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

(Received for publication, February 28, 1997, and in revised form, April 14, 1997)

Satoru Ebihara Dagger §, Tsukasa Sasaki Dagger , Wataru Hida Dagger , Yoshihiro Kikuchi Dagger , Takako Oshiro Dagger , Sanae Shimura Dagger , Shin Takasawa §, Hiroshi Okamoto , Akinori Nishiyama par , Norio Akaike ** and Kunio Shirato Dagger Dagger Dagger

From the Dagger  First Department of Internal Medicine, the  Department of Biochemistry, and the par  First Department of Physiology, Tohoku University School of Medicine, Sendai 980-77 and the ** Department of Physiology, Faculty of Medicine, Kyushu University, Fukuoka 812-82, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

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.


INTRODUCTION

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 Ca2+-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 Ca2+ store that is sensitive to cyclic ADP-ribose (cADPR),1 a newly discovered Ca2+-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-µm gauge nylon cloth. The filtrate was centrifuged at 200 × g for 10 min. The pelleted cells were resuspended and stored at 4 °C until use. The preparation yielded ~107 cells/rat, of which >97% 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 MgCl2, 2 CaCl2, 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+ were made by the substitution of equimolar Na+ with K+. The Ca2+-free external solution containing 2 mM EGTA was composed of (in mM): 150 NaCl, 5 KCl, 1 MgCl2, 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 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 µg/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 MgCl2, 5 Na2ATP, 0.5 Na2GTP, 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 µm 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 -400 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.

Chemicals

cADPR was prepared from NAD+ with ADP-ribosyl cyclase purified from ovotestes of Aplysia kurodai, a marine mollusk common around the Japanese coast, as described (8, 12). 8-Amino (NH2)-cADPR was synthesized and purified as described (13, 14). BAPTA was obtained from Dojin, Kumamoto, Japan. Charybdotoxin was from Peptide Institute, Osaka, Japan. All other chemicals used were purchased from Sigma.

Reverse Transcriptase-Polymerase Chain Reaction (PCR)

Total RNA was isolated (15), and 1 µg of total RNA was incubated with 500 units of SuperscriptTM (Life Technologies, Inc.) for 1 h at 42 °C in a total reaction volume of 20 µl containing 1 × reverse transcriptase buffer (50 mM Tris-HCl, pH 8.3, 40 mM KCl, 6 mM MgCl2, 1 mM dithiothreitol), a 0.5 mM concentration of each dNTP, 110 units of RNase inhibitor (Takara Shuzo, Otsu, Japan), and 1.5 ng/µl oligo(dT)12-18 (Pharmacia Biotech Inc.). The reverse transcriptase sample (1 µl) was used for PCR in a final volume of 50 µl as described (15, 16). The PCR primers correspond to nucleotides 168-187 and 822-842 (15) for rat CD38 mRNA, 138-157 and 598-617 for rat BST-1 mRNA (16), and 135-155 and 951-971 (17) for rat G3PDH mRNA. The nucleotide sequences of the resultant PCR products were confirmed by dideoxy sequencing.

Western Blot Analysis

Rat alveolar macrophages (7 × 106 cells) were homogenized in 0.4 ml of homogenizing buffer (50 mM MES, 1 mM EDTA, and 0.25 M sucrose, pH 7.2) supplemented with 0.5 mM phenylmethylsulfonyl fluoride. Western blot analysis was carried out as described (18, 19).

Enzyme Assays for cADPR Formation and Hydrolysis

Rat alveolar macrophages were homogenized as described above. ADP-ribosyl cyclase and cADPR hydrolase activities were measured essentially as described (18, 19). Briefly, the macrophage homogenate (50 µg of protein) was incubated for 1 h at 37 °C in 0.1 ml of phosphate-buffered saline (137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4, 1.5 mM KH2PO4, pH 7.4) with 0.2 mM NAD+ containing 5 µCi of [32P]NAD+ (DuPont NEN) for ADP-ribosyl cyclase or with 0.2 mM cADPR containing 5 µCi of [32P]cADPR for cADPR hydrolase. Reaction products were analyzed by high performance liquid chromatography using a flow scintillation analyzer (Flow-One Beta-525TR, Packard, Meriden, CT).


