The Impact of Oxytocin Gene Knockout on Sexual Behavior and Gene Expression Related to Neuroendocrine Systems in the Brain of Female Mice

Social relations are built and maintained from the interaction among individuals. The oxytocin (OT), vasopressin (VP), estrogen, dopamine, and their receptors are involved in the modulation of sexual behavior in females. This study aimed to analyze the impact of OT gene knockout (OTKO) on sexual behavior and the gene expression of oxytocin (OTR), estrogen alpha (ERα), estrogen beta (ERβ), vasopressin (V_1aR), and dopamine (D₂R) receptors in the olfactory bulb (OB), prefrontal cortex (PFC), hippocampus (HPC), and hypothalamus (HPT), as well as in the synthesis of VP in the HPT of female mice. Wild-type (WT) littermates were used for comparisons. The _CDNAs were synthesized by polymerase chain reaction and the gene expression was calculated with the 2^?ΔΔCt formula. Our results showed that the absence of OT caused an increase in the frequency and duration of non-receptive postures and a decrease in receptive postures in the OTKO. OTKO females showed a significant decrease in the gene expression of OTR in the HPC, V_1aR in the HPT, and ERα and ERβ in the PFC. There was no significant difference in the gene expression of D₂R of OTKO. However, OTKO showed an increased gene expression of V_1aR in the HPC. There is no significant difference in VP mRNA synthesis in the HPT between OTKO and WT. Our findings demonstrate that the absence of OT leads to significant changes in the expression of the studied genes (OTR, ERα, ERβ, V_1aR), and these changes may contribute to the decreased sexual behavior observed in OTKO females.     

Multiple alpha1-adrenergic receptor subtypes support synergistic stimulation of vasopressin and oxytocin release by ATP and phenylephrine

Simultaneous exposure of explants of the hypothalamo-neurohypophyseal system (HNS) to ATP and the α(1)-adrenergic receptor (α(1)-R) agonist, phenylephrine (ATP+PE) induces a synergistic stimulation of vasopressin and oxytocin (VP/OT) release that is sustained for hours. The current studies confirm that the synergism is dependent upon activation of α(1)-R by demonstrating that an α(1)-R antagonist prevents the response. The role of the α(1)A, B, and D-adrenergic receptor subtypes in the synergistic effect of ATP+PE on intracellular calcium ([Ca(2+)](i)) in supraoptic nucleus (SON) neurons and VP/OT release from neural lobe was evaluated. The increase in [Ca(2+)](i) induced by PE in SON predominantly reflects release from intracellular stores and is mediated by activation of the α(1)A adrenergic receptor subtype. The α(1)A subtype is also required for the sustained elevation in [Ca(2+)](i) induced by ATP+PE. In contrast, although synergistic stimulation of VP/OT release was eliminated by removal of PE and was blunted by benoxathian, an α(1)-R antagonist that is not subtype selective, no single α(1)-R subtype selective antagonist prevented sustained stimulation of VP/OT release by ATP+PE. Thus, sustained activation of α(1)-R is essential for the synergistic VP and OT response to ATP+PE, but multiple α(1)-R subtypes can support the response. Redundancy amongst the α(1)-R subunits in supporting this response is consistent with the predicted importance of the response for sustaining the elevated VP release required to prevent cardiovascular collapse during hemorrhage and sepsis.     

Synthesis and SAR studies of novel 2-(4-oxo-2-aryl-quinazolin-3(4H)-yl)acetamide vasopressin V1b receptor antagonists

Synthesis and structure-activity relationships (SAR) of a novel series of vasopressin V_1b (V₃) antagonists are described. 2-(4-Oxo-2-aryl-quinazolin-3(4H)-yl)acetamides have been identified with low nanomolar affinity for the V_1b receptor and good selectivity with respect to related receptors V_1a, V₂ and oxytocin (OT). Optimised compound 12j demonstrates a good pharmacokinetic profile and activity in a mechanistic model of HPA dysfunction.     

