Role of Cyclic ADP-Ribose in ATP-activated Potassium Currents in Alveolar Macrophages*

There is growing evidence that extracellular ATP causes a dramatic change in the membrane conductance of a variety of inflammatory cells. In the present study, using the nystatin perforated patch recording configuration, we found that ATP (0.3–30 μm) induced a transient outward current in a concentration-dependent manner and that the reversal potential of the ATP-induced outward current was close to the K+ equilibrium potential, indicating that the membrane behaves like a K+ electrode in the presence of ATP. The first application of ATP to alveolar macrophages perfused with Ca2+-free external solution could induce the outward current, but the response to ATP was diminished with successive applications. Intracellular perfusion with a Ca2+ chelator, 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid, also diminished the response. When cyclic ADP-ribose (cADPR) was applied to the macrophage cytoplasm, a transient outward current was elicited. Thereafter, the successive outward current was inhibited, suggesting the involvement of cADPR in the response. Intracellular perfusion with inositol 1,4,5-trisphosphate also induced a transient outward current, but the successive current was not inhibited. The ATP-induced outward current was abolished when 8-amino-cADPR (as a blocker of cADPR, 10−6–10−5 m) was introduced into the cytoplasm. Homogenates of alveolar macrophages showed both ADP-ribosyl cyclase and cADPR hydrolase activities, and CD38 (ADP-ribosyl cyclase/cADPR hydrolase) expression was confirmed by reverse transcriptase-polymerase chain reaction and Western blot analyses. These results indicate that ATP activates K+currents by releasing Ca2+ from cADPR-sensitive internal Ca2+ stores.

The alveolar macrophages, which are the most abundant nonparenchymal cells in the lung, play a central role in maintaining normal lung structure and function through their capacity to scavenge particulates, remove macromolecular debris, kill microorganisms, act as accessory cells in immune responses, and recruit and activate other inflammatory cells (1).
There is growing evidence that extracellular ATP causes a dramatic change in the membrane conductance of a variety of inflammatory cells (2). There are several reports concerning the actions of extracellular ATP in macrophage-like established cell lines and, to a lesser extent, macrophages that have been induced by the injection of protein-rich fluids, including patch-clamp studies that described only inward currents by activation of the ion-nonselective conductance (3) and a biphasic current that is composed of a nonselective conductance and a Ca 2ϩ -dependent potassium conductance (4,5).
In this study, using the nystatin perforated patch recording configurations (6,7), we found that extracellular ATP mainly induced an outward current in the nonelicited pulmonary alveolar macrophage. We then analyzed the intracellular mechanism of this response. Here, we also report the first evidence that the macrophage has a Ca 2ϩ store that is sensitive to cyclic ADP-ribose (cADPR), 1 a newly discovered Ca 2ϩ -releasing second messenger (8,9).

EXPERIMENTAL PROCEDURES
Preparation of Alveolar Macrophages-Alveolar macrophages were obtained by pulmonary lavage from specific pathogen-and virus-free, 250 -300-g Wistar rats using a modification of the method of Myrvik et al. (10). In brief, the trachea was cannulated, and the lungs were lavaged four times with 7 ml of standard external solution at 37°C. The lavage fluid was then filtered through a 37-m gauge nylon cloth. The filtrate was centrifuged at 200 ϫ g for 10 min. The pelleted cells were resuspended and stored at 4°C until use. The preparation yielded ϳ10 7 cells/rat, of which Ͼ97% were alveolar macrophages. Within 4 h after anesthetization, the cells were dropped into standard external solution in a Petri dish on the stage of a microscope, and the macrophages adhered to the bottom of the Petri dish within 7 min. In the present study, we adopted the data taken from the macrophages that were not morphologically different before and after the electrical measurements to avoid the possible influence of capacitance changes.
