ATP stimulates Na+-glucose cotransporter activity via cAMP and p38 MAPK in renal proximal tubule cells

Yun Jung Lee, Soo Hyun Park, and Ho Jae Han

Department of Veterinary Physiology, College of Veterinary Medicine, Chonnam National University, Gwangju, Korea

Submitted 3 January 2005 ; accepted in final form 6 July 2005


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Extracellular ATP plays an important role in the regulation of renal function. However, the effect of ATP on the Na+-glucose cotransporters (SGLTs) has not been elucidated in proximal tubule cells (PTCs). Therefore, this study was performed to examine the action of ATP on SGLTs and their related signal pathways in primary cultured rabbit renal PTCs. ATP increased [14C]-{alpha}-methyl-D-glucopyranoside ({alpha}-MG) uptake in a time-dependent (>1 h) and dose-dependent (>10–6 M) manner. ATP stimulated {alpha}-MG uptake by increasing in Vmax without affecting Km. ATP-induced increase of {alpha}-MG uptake was correlated with the increase in both SGLT1 and SGLT2 protein expression levels. ATP-induced stimulation of {alpha}-MG uptake was blocked by suramin (nonspecific P2 receptor antagonist), RB-2 (P2Y receptor antagonist), and MRS-2179 (P2Y1 receptor antagonist), suggesting a role for the P2Y receptor. ATP-induced stimulation of {alpha}-MG uptake was blocked by pertussis toxin (PTX, a Gi protein inhibitor), SQ-22536 (an adenylate cyclase inhibitor), and PKA inhibitor amide 14-22 (PKI). ATP also increased cAMP formation, which was blocked by PTX and RB-2. However, pretreatment of adenosine deaminase did not block ATP-induced cAMP formation. In addition, ATP-induced stimulation of {alpha}-MG uptake was blocked by SB-203580 (p38 MAPK inhibitor), but not by PD-98059 (p44/42 MAPK inhibitor) or SP-600125 (JNK inhibitor). Indeed, ATP induced phosphorylation of p38 MAPK. In conclusion, ATP increases {alpha}-MG uptake via cAMP and p38 MAPK in renal PTCs.

adenosine 5'-triphosphate; mitogen-activated protein kinase


ATP PLAYS A ROLE IN MEDIATING broad biological effects in many tissues and cells (21, 47). Two major classes of purinergic receptors have been identified: P1 receptors that are activated by adenosine and P2 receptors that are activated by ATP and ADP but not by adenosine or AMP (40). The membrane receptors for ATP and other nucleotides can be grouped into two major classes. The P2X receptors form ligand-gated, nonselective cation channels, whereas P2Y receptors are G protein-coupled membrane proteins (40). It is reported that P2 receptor subtypes were expressed in rat kidney and proximal tubule (33, 52). In the kidney, it has been suggested that ATP may be a mediator of tubuloglomerular feedback (53). This idea is based on the finding that nonspecific antagonists of P2 receptors interfere with the autoregulation of afferent arteriolar resistance. This phenomenon is mediated in part through the tubuloglomerular feedback mechanism (25). On the other hand, the regulation of epithelial transport via luminal P2 receptors has been reported (26, 33). Luminal nucleotides activate Cl secretion and inhibit Na+ and Ca2+ reabsorption in cultured cells of distal tubular origin (13, 30). A recent study identified luminal P2Y receptors in the rat proximal tubule that regulate HCO3 reabsorption (2). However, the role of ATP in regulating Na+-glucose cotransporters (SGLTs) of proximal tubule cells (PTCs) is poorly understood.

Glucose transporters, which incorporate glucose into the cell, can be divided into two groups: the facilitative type of glucose transporter and the SGLT. SGLTs are expressed in apical membranes of the PTCs, where they play a central role in the reabsorption of glucose from the glomerular filtrate (8). SGLT1 and SGLT2 are expressed in rabbit renal PTCs (36). The activation of cAMP by purinoceptors was reported in Madin-Darby canine kidney epithelial cells (38). cAMP is the second messenger in pathways that regulate SGLTs (57). The family of MAPKs includes ERK1/2, JNK, and p38 MAPK (9, 14). In mesangial cells, ERK1/2 and p38 MAPK mediate the ATP-induced cellular response (22, 54). Together, these studies suggest the possibility that these signaling molecules are involved in the effect of ATP on SGLTs in PTCs. However, the subtypes of ATP receptors and the ATP signal transduction mechanism inducing this important physiological effect on SGLTs remain unknown.

When grown in a hormonally defined medium, primary cultured renal PTCs form confluent monolayers of polarized cells, which retain a number of differentiated transport functions typical of renal PTCs (8). Included among these transport functions are a probenecid-sensitive PAH transport system, a Na+-dependent sugar transport system, and a Na+-dependent Pi transport system (8, 58). The results of studies concerning these membrane transport systems in PTCs are directly comparable to results obtained with original renal tissue (56). The PTCs respond to a number of hormones known to affect renal PTCs in vivo, including insulin (which inhibits phosphoenolpyruvate carboxykinase activity at physiological concentrations) (55), and parathyroid hormone (which is stimulatory to adenylate cyclase) (51). The PTCs lack a similar responsiveness to arginine vasopressin and calcitonin, indicating the PTC culture preparation is highly purified (8). More recently, we have reported a dose-dependent, biphasic effect of ANG II on Na+ uptake by the PTCs, consistent with results obtained with intact renal tissue (19). Therefore, PTCs in hormonally defined, serum-free culture conditions would be a powerful tool for studying the effect of ATP on [14C]-{alpha}-methyl-D-glucopyranoside ({alpha}-MG) uptake of renal PTCs. Thus we investigated the effect of ATP on {alpha}-MG uptake and its related signal cascades in PTCs.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. New Zealand White male rabbits (1.5–2.0 kg) were purchased from Dae Han Experimental Animal (Chungju, Korea). All procedures for animal management followed the standard operation protocols of Chonnam National University. An Institutional Review Board at Chonnam National University approved our research proposal and relevant experimental procedures, including animal care. Appropriate management of experimental samples and quality control of the laboratory facility and equipment were maintained. Class IV collagenase and soybean trypsin inhibitor were purchased from Life Technologies (Grand Island, NY). ATP, adenosine, UTP, {alpha},{beta}-methyleneadenosine 5'-triphosphate (AMP-CPP), 2-(methylthio) adenosine 5'-triphosphate (2-methylthio-ATP), MRS-2179, MRS-2159, cycloheximide, SB-203580, 8-bromoadenosine 3',5'-cyclic monophosphate, adenosine deaminase, apyrase, TPA, and {beta}-actin were obtained from Sigma Chemical (St. Louis, MO). SQ-22536, protein kinase inhibitor amide 14-22 (PKI), PD-98059, and SP-600125 were purchased from Calbiochem (La Jolla, CA). {alpha}-MG and myo-[3H]-inositol were purchased from Dupont NEN (Boston, MA). Rabbit anti-SGLT1 was purchased from Chemicon International (Temecula, CA), and rabbit anti-SGLT2 was obtained from Alpha Diagnostic International (San Antonio, TX). Phospho-p38 MAPK (Thr202/Tyr204) antibody and p44/42 MAPK antibody were purchased from Cell Signaling Technology (Beverly, MA). Goat anti-rabbit IgG was purchased from Jackson ImmunoResearch (West Grove, PA), and all other reagents were of the highest purity commercially available. Liquiscint was obtained from National Diagnostics (Atlanta, GA).

