Simplified drug efficacy evaluation system for vasopressin neurodegenerative disease using mouse disease-specific induced pluripotent stem cells.

Familial neurohypophyseal diabetes insipidus (FNDI) is a degenerative disorder in which vasopressin-secreting neurons degenerate over time due to the production of mutant proteins. We have demonstrated therapeutic effects of chemical chaperones in an FNDI mouse model, but the complexity and length of this evaluation were problematic. In this study, we established disease-specific mouse induced pluripotent stem cells (iPSCs) from FNDI-model mice and differentiated vasopressin neurons that produced mutant proteins. Fluorescence immunostaining showed that chemical chaperones appeared to protect vasopressin neurons generated from iPSCs derived from FNDI-model mice. Although KCL stimulation released vasopressin hormone from vasopressin neurons generated from FNDI-derived iPSCs, vasopressin hormone levels did not differ significantly between baseline and chaperone-added culture. Semi-quantification of vasopressin carrier protein and mutant protein volumes in vasopressin neurons confirmed that chaperones exerted a therapeutic effect. This research provides fundamental technology for creating in vitro disease models using human iPSCs and can be applied to therapeutic evaluation of various degenerative diseases that produce abnormal proteins.

Collecting duct water permeability inhibition by EGF is associated with decreased cAMP, PKA activity, and AQP2 phosphorylation at Ser269.

Prior studies showed that epidermal growth factor (EGF) inhibits vasopressin-stimulated osmotic water permeability in the renal collecting duct. Here, we investigated the underlying mechanism. Using isolated perfused rat inner medullary collecting ducts (IMCDs), we found that the addition of EGF to the peritubular bath significantly decreased 1-deamino-8-d-arginine vasopressin (dDAVP)-stimulated water permeability, confirming prior observations. The inhibitory effect of EGF on water permeability was associated with a reduction in intracellular cAMP levels and protein kinase A (PKA) activity. Using phospho-specific antibodies and immunoblotting in IMCD suspensions, we showed that EGF significantly reduces phosphorylation of AQP2 at Ser264 and Ser269. This effect was absent when 8-cpt-cAMP was used to induce AQP2 phosphorylation, suggesting that EGF's inhibitory effect was at a pre-cAMP step. Immunofluorescence labeling of microdissected IMCDs showed that EGF significantly reduced apical AQP2 abundance in the presence of dDAVP. To address what protein kinase might be responsible for Ser269 phosphorylation, we used Bayesian analysis to integrate multiple-omic datasets. Thirteen top-ranked protein kinases were subsequently tested by in vitro phosphorylation experiments for their ability to phosphorylate AQP2 peptides using a mass spectrometry readout. The results show that the PKA catalytic-α subunit increased phosphorylation at Ser256, Ser264, and Ser269. None of the other kinases tested phosphorylated Ser269. In addition, H-89 and PKI strongly inhibited dDAVP-stimulated AQP2 phosphorylation at Ser269. These results indicate that EGF decreases the water permeability of the IMCD by inhibiting cAMP production, thereby inhibiting PKA and decreasing AQP2 phosphorylation at Ser269, a site previously shown to regulate AQP2 endocytosis.NEW & NOTEWORTHY The authors used native rat collecting ducts to show that inhibition of vasopressin-stimulated water permeability by epidermal growth factor involves a reduction of aquaporin 2 phosphorylation at Ser269, a consequence of reduced cAMP production and PKA activity.

Vasopressin V2 receptor, tolvaptan, and ERK1/2 phosphorylation in the renal collecting duct.