Solutions-The ionic composition of the standard external solution was (in mM): 150 NaCl, 5 KCl, 1 MgCl 2 , 2 CaCl 2 , 10 glucose, and 10 HEPES; the pH was adjusted to 7.4 with Tris base. The external solutions containing 10, 30, and 60 mM K ϩ were made by the substitution of equimolar Na ϩ with K ϩ . The Ca 2ϩ -free external solution containing 2 mM EGTA was composed of (in mM): 150 NaCl, 5 KCl, 1 MgCl 2 , 2 EGTA, 10 glucose, and 10 HEPES, adjusted to pH 7.4 with Tris base. The composition of the patch-pipette (internal) solution for the nystatin * This study was supported in part by grants-in-aid from the Ministry of Education, Science, Sports and Culture, Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18  perforated patch was (in mM): 150 KCl and 10 HEPES, adjusted to pH 7.2 with Tris base. Nystatin was dissolved in methanol (10 mg/ml) and then diluted to a final concentration of 75 g/ml in the pipette solution as described (7). The composition of the pipette solution for the conventional whole-cell patch was (in mM): 150 KCl, 1 MgCl 2 , 5 Na 2 ATP, 0.5 Na 2 GTP, 0.5 EGTA, and 10 HEPES, adjusted to pH 7.2 with Tris base.
Electrical Measurements-The nystatin perforated patch recording configuration (6) was used to prevent the diffusion of intracellular constituents into the patch-pipette (7). Ionic currents were measured with a patch-clamp amplifier (EPC-7, List Electronic, Darmstadt, Germany), low pass filtered at 1 kHz (FV-665, NF Electronic Instruments, Yokohama, Japan), and monitored on both a storage oscilloscope (HS-5100A, Iwatsu, Tokyo, Japan) and a pen recorder (RECTI-HORIZ-8K21, Nippondenki San-ei, Tokyo, Japan). Patch-pipettes were made of glass capillary with an outer diameter of 1.5 mm using a vertical puller (PB-7, Narishige Scientific Instruments, Tokyo, Japan) and had a tip resistance of 4 -8 megohms. The junctional potential between the patch-pipette and bath solution was nulled by the amplifier circuitry.
Drug Application-Drugs were applied rapidly using the Y-tube method (11). In brief, the small orifice (about 40 m in diameter) of the Y-tube tip was placed near a macrophage in a Petri dish with continuous perfusion of the external solution. One of the other two ends was immersed in the external solution in a test tube, and the third was connected to a vacuum pump via an electromagnetic valve that was controlled with a stimulator (SEN-7103, Nihon Koden). With the opening of the valve for 1 s, the external solution was drawn from the test tube beyond the orifice by a negative pressure of Ϫ400 mmHg. Following closure of the valve, the external solution was flushed out from the Y-tube tip to the macrophage by gravity. The exchange of the external solution surrounding an alveolar macrophage was completed within 10 -20 ms.

Currents Induced by Externally
Applied ATP-Using the nystatin perforated patch recording mode, whole-cell currents were recorded from the alveolar macrophages. Under the current-clamp condition, the average resting membrane potential of the alveolar macrophage was Ϫ22.5 Ϯ 3.7 mV (n ϭ 21). The mean input capacitance was 19.8 Ϯ 2.5 picofarads (n ϭ 12). Of 138 alveolar macrophages treated with 10 M ATP at a holding potential (V H ) of Ϫ20 mV, 104 (75.4%) responded to ATP. In the 104 macrophages that responded to ATP, 10 M ATP evoked a transient outward current ( In the present experiments, we focused on elucidating the ATP-induced transient outward current (I ATP ) because this current constituted the major response in the alveolar macrophages. Using the nystatin perforated patch recording mode, successive applications of ATP at intervals of more than 3 min induced almost identical outward currents. The mean peak amplitude of I ATP was 122 Ϯ 7 pA (n ϭ 82). Therefore, in the present study, mainly the nystatin perforated patch recording mode was used to analyze I ATP . The conventional whole-cell patch technique was also used for intracellular perfusion of BAPTA, inositol 1,4,5-trisphosphate (IP 3 ), cADPR, and 8-NH 2 -cADPR.
Concentration-Response Relationships of I ATP - Fig. 2 shows the concentration dependence of I ATP elicited by various concentrations of ATP applied with the Y-tube method at a V H of Ϫ20 mV. The peak amplitude of I ATP increased in a concentrationdependent manner over the concentration range between 0.3 and 30 M. At concentrations of ATP above 100 M, ATP evoked an additional inward current that was superimposed on the I ATP (not shown), and those current traces resembled that in Fig. 1D.
