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J Biol Chem, Vol. 274, Issue 47, 33557-33564, November 19, 1999
,From the Laboratory of Adaptive Systems, NINDS, National Institutes of Health, Bethesda, Maryland 20817
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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In this report we investigated the
correlation between cell morphology and regulation of cytosolic calcium
homeostasis. Type I astrocytes were differentiated to stellate
process-bearing cells by a 100-min exposure to cAMP. Differentiation of
cortical astrocytes increased the magnitude and duration of calcium
transients elicited by phospholipase C-activating agents as measured by
single cell Fura-2-based imaging. Calcium imaging showed differences in
the spatial pattern of the response. In both differentiated and the control cells, the response originated in the periphery and gradually extended into the center of the cell. However, the elevation of cytosolic calcium concentration ([Ca2+]i)
was particularly evident within the processes and adjacent to the inner
cell membrane of the differentiated astrocytes. In addition,
differentiation significantly prolonged the duration of the
[Ca2+]i elevation. Potentiation of the calcium
transients was mimicked by forskolin-induced differentiation and
abolished by a specific protein kinase-A blocker. Conversely, the
enhancement of the calcium transients was not mimicked by brief
exposure to cAMP not causing morphological differentiation, and in PC12
cells that did not undergo morphological changes after 100 min of cAMP treatment. Impairing cAMP-induced cytoskeleton re-organization, by
means of cytochalasin D and nocodazole, prevented the potentiation of
the calcium transients in cAMP-treated astrocytes. Phospholipase C
activity and sensitivity to inositol (1,4,5)-trisphosphate were not
involved in the enhancement of the calcium responses. Also, potentiation of the calcium transients was dependent on extracellular calcium. Calcium storage and thapsigargin-depletable intracellular calcium reservoirs were analogously not increased in differentiated astrocytes. Rearrangement of the cell shape also caused a condensation of the endoplasmic reticulum and altered the spatial relationship between the endoplasmic reticulum and the cell membrane. In conclusion, morphological rearrangements of type I astrocytes increase the magnitude and the duration of agonist-induced calcium transients via
enhancement of capacitative calcium entry and is associated with a
spatial reorganization of the relationship between cell membrane and
the endoplasmic reticulum structures.
In this study, we investigated the possibility that changes in
cell morphology might result in changes in intracellular calcium signaling. A common means of inducing morphological changes in type I
astrocytes has been prolonged application of long lasting cAMP
analogues. Astrocytes, which differentiate after prolonged exposure to
cAMP analogues, changed from a flat polygonal form to a stellate
process-bearing appearance (1). Type I astrocytes differentiated with a
long term exposure to cAMP showed changes in biochemical properties
such as an increase in the production of inositol (1,4,5)-trisphosphate
(InsP3)1 in
response to both Cultured type I astrocytes express a wide array of second
messenger-coupled receptors (for review see Ref. 6). Among these receptors, P2Y, a subclass of purinergic receptors,
activated by ATP (7) and BK receptors (3, 8) are coupled to
phospholipase C (PLC). Their activation causes calcium mobilization
from intracellular calcium stores via PLC-induced InsP3
production (for review see Ref. 9).
Store emptying generates a putative signal (10) that induces the
opening of the store-operated calcium channel (SOC) at the level of the
cell membrane, also known as the calcium release-activated calcium
channel (CRAC) (for review see Ref. 11), which allows calcium into the
cells from the extracellular space. SOC/CRAC channels may be coupled to
intracellular calcium stores through a physical connection (9, 12-15).
Alternatively, the stores can refill by promptly capturing cytosolic
calcium, admitted in the cells after the opening of the SOC/CRAC (16).
Calcium entry through the SOC/CRAC, activated by the depletion of the
intracellular calcium stores, is also known as capacitative calcium
entry (CCE).
Disorganization of the cell shape induced by disrupting cytoskeleton
organization was previously found to differentially affect calcium
transients and CCE in different cell types (17, 18). In this study, we
provide evidence that active and rapid rearrangement of astrocyte
morphology induced by activation of the protein kinase A (PKA), is
responsible for enhancement of InsP3-induced cytosolic calcium concentration ([Ca2+]i) elevation via an
enhanced CCE. Enhanced CCE, in turn, is associated with reorganization
of the spatial relationship between the outer cell membrane and the
endoplasmic reticulum (ER).
Preparation of Primary Cultures of Rat Cortical Type I
Astrocytes--
Embryonic type I astrocyte cultures were obtained from
E-17 fetuses (19). Briefly, dissected cortex from 17-day-old fetuses, cut into small fragments, were exposed to Papain (Worthington) and
mechanically dissociated. The cell suspension was centrifuged and
plated onto noncoated 25-cm2 flasks in Dulbecco's modified
Eagle's medium (containing 10% fetal bovine serum (Hyclone, Logan,
UT) (10
Post-natal type I astrocytes were prepared as described previously
(20). Dissected cortex from 2-day-old Wistar rats were digested with
trypsin (ICN Biomedical Inc., Costa Mesa, CA) (0.125% in calcium-free
PBS), and then mechanically dissociated to single cells.
