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Am J Physiol Regul Integr Comp Physiol 293: R2382-R2389, 2007. First published September 12, 2007; doi:10.1152/ajpregu.00029.2007
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COMPARATIVE AND EVOLUTIONARY PHYSIOLOGY

Transport of a fluorescent cAMP analog in teleost proximal tubules

Valeska Reichel,1,2,4 Rosalinde Masereeuw,2 Jeroen J. M. W. van den Heuvel,2 David S. Miller,3,4 and Gert Fricker1,4

1Institute of Pharmacy and Molecular Biotechnology, University of Heidelberg, Heidelberg, Germany; 2Department of Pharmacology and Toxicology, Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands; 3Laboratory of Pharmacology and Chemistry, National Institutes of Health/National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina; and 4Mount Desert Island Biological Laboratory, Salisbury Cove, Maine


   ABSTRACT
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 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 
Previous studies have shown that killifish (Fundulus heteroclitus) renal proximal tubules express a luminal membrane transporter that is functionally and immunologically analogous to the mammalian multidrug resistance-associated protein isoform 2 (Mrp2, ABCC2). Here we used confocal microscopy to investigate in killifish tubules the transport of a fluorescent cAMP analog (fluo-cAMP), a putative substrate for Mrp2 and Mrp4 (ABCC4). Steady-state luminal accumulation of fluo-cAMP was concentrative, specific, and metabolism-dependent, but not reduced by high K+ medium or ouabain. Transport was not affected by p-aminohippurate (organic anion transporter inhibitor) or p-glycoprotein inhibitor (PSC833), but cell-to-lumen transport was reduced in a concentration-dependent manner by Mrp inhibitor MK571, leukotriene C4 (LTC4), azidothymidine (AZT), cAMP, and adefovir; the latter two compounds are Mrp4 substrates. Although MK571 and LTC4 reduced transport of the Mrp2 substrate fluorescein-methotrexate (FL-MTX), neither cAMP, adefovir, nor AZT affected FL-MTX transport. Fluo-cAMP transport was not reduced when tubules were exposed to endothelin-1, Na nitroprusside (an nitric oxide generator) or phorbol ester (PKC activator), all of which signal substantial reductions in cell-to-lumen FL-MTX transport. Fluo-cAMP transport was reduced by forskolin, and this reduction was blocked by the PKA inhibitor H-89. Finally, in membrane vesicles from Spodoptera frugiperda (Sf9) cells containing human MRP4, ATP-dependent and specific uptake of fluo-cAMP could be demonstrated. Thus, based on inhibitor specificity and regulatory signaling, cell-to-lumen transport of fluo-cAMP in killifish renal tubules is mediated by a transporter distinct from Mrp2, presumably a teleost form of Mrp4.

killifish; multidrug resistance-associated protein 4

THE VERTEBRATE RENAL PROXIMAL tubule is responsible for the active excretion of potentially toxic, negatively charged metabolic wastes, xenobiotics, and xenobiotic metabolites (27). To accomplish this task, this epithelium is polarized with the basolateral and luminal membranes containing a broad variety of organic anion transport proteins, including members of the multidrug resistance-associated protein (MRP), organic anion transporter (OAT), and organic anion transporting polypeptide (OATP) families (10). The MRPs, a subfamily of the ATP binding cassette protein superfamily, comprise at least nine members (MRP1–9). All are membrane-bound proteins and depend on ATP hydrolysis to drive substrate transport (3). In mammalian kidney, MRP2 and MRP4 are located on the apical membrane of proximal tubular epithelial cells and play an important role in the urinary excretion of a wide variety of compounds (22, 31, 32). Substrates for Mrp2 in rat and man include glutathione, glucuronide, and sulfate conjugates. Mrp2 also mediates transport of leukotriene C4 (LTC4), estradiol-17-(β-D-glucuronide), p-aminohippurate (PAH), S-glutathionyl 2,4-dinitrobenzene, and anticancer substances like methotrexate and anthracyclines (24) (see also, transporter database: http://www.tp-search.jp). Mrp4 transports the second messengers cAMP and cGMP (2, 32) and acyclic nucleoside phosphonates, e.g., 9-(2-phosphonyl-methoxyethyl)adenine (PMEA; adefovir) as well as monophosphorylated nucleoside analogs, such as azidothymidine-monophosphate and zidovudine-monophosphate (12). Mrp4 substrates also include PAH and methotrexate (12, 25).

