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Endocrinology, doi:10.1210/en.2006-1633
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Endocrinology Vol. 148, No. 9 4136-4146
Copyright © 2007 by The Endocrine Society

Pharmacological and Physiological Characterization of d[Leu4, Lys8]Vasopressin, the First V1b-Selective Agonist for Rat Vasopressin/Oxytocin Receptors

Ana Pena, Brigitte Murat, Miguel Trueba, Maria A. Ventura, Gyslaine Bertrand, Ling Ling Cheng, Stoytcho Stoev, Hazel H. Szeto, Nga Wo, Gabrielle Brossard, Claudine Serradeil-Le Gal, Maurice Manning and Gilles Guillon

Institut de Génomique Fonctionnelle, Départements d’Endocrinologie (A.P., B.M., G.G.) et de Neurophysiologie (G.Be.); Institut National de la Santé et de la Recherche Médicale (INSERM), Unité 661 (A.P., B.M., G.G., G.Be.), 34094 Montpellier, France; Centre National de la Recherche Scientifique (CNRS), Unité Mixte de Recherche (UMR) 5203 (A.P., B.M., G.G, G.Be.), 34094 Montpellier, France; Department of Biochemistry and Molecular Biology (M.T.), Faculty of Science and Technology, University of the Basque Country, 48080 Bilbao, Spain; Department of Biochemistry and Cancer Biology (L.L.C., S.S., M.M.), University of Toledo College of Medicine, Toledo, Ohio 43614; Department of Pharmacology (H.H.S., N.W.), Weill Medical College of Cornell University, New York, New York 10021; Sanofi-Aventis (G.Br., C.S.L), 31100 Toulouse, France; Institut Cochin (M.A.V.), Département d’Endocrinologie Metabolisme et Cancer, Université Paris Descartes, CNRS, UMR 8104, 75016 Paris, France; and INSERM (M.A.V.), Unité 567, 75012 Paris, France

Address all correspondence and requests for reprints to: Gilles Guillon, Institut de Génomique Fonctionnelle, 141 rue de la Cardonille, 34094 Montpellier Cedex 05, France. E-mail: gilles.guillon{at}igf.cnrs.fr.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Recently, we synthesized and characterized the first selective V1b vasopressin (VP)/oxytocin receptor agonist, d[Cha4]arginine vasopressin. However, this agonist was only selective for the human receptors. We thus decided to design a selective V1b agonist for the rodent species. We started from previous observations showing that modifying [deamino1,Arg8]VP in positions 4 and 8 altered the rat VP/oxytocin receptor selectivity. We synthesized a series of 13 [deamino1,Arg8]VP analogs modified in positions 4 and 8. Among them, one seemed very promising, d[Leu4, Lys8]VP. In this paper, we describe its pharmacological and physiological properties. This analog exhibited a nanomolar affinity for the rat, human, and mouse V1b VP receptors and a strong V1b selectivity for the rat species. On AtT20 cells stably transfected with the rat V1b receptor, d[Leu4, Lys8]VP behaved as a full agonist on both phospholipase C and MAPK assays. Additional experiments revealed its ability to induce the internalization of enhanced green fluorescent protein-tagged human and mouse V1b receptors as expected for a full agonist. Additional physiological experiments were performed to further confirm the selectivity of this peptide. Its antidiuretic, vasopressor, and in vitro oxytocic activities were weak compared with those of VP. In contrast, used at low doses, its efficiency to stimulate adrenocorticotropin or insulin release from mouse pituitary or perfused rat pancreas, respectively, was similar to that obtained with VP. In conclusion, d[Leu4, Lys8]VP is the first selective agonist available for the rat V1b VP receptor. It will allow a better understanding of V1b receptor-mediated effects in rodents.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
VASOPRESSIN (VP) IS A nonapeptide synthesized in the hypothalamus and responsible for various physiological effects in mammals. In vivo, it regulates blood pressure and diuresis but also, in synergism with CRH, the release of ACTH and thus ensures the control of the hypothalamo-pituitary-adrenal axis (for review, see Refs. 1, 2, 3).

Besides these well-known physiological effects, VP also controls the release of many hormones: insulin (4), glucagon (5), cortisol and aldosterone (6), neurotransmitters (7), and catecholamines (8) and regulates several behavioral functions, such as stress and anxiety (9) but also learning and memory processes (10). VP may also stimulate cell proliferation (11, 12) and neuronal maturation (13).

All of these diverse functions are triggered by three distinct VP receptor (R) subtypes named V1a, V1b (or V3), and V2 that are directly coupled to distinct second-messenger pathways: phospholipase C (PLC) and MAPK activation for V1a-R and V1b-R, and adenylyl cyclase and MAPK activation for the V2 subtype (for review, see Refs. 1 , 2 , 14). These three receptors, which belong to the G protein-coupled receptor (GPCR) family, have been sequenced and cloned (15) and exhibit strong sequence similarities but different pharmacological profiles for a series of peptidic or nonpeptidic analogs. These molecules have been widely used by the physiologists to ascribe a specific function to each VP-R subtype but also by the physicians for human therapeutic use (for review, see Refs. 2 , 16). Thus, antagonists of VP exhibiting a selective V1a, V1b, or V2 profile are the subject of a variety of clinical trials for many of them (for review, see Ref. 17). SR49059 ((2S) 1-[(2R 3S)-5-chloro-3(2-chlorophenyl)-1-(3,4-dimethoxybenzene-sulfonyl)-3-hydroxy-2,3-dihydro-1H-indole-2-carbonyl]-pyrrolidine-2-carboxamide), a selective nonpeptidic antagonist for human (h) V1a-R, showed potential clinical interest for the treatment of dysmenorrhea, preterm labor, and Raynaud’s disease. SR121463A (1-[4-(N-tert-butylcarbamoyl)-2-methoxybenzene- sulfonyl]-5-ethoxy-3-spiro-[4-(2-morpho-linoethoxy)cyclohexane]indol-2-one, fumarate), a selective V2 antagonist that possesses powerful oral aquaretic properties without affecting electrolyte balance in animals and humans could be of interest in several water-retaining diseases, such as the syndrome of inappropriate antidiuretic hormone secretion, liver cirrhosis, congestive heart failure, and dilutional hyponatremia (for review, see Ref. 18). SSR149415 ((2S,4R)-1-[5-chloro-1-[(2,4-dimethoxyphenyl)sulfonyl]-3-(2-methoxy-phenyl)-2-oxo-2,3-dihydro-1H-indol-3-yl]-4-hydroxy-N,N-dimethyl-2-pyrrolidine carboxamide), a V1b antagonist, belongs to a class of drugs with expected therapeutic interest mainly in various emotional diseases, such as stress-related disorders, anxiety, and depression (19, 20).