Synthesis and SAR studies of novel 2-(6-aminomethylaryl-2-aryl-4-oxo-quinazolin-3(4H)-yl)acetamide vasopressin V1b receptor antagonists

Synthesis and structure-activity relationships (SAR) of a novel series of vasopressin V_1b antagonists are described. 2-(6-Aminomethylaryl-2-aryl-4-oxo-quinazolin-3(4H)-yl)acetamide have been identified with low nanomolar affinity for the V_1b receptor and good selectivity with respect to related receptors V_1a, V₂ and OT. Optimised compound 16 shows a good pharmacokinetic profile and activity in a mechanistic model of HPA dysfunction.     

Potent and selective oxytocin receptor agonists without disulfide bridges

Oxytocin (OT) is a neuropeptide involved in a wide variety of physiological actions, both peripherally and centrally. Many human studies have revealed the potential of OT to treat autism spectrum disorders and schizophrenia. OT interacts with the OT receptor (OTR) as well as vasopressin 1a and 1b receptors (V_1aR, V_1bR) as an agonist, and agonistic activity for V_1aR and V_1bR may have a negative impact on the therapeutic effects of OTR agonism in the CNS. An OTR-selective agonistic peptide, FE 202767, in which the structural differences from OT are a sulfide bond instead of a disulfide bond, and N-alkylglycine replacement for Pro at position 7, was reported. However, the effects of amino acid substitutions in OT have not been comprehensively investigated to compare OTR, V_1aR, and V_1bR activities. This led us to obtain a new OTR-selective analog by comprehensive amino acid substitutions of OT and replacement of the disulfide bond. A systematic amino acid scanning (Ala, Leu, Phe, Ser, Glu, or Arg) of desamino OT (dOT) at positions 2, 3, 4, 5, 7, and 8 revealed the tolerability for the substitution at positions 7 and 8. Further detailed study showed that trans-4-hydroxyproline (trans-Hyp) at position 7 and γ-methylleucine [Leu(Me)] at position 8 were markedly effective for improving receptor selectivity without decreasing the potency at the OTR. Subsequently, a combination of these amino acid substitutions with the replacement of the disulfide bond of dOT analogs with a sulfide bond (carba analog) or an amide bond (lactam analog) yielded several promising analogs, including carba-1-[trans-Hyp⁷,Leu(Me)⁸]dOT (14) with a higher potency (7.2 pM) at OTR than that of OT and marked selectivity (>10,000-fold) over V_1aR and V_1bR. Hence, we investigated comprehensive modification of OT and obtained new OT analogs that exhibited high potency at OTR with marked selectivity. These OTR-selective agonists could be useful to investigate OTR-mediated effects on psychiatric disorders.     

V1 receptor in ENS mediates the excitatory effect of vasopressin on circular muscle strips of gastric body in vitro in rats

The purpose of this study was to localize vasopressin (VP) V_1a receptor in stomach and to characterize the role of VP in the regulation of gastric motility in rats. Double staining was used to locate the V_1a receptor in the gastric body of the rat. The contraction of the circular muscle strips of gastric body was monitored by a polygraph. V_1a receptor was expressed on the neurons of myenteric plexus of the gastric body. VP (10^− 10–10^− 6 M) caused a concentration-dependent contractile effect on the circular muscle strips of gastric body in vitro. V-1880 ([deamino-Pen¹, O-Me-Tyr², Arg⁸]-Vasopressin, 10^− 7 M), a V₁ receptor antagonist, inhibited the spontaneous contraction of the strips. Tetradotoxin (TTX, 10^− 6 M) and V-1880 (10^− 7 M) abolished the excitatory effect of VP. Atropine (10^− 6 M) partially inhibited VP-induced excitatory effect on the muscle strips but hexamethonium (10^− 4 M) did not influence it. These results suggest that V_1a receptor was expressed on the neurons of myenteric nerves. The cholinergic nerve was involved in the excitatory effect of VP on the contraction of gastric body.     