For a functional assessment of organic anion transport in the kidney we have used isolated killifish (Fundulus heteroclitus) renal proximal tubules. Kidneys of this teleost fish contain a high fraction of proximal tubules (4), which can be isolated easily and are viable for at least a day at room temperature. This comparative model is a powerful tool for the study of secretory transport in intact proximal tubules (20). Unlike mammalian proximal tubules, the broken ends of these tubules reseal after isolation, thus forming a closed fluid-filled luminal compartment. This enables study of cellular uptake and luminal accumulation of fluorescent compounds using imaging techniques.

We previously reported on the function and regulation of an Mrp2-like protein (7, 8, 17, 18) and the immunolocalization of a Mrp4-like protein (19) in killifish proximal tubules. Consistent with this, an Mrp4-like gene has been identified in the genome of zebrafish (Danio rerio), another teleost fish [European Molecular Biology Laboratory (EMBL) accession no. AL591370.10]. In the present study, we used confocal microscopy and the fluorescent cAMP analog fluo-cAMP to visualize transport across proximal tubular epithelium of the killifish. Our data indicate that tubular transport of fluo-cAMP is distinct from Mrp2-mediated transport and presumably mediated by a teleost form of Mrp4.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Chemicals. Fluo-cAMP was purchased from Biolog Life Science Institute (San Diego, CA and Bremen, Germany). Fluorescein-methotrexate (FL-MTX) and phorbol 12-myristate 13-acetate were obtained from Molecular Probes (Eugene, OR). Valspodar (PSC833) was from Novartis (Basle, Switzerland). All other chemicals were obtained from Sigma (St. Louis, MO; Zwijndrecht, the Netherlands). Primary Mrp4 antibody was a human Mrp4 antibody (pAb rM4-pl) raised in rabbit at the Nijmegen Centre for Molecular Life Sciences (Nijmegen, The Netherlands) as described in Van Aubel et al. (31). For this antibody, the linker region (amino acids 611–676) of human MRP4 was chosen as epitope for the polyclonal antibody we used in this study. We aligned human and zebrafish (AL591370) Mrp4 sequences and found 68% homology. Within the linker region (amino acids 611–676) we found 45% homology; at least 6 amino acids in line are conserved. This should be sufficient for some cross-reactivity. Goat-derived anti-rabbit IgG 488 was obtained from Molecular Probes.

Animals and tissue preparation. All animal studies were performed in accordance with institutional regulations for animal protection and had been approved by the animal care and use committee of Mount Desert Island Biological Laboratory. Killifish were collected in the vicinity of Mount Desert Island, Maine. Fish were held in tanks with natural flowing sea water at the Mount Desert Island Biological Laboratory. After death, masses of renal tubules were extracted and transferred to petri dishes containing a marine teleost saline buffer based on Taggart and Forster (28): 140 mM NaCl, 2.5 mM KCl, 1.5 mM CaCl2, 1.0 mM MgCl2, and 20 mM Tris, pH 8.0. All experiments were carried out at room temperature (18–22°C) under air.

Under a dissecting microscope, proximal tubules were isolated from the masses and transferred to foil-covered Teflon chambers (Bionique) with glass coverslip bottoms. The chamber contained 1 ml of marine teleost solution without (controls) or with added chemicals. These were added as stock solutions in marine teleost saline or in DMSO. The final DMSO concentration did not exceed 0.5%. This DMSO concentration had no effect on fluo-cAMP (present study, data not shown) or FL-MTX transport (1a). After 30-min preincubation, the fluorescent substrate (2 µM fluo-cAMP or 2 µM FL-MTX) was added, and tubules were incubated for 10–45 min.

Confocal microscopy. After incubation, tubules in the chambers were placed on an inverted confocal microscope (Olympus Fluoview) and viewed with a x40 water immersion objective. Under transmitted light, tubules with intact epithelia were selected and confocal fluorescent images were acquired using the 488-nm line of an argon ion laser, a 510-nm dichroic filter, and a 515-nm long-pass emission filter. Four scans, 4 s each, were averaged to provide a final image (512 x 512 x 12 bits). Images were saved to disk and analyzed using National Institutes of Health Scion Image software. For analysis, cellular and luminal regions of each tubule were selected, and average pixel intensity was computed. Laser intensity and photomultiplier gain were selected such that tissue autofluorescence and photobleaching were not detectable. Most of the experiment-to-experiment differences in fluorescence intensity for control tubules reflect changes in gain settings. All data are reported as fluorescence intensities after background subtraction. Detailed descriptions of image acquisition and analysis are given in Refs. 5, 7, 30.

