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MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS
Faculty of Life Sciences, Core Technology Facility, University of Manchester, Manchester, United Kingdom
Submitted 24 September 2007 ; accepted in final form 23 April 2008
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONREFERENCES |
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ubiquitin-proteasome pathway; urea transport; membrane localization
It has long been recognized that vasopressin regulates the urea permeability in the renal inner medullary collecting duct (IMCD) (15) and does so through regulation of specific urea transporters (9). UT-A1 and UT-A3 have been localized to the IMCD (12, 19, 21), and vasopressin has now been shown to regulate the localization of these transporters to the plasma membrane in rat IMCD cells (2, 8). The importance of regulating plasma membrane urea transporter localization to transepithelial IMCD urea permeability is therefore now evident.
In the renal collecting duct, vasopressin is known to regulate transporter plasma membrane expression by a number of different processes. For example, vasopressin stimulates the insertion of aquaporin-2 (AQP-2) water channels into the plasma membrane of IMCD cells (13), while it also regulates the apical membrane expression of the epithelial sodium channel by preventing ubiquitin-dependent endocytosis from the cell surface (18). Interestingly, while the nonmuscle myosin II inhibitor blebbistatin inhibits vasopressin-stimulated rat IMCD water permeability in vitro, it has no effect on urea permeability (4). This finding can be interpreted to suggest that vasopressin does not insert urea transporters into the IMCD plasma membrane by the nonmuscle myosin II-dependent mechanism, unlike AQP-2 channels. However, it has been previously reported that UT-A1 plasma membrane localization in the MDCK-rat (r)UT-A1 cell line is regulated by a ubiquitination-dependent pathway (3). The aim of the present study was therefore to further investigate the role of ubiquitination in the plasma membrane localization of renal UT-A urea transporters and its relevance to vasopressin regulation.
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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Immunoblotting.
MDCK cells were harvested 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 and 300 mM mannitol. Homogenates were initially centrifuged at 1,000 g for 5 min. The pellet was discarded, and the resulting supernatant was centrifuged at 17,000 g for 30 min. These 17,000 g pellets, containing the plasma membranes, were retained and resuspended in homogenization buffer. In some experiments, the 17,000 g supernatant underwent further centrifugation at 100,000 g for 30 min. The resulting pellet, containing intracellular membrane protein, was also resuspended in homogenization buffer. All centrifugal steps were performed at 4°C. Immunoblotting experiments were then employed using the protocol previously described (19). Briefly, SDS-PAGE was performed on minigels of 10% polyacrylamide by loading 20 µg/lane of protein. After transfer to nitrocellulose membranes, immunoblots were probed for 16 h at 4°C with either urea transporter antiserum ML-446 or ML-194, both previously shown to detect UT-A proteins (19), or anti-
Na-K-ATPase antiserum (no. 05–369, Upstate Biotechnology), or anti-AQP-2 antibody (a gift from Dr. R. Fenton), or anti-ubiquitin antiserum (U-5379, Sigma). Immunoblots were then washed and probed with 1:5,000 dilution of either goat anti-rabbit (for ML-446, ML-194, AQP-2, and ubiquitin antibodies) or goat anti-mouse (for Na-K-ATPase antibody) horseradish peroxidase-linked secondary antiserum (Dako) for 1 h at room temperature. After further washing, detection of protein was performed using the ECL Western Blotting Detection Reagents (Perkin Elmer) and ECL film (GE Healthcare). Images of developed film were then captured with the Image Reader LAS-1000 package.
Transepithelial urea flux measurements. For MDCK-rUT-A1 and MDCK-mUT-A2 cell lines, urea transport was measured using transepithelial urea flux experiments as previously described (14). Briefly, transepithelial urea flux was measured at 37 ± 0.2°C in an apical to basolateral direction, using [14C]urea (0.8 µCi per apical well) as a radiolabeled tracer. The basolateral solution was collected at 3-min intervals. These basolateral collections were then transferred into scintillation vials, 3 ml of Ecoscint A (National Diagnostics) scintillation fluid was added to each vial, and the radioactivity was counted using a 1900 TR liquid scintillation analyzer (Packard).
