1. Ingeniería

Journal of Neurochemistry
Lippincott Williams & Wilkins, Inc., Philadelphia
© 1999 International Society for Neurochemistry
ATP-Stimulated Ca2⫹ Influx and Phospholipase D Activities of
a Rat Brain-Derived Type-2 Astrocyte Cell Line, RBA-2, Are
Mediated Through P2X7 Receptors
Synthia H. Sun, Lian-Bin Lin, Amos C. Hung, and *Jon-Son Kuo
Institute of Neuroscience, National Yang Ming University, Taipei; and *Taichung Veteran General Hospital,
Taichung, Taiwan, R.O.C.
Abstract: This study characterizes and examines the P2
receptor-mediated signal transduction pathway of a rat
brain-derived type 2 astrocyte cell line, RBA-2. ATP induced Ca2⫹ influx and activated phospholipase D (PLD).
The ATP-stimulated Ca2⫹ influx was inhibited by
pretreating cells with P2 receptor antagonist, pyridoxalphosphate-6-azophenyl-2⬘,4⬘-disulfonic acid (PPADS),
in a concentration-dependent manner. The agonist 2⬘and 3⬘-O-(4-benzoylbenzoyl)adenosine 5⬘-triphosphate
(BzATP) stimulated the largest increases in intracellular
Ca2⫹ concentrations ([Ca2⫹]i); ATP, 2-methylthioadenosine triphosphate tetrasodium, and ATP␥S were much
less effective, whereas UTP, ADP, ␣,␤-methylene-ATP,
and ␤,␥-methylene-ATP were ineffective. Furthermore,
removal of extracellular Mg2⫹ enhanced the ATP- and
BzATP-stimulated increases in [Ca2⫹]i. BzATP stimulated
PLD in a concentration- and time-dependent manner that
could be abolished by removal of extracellular Ca2⫹ and
was inhibited by suramin, PPADS, and oxidized ATP. In
addition, PLD activities were activated by the Ca2⫹ mobilization agent, ionomycin, in an extracellular Ca2⫹ concentration-dependent manner. Both staurosporine and
prolonged phorbol ester treatment inhibited BzATP-stimulated PLD activity. Taken together, these data indicate
that activation of the P2X7 receptors induces Ca2⫹ influx
and stimulates a Ca2⫹-dependent PLD in RBA-2 astrocytes. Furthermore, protein kinase C regulates this PLD.
Key Words: ATP—2⬘- and 3⬘-O-(4-benzoylbenzoyl)adenosine 5⬘-triphosphate —Ca2⫹ influx—Phospholipase
D —P2X7 receptor—Type-2 astrocytes.
J. Neurochem. 73, 334 –343 (1999).
tion regarding the function and pharmacology of neurotransmitter receptor subtypes on type-2 astrocytes is very
limited.
The P2 receptors were classified initially into three
groups: (a) the G protein-coupled P2Y class, which
upon activation induces phosphoinositide hydrolysis
and Ca2⫹ release; (b) the cation channel P2X class,
which induces rapid depolarization of membranes; and
(c) the nonselective pore-forming P2Z class, which
induces cytolytic activities in macrophages and other
types of cells. These receptors have been shown to be
widely distributed in the CNS (Dubyak and ElMoatassim, 1993; Burnstock, 1997). In general, astrocytes possess metabotropic P2Y receptors, which induce Ca2⫹ release from intracellular stores (Pearce et
al., 1989; Kastritsis et al., 1992; King et al., 1996), and
P2Z receptors, which induce Ca2⫹ influx from extracellular space (Ballerini et al., 1996). Activation of
both types of receptors leads to increases in intracellular free Ca2⫹ concentration ([Ca2⫹]i). Thus, ATP,
which is recognized as a neurotransmitter that is coreleased from synapses (Burnstock, 1972), might play an
important role in neuron–astrocyte interaction.
It is known that activation of the P2Z receptor requires
a high concentration of ATP (El-Moatassim and Dubyak,
1993); the activation allows a bidirectional increase in
Received December 1, 1998; revised manuscript received February
12, 1999; accepted March 1, 1999.
Address correspondence and reprint requests to Dr. S. H. Sun at
Institute of Neuroscience, National Yang Ming University, #155, Section 2, Li-Non Street, Shi-Pai, Taipei, Taiwan, R.O.C.
Abbreviations used: ATP␥S, adenosine 5⬘-O-(3-thiotriphosphate);
BzATP, 2⬘- and 3⬘-O-(4-benzoylbenzoyl)adenosine 5⬘-triphosphate;
[Ca2⫹]i, intracellular free Ca2⫹ concentration; ECL, enhanced chemiluminescence; GFAP, glial fibrillary acidic protein; GS, glutamine
synthetase; 2-MeSATP, 2-methylthioadenosine triphosphate tetrasodium; ␣,␤-methylene-ATP, ␣,␤-methyleneadenosine 5⬘-triphosphate
dilithium; ␤,␥-methylene-ATP, ␤,␥-methyleneadenosine 5⬘-triphosphate tetrasodium; oATP, oxidized ATP; PBS, phosphate-buffered
saline; PEt, phosphatidylethanol; PKC, protein kinase C; PL, phospholipid; PLD, phospholipase D; PMA, phorbol 12-myristate 13-acetate;
PPADS, pyridoxalphosphate-6-azophenyl-2⬘,4⬘-disulfonic acid.
Type-2 astrocytes were first described in optic nerves.