Differentiation, Pretreatments, and Experimental
Protocols--
Astrocytes were exposed to 1 mM
Bt2cAMP for 30 min before Fura-2 loading. Successively, the
cells were loaded with Fura-2 always in the presence of the agent. In
all the other experiments, substances were added 10 min before
Bt2cAMP, and present throughout all the loading and washing
phases. All experiments were conducted in a saline solution (KRB)
containing in mM: NaCl, 125; KCl, 5; Na2HPO4, 1; MgSO4, 1;
CaCl2, 1; glucose, 5.5; HEPES, 20; calcium-free KRB
contained in mM: NaCl, 125; KCl, 5;
Na2HPO4, 1; MgSO4, 1; glucose, 5.5;
HEPES, 20; ethylene glycol-bis( Indirect Immunofluorescence--
Astrocytes plated on glass
coverslips were washed twice in PBS and fixed in 4% paraformaldehyde
in PBS. After a washing in PBS containing glycine, the cells were
permeabilized (0.1% Triton X-100). Nonspecific sites were saturated
with 0.2% bovine serum albumin. The preparations were exposed to
anti-GFAP monoclonal antibody 1:400 dilution (Sigma) raised in mouse.
After two washes in PBS-bovine serum albumin, the preparations were
exposed for 20 min to the secondary antibody 1:200 (anti-mouse IgG
conjugated with rhodamine). After two washes in PBS-bovine serum
albumin the coverslips were mounted on a glass slide with Vectashield (Vector Laboratories, Burlingame, CA). The preparations were observed with a conventional fluorescence microscope (Zeiss, Germany) equipped with a 63× magnification oil immersion objective (Zeiss).
ER Labeling and Living Cell Confocal Microscopy--
Astrocytes
were incubated for 15 min with 1 µM ER-Tracker (Molecular
Probes, Eugene, OR), washed for an additional 15 min, and then kept in
saline solution until observed. When ready, the coverslips were mounted
in saline solution on a glass slide and observed with a Zeiss confocal
microscope. We acquired a differential interference contrast image and
a series of fluorescence images at 0.5 µm intervals in the
z axis using 364 nm excitation and collecting with a 395 nm
long pass emission filter. A 63× lens, coupled to a software zooming
capability, was used to produce single cell images.
InsP Assays--
InsP1 accumulation was performed as
described previously (19). Near-confluent astrocytes were switched to
serum-free, myoinositol-free Dulbecco's modified Eagle's medium
containing 1 (3 for InsP3 assay) µCi/ml of
myo-[2-3H]inositol (30 Ci/mmol) (American Radiolabeled
Chemicals, Inc., St. Louis, MO), and labeled for 36 h. After
labeling, cells were washed in KRB containing in mM: NaCl,
125; KCl, 5; Na2HPO4, 1; MgSO4, 1;
CaCl2, 1; glucose, 5.5; HEPES, 20 mM. pH was
set at 7.2 at 22 °C. Cells were equilibrated in KRB containing 10 mM LiCl for 20 min and exposed to ATP for 90 min or 15 s (no lithium was included for InsP3 assay). The reaction
was terminated with 6% ice-cold perchloric acid and the acidity
neutralized. An anionic exchange chromatography was performed to
separate InsP1 (Dowex AG 1X-8, 100-200 mesh, mixed 1:3 in
water) (Bio-Rad). Water was used to elute the unbound. Total
InsP1 were eluted with 500 µl of a solution containing
1.2 M ammonium formate (AF) 0.1 M formic acid.
Alternatively, InsP1, InsP2, and
InsP3 were individually recovered from the resin with a
serial elution with increasing concentration of AF in 0.1 M
formic acid (0.4 M AF for InsP1, 0.8 M AF for InsP2, 1.2 M AF for
InsP3). Finally, collected samples were mixed with
appropriate volumes of scintillation mixture and counted for 4 min.
Single Cell [Ca2+]i
Measurements--
Nearly confluent astrocyte cultures seeded on glass
coverslips (Assistent, Germany) were washed once in KRB. After washing, cells were loaded with 2 µM Fura-2/AM (Molecular Probes)
for 22 min at room temperature to minimize dye compartmentalization in subcellular compartment, under continuous gentle agitation. After loading, the cells were washed once with fresh KRB and then kept for an
additional 22 min in Fura-2/AM-free KRB (19). Ratio measurements were
acquired every 2 s. The experimental data were analyzed with the
software MetaFluor (Universal Imaging, West Chester, PA). Briefly, an
area corresponding to the entire cell surface was delimited by using
the editing capability of the software. The experimental ratio values,
derived from the entire cytosolic area, obtained by delimiting the
profile of the cells and averaging the signals within the delimited
area, were converted in cytosolic calcium concentration using a
titration calibration curve obtained in living cells exposed to
known extracellular calcium concentration in the presence of 5 µM ionomycin (containing 5.2% calcium). The following
are the ratio values measured in astrocytes: 760 nM Caext R340/380 = 18; 1,260 µM Caext R340/380 = 32. R340/380 at 0 Caext = 0.29;
R340/380 at 10 mM Caext = 60.
Materials--
All substances were from Sigma unless stated otherwise.
Statistical Analyses--
Experiments were performed at least
three times on different cell preparations. The plots were created by
using the average ± S.E. of all the cells studied. The values
were analyzed by analysis of variance followed by t test.