Isolation of rabbit renal proximal tubules and culture conditions. Primary rabbit renal PTC cultures were prepared according to the method of Chung et al. (8). The PTCs were grown in DMEM-Ham's F-12 medium with 15 mM HEPES and 20 mM sodium bicarbonate (pH 7.4). Immediately before we used the medium, three growth supplements (5 µg/ml insulin, 5 µg/ml transferrin, and 5 x 10–8 M hydrocortisone) were added. The kidneys of a rabbit were perfused via the renal artery, first with PBS and then with medium containing 0.5% iron oxide. Renal cortical slices were prepared and homogenized. The homogenate was poured first through a 253-µm and then through an 83-µm mesh filter. Tubules and glomeruli on top of the 83-µm filter were transferred into sterile medium. Glomeruli (containing iron oxide) were removed with the stirring bar. The remaining tubules were incubated briefly in medium. The tubules were then washed by centrifugation, resuspended in medium containing the three supplements, and transferred into tissue culture dishes. Medium was changed 1 day after plating and every 2 days thereafter. PTCs were maintained in a 37°C, 5% CO2 humidified environment in serum-free basal medium supplemented with three growth supplements.

Uptake experiments. To study the effect of ATP on {alpha}-MG uptake, the confluent monolayers were incubated with ATP before [14C]-{alpha}-MG uptake. {alpha}-MG uptake experiments were conducted according to the method described previously by Sakhrani et al. (42). To study {alpha}-MG uptake, the culture medium was removed by aspiration and the monolayers were gently washed twice with the uptake buffer (in mM: 136 NaCl, 5.4 KC1, 0.41 MgSO4, 1.3 CaCl2, 0.44 Na2HPO4, 0.44 KH2PO4, 5 HEPES, and 2 glutamine, pH 7.4). After the washing procedure, the monolayers were incubated at 37°C for 30 min in an uptake buffer that contained 0.5 mM {alpha}-MG and 0.5 µCi/ml [14C]-{alpha}-MG. At the end of the incubation period, the monolayers were again washed three times with ice-cold uptake buffer and the cells were solubilized in 0.1% SDS (1 ml). To determine the [14C]-{alpha}-MG incorporated intracellularly, 900 µl of each sample were removed and counted in a liquid scintillation counter (LS6500; Beckman Instruments, Fullerton, CA). The remainder of each sample was used for protein determination. The protein content of each sample was determined using the Bradford method (6). The radioactivity counts in each sample were then normalized with respect to protein and were corrected for zero time uptake per milligram of protein. All uptake measurements were performed in triplicate.

RNA isolation and RT-PCR. Extraction of total RNA from PTCs was performed as described previously (7). PTCs were homogenated with STAT-60, a monophasic solution of phenol, and guanidine isothiocyanate obtained from Tel-Test (Friendswood, TX). Two micrograms of purified RNA were synthesized into cDNA using avian leukemia virus RT with oligo dT18 primers. PCR amplification was performed with 5 µl of RT product, 10 pmol of each primer, 1.25 U of Taq polymerase (Promega, Madison, WI), and 1 mM 2-deoxynucleotide 5'-triphosphate. After an initial incubation at 95°C for 5 min, 28 amplification cycles consisting of 95°C for 40 s, annealing at 55°C for 1 min, and extension at 72°C for 40 s were performed. Rabbit-specific sense and antisense primers used were as follows: P2Y1 sense, 5'-GCATCTCGGTGTACATGTTC-3', and antisense 5'-GCTGTTGAGACTTGCTAGACCT-3'; P2Y2 sense, 5'-TACAGCTCTGTCATGCTGGG-3', and antisense, 5'-GCCAGGAAGTAGAGCACAGG-3'; P2Y4 sense, 5'-CTTTGCAAGTTTGTCCGCTTTC-3', and antisense, 5'-CCGGGCCATGAGTCCATA-3'; and P2Y6 sense, 5'-CTGTGTCATCGCCCAGATATGC-3', and antisense, 5'-GGTTGCCGCCGGAACTTC-3'. This sequence was used to clone the P2Y receptors successfully from the rabbit (29). As a control for the amount of cDNA, RT-PCR was performed using {beta}-actin primers. PCR products were visualized using ethidium bromide staining.