They have stellate morphology, are glial fibrillary acidic
protein (GFAP)- and A2B5-positive, and extend processes to the nodes of Ranvier in vivo (Raff, 1989). They
are also found in other regions of the brain (Gallo et al.,
1989; Usowicz et al., 1989; Dave et al., 1991; Inagaki et
al., 1991). Although early reports revealed that type-2
astrocytes possess a variety of neurotransmitter receptors, due to the low yield of these cells obtained by
mechanical shaking from mixed glial cultures, informa334
P2X7 STIMULATES Ca2⫹ INFLUX AND PLD
plasma membrane permeability to molecules as large as
900 Da and induces release of endogenous molecules
(Steinberg et al., 1987). It is interesting that, in several
types of immunoresponsive and secretory cells, the P2Z
receptor-stimulated Ca2⫹ influx has been shown to be
closely associated with activation of phospholipase D
(PLD) in a ligand-occupied-dependent manner (ElMoatassim and Dubyak, 1992, 1993; Gargett et al., 1996;
Humphreys and Dubyak, 1996). Nevertheless, P2Z-associated PLD activity has never been characterized in the
nervous system. Recently, the P2Z receptor was cloned
from rat brain and showed sequence homology to the
P2X receptor, so it was renamed P2X7 (Surprenant et al.,
1996). The classification of P2 receptors is now grouped
preferentially into the metabotropic P2Y and ionotropic
P2X receptors (Fredholm et al., 1997).
In this study, we show that the extracellular ATPelicited Ca2⫹ influx is associated with activation of PLD
in a rat brain-derived type-2 astrocyte cell line, RBA-2,
which is positive to GFAP, glutamine synthetase (GS),
and A2B5. Pharmacological analysis suggests that these
responses are mediated through the 2⬘- and 3⬘-O-(4benzoylbenzoyl)adenosine 5⬘-triphosphate (BzATP)sensitive P2X7 (P2Z) receptors. In addition, we provide
evidence that protein kinase C (PKC) is involved in the
regulation of the P2X7-associated PLD in these astrocytes.
MATERIALS AND METHODS
Materials
ATP, adenosine 5⬘-O-(3-thiotriphosphate) (ATP␥S), BzATP,
nifedipine, goat anti-rabbit IgG, oxidized ATP (oATP), phorbol
12-myristate 13-acetate (PMA), pyridoxalphosphate-6-azophenyl-2⬘,4⬘-disulfonic acid (PPADS), secondary antibody rabbit
anti-mouse IgG conjugated with horseradish peroxidase, and
suramin were from Sigma (St. Louis, MO, U.S.A.). ␣,␤-Methyleneadenosine 5⬘-triphosphate dilithium (␣,␤-methyleneATP), ␤,␥-methyleneadenosine 5⬘-triphosphate tetrasodium
(␤,␥-methylene-ATP), 2-methylthioadenosine triphosphate tetrasodium (2-MeSATP), staurosporine, and thapsigargin were
from Research Biochemicals International (Natick, MA,
U.S.A.). Phosphatidylethanol (PEt) was from Biomol Research
Laboratories Inc. (Plymouth Meeting, PA, U.S.A.). Fetal bovine serum and gentamicin were from GibcoBRL (Gaithersburg, MD, U.S.A.). First antibody anti-A2B5 was from Boehringer Mannheim GmbH (Mannheim, Germany), and antiGFAP and anti-GS were from Chemicon International
(Temecula, CA, U.S.A.). Secondary antibody goat anti-mouse
IgM was from Jackson ImmunoResearch Laboratories, Inc.
(West Grove, PA, U.S.A.). Radioactively labeled [9,10(n)3
H]palmitic acid (specific activity 51.0 Ci/mmol) and the enhanced chemiluminescence (ECL) system were from Amersham Life Science (Buckinghamshire, U.K.). Culture flasks and
dishes were from Corning Laboratory Sciences Co. (Corning,
NY, U.S.A.). HPTLC plates (Kieselgel 60, 10 ⫻ 10 cm) and
organic solvents were from E. Merck (Darmstadt, Germany).
Medical x-ray film was from Fuji Photo Film Co., Ltd. (Tokyo,
Japan).
Cell culture of RBA-2 astrocyte cell line
The RBA-2 cell line developed for this study was a subclone
of the RBA-1 cell line, which was established originally
335
through continuous passages for 3 years of primary astrocyteenriched cultures isolated from brains of newborn JAR-2, F51
rats (Japanese Research Center of Tissue Culture, Dokkyo
University School of Medicine) by Dr. Teh-Cheng Jou (Jou and
Akimoto, 1983). Before this study, cells were cultured, and
morphological examination revealed that 66 ⫾ 7% of RBA-1
cells were small (15–20 ␮m), round, and process-bearing and
had the stellate morphology. These cells were then subjected to
a serial dilution method and cultured in 96-well plates. One
clone with small and stellate morphology was selected and
continuously maintained in F10 medium (pH 6.2, adjusted with
bicarbonate) supplemented with 10% fetal bovine serum and
gentamicin (50 ␮g/ml). The cells were maintained by using
0.25 mM EDTA in 0.02 M phosphate buffer and stock cultured
in T-75 flasks. All experiments were conducted within 10
passages of the cells used.
Immunocytochemical analysis of type-2
astrocyte markers
To analyze the expression of astrocyte marker proteins,
RBA-2 cells were subcultured at a density of 2 ⫻ 104 cells/cm2
on 9 ⫻ 24 mm rectangular glass coverslips (Matsunami, Japan)
and placed in a six-well plate for at least 3 days. Cells were
stained with first antibodies against anti-GFAP, anti-GS, or
anti-A2B5. After five washes with phosphate-buffered saline
(PBS) containing 0.05% Tween 20, the coverslips were treated
with a 1:250 dilution of a secondary antibody, rabbit antimouse IgG conjugated with horseradish peroxidase. Color determination was conducted by the 3,3⬘-diaminobenzidine
method with nuclei stained with hematoxylin. All negative
controls for each first antibody were conducted by using secondary antibody only. The photomicrographs were taken with
an Olympus BH2 microscope (Olympus, Japan).