Tolvaptan, a vasopressin antagonist selective for the V2-subtype vasopressin receptor (V2R), is widely used in the treatment of hyponatremia and autosomal-dominant polycystic kidney disease (ADPKD). Its effects on signaling in collecting duct cells have not been fully characterized. Here, we perform RNA-seq in a collecting duct cell line (mpkCCD). The data show that tolvaptan inhibits the expression of mRNAs that were previously shown to be increased in response to vasopressin including aquaporin-2, but also reveals mRNA changes that were not readily predictable and suggest off-target actions of tolvaptan. One such action is activation of the MAPK kinase (ERK1/ERK2) pathway. Prior studies have shown that ERK1/ERK2 activation is essential in the regulation of a variety of cellular and physiological processes and can be associated with cell proliferation. In immunoblotting experiments, we demonstrated that ERK1/ERK2 phosphorylation in mpkCCD cells was significantly reduced by vasopressin, in contrast to the increases seen in non-collecting-duct cells overexpressing V2R in prior studies. We also found that tolvaptan has a strong effect to increase ERK1/ERK2 phosphorylation in the presence of vasopressin and that tolvaptan's effect to increase ERK1/ERK2 phosphorylation is absent in mpkCCD cells in which both protein kinase A (PKA)-catalytic subunits have been deleted. Thus, it appears that the tolvaptan effect to increase ERK activation is PKA-dependent and is not due to an off-target effect of tolvaptan. We conclude that in cells expressing V2R at endogenous levels: 1) vasopressin decreases ERK1/ERK2 activation; 2) in the presence of vasopressin, tolvaptan increases ERK1/ERK2 activation; and 3) these effects are PKA-dependent.NEW & NOTEWORTHY Vasopressin is a key hormone that regulates the function of the collecting duct of the kidney. ERK1 and ERK2 are enzymes that play key roles in physiological regulation in all cells. The authors used collecting duct cell cultures to investigate the effects of vasopressin and the vasopressin receptor antagonist tolvaptan on ERK1 and ERK2 phosphorylation and activation.

Poly(ADP-ribose) polymerase-1 affects vasopressin-mediated AQP2 expression in collecting duct cells of the kidney.

Poly(ADP-ribosyl)ation (PARylation), as a posttranslational modification mediated by poly(ADP-ribose) polymerases (PARPs) catalyzing the transfer of ADP-ribose from NAD+ molecules to acceptor proteins, involves a number of cellular processes. As mice lacking the PARP-1 gene (Parp1) produce more urine, we investigated the role of PARP-1, the most prevalent member of the PARP family, in the vasopressin-responsive expression of aquaporin-2 (AQP2). In biotin-conjugated nicotinamide adenine dinucleotide (biotin-NAD+) pulldown and immunoprecipitation assays of poly(ADP)-ribose in mpkCCDc14 cells, immunoblots demonstrated that 1-deamino-8-D-arginine vasopressin (dDAVP) induced the PARylation of total proteins, associated with an increase in the cleavage of PARP-1 and cleaved caspase-3 expression. By inhibiting PARP-1 with siRNA, the abundance of dDAVP-induced AQP2 mRNA and protein was significantly diminished. In contrast, despite a substantial decrease in PARylation, the PARP-1 inhibitor (PJ34) had no effect on the dDAVP-induced regulation of AQP2 expression. The findings suggest that PARP-1 protein expression itself, and not PARP-1-mediated PARylation, is necessary for dDAVP-regulated AQP2 expression. Bioinformatic analysis revealed that 408 proteins interact with PARP-1 in the collecting duct (CD) cells of the kidney. Among them, the signaling pathway of the vasopressin V2 receptor was identified for 49 proteins. In particular, ß-catenin, which is phosphorylated at Ser552 by dDAVP, was identified as the PARP-1-interacting protein. A significant decrease of ß-catenin phosphorylation (Ser552) in response to dDAVP was associated with siRNA-mediated PARP-1 knockdown. Taken together, PARP-1 is likely to play a role in vasopressin-induced AQP2 expression by interacting with ß-catenin in renal CD cells.NEW & NOTEWORTHY The poly(ADP-ribose) polymerase (PARP) family catalyzes poly(ADP-ribosylation) (PARylation), which is one of the posttranslational modifications of largely undetermined physiological significance. This study investigated the role of PARP-1, the most prevalent member of the PARP family, in the vasopressin-responsive expression of aquaporin-2 (AQP2). The results demonstrated that PARP-1 protein expression itself, and not PARP-1-mediated PARylation, is necessary for dDAVP-regulated AQP2 expression. ß-Catenin, which is phosphorylated at Ser552 by dDAVP, was identified as the PARP-1-interacting protein.

Reducing sugar intake through chronic swimming training: Exploring palatability changes and central vasopressin mechanisms.

