Increased Renal Responsiveness to Vasopressin and Enhanced V2 Receptor Signaling in RGS2–/– Mice
Annie Mercier Zuber*,
Dustin Singer*,
Josef M. Penninger,
Bernard C. Rossier* and
Dmitri Firsov*
* Département de Pharmacologie et de Toxicologie, Université de Lausanne, Lausanne, Switzerland; and Institute of Molecular Biotechnology, Vienna, Austria
Address correspondence to: Dr. Dmitri Firsov, Département de Pharmacologie et de Toxicologie Université de Lausanne, 27, rue du Bugnon, CH-1005 Lausanne, Switzerland. Phone: +41-21-6925406; Fax: +41-21-692-5355; E-mail: dmitri.firsov{at}unil.ch
Received for publication January 9, 2007.
Accepted for publication March 16, 2007.
The antidiuretic effect of vasopressin is mediated by V2 receptors(V2R) that are located in kidney connecting tubules and collectingducts. This study provides evidence that V2R signaling is negativelyregulated by regulator of G protein signaling 2 (RGS2), a memberof the family of RGS proteins. This study demonstrates that(1) RGS2 expression in the kidney is restricted to the vasopressin-sensitivepart of the nephron (thick ascending limb, connecting tubule,and collecting duct); (2) expression of RGS2 is rapidly upregulatedby vasopressin; (3) the vasopressin-dependent accumulation ofcAMP, the principal messenger of V2R signaling, is significantlyhigher in collecting ducts that are microdissected from theRGS2–/– mice compared with their wild-type littermates;and (4) analysis of urine output of mice that were exposed towater restriction followed by acute water loading revealed thatRGS2–/– mice exhibit an increased renal responsivenessto vasopressin. It is proposed that RGS2 is involved in negativefeedback regulation of V2R signaling.
Vasopressin regulates body water balance by controlling waterpermeability in kidney connecting tubules (CNT) and collectingducts. In the CNT cells and the principal cells of the collectingduct, vasopressin binds to the V2 type of vasopressin receptor(V2R) located in the basolateral membrane. Vasopressin bindingto V2R activates adenylyl cyclase via a stimulatory G proteinGs, resulting in increased intracellular cAMP concentration(1). Elevation of intracellular cAMP induces a rapid (withinminutes) insertion of aquaporin-2 water channels into the apicalmembrane, thereby promoting the osmotically driven water reabsorptionfrom the tubular lumen into the blood (2). In humans, loss-of-functionmutations in V2R or aquaporin-2 genes leads to nephrogenic diabetesinsipidus, a disease that is characterized by the inabilityof the kidney to concentrate urine (reviewed in reference [3]).The recently identified gain-of-function mutations in the V2Rgene cause a nephrogenic syndrome that is characterized by hyponatremiaand undetectable vasopressin levels (4).
Vasopressin is also involved in the long-term genomic regulationof renal water handling (reviewed in reference [5]). A numberof vasopressin-dependent genes have been identified in variousexperimental settings and using various high-throughput techniques(6–8). Using gene expression profiling based on serialanalysis of gene expression, we recently characterized the vasopressin-dependentgene network of the principal cell of the mouse cortical collectingduct (mpkCCDcl4 cells) that was stimulated for 4 h with vasopressin(8). One of the identified vasopressin-induced transcripts,namely the regulator of G protein signaling 2 (RGS2), belongsto the family of RGS proteins that control both the strengthand the duration of signaling through G protein–coupledreceptors (GPCR). In vitro, RGS2 has been shown as a directinhibitor of at least four types of adenylyl cyclases (ACII,ACIII, ACV, and ACVI) as well as several GPCR. RGS2 has alsobeen shown to act as a GTPase activating protein for G proteinGq, thereby inhibiting Gq signaling (reviewed in reference [9]).In vivo, mice that are deficient in RGS2 exhibit a strong hypertensivephenotype as a result of abnormally prolonged signaling by Gq-coupledvasoconstrictor receptors (10).
