Introduction

Summary Introduction Procedures Results Discussion References

Adenosine, acting as a local hormone, regulates a number of renal functions, including renal hemodynamics, renin release, and ion transport, via several types of cell surface receptors that have been differentiated on the basis of their affinity for various adenosine analogues and their effect on adenylate cyclase (1, 2). The A₁ receptor has a high affinity for adenosine and inhibits adenylate cyclase while stimulating phosphoinositide turnover and mobilizing intracellular calcium; the A₂ receptor has a lower affinity for adenosine and stimulates adenylate cyclase (3). A₂ receptors are further divided into A_2A (high affinity for agonists) and A_2B (lower affinity) subtypes (4). Both A₁ and A₂ receptors are widely distributed throughout the nephrons as well as in several renal cell lines derived from different nephron segments (5-10). The ability of adenosine to couple to different effector systems is believed to account for its pleiotropic actions in renal cells. We recently demonstrated that A₆ cells, a cell line derived from the kidney of Xenopus laevis (11) that is commonly used as a model of the mammalian collecting duct, contain both A₁ and A₂ receptors (12). The A₁ receptors are located on the apical surface and regulate apical Cl secretion via intracellular calcium, whereas A₂ receptors are located on the basolateral surface and stimulate Na⁺ transport via an increase in cAMP intracellular levels. The expression of both adenosine receptors capable of regulating different ion transports on the same cell suggests a dual-control nature of adenosine as a regulator of kidney cell function.

Alterations in pH_i have been demonstrated to modulate the action of different hormones on apical Na⁺ conductive transport in tight epithelia (13-16). Na⁺/H⁺ exchange is a major determinant of pH_i and may be regulated by various hormones; it is mainly regulated by the phospholipase C/PKC and adenylate cyclase/PKA signaling pathways (17). We previously demonstrated that A₆-2F3 cell monolayers contain a basolaterally located Na⁺/H⁺ exchanger that is presumed to be involved primarily with cellular pH regulation (18). The current study was performed to determine whether changes in pH_i via regulation Na⁺/H⁺ exchange activity act as an intermediate in the second-messenger cascade initiated by adenosine and ending in a modification of apical Na⁺ conductance. The availability of potent selective adenosine A₁ and A₂ receptor antagonists and agonists provides the pharmacological probes by which the role of A₁ and A₂ receptors can be investigated in polarized A₆ cell monolayers. Our results indicate that adenosine most likely stimulates Na⁺/H⁺ exchange activity by increasing intracellular cAMP via activation of basolateral A₂ receptors. This intracellular alkalinization may play a permissive role in inducing the observed increase in transepithelial Na⁺ transport induced by basolateral adenosine.

	Experimental Procedures

Summary Introduction Procedures Results Discussion References

Cell culture. Experiments were performed with A₆ cells from the A₆-C1 subclone (passage 114-128). This subclone was obtained by ring-cloning of A₆-2F3 cells at passage 99 and was selected for its high transepithelial resistance and responsiveness to aldosterone and antidiuretic hormone (19).

Fluorescence measurements. pH_i was measured using the pH-sensitive fluorescent dye BCECF. Cells on permeable supports were loaded with the acetoxymethyl ester derivative of BCECF (10 µM) for 60 min at room temperature in sodium medium. To avoid dye leakage, BCECF loading was carried out in the presence of 50 µM probenecid. Coverslips with confluent monolayers were inserted at an angle of 60° in a fluorometer cuvette designed for independent perfusion of the apical and basolateral cell surfaces as previously described (20).

Adenosine agonists and antagonists. To distinguish between the involvement of the putative adenosine receptor subtypes in adenosine regulation of Na⁺/H⁺ exchange activity and cAMP generation in A₆ cells, we used various adenosine agonists and antagonists with the following K_i values reported in different mammalian tissues (for a review, see Ref. 4): CPA, A₁ = 0.6 nM versus A_2A = 460 nM; DPMA, A₁ = 140 nM versus A_2A = 4.4 nM, CPX, A₁ = 0.9 nM versus A_2A = 470 nM versus A_2B = 360 nM; and CSC, A₁ = 28,000 nM versus A_2A = 54 nM versus A_2B = 8,200 nM.

