(Stroke. 1995;26:1893-1900.)
© 1995 American Heart Association, Inc.
Articles |
Inositol Trisphosphate, Polyphosphoinositide Turnover, and High-Energy Metabolites in Focal Cerebral Ischemia and Reperfusion
From the Biochemistry Department, University of Missouri, Columbia (G.Y.S., J.-P.Z., T.A.L.), and the Neurology Department, Washington University School of Medicine, St Louis (T.-N.L., C.C.H., C.Y.H.), Mo.
Correspondence to Dr Grace Y. Sun, M121 Medical Sciences Bldg, Biochemistry Department, University of Missouri, Columbia, MO 65212.
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Background and Purpose Although the signaling pathway involving polyphosphoinositide (poly-PI) hydrolysis and release of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] is an important mechanism for regulation of neuronal calcium homeostasis, the effect of cerebral ischemia-reperfusion on this calcium signaling pathway is not well understood. Because activity of this pathway is dependent on availability of ATP, this study is aimed at examining the poly-PI signaling pathway and high-energy metabolites in a rat stroke model.
Methods Focal cerebral ischemia in rats was induced by temporary occlusion of the right middle cerebral artery and both common carotid arteries. Levels of Ins(1,4,5)P3 were determined by use of the radioreceptor binding assay. Poly-PI turnover in rat cortex was assessed with an in vivo protocol involving intracerebral injection of [3H]inositol and systemic administration of lithium. High-energy metabolites (ATP, ADP, and AMP) were analyzed by high-performance liquid chromatography.
Results Ischemia induced an increase in poly-PI turnover in the right middle cerebral artery cortex, but reperfusion led to a decline in this signaling activity. However, Ins(1,4,5)P3 levels decreased during ischemia, and these levels were not restored if ischemic insults were longer than 30 minutes. ATP levels decreased to 26% of control during ischemia and recovered to 80% of control during the initial 4 hours of reperfusion; these changes were followed by a second phase of decline.
Conclusions Results show an important relationship between ischemia-induced depletion of high-energy metabolites and poly-PI signaling activity. However, the uncoupling between Ins(1,4,5)P3 and ATP during reperfusion after severe ischemia suggests that metabolism of Ins(1,4,5)P3 is more stringently regulated than ATP.
Key Words: adenosine triphosphate • cerebral ischemia, focal • gene expression • inositol 1,4,5-trisphosphate • reperfusion
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Introduction |
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Cerebral ischemia is known to cause neuronal cell death and infarction. Excess influx of calcium due to stimulation of excitatory amino acid receptors has been regarded as an important mechanism underlying this type of injury.1 In the central nervous system, intracellular calcium homeostasis is controlled largely by the signal transduction pathway involving hydrolysis of PIP2 by phospholipase C (Fig 1
).2 3 Many
neurotransmitters, including acetylcholine, dopamine,
serotonin, and glutamate, transmit signals through
activation of receptors that are coupled to hydrolysis of
PIP2 and the release of Ins(1,4,5)P3, a
second messenger for mobilization of intracellular calcium stores.
Ins(1,4,5)P3 exerts its second messenger action by binding
to a specific intracellular receptor that is a calcium
channel.4 Upon release by phospholipase C,
Ins(1,4,5)P3 is enzymatically removed by the ATP-dependent
3-kinase and 5-phosphatase to inositol bisphosphates and IPs. In
turn, IPs are further hydrolyzed to inositol, which is reutilized for
synthesis of PI (Fig 1). PI is the substrate for the sequential
reaction catalyzing the biosynthesis of PIP and PIP2 by the
ATP-dependent enzymes PI kinase and PIP kinase, respectively (Fig 1).
Thus, several moles of ATP are required for sustaining the
receptor-mediated poly-PI signaling activity. Because of the active
metabolism of Ins(1,4,5)P3, this second
messenger is transiently increased within seconds upon stimulation by
agonists. However, studies with cultured cells and tissue slices have
successfully assayed poly-PI signaling activity by use of an incubation
system containing lithium, an uncompetitive inhibitor of
inositol monophosphatase.5 Systematic injection of lithium
in rats also resulted in a sustained increase in IP levels in
brain.5 Therefore, with rat brain labeled with
[3H]inositol, the extent of poly-PI turnover in brain can
be determined by measuring the amount of labeled IP accumulated after
lithium treatment.6 This in vivo protocol has been
successfully used to assess poly-PI turnover in brain after cholinergic
stimulation7 and acute ethanol
administration.8 In the mouse brain, it is possible to
observe a rapid and transient increase of Ins(1,4,5)P3
after global cerebral ischemia induced by
decapitation,9 suggesting that stimulation of the poly-PI
pathway is part of the early events of ischemia.
