INTRODUCTION

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IN MANY TISSUES, adenosine is involved in autoregulatory mechanisms affecting, for example, cardiac rate, lipolysis, smooth muscle tone, hemodynamics, hormone and neurotransmitter release, and renal electrolyte transport (4, 17). These actions of adenosine are mediated by cell surface receptors, which are coupled to guanine nucleotide binding proteins (G proteins). At present, three types of adenosine receptors are known which activate different effector systems (13-15). First, the A₁ receptor is linked to G_i to inhibit adenylyl cyclase (5, 13, 17, 21). In addition, binding of adenosine to A₁ receptors can result in activation of phospholipase C, leading to an increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) and protein kinase C (PKC) activity (1, 5, 9, 13, 20). Second, the A₂ receptor acts through G_s to activate adenylyl cyclase, leading to an increase in cAMP (9, 17, 19, 21). Third, the A₃ receptor is a novel receptor subtype, recently described in heart and nerve terminals (14). This latter receptor was demonstrated to inhibit adenylyl cyclase and to couple to phospholipase C.

In the kidney, adenosine is formed on the breakdown of adenosine nucleotides and extruded by the action of nucleoside transporters (8, 21). The presence of A₁ and A₂ receptors has been described in several segments of the nephron including glomeruli (17, 19), thick ascending limb of Henle's loop (6, 17), cortical collecting duct (1, 2, 17), and inner medullary collecting duct (17, 26, 27). Although the functional significance of A₁ and A₂ receptors in the distal part of the nephron is not clear, the ability of adenosine to regulate basal and hormone-stimulated cAMP production and to stimulate calcium and inositol phosphate turnover makes it a potentially important modulator of hormonally regulated solute and water transport in this part of the nephron (2). Recent studies using monolayers of A6 cells derived from the kidney of Xenopus laevis have implicated A₁ receptors in the regulation of sodium transport (9, 12). At present, however, there is limited knowledge on the role of these receptors in mammalian kidney functioning.

The aim of the present study was to investigate the effect of extracellular adenosine on Ca²⁺ reabsorption in the mammalian distal nephron. To this end, rabbit connecting tubule and cortical collecting duct cells were isolated by immunodissection and subsequently grown to confluence on permeable supports. Previous studies have demonstrated that these monolayers retain many characteristics of the original epithelium, including 1,25-dihydroxyvitamin D₃-, arginine vasopressin (AVP)-, and parathyroid hormone (PTH)-stimulated Ca²⁺ reabsorption, aldosterone-stimulated Na⁺ reabsorption, and K⁺ secretion (3, 23, 25). The data presented demonstrate that adenosine interacts with apical A₁ receptors to stimulate apical-to-basolateral Ca²⁺ transport.

	MATERIALS AND METHODS

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Chemicals. Collagenase A and hyaluronidase were obtained from Boehringer (Mannheim, Germany). 1,3-Dipropyl-8-cyclopenthylxanthine (DPCPX), 3,7-dimethyl-1-propargylxanthine (DMPX), CGS-21680 hydrochloride (CGS-21680), and the phosphodiesterase inhibitor, Ro 20-1724, were purchased from Research Biochemical International (Natick, MA). Fura 2-acetoxymethyl ester (fura 2-AM), 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA)-AM, and Pluronic F-127 were from Molecular Probes (Eugene, OR). The cAMP assay system was from Amersham. Thapsigargin was obtained from LC Services (Woburn, MA). All other chemicals, including adenosine, N⁶-cyclopentyladenosine (CPA), AVP, bovine parathyroid hormone [bPTH-(1-34)], forskolin, and 8-BrcAMP were obtained from Sigma (St. Louis, MO).

