Brattleboro Rats Research Articles

The role of vasopressin in hypothalamo-pituitary-adrenal axis activation during stress: an assessment of the evidence.

The hypothalamo-pituitary-adrenal (HPA) axis is a key component of the stress reaction. Most contemporary reviews mention the corticotropin-releasing hormone and arginine vasopressin (AVP)-containing parvocellular neurons of the hypothalamic paraventricular nucleus as the endocrinomotor component of the system. Although there are many studies about the role of AVP in the stress activation, there is evidence consistent and inconsistent with the general view on the importance of AVP. We propose a list of experiments that may provide critical evidence for or against the widely held opinion. The naturally AVP-deficient Brattleboro rat seems to be a good tool for studying the role of AVP. Our experiments on Brattleboro rats with restraint and ip hypertonic saline injection did not support the prominent role of AVP in acute stress, although in forced swim the lack of AVP influenced the HPA axis activation. Among different chronic stress situations (14 days' restraint, chronic morphine or ip hypertonic saline treatment, streptozotocin-induced diabetes mellitus), the role of AVP was not confirmed by changes in somatic parameter (i.e., body, thymus, and adrenal weight changes).

Effects of repeated restraint stress on hypothalamo-pituitary-adrenocortical function in vasopressin deficient Brattleboro rats

Arginine-vasopressin (AVP) has been proposed to be an important mediator during chronic stress in the regulation of the hypothalamo-pituitary-adrenal axis. In the present study we addressed the role of AVP in maintaining adrenocortical responsiveness during chronic stress using the AVP deficient mutant Brattleboro rat. Heterozygous Brattleboro rats (di/+) served as controls and were compared to homozygous rats (di/di) with diabetes insipidus. Sixty minutes daily restraint was repeated for 5, 8, 11 or 15 days and organ weights, plasma adrenocorticotropin (ACTH) and corticosterone levels and anterior pituitary proopiomelanocortin (POMC) mRNA and ACTH content were measured. The body, adrenal and thymus weight changes induced by chronic stress became significant between 5 and 8 repetition and AVP deficiency had no effect on these parameters. The first indication that AVP has a role to play appears after 11 repetitions. In the di/di group at the end of 11th restraint, the plasma ACTH was decreased when compared to the di/+ rats. In animals with indwelling cannulas some adaptation could be seen in ACTH response without any difference between di/+ and di/di rats after 15 restraints. The corticosterone- and prolactin-elevations induced by restraint did not habituate in the di/+ and the di/di rats. Chronic stress increased POMC mRNA in the anterior pituitary similarly in di/+ and di/di rats. Although AVP seems to be necessary for a full ACTH response, most of the other signs of chronic stress after repeated restraint occur unchanged in the absence of AVP in both genders. This suggests that either AVP is not indispensable for activating the hypothalamo-pituitary-adrenocortical system by chronic stress or the absence of AVP is compensated by other mediators in Brattleboro rats.

Membrane organization and function of M1 and M23 isoforms of aquaporin-4 in epithelial cells.

Aquaporin-4 (AQP4) water channels exist as heterotetramers of M1 and M23 splice variants and appear to be present in orthogonal arrays of intramembraneous particles (OAPs) visualized by freeze-fracture microscopy. We report that AQP4 forms OAPs in rat gastric parietal cells but not in parietal cells from the mouse or kangaroo rat. Furthermore, the organization of principal cell OAPs in Brattleboro rat kidney is perturbed by vasopressin (arginine vasopressin). Membranes of LLC-PK(1) cells expressing M23-AQP4 showed large, abundant OAPs, but none were detectable in cells expressing M1-AQP4. Measurements of osmotic swelling of transfected LLC-PK(1) cells using videomicroscopy, gave osmotic water permeability coefficient (P(f)) values (in cm/s) of 0.018 (M1-AQP4), 0.019 (M23-AQP4), and 0.003 (control). Quantitative immunoblot and immunofluorescence showed an eightfold greater expression of M1- over M23-AQP4 in the cell lines, suggesting that single-channel p(f) (cm(3)/s) is much greater for the M23 variant. Somatic fusion of M1- and M23-AQP4 cells (P(f) = 0.028 cm/s) yielded OAPs that were fewer and smaller than in M23 cells alone, and M1-to-M23 expression ratios ( approximately 1:4) normalized to AQP4 in M1 or M23 cells indicated a reduced single-channel p(f) for the M23 variant. Expression of an M23-AQP4-Ser(111E) mutant produced approximately 1.5-fold greater single-channel p(f) and OAPs that were up to 2.5-fold larger than wild-type M23-AQP4 OAPs, suggesting that a putative PKA phosphorylation site Ser(111) is involved in OAP formation. We conclude that the higher-order organization of AQP4 in OAPs increases single-channel osmotic water permeability by one order of magnitude and that differential cellular expression levels of the two isoforms could regulate this organization.

