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1Research Service, North Florida/South Georgia Veterans Health System, and 2Department of Medicine, University of Florida College of Medicine, Gainesville, Florida 32610-0224; and 3Department of Biochemistry, Brody School of Medicine, East Carolina University, Greenville, North Carolina 27858
Submitted 11 August 2003 ; accepted in final form 29 March 2004
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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S > ADP > UDP that is best fitted with the P2Y2 subtype profile. Although the P2Y agonist UTP induced a similar large transient increase in [Ca2+]i as did ATP, a small but sustained increase in [Ca2+]i occurred only in ATP-stimulated cells, suggesting the role of P2X receptors in Ca2+ influx. The sustained increase in [Ca2+]i could be blocked by either nonselective cation channel blockers Gd3+ or P2X antagonists PPADS and PPNDS. Furthermore, when either Gd3+ or PPNDS was applied to the bath solution before ATP application, the ATP-induced increase in [Ca2+]i was significantly reduced. Both RT-PCR and Western blotting corroborated the presence of P2X1 and P2Y2 receptors. These studies demonstrate that mIMCD-3 cells have both P2X and P2Y subtype receptors and that the activation of both P2X and P2Y receptors by extracellular ATP appears to be required to regulate intracellular Ca2+ signaling. epithelia; purinergic receptors; collecting duct; calcium channel; kidney
The inner medullary collecting duct (IMCD) is the final renal segment responsible for the regulation of solute and ionic composition of the urine. There is increasing evidence that extracellular nucleotides play an important role in the regulation of IMCD function. For example, extracellular ATP is involved in the inhibition of AVP-stimulated water permeability in rat IMCD cells (22), inhibition of Na+ short-circuit current, stimulation of Cl– short-circuit current in the mouse IMCD cell line mIMCD-K2 (5, 28), and modulation of whole cell Cl– current in the mouse IMCD cell line mIMCD-3 (41). Nucleotide actions in IMCD cells are Ca2+ dependent. Extracellular application of ATP has been shown to induce an increase in intracellular Ca2+ concentration ([Ca2+]i) (5, 15, 42) through cell membrane P2Y receptor subtypes. Several studies show that G protein-coupled P2Y2 receptors are widely expressed in mIMCD-K2 cells (28) and in rat IMCD cells (15, 23). In addition, G protein-coupled P2Y1 receptors are also expressed in mIMCD-K2 cells (28). In contrast, to date only one IMCD cell line, mIMCD-K2, is reported to have P2X receptor subtypes, i.e., P2X3 and P2X4 (28). The role of P2X receptors in the regulation of Ca2+ homeostasis in IMCD cells, however, is largely unknown. Moreover, the relationship between ionotropic P2X receptors, which may cause Ca2+ influx, and metabotropic P2Y G protein-coupled receptors, which can lead to the release of intracellular Ca2+, in the mobilization of intracellular Ca2+ in response to ATP stimulation has not been determined.
The mIMCD-3 cell line is widely used as a model in the study of renal transport physiology (2, 4, 12, 13, 29, 32, 33, 35, 36, 40, 41, 51). Although several laboratories have shown that ATP increases intracellular Ca2+ in mIMCD-3 cells (5, 41, 51), the source of Ca2+ that causes the increase has not been analyzed thoroughly. Moreover, the P2 receptor subtypes in response to extracellular ATP have not been defined.
The objective of this study was to determine whether P2 receptors were functionally expressed in mIMCD-3 cells, and if so, to examine their roles in cellular Ca2+ mobilization in response to extracellular ATP and to provide an initial characterization of the receptor subtypes.