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Depletion of high-energy metabolites such as phosphocreatine and ATP is known to occur during global10 11 12 13 14 and focal15 16 17 cerebral ischemia. In transient global cerebral ischemia induced in gerbils by ligation of bilateral common carotid arteries, these high-energy metabolites can be fully recovered after recirculation.12 13 14 However, in focal cerebral ischemia induced by MCA ligation, recovery of high-energy metabolites seems to be dependent on the region of the cortex and the duration of the insult.15 Despite recovery of high-energy metabolites during the early period of reperfusion, prolonged ischemia may result in development of infarction that occurs hours after reperfusion.18 19 Availability of ATP after reperfusion is a prerequisite for the induction of immediate early genes such as c-fos and junB.20 Although it is not well understood, the cell signaling pathway involving the poly-PI/Ins(1,4,5)P3 cascade is also implicated in the induction of these genes that subsequently upregulate or downregulate the expression of the late effector genes.20 21 22 Previous studies have demonstrated a rapid decrease in poly-PI levels in brain during the onset of global23 24 25 26 and focal ischemia.27 Despite the requirement for ATP for maintenance of the poly-PI signaling activity in brain, a temporal relationship between these two events during and after severe focal ischemic insult has not been examined in detail. In this study, a rat stroke model18 19 entailing focal cerebral ischemia in the MCA cortex was used to study ischemia-induced changes in Ins(1,4,5)P3 levels and poly-PI turnover in relation to high-energy metabolism.
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Induction of Focal Cerebral Ischemia in Rats
Focal cerebral ischemia was induced in rats by temporary occlusion of the right MCA together with the common carotid arteries according to the procedure described by Chen et al18 with minor modifications.28 In brief, adult male Long Evans rats (Harlan Sprague Dawley, Inc) weighing 250 to 300 g were allowed free access to water and lab chow. Animals were anesthetized with ketamine (100 mg/kg IP) and xylazine hydrochloride (6 mg/kg IM) (Sigma Chemical Co). Rectal temperature was monitored and maintained at 37.0±0.5°C by means of an electronic temperature controller (Versa-Therm 2156, Cole-Parmer Instrument Co). After the MCA trunk above the rhinal fissure was identified under a stereomicroscope (Zeiss Stemi SV6), the blood vessel was ligated with a 10-0 suture. Both common carotid arteries were then occluded with nontraumatic aneurysm clips. After the predetermined time (5 to 60 minutes), the aneurysm clips and the suture were removed and restoration of blood flow in the MCA was verified under the stereomicroscope. With this animal model, occlusion of the right MCA and both common carotid arteries reduced regional blood flow by 88% to 92%, whereas occlusion of the right common carotid artery alone resulted in a reduction of only 20%.18 Previous studies with this model indicated that infarction in the MCA cortex occurred after ischemia for 45 minutes or longer, whereas the left MCA cortex sustained only mild ischemia and no morphological evidence of ischemic injury up to 24 hours after the insult.19 28 29 After the surgical procedure, animals recovered from anesthesia and were kept under close observation with free access to food and water. Animal care and surgical procedures have been approved by the University of Missouri–Columbia Animal Care Committee (protocol number 1741).
At various times after ischemia and reperfusion, rats were killed by decapitation. The heads were immediately immersed in a liquid nitrogen bath. The skin in the frozen rat head was removed to expose the skull over the MCA cortices. The left and right MCA cortices were removed by use of a precooled metal plunger,27 and brain samples were stored at -70°C until analysis.
For analyses of Ins(1,4,5)P3 and high-energy metabolites,9 the frozen brain tissue (130 to 200 mg) was homogenized in 10 volumes (wt/vol) of ice-cold 1 mol/L TCA. The TCA homogenate was centrifuged at 4000 rpm for 15 minutes to sediment the denatured proteins. The supernatant was transferred to another tube and we removed the TCA in the supernatant by washing with two volumes of water-saturated diethyl ether three times. We then removed the residual ether in the supernatant by blowing the samples with nitrogen for 15 minutes. The supernatant was further centrifuged at 14 000 rpm for 7 minutes for further removal of proteins prior to its storage at -20°C until use. This tissue extract was either neutralized for Ins(1,4,5)P3 radioreceptor assay or used directly for analysis of ATP, ADP, and AMP by high-performance liquid chromatography (see below).