Effects of repeated restraint stress on hypothalamo-pituitary-adrenocortical function in vasopressin deficient Brattleboro rats

Arginine-vasopressin (AVP) has been proposed to be an important mediator during chronic stress in the regulation of the hypothalamo-pituitary-adrenal axis. In the present study we addressed the role of AVP in maintaining adrenocortical responsiveness during chronic stress using the AVP deficient mutant Brattleboro rat. Heterozygous Brattleboro rats (di/+) served as controls and were compared to homozygous rats (di/di) with diabetes insipidus. Sixty minutes daily restraint was repeated for 5, 8, 11 or 15 days and organ weights, plasma adrenocorticotropin (ACTH) and corticosterone levels and anterior pituitary proopiomelanocortin (POMC) mRNA and ACTH content were measured. The body, adrenal and thymus weight changes induced by chronic stress became significant between 5 and 8 repetition and AVP deficiency had no effect on these parameters. The first indication that AVP has a role to play appears after 11 repetitions. In the di/di group at the end of 11th restraint, the plasma ACTH was decreased when compared to the di/+ rats. In animals with indwelling cannulas some adaptation could be seen in ACTH response without any difference between di/+ and di/di rats after 15 restraints. The corticosterone- and prolactin-elevations induced by restraint did not habituate in the di/+ and the di/di rats. Chronic stress increased POMC mRNA in the anterior pituitary similarly in di/+ and di/di rats. Although AVP seems to be necessary for a full ACTH response, most of the other signs of chronic stress after repeated restraint occur unchanged in the absence of AVP in both genders. This suggests that either AVP is not indispensable for activating the hypothalamo-pituitary-adrenocortical system by chronic stress or the absence of AVP is compensated by other mediators in Brattleboro rats.

636. Reversal of Hypothalamic Diabetes Insipidus Using EIAV-Based Regulatable Lentiviral Vectors Expressing Arginine Vasopressin

Top of pageAbstract Lentiviral vectors, such as the Equine Infectious Anaemia Virus (EIAV)-based vectors, have the potential to be used in the clinic for a number of gene therapy applications. Efficient gene transfer using EIAV vectors and long-term expression has been demonstrated in vivo in the central nervous system. Hypothalamic diabetes insipidus (DI) is a disorder that affects water balance in humans. The cause of DI is the lack of the water retaining hormone arginine vasopressin (AVP) produced by magnocellular neurons of the supraoptic nuclei (SON) of the hypothalamus. AVP is secreted from the pituitary gland into the blood stream and acts on the kidney causing water reabsorption. In the AVP-deficient Brattleboro rat, as a result of a single mutation, AVP is not produced thus causing copious water intake and secretion of dilute urine. This genetic animal model allows the non-invasive monitoring of the efficacy of gene therapy strategies to treat DI (1). In this study, self-inactivating (SIN) minimal EIAV vector genomes with enhanced transduction efficiency (SMART vectors) have been used to express rat AVP. Vector particles were bilaterally injected into the (SON) in the hypothalamus of the Brattleboro rat model of DI. The EIAV-AVP treated animals show a dramatic reduction of daily water intake and urine volume output compared to control animals (injected with EIAV-GFP), reaching levels very similar to the ones observed in the Long Evans (wild type) rats. AVP is also detected in the urine of EIAV-AVP treated animals. These observations indicate that the hormone is now produced in the SON and released in the blood stream where it is correcting water balance. We observed reversal of the phenotype for one year, which is longer and more effective than that obtained with any previous gene therapy interventions. We have also developed tetracycline regulatable (single-vector) lentiviral systems and data showing that AVP expression can be increased following the addition of doxycycline will also be presented.