RESULTS

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 -22.5 ± 3.7 mV (n = 21). The mean input capacitance was 19.8 ± 2.5 picofarads (n = 12). Of 138 alveolar macrophages treated with 10 µM ATP at a holding potential (VH) of -20 mV, 104 (75.4%) responded to ATP. In the 104 macrophages that responded to ATP, 10 µM ATP evoked a transient outward current (Fig. 1A) in 82 cells (78.8%), inward currents (Fig. 1B) in 5 cells (4.8%), outward oscillations (Fig. 1C) in 3 cells (2.9%), and a mixture of outward and inward currents (Fig. 1D) in 14 cells (13.5%). No significant differences in morphology were observed among the four types of rat macrophages studied.


Fig. 1. Different patterns of currents evoked by 10 µM ATP at a VH of -20 mV using the nystatin perforated patch recording configuration. ATP was applied for the period indicated by a horizontal bar above each current trace. Panel A, ATP-induced transient outward current; panel B, ATP-induced inward current; panel C, ATP-induced outward oscillation; panel D, ATP-induced mixture of an outward and an inward current.
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In the present experiments, we focused on elucidating the ATP-induced transient outward current (IATP) 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 IATP was 122 ± 7 pA (n = 82). Therefore, in the present study, mainly the nystatin perforated patch recording mode was used to analyze IATP. The conventional whole-cell patch technique was also used for intracellular perfusion of BAPTA, inositol 1,4,5-trisphosphate (IP3), cADPR, and 8-NH2-cADPR.

Concentration-Response Relationships of IATP

Fig. 2 shows the concentration dependence of IATP elicited by various concentrations of ATP applied with the Y-tube method at a VH of -20 mV. The peak amplitude of IATP increased in a concentration-dependent manner over the concentration range between 0.3 and 30 µM. At concentrations of ATP above 100 µM, ATP evoked an additional inward current that was superimposed on the IATP (not shown), and those current traces resembled that in Fig. 1D.


Fig. 2. Concentration-response relationship of IATP. The macrophage was exposed to various concentrations of ATP in the same cell at VH = -20 mV. The IATP was measured at the peak of the response induced by ATP. All responses were normalized to the peak current induced by 3 µM ATP (*). Each point shows the mean ± S.E. of five to seven macrophages.
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The latent time from the application of ATP until the onset of the IATP (TL) was measured. In this experiment, the external solution surrounding an alveolar macrophage could be exchanged completely within 20 ms (10). Therefore, the time when the ATP reached the cell could be determined accurately by monitoring the closure of the electromagnetic valve. The TL values at 22 °C were 10.3 ± 2.2 s (n = 5), 8.5 ± 1.2 s (n = 7), 9.2 ± 1.2 s (n = 7), 8.2 ± 0.7 s (n = 7), and 9.1 ± 1.0 s (n = 7) for 0.3, 1, 3, 10, and 30 µM ATP, respectively, indicating that the TL was independent of the concentration range between 0.3 and 30 µM.

Current-Voltage Relationship for IATP

Fig. 3A shows the currents induced by 10 µM ATP at various VH values. In the current (I)-voltage (V) relationship, the ATP responses at various VH values were normalized to the peak response induced by ATP at a VH of -40 mV (*, Fig. 3B). The average reversal potential of the IATP (EATP) estimated from the intersection on the voltage axis in the I-V curves was -79.5 ± 1.2 mV (n = 6). This value was close to the K+ equilibrium potential (EK) of -85.9 mV calculated with the Nernst equation for the given extra- and intracellular K+ concentrations ([K+]o = 5 mM and [K+]i = 150 mM, respectively).


