INTRODUCTION

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THE ROLE OF INTRACELLULAR ATP as a ubiquitous cellular energy source has been recognized for many years. Although much is known about the regulation of ion transport across cell membranes by intracellular ATP (34, 36), less is known about the regulation of ion transport and cell function by extracellular nucleotides (1, 2). Extracellular ATP affects a wide variety of cells via purinergic membrane receptors (10). 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 (2). Recent molecular biological and functional studies indicate that there are two main types of P₂ receptors for extracellular ATP (11, 23, 28): G protein-coupled receptors, with seven transmembrane domains (P2T, P2Y, and P2U), and receptors with intrinsic ion channels (P2X). This latter class of receptors forms ATP-gated, calcium-permeable, cation-selective channels (13, 14).

The cloning of cDNAs encoding the P2X₁ receptor from rat pheochromocytoma P12 cells and P2X₂ receptor from vas deferens established a new family of related ion channels, termed ATP-gated channels or P2X receptors (4, 39). To date, seven members of this family have been cloned (3, 25, 35). These receptors have certain common functional features, such as an intermediate single channel conductance and nonselectivity among cations (15), but differ from one another with respect to agonist specificity, the presence or absence of desensitization, and their sensitivity to certain inhibitors (15, 33).

To date, the only well-documented P₂ receptors in LLC-PK₁ cells are P2Y receptors (22, 42). Previous studies have shown that ATP increases intracellular calcium concentration ([Ca²⁺]_i) in LLC-PK₁ cells acting, at least in part, via P2Y receptors coupled to G proteins (41). Although there is evidence that extracellular ATP acts via ionotropic receptors in some other epithelial cells, e.g., parathyroid acinar cells (32), there has been no direct evidence for such purinergic receptors in renal epithelial cells. The purpose of the present study was to examine, using molecular and functional assays, whether P2X receptors are expressed in the LLC-PK₁ renal epithelial cell line. This study provides the initial characterization of ionotropic purinergic receptors in the LLC-PK₁ renal epithelial cell line.

	MATERIALS AND METHODS

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Cell culture. LLC-PK₁ cells were obtained from American Type Culture Collection (CRL-1392; Rockville, MD) and grown in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal bovine serum and 2 mM glutamine (GIBCO-BRL; Life Technologies, Grand Island, NY) at 37°C under a 5% CO₂ atmosphere. For patch-clamp studies, cells were grown on 35-mm tissue culture dishes and studied 1-4 days after plating. To separate cells for patch-clamp studies without exposure to trypsin, which might affect ion channels, cells were incubated for 10-15 min in a Ca²⁺-/Mg²⁺-free phosphate-buffered saline solution at 37°C. After this treatment, cells became rounded and separate from adjacent cells but were still attached to the culture dish. During the patch-clamp experiments, the cells were superfused continuously via a micropipette positioned ~200 µM from the clamped cell. Delivery of the solutions was controlled by a remote-controlled manifold. Bath solutions were maintained at 37°C in a modified Leiden chamber (PDMI-2; Medical Systems, Greenvale, NY) mounted on an inverted microscope (Nikon Diaphot with Hoffman Modulation optics).

Solutions and chemicals. ATP, ATP analogues, and other compounds used in this study were dissolved directly in the bath solutions, and the pH of the resulting solution was readjusted to its original value. ATP was added as the Mg salt, and ATP-containing solutions were kept on ice until use, to minimize hydrolysis.

