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The Journal of Neuroscience, November 1, 1998, 18(21):8794-8804
Cytoskeletal Assembly and ATP Release Regulate Astrocytic Calcium
Signaling
Maria Luisa
Cotrina,
Jane H.-C.
Lin, and
Maiken
Nedergaard
Departments of Cell Biology and Anatomy, Pathology, and
Neurosurgery, New York Medical College, Valhalla, New York 10595
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ABSTRACT |
We have studied the role of actin fiber assembly on calcium
signaling in astrocytes. We found that (1) after astrocytes have been
placed in culture, it takes several hours for organization of the
definitive actin cytoskeleton. Actin organization and the number of
cells engaged in calcium signaling increased in parallel. (2)
Disruption of the actin cytoskeleton attenuated the calcium wave
propagation; cytochalasin D treatment reduced the number of astrocytes
engaged in calcium signaling. (3) Propagation of calcium waves depends
on cytoskeletal function; inhibition of myosin light chain kinase
suppressed wave activity. (4) Astrocytic calcium signaling is mediated
by release of ATP and purinergic receptor stimulation, because agents
that interfere with this cascade attenuated or reduced calcium
signaling. Because purinergic receptors are fully functional shortly
after plating and not affected by cytochalasin D, these observations
indicate that cytoskeleton organization is a prerequisite for
interastrocytic calcium signaling mediated by release of ATP.
Key words:
cytochalasin; actin; myosin; calcium waves; glioma
cells
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INTRODUCTION |
Calcium waves are slowly propagating
increments in cytosolic calcium levels among gap junction-coupled
cells. These long-distance waves may represent a mechanism by which
multicellular tissues coordinate responses to stimuli. In brain,
intercellular calcium waves occur among astrocytes and can evoke
prolonged elevation of calcium levels in nearby neurons. Hence,
astrocytes can modulate the calcium levels and thereby the excitability
of nerve cells and are thus an active participant in information
processing (Nedergaard, 1994 ; Parpura et al., 1994, Pasti et al., 1997;
Araque et al., 1998; Newman and Zahs, 1998).
Calcium waves are traditionally believed to be transferred from cell to
cell by gap junction-mediated diffusion of Ca2+ or
IP3 (Sanderson et al., 1994), but an extracellular
component may also participate in astrocytic signaling (Hassinger et
al., 1996). Cell lines deficient in gap junctions become capable of propagating intercellular calcium signals only after transfection and
overexpression of gap junction proteins (Charles et al., 1992; Elfgang
et al., 1995; Toyofuku et al., 1998). The present study was prompted by
the observation, originally reported by Naus et al. (1992), that forced
expression of gap junctions results not only in functional coupling but
also in distinct morphological alterations; poorly coupled cell lines
are typically composed of compact cells with few cellular contacts,
whereas the same cells reorganize into flat epithelioid layers after
expression of gap junctions. Thus, acquisition of signaling capability
and transformation of phenotype occur in parallel. Likewise, primary cells that transmit robust calcium waves, such as astrocytes and hepatocytes, are organized as flat confluent monolayers in cultures (Cornell-Bell et al., 1990). Because strict cellular organization is
critical for an array of cellular signaling mechanisms, such as
synaptic function (Prekeris et al., 1996) and receptor-mediated mitogenic signaling (Bohmer et al., 1996), we asked whether gap junction-coupled cells maintained the ability to propagate calcium waves when cellular organization was disrupted. We found that an intact
actin cytoskeleton is required for the propagation of astrocytic
calcium waves. Furthermore, inhibition of myosin light chain kinase
reduced the number of cells engaged in calcium signaling, suggesting
that wave propagation depends on cytoskeletal function.
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MATERIALS AND METHODS |
Astrocytic culture. The procedure for cultured
astrocytes has been described in detail elsewhere (Nedergaard et
al., 1991; Cotrina et al., 1998a). Briefly, newborn (1 d
postnatal) or prenatal (embryonic day 17) rat brains were trypsinized,
mechanically triturated, and plated on uncoated glass coverslips or on
plastic dishes (1 and 5 × 105 cells/ml,
respectively). Cultures were maintained in DMEM-F12 (Life Technologies,
Gaithersburg, MD) supplemented with 10% fetal bovine serum (Atlanta
Biologica), penicillin and streptomycin (Life Technologies), and 8%
glucose (Sigma, St. Louis, MO) in a 5% CO2 humidified
incubator at 37°C. Medium was changed every 2-3 d, and the cultures
were replated when confluent. Experiments were performed after 10-14 d
in vitro.
Mice with a null mutation of connexin 43. Heterozygotes of
the connexin 43 (Cx43) knock-out line from Jackson Laboratory
were used. Pregnant females were killed at 18-20 d after
gestation, and the embryonic brains were cultured as described above.
To identify homozygotes, heterozygotes, and wild type, we used PCR for
amplifying tail-blood genomic DNA flanking the null deletion, according
to the protocol of Jackson Laboratory. Also, immunohistochemical mapping of the extent of Cx43 was mapped in conjunction with dye transfer assays. The astrocytes from homozygotes and wild-type mice
used in this study were from three different litters.
Actin staining and immunocytochemistry. Cells were plated on
12 mm uncoated coverglasses (0.5-1 × 105
cells/ml) and fixed 1-3 d later with 4% paraformaldehyde for 10 min
at room temperature. For actin staining, cells were permeabilized with
0.1% Triton X-100 and incubated with Texas Red-phalloidin (Molecular
Probes, Eugene, OR) for 30 min. After several washes in PBS,
coverslips were mounted in glycerol and examined by confocal microscopy
(MRC1000; Bio-Rad, Hercules, CA). For immunodetection of Cx43 and glial
fibrillary acidic protein (GFAP), tubulin or myosin, cultures were
permeabilized with 0.1% Triton X-100 and blocked with 10% normal goat
serum. A polyclonal antibody against Cx43 (1:500) (kindly supplied by
Dr. Lau, University of Hawaii), a monoclonal antibody against GFAP
(1:100) (GAS; Sigma), a monoclonal antibody against  -tubulin
(1:50) (Boehringer-Mannheim, Indianapolis, IN), or a monoclonal
antibody against myosin light chain (1:50) (MY-21; Sigma) was applied
for 2 hr at room temperature or overnight at 4°C. After three washes
in PBS, FITC goat anti-rabbit or goat anti-mouse was applied for 1 hr
at room temperature. Reaction was completed by washing with PBS several
times and mounting the coverslips in Slow Fade (Molecular Probes).
