Novel linear antagonists of the antidiuretic (V2) and vasopressor (V1) responses to vasopressin

We report the solid phase synthesis of a series of 16 linear analogues of the cyclic antagonist of the antidiuretic (V2) and the vasopressor (V1) responses to arginine vasopressin (AVP), d(CH2)5[D-Tyr(Et)2, Val4]AVP(A). Peptide 1, the linear precursor of (A), (CH2)5(SH)-CH2-CO-D-Tyr(Et)-Phe-Val-Asn-Cys-Pro-Arg-Gly-NH2 was modified at position six with alpha-L-aminobutyric acid (Abu) to give peptide 2. Further modifications of the Abu6 analogue (No. 2) at position one by substituting cyclohexylacetic acid (Caa), cyclohexylpropionic acid (Cpa), 1-adamantaneacetic acid (Aaa), phenylacetic acid (Phaa), tert.-butylacetic acid (t-Baa), isovaleric acid (Iva), propionic acid (Pa), L-penicillamine (P), tert.-butoxycarbonyl (Boc) or omitting any substituent at this position, and/or in combination with Arg-NH2(9), Ala-NH2(9), D-Arg8-Arg-NH2(9), and desGly9 modifications yielded the remaining 14 peptides. All 16 peptides were examined for agonistic and antagonistic potencies in AVP V2 and V1 assays in rats. Apart from the Cpa analogue and the analogue lacking any substituent in the 1-position, all exhibit substantial V2 and V1 antagonism. A number are as potent as (A) as V2 antagonists. With an anti-V2 pA2 = 8.11 +/- 0.07, Aaa-D-Tyr(Et)-Phe-Val-Asn-Abu-Pro-Arg-Arg-NH2 (No. 6) is as potent as any cyclic AVP V2 antagonist reported to date. The PaI analogue of No. 6 exhibits promising anti-V2/anti-V1 selectivity. These findings prove conclusively that a ring structure is not a requirement for recognition of or for binding to AVP V2 or V1 receptors. This discovery thus offers a promising new approach to the design of peptide and non-peptide antagonists of AVP and perhaps also to other cyclic peptides such as somatostatin, atrial-natriuretic factor, insulin, and the recently discovered endothelin. Some of these linear antagonists may be of value as pharmacological tools and as therapeutic agents.     

Arginine vasopressin (AVP) replacement of helper cell requirement in IFN-gamma production. Evidence for a novel AVP receptor on mouse lymphocytes

Arginine vasopressin (AVP), a nine-amino acid neurohypophyseal hormone, is capable of replacing the helper cell requirement for IFN-gamma production by Lyt-2+ mouse splenic lymphocytes. We present data here showing that the AVP helper signal occurs via interaction with a novel R on splenic lymphocytes and involves primarily the N-terminal six-amino acid cyclic ring (pressinoic acid) with the C-terminal three-amino acid end of AVP playing a minor role. Pressinoic acid was capable of providing help at concentrations similar to those of AVP, whereas oxytocin and isoleucine pressinoic acid were 10- and 100-fold less effective, respectively. Isoleucine pressinoic acid has the same structure as pressinoic acid except for the substitution of isoleucine for phenylalanine in position 3 of the sequence. Consistent with the function data, R binding competitions with splenic lymphocyte membrane preparations showed that AVP and pressinoic acid competed similarly with [3H]AVP, whereas oxytocin and isoleucine pressinoic acid were much less effective competitors. Further characterization of the AVP lymphocyte R was performed using AVP analogues having well defined agonist and antagonist activities on either V1 (vasopressor) R or V2 (antidiuretic) R. The AVP helper signal was blocked by the V1 antagonist [d(CH2)1(5) Tyr(methyl)]AVP but not by another V1 antagonist, [d(CH2)1(5)D-Tyr(ethyl)2Val4]AVP. Both V1-R antagonists were able to block [3H]AVP binding to the V1-R on liver cells, whereas only the V1 antagonist that blocked AVP help was able to compete effectively for the spleen AVP-R. Neither a V2 agonist nor a V2 antagonist had any effect on AVP help in IFN-gamma production. These data strongly indicate the presence of a novel AVP-R on spleen lymphocytes, which is related to the classic V1-R on liver cell membranes.     

3H]AVP binding to rat renal tubular receptors during long-term treatment with an antagonist of arginine vasopressin

