Journal of Neurochemistry, 2007, 103, 569–579
ATP-sensitive potassium channel opener iptakalim protects against
MPP+-induced astrocytic apoptosis via mitochondria and
mitogen-activated protein kinase signal pathways
Shu Zhang,1 Fang Zhou,1 Jian-Hua Ding, Xi-Qiao Zhou, Xiu-Lan Sun and Gang Hu
Laboratory of Neuropharmacology, Institute of Neurosciences, Nanjing Medical University, Nanjing, Jiangsu, P. R. China
Abstract
Inhibition of astrocytic apoptosis has been regarded as a novel
prospective strategy for treating neurodegenerative disorders
such as Parkinson’s disease. In the present study, we dem-
onstrated that iptakalim (IPT), an ATP-sensitive potassium
channel (KATP channel) opener, exerted protective effect on
MPP+-induced astrocytic apoptosis, which was reversed by
selective mitochondrial KATP channel blocker 5-hydroxyde-
canoate. Further study revealed that IPT inhibited glutathione
(GSH) depletion, mitochondrial membrane potential loss and
subsequent release of pro-apoptotic factors (cytochrome c
and apoptosis-inducing factor (AIF), and c-Jun NH2-terminal
kinase/mitogen-activated protein kinases (MAPK) phos-
phorylation induced by MPP+. Meanwhile, extracellular signal-
regulated kinase (ERK) 1/2 inhibitor PD98059 inhibited the
Astrocytes, the major non-neuronal cells in the brain, have
been implicated in the segregation, maintenance, and support
of neurons, including clearance and release of extracellular
glutamate (Anderson and Swanson 2000; Mazzanti et al.
2001), scavenging oxygen free radicals (Chen et al. 2001),
homeostatic maintenance of extracellular ionic environment
and pH (Amiry-Moghaddam and Ottersen 2003; Simard and
Nedergaard 2004), provision of metabolic substrates for
neurons (Kasischke et al. 2004), and coupling of cerebral
blood flow to neuronal activity (Takano et al. 2006).
Accordingly, astrocyte dysfunction and even dysregulation
of astrocyte-specific functions can critically influence neur-
onal survival (Seifert et al. 2006). Traditionally, necrosis was
believed to be the predominant mechanism of astrocytes
death in brain injury models, but increasing evidence showed
that astrocytic apoptosis may contribute to pathogenesis of
many neurodegenerative disorders such as Alzheimer’s
disease and Parkinson’s disease (PD) (Kobayashi et al.
2002; Szydlowska et al. 2006). Thus, the advance in the
knowledge of molecular regulation of astrocytic apoptosis
 2007 The Authors
Journal Compilation  2007 International Society for Neurochemistry, J. Neurochem. (2007) 103, 569–579
doi:10.1111/j.1471-4159.2007.04775.
protective effect of IPT on MPP+-induced astrocytic apoptosis.
Furthermore, IPT could also activate ERK/MAPK and main-
tain increased phospho-ERK1/2 level after MPP+ exposure.
Taken together, these findings reveal for the first time that IPT
protects against MPP+-induced astrocytic apoptosis via inhi-
bition of mitochondria apoptotic pathway and regulating the
MAPK signal transduction pathways by opening mitochondrial
ATP-sensitive potassium (mitoKATP) channels in astrocytes.
And targeting KATP channels expressed in astrocytes may
provide a novel therapeutic strategy for neurodegenerative
disorders.
Keywords: apoptosis, astrocyte, ATP-sensitive potassium
channel opener, iptakalim, mitochondria, mitogen-activated
protein kinase.
J. Neurochem. (2007) 103, 569–579.
may lead to the development of novel therapeutic strategies
for neurodegenerative disorders (Takuma et al. 2004). And
the drugs that protect against astrocytic apoptosis are
promising neuroprotectants.
ATP-sensitive potassium (KATP) channels, which link cell
metabolism to its membrane potential, belong to a class of
Received January 16, 2007; revised manuscript received March 15,
2007; accepted May 30, 2007.
Address correspondence and reprint requests to Gang Hu, Laboratory
of Neuropharmacology, Institute of Neurosciences, Nanjing Medical
University, 140 Hanzhong Road, Nanjing, Jiangsu 210029, P. R. China.
E-mail: ghu@njmu.edu.cn
1
These authors contributed equally to this work.