Unidirectional basolateral urea flux measurements. In contrast, urea transport in MDCK-mUT-A3 cells was measured using basolateral urea uptake, as described previously (20). Briefly, unidirectional basolateral uptake urea flux experiments were performed at 37°C ± 0.2°C, using [14C]urea (1.5 µCi per basolateral well) as the radiolabeled tracer. MDCK-mUT-A3 cells were grown on semipermeable transwells (Corning) and were initially incubated in Hanks’ balanced salt solution (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 [14C]urea for 30 s. This was followed by 10 s in a basolateral solution consisting of standard 1x PBS plus 10 mM "cold" urea. Transwells were then removed from basolateral solution completely, and the apical HBSS solution was replaced with 500 µl 5% SDS solution. 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 scintillation fluid was added to each vial, and the radioactivity was counted with a 1900 TR liquid scintillation analyzer.
All test compounds were of certified grade and were made up either in sterile dH2O or anhydrous DMSO. Stocks solutions were as follows: 100 µM arginine vasopressin (AVP) in dH2O, 10 mM MG-132 in DMSO, and 10 mM proteasome inhibitor II in DMSO. Stocks were then diluted in HBSS containing 5 mM urea before use.
Immunolocalization studies. MDCK-mUT-A3 cells were grown on semipermeable transwells (Corning) until confluent epithelial monolayers were formed. After exposure to experimental agonists for 1 h (e.g., vasopressin), the cells were then fixed for 15 min using 4% paraformaldehyde diluted in 1x PBS. Fixed transwells were then placed in a solution containing 1% BSA, 0.2% gelatin, and 0.05% saponin, diluted in 1x PBS, for three 10-min washes. The transwells were then incubated overnight in 1:100 ML-446 antibody diluted in 0.1% BSA and 0.3% Triton X-100 in 1x PBS. After three 10-min washes in 0.1% BSA, 0.2% gelatine, and 0.05% saponin in 1x PBS, transwells were incubated in 1:100 dilution of FITC-conjugated AffiniPure goat anti-rabbit secondary antibody (Jackson ImmunoResearch) for 1 h. After three further 10-min washes in 0.1% BSA, 0.2% gelatine, and 0.05% saponin in 1x PBS, the nitrocellulose membranes containing the MDCK-mUT-A3 cells were removed and placed on slides, and coverslips were mounted with Eukitt mounting medium (Kindler, Freiburg, Germany). Slides were then viewed using an AxioPlan2 microscope (Zeiss), and images were recorded using a QICam Fast1394 camera and Q capture Pro software (Q Imaging).
Statistical analysis. All data values are shown as mean averages ± SE, with n representing the n number. One-way ANOVA was used for statistical analysis of all urea flux experiments. If the ANOVA indicated a difference, treatment comparison between groups with the Student-Newman-Keuls posthoc test was performed. Groups were deemed statistically significant if P < 0.05.
