INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

NUCLEOTIDES IN THE extracellular fluid act as autocrine and paracrine hormones to regulate a variety of physiological processes (1, 4, 9, 13, 21, 22). For example, nucleotides inhibit Na⁺ absorption and stimulate Cl secretion across epithelia derived from many tissues (25, 30, 31, 36, 42, 44). In addition, nucleotides activate K⁺ channels in Madin-Darby canine kidney (MDCK) cells (19) and inhibit Na⁺ absorption across the rabbit cortical collecting duct (CCD) (32). Despite evidence documenting the effects of nucleotides on MDCK cells, A6 cells, and CCD cells (19, 32, 36, 44), the effects of nucleotides on NaCl transport in the inner medullary collecting duct (IMCD), the final nephron segment responsible for fine adjustments in NaCl excretion, are not known.

Once released from cells, nucleotides interact with specific plasma membrane-resident purinoceptors that act as ion channels or couple with phospholipases and adenylyl cyclase-driven signal transduction pathways (1, 4, 6, 9, 13, 14, 18, 22). Purinoceptors are divided into two classes: P1 (activated by adenosine) and P2 (activated by ATP and ADP) (1, 4, 6, 9, 13, 14, 18, 22). P2 purinoceptors have been subdivided into two families: P2X and P2Y (1, 4, 6, 9, 13, 14, 18, 22). The P2X purinoceptor family contains at least seven distinct subtypes (P2X₁ through P2X₇), each of which is a two transmembrane-spanning, ion channel-forming protein (5, 9, 11, 33, 34, 43, 45, 47). P2X receptor channels bind ATP and form Ca²⁺-permeable, nonselective cation channels that, when activated, increase intracellular Ca²⁺ by allowing Ca²⁺ entry from the extracellular fluid. P2Y purinoceptors are seven transmembrane-spanning, G protein-coupled metabotropic receptors containing at least six distinct receptor subtypes (P2Y₁ through P2Y₆) (1, 4, 6, 7, 12). P2Y purinoceptors are coupled to effectors through heterotrimeric G proteins and either stimulate (40) or inhibit (1, 13, 22) adenylyl cyclase. P2Y receptors also stimulate phospholipase activity and, in turn, increase intracellular Ca²⁺ (1, 4, 13, 22). In addition, P2Y purinoceptors enhance cAMP production through receptor-mediated prostaglandin release (39, 40).

Previously, we demonstrated that a cell line derived from mouse IMCD (mIMCD-K2) absorbs Na⁺ and secretes Cl by electrogenic mechanisms (28, 29, 37, 48). Moreover, we demonstrated that the purine nucleoside, adenosine, through an interaction with adenosine A₁ receptors, inhibits arginine vasopressin-stimulated Cl secretion (37). Because ATP is released from IMCD cells (35) and is present in the urine (10) and because the effects of nucleotides on NaCl transport in the IMCD are not known, we tested the hypothesis that ATP and related nucleotides regulate NaCl transport across mIMCD-K2 cells. To this end, we examined the effect of selective P2Y or P2X agonists on electrogenic Cl and Na⁺ transport across monolayers of mIMCD-K2 cells mounted in an Ussing chamber. Our results suggest that Cl short-circuit current (I^Cl_sc) is stimulated by apical membrane-resident P2Y purinoceptors and P2X receptor channels, whereas Na⁺ short-circuit current (I^Na_sc) is inhibited by apical membrane-resident P2Y purinoceptors and P2X receptor channels. By RT-PCR, we also report that mIMCD-K2 cells express mRNA for P2X₃ and P2X₄ receptors and for P2Y₁ and P2Y₂ receptors that may transduce extracellular nucleotide signals. We conclude that nucleotide agonists stimulate I^Cl_sc while extracellular nucleotides inhibit I^Na_sc across mIMCD-K2 monolayers through interactions with P2X and P2Y purinoceptors expressed on the apical plasma membrane.

