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Am J Physiol Renal Physiol 293: F21-F27, 2007. First published February 27, 2007; doi:10.1152/ajprenal.00043.2007
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EDITORIAL FOCUS

Role of MAPK and PKA in regulation of rbOCT2-mediated renal organic cation transport

Sunhapas Soodvilai,1,2 Atip Chatsudthipong,2 and Varanuj Chatsudthipong2

1Faculty of Pharmaceutical Sciences, Ubon Rajathanee University, Ubon Ratchathani, and 2Department of Physiology, Faculty of Science, Mahidol University, Bangkok, Thailand

Submitted 25 January 2007 ; accepted in final form 23 February 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The effects of protein kinases MAPK and PKA on the regulation of organic cation transporter 2 (OCT2) were investigated both in a heterologous cell system [Chinese hamster ovary (CHO-K1) cells stably transfected with rabbit (rb)OCT2] and in native intact rabbit renal proximal S2 segments. Inhibition of MEK (by U-0126) or PKA (by H-89) reduced transport activity of rbOCT2 in CHO-K1 cells. The inhibitory effect of U-0126 combined with H-89 produced no additive effect, indicating that the action of PKA and MAPK in the regulation of rbOCT2 is in a common pathway. Activation of PKA by forskolin stimulated rbOCT2 activity, and this stimulatory effect was eliminated by H-89, indicating that the stimulation required PKA activation. In S2 segments of rabbit renal proximal tubules, activation of MAPK (by EGF) and PKA (by forskolin) stimulated activity of rbOCT2, and this activation was abolished by U-0126 and H-89, respectively. This is the first study to show that MAPK and PKA are involved, apparently in a common pathway, in the regulation of OCT2 activity in both a heterologous cell system and intact renal proximal tubules.

kidney; proximal tubule; epidermal growth factor; H-89; tetraethylammonium

PROXIMAL TUBULES PLAY AN ESSENTIAL role in the clearance of endogenous metabolites, xenobiotics, and therapeutic drugs from the body. Many of these compounds are organic cations (or weak organic bases) that have a net positive charge on the amine nitrogen at physiological pH and are transported by renal organic cation transporters (OCTs). Endogenous substrates such as epinephrine, histamine, dopamine, N-methylnicotinamide (NMN), and choline are transported by OCTs (1, 13, 29). Therapeutic drugs that are transported by OCTs include cimetidine, procainamide, metformin, amantadine, and cisplatin (4, 6, 13).

These renal OCTs play a major role in the detoxification and elimination of xenobiotics from systemic circulation (19, 27), and thus they are an important determinant of drug efficacy and toxicity. The process of renal tubular secretion of organic cations involves transport from the peritubular fluid across the basolateral membrane, accumulation in the renal epithelial cell, and subsequent extrusive transport across the apical membrane into the tubular fluid. Basolateral entry of organic cations into the cell is via electrogenic-facilitated diffusion mediated by OCTs. This step is driven by an inside-negative electrical potential difference. Extrusive transport occurs either by exchange for H+ or by ATP-driven multidrug resistance transporters (25, 30).

Several members of the OCT family located at the basolateral membrane have been cloned and characterized, including OCT1, OCT2, and OCT3 (6, 8, 9, 14, 17, 26, 32, 33). The molecular structure of all OCTs includes 12 transmembrane domains (TMDs) with a large hydrophilic extracellular loop between TMD1 and TMD2 and a number of intracellular protein kinase phosphorylation sites (5, 9).

In most species, OCT2 is extensively expressed in the kidney. Functional studies have shown that OCT2 is a very important player in basolateral organic cation transport in renal proximal tubules (24) and the dominant OCT in the basolateral membrane of the S2 segment (34). Although renal OCTs have been cloned and characterized, the regulation of organic cation transport, especially that mediated by OCT2, is still not clear and remains controversial. Protein kinases have been reported to regulate the OCT2-mediated organic cation transport. Activation of PKA by forskolin or the cAMP analog 8-Br-cAMP leads to a stimulation of 4-[4-(dimethylamino)styryl]-N-methylpyridinium (ASP+) uptake in IHKE-1 cells (12). In contrast, activation of PKA in isolated human renal proximal tubules as well as in HEK293 cells stably expressing human OCT2 (hOCT2) results in an inhibition of ASP+ uptake (2). Activation of PKC inhibits ASP+ uptake in LLC-PK1 cells and in isolated human renal proximal tubules (12, 18) but stimulates tetraethylammonium (TEA) and ASP+ uptake in the S2 segment of rabbit renal proximal tubules and in IHKE-1 cells, respectively (11, 12). Human OCT2 activity is inhibited by phosphatidylinositol-3-kinase (PI3-K) but is activated by a calmodulin-dependent protein kinase (2).