This study was undertaken to assess the role of RGS2 in waterhandling by the kidney. We hypothesized that stimulation ofRGS2 expression by vasopressin could interfere with V2R or otherGPCR signaling pathways and, thereby, influence water reabsorptionin the nephron. Here we demonstrate that RGS2–/–mice exhibit an enhanced V2R signaling and increased renal responsivenessto vasopressin. Thus, RGS2 is likely involved in the negativefeedback regulation of vasopressin effect in the kidney. Thisstudy provides the first evidence for the role of RGS2 in renalfunction.
Northern Blot Analysis
Northern blots were prepared with 2 µg of poly(A) RNAthat was extracted from mpkCCDcl4 cells that were grown on filters.Cell culture conditions for mpkCCDcl4 cells have been previouslydescribed (8).
Microdissection
Microdissection of different parts of the nephron was performedfrom collagenase-treated kidneys as described previously (11).
Qualitative Reverse Transcriptase–PCR Analysis
Experiments were performed on male C57BL/6 mice (8 to 12 wkold) that were maintained on a standard diet with free accessto water. RNA aliquots that originated from 1 mm of microdissectedtubule were used in reverse transcriptase–PCR (RT-PCR;Titan One Tube RT-PCR System, Roche, Rotkreuz, Switzerland).The sense and antisense primers that were designed to amplifya 449-bp fragment of RGS2 mRNA were 5'-GGCAACGGCCCCAAGGTCGAGG-3'and 5'-AAGCAGCCACTTGTAGCCTCTTG-3', respectively. The sense andantisense primers that were designed to amplify a 444-bp fragmentof V2R mRNA were 5'-CAGGAGGAGCTACTGGATGA-3' and 5'-CAGGCTGAGAAGGAGTGAGA-3',respectively. RT-PCR conditions were identical for both pairsof primers. The RT step was performed for 30 min at 50°C.The PCR conditions were as follows: 95°C for 30 s, 56°Cfor 30 s, and 68°C for 45 s (35 PCR cycles). The amplificationproducts were run on a 2.5% agarose gel and stained by ethidiumbromide. To check the specificity of the RGS2 primers, we clonedand sequenced the RT-PCR amplification product that originatedfrom the CCD RNA samples. Six independent clones were sequenced.All six clones contained the identical 449-bp insert that correspondedto the amplified part of the RGS2 cDNA sequence.
Quantitative Real-Time PCR Analysis
Experiments were performed on 6-wk-old male C57BL/6 mice. Inthe water-restricted group, mice were deprived of water for23 h. In the water-loaded group, mice were fed for 23 h with17 ml of a water-loaded gelled diet that consisted of 12.8 mlof water and the same amount of nutrients and electrolytes asthe water-restricted diet (4.5 g). The gelled diet was preparedas described previously (12). A total of 100 mm of both corticalthick ascending limb (CTAL) and CCD was microdissected fromeach mouse. Total RNA was extracted and reverse-transcribedusing Superscript II reverse transcriptase (Invitrogen, Basel,Switzerland). Real-time quantitative PCR analysis was performedusing the TaqMan system (Applied Biosystems, Foster City, CA).All reactions were performed in triplicate with the amount ofcDNA corresponding to 1 mm of microdissected tubule. All primersets that were used in this study were from Applied Biosystems:TaqMan Gene Expression Assay RGS2 Mm00501385_m1 (for RGS2),mouse ACTB (-actin) Endogenous Control (for -actin), and TaqManGene Expression Assay Hprt1 Mm00446968_m1 (for hypoxanthineguanine phosphoribosyl transferase I).
RGS2–/– Mice
A colony of RGS2–/– mice (C57BL/6 background) wasestablished from breeding pairs of RGS2 heterozygous mice originallydescribed by Oliveira-Dos-Santos et al. (13).
cAMP Accumulation Experiments
Experiments were performed on wild-type or RGS2–/–male mice (8 to 12 wk old) that were maintained on a standarddiet with free access to water. cAMP was measured in singlepieces of CTAL or CCD (0.5 to 1.0 mm of tubular length) in thepresence of 1 mM 3-isobutyl-1-methylxanthine, as described previously(14). Briefly, the microdissected nephron segments were kepton ice until analysis. After a preincubation step (10 min at30°C), each sample was incubated for 4 min at 35°C inthe presence of the agonist to be tested. Perkin Elmer cAMPRIA Kit (Waltham, MA) was used for measurement of cAMP levels.The amount of cAMP was calculated in femtomoles per millimeterof tubule length per 4-min incubation time at 35°C (fmol/mmper 4 min).