Determination of Ins(1,4,5)P3 by Radioreceptor
Binding Assay
An aliquot (450 µL) of the TCA extract was neutralized with 50
µL of 0.5 mol/L Tris-HCl (pH 8.4). The neutralized tissue extract was
diluted (10 to 20 times) with Tris buffer (50 mmol/L Tris-HCl, pH 8.4,
1 mmol/L EDTA, and 1 mmol/L 2-mercaptoethanol), and the diluted sample
was used for a radioreceptor assay similar to that described by Bredt
et al30 with minor modifications.9 Cerebellar
membranes isolated from adult Sprague-Dawley rats (Harlan
Sprague Dawley, Inc) were used as the source of
Ins(1,4,5)P3 receptor. The binding assay mixture contained
50 µL of the diluted sample, 30 µL of cerebellar membranes (90 µg
protein), and 195 µL of a binding buffer containing 50 mmol/L
Tris-HCl (pH 8.4), 1 nmol/L [3H]Ins(1,4,5)P3
(specific radioactivity, 17 Ci/mmol; Du Pont–New England Nuclear), 1
mmol/L EDTA, and 1 mmol/L 2-mercaptoethanol. The assay mixtures were
incubated at 4°C for 10 minutes and the binding reaction was
terminated by sedimentation of the membranes at 12 000g for
5 minutes followed by aspiration of the supernatant. The pellets were
suspended in 5 mL of scintillation fluid (Universol, Beckman Inc), and
radioactivity was determined by a Beckman LS5800 scintillation
spectrometer (Beckman Instrument Inc.). In each assay, we constructed a
standard curve for quantitation of Ins(1,4,5)P3 levels by
replacing the tissue extract with unlabeled Ins(1,4,5)P3
(final concentration, 1 to 100 nmol/L). The nonspecific binding, which
was determined by displacement of tissue extract with 2 µmol/L
Ins(1,4,5)P3, was subtracted for determination of
the data for specific binding.
Assessment of Poly-PI Turnover in MCA Cortex
For in vivo assessment of poly-PI turnover in brain, the
protocol described by Sun et al6 was followed with minor
modifications. In brief, rats were anesthetized with
isoflurane, and an incision was made between each rat's ears to expose
the midsagittal line. With a dental drill, a burred hole was made 1 mm
to the right of the midsagittal line and 3 mm posterior to the bregma.
Intracerebral injection of [3H]inositol
(20 µCi in 20 µL; specific radioactivity, 20
Ci/mmol · L-1 in sterile water; American Radiolabeled
Chemicals) was carried out with a 50-µL syringe adapted with a
polyethylene sheath to allow a penetration depth of 3 mm. After
injection, each rat's incision was sutured and the animals were
returned to the cage until ischemia induction.
In a preliminary study, rats were injected
intracerebrally with [3H]inositol for 20
hours and with lithium intraperitoneally for 4
hours. Analysis of labeled inositol metabolites (inositol
lipids and inositol phosphates) indicated no difference in distribution
between the left and right MCA cortices (data not shown). For the study
of poly-PI turnover in MCA cortex with respect to transient focal
cerebral ischemia and reperfusion, rats were injected
intracerebrally with [3H]inositol for 20
hours and subsequently injected intraperitoneally
with LiCl (8 meq/kg body weight) for 4 hours before they were killed.
The first experiment was designed to examine the amount of labeled IP
accumulation between the right and left MCA cortices after a 60-minute
ischemic insult. In this experimental protocol (protocol 1 in
Fig 2), rats were first injected
intracerebrally with [3H]inositol, and
after equilibration for 20 hours they were injected
intraperitoneally with LiCl. Three hours after LiCl
administration, rats were subjected to the ischemic insult for
60 minutes and killed after ischemia. In the second
experimental protocol, we examined poly-PI turnover in rats that
experienced a 60-minute ischemic insult followed by reperfusion
for 20 hours. In this experiment (protocol 2 in Fig 2), animals were
first subjected to a 60-minute ischemic insult and reperfused;
this procedure was followed immediately by
intracerebral injection with
[3H]inositol. At 20 hours after reperfusion, animals were
injected with LiCl and killed 4 hours later. The third experiment was
aimed at examining poly-PI turnover at different times after a
60-minute ischemic insult. This experiment was similar to
protocol 2 except that labeled inositol was injected
intracerebrally during reperfusion after 60 minutes of
ischemia and LiCl was injected
intraperitoneally 4 hours before decapitation at 8,
16, and 24 hours after reperfusion. In all experiments, frozen tissue
from left and right MCA cortices was dissected as described above and
brain samples were stored at -80°C until analysis.
