Nucleotides regulate NaCl transport in mIMCD-K2 cells via P2X
and P2Y purinergic receptors
David E.
McCoy1,
Amanda L.
Taylor1,
Brian A.
Kudlow2,
Katherine
Karlson1,
Margaret J.
Slattery1,
Lisa M.
Schwiebert2,
Erik M.
Schwiebert2, and
Bruce A.
Stanton1
1 Department of Physiology,
Dartmouth Medical School, Hanover, New Hampshire 03755; and
2 Department of Physiology and
Biophysics, University of Alabama at Birmingham, Birmingham, Alabama
35294
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ABSTRACT |
Extracellular nucleotides regulate NaCl transport in some
epithelia. However, the effects of nucleotide agonists on NaCl
transport in the renal inner medullary collecting duct (IMCD) are not
known. The objective of this study was to determine whether ATP and
related nucleotides regulate NaCl transport across mouse IMCD cell line (mIMCD-K2) epithelial monolayers and, if so, via what purinergic receptor subtypes. ATP and UTP inhibited
Na+ absorption [measured via
Na+ short-circuit current
(INasc)] and stimulated Cl
secretion
[measured via Cl
short-circuit current
(IClsc)].
Using selective P2 agonists, we report that P2X and P2Y purinoceptors
regulate INasc and
IClsc. By RT-PCR, two P2X
receptor channels (P2X3,
P2X4) and two P2Y G
protein-coupled receptors (P2Y1,
P2Y2) were
identified. Functional localization of P2 purinoceptors
suggest that IClsc is stimulated by apical membrane-resident P2Y purinoceptors and P2X receptor channels, whereas
INasc is inhibited by apical
membrane-resident P2Y purinoceptors and P2X receptor channels.
Together, we conclude that nucleotide agonists inhibit INasc across mIMCD-K2
monolayers through interactions with P2X and P2Y purinoceptors
expressed on the apical plasma membrane, whereas extracellular
nucleotides stimulate IClsc through interactions with P2X and P2Y purinoceptors expressed on the
apical plasma membrane.
inner medullary collecting duct; short-circuit current; kidney
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INTRODUCTION |
NUCLEOTIDES IN THE extracellular fluid act as autocrine
and paracrine hormones to regulate a variety of physiological processes (1, 4, 9, 13, 21, 22). For example, nucleotides inhibit
Na+ absorption and stimulate
Cl
secretion across
epithelia derived from many tissues (25, 30, 31, 36, 42, 44). In
addition, nucleotides activate K+
channels in Madin-Darby canine kidney (MDCK) cells (19) and inhibit
Na+ absorption across the rabbit
cortical collecting duct (CCD) (32). Despite evidence documenting the
effects of nucleotides on MDCK cells, A6 cells, and CCD cells (19, 32,
36, 44), the effects of nucleotides on NaCl transport in the inner
medullary collecting duct (IMCD), the final nephron segment responsible
for fine adjustments in NaCl excretion, are not known.
Once released from cells, nucleotides interact with specific plasma
membrane-resident purinoceptors that act as ion channels or couple with
phospholipases and adenylyl cyclase-driven signal transduction pathways
(1, 4, 6, 9, 13, 14, 18, 22). Purinoceptors are divided into two
classes: P1 (activated by adenosine) and P2 (activated by ATP and ADP)
(1, 4, 6, 9, 13, 14, 18, 22). P2 purinoceptors have been subdivided into two families: P2X and P2Y (1, 4, 6, 9, 13, 14, 18, 22). The P2X
purinoceptor family contains at least seven distinct subtypes
(P2X1 through
P2X7), each of which is a two
transmembrane-spanning, ion channel-forming protein (5, 9, 11, 33, 34,
43, 45, 47). P2X receptor channels bind ATP and form
Ca2+-permeable, nonselective
cation channels that, when activated, increase intracellular
Ca2+ by allowing
Ca2+ entry from the extracellular
fluid. P2Y purinoceptors are seven transmembrane-spanning, G
protein-coupled metabotropic receptors containing at least six distinct
receptor subtypes (P2Y1 through P2Y6) (1, 4, 6, 7, 12). P2Y
purinoceptors are coupled to effectors through heterotrimeric G
proteins and either stimulate (40) or inhibit (1, 13, 22) adenylyl
cyclase. P2Y receptors also stimulate phospholipase activity and, in
turn, increase intracellular Ca2+
(1, 4, 13, 22). In addition, P2Y purinoceptors enhance cAMP production
through receptor-mediated prostaglandin release (39, 40).
Previously, we demonstrated that a cell line derived from mouse IMCD
(mIMCD-K2) absorbs Na+ and
secretes Cl
by electrogenic
mechanisms (28, 29, 37, 48). Moreover, we demonstrated that the purine
nucleoside, adenosine, through an interaction with adenosine
A1 receptors, inhibits arginine vasopressin-stimulated Cl
secretion (37). Because ATP is released from IMCD cells (35) and is
present in the urine (10) and because the effects of nucleotides on
NaCl transport in the IMCD are not known, we tested the hypothesis that
ATP and related nucleotides regulate NaCl transport across mIMCD-K2
cells. To this end, we examined the effect of selective P2Y or P2X
agonists on electrogenic Cl
and Na+ transport across
monolayers of mIMCD-K2 cells mounted in an Ussing chamber. Our results
suggest that Cl
short-circuit current (IClsc)
is stimulated by apical membrane-resident P2Y purinoceptors and P2X
receptor channels, whereas Na+
short-circuit current (INasc)
is inhibited by apical membrane-resident P2Y purinoceptors and P2X
receptor channels. By RT-PCR, we also report that mIMCD-K2 cells
express mRNA for P2X3 and
P2X4 receptors and for
P2Y1 and
P2Y2 receptors that may transduce
extracellular nucleotide signals. We conclude that nucleotide agonists
stimulate IClsc while extracellular nucleotides inhibit
INasc across mIMCD-K2
monolayers through interactions with P2X and P2Y purinoceptors expressed on the apical plasma membrane.
