Adenosine modulates Mg2+ uptake in distal
convoluted tubule cells via A1 and A2
purinoceptors
Hyung Sub
Kang,
Dirk
Kerstan,
Long-Jun
Dai,
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 |
tk;1Adenosine plays a
role in the control of water and electrolyte reabsorption in the distal
tubule. As the distal convoluted tubule is important in the regulation
of renal Mg2+ balance, we determined the effects of
adenosine on cellular Mg2+ uptake in this segment. The
effect of adenosine was studied on immortalized mouse distal convoluted
tubule (MDCT) cells, a model of the intact distal convoluted tubule.
The rate of Mg2+ uptake was measured with fluorescence
techniques using mag-fura 2. To assess Mg2+ uptake, MDCT
cells were first Mg2+ depleted to 0.22 ± 0.01 mM by
being cultured in Mg2+-free media for 16 h and then
placed in 1.5 mM MgCl2; next, changes in intracellular
Mg2+ concentration ([Mg2+]i) were
determined. [Mg2+]i returned to basal levels,
0.53 ± 0.02 mM, with a mean refill rate,
d([Mg2+]i)/dt, of 137 ± 16 nM/s. Adenosine stimulates basal Mg2+ uptake by 41 ± 10%. The selective A1 purinoceptor agonist
N6-cyclopentyladenosine (CPA) increased
intracellular Ca2+ and decreased parathyroid hormone
(PTH)-stimulated cAMP formation and PTH-mediated Mg2+
uptake. On the other hand, the selective A2 receptor
agonist 2-[p-(2-carbonyl-ethyl)-phenylethylamino]-5'-N-ethylcarboxamidoadenosine (CGS) stimulated Mg2+ entry in a
concentration-dependent fashion. CGS increased cAMP formation and the
protein kinase A inhibitor RpcAMPS inhibited CGS-stimulated
Mg2+ uptake. Selective inhibition of phospholipase C,
protein kinase C, or mitogen-activated protein kinase enzyme cascades
with U-73122, Ro-31-8220, and PD-98059, respectively, diminished
A2 agonist-mediated Mg2+ entry. Aldosterone
potentiated CGS-mediated Mg2+ entry, and elevation of
extracellular Ca2+ diminished CGS-responsive cAMP formation
and Mg2+ uptake. Accordingly, MDCT cells possess both
A1 and A2 purinoceptor subtypes with
intracellular signaling typical of these respective receptors. We
conclude that adenosine has dual effects on Mg2+ uptake in
MDCT cells through separate A1 and A2
purinoceptor pathways.
intracellular magnesium; fluorescence; intracellular
calcium transients; intracellular adenosine 3',5'-cyclic monophosphate; immortalized mouse distal convoluted tubule cells
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INTRODUCTION |
ADENOSINE MODULATES A VARIETY of transport functions
in the distal tubule. Adenosine increases transepithelial resistance and diminishes osmotic water absorption and Na+ transport
in inner medullary collecting duct (IMCD) cells in culture and
Cl
secretion in distal and collecting tubule cells
(5, 15, 17, 18). On the other hand, adenosine
stimulates Na+ transport in amphibian kidney A6 cells, a
model of the distal tubule (8, 10), and Cl
conductance in rabbit distal convoluted tubule cells (16). Adenosine also stimulates Ca2+ reabsorption in a mixture of
rabbit primary connecting tubule and cortical collecting duct cells
(10). The diversity of responses might be due to
expression of different purine receptors in cells comprising the distal
tubule. Three receptor subtypes (A1, A2, A3) have been identified, all of which are coupled to G
proteins. The A1 receptor is coupled to Gi,
leading to inhibition of adenylate cyclase, and to Gq,
resulting in activation of phospholipase C, intracellular
Ca2+ release, and an increase in protein kinase C activity
(15). A2 receptors are coupled, through
Gq, to stimulate adenylate cyclase (15). The
A3 receptors, like the A1 receptor subtypes,
are coupled to a Gi and Gq but are only found
in the heart and nervous system (6). Thus adenosine may
have diverse effects on electrolyte reabsorption in the distal tubule.
