Regulation of human organic anion transporter 1 by ANG II: involvement of protein kinase Cα

Abstract

Human organic anion transporter 1 (hOAT1) belongs to a family of organic anion transporters that play critical roles in the body disposition of clinically important drugs, including anti-human immunodeficiency virus therapeutics, anti-tumor drugs, antibiotics, antihypertensives, and anti-inflammatories. hOAT1 is abundantly expressed in the kidney. In the current study, we examined the regulation of hOAT1 by ANG II in kidney COS-7 cells. ANG II induced a concentration- and time-dependent inhibition of hOAT1 transport activity. Such inhibition mainly resulted from a decreased cell surface expression without a change in total cell expression of the transporter, kinetically revealed as a decreased maximal velocity without significant change in Michaelis constant. ANG II-induced inhibition of hOAT1 activity could be prevented by treating hOAT1-expressing cells with staurosporine, a general protein kinase C (PKC) inhibitor. To obtain further information on which PKC isoform mediates ANG II regulation of hOAT1 activity, cellular distribution of various PKC isoforms was examined in cells treated with or without ANG II. We showed that ANG II treatment resulted in a significant translocation of PKCα from cytosol to membrane, and such translocation was blocked by treating hOAT1-expressing cells with Gö-6976, a PKCα-specific inhibitor. We further showed that ANG II-induced inhibition of hOAT1 activity and retrieval of hOAT1 from the cell surface could also be prevented by treating hOAT1-expressing cells with Gö-6976. We concluded that ANG II inhibited hOAT1 activity through activation of PKCα, which led to the redistribution of the transporter from the cell surface to the intracellular compartments.

the organic anion transporter (OAT) family mediates the body disposition of a diverse array of environmental toxins, and clinically important drugs, including anti-human immunodeficiency virus therapeutics, anti-tumor drugs, antibiotics, anti-hypertensives, and anti-inflammatories (35–37). Therefore, understanding the regulation of these transporters has profound clinical significance.

Several OATs have been cloned and their expressions identified in distinct tissues and cell membranes (2, 4, 5, 16, 18, 19, 21, 25–27, 29, 34, 39). OAT1 and OAT3 are predominantly expressed at the basolateral membrane of kidney proximal tubule cells and the apical membrane of brain choroid plexus. OAT4 is expressed at the apical membrane of kidney proximal tubule cells and the basolateral membrane of placental trophoblast. OAT2 is expressed at the basolateral membrane of hepatocytes and is expressed in the kidney. The cellular localization of OAT2 in the kidney is still controversial. OAT5 is expressed only in the kidney. OAT6 is expressed in the olfactory mucosa, and OAT7 was identified in the liver. The cellular localization of OAT5–7 has not been defined.

In the kidney, OAT1 and OAT3 utilize a tertiary transport mechanism to move organic anions across the basolateral membrane into the proximal tubule cells for subsequent exit across the apical membrane in the urine for elimination. Through this tertiary transport mechanism, Na⁺-K⁺-ATPase maintains an inwardly directed (blood-to-cell) Na⁺ gradient. The Na⁺ gradient then drives a sodium dicarboxylate cotransporter, sustaining an outwardly directed dicarboxylate gradient that is used by a dicarboxylate/organic anion exchanger, namely OAT, to move the organic anion substrate in the cell. This cascade of events indirectly links organic anion transport to metabolic energy and the Na⁺ gradient, allowing the entry of a negatively charged substrate against both its chemical concentration gradient and the electrical potential of the cell.

Studies from our laboratory and from others have shown that protein kinase C (PKC) activation resulted in a downregulation of organic anion transport in intact kidney tubules and in cells transfected with OAT1, OAT2, and OAT4 (6, 7, 10, 19, 20, 28, 30, 33, 38, 41, 42). Kinetic analysis (38, 42) revealed that the decreased transport activity of OAT1 and OAT4 caused by activation of PKC resulted from a decreased maximal transport velocity (V_max), without significant change in the substrate affinity (K_m) of the transporter. ANG II has been shown to exert its effects through PKC (28). However, the effects of this hormone on OATs have not been reported. Furthermore, the specific PKC isoform(s) involved in the regulation of OATs has not been explored. In the current study, we investigated the role of ANG II in the function of OAT1 and the specific PKC isoform(s) involved in this process.