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MATERIALS AND METHODS |
Cell culture.
The mIMCD-K2 cell line was established by clonal dilution of IMCD cells
isolated from transgenic mice [Tg(SV40E)Bri/7] and grown in
continuous culture as described previously (28). Briefly, cells were
cultured in tissue culture flasks (Costar, Cambridge, MA) coated with
Vitrogen plating media (VPM) containing DMEM (JRH Biosciences, Lenexa,
KS), human fibronectin (10 µg/ml; Collaborative Research Products,
Bedford, MA), 1% Vitrogen 100 (Collagen, Palo Alto, CA), and BSA (10 µg/ml; Sigma Chemical, St. Louis, MO) and placed in an incubator
maintained at 37°C and gassed with 5%
CO2-95% air. Every 48 h, 100% of
the medium, PC-1 (BioWhittaker, Walkersville, MD) supplemented with
PC-1 medium supplement (BioWhittaker), 5% fetal bovine serum (FBS;
Hyclone, Logan, UT), 2 mM
L-glutamine (Life Technologies,
Grand Island, NY), and 50 U/ml penicillin and 50 µg/ml streptomycin
(Life Technologies), was replaced. At confluence, cells were
subcultured by light trypsinization and reseeded in VPM-coated cell
culture flasks. In some studies, mIMCD-K2 cells were grown in
DMEM-Ham's F-12 medium (Life Technologies) supplemented with 10% FBS
(Hyclone), 10 µg/ml insulin (Sigma), 5.5 µg/ml transferrin (Sigma),
5.0 pg/ml sodium selenite (Sigma), 5 µM dexamethasone, 2 mM
L-glutamine, 50 U/ml penicillin,
and 50 µg/ml streptomycin, rather than PC-1 medium. Experimental
results were qualitatively and quantitatively similar regardless of the cell culture medium used. As demonstrated previously, mIMCD-K2 cells
display many of the phenotypic properties of the IMCD, including electrogenic Na+ absorption and
Cl
secretion (28, 29, 37,
48).
Measurement of short-circuit current in Ussing chambers.
To measure electrogenic Na+ and
Cl
currents, mIMCD-K2 cells
were seeded at a density of 1.2 × 105 cells/0.6
cm2 on permeable, VPM-coated
Cellagen filter supports (ICN Biomedicals, Aurora, OH) as described
previously (28). Every 24 h, 100% of the medium was replaced. Cells
between passages 11 and
20 were used for all experiments.
Short-circuit current
(Isc) was
measured by placing monolayers of mIMCD-K2 cells grown on Cellagen
filters into an Ussing-type chamber (Jim's Instrument, Iowa City, IA)
and voltage clamping the transepithelial voltage across the monolayer
to 0 mV with a VCC-600 four-electrode voltage clamp (Physiological
Instruments, Poway, CA) as described previously (28). Positive current
represents the net flow of cations from the apical to the basolateral
bath solution or the net flow of anions from the basolateral bath
solution to the apical bath solution. Bath solutions were maintained at 37°C and were stirred by bubbling with 5%
CO2-balance air. Electrical connections from the bath solutions to the voltage-clamp headstage were
made with 5.0 M NaCl-4% agar bridges and Ag-AgCl wires. Current output
from the clamp was digitized by a TL-1 DMA interface analog-to-digital converter (Axon Instruments, Foster City, CA). Data collection and
analysis were done using Axotape 2.0 software (Axon Instruments).
To measure secretory Cl
current, PC-1 medium containing 140 mM NaCl was used on both sides of
the monolayer. In these measurements, amiloride was added to block
absorptive Na+ current
(INasc) to measure
IClsc specifically. To measure
INasc, experiments were
performed in Cl
-free,
sodium gluconate bath solutions composed of (in mM) 116 sodium
gluconate, 3 potassium gluconate, 24 NaHCO3, 2 MgSO4, 8 HEPES, 8 glucose, and 2 CaSO4, adjusted to pH 7.4. Under
these conditions,
Isc is equivalent
to INasc (28, 29, 37, 48).
Isc data analysis.
Steady-state and peak values of the
Isc were
determined using Axotape software (Axon Instruments). Steady-state
Isc was recorded as the average of the current data over 1 min when individual current
values over that period differed by <1% of the mean current. Peak
values were reported as the highest single
Isc value
measured after stimulation with agonist. Data are expressed as means ± SE. Differences between means in unpaired experiments were
compared by repeated-measures ANOVA followed by Bonferroni's multiple
comparisons test. Differences between means in paired experiments were
compared by Student's t-test. All
statistical analyses were performed with InStat statistical software
(Graphpad, San Diego, CA). Statistical significance was ascribed for
P < 0.05 or lower.
Preparation of total RNA for PCR.
mIMCD-K2 cells were grown to confluence in collagen-coated flasks, and
RNA was extracted using TRIzol reagent (Life Technologies), according
to the manufacturer's instructions.
Preparation of cDNA.
