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Introduction
Stroke is one of the leading causes of death and long-term disability. Many neuroprotective agents targeting specific
pathways involved in ischemic neuronal death (eg, excitotoxicity,
[Ca2+]i overload and oxygen stress) have been tested in
stroke patients[1]. For the last two decades, a great deal of attention has been paid to neuroprotective therapies. Although
initial preclinical studies have demonstrated that numerous drugs are effective for treating acute stroke in animal models,
subsequent clinical trials have been frustrating, and almost none of the agents have been proven effective. The reason is that
such neuroprotective agents are not able to penetrate the blood-brain barrier and/or they are likely to be degraded into
nonbioactive fragments in the blood stream, mostly by the actions of
proteases[2]. Thus more efforts should be focused on
searching for novel targets so as to develop ideal neuroprotective drugs for stroke.
In 1983, Akinori Noma described a novel type of potassium channel in the membrane of cardiac myocytes characterized by
a pronounced inhibition of channel activity when the intracellular ATP concentration was
increased[3]. Subsequently, such ATP-sensitive potassium
(KATP) channels were found in a range of other tissues including vascular smooth muscle,
pancreatic b-cells, and neurons, etc.
KATP channels belonged to a class of inwardly-rectifying potassium channels activated by a
decrease in the ATP/ADP ratio, which could link cell metabolism to its membrane
potential[4]. Although KATP channels in
many brain cells were closed under normal conditions and did not contribute to resting membrane properties, membrane
hyperpolarization occurred when these channels were activated by selective openers or by decreased levels of oxygen or
glucose. Previous studies demonstrated that
KATP channels regulated neurotransmitter release and served a protective role
in reducing the release of excitatory amino acids during brain ischemia and
anoxia[5]. Opening of KATP channels hyperpolarized
presynaptic terminals, and thus prevented
Ca2+ influx and consequently glutamate release. Furthermore, activation of
KATP channels hyperpolarized glutamate-sensitive neurons thereby conferring resistance to the depolarization induced by various
glutamate receptors[6]. Therefore, the inhibitory modulatory effect of
KATP channels on neuronal excitability in the central
nervous system (CNS) might play an important intrinsic role in the development of ischemic brain damage. Abundant studies
have documented that KATP channel opener (KCO) exerted definite protective effects on ischemic myocardium, but few
studies of KCO as a systemic neuroprotective agent have been published until
now[7]. Molecular biological and
immunohistochemical studies have shown that some ischemia-sensitive brain areas including hippocampus, globus pallidus, substantia
nigra, striatum, and cortex were rich in KATP
channels[8], suggesting that
KATP channels might play a crucial functional
significance and might be an ideal target of neuroprotective agents. Theoretically, blood-brain barrier permeable KCO
should be expected to play a protective role against ischemic insult. However, there is not a KCO selectively targeting
KATP in CNS and exerting significant neuroprotection with less effect on normal blood pressure.
Recently, we designed and synthesized iptakalim (Ipt), a fatty para-amino compound with low molecular
weight[9]. Pharmacological, electrophysiological and biochemical studies have confirmed Ipt as a novel
KCO[5,7]. The blood-brain barrier permeability of Ipt provided us with an opportunity to evaluate its neuroprotective effects on CNS by systematic
administration[9]. Another superiority of Ipt is the minor adverse effect on normal animal blood pressure compared to
diazoxide, the classic mitochondrial ATP-sensitive potassium channel opener. In the present study, we investigated the
protective effects of Ipt in ischemic brain insult on gerbils using behavioral tests and histological study. We also report that
Ipt could alleviate excitotoxicity by enhancing the glutamate uptake of astrocytes and inhibiting glutamate release
in vivo and in vitro.
Materials and methods
Materials PC12 cells were purchased from the Shanghai Institute of Biochemistry, Chinese Academy of Sciences (Shanghai,
China). Ipt (99.9%) was synthesized by the Beijing Institute of Pharmacology and Toxicology (Beijing, China). Dulbecco¡¯s
modified Eagle¡¯s medium (DMEM) was obtained from Gibco RBL (Grand Island, NY, USA).
o-Phthalaldehyde (OPA) was provided by Fluka (Switzerland). All other chemicals and reagents were purchased from Sigma (St Louis, MO, USA).