MATERIALS AND METHODS

Materials.

para-[³H]aminohippurate (PAH) was purchased from Perkin-Elmer Life and Analytical Sciences (Boston, MA). Normal human serum (NHS)-SS-biotin and streptavidin-agarose beads were purchased from Pierce Chemical (Rockford, IL). PKC isoform-specific antibodies were from BD Biosciences (San Jose, CA). Gö-6976 was from LC Laboratories (Woburn, MA). All other reagents were from Sigma-Aldrich (St. Louis, MO).

Cell culture.

Parental COS-7 cells were grown in DMEM supplemented with 5% FBS, 100 U/ml penicillin/ streptomycin, and 100 mg/ml glucose in a 5% CO₂ atmosphere at 37°C. COS-7 cells stably expressing human OAT 1 (hOAT1) were maintained in the same medium containing 0.5 mg/ml geneticin (G418; Invitrogen, Carlsbad, CA).

Transport measurement.

Cells plated in 48-well plates were treated with each reagent at 37°C for certain time periods as indicated. For each well, uptake solution was added. The uptake solution consisted of PBS-Ca²⁺-Mg²⁺ (in mM: 137 NaCl, 2.7 KCl, 4.3 Na₂HPO₄, 1.4 KH₂PO₄, 1 CaCl₂, and 1 MgCl₂, pH 7.4) and [³H]PAH. At the times indicated, the uptake was stopped by aspirating off the uptake solution and rapidly washing the well with ice-cold PBS. The cells were then solubilized in 0.2 N NaOH, neutralized in 0.2 N HCl, and aliquotted for liquid scintillation counting. The uptake count was standardized by the amount of protein in each well. Values are means ± SE (n = 3).

Cell surface biotinylation.

Cell surface expression levels of hOAT1 were examined using the membrane-impermeant biotinylation reagent NHS-SS-biotin (Pierce Chemical). The cells were seeded on six-well plates at 8 × 10⁵ cells/well. After 24 h, the medium was removed, and the cells were washed two times with 3 ml of ice-cold PBS, pH 8.0. The plates were kept on ice, and all solutions were kept ice-cold for the rest of the procedure. Each well of cells was incubated with 1 ml of NHS-SS-biotin (0.5 mg/ml in PBS) in two successive 20-min incubations on ice with very gentle shaking. The reagent was freshly prepared for incubation. After biotinylation, each well was rinsed briefly with 3 ml of PBS containing 100 mM glycine and then incubated with the same solution for 20 min on ice to ensure complete quenching of the unreacted NHS-SS-biotin. The cells were then dissolved on ice for 1 h in 400 μl of lysis buffer [10 mM Tris, 150 mM NaCl, 1 mM EDTA, 0.1% SDS, 1% Triton X-100, and protease inhibitors (200 μg/ml phenylmethylsulfonyl fluoride and 3 μg/ml leupeptin), pH 7.4]. The unlysed cells were removed by centrifugation at 13,000 revolutions/min at 4°C. Streptavidin-agarose beads (50 μl; Pierce Chemical) were then added to the supernatant to isolate cell membrane protein. hOAT1 was detected in the pool of surface proteins by polyacrylamide gel electrophoresis and immunoblotting using an anti-myc antibody (1:500). Myc was tagged at the carboxyl terminus of hOAT1 for its immunodetection (8).

Subcellular fractionation.

The cells grown on a 100-mm dish were homogenized in isolation buffer (25 mM HEPES, pH 7.2, 10% glycerol, 1 mM EDTA, 1 mM EGTA, and 1 mM dithiothreitol) containing complete protease inhibitor mixture. The harvested cells were sonicated two times for 10 s each, and the cell lysate were then centrifuged at 1,000 g for 10 min at 4°C to remove unbroken cells. Ultracentrifugation was followed to separate the supernatant into cytosolic and crude membrane fractions at 120,000 g for 45 min at 4°C. The particular fraction was resuspended in the sample buffer.

Electrophoresis and Western blotting.