	MATERIALS AND METHODS

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Cell culture. The mIMCD-K2 cell line was established by clonal dilution of IMCD cells isolated from transgenic mice [Tg(SV40E)Bri/7] and grown in continuous culture as described previously (28). Briefly, cells were cultured in tissue culture flasks (Costar, Cambridge, MA) coated with Vitrogen plating media (VPM) containing DMEM (JRH Biosciences, Lenexa, KS), human fibronectin (10 µg/ml; Collaborative Research Products, Bedford, MA), 1% Vitrogen 100 (Collagen, Palo Alto, CA), and BSA (10 µg/ml; Sigma Chemical, St. Louis, MO) and placed in an incubator maintained at 37°C and gassed with 5% CO₂-95% air. Every 48 h, 100% of the medium, PC-1 (BioWhittaker, Walkersville, MD) supplemented with PC-1 medium supplement (BioWhittaker), 5% fetal bovine serum (FBS; Hyclone, Logan, UT), 2 mM L-glutamine (Life Technologies, Grand Island, NY), and 50 U/ml penicillin and 50 µg/ml streptomycin (Life Technologies), was replaced. At confluence, cells were subcultured by light trypsinization and reseeded in VPM-coated cell culture flasks. In some studies, mIMCD-K2 cells were grown in DMEM-Ham's F-12 medium (Life Technologies) supplemented with 10% FBS (Hyclone), 10 µg/ml insulin (Sigma), 5.5 µg/ml transferrin (Sigma), 5.0 pg/ml sodium selenite (Sigma), 5 µM dexamethasone, 2 mM L-glutamine, 50 U/ml penicillin, and 50 µg/ml streptomycin, rather than PC-1 medium. Experimental results were qualitatively and quantitatively similar regardless of the cell culture medium used. As demonstrated previously, mIMCD-K2 cells display many of the phenotypic properties of the IMCD, including electrogenic Na⁺ absorption and Cl secretion (28, 29, 37, 48).

Measurement of short-circuit current in Ussing chambers. To measure electrogenic Na⁺ and Cl currents, mIMCD-K2 cells were seeded at a density of 1.2 × 10⁵ cells/0.6 cm² on permeable, VPM-coated Cellagen filter supports (ICN Biomedicals, Aurora, OH) as described previously (28). Every 24 h, 100% of the medium was replaced. Cells between passages 11 and 20 were used for all experiments.

I_sc data analysis. Steady-state and peak values of the I_sc were determined using Axotape software (Axon Instruments). Steady-state I_sc was recorded as the average of the current data over 1 min when individual current values over that period differed by <1% of the mean current. Peak values were reported as the highest single I_sc value measured after stimulation with agonist. Data are expressed as means ± SE. Differences between means in unpaired experiments were compared by repeated-measures ANOVA followed by Bonferroni's multiple comparisons test. Differences between means in paired experiments were compared by Student's t-test. All statistical analyses were performed with InStat statistical software (Graphpad, San Diego, CA). Statistical significance was ascribed for P < 0.05 or lower.

Preparation of total RNA for PCR. mIMCD-K2 cells were grown to confluence in collagen-coated flasks, and RNA was extracted using TRIzol reagent (Life Technologies), according to the manufacturer's instructions.