Actin was quantified by analyzing astrocytic cultures stained
with Texas Red-phalloidin with a 20× objective of an IX-50 inverted fluorescence microscope (Olympus). At least 250 cells per field in
three different fields were counted in each culture and classified according to their pattern of actin organization. If actin was concentrated in a narrow band under the plasma membrane, the cell was
classified as lacking cytoskeletal organization. If the cell contained
parallel phalloidin-positive fibers, it was scored as organized.
Approximately 5% of the cells could not be classified according to
these criteria and were not included in the counting. Data summarize
results from three independent experiments scored blindly.
Intercellular calcium waves. Confluent monolayers of
astrocytes were loaded for 1 hr with 10 µM fluo-3
AM (Molecular Probes). Waves were elicited by mechanical
stimulation (briefly deforming the plasma membrane with an electrode
tip) using a hydraulic manipulator (MMO-220; Narishige, Tokyo, Japan).
All the experiments were performed in culture medium at room
temperature. Excitation was provided by the 488 nm line of the
krypton-argon laser of a Bio-Rad MRC1000 confocal-scanning microscope
attached to an inverted microscope (Diaphot; Nikon). Images were
acquired every 6-8 sec and recorded on an optical disk (LM-D702W;
Panasonic). Quantification of calcium waves was performed by measuring
the maximal distance traveled by the calcium wave from the point of
initiation (radius of the wave). Velocity was calculated by dividing
the distance (micrometers) by the time (seconds) from the initial point
of stimulation to the second round of cells that showed calcium
increases. In some cases, waves were quantified by counting the number
of cells in the field that showed calcium increases after stimulation
and were expressed as "number of cells per wave." Occurrence of
calcium waves was defined as a 50% increase in
 F/F (see Measurements of calcium responses to
agonists) that propagated for a minimum of 50 µm in at least
one direction or as more than six cells engaged in the response
to mechanical stimulation (nearest neighbors). Background counts were
subtracted from all measurements.
Pharmacological reagents. Stock solutions of cytochalasin D
(1 mg/ml; Sigma), nocodazole (1 mg/ml; Sigma), and ML7 (15 mM; Alexis) were prepared in DMSO. 18-Glycyrrhetinic
acid (-AGA; mM; Sigma) was prepared in ethanol. Treated
cultures were incubated for 5 or 10 min with cytochalasin D at 1 µg/ml or for 2 hr with nocodazole at 10 µg/ml at room temperature.
Inhibition of myosin light chain kinase activity (MLCK) was
accomplished by incubating astrocytes or C6 glioma cells for 1.5 hr
with the MLCK inhibitor ML7 (50 µM) (Shrode et al.,
1995). After each treatment, cultures were tested for the ability to
propagate calcium waves, fixed at the microscope stage, and processed
for actin or tubulin staining. Control cultures were incubated with
DMSO or ethanol alone (3-5 µl).
Measurement of gap junctional permeability. Gap junction
function was assessed by three different assays. (1) In the
scrape-loading assay (El-Fouly et al., 1987; Giaume et al., 1991),
briefly, several cuts were made with a scalpel while confluent
astrocytes were incubated in a calcium-free HBSS containing 10 µM carboxy-dichlorofluorescein (CDCF; Molecular Probes).
The solution was removed 90 sec later by several washes. Gap junctional
permeability was assessed by confocal microscopy after a 15 min
incubation in HBSS. Cytochalasin at 1 µg/ml (5-10 min) or nocodazole
at 10 µg/ml (2 hr) was added in selected cultures before the
scrape-loading assay. The extent of coupling was evaluated as rows of
CDCF-labeled cells visualized by confocal microscopy with the laser
adjusted to maximal power and gain and aperture at maximal settings.
(2) In the dye transfer technique (adapted from Goldberg et al., 1995),
cells were loaded with CDCF diacetate for 5 min, washed, and
trypsinized. After resuspension, cells were labeled with 10 µM DiIC18 (excitation, 648 nm;
Molecular Probes) for 10 min and mixed with unlabeled cells at a 1:250
ratio. One hour after plating on polylysine-coated dishes, dye transfer
from the CDCF- and DiIC18-labeled (donor) cells to
unlabeled (recipient) cells was evaluated using confocal-scanning microscopy. The coupling index was calculated as: the fraction of donor
cells transferring dye to surroundings × the mean number of
receiving cells. (3) In the fluorescence recovery after photobleach procedure (FRAP) (Wade et al., 1986), the cultures were loaded with
CDCF diacetate for 5 min, washed, and incubated at room temperature for
an additional 20 min. A baseline fluorescence image of the culture was
first obtained, and the area of laser scanning was then reduced by 10×
zooming. Complete photobleach was achieved after 3-5 scans at full
laser power (1 sec each). After returning the settings to initial mode,
fluorescence refill was evaluated after 2 min and calculated by:
%refill = (I2min  Io)/Io, where Io is pixel intensity of CDCF fluorescence
emission in the target cell before bleach and
I2min is pixel intensity in the same cell 2 min
after bleach.
Measurements of calcium responses to agonists. Astrocyte
cultures were loaded with 10 µM fluo-3 AM for 1 hr. After
fluo-3 resting signal was recorded, 25 µM ATP or 25 µM bradykinin (Sigma) was applied. Calcium responses were
monitored by confocal microscopy with fixed gain and maximal aperture.
For quantification, peak increments were measured, and relative changes
in fluorescence (F) were normalized against the
baseline fluorescence (F) by F/F.
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RESULTS |
Dynamics of actin organization in cultured brain astrocytes
Primary astrocytes undergo several morphological transitions,
which is paralleled by a complex redistribution of actin after plating.
Right after attachment to the substrate, astrocytes are round in shape
with few cellular contacts. At this stage, actin is typically found as
focal accumulations in the periphery of the cells (Fig.