Excessive sugar intake has been associated with the onset of several non-communicable chronic diseases seen in humans. Physical activity could affect sweet taste perception which may affect sugar intake. Therefore, it was investigated the chronic effects of swimming training on sucrose intake/preference, reactivity to sucrose taste, self-care in neurobehavioral stress, and the possible involvement of the vasopressin type V1 receptor in sucrose solution intake. Male Wistar rats, of from different cohorts were used, subjected to a sedentary lifestyle (SED) or swimming training (TR - 1 h/day, 5×/week, for 8 weeks, with no added load). Weekly intake was verified in SED and TR rats after access to a sucrose solution 1×/week, 2 h/day, for eight weeks. Chronic effects of swimming and/or a sedentary lifestyle were carried out three days after the end of the physical exercise protocol. Swimming training reduced the intake of sucrose solution from the third week onwards in the two-bottle test measured once a week for 8 weeks. After the ending of the swimming protocol, sucrose intake was also reduced as per its preference. This reduced intake is probably correlated with the carbohydrate aspect of sucrose since saccharin intake was not affected. In addition, chronic swimming training was shown to reduce ingestive responses, increase neutral responses, without interfering with aversive, in the sucrose solution taste reactivity test. In addition, these results are not related to a depressive-like behavior, nor to neurobehavioral stress. Furthermore, treatment with vasopressin V1 receptor antagonist abolished the reduced sucrose intake in trained rats. The results suggest that swimming performed chronically is capable of reducing intake and preference for sucrose by decreasing the palatability of sucrose without causing depressive-type behavior or stress. In addition, the results also suggest that central V1 vasopressin receptors are part of the mechanisms activated to reduce sucrose intake in trained rats.

ENDOCANNABINOID SYSTEM IN THE PARAVENTRICULAR NUCLEUS OF THE HYPOTHALAMUS MODULATES AUTONOMIC AND CARDIOVASCULAR CHANGES BUT NOT VASOPRESSIN RESPONSE IN A RAT HEMORRHAGIC SHOCK MODEL.

ABSTRACT: We evaluated the participation of the endocannabinoid system in the paraventricular nucleus of the hypothalamus (PVN) on the cardiovascular, autonomic, and plasma vasopressin (AVP) responses evoked by hemorrhagic shock in rats. For this, the PVN was bilaterally treated with either vehicle, the selective cannabinoid receptor type 1 antagonist AM251, the selective fatty acid amide hydrolase amide enzyme inhibitor URB597, the selective monoacylglycerol-lipase enzyme inhibitor JZL184, or the selective transient receptor potential vanilloid type 1 antagonist capsazepine. We evaluated changes on arterial pressure, heart rate, tail skin temperature (ST), and plasma AVP responses induced by bleeding, which started 10 min after PVN treatment. We observed that bilateral microinjection of AM251 into the PVN reduced the hypotension during the hemorrhage and prevented the return of blood pressure to baseline values in the posthemorrhagic period. Inhibition of local 2-arachidonoylglycerol metabolism by PVN treatment with JZL184 induced similar effects in relation to those observed in AM251-treated animals. Inhibition of local anandamide metabolism via PVN treatment with URB597 decreased the depressor effect and ST drop induced by the hemorrhagic stimulus. Bilateral microinjection of capsazepine mitigated the fall in blood pressure and ST. None of the PVN treatments altered the increased plasma concentration of AVP and tachycardia induced by hemorrhage. Taken together, present results suggest that endocannabinoid neurotransmission within the PVN plays a prominent role in cardiovascular and autonomic, but not neuroendocrine, responses evoked by hemorrhage.

Desmopressin enhances random-pattern skin flap survival in rats: Possible role of vasopressin Type-1a and 2 receptors.

BACKGROUND: Many drugs have been explored for their role in improving skin flap survival. 1-deamino-8-D-arginine vasopressin (DDAVP or desmopressin) is a synthesized form of anti-diuretic hormone (ADH) and a selective agonist for vasopressin type-2 receptors (V2 receptors). Desmopressin has been shown to improve endothelial function, induce vasodilation, and reduce inflammation. We aimed to evaluate its efficacy in enhancing flap survival and assess the role of vasopressin receptors in this process. MATERIALS AND METHODS: We randomly assigned six male Wistar rats to each study group. Different doses of desmopressin were injected intraperitoneally to find the most effective amount (8 µg/rat). SR-49059, a selective V1a receptor antagonist, was given at 2µg/rat before providing the most effective dose of desmopressin (8µg/rat). Histopathological assessments, quantitative measurements of interleukin-1ß (IL-1ß), Tumor necrosis factor-alpha (TNF-α), and Nuclear Factor-κB (NF-κB), optical imaging, and measurement of the expression levels of V2 receptor in the rat skin tissue were performed. RESULTS: Desmopressin (8µg/rat) significantly reduced the mean percentage of necrotic area compared to the control group (19.35% vs 73.57%). Histopathological evaluations revealed a notable reduction in tissue inflammation, edema, and degeneration following administration of desmopressin (8). The expression of the V2 receptor was increased following desmopressin administration. It also led to a reduction in IL-1ß, TNF-α, and NF-κB levels. The protective effect of desmopressin on flap survival was reversed upon giving SR-49059. The optical imaging revealed enhanced blood flow in the desmopressin group compared to the control group. CONCLUSIONS: Desmopressin could be repurposed to improve flap survival. V1a and V2 receptors probably mediate this effect.

