From the Department of Pharmacology and Toxicology, Medical College of Virginia, Virginia Commonwealth University, Richmond, Virginia 23298-0613
Received for publication, October 9, 2000
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ABSTRACT |
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Using an in vitro traumatic injury
model, we examined the effects of mechanical (stretch) injury on
intracellular Ca2+ store-mediated signaling in cultured
cortical neurons using fura-2. We previously found that elevation of
[Ca2+]i by the endoplasmic reticulum
Ca2+-ATPase inhibitor, thapsigargin, was abolished 15 min
post-injury. In the current studies, pre-injury inhibition of
phospholipase C with neomycin sulfate maintained
Ca2+-replete stores 15 min post-injury, suggesting that the
initial injury-induced store depletion may be due to increased inositol trisphosphate production. Thapsigargin-stimulated elevation of [Ca2+]i returned with time after injury and was
potentiated at 3 h. Stimulation with thapsigargin in
Ca2+-free media revealed that the size of the
Ca2+ stores was normal at 3 h post-injury. However,
Ca2+ influx triggered by depletion of intracellular
Ca2+ stores (capacitative Ca2+ influx) was
enhanced 3 h after injury. Enhancement was blocked by inhibitors
of cytosolic phospholipase A2 and cytochrome P450 epoxygenase. Since intracellular Ca2+ store-mediated
signaling plays an important role in neuronal function, the observed
changes may contribute to dysfunction produced by traumatic brain
injury. Additionally, our results suggest that capacitative
Ca2+ influx may be mediated by both conformational coupling
and a diffusible messenger synthesized by the combined action of
cytosolic PLA2 and P450.
Traumatic brain injury
(TBI)1 is a major cause of
death and disability (1). The pathophysiology of TBI is believed to
consist of two phases. A primary phase causes cellular damage at the
moment of insult by direct mechanical disruption of the brain. A
secondary, or delayed phase of damage, consists of biochemical and
cellular events that are initiated at the time of insult but do not
appear clinically for hours or days after injury. Clinically, this is often presented as patients who regain consciousness after head injury
and demonstrate the ability to speak and obey commands, only to die
hours or days later (2). At present, the biochemical and cellular
mechanisms that comprise secondary injury are not entirely understood.
A fundamental pathological mechanism underlying secondary cell injury
and death in the central nervous system after TBI is believed to be a
disruption of Ca2+ homeostasis (3). This "calcium
hypothesis" for TBI is based on the finding that calcium in the
cerebrospinal fluid decreases from 1 to 0.01 mM following
traumatic injury (4, 5). Concurrently, total tissue
45Ca2+ uptake is increased after in
vivo injury (6, 7). Therefore, substantial evidence supports the
notion that total intracellular calcium is elevated in cells of the
central nervous system after trauma. However, there are several pools
of intracellular calcium that comprise total cell calcium. These
include calcium bound to the plasma membrane, cytoskeletal elements,
and calcium-binding proteins; calcium stored in the endoplasmic
reticulum (ER), mitochondria, and nucleus; and intracellular free
calcium ([Ca2+]i) (8). The relative distribution
of calcium throughout the various cellular pools after traumatic injury
is unknown.
Maintenance of [Ca2+]i is of critical importance
to the cell because of the vital role that Ca2+ ions play
in regulation of cellular functions, especially in neurons (3). In
particular, [Ca2+]i plays a critical role in the
control of neuronal membrane excitability (8, 9), maintenance of
cytoskeletal integrity (10, 11), and control of synaptic activity and
neurotransmitter release (12-16). [Ca2+]i is
also an important signal transduction molecule in neurons and is
necessary for the activation of calcium-dependent enzymes
involved with normal cellular and physiological processes, including
several protein kinases and phospholipases (3). Alterations of these
important physiological mechanisms in neurons could have profound
effects on overall brain function.
Currently, there is a strong need to apply biochemical studies to the
pathology of TBI, particularly with respect to
[Ca2+]i dynamics. Due to technical limitations of
in vivo injury models, the mechanisms of altered
[Ca2+]i signaling after TBI are not well
documented. To investigate some of the possible mechanisms underlying
traumatic injury, our laboratory has utilized an in vitro
model that simulates a major component of TBI, that being tissue strain
or stretch (17). To produce strain, cells are grown in tissue culture
wells having a Silastic membrane bottom that is stretched by a 50-ms
pulse of pressurized gas. We have validated our model by demonstrating that in vitro stretch injury produces many of the
post-traumatic responses observed after in vivo TBI
(17-21). In addition, we have previously identified novel neuronal
alterations after in vitro injury, including a transient
stretch-induced delayed depolarization (22, 23), a stretch-induced
reduction in the Mg2+ blockade of the NMDA current (24),
and reduced 3-hydroxy-5-methyl-4-isoxazole receptor
desensitization (25).
Using our in vitro model of injury, we have identified
disruptions in store-controlled or capacitative calcium signaling in neurons (26). In the capacitative calcium influx pathway, G protein-coupled receptors linked to phospholipase C (PLC) catalyze the
hydrolysis of phosphatidylinositol bisphosphate yielding diacylglycerol and inositol 1,4,5-trisphosphate (IP3). IP3
binds to receptors on IP3-sensitive intracellular calcium
stores in the ER, resulting in the release of stored calcium (16, 27).
Depletion of calcium stores causes the production of a calcium influx
factor (CIF), which stimulates influx of extracellular calcium through
store-operated or second messenger-operated channels (SOCs/SMOCs) in
the plasma membrane (capacitative calcium influx), thereby further
increasing [Ca2+]i (8, 27-29). Although the
majority of data on capacitative calcium influx is derived from
nonexcitable cells, there is now strong evidence for the existence of
this pathway in neurons (30-32).
