Maitotoxin and P2Z/P2X7
purinergic receptor stimulation activate a common cytolytic
pore
William P.
Schilling,
Tanya
Wasylyna,
George R.
Dubyak,
Benjamin
D.
Humphreys, and
William G.
Sinkins
Rammelkamp Center for Education and Research and Department of
Physiology and Biophysics, Case Western Reserve University School of
Medicine, Cleveland, Ohio 44109
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ABSTRACT |
The effects of
maitotoxin (MTX) on plasmalemma permeability are similar to those
caused by stimulation of P2Z/P2X7
ionotropic receptors, suggesting that
1) MTX directly activates
P2Z/P2X7 receptors or
2) MTX and
P2Z/P2X7 receptor stimulation
activate a common cytolytic pore. To distinguish between these two
possibilities, the effect of MTX was examined in
1) THP-1 monocytic cells before and
after treatment with lipopolysaccharide and interferon-
, a maneuver
known to upregulate P2Z/P2X7
receptor, 2) wild-type HEK cells and
HEK cells stably expressing the
P2Z/P2X7 receptor, and
3) BW5147.3 lymphoma cells, a cell
line that expresses functional P2Z/P2X7 channels that are poorly
linked to pore formation. In control THP-1 monocytes, addition of MTX
produced a biphasic increase in the cytosolic free
Ca2+ concentration
([Ca2+]i);
the initial increase reflects MTX-induced
Ca2+ influx, whereas the second
phase correlates in time with the appearance of large pores and the
uptake of ethidium. MTX produced comparable increases in
[Ca2+]i
and ethidium uptake in THP-1 monocytes overexpressing the
P2Z/P2X7 receptor. In both
wild-type HEK and HEK cells stably expressing the
P2Z/P2X7 receptor, MTX-induced
increases in
[Ca2+]i
and ethidium uptake were virtually identical. The response of BW5147.3
cells to concentrations of MTX that produced large increases in
[Ca2+]i
had no effect on ethidium uptake. In both THP-1 and HEK cells, MTX- and
Bz-ATP-induced pores activate with similar kinetics and exhibit similar
size exclusion. Last, MTX-induced pore formation, but not channel
activation, is greatly attenuated by reducing the temperature to
22°C, a characteristic shared by the
P2Z/P2X7-induced pore. Together,
the results demonstrate that, although MTX activates channels that are
distinct from those activated by
P2Z/P2X7 receptor stimulation, the
cytolytic/oncotic pores activated by MTX- and Bz-ATP are indistinguishable.
THP-1 monocytes; HEK cells; heterologous expression; oncosis; vital
dyes
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INTRODUCTION |
IN THE ACCOMPANYING STUDY IN human skin fibroblasts
(31), we confirmed that maitotoxin (MTX), one of the most potent marine toxins known, produces a dramatic increase in cytosolic free
Ca2+ concentration
([Ca2+]i).
This rise in
[Ca2+]i
is unrelated to mobilization of
Ca2+ from internal stores but
rather results from a MTX-induced activation of nonselective cation
channels present in the plasmalemma. The activation of these
channels by MTX is followed closely in time by the formation of large
pores that allow the flux of vital dyes, such as ethidium and YO-PRO-1,
into the cell and the release of cell-associated fura 2. At much later
times, MTX causes the release of lactate dehydrogenase (LDH), an event
that presumably signals oncotic cell death. This sequence of
permeability changes is reminiscent of the changes observed in many
cells following stimulation of P2Z/P2X7 receptors (10, 12, 26,
43). The P2Z/P2X7 receptor is the
seventh member of the P2X receptor family (35). P2X receptors are
thought to form Ca2+-permeable,
nonselective cation channels activated by extracellular ATP. The
P2Z/P2X7 receptor has the novel
characteristic of inducing the formation of large pores, which, like
MTX, allow the entry of large organic vital dyes into the cell and
ultimately causes the release of LDH and cell death by oncosis.
Although it is clear that heterologous expression of the
P2Z/P2X7 receptor is associated
with expression of both functional channels and pores, the actual
structure of the pore-forming subunit remains unknown. Likewise, the
actual biophysical properties of the
P2Z/P2X7 channel that allow
formation of pores have not been defined. It has been suggested that
individual P2Z/P2X7 subunits may
aggregate to form pores of increasing size and hence allow larger and
larger molecules to cross the plasmalemma (26, 36). However, there is
little evidence to date that the pore is actually composed of
P2Z/P2X7 protein subunits, and it
remains possible that an endogenous pore structure present in the cells
employed for heterologous expression is activated following stimulation of P2Z/P2X7 channels by purinergic agonists.