Membrane preparation for Western blot analysis. The medium was removed, and the cells were then washed twice with ice-cold PBS, scraped, harvested by microcentrifugation, and resuspended in buffer A (137 mM NaCl, 8.1 mM Na2HPO4, 2.7 mM KCl, 1.5 mM KH2PO4, 2.5 mM EDTA, 1 mM DTT, 0.1 mM PMSF, and 10 µg/ml leupeptin, pH 7.5). The resuspended cells were then mechanically lysed on ice by performing trituration with a 21.1-gauge needle. The lysates were first centrifuged at 1,000 g for 10 min at 4°C. The supernatants were centrifuged at 100,000 g for 1 h at 4°C to prepare cytosolic and total particulate fractions. The particulate fractions, which contained the membrane fraction, were washed twice and resuspended in buffer A containing 1% Triton X-100. The protein in each fraction was quantified using a Bradford procedure (6).

Western blot analysis. Cell homogenates (20 µg of protein) were separated using 10% SDS-PAGE and transferred onto nitrocellulose paper. Blots were then washed with H2O, blocked with 5% skim milk powder in TBST (10 mM Tris·HCl, pH 7.6, 150 mM NaCl, and 0.05% Tween 20) for 2 h and incubated with the primary polyclonal antibody (SGLT1, SGLT2, or MAPK) at dilutions recommended by the supplier. SGLT1 is a rabbit polyclonal antibody that recognizes a synthetic peptide corresponding to amino acids 402–420 of the putative extracellular loop of rabbit SGLT1 (a sequence that differs by 1 residue from the complementary region in pig SGLT2). In the present study, we confirmed antibody specificity using control peptides. Subsequently, the membrane was washed and primary antibodies were detected with goat anti-rabbit-IgG conjugated to horseradish peroxidase, and the bands were visualized using ECL (Amersham Pharmacia Biotech, Little Chalfont, UK).

cAMP assay. Samples were prepared for intracellular cAMP determinations by performing homogenization in serum-free medium containing 4 mM EDTA using the Polytron PT 1200, followed by 5-min incubation at 100°C. After centrifugation at 890 g for 5 min, the supernatants was transferred into new tubes and stored at 4°C. These samples were used for cAMP assays using a [3H]cAMP assay system. Values were expressed as picomolar cAMP per milligram of protein.

Statistical analysis. Results were expressed as means ± SE. Statistical analysis was performed using Student's t-test or ANOVA. The difference was considered statistically significant when P < 0.05.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Time- and dose-dependent effect of ATP on {alpha}-MG uptake. To determine the time-dependent effect of ATP on {alpha}-MG uptake, PTCs were exposed to 10–4 M ATP for various intervals. ATP significantly stimulated {alpha}-MG uptake during the course of 1 h, whereas this effect was decreased to the control level during a 12-h period (Fig. 1A). We also determined the dose-dependent effect of ATP on {alpha}-MG uptake. Thus PTCs were exposed to various concentrations of ATP (0–10–2 M) for 6 h. ATP (>10–6 M) significantly increased {alpha}-MG uptake (Fig. 1B). The maximum effect was observed between 10–4 and 10–2 M ATP. Thus 10–4 M ATP for 6 h was used in the following experiments.



View larger version (16K):
[in this window]
[in a new window]
 
Fig. 1. Time-dependent (A) and dose-dependent responses (B) of ATP on [14C]-{alpha}-methyl-D-glucopyranoside ({alpha}-MG) uptake. Proximal tubule cells (PTCs) were treated with 10–4 M ATP for different times (0–48 h) or different dosages of ATP (0–10–2 M) for 6 h before uptake experiments. Values are means ± SE of 4 independent experiments with triplicate dishes. *P < 0.05 vs. control.

 
To examine the effect of ATP on the kinetic properties of {alpha}-MG uptake, the effect of ATP on {alpha}-MG uptake was examined as a function of increasing {alpha}-MG concentrations (0.25–4 mM). ATP increased the Vmax of {alpha}-MG uptake via a SGLT (control, 0.49 ± 0.02; ATP, 1.34 ± 0.02 nmol·mg of protein–1·min–1; P < 0.05) without affecting the Km (control, 1.63 ± 0.01; ATP, 1.61 ± 0.02 mM; P = NS) (Fig. 2). Furthermore, we examined the effect of ATP on SGLT protein levels. ATP (10–4 M for 6 h) significantly increased both SGLT1 and SGLT2 protein expression levels (Fig. 3A). On the other hand, ATP inhibits Pi uptake (data not shown). Thus, in the present study, we focused on only {alpha}-MG uptake. Pretreatment of cycloheximide (a protein synthesis inhibitor) prevented ATP-induced stimulation of {alpha}-MG uptake (Fig. 3B).



View larger version (19K):
[in this window]
[in a new window]
 
Fig. 2. Effects of ATP on the kinetic parameters of {alpha}-MG uptake. PTCs were treated with 10–4 M ATP for 6 h before uptake experiment. {alpha}-MG concentrations were varied from 0.25 to 4 mM, and 30-min uptake in the presence of 136 mM NaCl was determined. Values are the means ± SE of 9 separate experiments performed on 3 different cultures. *P < 0.05 vs. control.

 


View larger version (19K):
[in this window]
[in a new window]
 
Fig. 3. Effect of ATP on SGLT protein expression level. PTCs were incubated with ATP (10–4 M) for 6 h. A: SGLT1 and SGLT2 protein expression levels were determined using Western blot analysis with membrane protein obtained from PTCs. Blots were probed with SGLT1-, SGLT2-, or {beta}-actin-specific antibodies, and bands shown represent 70–77 kDa of SGLT1 or SGLT2 and 41 kDa of {beta}-actin, respectively. Bar graphs at right depict the values of relative protein concentration. The results represent 1 of at least 3 experiments. B: PTCs were treated with cycloheximide (4 x 10–5 M) for 30 min before the treatment of ATP (10–4 M) for 6 h. Values are means ± SE of 3 independent experiments with triplicate dishes. *P < 0.05 vs. control. **P < 0.05 vs. ATP alone.

