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MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS
1Department of Physiology and 2Renal Division, Department of Medicine, Emory University School of Medicine, Atlanta, Georgia
Submitted 15 August 2005 ; accepted in final form 13 April 2006
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ABSTRACT |
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10 min and at times even decreased again, followed by a delayed, strong increase over the next 30–40 min. Forskolin activated urea transport through activation of adenylyl cyclase; dideoxyforskolin was inactive. Vasopressin activated urea transport only from the basolateral side and was blocked by OPC-31260, indicating that its action was mediated by basolateral V2 receptors. In the presence of the phosphodiesterase inhibitor IBMX, vasopressin activated as strongly as forskolin. By itself, IBMX caused a slow increase over 50 min to 5 nmol·cm–2·min–1. 8-Bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP; 300 µM) activated urea flux only when added basolaterally. IBMX augmented the activation by basolateral 8-BrcAMP. Urea flux activation by vasopressin and forskolin were only partially blocked by the protein kinase A inhibitor H-89. Even at concentrations >10 µM, urea flux after 60 min of stimulation was reduced by <50%. The rapid-onset component appeared unaffected by the presence of H-89. These data suggest that activation of transepithelial urea transport across MDCK-UT-A1 cells by forskolin and vasopressin involves cAMP as a second messenger and that it is mediated by one or more signaling pathways separate from and in addition to protein kinase A. urea transporter; Madin-Darby canine kidney cells
UT-A1 is located in the apical membrane in the human (2) and rat (14) IMCD and in the apical and basolateral membranes in the mouse IMCD (6). The interstitial medullary urea concentration and, secondarily, the maximum urine concentrating ability are regulated by UT-A-mediated transport across the apical membrane (16). The active transport of NaCl in the thick ascending limb provides the power for concentrating urea in the IMCD luminal fluid by enabling movement of water down its thermodynamic gradient in the collecting duct. The physiological importance of UT-A1 for the urinary concentration mechanism has been demonstrated in knockout mice not expressing the UT-A gene (4, 5).
Along with increasing water permeability, vasopressin (arginine vasopressin, AVP) increases urea permeability in the terminal segment of the IMCD (17, 21) and allows the passive movement of concentrated urea from the tubular lumen into the papillary interstitium. The generally accepted scheme for this regulation includes steps in which AVP binds to the high-affinity V2 receptor. This receptor is coupled to the heterotrimeric G protein, Gs, through which it activates adenylyl cyclase, which in turn raises cAMP levels and activates protein kinase A (PKA) (9). The activation of PKA causes phosphorylation of UT-A1 (26) and coincidentally increases the urea flux, presumably mediated by UT-A1 (17).
To study the pathway or pathways by which urea permeability may be increased in the IMCD, we have developed (7) a Madin-Darby canine kidney (MDCK) cell culture model that heterologously expresses UT-A1. This model has the experimental advantage that it expresses only one UT-A isoform, eliminating the contributions of any of the other UT-A isoforms (UT-A3, UT-A4) that might be functional in vivo. It also lends itself to well-defined studies of transepithelial tracer urea fluxes. Using this cell system, we demonstrated (7) that UT-A1-mediated urea permeability is induced by AVP and forskolin and that UT-A1 protein is phosphorylated in response to these agonists.
In the present study we examined the stimulation of UT-A1-mediated transepithelial urea fluxes in more detail. We found that the time course of urea flux stimulation by AVP or forskolin consisted of two components, a low-amplitude and possibly transient component during the first 10–15 min and a large delayed component that took 40 min to reach maximum. The rapid component is not blocked by the PKA inhibitor H-89, and the slow component is only partially blocked by H-89. We conclude that the activation of UT-A1-mediated transport in MDCK cells is at least in part mediated by cAMP as second messenger. However, the cAMP responsible for flux activation may be compartmentalized by being generated and degraded in a restricted region or regions of the cell, and it may activate urea transport at least in part through non-PKA signaling pathways.
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METHODS |
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UT-A1-expressing MDCK cells.
Construction and selection of the MDCK-FLP recombinase target (FRT) and the MDCK-FRT-UT-A1 cells were performed as described previously (7). We have found that plating 2 x 105 cells per square centimeter of growth area and measuring the urea flux on the confluent high-resistance (>800 
·cm2) polarized epithelial membranes 4 days later give the most consistent results.
