Hormone-stimulated Ca2+ transport in rabbit kidney: multiple sites of inhibition by exogenous ATP

Jürgen van Baal1,2, Joost G. J. Hoenderop1,2, Maarten Groenendijk1, Carel H. van Os1, René J. M. Bindels1, and Peter H. G. M. Willems2

Departments of 1 Cell Physiology and 2 Biochemistry, University of Nijmegen, 6500 HB Nijmegen, The Netherlands


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Exogenous ATP markedly reduced 1-desamino-8-D-arginine vasopressin (dDAVP)-stimulated Ca2+ transport and cAMP accumulation in primary cultures of rabbit connecting tubule and cortical collecting duct cells. Similarly, ATP inhibited the stimulatory effect of 8-bromo-cAMP. At first sight, this is in agreement with the "classic" concept that dDAVP exerts its stimulatory effect via cAMP. However, dDAVP-stimulated Ca2+ transport was markedly reduced by the protein kinase C (PKC) inhibitor chelerythrine, reported previously to inhibit the cAMP-independent pathway responsible for parathyroid hormone-, [Arg8]vasopressin-, PGE2-, and adenosine-stimulated Ca2+ transport. Chelerythrine also inhibited the increase in Ca2+ transport evoked by the cAMP-independent A1 receptor agonist N6-cyclopentyladenosine (CPA). Downregulation of phorbol ester-sensitive PKC isoforms by chronic phorbol ester treatment has been shown before to be without effect on hormone-stimulated Ca2+ transport, indicating that the chelerythrine-inhibitable pathway consists of a phorbol ester-insensitive PKC isoform. Here, this maneuver did not affect ATP inhibition of dDAVP-stimulated Ca2+ transport and cAMP formation, while abolishing ATP inhibition of CPA-stimulated Ca2+ transport. These findings show that ATP acts via 1) a phorbol ester-sensitive PKC isoform to inhibit hormonal stimulation of Ca2+ transport at the level of the chelerythrine-inhibitable pathway involving a phorbol ester-insensitive PKC isoform and 2) a phorbol ester-insensitive mechanism to inhibit V2 receptor-mediated concomitant activation of this pathway and adenylyl cyclase.

connecting tubule; cortical collecting duct; adenosine 5'-triphosphate; protein kinase C; calcium transport


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE (PATHO)PHYSIOLOGICAL relevance of exogenous ATP has been studied extensively. Under physiological conditions, ATP is released from nerve terminals, endothelial cells, and muscle cells (7, 9). Recently, ATP release associated with the cystic fibrosis transmembrane conductance regulator has attracted a great deal of interest (14, 22, 28, 29, 31). Certain clinical syndromes are accompanied by the release of large quantities of ATP as a result of tissue injury (7, 9, 19). ATP binds to specific cell-surface receptors that are widely distributed throughout the body. Activation of these purinoceptors requires pericellular ATP concentrations in the high micromolar range (7, 9-11, 19, 21, 25, 30).

Purinoceptors are classified into two subtypes, designated P1 and P2 (9, 12, 19). P1 purinoceptors exhibit a greater sensitivity to adenosine and AMP and are subdivided into A1, A2, and A3 receptors (9, 12, 19, 24). A1 and A3 receptors have been demonstrated to inhibit adenylyl cyclase and to activate phospholipase C (23, 24), whereas A2 receptors activate adenylyl cyclase (25, 33). P2 purinoceptors are more sensitive to ATP and ADP and are subdivided into ionotropic P2X receptors and G protein-coupled P2Y receptors (12). P2X receptors are ligand-gated cation channels, whereas P2Y receptors increase the activity of phospholipase C.

Evidence for the involvement of P2 receptors in the regulation of renal electrolyte and water transport was first provided by the observation that exogenous ATP stimulated Na+-K+-Cl- cotransport activity in cultured A6 cells originally derived from the distal nephron of Xenopus laevis (25). More recently, ATP was shown to inhibit [Arg8]vasopressin (AVP)-stimulated water transport in perfused tubules of the rat inner medullary collecting duct (IMCD; see Ref. 20) and rabbit cortical collecting duct (CCD; see Ref. 30). Similarly, ATP was shown to inhibit Na+ and Ca2+ reabsorption in primary cultures of rabbit connecting tubule (CNT) and CCD cells (21). The involvement of P2Y receptors linked to the inositol 1,4,5-trisphosphate-mediated release of Ca2+ from the endoplasmic reticulum was concluded from the observation that ATP readily increased the cytosolic Ca2+ concentration in the absence of extracellular Ca2+. A similar observation was reached with rat IMCD (10) and mouse cortical thick ascending limb of Henle (27). In each case, the effect of ATP was shown to be mimicked by UTP, which is in agreement with the involvement of UTP-preferring P2Y receptors (12).

There is ample evidence that the distal nephron is the primary site of active Ca2+ reabsorption (1, 8, 13). To study the hormonal regulation of this process, we established a primary culture of immunodissected rabbit CNT and CCD cells (2-4, 16, 17, 21, 32, 33). We have shown that Ca2+ transport across these monolayers can be stimulated by parathyroid hormone (PTH; see Refs. 16 and 32), AVP (16, 32, 33), PGE2 (16, 32), and adenosine (16, 17). Importantly, we recently demonstrated that the adenosine analog N6-cyclopentyladenosine (CPA), which acts as an agonist at the A1 receptor, can maximally stimulate Ca2+ transport without detectably increasing the cytosolic cAMP concentration (17). This led us to postulate the existence of a novel cAMP-independent pathway leading to stimulated Ca2+ transport. Moreover, in a follow-up study, we showed that also PTH, PGE2, AVP, and adenosine act via this novel cAMP-independent pathway and that this pathway involves a chelerythrine-inhibitable protein kinase C (PKC) isotype that is not downregulated after chronic phorbol ester treatment (16).

