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Am J Physiol Renal Physiol 292: F1418-F1426, 2007. First published January 9, 2007; doi:10.1152/ajprenal.00358.2006
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Downregulation of vasopressin V2 receptor promoter activity via V1a receptor pathway

Yuichiro Izumi, Yushi Nakayama, Tomohiko Mori, Hiroki Miyazaki, Hideki Inoue, Yukimasa Kohda, Takeaki Inoue, Hiroshi Nonoguchi, and Kimio Tomita

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

Submitted 8 September 2006 ; accepted in final form 3 January 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Vasopressin V1a and V2 receptors (V1aR and V2R, respectively) distribute in the collecting duct of the kidney. Although the function of V2R mediating the antidiuretic effect of AVP has been investigated in detail, the role of V1aR in the collecting ducts has not been elucidated. In the present study, we have investigated the role of the V1aR pathway in V2R promoter activity. We cloned the 5'-flanking region of rat V2R (rV2R) and investigated rV2R promoter activity in the LLC-PK1 cell line transfected to express rat V1aR (rV1aR) dominantly (LLC-PK1/rV1aR). AVP induced a transient increase, followed by a sustained decrease, of rV2R promoter activity in these cells. This AVP-induced decrease of rV2R promoter activity was inhibited by V1aR, but not V2R, antagonist. PMA mimicked this decrease of rV2R promoter activity. On the contrary, 8-(4-chlorophenylthio)-cAMP increased rV2R promoter activity. These PMA- and 8-(4-chlorophenylthio)-cAMP-induced effects were not observed on the deletion segment of the 5'-flanking region lacking CAAT and SP1 sites. In conclusion, 1) expression of the V2R is downregulated via the V1aR pathway in LLC-PK1/rV1aR cells, and 2) expression of the V2R is downregulated by the PMA-induced PKC pathway and upregulated by the cAMP-PKA pathway. These opposite effects of PKC and PKA appear to be regulated by the same promoter region of CAAT and SP1.

transcription regulation; collecting ducts

THE MAIN ROLE OF THE KIDNEY is to maintain body fluid homeostasis by producing concentrated or diluted urine. Among many hormones, AVP plays a major role in the urine-concentrating mechanism. There are two types of AVP receptors in the kidney: vasopressin V1a and V2 receptors (V1aR and V2R, respectively).

The V2R is distributed at the basolateral membrane of the collecting ducts and mediates the antidiuretic effect of AVP (22, 25). The V2R is coupled to Gs proteins and increases cAMP in cytoplasm as a second messenger. Subsequently, PKA is activated. As a consequence of V2R stimulation from the basolateral membrane, excretion and reabsorption of water, electrolytes, and urea are induced through transporters such as aquaporin-2 (AQP2), Na-K-Cl cotransporter type 1, and renal urea transporter type A1 in the collecting ducts (16, 34, 37).

The V1aR is localized mainly in the vascular system and glomeruli and exerts a vasopressor effect (24). The V1aR is coupled to Gq/11 proteins and stimulates phospholipase C. Activation of phospholipase C releases Ca2+ from the endoplasmic reticulum, and subsequently PKC is activated. In the kidney, V1aR mRNA is expressed not only in the glomeruli but also in the collecting ducts (28, 30). Although the function of the V2R has been investigated in detail, the physiological role of the V1aR in the collecting ducts remains unclear.

Previous studies have suggested that the V1aR is present at the luminal membrane of the collecting ducts. Ando et al. (1) demonstrated that luminal AVP induces sustained hyperpolarization of transepithelial voltage. Naruse et al. (21) demonstrated that luminal AVP induces changes in luminal conductance. Our previous immunohistochemical study (22, 28) revealed that the V1aR was mainly localized at the luminal membrane and in the cytoplasm, whereas the V2R is localized at the basolateral membrane in the rat collecting duct.