Flux measurements. The measurement of [14C]urea flux with collagen-coated Costar Transwell inserts with 1-cm2 growth surface area was described previously, along with a number of controls to demonstrate that the methods are reliable (7). All fluxes were determined at 37°C in the apical-to-basolateral direction by adding the [14C]urea to the apical medium and collecting the radioactivity in the basolateral medium in 3-min intervals. Each experiment had internal controls: we initially measured the baseline flux in the absence of an activator, and, at the end of the experiment, we added the inhibitor dimethylurea (DMU) at 100 mM to the basolateral medium, which reduces the flux to close to the baseline level. The initial unstimulated baseline urea flux was 2.0 nmol·cm–2·min–1 (SD 1.7, n = 1,746), and the final flux after addition of the urea transport inhibitor DMU at 100 mM was 1.6 nmol·cm–2·min–1 (SD 0.8, n = 1,076), where three to six independent flux measurements from 405 different epithelial membranes were averaged. We used the DMU-inhibitable flux as a measure of the UT-A1-mediated urea flux. In the 5 mM urea solutions used in all experiments, a flux of 2.1 nmol·cm–2·min–1 corresponds to a permeability of 7 x 10–6 cm/s. All measurements were performed at pH 7.4 at 37°C in a medium containing (mM) 140 NaCl, 1.6 K2HPO4, 5.5 D-glucose, 1 CaCl2, 0.81 MgCl2, and 24 HEPES titrated with NaOH.
Immunofluorescence experiments. Cells were grown on Transwell filters to confluence as if they were to be used for flux experiments. Cells were washed three times with phosphate-buffered saline (PBS) and then exposed to 3.7% paraformaldehyde (PFA) for 15 min at room temperature. The PFA was replaced with 0.3% Triton X-100 in PBS for 5 min. The detergent was removed by two washes with PBS followed by three washes with PBS containing 1.5% bovine serum albumin (PBS-BSA). Cells were then incubated with primary antibody [anti-COOH-terminal UT-A (11) diluted 1:5,000 in PBS-BSA] for 1 h at room temperature. Unbound antibody was removed with five washes of PBS-BSA, and secondary antibody (anti-rabbit IgG-Alexa 546 diluted 1:250 in PBS-BSA) was added for an additional 1 h. If included, FITC-phalloidin diluted 1:250 in PBS was added with the secondary antibody. To remove secondary antibody, cells were washed three more times with PBS-BSA and one time with deionized water. The cells were mounted between slide and coverslip with Vectashield mounting medium and sealed with nail polish.
Data presentation and statistics. A typical experimental series consisted of parallel flux experiments with eight filter inserts. When data are presented in bar graphs, each bar represents one flux experiment and the error bar is the SD of the last three or four time point measurements of that flux. As one might expect, we found significantly less variability within an experimental series than among different series performed on different days, where maximally stimulated flux values could vary by 10–20%.
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RESULTS |
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25 nmol·cm–2·min–1 at saturating concentrations of forskolin (10 µM). The time course of activation appears to occur in two phases, an initial phase of 10–15 min that is characterized by a slow ramp of activation and a relatively small amplitude, and a second phase during which urea flux activation is accelerated before leveling off. Overall, it took 40–50 min for urea transport to reach this plateau level that we interpret as maximal stimulation under these conditions. At first glance, the initial phase of activation may appear like a lag period after which activation accelerates. However, close inspection (Fig. 2B) reveals that there is no lag. Instead, urea flux activation begins within the first time point (3 min) after exposure to forskolin, and the flux increases at a steady slope over the first 12 min, until it is accelerated during the second phase.
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10–15 nmol·cm–2·min–1, which is less than the activation achieved by forskolin. The modest activation (a slow continuous rise typically to 5 nmol·cm–2·min–1 after 50 min) by IBMX indicates that endogenous activities can accumulate cAMP to concentrations sufficient to activate urea flux if cAMP breakdown is inhibited. This suggests that even in the absence of an externally applied stimulus there was a low constitutive rate of cAMP production in these cells. Surprisingly, the cAMP analogs 8-bromoadenosine 3',5'-cyclic monophosphate 8-BrcAMP and 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate (CPT-cAMP) were poor activators even at the moderately high concentrations used (300 µM).
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In general, the mode of activation of a physiological process in the kidney by AVP is held to be mediated by activation of adenylyl cyclase, which produces the cAMP that activates PKA (9). To test for the direct activation of urea transport by cAMP in our MDCK-UT-A1 cells, we treated them with the membrane-permeant cAMP analog 8-BrcAMP. We found no effect on urea transport at 100 µM, but we did observe significant activation when 300 µM 8-BrcAMP was added to the basolateral side (Fig. 7). However, when added to the apical side, 300 µM 8-BrcAMP had no effect, and even in the presence of IBMX it did not activate urea transport beyond that observed for IBMX alone.
