Vasopressin V2 Receptor/Bioligand Interactions

We predict some essential interactions between the V2 vasopressin renal receptor (V2R) and its agonists [Arg⁸]vasopressin (AVP) and [D-Arg⁸]vasopressin (DAVP), and the non-peptide antagonist OPC-31260. V2R controls antidiuresis and belongs to the superfamily of heptahelical transmembrane (7TM) G-protein-coupled receptors (GPCRs). The receptor was built, the ligands were docked and the structures relaxed using advanced molecular modeling techniques. Docked agonists and antagonists appear to prefer similar V2R compartments. A number of receptor amino acid residues are indicated, mainly in the TM3–TM7 helices, as potentially important in ligand binding. Many of these residues are invariant for either the GPCR superfamily or the subfamily of related (vasopressin V2R, V1aR and V1bR and oxytocin OR) receptors. Moreover, some of the equivalent residues in V1aR have already been found critical for ligand affinity [Mouillac et al., J. Biol. Chem., 270 (1995) 25771].     

Vasopressin V2 Receptor/Bioligand Interactions

Summary We predict some essential interactions between the V2 vasopressin renal receptor (V2R) and its agonists [Arg⁸]vasopressin (AVP) and [D-Arg⁸]vasopressin (DAVP), and the non-peptide antagonist OPC-31260. V2R controls antidiuresis and belongs to the superfamily of heptahelical transmembrane (7TM) G-protein-coupled receptors (GPCRs). The receptor was built, the ligands were docked and the structures relaxed using advanced molecular modeling techniques. Docked agonists and antagonists appear to prefer similar V2R compartments. A number of receptor amino acid residues are indicated, mainly in the TM3-TM7 helices, as potentially important in ligand binding. Many of these residues are invariant for either the GPCR superfamily or the subfamily of related (vasopressin V2R, V1aR and V1bR and oxytocin OR) receptors. Moreover, some of the equivalent residues in V1aR have already been found critical for ligand affinity [Mouillac et al., J. Biol. Chem., 270 (1995) 25771].     

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.     

Lateral mobility of the phospholipase C-activating vasopressin V1-type receptor in A7r5 smooth muscle cells: a comparison with the adenylate cyclase-coupled V2-receptor.

The present work examines lateral mobility of the vasopressin V1-type receptor, representing the first determination of lateral mobility of a hormone receptor coupled to phospholipase C activation. The V1-receptor of A7r5 smooth muscle cells was characterized for [Arg8] vasopressin (AVP) binding properties and affinity for the fluorescent vasopressin analogue 1-deamino[8-lysine (N6-tetramethylrhodamylaminothiocarbonyl)] vasopressin (TR-LVP). TR-LVP was biologically active in A7r5 cells, inducing inositol 1,4,5-trisphosphate turnover in similar fashion to AVP. TR-LVP was used to specifically label the V1-receptor of living A7r5 cells, and lateral mobility of the V1-receptor was measured using the technique of fluorescence microphotolysis. The apparent lateral diffusion coefficient (D) at 37 degrees C was 5.1 x 10(-10) cm2/s, falling to 2.9 x 10(-10) cm2/s at 13 degrees C. These D values are higher than comparable values for the adenylate cyclase-activating vasopressin V2-receptor of LLC-PK1 renal epithelial cells analysed with the same fluorescent ligand. In contrast to the V2-receptor, no marked temperature dependence was observed for the V1-receptor mobile fraction (f). From 37 degrees C to 13 degrees C, f was relatively low (between 0.4 and 0.5) consistent with V1-receptor immobilization through internalization, which is rapid even at room temperature in A7r5 cells. These differences between V1- and V2-receptor lateral mobility are discussed in terms of the implications for their respective signal transduction systems.     

Renal function in transgenic rats expressing an angiotensin-(1-7)-producing fusion protein

Transgenic rats [TGR(A1-7)3292] present a chronic 2.5-fold increase in plasma Angiotensin-(1-7) [Ang-(1-7)] concentration. In the present study, we investigated the effects of this chronic elevation on renal function, vasopressin levels, kidney morphology, expression of Ang-(1-7) and vasopressin receptors in TGR(A1-7)3292. Urine volume and water intake were measured for 24 h. At the end of this period, plasma and urine samples were collected to evaluate renal function parameters and circulating vasopressin levels. Expression of renal V2 receptors and Mas was assessed by ribonuclease protection assay. Renal slices were processed for histological analysis. The urine flow of TGR(A1-7)3292 was significantly lower in comparison with Sprague-Dawley rats. The reduced urine volume of TGR(A1-7)3292 was accompanied by a significant increase in urinary osmolality and decrease free water clearance. Glomerular filtration rate, urinary sodium and potassium excretion were similar in both strains. No significant changes were observed in vasopressin levels as well as in V2 receptor and Mas mRNA expression in renal tissue. No changes in kidney structure of TGR(A1-7)3292 were detected. These data suggest that changes in circulating renin-angiotensin system produced by chronic increase of Ang-(1-7) levels can lead to adjustments in the water balance that are independent of vasopressin release and V2 receptor expression.     