Regulation of cyclooxygenase expression by vasopressin in rat renal medulla.

The antagonism between prostaglandin and vasopressin represents a classic negative feedback loop. It is not clear whether cyclooxygenase (COX)-2 and/or COX-1 expression is involved in elevated prostaglandin production stimulated by vasopressin in vivo. In the present study, we explored vasopressin regulation of medullary COX-2 and COX-1 expression acutely and chronically in rats. Medullary COX-1 expression was moderately lower and COX-2 expression was significantly lower in adult male Brattleboro rats than age-matched Long-Evans controls. Chronic treatment of Brattleboro rats with vasopressin for 1 wk led to a decrease in urine volume and a moderate increase in medullary COX-1; in contrast, medullary COX-2 expression was almost undetectable in untreated rats but was dramatically up-regulated with vasopressin treatment and was accompanied by increased urinary prostaglandin E(2) excretion. Further investigation revealed that both V1 and V2 receptors were involved in chronic medullary COX-1 and COX-2 up-regulation. Acute treatment with specific V1 or V2 receptor agonists resulted in specific increases in medullary COX-2, which was prevented by furosemide. Vasopressin did not affect COX-2 expression in cultured renomedullary interstitial cells. These data demonstrate that vasopressin stimulates medullary COX-2 expression through activation of both V1 and V2 receptors, and this stimulation is indirect and probably involves increased medullary electrolyte tonicity.

Persistent phenotypic correction of central diabetes insipidus using adeno-associated virus vector expressing Arginine–Vasopressin in brattleboro rats

Adeno-associated virus (AAV) vector is suitable for gene transfer to the central nervous system. However, the efficacy of gene therapy for neuroendocrine disease is still unknown. In this study, we injected AAV vector encoding arginine–vasopressin (AVP) stereotaxically into the bilateral hypothalamus of Brattleboro rats. Brattleboro rats show a central diabetes insipidus (CDI) phenotype and growth retardation due to a complete deficiency of AVP. Following injection, both urine volume and urine osmolality normalized, and these therapeutic effects persisted for more than 50 weeks. In addition to phenotypic correction, secretion of transgene-derived AVP was enhanced after 24 h water deprivation or hypertonic saline injection, and water diuresis was demonstrated after acute water loading. Also, reduced body weight and low plasma insulin-like growth factor I levels of Brattleboro rats were restored after AVP gene transduction, suggesting the importance of AVP in growth. These findings indicate that hypothalamic neurons of Brattleboro rats can produce and release mature AVP following AAV-mediated gene transduction, resulting in long-term phenotypic correction of CDI. Moreover, the fact that transgene-derived AVP was secreted adequately in response to stimuli, even if it was expressed constitutively, suggests advantages of gene therapy for neuroendocrine diseases and offers a basis to investigate AVP function.

Role of vasopressin in diabetes mellitus-induced changes in medullary transport proteins involved in urine concentration in Brattleboro rats.

In rats with streptozotocin-induced diabetes mellitus for 10-20 days, we showed that the abundance of the major medullary transport proteins involved in the urinary concentrating mechanism, urea transporter (UT-A1), aquaporin-2 (AQP2), and the Na+-K+-2Cl- cotransporter (NKCC2/BSC1), is increased, despite the ongoing osmotic diuresis. To test whether vasopressin is necessary for these diabetes mellitus-induced changes in UT-A1, AQP2, or NKCC2/BSC1, we studied Brattleboro rats because they lack vasopressin. Brattleboro rats were given vasopressin (2.4 microg/day via osmotic minipump) for 5 or 12 days. At 5 days, vasopressin increased AQP2 protein abundance but decreased UT-A1 abundance compared with untreated Brattleboro rats. At 12 days, vasopressin increased the abundance of both UT-A1 and AQP2 proteins but did not alter NKCC2/BSC1. Next, untreated Brattleboro rats were made diabetic for 10 days by injecting them with streptozotocin (40 mg/kg). Diabetes mellitus increased the abundance of AQP2 and NKCC2/BSC1 proteins, but UT-A1 protein abundance did not increase. Third, vasopressin-treated Brattleboro rats were made diabetic with streptozotocin for 10 days. In vasopressin-treated Brattleboro rats, diabetes mellitus increased UT-A1, AQP2, and NKCC2/BSC1 protein abundances. Vasopressin significantly increased UT-A1 phosphorylation in vasopressin-treated diabetic Brattleboro rats but not in the other groups of Brattleboro rats. We conclude that 1) administering vasopressin to Brattleboro rats for 12 days, but not for 5 days, increases UT-A1 protein abundance and 2) vasopressin is necessary for the increase in UT-A1 protein in diabetic rats but is not necessary for the increase in AQP2 or NKCC2 proteins.