 
Receptor dependency of ATP on {alpha}-MG uptake. To determine which purinergic receptor is involved in the stimulatory effect of {alpha}-MG uptake, PTCs were treated with suramin (a P2 receptor antagonist), RB2 (P2Y receptor antagonist), MRS-2179 (a P2Y1 receptor antagonist), or MRS-2159 (a P2X1 receptor antagonist) before the treatment of ATP, respectively. Pretreament with suramin, RB2, and MRS-2179 completely blocked ATP-induced stimulation of {alpha}-MG uptake (Fig. 4A). In addition, PTCs were treated with UTP (a P2Y receptor agonist), AMP-CPP (a P2X receptor agonist), or 2-methylthio-ATP (a P2Y receptor agonist). UTP and 2-methylthio-ATP, but not AMP-CPP, stimulated {alpha}-MG uptake. RT-PCR results also showed that the P2Y1, P2Y2, P2Y4, and P2Y6 receptors are expressed in rabbit PTCs (Fig. 4B). Adenosine did not affect {alpha}-MG uptake, and pretreatment of adenosine deaminase (ADA; ~0.15–2 U/ml) did not block the ATP-induced stimulation of {alpha}-MG uptake (Fig. 5A). ADA and apyrase were also added to the incubation medium to degrade endogenous adenosine or ATP, respectively, without the addition of ATP. Neither ADA nor apyrase did not affect {alpha}-MG uptake (Fig. 5B).



View larger version (32K):
[in this window]
[in a new window]
 
Fig. 4. Effect of purinoceptor analogs on ATP-induced stimulation of {alpha}-MG uptake. A: PTCs were incubated with suramin, RB2, MRS-2179, or MRS-2159 (10–7 M) for 30 min before the treatment of ATP for 6 h. UTP (10–4 M), AMP-CPP, or 2-methylthio-ATP (10–6 M) was added to PTCs for 6 h. *P < 0.05 vs. control. **P < 0.05 vs. ATP alone. B: expression of P2Y1, P2Y2, P2Y4, and P2Y6 receptor subtypes in PTCs. RT-PCR was performed as described in MATERIALS AND METHODS. The results represent 1 of at least 3 experiments.

 


View larger version (33K):
[in this window]
[in a new window]
 
Fig. 5. Effect of adenosine deaminase (ADA) and apyrase on {alpha}-MG uptake. A: adenosine (10–4 M) and ADA (0.15–2 U/ml) were added for 30 min before the treatment of ATP for 6 h. *P < 0.05 vs. control. B: PTCs were incubated with ADA, apyrase (2 U/ml), and a combination of ADA and apyrase for 6 h. Values in A and B are means ± SE of 4 independent experiments with triplicate dishes.

 
Involvement of cAMP-PKA pathway in ATP-induced stimulation of {alpha}-MG uptake. To examine whether ATP stimulates G protein of the renal PTCs, pertussis toxin (PTX; 1 µg/ml), an agent that prevents receptor-mediated processes that use G protein (17), was used to treat the PTCs before administration of ATP. As shown in Fig. 6, PTX prevented ATP-induced stimulation of {alpha}-MG uptake. To study the involvement of cAMP in the ATP-induced stimulation of {alpha}-MG uptake, SQ-22536 (10–6 M, an adenylate cyclase inhibitor) or PKA inhibitor amide 14-22 (PKI; 10–6 M) was used to treat PTCs for 30 min before the treatment of ATP. SQ-22536 and PKI blocked ATP-induced stimulation of {alpha}-MG uptake. To confirm these results, we measured intracellular cAMP content after the treatment of ATP in the presence of IBMX. Indeed, ATP (10–4 M) increased the production of cAMP, which was blocked by PTX and RB-2 (Fig. 7A). On the other hand, ADA did not affect ATP-induced increase of cAMP formation (Fig. 7B).



View larger version (16K):
[in this window]
[in a new window]
 
Fig. 6. Effect of pertussis toxin (PTX), SQ-22536, and PKA inhibitor amide 14-22 (PKI) on ATP-induced stimulation of {alpha}-MG uptake. PTCs were treated with PTX (10 ng/ml), SQ-22536 (10–6 M), or PKI (10–6 M) for 30 min before treatment of ATP. Values are means ± SE of 3 independent experiments with triplicate dishes. Open bars, control; closed bars, ATP. *P < 0.05 vs. control. **P < 0.05 vs. ATP alone.

 


View larger version (14K):
[in this window]
[in a new window]
 
Fig. 7. Effect of PTX, RB2, and ADA on ATP-induced increase of cAMP formation. PTCs were preincubated with PTX (10 ng/ml) or RB2 (10–7 M) (A), or with ADA (0.15–1 U/ml) (B) for 30 min before treatment of ATP for 6 h. PTCs were preincubated to IBMX (10–4 M) for 30 min to prevent the degradation of cAMP into AMP before exposure to ATP. Values are means ± SE of 3 independent experiments with triplicate dishes. Open bars, control; closed bars, ATP. *P < 0.05 vs. control. **P < 0.05 vs. ATP alone.

 
Involvement of MAPKs in ATP-induced stimulation of {alpha}-MG uptake. To investigate whether MAPKs are involved in ATP-induced stimulation of {alpha}-MG uptake, PD-98059 (10–6 M; a p44/42 MAPK inhibitor), SB-203580 (10–6 M; a p38 MAPK inhibitor), or SP-600125 (10–6 M; a JNK/SAPK inhibitor) was used to treat the PTCs. Figure 8A shows that SB-203580 prevented the ATP-induced stimulation of {alpha}-MG uptake (P < 0.05). However, PD-98059 and SP-600125 did not affect {alpha}-MG uptake. Next, we confirmed these results using Western blot analysis. ATP enhanced phosphorylation of p38 MAPK, which peaked ~30 min after ATP treatment and decreased gradually thereafter (Fig. 8B). In addition, pretreatment of SB-203580 blocked ATP-induced phosphorylation of p38 MAPK, which is parallel to the result for {alpha}-MG uptake (Fig. 8C). To investigate the relationship between cAMP and MAPKs in ATP-induced stimulation of {alpha}-MG uptake, a cAMP assay was conducted in the presence of SB-203580. ATP-induced increase of cAMP formation was not blocked by SB-203580 (data not shown). However, SQ-22536 or PKI prevented ATP-induced phosphorylation of p38 MAPK (Fig. 9).



