Global analysis of the effects of the V2 receptor antagonist satavaptan on protein phosphorylation in collecting duct.
ABSTRACT: Satavaptan (SR121463) is a vasopressin V2 receptor antagonist that has been shown to improve hyponatremia in patients with cirrhosis, congestive heart failure, and syndrome of inappropriate antidiuresis. While known to inhibit adenylyl cyclase-mediated accumulation of intracellular cyclic AMP and potentially recruit ?-arrestin in kidney cell lines, very little is known regarding the signaling pathways that are affected by this drug. To this end, we carried out a global quantitative phosphoproteomic analysis of native rat inner medullary collecting duct cells pretreated with satavaptan or vehicle control followed by the V2 receptor agonist desmopressin (dDAVP) for 0.5, 2, 5, or 15 min. A total of 2,449 unique phosphopeptides from 1,160 proteins were identified. Phosphopeptides significantly changed by satavaptan included many of the same kinases [protein kinase A, phosphoinositide 3-kinase, mitogen-activated protein kinase kinase kinase 7 (TAK1), and calcium/calmodulin-dependent kinase kinase 2] and channels (aquaporin-2 and urea transporter UT-A1) regulated by vasopressin. Time course clustering and kinase motif analysis suggest that satavaptan blocks dDAVP-mediated activation of basophilic kinases, while also blocking dDAVP-mediated inhibition of proline-directed kinases. Satavaptan affects a variety of dDAVP-mediated processes including regulation of cell-cell junctions, actin cytoskeleton dynamics, and signaling through Rho GTPases. These results demonstrate that, overall, satavaptan acts as a selective V2 receptor antagonist and affects many of the same signaling pathways regulated by vasopressin. This study represents the first "systems-wide" analysis of a "vaptan"-class drug and provides a wealth of new data regarding the effects of satavaptan on vasopressin-mediated phosphorylation events.
Project description:Vasopressin's action in renal cells to regulate water transport depends on protein phosphorylation. Here we used mass spectrometry-based quantitative phosphoproteomics to identify signaling pathways involved in the short-term V2-receptor-mediated response in cultured collecting duct cells (mpkCCD) from mouse. Using Stable Isotope Labeling by Amino acids in Cell culture (SILAC) with two treatment groups (0.1 nM dDAVP or vehicle for 30 min), we carried out quantification of 2884 phosphopeptides. The majority (82%) of quantified phosphopeptides did not change in abundance in response to dDAVP. Analysis of the 273 phosphopeptides increased by dDAVP showed a predominance of so-called "basophilic" motifs consistent with activation of kinases of the AGC family. Increases in phosphorylation of several known protein kinase A targets were found. In addition, increased phosphorylation of targets of the calmodulin-dependent kinase family was seen, including autophosphorylation of calmodulin-dependent kinase 2 at T286. Analysis of the 254 phosphopeptides decreased in abundance by dDAVP showed a predominance of so-called "proline-directed" motifs, consistent with down-regulation of mitogen-activated or cyclin-dependent kinases. dDAVP decreased phosphorylation of both JNK1/2 (T183/Y185) and ERK1/2 (T183/Y185; T203/Y205), consistent with a decrease in activation of these proline-directed kinases in response to dDAVP. Both ERK and JNK were able to phosphorylate residue S261of aquaporin-2 in vitro, a site showing a decrease in phosphorylation in response to dDAVP in vivo. The data support roles for multiple vasopressin V2-receptor-dependent signaling pathways in the vasopressin signaling network of collecting duct cells, involving several kinases not generally accepted to regulate collecting duct function.
Project description:Activation of V2 receptors (V2R) during antidiuresis increases the permeability of the inner medullary collecting duct to urea and water. Extracellular osmolality is elevated as the concentrating capacity of the kidney increases. Osmolality is known to contribute to the regulation of collecting duct water (aquaporin-2; AQP2) and urea transporter (UT-A1, UT-A3) regulation. AQP1KO mice are a concentrating mechanism knockout, a defect attributed to the loss of high interstitial osmolality. A V2R-specific agonist, deamino-8-D-arginine vasopressin (dDAVP), was infused into wild-type and AQP1KO mice for 7 days. UT-A1 mRNA and protein abundance were significantly increased in the medullas of wild-type and AQP1KO mice following dDAVP infusion. The mRNA and protein abundance of UT-A3, the basolateral urea transporter, was significantly increased by dDAVP in both wild-type and AQP1KO mice. Semiquantitative immunoblots revealed that dDAVP infusion induced a significant increase in the medullary expression of the endoplasmic reticulum (ER) chaperone GRP78. Immunofluorescence studies demonstrated that GRP78 expression colocalized with AQP2 in principal cells of the papillary tip of the renal medulla. Using immunohistochemistry and immunogold electron microscopy, we demonstrate that vasopressin induced a marked apical targeting of GRP78 in medullary principal cells. Urea-sensitive genes, GADD153 and ATF4 (components of the ER stress pathway), were significantly increased in AQP1KO mice by dDAVP infusion. These findings strongly support an important role of vasopressin in the activation of an ER stress response in renal collecting duct cells, in addition to its role in activating an increase in UT-A1 and UT-A3 abundance.