Using this cell model, we previously reported that ATP can markedly inhibit Ca2+ transport, and evidence was provided for the involvement of PKC in the mechanism of action of ATP (2, 21). However, of eminent importance for a correct interpretation of the mechanism of action of ATP is our recent finding that primary cultures of CNT and CCD cells produce prostanoids that stimulate Ca2+ transport (32). This latter finding leaves the possibility that ATP might act by inhibiting this autostimulatory pathway.

The first aim of the present study is to assess whether the previously reported inhibitory action of ATP on Ca2+ transport is due to inhibition of the autostimulatory pathway. The second aim is to investigate whether ATP also inhibits the stimulatory effects of other hormones such as AVP and adenosine. Finally, the third aim of the present study is to investigate whether PKC is indeed involved in the inhibitory actions of ATP, as suggested by our previous work, and, if so, to elucidate the level(s) at which PKC exerts its inhibitory effects.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Drugs. Collagenase A and hyaluronidase were obtained from Boehringer (Mannheim, Germany). 1-Desamino-8-D-arginine vasopressin (dDAVP) was from Bachem Feinchemikalien (Bubendorf, Switzerland). Pertussis toxin (PTX), chelerythrine, and Ro-20-1724 were purchased from Research Biochemicals International (Natick, MA). All other chemicals, including AVP, adenosine, CPA, PGE2, 8-bromo-cAMP (8-BrcAMP), and 12-O-tetradecanoylphorbol 13-acetate (PMA), were obtained from Sigma (St. Louis, MO).

Primary cultures of rabbit CNT and CCD cells. New Zealand White rabbits weighing ~0.5 kg were killed by cervical dislocation. Kidney CNT and CCD cells were immunodissected with monoclonal antibody R2G9 and set in primary culture on permeable filter supports (0.33 cm2; Costar, Cambridge, MA), as described previously (32). The cells were cultured in a 1:1 mixture of Dulbecco's modified Eagle's medium-Ham's F-12 (GIBCO, Paisley, UK), supplemented with 5% (vol/vol) decomplemented FCS (Serva, Heidelberg, Germany), 50 µg/ml gentamicin, 0.5% (vol/vol) of a 100× mixture of nonessential amino acids (GIBCO), 5 µg/ml insulin, 5 µg/ml transferrin, 50 nM hydrocortisone, 70 ng/ml PGE1, 50 nM Na2SeO3, and 5 pM triiodothyronine, equilibrated with 5% CO2-95% O2 at 37°C. Cell monolayers reached confluency at day 3, and experiments were performed between 5 and 8 days after seeding the cells. Confluency of the monolayers was routinely checked by determining transepithelial potential difference and resistance with two "chopstick"-like electrodes connected to a Millicell-ERS meter (Millipore, Bedford, MA).

Measurement of transepithelial Ca2+ transport. Transepithelial Ca2+ transport was measured as described previously (32). Briefly, confluent monolayers were washed three times and preincubated in a physiological salt solution (PSS) containing (in mM): 140 NaCl, 2 KCl, 1 K2HPO4, 1 KH2PO4, 1 MgCl2, 1 CaCl2, 5 glucose, 5 L-alanine, and 10 HEPES (adjusted to pH 7.40 with Tris) for 15 min at 37°C. Long-term treatments with drugs were performed in culture medium. Subsequently, the monolayers were washed three times and incubated in PSS for another 90 min at 37°C to measure transepithelial Ca2+ transport. Drugs and hormones were added to either the apical and/or basolateral compartment, as indicated in the text. At the end of the incubation period, 25-µl samples were removed in duplicate from the apical compartment and assayed for Ca2+ with a colorimetric test kit (Boehringer). Ca2+ transport is expressed in nanomoles per hour per square centimeter.

Measurement of cAMP accumulation. To assess the effect of ATP on dDAVP-stimulated adenylyl cyclase activity, accumulation of cAMP was measured in the presence of an inhibitor of cyclic nucleotide phosphodiesterase activity, Ro-20-1724. Confluent monolayers were preincubated in PSS containing 0.1 mM Ro-20-1724 for 15 min at 37°C. Subsequently, hormones were added to either the apical and/or basolateral compartment as indicated in the text, and the monolayers were incubated for another 15 min. At 15 min, the filters were excised and immediately transferred to Eppendorf microtubes containing 500 µl of 5% (wt/vol) trichloroacetic acid. The samples were vigorously mixed and rapidly frozen in liquid nitrogen. Samples were frozen and thawed three times and centrifuged for 4 min at 10,000 g (Eppendorf minifuge). A 400-µl aliquot of the supernatant was removed and extracted three times with water-saturated diethyl ether. The aqueous phase was blown to dryness with nitrogen, after which the residue was dissolved in 500 µl of sodium acetate buffer (pH 6.2). Samples and standards were acetylated, and the cAMP content was determined by RIA ([I125]cAMP Assay System; Amersham, Arlington Heights, IL). The amount of cAMP is expressed in nanomoles per 15 min per filter.

Statistics. Results are given as means ± SE. Overall statistical significance was determined by ANOVA and, in the case of significance, individual groups were compared by contrast analysis according to Scheffé. P values <0.05 were considered significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

ATP inhibition of Ca2+ transport stimulated by endogenously produced prostanoids. We have shown that ATP interacts with apical and basolateral P2Y purinoceptors to inhibit net apical-to-basolateral Ca2+ transport across monolayers of rabbit CNT and CCD cells (21). However, our recent finding that endogenously produced prostanoids potently stimulate this transport process (32) urged us to investigate the possibility that ATP might act by inhibiting this autostimulatory pathway. For maximal inhibition, ATP was added to both sides of the monolayer (21). Figure 1 shows the inhibitory action of ATP (100 µM; both compartments) on Ca2+ transport (P < 0.05; n = 8 filters). However, ATP did not have an effect in monolayers in which Ca2+ transport was reduced as a result of the inhibition of endogenous prostanoid production by the cyclooxygenase inhibitor indomethacin (5 µM; both compartments). To investigate whether ATP exerts its inhibitory action on the autostimulatory pathway at or beyond the receptor level, its effect on PGE2-stimulated Ca2+ transport was studied in indomethacin-treated monolayers. Figure 1 shows that ATP significantly (P < 0.005; n = 8 filters) reduced the stimulatory effect of exogenous PGE2 (10 nM). The percentage inhibition was calculated to be 51%. These findings demonstrate that ATP inhibits the autostimulatory pathway at least at or beyond the level of the prostanoid receptor. Subsequent experiments were routinely performed with indomethacin-treated monolayers.


