ATP inhibits Mg2+ uptake in MDCT cells via P2X
purinoceptors
Long-Jun
Dai,
Hyung Sub
Kang,
Dirk
Kerstan,
Gordon
Ritchie, and
Gary A.
Quamme
Department of Medicine, University of British Columbia, Vancouver
Hospital and Health Sciences Centre, Vancouver, British Columbia,
Canada V6T 1Z3
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ABSTRACT |
First published July 12, 2001;
10.1152/ajprenal.00349.2001.
Nucleotides have diverse effects on water
and electrolyte reabsorption within the distal tubule of the nephron.
As the distal tubule is important in control of renal Mg2+
balance, we determined the effects of ATP on cellular Mg2+
uptake in this segment. The effects of ATP on immortalized mouse distal
convoluted tubule (MDCT) cells were studied by measuring Mg2+ uptake with fluorescence techniques. The mean basal
Mg2+ uptake rate was 165 ± 6 nM/s. ATP inhibited
basal Mg2+ uptake and hormone-stimulated Mg2+
entry by 40%. Both P2X (P2X1-P2X5 subtypes) and P2Y2 receptor subtypes were identified in MDCT cells using differential RT-PCR. Activation of both receptor subtypes with selective agonists increased intracellular Ca2+ concentration, P2X purinoceptors by
ionotropic-gated channels, and P2Y receptors via G protein-mediated
intracellular Ca2+ release. The more relatively selective
P2X agonists [
,
-methylene ATP (
,
-Me-ATP) and 2'- and
-3'-O-(4-benzoyl-benzoyl)-ATP] inhibited arginine
vasopressin (AVP)- and parathyroid hormone (PTH)-mediated Mg2+ uptake whereas agonists more selective for P2Y
purinoceptors (UTP, ADP, and 2-methylthio-ATP) were without
effect. Removal of extracellular Ca2+ diminished
,
-Me-ATP-mediated increase in intracellular Ca2+ and
inhibition of AVP-stimulated Mg2+ entry. We conclude from
this information that ATP inhibited Mg2+ uptake in MDCT
cells through P2X purinoceptors expressed in this distal convoluted
tubule cell line.
intracellular magnesium, fluorescence; adenosine triphosphate; P2Y
purinoceptors; prostanoids; intracellular calcium transients; intracellular adenosine 3',5'-cyclic monophosphate; immortalized mouse
distal convoluted tubule cells
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INTRODUCTION |
EXTRACELLULAR
NUCLEOTIDES have important effects on water and electrolyte
transport in the distal tubule of the kidney (3, 18).
Extracellular ATP inhibits arginine vasopressin (AVP)-stimulated water
transport in rabbit cortical collecting ducts (CCD) and rat inner
medullary collecting ducts (IMCD) and sodium absorption in CCD and
medullary collecting duct cells (13, 15, 22). Nucleotides
activate K+ channels and stimulate Cl
secretion in Madin-Darby canine kidney (MDCK) cells and Xenopus laevis renal (A6) cells, which are models for distal tubular
function (12, 23). Nucleotides also regulate NaCl
transport in IMCD, the final nephron segment responsible for fine
adjustments in NaCl excretion (17). Extracellular ATP has
also been reported to inhibit Ca2+ absorption in a mixture
of isolated rabbit cortical connecting tubules and CCD cells (15,
26). Accordingly, purinoceptor agonists play an important role
in determining water and electrolyte reabsorption in the final segments
of the nephron. Unlike sodium and Ca2+, the terminal
segments involved with tubular Mg2+ reabsorption comprise
only the convoluted portion of the distal tubule. The distal convoluted
tubule reabsorbs ~15% of the filtered Mg2+ or 90% of
the Mg2+ delivered to it from the loop of Henle
(20). As there is no Mg2+ reabsorbed beyond
this segment, the distal tubule acts as the final control in
determining the final urinary excretion. Accordingly, characterization
of the effects of purinergic nucleotides on Mg2+ uptake in
distal convoluted tubule cells would be important to understanding its
role in overall renal Mg2+ handling.
Extracellular ATP acts through P2 purinoceptors, which have been
divided into P2X and P2Y receptor families (1). The P2X receptor family contains a least seven distinct subtypes, all of which
are ATP-gated ion channels that on activation form
Ca2+-permeable channels permitting extracellular
Ca2+ entry into the intracellular fluid (21).
