Increased anxiety and decreased sociability induced by paternal deprivation involve the PVN-PrL OTergic pathway.

Early adverse experiences often have devastating consequences. However, whether preweaning paternal deprivation (PD) affects emotional and social behaviors and their underlying neural mechanisms remain unexplored. Using monogamous mandarin voles, we found that PD increased anxiety-like behavior and attenuated social preference in adulthood. PD also decreased the number of oxytocin (OT)-positive neurons projecting from the paraventricular nucleus (PVN) and reduced the levels of the medial prefrontal cortex OT receptor protein in females and of the OT receptor and V1a receptor proteins in males. Intra-prelimbic cortical OT injections reversed the PD-induced changes in anxiety-like behavior and social preferences. Optogenetic activation of the prelimbic cortex OT terminals from PVN OT neurons reversed the PD-induced changes in emotion and social preference behaviors, whereas optogenetic inhibition was anxiogenic and impaired social preference in naive voles. These findings demonstrate that PD increases anxiety-like behavior and attenuates social preferences through the involvement of PVN OT neuron projections to the prelimbic cortex.

Biomarker discovery for disease status and symptom severity in children with autism.

Autism spectrum disorder (ASD) is characterized by social impairments and repetitive behaviors, and affects 1 in 68 US children. Despite ASD's societal impact, its disease mechanisms remain poorly understood. Recent preclinical ASD biomarker discovery research has implicated the neuropeptides oxytocin (OXT) and arginine vasopressin (AVP), and their receptors, OXTR and AVPR1A, in animal models. Efforts to translate these findings to individuals with ASD have typically involved evaluating single neuropeptide measures as biomarkers of ASD and/or behavioral functioning. Given that ASD is a heterogeneous disorder, and unidimensional ASD biomarker studies have been challenging to reproduce, here we employed a multidimensional neuropeptide biomarker analysis to more powerfully interrogate disease status and symptom severity in a well characterized child cohort comprised of ASD patients and neurotypical controls. These blood-based neuropeptide measures, considered as a whole, correctly predicted disease status for 57 out of 68 (i.e., 84%) participants. Further analysis revealed that a composite measure of OXTR and AVPR1A gene expression was the key driver of group classification, and that children with ASD had lower neuropeptide receptor mRNA levels compared to controls. Lower neuropeptide receptor mRNA levels also predicted greater symptom severity for core ASD features (i.e., social impairments and stereotyped behaviors), but were unrelated to intellectual impairment, an associated feature of ASD. Findings from this research highlight the value of assessing multiple related biological measures, and their relative contributions, in the same study, and suggest that low blood neuropeptide receptor availability may be a promising biomarker of disease presence and symptom severity in ASD.

The potential repertoire of the innate immune system in the bladder: expression of pattern recognition receptors in the rat bladder and a rat urothelial cell line (MYP3 cells).

PURPOSE: The urothelium is a frontline sensor of the lower urinary tract, sampling the bladder lumen and stimulating an immune response to infectious and noxious agents. Pattern recognition receptors (PRRs) recognize such agents and coordinate the innate response, often by forming inflammasomes that activate caspase-1 and the release of interleukin-1. We have shown the presence of one PRR (NLRP3) in the urothelia and its central role in the inflammatory response to cyclophosphamide. The purpose of this study was to (1) assess the likely range of the PPR response by assessing the repertoire present in the rat bladder and (2) determine the utility of the MYP3 rat urothelia cell line for in vitro studies by assessing its PPR repertoire and functional responsiveness. METHODS: Immunohistochemistry was performed for seven PPRs (NLRP1, NLRP3, NLRP6, NLRP7, NLRP12, NLRC4 and AIM2) on bladder sections and MYP3 cells. For functionality, MYP3 cells were challenged with the quintessential NLRP3 activator ATP and assessed for caspase-1 activation. RESULTS: All PPRs examined were expressed in the bladder and localized to the urothelial layer with several also in the detrusor (none in the interstitia). MYP3 cells also expressed all PRRs with a variable intracellular location. ATP-stimulated caspase-1 activity in MYP3 cells in a dose-dependent manner was reduced by knockdown of NLRP3 expression. CONCLUSION: The results suggest that the bladder possesses the capacity to initiate an innate immune response to a wide array of uropathological agents and the MYP3 cells will provide an excellent investigational tool for this field.