Metabolic Cage Experiments
Mice were housed in individual metabolic cages (Tecniplast,Buguggiate, Italy). All experiments were performed after 3 dof adaptation. Urine and plasma osmolarity as well as ioniccomposition were analyzed in the Laboratoire Central de ChimieClinique, Centre Hospitalier Universitaire Vaudoise (CHUV) UniversityHospital (Lausanne, Switzerland).
Regulation of RGS2 Expression by Vasopressin in mpkCCDcl4 cells
The time-course and dosage-response effects of vasopressin onRGS2 mRNA expression were studied by Northern hybridizationon RNA that were prepared from mpkCCDcl4 cells. As shown inFigure 1A, RGS2 mRNA levels were already increased at 30 minof vasopressin stimulation, reached a maximum at 1 h, and rapidlydeclined after 4 h. The dosage-response analysis (Figure 1B)revealed that RGS2 mRNA expression could be stimulated withinthe low physiologic range of hormone concentration (10–12to 10–11 M). As shown in Figure 2, RGS2 expression wasalso upregulated by vasopressin at the protein level. Collectively,these results characterize RGS2 as a rapidly but transientlyupregulated vasopressin-induced gene.
Figure 1. Vasopressin regulates regulator of G protein signaling 2 (RGS2) mRNA expression in principal cells of the mouse cortical collecting duct (mpkCCDcl4 cells). (A) Time course. Northern blot analysis with RGS2 probe was performed on mRNA that were extracted from untreated (control) or vasopressin-stimulated (10–8 M) mpkCCDcl4 cells after the indicated period of time. (B) Dosage-response. Northern blot analysis was performed on mRNA that were extracted from untreated mpkCCDcl4 cells (control) or mpkCCDcl4 cells that were stimulated for 4 h with vasopressin at various concentrations.
Figure 2. Vasopressin stimulates RGS2 protein expression in mpkCCDcl4 cells. Protein extracts that were prepared from untreated mpkCCDcl4 cells (control) or mpkCCDcl4 cells that were stimulated for 4 h with vasopressin (10–8 M) were run on a 7 to 13% gradient SDS-PAGE. Western blot was probed with an anti-RGS2 antibody (Santa Cruz Biotechnology, Santa Cruz, CA) or with an anti-actin antibody (Sigma, St. Louis, MO).
RGS2 mRNA Expression along the Nephron
To map the distribution of RGS2 along the mouse nephron, weperformed RT-PCR analysis of RGS2 mRNA expression on RNA thatwas extracted from the microdissected nephron segments. Micethat were used in these experiments were allowed ad libitumaccess to food and water. As shown in Figure 3A, RGS2 mRNA isstrongly expressed in the medullary thick ascending limb, CTAL,distal convoluted tubule (DCT), CNT, CCD, and outer medullarycollecting duct. The RGS2 mRNA was barely detectable in theproximal convoluted tubule, proximal straight tubule, an thindescending and ascending limbs. This distribution was very similarto that of V2R mRNA expression (Figure 3B). It should be notedthat although in our experiments the DCT was positive for V2RmRNA expression, the sensitivity of this nephron segment tovasopressin in the mouse is debated. In the rat, the V2R isexpressed only in the late DCT (15). It is interesting thatthe V2R RT-PCR amplification products in the medullary thickascending limb and CTAL as well as in the total kidney extractcontained an additional band of a higher size. The upstreamand downstream primers for V2R mRNA amplification were selectedin the coding sequence of V2R cDNA, suggesting the possibleexistence of a thick ascending limb–specific V2R isoformin the mouse. The absence of cross-contamination between microdissectedsamples was validated by RT-PCR amplification of several additionalmRNA species that are characteristic to the different partsof the nephron (see Supplementary Figure 1). Collectively, theseresults demonstrate that RGS2 expression in the kidney is restrictedto the vasopressin-sensitive parts of the nephron.