Total RNA was recovered by centrifugation, resuspended in diethyl
pyrocarbonate (DEPC)-treated water (10-30 µl
depending on size of pellet), and treated with RNase-free DNase (Life
Technologies) to remove contaminating genomic DNA. Total RNA (2-4
µl) was mixed with 12-14 µl
DEPC-H2O, 2 µl DNase buffer, and
2 µl RNase-free DNase I and incubated at room temperature for 15 min.
Two microliters of 25 mM EDTA were added to chelate
Ca2+ and
Mg2+ to stop the reaction, and the
DNase-treated samples were heated at 65°C for 10 min to inactivate
DNase I activity. Moloney murine leukemia virus (MMLV)-RT (Life
Technologies) was used to reverse transcribe total RNA to cDNA. Each
reaction contained 20-22 µl of total RNA template (DNase-treated
sample from above), 2.5 µl dNTP mix (mixture of dATP, dCTP, dGTP, and
dTTP; Pharmacia, 2 µM each), 2 µl
oligo(dT)12-18 primer (Life
Technologies), 10 µl of 5× RT buffer, 2.5 µl dithiothreitol
(0.1 M), 9.8 µl DEPC-H2O, and 2 µl MMLV-RT enzyme (Life Technologies). Oligo(dT) primer ensures that
all messenger RNA species were reverse transcribed to cDNA. The
reaction was incubated for 1 h at 37°C in a thermocycler. Complementary DNA (cDNA) samples were then incubated at 90°C for 5 min in the thermocycler to inactivate the RT enzyme.
PCR of epithelial cDNA.
We used degenerate and specific PCR primers designed to amplify the P2X
gene family and specific members of the P2Y receptor gene family,
P2Y1 and
P2Y2. P2X degenerate primers were
designed to an alignment of rat
P2X1,
P2X2, and
P2X3 (16).
P2Y1 and
P2Y2 primers were designed to the
human cDNAs. Each PCR reaction contained 3-5 µl cDNA template,
33.8-35.8 µl ultraviolet-sterilized, double-distilled H2O, 1 µl dNTP mix (Pharmacia, 2 µM), 1 µl forward primer (A) and
reverse primer (B) (2 µM final
concentration; see primers below), 0.2 µl
Taq polymerase (Perkin-Elmer), 5 µl
of 10× PCR buffer, and 3 µl of
MgCl2 solution (1.5 mM final
concentration). The cycling parameters were established in 3 steps:
1) a 5-min, 94°C "hot
start"; 2) 40 cycles of 30 s at
94°C, 1 min at appropriate "annealing" temperature (indicated
with each primer pair below), and 1 min at 72°C; and
3) a 10-min "elongation" at
72°C to end the reaction. PCR products were visualized on a 1.5%
agarose gel run with a 100-bp DNA ladder. PCR primers are shown below
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-Actin
PCR amplification was performed to authenticate that cDNA synthesis was
successful in each sample using a housekeeping gene. Once
-actin
cDNA was detected and amplified, amplification with P2X or P2Y primer
pairs could proceed on the authenticated cDNA samples.
DNA sequencing of PCR products.
PCR products were isolated from agarose gel slices using the Qiaquick
gel extraction kit (Qiagen). PCR products were ligated into the pGEM-T
vector system (Promega) and were then transformed into JM109
high-efficiency competent cells (Promega). The grow-ups of the
transformations were then plated on LB agar plates containing ampicillin (100 µg/ml),
5-bromo-4-chloro-3-indolyl-D-galactopyranoside (X-Gal; 40 µg/ml), and isopropyl-
-thiogalactopyranoside (IPTG; 100 µg/ml)
for ampicillin selection of successful transformants and for blue/white
selection of successful PCR product ligation. White colonies were
picked off the plate to inoculate a 6-ml LB grow-up with continued
ampicillin selection. The pGEM-T plasmid with insert was purified from
the grow-up using a PerfectPrep miniprep kit (5 Prime
3 Prime,
Boulder, CO). The purified DNA was denatured in 0.2 N NaOH,
precipitated in 7.5 M NH4Cl and
100% ethanol, and sequenced using the Sequenase dideoxy termination method (Amersham/US Biochemicals) using
[
-35S]dATP (DuPont
NEN) incorporation by our laboratories. The resulting DNA sequence was
read by two independent investigators and screened with the basic local
alignment research tool (BLAST) algorithm (2).
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RESULTS |
Nucleotides regulate
INasc
and
IClsc.
To determine whether extracellular nucleotides regulate electrogenic
Na+ and/or
Cl
transport, we measured
INasc and
IClsc across monolayers of
mIMCD-K2 cells mounted in Ussing chambers. ATP and UTP (100 µM)
induced a small, insignificant increase of INasc followed by prolonged
inhibition of INasc (Figs.
1 and 2). ATP
and UTP transiently, but significantly, increased
IClsc (Figs. 3 and 4). After
the transient increase, IClsc returned gradually (~10 min) to levels not significantly different from Isc before
the addition of ATP and UTP (Figs. 3 and 4). It is important to note
that physiologically relevant concentrations of ATP (1 µM) also
reduced INasc (
4.8 ± 1.5 µA/cm2;
P < 0.05) and stimulated
IClsc (4.2 ± 0.1 µA/cm2;
P < 0.05).

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Fig. 1.
ATP inhibits Na+ short-circuit
current (INasc).
Representative current record illustrating the effects of ATP on
INasc is shown. ATP (100 µM)
was added to the apical and basolateral bath solutions. Amiloride (10 µM) was added to the apical bath solution to inhibit
Na+ absorption. Effects of UTP on
Isc were
qualitatively similar (representative record not shown). Decline in
Isc at
time 0 to steady-state (~10-12
min) has been described previously (28, 29, 37, 48).
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Fig. 2.