Previous studies have demonstrated the involvement of PKA and mitogen-activated protein kinase (MAPK) in the regulation of organic anion transporter 1 (OAT1)- and organic anion transporter 3 (OAT3)-mediated organic anion transport in S2 segments of rabbit renal proximal tubules (20, 22). Because PKA is apparently involved in the regulation of OCT2 in a manner similar to its regulation of OAT1 and OAT3 and MAPK is involved in the regulation of OAT1 and OAT3 by interaction with the PKA signaling pathway, we were interested in examining whether regulation of OCT2-mediated organic cation transport involved a pathway similar to that of OAT-mediated organic anion transport. We investigated the roles of PKA and MAPK in the regulation of organic cation transport by rabbit OCT2 (rbOCT2) stably expressed in CHO-K1 cells and by native rabbit proximal tubules. The present study indicates that activation of PKA and MAPK lead to stimulation of OCT2 activity.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials

CHO-K1 cells transfected with rabbit OCT2 were obtained from Dr. Stephen H. Wright (Dept. of Physiology, Univ. of Arizona). [3H]tetraethylammonium (TEA; 20.0 Ci/mmol) was from the Synthesis Core of the Southwest Environmental Health Science Center, University of Arizona. EGF and forskolin were purchased from Sigma (St. Louis, MO), H-89 and dibutyryl-cAMP (db-cAMP) from Calbiochem (San Diego, CA), and U-0126 from Promega (Madison, WI). Cell culture media and molecular biology reagents were purchased from Sigma or Life Technologies (Gaithersburg, MD).

Cell Culture and Transport Assay

CHO-K1 cells transfected with rbOCT2 were maintained in culture at 37°C under a humidified 5% CO2-95% air atmosphere. After removal of the culture media, cells were washed twice with Waymouth's buffer [WB; (in mM) 135 NaCl, 13 HEPES, 28 D-glucose, 5 KCl, 1.2 MgCl2, 2.5 CaCl2, 0.8 MgSO4, pH adjusted to 7.4 with NaOH] and then incubated for 30 min in WB. Cells were then treated with protein kinase inhibitors or activators as indicated in the figure legends and then washed three times with WB. Then, the cells were incubated with WB containing [3H]TEA (~0.05 µM) for 1 min. Uptake was stopped by removing the transport buffer and then rinsing the cells with three successive washes of 2 ml ice-cold WB. Cells were solubilized with 1% SDS in 0.2 M NaOH, neutralized with 0.2 M HCl, and transferred to scintillation vials for measurement of accumulated radioactivity. Uptake rates are expressed as moles per square centimeter of surface area of the confluent monolayer.

Kinetics of rbOCT2 Activity Following Inhibition of MAPK

Transfected CHO-K1 cells were preincubated with the MAPK inhibitor U-0126 at 10 µM for 30 min. The cells were washed twice with WB. Then, the WB containing ~0.06 µM [3H]TEA and unlabeled TEA of varying concentrations was added and the cells were incubated for 1 min. Uptake was stopped by removing the transport buffer and then rinsing the cells with three successive 2-ml washes of ice-cold WB. The cells were solubilized with 1% SDS in 0.2 M NaOH, neutralized with 0.2 M HCl, and transferred to scintillation vials for measurement of accumulated amounts of [3H]TEA as described above. Uptake rates are expressed as moles per square centimeter of surface area of the confluent monolayer. The hyperbolic inhibition of [3H]TEA transport was described by Michaelis-Menten kinetics of competitive interaction between labeled and unlabeled TEA as defined by the following equation (16)

Formula
where J is the rate of [3H]TEA transport from the concentration of labeled substance [*T]; Jmax is the maximum rate of TEA transport; Kt is the TEA concentration that results in half-maximum transport (Michaelis constant); [T] is the concentration of unlabeled TEA in the transport reaction; and C is a constant that represents the component of total TEA uptake that is not saturated (over the range of substrate concentration tested) and presumably reflects the combined influence of diffusive flux, nonspecific binding, and/or incomplete rinsing of the cell layer.