P2X agonists include
,
-methylene-ATP (
,
-Me-ATP) and the
more selective
,
-methylene-ATP (
,
-Me-ATP) and [2'- and
-3'-O-(4-benzoyl-benzoyl)-ATP] (benzoyl-benzoyl-ATP). The P2Y receptors, comprising at least six subtypes, are widely expressed and differ in their specificity for nucleotides. P2Y1 receptors bind ATP and the synthetic agonist
,
-Me-ATP whereas P2Y2 receptors respond to both ATP and UTP (21). P2Y
receptors are coupled to G proteins that increase the activity of
phospholipase C, D, and A2, release intracellular
Ca2+, and activate protein kinase C (21). P2Y
receptors may also stimulate or inhibit adenylate cyclase depending on
the cell type (21). In those cells in which adenylate
cyclase is stimulated, the increase in intracellular cAMP production is
sensitive to indomethacin, suggesting that cyclooxygenase-derived
products mediate this response (17, 19). Additionally, ATP
can be metabolized by ectoATPases to adenosine, the natural agonist for
adenosine, or P1 receptors, all of which couple to G proteins
(21). We have shown that hormone receptor-mediated
intracellular cAMP formation, stimulation of protein kinase A and
phospholipase C, or protein kinase C activation importantly affects
Mg2+ entry in mouse DCT (MDCT) cells (5, 7).
As ATP might act through these intracellular pathways, we determined
the effect of purinergic nucleotides on hormone-stimulated
Mg2+ uptake in distal tubule cells.
In the present studies, we determined the effect of ATP on
Mg2+ uptake in immortalized MDCT cells (11).
The MDCT cell line possesses many of the properties of the intact
distal convoluted tubule. Parathyroid hormone (PTH) and calcitonin
stimulate Ca2+ and Mg2+ uptake whereas glucagon
and AVP increase Mg2+ entry in MDCT cells (5, 7,
11). The distal convoluted tubule has not been extensively
studied because it is difficult to perform in vitro perfusion
experiments, so we used this cell line as a model to investigate the
actions of ATP on Mg2+ uptake in this segment.
Mg2+ uptake rate, measured with microfluorescence using
mag-fura, is concentration dependent and selective for Mg2+
(8). Moreover, the influx rate is rapid and reproducible
so that it is possible to determine the effects of extracellular influences on transport rates. In the present study, we show that ATP
inhibits basal and hormone-stimulated Mg2+ entry in MDCT
cells via P2X purinoceptors.
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MATERIALS AND METHODS |
Cell culture.
Distal convoluted tubule cells were isolated from mice, immortalized,
and functionally characterized as previously described by Friedman and
Gesek et al. (11). The MDCT cell line was grown on 60-mm
plastic culture dishes (Corning Glass Works, Corning Medical and
Scientific, Corning, NY) in basal DMEM/Ham's-F-12, 1:1, media (GIBCO)
supplemented with 10% FCS (Flow Laboratories, McLean, VA.), 1 mM
glucose, 5 mM L-glutamine, 50 U/ml penicillin, and 50 µg/ml streptomycin in a humidified environment of 5%
CO2-95% air at 37°C. For the fluorescence studies,
confluent cells were washed three times with PBS containing 5 mM EGTA,
trypsinized, and seeded on glass coverslips. Aliquots of harvested
cells were allowed to settle onto sterile glass coverslips in 100-mm
Corning tissue culture dishes, and the cells were grown to
subconfluence over 1-2 days in supplemented media as described
above. The normal media contained 0.6 mM Mg2+ and 1.0 mM
Ca2+. In the experiments indicated, MDCT cells were
cultured in Mg2+-free media (<0.01 mM) where indicated for
16-24 h before study. Other constituents of the
Mg2+-free culture media were similar to those of the
complete media. Customized Mg2+-free media were purchased
from Stem Cell Technologies (Vancouver, BC). These media contained
0.2% BSA rather than FCS.
Determination of cAMP concentration.
cAMP was determined in confluent MDCT cell monolayers cultured in
24-well plates in DMEM-Ham's F-12 media without serum but with 0.1%
BSA. The media contained 0.6 mM Mg2+ or zero
Mg2+ where indicated. After addition of either PTH or AVP,
MDCT cells were incubated at 37°C for 5 min in the presence of 0.1 mM
IBMX. The hormones were obtained from Sigma. The cAMP was extracted with 5% trichloroacetic acid that was removed with ether, and the
extract was acidified with 0.1 N HCl. The aqueous phase was dried and
then dissolved in Tris-EDTA buffer, and cAMP was measured with a
radioimmunoassay kit (Diagnostic Products, Los Angeles, CA).
Cytoplasmic Mg2+ and
Ca2+ measurements.
Coverslips were mounted into a perfusion chamber, and intracellular
Mg2+ concentration ([Mg2+]i) and
intracellular Ca2+ concentrations
([Ca2+]i) were determined with the use of the
Mg2+-sensitive and Ca2+-sensitive fluorescent
dyes mag-fura 2 and fura 2, respectively (Molecular Probes, Eugene,
OR). The cell-permeant acetoxymethyl ester form of the dye was
dissolved in DMSO to a stock concentration of 5 mM and then diluted to
5 µM mag-fura 2-AM or 10 µM fura 2-AM in media for 20 min at
37°C. Cells were subsequently washed three times with buffered salt
solution containing (in mM) 145 NaCl, 4.0 KCl, 0.8 Na2HPO4, 0.2 KH2PO4,
1.0 CaCl2, 5 glucose, and 20 HEPES-Tris, at pH 7.4. The
MDCT cells were incubated for a further 20 min to allow for complete
deesterfication and washed once before measurement of fluorescence.