The finding that MTX induces formation of large pores with
characteristics similar to
P2Z/P2X7 receptor-induced pores
suggests two possibilities: 1) MTX
is a high-affinity ligand for the
P2Z/P2X7 receptor or
2) MTX and
P2Z/P2X7 receptor stimulation
activate distinct channels but a common cytolytic pore. To distinguish between these two possibilities, the effect of MTX was examined in
cells in which the expression of functional
P2Z/P2X7 channels and pores could
be varied either by alterations in the growth conditions or by
heterologous expression. In the first set of experiments, the effect of
MTX was examined in THP-1 monocytes. These cells express low levels of
P2Z/P2X7 receptor under normal growth conditions, but pretreatment of these cells with bacterial lipopolysaccharide (LPS) and interferon-
(IFN-
) dramatically upregulates P2Z/P2X7 message and
function (18). In the second set of experiments, the effect of MTX was
examined in control HEK cells and in HEK cells stably expressing the
P2Z/P2X7 receptor. Last, the
effect of MTX on BW5147.3 cells, a cell line that expresses P2Z/P2X7 receptors that are poorly
coupled to pore formation (20), was examined. The results suggest that
MTX activates channels that are distinct from those activated by
stimulation of P2Z/P2X7 receptors;
however, the characteristics of the MTX-induced pores are
indistinguishable from the pores activated by
P2Z/P2X7 receptor stimulation. A
physical model is proposed in which MTX-activated channels and
P2Z/P2X7 receptor-activated
channels compete for a common cytolytic pore.
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MATERIALS AND METHODS |
Solutions and reagents.
Unless otherwise indicated, HEPES-buffered saline (HBS) contained 140 mM NaCl, 5 mM KCl, 1 mM MgCl2, 10 mM D-glucose, 1.8 mM
CaCl2, 15 mM HEPES, and 0.1% BSA,
pH adjusted to 7.40 at 37°C with NaOH.
Ca2+-free HBS contained 0.3 mM
EGTA and the same salts as HBS without added
CaCl2. Fura 2-AM, ethidium
bromide, YO-PRO-1, and POPO-3 were obtained from Molecular Probes
(Eugene, OR). MTX was obtained from Calbiochem (San Diego, CA) or from
LC Laboratories (Woburn, MA) and was stored in amber-colored glass or
plastic vials at
20°C as an aqueous stock solution (>1
µM) with 0.1% BSA. 2' and 3'-O-(4-benzoylbenzoyl)-ATP
(Bz-ATP) was obtained from Sigma Chemical (St. Louis, MO). All other
salts and chemicals were of reagent grade.
Cell culture.
The THP-1 monocytic cell line was obtained from American Type Culture
Collection (ATCC) and grown in RPMI 1640 containing 10%
heat-inactivated fetal bovine serum (FBS), 1%
penicillin-streptomycin-neomycin (PSN) antibiotic mixture,
and 2 mM L-glutamine in spinner
flasks as previously described (18). In some experiments, the
P2Z/P2X7 receptor was upregulated
by pretreatment of THP-1 cells for 48 h in complete RPMI 1640 medium
containing 1 µg/ml bacterial LPS and 1,000 U/ml IFN-
. Wild-type
HEK cells were obtained from ATCC and grown in MEM supplemented with
10% FBS, 1% PSN antibiotic mixture, and 2 mM
L-glutamine. For passage, cells
were dispersed by trypsin treatment and seeded at a density of ~3 × 103
cells/cm3. The medium was changed
every 2-3 days after seeding. HEK cells were harvested for
experimentation when the cells had reached confluence. HEK cells stably
expressing the human P2Z/P2X7
receptor were generated as previously described (19) and cultured in a
fashion identical to wild-type HEK. BW5147.3, a murine lymphoma cell
line, was obtained from ATCC and grown in spinner flasks in
DMEM supplemented with 10% FBS, 1% PSN antibiotic
mixture, and 2 mM L-glutamine.
Uptake of vital dyes.
The uptake of ethidium, YO-PRO-1, and POPO-3 was determined in the
absence and presence of MTX or Bz-ATP as previously described (31).
Measurement of the apparent
[Ca2+]i.
[Ca2+]i
was measured using the fluorescent indicator fura 2 as previously
described (30, 31). Unless otherwise indicated, all measurements were
performed at 37°C. Calibration of the fura 2 associated with the
cells was accomplished using Triton X-100 lysis in the
presence of saturating divalent cation concentrations followed by
addition of EGTA (pH 8.5).
[Ca2+]i
was calculated by the equation of Grynkiewicz et al. (15) using a
dissociation constant value for
Ca2+ binding to fura 2 of 224 nM.
As reported in the accompanying paper (31), MTX causes fura 2 efflux
from cells. Therefore, where indicated,
[Ca2+]i
values are reported as "apparent" changes in
[Ca2+]i.
Data presentation and statistical treatment.
Figures 1-12 show representative traces from experiments performed
at least three times. For mean values, traces recorded
under identical conditions on one day were pooled yielding an
n of 1. Where indicated,
n equals the number of independent
experiments and statistical differences between means were determined
by Student's t-test, with
P < 0.05 considered significant. To
control for cell-to-cell and day-to-day variation, all traces shown
within each panel of Figs. 1-12 were obtained on the same day and
on the same batch of cells. Where indicated, traces in multiple panels
of a figure were also paired. For example, the traces shown in both
A and B of Fig. 3 were obtained on the same
day on the same batch of THP-1 cells, divided into control and treated groups.
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RESULTS |
Effect of MTX on THP-1 monocytes.