We have previously found that, immediately after injury, the
[Ca2+]i elevation elicited by the metabotropic
glutamate receptor (mGluR) agonist,
trans-(1S,3R)-1-amino-1,3-cyclopentanedicarboxylic acid (tACPD) was abolished, suggesting alterations to mGluRs, IP3 receptors, IP3 production, or capacitative
calcium influx (26). To dissect the various components of the tACPD
response, we utilized the pharmacological agent, thapsigargin, which
inhibits the sarcoplasmic-endoplasmic reticulum Ca2+-ATPase
(SERCA) of intracellular calcium stores (33). Thapsigargin-stimulated elevation of [Ca2+]i was also inhibited 15 min
after neuronal injury (26), suggesting that intracellular calcium
stores are either empty or can no longer sequester calcium. However, 3 and 24 h after injury, intracellular calcium stores responded to
tACPD and thapsigargin with significantly enhanced elevation of
[Ca2+]i. Therefore, we observe a primary phase in
which intracellular calcium stores elicit a depressed response to
stimuli after injury, followed by a secondary phase in which
store-mediated signaling appears to be potentiated in neurons.
The initial phase, in which IP3-mediated signaling is
diminished in injured neurons, is similar to other findings in which disruption of the cytoskeleton results in inhibition of agonist-induced [Ca2+]i signaling (34). Therefore, the initial
phase of decreased [Ca2+]i signaling after
neuronal injury may correlate with the primary phase of TBI and be due
to direct mechanical disruption of the cytoskeleton. The delayed
response in which we observe a potentiation in calcium store-mediated
signaling may correlate with the secondary phase of TBI due to altered
biochemical mechanisms. In the current report, we describe the apparent
underlying mechanisms of the observed changes in intracellular calcium
store-mediated signaling after mechanical injury. We report, for the
first time, a delayed enhancement in capacitative calcium influx in
mechanically injured cortical neurons. Despite several recent findings
that are in strong support of a physical coupling and the possible involvement of a secretory mechanism in capacitative calcium influx (35-38), the delayed enhancement in capacitative calcium influx that
we observe in injured neurons appears to also involve activation of
cytosolic phospholipase A2 (cPLA2) and a P450
monooxygenase system. These findings are consistent with the production
of a diffusible signaling molecule in neurons, which is similar to our
previous findings in cortical astrocytes (29). Therefore, alterations
in capacitative calcium influx could involve both structural disruption
in neurons as well as changes in CIF-mediated signaling. We hypothesize
that these alterations in IP3-mediated calcium signaling
pathways and intracellular calcium stores may play a role in the
neuronal dysfunction that follows TBI.
Cell Culture--
Mixed cultures of neuronal plus glial cells
were prepared from 1- to 2-day-old neonatal Harlan Sprague-Dawley rat
pups (Hilltop Lab Animals, Scottdale, PA) as described previously (18).
In brief, brains were removed aseptically and placed in sterile
dissecting saline. Cortices were dissected from the brain and cleaned
of white matter and meninges, leaving the neocortex. The neocortex was
placed in 5 ml of dissecting saline that contained 0.125% trypsin
(Sigma) for 10 min and was transferred to a tube containing 5 ml of
Dulbecco's modified Eagle's medium (DMEM, Mediatech, Herndon, VA)
supplemented with 20 mM glucose, 10% fetal bovine serum
(Summit Biotechnology, Fort Collins, CO), 100 units/ml penicillin, 100 µg/ml streptomycin, and 2 mM L-glutamine. The
tissue was washed, the supernatant removed, and 5 ml of culture medium
added for a second wash. After washing, the supernatant was removed,
and the tissue fragments were dissociated by trituration with a glass pipette in 5 ml of culture medium. The suspension was centrifuged for
10 min at 200 × g. The supernatant was removed, and 5 ml of culture medium was added. The suspension was triturated, filtered with a 70-µm nylon cell strainer, diluted with DMEM, and counted with
a hemocytometer. One-ml aliquots of the cell suspension containing 1 × 106 cells were seeded into each well of a
collagen-coated Flex Plate (Flexcell International, McKeesport, PA).
Cells were cultured in a 5% CO2 incubator at 37 °C. One
day after plating, the media were removed and replaced with 1 ml of
fresh medium. Three days later, 0.5 ml of medium was removed from each
well and replaced with 0.5 ml of neuronal growth medium. Neuronal
growth medium contained DMEM, 10 mM glucose, 100 units/ml
penicillin, 100 µg/ml streptomycin, and 5% horse serum (Life
Technologies, Inc.). Cells were used for experiments within 10-15 days
after removal from the rat. Glial cells typically adhered to the
membrane substrate, and the neurons adhered to the glial cells.
Neuronal processes and interconnections between individual neuronal
cells and neuronal cell clusters were readily apparent.
Fura-2 Loading--
[Ca2+]i was measured
with the ratiometric dye, fura-2. Mixed cultures of neuronal
plus glial cells were loaded with fura-2 AM as described previously
(24, 26). Briefly, cells were washed once and placed in 1 ml of
Dulbecco's phosphate-buffered saline (DPBS) containing 1 mM Ca2+ and Mg2+ and supplemented
with 1 mM glucose and 1% fatty acid-free bovine serum
albumin. Fura-2 AM (5 µM, Molecular Probes, Eugene, OR) was added, and the cells were incubated in the dark at 37 °C for 1 h. Under these conditions, the astrocytes in the co-cultures retain fura-2 only weakly in the cytoplasm. In our studies,
astrocytes required room temperature conditions for adequate fura-2
loading since the cells appeared to actively metabolize and
compartmentalize fura-2, and lose fluorescence when maintained at
37 °C (20, 26, 29). Thus, loading of mixed neuronal plus glial
cultures at 37 °C resulted in a more selective loading of neurons.
The concentration of intracellular fura-2, the presence of unhydrolyzed fura-2 AM derivatives, and dye compartmentalization were routinely monitored (39-41). We have found that these fura-2 loading techniques produce high concentrations of intracellular fura-2 in neurons, negligible dye compartmentalization, and minimal incomplete AM ester
hydrolysis as determined by scanning the excitation spectra of loaded
cells. After loading, mixed cultures were washed once with supplemented
DPBS and were placed in a final volume of 2 ml of DPBS for measurement
of [Ca2+]i.