Western blot analysis of GFAP and GS
RBA-2 cells were subcultured into 100-mm dishes, washed,
scraped, centrifuged, and resuspended by the addition of 400 ␮l
of wash buffer (0.02 M phosphate buffer containing 0.1%
glucose) and 100 ␮l of lysis buffer (5 mM Tris-HCl, 5 mM
EDTA, 2 mM phenylmethanesulfonyl fluoride, 10 mM Nethylmaleimide, 0.6% sodium dodecyl sulfate, and 200 ␮M
leupeptin, pH 8.0) in an ice-cold water bath. Cells were homogenized with a Polytron. Protein levels were analyzed by the
method of Lowry et al. (1951) using bovine serum albumin as
the standard, and aliquots of homogenate protein (50 ␮g) were
loaded onto each lane of 12.5% sodium dodecyl sulfate–polyacrylamide gel for electrophoresis according to Laemmli
(1970). After separation, the proteins were transferred to nitrocellulose sheets using a Semi-Dry Trans-Blot (Pharmacia LKB
Multiphor II, U.S.A.). For detection of GFAP and GS, nonspecific binding sites were blocked by soaking the protein-loaded
nitrocellulose sheets for 30 min in a solution of PBS containing
0.05% Tween 20 and 5% dried skim milk. The sheet was then
reacted with a 1:250 dilution of monoclonal anti-GFAP or
anti-GS antibody. Then after five washes with PBS containing
0.05% Tween 20, the sheets were treated with a 1:10,000
dilution of a secondary antibody, rabbit anti-mouse IgG, conjugated with horseradish peroxidase (Sigma). The blots were
then washed thoroughly, dried, reacted with ECL immunodetection reagents (Amersham), and visualized by autoradiography using Fuji medical x-ray film.
Measurement of [Ca2ⴙ]i
Increases in [Ca2⫹]i were determined by using the fluorescent Ca2⫹ indicator fura-2 methods described by Grynkiewicz
J. Neurochem., Vol. 73, No. 1, 1999
336
S. H. SUN ET AL.
et al. (1985) and Ou et al. (1997). In brief, RBA-2 cells were
suspended at a density of 1 ⫻ 107 cells/ml in serum-free F10
medium containing fura-2 acetoxymethyl ester (5 ␮M) and
incubated for 30 min at 37°C. The cells were then centrifuged
at 700 g for 5 min, rinsed twice with serum-free F10 medium
to remove the excess fura-2 acetoxymethyl ester, resuspended
in serum supplemented with F10 culture medium at a density of
4 ⫻ 106/ml, and left at room temperature until use. Before
measurement, the cell suspension (0.5 ml) was diluted with 0.5
ml of loading buffer (150 mM NaCl, 5 mM KCl, 1 mM MgCl2,
2.2 mM CaCl2, 5 mM glucose, 10 mM HEPES at pH 7.4),
centrifuged at 700 g at room temperature for 3 min, rinsed
twice with 1 ml of loading buffer, resuspended in 2.5 ml of
loading buffer, and then transferred to a 3-ml cuvette positioned
in a thermostat-regulated (37°C) sample chamber of a dualexcitation beam spectrofluorometer (SPEX, model CM1T111,
Edison, NJ, U.S.A.) with continuous stirring. Fluorescence
ratios were measured by an alternative wavelength time scanning at 340 and 380 nm; emission was at 505 nm. Calibration
of the fluorescent signal in terms of [Ca2⫹]i was performed as
described by Grynkiewicz et al. (1985).
To elucidate whether ATP stimulates Ca2⫹ release from
intracellular stores, [Ca2⫹]i was measured by pretreating the
cells with thapsigargin (1 ␮M) for 3 min at room temperature.
To elucidate whether ATP stimulates Ca2⫹ influx, [Ca2⫹]i was
measured by resuspending the cells in a nominally Ca2⫹-free
loading buffer system (150 mM NaCl, 5 mM KCl, 1 mM
MgCl2, 5 mM glucose, 10 mM HEPES at pH 7.4, in the absence
of EDTA). To elucidate whether ATP activates voltage-sensitive Ca2⫹ channels, [Ca2⫹]i was measured by resuspending
cells in loading buffer containing nifedipine (a voltage-sensitive Ca2⫹-channel blocker). The cell suspension was then
placed inside the thermostat-regulated sample chamber. To
characterize the P2 receptor, [Ca2⫹]i was measured in the
presence of eight P2 agonists: BzATP, ATP, 2-MeSATP,
ATP␥S, ADP, UTP, ␣,␤-methylene-ATP, and ␤,␥-methyleneATP. These agents were added to the cell suspension at the
times indicated. To measure the effect of the P2 receptor
antagonist, the cells were resuspended in loading buffer in the
presence of 1–100 ␮M PPADS, incubated at room temperature
for 1 min, centrifuged, rinsed one time with loading buffer, and
resuspended in fresh loading buffer. The sample was then
placed inside the sample chamber, and the effect of ATP was
investigated. ATP-stimulated [Ca2⫹]i was also investigated using cells that had been resuspended in a Mg2⫹-free loading
buffer system (150 mM NaCl, 5 mM KCl, 2.2 mM CaCl2, 5
mM glucose, 10 mM HEPES at pH 7.4) in separate experiments.
To calculate the net increase in [Ca2⫹]i, the basal level was
recorded 60 s after the sample was placed in the sample
chamber and before the addition of agonist. The peak level was
recorded ⬃10 – 40 s after the addition of the agonist. Net
increases in [Ca2⫹]i were calculated by subtracting the basal
level of [Ca2⫹]i from peak levels.
PLD assay
PLD activity was measured according to the original
method of Kobayashi and Kanfer (1987) and Liscovitch
(1989) by analyzing the accumulation of PEt in the presence
of 300 mM ethanol. In brief, RBA-2 astrocytes (1 ⫻ 106
cells/dish) were subcultured into 60-mm dishes and cultured
for 2 days. Cells were then labeled with 1 ␮Ci/ml [9,10(n)3
H]palmitic acid (specific activity 51.0 Ci/mmol) in F10
medium, pH 6.2, and supplemented with 2.5% fetal bovine
serum for 18 h. After the labeled medium was removed, the
J. Neurochem., Vol. 73, No. 1, 1999
cells were rinsed with wash buffer and cultured in 2 ml of
loading buffer supplemented with 300 mM ethanol in the
presence of the agonist at 37°C for the lengths of time
indicated. The reactions were stopped by aspiration, followed by addition of 1.3 ml of ice-cold methanol. The cells
were scraped from the culture dish and transferred to a
borosilicate glass test tube (13 ⫻ 100 mm), and the dish was
scraped again after the addition of 1 ml of water. The lipids
were extracted by the addition of 2.7 ml of chloroform, after
which the sample was mixed by vortexing and centrifuging
at 400 g for 5 min to allow phase separation. The lower
organic phases were transferred to new test tubes, where
they were evaporated to dryness (Sun et al., 1997). The
lipids were then redissolved in 200 ␮l of chloroform and
applied to 1% potassium oxalate-preimpregnated 10 ⫻ 10
cm Kieselgel 60 HPTLC plates (E. Merck). Additional unlabeled PEt was also applied to each sample, and the plates
were separated by a one-dimensional solvent system using
the upper layer of a mixture of ethyl acetate/isooctane/acetic
acid/H2O (65:10:10:50, by volume). After development, the
plates were dried, the lipid bands were visualized by exposure to iodine vapor, and the PEt and phospholipid (PL)
bands were scraped into scintillation vials for counting by
scintillation spectrometry. The radioactivities of PEt were
standardized as dpm PEt/100,000 dpm in PL (El-Moatassim
and Dubyak, 1993).