Fluoromodule-based reporter/probes designed for in vivo fluorescence imaging.

Optical imaging of whole, living animals has proven to be a powerful tool in multiple areas of preclinical research and has allowed noninvasive monitoring of immune responses, tumor and pathogen growth, and treatment responses in longitudinal studies. However, fluorescence-based studies in animals are challenging because tissue absorbs and autofluoresces strongly in the visible light spectrum. These optical properties drive development and use of fluorescent labels that absorb and emit at longer wavelengths. Here, we present a far-red absorbing fluoromodule-based reporter/probe system and show that this system can be used for imaging in living mice. The probe we developed is a fluorogenic dye called SC1 that is dark in solution but highly fluorescent when bound to its cognate reporter, Mars1. The reporter/probe complex, or fluoromodule, produced peak emission near 730 nm. Mars1 was able to bind a variety of structurally similar probes that differ in color and membrane permeability. We demonstrated that a tool kit of multiple probes can be used to label extracellular and intracellular reporter-tagged receptor pools with 2 colors. Imaging studies may benefit from this far-red excited reporter/probe system, which features tight coupling between probe fluorescence and reporter binding and offers the option of using an expandable family of fluorogenic probes with a single reporter gene.

Mutation in the V2 vasopressin receptor gene, AVPR2, causes nephrogenic syndrome of inappropriate diuresis.

Nephrogenic syndrome of inappropriate antidiuresis (NSIAD) is a recently discovered rare disease caused by gain-of-function mutations of the V2 vasopressin receptor gene, AVPR2. To date, mutations of Phe229 and Arg137 have been identified as gain-of-function in the V2 vasopressin receptor (V2R). These receptor mutations lead to hyponatremia, which may lead to clinical symptoms in infants. Here we present a newly identified I130N substitution in exon 2 of the V2R gene in a family, causing NSIAD. This I130N mutation resulted in constitutive activity of the V2R with constitutive cyclic adenosine monophosphate (cAMP) generation in HEK293 cells. This basal activity could be blocked by the inverse agonist tolvaptan and arginine-vasopressin stimulation enhanced the cAMP production of I130N-V2R. The mutation causes a biased receptor conformation as the basal cAMP generation activity of I130N does not lead to interaction with ß-arrestin. The constitutive activity of the mutant receptor caused constitutive dynamin-dependent and ß-arrestin-independent internalization. The inhibition of basal internalization using dominant-negative dynamin resulted in an increased cell surface expression. In contrast to the constitutive internalization, agonist-induced endocytosis was ß-arrestin dependent. Thus, tolvaptan could be used for treatment of hyponatremia in patients with NSIAD who carry the I130N-V2R mutation.

VACM-1/cul5 expression in vascular tissue in vivo is induced by water deprivation and its expression in vitro regulates aquaporin-1 concentrations.

VACM-1, a cul5 gene product, when overexpressed in vitro, has an antiproliferative effect. In vivo, VACM-1/cul5 is present in tissues involved in the regulation of water balance. Neither proteins targeted for VACM-1/cul5-specific degradation nor factors that may regulate its expression in those tissues have been studied. To identify genes that may be misregulated by VACM-1 cDNA, we performed microarray analysis. Our results indicate that in cos-1 cells transfected with VACM-1 cDNA, mRNA levels for several genes, including AQP1, were decreased when compared to the control group. Our results also indicate that in cos-1 cells transfected with VACM-1 cDNA, endogenous AQP1 protein was decreased about 6-fold when compared to the controls. To test the hypothesis that VACM-1/cul5 may be regulated by conditions that compromise water homeostasis in vivo, we determined if 24 h of water deprivation affects VACM-1/cul5 levels or the effect of VACM-1/cul5 on AQP1. VACM-1 mRNA and protein levels were significantly higher in rat mesenteric arteries, skeletal muscle and the heart ventricle but not in the heart atrium from 24-h water-deprived rats when compared to the controls. Interestingly, 24 h of water deprivation increased modification of VACM-1 by an ubiquitin-like protein, Nedd8, essential for cullin-dependent E3 ligase activity. Although water deprivation did not significantly change AQP1 levels in the mesenteric arteries, AQP1 protein concentrations were inversely correlated with the ratio of the VACM-1 to Nedd8-modified VACM-1. These results suggest that VACM-1/cul5 may regulate endothelial AQP1 concentration both in vivo and in vitro.