Fig. 3. Current-voltage (I-V) relationship of the IATP. Panel A, ATP-induced currents at various VH values in the same macrophage perfused with external and internal solutions containing 5 and 150 mM K+, respectively. Panel B, I-V relationship for ATP. Each point is the average of six macrophages. Panel C, effect of [K+]o on EATP. Macrophages were perfused with external solutions containing various concentrations of K+. The [K+]i was 150 mM throughout this experiment. Each point is the mean ± S.E. of five to six macrophages.
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When the macrophages were perfused with a normal internal solution and an external solution containing various concentrations of K+ ([K+]o = 10, 30 and 60 mM), the EATP values were -63.1 ± 3.1 mV (n = 5), -43.8 ± 1.8 mV (n = 6), and -23.5 ± 1.2 mV (n = 5) for 10, 30, and 60 mM [K+]o, respectively. The change in EATP for a 10-fold change in [K+]o was 51.9 mV (Fig. 3C), indicating that the cell membrane behaves like a K+ electrode in the presence of ATP.

Effect of Various K+ Channel Blockers on IATP

To elucidate the pharmacological properties of the K+ channel activated by ATP, the effects of K+ channel blockers such as apamin, charybdotoxin, tetraethylammonium, and quinidine on IATP were tested at a VH of -20 mV. The macrophages were pretreated for 2 min with the normal external solution containing various K+ channel blockers. Then 10 µM ATP and one of the blockers were applied simultaneously. IATP was almost completely inhibited by 0.5 mM quinidine (Fig. 4). In contrast, blockers of two types of Ca2+-dependent K+ channels, 1 µM apamin, which blocks the small K+ conductance (20), and 1 µM charybdotoxin, which blocks the large and intermediate K+ conductances (21), were ineffective. 10 mM tetraethylammonium also had no effect on the IATP (Fig. 4B).


Fig. 4. Effect of K+ channel blockers on IATP. Panel A, effect of apamin (upper panel) and quinidine (lower panel) on IATP at a VH of -20 mV. Quinidine reversibly blocked IATP, whereas apamin did not. Panel B, effects of 10-6 M apamin (n = 6), 10-6 M charybdotoxin (n = 5), 10-2 M tetraethylammonium (TEA, n = 7), and 5 × 10-4 M quinidine (n = 8) on IATP. All responses were normalized to the peak current induced by 10-5 M ATP alone. Each column and vertical bar represent mean ± S.E., respectively.
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Ca2+ Dependence of IATP

To determine whether a rise in the intracellular free Ca2+ concentration ([Ca2+]i) is necessary for the IATP, the following studies were performed. In the first study, the effect of intracellular Ca2+ chelation by BAPTA on IATP was examined. After the IATP 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 IATP in all macrophages tested (n = 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 IATP (84 ± 16% of control, n = 5). The second study was performed with the nystatin perforated patch recording configuration. ATP was applied to the macrophages before and during continuous superfusion with Ca2+-free external solution containing 2 mM EGTA. The first application of ATP during the perfusion with Ca2+-free external solution could induce an IATP similar to that in the standard external solution. However, the second application of ATP after a 3-min interval induced little IATP, 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 Ca2+, the IATP was completely restored. These results indicate that the increase of [Ca2+]i is an important factor for inducing the IATP and that Ca2+ is released from the intracellular Ca2+ stores in the presence of ATP.


Fig. 5. Involvement of Ca2+ in IATP. 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 IATP at a VH of -20 mV. ATP (10-5 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. IATP was blocked completely by intracellular perfusion with 10 mM BAPTA. The results are typical of five reproducible observations. Panel B, effect of removing extracellular Ca2+ on IATP. Current traces were obtained from the same macrophage at a VH of -20 mV. The ATP (10-5 M) was applied repeatedly at an interval of 3 min before and during the superfusion with Ca2+-free external solution containing 2 mM EGTA. The first application of ATP in Ca2+-free solution induced an IATP that was almost identical to that in the standard external solution, but the IATP values in subsequent applications were inhibited. Restoration to the standard external solution resulted in complete recovery of the IATP. The results are typical of five reproducible observations.
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Ca2+-releasing Mechanisms Involved in IATP