Global ischemic model Mongolian gerbils (60±5 g, Chinese Academy of Science, Shanghai, China) of both sexes were
housed in the animal care facility with a 12-h light, 12-h dark photoperiod and free access to tap water. After acclimation
(5-10 d), the animals were anesthetized with sodium amylbarbital (45 mg/kg, ip), the common carotid arteries were visualized via
a mid-line neck incision and both arteries were simultaneously occluded for 5 min with non-traumatic micro-aneurysm clips.
After occlusion, the clips were removed to allow cerebral reperfusion and the neck incision closed with wound clips
throughout the surgery, rectal temperature was maintained at 37±0.5
ºC with a heating blanket with feedback control. Room
temperature was regulated to 22-25 ºC by air
conditioning[10]. Sham-operated gerbils were killed just after exposing the carotid artery
without clamping the vessels. The animals were divided into six groups: sham-operated group, ischemia group, and Ipt
groups (0.5, 1.0, 2.0, or 4.0 mg/kg per day, ip). Ipt and saline were administered 40 min prior to carotid artery ligation and
subsequently delivered at the same doses (once every day) up to d 7 after surgery.
Behavioral assessment Assessment of locomotor activity was commonly used as behavioral test following global
ischemia in the gerbil. On d 1 and d 5 following surgery, all gerbils were placed in an open field maze for 15 min while their
locomotor activities were recorded. Locomotor activity included movements across squares and rears (rearing up on haunches),
which represented the exploring activities in horizontal and vertical orientation, respectively. On d 2 and d 3, the gerbils were
trained in the T-maze (15 pairs of trials/day). On d 4 and d 6, the gerbils were given 15 pairs of trials/day and the percentages
of correct choices were determined to measure the working memory of the animals.
Determination of pyramidal cell counts in the hippocampal CA1 region
Histological endpoints (counting the remaining
number of survival cells or calculating infarct volumes) determined up to 7 d after gerbil global ischemia were customarily
used to assess the efficacy of potential therapeutic interventions. For histological endpoints
detected, global ischemic gerbils treated with drugs up to
d 7 were anesthetized by an overdose of sodium amylbarbital and were then transcardially
perfused with 50 mL of saline and 50 mL of 4% buffered polyformaldehyde. The brains were then coronally
sectioned at 6 mm. Sections were stained with hematoxylin and eosin, and
the cells in the CA1 region of hippocampus were counted.
Measurement of amino acid levels Gerbils were decapitated and the hippocampus was immediately removed and then
homogenized in ice-cold homogenizing medium
(HClO4 0.4 mol/L). The homogenate was centrifuged (10
000×g, 4 ºC) for 15 min. The supernatant was neutralized with 1.33 volumes of 2.0 mol/L
KHCO3. After re-centrifuging at
3000×g at 4 ºC for 5 min, the resulting supernatant was frozen at -80 °C and was used to determine the levels of glutamate by high performance liquid
chromatography (HPLC) with fluorescent detection. The HPLC-fluorescent detector system (Shimadzu, Tokyo, Japan)
consisted of Shimadzu HPLC, a reverse phase C18 column (Ultrasphere ODS 4.6 mm×250 mm, 5 µm, Japan), fluorescence
HPLC monitor RF-530 and a liquid chromatograph LC-6A. The mobile phase consisted of 0.1 mol/L
Na2HPO4×12H2O, 0.1
mmol/L edetic acid, methanol 30%, pH 6.04. The emission and excitation wavelengths were set at 425 nm and 338 nm,
respectively. The pre-column derivation solution contained OPA (20 mmol/L),
b-mercaptoethanol (2 mmol/L), tetraborate (25 mmol/L), 50% methanol, pH 9.6. Samples were mixed with an equivalent volume of derivation solution and were incubated at
room temperature (21±1 °C) for 4 min. The derived reaction solutions were used to assay glutamate concentrations at 37 °C
with a flow rate of 0.8 mL/min[17].
PC12 cell culture and viability assay PC12 cell were cultured in DMEM supplemented with 10%
(v/v) heat-inactivated fetal bovine serum, penicillin 200 kU/L and streptomycin 100 mg/L at 37 °C in atmosphere with 5%
CO2. The PC12 cells were then seeded in 96-well plates and grown to 80% confluence in DMEM medium. The cultures were then rinsed with phenol
red-free DMEM medium, and 20 µL of MTT solution (dissolved at 5 g/L in PBS) was added to each well. The reaction was
stopped after incubation at 37 °C for 4 h by discarding the supernatants, and then 100 µL of dimethylsulfoxide
(Me2SO) was added to each well in order to dissolve the resultant dark blue crystal. The absorbance in each well was determined at 570 nm
wavelength with an automatic plate reader (TECAN-SUNRISE, F039246A, Austria).