Cell Injury--
Fura-2-loaded neuronal plus glial cultures were
injured as described previously, using a commercially available model
94A Cell Injury Controller (Bioengineering Facility, Virginia
Commonwealth University, Richmond, VA) (17). In brief, the Silastic
membrane of the Flex Plate well is rapidly and transiently deformed by a 50-ms pulse of compressed gas, which deforms the Silastic membrane and adherent cells to varying degrees controlled by the pulse pressure.
The extent of cell injury, as measured by uptake of propidium iodide
and release of lactate dehydrogenase, is dependent on the degree of
Silastic membrane deformation (17, 18, 26). Based on our previous
work we have arbitrarily defined three levels of cell injury,
mild, moderate, and severe, produced by deforming the silastic membrane
on which the cells are grown by 5.5, 6.5, and 7.5 mm, respectively
(17-20). These degrees of membrane deformation result in a biaxial
strain or "stretch" of 31, 38, and 54%, respectively (17). This
range of cell stretch has been shown to be relevant to what would occur
in humans after rotational acceleration/deceleration injury (42). The
current experiments were all performed at a mild level of injury (5.5 mm deformation, 31% stretch). Correlation of degree of strain produced
in models to strain-induced deficits in humans has shown that greater
than 30% stretch produces persistent behavioral deficits. It is
important to note that with the cell injury model, as currently used,
there is no hypoxia superimposed on the injured cells.
Microspectrophotometry--
[Ca2+]i was
measured at room temperature using a Ratiomaster (Photon Technologies
International, South Brunswick, NJ) microspectrophotometry system.
Excitation light was provided by a xenon arc lamp coupled to a RAM
scanner that alternated excitation light between 350 and 380 nm. Band
pass was set at ±1 nm. Excitation light was delivered to the cells via
fiber optics through the epifluorescence port of a Zeiss Standard 16 microscope coupled to a Zeiss Achroplan 40× water immersion lens. Use
of the water immersion lens allowed in situ measurements and
easy manipulation of the Flex Plates during injury. However, the
optical properties of the lens necessitated the ratioing of fura-2 at
350 and 380 nm. Emission was measured at 510 nm via a microphotometer.
The entire system for data collection and analysis was
computer-driven.
Flex plates containing neuronal plus glial cultures were placed on the
microscope stage, and single neurons were selected for
[Ca2+]i measurement using slit width adjustments
on the microphotometer. Basal [Ca2+]i was
recorded for several minutes at a sampling rate of one ratio every
0.2 s. In experiments utilizing neomycin sulfate (Sigma), an
inhibitor of PLC, cells were pretreated with neomycin for 3 h
prior to injury. Thapsigargin (Calbiochem) and tACPD (Research Biochemicals International, Natrick, MA) were added directly to culture
wells at the indicated time after injury. In the indicated experiments,
the SOC/SMOC antagonist SKF 96365 (Calbiochem) was added to cultures
for 10 min prior to application of thapsigargin. The cytochrome P450
inhibitor, econazole (Calbiochem), and the cPLA2 inhibitor,
arachidonyltrifluoromethyl ketone (AACOCF3, Calbiochem), were added to cultures for 2 min prior to thapsigargin stimulation. An
additional inhibitor of cytochrome P450,
N-methylsulfonyl-6-(2-propargyloxyphenyl) hexanimide
(MS-PPOH, synthesized by Dr. J. R. Falck, University of Texas
Southwestern Medical Center, Dallas) (29) was also used and was added
to cultures 2 min prior to thapsigargin stimulation. MS-PPOH is a
"suicide substrate" inhibitor of P450 arachidonic acid epoxygenase,
designed to resemble the substrate arachidonic acid and inactivate the
enzyme (43). All agents were added directly to culture wells in a
volume of 200 µl at the indicated time after injury. This volume
ensured almost immediate mixing with the buffer, as determined by dye diffusion.
In experiments in which [Ca2+]i was measured at
3 h post-injury, the cells were injured in DMEM without serum or
phenol red. After injury, serum was added back to the media, and cells were incubated for the 3-h interval. Uninjured controls were incubated under the same conditions. Cells were loaded with fura-2 AM 1 h
prior to the end of the incubation period. The concentration of
intracellular fura-2 was routinely monitored (39) at 3 h post-injury and did not significantly differ from controls.
In Ca2+-free experiments, supplemented DPBS was replaced
with Ca2+-free DPBS. Nominally Ca2+-free
conditions caused a small but nonsignificant drop in basal [Ca2+]i as previously reported by others (30, 44,
45). After basal [Ca2+]i had stabilized,
thapsigargin was applied to culture wells. To preserve the original
status of intracellular calcium stores, neurons were maintained in
nominally Ca2+-free conditions for a maximum of 2 min
before stimulation with thapsigargin.
[Ca2+]i was calculated as described previously
(46) using a correction for intracellular viscosity (39).
Autofluorescence at 350 and 380 nm was recorded from neurons of
neuronal plus glial cultures that were not loaded with fura-2 and was
subtracted from all measurements. Leakage of fura-2 into the medium
after injury was monitored by measuring intracellular fura-2
concentration at the isosbestic wavelength, 362 nm (46). Additional
measurements were made to assess fura-2 fluorescence directly in the
medium. Consistent with the lack of propidium iodide uptake that we
have previously reported immediately after injury in neurons (26), no
significant leakage of fura-2 was observed following injury.
Statistical Analysis--
Data are mean ± S.E. values.
Statistical significance was established by analysis of variance
followed by Fisher's protected least significant difference test.
We have previously reported that tACPD and thapsigargin-stimulated
elevation of [Ca2+]i is almost completely
abolished 15 min after injury, suggesting that intracellular calcium
stores are depleted (26). However, intracellular calcium stores elicit
a delayed enhancement in response to stimuli at 3 h post-injury.
Therefore, we observe two phases of altered calcium store-mediated
signaling after injury, an early or immediate phase in which the stores
do not respond to stimuli and a late or delayed phase in which
store-mediated signaling is potentiated. The current experiments were
designed to decipher the mechanisms of the two different phases of
calcium store-mediated signaling that are observed after traumatic
injury of cortical neurons.