Preparation of cDNA. Total RNA was recovered by centrifugation, resuspended in diethyl pyrocarbonate (DEPC)-treated water (10-30 µl depending on size of pellet), and treated with RNase-free DNase (Life Technologies) to remove contaminating genomic DNA. Total RNA (2-4 µl) was mixed with 12-14 µl DEPC-H₂O, 2 µl DNase buffer, and 2 µl RNase-free DNase I and incubated at room temperature for 15 min. Two microliters of 25 mM EDTA were added to chelate Ca²⁺ and Mg²⁺ to stop the reaction, and the DNase-treated samples were heated at 65°C for 10 min to inactivate DNase I activity. Moloney murine leukemia virus (MMLV)-RT (Life Technologies) was used to reverse transcribe total RNA to cDNA. Each reaction contained 20-22 µl of total RNA template (DNase-treated sample from above), 2.5 µl dNTP mix (mixture of dATP, dCTP, dGTP, and dTTP; Pharmacia, 2 µM each), 2 µl oligo(dT)_12-18 primer (Life Technologies), 10 µl of 5× RT buffer, 2.5 µl dithiothreitol (0.1 M), 9.8 µl DEPC-H₂O, and 2 µl MMLV-RT enzyme (Life Technologies). Oligo(dT) primer ensures that all messenger RNA species were reverse transcribed to cDNA. The reaction was incubated for 1 h at 37°C in a thermocycler. Complementary DNA (cDNA) samples were then incubated at 90°C for 5 min in the thermocycler to inactivate the RT enzyme.

PCR of epithelial cDNA. We used degenerate and specific PCR primers designed to amplify the P2X gene family and specific members of the P2Y receptor gene family, P2Y₁ and P2Y₂. P2X degenerate primers were designed to an alignment of rat P2X₁, P2X₂, and P2X₃ (16). P2Y₁ and P2Y₂ primers were designed to the human cDNAs. Each PCR reaction contained 3-5 µl cDNA template, 33.8-35.8 µl ultraviolet-sterilized, double-distilled H₂O, 1 µl dNTP mix (Pharmacia, 2 µM), 1 µl forward primer (A) and reverse primer (B) (2 µM final concentration; see primers below), 0.2 µl Taq polymerase (Perkin-Elmer), 5 µl of 10× PCR buffer, and 3 µl of MgCl₂ solution (1.5 mM final concentration). The cycling parameters were established in 3 steps: 1) a 5-min, 94°C "hot start"; 2) 40 cycles of 30 s at 94°C, 1 min at appropriate "annealing" temperature (indicated with each primer pair below), and 1 min at 72°C; and 3) a 10-min "elongation" at 72°C to end the reaction. PCR products were visualized on a 1.5% agarose gel run with a 100-bp DNA ladder. PCR primers are shown below -Actin PCR amplification was performed to authenticate that cDNA synthesis was successful in each sample using a housekeeping gene. Once -actin cDNA was detected and amplified, amplification with P2X or P2Y primer pairs could proceed on the authenticated cDNA samples.

DNA sequencing of PCR products. PCR products were isolated from agarose gel slices using the Qiaquick gel extraction kit (Qiagen). PCR products were ligated into the pGEM-T vector system (Promega) and were then transformed into JM109 high-efficiency competent cells (Promega). The grow-ups of the transformations were then plated on LB agar plates containing ampicillin (100 µg/ml), 5-bromo-4-chloro-3-indolyl-D-galactopyranoside (X-Gal; 40 µg/ml), and isopropyl--thiogalactopyranoside (IPTG; 100 µg/ml) for ampicillin selection of successful transformants and for blue/white selection of successful PCR product ligation. White colonies were picked off the plate to inoculate a 6-ml LB grow-up with continued ampicillin selection. The pGEM-T plasmid with insert was purified from the grow-up using a PerfectPrep miniprep kit (5 Prime3 Prime, Boulder, CO). The purified DNA was denatured in 0.2 N NaOH, precipitated in 7.5 M NH₄Cl and 100% ethanol, and sequenced using the Sequenase dideoxy termination method (Amersham/US Biochemicals) using [-³⁵S]dATP (DuPont NEN) incorporation by our laboratories. The resulting DNA sequence was read by two independent investigators and screened with the basic local alignment research tool (BLAST) algorithm (2).