Maternal genotype influences stress reactivity of vasopressin-deficient brattleboro rats.

The role of vasopressin, cosecreted with corticotropin-releasing hormone (CRH), in stress is debated, because both normal as well as reduced adrenocorticotropin hormone (ACTH) rise to an acute challenge has been reported in Brattleboro rats genetically lacking vasopressin (di/di). Because di/di pups could be born either from di/+ (heterozygous) or from di/di mothers, and maternal influence is known to modify adult responsiveness, we investigated whether the influence of maternal genotype could explain the variability. Adult rats from mothers with different genotypes were stressed with 60 min restraint and trunk blood was collected for measuring hormone content by radioimmunoassay at the end of stress. All offspring of di/+ mothers had similar ACTH responses to restraint, while the di/di rats born to, and raised by di/di mothers showed reduced ACTH reactivity to restraint. The di/di rats showed elevated water turnover and required a daily cage cleaning every day, which meant frequent handling. To offset the role of handling, all rats had daily cage cleaning in the next series, but the results were the same as in the first series. To investigate whether lactation, the behaviour of the mother or some other factor during the pregnancy is responsible for the differences, pups from di/+ dams were raised by di/di foster mothers and vice versa. We found that the genotype of parental mother is more important than that of the foster mother. The corticosterone and prolactin elevation normally seen after acute stress was unchanged by family history, maternal or personal genotype. Furthermore, in studies with mutant animals, the rearing conditions should be controlled by the experimenter. In experiments with Brattleboro rats, the use of homozygous and heterozygous rats from the same litters of di/+ dams and di/di males is recommended. Our results suggest that vasopressin is not indispensable for ACTH release, and that the di/di genotype of the parental mother can decrease the stress reactivity of the di/di Brattleboro rats.

Immune system in vasopressin-deficient rats during ontogeny.

Morphofunctional immune disorders were revealed in vasopressin-deficient Brattleboro rats with diabetes insipidus during ontogeny. We observed a permanent decrease in the number of blood lymphocytes, increase in neutrophil count, reduced activity of macrophages, early involution of the thymus and spleen, and suppression of antibody production. These changes reflect impaired general resistance of these animals.

Proteomic analysis of long-term vasopressin action in the inner medullary collecting duct of the Brattleboro rat.

Vasopressin regulates water and solute transport in the renal collecting duct. In addition to short-term regulation of aquaporin-2 trafficking, vasopressin also has long-term effects to regulate the abundances of aquaporins-2 and -3 and beta- and gamma-subunits of the epithelial sodium channel in collecting duct principal cells. To investigate further the direct and indirect long-term regulatory actions of vasopressin in the inner medullary collecting duct (IMCD), we used a proteomic approach [difference gel electrophoresis (DIGE) coupled with MALDI-TOF identification of differentially expressed protein spots]. DDAVP or vehicle was infused subcutaneously in Brattleboro rats for 3 days, and IMCD cells were purified from the inner medullas for proteomic analysis. Forty-three proteins were found to be regulated in response to vasopressin infusion, including 18 that were increased in abundance, 22 that were decreased, and 3 that were shifted in the gel, presumably because of posttranslational modification. Immunocytochemistry confirmed collecting duct expression of several of the proteins that were identified. Immunoblot analysis of nine of the proteins confirmed the changes seen by the DIGE method. Of these nine proteins, six were increased in response to DDAVP infusion: nitric oxide synthase-2 (NOS2), GRP78, heat shock protein-70, annexin II, glutaminase, and cathepsin D. The remaining three were decreased in response to DDAVP: aldehyde reductase I, adenylyl cyclase VI, and carbonic anhydrase II. The findings point to a role for vasopressin in the coordinate regulation of several determinants of nitric oxide levels (NOS2, arginase II, NADPH oxidase) and of proteins potentially involved in vasopressin escape (adenylyl cyclase VI and G protein-coupled receptor kinase 4).

