Acute regulation of mUT-A3 urea transporter expressed in a MDCK cell line - 《American Journal of Physiology》美国生理学杂志干细胞之家



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发表于 2009-4-22 09:42 |只看该作者 |倒序浏览 |打印

作者：Gavin S. Stewart, Sarah L. King, Elizabeth A. Potter, and Craig P. Smith作者单位：Faculty of Life Sciences, Core Technology Facility, The University of Manchester, Manchester, United Kingdom 9 f; v* R; m0 q, Z



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      【摘要】
   Renal facilitative urea transporters play a vital role in the urinary concentrating mechanism. UT-A3 is a phloretin-sensitive urea transporter that in the mouse is expressed on the basolateral membrane of renal inner medullary collecting duct (IMCD) cells. In this study, we engineered a Madin-Darby canine kidney (MDCK) I cell line that stably expresses mouse UT-A3 (MDCK-mUT-A3). Immunoblotting using the UT-A-targeted antibody ML446 detected a 40-kDa signal in MDCK-mUT-A3 protein that corresponds to mUT-A3. Using cultured epithelial monolayers, radioactive 14 C-urea flux experiments determined that basolateral urea transport was no different between MDCK-mUT-A3 and control MDCK-FLZ cells under basal conditions [not significant (NS), ANOVA]. However, exposure to arginine vasopressin (AVP) significantly stimulated basolateral urea flux in MDCK-mUT-A3 monolayers ( P < 0.05, ANOVA), while it had no effect in control MDCK-FLZ monolayers (NS, ANOVA). The AVP-stimulated basolateral urea transport in MDCK-mUT-A3 was inhibited by 1,3 dimethyl urea ( P < 0.05, ANOVA) or phloretin ( P < 0.05, ANOVA), both known inhibitors of facilitative urea transporters. MDCK-mUT-A3 basolateral urea flux was also stimulated by increasing intracellular levels of cAMP, via forskolin ( P < 0.05, ANOVA), or intracellular calcium, via ATP ( P < 0.05, ANOVA). Finally, 1-h preincubation with a specific PKA inhibitor, H89, significantly inhibited the increase in urea transport produced by AVP ( P < 0.05, ANOVA). In conclusion, we have produced the first renal cell line to stably express the mUT-A3 urea transporter. Our results indicate that mUT-A3 is acutely regulated by AVP, via a PKA-dependent pathway. These findings have important implications for the regulation of urea transport in the renal IMCD and the urinary concentrating mechanism.
      【关键词】 arginine vasopressin urine concentration protein kinase A cAMP intracellular calcium
   THE REGULATED PASSIVE MOVEMENT OF UREA across biological membranes occurs via facilitative urea transporters. In mammals, these proteins are derived from two distinct genes, namely, UT-A ( Slc14a2 ) and UT-B ( Slc14a1 ) ( 11 ). There are six major isoforms derived from the UT-A gene by alternative splicing (UT-A1 to UT-A6) compared with two major UT-B isoforms (UT-B1 and UT-B2) ( 14, 16, 19 ). UT-A1, UT-A2, UT-A3, and UT-B1 are the major renal isoforms, and it has recently been shown that these proteins play a vital role in the urinary concentrating mechanism. Indeed, mouse knockout models have shown that both UT-A1 and UT-A3 ( 3 ) and UT-B ( 1, 23 ) are important in maintaining the high inner medullary solute concentration required for the production of concentrated urine.