Protein samples (100 μg) were resolved on 7.5% SDS-PAGE minigels and electroblotted on polyvinylidene difluoride membranes. The blots were blocked for 1 h with 5% nonfat dry milk in PBS-0.05% Tween, washed, and incubated overnight at 4°C with polyclonal anti-myc antibody (1:500). The membranes were washed and then incubated with goat anti-rabbit IgG conjugated to horseradish peroxidase (1:5,000), and signals were detected using a SuperSignal West Dura extended duration substrate kit (Pierce Chemical).

Data analysis.

Statistical analysis was conducted using Student's paired t-test for comparing two treatments. A one-way ANOVA followed by a Dunnett's post hoc test was used for comparing among more than two treatments. A P value <0.05 was considered significant.

RESULTS

Effects of ANG II on hOAT1 activity.

We examined whether treatment with ANG II could affect hOAT1 transport activity in COS-7 cells. Because the hOAT1 expression vector for the current study does not contain the promoter region of hOAT1, the long-term regulation at the transcriptional level cannot be investigated. We only focused on the short-term regulation of the transporter (within a time frame of 2 h). ANG II induced a concentration-dependent inhibition of PAH uptake with the concentration of maximum inhibition at 500 nM (Fig. 1A). With 500 nM as the working concentration, we then performed study on time-dependent inhibition of PAH uptake (Fig. 1B). ANG II seemed to cause maximum inhibition after 30 min treatment. Longer treatment with ANG II at 1 and 2 h did not result in further inhibition. To examine the mechanism of ANG II-induced inhibition of hOAT1 activity, we determined [³H]PAH uptake at different substrate concentrations. An Eadie-Hofstee analysis of the derived data (Fig. 1C) showed that pretreatment with ANG II resulted in a decreased V_max (0.569 ± 0.042 pmol·μg⁻¹·3 min⁻¹ with untreated cells and 0.293 ± 0.011 pmol·μg⁻¹·3 min⁻¹ in the presence of ANG II) with no significant change in K_m for PAH (20.2 ± 1.5 μM with untreated cells and 18.3 ± 0.08 μM in the presence of ANG II). Determination of the protein concentrations in control cells confirmed that ANG II treatment did not change the total protein content of the cultures (data not shown).

Fig. 1.

Effect of ANG II on human organic anion transporter 1 (hOAT1). hOAT1-expressing cells were treated with ANG II at various concentrations (A) or at various periods of time (B), followed by para-[³H]-aminohippurate (PAH) uptake (3 min, 20 μM). Uptake activity was expressed as a percentage of the uptake measured in untreated cells. The results represent data from 3 experiments. The uptake values in mock cells (parental COS-7 cells) were subtracted. Values are means ± SE (n = 3 experiments). C: effect of ANG II on the kinetics of PAH transport. COS-7 cells expressing hOAT1 were pretreated with or without ANG II (500 nM) for 1 h, and initial uptake (3 min) of [³H]PAH was measured at 8–800 μM PAH. Data represent uptake in hOAT1-transfected cells minus uptake in mock cells (parental COS-7 cells). Values are means ± SE (n = 3). V, velocity; S, substrate concentration.

Effect of ANG II on hOAT1 expression.

A decreased V_max could be affected by either a reduced number of the transporter at the cell surface or a reduced transporter turnover number (1, 9, 12, 31, 43). To differentiate between these possibilities, we determined transporter expression both at the cell surface and in the total cell lysates. We showed that ANG II treatment resulted in a reduced cell surface expression of hOAT1 without affecting the total cell expression of the transporter (Fig. 2).

Fig. 2.

Effect of ANG II on cell surface and total cell expression of hOAT1. A: Western blot analysis of cell surface expression of hOAT1. Top: COS-7 cells stably expressing hOAT1 were treated with or without ANG II (500 nM, for 15 and 30 min) and biotinylated, and the labeled cell surface proteins were precipitated with streptavidin beads and separated by SDS-PAGE, followed by Western blotting with anti-myc antibody (1:500). Bottom: the intensity of the transporter expression from the experiment shown at top and other experiments was quantified. *P < 0.05, significantly different from untreated cells. B: Western blot analysis of total cell expression of hOAT1 in cells treated with or without ANG II (500 nM, for 15 and 30 min). Top: COS-7 cells stably expressing hOAT1 were treated with or without ANG II (500 nM, for 15 and 30 min). Cells were lysed, and their proteins were separated by SDS-PAGE, followed by Western blotting with anti-myc antibody. Bottom: the intensity of the transporter expression from the experiment shown at top and other experiments was quantified.