Differences were considered significant for p Differentiation of Rat Cortical Type I Astrocytes with a Brief
Treatment of Bt2-cAMP--
Flat polygonal type I
astrocytes (Fig. 1a) exposed
to 1 mM Bt2cAMP (Fig. 1b) or 100 µM forskolin (not shown) for 100 min, exhibited a
process-bearing cell phenotype, as shown by imaging of GFAP
immunoreactivity.
Agonist-induced Calcium Responses in Undifferentiated and
Differentiated Astrocytes--
ATP- and BK-induced statistically
significant responses in astrocytes (Fig.
2a and b,
insets I, respectively). [Ca2+]i
elevation induced by 10 µM ATP (Fig. 2a,
main and inset I), 100 nM BK (Fig.
2b, main and inset I), norephinephrine or carbamylcholine (not shown) was greatly enhanced in differentiated astrocytes. As previously reported, ATP responses were elicited in
virtually all astrocytes studied (7) (see Fig. 4, panels r
and s for typical experimental traces of ATP responses),
whereas about 75% of the astrocytes responded to BK (Fig. 2,
panel b, inset II) (9). In differentiated
astrocytes, we observed a potentiation of ATP responses at all
concentrations tested (Fig. 2,
panel a, open circles versus closed
circles). Both the initial, "spike-like", phase and the
sustained phase of intracellular calcium elevation were increased in
differentiated cells, as compared with undifferentiated cells (Fig. 3,
panels a and b, closed circles versus
open circles). The spatial development of ATP response was
also analyzed. In control astrocytes, the responses began in the
periphery of the cell body and extended into the perinuclear area (Fig.
4, panels a-d).
The decay of the responses began from the perinuclear area and
subsequently extended into the periphery of the cell. The entire
response was very fast (8 s in toto). In differentiated
astrocytes [Ca2+]i increased initially in the
astrocyte prolongment and in the periphery of the cells and extended to
the soma (panels e-n). Astrocyte processes and
the regions immediately adjacent to the inner cell membrane showed a
more marked [Ca2+]i elevation. In addition,
differentiation significantly prolonged the duration of the
[Ca2+]i elevation. In fact, as shown in Fig. 4,
the response lasted 4 frames (panels a-d) in
control, and 8 frames (panels e-n) in
differentiated astrocytes (given the 2 s interval between each
frame acquisition, the response lasted 8 s in controls and 16 s in differentiated astrocytes (Fig. 4)).
Statistically significant BK responses (Fig. 2b, inset
I) were recorded in 70-80% of the cells studied as already
described by other authors (Fig. 2b, inset II)
(9). The percentage of cells responding to BK was not affected by
differentiation (Fig. 2b, inset II). ATP
responses were also increased when differentiation of astrocytes was
achieved by exposure to 100 µM forskolin for 100 min
([Ca2+]i peak in undifferentiated astrocytes was
620 ± 35 nM versus 1.650 ± 120 µM in forskolin differentiated astrocytes, n = 76, p PLC Activity in Undifferentiated and Differentiated
Astrocytes--
ATP-induced InsP1 accumulation was similar
in both undifferentiated and differentiated cells (Fig.
6a). However, in
differentiated astrocytes the stimulation reached a plateau at a lower
concentration of ATP than in undifferentiated cells. Although there
were no significant changes in the accumulation of InsP1,
we also tested whether the production of InsP3 was changed
in the two cell types. InsP3 production after a 15 s
stimulation with ATP was unaffected by differentiation (Fig. 6,
inset to panel a). Because it has been reported
that long term treatment with cAMP analogues can affect both BK- and
Effect of Differentiation on Calcium Release from Intracellular
Stores--
In the absence of extracellular calcium, the release of
calcium from intracellular stores induced by 20 µM
thapsigargin (an irreversible inhibitor of the smooth endoplasmic
reticulum Ca-ATPase; for review see Ref. 22) was identical in both
undifferentiated and differentiated astrocytes (Fig.
7a). However, in the presence of extracellular calcium, the increase of [Ca2+]i
caused by 20 µM thapsigargin was significantly larger (see Fig. 7c) in differentiated astrocytes than in
undifferentiated cells (Fig. 7b).
Effect of Actin Stress Fiber Depolymerization on the Potentiation
of ATP- and Thapsigargin-induced [Ca2+]i
Elevation--
Treatment of astrocytes with 10 µM
cytochalasin D (CytD) did not significantly affect morphology (not
shown) and slightly increased ATP responses in undifferentiated cells
(Fig. 8a). Treatment with 10 µM CytD, however, prevented the morphological changes induced by Bt2cAMP (Fig. 1d), and the
potentiation of ATP-induced [Ca2+]i elevation
(Fig. 8a). Thapsigargin-induced
[Ca2+]i elevation in undifferentiated cells was
decreased by 10 µM CytD (Fig. 8b). In
differentiated cells the potentiation of thapsigargin-induced
[Ca2+]i elevation was abolished by actin
depolymerization (Fig. 8b).
Effect of the Anti-tubulin Drug Nocodazole on the Potentiation of
ATP- and Thapsigargin-induced [Ca2+]i
Elevation--
Treatment of astrocytes with the anti-tubulin drug
nocodazole, at 10 µM, prevented cAMP-induced
morphological differentiation of type I astrocytes (Fig.
1e). Nocodazole treatment increased ATP-induced
[Ca2+]i elevation in control cells (Fig.