The largest renal UT-A isoform is UT-A1, consisting of 930 amino acids in the mouse (929 in the rat), while UT-A2 consists of the COOH-terminal 397 amino acids of UT-A1, and UT-A3 consists of the NH 2 -terminal 461 amino acids (460 in rat). UT-A1 and UT-A3 are located in the inner medullary collecting duct (IMCD), whereas UT-A2 has been localized to type 1 and type 3 thin descending limbs ( 2, 12, 21 ). In terms of subcellular localization, UT-A1 has been located to the apical membrane of IMCD cells ( 9 ), while UT-A2 is found on both apical and basolateral membranes of the thin descending limbs ( 2 ). In contrast, UT-A3 has been localized to the basolateral membrane of IMCD cells in both the mouse ( 18 ) and rat ( 8, 13 ), and also apically in the rat ( 20 ).
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UT-A1 and UT-A3 transporters localized in the IMCD regulate urea reabsorption into the interstitium, under the control of arginine vasopressin (AVP) ( 3 ). In rat perfused, isolated IMCD, AVP has been shown to increase intracellular levels of cAMP and calcium, as well as stimulating trans -epithelial phloretin-sensitive urea transport ( 17, 22 ). Recent studies of rat UT-A1 ( 4 ) and mouse UT-A2 ( 10 ) expressed in Madin-Darby canine kidney (MDCK) type I cell lines showed that both isoforms can be acutely regulated by AVP, probably via a cAMP-dependent pathway. Interestingly, while the cAMP stimulation of UT-A1 is known to be sensitive to a PKA inhibitor, N -[2-( p -bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide.2HCl (H89) ( 24 ), our recent study suggests that cAMP activation of mUT-A2 is in fact PKA independent ( 10 ).
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When expressed in HEK-293 cells or Xenopus laevis oocytes, UT-A3 is activated by cAMP ( 2, 7 ), although nothing is yet known about the mechanism of this activation. In this study, we have engineered an MDCK type I cell line that stably expresses mouse UT-A3 protein (i.e., MDCK-mUT-A3) and used this to show that mUT-A3 is sensitive to AVP. Furthermore, we have shown that mUT-A3 is also sensitive to increases in cAMP or calcium and that H89 blocks the AVP response but not the calcium response.# P8 x3 T) U3 Z+ t

METHODS, `9 y1 z% @. r8 G$ q, P7 d* H
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Establishment of MDCK-mUT-A3 cells. MDCK cells stably transfected with pFRT/ lac Zeo (Invitrogen; termed "MDCK-FLZ" cells) and MDCK-FLZ cells stably expressing rat UT-A1 (termed MDCK-rUT-A1) were the kind gift of the late Dr. Robert Gunn (Emory University, Atlanta, GA). Cells were cultured as previously described ( 4 ). MDCK-FLZ cells stably expressing mouse mUT-A3 (termed MDCK-mUT-A3) were derived using the Flp-In system (Invitrogen) as follows. Sequence encoding a c- myc tag was incorporated into the open reading frame of mUT-A3 cDNA immediately 5' to the stop codon by PCR/plasmid subcloning. This incorporated sequence encoded for 10 amino acids (EQKLISEEDL), a stop codon, and a Spe 1 restriction site. To verify correct incorporation, restriction digests and nucleotide sequencing were performed.
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The UT-A3 c- myc construct was subcloned into pcDNA5/FRT (Invitrogen) and cotransfected with pOG44 into the MDCK-FLZ cells using an Amaxa nucleofector (Amaxa, Cologne, Germany) according to the manufacturer's instructions. Cells were selected with 300 µg/ml hygromycin after 24 h, and individual clones were isolated after 2 wk. Three clonal cell lines were isolated (termed EP15, EP16, and EP17), and protein expression was assessed using semiquantitative immunoblotting (see below for details).# K( A; f7 V+ X+ `
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Epithelial monolayers were cultured on semipermeable polyester supports (0.4-µm pore, Transwell, Corning) as described elsewhere ( 4 ). Monolayers were fed on day 2 following seeding, then daily until use. The resistance of each monolayer was measured using an EVOM resistance meter (World Precision Instruments). After confluence, membranes developed a transmembrane resistance 1.0 k. Only membranes that showed sequential increases in transmembrane resistance to 1.0 k were used for flux experiments.

Immunoblotting. Cells from various MDCK cell lines (MDCK-FLZ, MDCK-rUT-A1, MDCK-mUT-A2, MDCK-mUT-A3) were harvested separately using 0.05% trypsin-EDTA (GIBCO) and washed twice with PBS. Cell protein was homogenized with a handheld dounce homogenizer, using a standard homogenization buffer (pH 7.6) containing 12 mM HEPES, 300 mM mannitol, and peptidase inhibitors added immediately before use (1 µg/ml pepstatin, 2 µl/ml leupeptin, and 1 µg/ml phenylmethylsulfonyl fluoride, Sigma). Homogenates were initially centrifuged at 2,500 g for 15 min at 4°C, and then the resulting supernatant was centrifuged at 200,000 g for another 30 min at 4°C. These plasma membrane-enriched pellets were retained and resuspended in homogenization buffer. Immunoblotting experiments were then performed using the protocol previously described ( 18 ). Briefly, SDS-PAGE was performed on minigels of 10% polyacrylamide by loading 20 µg protein/lane. After transfer to nitrocellulose membranes, immunoblots were probed for 16 h at 4°C with an affinity-purified antiserum, ML446, previously shown to detect UT-A1 and UT-A3 ( 18 ). Immunoblots were then washed and probed with 1:5,000 dilution of goat anti-rabbit horseradish peroxidase (HRP)-linked secondary antiserum (Dako) for 1 h at room temperature. After further washing, detection of protein was performed using ECL Western Blotting Detection Reagents and ECL film (Amersham Pharmacia).

