A cAMP-biosensor-based assay for measuring plasma arginine-vasopressin levels.

Arginine-vasopressin (AVP), a cyclic peptide hormone composed of nine amino acids, regulates water reabsorption by increasing intracellular cyclic adenosine monophosphate (cAMP) concentrations via the vasopressin V2 receptor (V2R). Plasma AVP is a valuable biomarker for the diagnosis of central diabetes insipidus (CDI) and is commonly measured using radioimmunoassay (RIA). However, RIA has several drawbacks, including a long hands-on time, complex procedures, and handling of radioisotopes with special equipment and facilities. In this study, we developed a bioassay to measure plasma AVP levels using HEK293 cells expressing an engineered V2R and a cAMP biosensor. To achieve high sensitivity, we screened V2R orthologs from 11 various mammalian species and found that the platypus V2R (pV2R) responded to AVP with approximately six-fold higher sensitivity than that observed by the human V2R. Furthermore, to reduce cross-reactivity with desmopressin (DDAVP), a V2R agonist used for CDI treatment, we introduced a previously described point mutation into pV2R, yielding an approximately 20-fold reduction of responsiveness to DDAVP while maintaining responsiveness to AVP. Finally, a comparison of plasma samples from 12 healthy individuals demonstrated a strong correlation (Pearson's correlation value: 0.90) between our bioassay and RIA. Overall, our assay offers a more rapid and convenient method for quantifying plasma AVP concentrations than existing techniques.

Social regulation of arginine vasopressin and oxytocin systems in a wild group-living fish.

The neuropeptides arginine vasopressin (AVP) and oxytocin (OXT) are key regulators of social behaviour across vertebrates. However, much of our understanding of how these neuropeptide systems interact with social behaviour is centred around laboratory studies which fail to capture the social and physiological challenges of living in the wild. To evaluate relationships between these neuropeptide systems and social behaviour in the wild, we studied social groups of the cichlid fish Neolamprologus pulcher in Lake Tanganyika, Africa. We first used SCUBA to observe the behaviour of focal group members and then measured transcript abundance of key components of the AVP and OXT systems across different brain regions. While AVP is often associated with male-typical behaviours, we found that dominant females had higher expression of avp and its receptor (avpr1a2) in the preoptic area of the brain compared to either dominant males or subordinates of either sex. Dominant females also generally had the highest levels of leucyl-cystinyl aminopeptidase (lnpep)-which inactivates AVP and OXT-throughout the brain, potentially indicating greater overall activity (i.e., production, release, and turnover) of the AVP system in dominant females. Expression of OXT and its receptors did not differ across social ranks. However, dominant males that visited the brood chamber more often had lower preoptic expression of OXT receptor a (oxtra) suggesting a negative relationship between OXT signalling and parental care in males of this species. Overall, these results advance our understanding of the relationships between complex social behaviours and neuroendocrine systems under natural settings.

Methylation of the Oxytocin, Oxytocin Receptor, and Vasopressin Gene Promoters in Tobacco Use Disorder during Cessation.

INTRODUCTION: Vasopressin (AVP) and oxytocin (OT) exert sex-specific effects on social pair bonding and stress reactions while also influencing craving in substance use disorders. In this regard, intranasal oxytocin (OT) and AVP antagonists present potential treatments for tobacco use disorder (TUD). Since transcription of both hormones is also regulated by gene methylation, we hypothesized sex-specific changes in methylation levels of the AVP, OT, and OT receptor (OXTR) gene during nicotine withdrawal. METHODS: The study population consisted of 49 smokers (29 males, 20 females) and 51 healthy non-smokers (25 males, 26 females). Blood was drawn at day 1, day 7, and day 14 of smoking cessation. Craving was assessed with the questionnaire on smoking urges (QSU). RESULTS: Throughout cessation, mean methylation of the OT promoter gene increased in males and decreased in females. OXTR receptor methylation decreased in females, while in males it was significantly lower at day 7. Regarding the AVP promoter, mean methylation increased in males while there were no changes in females. Using mixed linear modeling, CpG position, time point, sex, and the interaction of time point and sex as well as time point, sex, and QSU had a significant fixed effect on OT and AVP gene methylation. The interaction effect suggests that sex, time point, and QSU are interrelated, meaning that, depending on the sex, methylation could be different at different time points and vice versa. There was no significant effect of QSU on mean OXTR methylation. DISCUSSION: We identified differences at specific CpGs between controls and smokers in OT and AVP and in overall methylation of the AVP gene. Furthermore, we found sex-specific changes in mean methylation levels of the mentioned genes throughout smoking cessation, underlining the relevance of sex in the OT and vasopressin system. This is the first study on epigenetic regulation of the OT promoter in TUD. Our results have implications for research on the utility of the AVP and OT system for treating substance craving. Future studies on both targets need to analyze their effect in the context of sex, social factors, and gene regulation.