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For analysis of labeled inositol metabolites, brain tissue was homogenized in 10 volumes of chloroform/methanol (2:1, vol/vol) and subsequently partitioned with 1/4 volumes of 1% glacial acetic acid to allow separation of the aqueous phase from the organic phase. Aliquots of the organic phase were removed for determination of radioactivity representing labeled phosphoinositides (PI, PIP, and PIP2). The aqueous phase was recovered and separated by a Dowex ion exchange column (AG1x8, formate form; Bio-Rad Laboratories) according to the procedure described by Sun et al.6 After sample application, we separated IPs by eluting [3H]inositol with 10 mL of 5 mmol/L myo-inositol followed by 20 mL of water, glycerophosphoinositol with 10 mL of 5 mmol/L disodium tetraborate in 60 mmol/L sodium formate, and IPs with 10 mL of 0.2 mol/L ammonium formate in 0.1 mol/L formic acid. Aliquots of the eluant were mixed with scintillation fluid (Cytoscint, ICN) and radioactivity was determined in a Beckman LS 5800 liquid scintillation counter (Beckman Instrument, Inc).
Analysis of ATP, ADP, and AMP by High-Performance
Liquid Chromatography
The procedure for analysis of ATP, ADP, and AMP in the
TCA extract was similar to that described by Lin et al.9
This analysis was carried out by high-performance
liquid chromatography by use of a Dionex BioLC system
with an OmniPac PAX-100 column (Dionex). An aliquot of 20 µL was
injected into the sample injection loop and isocratic elution was
carried out with H2O/200 mmol/L NaOH/50% isopropyl alcohol
(42:56:2 by volume). Detection of the eluant was carried out by
monitoring of the output at 260 nm with a Perkin-Elmer LC-95
spectrophotometric detector. For each series of analysis,
standard curves for ATP, ADP, and AMP were individually constructed
before calculation of the data.
Statistical Analysis of Data
Analysis of nucleotide levels in MCA cortex
by ANOVA was done with a two-factorial experimental design in which
comparisons were made among groups and between the left and right
sides. After the two-way ANOVA, we examined whether there were
interactions among groups, and multiple comparisons were used to assess
group differences (Biostatistics Core, University of Missouri).
Student's t test was used for comparison between left and
right MCA cortices. Statistical significance was set at
P<.05.
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Results |
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Effects of Transient Focal Cerebral Ischemia and Reperfusion on Ins(1,4,5)P3 Levels in Rat MCA Cortex
Measurement of the Ins(1,4,5)P3 in rat MCA cortex indicated a rapid decrease in levels of this second messenger in the right MCA cortex during the ischemia period, whereas the levels in the left MCA cortex were not appreciably altered (Fig 3
). There were no differences in
Ins(1,4,5)P3 levels between the left and right MCA cortices
of the sham-operated group and the left MCA cortices of the
ischemic and sham-operated groups. The
ischemia-induced decrease in Ins(1,4,5)P3
levels was rapid, and levels were maintained at 25% of those in the
left MCA cortex during the entire period of ischemic
insult.
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indicates data from the right MCA cortices; 
, data from the left
MCA cortices.
To examine whether the ischemia-induced decrease in
Ins(1,4,5)P3 levels could be recovered after reperfusion,
we subjected rats to transient ischemic insult for 15, 30, and
60 minutes followed by reperfusion for 4 or 24 hours. Results in Fig 4 indicate that with a transient ischemic insult
for 15 minutes, Ins(1,4,5)P3 levels readily returned to
control levels in the left MCA cortex after 4 hours. However, after a
60-minute ischemic insult, no recovery in
Ins(1,4,5)P3 could be observed either at 4 or 24 hours
after reperfusion (Fig 4).
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Assessment of Poly-PI Turnover In Vivo in MCA Cortex
Using experimental protocol 1 described in Fig 2
, we
examined the effects of focal ischemia on poly-PI turnover in
the left and right MCA cortices. In this experiment, rat brains were
prelabeled with [3H]inositol for 20 hours before
injection with lithium and surgical administration of ischemia
for 60 minutes (total, 24 hours). Under this condition,
ischemia for 60 minutes resulted in a threefold increase in
labeled IP in the right MCA cortex compared with that in the left
cortex (Table 1). The increase in labeled IPs was marked
by a significant decrease in labeled inositol but not in the organic
layer composed of all inositol phospholipids. Experimental protocol 2
(Fig 2) was designed to assess poly-PI signaling activity 24 hours
after ischemia for 60 minutes. In this experiment, rats were
first subjected to a 60-minute ischemia followed by injection
of [3H]inositol and reperfusion. After 20 hours they were
injected with lithium and killed 4 hours later. Under this condition,
labeled IP in the left MCA cortex was 5.2-fold higher than that in the
same side in protocol 1, but the proportion of labeled IP in the right
MCA cortex was significantly lower than that in the left MCA cortex
(Table 1). Furthermore, the decrease in labeled IP in the right MCA
cortex was marked by significant increases in labeling in the organic
phase and inositol (Table 1).