9a). However,
Bt2cAMP-treated astrocytes exposed to nocodazole showed a
reduction of the calcium transient evoked by ATP as compared with
differentiated astrocytes not treated with nocodazole (Fig.
9a). In control cells, thapsigargin-induced calcium
mobilization was not affected by nocodazole (Fig. 9b). In
differentiated cells the treatment with nocodazole eliminated the
potentiation of [Ca2+]i elevation observed after
exposure to thapsigargin (Fig. 9b).
Distribution of the Endoplasmic Reticulum in Polygonal and
Differentiated Type I Astrocytes--
In flat polygonal type I
astrocytes, the internal membrane-delimited compartments are well
visible in the periphery of the cell, whereas in the perinuclear space
ER structures are grouped with other intracellular membrane delimited
compartments such as the Golgi apparatus (Fig.
10, a and b).
Isolated and rare single tubular structures, reaching out of the cell
membrane border, were present in the periphery of the cell
(arrowheads, Fig. 10, a and b). Fully
differentiated astrocytes were smaller, thicker, and rounder and had
extended large processes (Fig. 10c). In this reconfigured
morphology, the cells showed a marked condensation of the internal
membrane-delimited compartments all across the cell (Fig.
10d).
Capacitative calcium entry is a mechanism whereby intracellular
calcium stores are refilled (9, 12-15, 16). The maintenance of the
filled state of intracellular stores is involved in cell survival as
the prolonged depletion of calcium reservoirs causes cell death and
apoptosis (23).
Our data indicated that the fast occurring morphological rearrangement
of type I astrocytes from a flat polygonal appearance to a stellate
process-bearing one, induced by exposure to Bt2cAMP or
forskolin, was associated with larger and longer calcium transients in
response to neurotransmitters activating PLC, particularly within the
newly created processes and in regions immediately adjacent to the cell
membrane. Potentiation of the response to InsP3-generating
agents affected both the spike and the plateau phase of the
agonist-induced calcium mobilization. Our data indicated that even if
the cAMP-PKA system is activated with exogenous cAMP, in the absence of
morphological changes, calcium transients are unaffected. In fact, a
brief exposure to Bt2cAMP, was not able to induce
morphological changes, and did not change the intensity of calcium
transients. This view is supported by the fact that PC12 cells treated
for 100 min with Bt2cAMP did not change their morphology or
their response to ATP (data not shown). The blockade of PKA, with
KT-5720, a fairly specific inhibitor of the cAMP-activated protein
kinase (20, 21), prevented the morphological changes and returned the
calcium responses to the values of the undifferentiated cells. Thus,
activity of PKA was required to induce potentiation of the calcium
transients, although intermediate steps may have occurred between
activation of the kinase and the enhancement of the calcium transient.
Therefore, we hypothesized that PKA activation might have an effect on
intermediate targets, the modification of which, in turn, potentiated
both agonist- and thapsigargin-induced calcium transients. Involvement
of the membrane receptor-G protein-PLC system, as a target of PKA
action, was ruled out as a consequence of the fact that agonist-induced
InsP1 accumulation and InsP3-production were
both unaffected by differentiation. Although PLC activity was not
changed by differentiation, differences in InsP3-receptor sensitivity, and direct modulation of the InsP3-activated
calcium channel on the ER could have played a role in the enhancement of calcium transients in differentiated cells. However, the fact that
thapsigargin-induced calcium transients, which do not rely upon
InsP3 receptor activation, were also enhanced by astrocyte differentiation, supports the involvement of a mechanism downstream to
InsP3 receptor activation. To further characterize the
mechanism underlying enhancement of the calcium transients in
differentiated cells, we analyzed the contribution of extracellular
calcium to the potentiation of calcium transients in differentiated
astrocytes. In the absence of extracellular calcium, ATP- and
thapsigargin-induced [Ca2+]i elevation were
similar in control and differentiated cells. This finding strongly
suggests that the sensitivity to InsP3, the amount of
calcium stored in the intracellular compartments, and the amount of
calcium stored in other intracellular calcium reservoirs (that were not
sensitive to InsP3 but could have been released by calcium
itself) were not involved in the enhancement of the calcium transients
in differentiated cells. On the other hand, the absolute dependence on
extracellular calcium for the enhancement of calcium transients,
observed in differentiated astrocytes, indicates that an increase of
calcium entry was clearly involved in the potentiation of calcium
responses in differentiated cells. Thus, our data suggests that calcium
entrance from the extracellular space was increased in
Bt2cAMP-treated astrocytes, only if they had undergone
shape changes.
Cell morphology has been shown to play a role in agonist-induced
calcium mobilization in fibroblasts (17), in thapsigargin-induced store
depletion, and in the associated CCE in endothelial cells (18). Because
cAMP-induced differentiation causes the rearrangement of the
actin-formed stress fiber (1), we induced actin to depolymerize with
CytD before exposing the cells to Bt2cAMP. As expected, in the presence of CytD, the cells did not reshape and maintained their
flat polygonal appearance, although PKA was being activated by cAMP.