Two major mechanisms are known to mediate Ca2+ release from intracellular Ca2+ stores (22). One is the IP3-dependent pathway, whereas the other is Ca2+-induced Ca2+ release (CICR) (23). The CICR is believed to be mediated by the ryanodine receptor, which is also a Ca2+-sensitive Ca2+ 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 IATP, we perfused IP3 or cADPR intracellularly. Using the nystatin perforated patch mode, the first application of 10-5 M ATP markedly inhibited the response to the second application of 10-5 M ATP 20 s after the current traces returned to the base line (Fig. 6A). Intracellular perfusion with 10-5 M IP3 induced a transient outward current (88.2 ± 8.2 pA, n = 5), and the application of 10-5 M ATP 20 s after the current trace returned to the base line induced an IATP of ordinary size (92.6 ± 9.5 pA, n = 5, Fig. 6B). On the other hand, the intracellular perfusion with cADPR also induced a transient outward current, but successive applications of 10-5 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-NH2-cADPR (10-6-10-5 M), abolished IATP 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-NH2-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+ current. These results suggested that cADPR-sensitive Ca2+ stores might be involved in the IATP.


Fig. 6. Involvement of cADPR in IATP. The current trace in panel A was obtained by the nystatin perforated patch mode at a VH of -20 mV. The current traces in panels B, C, and D were obtained by the conventional whole-cell patch recording mode at a VH of -20 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 10-5 M ATP markedly diminished the successive ATP response. Panel B, intracellular perfusion of 10-5 M IP3 induced an outward current, and the successive application of ATP also induced an ordinary outward current. Panel C, intracellular perfusion of 10-6 M cADPR mimicked the ATP response, but the successive ATP response was inhibited dramatically. Panel D, intracellular perfusion of 10-5 M 8-NH2-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-NH2-cADPR sufficiently. Panel E, the concentration dependence of cADPR regulation of the K+ 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 ± S.E. of five to eight macrophages.
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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 ± 3.99 pmol/min/mg of protein, n = 3) and cADPR hydrolase (327.7 ± 51.5 pmol/min/mg of protein, n = 3) activities.


Fig. 7. Expression of cADPR-metabolizing enzymes in rat alveolar macrophages. Panel A, reverse transcriptase-PCR analyses of CD38 and BST-1 mRNA. reverse transcriptase-PCR was performed using cDNA converted from 1 µg of total RNA as a template. Lane 1, liver; lane 2, spleen; lane 3, thymus; lane 4, alveolar macrophage; lane 5, islets; lane 6, streptozotocin/nicotinamide-induced insulinomas; lane 7, RINm5F cells; lane 8, cerebellum; lane 9, cerebrum; lane 10, heart; lane 11, skeletal muscle; lane 12, salivary gland; lane 13, kidney. Rat CD38-specific primers (CD38) or rat BST-1-specific primers (BST-1) are used for PCR as described (16). As an internal control, PCR products using glyceraldehyde-3-phosphate dehydrogenase-specific primers are also shown (G3PDH). Panel B, Western blot analysis of CD38. Lane 1, homogenate of COS-7 cells into which the pSV2 vector (18) had been introduced (50 µg of protein); lane 2, homogenate of COS-7 cells into which the human CD38 expression vector (18) had been introduced (50 µg of protein); lane 3, homogenate of COS-7 cells into which the rat CD38 expression vector (15) had been introduced (50 µg of protein); lane 4, homogenate of rat alveolar macrophages (100 µg of protein); lane 5, homogenate of rat alveolar macrophages (50 µg of protein).
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DISCUSSION

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 VH of -20 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+ current, which is mediated via the release of Ca2+ from internal stores. Using the patch-clamp technique, evidence for the existence of two types of Ca2+-dependent K+ channels (delayed rectifier K+ channel and inward rectifying K+ 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, 27-30). Recently, extracellular ATP-induced Ca2+-dependent K+ 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+ 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+ conductance (KG) (30). The current-voltage relationship of IATP shows an outward rectification similar to that of KG. In KG, the mechanism of K+ channel activation after G-protein activation has not been elucidated except for the evidence that cyclic AMP and IP3 are not involved in KG. Therefore, KG is one of the candidate channels responsible for IATP.