The results of our accompanying study (31) on fibroblasts suggested two
possibilities: 1) MTX activates
P2Z/P2X7 or
2) MTX and the
P2Z/P2X7 receptor activate
distinct channels but a common cytolytic pore. If MTX activates the
P2Z/P2X7 receptor, then the response to MTX should be proportional to the level of expression of
the P2Z/P2X7 receptor. Previous
studies have shown that P2Z/P2X7 expression is low in control THP-1 monocytes but can be dramatically upregulated by pretreatment of the cells with LPS and IFN-
for 24-48 h (18). The effect of MTX on
[Ca2+]i
in control THP-1 cells is shown in Fig. 1.
MTX produced a concentration-dependent, biphasic change in
[Ca2+]i.
At the lowest concentration tested (2 pM), MTX initially produced a
significant 1.8-fold increase in
[Ca2+]i
from a resting level of 71.8 ± 6.7 nM to a sustained level of 129.6 ± 20 nM (means ± SE, n = 7, P < 0.01). After a delay of ~5 min,
[Ca2+]i
began to increase again with time. Increasing the concentration of MTX
produced a decrease in the delay time between the first and second
phase with little change in the subsequent rate of increase of
[Ca2+]i
(Fig. 1A). Thus the first phase
appears to be concentration dependent, whereas the second phase appears
to be all or nothing. As the concentration of MTX increased from 25 pM
to 2 nM, the first and second phase gradually merged into a single
response (Fig. 1B). Addition of MTX
in the absence of extracellular
Ca2+ had no effect, but a large
increase in
[Ca2+]i
was observed on subsequent addition of the
Ca2+ to the bath solution (data
not shown). Thus, as previously shown, MTX does not release
Ca2+ from internal stores but
initially stimulates Ca2+ influx
from the extracellular space. Note that there is a "crossover" phenomenon seen in this experiment at the three highest concentrations of MTX tested. This was a consistent finding (see Figs.
3A and 8A); the maximum level of apparent
[Ca2+]i
was generally greater for the lower concentrations vs. the higher
concentrations of MTX, at least over this time frame.

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Fig. 1.
Effect of maitotoxin (MTX) on fura 2 fluorescence in THP-1 monocytes.
Fura 2-loaded THP-1 cells were suspended in HEPES-buffered saline
(HBS), and fluorescence ratio was recorded as described in
MATERIALS AND METHODS as a function of
time. A: at arrow, MTX was added
during the individual recordings at the final concentrations indicated.
Six traces are shown superimposed. B:
same as A but with higher
concentrations of MTX. Traces in A and
B were obtained on the same day using
the same batch of THP-1 cells.
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Previous studies in fibroblasts showed that the
[Ca2+]i
response to MTX was composed of two components; the first phase
reflects the influx of Ca2+ from
the extracellular space, whereas the second phase reflects pore
formation and the concomitant efflux of fura 2. To test the hypothesis
that the second phase of the
[Ca2+]i
response to MTX in control THP-1 cells reflects pore formation, the
effect of MTX on ethidium uptake was examined (Fig.
2). MTX produced an increase in ethidium
uptake following a time delay that decreased as the concentration of
MTX increased; the uptake of ethidium correlated closely in time with
the second phase of the
[Ca2+]i
response. Thus, as was seen in fibroblasts (31), the fura 2 response
shown in Fig. 1 at early times reflects
Ca2+ influx via MTX-induced
channels but at later times probably reflects fura 2 efflux following
pore formation. At intermediate concentrations of MTX, the maximum
level of ethidium uptake appeared to be attenuated as evidenced by the
crossover of the lower concentrations (Fig. 2A, traces
c and d). At higher
MTX concentrations (>50 pM), there was a progressive decrease in the
time delay and an increase in the maximum level of ethidium uptake (Fig
2B). The crossover phenomenon observed in the ethidium uptake experiments was a consistent finding (see Figs. 4A and
9A).

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Fig. 2.
Effect of MTX on ethidium uptake in THP-1 monocytes.
A: ethidium uptake was determined as
an increase in fluorescence as described in materials
and methods as a function of time. Four traces are
shown superimposed. Ethidium bromide (5 µM) was added at 50 s. At
time indicated by arrow, MTX was added at the final concentrations
indicated. In this figure and in Figs. 4, 5B, 7, 9, 11, and 12,
values of vital dye uptake were normalized to the maximum fluorescence
obtained following permeabilization of the cells with either saponin or
digitonin at the end of each trace. B:
same as A but with higher
concentrations of MTX. C: same as in
A but over a longer time course.
Traces in A and
B were obtained on the same day using
the same batch of THP-1 cells.
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To more closely examine the extent of the crossover phenomenon, longer
time courses were performed. As seen in Fig.
2C, ethidium uptake at 20 and 50 pM
produced almost maximum ethidium uptake in this batch of THP-1 cells.
Ethidium uptake occurred with a significant time delay that decreased
as the concentration of MTX increased. Once activated, however,
ethidium uptake proceeded in an all-or-nothing fashion, reaching
>80% within 8 min of activation. In contrast, ethidium uptake
induced by 300 pM MTX occurred with shorter delay but was clearly
attenuated, giving rise to a prominent crossover phenomenon. This
attenuation was sustained, but ethidium uptake continued to increase
slowly with time, reaching ~60% over this time frame.
