Basolateral and apical A1 adenosine receptors mediate sodium transport in cultured renal epithelial (A6) cells

Lawrence J. Macala and John P. Hayslett

Section of Nephrology, Department of Internal Medicine, Yale University School of Medicine, New Haven, Connecticut 06520-8029


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
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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 <10-7 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


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 · 10-8 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 · 10-5 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; 10-7 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.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
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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 Omega  · 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.

Statistical analyses are described in the figure legends.

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).


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
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REFERENCES

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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Table 1.   Concentration-dependent effect of DMSO on A6S2 cells

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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Fig. 1.   Response of the A6S2 subclone to the apical and basal addition of agonists that stimulate sodium transport. Equivalent short-circuit current (Ieq) was measured before (t = 0) and 30 min after addition of the agonists to the apical or basal surfaces. Agonist concentrations were 10-7 M N6-cyclohexyladenosine (CHA), 10-7 M insulin (INS), and 10-6 M AVP. Values are the mean Ieq ± SE (n = 3). CONT, control.

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 10-6 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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Fig. 2.   Determination whether CHA stimulates an amiloride-insensitive current in the A6S2 subclone. A: basal CHA time course. CHA (10-6 M) or DMSO was added to the basal surface at 8 min (open arrow). Amiloride (10-4 M) was added to the apical cell membrane 3 min before adding CHA (open circle ) or DMSO (triangle ) or 30 min after adding CHA () or DMSO (black-triangle). B: apical CHA time course. CHA (10-6 M) or DMSO was added to the apical surface at 8 min (open arrow). Amiloride (10-4 M) was added to the apical cell membrane 3 min before adding CHA (open circle ) or DMSO (triangle ) or 30 min after adding CHA () or DMSO (black-triangle). For clarity, symbols are not shown for all data points. Error bars are plotted for each data point but are not evident when the SE is smaller than the graph symbol or line. Values are the mean Ieq ± SE (n = 4 for CHA and n = 3 for DMSO).

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 · 10-8-10-7 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.

Figure 3A shows that basolateral CHA, with an A1 affinity constant in the low nanomolar range and an A2 affinity constant of 3 · 10-5 M (10), dose-dependently stimulated an amiloride-sensitive current. The threshold concentration between 10-9 and 10-8 M is consistent with A1 receptor activation.


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Fig. 3.   Dose-response curves for CHA added to the apical or basal cell surfaces of the A6S2 subclone. Ieq was measured before (t = 0) and 10 and 30 min after adding CHA (in the concentrations shown) or DMSO (CONT) to the basal (A) or apical (B) surfaces and after adding 10-4 M apical amiloride at 30 min. Values are the mean Ieq ± SE (n = 4).

Figure 3B shows that apical CHA also dose dependently stimulated an amiloride-sensitive current. The threshold concentration between 10-8 and 10-7 M, although slightly higher compared with basolateral CHA, is also consistent with activation of an A1 receptor. The rapid onset of the increase in sodium current, shown in Fig. 2B, indicates that it is unlikely that apical CHA stimulated basolateral receptors after diffusing through the monolayer. In neither the basolateral nor the apical response to CHA was the threshold concentration near the level required for activation of the A2 receptor. As in Fig. 2, A and B, Fig. 3, A and B, also demonstrates that 30 min after adding CHA, there was no evidence of an amiloride-insensitive current. Together, these data show that basolateral and apical adenosine stimulate sodium transport via an A1 receptor in the A6S2 subclone.

Furthermore, the dose-dependent amiloride inhibition (Fig. 4A) strongly implies that CHA-stimulated sodium transport is mediated by the same sodium channel when applied to either cell surface, because the concentrations that produce half-maximal inhibition are nearly identical (3 · 10-7 M). This inhibition is comparable to values observed in oocytes transfected with the cDNA for each of the subunits of the epithelial sodium channel (29), the native apical membrane of rat cortical collecting tubule (CCT) (27), and the A6 cell line from ATCC (33).


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Fig. 4.   Dose-dependent amiloride inhibition of Ieq induced by apical or basal CHA. A: A6S2 subclone; n = 4 for basal CHA, n = 3 for apical CHA. B: A6C1 subclone; n = 4 for both basal and apical CHA. CHA (10-6 M) was added to the basal () or apical (open circle ) surface, and Ieq was measured after 30 min. Amiloride was then sequentially added to the apical side in increasing amounts to give the indicated final concentrations, and Ieq was measured several minutes after each addition. Values are the mean Ieq ± SE and are reported as a fraction of the Ieq at 30 min, before the addition of amiloride.