1). Functional coupling indicated by
extensive dye diffusion is already evident 1 hr after plating (Fig. 1). During the following hours, the cells lose their initial stage of
compaction, flatten, and establish contact with neighbors. Actin fibers
form, and parallel arrays of phalloidin-positive fibers radiate in many
different directions, both within and between individual cells. Less
than 5% of astrocytes have actin fibers at 1 and 2 hr, increasing to
40-50% at 5 hr, whereas essentially all viable astrocytes have
organized their cytoskeleton by 24 hr. Some of the actin fibers are
very long, spanning >1 mm. These fibers are often arranged in parallel
arrays (Fig. 1). Not all parts of a culture are equally well organized,
but it is in these well-organized areas that intercellular calcium
signaling is most strongly expressed (see below).
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Figure 1.
Left. Temporal pattern of cytoskeletal
organization after replating of cortical astrocytes. Differential
interference contrast and Texas Red-phalloidin staining detected by
confocal microscopy were combined to visualize the cellular arrangement
of actin at 1, 8, and 24 hr after plating. At 1 hr, most cells have
attached to the substrate but have not yet flattened. Actin aggregates
in these rounded compact cells are accumulated in a cortical mantle. At
8 hr, the majority of the cells have flattened, and actin has organized
into stress fiber bundles crossing the cells in many different
directions. One day after plating, actin is arranged in parallel arrays
of long and continuous stress fibers. Inset, Extensive
dye coupling is evident 1 hr after plating. The donor astrocytes were
prelabeled with the membrane dye DiIC18
(red) and CDCF (green; gap
junction permeable) and mixed with unlabeled astrocytes. The extent of
CDCF diffusion to neighboring recipient cells was visualized 1 hr
later. The donor cells appear yellow because of the
merge of red and green. In the example
shown, three out of six donor cells transferred dye to surrounding
cells. Scale bars, 25 µm.
Figure 2.
Right. Multiple astrocytes contribute to the
formation of long actin fibers. Texas Red-phalloidin staining of
stress fibers was combined with outlining of cell-to-cell boundaries to
visualize the cellular organization of actin fibers. A,
Individual cells were profiled by differential interference
contrast microscopy. Two neighboring astrocytes contributed the
formation of phalloidin-positive fibers. B, Staining
against the astrocytic gap junctional protein Cs43
(fluorescein-labeled) is shown. Cx43-immunoreactive plaques localized
in the plasma membrane specially at regions of cell-to-cell
appositions. The double staining revealed that actin fibers did not
respect Cx43-positive cell borders but spanned uninterrupted from cell
to cell. Nuclei were counterstained with Hoechst 33342 (purple). Scale bar, 10 µm.
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To establish whether multiple astrocytes contribute to the formation of
long actin fibers or whether these fibers were contained within
individual elongated astrocytes, we combined actin staining with
profiling of individual cells by differential interference microscopy.
As illustrated in Figure
2A, two neighboring
astrocytes contributed to the formation of actin fibers. Another
approach gave similar results; the astrocytic gap junction protein
Cx43 is localized in the plasma membrane, especially at regions
of cell-to-cell appositions, and may therefore outline individual astrocytes (Dermietzel et al., 1991). We found that phalloidin-stained fibers were not interrupted at the Cx43-positive cell borders (Fig.
2B), supporting the notion that multiple astrocytes
contribute to the construction of parallel-arrayed bundles of actin
fibers (Abd-El-Basset and Fedoroff, 1994).
Astrocytes cannot propagate continuous calcium waves immediately
after plating despite extensive gap junction coupling
We next determined when astrocytes became capable of propagating
intercellular calcium waves. To this end, calcium waves were evoked by
mechanical stimulation in fluo-3-loaded astrocytic cultures at 1, 2, 5, 8, and 24 hr after plating. Calcium wave activity was first observed
5 hr after plating, and the wave radius increased at 8 and 24 hr (Fig.
3). No further increase in wave radius
was observed at 48-72 hr (data not shown). At the earlier time points studied, calcium signaling was poor or absent (Fig. 3), although extensive cell coupling was established as early as 1 hr after plating
(Fig. 1, inset). Presence of functional gap junctions therefore was not sufficient for expression of calcium waves. We can,
however, not exclude that coupling increased as a function of time in
culture.
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Figure 3.
Astrocytic calcium signaling increases as a
function of time in culture. Upper row, One hour after
plating, mechanical stimulation triggers a robust calcium increase only
in the stimulated cell (red arrows) but fails to
initiate a calcium wave. Middle row, Eight hours after
plating, calcium waves propagated to include several neighboring cells.
Lower row, Twenty-four hours after plating, the wave
included astrocytes located as far as 300 µm from the stimulation
site. Confluent astrocytic cultures were loaded with fluo-3, and
calcium waves were evoked by mechanical stimulation. Fluorescence
intensities of fluo-3 were monitored 1, 7, 15, and 23 sec after
stimulation by confocal microscopy. In all frames, background counts
were subtracted. Scale bar, 50 µm.
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Formation of actin stress fibers correlates with the
occurrence of propagating calcium waves
The extent of calcium signaling was next correlated with
cytoskeletal organization by fixing the cultures after calcium imaging and subsequent staining with Texas Red-phalloidin. Analysis of the
actin distribution in these cultures revealed that the extent of wave
propagation increased as a linear function of the extent of actin
stress fiber formation (Fig.
4A; r = 0.88). Thus, newly plated astrocytes acquired the capability for
propagating calcium signal in parallel with the organization of their
cytoskeleton.
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Figure 4.
A, Calcium wave activity correlates
with the extent of actin fiber organization after plating of cortical
astrocytes. Comparison of the maximal distance traveled by the calcium
waves and the fraction of cells with stress fibers 1, 2, 5, 8, or 24 hr
after plating is shown. The extent of calcium wave activity and the
organization of actin stress fibers increased in parallel. A
first-order regression of wave radius as a function of percent actin
organization demonstrated that wave radius (micrometers) = 28.6 µm + 2.6 µm × percent actin organization; r = 0.88. The cultures were fixed immediately after calcium imaging and
stained with Texas Red-phalloidin to correlate directly the activity
of calcium signaling with the extent of actin organization.
B, Number of cells engaged in non-contacting calcium
signaling as a function of time after plating is shown. Jumping calcium
signaling is most pronounced shortly after plating. C,
Non-contacting calcium signaling at 2 hr is almost abolished by the
ATPase apyrase (40 U/ml); p < 0.01 (Student's t test). D, The radius of the
continuous wave evoked 24 hr after plating is also reduced by apyrase
(40 U/ml).