The interaction of an antagonist of arginine vasopressin (AVP), d(CH2)5-D-Tyr(Et)VAVP, with renal tubular V2 receptors were studied in medullary membrane preparations from kidneys of Sprague-Dawley and Brattleboro rats. In both rat strains, V2 receptors had comparable KD and Bmax values for binding of [3H]AVP. In vitro studies revealed that the V2-antagonist was more potent than cold AVP in displacing [3H]AVP. In vivo treatment of Sprague-Dawley rats with the antagonist over one week resulted only in a transient state of diabetes insipidus (DI). No specific [3H]AVP binding was detectable throughout the period of administration. Chronic treatment of Brattleboro rats resulted in a complete normalization of water intake. This agonistic effect was also associated with undetectable [3H]AVP binding. After stopping the infusion of d(CH2)5-D-Tyr(Et)VAVP, Bmax values tended to rise but had still not reached base line values after 6 days. In contrast, the chronic infusion of AVP in Brattleboro rats resulted in a reduction in water intake which was accompanied by a decreased Bmax. [3H]AVP binding remained detectable during the entire treatment period. Thereafter Bmax was restored to base line values within 2 days of stopping the infusion. These results suggest that d(CH2)5-D-Tyr(Et)VAVP has a high affinity for V2 receptors in both Sprague-Dawley and Brattleboro rats. Its rate of dissociation from the receptor appears to be much slower than that of AVP. In Brattleboro rats, the binding of d(CH2)5-D-Tyr(Et)VAVP leads to an antidiuretic response. In Sprague-Dawley rats, a transient diuretic response is followed by a progressive normalization in water intake. This occurs despite persistent and complete blockade of renal medullary V2 receptors.     

Discovery and design of novel vasopressin hypotensive peptide agonists

This presentation will trace the serendipitous discovery of novel vasopressin (VP) hypotensive agonists d(CH2)5[D-Tyr(Et)2,X3]VAVP (where X = Arg, Lys). These peptides were uncovered as part of an ongoing program aimed at the design of potent and selective VP antidiuretic (V2 receptor) antagonists. We will also present highlights of our subsequent preliminary studies seeking (i) to design high affinity radioiodinatable ligands for the localization and characterization of the putative VP vasodilatory (V1c?) receptor; (ii) to identify the structural features of selective and non-selective cyclic and linear VP and oxytocin (OT) antagonists of the V2 receptor, the vascular (V1a) receptor and of the uterine (OT) receptor required for hypotensive agonism and; (iii) to enhance hypotensive potency. These novel VP hypotensive agonists could serve as valuable research tools in studies on the roles of VP in blood pressure regulation and may also lead to the development of a new class of therapeutically useful antihypertensives.     

Vascular vasopressin receptors mediate phosphatidylinositol turnover and calcium efflux in an established smooth muscle cell line

Vasopressin-induced phosphatidylinositol turnover and mobilization of intracellular Ca2+ was studied using an established smooth muscle cell line (A-10). The cells were subcloned to ensure a monoclonal cell population. The accumulation of inositol mono-, di-, and tris-phosphates (IP1, IP2, and IP3, respectively), and the mobilization of intracellular Ca2+ were dependent on the time of incubation and the concentration of arginine vasopressin (AVP). IP1, IP2, and IP3 were significantly elevated after 15 sec and remained elevated for up to 2 hr. The concentrations of AVP required for half-maximal stimulation of IP1, IP2, and IP3 formation were 2, 12, and 4 nM, respectively. LiCl was required to observe the accumulation of inositol phosphates in response to AVP. Significant 45Ca2+ efflux was observed within 15 sec after exposure to AVP. By employing the vasopressin receptor subtype selective antagonists [d(CH2)5Tyr(Me)AVP, V1; d(CH2)5D-Tyr(Et)VAVP,V1/V2; d(CH2) 5D-IleVAVP,V2] and agonists [AVP, V1/V2; dDAVP, V2; dVDAVP, V2], we found that the vasopressin-induced stimulation of phosphatidylinositol turnover and 45Ca2+ efflux were mediated by receptors of the vascular V1 subtype. Pertussis toxin pretreatment partially inhibited vasopressin-induced phosphatidylinositol turnover. These data demonstrate that activation of V1 receptors of vascular smooth muscle cells resulted in enhanced phosphatidylinositol turnover and mobilization of intracellular Ca2+.     

Novel approach to the design of synthetic radioiodinated linear V1A receptor antagonists of vasopressin.

We report the solid phase synthesis of six analogs of the potent and selective linear AVP vasopressor (V1a receptor) antagonist: Phaa1-D-Tyr(Et)2-Phe3-Gln4-Asn5-Lys6-Pro7-Arg-NH(8)2(A) (where Phaa = phenylacetyl) in which the Phaa1 residue is replaced by hydroxyphenylacetyl (HO-Phaa), hydroxyphenylpropionyl (HO-Phpa) and phenylpropionyl (Phpa) and the D-Tyr(Et)2 and Lys6 residues by D-Tyr(Me)2 and Arg6 substituents. The phenolic-containing peptides were synthesized to test the feasibility of using this approach for the design of high affinity selective ligands for AVP V1a receptors. The following analogs of A were synthesized: 11 [(HO)Phaa1]; 2. [(HO)Phaa1,D-Tyr(Me)2]; 3. [(HO)Phaa1,D-Tyr(Me)2, Arg6]; 4. [(HO)Phaa1,Arg6]; 5. [Phpa1]; 6. [(HO)Phpa1]. All six peptides were examined for agonistic and antagonistic potencies in vasopressor (V1a-receptor) and antidiuretic (V2-receptor) and in vitro oxytocic assays in rats. The affinities of the phenolic-containing peptides for hepatic V1a and uterine receptors were also determined. The phenolic-containing peptides all exhibit potent V1a antagonism. Their anti-V1a pA2 values range from 8.23 to 8.63 (the anti-V1a pA2 value of A = 8.69). Their inhibition constants (Ki in nM) range 0.4 to 1.0. They are weak antidiuretic agonists with activities ranging from 0.022 U/mg to 0.13 U/mg (A = 0.033 U/mg). They all exhibit OT antagonism in vitro. Their anti-OT pA2 values range from 7.28 to 7.71 (A = 7.62). All five phenolic compounds were iodinated using iodine chloride and tested in the same in vivo and in vitro assay system.(ABSTRACT TRUNCATED AT 250 WORDS)     

Synthesis and structure-activity investigation of novel vasopressin hypotensive peptide agonists.