Figure 3. RGS2 (A) and V2R (B) mRNA expression along the mouse nephron. Ethidium bromide–stained gel that contained reverse transcriptase–PCR (RT-PCR) amplification products of RGS2 and V2R mRNA in different parts of the mouse nephron is shown. RT-PCR was performed on total RNA that were extracted from microdissected tubules. RNA aliquots that originated from 1 mm of microdissected tubule were used in RT-PCR. Thirty-five PCR cycles were performed for all samples.
In Vivo Regulation of RGS2 mRNA Expression
Water restriction is a highly potent stimulus for vasopressinsecretion, whereas water loading efficiently suppresses plasmahormone concentration. To determine whether RGS2 expressioncorrelates with circulating vasopressin levels, we performedreal-time quantitative PCR analysis of RGS2 mRNA expressionin CTAL and CCD that were microdissected either from water-loadedor from water-restricted mice. In the water-restricted group,mice were deprived of water for 23 h before kidney perfusionand microdissection. In the water-loaded group, mice were fedfor 23 h with water-saturated gelled diet (see the Materialsand Methods section). RGS2 mRNA expression levels were quantifiedusing two independent internal standards, namely -actin andhypoxanthine guanine phosphoribosyl transferase I. As shownin Figure 4, the RGS2 mRNA expression was significantly higherin both CTAL and CCD that were microdissected from water-restrictedmice using both internal standards.
Figure 4.In vivo regulation of RGS2 mRNA expression. (A) RGS2 mRNA expression in cortical thick ascending limb (CTAL) that was microdissected from either water-loaded (23 h) or water-restricted (23 h) mice. Data are means ± SEM of values that were obtained from five mice. (B) RGS2 mRNA expression in CCD that were microdissected from either water-loaded or water-restricted mice. Data are means ± SEM of values that were obtained from six mice. RGS2 expression levels were normalized either to -actin or to hypoxanthine guanine phosphoribosyl transferase I (Hprt1) expression levels. Statistical significance was calculated using unpaired t test.
Quantitatively, the increase in RGS2 mRNA expression was higherin the CTAL compared with the CCD. It should be noted, however,that this quantification was performed at a single time point(23-h water restriction compared with 23-h water loading). Itis possible that RGS2 mRNA, which is strongly but transientlyinduced by vasopressin in in vitro systems, does not followthe same accumulation/degradation kinetics between the CTALand the CCD in vivo.
Agonist-Stimulated cAMP Levels in Wild-Type and RGS2–/– Mice
To assess directly the role of RGS2 in V2R signaling, we measuredthe cAMP levels in CCD that were microdissected from wild-typeand RGS2–/– mice. All measurements of cAMP wereperformed in the presence of 1 mM 3-isobutyl-1-methylxanthine,a phosphodiesterase inhibitor. The cAMP levels were measuredin unstimulated CCD (basal cAMP levels) and in CCD that weretreated with 10–9 or 10–8 M vasopressin for 4 minat 35°C. Basal cAMP levels were not different between wild-typeand RGS2–/– mice (data not shown). Vasopressin-stimulatedcAMP levels were higher in CCD that were microdissected fromRGS2–/– mice at both hormone concentrations (Figure 5A).
