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ATP stimulates Cl^- secretion and reduces amiloride-sensitive Na⁺ absorption in M-1 mouse cortical collecting duct cells

John E. Cuffe*, Andreas Bielfeld-Ackermann*, Jörg Thomas¹, Jens Leipziger¹ and Christoph Korbmacher*

MS 0111 Received 15 September 1999; accepted after revision 22 December 1999.

	ABSTRACT

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1. Using equivalent short circuit current (I_SC) measurements we examined the effect of extracellular ATP on transepithelial ion transport in M-1 mouse cortical collecting duct cells. Apical addition of ATP produced a rapid transient peak increase in I_SC. This was followed by a fall below basal I_SC due to a reduction in the amiloride-sensitive I_SC component.

2. The ATP-induced I_SC increase was preserved in the presence of apical amiloride while it was reduced in the absence of extracellular Cl^- and in the presence of the apical Cl^- channel blockers diphenylamine-2-carboxylic acid (DPC, 1 mM), DIDS (300 µM) and niflumic acid (100 µM).

3. The stimulatory effect of apical ATP on I_SC was concentration dependent with an EC₅₀ of about 0·6 µM. Basolateral ATP elicited a similar I_SC response. Experiments using the ATP scavenger hexokinase demonstrated that the ATP effects were elicited via separate apical and basolateral receptors.

4. ATP and UTP applied to either the apical or the basolateral bath equi-potently stimulated I_SC while 'purified' ADP and UDP had no effect consistent with P2Y₂ purinoceptors, the expression of which was confirmed using RT-PCR.

5. Intracellular calcium concentration ([Ca²⁺]_i) measurements using fura-2 demonstrated that ATP and UTP elicited a rise in [Ca²⁺]_i with EC₅₀ values of 1·1 and 0·6 µM, respectively. The shape and time course of the calcium response were similar to those of the I_SC response. The peak I_SC response was preserved in the nominal absence of extracellular calcium but was significantly reduced in cells pre-incubated with the calcium chelator BAPTA AM.

6. We conclude that in M-1 cells extracellular ATP reduces amiloride-sensitive Na⁺ absorption and stimulates Cl^- secretion via calcium-activated Cl^- channels through activation of P2Y₂ purinoreceptors located in the apical and basolateral membrane.

	INTRODUCTION

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Over the past decade, extracellular ATP has been implicated in the regulation of epithelial cell function through changes in intracellular second messenger activity and electrogenic ion transport. ATP may be released from nerve endings, from immune cells, and also from epithelial cells under physiological and pathophysiological conditions (Schwiebert, 1999). The released ATP may act in an autocrine or paracrine fashion on purinoceptors associated with these or neighbouring cells. There are two main families of purinoceptors, adenosine or P1 receptors, and P2 receptors, recognizing ATP and related nucleotides. P2 receptors divide into two families of ionotropic P2X receptors and metabotropic G protein-coupled P2Y receptors which display distinctive nucleotide selectivity sequences (Ralevic & Burnstock, 1998).

The kidney is a rich source of purinoceptors; however, the physiological role of the purinoceptors is still largely unknown. P2Y₁ and P2X receptors are found mainly on the smooth muscle of intrarenal arteries and may contribute to the regulation of renal haemodynamics. On the other hand, P2Y₂ purinoceptors have been identified along the length of the nephron, including the cortical collecting duct (CCD). Thus it has been speculated that extracellular ATP may be a regulator of renal tubular transport (Chan et al. 1998).

Various renal cell lines have been employed to elucidate the physiological role of extracellular ATP in the kidney. In A6 cells, an amphibian distal renal cell line, ATP stimulates an increase in intracellular calcium concentration ([Ca²⁺]_i) and activates Cl^-, K⁺ and non-selective cation channels (Middleton et al. 1993; Nilius et al. 1995; Mori et al. 1996; Atia et al. 1999). In Madin-Darby canine kidney (MDCK) cells, which display characteristics of distal nephron epithelial cells, ATP stimulates K⁺ channels (Jungwirth et al. 1989), basolateral capacitive Ca²⁺ entry (Gordjani et al. 1997) and electrogenic chloride secretion (Simmons, 1981; Woo et al. 1998). However, functional data from differentiated mammalian tissues of characterised renal origin are sparse.

In the present study we used M-1 mouse CCD cells, which express properties typical of CCD principal cells in vivo including the amiloride-sensitive epithelial sodium channel ENaC (Stoos et al. 1991; Korbmacher et al. 1993; Letz et al. 1995). Preliminary experiments have shown that ATP stimulates an increase in intracellular Ca²⁺ concentration in M-1 cells (Higgins & Harvey, 1996). In this paper, we examined the effects of extracellular ATP on electrogenic transepithelial ion transport of M-1 cells using Ussing-type chambers. In addition, intracellular calcium measurements and PCR experiments were performed to further characterise the purinoceptor subtype mediating the effect of ATP on transepithelial ion transport in M-1 cells.

Part of this work has been published in abstract form (Cuffe et al. 1999).

	METHODS

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Cell culture

The M-1 cell line (ATCC 2038-CRL, American Type Culture Collection, Rockville, MD, USA) was originally obtained from Dr G. Fejes-Tóth (Stoos et al. 1991). Cells were used from passage 25 to 46 and handled as previously described (Korbmacher et al. 1993; Letz et al. 1995; Bertog et al. 1999). For transepithelial studies cells were seeded onto 12 mm diameter Millicell-HA culture plate inserts (Millipore Corporation, Bedford, MA, USA) and used 9-11 days after seeding.