Primary cultures of rabbit kidney cortical collecting system. Rabbit kidney connecting tubule and cortical collecting duct cells were immunodissected from kidney cortex of New Zealand White rabbits (±0.5 kg) with antibody R2G9 and set in primary culture on permeable supports (0.33 cm²; Costar, Cambridge, MA), as described in detail previously (3). The culture medium was a 1:1 mixture of Dulbecco's modified Eagle's medium and Ham's F-12 medium (DME/F12, GIBCO; Life Technologies, Paisley, UK) supplemented with 5% (vol/vol) decomplemented fetal calf serum, 50 µg/ml gentamicin, 10 µl/ml nonessential amino acids (GIBCO), 5 µg/ml insulin, 5 µg/ml transferrin, 50 nM hydrocortisone, 70 ng/ml prostaglandin E₁, 50 nM Na₂SeO₃, and 5 pM triiodothyronine, equilibrated with 5% CO₂-95% air at 37°C. All experiments were performed with confluent monolayers between 5 and 8 days after seeding the cells. Transepithelial potential difference and resistance were routinely checked before and after every experiment to confirm confluency and intactness of the monolayer.

Determination of transcellular Ca²⁺ transport. Confluent monolayers of rabbit cortical collecting system cells, grown on permeable filters, were washed twice and preincubated in physiological salt solution (PSS) containing (in mM) 140 NaCl, 2 KCl, 1 K₂HPO₄, 1 MgCl₂, 1 CaCl₂, 5 glucose, 5 L-alanine, 5 µM indometacin, and 10 mM HEPES-Tris (pH 7.4) for 15 min at 37°C. Subsequently, the monolayers were incubated in PSS (100 µl apical and 600 µl basolateral) for another 90 min to measure transepithelial Ca²⁺ transport. Drugs and hormones were added to either the apical or basolateral compartment, as indicated in the text. Appropriate stocks (×1,000) of these compounds were dissolved in ethanol or dimethyl sulfoxide. Identical concentrations of solvent were used as controls. At the end of the incubation period, 25-µl samples were removed in triplicate from the apical compartment and assayed for Ca²⁺, using a colorimetric assay kit (Boehringer). Under these experimental conditions, net apical-to-basolateral Ca²⁺ flux is linear with time for at least 3 h (3). Ca²⁺ reabsorption is expressed in nmol · h¹ · cm².

Determination of intracellular cAMP accumulation. To assess the effects of hormones and drugs on intracellular cAMP accumulation, confluent monolayers were preincubated in PSS containing the phosphodiesterase inhibitor, Ro 20-1724 (100 µM), for 15 min at 37°C. 3-Isobutyl-1-methylxanthine was not used, since it was shown to act as an adenosine receptor antagonist (1, 22). Hormones and drugs were added to either the apical or basolateral compartment, as indicated in the text, and the monolayers were incubated in PSS/Ro 20-1724 for another 15 min. At 15 min, both the apical and basolateral medium were discarded, and the filter was excised and rapidly transferred to 500 µl 5% (wt/vol) trichloroacetic acid to terminate the generation of cAMP. After three cycles of freezing and thawing, samples were centrifuged at 12,000 g to remove precipitated material, and trichloroacetic acid was repeatedly extracted with water-saturated diethyl ether. cAMP was assayed by radioligand binding using a cAMP assay system from Amersham.

Measurements of the cytosolic free Ca²⁺ concentration. Cortical collecting system cells, grown to confluence on permeable transparent supports, were loaded with fura 2-AM in DME/F12 medium containing 10 µM fura 2-AM, 0.01% (wt/vol) Pluronic F-127, 0.5 mM probenicid, and 4% (vol/vol) decomplemented fetal calf serum for 60 min at 37°C. Subsequently, the monolayers were washed twice with PSS and transferred to a temperature-controlled (37°C) perfusion chamber placed on the stage of an inverted microscope (Nikon Diaphot, Tokyo, Japan). The apical and basolateral compartments were perfused separately with PSS by means of a gravity-controlled superfusion system. Apical and basolateral flow rates were set at 1 and 5 ml/min, respectively. Fluorescence measurements were performed with a long-working-distance objective (Fluor ×60, N.A. = 0.7; Nikon). Dynamic video imaging was carried out with the MagiCal system and TARDIS software provided by Joyce Loebl (Tyne and Wear, UK), as described previously (10, 11, 18, 25). The fluorescence emission ratio at 492 nm after excitation at 340 and 380 nm was monitored. The interframe interval between two ratio frames was 6.4 s. Because absolute Ca²⁺ concentrations were difficult to obtain, due to technical problems with ionomycin calibration, fluorescence ratios are reported as a measure of [Ca²⁺]_i.