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Nucleotides regulate I^Na_sc and I^Cl_sc. To determine whether extracellular nucleotides regulate electrogenic Na⁺ and/or Cl transport, we measured I^Na_sc and I^Cl_sc across monolayers of mIMCD-K2 cells mounted in Ussing chambers. ATP and UTP (100 µM) induced a small, insignificant increase of I^Na_sc followed by prolonged inhibition of I^Na_sc (Figs. 1 and 2). ATP and UTP transiently, but significantly, increased I^Cl_sc (Figs. 3 and 4). After the transient increase, I^Cl_sc returned gradually (~10 min) to levels not significantly different from I_sc before the addition of ATP and UTP (Figs. 3 and 4). It is important to note that physiologically relevant concentrations of ATP (1 µM) also reduced I^Na_sc (4.8 ± 1.5 µA/cm²; P < 0.05) and stimulated I^Cl_sc (4.2 ± 0.1 µA/cm²; P < 0.05).

View larger version (14K): [in this window] [in a new window]   Fig. 1.   ATP inhibits Na+ short-circuit current (INasc). Representative current record illustrating the effects of ATP on INasc is shown. ATP (100 µM) was added to the apical and basolateral bath solutions. Amiloride (10 µM) was added to the apical bath solution to inhibit Na+ absorption. Effects of UTP on Isc were qualitatively similar (representative record not shown). Decline in Isc at time 0 to steady-state (~10-12 min) has been described previously (28, 29, 37, 48).

View larger version (33K): [in this window] [in a new window]   Fig. 2.   ATP and UTP inhibit INasc. Data summary of the effects of ATP and UTP on INasc is given. ATP (100 µM) and UTP (100 µM) induced a small, transient increase in INasc (not significantly different from basal; P < 0.05), followed by a prolonged steady-state inhibition of INasc. After administration of amiloride, Isc was not significantly different from 0 µA/cm2, thereby indicating that Isc is referable to Na+ absorption. * P < 0.05 vs. basal.

View larger version (14K): [in this window] [in a new window]   Fig. 3.   ATP stimulates Cl short-circuit current (IClsc). Representative current record illustrating the effects of ATP on IClsc is shown. ATP (100 µM) was added to the apical and basolateral bath solutions. Amiloride (10 µM) and diphenylamine-2-carboxylic acid (DPC; 10 mM), an inhibitor of Cl channels, were added to the apical bath solution. Effects of UTP on Isc were qualitatively similar (representative record not shown).

View larger version (20K): [in this window] [in a new window]   Fig. 4.   ATP and UTP stimulate IClsc. Data summary of the effects of ATP and UTP on IClsc is given. ATP (100 µM) and UTP (100 µM) induced a transient, significant increase in IClsc, followed by a slow decline to a steady-state value significantly less than the peak value following administration of ATP or UTP. In the presence of amiloride and following administration of DPC, Isc was not significantly different from 0 µA/cm2, thereby indicating that Isc in amiloride-treated monolayers is referable to Cl secretion. * P < 0.05 vs. amiloride-inhibited Isc.

Pharmacological identification of purinoceptors. To determine if P2X and/or P2Y receptors regulate I^Na_sc and I^Cl_sc, we used a pharmacological approach. ATP binds to both P2X and P2Y receptors (1 µM); thus ATP cannot be used to discriminate between P2X and P2Y receptors. In contrast, UTP (1-100 µM) binds selectively to P2Y receptors and, in particular, to the P2U or P2Y₂ receptors, whereas ,-methylene-ATP (,-MeATP; 1-100 µM) and ,-methylene-ATP (,-MeATP; 1-100 µM) bind selectively to P2X receptors (1, 11, 21). Therefore, UTP can be used to identify P2Y receptors and ,-MeATP and ,-MeATP can be used to identify P2X receptors. UTP (10 µM) reduced I^Na_sc by 4.8 ± 1.0 µA/cm² (P < 0.05). ,-MeATP (100 µM) and ,-MeATP (100 µM) also reduced I^Na_sc by 2.0 ± 0.8 µA/cm² (P < 0.05) and by 3.9 ± 0.9 µA/cm² (P < 0.05), respectively. These results indicate that nucleotide inhibition of I^Na_sc across mIMCD-K2 cells is mediated by P2X and P2Y purinoceptors.