Preparation of Isolated Tubules

New Zealand White rabbits [1.5–2.0 kg, National Laboratory Animal Center (NLAC), Bangkok, Thailand] were killed by intravenous injection of pentobarbital sodium in accordance with the principles and guidelines of the Laboratory Animal Ethical Committee of Mahidol University (Bangkok, Thailand). Kidneys were flushed via the renal artery with an ice-chilled HEPES-sucrose buffer containing 250 mM sucrose and 10 mM HEPES, adjusted to pH 7.4 with Tris, and bubbled with 100% O2 before use. Kidneys were then gently removed and sliced transversely using a single-edge razor. A kidney slice was transferred to a petri dish on ice that contained a standard buffer solution (in mM: 110 NaCl, 25 NaHCO3, 5 KCl, 2 NaH2PO4, 1 MgSO4, 1.8 CaCl2, 10 Na-acetate, 8.3 D-glucose, 5 L-alanine, 0.9 glycine, 1.5 lactate, 1 malate, and 1 sodium citrate). This buffer solution was aerated continuously with 95% O2-5% CO2 to maintain pH at 7.4. The osmolality of the solutions averaged ~290 mosmol/kgH2O. S2 segments of proximal tubules were individually dissected from the cortical zone without the aid of enzymatic agents, as reported previously (3). These segments were isolated from proximal tubules by teasing out a 1.0- to 1.4-mm length of straight tubule starting from the cortical surface of the kidney. All dissections were performed at 4°C.

Measurement of Transport of [3H]TEA in S2 Segments of Nonperfused, Isolated Renal Proximal Tubules

These experiments were performed in a manner similar to that reported previously (3). Briefly, an appropriate number of tubule segments (3–5 for each condition to be studied) were teased from fresh renal tissue and maintained at 4°C under 95% O2-5% CO2 in standard buffer solution covered with a layer of mineral oil to prevent evaporation before use. The tubules were then transferred to bathing solutions containing different activators or inhibitors of protein kinases at 37°C and preincubated for 20 min. At the end of the preincubation period, each tubule was transferred to new bathing medium containing [3H]TEA (0.5 µM) for 1 min, a time period chosen to permit an adequate approximation of the initial rate of TEA uptake. Uptake was stopped by transferring each tubule into an individual well containing 7 µl of 1 M NaOH. Substrate accumulated by each tubule was determined by liquid scintillation counting of the total NaOH extraction solution. In each experiment, at least three tubules were used to determine transport for each condition tested. Transport rates were normalized to tubule surface area based on tubule lengths and average diameters. Control and experimental uptake studies were determined alternately and sequentially in tubules from the same kidney.

Data Analysis

Data are reported as means ± SE. For cell culture studies, n represents the number of experiments. In each experiment using CHO-K1 cells, a minimum of three wells was used to generate each data point. For isolated tubule experiments, n represents the number of experiments (each one using tubules from a different rabbit). The differences between rates of uptake for the different regimens were analyzed using one-way ANOVA. Differences were considered statistically significant when P < 0.05.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Studies Employing rbOCT2 Expressed in CHO-K1 Cells

Effect of MAPK on initial rate of TEA uptake. Previously, we reported that MAPK is involved in the regulation of OAT3-mediated organic anion transport (22). To examine the involvement of MAPK on rbOCT2-mediated TEA transport, we tested the effect of U-0126, an inhibitor of MEK. As shown in Fig. 1A, preincubation of CHO-K1 cells with U-0126 for 30 min resulted in a concentration-dependent inhibition of TEA uptake over the concentration range of 1–100 µM.


Figure 1
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  		Fig. 1. Effect of U-0126 (A) and H-89 (B) on initial rate of uptake of tetraethylammonium (TEA) in Chinese hamster ovary (CHO-K1) cells expressing rabbit organic anion transporter 2 (rbOCT2). Cells were preincubated with medium containing various concentrations of U-0126 or H-89 for 30 min and then incubated with medium containing [3H]TEA for 1 min. Each point represents the mean ± SE of uptake measured in triplicate from 3 independent experiments, expressed as a percentage of control uptake. *P < 0.05 compared with control value.