View larger version (16K):
[in this window]
[in a new window]
 
Fig. 1.   Inhibitory effect of ATP on Ca2+ transport stimulated by endogenously produced prostanoids and exogenous PGE2 in monolayers of rabbit connecting tubule (CNT) and cortical collecting duct (CCD) cells. Confluent monolayers were preincubated in the absence (open bars) and presence (filled bars) of ATP (100 µM; both compartments) and/or indomethacin (5 µM; both compartments) for 15 min at 37°C. Subsequently, either vehicle or PGE2 (10 nM; both compartments) was added, and Ca2+ transport was measured for another 90 min. Data presented are means ± SE of 8 filters. * Significantly different from corresponding unstimulated monolayers (P < 0.05). # Significantly different from corresponding monolayers incubated (untreated) or stimulated (indomethacin treated) in the absence of ATP (P < 0.05). & Significantly different from corresponding monolayers incubated in the absence of indomethacin (P < 0.05).

ATP inhibition of AVP- and adenosine-stimulated Ca2+ transport. Both AVP and adenosine have been demonstrated to potently stimulate Ca2+ transport across monolayers of rabbit CNT and CCD cells (16, 17, 32, 33). Table 1 shows that ATP (100 µM; both compartments) significantly (P < 0.05) reduced the stimulatory effect of AVP (1 nM; basolateral compartment) and adenosine (10 µM; apical compartment) by 37 and 63%, respectively.

                              
View this table:
[in this window]
[in a new window]
 
Table 1.   Inhibitory effect of exogenous ATP on AVP- and adenosine-stimulated Ca2+ transport across confluent monolayers of rabbit CNT and CCD cells

ATP inhibition of dDAVP- and CPA-stimulated Ca2+ transport. The stimulatory effect of AVP was shown to be mimicked by dDAVP (33), which suggests the involvement of the V2 receptor. Figure 2 shows that dDAVP (1 nM; basolateral compartment) increased Ca2+ transport by 71 ± 5 nmol · h-1 · cm-2 (P < 0.05; n = 12 filters) and that ATP (100 µM; both compartments) significantly decreased this value to 36 ± 4 nmol · h-1 · cm-2 (P < 0.05; n = 12 filters). The percentage inhibition was calculated to be 50%. ATP did not affect basal Ca2+ transport. As cAMP is generally implicated in the mechanism of action of dDAVP (5), we investigated the effect of ATP on cAMP-stimulated Ca2+ transport. Figure 2 shows that the membrane-permeable cAMP analog 8-BrcAMP (0.1 mM; basolateral compartment) markedly increased Ca2+ transport by 71 ± 5 nmol · h-1 · cm-2 (P < 0.05; n = 9 filters) and that ATP significantly decreased this value to 39 ± 6 nmol · h-1 · cm-2 (P < 0.05; n = 9 filters). The percentage inhibition was calculated to be 45%. This demonstrates that, in case a hormone acts through cAMP, ATP acts, at least in part, at or beyond the level of protein kinase A. We recently showed that the adenosine analog CPA, which acts as an agonist at the A1 receptor (34), can maximally stimulate Ca2+ transport without detectably increasing the cytosolic cAMP concentration (17). This led us to investigate whether ATP can also inhibit this novel cAMP-independent stimulatory pathway. Figure 2 shows that CPA (10 µM; apical compartment) increased Ca2+ transport by 48 ± 3 nmol · h-1 · cm-2 (P < 0.05; n = 5 filters) and that ATP decreased this value to 14 ± 3 nmol · h-1 · cm-2 (P < 0.05; n = 5 filters). The percentage inhibition was calculated to be 71%.


View larger version (15K):
[in this window]
[in a new window]
 
Fig. 2.   Inhibitory effect of ATP on 1-desamino-8-D-arginine vasopressin (dDAVP)-, 8-bromo-cAMP (8-BrcAMP)-, and N6-cyclopentyladenosine (CPA)-stimulated Ca2+ transport in monolayers of rabbit CNT and CCD cells. Confluent monolayers were preincubated with indomethacin (5 µM; both compartments) in the absence (open bars) or presence (filled bars) of ATP (100 µM; both compartments) for 15 min before stimulation with dDAVP, 8-BrcAMP, or CPA. CPA (10 µM) was added to the apical compartment, whereas dDAVP (1 nM) and 8-BrcAMP (100 µM) were applied basolaterally. Ca2+ transport was determined at 90 min after the onset of stimulation. Data presented are means ± SE of 6 (CPA) and 12 (dDAVP and 8-BrcAMP) filters. * Significantly different from corresponding unstimulated monolayers (P < 0.05). # Significantly different from corresponding monolayers stimulated in the absence of ATP (P < 0.05).

Chelerythrine inhibition of dDAVP- and CPA-stimulated Ca2+ transport. In a previous study, we demonstrated that chelerythrine, a potent and selective inhibitor of PKC (15), markedly reduced PTH-, PGE2-, AVP-, and adenosine-stimulated Ca2+ transport (16). Table 2 shows that chelerythrine (5 µM; both sides) abolished the stimulatory effect of CPA (10 µM) and markedly reduced the increase in Ca2+ transport evoked by dDAVP (10 nM) from 77 ± 4 to 37 ± 9 nmol · h-1 · cm-2 (P < 0.05; n = 4 filters).