ANG II regulation of hOAT1 activity through PKC.

ANG II was shown to exert its effects through PKC. To determine whether ANG II regulates hOAT1 function through PKC, we treated hOAT1-expressing COS-7 cells with ANG II in the presence of staurosporine, a general PKC inhibitor. As shown in Fig. 3, staurosporine efficiently reversed the inhibitory effect of ANG II on hOAT1 activity.

Fig. 3.

Effect of protein kinase C (PKC) inhibitor staurosporine (St) on ANG II-induced inhibition of hOAT1 activity. hOAT1-expressing cells were pretreated with staurosporine (2 μM, 5 min) followed by incubation with ANG II (500 nM, 30 min) in the presence or absence of staurosporine (2 μM). The uptake of [³H]PAH (3 min, 20 μM) was then performed. Results represent data from three experiments. The uptake values in mock cells (parental COS-7 cells) were subtracted. Values are means ± SE (n = 3).

Identification of PKC isoforms involved in ANG II effect.

The PKC isoforms are divided into three categories based upon the cofactors that are required for optimal catalytic activity. Conventional PKCs (α, β, and γ) are calcium-dependent and are stimulated by a second messenger, diacylglycerol. Novel PKCs (δ, ɛ, η, and θ) are also activated by diacylglycerol but are calcium independent. Atypical PKCs (ζ and λ/ι) require neither calcium nor diacylglycerol for optimal activity. Although most cells express more than one type of PKC, differences among the isoforms with respect to activation conditions and subcellular locations suggest that individual PKC isoforms mediate distinct cellular processes in a cell type-dependent manner. In COS-7 cells, it was shown that PKCα, PKCδ, PKCι, and PKCɛ were abundantly expressed (3). Our Western blotting showed that, in contrast to other PKC isoforms, treatment of cells with ANG II resulted in a significant translocation of PKCα from cytosol to membrane fraction (Fig. 4A), and such translocation was blocked by treating the cells with Gö-6976, a PKCα-specific inhibitor (24) (Fig. 4B).

Fig. 4.

Western blot analysis of subcellular fraction expression of PKC isoforms in cells treated with or without ANG II (500 nM, 30 min). A: COS-7 cells stably expressing hOAT1 treated with or without ANG II were lysed and fractionated in crude membrane and cytosol, and the proteins were separated by SDS-PAGE, followed by Western blotting with PKC isoform-specific antibodies. Total cell lysate was also prepared. B: COS-7 cells stably expressing hOAT1 were treated with or without ANG II in the presence and absence of PKCα specific inhibitor Gö-6976. Cells were then lysed and fractionated in crude membrane and cytosol, and the proteins were separated by SDS-PAGE, followed by Western blotting with PKCα-specific antibody. Total cell lysate was also prepared.

ANG II regulation of hOAT1 activity and cell surface expression through PKCα.

The involvement of PKCα in ANG II regulation of hOAT1 was further investigated through functional assay in conjunction with cell surface biotinylation study. We treated hOAT1-expressing COS-7 cells with ANG II in the presence of PKCα-specific inhibitor Gö-6976. As shown in Fig. 5, both ANG II-induced inhibition of hOAT1 activity and the retrieval of hOAT1 from cell surface were reversed by Gö-6976.

Fig. 5.

Effect of PKCα specific inhibitor Gö-6976 on ANG II-mediated hOAT1 activity and cell surface expression. A: effect of Gö-6976 on ANG II-induced inhibition of hOAT1 activity. hOAT1-expressing cells were pretreated with Gö-6976 (1 μM, 20 min) followed by incubation with ANG II (500 nM, 30 min). Uptake of [³H]PAH (3 min, 20 μM) was then performed. results represent data from three experiments. Uptake values in mock cells (parental COS-7 cells) were subtracted. *P < 0.05, significantly different from untreated cells. Values are means ± SE (n = 3). B: effect of Gö-6976 on ANG II-induced retrieval of hOAT1 from cell surface. hOAT1-expressing cells were pretreated with Gö-6976 (1 μM, 20 min) followed by incubation with ANG II (500 nM, 30 min). Cells were then biotinylated, and the labeled cell surface proteins were precipitated with streptavidin beads and separated by SDS-PAGE, followed by Western blotting with anti-myc antibody (1:500). Total cell expression was also shown as control.