The prevention of the shape changes in Bt2cAMP-treated cells, by CytD, avoided the potentiation of ATP- and
thapsigargin-induced [Ca2+]i elevation. Both PKA
activity in the cell cytosol and PKA localization at level of the cell
membrane, where CRAC channels are localized, have been reported not to
be affected by CytD pretreatment (24-26). Therefore, it seems likely
that morphological changes involving the rearrangement of stress fibers
play a critical role in the potentiation of the CCE. CytD was reported
to decrease CCE in endothelial cells without affecting agonist-induced
calcium transients (18). However, it was also reported that CytD was able to induce shape changes in fibroblasts that were associated with
the loss of the sensitivity to ATP in the presence of preserved thapsigargin and CCE responses (17). We did observe both a slight potentiation of ATP response and a more pronounced inhibition of
thapsigargin-induced calcium mobilization, following CytD treatment in
undifferentiated cells, the morphology of which did not undergo appreciable changes after CytD pretreatment (not shown), although actin
filaments were disassembled by the drug (not shown). This suggests
that, in type I astrocytes, disrupting stress fibers with CytD is not
sufficient to abolish calcium transients, unless a dramatic change of
the shape of the cells is achieved. However, the specific inhibition of
the stress fiber reorganization was able to block the cAMP-induced
differentiation and the associated potentiation of the calcium transients.
To confirm this interpretation, we tested the effect of nocodazole, an
agent that impairs tubulin cytoskeleton organization. Nocodazole
pre-exposure blocked the differentiation of the astrocytes obtained
with cAMP. Noticeably, nocodazole itself caused a consistent increase
of the calcium transients elicited by ATP in control astrocytes,
although it did not affect thapsigargin-induced calcium mobilization.
This unexpected and selective increase in ATP response by nocodazole,
without affecting thapsigargin action, may be due to an interference of
the drug with the ATP system, unrelated to its activity as a tubulin
depolymerizer. However, nocodazole-treated cells in the absence or in
the presence of Bt2cAMP showed similar responses,
suggesting that although the response to ATP was increased by
nocodazole itself, nocodazole treatment was able to prevent calcium
signaling changes due to shape changes. In addition, the ability of
nocodazole to block the potentiation of thapsigargin-induced calcium
mobilization, without affecting thapsigargin effect, in cAMP-treated
astrocytes showed clearly the dependence of the calcium transient
enhancement on the acquisition of the new morphology.
Two hypotheses have been proposed to explain how calcium flowing
through the SOC/CRAC during CCE can be captured and stored in the ER
compartments and thus contribute to the spike or the plateau phase of
the single calcium transient. Calcium, according to the preferential
pathway hypotheses, could be directly sequestered within intracellular
stores because of a physical association between the SOC/CRAC and the
SERCA-ER complex (9, 12-15). Alternatively, calcium ions, once
admitted into the cytosol by SOC/CRAC opening, might be quickly removed
from the cytosol and accumulated in the ER by the SERCA (diffuse
pathway) (16). Regardless of which of the two mechanisms is operating
in type I astrocytes, it is likely that the reorganization of the
spatial relationship between ER structures and cell membrane may cause
a change in the efficiency of calcium mobilization as results of the
improved refilling of the intracellular calcium stores. This would, in
turn, result in larger calcium transients. We assessed the arrangement
of the ER in undifferentiated and differentiated cells. In
undifferentiated astrocytes, ER structures were largely evident in the
periphery of the cells where a tubular network of membrane delimited
structures were identifiable. In addition, grouped in the perinuclear
region of the astrocytes, ER structures and the Golgi complex were
identified. In differentiated cells, at least as a consequence of the
marked reorganization of the cell shape, the ER was so condensed that areas of the cytoplasm free of ER structures were virtually absent. Such a marked rearrangement may, in turn, increase the availability of
calcium flowing from the extracellular space to provide for refilling
of the stores during CCE, through a closer association of the outer
cell membrane and ER structures.
In conclusion, our data indicates that an active and rapid remodeling
of type I astrocyte morphology from an expanded epithelial-like to a
condensed process-bearing shape is induced by the activation of the
cAMP-PKA system. This remodeling appears to cause a cytoskeleton-driven spatial reorganization of the relationship between the plasma membrane
and ER structures, thereby increasing calcium flow into the cells
during CCE, that, in turn, enhances agonist-induced [Ca2+]i elevation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1-adrenergic agonists (2) and
bradykinin (BK) (3). In addition, long-term cAMP-induced
differentiation modifies membrane ionic conductance (4, 5).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
6 cells/flask)(Corning-Costar, NJ). After 6-8 h,
unattached cells, mainly neurons, were washed away. This protocol
yielded at least 95% pure type I astrocytes as characterized by glial
fibrillary acid protein (GFAP).
-aminoethyl ether)-tetraacetic acid,
0.1 pH was set at 7.2.
0.05.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (112K):
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Fig. 1.
Pharmacological manipulation of type I
astrocytes morphology. Astrocytes were treated for 100 min with 1 mM Bt2cAMP in the absence or presence of 300 nM KT-5720, 10 µM CytD or nocodazole,
respectively. Next, the cells were fixed and immunostained for GFAP
(a-e) and observed with an inverted
epifluorescence microscope equipped with a 63× lens. Panel
a displays GFAP immunoreactivity in a control astrocyte culture.
Panel b shows GFAP immunoreactivity in
Bt2cAMP-treated astrocytes. Panels c,
d, and e illustrate the effect of 300 nM KT-5720, 10 µM CytD, and 10 µM nocodazole, respectively, in the presence of 1 mM Bt2cAMP.
View larger version (13K):
[in a new window]
Fig. 2.
