Section of Nephrology, Department of Internal Medicine, Yale University School of Medicine, New Haven, Connecticut 06520-8029
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ABSTRACT |
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There are
conflicting reports in the literature regarding the adenosine receptor
that mediates the increase in sodium transport in the A6 cell. In this
study we used specific A1 and A2 adenosine receptor agonists and antagonists, as well as two different subclones of the A6 cell, to determine which adenosine receptor mediates the
increase in sodium transport. In the A6S2 subclone, basolateral and
apical N6-cyclohexyladenosine (CHA), a selective
A1 receptor agonist, stimulated sodium transport at a
threshold concentration <107 M, whereas CGS-21680,
a selective A2 receptor agonist, had a threshold
concentration that was at least 10
5 M. The
A1 receptor antagonist
8-cyclopentyl-1,3-dipropylxanthine (DPCPX) was found to have a
nonspecific effect on CHA-stimulated sodium transport, whereas the
A2 receptor antagonist 8-(3-chlorostyryl)caffeine (CSC) had
no effect. As with the A6S2 subclone, basolateral and apical CHA
stimulated sodium transport at a nanomolar concentration in the A6C1
subclone and the threshold concentration for CGS-21680 was in the high
micromolar range. Concurrent with the increase in sodium transport, CHA
also stimulated anion secretion in the A6C1 subclone. These data
demonstrate that adenosine increases sodium transport via the
A1 receptor in different subclones of the A6 cell,
including a subclone capable of anion secretion.
epithelial sodium channel; chloride transport; A1 receptors; A2 receptors; kidney
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INTRODUCTION |
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ADENOSINE EXERTS MANY TYPES of cell-specific effects after binding to two general classes of receptors, classified as A1 and A2. As proposed by Londos et al. (24), these receptors are defined by their functional properties; activation of the A1 receptor inhibits adenylate cyclase via the mediation of a pertussis toxin-sensitive G protein, whereas activation of the A2 receptor stimulates the enzyme. The A1 receptor was subsequently linked to a second messenger system that involves release of intracellular calcium and phospholipid turnover (1, 2).
The A6 cell line [obtained from the American Type Culture
Collection (ATCC)] and various subclones derived from the A6 cell line
have been widely used to study electrogenic sodium transport in tight
epithelium and, more recently, the properties of the epithelial sodium
channel. Lang et al. (20) were the first to discover that
basolateral adenosine and its analogs stimulate sodium transport in A6
cells. In their effort to identify the mechanism that mediates sodium
transport, they found no evidence that cAMP was the second messenger
because the threshold concentration at which 2-chloroadenosine
increased short-circuit current (Isc; 5 · 108 M) was several orders of magnitude lower
than the concentration required to increase cellular levels of cAMP
(10
4 M). They did, however, demonstrate that the
A2 receptor was present in the basolateral membrane of A6 cells.
A decade later in our laboratory, we aimed to determine, alternatively,
whether adenosine-stimulated sodium transport was mediated by
activation of an A1 receptor and a calcium-dependent mechanism. Using N6-cyclohexyladenosine (CHA), a
highly selective A1 agonist reported not to increase cAMP
content when used in a concentration <3 · 105 M
(3), we identified the presence of an A1
receptor in the basolateral membrane by showing that CHA inhibited
adenylate cyclase via a pertussis toxin-sensitive mechanism. We
also demonstrated that adenosine-stimulated sodium transport, as
first reported by Lang et al. (20), was linked to the
basolateral A1 receptor (14). This conclusion
was based on several independent strategies as follows: 1)
CHA stimulation of sodium transport was not associated with detectable
increases in cAMP nor was it reduced after abolishment of adenylate
cyclase activity with 2',5'-dideoxyadenosine; 2) the minimal
stimulatory concentration of CHA was several orders of magnitude lower
than that of the selective A2 agonist CGS-21680; 3) CHA-stimulated sodium transport correlated in a time- and
dose-dependent manner with increases in inositol trisphosphate and
intracellular Ca2+; and 4) chelation of
intracellular Ca2+ with BAPTA dose-dependently blocked CHA
stimulation of sodium transport.