Isolation of crude membrane vesicles from Spodoptera frugiperda 9 cells. Cells from Spodoptera frugiperda (Sf9) expressing human MRP4 were generated by infection using a recombinant baculovirus encoding MRP4 or, as a control, opsine, as described previously (31). Infected Sf9 cells were resuspended in ice-cold homogenization buffer (0.5 mM sodium phosphate, 0.1 mM EDTA) supplemented with protease inhibitors (100 µM PMSF, 5 µg/ml aprotinin, 5 µg/ml leupeptin, 1 µg/ml pepstatin, 1 µM E64), and shaken on ice for 1 h. Lysed cells were centrifuged at 100,000 g, and the resulting pellet was homogenized in ice-cold Tris-sucrose (TS) buffer (10 mM Tris·HEPES, 250 mM sucrose, pH 7.4) with a tight-fitting Dounce glass tissue grinder. The homogenate was centrifuged at 500 g, and the resulting supernatant was centrifuged at 100,000 g. The resulting pellet was resuspended in TS buffer and passed 30 times through a 27-gauge needle. Aliquots of crude membrane vesicles were frozen in liquid nitrogen and stored at –80°C until use.

Vesicle transport studies. Uptake studies were performed using fluo-cAMP as substrate as described in Smeets et al. (25). Vesicles stored at –80°C were prewarmed for 1 min at 37°C and then added to a mixture of TS buffer, 10 mM MgCl2, an ATP-regenerating system (4 mM ATP, 10 mM creatine phosphate, 100 µg/ml creatine kinase), and fluo-cAMP. For control experiments, ATP was replaced by 5'-AMP. At indicated time points, samples were taken and diluted in 900 µl ice-cold TS buffer to stop transport. Samples were filtered through NC45 filters (Whatman, Maidstone, UK) on a sampling manifold (Millipore, Billerica, MA). Filters were extracted in SDS/HEPES buffer (1% SDS, 7.5 mM HEPES) for at least 30 min. Afterward fluid fluorescence was measured using a Shimadzu RF-5301PC spectrofluorophotometer (excitation: 480 nm, emission: 520 nm). Experiments were performed in triplicate.

ATP-dependent transport was calculated by subtracting values obtained in the presence of 5'-AMP from those obtained in the presence of ATP. To calculate MRP4-specific transport, respectively, values for ATP-dependent transport of control vesicles were subtracted from those of infected cell membrane vesicles.

Statistics. Values are presented as means ± SE. Means of control and treated groups were compared using one-way ANOVA and Dunnett's post hoc test. Differences were considered statistically significant when P < 0.05.


   RESULTS
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 MATERIALS AND METHODS
 RESULTS
DISCUSSION
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Figure 1 shows a typical confocal image of a control killifish tubule after 30 min (steady-state) incubation in medium containing 2 µM fluo-cAMP. The distribution of fluorescence is similar to that seen for other fluorescent organic anions, i.e., the Mrp2-substrate Fl-MTX and the Oat substrate, fluorescein (13, 16). That is, fluorescence intensity in the lumens > cells > medium. This pattern suggests a two-step process involving uptake at the basolateral membrane and efflux into the lumen.


Figure 1
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  		Fig. 1. Confocal micrograph of a killifish tubule incubated in medium containing 2 µM fluo-cAMP. The image shows a tubule at steady state (30-min incubation). The white bar indicates 50 µm.

 
Quantitation of confocal images showed that luminal accumulation of fluo-cAMP increased rapidly over the first 20 min and reached a steady-state level within 25–30 min (Fig. 2A). Cellular fluorescence remained constant from 10 min on. At steady state, luminal fluorescence exceeded cellular fluorescence by a factor of 5–8. Steady-state luminal fluo-cAMP accumulation was reduced when cellular energy metabolism was inhibited by 1 mM NaCN (Fig. 2B). In contrast, cellular fluorescence was not affected by NaCN. Exposing tubules to 1 mM ouabain did not affect steady-state fluo-cAMP accumulation (Fig. 2C). In killifish tubules, this concentration of ouabain abolishes Na-dependent transport of the fluorescent small organic anion, fluorescein (13). Similarly, increasing medium K+ by a factor of 10 was without effect (Fig. 2D); this treatment depolarizes killifish renal epithelial cells by about 40 mV and substantially inhibits uptake of organic cations (26). These results indicate that concentrative fluo-cAMP transport from cell to lumen was energy-dependent but not sensitive to changes in ion gradients or the membrane electrical potential.