Flux measurements. Trans -epithelial urea flux experiments were performed as previously described ( 10 ) at 37 ± 0.2°C, in an apical-to-basolateral direction, using [ 14 C]urea (0.8 µCi/apical well) as the radiolabeled tracer. The basolateral solution was collected at 3-min intervals.

"Unidirectional basolateral uptake" urea flux experiments were performed at 37 ± 0.2°C, using [ 14 C]urea (1.0 µCi/basolateral well) as the radiolabeled tracer. Transwells were initially incubated in HBSS media (GIBCO) containing 5 mM urea, 12 mM HEPES, and the specific test compounds. After the relevant incubation time period, they were then placed in HBSS basolateral solution containing [ 14 C]urea for between 0.5 and 5 min, with or without test compounds present. This was followed by 10 s in a basolateral solution consisting of standard 1 x PBS containing 10 mM cold urea. Transwells were then removed from the basolateral solution completely, and the apical HBSS solution was replaced with 500 µl 5% SDS solution (see Fig. 3 ). Cells were allowed to dissolve for 45 min on a horizontal shaker. SDS-cell suspensions were then transferred into scintillation vials, 3 ml of Ecoscint A (National Diagnostics) scintillation fluid were added to each vial, and the radioactivity was counted using a 1900 TR liquid scintillation analyzer (Packard, Canberra, Australia).- y8 J9 M9 r9 p
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Fig. 1. A : immunoblot of stable Madin-Darby canine kidney (MDCK) cell lines probed with ML446 antibody (20 µg of protein/lane). The expected 85-kDa signal was detected in MDCK-rUT-A1 cells, while a 40-kDa doublet signal was detected in MDCK-mUT-A3 cells. See text for definitions. As predicted, no signals were present in control MDCK-FLZ or MDCK-mUT-A2 cells. B : immunoblot comparing three different MDCK-mUT-A3 cell lines (EP15-17), probed with ML446 antibody (20 µg of protein/lane), showing EP16 expresses the highest level of 40-kDa UT-A3 protein.: Z2 h9 z+ C! S! x1 U

Fig. 2. AVP stimulates urea transport mediated by wild-type mUT-A3 expressed in Xenopus laevis oocytes. Radioactive 14 C-labeled urea uptakes into oocytes were measured over a 90-s time period. * P
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Fig. 3. A : summary of trans -epithelial 14 C-labeled urea flux across monolayers of stable MDCK cell lines (all n = 4). B : addition of 10 -6 M AVP has no effect on trans -epithelial urea flux in MDCK-mUT-A3 monolayers. *** P
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All test compounds were of certified grade and were made up either in sterile dH 2 O or anhydrous DMSO. Stocks solutions were 100 µM AVP in dH 2 O; 50 µM forskolin in DMSO; 50 mM 8-bromo-cAMP in dH 2 O; 1 mM ATP in dH 2 O; 10 mM H89 in DMSO; 500 mM 1,3 dimethyl urea (DMU) in dH 2 O; and 100 mM phloretin in DMSO. Stocks were then diluted in Hank's balanced salt solution (GIBCO) containing 5 mM urea and 12 mM HEPES before use. Control solutions were prepared by adding the corresponding solvent minus the test compound.0 j& k9 P  g3 s) _
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X. laevis oocyte expression experiments. These were performed as previously described ( 15 ). Briefly, a plasmid containing mUT-A3 was linearized, and cRNA was prepared using the T7 mMessage mMachine (Ambion). Defolliculated oocytes were injected with either 0.6 ng mUT-A3 cRNA or deionized water and incubated for 3 days at 18°C. To investigate the effect of vasopressin, [ 14 C]urea uptakes were measured with or without 1-h preincubation in 10 -6 M AVP.8 h1 ^4 w* s% w0 s) c6 W0 k5 G8 Y+ Z
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Statistical analysis. All data values are shown as means ± SE, with n representing the number. For statistical analysis of all urea flux and oocyte expression experiments, one-way ANOVA was used. If the ANOVA indicated a difference, treatment comparison between groups with the Student-Newman-Keuls post hoc test was performed. Groups were deemed statistically significant if P