LRBA signalosomes activate vasopressin-induced AQP2 trafficking at recycling endosomes.

Aquaporin-2 (AQP2) water channels are proteins that are recycled between intracellular vesicles and the apical plasma membrane in renal collecting ducts. Lipopolysaccharide-responsive beige-like anchor protein (LRBA) is a protein kinase A (PKA) anchoring protein that creates compartmentalized PKA signalling responsible for AQP2 phosphorylation. In response to increased plasma osmolality, vasopressin/cyclic adenosine monophosphate (cAMP)/PKA signalling phosphorylates AQP2, promoting AQP2 trafficking into the apical plasma membrane and increasing water reabsorption from urine. However, the molecular mechanisms by which LRBA mediates vasopressin-induced AQP2 phosphorylation remain unknown. To investigate AQP2 intracellular localization and phosphorylation status in vivo, a density gradient ultracentrifugation technique was combined with an in situ proximity ligation assay, super-resolution structured illumination microscopy and immunoelectron microscopy. Most of the AQP2 was localized on the recycling endosome in the presence of tolvaptan, a vasopressin type 2 receptor (V2R) antagonist. Desmopressin, a V2R agonist, phosphorylated AQP2, translocating it from the recycling endosome to the apical plasma membrane. In contrast, LRBA was constitutively localized at the recycling endosome. Therefore, LRBA and AQP2 were well colocalized in the absence of vasopressin stimulation. The loss of LRBA/PKA signalling by Lrba knockout impaired vasopressin-induced AQP2 phosphorylation, resulting in AQP2 retention at the recycling endosome. Defective AQP2 trafficking caused low urinary concentrating ability in Lrba-/- mice. The LRBA-PKA complex created compartmentalized PKA signalling at the recycling endosome, which facilitated AQP2 phosphorylation in response to vasopressin. KEY POINTS: Membrane proteins are continuously internalized into the endosomal system via endocytosis, after which they are either recycled back to the plasma membrane or degraded at the lysosome. In T cells, lipopolysaccharide-responsive beige-like anchor protein (LRBA) binds directly to the cytotoxic T lymphocyte antigen 4 (CTLA-4), a checkpoint immune molecule, to prevent CTLA-4 lysosomal degradation and promote its vesicle recycling. LRBA has different physiological functions in renal collecting ducts. LRBA and aquaporin-2 (AQP2) water channels were colocalized on the recycling endosome in vivo in the absence of the anti-diuretic hormone vasopressin. LRBA promoted vasopressin-induced AQP2 trafficking, increasing water reabsorption from urine via AQP2. LRBA determined renal responsiveness to vasopressin at recycling endosomes. LRBA is a ubiquitously expressed anchor protein. LRBA signalosomes might regulate membrane trafficking of several constitutively recycled proteins at recycling endosomes.

Photoswitchable Probes of Oxytocin and Vasopressin.

Oxytocin (OT) and vasopressin (VP) are related neuropeptides that regulate many biological processes. In humans, OT and VP act via four G protein-coupled receptors, OTR, V1aR, V1bR, and V2R (VPRs), which are associated with several disorders. To investigate the therapeutic potential of these receptors, particularly in the receptor-dense areas of the brain, molecular probes with a high temporal and spatial resolution are required. Such a spatiotemporal resolution can be achieved by incorporating photochromic moieties into OT and VP. Here, we report the design, synthesis, and (photo)pharmacological characterization of 12 OT- and VP-derived photoprobes using different modification strategies. Despite OT's and VP's sensitivity toward structural changes, we identified two photoprobes with good potency and photoswitch window for investigating the OTR and V1bR. These photoprobes should be of high value for producing cutting-edge photocontrollable peptide probes for the study of dynamic and kinetic receptor activation processes in specific regions of the brain.

The Vasopressin Loading for Refractory septic shock (VALOR) study: a prospective observational study.