AVPR2 is a potential prognostic biomarker and correlated with immune infiltration in head and neck squamous cell carcinoma.

PURPOSE: To explore the potential of AVPR2 in the immunotherapy of head and neck squamous cell carcinoma (HNSCC), thus providing insights into a novel antitumour strategy. METHODS: In this study, we performed a comprehensive analysis of the AVPR2 gene in HNSCC using public datasets from The Cancer Genome Atlas and Gene Expression Omnibus. We explored the potential molecular mechanism of HNSCC in clinical prognosis and tumour immunity from the aspects of gene expression, prognosis, immune subtypes, and immune infiltration. RESULTS: AVPR2 expression was significantly downregulated in primary HNSCC tissue compared with normal tissue. HNSCC patients with high AVPR2 expression had a better prognosis. Moreover, the results of GSEA showed that immune subtype surface AVPR2 is involved in immune modulation. Furthermore, significant strong correlations between AVPR2 expression and infiltrating immune cells existed in HNSCC, and marker genes of infiltrating immune cells were also significantly related to AVPR2 expression in HNSCC. These results suggest that AVPR2 expression can influence the infiltration of tumour immune cells. Finally, we found that only high levels of B-cell infiltration, rather than those of other immune cells, can predict a longer overall survival in patients with HNSCC. Future studies are needed to explore the role of AVPR2 and tumour-infiltrating B cells in HNSCC. CONCLUSIONS: The AVPR2 gene may be a prognostic biomarker of HNSCC. Moreover, AVPR2 may play a role in HNSCC immune modulation, and the regulation of tumour-infiltrating B cells by AVPR2 may be a key link.

Arginine vasopressin deficiency and conivaptan (a V1a-V2 receptor antagonist) treatment reverses liver damage and fibrosis in rats with chronic portocaval anastomosis.

Arginine vasopressin (AVP) is a naturally occurring hormone synthesized in the hypothalamus. AVP demonstrates pro-fibrotic effects as it stimulates hepatic stellate cells to secrete transforming growth factor-ß (TGF-ß) and collagen. Previous work in liver cirrhotic (CCL4 -induced) hamsters demonstrated that AVP deficiency induced by neurointermediate pituitary lobectomy (NIL) can restore liver function. Therefore, we hypothesized that liver fibrosis would decrease in portocaval anastomosis (PCA) rats, which model chronic liver diseases, when they are treated with the V1a-V2 AVP receptor antagonist conivaptan (CV). In this study, changes in liver histology and gene expression were analysed in five experimental groups: control, PCA, NIL, PCA + NIL and PCA + CV, with NIL surgery or CV treatment administered 8 weeks after PCA surgery. Body weight gain was assessed on a weekly basis, and serum liver function, liver weight and liver glycogen content were assessed following euthanasia. Most PCA-induced phenotypes were reverted to normal levels following AVP-modelled deficiency, though hypoglycemia and ammonium levels remained elevated in the PCA + CV group. Liver histopathological findings showed a significant reversal in collagen content, less fibrosis in the triad and liver septa and increased regenerative nodules. Molecular analyses showed that the expression of fibrogenic genes (TGF-ß and collagen type I) decreased in the PCA + CV group. Our findings strongly suggest that chronic NIL or CV treatment can induce a favourable microenvironment to decrease liver fibrosis and support CV as an alternative treatment for liver fibrosis.