Effect of P2Z/P2X7 receptor upregulation
on the response to MTX.
THP-1 monocytes were incubated with LPS and IFN-
for 48 h to
upregulate P2Z/P2X7 as previously
described (18). Increased expression of
P2Z/P2X7 was confirmed in the
present study by primer-specific amplification using RT-PCR (data not
shown). Compared with matched controls, LPS and IFN-
pretreatment
increased basal resting
[Ca2+]i
from 65.2 ± 12 to 115 ± 27 nM (means ± SE,
n = 3, P = 0.085) and produced an attenuation
the response of the cells to MTX (Fig. 3).
The primary effect of LPS and IFN-
pretreatment was on the second
phase of the response to MTX (e.g., compare the
[Ca2+]i
response to 20 pM MTX in control and LPS- and IFN-
-treated cells;
Fig. 3, trace c in
A and
B). This result suggests that upregulation of P2Z/P2X7 may
reduce MTX-induced pore formation. Consistent with this hypothesis, LPS
and IFN-
pretreatment slightly inhibited MTX-induced ethidium uptake
at concentrations <0.5 nM (Fig. 4). At
0.5 nM MTX, ethidium uptake in LPS- and IFN-
-treated cells was
actually greater than in controls and the crossover phenomenon was not
observed over this concentration range. This suggests that the
crossover may be related to
[Ca2+]i,
which is clearly attenuated in the cells expressing the
P2Z/P2X7 receptor. Importantly,
control THP-1 cells, which essentially lack the
P2Z/P2X7 receptor, and pretreated
cells, which have a high level of receptor expression, both respond to
MTX. This result supports the hypothesis that the channels activated by
MTX are distinct from P2Z/P2X7.

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Fig. 3.
Effect of lipopolysaccharide (LPS) and interferon- (IFN- )
pretreatment on MTX-induced change in cytosolic free
Ca2+ concentration
([Ca2+]i)
in THP-1 monocytes. A: fura 2-loaded
THP-1 cells were suspended in HBS, and fluorescence ratio was recorded.
Six traces are shown superimposed. At arrow, MTX was added during the
individual recordings at the final concentrations indicated.
B: same as
A but using THP-1 monocytes pretreated
with 1 µg/ml bacterial LPS and 1,000 U/ml IFN- for 48 h. Traces in
A and
B were obtained on the same day using
the same batch of THP-1 cells.
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Fig. 4.
Effect of LPS and IFN- pretreatment on MTX-induced ethidium uptake
in THP-1 monocytes. A: ethidium
bromide (5 µM) was added to THP-1 monocytes at 50 s. At time
indicated by arrow, MTX was added at the final concentrations
indicated. Six traces are shown superimposed.
B: same as
A but using THP-1 monocytes pretreated
with 1 µg/ml LPS and 1,000 U/ml IFN- for 48 h. Traces in
A and
B were obtained on the same day using
the same batch of THP-1 cells.
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Effect of MTX on heterologously expressed
P2Z/P2X7 receptor.
To further test the hypothesis that MTX activates the
P2Z/P2X7 receptor, the effect of
MTX on
[Ca2+]i
and ethidium uptake was examined in wild-type HEK cells and in HEK
cells stably expressing the
P2Z/P2X7 receptor. Basal,
nonstimulated [Ca2+]i
was 56.4 ± 6.9 and 50.2 ± 3.8 nM (means ± SE;
n = 3) in wild-type HEK
and P2Z/P2X7-expressing cells
(Fig. 5). The wild-type HEK cells were
essentially unresponsive to Bz-ATP (up to 200 µM), a specific
P2Z/P2X7 receptor agonist. In
sharp contrast, addition of Bz-ATP to HEK cells expressing the
P2Z/P2X7 receptor produced a
concentration-dependent increase in both
[Ca2+]i
(Fig. 5A) and ethidium uptake (Fig.
5B), consistent with both channel
and pore formation. The responses of these two cell types to MTX are
shown in Figs. 6 and
7. MTX produced a time- and
concentration-dependent increase in both apparent
[Ca2+]i
and ethidium uptake. The response was virtually identical for wild-type
HEK cells and the
P2Z/P2X7-expressing cells.
Together with the results obtained in the THP-1 monocytes, these
results strongly suggest that expression of the
P2Z/P2X7 receptor has little
effect on MTX-induced channel activation, a result that is inconsistent
with MTX acting as a high-affinity ligand for the
P2Z/P2X7 receptor.

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Fig. 5.
Effect of P2Z/P2X7 receptor
expression on 2' and
3'-O-(4-benzoylbenzoyl)-ATP
(Bz-ATP)-induced change in
[Ca2+]i
and ethidium uptake in HEK cells. A:
fura 2-loaded wild-type HEK cells (trace
a) or HEK cells stably expressing the
P2Z/P2X7 receptor
(traces b-d) were suspended in
HBS. At arrow, Bz-ATP was added to a final concentration of 20 (trace b), 50 (trace c) or 100 (traces a and
d) µM. Four traces are shown
superimposed. B: ethidium uptake in
wild-type HEK cells (trace a) and in
HEK cells stably expressing the
P2Z/P2X7 receptor
(traces b and
c) were recorded in response to 100 µM (trace b) or 200 µM
(traces a and
c) Bz-ATP added at the arrow. Three
traces are shown superimposed. Values were corrected for a small
quenching of ethidium fluorescence by Bz-ATP. Traces in
A and
B were obtained on the same day in
each cell type.