Building bridges for highly selective,potent and stable oxytocin and vasopressin analogs

Oxytocin (OT) is an exciting potential therapeutic agent, but it is highly sensitive to modification and suffers extensive degradation at elevated temperature and in vivo. Here we report studies towards OT analogs with favorable selectivity, affinity and potency towards the oxytocin receptor (OTR), in addition to improving stability of the peptide by bridging the disulfide region with substituted dibromo-xylene analogs. We found a sensitive structure-activity relationship in which meta-cyclized analogs (dOT_meta) gave highest affinity (50?nM K_i), selectivity (34-fold), and agonist potency (34?nM EC₅₀, 87-fold selectivity) towards OTR. Surprisingly, ortho-cyclized analogs demonstrated OTR and vasopressin V_1a receptor subtype affinity (220?nM and 69?nM, respectively) and pharmacological activity (294?nM and 35?nM, respectively). V_1a binding and selectivity for ortho-cyclized peptides could be improved 6-fold by substituting a neutral residue at position 8 with a basic amino acid, providing potent antagonists (14?nM IC₅₀) that displayed no activation of the OTR. Furthermore, xylene-bridged analogs demonstrated increased stability compared to OT at elevated temperature, demonstrating promising therapeutic potential for these analogs which warrants further study.     

Characterization of knowledge-based volumetric modulated arc therapy plans created by three different institutions’ models for prostate cancer

BackgroundThe aim of this study was to clarify factors predicting the performance of knowledge-based planning (KBP) models in volume modulated arc therapy for prostate cancer in terms of sparing the organ at risk (OAR).Materials and methodsIn three institutions, each KBP model was trained by more than 20 library plans (LP) per model. To validate the characterization of each KBP model, 45 validation plans (VP) were calculated by the KBP system. The ratios of overlap between the OAR volume and the planning target volume (PTV) to the whole organ volume (V_overlap/V_whole) were analyzed for each LP and VP. Regression lines between dose–volume parameters (V₉₀, V₇₅, and V₅₀) and V_overlap/V_whole were evaluated. The mean OAR dose, V₉₀, V₇₅, and V₅₀ of LP did not necessarily match those of VP.ResultsIn both the rectum and bladder, the dose–volume parameters for VP were strongly correlated with V_overlap/V_whole at institutes A, B, and C (R > 0.74, 0.85, and 0.56, respectively). Except in the rectum at institute B, the slopes of the regression lines for LP corresponded to those for VP. For dose–volume parameters for the rectum, the ratios of slopes of the regression lines in VP to those in LP ranged 0.51–1.26. In the bladder, most ratios were less than 1.0 (mean: 0.77).ConclusionFor each OAR, each model made distinct dosimetric characterizations in terms of V_overlap/V_whole. The relationship between dose–volume parameters and V_overlap/V_whole of OARs in LP predicts the KBP models’ performance sparing OARs.     

Oxytocin and vasopressin involved in restraint water-immersion stress mediated by oxytocin receptor and vasopressin 1b receptor in rat brain

Aims

Vasopressin (AVP) and oxytocin (OT) are considered to be related to gastric functions and the regulation of stress response. The present study was to study the role of vasopressinergic and oxytocinergic neurons during the restraint water-immersion stress.

Methods

Ten male Wistar rats were divided into two groups, control and RWIS for 1h. The brain sections were treated with a dual immunohistochemistry of Fos and oxytocin (OT) or vasopressin (AVP) or OT receptor or AVP 1b receptor (V_1bR).

Results

(1) Fos-immunoreactive (Fos-IR) neurons dramatically increased in the hypothalamic paraventricular nucleus (PVN), the supraoptic nucleus (SON), the neucleus of solitary tract (NTS) and motor nucleus of the vagus (DMV) in the RWIS rats; (2) OT-immunoreactive (OT-IR) neurons were mainly observed in the medial magnocellular part of the PVN and the dorsal portion of the SON, while AVP-immunoreactive (AVP-IR) neurons mainly distributed in the magnocellular part of the PVN and the ventral portion of the SON. In the RWIS rats, Fos-IR neurons were indentified in 31% of OT-IR neurons and 40% of AVP-IR neurons in the PVN, while in the SON it represented 28%, 53% respectively; (3) V_1bR-IR and OTR-IR neurons occupied all portions of the NTS and DMV. In the RWIS rats, more than 10% of OTR-IR and V_1bR-IR neurons were activated in the DMV, while lower ratio in the NTS.

