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Endocrinology Vol. 143, No. 12 4655-4664
Copyright © 2002 by The Endocrine Society

ARTICLE

[1-Deamino-4-Cyclohexylalanine] Arginine Vasopressin: A Potent and Specific Agonist for Vasopressin V1b Receptors

S. Derick, L. L. Cheng, M. J. Voirol, S. Stoev, M. Giacomini, N. C. Wo, H. H. Szeto, M. Ben Mimoun, M. Andres, R. C. Gaillard, G. Guillon and M. Manning

Institut National de la Santé et de la Recherche Médicale, Unité 469 (S.D., M.B.M., M.A., G.G.), 34094 Montpellier Cedex 05, France; Medical College of Ohio (L.L.C., S.S., M.M.), Department of Biochemistry and Molecular Biology, Toledo, Ohio 43614; Division d’Endocrinologie, Diabétologie et Métabolisme, Département de Médecine (M.J.V., M.G., R.C.G.), Centre Hospitalier Universitaire Vaudois, CH-1011 Lausanne, Switzerland; and Department of Pharmacology (N.C.W., H.H.S.), Joan and Sanford I. Weill Medical College of Cornell University, New York, New York 10021

Address all correspondence and requests for reprints to: Guillon Gilles, Institut National de la Santé et de la Recherche Médicale, Unité 469, 141 rue de la Cardonille, 34094 Montpellier Cedex 05, France. E-mail: guillon{at}u469.montp.inserm.fr.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
To date, there are no vasopressin (VP) agonists that exhibit a high affinity and selectivity for the VP V1b receptor with respect to the V1a, V2, and oxytocin receptors. In this study, we describe the synthesis and pharmacological properties of [1-deamino-4-cyclohexylalanine] arginine vasopressin (d[Cha4]AVP). Binding experiments performed on various membrane preparations revealed that d[Cha4]AVP exhibits a nanomolar affinity for V1b receptors from various mammalian species (rat, bovine, human). It exhibits high V1b/V1a and V1b/oxytocin selectivity for rat, human, and bovine receptors. Furthermore, it exhibits high V1b/V2 specificity for both bovine and human vasopressin receptors. Functional studies performed on biological models that naturally express V1b receptors indicate that d[Cha4]AVP is an agonist. Like VP, it stimulated basal and corticotropin-releasing factor-stimulated ACTH secretion and basal catecholamine release from rat anterior pituitary and bovine chromaffin cells, respectively. In vivo experiments performed in rat revealed that d[Cha4]AVP was able to stimulate both ACTH and corticosterone secretion and exhibits negligible vasopressor activity. It retains about 30% of the antidiuretic activity of VP. This long-sought selective VP V1b receptor ligand with nanomolar affinity will allow a better understanding of V1b-mediated VP physiological effects and is a promising new tool for V1b receptor structure-function studies.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IN MAMMALS, VASOPRESSIN (VP) is a nonapeptide synthesized by supraoptic and paraventricular hypothalamic neurons. Released in the general circulation and in the hypophysial portal system, it exerts three main physiological actions: vasoconstriction, antidiuresis, and ACTH secretion (1). Besides these well-documented effects, VP also regulates other functions like liver glycogenolysis (1), steroid and catecholamine secretion by the adrenals (2), insulin and glucagon release by the pancreas (3), and heart atrial natriuretic factor secretion (4). VP is also supposed to regulate numerous central functions like synaptic transmission, central control of body temperature, memory, and more recently, anxiety and depression behaviors in the rat (for review see Refs. 5, 6).

All these effects are triggered by specific VP receptors belonging to the G protein-coupled receptor family. Only three distinct isoforms have been described and cloned in numerous mammalian species (for review see Ref. 7). Historically, classification of VP receptors was established in 1978 by Michell on the basis of their second messenger pathways (8). VP receptors from liver positively coupled to phospholipase Cß via {alpha}q/11 heterotrimeric G proteins are called V1 receptors. Renal VP receptors activate adenylyl cyclase (AC) via {alpha}s G protein and are called V2 receptors. They exhibit a pharmacological profile distinct from those of V1 receptors (for review see Ref. 1).

The classification of VP receptor isoforms has been improved by the availability of numerous VP radioligands and VP structural analogs (7, 9). Thus, two distinct V1 receptor subtypes have been characterized on the basis of their affinities for cyclopentamethylene VP antagonists: V1a-R are those responsible for the pressor and the glycogenolytic effects; V1b-R present principally at the pituitary level and induce ACTH secretion (10). At the present time, peptide and nonpeptide agonists or antagonists of V1a and V2 receptors exhibiting a good affinity have been described (7, 9). For example, SR49059 and d[(CH2)5Tyr(Me2)]AVP are both potent and selective V1a antagonists with nanomolar affinities in various species (7). Similarly, SR121463 is the best selective V2 antagonist described, exhibiting a good affinity (7). The design of such selective compounds has led to the development of useful tools for physiological studies and are also of potential therapeutic interest. Thus, several specific non-peptide antagonists of human V2 and V1a receptors are currently in clinical trials (7, 9).