We report the solid phase synthesis and vasodepressor potencies of the novel hypotensive peptide [1(-beta-mercapto-beta,beta-pentamethylene propionic acid)-2-O-ethyl-D-tyrosine, 3-arginine, 4-valine] arginine vasopressin, d(CH2)5[D-Tyr(Et)2, Arg3, Val4]AVP (A), its related Lys3 (B), Tyr-NH(9)2 (C), [Lys3, Tyr-NH(9)2 (D) analogs and in a preliminary structure-activity study of positions 2-4 and 7-9, 24 analogs (1-24) of A-C. Peptides 1-6, 9-14 have the following single substituents at positions 2, 3, 4, 8 and 9 in (A): 1, D-Tyr(Me)2; 2, L-Tyr(Et)2; 3, Orn3; 4, N-Me-Arg3; 5, Glu3; 6, Arg4; 9, D-Arg8; 10, Eda9; 11, Arg-NH(9)2; 12, Ala-NH(9)2; 13, desGly9; 14, desGly-NH(9)2. Peptides 15 and 16 are analogs of B which possess the following single modifications: 15, Arg-NH(9)2; 16, desGly9. Peptides 7 and 8 are analogs of (C) with the following single modification: 7, Gln4; 8, Lys8. Peptides 17-24 are analogs of A possessing the following multiple modifications: 17, [Sar7, Eda9]; 18, [Arg7, Eda9]; 19, [Arg7, Eda9<--Tyr10]; 20, [Arg4, Arg-NH(9)2]; 21, [Ile4, desGly9]; 22, [Arg4, desGly9]l; 23, [Arg7, desGly9]; 24, [Arg7, Lys8, desGly9]. All 24 new peptides were evaluated for agonistic and antagonistic activities in in vivo antidiuretic (V2-receptor), vasopressor (V1a-receptor) and in in vitro (no Mg2+) oxytocic (OT-receptor) assays and like the parent peptides (A-D) (Chan et al. Br. J. Pharmacol. 1998; 125: 803-811) were found to exhibit no or negligible activities in these assays. Vasodepressor potencies were determined in anesthetized male rats with baseline mean arterial blood pressure maintained at 110-120 mmHg. The effective dose (ED), in microg 100 g(-1) i.v., required to produce a vasodepressor response of 5 cm2, area under the vasodepressor response curve (AUC) during the 5-min period following the injection of the test peptide, was determined. Therefore, the EDs measure the relative vasodepressor potencies of the hypotensive peptides. The following ED values were obtained for A-D and for peptides 1-24: A, 4.66; B, 5.75; C, 10.56; D, 11.60; 1, approximately 20; 2, approximately 30; 3, 6.78; 4, non-detectable (ND); 5, ND; 6, approximately 32; 7, ND; 8, 8.67; 9, ND; 10, 2.43; 11, 3.54; 12, 10.57; 13, 4.81; 14, ND; 15, 4.47; 16, 9.78; 17, 5.72; 18, 1.10; 19, 1.05; 20, 10.41; 21, 9.13; 22, approximately 33; 23, 3.01; 24, 1.71. A is clearly the most potent of the four original hypotensive peptides A-D. These data provide insights to which modification of A enhance, retain or abolish hypotensive potencies. Six of the new hypotensive peptides are significantly more potent than A. These are peptides 10, 11, 18, 19, 23 and 24. Peptide 19, a radioiodinatable ligand, is ten times more potent than C or D. The Gln4 modification of C and the N-Me-Arg3, Glu3, D-Arg8 and desGly-NH(9)2 modifications of A abolished hypotensive potency. By contrast, the Eda9, Arg-NH(9)2, [Sar7, Eda9], [Arg7, Eda9<- -Tyr10], [Arg7, desGly9], [Arg7, Lys8, desGly9] modifications of A all led to enhancements of hypotensive potency. This initial structure-activity exploration provides useful clues to the design of (a) more potent vasodepressor peptides and (b) high affinity radioiodinatable ligands for the putative AVP vasodilating receptor. Some of the peptides here may be of value as pharmacological tools for studies on the complex cardiovascular actions of AVP and may lead to the development of a new class of anti-hypertensive agents.     

