Adenylyl cyclase is involved in desensitization and recovery of ATP-stimulated Clminus secretion in MDCK cells

Jae Suk Woo1, Chiyoko N. Inoue1, Kazushige Hanaoka1, Erik M. Schwiebert1, Sandra E. Guggino2, and William B. Guggino1

1 Department of Physiology and Pediatrics and 2 Division of Gastroenterology, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

We investigated the process of and recovery from desensitization of the P2 receptor-mediated stimulation of Cl- secretion in Madin-Darby canine kidney (MDCK) cell monolayers by assaying the response of short-circuit current (Isc). When the cells were exposed to repeated 3-min challenges of ATP or UTP interspersed with 5-min washes, the response of Isc desensitized rapidly followed by spontaneous recovery. The pattern of inhibition by various channel blockers or enzyme inhibitors revealed that both the initial and recovered responses of Isc have the same ionic and signaling mechanisms. The desensitization and recovery processes were confined to the membrane exposed to the repeated challenges. When added during the desensitized phase, 8-bromoadenosine 3',5'-cyclic monophosphate enhanced the ATP-stimulated Isc response, whereas it did not during the initial or recovered phases. ATP-induced increases of intracellular adenosine 3',5'-cyclic monophosphate showed similar desensitization and recovery in parallel with the changes in the responses of Isc. The desensitization process was attenuated by pretreatment with cholera toxin or pertussis toxin. Taken together, our results suggest that the adenylyl cyclase system plays a role in the desensitization and recovery mechanism of the ATP-stimulated Cl- secretion in MDCK cells.

P2 receptor; short-circuit current; adenosine 3',5'-cyclic monophosphate; cholera toxin; pertussis toxin

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

EXTRACELLULAR ATP ACTS as an agonist to regulate a broad range of physiological processes by interacting with the P2 receptors, which can be grouped into two subfamilies (for reviews, refer to Refs. 2, 7, 10, 11, 15). The P2x receptors are ligand-gated cation channels with a higher affinity to alpha ,beta - or beta ,gamma -methylene ATP. The G protein-coupled receptor superfamily includes the P2y (or P2y1), P2u (or P2y2), and P2t (or P2y3) receptors. Among them, the P2t subtype, which is activated by ADP, is expressed in a very limited number of cell types. The P2y and P2u receptors are widely expressed in various tissues. The latter can be discriminated pharmacologically by the agonist selectivity: 2-methylthio-ATP (2-MeSATP) > ATP > ADP >> UTP for P2y and UTP = ATP > adenosine 5'-O-(3-thiotriphosphate) (ATPgamma S) >> 2-MeSATP = ADP for P2u.

Desensitization of the G protein-linked receptor-mediated responses is a widespread phenomenon occurring upon prolonged or repeated exposure(s) to agonists (24). The P2 receptors show both heterologous and homologous desensitization (33). The molecular events involved in the desensitization are not entirely clear, but it has been suggested that the phospholipase C (PLC) products, Ca2+ or protein kinase C, act as negative-feedback modulators of receptor-G protein or G protein-PLC interaction (6, 8, 25).

In epithelial cells found in the kidney (29), airway (18, 20), and intestine (9), ATP stimulates Cl- secretion through multiple purinoceptors and signaling mechanisms. G protein-dependent activation of PLC and subsequent increase in intracellular Ca2+ is a major signaling mechanism that stimulates Cl- secretion. Madin-Darby canine kidney (MDCK) cells are a well-differentiated cell line and have many characteristics of the distal nephron (14, 32). They are widely used as a model system for studying the regulation of epithelial cell function. They are known to have an ATP-stimulated Cl- secretory mechanism (28). Recently, Post et al. (23) demonstrated that the P2 purinergic agonists enhance adenosine 3',5'-cyclic monophosphate (cAMP) production in these cells.

In the course of preliminary experiments on ATP-regulated Cl- secretion in MDCK cells, we also observed the desensitization of the ATP-stimulated increases in short-circuit current (Isc), but with an interesting and unexpected phenomenon, spontaneous and gradual recovery from desensitization with repeated challenges. This initial result prompted us to investigate further the mechanisms of the desensitization and recovery of the ATP-stimulated Cl- secretion in MDCK cells.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Materials. ATP, ATPgamma S, UTP, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), amiloride, quinine, indomethacin, and bumetanide were purchased from Sigma Chemical (St. Louis, MO). 2-MeSATP and adenosine were obtained from Research Biochemicals International (Natick, MA). Forskolin, 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP), N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89), cholera toxin, pertussis toxin, and 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) acetoxymethyl ester (AM) were obtained from Calbiochem (La Jolla, CA). Diphenylamine-2-carboxylate (DPC) was from Fluka Chemie (Buchs, Switzerland). Eagle's minimal essential medium (MEM) and fetal bovine serum (FBS) were purchased from GIBCO (Grand Island, NY).

