Iodinated photoreactive vasopressin antagonists: labelling of hepatic vasopressin receptor subunits

To identify and characterize V1 vasopressin receptors, photoreactive antagonists of the glycogenolytic and vasoconstrictor activity of vasopressin have been synthesized. The following analogues with 3-mercapto-3,3-cyclopentamethylene-propionic acid (Mca) and N-methylalanine (MeAla) in position 1 and 7 of vasopressin (VP) were effective V1 antagonists: [Mca1, D-Tyr2, MeAla7, Lys8]VP (1), [Mca1, MeAla7, Arg8, Lys9]VP (2), [Mca1, MeAla7, Arg8, D-Lys9]VP (3). Introduction of the photoreactive 4-azidophenylamidino group into the side-chain of Lys8 in analogue 1 or into Lys9 in analogues 2 and 3 increased the potency (for analogue 1 a tenfold increase in the antiglycogenolytic effect and a fivefold increase in the antivasopressor effect) and binding affinity for the rat hepatic V1 receptor. Mono-iodination at Tyr2 with 125I resulted in photoreactive antagonists of high specific radioactivity, which had roughly the same binding affinity as vasopressin for the rat hepatic V1 receptor (Kd = 0.9-1.8 nM). In photoaffinity labelling experiments with purified rat liver membranes, containing 2--3 pmol V1 receptor/mg protein, the analogues labelled specifically two proteins with the relative molecular masses (Mr) of 30,000 and 38,000. These results and the results of a recent study using 3H-labelled photoreactive vasopressin agonists [Boer, R. and Fahrenholz, F. (1985) J. Biol. Chem. 260, 15051-15054] provide evidence that both vasopressin agonists and antagonists can interact with the same two subunits of the heterodimeric hepatic V1 receptor. Furthermore the radioiodinated photoreactive V1 antagonists should be helpful to identify V1 receptor proteins in membranes of other cell types.     

Structure-activity study of novel tricyclic benzazepine arginine vasopressin antagonists

Novel tricyclic benzazepine derivatives were synthesized as arginine vasopressin (AVP) antagonists. Several tricyclic compounds showed potent antagonistic activity in rat AVP receptors V(1a) and V(2). Derivatives containing pyrrolo-tricyclic amines, 13i-k, 30, and 31 also showed selectivity for the V(2) receptor.     

Pyridobenzodiazepines: a novel class of orally active, vasopressin V2 receptor selective agonists

Our efforts in seeking low molecular weight agonists of the antidiuretic peptide hormone arginine vasopressin (AVP) have led to the identification of the clinical candidate WAY-151932 (VNA-932). Further exploration of the structural requirements for agonist activity has provided another class of potent, orally active, non-peptidic vasopressin V2 receptor selective agonists exemplified by the 5,11-dihydro-pyrido[2,3-b][1,5]benzodiazepine as a candidate for further development.     

Arginine vasopressin stimulation of cerebral microvascular endothelial cell Na-K-Cl cotransporter activity is V1 receptor and [Ca] dependent

Ischemia-induced brain edema formation is mediated by increased transport of Na and Cl across an intact blood-brain barrier (BBB). Our previous studies have provided evidence that a luminally located BBB Na-K-Cl cotransporter is stimulated during cerebral ischemia to increase transport of Na and Cl into the brain. The main focus of the present study was to evaluate the effects of arginine vasopressin (AVP), previously shown to be increased in the brain during ischemia and to promote edema formation, on activity of the BBB cotransporter. Cerebral microvascular endothelial cell (CMEC) monolayers were cultured in astroglial cell conditioned medium, and Na-K-Cl cotransporter activity was assessed as bumetanide-sensitive ⁸⁶Rb influx. In both human and bovine CMECs, as well as in freshly isolated microvessels, AVP stimulated cotransport activity. This stimulatory effect was mimicked by V₁ but not V₂ vasopressin agonists and was blocked by V₁ but not V₂ vasopressin antagonists. Consistent with a V₁ vasopressin receptor mechanism of action, AVP caused an increase in CMEC intracellular [Ca] that was blocked by a V₁ antagonist. Exposing the cells to [Ca]-free media and/or reducing intracellular [Ca] by BAPTA also blocked AVP stimulation of CMEC cotransporter activity, as did the phospholipase C inhibitor U-73122. Finally, we found that while stimulation of CMEC cotransporter activity by AVP occurred within minutes, it was also sustained for hours in the continued presence of AVP. These findings support the hypothesis that AVP, through a V₁ receptor- and [Ca]-dependent mechanism, stimulates the BBB Na-K-Cl cotransporter to participate in ischemia-induced edema formation. blood-brain barrier; stroke; cerebral ischemia; brain edema     

An arginyl in the N-terminus of the V1a vasopressin receptor is part of the conformational switch controlling activation by agonist.