Figure 5. Agonist-simulated cAMP accumulation in CCD that were microdissected from wild-type or RGS2–/– mice. (A) cAMP accumulation in wild-type and RGS2–/– mice in response to 10–9 or 10–8 M vasopressin. The effect of 10–9 M vasopressin was tested in CCD that were microdissected from 15 wild-type and 15 RGS2–/– mice (n = 138 and n = 139, respectively). The effect of 10–8 M vasopressin was tested in CCD that were microdissected from nine wild-type and 9 RGS2–/– mice (n = 85 and n = 92, respectively). The absolute cAMP levels in nonstimulated CCD were 0.92 ± 0.11 and 1.09 ± 0.08 fmol/mm per 4 min, in the wild-type and RGS2–/– mice, respectively. The absolute values of cAMP levels that were induced by 10–9 M vasopressin were 57.28 ± 2.04 and 66.11 ± 2.20 fmol/mm per 4 min, respectively (P < 0.005). The absolute values of cAMP levels that were induced by 10–8 M vasopressin were 114.01 ± 5.61 and 128.43 ± 4.05 fmol/mm per 4 min, respectively (P < 0.05). (B) Vasopressin (10–9 M) was added simultaneously with either endothelin-1 (ET-1; 10–8 M) or phenylephrine (PE; 2 x 10–4 M). For ET-1 experiments, the CCD were microdissected from four wild-type mice (n = 31 in vasopressin group and n = 32 in vasopressin + ET-1 group) or four RGS2–/– mice (n = 29 in vasopressin group and n = 30 in vasopressin + ET-1 group). For PE experiments, the CCD were microdissected from three wild-type mice (n = 23 in vasopressin group and n = 24 in vasopressin + PE group) or three RGS2–/– mice (n = 19 in vasopressin group and n = 21 in vasopressin + PE group). Results are presented as the percentage of cAMP accumulation produced by vasopressin (10–9 M) alone. Statistical significance was calculated using unpaired t test. *P < 0.05; **P < 0.01.
It was previously demonstrated that mouse CCD cells expressat least two Gq-coupled GPCR, namely endothelin-1 (ET-1) receptorB and (1)-adrenergic receptor (12,16). Because RGS2 has alsobeen shown to interfere with Gq-mediated signaling pathways,we measured the effects of ET-1 and phenylephrine on cAMP accumulationthat was induced by vasopressin. As shown in Figure 5B, bothET-1 and phenylephrine provoked a significant inhibition ofvasopressin-stimulated cAMP levels. However, there was no significantdifference in the inhibitory effects between wild-type and RGS2–/–mice.
The increased cAMP levels in CCD that were microdissected fromthe RGS2–/– mice together with the rapid but transientupregulation of RGS2 expression by vasopressin suggest thatvasopressin-stimulated RGS2 expression could be involved inthe mean-term (30 min to several hours) negative feedback regulationof V2R signaling. The exact molecular targets of RGS2 in vasopressin-sensitiveparts of the nephron remain unknown. However, the V2R itselfor adenylyl cyclases ACIII, ACV, and ACVI, which are abundantlyexpressed in the principal cell of the collecting duct, arelikely candidates for future studies (7,18).
Renal Function in RGS2–/– Mice
From our cAMP experiments, we hypothesized that RGS2–/–mice should exhibit an increased functional responsiveness tovasopressin in the collecting ducts. To test this hypothesis,we assessed renal water handling of RGS2–/– micein two different experimental settings. In the first set ofexperiments, RGS2–/– mice or their wild-type littermateswere housed individually in metabolic cages and allowed freeaccess to food and water. In these baseline conditions, therewas no difference in body weight; urine volume; urine and plasmaosmolality; plasma Na concentration; and urine Na, K, Cl, PO4,Ca, and Mg excretion (Table 1). In addition, we did not findany significant difference in expression of several vasopressin-sensitivetransporters (aquaporin-2, and subunits of the amiloride-sensitiveepithelial sodium channel, and 1 and 1 subunits of the Na, K-ATPase),as tested by Western blot analysis that was performed on microdissectednephron segments (Figure 6).