Vasopressin Receptor Type-2 Mediated Signaling in Renal Cell Carcinoma Stimulates Stromal Fibroblast Activation.

Vasopressin type-2 receptor (V2R) is ectopically expressed and plays a pathogenic role in clear cell renal cell carcinoma (ccRCC) tumor cells. Here we examined how V2R signaling within human ccRCC tumor cells (Caki1 cells) stimulates stromal cancer-associated fibroblasts (CAFs). We found that cell culture conditioned media from Caki1 cells increased activation, migration, and proliferation of fibroblasts in vitro, which was inhibited by V2R gene silencing in Caki1 cells. Analysis of the conditioned media and mRNA of the V2R gene silenced and control Caki1 cells showed that V2R regulates the production of CAF-activating factors. Some of these factors were also found to be regulated by YAP in these Caki1 cells. YAP expression colocalized and correlated with V2R expression in ccRCC tumor tissue. V2R gene silencing or V2R antagonist significantly reduced YAP in Caki1 cells. Moreover, the V2R antagonist reduced YAP expression and myofibroblasts in mouse xenograft tumors. These results suggest that V2R plays an important role in secreting pro-fibrotic factors that stimulate fibroblast activation by a YAP-dependent mechanism in ccRCC tumors. Our results demonstrate a novel role for the V2R-YAP axis in the regulation of myofibroblasts in ccRCC and a potential therapeutic target.

Exploiting Dependence of Castration-Resistant Prostate Cancer on the Arginine Vasopressin Signaling Axis by Repurposing Vaptans.

Men with advanced prostate cancer are treated by androgen deprivation therapy but the disease recurs as incurable castration-resistant prostate cancer (CRPC), requiring new treatment options. We previously demonstrated that the G protein-coupled receptor (GPCR) arginine vasopressin receptor type1A (AVPR1A) is expressed in CRPC and promotes castration-resistant growth in vitro and in vivo. AVPR1A is part of a family of GPCR's including arginine vasopressin receptor type 2 (AVPR2). Interrogation of prostate cancer patient sample data revealed that coexpression of AVPR1A and AVPR2 is highly correlated with disease progression. Stimulation of AVPR2 with a selective agonist desmopressin promoted CRPC cell proliferation through cAMP/protein kinase A signaling, consistent with AVPR2 coupling to the G protein subunit alpha s. In contrast, blocking AVPR2 with a selective FDA-approved antagonist, tolvaptan, reduced cell growth. In CRPC xenografts, antagonizing AVPR2, AVPR1A, or both significantly reduced CRPC tumor growth as well as decreased on-target markers of tumor burden. Combinatorial use of AVPR1A and AVPR2 antagonists promoted apoptosis synergistically in CRPC cells. Furthermore, we found that castration-resistant cells produced AVP, the endogenous ligand for arginine vasopressin receptors, and knockout of AVP in CRPC cells significantly reduced proliferation suggesting possible AVP autocrine signaling. These data indicate that the AVP/arginine vasopressin receptor signaling axis represents a promising and clinically actionable target for CRPC. IMPLICATIONS: The arginine vasopressin signaling axis in CRPC provides a therapeutic window that is targetable through repurposing safe and effective AVPR1A and AVPR2 antagonists.

Responsiveness to DDAVP in Cushing's disease is associated with USP8 mutations through enhancing AVPR1B promoter activity.