Patch-clamp studies. Whole cell patch-clamp studies were performed using the conventional, whole cell patch-clamp technique, as described in previous studies (17). Patch pipettes were fabricated from Corning 7052 glass (Garner Glass, Claremont, CA), using a Flaming Brown micropipette puller (model P-87; Sutter Instrument, Novato, CA) and coated with Sylgard 184 (Dow Corning, Midland, MI) to within 500 µm of the tip and fire polished prior to use. The pipette resistance averaged 4-6 M when filled and immersed in the standard solutions. The pipette was connected to the head stage of an Axopatch 1D amplifier (Axon Instruments, Foster City, CA) via a Ag-AgCl pellet. The bath solution was connected to ground via a KCl-agar bridge. The pipette solution contained (in mM) 140 potassium methanesulfonate, 10 NaCl, 10 EGTA, 10 HEPES, pH 7.2. The standard bath solution contained (in mM) 140 NaCl, 5 KCl, 1 CaCl₂, 2 MgCl₂, and 10 HEPES, pH 7.4. To study ion selectivity, Na⁺ in the bath solution was replaced by either K⁺ or N-methyl-D-glucamine (NMDG), and Cl was replaced with methanesulfonate. To obtain current-voltage relations, cells were voltage clamped at a holding potential of 60 mV and then clamped for 400 ms to voltages ranging from 100 mV to +40 mV, in 10-mV steps. Currents were digitized (usually at 5 kHz) and stored on a hard disk for later analysis. The LLC-PK₁ cells in this study had an average capacitance of 30 ± 2 pF (n = 28). All experiments were performed at 37°C. All potentials are expressed as pipette relative to the bath. Currents are defined as positive when the direction of flow of positive charges is out of the pipette and cell.

Fluorescence measurements of [Ca²⁺]_i. Subconfluent LLC-PK₁ cells grown on glass coverslips were loaded with 5 µM fura 2-AM (Molecular Probes, Eugene, OR) in bicarbonate-buffered saline containing 10 mM probenecid for 1 h at 37°C, washed, and then incubated for 30 min to allow for hydrolysis of fura 2. This protocol gave good loading in a homogenous pattern. The coverslip was then transferred to a temperature-regulated, continuously perfused chamber on the stage of an inverted microscope. Cells were excited alternately at 340 and 380 nm, and the emitted light (>510 nm) intensity was determined by photometry. Fluorescence was measured from a group of two to three cells, using an adjustable window in the emission light path. Background fluorescence was measured before each experiment, using the same-size field containing unloaded cells, and automatically subtracted from the final trace. Fluorescence ratios were acquired at 1-s intervals during the experiments. In situ calibration of the ratios was carried out at the end of each experiment by application of 5 µM ionomycin and either saturating levels of Ca²⁺ (1.25 mM), to give maximal ratio (R_max) of 340 to 380, or very low levels of Ca²⁺ (10 mM EGTA and no added Ca²⁺), to give a minimum ratio (R_min) of 340 to 380. Cytosolic calcium was calculated from the fluorescence ratios using the equation where K_d = 224 nM is dissociation constant for Ca²⁺ binding to fura 2, and  is the ratio of the fluorescence signal at 380 nm in zero Ca²⁺ and saturating levels of Ca²⁺ obtained with ionomycin (20).

Reverse transcription-polymerase chain reaction. Total RNA was isolated from LLC-PK₁ cells, using a guanidine thiocyanate-phenol extraction method (TriReagent; Molecular Research Center, Cincinnati, OH). RT-PCR was performed using degenerate primers corresponding to conserved regions of the P2X_1-3 rat cDNA sequences. The forward primer was 5' TTC ACC (C/A)T(T/C) (T/C)TC ATC AA(G/A) AAC AGC ATC 3', corresponding to nucleotides 547-574 of the P2X₁ open reading frame (4). The reverse primer, 5' TGG CAA A(C/T)C TGA AGT TG(A/T) AGC C 3', corresponded to nucleotides 855-877 of the P2X₁ open reading frame. Reverse transcription was performed on 1 µg total RNA at 42°C for 30 min in a total reaction volume of 20 µl containing 1 µM of the reverse primer and 50 U of Moloney murine leukemia virus reverse transcriptase (GIBCO-BRL, Life Technologies). A control reaction was performed in the absence of reverse transcriptase. PCR amplification of the resulting cDNA was performed in a total volume of 100 µl. The PCR mixture, containing 50 mM KCl, 2.5 mM MgCl₂, 0.2 mM dNTP, 20 pmol of each primer, 10 mM Tris · HCl, and 2 U Taq polymerase, was subjected to 35 cycles at 94°C for 30 s, 55°C for 30 s, and 72°C for 1 min. The PCR products were separated by electrophoresis through a 2% agarose gel. A single band of the anticipated size was obtained. The DNA band was purified from the gel (QiaQuick Gel Extraction Kit; Qiagen, Chatsworth, CA), ligated into the pGEM-T vector (Promega, Madison, WI), and transformed into INVF' cells (Invitrogen, San Diego, CA). Plasmid DNA was isolated from the resulting colonies, using an alkaline extraction mini-prep (PerfectPrep; 5'  3', Boulder, CO), and sequenced using an automated fluorescent sequencing system (Prism DyeDeoxy Terminator sequencing kit and ABI model 373 sequencer; Perkin-Elmer, Foster City, CA).