The panoply of VP-selective agonists or antagonists available is still incomplete (for review, see Ref. 16). This is mainly attributable to 1) the number of VP-R subtypes concerned (V1a-R, V1b-R, and V2-R) to which we generally include the closely related oxytocin (OT)-R because of its strong amino acid sequence homology with the VP-R family and its excellent affinity for AVP, the natural hormone of V1a-R, V1b-R, and V2-R (21) and 2) the different pharmacological profiles observed for the VP/OT-Rs from various species. Numerous examples of marked species differences have been reported previously (22, 23).

Recently, we synthesized the first selective V1b agonist d[Cha4]arginine vasopressin (d[Cha4]AVP) (24). This peptide exhibits a nanomolar affinity for all of the V1b-Rs tested whatever the species considered but was found to be selective only for the hV1b-R and bovine V1b-R. At variance with the hV2-R or bovine V2-R, this compound has a good affinity for the rat (r) V2-R and was a potent V2 agonist in the rat antidiuretic assay (24). Thus, it could not be considered as a rat-specific V1b agonist. To design a more selective peptide, we started from our previous studies showing that replacing the glutamine residue in position 4 of [deamino1,Arg8]AVP (dAVP) by a hydrophobic residue of at least four carbons (cyclohexylalanine or leucine) increased the V1a or OT selectivity (25) and from a study by Dyckes et al. showing that d[Leu4,Lys8]VP has weak antidiuretic and vasopressor activities (26). Such observations suggest that modifications of dAVP in positions 4 and 8 may lead to rV1b-selective analogs. We thus synthesized 13 analogs modified in positions 4 and 8 and determined their binding properties (27). On the basis of these data, four of them exhibited a good V1b selectivity and a nanomolar affinity for this receptor subtype.

In this paper, we characterize both the functional and physiological properties of the most promising of these peptides, d[Leu4,Lys8]VP. Extensive characterizations of this VP analog reported here demonstrate that it is in fact the first selective agonist available for the rV1b-R described so far.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Chemicals
Most standard chemicals were purchased from Sigma (St. Louis, MO), Roche Molecular Biochemicals (Mannheim, Germany), or Merck (Darmstadt, Germany), unless otherwise indicated. AVP and OT came from Bachem (Bubendorf, Switzerland). Fetal calf serum came from Sigma (Saint-Quentin Fallavier, France). Nu-serum came from BD Biosciences (via Ozyme, Saint-Quentin en Yvelines, France) and FetalClone III from Hyclone (via Perbio, Brévières, France). [3H]AVP (60–80 Ci/mmol), [3H]cAMP (31.7 Ci/mmol), myo-[2-3H]inositol, and [{alpha}-32P]ATP (30 Ci/mmol) were from PerkinElmer (Courtaboeuf, France). MEM, DMEM, DMEM/F-12 medium, penicillin-streptomycin, geneticin (G-418), and Lipofectamine Reagent were purchased from Invitrogen (Cergy Pontoise, France). Inositol-free DMEM came from ICN Biochemicals (Orsay, France). Dowex AG1-X8 formate form 200–400 mesh was purchased from Bio-Rad (Munich, Germany). Mouse corticotroph AtT20 cells (CRL-1795) were from American Type Culture Collection (via LGC Promochem, Molsheim, France). Enzymes for molecular cloning were from New England Biolabs (via Ozyme, Saint-Quentin en Yvelines, France). Oligonucleotides were synthesized by Invitrogen.

Membrane preparations from liver, kidney, anterior pituitaries, and lactating mammary glands
Membranes were obtained as described previously (22) from adult Wistar rats (175–200 g) or adult C57BL/6 mice purchased from Janvier (l’Arbresle, France). Male OF1 mice (30–35 g) used for in vivo experiments were purchased from Charles River Laboratories (Saint Aubin Les Elbeufs, France). Animal manipulations were performed according to the recommendations of the French Ethical Committee and under the supervision of authorized investigators. All protocols performed have been approved by the local Animal Care and Use Committee.

DNA constructs
The cDNAs coding for the rV1b-R (28), the hV1b-R (29), and the mouse (m) V1b-R (30) were amplified by PCR, inserted into the HindIII-XbaI (rat and human) or EcoRI-XbaI (mouse) sites of the expression vector pcDNA3, and named pcDNA–rV3, pcDNA–hV3, and pcDNA–mV3, respectively. The constructs were verified by restriction enzyme digestion and DNA sequencing.

