Serge Jard

Localization and pharmacological characterization of high affinity binding sites for vasopressin and oxytocin in the rat brain by light microscopic autoradiography

Sites which bind tritiated vasopressin (AVP) with high affinity were detected in the brain of male, adult rats, by light microscopic autoradiography. Their anatomical localization differed markedly from that of high affinity binding sites for tritiated oxytocin (OT) determined in the same animal. Co-labelling was minimized by using low concentrations of [3H]AVP and [3H]OT. Binding of the former occurred predominantly in several structures of the limbic system (septum, amygdala, bed nucleus of the stria terminalis, accumbens nucleus), in two hypothalamic nuclei (suprachiasmatic and dorsal tuber) and in the area of the nucleus of the solitary tract. Binding of OT was evidenced in the olfactory tubercle, the ventromedial hypothalamic nucleus, the central amygdaloid nucleus and the ventral hippocampus. The ligand specificity of the binding sites was assessed in competition experiments. Synthetic structural analogues were used, allowing to discriminate OT receptors (OH[Thr4,Gly7]OT) from V2 receptors (dDAVP and d[Tyr(Me)2]VDAVP), V1 receptors ([Phe2,Orn8]VT) and V1b receptors (desGly9d(CH2)5AVP). Our main conclusions are, firstly, that AVP and OT binding sites can be readily distinguished, and that there is virtually no overlap in their distribution in the rat brain. Second, we showed that the sites which bind AVP with high affinity in the brain are V1 receptors, different both from the renal V2 receptors and from the anterior pituitary V1b receptors. Our results support the conjecture that AVP and OT play a role in interneuronal communication in the brain.

125I-labelled d(CH2)5[Tyr(Me)2,Thr4,Tyr-NH29]OVT: a selective oxytocin receptor ligand

An oxytocic antagonist, [1-(beta-mercapto-beta, beta-cyclopentamethylenepropionic acid,2-O-methyltyrosine,4-threonine, 8-ornithine,9-tyrosylamide]vasotocin (d(CH2)5[Tyr(Me)2, Thr4,Tyr-NH2(9)]OVT [corrected], was monoiodinated at the phenyl moiety of the tyrosylamide residue at position 9. 125I-labelling was performed with 1,3,4,6-tetrachloro-3 alpha,6 alpha-diphenyl-glycoluril. Iodination resulted in an increased affinity for rat uterine oxytocin receptors. A considerably lower affinity for rat vascular V1- and renal V2-receptors was found, resulting in a highly specific oxytocin receptor ligand. 125I-labelled d(CH2)5[Tyr(Me)2,Thr4,Tyr-NH2(9)]OVT [corrected] was demonstrated to bind selectively to one population of binding sites in rat uterus and ventral hippocampal membrane preparations. Dissociation constants ranged between 0.03 and 0.06 nM. After 3 days of exposure autoradiography revealed binding in regions known to contain oxytocin receptors as well as labelling in some new regions, while no binding was found in the lateral septum, a structure containing mainly [8-arginine]vasopressin receptors. The high specific radioactivity of 125I-labelling allowed important reductions in membrane protein amount, gain in precision of binding analysis as well as considerably lower exposure times for autoradiography.

The Binding Site of Neuropeptide Vasopressin V1a Receptor EVIDENCE FOR A MAJOR LOCALIZATION WITHIN TRANSMEMBRANE REGIONS

To identify receptor functional domains underlying binding of the neurohypophysial hormones vasopressin (AVP) and oxytocin (OT), we have constructed a three-dimensional (3D) model of the V1a vasopressin receptor subtype and docked the endogenous ligand AVP. To verify and to refine the 3D model, residues likely to be involved in agonist binding were selected for site-directed mutagenesis. Our experimental results suggest that AVP, which is characterized by a cyclic structure, could be completely buried into a 15-20-Å deep cleft defined by the transmembrane helices of the receptor and interact with amino acids located within this region. Moreover, the AVP-binding site is situated in a position equivalent to that described for the cationic neurotransmitters. Since all mutated residues are highly conserved in AVP and OT receptors, we propose that the same agonist-binding site is shared by all members of this receptor family. In contrast, the affinity for the antagonists tested, including those with a structure closely related to AVP, is not affected by mutations. This indicates a different binding mode for agonists and antagonists in the vasopressin receptor.