Intracellular calcium measurement The membrane-permeable
Ca2+ indicator dye Fluo-3 AM (Molecularprobes, Eugene,
OR, USA) was dissolved in Me2SO to produce 1 mmol/L stock solution. For dye loading, cells were incubated with 5
µmol/L Fluo-3 AM diluent for 30 min at 37 °C, followed by 2-3 washes with hydroxyethyl piperazine ethanesulfonic acid solution,
with 0.5 mL of this solution for the determination. A Bio-Rad Radiance 2100TM confocal system in conjunction with a Nikon
TE300 microscope was used and images were viewed through a 40×magnification lens with a factor N computer zoomed
image in a single optical plane. The pixel intensity was used to determine the density value for calcium measures, and ten
fields of view were randomly selected for each group.
Measurement of extracellular glutamate of PC12 cell
The extracellular glutamate levels of PC12 cell exposure to 80
mmol/L KCl were measured by HPLC with fluorescent detection. An aliquot of culture supernatant was deproteinized with
0.4 mol/L perchloric acid and centrifuged at 12
000×g for 10 min at 4 °C. The supernatants were analyzed by HPLC combined
with fluorescent detector analysis after pre-column derivation with OPA as shown above.
Primary culture of astrocytes and assay of glutamate
uptake Rat primary astrocytes were prepared from the whole brains
of postnatal (1-2-day-old) Sprague-Dawley rats and were plated on
poly-D-lysine precoated cell culture flasks containing
DMEM (10% fetal calf serum, 100 U/mL penicillin, and 100 µg/mL streptomycin). The cells were grown in a humidified
atmosphere of 5% CO2/95% air at 37 °C. When the astrocytes reached confluence, the cells were passaged by trypsinization
and plated at a density of 1×106 cells/well on 24-well culture plates in a final volume of 1 mL DMEM, and were grown in a
humidified atmosphere of 5% CO2/95% air at 37 °C. Two days later, the astrocytes were used for experimentation.
Immunocytochemical analyses have shown that this method produces cultures comprising >95% glial fibrillary acid protein-positive
astrocytes.
Glutamate uptake measurements were performed as described previously by Pines and Kanner with little
modification[11]. The culture medium was removed and HEPES buffered saline containing
[3H]glutamate was added to each well (final
concentration was 50 µmol/L). Fifteen minutes later, cells were rapidly washed three times with ice-cold NaCl
(0.9%) to terminate glutamate uptake. Cells were then harvested and solubilized in 0.2 mL NaOH (0.3 mol/L) followed by
immediate centrifugation (5000×g for 5 min at 4 °C). Radioactivity and protein contents of the supernatant were determined
by the liquid scintillation counting and Bradford methods, respectively.
Statistical analysis Data were expressed as
mean±SD.Significance of difference between control and samples treated
with various drugs was determined by one-way ANOVA followed
by post hoc least significant difference (LSD) test. Values
of P<0.05 were taken as statistically significant.
Results
Protective effects of Ipt on high locomotor activity of gerbils with global cerebral
ischemia The open field maze was a valuable functional test that reflected the degree of CA1 pyramidal neuronal injury induced by global cerebral ischemia.
Increased locomotor activity in a novel open field correlates with the severity of ischemia-induced cell death in the CA1
pyramidal layer neuron of the hippocampus. On d 1 after operation, square crossed and rears counted of ischemic gerbils
were increased by 40% and 31%, respectively. Treatment with Ipt (0.5, 1.0 mg/kg per day, ip) failed to change
ischemia-evoked increase of locomotor activity. However, Ipt (2.0, 4.0 mg/kg per day, ip) could decrease the increases of square
crossed and rears counted evoked by the global cerebral ischemia. On d 5, square crossed and rears counted of ischemic
gerbils were still significantly higher than that of the sham group. But Ipt (0.5-4.0 mg/kg per day, ip) decreased the increase
of square crossed and rears counted induced by the global cerebral ischemia (Figure 1).
Ipt improved working memory of gerbils with global cerebral
ischemia The T-maze task provided a valuable method to
assess the function of working memory. On d 2 and d 3, all groups were trained in the T-maze (15 pairs of trials/day). On d 4
and d 6, gerbils were given 15 pairs of trial/day and the % correct to measure working memory of gerbils was calculated. The
gerbils with global cerebral ischemia exhibited significant working memory impairments, which could not be
improved by Ipt (0.5, 1.0 mg/kg, ip). However, Ipt (2.0, 4.0
mg/kg, ip) could increase the % correct of ischemic gerbils so as to obtain the food
reward (Figure 2).