Bankir (2) proposed a role for urinary AVP via the V1aR and the interaction between V1aR- and V2R-mediated effects. We previously demonstrated that fractional extraction of AVP is closely related to fractional excretion of Na+ in patients with chronic renal failure (23). Our previous study also showed that luminal AVP decreases osmotic water permeability in the presence of basolateral AVP in the inner medullary collecting ducts (22). In addition, the increase of V1aR mRNA and decrease of V2R mRNA were simultaneously observed during metabolic acidosis (28). These studies strongly indicate that body fluid homeostasis is maintained not only by basolateral AVP, but also by luminal AVP, presumably via the V1aR pathway.

On the basis of previous reports, we hypothesized that V1aR stimulation by luminal AVP might modulate the basolateral AVP-induced activity by regulating V2R expression at the transcriptional level. To address this hypothesis, we characterized the rat V2R (rV2R) promoter region and investigated the regulation of rV2R promoter activity in LLC-PK1 cells that were transfected to express rat V1aR (rV1aR) dominantly. Furthermore, we analyzed the effects of the PKC and PKA pathways on V2R promoter activity and investigated the cross talk between V1aR and V2R signaling pathways in LLC-PK1 cells.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Reagents. [Arg8]-vasopressin (AVP) was obtained from Sigma-Aldrich (St. Louis, MO). RO 31-8220 and 8-(4-chlorophenylthio)-cAMP (cpt-cAMP) were obtained from Sigma-Aldrich (Steinheim, Germany). PMA and 4{alpha}-PMA were purchased from Promega (Madison, WI). OPC-21268 and OPC-31260 were kind gifts from Otsuka Pharmaceutical (Tokushima, Japan).

Cloning of the 5'-flanking region of the rV2R gene. The 1,092-bp 5'-flanking region of the rV2R gene was cloned by PCR amplification, with rat genomic DNA used as a template. 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 (17).

Reporter plasmid construction. For analysis of rV2R promoter activity, a series of deletion mutants were synthesized by PCR using sense primers designed to downstream (–664, –345, –245, –225, –165, –155, –124, –92, and –46 bp) and antisense primers on exon 1 (+57 bp). A deletion mutant, lacking –164 to 47 bp from the 5'-flanking region, was synthesized by the in vitro overlap-extension PCR method, as described previously (20). PCR-amplified products were subcloned into a pGL3-basic luciferase reporter vector (Promega).

Cell culture, transfection, and determination of promoter activity. LLC-PK1 cells (catalog no. JCRB0060, Health Science Research Resources Bank, Tokyo, Japan) were maintained in DMEM supplemented with 100 IU/ml penicillin-streptomycin and 10% fetal bovine serum (Invitrogen, Tokyo, Japan).

For transfection studies, cells were seeded in 12-well clusters (Corning) and grown to reach 50% confluence. FuGENE6 transfection reagent (Roche Applied Science, Indianapolis, IN) was used to cotransfect 0.314 pmol of pGL3-rV2R promoter constructs and 3.75 fmol of pRL-TK constructs into the cells. Promoter activity was examined after treatment with AVP, cpt-cAMP, PMA, and vehicle (DMSO).

For determination of reporter activity, firefly luciferase activity from the pGL3 reporter vector and Renilla luciferese activity from the pRL-TK vector were measured by the Dual-Luciferase Assay System (Promega) on a luminometer (model TD-20/20, Turner Design).

Establishment of a stable LLC-PK1 cell line expressing rV1aR. The rV1aR gene (GenBank accession no. BC088095) was synthesized by PCR amplification from a pExpress1 vector containing rV1aR cDNA (Open Biosystems) and subcloned into a pcDNA5/FRT vector. Gene-specific sense and antisense primers were designed on the basis of the published cDNA sequence of the rV1R gene (19).