Neurohypophyseal hormone receptor systems in brain and periphery.

Publisher Summary This chapter focuses on the similarities and differences between vasopressin or oxytocin receptors in the brain and peripheral receptors for these peptides. The chapter also discusses the transduction mechanisms activated by vasopressin and oxytocin receptors in the brain and peripheral nervous system. Vasopressin and oxytocin meet most of the criteria that are applied to assign a role as a neurotransmitter or a neuromodulator to a given compound. It is recognized that vasopressin and oxytocin are synthesized at multiple sites in the brain in neurons projecting to several areas. A calcium-dependent release of oxytocin and (or) vasopressin can be evoked by potassium or veratridine in various extrahypothalamic areas of the brain. No direct effect of vasopressin on the adenylate cyclase activity of nervous tissue can be demonstrated. Vasopressin stimulates inositol lipid breakdown in hippocampal slices and isolated sympathetic ganglia.

Vasopressin Isoreceptors in Mammals: Relation to Cyclic AMP-Dependent and Cyclic AMP-Independent Transduction Mechanisms

Publisher Summary The chapter reviews available pharmacological and biochemical data on vasopressin receptors in mammals and discusses the validity of the different criteria that can be used to distinguish vasopressin isoreceptors. A comparison of vasopressin receptors in mammals is presented with respect to (1) the kinetics of hormonal binding, (2) the nature of their primary effectors, (3) the nature of the putative modulators of receptor-effector coupling, (4) the known physiocochemical characteristics of solubilized receptors, and (5) respective recognition patterns. There are two types of vasopressin receptors, V1 and V2, distinguished on the basis of their functional coupling either to adenylate cyclase (V2 receptors) or to the cellular mechanisms involved in the regulation of calcium entry in the cell and (or) mobilization of cellular calcium (V1 receptors). Specific vasopressin binding sites are characterized in isolated cells or membrane fractions derived from three vasopressin-responsive tissues: kidney, liver, and blood vessels. Unlike vasopressin receptors triggering the tubular effects (increase in water and solute transport) of vasopressin on the kidney, the receptors present on hepatocytes and vascular smooth muscle cells are not functionally coupled to adenylate cyclase. The primary involvement of a rise in cytosolic cell calcium in the glycogenolytic response of hepatocytes and in the contractile response of vascular smooth muscle cells to vasopressin is established. Several other biological effects of vasopressin such as a platelet aggregation and corticotropin release by the adenohypophysis are also elicited by calcium-dependent, CAMP-independent mechanisms.

Two aromatic residues regulate the response of the human oxytocin receptor to the partial agonist arginine vasopressin

We investigated the mechanisms that regulate the efficacy of agonists in the arginine‐vasopressin (AVP)/oxytocin (OT) receptor system. In this paper, we present evidence that AVP, a full agonist of the vasopressin receptors, acts as a partial agonist on the oxytocin receptor. We also found that AVP becomes a full agonist when two aromatic residues of the oxytocin receptor are replaced by the residues present at equivalent positions in the vasopressin receptor subtypes. Our results indicate that these two residues modulate the response of the oxytocin receptor to the partial agonist AVP.