RESULTS
RBA-2 is a type-2 astrocyte cell line
RBA-2 cells were first characterized by immunocytochemical analysis of astrocyte markers. As shown in Fig.
1A, these cells were positive for GFAP, GS, and A2B5.
Morphological examination of a total of 4,696 cells from
20 randomly taken photomicrographs revealed that 97
⫾ 2% of RBA-2 cells had small (15–20 ␮m), processbearing morphology typical of type-2 astrocytes,
whereas 2.9 ⫾ 0.8% were flat and were microglial cells,
macrophages, or flat astrocytes. A comparison of the
astrocyte-specific protein expression in C6 glioma cells
and RBA-2 cells is indicated in Fig. 1B, which shows
that the antibody against GFAP revealed a single band at
50 kDa, and the antibody against GS revealed a single
band at 45 kDa in RBA-2 astrocytes. As shown in Fig.
1B, RBA-2 cells were highly enriched in GS. Together,
these results indicate that RBA-2 cells exhibit the characteristics of type-2 astrocytes.
ATP-stimulated Ca2ⴙ influx is mediated through
P2 receptors
As indicated in Fig. 2A, 1 mM ATP stimulated a
sustained increase in [Ca2⫹]i. Treatment of these cells
with 1 ␮M thapsigargin raised the basal level of [Ca2⫹]i,
and ATP further stimulated the increase in [Ca2⫹]i in
these cells. As shown in Fig. 2B, when cells were incubated in a nominally Ca2⫹-free loading buffer system
(⫺Ca2⫹), the addition of ATP failed to elicit an increase
in [Ca2⫹]i, but the reintroduction of 2 mM extracellular
Ca2⫹ (⫹Ca2⫹) led to an increase in [Ca2⫹]i. Addition of
ATP (1 mM) to cells treated with a 5 ␮M concentration
of the voltage-sensitive Ca2⫹-channel blocker, nifedipine, stimulated increases in [Ca2⫹]i (Fig. 2C). Together,
P2X7 STIMULATES Ca2⫹ INFLUX AND PLD
337
FIG. 1. Characterization of RBA-2 astrocytes. A:
RBA-2 expressed GFAP, GS, and A2B5 antigens.
Cells were subcultured on glass coverslips, placed
in a six-well plate for 3 days, and then stained with
first antibodies: (1) anti-GFAP, (2) anti-GS, (3) antiA2B5, or (4) none as indicated. After washing, the
coverslips were treated with a 1:250 dilution of a
secondary antibody, rabbit anti-mouse IgG conjugated with horseradish peroxidase. Color determination was conducted by the 3,3⬘-diaminobenzidine method with nuclei counterstained with hematoxylin. Scale bar ⫽ 20 ␮m. B: Western blot
analysis of the expression of GFAP and GS in
RBA-2 astrocytes (lanes 1) and C6 glioma cells
(lanes 2). Aliquots of proteins (50 ␮g of protein)
from cell lysates were separated by gel electrophoresis, transblotted to nitrocellulose membrane.
Detection of GFAP (50 kDa) and GS (45 kDa) was
performed by reacting with anti-GFAP or anti-GS
antibodies visualized by the ECL method as indicated by the arrows.
the extracellular [Ca2⫹]-dependent ATP-stimulated increases in [Ca2⫹]i did not involve Ca2⫹ release from
intracellular stores, nor were they mediated through voltage-sensitive Ca2⫹ channels.
To determine whether the ATP-stimulated Ca2⫹ influx
is mediated through P2 receptors, the cells were pretreated with a common P2 receptor antagonist, PPADS,
for 1 min, washed, and then analyzed for ATP-stimulated
increases in [Ca2⫹]i. This treatment did not affect the
basal level of [Ca2⫹]i nor those of thapsigargin- and
ionomycin-raised [Ca2⫹]i, indicating that the pretreatment may not affect Ca2⫹ measurement (data not
shown). As shown in Fig. 2D, the ATP-stimulated Ca2⫹
influx was inhibited by 10 –100 ␮M PPADS in a concentration-dependent manner. PPADS at 1, 3, 10, 30, and
100 ␮M inhibited ATP-stimulated increases in [Ca2⫹]i
by 23 ⫾ 9%, 39 ⫾ 11%, 57 ⫾ 6%, 86 ⫾ 3%, and 95
⫾ 4%, respectively (n ⫽ 3). The IC50 of the effect of
PPADS was ⬃10 ␮M. We concluded that ATP stimulates Ca2⫹ influx through a P2 receptor.