Project description:Ascending urinary tract infection (UTI) and pyelonephritis caused by uropathogenic Escherichia coli (UPEC) are very common infections that can cause severe kidney damage. Collecting duct cells, the site of hormonally regulated ion transport and water absorption controlled by vasopressin, are the preferential intrarenal site of bacterial adhesion and initiation of inflammatory response. We investigated the effect of the potent V2 receptor (V2R) agonist deamino-8-D-arginine vasopressin (dDAVP) on the activation of the innate immune response using established and primary cultured collecting duct cells and an experimental model of ascending UTI. dDAVP inhibited Toll-like receptor 4-mediated nuclear factor kappaB activation and chemokine secretion in a V2R-specific manner. The dDAVP-mediated suppression involved activation of protein phosphatase 2A and required an intact cystic fibrosis transmembrane conductance regulator Cl- channel. In vivo infusion of dDAVP induced a marked fall in proinflammatory mediators and neutrophil recruitment, and a dramatic rise in the renal bacterial burden in mice inoculated with UPECs. Conversely, administration of the V2R antagonist SR121463B to UPEC-infected mice stimulated both the local innate response and the antibacterial host defense. These findings evidenced a novel hormonal regulation of innate immune cellular activation and demonstrate that dDAVP is a potent modulator of microbial-induced inflammation in the kidney.
Project description:Vasopressin regulates renal water excretion by binding to the Gs-coupled vasopressin receptor (V2R) in collecting duct cells, resulting in cyclic AMP-dependent increases in epithelial water permeability through regulation of the aquaporin-2 (AQP2) water channel. Our prior studies showed that CRISPR-mediated deletion of protein kinase A (PKA) in cultured mpkCCD cells largely eliminates these regulatory events. These PKA-null cells provide a means of identifying PKA-independent signaling downstream from the V2 receptor. We carried out large-scale quantitative protein mass spectrometry (SILAC) to identify PKA-independent phosphorylation changes in response to V2R-selective vasopressin analog, dDAVP. The results show that V2R-mediated vasopressin signaling is predominantly, but not entirely, PKA-dependent. Target motif analysis of the phosphopeptides increased in response to dDAVP in PKA-null cells indicates that the vasopressin activates of one or more members of the AMPK/SNF1 subfamily of basophilic protein kinases. Among the upregulated phosphorylation sites were three known targets of SNF1-subfamily kinases, namely Lipe (S559), Crtc1 (S151) and Arhgef2 (S151). One of the phosphorylation sites that increased in occupancy in PKA-null cells was Ser256 of AQP2, a site critical for vasopressin-mediated trafficking of AQP2 to the cell surface. Beyond this, PKA-independent active site phosphorylation changes were also seen for protein kinases Stk39 (SPAK) and Prkci (Protein kinase C iota). Cyclic AMP levels were ~10-fold higher in PKA-null than in PKA-intact cells in the presence of phosphodiesterase inhibitor IBMX, consistent with a marked acceleration of cAMP production in PKA-null cells. The findings are indicative of substantial PKA-independent signaling downstream from the Gs-coupled V2 receptor.
Project description:Vasopressin regulates transport across the collecting duct epithelium in part via effects on gene transcription. Transcriptional regulation occurs partially via changes in phosphorylation of transcription factors, transcriptional coactivators, and protein kinases in the nucleus. To test whether vasopressin alters the nuclear phosphoproteome of vasopressin-sensitive cultured mouse mpkCCD cells, we used stable isotope labeling and mass spectrometry to quantify thousands of phosphorylation sites in nuclear extracts and nuclear pellet fractions. Measurements were made in the presence and absence of the vasopressin analog dDAVP. Of the 1,251 sites quantified, 39 changed significantly in response to dDAVP. Network analysis of the regulated proteins revealed two major clusters ("cell-cell adhesion" and "transcriptional regulation") that were connected to known elements of the vasopressin signaling pathway. The hub proteins for these two clusters were the transcriptional coactivator ?-catenin and the transcription factor c-Jun. Phosphorylation of ?-catenin at Ser552 was increased by dDAVP [log(2)(dDAVP/vehicle) = 1.79], and phosphorylation of c-Jun at Ser73 was decreased [log(2)(dDAVP/vehicle) = -0.53]. The ?-catenin site is known to be targeted by either protein kinase A or Akt, both of which are activated in response to vasopressin. The c-Jun site is a canonical target for the MAP kinase Jnk2, which is downregulated in response to vasopressin in the collecting duct. The data support the idea that vasopressin-mediated control of transcription in collecting duct cells involves selective changes in the nuclear phosphoproteome. All data are available to users at http://helixweb.nih.gov/ESBL/Database/mNPPD/.