Functional localization of P2Y and P2X purinoceptors. Experiments were conducted to determine if P2Y and P2X purinoceptors are expressed in the apical and/or basolateral plasma membranes of mIMCD-K2 cells. Because antibodies that recognize these receptors are not yet available, we used a pharmacological approach to determine if P2Y and P2X purinoceptors are expressed in the apical and/or basolateral membranes. UTP (100 µM) reduced I^Na_sc when added to the apical solution (Fig. 5). UTP also stimulated I^Cl_sc when added to the apical solution (Fig. 6). P2U purinoceptor-mediated effects in the basolateral membrane were not statistically significant. Therefore, P2Y purinoceptors are expressed predominantly in the apical plasma membrane.

View larger version (17K): [in this window] [in a new window]   Fig. 5.   Polarity of nucleotide receptor agonist effects on INasc. Sidedness of the effects of UTP, ,-methylene-ATP (MeATP), and ,-MeATP on INasc is shown. All 3 agonists had a significant effect when added to the apical solution. * P < 0.05 vs. amiloride-inhibited current.

View larger version (19K): [in this window] [in a new window]   Fig. 6.   Polarity of nucleotide receptor agonist effects on IClsc. Sidedness of the effects of UTP, ,-MeATP, and ,-MeATP on IClsc is shown. Although all 3 agonists increased IClsc significantly when added to the apical solution, they had no significant effect when added to the basolateral solution. * P < 0.05 vs. 0 µA/cm2.

Molecular identification of P2X and P2Y purinoceptors. Little is known about P2X and P2Y receptor expression in IMCD cells. Accordingly, to begin to identify which P2X and P2Y receptors are expressed in mIMCD-K2 cells, we identified P2X and P2Y receptor subtype mRNA expression by RT-PCR. RT-PCR for the housekeeping gene, -actin (750-bp product), was performed initially to authenticate a successful cDNA synthesis (Fig. 7A). The "no cDNA" and "no RT" negative PCR controls had no PCR product for all genes amplified (Fig. 7). PCR products of the expected size (330 bp) for P2X receptors were identified in mIMCD-K2 cells (Fig. 7B). A positive control was run in parallel that included a human pancreatic epithelial cell line (CFPAC-1). To confirm that the PCR products were indeed derived from P2X receptor mRNA and to determine which P2X receptor mRNAs are expressed in mIMCD-K2 cells, PCR products were sequenced. Of 32 minipreps processed from two different degenerate mIMCD-K2 P2X receptor PCR reactions, 14 sequences were identified through BLAST analysis as P2X₃ and 18 were identified as P2X₄ (Table 1). Representative sequence alignments for our mouse P2X₃ and P2X₄ PCR product with cloned rat cDNA sequences are shown in Fig. 8, A and B. These results suggest that P2X₃ and P2X₄ receptors may regulate transepithelial NaCl transport by mIMCD-K2 monolayers.

View larger version (55K): [in this window] [in a new window]   Fig. 7.   P2X and P2Y receptors are expressed by a mouse inner medullary collecting duct cell line (mIMCD-K2 cells). A: amplification of -actin (750-bp PCR product) to verify a successful cDNA synthesis (CFPAC-1 cells used as a positive control for P2X receptors). Amplification of P2 purinoceptors was conducted using degenerate primers for the P2X receptor gene family (330-bp PCR product) in B, using specific primers for the P2Y1 gene (750-bp PCR product) in C, and using specific primers for the P2Y2 (or P2U) gene (500-bp PCR product) in D (16HBE14o cells used as a positive control for P2Y2 receptors). NC, "no cDNA," negative PCR control. "No RT" controls were also negative (data not shown). L, 100-bp DNA ladder. These gels are representative of at least 3 PCR amplification with each set of PCR primers.