The convoluted portion of the distal tubule provides the final control
of urinary Mg2+ excretion, as there is no Mg2+
reabsorption beyond this segment (14). Accordingly, any
influence of adenosine on distal magnesium transport would be expected
to alter renal Mg2+ excretion.
In the present studies, we determined the effect of adenosine on
Mg2+ uptake into immortalized mouse distal convoluted
tubule (MDCT) cells, a model we have extensively used to study
Mg2+ handling in the distal convoluted tubule
(4). The distal convoluted tubule has not been extensively
studied because performing in vivo or in vitro perfusion experiments is
difficult. Our studies using MDCT cells suggest that the rate of
Mg2+ entry reflects overall transepithelial reabsorption
(4). The MDCT cell line possesses many of the properties
of the intact distal convoluted tubule, including many hormone
receptors and extracellular divalent cation-sensing receptors (CaSR).
We have reported that hormones such as parathyroid hormone (PTH),
glucagon, and arginine vasopressin (AVP) stimulate Mg2+
uptake (2, 4). Aldosterone potentiates hormone-mediated Mg2+ entry (3) and high extracellular
Ca2+ and Mg2+ levels inhibit hormone-responsive
uptake (1). Accordingly, MDCT cells are a useful model to
study controls of Mg2+ transport. In the present study, we
show that adenosine may stimulate Mg2+ entry or inhibit
hormone-mediated Mg2+ uptake in MDCT cells via
A2 and A1 receptors, respectively. We infer
from these studies that adenosine might modulate Mg2+
transport in the intact distal convoluted tubule.
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MATERIALS AND METHODS |
Cell culture.
Distal convoluted tubule cells were isolated from mice and immortalized
by Pizzonia et al. (13) and functionally characterized as
described by Friedman and Gesek and their colleagues (7). 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% fetal calf
serum (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) for 16-24 h before study. Other constituents of the
Mg2+-free culture media were similar to the complete media.
Customized Mg2+-free media were purchased from Stem Cell
Technologies (Vancouver, BC). These media contained 0.2% bovine serum
albumin rather than fetal calf serum.
Cytoplasmic Mg2+ and
Ca2+ measurements.
Coverslips were mounted in a perfusion chamber, and intracellular
Mg2+ and Ca2+ concentration
([Mg2+]i and
[Ca2+]i, respectively) were determined with
the use of the Mg2+- and Ca2+-sensitive
fluorescent dyes mag-fura 2 and fura 2, respectively (Molecular Probes,
Eugene, OR). The cell-permeant acetoxymethyl ester (AM) form of the dye
was dissolved in DMSO to a stock concentration of 5 mM and then diluted
to 5 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 single MDCT cells cultured in monolayers.
The chamber was mounted on an inverted Nikon Diaphot-TMD microscope,
with a Fluor ×100 objective, and fluorescence within a single cell was
monitored under oil immersion over the course of the 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 emission
at 505 nm. All experiments were performed at 21°C because the
mag-fura 2 and fura 2 responses were found to be identical at room
temperature and 37°C. Media changes were made without an interruption
in recording.
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 (2). 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 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 (2).
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 or 0 Mg2+ where indicated.
After addition of the agonist to be tested, MDCT cells were incubated
at 37°C for 5 min. cAMP was extracted with 5% trichloroacetic acid,
which was removed with ether, and the extract was acidified with 0.1 N
HCl. The aqueous phase was dried, dissolved in Tris-EDTA buffer, and
then cAMP was measured with a radioimmunoassay kit (Diagnostic
Products, Los Angeles, CA).
Statistical analysis.
Representative tracings of fluorescent intensities are given, and
significance was determined by Student's t-test or Tukey's analysis of variance as appropriate. All results are expressed as
means ± SE where indicated.
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RESULTS |
Adenosine alters 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 (4). 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,
[Mg2+]i increased with time and plateaued at
0.50 ± 0.07 mM, n = 7, which was similar to that
observed in normal cells (4). The mean rate of refill,
d([Mg2+]i)/dt, measured as the
change in [Mg2+]i with time, was 137 ± 16 nM/s, n = 7, experiments, as determined over the
first 500 s after addition of Mg2+. We have previously
reported data that indicate the Mg2+ uptake is
concentration dependent and selective for Mg2+
(4).
