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Am J Physiol Renal Physiol 295: F1170-F1176, 2008. First published August 13, 2008; doi:10.1152/ajprenal.00119.2008
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Regulation of V2R transcription by hypertonicity and V1aR-V2R signal interaction

Yuichiro Izumi, Yushi Nakayama, Hasiyet Memetimin, Takeaki Inoue, Yukimasa Kohda, Hiroshi Nonoguchi, and Kimio Tomita

Department of Nephrology, Kumamoto University, Graduate School of Medical Sciences. Kumamoto, Japan

Submitted 7 March 2008 ; accepted in final form 11 August 2008


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Arginine vasopressin (AVP) and hypertonicity in the renal medulla play a major role in the urine concentration mechanism. Previously, we showed that rat vasopressin V2 receptor (rV2R) promoter activity was increased by vasopressin V2R stimulation and decreased by vasopressin V1a receptor (V1aR) stimulation in a LLC-PK1 cell line stably expressing rat V1aR (LLC-PK1/rV1aR). In the present study, we investigated the effects of hypertonicity on the rV2R promoter activity and on the suppression of rV2R promoter activity by V1aR stimulation in LLC-PK1/rV1aR cells. rV2R promoter activity was increased in NaCl- or mannitol-induced hypertonicity. The hypertonicity-responsive site in the rV2R promoter region was limited to 10 bp, including the Sp1 motif. The increase of V2R promoter activity by hypertonicity was significantly inhibited by a JNK inhibitor (SP600125) and PKA inhibitor (H89). In contrast, rV2R promoter activity was remarkably suppressed by V1aR stimulation in the hypertonic condition rather than in the isotonic condition. The AVP-stimulated intracellular Ca2+ concentration was increased in the hypertonic condition, suggesting the functional activation of V1aR by hypertonicity. In conclusion, 1) V2R promoter activity is increased by hypertonicity via the JNK and PKA pathways, 2) suppression of V2R expression by the V1aR-Ca2+ pathway is enhanced by hypertonicity, and 3) hypertonicity enhances the V1aR-Ca2+ pathway. The counteractivity of V2R and V1aR could be required to maintain minimum urine volume in the dehydrated state.

collecting ducts; transcription regulation; cell signaling

THE MAIN ROLE OF THE KIDNEY is to maintain body fluid homeostasis by the production of concentrated or diluted urine under different conditions. Among many hormones involved, arginine vasopressin (AVP) plays the major role in the urine-concentrating mechanism. Water reabsorption is regulated by the vasopressin V2 receptor (V2R)-aquaporin 2 (AQP2) system in the collecting ducts. The V2R is distributed at the basolateral membrane and subcellular region of the collecting ducts (9, 28). The V2R is coupled to Gs proteins and increases cyclic adenosine monophosphate (cAMP) in the cytoplasm and elicits Ca2+ transient increase upon activation. Subsequently, protein kinase A (PKA) is activated through the V2R-cAMP pathway. In the dehydrated state, AVP is excreted into plasma from neurohypophysis and stimulates the V2R in the collecting ducts. The V2R activates the trafficking of AQP2 in the intracellular vesicles to the surface of the luminal membrane and increases the expression of AQP2 through the cAMP-PKA pathway (27).

The tonicity gradient in the renal interstitium, which is mainly created by NaCl and urea, is also crucial for urine concentration in the kidney (21). Hypertonicity is known to activate AQP2 promoter activity via the TonEBP pathway and increases the mRNA and protein expression of AQP2 in vitro (14, 19). The tonicity of the interstitium in the kidney is augmented in the dehydrated state. Thus it is clear that the expression of AQP2 is increased in the dehydrated state. We previously reported that hypertonicity increased the expression of V2R in the inner medullary collecting ducts (23). Although the modulation of AQP2 expression has been investigated in detail, the regulatory mechanism of V2R expression remains unclear.