PURPOSE: To clarify the characteristics of Cushing's disease (CD) patients who respond to the desmopressin (DDAVP) test and its underlying mechanisms. METHODS: Forty-seven patients with CD who underwent DDAVP testing were included. Patients were divided into two groups: DDAVP test (+) (adrenocorticotropic hormone [ACTH] levels increased by ≥ 1.5-fold during the DDAVP test) and DDAVP test (-) (ACTH levels increased by < 1.5-fold). AVP receptor expression levels in these tumors were quantified using quantitative RT-PCR and immunohistochemistry. AVP receptor promoter activity was analyzed using a dual-luciferase reporter assay system. RESULTS: Females (96.9%) and USP8 mutants (85.7%) were more prevalent in the DDAVP test (+) than in the DDAVP test (-). Indeed, the ACTH and cortisol responsiveness to DDAVP was greater in USP8 mutation positive tumors than that in USP8 wild type tumors (3.0-fold vs. 1.3-fold, 1.6-fold vs. 1.1-fold, respectively). Responsiveness to DDAVP was correlated with the expression levels of AVPR1B, but not with those of AVPR2. Comparably, Avpr1b promoter activity was enhanced by the overexpression of mutant USP8 compared to the wild type. CONCLUSIONS: We found that the responsiveness of ACTH to DDAVP in CD was greater in tumors with USP8 mutations. The present data suggest that USP8 mutations upregulate the AVPR1B promoter activity. Additionally, we showed that the DDAVP test can predict the presence of USP8 mutations.

Single-cell transcriptomics of suprachiasmatic nuclei reveal a Prokineticin-driven circadian network.

Circadian rhythms in mammals are governed by the hypothalamic suprachiasmatic nucleus (SCN), in which 20,000 clock cells are connected together into a powerful time-keeping network. In the absence of network-level cellular interactions, the SCN fails as a clock. The topology and specific roles of its distinct cell populations (nodes) that direct network functions are, however, not understood. To characterise its component cells and network structure, we conducted single-cell sequencing of SCN organotypic slices and identified eleven distinct neuronal sub-populations across circadian day and night. We defined neuropeptidergic signalling axes between these nodes, and built neuropeptide-specific network topologies. This revealed their temporal plasticity, being up-regulated in circadian day. Through intersectional genetics and real-time imaging, we interrogated the contribution of the Prok2-ProkR2 neuropeptidergic axis to network-wide time-keeping. We showed that Prok2-ProkR2 signalling acts as a key regulator of SCN period and rhythmicity and contributes to defining the network-level properties that underpin robust circadian co-ordination. These results highlight the diverse and distinct contributions of neuropeptide-modulated communication of temporal information across the SCN.

Activation of AQP2 water channels by protein kinase A: therapeutic strategies for congenital nephrogenic diabetes insipidus.

BACKGROUND: Congenital nephrogenic diabetes insipidus (NDI) is primarily caused by loss-of-function mutations in the vasopressin type 2 receptor (V2R). Renal unresponsiveness to the antidiuretic hormone vasopressin impairs aquaporin-2 (AQP2) water channel activity and water reabsorption from urine, resulting in polyuria. Currently available symptomatic treatments inadequately reduce patients' excessive amounts of urine excretion, threatening their quality of life. In the past 25 years, vasopressin/cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) has been believed to be the most important signaling pathway for AQP2 activation. Although cAMP production without vasopressin is the reasonable therapeutic strategy for congenital NDI caused by V2R mutations, the efficacy of candidate drugs on AQP2 activation is far less than that of vasopressin. RESULTS: Intracellular distribution and activity of PKA are largely controlled by its scaffold proteins, A-kinase anchoring proteins (AKAPs). Dissociating the binding of AKAPs and PKA significantly increased PKA activity in the renal collecting ducts and activated AQP2 phosphorylation and trafficking. Remarkably, the AKAPs-PKA disruptor FMP-API-1 increased transcellular water permeability in isolated renal collecting ducts to the same extent as vasopressin. Moreover, derivatives of FMP-API-1 possessed much more high potency. FMP-API-1/27 is the first low-molecular-weight compound to be discovered that can phosphorylate AQP2 more effectively than preexisting drug candidates. CONCLUSION: AKAP-PKA disruptors are a promising therapeutic target for congenital NDI. In this article, we shall discuss the pathophysiological roles of PKA and novel strategies to activate PKA in renal collecting ducts.