The latent time from the application of ATP until the onset of the I ATP (T L ) was measured. In this experiment, the external solution surrounding an alveolar macrophage could be exchanged completely within 20 ms (10). Therefore  Fig. 3B). The average reversal potential of the I ATP (E ATP ) estimated from the intersection on the voltage axis in the I-V curves was Ϫ79.5 Ϯ 1.2 mV (n ϭ 6). This value was close to the K ϩ equilibrium potential (E K ) of Ϫ85.9 mV calculated with the Nernst equation for the given extra-and intracellular K ϩ concentrations ([K ϩ ] o ϭ 5 mM and [K ϩ ] i ϭ 150 mM, respectively).
Effect of Various K ϩ Channel Blockers on I ATP -To elucidate the pharmacological properties of the K ϩ channel activated by ATP, the effects of K ϩ channel blockers such as apamin, charybdotoxin, tetraethylammonium, and quinidine on I ATP were tested at a V H of Ϫ20 mV. The macrophages were pretreated for 2 min with the normal external solution containing various K ϩ channel blockers. Then 10 M ATP and one of the blockers were applied simultaneously. I ATP was almost completely inhibited by 0.5 mM quinidine (Fig. 4). In contrast, blockers of two types of Ca 2ϩ -dependent K ϩ channels, 1 M apamin, which blocks the small K ϩ conductance (20), and 1 M charybdotoxin, which blocks the large and intermediate K ϩ conductances (21), were ineffective. 10 mM tetraethylammonium also had no effect on the I ATP (Fig. 4B).
Ca 2ϩ Dependence of I ATP -To determine whether a rise in the intracellular free Ca 2ϩ concentration ([Ca 2ϩ ] i ) is necessary for the I ATP , the following studies were performed. In the first study, the effect of intracellular Ca 2ϩ chelation by BAPTA on I ATP was examined. After the I ATP was recorded by the nystatin perforated patch recording mode with the patch-pipette filled with solution containing 10 mM BAPTA, the patched membrane was ruptured by applying negative pressure. The recording that followed was made in the conventional whole-cell mode. Intracellular perfusion with BAPTA caused a complete abolition of the I ATP in all macrophages tested (n ϭ 7) (Fig. 5). On the other hand, 6-min intracellular perfusion with the patch-pipette solution without BAPTA in the conventional whole-cell patch recording mode after membrane rupture had little effect on the I ATP (84 Ϯ 16% of control, n ϭ 5). The second FIG. 2. Concentration-response relationship of I ATP . The macrophage was exposed to various concentrations of ATP in the same cell at V H ϭ Ϫ20 mV. The I ATP was measured at the peak of the response induced by ATP. All responses were normalized to the peak current induced by 3 M ATP (*). Each point shows the mean Ϯ S.E. of five to seven macrophages. study was performed with the nystatin perforated patch recording configuration. ATP was applied to the macrophages before and during continuous superfusion with Ca 2ϩ -free external solution containing 2 mM EGTA. The first application of ATP during the perfusion with Ca 2ϩ -free external solution could induce an I ATP similar to that in the standard external solution. However, the second application of ATP after a 3-min interval induced little I ATP , and the fourth and subsequent applications could not induce any response, as shown in Fig.  5B. 3 min after the return to the standard external solution containing 2 mM Ca 2ϩ , the I ATP was completely restored. These results indicate that the increase of [Ca 2ϩ ] i is an important factor for inducing the I ATP and that Ca 2ϩ is released from the intracellular Ca 2ϩ stores in the presence of ATP.