 
Effect of PKA on rbOCT2-mediated TEA uptake. To investigate the involvement of the PKA in the regulation of rbOCT2, we examined the effects of both PKA inhibitor (H-89) and PKA activator (forskolin) on the initial rate of TEA uptake in CHO-K1 cells stably expressing rbOCT2. Figure 1B shows that the inhibitory effect of H-89 was concentration dependent at the concentrations between 0.1 and 10 µM. Figure 2A shows that forskolin (1 µM) significantly increased the initial rate uptake of TEA (~20% of control level). Increasing the concentration of foskolin up to 100 µM did not produce any further increase in TEA uptake. The cAMP analog db-cAMP (100 µM) produced a stimulatory effect similar to that of forskolin (1 µM). To examine whether the stimulatory effect produced by forskolin reflected activation of PKA, we examined the effect of H-89 on the forskolin-induced stimulation of basolateral TEA uptake. Cells were preincubated with 1 µM H-89 for 20 min followed by 10 µM forskolin plus 1 µM H-89 for 10 min. Uptake of TEA in the presence of both forskolin and H-89 was significantly less than that of forskolin alone (Fig. 2B). These results support the notion that upregulation of rbOCT2-mediated organic cation transport induced by forskolin involves PKA activation.


Figure 2
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  		Fig. 2. Effect of protein kinase A activators [forskolin and dibutyryl (db)-cAMP; A] and forskolin and H-89 (B) on initial rate of uptake of TEA in CHO-K1 cells expressing rbOCT2. Cells were preincubated for 30 min under the following conditions: 1) control medium; 2) control medium for 20 min followed by medium containing forskolin for 10 min; 3) control medium for 20 min followed by medium containing db-cAMP for 10 min; 4) control medium containing H-89 for 30 min; and 5) control medium for 20 min followed by medium containing forskolin plus H-89 for 10 min. They were then incubated with medium containing [3H]TEA for 1 min. The conditions of preincubation are given under each bar. The rates of TEA uptake were based on 1-min uptake. Results are means ± SE of uptake measured in triplicate wells from 3 independent experiments, expressed as a percentage of control uptake. *P < 0.05 compared with control. **P < 0.05 compared with forskolin-treated group.

 
Effect of MAPK and PKA inhibition on initial rate of TEA uptake. We further investigated whether the action of PKA occurred via the MAPK pathway. Preincubation of cells with U-0126 and H-89 alone inhibited TEA uptake by 20 and 25% of control, respectively (Fig. 3). Exposing the cells to both U-0126 and H-89 simultaneously, at concentrations that produced a maximum response, did not reduce TEA uptake more than exposure to U-0126 or H-89 alone (data not shown). Because the lack of an additive effect might have resulted from complete inhibition by one of these inhibitors alone (especially H-89 as shown in Fig. 1B), we also exposed the cells to both U-0126 and H-89 simultaneously at concentrations at which each produced submaximal inhibition, but the combination still did not reduce TEA uptake more than with U-0126 or H-89 alone. The lack of additive effect suggests that the actions of PKA and MAPK in the regulation of OCT2-mediated organic cation transport are in the same pathway. We next determined whether the inhibition of MAPK by U-0126 is involved in foskolin-induced stimulation of TEA uptake by adding U-0126 in the presence of forskolin. TEA uptake with forskolin plus U-0126 was not significantly different from that with forskolin alone (Fig. 4). These results suggest that MAPK may be activated upstream of PKA in the regulation of rbOCT2-mediated TEA transport.


Figure 3
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  		Fig. 3. Effect of U-0126 and H-89 on initial rate of uptake of TEA in CHO-K1 cells expressing rbOCT2. The conditions of preincubations are given under each bar (see details in text). Results are means ± SE for 3 experiments (triplicate measurements) and expressed as a percentage of control. *P < 0.05 compared with control value.

 

Figure 4
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  		Fig. 4. Effect of U-0126 on the stimulatory effect of forskolin (Forsk) on initial rate of uptake of TEA in CHO-K1 cells expressing rbOCT2. The conditions of preincubations are given under each bar. The cells were preincubated for 30 min under four conditions: 1) control medium; 2) control medium for 20 min followed by medium containing 10 µM forskolin for 10 min; 3) control medium containing 10 µM U-0126 for 30 min; and 4) control medium containing 10 µM U-0126 for 20 min followed by medium containing 10 µM forskolin plus 10 µM U-0126 for 10 min. The rates of TEA uptakes were based on 1-min uptake. Results are means ± SE of uptake measured in triplicate wells from 3 independent experiments, expressed as a percentage of control uptake. *P < 0.05 compared with control.