Immediately after Injury IP3-sensitive Calcium Stores
Are Depleted--
One possible explanation for the diminished response
to tACPD and thapsigargin that we observe initially after injury is
increased activation of PLC, resulting in elevated IP3.
High levels of IP3 may stimulate IP3 receptors
on intracellular calcium stores, possibly maintaining them in an empty
state (47). Therefore, we used the PLC inhibitor, neomycin sulfate (48,
49), to evaluate the effect of PLC activation on store-mediated calcium
signaling in stretch-injured neurons.
Pretreatment of uninjured neurons with 100 µM neomycin
sulfate completely blocked the elevation of
[Ca2+]i in response to 100 µM
tACPD, suggesting that neomycin inhibits PLC activation in cortical
neurons (data not shown). Fig.
1A is a representative tracing
of an uninjured neuron stimulated with 1 µM thapsigargin.
Basal [Ca2+]i was 87 ± 2 nM in
uninjured neurons. Thapsigargin increased [Ca2+]i
43 ± 6 nM above basal in uninjured cells. Fig.
1B shows representative tracings of an injured neuron and an
injured neuron pretreated with neomycin and stimulated with 1 µM thapsigargin 15 min after injury. Basal
[Ca2+]i levels before injury are shown as the two
lower tracings in Fig. 1B. Basal
[Ca2+]i was 84 ± 2 nM in
neurons treated with 100 µM neomycin sulfate, suggesting
that neomycin does not significantly alter basal
[Ca2+]i. The representative tracings in Fig.
1B show that [Ca2+]i increases to 175 nM 15 min after injury. Injured neurons pretreated with
neomycin had a similar elevation in [Ca2+]i after
injury. Thapsigargin did not elicit an elevation of
[Ca2+]i in injured neurons at 15 min post-injury.
However, when neurons are pretreated with 100 µM neomycin
sulfate 3 h prior to injury, the thapsigargin-stimulated elevation
of [Ca2+]i was 50 ± 8 nM,
suggesting that blockade of PLC maintains calcium-replete intracellular
calcium stores after injury. These results suggest that activation of
PLC may cause intracellular Ca2+ stores to remain in an
empty state after stretch injury.
Thapsigargin-stimulated Elevation of [Ca2+]i
Is Restored with Time after Injury--
We have previously found that
the [Ca2+]i elevation elicited by tACPD or
thapsigargin is potentiated at 3 h post-injury (26) suggesting
that intracellular calcium stores undergo a delayed enhancement in
their response to stimuli after injury. However, the status of
intracellular calcium stores between 15 min and 3 h post-injury is
unknown. Therefore, we investigated the time course of
thapsigargin-stimulated elevation of [Ca2+]i
after injury (Fig. 2). Application of 1 µM thapsigargin increased [Ca2+]i
43 ± 6 nM above basal in uninjured neurons. Fifteen minutes after injury thapsigargin-stimulated elevation of
[Ca2+]i is almost completely abolished, being
only 8 ± 4 nM above basal. The elevation of
[Ca2+]i elicited by 1 µM
thapsigargin increased with time after injury, being 34 ± 18 nM 1 h after injury, 61 ± 20 nM
2 h after injury, and 87 ± 19 nM 3 h after
injury, at which time the response was potentiated as compared with
uninjured neurons. Therefore, after injury we observe a primary phase
in which calcium signaling mediated through intracellular stores is
depressed, followed by a secondary phase in which store-mediated
signaling appears to be potentiated.
The Size of Intracellular Calcium Stores Is Unaltered after
Injury--
There are several possible explanations for the mechanism
underlying enhanced thapsigargin-stimulated elevation of
[Ca2+]i 3 h after injury. One possibility is
that calcium is released from the stores more rapidly in injured
neurons. However, we have previously found that the release of calcium
from intracellular calcium stores is actually slower in injured neurons
when compared with uninjured cells (26). Since we have also found that
[Ca2+]i is elevated immediately after mild injury
of neurons but returns to basal levels by 3 h post-injury (26)
intracellular calcium stores may play a role in restoring basal
[Ca2+]i by sequestering additional calcium after
injury. An increased uptake of calcium after injury could result in an
enlargement of the intracellular calcium store pool and may explain the
potentiated store-mediated signaling 3 h post-injury.
To investigate the possibility that the size of intracellular calcium
stores had increased at 3 h after injury, neurons were stimulated
with 1 µM thapsigargin in Ca2+-free DPBS
(Fig. 3). Basal
[Ca2+]i in uninjured neurons placed in nominally
Ca2+-free conditions was 81 ± 8 nM.
Stimulation of uninjured neurons with 1 µM thapsigargin
in Ca2+-free DPBS resulted in an increase in
[Ca2+]i of 24 ± 6 nM,
suggesting that approximately half of the thapsigargin-stimulated
elevation of [Ca2+]i is due to release of calcium
from intracellular stores. The thapsigargin-stimulated elevation of
[Ca2+]i in Ca2+-free DBPS was 32 ± 17 nM at 3 h post-injury, similar to uninjured neurons. Thus, the size of the intracellular calcium stores is unchanged 3 h after injury. This implies that the increased
[Ca2+]i response at 3 h post-injury is
dependent on influx of extracellular calcium.
Injury Causes a Delayed Enhancement in Capacitative Calcium
Influx--
Since the size of the intracellular calcium stores was
apparently unaltered 3 h after injury, we tested whether the
enhancement in thapsigargin-stimulated elevation of
[Ca2+]i was due to an increase in capacitative
calcium influx. To do this, we utilized the SOC/SMOC antagonist, SKF
96365 (50). In Fig. 4, 1 µM
SKF 96365 was added to cultures 10 min prior to stimulation with 1 µM thapsigargin. Application of SKF 96365 did not
significantly alter basal [Ca2+]i. Stimulation
with 1 µM thapsigargin in the presence of 1 µM SKF 96365 increased [Ca2+]i
33 ± 7 nM above basal in uninjured neurons and
32 ± 7 nm in neurons at 3 h post-injury. Therefore,
treatment with 1 µM SKF 96365 at 3 h post-injury
completely blocked the potentiated [Ca2+]i
elevation elicited by thapsigargin. These results further support the
enhanced thapsigargin-stimulated elevation of
[Ca2+]i at 3 h post-injury is due to an
increase in capacitative calcium influx and not to a change in the size
of the intracellular calcium store pool. Application of 1 µM SKF 96365 had no effect on the
[Ca2+]i elevation elicited by 50 mM
KCl, indicating that SKF 96365 had no effect on voltage-gated calcium
channels at the concentration used in these experiments.