                              
View this table:
[in this window]
[in a new window]
 
Table 2.   Inhibitory effect of chelerythrine on dDAVP- and CPA-stimulated Ca2+ transport across confluent monolayers of rabbit CNT and CCD cells

Effect of PKC downregulation on ATP inhibition of dDAVP-, CPA-, and 8-BrcAMP-stimulated Ca2+ transport. Short-term activation of PKC by the phorbol ester PMA was shown to result in a rapid and marked inhibition of Ca2+ transport (2). In the continuous presence of PMA, however, Ca2+ transport gradually recovered. This recovery reflects the disappearance or downregulation of PMA-sensitive PKC isoforms. It was also demonstrated that ATP failed to inhibit Ca2+ transport in monolayers treated with PMA for a prolonged period of time (21). Because Ca2+ transport measurements were performed in the absence of indomethacin, the above findings can be taken as evidence that ATP acts via one or more PMA-sensitive PKC isoforms to inhibit the stimulatory effect of endogenously produced prostanoids. Figure 3 shows that dDAVP increased Ca2+ transport by 67 ± 4 nmol · h-1 · cm-2 (P < 0.05; n = 9 filters) in monolayers pretreated with PMA (0.1 µM; both compartments) for 120 h. This value was not different from that obtained with untreated monolayers (P > 0.9). ATP still significantly reduced the dDAVP-induced increase to a value of 44 ± 2 nmol · h-1 · cm-2 (P < 0.05; n = 9 filters), and also this value was not different from that obtained with untreated monolayers (P > 0.6). Chronic PMA treatment did not significantly affect the increase in Ca2+ transport in response to 8-BrcAMP (P > 0.1) but abolished the inhibitory action of ATP thereupon. Similarly, this treatment abolished ATP inhibition of CPA-stimulated Ca2+ transport without altering the response to CPA (P > 0.9).


View larger version (15K):
[in this window]
[in a new window]
 
Fig. 3.   Effect of chronic phorbol ester treatment on ATP inhibition of dDAVP-, 8-BrcAMP-, and CPA-stimulated Ca2+ transport in monolayers of rabbit CNT and CCD cells. Confluent monolayers were preincubated with PMA (0.1 µM; both compartments) for 120 h. Indomethacin (5 µM; both compartments) was added without (open bars) or with (filled bars) ATP (100 µM; both compartments) 15 min before stimulation. CPA (10 µM) was added to the apical compartment, whereas dDAVP (1 nM) and 8-BrcAMP (100 µM) were applied basolaterally. Ca2+ transport was determined at 90 min after the onset of stimulation. Data presented are means ± SE of 6 (CPA) and 12 (dDAVP and 8-BrcAMP) filters. * Significantly different from corresponding unstimulated monolayers (P < 0.05). # Significantly different from corresponding monolayers stimulated in the absence of ATP (P < 0.05).

ATP inhibition of the dDAVP-induced increase in adenylyl cyclase activity. To evaluate the effect of ATP on receptor-mediated adenylyl cyclase activation, the accumulation of cAMP was measured in the presence of the inhibitor of cyclic nucleotide phosphodiesterase activity, Ro-20-1724 (0.1 mM; both compartments). Under these conditions, cAMP accumulation in unstimulated monolayers amounted to 1.6 ± 0.1 nmol · 15 min-1 · filter-1 (n = 4 filters). dDAVP (1 nM; basolateral compartment) increased the cAMP content by 6.3 ± 0.6 nmol · 15 min-1 · filter-1 (P < 0.05; n = 4 filters), and ATP (100 µM; both compartments) significantly reduced this increase to 3.3 ± 0.6 pmol · 15 min-1 · filter-1 (P < 0.05; n = 4 filters; Fig. 4). The percentage inhibition was calculated to be 48%. ATP did not affect basal cAMP accumulation (see also, Fig. 5).


View larger version (13K):
[in this window]
[in a new window]
 
Fig. 4.   Effect of chronic phorbol ester treatment on ATP inhibition of dDAVP-induced cAMP accumulation in monolayers of rabbit CNT and CCD cells. Confluent monolayers were pretreated without (untreated) or with (0.1 µM; both compartments) 12-O-tetradecanoylphorbol 13-acetate (PMA) for 120 h at 37°C. Indomethacin (5 µM; both compartments) and Ro-20-1724 (100 µM; both compartments) were added without (open bars) or with (filled bars) ATP (100 µM; both compartments) at 15 min before stimulation with dDAVP (1 nM; basolateral compartment). Accumulation of cAMP was measured for another 15 min. Basal cAMP accumulation in unstimulated monolayers amounted to 1.6 ± 0.1 nmol · 15 min-1 · filter-1 (n = 4 filters). Data presented show the increase in cAMP accumulation above basal and are means ± SE of 4 (untreated) and 9 (PMA-treated) filters. * Significantly different from corresponding untreated monolayers (P < 0.05). # Significantly different from corresponding monolayers stimulated in the absence of ATP (P < 0.05).

Effect of PKC downregulation on ATP inhibition of dDAVP-induced adenylyl cyclase activation. To test the possibility that ATP acts via a phorbol ester-sensitive PKC isotype to inhibit dDAVP-induced adenylyl cyclase activation, monolayers were pretreated with PMA (0.1 µM; both compartments) for 120 h. Figure 4 shows that dDAVP readily increased cAMP by 4.9 ± 0.3 nmol · 15 min-1 · filter-1 and that ATP still significantly lowered this increase to 2.6 ± 0.2 nmol · 15 min-1 · filter-1 in PMA-treated monolayers (P < 0.05; n = 9). In the absence of ATP, the dDAVP-induced increase in cAMP was slightly but significantly (P < 0.04) reduced after chronic PMA treatment, whereas, in the presence of ATP, no difference (P > 0.2) between the two groups was observed.

