Fluorescent agonists and antagonists for vasopressin/oxytocin G protein-coupled receptors: usefulness in ligand screening assays and receptor studies.

Different series of fluorescent agonists and antagonists have been developed and characterized for arginine-vasopressin and oxytocin G protein-coupled receptors. Both cyclic and linear peptide analogs of the neurohypophysial hormones are useful tools for investigating receptor localization and trafficking, analysing receptor structural organization, and developing new receptor-selective high-throughput ligand screening assays.

Key amino acids located within the transmembrane domains 5 and 7 account for the pharmacological specificity of the human V1b vasopressin receptor.

In mammals, the vasopressin V(1b) receptor (V(1b)-R) is known to regulate ACTH secretion and, more recently, stress and anxiety. The characterization of the molecular determinant responsible for its pharmacological selectivity was made possible by the recent discovery of the first V(1b) antagonist, SSR149415. Based upon the structure of the crystallized bovine rhodopsin, we established a three-dimensional molecular model of interaction between the human V(1b)-R (hV(1b)-R) and SSR149415. Four amino acids located in distinct transmembrane helices (fourth, fifth, and seventh) were found potentially responsible for the hV(1b)-R selectivity. To validate these assumptions, we selectively replaced the leucine 181, methionine 220, alanine 334, and serine 338 residues of hV(1a)-R by their corresponding amino acids present in the hV(1b)-R (phenylalanine 164, threonine 203, methionine 324, and asparagine 328, respectively). Four mutants, which all exhibited nanomolar affinities for vasopressin and good coupling to phospholipase C pathway, were generated. hV(1a) receptors mutated at position 220 and 334 exhibited striking increase in affinity for SSR149415 both in binding and phospholipase C assays at variance with the hV(1a)-R modified at position 181 or 338. In conclusion, this study provides the first structural features concerning the hV(1b)-R and highlights the role of few specific residues in its pharmacological selectivity.

Effects of adrenocorticotropin on action potential and calcium currents in cultured rat and bovine glomerulosa cells.

Action potentials (APs) and ionic currents were recorded in primary cultured rat and bovine glomerulosa cells by using the whole-cell recording technique. Switching from the current-clamp mode to the voltage-clamp mode allowed recordings of APs and currents in the same cell. APs can be elicited by appropriate stimulation in conditions where the excitability of the cell is increased by blocking a transient outward current. A T-current or a N-current was always present in cells in which APs were recorded; an L-current could also be recorded, but a cell presenting only an L-current was not able to fire an AP. The addition of Bay K 8644 (10(-8) M) induced a dramatic increase in the action potential duration. In the same cells, the analysis of the currents showed that the L-current was increased, whereas the T-current was not significantly affected. The effects of ACTH (10(-8) M) were analysed on APs and currents. On APs, at least two phases could be distinguished, the first corresponded to the reduction of the action potential duration, whereas the second was a huge increase of the plateau duration. The T-current was strongly affected by ACTH as a great inhibition took place in the first seconds after the superfusion with a 10(-8) M ACTH medium. Then a partial recovery of the T-current appeared. The effects of ACTh were reversible on washing. On the contrary, the L-current was increased by ACTH, but this effect was not reversible. The effects of ACTH were mimicked by 8 Bromo cAMP (10(-3) M). Similar results were found in rat and bovine glomerulosa cells. These results suggest that second messengers generated by ACTH would regulate Ca2+ entrance by nondetermined phosphorylation process in the sense of an increase in intracellular Ca2+.

Characterization of K+ and Ca2+ ionic currents in glomerulosa cells from human adrenal glands.

Ionic currents of primary cultured glomerulosa cells from human adrenal glands were studied with the patch-clamp technique. Two types of outward K+ currents and two types of inward Ca2+ currents were described. The transient outward K+ current activated at potential positive to -40 mV and demonstrated a marked time-dependent inactivation. It was blocked by 4-aminopyridine but not tetraethylammonium. A second type of outward current activated rapidly at the depolarization onset and then increased slowly with no time-dependent inactivation. The transient inward T-type Ca2+ current was activated for potential positive to -60 mV with a maximal current amplitude at -30 mV and zero current voltage at +40 mV; it was completely inactivated for membrane potential positive to -40 mV. The pharmacological studies of the T-type channel showed that Ni2+ was a potent blocker but that the channel was not sensitive to dihydropyridine. The long-lasting inward Ca2+ current was activated for potentials positive to -20 mV with a maximum current amplitude at +70 mV. This current was increased by the agonist Bay K 8644 and blocked by the antagonist nifedipine; in addition, it was blocked by Cd2+ but less sensitive to Ni2+. This study revealed that glomerulosa cells from human adrenal demonstrated the presence of K+ and Ca2+ currents similar to those found in rat and bovine cells. Moreover, the main stimuli of aldosterone secretion, ACTH and angiotensin II, induce an increase in aldosterone secretion which is inhibited in a Ca(2+)-free external medium.