LLC-PK1 stably expressing the rV1aR was constructed using the Flp-In System (Invitrogen Life Technologies, Carlsbad, CA), which is able to create isogenic cell lines with one or more Flp recombination targets (FRT). To prepare a new LLC-PK1/FRT cell line, LLC-PK1 cells were transfected with pFRT/lacZeo and selected in Zeocin (250 µg/ml) for 2 wk. Cells were confirmed to have the FRT site by beta-galactosidase staining assay (Invitrogen Life Technologies). Positive cells were named LLC-PK1/FRT.

The LLC-PK1/FRT cell line was cotransfected with pOG44, a vector for transient expression of Flp recombinase, and pcDNA5/FRT/rV1aR, an expression vector containing the coding region for the rV1aR protein, and possesses the FRT site for homologous recombination. Since the pcDNA5/FRT vector also contained the hygromycin B-resistant gene, LLC-PK1/FRT cells stably transfected with rV1aR (LLC-PK1/rV1aR) were selected in 800 µg/ml hygromycin B. Single-cell colonies were used to analyze the interaction between rV1aR stimulation and rV2R promoter activity.

mRNA isolation and cDNA synthesis. MgNA Pure LC mRNA Isolation Kit II (Roche Diagnostics, Tokyo, Japan) was used for mRNA extraction. The cell pellets were diluted with 300 µl of lysis buffer and dissolved in a buffer containing a chaotropic salt and an RNase inactivator. The 3'-poly(A+) from the released mRNA hybridizes to the added biotin-labeled oligo(dT). This complex is immobilized onto the surface of streptavidin-coated magnetic beads. After a DNase digestion step, unbound substances were removed by three washing steps, and purified mRNA was eluted with a low-salt buffer. cDNA was synthesized using the High-Capacity cDNA Archive Kit (Applied Biosystems).

Intracellular Ca2+ measurement. LLC-PK1 cells expressing the rV1aR 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 containing 1.25 mM probenecid. Fluorescence intensity was measured as the Ca2+ concentration for 5 min after AVP treatment with use of a plate reader (Mulch Micro MTP-800Lab, Corona, Niigata, Japan).

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


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Analysis and characterization of the 1,092-bp segment from the 5'-flanking region of the rV2R gene. The sequence up to 1,092 bp upstream from the transcriptional initiation site of the rV2R was identified by Mandon et al. (17) (Fig. 1). We confirmed the whole sequence using our PCR product. This region contains multiple motif sequences that may play an important role in transcriptional regulation. This gene has no TATA box, but it does have a CAAT box at –82 bp from the transcription initiation site. There are seven binding sites for PEA3 and one each for AP1, AP3, and SP1. As is the case for other genes, these consensus sequences are located close to the transcription initiation site.


Figure 1
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  		Fig. 1. Nucleotide sequence of the 5'-flanking region of the rat vasopressin V2 receptor (rV2R) gene. Inverse PCR was used to clone the 5'-flanking region. Possible promoter regulatory elements are shown in boxes.

 
Basal promoter activity of the rV2R is shown in Fig. 2. Deletion of the 1,092-bp 5'-flanking region to 345 bp significantly increased promoter activity: 25- to 30-fold greater in the 345-bp promoter segment than in control pGL3-basic. However, deletion of the segment further downstream decreased promoter activity. By deletion to 46 bp, the promoter activity was completely abolished.


Figure 2
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  		Fig. 2. Promoter activity of the rV2R in LLC-PK1 cells with deletion analysis. LLC-PK1 cells were cotransfected with pGL3-rV2R promoter constructs and pRL-TK constructs. Luciferase activity was measured 42 h after transfection. The 345-bp 5'-flanking region of rV2R showed the highest promoter activity among deletion segments. Values are means ± SE (n = 6). *P < 0.05 vs. pGL3 basic.