Composition of perfusion fluids. All pH_i measurements were performed at room temperature in HEPES-buffered media. Sodium medium contained 110 mM NaCl, 3 mM KCl, 1 mM CaCl₂, 0.5 mM MgSO₄, 1 mM KH₂PO₄, 5 mM glucose, and 10 mM HEPES buffered with Tris to final pH 7.5. For cellular acidification, we used an NH₄Cl solution identical to Na⁺ medium plus 20 mM NH₄Cl. A sodium-free (TMA medium) was obtained by complete replacement of sodium with TMA.

cAMP determination. Intracellular cAMP levels were analyzed as previously reported (12, 22, 23). Cell monolayers grown on filter inserts were placed in the A₆ Ringer's solution described above and exposed to hormones for 15 min in the presence of 1 mM rolipram, a phosphodiesterase inhibitor that is not an adenosine receptor antagonist. When used, the adenosine antagonists were added 5 min before the addition of adenosine. The monolayers were rapidly rinsed twice with ice-cold assay buffer (50 mM Tris·HCl, 16 mM 2-mercaptoethanol, 8 mM theophylline, pH 7.4) and immediately immersed in liquid nitrogen. The filter apparatus was stored at 20° until assayed. For assay, the filters were cut out of the filter apparatus while still frozen and immersed in 100 µl of the above assay buffer plus 10 µl of 0.1 M HCl in an Eppendorff tube. Cells were disrupted by two 5-sec pulses with a probe sonicator (Branson, Zurich, Switzerland), the sample was neutralized with 10 µl of 0.1 N NaOH, and the filter and cell debris were removed by centrifugation at 14,000 rpm for 15 sec in an Eppendorff centrifuge. The cAMP concentration was determined of a 50-µl aliquot of the supernatant using the test kit from DuPont-New England Nuclear (Boston, MA) based on a competitive protein-binding assay (24).

Measurements of transepithelial I_sc Measurements of transepithelial potential difference (mV) and I_sc (µA/cm²) were performed in a modified chamber according to published methods (12). Transepithelial resistance (1/2 × cm²) was calculated according to Ohm's law; the electrical parameters were measured in Na⁺ medium used for pH_i measurements at room temperature.

Materials. BCECF-AM was purchased from Molecular Probes (Eugene, OR). HOE-694 was kindly provided by Dr. H. J. Lang (Hoerchst AG, Frankfurt, Germany). H-89 was purchased from BIOMOL Research Laboratories (Plymouth Meeting, PA) and calphostin C from Calbiochem (San Diego, CA)and activated according to the manufacturer's instructions. All other substances were obtained from Sigma Chemical (St. Louis, MO).

Data analysis and statistics. Data are expressed as mean ± standard error. Statistical comparisons were made using the paired and unpaired Student's t tests, and p < 0.05 indicated a statistical difference.

	Results

Summary Introduction Procedures Results Discussion References

Electrical parameters. The effect of adenosine was studied in confluent A₆ monolayers grown onto porous filters, which allows selective access to the basolateral and apical sides of the epithelial cell. After 10-14 days of growth, A₆ monolayers displayed a high transepithelial resistance (9850 ± 467 1/2 × cm², 40 experiments) and substantial I_sc (5-10 µA/cm²). These parameters were always controlled before Na⁺/H⁺ exchange activity was measured to ensure that the monolayers examined were "tight."

Effect of CPA on Na⁺/H⁺ exchange activity. In a previous study, we found that A₆ cells recover from an acid load by activation of a Na⁺/H⁺ exchanger located exclusively on the basolateral side that was highly sensitive to the addition of 5-(N-ethyl-N-isopropyl)-amiloride (18). To study the effect of adenosine on Na⁺/H⁺ exchange activity of the A₆ cell monolayers, we used CPA, a metabolically stable adenosine analogue poorly taken up by cells that binds both A₁ and A_2A adenosine receptor subtypes but has a higher affinity for the A₁ receptor (25).

6

6

View larger version (21K): [in this window] [in a new window]   Fig. 1.   Representative traces depicting the effect of 106 M CPA on Na+/H+ exchange activity in A6 monolayers. Na+/H+ exchange activity was assayed as the initial rate of Na+-dependent pHi recovery induced by the basolateral addition of 22 mM Na+ after intracellular acidification via superfusion with NH4+ medium followed by Na+-free TMA medium (see Experimental Procedures). First trace, absence of CPA. Second trace, obtained in the same monolayer after a 20-min preincubation with CPA. A, CPA was added only to the apical side. B, CPA application was only to the basolateral side of the monolayer. pHi corresponds to the nigericin calibration performed as described in Experimental Procedures.