Conclusion

RWIS activates both oxytocinergic and vasopressinergic neurons in the PVN and SON, which may project to the NTS or DMV mediating the activity of the neurons by OTR and V_1bR.     

Oxytocin antagonist blocks the vasodepressor but not the vasopressor effect of neurohypophysial peptides in chickens

Cockerels with permanent cannulas in the brachial artery and vein were put into isolated slings. Arterial pressure and heart rate were continuously recorded. Following habituation, tests were initiated. In each cockerel 2 nmol/kg of the tested neurohypophysial peptide (NPs) or analogue was IV injected six times at 6-min intervals. Arginine vasotocin (AVT) caused an immediate vasodepressor (VDP) effect and tachycardia. These subsided within 20–30 s and were followed by a vasopressor (VP) response and bradycardia. On repeated injections of AVT, the VDP response declined and bradycardia intensified. Arginine vasopressin (AVP), oxytocin (OT), and mesotocin (MT) had short-lasting VDP effect in the following order of potency: OT = MT > AVT > AVP. Only AVT and, more effectively, AVP, caused a VP response. The VDP effect of MT and OT declined on repeated injections. When AVT was injected after three injections of MT, it had mostly an immediate VP effect. Although the V₁ agonist is VP in chickens, at the dose used the V₁ antagonist, [d(CH₂)₅,O-Me-Tyr²]AVP, had no effect on cardiovascular responses to AVT. Pretreatment with OT antagonist, [d(CH₂)₅-O-Me-Tyr^2,Thr^4,Tyr^9,Orn⁸]VT, abolished the VDP effect of all NPs. Thus, MT had no effect on blood pressure, whereas AVP and, more effectively, AVT, had a marked immediate VP action. In chickens the VDP effect of NPs is probably mediated by an OT/MT-like receptor, wherein the peptide's ring structure, shared by AVT, OT, and MT, is important. The VP effect is mediated by a receptor only partially similar to the mammalian V₁ receptor, where arginine in position 8, shared only by AVT and AVP, is necessary for action, and the native AVT is more effective than the mammalian AVP. This receptor reacts to the V₁ agonist but probably not to the V₁ antagonist.     

Exposure to chronic isolation modulates receptors mRNAs for oxytocin and vasopressin in the hypothalamus and heart

The goal of our study was to explore the effect of social isolation stress of varying durations on the plasma oxytocin (OT), messenger ribonucleic acid (mRNA) for oxytocin receptor (OTR), plasma arginine vasopressin (AVP) and mRNA for V_1a receptor of AVP (V_1aR) expression in the hypothalamus and heart of socially monogamous female and male prairie voles (Microtus ochrogaster). Continuous isolation for 4 weeks (chronic isolation) increased plasma OT level in females, but not in males. One hour of isolation every day for 4 weeks (repeated isolation) was followed by a significant increase in plasma AVP level. Chronic isolation, but not repeated isolation, significantly decreased OTR mRNA in the hypothalamus and heart in both sexes. Chronic isolation significantly decreased cardiac V_1aR mRNA, but no effect on hypothalamic V_1aR mRNA expression. We did not find a gender difference within repeated social isolation groups. The results of the present study reveal that although chronic social isolation can down-regulate gene expression for the OTR in both sexes, the release of the OT peptide was increased after chronic isolation only in females, possibly somewhat protecting females from the negative consequences of isolation. In both sexes repeated, but not chronic, isolation increased plasma AVP, which could be permissive for mobilization and thus adaptive in response to a repeated stressor. The differential effects of isolation on OT and AVP systems may help in understanding mechanisms through social interactions can be protective against emotional and cardiovascular disorders.     