Figure 6. Assessment of expression of vasopressin-sensitive transporters in the CCD that were microdissected from the wild-type or RGS2–/– mice. Proximal convoluted tubule (PCT), CTAL, and CCD were microdissected from RGS2–/– mice or their wild-type littermates that were allowed ad libitum access to food and water. Protein extracts from 10 mm of microdissected nephron segments were loaded and electrophoresed on a 13% SDS-PAGE. (A) Western blot was probed with an anti–1Na,K-ATPase antibody, an anti–1Na,K-ATPase antibody, and an anti-actin antibody (Sigma). It is interesting that the apparent molecular weight of the 1 subunit of the Na,K-ATPase is lower in the PCT compared with CTAL and CCD. This observation was confirmed in four independent experiments. (B) Western blot was probed with an anti– epithelial sodium channel (anti–-ENaC) antibody, an anti–-ENaC antibody, and an anti-actin antibody (Sigma). (C) Western blot was probed with an anti–aquaporin 2 (anti-aqp2) antibody and an anti-actin antibody (Sigma). fg, fully glycosylated form of aqp2; cg, core glycosylated form of aqp2. Western blots were quantified using a Phosphoimager system (BioRad, Hercules, CA). Protein bands in a given lane were normalized to actin (for aqp2, the signals from both fg and cg bands were summarized). Then, the ratio of normalized signals that were obtained from the wild-type and RGS2–/– samples was calculated. None of probed proteins revealed a significant difference of expression between RGS2–/– mice and their wild-type littermates: 1Na,K-ATPase: PCT 1.16 ± 0.08 (n = 5; NS), CTAL 0.75 ± 0.09 (n = 4; NS), CCD 1.18 ± 0.08 (n = 6; NS); 1Na,K-ATPase: PCT 1.15 ± 0.29 (n = 4; NS), CTAL 0.90 ± 0.13 (n = 4; NS), CCD 1.10 ± 0.09 (n = 3; NS); -ENaC: CCD 1.39 ± 0.33 (n = 5; NS); -ENaC: CCD 1.53 ± 0.50 (n = 5; NS); aqp2: CCD 0.96 ± 0.10 (n = 5; NS).
The second experimental setting examined acute differences inwater handling between RGS2–/– and wild-type mice.In this protocol, we measured urine output in mice that wereexposed to 23 h of water restriction followed by acute waterloading (2 ml of intraperitoneal water injection). Urine wascollected hourly during the first 5 h followed by water loading.The last fraction was collected between 5 and 7.5 h after waterloading. As shown in Figure 7A, RGS2–/– mice excretedless urine in the beginning of the collection period than theirwild-type littermates and more urine at the end of the collectionperiod. Importantly, the total urine volume at the end of thecollection period was not different between RGS2–/–and wild-type mice (0.57 ± 0.06 ml [n = 15] versus 0.72± 0.07 ml [n = 11], respectively). To check whether theobserved difference is due to the difference in V2R signaling,we performed a similar water-loading experiment in which weadded SR121463B, a V2R-specific antagonist, to the injectedwater. As shown in Figure 7B, in the presence of SR121463B,there was no difference in urine excretion between RGS2–/–and wild-type mice. Comparison of urine flow in the presenceor absence of SR121463B also demonstrated in our experimentalsettings (23-h water deprivation followed by a 2 ml of intraperitonealwater injection with or without SR121463B) that the circulatingvasopressin levels remain significant, at least in the beginningof the collection period. Indeed, during the 1- to 2- and 2-to 3-h collection periods, the mice that were administered aninjection only of water excreted three to four times less urinethan mice that were administered an injection of water complementedwith SR1212463B. These results clearly indicate that the vasopressin/V2R-mediatedsignaling pathway is responsible for the observed differencein water excretion between the wild-type and RGS2–/–mice.
Figure 7. Time course of urine excretion in wild-type or RGS2–/– mice after acute water loading. (A) Mice were water deprived for 23-h before acute water loading (2 ml of intraperitoneal tap water). (B) After 23 h of water deprivation, mice were administered an intraperitoneal injection of 2 ml of tap water that contained SR121463B (1 mg/kg body wt). Statistical significance was calculated using unpaired t test. *P < 0.05; **P < 0.01.
