Effects of the renal medullary pH and ionic environment on vasopressin binding and signaling
2008; Elsevier BV; Volume: 74; Issue: 12 Linguagem: Inglês
10.1038/ki.2008.412
ISSN1523-1755
AutoresElena A. Zalyapin, Richard Bouley, Udo Hasler, Jean‐Pierre Vilardaga, Herbert Y. Lin, Dennis Brown, Dennis A. Ausiello,
Tópico(s)Electrolyte and hormonal disorders
ResumoThe kidney has a cortico-medullary interstitial gradient of decreasing pH and increasing concentrations of sodium chloride and urea, but the influence of these gradients on receptor signaling is largely unknown. Here, we measured G-protein coupled receptor function in LLC-PK1 cells acutely exposed to conditions mimicking different kidney regions. Signaling through the parathyroid hormone receptor, normally expressed in the cortex, was greatly reduced at an acidic pH similar to that of the inner medulla. Parathyroid hormone receptor, tagged with green fluorescent protein, showed no ligand-induced internalization. In contrast, under both acidic and hyperosmotic conditions, vasopressin increased intracellular cAMP, and upon binding to its type 2 receptor (V2R) was internalized and degraded. Dose-displacement binding assays with selective vasopressin/oxytocin receptor ligands under inner medullary conditions indicated a shift in the V2R pharmacological profile. Oxytocin did not bind to the V2R, as it does under normal conditions and the vasopressin type 1 receptor (V1R) had reduced affinity for vasopressin compared to the V2R in low pH and high osmolality. We suggest that the cortico-medullary gradient causes a receptor-specific selectivity in ligand binding that is of functional significance to the kidney. While the gradient is important for urinary concentration, it may also play a substantial role in fine-tuning of the vasopressin response through the V2R. The kidney has a cortico-medullary interstitial gradient of decreasing pH and increasing concentrations of sodium chloride and urea, but the influence of these gradients on receptor signaling is largely unknown. Here, we measured G-protein coupled receptor function in LLC-PK1 cells acutely exposed to conditions mimicking different kidney regions. Signaling through the parathyroid hormone receptor, normally expressed in the cortex, was greatly reduced at an acidic pH similar to that of the inner medulla. Parathyroid hormone receptor, tagged with green fluorescent protein, showed no ligand-induced internalization. In contrast, under both acidic and hyperosmotic conditions, vasopressin increased intracellular cAMP, and upon binding to its type 2 receptor (V2R) was internalized and degraded. Dose-displacement binding assays with selective vasopressin/oxytocin receptor ligands under inner medullary conditions indicated a shift in the V2R pharmacological profile. Oxytocin did not bind to the V2R, as it does under normal conditions and the vasopressin type 1 receptor (V1R) had reduced affinity for vasopressin compared to the V2R in low pH and high osmolality. We suggest that the cortico-medullary gradient causes a receptor-specific selectivity in ligand binding that is of functional significance to the kidney. While the gradient is important for urinary concentration, it may also play a substantial role in fine-tuning of the vasopressin response through the V2R. The antidiuretic hormone vasopressin (VP) binds to VP receptor type 1 (V1R), VP receptor type 2 (V2R), as well as the oxytocin receptor,1.Birnbaumer M. Seibold A. Gilbert S. et al.Molecular cloning of the receptor for human antidiuretic hormone.Nature. 1992; 357: 333-335Crossref PubMed Scopus (465) Google Scholar,2.Lolait S.J. O'Carroll A.M. McBride O.W. et al.Cloning and characterization of a vasopressin V2 receptor and possible link to nephrogenic diabetes insipidus.Nature. 1992; 357: 336-339Crossref PubMed Scopus (463) Google Scholar,3.Morel A. O′Carroll A.M. Brownstein M.J. et al.Molecular cloning and expression of a rat V1a arginine vasopressin receptor.Nature. 1992; 356: 523-526Crossref PubMed Scopus (441) Google Scholar,4.Sugimoto T. Saito M. 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Nonoguchi H. et al.Different localization and regulation of two types of vasopressin receptor messenger RNA in microdissected rat nephron segments using reverse transcription polymerase chain reaction.J Clin Invest. 