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"None-contacting" calcium signaling is prominent
shortly after plating but not at later time points
Immediately after plating, calcium increments were often observed
in astrocytes that were not in contact with the stimulated cell.
Mechanical stimulation evoked "jumping" calcium signaling in
approximately five to seven cells the first few hours after plating,
one to three cells at 5-8 hr, and none at 24 hr (Fig. 4B). However, the number of astrocytes engaged in
none-contacting calcium signaling was considerably lower than
the number of cells that participated in calcium waves. In comparison,
continuous calcium waves at 24 hr engaged 20-60 cells. Of interest,
jumping calcium signaling was often observed far away (>80-100 µm)
from the contiguous calcium wave. These observations are in accordance with those of Hassinger et al. (1996) who found that calcium waves crossed cell-free lanes (produced by scraping with a glass pipette 4-8
hr earlier).
Astrocytic calcium signaling is reduced by apyrase
and suramin
In other cell types, including hepatocytes (Schlosser et
al., 1996), neuroepithelial cells (Palmer et al., 1996), osteoblastic cells (Jørgensen et al., 1997), and insulin-secreting cells (Cao et
al., 1997), ATP or a related compound has been identified as the
diffusable messenger in intercellular signaling. Furthermore, ATP is
known to elicit elevation of astrocytic
Ca2+i (Kastritsis et al., 1992; Walz et
al., 1994; Salter and Hicks, 1995; King et al., 1996; Centemeri et al.,
1997). Accordingly, addition of the ATPase apyrase (40 U/ml)
attenuated jumping calcium signaling at 2 hr (Fig. 40). Apyrase also
reduced the radius of continuous calcium waves at 24 hr, suggesting
that ATP participates in both types of calcium signaling (Fig.
4D). Apyrase does not interfere with intracellular
ATP content or resting membrane potential (data not shown). In
support of the notion that ATP participates in astrocytic calcium
signaling, the purinergic receptor blocker suramin (100 µM) reduced the extent of astrocytic signaling by 78%
(from 27 ± 2 cells per control wave to 6 ± 1 cells per wave in the presence of suramin; n = 5).
Calcium mobilization in response to ATP is not compromised
in newly plated astrocytes
Because mechanical stimulation engaged fewer astrocytes
shortly after plating than at later time points and apyrase reduced the
extent of signaling at both time points, a possible explanation is that
the number of functional purinergic receptors is reduced or that the
cascade by which receptor activation mobilizes calcium is compromised
shortly after plating. However, ATP exposure (25 µM)
evoked a calcium increase, expressed as F/F
that averaged 145 ± 11% 2 hr after plating (n = 18) compared with 138 ± 6% at 24 hr (n = 8).
Thus, the astrocytic Ca2+ response to ATP exposure
did not change as a function of time after plating.
Disruption of the cytoskeleton attenuates interastrocytic
calcium signaling
We then tested whether cytoskeletal disruption in previously
established cultures affected their calcium signaling. Calcium waves were evoked in the presence of cytochalasin D (CD), which binds to actin and is associated with a rapid depolymerization of
stress fibers (Cooper, 1987). Whereas under control conditions calcium
waves propagated at a mean velocity of 12 ± 2 µm/sec and included 25 ± 4 astrocytes (n = 9) (Fig.
5, upper row), after a 5 min
treatment with CD (1 µg/ml), calcium waves spread to only a few
astrocytes in the field, 8 ± 1 cells per wave
(p < 0.01; n = 7) (Fig. 5,
middle row). A 10 min treatment with CD almost completely
blocked the propagation of calcium waves (Fig. 5, bottom row). Only 6 ± 1 astrocytes (p < 0.003; n = 8) exhibited calcium increments after
stimulation. The velocity of calcium waves was not significantly
affected by CD treatment (12 ± 0.3 or 11 ± 1 µm/sec after
5 or 10 min treatment, respectively). To correlate the extent of
calcium signaling directly with the effect of CD on the actin
cytoskeleton, we used Texas-Red-phalloidin staining to visualize actin
organization. In accordance with previous studies (Yahara et al.,
1982), the doses of CD used here caused profound disintegration of
actin fibers. Only a few intact stress fibers were found after a 5 min
treatment, and essentially none were found after a 10 min CD treatment.
Concurrently, actin accumulated in the periphery of the cells (Fig. 5,
right).
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Figure 5.
Calcium wave activity is suppressed by
cytochalasin D in established astrocytic cultures. Confluent astrocytic
cultures were loaded with fluo-3, and calcium waves were evoked by
mechanical stimulation. Upper row, A control untreated
culture propagates a calcium wave that includes >25 cells.
Middle row, Astrocytes pretreated with cytochalasin D
for 5 min only propagate calcium increases to a few astrocytes.
Lower row, Culture treated with cytochalasin D for 10 min was not capable of propagating a calcium wave, despite a robust
calcium increment in the stimulated cell. Actin fibers were visualized
in the same field (right, in red) by
fixing and staining with Texas Red-phalloidin immediately after
analysis of calcium signaling. Note the gradual disappearance of stress
fibers in cytochalasin D-treated cultures. Fluorescence intensities of
the fluo-3 probe corresponding to relative calcium levels were
monitored 1, 7, 15, and 23 sec after stimulation by confocal
microscopy. In all frames, background counts were subtracted. Scale
bar, 10 µm.
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Calcium responses remain unchanged in cytochalasin
D-treated astrocytes
To examine the possibility that CD affected cellular
calcium regulation and inhibited calcium signaling by impairing
mobilization of calcium, we compared calcium responses in the
directly stimulated cell in CD-treated astrocytes versus that in
control. The initial increase in intracellular calcium after
mechanical stimulation reflects primarily calcium influx (Sanderson
et al., 1994; Venance et al., 1997). In control astrocytes, the calcium
increase in the stimulated cell, expressed as
F/F, was 139 ± 25%, a value not
significantly different from those obtained in astrocytes treated with
CD for 5 min, 146 ± 25%, or 10 min, 114 ± 19% (Fig. 6A). The spread or
propagation of calcium waves requires release of calcium from
intracellular stores (Venance et al., 1997). Also, changes in calcium
levels from activation of receptors that promote IP3-dependent intracellular calcium release showed no
significant difference between control calcium responses to ATP-,
97 ± 32%, and CD-treated astrocytic responses, 143 ± 9%
(5 min) and 107 ± 2% (10 min). Similar results were obtained
when calcium responses were evoked by bradykinin: 99 ± 2% in
control versus 96 ± 14% and 86 ± 20% in astrocytes after
a 5 or 10 min CD treatment, respectively (Fig. 6B).