Subsequently, Casavola et al. (7) reported that
N6-cyclopentyladenosine (CPA), a selective
A1 receptor agonist, stimulated sodium transport in another
subclone of the A6 cell (A6C1) by activation of a basolateral
A2 receptor. This conclusion was based on the action of two
inhibitors: 1) the selective A1 inhibitor 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; 107 M)
inhibited apical but not basolateral CPA-stimulated
Isc; and 2) the selective
A2 inhibitor 8-(3-chlorostyryl)caffeine (CSC; 10
6 M) inhibited basolateral CPA-stimulated
Isc. This study also included the new
observation that apical adenosine stimulated an
amiloride-insensitive current in the A6C1 subclone. The authors proposed that this current reflected chloride secretion mediated by
activation of an apical A1 receptor.
The primary aim of this study was to reconcile the qualitative differences between our previous study (14) and that of Casavola et al. (7) concerning the signal mechanism for basolateral adenosine-stimulated sodium transport. A second aim was to determine whether CHA stimulated anion secretion in the A6S2 subclone under the conditions we previously used to study sodium transport, because such an occurrence may have confounded those results (14). Last, we wished to determine whether the apical A1 receptor identified in the A6C1 subclone also existed in the A6S2 subclone and, if so, whether it mediated sodium transport in either or both subclones.
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METHODS |
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Cell culture. Experiments were performed on two subclones of A6 cells (A6S2 and A6C1) derived from the kidney of Xenopus laevis. A6S2 cells were maintained, as previously described (14), in media at 27°C in a humidified incubator gassed with 1% CO2 and were passaged at intervals of 7-8 days. The culture medium contained DMEM, glutamine (2 mM), 10% FBS (GIBCO-BRL, Grand Island, New York), and NaHCO3 (8 mM). The final composition of the media contained (mM) 94 Na, 94 Cl, 4 K, 8 HCO3, and 1.7 Ca, pH 7.6. Because we confirmed previous findings demonstrating that FBS can be removed from the media a number of days after subculture without altering basal function or hormone responsiveness (5, 36), FBS was used for only 6 days after seeding cell culture inserts. For transport studies, cells were seeded at a density of 105 cells/cm2 on Millicell-HA cell culture inserts (12-mm diameter, 0.45-µm pore size, 0.6-cm2 growth area; Millipore, Bedford, MA), and experiments were performed 10-14 days after seeding, when transmembrane resistance was maximal.
Regarding the A6C1 subclone, Prof. François Verrey kindly provided cells from the same stock used in the Casavola et al. study (7), along with detailed methods and a sample of FBS to conduct our experiments. It should be noted that those methods, as used in this study, are referenced to an earlier report from the same laboratory (35). Accordingly, A6C1 cells were grown in DMEM (diluted to 80% with deionized H2O) containing 25 mM NaHCO3, 10% heat-inactivated FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin (each obtained from GIBCO-BRL). The cells were maintained at 28°C in a humidified incubator gassed with 5% CO2 and were passaged every 7-10 days. For transport studies, they were seeded at a density of 105 cells/cm2 on Transwell cell culture inserts (6.5-mm diameter, 0.4-µm pore size, 0.33-cm2 growth area; Costar, Cambridge, MA) coated with Vitrogen 100 (Collagen, Palo Alto, CA) as described (35). The apical media was replaced the following day and 7 days after seeding both the apical and the basolateral media were replaced. Ten days after seeding the apical and basolateral media were replaced with DMEM (diluted to 80% with deionized H2O) without FBS and buffered with 20 mM HEPES (pH 7.4), and the cells were incubated at 28°C without CO2. The media was again changed 14 days after seeding and the day before an experiment. All experiments were done in HEPES-buffered media except for three individual time course studies. In those experiments the cells were not transferred into HEPES-buffered media on day 10 but were kept in bicarbonate media throughout. Their response to CHA was similar to those cells transferred into HEPES media (data not shown). All experiments were done, 15 or 16 days after seeding, under sterile conditions in a culture hood at room temperature. Cells were not treated with aldosterone at any time during culture or for experiments.Cell incubations. Adenosine agonists and antagonists were dissolved in DMSO. All DMSO solutions were added to the apical surface by exchanging 40% of the apical media with an equal volume of media containing the agonists or antagonists in concentrations that yielded the desired final concentration of each. For the time course studies with basolateral CHA solutions were added in the same manner, for rapid dissolution, to detect early changes in transport. In all experiments, the final concentration of DMSO did not exceed 1%. Cells were preincubated with antagonists for time periods indicated in the figure legends before addition of agonists. Controls had DMSO alone added in the same concentration as the experimental groups.
Measurement of ion transport.