A radioiodinated linear vasopressin antagonist: A ligand with high affinity and specificity for V1a receptors

A linear vasopressin antagonist, Phaa‐D‐Tyr(Me)‐Phe‐Gin‐Asn‐Arg‐Pro‐Arg‐Tyr‐NH2 (Linear AVP Antag) (Phaa = Phenylacetyl), was monoiodinated at the phenyl moiety of the tyrosylamide residue at position 9. This antagonist appeared to be a highly potent anti‐vasopressor peptide with a pA2 value in vivo of 8.94. It was demonstrated to bind to rat liver membrane preparations with a very high affinity (K d = 0.06 nM). The affinity for the rat uterus oxytocin receptor was lower (K i = 2.1 nM), and affinities for the rat kidney‐ and adenohypophysis‐vasopressin receptors were much lower (K i 47 nM and 92 nM, respectively), resulting in a highly specific vasopressin V18 receptor ligand. Autoradiographical studies using rat brain slices showed that this ligand is a good tool for studies on vasopressin receptor localization and characterization.

Characterization of a novel, linear radioiodinated vasopressin antagonist: an excellent radioligand for vasopressin V1a receptors.

We report on the pharmacological properties of a potent and selective linear vasopressin (AVP) V1a receptor antagonist HO-Phenylacetyl1-D-Tyr(Me)2-Phe3-Gln4-Asn5-Arg6-Pro7-Arg8-NH2 (HO-LVA). Iodinated on the phenolic substituent at position 1, [125I]-HO-LVA displayed the highest affinity for rat liver V1a receptors (8 pM) ever reported. Furthermore, affinities of HO-LVA and I-HO-LVA for V1b, V2 and oxytocin (OT) receptors was 400- to 1,000-fold lower than for V1a receptors, rendering it a highly selective ligand. Both HO-LVA and its iodinated derivative are V1 antagonists, they potently inhibited AVP-induced inositol-phosphate accumulation in WRK1 cells, and also, although with a much lower potency, the AVP-induced ACTH release from freshly prepared pituitary cells. Using autoradiography [125I]-HO-LVA appeared to be the first radioligand to successfully identify and localize the presence of V1a receptors in rat liver and blood vessel walls. Moreover, several new brain regions expressing V1a receptors could be identified, in addition to those brain regions that were previously identified with other radiolabelled AVP analogues.

Vasoactive intestinal polypeptide increases inositol phospholipid breakdown in the rat superior cervical ganglion.

The effects of VIP and of peptides of the VIP family: secretin, glucagon, the porcine histidine isoleucine containing peptide (PHI) and the rat hypothalamic growth hormone-releasing hormone (rhGRF) on the cyclic AMP and inositol phosphate contents of isolated rat superior cervical ganglia were investigated. We demonstrate that VIP is able to provoke a large inositol lipid breakdown by acting directly on ganglionic cells. This observation suggests the presence in rat superior cervical ganglia of a new type of receptors for VIP or for an unidentified peptide structurally related to VIP.

Localization and characterization of vasopressin binding sites in the rat brain using an iodinated linear AVP antagonist

The binding characteristics and central distribution of 125I-Linear AVP antagonist, a new ligand for vasopressin binding sites, are described in the following studies. Saturation studies performed on rat brain septal membranes demonstrated that 125I-Linear AVP antagonist binds to a single class of sites with high affinity (55 pM) and limited capacity (88 fmol/mg protein). In autoradiographic studies, 125I-Linear AVP antagonist labeled brain areas known to contain vasopressin receptors without binding to neurophysins. 125I-Linear AVP antagonist also labeled sites in cortex, hypothalamus, ventral tegmental area and substantia nigra. In competition studies, 125I-Linear AVP antagonist binding was most readily blocked by AVP and a selective V1a agonist. Oxytocin and a selective V2 ligand were effective only in micromolar concentrations. A selective oxytocin agonist was virtually ineffective in blocking 125I-Linear AVP antagonist binding. In regions that contain a high density of oxytocin binding sites, however, oxytocin-displaceable binding was observed. In agreement with studies on peripheral tissues, the binding profile generated from these studies indicates that 125I-Linear AVP antagonist binds to vasopressin receptors of the V1a subtype. These results suggest that 125I-Linear AVP antagonist is a valuable ligand for the study of central AVP receptors.