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Fig. 6.
Effect of P2Z/P2X7 receptor
expression on MTX-induced change in
[Ca2+]i
in HEK cells. A: fura 2-loaded
wild-type HEK cells were suspended in HBS, and fluorescence ratio was
recorded. Six traces are shown superimposed. At arrow, MTX was added
during the individual recordings at the final concentrations indicated.
B: same as
A but using HEK cells stably
expressing the P2Z/P2X7 receptor.
Traces in A and
B were obtained on the same day.
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Fig. 7.
Effect of P2Z/P2X7 receptor
expression on MTX-induced change in ethidium uptake in HEK cells.
A: ethidium uptake was determined as
the increase in fluorescence as a function of time. Five traces are
shown superimposed. Ethidium (5 µM) was added at 50 s. At time
indicated by arrow, MTX was added at the final concentrations
indicated. B: same as
A but using HEK cells stably
expressing the P2Z/P2X7 receptor.
Traces in A and
B were obtained on the same day.
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Effect of temperature on MTX-induced pore formation.
The above results provide evidence that MTX activates a
Ca2+-permeable channel that is
distinct from the P2Z/P2X7
receptor channels. However, the experiments do not eliminate the
possibility that activation of the MTX-sensitive channel and the
P2Z/P2X7 channel induce the
formation of a common cytolytic pore. To test this hypothesis, we have
compared the properties of the
P2Z/P2X7-induced pores to those
activated by MTX. Previous studies have shown that a decrease in
temperature has little effect on
P2Z/P2X7-induced channel activity
(i.e., the change in
[Ca2+]i)
but substantially attenuates pore formation (i.e., dye uptake) (26,
33). To determine if the MTX-induced pores have the same characteristic, we compared the MTX response of the control THP-1 monocytes at 37 and 22°C (Figs. 8 and
9). Although there is a shift in the
apparent ED50 for MTX-induced
change in
[Ca2+]i,
large increases in
[Ca2+]i
were clearly observed at 0.2 and 0.5 nM MTX at 22°C (Fig.
8B). The response, however, lacked
the biphasic characteristic seen at 37°C, suggesting that the
second phase of the response was attenuated. Consistent with this
hypothesis, ethidium uptake was dramatically inhibited at all
concentrations of MTX when examined at 22°C (Fig. 9). Thus
MTX-induced pore formation has a temperature sensitivity similar to
that previously reported for the
P2Z/P2X7 pore.

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Fig. 8.
Effect of temperature on MTX-induced change in
[Ca2+]i
in THP-1 monocytes. A: fura 2-loaded
THP-1 cells were suspended in HBS at 37°C, and fluorescence ratio
was recorded as a function of time. Six traces are shown superimposed.
At time indicated by arrow, MTX was added to the cuvette at the final
concentrations indicated. B: same as
A but at 22°C. Traces in
A and
B were obtained on the same day on the
same batch of THP-1 cells.
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Fig. 9.
Effect of temperature on MTX-induced uptake of ethidium uptake in THP-1
monocytes. A: ethidium bromide (5 µM) was added at 50 s to THP-1 cells suspended in HBS at 37°C. At
time indicated by arrow, MTX was added at the final concentrations
indicated. Six traces are shown superimposed.
B: same as
A but at 22°C. Traces in
A and
B were obtained on the same day on the
same batch of THP-1 cells.
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Effect of MTX on
[Ca2+]i
and ethidium uptake in BW5147.3 lymphoma cells.
Previous studies suggest that BW5147.3 lymphoma cells express
functional P2Z/P2X7 channels that
are poorly coupled to pore formation (20). The effect of MTX in this
cell line is shown in Fig. 10. Basal,
nonstimulated
[Ca2+]i
in BW5147.3 cells was 197 ± 16 nM (means ± SE;
n = 4), a value that is approximately
threefold greater than that measured in either control THP-1 monocytes
(P < 0.002) or in wild-type HEK cells (P < 0.001). MTX (0.05 and 0.2 nM) produced a large, concentration-dependent increase in
[Ca2+]i
in BW5147.3 lymphoma cells that increased gradually with time over 6 min. In contrast, uptake of ethidium was unaffected by MTX at these
concentrations, even when examined for extended periods of time (13 min; see Fig. 10, inset). Ethidium
uptake was, however, observed in BW5147.3 cells in response to
supermaximal concentrations (2-20 nM) of MTX (not shown). Thus
BW5147.3 cells apparently have MTX-activated channels that are poorly
coupled to pore formation, as previously noted for the
P2Z/P2X7 response in this cell
line.

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Fig. 10.