1993; 92: 2339-2345Crossref PubMed Scopus (119) Google Scholar The V2R is expressed in principal cells of all parts of the kidney-collecting duct, from the cortex to the tip of the papilla32.Edwards R.M. Jackson B.A. Dousa T.P. ADH-sensitive cAMP system in papillary collecting duct: effect of osmolality and PGE2.Am J Physiol. 1981; 240: F311-F318PubMed Google Scholar and the composition of the kidney interstitium is known to vary in pH and osmolarity from cortex to the outer and inner medulla. The cortical interstitium has a neutral pH (7.4) and is isoosmotic (300 mOsm/kg). However, these conditions gradually change along the collecting duct to produce a more extreme environment in which the renal tubules function. At the tip of the papilla, the interstitium is acidic (pH 5.7±0.2)33.Kersting U. Dantzler D.W. Oberleithner H. et al.Evidence for an acid pH in rat renal inner medulla: paired measurements with liquid ion-exchange microelectrodes on collecting ducts and vasa recta.Pflugers Arch. 1994; 426: 354-356Crossref PubMed Scopus (23) Google Scholar,34.Good D.W. Knepper M.A. Ammonia transport in the mammalian kidney.Am J Physiol. 1985; 248: F459-F471PubMed Google Scholar,35.Hamm L.L. Brenner B.M. Levine S.A. Renal acidification in Kidney. 1. Saunders, Philadelphia2004: 497-534Google Scholar and hyperosmotic (up to 1200 mOsm/kg in humans and >1200 mOsm/kg in rodents and many other mammals).36.Cohen D.M. Signalling and gene regulation by urea and NaCl in the renal medulla.Clin Exp Pharmacol Physiol. 1999; 26: 69-73Crossref PubMed Scopus (20) Google Scholar,37.Jamison R.L. Robertson C.R. Editorial review. Recent formulations of the urinary concentrating mechanism: a status report.Kidney Int. 1979; 16: 537-545Abstract Full Text PDF PubMed Scopus (14) Google Scholar,38.Knepper M.A. Gamba G. Brenner B.M. Levine S.A. Urine Concentration and Dilution in Kidney. 1. Saunders, Philadelphia2004: 599-636Google Scholar Thus, VP must be able to bind to the V2R in environments ranging from isoosmotic/neutral pH to hyperosmotic/acidic pH. The functional requirement of the V2R to be active under the increasingly acidic and hyperosmotic conditions with high urea concentrations that are encountered as the collecting duct descends into the inner medulla, contrasts with that of several other receptors that are present mainly in the renal cortex and outer medulla. These include the parathyroid hormone receptor (PTHR) and the angiotensin II receptor. Interestingly, the V1R, which is present in the renal vasculature, is also found in collecting ducts but is more abundant in the cortex than in the outer or inner medulla. The role of the medullary environment on VP binding has not been studied in detail. Earlier work from our lab showed that at neutral pH, intracellular cAMP accumulation following VP-induced V2R activation is further increased by hypertoncity.39.Skorecki K.L. Conte J.M. Ausiello D.A. Effects of hypertonicity on cAMP production in cultured renal epithelial cells (LLC-PK1).Miner Electrolyte Metab. 1987; 13: 165-172PubMed Google Scholar cAMP production decreased following addition of urea, but when both sodium chloride (NaCl) and urea were added at gradually increasing concentrations, intracellular cAMP content increased.39.Skorecki K.L. Conte J.M. Ausiello D.A. Effects of hypertonicity on cAMP production in cultured renal epithelial cells (LLC-PK1).Miner Electrolyte Metab. 1987; 13: 165-172PubMed Google Scholar The combined effect of acidification, NaCl and urea, however, remains unknown. Recent work from our group and others has shown that following VP binding at neutral pH, V2R activity is downregulated by lysosomal degradation.24.Bouley R. Lin H.Y. Raychowdhury M.K. et al.Downregulation of the vasopressin type 2 receptor after vasopressin-induced internalization: involvement of a lysosomal degradation pathway.Am J Physiol Cell Physiol. 