DISCUSSION

Previously, we and others (6, 7, 10, 19, 20, 28, 30, 33, 38, 41, 42) showed that OAT1 activity was inhibited by activation of PKC in cultured cells and in proximal tubules. ANG II was shown to exert its various actions through PKC such as its regulation of ATP-sensitive potassium channels (23). In the current study, we showed that ANG II regulated OAT1 activity through PKC. Furthermore, we showed that the specific PKC isoform involved in such regulation is PKCα.

ANG II induced a dose- and time-dependent inhibition of uptake of PAH mediated by hOAT1 in COS-7 cells (Fig. 1, A and B). Our kinetic analysis of the inhibition of hOAT1 activity by ANG II (Fig. 1C) showed that the reduced transport activity was contributed by a reduced V_max without affecting the K_m for the substrates. V_max can be affected by either the number of the transporter at the cell surface or the transporter turnover number (1, 9, 12, 31, 43). To differentiate between these possibilities, we determined the effect of ANG II on hOAT1 expression both at the cell surface and in the total cell lysates. Our results showed that ANG II treatment resulted in a reduced cell surface expression of hOAT1 without affecting its total cell expression (Fig. 2), suggesting that a redistribution of hOAT1 from cell surface to the intracellular compartments occurred during such treatment. Such redistribution was observed previously from other membrane transporters (11, 17). In response to stimuli, these transporters were removed from the cell surface to intracellular compartments, where they waited for the next signal to recycle back to the cell surface. We further showed that ANG II-induced inhibition of hOAT1 activity could be prevented by treating hOAT1-expressing cells with staurosporine (Fig. 3). Staurosporine exerts its inhibition effect on PKC by binding to the ATP-binding site on the catalytic domain of the kinase (22). Unfortunately, the ATP-binding site of PKC shares great homology with the ATP-binding sites of other protein kinases, making it difficult for staurosporine to achieve high selectivity for PKC (22). However, our further studies provided strong evidence that the PKC mediated the effect of ANG II. This conclusion is based on the following points. First, ANG II induced significant translocation of PKCα from cytosol to plasma membrane (Fig. 4A), and such translocation was blocked in the presence of PKCα-specific inhibitor Gö-6976 (Fig. 4B). Second, ANG II-induced inhibition of hOAT1 activity could be reversed by treating hOAT1-expressing cells with Gö-6976 (Fig. 5A). Third, ANG II-induced retrieval of hOAT1 from the cell surface was prevented by treating hOAT1-expressing cells with Gö-6976 (Fig. 5B). Therefore, this is the first study to demonstrate that ANG II inhibited hOAT1 activity through activation of PKCα, which led to the redistribution of the transporter from the cell surface to the intracellular compartments.

Our recently published work (40) demonstrated that hOAT1 undergoes constitutive trafficking between the cell surface and intracellular compartments with both the internalization rate and the recycling rate of 10%/5 min. Activation of PKC only accelerated hOAT1 internalization without affecting its recycling. Therefore, there is always a certain amount of hOAT1 at the cell surface because of the recycling, even with the maximum stimulation of PKC. This is consistent with the observation in our current studies that the ANG II could not completely abolish hOAT1 activity but showed ∼30% inhibition. In vivo, regulation at such a moderate scale may play an important role in providing quick and efficient fine tuning in the body response to environmental changes.

It was shown (32) that, in rats with bilateral ureteral obstruction (BUO), elimination of drugs was impaired partly because of a redistribution of OAT1 from the cell surface to the intracellular compartment. BUO is a serious and common clinical condition and an important cause of acute renal failure (13, 32). In BUO, ANG II has elevated levels of expression (13–15). Therefore, ANG II may affect OAT1 cellular distribution through such a signaling pathway. Our current studies may provide important insight in the molecular, cellular, and clinical bases underlying BUO.

GRANTS

This work was supported by National Institutes of Health Grants R01-DK 60034 and R01-GM-079123 (to G. You).