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Because results from experimental protocol 2 indicated a decrease in
poly-PI turnover in the right MCA cortex at 24 hours after
ischemia for 60 minutes, an experiment was carried out to
examine the time course for this event after reperfusion. There was a
small increase (not significant) in labeling of organic layer
(representing PI) in both the left and right MCA cortices
with time after injection of labeled inositol, but labeling of IP in
the left MCA cortex was not different (Fig 5). However,
significant differences in labeled IP were found between the left and
right MCA cortices in the 16- and 24-hour groups (Fig 5).
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Levels of ATP, ADP, and AMP
A preliminary experiment indicated no significant difference
in ATP levels in left and right MCA cortices in the control and sham
groups, although higher ATP levels were found in the left MCA cortex
(Table 2). There were no differences in ATP levels in
the left MCA cortex among different groups both during ischemia
and at different reperfusion times after a 60-minute ischemia
(Table 2). However, focal cerebral ischemia resulted in a
decrease in ATP levels in the right MCA cortex, and the levels were
maintained at 26.5% of those in sham controls even 60 minutes after
ischemia (Table 2). ADP levels showed a decline during
ischemia similar to that of ATP. The decline in ATP and ADP
levels was marked by an increase in AMP levels. After 4 hours of
reperfusion following a 60-minute ischemic insult, ATP in the
right MCA cortex showed a substantial recovery but reached only 80% of
the levels of the sham controls (group 1). However, ADP and AMP levels
appear to return completely to the sham levels by 4 hours after a
60-minute ischemic insult (Table 2). Beyond the first 4 hours
of reperfusion after a 60-minute ischemia, ATP levels in the
right MCA cortex showed a second phase of decline. By 24 hours ATP had
already declined to levels similar to those at 60 minutes of
ischemia (Table 2). During this period, AMP levels were no
longer elevated.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In this study, we used a sensitive radioreceptor binding procedure30 to measure Ins(1,4,5)P3 levels in MCA cortex and an in vivo protocol6 to assess the poly-PI turnover activity after focal cerebral ischemia and reperfusion. Because the release of Ins(1,4,5)P3 from receptor-mediated hydrolysis of PIP2 is part of a cyclic event requiring ATP (Fig 1
), levels of high-energy
metabolites (ATP, ADP, and AMP) were also measured. Results indicate
specific changes in poly-PI turnover and levels of
Ins(1,4,5)P3 as well as in high-energy metabolites in
the right ischemic MCA cortex both during ischemia and
after reperfusion. In this study ketamine and xylazine were
used as anesthetic agents. Although ketamine can have an effect
antagonistic to the NMDA receptor, it apparently did not
alter the biochemical analyses in this study. Alteration in Ins(1,4,5)P3 levels was most drastic in response to cerebral ischemia and reperfusion. A rapid decrease in Ins(1,4,5)P3 levels could be observed during the early onset of ischemia. Although ischemia also led to a decrease in ATP levels, initiation of the decline in Ins(1,4,5)P3 appeared to precede ATP depletion. These results illustrate the rapid response of the poly-PI signaling pathway to ischemia. Previous studies have demonstrated the rapid decrease in PIP2 levels in brain during the initial period of both global and focal cerebral ischemia.23 24 25 26 27 Thus, the decrease in Ins(1,4,5)P3 after ischemia is likely due to substrate depletion. Nevertheless, the possibility remains that the decreased level of Ins(1,4,5)P3 during ischemia is due to a more rapid removal or an inhibition of the phospholipase C that mediates the hydrolysis of PIP2. These results clearly illustrate the presence of an intricate interdependent mechanism for regulation of Ins(1,4,5)P3 homeostasis in the brain.
Ischemia for 60 minutes in this model is associated with consistent infarction formed in the right MCA territory. Under this condition, we noted an uncoupling between ATP and Ins(1,4,5)P3 recovery after ischemia. Whereas ATP levels returned to 80% of control at 4 hours after ischemia, Ins(1,4,5)P3 levels failed to show recovery. These results confirm the notion that Ins(1,4,5)P3 metabolism is more sensitive to the effects of ischemia-reperfusion than that of ATP. It is possible that this signaling cascade requires a threshold level of ATP for proper functioning and that partial recovery of ATP after reperfusion is not sufficient for the recovery of Ins(1,4,5)P3. However, it is also possible that ischemic insult inhibits the receptor-mediated signaling activity that leads to Ins(1,4,5)P3 release. The irreversible recovery of Ins(1,4,5)P3 after prolonged ischemia correlates well with our earlier observation showing an irreversible alteration of the Ins(1,4,5)P3 3-kinase activity under similar conditions.31 These results clearly demonstrate the complex regulatory mechanisms governing different steps of the poly-PI signaling pathway in the brain. Furthermore, results from the present study reaffirm the previous notion that recovery of high-energy metabolism after ischemia reflects neither recovery of cell function nor the ultimate fate of the ischemic neurons.