The potential difference (VT) across confluent
monolayers was measured with Ag/AgCl2 electrodes. Current
was passed with a model DVC-1000 voltage/current clamp apparatus (WPI
Industries, Sarasota, FL) to estimate transepithelial resistance
(RT), which generally ranged between 6,000 and
10,000 · cm2 for both subclones. Equivalent
Isc (Ieq) was calculated
from the open-circuit measurements of VT and
RT, using Ohm's law, and represents the net
current associated with active ion flow when VT = 0 mV. Compared with the
Isc, this measure avoids alterations in ion flow
across the apical membrane due to hyperpolarization.
Reagents. AVP, insulin, and amiloride were obtained from Sigma (St. Louis, MO). CHA was obtained from Boehringer Mannheim (Indianapolis, IN), and CGS-21680, DPCPX, and CSC were purchased from RBI (Natick, MA).
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RESULTS |
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Determination of the effect of DMSO on A6S2 cells.
Many synthetic adenosine analogs are highly lipophilic and are often
dissolved in DMSO. Therefore, we tested the effect of DMSO on the
basolateral CHA-stimulated increase in Ieq by
exchanging 40% of the apical media with an equal volume of media
containing DMSO in concentrations that yielded the desired final
concentration of DMSO (1, 2, or 10%). We found that DMSO did not
significantly affect Ieq or
RT when added to A6S2 cells in concentrations
2%, as shown in Table 1.
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Examination of the apical membrane for receptors of agonists that
stimulate sodium transport in the A6S2 subclone.
Three surface binding agonists, vasopressin (15), insulin
(32), and adenosine (14), have been shown to
stimulate electrogenic sodium transport when added to the basolateral
surface of A6S2 cells. In another subclone (A6C1), the addition of an
adenosine agonist to the apical membrane induced an
amiloride-insensitive Isc (7). We
determined whether apical CHA, AVP, or insulin stimulated electrogenic
transport in the A6S2 subclone and found that only apical CHA
increased Ieq (Fig.
1).
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Studies to determine amiloride-insensitive current in the A6S2
subclone.
Initial studies demonstrated that Isc equaled
net sodium transport in A6 cells under basal and agonist-stimulated
conditions (28), and many other studies have not found an
amiloride-insensitive current when sodium transport is stimulated
(30, 31, 33, 36). In contrast, some laboratories have
demonstrated concurrent active chloride secretion and sodium
reabsorption when the basolateral surface of A6 cells is exposed to AVP
(8, 25, 34, 37, 38). To determine whether CHA induced an
amiloride-insensitive current in the A6S2 subclone, we examined the
effect of amiloride on the increase in Ieq when
CHA was added to either the basolateral or the apical cell
surfaces. Figure 2A shows that
after addition of 106 M CHA to the basolateral surface
the rise in Ieq was abolished by
10
4 M amiloride added to the apical surface. When
amiloride was applied before the addition of CHA to the basolateral
surface there was no subsequent increase in Ieq.
Similar results (Fig. 2B) were obtained when CHA was added
to the apical surface. Therefore, under our experimental conditions as
reported earlier from this laboratory (14), CHA-stimulated
Ieq reflects only electrogenic sodium transport
in A6S2 cells.
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Basolateral and apical dose response to CHA in the A6S2 subclone.
Based on adenylate cyclase activity, the A1
receptor has a higher affinity for adenosine
(1.5 · 108-10
7 M) than does
the A2 receptor
(5 · 10
7-2 · 10
5 M)
(26). In renal cells containing both types of receptors, such as the A6 cell, higher concentrations of the parent compound, adenosine, may result in a biological response that reflects the concurrent action of both activated receptors. Therefore, to link each
type of receptor to a specific biological response there is a clear
advantage in the use of highly selective synthetic agonists.
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Action of CGS-21680 on basolateral and apical cell surfaces of the
A6S2 subclone.
We also used the A2 agonist CGS-21680 to determine whether
the A2 receptor is involved in basolateral CHA-stimulated
sodium transport in the A6S2 subclone. Because IC50's for
the A1 and A2 receptors are in the range of
3 · 106 and 2 · 10
8 M,
respectively (17), CGS-21680 should stimulate sodium
transport at a lower threshold concentration than CHA if the
A2 receptor mediates this transport. However, the threshold
concentration of CGS-21680 applied to the basolateral surface was
>10
5 M (Fig.
5A), which is at least three
orders of magnitude higher than the value for CHA shown in Fig.