Collectively, our findings are compatible with a negative regulatoryrole of RGS2 in V2R signaling. Thus, RGS2–/– micerepresent the first mouse model to exhibit a gain-of-functionphenotype in water handling by the kidney. In humans, two gain-of-functionmissense mutations in the V2R gene were recently shown to causea nephrogenic disorder that is characterized by hyponatremia,serum hyposmolarity, and suppressed vasopressin secretion. Asproposed by Feldman et al. (4), these mutations result in constitutiveV2R activation in the absence of antagonist, thereby leadingto dysregulation of receptor function. This disease was labeledas the nephrogenic syndrome of inappropriate diuresis (4) oras the pseudo-SIADH (19) to emphasize that it represents a subtypeof a relatively common syndrome of inappropriate antidiuretichormone secretion (SIADH). In general, the pseudo-SIADH andSIADH have identical clinical features with the exception ofhigh and low vasopressin levels, respectively. As discussedby Feldman et al. (4), cases of SIADH with low or undetectablevasopressin levels represent as much as 10 to 20% of affectedpatients. Our study indicates that in addition to V2R-activatingmutations, the inactivating mutations of the RGS2 gene shouldbe considered in these patients. The pseudo-SIADH was studiedonly in a very limited number of patients (two children of <3mo of age). In addition to the species difference, it is difficultto compare the pseudo-SIADH clinical features with the phenotypeof the RGS2–/– mice. However, the gain of functionin the V2R signaling pathway and the inability to excrete afree water load (at least transiently) remain common featuresbetween patients with pseudo-SIADH and the RGS2–/–mouse model.
This work was supported by the Swiss National Fund for ScientificResearch, grant 3100A0–105592/1 (D.F.).
Some of the data were presented in abstract form at the annualmeeting of the American Society of Nephrology; November 14 through19, 2006; San Diego, CA.
We thank Dr. Claudine Serradeil-Le Gal (Sanofi Synthélabo,Toulose, France) for the generous gift of SR121463B.
Footnotes
Published online ahead of print. Publication date availableat www.jasn.org.
Nielsen S, Chou CL, Marples D, Christensen EI, Kishore BK, Knepper MA: Vasopressin increases water permeability of kidney collecting duct by inducing translocation of aquaporin-cd water channels to plasma membrane.
Proc Natl Acad Sci U S A 92
: 1013
–1017, 1995[Abstract/Free Full Text]
Knoers NV, Deen PM: Molecular and cellular defects in nephrogenic diabetes insipidus.
Pediatr Nephrol 16
: 1146
–1152, 2001[CrossRef][Medline]
Feldman BJ, Rosenthal SM, Vargas GA, Fenwick RG, Huang EA, Matsuda-Abedini M, Lustig RH, Mathias RS, Portale AA, Miller WL, Gitelman SE: Nephrogenic syndrome of inappropriate antidiuresis.
N Engl J Med 352
: 1884
–1890, 2005[Abstract/Free Full Text]
Firsov D: Revisiting sodium and water reabsorption with functional genomics tools.
Curr Opin Nephrol Hypertens 13
: 59
–65, 2004[CrossRef][Medline]
Brooks HL, Ageloff S, Kwon TH, Brandt W, Terris JM, Seth A, Michea L, Nielsen S, Fenton R, Knepper MA: cDNA array identification of genes regulated in rat renal medulla in response to vasopressin infusion.
Am J Physiol Renal Physiol 284
: F218
–F228, 2003[Abstract/Free Full Text]
Cai Q, Keck M, McReynolds MR, Klein JD, Greer K, Sharma K, Hoying JB, Sands JM, Brooks HL: Effects of water restriction on gene expression in mouse renal medulla: Identification of 3betaHSD4 as a collecting duct protein.
Am J Physiol Renal Physiol 291
: F218
–F224, 2006[Abstract/Free Full Text]
Robert-Nicoud M, Flahaut M, Elalouf JM, Nicod M, Salinas M, Bens M, Doucet A, Wincker P, Artiguenave F, Horisberger JD, Vandewalle A, Rossier BC, Firsov D: Transcriptome of a mouse kidney cortical collecting duct cell line: Effects of aldosterone and vasopressin.