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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MDCK-rUT-A1. First, we investigated the effect of 50 µM MG-132 on transepithelial urea transport in MDCK-rUT-A1 cells. Representative traces of the effects of preincubation in MG-132 for 1, 4, 8, and 24 h are shown in Fig. 1, A and, B. All increases in transepithelial flux that were subsequently inhibited by 1,3-dimethyl urea (DMU), a known inhibitor of UT-A urea transporters, were interpreted as being due to stimulation of UT-A1-dependent urea flux. As a result, only the 24-h exposure to MG-132 gave nonspecific increases and was therefore discounted from further evaluation. Preincubation of MDCK-rUT-A1 monolayers in MG-132 for 4 and 8 h stimulated basal urea flux (P < 0.01, n = 3, ANOVA) (Fig. 2A). As had previously been reported for MDCK-rUT-A1 monolayers (6, 14), the addition of 10–8 M AVP gave a biphasic response under control conditions (Fig. 1A). There was an initial peak after 9 min, followed by a plateau and then a larger, prolonged increase up to 30 min. Preexposure to MG-132 for either 4 or 8 h greatly increased the initial AVP response after 9 min (P < 0.01, n = 3, ANOVA) (Fig. 2B). In contrast, an increased 30 min AVP stimulation only occurred after 1 h MG-132 preexposure (P < 0.05, n = 3, ANOVA), with a significant decrease in this response after 4 or 8 h MG-132 exposure (P < 0.05, n = 3, ANOVA) (Fig. 2B). Prolonged exposure to MG-132 also produced an increase in the DMU-sensitive flux that was present after AVP stimulation (Fig. 2C), with significant increases after both 4 h (P < 0.05, n = 3, ANOVA) and 8 h (P < 0.001, n = 3, ANOVA). The effect of MG-132 was confirmed by experiments using 50 µM proteasome inhibitor (see Fig. 2D), which also stimulated basal urea flux in MDCK-rUT-A1 monolayers after 4 or 8 h exposure (P < 0.01, n = 3, ANOVA).
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160 kDa Na-K-ATPase -β-subunit complex (Fig. 3A). It is known that the Na-K-ATPase -β-subunit complex only exists at the plasma membrane (10), and indeed our data showed it was not present in either 100,000 g pellet or 100,000 g supernatant samples. In contrast, the 100-kDa signal representing the isolated -subunit was detected in both 17,000 g and 100,000 g pellet samples. For MDCK-rUT-A1 cells, immunoblot analysis showed that both 1 and 4 h exposure to MG-132 increased the UT-A1 signal in 17,000 g MDCK-rUT-A1 protein samples while having no effect on Na-K-ATPase expression (Fig. 3B). In addition, it was confirmed that exposure to MG-132 did indeed increase the amount of ubiquitinated proteins present in MDCK-rUT-A1 protein samples (Fig. 3C).
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45- to 50-kDa UT-A3 signal from the 100,000 g sample (containing mainly intracellular membranes) to the 17,000 g sample (containing mainly plasma membranes) (Fig. 9B). Further experiments confirmed that this increase in the 17,000 g UT-A3 signal was both significant (P < 0.05, n = 3, ANOVA) (Fig. 9C) and specific for UT-A3, since it did not occur for the Na-K-ATPase signal in the same samples (NS, n = 3, ANOVA) (Fig. 9D). Next, the effect on MDCK-mUT-A3 basolateral urea transport of simultaneously adding 10–6 M AVP and 50 µM MG-132 was compared to adding either AVP or MG-132 alone (Fig. 10A). While both AVP and MG-132 individually increased urea transport as expected after 60 min (P < 0.05, n = 4, ANOVA), combining the two produced a larger increase (P < 0.01, n = 4, ANOVA). A similar pattern was also observed after an incubation period of just 10 min, where only the combination of AVP and MG-132 produced a significant increase in basolateral urea flux (P < 0.05, n = 4, ANOVA). To further confirm these additive effects of MG-132 and AVP, we investigated the effects on MDCK-mUT-A3 immunoblot signals. Immunoblots of plasma membrane-containing 17,000 g MDCK-mUT-A3 samples showed that combining MG-132 and AVP produced a greater increase in the UT-A3 signal (P < 0.05, n = 3, ANOVA) than either MG-132 or AVP alone (Fig. 10B). In addition, a combination of both MG-132 and AVP also produced a corresponding greater decrease in the UT-A3 signal in the intracellular membrane-containing 100,000g samples (P < 0.01, n = 3, ANOVA) than either MG-132 or AVP alone (P < 0.05, n = 3, ANOVA) (Fig. 10B). Importantly, no such changes were apparent in Na-K-ATPase signals from the same 17,000 g and 100,000 g samples. Further immunoblot analysis confirmed that while MG-132 exposure increased the amount of ubiquitinated protein in MDCK-mUT-A3 cells, AVP exposure had no such effect (Fig. 10C). Finally, immunolocalization studies showed that exposure to AVP for 1 h increased the UT-A3 signal obtained at the basolateral membranes of MDCK-mUT-A3 cells (Fig. 11). In contrast, while exposure to MG-132 did indeed increase UT-A3 basolateral signal as expected, it also increased the intracellular UT-A3 signal.