 
Effect of AVP on intracellular Ca2+ concentration in LLC-PK1 cells transfected with rV1aR. RT-PCR was used to confirm mRNA expression of the rV1aR in LLC-PK1 cells transfected with rV1aR by the Flp-In System. cDNA (2 µg) obtained by reverse transcription from cell lysate was amplified for 35 cycles by PCR. Agarose gel electrophoresis showed the PCR products at the appropriate molecular weight, as well as the rV1aR-positive control (Fig. 3). These findings indicate that the rV1aR is expressed in LLC-PK1/rV1aR cells at the mRNA level. To demonstrate the function of the rV1aR in this cell line, we investigated the accumulation of intracellular Ca2+ by measuring the change of fluo 3 fluorescence intensity (Fig. 4). In LLC-PK1/rV1aR cells, treatment with 10–9 M AVP increased fluorescence intensity, indicating an increase of the intracellular Ca2+ concentration. Maximal intensity was achieved <30 s after addition of AVP (Fig. 4A). AVP dose dependently increased intracellular Ca2+ (Fig. 4B). These results indicate that rV1aR protein is present on the cell membrane and is functionally active in LLC-PK1/rV1aR cells. In contrast, in non-rV1aR-transfected LLC-PK1 (LLC-PK1/FRT) cells, only a high dose (10–7 M) AVP increased Ca2+ concentration.


Figure 3
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  		Fig. 3. Agarose gel electrophoresis for detection of rat vasopressin V1a receptor (rV1R) mRNA in LLC-PK1/rV1aR cells. The 1.5-kbp bands indicate rV1aR. cDNA (2 µg) obtained by RT with cell as template was amplified for 35 cycles by PCR. Lane 2, amplification by cDNA in LLC-PK1/rV1aR cells (transfected with rV1aR using Flp-In System); lane 3, negative control amplified by purified mRNA in LLC-PK1/rV1aR cells; lane 4, positive control amplified by pcDNA5 plasmid with rV1aR as template; lane 5, negative control amplified by cDNA in LLC-PK1/FRT cells; lane 6, negative control amplified by purified mRNA in LLC-PK1/FRT cells; lanes 1 and 7, molecular markers.

 

Figure 4
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  		Fig. 4. Alteration of relative fluorescence intensity for accumulation of intracellular Ca2+. Fluorescence intensity for fluo 3, indicating accumulation of intracellular Ca2+, was measured in LLC-PK1/FRT and LLC-PK1/rV1aR cells at 1-s intervals for 5 min after treatment with AVP or DMSO (vehicle). A: alteration of fluorescence intensity relative to mean basic intensity (before AVP stimulation). AVP at 10–9 M increased intracellular Ca2+ in LLC-PK1/rV1aR, but not LLC-PK1/FRT, cells. B: dose-response effect of AVP on intracellular Ca2+ in LLC-PK1/rV1aR and LLC-PK1/FRT cells shown as mean fluorescence intensity 30 s after treatment with AVP or vehicle. AVP dose dependently increased intracellular Ca2+ in LLC-PK1/rV1aR cells. Values are means ± SE (n = 6). *P < 0.05 vs. vehicle. {dagger}P < 0.05 vs. LLC-PK1/FRT.

 
Downregulation of rV2R promoter activity via rV1aR stimulation. Deletion study of the promoter element demonstrated the greatest activity at a 345-bp segment of the rV2R promoter (Fig. 2). Therefore, we transfected the 345-bp 5'-flanking region of the rV2R in LLC-PK1/rV1aR cells to investigate the interaction between V2R promoter activity and V1aR stimulation. First, we examined the time course of the effect of 10–8 M AVP on rV2R promoter activity (Fig. 5A). AVP transiently increased rV2R promoter activity 2 h after AVP treatment. Sustained suppression of rV2R promoter activity was observed 12–24 h after AVP treatment. Although rV2R promoter activity was transiently increased 2–4 h after AVP treatment in LLC-PK1/FRT cells, a significant decrease of rV2R promoter activity was not observed at 12–24 h (Fig. 5B). In addition, rV2R promoter activity was decreased over a wide range of AVP concentrations in LLC-PK1/rV1aR cells (Fig. 6). The decrease of rV2R promoter activity by 10–10 M AVP was inhibited by a V1aR-specific antagonist (OPC-21268) in LLC-PK1/rV1aR cells (Fig. 7A). This restoration of rV2R promoter activity by a V1aR-specific antagonist was more distinct when the cells were treated with high-dose (10–8 M) AVP (Fig. 7C). In contrast, a V2R-specific antagonist (OPC-31260) did not restore V2R promoter activity. A PKC inhibitor (RO 31-8220), as well as the V1aR-specific antagonist, inhibited the decrease of rV2R promoter activity. Although AVP slightly decreased rV2R promoter activity in LLC-PK1/FRT cells (Fig. 7B), the change was not statistically significant, and neither V1aR nor V2R antagonists restored V2R promoter activity.