Novel AVPR2 mutations and clinical characteristics in 28 Chinese families with congenital nephrogenic diabetes insipidus.

AIMS: To investigate genotype and phenotype of congenital nephrogenic diabetes insipidus caused by AVPR2 mutations, which is rare and limitedly studied in Chinese population. METHODS: 88 subjects from 28 families with NDI in a department (Beijing, PUMCH) were screened for AVPR2 mutations. Medical records were retrospectively reviewed and characterized. Genotype and phenotype analysis was performed. RESULTS: 23 AVPR2 mutations were identified, including six novel mutations (p.Y117D, p.W208R, p.L313R, p.S127del, p.V162Sfs*30 and p.G251Pfs*96). The onset-age ranged from 1 week to 3 years. Common presentations were polydipsia and polyuria (100%) and intermittent fever (57%). 21% and 14% of patients had short stature and mental impairment. Urine SG and osmolality were decreased, while serum osmolality and sodium were high. Urological ultrasonography results showed hydronephrosis of the kidney (52%), dilation of the ureter (48%), and thickened bladder wall or increased residual urine (32%), led to intermittent urethral catheterization (7%), cystostomy (11%) and binary nephrostomy (4%). Urological defects were developed in older patients. Genotype and phenotype analysis revealed patients with non-missense mutations had higher levels of serum sodium than missense mutations. CONCLUSION: In the first and largest case series of NDI caused by AVPR2 mutations in Chinese population, we established genetic profile and characterized clinical data, reporting six novel mutations. Further, we found genotype was associated with phenotype. This knowledge broadens genotype and phenotype spectrum of rare congenital NDI caused by AVPR2 mutations, and provides basis for studying molecular biology of AVPR2.

Functional characterization of a loss-of-function mutant I324M of arginine vasopressin receptor 2 in X-linked nephrogenic diabetes insipidus.

X-linked nephrogenic diabetes insipidus (X-linked NDI) is a rare inherited disease mainly caused by lost-of-function mutations in human AVPR2 gene encoding arginine vasopressin receptor 2 (V2R). Our focus of the current study is on exploration of the functional and biochemical properties of Ile324Met (I324M) mutation identified in a pedigree showing as typical recessive X-linked NDI. We demonstrated that I324M mutation interfered with the conformation of complex glycosylation of V2R. Moreover, almost all of the I324M-V2R failed to express on the cell surface due to being captured by the endoplasmic reticulum control system. We further examined the signaling activity of DDAVP-medicated cAMP and ERK1/2 pathways and the results revealed that the mutant receptor lost the ability in response to DDAVP stimulation contributed to the failure of accumulation of cAMP and phosphorylated ERK1/2. Based on the characteristics of molecular defects of I324M mutant, we selected two reagents (SR49059 and alvespimycin) to determine whether the functions of I324M-V2R can be restored and we found that both compounds can significantly "rescue" I324M mutation. Our findings may provide further insights for understanding the pathogenic mechanism of AVPR2 gene mutations and may offer some implications on development of promising treatments for patients with X-linked NDI.

Psychotropic drugs upregulate aquaporin-2 via vasopressin-2 receptor/cAMP/protein kinase A signaling in inner medullary collecting duct cells.