Adenosine stimulated Mg2+ uptake in
Mg2+-depleted MDCT cells by 41 ± 10% of control
values (Fig. 1). The adenosine/P1
receptor family in epithelial cells comprises A1 and
A2 adenosine receptors that have been identified by
molecular and pharmacological studies (15). We used
N6-cyclopentyladenosine (CPA), a selective
A1 agonist, and
2-[p-(2-carbonyl-ethyl)-phenylethylamino]-5'-N-ethylcarboxamidoadenosine (CGS), an A2 agonist, to determine the P1 subtype by
which adenosine alters Mg2+ entry. The agonists, CPA and
CGS, were from RBI (Sigma, St. Louis, MO). The A1 agonist,
CPA (10 µM) did not alter basal Mg2+ uptake, 159 ± 17 nM/s, n = 5, but the A2 receptor agonist
CGS nearly doubled the entry rate to 252 ± 18 nM/s,
n = 5.

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Fig. 1.
Adenosine alters 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 magnesium, 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.
N6-cyclopentyladenosine (CPA), a selective
A1 agonist, and
2-[p-(2-carbonyl-ethyl)-phenylethylamino]-5'-N-ethylcarboxamidoadenosine
(CGS), an A2 agonist, were added at concentrations of 10 µM. Fluorescence was measured at 1 data point/s with 25-point signal
averaging, and the tracing was smoothed according to methods previously
described (2). Values are means ± SE for 5-7
cells. *P < 0.01, Mg2+ entry rates vs.
control values.
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The relatively selective antagonist of A1 receptors,
8-cyclopentyl-1,3-diproxylxanthine (DPCPX), and of A2
receptors, 3,7-dimethyl-1-propargylxanthine (DMPX), were used to
confirm that adenosine stimulated Mg2+ entry by
A2 purinoceptors. The antagonists were from RBI. The A2 receptor antagonist DMPX inhibited adenosine-stimulated
Mg2+ uptake from 252 ± 18 to 120 ± 15 nM/s,
n = 4; the latter value was not different from basal
values (Fig. 2). DPCPX did not alter adenosine responses (Fig. 2). These findings support the conclusion that adenosine increases Mg2+ entry via A2
purinoceptors.

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Fig. 2.
Antagonists of A2 purinoceptors inhibit
adenosine-mediated Mg2+ uptake. Relatively selective
antagonists of A1 receptors,
8-cyclopentyl-1,3-diproxylxanthine (DPCPX), and A2
receptors, 3,7-dimethyl-1-propargylxanthine (DMPX), were added where
indicated 15 min before the addition of adenosine at concentrations of
10 µM. *, +: P < 0.01, Mg2+
entry rates vs. control and adenosine values, respectively.
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Next, we determined some of the receptor-mediated signaling mechanisms
involved with the adenosine-mediated Mg2+ uptake. The most
commonly recognized signal transduction mechanism for the
A2 receptor is activation of adenylate cyclase. This
implies coupling with the G protein Gs, although other G
proteins may also be involved (15). A1
purinoceptors, on the other hand, mediate a broad range of signaling
responses caused by its coupling to different G proteins within the
Gi/o family (15). This signaling pathway leads
to diminished cAMP, activation of phospholipase C, which, in turn,
leads to membrane phosphoinositide metabolism and increased production
of inositol triphosphate and Ca2+ mobilization. First, we
determined receptor-mediated [Ca2+]i with
fluorescence. Adenosine resulted in a transient increase in
Ca2+ concentration, from basal concentrations of 95 ± 7 to 516 ± 35 nM, which is typical of receptor-mediated
intracellular Ca2+ release (Table
1). The A1 agonist CPA also
initiated a transient increase in intracellular Ca2+ of a
magnitude similar to that for adenosine. However, the A2 agonist CGS did not illicit large changes in Ca2+ signaling
(129 ± 6 nM). The observation that CPA induced Ca2+
signaling supports the idea that there are A1 purinoceptors
present in MDCT cells. Second, we determined the effect of the
A1 and A2 receptor agonists on intracellular
cAMP formation. Adenosine and CGS increased intracellular cAMP
production by about threefold, from 20 ± 2 to 54 ± 9 and
65 ± 10 pmol · mg protein
1 · 5 min
1, respectively, whereas CPA had no effect (25 ± 2 pmol · mg protein
1 · 5 min
1) (Table 2). PTH
stimulated cAMP in control cells (42 ± 6 pmol · mg
protein
1 · 5 min
1). CPA diminished
PTH-stimulated cAMP (35 ± 2 pmol · mg
protein
1 · 5 min
1), whereas the
A2 receptor agonist CGS increased PTH-stimulated cAMP
formation (69 ± 9 pmol · mg
protein
1 · 5 min
1), which was
similar to the results for CGS alone; i.e., no additive effect. These
findings indicate that MDCT cells possess both A1 and
A2 purinoceptors that have the classic signaling pathways of each of the respective receptor families.