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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Our initial findings on the effect of the ubiquitin-proteasome inhibitor MG-132 on MDCK-rUT-A1 transepithelial urea transport confirmed preliminary reports by Chen et al. (3). Up to 8 h exposure to MG-132 led to a dramatic increase in both the initial urea flux observed under control conditions and the subsequent DMU-sensitive flux following AVP stimulation (Figs. 1 and 2). Similar effects on rUT-A1 urea transport were observed after exposure to proteasome inhibitor II (Fig. 2D), confirming that the effects of MG-132 were indeed due to its inhibitory action on of the proteasome-ubiquitin pathway. These observations suggest that MG-132 increased UT-A1 expression at the plasma membrane, and this was confirmed by immunoblot analysis of plasma membrane-containing 17,000 g samples (Fig. 3B). Further immunoblot analysis then confirmed that 4 h exposure to MG-132 did indeed cause a large increase in ubiquitinated protein in MDCK-rUT-A1 cells (Fig. 3C).
The biphasic AVP response observed for MDCK-rUT-A1 cells is similar to that reported for vasopressin-stimulated changes in IMCD urea permeability in vitro (23). The simplest explanation of this biphasic response is that the initial peak (i.e., <10 min) is due to the direct phosphorylation and activation of UT-A1 transporters already present at or near the plasma membrane (25), while the latter peak (i.e., >20 min) is due to the trafficking of UT-A1 transporters from intracellular compartments to the plasma membrane. Our results agree with this hypothesis, since they showed that, as MG-132 increased the plasma membrane expression of UT-A1, we observed a large increase in the initial AVP response (0 to 9 min; Fig. 2B). In contrast, the later AVP response (9 to 30 min) actually decreased after prolonged MG-132 exposure, suggesting that there was already a high concentration of UT-A1 urea transporters present at the plasma membrane, and hence only minimal trafficking could occur. Finally, the lack of DMU sensitivity of the large urea flux after 24 h MG-132 can perhaps be attributed to damage to the epithelial monolayers (i.e., simple leak of urea), due to the toxic effects of prolonged MG-132 exposure.
Investigation of the effects of MG-132 on the transepithelial urea flux in MDCK-mUT-A2 monolayers produced results similar to those observed for MDCK-rUT-A1. Exposure to MG-132 for 4 or 8 h increased the basal DMU-sensitive urea flux and UT-A2 plasma membrane expression (see Figs. 4, 5, and 6). As previously reported (14), the time course of AVP stimulation of UT-A2 was different from that of UT-A1 because it consisted of a single, transient response. Nevertheless, the effect of MG-132 on the AVP response was very similar to that observed for the latter UT-A1 9 to 30 min response; there was an initial increase after 1 h preexposure MG-132, but a decrease after 8 h MG-132 preexposure. Again, this is most readily interpreted as being due to a high concentration of UT-A2 transporters already being present in the plasma membrane.