Transepithelial measurements

The Millicell inserts with confluent M-1 cells were transferred to purpose-built Ussing chambers for continuous equivalent short-circuit current (I_SC) measurements, as previously described (Bertog et al. 1999). Transepithelial resistance (R_te) was evaluated every second by measuring the voltage deflections induced by 100 ms symmetrical square current pulses of ±10 µA cm^-2. Open circuit transepithelial potential difference (V_te) was also measured and I_SC was calculated according to Ohm's law. Conventionally a lumen negative V_te corresponds to a positive I_SC which may be due to electrogenic cation absorption or electrogenic anion secretion or a combination of both. A standard bath solution (containing (mM): 140 Na⁺, 4 K⁺, 1 Ca²⁺, 1 Mg²⁺, 124 Cl^-, 24 HCO₃^-, 5 glucose) was used on both the apical and basolateral side of the epithelial monolayer. Bath solution was maintained at 37°C and gassed with 95 % O₂ and 5 % CO₂ maintaining pH at 7·4. Chloride-free solutions were achieved by replacing Cl^- with gluconate and contained 6 mM Ca²⁺ gluconate to compensate for the Ca²⁺ buffering properties of gluconate. In order to investigate the role played by intracellular Ca²⁺ in the ATP response, cells were loaded with the Ca²⁺ chelator BAPTA AM (50 µM) for 60 min.

Intracellular calcium measurements

The 'fluorescence Ussing chamber' was designed to measure [Ca²⁺]_i using fura-2 while separately superfusing apical and basolateral sides of an epithelial monolayer of M-1 cells grown on filters. A filter with M-1 cells was positioned in this chamber with the apical side of the cells facing down thus allowing an improved optical access in the inverted microscope. M-1 cells grown on Transwell Col (12 mm diameter, 0·4 µm pore size, Costar, Bodenheim, Germany) filters were used on days 4-6. A long-distance objective lens (LD-Achroplan ×40/0·6, Zeiss, Germany) was used to visualise the cells. The distance from the glass base to the cell layer was 2 mm. The cells were loaded with fura-2 AM (10 µmol l^-1, 60 min, room temperature) in Ringer solution to which 1·6 µmol l^-1 Pluronic F127 had been added. Pluronic F127 is a surfactant polyol helping to solubilise water-insoluble dyes like fura-2 AM. As a measure of [Ca²⁺]_i the fluorescence emission ratio at 340 nm/380 nm excitation was calculated. In each experiment the fluorescence signal was recorded from approximately 10 cells (30 µm²). Autofluorescence was measured in separate experiments in M-1 cells grown on Transwell Col filters without fura-2 dye loading. The mean of the measured autofluorescence signal (n = 6) was subtracted from all fluorescence values measured in fura-2 loaded cells. The fluorescence Ussing chamber had a basolateral chamber volume of 200 µl and an apical volume of 500 µl. Both sides were continuously perfused with a flow rate of approximately 2 ml min^-1 with a bath solution containing (mM): 145 NaCl, 0·4 KH₂PO₄, 1·6 K₂HPO₄, 5 glucose, 1 MgCl₂, 1·3 calcium gluconate, 5 probenecid. Probenecid was used to minimise extrusion of fura-2 from dye loaded cells (Di Virgilio et al. 1990) and had no noticeable effect on the [Ca²⁺]_i response elicited by ATP. All experiments were performed at 37°C. The [Ca²⁺]_i data are shown qualitatively as a change in fluorescence ratio.

RT-PCR

Confluent M-1 cells were harvested with a cell scraper. Total RNA was isolated by acid guanidinium thiocyanate-phenol-chloroform extraction (Chomczynski & Sacchi, 1987) and subsequently poly(A)⁺ RNA was obtained using oligo(dT) cellulose columns (Life Technologies, Paisley, UK). M-1 poly(A)⁺ RNA was then reverse transcribed using Superscript reverse transcriptase (Life Technologies) and random hexamer primers (Pharmacia, Freiburg, Germany). P2Y₂ specific primers were chosen according to Clarke et al. (1999) who designed their primers to be receptor subtype specific but to recognise regions of high homology among species (based on the rat, human and mouse sequences). We made a two nucleotide exchange at the 3' end of the sense primer so that the primers used (sense 5' primer: 5'-CTTCAACGAGGACTTCAAGTACGTGC-3'; antisense 3' primer: 5'-CATGTTGATGGCGTTGAGGGTGTGG-3') corresponded exactly to the published mouse sequence (GenBank accession no. L 14751; Lustig et al. 1993). The resulting PCR fragment was 781 bp long (bp 347-1127 of the mouse sequence). The PCR protocol was 95°C for 3 min 45 s, then 30 cycles of 95°C for 1 min 15 s, 60°C for 1 min, and 72°C for 2 min, followed by 72°C for 8 min. Negative controls were performed without reverse transcriptase and without RNA. The obtained PCR product was sequenced using the same primers (DNA Sequencing Facility, Department of Biochemistry, Oxford University, UK).

Drugs used

Amiloride hydrochloride, adenosine 5'-triphosphate (ATP) disodium salt, adenosine 5'-diphosphate (ADP) sodium salt, adenosine 5'-monophosphate (AMP) sodium salt, uridine 5'-triphosphate (UTP) sodium salt, uridine 5'-diphosphate (UDP) sodium salt, 2-methylthioATP (2-MeSATP) tetrasodium salt, bumetanide, 4,4'-diisothiocyanato-stilbene-2,2'-disulphonic acid disodium salt (DIDS), niflumic acid and hexokinase were purchased from Sigma-Aldrich (Steinheim, Germany), diphenylamine-2-carboxylic acid (DPC) was obtained from Fluka (Neu-Ulm, Germany). 1,2-Bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra (acetoxymethyl)ester (BAPTA AM) was supplied by Calbiochem-Novabiochem (UK) Ltd (Nottingham, UK). Amiloride was either directly dissolved in standard bicarbonate solution or added from a 100 mM methanol stock solution to give a final concentration of 100 µM. DPC (100 mM) and BAPTA AM (10 mM) were dissolved in DMSO and stock solutions were freshly prepared on the day of the experiment. All chemicals used were of the highest grade commercially available. Commercial preparations of nucleotide diphosphates ('non-purified' ADP or UDP) are known to be contaminated by nucleotide triphosphates (Nicholas et al. 1996). To circumvent this problem, we performed control experiments using 'purified' stock solutions of UDP or ADP (1 mM) which had been pretreated for 1 h at room temperature with hexokinase (10 units ml^-1) in the presence of 25 mM glucose to eliminate nucleotide triphosphates.