To date, no specific VP agonists with both high affinity and selectivity for the V1b receptor with respect to either V1a or V2 receptors are available. On the basis of in vivo bioassays, Schwartz and collaborators (11) designed a synthetic VP analog modified in position 2: d[D-3-Pal2]AVP. In the rat, this compound is a weak antidiuretic agonist and a weak antagonist of the vasoconstrictor responses. In dissociated ovine pituitary cells, it maximally stimulates ACTH release with the same efficiency but with a reduced affinity compared with VP. Yet, binding studies performed on human VP receptors indicate that this V1b agonist is totally nonselective with respect to V1a receptors (for review see Ref. 7). Very recently, Serradeil le-Gal and collaborators (6) described a novel nonpeptide antagonist, SSR149415, exhibiting a good affinity and selectivity for both human and rat V1b receptors. Using this V1b antagonist, they demonstrated that VP, by acting on central V1b receptors, may control anxiety and depression behaviors in rats.

In this study, we describe both on native and transfected biological models, the binding and functional properties of d[Cha4]AVP, the first specific V1b/V1a agonist in rat, human, and bovine species. This peptide is also the first specific V1b/V2 agonist in human and bovine species. We also present affinity data for the rat, bovine, and human V1a, V1b, V2, and oxytocin (OT) receptors for d[D-3-Pal2]AVP and d[D-Phe2]AVP.


   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 \|[amp ]\| Co., Inc. (Darmstadt, Germany), unless otherwise indicated. Arginine vasopressin (AVP) and OT came from Bachem (Bubendorf, Switzerland). d[D-3-Pal2]AVP, deoxyribonuclease (DNase) IV, and fetal calf serum came from Sigma (Saint-Quentin Fallavier, France). Deamino-AVP (dAVP), [Val4]AVP, and d[Val4]AVP (dVAVP) were synthesized in our laboratory as previously described (12, 13). [3H]-AVP (60–80 Ci/mmol), [3H]-norepinephrine (40–80 Ci/mmol), [3H]cAMP (31.7 Ci/mmol) and {alpha}[32P]ATP (30 Ci/mmol) were from Perkin-Elmer Life Sciences (Courtaboeuf, France). d(CH2)5[Tyr(Me)2, Thr4, Tyr-NH2 9]ornithine vasotocin (OTA) was iodinated in our laboratory to give [125I]-OTA with a specific radioactivity of 2000 Ci/mmol as previously described (14). MEM, DMEM, DMEM/F-12 medium, penicillin-streptomycin, and collagenase type I were purchased from Life Technologies, Inc. (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 Laboratories, Inc. (München, Germany). The synthesis of d[Cha4]AVP and d[D-Phe2]AVP is described below.

Membrane preparations from liver, kidney, and anterior pituitaries
These were obtained as previously described (15) from adult female Wistar rats (175–200 g) purchased from Iffa Credo (l’Arbresle France) and from tissues of young bovines freshly collected in a slaughterhouse. Animal manipulations were performed according to the recommendations of the French Ethical Committee and under the supervision of authorized investigators.

Chinese hamster ovary (CHO) cell culture and membrane preparation
Membranes from CHO cell lines stably transfected with the human V1a, V1b, V2, and OT receptors (hV1a-R, hV1b-R, hV2-R, and hOT-R, respectively) were prepared as previously described (16).

Binding assays
Membrane incubations with VP radioligands were performed as previously described (15). Briefly, 1–20 µg of membrane protein were incubated 60 min at 30 C (membranes from CHO cells) or 37 C (membranes from native tissues) in a medium containing: 50 mM Tris-HCl (pH 7.4), 3 mM MgCl2, 1 mg·ml-1 BSA, 0.01 mg·l-1 leupeptine, and 0.1 mM phenylmethylsulfonylfluoride. A quantity of 0.5–3 nM of [3H]AVP or 150 pM [125I]-OTA were added in the incubation medium for V1a, V1b, V2, or OT receptor measurements, respectively, with (nonspecific binding) or without (total binding) 1 µM of unlabeled AVP or OT and increasing amounts of the unlabeled analogs to be tested. Radioactivity found associated to plasma membranes was determined by filtration through GF/C filters as previously described (15). Specific binding was calculated in each condition and expressed as percent of the maximal binding capacity determined without unlabeled analog.

Chromaffin cells primary culture and catecholamine release assays
Culture of bovine chromaffin cells were cultured according to Wilson and Viveros (17). Briefly, adrenal glands from young bovine animals (1–3 months) were perfused through the adrenal vein with a Locke buffer complemented with 1 mg·ml-1 collagenase I and 0.1 mg·ml-1 DNase IV. The medulla part of the adrenal gland was separated from cortex, minced, and digested in the same Locke/collagenase/DNase medium. Cells subsequently dissociated were purified on a Percoll density gradient and plated on collagen-coated dishes. Cells were maintained in a DMEM/F-12 nutrient medium in a 95%O2/5%CO2 atmosphere and used for experiments 1 d after. The purity of this primary culture (80% of chromaffin cells) was estimated by morphological and functional criteria (calcium mobilization induced by nicotine application).

Catecholamines release was measured as previously described (18). Briefly, chromaffin cells were loaded with 4 µCi·ml-1 [3H]-norepinephrine in the DMEM/F-12 medium complemented with 5 mM ascorbic acid. After a 2- to 4-h loading period, cells were washed in a Hanks’ buffered saline (HBS), further incubated for 6 min at 25 C with or without the analogs to be tested. The incubation medium was collected, and [3H]-norepinephrine release measured (A). Cells were then lysed by addition of 5% trichloroacetic acid to determine cellular [3H]-norepinephrine content (B). Catecholamine release was calculated as follow: [A/(A + B)] x 100.