Renal vasopressin receptor expression and function in rats following spaceflight.

It has been suggested there is a decreased renal responsiveness to vasopressin following spaceflight and that this may be the mechanism for the increased urine flow that is observed following return to normal gravity. In the present study, we have therefore measured vasopressin receptor expression and activity in kidneys taken from rats 1 and 14 days following spaceflight of 15 days duration. Measurements of renal vasopressin V(2) and V(1a) receptor mRNA expression by quantitative RT-PCR demonstrated little difference at either 1 day or at 14 days following return from space. Evaluation of (3)H-labeled arginine vasopressin binding to membranes prepared from kidneys indicated that the majority of the vasopressin receptors were V(2) receptors. Furthermore, the data suggested that binding to vasopressin V(2) or V(1a) receptors was unaltered at 1 day and 14 days following spaceflight. Similarly, the ability of vasopressin to stimulate adenylate cyclase suggested no change in vasopressin V(2) receptor activity in these animals. These data suggest that, whatever changes in fluid and electrolyte metabolism are observed following spaceflight, they are not mediated by changes in vasopressin receptor number or vasopressin-induced stimulation of adenylate cyclase.     

New V1a receptor antagonist. Part 1. Synthesis and SAR development of urea derivatives

Solid preclinical evidence links vasopressin to social behavior in animals, so, extensive work has been initiated to find new vasopressin V1a receptor antagonists which can improve deteriorated social behavior in humans and can treat the core symptoms of autistic behavior, as well. Our aim was to identify new chemical entities with antagonizing effects on vasopressin V1a receptors. Starting from a moderately potent HTS hit (7), we identified a molecule (49) having nanomolar binding strength and functional activity, which is in the same range as the potency of clinically tested V1a antagonists.     

MCF-7 breast cancer cells express normal forms of all vasopressin receptors plus an abnormal V2R.

We have previously provided evidence that an autocrine loop involving vasopressin is present in perhaps all breast cancers. This study now shows MCF-7 breast cancer cells express mRNAs for all currently recognized vasopressin receptor subtypes (V1a, V1b, and V2). Cloning and DNA sequencing over the entire open reading frame of each mRNA revealed that normal sequences representing each receptor were present. However, in addition, an abnormal mRNA for the V2 receptor, expected to give rise to a truncated 'diabetic' protein, was also expressed. Western analysis revealed that all three normal mRNAs gave rise to proteins of sizes compatible with them being functional receptors. The abnormal V2 receptor mRNA also gave rise to proteins.     

Vasopressin antisense peptide interactions with the V1 receptor

The molecular recognition hypothesis, that peptide ligands and their receptor binding sites are encoded by complementary nucleotide sequences, was tested for arginine vasopressin (AVP) and its V1 receptor. Binding of [125I] [d(CH2)5,Sar7]AVP (a selective V1 vasopressin antagonist radioligand) or [3H]AVP to rat liver plasma membranes was inhibited by peptides known to bind to V1 receptors but not by the AVP complementary peptide (Ser-Ser-Trp-Ala-Val-Leu-Glu-Val-Ala) (PVA). Rabbit anti-PVA antibodies were nonimmunoreactive with any protein in rat liver membranes or in a partially purified preparation from rat liver containing reconstitutable vasopressin binding activity. Furthermore, there was no suppression of the AVP pressor effect by PVA in vivo using a rat blood pressure bioassay. These findings do not support the hypothesis that the V1 receptor binding site is encoded by the antisense DNA strand to AVP.     

Generation of anti-idiotypic monoclonal antibodies recognizing vasopressin receptors in cultured cells and kidney sections.