Protective effects of Ipt on the CA1 neurons of gerbils with global cerebral
ischemia CA1 neurons are highly susceptible to global ischemic injury and their laminar distribution and large size made them relatively easy to quantify. The results
from HE staining showed extensive losses (overall counted averaged ~15% of sham group) of hippocampus CA1 zone
pyramidal neurons in gerbils with global cerebral ischemia for 5 min. Ipt (0.5-4.0 mg/kg, ip) could increase the remaining
number of healthy neurons in hippocampus CA1 zone in a dose-dependent manner (Figure 3).
Effects of Ipt on the contents of amino acids in the hippocampus of gerbils with global cerebral
ischemia After the ligation of bilateral carotid artery for 5 min and reperfusion for 60 min, the contents of glutamate from hippocampus in gerbils
increased by 40%. Pretreatment with Ipt (1.0, 2.0, 4.0 mg/kg, ip) 40 min prior to carotid artery ligation could reverse
ischemia-evoked increases of glutamate in hippocampus of gerbils (Figure 4).
Protective effect of Ipt on glutamate-induced
cytotoxicity After pre-treatment of PC12
cells with 10 µmol/L Ipt for 15 min, cells were co-exposed to 10 µmol/L
Ipt and 10 mmol/L glutamate for 24 h, after which the cell viability was measured.
The viability of Ipt treated cells was 95%, which was significantly higher than that of glutamate treated cells (79%). Application of
the classic KATP channels opener pinacidil showed similar protective effects. Pre-incubation of PC12 cells with glibenclamide, a
KATP channels blocker, for 15 min, could abolish the protective effects of Ipt and pinacidil (Figure 5).
Effects of Ipt on the elevation of intracellular calcium induced by glutamate
Fluorometric Ca2+ measurements
demonstrated that cells pre-treated with 10 µmol/L
Ipt or pinacidil had lower Ca2+ in PC 12 cells compared to that treated with 10
mmol/L glutamate for 24 h (Figure 6). Likewise, the reduction of intracellular
Ca2+ overload was attenuated by glibenclamide.
Effects of Ipt on extracellular glutamate levels of PC12 cell incubated with high extracellular
K+ PC12 cell were pre-incubated with different concentrations of Ipt before high extracellular
K+ treatments. As shown in Figure 7, pre-treatment
with different concentrations of Ipt for 15 min reduced extracellular glutamate levels of PC12 cell induced by high extracellular
K+ in a concentration-dependent manner. Glibenclamide reversed the inhibitory effects of Ipt (Figure 7).
Effects of Ipt on astrocytic glutamate uptake
Primary cultured astrocytes were incubated with different concentrations of
Ipt for 24 h. Astrocytic glutamate uptake was determined by measuring intracellular concentrations of
[3H]D,L-glutamate using isotope techniques. Ipt (10, 100
µmol/L) could enhance astrocytic glutamate uptake
(Figure 8).
Discussion
Our studies indicated that Ipt (0.5-4.0 mg/kg, ip) could reduce the high locomotor activity evoked by ischemia and
improve global cerebral ischemia-induced working memory impairments. Ipt did not affect the locomotor activity and working
memory of normal gerbils in the preliminary experiments. Histological study revealed that Ipt (0.5-4.0 mg/kg per day, ip) could
increase the survival neuron in hippocampus CA1 zone in a dose-dependent manner. Moreover, Ipt (1.0, 2.0, 4.0 mg/kg, ip)
40 min prior to carotid artery ligation could reverse ischemia-evoked increases of glutamate in the hippocampus of gerbils.
According to previous studies, hippocampal glutamate levels were thought to represent the extracellular released
glutamate[12]. Ipt could protect PC12 cells against glutamate induced cytotoxicity by reducing the
[Ca2+]i and extracellular glutamate
levels of PC12 cells and enhancing the glutamate uptake activity of primary cultured astrocytes.
Brief periods of global brain ischemia in rodents caused delayed cell death in hippocampal CA1 pyramidal neurons days
after reperfusion. A similar phenomenon occurred after ischemic injury in
humans[13]. The cause of such delayed neuronal
damage in the CA1 neurons has not been fully understood, although many mechanisms have been proposed. Among them,
glutamate excitotoxicity has played a critical
role[14].