Thus, cytochalasin D does not alter astrocytic calcium responses after
either mechanical stimulation or ATP and bradykinin exposures,
supporting the notion that it is one of the most specific cytochalasins for actin (Cooper, 1987).
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Figure 6.
Astrocytic calcium responses to mechanical
stimulation or to addition of calcium-mobilizing agonists are not
suppressed by cytochalasin D treatment. A, The initial
calcium response of the mechanically stimulated cell was compared in
control versus cytochalasin D- (5 or 10 min) treated astrocytes.
B, Relative calcium increments after application of 25 µM ATP or 25 µM bradykinin are shown. No
difference between control and cytochalasin D-treated cultures were
observed. Relative calcium levels are indicated as
F/F; n = 10.
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Cytochalasin D does not alter gap
junction permeability
Immunocytochemical studies localized numerous Cx43 gap junctional
plaques in areas of cell-to-cell contact together with abundant actin
stress fibers in control cultures (Fig.
7A). Strong immunoreactivity was also present in astrocytes treated with CD for 5 min (Fig. 7C), even after loss of stress fibers. Numerous, although
somewhat smaller, immunoreactive plaques at cell membranes were found
in cultures treated with CD for 10 min when actin stress fibers were primarily absent (Fig. 7E). The functionality of these gap
junctional plaques, assessed by the scrape-loading method, was not
altered by a 5 or 10 min exposure to CD (Fig.
7B,D,F). Also,
fluorescence recovery after the photobleach (FRAP) technique did not
reveal any significant changes in gap junction coupling after
cytochalasin D treatment; fluorescence recovered to 61 ± 6%
(n = 14) in control cultures, whereas recoveries of
59 ± 8% (n = 14) and 56 ± 9%
(n = 12) were observed in cultures treated with CD for
5 and 10 min, respectively.
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Figure 7.
Cytochalasin D does not alter the pattern of
connexin 43 immunoreactivity or the extent of functional coupling.
Control cultures (A, B) or cultures
treated for 5 min (C, D) or 10 min
(E, F) with cytochalasin D were
stained with Texas-Red phalloidin and counterstained with an anti-Cx43
antibody to visualize actin stress fibers (red) and Cx43
plaques (green). Disintegration of actin stress
fibers is evident in cytochalasin D-treated cultures, whereas
Cx43-immunoreactive plaques remain in the plasma membrane. In sister
cultures, the scrape-loading assay revealed that the gap junction
plaques were functional because dye diffusion was not reduced by
cytochalasin-D when compared with control cells. Scale bars:
A, C, E, 10 µm;
B, D, F, 50 µm.
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Astrocytic calcium waves do not require
microtubule organization
A requirement for both microfilaments and microtubules has been
reported for shape change in astrocytes or growth cone movement and
exocytosis in neurons (Goldman and Abramson, 1990; Shain et al., 1992;
Ashton and Dolly, 1997; Gavin, 1997). In our cultures, -tubulin
immunostaining only stained a fraction of the cells. In these cells,
microtubules organized as a fine meshwork of fibers throughout the
entire astrocytic cell body (Fig.
8B, left).
When cultures were treated with nocodazole for 2 hr, tubulin staining was completely absent (Fig. 8B, right).
Despite the absence of intact microtubules, nocodazole-treated cultures
did not show a significant difference either in the extent of
propagation or in the velocity of calcium waves (25 ± 6 cells/wave and 16 ± 3 µm/sec) when compared with control
untreated astrocytes (Fig 8A, 33 ± 5 cells/wave and
16 ± 2 µm/sec; p = 0.324, Student's
t test).
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Figure 8.
Nocodazole treatment does not alter either the
extent or the velocity of astrocytic calcium waves. A,
Calcium waves in control astrocytes were compared with waves in
astrocytes pretreated for 2 hr with nocodazole. B,
Microtubule organization was assessed by immunostaining against
-tubulin. Disappearance of polymerized tubulin is observed in
nocodazole-treated astrocytes (right) but not in control
astrocytes treated with DMSO alone (left). Scale bar, 10 µm.
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Inhibition of myosin light chain kinase activity reduces
wave propagation
Actin-myosin complexes mediate contractility events in muscle and
nonmuscle cells (Kamm and Stull, 1985). Contractility requires phosphorylation of myosin light chain by its kinase MLCK (for review,
see Adelstein and Eisenberg, 1980). To test the requirement for
myosin-actin interactions in intercellular signaling, we evoked calcium waves in primary astrocytes that had been preincubated with the
MLCK inhibitor ML7 (50 µM). ML7 is specific because it is
a 100-fold more potent inhibitor of MLCK than are other kinases (Saitoh
et al., 1987; Shrode et al., 1995). ML7 treatment reduced the radius of
mechanically induced calcium waves by 36 ± 11%
(n = 10) compared with that of DMSO-treated astrocytic
cultures (n = 10; p < 0.01). Gap
junctional permeability was not altered in ML7-treated cultures when
evaluated by the scrape-loading assay (data not shown), and the initial
calcium increase in stimulated astrocytes was not reduced compared with
control values (185 ± 27 vs 187 ± 21%; n = 10). Thus, MLCK inhibition suppresses calcium signaling without
affecting coupling or calcium mobilization.