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Solutions. For intracellular Ca2+ measurement, a Ca2+-containing Ringer solution contained (in mM) 140 NaCl, 5 KCl, 1.5 CaCl2, 1 MgCl2, 10 glucose, and 10 HEPES. A Ca2+-free Ringer solution contained (in mM) 140 NaCl, 5 KCl, 1 MgCl2, 10 glucose, 10 HEPES, and 1–2 EGTA. Cells were continuously superfused with experimental solutions through a gravity-fed system at a rate of 3–6 ml/min. Solutions were evacuated by suction. All solutions were adjusted to pH 7.4 with NaOH. The dyes and drugs, as needed, were dissolved in DMSO, and the final concentration of DMSO in the loading or experimental solutions was <5% (vol/vol). Chemicals used in this study were purchased from Sigma (St. Louis, MO) or as indicated.
Fura 2-AM loading and intracellular Ca2+ measurement.
Cells were loaded at room temperature for 
2 h in HEPES-buffered Ringer solution containing 5–10 µM Ca2+ indicator fura 2-AM (Molecular Probes, Eugene, OR), then washed at least three times, and incubated for an additional 15–20 min in dye-free Ringer solution to reduce the possibility of incomplete hydrolysis of the AM esters by intracellular esterases. In some experiments, the detergent Pluronic F-127 (20% solution in DMSO) was added to the loading solution (0.05% vol/vol) to facilitate the uptake of fura 2. The cells in this group had no detectable difference in Ca2+ responses to ATP compared with cells in the group without the detergent.
The [Ca2+]i measurements were made with a ratiometric imaging system (InCyt Im2, Intracellular Imaging, Cincinnati, OH), including a PC, a filter wheel of conventional design, a charge-coupled device camera, and a Nikon TE 300 microscope with x40 air objective (0.9 numerical aperture). In each experiment, either a number of single cells or a group of cells [i.e., region of interest (ROI)] was selected using the software. The fluorescent emissions as paired signals, at a wavelength of 510 nm from the ROI, were measured accordingly to excitation wavelengths of 340 and 380 nm at a time interval of every 3 s. Background fluorescence was subtracted online from F340 and F380 signals. Changes in [Ca2+]i are reported as the mean F340/F380 fluorescence ratio over time from a number of ROIs that were generated offline and expressed as R/R0, with R being the fluorescence ratio change over time and R0 the averaged fluorescence ratio of a period of 100–120 s before the first drug addition.
The change in single-cell Ca2+ was estimated in some experiments using standard in situ calibration and the equation as described previously (19, 49), with a Kd of 230 nM for fura 2.
Total RNA extraction. Confluent mIMCD-3 cells and mIMCD-K2 cells were harvested by carefully scraping off the cell monolayer with a rubber policeman into 10 ml of ice-cold PBS solution (150 mM NaCl, 4.52 mM NaH2PO4, 15.48 mM Na2HPO4, pH 7.4). Cells were then spun at 2,500 rpm for 10 min at 4°C. RNA was extracted from the cell pellet using an Eppendorf Perfect RNA Extraction Kit (Brinkmann, Westbury, NY) according to the manufacturer's instructions. Quantification of the purified RNA was performed using a Bio-Rad SmartSpec 3000 spectrophometer with wavelengths of 260 and 280 nm (Bio-Rad, Hercules, CA).
Preparation of cDNA. The first-strand RT reaction for mIMCD-3 cells and mIMCD-K2 cells was performed with the SuperScript First-Strand Synthesis System for RT-PCR (Invitrogen, Carlsbad, CA) using oligo(dT). The second-strand synthesis was performed with two gene-specific primers (see below) designed to specific sequences in the coding region of both genes.