Gene therapy for central diabetes insipidus: effective antidiuresis by muscle-targeted gene transfer.

Central diabetes insipidus, characterized by severe polyuria and polydipsia, is a disorder resulting from deficient secretion of the small neuropeptide hormone vasopressin in the neurohypophysis. The standard therapy is daily and life-long administration of vasopressin analog (desmopressin acetate), but gene therapy is potentially alternative to the conventional replacement therapy. To obtain the therapeutic neuropeptide more feasibly, we tried to express vasopressin in nonneuronal tissues using nonviral gene transfer techniques. We found that the unprocessed large precursor form, provasopressin, was predominantly produced in nonendocrine cells transfected with the wild-type vasopressin gene, because of the lack of neuroendocrine cell-specific endopeptidases. In sharp contrast, appropriately processed bioactive vasopressin can be efficiently produced even in nonendocrine cells with a modified vasopressin gene containing a ubiquitous endoprotease furin cleavage site. We also succeeded in maintaining a long-term antidiuretic effect on vasopressin-deficient (Brattleboro) rats by direct introduction of the furin-processible gene into skeletal muscle by electroporation. Altogether, our data clearly show that skeletal muscle is a useful target tissue for continuous delivery of bioactive neuropeptide. Furthermore, our strategies may be applicable to future gene therapies for central diabetes insipidus and other peptide hormone deficiencies.

Vasopressin-independent renal urinary concentration: Increased rBSC1 and enhanced countercurrent multiplication

A close association between the expression of the sodium transporter, rat bumetanide sensitive cotransporter (rBSC), in thick ascending limb of Henle and urinary concentration has been reported. However, direct evidence for this association and the mechanism of rBSC1 expression are still to be elucidated. Brattleboro (BB) rats weighing approximately 200 g were dehydrated by water restriction for 4 hours, which induced around a 5% body weight reduction. Although plasma arginine vasopressin (AVP) was undetectable even after the water restriction, BB rats concentrated urine from 182 +/- 23 (mean +/- SD) at baseline to 404 +/- 65 mOsm/kg. H2O. Urinary volume was reduced from 5.8 +/- 1.8 to 1.4 +/- 0.6 mL/h. This treatment significantly increased sodium and urea accumulation in the renal medulla and reduced urinary sodium excretion. rBSC1 signals for both mRNA and protein were increased in dehydrated rats, although aquaporin type 2 (AQP2) expression was not enhanced in dehydrated BB rats. Subcutaneous infusion of desmopressin acetate (DDAVP) intensified rBSC1 signals of BB rats more than those in dehydrated condition. Dehydration increased rBSC1 expression and enhanced countercurrent multiplication even in AVP deficiency. These results supply strong evidence for the association between rBSC1 expression and urinary concentration, and indicate the presence of an AVP-independent mechanism for urine concentration.

Local synthesis of the rat Vasopressin precursor in dendrites of in vitro cultured nerve cells

Vasopressin (VP) mRNA is subject to dendritic targeting both in vivo and in primary cultured neurons microinjected with an appropriate expression vector. We have constructed a vector encoding the mutant Brattleboro rat VP precursor which is non-diffusable, because it cannot leave the site of its synthesis, the rough endoplasmic reticulum. Expression of this construct in cultured nerve cells shows that the mutant protein is readily detectable in dendrites when mRNA transport has occurred, while dendrites devoid of the mRNA lack the protein. These results demonstrate that neurons have the capacity to locally synthesize secretory proteins in the dendritic compartment.