Figure 2
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  		Fig. 2. A: time course of fluo-cAMP accumulation in killifish renal proximal tubules. Tubular tissue was incubated with 2 µM fluo-cAMP in teleost Ringer solution for the indicated times. B: effect of 1 mM NaCN on the steady-state (30-min) accumulation of fluo-cAMP. C: effect of 1 mM ouabain on steady-state accumulation of fluo-cAMP. D: effect of 10-fold increased medium K+concentration on steady-state fluo-cAMP accumulation. Data are presented as the mean values for 12 tubules; variability is shown as SE bars. *Significantly different from controls, P < 0.05; **significantly different from controls, P < 0.01.

 
To determine the characteristics of cellular and luminal accumulation of fluo-cAMP, we examined the effects of a number of chemicals on tubular transport. These chemicals were chosen because of known interactions with specific transporters or groups of related transporters. Previous studies with killifish tubules showed that cell-to-lumen transport of the Mrp2 substrate, FL-MTX, was sensitive to inhibition by MK571 and LTC4, both inhibitors of MRPs (6, 11). Figure 3, A and B shows that MK571 and LTC4 reduced luminal accumulation of fluo-cAMP in a concentration-dependent manner; neither compound affected cellular accumulation of fluo-cAMP. In contrast, the inhibitor of transport mediated by Oats, PAH, and the p-glycoprotein inhibitor (PSC833) were without effect (Fig. 3, C and D), even at concentrations known to block Oat-mediated transport of fluorescein and p-glycoprotein-mediated transport of a fluorescent CSA derivative, respectively, in killifish tubules (15, 23).


Figure 3
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  		Fig. 3. Inhibition of fluo-cAMP transport. Killifish tubules were incubated for 30 min in medium containing 2 µM fluo-cAMP without (control) or with the indicated additions. MK571, Mrp inhibitor; LTC4, leukotriene C4; PAH, p-aminohippurate; PSC833, p-glycoprotein inhibitor. Images were collected and analyzed as described in MATERIALS AND METHODS. Data are presented as the mean values for 12 tubules; variability is shown as SE bars. **Significantly different from controls, P < 0.01.

 
The MRP4 substrates, cAMP and adefovir (PMEA) caused concentration-dependent inhibition of steady-state luminal accumulation of fluo-cAMP with no change in cellular accumulation (Fig. 4). The same was observed with azidothymidine (AZT), which inhibits MRP4 in its monophosphate form (12, 21).


Figure 4
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  		Fig. 4. Inhibition of organic anion transport. Killifish tubules were incubated for 30 min in medium containing 2 µM fluo-cAMP or 2 µM fluorescein-methotrexate (FL-MTX), without (control) or with the indicated additions. PMEA, 9-(2-phosphonyl-methoxyethyl)adenine; AZT, azidothymidine; 8-Br-cGMP, 8-Bromo-cGMP. Data are presented as the mean values for 12–15 tubules; variability is shown as SE bars. **Significantly different from controls, P < 0.01.

 
None of these latter compounds affected transport of FL-MTX (Fig. 4), indicating that they do not interact with teleost Mrp2. These results indicate that cell-to-lumen transport of fluo-cAMP and FL-MTX are mediated by different transporters, likely different teleost Mrp isoforms.

The stable cGMP analog, 8-bromo-cGMP, also decreased luminal accumulation of fluo-cAMP (Fig. 4). 8-Bromo-cGMP inhibits cAMP-specific phosphodiesterases, causing intracellular cAMP levels to increase. As a consequence, excretion of fluo-cAMP was inhibited.

Masereeuw and colleagues (13, 14) showed that cell-to-lumen transport of FL-MTX in killifish proximal tubules is mediated by a teleost homolog of Mrp2. Activity of this transporter is regulated by a signaling pathway involving endothelin-1 (ET-1) signaling through an endothelin B (ETB) receptor, nitric oxide synthase (NOS), and PKC (14, 17). Activating this pathway by exposing killifish tubules to 1–100 nM ET-1, 10–100 µM Na nitroprusside (an NO donor), or 1–100 nM phorbol ester (activates PKC) substantially reduces luminal accumulation of FL-MTX. Figure 5 shows that neither ET-1, Na nitroprusside, nor phorbol-12-myristate-13-acetate had any effect on the transport of fluo-cAMP in killifish tubules.