Capacitative Calcium Influx May Be Enhanced Due to Alterations in
the Production of Calcium Influx Factor--
The effects of
cPLA2 on capacitative calcium influx in neurons was
assessed with AACOCF3, an inhibitor of cPLA2.
As shown in Fig. 5, 5 µM
AACOCF3 had no effect on basal
[Ca2+]i. Application of 5 µM
AACOCF3 did not alter the thapsigargin-stimulated elevation
of [Ca2+]i in calcium-free medium, suggesting
that AACOCF3 had no effect on intracellular calcium stores
(data not shown). AACOCF3 also had no effect on the
[Ca2+]i elevation induced by 50 mM
KCl, suggesting that voltage-gated calcium signaling was unaffected
(data not shown). However, consistent with our published results in
astrocytes (29), AACOCF3 significantly decreased
thapsigargin-stimulated elevation of [Ca2+]i in
uninjured neurons (Fig. 5, light gray bars), to a level
consistent with blockade of capacitative calcium influx. In injured
neurons, AACOCF3 effectively blocked the enhancement in
thapsigargin-stimulated calcium influx observed at 3 h post-injury (Fig. 5, dark bars). These results suggest that activation
of cPLA2 may be involved in the delayed enhancement of
capacitative calcium influx in stretch-injured neurons.
To dissect the effects of cytochrome P450 on the enhancement of
capacitative calcium influx in injured neurons, we utilized two P450
inhibitors, MS-PPOH and econazole. The cytochrome P450 enzymes comprise
a large family of isoforms (51) of which econazole is a generalized
inhibitor. MS-PPOH is a specific suicide substrate inhibitor of
P450 AA epoxygenase, designed to resemble the substrate AA and
inactivate the enzyme (43). Neither 1 µM MS-PPOH nor 10 µM econazole had any effect on the
thapsigargin-stimulated elevation of [Ca2+]i in
calcium-free medium, suggesting that the intracellular calcium stores
were unaffected by these agents. Additionally, MS-PPOH and econazole
did not alter the [Ca2+]i elevation induced by 50 mM KCl, suggesting that voltage-gated calcium channels were
not affected at the concentrations utilized in these experiments (data
not shown).
As shown in Fig. 5, MS-PPOH significantly decreased the
thapsigargin-stimulated [Ca2+]i elevation in
uninjured neurons, consistent with blockade of capacitative calcium
influx and similar to our previous observations in astrocytes (29).
Econazole did not significantly attenuate thapsigargin-stimulated
elevation of [Ca2+]i in uninjured cells; however,
it did block the enhanced thapsigargin-stimulated elevation of
[Ca2+]i at 3 h post-injury. MS-PPOH
attenuated thapsigargin-stimulated elevation of
[Ca2+]i at 3 h post-injury in two of five
neurons examined but was ineffective in blocking the potentiated
response in three neurons, hence the large error bar in Fig. 5. Taken
together, the results suggest the involvement of cytochrome P450 in the delayed enhancement of capacitative calcium influx observed in injured neurons.
A fundamental pathological mechanism underlying secondary cell
injury in the central nervous system following TBI is proposed to be a
disruption of Ca2+ homeostasis (3). Although total
intracellular calcium is elevated in the brain after traumatic injury,
the relative distribution of calcium throughout the various cellular
pools is unknown. For example, much of the calcium influx after injury
may be buffered by the plasma membrane, cytoskeletal elements, and by
cytoplasmic proteins such as calmodulin, calbindin, and parvalbumin
(52). These processes may protect against dramatic changes in
[Ca2+]i levels. Concurrently, additional uptake
of calcium by the mitochondria could be detrimental, based on reports
that calcium overload may disrupt energy metabolism (53-55). Also,
greater uptake of calcium by the nucleus could adversely affect genetic mechanisms by activating calcium-dependent endonucleases
(56, 57). In addition, [Ca2+]i plays a critical
role in regulating several important neuronal functions (8-16).
Therefore, alterations in [Ca2+]i homeostasis
could trigger neuronal degeneration and cell death which could
ultimately result in clinical disability. We have previously described
alterations in IP3-mediated signaling and intracellular
calcium stores in cortical neurons after stretch-induced traumatic
injury (26). The current report seeks to elucidate some of the
underlying biochemical mechanisms of the injury-induced changes in
[Ca2+]i-mediated signaling in neurons.
We previously found that NMDA-stimulated elevation of
[Ca2+]i was potentiated 15 min after injury but
was normal by 3 h post-injury in neurons of neuronal plus glial
cultures (26). Glutamate-stimulated elevation of
[Ca2+]i was also enhanced 15 min after injury and
remained enhanced at 3 h post-injury. The discrepancy in the
responses to NMDA and glutamate 3 h after injury suggested a
possible alteration in mGluRs coupled to intracellular calcium stores.
We further found that elevation of [Ca2+]i
elicited by the mGluR agonist, tACPD, or by thapsigargin was nearly
abolished 15 min after injury (26). This observation may be due to the
elevated [Ca2+]i at 15 min after injury, which
could desensitize the IP3 receptor (58). However,
thapsigargin acts independently of the IP3 receptor,
suggesting that the stores are either empty or can no longer sequester calcium.
Using the PLC inhibitor, neomycin sulfate (48, 49), we were able to
investigate the effect of PLC blockade on the status of intracellular
calcium stores after injury (see Fig. 6).