ATP and UTP inhibit INasc.
Data summary of the effects of ATP and UTP on
INasc is given. ATP (100 µM)
and UTP (100 µM) induced a small, transient increase in
INasc (not significantly
different from basal; P < 0.05),
followed by a prolonged steady-state inhibition of
INasc. After administration of
amiloride, Isc
was not significantly different from 0 µA/cm2, thereby indicating that
Isc is referable
to Na+ absorption.
* P < 0.05 vs.
basal.
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Fig. 3.
ATP stimulates Cl
short-circuit current (IClsc).
Representative current record illustrating the effects of ATP on
IClsc is shown. ATP (100 µM)
was added to the apical and basolateral bath solutions. Amiloride (10 µM) and diphenylamine-2-carboxylic acid (DPC; 10 mM), an inhibitor of
Cl channels, were added to
the apical bath solution. Effects of UTP on
Isc were
qualitatively similar (representative record not shown).
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Fig. 4.
ATP and UTP stimulate IClsc.
Data summary of the effects of ATP and UTP on
IClsc is given. ATP (100 µM)
and UTP (100 µM) induced a transient, significant increase in
IClsc, followed by a slow
decline to a steady-state value significantly less than the peak value
following administration of ATP or UTP. In the presence of amiloride
and following administration of DPC,
Isc was not
significantly different from 0 µA/cm2, thereby indicating that
Isc in
amiloride-treated monolayers is referable to
Cl secretion.
* P < 0.05 vs.
amiloride-inhibited
Isc.
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Pharmacological identification of purinoceptors.
To determine if P2X and/or P2Y receptors regulate
INasc and
IClsc, we used a
pharmacological approach. ATP binds to both P2X and P2Y receptors (
1
µM); thus ATP cannot be used to discriminate between P2X and P2Y
receptors. In contrast, UTP (1-100 µM) binds selectively to P2Y
receptors and, in particular, to the P2U or
P2Y2 receptors, whereas
,
-methylene-ATP (
,
-MeATP; 1-100 µM) and
,
-methylene-ATP (
,
-MeATP; 1-100 µM) bind selectively
to P2X receptors (1, 11, 21). Therefore, UTP can be used to identify
P2Y receptors and
,
-MeATP and
,
-MeATP can be used to
identify P2X receptors. UTP (10 µM) reduced
INasc by 4.8 ± 1.0 µA/cm2
(P < 0.05).
,
-MeATP (100 µM)
and
,
-MeATP (100 µM) also reduced INasc by 2.0 ± 0.8 µA/cm2
(P < 0.05) and by 3.9 ± 0.9 µA/cm2
(P < 0.05), respectively. These
results indicate that nucleotide inhibition of
INasc across mIMCD-K2 cells is
mediated by P2X and P2Y purinoceptors.
To determine whether P2X and/or P2Y purinoceptors regulate
IClsc, we examined the effects
of UTP,
,
-MeATP, and
,
-MeATP on
IClsc. UTP (1 µM)
transiently stimulated IClsc
by 6.4 ± 0.4 µA/cm2
(P < 0.05).
,
-MeATP and
,
-MeATP (50 µM) transiently increased IClsc by 0.5 ± 0.1 µA/cm2
(P < 0.05) and 2.4 ± 0.2 µA/cm2
(P < 0.05), respectively. These
results suggest that nucleotide stimulation of
IClsc is mediated by P2Y and P2X purinoceptors.
Functional localization of P2Y and P2X purinoceptors.
Experiments were conducted to determine if P2Y and P2X purinoceptors
are expressed in the apical and/or basolateral plasma membranes of
mIMCD-K2 cells. Because antibodies that recognize these receptors are
not yet available, we used a pharmacological approach to determine if
P2Y and P2X purinoceptors are expressed in the apical and/or
basolateral membranes. UTP (100 µM) reduced INasc when added to the apical
solution (Fig. 5). UTP also stimulated
IClsc when added to the apical
solution (Fig. 6). P2U
purinoceptor-mediated effects in the basolateral membrane were not
statistically significant. Therefore, P2Y purinoceptors are expressed
predominantly in the apical plasma membrane.

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Fig. 5.
Polarity of nucleotide receptor agonist effects on
INasc. Sidedness of the
effects of UTP, , -methylene-ATP (MeATP), and , -MeATP on
INasc is shown. All 3 agonists
had a significant effect when added to the apical solution.
* P < 0.05 vs.
amiloride-inhibited current.
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Fig. 6.
Polarity of nucleotide receptor agonist effects on
IClsc. Sidedness of the
effects of UTP, , -MeATP, and , -MeATP on
IClsc is shown. Although all 3 agonists increased IClsc
significantly when added to the apical solution, they had no
significant effect when added to the basolateral solution.
* P < 0.05 vs. 0 µA/cm2.
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,
-MeATP and
,
-MeATP (100 µM) inhibited
INasc and stimulated
IClsc when added to the apical
solution (Fig. 6). P2X purinoceptor-mediated effects in the basolateral
membrane were not statistically significant. Thus P2X purinoceptors in
the apical membrane inhibit
INasc and stimulate
IClsc.
Molecular identification of P2X and P2Y purinoceptors.
Little is known about P2X and P2Y receptor expression in IMCD cells.
Accordingly, to begin to identify which P2X and P2Y receptors are
expressed in mIMCD-K2 cells, we identified P2X and P2Y receptor subtype
mRNA expression by RT-PCR. RT-PCR for the housekeeping gene,
-actin
(750-bp product), was performed initially to authenticate a successful
cDNA synthesis (Fig.