Cell culture. MDCK cells obtained from American Type Culture Collection were routinely maintained on plastic culture flasks in MEM supplemented with 10% FBS, 50 IU/ml penicillin G, and 50 µg/ml streptomycin. Cells were trypsinized when cells became confluent (approximately every 4-5 days) using 0.05% trypsin-0.53 mM EDTA solution and reseeded at one-sixth the original density. For Isc measurement, cells were subcultured at a density of 5 × 105 cells on 12-mm polycarbonate membrane filters (Snapwell, Costar, Cambridge, MA). The cells were fed with fresh media every other day and the day before the experiments. The cells used for this study were between passages 86 and 110.

Isc measurements. Isc measurement was performed in a modified Ussing chamber designed to accept Snapwell filters (World Precision Instruments, Sarasota, FL). The transepithelial potential difference was short circuited with a voltage clamper (model DVC-1000, World Precision Instruments) connected to apical and basolateral chambers via Ag/AgCl electrodes. The experiments were carried out in bicarbonate-free Ringer solution that was composed of (in mM) 140 NaCl, 2.3 K2HPO4, 0.4 KH2PO4, 1.5 CaCl2, 1.5 MgCl2, 10 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, and 5 glucose (pH 7.4). Both the apical and basolateral bathing solutions were maintained at 37°C, oxygenated with 100% O2, and subject to constant circulation. Before the stimulation by agonists, the cell monolayers were equilibrated in Ringer solution for 30 min.

Measurement of intracellular cAMP content. Cells were grown on Snapwell filters and subject to the same procedure as in measurement of Isc. After 3-min exposure to the agonists, the Snapwells were rapidly removed from the Ussing chambers and immersed in ice-cold ethanol HCl (ethanol solution containing 20 mM HCl). The membrane filters were cut off the Snapwell support using sharp tuberculin needles. Cells attached to the membrane filters in ethanol HCl solution were then transferred to microcentrifuge tubes and sonicated to disrupt the cell membrane and complete extraction of intracellular cAMP. The cell suspension was then centrifuged (12,000 g) for 10 min at 4°C to precipitate the protein, and the supernatant was collected. The supernatant was freeze-dried and dissolved in an adequate volume of 50 mM tris(hydroxymethyl)aminomethane/1 mM EDTA (pH 7.5). cAMP content was determined by radioimmunoassay using [3H]cAMP assay kit from Amersham (Arlington Heights, IL). Protein concentration was determined using the Bio-Rad protein assay kit with bovine serum albumin as standard.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Desensitization and recovery of ATP stimulation of Isc . Figure 1 shows a typical response of Isc in MDCK cells exposed to repeated doses of 10 and 100 µM ATP added into the apical bathing solution. The protocol involved repeated 3-min exposures punctuated by a 5-min wash period. During the wash period, previously added ATP was carefully washed out by irrigation with Ringer solution four or five times so that it did not affect the integrity of tight junctions. With this maneuver, the Isc and transepithelial resistance returned to the basal level usually 2-3 min before the next application of ATP. The basal Isc and transepithelial resistance in the MDCK cell monolayer used for this study were in the range from 0 to 4 µA (mean = 2.7 µA) and 2,450 to 5,230 Omega  · cm2 (mean = 4,310 Omega  · cm2), respectively.


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Fig. 1.   Changes in responses of short-circuit current (Isc) to repeated doses of ATP added into apical bathing solution. Cells were exposed to repeated 3-min exposures to ATP punctuated by a 5-min washout period. A: a representative tracing illustrating desensitization and recovery of Isc stimulated by repeated challenges of ATP (100 µM). B: responses of Isc to repeated challenges of 10 and 100 µM ATP are presented as means ± SE (n = number of experiments).

As expected, the first application of 100 µM ATP stimulated Isc, but repeated applications elicited a lessened response typical of desensitization. The maximum desensitization usually occurred after four or five repeated challenges, which elicited responses that were 23.9 ± 7.5% (n = 16) as high as the initial peak responses. Interestingly, the magnitude of the ATP stimulation recovered gradually. Typically, the recovery process began after 5 or 6 challenges and reached a maximum recovery after the 8th to 10th exposure. At this point, the stimulation recovered to 80.1 ± 9.5% (n = 16) of the initial peak response. The peak and plateau phases of the response underwent parallel changes during the desensitization and recovery process. Therefore, in this paper, we present the changes of the peak response only. When we lowered the concentration of ATP to 10 µM, the cells showed similar course of desensitization, but the recovery process was much delayed (Fig. 1B).