Effect of ATP on dDAVP stimulation of Ca2+ transport and activation of adenylyl cyclase in PTX-treated monolayers. To investigate the possibility that ATP might act via the inhibitory GTP-binding protein (Gi) to inhibit dDAVP stimulation of Ca2+ transport and activation of adenylyl cyclase, monolayers were pretreated with PTX (170 ng/ml) for 24 h. Figure 5 shows that this treatment did not interfere with the inhibitory effect of ATP on dDAVP-stimulated Ca2+ transport (A; P < 0.05; n = 4 filters) and cAMP accumulation (B; P < 0.05; n = 4 filters). This demonstrates that ATP does not act via Gi to exert its inhibitory effect. PTX did not affect basal cAMP accumulation but significantly potentiated the stimulatory effect of dDAVP both in the absence (P < 0.05; n = 4 filters) and presence (P < 0.05; n = 4 filters) of ATP. By contrast, PTX did not affect the dDAVP-induced increase in Ca2+ transport either in the absence (P > 0.8; n = 4 filters) or presence (P > 0.9; n = 4 filters) of ATP. This is in agreement with the idea that cAMP does not play a role in dDAVP-stimulated Ca2+ transport (16). As a control, we tested the effect of PTX on CPA inhibition of AVP-induced cAMP formation. CPA inhibited AVP-induced cAMP accumulation by 79% (see also, Ref. 17), and PTX abolished this inhibitory effect of CPA. PTX did not change CPA-stimulated Ca2+ transport (transport rates of 110 ± 5 and 116 ± 8 nmol · h-1 · cm-2 for untreated and PTX-treated monolayers, respectively, n = 3 filters, P > 0.2). This demonstrates that CPA does not interact with a Gi protein to activate the novel cAMP-independent pathway leading to Ca2+ transport.



View larger version (1413K):
[in this window]
[in a new window]
 
Fig. 5.   Effect of pertussis toxin (PTX) on ATP inhibition of dDAVP-stimulated Ca2+ transport and cAMP accumulation in monolayers of rabbit CNT and CCD cells. Confluent monolayers were pretreated without (open bars) or with (filled bars) PTX (170 ng/ml) for 24 h at 37°C. A: monolayers were preincubated with indomethacin (5 µM; both compartments) and ATP (100 µM; both compartments) for 15 min before stimulation with dDAVP (1 nM; basolateral compartment). Ca2+ transport was determined at 90 min after the onset of stimulation. B: indomethacin (5 µM; both compartments), Ro-20-1724 (100 µM; both compartments), and ATP (100 µM; both compartments) were added 15 min before stimulation with dDAVP (1 nM; basolateral compartment), and accumulation of cAMP was measured for another 15 min. Data presented are means ± SE of 4 filters. * Significantly different from corresponding unstimulated monolayers (P < 0.05). # Significantly different from corresponding monolayers stimulated in the absence of ATP (P < 0.05). $ Significantly different from corresponding monolayers not treated with PTX (P < 0.05).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The data obtained in the present study are incorporated in a model of the hormonal regulation of active Ca2+ reabsorption in rabbit CNT and CCD. The model, schematized in Fig. 6, includes the interaction of ATP with apical and basolateral receptors to inhibit hormone-stimulated Ca2+ transport via a phorbol ester-sensitive PKC isoform and vasopressin-stimulated Ca2+ transport via a phorbol ester-insensitive mechanism. Of note, the percentage inhibition reached with a maximally effective ATP concentration in combination with a maximally effective concentration of stimulant ranged from 37% (AVP) to 71% (CPA), indicating that ATP can only partly inhibit maximally stimulated Ca2+ transport.


View larger version (50K):
[in this window]
[in a new window]
 
Fig. 6.   Schematic presentation of signal pathways involved in ATP inhibition of dDAVP- and CPA-stimulated Ca2+ transport in rabbit CNT and CCD cells. AC, adenylate cyclase; PLC, phospholipase C; DAG, diacylglycerol; IP3, inositol 1,4,5-trisphosphate; CaBP, Ca2+-binding protein. For explanation, see text.

Inhibition of the autostimulatory pathway. The initial observation that exogenous ATP can markedly inhibit Ca2+ transport across confluent monolayers of rabbit CNT and CCD cells (21) took a new turn when it was found that basal transport was in fact increased by the action of endogenously produced prostanoids (32). Possible mechanisms of action of ATP are therefore 1) inhibition of the production and/or release of the autostimulatory prostanoids and 2) inhibition of the stimulatory action of these prostanoids. The present finding that ATP markedly inhibited PGE2-stimulated Ca2+ transport in indomethacin-treated monolayers is in agreement with the latter explanation. Obviously, this finding does not exclude that ATP may also interfere with the production and/or release of autostimulatory prostanoids.

Inhibition of AVP- and dDAVP-stimulated Ca2+ transport. This work shows that ATP can markedly inhibit AVP- and dDAVP-stimulated Ca2+ transport in a primary culture of rabbit CCD and CNT cells. Inhibitory effects of ATP have also been reported for AVP-stimulated water transport in perfused rabbit CCD (30) and rat IMCD (20). ATP inhibition of AVP- and dDAVP-stimulated Ca2+ transport was found to be paralleled by a reduction of dDAVP-stimulated cAMP accumulation. At first sight, the latter finding supports the "classic" concept that dDAVP interacts with V2 receptors to stimulate Ca2+ transport in a cAMP-dependent fashion. However, in a recent study, we provided evidence that cAMP is not involved in AVP-stimulated Ca2+ transport (16). Moreover, we showed that AVP-stimulated Ca2+ transport, although insensitive to PKC downregulation by chronic phorbol ester treatment, was inhibited by chelerythrine. Similarly, chelerythrine inhibited the effect of dDAVP in the present study. These findings suggest the involvement of a chelerythrine-inhibitable, phorbol ester-insensitive PKC isoform in the mechanism of action of AVP and dDAVP. In this context, the present observation that ATP reduces AVP- and dDAVP-stimulated Ca2+ transport indicates that the nucleotide inhibits the V2 receptor-mediated activation and/or action of this "novel" chelerythrine-inhibitable pathway.