Fluorescent pseudo-peptide linear vasopressin antagonists: design, synthesis, and applications.

Fluoresceinyl and rhodamyl groups have been coupled by an amide link to side-chain amino groups at positions 1, 6, and 8 of pseudo-peptide linear vasopressin antagonists (Manning et al. Int. J. Pept. Protein Res. 1992, 40, 261-267) through different positions on the fluorophore, to give tetraethylrhodamyl-DTyr(Me)-Phe-Gln-Asn-Arg-Pro-Arg-Tyr-NH2 (2), 4-HOPh(CH2)2CO-DTyr(Me)-Phe-Gln-Asn-Lys(5-carboxyfl uoresceinyl)-Pro-A rg-NH2 (4), 4-HOPh(CH2)2CO-DTyr(Me)-Phe-Gln-Asn-Lys(5- or 6-carboxytetramethylrhodamyl)-Pro-Arg-NH2 (5, 6), 4-HOPh(CH2)2CO-DTyr(Me)-Phe-Gln-Asn-Arg-Pro-Lys(5- or 6- carboxyfluoresceinyl)-NH2 (8, 9), and 4-HOPh(CH2)2CO-DTyr(Me)-Phe-Gln-Asn-Arg-Pro-Lys(5- or 6- carboxytetramethylrhodamyl)-NH2 (10, 11). The closer to the C-terminus the fluorophore, the higher the affinities of the fluorescent derivatives for the human vasopressin V1a receptor transfected in CHO cells. The compound 10 has a Ki of 70 pM, as determined by competition experiments with [125I]-4-HOPhCH2CO-DTyr(Me)-Phe-Gln-Asn-Arg-Pro-Arg-NH2. It showed a good selectivity for human V1a receptor versus human OT (Ki = 1.2 nM), human vasopressin V1b (Ki approximately 27 nM), and human vasopressin V2 (Ki > 5000 nM) receptor subtypes. All fluorescent analogues were antagonists as shown by the inhibition of vasopressin induced inositol phosphate accumulation. These fluorescent ligands are efficient for labeling cells expressing the human V1a receptor subtype, as shown by flow cytofluorometric experiments or fluorescence microscopy. They are also appropriate tools for structural analysis of the vasopressin receptors by fluorescence.

Visualization of cell surface vasopressin V1a receptors in rat hepatocytes with a fluorescent linear antagonist.

To visualize cell surface V1a vasopressin receptors in rat hepatocytes in the absence of receptor-mediated endocytosis, we used a high-affinity fluorescent linear antagonist, Rhm8-PVA. Epifluorescence microscopy (3CCD camera) and fluorescence spectroscopy were used. Rhm8-PVA alone did not stimulate Ca2+ signals and competitively blocked Ca2+ signals (Kinact of 3.0 nM) evoked by arginine vasopressin (vasopressin). When rat hepatocytes were incubated with 10 nM of Rhm8-PVA for 30 min at 4C, the fluorescent antagonist bound to the surface of cells, presumably the plasma membrane. The V1a receptor specificity of Rhm8-PVA binding was confirmed by its displacement by the nonfluorescent antagonist V4253 and by the natural hormone vasopressin at 4C. Prior vasopressin-mediated endocytosis of V1a receptors at 37C abolished binding of the labeled antagonist, whereas in non-preincubated cells, Rhm8-PVA labeled the cell surface of rat hepatocytes. When cells labeled with Rhm8-PVA at 4C were warmed to 37C to initiate receptor-mediated internalization of the fluorescent complex, Rhm8-PVA remained at the cell surface. Incubation temperature at 4C or 37C had little effect on binding of Rhm8-PVA. We conclude that Rhm8-PVA is unable to evoke receptor-mediated endocytosis and can readily be used to visualize cell surface receptors in living cells.

Distribution of signaling molecules involved in vasopressin-induced Ca2+ mobilization in rat hepatocyte multiplets.