Culture of rat adenohypophysial cells and ACTH release measurements
Adenohypophysial cells from 12–20 pituitaries were prepared as previously described (10). Briefly, cells were dissociated using a collagenase/dispase medium, washed, and plated at a density of 0.6–0.8 106 cells per well in a culture medium containing a DMEM/F-12/BGJB nutrient medium supplemented with 2 mg·ml-1 BSA, 2.38 mg·ml-1 HEPES, 50 mg·liter-1 gentamycin, 10 mg·liter-1 transferrin, and 2.5% fetal calf serum. After 4 d in primary culture, cells were washed with a serum-free culture medium and then incubated for 3 h in 1 ml of this medium, with or without (control) the analogs to be tested. The medium was then collected and stored frozen at –20 C until ACTH determination. ACTH secretion was determined by RIA as previously published (10).

Inositol phosphate (IP) assays
IP accumulation was determined as previously described (19). Briefly, CHO cells stably transfected with the human V1b-R or WRK1 cells that naturally expressed rat V1a-R (20) were plated at 20,000 cells/dishes. Cells were grown for 48 h in DMEM supplemented with 10% fetal calf serum (CHO cells) and MEM supplemented with 5% fetal calf serum/2% rat serum (WRK1 cells). Cells were further incubated for another 24-h period in a serum and inositol-free medium supplemented with 1 µCi·ml-1 myo-[2-3H]-inositol. Cells were then washed twice with HBS, equilibrated at 37 C in HBS for 30 min, and incubated for 15 min in HBS supplemented with 10 mM LiCl. Cells were then stimulated for 15 min with increasing concentrations of analogs to be tested. Reaction was stopped with 5% (vol/vol) perchloric acid. Total inositol IPs accumulated were extracted and purified on Dowex AG1-X8 anion exchange chromatography column and counted.

Adenylate cyclase assays
Adenylate cyclase activity was assessed as previously described (21) on plasma membranes from rat kidney and was determined by measuring the conversion of {alpha}[32P]-ATP to {alpha}[32P]-cAMP. Briefly, membranes were preincubated for 15 min at 37 C in a 200-µl reaction volume containing: 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-1 creatine kinase, and 1.3 mg·ml-1 creatine phosphate, with or without the compounds to be tested. A quantity of 1 µCi {alpha}[32P]-ATP per assay was then added for a further 6-min period. [3H]-cAMP (10,000 cpm) was 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 in each sample.

In vivo bioassays
The influence of d[Cha4]AVP injections in anesthetized rats was tested on vasopressor and antidiuretic responses and on its ability to stimulate ACTH and corticosterone secretion. Antidiuretic assays were performed on water-loaded rats under anesthesia as described by Sawyer (22). Vasopressor assays were performed on urethane-anesthetized and phenoxybenzamine-treated rats as described by Dekanski (23). Synthetic AVP, which had been standardized in vasopressor units against the USP Posterior Pituitary Reference Standard, was used as a working standard in these two bioassays.

In vivo ACTH and corticosterone secretion measurements were performed as previously described by Aizawa et al. (24) with some minor modifications. Rats were anesthetized with Ketalar (17.5 mg ketamine plus 0.5 mg xylasin/100 g body weight, BW). Analogs to be tested were injected in the jugular vein 1 h after anesthesia. Blood samples (0.3 ml) were collected from the jugular vein just before (0) and at 5, 15, and 30 min after injection of the analogs tested. ACTH and corticosterone secretions were determined by immunoradiometric as previously modified and validated for the rat (25).