3A. In addition, the threshold concentration observed for
apical CGS-21680 (Fig. 5B), where A2 receptors
are reported to be absent in the A6 cell (7), was also in
the micromolar range, suggesting that the action of this agonist on
both cell surfaces reflected crossover activation of the A1
receptor. These data are consistent with CHA-stimulated sodium
transport via activation of A1 receptors on both the apical and the basolateral membranes.
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The effect of selective adenosine receptor antagonists on
CHA-stimulated sodium transport in the A6S2 subclone.
DPCPX is reported to be at least 500-fold more selective for the
A1 than the A2 receptor (9, 22),
so we tested its effect on CHA-stimulated Ieq.
Basolateral CHA-stimulated Ieq was inhibited ~50% by an equal concentration of DPCPX (108 M), but
there was complete inhibition at all higher concentrations of DPCPX
(Fig. 6A).
Fig. 6C shows that apical CHA-stimulated
Ieq was completely inhibited by all
concentrations of DPCPX. We therefore sought to determine whether
DPCPX, in addition to competitive inhibition at the site of the
A1 receptor, also inhibited sodium transport
nonspecifically. DPCPX and CHA were applied together to the same cell
surface and also to opposite cell surfaces. When DPCPX and CHA were
added in equimolar concentrations to opposite cell surfaces, both
basolateral and apical CHA-stimulated Ieq were
completely inhibited (Fig. 6, B and D),
indicating a nonspecific effect of the antagonist.
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Evaluation of CHA-stimulated ion transport in the A6C1 subclone.
This A6 cell subclone has been previously reported to exhibit both
chloride secretion and sodium reabsorption (7, 34). Figure
8A shows the response of A6C1
cells to apical and basolateral CHA with and without amiloride
pretreatment. In the absence of amiloride pretreatment, apical CHA
induced an immediate increase in Ieq to a
maximal level at ~1 min, followed by a rapid fall to a stable lower
level between 10 and 15 min, which persisted until 30 min when
amiloride abolished transport. The effect of basolateral CHA was almost
identical. Pretreatment with amiloride before adding CHA to either cell
surface reduced the peak level but not the early rise in
Ieq, which then fell within 10-15 min to a
level not statistically different from control cells. These findings
contrast with the Ieq profile for the A6S2
subclone, where neither apical nor basolateral CHA induced an
amiloride-insensitive current (Fig. 2, A and B).
It is unlikely that when CHA was added apically it diffused through the
monolayer to activate receptors on the basolateral membrane because
transport was stimulated immediately and there was no difference in the
peak effect compared with basolateral CHA (Fig. 8, A and
B).
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Action of CGS-21680 on basolateral and apical cell surfaces of the
A6C1 subclone.
As with the A6S2 subclone, we used the A2 agonist CGS-21680
to determine whether the basolateral A2 receptor is
involved in CHA-stimulated sodium transport in the A6C1 subclone.
Figure 9, A and B,
indicate that both basolateral and apical CGS-21680 stimulated sodium
transport at threshold concentrations that were micromolar. In
contrast, 107 M CHA stimulated sodium transport to a
greater or equal degree than the highest dose of CGS-21680 used
(10
4 M). With both agonists the stimulated current was
inhibited almost completely by 10
4 M amiloride,
indicating that both agonists stimulate sodium transport. These results
strongly suggest that stimulation of sodium reabsorption by CGS-21680
at micromolar concentrations is probably due to crossover binding to
the A1 receptor. These data also provide evidence that both
basolateral and apical CHA stimulate sodium transport via an
A1 receptor in the A6C1 subclone.
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DISCUSSION |
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An earlier report, from this laboratory, showed that basolateral CHA-stimulated sodium transport in the A6S2 subclone was mediated by a calcium-dependent mechanism linked to the activation of an A1 receptor and did not depend on activation of adenylate cyclase and increases in intracellular cAMP (14). In this study we sought to determine whether qualitatively different signaling mechanisms for sodium transport could exist in different subclones of the A6 cell (A6S2 and A6C1).
In the A6C1 subclone apical CPA was previously reported to stimulate chloride secretion (7). In our earlier study of the A6S2 subclone, we did not examine the apical surface for adenosine receptors nor did we determine whether CHA could induce anion secretion (14). In this study we found that apical CHA stimulates a sodium current in the A6S2 subclone, but neither apical nor basolateral CHA stimulated anion secretion. Therefore Ieq reflects net electrogenic sodium transport in the A6S2 subclone.