Psychotropic drugs may be associated with hyponatremia, but an understanding of how they induce water retention in the kidney remains elusive. Previous studies have postulated that they may increase vasopressin production in the hypothalamus without supporting evidence. In this study, we investigated the possibility of drug-induced nephrogenic syndrome of inappropriate antidiuresis using haloperidol, sertraline, and carbamazepine. Haloperidol, sertraline, or carbamazepine were treated in inner medullary collecting duct (IMCD) suspensions and primary cultured IMCD cells prepared from male Sprague-Dawley rats. The responses of intracellular cAMP production, aquaporin-2 (AQP2) protein expression and localization, vasopressin-2 receptor (V2R) and AQP2 mRNA, and cAMP-responsive element-binding protein (CREB) were tested with and without tolvaptan and the protein kinase A (PKA) inhibitors H89 and Rp-cAMPS. In IMCD suspensions, cAMP production was increased by haloperidol, sertraline, or carbamazepine and was relieved by tolvaptan cotreatment. In primary cultured IMCD cells, haloperidol, sertraline, or carbamazepine treatment increased total AQP2 and decreased phosphorylated Ser261-AQP2 protein expression. Notably, these responses were reversed by cotreatment with tolvaptan or a PKA inhibitor. AQP2 membrane trafficking was induced by haloperidol, sertraline, or carbamazepine and was also blocked by cotreatment with tolvaptan or a PKA inhibitor. Furthermore, upregulation of V2R and AQP2 mRNA and phosphorylated CREB was induced by haloperidol, sertraline, or carbamazepine and was blocked by tolvaptan cotreatment. We conclude that, in the rat IMCD, psychotropic drugs upregulate AQP2 via V2R-cAMP-PKA signaling in the absence of vasopressin stimulation. The vasopressin-like action on the kidney appears to accelerate AQP2 transcription and dephosphorylate AQP2 at Ser261.NEW & NOTEWORTHY It is unclear whether antipsychotic drugs can retain water in the kidney in the absence of vasopressin. This study demonstrates that haloperidol, sertraline, and carbamazepine can produce nephrogenic syndrome of inappropriate antidiuresis because they directly upregulate vasopressin-2 receptor and aquaporin-2 (AQP2) via cAMP/PKA signaling. We showed that, in addition to AQP2 trafficking, AQP2 protein abundance was rapidly increased by treatment with antipsychotic drugs in association with dephosphorylation of AQP2 at Ser261 and accelerated AQP2 transcription.

Congenital nephrogenic diabetes insipidus accompanied with central nephrogenic diabetes secondary to pituitary surgery -a case report.

BACKGROUND: Diabetes insipidus (DI) can be a common cause of polydipsia and polyuria. Here, we present a case of congenital nephrogenic diabetes insipidus (CNDI) accompanied with central diabetes insipidus (CDI) secondary to pituitary surgery. CASE PRESENTATION: A 24-year-old Chinese woman came to our hospital with the complaints of polydipsia and polyuria for 6 months. Six months ago, she was detected with pituitary apoplexy, and thereby getting pituitary surgery. However, the water deprivation test demonstrated no significant changes in urine volume and urine gravity in response to fluid depression or AVP administration. In addition, the genetic results confirmed a heterozygous mutation in arginine vasopressin receptor type 2 (AVPR2) genes. CONCLUSIONS: She was considered with CNDI as well as acquired CDI secondary to pituitary surgery. She was given with hydrochlorothiazide (HCTZ) 25 mg twice a day as well as desmopressin (DDAVP, Minirin) 0.1 mg three times a day. There is no recurrence of polyuria or polydipsia observed for more than 6 months. It can be hard to consider AVPR2 mutation in female carriers, especially in those with subtle clinical presentation. Hence, direct detection of DNA sequencing with AVPR2 is a convenient and accurate method in CNDI diagnosis.

An AMPK activator as a therapeutic option for congenital nephrogenic diabetes insipidus.

Nephrogenic diabetes insipidus (NDI) patients produce large amounts of dilute urine. NDI can be congenital, resulting from mutations in the type-2 vasopressin receptor (V2R), or acquired, resulting from medications such as lithium. There are no effective treatment options for NDI. Activation of PKA is disrupted in both congenital and acquired NDI, resulting in decreased aquaporin-2 phosphorylation and water reabsorption. We show that adenosine monophosphate-activated protein kinase (AMPK) also phosphorylates aquaporin-2. We identified an activator of AMPK, NDI-5033, and we tested its ability to increase urine concentration in animal models of NDI. NDI-5033 increased AMPK phosphorylation by 2.5-fold, confirming activation. It increased urine osmolality in tolvaptan-treated NDI rats by 30%-50% and in V2R-KO mice by 50%. Metformin, another AMPK activator, can cause hypoglycemia, which makes it a risky option for treating NDI patients, especially children. Rats with NDI receiving NDI-5033 showed no hypoglycemia in a calorie-restricted, exercise protocol. Congenital NDI therapy needs to be effective long-term. We administered NDI-5033 for 3 weeks and saw no reduction in efficacy. We conclude that NDI-5033 can improve urine concentration in animals with NDI and holds promise as a potential therapy for patients with congenital NDI due to V2R mutations.