2005; 288: C1390-C1401Crossref PubMed Scopus (40) Google Scholar,25.Robben J.H. Knoers N.V. Deen P.M. Regulation of the vasopressin V2 receptor by vasopressin in polarized renal collecting duct cells.Mol Biol Cell. 2004; 15: 5693-5699Crossref PubMed Scopus (60) Google Scholar We suggested that this degradation results from the inability of V2R to dissociate from its ligand, VP, in acidic early endosomes. The association between the V2R and VP may have adapted and evolved to allow a tight ligand–receptor interaction that is maintained even under acidic conditions such as those of the inner medulla. It is well established that in contrast to V2R, most ligands detach from their cognate receptor in acidic endosomes before their subsequent lysosomal degradation, whereas the receptor is recycled back to the cell surface.40.Innamorati G. Le Gouill C. Balamotis M. et al.The long and the short cycle. Alternative intracellular routes for trafficking of G-protein-coupled receptors.J Biol Chem. 2001; 276: 13096-13103Crossref PubMed Scopus (133) Google Scholar Thus, it is important to understand the pharmacology of the V2R under conditions that reflect the prevailing environment of the inner medulla (low pH, high tonicity, and high concentrations of urea) as well as those of other kidney regions. The purpose of this study was to characterize the pharmacology and downregulation of V2R under conditions mimicking those occurring in the renal cortex and medulla. We show that V2R indeed has properties that allow it to function in the inner medullary environment, in contrast to the PTHR, which is located mostly in the cortex and outer medulla. Our data also show that of all selective VP/oxytocin receptor ligands tested, VP displays the highest affinity for V2R under all conditions tested. [3H]-VP saturation of binding sites in LLC-FLAG-V2R cells was dose dependent at both pH 7.4 and 5.5 (Figure 2), reaching maximal binding capacity under both conditions (Figure 2a). Scatchard analysis revealed one class of high-affinity binding sites (Figure 2b). The affinity constant (KD) of [3H]-VP for V2R under acidic conditions was 10-fold lower than under conditions of neutral pH (78±34 and 7±3 nM, respectively) but maximal V2R-binding capacity under both conditions was not significantly different (78,000±6000 and 49,800±13,400 binding sites per cell, respectively). A similar result was observed by dose–displacement of [3H]-VP with VP on COS cells transfected with either V1R or V2R (Table 1). Thus, V2R displayed a reduced affinity for VP under acidic conditions, whereas an increase of osmolality had no effect on V2R affinity for VP. In contrast, a combination of acidity and increased osmolality reduced V1R affinity for VP.Table 1The effect of pH and hypertonicity on VP binding to V1R and V2RIC50 (nM) (n)Receptor7.47.4 NaCl5.55.5 NaClV1R6.0±1.1 (3)8.8±1.2 (3)7.3±1.8 (3)29.3±15.0 (3)V2R1.5±0.4 (3)2.6±0.6 (3)9.1±1.3 (3)7.8±2.1 (3)VP, vasopressin; V1R and V2R, VP receptor types 1 and 2.The results shown represent three independent experiments (mean±s.d.). Open table in a new tab VP, vasopressin; V1R and V2R, VP receptor types 1 and 2. The results shown represent three independent experiments (mean±s.d.). The effect of an acidic buffer and hyperosmolality on the binding of VP analogs to the V2R was studied by performing [3H]-VP dose–displacement experiments using VP, [deamino-Cys1, D-Arg8]-VP (DDAVP), Manning's compound, and oxytocin at pH 7.4 or 5.5 in the presence or absence of hypertonic NaCl. In all cases, [3H]-VP was displaced in a dose-dependent manner. At neutral pH, V2R affinity for VP was high (KD of 3.67±0.43 nM) (Table 2). V2R affinity for DDAVP was 10-fold lower than for VP whereas its affinity for the Manning's compound and oxytocin was 46- and 179-fold lower than for VP, respectively (Table 2). This pharmacological profile is different from that observed under conditions where either osmolality or pH was modified (VP > Manning's compound > DDAVP). Oxytocin, which has 163 times less affinity for V2R as compared with VP, was unable to displace [3H]-VP from V2R under acidic conditions. This modified pharmacological profile was even more significant when both pH and osmolality were simultaneously changed (Manning's compound > VP > DDAVP).Table 2Affinity of VP analogs for the V2R at pHs 7.4 and 5.5 under isoosmotic and hyperosmotic conditionsVP analogKD (nM) at pH 7.4, 300 mOsmKD (nM) at pH 7.4, 600 mOsmKD (nM) at pH 5.5, 300 mOsmKD (nM) at pH 5.5, 600 mOsmVP3.67±0.43 (3)20.34±9.19 (3)88.22±34.08 (3)142.40±59.06 (3)DDAVP32.21±5.00 (3)81.44±44.53 (3)600.90±160.71 (3)754.92±407.25 (3)Manning's compound178.00±54.19 (3)42.47±44.53 (3)144.40±50.00 (3)34.93±12.38 (3)OT678.90±192.71 (3)ND>100,000 (3)NDDDAVP, [deamino-Cys1, D-Arg8]-VP; ND, not defined; OT, oxytocin; VP, vasopressin.The results shown represent three