Data analysis
The radioligand binding data were analyzed by GraphPad Software, Inc. Prism (GraphPad Software, Inc., San Diego, CA). The inhibitory dissociation constants (Ki) for unlabeled AVP analogs were calculated from binding competition experiments according to the Cheng and Prusoff (25A ) equation: Ki = IC50(1+[L]/KD), where IC50 is the concentration of unlabeled analog leading to half-maximal inhibition of specific binding, [L] the concentration of the radioligand present in the assay, and KD its affinity for the VP receptor studied. Concentrations of VP analog leading to half-maximal stimulation of second messenger accumulations (Kact), were calculated from functional studies using GraphPad Software, Inc. PRISM. Results are expressed as the mean ± SEM of the number of distinct experiments indicated.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Synthesis of d[Cha4]AVP and d[D-Phe2]AVP
The protected precursor required for the synthesis of d[Cha4]AVP was prepared by the manual solid-phase method (26) starting from Boc-Gly-resin, following previously described procedures (13, 27, 28). Successive cycles of deprotection, neutralization, and coupling gave the protected acyloctapeptide resin. Ammonolysis in methanol (13, 27, 28) yielded crude Mpr(Bzl)-Tyr(Bzl)-Phe-Cha-Asn-Cys(Bzl)-Pro-Arg(Tos)-Gly-NH2 (A), (Mpr, 3-mercaptopropionic acid; Bzl, benzyl; Tos, tosyl), which was purified by precipitations from DMF (dimethylformamide)/water and DMF/ether; yield 91.5%, m.p. 219–21 C, [{alpha}]D25 = -34.5 (c = 1, DMF). Thin layer chromatography: rate of flow (RF) (A) = 0.72, RF(B) = 0.67, RF(C) = 0.72. A = 1-butanol/acetic acid/water, 4:1:5 (upper phase); B = 1-butanol/acetic acid/water, 4:1:1; C = 1-butanol/acetic acid/water/pyridine, 15:3:3:10. An aliquot of the protected precursor (A) (150 mg) was reduced with Na/liquid NH3 (29), cyclized with potassium ferricyanide by the reverse procedure (30) and the crude peptide was purified in a two-step procedure involving gel filtration on Sephadex G-15 (31) in 50% acetic acid, and on Sephadex LH-20 in 0.2 N acetic acid as previously described (28). The final lyophilization gave the desired product Mpr-Tyr-Phe-Cha-Asn-Cys-Pro-Arg-Gly-NH2 (d[Cha4]AVP) as a white powder, 28 mg (26.5% yield); [{alpha}]D25 = -96.0° (c = 0.1, 1 N acetic acid), RF(A) = 0.41, RF(B) = 0.34, RF(C) = 0.59. The homogeneity of the product was additionally confirmed by HPLC. Analytical HPLC was performed on a Waters 810 instrument under the following conditions: 90:10 to 30:70 0.05% aqueous TFA (trifluoroacetic acid):0.05% TFA in acetonitrile, linear gradient over 60 min at 1.0 ml/min ({lambda} = 210 nm) on a Microsorb C18 column (Rainin Instrument Co., Inc.); time of retention found 32.9 min. The structure of the peptide was confirmed by electron spray mass spectrometry, which was performed by the University of Oklahoma Health Sciences Center Molecular Biology Resource Facility on a PE Sciex Q-STAR Quadropole TOF Mass Spectrometer using 50:50 MeCN/water with 0.5% AcOH as a solvent. MW calculated 1094.3; MW found 1094.5.

d[D-Phe2]AVP was synthesized as follows. The protected precursor Mpr(Meb)-D-Phe-Phe-Gln-Asn-Cys(Bzl)-Pro-Arg(Tos)-Gly-NH2 (Meb, p-methylbenzyl) of the free peptide d[D-Phe2]AVP was synthesized and purified as described above. Yield 90%, m.p. 192–194 C, [{alpha}]D25 = -21 (c = 1, DMF). RF(A) = 0.65, RF(B) = 0.48, RF(C) = 0.66. Its deprotection, purification, and characterization were as described above for d[Cha4]AVP. Yield 34.5%, [{alpha}]D25 = -108 (c = 0.1, 1 N acetic acid). RF(A) = 0.24, RF(B) = 0.12, RF(C) = 0.26; time of retention = 27.6 min; MW calculated 1053.3, MW found 1053.2.

Rationale for designing selective V1b VP analogs
As illustrated in Table 1Go the affinities of VP for V1a, V1b, and V2 receptors are in a nanomolar range. These values corroborated previous studies (for review see Ref. 7) and validate the binding assays performed in this work. Deamination of AVP at position 1 to give dAVP resulted in a slight gain in selectivity for the rat and human V1b VP receptors (Table 1Go). To increase this selectivity, Schwartz et al. (11) modified dAVP at position 2 to give d[D-3-pyridylalanine2]AVP (d[D-3-Pal2]AVP). This compound was claimed to be relatively specific for mammalian V1b receptors. Yet as described earlier (for review see Ref. 7) and verified in this study for human VP receptors, this compound has a weak affinity for human V1b-R and no V1b/V1a selectivity (Table 1Go). Similar results were obtained for rat V1b receptors isoforms. The Ki of d[D-3-Pal2]AVP was 10-fold higher than that measured for human V1b-R (this study). We had obtained preliminary findings showing that replacement of the Tyr2 residue by a D-Phe2 residue to give d[D-Phe2]AVP resulted in full retention of affinities for the rat V1a, V1b, and V2 receptors (32). In the rat, dAVP and d[D-Phe2]AVP exhibit almost identical pharmacological profiles (Table 1Go). d[D-Phe2]AVP also exhibits very high affinity for the human V1a and V1b receptors (Table 1Go). However, in a striking example of species differences between the rat and human V2 receptors, d[D-Phe2]AVP exhibits a significant reduction in affinity for the human V2 receptor compared with that of dAVP. Thus, for human VP receptors d[D-Phe2]AVP exhibits very high V1b/V2 receptor selectivity but is not selective for human or rat V1b/V1a receptors. In the same preliminary studies (32), we had also found that one of the previously published selective antidiuretic agonists, [Val4]AVP (13) exhibited somewhat higher affinities for the rat and human V1b receptors compared with their respective V1a receptors (32). It should also be noted that this peptide had earlier been shown to exhibit corticotropin-releasing factor (CRF) activity in the rat (24). These findings prompted our examination of the receptor affinities of the previously published selective antidiuretic agonist d[Val4]AVP (13) reported here (Table 2Go). d[Val4]AVP was found to exhibit high affinities for the human and rat V1b receptors, whereas it exhibited diminished affinities for the human and rat V1a receptors. These findings prompted a further investigation of position 4 in dAVP with a variety of aliphatic amino acids (Manning, M., S. Derick, and G. Guillon, unpublished results). These studies led to the discovery of d[Cha4]AVP being reported here.