Desensitization of renal vasopressin-stimulated adenylate cyclase by a vasopressor and antidiuretic antagonist, [1-(beta-mercapto-beta,beta-cyclopentamethylenepropionic acid),2-(O-ethyl)-D-tyrosine, 4-valine]arginine vasopressin

Desensitization of the renal antidiuretic (V2) receptor has been regarded as agonist-specific. We found that a vasopressor and antidiuretic (V1V2) antagonist d(CH2)5DTyr(Et)VAVP induced desensitization of the receptor and that a vasopressor (V1) antagonist d(CH2)5Tyr(Me)AVP, which is a weak antidiuretic (V2) agonist, counteracted the potent desensitizing action of 1-deamino-8-D-arginine-vasopressin and endogenous arginine vasopressin.     

Design of oxytocin antagonists, which are more selective than atosiban.

We report the solid phase synthesis of four pairs of L- and D-thienylalanine (Thi/D-Thi) position two modified analogues of the following four oxytocin (OT) antagonists: des-9-glycinamide [1-(beta-mercapto-beta,beta-pentamethylene propionic acid), 2-O-methyltyrosine, 4-threonine]ornithine-vasotocin (desGly(NH2)9,d (CH2)5[Tyr(Me)2,Thr4]OVT) (A); the Tyr-(NH2)9 analogue of (A), d(CH2)5[Tyr(Me)2,Thr4,Tyr-(NH2)9]OVT (B); the Eda9 analogue (where Eda = ethylenediamine) of (A), d(CH2)5[Tyr(Me)2, Thr4, Eda9]OVT (C); and the retro Tyr10 modified analogue of (C), d(CH2)5[Tyr(Me)2, Thr4, Eda9<--Tyr10]OVT (D). The eight new analogues of A-D are (1) desGly(NH2),d(CH2)5[Thi2,Thr4]OVT, (2) desGly(NH2),d(CH2)5[D-Thi2,Thr4]OVT, (3) d(CH2)5[Thi2, Thr4,Tyr-(NH2)9]OVT, (4) d(CH2)5[D-Thi2,Thr4,Tyr-(NH2)9]OVT (5) d(CH2)5[Thi2,Thr4Eda9]OVT, (6) d(CH2)5[D-Thi2,Thr4,Eda9]OVT, (7) d(CH2) [Thi2,Thr4,Eda9<--Tyr10]OVT, (8) d(CH2),[D-Thi2,Thr4,Eda9<--Tyr10]OVT. We also report the synthesis of (C). Peptides 1-8 and C were evaluated for agonistic and antagonistic activities in in vitro and in vivo OT assays, in in vivo vasopressor (V1a receptor) assays and in in vivo antidiuretic (V2 receptor) assays. None of the eight peptides nor C exhibit oxytocic or vasopressor agonism. Peptides 1-8 are extremely weak V2 agonists (antidiuretic activities range from < 0.0005 to 0.20 U/mg). Peptide C is a weak mixed V2 agonist/antagonist. Peptides 1-8 and C exhibit potent in intro (no Mg2+) OT antagonism (anti-OT pA2 values range from 7.76 to 8.05). Peptides 1-8 are all OT antagonists in vivo (estimated in vivo anti-OT pA2 values range from 6.54-7.19). With anti-V1a pA2 values of approximately 5-5.80, peptides 1-8 exhibit marked reductions in anti-V1a potencies relative to those of the parent peptides A-D (anti-V1a pA2 range from 6.48 to 7.10) and to l-deamino[D-Tyr(Et)2, Thr4]OVT (Atosiban, trade name Tractocile) (anti-V1a pA2-6.14). Atosiban has recently been approved in Europe for clinical use for the prevention of premature labour (Pharm. J. 264(7-100): 871). Peptides 1-8 exhibit striking gains in in vitro anti-OT/anti-V1a selectivities with respect to the parent peptides A, B, C and D and to Atosiban. Peptides 1-8 exhibit anti-OT (in vitro)/anti-V1a selectivities of 450, 525, 550, 450, approximately 1080, 116, 355, 227 respectively. The corresponding values for A-D and Atosiban are 30, 4.2, 4.3, 2.6 and 37. With the exception of peptide 6, the remaining seven peptides exhibit 3-18-fold gains in anti-OT (in vivo)/anti-V1a selectivity with respect to Atosiban, peptides 1-8 exhibit anti-OT (in vivo)/anti-V1a selectivities of 22, approximately 82, approximately 82, 147, approximately 83, 11, 31 and 42. By comparison, Atosiban exhibits an anti-OT (in vivo)/anti-V1a selectivity = 8. With an estimated in vivo anti-OT pA2 value = 7.19+/-0.06, peptide 4 is equipotent with Atosiban (pA2 = 7.05+/-0.05). However, with its significantly reduced anti-vasopressor potency, pA2 = approximately 5, it is approximately 18 times more selective for OT receptors with respect to VP V1a receptors than Atosiban. Since we have shown that V1a antagonism could be an unwanted side-effect in tocolytics, peptide 4 and some of the OT antagonists reported here have advantages over Atosiban and thus may be suitable candidates for evaluation as potential tocolytic agents for the treatment of preterm labour.     

Antipyretic effect of central arginine8-vasopressin treatment: V1 receptors specifically involved?