Long-term replacement of a mutated nonfunctional CNS gene: reversal of hypothalamic diabetes insipidus using an EIAV-based lentiviral vector expressing arginine vasopressin

Due to the complexity of brain function and the difficulty in monitoring alterations in neuronal gene expression, the potential of lentiviral gene therapy vectors to treat disorders of the CNS has been difficult to fully assess. In this study, we have assessed the utility of a third-generation equine infectious anemia virus (EIAV) in the Brattleboro rat model of diabetes insipidus, in which a mutation in the arginine vasopressin (AVP) gene results in the production of nonfunctional mutant AVP precursor protein. Importantly, by using this model it is possible to monitor the success of the gene therapy treatment by noninvasive assays. Injection of an EIAV-CMV-AVP vector into the supraoptic nuclei of the hypothalamus resulted in expression of functional AVP peptide in magnocellular neurons. This was accompanied by a 100% recovery in water homeostasis as assessed by daily water intake, urine production, and urine osmolality lasting for a 1-year measurement period. These data show that a single gene defect leading to a neurological disorder can be corrected with a lentiviral-based strategy. This study highlights the potential of using viral gene therapy for the long-term treatment of disorders of the CNS.

Genetic Vasopressin Deficiency Facilitates Performance of a Lateralized Reaction-Time Task: Altered Attention and Motor Processes

Brattleboro rats are a variety of the outbred Long-Evans strain that possess a single nucleotide deletion in the second exon of the arginine vasopressin gene, resulting in the synthesis of an altered protein that does not enter the normal secretory pathway. Rats heterozygous (di/+) for the deletion have a partial vasopressin deficiency and exhibit a variety of behavioral and neurochemical alterations compared with normal wild-type Long-Evans rats, which provide evidence for a CNS function for vasopressin. Here, we examined the acquisition and performance of a test of visuospatial attention by di/+ Brattleboro rats and their wild-type Long-Evans control counterparts. Surprisingly, di/+ rats exhibited superior performance of the task compared with wild-type controls; performance differences included greater accuracy of detection of visual target stimuli, faster overall reaction times, and fewer trial omissions. Di/+ rats also exhibited more approaches to the reinforcer receptacle at nonreinforcement times. These results indicate that alterations of vasopressin signaling result in a clear cognitive phenotype, including faster motor initiations and superior choice accuracy in a test of visual attention.

Downregulation of renal aquaporins in response to unilateral ureteral obstruction.

The expression of aquaporin-2 (AQP2) is decreased in rats with bilateral ureteral obstruction (BUO) and unilateral ureteral obstruction (UUO). Therefore, the expression of additional renal aquaporins (AQP1-4) and phosphorylated AQP2 (p-AQP2), known to play a role in urinary concentration, was examined in a Wistar rat model with 24 h of UUO. In obstructed kidneys, immunoblotting revealed a significant decrease in the expression of inner medullary AQP2 to 42 +/- 4, p-AQP2 to 23 +/- 5, AQP3 to 19 +/- 6, AQP4 to 11 +/- 5, and AQP1 to 64 +/- 8% of sham levels. AQP1 expression located in the proximal tubule decreased to 74 +/- 4% of sham levels (P < 0.05). Immunocytochemistry confirmed the downregulation of AQP3, AQP4, and p-AQP2. In contralateral nonobstructed kidneys, immunoblotting also revealed significant reductions of AQP1 in the inner medulla, outer medulla, and cortex, whereas expression of AQP2, AQP3, AQP4, and p-AQP2 was unchanged. Furthermore, we collected the urine from both obstructed and nonobstructed kidneys for 2 h, respectively, after 24 h of UUO. Urine collection from obstructed kidneys during 2 h after release of UUO revealed a significant reduction in urine osmolality and solute-free water reabsorption (T(c)H(2)O). Moreover, an increase in urine production and T(c)H(2)O was observed in contralateral kidneys. To examine whether vasopressin-independent mechanisms are involved in AQP2 regulation, vasopressin-deficient Brattleboro (BB) rats with 24 h of UUO were examined. Immunoblotting revealed downregulation of AQP2, p-AQP2, AQP3, and AQP1 in obstructed kidneys and downregulation of p-AQP2 and AQP1 in nonobstructed kidneys. In conclusion, 1) UUO is associated with severe downregulation of AQP2, AQP3, AQP4, and AQP1; thus all of these AQPs may play important roles in the impaired urinary concentrating capacity in the obstructed kidney; 2) the reduced levels of AQP1 in the nonobstructed kidney may contribute to the compensatory increase in urine production; and 3) downregulation of AQPs in BB rats supports the view that vasopressin-independent pathways may be involved in AQP2 and AQP3 regulation in the obstructed kidney.