Epifluorescence microscopy was used to monitor changes in the mag-fura
2 or fura 2 fluorescence of the MDCT cell monolayer. The chamber was
mounted on an inverted Nikon Diaphot-TMD microscope with a Fluor ×100
objective, and fluorescence was monitored under oil immersion within a
single cell over the course of study. Fluorescence was recorded at 1-s
intervals using a dual-excitation wavelength spectrofluorometer
(Delta-scan, Photon Technologies, Princeton, NJ) with excitation for
mag-fura 2 at 335 and 385 nm, for fura 2 at 340 and 380 nm (chopper
speed set at 100 Hz/s), and for emission at 505 nm. Media changes were
made without an interruption in recording.
The free [Mg2+]i and
[Ca2+]i were calculated from the ratio of the
fluorescence at the two excitation wavelengths as described, using a
dissociation constant (Kd) of 1.4 mM and 224 nM,
respectively, for the mag-fura 2-Mg2+ and fura
2-Ca2+ complexes (5). The minimum
(Rmin) and maximum (Rmax) ratios were
determined for the cells at the end of each experiment using 20 µM
digitonin. Rmax for mag-fura 2 was found by the addition of
50 mM MgCl2 in the absence of Ca2+, and
Rmin was obtained by removal of Mg2+ and
addition of 100.0 mM EDTA, pH 7.2. The excitation spectrum of the
cellular mag-fura 2 under these conditions was similar to that of free
mag-fura 2 in the same solutions. Rmax and Rmin for fura 2 were obtained with Ca2+ and EGTA by previously
published techniques (5).
RT-PCR analysis and differential DNA sequencing.
Total RNA was extracted from confluent MDCT cells using TRIzol reagent
(GIBCO BRL) according to the manufacturer's instructions. Briefly,
cells from a 150-cm2 flask were rinsed in 5 ml PBS,
pelleted, and lysed with a 5-min incubation in 2 ml TRIzol reagent. The
mixture was shaken vigorously for 60 s after 0.4 ml chloroform was
added. The mixture was centrifuged at 14,000 g for 15 min,
and the upper aqueous phase containing the extracted RNA was aspirated.
RNA was precipitated from the aqueous phase by an equal volume of
isopropanol and pelleted with centrifugation at 14,000 g for
15 min at 4°C. The RNA precipitate was washed two times with 75%
ice-cold ethanol, dried, and taken up in 200 µl
diethylpyrocarbonate-treated distilled H2O. Ten micrograms of RNA were incubated with RNase-free DNase (134 U) in the presence of
5 mM MgCl2 at 37°C for 10 min. The DNase was heat
inactivated with a 5-min incubation at 99°C, and the product was
stored at
80°C. The same procedure was used to prepare total RNA
from mouse cortical kidney.
RT-PCR was carried out as follows. Total RNA (5 µg) was
reverse-transcribed with the use of Superscript II RT from (GIBCO BRL
Life Technologies). One microliter of cDNA was used for PCR amplification. We used degenerate primers for rat P2X1, P2X2, and P2X3
receptors as described by Filipovic et al. (10) and McCoy
et al. (17). The forward primer was 5'-TTC ACC (C/A)T(T/C) (T/C)TC ATC AA(G/A) AAC AGC ATC-3', and the reverse primer was 5'-TGG
CAA A(C/T)C TGA AGT TG(A/T) AGC C-3'. The PCR reaction (total volume 50 µl) consisted of 32 cycles at 94°C for 30 s, annealing at
52°C for 30 s, and polymerization at 72°C for 30 s using
a GeneAmp PCR system thermocycler (model 2400, PerkinElmer, Branchburg, NJ). The PCR products were extracted from agarose gels and
ligated into pGem-T Easy Vector systems (Promega). Ligations were
transfected into DH5
-competent cells, and successful insertions were
selected from colonies grown on LB-agar plates. Specific primers were
designed from mouse sequences to amplify P2X4-P2X7 receptors. The
primers were for the following: P2X4, forward primer, 5'-GAG AAT GAC
GCT GGT GTG CC-3'; reverse primer, 5'-TTG GTG AGT GTG CGT TGC TC-3';
for P2X5, forward primer, 5'-TCC ACC AAT CTC TAC TGC-3'; reverse
primer, 5'-CCA GGT CAC AGA AGA AAG-3'; for P2X6, forward primer, 5'-TAC
GTA CTA ACA GAC GCA-3'; reverse primer, 5'-ATA TCA GGG TTC TTT GGG-3';
and for P2X7, forward primer, 5'-AAG TCT CTG CCT GGT GTC-3', and
reverse primer, 5'-GGC ATA TCT GAA GTT GTA GC-3'. The primers for P2Y2
mRNA comprised 5'-CGT CAT CCT TGT CTG TTA CGT GCT-3' and 5'-CTA CAG CCG
AAT GTC CTT AGT G-3' (17). The PCR reaction for P2X4/P2X5
consisted of 35 cycles at 94°C for 30 s, annealing at 62°C for
30 s, and polymerization at 72°C for 30 s and for P2X6/P2X7
consisted of 35 cycles at 94°C for 30 s, annealing at 50°C for
30 s, and polymerization at 72°C for 30 s. Aliquots (8 µl) of the PCR reaction were electrophoresed through ethidium
bromide-stained 1% agarose gels run with a 100-bp DNA ladder. The PCR
products were extracted from agarose gels by established techniques and
sequenced by Amplicon Express (Pullman, WA). The DNA sequence was
screened with the basic local alignment search tool algorithm to
compare with known P2X and P2Y2 receptor sequences.