Project description:1. Effects of neuropeptides of the vasopressin family on Cl(-) secretion have not yet been reported in lung. Using the 16HBE14o- bronchial epithelial cell line, we investigated their action on Cl(-) secretion. 2. In symmetrical Cl(-) solutions, basolateral application of arginine vasotocin (AVT), oxytocin or isotocin induced a transient I(sc) stimulation (I(peak)), whereas arginine vasopressin (AVP) did not. The effects of different Cl(-) channel blockers and of a protein kinase C (PKC) inhibitor suggest that CFTR is involved in I(peak). The calcium-activated K(+) channel (SK4) and the Cl(-)/HCO(-)(3) exchanger favor the driving force for AVT-mediated Cl(-) secretion. The antagonists of V1a (SR49059)- and V1b (SSR149415)-receptors blocked I(peak), while SR121463B, a V2 receptor antagonist, did not. These results point to the stimulation of a V1-like receptor mediating I(peak) and presenting an efficacy order, AVT>oxytocin>isotocin>>AVP. 3. When a serosal to mucosal Cl(-) gradient was applied, AVT and AVP both stimulated I(sc) according to a biphasic profile, I(peak) being followed by a plateau phase (I(plateau)). The pharmacology of I(plateau) suggests that CFTR channels are involved and that Na(+)/K(+)/2Cl(-) is the only transporter associated with I(plateau). dDAVP, a V2 receptor agonist-induced I(plateau) with the same potency as AVP, suggesting the involvement of V2 receptors in the AVP-induced I(plateau). V2 receptors are present on both opposite membranes, while V1-like receptors are mainly expressed on the basolateral membranes. RT-PCR experiments show the expression of V1a, V1b, V2 and vasopressin-activated calcium-mobilizing (VACM) receptors mRNAs.
Project description:Vasopressin regulates water excretion through effects on the renal collecting duct. Vasopressin signaling in the inner medullary collecting duct (IMCD) is mediated by V2 receptor occupation coupled to the generation of cyclic AMP. Here, we employ a "systems" approach to analysis of vasopressin signaling. The objective is to investigate roles of activation of the Akt and ERK1/2 MAP kinase pathways, as well as Ca2+ mobilization, in IMCD cells isolated from rat kidney. The V2 receptor-selective vasopressin analog dDAVP increased the state of Akt activation (increased phosphorylation at T308 and S473) and decreased the state of ERK1/2 activation (decreased phosphorylation at T202 and Y204). Akt activation was blocked by an inhibitor of PI3K, LY294002. In microdissected IMCD segments, nonperiodic spike-like increases in intracellular Ca2+ (FLUO-4) were accelerated by vasopressin. Chelation of Ca2+ or calmodulin inhibition markedly decreased Akt phosphorylation. Decreased ERK1/2 phosphorylation was associated with a decrease in MEK1/2 phosphorylation and an increase in c-Raf phosphorylation at S259 (an inhibitory site). Based on the current findings integrated with previous findings in the IMCD, we now report a 33-node vasopressin signaling network involved in vasopressin regulation of IMCD function.
Project description:Vasopressin controls water balance largely through PKA-dependent effects to regulate the collecting duct water channel aquaporin-2 (AQP2). Although considerable information has accrued regarding the regulation of water and solute transport in collecting duct cells, information is sparse regarding the signaling connections between PKA and transport responses. Here, we exploited recent advancements in protein mass spectrometry to perform a comprehensive, multiple-replicate analysis of changes in the phosphoproteome of native rat inner medullary collecting duct cells in response to the vasopressin V2 receptor-selective agonist 1-desamino-8D-arginine vasopressin. Of the 10,738 phosphopeptides quantified, only 156 phosphopeptides were significantly increased in abundance, and only 63 phosphopeptides were decreased, indicative of a highly selective response to vasopressin. The list of upregulated phosphosites showed several general characteristics: 1) a preponderance of sites with basic (positively charged) amino acids arginine (R) and lysine (K) in position -2 and -3 relative to the phosphorylated amino acid, consistent with phosphorylation by PKA and/or other basophilic kinases; 2) a greater-than-random likelihood of sites previously demonstrated to be phosphorylated by PKA; 3) a preponderance of sites in membrane proteins, consistent with regulation by membrane association; and 4) a greater-than-random likelihood of sites in proteins with class I COOH-terminal PDZ ligand motifs. The list of downregulated phosphosites showed a preponderance of those with proline in position +1 relative to the phosphorylated amino acid, consistent with either downregulation of proline-directed kinases (e.g., MAPKs or cyclin-dependent kinases) or upregulation of one or more protein phosphatases that selectively dephosphorylate such sites (e.g., protein phosphatase 2A). The phosphoproteomic data were used to create a web resource for the investigation of G protein-coupled receptor signaling and regulation of AQP2-mediated water transport.