Neomycin pretreatment restored thapsigargin-stimulated elevation of
[Ca2+]i in injured neurons. In
neomycin-pretreated cells, the [Ca2+]i elevation
elicited by thapsigargin was restored despite a significant
injury-induced elevation of [Ca2+]i at 15 min post-injury. These results show that pre-injury blockade of PLC
maintains calcium-replete intracellular stores after injury and suggest
that G protein-coupled receptors linked to PLC (i.e. Group I
mGluRs) may be aberrantly activated after mechanical injury. Activation
of PLC and subsequent generation of IP3 appear to be
responsible for the decreased calcium store-mediated signaling 15 min
after injury. These findings are consistent with reports following
in vivo TBI in which IP3 levels are elevated (59, 60) and with our previous findings in cortical astrocytes demonstrating that inositol polyphosphates are elevated 10-fold after
stretch injury (61).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Blockade of phospholipase C inhibits
depletion of intracellular calcium stores after injury in neurons of
neuronal plus glial cultures. A, the tracing shown is
representative of the average response of 22 uninjured neurons
administered 1 µM thapsigargin. Basal
[Ca2+]i in uninjured neurons was 87 ± 2 nM. The addition of 1 µM thapsigargin
elicited an increase in [Ca2+]i of 43 ± 6 nM above basal. B, the tracings shown are
representative of four separate experiments. Fifteen minutes after
injury [Ca2+]i remains elevated, and thapsigargin
does not elicit an increase in [Ca2+]i. However,
if neurons are pretreated with 100 µM neomycin sulfate,
an inhibitor of PLC, application of 1 µM thapsigargin
increases [Ca2+]i 50 ± 8 nM.
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Fig. 2.
Thapsigargin-stimulated elevation of
[Ca2+]i changes with time after injury.
Neurons were stimulated with 1 µM thapsigargin at 15 min
and 1-3 h post-injury. The maximal change in
[Ca2+]i produced by thapsigargin stimulation was
determined. Basal [Ca2+]i values prior to
stimulation are listed on the x axis. Controls consisted of
uninjured cells stimulated with 1 µM thapsigargin. Data
represent mean ± S.E. for 5-12 separate experiments. *,
p < 0.05 versus uninjured.
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Fig. 3.
The size of intracellular calcium stores is
unaltered 3 h after injury in neurons. A, to
examine the size of intracellular calcium stores, neurons were
stimulated with 1 µM thapsigargin in
Ca2+-free DPBS. Tracings are representative of neurons
stimulated with 1 µM thapsigargin under normal (1 mM Ca2+) and Ca2+-free conditions.
The arrow represents the application of 1 µM
thapsigargin in uninjured neurons and in neurons 3 h post-injury.
For experiments at 3 h post-injury, Ca2+-containing
DPBS was replaced with Ca2+-free DPBS just prior to
stimulation with 1 µM thapsigargin. B
represents mean ± S.E. for the representative tracings shown in
A. The maximal change in [Ca2+]i
produced by thapsigargin stimulation was determined. Basal
[Ca2+]i values prior to stimulation with
thapsigargin are listed on the x axis. Data represent
mean ± S.E. for five to seven separate experiments. a,
p < 0.05 versus uninjured; b,
p < 0.05 versus injured in 1 mM
Ca2+ DPBS.
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Fig. 4.
Capacitative calcium influx is enhanced
3 h after injury in neurons. A, tracings are
representative of neurons stimulated with 1 µM
thapsigargin with or without SKF 96365 pretreatment. The
arrow represents the application of 1 µM
thapsigargin. Where indicated,1 µM SKF 96365 was added 10 min prior to initiation of recording. B, mean ± S.E.
of all responses described in A. Basal
[Ca2+]i values prior to stimulation with
thapsigargin are listed on the x axis. Data represent
mean ± S.E. of five separate experiments. a,
p < 0.05 versus uninjured; b,
p < 0.05 versus injured without SKF
96365.
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Fig. 5.
Enhanced capacitative calcium influx at
3 h post-injury involves cytosolic phospholipase A2
and P450 monooxygenase. The P450 monooxygenase inhibitors,
econazole and MS-PPOH, and the cytosolic phospholipase A2
inhibitor, AACOCF3, were added to cultures 2 min prior to
application of 1 µM thapsigargin. The maximal change in
[Ca2+]i produced by thapsigargin was determined.
Basal [Ca2+]i prior to stimulation with
thapsigargin is listed on the x axis. Econazole, MS-PPOH,
and AACOCF3 did not significantly alter basal
[Ca2+]i. Data are mean ± S.E. of five
separate experiments. a, p < 0.05 versus 1 µM thapsigargin uninjured;
b, p < 0.05 versus 1 µM thapsigargin 3 h post-injury.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 6.
Status of intracellular calcium stores and
capacitative calcium influx in neurons after injury. To
investigate the status of intracellular calcium stores after injury, we
utilized the SERCA inhibitor, thapsigargin. Fifteen min after injury
thapsigargin-stimulated elevation of [Ca2+]i is
abolished suggesting intracellular calcium stores are depleted.
However, pretreatment of neurons with the PLC inhibitor, neomycin
sulfate, maintained calcium-replete stores after injury, suggesting
elevated levels of IP3 may be responsible for empty calcium
stores. Three hours after injury, thapsigargin-stimulated elevation of
[Ca2+]i is potentiated. This enhanced response is
not evident in Ca2+-free media and is blocked by the
SOC/SMOC antagonist, SKF 96365, suggesting capacitative calcium influx
is enhanced. Enhanced thapsigargin-stimulated elevation of
[Ca2+]i is also blocked by econazole and MS-PPOH,
inhibitors of P450 metabolism of AA, and by the cPLA2
inhibitor, AACOCF3, suggesting alterations in the
production of CIF or changes in the sensitivity of SOCs/SMOCs. A
disrupted physical coupling between IP3 receptors and
SOCs/SMOCs may also contribute to the observed effects. In addition,
P450 and cPLA2 may be part of a physical connection.
PM, plasma membrane; IP3R,
IP3 receptor.