Statistical analysis.
Representative tracings of fluorescence intensities are given, and
significance was determined by Student's t-test or Tukey's ANOVA as appropriate. A probability of P < 0.05 was
taken to be statistically significant. All results are means ± SE
where indicated.
 |
RESULTS |
Extracellular ATP diminishes basal and
prostaglandin-stimulated Mg2+
uptake into Mg2+-depleted MDCT cells.
Because there is not an appropriate radioisotope for Mg2+
to directly measure Mg2+ transport rates, we developed the
following model to assess Mg2+ influx into single MDCT
cells (7). Subconfluent MDCT monolayers were cultured in
Mg2+-free medium for 16 h. These cells possessed a
significantly lower [Mg2+]i, 0.22 ± 0.01 mM, than that observed in normal MDCT cells, 0.53 ± 0.02 mM.
When the Mg2+-depleted MDCT cells were placed in a bathing
solution containing 1.5 mM MgCl2, the
[Mg2+]i increased with time and plateaued at
0.52 ± 0.06 mM, n = 9, which was similar to that
observed for normal cells (7). The mean rate of refill,
d([Mg2+]i)/dt, measured as the
change in [Mg2+]i with time, was 165 ± 6 nM/s, n = 6 experiments, as determined over the first
500 s after the addition of Mg2+. We have previously
reported data that indicate the Mg2+ uptake is
concentration dependent and selective for Mg2+
(8).
We determined the effect of ATP on basal and hormone-stimulated
Mg2+ uptake in MDCT cells (Fig.
1). ATP (10
4 M)
significantly inhibited the basal mean Mg2+ entry
rate, 109 ± 4 nM/s, n = 3. We have reported
that endogenously produced prostanoids stimulate Mg2+
uptake in MDCT cells so that the addition of indomethacin, a cycloxygenase inhibitor, diminishes basal Mg2+ uptake
(6). To test the idea that ATP may decrease basal
Mg2+ uptake by inhibiting this autostimulatory pathway, we
treated the MDCT cells with indomethacin and ATP. The percent
inhibition was similar with indomethacin alone, 100 ± 20 nM/s,
n = 4, as with indomethacin plus ATP, 83 ± 17 nM/s, n = 5 (Fig. 1). We have also shown that
exogenously administered PGE2 stimulates Mg2+
entry into MDCT cells by 49 ± 9% (6). Accordingly,
we tested the effect of ATP on PGE2-mediated
Mg2+ uptake. Pretreatment of cells with ATP diminishes
PGE2-stimulated Mg2+ entry from 245 ± 23 nM/s, n = 4, to 89 ± 11 nM/s, n = 3 (Fig. 1). These studies suggest that ATP inhibits endogenously
generated prostaglandins and exogenously applied PGE2. In
those studies indicated, we performed the experiments with
indomethacin-treated cells to avoid the effects of autostimulation of
prostaglandin formation.

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Fig. 1.
ATP inhibits basal and prostaglandin-mediated
Mg2+ uptake in Mg2+-depleted mouse distal
convoluted tubule (MDCT) cells. MDCT cells were cultured in
Mg2+-free media (<0.01 mM) for 16 h. Fluorescence
studies were performed in buffer solutions in absence of external
Mg2+, and where indicated, MgCl2 (1.5 mM final
concentration) was added to observe changes in intracellular
Mg2+ concentration ([Mg2+]i). The
buffer solutions contained (in mM) 145 NaCl, 4.0 KCl, 0.8 K2HPO4, 0.2 KH2PO4, 1.0 CaCl2, 5.0 glucose, and 10 HEPES-Tris, pH 7.4, with and
without 1.5 mM MgCl2. ATP, 10 4 M, was added
to this buffer solution from a stock ethanol solution. Where indicated,
MDCT cells were treated with 5 µM indomethacin (Indo) 15 min before
experimentation according to the methods given in Ref. 4.