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  		Table 1. Rationale for V1b selective analogs design

 

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  		Table 2. Binding properties of AVP and dAVP peptides modified in position 4 for mammalian AVP/OT receptor subtypes

 
Binding properties of d[Cha4]AVP on vasopressin and oxytocin receptors
The binding affinities for human, rat and bovine VP and OT receptors are presented in Table 2Go. As shown in Fig. 1Go, whatever the rat VP/OT receptor isoforms considered, d[Cha4]AVP completely displaced [3H]AVP or [125I]-OTA specific binding in a dose-dependent manner. The dose-displacement curves obtained with d[Cha4]AVP could be fitted using the Cheng-Prusoff (25A ) equation suggesting a competitive interaction between radioligands and the unlabeled VP analogs at the same binding site. Dissociation constant values (Ki) deduced from these calculations are summarized in Table 2Go. d[Cha4]AVP exhibited a good affinity for rat V1b receptor (Ki = 2.8 nM). Its pharmacological selectivity for rat V1b receptors vs. V1a or OT receptors was excellent (see Table 2Go) but less pronounced concerning the rat V2 isoform. We also measured the Ki of d[Cha4]AVP for VP receptors from other mammalian species. Values from Table 2Go indicate that d[Cha4]AVP exhibited a good affinity for human and bovine V1b receptors. Moreover, its selectivity for the V1b receptor isoform vs. V1a, V2, or OT receptors was excellent for both of these species.



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  		Figure 1. Binding specificity of d[Cha4]AVP for the different rat vasopressin receptors. Plasma membrane preparations from rat liver, kidney, and anterior pituitary known to express specifically V1a-R, V2-R, V1b-R and from CHO cells expressing rat OT-R were incubated either with [3H]AVP (V1a-R, V2-R, V1b-R) or [125I]-OTA (OT-R) in the presence or absence (control) of increasing amounts of AVP ({circ}), OT ({square}), d[D-3-Pal2]AVP ({blacktriangleup}), or d[Cha4]AVP ({bullet}). Specific binding were calculated in each condition and expressed as percent of corresponding control specific binding (see Materials and Methods). Results are the mean ± SEM of three to seven distinct experiments, each performed in triplicate.

 
Functional properties of d[Cha4]AVP on native biological models and CHO cells expressing V1b receptors
We decided to determine the functional properties of d[Cha4]AVP on rat pituitary cells, which are known to secrete ACTH upon V1b receptor stimulation (10). As shown in Fig. 2AGo, d[Cha4]AVP and AVP dose dependently stimulated ACTH secretion with similar Kact values. The efficiency of d[Cha4]AVP was slightly weaker than AVP (maximal ACTH stimulation observed with d[Cha4]AVP= 83% of maximal AVP effect, n = 5) (Table 3Go). AVP is also known to potentiate CRF-induced ACTH secretion by corticotrophs (33). Thus, we verified if such synergism occurred using our selective V1b agonist. Costimulation of pituitary cells with 0.1 nM CRF and 10 nM d[Cha4]AVP or AVP leads to a larger increase of ACTH secretion than the addition of the one obtained by CRF, d[Cha4]AVP or AVP added separately. This potentiation was 41 ± 7 and 40 ± 10% for d[Cha4]AVP and AVP, respectively (Fig. 2BGo).



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  		Figure 2. In vitro properties of AVP and d[Cha4]AVP on mammalian V1b expressing models. Rat anterior pituitary cells, CHO cells expressing human V1b-R and bovine adrenomedullary cells were cultured as described in methods. For dose-response curves illustrated on panels A, C, and D, cells were incubated without (basal) or with increasing amounts of AVP ({circ}) or d[Cha4]AVP ({bullet}). A, ACTH secretion by rat anterior pituitary cells primary culture was measured by RIA and expressed as percent of basal secretion (100% = 34.8 ± 3.3 ng ACTH/106 cells). B, ACTH secretion by rat anterior pituitary cells primary culture was determined after incubation of the cells with either 0.3 or 10 nM AVP or d[Cha4]AVP in the absence or presence of 0.1 nM CRF. C, Total IPs accumulated by CHO cells expressing human V1bR were determined as described in Materials and Methods and expressed as percent of maximal vasopressin effect (100% = 30.8 ± 1.2-fold stimulation over basal production). D, [3H]-norepinephrine secreted by bovine adrenomedullary cells primary culture were measured and expressed as percent of total [3H]norepinephrine cellular content (see Materials and Methods). Results are expressed as percent of control basal secretion (2.8 ± 0.4%). Results illustrated in panels A, C, and D are the mean ± SEM of three to five distinct experiments, each performed in triplicate. Results from panel B are the mean ± SEM of quadruplicate determination from one experiment representative of three. **, P < 0.025; *, P < 0.05 AVP effect vs. corresponding dose of d[Cha4]AVP.

 

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  		Table 3. Functional properties of AVP and d[Cha4]AVP peptides for mammalian V1b receptors: in vitro studies

 
We also verified the agonist properties of d[Cha4]AVP on CHO cell lines stably transfected with human V1b receptors. Both AVP and d[Cha4]AVP stimulated total IPs accumulation in a dose-dependent manner (Fig. 2CGo). On these cells, d[Cha4]AVP was slightly less efficient than AVP to induce IPs accumulation (Table 3Go) (maximal d[Cha4]AVP–induced IPs accumulation = 88% of maximal AVP effect; n = 4).