Astrocytes from Cx43 "knock-out" mice are morphologically
indistinguishable from wild type and propagated robust calcium
waves
We found that astrocytes derived from transgenic Cx43 null-mutant
mice are capable of propagating intercellular calcium waves that are
reduced only minimally, if at all, from those propagated by wild-type
astrocytes (Fig. 9). Naus et al. (1997)
and Spray (1998) have reported previously that astrocytes deficient in
Cx43 can propagate Ca2+ waves, although the radius
of propagation was less than that from wild-type mice. Lack of Cx43 is
not associated with uncoupling, because endogenous expression of at
least three other gap junction proteins, Cx40, Cx45, and Cx46, is high
enough to maintain 5% residual coupling (Spray, 1998) (data not
shown). In this regard, the morphological phenotype of astrocytes from
Cx43 knock-out mice is indistinguishable from that of wild-type
astrocytes; both are characterized by highly organized arrays of actin
filaments (Fig. 9). Possibly, the endogenous expression of connexins
other than Cx43 is sufficient to act as a central organizer of the
cytoskeleton preserving the normal astrocytic phenotype.
View larger version (64K):
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|
Figure 9.
Astrocytes from mice with a Cx43 null mutation
propagate robust Ca2+ waves. Upper
row, Mechanical stimulation induced a calcium wave that
propagated >300 µm from the stimulated cell in astrocytes from a
Cx43 knock-out mouse. The culture was loaded with fluo-3. Lower
row, Texas Red-phalloidin staining of a sister culture
revealed well-organized actin fiber bundles. Actin organization in Cx43
knock-out mice did not differ from wild-type astrocytes.
Scale bar: Upper row, 75 µm; lower row,
7 µm.
|
|
| |
DISCUSSION |
This study evaluated the role of actin assembly in
astrocytic calcium signaling and found that cytoskeletal organization
seems to be a key requirement for the generation of long-distance
calcium waves. First, cytoskeletal organization coincided temporally
with the number of astrocytes engaged in calcium signaling after
plating. Second, disruption of the actin cytoskeleton attenuated
calcium signaling in established cultures. The spatial expansion of
calcium waves decreased gradually and paralleled the loss of actin
fibers after exposure to cytochalasin D. Gap junction coupling or
astrocytic capability to mobilize calcium in response to ATP was not
compromised by identical treatment, suggesting that the effect was
specific to cytoskeletal organization. Several lines of evidence
suggest that release of a polyphosphate, possible ATP, is a necessary intermediate in astrocytic signaling. Focal stimulation
consistently engaged astrocytes not in contact with the
stimulated cell, and the ATPase apyrase attenuated both jumping calcium
signaling as well as continuous calcium waves. An inhibitor of
purinergic receptors, suramin, also attenuated astrocytic signaling.
Combined, these observations support the notion that gap junctions are
required for calcium signaling because of their effects on cellular
organization, rather than as a route for the exchange of intercellular
messengers. As such, the persistence of calcium waves in mice
with Cx43 null mutations is a function of their preserved actin
superstructure, rather than intercellular gap junction coupling.
At which step do actin stress fibers contribute to the generation of
calcium waves? One possibility is that wave propagation is critically
dependent on the function of the cytoskeleton as a scaffold for
signaling proteins. Actin stress fibers physically bind a number of
signaling molecules either directly or via other cytoskeletal-associated proteins (Sastry and Horwitz, 1993). In this
regard, phosphatidyl 4,5-bisphosphate (PIP2) and
IP3 receptors associate with actin in several systems (Feng
and Kraus-Friedmann, 1993; Miki et al., 1996). Possibly, cytochalasin D
treatment interferes with the signaling cascade that leads to the
generation of IP3 and Ca2+ release from
intracellular stores. The observation that Ca2+
responses to ATP and bradykinin, agonists that both promote
Ca2+ release via the IP3 signaling
pathway, were not significantly altered in cytochalasin D-treated
astrocytes does not support such a mechanism, but neither does this
observation exclude a local effect on sites of Ca2+
amplification that may be critical for wave propagation.
Traditionally, actin stress fibers have been implicated mainly in cell
shape changes and cell motility by modulation of cell-cell and
cell-matrix contacts (Pavalko and Otey, 1994). Assembly of stress
fibers is initiated by formation of focal adhesion complexes. These
complexes are composed of aggregated adhesion molecules that span the
plasma membrane and interact on the outside with other cells or the
extracellular matrix and on the inside with bundles of actin fibers and
actin-associated components. Numerous observations demonstrate that
these complexes, in addition to their functional role in adhesion, also
act as "outside-in" signal transduction receptors triggering a
number of intracellular cascades, including mobilization of
intracellular Ca2+. Cytosolic
Ca2+ elevations can in turn feed back as
"inside-out" signaling to regulate receptor function. Indeed,
phospholipase C- and IP3-dependent pathways have been described in
several of these systems including astrocytes (McNamme et al., 1993),
and both astrocytes and C6 glioma cells express multiple N-CAMs and
integrins (Noble et al., 1985; Bhat and Silberberg, 1987; Malek-Hedayat
and Rome, 1992; Tawill et al., 1993). However, although it is
established that Ca2+ signaling can be activated
after receptor binding of ligands and modulated by an array of growth
factors and cytokines, it is not clear how adhesion complexes can
contribute to intercellular calcium signaling. One possibility is that
adhesion complexes can directly transmit "inside-out-inside"
calcium signals and thereby function as a pathway that mediates or
supports the propagation of Ca2+ waves. Experimental
support for the existence of such a mechanism is lacking, but
cell-to-cell signaling has been linked to adhesion complexes in other
systems. It is believed that integrin bonds mediate the stretch reflex
by which muscle modulates transmitter release from its own motor nerve
terminals. A blocking peptide, RGD, that mimics the main integrin
binding sites effectively prevents stress-induced changes in
transmitter release (Chen and Grinnell, 1995). Integrin bonds might, as
a physical link, provide a pathway by which the presynaptic neurons can
almost immediately sense changes in muscle length and explain
that changes in transmitter release are induced within 10-20
msec of muscle stress. The stress-induced modulation of transmitter
release is dependent on intracellular Ca2+ stores,
suggesting that integrin bonds mediate an inside-out-inside Ca2+ signal in this system.