Abbreviations used: DYm, mitochondrial membrane potential; 5-HD,
5-hydroxydecanoate; AIF, apoptosis-inducing factor; DZ, diazoxide;
ERK, extracellular signal-regulated kinase; IPT, iptakalim; JNK, c-Jun
NH2-terminal kinase; KATP channels, ATP-sensitive potassium channels;
KCO, KATP channel opener; Kir, inwardly rectifying potassium subunit;
MAPK, mitogen-activated protein kinase; PBS, phosphate-buffered
saline; PD, Parkinson’s disease; PI, propidium iodide; SUR, sulfonylurea
receptors.
569
570 S. Zhang et al.
inwardly rectifying potassium channels that are widely
distributed in the brain (Dunn-Meynell et al. 1998; Zawar
et al. 1999). Functional KATP channels are heteroctameric
complexes, consisting of discrete pore-forming (inwardly
rectifying potassium subunit; Kir6.1/Kir6.2) and regulatory
subunits (sulfonylurea receptors; SUR1/SUR2). The chan-
nels are usually closed in normal conditions, but are activated
rapidly in response to decrease in intracellular ATP/ADP
ratio under metabolic stress, which results in hyperpolariza-
tion of the cell membrane and limitation of Ca2+ influx, thus
blocking subsequent neurotoxic biochemical cascades (Seino
and Miki 2003). Emerging evidence including from our
laboratory has revealed a novel neuronal protective role of
KATP channel openers (KCOs) in PD models both in vivo and
in vitro (Tai et al. 2003; Wang et al. 2005; Yang et al. 2004,
2006b). Therefore, KCOs that can freely cross the blood-
brain barrier are considered as potential neuroprotectants
(Wang et al. 2006; Yang et al. 2005). Accumulated evidence
shows that modulation of KATP channels expressed in
neurons exerts neuroprotective effect (Liss et al. 2005;
Nakagawa et al. 2005). Nevertheless, little is known about
the role of regulating astrocytic KATP channels in neurode-
generative disorders.
Iptakalim (IPT) is a lipophilic para-amino compound with
low molecular weight, which can freely cross the blood-brain
barrier and has been demonstrated to be a novel KCO by
pharmacological, electrophysiological, and biochemical stud-
ies, and receptor binding test (Wang 2003; Wang et al. 2004;
Xie et al. 2005). Our previous studies provided compelling
supports for the neuroprotective effects of IPT on behavioral
recovery and neuronal survival in various animal models
such as stroke and PD, as well as in cultured cells (Wang
et al. 2004, 2005; Yang et al. 2004). IPT is not only a useful
pharmacological tool for KATP channel investigation, but it
also serves as a novel, high-potent, low-toxic, therapeutic
agent that protects brain neurons against a variety of
neurodegenerative diseases (Wu et al. 2005). As KATP
channels are expressed in astrocytes (Thomzig et al. 2001),
whether IPT exerts neuroprotective effects via regulating
KATP channels in astrocytes needs to be further investigated.
The neurotoxin MPP+, a high affinity inhibitor of
mitochondrial complex I, which is metabolite formed by
monoamine oxidase-B oxidation of MPTP in astrocytes
(Tipton and Singer 1993), can induce selective dopaminer-
gic neuronal degeneration in substantia nigra (Chiueh and
Rauhala 1998). In addition, MPP+ is a common neurotoxin
used to explore the alteration of astrocytic function (Henze
et al. 2005; Tripanichkul et al. 2006). And KATP channel is
considered as a potential downstream target of mitochond-
rial complex I inhibition (Liss et al. 2005). Therefore, in the
present study, we try to investigate whether IPT offers
protective effect on MPP+-induced astrocytic apoptosis by
opening KATP channels and to elucidate the anti-apoptosis
mechanisms of IPT.
 2007 The Authors
Journal Compilation  2007 International Society for Neurochemistry, J. Neurochem. (2007) 103, 569–579
Materials and methods
Astrocyte cell culture
Rat primary astrocytes were prepared according to previously
described protocol with slight modifications (Hamprecht and Loffler
1985). Briefly, tissues from whole brains of postnatal (P1–P2)
Sprague–Dawley (SD) rats were triturated and then cells were plated
on poly-D-lysine pre-coated cell culture flasks in Dulbecco’s
modified Eagle’s medium containing 10% fetal calf serum, 100 U/
mL penicillin and 100 lg/mL streptomycin. Cultures were main-
tained at 37C in a humidified atmosphere of 5% CO2/95% air. After
reaching a confluent monolayer of glial cells (10–14 days),
microglia were separated from astrocytes by shaking off for 5 h at
100 rpm. The enriched astrocytes were >96% positive for glial
fibrillary acidic protein as assessed by immunocytochemical
staining.