Statistics. In all experiments, the data are expressed as means ± SE. Overall statistical significance was determined by analysis of variance. In the case of significance (P < 0.05), individual groups were compared by contrast analysis according to Scheffé. P < 0.05 were considered significant.

	RESULTS

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Effect of adenosine on Ca²⁺ absorption across rabbit cortical collecting system. Cultured cells of the rabbit cortical collecting system, grown to confluence on permeable supports and incubated in the absence of any stimulus, exhibited a net apical-to-basolateral Ca²⁺ flux of 48 ± 3 nmol · h¹ · cm² (n = 50) (Fig. 1). Ca²⁺ absorption was significantly stimulated by 10 µM adenosine when added to the apical side (110 ± 4 nmol · h¹ · cm²; n = 30, P < 0.05), whereas basolateral addition did not significantly affect Ca²⁺ absorption (51 ± 2 nmol · h¹ · cm², n = 6). The stimulatory effect of adenosine was comparable to that of bPTH (100 nM, basolateral), AVP (10 nM, basolateral), the adenylyl cyclase activator forskolin (1 µM, both sides), and the cell-permeable cAMP analog, 8-BrcAMP (100 µM, both sides), which increased Ca²⁺ reabsorption to 204 ± 9, 239 ± 8, 217 ± 4, and 241 ± 7%, respectively (Fig. 1). Figure 2A shows that apical adenosine stimulated transcellular Ca²⁺ transport in a dose-dependent manner with an EC₅₀ of 0.7 ± 0.2 µM, a threshold concentration of 10-100 nM, and a maximally effective concentration of 10 µM.

View larger version (24K): [in this window] [in a new window]   Fig. 1.   Effect of adenosine, arginine vasopressin (AVP), bovine parathyroid hormone-(1-34) (PTH), forskolin, and 8-BrcAMP on Ca2+ transport. Adenosine was added at a concentration of 10 µM to the apical and/or basolateral compartment. AVP, PTH, forskolin, and 8-BrcAMP were added at concentrations of 10 nM, 100 nM, 10 µM, and 100 µM, respectively, to the basolateral compartment. Values are presented as means ± SE (n = 6). * P < 0.05, significantly different from the control value.

View larger version (13K): [in this window] [in a new window]   Fig. 2.   Dose dependence of the stimulatory effect of adenosine on Ca2+ transport and cAMP accumulation. A: Ca2+ transport was measured in the presence of indicated apical concentrations of adenosine for 90 min at 37°C. At the end of the incubation period, apical medium was collected to determine the amount of Ca2+ transported across the monolayer. Data are means ± SE (n = 6). B: confluent monolayers were preincubated in the presence of 100 µM Ro 20-1724 for 15 min at 37°C and subsequently stimulated with indicated apical concentrations of adenosine for another 15 min. In each experiment, basal values were set at 100% (4.04 ± 0.24 pmol · 15 min1 · cm2). Data are means ± SE (n = 3).

Characterization of receptor type involved in adenosine-stimulated Ca²⁺ absorption. As shown in Fig. 2B, addition of adenosine to the apical side of monolayers incubated in the presence of 100 µM Ro 20-1724 to inhibit cAMP breakdown resulted in a dose-dependent accumulation of intracellular cAMP. At 100 µM, the maximal concentration used in this study, adenosine increased cAMP to 314 ± 20%. This observation suggests the involvement of an A₂ receptor. To further characterize the type of receptor involved in adenosine-stimulated Ca²⁺ transport, the effect of specific agonists and antagonists was tested. Addition of the A₂ receptor agonist CGS-21680 to the apical compartment did not stimulate Ca²⁺ transport, unless added at a relatively high concentration of 100 µM. However, at this latter concentration, the A₂ agonist produced only one-third of the maximal effect of adenosine (Fig. 3A). By contrast, the A₁ receptor agonist CPA progressively increased Ca²⁺ transport to a maximum of 228 ± 8%.