Effect of various channel blockers or enzyme inhibitors. The pattern of inhibition by various channel blockers or enzyme inhibitors in Fig. 2 showed that the same ionic and signaling mechanisms are involved in the ATP-stimulated Isc responses in both the initial and recovered phases. Although we did not present the result here, when the Cl- in the experimental solution was replaced with gluconate, the ATP-stimulated Isc response disappeared almost completely. This result, together with the significant inhibition by bumetanide in the basolateral bathing solution (Fig. 2), suggests that the ATP-stimulated Isc responses during both the initial and recovered phases can be ascribed to basolateral to apical secretion of Cl-. Both the initial and recovered responses of Isc to ATP were significantly inhibited by the Cl- channel blocker (1) DPC (200 µM, apical side). In contrast, DIDS (200 µM, apical side), which is known to block some Ca2+-activated Cl- channels (5), or glibenclamide (200 µM, apical side), which blocks cystic fibrosis transmembrane conductance regulator (CFTR) (27), was without effect. This result suggests that the Ca2+-activated Cl- channel or CFTR is not likely to be involved in the apical Cl- conductance. However, significant inhibition by the protein kinase A inhibitor (4) H-89 (20 µM, both sides) suggests that the stimulation of Cl- conductance is dependent on protein kinase A. The inhibition by the intracellular Ca2+ chelator (16) BAPTA-AM (10 µM, both sides) and the nonspecific K+ channel blocker (30) quinine (500 µM, basolateral side) suggests that activation of basolateral Ca2+-dependent K+ channels is also very important to the ATP-induced stimulation of Isc. The ATP-stimulated Isc response was inhibited almost completely by indomethacin (10 µM, both sides). This result strongly suggests that the metabolites of arachidonic acid are involved in the signaling mechanism.


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Fig. 2.   Effects of various channel or transporter blockers, enzyme inhibitors, and intracellular Ca2+ chelator on initial (A) and recovered (B) responses of Isc to ATP. A: after basal Isc was stabilized, Isc response to the first application of ATP (100 µM) added into apical bathing solution was recorded in presence of each drug. B: cells were exposed first to repeated doses of ATP added into apical bathing solution, and after we confirmed that ATP-stimulated Isc was recovered from desensitization (usually after washout of 10th application), final responses to ATP in presence of each drug were determined. DIDS (200 µM), glibenclamide (Glyb; 200 µM), diphenylamine-2-carboxylate (DPC; 200 µM), amiloride (Amil, 10 µM), and indomethacin (Indomet, 10 µM) were added into apical; quinine (500 µM) and bumetanide (Bumet, 100 µM) into basolateral bathing solution 5 min before final exposure to ATP. H-89 (20 µM) and BAPTA acetoxymethyl ester (BAPTA, 10 µM) were added into both apical and basolateral bathing solution 20 min before final exposure to ATP (100 µM). Effect of H-89 was partially reversible. After treatment with H-89 for 20 min followed by washout for 10 min, response recovered over 60% of control response. Each point and bar represents mean ± SE (n = 4-6). * P < 0.01 vs. control.

Responses to apical vs. basolateral stimulation. A similar pattern of desensitization followed by recovery occurred with exposures to ATP in the basolateral bathing solution (Fig. 3, top right). To address whether this phenomenon occurs in the exposed membrane only, we determined whether the responses to ATP in the contralateral side are affected during the desensitization and recovery phases induced by repeated exposures to ATP in the apical or basolateral side. Figure 3 shows the typical responses when the epithelium is exposed repeatedly to apical ATP (top left). During the initial, desensitized, and recovery phases of the response to apical application, a single dose of ATP was applied to the basolateral cell membrane (Fig. 3, bottom left). The results show that repeated application of ATP to the apical solution does not affect the response to basolateral application. Figure 3 also shows the typical response when the epithelium is exposed repeatedly to basolateral ATP (top right). During the initial, desensitized, and recovery phases of the response to basolateral application, a single dose of ATP was applied to the apical membrane (Fig. 3, bottom right). The results show that repeated application of ATP to the basolateral solution does not affect the response to apical application. These results demonstrate that the desensitization and recovery process is confined to the membrane that is exposed to the repeated challenges and does not affect the responses to ATP in the contralateral side.


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Fig. 3.   Changes in responses of Isc to repeated doses of ATP applied into apical or basolateral bathing solution, and their effects on responses to ATP at contralateral side. Cells were exposed to repeated doses of ATP (100 µM) that were applied into apical (top left) or basolateral (top right) bathing solution. In parallel with recording changes in responses of Isc to repeated doses of ATP at each side, single doses of ATP were applied into contralateral bathing solution, and responses were determined during initial phase (I phase, before exposure to repeated applications of ATP), desensitized phase (D phase, after washout of 4 repeated applications of ATP), and recovered phase (R phase, after washout of 10 repeated applications of ATP). Each point and bar represents mean ± SE (n = 4-6).