To then determine whether qualitatively different signaling mechanisms
for sodium transport exist in the A6S2 and A6C1 subclones, we directly
compared them using the same cell culture and cell-handling methods
previously employed for each subclone (7, 14, 35). We also
used highly selective A1 and A2 receptor
agonists to correlate their effect on sodium transport with their
affinity for each receptor. To avoid crossover binding to the
A2 receptor we used CHA because it does not stimulate
adenylate cyclase in cultured rabbit CCT cells (3) nor in
A6S2 cells (14) at concentrations 10
6
M. Conversely, we used CGS-21680 because its affinity for the A2 receptor is also in the low nanomolar range, whereas the
IC50 for the A1 receptor is
3 · 10
6 M (17).
In the A6S2 subclone, basolateral CHA stimulated sodium transport at a
threshold of 109-10
8 M, the
approximate binding affinity of the A1 receptor
(10). In contrast, the highly selective A2
receptor agonist CGS-21680 did not stimulate sodium transport at a
concentration
10
5 M. These results clearly demonstrate
that the A1 receptor mediates basolateral CHA-stimulated
Ieq.
In the A6C1 subclone, basolateral CHA also stimulated sodium transport at a nanomolar concentration, whereas the threshold concentration for CGS-21680 was at least two orders of magnitude higher than for CHA. Taken together, these results indicate that basolateral CHA stimulates sodium transport by activation of the A1 receptor in both the A6S2 and the A6C1 subclones.
We also used A1 and A2 adenosine
receptor-specific antagonists to further elucidate which receptor
mediates the CHA-stimulated increase in Ieq.
DPCPX (108 M), an A1 receptor antagonist,
inhibited CHA-stimulated Ieq by ~50% at a
concentration equal to the concentration of basolateral CHA
(10
8 M), suggesting competition with CHA at the
A1 receptor, but at higher concentrations of DPCPX, there
was complete inhibition. In similar experiments with apical CHA, all
concentrations of DPCPX used completely inhibited CHA-stimulated
Ieq. This lack of a dose response suggested that
the inhibition of CHA-stimulated Ieq by DPCPX
may not be solely due to competitive binding to the A1
receptor. DPCPX was therefore applied to the opposite cell surface to
which CHA was added, in an equimolar concentration, and both
basolateral and apical CHA-stimulated Ieq were
completely inhibited. Together, these results suggest that DPCPX may
act concurrently by competitive and nonspecific mechanisms, making any
interpretation of its antagonistic action against CHA ambiguous. Therefore, it was not further used to determine the role of the A1 receptor in the A6S2 subclone.
When CSC, an A2 receptor antagonist, was applied to the
basolateral cell surface, CHA-stimulated Ieq was
not inhibited compared with control. Because the affinity of the
A2 receptor for CHA (3 · 105 M)
(10) is much lower than for CSC (Ki = 5.4 · 10
8 M) (19), it can be
expected that stimulation of sodium transport by CHA (10
8
M) would have been inhibited by the 10-fold higher concentration of CSC
(10
7 M) if CHA-stimulated sodium transport was mediated
by the A2 receptor. Therefore, the absence of inhibition by
CSC is consistent with the notion that CHA-stimulated transport was not
mediated by the A2 receptor.
It has been previously reported that basolateral vasopressin (34) and apical CPA (7) stimulate chloride secretion in the A6C1 subclone. In the present study, we found that CHA stimulated an amiloride-insensitive current when applied to either side of the A6C1 subclone, in contrast to the A6S2 subclone where anion secretion was not observed in either condition. In addition, however, we found that apical CHA also stimulated sodium transport in the A6C1 subclone. Using the same strategy employed to examine the mechanism responsible for basolateral CHA-stimulated sodium transport, the results showed that apical CHA stimulated both sodium transport, via an A1 receptor, and anion secretion, apparently by the same receptor mechanism. As in the experiments designed to examine the effect of basolateral CHA, we found that apical CHA stimulated sodium transport at a concentration at least two orders of magnitude less than the threshold concentration of CGS-21680.