ATP-stimulated increases in [Ca2ⴙ]i are mediated
through a BzATP-sensitive P2X7 (P2Z) receptor
To determine which of the P2 receptors mediates the
ATP-stimulated Ca2⫹ influx in RBA-2 astrocytes, we
tested the response of these cells to eight ATP analogues
(El-Moatassim and Dubyak, 1993; Harden et al., 1995;
Burnstock, 1997; Boarder and Hourani, 1998). As indicated in Fig. 3A, ATP, 2-MeSATP, and ATP␥S induced
moderate increases in [Ca2⫹]i, and BzATP stimulated a
marked increase in [Ca2⫹]i in these cells. ATP, 2-MeSATP, and ATP␥S induced net increases in [Ca2⫹]i of 70
⫾ 16, 38 ⫾ 4, and 27 ⫾ 1 nM, respectively (Fig. 3B). By
contrast, a 1 mM concentration of ADP, UTP, ␣,␤methylene-ATP, or ␤,␥-methylene-ATP was ineffective
(data not shown). Figure 3C demonstrates the concentration dependence of increases in [Ca2⫹]i by ATP (0.3–2
mM) and BzATP (10 ␮M to 1 mM) stimulation; marked
effects of BzATP were observed. The maximal effect of
BzATP was at 500 ␮M, which elicited a net increase in
[Ca2⫹]i of almost 1 ␮M. EC50 values of ATP and BzATP
J. Neurochem., Vol. 73, No. 1, 1999
338
S. H. SUN ET AL.
dylcholine, the major PLD analyzed by this method
could be attributed to phosphatidylcholine-PLD. Effects
of ATP and BzATP on PLD activity were examined by
measuring the accumulation of PEt in the presence of
300 mM ethanol. As shown in Fig. 5A, BzATP was
much more potent than ATP in the stimulation of PEt
accumulation in RBA-2 astrocytes. The BzATP-stimulated PLD activities were concentration-dependent with
a maximal effect at 100 ␮M. The EC50 of BzATPstimulated PLD was ⬃40 ␮M. As shown in Fig. 5B, the
FIG. 2. ATP stimulation of Ca2⫹ influx, but not Ca2⫹ release
through P2 receptors. RBA-2 astrocytes were preloaded with
fura-2, rinsed, and resuspended (A) in loading buffer in the
presence or the absence of 1 ␮M thapsigargin (TG) and incubated for 3 min, (B) in nominally Ca2⫹-free loading buffer, or (C)
in loading buffer containing 5 ␮M nifedipine (NF). D: In the case
of P2 antagonist, the fura-2-preloaded cells were resuspended
in loading buffer containing 0 ␮M (trace 1), 10 ␮M (trace 2), 30
␮M (trace 3), and 100 ␮M (trace 4) PPADS, incubated for 1 min,
washed once with loading buffer, and resuspended in fresh
loading buffer. The cell suspensions were then placed inside a
thermostat-regulated sample chamber. The addition of ATP (1
mM) or CaCl2 (2 mM) is indicated by an arrow, and the fluorescences of fura-2 and fura-2-Ca2⫹ were recorded and [Ca2⫹]i
calculated as described in Materials and Methods. The experiments were performed at least three times, and results were
reproducible.
were ⬃450 and ⬃170 ␮M, respectively. Taken together,
the ATP-stimulated Ca2⫹ influx was mediated probably
through the BzATP-sensitive P2X7 receptors in these
cells.
As it is known that extracellular [Mg2⫹] affects poreforming activities and Ca2⫹ influx of P2X7 receptors
(Wiley et al., 1993; Song and Chueh, 1996; Ross et al.,
1997), we then tested ATP- or BzATP-stimulated
[Ca2⫹]i in the absence of extracellular Mg2⫹. By incubating cells in a Mg2⫹-free loading buffer system, the
ATP- (Fig. 4A) and BzATP-stimulated (Fig. 4B) increases in [Ca2⫹]i were enhanced as compared with those
using the Mg2⫹-supplemented loading buffer system.
These results combined confirm that ATP-stimulated
Ca2⫹ influx is mediated through P2X7 receptors in
RBA-2 astrocytes.
Stimulation of the P2X7 receptor is associated with
activation of PLD in RBA-2 astrocytes
To analyze the signal transduction pathway mediation
of the P2X7 receptor in RBA-2 astrocytes, we determined if ATP and BzATP stimulate PLD activity in
these cells. RBA-2 astrocytes were prelabeled with
[3H]palmitic acid for 18 h. As most of the labeling (74.0
⫾ 0.8%; n ⫽ 3) was distributed in membrane phosphatiJ. Neurochem., Vol. 73, No. 1, 1999
FIG. 3. ATP analogue-stimulated increases in [Ca2⫹]i. The fura2-preloaded RBA-2 astrocytes were rinsed, suspended in loading buffer, and stimulated with ATP analogues. Then the fluorescences of fura-2 and fura-2-Ca2⫹ were recorded and [Ca2⫹]i
calculated as described in Materials and Methods. A: Traces of
increases in [Ca2⫹]i stimulated by 1 mM each of BzATP (trace 1),
ATP (trace 2), 2-MeSATP (trace 3), and ATP␥S (trace 4). B: Net
increases in [Ca2⫹]i stimulated by 1 mM each of ATP, 2MeSATP, and ATP␥S (n ⫽ 3). C: The concentration dependence
of (䊐) ATP- or (■) BzATP-stimulated net increase in [Ca2⫹]i. The
net increases in [Ca2⫹]i were calculated by subtracting the basal
level of [Ca2⫹]i from peak levels. Data represent the means ⫾ SD
from three determinations.
P2X7 STIMULATES Ca2⫹ INFLUX AND PLD
339
PEt. In addition, 100 – 600 ␮M oATP inhibited the
BzATP-stimulated PLD activities (Fig. 5D), but 1–10
␮M oATP was ineffective. Furthermore, oATP also inhibited the ATP-stimulated PLD activities and the
BzATP-stimulated phosphatidic acid accumulation in
these cells (data not shown). Thus, the P2X7 receptor is
responsible for the action of BzATP-mediated PLD activity in RBA-2 astrocytes.
FIG. 4. Effect of extracellular Mg2⫹ on (A) ATP- and (B) BzATPstimulated increases in [Ca2⫹]i. The fura-2-preloaded RBA-2
astrocytes were suspended in loading buffer supplemented with
(䊐) 0 mM Mg2⫹ or (■) 1 mM Mg2⫹ and stimulated with various
concentrations of ATP or BzATP. The fluorescences of fura-2
and fura-2-Ca2⫹ were recorded and [Ca2⫹]i calculated as described in Materials and Methods. Net increases in [Ca2⫹]i were
calculated by subtracting the basal level of [Ca2⫹]i from peak
levels. Data represent the means ⫾ SD from three determinations.