Ca 2ϩ -releasing Mechanisms Involved in I ATP -Two major mechanisms are known to mediate Ca 2ϩ release from intracellular Ca 2ϩ stores (22). One is the IP 3 -dependent pathway, whereas the other is Ca 2ϩ -induced Ca 2ϩ release (CICR) (23). The CICR is believed to be mediated by the ryanodine receptor, which is also a Ca 2ϩ -sensitive Ca 2ϩ channel (24,25). Accumulating evidence indicates that CICR may be regulated by cADPR, a newly discovered cyclic nucleotide (9). To evaluate which mechanism is involved in I ATP , we perfused IP 3 or cADPR intracellularly. Using the nystatin perforated patch mode, the first application of 10 Ϫ5 M ATP markedly inhibited the response to the second application of 10 Ϫ5 M ATP 20 s after the current traces returned to the base line (Fig. 6A). Intracellular perfusion with 10 Ϫ5 M IP 3 induced a transient outward current (88.2 Ϯ 8.2 pA, n ϭ 5), and the application of 10 Ϫ5 M ATP 20 s after the current trace returned to the base line induced an I ATP of ordinary size (92.6 Ϯ 9.5 pA, n ϭ 5, Fig. 6B). On the other hand, the intracellular perfusion with cADPR also induced a transient outward current, but successive applications of 10 Ϫ5 M ATP of 20 s after the current trace returned to the base line could induce only a small current (Fig. 6C). Moreover, an antagonist of cADPR, 8-NH 2 -cADPR (10 Ϫ6 -10 Ϫ5 M), abolished I ATP when introduced into the cell interior via patch-pipette using a conventional whole-cell configuration (Fig. 6D). In 12 out of 13 cells investigated, exogenous ATP elicited essentially no response in the presence of 8-NH 2 -cADPR. Only one cell responded to ATP with a transient inward current (in a manner quite similar to Fig. 1B). Fig. 6E shows the concentration dependence of cADPR regulation on the K ϩ current. These results suggested that cADPR-sensitive Ca 2ϩ stores might be involved in the I ATP .
Detection of cADPR Metabolic Enzyme in Macrophages-To confirm the presence of a cADPR-mediated signaling system in macrophages, we prepared RNAs and performed reverse transcriptase-PCR analysis of mRNAs for CD38 and BST-1 which catalyze the synthesis and degradation of cADPR (15,18,26). As shown in Fig. 7A, BST-1 mRNA was detected in almost all of the tissues examined except for salivary gland. CD38 mRNA was detected in liver, spleen, thymus, islets, cerebellum, cerebrum, heart, salivary gland, and alveolar macrophages. Moreover, CD38 was detected in alveolar macrophages by Western blot analysis (Fig. 7B), and the macrophage homogenate exhibited both ADP-ribosyl cyclase (14.03 Ϯ 3.99 pmol/min/mg of protein, n ϭ 3) and cADPR hydrolase (327.7 Ϯ 51.5 pmol/ min/mg of protein, n ϭ 3) activities.

Effects of Extracellular ATP in Alveolar Macrophages-We
have studied the electrophysiological effects of extracellular ATP on the membrane properties of rat alveolar macrophages. Under the whole-cell voltage-clamp condition, the typical current response to extracellular ATP is a transient outward current at a V H of Ϫ20 mV, which is close to the resting membrane potential. This resting membrane potential was more positive than that measured in cultured macrophages (3,24). In cultured cells, there is a possibility that the membrane properties are changed by the protein-rich solution of the culture. However, in our present preparation, there is also a possibility that the cells were damaged by the collecting procedure. It is difficult to determine which preparation is more physiological. At the resting membrane potential of our preparation, we clearly showed that ATP induced mainly an outward K ϩ current, which is mediated via the release of Ca 2ϩ from internal stores. Using the patch-clamp technique, evidence for the existence of two types of Ca 2ϩ -dependent K ϩ channels (delayed rectifier K ϩ channel and inward rectifying K ϩ channel), an outwardly rectifying noninactivating channel, a large conductance anion channel, ligand-dependent Fc receptor-associated channel, and an ATP-activated cation-selective conductance have been reported in macrophages (3,(27)(28)(29)(30). Recently, extracellular ATPinduced Ca 2ϩ -dependent K ϩ channels were reported in thioglycolate-elicited mouse peritoneal macrophages (4,5) and macrophage polykaryons and in human monocyte-derived macrophages (4). In rat alveolar macrophages, the pharmacological properties of the K ϩ current activated by ATP were not only similar to those in mouse peritoneal macrophage (5) in terms of the sensitivity to quinidine and tetraethylammonium, but also to those of G-protein activator-induced K ϩ conduct- ance (K G ) (30). The current-voltage relationship of I ATP shows an outward rectification similar to that of K G . In K G , the mechanism of K ϩ channel activation after G-protein activation has not been elucidated except for the evidence that cyclic AMP and IP 3 are not involved in K G . Therefore, K G is one of the candidate channels responsible for I ATP .