Cell culture, stable transfection, and membrane preparation
CHO cell lines stably transfected with the hV1a-R, hV1b-R, hV2-R, and hOT-R or with the rOT-R and WRK1 cells naturally expressing the rV1a-R were grown as previously described (24, 31). AtT20 wild-type cells were transfected with the rV1b-R, hV1b-R, or mV1b-R using the pcDNA-rV3, -hV3, or -mV3 plasmids or with the C-terminal enhanced green fluorescent protein (EGFP)-tagged version of the hV1b-R, named phV3–EGFP (32) using the Lipofectamine Plus Reagent according to the instructions of the manufacturer. Cells stably expressing the wild-type receptors were selected with geneticin (G-418). The clones were screened by [3H]AVP binding assay and purified by the limited dilution technique. The Kd and Bmax of the clones selected for this study for [3H]AVP were 0.29 nM and 2.08 pmol/mg protein for the rat, 0.95 nM and 3.35 pmol/mg protein for the human and 0.55 nM and 8.85 pmol/mg protein for the mV1b-R isoforms, respectively. Cells stably expressing phV3–EGFP were selected with geneticin and purified by fluorescence-activated cell sorting, and the clones were screened by [3H]AVP binding assay. The EGFP tag did not alter the affinity of the hV1b-R, because the Kd measured in membrane preparations using [3H]AVP as ligand was in the range of that of the wild type, 0.23 vs. 0.95 nM. The Bmax of the clone selected for these experiments was 0.69 pmol/mg protein. The cell line expressing the C-terminal EGFP-tagged version of the mV1b-R was described by C. Serradeil-le Gal (unpublished observations). The cells were cultured in DMEM/F12 supplemented with 7.5% Fetalclone, 7.5% Nu-serum, 0.5 mM glutamine, and 0.2 mg/ml geneticin at 37 C in a 5% CO2 atmosphere. AtT20 cell membranes were prepared as described previously for CHO cell lines (see above).

Binding assays
Membrane incubations with VP radioligands were performed as described previously (24). Briefly, 1–20 µg membrane protein were incubated 60 min at 30 C (membranes from CHO or AtT20 cells) or 37 C (membranes from native tissues) in a medium containing the following: 50 mM Tris-HCI (pH 7.4), 3 mM MgCl2, 1 mg/ml BSA, and 0.01 mg/liter leupeptin. [3H]AVP at 0.5–3 nM was added in the incubation medium depending on the receptors studied with (nonspecific binding) or without (total binding) 1 µM unlabeled AVP and increasing amounts of the unlabeled analogs to be tested. Radioactivity found associated with plasma membranes was determined by filtration through GF/C filters as described previously (24). Specific binding was calculated in each condition and expressed as percentage of the specific binding determined without unlabeled analog.

Inositol phosphate assays
Inositol phosphate (InsP) accumulation was determined as described previously (24, 31). Briefly, CHO or AtT20 cells stably transfected with VP/OT-Rs, or WRK1 cells that naturally expressed the rV1a-R were plated at 100,000 cells per well. Cells were grown for 24 h in their respective culture medium (see above) and further incubated for another 24 h period in a serum and inositol-free medium supplemented with 1 µCi/ml myo-[2-3H]inositol. Cells were then washed twice with an Hank’s buffered saline (HBS) medium, incubated for 15 min in this medium supplemented with 20 mM LiCl, and further stimulated for 15 min with increasing concentrations of analogs to be tested. Reaction was stopped by adding perchloric acid (5% vol/vol). Total InsPs accumulated were extracted and purified on Dowex AGI-X8 anion exchange chromatography column as described previously (24) and counted.

Adenylyl cyclase assays
Adenylyl cyclase activity was assessed as described previously on rat kidney plasma membranes by measuring the conversion of [{alpha}-32P]ATP to [{alpha}-32P]cAMP (24). Briefly, membranes were preincubated for 15 min at 37 C in a 200 µl reaction volume containing the following: 50 mM Tris-HCl (pH 7.4), 3 mM MgCl2, 1 µM GTP, 100 nM free-Ca2+, 0.1 mM cAMP, 0.1 mM ATP, 0.25 mg/ml creatine kinase, and 1.3 mg/ml creatine phosphate, with or without the compounds to be tested. [{alpha}-32P]ATP at 1 µCi per assay was then added in the incubation medium for an additional 6-min period. [3H]cAMP (10,000 cpm) was also added in each sample and used as an internal standard to measure overall recovery. cAMP levels were determined by measuring the formation of [{alpha}-32P]cAMP from the prelabeled [{alpha}-32P]ATP. Labeled cAMP and ATP were separated by sequential chromatography on Dowex and alumina columns. [32P] and [3H] radioactivity present in the cAMP fractions were determined. [{alpha}-32P]cAMP content was normalized according to the recovery of exogenous [3H]cAMP added to each sample.

MAPK assays
AtT20 cells (250,000 cells per dish) stably transfected with the rV1b-R were seeded into 24-well culture plates and cultured for 48 h in a DMEM/F12 medium supplemented with serum, which was removed for the last 18 h. The cells were then incubated for 3 min at 37 C in a serum-free DMEM/F12 medium containing various concentrations of the analog to be tested. Reaction was stopped by aspirating the medium and adding 160 µl Laemmli’s solution (Tris-HCl 50 mM, 6% glycerol, 4% SDS, 1% ß-mercaptoethanol, and 0.2% bromophenol blue). Lysed cells were sonicated three times for 15 sec and centrifuged for 10 min at 18,000 x g. Proteins were transferred onto a nitrocellulose sheet, and Western blot analysis was performed by using a 1:2000 dilution of mouse monoclonal antiphospho-ERK1/2 antibody (phospho-p44/42 MAPK Thr202/Tyr204; Cell Signaling Technology, Beverly, MA). After stripping, total ERK proteins was determined by using a 1:750 dilution of anti-ERK2 (C14) (sc-154 antibody; Santa Cruz Biotechnology, Santa Cruz, CA). Blots were then probed with antimouse (goat antimouse IgG HRP, sc-2005; Santa Cruz Biotechnology) or antirabbit (enhanced chemiluminescence rabbit IgG, HRP linked whole antibody; GE Healthcare, Little Chalfont, UK) secondary antibodies and visualized by using enhanced chemiluminescence detection reagent (GE Healthcare). Unsaturated films were scanned, and the intensity of each band was determined using the NIH Image J program. The ERK phosphorylation signal, normalized in each experimental condition to the relative amount of corresponding total ERK, was expressed as percentage of the normalized ERK phosphorylation signal obtained for 100 nM AVP.

Time-lapse confocal video microscopy
Cells expressing the human or mouse EGFP-tagged V1b-R were grown for 48 h in Labtek (Nalge Nunc, Rochester, NY) chambered cover glasses. The day of the experiment, the culture medium was replaced by DMEM/F12 medium without phenol red, containing 15 mM HEPES and 0.2% dialyzed BSA. Fluorescence was detected using a Leica (Nussloch, Germany) TCS SP2 AOBS confocal microscope with a thermostated chamber at 37 C. Images were collected every 30 sec for 15 min, and the drugs were added to the cells directly on the stage of the microscope when required.