PGE2 was added from stock solutions at final concentrations
of 10 7 M. Fluorescence was measured at 1 data point/s
with 25-point signal averaging, and tracing was smoothed according to
methods previously described (4). Values are means ± SE for 3-7 cells. *P < 0.01, significance of
Mg2+ entry rates compared with control values.
Significance of ATP+PGE2 vs. ATP.
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ATP inhibits hormone-stimulated Mg2+
entry in MDCT cells.
We have shown that PTH and AVP stimulate Mg2+ uptake into
Mg2+-depleted MDCT cells by 27 ± 4 and 21 ± 5%, respectively, above basal entry rates (7, 8).
Pretreatment of MDCT cells with ATP reduced PTH-stimulated
Mg2+ uptake by 19 ± 3%, n = 5 (Fig.
2). ATP diminished AVP-stimulated uptake
from 201 ± 9 to 102 ± 8 nM/s, n = 5, which
was similar to ATP-treated cells, 109 ± 4 nM/s, n = 3 (Fig. 2). The reason for these differences in inhibition of ATP on
PTH and AVP is not known but may suggest differences in intracellular
hormone receptor-mediated signaling pathways. As mentioned above, ATP
inhibited PGE2-responsive Mg2+ influx to
89 ± 11 nM/s, n = 3, similar to that observed for
ATP plus AVP.

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Fig. 2.
ATP inhibits hormone-stimulated Mg2+ entry in
MDCT cells. Where indicated parathyroid hormone (PTH),
10 7 M, or arginine vasopressin (AVP), 10 7
M, was added 5 min after ATP, 10 4 M, treatment. The rate
of Mg2+ influx as determined by
d([Mg2+]i)/dt was measured using
fluorescence techniques performed according to that given in legend to
Fig. 1. d([Mg2+]i)/dt values were
determined over the first 500 s of fluorescence measurements.
Values are means ± SE for 3-6 cells. *P < 0.01, significance of Mg2+ entry rates compared with
control values. Significance of ATP+PTH vs. PTH and
ATP+AVP vs. AVP.
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MDCT cells possess P2X and P2Y purinoceptor subtypes.
To characterize the purinogenic response, we determined both
biochemical and functional expression of purinoceptors in MDCT cells.
Expression of P2X receptor mRNA in MDCT cells was determined by RT-PCR
using degenerate primers of P2X1-P2X3 and specific primers for
each of P2X4-P2X7. As shown in Fig.
3, amplified PCR products of the expected
size were identified for P2X1-P2X3 (500 bp). Twenty-seven colony
picks were sequenced with the following results (number of positive
picks given in brackets): P2X1 (1); P2X2
(10); and P2X3 (13). PCR products of the
correct size were also observed for P2X4 and P2X5 in three separate PCR
reactions using specific primers (Fig. 3). In addition, the MDCT cells
possessed P2Y2 receptor mRNA (Fig. 3). The negative control (no cDNA)
had no PCR products, and the positive control, cortical renal tissue,
possessed a number of bands of the expected sizes (not shown). The
identity of all RT-PCR products was verified by sequencing. The P2X and
P2Y fragments identified in MDCT cells had the same sequence alignment
reported for mouse cDNA. These results indicate that five members of
the P2X receptor family and at least one member of the P2Y receptor family are represented in MDCT cells. Taylor et al. (25)
reported a high incidence of multiple coexpression of P2X purinoceptor isoforms in a large number of different pulmonary- and
gastrointestinal-derived epithelial cells. The most common isoforms
were P2X2-P2X7, with P2X6 being noticeably absent from these cells
(25). The observation that multiple subtypes are
represented in the MDCT cell line used here is consonant with these
findings in other epithelial cells.

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Fig. 3.
RT-PCR analysis of P2X and P2Y2 purinoceptors in MDCT
cells. Amplification of P2 receptors was performed using degenerate
primers for P2X1-3 and specific primers for P2Y2 and P2X4-7
DNA fragments. Differential sequencing of the P2X1-P2X3 PCR
products resulted in (positive colony picks in parentheses) P2X1
(1), P2X2 (10), and P2X3 (13).
Renal cortical tissue was used as the positive control (not shown), and
"no cDNA" was used as negative PCR control.
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Next, we determined whether the purinoceptors identified by PCR were
functionally expressed in MDCT cells. Receptor-mediated intracellular
Ca2+ signaling was assessed by fluorescence measurements
using relatively selective agonists of the P2X and P2Y receptors. At
present, there are no agonists or antagonists that discriminate
adequately between the P2X and P2Y receptor families or between
subtypes of receptors within each of these groups (21).