Receptors involved. In Fig. 4, we determined whether the responses to UTP and 2-MeSATP, relatively specific agonists of the P2u and P2y receptors, undergo the similar course of desensitization and recovery as in the ATP-stimulated responses. In addition, we determined whether the desensitization and recovery induced by each agonist can cross-affect the responses to the other. The ATP- and UTP-induced responses showed the same course of desensitization (Fig. 4, top left and middle). The pattern of the response to single UTP exposures during the initial, desensitized, and recovery phases of the response to repeated exposures to ATP mimicked those of ATP (Fig. 4, bottom left). Likewise, the pattern of the response to single ATP exposures during the initial, desensitized, and recovery phases of repeated UTP exposures mimicked those of UTP (Fig. 4, bottom middle). In contrast, the response to 2-MeSATP was small (usually <30% of the responses to the same concentration of ATP or UTP), failed to show the recovery process after desensitization, and did not affect the responses to ATP or UTP. These results indicate that the desensitization and recovery processes are mainly associated with the P2u or a P2u-like receptor-dependent mechanism.


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Fig. 4.   Changes in responses of Isc to repeated doses of ATP, UTP, or 2-methylthio-ATP (2-MeSATP) and their cross-effects on responses to each nucleotide. Cells were exposed to repeated challenges of each 100 µM of ATP (left), UTP (middle), or 2-MeSATP (right) which was added into apical bathing solution. In parallel with recording changes in responses of Isc to repeated applications of each nucleotide, cells were exposed to single doses of other two nucleotides, and responses were determined during initial phase (I phase, before exposure to repeated applications of each nucleotide), desensitized phase (D phase, after washout of 4 repeated applications of each nucleotide), and recovered phase (R phase, after washout of 10 repeated applications of each nucleotide). Each point and bar represents mean ± SE (n = 4).

Role of cAMP and adenylyl cyclase. The effect of the protein kinase A inhibitor H-89 (4) in Fig. 2 indicates that the production of cAMP and activation of protein kinase A plays a critical role in the stimulation of Isc by ATP. To elucidate further the role of cAMP-dependent mechanism in the desensitization and recovery of the ATP-stimulated Isc responses, we evaluated the effect of single doses of the membrane-permeable cAMP analog 8-BrcAMP during the initial, desensitized, and recovered phases induced by repeated doses of ATP. The magnitude of the response of Isc to 8-BrcAMP itself was relatively small (18.1 ± 3.1% of ATP-stimulated responses) and remained unchanged during the desensitization and recovery phases induced by repeated exposures to ATP. Importantly, the presence of 8-BrcAMP did not add to the ATP-stimulated responses during the initial or recovered phase (Fig. 5B). In sharp contrast, when added during the desensitized phase, 8-BrcAMP enhanced the ATP-stimulated Isc significantly (Fig. 5, A and B). These data lead us to hypothesize that the desensitization process of the Isc responses to repeated doses of ATP occurs via a second messenger system involving adenylyl cyclase-dependent cAMP production.


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Fig. 5.   Interaction of ATP, 8-bromo-cAMP (8-BrcAMP), and ionomycin on responses of Isc during initial, desensitized, and recovered phases that were induced by repeated applications of ATP. Cells were exposed to repeated challenges of ATP (100 µM) added into apical bathing solution. In parallel with recording changes in responses of Isc to repeated doses of ATP, single dose of 8-BrcAMP (100 µM) or ionomycin (1 µM) was applied into both apical and basolateral bathing solution during initial (before exposure to repeated applications of ATP), desensitized (after washout of 4 repeated applications of ATP), and recovered (after washout of 10 repeated applications of ATP) phases, and its effect on ATP-stimulated response was determined. A: a representative tracing illustrating recovery of ATP-stimulated Isc from desensitization in presence of 8-BrcAMP. B: responses to 8-BrcAMP and ionomycin and its effect on ATP-stimulated responses during initial, desensitized, and recovered phases. Each point and bar represents mean ± SE (n = 5). * P < 0.05 vs. response to ATP alone.

The addition of ionomycin failed to recover the desensitized responses to ATP (Fig. 5B), suggesting that intracellular Ca2+ concentration is not an important factor for the desensitization mechanism. Combined application of ionomycin and 8-BrcAMP elicited a response the magnitude of which was comparable to that by ATP, and it remained unchanged during the desensitization phase by repeated applications of ATP (Fig. 5B). This result is consistent again with our hypothesis that the stimulation of Isc by ATP occurs via both cAMP and Ca2+-dependent mechanisms, and a decreased cAMP production is responsible for the desensitization process. Our data also suggest that the reason that the magnitude of the response to 8-BrcAMP is relatively small is because the electrochemical gradient for Cl- across the apical membrane, which is the driving force for the secretion, is not large in quiescent cells. Therefore, opening of the apical cAMP-dependent Cl- channel alone elicits only a small response. On the other hand, ATP can evoke a full response by activating both the apical Cl- and basolateral K+ conductances via cAMP- and Ca2+-dependent mechanisms.