Why did inhibition of MLCK suppress wave activity? Forced
expression of gap junctions in glioma cells resulted in a profound reorganization, not only of actin, but also of myosin (Cotrina et al.,
1998b). Actin and myosin were intimately colocalized in a
manner identical to what has been described previously in astrocytes (Abd-El-Basset and Fedoroff, 1994). Isometric contraction is activated by high Ca2+ in both smooth muscle and nonmuscle
cells. Formation of a Ca2+-calmodulin complex
activates MLCK, which subsequently phosphorylates several sites on
myosin light chain, leading to an increase in actin-activated ATPase
the activity of which triggers contraction (for review, see Korn and
Hammer, 1988; Citi and Kendrick-Jones, 1996). Because
astrocytes contain all the elements required for isometric contraction,
it is intriguing to speculate that calcium-induced contractility
contributes to wave propagation. However, visible manifestation of
stress fiber movements has been difficult to prove in other systems.
Rather, a large body of evidence supports the idea that isometric
tension induced by contractility drives the formation of stress fibers
and focal adhesion complexes. Agents that inhibit MLCK, in turn, lead
to stress fiber and focal adhesion complex disassembly. Inhibition of
MLCK in this study did not result in visible degradation of stress
fibers (data not shown), suggesting that ML-7 suppression of wave
activity was not secondary to actin disassembly, but further studies
are required to establish the role of contractility in the formation of
calcium waves.
To date, no clear relationship between actin microfilaments and gap
junctional proteins has been established. As for many other ion channel
precursors, several observations suggest that connexon
precursors are transported to the plasma membrane by actin fibers; an
association between connexon precursors and actin fibers has
been identified in epithelial cells of the prostate (Tadvalkar and
Pinto da Silva, 1983), in outer horizontal cells of the goldfish retina
(Kurz-Isler and Worburg, 1988), and in the lens of primates (Lo et al.,
1994). However, studies by Wang and Rose (1995) indicate that although
cytochalasin suppressed clustering of gap junction hemichannels into
functional plaques, the treatment did not affect established plaques.
Assuming that the punctate staining observed by immunocytochemistry
corresponds to gap junction channels previously assembled in gap
junctional plaques (Paul, 1986; Dermietzel et al., 1987; Kumar and
Gilula, 1996), our experiments also suggest that existing channels were not affected by cytochalasin treatment. Cytochalasin-treated astrocytes also maintained fully functional gap junctions, as assessed by the
scrape-loading method. Consistent with this observation, Vaughan and
Lasater (1990) found no effect of cytochalasin on the electrical coupling of fish horizontal cells. Of note, stress fibers and gap
junctional plaques did, as a rule, not colocalize in either astrocytes
or transfected glioma cells (data not shown).
Extracellular ATP mediates calcium signaling among rat basophilic
leukemia cells, hepatocytes, and neuroepithelioma cells (Osipchuk and
Cahalan, 1992; Palmer et al., 1996; Schlosser et al., 1996) and
possibly also among astrocytes (Hassinger et al., 1996).
Transmitter released from astrocytes has primarily been associated with transport systems (Szatkowski et al., 1990; Parpura et
al., 1994), but glutamate release via a vesicular-like process in
cortical astrocytes has been proposed (Parpura et al., 1995a). The fact
that an intact actin cytoskeleton is necessary for calcium-dependent secretion in both neurons and secretory cells (for review, see Trifaró and Vitale, 1993) and proteins generally involved in regulated exocytosis are expressed in cultured astrocytes (Parpura et
al., 1995b; Madison et al., 1996) provides a link between an extracellular component of calcium wave activity and the cytoskeleton. However, the cytoskeleton may also regulate the activity and number of
transport proteins in the membrane (Mills and Mandel, 1994).
This study established that cellular organization is a critical
constituent for the propagation of intercellular calcium signaling. Astrocytes in culture lost their capability for propagating calcium waves when their cytoskeleton was disrupted by cytochalasin D. Although
we do not know at which level stress fibers act, the fact that
inhibition of myosin light chain kinase suppressed calcium signaling
suggests that a functional cytoskeleton is a prerequisite for
interastrocytic calcium signaling.
| |
FOOTNOTES |
Received May 8, 1998; revised Aug. 5, 1998; accepted Aug. 14, 1998.
This study was supported by National Institute of Neurological
Disorders and Stroke and National Institutes of Health Grants NS130007
and NS135011. M.N. is an Established Investigator sponsored by The
American Heart Association. We thank B. Grafstein for comments on this
manuscript.
Correspondence should be addressed to Dr. Maiken Nedergaard, Department
of Cell Biology and Anatomy, New York Medical College, Valhalla, NY
10595.
| |
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D. A. Prosdocimo, D. C. Douglas, A. M. Romani, W. C. O'Neill, and G. R. Dubyak
Autocrine ATP release coupled to extracellular pyrophosphate accumulation in vascular smooth muscle cells
Am J Physiol Cell Physiol,
April 1, 2009;
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[Abstract]
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T. M. Hoogland, B. Kuhn, W. Gobel, W. Huang, J. Nakai, F. Helmchen, J. Flint, and S. S.-H. Wang
Radially expanding transglial calcium waves in the intact cerebellum
PNAS,
March 3, 2009;
106(9):
3496 - 3501.
[Abstract]
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R. Ponsaerts, C. D'hondt, G. Bultynck, S. P. Srinivas, J. Vereecke, and B. Himpens
The Myosin II ATPase Inhibitor Blebbistatin Prevents Thrombin-Induced Inhibition of Intercellular Calcium Wave Propagation in Corneal Endothelial Cells
Invest. Ophthalmol. Vis. Sci.,
November 1, 2008;
49(11):
4816 - 4827.
[Abstract]
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A. E. Blum, S. M. Joseph, R. J. Przybylski, and G. R. Dubyak
Rho-family GTPases modulate Ca2+-dependent ATP release from astrocytes
Am J Physiol Cell Physiol,
July 1, 2008;
295(1):
C231 - C241.
[Abstract]
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U. Lalo, Y. Pankratov, S. P. Wichert, M. J. Rossner, R. A. North, F. Kirchhoff, and A. Verkhratsky
P2X1 and P2X5 Subunits Form the Functional P2X Receptor in Mouse Cortical Astrocytes
J. Neurosci.,
May 21, 2008;
28(21):
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[Abstract]
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H. C. Unsworth, T. Aasen, S. McElwaine, and D. P. Kelsell
Tissue-specific effects of wild-type and mutant connexin 31: a role in neurite outgrowth
Hum. Mol. Genet.,
January 15, 2007;
16(2):
165 - 172.