7A). The
"no cDNA" and "no RT" negative PCR controls had no PCR
product for all genes amplified (Fig. 7). PCR products of the expected
size (330 bp) for P2X receptors were identified in mIMCD-K2 cells (Fig.
7B). A positive control was run in
parallel that included a human pancreatic epithelial cell line
(CFPAC-1). To confirm that the PCR products were indeed derived from
P2X receptor mRNA and to determine which P2X receptor mRNAs are
expressed in mIMCD-K2 cells, PCR products were sequenced. Of 32 minipreps processed from two different degenerate mIMCD-K2 P2X receptor
PCR reactions, 14 sequences were identified through BLAST analysis as
P2X3 and 18 were identified as
P2X4 (Table
1). Representative sequence alignments for
our mouse P2X3 and
P2X4 PCR product with cloned rat
cDNA sequences are shown in Fig. 8, A and
B. These results suggest that
P2X3 and
P2X4 receptors may regulate
transepithelial NaCl transport by mIMCD-K2 monolayers.

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Fig. 7.
P2X and P2Y receptors are expressed by a mouse inner medullary
collecting duct cell line (mIMCD-K2 cells).
A: amplification of -actin (750-bp
PCR product) to verify a successful cDNA synthesis (CFPAC-1 cells used
as a positive control for P2X receptors). Amplification of P2
purinoceptors was conducted using degenerate primers for the P2X
receptor gene family (330-bp PCR product) in
B, using specific primers for the
P2Y1 gene (750-bp PCR product) in
C, and using specific primers for the
P2Y2 (or P2U) gene (500-bp PCR
product) in D
(16HBE14o cells used as a
positive control for P2Y2
receptors). NC, "no cDNA," negative PCR control. "No RT"
controls were also negative (data not shown). L, 100-bp DNA ladder.
These gels are representative of at least 3 PCR amplification with each
set of PCR primers.
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Table 1.
P2X receptor channel mRNA expression in the mIMCD-K2 epithelial cell
line as determined by degenerate RT-PCR
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Fig. 8.
cDNA sequences confirming the authenticity of mIMCD-K2 P2X and P2Y PCR
products. BLAST alignments of P2 purinoceptor mIMCD-K2 PCR products
with the closest cloned cDNA sequences are shown.
A:
P2X3 PCR product alignment with
the cloned rat P2X3 receptor
(rP2X3R) cDNA sequence.
B:
P2X4 PCR product alignment with
the cloned rat P2X4 receptor
(rP2X4R) cDNA sequence.
C:
P2Y1 PCR product alignment with
the cloned mouse P2Y1 receptor
(mP2Y1R) cDNA sequence.
D:
P2Y2 PCR product with the cloned
mouse P2Y2 receptor
(mP2Y2R) cDNA sequence.
Nucleotides in bold show differences in identity; gaps in sequence are
shown by the dashed or dotted lines or where indicated.
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mIMCD-K2 cells also express P2Y purinoceptor mRNA. Figure 7,
C and
D, shows representative agarose gels
demonstrating positive amplification of a
P2Y1 PCR product (750 bp) and
P2Y2 PCR product (500 bp),
respectively. DNA sequencing of both
P2Y2 PCR products confirmed that
these PCR products were indeed derived from
P2Y2 and
P2Y1 mRNA (Fig. 8,
C and
D). Together, these results suggest that P2Y1 and
P2Y2 receptors may regulate
transepithelial NaCl transport by mIMCD-K2 monolayers.
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DISCUSSION |
The goal of the present study was to determine if nucleotides regulate
NaCl transport across mIMCD-K2 cells. Our results demonstrate that
nucleotides inhibit INasc
through interaction with P2Y purinoceptors expressed in the apical
plasma membrane. P2X purinoceptors in the apical membrane also inhibit
INasc. In contrast,
nucleotides stimulate IClsc
across mIMCD-K2 cells through interactions with both P2X and P2Y
purinoceptors expressed in the apical plasma membrane. Finally, by
RT-PCR, we demonstrated that mIMCD-K2 cells express mRNA for
P2X3 and
P2X4 purinergic receptor channels
and P2Y1 and
P2Y2 G protein-coupled purinergic receptors.
Previous research has demonstrated that
P2Y2 (or P2U) purinoceptors are
expressed in the proximal tubule (8, 26, 46, 49), MDCK cells (17, 39,
40), LLC-PK1 cells (3), A6 cells
(36), CCD (32, 41), and IMCD (15, 20, 27). Little is known about P2X
purinoceptor expression in renal epithelia. P2X1-like purinoceptors are
expressed in LLC-PK1 cells (16). Together, our results provide novel data demonstrating that
P2Y1 and
P2Y2 receptors and
P2X3 and
P2X4 receptors expressed in the apical plasma membrane inhibit
INasc and stimulate IClsc across mIMCD-K2 cells.
Our data are consistent with the hypothesis that nucleotides released
from IMCD cells themselves or from other upstream renal epithelial cells activate P2 purinoceptors that regulate
Na+ absorption and
Cl
secretion. It is
important to note that nucleotides are present in the urine and blood
in nanomolar to low micromolar (10 µM) concentrations (10, 21, 23,
24), amounts that would stimulate purinoceptors and regulate
Na+ and
Cl
transport in mIMCD-K2
cells. Recent preliminary results showed that mIMCD-K2 cells release
ATP under basal and stimulated conditions into the apical and
basolateral solution (35).