Figure 5
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  		Fig. 5. Lack of effect of endothelin (ET), Na nitroprusside, and phorbol-12-myristate-13-acetate (PMA) on fluo-cAMP transport in killifish tubules. Tubules were incubated for 30 min in medium containing 2 µM fluo-cAMP without (control) the indicated additions. Data are presented as the mean values for 10–15 tubules; variability is shown as SE bars.

 
FL-MTX transport in killifish tubules is not affected by forskolin, an indirect activator of PKA (14). Figure 6 shows that 10 µM forskolin substantially reduced steady-state luminal accumulation of fluo-cAMP. This effect was blocked by the PKA inhibitor H-89, which by itself did not affect fluo-cAMP transport.


Figure 6
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  		Fig. 6. Effects of modulation of PKA activity on fluo-cAMP transport. Tubules were incubated 30 min in medium containing 2 µM fluo-cAMP without (control) or with the indicated additions. H-89, PKA inhibitor. Data are presented as the mean values for 15–16 tubules; variability is shown as SE bars. **Significantly different from controls, P < 0.01.

 
To determine whether fluo-cAMP was a substrate for human MRP4, transport studies were performed using membrane vesicles isolated from Sf9 cells transfected with human MRP4. We found ATP-dependent and specific uptake of fluo-cAMP into MRP4 expressing vesicles. The time course study showed that uptake of 20 µM fluo-cAMP in Sf9-MRP4 membrane vesicles started to reach a plateau after 20–30 min (Fig. 7A). Uptake clearly saturated with increasing fluo-cAMP concentration (Fig. 7B); apparent Km and Vmax values were 5.3 ± 2.0 µM and 1.1 ± 0.2 nmol·mg–1·min–1, respectively. Both MK571 and LTC4 caused concentration-dependent inhibition of fluo-cAMP uptake in vesicles from MRP4-infected cells (Figs. 7, C and D).


Figure 7
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  		Fig. 7. Uptake of fluo-cAMP into membrane vesicles of Spodoptera frugiperda 9 (Sf9) cells transfected with human multidrug resistance-associated protein 4 (MRP4). A: time course of uptake. B: concentration dependence. C: LTC4 dose response. D: MK571 dose response. Data are expressed as means ± SE from 3 independent experiments, each measured in triplicate. *Significantly different from controls, P < 0.05; **significantly different from controls, P < 0.01.

 

   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
With the use of teleost renal tubules, fluorescent substrates and confocal microscopy can be applied to both visualize and measure all steps in the excretory pathway. For organic anions, previous imaging studies with killifish tubules have characterized an Oat-like uptake transporter on the basolateral membrane and an Mrp2-like efflux transporter on the luminal membrane. The basolateral teleost transporter drives the concentrative uptake of small organic anions (PAH, 2,4-D and fluorescein, not FL-MTX) through indirect coupling to the Na+ gradient. It is functionally analogous to mammalian Oat1 and Oat3 and a recent report suggests that the mammalian Oat1/Oat3 dichotomy is a result of an early gene duplication in lower vertebrates (1). The efflux transporter in killifish tubules drives concentrative accumulation of larger organic anions (FL-MTX, Texas Red, not fluorescein or PAH) into the tubular lumen (13). This uphill step is Na+-independent, ouabain insensitive, and insensitive to changes in membrane electrical potential, suggesting that it is ATP-driven.

Both the Oat-like and Mrp2-like transporters appear to be regulated by PKC-dependent signals, with PKC activation rapidly (minutes) reducing transport for both (14, 16). In this regard, PKC activation is known to reduce Na+-dependent organic anion uptake in mammalian renal slices and tubules and a number of mammalian renal cell lines as well as in killifish tubules (16, 29, 34). Experiments with Xenopus oocytes expressing hOAT1 or flOat show both reduced specific transport and transporter internalization following PKC activation (9, 34). For the efflux transporter, both ET-1 and calciotropic hormones act through PKC to reduce transport (14, 17, 33).