View larger version (15K): [in this window] [in a new window]   Fig. 3.   Dose dependence of stimulatory effects of adenosine agonists on Ca2+ transport and cAMP accumulation. A: Ca2+ transport was measured in the presence of indicated concentrations of either N6-cyclopentyladenosine (CPA) or phosphodiesterase inhibitor CGS-21680 for 90 min at 37°C. At the end of the incubation period, apical medium was collected to determine the amount of Ca2+ transported across the monolayer. Data are means ± SE (n = 6). B: confluent monolayers were preincubated in the presence of 100 µM Ro 20-1724 for 15 min at 37°C and stimulated with indicated apical concentrations of either CPA or CGS-21680 for another 15 min. In each experiment, basal values were set at 100% (4.04 ± 0.24 pmol · 15 min1 · cm2). Data are presented as means ± SE (n = 3).

CGS-21680 increased cAMP formation dose dependently (Fig. 3B). Remarkably, comparison of Fig. 3, A and B, reveals that the effect of CGS-21680 on cAMP formation was already significant (150% over basal) at a concentration (10 µM) at which Ca²⁺ transport was not stimulated. By contrast, CPA, at concentrations at which it effectively stimulated Ca²⁺ reabsorption, only poorly (117 ± 8%, n = 3) increased cAMP. Moreover, the effect of CPA on cAMP accumulation appeared to be all but dose dependent. These observations suggest that cAMP is not the intracellular messenger involved in adenosine-stimulated Ca²⁺ absorption.

Figure 3 shows that CGS-21680 stimulates cAMP accumulation at least 10 times better than Ca²⁺ transport. Taking into account that the affinity of CGS-21680 for the A₂ receptor is ~100 times higher than that for the A₁ receptor (4), it is most likely that the stimulatory effect of 100 µM CGS-21680 on Ca²⁺ transport occurs through the A₁ receptor.

To confirm that the stimulatory action of adenosine on Ca²⁺ absorption was mediated by an A₁ receptor, additional experiments were performed in the presence of the specific A₁ antagonist, DPCPX. As shown in Fig. 4, pretreatment of the monolayers with DPCPX (10 µM) completely inhibited the action of 3 µM adenosine. By contrast, the A₂ antagonist DMPX did not affect the stimulatory effect of adenosine.

View larger version (16K): [in this window] [in a new window]   Fig. 4.   Effect of adenosine antagonists on adenosine-stimulated Ca2+ transport. Confluent monolayers of rabbit cortical collecting system cells in primary culture were preincubated in the presence of the indicated concentrations of either the A1 antagonist 1,3-dipropyl-8-cyclopenthylxanthine (DPCPX) or the A2 antagonist 3,7-dimethyl-1-propargylxanthine (DMPX) for 15 min at 37°C and subsequently stimulated with 3 µM adenosine for another 90 min in the continuous presence of antagonist. At the end of the incubation period, apical medium was collected to determine amount of Ca2+ transported across the monolayer. Antagonists and adenosine were added to the apical side of the monolayer only. Data are presented as means ± SE (n = 3).

As shown in Fig. 5A, apically added adenosine (10 µM) induced a transient increase in [Ca²⁺]_i within seconds after the onset of stimulation. Removal of extracellular Ca²⁺ did not significantly affect the adenosine-induced [Ca²⁺]_i response (data not shown). A similar Ca²⁺ transient increase was observed when the monolayers were perfused at the apical side with the A₁ receptor agonist CPA (Fig. 5B). By contrast, the A₂ agonist CGS-21680, added at a concentration of 100 µM at the apical side, did not evoke an increase in [Ca²⁺]_i (Fig. 5C). Subsequent addition of 100 µM ATP to the basolateral side still induced an increase in [Ca²⁺]_i, indicating the responsiveness of the cells. The adenosine-induced increase in [Ca²⁺]_i confirms A₁ receptor activation of phospholipase C, resulting in enhanced inositol 1,4,5-trisphosphate and diacylglycerol formation.