Data analysis

Data are expressed as means ± standard error of the mean and significance of difference was estimated using the appropriate version of Student's t test. Unless otherwise stated in the text, control responses to nucleotides were elicited at the beginning and end of an experiment, and the average of these values was compared with the test response using paired analysis. In experiments where internal controls were not possible, monolayers treated with a particular inhibitor were matched with an untreated control from the same batch of cells. Throughout n refers to the number of monolayers.

	RESULTS

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Baseline parameters

After an equilibration period of about 30 min after transfer into the Ussing-type chambers confluent monolayers of M-1 cells displayed mean transepithelial voltage (V_te), resistance (R_te), and equivalent short-circuit current (I_SC) values of -40·6 ± 1·3 mV (lumen negative), 708 ± 16 cm², and 64·1 ± 2·4 µA cm^-2 (n = 196), respectively. Apical application of 100 µM amiloride decreased V_te to -6·7 ± 0·3 mV, increased R_te to 1403 ± 34 cm² and reduced I_SC to 5·1 ± 0·3 µA cm^-2 (n = 196). This corresponds to a 92 % inhibition of I_SC and confirms that the predominant electrogenic ion transport across M-1 monolayers is sodium absorption via the amiloride-sensitive epithelial sodium channel (ENaC) known to be expressed in M-1 cells (Korbmacher et al. 1993; Letz et al. 1995). A continuous I_SC recording is shown in Fig. 1A which demonstrates the stability of the preparation with an average spontaneous I_SC rundown of only 12·5 ± 2 % within 1 h (n = 13).

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Figure 1. Effect of apical ATP on the ISC of M-1 mouse CCD cells A, continuous ISC recording in a control experiment demonstrating long term stability of ISC and the effect of apical (ap) amiloride (100 µM) and DPC (1 mM) on resting ISC. Drugs were applied as indicated by the horizontal bars. Positive ISC corresponds to electrogenic cation absorption or anion secretion or a combination of both. B, effect of apical application of ATP (100 µM) on ISC of M-1 cells from the same batch of cells as in A; amiloride (100 µM) and DPC (1 mM) were apically applied in the continuous presence of ATP.

Biphasic effect of apical ATP on I_SC

Figure 1B shows an experiment in which the effect of apical application of ATP on I_SC was tested. ATP (100 µM) induced an initial transient increase in I_SC with a sharp peak reaching its maximum within 30 s after addition. The peak amplitude averaged 67·0 ± 4·9 µA cm^-2 (n = 12). The shape of the ATP-induced initial I_SC increase was somewhat variable with either a single peak or an occasional second peak as shown in Fig. 1B. The initial transient I_SC increase was followed by a subsequent sustained decrease below the steady state current level. This secondary decrease suggested that the amiloride-sensitive baseline I_SC was affected by ATP.

ATP reduces the amiloride-sensitive I_SC component

Indeed, in monolayers exposed to ATP (Fig. 1B) apical addition of 100 µM amiloride reduced I_SC significantly less than in time-matched control experiments without ATP (Fig. 1A). In similar experiments to that shown shown in Fig. 1 the amiloride-sensitive I_SC component after exposure to apical ATP for 40 min was reduced to 26·2 ± 1·8 µA cm^-2 (n = 13) compared with 37·8 ± 2·4 µA cm^-2 in matched control experiments (n = 13, P < 0·01). In contrast, the initial steady state I_SC of the control monolayers was not significantly different from that of the monolayers subsequently exposed to ATP, averaging 48·0 ± 2·3 µA cm^-2 (n = 13) and 49·1 ± 2·1 µA cm^-2 (n = 13), respectively. Thus, exposure to ATP significantly reduced the amiloride-sensitive I_SC component of M-1 cells by about 16 %. Interestingly, ATP-induced inhibition of the amiloride-sensitive steady state I_SC was not readily reversible upon washout of ATP (Fig. 2) and 30 min after a 15 min exposure to ATP the inhibition averaged 15 % (n = 4, P < 0·01).

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Figure 2. The ISC response to ATP is preserved in the presence of amiloride M-1 cells were exposed to apical ATP (100 µM) in the absence and presence of apical amiloride (100 µM) as indicated by the horizontal bars. The noisy ISC trace after the first ATP application is due to washout of ATP.

ATP stimulates an additional DPC-sensitive I_SC component

Interestingly, the remaining amiloride-insensitive I_SC was significantly larger in monolayers exposed to ATP (Fig. 1B) than in control experiments (Fig. 1A) averaging 8·9 ± 0·7 µA cm^-2 (n = 13) and 3·3 ± 0·5 µA cm^-2 (n = 13, P < 0·01), respectively. A positive I_SC may be due to either electrogenic cation absorption or anion secretion or a combination of both. As shown in Fig. 1B, apical application of the known chloride channel blocker DPC (1 mM) to the ATP-treated monolayers inhibited the remaining amiloride-insensitive I_SC from 8·9 ± 0·7 to 2·8 ± 0·2 µA cm^-2 (n = 13) which corresponds to a DPC-sensitive I_SC component of 6·1 ± 0·6 µA cm^-2. In contrast, in the control experiments (Fig. 1A) the DPC-sensitive I_SC component was significantly smaller averaging 0·8 ± 0·2 µA cm^-2 (n = 13, P < 0·01). These findings suggest that in addition to its inhibitory effect on the amiloride-sensitive I_SC component ATP stimulates a DPC-sensitive I_SC component possibly due to electrogenic anion secretion via DPC-sensitive apical anion channels.

The stimulatory effect of ATP is preserved in the presence of amiloride

As shown in Fig. 2 the initial I_SC peak induced by apical ATP (averaging 57·2 ± 2·5 µA cm^-2, n = 40) was preserved in the presence of 100 µM amiloride in the apical bath. This lack of effect of amiloride rules out the possibility that the transient initial I_SC increase is mediated by stimulation of electrogenic Na⁺ absorption. Thus, the initial transient I_SC stimulation is likely to be due to an anion secretory I_SC component. In addition, the experiment shown in Fig. 2 demonstrates that after washout of ATP a second response to ATP may be elicited suggesting that little desensitisation of the underlying receptor and signal transduction pathway occurs (see below).