Vasopressin mediates fructose-induced metabolic syndrome by activating the V1b receptor.

Subjects with obesity frequently have elevated serum vasopressin levels, noted by measuring the stable analog, copeptin. Vasopressin acts primarily to reabsorb water via urinary concentration. However, fat is also a source of metabolic water, raising the possibility that vasopressin might have a role in fat accumulation. Fructose has also been reported to stimulate vasopressin. Here, we tested the hypothesis that fructose-induced metabolic syndrome is mediated by vasopressin. Orally administered fructose, glucose, or high-fructose corn syrup increased vasopressin (copeptin) concentrations and was mediated by fructokinase, an enzyme specific for fructose metabolism. Suppressing vasopressin with hydration both prevented and ameliorated fructose-induced metabolic syndrome. The vasopressin effects were mediated by the vasopressin 1b receptor (V1bR), as V1bR-KO mice were completely protected, whereas V1a-KO mice paradoxically showed worse metabolic syndrome. The mechanism is likely mediated in part by de novo expression of V1bR in the liver that amplifies fructokinase expression in response to fructose. Thus, our studies document a role for vasopressin in water conservation via the accumulation of fat as a source of metabolic water. Clinically, they also suggest that increased water intake may be a beneficial way to both prevent or treat metabolic syndrome.

Direct evidence that the GPCR CysLTR2 mutant causative of uveal melanoma is constitutively active with highly biased signaling.

Uveal melanoma is the most common eye cancer in adults and is clinically and genetically distinct from skin cutaneous melanoma. In a subset of cases, the oncogenic driver is an activating mutation in CYSLTR2, the gene encoding the G protein-coupled receptor cysteinyl-leukotriene receptor 2 (CysLTR2). The mutant CYSLTR2 encodes for the CysLTR2-L129Q receptor, with the substitution of Leu to Gln at position 129 (3.43). The ability of CysLTR2-L129Q to cause malignant transformation has been hypothesized to result from constitutive activity, but how the receptor could escape desensitization is unknown. Here, we characterize the functional properties of CysLTR2-L129Q. We show that CysLTR2-L129Q is a constitutively active mutant that strongly drives Gq/11 signaling pathways. However, CysLTR2-L129Q only poorly recruits ß-arrestin. Using a modified Slack-Hall operational model, we quantified the constitutive activity for both pathways and conclude that CysLTR2-L129Q displays profound signaling bias for Gq/11 signaling pathways while escaping ß-arrestin-mediated downregulation. CYSLTR2 is the first known example of a G protein-coupled receptor driver oncogene that encodes a highly biased constitutively active mutant receptor. These results provide new insights into the mechanism of CysLTR2-L129Q oncoprotein signaling and suggest CYSLTR2 as a promising potential therapeutic target in uveal melanoma.

Hypoxia in utero increases the risk of pulmonary hypertension in rat offspring and is associated with vasopressin type­2 receptor upregulation.

Pulmonary hypertension (PH) in newborns and adults is a disease that can lead to right heart failure and result in a shorter lifespan. PH was induced by maintaining pregnant rats in a hypoxic chamber for 4 h twice a day, from days 7­21 of pregnancy. Hypoxia was confirmed by a decrease in the partial pressure of oxygen (PaO2) and the oxygen saturation (SaO2) of arterial blood in the aorta. The body weight of newborns from hypoxic rats was ~20% decreased compared with the control newborns of normoxic rats. The vascular wall thickness/vascular diameter values of hypoxia treated pubs were increased compared with that of control newborns 7 days after birth; however, it decreased to similar levels than in the control group after 3 months, and then further decreased to significantly lower levels than in the control group at 6 months after birth. At birth, the lung tissues of newborns from hypoxic rats exhibited an increase in the levels of mRNA and proteins associated with PH such as HIF­1α, HIF­2α, V2R, TGF­ß, TNF­α, Ang­2 and α­SMA. At 3 and 6 months after birth, the levels of both V2R mRNA and protein in offspring from hypoxic rats were at least 2­fold higher, whereas the expression of all other factors decreased compared with the control offspring. By contrast, HIF­2α and Ang­2 expression levels were significantly increased in the 6­month­old control offspring from normoxic rats. V2R overexpression in pups induced by hypoxia in maternal rats was sustained until their adulthood. V2R may be a marker for detecting PH.