BACKGROUND: Vasopressin is a second-line vasoactive agent for refractory septic shock. Vasopressin loading is not generally performed because of the lack of evidence for its effects and safety. However, based on our previous findings, we hypothesized it can predict the responsibility to vasopressin infusion with safety, and prospectively examined it in the present study. METHODS: Vasopressin loading was performed via the intravenous administration of a bolus of 1 U, followed by its continuous infusion at 1U/h in patients with septic shock treated with ≥ 0.2 µg/kg/min noradrenaline. An arterial pressure wave analysis was conducted, and endocrinological tests were performed immediately prior to vasopressin loading. We classified patients into responders/non-responders based on mean arterial pressure (MAP) changes after vasopressin loading. Based on our previous findings, the lower tertile of MAP changes was selected as the cut-off. The change in the catecholamine index (CAI) after 6 h was assigned as the primary outcome. Digital ischemia, mesenteric ischemia, and myocardial ischemia during the admission period were prospectively and systematically recorded as adverse events. RESULTS: Ninety-two patients were registered during the study period and examined. Sixty-two patients with a MAP change > 22 mmHg were assigned as responders and the others as non-responders. Blood adrenocorticotropic hormone levels were significantly higher in non-responders. Stroke volume variations were higher in responders before loading, while stroke volume and dP/dtmax were higher in responders after loading. Median CAI changes were - 10 in responders and 0 in non-responders, which was significantly lower in the former (p < 0.0001). AUROC of MAP change with vasopressin loading to predict CAI change < 0 after continuous infusion was 0.843 with sensitivity of 0.92 and specificity of 0.77. Ischemia events were observed in 5 cases (5.4%). CONCLUSIONS: Vasopressin loading may be safely introduced for septic shock. Vasopressin loading may be used to predict responses to its continuous infusion and select appropriate strategies to increase blood pressure.

Interactions between the Astrocytic Volume-Regulated Anion Channel and Aquaporin 4 in Hyposmotic Regulation of Vasopressin Neuronal Activity in the Supraoptic Nucleus.

We assessed interactions between the astrocytic volume-regulated anion channel (VRAC) and aquaporin 4 (AQP4) in the supraoptic nucleus (SON). Acute SON slices and cultures of hypothalamic astrocytes prepared from rats received hyposmotic challenge (HOC) with/without VRAC or AQP4 blockers. In acute slices, HOC caused an early decrease with a late rebound in the neuronal firing rate of vasopressin neurons, which required activity of astrocytic AQP4 and VRAC. HOC also caused a persistent decrease in the excitatory postsynaptic current frequency, supported by VRAC and AQP4 activity in early HOC; late HOC required only VRAC activity. These events were associated with the dynamics of glial fibrillary acidic protein (GFAP) filaments, the late retraction of which was mediated by VRAC activity; this activity also mediated an HOC-evoked early increase in AQP4 expression and late subside in GFAP-AQP4 colocalization. AQP4 activity supported an early HOC-evoked increase in VRAC levels and its colocalization with GFAP. In cultured astrocytes, late HOC augmented VRAC currents, the activation of which depended on AQP4 pre-HOC/HOC activity. HOC caused an early increase in VRAC expression followed by a late rebound, requiring AQP4 and VRAC, or only AQP4 activity, respectively. Astrocytic swelling in early HOC depended on AQP4 activity, and so did the early extension of GFAP filaments. VRAC and AQP4 activity supported late regulatory volume decrease, the retraction of GFAP filaments, and subside in GFAP-VRAC colocalization. Taken together, astrocytic morphological plasticity relies on the coordinated activities of VRAC and AQP4, which are mutually regulated in the astrocytic mediation of HOC-evoked modulation of vasopressin neuronal activity.

Acute Hemodynamics in the Fontan Circulation: Open-Label Study of Vasopressin.