Quantitative proteomics identifies vasopressin-responsive nuclear proteins in collecting duct cells.

Vasopressin controls transport in the renal collecting duct, in part, by regulating transcription. This complex process, which can involve translocation and/or modification of transcriptional regulators, is not completely understood. Here, we applied a method for large-scale profiling of nuclear proteins to quantify vasopressin-induced changes in the nuclear proteome of cortical collecting duct (mpkCCD) cells. Using stable isotope labeling and tandem mass spectrometry, we quantified 3987 nuclear proteins and identified significant changes in the abundance of 65, including previously established targets of vasopressin signaling in the collecting duct. Vasopressin-induced changes in the abundance of the transcription factors JunB, Elf3, Gatad2b, and Hmbox1; transcriptional co-regulators Ctnnb1 (ß-catenin) and Crebbp; subunits of the Mediator complex; E3 ubiquitin ligase Nedd4; nuclear transport regulator RanGap1; and several proteins associated with tight junctions and adherens junctions. Bioinformatic analysis showed that many of the quantified transcription factors have putative binding sites in the 5'-flanking regions of genes coding for the channel proteins Aqp2, Aqp3, Scnn1b (ENaCß), and Scnn1g (ENaCγ), which are known targets of vasopressin. Immunoblotting demonstrated that the increase in ß-catenin in nuclear fractions was accompanied by an even larger increase in its phosphorylated form (pSer552). The findings provide a new online database resource for nuclear proteomics (http://helixweb.nih.gov/ESBL/Database/mNPD/) and generate new hypotheses regarding vasopressin-mediated transcriptional regulation in the collecting duct.

Synthesis and evaluation of potent and selective human V1a receptor antagonists as potential ligands for PET or SPECT imaging.

SRX246 is a potent, highly selective human vasopressin V1a antagonist that crosses the blood-brain barrier in rats. CNS penetration makes SRX246 an ideal candidate for potential radiolabeling and use in visualization and characterization of the role of the V1a receptor in multiple stress-related disorders. Before radiolabeling studies, cold reference analogs of SRX246 were prepared. This study describes the synthesis and in vitro screening for human V1a receptor binding and permeability of fluoro, iodo, and methyl reference compounds for SRX246 and the preparation of a tin precursor. For each compound, the potential utility of corresponding radiolabeled analogs for PET and SPECT imaging is discussed.

Plasma corticotrophin response to desmopressin in patients with Cushing's disease correlates with the expression of vasopressin receptor 2, but not with that of vasopressin receptor 1 or 3, in their pituitary tumours.