Histamine-induced enhancement of vasopressin and oxytocin secretion in rat neurohypophyseal tissue cultures

The effects of histamine (HA) on vasopressin (VP) and oxytocin (OT) secretion were studied in 13-14-day cultures of isolated rat neurohypophyseal (NH) tissue. The VP and OT contents of the supernatant were determined by radioimmunoassay (RIA) after a 1 or 2-h incubation. Significantly increased levels of VP and OT production were detected in the tissue culture media following HA administration, depending on the HA dose. The elevation of NH hormone secretion could be partially blocked by previous administration of the HA antagonist mepyramine (MEP, an H1 receptor antagonist) or cimetidine (CIM, an H2 receptor antagonist). Thioperamide (TPE, an H3-H4 receptor antagonist) did not influence the VP or OT secretion increase induced by HA. The application of MEP, CIM or TPE after HA administration proved ineffective. The H1 and H2 receptors are mainly involved in the HA-induced increase of both VP and OT secretion in isolated NH tissue cultures. The results indicate that NH hormone release is influenced directly by the histaminergic system, and the histaminergic control of VP and OT secretion from the NH tissue in rats can occur at the level of the posterior pituitary.     

Noncanonical Control of Vasopressin Receptor Type 2 Signaling by Retromer and Arrestin

The vasopressin type 2 receptor (V2R) is a critical G protein-coupled receptor (GPCR) for vertebrate physiology, including the balance of water and sodium ions. It is unclear how its two native hormones, vasopressin (VP) and oxytocin (OT), both stimulate the same cAMP/PKA pathway yet produce divergent antinatriuretic and antidiuretic effects that are either strong (VP) or weak (OT). Here, we present a new mechanism that differentiates the action of VP and OT on V2R signaling. We found that vasopressin, as opposed to OT, continued to generate cAMP and promote PKA activation for prolonged periods after ligand washout and receptor internalization in endosomes. Contrary to the classical model of arrestin-mediated GPCR desensitization, arrestins bind the VP-V2R complex yet extend rather than shorten the generation of cAMP. Signaling is instead turned off by the endosomal retromer complex. We propose that this mechanism explains how VP sustains water and Na⁺ transport in renal collecting duct cells. Together with recent work on the parathyroid hormone receptor, these data support the existence of a novel “noncanonical” regulatory pathway for GPCR activation and response termination, via the sequential action of β-arrestin and the retromer complex.     

Oxytocin and vasopressin stimulate anion secretion by human and porcine vas deferens epithelia

Experiments were conducted to characterize the effects of oxytocin (OT) and vasopressin (VP) on epithelial cells isolated from human (1 degree HVD) and porcine (1 degree PVD) vas deferens and an immortalized epithelial cell line derived from porcine vas deferens (PVD9902 cells). Cultured monolayers were assessed in modified Ussing flux chambers and the OT- or VP-induced change in short circuit current (I(SC)) was recorded. All cell types responded to basolateral OT or VP with a transient increase in I(SC) that reached a peak of 3-5 microA cm(-2). Concentration-response curves constructed with 1 degree PVD and PVD9902 cells revealed that the apparent K(D) (k(app)) for OT was approximately 100-fold less than the k(app) for VP. Amplicons for the OT receptor (OXTR) and vasopressin type 2 and type 1a receptors (AVPR2 and AVPR1A) were generated with RT-PCR and the identification of each amplicon confirmed by sequence analysis. A selective antagonist for OXTR and AVPR1A fully blocked the effects of OT and partially blocked the effects of VP when assessed in both 1 degree PVD and PVD9902 monolayers. APVR2 antagonists blocked the effects of low (< or =30 nM) but not high concentrations of VP, indicating that VP was affecting both AVPR2 and a second receptor subtype, likely OXTR or AVPR1A. Experiments employing chelerythrine demonstrated that OT stimulation of vas deferens monolayers requires PKC activity. Alternatively, VP (but not OT) increased the accumulation of cytosolic cAMP in vas deferens epithelial cells. Results from this study demonstrate that OT and VP can modulate ion transport across vas deferens epithelia by independent mechanisms. OT and VP have the potential to acutely change the environment to which sperm are exposed and thus, have the potential to affect male fertility.     