Regarding apical CHA stimulation of sodium transport, independent
approaches have not detected adenylate cyclase in the apical membrane
of the A6 cell. In one study a high concentration of apical CPA
(105 M) did not increase intracellular cAMP, whereas a
much lower concentration of basolateral CPA (10
7 M) did
increase cAMP (7). In another study adenylate cyclase was
not detected in the apical membrane using a cytochemical method (12). Because apical CHA stimulated sodium transport in
both the A6S2 and the A6C1 subclones, the absence of adenylate cyclase and, therefore, the A2 receptor activity in the apical cell
membrane further supports the notion that apical CHA must have
stimulated sodium transport via an A1 receptor. Moreover,
the absence of an apical A2 receptor activity provides
insight into the mechanism by which apical CGS-21680 stimulates sodium
transport in both subclones. Because the threshold concentration of
apical CGS-21680 was in the range of
10
5-10
4 M, approximating its affinity
for the A1 receptor, and the only apparent adenosine
receptor activity in the apical membrane is the A1
receptor, it seems highly likely that activation of the A1
receptor mediated the action of CGS-21680 to stimulate sodium transport. Therefore, it is also probable that basolateral CGS-21680, which stimulated sodium transport between 10
5 and
10
4 M, had a similar mechanism of action.
Adenosine receptors mostly exist as a single subtype in a wide variety of cells (18). In epithelial cells, in contrast, there is evidence that A1 and A2 receptors can exist together in the same cell and/or in different spatial relationships. Examples include both A1 and A2 receptors in rabbit cortical collecting tubule cells (RCCT) (3), T84 human colonic cells (4), the LLC-PK1 renal cell line (23), and cultured rabbit medullary thick ascending limb cells (6). Although in most cases the receptor subtype was identified from the signal transduction pathway without a functional role being determined, some studies have linked function with specific receptors. Some examples include a basolateral A1 receptor in rabbit inner medullary collecting duct cells that inhibits the action of vasopressin (11), an apical A1 receptor that stimulates Ca2+ absorption in cultured rabbit cortical collecting system cells (16), and adenosine stimulation of the Na-K-Cl cotransporter in A6 cells from ATCC (13).
The widespread distribution of adenosine in renal tissue, including the thick ascending limb and distal nephron, suggests that adenosine may play an important role in the regulation of renal function. Of note, the CHA-induced increase in intracellular Ca2+, reported in A6S2 cells from this laboratory (14), is not unique to amphibian renal epithelial cells. Arend et al. (3) demonstrated the presence of both A1 and A2 receptors in primary cultures of RCCT cells and showed that analogs of adenosine stimulated increases in intracellular calcium (1) and phosphoinositide turnover (2), which were blocked by pertussis toxin. These findings, and the results obtained with the A6 cell subclones, suggest that electrogenic sodium transport in the mammalian kidney may be controlled by adenosine locally produced in the kidney acting as an autacoid. For example, because adenosine is derived from ATP it could augment the action of aldosterone to stimulate sodium transport when turnover of Na-K-ATPase, ATP consumption, and production of adenosine rise in parallel with sodium entry across the apical membrane. Adenosine diffuses out of the cell and could bind to local adenosine receptors to further increase apical membrane permeability for sodium. In addition, there is ample evidence that nucleotides released from nerve endings can be converted to adenosine by ecto-5'-nucleotidase present at extracellular sites along the collecting duct (21).
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ACKNOWLEDGEMENTS |
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This work was supported by a Veterans Administration Merit Award and by a grant from the American Diabetes Association.
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FOOTNOTES |
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Address for reprint requests and other correspondence: J. P. Hayslett, Section of Nephrology, Dept. of Internal Medicine, Yale Univ. School of Medicine, PO Box 208029, New Haven, CT 06520-8029 (E-mail: john.hayslett{at}yale.edu).
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.
August 6, 2002;10.1152/ajprenal.00085.2002
Received 4 March 2002; accepted in final form 23 July 2002.
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REFERENCES |
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![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Arend, LJ,
Burnatowska-Hledin MA,
and
Spielman WS.
Adenosine receptor-mediated calcium mobilization in cortical collecting tubule cells.
Am J Physiol Cell Physiol
255:
C581-C588,
1988
2.
Arend, LJ,
Handler JS,
Rhim JS,
Gusovsky F,
and
Spielman WS.
Adenosine-sensitive phosphoinositide turnover in a newly established renal cell line.
Am J Physiol Renal Fluid Electrolyte Physiol
256:
F1067-F1074,
1989
3.
Arend, LJ,
Sonnenburg WK,
Smith WL,
and
Spielman WS.
A1 and A2 adenosine receptors in rabbit cortical collecting tubule cells. Modulation of hormone-stimulated cAMP.
J Clin Invest
79:
710-714,
1987[ISI][Medline].
4.