View larger version (39K): [in this window] [in a new window]   Fig. 2.   Effect of apical or basolateral CPA addition on the Na+/H+ exchange activity. Experiments were performed according to the protocol described in the legend to Fig. 1. Data are presented as percentage of control values. Columns, mean ± standard error of five experiments. ***, p < 0.001.

8

6

9

6

View larger version (38K): [in this window] [in a new window]   Fig. 3.   Effect of increasing concentration of CPA on Na+/H+ exchange activity. Experiments were performed at increasing concentrations of CPA according to the protocol described in the legend to Fig. 1. The basolateral side of A6 monolayers was exposed to CPA for 20 min before analysis of pHi recovery from an acid load. Columns, mean ± standard error of the experiments made at different CPA concentrations (106 M, seven experiments; 107 M, three experiments; 108 M, three experiments; 109 M, four experiments). **, p < 0.01; ***, p < 0.001.

6

View larger version (15K): [in this window] [in a new window]   Fig. 4.   Effect of various Na concentrations on CPA-dependent stimulation of Na+/H+ exchange. Experiments were performed according to the protocol described in the legend to Fig. 1 at different sodium concentrations (11 mM, seven experiments; 22 mM, five experiments; 37 mM, three experiments; 55 mM, seven experiments; 110 mM, six experiments) in the absence () or presence () of 106 M CPA. Na+ activation of basolateral Na+/H+ exchange followed simple Michaelis-Menten kinetics. The apparent Km values were 40 ± 5.6 mM Na+ in the absence and 12 ± 3.9 mM Na+ in the presence of CPA .

Effects of A₁ and A₂ antagonists on CPA action. To determine whether the stimulatory effect of basolateral CPA occurred via a receptor-mediated mechanism, we examined the effect on CPA action of either the A₁-selective receptor antagonist CPX (4, 25) or the A_2A-selective antagonist CSC (4, 26). Neither 10⁷ M CPX nor 10⁷ M CSC added alone to the basolateral side of the monolayers altered the Na⁺/H⁺ exchange rate (data not shown). The basolateral CPA-dependent stimulation of exchange activity was completely prevented by CSC preincubation, whereas it was not affected by CPX preincubation (Fig. 5). These data suggest that the stimulatory effect of basolateral CPA on Na⁺/H⁺ exchanger occurs through an A_2A-like adenosine receptor localized on the basolateral membrane. Further support for this hypothesis comes from the observation that the A_2A-selective agonist DPMA (27), when added to the basal side solution, stimulated Na⁺/H⁺ exchange activity (0.650 ± 0.069 versus 0.818 ± 0.083 pH/min before and after 10⁶ M DPMA addition, respectively, p < 0.05, four experiments), whereas it had no effect when added to the apical side (data not shown).

View larger version (42K): [in this window] [in a new window]   Fig. 5.   Effect of the A1-selective antagonist CPX and the A2A-selective antagonist CSC on basolateral CPA stimulation of the Na+/H+ exchanger. Experiments were performed according to the protocol described in the legend to Fig. 1. The A1 adenosine-selective antagonist CPX (107 M) or the A2A antagonist CSC (107 M) was added to the basal solution 5 min before stimulation with CPA (106 M). Columns, mean ± standard error of five experiments. ***, p < 0.001, compared with the value for CPA alone.

7

Second messengers involved in CPA action. To elucidate the signal transduction pathway by which CPA affects pH_i, we analyzed the effect of the pharmacological activation of several regulatory pathways on Na⁺/H⁺ exchange activity. As shown in Fig. 6, a 5-min preincubation with TPA (10⁷ M), a phorbol ester known to activate PKC, induced a small but significant inhibition of Na⁺/H⁺ exchange rate (from 0.506 ± 0.043 to 0.366 ± 0.031 pH/min before and after TPA treatment, respectively, p < 0.01, three experiments). Similarly, preincubation with 10⁶ M ionomycin caused a small inhibition (0.609 ± 0.117 versus 0.493 ± 0.098 pH/min before and after ionomycin, respectively, p < 0.01, four experiments), suggesting that Na⁺/H⁺ exchange in A₆ cells is negatively regulated by both calcium-dependent PKC and intracellular Ca²⁺. To verify whether PKC is involved in the inhibitory effect of apical CPA on Na⁺/H⁺ exchange, we analyzed the effect of CPA after pretreatment with calphostin C, a known inhibitor of PKC activity (28). A 5-min preincubation with calphostin C (10⁸ M) completely prevented the inhibitory action of apical CPA (0.768 ± 0.071 versus 0.774 ± 0.075 pH/min in the absence and presence of CPA plus calphostin C, respectively, p = NS, three experiments) while having no effect on basal Na⁺/H⁺ exchange rate when added alone to both sides of the monolayers (data not shown). These data support the hypothesis that apical CPA action may be dependent on a functional PKC.