OBJECTIVE: To describe the acute hemodynamic effect of vasopressin on the Fontan circulation, including systemic and pulmonary pressures and resistances, left atrial pressure, and cardiac index. DESIGN: Prospective, open-label, nonrandomized study (NCT04463394). SETTING: Cardiac catheterization laboratory at Lucile Packard Children's Hospital, Stanford. PATIENTS: Patients 3-50 years old with a Fontan circulation who were referred to the cardiac catheterization laboratory for hemodynamic assessment and/or intervention. INTERVENTIONS: A 0.03 U/kg IV (maximum dose 1 unit) bolus of vasopressin was administered over 5 minutes, followed by a maintenance infusion of 0.3 mU/kg/min (maximum dose 0.03 U/min). MEASUREMENTS AND MAIN RESULTS: Comprehensive cardiac catheterization measurements before and after vasopressin administration. Measurements included pulmonary artery, atrial, and systemic arterial pressures, oxygen saturations, and systemic and pulmonary flows and resistances. There were 28 patients studied. Median age was 13.5 (9.1, 17) years, and 16 (57%) patients had a single or dominant right ventricle. Following vasopressin administration, systolic blood pressure and systemic vascular resistance (SVR) increased by 17.5 (13.0, 22.8) mm Hg ( Z value -4.6, p < 0.001) and 3.8 (1.8, 7.5) Wood Units ( Z value -4.6, p < 0.001), respectively. The pulmonary vascular resistance (PVR) decreased by 0.4 ± 0.4 WU ( t statistic 6.2, p < 0.001), and the left atrial pressure increased by 1.0 (0.0, 2.0) mm Hg ( Z value -3.5, p < 0.001). The PVR:SVR decreased by 0.04 ± 0.03 ( t statistic 8.1, p < 0.001). Neither the pulmonary artery pressure (median difference 0.0 [-1.0, 1.0], Z value -0.4, p = 0.69) nor cardiac index (0.1 ± 0.3, t statistic -1.4, p = 0.18) changed significantly. There were no adverse events. CONCLUSIONS: In Fontan patients undergoing cardiac catheterization, vasopressin administration resulted in a significant increase in systolic blood pressure, SVR, and left atrial pressure, decrease in PVR, and no change in cardiac index or pulmonary artery pressure. These findings suggest that in Fontan patients vasopressin may be an option for treating systemic hypotension during sedation or general anesthesia.

Null results of oxytocin and vasopressin administration on mentalizing in a large fMRI sample: evidence from a randomized controlled trial.

BACKGROUND: Although potential links between oxytocin (OT), vasopressin (AVP), and social cognition are well-grounded theoretically, most studies have included all male samples, and few have demonstrated consistent effects of either neuropeptide on mentalizing (i.e. understanding the mental states of others). To understand the potential of either neuropeptide as a pharmacological treatment for individuals with impairments in social cognition, it is important to demonstrate the beneficial effects of OT and AVP on mentalizing in healthy individuals. METHODS: In the present randomized, double-blind, placebo-controlled study (n = 186) of healthy individuals, we examined the effects of OT and AVP administration on behavioral responses and neural activity in response to a mentalizing task. RESULTS: Relative to placebo, neither drug showed an effect on task reaction time or accuracy, nor on whole-brain neural activation or functional connectivity observed within brain networks associated with mentalizing. Exploratory analyses included several variables previously shown to moderate OT's effects on social processes (e.g., self-reported empathy, alexithymia) but resulted in no significant interaction effects. CONCLUSIONS: Results add to a growing literature demonstrating that intranasal administration of OT and AVP may have a more limited effect on social cognition, at both the behavioral and neural level, than initially assumed. Randomized controlled trial registrations: ClinicalTrials.gov; NCT02393443; NCT02393456; NCT02394054.

Slow Intravenous Infusion of a Novel Damage Control Cocktail Decreases Blood Loss in a Pig Polytrauma Model.

BACKGROUND: Our objective was to optimize a novel damage control resuscitation (DCR) cocktail composed of hydroxyethyl starch, vasopressin, and fibrinogen concentrate for the polytraumatized casualty. We hypothesized that slow intravenous infusion of the DCR cocktail in a pig polytrauma model would decrease internal hemorrhage and improve survival compared with bolus administration. METHODS: We induced polytrauma, including traumatic brain injury (TBI), femoral fracture, hemorrhagic shock, and free bleeding from aortic tear injury, in 18 farm pigs. The DCR cocktail consisted of 6% hydroxyethyl starch in Ringer's lactate solution (14mL/kg), vasopressin (0.8U/kg), and fibrinogen concentrate (100mg/kg) in a total fluid volume of 20mL/kg that was either divided in half and given as two boluses separated by 30 minutes as control or given as a continuous slow infusion over 60 minutes. Nine animals were studied per group and monitored for up to 3 hours. Outcomes included internal blood loss, survival, hemodynamics, lactate concentration, and organ blood flow obtained by colored microsphere injection. RESULTS: Mean internal blood loss was significantly decreased by 11.1mL/kg with infusion compared with the bolus group (p = .038). Survival to 3 hours was 80% with infusion and 40% with bolus, which was not statistically different (Kaplan Meier log-rank test, p = .17). Overall blood pressure was increased (p < .001), and blood lactate concentration was decreased (p < .001) with infusion compared with bolus. There were no differences in organ blood flow (p > .09). CONCLUSION: Controlled infusion of a novel DCR cocktail decreased hemorrhage and improved resuscitation in this polytrauma model compared with bolus. The rate of infusion of intravenous fluids should be considered as an important aspect of DCR.