Changes in intracellular cAMP accumulation. We determined intracellular cAMP accumulation to decipher directly the involvement of adenylyl cyclase in the desensitization and recovery of ATP stimulation of Isc (Fig. 6). We measured intracellular cAMP content in cells prepared in parallel with those used to record Isc in the Ussing chambers. Exposure to ATP for 3 min during the initial phase increased intracellular cAMP content 4.1-fold. The stimulatory effect of ATP on cAMP accumulation was attenuated to 1.6-fold in cells desensitized by four repeated exposures to ATP (desensitized phase), but increased again to 3.2-fold after recovery of the stimulation of Isc by ATP (recovered phase). This result indicates adenylyl cyclase activity changes during the ATP-induced desensitization and recovery process. Equipotent effects of the nonhydrolyzable ATP analog ATPgamma S and failure to stimulate cAMP accumulation by the P1 receptor agonist adenosine indicates that the P1 receptor is not likely to be involved in the stimulation of cAMP production by ATP. Addition of ATP into the basolateral bathing solution also stimulated the production of cAMP, and it was not affected by the desensitization and recovery process induced by the apically added ATP. This is consistent with the result in the Isc experiments (Fig. 3) and confirms our hypothesis that the desensitization and recovery process is limited to the one membrane that is exposed to the repeated challenges. Figure 6 also shows that ATP-stimulated production of cAMP is completely blocked in the presence of indomethacin, a cyclooxygenase inhibitor, suggesting that the metabolic product of arachidonic acid is involved importantly in the signaling mechanism to stimulate adenylyl cyclase activity.


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Fig. 6.   Effect of ATP, adenosine 5'-O-(3-thiotriphosphate) (ATPgamma S), and adenosine on cAMP production during initial, desensitized, and recovered phases of ATP-stimulated Isc. Cells were exposed to repeated challenges of ATP (100 µM) added into apical bathing solution in same manner as in Isc experiments. Change in cAMP production by 3-min single exposure to each nucleotide was determined during initial phase (before exposure to repeated applications of ATP), desensitized phase (after washout of 4 repeated applications of ATP), and recovered phase (after washout of 10 repeated applications of ATP) of ATP-stimulated Isc. To examine involvement of metabolic product of arachidonic acid in ATP-stimulated cAMP production, indomethacin (10 µM) was added into apical bathing solution 5 min before final exposure to ATP, and effect was determined. Data represent means ± SE (n = 6).

Effect of cholera toxin and pertussis toxin. To assess the involvement of G proteins in the cascade of events, we treated cells with cholera toxin or pertussis toxin (1 µg/ml for 3 h, apical side in the Ussing chambers) and observed the responses to repeated doses of ATP (Fig. 7A). The desensitization process was attenuated in cholera toxin-treated cells, supporting the idea that the decrease in adenylyl cyclase-dependent cAMP production is responsible for the desensitization of ATP-induced Isc responses. The desensitization process was also attenuated significantly in pertussis toxin-treated cells. We also evaluated the effect of pertussis toxin on the basal and ATP-stimulated cAMP production. Treatment of the cells with pertussis toxin itself did not affect the basal production of cAMP. It did, however, block the desensitization that occurs after repeated exposure to ATP (Fig. 7B). This suggests that a pertussis toxin-sensitive Gi protein-dependent mechanism might be involved.


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Fig. 7.   Changes in responses of Isc (A) and intracellular cAMP production (B) to repeated doses of ATP in cholera toxin- and pertussis toxin-treated cells. A: cells were treated with 1 µg/ml cholera toxin (CTX) or pertussis toxin (PTX) in apical bathing solution for 3 h, and responses of Isc to repeated challenges of ATP (100 µM) added into apical bathing solution were recorded. B: control and pertussis toxin-treated cells were exposed to repeated challenges of ATP (100 µM) added into apical bathing solution in same manner as in Isc experiments. Changes in basal and stimulated cAMP production by 3-min single exposure to ATP were determined during initial phase (before exposure to repeated applications of ATP), desensitized phase (after washout of 4 repeated applications of ATP), and recovered phase (after washout of 10 repeated applications of ATP) of ATP-stimulated Isc. Each point and bar represent mean ± SE (n = 4). * P < 0.01 vs. basal. # P < 0.01 vs. ATP alone.

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

It is well known (29) and confirmed in our results that in MDCK cells stimulation of Isc by various agonists including ATP occurs via an increase in net basolateral to apical Cl- secretion. In addition, we have shown that ATP stimulates Isc by mechanisms involving both cAMP and intracellular Ca2+ through the activation of apical Cl- and basolateral K+ conductances. The nature of the Cl- secretory pathway is not completely understood. CFTR or Ca2+-activated Cl- channels are not likely to be involved primarily because neither glibenclamide nor DIDS affects the ATP-stimulated Isc response. Our results are also consistent with the report showing that CFTR is not expressed in these cells (31).