 
Kinetic studies. To determine whether MAPK and PKA regulated rbOCT2-mediated organic cation transport by altering the affinity of the transporter for the substrate (Kt) or the number of rbOCT2 transporters (Jmax) functionally expressed in the membrane, Kt and Jmax of rbOCT2 were determined after the cells were preincubated with U-0126. Because MAPK and PKA appear to be in the same pathway in the regulation of OCT2, we chose to use just one inhibitor (U-0126) that apparently inhibits upstream of H-89 in the pathway. As shown in Fig. 5, U-0126 significantly decreased Jmax from 66.65 ± 1.21 to 34.19 ± 1.04 pmol·min–1·cm–2, whereas it had no effect on Kt, suggesting that U-0126-induced inhibition of the organic cation uptake is due to a decrease in the membrane expression of rbOCT2 not in the affinity of the transporter for the substrate.


Figure 5
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  		Fig. 5. Kinetics of TEA transport by rbOCT2 expressed in CHO-K1 cells. Uptake of radiolabeled TEA ([3H]TEA) is shown on the ordinate vs. concentrations of unlabeled TEA in bathing medium on the abscissa. bullet, rbOCT2-mediated TEA transport in control cells; {circ}, transport in cells pretreated with U-0126 for 30 min; Jmax, maximum rate of TEA transport; Kt, TEA concentration that results in half-maximum transport.

 
Studies Employing S2 Segment of Rabbit Renal Proximal Tubules

Effect of MAPK and PKA on initial rate of TEA uptake. To verify that the regulatory mechanism obtained using heterologous cells in culture was applicable to intact native tissue, we examined the effect of EGF, a MAPK activator, and forskolin on the initial rate of TEA uptake in S2 segments of rabbit renal proximal tubules. The tubules were preincubated in medium containing 50 ng/ml of EGF or 10 µM forskolin for 10 min followed by a 1-min incubation with [3H]TEA. Exposure to EGF or forskolin resulted in increased TEA uptake by 30 or 20%, respectively (Fig. 6). We also determined the effect on rbOCT2-mediated TEA uptake of MAPK and PKA inhibition by U-0126 and H-89, respectively. Tubules were preincubated with U-0126 or H-89 for 20 min followed by incubation with [3H]TEA for 1 min. As shown in Fig. 6, exposure to U-0126 led to significant inhibition of TEA uptake by ~30% from control. Preincubation with H-89 also led to significant inhibition of TEA uptake (~50% of control).


Figure 6
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  		Fig. 6. Effect of protein kinase activators and inhibitors on initial rate of TEA uptake. The conditions of preincubations are given under each bar (see details in text). Results are means ± SE expressed as a percentage of control. Mean control initial rate of TEA uptake was 138 ± 30. The numbers inside the bars indicate number of experiments. *P < 0.05 compared with control value.

 
Effect of inhibition of MAPK on initial rate of TEA uptake in the presence of EGF. We hypothesized that the stimulatory effect of EGF occurred via the MAPK pathway as it does in the regulation of OAT3. To determine whether this was the case, we examined the effect U-0126 on the initial rate of TEA uptake in the presence of EGF. Tubules were preincubated with 10 µM U-0126 for 10 min followed by 50 ng/ml of EGF plus 10 µM U-0126 for 10 min. As shown in Fig. 7A, exposure to EGF alone stimulated rbOCT2-mediated TEA uptake, whereas exposure to U-0126 alone inhibited TEA uptake. The stimulatory effect of EGF was antagonized by U-0126. These results support the concept that EGF stimulates TEA uptake by activation of the MAPK pathway.


Figure 7
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  		Fig. 7. Effect of EGF and U-0126 (A) and forskolin (B) on initial rate of TEA uptake in nonperfused S2 segments of rabbit renal proximal tubule. The concentrations of EGF and U-0126 were 50 ng/ml and 10 µM, respectively. The conditions of preincubation are given under each bar. For EGF and U-0126 treatment, the tubules were preincubated for 10 and 20 min, respectively. For forskolin, tubules were preincubated for 10 min. For EGF plus U-0126 treatment, the tubules were first preincubated for 10 min in 10 µM U-0126, followed by 10 min in both EGF and U-0126, followed by a 1-min incubation in control medium containing [3H]TEA. Results are means ± SE expressed as a percentage of control for 6 (A) and 4 experiments (B). Mean control initial rate of TEA uptake was 101 ± 14 (A) and 92 ± 6 fmol·min–1·mm–1 (B). *P < 0.01 compared with control. **P < 0.05 compared with treated group.