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 · 10-6 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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Fig. 5.   Dose-response curves for CGS-21680 added to the apical or basal cell surfaces of the A6S2 subclone. CGS-21680 was added to the basal (A) or apical (B) surface. Ieq was measured before (t = 0) and 30 min after adding CGS-21680 (in the concentrations shown) or DMSO (CONT). Values are the mean Ieq ± SE; n = 3 or 4 for A and n = 4 for B. *P < 0.05, significantly different from the control as determined by Dunnett's test.

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 (10-8 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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Fig. 6.   Inhibition of CHA-stimulated Ieq by the A1 antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) in the A6S2 subclone. A: both CHA (10-8 M) and DPCPX, in various concentrations, were added to the basal surface. Ieq was measured before (t = 0) and 30 min after adding CHA (or DMSO where CHA was not added). B: CHA (5 · 10-8 M) was added to the basal surface and DPCPX (5 · 10-8 M) was added to either the apical or the basal surface. Ieq was measured before (t = 0) and 10 min after adding CHA (or DMSO where CHA was not added). C: both CHA (10-7 M) and DPCPX, in various concentrations, were added to the apical surface. Ieq was measured before (t = 0) and 30 min after adding CHA (or DMSO where CHA was not added). D: CHA (5 · 10-8 M) was added to the apical surface and DPCPX (5 · 10-8 M) to either the apical or the basal surface. Ieq was measured before (t = 0) and 10 min after adding CHA (or DMSO where CHA was not added). CHA and DPCPX were added simultaneously (except in A, where DPCPX was added 10 min before CHA). Values are the mean Ieq ± SE (n = 4).

CSC is reported to have a 500-fold greater selectivity for the A2 compared with the A1 receptor (19). When applied to the basolateral cell surface in a concentration 10-fold higher than that of CHA, sodium transport was not inhibited compared with control, implying that the action of basolateral CHA was not dependent on activation of the A2 receptor (Fig. 7). As expected, the increase in Ieq with basolateral CHA was not reduced when CSC was added to the apical cell surface.


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Fig. 7.   Effect of the A2 antagonist CSC on basal CHA-stimulated Ieq in the A6S2 subclone. CHA (10-8 M) was added to the basal surface and CSC (10-7 M) was added simultaneously to either the apical or the basal surface. Ieq was measured before (t = 0) and 30 min after adding CHA (or DMSO where CHA was not added). Values are the mean Ieq ± SE (n = 4).

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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Fig. 8.   Effect of basal and apical CHA on ion transport in the A6C1 subclone. A: total and amiloride-insensitive Ieq; total Ieq stimulated by 10-6 M basal () or apical () CHA. CHA was applied at 0 min and amiloride (10-4 M) was applied to the apical surface 30 min after CHA. Shown is amiloride-insensitive Ieq stimulated by 10-6 M basal () or apical (open circle ) CHA. Amiloride (10-4 M) was applied to the apical surface 3 min before CHA was added at 0 min. Values, at each time point, are the means for CHA-stimulated Ieq (n = 3 or 4) minus control (DMSO) Ieq (n = 3). *P > 0.05, not significantly different from respective control means as determined by the unpaired t-test (one-tailed). B: amiloride-sensitive Ieq derived from the experiment shown in A. The component of the total basal () and apical () CHA-stimulated current that represents sodium transport, which was obtained by subtracting, at each time point in A, the Ieq for amiloride-pretreated cells from the Ieq observed without pretreatment.

The component of the total CHA-stimulated current that represents sodium transport is shown in Fig. 8B. These results demonstrate that both basolateral and apical CHA stimulate sodium transport in the A6C1 subclone. A low level of unstimulated and agonist-stimulated sodium current for this subclone (without aldosterone pretreatment) was shown previously (34, 35) and contrasts with the more robust response of the A6S2 subclone. In addition, the dose-dependent amiloride inhibition, shown in Fig. 4B, is similar to that found in the A6S2 subclone. These data together show that the A1 receptor-specific agonist CHA stimulates both a transient amiloride-insensitive current (presumably chloride) and sustained sodium transport when added to either cell surface of the A6C1 subclone.

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, 10-7 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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Fig. 9.   Dose-response curves for CGS-21680 added to the apical or basal cell surfaces of the A6C1 subclone compared with 10-7 M CHA. Ieq was measured before (t = 0) and 30 min after adding the agonist (CGS-21680 or CHA, in the concentrations shown), DMSO, or MEDIA to the basal (A) or apical (B) surfaces and after adding 10-4 M apical amiloride at 30 min. Values are the mean Ieq ± SE (n = 3). *P < 0.05, significantly different from the control as determined by Dunnett's test.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 10-9-10-8 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 (10-8 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 · 10-5 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 (10-5 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).


    ACKNOWLEDGEMENTS

This work was supported by a Veterans Administration Merit Award and by a grant from the American Diabetes Association.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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