Barrett, KE,
Huott PA,
Shah SS,
Dharmsathaphorn K,
and
Wasserman SI.
Differing effects of apical and basolateral adenosine on colonic epithelial cell line T84.
Am J Physiol Cell Physiol
256:
C197-C203,
1989
5.
Bindels, RJ,
Schafer JA,
and
Reif MC.
Stimulation of sodium transport by aldosterone and arginine vasotocin in A6 cells.
Biochim Biophys Acta
972:
320-330,
1988[ISI][Medline].
6.
Burnatowska-Hledin, MA,
and
Spielman WS.
Effects of adenosine on cAMP production and cytosolic Ca2+ in cultured rabbit medullary thick limb cells.
Am J Physiol Cell Physiol
260:
C143-C150,
1991
7.
Casavola, V,
Guerra L,
Reshkin SJ,
Jacobson KA,
Verrey F,
and
Murer H.
Effect of adenosine on Na+ and Cl currents in A6 monolayers. Receptor localization and messenger involvement.
J Membr Biol
151:
237-245,
1996[ISI][Medline].
8.
Chalfant, ML,
Coupaye-Gerard B,
and
Kleyman TR.
Distinct regulation of Na+ reabsorption and Cl secretion by arginine vasopressin in the amphibian cell line A6.
Am J Physiol Cell Physiol
264:
C1480-C1488,
1993
9.
Coates, J,
Sheehan MJ,
and
Strong P.
1,3-Dipropyl-8-cyclopentyl xanthine (DPCPX): a useful tool for pharmacologists and physiologists?
Gen Pharmacol
25:
387-394,
1994[Medline].
10.
Daly, JW.
Adenosine receptors: targets for future drugs.
J Med Chem
25:
197-207,
1982[ISI][Medline].
11.
Edwards, RM,
and
Spielman WS.
Adenosine A1 receptor-mediated inhibition of vasopressin action in inner medullary collecting duct.
Am J Physiol Renal Fluid Electrolyte Physiol
266:
F791-F796,
1994
12.
Els, WJ,
and
Butterworth MB.
Cytochemical localization of adenylate cyclase in cultured renal epithelial (A6) cells.
Microsc Res Tech
40:
455-462,
1998[ISI][Medline].
13.
Fan, PY,
Haas M,
and
Middleton JP.
Identification of a regulated Na/K/Cl cotransport system in a distal nephron cell line.
Biochim Biophys Acta
1111:
75-80,
1992[ISI][Medline].
14.
Hayslett, JP,
Macala LJ,
Smallwood JI,
Kalghatgi L,
Gasalla-Herraiz J,
and
Isales C.
Adenosine stimulation of Na+ transport is mediated by an A1 receptor and a [Ca2+]i-dependent mechanism.
Kidney Int
47:
1576-1584,
1995[ISI][Medline].
15.
Hayslett, JP,
Macala LJ,
Smallwood JI,
Kalghatgi L,
Gassala-Herraiz J,
and
Isales C.
Vasopressin-stimulated electrogenic sodium transport in A6 cells is linked to a Ca(2+)-mobilizing signal mechanism.
J Biol Chem
270:
16082-16088,
1995
16.
Hoenderop, JG,
Hartog A,
Willems PH,
and
Bindels RJ.
Adenosine-stimulated Ca2+ reabsorption is mediated by apical A1 receptors in rabbit cortical collecting system.
Am J Physiol Renal Physiol
274:
F736-F743,
1998
17.
Hutchison, AJ,
Webb RL,
Oei HH,
Ghai GR,
Zimmerman MB,
and
Williams M.
CGS 21680C, an A2 selective adenosine receptor agonist with preferential hypotensive activity.
J Pharmacol Exp Ther
251:
47-55,
1989[Abstract].
18.
Jacobson, KA.
Chemical approaches to the definition of adenosine receptors.
In: Adenosine Receptors, edited by Cooper DMF,
and Londos C.. New York: Alan R. Liss, 1988, p. 43-62.
19.
Jacobson, KA,
Gallo-Rodriguez C,
Melman N,
Fischer B,
Maillard M,
van Bergen A,
van Galen PJ,
and
Karton Y.
Structure-activity relationships of 8-styrylxanthines as A2-selective adenosine antagonists.
J Med Chem
36:
1333-1342,
1993[ISI][Medline].
20.
Lang, MA,
Preston AS,
Handler JS,
and
Forrest JN, Jr.