View larger version (108K): [in this window] [in a new window]   Fig. 8.   cDNA sequences confirming the authenticity of mIMCD-K2 P2X and P2Y PCR products. BLAST alignments of P2 purinoceptor mIMCD-K2 PCR products with the closest cloned cDNA sequences are shown. A: P2X3 PCR product alignment with the cloned rat P2X3 receptor (rP2X3R) cDNA sequence. B: P2X4 PCR product alignment with the cloned rat P2X4 receptor (rP2X4R) cDNA sequence. C: P2Y1 PCR product alignment with the cloned mouse P2Y1 receptor (mP2Y1R) cDNA sequence. D: P2Y2 PCR product with the cloned mouse P2Y2 receptor (mP2Y2R) cDNA sequence. Nucleotides in bold show differences in identity; gaps in sequence are shown by the dashed or dotted lines or where indicated.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The goal of the present study was to determine if nucleotides regulate NaCl transport across mIMCD-K2 cells. Our results demonstrate that nucleotides inhibit I^Na_sc through interaction with P2Y purinoceptors expressed in the apical plasma membrane. P2X purinoceptors in the apical membrane also inhibit I^Na_sc. In contrast, nucleotides stimulate I^Cl_sc across mIMCD-K2 cells through interactions with both P2X and P2Y purinoceptors expressed in the apical plasma membrane. Finally, by RT-PCR, we demonstrated that mIMCD-K2 cells express mRNA for P2X₃ and P2X₄ purinergic receptor channels and P2Y₁ and P2Y₂ G protein-coupled purinergic receptors.

Previous research has demonstrated that P2Y₂ (or P2U) purinoceptors are expressed in the proximal tubule (8, 26, 46, 49), MDCK cells (17, 39, 40), LLC-PK₁ cells (3), A6 cells (36), CCD (32, 41), and IMCD (15, 20, 27). Little is known about P2X purinoceptor expression in renal epithelia. P2X₁-like purinoceptors are expressed in LLC-PK₁ cells (16). Together, our results provide novel data demonstrating that P2Y₁ and P2Y₂ receptors and P2X₃ and P2X₄ receptors expressed in the apical plasma membrane inhibit I^Na_sc and stimulate I^Cl_sc across mIMCD-K2 cells. Our data are consistent with the hypothesis that nucleotides released from IMCD cells themselves or from other upstream renal epithelial cells activate P2 purinoceptors that regulate Na⁺ absorption and Cl secretion. It is important to note that nucleotides are present in the urine and blood in nanomolar to low micromolar (10 µM) concentrations (10, 21, 23, 24), amounts that would stimulate purinoceptors and regulate Na⁺ and Cl transport in mIMCD-K2 cells. Recent preliminary results showed that mIMCD-K2 cells release ATP under basal and stimulated conditions into the apical and basolateral solution (35).

The IMCD, and perhaps other nephron segments or cell types in the cortex and/or medulla, may be a source of nucleotides that, once released, can regulate NaCl transport in an autocrine and/or paracrine fashion. Interestingly, our results demonstrate that ATP would affect NaCl transport by the IMCD directly by inhibiting Na⁺ absorption and stimulating Cl secretion. Thus ATP would convert NaCl absorption by the IMCD to NaCl secretion (28, 29), thereby enhancing NaCl excretion. It appears, therefore, that ATP and other nucleotides may play an important role in the fine tuning of NaCl transport by the IMCD. The effects of ATP (and, possibly, its metabolites) on glomerular afferent and efferent arterioles may also affect NaCl handling indirectly by affecting glomerular filtration rate (GFR) and renal blood flow. These effects are likely to be complex; for example, ATP is known to stimulate vasoconstriction of afferent, but not efferent, arterioles and to decrease glomerular pressure (10, 23). These effects, in turn, would result in a decrease in GFR and reduced NaCl excretion.

In conclusion, our data demonstrate that nucleotides inhibit Na⁺ absorption and stimulate Cl secretion through interactions with P2X purinergic receptors (P2X₃, P2X₄) and P2Y G protein-coupled receptors (P2Y₁, P2Y₂) expressed in the apical membrane of mIMCD-K2 cells.