The vasopressin V1a receptor (V1aR) is mainly expressed in the brain, vascular system, liver, and kidney (29). The V1aR is coupled to Gq/11 proteins and increases intracellular Ca2+ concentration via phospholipase C, and subsequently, protein kinase C (PKC) is activated. The V1aR is involved in blood pressure, circulating volume, and glucose homeostasis (2, 22). In the kidney, the V1aR has been suggested to be distributed in the luminal membrane and cytoplasm in the collecting ducts by immunohistochemical, in situ hybridization, and functional studies (1, 6, 26, 37). Recently, we reported the regulation of the renin-angiotensin-aldosterone system and the V2R-AQP2 system through the V1aR in macula densa cells (3). Although the physiological importance of the V1aR is becoming clearer, the role of V1aR in the collecting ducts still remains unclear. In a recent study, we investigated the effects of V1aR and V2R stimulation on V2R promoter activity using LLC-PK1 cells dominantly expressing rat V1aR (LLC-PK1/rV1aR cells) (17). V2R promoter activity was not decreased by AVP in LLC-PK1 cells but was decreased in LLC-PK1/rV1aR cells, indicating that V1aR stimulation decreased V2R promoter activity. We previously reported that luminal AVP decreased osmotic water permeability in the presence of basolateral AVP in the inner medullary collecting duct (IMCD) (28). These findings suggest that luminal AVP inhibits the function of V2R through the V1aR on the luminal side of the collecting ducts. A number of reports have indicated that body fluid homeostasis is maintained not only by basolateral AVP but also by luminal AVP, presumably through the V1aR pathway (1, 4, 26). However, it is unclear how V1aR in the collecting ducts physiologically contributes to body fluid homeostasis.

The purpose of the present study was to elucidate the regulation mechanisms of V2R promoter activity by hypertonicity. We investigated the involvement of mitogen-activated protein (MAP) kinases and PKA with V2R expression under hypertonic conditions. We also examined the functional importance of V1aR stimulation on the regulation of V2R under hypertonicity.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Reagents. [Arg8]-vasopressin (AVP), SP600125, SB203580, U0126, H-89, and 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate (CPT-cAMP) were obtained from Sigma-Aldrich (St. Louis, MO). Phorbol 12-myristate 13-acetate (PMA) was purchased from Promega (Madison, WI).

Preparation of rat V2R promoter-luciferase reporter constructs. The 1,092-bp 5'-flanking region of the rat V2R (rV2R) gene was cloned by PCR amplification and was subcloned into a pGL3 luciferase reporter vector as previously described (17). An Expand High FidelityPLUS PCR system (Roche Applied Science, Penzberg, Germany) was used for PCR amplification. Gene-specific sense and antisense primers were designed on the basis of the published cDNA sequence of the rV2R gene (24). The series of deletion mutants were synthesized by PCR using sense primers designed to downstream and antisense primers on exon 1 (+57 bp). The –664, –345, –245, –165, –124, –92, –62, and –46 bp of the V2R promoter-pGL3 constructs were prepared in the previous study (17). The –82, –72, and –52 bp of the V2R promoter-pGL3 constructs were newly synthesized for the present study.

Cell culture, transfection, and determination of promoter activity. LLC-PK1/rV1aR cells were established using the Flp-In system (Invitrogen Life Technologies, Carlsbad, CA) as previously described (17). Cells were maintained in 290 mosmol/kgH2O Dulbecco's modified Eagle's medium (DMEM) supplemented with 800 µg/ml hygromycin B and 10% fetal bovine serum (Invitrogen, Tokyo, Japan). For transfection studies, cells were seeded in 12-well clusters (Corning, NJ) and grown to reach 50% confluence. We cotransfected 0.314 pmol of the 5'-flanking region of the rV2R-pGL3 constructs and 3.75 fmol of the PRL-TK constructs into cells using FuGENE6 transfection reagent (Roche Applied Science, Indianapolis, IN). After the transfection, LLC-PK1/rV1aR cells were incubated in 290 mosmol/kgH2O DMEM at 37°C for 18 h. Promoter activity was examined after incubation in the indicated tonicity. Hypertonic medium was prepared by adding NaCl or mannitol to the isotonic medium. The tonicity of the medium was confirmed by the measurement of osmolality using a micro-osmometer (Fiske, Needham Heights, MA).

To investigate the time-dependent effect of hypertonicity on rV2R promoter activity, we cotransfected LLC-PK1/rV1aR cells with the –1,092-bp 5'-flanking region of the rV2R-pGL3 construct and incubated cells in NaCl- and mannitol-induced hypertonic (490 mosmol/kgH2O) or isotonic (290 mosmol/kgH2O) condition. The effect of urea with an osmolality of 490 mosmol/kgH2O on rV2R promoter activity was also examined. Promoter activity was examined at 3, 6, and 12 h after the replacement with hypertonic or isotonic medium. To assess the tonicity-dependent effect on rV2R promoter activity, we cotransfected LLC-PK1/rV1aR cells with the –1,092-bp 5'-flanking region of the rV2R-pGL3 construct and incubated cells in the NaCl-induced hypertonic (490 or 690 mosmol/kgH2O) or isotonic condition for 12 h.