Results are presented as means ± SE. Statistical significance was determined using the t-test. One-way analysis of variance with Bonferroni correction was applied to multiple comparisions.

	RESULTS

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Whole cell patch-clamp studies of ATP-induced currents in LLC-PK₁ cells. Although ATP-induced whole cell currents have been characterized in a variety of excitable and nonexcitable cells, the effects of extracellular ATP on whole cell currents in LLC-PK₁ cells have not been reported. Figure 1 depicts the whole cell current response to the application of extracellular ATP and suramin, a P2 receptor antagonist. For these experiments, the cells were clamped at a voltage of 60 mV, and current was measured continuously while cells were superfused with solutions containing the indicated substances. ATP caused a prompt, sustained increase in the inward whole cell current, which reflected an increase in the whole cell conductance (see Fig. 3). This response was concentration dependent and reversible. ATP (10 µM) and 100 µM ATP increased the average whole cell conductance from 1.7 ± 0.1 (n = 24) to 4.2 ± 0.4 nS (P < 0.001, n = 6) and 12.6 ± 0.7 nS (P < 0.0001, n = 19), respectively. As shown in Fig. 1, D-F, suramin had no effect on basal whole cell current but completely and reversibly inhibited the current response to 100 µM ATP (1.6 ± 0.2 nS, n = 4).

View larger version (15K): [in this window] [in a new window]   Fig. 1.   Concentration-dependent effects of ATP on whole cell currents in LLC-PK1 cells. Membrane currents were recorded at 60 mV, using a conventional whole cell patch configuration. ATP-containing solutions were applied during the intervals indicated by the bars. Application of extracellular ATP caused a sustained inward current, which was concentration dependent (A-C) and could be inhibited reversibly by suramin (D-F). Small spikes in the control tracing are electrical artifacts resulting from the solution-changing device. Pipette solution contained (in mM) 140 potassium methanesulfonate, 10 NaCl, 10 EGTA, and 10 HEPES, and bath solution contained (in mM) 140 NaCl, 5 KCl, 1 CaCl2, 2 MgCl2, and 10 HEPES. Traces are representative of 4-24 experiments for each condition.

Figure 2 shows continuous current traces from a cell during exposure to the indicated agents, all at concentrations of 100 µM. As in Fig. 1, membrane current was recorded at 60 mV. Adenosine, ADP, ,-methylene- ATP, and ,-methylene-ATP had little or no effect on the whole cell conductance. ATP and 2-methylthio-ATP, a P2X receptor agonist, stimulated inward currents at 60 mV (data not shown). The sustained inward current during ATP exposure (5-10 min, Figs. 1 and 2) indicates that the ATP-activated currents showed no sign of desensitization during repeated or continued exposure to extracellular ATP. As will be discussed below, these functional properties are similar to those of the P2X₂ receptor found in rat PC12 pheochromocytoma cells (4).