Defining how the agonist-receptor interaction differs from that of the antagonist-receptor and understanding the mechanisms of receptor activation are fundamental issues in cell signalling. The V1a vasopressin receptor (V1aR) is a member of a family of related G-protein coupled receptors that are activated by neurohypophysial peptide hormones, including vasopressin (AVP). It has recently been reported that an arginyl in the distal N-terminus of the V1aR is critical for binding agonists but not antagonists. To determine specific features required at this locus to support high affinity agonist binding and second messenger generation, Arg46 was substituted by all other 19 encoded amino acids. Our data establish that there is an absolute requirement for arginyl, as none of the [R46X]V1aR mutant constructs supported high affinity agonist binding and all 19 had defective signalling. In contrast, all of the mutant receptors possessed wildtype binding for both peptide and nonpeptide antagonists. The ratio of Ki to EC50, an indicator of efficacy, was increased for all substitutions. Consequently, although [R46X]V1aR constructs have a lower affinity for agonist, once AVP has bound all 19 are more likely than the wildtype V1aR to become activated. Therefore, in the wildtype V1aR, Arg46 constrains the inactive conformation of the receptor. On binding AVP this constraint is alleviated, promoting the transition to active V1aR. Our findings explain why arginyl is conserved at this locus throughout the evolutionary lineage of the neurohypophysial peptide hormone receptor family of G-protein coupled receptors.     

A novel vasopressin receptor in rat early proximal tubule

In order to evaluate the receptor subtypes of arginine vasopressin (AVP) in early proximal tubule (S1), outer medullary thick ascending limb of Henle's loop (MTAL) and collecting tubule (OMCT), the effect of AVP on intracellular free calcium ([Ca++]i) was determined using the fluorescence indicator Fura-2. Physiological concentration (greater than or equal to 10(-12) M) of AVP in MTAL and OMCT mobilized [Ca++]i in a dose-dependent manner, but relatively high concentration (greater than or equal to 10(-9) M) of AVP in S1 increased [Ca++]i. Moreover, pretreatment with both V1 and V2 antagonists in MTAL or OMCT completely inhibited the AVP-induced [Ca++]i transient, but in S1 partially blocked it. Using several AVP analogues, a relative distribution of AVP receptor subtypes was tentatively calculated in each nephron segment, indicating that although these nephron segments possess V1, its density was very low (about 10%). The majority (about 90%) of AVP receptor in MTAL and OMCT was V2, while that in S1 was a new subtype (named Vp) which is insensitive to V1 and V2 antagonists. To evaluate physiological significance of Vp receptor, AVP-mediated cellular ATP change was measured. Cellular ATP content in S1 was significantly increased by 10(-7) M AVP, but in MTAL it was significantly decreased by the same concentration of AVP. This study suggests that a novel AVP receptor exists in isolated rat S1, and its physiological significance may be the inhibition of ATP-consuming ion transport system.     

V2 vasopressin receptor (V2R) mutations in partial nephrogenic diabetes insipidus highlight protean agonism of V2R antagonists

Inactivating mutations of the V2 vasopressin receptor (V2R) cause cross-linked congenital nephrogenic diabetes insipidus (NDI), resulting in renal resistance to the antidiuretic hormone AVP. In two families showing partial NDI, characterized by an apparently normal response to diagnostic tests and an increase in the basal ADH levels suggesting AVP resistance, we have identified two V2R mutations, Ser-333del and Y128S. Both mutant V2Rs, when expressed in COS-7 cells, show partial defects in vasopressin-stimulated cAMP accumulation and intracellular localization. The inhibition of internalization does not rescue their localization. In contrast, the non-peptide V2R antagonists OPC41061 and OPC31260 partially rescue the membrane localization and basal function of these V2R mutants, whereas they inhibit the basal activity of the wild-type V2R. These results indicate that a partial loss of function of Ser-333del and Y128S mutant V2Rs results from defective membrane trafficking. These findings further indicate that V2R antagonists can act as protean agonists, serving as pharmacological chaperones for inactivating V2R mutants and also as inverse agonists of wild-type receptors. We speculate that this protean agonism could underlie the possible dual beneficial effects of the V2R antagonist: improvement of hyponatremia with heart failure or polycystic kidney disease and potential rescue of NDI.     