Nevertheless, some of the most useful agonists are the stable ATP
analogs
,
-Me-ATP and
,
-Me-ATP that, if effective, strongly
imply actions at P2X receptors and are generally inactive at P2Y
receptors. ADP, UTP, UDP, and 2-methylthio ATP (2-MeS-ATP) are more
selective for P2Y purinoceptors. All of the nucleotides tested resulted
in transient increases in [Ca2+]i, but the
concentration profiles were different (Table
1). ATP, ADP, UTP, and 2-MeS-ATP produced
Ca2+ transients that were characterized by rapid
basal-to-peak increases in Ca2+ concentration, in excess of
500 nM, which then rapidly subsided to a level that was 20-40 nM
higher than basal concentrations for 5 min before returning to basal
levels (Fig. 4, top). This profile is typical of receptor-mediated intracellular Ca2+
signaling characteristic of P2Y purinoceptors. The more selective P2X
purinoceptor agonist,
,
-Me-ATP, resulted in a relatively delayed
increase in Ca2+ that did not attain the maximal levels
observed with the other nucleotides used (Fig. 4, bottom).
This was more characteristic of receptor-gated Ca2+
channels, as would be expected of P2X purinoceptors. In support of
these conclusions, the increase in intracellular Ca2+ by
,
-Me-ATP was abolished by removal of extracellular
Ca2+ whereas the initial rapid Ca2+ transients
of ATP, ADP, UTP, and 2-MeS-ATP remained evident after the removal of
bath Ca2+ (Fig. 4 and Table 1). These biochemical and
functional studies suggest that both P2Y and P2X receptor subtypes are
represented in MDCT cells: activation of P2Y leads to receptor-mediated
intracellular Ca2+ signaling and Ca2+ entry,
and P2X results in increases in intracellular Ca2+
concentration via receptor-gated Ca2+ channels.

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Fig. 4.
Nucleotide-mediated Ca2+ signals in
immortalized MDCT cells. The P2Y and P2X agonists 2-methylthio-ATP
(2-MeS-ATP; top) and , -methylene ATP ( , -Me-ATP;
bottom) were added where indicated at concentrations of
10 4 M. Intracellular free Ca2+ concentration
([Ca2+]i) was determined by microfluorescence
on single subconfluent MDCT cells using fura 2. Also shown is the
effect of removal of the external Ca2+
(Ca ) from bath buffer solution with the addition of
0.5 mM EGTA. The mean Ca2+ values are given in Table 1.
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ATP inhibits hormone-mediated
Mg2+ uptake through P2X
purinoceptors.
Next, we tested the effect of the selective nucleotides on
AVP-stimulated Mg2+ uptake in MDCT cells. The addition of
ADP, 99 ± 26 nM/s, n = 3, or UTP, 134 ± 16 nM/s, n = 5, did not change AVP-stimulated Mg2+ uptake, 214 ± 6 nM/s, n = 3 (Fig. 5). The more selective P2Y receptor
agonist, 2-MeS-ATP, also did not change AVP-mediated Mg2+
entry rates, 234 ± 29 nM/s (Fig.
6). In these latter studies, the cells
were not treated with indomethacin. The P2X agonist
,
-Me-ATP, on
the other hand, inhibited AVP-responsive Mg2+ entry,
79 ± 23 nM/s (Fig. 7). In support
of the observation that P2X purinoceptor agonists inhibit
hormone-stimulated Mg2+ entry, we showed that another P2X
agonist, benzoyl-benzoyl-ATP, diminished PTH-stimulated
Mg2+ uptake from 209 ± 8 nM/s, n = 5, to 89 ± 19 nM/s, n = 4. Accordingly, P2X
purinoceptor-selective agonists inhibit hormone-stimulated Mg2+ entry in MDCT cells. It was of interest that P2Y
receptor agonists increase intracellular Ca2+ release and
Ca2+ entry through coupling to G proteins whereas PX2
receptor agonists increase intracellular Ca2+ via
receptor-gated Ca2+ entry. To evaluate the significance of
extracellular Ca2+ entry in P2X purinoceptor responses, we
performed studies in the absence of an extracellular Ca2+
that abolishes P2X receptor-mediated increases in [Ca2+
]i (Fig. 5). Removal of the extracellular Ca2+
and the addition of EGTA to the bathing solution prevented
,
-Me-ATP inhibition of AVP-stimulated Mg2+ uptake,
270 ± 23 nM, n = 3, (Fig. 7). Accordingly, gated
Ca2+ entry appears to be necessary, if not essential, for
P2X receptor agonist inhibition of hormone-stimulated Mg2+
uptake. On balance, these observations indicate that ATP affects Mg2+ entry through P2X purinoceptors that are associated
with ionotropic responses.

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Fig. 5.
P2Y purinoceptor agonists do not inhibit Mg2+ uptake in
MDCT cells. The cells were pretreated with indomethacin before study.
UTP or ADP, at concentrations of 10 4 M, were added where
indicated. Arginine vasopressin (AVP), 10 7 M, was added 5 min after treatment with the individual nucleotide. Mg2+
uptake was determined by techniques outlined in legend to Fig. 1.
d([Mg2+]i)/dt was measured over
the first 500 s after addition of AVP and 1.5 mM
MgCl2. Values are means ± SE for 4-5 cells.