Proc Natl Acad Sci U S A 98
: 2712
–2716, 2001[Abstract/Free Full Text]
Abramow-Newerly M, Roy AA, Nunn C, Chidiac P: RGS proteins have a signalling complex: Interactions between RGS proteins and GPCRs, effectors, and auxiliary proteins.
Cell Signal 18
: 579
–591, 2006[CrossRef][Medline]
Heximer SP, Knutsen RH, Sun X, Kaltenbronn KM, Rhee MH, Peng N, Oliveira-dos-Santos A, Penninger JM, Muslin AJ, Steinberg TH, Wyss JM, Mecham RP, Blumer KJ: Hypertension and prolonged vasoconstrictor signaling in RGS2-deficient mice.
J Clin Invest 111
: 445
–452, 2003[CrossRef][Medline]
Firsov D, Mandon B, Morel A, Merot J, Le MS, Bellanger AC, De Rouffignac C, Elalouf JM, Buhler JM: Molecular analysis of vasopressin receptors in the rat nephron. Evidence for alternative splicing of the V2 receptor.
Pflugers Arch 429
: 79
–89, 1994[Medline]
Ahn D, Ge Y, Stricklett PK, Gill P, Taylor D, Hughes AK, Yanagisawa M, Miller L, Nelson RD, Kohan DE: Collecting duct-specific knockout of endothelin-1 causes hypertension and sodium retention.
J Clin Invest 114
: 504
–511, 2004[CrossRef][Medline]
Oliveira-Dos-Santos AJ, Matsumoto G, Snow BE, Bai D, Houston FP, Whishaw IQ, Mariathasan S, Sasaki T, Wakeham A, Ohashi PS, Roder JC, Barnes CA, Siderovski DP, Penninger JM: Regulation of T cell activation, anxiety, and male aggression by RGS2.
Proc Natl Acad Sci U S A 97
: 12272
–12277, 2000[Abstract/Free Full Text]
Firsov D, Aarab L, Mandon B, Siaume-Perez S, De Rouffignac C, Chabardes D: Arachidonic acid inhibits hormone-stimulated cAMP accumulation in the medullary thick ascending limb of the rat kidney by a mechanism sensitive to pertussis toxin.
Pflugers Arch 429
: 636
–646, 1995[CrossRef][Medline]
Morel F, Doucet A: Hormonal control of kidney functions at the cell level.
Physiol Rev 66
: 377
–468, 1986[Free Full Text]
Cuffe JE, Howard DP, Bertog M, Korbmacher C: Basolateral adrenoceptor activation mediates noradrenaline-induced Cl- secretion in M-1 mouse cortical collecting duct cells.
Pflugers Arch 445
: 381
–389, 2002[CrossRef][Medline]
Chabardes D, Firsov D, Aarab L, Clabecq A, Bellanger AC, Siaume-Perez S, Elalouf JM: Localization of mRNAs encoding Ca2+-inhibitable adenylyl cyclases along the renal tubule. Functional consequences for regulation of the cAMP content.
J Biol Chem 271
: 19264
–19271, 1996[Abstract/Free Full Text]
Hoffert JD, Chou CL, Fenton RA, Knepper MA: Calmodulin is required for vasopressin-stimulated increase in cyclic AMP production in inner medullary collecting duct.
J Biol Chem 280
: 13624
–13630, 2005[Abstract/Free Full Text]
Segal A: Nephrogenic syndrome of inappropriate antidiuresis.
N Engl J Med 353
: 529
–530, 2005[Free Full Text]
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Abstract
Introduction
-actin) Endogenous Control (for 
(1)-adrenergic receptor (12,16). Because RGS2 has also been shown to interfere with Gq-mediated signaling pathways, we measured the effects of ET-1 and phenylephrine on cAMP accumulation that was induced by vasopressin. As shown in Figure 5B, both ET-1 and phenylephrine provoked a significant inhibition of vasopressin-stimulated cAMP levels. However, there was no significant difference in the inhibitory effects between wild-type and RGS2–/– mice. 
subunits of the amiloride-sensitive epithelial sodium channel, and 