The IMCD, and perhaps other nephron segments or cell types in the
cortex and/or medulla, may be a source of nucleotides that, once
released, can regulate NaCl transport in an autocrine and/or paracrine
fashion. Interestingly, our results demonstrate that ATP would affect
NaCl transport by the IMCD directly by inhibiting Na+ absorption and stimulating
Cl
secretion. Thus ATP
would convert NaCl absorption by the IMCD to NaCl secretion (28, 29),
thereby enhancing NaCl excretion. It appears, therefore, that ATP and
other nucleotides may play an important role in the fine tuning of NaCl
transport by the IMCD. The effects of ATP (and, possibly, its
metabolites) on glomerular afferent and efferent arterioles may also
affect NaCl handling indirectly by affecting glomerular filtration rate
(GFR) and renal blood flow. These effects are likely to be complex; for
example, ATP is known to stimulate vasoconstriction of afferent, but
not efferent, arterioles and to decrease glomerular pressure (10, 23).
These effects, in turn, would result in a decrease in GFR and reduced
NaCl excretion.
In conclusion, our data demonstrate that nucleotides inhibit
Na+ absorption and stimulate
Cl
secretion through
interactions with P2X purinergic receptors (P2X3,
P2X4) and P2Y G protein-coupled
receptors (P2Y1,
P2Y2) expressed in the apical
membrane of mIMCD-K2 cells.
 |
ACKNOWLEDGEMENTS |
We thank Stephanie Kelley and Melissa Levack for excellent technical assistance.
 |
FOOTNOTES |
This work was supported, in part, by the National Institutes of Health
(DK-34533 and DK/HL-45881 to B. A. Stanton), the Cystic Fibrosis
Foundation (CFF-F992 to D. E. McCoy), and the American Heart
Association (New Hampshire Chapter to D. E. McCoy). E. M. Schwiebert is
supported by a New Investigator Grant from the American Heart
Association (Southern Research Consortium). L. M. Schwiebert is
supported by a separate New Investigator Grant from the American Heart
Association (Southern Research Consortium).
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: B. A. Stanton,
Dept. of Physiology, Dartmouth Medical School, Hanover, NH 03755 (E-mail: bas{at}dartmouth.edu).
Received 26 March 1998; accepted in final form 4 June 1999.
 |
REFERENCES |
1.
Abbracchio, M. P.,
and
G. Burnstock.
Purinoceptors: are there families of P2X and P2Y purinoceptors?
Pharmacol. Ther.
64:
445-475,
1994[Medline].
2.
Altschul, S. F.,
W. Gish,
W. Miller,
E. W. Myers,
and
D. J. Lipman.
Basic local alignment search tool (BLAST).
J. Mol. Biol.
215:
403-410,
1990[Medline].
3.
Anderson, R. J.,
R. Breckon,
R.,
and
B. S. Dixon.
ATP receptor regulation of adenylate cyclase and protein kinase C activity in cultured renal LLC-PK1 cells.
J. Clin. Invest.
87:
1732-1738,
1991[Medline].
4.
Barnard, E. A.,
G. Burnstock,
and
T. E. Webb.
G protein-coupled receptors for ATP and other nucleotides: a new receptor family.
Trends Pharmacol. Sci.
15:
67-70,
1994[Medline].
5.
Bean, B. P.
Pharmacology and electrophysiology of ATP-activated ion channels.
Trends Pharmacol. Sci.
13:
87-90,
1992[Medline].
6.
Boarder, M. R.,
G. A. Weisman,
J. T. Turner,
and
G. F. Wilkinson.
G protein-coupled P2 purinoceptors: from molecular biology to functional responses.
Trends Pharmacol. Sci.
16:
133-139,
1995[Medline].
7.
Bogdanov, Y. D.,
L. Dale,
B. F. King,
N. Whittock,
and
G. Burnstock.
Early expression of a novel nucleotide receptor in the neural plate of Xenopus embryos.
J. Biol. Chem.
272:
12583-12590,
1997[Abstract/Free Full Text].
8.
Bouyer, P.,
M. Palais,
M. Cougnon,
P. Hulin,
T. Anagnostopoulos,
and
G. Planelles.
Extracellular ATP raises cytosolic calcium and activates basolateral chloride conductance in Necturus proximal tubule.
J. Physiol. (Lond.)
510:
535-548,
1998[Abstract/Free Full Text].
9.
Buell, G.,
G. Collo,
and
F. Rassendren.
P2X receptors: an emerging channel family.
Eur. J. Neurosci.
8:
2221-2228,
1996[Medline].
10.
Chan, C. M.,
R. J. Unwin,
and
G. Burnstock.
Potential functional roles of extracellular ATP in kidney and urinary tract.
Exp. Nephrol.
6:
200-207,
1998[Medline].
11.
Collo, G.,
R. A. North,
E. Kawashima,
E. Merlo-Rich,
S. Neidhart,
A. Suprenant,
and
G. Buell.
Cloning of P2X5 and P2X6 receptors and the distribution and properties of an extended family of ATP-gated ion channels.
J. Neurosci.
16:
2495-2507,
1996[Abstract].
12.
Communi, D.,
C. Govaerts,
M. Parmentier,
M.,
and
J.-M. Boeynaems.
Cloning of a human purinergic P2Y receptor coupled to phospholipase C and adenylyl cyclase.
J. Biol. Chem.
272:
31969-31973,
1997[Abstract/Free Full Text].
13.
Dalziel, H. H.,
and
D. P. Westfall.
Receptors for adenine nucleotides and nucleosides: subclassification, distribution, and molecular characterization.
Pharmacol. Rev.
46:
449-466,
1994[Medline].
14.
Dubyak, G.,
and
C. El-Moatassim.
Signal transduction via P2 purinoceptors for extracellular ATP and other nucleotides.