For the determination of the tonicity-responsive site, we cotransfected cells with a deletion series of the 5'-flanking region of the rV2R-pGL3 constructs and incubated cells in 290 or 490 mosmol/kgH2O medium, which was prepared by the addition of NaCl. rV2R promoter activity was examined at 12 h after the incubation in the indicated tonicity.

Based on the results of deletion study, cells were cotransfected with –62 bp of the 5'-flanking region of the rV2R-pGL3 constructs and incubated in NaCl-induced hypertonic medium (490 mosmol/kgH2O) with SP600125 (JNK inhibitor), SB203580 (p38 inhibitor), U0126 (MEK inhibitor), H-89 (PKA inhibitor), AVP, CPT-cAMP, PMA, or vehicle (DMSO or H2O) to investigate the effects of hypertonicity, the V1aR pathway, and the V2R pathway on V2R promoter activity. rV2R promoter activity was examined 12 h after incubation in the hypertonic condition.

All promoter activity was determined as the ratio of firefly luciferase activity to Renilla luciferase activity for the revision of the cell population. Firefly luciferase activity from the pGL3 reporter vector and Renilla luciferase activity from the PRL-TK vector were measured using the Dual Luciferase Assay System (Promega) on a TD-20/20 luminometer (Turner Design, CA).

Intracellular calcium measurement. LLC-PK1/rV1aR cells were cultured in 96-well black plates to reach 100% confluence. According to the protocol for the Calcium Kit Fluo-3 (Dojindo Laboratories, Kumamoto, Japan), cells were preincubated in 100 µl of loading buffer containing 5 mg/l fluo-3 AM, 1.25 mM probenecid, and 0.04% Pluronic F-127 at 37°C for 1 h. After preincubation, loading buffer was replaced with 100 µl of recording medium with 1.25 mM probenecid with various tonicities induced by the addition of NaCl. Fluorescence intensity was continuously measured as the intracellular Ca2+ concentration at 1-s intervals for 50 s after the addition of 100 µl of recording medium with AVP (10–8 M) using a plate reader (Fluoroskan Ascent; DS Pharma Biomedical, Osaka, Japan). To assess the effect of hypertonicity on V1aR stimulation, we compared the mean of the fluorescence intensity 15 s after the treatment with AVP, which was adjusted with mean basic intensity before AVP stimulation, between the hypertonic and isotonic conditions.

Data analysis. Values are means ± SE. Statistical analysis was performed by analysis of variance (ANOVA) and multiple comparison (Bonferroni or Sheffé's test) or by Student's t-test. P < 0.05 was considered significant.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Effect of tonicity on V2R promoter activity. LLC-PK1/rV1aR cells were cotransfected with the –1,092-bp 5'-flanking region of rV2R-pGL3 construct and incubated in the indicated tonicity. rV2R promoter activity was significantly increased at 3 and 12 h after the incubation in NaCl-induced hypertonic condition (490 mosmol/kgH2O) compared with the isotonic condition (290 mosmol/kgH2O) (Fig. 1A). No significant difference of rV2R promoter activity was observed between 490 and 690 mosmol/kgH2O after the 12-h incubation in the NaCl-induced hypertonicity condition (Fig. 1B). The increase of rV2R promoter activity was also observed in short-term incubation (3 h) in a mannitol-induced hypertonic condition (490 mosmol/kgH2O) (Fig. 1C). In contrast, rV2R promoter activity was decreased after 12 h of treatment with urea (490 mosmol/kgH2O).


Figure 1
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  		Fig. 1. Effects of hypertonicity on rat vasopressin V2 receptor (rV2R) promoter activity. A: time-dependent effect of NaCl-induced hypertonicity on rV2R promoter activity. B: osmolality higher than 490 mosmol/kgH2O (690 mosmol/kgH2O) did not further enhance rV2R promoter activity. C: time course of rV2R promoter activity in isotonic or the mannitol- or urea-added hypertonic medium (490 mosmol/kgH2O). Luciferase activity is shown as a ratio to pGL3-Basic. Values are means ± SE (n = 6–9). *P < 0.05 vs. 290 mosmol/kgH2O.