OBJECTIVES: Most patients with Cushing's disease (CD) respond to corticotrophin-releasing hormone (CRH) or desmopressin with increased corticotrophin (ACTH) and cortisol levels. Although the vasopressin receptor subtype located on normal corticotrophs is the V3 receptor (V3R), desmopressin is a selective V2 receptor (V2R) agonist and it is unclear whether corticotrophinomas exhibit aberrant V2R expression. Furthermore, no studies have determined the relationship between the in vivo response of CD patients to desmopressin and vasopressin receptor expression, or between the response to CRH and CRH receptor (CRHR) expression. Therefore, the aim of this study was to investigate the expression of vasopressin receptors (V1R, V2R, and V3R) and CRHR on corticotroph tumours and its possible relation to the in vivo response. DESIGNS: A prospective study of 29 patients with CD. METHODS: Patients underwent desmopressin and CRH stimulation tests before surgery. The expression of vasopressin receptors and CRHR on corticotrophinomas was determined by immunocytochemistry. RESULTS: Most of the corticotrophinomas exhibited abundant expression of V1R, V3R, and CRHR, whereas the expression of V2R varied greatly and was lower in macroadenomas than in microadenomas. Both the percentage increment of ACTH and net area under the curve (AUC) of ACTH in the desmopressin stimulation test were found to be correlated with tumour volume. After adjustment for tumour volume, a positive correlation was found between the percentage increment of ACTH and the degree of V2R expression, but not between that of V1R or V3R. No relationship between the level of expression of CRHR on tumour tissues and the percentage increment or netAUC of ACTH to CRH was observed in CD patients. CONCLUSIONS: We concluded that V2R was expressed on corticotrophinomas and that the level of its expression correlated well with the ACTH response to desmopressin in CD patients, although abundant expression of V1R and V3R was also found in almost all corticotroph tumours. Further studies are needed to elucidate the role of these receptors in the pathogenesis of CD.

Photoperiodicity and increasing salinity as environmental cues for reproduction in desert adapted rodents.

Understanding the ways environmental signals, regulate reproduction and reproductive behavior of desert adapted rodents is a major gap in our knowledge. In this study, we assessed the roles of photoperiod and diet salinity, as signals for reproduction. We challenged desert adapted common spiny mice, Acomys cahirinus, males and females with osmotic stress, by gradually increasing salinity in their water source - from 0.9% to 5% NaCl under short and long days (SD and LD, respectively). Photoperiodicity affected testosterone levels, as under LD-acclimation, levels were significantly (p<0.05) higher than under SD-acclimation. Salinity treatment (ST) significantly reduced SD-acclimated male body mass (W(b)) and testis mass (p<0.005; normalized to W(b)). ST-LD-females significantly (p<0.005) decreased progesterone levels and the numbers of estrous cycles. A reduction in white adipose tissue (WAT) to an undetectable level was noted in ST-mice of both sexes under both photoperiod regimes. Receptors for vasopressin (VP) and aldosterone were revealed on testes of all male groups and on WAT in control groups. Our results suggest that photoperiod serves as an initial signal while water availability, expressed by increased salinity in the water source, is an ultimate cue for regulation of reproduction, in both sexes of desert-adapted A. cahirinus. We assume that environmental changes also affect behavior, as water seeking behavior by selecting food items, or locomotor activity may change in extreme environment, and thus indirectly affect reproduction and reproductive behavior. The existence of VP and aldosterone receptors in the gonads and WAT suggests the involvement of osmoregulatory hormones in reproductive control of desert adapted rodents.

COX-2 disruption leads to increased central vasopressin stores and impaired urine concentrating ability in mice.

It was hypothesized that cyclooxygenase-2 (COX-2) activity promotes urine concentrating ability through stimulation of vasopressin (AVP) release after water deprivation (WD). COX-2-deficient (COX-2(-/-), C57BL/6) and wild-type (WT) mice were water deprived for 24 h, and water balance, central AVP mRNA and peptide level, AVP plasma concentration, and AVP-regulated renal transport protein abundances were measured. In male COX-2(-/-), basal urine output and water intake were elevated while urine osmolality was decreased compared with WT. Water deprivation resulted in lower urine osmolality, higher plasma osmolality in COX-2(-/-) mice irrespective of gender. Hypothalamic AVP mRNA level increased and was unchanged between COX-2(-/-) and WT after WD. AVP peptide content was higher in COX-2(-/-) compared with WT. At baseline, plasma AVP concentration was elevated in conscious chronically catheterized COX-2(-/-) mice, but after WD plasma AVP was unchanged between COX-2(-/-) and WT mice (43 ± 11 vs. 70 ± 16 pg/ml). Renal V2 receptor abundance was downregulated in COX-2(-/-) mice. Medullary interstitial osmolality increased and did not differ between COX-2(-/-) and WT after WD. Aquaporin-2 (AQP2; cortex-outer medulla), AQP3 (all regions), and UT-A1 (inner medulla) protein abundances were elevated in COX-2(-/-) at baseline and further increased after WD. COX-2(-/-) mice had elevated plasma urea and creatinine and accumulation of small subcapsular glomeruli. In conclusion, hypothalamic COX-2 activity is not necessary for enhanced AVP expression and secretion in response to water deprivation. Renal medullary COX-2 activity negatively regulates AQP2 and -3. The urine concentrating defect in COX-2(-/-) is likely caused by developmental glomerular injury and not dysregulation of AVP or collecting duct aquaporins.