To complete the analysis of major renal UT-A isoforms, we investigated the effect of inhibiting the ubiquitination-proteasome degradation process on basolateral urea transport in MDCK-mUT-A3 monolayers. Our initial results indicated that MG-132 increased urea flux through a DMU-sensitive, UT-A3-dependent pathway in a concentration-dependent manner (see Fig. 7) by increasing UT-A3 plasma membrane expression (Fig. 8A). These MG-132 effects on UT-A3 basolateral urea transport were again confirmed by the similar effect of proteasome inhibitor II (Fig. 7D). The rapid timescale of the MG-132-dependent increase in UT-A expression, although suggesting that UT-A turnover rate may be relatively high [e.g., compared with that for the epithelial sodium channel (18)], was merely a feature of the MDCK expression system used in the present study.
The exact mechanism by which UT-A urea transporters are ubiquitinated and undergo the process of endocytosis to be removed from the plasma membrane remains to be elucidated. There are a number of candidate sites within the UT-A amino acid sequences that are possible sites for involvement in these steps. For example, the key amino acid known to be involved in the short-chain ubiquitination of the AQP-2 water channel, K270 (7), is part of a class I PDZ domain: X-S/T-X-
(where X = any amino acid, S = serine, T = threonine, X = any amino acid, and = hydrophobic amino acid). Interestingly, a class I PDZ domain, ITKY, is present in UT-A1 at amino acids 920–923 and hence in UT-A2 (at amino acids 388–391), although there are none in the COOH terminus of UT-A3. In addition, it has been suggested that YXX (where Y is tyrosine) represents a "tyrosine endocytosis motif," usually located in the COOH-terminal sequence (18). Such tyrosine motifs are present in UT-A transporter COOH-terminal sequences, YQAY in UT-A1 at amino acids 923–926 (and hence amino acids 391–394 in UT-A2) and YPEA in mUT-A3 at amino acids 439–442, so as such require further investigation.
Next, we studied the possible role of the ubiquitin-proteasome process in vasopressin regulation of UT-A subcellular localization by further investigating the MDCK-mUT-A3 cell line. Initially, we confirmed recent reports that UT-A3 localization can be regulated (2), by exposing MDCK-mUT-A3 monolayers to AVP (see Fig. 9A). One hour AVP exposure dramatically shifted the predominant UT-A3 signal from intracellular membrane samples to plasma membrane samples in a significant and specific manner (Fig. 9, B–D). However, upon simultaneous exposure to MG-132 and AVP, we observed that the effects on basolateral urea flux were additive, after either 10 or 60 min (Fig. 10A). This was confirmed by immunoblot analysis, which demonstrated that combining MG-132 and AVP increased the amount at which the predominant UT-A3 signal shifted from intracellular membranes to the plasma membrane (Fig. 10B). The presence of separate responses to AVP and MG-132 was further confirmed by immunoblot analysis showing that, unlike MG-132, AVP had no effect on the levels of ubiquitinated protein present in MDCK-mUT-A3 samples (Fig. 10C). Taken together, these results are strong evidence that MG-132 and AVP are acting on UT-A3 plasma membrane expression levels through two separate mechanisms. Lastly, immunolocalization data from fixed MDCK-mUT-A3 cells using ML-446 antibody supported a role for ubiquitination in UT-A3 regulation, since MG-132 increased UT-A3 signals (Fig. 11). Interestingly, while AVP specifically increased UT-A3 signal at the basolateral membrane, exposure to MG-132 actually increased UT-A3 signals throughout MDCK-mUT-A3 cells. These data therefore further illustrate differences in the actions of AVP and MG-132 and hence confirm that it is highly unlikely that ubiquitination plays a major role in vasopressin regulation of renal UT-A transporters in vivo.
In conclusion, the present study has shown that the ubiquitin-proteasome pathway regulates the plasma membrane expression levels of all three major renal UT-A urea transporters when expressed in MDCK cell lines. These findings suggest that the ubiquitination process therefore plays an important role in regulating renal transepithelial urea transport and hence in the urinary concentrating mechanism. However, our data also suggest that alteration of this ubiquitin-proteasome pathway is not the mechanism by which vasopressin primarily regulates UT-A3 cellular localization.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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.
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REFERENCES |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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