In freshly isolated rat hepatocyte multiplets, Ca2+ signals in response to vasopressin are highly organized. In this study we used specific probes to visualize, by fluorescence and confocal microscopy, the main signaling molecules involved in vasopressin-mediated Ca2+ responses. V1a receptors were detected with a novel fluorescent antagonist, Rhm8-PVA. The Galphaq/Galpha11, PLCbeta3, PIP2, and InsP3 receptors were detected with specific antibodies. V1a vasopressin receptors and PIP2 were associated with the basolateral membrane and were not detected in the bile canalicular domain. Galphaq/Galpha11, PLCbeta3, and InsP3 receptors were associated with the basolateral membrane and also with other intracellular structures. We used double labeling, Western blotting, and drugs (cytochalasin D, colchicine) known to disorganize the cytoskeleton to demonstrate the partial co-localization of Galphaq/Galpha11 with F-actin.

Comparison of vasopressin and oxytocin receptors in the rat uterus and vascular tissue.

Several studies indicate that oxytocin and vasopressin receptors in the human uterus are heterogeneous. We have investigated whether oxytocin and vasopressin bind to separate receptors in the day 21 and day 22 pregnant rat uterus and whether uterine vasopressin receptors are the same as the vascular V1A subtype. In isolated organ bath experiments we showed that the potency of d(CH2)5[Tyr(Me)2]vasopressin to inhibit vasopressin contraction in rat aorta was different from that in the day 21 pregnant uterus. Saturation curves of [3H]vasopressin in membranes from cultured aortic myocytes and pregnant uterus were linear and yielded the same 1 nM Kd values. However, the potency of d(CH2)5[Tyr(Me)2]vasopressin and of [Thr4,Gly7]oxytocin at antagonizing [3H]vasopressin confirmed the differences between the vascular smooth muscle and uterine vasopressin receptor. The peptides had respectively higher and lower affinity for aortic cell sites than for uterine sites. It was more difficult to distinguish pharmacological differences for oxytocin and vasopressin receptors in the uterus. On day 22, the high affinity of [Thr4,Gly7]oxytocin and oxytocin for both [3H]oxytocin and [3H]vasopressin binding sites was consistent with the notion that the uterus expresses essentially oxytocin receptors at this stage of gestation. However, oxytocin, vasopressin and three analogs showed a different potency for inhibiting [3H]oxytocin and [3H]vasopressin binding on day 21 versus day 22 of gestation. We conclude that in the rat uterus vasopressin binds to a receptor that is different from the vascular V1A subtype. Also, the binding sites for [3H]vasopressin and [3H]oxytocin on day 21 uterus membranes do not resemble the classical oxytocin receptor as described in the literature suggesting that on day 21 vasopressin and oxytocin bind in the uterus to a receptor that might be different from those currently characterized.

Vasoactive intestinal polypeptide and carbachol act synergistically to induce the hydrolysis of inositol containing phospholipids in the rat superior cervical ganglion.

The effects of vasoactive intestinal polypeptide (VIP) and carbachol on inositol lipid breakdown were assayed in isolated rat superior cervical ganglia. We report here that VIP and carbachol act synergistically to stimulate the formation of inositol phosphates. This synergistic interaction may explain the modulatory effect of VIP on the muscarinic transmission in the sympathetic ganglia.

Vasopressin regulates adrenal functions by acting through different vasopressin receptor subtypes.

In mammals, vasopressin is known to be synthesized in the hypothalamus and released in the blood stream at the pituitary level. This neuropeptide is also synthesized and secreted by the adrenal medulla in many species including human. Moreover, agents like acetylcholine and corticotropin releasing factor stimulates its basal secretion. V1a vasopressin receptors are present in the adrenal cortex and are involved in steroids secretion (aldosterone in the zona glomerulosa and glucocorticoids in the zona fasciculata of some species). These receptors are coupled to phospholipase C beta and to dihydropyridine-sensitive calcium channels via heterotrimeric G proteins differing by their sensitivities to pertussis toxin. The adrenal medulla, from many species, exhibits V1a vasopressin receptors. In rat adrenal medulla, functional V1b vasopressin receptors could also be characterized. These receptors stimulate catecholamines secretion via activation of phospholipase C beta and subsequent mobilization of intracellular calcium. The adrenal medulla secretes AVP and exhibits functional vasopressin receptors. The adrenal cortex also possesses functional vasopressin receptors and is in contact with adrenal medulla via "medullary rays". We may thus reasonably conclude that AVP physiologically regulates adrenal gland functions via autocrine/paracrine mechanisms.

Nephrogenic syndrome of inappropriate antidiuresis.