Intracerebroventricular (i.c.v.) administration of the neurohypophyseal neuropeptide arginine8-vasopressin (AVP) results in a dose-dependent attenuation of endotoxin-induced fever (EIF) in rats. Specific antagonists of the neuropeptided(CH2)5[Tyr(Me)2]AVP for V1 receptors, d(CH2)5[dlle2lle4]AVP for the V2 receptors and Des-Gly,NH2d(CH2)5[Tyr)Me2)Thr4Orn8]vasotocin, an antagonist of the oxytocin receptors (AOXT), failed to modify EIF when administered i.c.v. Relatively high doses (100 ng) of all three peptide antagonists effectively blocked the antipyretic effect of AVP. Administered in smaller doses (10 or 30 ng), however, a more specific interaction was observed, i.e. the V1 antagonist being the only effective compound in preventing the effect of AVP. Although the data indicate that peptide-antagonist interactions should be interpreted carefully, the present experiments confirm previous observations on the involvement of V1-type receptors in the antipyretic action of AVP and suggest additional interactions with V2 vasopressinergic and oxytocinergic receptors.     

Design and synthesis of potent, highly selective vasopressin hypotensive agonists.

We report here the solid-phase synthesis and vasodepressor potencies of a new lead vasopressin (VP) hypotensive peptide [1(beta-mercapto-beta,beta-pentamethylenepropionic acid)-2-0-ethyl-D-tyrosine, 3-arginine, 4-valine, 7-lysine, 9-ethylenediamine] lysine vasopressin, d(CH(2))(5)[D-Tyr(Et)(2), Arg(3), Val(4), Lys(7), Eda(9)]LVP (C) and 21 analogues of C with single modifications at positions 9 (1-13), 6 (14), 2 (16-20) and combined modifications at positions 6 and 10 (15) and 2 and 10 (21). Peptides 1-13 have the following replacements for the Eda residue at position 9 in C: (1) Gly-NH(2); (2) Gly-NH-CH(3); (3) Ala-NH(2); (4) Ala-NH-CH(3), (5) Val-NH(2); (6) Cha-NH(2); (7) Thr-NH(2); (8) Phe-NH(2); (9) Tyr-NH(2); (10) Orn-NH(2); (11) Lys-NH(2); (12) D-Lys-NH(2); (13) Arg-NH(2). Peptide 14 has the Cys residue at position 6 replaced by Pen. Peptide 15 is the retro-Tyr(10) analogue of peptide 14. Peptides 16-20 have the D-Tyr(Et) residue at position 2 in C replaced by the following substituents: D-Trp (16); D-2-Nal (17); D-Tyr(Bu(t))(18); D-Tyr(Pr(n)) (19); D-Tyr(Pr(i)) (20). Peptide 21 is the retro-Tyr(10) analogue of peptide 20. C and peptides 1-21 were evaluated for agonistic and antagonistic activities in in vivo vasopressor (V(1a)-receptor), antidiuretic (V(2)-receptor), and in in vitro (no Mg(2+)) oxytocic (OT-receptor) assays in the rat, and, like the original hypotensive peptide, d(CH(2))(5)[D-Tyr(Et)(2), Arg(3), Val(4)]AVP (A) (Manning et al., J. Peptide Science 1999, 5:472-490), were found to exhibit no or negligible activities in these assays. Vasodepressor potencies were determined in anesthetized male rats with baseline mean arterial blood pressure (BP) maintained at 100-120 mmHg. The effective dose (ED), in microg/100 g i.v., the dose required to produce a vasodepressor response of 5 cm(2) area under the vasodepressor response curve (AUC) during the 5-min period following the injection of the test peptide, was determined. The EDs measure the vasodepressor potencies of the hypotensive peptides C and 1-21 relative to that of A (ED = 4.66 microg/100 g) and to each other. The following ED values in microg/100 g were obtained for C and for peptides 1-21; C 0.53; (1) 2.41; (2) 1.13; (3) 1.62; (4) 0.80; (5) 1.83; (6) 1.56; (7) 2.12, (8) 2.58; (9) 1.40; (10) 0.88; (11) 0.90; (12) 0.85; (13) 0.68; (14) 0.99; (15) 1.05; (16) 0.66; (17) 0.54; (18) 0.33; (19) 0.18; (20) 0.15; (21) 0.14. All of the hypotensive peptides reported here are more potent than A. Peptides 20 and 21 exhibit a striking 30-fold enhancement in vasodepressor potencies relative to A. With a vasodepressor ED = 0.14, peptide 21 is the most potent VP vasodepressor agonist reported to date. Because it contains a retro-Tyr(10) residue, it is a promising new radioiodinatable ligand for the putative VP vasodilating receptor. Some of these new hypotensive peptides may be of value as research tools for studies on the complex cardiovascular actions of VP and may lead to the development of a new class of antihypertensive agents.     

Conserved aromatic residues in the transmembrane region VI of the V1a vasopressin receptor differentiate agonist vs. antagonist ligand binding.