Project description:Transient receptor potential channels TRPC3 and TRPC6 are expressed in principal cells of the collecting duct (CD) along with the water channel aquaporin-2 (AQP2) both in vivo and in the cultured mouse CD cell line IMCD-3. The channels are primarily localized to intracellular vesicles, but upon stimulation with the antidiuretic hormone arginine vasopressin (AVP), TRPC3 and AQP2 translocate to the apical membrane. In the present study, the effect of various activators and inhibitors of the adenylyl cyclase (AC)/cAMP/PKA signaling cascade on channel trafficking was examined using immunohistochemical techniques and by biotinylation of surface membrane proteins. Both in vivo in rat kidney and in IMCD-3 cells, translocation of AQP2 and TRPC3 (but not TRPC6) was stimulated by [deamino-Cys(1), d-Arg(8)]-vasopressin (dDAVP), a specific V2-receptor agonist, and blocked by [adamantaneacetyl(1), O-Et-d-Tyr(2), Val(4), aminobutyryl(6), Arg(8,9)]-vasopressin (AEAVP), a specific V2-receptor antagonist. In IMCD-3 cells, translocation of TRPC3 and AQP2 was activated by forskolin, a direct activator of AC, or by dibutyryl-cAMP, a membrane-permeable cAMP analog. AVP-, dDAVP-, and forskolin-induced translocation in IMCD-3 cells was blocked by SQ22536 and H89, specific inhibitors of AC and PKA, respectively. Translocation stimulated by dibutyryl-cAMP was unaffected by AEAVP but could be blocked by H89. AVP- and forskolin-induced translocation of TRPC3 in IMCD-3 cells was also blocked by two additional inhibitors of PKA, specifically Rp-cAMPS and the myristoylated inhibitor of PKA (m-PKI). Quantification of TRPC3 membrane insertion in IMCD-3 cells under each assay condition using a surface membrane biotinylation assay, confirmed the translocation results observed by immunofluorescence. Importantly, AVP-induced translocation of TRPC3 as estimated by biotinylation was blocked on average 95.2 +/- 1.0% by H89, Rp-cAMPS, or m-PKI. Taken together, these results demonstrate that AVP stimulation of V2 receptors in principal cells of the CD causes translocation of TRPC3 to the apical membrane via stimulation of the AC/cAMP/PKA signaling cascade.
Project description:The action of vasopressin in rodent collecting ducts to regulate water permeability depends in part on increases in phosphorylation of the water channel aquaporin-2 (AQP2) at three sites: Ser256, Ser264, and Ser269. Previous studies of AQP2 phosphorylation have depended largely on qualitative data using protein mass spectrometry and phospho-specific antibodies. Here, we use a new method employing phospho-specific antibodies to determine the percentage of total AQP2 phosphorylated at each site in the presence and absence of the V2-receptor-selective vasopressin analog dDAVP in rat renal inner medullary collecting duct (IMCD) and cultured mpkCCD cells. Phosphorylation of Ser269, a site previously implicated in plasma membrane retention, was found to increase from 3 to 26% of total AQP2 in rat IMCD cells following dDAVP. Quantification of immunogold labeling of the opposite kidneys from the same rats estimated that 11% of total AQP2 is present in the apical plasma membrane (APM) without injection of dDAVP and 25% is present in the APM after dDAVP. Surprisingly, the baseline level of Ser256 phosphorylation was constitutively high, and there was no increase with dDAVP (confirmed in 2 more sets of rats). In general, Ser264 phosphorylation remained below 5% of total. The pattern of response was similar in cultured mpkCCD cells (large increase in Ser269 phosphorylation following dDAVP, but constitutively high levels of Ser256 phosphorylation). We suggest from these studies that Ser269 phosphorylation may be a more consistent indicator of vasopressin action and AQP2 membrane abundance than is Ser256 phosphorylation.