Effect of MTX on
[Ca2+]i
and ethidium uptake in BW5147.3 lymphoma cells. Fura 2-loaded BW5147.3
lymphoma cells were suspended in HBS at 37°C. Two traces are shown
superimposed. MTX was added at a final concentration of 0.05 nM
(trace a) or 0.2 nM
(trace b) at time indicated by
arrow. Inset: ethidium uptake (3 traces superimposed) as a function of time in BW5147.3 cells in the
absence or presence of 0.2 or 0.5 nM MTX.
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Comparison of MTX- and Bz-ATP-induced pore size.
Another characteristic of the
P2Z/P2X7-induced pore is that the
apparent size exclusion varies with cell type (10). We, therefore,
compared MTX- and Bz-ATP-induced ethidium (314 Da), YO-PRO (375 Da),
and POPO-3 (715 Da) uptake in HEK cells and THP-1 monocytes expressing
the P2Z/P2X7 receptor. In HEK
cells, both Bz-ATP (200 µM) and MTX (1 nM) produced a time-dependent
increase in ethidium uptake (Fig. 11);
however, uptake of YO-PRO was much slower, and uptake of POPO-3 was
essentially zero over this time frame. Thus MTX- and Bz-ATP-induced
pores activate with a similar time course and have the same relative
permeability in HEK cells, i.e., ethidium > YO-PRO
POPO-3. In
sharp contrast to the HEK cells, MTX (50 pM) and Bz-ATP (200 µM)
increased the uptake of all three dyes into THP-1 monocytes (Fig.
12). Thus the pores formed in THP-1
monocytes appear to be larger than those seen in HEK cells and, as
suggested above, seem to activate in an all-or-nothing fashion. One
note of caution, release of LDH in response to Bz-ATP occurs very soon
after ethidium uptake in THP-1 cells pretreated with LPS and IFN-
(18). This could explain, in part, the differences between HEK and
THP-1 cells with respect to apparent size exclusion. Importantly,
however, the profiles of permeability change induced by MTX and Bz-ATP
are essentially identical in the two cell types with respect to
kinetics of activation and apparent size exclusion.

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|
Fig. 11.
Effect of MTX and Bz-ATP on vital dye uptake in HEK cells expressing
the P2Z/P2X7 receptor. In
A and
B, 3 traces are superimposed. MTX (1 nM; A) or Bz-ATP (200 µM;
B) was added to HEK cells stably
expressing the P2Z/P2X7 receptor
suspended in HBS at time indicated by arrow. Vital dyes were added at
50 s, and uptake of ethidium (trace
a), YO-PRO-1 (trace
b), or POPO-3 (trace
c) was followed as a function of time.
|
|

View larger version (23K):
[in this window]
[in a new window]
|
Fig. 12.
Effect of MTX and Bz-ATP on vital dye uptake in THP-1 monocytes
overexpressing the P2Z/P2X7
receptor. In A and
B, 3 traces are superimposed. MTX (50 pM; A) or Bz-ATP (100 µM;
B) was added at time indicated by
arrow to THP-1 cells pretreated with 1 µg/ml LPS and 1,000 U/ml
IFN- for 48 h. Vital dyes were added at 50 s, and uptake of POPO-3,
YO-PRO-1, or ethidium (EB) was followed as a function of
time.
|
|
 |
DISCUSSION |
MTX is one of the most potent marine toxins isolated to date. This
toxin produces increases in
[Ca2+]i
in all cell tested via the activation of
Ca2+-permeable, nonselective
cation channels. The results of the present study demonstrate that
THP-1 monocytes are extremely sensitive to MTX with apparent
ED50 of ~10 pM, estimated from
the initial change in
[Ca2+]i.
In our accompanying study using human skin fibroblasts (31), we showed
for the first time that MTX also activates large pores that allow
molecules with molecular masses up to 800 Da to enter or exit the cell.
In fibroblasts, this pore formation occurred with a short delay
following channel activation, and a similar result was obtained in the
present study in HEK cells. However, in THP-1 monocytes, a considerable
lag was observed between the initial activation of the channel and
subsequent pore formation. Once pore formation was initiated in the
THP-1 monocytes, it occurred in an all-or-nothing fashion, causing
large increases in ethidium, YO-PRO-1, and POPO-3 uptake into the
cells. The lag phase would suggest that there may be several
biochemical steps between channel activation and pore formation or that
the concentration of a critical messenger or factor may need to exceed
a threshold value before activation of the pore is observed. An
enzymatic step(s) between channel activation and pore formation is also
supported by the sensitivity of pore formation to temperature. As shown
in our previous studies on fibroblasts, reducing the temperature
substantially increased the lag time between channel activation and
pore formation, and the results of the present study on THP-1 monocytes
clearly shows that, at concentrations of MTX that substantially
increase [Ca2+]i,
pore formation is essentially reduced to zero at 22°C. The biochemical events responsible for coupling of channel activation to
pore formation remain unknown. In this regard, the crossover phenomenon
seen in the THP-1 monocytes may provide a clue. At low concentrations
of MTX, pore formation appears to occur in an all-or-nothing fashion
after a considerable lag time. However, at the higher concentrations of
MTX associated with the crossover phenomenon, (i.e., inhibition of pore
formation), a rapid increase in
[Ca2+]i
was observed. Thus an increase in
[Ca2+]i
may produce feedback inhibition. Interestingly, the resting [Ca2+]i
in the BW5147.3 lymphoma cells is approximately threefold higher than
in THP-1 cells and pore formation induced by either Bz-ATP or MTX is
substantially attenuated, supporting this hypothesis. Understanding
this regulation will require greater knowledge of the
molecular/biochemical link between channel activation and pore formation.