Despite their opposite effects on signal transduction, the nonapeptide hormone arginine-vasopressin (AVP) and its V1a receptor-selective cyclic peptide antagonist d(CH2)5[Tyr(Me)2]AVP display homologous primary structures, differing only at residues 1 and 2. These structural similarities led us to hypothesize that both ligands could interact with the same binding pocket in the V1a receptor. To determine receptor residues responsible for discriminating binding of agonist and antagonist ligands, we performed site-directed mutagenesis of conserved aromatic and hydrophilic residues as well as nonconserved residues, all located in the transmembrane binding pocket of the V1a receptor. Mutation of aromatic residues of transmembrane region VI (W304, F307, F308) reduced affinity for the d(CH2)5[Tyr(Me)2]AVP and markedly decreased affinity for the unrelated strongly hydrophobic V1a-selective nonpeptide antagonist SR 49059. Replacement of these aromatic residues had no effect on AVP binding, but increased AVP-induced coupling efficacy of the receptor for its G protein. Mutating hydrophilic residues Q108, K128 and Q185 in transmembrane regions II, III and IV, respectively, led to a decrease in affinity for both agonists and antagonists. Finally, the nonconserved residues T333 and A334 in transmembrane region VII, controlled the V1a/V2 binding selectivity for both nonpeptide and cyclic peptide antagonists. Thus, because conserved aromatic residues of the V1a receptor binding pocket seem essential for antagonists and do not contribute at all to the binding of agonists, we propose that these residues differentiate agonist vs. antagonist ligand binding.     

Expression of vasopressin V2 receptor in Xenopus laevis oocytes by porcine kidney cell line (LLC-PK1) messenger RNA.

Vasopressin V2 receptor was expressed in Xenopus laevis oocytes which were injected with poly(A) +RNA from porcine kidney cell line LLC-PK1. Pharmacological antagonism of the expressed V2 receptor was observed between arginine vasopressin and two potent and selective vasopressin antagonists: [d(CH2)5, D2-Phe2 Ile4, Ala9-NH2]arginine vasopressin and [d(CH2)5,D-Ile2, Ile4]arginine vasopressin. Activation constant for arginine vasopressin concentration was 1.32 x 10(-10)M. The nucleotide length of the mRNA encoding for vasopressin V2 receptor was deduced to be approximately 2 kilobases.     

Position 4 analogues of [deamino-Cys(1)] arginine vasopressin exhibit striking species differences for human and rat V(2)/V(1b) receptor selectivity.

Arginine vasopressin (AVP) mediates a wide variety of biological actions by acting on three distinct G-protein coupled receptors, termed V(1a) (vascular), V(1b) (pituitary) and V(2) (renal). It also binds to the oxytocin (OT) receptor. As part of a program aimed at the design of selective agonists for the human V(1b) receptor, we recently reported the human V(1b), V(1a), V(2) and OT receptor affinities of the following position 4 substituted analogues of [deamino-Cys(1)] arginine vasopressin (dAVP)-(1) d[Leu(4)]AVP, (2) d[Orn(4)]AVP, (3) d[Lys(4)]AVP, (4) d[Har(4)]AVP, (5) d[Arg(4)]AVP, (6) d[Val(4)]AVP, (7) d[Ala(4)]AVP, (8) d[Abu(4)]AVP, (9) d[Nva(4)]AVP, (10) d[Nle(4)]AVP, (11) d[Ile(4)]AVP, (12) d[Phe(4)]AVP, (13) d[Asn(4)]AVP, (14) d[Thr(4)]AVP: (15) d[Dap(4)]AVP. With the exception of Nos. 7 and 12, all peptides exhibit very high affinities for the human V(1b) receptor. Furthermore, peptides 1-4 exhibit high selectivities for the human V(1b) receptor with respect to the V(1a), V(2) and OT receptors and, with d[Cha(4)]AVP, in functional tests, are the first high affinity selective agonists for the human V(1b) receptor (Cheng LL et al., J. Med. Chem. 47: 2375-2388, 2004). We report here the pharmacological properties of peptides 1-4, 5 (from a resynthesis), 7, 9-13, 15 in rat bioassays (antidiuretic, vasopressor and oxytocic) (in vitro: no Mg(++)) with those previously reported for peptides 5, 6, 8, 14. We also report the rat V(1b), V(1a), V(2) and OT receptor affinities of peptides 1-5 and the rat V(2) receptor affinities for peptides: 7-15.The antidiuretic activities in units/mg of peptides 1-15, are: 1=378; 2=260; 3=35; 4=505; 5=748; 6=1150; 7=841; 8=1020; 9=877; 10=1141; 11=819, 12=110; 13=996; 14=758; 15=1053. Peptides 1-4 exhibit respectively the following rat and human (in brackets) V(2) receptor affinities: 1=3.1 nm (245 nm); 2=3.4 nm (1125 nm); 3=24.6 nm (11,170 nm); 4=0.6 nm (1386 nm). Their rat V(1b) receptor affinities are 1=0.02 nm; 2=0.45 nm; 3=9.8 nm; 4=0.32 nm. Their rat V(1a) receptor affinities are 1=1252 nm; 2=900 nm; 3=1478 nm; 4=32 nm. Their rat oxytocin (OT) receptor affinities are 1=481 nm; 2=997 nm; 3=5042 nm; 4=2996 nm. All four peptides have high affinities and selectivities for the rat V(1b) receptor with respect to the rat V(1a) and OT receptors. However, in contrast to their high selectivity for the human V(1b) receptor with respect to the human V(2) receptor, they are not selective for the V(1b) receptor with respect to the V(2) receptor in the rat. These findings confirm previous observations of profound species differences between the rat and human V(2) receptors. Peptides 1-4 are promising leads to the design of the first high affinity selective agonists for the rat V(1b) receptor.     