Hiponatremia: conceitos básicos e abordagem prática / Hyponatremia: basic concepts and practical approach

Hyponatremia is the most common electrolyte imbalance in hospitalized patients. It is associated with several unfavorable endpoints such as: the need for intensive care, longer hospital stay, higher hospitalization costs, discharge to long-term care facilities, and mortality. It is still not clear if there is a direct causal relationship or if hyponatremia is simply a marker of disease severity. Nevertheless, it is quite clear that improper management of a hyponatremic patient may result in severe neurologic damage or death. This paper addresses the basic pathophysiologic concepts about hyponatremia followed by a practical approach to its diagnosis and management.

Hiponatremia é o distúrbio hidroeletrolítico mais comum em pacientes hospitalizados. A presença de hiponatremia está associada a uma série de desfechos desfavoráveis, tais como: necessidade de internamento em unidade de terapia intensiva, hospitalização prolongada e de maior custo, transferência para abrigos e mortalidade. Ainda não está claro se existe relação de causalidade direta ou se a hiponatremia é apenas um marcador de gravidade da doença de base. No entanto, sabe-se que o manejo inadequado de um paciente hiponatrêmico pode causar graves danos neurológicos ou até mesmo a morte. Neste manuscrito, os conceitos básicos sobre a fisiopatologia da hiponatremia serão revisados, seguido de uma abordagem prática sobre sua investigação e tratamento.

Sex differences in vasopressin V2 receptor expression and vasopressin-induced antidiuresis.

The renal vasopressin V(2) receptor (V(2)R) plays a critical role in physiological and pathophysiological processes associated with arginine vasopressin (AVP)-induced antidiuresis. Because clinical data suggests that females may be more prone to hyponatremia from AVP-mediated antidiuresis, we investigated whether there are sex differences in the expression and function of the renal V(2)R. In normal Sprague-Dawley rat kidneys, V(2)R mRNA and protein expression was 2.6- and 1.7-fold higher, respectively, in females compared with males. To investigate the potential physiological implications of this sex difference, we studied changes in urine osmolality induced by the AVP V(2)R agonist desmopressin. In response to different doses of desmopressin, there was a graded increase in urine osmolality and decrease in urine volume during a 24-h infusion. Females showed greater mean increases in urine osmolality and greater mean decreases in urine volume at 0.5 and 5.0 ng/h infusion rates. We also studied renal escape from antidiuresis produced by water loading in rats infused with desmopressin (5.0 ng/h). After 5 days of water loading, urine osmolality of both female and male rats escaped to the same degree physiologically, but V(2)R mRNA and protein in female kidneys was reduced to a greater degree (-63% and -73%, respectively) than in males (-32% and -48%, respectively). By the end of the 5-day escape period, renal V(2)R mRNA and protein expression were reduced to the same relative levels in males and females, thereby abolishing the sex differences in V(2)R expression seen in the basal state. Our results demonstrate that female rats express significantly more V(2)R mRNA and protein in kidneys than males, and that this results physiologically in a greater sensitivity to V(2)R agonist administration. The potential pathophysiological implications of these results are that females may be more susceptible to the development of dilutional hyponatremia because of a greater sensitivity to endogenously secreted AVP.

Localization of three types of arginine vasotocin receptors in the brain and pituitary of the newt Cynops pyrrhogaster.