*P < 0.01, significance of control uptake rates.
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Fig. 6.
The P2Y agonist 2-MeS-ATP does not inhibit AVP-mediated
Mg2+ uptake. The more P2Y-selective agonist 2-MeS-ATP
(10 4 M) was added where indicated ~5 min before AVP,
10 7 M. Cells were not pretreated with indomethacin in
these studies. Values are means ± SE for 4-5 cells.
*P < 0.01, significance of control uptake rates.
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Fig. 7.
P2X purinoceptor agonists inhibit hormone-mediated
Mg2+ uptake. The more P2X-selective agonist , -Me-ATP
was added where indicated at concentrations of 10 4 M, and
AVP was added at 10 7 M. Where indicated, extracellular
Ca2+ was removed from the buffer solution with the addition
of 0.5 mM EGTA 10 min before the addition of , -Me-ATP,
10 4 M, and AVP, 10 7 M. Values are
means ± SE for 4-5 cells. *P < 0.01, significance of control uptake rates.
|
|
Finally, as P2Y receptors are coupled to G proteins that commonly
affect intracellular cAMP formation, we determined the effect of the
purinoceptor agonists on cAMP. The nucleotides ATP, ADP, UTP,
2-MeS-ATP, and
,
-Me-ATP do not stimulate intracellular cAMP
formation in MDCT cells (Table 2). The
nucleotides also did not alter PTH-responsive cAMP generation (Table
2). Accordingly, the P2Y purinoceptors do not appear to be coupled to
G
s or G
i proteins involved with mediation
of intracellular cAMP accumulation. Again, these results support the
notion that ATP inhibits Mg2+ uptake through the P2X
purinoceptor.
We have shown that adenosine increases Mg2+ uptake via
A2 receptors (12a). We also found in
these studies that ATP inhibits adenosine-stimulated Mg2+
entry. Accordingly, it is unlikely that degradation of extracellular ATP to ADP, AMP, and adenosine by ectonucleotidases plays a role in ATP
inhibition of basal and hormone-stimulated Mg2+ uptake in
MDCT cells.
 |
DISCUSSION |
This study shows that ATP inhibits basal and hormone-stimulated
Mg2+ transport in MDCT cells. There are two major subtypes
of ATP purinoceptors, P2X and P2Y, that have been characterized
pharmacologically according to their respective rank order of responses
to selected purine and pyrimidine agonists (21). More
recently, these receptors have now been further divided into
P2X1-7 and P2Y1-8 subtypes according to their molecular
identity and their intracellular signal transduction pathways
(21). Biochemical and functional studies using RT-PCR and
fluorescence measurements, respectively, provided evidence that both
P2X and P2Y receptor families are present in MDCT cells. Our study
using RT-PCR analysis showed that P2X1, P2X2, P2X3, P2X4, and P2X5
isoforms are represented in MDCT cells. We did not fully characterize
the P2Y subtypes because our evidence indicates that this receptor
family does not affect Mg2+ uptake. Nor did we attempt to
identify which P2X purinoceptor subtype inhibits Mg2+ entry
because of the pharmacological uncertainty of identifying P2 receptors
when more than one subtype is expressed in the same cell type. Agonists
and antagonists show cross-reactivity, similar affinities, and
different responses based on the specific cell types. Furthermore, the
functional classification of P2 receptors is made even more complex by
the likelihood that P2X and P2Y receptor subtypes may combine to form
heteromultimeric receptors, whose functional properties are distinct
from the homomultimeric receptors (21). However, the
evidence that ATP acts through P2X and not P2Y purinoceptor subtypes in
MDCT cells is persuasive. The nucleotides ADP, UTP, and 2-MeS-ATP,
which have a preference for the P2Y receptor, did not alter basal or
hormone-stimulated Mg2+ uptake (Fig. 5). P2Y purinoceptors
are coupled to heterotrimeric G proteins that either stimulate or
inhibit adenylate cyclase (21). The nucleotides used in
this study did not alter basal cAMP concentration nor inhibit AVP
receptor-mediated cAMP generation (Table 2). Accordingly, P2Y receptors
are represented in MDCT cells as indicated by RT-PCR and
agonist-elicited intracellular Ca2+ signals, but the
physiological function of P2Y purinoceptors in MDCT cells is not
apparent from these studies. Conversely, the P2X receptor agonists
,
-Me-ATP and benzoyl-benzoyl-ATP inhibited Mg2+ entry
in a similar fashion as that observed with ATP (Fig. 7). P2X
purinoceptors are coupled to ATP-gated channels that on activation let
Ca2+ enter the cell. Removal of extracellular
Ca2+ blocked the rise in intracellular Ca2+
(Fig. 5) and prevented ATP inhibition of AVP-stimulated
Mg2+ uptake (Fig. 7), indicating that gated channels
typical of P2X receptors are involved in mediating ATP effects rather
than receptor-coupled G proteins characteristic of P2Y purinoceptors.