Figure 5
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  		Fig. 5. Time course of effect of AVP on rV2R promoter activity in LLC-PK1/rV1aR and LLC-PK1/FRT cells. Each cell was cotransfected with the 5'-flanking region (–345 to +57 bp) of rV2R. At 18 h after transfection, cells were incubated with 10–8 M AVP. Luciferase assay was performed 0, 2, 4, 12, and 24 h after AVP stimulation. A: luciferase activity in LLC-PK1/rV1aR cells. AVP transiently increased rV2R promoter activity 2 h after AVP treatment. Sustained decrease of activity was observed at 12–24 h. B: luciferase activity in LLC-PK1/FRT cells. AVP transiently increased rV2R promoter activity. Values are means ± SE (n = 6). *P < 0.05 vs. vehicle.

 

Figure 6
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  		Fig. 6. Dose-dependent effect of AVP on rV2R promoter activity in LLC-PK1/rV1aR cells. LLC-PK1/rV1aR cells were cotransfected with the 5'-flanking region (–345 to +57 bp) of rV2R and incubated with 10–11–10–7 M AVP for 24 h. rV2R promoter activity was decreased over a wide range of AVP concentrations. Values are means ± SE (n = 6). *P < 0.05 vs. vehicle.

 

Figure 7
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  		Fig. 7. Effect of AVP on rV2R promoter activity in LLC-PK1/rV1aR and LLC-PK1/FRT cells. Each cell was cotransfected with the 5'-flanking region (–345 to +57 bp) of rV2R and incubated with AVP, antagonists, and PKC inhibitor for 12 or 24 h. Final concentration of each solution is as follows: 10–10 or 10–8 M AVP, 10–6 M OPC-21268 (a V1aR-specific antagonist), 10–7 M OPC-31260 (a V2R-specific antagonist), and 10–7 M RO 31-8220 (a PKC inhibitor). A: luciferase activity in LLC-PK1/rV1aR cells treated with 10–10 M AVP for 12 h. B: luciferase activity in LLC-PK1/FRT cells treated with 10–10 M AVP for 12 h. AVP significantly decreased rV2R promoter activity in LLC-PK1/rV1aR, but not LLC-PK1/FRT, cells. AVP effect was abolished by OPC-21268 and RO 31-8220, but not by OPC-31260. C: luciferase activity in LLC-PK1/rV1aR cells treated with 10–8 M AVP for 24 h. Note distinct restoration of rV2R promoter activity by OPC-21268. Values are means ± SE (n = 6). *P < 0.05 vs. AVP.

 
Identification of PKC and PKA response element in the rV2R promoter region. To analyze signal transduction, we treated LLC-PK1 cells with PMA, a PKC activator. First, we used the 1,092-bp 5'-flanking region of the rV2R to examine the time course of the effect of PMA on rV2R promoter activity (Fig. 8A). rV2R promoter activity was decreased 6–12 h after treatment with PMA. rV2R promoter activity was suppressed by PMA in a dose-dependent manner (Fig. 8B). On the basis of this finding, we performed additional experiments on the cells 12 h after they were treated with 10–6 M PMA. In contrast to PMA, 4{alpha}-PMA, a PMA analog, did not change rV2R promoter activity (Fig. 9). rV2R promoter activity was downregulated by PMA, even in short segments (Fig. 10). However, this downregulation was abolished by deletion of the promoter region to 46 bp.