RESULTS2 S/ U# u9 Y" Z% n# h1 _
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Semiquantitative immunoblotting of MDCK-mUT-A3 cell lines. Following transfection of MDCK-FLZ cells with the pcDNA5/FRT/mUT-A3 construct, a hygromycin-resistant clone, termed EP16, was selected and expanded. Using the previously characterized UT-A1/UT-A3-selective antiserum ML446 ( 18 ), immunoblotting analysis of the EP16 mUT-A3 cell line, a MDCK-rUT-A1 cell line ( 4 ), a MDCK-mUT-A2 cell line ( 10 ), and a MDCK-FLZ control cell line was performed. A signal of 85 kDa was detected in the MDCK-rUT-A1 cell line, corresponding to unglycosylated rUT-A1, while no signals were detected for the MDCK-FLZ or MDCK-mUT-A2 cell lines (see Fig. 1 A ). In contrast, a doublet signal centered at 40 kDa was detected for MDCK-mUT-A3, similar to that previously reported for unglycosylated mUT-A3 protein ( 18 ). Immunoblotting of EP16 and two other UT-A3 cell lines (EP15 and EP17) confirmed the presence of a 40-kDa signal in all MDCK-mUT-A3 cell lines and showed that EP16 displayed the highest level of protein expression (see Fig. 1 B ). The EP16 cell line was subsequently utilized for all MDCK-mUT-A3 urea flux experiments.8 S- o" W4 c( @' V

X. laevis oocyte expression experiments. To test the hypothesis that mUT-A3 function could be regulated by vasopressin, preliminary tests were performed in X. laevis oocytes. Under control conditions, oocytes injected with 0.6 ng of mUT-A3 cRNA displayed a twofold increase in urea uptake compared with water-injected controls (27 ± 3 pmol·oocyte -1 ·90 s -1, n = 7, cf. 13 ± 3 pmol·oocyte -1 ·90 s -1, n = 8, P

Functional analysis of MDCK-mUT-A3 monolayers: trans-epithelial flux. Trans -epithelial urea flux rates were measured for confluent monolayers of different MDCK cell lines grown separately on Transwell membranes, as previously described by Potter et al. ( 10 ). Control MDCK-FLZ monolayers had a trans -epithelial urea flux of 0.4 ± 0.1 nmol·min -1 ·cm -2 ( n = 4). MDCK-mUT-A2 monolayers had a significantly higher urea flux rate of 3.2 ± 0.2 nmol·min -1 ·cm -2 ( n = 4, P : p: g5 ]) }) c: j1 p* c
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Functional analysis of MDCK-mUT-A3 monolayers: basolateral flux. The lack of AVP-stimulated trans -epithelial urea transport in MDCK-mUT-A3 monolayers suggested that functional mUT-A3 transporters were not present on both apical and basolateral membranes. Since mUT-A3 has previously been shown to localize to the basolateral membrane ( 18 ), specific unidirectional urea flux rates across the basolateral membrane were measured for the different MDCK cell lines (see Fig. 4 ). The time course of 14 C-labeled urea basolateral uptake into MDCK-mUT-A3 monolayers was calculated by measuring the cellular urea content after uptake periods of between 0.5 and 5 min (see Fig. 5 A ). These data illustrated that the uptake of urea was reasonably linear within the first minute and had not reached a maximum; hence simpler basolateral uptake experiments could be performed using a 1-min uptake period. Under basal conditions, basolateral urea flux in MDCK-mUT-A3 cells (2.0 ± 0.2 nmol·min -1 ·cm -2, n = 16) was not significantly different to that measured in MDCK-FLZ monolayers (1.7 ± 0.2 nmol·min -1 ·cm -2, n = 16, NS, ANOVA) (see Fig. 5 B ), in contrast to that observed in MDCK-mUT-A2 cells (2.5 ± 0.1 nmol·min -1 ·cm -2, n = 20, P