View larger version (31K):
[in this window]
[in a new window]
 
Fig. 8. Effect of MAPK inhibitors on ATP-induced stimulation of {alpha}-MG uptake. A: PD-98059, SB-203580, or SP-600125 (10–6 M) was was used to treat the PTCs for 30 min before the treatment of ATP. B: representative effect of ATP on p38 MAPK activation. PTCs were treated with ATP (10–4 M) for different times (0–240 min). C: SB-203580, PD-98059, or SP-600125 was used to pretreat the PTCs before the treatment of ATP (10–4 M for 30 min), which were then examined using Western blot analysis. The results represent 1 of at least 3 experiments. Graphs below histograms in B and C show the relative protein concentration values. Values are means ± SE of 3 independent experiments with triplicate dishes. Open bars, control; solid bars, ATP. *P < 0.05 vs. control. **P < 0.05 vs. ATP alone.

 


View larger version (27K):
[in this window]
[in a new window]
 
Fig. 9. Effect of cAMP-PKA pathway inhibitors on ATP-induced stimulation of p38 MAPK. SQ-22536, or PKI (10–6 M) was used to treat the PTCs for 30 min before the treatment of ATP (10–4 M). The results represent 1 of at least 3 experiments. Graph below histograms shows the relative protein concentration values. Values are the means ± SE of 3 independent experiments with triplicate dishes. *P < 0.05 vs. control. **P < 0.05 vs. ATP alone.

 

    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The role of ATP and other nucleotides as signaling molecules in a large number of tissues is well documented (40, 45). The present study has shown that ATP is associated with the stimulation of {alpha}-MG uptake in primary cultured renal PTCs. ATP produced a significant increase in Vmax without changing the Km value. This result suggests that ATP-induced stimulation of {alpha}-MG uptake may be attributable to increased activity of existing glucose carriers and/or to an increase in the number of glucose transport sites. This result is supported by the evidence that ATP increased Na+ uptake because of the increase in Vmax but not because of the influence of Km values, although the effect of ATP is applicable to distal tubules (37). However, a contradictory report has been published indicating that ATP inhibits glucose transport in cardiomyocytes (16), although our present observation is similar to that reported with regard to skeletal muscle cells (28). The concentrations of ATP needed to activate the P2 receptors are at micromolar levels (3), and the proximal tubule is the richest source of ATP under basal conditions at concentrations that reach 10–6 M. Upon stimulation, it can reach up to 5–10 µM (44). Consistent with the previous reports (3, 44), in the present study, the minimal effective dosage of ATP required to induce stimulation of {alpha}-MG uptake is 10–6 M. This result suggests that the ATP concentration used in the present study has the physiological action of ATP on P2 receptor activation. Solini et al. (46) reported similar results that P2Y receptor stimulation in human fibroblasts increased glucose uptake over a range ATP concentrations from 10–4 to 10–3 M, although isoforms of glucose transporters are different.

Previous studies have focused on the roles of purinergic receptors (P2 receptors and adenosine receptors) in glucose transport (15, 18, 49). Adenosine was reported to stimulate SGLT in opossum kidney cells, a PTC line (12). However, in the present study, adenosine did not stimulate {alpha}-MG uptake, suggesting the role of P2 receptors but not P1 receptors in the regulation of SGLT. In addition, pretreatment with ADA did not block ATP-induced increase of {alpha}-MG uptake. Herein we have demonstrated that ATP-induced stimulation of {alpha}-MG uptake is mediated by P2Y receptors. These results are in agreement with a recent report that P2Y1 receptors mediate HCO3 reabsorption in the apical membrane of rat PTCs (2). P2Y-dependent GLUT1 activation is deficient in fibroblasts from individuals type 2 diabetes mellitus, suggesting the physiological and clinical role of extracellular ATP in the modulation of glucose transport (46). Our result firstly revealed that activity of SGLT is physiologically upregulated by P2Y receptor activation. However, it is not clear which specific subtypes of P2Y receptors are involved in the effect of ATP on {alpha}-MG uptake. Herein we have demonstrated that P2Y1, P2Y2, P2Y4, and P2Y6 receptors are expressed in rabbit PTCs. Some of them may mediate the effect of ATP on {alpha}-MG uptake. In the present study, we showed that AMP-CPP (a P2X receptor agonist) did not affect {alpha}-MG uptake but UTP (a P2Y receptor agonist) increased it. However, we cannot rule out the possibility that the P2X receptors are involved in the effect of ATP, because MRS-2159 is a partial antagonist against the effect of ATP and UTP and 2-methylthio-ATP are only partially effective. Therefore, molecular identification of P2X and P2Y receptor subtypes remains to be examined in rabbit PTCs.

P2 purinoceptors were previously reported to be coupled to Gi protein in proximal tubule (31). In the present study, ATP-induced stimulation of {alpha}-MG uptake was blocked by 100 ng/ml PTX, indicating the involvement of a PTX-sensitive G protein. Thus extracellular ATP may suffice to trigger the alterations in G proteins that may activate SGLT. In addition, P2 purinoceptors have been reported to couple to adenylyl cyclase in several systems (11, 39). Several lines of evidence suggested that activation of P2Y receptor inhibits adenylyl cyclase (4, 5). However, in the present study, cAMP/PKA inhibitors blocked the effect of ATP on {alpha}-MG uptake, and ATP-induced increase of cAMP formation was prevented by P2Y receptor antagonists. These results suggest that the P2Y purinoceptor is coupled to the adenylyl cyclase-cAMP pathway, which induces stimulation of SGLT in PTCs. Our hypothesis is consistent with the report that the ADP-sensitive P2Y receptor is linked to cAMP accumulation with PKA, one of the activation systems in bovine adrenocortical fasciculata cells (34). Although the reasons why contradictory effects of ATP on the cAMP pathway are observed between astrocytomas and PTCs are not clear, it may be due to the different subtypes of receptors involved in the effect of ATP.