In this study, an in vivo protocol was used to assess poly-PI turnover
in MCA cortex during and after the ischemic insult (Fig 1). Our
previous study had indicated that upon injection of
[3H]inositol into the brain, a major proportion of the
label was incorporated into PI and that equilibration of radioactivity
among the inositol metabolites occurred by 6 to 8 hours after
injection.6 In the absence of lithium, labeled IP
accounted for less than 1% of total label in the brain but increased
dramatically with time after administration of LiCl.6 In
this study, a 4-hour window of LiCl administration was used to assess
the accumulation of labeled IP (due to poly-PI turnover) under
different conditions of ischemia and reperfusion. An apparent
limitation with this in vivo protocol is variance between individual
animals due to multiple injections and different activity states of the
animals during the 4-hour period after LiCl administration. The data in
Table 1 further indicate that when rats were anesthetized
during the surgical procedure as well as the ischemia period
(protocol 1), a relatively small amount of labeled IP accumulated in
the left MCA cortex compared with that seen in the group killed without
anesthesia at 24 hours after reperfusion (protocol 2).
However, by a comparison of labeled IP in the right MCA cortex with
that in the left MCA cortex, it can be shown that ischemia
elicited an increase in poly-PI turnover in the right MCA cortex. We
believe that the increased poly-PI turnover probably occurred during
the early time period prior to depletion of high-energy
metabolites. This experiment also showed a decrease in poly-PI turnover
at 24 hours after reperfusion following 60 minutes of ischemia.
This is not surprising because this time period is correlated with
development of cortical infarction with extensive cell
death.19 28 29 The decline in poly-PI turnover after
reperfusion also correlated well with the second phase of ATP
depletion, suggesting a close link between ATP availability and poly-PI
turnover.
Previous studies with brain slices had suggested an increase in stimulation of metabotropic glutamate receptors coupled to poly-PI hydrolysis after severe ischemia.32 33 However, our data on Ins(1,4,5)P3 recovery and on poly-PI turnover in vivo do not support the notion of an increased metabotropic event in the insulted cortex after reperfusion. These discrepancies may be due to the previous studies' use of an in vitro system rather than the in vivo system we used.
As seen in other forms of ischemia,12 13 14 our results as well as others'15 16 17 indicate a rapid decline in ATP levels due to focal cerebral ischemia. In agreement with results from previous studies,17 ATP decline in the ischemic MCA cortex was rapid but not complete (26% of the left). The results of a study by Nowicki et al16 further indicated temporal regional differences in ATP levels after focal cerebral ischemic insult. The residual levels of ATP in the right MCA cortex may be due to an incomplete reduction of blood flow (by 88%) in this model.18 However, because glial cells may contain a larger glycogen store and are more resistant to ischemic insult,34 it is also possible that under the anoxic condition small amounts of ATP are formed in the glial cells because of anaerobic glycolysis. Although ATP levels recovered to 80% of control upon reperfusion (4 hours) after a 60-minute ischemic insult, this recovery was not sustained and was followed by a second phase of decline. These results suggest a perturbation of the intrinsic mechanism for oxidative phosphorylation and ATP production in mitochondria after prolonged ischemia. Similar changes in respiratory function in brain mitochondria have been reported after an ischemic insult.35 Free fatty acids are potent uncouplers of mitochondrial oxidative phosphorylation; therefore, the increase in free fatty acids during the ischemia period36 may also contribute to the decline in mitochondrial function.37 38 Because ATP depletion is directly correlated with the increase in lactate level,16 studies to further investigate factors underlying the second decline in ATP are warranted.
In summary, the present studies demonstrate a sensitive response of the poly-PI signaling pathway and its second messenger, Ins(1,4,5)P3, to focal cerebral ischemia and reperfusion. The close relationship between poly-PI turnover and high-energy metabolism suggests that different levels of the signaling cascade may be disturbed at different phases of ischemia-reperfusion. However, although ATP is needed for synthesis of poly-PI, the uncoupling between ATP and Ins(1,4,5)P3 during ischemia and reperfusion suggests that more intricate factors are present for regulating the metabolism of this second messenger. It can be envisaged that disturbance of the receptor-mediated poly-PI signaling pathway and metabolism of Ins(1,4,5)P3 during the ischemia-reperfusion period can be important factors preventing functional recovery of neurons. Although the pathophysiological significance of the alteration of this signaling cascade remains to be determined, results from this study have provided a better understanding of the biochemical mechanism that underlies the disturbance of calcium homeostasis and induction of immediate early genes that precede tissue damage and cell death.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This study was supported in part by US Public Health Service grants NS-30178, NS-25545, and NS-28995 from the National Institute of Neurological Disorders and Stroke.
Received January 9, 1995; revision received April 7, 1995; accepted May 25, 1995.