View larger version (37K): [in this window] [in a new window]   Fig. 6.   Effect of the the pharmacological activation of several regulatory pathways on Na+/H+ exchange activity. Paired measurements of initial rate of pHi increase induced by the basolateral superfusion of 22 mM Na+ medium to acid-loaded cells in the absence (control; open columns) or presence of the indicated agents were performed as described in the legend to Fig. 1. Cell monolayers were exposed to various agents from both the apical and basolateral side for different times: forskolin (FSK) and 8-bromo-cAMP for 10 min, ionomycin (Iono) for 2 min, and TPA for 5 min. Values are mean ± standard error of five experiments for the forskolin, four experiments for 8-bromo-cAMP, three experiments for TPA, and four experiments for ionomycin determinations. **, p < 0.01; ***, p < 0.001.

4

5

6

6

View larger version (52K): [in this window] [in a new window]   Fig. 7.   Effect of preincubation with the antagonists CPX and CSC on the basolateral CPA-induced cAMP generation. A6 monolayers were preincubated with antagonists 5 min before the addition of CPA (106 M) to the basolateral side of the monolayers. Levels of intracellular cAMP was determined as described in Experimental Procedures. Columns, mean ± standard error of three experiments. ***, p < 0.001, compared with the value for CPA alone.

Coupling between the pH_i and the transepithelial Na⁺ transport. Alterations in pH_i has been reported to directly influence apical Na⁺ conductance and/or indirectly Na⁺/K⁺-ATPase (13). Our previous observations that basolateral CPA increases Na⁺ transepithelial transport in A₆ cell monolayers (12) together with the current findings suggest that the intracellular signal by which adenosine stimulates transepithelial Na⁺ transport could be cellular alkalinization via a cAMP-dependent stimulation of Na⁺/H⁺ exchange. To assess the role of pH_i changes in mediating the action of basolateral CPA on Na⁺ transport, we first analyzed the effect of the specific Na⁺/H⁺ exchanger inhibitor HOE-694 (31) on resting pH_i. We found that although basolateral CPA (10⁶ M) slightly increased the resting pH_i by 0.16 ± 0.02 units (three experiments), as might be expected from the demonstrated stimulatory action on the Na⁺/H⁺ exchanger, HOE-694 (2 × 10⁷ M) added to the same side of the monolayer induced an intracellular acidification of 0.23 ± 0.02 units (three experiments). We then analyzed the effect of basolateral CPA addition either alone or after HOE-694 preincubation on the transepithelial Na⁺ transport measured as I_sc in HEPES-buffered solutions (Table 1). Basolateral CPA, as previously observed (12), produced a late and sustained increase in I_sc that reached a maximum effect 20 min after the addition of the hormone. The addition of 2 × 10⁷ M HOE-694 to the the basal surface of the A₆ monolayers 5 min before the subsequent addition of CPA did not alter basal I_sc (9.63 ± 2.51 versus 9.78 ± 2.43 µA/cm² before and after HOE-694 addition, respectively, p = NS, six experiments) while significantly inhibiting the CPA-induced Isc increase. The action of HOE-694 in preventing the CPA-induced I_sc increase seems to be due to its specific inhibitory action on the Na⁺/H⁺ exchanger and not to an interference with A₂ adenosine receptors; CPA-induced intracellular levels of cAMP were not significantly affected by HOE-694 preincubation (1.77 ± 0.2 versus 1.70 ± 0.3 pmol/filter/10 min after CPA plus HOE-694 pretreatment, three experiments).

View this table: [in this window] [in a new window]   TABLE 1 Basolateral CPA effect on short-circuit current in A6 monolayers Values are mean ± standard error. CPA (106 M) was added to the basolateral side of the monolayer. The value reported corresponds to the maximal effect recorder after 20 min of treatment with CPA. HOE-694 (2 × 107 M) was added 5 min before CPA treatment. Isc, short-circuit current.