View larger version (12K): [in this window] [in a new window]   Fig. 2.   Effect of different nucleotides, all applied at 100 µM concentrations, on whole cell current. Adenosine, ADP, ,-methylene-ATP (--ATP), and ,-methylene-ATP (--ATP) had little or no effect on the whole cell currents recorded at 60 mV. Pipette and bath solutions were the same as described in Fig. 1. Traces are representative of 3-5 experiments for each agent.

The ionic basis of the ATP-dependent inward current was examined. Figure 3A shows the current-voltage relationships of a cell before (solid squares) and during (open triangles) the application of 100 µM ATP in a bath solution containing Na⁺. Application of ATP resulted in an increased whole cell conductance with a reversal potential of +5 mV, which was far from the reversal potentials (E) for either Cl (E_Cl = 72 mV), Na⁺ (E_Na = 70 mV), or K⁺ (E_K = 88 mV). Overall, exposure of cells to 100 µM ATP depolarized the reversal potential to 2.6 ± 0.8 mV (n = 19). UTP (100 µM), a P2Y₂ receptor agonist, elicited no change in the whole cell conductance (open circles, n = 3). When Cl in the bath solution was replaced by methanesulfonate, the reversal potential did not change (data not shown), indicating that Cl did not serve as the charge carrier. In contrast, the effects of substitution of Na⁺ and K⁺ were consistent with cations being the major conducting ions. Thus, when Na⁺ was replaced by NMDG (Fig. 3A, solid triangles), the reversal potential shifted from +5 mV to 72 mV, consistent with a Na⁺ permeability. However, when the bath Na⁺ was replaced by K⁺ (Fig. 3B, open triangles), the reversal potential did not shift, indicating that the ATP-induced conductance was not selective for Na⁺ over K⁺. The ATP-induced conductance was not inhibited by either 500 µM glibenclamide (Fig. 3B, open inverted triangles) or 5 mM barium (Fig. 3B, solid diamonds), indicating that it was not mediated by potassium channels.

View larger version (15K): [in this window] [in a new window]   View larger version (17K): [in this window] [in a new window]   Fig. 3.   A: current-voltage relations of a cell before and during application of 100 µM ATP. Application of ATP resulted in an increase in the conductance from 1 to 10 nS, with a reversal potential close to 5 mV. Replacement of Na+ by N-methyl-D-glucamine (NMDG) shifted the reversal potential to 75 mV. UTP (100 µM) had no effect on the whole cell current-voltage relations. B: when Na+ was deleted from the bath and replaced with a symmetric, high-K+ solution, the reversal potential did not shift, indicating that the conductance does not discriminate between Na+ and K+. ATP-induced conductance was not inhibited by either 500 µM glibenclamide (Glib) or 5 mM Ba2+.

Activation of P2Y receptors causes the mobilization of calcium from intracellular stores (10, 23). Mobilization of intracellular calcium, with depletion of calcium stores, activates a conductive pathway for calcium in the plasma membrane, the so-called calcium release-activated conductance (7). To determine whether the membrane currents activated by ATP were the result of the depletion of intracellular calcium stores, we examined the effects of thapsigargin on whole cell currents. In these experiments, the pipette solution contained 10 mM EGTA to chelate any free cytosolic calcium. As shown in Fig. 4, A-C, ATP reversibly increased the whole cell conductance. In D-F, the cell was superfused with a calcium-free bath solution, which also contained 5 µM thapsigargin for 5 min, to deplete the intracellular stores. Depletion of the intracellular calcium stores (Fig. 4D) did not result in an increase in the whole cell conductance. However, ATP still activated the whole cell conductance reversibly, even when calcium stores were depleted (Fig. 4, E and F). In separate studies, intracellular calcium was measured to confirm that thapsigargin depleted intracellular calcium stores and activated capacitative calcium influx. Figure 4G shows a trace from such an experiment, indicating that, in the absence of extracellular calcium, thapsigargin caused a transient increase in [Ca²⁺]_i (due to release of calcium from stores) and that, when calcium was returned to the extracellular solution, [Ca²⁺]_i increased promptly due to capacitative entry. The findings that thapsigargin did not increase the whole cell conductance by itself and did not inhibit the response to ATP indicate that the effects of ATP on the whole cell conductance were not mediated via ATP-induced depletion of intracellular calcium stores.