As V1b receptors are also expressed in peripheral organs, we decided to validate the properties of d[Cha4]AVP on a native extrapituitary V1b receptor model: the adrenal medulla. We previously demonstrated that V1b receptors are present in rat and human adrenal medulla and involved in catecholamine release (34). Such receptors are also present in bovine adrenomedullary chromaffin cells and modulate the same biological activity (Andrès, M., unpublished results). d[Cha4]AVP, like AVP, dose dependently stimulated catecholamine release, and its maximal activity was 80% (n = 4) of those measured with maximal doses of AVP (Fig. 2DGo and Table 3Go).

Functional properties of d[Cha4]AVP on native biological models expressing rat V1a and V2 vasopressin receptors
To further characterize the functional properties of d[Cha4]AVP on other rat VP receptor isoforms and evaluate its V1b selectivity, we measured its ability to stimulate the V1a and V2 receptors transduction pathways.

On WRK1 cells, a rat mammary tumor cell line that expresses V1a receptors (20), AVP dose dependently stimulated IPs accumulation with a Kact of 0.94 ± 0.01 nM (n = 3) (Fig. 3Go, left panel). By contrast, higher concentrations of d[Cha4]AVP were necessary to induce maximal IPs accumulation. At 10 µM, this analog only stimulated IPs accumulation by 41% compared with VP maximal effect with a Kact of 735 ± 150 nM (n = 3). On rat kidney plasma membranes derived from the medulla, a structure known to express V2 receptors physiologically involved in water reabsorption (21), AVP stimulated AC activity in a dose-dependent manner with a Kact of 0.54 ± 0.20 nM (n = 3) (Fig. 3Go, right panel). In the same experimental conditions, d[Cha4]AVP also stimulated AC activity but with a Kact of 117 ± 15 nM and an efficiency of 86% of VP maximal effect (n = 3).



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  		Figure 3. In vitro properties of AVP and d[Cha4]AVP for rat V1a and V2 receptors. WRK1 cells and membrane fractions from rat kidney medulla naturally expressing V1a-R and V2-R, respectively, were incubated without (basal conditions; B) or with increasing amounts of AVP ({circ}) or d[Cha4]AVP ({bullet}). Total IPs accumulated in WRK1 cells (left panel) and {alpha}[32P]cAMP produced by kidney membrane fraction (right panel) were measured as described in Materials and Methods. In each case, results are expressed as percent of maximal response obtained with 1 µM VP (100% = 14.4 ± 2.6-fold and 2.2 ± 0.1-fold stimulation over basal for IPs and cAMP production, respectively). Results are the mean ± SEM of three distinct experiments, each performed in triplicate.

 
In vivo properties of d[Cha4]AVP in rat
The antidiuretic properties of d[Cha4]AVP was evaluated as described in Materials and Methods. The value measured for d[Cha4]AVP (133 U·mg-1) was 95 and 60% lower than that obtained for its precursor dAVP and for AVP, respectively (Table 4Go).


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  		Table 4. Functional properties of AVP and AVP modified peptides for rat vasopressin receptor subtypes: in vivo studies

 
Similarly, the vasopressor activity of d[Cha4]AVP was measured (see Materials and Methods). Compared with AVP and dAVP, this peptide exhibited drastic reductions in vasopressor activity from around 370 U/mg to 0.067 U/mg.

To evaluate the ability of d[Cha4]AVP to stimulate the hypothalamo-pituitary-adrenal axis, we decided to measure the influence of AVP or d[Cha4]AVP injections on ACTH and corticosterone plasma concentrations. As illustrated in Fig. 4Go, left panel, both AVP and d[Cha4]AVP induced a rapid increase of ACTH concentration. Used at the same dose (1 µg peptide injected/200 g BW), these two peptides that exhibit the same Ki for rat V1b receptor (Table 2Go) and have approximately the same molecular weight did not produce the same maximal effect (maximal d[Cha4]AVP effect = 22% compared with maximal VP effect). By increasing the dose of d[Cha4]AVP injected from 1 to 5 µg/200 g BW, we slightly increased the ACTH secretion (maximal effect = 37%). No further significant increase was observed between 5 and 10 µg d[Cha4]AVP (data not shown). By contrast, the corticosterone responses were found to be similar whichever peptide was tested (7-fold stimulation compared with basal control values) (Fig. 4Go, right panel). Upon AVP or d[Cha4]AVP injections, the kinetics of corticosterone level increase were delayed compared with those of ACTH.



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  		Figure 4. In vivo properties of AVP and d[Cha4]AVP on ACTH and corticosterone secretion. Anesthetized rats were injected with 1 µg AVP ({circ}), 1 µg ({bullet}) or 5 µg ({diamondsuit}) d[Cha4]AVP/200 g BW or vehicle ({square}). Blood samples (0.3 ml) were collected just before (0) or 5, 15, and 30 min and after analog injection of the analogs. ACTH (left panel) and corticosterone (right panel) plasma levels were determined as described in Materials and Methods. In both cases, results are the mean ± SEM of four to nine different animals from three distinct experiments. *, P < 0.01, AVP effects vs. d[Cha4]AVP used at 5 µg.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We report in this study the pharmacological properties of d[Cha4]AVP, a structural analog of dAVP modified at position 4. d[Cha4]AVP is a highly promising new and specific ligand for V1b receptor studies.