Solubilization of human platelet vasopressin receptors

The human platelet membrane receptor for vasopressin (AVP) has been solubilized with the cholic acid derivative detergent 3-( [3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate. Rapid and simple separation of free tritiated AVP ( [3H]AVP) from the solubilized receptor-hormone complex was done by filtration through polyethylenimine-treated filters. [3H]AVP binds to this soluble receptor with an equilibrium dissociation constant of 11.03 +/- 1.86 nM and a maximal number of binding sites = 288 +/- 66 fmol/mg protein while the corresponding values of the membrane-bound receptor are 1.62 +/- 0.21 nM and 237 +/- 38 fmol/mg of protein, respectively. The Ki value for native AVP derived from competition experiments is 11.02 +/- 2.05 nM for the soluble receptor. Competition experiments with specific vascular and renal antagonists confirm that the solubilized receptor belongs to the V1-vascular subtype.     

Central arginine vasopressin and endogenous antipyresis.

Arginine vasopressin (AVP) is a centrally synthesized nonapeptide that exerts classical endocrine effects as well as a host of centrally mediated actions. A strong case can be argued in support of a neurotransmitter-neuromodulator role for AVP. Acting within the central nervous system (CNS), AVP has been demonstrated to be involved in the modulation of febrile body temperature. Because AVP acts to reduce pyrogen-induced fevers, but not normal body temperature, its actions are deemed to be antipyretic. However, to demonstrate an endogenous antipyretic function, AVP must be shown to be active during conditions where fever is naturally suppressed. This review will focus on five such conditions where the absence of pyrogen-induced fever can be linked to the endogenous activity of AVP within the brain. In the neonatal rat pup, the use of specific antagonists to the AVP receptor has revealed a role for CNS AVP in the absence of fever following peripheral injections of bacterial endotoxin. These results may help to explain a similar lack of fever in other newborn species. In parturient animals a reduced or absent febrile response has been linked to the increased presence of AVP within the septal area of the brain. The combined use of AVP receptor antagonism as well as immunohistochemistry has shown enhanced AVP activity within the ventral septal area of the rat and guinea pig brain during tolerance to intravenous pyrogens. These results suggest that the mechanism of fever suppression following repeated systemic injections of bacterial pyrogen includes centrally acting AVP.(ABSTRACT TRUNCATED AT 250 WORDS)     

A single residue (arg46) located within the N-terminus of the V1a vasopressin receptor is critical for binding vasopressin but not peptide or nonpeptide antagonists

A fundamental issue in molecular endocrinology is to define how agonist:receptor interaction differs from antagonist:receptor interaction. The vasopressin V1a receptor (V1aR) is a member of a subfamily of related G protein-coupled receptors that are activated by the hormone AVP or related peptides. The N-terminus of the V1aR has recently been shown to be critical for binding agonists but not antagonists. Using a combination of N-terminally truncated constructs and alanine-scanning mutagenesis, individual residues that provide these agonist-specific binding epitopes have now been identified in this study. Our data establish that a single residue, Arg46, is critical for AVP binding to the V1aR. Systematic substitution revealed that Arg was required at this locus and could not be substituted by Lys, Glu, Leu, or Ala. In contrast, antagonist binding (cyclic or linear, peptide or nonpeptide) was unaffected. Disruption of Arg46 also resulted in defective intracellular signaling. Arginine is conserved at this locus in all members of the neurohypophysial peptide hormone receptor family cloned to date, indicative of a fundamental role in receptor function. In addition to Arg46, the residues Leu42, Gly43, Asp45 form a patch contributing to AVP binding. This study provides molecular insight into the role of the V1aR N-terminus and key differences between agonist and antagonist binding requirements.     

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.     

Theoretical conformational analysis of six arginine vasopressin analogs with the L-naphthylalanine in position 3.

Introduction of the naphthylalanine residue into either position 3 of arginine vasopressin (AVP), or its analogs results in peptides with interesting pharmacological properties. The single substituted analog of AVP with L-2-Nal in position 3 causes moderate antiduretic activity, whereas [Mpa1, (L-1-Nal)3, (D-Arg)8] VP and [Mpa1, (L-2-Nal)3, (D-Arg)8] VP are potent and selective V2 agonists. Moreover [(L-2-Nal)3, (D-Arg)8] VP is among the most potent and selective antagonists of V1a receptors. In this study we carried out conformational calculations on [(L-1-Nal)3] AVP, [(L-2-Nal)3] AVP, [(L-1-Nal)3, (D-Arg)8] VP, [(L-2-Nal)3, (D-Arg)8] VP, [Mpa1, (L-1-Nal)3, (D-Arg)8] VP, [Mpa1, (L-2-Nal)3, (D-Arg)8] VP, using the ECEPP/3 force field with and without including hydration to simulate aqueous and nonpolar environments. It was found that in all six compound studied, the low-energy conformations have common geometry and relative energies. Therefore, the modifications of the Phe in position 3 influence the binding to the receptor by changing the size of the third residue, rather than by changing the conformational space. The lowest-energy conformations in the presence and absence of water had beta-turns at residues Phe3-Gln4 and Gln4-Asn5 and Gln4-Asn5, respectively. The conformation at the Gln4-Asn5 turn was most similar to the crystal structure of the pressinoic acid (the cyclic moiety of vasopressin).     