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References |
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1. Choi DW, Rothman SM. The role of glutamate neurotoxicity in hypoxic-ischemic neuronal death. Annu Rev Neurosci. 1990;13:171-182. [Medline] [Order article via Infotrieve]
2. Berridge MJ. Inositol trisphosphate and diacylglycerol: two interacting second messengers. Annu Rev Biochem. 1987;56:156-193.
3. Berridge MJ. Inositol trisphosphate and calcium signalling. Nature. 1993;361:315-325. [Medline] [Order article via Infotrieve]
4. Ferris CD, Snyder SH. Inositol phosphate receptors and calcium disposition in the brain. J Neurosci. 1992;12:1567-1574. [Medline] [Order article via Infotrieve]
5. Sherman WE, Munsell LY, Gish BG, Honchar MP. The effect of systemic administration of lithium on phosphoinositide metabolism in rat brain, kidney and testis. J Neurochem. 1985;44:798-804. [Medline] [Order article via Infotrieve]
6. Sun GY, Navidi M, Yoa FG, Lin TN, Orth OE, Stubbs EB, MacQuarrie RA. Lithium effects on inositol phosphate and inositol phospholipids in rat brain: studies with radiotracer technique and ion chromatography. J Neurochem. 1992;58:290-297. [Medline] [Order article via Infotrieve]
7. Lin TA, Zhang JP, Sun GY. The cholinergic receptor linked phosphoinositide metabolism in mouse cerebrum and cerebellum in vivo. Brain Res. 1993;622:169-176. [Medline] [Order article via Infotrieve]
8. Lin TA, Navidi M, James W, Lin TN, Sun GY. Effects of acute ethanol administration on poly-phosphoinositide turnover and levels of inositol 1,4,5-trisphosphate in the mouse brain. Alcohol Clin Exp Res. 1993;17:401-405. [Medline] [Order article via Infotrieve]
9. Lin TA, Zhang JP, Sun GY. Metabolism of inositol 1,4,5-trisphosphate in mouse brain due to decapitation ischemic insult: effects of acute lithium administration and temporal relationship to diacylglycerols, free fatty acids and energy metabolites. Brain Res. 1993;606:200-206. [Medline] [Order article via Infotrieve]
10. Hashimoto K, Kikuchi H, Ishikawa M, Kobayashi S. Changes in cerebral energy metabolism and calcium levels in relation to delayed neuronal death after ischemia. Neurosci Lett. 1992;137:165-168. [Medline] [Order article via Infotrieve]
11.
Lowry OH, Passonneau JV, Hasselberger FY, Schulz DW.
Effect of ischemia on known substrates and cofactors of
the glycolytic pathway in brain. J Biol
Chem. 1964;239:18-30.
12. Kobayashi H, Liest WD, Passonneau JV. Concentrations of energy metabolites and cyclic nucleotides during and after bilateral ischemia in the gerbil cerebral cortex. J Neurochem. 1977;29:53-59. [Medline] [Order article via Infotrieve]
13. Levy DE, Duffy TE. Cerebral energy metabolism during transient ischemia and recovery in the gerbil. J Neurochem. 1977;28:63-70. [Medline] [Order article via Infotrieve]
14. Nowak TS, Fried RL, Lust WD, Passonneau JV. Changes in brain energy metabolism and protein synthesis following transient bilateral ischemia in the gerbil. J Neurochem. 1985;44:487-494. [Medline] [Order article via Infotrieve]
15.
Selman WR, Crumrine RC, Ricci AJ, LaManna JC, Ratcheson
RA, Lust WD. Impairment of metabolic recovery with
increasing periods of middle cerebral artery occlusion in rats.
Stroke. 1990;21:467-471.
16. Nowicki JP, Assumel-Lurdin C, Duverger D, MacKenzie ET. Temporal evolution of regional energy metabolism following focal cerebral ischemia in the rat. J Cereb Blood Flow Metab. 1988;8:462-473. [Medline] [Order article via Infotrieve]
17. Folbergrova J, Memezawa H, Smith ML, Siesjo BK. Focal and perifocal changes in tissue energy state during middle cerebral artery occlusion in normo- and hyperglycemic rats. J Cereb Blood Flow Metab. 1992;12:25-33. [Medline] [Order article via Infotrieve]
18.
Chen ST, Hsu CY, Hogan EL, Maricq H, Balentine JP.
A model of focal ischemic stroke in the rat:
reproducible extensive cortical infarction. Stroke. 1986;17:738-743.
19.
Liu TH, Beckman JS, Freeman BA, Hogan EL, Hsu CY.
Polyethyleneglycol-conjugated superoxide dismutase and
catalase reduce ischemic brain injury. Am J
Physiol. 1989;256:H589-H593.