Effects of nonapeptide antagonists on oxytocin- and arginine-vasopressin-induced analgesia in mice

Several peptides, including arginine-vasopressin (AVP), neurotensin, and substance P, produce analgesia that is not mediated by opiate systems. Using the hot plate test, we studied the analgesic effects of intracisternal (i.c.) administration of various doses of the nonapeptide oxytocin (OXY) in Swiss-Webster mice. We found that OXY (1-4 micrograms) significantly increased the latency of animals to jump or lick their paws after placement on a hot plate. This effect was not blocked by naloxone pretreatment, which suggests that it is not opiate dependent. Using the hot plate test, we confirmed that AVP (1 and 4 micrograms) also produces analgesia. We then studied the analgesia produced by OXY and by AVP using 3 nonapeptide analogues with antagonist properties: [Pen1, LpMePhe2, Thr4, Orn8]OXY (PLMPTO-OXY) that has anti-oxytocic properties in the uterine contraction assay, d(CH2)5Tyr(Me)AVP(dTM-AVP) which antagonizes the antidiuretic properties of AVP and d(CH2)5D-Ile2,Abu4-AVP (dIA-AVP) which antagonizes the vasopressor effects of AVP. Simultaneous administration of PLMPTO-OXY completely blocked the analgesia produced by OXY whereas the antidiuretic antagonist dIA-AVP partially blocked OXY-induced analgesia and dTM-AVP had no effect. None of the antagonists used blocked AVP-induced analgesia. We concluded that the neural systems mediating the analgesic effects of i.c. OXY differ from those for AVP.     

Evidence that the effects of arginine-8-vasopressin (AVP) on pituitary corticotropin (ACTH) release are mediated by a novel type of receptor

Ovine corticotropin releasing factor (oCRF-41) and AVP act synergistically to stimulate pituitary ACTH secretion. In the present study we have investigated whether the effect of AVP, either in the presence or in the absence of oCRF-41 (0.5 nmol/l), could be blocked by V1 (pressor)-antagonists. Furthermore, oxytocin, and [1-deamino,8-D-arginine] vasopressin (dDAVP) were tested for their ability to release ACTH. All experiments were carried out in vitro, using segments of rat anterior pituitary glands. The V1-antagonist [1-deamino,penicillamine(o-methyl-tyrosine)]AVP inhibited ACTH release induced by AVP or AVP + oCRF-41. However, it also had some agonistic activity which was more pronounced in the presence of oCRF-41. An equally potent V1-antagonist, [1-beta-mercapto-beta, beta-cyclopentamethyleneproprionic acid (o-methyl-tyrosine)]AVP, failed to inhibit AVP-stimulated ACTH secretion, and also had weak agonist potency. The relatively selective V2 (antidiuretic)-agonist dDAVP was 20-30 fold less potent than AVP. Oxytocin, a weak V1- and V2-agonist was only 4-8 fold less potent than AVP. These data are compatible with the suggestion that AVP receptors on pituitary corticotrope cells are neither classical V1- nor V2-receptors.     

Characterization of rodent liver and kidney AVP receptors: pharmacologic evidence for species differences.

Radioligand binding studies with [3H]vasopressin (AVP) were used to determine the affinities of AVP receptor agonists and antagonists for mouse liver and kidney plasma membrane preparations. Both membrane preparations exhibited one class of high-affinity binding site. AVP ligand binding inhibition studies confirmed that mouse liver binding sites belong to the V1A subtype while kidney binding sites belong to the V2 receptor subtype. The affinity of each ligand for mouse V1A receptors was very similar to that for rat V1A receptors, showing differences in Ki values of less than 3-fold. In contrast, several peptide (d(CH2)5Tyr(Me)AVP) and nonpeptide (OPC-21268 and SR 49059) ligands had different affinities for mouse and rat kidney V2 receptors, with differences in Ki values ranging from 14- to 17-fold. These results indicate that mouse and rat kidney V2 receptors show significant pharmacologic differences.     