The primers used to amplify a fragment of mouse P2Y2 (P35383 [GenBank] ) were designed to anneal to base pairs 411–431 (5'-GGA AGC CTC TTT AGG GAT GG-3') on the sense strand and base pairs 1365–1384 (5'-GCT CAC CCA CCT TGT TTT GT-3') on the antisense strand. The primers for amplifying mouse P2X1 (AAF68968 [GenBank] were designed to anneal to base pairs 99–128 (5'-CCG TCT GAT CCA GTT GGT GGT TCT GGT C-3') on the sense strand and 993–1024 (5'-AGA TGC CAA TCC CAG AGC CGA TGG TAG T-3') on the antisense strand. All of the primers are from sequences within the coding region. Each PCR reaction was set up in a 0.2-ml Thin Wall Tube (Bio-Rad) with 36 µl of autoclaved water, 5 µl of 10x cDNA PCR buffer (BD Biosciences, Palo Alto, CA), 5 µl of template, 1 µl each of forward and reverse primers at a concentration of 1 µM, 1 µl of 10 mM dNTP, and 1 µl of advantage cDNA polymerase mix (BD Biosciences). The amplification was done in a Bio-Rad iCycler thermal cycler for 1 cycle of denaturation at 94°C for 4 min; 30 cycles at 94°C denaturation for 30 s, 60 and 68°C annealing for 30 s for P2Y2 and P2X1, respectively, 72°C extension for 1 min; and 1 cycle for a final extension at 72°C for 3 min.
Cloning and sequencing of purinergic receptors. Gel-excised PCR products were purified using a QIAquick Gel Extraction Kit (Qiagen, Valencia, CA) following the manufacturer's instructions. The PCR products were then ligated into the TA cloning site of pCR 2.1 using a TA Cloning Kit (Invitrogen) according to the manufacturer's instructions. The transformation was performed using One Shot TOP10 Chemically Competent Escherichia coli (Invitrogen). The transformants were plated on LB ampicillin plates (50 µg/ml) containing 5-bromo-4-chloro-3-indolyl-D-galactopyranside (40 µg/ml) to allow blue/white screening of positives containing the insert of interest. The following day, white colonies were picked and each was incubated in 2 ml of LB media containing ampicillin (50 µg/ml) overnight at 240 rpm at 37°C. One milliliter of the overnight culture was pipetted into a 1.5-ml Eppendorf tube for plasmid isolation using a PerfectPrep Miniprep Kit (Brinkmann) according to the manufacturer's instructions. The purified DNA was visualized under 0.1% ethidium bromide on a 0.8% agarose gel using a 500-base pair ladder as a standard. Two distinct positive clones were sent for sequencing (DNA Sequencing Core Laboratory, Gainesville, FL). The resulting sequences were compared with the published sequences using the basic alignment research tool algorithm (1).
Preparation of protein samples and Western blot analysis. A confluent monolayer of mIMCD-3 cells was rinsed twice with ice-cold PBS (150 mM NaCl, 4.52 mM NaH2PO4, 15.48 mM Na2HPO4, pH 7.4) and then harvested using a rubber policeman. After a brief centrifugation at 660 g (Eppendorf 5417R; 2,500 rpm) for 10 min, the cells were resuspended in a hypertonic lysis buffer containing 20 mM NaH2PO4, 0.5 M NaCl (pH 7.4), and a protease inhibitor cocktail (Complete, Roche Molecular Biochemicals). The mixture was left on ice for 50 min, with gentle shaking every 10 min. The cells were disrupted by dounce homogenization using glass douncers, and the undisrupted cells and cellular debris were removed by centrifugation at 2,060 g (4,400 rpm) for 15 min at 4°C. The supernatant was then diluted fivefold with lysis buffer and subjected to high-speed centrifugation at 92,500 g in a 70.1 Ti rotor (Beckman Optima LE-80K; 30,000 rpm) for 1 h at 4°C. The resulting pellet was resuspended in 500 µl of lysis buffer. The sample was then diluted 1:1 with Laemmli sample buffer (62.5 mM Tris·HCl, pH 6.8, 2% SDS, 25% glycerol, 0.01% bromophenol blue) containing 20 mM DTT and incubated at 95°C for 5 min before being loaded onto a gel.