 
Effect of inhibition of PKA on initial rate of TEA uptake in the presence of forskolin. To determine whether the stimulatory effect of forskolin on rbOCT2-mediated TEA transport in native tissue was mediated by PKA activation, the tubules were preincubated with 5 µM H-89 for 10 min followed by 10 µM forskolin plus 5 µM H-89 for 10 min. Forskolin stimulated TEA uptake by 40% of control, and this stimulation was antagonized by H-89 (Fig. 7B). These data indicate that the stimulatory effect of forskolin is mediated by the activation of PKA.


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
OCT2 plays a major role in the renal tubular secretion of endogenous and therapeutic xenobiotic compounds. Several studies have shown that OCT2 is the major determinant of therapeutic cationic drug efficacy and toxicity (13, 30, 34). Therefore, knowledge of OCT2 regulation is important because modulation of OCT2 transporter activity will influence renal clearance of therapeutic drugs. Although some aspects of regulation of OCT2 have been studied, the results remain controversial. Differences in the local environment of immortalized cells and experimental protocols may markedly alter regulation in these systems. Therefore, regulation in heterologous cell systems needs to be confirmed with studies in native tissues. The rabbit offers an excellent experimental system for such studies because rbOCT2, stably expressed in CHO-K1 cells, is available and results of studies with these cells can be compared with results of similar studies with intact isolated rabbit renal proximal tubules.

Mediators, including PKC, PKA, MAPK, PI3-K, and calmodulin-dependent protein kinase have been reported to be involved in the regulation of OCT2 (5). These protein kinases are also mediators in the regulation of OAT1- and OAT3-mediated organic anion transport (20–23). Because of the structural similarity between OCT2 and OAT (12 TMDs and various phosphorylation sites at intracellular loop), it appeared possible that the regulatory mechanisms of OCT2-mediated organic cation transport were similar to those of OAT1- and OAT3-mediated organic anion transport. Previous studies reported that PKA and MAPK play a crucial role in the regulation of OAT1- and OAT3-mediated organic anion transport in heterologous cell systems and in intact renal proximal tubules (20, 22). MAPK has been reported to regulate the activity of several transport proteins, including not only OAT1 and OAT3 but also the epithelium sodium channel (ENaC) and sodium/hydrogen exchanger-3 (NHE3) (7, 15, 20, 22). The aim of the present study was to determine whether regulation of basolateral organic cation transport mediated by OCT2 involved the activities of PKA and MAPK. Therefore, we investigated the effects of modulation of PKA and MAPK activity on rbOCT2-mediated TEA transport in both a heterologous cell system (CHO-K1 cells stably expressing rbOCT2) and S2 segments of rabbit renal proximal tubules.

Inhibition of MEK with its specific inhibitor, U-0126, reduced TEA uptake in CHO-K1 cells stably expressing rbOCT2 and in S2 segments of rabbit proximal tubules, in which organic cation transport activity is dominated by OCT2 (13, 31, 33). These data suggest that the MAPK pathway is involved in setting the basal level of transport activity in OCT2, whether it is expressed in a heterologous cell system or in native tissue. A previous study reported that MAPK is not involved in the regulation of OCT2 activity, as evidence by the lack of effect of 1 µM U-0126 on basal ASP+ transport by hOCT2 expressed in HEK-293 cells (2). However, in the current study, we found that increasing the concentration of U-0126 from 1 to 10 µM decreased the basal activity of rbOCT2 in CHO-K1 cells (Fig. 1A). The concentration used in the previous study may have been too low to inhibit MEK activity, as 1 µM U-0126 is the IC50 for inhibition of MEK.