View larger version (25K): [in this window] [in a new window]   Fig. 4.   Effects of calcium store depletion on whole cell currents. Current-voltage profiles were obtained from the same cell under the indicated conditions. Intracellular calcium was chelated with 10 mM EGTA in the pipette solution. A-C: reversible activation of the whole cell conductance by 100 µM ATP (B). D-F: cell was bathed in a calcium-free bath containing 5 µM thapsigargin (TG). Thapsigargin had no effect on the whole cell conductance (D) and did not affect the response to 100 µM ATP (E). G: intracellular calcium measured using fura 2 during exposure of cells to 5 µM thapsigargin in the absence and presence of extracellular calcium. Trace is representative of 3 such experiments. Voltage steps were from 100 mV to +40 mV, in 10-mV increments, from a holding potential of 60 mV. R 340/380, 340- to 380-nm ratio.

Effects of ATP on [Ca²⁺]_i in LLC-PK₁ cells. P2X receptors form Ca²⁺-permeable channels in some cells (3, 4, 39). To determine whether extracellular ATP induces an influx of extracellular calcium in LLC-PK₁ cells, [Ca²⁺]_i was measured, using the calcium indicator dye fura 2-AM. As shown by a representative trace in Fig. 5, the application of 10 µM ATP caused a rapid and sustained rise in [Ca²⁺]_i in LLC-PK₁ cells, from 48 ± 3 to 380 ± 67 nM (n = 16). To determine the source of the intracellular calcium, ATP was applied to cells in a nominally calcium-free bath solution. When extracellular calcium was chelated, ATP produced only a small, transient rise in [Ca²⁺]_i. Overall, in the absence of extracellular Ca²⁺ ATP increased [Ca²⁺]_i from 34 ± 4 to only 78 ± 13 nM (n = 9), indicating that the majority of the calcium increase was due to influx of extracellular Ca²⁺. The effect of ATP on [Ca²⁺]_i, like the effect of ATP on whole cell current (Fig. 1), was inhibited by 100 µM suramin (Fig. 6). Figure 7 illustrates effects of other nucleotides on [Ca²⁺]_i. Adenosine (10 µM, n = 2; data not shown), ADP (10 µM), and ,-methylene ATP (10 µM) had little or no effect on [Ca²⁺]_i. Consistent with previous studies demonstrating P2Y₂ receptors in LLC-PK₁ cells, we found that 10 µM UTP increased [Ca²⁺]_i (not shown, n = 3). Finally, since P2X receptors are pertussis toxin insensitive, we measured [Ca²⁺]_i in cells treated overnight with 0.5 µg/ml pertussis toxin. The ATP-induced increase in [Ca²⁺]_i in pertussis toxin-treated cells, from 51 ± 6 to 264 ± 42 nM (n = 3), was essentially the same magnitude as that seen in control cells. Thus extracellular ATP induces a suramin-sensitive, pertussis toxin-insensitive increase in calcium influx in LLC-PK₁ cells.

View larger version (15K): [in this window] [in a new window]   Fig. 5.   Original record showing the effect of 10 µM ATP on intracellular Ca2+ concentration ([Ca2+]i) measured using fura 2 in the presence and absence of extracellular Ca2+. Application of ATP produced a large and sustained increase in intracellular Ca2+. However, only a partial and transient increase of intracellular Ca2+ was observed when ATP was applied in a Ca2+-free, EGTA-buffered solution (see also Fig. 4G).