The only documented example of channels linked to activation of large
pores are members of the P2X ionotropic receptor family (21, 40), of
which P2Z/P2X7 is the best-studied
example. The P2Z/P2X7 receptor is
a protein of 595 amino acids in length possessing two putative
transmembrane segments with cytoplasmic
NH2- and COOH-terminal domains
(35). It has a structure similar to the inwardly rectifying
K+ channels, amiloride-sensitive
Na+ channels and the
mechanosensitive Mec channels of Caenorhabditis elegans (2). Each of these channel families has a
cytoplasmic NH2 and COOH terminus,
two membrane spanning segments, and a permeation pathway formed by a
reentrance loop immediately preceding the second
transmembrane segment. These channels are thought to form homo- and
heteromeric channel structures. On the basis of structural similarity,
the P2X purinergic receptor family may form channels in a similar
fashion, i.e., by homo- or heteromeric assembly (39), and it has been
suggested that P2Z/P2X7-induced
pore formation reflects the progressive association/aggregation of
channel-forming subunits into a large pore structure (26, 36), although
evidence to support such a biophysical model is scant. It became clear early in the present study that MTX-induced pores share many
characteristics with the pores formed following activation of
P2Z/P2X7 receptors/channels. Therefore, we initially focused on the intriguing possibility that MTX
was a high-affinity ligand for
P2Z/P2X7. The results of the
present study however, demonstrate that this is not the case.
First, untreated THP-1 cells and wild-type HEK-293 cells show little response to Bz-ATP, suggesting that functional
P2Z/P2X7 receptors are not
expressed or are expressed at very low levels, yet both cell types are
sensitive to MTX, exhibiting both large increases in
[Ca2+]i
and fluorescent dye uptake. Thus the MTX-activated channel and pore are
functional in the apparent absence of
P2Z/P2X7 receptor. Second,
upregulation of the P2Z/P2X7 in
THP-1 cells by pretreatment with LPS and IFN-
greatly enhances both
the rise in
[Ca2+]i
and the uptake of fluorescent dyes in response to Bz-ATP (18) but
slightly attenuates the response of the cells to MTX. Third, heterologous expression of
P2Z/P2X7 in HEK-293 cells
dramatically increases the response of the cells to Bz-ATP but has
little or no effect on the response to MTX. Together, these results
demonstrate that expression of
P2Z/P2X7 does not correlate with
MTX responsiveness, and provide strong support for the conclusion that
P2Z/P2X7 is not the MTX-activated channel.
Although the channels appear to be different, the MTX- and
Bz-ATP-induced pores are indistinguishable, leading to the hypothesis that MTX and P2Z/P2X7 activate a
common cytolytic pore. This conclusion is based on the following
results. First, all cells examined in the present study that exhibit
Bz-ATP-induced pores also possess the MTX-induced pore. Second, the
pores for both agonists activate with a similar time course following
stimulation of the channel activity and this time course of activation
is cell specific. Thus, as discussed above, MTX- and Bz-ATP-induced
channel activation is followed closely in time by the formation of
pores in HEK cells, but this activation requires a considerable lag
time in THP-1 cells. Third, the MTX and Bz-ATP-induced pores have
similar size exclusions. In HEK, there is a clear difference in
ethidium, YO-PRO-1, and POPO-3 uptake for both MTX and Bz-ATP, whereas,
in THP-1 cells, uptake of each dye is observed for both agonists.
Fourth, BW5147.3 lymphoma cells appear to have Bz-ATP- (20) and
MTX-induced channels that are poorly coupled to pore formation.
Finally, fifth, both MTX- and Bz-ATP-induced pores have similar
temperature sensitivity. Overall, we could find no characteristic that
would distinguish between these two pores. Although we cannot eliminate
the possibility that each agonist activates different channels and
different pores, it is difficult to reconcile the similarities with
such a model; even cell-specific characteristics such as kinetics of
activation, size exclusion, and the inefficient coupling between
channel and pore seen in BW5147.3 cells are essentially identical for
MTX- and Bz-ATP-induced pores. Thus the simplest explanation for these results is that MTX and Bz-ATP activate distinct channels but a common
cytolytic pore.
Evidence in the literature demonstrating identity between
P2Z/P2X7 channels and pores is
very limited and relies on heterologous expression. These reports show
that P2X receptor expression is required for responsiveness to
externally applied purinergic receptors agonists and that channels
formed by different P2X subunits, either alone or in various
combinations, possess unique biophysical properties (13, 14, 21, 24,
26, 28, 34, 39, 40). Thus it is likely that the P2X protein forms at
least part of both the agonist binding domain and the channel
permeation pathway. The results of the present study support this
hypothesis. However, it remains possible that heterologously expressed
P2Z/P2X7 protein does not form the
pore but rather is coupling (either physically or biochemically) to
endogenous pores in the cell types used for expression. The most common
cell types used for heterologous expression of
P2Z/P2X7 receptors are the HEK
cell and frog oocyte. In this regard, it is clear from the present
study that HEK cells have an endogenous pore that is activated by MTX.