The distribution of three types of arginine vasotocin (AVT) receptors in the brain and pituitary of the newt Cynops pyrrhogaster, namely, the V1a-, V2-, and V3/V1b-type receptors, was studied by means of in situ hybridization and immunohistochemistry. mRNA signals and immunoreactive cells for the V1a-type receptor were observed in the telencephalon (mitral layer of the olfactory bulb, dorsal and medial pallium, lateral and medial amygdala, bed nucleus of the decussation of the fasciculus telencephali, bed nucleus of the stria terminalis), diencephalon (anterior preoptic area, magnocellular preoptic nucleus, suprachiasmatic nucleus, ventral thalamus, dorsal and ventral hypothalamic nucleus), mesencephalon (tegmentum, interpeduncular nucleus), and medulla oblongata (median reticular formation, nucleus motorius tegmenti). Cells expressing the V2-type receptor were found in the telencephalon (medial pallium, lateral and medial amygdala, bed nucleus of the decussation of the fasciculus telencephali), and mesencephalon (tegmentum trigemini and facialis). In the paraphysis (possibly the main site of cerebrospinal fluid production), only V2-type receptor mRNA signal and immunoreactivity were detected. V3/V1b-type receptor mRNA was expressed in the diencephalon (dorsal hypothalamic nucleus, nucleus tuberculi posterioris), mesencephalon (tegmentum, interpeduncular nucleus), and medulla oblongata (raphe nucleus), whereas V3/V1b-type-receptor-like immunoreactivity was scarcely detectable in the entire brain. The V3/V1b-type receptor was predominantly expressed in the anterior pituitary. V3/V1b-type receptor and proopiomelanocortin mRNAs were co-localized in the distal lobe of the pituitary. This is the first report of the distribution of three types of AVT receptor in the brain and pituitary of non-mammalian vertebrates.

Expression of receptors for luteinizing hormone, gastric-inhibitory polypeptide, and vasopressin in normal adrenal glands and cortisol-secreting adrenocortical tumors in dogs.

Hypercortisolism caused by an adrenocortical tumor (AT) results from adrenocorticotropic hormone (ACTH)-independent hypersecretion of glucocorticoids. Studies in humans demonstrate that steroidogenesis in ATs may be stimulated by ectopic or overexpressed eutopic G protein-coupled receptors. We report on a screening of 23 surgically removed, cortisol-secreting ATs for the expression of receptors for luteinizing hormone (LH), gastric-inhibitory polypeptide (GIP), and vasopressin (V(1a), V(1b), and V(2)). Normal adrenal glands served as control tissues. Abundance of mRNA for these receptors was quantified using quantitative polymerase chain reaction (QPCR), and the presence and localization of these receptors were determined by immunohistochemistry. In both normal adrenal glands and ATs, mRNA encoding for all receptors was present, although the expression abundance of the V(1b) receptor was very low. The mRNA expression abundance for GIP and V(2) receptors in ATs were significantly lower (0.03 and 0.01, respectively) than in normal adrenal glands. The zona fasciculata of normal adrenal glands stained immunonegative for the GIP receptor. In contrast, islands of GIP receptor-immunopositive cells were detected in about half of the ATs. The zona fasciculata of both normal adrenal glands and AT tissue were immunopositive for LH receptor; in ATs in a homogenous or heterogenous pattern. In normal adrenal glands, no immunolabeling for V(1b)R and V(2) receptor was present, but in ATs, V(2) receptor-immunopositive cells were detected. In conclusion, QPCR analysis did not reveal overexpression of LH, GIP, V(1a), V(1b), or V(2) receptors in the ATs. However, the ectopic expression of GIP and V(2) receptor proteins in tumorous zona fasciculata tissue may play a role in the pathogenesis of canine cortisol-secreting ATs.

Expression of aquaporins and vasopressin type 2 receptor in the stria vascularis of the cochlea.