Pancreas perfusion and insulin release measurement
Experiments were performed with Wistar rats fed ad libitum and weighing 320–350 g. The surgical procedure for the isolated perfused rat pancreas has already been described (33). After total isolation from all neighboring tissues, the pancreas was perfused at 37.5 C through its own arterial system and at a constant pressure with a Krebs-Ringer bicarbonate buffer containing 2 g/liter BSA (fraction V) and 8.3 mM glucose. Medium was continuously gassed with O2/CO2 (95/5%) leading to a pH of 7.4. The pressure of perfusion was selected to give a flow rate of 2.5 ml/min during the first 30 min of the stabilization period. The pancreatic flow rate was measured by collecting and measuring pancreas effluent every minute to detect any change in pancreatic vascular bed resistance. Aliquots of perfusate were immediately frozen for insulin determination. AVP or d[Leu4,Lys8]VP were added in the perfusion medium for 30 min after a 45 min stabilization period. In some experiments, VP-selective antagonists were added in the perfusion medium 15 min before agonist addition. Insulin was determined by RIA as reported previously (34) using charcoal separation, an antiporcine insulin antibody (ICN Biochemicals, Orsay, France), and rat insulin as standard (Novo Nordisk, Bagsværd, Denmark).

Bioassays
d[Leu4,Lys8]VP was assayed in the rat antidiuretic assay, rat vasopressor assay, and in vitro rat oxytocic assay using the four-point assay design (35). All experimental procedures were approved by the Institutional Committee for the Care and Use of Animals at Weill Medical College of Cornell University. Synthetic AVP and OT, which had been standardized in vasopressor and oxytocic units against the United States Pharmacopeia Posterior Pituitary Reference Standard, were used as working standards in all bioassays. Antidiuretic assays were performed on water-loaded rats under ethanol anesthesia as described by Sawyer et al. (36). Vasopressor assays were performed on urethane-anesthetized and phenoxybenzamine-treated rats as described by Dekanski (37). Oxytocic assays were performed on isolated uteri from diethylstilbestrol-primed rats in a Mg2+-free van Dyke-Hasting’s solution (38). When SEs are presented in the tables, the means reflect results from at least four independent assay groups.

In vivo assay for ACTH release
In vivo plasma ACTH measurements were performed in conscious mice (five to seven animals per group) as described previously (19). In a first set of experiments, the dose effect of d[Leu4, Lys8]VP (0.03, 0.1, and 0.3 µg/kg iv) on ACTH secretion was studied compared with AVP (0.3 µg/kg iv). In a second set of studies, mice were pretreated either with the vehicle (5% dimethylsulfoxide, 5% cremophor in saline) or SSR149415 [30 mg/kg orally (po)] 2 h before the d[Leu4, Lys8]VP (0.3 µg/kg iv) challenge. Ten minutes after agonist administration, animals were killed and blood samples of 0.5 ml were collected on EDTA (1 mg/ml final concentration). After centrifugation (760 x g for 10 min, 4 C), plasma was collected and stored as aliquots at – 20 C until ACTH measurements by RIA (DiaSorin, Stillwater, MN) as described previously (19).

Data analysis
The radioligand binding data were analyzed with Prism (GraphPad Software, San Diego, CA). The inhibitory dissociation constants (Ki) for unlabeled AVP analogs were calculated from binding competition experiments according to the following Cheng and Prusoff equation: Ki = IC50(1 + [L]/Kd), where IC50 is the concentration of unlabeled analog leading to half-maximal inhibition of specific binding, [L] is the concentration of the radioligand present in the assay, and Kd is its affinity for the VP-R studied. Concentration of VP analogs leading to half-maximal stimulation of second-messenger accumulation (Kact) was calculated from functional studies using Prism. Results are expressed as the mean ± SEM of the number of distinct experiments indicated.

Statistical differences were assessed by paired and unpaired two-tailed Student’s t test. Differences were considered significant when P < 0.05. Statistical analysis for in vivo ACTH secretion was performed using a one-way ANOVA followed by a Dunnett’s test, and the level of significance was taken as *, P < 0.05 or **, P < 0.01.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Synthesis of d[Leu4,Lys8]VP
d[Leu4,Lys8]VP (26) was resynthesized by the Merrifield solid-phase method (39), following previously described procedures (25, 27). The purity of this peptide, assayed by HPLC, was 99.0%. Its molecular weight determined by mass spectrometry was 1026.0, a value very close to the calculated theoretical value (1026.3).

Binding properties of d[Leu4,Lys8]VP for VP-R and OT-R
On plasma membranes derived from AtT20 cells stably transfected with the rV1b-R, d[Leu4,Lys8]VP fully inhibited [3H]AVP binding in a dose-dependant manner (Fig. 1AGo) with a Ki of 0.16 nM (Table 1Go). When similar experiments were performed on crude plasma membranes derived from rat tissues expressing the native V2-R, V1a-R, or OT-R subtype (kidney, liver, or lactating mammary gland, respectively), d[Leu4,Lys8]VP was found to be less efficient than AVP or OT (Table 1Go). Binding experiments were also performed on crude plasma membranes from rat pituitary preparations known to naturally express the rV1b-R. The Ki value obtained for d[Leu4,Lys8]VP on these membranes was also in the nanomolar range. Whatever the affinity of d[Leu4,Lys8]VP chosen as reference for calculating the selectivity index (AtT20 or pituitary membrane preparation), this analog is found selective for the rV1b-R (normalized selectivity index higher than 176 for other rVP/OT-Rs) (Table 1Go). Similar experiments were also performed on mVP/OT-Rs. As for rat, d[Leu4,Lys8]VP exhibited a nanomolar affinity for the mV1b-R but seemed less selective for this receptor isoform (normalized selectivity index for the rOT-R of 10) (Table 1Go). The pharmacological properties of d[Leu4,Lys8]VP for hVP/OT-Rs were quite different from those determined in the rat and mouse. It exhibited a very good affinity for the hV1b-R (Ki of 0.52 nM), an intermediate affinity for the hOT-R and the hV1a-R, and a much lower affinity for the hV2-R (Table 1Go).