The desensitization of the ATP-stimulated Isc responses in our study was expected, but subsequent spontaneous recovery from desensitization with repeated challenges of ATP either in the apical or basolateral solution was an unexpected and interesting phenomenon. Our data showed that repeated challenges of ATP to a particular membrane side did not desensitize the response to contralateral application of ATP. The results indicate strongly that a membrane-specific mechanism, possibly the adenylyl cyclase system itself, is responsible for the desensitization and recovery process.

In a recent report (35), it was suggested that both the P2u and P2y receptors are involved in the stimulation of Isc by apically added ATP in MDCK cells. In our study, the responses to ATP or UTP displayed a similar course of desensitization and recovery and were similarly cross-affected during the desensitization or recovery phase induced by the other. In contrast, the specific P2y agonist 2-MeSATP evoked a relatively small and transient effect that did not cross-affect the responses to either ATP or UTP. These results suggest that the P2u or a P2u-like receptor is the major transducer for the process of stimulation, desensitization, and the recovery of Isc induced by repeated exposure to ATP. The role of the adenylyl cyclase system in the P2 purinergic receptor-mediated response is still controversial. In this regard, some contradictory results have been reported. Inhibition of cAMP accumulation by activation of the P2 purinergic receptor was reported in rat hepatocytes (21), mouse ventricular myocytes (34), FRTL-5 thyroid cells (26), and C6 glioma cells (19, 22). In contrast, several reports suggested that the P2-purinergic receptors could lead to increases in intracellular cAMP accumulation (12, 13, 17). Recently, Post et al. (23) demonstrated the enhancement of cAMP production by the P2 purinergic agonists in MDCK cells. Our results also suggest the involvement of cAMP in the signaling of ATP-induced stimulation of Isc. Inhibition by H-89 of both the initial and recovered responses of Isc to ATP suggests that a protein kinase A-dependent mechanism is involved in both phases of stimulation. Determination of the changes in intracellular cAMP production in response to various purinergic agonists also supported our hypothesis. We can rule out possible involvement of the P1 receptor in ATP-stimulated cAMP production because the nonhydrolyzable ATP analog ATPgamma S as well as ATP stimulated Isc, whereas the P1 receptor agonist adenosine failed to stimulate cAMP production. The action of ATP to stimulate cAMP production may be mediated by cyclooxygenase-dependent metabolism of arachidonic acid, because indomethacin inhibited almost completely the ATP-stimulated Isc and cAMP production in both the initial and recovered phases. Prostaglandin E2 is known to be a major product of arachidonic acid metabolism in the kidney (3) and is a likely candidate as the agonist in this stimulatory pathway.

We have provided several lines of evidence that the adenylyl cyclase system is involved in the desensitization and recovery process of ATP-stimulated Isc. First, addition of the membrane-permeable cAMP during the desensitized phase enhanced the ATP-stimulated responses significantly, whereas it did not during the initial or recovered phase. In addition, the desensitization process was significantly attenuated in cholera toxin-treated cells. These results suggest that the desensitization process is closely linked with the decrease in intracellular cAMP concentration. The absence of additive effect of the membrane-permeable cAMP on the ATP-stimulated responses during the initial and recovered phases implies that ATP can stimulate adenylyl cyclase sufficiently, resulting in maximal activation of cAMP-dependent Cl- conductance in nondesensitized cells. Second, the response to the membrane-permeable cAMP remained unchanged during the ATP-induced desensitization and recovery process. This indicates that Cl- channels or another mechanism that is distal to the production of cAMP is not affected during the desensitization phase. This result, together with the localization to the exposed membrane only of the desensitization process of ATP-stimulated Isc and cAMP production, suggests strongly that a membrane-bound adenylyl cyclase is affected during the desensitization process. Finally, determination of ATP-stimulated cAMP production resulted in a similar course of desensitization and recovery in parallel with the changes in the responses of Isc. This result confirms clearly our hypothesis that modification of the adenylyl cyclase system is involved in the mechanism of the desensitization and recovery of the ATP-induced stimulation of Isc.

The molecular basis underlying the modification of the adenylyl cyclase activity during the ATP-induced desensitization and recovery is not clear. Attenuation of the desensitization process in pertussis toxin-treated cells suggests that a Gi protein might be involved in the desensitization mechanism. The effect of pertussis toxin is not likely to result from a nonspecific increase in intracellular cAMP, because it did not stimulate basal cAMP production by itself. So, it is suggested that the modification of Gi protein during the first several exposures to ATP attenuates the responsiveness of the adenylyl cyclase, which then recovers again with increasing number of repeated exposures.

In summary, our results suggest that the adenylyl cyclase system is involved in the signaling mechanism of the ATP-stimulated of Cl- secretion and its desensitization and recovery in MDCK cells. More detailed studies will give us an insight into the understanding of purinergic receptor-mediated responses and their signaling mechanism.