Recently, considerable evidence has been accumulated to support the novel view that water homeostasis in the inner ear is regulated via the vasopressin-aquaporin 2 (VP-AQP2) system in the same fashion as in the kidney. Indeed, multiple subtypes of AQPs including AQP-2 are reported to be expressed in the cochlea. However, the mechanism that underlies VP-AQP-2 mediated water homeostasis remains to be elucidated. In the present study, the localizations of AQP-1, -2, -3, -4, -5, -7, -8, -9, and vasopressin type 2 receptor (V2-R) in the stria vascularis (SV) were molecular biologically and immunohistochemically examined to evaluate the role of the AQP water channel system in water homeostasis of the SV. A RT-PCR study revealed that AQPs and V2-R mRNA are expressed in the cochlea. As for their immunohistochemical localization, the AQP-2 protein is expressed on the basal side of the basal cells of the SV, and proteins of AQP-3 and V2-R are expressed on the apical side of the basal cells. AQP-7 and -9 proteins are expressed on the apical side of marginal cells. AQP-4, -5, and -8 protein expressions could not be detected in the lateral wall of the cochlea. From the present results, water flux in the SV is thought to be regulated at the level of the basal cells by vasopressin. Furthermore, such a distribution of AQP-2, -3, and V2-R suggests that VP-AQP-2 mediated water transport might work actively in the basal cells from perilymph towards endolymph containing AQP-1, -7 and -9.

AT1a receptor knockout in mice impairs urine concentration by reducing basal vasopressin levels and its receptor signaling proteins in the inner medulla.

Angiotensin II plays an important role in the regulation of blood pressure, body salt and fluid balance, and urine concentration. Mice with deletion of the AT(1a) receptor develop polyuria and urine concentration defects. We studied the mechanisms of these urine concentration defects by treating wild-type and AT(1a)-knockout mice with arginine vasopressin (AVP) for 2 weeks, controlling their water intake, or giving them an osmotic diuretic (sucrose) in order to determine whether central or nephrogenic mechanisms were involved. Under basal conditions, AT(1a)-knockout mice were hypotensive, had lower plasma AVP, and excreted more urine with a markedly reduced osmolality compared with wild-type mice. However, basal glomerular filtration rates were similar in both strains of mice. We isolated total lysate and membrane proteins from the inner medulla of wild-type and mutant mouse kidneys, and found that the amounts of aquaporin 2 (AQP2), adenylyl cyclases III and V/VI, and phosphorylated MAP kinases ERK 1/2 proteins were all reduced in the inner medulla of the knockout mice. Infusion of AVP raised plasma levels and blood pressure proportionally in both strains, but polyuria persisted and urine osmolality remained significantly lower in the knockout mice. Although AVP increased urine osmolality slightly in water-deprived knockout mice, this was well below the basal osmolality of wild-type mice. The diuretic response to the hyperosmotic sucrose was also impaired in the knockout mice. Neither AVP nor water rationing restored the levels of the inner medullary signaling proteins and membrane AQP2 proteins in the knockout mice. We suggest that AT(1a) receptor deletion causes polyuria and urine concentration defects by decreasing basal AVP release and impairing AVP-induced receptor signaling in the inner medulla.

Modulation of aquaporin-2/vasopressin2 receptor kidney expression and tubular injury after endotoxin (lipopolysaccharide) challenge.

OBJECTIVE: Sepsis-induced organ dysfunctions remain prevalent and account for >50% of intensive care unit admissions for acute renal failure with a mortality rate nearing 75%. In addition to the fact that the mechanisms underlying the pathophysiology of sepsis-related acute renal failure are unclear, the impact on septic-induced acute renal failure of either norepinephrine, a gold-standard vasopressor, and arginine vasopressin, a candidate alternative, are not well understood. DESIGN: Randomized and controlled in vivo study. SETTING: Research laboratory and animal facilities. SUBJECTS: Adult rats treated with endotoxin (lipopolysaccharide) and/or vasopressors. INTERVENTIONS: Rats were intraperitoneally injected with lipopolysaccharide (12 mg/kg) or saline and then infused with either saline, 0.375 microg/microL arginine vasopressin, or 32.5 microg/microL norepinephrine for 18 hrs. These vasopressor rates yielded respective targeted blood levels observed in human septic shock. MEASUREMENTS AND MAIN RESULTS: Renal function, including glomerular filtration rate and fraction, renal blood flow, aquaporin-2, and arginine vasopressin-2 (V2 receptor) networking, water and salt handling, and urinary protein excretion, were evaluated. After lipopolysaccharide challenge arginine vasopressin infusion: 1) impaired creatinine clearance without affecting renal blood flow, glomerular filtration rate, and fraction but reduced free-water clearance, both of which being partially restored by the V2 receptor antagonist SR-121463B; 2) decreased the recognized ability of arginine vasopressin alone to recruit aquaporin-2 to the apical membrane increase its mRNA expression and urinary release; 3) increased urinary protein content but decreased specific kidney injury molecule-1, and Clara cell protein-16 release (p < 0.05 vs. lipopolysaccharide alone). Conversely, norepinephrine infusion did not add to lipopolysaccharide-induced alteration of urine biochemistry, except for improved creatinine clearance and increased microalbuminuria. CONCLUSION: In this endotoxic model, dose-targeted arginine vasopressin infusion increased lipopolysaccharide-induced renal dysfunction without affecting renal blood flow and glomerular function, but with particular disruption of aquaporin-2/V2 receptor networking, consecutive decreased salt and water handling ability. This is in clear contrast with norepinephrine infusion and suggests specific arginine vasopressin-induced "tubular epithelial dysfunction."