Effects of the differentiation on
agonist-induced calcium transients. [Ca2+]i
in single cells, expressed as average of the entire cell area, were
measured. Thereafter the values derived from all the cells were
averaged and graphed as average ± S.E. Panel a shows
the effect of ATP-induced [Ca2+]i elevation in
undifferentiated (open circles) and differentiated
(closed circles) astrocytes. The inset to
panel a displays the statistical analysis of the
experimental data at the maximal value of the spike. In panel
b the response to BK in undifferentiated (open
triangles) and differentiated (closed triangles)
astrocytes is shown. Inset II to panel b is a
plot of the responding (open bars) versus
nonresponding (hatched bars) cells. Inset I to
panel b displays the statistical analysis of the
experimental data at the maximal value of the spike. The
first arrowhead indicates the time at which ATP
or BK was added. The second arrowhead indicates the washout
of the agonist. * p 
0.05, versus value in
undifferentiated cells.
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Fig. 3.
Effect of differentiation on the properties
of ATP response. In panel a the effect of increasing
concentrations of ATP in undifferentiated astrocytes (open
circles) and differentiated astrocytes (closed circles)
at the peak of the [Ca2+]i elevation is shown, as
average ± S.E. of the response measured in all the cells studied.
In panel b the effect of increasing concentration of ATP in
undifferentiated (open circles) and differentiated
(closed circles) astrocytes on
[Ca2+]i, 20 s after the spike, is displayed
([Ca2+]i values 20 s after the spike were
averaged and graphed as curve). Each concentration was tested in
different sets of coverslips. *p 0.05, versus value in undifferentiated cells.
View larger version (74K):
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Fig. 4.
Space- and time-resolved imaging of ATP
response. Fura-2 fluorescence at 340 and 380 was imaged using a
40× lens every 2 s, ratioed and converted into
[Ca2+]i. [Ca2+]i was
expressed as pseudocolor ranging from blue to red
as shown by the color bar in the top right corner of the
figure. The arrowheads in panels a and
e indicate the direction of perfusion flow. Panels
a-d show the responses to 10 µM ATP in
undifferentiated astrocytes. ATP reached the cells at the image
a. The successive ratio images display the development of
the [Ca2+]i elevation. Panels
e-n show responses to 10 µM ATP in
differentiated astrocytes. ATP reached the cells in image e.
The arrows in panel f indicate the early and high
increase of [Ca2+]i in the processes
(orange) and in the periphery (white) of the
cell, respectively. The white arrow in panel h
indicates the initial increase of [Ca2+]i in the
cell soma. Panels r and s display traces of the
response to ATP in two representative experiments in control and
differentiated astrocytes, respectively. Each line represents the trace
derived from the entire cell imaged.
0.05). A few minutes
exposure to Bt2cAMP, which was unable to induce
morphological changes, did not increase ATP responses ([Ca2+]i = 580 ± 50 nM,
n = 46). In the absence of extracellular calcium,
however, both undifferentiated and differentiated astrocytes displayed
similar [Ca2+]i elevation in response to ATP
([Ca2+]i in undifferentiated astrocytes in
absence of extracellular calcium and 100 µM EGTA = 420 ± 70, n = 65; [Ca2+]i
in differentiated astrocytes in absence of extracellular calcium and
100 µM EGTA 480 ± 100 nM,
n = 66). Blockade of PKA with 300 nM
KT-5720 (for review see Refs. 20 and 21) (a gift from Kamiya Biomedical
Co., Seattle, WA), although did not decrease calcium transients evoked
by ATP (Fig. 5), prevented the
morphological changes induced by cAMP (Fig. 1c) and the
enhancement of the [Ca2+]i elevation in response
to ATP (Fig. 5).
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Fig. 5.
Effect of PKA blockade on ATP response.
The inhibition of PKA activity by KT-5720 blocked the increase of
ATP-induced [Ca2+]i elevation in cAMP-treated
astrocytes. The response to ATP was measured in undifferentiated
astrocytes (open bar), in undifferentiated astrocytes in the
presence of 300 nM of KT5720 (hatched bar), in
differentiated astrocytes (solid bar), and in
Bt2cAMP-treated astrocytes pre-exposed to 300 nM KT-5720 (square-filled bar). Thereafter, the
peak value of [Ca2+]i from individual treatments
was averaged and expressed as a bar graph and used to perform
statistical analysis. * p 0.05 versus
value in control cells; ** p 0.05 versus
value in differentiated cells.
1-adrenergic-induced InsP1 production in post-natal astrocytes (2, 3), we also studied InsP1
production in post-natal astrocytes (Fig. 6b). In post-natal
astrocytes, the treatment with Bt2cAMP for 100 min caused
cell differentiation (not shown) and potentiated ATP-induced
[Ca2+]i elevation ([Ca2+]i
after ATP stimulation in undifferentiated astrocytes = 654 ± 22 nM, n = 58;
[Ca2+]i after ATP stimulation in differentiated
astrocytes = 1,753 ± 200 nM, n = 77; p 0.05). As was observed in embryonic astrocytes, InsP1 generation was not affected by
differentiation in post-natal astrocytes either (Fig.
6b).
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Fig. 6.
Effect of differentiation on
InsP1 and InsP3 production induced by ATP.