[Abstract]
[Full Text]
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C. D'hondt, R. Ponsaerts, S. P. Srinivas, J. Vereecke, and B. Himpens
Thrombin Inhibits Intercellular Calcium Wave Propagation in Corneal Endothelial Cells by Modulation of Hemichannels and Gap Junctions
Invest. Ophthalmol. Vis. Sci.,
January 1, 2007;
48(1):
120 - 133.
[Abstract]
[Full Text]
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K. Furuya, M. Sokabe, and S. Furuya
Characteristics of subepithelial fibroblasts as a mechano-sensor in the intestine: cell-shape-dependent ATP release and P2Y1 signaling
J. Cell Sci.,
August 1, 2005;
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3289 - 3304.
[Abstract]
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S. F. Okada, W. K. O'Neal, P. Huang, R. A. Nicholas, L. E. Ostrowski, W. J. Craigen, E. R. Lazarowski, and R. C. Boucher
Voltage-dependent Anion Channel-1 (VDAC-1) Contributes to ATP Release and Cell Volume Regulation in Murine Cells
J. Gen. Physiol.,
October 25, 2004;
124(5):
513 - 526.
[Abstract]
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C. J. Gallagher and M. W. Salter
Differential Properties of Astrocyte Calcium Waves Mediated by P2Y1 and P2Y2 Receptors
J. Neurosci.,
July 30, 2003;
23(17):
6728 - 6739.
[Abstract]
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E. D. Martin and W. Buno
Caffeine-Mediated Presynaptic Long-Term Potentiation in Hippocampal CA1 Pyramidal Neurons
J Neurophysiol,
June 1, 2003;
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3029 - 3038.
[Abstract]
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S. R. Fam, C. J. Gallagher, L. V. Kalia, and M. W. Salter
Differential Frequency Dependence of P2Y1- and P2Y2- Mediated Ca 2+ Signaling in Astrocytes
J. Neurosci.,
June 1, 2003;
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M. Darby, J. B. Kuzmiski, W. Panenka, D. Feighan, and B. A. MacVicar
ATP Released From Astrocytes During Swelling Activates Chloride Channels
J Neurophysiol,
April 1, 2003;
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[Abstract]
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J. Aleu, M. Martin-Satue, P. Navarro, I. P. de Lara, L. Bahima, J. Marsal, and C. Solsona
Release of ATP induced by hypertonic solutions in Xenopus oocytes
J. Physiol.,
February 15, 2003;
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[Abstract]
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J. H.-C. Lin, J. Yang, S. Liu, T. Takano, X. Wang, Q. Gao, K. Willecke, and M. Nedergaard
Connexin Mediates Gap Junction-Independent Resistance to Cellular Injury
J. Neurosci.,
January 15, 2003;
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[Abstract]
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X. Peng, J. R. Carhuapoma, A. Bhardwaj, N. J. Alkayed, J. R. Falck, D. R. Harder, R. J. Traystman, and R. C. Koehler
Suppression of cortical functional hyperemia to vibrissal stimulation in the rat by epoxygenase inhibitors
Am J Physiol Heart Circ Physiol,
November 1, 2002;
283(5):
H2029 - H2037.
[Abstract]
[Full Text]
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A. A. Mongin and H. K. Kimelberg
ATP potently modulates anion channel-mediated excitatory amino acid release from cultured astrocytes
Am J Physiol Cell Physiol,
August 1, 2002;
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C569 - C578.
[Abstract]
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W. J. Nett, S. H. Oloff, and K. D. McCarthy
Hippocampal Astrocytes In Situ Exhibit Calcium Oscillations That Occur Independent of Neuronal Activity
J Neurophysiol,
January 1, 2002;
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[Abstract]
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E. A. Newman
Propagation of Intercellular Calcium Waves in Retinal Astrocytes and Muller Cells
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April 1, 2001;
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[Abstract]
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M. E. Rubio and F. Soto
Distinct Localization of P2X Receptors at Excitatory Postsynaptic Specializations
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January 15, 2001;
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A. Bhardwaj, F. J. Northington, J. R. Carhuapoma, J. R. Falck, D. R. Harder, R. J. Traystman, and R. C. Koehler
P-450 epoxygenase and NO synthase inhibitors reduce cerebral blood flow response to N-methyl-D-aspartate
Am J Physiol Heart Circ Physiol,
October 1, 2000;
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[Abstract]
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J. A. Felix, E. R. Dirksen, and M. L. Woodruff
Physiology of a Microgravity Environment: Selected Contribution: PKC activation inhibits Ca2+ signaling in tracheal epithelial cells kept in simulated microgravity
J Appl Physiol,
August 1, 2000;
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[Abstract]
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S. R. Fam, C. J. Gallagher, and M. W. Salter
P2Y1 Purinoceptor-Mediated Ca2+ Signaling and Ca2+ Wave Propagation in Dorsal Spinal Cord Astrocytes
J. Neurosci.,
April 15, 2000;
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M. L. Cotrina, J. H.-C. Lin, J. C. Lopez-Garcia, C. C. G. Naus, and M. Nedergaard
ATP-Mediated Glia Signaling
J. Neurosci.,
April 15, 2000;
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[Abstract]
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R. S. Ostrom, C. Gregorian, and P. A. Insel
Cellular Release of and Response to ATP as Key Determinants of the Set-Point of Signal Transduction Pathways
J. Biol. Chem.,
April 14, 2000;
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11735 - 11739.
[Abstract]
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M. L. Cotrina and M. Nedergaard
ATP as a Messenger in Astrocyte-Neuronal Communication
Neuroscientist,
April 1, 2000;
6(2):
120 - 126.
[Abstract]
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N. J. Willmott, K. Wong, and A. J. Strong
A Fundamental Role for the Nitric Oxide-G-Kinase Signaling Pathway in Mediating Intercellular Ca2+ Waves in Glia
J. Neurosci.,
March 1, 2000;
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[Abstract]
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M. L. Cotrina, J. H.-C. Lin, A. Alves-Rodrigues, S. Liu, J. Li, H. Azmi-Ghadimi, J. Kang, C. C. G. Naus, and M. Nedergaard
Connexins regulate calcium signaling by controlling ATP release
PNAS,
December 22, 1998;
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[Abstract]
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Top
Abstract
Introduction