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70–75%. This suggests that other signaling pathways besides that involving PKA play a role in the activation of urea transport by AVP or forskolin.
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DISCUSSION |
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Urea permeability was strongly increased in the presence of forskolin. Although subject to some fluctuations between experiments, the maximal urea flux at saturating concentrations of forskolin (10 µM) was typically around 25 nmol·cm–2·min–1. This corresponded to a 12-fold stimulation over the fluxes observed in the absence of an externally applied stimulus. AVP activated urea fluxes to a lesser extent than forskolin, reaching typical values of 10–15 nmol·cm–2·min–1 in the presence of 10–10–10–8 M AVP. However, in the presence of the phosphodiesterase inhibitor IBMX the activation by AVP was as strong as that by forskolin alone, whereas the activation by forskolin was not further enhanced by IBMX (data not shown). The fact that IBMX by itself caused a slow upward drift in the urea flux that reached 5–10 nmol·cm–2·min–1 after 50 min indicates that even in the absence of an externally applied stimulus there is a constitutively active rate of cAMP formation. It also indicates that this continually formed cAMP is continually degraded and kept to a basal level by a strong phosphodiesterase activity. In fact, the phosphodiesterase activity in the absence of IBMX appears to be so strong that a 300 µM basolateral concentration of 8-BrcAMP was required to achieve significant activation of urea transport. In comparison, in the presence of IBMX the same concentration of 8-BrcAMP caused a robust activation of urea transport to as high as 15–20 nmol·cm–2·min–1 after 50 min. It is interesting that when added to the apical side, 300 µM 8-BrcAMP did not activate urea transport even in the presence of (basolaterally added) IBMX. Virtually identical observations were made with CPT-cAMP (data not shown). This asymmetric response to externally added cAMP analogs could be due to different permeabilities or surface areas of the apical and basolateral membranes, or it could be due to an asymmetric distribution of cAMP-degrading enzymes within the cell or of other components of the cAMP signaling pathway. In control experiments, we observed that in the range of submaximal activation it took about twice as much apical forskolin to achieve the same activation of urea transport as achieved by basolateral forskolin (data not shown).
The response of urea transport to added AVP was also asymmetric. Basolateral AVP elicited a strong activation, whereas apical AVP had no noticeable effect (data not shown). This is most readily explained by a nearly exclusive presence of V2 receptors on the basolateral side. The functional asymmetry is certainly much stronger than could be caused by the differences in membrane area (at constant receptor densities) on the two sides. A strong asymmetry of V2 receptor density in MDCK cells was demonstrated previously by immunologic techniques (1). However, the present flux data suggest that the observed asymmetric distribution of overexpressed, epitope-labeled V2 receptors in receptor-transfected cells is still less than the asymmetric distribution of natively expressed receptor proteins in MDCK cells, which is similar to findings in isolated renal tubules (15).
Our data suggest that the activation of UT-A1-mediated urea transport in our MDCK cells by forskolin and AVP occurs in at least two different phases. We observed a rapid response of low amplitude whose onset occurred within the first time point taken after addition of the stimulant (3 min), followed by a slower component that exhibited a lag of up to 15 min. When urea transport was activated by forskolin, the rapid response component was visible only as a weak shoulder riding on the onset of the slower component with its much larger amplitude. In contrast, when urea transport was activated by AVP, the early component was much more distinct, probably because of the smaller amplitude of the slower component in the presence of AVP. Under these conditions, urea flux briefly decreased, suggesting that the early component may be a transient response to the stimulus.
This biphasic response is reminiscent of the response of urea and water permeability to AVP in isolated rat terminal IMCD tubules (24). In these experiments, urea and water permeabilities exhibited a rapid phase that reached completion around 10 min, followed by a slower component over the next 30 min. The difference between the two cell systems, however, is that in the IMCD the early component had the larger amplitude, contributing 80–90% to the maximal response, whereas in the MDCK cells it contributes 10% to the response to forskolin and up to 30% to the response to AVP. The nature of this quantitative difference is not clear at this time. There are several differences between the rodent tubule and the canine cell line beyond the difference in species that could account for possible differences in the regulation of renal urea transport. One possibility would be that through the long history of culture of the MDCK cells the relative strength of signaling or regulatory pathways may have shifted; a component could even have been lost. Because it is likely that the MDCK-UT-A1 cells overexpress the UT-A1 protein, the elevated level of heterologous protein could be recruited into a membrane pool that is differently regulated. Nonetheless, at this time the MDCK-UT-A1 cells represent the best available cell culture model for studying the regulation of inner medullary urea transport.