We studied InsP1 production in both embryonic and
post-natal astrocytes. Panel a displays the
concentration-response curve of InsP1 generation in
response to ATP in undifferentiated (open circle) and
differentiated (closed circle) cells in embryonic
astrocytes. The inset to panel a displays
InsP3 production in basal conditions (open bars)
and after 15 s of stimulation with 10 µM ATP
(solid bars) in undifferentiated and differentiated cells,
respectively, in embryonic astrocytes. Panel b displays the
accumulation of InsP1 in basal conditions (open
bars) and after stimulation with 10 µM ATP
(solid bars) in undifferentiated and differentiated cells,
respectively, in astrocytes prepared from a 2-day-old rat.
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Fig. 7.
Effect of differentiation on
thapsigargin-induced mobilization of calcium from intracellular stores.
Panel a displays the effect of 20 µM
thapsigargin on both differentiated and undifferentiated astrocytes in
calcium-free KRB. Undifferentiated (open circles) and
differentiated (closed circles) astrocytes released the same
amount of calcium from intracellular stores in the absence of
extracellular calcium. Panel b shows the effect of 20 µM thapsigargin in control (open circles) and
differentiated (closed circles) astrocytes in the presence
of extracellular calcium. Lines were obtained by averaging
[Ca2+]i values obtained in all the cells
imaged ± S.E. Panel c shows the statistical analysis
of the experimental results at the highest
[Ca2+]i obtained in the presence and absence of
extracellular calcium in undifferentiated (open bars) and
differentiated (solid bars) astrocytes (the
arrowheads indicate the addition of thapsigargin to the
perfusion chamber; 
- indicates the perfusion of calcium-free
KRB). * p 0.05 versus value in control
cells; ** p 0.05 versus value in
differentiated cells.
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Fig. 8.
Effect of stress fiber depolymerization on
ATP- and thapsigargin-induced [Ca2+]i
elevation. The effect of stress fiber disassembly by means of 10 µM CytD was studied. [Ca2+]i values
obtained at the peak of the response was averaged and graphed as a
bar ± S.E. In panel a, ATP-induced intracellular
calcium mobilization in undifferentiated and
Bt2cAMP-treated astrocytes. Undifferentiated (open
bar) and 10 µM CytD pretreated (hatched
bar) astrocytes display a similar response to ATP. However, the
potentiation of ATP stimulation in differentiated astrocytes
(solid bar versus open bar) was reversed by CytD
pretreatment (square-filled bar). * p 0.05 versus value in undifferentiated cells; **
p 0.05 versus value in differentiated
cells.
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Fig. 9.
Effect of the anti-tubulin drug nocodazole on
ATP- and thapsigargin-induced [Ca2+]i
elevation. [Ca2+]i values obtained at the
peak of the response were averaged and graphed as bars ± S.E. In
panel a, the effect of anti-tubulin drug nocodazole on
ATP-induced intracellular calcium mobilization in undifferentiated and
differentiated astrocytes is displayed. Nocodazole-treated astrocytes
(hatched bar) showed a higher response to ATP than untreated
undifferentiated astrocytes (open bar). However, nocodazole
pretreatment (square-filled bar) reversed the potentiation
of ATP response in differentiated astrocytes (solid bar) to
the values observed in the nocodazole-treated undifferentiated cells.
In panel b, the effect of nocodazole on thapsigargin-induced
intracellular calcium mobilization in undifferentiated and
differentiated astrocytes is displayed. Nocodazole-treated
undifferentiated astrocytes (hatched bar) showed a similar
response to thapsigargin as undifferentiated cells (open
bar). Nocodazole pretreatment (square-filled bar)
completely reversed the potentiation of thapsigargin response in
differentiated astrocytes (solid bar). * p 0.05 versus value in undifferentiated cells; **
p 0.05 versus value in differentiated
cells.
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Fig. 10.
Analysis of the ER distribution and
association with cell membrane in control and differentiated
astrocytes. ER was labeled in living cells by means of the ER
tracker and analyzed with a confocal microscope, equipped with a 63×
lens for ER-associated fluorescence. Images were then software zoomed
to resolve single astrocytes. Undifferentiated astrocytes (panel
a) present a higher density of ER membranes in the perinuclear
area, whereas the density of the tubular network is decreased in the
peripheral part of the cells (panel b). The
arrowheads indicate some connection between the tubular ER
structures and the cell membrane. Differentiated astrocytes
(panel c) showed a more condensed ER organization
(panel d) without appreciable areas free of fluorescence
signal across the cell.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
| |
ACKNOWLEDGEMENTS |
|---|
We gratefully acknowledge Dr. Alessandro Fatatis for critical discussion of the data. We thank Deanna L. Buck for comments on the manuscript. David Ide is also acknowledged for help in fabricating the perfusion chamber used for calcium imaging experiments. We acknowledge Dr. Carolyn Smith and NINDS Light Imaging Facility for assistance provided in production of confocal images.
| |
FOOTNOTES |
|---|
* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Laboratory of Adaptive
Systems, NINDS, National Institutes of Health, 36 Convent Dr., Bldg.
36, Rm. 4A22, Bethesda, MD 20817. Tel.: 301-402-0514; Fax:
301-402-5360; E-mail: maurizio@codon.nih.gov.