Previous imaging studies with killifish tubules have suggested that there are additional OATs at the luminal membrane. First, Miller et al. (15) presented evidence for a potential-insensitive efflux pathway for fluorescein, carboxyfluorescein and bimane-S-conjugates. This pathway was sensitive to inhibition by PAH and it was assumed to be the major efflux step for small organic anions; it has not been investigated further. Second, Notenboom et al. (19) recently demonstrated that an antibody raised against human MRP4 immunolocalized to the luminal membrane of killifish renal proximal tubules. In that study, prolonged exposure (12–24 h) of tubules to gentamicin or ET-1 upregulated FL-MTX transport into the tubular lumen and increased luminal membrane immunoreactivity to an antibody raised to rabbit Mrp2. No changes in Mrp4 immunoreactivity were seen, suggesting the presence of a second Mrp isoform in the luminal membrane. Consistent with this, recent mining of the zebrafish genome indicated the presence of a sequence coding for a teleost Mrp4 homolog (EMBL accession no. AL591370.10).

Taken together, the results of the present study are consistent with the involvement of a teleost Mrp4 in the cell-to-lumen transport of fluo-cAMP. Measurements of steady-state fluo-cAMP accumulation in tubules suggested two concentrative steps, one at the basolateral membrane and the other at the luminal membrane. However, no treatment used (NaCN, ouabain and high medium K+, competitor organic anions) had any effect on cellular fluo-cAMP accumulation. Thus, it is unlikely that the uptake step is mediated and cellular accumulation must reflect simple diffusion plus binding of the dye to cellular components.

This was not the case for luminal accumulation of fluo-cAMP, which was substantially reduced when metabolism was inhibited by NaCN and when a number of organic anions were added to the medium. Clearly, cell-to-lumen transport was mediated and directly or indirectly coupled to ATP. This step was not affected by ouabain or high K+, suggesting that it was independent of ion gradients and membrane electrical potential. Luminal accumulation was not affected by millimolar concentrations of PAH nor by micromolar concentrations of PSC833, ruling out involvement of the efflux pathway for small organic anions (15) and p-glycoprotein. Luminal accumulation was reduced by MK571, LTC4, cAMP, PMEA, and AZT. Although MK571 and LTC4 have been shown to reduce luminal accumulation of FL-MTX in killifish tubules (14), neither cAMP, PMEA, nor AZT had any effect on FL-MTX transport (present study). Finally, luminal accumulation of fluo-cAMP was not affected when ET-1/NOS/PKC signaling was activated (present study); this signaling pathway reduces luminal accumulation of FL-MTX (14, 17). Fluo-cAMP transport was reduced by PKA activation (present study), which does not affect FL-MTX transport (14).

Thus, based on inhibitor specificity and regulatory mechanisms, cell-to-lumen transport of FL-MTX and fluo-cAMP appear to occur through separate and distinct luminal membrane transporters. For fluo-cAMP, sensitivity of transport to cAMP, PMEA, and AZT suggests involvement of a teleost Mrp4. Consistent with this, fluo-cAMP proved to be a substrate for transport by human MRP4 with a 10-fold higher apparent affinity than cAMP (present study and Ref. 2). Thus, it is likely that a teleost Mrp4 mediates cell-to-lumen transport of fluo-cAMP in killifish renal tubules.

Perspectives and Significance

Isolated teleost renal proximal tubules provide an important comparative model for the study of renal excretory transport. The present study gives functional evidence for a teleost isoform of Mrp4 and suggests that Mrp4 isoforms may have a regulatory impact on cellular nucleotides. In addition, the study demonstrates that fluo-cAMP may be a useful tool to investigate Mrp4 regulation and function.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported by the Boehringer Ingelheim Fonds, DFG Grants GF1211/12-1 and GF1211/13-1, National Institutes of Health Grant MDIBL-CMTS (ES-03828), the Federation of European Biochemical Societies, GlaxoSmithKline Foundation, and the Intramural Research Program of the National Institute of Environmental Health Sciences, National Institutes of Health.


   FOOTNOTES
 

Address for reprint requests and other correspondence: G. Fricker, Institute of Pharmacy and Molecular Biotechnology, INF 366, 69120 Heidelberg, Germany (e-mail: gert.fricker{at}uni-hd.de)