20. An G, Lin TN, Lin JS, Xue JJ, He TY, Hsu CY. Expression of c-fos and c-jun family genes after focal cerebral ischemia. Ann Neurol. 1993;33:457-464. [Medline] [Order article via Infotrieve]
21. Morley P, Hogan MJ, Hakim AM. Calcium-mediated mechanisms of ischemic injury and protection. Brain Pathol. 1994;4:37-47. [Medline] [Order article via Infotrieve]
22. Kiessling M, Gass P. Stimulus-transcription coupling in focal cerebral ischemia. Brain Pathol. 1994;4:77-83. [Medline] [Order article via Infotrieve]
23. Ikeda M, Yoshida S, Busto R, Santiso M, Ginsberg MD. Poly-phosphoinositides as a probable source of brain free fatty acids accumulated at the onset of ischemia. J Neurochem. 1986;47:123-132. [Medline] [Order article via Infotrieve]
24. Yoshida S, Ikeda M, Busto R, Santiso M, Martinez E, Ginsberg MD. Cerebral phosphoinositide, triacylglycerol and energy metabolism in reversible ischemia: origin and fate of free fatty acids. J Neurochem. 1986;47:744-757. [Medline] [Order article via Infotrieve]
25. Sun GY, Yoa F-G, Lin TN. Degradation of poly-phosphoinositides in brain subcellular membranes in response to decapitation insult. Neurochem Int. 1990;17:529-535.
26. Sun GY. Cerebral ischemia and polyphosphoinositide metabolism. In: Bazan NG, Braquet P, Ginsberg MD, eds. Neurochemical Correlates of Cerebral Ischemia: Advances in Neurochemistry. New York, NY: Plenum Press; 1992;7:219-244.
27.
Lin TN, Liu TH, Xu J, Hsu CY, Sun GY. Brain
polyphosphoinositide metabolism during
focal ischemia in rat cortex. Stroke. 1991;22:495-498.
28.
He YY, Hsu CY, Ezrin AM, Miller MS. Polyethylene
glycol-conjugated superoxide dismutase in focal cerebral
ischemia-reperfusion. Am J Physiol. 1993;265:H252-H256.
29.
Yip PK, He YY, Hsu CY, Garg N, Marangos P, Hogan
EL. Effect of plasma glucose on infarct size in focal cerebral
ischemia-reperfusion. Neurology. 1991;41:899-905.
30. Bredt DS, Mourey RJ, Snyder SH. A simple, sensitive, and specific radioreceptor assay for inositol 1,4,5-trisphosphate in biological tissues. Biochem Biophys Res Commun. 1989;159:976-982. [Medline] [Order article via Infotrieve]
31. Lin TA, Lin TN, He YY, Hsu CY, Sun GY. Effects of focal cerebral ischemia on inositol 1,4,5-trisphosphate 3-kinase and 5-phosphatase activities in rat cortex. Biochem Biophys Res Commun. 1992;184:871-877. [Medline] [Order article via Infotrieve]
32. Chen CK, Silverstein FS, Fisher SK, Statman D, Johnston MV. Perinatal hypoxic-ischemic brain injury enhances quisqualic acid-stimulated phosphoinositide turnover. J Neurochem. 1988;51:353-359. [Medline] [Order article via Infotrieve]
33. Seren MS, Aldinio C, Zanoni R, Leon A, Nicoletti F. Stimulation of inositol phospholipid hydrolysis by excitatory amino acids is enhanced in brain slices from vulnerable regions after transient global ischemia. J Neurochem. 1989;53:1700-1705. [Medline] [Order article via Infotrieve]
34. Swanson RA, Choi DW. Glial glycogen stores affect neuronal survival during glucose deprivation in vitro. J Cereb Blood Flow Metab. 1993;13:162-169. [Medline] [Order article via Infotrieve]
35. Sims NR, Finegan JM, Blass JP. Effects of postdecapitative ischemia on mitochondrial respiration in brain tissue homogenates. J Neurochem. 1986;47:506-511. [Medline] [Order article via Infotrieve]
36. Zhang JP, Sun GY. Free fatty acids, neutral glycerides and phosphoglycerides in transient focal cerebral ischemia. J Neurochem. 1995;64:1688-1695. [Medline] [Order article via Infotrieve]
37. Hillered L, Chan PH. Role of arachidonic acid and other free fatty acids in mitochondrial dysfunction in brain ischemia. J Neurosci Res. 1988;20:451-456. [Medline] [Order article via Infotrieve]
38. Hillered L, Chan PH. Effects of arachidonic acid on respiratory activities in isolated brain mitochondria. J Neurosci Res. 1988;19:94-100. [Medline] [Order article via Infotrieve]
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