View larger version (13K): [in this window] [in a new window]   Fig. 5.   Effect of adenosine and receptor agonists on intracellular Ca2+ concentration ([Ca2+]i). Monolayers cultured on transparent supports and loaded with the fluorescent Ca2+ indicator fura 2 were stimulated with either 10 µM adenosine (A), 100 µM CPA (B), or 100 µM CGS-21680 (C). Stimuli were present in the apical superfusion medium for indicated period of time. After removal of stimulus from the superfusion medium, cells were stimulated with 100 µM ATP added to the basolateral compartment. Both the apical and basolateral compartment were superfused continuously, and measurements were performed at 37°C. Temporal dynamics of [Ca2+]i were analyzed simultaneously in ~20 individual cells by digital-imaging microscopy. Fluorescence emission ratio at 492 nm is monitored as a measure of [Ca2+]i after excitation at 340 and 380 nm. Recordings are from single cells of a representative experiment.

It is well established that the A₁ receptor is coupled to G_i to inhibit adenylyl cyclase activation (5, 13, 17, 21). To provide additional evidence for the presence of a functional A₁ receptor, we investigated the effect of CPA on AVP-induced cAMP accumulation. Figure 6 clearly demonstrates that CPA (100 µM) indeed inhibits the increase in cAMP evoked by 1 nM AVP.

View larger version (14K): [in this window] [in a new window]   Fig. 6.   Effect of CPA on AVP-induced cAMP accumulation. Confluent monolayers were preincubated for 15 min at 37°C in the presence of 100 µM Ro 20-1724 and in the absence or presence of 100 µM CPA in the apical compartment. Subsequently, either saline (solid bars) or 1 nM AVP (open bars) was added to the basolateral side, and cAMP accumulation was measured for another 15 min. In each experiment, basal values were set at 100% (4.40 ± 0.25 pmol · 15 min1 · cm2). Data are presented as means ± SE (n = 3). * P < 0.05, significantly different from control value.

In view of the above finding that the stimulatory effect of adenosine on Ca²⁺ transport is not paralleled by an increase in cAMP formation, it was tested whether the transient rise in [Ca²⁺]_i and/or the increase in PKC activity mediate adenosine-stimulated Ca²⁺ absorption. To this end, monolayers were cultured in the presence of the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA, 0.1 µM) for 5 days to downregulate PKC (24), after which the effect of adenosine on Ca²⁺ absorption was determined. Adenosine-stimulated Ca²⁺ transport in PKC-downregulated cells occurred with an equal potency as in untreated monolayers (81 ± 4 vs. 88 ± 3 nmol · h¹ · cm²; n = 3) (Fig. 7). Buffering [Ca²⁺]_i by loading the cells with 15 µM BAPTA-AM abolished the adenosine-induced transient increase in [Ca²⁺]_i but had no effect on the stimulatory action of adenosine on Ca²⁺ absorption (102 ± 2 vs. 99 ± 3 nmol · h¹ · cm²) (Fig. 8). On the other hand, increasing of [Ca²⁺]_i by addition of the inhibitor of intracellular Ca²⁺-ATPase activity, thapsigargin (11, 16), did not affect the basal rate of Ca²⁺ reabsorption (49.3 ± 2.7 vs. 45.3 ± 2.6 nmol · h¹ · cm²; n = 9, for control and thapsigargin-treated monolayers, respectively). Taken together, these results suggest that adenosine-stimulated Ca²⁺ transport is not mediated by a downregulatable PKC isotype or transient elevation of [Ca²⁺]_i.