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


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Aslamkhan AG, Thompson DM, Perry JL, Bleasby K, Wolff NA, Barros S, Miller DS, Pritchard JB. The flounder organic anion transporter fOat has sequence, function, and substrate specificity similarity to both mammalian Oat1 and Oat3. Am J Physiol Regul Integr Comp Physiol 291: R1773–R1780, 2006.[Abstract/Free Full Text]
  2. Breen CM, Sykes DB, Baehr C, Fricker G, Miller DS. Fluorescein-methotrexate transport in rat choroid plexus using confocal microscopy. Am J Physiol Renal Physiol 287: F562–F569, 2004.[Abstract/Free Full Text]
  3. Chen ZS, Lee K, Kruh GD. Transport of cyclic nucleotides and estradiol-17-β-D-glucuronide by multidrug resistance protein 4. Resistance to 6-mercaptopurine and 6-thioguanine. J Biol Chem 276: 33747–33754, 2001.[Abstract/Free Full Text]
  4. Dean M, Allikmets R. Complete characterization of the human ABC Gene family. J Bioenerg Biomembr 33: 475–479, 2001.[CrossRef][Web of Science][Medline]
  5. Evans DH. Physiology of Fishes. Boca Raton, FL: CRC, 1975.
  6. Fricker G, Gutmann H, Droulle A, Drewe J, Miller DS. Epithelial transport of anthelmintic ivermectin in a novel model of isolated proximal kidney tubules. Pharm Res 16: 1570–1575, 1999.[CrossRef][Web of Science][Medline]
  7. Gekeler V, Ise W, Sanders KH, Ulrich WR, Beck J. The leukotriene LTD4 receptor antagonist MK571 specifically modulates MRP associated multidrug resistance. Biochem Biophys Res Commun 208: 345–352, 1995.[CrossRef][Web of Science][Medline]
  8. Gutmann H, Fricker G, Drewe J, Toeroek M, Miller DS. Interactions of HIV protease inhibitors with ATP-dependent drug export proteins. Mol Pharmacol 56: 383–389, 1999.[Abstract/Free Full Text]
  9. Gutmann H, Miller DS, Droulle A, Drewe J, Fahr A, Fricker G. P-glycoprotein- and mrp2-mediated octreotide transport in proximal tubule. Br J Pharmacol 129: 251–256, 2000.[CrossRef][Web of Science][Medline]
  10. Hagos Y, Burckhardt BC, Larsen A, Mathys C, Gronow T, Bahn A, Wolff NA, Burckhardt G, Steffgen J. Regulation of sodium-dicarboxylate cotransporter-3 from winter flounder kidney by protein kinase C. Am J Physiol Renal Physiol 286: F86–F93, 2004.[Abstract/Free Full Text]
  11. Ito K, Suzuki Horie TH, Sugiyama Y. Apical/basolateral surface expression of drug transporters and its role in vectorial drug transport. Pharm Res 22: 1559–1577, 2005.[CrossRef][Web of Science][Medline]
  12. Jedlitschky G, Keppler D. Transport of leukotriene C4 and structurally related conjugates. Vitam Horm 64: 153–184, 2002.[Web of Science][Medline]
  13. Lee K, Klein-Szanto AJP, Kruh G. Analysis of the MRP4 drug resistance profile in transfected NIH3T3 cells. J Natl Cancer Inst 92: 1934–1940, 2000.[Abstract/Free Full Text]
  14. Masereeuw R, Russel FG, Miller DS. Multiple pathways of organic anion secretion in renal proximal tubulus revealed by confocal microscopy. Am J Physiol Renal Fluid Electrolyte Physiol 271: F1173–F1182, 1996.[Abstract/Free Full Text]
  15. Masereeuw R, Terlouw SA, Van Aubel RAMH, Russel FGM, Miller DS. Endothelin B receptor-mediated regulation of ATP-driven drug secretion in renal proximal tubule. Mol Pharmacol 57: 59–67, 2000.[Abstract/Free Full Text]
  16. Miller DS, Letcher S, Barnes DM. Fluorescence imaging study of organic anion transport from renal proximal tubule cell to lumen. Am J Physiol Renal Fluid Electrolyte Physiol 271: F508–F520, 1996.[Abstract/Free Full Text]
  17. Miller DS. Protein kinase C regulation of organic anion transport in renal proximal tubule. Am J Physiol Renal Physiol 274: F156–F164, 1998.[Abstract/Free Full Text]
  18. Notenboom S, Miller DS, Smits P, Russel FMG, Masereeuw R. Role of NO in endothelin-regulated drug transport in the renal proximal tubulus. Am J Physiol Renal Physiol 282: F458–F462, 2002.[Abstract/Free Full Text]