A1 purinoceptor agonists inhibit receptor-stimulated
Mg2+ uptake.
As the A1 purinoceptor agonists inhibit PTH-stimulated cAMP
formation (Table 2), we tested whether CPA might inhibit
hormone-mediated Mg2+ uptake. Pretreatment of MDCT cells
with CPA diminished PTH-stimulated Mg2+ entry rate by
27 ± 9% (Fig. 3). The pretreatment
of MDCT cells with CPA also inhibited CSG-stimulated Mg2+
uptake from 252 ± 18, n = 4, to 196 ± 14, n = 3, nM/s (Fig. 3). These observations indicate that
A1 purinoceptor agonists inhibit hormone-stimulated
Mg2+ entry and modulate the actions of A2
purinoceptor agonists in MDCT cells.

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Fig. 3.
A1 purinoceptors inhibit parathyroid hormone
(PTH)- and A2 agonist-mediated cAMP formation and
Mg2+ uptake in MDCT cells. Cells were pretreated with the
A1 agonist CPA (10 µM, 10 7 M) or the
A2 agonist CGS (10 µM) 5 min before the addition of PTH.
Values are means ± SE for 3-5 observations. *,
+, : P < 0.01, mean
Mg2+ entry rates and cAMP determinations of either PTH or
CGS vs. respective control values, of PTH+CPA vs. PTH alone, and of
CGS+CPA vs. CGS alone, respectively.
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Characterization of A2 purinoceptor agonist-stimulation
of Mg2+ uptake in MDCT cells.
CGS increased Mg2+ uptake in a concentration-dependent
fashion with the maximal dose of ~10 µM (Fig.
4). These data for selective P1 receptor
agonists indicate that adenosine stimulates Mg2+ uptake by
the A2 purinoceptor.

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Fig. 4.
Concentration dependence of CGS-stimulation of
Mg2+ entry in MDCT cells. The rate of Mg2+
influx as determined by uptake rate
{d([Mg2+]i)/dt} was measured
with the CGS concentrations shown, using fluorescence techniques
performed according to procedures 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. Cont, control.
*P < 0.01 for Mg2+ entry rates.
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To characterize some of the A2-mediated signaling pathways
that adenosine uses to stimulate Mg2+ uptake, we pretreated
the MDCT cells with a number of well-known inhibitors of kinases
involved in G protein transduction. RpcAMPS, an inhibitor of protein
kinase A, inhibited CGS agonist-stimulated Mg2+ entry rates
(143 ± 16 nmol/s, n = 3), supporting the above
observation that receptor-mediated cAMP formation is involved in
A2-receptor agonist-mediated actions (Fig.
5). Pretreatment of MDCT cells with the
phospholipase C inhibitor U-73122 inhibited A2-adrenergic agonist-stimulated Mg2+ uptake (124 ± 25 nmol/s,
n = 5), and the protein kinase C inhibitor Ro-31-822
diminished CGS agonist-stimulated uptake, from 252 ± 18, n = 5, to 178 ± 18 nmol/s, n = 4 (Fig. 5). Inhibition of the MAP kinase cascade
(extracellular-signal-regulated kinase, c-jun NH2-terminal
kinase, p38) with PD-98059 also decreased Mg2+
uptake (125 ± 22 nmol/s, n = 4). Accordingly,
A2 receptors act through a number of
receptor-mediated signaling pathways to affect changes in
Mg2+ transport. These findings are similar to those we have
reported for hormone (PTH, glucagon, AVP) receptor-mediated
Mg2+ transport in MDCT cells (4). Accordingly,
we would expect that aldosterone and high extracellular
Ca2+ concentration might alter CSG-stimulated
Mg2+ uptake.