Figure 8
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  		Fig. 8. Dose- and time-dependent effect of PMA on rV2R promoter activity. A: time-dependent effect of PMA and 8-(4-chlorophenylthio)-cAMP (cpt-cAMP) on rV2R promoter activity in LLC-PK1 cells. LLC-PK1 cells were cotransfected with the 5'-flanking region (–1,092 to +57 bp) of rV2R. At 18 h after transfection with promoter construct, cells were exposed to 10–6 M PMA or 4 x 10–4 M cpt-cAMP. Luciferase activity was assayed 0, 2, 4, 6, and 12 h after PMA or cpt-cAMP stimulation. rV2R promoter activity was increased 2–12 h after cpt-cAMP and decreased 6–12 h after PMA. B: dose-dependent effect of PMA on rV2R promoter activity in LLC-PK1 cells. LLC-PK1 cells were cotransfected with the 5'-flanking region (–1,092 to +57 bp) of rV2R and exposed to 10–8–10–6 M PMA for 12 h. PMA dose dependently decreased rV2R promoter activity. Values are means ± SE (n = 6). *P < 0.05 vs. vehicle.

 

Figure 9
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  		Fig. 9. Effect of PMA, cpt-cAMP, and 4{alpha}-PMA on rV2R promoter activity in LLC-PK1 cells. LLC-PK1 cells were cotransfected with the 5'-flanking region (–1,092 to +57 bp) of rV2R and exposed to DMSO, 10–6 M PMA, 4 x 10–4 M cpt-cAMP, or 10–6 M 4{alpha}-PMA for 12 h. rV2R promoter activity was significantly decreased by PMA and decreased by cpt-cAMP. 4{alpha}-PMA did not change rV2R promoter activity. Values are means ± SE (n = 6). *P < 0.05 vs. vehicle.

 

Figure 10
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  		Fig. 10. Deletion series of rV2R promoter element and promoter activity. LLC-PK1 cells were cotransfected with a deletion series of the 5'-flanking region from –1,092 to –46 bp of rV2R and exposed to 10–6 M PMA for 12 h. PMA suppressed rV2R promoter activity, even in short segments. Effect of PMA was abolished in the deletion mutant lacking CAAT and SP1 elements (–345, –165/–46, and +57). Values are means ± SE (n = 6). *P < 0.05.

 
To examine the role of the PKA pathway in V2R signal transduction, we added cpt-cAMP to LLC-PK1 cells. rV2R promoter activity was significantly increased 2–12 h after treatment with cpt-cAMP (Fig. 8A). These opposite effects caused by PMA and cpt-cAMP were abolished by deletion at –164 to –47 bp on the 345-bp 5'-flanking region of rV2R (Fig. 11B). This mutant lacks consensus sequences of CAAT and Sp1 from the original 5'-flanking region.