Calcium stores appear to refill with time after injury as evidenced by the ability of thapsigargin to elicit an increase in [Ca2+]i at 1 and 2 h after injury. Three hours after injury, we observed an enhancement of thapsigargin-stimulated elevation of [Ca2+]i, consistent with our previous findings (26). We have previously found that the enhancement in thapsigargin-stimulated elevation of [Ca2+]i is not due to a more rapid release of Ca2+ from intracellular stores (26). An alternative mechanism for the enhanced response to thapsigargin is that calcium stores sequester additional calcium following injury. Further uptake of calcium could result in an increase in the size of the intracellular calcium store pool and may explain the enhanced thapsigargin-stimulated elevation of [Ca2+]i observed 3 h post-injury. However, neurons that were stimulated with thapsigargin under Ca2+-free conditions 3 h post-injury elicited an elevation of [Ca2+]i that was similar to uninjured controls. These findings suggest that the size of calcium stores does not increase after injury and implies that extracellular Ca2+ is necessary for the enhanced thapsigargin-stimulated elevation of [Ca2+]i observed 3 h post-injury.
Since an increase in the size of intracellular calcium stores did not seem to be the cause of the delayed enhancement in calcium store-mediated signaling, this suggests that capacitative calcium influx may be enhanced 3 h after injury. We investigated the possibility that capacitative calcium influx was enhanced by stimulating neurons with thapsigargin in the presence of the SOC/SMOC antagonist, SKF 96365, at 3 h post-injury. Application of SKF 96365 blocked the enhanced thapsigargin-stimulated elevation of [Ca2+]i at 3 h post-injury. Taken together with the results observed under Ca2+-free conditions, our findings strongly suggest that an increase in capacitative calcium influx is responsible for the potentiated thapsigargin-stimulated elevation of [Ca2+]i observed 3 h after injury. These results imply that the biochemical mechanisms that link the status of calcium stores with influx of calcium through the plasma membrane can be disrupted by mechanical trauma.
The exact mechanism for the activation of capacitative calcium influx is still a topic of considerable debate. The "conformational coupling" hypothesis as first proposed by Irvine (62) suggests a direct physical connection between the IP3 receptor and plasma membrane channels that control capacitative calcium influx. When the IP3 receptor is activated, it is proposed that an alteration in the physical coupling causes influx of calcium into the cell via plasma membrane channels. The "diffusible messenger" hypothesis suggests that when IP3-sensitive stores are activated and depleted of calcium, a diffusible messenger (CIF) is released from the stores that binds to SOCs/SMOCs on the plasma membrane and activates capacitative calcium influx (see Fig. 6). There have been several recent findings that are in strong support of a physical coupling and/or a secretory mechanism in capacitative calcium influx (35-38). However, our previous work also suggests that CIF, possibly an epoxide metabolite of AA, may be a component of conformational coupling and/or secretory models.
We have previously reported that in astrocytes, cPLA2 and cytochrome P450 are coupled to activation of capacitative calcium influx through the epoxide metabolite of AA, 5,6-EET (29). Production of CIF has not been evaluated in cortical neurons. To investigate the possibility that the enhancement in thapsigargin-stimulated elevation of [Ca2+]i at 3 h post-injury is due to an alteration in CIF production, we used inhibitors of two critical steps in 5,6-EET synthesis, release of AA from phospholipid stores by cPLA2 and cytochrome P450 metabolism of AA. The enhancement in thapsigargin-stimulated elevation of [Ca2+]i at 3 h post-injury was blocked by AACOCF3 and econazole, suggesting that both cPLA2 activation and cytochrome P450 are involved in this enhanced response. The fact that MS-PPOH was not able to more effectively block the enhanced elevation of [Ca2+]i elicited by thapsigargin 3 h after injury in all neurons is difficult to interpret, especially since this compound is reported to be a potent and specific inhibitor of the AA epoxygenase that produces 5,6-EET (29, 43). However, there are several isoforms of cytochrome P450 enzymes that differ among cell types (51); therefore, it is possible that MS-PPOH is not a specific inhibitor of CIF production in all cortical neurons. MS-PPOH did significantly decrease thapsigargin-stimulated elevation of [Ca2+]i in uninjured neurons to a level that was consistent with thapsigargin stimulation under Ca2+-free conditions, suggesting that it does inhibit capacitative calcium influx in uninjured neurons. It is possible that injury induces expression of additional P450 enzymes that are differentially selective to pharmacological inhibition. Nonetheless, the overall results suggest that cPLA2 and cytochrome P450 may be components of CIF in cortical neurons and that these components are involved in the enhanced thapsigargin-stimulated elevation of [Ca2+]i observed 3 h after injury (Fig. 6).
The injury we are administering to neurons in the current study is mechanical stretch; therefore it is likely that there is disruption of cytoskeletal elements after injury. Previous examination of light microscopy and transmission electron micrographs of neurons in neuronal plus glial cultures has revealed only minor ultrastructural changes in neurons after mild stretch injury.2 However, by using rhodamine-phalloidin staining for actin, we have recently observed substantial disorganization of actin filaments in injured astrocytes and neurons.2 It is possible that even minor disruptions of cytoskeletal elements may contribute to some of the effects we report. For example, Pedrosa Ribeiro et al. (34) have shown that cytoskeletal disruption inhibits agonist-elicited mobilization of calcium from IP3-sensitive stores but does not interfere with thapsigargin-stimulated calcium influx. Although our data suggest that elevated levels of IP3 are most likely responsible for the attenuated calcium-mediated signaling in the immediate phase after injury, a disrupted cytoskeleton caused by the mechanical insult may also contribute to this observation. Also, it is possible that capacitative calcium influx involves both CIF and a physical interaction between the IP3-sensitive stores and plasma membrane channels. A strong physical connection with membrane channels as well as CIF may be necessary to activate capacitative calcium influx. For example, cPLA2 may alter the membrane in the vicinity of SOCs/SMOCs. Likewise, the presence of 5,6-EET, with its epoxide group, may also alter membrane characteristics and impact the tertiary conformation of membrane proteins. Therefore, the enhancement in store-mediated signaling 3 h post-injury may be due to alterations in CIF production, the sensitivity of SOCs/SMOCs to CIF, a damaged physical connection of the stores with the plasma membrane, or to a combination of these factors. Since structural alterations are likely to occur after mechanical injury, a more thorough examination of cytoskeletal abnormalities associated with stretch injury in neurons may shed further light on this issue.