Mutations in the vasopressin V2 receptor gene are responsible for two human tubular disorders: X-linked congenital nephrogenic diabetes insipidus, due to a loss of function of the mutant V2 receptor, and the nephrogenic syndrome of inappropriate antidiuresis, due to a constitutive activation of the mutant V2 receptor. This latter recently described disease may be diagnosed from infancy to adulthood, as some carriers remain asymptomatic for many years. Symptomatic children, however, typically present with clinical and biological features suggesting inappropriate antidiuretic hormone secretion with severe hyponatremia and high urine osmolality, but a low plasma arginine vasopressin level. To date, only two missense mutations in the vasopressin V2 receptor gene have been found in the reported patients. The pathophysiology of the disease requires fuller elucidation as the phenotypic variability observed in patients bearing the same mutations remains unexplained. The treatment is mainly preventive with fluid restriction, but urea may also be proposed.

Oxytocin and vasopressin agonists and antagonists as research tools and potential therapeutics.

We recently reviewed the status of peptide and nonpeptide agonists and antagonists for the V(1a), V(1b) and V(2) receptors for arginine vasopressin (AVP) and the oxytocin receptor for oxytocin (OT). In the present review, we update the status of peptides and nonpeptides as: (i) research tools and (ii) therapeutic agents. We also present our recent findings on the design of fluorescent ligands for V(1b) receptor localisation and for OT receptor dimerisation. We note the exciting discoveries regarding two novel naturally occurring analogues of OT. Recent reports of a selective VP V(1a) agonist and a selective OT agonist point to the continued therapeutic potential of peptides in this field. To date, only two nonpeptides, the V(2) /V(1a) antagonist, conivaptan and the V(2) antagonist tolvaptan have received Food and Drug Administration approval for clinical use. The development of nonpeptide AVP V(1a), V(1b) and V(2) antagonists and OT agonists and antagonists has recently been abandoned by Merck, Sanofi and Pfizer. A promising OT antagonist, Retosiban, developed at Glaxo SmithKline is currently in a Phase II clinical trial for the prevention of premature labour. A number of the nonpeptide ligands that were not successful in clinical trials are proving to be valuable as research tools. Peptide agonists and antagonists continue to be very widely used as research tools in this field. In this regard, we present receptor data on some of the most widely used peptide and nonpeptide ligands, as a guide for their use, especially with regard to receptor selectivity and species differences.

Past, present and future of vasopressin and oxytocin receptor oligomers, prototypical GPCR models to study dimerization processes.

The G protein-coupled receptors (GPCRs) play a major role in the regulation of physiological function. The emergence of the concept of GPCR oligomerization deeply modifies our understanding of their functioning. Much more than a simple association leading to an independent functioning, the GPCR oligomerization affects various steps such as membrane targeting of the receptors, binding of ligands, coupling to the intracellular pathways and internalization. Although significant advances have been performed in proving the existence of GPCR oligomers, its physiological impact remains to be established. Vasopressin and oxytocin receptors have constituted interesting experimental models in oligomer analysis. Because of the pharmacological tools available regarding these receptors and their expression at a high level in various tissues they can constitute very promising models to study the consequences of oligomerization in physiology.

Three components of the calcium current in cultured glomerulosa cells from rat adrenal gland.

1. Ca2+ channels were studied in cultured glomerulosa cells from the rat adrenal gland. The whole-cell configuration of the patch-clamp technique was used. Cs+-filled pipettes were used in order to block K+ channels. 2. Three Ca2+ components were found, namely, T, L and N, according to the nomenclature proposed by Nowycky, Fox & Tsien (1985). The T-component was a fast transient component activated in the range -60 to -40 mV; the L-component did not inactivate for a sustained depolarization and activated at voltages around -30 mV; the third component, the N-component, was transient and was activated at voltages close to -20 mV. 3. A statistical analysis made on seventy-one experiments showed that the L-component was the most frequent (65% of the experiments), followed by the T- and finally the N- components (59 and 29% of the experiments, respectively). 4. The substitution of Ba2+ ions for Ca2+ ions greatly enhanced the L-component's amplitude (iBa/iCa = 4) while the N-component was unaffected and the T-component was reduced (iBa/iCa = 0.4). 5. A comparison of the voltage-dependent steady-state inactivation of the three components showed that the T-component was inactivated at -60 mV while the inactivation of the L- and N-components was complete at -25 and 0 mV, respectively. 6. A run-down effect was detected in some cells. The time stability of the L-component was lower than that of the T-component. The N-component seemed to be insensitive for at least 1 h. The results for the L- and T-components were obtained in cells which presented no run-down of the current or only a weak one. 7. Cd2+ ions (5 x 10(-5)M) completely blocked the long-lasting component (L-component) and slightly decreased the T-component. 8. Bay K 8644, a dihydropyridine agonist, enhanced the L-component at a concentration of 2.5 microM but decreased it for a higher concentration (5 microM). The T-component was decreased in a reversible way by 1 microM-Bay K 8644. Nifedipine, a well-known antagonist, blocked completely the L-component. This effect was reversed by the addition of Bay K 8644 to the perfusion medium. The T-component was also blocked by nifedipine, a result which is in keeping with the fact that Bay K 8644 has a weak effect on this current.