Intracellular Mechanism of I ATP -I ATP was diminished by intracellular perfusion with BAPTA (Fig. 5A). In rat alveolar macrophage, using fura-2/AM, Hagenlocker et al. (31) showed that extracellular ATP increased intracellular Ca 2ϩ . The evidence showed that the I ATP was most likely due to a rise in the intracellular Ca 2ϩ . Since removal of extracellular Ca 2ϩ from the external medium did not block the response by the first application of ATP, the action of ATP appears to be mediated via the mobilization of Ca 2ϩ from internal stores. However, subsequent attenuation of the response in the Ca 2ϩ -deficient solution suggests that internal Ca 2ϩ stores are depleted during the response and are not subsequently replenished in a Ca 2ϩfree solution. Thus, a refilling of internal stores by external Ca 2ϩ is important for maintaining the vigorous response to ATP. Fig. 6A shows that 20 s is too short for refilling the Ca 2ϩ stores after the maximal ATP response.
There is evidence that stimulation of macrophages by platelet-activating factor (32) or bacterial lipopolysaccharide (33) leads to the activation of the phospholipase C cascade and to a subsequent increase in intracellular Ca 2ϩ . Moreover, Pfeilschifter et al. (34) reported that extracellular ATP stimulates poly(inositol phospholipid) hydrolysis in mouse peritoneal macrophages in culture. Therefore, the most likely possibility is that ATP stimulates the production of IP 3 , resulting in a rise in the intracellular Ca 2ϩ and activation of Ca 2ϩ -dependent K ϩ channels. However, in this study, Ca 2ϩ store depletion by IP 3 did not inhibit the subsequent ATP response, implying that the IP 3 -sensitive store might not be the sole Ca 2ϩ store responsible for I ATP . An interesting candidate for this purinergic signaling pathway is the newly discovered Ca 2ϩ -releasing compound cADPR, which is synthesized from NAD ϩ by ADP-ribosyl cyclase (35,36) and which is reported to be dependent upon cyclic GMP (22,37). It is interesting to note that stimulation of macrophages with ATP causes synthesis of cGMP (38). This cyclic GMP production could be implicated in the Ca 2ϩ release through the cADPR pathway. Our finding that Ca 2ϩ store stimulation by cADPR inhibited the successive ATP response suggests the possibility that cADPR acts as an intracellular Ca 2ϩ -releasing messenger following ATP stimulation. The experiment using 8-NH 2 -cADPR, a blocker of cADPR, further supported the idea. This insight was also evidenced by the fact that CD38 (ADP-ribosyl cyclase/cADPR hydrolase) and its mRNA were detected and that cADPR metabolizing activities were detected in macrophages. Furthermore, FK506-binding proteins were recently shown to be essential for the Ca 2ϩ release by cADPR from the ryanodine receptor and also detected in macrophages (39).
The onset of I ATP was abrupt, following about 10 s of total quiescence after the application of ATP, thereby indicating the existence of a definite latency in the appearance of the ATP response. In some recent reports the responses mediated by cADPR followed the responses mediated by IP 3 in lacrimal acinar cells (40) and tracheal mucosal gland (12). The latency of I ATP in the alveolar macrophage is quite similar to the time required for lacrimal acinar cells to reach the peaks in the intracellular Ca 2ϩ transient induced by ␤-adrenergic stimulation and for the muscarinic receptor-coupled K ϩ (M) channel inhibition by acetylcholine in rodent NG108 -15 cells (41). Because the involvement of the cADPR-mediated signaling pathway has been reported in these cell types, the evidence might also suggest that the I ATP was mediated by cADPR. The symbols depicted on the left in panels A and B represent a nystatin perforated patch configuration, and that on the right in panel A represents a conventional whole-cell patch. The nystatin perforated patch is thought to form monovalent ion paths in the membrane just beneath the patch-pipette, preventing washout of cellular factors by the pipette solution. The conventional whole-cell configuration was employed to introduce molecules such as BAPTA into the cytoplasm via the patch-pipette through a rupture made by repetitive negative pressure applied to the pipette tip. Panel A, effect of intracellular BAPTA on I ATP at a V H of Ϫ20 mV. ATP (10 Ϫ5 M) was applied before (left) and 6 min after (right) the rupture of the patch membrane with a pipette filled with a solution containing 10 mM BAPTA. I ATP was blocked completely by intracellular