Stimulatory effects of vasopressin on progesterone production and BMP signaling by ovarian granulosa cells.

The aim of the present study was to clarify the effects of arginine vasopressin (AVP) on ovarian steroid production and its functional relationship to the ovarian bone morphogenetic protein (BMP) system. The results showed that AVP treatment significantly increased gonadotropin- and forskolin-induced progesterone synthesis by primary culture of rat granulosa cells and human granulosa cells, respectively. In contrast, estradiol production was not significantly affected by AVP. Treatment with AVP significantly increased forskolin-induced cAMP synthesis by human granulosa cells and mRNA levels of the progesterogenic enzymes CYP11A1 and HSD3B2 in the cells. On the other hand, AVP also enhanced BMP-15-induced phosphorylation of SMAD1/5/9 and ID1 transcription. It was further revealed that the expression levels of BMP receptors, including ALK3, ALK6 and BMPR2, were upregulated by AVP. Collectively, the results indicate that AVP stimulates progesterone production via the cAMP-PKA pathway with upregulation of BMP signaling that inhibits progesterone production, which may lead to fine adjustment of progesterone biosynthesis by granulosa cells.

Insulin-regulated aminopeptidase is required for water excretion in response to acute hypotonic stress.

The objective of this study was to understand the response of mice lacking insulin-regulated aminopeptidase (IRAP) to an acute water load. For mammals to respond appropriately to acute water loading, vasopressin activity needs to decrease. IRAP degrades vasopressin in vivo. Therefore, we hypothesized that mice lacking IRAP have an impaired ability to degrade vasopressin and, thus, have persistent urinary concentration. Age-matched 8- to 12-wk-old IRAP wild-type (WT) and knockout (KO) male mice were used for all experiments. Blood electrolytes and urine osmolality were measured before and 1 h after water load (∼2 mL sterile water via intraperitoneal injection). Urine was collected from IRAP WT and KO mice for urine osmolality measurements at baseline and after 1 h administration of the vasopressin type 2 receptor antagonist OPC-31260 (10 mg/kg ip). Immunofluorescence and immunoblot analysis were performed on kidneys at baseline and after 1 h acute water load. IRAP was expressed in the glomerulus, thick ascending loop of Henle, distal tubule, connecting duct, and collecting duct. IRAP KO mice had elevated urine osmolality compared with WT mice due to higher membrane expression of aquaporin 2 (AQP2), which was restored to that of controls after administration of OPC-31260. IRAP KO mice developed hyponatremia after an acute water load because they were unable to increase free water excretion due to increased surface expression of AQP2. In conclusion, IRAP is required to increase water excretion in response to an acute water load due to persistent vasopressin stimulation of AQP2.NEW & NOTEWORTHY Insulin-regulated aminopeptidase (IRAP) degrades vasopressin, but its role in urinary concentration and dilution is unknown. Here, we show that IRAP-deficient mice have a high urinary osmolality at baseline and are unable to excrete free water in response to water loading. These results reveal a novel regulatory role for IRAP in urine concentration and dilution.

The Role of Oxytocin and Vasopressin in Drug-Induced Reward-Implications for Social and Non-Social Factors.

Drug abuse is a worldwide problem that leads to negative physical, mental, and economic consequences. Although pharmacological strategies for drug addiction management have been widely studied, therapeutic options with high efficacy and a low side-effects profile are still limited. Recently, there has been a growing interest in oxytocin (OT) and vasopressin (AVP) systems as potential therapeutic targets for the treatment of drug abuse. OT and AVP are hypothalamic neuropeptides involved in numerous physiological processes. Additionally, studies show that these neurohormones are highly implicated in the modulation of a wide range of behaviors. Interestingly, ample evidence has shown that both, OT and AVP are able to decrease the consumption of different drugs of abuse, as well as to ameliorate their rewarding and reinforcing effects. Furthermore, OT and AVP have been strongly involved in prosocial effects and social reward. In particular, OT has been shown to be able to shift drug-induced reward into social-induced reward, mainly due to its interaction with the dopaminergic system. This phenomenon is also reflected in the results of clinical trials where intranasal OT shows promising efficacy in managing substance use disorder. Therefore, the aim of this review is to comprehensively characterize the involvement of OT and AVP in the rewarding and other behavioral effects of drugs of abuse in animal models, with a particular highlight on the impact of social factors on the observed effects. Understanding this relationship may contribute to higher drug development success rates, as a result of a more profound and deliberate studies design.