To produce anti-idiotypic antibodies against receptors for the neurohypophyseal hormone vasopressin, an anti-vasopressin monoclonal antibody with a ligand specificity similar to that of vasopressin receptors was employed for immunization. Three anti-idiotypic monoclonal antibodies were obtained which induced, like vasopressin, plasminogen activator production in the renal epithelial cell line LLC-PK1 (expressing V2-receptors). Induction of plasminogen activator synthesis by the anti-idiotypic antibodies could be inhibited by coincubation with a vasopressin antagonist. In a fashion similar to that of vasopressin itself, the anti-idiotypic antibodies induced receptor down-regulation. The anti-idiotypic antibodies were employed to visualize vasopressin receptors on LLC-PK1 and A7r5 (V1-receptor-expressing) smooth muscle cells by immunofluorescence. Antibody-mediated fluorescence was not observed in receptor-deficient mutant cell lines or vasopressin-receptor-down-regulated cells. Furthermore, these antibodies were used for immunohistochemical localization of vasopressin receptors in rat and bovine kidney preparations. In accordance with earlier physiological and biochemical observations, vasopressin receptors were detected predominantly in collecting ducts in cortex and medulla. On the cellular level, a differential staining pattern was observed.     

Conformation of [8-arginine]vasopressin and V1 antagonists in dimethyl sulfoxide solution derived from two-dimensional NMR spectroscopy and molecular dynamics simulation

Structural and dynamic properties of [8-arginine]vasopressin and a class of highly potent vasopressin V1 antagonists which contain 3-mercapto-3,3-cyclopentamethylene propionic acid (Mca) in position 1 of the vasopressin sequence have been determined. On the basis of two-dimensional NMR experiments in dimethyl sulfoxide solution, interproton distances were derived according to which model conformations were built and refined using molecular dynamics simulations. The conformation of vasopressin and the V1 antagonists differ mainly in the region of the mutated residue. The antagonistic property was found to be related to an inversed chirality of the disulfide bridge. In all investigated molecules, characteristic beta-turn structure elements were found for the backbone conformation of the endocyclic residues Tyr2-Asn5. For this portion of the peptide sequence, various conformational equilibria were detected which matched different time scales. For [Arg8]vasopressin, averaged NMR parameters were obtained which could be explained by rapid interconversion between different beta-turn geometries, whereas multiple slowly exchanging conformations were observed for the V1 antagonists. V1 antagonists containing sarcosine in position 7 exhibited multiple spectral patterns for the exocyclic part attributed to cis/trans isomerization. The X-ray structure of deamino-oxytocin [Wood, S. P., Tickle, I. J., Treharne, A. M., Pitts, J. E., Mascarenhas, Y., Li, J. Y., Husain, J., Cooper, S., Blundell, T. L., Hruby, V. J., Buku, A., Fischman, A. J. & Wyssbrod, H. R. (1986) Science 232, 633-636] was found to represent one sample of the conformational space covered by the multiple conformations found for [Mca1, Arg8]vasopressin.     

Desensitization of the human V2 vasopressin receptor. Homologous effects in the absence of heterologous desensitization.

Three main pathways have been implicated in desensitization of receptors that stimulate adenylylcyclase (AC): cAMP-mediated phosphorylation; cAMP-independent phosphorylation, and receptor internalization. Cell lines derived from the murine Ltk- cell were found useful in exploring the contribution of cAMP-dependent phosphorylation in V2 vasopressin receptor desensitization. The HTB-2 cell expresses the human V2 vasopressin receptor, introduced by transfection of human genomic DNA, and the prostaglandin E1 (PGE1) receptor, endogenous to the Ltk- cell. The A7 cell expresses the hamster beta 2-adrenoceptor, which undergoes the above-mentioned desensitization processes. Treatment of HTB-2 cells with arginine-vasopressin (AVP) had no effect on AC responsiveness to PGE1, but promoted desensitization of the AVP response. This was seen as a 5-6-fold right shift in the dose-response curves for AVP action (cAMP accumulation in intact cells and AC stimulation in homogenates and isolated membranes) and in a decrease in the maximum effect of AVP on these parameters. AVP treatment caused a decrease in cell surface receptors to approximately 75% of control without changes in KD, as determined by Scatchard analysis. When cAMP was increased by treatment with 10 microM PGE1 and isobutylmethylxanthine, desensitization of the PGE1 receptor was observed but not of the AVP receptor. In A7 cells the same treatment caused, as expected, a 3-fold right shift in the dose-response curve for AC stimulation by isoproterenol, indicating that L cells can mediate heterologous desensitization. These data demonstrate that the V2 vasopressin and the PGE1 receptors undergo homologous desensitization in the absence of cAMP-mediated phosphorylation and that this component is not required for vasopressin receptor internalization.     