A disruption in [Ca2+]i homeostasis mediated by intracellular calcium stores could trigger cellular degeneration and cell death, resulting ultimately in behavioral dysfunction. For example, a depletion of ER calcium stores is reported to suppress protein synthesis (63, 64), which may contribute to neuronal injury and death. Although we have not directly evaluated cell death in the current studies, we have found that uptake of propidium iodide is minimal immediately after stretch injury of neurons (26) but is increased with time post-injury, suggesting delayed neuronal injury. Previous reports have shown that blocking the release of calcium from intracellular stores with dantrolene protects against NMDA and glutamate-induced neurotoxicity even when [Ca2+]i levels are elevated (65, 66), suggesting a role for calcium stores in neuronal death. It is also possible that the potentiation of intracellular store signaling observed at 3 h post-injury may be an eventual signal for delayed neuronal death. Although the present study demonstrates enhancement of thapsigargin-stimulated capacitative calcium signaling at 3 h post-injury, we have previously demonstrated that glutamate and tACPD signaling are also enhanced, suggesting that IP3-mediated signaling as a whole is potentiated. Initial depletion of intracellular calcium stores may trigger an apoptotic pathway, and enhanced IP3-mediated signaling 3 h post-injury could lead to the eventual increase in the synthesis of apoptotic enzymes such as proteases and endonucleases. Recent evidence from non-neuronal cells suggests the involvement of the IP3 pathway in delayed cell death in that cells undergoing apoptosis have an increased amount of IP3 receptors (67) and cells that are deficient in IP3 receptors are resistant to apoptosis (68).
Our findings suggest that IP3-mediated signaling, intracellular calcium stores, and SOC/SMOC may be potential targets for therapeutic intervention in TBI. A recent clinical study has indicated that treatment of patients suffering from ischemic stroke within 48 h with conventional heparin, an anticoagulant that cannot cross cell membranes, had no beneficial effects on outcome at 6 months (69). However, treatment within 48 h with low molecular weight heparin, which can cross the cell membrane and inhibit the release of stored calcium, did improve outcome at 6 months. These findings were attributed to the intracellular effects of low molecular weight heparin, most likely antagonism of IP3 receptors. IP3 receptor antagonists may offer a considerable advantage over drugs such as mGluR antagonists for the treatment of TBI because several neurotransmitters signal through IP3-linked pathways, including glutamate and acetylcholine. Rather than using a mixture of pharmaceutical agents to block several types of mGluRs and acetylcholine receptors, IP3 receptor antagonists would elicit their action downstream of receptor activation, regardless of the type of receptors being activated. Therefore, IP3 receptor antagonists may be more efficient than a mixture designed to block IP3-linked signaling at the receptor level. In addition, carboxyamidotriazole, an agent which blocks calcium influx primarily by action at SOCs/SMOCs, has shown beneficial effects in clinical trials for the treatment of refractory cancers (70). Therefore, agents that can block the release of calcium from intracellular stores or capacitative calcium influx have shown promise in certain disease conditions.
To our knowledge, this is the first report on the mechanisms of
traumatically induced changes in calcium store-mediated signaling in
neurons. Although in vitro results do not always correlate with the in vivo situation, we have shown that many of the
post-traumatic occurrences of in vivo TBI also occur in our
model of in vitro stretch injury, suggesting that similar
pathologies do occur in vivo and in vitro. Our
results suggest that blockade of SOCs/SMOCs with agents such as SKF
96365 may have potential benefits in restoring normal
Ca2+-mediated signaling processes in neurons after a
traumatic insult. Also, inhibition of cytochrome P450 with drugs
similar to the antifungal agent, econazole, may aid in maintaining
Ca2+ homeostasis following trauma. Although further
experimentation in in vitro and in vivo models of
trauma is necessary, capacitative calcium influx and IP3
receptors may serve as useful targets for pharmaceutical intervention
in the future treatment of TBI. Finally, the present work suggests that
both the conformational coupling and the diffusible messenger models
for capacitative calcium influx may be intertwined in the mechanism
underlying this important signal transduction pathway.
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ACKNOWLEDGEMENTS |
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We are extremely grateful to Dr. J. R. Falck from the University of Texas Southwestern Medical Center in Dallas for supplying us with MS-PPOH and to Heather A. Sitterding and Karen A. Willoughby for culturing of the cells. We also thank Sallie Holt and Jennifer E. Slemmer for assistance in preparing the manuscript.
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FOOTNOTES |
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* This work was supported by Grants NS-27214 and HL57869 from the National Institutes of Health and a Center of Excellence Award from the Commonwealth of Virginia.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: Dept. of Pharmacology
and Toxicology, Medical College of Virginia, Virginia Commonwealth University, Box 980613, Richmond, VA 23298-0613. Tel.: 804-828-8465; Fax: 804-828-5502; E-mail: brzigali@hsc.vcu.edu.
Published, JBC Papers in Press, October 24, 2000, DOI 10.1074/jbc.M009209200
2 B. A. Rzigalinski, and E. F. Ellis, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are: TBI, traumatic brain injury; AA, arachidonic acid; [Ca2+]i, intracellular free calcium; CIF, calcium influx factor; cPLA2, cytosolic phospholipase A2; DMEM, Dulbecco's modified Eagle's medium; DPBS, Dulbecco's phosphate-buffered saline; ER, endoplasmic reticulum; 5, 6-EET, 5,6-epoxyeicosatrienoic acid; IP3, inositol 1,4,5-trisphosphate; mGluR, metabotropic glutamate receptor; PLC, phospholipase C; SERCA, sarcoplasmic-endoplasmic reticulum Ca2+-ATPase; SMOCs, second messenger-operated channels; SOCs, store-operated channels; tACPD, trans-(1S,3R)-1-amino-1,3-cyclopentanedicarboxylic acid; NMDA, N-methyl-D-aspartate; AACOCF3, arachidonyltrifluoromethyl ketone; MS-PPOH, N-methylsulfonyl-6-(2-propargyloxyphenyl)hexanimide.
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