In order to characterize the number of copies of osst5 gene, a cDNA sequence of osst5 was used to probe a Southern blot of ovine cortex DNA digested with five restriction endonucleases that do not cleave within the region encompassed by the probe. This osst5 cDNA probe

Fig. 6. Displacement of specific [125 I]Tyr0 -DTrp8 -SRIH 14 binding to osst5 transiently expressed in CHO cells or to nontransfected CHO cells by SRIH 14. [125 I]Tyr0 -DTrp8 -SRIH 14 (0.25 nM) was incubated at 25◦ C for 30 min with 106 cells/tube in presence of increasing concentration of SRIH 14. Values are the means of three determinations. Results are expressed as the percentage of displacement as described in Section 2.

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Fig. 7. RT-PCR analysis of sst5 mRNA expression in ovine tissues. A 5 ␮g of total RNA from various tissues was subjected to RT-PCR using osst5-specific primers, as described in Section 2. The PCR products were separated on an agarose gel, stained with ethidium bromide, and photographed under UV illumination.

hybridized to a single fragment of sheep cortex DNA digested with each of the five enzymes (Fig. 5). These data indicate that sst5 is encoded by a single sequence in the ovine genome. Osst5 transiently expressed in CHO cells exhibited high affinity for SRIH 14, with a KD value of 3.57 × 10−8 M and Bmax value was 110 fmol/106 cells. No specific binding was observed in CHO cells transfected with an empty vector (Fig. 6). Analysis of osst5 mRNA expression by RT-PCR revealed expression of transcripts in sheep pituitary, brain (cortex, hypothalamus, hippocampus) and peripheral tissues: colon, adrenal gland and kidney but not in liver (Fig. 7).

4. Discussion We isolated ovine sst5 cDNA which displays high nucleotide sequence homology with other sst receptors, particularly the hsst5 receptor. Its tissue distribution, its size, the presence of seven hydrophobic domains typical of G protein-coupled receptors [36], and the presence of a variety of motifs characteristics of sst receptor, such as YANSCANP sequence in TM7 and the cluster of phosphorylation sites in the third intracellular loop, clearly link this cDNA to the somatostatin receptor family. This was confirmed by the high affinity for [125 I]Tyr0 -DTrp8 -SRIH 14 of osst5 transiently expressed in CHO cells. Other sequence properties, such as the presence of a N-glycosylation site in the second EX2, the lack of a glutamate residue in TM2, and the substitution of Ile by Cys in the YANSCANPILY motif in TM7, are specific features of type 5 somatostatin receptors. The osst5 sequence is closer to the hsst5 sequence than to rsst5 or msst5 sequence. The high homology between osst5 and hsst5 gives further support to our proposal of the sheep as a good animal model to approach human GH neuroregulation. In the mouse, two different lengths of the msst5 N-terminal fragment have been reported, the longer one being due to an extension of the open reading frame to the 5 direction by 23 amino acids ([19], direct submission in GenBank by O’Carroll) and the shorter corresponding to the human and rat sst5 length [18,20]. The N-terminal fragment of osst5 has the same length as the hsst5 and rsst5.

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Within TM7, the sequence YANSCANPILY is completely conserved in each of the rat, mouse, and human forms of sst1–4. This sequence is not present in other seven transmembrane domain receptors, suggesting that this motif may serve as signature sequence for the sst family [17,41]. However, this motif is not completely conserved in rodent and human sst5; Ile is substituted by Val in hsst5 and by Leu in rsst5 and msst5 [10,17]. These substitutions seem to be characteristic of the somatostatin receptor subtype 5 and are also observed in the ovine sst5 receptor (substitution of Ile by Cys). The EX3 length seems important in the pharmacological properties of the sst receptors. Indeed, Fitzpatrick and Vandlen [42] observed that sst1–5 can be separated into two subgroups according to their EX3 length, sst1 and 4 having EX3 regions that are four amino acids shorter than those seen in sst2, 3, and 5. Different EX3 lengths have a strong correlation with affinity for MK678 SRIH analogue, suggesting functional significance. We showed that osst5 displays a long EX3 that links it to the sst2, 3, 5 subclass of somatostatin receptors. Furthermore, Ozenberger and Hadcok [43] have found that phenylalanine at position 265 is important for sst5 pharmacological selectivity; the replacement of phenylalanine at position 265 in sst5 with tyrosine (the corresponding residue of other ssts) can modify ligand binding selectivity and abolish the preference for SRIH 28 over SRIH 14. In ovine sst5, this phenylalanine is conserved. When osst5 mRNA expression was analyzed using RT-PCR in various ovine tissues, we observed a widespread distribution (in cerebral cortex, hypothalamus, hippocampus, pituitary, colon, adrenal gland, and kidney), consistent with the results obtained in mice using the same highly sensitive technique [19]. However, osst5 mRNAs are probably of low abundance in these tissues, preventing their detection by Northern blot analysis. This low level of sst5 mRNAs seems to be a common finding by investigators using less sensitive techniques than RT-PCR [10,21,44,45]. However, it is difficult to compare our results to the literature because of the various tissues and/or techniques used. Nevertheless, the recurring observation of the presence of sst5 mRNAs in the pituitary, and their lack in the liver, is consistent with our results [10,19,22,23,44,45]. Since normal human tissue is not easily available, the tissue distribution of hsst5 has not been extensively studied. Hsst5 is expressed in adult pituitary and cerebellum, in fetal pituitary and hypothalamus but not in cerebral cortex [17,46]. Outside the brain it is highly expressed in the colon and the stomach [33,34,47]. Sst5 mRNA distribution described in human and in rodents concords with results obtained in sheep. To conclude, the ovine sst5 receptor was cloned and was shown to display greater similarity with the human sst5 than with the two rodent sst5. This could suggest similar physiological functions of sst5 in humans and sheep, and gives further support to the use of sheep as an animal model to approach human GH neuroregulation.

Acknowledgments N. Debus was supported by a fellowship from IPSEN France and by Regional Council Provence Alpes Cˆote d’Azur. The scientific interest and continuous support of K. Drieu (Ipsen-Beaufour, Paris, France) and B Tissier (Pharma-Biotech, Signes, France) are gratefully acknowledged. The authors express their thanks to Dr. F. Dadoun (INSERM U501, Marseilles,

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