Solubilized proteins were separated on a 10% Tris·HCl Ready Gel (Bio-Rad) for 90 min at 20 mA and electrophoretically transferred to polyvinylidene difluoride membranes for 60 min at 225 mA. The membranes were blocked with 5% nonfat dry milk in PBST buffer (50 mM NaH2PO4, 150 mM NaCl, 0.1% Tween 20) for 1 h and then immediately incubated for 1 h with either anti-rP2X1 polyclonal antibody (1:400) or anti-rP2Y2 polyclonal antibody (1:400, Alomone Labs) dissolved in antibody buffer (50 mM NaH2PO4, 150 mM NaCl, 0.1% Tween 20, 3% BSA, 0.01% sodium azide). The antibody used for peptide blocking was treated with 1 µg peptide/µg antibody for 1 h at room temperature immediately before being incubated with the membrane. Dilutions of the antibodies were similar in the presence and absence of peptide. After being washed for 15 min with PBST, the membranes were incubated for 1 h with peroxidase-conjugated goat anti-rabbit antibody (Jackson ImmunoLabs, West Grove, PA) at a 1:2,000 dilution. Membranes were washed and then detected using ECL Plus Western Blotting Detection Reagents (Amersham, Piscataway, NJ) following the manufacturer's instructions.
The separation of each protein sample was repeated at least three times with the same amount of protein (20 µg) loaded onto each lane. The Lowry protein assay was used to determine protein concentration (27).
Data analysis. Origin 6.0 (Microcal Software, Northampton, MA) was used for data analysis and graphics. The peak response of each experiment was determined by calculating three to six peak ratios from the ROIs within the experiment. The final results from each group of experiments (n) are reported as the mean peak response (means ± SE). Statistical significance was examined using Student's t-test. A value of P < 0.05 was considered significant.
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RESULTS |
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Evidence for the presence of purinergic P2Y receptors. In the absence of extracellular Ca2+, cells were treated with 5–10 µM thapsigargin, an agent that mobilizes Ca2+ specifically from IP3-sensitive Ca2+ stores by inhibiting endoplasmic reticulum Ca2+-ATPase activity. A transient increase in [Ca2+]i indicates the release of Ca2+ from intracellular Ca2+ stores (see Fig. 4A). Subsequently, the thapsigargin-treated cells were treated with 10–100 µM ATP, which failed to induce a transient increase in [Ca2+]i. In reverse order, a transient increase in [Ca2+]i was observed with the addition of ATP, but no further increase was observed after the addition of thapsigargin (Fig. 4B). In both cases, the addition of the second reagent failed to cause an increase in [Ca2+]i, indicating that each agent released the full amount of Ca2+ from the same intracellular Ca2+ store. Because it has been well established that thapsigargin releases intracellular Ca2+ from IP3-sensitive Ca2+ stores (34), our results would support the involvement of the P2Y receptor subtype in response to ATP stimulation.
We further examined the presence of G protein-coupled P2Y receptors by blocking the activation of PLC and IP3 generation with a membrane-permeable PLC inhibitor, U-73122. A 5- to 10-min pretreatment of cells with 10 µM U-73122 blocked the intracellular Ca2+ increase by 10 µM ATP (Fig. 5A), consistent with the blockade of ATP-activated PLC activity. In five such experiments, only a small recovery was observed after the washout of U-73122.
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S (47 ± 8.6%; n = 4), and ADP (40 ± 4.7%; n = 4) produced different levels of increase in [Ca2+]i, whereas UDP (n = 3) had no detectable responses. The functional potencies of P2-receptor agonists in the order of UTP = ATP > ATPS > ADP >> UDP are best fitted with the established profile of mammalian P2 receptor subtype P2Y2 (38). Plasma membrane Ca2+ entry via P2X receptors occurs during ATP stimulation. In the Ca2+-containing solution, a long exposure of the cells to ATP produced a small but sustained increase in [Ca2+]i after the large, transient increase in [Ca2+]i (Fig. 6A). This small, sustained increase in [Ca2+]i occurred as long as ATP was present (up to 15 min in our test).