Fig. 4. Schematic diagram representing the experimental protocol for unidirectional basolateral urea uptakes. A : after preincubation stage, Transwell membrane was placed in basolateral solution containing [ 14 C]urea and test compounds for between 0.5 and 5 min. B : Transwell membrane was then washed in basolateral solution of 1 x PBS plus 10 mM cold urea for 10 s. C : after basolateral and apical solutions were removed, the apical chamber was filled with 500 µl 5% SDS solution and placed on platform shaker for 45 min. The level of radioactive [ 14 C]urea in each cell suspension was then counted using a scintillation counter.* L. R! w" q) T9 f# A8 u7 }# B
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Fig. 5. A : time course of basolateral 14 C-labeled urea unidirectional flux in MDCK-mUT-A3 monolayers, using uptake periods of 0.5-5 min and measuring total cellular urea content. B : summary of basolateral 14 C-labeled urea unidirectional flux in monolayers of MDCK cell lines under basal conditions, using 1-min uptake periods. The numbers in brackets represent n. * P 1 j; ~  |$ X5 Q1 J* Z. F
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The effect of AVP on basolateral urea uptake was then investigated. Pretreatment of MDCK-mUT-A3 monolayers to 10 -8 M AVP for 1 h significantly increased basolateral urea uptake ( n = 4, P   U; Z, T' D$ w. j5 u
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Fig. 6. Comparison of the stimulation of MDCK-mUT-A3 basolateral urea flux after exposure to 10 -12, 10 -10, or 10 -6 M AVP. Urea fluxes are shown for both 10- and 60-min exposures for each AVP concentration, and all concentrations produced a significant increase in urea flux. * P

To confirm that the AVP-stimulated increase in urea transport was indeed due to regulation of mUT-A3, the effects of known inhibitors of facilitative urea transporters were investigated. Addition of DMU had no effect on basal urea transport in MDCK-mUT-A3 cells ( n = 4, NS, ANOVA). However, the increased urea transport induced by 10-min exposure to AVP was completely inhibited in the presence of 50 mM DMU ( n = 4, P
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Fig. 7. Effect of the urea analog DMU on basolateral urea flux in MDCK-mUT-A3 monolayers. 1,3 Dimethyl urea (DMU; 50 mM) had no effect on basolateral urea flux under basal conditions, but completely inhibited the significant increase observed with 10-min exposure to 10 -6 M AVP. * P
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Fig. 8. Effect of phloretin, an inhibitor of facilitative urea transporters, on basolateral urea flux in MDCK-mUT-A3 monolayers. Phloretin (500 µM) had no effect on basolateral urea flux under basal conditions but significantly inhibited the substantial increase observed with 60-min exposure to 10 -6 M AVP. * P ) q0 k2 C( l% X+ \
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It is known that exposure of the IMCD to AVP can lead to increases in intracellular levels of both cAMP and calcium, e.g., in rat isolated, perfused IMCD ( 17 ). The effects of increasing either intracellular cAMP or calcium on MDCK-mUT-A3 urea transport were therefore investigated. The application of 30 µM forskolin was used to increase intracellular cAMP and resulted in a significant increase in urea transport after 10 min ( n = 4, P
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Fig. 9. A : effect of increasing intracellular cAMP levels with forskolin on basolateral urea flux in MDCK-mUT-A3 monolayers. The addition of 30 µM forskolin for 10 min significantly stimulated urea flux. B : effect of increasing intracellular calcium levels with ATP on basolateral urea flux in MDCK-mUT-A3 monolayers. The addition of 1 mM ATP for 10 min significantly stimulated urea flux. * P $ O5 @' _9 o  A7 G* P

Since PKA has previously been indicated to play a role in vasopressin regulation of rat UT-A1 ( 24 ), the effect of a specific PKA inhibitor, H89, was investigated. Interestingly, 1-h preincubation with 10 µM H89 completely prevented the increase in urea transport observed with 10-min exposure to AVP ( n = 4, P # v. y" U) x: S

Fig. 10. A : effect of H89, an inhibitor of PKA, on AVP-stimulated basolateral urea flux in MDCK-mUT-A3 monolayers. Although preincubation for 1 h with 10 µM H89 had no effect on basolateral urea flux under basal conditions, it completely inhibited the significant increase observed with 10-min exposure to 10 -6 M AVP. B : in contrast, H89 had no effect on the stimulation of urea transport observed with 10-min exposure to 1 mM ATP, after it again had no effect under basal conditions. * P 9 I. x9 C. W2 B( b9 p  u