In the presence of amiloride ATP not only induced a transient I_SC peak but I_SC remained at a slightly elevated level above baseline (Fig. 2). This is in contrast to the secondary ATP-induced I_SC decline observed in the absence of amiloride (Fig. 1B and Fig. 2). However, the data are consistent with the interpretation that ATP has a dual effect of inhibiting the amiloride-sensitive I_SC component and stimulating an anion secretory I_SC component which becomes apparent in the presence of amiloride. Taken together our data indicate that the initial stimulatory ATP effect on I_SC is not mediated by a stimulation of amiloride-sensitive Na⁺ absorption and that both the initial transient I_SC increase as well as the prolonged plateau increase of I_SC are due to an anion secretory response which is at least partially inhibitable by the chloride channel blocker DPC (Fig. 1B).

Effect of basolateral ATP on I_SC, V_te and R_te

Basolateral application of ATP elicited an almost identical I_SC response to that observed with apical application of ATP (Fig. 3A). Following basolateral application of 100 µM ATP the peak I_SC increase averaged 31·0 ± 3·2 µA cm^-2 (n = 8). The peak response to basolateral ATP was preserved in the presence of apical amiloride with a peak I_SC increase of 34·8 ± 2·5 µA cm^-2 (n = 33). On average the peak response to basolateral ATP (100 µM) was 32 % smaller than the average peak response to apical ATP (P < 0·001; see above). In the absence of amiloride steady state I_SC before the transient I_SC peak induced by basolateral ATP application averaged 39·0 ± 1·4 µA cm^-2 while 45 min after ATP application I_SC was reduced to 31·5 ± 1·1 µA cm^-2 (n = 4, P < 0·01). In contrast, in the presence of amiloride there was a sustained I_SC stimulation of 2·7 ± 0·3 µA cm^-2 (n = 33, P < 0·01) by basolateral ATP 10 min after its application. These findings indicate that basolateral ATP also has a dual effect of inhibiting the amiloride-sensitive steady state I_SC component and stimulating an anion secretory I_SC component which is revealed in the presence of amiloride.

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Figure 3. Basolateral ATP has a similar effect to apical ATP Corresponding traces of ISC (A), Vte (B) and Rte (C) from an experiment during which ATP (100 µM) was added basolaterally (bl) in the absence and presence of apical amiloride (100 µM) are shown. The noisy ISC trace after the first ATP application is due to washout of ATP.

In addition to the I_SC trace Fig. 3 also shows the corresponding V_te (Fig. 3B) and R_te (Fig. 3C) traces. In the absence of amiloride, application of ATP caused an initial peak depolarisation of V_te from -28·5 ± 3·0 to -14·0 ± 2·2 mV (n = 8) while it caused a hyperpolarisation from -4·2 ± 0·5 to -17·6 ± 1·2 mV (n = 33) in the presence of amiloride. The corresponding R_te trace indicates that in both cases ATP caused a dramatic transient decrease in R_te from 535 ± 56 to 176 ± 26 cm² (n = 8) in the absence and from 1445 ± 137 to 360 ± 32 cm² (n = 33) in the presence of amiloride.

M-1 CCD cells form a relatively tight epithelium in which changes of the apical cell membrane resistance dominate R_te as evidenced by the substantial increase of R_te observed upon inhibition of the apical Na⁺ conductance by amiloride (see above). Therefore, the ATP-induced decrease in R_te is most probably due to an increase in apical cell membrane conductance. Our findings indicate that at least transiently the apical conductance activated by ATP becomes the dominating conductance component with a reversal potential more negative than that of the apical Na⁺ conductance but more positive than that of the basolateral K⁺ conductance. Assuming a constantly high basolateral K⁺ conductance and an intracellular chloride concentration above electrochemical equilibrium the observed V_te and R_te changes are compatible with an ATP-induced activation of apical Cl^- channels. Stimulation of an apical Cl^- conductance that becomes the dominating conductance of the apical membrane would explain why during the peak response to ATP a similar V_te is reached in the presence or absence of amiloride by either depolarising or hyperpolarising the apical membrane, respectively (Fig. 3B).

Cl^- dependence of the stimulatory ATP response

To test whether the ATP-induced increase in I_SC was due to Cl^- secretion, we examined the Cl^- dependence of the ATP response by replacing extracellular Cl^- ions (apically and basolaterally) with gluconate in experiments as shown in Fig. 4. An initial application of 100 µM ATP elicited the usual response while removal of extracellular Cl^- largely reduced the stimulatory effect of a second ATP application. Subsequent readdition of bath chloride restored the responsiveness to ATP. In similar experiments extracellular Cl^- removal reduced the peak response to apical ATP from 51·3 ± 3 to 17·1 ± 1·3 µA cm^-2 (n = 15, P < 0·01) and reduced the peak response to basolateral ATP from 25·7 ± 2·6 to 6·3 ± 1 µA cm^-2 (n = 16, P < 0·01). Moreover, the shape of the response was altered. In the absence of Cl^- the I_SC response to ATP was very short-lived without a prominent plateau phase (Fig. 4). Thus, the overall inhibitory effect of Cl^- removal on the I_SC response to ATP was probably even larger than the observed reduction of the peak response.

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Figure 4. Removal of extracellular Cl- inhibited the response to apical ATP M-1 cells were exposed to apical ATP (100 µM) in the presence and absence of extracellular Cl-. The experiment was conducted in the continuous presence of 100 µM amiloride.