    ACKNOWLEDGEMENTS

This work was funded by National Institutes of Health Grants DK-32753, DK-48977, and HL-47122 and the Cystic Fibrosis Foundation Research Development Program.

    FOOTNOTES

Address for reprint requests: W. B. Guggino, Professor of Physiology and Pediatrics, Johns Hopkins University School of Medicine, 725 North Wolfe St., Baltimore, MD 21205.

Received 18 December 1996; accepted in final form 29 October 1997.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

1.   Anderson, M. P., D. N. Sheppard, H. A. Berger, and M. J. Welsh. Chloride channels in the apical membrane of normal and cystic fibrosis airway and intestinal epithelia. Am. J. Physiol. 263 (Lung Cell. Mol. Physiol. 7): L1-L14, 1992[Abstract/Free Full Text].

2.   Boarder, M. R., G. A. Weisman, J. T. Turner, and G. F. Wilkinson. G protein-coupled P2 purinoceptors: from molecular biology to functional responses. Trends Pharmacol. Sci. 16: 133-139, 1995[Medline].

3.   Bonvalet, J.-P., P. Pradelles, and N. Farman. Segmental synthesis and actions of prostaglandins along the nephron. Am. J. Physiol. 253 (Renal Fluid Electrolyte Physiol. 22): F377-F387, 1987[Abstract/Free Full Text].

4.   Chijiwa, T., A. Mishima, M. Hagiwara, M. Sano, K. Hayashi, T. Inoue, K. Naito, T. Toshioka, and H. Hidaka. Inhibition of forskolin-induced neurite outgrowth and protein phosphorylation by a newly synthesized selective inhibitor of cyclic AMP-dependent protein kinase, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89), of PC12D pheochromocytoma cells. J. Biol. Chem. 265: 5267-5272, 1990[Abstract/Free Full Text].

5.   Cliff, W. H., and R. A. Frizzell. Separate Cl- conductances activated by cAMP and Ca2+ in Cl--secreting epithelial cells. Proc. Natl. Acad. Sci. USA 87: 4956-4960, 1990[Abstract].

6.   Connolly, T. M., W. J. Lawing, Jr., and P. W. Majerus. Protein kinase C phosphorylates human platelet inositol trisphosphate 5'-phosphomonoesterase, increasing the phosphatase activity. Cell 46: 951-958, 1986[Medline].

7.   Dalziel, H. H., and D. P. Westfall. Receptors for adenine nucleotides and nucleosides: subclassification, distribution, and molecular characterization. Pharmacol. Rev. 46: 449-466, 1994[Medline].

8.   Demolle, D., M. Lecomte, and J. M. Boeynaems. Pattern of protein phosphorylation in aortic endothelial cells. Modulation by adenine nucleotides and bradykinin. J. Biol. Chem. 263: 18459-18465, 1988[Abstract/Free Full Text].

9.   Dho, S., K. Stewart, and J. K. Foskett. Purinergic receptor activation of Cl- secretion in T84 cells. Am. J. Physiol. 262 (Cell Physiol. 31): C67-C74, 1992[Abstract/Free Full Text].

10.   Dubyak, G. R., and C. El-Moatassim. Signal transduction via P2-purinergic receptors for extracelluar ATP and other nucleotides. Am. J. Physiol. 265 (Cell Physiol. 34): C577-C606, 1993[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.   Gailly, P., B. Boland, C. Paques, B. Himpens, R. Casteels, and J. M. Gillis. Post-receptor pathway of the ATP-induced relaxation in smooth muscle of the mouse vas deferens. Br. J. Pharmacol. 110: 326-330, 1993[Abstract].

13.   Griese, M., L. I. Gobran, and S. A. Rooney. A2 and P2 purine receptor interactions and surfactant secretion in primary cultures of type II cells. Am. J. Physiol. 261 (Lung Cell. Mol. Physiol. 5): L140-L147, 1991[Abstract/Free Full Text].

14.   Gstraunthaler, G., W. Pfaller, and P. Kotanko. Biochemical characterization of renal epithelial cell cultures (LLC-PK1 and MDCK). Am. J. Physiol. 248 (Renal Fluid Electrolyte Physiol. 17): F536-F544, 1985[Abstract/Free Full Text].

15.   Harden, T. K., J. L. Boyer, and R. A. Nicholas. P2-purinergic receptors: subtype-associated signaling responses and structure. Annu. Rev. Pharmacol. Toxicol. 35: 541-579, 1995[Medline].

16.   Harrison, S. M., and D. M. Bers. The effect of temperature and ionic strength on the apparent Ca-affinity of EGTA and the analogous Ca-chelators BAPTA and dibromo-BAPTA. Biochim. Biophys. Acta 925: 133-143, 1987[Medline].