ATP- and BzATP-stimulated PLD activities were timedependent. The time-course studies revealed that a fourfold increase in BzATP-stimulated PEt was observed
at 1 min, and the maximal effect was found at 10 min
(Fig. 5B).
To elucidate whether BzATP-stimulated PLD activity
is associated with the activation of P2X7 receptors, we
analyzed the effect of two common P2 antagonists,
PPADS and suramin, on BzATP-stimulated PEt accumulation. As shown in Fig. 5C, PPADS and suramin both
caused concentration-dependent inhibitions of BzATPstimulated PEt accumulation. Values of IC50 for PPAD
and suramin were ⬃5 and ⬃40 ␮M, respectively. These
results confirm that the BzATP-stimulated PLD activities
are mediated through P2 receptors. To confirm whether
BzATP-stimulated PLD activities are mediated through
the P2X7 receptor, we measured the PEt accumulation in
cells pretreated with the selective P2X7 receptor antagonist, oATP. Pretreatment of cells with 600 ␮M oATP in
labeled medium for 2 h did not alter the basal levels of
Extracellular Ca2ⴙ is required for the activation of
PLD activity
To confirm whether nucleotide-stimulated PLD is associated with P2X7-induced Ca2⫹ influx, we then analyzed the ATP- or BzATP-stimulated PEt accumulation
in a nominally Ca2⫹-free buffer system. As shown in
Fig. 6A, the ATP- or BzATP-stimulated PEt accumulations were abolished when cells were incubated in a
nominally Ca2⫹-free buffer system, suggesting that the
nucleotide-induced Ca2⫹ influx is required for the activation of PLD in these cells.
To establish the linkage between activation of PLD
and increases in [Ca2⫹]i, we analyzed ionomycin-stimulated PEt accumulations in the presence of various concentrations of extracellular Ca2⫹ (0 –2.2 mM). As indicated in Fig. 6B, a minimum of 0.44 mM extracellular
Ca2⫹ was required for ionomycin to induce PEt accumulation; thereafter, PLD activity increased with increasing extracellular [Ca2⫹]. In parallel with this observation, ionomycin-induced [Ca2⫹]i also increased with
increasing medium [Ca2⫹]i (Fig. 6C). The average (n
⫽ 2) ionomycin-induced net increases in [Ca2⫹]i in the
presence of 0, 0.44, 1.1, and 2.2 mM extracellular Ca2⫹
were 39, 196, 808, and 6,258 nM, respectively. Taken
together, these results suggest that activation of PLD
requires an increase in [Ca2⫹]i.
PKC is involved in the activation of
P2X7-associated PLD
To examine whether PKC regulates the P2X7-associated PLD activation in RBA-2 cells, we analyzed
BzATP-stimulated PEt accumulation in the presence of
the PKC inhibitor, staurosporine, or in cells pretreated
with PMA for 4 h. An initial time-course study indicated
that the PKC activator, PMA, stimulated increases in PEt
accumulations from 5 to 30 min, with a maximal effect
observed at 30 min; but the effect diminished with prolonged PMA treatment, and after 4 h PMA-induced PLD
activity was abolished (data not shown). BzATP-stimulated PEt accumulation was inhibited by 50% in the
presence of 100 and 1,000 nM staurosporine (Fig. 7A)
and also in cells pretreated with 100 nM PMA for 4 h
(Fig. 7B), suggesting that PKC regulates, in part if not
all, the BzATP-stimulated PLD activities in RBA-2 astrocytes.
DISCUSSION
In the present study, a permanent type-2 astrocyte cell
line, RBA-2, was established with a purity of 97% and
used for repetitive and continuous biochemical analyses.
J. Neurochem., Vol. 73, No. 1, 1999
340
S. H. SUN ET AL.
FIG. 5. Activation of P2X7 receptor stimulation of PLD activities. RBA-2 astrocytes were subcultured in 60-mm dishes
(1 ⫻ 106 cells/dish), labeled with
[3H]palmitate for 18 h, washed, and incubated in loading buffer containing 300
mM ethanol (A) supplemented with various concentrations of (䊐) ATP or (■)
BzATP and incubated for 15 min, or (B)
supplemented with 1 mM ATP or 100
␮M BzATP and incubated for various
lengths of time at 37°C. C: In the case of
P2 receptor antagonist, the prelabeled
cells were incubated in loading buffer
containing 300 mM ethanol and supplemented with various concentrations of
the common P2 antagonists, PPADS (F)
or suramin (■), and incubated for 5 min
at 37°C. BzATP at 40 ␮M was then
added, and the cells were incubated further at 37°C for 10 min. D: In the case of
the selective P2X7 receptor antagonist,
cells were subcultured into 30-mm
dishes (5 ⫻ 105 cells/dish), labeled with
[3H]palmitic acid for 18 h, and treated
with the indicated concentrations of
oATP for 2 h. Cells were then washed
and incubated in loading buffer containing 300 mM ethanol in the presence or
absence of 40 ␮M BzATP for 10 min.
PLD was assayed by measuring the accumulation of PEt and standardized with
105 dpm in PL as described in Materials and Methods. Data represent the means ⫾ SD of dpm PEt/100,000 dpm in PL from three
determinations. *Significantly different from the controls (nonpaired Student’s t test, p ⱕ 0.05). The experiments were performed twice,
and results were reproducible.
This cell line provides an accessible system for the
analysis of neurotransmitter-stimulated second messenger formation that may provide insight into physiological
functioning of these astrocytes. The RBA-2 cell line was
established in a similar way to another permanent type-2
lineage cell culture, L3, which was established through
repetitive passaging and selection from mixed glial cultures (Aloisi et al., 1990). Previous studies have shown
that type-2 astrocytes, but not type-1 astrocytes, accumulate GABA (Aloisi et al., 1988; Gallo et al., 1989).