DISCUSSION. _2 T/ x3 M% k" K; Q( e
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The regulation of renal UT-A urea transporters by the central antidiuretic hormone vasopressin is vital to the urinary concentrating mechanism and hence overall water balance. There are three major renal UT-A isoforms involved in this process: UT-A1, UT-A2, and UT-A3. Previous studies utilizing MDCK cells stably expressing rUT-A1 ( 4 ) and mUT-A2 ( 10 ) have shown both of these isoforms are acutely regulated by AVP. In the present study, we have engineered an MDCK type I cell line that expresses mUT-A3 and investigated its functional properties, including acute AVP regulation.
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Immunoblotting of clonally selected mUT-A3-transfected MDCK cells confirmed that all three of the cell lines (EP15-17) expressed significant amounts of mUT-A3 protein. The 40-kDa signal detected by the ML446 antibody in MDCK-mUT-A3 cells corresponds to the size of unglycosylated mUT-A3 previously reported in mouse IMCD tissue, where a 45- to 65-kDa mUT-A3 signal was deglycosylated to 40 kDa ( 18 ). Since EP16 expressed the highest level of mUT-A3 protein, this cell line was utilized for all further experiments. Importantly, these data indicated that we successfully engineered the first renal epithelial cell line to stably, heterologously express mUT-A3. 1.0 k /cm 2 ) tight epithelial monolayers that under basal nonstimulated conditions had the same trans -epithelial urea flux as untransfected MDCK-FLZ control monolayers. Preliminary data had shown that wild-type mUT-A3 expressed in X. laevis oocytes was significantly stimulated by acute exposure to AVP, but exposure to AVP had no effect on the MDCK-mUT-A3 trans -epithelial urea flux. This suggests that functional mUT-A3 transporters were not present on both apical and basolateral membranes. Since we have previously reported that mUT-A3 transiently expressed in MDCK type II cells is localized to the basolateral membrane ( 18 ), we investigated the unidirectional basolateral membrane urea permeability in MDCK-mUT-A3 monolayers, but this was again no different from control levels. This suggests that mUT-A3 protein exhibits a lack of activity under basal conditions when expressed in MDCK cells. In contrast, high basal urea transporter activity has previously been observed in MDCK-mUT-A2 cells ( 10 ).6 h3 ^8 C4 y+ Y8 l/ v
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We next investigated the effects of AVP on unidirectional basolateral urea uptake. The addition of AVP had no effect on MDCK-FLZ basolateral urea uptake whatsoever, and only a small stimulatory effect on MDCK-mUT-A2 cells. In contrast, AVP significantly increased the urea transport in MDCK-mUT-A3 cells, indicating stimulated urea transport via basolateral mUT-A3 transporters. The possibility that AVP stimulation was due to the c- myc tag present in the mUT-A3 protein can be ruled out since wild-type UT-A3 expressed in X. laevis oocytes also responded to AVP. The AVP regulation of urea transport observed in MDCK-mUT-A3 cells occurred within 5 min, initially peaked at 10 min, and was maintained for at least 60 min (see Fig. 5 D ). Importantly, similar increases were observed with both high (10 -6 M) and low (10 -12 M) doses of AVP. Since circulating concentrations of AVP in vivo are 10 -11 to 10 -12 M, this finding strongly suggests the response is physiologically relevant. It is important to note that the precise nature of AVP's action on UT-A3 urea transport remains unclear. While AVP does significantly increase initial basolateral urea flux in MDCK-mUT-A3 cells, the fact that it also increases final cellular urea content after 5 min (see Fig. 5 C ) suggests an additional effect is also occurring. There are two distinct explanations for the increase in cellular urea content: First, AVP may stimulate active urea transport in MDCK cells. However, whether MDCK cells endogenously express active urea transporters has not been determined. Second, AVP treatment may cause an increase in cell volume either by its effect on urea transporter proteins or independently of this action. Clearly, these observations provide fuel for future experiments.: x) j1 @, v3 }! ^1 D2 c