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We further examined whether the observed sustained increase in [Ca2+]i in the Ca2+-containing solution could be blocked by P2X-receptor antagonists and Ca2+ channel blockers. Because a P2X1-like transcript was detected in mIMCD-3 cells (Gumz ML and Cain BD, personal communication), pyridoxal-5'-phosphate-6-(2'-naphthylazo-6-nitro-4',8'-disulfonate) (PPNDS), a P2X1-receptor antagonist (24), was used to test the functional effect. When cells were exposed to PPNDS (10 µM), the ATP-induced sustained increase in [Ca2+]i was blocked (Fig. 6C; n = 4 of 5). P2X-receptor antagonist PPADS (10 µM) had a similar effect (n = 4 of 5).
When cells were exposed to the trivalent cation gadolinium (Gd3+; 10–50 µM), a nonselective Ca2+ channel blocker that can inhibit ATP-gated channels (30), the ATP-induced sustained increase in [Ca2+]i was blocked (Fig. 6D; n = 3). The blockade process could be reversed after washout. The trivalent cation lanthanum had a similar effect (La3+; 50–100 µM; data not shown).
Because mIMCD-3 cells have been reported to have voltage-dependent Ca2+ channels (3), nifadipine (10 µM), an L-type Ca2+ channel blocker, and pimozide (15 µM) and amiloride (10 µM), T-type Ca2+ channel blockers, were also tested, but no detectable effect was found.
Taken together, our results suggest that Ca2+ entry generated by ATP stimulation occurs via P2X receptors (i.e., ATP-gated cation channels), not voltage-dependent Ca2+ channels.
Further evidence for the presence of purinergic P2X receptors. Figure 7, A and B, shows typical experiments with the ATP-gated channels blockers La3+ and Gd3+, respectively. When cells were exposed to the bath solution with La3+ (50–100 µM; n = 3) or Gd3+ (10–50 µM; n = 8), ATP failed to induce a significant increase in [Ca2+]i. The effect on [Ca2+]i blocking the action of ATP was reversible, as demonstrated by the [Ca2+]i response to ATP application after washout of the trivalent cations.
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Following a protocol similar to the above-mentioned Gd3+ experiments, we further tested the functional effect of P2X-receptor antagonists. As shown in Fig. 8A, in the presence of 10 µM PPNDS, the ATP-stimulated increase in [Ca2+]i was largely inhibited. A partial recovery could be observed after the washout of the antagonist. A summary of four similar experiments is shown in Fig. 8B. The data presented here indicate that the action of ATP through P2X receptor subtypes could be an early event that is necessary for purinergic Ca2+ signaling.
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-methylene-ATP (,-MeATP; 10–100 µM), a significant increase in [Ca2+]i was observed in four of them. We also tested the P2Y-receptor agonist 2-methylthioadenosine 5'-triphosphate (2-MeSATP; 10–100 µM), and no consistent increase in [Ca2+]i was found. The P2X1 antagonist NF-279 (10 µM), a suramin analog, not only failed to inhibit the ATP-induced increase in [Ca2+]i but also slightly enhanced the response (n = 3); so did TNP-ATP (5 µM; n = 3). mRNA expression of purinergic P2X1 and P2Y2 receptors using RT-PCR. We performed RT-PCR experiments to identify the presence of P2X1 and P2Y2 purinoceptors in mIMCD-3 cells. Previously, it has been established that mIMCD-K2 cells express P2Y2 but not P2X1 receptors (28), so these experiments were conducted in parallel as controls. The P2X1 cDNA with the expected 926-bp band was observed in the mIMCD-3 cells, but not the mIMCD-K2 cells (Fig. 9A). In the second set of experiments regarding P2Y2, a band was observed at the expected size of 973 bp in mIMCD-3 cells, and, as anticipated, in mIMCD-K2 cells (Fig. 9B). The sequencing data from the positive clones confirmed the expression of both P2X1 and P2Y2 receptors in mIMCD-3 cells (Fig. 9, C and D).