 
Determination of the tonicity-responsive site in the V2R promoter region. To determine the tonicity-responsive site in the rV2R promoter region, we cotransfected LLC-PK1/rV1aR cells with a deletion series of the 5'-flanking region of the rV2R-pGL3 construct and incubated in isotonicity (290 mosmol/kgH2O) or NaCl-induced hypertonicity (490 mosmol/kgH2O) for 12 h. The increase of rV2R promoter activity was abolished by deletion of 10 bp located at –62 to –52 bp upstream from the transcriptional initiation site, which includes the consensus sequence of SP1 (–59 to –54 bp) (Fig. 2). Therefore, this 10-bp segment, including the SP1 motif, was considered to be the NaCl-induced tonicity-responsive site.


Figure 2
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  		Fig. 2. Determination of the tonicity-responsive site in the 5'-flanking region of rV2R by deletion analysis. The tonicity-responsive site was limited to 10 bp, including the Sp1 consensus sequence, which was located at –59 to –54 bp from the transcriptional initiation site of 5'-flanking region of rV2R (n = 6–9). *P < 0.05.

 
Involvement of MAP kinase and PKA pathways in the hypertonicity-induced increase of V2R promoter activity. SP600125, SB203580, U0126, and H-89 were used to analyze the signal transduction mechanism of V2R promoter activity. SP600125 dose-dependently suppressed the increase of rV2R promoter activity by hypertonicity, as shown in Fig. 3A. SB 203580 decreased rV2R promoter activity only at a high dose (Fig. 3B). U0126 did not decrease rV2R promoter activity (Fig. 3C). In addition, rV2R promoter activity was inhibited by H-89 (Fig. 4). The suppression of rV2R promoter activity by SP600125 and H-89 was observed only in hypertonic conditions, indicating that V2R promoter activity was activated by hypertonicity via the JNK and PKA pathways (Fig. 5).


Figure 3
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  		Fig. 3. Effects of JNK, p38, and ERK inhibitors on rV2R promoter activity in the hypertonic condition. SP600125 (JNK inhibitor) dose-dependently decreased (A) and only high-dose SB203580 (p38 inhibitor) decreased (B) rV2R promoter activity in the hypertonic condition. U0126 (ERK inhibitor) did not affect rV2R promoter activity (C) (n = 6). *P < 0.05 vs. vehicle in 490 mosmol/kgH2O.

 

Figure 4
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  		Fig. 4. Effect of H-89 (PKA inhibitor) on V2R promoter activity in the hypertonic condition (n = 6–9). *P < 0.05 vs. vehicle in 490 mosmol/kgH2O.

 

Figure 5
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  		Fig. 5. Suppression of hypertonicity-induced rV2R promoter activity by SP600125 (JNK inhibitor; A) and H-89 (PKA inhibitor; B) (n = 6). *P < 0.05.

 
Effect of V1aR stimulation on V2R promoter activity under hypertonicity. We previously demonstrated that rV2R promoter activity was decreased by AVP in LLC-PK1/rV1aR cells but not in LLC-PK1 cells, indicating that AVP decreased V2R promoter activity through V1aR stimulation (17). In the present study, we tested the effect of AVP on V2R promoter activity under a hypertonic condition. The AVP-induced decrease of rV2R promoter activity was significantly larger in the hypertonic than isotonic condition (Fig. 6A). In LLC-PK1/rV1aR cells, treatment with 10–8 M AVP rapidly increased fluo-3 fluorescence intensity, indicating an increased intracellular Ca2+ concentration (Fig. 6B). The intracellular Ca2+ concentration was remarkably increased by the addition of AVP in the hypertonic rather than isotonic condition (Fig. 6C). This suggests the function of V1aR is enhanced by hypertonicity.


Figure 6
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  		Fig. 6. A: tonicity-dependent effect of V1aR stimulation on V2R promoter activity (n = 6). *P < 0.05. B: effects of arginine vasopressin (AVP) on intracellular Ca2+ concentration in isotonic or hypertonic conditions. C: AVP-stimulated intracellular Ca2+ concentration in isotonic or hypertonic condition. Values are shown as a ratio to that in vehicle (n = 6). *P < 0.05.