Mapping the binding site of arginine vasopressin to V1a and V1b vasopressin receptors

Starting from the 2.8-A resolution x-ray structure of bovine rhodopsin, three-dimensional molecular models of the complexes between arginine vasopressin and two receptor subtypes (V1a, V1b) have been built. Amino acid sequence alignment and docking studies suggest that four key residues (1.35, 2.65, 4.61, and 5.35) fine tune the binding of vasopressin and related peptide agonists to both receptor subtypes. To validate these predictions, a series of single or double mutants were engineered at V1a and V1b receptor subtypes and tested for their binding and functional properties. Two negatively charged amino acids at positions 1.35 and 2.65 are key anchoring residues to the Arg8 residue of arginine vasopressin. Moreover, two amino acids (V(4.61) and P(5.35)) delineating a hydrophobic subsite at the human V1b receptor are responsible for the recognition of V1b selective peptide agonists. Last, one of the latter positions (5.35) is hypothesized to explain the pharmacological species differences between rat and human vasopressin receptors for a V1b peptide agonist. Altogether these refined three-dimensional models of V1a and V1b human receptors should enable the identification of further new selective V1a and V1b agonists as pharmacological but also therapeutic tools.     

Cytokine-mediated downregulation of vasopressin V(1A) receptors during acute endotoxemia in rats

The reduced pressure response to vasopressin during acute sepsis has directed our interest to the regulation of vasopressin V(1A) receptors. Rats were injected with lipopolysaccharide for induction of experimental gram-negative sepsis. V(1A) receptor gene expression was downregulated in the liver, lung, kidney, and heart during endotoxemia. Inasmuch as the concentrations of proinflammatory cytokines such as interleukin-1beta, tumor necrosis factor-alpha, and interferon-gamma were highly increased during sepsis, the influence of these cytokines on V(1A) receptor expression was investigated in primary cultures of hepatocytes and in the aortic vascular smooth muscle cell line A7r5. V(1A) receptor expression was downregulated by the cytokines in a nitric oxide-independent manner. Blood pressure dose-response studies after injection of endotoxin showed a diminished responsiveness to the selective V(1) receptor agonist Phe(2),Ile(3),Orn(8)-vasopressin. Our data show that sepsis causes a downregulation of V(1A) receptors and suggest that this effect is likely mediated by proinflammatory cytokines. We propose that this downregulation of V(1A) receptors contributes to the attenuated responsiveness of blood pressure in response to vasopressin and, therefore, contributes to the circulatory failure in septic shock.     

Conopressin-T from Conus tulipa reveals an antagonist switch in vasopressin-like peptides

We report the discovery of conopressin-T, a novel bioactive peptide isolated from Conus tulipa venom. Conopressin-T belongs to the vasopressin-like peptide family and displays high sequence homology to the mammalian hormone oxytocin (OT) and to vasotocin, the endogenous vasopressin analogue found in teleost fish, the cone snail's prey. Conopressin-T was found to act as a selective antagonist at the human V 1a receptor. All peptides in this family contain two conserved amino acids within the exocyclic tripeptide (Pro7 and Gly9), which are replaced with Leu7 and Val9 in conopressin-T. Whereas conopressin-T binds only to OT and V 1a receptors, an L7P analogue had increased affinity for the V 1a receptor and weak V2 receptor binding. Surprisingly, replacing Gly9 with Val9 in OT and vasopressin revealed that this position can function as an agonist/antagonist switch at the V 1a receptor. NMR structures of both conopressin-T and L7P analogue revealed a marked difference in the orientation of the exocyclic tripeptide that may serve as templates for the design of novel ligands with enhanced affinity for the V 1a receptor.     

Investigation of mechanism of desmopressin binding in vasopressin V2 receptor versus vasopressin V1a and oxytocin receptors: molecular dynamics simulation of the agonist-bound state in the membrane-aqueous system

The vasopressin V2 receptor (V2R) belongs to the Class A G protein-coupled receptors (GPCRs). V2R is expressed in the renal collecting duct (CD), where it mediates the antidiuretic action of the neurohypophyseal hormone arginine vasopressin (CYFQNCPRG-NH2, AVP). Desmopressin ([1-deamino, 8-D]AVP, dDAVP) is strong selective V2R agonist with negligible pressor and uterotonic activity. In this paper, the interactions responsible for binding of dDAVP to vasopressin V2 receptor versus vasopressin V1a and oxytocin receptors has been examined. Three-dimensional activated models of the receptors were constructed using the multiple sequence alignment and the complex of activated rhodopsin with Gt(alpha) C-terminal peptide of transducin MII-Gt(alpha) (338-350) prototype (Slusarz, R.; Ciarkowski, J. Acta Biochim Pol 2004 51, 129-136) as a template. The 1-ns unconstrained molecular dynamics (MD) of receptor-dDAVP complexes immersed in the fully hydrated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) membrane model was conducted in an Amber 7.0 force field. Highly conserved transmembrane residues have been proposed as being responsible for V2R activation and G protein coupling. Molecular mechanism of the dDAVP binding has been suggested. The internal water molecules involved in an intricate network of the hydrogen bonds inside the receptor cavity have been identified and their role in the stabilization of the agonist-bound state proposed.     