Three principal MAPKs are expressed in whole kidney: ERK1/2, JNK, and p38 MAPK (49). The ATP-induced activation of MAPKs, including ERK1/2, JNK, and p38 MAPK, was shown in mesangial cells and distal tubule cells (23, 24, 35, 51). Our previous report (20) showed that MAPK activation is involved in the regulation of SGLTs in PTCs, in which p44/42 MAPK activation is responsible for the inhibition of SGLTs. Unlike our previous report (20), our present results show that p38 MAPK, but not p44/42 MAPK and JNK, is an important signaling molecule in ATP-induced stimulation of {alpha}-MG uptake of PTCs. Recently, the cross-talk between cAMP and MAPK activation has been documented in various cells (41, 48). In the present study, ATP-induced activation of p38 MAPK was significantly blocked by PTX (a Gi protein inhibitor) and inhibitors of cAMP-PKA pathways. This result suggests that Gi protein-dependent pathways are molecules that are upstream from the activation of p38 MAPK. This result is consistent with the report of Aimond et al. (1) that ATP-induced activation of p38 MAPK is dependent on the primary activation of the PKA pathway and is prevented by inhibition of adenylyl cyclase and PKA. The regulatory role of PKA in the p38 MAPK signaling cascade is presently unclear. However, it might be involved in the activation of intermediate molecules, such as protein tyrosine phosphatases containing a PKA consensus sequence that shares a common function, as negative regulators of the p38 MAPK pathways (43). In con-clusion, ATP stimulates {alpha}-MG uptake via cAMP and p38 MAPK in renal PTCs.


    GRANTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
This research was supported by Grant SC 2210 from the Stem Cell Research Center of 21st Century Frontier Research Program funded by the Ministry of Science and Technology, and Korean Rural Development Administration (BioGreen 21 Program), Republic of Korea.


    FOOTNOTES
 

Address for reprint requests and other correspondence: H. J. Han, Dept. of Veterinary Physiology, College of Veterinary Medicine, Chonnam National Univ., Gwangju 500-757, Korea (e-mail: hjhan{at}chonnam.ac.kr)

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
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
1. Aimond F, Rauzier JM, Bony C, and Vassort G. Simultaneous activation of p38 MAPK and p42/44 MAPK by ATP stimulates the K+ current ITREK in cardiomyocytes. J Biol Chem 275: 39110–39116, 2000.[Abstract/Free Full Text]

2. Bailey MA. Inhibition of bicarbonate reabsorption in the rat proximal tubule by activation of luminal P2Y1 receptors. Am J Physiol Renal Physiol 287: F789–F796, 2004.[Abstract/Free Full Text]

3. Born GV and Kratzer MA. Source and concentration of extracellular adenosine triphosphate during haemostasis in rats, rabbits and man. J Physiol 354: 419–429, 1984.[Abstract]

4. Boyer JL, Delaney SM, Villanueva D, and Harden TK. A molecularly identified P2Y receptor simultaneously activates phospholipase C and inhibits adenylyl cyclase and is nonselectively activated by all nucleoside triphosphates. Mol Pharmacol 57: 805–810, 2000.[Abstract/Free Full Text]

5. Boyer JL, Lazarowski ER, Chen XH, and Harden TK. Identification of a P2Y-purinergic receptor that inhibits adenylyl cyclase. J Pharmacol Exp Ther 267: 1140–1146, 1993.[Abstract/Free Full Text]

6. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248–254, 1976.[CrossRef][ISI][Medline]

7. Chomczynski P and Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162: 156–159, 1987.[CrossRef][ISI][Medline]

8. Chung SD, Alavi N, Livingston D, Hiller S, and Taub M. Characterization of primary rabbit kidney cultures that express proximal tubule functions in hormonally defined medium. J Cell Biol 95: 118–126, 1982.[Abstract]

9. Cobb MH and Goldsmith EJ. How MAP kinases are regulated. J Biol Chem 270: 14843–14846, 1995.[Free Full Text]

10. Communi D, Janssens R, Suarez-Huerta N, Robaye B, and Boeynaems JM. Advances in signalling by extracellular nucleotides: the role and transduction mechanisms of P2Y receptors. Cell Signal 12: 351–360, 2000.[CrossRef][ISI][Medline]

11. Conigrave AD, Lee JY, Weyden L, Jiang L, Ward P, Tasevski V, Luttrell BM, and Morris MB. Pharmacological profile of a novel cyclic AMP-linked P2 receptor on undifferentiated HL-60 leukemia cells. Br J Pharmacol 124: 1580–1585, 1998.[CrossRef][ISI][Medline]

12. Coulson R, Johnson RA, Olsson RA, Cooper DR, and Scheinman SJ. Adenosine stimulates phosphate and glucose transport in opossum kidney epithelial cells. Am J Physiol Renal Fluid Electrolyte Physiol 260: F921–F928, 1991.[Abstract/Free Full Text]

13. Cuffe JE, Bielfeld-Ackermann A, Thomas J, Leipziger J, and Korbmacher C. ATP stimulates Cl secretion and reduces amiloride-sensitive Na+ absorption in M-1 mouse cortical collecting duct cells. J Physiol 524: 77–90, 2000.[Abstract/Free Full Text]

14. Davis RJ. MAPKs: new JNK expands the group. Trends Biochem Sci 19: 470–473, 1994.[CrossRef][ISI][Medline]

15. Derave W and Hespel P. Role of adenosine in regulating glucose uptake during contractions and hypoxia in rat skeletal muscle. J Physiol 515: 255–263, 1999.[Abstract/Free Full Text]

16. Fischer B, Yefidoff R, Major DT, Rutman-Halili I, Shneyvays V, Zinman T, Jacobson KA, and Shainberg A. Characterization of "mini-nucleotides" as P2X receptor agonists in rat cardiomyocyte cultures: an integrated synthetic, biochemical, and theoretical study. J Med Chem 42: 2685–2696, 1999.[CrossRef][ISI][Medline]