independent experiments (three) done in triplicate (mean±s.d.). Open table in a new tab DDAVP, [deamino-Cys1, D-Arg8]-VP; ND, not defined; OT, oxytocin; VP, vasopressin. The results shown represent three independent experiments (three) done in triplicate (mean±s.d.). cAMP accumulation induced by forskolin decreased 1.5-fold under acidic conditions, as compared with that induced under conditions of neutral pH (data not shown). Intracellular cAMP was increased by VP stimulation under both normal and acidic conditions, and VP-induced cAMP accumulation was enhanced by tonicity (Figure 3a and b). Similar experiments performed in the presence of sucrose suggest that the effect of NaCl on cAMP is not due to a specific effect of either sodium or chloride. In contrast to the positive effect of hypertonicity on V2R, intracellular cAMP levels in cells expressing PTHR were reduced in hypertonic medium containing PTH under both normal and acidic conditions (Figure 3c or d). Thus, in contrast to VP signaling, PTH signaling was significantly inhibited by hypertonicity (Figure 3c and d). We then investigated the combined effect of NaCl and urea on cAMP generation under conditions of varying pH. For this purpose, we used neutral pH medium mimicking the kidney outer medullary environment and a second medium of pH 5.5, mimicking the kidney inner medulla (Figure 4). We found that in ‘outer medullary’ conditions (600 mOsm/kg, pH 7.4), cAMP generation was increased (Figure 4). Results of the present study (Figure 3a) and our previous study39.Skorecki K.L. Conte J.M. Ausiello D.A. Effects of hypertonicity on cAMP production in cultured renal epithelial cells (LLC-PK1).Miner Electrolyte Metab. 1987; 13: 165-172PubMed Google Scholar show that NaCl added at pH 7.4 increases VP-induced cAMP signaling. We now conclude that 60 mOsm/kg urea does not diminish this effect at a neutral pH. Furthermore, VP causes cAMP content to significantly increase even under conditions mimicking the inner medulla (1200 mOsm/kg, pH 5.5), although this effect was reduced as compared with the signal generated under less extreme ‘outer medullary’ conditions, but did not change in comparison to control conditions (pH 7.4, 300 mOsm/kg).Figure 4cAMP accumulation following V2R stimulation by VP occurs under ‘harsh’ conditions mimicking those of the inner medulla. cAMP accumulation was studied in the presence or absence of 1 μM VP (open bars and closed bars, respectively). VP was diluted in normal and acidic buffer in the presence of increasing amounts of sodium and urea. Experiments shown represent an average of three different experiments performed in triplicate (mean±s.e.m.). Maximal forskolin response at pH 7.4, 300 mOsm is 12,270±467 fmol/106 cells.View Large Image Figure ViewerDownload (PPT) We have shown that even under harsh conditions mimicking the inner medulla, VP significantly increases cAMP generation (Figure 4). To further investigate V2R function, we examined ligand-induced V2R internalization occurring under these conditions. As shown in Figure 5 and confirmed by quantification (Figure 5g), VP induced an internalization of V2R-green fluorescent protein (GFP) under all conditions tested (Figure 5). Similar results were found with two other LLC-PK1 cell lines expressing V2R (data not shown), indicating that this is not a clonal effect. Similar experiments were performed on LLC-PK1 cells stably expressing GFP coupled to either the PTHR (PTHR–GFP) or the angiotensin II receptor (AT1R–GFP) (Figure 6). In the absence of ligands, both receptors were localized at the plasma membrane under normal (Figure 6a and e) or acidic and hyperosmotic conditions (Figure 6c and g). In normal isoosmotic medium at neutral pH, all receptors were internalized in the presence of their respective ligands (Figure 6b and f). In the absence of ligands, an acidic pH together with the addition of both NaCl and urea did not affect the baseline localization of these receptors. However, although addition of PTH did not induce PTHR–GFP internalization under these same conditions, AT1R–GFP was internalized on angiotensin II addition (Figure 6d and h). Quantification of PTHR–GFP fluorescence showed a reduction of membrane fluorescence and an increase of cytoplasmic fluorescence resulting from PTH internalization under conditions mimicking the outer medullary environment. The