Background calcium permeable channels in glomerulosa cells from adrenal gland.

The cell-attached recording mode of the patch-clamp technique was used to study Ca2+ permeable background currents of glomerulosa cells from rat and bovine adrenal gland. With a pipette filled with 110 mM BaCl2 or 90 mM CaCl2, three different types of unitary currents were detected. The B1 channel demonstrates a nonlinear I-V curve. The conductances are 4 and 7 pS at -40 and -70 mV, respectively. The curve of the opening probability vs. membrane potential is bell shaped with its maximum at -70 mV. The B2 channel has a conductance of 6 pS, while the B3 channel shows a nonlinear I-V relationship with conductances close to 17 and 10 pS at HPs of -60 and -20 mV. The three types of currents are insensitive to dihydropyridines. We suggest that these background currents could be responsible for the basal calcium influx and aldosterone secretion previously observed in nonstimulated glomerulosa cells.

Membrane-delimited G protein-mediated coupling between V1a vasopressin receptor and dihydropyridine binding sites in rat glomerulosa cells.

In rat glomerulosa cells, vasopressin stimulates intracellular calcium mobilization via at least two distinct mechanisms: the release of calcium from inositol-1,4,5-P3-sensitive stores and the activation of transmembrane calcium influx. In this study, we focused on the second mechanism through three experimental approaches. By videomicroscopically examining Fura-2-loaded cells, we demonstrate that vasopressin induces a dose-dependent and receptor-mediated calcium influx fully inhibited by either 1 microM nifedipine or a pertussis toxin pretreatment and potentiated by 1 microM BAY K 8644. Patch-clamp experiments also indicate that vasopressin stimulates L-type calcium current by 87% and only weakly inhibits T-type calcium current. To further characterize the coupling between the vasopressin receptor and the dihydropyridine calcium channel, we performed binding studies using tritiated nitrendipine. With this technique, we showed that on intact cells, vasopressin is able to increase the specific binding of tritiated nitrendipine in a dose-dependent manner (Kact = 2 nM). Pharmacological studies using a series of vasopressin analogs revealed that this effect is mediated via a V1a vasopressin receptor subtype. Furthermore, the vasopressin-stimulated nitrendipine binding was sensitive to pertussis toxin pretreatment, which affected only the maximum binding capacity of nitrendipine-binding sites. More interestingly, we demonstrate that vasopressin still increases nitrendipine binding to plasma membrane preparation and that GTP is absolutely necessary for such a hormonal effect. Altogether, these data confirm the existence of a tight and direct coupling between the V1a vasopressin receptor and a dihydropyridine calcium channel via a pertussis toxin-sensitive G protein.

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.

Direct identification of human oxytocin receptor-binding domains using a photoactivatable cyclic peptide antagonist: comparison with the human V1a vasopressin receptor.

Understanding of the molecular determinants responsible for antagonist binding to the oxytocin receptor should provide important insights that facilitate rational design of potential therapeutic agents for the treatment of preterm labor. To study ligand/receptor interactions, we used a novel photosensitive radioiodinated antagonist of the human oxytocin receptor, d(CH(2))(5) [Tyr(Me)(2),Thr(4),Orn(8),Phe(3(125)I,4N(3))-NH(2)9]vasotocin. This ligand had an equivalent high affinity for human oxytocin and V(1a) vasopressin receptors expressed in Chinese hamster ovary cells. Taking advantage of this dual specificity, we conducted photoaffinity labeling experiments on both receptors. Photolabeled oxytocin and V(1a) receptors appeared as a unique protein band at 70-75 kDa and two labeled protein bands at 85-90 and 46 kDa, respectively. To identify contact sites between the antagonist and the receptors, the labeled 70-75- and the 46-kDa proteins were cleaved with CNBr and digested with Lys-C and Arg-C endoproteinases. The fragmentation patterns allowed the identification of a covalently labeled region in the oxytocin receptor transmembrane domain III consisting of the residues Leu(114)-Val(115)-Lys(116). Analysis of contact sites in the V(1a) receptor led to the identification of the homologous region consisting of the residues Val(126)-Val(127)-Lys(128). Binding domains were confirmed by mutation of several CNBr cleavage sites in the oxytocin receptor and of one Lys-C cleavage site in the V(1a) receptor. The results are in agreement with previous experimental data and three-dimensional models of agonist and antagonist binding to members of the oxytocin/vasopressin receptor family.