 
In the previous study, CAAT and/or the Sp1 site was indicated to be involved in the opposite effects of V1aR and V2R stimulation on V2R promoter activity (17). We examined the effect of AVP, PKC, and cAMP on the promoter activity of the –62-bp 5'-flanking region of rV2R including the Sp1 site, which was the minimum fragment responsive to tonicity. As shown in Fig. 7, AVP and PMA decreased and CPT-cAMP increased rV2R promoter activity in the hypertonic condition. Therefore, it is not CAAT but the Sp1 site that is crucial for the modulation of V2R promoter activity through tonicity in the V1aR and V2R pathways.


Figure 7
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  		Fig. 7. Effect of V1aR, PKC, and cAMP stimulation on V2R promoter activity of –62-bp 5'-flanking region of rV2R including the Sp1 site in the hypertonic condition (n = 6). *P < 0.05 vs. vehicle in 490 mosmol/kgH2O. PMA, phorbol 12-myristate 13-acetate; CPT-cAMP, 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Recently, we demonstrated the suppression of V2R promoter activity by V1aR stimulation (17). After the transient increase of rV2R promoter activity in both LLC-PK1 and LLC-PK1/rV1aR cells, a sustained decrease of rV2R promoter activity was observed only in LLC-PK1/rV1aR cells by the treatment with AVP, suggesting a possible mechanism of the downregulation of V2R expression by V1aR in the dehydrated state. Since urinary AVP concentration and the tonicity gradient of renal interstitium are increased in the dehydrated state, in the present study we investigated the effect of hypertonicity on V2R promoter activity and the influence of V1aR stimulation. V2R promoter activity was upregulated by hypertonicity through the JNK and PKA pathways (Fig. 5). Hypertonicity enhanced the suppression of V2R promoter activity by V1aR stimulation (Fig. 6A). The AVP-stimulated intracellular Ca2+ concentration was increased by hypertonicity (Fig. 6, B and C). Thus hypertonicity increased not only V2R promoter activity but also its suppression by V1aR stimulation.

As shown in Fig. 1, hypertonicity increased V2R promoter activity. These results are consistent with the increases of V2R mRNA and protein under NaCl-induced hypertonicity in microdissected collecting ducts, as reported previously (23). Mannitol-induced increase of V2R promoter activity also supported the requirement of hypertonicity for the expression of V2R (Fig. 1C). On the other hand, the treatment with urea decreased rV2R promoter activity. Since urea is essentially permeable through the membrane, especially in the presence of vasopressin (32), the hypertonic effect of urea would be small. The expression of V2R was showed to be decreased by dehydration (23, 30). The urea signaling pathway may participate in the downregulation of V2R expression in dehydration.

Although TonEBP is well known to upregulate the expression of tonicity-responsive proteins (7, 10, 18, 25), there was no TonEBP binding site in the 5'-flanking region of rV2R. However, the Sp1 consensus sequence was considered to be the tonicity-responsive site in the rV2R promoter region in the present study (Fig. 2). Umenishi et al. (41) reported a novel hypertonicity-responsive element (HRE) in the human AQP1 promoter region. Although this HRE is a GC-rich sequence like the Sp1 consensus sequence, electrophoretic mobility shift assay (EMSA) demonstrated that the DNA binding proteins bound to HRE were not Sp1 family transcription factors. On the other hand, Hwang et al. (16) demonstrated that glucose transporter 1 promoter activity was increased by hypertonicity via Sp1 transcription factor by promoter analysis and EMSA. As shown in Fig. 7, the hypertonicity-responsive site in the V2R promoter region was also responsive to V1aR, PMA, and CPT-cAMP stimulation. It is still unclear whether the Sp1 transcription factor is involved in the regulation of V2R promoter activity. However, it is interesting that V1aR-PKC, V2R-cAMP, and hypertonicity-induced signaling pathways modulate V2R promoter activity through the same responsive site in the V2R promoter region.