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55 kDa for the P2X1 receptors (Fig. 10A, left), and the specificity of the band was demonstrated by peptide-blocking experiments (Fig. 10A, right). The electrophoretic mobility of this band is similar to that reported in human salivary gland epithelial cells as well as transfected human embryonic kidney cells (HEK 293 cells) (44, 48). The calculated molecular mass of mouse P2X1, 44.9 kDa (AAF68968
[GenBank]
, is lower than our observed molecular mass as well as that in previous reports (43, 44, 48). The slower electrophoretic migration has been attributed to glycosylation of the receptor in the extracellular domain, like the 70-kDa P2X1 immunoband found in human platelets (43).
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40, 55, and 67 kDa for mIMCD-3 cells, as were two other bands at 35 and 45 kDa (Fig. 10B, left). Peptide-blocking experiments indicate that the 40-, 55-, and 67-kDa bands are those recognized by the primary antibody (Fig. 10B, right). The calculated molecular mass for the P2Y2 receptor is 42 kDa (P35383
[GenBank]
), which is close to the observed 40-kDa band in mIMCD-3 cells. In the technical information provided by Alomone Labs, the P2Y2 receptor antibody has been shown by the manufacturer to detect a single immunoband near 60 kDa in rat brain membranes and two immunobands of 42 and 60 kDa in rat lung membranes. Therefore, it is possible that the more slowly migrating bands (55 and 67 kDa) are modified forms of P2Y2 or that the antibody cross-reacts with different proteins. The band pattern of interest in mIMCD3 cells was similar to that in mIMCD-K2 cells, and in both cases the 40-kDa band appears to be the most prominent. The calculated mass, peptide-blocking studies, and the presence of a 40-kDa band in both mIMCD-3 and mIMCD-K2 cells would suggest that the 40-kDa band is most likely P2Y2. |
DISCUSSION |
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It is well known that intracellular Ca2+ signaling is a focal point of many cellular signal transduction pathways and that it regulates a variety of physiological activities (6). The activation of different P2 receptors (i.e., P2X and P2Y) has been shown to cause different physiological responses. For example, extracellular ATP can influence either vasoconstriction via a P2X-dependent mechanism or vasodilatation via a P2Y-dependent mechanism in renal blood vessels (11, 16). More recently, it has been demonstrated that activation of P2X can induce cell apoptotic death, whereas activation of P2Y can induce cell proliferation (20). It is clear that a full understanding of ionotropic P2X and metabotropic P2Y signal transduction pathways in the renal epithelium will help to better define the roles of P2 receptors in the regulation of electrolyte, water, and acid-base in renal tubules.
In the kidney, P2Y receptors have been shown in almost all cells, and P2Y2 appears to be ubiquitous (26, 38). Studies using native cells or cultured cells from rats, rabbits, and mice show that the renal collecting duct has P2Y2 subtype receptors (8, 15, 23, 24, 28, 45, 46). The order of P2-receptor agonist potency in those studies is very similar to that in the current study (Fig. 5B). In addition, previous studies have also shown that P2Y1 receptors are expressed in rat IMCD cells (15, 23), rat outer medullary collecting tubule (8), mIMCD-K2 cells (28), and rabbit cortical collecting duct (47). Our data that the Ca2+ response induced by ATP could be inhibited by PPADS (Fig. 3A) and that the cells had a reasonable Ca2+ response to ADP at a dose of 10 µM (Fig. 5B) suggest that other P2Y subtype receptors, P2Y1 and P2Y4, may be present.