ATP also inhibited the stimulatory effect of 8-BrcAMP on Ca2+ transport. This demonstrates the potential of ATP to inhibit also the classic cAMP-dependent pathway and that inhibition occurs at a level at or beyond protein kinase A. As far as the AVP-induced increase in cAMP is concerned, we previously speculated that it occurs in a compartment that does not affect Ca2+ transport, whereas a generalized increase in cAMP, as produced by 8-BrcAMP, does stimulate Ca2+ transport (16).

Inhibition of adenosine- and CPA-stimulated Ca2+ transport. We recently demonstrated that the adenosine analog CPA, which specifically activates the A1 receptor (34), can maximally stimulate Ca2+ transport without having an effect on cAMP accumulation (17). Moreover, we showed that chelerythrine completely inhibited the effect of adenosine (16). Together with the present finding that chelerythrine abolished the stimulatory action of CPA, this suggests that adenosine and CPA, similar to AVP and dDAVP, exert their stimulatory effect via the chelerythrine-inhibitable pathway. The present study shows that ATP also partly inhibits adenosine- and CPA-stimulated Ca2+ transport. This is in agreement with the idea that ATP inhibits the chelerythrine-inhibitable pathway itself and/or its activation via the adenosine A1 receptor. Of note, CPA-stimulated Ca2+ transport was not affected by PTX. This excludes the possibility that a G protein of the Gi family couples the A1 receptor to this novel pathway.

Involvement of PKC in the mechanism of action of ATP. In a previous study, we demonstrated that ATP acts through apical and basolateral P2Y receptors to inhibit Ca2+ transport (21). P2Y purinoceptors are generally believed to couple to a PTX-insensitive G protein of the Gq family to increase the activity of phospholipase C (12). In agreement with this, preliminary results revealed that PTX did not affect the ATP-induced increase in cytosolic Ca2+ concentration, while readily inhibiting the effect of adenosine thereupon (H. P. G. Koster, A. Hartog, C. H. Van Os, and R. J. M. Bindels, unpublished observations).

Activation of P2Y receptors leads to the increased production of 1,2-diacylglycerol, which functions as the physiological activator of PKC (9, 26). The ability of PKC to inhibit Ca2+ transport was demonstrated previously by means of the membrane-permeable diacylglycerol analog 1,2-dioctanoylglycerol (21) and the potent PKC activator PMA (2). Both PKC activators instantaneously inhibit dDAVP-stimulated Ca2+ transport (J. van Baal, S. E. van Emst-de Vries, R. J. M. Bindels, and P. H. G. M. Willems, unpublished observations) and Ca2+ transport stimulated via the autostimulatory pathway (2). However, this inhibition was only temporary and gradually diminished during continued PMA treatment. Finally, we showed that ATP inhibition of the autostimulatory pathway was abolished when phorbol ester-sensitive PKC isoforms were downregulated by chronic PMA treatment (21). Similarly, the present study shows that chronic PMA treatment abolished ATP inhibition of CPA-stimulated Ca2+ transport. These findings demonstrate that ATP acts via a phorbol ester-sensitive PKC isoform to inhibit stimulation of Ca2+ transport through the prostanoid receptor and the adenosine A1 receptor and, as can be deduced from the instantaneous inhibition of dDAVP-stimulated Ca2+ transport by PMA (see above), also through the vasopressin V2 receptor. Because all three receptors act via the novel chelerythrine-inhibitable pathway, these findings suggest that ATP acts via the phorbol ester-sensitive mechanism to inhibit the action of this novel pathway. However, chronic PMA treatment did not affect ATP inhibition of dDAVP-stimulated Ca2+ transport. This indicates that ATP acts in addition via a phorbol ester-insensitive mechanism to inhibit stimulation of Ca2+ transport through the vasopressin V2 receptor.

Chronic PMA treatment did not affect ATP inhibition of dDAVP-evoked cAMP formation. This suggests that ATP acts via a phorbol ester-insensitive mechanism to inhibit dDAVP-induced cAMP formation. It is tempting to speculate that the same phorbol ester-insensitive mechanism attenuates both V2 receptor-mediated adenylyl cyclase activation and activation of the chelerythrine-inhibitable pathway and therefore acts at the V2 receptor itself. Such a mechanism would also fit with the idea that the measured changes in cAMP are an epi-phenomenon unrelated to the stimulatory action of dDAVP on Ca2+ transport (16).

Chronic PMA treatment abolished ATP inhibition of 8-BrcAMP-stimulated Ca2+ transport. This indicates that ATP acts via a phorbol ester-sensitive PKC isoform to inhibit stimulation of Ca2+ transport via the classic cAMP-dependent pathway at or beyond the level of protein kinase A.

ATP inhibition does not involve a PTX-sensitive G protein. The present work shows that PTX did not interfere with the inhibitory action of ATP on dDAVP-stimulated cAMP accumulation and Ca2+ transport. This lack of effect of the toxin on dDAVP-induced cAMP formation excludes the possibility that ATP interacts with a receptor that couples to Gi to inhibit adenylyl cyclase. By contrast, Rouse et al. (30) demonstrated that ATP inhibits AVP-stimulated water transport in rabbit CCD via the activation of both PKC and Gi.