	Discussion

Summary Introduction Procedures Results Discussion References

In the current study, we characterized the effect of adenosine on Na⁺/H⁺ exchange activity in A₆ cell monolayers. We used A₆ cell monolayers as a model to study the mechanism of adenosine action for two reasons. First, the vectorial sodium ion transport in A₆ cells, as in native epithelia such as the mammalian collecting duct or the amphibian skin, is mediated by amiloride-sensitive Na⁺ channels at the apical surface and Na⁺/K⁺-ATPase at the basolateral cell surface (19, 32-34). Second, due to their high tightness (transepithelial resistance, 9850 ± 467 1/2 × cm²), A₆ cell monolayers provide a useful model for the study of the functional localization of adenosine receptor action to the two cell surfaces. In addition, the location and characteristics of Na⁺/H⁺ exchange activity in A₆ cells have been previously described (18, 22); the Na⁺/H⁺ exchanger, which is highly sensitive to amiloride inhibition and stimulated by vasopressin, is confined to the basolateral cell surface of A₆ monolayers as observed in cells of rabbit cortical collecting tubule (16, 33, 34).

We assessed adenosine regulation of pH_i by spatially stimulating the A₆ cell monolayers with CPA, a metabolically stable adenosine analogue with higher affinity for the A₁ receptor, used in conjunction with A₁ and A_2A receptor-selective antagonists for which the K_i value has been determined in mammalian tissues (see Experimental Procedures). Pharmacological identification of adenosine receptor subtypes has not been fully explored in amphibians (6, 12, 35); therefore, there is the possibility that the selectivity of these agonists and antagonists in A₆ cells could be different than that in mammalian tissues.

The results of the current study demonstrate that basolaterally added CPA caused a consistent stimulation of Na⁺/H⁺ exchange activity with a consequent significant alkalinization of pH_i. We also observed a small but significant inhibitory effect of CPA on Na⁺/H⁺ exchange activity when added to the apical side (Figs. 1 and 2). The fact that CPA exerts different effects depending on the side of the addition confirms and extends previous results that demonstrated that A₆ cell monolayers express A₁- and A₂-like adenosine receptors on different cell surface membranes of the same cell that regulate different cell processes: the A₁-like receptors located on the apical membrane regulate chloride secretion via a rapid increase in intracellular calcium, whereas the A₂-like receptors located on the basolateral membrane regulate transepithelial sodium transport via an increase in cAMP (12).

Basolateral CPA action on Na⁺/H⁺ exchange. We found that the stimulatory effect of basolateral CPA on Na⁺/H⁺ exchange was dose dependent, with its major effect occurring at a relatively high concentration (10⁶ M). At this concentration, CPA increased the Na⁺/H⁺ exchanger's affinity for external sodium without affecting V_max. Consequently, it seems that at least a part of the observed basolateral effect of CPA on the pH_i recovery rate can be accounted for by an increased affinity for external sodium. A similar mechanism underlying the vasopressin-dependent increase in Na⁺/H⁺ exchange activity in A₆ monolayers has also been reported (18).

Apical CPA action on Na⁺/H⁺ exchange. A slight but significant inhibition of Na⁺/H⁺ exchange was observed on apical application of CPA to A₆ monolayers (Figs. 1 and 2). The hypothesis that this inhibition was due to the action of an apically located adenosine receptor related to the mammalian A₁ receptor is supported by the observation that apical CPA-dependent inhibition of Na⁺/H⁺ exchange activity was significantly reduced by the A₁ receptor-selective antagonist CPX (25).

Coupling between pH_i and the transepithelial Na⁺ transport. The dual-control regulation of the Na⁺/H⁺ exchanger and the presence of two types of adenosine receptors linked to distinct postreceptor mechanisms suggest that adenosine can exert different effects on pH_i depending on both the external adenosine concentration and the site of action. It has been well documented that pH_i seems to be a possible mediator coupling the rate of Na⁺ transport across both the apical and basolateral membranes. Alterations in pH_i has been reported to directly influence apical Na⁺ conductance and/or indirectly the basolateral Na⁺/K⁺-ATPase (13-16). The relationship between pH_i and Na⁺ transport seemed to be reciprocal: an increase in H⁺ concentration was reported to cause a decrease in transepithelial Na⁺ transport. The current findings together with our previous observations that basolateral CPA increases Na⁺ transepithelial transport via A₂ adenosine receptors (12) suggest that the intracellular signal by which adenosine stimulates transepithelial Na⁺ transport could be cellular alkalinization via cAMP-dependent stimulation of Na⁺/H⁺ exchange. In line with this hypothesis are the results that we obtained in A₆ monolayers treated with the specific inhibitor of the Na⁺/H⁺ exchanger, HOE-694 (31).