Sensitivity of the stimulatory ATP response to apical Cl^- channel blockers

To confirm the involvement of apical Cl^- channels we investigated the effects of apically administered Cl^- channel inhibitors on the I_SC peak response to ATP. Representative experiments are shown in Fig. 5. In the presence of apical DPC (1 mM, Fig. 5A) the peak I_SC response elicited by apical ATP (100 µM) was significantly reduced by 61 % from 70·9 ± 4·7 to 27·6 ± 2·6 µA cm^-2 (n = 12, P < 0·01). This finding is consistent with the inhibitory effect of DPC during the plateau phase shown in Fig. 1B. Likewise, apical DIDS (300 µM, Fig. 5B) significantly reduced the I_SC peak response to apical ATP (100 µM) by 82 % from 52·1 ± 3·7 to 9·6 ± 1·7 µA cm^-2 (n = 9, P < 0·01). Note that the effect of DIDS is not fully reversible. To rule out the possibility that the inhibitory effect of apical DIDS on the ATP response was due to a non-specific inhibitory effect of DIDS on purinergic receptors (Ralevic & Burnstock, 1998), we also examined the effects of apical DIDS (300 µM) on the I_SC response to basolateral ATP. Apical DIDS also reduced the peak I_SC response to basolateral ATP (100 µM) which averaged 60·2 ± 2·5 and 23·7 ± 4·0 µA cm^-2 (n = 9) in the absence and presence of DIDS, respectively. This suggests that the inhibitory effect of DIDS is indeed mediated by inhibition of apical Cl^- channels and not through interference with purinergic receptors.

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Figure 5. Apical Cl- channel inhibitors significantly reduced the apical ISC response to ATP Online recording of the ISC responses to apical ATP (100 µM) before, during and after apical application of 1 mM DPC (A), 300 µM DIDS (B) and 100 µM niflumic acid (C). Apical amiloride (100 µM) was present throughout.

We also tested the effect of niflumic acid, a known inhibitor of calcium-activated Cl^- channels (Fig. 5C). In the presence of apical niflumic acid (100 µM) the ATP-stimulated peak increase in I_SC was reduced by 52 % from 71·2 ± 3·6 to 33·9 ± 2·1 µA cm^-2 (n = 6). Taken together these results indicate that the ATP-mediated increase in I_SC is due to electrogenic Cl^- secretion via apical Cl^- channels.

Effect of basolateral bumetanide and DIDS

For Cl^- secretion to occur a Cl^- uptake mechanism must be present to accumulate intracellular Cl^- above electrochemical equilibrium. The Na⁺-K⁺-2Cl^- cotransport inhibitor bumetanide and the Cl^--HCO₃^- exchange inhibitor DIDS were used to investigate a possible role of these transporters as basolateral Cl^- entry pathways in M-1 cells. Basolateral application of these agents has been shown to inhibit Cl^- secretion in a variety of epithelia. However, basolateral bumetanide (100 µM) had no significant effect on the stimulatory ATP response which averaged 61·1 ± 4·1 (n = 12) and 56·5 ± 7·5 µA cm^-2 (n = 9) in the absence and presence of bumetanide, respectively. Basolateral DIDS (300 µM) increased rather than decreased the response to apical ATP from 50·2 ± 1·5 µA cm^-2 (n = 9) in the absence to 179·3 ± 11·6 µA cm^-2 (n = 9) in the presence of DIDS. Thus, the basolateral Cl^- entry pathway remains unclear at present.

ATP effect is concentration dependent

We investigated the concentration dependence of the ATP effect (Fig. 6). The threshold concentration was 100 nM with a maximal I_SC peak occurring at 100 µM. Therefore, 100 µM ATP was the standard concentration used throughout this study. The response to 100 nM ATP was qualitatively different from that to higher concentrations, as it was characterised by a very short-lived peak increase in I_SC without the prominent plateau stimulation observed with higher concentrations. Interestingly, the plateau was highest with 10 µM ATP while peak and plateau elicited by 1 mM ATP were slightly smaller than the response elicited with 100 µM ATP (Fig. 6). This may indicate that some degree of desensitisation occurs following ATP application at high concentrations which may affect the peak and plateau phase differently. Using a Michaelis-Menten fit of the data we estimated an EC₅₀ for ATP of 0·6 µM (Fig. 6B, n = 12).

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Figure 6. The peak ISC increase induced by apical ATP was concentration dependent A, ATP was applied for periods of 5 min in increasing concentrations, interspersed with 10 min washouts, over the range 10 nM to 1 mM. Experiments were conducted with 100 µM amiloride present in the apical bath. B, summary of ISC data of 12 experiments similar to that shown in A. For the duration of each ATP application the area under the ISC curve above baseline (ISC in µA cm-2 s) was estimated and means ± S.E.M. are given for each ATP concentration. A Michaelis-Menten fit of the data points representing the mean values reveals an EC50 of 0·6 µM. The data point for 1 mM ATP was not included in the fit since the ATP response was reduced at this high concentration possibly due to receptor desensitisation.

Independence of apical and basolateral purinergic receptors

The responses to apical and basolateral ATP appeared to operate independently of each other as apical ATP could elicit an I_SC increase in the presence of ATP in the basolateral bath with an average peak response of 47·9 ± 3·2 µA cm^-2(n = 8). Similarly, basolateral ATP applied in the presence of apical ATP elicited an average peak response of 26·5 ± 2·7 µA cm^-2. In contrast a second application of ATP in the continuous presence of apical ATP was essentially without effect (1·3 ± 0·6 µA cm^-2, n = 9). To rule out the possibility that the response to apical or basolateral ATP was due to diffusion of ATP to the opposite bath the ATP-scavenging enzyme hexokinase was used which catalyses the conversion of 1 mole of ATP to ADP per mole of glucose. As shown in Fig. 7 the peak response to apical ATP was almost completely abolished (4·6 ± 1·9 µA cm^-2, n = 11) in the presence of 126 units ml^-1 hexokinase on the apical side. In contrast, ATP elicited a normal response before addition of hexokinase and after its washout (Fig. 7A). This small and short lived peak response observed in the presence of hexokinase in five of 11 experiments (e.g. Fig. 7A) is similar to the response observed with very low concentrations of ATP (see Fig. 6). This demonstrated that hexokinase was efficient in scavenging most of the apical ATP. Despite the presence of apical hexokinase basolateral application of ATP still elicited the usual I_SC increase which averaged 37·4 ± 6·8 µA cm^-2 (n = 8) (Fig. 7A). Likewise, in the presence of hexokinase on the basolateral side, apical ATP stimulated an increase in I_SC of 47·8 ± 10 µA cm^-2 (n = 5) whereas the response to basolateral ATP was completely abolished (n = 11) (Fig. 7B). These findings suggest that purinergic receptors are present in both the apical and basolateral membrane and are capable of acting independently of each other.