PTX markedly potentiated dDAVP-induced cAMP accumulation without affecting, however, dDAVP-stimulated Ca2+ transport. This is in agreement with the idea that dDAVP does not act via cAMP to increase Ca2+ transport. Presently, the mechanism underlying the potentiating effect of PTX on dDAVP-induced cAMP accumulation is unclear. One possibility is that dDAVP not only stimulates adenylyl cyclase via Gs but also inhibits the enzyme via Gi.

In conclusion, the present study shows that ATP acts primarily via a phorbol ester-sensitive PKC isoform to inhibit hormonal stimulation of Ca2+ transport at the level of the novel chelerythrine-inhibitable pathway involving a phorbol ester-insensitive PKC isoform. Presently, it is unknown at which of the steps involved in transcellular Ca2+ transport, i.e., apical Ca2+ influx, cytosolic diffusion of Ca2+ bound to Ca2+-binding protein, and basolateral Ca2+ extrusion, the chelerythrine-inhibitable pathway acts to exert its stimulatory effect. However, it is tempting to speculate that the Ca2+ influx step is rate limiting and that regulation takes place at this level. Recent elucidation of the primary structure of the apical Ca2+ influx channel (18) reveals the presence of seven potential PKC phosphorylation sites, and future studies may decide whether or not these sites are involved in the hormonal regulation of Ca2+ reabsorption. In addition, ATP acts via a phorbol ester-insensitive mechanism to inhibit V2 receptor-mediated concomitant activation of this novel pathway and adenylyl cyclase. Finally, ATP acts through a phorbol ester-sensitive PKC isoform to inhibit Ca2+ transport evoked by a generalized increase in cAMP. The latter finding demonstrates the potential of ATP to inhibit the classic cAMP-dependent pathway at a level at or beyond protein kinase A.


    FOOTNOTES

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. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: R. J. M. Bindels, 162 Cell Physiology, Univ. of Nijmegen, PO Box 9101, NL-6500 HB Nijmegen, The Netherlands (E-mail: Reneb{at}sci.kun.nl).

Received 20 July 1998; accepted in final form 22 July 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Bindels, R. J. M. Calcium handling by the mammalian kidney. J. Exp. Biol. 184: 89-104, 1993[Abstract/Free Full Text].

2.   Bindels, R. J. M., J. A. Dempster, P. L. M. Ramakers, P. H. G. M. Willems, and C. H. Van Os. Effect of protein kinase C activation and down-regulation on active calcium transport. Kidney Int. 43: 295-300, 1993[Medline].

3.   Bindels, R. J. M., A. Hartog, S. L. Abrahamse, and C. H. Van Os. Effect of pH on apical calcium entry and active calcium transport in rabbit cortical collecting system. Am. J. Physiol. 266 (Renal Fluid Electrolyte Physiol. 35): F620-F627, 1994[Abstract/Free Full Text].

4.   Bindels, R. J. M., A. Hartog, J. A. H. Timmermans, and C. H. Van Os. Active Ca2+ transport in primary cultures of rabbit kidney CCD: stimulation by 1,25-dihydroxyvitamin D3 and PTH. Am. J. Physiol. 261 (Renal Fluid Electrolyte Physiol. 30): F799-F807, 1991[Abstract/Free Full Text].

5.   Breyer, M. D., and Y. Ando. Hormonal signaling and regulation of salt and water transport in the collecting duct. Annu. Rev. Physiol. 56: 711-739, 1994[Medline].

6.   Chabardes, D., D. Firsov, L. Aarab, A. Clabecq, A. C. Bellanger, S. Siaume-Perez, and J. M. Elalouf. Localization of mRNAs encoding Ca2+-inhibitable adenylyl cyclases along the renal tubule. Functional consequences for regulation of the cAMP content. J. Biol. Chem. 271: 19264-19271, 1996[Abstract/Free Full Text].

7.   Conigrave, A. D., and L. Jiang. Ca2+-mobilizing receptors for ATP and UTP. Cell Calcium 17: 111-119, 1995[Medline].

8.   Costanzo, L. S., and E. E. Windhager. Renal regulation of calcium balance. In: The Kidney: Physiology and Pathology, edited by D. W. Seldin, and G. Giebisch. New York: Raven, 1992, p. 2375-2393.

9.   Dubyak, G. R., and C. El-Moatassim. Signal transduction via P2-purinergic receptors for extracellular ATP and other nucleotides. Am. J. Physiol. 265 (Cell Physiol. 34): C577-C606, 1993[Abstract/Free Full Text].

10.   Ecelbarger, C. A., Y. Maeda, C. C. Gibson, and M. A. Knepper. Extracellular ATP increases intracellular calium in rat terminal collecting duct via a nucleotide receptor. Am. J. Physiol. 267 (Renal Fluid Electrolyte Physiol. 36): F998-F1006, 1994[Abstract/Free Full Text].

11.   El-Moatassim, C., J. Dornand, and J. C. Mani. Extracellular ATP and cell signalling. Biochim. Biophys. Acta 1134: 31-45, 1992[Medline].

12.   Fredholm, B. B., M. P. Abbracchio, G. Burnstock, G. R. Dubyak, T. K. Harden, K. A. Jacobson, U. Schwabe, and M. Williams. Towards a revised nomenclature for P1 and P2 receptors. Trends Biochem. Sci. 18: 79-82, 1997.

13.   Friedman, P. A., and F. A. Gesek. Cellular calcium transport in renal epithelia: measurements, mechanisms, and regulation. Physiol. Rev. 75: 429-471, 1995[Abstract/Free Full Text].

14.   Grygorczyk, R., J. A. Tabcharani, and J. W. Hanrahan. CFTR channels expressed in CHO cells do not have detectable ATP conductance. J. Membr. Biol. 151: 139-148, 1996[Medline].

15.   Hebert, J. M., J. M. Augereau, J. Gleye, and J. P. Maffrand. Chelerythrine is a potent and a specific inhibitor of protein kinase C. Biochem. Biophys. Res. Commun. 172: 993-999, 1990[Medline].