The AVP-stimulated increase in urea transport was confirmed to be mUT-A3 mediated by the fact that it was significantly inhibited by DMU and phloretin, both known inhibitors of facilitative urea transporters. These results strongly suggest that mUT-A3 localizes to the basolateral membrane in MDCK type I cells, in agreement with previous localization in MDCK type II cells, mouse IMCD ( 18 ), and rat IMCD ( 8 ). The lack of effect of both phloretin and DMU under basal conditions also confirmed that UT-A3 remained completely inactive in the absence of AVP.
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Similar to the results observed for mUT-A2 ( 10 ), increases in intracellular levels of either cAMP or calcium were able to stimulate UT-A3 urea transport. Although further investigations are required to elucidate the exact mechanism of AVP stimulation of UT-A3, it is highly likely that the response involves increases in intracellular cAMP, after the activation of basolateral V2 vasopressin receptors and involving the actions of calmodulin ( 6 ). Although increases in intracellular calcium activate mUT-A3, as was also observed with rUT-A1 and mUT-A2 ( 10 ), it has previously been shown in the rat terminal IMCD that the AVP-elicited calcium transient is not involved in the stimulation of urea transport ( 5 ). In addition, a very large dose of ATP (1 mM) is required to elicit a significant response, both for UT-A3 in the present study and previously for UT-A2 ( 10 ), where 10 µM ATP had no effect (Stewart GS and Smith CP, unpublished observations). This finding suggests that while urea transporters can be stimulated by large increases in calcium, their acute regulation does not primarily involve whole-scale changes in intracellular calcium levels. However, it must also be considered that calmodulin, a calcium binding protein, is required for vasopressin stimulation of adenylate cyclase-dependent cAMP production in the IMCD ( 6 )., Y7 g8 z" h$ I# F( x( |
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Importantly, acute AVP stimulation of mUT-A3 urea transport was shown to be PKA dependent, since it was inhibited by H89, a PKA inhibitor. In this manner, AVP regulation of mUT-A3 appears similar to that of rUT-A1 ( 24 ), rather than that of mUT-A2 ( 10 ). This also suggests that the PKA-responsive element in UT-A1 regulated by AVP lies within the 461 NH 2 -terminal amino acids. The fact that ATP-induced activation of mUT-A3 was not dependent on PKA is further evidence that AVP is primarily stimulating urea transport through a cAMP-dependent pathway, rather than a calcium-dependent pathway. It is therefore also likely that AVP facilitates the phosphorylation of mUT-A3 in a manner similar to that observed for UT-A1, where AVP has been shown to phosphorylate both UT-A1 in rat IMCD, via a PKA-dependent pathway ( 24 ), and UT-A1 expressed in MDCK cells ( 4 ).

At present, it remains unclear whether AVP activates mUT-A3 transporters already present in the basolateral membrane, whether it facilitates acute trafficking of mUT-A3 to the membrane, or a combination of both. The AVP stimulation of urea transport in MDCK-mUT-A3 cells appears similar to the response previously observed for AVP stimulation of urea transport in rat IMCD, where an initial rapid increase occurred within 10 min, followed by a slower increase over the next 30 min ( 17, 22 ). However, due to the possible limitations of basolateral uptake measurements, caution must be taken not to draw definitive conclusions concerning the nature of AVP activation of UT-A3 at this stage. Interestingly, however, while the rat IMCD responses were observed with 10 -11 M AVP, maximal stimulation only occurred with 10 -8 M AVP ( 17 ), again similar to the dose response for mUT-A3 stimulation observed in this report. Our data concerning acute regulation of mUT-A3 urea transport are therefore consistent with results observed for urea transport in the IMCD, the tissue that expresses UT-A3 transporters. It is interesting to consider that AVP could be increasing IMCD urea permeability by activating urea transporters on both membranes, namely, UT-A1 on the apical membrane and UT-A3 on the basolateral membrane. The overall effect would therefore be a composite of both UT-A1 and UT-A3 activation and future study of individual UT-A isoforms, expressed in MDCK cells, may lead to understanding the exact components of vasopressin-regulated IMCD urea permeability.
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In conclusion, we have engineered the first renal cell line that heterologously expresses mUT-A3. When expressed in MDCK type I cells, mUT-A3 localizes to the basolateral membrane and is responsible for a urea flux that can be acutely activated by AVP. This AVP-stimulated urea transport is sensitive to DMU and phloretin, and is also PKA dependent. Regulation of mUT-A3 also occurs with increases in intracellular levels of either cAMP or calcium. These findings have important implications for the regulation of urea transport in the renal IMCD and the urinary concentrating mechanism.

GRANTS; ]! I+ m" H& G8 I( t, W  ?% M

This work was funded by Kidney Research UK and the Biotechnology and Biological Sciences Research Council.' H1 }& ]" R7 q. R- |
      【参考文献】9 P! R8 U. X* d/ Z+ x+ z0 [
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