membrane/cytosolic fluorescence ratio was reduced from 0.56±0.01–0.48 (cortical environment) and 0.38 (outer medullary environment) ±0.01, whereas no significant decrease was observed under conditions mimicking the inner medullary environment. This result suggests that corticomedullary gradients differentially affect receptor internalization. Western blot analysis showed that internalized V2R is degraded under all conditions (Figure 7). Thus, after internalization, V2R is degraded in a similar quantitative manner to that previously reported.24.Bouley R. Lin H.Y. Raychowdhury M.K. et al.Downregulation of the vasopressin type 2 receptor after vasopressin-induced internalization: involvement of a lysosomal degradation pathway.Am J Physiol Cell Physiol. 2005; 288: C1390-C1401Crossref PubMed Scopus (40) Google Scholar,25.Robben J.H. Knoers N.V. Deen P.M. Regulation of the vasopressin V2 receptor by vasopressin in polarized renal collecting duct cells.Mol Biol Cell. 2004; 15: 5693-5699Crossref PubMed Scopus (60) Google ScholarFigure 6AT1R receptors internalize following AngII stimulation under ‘harsh’ inner medullary conditions, whereas PTH-stimulated PTHR does not. LLC-PTHR-GFP (a–d) and LLC-AT1R-GFP (e–h) cells, stably expressing either PTHR-GFP or AT1R-GFP were incubated in the absence (a, c and e, g) or presence of 1 μM of their respective agonists (b, d and f, h). Cells were incubated with either neutral (a, b and e, f) or acidic hyperosmotic medium (c, d and g, h). Sequential images were acquired using spinning disk confocal microscopy. Acquired images of cells taken before and after 30 min of agonist stimulation are representative of four independent experiments.View Large Image Figure ViewerDownload (PPT)Figure 7VP stimulated V2R-GFP degrades under conditions of both acidic and neutral pH. V2R-GFP degradation after 4 hours of VP stimulation (at 37 °C) under both neutral and acidic conditions was studied by Western blot analysis (a). LLC-V2R-GFP cells were incubated in the absence (lanes 1 and 3) or presence (lanes 2 and 4) of VP. In the absence of VP, 71 and 55 kDa bands are observed corresponding to intact, glycosylated V2R and to a degraded fragment of this receptor, respectively. The presence of VP reduced the intensity of the 71 kDa band (lane 2). Similar results were observed under acidic conditions (lanes 4–6). Disappearance of the upper band intensity was quantified in relation to the intensity of corresponding β-actin bands, used as a loading control (b). This is a representative blot from three independent experiments; Mean±SD. ***P>0.005 between VP treated and untreated conditions, ***P>0.001 between sample incubated at pH 5 in the presence of VP and in the presence of either NaCl or Urea.View Large Image Figure ViewerDownload (PPT) The interstitium of the kidney papilla is a harsh environment in which VP and V2R interact to modify the behavior of specific target cells. Cell signaling becomes a challenge when ligands and their cognate receptors face conditions that are known to affect protein–protein interactions. These conditions include an acidic pH, high concentrations of NaCl, and high concentrations of urea, a compound that affects protein conformation. On the basis of physiological data, it is clear that VP and V2R can indeed interact in such environments, which occur in parts of the kidney medulla. Here, we present data quantifying this interaction under various conditions that partially reflect the in vivo milieu in which V2R ligand stimulation, signaling, and downregulation occur. In this study, we focused our attention on major components of the interstitial environment (NaCl, urea, and pH). However, the presence of other constituents such as reactive oxygen species,41.Mori T. Cowley A.W. Angiotensin II-NAD(P)H oxidase-stimulated superoxide modifies tubulovascular nitric oxide cross-talk in renal outer medulla.Hypertension. 2003; 42: 588-593Crossref PubMed Scopus (86) Google Scholar,42.Zhang Z. Rhinehart K. Kwon W. et al.ANG II signaling in vasa recta pericytes by PKC and reactive oxygen species.Am J Physiol Heart Circ Physiol. 2004; 287: H773-H781Crossref PubMed Scopus (33) Google Scholar amino acids,43.Silbernagl S. Volker K. Dantzler W.H. 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We show that VP b
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