Figure 1
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  		FIG. 1. Binding properties of d[Leu4,Lys8]VP for rVP/OT-R and hVP/OT-R. Plasma membranes obtained from stably transfected AtT20 cells expressing rV1b-R, rat liver, kidney, and lactating mammary gland naturally expressing V1a-R, V2-R, and OT-R, respectively (A), and from CHO cells stably expressing hVP/OT-Rs (B) were incubated with 0.5–1.2 nM [3H]AVP in the presence or absence (C) of increasing concentrations of d[Leu4,Lys8]VP as described in Materials and Methods. Nonspecific and total binding were determined with or without 1 µM unlabeled AVP. Results, expressed as percentage of the maximal control specific binding, are the mean ± SEM of at least three independent experiments each performed in triplicate.

 

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  		TABLE 1. Affinity of d[Leu4,Lys8]VP for vasopressin and oxytocin receptors

 
Effects of d[Leu4,Lys8]VP on cAMP and IP accumulation in intact cells expressing VP-Rs or OT-Rs
As illustrated in Fig. 2AGo, d[Leu4,Lys8]VP dose-dependently stimulated total InsP accumulation in AtT20 cells stably transfected with the rV1b-R. Maximal efficiency was significantly higher than that obtained with AVP (P < 0.05). As summarized in Table 2Go, the concentration of d[Leu4, Lys8]VP leading to half-maximal IP accumulation (Kact) was slightly lower than that of AVP. As illustrated in Fig. 2BGo, cell preincubation with increasing doses of SSR149415, a selective V1b antagonist (19), fully and dose dependently inhibited d[Leu4,Lys8]VP-stimulated InsP accumulation. The Ki of SSR149415 deduced from this experiment was well correlated with its affinity for the rV1b-R (Ki of 3.4 ± 0.8 nM; n = 4). Similar results were obtained using CHO cells stably transfected with the hV1b-R (Fig. 2CGo) (our unpublished results).


Figure 2
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  		FIG. 2. Agonist properties of d[Leu4,Lys8]VP on V1b-stimulated PLC activity. AtT20 cells stably transfected with the rV1b-R and CHO cells stably transfected with the hV1b-R were prelabeled with myo-[2-3H]inositol. In A and C, cells were further incubated 15 min at 37 C in an HBS medium with or without (C) increasing concentrations of d[Leu4,Lys8]VP. In B, cells were preincubated 15 min at 37 C with or without (C) increasing amounts of SSR149415 and further stimulated for another 15 min with 1 nM d[Leu4,Lys8]VP. InsP accumulation was measured as described in Materials and Methods. Results, expressed as percentage of the maximal second-messenger accumulation induced by 0.1 µM AVP (A and C) or 1 nM d[Leu4,Lys8]VP in the absence of SSR149415 (B), are the mean ± SEM of at least three independent experiments each performed in triplicate.

 

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  		TABLE 2. Functional and physiological properties of d[Leu4,Lys8]VP for rat vasopressin and oxytocin receptors

 
The functional selectivity of d[Leu4,Lys8]VP for the rV1b-R was further examined by studying its effects on cells or tissues expressing the rV1a-R, rV2-R, and rOT-R subtypes. As illustrated in Fig. 3AGo, d[Leu4,Lys8]VP was a full V1a agonist on WRK1 cells because it maximally stimulated InsP accumulation, as did AVP. However, its Kact was found 1800-fold higher compared with that exhibited by AVP (Table 2Go). For the rOT-R or rV2-R, d[Leu4,Lys8]VP stimulated InsPs or cAMP accumulation with much lower efficiency than AVP (only 32 and 76% of maximal effect obtained with 100 nM AVP, respectively) (Fig. 3Go, B and C). Moreover, the Kact found for these two receptors were higher than 100 nM (Table 2Go).


Figure 3
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  		FIG. 3. Agonist properties of d[Leu4,Lys8]VP for rV1a-R, rV2-R, and rOT-R. WRK1 cells expressing the rV1a-R or CHO cells expressing the rOT-R were prelabeled with myo-[2-3H]inositol and further incubated at 37 C in an HBS medium with or without (C) increasing concentrations of d[Leu4,Lys8]VP, [Thr4,Gly7]OT, or AVP. Rat kidney membranes expressing the V2-R were incubated with [{alpha}-32P]ATP in the presence of increasing amounts of AVP or d[Leu4,Lys8]VP or with vehicle (C). Second messengers accumulated (InsPs for WRK1 and CHO cells or cAMP for rat kidney) were measured as described in Materials and Methods. Results, expressed as percentage of the maximal second-messenger accumulation induced by 0.1 µM AVP, are the mean ± SEM of at least three independent experiments each performed in triplicate.

 
Effects of d[Leu4,Lys8]VP on MAPK activity in AtT20 cells expressing V1b-Rs
The rate of ERK1/2 phosphorylation on d[Leu4,Lys8]VP stimulation was measured in AtT20 cells stably transfected with the rV1b-R. As illustrated in Fig. 4AGo, this peptide dose dependently stimulated the phosphorylation of p42 and p44 proteins revealed using an antiphospho-ERK1/2 antibody. Quantification of these Western blots indicated that this effect was saturable. The Kact deduced for d[Leu4,Lys8]VP was 5.6-fold lower than that found for AVP, and maximal MAPK stimulation was found similar to that obtained with maximal doses of AVP (Table 2Go). Stimulation of MAPK by 10 nM d[Leu4, Lys8]VP was fully blocked by preincubation of the cells with 100 nM SSR149415 (Fig. 4BGo).