Hypertonicity Is Involved in Redirecting the Aquaporin-2 Water Channel into the Basolateral, Instead of the Apical, Plasma Membrane of Renal Epithelial Cells

In renal collecting ducts, vasopressin increases the expression of and redistributes aquaporin-2 (AQP2) water channels from intracellular vesicles to the apical membrane, leading to urine concentration. However, basolateral membrane expression of AQP2, in addition to AQP3 and AQP4, is often detected in inner medullary principal cells in vivo. Here, potential mechanisms that regulate apical versus basolateral targeting of AQP2 were examined. The lack of AQP2-4 association into heterotetramers and the complete apical expression of AQP2 when highly expressed in Madin-Darby canine kidney cells indicated that neither heterotetramerization of AQP2 with AQP3 and/or AQP4, nor high expression levels of AQP2 explained the basolateral AQP2 localization. However, long term hypertonicity, a feature of the inner medullary interstitium, resulted in an insertion of AQP2 into the basolateral membrane of Madin-Darby canine kidney cells after acute forskolin stimulation. Similarly, a marked insertion of AQP2 into the basolateral membrane of principal cells was observed in the distal inner medulla from normal rats and Brattleboro rats after acute vasopressin treatment of tissue slices that had been chronically treated with vasopressin to increase interstitial osmolality in the medulla, but not in tissues from vasopressin-deficient Brattleboro rats. These data reveal for the first time that chronic hypertonicity can program cells in vitro and in vivo to change the insertion of a protein into the basolateral membrane instead of the apical membrane.

Interaction of the diabetes insipidus locus alleles with the renal 120 kDa protein-encoding gene in rat development

Age-dependant dynamics of the kidney inner medullary 120-kDa protein content in vasopressin-deficient Brattleboro rats with di/di mutant genotype was studied in comparison with WAG rats with genotype and normal vasopressin expression. Age-dependant dynamics of vasopressin content in neurohypophysis of WAG rats was also examined. It was shown that 10-day-old WAG rats were unable to elevate the synthesis of the 120 kDa protein in respond to long-term dehydration, while this tendency was clearly observed in the 15-day-old rats and later in the development. In WAG rats the onset of this specific feature was time correlated with the development of the respond to hydration by the elevation of vasopressin synthesis and release from neurohypophysis into blood. In the di/di rats dehydration had no effect on the kidney 120-kDa protein synthesis in all ages examined. These results point to the interaction between the di alleles and the 120-kDa protein-encoding gene in the course of development.

Vasopressin induces vascular superoxide via endothelin-1 in mineralocorticoid hypertension.

We have recently reported that endothelin-1 (ET-1), which is increased in the arteries of deoxycorticosterone acetate (DOCA)-salt hypertensive rats, stimulates superoxide production. However, the humoral mechanisms responsible for ET-1-induced superoxide formation in low-renin models of hypertension, such as DOCA-salt hypertension, remain undefined. Vasopressin is known to upregulate vascular preproET-1 gene expression in DOCA-salt rats, an effect that is absent in vasopressin-deficient Brattleboro rats treated with DOCA-salt. The present study tested the hypothesis that vasopressin contributes to ET-1-induced vascular superoxide production in DOCA-salt hypertensive rats. Carotid arterial segments of DOCA, sham (uninephrectomized), or normal (untreated) rats were used for the study. In vitro vasopressin treatment of carotid arteries from normal rats for 24 hours, but not 4 hours, increased both ET-1 and superoxide levels. The increase of vasopressin-induced superoxide was reduced by pretreatment of the vessels with ABT627, a selective ETA receptor antagonist ABT627. Vasopressin, ET-1, and superoxide levels were significant elevated in carotid arteries of DOCA-salt rats compared with sham controls. The selective V1-vasopressin receptor antagonist (beta-Mercapto-beta, beta-cyclopentamethylenepropiony1, O-Me-Tyr2, Arg8 vasopressin, ME-AVP), decreased superoxide both in vasopressin-treated vessels of normal rats and in vessels of DOCA-salt rats, with a concomitant reduction of ET-1 content. These results suggest that vasopressin increases vascular superoxide levels by stimulating ET-1 formation in mineralocorticoid hypertension, and that V1-vasopressin receptors play an important role in this process.