In contrast to the P2Y receptor, however, few studies have documented the expression of P2X receptors within the kidney. Filipovic et al. (17) demonstrated a P2X1-like purinergic receptor in LLC-PK1 cells, a widely used renal epithelial cell line. Schulze-Lohoff et al. (37) showed the expression of P2X7 in cultured mesangial cells. Chan et al. (9) investigated localization of the P2X1 receptor subtype in rat kidneys, and the receptor subtype has been detected in the vascular smooth muscle of intrarenal arteries, but not renal tubules. McCoy et al. (28) detected the presence of P2X3 and P2X4 receptors in mIMCD-K2 cells, but not other P2X receptor subtypes, including P2X1 receptors. More recently, Schwiebert et al. (39) reported that the P2X1 subtype sequence was detected by degenerate RT-PCR in mouse B6 collecting duct primary cultures. In the current study, the functional expression of P2X1 receptors in mIMCD-3 cells has been shown by pharmacological analysis (Figs. 6–8) and molecular analysis (Figs. 9 and 10). Although P2X1 (as well as P2X3) receptors are generally thought to rapidly desensitize after binding of ATP (usually within 1–2 s in excitable cells) (31), the actual desensitization time in different cell types, such as nonexcitable renal cells, could be prolonged. For example, a recent study, using simultaneous patch-clamp and intracellular Ca2+ measurements in P2X1 ion channel transfected HEK 293 cells, shows that ATP-elicited whole cell currents could be sustained over 20 s (44). The same study also shows that the intracellular Ca2+ response to ATP continues even longer than the aforementioned elicited whole cell current response. In conjunction, the P2X channels (perhaps more than 1 subtype) in mIMCD-3 cells appear to provide an appropriate Ca2+ influx in response to ATP (see Fig. 6).
Ca2+ influx plays an important role in signal transduction and in replenishing depleted Ca2+ stores. Voltage-dependent Ca2+ channels can provide a Ca2+ influx, and T-type Ca2+ channel subunit-1G mRNA is actually expressed in mIMCD-3 cells (3). However, it is unlikely that ATP-elicited Ca2+ influx occurs through this mechanism, because of the lack of effect of nifadipine and pimozide, L- and T-type Ca2+ channel blockers, respectively. Nonexcitable mIMCD-3 cells may employ other Ca2+ influx mechanisms, such as capacitative Ca2+ entry (50) and Ca2+ entry via ATP-gated nonspecific cation channels (the current study). The former mechanism is activated by the depletion of internal Ca2+ stores, resulting in activation of a Ca2+ influx (34). The latter mechanism is provided by agonist binding to a receptor to form ion channels (i.e., ATP-gated P2X ion channels) (10, 31). The two mechanisms may work together, but dissection of the separate actions is difficult. However, it is conceivable that each Ca2+ influx pathway has played its role in response to ATP. For example, our data show that Ca2+ entry to refill intracellular Ca2+ stores is essential for the cells to maintain the consecutive responses to repeated ATP stimulations (compare Figs. 1 and 4). The process of refilling most of the intracellular Ca2+ stores that are releasable via P2Y signaling pathways may be very rapid, because the repeated Ca2+ responses could be achieved within a few minutes (to recover the desensitization of the ATP receptor and to replenish the stores with Ca2+; see Fig. 1). In contrast, the long, sustained phase of the [Ca2+]i response in the presence of ATP could be contributed to by the combination of capacitative Ca2+ entry and Ca2+ entry through ATP-gated nonspecific cation channels. However, the sustained phase that has not been observed in the presence of UTP (Fig. 6B) supports that notion that the sustained phase of the [Ca2+]i is due to Ca2+ entry largely via ATP-gated ion channels.
The precise function of coexisting multiple P2X and P2Y receptor subtypes remains to be understood. Although P2X channels may provide an appropriate Ca2+ influx to facilitate the intracellular Ca2+ response to ATP, the data on ATP-stimulated intracellular Ca2+ release in a Ca2+-free bath solution (see Fig. 4B) indicate that the Ca2+ influx is not a prerequisite for internal Ca2+ release. While it might be possible that other ions in the Ca2+-free bath solution could still move in or out of the cell through ATP-gated nonselective cation channels and positively affect G protein-coupled P2Y receptor signaling pathways (needs to be determined), our data from Figs. 7 and 8 suggest that the functional P2X receptor itself plays a pivotal role in the P2Y receptor signaling pathway, at least in the intracellular Ca2+ in response to ATP action. However, the data cannot rule out the possibility that the P2X antagonists used in the study also affected P2Y receptors. A previous study shows that in the presence of trivalent cations, the ATP-elicited nonselective cation current became much smaller (30). Exactly how P2X-receptor antagonists block the increase in intracellular Ca2+ by ATP is subject to future investigation.