16.   Hoenderop, J. G. J., J. J. H. H. M. De Pont, R. J. M. Bindels, and P. H. G. M. Willems. Hormone-stimulated Ca2+ reabsorption in rabbit kidney cortical collecting system is cAMP-independent and involves a phorbol ester-insensitive PKC isotype. Kidney Int. 55: 225-233, 1999[Medline].

17.   Hoenderop, J. G. J., A. Hartog, P. H. G. M. Willems, and R. J. M. Bindels. Adenosine-stimulated Ca2+ reabsorption is mediated by apical A1 receptors in rabbit cortical collecting system. Am. J. Physiol. 274 (Renal Physiol. 43): F736-F743, 1998[Abstract/Free Full Text].

18.   Hoenderop, J. G. J., A. W. C. M. Van der Kemp, A. Hartog, S. F. J. Van de Graaf, C. H. Van Os, P. H. G. M. Willems, and R. J. M. Bindels. Molecular identification of the apical Ca2+channel in 1,25-dihydroxyvitamin D3-responsive epithelia. J. Biol. Chem. 274: 8375-8378, 1999[Abstract/Free Full Text].

19.   Inscho, E. W., K. D. Mitchell, and L. G. Navar. Extracellular ATP in the regulation of renal microvascular function. FASEB J. 8: 319-328, 1994[Abstract/Free Full Text].

20.   Kishore, B. K., C.-L. Chou, and M. A. Knepper. Extracellular nucleotide receptor inhibits AVP-stimulated water permeability in inner medullary collecting duct. Am. J. Physiol. 269 (Renal Fluid Electrolyte Physiol. 38): F863-F869, 1995[Abstract/Free Full Text].

21.   Koster, H. P. G., A. Hartog, C. H. Van Os, and R. J. M. Bindels. Inhibition of Na+ and Ca2+ reabsorption by P2u-purinoceptors requires protein kinase C but not Ca2+ signaling. Am. J. Physiol. 270 (Renal Fluid Electrolyte Physiol. 39): F53-F60, 1996[Abstract/Free Full Text].

22.   Li, C., M. Ramjeesingh, and C. E. Bear. Purified cystic fibrosis transmembrane conductance regulator (CFTR) does not function as an ATP channel. J. Biol. Chem. 271: 11623-11626, 1996[Abstract/Free Full Text].

23.   Linden, J. Structure and function of A1 receptors. FASEB J. 5: 2668-2676, 1991[Abstract/Free Full Text].

24.   Linden, J. Cloned adenosine A3 receptors: pharmacological properties, species differences and receptor functions. Trends Pharmacol. Sci. 15: 298-306, 1994[Medline].

25.   Middleton, J. P., A. W. Mangel, S. Basavappa, and J. G. Fitz. Nucleotide receptors regulate membrane ion transport in renal epithelial cells. Am. J. Physiol. 264 (Renal Fluid Electrolyte Physiol. 33): F867-F873, 1993[Abstract/Free Full Text].

26.   Nishizuka, Y. Protein kinase C and lipid signaling for sustained cellular responses. FASEB J. 9: 484-496, 1995[Abstract/Free Full Text].

27.   Paulais, M., M. Baudouin-Legros, and J. Teulon. Extracellular ATP and UTP trigger calcium entry in mouse cortical thick ascending limbs. Am. J. Physiol. 268 (Renal Fluid Electrolyte Physiol. 37): F496-F502, 1995[Abstract/Free Full Text].

28.   Reddy, M. M., P. M. Quinton, C. Haws, J. J. Wine, R. Grygorczyk, J. A. Tabcharani, J. W. Hanrahan, K. L. Gunderson, and R. R. Kopito. Failure of the cystic fibrosis transmembrane conductance regulator to conduct ATP. Science 271: 1876-1879, 1996[Abstract].

29.   Reisin, I. L., A. G. Prat, E. H. Abraham, J. F. Amara, R. J. Gregory, D. A. Ausiello, and H. F. Cantiello. The cystic fibrosis transmembrane conductance regulator is a dual ATP and chloride channel. J. Biol. Chem. 269: 20584-20591, 1994[Abstract/Free Full Text].

30.   Rouse, D., M. Leite, and W. N. Suki. ATP inhibits the hydrosmotic effect of AVP in rabbit CCT: evidence for a nucleotide P2u receptor. Am. J. Physiol. 267 (Renal Fluid Electrolyte Physiol. 36): F289-F295, 1994[Abstract/Free Full Text].

31.   Schwiebert, E. M., M. E. Egan, T. H. Hwang, S. B. Fulmer, S. A. Allen, G. R. Cutting, and W. B. Guggino. CFTR regulates outwardly rectifying chloride channels through an autocrine mechanism involving ATP. Cell 81: 1063-1073, 1995[Medline].

32.   Van Baal, J., M. D. De Jong, F. J. Zijlstra, P. H. G. M. Willems, and R. J. M. Bindels. Endogenously produced prostanoids stimulate calcium reabsorption in rabbit cortical collecting system. J. Physiol. (Lond.) 497: 229-239, 1996[Abstract].

33.   Van Baal, J., G. Raber, J. De Slegte, R. Pieters, R. J. M. Bindels, and P. H. G. M. Willems. Vasopressin-stimulated Ca2+ reabsorption in rabbit cortical collecting system: effects on cAMP and cytosolic Ca2+. Pflügers Arch. 433: 109-115, 1996[Medline].

34.   Van Galen, P. J. M., G. L. Stiles, G. Michaels, and K. A. Jacobson. Adenosine A1 and A2 receptors: structure-function relationships. Med. Res. Rev. 12: 423-471, 1992[Medline].


Am J Physiol Renal Physiol 277(6):F899-F906
0002-9513/99 $5.00 Copyright © 1999 the American Physiological Society