View larger version (14K): [in this window] [in a new window]   Fig. 6.   Effects of ATP and suramin on intracellular Ca2+. After the first application of 10 µM ATP, which produced a large increase in intracellular Ca2+, cells were superfused with 100 µM suramin, and 10 µM ATP was added in the presence of suramin (bar). ATP-induced rise in intracellular Ca2+ was almost completely abolished in the presence of suramin. Thereafter, cells were rinsed with control solution, and ATP was added, producing again a prompt rise in intracellular Ca2+. Trace is representative of 4 paired experiments.

View larger version (13K): [in this window] [in a new window]   Fig. 7.   Effects of different nucleotides on intracellular Ca2+. Cells were superfused with the indicated agents during time intervals indicated by bars. ADP (n = 4) produced a small rise in intracellular Ca2+, whereas ,-methylene ATP (n = 3) produced no response.

Identification of P2X transcripts in LLC-PK₁ cells by RT-PCR. The functional results described above are consistent with the ATP activation of P2X receptors in LLC-PK₁ cells. However, P2X receptors have not been described previously in renal epithelial cells and in only a limited number of nonrenal epithelial cells (16). To determine whether LLC-PK₁ cells express a P2X receptor related to other cloned P2X receptors, RT-PCR was performed using degenerate primers based on conserved regions of the rat P2X₁, P2X₂, and P2X₃ sequences. As shown in Fig. 8, RT-PCR produced a 330-bp fragment from total RNA isolated from LLC-PK₁ cells. The product was generated only in the presence of reverse transcriptase. This PCR product was cloned and sequenced. Figure 9 shows an alignment of the predicted amino acid sequence of the PCR fragment with the known P2X receptors cloned in the rat. The sequence of the LLC-PK₁ fragment was 80% identical to the rat P2X₁ receptor cloned from vas deferens smooth muscle, confirming that the amplified product corresponds to a porcine member of the P2X receptor family. Homology to the other rat P2X receptors was 30-50%.

View larger version (67K): [in this window] [in a new window]   Fig. 8.   Agarose gel showing the product of RT-PCR amplification. Product was amplified by RT-PCR of LLC-PK1 RNA, using degenerate primers for the rat P2X1-3 receptors. A fragment of the expected size (330 bp) was produced in the presence but not absence of reverse transcriptase (RT). Lanes, from left to right: 100-bp DNA ladder, reaction with (+) RT, and control (RT).

View larger version (56K): [in this window] [in a new window]   Fig. 9.   Amino acid sequences of the rat P2X receptors aligned with the sequence deduced from the nucleotide sequence of the LLC-PK1 PCR product (Fig. 8). Bold letters denote identical amino acids. Numbers in parentheses indicate the position of the amino acids.

	DISCUSSION

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Our results indicate that, in LLC-PK₁ cells, 1) ATP activates a suramin-sensitive, nondesensitizing, cation-selective current; 2) extracellular ATP induces an influx of extracellular Ca²⁺, which is blocked by suramin but not by pretreatment with pertussis toxin; and 3) a product having 80% amino acid homology with the rat vas deferens P2X₁ receptor is expressed. Taken together, these results provide strong support for the view that LLC-PK₁ cells possess a subtype of ionotropic P2X receptors. P2X receptors have been characterized in a number of tissues (2, 23), and seven members of the family have been cloned (35). The various P2X receptors differ from one another in their tissue distribution and also in their pharmacological profiles (5, 8, 33). With respect to our results, the rat P2X₁ and P2X₃ receptors are sensitive to ,-methylene-ATP, whereas P2X₂, P2X₄, P2X₅, and P2X₆ are not. The P2X₄ and P2X₆ receptors are insensitive to suramin, whereas the others are sensitive. Finally, the P2X₁ and P2X₃ receptors desensitize rapidly during prolonged exposure to ATP. Thus the pharmacological properties we observed in the LLC-PK₁ receptor, i.e., insensitivity to ,-methylene ATP, sensitivity to suramin, and no desensitization, are most typical of a P2X₂ receptor. However, the sequence of the LLC-PK₁ P2X receptor fragment most closely resembled the rat P2X₁ receptor. Thus the LLC-PK₁ receptor appears to have pharmacological properties distinct from those of the corresponding rat P2X receptors. It is unclear, at present, whether such functional variations might reflect species differences among the gene products, the expression pattern of different subunits, or cell type-specific microenviromental modulations of the same subunit assembly (44). The functional classification of P2X receptors is made even more complex by the likelihood that P2X receptor subtypes may combine to form heteromultimeric receptors, whose functional properties are distinct from the homomultimeric receptors (16). Thus it is possible that, in LLC-PK₁ cells, the P2X receptor is composed of several different subunits, only one of which is represented by the product obtained by RT-PCR. Cloning and functional analysis of the full-length LLC-PK₁ receptor will be required to address this point.