In the present study, stimulation of MAPK by EGF increased TEA uptake via OCT2 in rabbit proximal renal tubules, and this effect was blocked by inhibition of MEK with 10 µM U-0126 (Fig. 7A). These results support the contention that the MAPK pathway regulates OCT2-mediated organic cation transport in intact renal proximal tubules. Although TEA is not a specific substrate for OCT2 and rabbit renal proximal tubules express several OCTs (including OCT1, OCT2, and OCT3), the activity of OCT2 is functionally dominant in the S2 segment compared with other OCTs (34). Thus TEA transport in the S2 segment of rabbit renal proximal tubules is mediated mainly by OCT2, rather than OCT1 or OCT3. Nevertheless, we cannot rule out small contributions of OCT1 and OCT3 in mediating of TEA transport. The effect of EGF on TEA uptake in the CHO-K1 cells expressing rbOCT2 was not determined because the EGF receptor is not expressed in this cell line (10).

In the current study, the specific PKA inhibitor, H-89, reduced TEA uptake by rbOCT2 stably expressed in CHO-K1 cells and by intact S2 segments of rabbit renal proximal tubules. We also examined the effect of PKA stimulation on OCT2 activity in both CHO-K1 cells and intact tubules. Stimulation of PKA with forskolin led to stimulation of OCT2 activity in both systems (compare Fig. 1B with Fig. 6). The stimulatory effect of forskolin directly reflects the activity of PKA rather than a nonspecific effect of forskolin is evidenced by the fact that H-89 eliminated the stimulatory effect of forskolin in both CHO-K1 cells expressing rbOCT2 and intact tubules (Figs. 2B and 7B). These results are consistent with those in the study on IHKE-1 cells expressing hOCT2 showing that activation of PKA stimulates ASP+ uptake (12). However, the present results are different from studies with HEK-293 cells and with isolated human renal proximal tubules (2, 18). The reasons for such differences remain unknown.

The inhibitory effects of U-0126 and H-89 OCT2-mediated TEA uptake were not additive, suggesting that regulation of OCT2 by MAPK and PKA involves a single pathway. Moreover, MAPK appears to be upstream of PKA in the regulation of OCT2-mediated TEA uptake because inhibition of MAPK by U-0126 did not block the increase in TEA uptake that occurs when PKA is stimulated by forskolin (Fig. 4).

Kinetic analysis revealed that U-0126 treatment resulted in a decrease in Jmax but not in Kt (Fig. 5). These results suggest that the mechanism by which inhibition of MAPK inhibits organic cation transport could involve a decrease in membrane expression of the rbOCT2 transporter. It is unlikely that the reduction of rbOCT2 at the cell membrane is associated with protein synthesis because the 30-min preincubation period would not be long enough to reduce the amount of transporter by inhibition of its synthesis. Instead, the most likely explanation is that inhibition of MAPK increased movement of rbOCT2 from the membrane to some intracellular compartment and/or inhibited translocation of rbOCT2 from an intracellular compartment to the membrane. A recent study of PKC regulation of human OAT1 expressed in Xenopus laevis oocytes supports this possibility (28).

In summary, rbOCT2-mediated organic cation transport in both a heterologous cell system and intact tubules was influenced by MAPK and PKA activity. Activation of these protein kinases resulted in stimulation of OCT2 activity whereas inhibition led to depression of OCT2 activity. Regulation of OCT2 was similar to that observed for OAT1 and OAT3, suggesting that regulation of renal tubular transport of organic anions and cations involves a common MAPK and PKA pathway. The precise cellular processes involved in OCT2 regulation by activation or inhibition of MAPK and PKA have yet to be determined. However, this study provides the first evidence that regulation of OCT2 when it is expressed alone in a heterologous cell system is comparable to that in intact tubules where it is coexpressed with other OCTs. As modulation of OCT2 activity may influence the clearance of therapeutic cationic drugs from the body, manipulation of this transporter activity may provide the means to improve the therapeutic efficacy of these drugs.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by Mahidol University Grant 2549, Thailand Research Fund Grant 00165/2543, and National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency, Thailand Grant 3-2548.


   ACKNOWLEDGMENTS
 
We express great appreciation to Prof. William H. Dantzler for valuable comments on this manuscript and special thanks to Prof. Stephen H. Wright for providing CHO-K1 cells and radiolabeled TEA. The technical support of Chutima Srimaroeng is also gratefully acknowledged.


   FOOTNOTES
 

Address for reprint requests and other correspondence: V. Chatsudthipong, Dept. of Physiology, Faculty of Science, Mahidol Univ., Bangkok, Thailand 10400 (e-mail: scvcs{at}mahidol.ac.th)

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
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