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Fig. 5.
CGS stimulates Mg2+ uptake though
A2 receptor-mediated signaling pathways. Inhibitors for
protein kinase A [Rp-cAMPS (0.5 µM)], phospholipase C [U-73122 (15 µM)], and protein kinase C [Ro-31-822 (0.1 µM)] were added to
Mg2+-depleted MDCT cells 5 min before the addition of CGS
(10 µM). The MAP kinase inhibitor PD-98059 (1.0 µM) was added 10 min before CGS. Values are means ± SE for 3-5 cells.
* P < 0.001, CGS vs. control uptake
rates. P < 0.01, inhibitor + CGS vs. CGS alone.
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Aldosterone potentiates A2 receptor agonist-stimulated
Mg2+ uptake in MDCT cells.
We have previously shown that aldosterone, applied 16 h before
experimentation, increases PTH-, glucagon-, and AVP-mediated cAMP
generation and potentiates hormone-mediated Mg2+
uptake (4). Although the cellular mechanisms are not
known, it has been speculated that aldosterone-induced proteins
modulate receptor signaling in epithelial cells (11). In
the present study, we determined whether pretreatment of MDCT cells
with aldosterone for 16 h potentiated the actions of the
A2 receptor agonist CGS. Treatment of cells with
aldosterone, for 16 h before the study, did not significantly
affect basal Mg2+ uptake (142 ± 11 nM/s,
n = 3) but potentiated CGS-stimulated Mg2+
entry, from 251 ± 18 nM/s, n = 4, to 305 ± 17 nM/s, n = 6 (Fig. 6).
Interestingly, aldosterone did not potentiate CGS-responsive cAMP
production (62 ± 16 pmol · mg
protein
1 · 5 min
1), suggesting that
the actions are downstream of the generation of this second message
(Fig. 6).

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Fig. 6.
Aldosterone (Aldo) potentiates CGS-mediated Mg2+
uptake. MDCT cells were incubated for 16 h in magnesium-free
buffer solution containing aldosterone (10 7 M). CGS (10 µM) was added where indicated, and Mg2+ uptake was
determined after 500 s in 1.5 mM MgCl2 or cAMP was
measured after 5 min. Values are means ± SE for 4-7
observations. *, +, , :
P < 0.01, mean Mg2+ entry rates and cAMP
determinations vs. respective control values, CGS+aldosterone vs. CGS,
CPA+CGS vs. CGS, and aldosterone+CPA+CGS vs. aldosterone+CGS,
respectively.
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Elevation of extracellular Ca2+
inhibits A2 receptor agonist-stimulated cAMP generation and
Mg2+ uptake.
MDCT cells possess an extracellular CaSR that, on activation with
polyvalent cations such as Ca2+, Mg2+, or
neomycin, inhibits PTH-, glucagon-, and AVP-mediated cAMP generation
and glucagon- and AVP-stimulated Mg2+ uptake
(1). To determine whether activation of the CaSR alters A2 receptor agonist actions, we pretreated cells for 5 min
with 5.0 mM CaCl2 before the addition of CGS. Elevation of
extracellular Ca2+ did not have any effects on basal
Mg2+ entry (147 ± 10 nM/s, n = 4) but
abolished CGS stimulation of cAMP generation (21 ± 2 pmol · mg protein
1 · 5 min
1, n = 4) and Mg2+ uptake
(128 ± 13 nM/s, n = 5) (Fig. 7). The mechanisms
by which the CaSR inhibits CGS actions remain unclear, but the receptor is coupled to G
i proteins, which is consistent with the
conclusion that CGS responses in MDCT cells are dependent, in part, on
cAMP-mediated signaling pathways.

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Fig. 7.