Several reports have suggested that the expression of tonicity-responsive proteins is involved in the MAP kinase (5. 12, 40) or PKA pathways (11). In the present study, the JNK and PKA pathways were suggested to be mainly involved with the hypertonicity-induced V2R promoter activity. Previously, we demonstrated that PMA, which activates PKC and inhibits AVP-induced cAMP accumulation (8, 38), decreased V2R promoter activity in an isotonic condition (17). The current results showed the suppression of V2R promoter activity by PMA also in the hypertonic condition (Fig. 7). H-89 also reduced the hypertonicity-induced increase of V2R promoter activity (Fig. 4). AVP-stimulated cAMP accumulation increased in the hypertonic condition (31, 34). These results seem consistent with the inhibition of cAMP accumulation by the V1aR-PKC pathway under hypertonicity, as we have suggested. However, the inhibition of V2R promoter activity by H-89 was observed only in the hypertonic condition, in contrast to the inhibition by V1aR or PMA in the isotonic and hypertonic conditions (Figs. 5B, 6A, and 7). There is a discrepancy between the PKA and cAMP pathways in the isotonic condition. The cAMP-PKA pathway is known to be a common pathway (33). Although a number of studies have reported the effect of cAMP-PKA pathway on the expression of AQP2 (20, 35), several reports also have suggested cAMP-independent regulation of AQP2 expression under hypertonicity (11, 36). In contrast, PKA-independent cAMP regulation of AQP2 expression has been suggested (39). Our results suggest that V2R promoter activity was increased by hypertonicity via the PKA pathway. These LLC-PK1/rV1aR cells should prove to be useful for elucidating the complicated steps in the cAMP-PKA pathway.

With regard to the effect of V1aR, the suppression of V2R promoter activity by V1aR stimulation (Fig. 7) was larger than the suppression by H-89 (Fig. 5B) and PMA (Fig. 7) (66, 48, and 39% decrease by V1aR stimulation, H-89, and PMA, respectively, compared with vehicle in the hypertonic condition). V1aR stimulation might influence not only the cAMP and PKA pathways but also the JNK pathway under the hypertonic condition. The V1aR has been suggested to activate MAP kinases (13, 15, 42). Other signaling pathways also could be involved in the downregulation of V2R promoter activity by V1aR stimulation. Furthermore, certain other issues currently remain unsolved. It is uncertain how the transient increase of Ca2+ by V1aR stimulation could convert to a sustained signal for V2R transcription. There might be other factors influencing the expression of V2R and V1aR stimulation. Further experiments are needed to investigate in detail the relationship between tonicity and AVP stimulation on V2R expression.

During thirst, there is a gradual buildup of medullary hypertonicity, and the collecting ducts become very sensitive to the AVP released from the neurohypophysis by blood volume and tonicity changes. The increase of V2R expression by hypertonicity thus contributes to the sensitive response to AVP in the dehydrated state. On the other hand, the suppression of V2R by V1aR stimulation could be required for the maintenance of minimum urine volume. The suppression of V2R promoter activity by V1aR stimulation is long-term regulation of vasopressin activity. However, in acute volume loss, there would be an urgent demand for an acute response to retain blood volume. V1aR in macula densa cells is suggested to activate the renin-angiotensin-aldosterone system and, subsequently, the V2R-AQP2 system (3). There are different roles of luminal AVP between the conditions of acute and chronic volume loss through the V1aR in macula densa cells and the collecting duct cells. Recently, Carmosino et al. (6) demonstrated the homogeneous distribution of V1aR along the cortical collecting ducts, in contrast to the intercalated cell restricted expression of V1aR in the medullary collecting ducts. V1aR has been suggested to be involved in acid-base balance regulation. We (37) previously reported the increase of V1aR expression in chronic metabolic acidosis. The role of V1aR could be different along different regions of the nephron.

In summary, our data show that 1) V2R promoter activity is increased by hypertonicity via the JNK and PKA pathways, 2) suppression of V2R expression by the V1aR-Ca2+ pathway is enhanced by hypertonicity, and 3) hypertonicity enhances the V1aR-Ca2+ pathway. The suppression of V2R promoter activity by V1aR stimulation could be required to maintain the minimum urine volume in the dehydrated state.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported by the Grants-in-Aid for Scientific Research 19590955, 18590895, 17590833, 167904660, 16590791, 16590792, 16390246, 15590852, and 14370321 from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.


   ACKNOWLEDGMENTS
 
We thank Noriko Teramoto for excellent technical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: Y. Izumi, 1-1-1 Honjo, Kumamoto City, Kumamoto 860-8556, Japan (e-mail: 053r5110{at}med.stud.kumamoto-u.ac.jp)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 

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H. Memetimin, Y. Izumi, Y. Nakayama, Y. Kohda, H. Inoue, H. Nonoguchi, and K. Tomita
Low pH stimulates vasopressin V2 receptor promoter activity and enhances downregulation induced by V1a receptor stimulation
Am J Physiol Renal Physiol, September 1, 2009; 297(3): F620 - F628.
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