The mechanisms underlying urea flux activation in the presence of forskolin and AVP in the MDCK cells are also not clear. The generally held notion is that the activation of urea transport in the IMCD by AVP occurs through activation of adenylyl cyclase, cAMP formation, and activation of PKA (9), followed by phosphorylation of the UT-A1 urea transporter (26). If phosphorylation of UT-A1 is mediated solely through activation of PKA, one would expect the activation of urea flux to be nearly completely inhibited in the presence of 1–5 µM of the PKA inhibitor H-89. However, the activated urea flux was only partially suppressed even in the presence of 30 µM H-89. This strongly suggests that in the MDCK cells the slow activation of urea transport is mediated by at least one additional signaling pathway that does not involve PKA.
The observed incomplete block of urea transport activation by H-89 is consistent with previous work on the effect of PKA-specific inhibitors on the phosphorylation of UT-A1 in IMCD suspensions. Zhang et al. (26) found that H-89 and the specific peptide PKA inhibitor 14-22 amide only partially blocked the phosphorylation of UT-A1 protein induced by AVP. This parallel behavior of UT-A1 protein phosphorylation and urea transport activity suggests that UT-A1 protein phosphorylation in the isolated IMCD and urea transport activation in cultured MDCK cells are subject to the same set of signaling pathways.
Not only was H-89 an incomplete inhibitor of the activated urea transport at the (maximum) plateau phase of urea flux activation, we observed no inhibitory effect at all during the early phase (Fig. 8). During this early phase, which comprises the first 10–15 min of stimulation, there was no difference whether the cells had been preincubated with H-89 for 27 min or not. This suggests that during this early phase of activation PKA is not involved at all. That PKA might not be the sole mediator of urea transport activation is more readily understood for AVP because in addition to stimulating cAMP production AVP also raises intracellular Ca2+ levels (21, 25) and thus activates additional pathways that can lead to UT-A1 phosphorylation and activation. However, no such behavior is known for forskolin, at least not stimulation that is specific to forskolin over dideoxyforskolin.
In recent years a new family of related proteins that can function as nonkinase effectors of several second messengers has been identified (20). The subfamily of Epac/cAMP-GEF proteins can bind cAMP and in response activate a small G protein, Rap1, which in turn can trigger downstream kinase cascades such as MEK/Erk. Thus Epacs can transduce signals to the same effector molecules as PKA. Epac-1 is ubiquitously expressed, including in the kidney, where it has been shown to mediate the activation of H-K-ATPase by cAMP (12). It will be of interest to explore whether Epac-1 also participates in the activation of urea transport in response to AVP.
In summary, we have demonstrated that constitutively expressed UT-A1 protein in transfected MDCK-UT-A1 cells mediates urea transport only after activation by forskolin and AVP. Much of this activation is expected, based on the action of these agents in other systems including isolated IMCD tubules. However, there are also significant quantitative differences in the time course of activation. It is possible that the MDCK-UT-A1 cells provide a model to dissect out signaling pathway components that are not as readily observed in intact tubules.
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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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2. Bagnasco SM, Peng T, Janech MG, Karakashian A, and Sands JM. Cloning and characterization of the human urea transporter UT-A1 and mapping of the human Slc14a2 gene. Am J Physiol Renal Physiol 281: F400–F406, 2001.
3. Christensen BM, Marples D, Jensen UB, Frøkiaer J, Sheikh-Hamad D, Knepper M, and Nielsen S. Acute effects of vasopressin V2-receptor antagonist on kidney AQP2 expression and subcellular distribution. Am J Physiol Renal Physiol 275: F285–F297, 1998.
4. Fenton RA, Chou CL, Stewart GS, Smith CP, and Knepper MA. Urinary concentrating defect in mice with selective deletion of phloretin-sensitive urea transporters in the renal collecting duct. Proc Natl Acad Sci USA 101: 7469–7474, 2004.
5. Fenton RA, Flynn A, Shodeinde A, Smith CP, Schnermann J, and Knepper MA. Renal phenotype of UT-A urea transporter knockout mice. J Am Soc Nephrol 16: 1583–1592, 2005.