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Figure 7. Use of the ATP scavenger hexokinase demonstrates the presence of separate apical and basolateral ATP receptors In these experiments hexokinase (126 units ml-1) was added to the apical (A) or basolateral (B) bath solution prior to sequential application of apical and basolateral ATP (100 µM). Apical amiloride (100 µM) was present throughout the experiment.

Characterisation of the underlying purinoceptor subtype

We compared the effects of different nucleotides on I_SC in the presence of apical amiloride to define the receptor subtype mediating the ATP response. In this series of experiments 100 µM ATP and 100 µM UTP had a similar effect with an average peak response of 72·7 ± 2·8 (n = 9) and 71·9 ± 3·4 µA cm^-2 (n = 9), respectively. Application of 'non-purified' UDP or ADP produced substantial I_SC peaks of 47·8 ± 4·3 (n = 9) or 46·2 ± 3·2 µA cm^-2 (n = 9), respectively. In contrast, 'purified' UDP or ADP (see Methods) did not elicit a significant response (-0·5 ± 0·3 or 0·8 ± 0·4 µA cm^-2, respectively) while subsequent application of the 'non-purified' UDP or ADP elicited a peak I_SC increase of 84·2 ± 4·7 (n = 6) or 76·7 ± 3·3 µA cm^-2 (n = 6), respectively. In the same experiments UTP and ATP elicited peak responses of 104·8 ± 3·0 (n = 6) and 99·9 ± 2·9 µA cm^-2 (n = 6), respectively. These findings suggest that the responses observed with the 'non-purified' nucleotide diphosphates are due to contamination with nucleotide triphosphates and not to receptor activation by ADP or UDP (Nicholas et al. 1996). Application of AMP (n = 9) and 2-MeSATP (n = 9), an agent specific for P2Y₁ receptors, had no effect. In the presence of UTP the ATP-induced increase in I_SC was almost completely inhibited (0·3 ± 0·3 µA cm^-2; n = 3). Likewise, the UTP response was almost completely abolished (1·6 ± 0·5 µA cm^-2; n = 6) following the application of ATP. These findings indicate a common pathway of the ATP and UTP response. When added to the basolateral membrane ATP and UTP had similar effects with an average peak I_SC increase of 31·1 ± 3·0 (n = 8) and 23·2 ± 6·7 µA cm^-2(n = 3), respectively.

Taken together, these data indicate that the ATP-induced increase in I_SC is mediated by the same receptor subtype located on both membranes, possibly P2Y₂. Indeed, RT-PCR experiments confirmed that M-1 cells express P2Y₂ transcripts. Figure 8 shows an agarose gel electrophoresis analysis of a product obtained from a RT-PCR reaction using P2Y₂ specific primers. A band of the expected size is seen in the RT-PCR reaction using M-1 mRNA while the two controls (no RT and no RNA) are negative. Sequencing of the PCR product confirmed that the sequence of the PCR product was identical to the corresponding part of the sequence of the P2Y₂ receptor. This finding is consistent with the conclusion that the ATP effect is mediated by a P2Y₂ receptor.

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Figure 8. P2Y2 mRNA is present in M-1 mouse CCD cells Agarose gel electrophoresis of products from RT-PCR reactions performed with primers based on mouse P2Y2 (predicted product size 781 bp) using RNA from M-1 cells (lane 2). Products from control reactions without RT or without RNA are shown in lane 3 and 4, respectively. A size marker is shown in lane 1.

Effect of ATP on [Ca²⁺]_i

P2Y₂ receptors are known to mediate an increase in [Ca²⁺]_i and we therefore performed [Ca²⁺]_i measurements in confluent M-1 monolayers grown on filters using fura-2. Figure 9 (upper panel) shows a representative experiment in which [Ca²⁺]_i was continuously monitored while the apical side of the M-1 monolayer was superfused with solutions containing different concentrations of ATP as indicated. Application of ATP elicited a concentration-dependent transient peak increase of [Ca²⁺]_i. Application of UTP had a similar effect and the ATP and UTP data are summarised in Fig. 9 (lower panel). A Michaelis-Menten fit of the data reveals EC₅₀ values of 0·6 and 1·1 µM for UTP and ATP, respectively. Similarly, basolateral ATP and UTP increased [Ca²⁺]_i in a concentration-dependent manner (data not shown).

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Figure 9. Extracellular ATP stimulates a concentration-dependent increase in [Ca2+]i in polarised M-1 cells A, continuous recording of the fluorescence ratio 340nm/380 nm in fura-2 loaded confluent M-1 monolayers which were stimulated by increasing concentrations of apical ATP (100 nM to 100 µM). B, average peak responses to different concentrations of apical UTP (; n = 6) and ATP (; n = 3-6). A Michaelis-Menten fit of the data points representing the mean values reveals EC50 values of 0·6 and 1·1 µM for UTP and ATP, respectively.

A rise in [Ca²⁺]_i mediates the I_SC peak response to ATP

The similar time course of the ATP-induced [Ca²⁺]_i peak response and the I_SC peak response suggested that the intracellular calcium signal mediates the electrophysiological response. An increase in [Ca²⁺]_i may be due to extracellular calcium entry or to calcium release from intracellular stores.

We first examined the importance of the extracellular calcium concentration on the I_SC response to ATP. The apical bathing solution containing 1 mM Ca²⁺ was changed to a nominally Ca²⁺-free solution containing 0·1 mM EGTA. In the absence of apical Ca²⁺ the average increase in I_SC was 87·1 ± 4·8 µA cm^-2 (n = 6). These data suggest that apical Ca²⁺ entry does not mediate the response. However, this does not rule out a basolateral Ca²⁺ entry path or a combination of both. Therefore we incubated filters initially in solutions containing 10 µM Ca²⁺ and subsequently in nominally Ca²⁺-free solution without EGTA on both sides. The I_SC peak responses elicited by apical ATP were preserved in the presence of 10 µM bath calcium or in the nominal absence of bath calcium (apically and basolaterally) and averaged 76·5 ± 6·6 and 69·5 ± 13·8 µA cm^-2 (n = 3), respectively. Thus, the ATP peak response is not dependent on extracellular calcium but most probably due to calcium release from intracellular stores. However, the plateau phase of I_SC was reduced in nominally calcium-free solution (n = 3, data not shown). Thus, calcium entry may contribute to the sustained effect of ATP. When both sides of the M-1 monolayer were bathed in nominally Ca²⁺-free solutions containing 0·1 mM EGTA to further lower the extracellular calcium concentration, V_te and R_te rapidly decreased towards zero presumably due to breakdown of tight junctions. This made it impossible to obtain reliable I_SC recordings under these conditions.