Summary of the effects of extracellular Ca2+
on CGS-stimulated cAMP formation and Mg2+ uptake. cAMP was
measured by radioimmunoassay, and
d([Mg2+]i)/dt was determined with
1.5 mM extracellular Mg2+ in the absence and presence of
5.0 mM CaCl2 as indicated. CaCl2 was added 5 min before the addition of 10 µM CGS.
d([Mg2+]i)/dt was determined over
the initial 500 s after addition of CGS. Values are means ± SE for 4-7 cells. *, +: P < 0.01, mean Mg2+ entry rates and cAMP determinations vs.
respective control values and CGS vs. CGS+5.0 mM extracellular
Ca2+, respectively.
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ATP inhibits A2 receptor agonist-stimulated
Mg2+ uptake in MDCT cells.
The relationship between the purines ATP and adenosine is complex. We
have recently determined that ATP inhibits basal and hormone-stimulated
Mg2+ transport by 21% in MDCT cells
(2a). Our studies showed that this inhibition was
via P2X purinoceptors as the selective P2X agonist
,
-methylene-ATP (
,
-Me-ATP) inhibited Mg2+
uptake, but the more P2Y selective agonists UTP, ADP, and 2-methylthio ATP were without effect. Accordingly, it was of interest to see whether
ATP would have any effect on Mg2+-conserving actions of
adenosine. Pretreatment of MDCT cells with
,
-Me-ATP prevented the
stimulation of Mg2+ uptake by the A2 receptor
agonist (160 ± 9 nM/s, n = 4) (Fig. 8). These
observations suggest that the purines may modulate Mg2+
uptake in MDCT cells by diverse receptor-mediated mechanisms.

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Fig. 8.
Extracellular ATP inhibits A2 receptor
agonist-stimulated Mg2+ uptake in MDCT cells. P2X
purinoceptor agonists, such as , -methylene-ATP ( , -Me-ATP)
inhibit hormone-mediated Mg2+ uptake (unpublished
observations). The more selective P2X agonist, , -Me-ATP
(10 4 M) was added 5 min before 10 µM CGS where
indicated. Values are means ± SE for 4-7 cells. *,
+: P < 0.01, mean Mg2+ entry
rates and cAMP determinations vs. respective control values and CGS vs.
CGS+ , -Me-ATP, respectively.
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DISCUSSION |
The findings in this study indicate that adenosine modulates
Mg2+ uptake in MDCT cells, a model for the intact distal
convoluted tubule. Our data show that both A1 and
A2 purinoceptor subtypes are present in MDCT cells.
Adenosine and the selective A2 agonist CPA elicited
receptor-mediated intracellular Ca2+ signaling, whereas
adenosine and the A1 receptor agonist CGS increase cAMP
formation (Tables 1 and 2). CPA inhibited PTH- and CGS-stimulated cAMP
formation typical of A1 purinoceptor-induced signaling
involving Gi-coupled proteins (Fig. 3). This was associated with diminished Mg2+ uptake (Fig. 3). On the other hand,
CGS stimulated cAMP formation in a manner characteristic of
A2 purinoceptor signal transduction. CGS increased
Mg2+ entry (Fig. 3). Moreover, CGS-stimulate
Mg2+ uptake was decreased by protein kinase A inhibition,
supporting the notion that A2 receptors modulate
Mg2+ entry in MDCT cells via the Gs-coupled
proteins (Fig. 6). A1 and A2 receptors are
often polarized to either the apical or basolateral membrane so that
adenosine may have divergent effects depending on the concentration at
these two sides (15). Unfortunately, we are not able to
determine the polarity of A1 and A2 receptors in MDCT cells; nevertheless, these observations demonstrate the multipotency of the effect of adenosine on Mg2+ transport.
Polarization of the purinoceptors remains to be determined in the
intact distal convoluted tubule.
Aldosterone modulates adenosine-stimulated
Mg2+ entry in MDCT cells.
We have shown that aldosterone potentiates hormone-responsive
Mg2+ transport in MDCT cells (3). The
prominent mechanism of steroids, which operate through nuclear
receptors, is to control transcriptional regulation, expression, and
posttranslational targeting of heterotrimeric G proteins such as
G
s , G
i, G
, G
, and phospholipase C
(11). Pretreatment of MDCT cells with aldosterone
potentiated CGS-stimulated Mg2+ entry (Fig. 6). Aldosterone
may increase any of the above pathways or others that ultimately lead
to increased adenosine-stimulated Mg2+ entry in MDCT cells.