Figure 4
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  		FIG. 4. Agonist properties of d[Leu4,Lys8]VP on V1b-stimulated MAPK activity. A, AtT20 cells stably transfected with the rV1b-R were incubated at 37 C for 3 min in serum-free DMEM/F12 medium with or without increasing concentrations of AVP or d[Leu4,Lys8]VP. Reaction was stopped by adding Laemmli’s buffer, and samples were submitted to SDS-PAGE. Western blots were first revealed using a phospho-p44/42 MAPK antibody. Blots were then stripped and further revealed using an ERK1/2 antibody. Quantification of MAPK phosphorylation experiments was performed as described in Materials and Methods. B, AtT20 cells were preincubated for 15 min at 37 C with or without increasing amounts of SSR149415 and further incubated 3 min with or without 10 nM d[Leu4,Lys8]VP. The degree of MAPK phosphorylation was quantified by Western blot analysis as described above. Results, expressed as percentage of the maximal phosphorylation induced by 0.1 µM AVP, are the mean ± SEM of at least three independent experiments each performed in duplicate.

 
Effect of d[Leu4,Lys8]VP on hV1b-R internalization
It is well known that stimulation of a GPCR by an agonist rapidly induces its internalization by endocytosis (40). To further document this property, we took advantage of AtT20 cell lines that stably express the hV1b-R and mV1b-R tagged with the fluorescent protein EGFP at their C-terminus. As shown in Fig. 5Go, under basal conditions, both hV1b-R and mV1b-R were almost exclusively located at the plasma membrane. After a 6 min stimulation period with 100 nM d[Leu4,Lys8]VP or AVP, a rapid internalization of both V1b-R was observed, as shown by the decrease of the fluorescent signal from the plasma membrane and the appearance of a punctate signal in intracellular compartments. These effects were fully prevented when the cells were preincubated with 1 µM SSR149415 before d[Leu4,Lys8]VP or AVP stimulation.


Figure 5
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  		FIG. 5. d[Leu4,Lys8]VP-induced hV1b-R internalization. AtT20 cells stably expressing the EGFP-tagged hV1b-R (top rows) or mV1b-R (bottom rows) were grown at 37 C in Labtek chambered cover glasses. Images were acquired every 30 sec using a confocal microscope. Left columns, The images shown were captured just before and 6 min after addition of either 100 nM AVP or d[Leu4,Lys8]VP as indicated. Right columns, The images shown were captured just before and 3 min after addition of 1 mM SSR149415. Then, AVP or d[Leu4,Lys8]VP were added as indicated, and the images were captured 6 min later.

 
Effects of d[Leu4,Lys8]VP on rat pancreatic functions
The isolated perfused pancreas model preserves the anatomical integrity of the islet of Langherhans and the pancreatic vessels. Thus, we tested the effects of d[Leu4,Lys8]VP both on insulin secretion and pancreatic perfusion flow rate and compared these effects with those obtained with AVP. As illustrated on Fig. 6AGo, 1 nM AVP induced an immediate, strong and sustained decrease in pancreatic flow rate (55 ± 8% inhibition of basal pancreatic flow rate measured 2 min after AVP infusion; n = 4). This effect was mediated via a V1a-R because it was completely blocked by a preinfusion with 30 nM SR49059, a selective V1a antagonist (Fig. 6AGo), and not by 30 nM SSR149415, a selective V1b antagonist (data not shown). Added alone, 1 nM AVP did not stimulate insulin release. Such secretion was only observed in the presence of 30 nM SR49059 (Fig. 6BGo). Moreover, in the presence of 50 nM SSR149415 plus 30 nM SR49059, the secretory response was abolished (data not shown). When similar experiments were performed with 1 nM d[Leu4,Lys8]VP, no modification of pancreatic blood flow was observed (Fig. 6CGo), yet this analog alone induced insulin release (Fig. 6DGo). If the pancreas was perfused with 50 nM SSR149415 before adding d[Leu4,Lys8]VP, insulin secretion was completely prevented, in good agreement with a specific V1b-mediated effect (Fig. 6DGo).


Figure 6
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  		FIG. 6. Effects of AVP and d[Leu4,Lys8]VP on pancreatic blood flow and insulin release. Left column, Rat pancreas were preequilibrated at 37 C with ({circ}) or without (bullet) 30 nM SR49059 and further perfused with 1 nM AVP, and pancreas flow rate (A) and insulin secretion (B) were measured. Right column, Rat pancreas were preequilibrated at 37 C with ({triangleup}) or without ({blacktriangleup}) 50 nM SSR149415 and further perfused with 1 nM d[Leu4,Lys8]VP, and pancreas flow rate (C) or insulin release (D) were measured. Results are representative of at least three different experiments.

 
In vivo properties of d[Leu4,Lys8]VP
In vivo experiments were performed to verify the efficiency of d[Leu4,Lys8]VP to increase plasma concentration of circulating ACTH. As illustrated in Fig. 7AGo, iv injection of d[Leu4,Lys8]VP dose dependently increased plasma ACTH concentrations up to 2-fold at the highest dose tested compared with control animals. The effect observed at 0.3 µg/kg d[Leu4,Lys8]VP was similar to that obtained for the same dose of AVP. This stimulation was blunted if animals were pretreated 2 h before by a single oral administration of SSR149415 (30 mg/kg po) (Fig. 7BGo).


Figure 7
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  		FIG. 7. Effect of d[Leu4,Lys8]VP on plasma ACTH secretion in mice. In A, mice were injected with increasing amounts of d[Leu4,Lys8]VP or 0.3 µg/kg AVP as control; in B, animals were pretreated 2 h before by SSR149415 (30 mg/kg po) or vehicle before the d[Leu4,Lys8]VP challenge. Blood samples (0.5 ml) were collected 10 min after agonist injection, and ACTH was determined by RIA as described in Materials and Methods. Results are the mean ± SEM of five to seven animals per group. Statistical analysis was performed using a one-way ANOVA followed by a Dunnett’s test, and the level of significance was taken as *, P < 0.05 or **, P < 0.01 for comparison with the vehicle-treated control group in A or with the d[Leu4,Lys8]VP (0.3 µg/kg)-treated control group in B.