Furthermore, it has recently been shown (1) that frog oocytes have an
MTX-induced nonselective cation current that initially excludes the
large cation N-methyl-D-glucamine (NMDG) (167 Da). Although these authors did not specifically test for pore
formation, it is clear from their current recordings that a conductance
to NMDG developed at later times following MTX addition to the external
bath, consistent with pore formation. Furthermore, all cells examined
to date respond to MTX with large increases in
[Ca2+]i
and/or downstream Ca2+-dependent
signaling events (1, 6, 8, 9, 16, 17, 22, 25, 29). Thus it seems likely
that most, if not all cells, have an endogenous MTX-activated cytolytic
pore, including cell types used for heterologous expression of
P2Z/P2X7 receptors.
The model shown in Fig. 13 proposes that
the P2X purinergic ionotropic receptors and the channels activated by
MTX interact with a common cytolytic/oncotic pore, or COP. Although the
model suggests that the coupling between channel and COP is membrane delimited, cytosolic proteins or factors may play a role in activation of COP. As was seen in Fig. 4, however, overexpression of
P2Z/P2X7 in THP-1 cells causes an
attenuation of MTX-induced pore formation, which could be overcome by
increasing the concentration of MTX. Thus it appears that
P2Z/P2X7 may compete with the
MTX-induced channel for activation of COP, suggesting a preexisting
channel-pore complex. In this regard, attenuation of the MTX response
by expression of P2Z/P2X7 in THP-1
cells may reflect a greater affinity of
P2Z/P2X7 for COP relative to the
MTX channel or alternatively may reflect the amount and/or
number of P2Z/P2X7
receptors relative to MTX channels in these cells. Irrespective of the
exact mechanism of coupling between channel and COP, MTX should provide
a useful tool for understanding this interaction.
Although the experiments of the present study focused on
P2Z/P2X7-mediated and MTX-induced
COP activation, two recent studies demonstrate that other members of
the P2X family can, to various extents, initiate pore formation and
that mutations at specific residues within the second membrane spanning
segment can differentially alter transition from channel to pore (21,
40). These experiments were performed in both frog oocytes and HEK
cells heterologously expressing the various P2X receptor subtypes and
mutants. Thus, although these investigators interpreted their results
as reflecting a conformational change or dilation of the P2X permeation
pathway, the findings are not inconsistent with differential activation of the endogenous COP in these cell types. Furthermore, P2X- and MTX-activated channels may not be the only channels linked to COP.
Indeed, activation of a number of different channel types has been
shown to produce oncotic cell death. These include the VR1 vanilloid receptor, which is
responsible for pain sensation associated with stimulation by
capsaicin, the active compound in chili peppers (3), the
NR1-NR2
N-methyl-D-aspartate
glutamate receptor, which has been implicated in excitatory
neurotoxicity (5, 7, 37, 38), and the degenerins family of ion
channels, responsible for hereditary neurodegeneration in
C. elegans (4). Interestingly,
heterologous expression in HEK cells of the mammalian degenerin MDEG,
with the same mutation as that found in C. elegans neurodegeneration phenotypes, gives rise to
constitutively active channels that cause cell death by swelling (41).
Thus it is possible that the activity of a number of different channels
may be coupled to oncosis by activation of COP.
Last, activation of COP need not always proceed to cell death by
oncosis. Substantial evidence supports the hypothesis that apoptosis
and oncosis reflect different endpoints of a common cell death pathway
(11, 23, 27, 37). In this regard, activation of COP by extracellular
ATP is associated with both apoptosis and oncosis in mesangial cells
(32) and thymocytes (44). Furthermore, Warny and Kelly (42) recently
showed that oncosis in THP-1 monocytes is mediated by loss of cellular
K+ and activation of caspase-like
proteases (42), a hallmark of apoptotic cell death. Under normal ionic
conditions, it seems likely that activation of COP will lead to
pronounced loss of cellular K+ in
exchange for extracellular Na+ and
Ca2+, which may lead to either
apoptosis or oncosis, depending possibly on the metabolic status of the
cell. In any event, it will be important to define the role of COP in
both types of cell death and to identify and characterize the COP
protein at the molecular level.
 |
ACKNOWLEDGEMENTS |
We thank Drs. Diana L. Kunze and Mark Estacion for helpful discussions
and acknowledge the technical assistance of Erin McGowan.
 |
FOOTNOTES |
This work was supported by National Institute of General Medical
Sciences Grants GM-52019 (W. P. Schilling) and GM-36387 (G. R. Dubyak)
and by a Postdoctoral Fellowship awarded to W. G. Sinkins by the
American Heart Association, Northeast Ohio Affiliate.
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: W. P. Schilling,
Rammelkamp Center for Education and Research, MetroHealth Medical
Center, Rm. R322, 2500 MetroHealth Dr., Cleveland, OH 44109-1998 (E-mail: wschilling{at}metrohealth.org).
Received 11 May 1999; accepted in final form 24 June 1999.
 |
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