In support of this notion, aldosterone potentiates hormone-stimulated
Mg2+ uptake without increasing cAMP formation so that other
processes downstream of cAMP generation are involved.
These studies indicate that MDCT cell Mg2+ uptake is
regulated at two levels: first by membrane-receptor (adenosine)
signaling and, second, by nuclear transcription-dependent receptor
(aldosterone) signaling.
Extracellular Ca2+ affects
A2 agonist-mediated Mg2+
uptake in MDCT cells.
The CaSR within the distal tubule is important in controlling
Mg2+ entry in MDCT cells (1, 4). The
extracellular Ca2+- and Mg2+-sensing mechanisms
provide a negative-feedback loop to diminish the renal conserving
actions of the circulating hormones like PTH, glucagon, and AVP
(4). We have reported that elevation of extracellular
Ca2+ or Mg2+, or the addition of the polyvalent
cation neomycin, inhibits peptide hormone-stimulated cAMP formation and
hormone-responsive Mg2+ uptake in MDCT cells
(1). Activation of CaSR inhibits A2 agonist stimulation of Mg2+ uptake in MDCT cells (Fig.
7). The responses likely involve
diminished A2 agonist-mediated cAMP formation,
phospholipase C, protein kinase C, or MAP kinase cascades (Fig. 5).
These findings show that adenosine-mediated effects may be modulated by
extracellular Ca2+ and Mg2+ concentration.
ATP inhibits adenosine-stimulated
Mg2+ uptake.
We have shown that ATP inhibits hormone-stimulated Mg2+ via
P2X purinoceptors (2a). These receptors are
coupled to ATP-gated channels that activate nonselective cation
channels. In these studies, the selective P2X receptor agonist
,
-Me-ATP inhibited basal and hormone-stimulated Mg2+
uptake by 32%. In the present study,
,
-Me-ATP inhibited
adenosine-stimulated Mg2+ uptake in MDCT cells (Fig.
8). The pathophysiological implications of these interactions are unclear, but autocrine or paracrine secretion
or tissue damage leading to cellular ATP release and its degradation to
adenosine may be sufficient to alter Mg2+ handling in the
distal tubule.
Role of adenosine in distal tubular
Mg2+ handling.
We infer from our data that adenosine modulates magnesium transport in
the distal convoluted tubule of the nephron. Adenosine via
A1 receptors may inhibit hormone-stimulated
Mg2+ uptake or, via A2, may stimulate
Mg2+ entry. Accordingly, hormones such as PTH, vasopressin,
and calcitonin stimulate distal Mg2+, in part, through
intracellular generation of cAMP that may be metabolized to 5'-AMP and
adenosine within the cell (17). Adenosine is transported
out of the cell by a nucleoside transporter. In addition, intracellular
5'-AMP may be transported out of the cell by a nucleotide transporter
and further metabolized to adenosine by a membrane
ecto-5'-nucleotidase, as summarized by Schwiebert et al.
(17). Extracellular adenosine may act at cell-surface A1 receptors to diminish hormone-mediated cAMP formation,
leading to termination of the hormone stimulus. Alternatively,
extracellular adenosine may act at cell-surface A2
receptors to further increase cAMP, leading to a propagation of the
hormone stimulus so that adenosine may act as an autocoid to stimulate
Mg2+ reabsorption in conjunction with the known
Mg2+-conserving circulating hormones (4).
Further studies are required to identify and locate nucleotide
transporters and cell-surface purinoceptors in the distal convoluted tubule.
 |
ACKNOWLEDGEMENTS |
We thank Dr. Peter A. Friedman for providing the MDCT cell line.
 |
FOOTNOTES |
Dr. Hyung Sub Kang is a Postdoctoral Fellow of the Korean Science and
Engineering Foundation. This work was supported by research grants from
the Canadian Institutes of Health Research (MT-5793) and Kidney
Foundation of Canada.
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 16 March 2001; accepted in final form 25 July 2001.
 |
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