Adenosine stimulates sodium transport in kidney A6 epithelia in culture.
Am J Physiol Cell Physiol
249:
C330-C336,
1985[Abstract].
21.
Le Hir, M,
and
Kaissling B.
Distribution and regulation of renal ecto-5'-nucleotidase: implications for physiological functions of adenosine.
Am J Physiol Renal Fluid Electrolyte Physiol
264:
F377-F387,
1993
22.
Lee, KS,
and
Reddington M.
1,3-Dipropyl-8-cyclopentylxanthine (DPCPX) inhibition of [3H]N- ethylcarboxamidoadenosine (NECA) binding allows the visualization of putative non-A1 adenosine receptors.
Brain Res
368:
394-398,
1986[ISI][Medline].
23.
LeVier, DG,
McCoy DE,
and
Spielman WS.
Functional localization of adenosine receptor-mediated pathways in the LLC-PK1 renal cell line.
Am J Physiol Cell Physiol
263:
C729-C735,
1992
24.
Londos, C,
Cooper DM,
and
Wolff J.
Subclasses of external adenosine receptors.
Proc Natl Acad Sci USA
77:
2551-2554,
1980[Abstract].
25.
Marunaka, Y,
and
Tohda H.
Effects of vasopressin on single Cl channels in the apical membrane of distal nephron cells (A6).
Biochim Biophys Acta
1153:
105-110,
1993[ISI][Medline].
26.
Olah, ME,
and
Stiles GL.
Adenosine receptors.
Annu Rev Physiol
54:
211-225,
1992[ISI][Medline].
27.
Palmer, LG,
and
Frindt G.
Amiloride-sensitive Na channels from the apical membrane of the rat cortical collecting tubule.
Proc Natl Acad Sci USA
83:
2767-2770,
1986[Abstract].
28.
Perkins, FM,
and
Handler JS.
Transport properties of toad kidney epithelia in culture.
Am J Physiol Cell Physiol
241:
C154-C159,
1981[Abstract].
29.
Puoti, A,
May A,
Canessa CM,
Horisberger JD,
Schild L,
and
Rossier BC.
The highly selective low-conductance epithelial Na channel of Xenopus laevis A6 kidney cells.
Am J Physiol Cell Physiol
269:
C188-C197,
1995
30.
Record, RD,
Johnson M,
Lee S,
and
Blazer-Yost BL.
Aldosterone and insulin stimulate amiloride-sensitive sodium transport in A6 cells by additive mechanisms.
Am J Physiol Cell Physiol
271:
C1079-C1084,
1996
31.
Rehn, M,
Weber WM,
and
Clauss W.
Role of the cytoskeleton in stimulation of Na+ channels in A6 cells by changes in osmolality.
Pflügers Arch
436:
270-279,
1998[ISI][Medline].
32.
Rodriguez-Commes, J,
Isales C,
Kalghati L,
GasallaHerraiz J,
and
Hayslett JP.
Mechanism of insulin-stimulated electrogenic sodium transport.
Kidney Int
46:
666-674,
1994[ISI][Medline].
33.
Sariban-Sohraby, S,
Burg MB,
and
Turner RJ.
Apical sodium uptake in toad kidney epithelial cell line A6.
Am J Physiol Cell Physiol
245:
C167-C171,
1983[Abstract].
34.
Verrey, F.
Antidiuretic hormone action in A6 cells: effect on apical Cl and Na conductances and synergism with aldosterone for NaCl reabsorption.
J Membr Biol
138:
65-76,
1994[ISI][Medline].
35.
Verrey, F,
Digicaylioglu M,
and
Bolliger U.
Polarized membrane movements in A6 kidney cells are regulated by aldosterone and vasopressin/vasotocin.
J Membr Biol
133:
213-226,
1993[ISI][Medline].
36.
Wills, NK,
and
Millinoff LP.
Amiloride-sensitive Na+ transport across cultured renal (A6) epithelium: evidence for large currents and high Na:K selectivity.
Pflügers Arch
416:
481-492,
1990[ISI][Medline].
37.
Yanase, M,
and
Handler JS.
Adenosine 3',5'-cyclic monophosphate stimulates chloride secretion in A6 epithelia.
Am J Physiol Cell Physiol
251:
C810-C814,
1986
38.
Zeiske, W,
Atia F,
and
Van Driessche W.
Apical Cl channels in A6 cells.
J Membr Biol
166:
169-178,
1998[ISI][Medline].