Role of non-NMDA receptors in vasopressin and oxytocin release from rat hypothalamo-neurohypophysial explants

Glutamate is recognized as a prominent excitatory transmitter in the supraoptic nucleus (SON) and is involved in transmission of osmoregulatory information from the osmoreceptors to the vasopressin (VP) and oxytocin (OT) neurons. Explants of the hypothalamo-neurohypophysial system were utilized to characterize the roles of the non-N-methyl-D-aspartate (NMDA) glutamate receptor subtypes (non-NMDA-Rs), kainic acid receptors (KA-Rs), and aminopropionic acid receptors (AMPA-Rs) and to evaluate the interdependence of NMDA-Rs and non-NMDA-Rs in eliciting hormone release. Although both KA and AMPA increased hormone release, a specific agonist of the KA-Rs, SYM-2081, was not effective. This combined with the finding that cyclothiazide, an agent that inhibits the desensitization of AMPA-Rs, increased the VP response to both KA and AMPA indicates that the increase in hormone release induced by the non-NMDA agonists is mediated via AMPA-Rs, rather than KA-Rs. Inhibition of osmotically stimulated VP and OT release by a specific AMPA-R antagonist indicated that AMPA-Rs are essential for mediating osmotically stimulated hormone release. NMDA-stimulated VP but not OT release was prevented by blockade of non-NMDA-Rs, but AMPA-stimulated VP/OT release was not prevented by NMDA-R blockade.     

Regional expression of P2Y4 receptors in the rat central nervous system

P2Y receptors are G protein-coupled receptors composed of eight known subunits (P2Y_{1, 2, 4, 6, 11, 12, 13, 14}), which are involved in different functions in neural tissue. The present study investigates the expression pattern of P2Y₄ receptors in the rat central nervous system (CNS) using immunohistochemistry and in situ hybridization. The specificity of the immunostaining has been verified by preabsorption, Western blot, and combined use of immunohistochemistry and in situ hybridization. Neurons expressing P2Y₄ receptors were distributed widely in the rat CNS. Heavy P2Y₄ receptor immunostaining was observed in the magnocellular neuroendocrine neurons of the hypothalamus, red nucleus, pontine nuclei, mesencephalic trigeminal nucleus, motor trigeminal nucleus, ambiguous nucleus, inferior olive, hypoglossal nucleus, and dorsal motor vagus nucleus. Both neurons and astrocytes express P2Y₄ receptors. P2Y₄ receptor immunostaining signals were mainly confined to cell bodies and dendrites of neurons, suggesting that P2Y₄ receptors are mainly involved in regulating postsynaptic events. In the hypothalamus, all the vasopressin (VP) and oxytocin (OT) neurons and all the orexin A neurons were immunoreactive for P2Y₄ receptors. All the neurons expressing P2Y₄ receptors were found to express N-methyl-d-aspartate receptor 1 (NR1). These data suggest that purines and pyrimidines might be involved in regulation of the release of the neuropeptides VP, OT, and orexin in the rat hypothalamus via P2Y₄ receptors. Further, the physiological and pathophysiological functions of the neurons may operate through coupling between P2Y₄ receptors and NR1.     