These findings indicate that ATP modifies Mg2+ uptake via
P2X purinoceptors.
ATP acting via P2X purinoceptors plays an important role in control of
epithelial transport. Filipovic et al. (10) have provided
functional and molecular evidence for P2X receptors, possibly the P2X1
isoform, in LLC-PK1 cells. McCoy et al. (17) have reported that P2X purinoceptors that inhibit Na+
absorption and Cl
secretion are present in a mouse IMCD
cell line. Using RT-PCR, they identified two isoforms, P2X3 and P2X4
receptors, in these cells. Luo et al. (16) demonstrated
P2X receptors, comprising P2X1, P2X4, and P2X7 subtypes, in luminal and
basolateral membranes of pancreatic duct cells that were capable of
activating Cl
channels. Taylor et al. (26)
surveyed the P2X isoforms expressed in a range of pulmonary- and
gastroinestinal-derived epithelial cells. Their studies
identified multiple isoforms that encompassed P2X1-P2X7 in various
epithelial cells, with P2X6 being notably absent. Their studies suggest
that a large number of P2X isoforms may mediate ATP signaling and
Cl
transport in epithelium disturbed in cystic fibrosis
(26). In the present study, we demonstrate that
P2X1-P2X5 subtypes are represented in MDCT cells, so that ATP may
influence Mg2+ entry via any or all of these isoforms.
We have previously shown that endogenous production of prostanoids may
stimulate basal Mg2+ entry in MDCT cells, as indomethacin,
a cyclooxygenase inhibitor, diminished Mg2+ uptake
(6). It was of interest therefore that ATP also inhibited basal Mg2+ entry to a similar extent as indomethacin (Fig.
2). ATP also inhibited exogenous PGE2-stimulated uptake
(6). Accordingly, we speculate that ATP may inhibit the
autostimulatory prostanoid pathway. Van Baal et al. (27)
also showed that ATP diminished endogenously generated prostanoids in
primary rabbit cortical connecting tubule and collecting duct cells,
but in their studies ATP did so via P2Y receptors. The mechanism by
which ATP inhibits endogenous prostanoid release is unknown. ATP, by
the P2Y purinoceptor, has been reported to increase prostaglandin
release that, in turn, enhances cAMP production in MDCK cells
(19). ATP did not alter cAMP formation in MDCT cells
(Table 2) so that is not evident how ATP inhibits the autostimulatory
prostanoid pathway in MDCT cells.
The distal tubule reabsorbs significant amounts of Mg2+ and
plays an important role in determining the final urinary excretion rate
(20). In contrast to more proximal segments of the
nephron, distal Mg2+ transport processes are postulated to
be active and transcellular in nature (8). Hormonal
control of Mg2+ transport in this segment provides the
fine-tuning of renal conservation, contributing to whole body
Mg2+ balance. ATP plays a physiological/pathological role
in control of water in electrolyte reabsorption by the terminal
segments of the nephron (13, 15, 17, 22). The present
study suggests that these nucleotides may also affect Mg2+
transport in the distal convoluted tubule. Local concentrations of ATP
and its metabolites may be high enough to affect autocrine-paracrine function in the distal tubule (3). A number of models of
cellular ATP release, metabolism, and luminal and basolateral functions have been postulated (9, 25-28). Although it is
difficult to envision a role for nucleotides in the physiological
regulation of renal Mg2+ balance, Mg2+
excretion, like that of other electrolytes, is dependent on the filtered load. ATP, through its actions on the afferent and efferent arterioles, decreases glomerular filtration rate and filtered Mg2+ (2). Accordingly, it may be appropriate
to have reduced distal Mg2+ reabsorption in this condition
to balance diminished filtered load. Whatever the rationale for
nucleotide actions, it appears that ATP-mediated inhibition of NaCl and
Ca2+ reabsorption is associated with similar changes in
Mg2+ transport in the distal nephron.
 |
ACKNOWLEDGEMENTS |
We thank Dr. Peter A. Friedman for providing the MDCT cell line.
Dr. Hyung Sub Kang is a Postdoctoral Fellow of the Korean Science and
Engineering Foundation.
 |
FOOTNOTES |
First published July 12, 2001; 10.1152/ajprenal.00349.2001
This work was supported by a research grant from the Canadian
Institutes of Health Research (MT-5793).
Address for reprint requests and other correspondence: G. A. Quamme, Dept. of Medicine, Vancouver Hospital and Health Sciences Centre, Koerner Pavilion, 2211 Wesbrook Mall, Vancouver, BC Canada, V6T
1Z3 (E-mail:quamme{at}interchange.ubc.ca).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 21 December 2000; accepted in final form 18 June 2001.
 |
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