Figure 11
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  		Fig. 11. Effect of PMA and cpt-cAMP on rV2R promoter activity of the deletion mutant in LLC-PK1 cells. LLC-PK1 cells were cotransfected with the deletion mutant lacking CAAT and SP1 elements (from –164 to –47 bp) and exposed to 10–6 M PMA or 4 x 10–4 M cpt-cAMP or DMSO for 12 h. A: effect of PMA and cpt-cAMP on the 5'-flanking region (–345 to +57 bp). B: effect of PMA and cpt-cAMP on the deletion mutant (–345, –165/–46, and +57). Opposite effects of PMA and cpt-cAMP on rV2R promoter activity were abolished on the deletion mutant. Values are means ± SE (n = 6). *P < 0.05 vs. vehicle.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In the present study, we used an LLC-PK1 cell line to investigate the interaction between V2R promoter activity and the V1aR pathway. The LLC-PK1 cell line, which is derived from the proximal tubule of porcine kidney, has been widely used to investigate the kinetics of the V2R and AQP2 because of its response to vasopressin stimulation (12, 13). Moreover, the promoter analysis, trafficking, and recycling of AQP2 have been investigated in LLC-PK1 cells (5, 9, 13). Preliminarily, we also measured rV2R promoter activity in Madin-Darby canine kidney, COS-7, and HEK-293 cells, but the greatest increase of V2R promoter activity was observed in LLC-PK1 cells. On the basis of these reports and our preliminarily data, we considered the LLC-PK1 cell line to be suitable for our purpose.

In previous studies, stimulation of LLC-PK1 cells with 10–10–10–7 M AVP increased cAMP, indicating expression of the V2R in LLC-PK1 cells (11). In contrast, the increase of intracellular Ca2+ in response to V1aR stimulation required the high dose (10–7–5 x 10–6 M) of AVP in LLC-PK1 cells (3, 7, 27). Our present results also showed an increase of intracellular Ca2+ in LLC-PK1/FRT cells in response to only high-dose AVP (Fig. 4B). It is considered that LLC-PK1 cells might express endogenous V1aR; however, this functional role could be small at physiological AVP concentration.

In the present study, using the Flp-In System, we have established an LLC-PK1 cell line stably expressing the rV1aR. This cell line expresses rV1aR mRNA and shows an increase of intracellular Ca2+ in response to 10–10–10–7 M AVP, suggesting functional expression of the rV1aR (Figs. 3 and 4). Since the host cell line (LLC-PK1/FRT) can be transfected with any other genes of interest in FRT sites, this cell line could be useful for further study. It should be possible to compare the kinetics of plural genes or proteins in isogenic cell conditions. For example, it might be interesting to analyze a functional interaction of the V1aR with the oxytocin receptor, which has been demonstrated to cross-react with vasopressin (6, 10, 32, 33).

AVP caused a sustained decrease of rV2R promoter activity in LLC-PK1/rV1aR cells (Fig. 5). In addition, suppression of the AVP-induced V2R was inhibited by a V1aR-specific antagonist and a PKC inhibitor (Fig. 7, A and C). These findings strongly indicate that V2R promoter activity could be downregulated via the V1aR-PKC pathway. It is known that the physiological concentration of urinary AVP usually ranges from 10–11 to 10–9 M (18). Our results were obtained within this range of AVP concentrations, indicating that V1aR stimulation affects V2R promoter activity under physiological conditions.

However, some issues remain unresolved. In contrast to the dose-dependent increase of intracellular Ca2+ by AVP, rV2R promoter activity showed the two-phased decrease over a wide range of AVP concentrations (Fig. 6). In addition, the V1aR antagonist and the PKC inhibitor did not completely restore V2R promoter activity. Perhaps a pathway other than the rV1aR-PKC pathway decreases V2R promoter activity in LLC-PK1 cells.

rV2R promoter activity was increased in the early phase by addition of AVP to LLC-PK1/rV1aR and LLC-PK1/FRT cells (Fig. 5). It is suggested that AVP increased rV2R promoter activity via the V2R pathway. In the late phase, rV2R promoter activity was slightly decreased, even in LLC-PK1/FRT cells (Fig. 7B). However, this slight decrease in LLC-PK1/FRT cells was also observed after treatment with V1aR antagonist, V2R antagonist, and PKC inhibitor (not shown) and was not statistically significant.

Wong and Tsui (35) showed that V2R mRNA is upregulated by PKA stimulation and downregulated by PKC stimulation in the rat inner medullary collecting duct. As shown in Fig. 9, rV2R promoter activity was decreased by PMA and increased by cpt-cAMP. These results indicate that rV2R promoter activity is modulated by PKC and PKA pathways.