Am. J. Physiol.
265 (Cell Physiol. 34):
C577-C606,
1993[Abstract/Free Full Text].
15.
Ecelbarger, C. A.,
Y. Maeda,
C. C. Gibson,
and
M. A. Knepper.
Extracellular ATP increases intracellular calcium in rat terminal collecting duct via a nucleotide receptor.
Am. J. Physiol.
267 (Renal Fluid Electrolyte Physiol. 36):
F998-F1006,
1994[Abstract/Free Full Text].
16.
Filipovic, D. M.,
O. A. Adebanjo,
M. Zaidi,
and
W. B. Reeves.
Functional and molecular evidence for P2X receptors in LLC-PK1 cells.
Am. J. Physiol.
274 (Renal Physiol. 43):
F1070-F1077,
1998[Abstract/Free Full Text].
17.
Firestein, B. L.,
M. Xing,
R. J. Hughes,
C. U. Corvera,
and
P. A. Insel.
Heterogeneity of P2U and P2Y purinoceptor regulation of phospholipases in MDCK cells.
Am. J. Physiol.
271 (Renal Fluid Electrolyte Physiol. 40):
F610-F618,
1996[Abstract/Free Full Text].
18.
Fredholm, B. B.,
M. P. Abbracchio,
G. Burnstock,
J. W. Daly,
T. K. Harden,
K. J. Jacobson,
P. Leff,
and
M. Williams.
Nomenclature and classification of purinoceptors: a report from the IUPHAR subcommittee.
Pharmacol. Rev.
46:
143-156,
1994[Medline].
19.
Friedrich, F.,
H. Weiss,
M. Paulmichl,
E. Woll,
S. Waldegger,
and
F. Lang.
Further analysis of ATP-mediated activation of K+ channels in renal epitheloid Madin Darby canine kidney (MDCK) cells.
Eur. J. Physiol.
418:
551-555,
1991[Medline].
20.
Ginns, S. N.,
S. Nielsen,
M. A. Knepper,
and
B. K. Kishore.
Immunolocalization of extracellular nucleotide receptor (P2U) in renal inner medulla (Abstract).
FASEB J.
11:
A9,
1997.
21.
Gordon, J. L.
Extracellular ATP: effects, sources and fate.
Biochem. J.
233:
309-319,
1986[Medline].
22.
Harden, T. K.,
J. L. Boyer,
and
R. A. Nicholas.
P2 purinoceptors: subtype-associated signaling responses and structure.
Annu. Rev. Pharmacol. Toxicol.
35:
541-579,
1995[Medline].
23.
Inscho, E. W.,
K. D. Mitchell,
and
L. G. Navar.
Extracellular ATP in the regulation of renal microvascular function.
FASEB J.
8:
319-328,
1994[Abstract/Free Full Text].
24.
Ishikawa, S.,
M. Higashiyama,
I. Kusaka,
T. Saito,
S. Nagasaka,
and
S. Fukuda.
Extracellular ATP promotes cellular growth of renal inner medullary collecting duct cells mediated by P2U receptors.
Nephron
76:
208-214,
1997[Medline].
25.
Iwase, N.,
T. Sasaki,
S. Shimura,
M. Yamamoto,
S. Suzuki,
and
K. Sharito.
ATP-induced Cl
secretion with suppressed Na+ absorption in rabbit tracheal epithelium.
Respir. Physiol.
107:
173-180,
1997[Medline].
26.
Jin, W.,
and
U. Hopfer.
Purinergic-mediated inhibition of Na+-K+-ATPase in proximal tubule cells: elevated cytosolic calcium is not required.
Am. J. Physiol.
272 (Cell Physiol. 41):
C1169-C1177,
1997[Abstract/Free Full Text].
27.
Kishore, B. K.,
C. L. Chou,
and
M. A. Knepper.
Extracellular nucleotide receptor inhibits AVP-stimulated water permeability in inner medullary collecting duct.
Am. J. Physiol.
269 (Renal Fluid Electrolyte Physiol. 38):
F863-F869,
1995[Abstract/Free Full Text].
28.
Kizer, N. L.,
B. Lewis,
and
B. A. Stanton.
Electrogenic sodium absorption and chloride secretion by an inner medullary collecting duct cell line (mIMCD-K2).
Am. J. Physiol.
268 (Renal Fluid Electrolyte Physiol. 37):
F347-F355,
1995[Abstract/Free Full Text].
29.
Kizer, N. L.,
D. Vandorpe,
B. Lewis,
B. Bunting,
J. Russell,
and
B. A. Stanton.
Vasopressin and cAMP stimulate electrogenic chloride secretion in an IMCD cell line.
Am. J. Physiol.
268 (Renal Fluid Electrolyte Physiol. 37):
F854-F861,
1995[Abstract/Free Full Text].
30.
Knowles, M. R.,
L. L. Clarke,
and
R. C. Boucher.
Activation by extracellular nucleotides of chloride secretion in airway epithelia of patients with cystic fibrosis.
N. Engl. J. Med.
325:
533-538,
1991[Abstract].
31.
Knowles, M. R.,
L. L. Clarke,
and
R. C. Boucher.
Extracellular ATP and UTP induce chloride secretion in nasal epithelia of cystic fibrosis patients and normal subjects in vivo.
Chest
101:
60-63,
1992.
32.
Koster, H. P.,
A. Hartog,
C. H. van Os,
and
R. J. M. Bindels.
Inhibition of Na+ and Ca2+ reabsorption by P2U purinoceptors requires PKC but not calcium signaling.
Am. J. Physiol.