View larger version (17K): [in this window] [in a new window]   Fig. 7.   Effect of adenosine on Ca2+ transport across 12-O-tetradecanoylphorbol 13-acetate (TPA)-treated cortical collecting system cells. Cells were cultured in the presence of 0.1 µM TPA (open bars) for 120 h to downregulate protein kinase C (PKC), as described previously (24). Control cells (solid bars) were cultured during the same time period in the presence of vehicle only. Subsequently, the PKC downregulated, and untreated cells were exposed to 10 µM apical adenosine (+) or to vehicle only (). Values are means ± SE (n = 4). * P < 0.05, significantly different from control values.

View larger version (18K): [in this window] [in a new window]   Fig. 8.   Effect of 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) on adenosine-stimulated Ca2+ transport and intracellular Ca2+ mobilization. A: cells preincubated in the presence of 15 µM BAPTA-AM (open bars) for 30 min were stimulated apically with 10 µM adenosine for another 90 min at 37°C. At the end of the incubation period, apical medium was collected and assayed for the amount of Ca2+. Data are means ± SE (n = 4). * P < 0.05, significantly different from corresponding control. B: rabbit cortical collecting system cells, grown to confluence on transparent supports, were loaded with the fluorescent Ca2+ indicator fura 2 in the absence (top trace) and presence (bottom trace) of 15 µM BAPTA-AM and subsequently stimulated apically with 10 µM adenosine. Digital-imaging microscopy was performed, as described in Fig. 5. Recordings shown are from a representative experiment.

To investigate the relationship between the classic cAMP-mediated pathway and the novel cAMP-independent pathway, we investigated the stimulatory effect of CPA and 8-BrcAMP, alone and in combination, on Ca²⁺ transport. Combination of maximally effective doses of both stimulants did not further increase the rate of Ca²⁺ transport. The most likely explanation is that the activity of one of the Ca²⁺ transporters is rate limiting. However, half maximally effective doses of CPA (3 µM) and 8-BrcAMP (3 µM) were clearly additive (Fig. 9). Because CPA, even in the presence of an inhibitor of cyclic nucleotide phosphodiesterase activity, only marginally increases cAMP (Fig. 3B), this additivity convincingly demonstrates that CPA acts through a separate cAMP-independent pathway and that this pathway and the cAMP-dependent pathway converge distally to stimulate Ca²⁺ transport.

View larger version (16K): [in this window] [in a new window]   Fig. 9.   Additive stimulatory effect of CPA and 8-BrcAMP on Ca2+ transport. Confluent monolayers were stimulated with 3 µM 8-BrcAMP and/or 3 µM CPA added to both sides and the apical side, respectively. Ca2+ transport was measured for 90 min at 37°C. At the end of the incubation period, apical medium was collected to determine the amount of Ca2+ transported across the monolayer. Data are means ± SE (n = 3). * P < 0.05, significantly different from control values.

	DISCUSSION

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The present study demonstrates that adenosine stimulates Ca²⁺ reabsorption in primary cultures of the rabbit cortical collecting system via an apical A₁ receptor. This conclusion is based on the following observations: 1) stimulation of Ca²⁺ transport was only observed on apical addition of adenosine; 2) the selective A₁ agonist CPA stimulated Ca²⁺ reabsorption at least 100 times more potently than the selective A₂ agonist CGS-21680; 3) adenosine-stimulated Ca²⁺ transport was completely inhibited by the selective A₁ antagonist DPCPX and not by the selective A₂ antagonist DMPX; 4) both CPA and adenosine, but not CGS-21680, induced an extracellular Ca²⁺-independent transient rise in [Ca²⁺]_i, which is typical for A₁ receptor activation; 5) CPA effectively inhibited AVP-induced cAMP accumulation, which implies the action of a G_i-coupled A₁ receptor (5, 13, 17, 21); and 6) the minimal effective concentration achieved with adenosine was 10-100 nM, which is in agreement with previously published values for the A₁ receptor (7).