perfusion with 10 mM BAPTA. The results are typical of five reproducible observations. Panel B, effect of removing extracellular Ca 2ϩ on I ATP . Current traces were obtained from the same macrophage at a V H of Ϫ20 mV. The ATP (10 Ϫ5 M) was applied repeatedly at an interval of 3 min before and during the superfusion with Ca 2ϩ -free external solution containing 2 mM EGTA. The first application of ATP in Ca 2ϩ -free solution induced an I ATP that was almost identical to that in the standard external solution, but the I ATP values in subsequent applications were inhibited. Restoration to the standard external solution resulted in complete recovery of the I ATP . The results are typical of five reproducible observations. We report here the first evidence that alveolar macrophages have a cADPR-sensitive Ca 2ϩ store. Accumulating evidence suggests that cADPR may be an endogenous modulator of the CICR mechanism (9). The CICR is believed to be mediated by the ryanodine receptor, which is also a Ca 2ϩ -sensitive Ca 2ϩ channel (24,25). It was originally thought that caffeine acts solely as an agonist for the ryanodine receptor. In this study, caffeine and ryanodine could not induce any Ca 2ϩ -dependent K ϩ current, implying that the rat alveolar macrophages have no CICR mechanism. 2 However, recent experiments indicate that these compounds have multiple pharmacological effects, confusing the classification of intracellular Ca 2ϩ release channels (42). Therefore, further studies are needed to evaluate the Ca 2ϩ -releasing mechanism in rat alveolar macrophages.
Possible Physiological roles of I ATP -A concentration of 10 M extracellular ATP sufficed to induce clearly detectable outward currents. This value is less than that detected in the plasma after degranulation of platelets (43), suggesting that the phenomena we described here can be of physiological significance. The ability of ATP to increase the intracellular free Ca 2ϩ concentration raises the interesting possibility that some of the secretory and immune actions of macrophages are regulated by extracellular ATP (2). The physiological roles of the activation of Ca 2ϩ -dependent K ϩ channels by ATP in alveolar macrophages are uncertain, but they could amplify the Ca 2ϩ signal by keeping the cell membrane at a hyperpolarized potential to sustain the driving force for Ca 2ϩ influx during macrophage activation. Recently, a Ca 2ϩ current activated by intracellular Ca 2ϩ store depletion has been characterized in a variety of cells including macrophages (44 -46), and this current has been called the calcium release-activated current (I CRAC ). I CRAC is activated by molecules, called Ca 2ϩ influx factor (CIF), released from depleted Ca 2ϩ stores (45,46) and is also facilitated when the membrane is hyperpolarized (44). Therefore, in alveolar macrophages stimulated by ATP, both CIF and Ca 2ϩ -depend-FIG. 6. Involvement of cADPR in I ATP . The current trace in panel A was obtained by the nystatin perforated patch mode at a V H of Ϫ20 mV. The current traces in panels B, C, and D were obtained by the conventional whole-cell patch recording mode at a V H of Ϫ20 mV, and whole-cell current recordings were started at the point indicated by arrows; the traces to the left of the arrows were obtained by the cell-attached configuration. The symbols at the left side of each record are as described in the legend for Fig. 5. The results are typical of 4 to 12 reproducible observations. Panel A, the application of 10 Ϫ5 M ATP markedly diminished the successive ATP response. Panel B, intracellular perfusion of 10 Ϫ5 M IP 3 induced an outward current, and the successive application of ATP also induced an ordinary outward current. Panel C, intracellular perfusion of 10 Ϫ6 M cADPR mimicked the ATP response, but the successive ATP response was inhibited dramatically. Panel D, intracellular perfusion of 10 Ϫ5 M 8-NH 2 -cADPR, an antagonist of cADPR, abolished the response to exogenous ATP. In this series of experiments, ATP was applied 3 min after the establishment of the whole-cell configuration to perfuse the cell interior with 8-NH 2 -cADPR sufficiently. Panel E, the concentration dependence of cADPR regulation of the K ϩ current. The current was obtained with the conventional whole-cell configuration as shown in Fig.  6C in the presence of various concentrations of cADPR in pipette. Each point is the mean Ϯ S.E. of five to eight macrophages. ent K ϩ current might act synergistically to replenish the Ca 2ϩ store.