5-fluoro-2-methyl-N-[5-(5H-pyrrolo[2,1-c][1,4]benzodiazepine-10(11H)-yl carbonyl)-2-pyridinyl]benzamide (CL-385004) and analogs as orally active arginine vasopressin receptor antagonists.

Synthesis and structure-activity relationships (SAR) of orally active arginine vasopressin (AVP) receptor antagonists are discussed. Potent and orally active AVP receptor antagonists are produced when ring A of VPA-985 (1) is replaced with a 3-pyridinyl unit (2b).     

Effect of vasopressin on type IV collagen production in human mesangial cells

Production of extracellular matrix proteins, such as type IV collagen and fibronectin, by mesangial cells contributes to progressive glomerulosclerosis. In this study, the ability of vasopressin (AVP), which causes mesangial cell proliferation and hypertrophy, to stimulate type IV collagen production by cultured human mesangial cells was examined using an enzyme-linked immunosorbent assay. AVP induced a concentration-dependent increase in the production of type IV collagen and this effect was potently and concentration-dependently inhibited by AVP V_1A receptor antagonists, including YM218. AVP also induced a concentration-dependent increase in transforming growth factor (TGF)-β secretion by human mesangial cells and this effect was inhibited by V_1A receptor antagonists. Furthermore, TGF-β also induced an increase in the production of type IV collagen; the AVP-enhanced production of type IV collagen was inhibited by an anti-TGF-β antibody. These findings indicate that AVP stimulates synthesis of type IV collagen by cultured human mesangial cells through the induction of TGF-β synthesis mediated by V_1A receptors; consequently, AVP contributes to glomerular remodeling and extracellular matrix accumulation observed in glomerular diseases.     

Desensitization of vasopressin sensitive adenylate cyclase by vasopressin and phorbol esters

Desensitization of vasopressin V2 receptor-mediated adenylate cyclase was studied in canine kidney cell line, MDCK cells. Overnight treatment of MDCK cells with arginine vasopressin (AVP) resulted in a loss of vasopressin receptors and an inhibition of cAMP accumulation in response to AVP. Both the loss of receptor and reduction in cAMP accumulation were time- and AVP concentration-dependent. Desensitization was selective for AVP because cAMP formation in response to isoproterenol, prostaglandin E1 (PGE1) and forskolin was not affected by AVP pre-treatment. Pre-treatment of MDCK cells with phorbol dibutyrate (PDBu) also caused a dose-dependent inhibition of AVP mediated cAMP accumulation, but not of isoproterenol-, PGE1- and forskolin-induced cAMP accumulation. PDBu pre-treatment did not cause loss of vasopressin receptors. Instead, the affinity for vasopressin was changed by PDBu treatment. Pre-treatment of the cells with pertussis toxin (PT) had no effect on the desensitization and downregulation of vasopressin (V2) receptors, suggesting that the desensitization may not be mediated by pertussis toxin sensitive G-protein. Our data suggest that pre-treatment of MDCK cells with AVP or PDBu caused desensitization of AVP-mediated cAMP accumulation and that downregulation of V2 receptors required agonist occupancy of the receptors, whereas the affinity of the receptors was changed by phorbol ester treatment.     