Immunocytochemical and western blot analysis revealed
that RBA-2 astrocytes are highly enriched in GS (Fig. 1),
which is an important astrocyte enzyme for metabolizing
glutamate and GABA in the brain (Norenberg and Martinez-Hernandez, 1979; Patel and Hunt, 1985). Thus, the
high level of GS expression suggests that one of the
important physiological functions of RBA-2 astrocytes is
probably to metabolize these neurotransmitters.
In the present study, we demonstrate ATP-stimulated
Ca2⫹ influx in RBA-2 astrocytes, which is blocked by
PPADS. P2 receptor agonists (ADP, UTP, ␣,␤-methylene-ATP, and ␤,␥-methylene-ATP) were ineffective,
whereas BzATP was the most effective in raising
[Ca2⫹]i. Thus, RBA-2 appears to possess only a P2X7
receptor but not a P2Y receptor, nor the P2X1– 6 receptors. The pharmacological profile of RBA-2 P2X7 resembles that of the cloned rat brain P2X7 receptor (Surprenant et al., 1996). In separate experiments, we demJ. Neurochem., Vol. 73, No. 1, 1999
onstrate that ATP and BzATP stimulate PLD activities in
RBA-2 astrocytes, and that suramin and PPADS antagonize BzATP-induced PLD activity. In addition, pretreatment of cells with the selective P2X7 receptor
blocker, oATP, blocks BzATP-stimulated PLD activity.
Furthermore, BzATP stimulates lucifer yellow admission
into RBA-2 astrocytes (data not shown), confirming that
BzATP induces characteristic pore-forming activities of
P2X7 receptors in these cells. Together, these results are
in agreement with the initial findings in immunoresponsive cells that stimulation of the P2X7 receptor is closely
associated with the activation of PLD (El-Moatassim and
Dubyak, 1992, 1993; Gargett et al., 1996; Humphreys
and Dubyak, 1996).
In the present study, the BzATP-stimulated increase in
[Ca2⫹]i is 10 times greater than that of ATP. This result
is consistent with a common notion that BzATP is much
more potent than ATP in activation of the P2X7 receptor
(Dubyak and El-Moatassim, 1993). Pharmacological
selectivity for the P2X7 receptor is ATP4⫺ ⫽ BzATP
Ⰷ ATP (Harden et al., 1995), and therefore ATP is a
partial agonist with lower efficiency. At concentrations
of ATP higher than 1 mM and BzATP concentrations
higher than 500 ␮M, the increases in [Ca2⫹]i become
smaller, suggesting that high concentrations of ATP and
BzATP may chelate some Ca2⫹ and interfere with
[Ca2⫹]i measurement (Fig. 3C). In addition, high concentrations of BzATP (ⱖ1 mM) slightly quenched fluo-
P2X7 STIMULATES Ca2⫹ INFLUX AND PLD
341
rescence of fura-2 in the medium (data not shown). Thus,
the lowering of the maximum of BzATP-increased
[Ca2⫹]i could be due to interference of fura-2 released
from pores or lysed cells.
Activation of PLD is known to be regulated via multiple pathways involving Ca2⫹, PKC, G protein, and
protein tyrosine kinase (Billah, 1993; Briscoe et al.,
1995; Klein et al., 1995; Exton, 1997). A direct relationship between [Ca2⫹]i and PLD activation has been demonstrated (Wu et al., 1992). Early results indicated that
ATP-activated PLD was dependent on extracellular
Ca2⫹ and PKC in endothelial cells (Purkiss and Boarder,
1992). However, activation of PLD may be a secondary
response of the P2X7 signal pathways (Harden et al.,
1995). Nevertheless, as our results reveal that BzATPactivated PLD is inhibited by suramin, PPADS, and
oATP and is abolished in nominally Ca2⫹-free buffer
conditions, we can conclude that activation of PLD is
closely associated with the activation of the P2X7 receptor in these astrocytes.
We have not completely ruled out the possibility that
RBA-2 may possess other P2 receptors, because there is
a discrepancy between the concentrations evoking in-
FIG. 6. Roles of Ca2⫹ in the activation of PLD. A: The
[3H]palmitic acid-prelabeled RBA-2 astrocytes were washed
and incubated in loading buffer containing 300 mM ethanol
and supplemented with 2.2 mM Ca2⫹ (⫹Ca2⫹) or without Ca2⫹
(⫺Ca2⫹) and with (䊐) 1 mM ATP or (■) 100 ␮M BzATP or
without nucleotides (basal) at 37°C for 15 min. B: The prelabeled cells were incubated in loading buffer containing 300
mM ethanol, 0 –2.2 mM Ca2⫹ as indicated, and 2 ␮M ionomycin at 37°C for 15 min. The reactions were stopped by aspiration; lipids were extracted, applied to HPTLC, and separated by a one-dimensional solvent system. After lipid separation, the bands corresponding to PEt and PL were scraped,
and the radioactivities determined by scintillation spectrophotometry. Data represent the means ⫾ SD of dpm PEt/100,000
dpm in PL from three determinations. *Significantly different
from the respective controls (nonpaired Student’s t test, p
ⱕ 0.05). C: Traces of increases in [Ca2⫹]i, which were assayed
by incubating the fura-2-preloaded cells in loading buffer
supplemented with 0 –2.2 mM Ca2⫹ with ionomycin (2 ␮M ).
The experiments were performed twice, and the results were
reproducible.
FIG. 7. Involvement of PKC in BzATP-stimulated PLD activity. A:
RBA-2 astrocytes were prelabeled with [3H]palmitic acid, incubated in loading buffer in the presence or absence of the PKC
inhibitor staurosporine (100 and 1,000 nM), and stimulated with
BzATP (100 ␮M) in the presence of 300 mM ethanol for 15 min.
B: The prelabeled cells were pretreated (■) with or (䊐) without
PMA (100 nM) for 4 h, and then stimulated with 100 or 200 ␮M
BzATP in loading buffer in the presence of 300 mM ethanol for
15 min. Reactions were stopped by aspiration; the lipids were
extracted and applied to HPTLC, and PEt and PL were separated by a one-dimensional solvent system. Radioactivities of
PEt and PL were determined by scintillation spectrophotometry.