The design of VP agonists, which exhibit a good selectivity for rat and human V1b receptors with respect to both the vasopressin V1a and V2 receptors, has proved to be a challenging and an elusive goal. Modification of AVP by deamination of cysteine at position 1 led, on the basis of binding pharmacological tests, to a compound, dAVP, which exhibits some V1b/V1a selectivity for rat and human VP receptors (Table 1Go). Modification of dAVP performed at position 2 led to d[D-Phe2]AVP, exhibiting a nanomolar affinity for the human and rat V1b receptors and a good selectivity vs. the human V2 receptors. Yet, it did not discriminate between rat or human V1a and V1b receptors. Similarly d[D-3-Pal2]AVP, also called Schwartz compound, a VP analog modified in position 2, is not selective for the human or rat V1b receptors (Ref. 7 and this study). Binding studies performed on native rat tissues reported here, reveal that d[D-3-Pal2]AVP exhibits a weak affinity for the rat pituitary receptor and a very low V1b/V1a selectivity (Table 1Go). Thus, as previously described for the human V1b receptor, this compound is not a selective V1b analog. Replacement of the glutamine residue by a valine at position 4 in AVP or in dAVP leads to [Val4]AVP and d[Val4]AVP, analogs, which exhibit a good V1b affinity for rat and human VP receptors and some V1b/V1a selectivity (Table 2Go). Not surprisingly, these previously reported potent antidiuretic agonists (13) exhibit high affinities for the rat and human V2 receptors (Table 2Go). By contrast, our new peptide, d[Cha4]AVP, exhibits high V1b/V1a selectivities for rat, human, and bovine receptors. In all three species, the affinity of this analog for V1b receptors is in the nanomolar range. d[Cha4]AVP behaves like a VP agonist both in biological models which naturally express V1b receptors (rat pituitary and bovine chromaffin cells in primary culture) and on transfected cell lines (CHO stably transfected with the human V1b receptor). Like VP, the concentration of d[Cha4]AVP leading to half-maximal ACTH secretion (Kact) is lower than that necessary to half-occupy V1b receptors (coupling ratio Ki/Kact larger than 10 for both the two peptides). These data appear to indicate that a strong amplification phenomenon occurs between the hormone-receptor interaction and the secretory processes. Such amplification mechanism was previously observed for adrenocortical cells, known to secrete cortisol and corticosterone upon vasopressin stimulation (19). Finally, as previously described for AVP, d[Cha4]AVP is also able to potentiate CRF-stimulated ACTH secretion by rat pituitary cells in primary culture (Ref. 33 and Fig. 2BGo).

As previously observed for other VP structural analogs like DDAVP, dVDAVP, and F-180 (7, 15, 35, 36), the pharmacological properties of d[Cha4]AVP vary from one species to another. Thus, d[Cha4]AVP exhibits a relative good affinity for the rat V2 receptor and a very weak affinity for the human V2 receptor. Thus, in the rat it exhibits reduced V1b/V2 selectivity compared with the human or bovine isoforms (Table 2Go). To further analyze its functional properties, we calculated its Ki/Kact ratio for the rat V1a and V2 receptors. d[Cha4]AVP exhibits a coupling ratio for the rat V1a receptor similar to AVP (3.3 and 2.8 respectively; data from Table 2Go and Fig. 3Go, left panel) suggesting, as previously described, a linear coupling between receptor occupancy and phospholipase Cß activation (37). For the rat V2 receptor, the coupling ratio of AVP is 0.9. By contrast that of d[Cha4]AVP is 10-fold lower, indicating that, at variance with AVP, low doses of d[Cha4]AVP (<10 nM) may interact with V2 receptors without activating AC (compare Figs. 1CGo and 3Go, right panel). d[Cha4]AVP may interact and/or stabilize an intermediate V2 receptor complex not or weakly coupled to heterotrimeric G proteins and thus to AC. This phenomenon has been previously observed for the neurokinin receptor and some specific agonists (Galzi, J. L., Faculté de Pharmacie, Strasbourg, France; personal communication). In summary, on the basis of binding experiments, d[Cha4]AVP is a weak selective V1b/V2 analog for rat VP receptors. It is, however, the first V1b/V1a and V1b/OT selective agonist available for the study of rat VP/OT receptors. For functional studies performed in vitro, this compound is a highly selective V1b agonist (Kact = 0.09, 743, and 117 nM for rat V1b, V1a, and V2 receptors, respectively; Figs. 2AGo and 3Go).