Intracellular Mechanism of IATP

IATP 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 Ca2+. The evidence showed that the IATP was most likely due to a rise in the intracellular Ca2+. Since removal of extracellular Ca2+ 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 Ca2+ from internal stores. However, subsequent attenuation of the response in the Ca2+-deficient solution suggests that internal Ca2+ stores are depleted during the response and are not subsequently replenished in a Ca2+-free solution. Thus, a refilling of internal stores by external Ca2+ is important for maintaining the vigorous response to ATP. Fig. 6A shows that 20 s is too short for refilling the Ca2+ 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 Ca2+. 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 IP3, resulting in a rise in the intracellular Ca2+ and activation of Ca2+-dependent K+ channels. However, in this study, Ca2+ store depletion by IP3 did not inhibit the subsequent ATP response, implying that the IP3-sensitive store might not be the sole Ca2+ store responsible for IATP. An interesting candidate for this purinergic signaling pathway is the newly discovered Ca2+-releasing compound cADPR, which is synthesized from NAD+ 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 Ca2+ release through the cADPR pathway. Our finding that Ca2+ store stimulation by cADPR inhibited the successive ATP response suggests the possibility that cADPR acts as an intracellular Ca2+-releasing messenger following ATP stimulation. The experiment using 8-NH2-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 Ca2+ release by cADPR from the ryanodine receptor and also detected in macrophages (39).

The onset of IATP 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 IP3 in lacrimal acinar cells (40) and tracheal mucosal gland (12). The latency of IATP in the alveolar macrophage is quite similar to the time required for lacrimal acinar cells to reach the peaks in the intracellular Ca2+ transient induced by beta -adrenergic stimulation and for the muscarinic receptor-coupled K+ (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 IATP was mediated by cADPR.

We report here the first evidence that alveolar macrophages have a cADPR-sensitive Ca2+ 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 Ca2+-sensitive Ca2+ 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 Ca2+-dependent K+ 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 Ca2+ release channels (42). Therefore, further studies are needed to evaluate the Ca2+-releasing mechanism in rat alveolar macrophages.

Possible Physiological roles of IATP

A concentration of 10 µM 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 Ca2+ 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 Ca2+-dependent K+ channels by ATP in alveolar macrophages are uncertain, but they could amplify the Ca2+ signal by keeping the cell membrane at a hyperpolarized potential to sustain the driving force for Ca2+ influx during macrophage activation. Recently, a Ca2+ current activated by intracellular Ca2+ 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 (ICRAC). ICRAC is activated by molecules, called Ca2+ influx factor (CIF), released from depleted Ca2+ stores (45, 46) and is also facilitated when the membrane is hyperpolarized (44). Therefore, in alveolar macrophages stimulated by ATP, both CIF and Ca2+-dependent K+ current might act synergistically to replenish the Ca2+ store.


FOOTNOTES

*   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. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§   Supported by a grant from Kanae Foundation of Research for New Medicine.
Dagger Dagger    To whom correspondence should be addressed: First Department of Internal Medicine, Tohoku University School of Medicine, Seiryo-machi 1-1, Aoba-ku, Sendai 980-77, Japan. Tel.: 81-22-717-7153; Fax: 81-22-717-7156.
1   The abbreviations used are: cADPR, cyclic ADP-ribose; 8-NH2-cADPR, 8-amino-cADPR; BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; PCR; polymerase chain reaction; MES, 4-morpholineethanesulfonic acid; VH, holding potential; IATP, ATP-induced transient outward current; IP3, inositol 1,4,5-trisphosphate; TL, latent time from the application of ATP until the onset of the IATP; I, current; V, voltage; EATP, average reversal potential of the IATP; EK, K+ equilibrium potential; [Ca2+]i, intracellular free Ca2+ concentration; CICR, Ca2+-induced Ca2+ release; KG, G-protein activator-induced K+ conductance; ICRAC, calcium release-activated current; CIF, Ca2+-influx factor.
2   We used caffeine and ryanodine to try to activate the CICR process. However, using a perforated patch mode, neither 10-2 M caffeine (n = 21) nor 10 µM ryanodine (n = 10) could induce any currents in the alveolar macrophages tested. Moreover, a mixture of caffeine (10-2 M) and ryanodine (10 µM) also could not induce any currents (n = 5).

ACKNOWLEDGEMENTS

We thank Brent Bell for reading the manuscript.


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