Data represent the means ⫾ SD of dpm PEt/100,000 dpm in PL
from three determinations. *Significantly different from the respective control (nonpaired Student’s t test, p ⱕ 0.05).
J. Neurochem., Vol. 73, No. 1, 1999
342
S. H. SUN ET AL.
creases in [Ca2⫹]i and the accumulation of PEt. BzATP
maximally stimulated the accumulation of PEt at a concentration of 100 ␮M (Fig. 5A). In contrast, at the same
concentration, BzATP evoked a submaximal increase in
[Ca2⫹]i (Fig. 3C). Nevertheless, RBA-2 astrocytes appear to resemble human lymphocytes, which possess
only the P2X7 (P2Z) receptor (Gargett et al., 1996). In
human lymphocytes, P2X7-mediated PLD activity is directly proportional to the influx of divalent cations; thapsigargin raised [Ca2⫹]i, but failed to stimulate PLD activity in these cells. In the present study, ionomycin
induced significant increases in PEt accumulation, and at
an extracellular [Ca2⫹] of ⱖ0.44 mM, the ionomycinstimulated PEt accumulations were proportional to
extracellular [Ca2⫹]. This result suggests that the ionomycin-increased [Ca2⫹]i must reach a threshold concentration before eliciting PLD activation. However, ionomycin caused a tremendous increase of [Ca2⫹]i (⬃6
␮M), far more than that by 100 ␮M BzATP (364 ⫾ 81
nM), but ionomycin-induced PEt accumulation was less
than that of BzATP. Thus, although the activation of
PLD is associated with P2X7-induced Ca2⫹ influx, it is
not crucially related to [Ca2⫹]i. Gargett et al. (1996)
suggested that Ca2⫹ influx accumulates in the microenvironment near the P2X7 pore, and that this is important
to the activation of PLD; local increases in Ca2⫹ may
stimulate translocation of PKC to the membrane to regulate the PLD. Therefore, PKC may be the limiting
factor in the activation of PLD in RBA-2 astrocytes.
Taken together, these results suggest that P2X7-induced
Ca2⫹ influx is required for PLD, and the regulation of
PLD may depend on a mechanism that is closely associated with the P2X7 receptor. Possibly, PKC plays a
rate-limiting regulatory role in P2X7-activated PLD in
these cells.
By using the PKC activator O-tetradecanoylphorbol
13-acetate and inhibitor H-7, Gustavsson and Hansson
(1990) revealed that PKC regulates PLD activities in
cultured astrocytes. Our results indicate that PKC is
involved in the activation of a P2X7-associated PLD in
RBA-2 astrocytes. Recently, PKC⑀ was found to mediate
the ATP- and UTP-activated PLD in renal mesangial
cells (Pfeilschifter and Merriweather, 1993), and PKC␣
was found to exert a synergistic effect with RhoA in the
activation of membrane-bound PLD in HL60 cells
(Ohguchi et al., 1996). We have found that RBA-2
astrocytes express at least seven PKC isozymes (data not
shown). Thus, P2X7-activated PLD could be regulated
by one or more of the PKC isozymes or may involve
some other Ca2⫹-dependent mechanism. Earlier, the
Ca2⫹/calmodulin protein kinase II-activated pathway,
distinct from that of PKC, was shown to be a potential
mediator in P2X7-mediated PLD activation in human
lymphocytes (Humphreys and Dubyak, 1996). Nevertheless, Gargett and Wiley (1997) found that inhibition by
KN-62 is not mediated by Ca2⫹/calmodulin protein kinase II, but occurs directly at the receptor. Recently, a
335-amino acid sequence on an extracellular domain of
the human lymphocyte P2X7 receptor, but not of the rat
J. Neurochem., Vol. 73, No. 1, 1999
P2X7 receptor, has been identified as the domain sensitive to KN-62 (Humphreys et al., 1998). In the present
study, KN-62 (0.5–5 ␮M) did not inhibit BzATP-stimulated PLD activities in RBA-2 astrocytes (data not
shown). Although the molecular identity of the RBA-2
P2X7 receptor is not known, this result indicates that
RBA-2 P2X7 closely resembles the cloned rat P2X7
receptor (Surprenant et al., 1996).
The exact physiological function of P2X7 in type-2
astrocytes is not clear at this moment. Recently, Ballerini
et al. (1996) demonstrated that activation of the P2X7
receptor-induced Ca2⫹ influx is correlated with efflux of
purine in cultured rat astrocytes. In microglia, activation
of the P2X7 receptor triggers interleukin-1␤ release (Ferrari et al., 1996). However, both P2X7-associated increases in [Ca2⫹]i and activation of PLD require high
concentrations of ATP. The amount of ATP released
from synaptic terminals during neuronal transmission,
which then diffuses to the astrocyte, probably falls below
these high thresholds. Therefore, additional extracellular
ATP would have to be provided by (a) the release of
cytosolic ATP via intrinsic plasma channels or pores in
the absence of irreversible cytolysis, or (b) stimulationinduced release of cytosolic ATP upon sudden breakage
of intact cells (Dubyak and El-Moatassim, 1993). Recently, P2X7 immunoreactivities were shown to appear
in a necrotic area in the brain by prior occlusion of the
middle cerebral artery (Collo et al., 1997). Thus, with
pathophysiological conditions, ATP may act via the
P2X7 receptor to regulate type-2 astrocytes in the CNS.
In conclusion, we demonstrate that P2X7 is the major
P2 receptor in a permanent type-2 astrocyte cell line,
RBA-2, and that stimulation of P2X7 is closely associated with activation of PLD in these astrocytes. The
P2X7-activated PLD is dependent on Ca2⫹ influx and is
regulated by PKC.
Acknowledgment: We would like to thank Dr. S.-H. Chueh
and Dr. F. F. Wang for critical reading of the manuscript. This
work was supported by a grant from Taichung Veterans General Hospital, Taiwan, R.O.C. (TVGH-8674011D) and a grant
from the National Science Council (NSC 87-2316-B-010-009).
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