Extracellular loop 3 (ECL3) and ECL3-proximal transmembrane domains VI and VII of the mesotocin and vasotocin receptors confer differential ligand selectivity and signaling activity.

Mesotocin (MT) and vasotocin (VT) are the nonmammalian orthologs of mammalian oxytocin (OT) and arginine vasopressin (AVP), respectively. The OT/AVP family of peptides has arisen from gene duplication but has evolved to possess high selectivity toward their cognate receptors. The process of molecular evolution of receptors to confer high selectivity to their cognate ligands, however, is poorly understood. We constructed a series of reciprocal chimeras using a pair of bullfrog MT receptor (MTR) and VT1 receptor (VT1R) DNA fragments. Among the MTR/VT1R chimeras, the MTR chimera containing a region from transmembrane domain (TMD) VI to the carboxyl-terminal tail (C-tail) of VT1R showed an increased sensitivity to VT, while a chimeric VT1R containing TMD VI to C-tail of MTR showed an increased sensitivity to MT. Further dissection of domains using additional chimeras demonstrated that the receptor with the fragment containing extracellular loop 3 (ECL3) and ECL3-proximal TMDs VI and VII of MTR increased MT selectivity. This fragment is also important for receptor conformation that permits the signaling ability of the receptor. Particularly, the amino acids Val/Ile(6.54) in TMD VI and Pro/Glu(7.29) in ECL3 appear to be involved in this activity, since double mutation of these amino acids completely blocked signaling activity while maintaining ligand binding activity. Mutations at these residues in human OT and AVP 1a receptors markedly decreased receptor signaling activity. This study provides clues for understanding molecular coevolution of the OT/AVP peptides and their receptors with regard to receptor-ligand binding and receptor signaling activity.

Remodelling of calcium signalling during liver regeneration in the rat.

BACKGROUND/AIMS: During liver regeneration, a network of cytokines and growth factors interact with hepatocytes, helping to restore the liver mass and functions after partial tissue loss. Agonists that trigger Ca2+ signals in the liver contribute to this process, although little is known about calcium signalling during liver regeneration. RESULTS: We observed two phases in which the hepatocyte response to calcium-mobilising agonists was greatly reduced versus control cells at 24h and five days after partial hepatectomy. We found that both phases of hepatocyte desensitisation involved the down-regulation of cell surface receptors and the type II InsP3 receptor. Single cell studies with flash photolysis of caged InsP3 revealed that InsP3-mediated Ca2+ release was slower in regenerating hepatocytes at 24, 48 h and 5 days than in control cells. Also, the temporal pattern of vasopressin-elicited intracellular calcium oscillations studied on fura2-loaded cells was altered, with the duration of each Ca2+ peak being longer. Finally, we showed an association between hepatocyte desensitisation and progression through the cell cycle towards the S phase at 24 h after hepatectomy. CONCLUSIONS: Our study supports the remodelling of hepatocyte calcium signalling during liver regeneration, and that this change is partly linked with cell cycle progression.