Apelin, a neuropeptide that counteracts vasopressin secretion

Apelin is a peptide that was recently isolated as the endogenous ligand for the human orphan APJ receptor, a G protein-coupled receptor which shares 31 % amino-acid sequence identity with the angiotensin type 1 receptor. Apelin naturally occurs in the brain and plasma as 13 (pE13F) and 17 amino-acid (K17F) fragments of a single pro-peptide precursor. In transfected CHO cells, K17F and pE13F bind with high affinity to the rat APJ receptor, promote receptor internalization, and inhibit forskolin-induced cAMP formation. In the same cells, pE13F activates MAP kinase and PI3 kinase pathways. Apelin and APJ receptors are both widely distributed in the brain but are particularly highly expressed in the supraoptic (SON) and paraventricular (PVN) hypothalamic nuclei. Dual labeling studies demonstrate that within these two nuclei, apelin and its receptor are colocalized with vasopressin (AVP) in a subset of magnocellular neurons. In lactating rats, characterized by increases in both synthesis and release of AVP, central injection of apelin inhibits the phasic electrical activity of AVP neurons, reduces plasma AVP levels, and increases aqueous diuresis. Moreover, water deprivation, while increasing the activity of AVP neurons, reduces plasma apelin concentrations and induces an intra-neuronal pile up of the peptide, thereby decreasing the inhibitory effect of apelin on AVP release and preventing additional water loss at the kidney level. Taken together, these data demonstrate that apelin counteracts the effects of AVP in the maintenance of body fluid homeostasis. In addition, apelin and its receptor are present in the cardiovascular system, i.e. heart, kidney and vessels. Systemically administered apelin reduces arterial blood pressure, increases cardiac contractility and reduces cardiac loading. The development of non peptidic analogs of apelin may therefore offer new therapeutic avenues for the treatment of cardiovascular disorders.     

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+.     

Centrally administered vasopressin antagonizes pentobarbital-induced narcosis and depression of hippocampal cholinergic activity

Intracerebroventricular (ICV) microinjection of arginine vasopressin (AVP) to pentobarbital-anesthetized rats produced shortening of the duration of narcosis. This analeptic effect was blocked by atropine, indicating the central cholinergic nature of the response. AVP also increased hippocampal sodium-dependent high affinity choline uptake activity that had been depressed by the barbiturate. The AVP analeptic effect was blocked by pretreatment with a V-1 (vasopressor), but not a V-2 (antidiuretic), vasopressin receptor antagonist. These results suggest that ICV AVP produces its analeptic effect by interacting with central V-1 receptors to activate a hippocampal cholinergic arousal system. The cholinergic arousal effect may be a factor in the memory enhancing property of AVP.     

Thirst impairment elicited by intraventricular administration of vasopressin antagonists

To determine whether centrally released vasopressin influences thirst, observations of osmotic thirst threshold, osmotic load excretion and postloading restitution of plasma osmolality were made in dogs in control experiments and during infusion of AVP antagonists into the third ventricle. Significant elevation of osmotic thirst threshold was elicited by infusion of d(CH₂)₅AVP at a rate of 0.2–2.0 μg·min⁻¹ and of d(Et₂)AVP at a rate of 0.3 μg·min⁻¹ (V₁ antagonists, weak V₂ agonists) as well as by administration of d(CH₂)₅[D-Ile²,Abu⁴]AVP at a rate of 0.4 μg·min⁻¹ (potent V₂ antagonist, weak V₁ antagonist). Administration of d(CH₂)₅AVP at a rate of 2.0 μg·min⁻¹ was associated with a significant suppression of the postloading water intake and osmotic load excretion and with a delay in restitution of plasma osmolality. These findings indicate that centrally released vasopressin may participate in the control of thirst.     

Effects of i.c.v. losartan on the angiotensin II-mediated vasopressin release and hypothalamic fos expression in near-term ovine fetuses

Our previous studies have shown that central administration of angiotensin (ANG II) causes arginine vasopressin (AVP) release in the fetus at 70-90% gestation. This is evidence that the hypothalamic-neurohypophysial system is relatively mature before birth. However, few data exist regarding central ANG receptor mechanisms-mediated AVP response during fetal life. To determine roles of brain ANG receptor subtypes in this response, AT1 and AT2 receptor antagonists, losartan and PD123319, were investigated in the brain in chronically prepared ovine fetuses at the last third of gestation. Application of losartan intracerebroventricularly (i.c.v.) at 0.5 mg/kg suppressed central ANG II-stimulated plasma AVP release. Losartan at 5 mg/kg (i.c.v.) demonstrated a significant enhancement of AVP increase to i.c.v. ANG II. Associated with the increase of plasma vasopressin levels, c-fos expression in the hypothalamic neurons was significantly different between the low and high doses of losartan. The low dose losartan markedly reduced the dual immunoreactivity for FOS and AVP in the supraoptic nuclei and paraventricular nuclei after i.c.v. ANG II, whereas the high dose losartan together with ANG II, significantly increased the co-localization of positive FOS in the AVP-containing neurons than that induced by i.c.v. ANG II alone. Central ANG II induced fetal plasma vasopressin increase was not altered by PD123319. The data suggest that losartan in the fetal brain has remarkably different effects based on the doses administrated on central ANG II-related neuroendocrine effects at the late gestation, and that the AT1 mechanism is critical in the regulation of fetal body fluid homeostasis related to plasma AVP levels.