6. Fenton RA, Stewart GS, Carpenter B, Howorth A, Potter EA, Cooper GJ, and Smith CP. Characterization of mouse urea transporters UT-A1 and UT-A2. Am J Physiol Renal Physiol 283: F817–F825, 2002.
7. Fröhlich O, Klein JD, Smith PM, Sands JM, and Gunn RB. Urea transport in MDCK cells that are stably transfected with UT-A1. Am J Physiol Cell Physiol 286: C1264–C1270, 2004.
8. Gillin AG and Sands JM. Characteristics of osmolarity-stimulated urea transport in rat IMCD. Am J Physiol Renal Fluid Electrolyte Physiol 262: F1061–F1067, 1992.
9. Ishikawa S. Cellular actions of arginine vasopressin in the kidney. Endocr J 40: 373–386, 1993.[Web of Science][Medline]
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11. Klein JD, Rouillard P, Roberts BR, and Sands JM. Acidosis mediates the upregulation of UT-A protein in livers from uremic rats. J Am Soc Nephrol 13: 581–587, 2002.
12. Laroche-Joubert N, Marsy S, Michelet S, Imbert-Teboul M, and Doucet A. Protein kinase A-independent activation of ERK and H,K-ATPase by cAMP in native kidney cells: role of Epac I. J Biol Chem 277: 18598–18604, 2002.
13. Lavelle JP, Negrete HO, Poland PA, Kinlough CL, Meyers SD, Hughey RP, and Zeidel ML. Low permeabilities of MDCK cell monolayers: a model barrier epithelium. Am J Physiol Renal Physiol 273: F67–F75, 1997.
14. Nielsen S, Terris J, Smith CP, Hediger MA, Ecelbarger CA, and Knepper MA. Cellular and subcellular localization of the vasopressin-regulated urea transporter in rat kidney. Proc Natl Acad Sci USA 93: 5495–5500, 1996.
15. Nonoguchi H, Owada A, Kobayashi N, Takayama M, Terada Y, Koike J, Ujiie K, Marumo F, Sakai T, and Tomita K. Immunohistochemical localization of V2 vasopressin receptor along the nephron and functional role of luminal V2 receptor in terminal inner medullary collecting ducts. J Clin Invest 96: 1768–1778, 1995.[Web of Science][Medline]
16. Sands JM. Mammalian urea transporters. Annu Rev Physiol 65: 543–566, 2003.[CrossRef][Web of Science][Medline]
17. Sands JM, Nonoguchi H, and Knepper MA. Vasopressin effects on urea and H2O transport in inner medullary collecting duct subsegments. Am J Physiol Renal Fluid Electrolyte Physiol 253: F823–F832, 1987.
18. Seamon KB, Padgett W, and Daly JW. Forskolin: unique diterpene activator of adenylate cyclase in membranes and in intact cells. Proc Natl Acad Sci USA 78: 3363–3367, 1981.
19. Serradeil-Le Gal C, Lacour C, Valette G, Garcia G, Foulon L, Galindo G, Bankir L, Pouzet B, Guillon G, Barberis C, Chicot D, Jard S, Vilain P, Garcia C, Marty E, Raufaste D, Brossard G, Nisato D, Maffrand JP, and Le Fur G. Characterization of SR 121463A, a highly potent and selective, orally active vasopressin V2 receptor antagonist. J Clin Invest 98: 2729–2738, 1996.[Web of Science][Medline]
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21. Star RA, Nonoguchi H, Balaban R, and Knepper MA. Calcium and cyclic adenosine monophosphate as second messengers for vasopressin in the rat inner medullary collecting duct. J Clin Invest 81: 1879–1888, 1988.[Web of Science][Medline]
22. Stewart GS, Fenton RA, Wang W, Kwon TH, White SJ, Collins VM, Cooper G, Nielsen S, and Smith CP. The basolateral expression of mUT-A3 in the mouse kidney. Am J Physiol Renal Physiol 286: F979–F987, 2004.
23. Terris JM, Knepper MA, and Wade JB. UT-A3: localization and characterization of an additional urea transporter isoform in the IMCD. Am J Physiol Renal Physiol 280: F325–F332, 2001.
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25. Yip KP. Coupling of vasopressin-induced intracellular Ca2+ mobilization and apical exocytosis in perfused rat kidney collecting duct. J Physiol 538: 891–899, 2002.
26. Zhang C, Sands JM, and Klein JD. Vasopressin rapidly increases phosphorylation of UT-A1 urea transporter in rat IMCDs through PKA. Am J Physiol Renal Physiol 282: F85–F90, 2002.
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