Glutamate, the major excitory neurotransmitter in the CNS, is the main cause of excitotoxicity in CNS
pathology[15]. As glutamate neurotoxicity was considered to be initiated by excessive
Ca2+ influx resulting from the overactivation of the
NMDA receptor, it was generally accepted that one of the most important pathophysiological factors underlying
glutamate-induced cellular damage was the failure of intracellular
Ca2+ homeostasis[16]. Excessive activation of the NMDA receptor
could induce a massive influx of extracellular calcium, which further activated a series of calcium-dependent enzymes
involved in the catabolism of proteins and eventually resulted in neuronal
apoptosis[17]. Therapeutic strategies for cerebral
ischemia have been developed to inhibit the downstream occurrence of glutamate receptor overactivation-induced
pathophysiological reactions. However, more attention should been given to reducing the glutamate levels in the synaptic cleft.
Two main processes are involved in maintaining physiological glutamate concentrations at the synaptic cleft: one regulates
glutamate release from the presynaptic membrane, and the other regulates glutamate uptake, which is mainly mediated by
high affinity glutamate transporters located in
astrocytes[6,18].
Ipt has been demonstrated as a novel KCO and a promising neuroprotectant with previous results showing that Ipt
protected substantia nigra dopaminergic neurons against a variety of neurotoxins (eg,
MPP+, 6-OHDA or rotenone) in
vitro and in vivo[6,19-21]. In this study, we assessed the protective effect of Ipt on global cerebral ischemia through systematic
administration. To explore the protective mechanism regarding excitotoxicity, we found that Ipt could reverse global cerebral
ischemia induced increase of glutamate levels in the hippocampus of gerbils, which may be an important neuronal mechanism
of its neuroprotective role. Furthermore, Ipt inhibited glutamate release from PC12 cells evoked by high extracellular
K+ and reduced intracellular
Ca2+ of PC12 cells, which could be abolished by
KATP channel blocker glibenclamide, suggesting it was
the consequence of, at least partly, activation of
KATP channels. Meanwhile, Ipt exhibited potent neuroprotective capacities
in PC12 cells exposed to glutamate. Several lines of evidence indicated that the activation of
KATP channels inhibited neurotransmitter releases such as acetylcholine from rat striatal slices, catecholamide from PC12
cell[5] and glutamate release from embryonic chick telencephalon neuron by causing
hyperpolarization[22]. Combined with the results of the present
study, we hypothesized that the inhibitory regulation of glutamate release was involved in the neuroprotection of Ipt on
ischemia-evoked neuronal death.
KATP channels exist both in astrocytes in adult rats and newborn rat primary
astrocytes[18, 23, 24]. In the present study we
revealed that Ipt could enhance the glutamate uptake of primary cultured astrocytes. Our previous
in vitro analysis and in vivo microdialysis experiments indicated that Ipt could reduce extracellular glutamate
levels[18,21,25]. Therefore, we hypothesized that Ipt alleviated glutamate excitotoxicity in two ways: one was to inhibit glutamate release and the other was to
enhance glutamate uptake of astrocytes. How did Ipt regulate the activity of astrocytic glutamate uptake? The activity of
Na+-dependent glutamate transporters was governed by both thermodynamic and kinetic factors, such as
Na+/K+
gradients[26]. As the driving forces were all supplied by ATP hydrolysis, opening of
KATP channels resulted in the preservation of
intracellular ATP production, hence, subsequently elevated activity of glutamate transporter. Moreover, the activation of
KATP channels lead to the almost immediate activation of protein kinase
C[27]. Activators of protein kinase C, such as phorbol
esters, rapidly stimulate glutamate transporter
activity[28]. So it is proposed that Ipt may enhance glutamate uptake mediated
by glutamate transporters by the opening of
KATP channels, subsequently augmenting ATP production and PKC-mediated
phosphorylation.
In conclusion, Ipt, a novel and blood-brain barrier permeable KCO, could effectively protect brain neurons against
glutamate neurotoxicity in in vitro and in
vivo animal ischemic models. The mechanisms underlying the neuroprotection of
Ipt may involve the opening of KATP, diminishing glutamatergic synaptic transmission by inhibiting presynaptic glutamate
release, alleviating postsynaptic ionotropic glutamate receptor such as NMDA receptor overdepolarization-induced events,
enhancing glutamate uptake of astrocytes, and reducing intracellular
Ca2+ stores. Accordingly, Ipt is not only a useful
pharmacological tool for systemic investigation on
KATP channels in CNS but also a promising therapeutic agent with few
side-effects for stroke.
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