Vasopressin and oxytocin receptors on plasma membranes from rat mammary gland. Demonstration of vasopressin receptors by stimulation of inositol phosphate formation, and oxytocin receptors by binding of a specific 125I-labeled oxytocin antagonist, d(CH2)5(1)[Tyr(Me)2, Thr4,Tyr-NH2(9)]OVT

The addition of oxytocin to minces of rat mammary gland preincubated with (3H)myo-inositol stimulated the formation of inositol phosphate (IP) in both lactating and regressed glands. Stimulation was about 4 times greater in regressed tissue, consistent with an oxytocin effect on myoepithelial cells, which are enriched relative to epithelial cells during regression. The stimulation of IP formation was agonist specific, as shown with several oxytocin analogs. Arginine vasopressin (AVP), however, was more than twice as potent as oxytocin in stimulating IP formation in regressed tissue. Both V1- and V2-selective AVP receptor antagonists inhibited the stimulation of IP formation by oxytocin. The V1-selective antagonist was about 10 times more inhibitory than the V2-selective antagonist. [3H]AVP was bound to plasma membranes from the mammary gland of the lactating rat with an apparent Kd of about 0.7 nM and Bmax of 54.6 fmol/mg protein. These values were comparable with those found for AVP receptors of kidney plasma membranes. Our results suggest that the stimulation of IP formation in rat mammary gland by oxytocin occurs through occupancy of AVP, and not oxytocin, receptor sites. A second aspect of these studies was to determine if a recently developed iodinated antagonist of oxytocin-induced uterine contractions could be used as a specific probe for oxytocin receptors in the rat mammary gland. Under steady state conditions, [125I]d(CH2)5(1)[Tyr(Me)2,Thr4,Tyr-NH2(9)]OVT was bound to a single class of independent binding sites in mammary gland plasma membrane from lactating rats with an apparent Kd of 65 pM and Bmax of 225 fmol/mg protein. Noniodinated antagonist had an affinity about 150 times less than the monoiodinated form. The affinity of binding sites for AVP was 10 times greater than the noniodinated antagonist and 2.4 times greater than oxytocin. In view of the presence of AVP receptors in mammary tissue, these findings suggested that the iodinated antagonist binds to AVP receptors. However, comparison of the binding of iodinated antagonist to plasma membranes from the lactating mammary gland with kidney medulla and liver, target sites for AVP, showed that binding was specific for the mammary gland and hence oxytocin receptors. The concentration of oxytocin receptors in mammary gland, as determined by [125I]d(CH2)5(1)[Tyr(Me)2,Thr4,Tyr-NH2(9)]OVT binding, was 4 times greater than the concentration of high-affinity AVP receptors, as determined by [3H]AVP binding.(ABSTRACT TRUNCATED AT 250 WORDS)     

Spinal vasopressin modulates the reflex cardiovascular response to static contraction.

Recent evidence has demonstrated that arginine vasopressin (AVP) may modulate primary afferent activity of nociceptors in the dorsal horn of the spinal cord. Because nociceptors are group III and IV afferents, spinal AVP also may modulate the activity of group III and IV afferents that cause reflex cardiovascular responses to muscle contraction. Thus, we compared the pressor (mean arterial pressure), myocardial contractile (dP/dt), and heart rate (HR) responses to electrically induced static contraction of the cat hindlimb before and after lumbar intrathecal (IT) injection (L1-L7) of AVP (n = 9), the V1 receptor antagonist d(CH2)5Tyr(Me)AVP (n = 6), the V2 receptor antagonist d(CH2)5[D-Ile2,Ile4,Ala-NH2(9)]AVP (n = 6), and the V2 agonist [Val4,D]AVP (n = 8). After IT injection of AVP (0.1 or 1 nmol) the pressor and contractile responses to static contraction were attenuated by 55 and 44%, respectively. HR was unchanged. Forty-five to 60 min after AVP injection, the contraction-induced pressor and contractile responses were restored to control levels. V1 receptor blockade augmented contraction-induced increases in mean arterial pressure (36%) and dP/dt (49%) but not HR. V2 receptor blockade had no effect on the cardiovascular response to contraction, whereas selective V2 stimulation attenuated the dP/dt (-20%) and HR (-33%) responses but not the pressor response. These results suggest that AVP attenuates the reflex cardiovascular response to contraction by modulating sensory nerve transmission from contracting muscle primarily via a V1 receptor mechanism in the lumbar spinal cord.     

Through V2, not V1 receptor relating to endogenous opiate peptides, arginine vasopressin in periaqueductal gray regulates antinociception in the rat

Our previous study has proven that central arginine vasopressin (AVP) plays an important role in antinociception, and pain stimulation raises AVP concentration in the periaqueductal gray (PAG). The nociceptive effect of AVP in PAG was investigated in the rat. The results showed that microinjection of AVP into PAG increased pain threshold, whereas microinjection of V2 receptor antagonist-d(CH2)5[d-Ile2, Ile4, Ala9-NH2]AVP into PAG decreased pain threshold in a dose-dependent manner, but local administration of V1 receptor antagonist-d(CH2)5Tyr(Me)AVP did not change pain threshold; Pain stimulation elevated AVP, Leucine-enkephalin (L-Ek), Methionine-enkephalin (M-Ek) and beta-endorphin (beta-Ep), not dynorphinA(1-13) (DynA(1-13)) concentrations in PAG perfuse liquid; PAG pre-treatment with naloxone, an opiate receptor antagonist or V2 receptor antagonist completely reversed AVP-induced increase in pain threshold, however, PAG pre-treatment with V1 receptor antagonist did not influence this effect of AVP administration. The data suggest that AVP in the PAG, through V2 rather than V1 receptor, regulates antinociception, which progress relates to enkephalin and endorphin.