To further investigate the role of [Ca²⁺]_i changes in the I_SC response we pretreated filters for 60 min with the Ca²⁺ chelator BAPTA AM (50 µM). When compared with matched controls (Fig. 10A) with an average peak increase of 66·9 ± 3·5 (n = 10), pretreatment with BAPTA AM (Fig. 10B) significantly reduced the apical ATP response to 27·6 ± 3·3 µA cm^-2 (n = 10). BAPTA AM also blunted the response to apical ionomycin (1 µM), a Ca²⁺ ionophore, which confirms the increased calcium buffer capacity of the BAPTA pretreated cells (n = 3, data not shown). Taken together, these data imply that changes in [Ca²⁺]_i which most likely involve calcium release from intracellular calcium stores mediate the ATP-induced Cl^- secretory response in confluent M-1 cells.

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Figure 10. Pretreatment with extracellular BAPTA AM blunted the response to apical ATP A, control response to apical ATP (100 µM). B, response to apical ATP (100 µM) in cells pretreated with 50 µM BAPTA AM for 60 min. Amiloride (100 µM) was present throughout the experiment and 1 mM DPC was added apically as indicated.

	DISCUSSION

Top Abstract Introduction Methods Results Discussion References

The present study demonstrates that extracellular ATP stimulates electrogenic transepithelial Cl^- secretion in differentiated M-1 mouse cortical collecting duct cells. In addition to its stimulatory effect on electrogenic Cl^- secretion extracellular ATP partially inhibits the amiloride-sensitive I_SC in M-1 cells.

The response to extracellular ATP is mediated through activation of independent apical or basolateral receptors. ATP and UTP were found to be equi-potent while ADP (pure) had no effect consistent with P2Y₂ receptors. The presence of P2Y₂ receptors is further supported by RT-PCR data showing that M-1 cells express P2Y₂ mRNA. It has recently been shown (Bogdanov et al. 1998) that unlike the human P2Y₄ receptor (Nicholas et al. 1996) the rat P2Y₄ homologue is equi-sensitive to ATP and UTP which makes it difficult to distinguish functionally between these two receptor types. Since the nucleotide selectivity of the murine P2Y₄ receptor is not known the possibility of its involvement in the response of M-1 cells to ATP and UTP cannot be ruled out.

The intracellular calcium measurements reported in the present study confirm that polarised M-1 cells performing amiloride-sensitive electrogenic Na⁺ transport respond to extracellular ATP with an increase in [Ca²⁺]_i. This is consistent with previous findings that stimulation of P2Y₂ receptors mediates an increase in [Ca²⁺]_i in microdissected rat (Ecelbarger et al. 1994; Cha et al. 1998; Bailey et al. 1999) and rabbit (Rouse et al. 1994) collecting ducts. These findings suggest that extracellular ATP is likely to affect mammalian collecting duct function in vivo (Chan et al. 1998). The similar time course of the [Ca²⁺]_i and I_SC peak response to ATP and the inhibitory effect of BAPTA AM suggest that the intracellular calcium signal is important in mediating the Cl^- secretory response. An ATP-induced rise in [Ca²⁺]_i with a concomitant chloride secretory response similar to that observed in M-1 cells has previously been reported in various epithelial tissues including, for example, airway epithelium (Mason et al. 1991), pancreatic duct epithelium (Chan et al. 1996), and rat distal colon (Leipziger et al. 1997).

The ATP-induced peak I_SC response in M-1 cells was largely independent of extracellular calcium consistent with calcium release from intracellular stores. Indeed, the G protein-coupled P2Y₂ receptors are known to stimulate phospholipase C, IP₃ formation, and Ca²⁺ mobilisation from intracellular stores (Ralevic & Burnstock, 1998). In addition, the suppression of the plateau phase of the Cl^- secretory response in the nominal absence of extracellular calcium suggests that ATP also activates a calcium entry mechanism in M-1 cells. In MDCK cells it has been demonstrated that capacitive Ca²⁺ entry induced by luminal and basolateral ATP is restricted to the basolateral membrane (Gordjani et al. 1997) which is consistent with the lack of effect of apical calcium removal observed in M-1 cells (data not shown).

Different classes of Cl^- channels have been identified in M-1 cells, including cystic fibrosis transmembrane conductance regulator (CFTR) (Todd-Turla et al. 1996; Letz & Korbmacher, 1997), members of the ClC gene family of voltage-gated Cl^- channels (Korbmacher et al. 1996), ATP-dependent swelling-activated Cl^- channels, and calcium-activated Cl^- channels (Meyer & Korbmacher, 1996). These latter channels are likely candidates to mediate the Cl^- secretory response induced by ATP. Indeed, the inhibitory effect of niflumic acid, DPC and DIDS on the ATP-induced response is consistent with an involvement of apical calcium-activated Cl^- channels. A recent study in A6 cells concludes from current fluctuation analysis that the apical chloride channel activated by ATP is identical to the cAMP-activated Cl^- channel and possibly corresponds to CFTR (Atia et al. 1999). In contrast to observations in some other cell systems in which ATP via P2Y₂ receptors reduces intracellular cAMP levels (Ralevic & Burnstock, 1998), extracellular ATP has been shown to stimulate intracellular cAMP production in MDCK renal epithelial cells (Post et al. 1996). Moreover, the cloning of a novel human P2Y receptor coupled to phospholipase C and adenylyl cyclase has recently been described (Communi et al. 1997). Thus, it is conceivable that the cAMP-activated CFTR-like chloride channels previously described in M-1 cells (Letz & Korbmacher, 1997) may contribute to the ATP-induced chloride secretory response. However, a major contribution of CFTR Cl^- channels seems unlikely because of the substantial inhibition of the ATP response by DIDS (Anderson & Welsh, 1991).