The mIMCD-3 cell line retains many cellular characteristics of the intact collecting duct, including formation of polarized functional epithelia (35), and has been used as a model for cellular biological and physiological studies (13, 36, 40, 41). The current study shows that cells were responsive to even submicromolar concentrations of ATP (Fig. 2), which is very close to the in vivo condition of the collecting duct (38). Our study also provides evidence of the presence of P2X and P2Y receptor subtypes in the cell line. Taking these results into consideration, the mIMCD-3 cell line can be used as a valuable model for defining nucleotides in the physiological and pathological processes of renal epithelial cells.
In summary, the present study has showed that Ca2+ entry to refill intracellular Ca2+ stores is an essential process for the cells to maintain the consecutive responses to repeated ATP stimulations. The ATP-induced increase in [Ca2+]i involves both P2X and P2Y subtype receptor signaling pathways. ATP failed to induce a significant increase in [Ca2+]i in the presence of P2X-receptor antagonists, indicating that P2X receptor-related signaling pathways, which usually cause extracellular Ca2+ influx, could interact with P2Y receptor-related signaling pathways that usually lead to internal Ca2+ release. The finding that inhibition of P2X signaling pathways affects the P2Y signaling pathways may provide a new regulatory mechanism of intracellular Ca2+ homeostasis in mIMCD-3 cells.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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1G-subunit of a voltage-dependent Ca2+ channel is localized in rat distal nephron and collecting duct. Am J Physiol Renal Physiol 279: F997–F1005, 2000.
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 J. S. Heo and H. J. Han ATP Stimulates Mouse Embryonic Stem Cell Proliferation via Protein Kinase C, Phosphatidylinositol 3-Kinase/Akt, and Mitogen-Activated Protein Kinase Signaling Pathways Stem Cells, December 1, 2006; 24(12): 2637 - 2648. [Abstract] [Full Text] [PDF]
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B. Kadereit, P. Fustier, K. Shojaati, B. M. Frey, F. J. Frey, and M. G. Mohaupt Extracellular ATP Determines 11{beta}-Hydroxysteroid Dehydrogenase Type 2 Activity via Purinergic Receptors J. Am. Soc. Nephrol., December 1, 2005; 16(12): 3507 - 3516. [Abstract] [Full Text] [PDF]
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S. S. Wildman, J. Marks, L. J. Churchill, C. M. Peppiatt, A. Chraibi, D. G. Shirley, J.-D. Horisberger, B. F. King, and R. J. Unwin Regulatory Interdependence of Cloned Epithelial Na+ Channels and P2X Receptors J. Am. Soc. Nephrol., September 1, 2005; 16(9): 2586 - 2597. [Abstract] [Full Text] [PDF]
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R. Sun, N. G. Carlson, A. C. Hemmert, and B. K. Kishore P2Y2 receptor-mediated release of prostaglandin E2 by IMCD is altered in hydrated and dehydrated rats: relevance to AVP-independent regulation of IMCD function Am J Physiol Renal Physiol, September 1, 2005; 289(3): F585 - F592. [Abstract] [Full Text] [PDF]
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) and SE (vertical bar) for each experiment are shown. The number of experiments (each experiment represents the response from at least 60 cells) varied from 4 to 18, as indicated in parentheses next to each data point. The dashed line is the sigmoidal curve fitted to the ATP doses, with an EC50 of 2.5 µM. Inset: 10 µM ATP raised [Ca2+]i 