17. Gilman AG. G proteins: transducers of receptor-generated signals. Annu Rev Biochem 56: 615–649, 1987.[CrossRef][ISI][Medline]

18. Han DH, Hansen PA, Nolte LA, and Holloszy JO. Removal of adenosine decreases the responsiveness of muscle glucose transport to insulin and contractions. Diabetes 47: 1671–1675, 1998.[Abstract]

19. Han HJ, Park SH, Koh HJ, and Taub M. Mechanism of regulation of Na+ transport by angiotensin II in primary renal cells. Kidney Int 57: 2457–2467, 2000.[CrossRef][ISI][Medline]

20. Han HJ, Park SH, and Lee YJ. Signaling cascade of ANG II-induced inhibition of {alpha}-MG uptake in renal proximal tubule cells. Am J Physiol Renal Physiol 286: F634–F642, 2004.[Abstract/Free Full Text]

21. Harden TK, Boyer JL, and Nicholas RA. P2-purinergic receptors: subtype-associated signaling responses and structure. Annu Rev Pharmacol Toxicol 35: 541–579, 1995.[CrossRef][ISI][Medline]

22. Huwiler A, Rölz W, Dorsch S, Ren S, and Pfeilschifter J. Extracellular ATP and UTP activate the protein kinase B/Akt cascade via the P2Y2 purinoceptor in renal mesangial cells. Br J Pharmacol 136: 520–529, 2002.[CrossRef][ISI][Medline]

23. Huwiler A, van Rossum G, Wartmann M, and Pfeilschifter J. Stimulation by extracellular ATP and UTP of the stress-activated protein kinase cascade in rat renal mesangial cells. Br J Pharmacol 120: 807–812, 1997.[ISI][Medline]

24. Huwiler A, Wartmann M, van den Bosch H, and Pfeilschifter J. Extracellular nucleotides activate the p38-stress-activated protein kinase cascade in glomerular mesangial cells. Br J Pharmacol 129: 612–618, 2000.[CrossRef][ISI][Medline]

25. Inscho EW, Cook AK, and Navar LG. Pressure-mediated vasoconstriction of juxtamedullary afferent arterioles involves P2-purinoceptor activation. Am J Physiol Renal Fluid Electrolyte Physiol 271: F1077–F1085, 1996.[Abstract/Free Full Text]

26. Inscho EW, Cook AK, Imig JD, Vial C, and Evans RJ. Renal autoregulation in P2X1 knockout mice. Acta Physiol Scand 181: 445–453, 2004.[CrossRef][ISI][Medline]

27. Jin W and Hopfer U. Purinergic-mediated inhibition of Na+-K+-ATPase in proximal tubule cells: elevated cytosolic Ca2+ is not required. Am J Physiol Cell Physiol 272: C1169–C1177, 1997.[Abstract/Free Full Text]

28. Kim MS, Lee J, Ha J, Kim SS, Kong Y, Cho YH, Baik HH, and Kang I. ATP stimulates glucose transport through activation of P2 purinergic receptors in C2C12 skeletal muscle cells. Arch Biochem Biophys 401: 205–214, 2002.[CrossRef][ISI][Medline]

29. Konduri GG, Bakhutashvili I, Frenn R, Chandrasekhar I, Jacobs ER, and Khanna AK. P2Y purine receptor responses and expression in the pulmonary circulation of juvenile rabbits. Am J Physiol Heart Circ Physiol 287: H157–H164, 2004.[Abstract/Free Full Text]

30. Koster HPG, Hartog A, van Os CH, and Bindels RJM. Inhibition of Na+ and Ca2+ reabsorption by P2u purinoceptors requires PKC but not Ca2+ signaling. Am J Physiol Renal Fluid Electrolyte Physiol 270: F53–F60, 1996.[Abstract/Free Full Text]

31. Lederer ED and McLeish KR. P2 purinoceptor stimulation attenuates PTH inhibition of phosphate uptake by a G protein-dependent mechanism. Am J Physiol Renal Fluid Electrolyte Physiol 269: F309–F316, 1995.[Abstract/Free Full Text]

32. Lee WS, Kanai Y, Wells RG, and Hediger MA. The high affinity Na+/glucose cotransporter: re-evaluation of function and distribution of expression. J Biol Chem 269: 12032–12039, 1994.[Abstract/Free Full Text]

33. Leipziger J. Control of epithelial transport via luminal P2 receptors. Am J Physiol Renal Physiol 284: F419–F432, 2003.[Abstract/Free Full Text]

34. Nishi H, Kato F, Masaki E, and Kawamura M. ADP-sensitive purinoceptors induce steroidogenesis via adenylyl cyclase activation in bovine adrenocortical fasciculata cells. Br J Pharmacol 137: 177–184, 2002.[CrossRef][ISI][Medline]

35. Orlov SN, Dulin NO, Gagnon F, Gekle M, Douglas JG, Schwartz JH, and Hamet P. Purinergic modulation of Na+,K+,Cl cotransport and MAP kinases is limited to C11-MDCK cells resembling intercalated cells from collecting ducts. J Membr Biol 172: 225–234, 1999.[CrossRef][ISI][Medline]

36. Panayotova-Heiermann M, Loo DDF, Kong CT, Lever JE, and Wright EM. Sugar binding to Na+/glucose cotransporters is determined by the carboxyl-terminal half of the protein. J Biol Chem 271: 10029–10034, 1996.[Abstract/Free Full Text]

37. Pellerin I, Leclerc M, Claveau D, Mailloux J, and Brunette MG. Roles of ATP and cytoskeleton in the regulation of Na+/H+ exchanger along the nephron luminal membrane. J Cell Physiol 187: 109–116, 2001.[CrossRef][ISI][Medline]