While amiloride-sensitive Na⁺ absorption and K⁺ secretion are the predominant electrogenic transport features of CCD principal cells, active Cl^- secretion has been demonstrated in microdissected, microperfused rabbit CCD after the animals were fed a K⁺-rich diet (Wingo et al. 1990). There is considerable evidence that various types of apical Cl^- channels are present in CCD cells which, under favourable transepithelial electrochemical gradients, may mediate Cl^- secretion (Christine et al. 1991; Superdock et al. 1993; Ling et al. 1994; Letz & Korbmacher, 1997). Moreover, it has been found that the intracellular Cl^- activity for rabbit CCD principal cells is significantly elevated above electrochemical equilibrium (Sauer et al. 1989; Simmons, 1993), favouring Cl^- secretion. This is consistent with our findings in M-1 cells which demonstrate that Cl^- must be distributed above its electrochemical equilibrium since Cl^- secretion occurs. However, the possible involvement of apical Cl^- channels in Cl^- secretion and the physiological relevance of the Cl^- secretory capacity of the CCD in vivo are still a matter of debate (Schlatter et al. 1990; Simmons, 1993).

Our experiments demonstrate that a full response to apical ATP can be elicited in the presence of basolateral ATP and vice versa. Experiments using the ATP-scavenger hexokinase confirmed that the response to ATP was not due to diffusion of molecules across the epithelial layer to the contralateral membrane. This dual sidedness of the ATP response has also been observed in cultured rabbit cortical collecting duct cells (Koster et al. 1996) and in the MDCK cell line where ATP stimulated an equipotent increase in [Ca²⁺]_i and I_SC from both the apical and basolateral side (Gordjani et al. 1997; Woo et al. 1998).

The micromolar EC₅₀ values estimated for ATP and UTP in the present study are consistent with values previously reported for P2Y₂ receptors in other preparations (Mason et al. 1991; Middleton et al. 1993; Chan et al. 1996; Gordjani et al. 1997) and indicate that CCD cells are highly responsive to extracellular ATP. It is conceivable that under physiological and/or pathophysiological conditions luminal and basolateral ATP concentrations may reach levels high enough to activate apical and basolateral renal tubular P2Y₂ receptors (Wilson et al. 1999). There are many potential sources of extracellular ATP including controlled release from epithelia themselves. However, the mechanisms involved in epithelial ATP release are not yet clear (Schwiebert, 1999).

The finding that extracellular ATP inhibits amiloride-sensitive Na⁺ absorption in M-1 cells is reminiscent of the reported inhibitory effect of ATP on Na⁺ and Ca²⁺ absorption in cultured rabbit connecting tubule and cortical collecting duct cells which is thought to be mediated by PKC activation (Koster et al. 1996). An inhibitory effect of ATP on Na⁺ transport has been observed in a variety of tissues including airway epithelia (Mason et al. 1991; Iwase et al. 1997; Devor & Pilewski, 1999; Ramminger et al. 1999; Inglis et al. 1999), and thyroid cells (Bourke et al. 1999). Thus, the reciprocal regulation of Cl^- secretion and Na⁺ absorption by ATP may be a common theme in epithelia. However, the molecular mechanism by which ATP inhibits amiloride-sensitive Na⁺ absorption remains poorly understood. The inhibitory effect of ATP on transepithelial Na⁺ transport may be due to a reduction of basolateral Na⁺-K⁺-ATPase activity, or a decrease of basolateral K⁺ conductance, or an inhibition of apical Na⁺ entry via the epithelial sodium channel (ENaC), or a combination of these factors.

In this context it is interesting that a reciprocal regulation of cAMP-activated Cl^- channels and ENaC has been found in several different preparations including M-1 cells (Letz & Korbmacher, 1997). Indeed, it has been suggested that a regulatory relationship exists between ENaC and CFTR (Stutts et al. 1995), but the molecular mechanism of this regulatory relationship and its relevance to cystic fibrosis pathophysiology is not yet fully understood (Kunzelmann & Schreiber, 1999). As discussed above, in M-1 cells the chloride secretory response elicited by ATP is likely to involve calcium-activated Cl^- channels. Thus, reciprocal regulation of amiloride-sensitive Na⁺ absorption and Cl^- secretion in epithelia may not be limited to an interaction between ENaC and CFTR Cl^- channels but may be a more general phenomenon.

Extracellular ATP may play a role in certain pathophysiological states such as polycystic kidney disease, where ATP release into the cyst lumen secondary to cyst formation has been linked to the increase in fluid secretion and cyst volume which characterises the disease (Wilson et al. 1999). On the other hand the inhibitory effect of ATP on amiloride-sensitive Na⁺ absorption may be a protective mechanism after ischaemic tissue damage since release of ATP from hypoxic cells may reduce O₂ consumption and further tissue damage by reducing active transport. Similarly, ATP release due to cell swelling may stimulate Cl^- secretion and inhibit Na⁺ absorption which may contribute to regulatory volume decrease. Finally, the short-lived nature of the peak [Ca²⁺]_i and chloride secretory response to extracellular ATP suggests that the initial response to ATP may act as a trigger mechanism for additional downstream events involving a range of signal transduction pathways (Ralevic & Burnstock, 1998).

In summary, our findings in M-1 cells suggest that extracellular ATP may have a dual effect on ion transport in the CCD. This dual effect is mediated through activation of P2Y₂ receptors located in the apical and basolateral membrane and consists of an inhibition of amiloride-sensitive Na⁺ absorption via ENaCs and stimulation of Cl^- secretion via calcium-activated Cl^- channels.

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Acknowledgements

We thank Dr Marko Bertog, Philipp Deetjen and Dr Maria A. Higgins for their valuable assistance. This work was supported by the Wellcome Trust and the Deutsche Forschungsgemeinschaft (grant LE 942/1-2).

Corresponding author

C. Korbmacher: University Laboratory of Physiology, Parks Road, Oxford OX1 3PT, UK.

Email: christoph.korbmacher{at}physiol.ox.ac.uk

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