 
To validate the pharmacological specificity of this peptide in vivo and in vitro, we measured its antidiuretic, vasopressor, and in vitro oxytocic activities in classical bioassays as described previously (36, 37, 38). As illustrated in Table 2Go, d[Leu4,Lys8]VP exhibited only 3% of the antidiuretic activity of the natural hormone AVP and was without significant vasopressor or oxytocic activity compared with AVP or OT, respectively.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this paper, we report the pharmacological and physiological properties of d[Leu4,Lys8]VP, an analog of dAVP modified in positions 4 and 8. This peptide had been reported previously to be a weak antidiuretic/vasopressor agonist (26). Its pharmacological properties and its ability to stimulate ACTH release had never been examined before this study.

Previous studies of our group showed that replacement of the glutamine residue in position 4 of dAVP by an aliphatic residue of at least three carbons, such as leucine, isoleucine, or cyclohexylalanine, leads to selective hV1b agonists (25). These new compounds and especially d[Cha4]AVP (24) are not selective for the rV1b-R because they also exhibit a good affinity for the rV2-R isoform and are potent antidiuretic agonists in rat bioassays (23). Because Dyckes et al. showed 30 yr ago that d[Leu4,Lys8]VP had neither antidiuretic nor vasopressor activities (26), we hypothesized that modifying dAVP in positions 4 and 8 might lead to selective rV1b analogs. We synthesized a series of dAVP analogs modified in positions 4 and 8. Among them, d[Leu4,Lys8]VP seemed very promising on the basis of binding experiments (27). This peptide exhibits a nanomolar affinity for the rV1b-R, hV1b-R, and mV1b-R. Concerning its selectivity, once again, this study emphasized the importance of the species for studying VP/OT-R pharmacology. Thus, d[Leu4,Lys8]VP is an excellent rat-selective V1b analog (normalized selectivity index at least > 176) but a weak human- and mouse-selective analog (Table 1Go). In contrast, we showed previously that d[Cha4]AVP is a human but not a rat-selective V1b agonist (23, 25). These subtle structural modifications leading to profound pharmacological profile alterations could be explained by the existence of a few amino acid residues located in the IVth and Vth transmembrane helices, which are different in hVP-R, rVP-R, and mVP-R and in close interaction with the fourth residue of dAVP analogs. Experiments are in progress to demonstrate that such species differences are attributable to different hydrophobic environments of this particular residue (21 and our unpublished data).

SSR149415 was described as a unique selective antagonist for animal (rat, bovine) and hV1b (19) receptors, but recent data reported nanomolar affinity for recombinant hV1b-R and also OT-R in CHO cells (41). In our hands, using [3H]AVP as radioligand, an affinity value (Ki) of 30 ± 8 nM (n = 4) was obtained for the rOT-R vs. a Ki ranging from 0.44 to 1 nM for the natural hormone OT (data not shown). Such results indicate that SSR149415 is a relatively good rat-selective V1b antagonist. However, on the basis of normalized selectivity indexes (Table 1Go), d[Leu4,Lys8]VP remains the best selective pharmacological tool for studying rV1b-R functions.

The characterization of the functional properties of d[Leu4,Lys8]VP on appropriate cellular models remained a complex goal. On rat pituitary cells in primary culture, the percentage of cells naturally expressing the V1b-R is low, and this leads to a weak stimulation of PLC activity (42). Better results were obtained on V1b-R transfected CHO cells (24), but we lost the specificity of a corticotroph cell context. We decided to generate a more suitable cellular model. We chose the AtT20 cell line derived from a murine corticotropic tumor that secretes ACTH (43) but does not express the V1b-R (30), and we established clones that express the rV1b-R isoform. On this cell line, d[Leu4,Lys8]VP behaves as a full V1b agonist. It stimulates PLC activity in a dose-dependent manner with a Kact similar to its Ki determined in binding experiments (Tables 1Go and 2Go). Maximal PLC stimulation obtained with this analog is similar or slightly higher than that observed with AVP. Functional studies concerning VP-R or OT-R show that they can be coupled to different second-messenger pathways via distinct G proteins and also depend on the G protein densities (44) or the agonist or antagonist used (45). Thus, we studied the coupling of the rV1b-R to the MAPK pathway because it has been shown in CHO cells transfected with the hV1b-R that AVP activates phosphorylation of ERK1/2 protein. In AtT20 cells stably transfected with the rV1b-R, d[Leu4,Lys8]VP dose dependently stimulates MAPK phosphorylation with a nanomolar Kact in the range of that found for PLC activation (Table 2Go). Maximal stimulation of this second-messenger pathway was found similar to that observed with the natural hormone. The agonist properties of d[Leu4,Lys8]VP were further confirmed by its ability to induce V1b-R internalization. Like AVP, it rapidly stimulated the internalization of the hV1b–EGFP and mV1b–EGFP receptor expressed in AtT20 cells. The ability of d[Leu4,Lys8]VP to internalize the V1b-R may be related to its stimulating effect on the MAPK pathway because it was described for numerous GPCRs that receptor internalization represents a crucial step for such stimulation (for review, see Ref. 46). However, the precise mechanisms by which V1b-R stimulates MAPK pathways seem complex and may involve different biochemical cascades as reported previously for the OT-R (45). Altogether, on the basis of the in vitro functional tests performed on the AtT20 cell line expressing the V1b-R, we conclude that d[Leu4,Lys8]VP is a full V1b agonist. Additional functional experiments performed on cells or tissues expressing the other rVP/OT-R isoforms confirm its specificity already observed in binding experiments. d[Leu4,Lys8]VP is a partial agonist for rV2-R and OT-R, a full agonist of rV1a-R, but it exhibits a very low Kact for each of these three receptor isoforms.