The time-dependent effects of AVP, PMA, and cpt-cAMP on rV2R promoter activity demonstrate that differences in time course between V1aR and V2R stimulation affect rV2R promoter activity. rV2R promoter activity was increased in the early phase and decreased in the late phase by AVP (Fig. 5A). The increase of rV2R promoter activity by cpt-cAMP was observed 2 h after AVP treatment, whereas the decrease of rV2R promoter activity appeared 6 h after addition of AVP (Fig. 8A). Although intracellular Ca2+ was rapidly increased to maximal concentration via V1aR stimulation (Fig. 4), the effect of cAMP on V2R promoter activity via V2R stimulation could be more potent and could occur before the effect of V1aR stimulation. It is suggested that the early AVP-induced increase of rV2R promoter activity is caused by V2R stimulation and the delayed AVP-induced decrease is mediated by V1aR stimulation. As discussed in previous studies, a 10- to 100-fold higher dose of AVP seems to be required for V1aR than for V2R activation (2, 26). The response of the V1aR to AVP might be slower than the response of the V2R.

By the deletion mutant study of the rV2R promoter, consensus sequences of CAAT and SP1 were speculated to be the PKC-responsive element. Interestingly, our results showed that the same element was also crucial for cAMP-induced V2R promoter activity. It is suggested that the opposite effects of PKC and PKA on regulation could affect the same consensus sequences, such as CAAT and/or SP1, in this gene. Several reports have demonstrated that CAAT and/or SP1 are related to cAMP-inducible promoter activity (8, 9, 14). However, a direct correlation between these consensus sequences and PKC has not been reported. An indirect mechanism for suppression of V2R expression via the PKC pathway at the transcriptional level could be supposed.

Klingler et al. (15) demonstrated that long-term (5 h) exposure to PMA decreased cAMP accumulation in Chinese hamster ovary cells stably transfected with the V1aR and V2R, suggesting an interaction between PMA stimulation and cAMP accumulation. Previous reports have indicated a connection between PKC and cAMP in cultured collecting tubular cells (4, 29). These studies have suggested that PKC caused phosphorylation of adenylyl cyclase or, alternatively, phosphorylation of Gi protein. We confirmed the PMA-induced decrease of cAMP accumulation in LLC-PK1 cells (not shown). The interaction between PMA-induced PKC and AVP-induced cAMP could support our hypothesis of cross talk between the V1aR and V2R signaling pathways.

It is well known that expression of V2R mRNA and protein is decreased when plasma AVP increases in vivo (31, 36). We have demonstrated that inhibition of V1aR increased V2R mRNA in the rat outer medullary collecting duct (28). However, downregulation of V2R expression has not been investigated in detail. Our present study could support this downregulation of the V2R at the transcriptional level.

Our results can be summarized as follows. 1) Expression of the V2R is downregulated via the V1aR pathway and upregulated via the V2R pathway. 2) Expression of the V2R is downregulated by the PMA-induced PKC signaling pathway and upregulated by the cAMP-PKA signaling pathway. These opposite effects of PKC and PKA stimulation could be exerted on the same consensus sequences in the V2R promoter region, such as CAAT and SP1. 3) Intracellular cross talk between the V1aR and V2R pathways in the collecting duct cells appears to strictly and sensitively maintain body fluid homeostasis.


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


   ACKNOWLEDGMENTS
 
We thank Dr. Tetsushi Kagawa for valuable suggestions regarding the luciferase assay and Takanobu Matsuzaki and Noriko Teramoto for technical assistance. We are grateful for support by the staff at the Gene Technology Center at Kumamoto University. We also thank Dr. Mark A. Knepper for critical reading of the manuscript.


   FOOTNOTES
 

Address for reprint requests and other correspondence: Y. Izumi, 1-1-1 Honjo, Kumamoto, 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
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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