Several previous studies have addressed the effects of nucleotides on intracellular calcium in renal epithelial cells (6, 12, 26, 30). In LLC-PK₁ cells, Weinberg et al. (41) and Harada et al. (21) concluded that ATP increased intracellular calcium via a pertussis toxin-insensitive receptor, likely P2U (now referred to as P2Y₂) or P2Y, coupled to phospholipase C. In those studies, ATP elicited a large, sustained increase in intracellular calcium, which depended on both release from intracellular stores and influx from extracellular solutions. In agreement, we also found that in the absence of extracellular calcium ATP did produce a small increase in intracellular calcium (Fig. 5) and that UTP produced an increase in [Ca²⁺]_i, consistent with the presence of a P2Y₂ receptor. Expression of P2Y receptors has also been noted in renal Madin-Darby canine kidney cells (18). However, the failure of UTP to increase the whole cell conductance (Fig. 3A) indicates that P2Y receptors do not mediate the ATP-dependent changes in cell conductance observed in the present study (Figs. 1-4). In freshly isolated rabbit proximal tubules, the ATP-induced increase in intracellular calcium was attributed to activation of a P2Y receptor, leading to both calcium mobilization and calcium influx (43), although a role for a P2X receptor could not be excluded. The results of the present study are consistent with the expression of both P2X receptors and P2Y receptors, with the latter receptors causing calcium mobilization while the P2X receptors lead to calcium influx (Figs. 5-7) and membrane depolarization (Figs. 1-4).

Agents that mobilize intracellular calcium often lead to an increase in calcium influx. This response, termed capacitative calcium influx, refers to the increase in plasma membrane calcium permeability on depletion of the intracellular calcium stores. Two observations support the view that the increase in the whole cell currents observed in this study does not represent capacitative calcium entry. First, in contrast to capacitative calcium entry, which is highly calcium selective (45), the conductance activated by ATP in this study was cation nonselective. Second, depletion of intracellular calcium stores by thapsigargin did not, by itself, induce a large increase in the whole cell conductance (Fig. 4), whereas subsequent exposure to ATP did increase the conductance. This observation is consistent with the extremely small unitary conductance of the calcium release-activated calcium channel (7).

The physiological role of purinergic receptors in proximal tubule epithelial cells is unclear, as is the source of the extracellular nucleotides. Whether nucleotides may be released through P-glycoprotein or cystic fibrosis transmembrane conductance regulator channels, both of which are present in the proximal tubule (9, 37), remains controversial (31). Regardless of the mechanism, ATP is released into the extracellular space during cell injury (19, 29). Accordingly, P2X receptors, which act as Ca²⁺-permeable ion channels when gated by ATP, could contribute to increases in [Ca²⁺]_i and activation of cytotoxic mechanisms associated with various types of cell injury, including apoptosis (38). However, earlier studies suggest that extracellular nucleotides or their metabolites protect renal cells from cell injury (40) rather than exacerbate injury. It has also been suggested that P₂ receptors may be involved in cell proliferation (22, 24), control of renal circulation (27), and possibly in cell volume regulation (11). Clearly, further studies are required to establish the physiological and pathophysiological roles of purinergic receptors and, more specifically, P2X receptors in renal epithelial cells.