Sex-specific activity and function of hypothalamic nonapeptide neurons during nest-building in zebra finches

Vertebrate species from fish to humans engage in a complex set of preparatory behaviors referred to as nesting; yet despite its phylogenetic ubiquity, the physiological and neural mechanisms that underlie nesting are not well known. We here test the hypothesis that nesting behavior is influenced by the vasopressin–oxytocin (VP–OT) peptides, based upon the roles they play in parental behavior in mammals. We quantified nesting behavior in male and female zebra finches following both peripheral and central administrations of OT and V_1a receptor (OTR and V1aR, respectively) antagonists. Peripheral injections of the OTR antagonist profoundly reduce nesting behavior in females, but not males, whereas comparable injections of V1aR antagonist produce relatively modest effects in both sexes. However, central antagonist infusions produce no effects on nesting, and OTR antagonist injections into the breast produce significantly weaker effects than those into the inguinal area, suggesting that antagonist effects are mediated peripherally, likely via the oviduct. Finally, immunocytochemistry was used to quantify nesting-induced Fos activation of nonapeptide neurons in the paraventricular and supraoptic nuclei of the hypothalamus and the medial bed nucleus of the stria terminalis. Nest-building induced Fos expression within paraventricular VP neurons of females but not males. Because the avian forms of OT (Ile⁸-OT; mesotocin) and VP (Ile³-VP; vasotocin) exhibit high affinity for the avian OTR, and because both peptide forms modulate uterine contractility, we hypothesize that nesting-related stimuli induce peptide release from paraventricular vasotocin neurons, which then promote female nesting via peripheral feedback from OTR binding in the oviduct uterus.     

Position Three in Vasopressin Antagonist Tolerates Conformationally Restricted and Aromatic Amino Acid Substitutions: A Striking Contrast with Vasopressin Agonists

We report the solid-phase synthesis and some pharmacological properties of 12 position three modified analogues (peptides 1–12) of the potent non-selective antagonist of the antidiuretic (V₂-receptor), vasopressor (V_1a-receptor) responses to arginine vasopressin (AVP) and of the uterine contracting (OT-receptor) responses to oxytocin (OT), [1(-β mercapto-β,β-pentamethy lenepropionic acid)-2-O-ethyl-d -tyrosine 4-valine] arginine vasopressin [d(CH₂)₅D -Tyr(Et) ²VAVP] (A) and two analogues of ( B ) (peptides 13,14), the 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid³ (Tic³) analogue of ( A ). Peptides 1–12 have the following substituents at position three in ( A ): (1) Pro; (2) Oic; (3) Atc; (4) D -Atc; (5) Aic; (6) D -Phe; (7) Ile; (8) Leu; (9) Tyr; (10) Trp; (11) Hphe; (12) [HO]Tic; Peptide (13) is the Tyr-NH₂ ⁹ analogue of ( B ): Peptide (14) is the D -Cys ⁶ analogue of ( B ). All 14 new peptides were evaluated for agonistic and antagonistic activities in in vivo V₂ and V_1a assays and in in vitro (no Mg²⁺) _n oxytocic assays. With the exception of the D -Phe³ peptide (No. 6), which exhibits very weak V₂ agonism (…0.0017 u/mg), none of the remaining 13 peptides exhibit any agonistic activities in these assays. In striking contrast to their deleterious effects on agonistic activities in AVP, the Pro³, Oic³, Tyr³, Trp³ and Hphe³ substitutions in ( A ) are very well tolerated, leading to excellent retention of V₂, V_1a and OT antagonistic potencies. All are more potent as V₂ antagonists than the Ile³ and Leu³ analogues of ( A ). The Tyr-NH₂⁹ and D -Cys⁶ substitutions in ( B ) are also well tolerated. The anti-V₂ pA₂ values of peptides 1–5 and 7–14 are as follows (1) 7.77±0.03; (2) 7.41± 0.05; (3) 6.86±0.02; (4) 5.66±0.09; (5) …5.2; (7) 7.25± 0.08; (8) 6.82±0.06; (9) 7.58±0.05; (10) 7.61±0.08; (11) 7.59±0.07; (12) 7.20±0.05; (13) 7.57±0.1; (14) 7.52± 0.06. All analogues antagonize the vasopressor responses to AVP, with anti-V _1a pA₂ values ranging from 5.62 to 7.64, and the in vitro responses to OT, with anti-OT pA₂ values ranging from 5.79 to 7.94. With an anti-V₂ potency of 7.77±0.03, the Pro³ analogue of ( A ) is surprisingly equipotent with ( A ), (anti-V₂ pA₂=7.81±0.07). These findings clearly indicate that position three in AVP V₂/V_1a antagonists, in contrast to position three in AVP agonists, is much more amenable to structural modification than had heretofore been anticipated. Furthermore, the surprising retention of V₂ antagonism exhibited by the Pro³, Oic³, Tyr³, Trp³ and Hphe³ analogues of ( A ), together with the excellent retention of V₂ antagonism by the Tyr-NH₂⁹ and D -Cys⁶ analogues of ( B ) are promising new leads to the design of potent and possibly orally active V₂ antagonists for use as pharmacological tools and/or as radioiodinatable ligands and for development as potential therapeutic agents for the treatment of the hyponatremia caused by the syndrome of the inappropriate secretion of the antidiuretic hormone (SIADH). © 1997 European Peptide Society and John Wiley & Sons, Ltd.     