High specific activity 125I- and 35S-labeled vasopressin analogues with high affinity for the V1 and V2 vasopressin isoreceptors.

Since iodination of the tyrosine residue in the pressin ring of vasopressins abolishes binding to the V2 (renal) isoreceptor, the low specific activity tritiated vasopressins have been the only radioligands available for this receptor. Alternative vasopressin radioligands are described in the present study. N-tert-Butoxycarbonyl- (N-t-Boc) 125I-tyrosine or [35S]methionine were conjugated to the 8th amino acid of lysine- (LVP) or deamino-ornithine-vasopressin via active succinimidyl esters. Following the purification on C-18 reverse-phase high pressure liquid chromatography, t-Boc removal, and a second high pressure liquid chromatography purification, specific activities of 2200 and 1300 Ci/mmol were obtained for the 125I- and the 35S-labeled ligands, respectively. These vasopressin analogues, conjugated outside the pressin ring, were found to bind with high affinity to the V1A (vascular) and V2 vasopressin isoreceptors (Kd less than or equal to 10(-9) M) and to retain the full biological activity of intact vasopressin. The present study demonstrates the possibility of producing high specific activity radioligands with high affinity for the V1A and V2 vasopressin isoreceptors by conjugating labeled moieties to the 8th amino acid of vasopressin analogues. Since these new radioligands have specific activities much higher than the tritiated ligands (1300-2200 versus 10-30 Ci/mmol), they should provide considerable advantages in the future study of the physiology and biochemistry of the AVP receptors.     

Vasopressin and fever: evidence supporting the existence of an endogenous antipyretic system in the brain

Vasopressin administered into the ventral septum exerts a dose-related antipyresis. This site of action is similar in a number of species. The fever-reducing properties of vasopressin are both site and neuropeptide specific. Evidence supporting a role for endogenous vasopressin in fever suppression is the demonstration that the release of the peptide from the ventral septal area is altered during fever: the amount released correlates negatively with febrile changes in body temperature. In addition, changes in the concentration of vasopressin in the septum and amygdala have been demonstrated immunocytochemically during fever: an activation of vasopressinergic neurons occurs which is similar to that observed in pregnant animals at term when fever is absent. Specific antibodies directed against vasopressin or specific vasopressin antagonist analogues (e.g., d(CH2)5Tyr(Me)AVP) enhanced the febrile response to a pyrogen challenge when injected into the ventral septum. The same antagonist also can antagonize the antipyretic effect of exogenously administered vasopressin. The use of relatively specific antagonists and agonists of vasopressin, directed against the V1 and V2 subtypes of the peripheral vasopressin receptor, suggests that the central receptor responsible for the antipyretic effect of vasopressin may resemble the V1 subtype. Recent experiments using electrophysiological techniques have demonstrated the existence of thermoresponsive units in the ventral septal area whose activity may be altered by vasopressin which is possibly derived from the paraventricular nucleus and bed nucleus of the stria terminalis.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Derivatives of somatic cell hybrids which carry the human gene locus for nephrogenic diabetes insipidus (NDI) express functional vasopressin renal V2-type receptors

The gene responsible for familial vasopressin-resistant nephrogenic diabetes insipidus (NDI) has been localized to a small region of the human X-chromosome (Xq28). A series of hamster lung fibroblast and mouse lymphocyte cell lines carrying fragments of the wild type human X-chromosome was analyzed for vasopressin renal-type V2 receptor expression, to test the hypothesis that the NDI locus may have identity with the V2 receptor gene. V2 receptor binding activity and induction of cAMP production in response to [Arg8] vasopressin (AVP) were exhibited by all cell lines carrying the wild type NDI locus, in contrast to control cell lines. AVP stimulation of cAMP production was concentration-dependent and could be almost completely inhibited by co-incubation with a V2-V1 receptor-specific antagonist. The V2-specific agonist [Mpa1,Val4,Sar7]AVP was as potent as AVP in inducing cAMP production by NDI-DNA-carrying cells, whereas no response was shown to other hormones such as calcitonin, oxytocin (less than 10(-8) M), isoproterenol, or an oxytocin-specific agonist. All results were consistent with the hypothesis that the V2 receptor gene co-localized with the NDI locus, supporting the view that the loci are one and the same.