The presence of adenosine receptors in the cortical collecting tubule, which forms part of the cortical collecting system used in the present study, has been demonstrated by Arend and co-workers (1, 2). At present, the functional relevance of these receptors is largely unknown. We now show, however, that apical adenosine receptors may play a regulatory role in Ca²⁺ homeostasis in the distal nephron. In the kidney, adenosine is produced on breakdown of adenosine nucleotides and transported to the lumen by the action of nucleoside transporters demonstrated to be present in proximal tubules, connecting tubules, and cortical collecting ducts (8, 21). Using monolayers of amphibian kidney A6 cells, Hayslett et al. (9) and Lang et al. (12) provided evidence for the involvement of adenosine receptors in the regulation of Na⁺ transport. It remains, however, to be elucidated whether this is also the case in the mammalian kidney.

It is generally accepted that transepithelial Ca²⁺ transport is triggered by an increase in cAMP, as classically observed with parathyroid hormone (3). However, the present study provides evidence that Ca²⁺ transport can be stimulated in a cAMP-independent manner. First, the A₁ agonist CPA stimulated Ca²⁺ transport at concentrations at which it did not increase cAMP. Second, the selective A₂ agonist CGS-21680 increased cAMP without affecting Ca²⁺ transport. This lack of effect of CGS-21680 on Ca²⁺ reabsorption is explained by the fact that, even in the presence of the cyclic nucleotide phosphodiesterase inhibitor, the increase in cAMP is marginal compared with that evoked by AVP (compare Figs. 3B and 6) and apparently does not exceed the threshold for Ca²⁺ transport. Further evidence that adenosine does not primarily act through cAMP comes from the observation that, at 1 µM, it markedly stimulated Ca²⁺ transport while increasing cAMP to a level (150% over basal) at which no Ca²⁺ transport was observed in the case of 10 µM CGS-21680. Together with the finding that both 8-BrcAMP and forskolin effectively stimulated Ca²⁺ reabsorption, representing the classic cAMP-dependent pathway, this suggests that cortical collecting system cells possess a separate cAMP-independent pathway leading to increased Ca²⁺ reabsorption. This idea is further supported by the fact that the stimulatory effects of CPA and 8-BrcAMP were additive.

Although the primary cell culture used in this study is heterogenous by nature and consists of at least three cell types, designated connecting tubules and principal and intercalating cells, we have previously demonstrated that the majority (±80%) of the cultured cells are calbindin-D_28K positive and therefore involved in transepithelial Ca²⁺ transport (25). In addition, the present finding that adenosine and CPA increase [Ca²⁺]_i in at least 80% of the individually analyzed cells suggests the functional presence of A₁ receptors on the majority of the calbindin-D_28K-containing cells.

In an attempt to elucidate the nature of the cAMP-independent pathway, we investigated whether the transient increase in [Ca²⁺]_i and/or the activation of PKC in response to A₁ receptor activation are involved in the stimulatory action of adenosine in Ca²⁺ reabsorption. Downregulation of PKC by prolonged (120 h) exposure to TPA did not affect adenosine-stimulated Ca²⁺ reabsorption. This finding suggests that PKC isotypes, sensitive to downregulation, are not involved in the mechanism of action of adenosine. The involvement of the transient increase in [Ca²⁺]_i was excluded by the observation that adenosine-stimulated Ca²⁺ transport was not affected in monolayers loaded with the calcium chelator BAPTA. Alternatively, increasing [Ca²⁺]_i by means of the selective inhibitor of endoplasmic reticulum Ca²⁺-ATPase activity, thapsigargin (1 µM), did not alter basal Ca²⁺ reabsorption. Taken together, these findings suggest that neither PKC nor [Ca²⁺]_i represent the cAMP-independent pathway used by adenosine to stimulate Ca²⁺ reabsorption in this nephron segment.

In conclusion, our study provides evidence for the presence of an apical A₁ receptor coupled to a hitherto unknown cAMP-independent pathway to stimulate apical-to-basolateral Ca²⁺ transport in cultured cortical collecting system cells. In terms of kidney physiology, adenosine may act as an autacoid to stimulate Ca²⁺ reabsorption in conjunction with circulating hormones.