Structure-Affinity and Structure-Kinetic Relationship Studies of Benzodiazepine Derivatives for the Development of Efficacious Vasopressin V2 Receptor Antagonists.

Vasopressin V2 receptors (V2R) are a promising drug target for autosomal dominant polycystic kidney disease (ADPKD). As previous research demonstrated that the residence time of V2R antagonists is critical to their efficacy in both ex vivo and in vivo models of ADPKD, we performed extensive structure-kinetic relationship (SKR) analyses on a series of benzodiazepine derivatives. We found that subtle structural modifications of the benzodiazepine derivatives dramatically changed their binding kinetics but not their affinity. Compound 18 exhibited a residence time of 77 min, which was 7.7-fold longer than that of the reference compound tolvaptan (TVP). Accordingly, compound 18 exhibited higher efficacy compared to TVP in an in vivo model of ADPKD. Overall, our study exemplifies a kinetics-directed medicinal chemistry effort for the development of efficacious V2R antagonists. We envision that this strategy may also have general applicability in other therapeutic areas.

Norepinephrine and Vasopressin in Hemorrhagic Shock: A Focus on Renal Hemodynamics.

During hemorrhagic shock, blood loss causes a fall in blood pressure, decreases cardiac output, and, consequently, O2 transport. The current guidelines recommend the administration of vasopressors in addition to fluids to maintain arterial pressure when life-threatening hypotension occurs in order to prevent the risk of organ failure, especially acute kidney injury. However, different vasopressors exert variable effects on the kidney, depending on the nature and dose of the substance chosen as follows: Norepinephrine increases mean arterial pressure both via its α-1-mediated vasoconstriction leading to increased systemic vascular resistance and its ß1-related increase in cardiac output. Vasopressin, through activation of V1-a receptors, induces vasoconstriction, thus increasing mean arterial pressure. In addition, these vasopressors have the following different effects on renal hemodynamics: Norepinephrine constricts both the afferent and efferent arterioles, whereas vasopressin exerts its vasoconstrictor properties mainly on the efferent arteriole. Therefore, this narrative review discusses the current knowledge of the renal hemodynamic effects of norepinephrine and vasopressin during hemorrhagic shock.

The role of glucagon-like peptide-1 (GLP-1) in fluid and food intakes in vasopressin-deficient Brattleboro rats.

Eating and drinking co-occur and many of the same mechanisms that control one are involved in the control of the other, making it difficult to isolate specific mechanisms for the control of fluid intake. Glucagon-like peptide-1 (GLP-1) is a peptide that seems to be involved in the endogenous control of both ingestive behaviors, but we lack a thorough understanding of how and where GLP-1 is acting to control fluid intake. Vasopressin-deficient Brattleboro rats are a model of hereditary hypothalamic diabetes insipidus that have been used extensively for the study of vasopressin actions in behavior and physiology. Here, we propose that these rats, that eat normally but drink excessively, provide a useful model to dissociate central controls of food and fluid intakes. As an initial step toward establishing this model for these purposes, we focused on GLP-1. Similar to the effect observed after treatment with a GLP-1 receptor (GLP-1R) agonist, the intake difference between wildtype and Brattleboro rats was largely a function in the number of licking bursts, indicating differences in post-ingestive feedback (e.g., satiation). When given central injections of a GLP-1R agonist, the effect on feeding was comparable between wildtype and Brattleboro rats, but the effect of drug on fluid intake was markedly exaggerated in Brattleboro rats. Additionally, Brattleboro rats did not respond to GLP-1R antagonism, whereas wildtype rats did. Taken together, these results suggest that Brattleboro rats exhibit a selective disruption to GLP-1's control of water intake. Overall, these experiments provide foundational studies of the ingestive behavior of Brattleboro rats and demonstrate the potential to use these rats to disentangle the effects of GLP-1 on food and fluid intakes.