270 (Renal Fluid Electrolyte Physiol. 39):
F53-F60,
1996[Abstract/Free Full Text].
33.
Le, K. T.,
K. Babinski,
and
P. Seguela.
Central P2X4 and P2X6 channel subunits coassemble into a novel heteromeric ATP receptor.
J. Neurosci.
18:
7152-7159,
1998[Abstract].
34.
Lewis, C.,
S. Neidhart,
C. Holy,
R. A. North,
G. Buell,
and
A. Surprenant.
Coexpression of P2X2 and P2X3 receptor subunits can account for ATP-gated currents in sensory neurons.
Nature
377:
432-435,
1995[Medline].
35.
McCoy, D. E.,
B. A. Stanton,
W. B. Guggino,
and
E. M. Schwiebert.
ATP release and nucleotide regulation of Na+ and Cl
transport in mouse mIMCD-K2 cells (Abstract).
J. Am. Soc. Nephrol.
7:
1285,
1996.
36.
Middleton, J. P.,
A. W. Mangel,
S. Basavappa,
S.,
and
J. G. Fitz.
Nucleotide receptors regulate membrane ion transport in renal epithelial cells.
Am. J. Physiol.
264 (Renal Fluid Electrolyte Physiol. 33):
F867-F873,
1993[Abstract/Free Full Text].
37.
Moyer, B. D.,
D. E. McCoy,
B. Lee,
N. Kizer,
and
B. A. Stanton.
Adenosine inhibits arginine vasopressin-stimulated chloride secretion in a mouse IMCD cell line (mIMCD-K2).
Am. J. Physiol.
269 (Renal Fluid Electrolyte Physiol. 38):
F884-F891,
1995[Abstract/Free Full Text].
38.
Nicholas, R. A.,
W. C. Watt,
E. R. Lazarowski,
Q. Li,
and
T. K. Harden.
Uridine nucleotide selectivity of three phospholipase C-activating P2 receptors: Identification of a UDP-selective, and an ATP- and UTP-specific receptor.
Mol. Pharmacol.
50:
224-229,
1996[Abstract].
39.
Post, S. R.,
J. P. Jacobson,
and
P. A. Insel.
P2 purinergic receptor agonists enhance cAMP production in Madin-Darby canine kidney epithelial cells via an autocrine/paracrine mechanism.
J. Biol. Chem.
271:
2029-2032,
1996[Abstract/Free Full Text].
40.
Post, S. R.,
L. C. Rump,
A. Zambon,
R. J. Hughes,
M. D. Buda,
J. P. Jacobson,
C. C. Kao,
and
P. A. Insel.
ATP activates cAMP production via multiple purinergic receptors in MDCK-D1 epithelial cells. Blockade of an autocrine/paracrine pathway to define receptor preference of an agonist.
J. Biol. Chem.
273:
23093-23097,
1998[Abstract/Free Full Text].
41.
Rouse, D.,
M. Leite,
and
W. N. Suki.
ATP inhibits the hydrosmotic effect of AVP in rabbit CCT: evidence for a nucleotide P2U receptor.
Am. J. Physiol.
267 (Renal Fluid Electrolyte Physiol. 36):
F289-F295,
1994[Abstract/Free Full Text].
42.
Schwiebert, E. M.,
M. E. Egan,
T.-H. Hwang,
S. B. Fulmer,
S. S. Allen,
G. R. Cutting,
and
W. B. Guggino.
CFTR regulates outwardly rectifying chloride channels through an autocrine mechanism involving ATP.
Cell
81:
1063-1073,
1995[Medline].
43.
Seguela, P.,
A. Haghighi,
J. J. Soghomonian,
and
E. Cooper.
A novel neuronal P2X ATP receptor ion channel with widespread distribution in the brain.
J. Neurosci.
16:
448-455,
1996[Abstract].
44.
Simmons, N. L.
Stimulation of Cl
secretion by exogenous ATP in cultured MDCK epithelial monolayers.
Biochim. Biophys. Acta
646:
231-242,
1981[Medline].
45.
Suprenant, A.,
F. Rassendren,
E. Kawashima,
R. A. North,
and
G. Buell.
The cytolytic P2Z receptor for extracellular ATP identified as a P2X receptor (P2X7).
Science
272:
735-738,
1996[Abstract].
46.
Takeda, M.,
M. Kobayashi,
and
H. Endou.
Establishment of a mouse clonal early proximal tubule cell line and outer medullary collecting duct cells expressing P2 purinoceptors.
Biochem. Mol. Biol. Int.
44:
657-664,
1998[Medline].
47.
Valera, S.,
N. Hussy,
R. J. Evans,
N. Adami,
R. A. North,
A. Surprenant,
and
G. Buell.
A new class of ligand-gated ion channel defined by P2X receptor for extracellular ATP.
Nature
371:
516-519,
1994[Medline].
48.
Vandorpe, D.,
N. Kizer,
F. Ciampolillo,
V. A. Memoli,
W. B. Guggino,
and
B. A. Stanton.
CFTR mediate electrogenic chloride secretion in mouse inner medullary collecting duct (mIMCD-K2) cells.
Am. J. Physiol.
269 (Cell Physiol. 38):
C683-C689,
1995[Abstract].
49.
Yamada, H.,
G. Seki,
S. Taniguchi,
S. Uwatoko,
K. Suzuki,
and
K. Kurokawa.
Mechanism of [Ca2+]i increase by extracellular ATP in isolated rabbit renal proximal tubules.
Am. J. Physiol.
270 (Cell Physiol. 39):
C1096-C1104,
1996[Abstract/Free Full Text].
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