  19. Notenboom S, Miller DS, Smits P, Russel FG, Masereeuw R. Involvement of guanylyl cyclase and cGMP in the regulation of Mrp2-mediated transport in the proximal tubule. Am J Physiol Renal Physiol 287: F33–F38. 2004.[Abstract/Free Full Text]
  20. Notenboom S, Miller DS, Kuik LH, Smits P, Russel FGM, Masereeuw R. Short-term exposure of renal proximal tubules to gentamicin increases long-term Mrp2 (ABCC2) transport function and reduces nephrotoxicant sensitivity. J Pharmacol Exp Ther 315: 912–920, 2005.[Abstract/Free Full Text]
  21. Pritchard JB, Miller DS. Comparative insights into the mechanisms of renal organic anion and cation secretion. Am J Physiol Regul Integr Comp Physiol 261: R1329–R1340, 1991.[Abstract/Free Full Text]
  22. Sampath J, Adachi M, Hatse S, Naesens L, Balzarini J, Flatley RM, Matherly LH, Schuetz JD. Role of MRP4 and MRP5 in biology and chemotherapy. AAPS Pharm Sci 4: E14, 2002.[CrossRef]
  23. Schaub TP, Kartenbeck J, Konig J, Voge O, Witzgall R, Kritz W, Keppler D. Expression of the conjugate export pump encoded by the mrp2 gene in the apical membrane of kidney proximal tubulus. J Am Soc Nephrol 8: 1213–1221, 1997.[Abstract]
  24. Schramm U, Fricker G, Wenger R, Miller DS. P-glycoprotein-mediated secretion of a fluorescent cyclosporin analogue by teleost renal proximal tubules. Am J Physiol Renal Physiol 268, F46–F52, 1995.[Abstract/Free Full Text]
  25. Schinkel AH, Jonker JW. Mammalian drug efflux transporters of the ATP binding cassette (ABC) family: an overview. Adv Drug Deliv Rev 55: 3–29, 2003.[CrossRef][Web of Science][Medline]
  26. Smeets PHE, Van Aubel RAMH, Wouterse AC, Van den Heuvel JJMW, Russel FGM. Contribution of multidrug resistance protein 2 (MRP2/ABCC2) to the renal excretion of p-aminohippurate (PAH) and identification of MRP4 (ABCC4) as a novel PAH transporter. J Am Soc Nephrol 15: 2828–2835, 2004.[Abstract/Free Full Text]
  27. Smith PM, Pritchard JB, Miller DS. Membrane potential drives organic cation transport into teleost renal proximal tubules. Am J Physiol Regul Integr Comp Physiol 255: R492–R499, 1988.[Abstract/Free Full Text]
  28. Sweet DH. Organic anion transporter (Slc22a) family members as mediators of toxicity. Toxicol Appl Pharmacol 204: 198–215, 2005.[CrossRef][Web of Science][Medline]
  29. Taggart JV, Forster RP. Renal tubular transport: effect of 2,4-dinitrophenol and related compounds on phenol red transport in the isolated tubules of the flounder. Am J Physiol 161: 167–172, 1950.[Free Full Text]
  30. Takeda M, Sekine T, Endou H. Regulation by protein kinase C of organic anion transport driven by rat organic anion transporter 3 (rOAT3). Life Sci 67: 1087–1093, 2000.[CrossRef][Medline]
  31. Terlouw SA, Graeff C, Smeets PH, Fricker G, Russel FG, Masereeuw R, Miller DS. Short- and long-term influences of heavy metals on anionic drug efflux from renal proximal tubule. J Pharmacol Exp Ther 301: 578–585, 2002.[Abstract/Free Full Text]
  32. Van Aubel RAMH, Smeets PHE, Peters JGP, Bindels RJM, Russel FGM. The MRP4/ABCC4 gene encodes a novel apical organic anion transporter in human kidney proximal tubulus: putative efflux pump for urinary cAMP and cGMP. J Am Soc Nephrol 13: 595–603, 2002.[Abstract/Free Full Text]
  33. van de Water FM, Masereeuw R, Russel FG. Function and regulation of multidrug resistance proteins (MRPs) in the renal elimination of organic anions. Drug Metab Rev 37: 443–471, 2005.[CrossRef][Web of Science][Medline]
  34. Wever KE, Masereeuw R, Miller DS, Hang XM, Flik G. Endothelin and calciotropic hormones share regulatory pathways in multidrug resistance protein 2 (Mrp2-) mediated transport. Am J Physiol Renal Physiol 292: F38–F46, 2007.[Abstract/Free Full Text]
  35. Wolff NA, Thies K, Kuhnke N, Reid G, Friedrich B, Lang F, Burckhardt G. Protein kinase C activation downregulates human organic anion transporter 1-mediated transport through carrier internalization. J Am Soc Nephrol 14: 1959–1968, 2003.[Abstract/Free Full Text]



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