§ Present address: Dept. of Neurology, USUHS, National Institutes of Health, Bethesda, MD 20817.
| |
ABBREVIATIONS |
|---|
The abbreviations used are: InsP3, inositol (1,4,5)-trisphosphate; Bt2cAMP, dibutyryl-cAMP; PKA, protein kinase A; [Ca2+]i, cytosolic calcium concentration; ER, endoplasmic reticulum; BK, bradykinin; PLC, phospholipase C; CCE, capacitative calcium entry; CytD, cytochalasin D; GFAP, glial fibrillary acidic protein; CRAC, calcium release-activated calcium channel; SOCC, store-operated calcium channel; PBS, phosphate-buffered saline; AF, ammonium formate.
| |
REFERENCES |
|---|
|
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
|
|---|
| 1. | Goldman, J. E., and Chiu, F. C. (1984) Brain Res. 306, 85-95[CrossRef][Medline] [Order article via Infotrieve] |
| 2. | Fahrig, T., and Sommermeyer, H. (1993) Brain Res. 602, 318-324[CrossRef][Medline] [Order article via Infotrieve] |
| 3. | Chen, C. C., Chang, J., and Chen, W. C. (1996) Glia 16, 210-217[CrossRef][Medline] [Order article via Infotrieve] |
| 4. | Barres, B. A., Chun, L. L., and Corey, D. P. (1989) J. Neurosci. 9, 3169-3175[Abstract] |
| 5. |
Lascola, C. D.,
and Kraig, R. P.
(1996)
J. Neurosci.
16,
2532-2545 |
| 6. | Bhat, N. R. (1995) Dev. Neurosci. 17, 267-284[Medline] [Order article via Infotrieve] |
| 7. | Shao, Y., and McCarthy, K. D. (1993) Neuroscience 55, 991-1001[CrossRef][Medline] [Order article via Infotrieve] |
| 8. | Stephens, G. J., Cholewinski, A. J., Wilkin, G. P., and Djamgoz, M. B. A. (1993) Glia 9, 269-279[CrossRef][Medline] [Order article via Infotrieve] |
| 9. | Berridge, M. J. (1995) Biochem. J. 312, 1-11 |
| 10. |
Rzigalinski, B. A.,
Willoughby, K. A.,
Hoffman, S. W.,
Falck, J. R.,
and Ellis, E. F.
(1999)
J. Biol. Chem.
274,
175-182 |
| 11. |
Parekh, A. B.,
and Penner, R.
(1997)
Physiol. Rev.
77,
901-930 |
| 12. | Putney, J. W., Jr. (1986) Cell Calcium 7, 1-12[CrossRef][Medline] [Order article via Infotrieve] |
| 13. | Putney, J. W., Jr. (1990) Cell Calcium 11, 611-624[CrossRef][Medline] [Order article via Infotrieve] |
| 14. | Putney, J. W., Jr. (1997) Cell Calcium 21, 257-261[CrossRef][Medline] [Order article via Infotrieve] |
| 15. |
Putney, J. W., Jr.
(1998)
Science
279,
191-192 |
| 16. | Hofer, A. M., Landolfi, B., Debellis, L., Pozzan, T., and Curci, S. (1998) EMBO J. 17, 1986-1995[CrossRef][Medline] [Order article via Infotrieve] |
| 17. |
Ribeiro, C. M.,
Reece, J.,
and Putney, J. W., Jr.
(1997)
J. Biol. Chem.
272,
26555-26561 |
| 18. | Holda, J. R., and Blatter, L. A. (1997) FEBS Lett. 403, 191-196[CrossRef][Medline] [Order article via Infotrieve] |
| 19. | Grimaldi, M., and Cavallaro, S. (1999) Eur. J. Neurosci. 11, 1-7[CrossRef][Medline] [Order article via Infotrieve] |
| 20. | Grimaldi, M., Pozzoli, G., Navarra, P., Preziosi, P., and Schettini, G. (1994) J. Neurochem. 63, 344-350[Medline] [Order article via Infotrieve] |
| 21. | Kase, H., Iwahashi, K., Nakanishi, S., Matsuda, Y., Yamada, K., Takahashi, M., Murakata, C., Sato, A., and Kaneko, M. (1987) Biochem. Biophys. Res. Commun. 142, 436-440[CrossRef][Medline] [Order article via Infotrieve] |
| 22. |
Thastrup, O.,
Cullen, P.,
Drobak, B. K.,
Hanley, M. R.,
and Dawson, A. P.
(1990)
Proc. Natl. Acad. Sci. U. S. A.
87,
2466-2470 |
| 23. | Zhou, Y. P., Teng, D., Dralyuk, F., Ostrega, D., Roe, M. W., Philipson, L., and Polonsky, K. S. (1998) J. Clin. Invest. 101, 1623-1632[Medline] [Order article via Infotrieve] |
| 24. | Iwasaki, H., Eguchi, S., Shichiri, M., Marumo, F., and Hirata, Y. (1998) Eur. J. Pharmacol. 352, 131-134[CrossRef][Medline] [Order article via Infotrieve] |
| 25. | Kovbasnjuk, O. N., Szmulowicz, U., and Spring, K. R. (1998) J. Membr. Biol. 161, 93-104[CrossRef][Medline] [Order article via Infotrieve] |
| 26. |
Li, Y.,
Ndubuka, C.,
and Rubin, C. S.
(1996)
J. Biol. Chem.
271,
16862-16869 |
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