(Received for publication, February 28, 1997, and in revised form, April 14, 1997)
From the 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 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).
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.
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.
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.
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
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.
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.
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).
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).
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
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.
Fig.
2 shows the concentration dependence of
IATP elicited by various concentrations of ATP
applied with the Y-tube method at a VH of
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.
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
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
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
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.
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
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.
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
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 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.
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.
We thank Brent Bell for reading the
manuscript.
First Department of Internal Medicine, the
¶ Department of Biochemistry, and the
First Department
of Physiology,
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
,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.
Preparation of Alveolar Macrophages
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.
Currents Induced by Externally Applied ATP
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.
[View Larger Version of this Image (7K GIF file)]
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.
[View Larger Version of this Image (14K GIF file)]
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.
[View Larger Version of this Image (17K GIF file)]
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.
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.
[View Larger Version of this Image (26K GIF file)]
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.
[View Larger Version of this Image (16K GIF file)]
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.
[View Larger Version of this Image (14K GIF file)]
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).
[View Larger Version of this Image (34K GIF file)]
Effects of Extracellular ATP in Alveolar Macrophages
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.
-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.
*
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.
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 102 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).
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.