Characterization of a novel vasopressin/oxytocin superfamily peptide and its receptor from an ascidian, Ciona intestinalis

The vasopressin (VP)/oxytocin (OT) superfamily peptides are one of the most widely distributed neuropeptides and/or neurohypophysial hormones, but have ever not been characterized from any deuterostome invertebrates including protochordates, ascidians. In the present study, we show the identification of a novel VP/OT superfamily peptide and its receptor in the ascidian, Ciona intestinalis. Intriguingly, the Ciona VP/OT-related peptide (Ci-VP), unlike other 9-amino acid and C-terminally amidated VP/OT superfamily peptides, consists of 13 amino acids and lacks a C-terminal amidation. Mass spectrometry confirmed the presence of the 13-residue Ci-VP in the neural complex. Furthermore, 10 of 14 cysteines are conserved in the neurophysin domain, compared with other VP/OT counterparts. These results revealed that the VP/OT superfamily is conserved in ascidians, but the Ci-VP gene encodes an unprecedented VP/OT-related peptide and neurophysin protein. Ci-VP was also shown to activate its endogenous receptor, Ci-VP-R, at physiological concentrations, confirming the functionality of Ci-VP as an endogenous ligand. The Ci-VP gene was expressed exclusively in neurons of the brain, whereas the Ci-TK-R mRNA was distributed in various tissues including the neural complex, alimentary tract, gonad, and heart. These expression profiles suggest that Ci-VP, like other VP/OT superfamily peptides, serves as a multifunctional neuropeptides. Altogether, our data revealed both evolutionary conservation and specific divergence of the VP/OT superfamily in protochordates. This is the first molecular characterization of a VP/OT superfamily peptide and its cognate receptor from not only ascidians but also deuterostome invertebrates.     

Conformational Aspects of Differences in Requirements for Oxytocin and Vasopressin Receptors

Abstract

Conformational energy calculations were carried out on three non-peptide antagonists of oxytocin and vasopressin: penicilide (compound 1; selective for oxytocin receptors), 1- {1-[4-(3-acetylaminopropoxy (benzoyl]-4-piperidyl}-3,4-dihydro-2(1H)-quinoline (compound 2; selective for vasopressin V₁ receptors) and 5-dimethylamino-1-{(2-methylbenzylamino)-benzoyl}-2,3,4,5–tetrahydro-1H-benzapine (compound 3; selective for vasopressin V₂ receptors). The obtained low-energy conformations of compound 1 were compared with low-energy conformations of oxytocin (OT) and low-energy conformations of compounds 2 and 3 were compared with low-energy conformations of arginine vasopressin (AVP). It was found that the affinity of the non-peptide antagonists and their selectivity for vasopressin and oxytocin receptors is probably connected with mimicking the aromatic rings of the Tyr² and the Phe³ residues of AVP in the case of compounds 2 and 3 and with mimicking the Tyr² residue and the Ile³ or Leu⁸ residues of OT by the outer benzene ring and the isobutyl group of compound 1. Application of the results in the design of more potent non-peptide antagonists of OT and VP is also discussed.