GENETICS IN ENDOCRINOLOGY Pathophysiology, diagnosis and treatment of familial nephrogenic diabetes insipidus.

For an endocrinologist, nephrogenic diabetes insipidus (NDI) is an end-organ disease, that is the antidiuretic hormone, arginine-vasopressin (AVP) is normally produced but not recognized by the kidney with an inability to concentrate urine despite elevated plasma concentrations of AVP. Polyuria with hyposthenuria and polydipsia are the cardinal clinical manifestations of the disease. For a geneticist, hereditary NDI is a rare disease with a prevalence of five per million males secondary to loss of function of the vasopressin V2 receptor, an X-linked gene, or loss of function of the water channel AQP2. These are small genes, easily sequenced, with a number of both recurrent and private mutations described as disease causing. Other inherited disorders with mild, moderate or severe inability to concentrate urine include Bartter's syndrome and cystinosis. MAGED2 mutations are responsible for a transient form of Bartter's syndrome with severe polyhydramnios. The purpose of this review is to describe classical phenotype findings that will help physicians to identify early, before dehydration episodes with hypernatremia, patients with familial NDI. A number of patients are still diagnosed late with repeated dehydration episodes and large dilations of the urinary tract leading to a flow obstructive nephropathy with progressive deterioration of glomerular function. Families with ancestral X-linked AVPR2 mutations could be reconstructed and all female heterozygote patients identified with subsequent perinatal genetic testing to recognize affected males within 2 weeks of birth. Prevention of dehydration episodes is of critical importance in early life and beyond and decreasing solute intake will diminish total urine output.

Cumulative Risk on Oxytocin-Pathway Genes Impairs Default Mode Network Connectivity in Trauma-Exposed Youth.

Background: Although the default mode network (DMN) is a core network essential for brain functioning, little is known about its developmental trajectory, particularly on factors associated with its coherence into a functional network. In light of adult studies indicating DMN's susceptibility to stress-related conditions, we examined links between variability on oxytocin-pathway genes and DMN connectivity in youth exposed to chronic war-related trauma Methods: Following a cohort of war-exposed children from early childhood, we imaged the brains of 74 preadolescents (age 11-13 years; 39 war-exposed) during rest using magnetoencephalography (MEG). A cumulative risk index on oxytocin-pathway genes was constructed by combining single nucleotide polymorphisms on five genes previously linked with social deficits and psychopathology; OXTR rs1042778, OXTR rs2254298, OXTRrs53576, CD38 rs3796863, and AVPR1A RS3. Avoidant response to trauma reminders in early childhood and anxiety disorders in late childhood were assessed as predictors of disruptions to DMN theta connectivity. Results: Higher vulnerability on oxytocin-pathway genes predicted greater disruptions to DMN theta connectivity. Avoidant symptoms in early childhood and generalized anxiety disorder in later childhood were related to impaired DMN connectivity. In combination, stress exposure, oxytocin-pathway genes, and stress-related symptoms explained 24.6% of the variance in DMN connectivity, highlighting the significant effect of stress on the maturing brain. Conclusions: Findings are the first to link the oxytocin system and maturation of the DMN, a core system sustaining autobiographical memories, alteration of intrinsic and extrinsic attention, mentalization, and sense of self. Results suggest that oxytocin may buffer the effects of chronic early stress on the DMN, particularly theta rhythms that typify the developing brain.