Pharmacological characterization of signaling molecules in transfected cells is necessary but insufficient to ascertain their specificity or efficiency in vivo. For instance, d[Cha4,Arg8]VP is a selective full V1b agonist in transfected cells expressing the rV1b-R or hV1b-R but behaves as a weak agonist in vivo when measuring ACTH secretion in rat (24). Similarly, AVP and dAVP, which have similar nanomolar affinities for the rV2-R, exhibited significantly different antidiuretic activities in vivo: 332 and 1745 U/mg, respectively (36). Experiments performed on perfused organs or in vivo were conducted to further characterize d[Leu4,Lys8]VP properties. On isolated perfused rat pancreas, AVP alone has no significant effect on insulin release. This is probably attributable to simultaneous and opposite effects of the hormone on V1a-R and V1b-R. AVP induces a strong vasoconstriction effect attributable to the V1a-R subtype present on vascular smooth muscle cells of pancreatic vessels because this effect was completely blocked by SR49059. The presence of this antagonist together with AVP revealed the ability of the natural hormone to induce insulin secretion. This last effect is mediated by a V1b-R, probably located on ß-cells of Langherhans islets, because SSR149415 fully prevents AVP-mediated insulin secretion. Such results confirm previous studies on mouse, rat, and human pancreas indicating the presence of the V1b-R involved in insulin release (4, 47, 48, 49). Experiments performed with d[Leu4,Lys8]VP also validate this assumption. Used at 1 nM, a concentration eliciting a maximal V1b response on both PLC and MAPK bioassays, d[Leu4,Lys8]VP has no significant effect on pancreatic perfusion flow rate at variance with a similar concentration of AVP but is able to stimulate insulin secretion. Such observations confirm the V1a/V1b selectivity of this peptide and are consistent with its low affinity for the rV1a-R and its very low vasopressor activity (Table 2Go). This compound also presents a very weak antidiuretic activity compared with AVP. In contrast, even injected at a low dose (0.13 µg/kg), it induces maximal significant ACTH release similar to that obtained with equivalent doses of AVP. However, the secretory response obtained with d[Leu4,Lys8]VP was more sustained compared with that observed with the natural peptide (data not shown). As expected, this effect is blocked by the V1b antagonist SSR149415. Altogether, these data demonstrate that d[Leu4,Lys8]VP can be considered as a full V1b agonist for in vivo experiments. Its full agonist properties probably arise from improved physicochemical properties yielding a better bioavailability compared with d[Cha4,Arg8]VP, which was described as a partial agonist in vivo (24), yet replacing a cyclohexylalanine residue by a leucine in position 4 reduces the hydrophobicity of the peptide and probably allows its better diffusion within the animal once injected. Similarly, the presence of a deamino cysteine in position 1 prevents its degradation from proteases and increases its efficiency (36).

Knockout experiments represent a good strategy to ascertain the functional role of V1b-Rs (50, 51). However, the absence of a receptor at the first steps of development may be compensated by regulatory mechanisms. The treatment of wild-type animals with V1b-R-specific analogs is a complementary approach, successfully used to discover the antidepressive and anxiolytic properties of SSR149415, the first selective V1b antagonist (19). The selective agonist d[Leu4,Lys8]VP could help V1b-R function studies at least along three axes. First, the identification of V1b-Rs involved in stress and anxiety could be addressed by mapping their central nervous system distribution, because the data based on in situ hybridization or immunohistochemistry using weak selective receptor antibodies (52, 53) are not in good agreement. The use of a radiolabeled V1b agonist or antagonist for autoradiography has failed up to now attributable to their hydrophobic nature (9, 16). The more hydrophilic d[Leu4,Lys8]VP molecule could be a good candidate. Second, the role of V1b-R in insulin secretion was validated recently in mouse by knockout experiments (50) and confirmed in the rat by our results (Fig. 6Go). More interestingly, we show that d[Leu4,Lys8]VP triggers a rapid and transient phase of insulin release. As in human, an alteration of the first phase of insulin secretion was often associated with diabetes type 2 (54), and selective V1b agonists may be considered as potential therapeutic tools for such disease. Third, the controversy concerning the V1a or V1b nature of the VP-Rs involved in the autocontrol of magnocellular VPergic neurons in the supraoptic nucleus could be solved, helping to clarify the control of VP secretion and thus the regulation of water homeostasis (55, 56).

In conclusion, d[Leu4,Lys8]VP is the first selective V1b agonist in the rat suitable for both in vitro and in vivo studies. Its reduced hydrophobicity and excellent selective V1b agonist properties confer to this peptide a promising role to study V1b-regulated physiological functions.


   Acknowledgments
 
The rV1b cDNA was provided by Dr. M. Saito (Japan) to whom we are greatly indebted, as well as to M. C. Gendron (Service de Cytométrie of the Institut Jacques Monod, Paris, France) for the sorting of EGFP-transfected cells, and F. Letourneur from the Sequencing Facility and M. Garfa and P. Bourdoncle from the Confocal Facility of the Institut Cochin. We are thankful to M. Passama for her excellent work in preparing the illustrations, A. Chlebowski for her assistance in the preparation of this manuscript, and M. Desarménien for helpful discussions.


   Footnotes
 
This work was supported by Centre National de la Recherche Scientifique, Institut National de la Santé et de la Recherche Médicale, Universités Montpellier I and II (to A.P., G.G., B.M., G.Be.), Université Paris Descartes (to M.A.V.), National Institutes of Health Grant GM25280 (to M.M.), Basque Country University (Bilboa, Spain) Grant 9/UPV-00042.31015852/2004, and "Secretaria de Estado de Educacion y Universidades" (Madrid, Spain) for the fellowship (to M.T.).

Disclosure Summary: G.Br. and C.S.L.G are employed by Sanofi-Aventis. All other authors have nothing to declare.

First Published Online May 10, 2007

Abbreviations: AVP, Arginine vasopressin; dAVP, [deamino1,Arg8]arginine vasopressin; EGFP, enhanced green fluorescent protein; GPCR, G protein-coupled receptor; h, human; HBS, Hank’s buffered saline; InsP, inositol phosphate; m, mouse; OT, oxytocin; PLC, phospholipase C; po, orally; r, rat; R, receptor; VP, vasopressin.

Received December 5, 2006.

Accepted for publication May 2, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
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