38. Post SR, Jacobson JP, and Insel PA. P2 purinergic receptor agonists enhance cAMP production in Madin-Darby canine kidney epithelial cells via an autocrine/paracrine mechanism. J Biol Chem 271: 2029–2032, 1996.[Abstract/Free Full Text]

39. Post SR, Rump LC, Zambon A, Hughes RJ, Buda MD, Jacobson JP, Kao CC, and Insel PA. ATP activates cAMP production via multiple purinergic receptors in MDCK-D1 epithelial cells: blockade of an autocrine/paracrine pathway to define receptor preference of an agonist. J Biol Chem 273: 23093–23097, 1998.[Abstract/Free Full Text]

40. Ralevic V and Burnstock G. Receptors for purines and pyrimidines. Pharmacol Rev 50: 413–492, 1998.[Abstract/Free Full Text]

41. Robinson-White A and Stratakis CA. Protein kinase A signaling: "cross-talk" with other pathways in endocrine cells. Ann NY Acad Sci 968: 256–270, 2002.[Abstract/Free Full Text]

42. Sakhrani LM, Badie-Dezfooly B, Trizna W, Mikhail N, Lowe AG, Taub M, and Fine LG. Transport and metabolism of glucose by renal proximal tubular cells in primary culture. Am J Physiol Renal Fluid Electrolyte Physiol 246: F757–F764, 1984.[Abstract/Free Full Text]

43. Saxena M, Williams S, Taskén K, and Mustelin T. Crosstalk between cAMP-dependent kinase and MAP kinase through a protein tyrosine phosphatase. Nat Cell Biol 1: 305–311, 1999.[CrossRef][ISI][Medline]

44. Schwiebert EM and Kishore BK. Extracellular nucleotide signaling along the renal epithelium. Am J Physiol Renal Physiol 280: F945–F963, 2001.[Abstract/Free Full Text]

45. Schwiebert LM, Rice WC, Kudlow BA, Taylor AL, and Schwiebert EM. Extracellular ATP signaling and P2X nucleotide receptors in monolayers of primary human vascular endothelial cells. Am J Physiol Cell Physiol 282: C289–C301, 2002.[Abstract/Free Full Text]

46. Solini A, Chiozzi P, Morelli A, Passaro A, Fellin R, and Di Virgilio F. Defective P2Y purinergic receptor function: a possible novel mechanism for impaired glucose transport. J Cell Physiol 197: 435–444, 2003.[CrossRef][ISI][Medline]

47. Spyer KM, Dale N, and Gourine AV. ATP is a key mediator of central and peripheral chemosensory transduction. Exp Physiol 89: 53–59, 2004.[Abstract/Free Full Text]

48. Stork PJS and Schmitt JM. Crosstalk between cAMP and MAP kinase signaling in the regulation of cell proliferation. Trends Cell Biol 12: 258–266, 2002.[CrossRef][ISI][Medline]

49. Takasuga S, Katada T, Ui M, and Hazeki O. Enhancement by adenosine of insulin-induced activation of phosphoinositide 3-kinase and protein kinase B in rat adipocytes. J Biol Chem 274: 19545–19550, 1999.[Abstract/Free Full Text]

50. Tian W, Zhang Z, and Cohen DM. MAPK signaling and the kidney. Am J Physiol Renal Physiol 279: F593–F604, 2000.[Abstract/Free Full Text]

51. Toutain H, Vauclin-Jacques N, Fillastre JP, and Morin JP. Biochemical, functional, and morphological characterization of a primary culture of rabbit proximal tubule cells. Exp Cell Res 194: 9–18. 1991.[CrossRef][ISI][Medline]

52. Turner CM, Vonend O, Chan C, Burnstock G, and Unwin RJ. The pattern of distribution of selected ATP-sensitive P2 receptor subtypes in normal rat kidney: an immunohistological study. Cells Tissues Organs 175: 105–117, 2003.[CrossRef][ISI][Medline]

53. Vallon V, Richter K, Huang DY, Rieg T, and Schnermann J. Functional consequences at the single-nephron level of the lack of adenosine A1 receptors and tubuloglomerular feedback in mice. Pflügers Arch 448: 214–221, 2004.[CrossRef][ISI][Medline]

54. Vonend O, Grote T, Oberhauser V, Von Kugelgen I, and Rump LC. P2Y-receptors stimulating the proliferation of human mesangial cells through the MAPK42/44 pathway. Br J Pharmacol 139: 1119–1126, 2003.[CrossRef][ISI][Medline]

55. Wang W and Taub M. Insulin and other regulatory factors modulate the growth and the phosphoenolpyruvate carboxykinase (PEPCK) activity of primary rabbit kidney proximal tubule cells in serum free medium. J Cell Physiol 147: 374–382, 1991.[CrossRef][ISI][Medline]

56. Waqar MA, Seto J, Chung SD, Hiller-Grohol S, and Taub M. Phosphate uptake by primary renal proximal tubule cell culture grown in hormonally defined medium. J Cell Physiol 124: 411–423, 1985.[CrossRef][ISI][Medline]

57. Wright EM, Hirsch JR, Loo DD, and Zampighi GA. Regulation of Na+/glucose cotransporters. J Exp Biol 200: 287–293, 1997.[Abstract/Free Full Text]

58. Yang IS, Goldinger J, Hong SK, and Taub M. Preparation of basolateral membranes that transport p-aminohippurate from primary cultures of rabbit kidney proximal tubule cells. J Cell Physiol 135: 481–487, 1988.[CrossRef][ISI][Medline]





This Article
Abstract
Full Text (PDF)
All Versions of this Article:
289/5/C1268    most recent
00002.2005v1
Alert me when this article is cited
Alert me if a correction is posted
Citation Map
Services
Email this article to a friend
Similar articles in this journal
Similar articles in ISI Web of Science
Similar articles in PubMed
Alert me to new issues of the journal
Download to citation manager
Google Scholar
Articles by Lee, Y. J.
Articles by Han, H. J.
PubMed
PubMed Citation
Articles by Lee, Y. J.
Articles by Han, H. J.


HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2005 by the American Physiological Society.