17.   Henning, R. H., M. Duin, den A. Hertog, and A. Nelemans. Characterization of P2-purinoceptor mediated cyclic AMP formation in mouse C2C12 myotubes. Br. J. Pharmacol. 110: 133-138, 1993[Abstract].

18.   Hwang, T. H., E. M. Schwiebert, and W. B. Guggino. Apical and basolateral ATP stimulate tracheal epithelial chloride secretion via multiple purinergic receptors. Am. J. Physiol. 270 (Cell Physiol. 39): C1611-C1623, 1996[Abstract/Free Full Text].

19.   Lin, W.-W., and D. M. Chuang. Endothelin- and ATP-induced inhibition of adenylyl cyclase activity in C6 glioma cells: role of Gi and calcium. Mol. Pharmacol. 44: 158-165, 1993[Abstract].

20.   Mason, S. J., A. M. Paradiso, and R. C. Boucher. Regulation of transepithelial ion transport and intracellular calcium by extracellular ATP in human normal and cystic fibrosis airway epithelium. Br. J. Pharmacol. 103: 1649-1656, 1991[Abstract].

21.   Okajima, F., Y. Tokumitsu, Y. Kondo, and M. Ui. P2-purinergic receptors are coupled to two signal transduction systems leading to inhibition of cAMP generation and to production of inositol triphosphate in rat hepatocytes. J. Biol. Chem. 262: 13483-13490, 1987[Abstract/Free Full Text].

22.   Pianet, I., M. Merle, and J. Labouesse. ADP and, indirectly, ATP are potent inhibitors of cAMP production in intact isoproterenol-stimulated C6 glioma cells. Biochem. Biophys. Res. Commun. 163: 1150-1157, 1989[Medline].

23.   Post, S. R., J. P. Jacobson, and P. A. Insel. P2 purinergic receptor agonists enhance cAMP production in Madin-Darby canine kidney epithelial cells via autocrine/paracrine mechanism. J. Biol. Chem. 271: 2029-2032, 1996[Abstract/Free Full Text].

24.   Raymond, J. R. Multiple mechanisms of receptor-G protein signaling specificity. Am. J. Physiol. 269 (Renal Fluid Electrolyte Physiol. 38): F141-F158, 1995[Abstract/Free Full Text].

25.   Ryu, S.-H., U.-H. Kim, M. I. Wahl, A. B. Brown, G. Carpenter, K.-P. Huang, and S. G. Rhee. Feedback regulation of phospholipase C-beta by protein kinase C. J. Biol. Chem. 265: 17941-17945, 1990[Abstract/Free Full Text].

26.   Sato, K., F. Okajima, and Y. Kondo. Extracellular ATP stimulates three different receptor-signal transduction systems in FRTL-5 thyroid cells. Biochem. J. 283: 282-287, 1992.

27.   Sheppard, D. N., and M. J. Welsh. Inhibition of the cystic fibrosis transmembrane conductance regulator by ATP-sensitive K+ channel regulators. Ann. NY Acad. Sci. 707: 275-284, 1993[Medline].

28.   Simmons, N. L. Stimulation of Cl- secretion by exogenous ATP in cultured MDCK epithelial monolayers. Biochim. Biophys. Acta 646: 231-242, 1981[Medline].

29.   Simmons, N. L. Renal epithelial Cl- secretion. Exp. Physiol. 78: 117-137, 1993[Medline].

30.   Strabel, D., and M. Diener. Evidence against direct activation of chloride secretion by carbachol in the rat distal colon. Eur. J. Pharmacol. 274: 181-191, 1995[Medline].

31.   Stutts, M. J., C. M. Canessa, J. C. Olsen, M. Hamrick, J. A. Cohn, B. C. Rossier, and R. C. Boucher. CFTR as a cAMP-dependent regulator of sodium channels. Science 269: 847-850, 1995[Medline].

32.   Valentich, J. D. Morphological similarities between the dog kidney cell line MDCK and the mammalian cortical collecting tubule. Ann. NY Acad. Sci. 372: 394-405, 1981.

33.   Wilkinson, G. F., J. R. Purkiss, and M. R. Boarder. Differential heterologous and homologous desensitization of two receptors for ATP (P2Y purinoceptors and nucleotide receptors) coexisting on endothelial cells. Mol. Pharmacol. 45: 731-736, 1994[Abstract].

34.   Yamada, M., Y. Hamamori, H. Akita, and M. Yokoyama. P2-purinoceptor activation stimulates phosphoinositide hydrolysis and inhibits accumulation of cAMP in cultured ventricular myocytes. Circ. Res. 70: 477-485, 1992[Abstract].

35.   Zegra-Moran, O., G. Romeo, and L. J. V. Galietta. Regulation of transepithelial ion transport by two different purinoceptors in the apical membrane of canine kidney (MDCK) cells. Br. J. Pharmacol. 114: 1052-1056, 1995[Abstract].


AJP Cell Physiol 274(2):C371-C378
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