The selectivity of d[Cha4]AVP was further studied in vivo on anesthetized rats. d[Cha4]AVP, despite a weak agonist potency observed in vitro on WRK1 cells, exhibits negligible pressor activity (0.067 U·mg-1) (Table 4Go). This is probably due to its very low affinity for the rat V1a receptor (Ki=2.4 µM; Table 2Go) with respect to the doses of peptide injected. Interestingly, this loss of pressor activity seems due to the nature of the residue in position 4. Substitution of the glutamine residue by a valine at position 4 led to a significant decrease of the pressor activity of dAVP (13); and this effect is more pronounced with a cyclohexylalanine residue (Table 4Go). Similarly, replacement of the glutamine in dAVP by a cyclohexylalanine led to a 95% reduction of the antidiuretic activity (Table 4Go). Yet, compared with the natural hormone AVP, d[Cha4]AVP retains a significant antidiuretic activity: 133 U·mg-1 compared with 332 U·mg-1 for AVP. d[Cha4]AVP also stimulates in vivo ACTH and corticosterone secretion. Compared with AVP, d[Cha4]AVP behaves as a partial agonist for ACTH release (Fig. 4Go, left panel). Such properties have been previously observed on in vitro models but to a lesser extent (Fig. 2AGo and Table 3Go) suggesting that on these models d[Cha4]AVP could be considered as a good V1b receptor agonist. These discrepancies between in vivo and in vitro data are probably due to a weaker bioavailability of d[Cha4]AVP especially at the pituitary level and/or pharmacokinetic differences compared with AVP. Unlike ACTH secretion, d[Cha4]AVP and AVP stimulate corticosterone release with the same maximal efficiency. Such results appear to indicate that only a fraction of ACTH release by the pituitary under hormonal stimulation is necessary to induce a full adrenal response and that AVP and d[Cha4]AVP stimulate the hypothalamo-pituitary-adrenal axis to the same extent. d[Cha4]AVP is thus as efficient that AVP to induce a stress response. Interestingly, modification of dAVP, which exhibits the same CRF activity as AVP (24), at position 4 by cyclohexylalanine, does not alter its CRF activity. This is at variance with the effects of this modification on antidiuretic and vasopressor activities (Table 4Go). The high V1b/V1a selectivity in vivo and in vitro for rat receptors makes d[Cha4]AVP a valuable research tool for discriminating between both receptor isoforms in this species.

Up to now, studies concerning potential new physiological effects of VP mediated by V1b receptors have been hampered by the lack of specific V1b agonists or antagonists. The presence of V1b receptors in various regions of rat brain has been demonstrated using the in situ hybridization method (38) or more recently, with a V1b receptor polyclonal antibody (39). Their physiological involvement remains to be elucidated. Using a new specific V1b antagonist, a major role of VP and central V1b receptors in depression and anxiety has recently been strongly suggested (6). Intracerebroventricular injection of SSR149415 in rat brain was shown to reduce the depressive and anxious behaviors of animals treated with AVP. These striking observations confirmed previous studies obtained in humans and rats. In depressed patients, it was shown that the number of hypothalamic neurons coexpressing VP and CRF is strongly increased (40). This observation seems correlated to the high percentage of depressed patients presenting with high levels of circulating ACTH and corticotrope axis hyperactivity (41). Moreover, VP plasma concentration is strongly increased in this disease (42). Similarly, in the rat, induced-chronic stress increases the circulating level of ACTH and corticosterone and also the density of pituitary VP receptors (43, 44). The availability of the selective V1b ligand d[Cha4]AVP as a new pharmacological tool now makes possible the study of these physiological effects in the rat.

In conclusion, our studies demonstrate that d[Cha4]AVP is the first specific VP agonist that exhibits a nanomolar affinity for mammalian V1b receptors. Furthermore, it exhibits a good V1b/V1a selectivity for the rat VP receptors and excellent V1b/V1a and V1b/V2 selectivities for the human and bovine VP receptors. d[Cha4]AVP is thus a promising new pharmacological tool. Together with the recently reported nonpeptide selective V1b antagonist SSR149415 (6), d[Cha4]AVP could provide important new insights for V1b receptor structure/function investigations and may lead to a better understanding of V1b-mediated vasopressin actions, especially in the field of depression.


   Acknowledgments
 
We thank Ms. Suzanne Payne and Ms. Ann Chlebowski for their expert assistance in the preparation of this manuscript, Ms. Mireille Passama for drawing the illustrations and Dr. Claude Barberis for very stimulating discussions, and Bernard Mouillac for providing CHO cell line expressing VP receptors. The human vasopressin V1b receptor was a gift from Hiroyuki Kawashima (Yamanouchi Pharmaceutical Co. Ltd., Tokyo, Japan).


   Footnotes
 
This work was supported by the Institut National de la Santé et de la Recherche Médicale (to G.G.), NIH Grant GM-25280 (to M.M.), and Fond National Suisse de la Recherche Scientifique Grant 32-064-10700 (to R.C.G.).

Abbreviations: AC, Adenylyl cyclase; AVP, arginine vasopressin; BW, body weight; CHO, Chinese hamster ovary; CRF, corticotropin-releasing factor; d[Cha4]AVP, [1-deamino-4-cyclohexylalanine] arginine vasopressin (d[Cha4]AVP); dAVP, deamino AVP; DMF, dimethylformamide; DNase, deoxyribonuclease; HBS, Hanks’ buffered saline; IP, inositol phosphate; Kact, half-maximal stimulation of second messenger accumulations; Ki, inhibitory dissociation constant; OT, oxytocin; OTA, d(CH2)5[Tyr(Me)2, Thr4, Tyr-NH2 9]ornithine vasotocin; RF, rate of flow; VP, vasopressin.

Received April 1, 2002.

Accepted for publication August 5, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
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