GSx assay
GSH was assayed as total glutathione (GSx), which was the sum of
the reduced and oxidized forms [GSH+2·glutathione disulfide
(GSSG)], by a modified enzymatic recycling method of Tietze
(Tietze 1969). Astrocytes were washed in phosphate-buffered saline
(PBS), dissolved in 200 lL PBS buffer (0.05 mmol/L edetic acid;
0.05% Triton-X 100, pH 8.0), and centrifuged to remove protein.
According to the Bradford method (Kruger 1994), a volume of
50 lL of supernatant was used to assess protein by using bovine
serum albumin as a standard. A volume of 100 lL of supernatant
was neutralized with 50 lL of a 5% (w/v) salicylsalicylic acid
solution. The solution was centrifuged, and then the GSH content
was determined after the addition of 4 lL 10 mmol/L b-NADPH,
3 lL 10 mmol/L 5, 5¢- dithio-bis-2-nitrobenzoic acid, 2 kU/L GSH
reductase. The GSH content was determined by kinetic measure-
ment of the absorbance changes at 412 nm for 5 min and calculated
by comparison with standards.
Determination of protein
The cells were dissolved in 1% dodecylsulfate in the presence of
0.1 mol/L NaOH. The amount of protein was determined by
applying the method of Bradford assay (Kruger 1994) using bovine
serum albumin as the standard.
Morphological assessment and quantification of apoptotic
astrocytes
Hoechst staining
To quantify apoptotic astrocytes, astrocytic monolayer was fixed and
stained with Hoechst 33324 (Sigma, St Louis, MO, USA). The
morphological features of apoptosis (cell shrinkage, chromatin
condensation, and fragmentation) were monitored by fluorescence
microscopy (Olympus BX 60, Tokyo, Japan). At least 400 cells
from 12 randomly selected fields per dish were counted, and each
treatment was performed in triplicate.
Flow cytometry
Astrocytic apoptosis was estimated using the Annexin-V Fluoresc-
ein (FITC) apoptosis detection kit (Oncogene Research Products,
San Diego, CA, USA) according to the kit instructions. The cell
samples were analyzed in a flow cytometry apparatus (Becton

Dickinson FACSVantage SE, San Jose, CA, USA). Annexin V binds
to phosphatidylserine that is translocated during apoptosis from the
inner to the outer leaflet of the plasma membrane. Live cells with
intact membranes are distinguished by their ability to exclude
propidium iodide (PI), which readily penetrates dead or damaged
cells. Dual analysis was introduced using a quadrant dot plot, in
which necrotic cells were identified as single PI-positive, early
apoptotic cells were annexin V-FITC-positive only, and cells in late
apoptosis were recognized as double-positive for annexin V-FITC
and PI. Cells that stained negative for both annexin V-FITC and PI
were classified as live cells. Finally, the number of cells in each
category was expressed as a percentage of the total number of
stained cells counted.
Measurement of astrocyte mitochondrial membrane potential
Astrocyte mitochondrial membrane potential (DYm) was assessed
with the fluorescent probe JC-1. At 490 nm, cells with depolarized
mitochondria contained JC-1 predominantly in monomeric form and
fluoresced green. Cells with polarized mitochondria predominantly
contained JC-1 in aggregate form, and show fluoresced red-orange.
Astrocytes were incubated with 5 lmol/L of JC-1 (Molecular
Probes, Eugene, OR, USA) for 10 min at 37C, washed, and placed
on a thermostatted stage at 37C. Fluorescent images were
visualized by a Nikcon Optical TE2000-S inverted fluorescence
microscope with excitation at 490 nm and emission at >520 nm.
Astrocytes with polarized mitochondria were seen with distinct
mitochondria fluorescing red-orange, and, in astrocytes with
depolarized mitochondria, the cytoplasm and mitochondria appeared
green. Acquired signal was analyzed with image-analysis software
(Simple PCI; Compix Inc., Cranberry Township, PA, USA). A
minimum of six fields were selected and average intensity for each
region was quantified. The ratio of J-aggregate to JC-1 monomer
intensity for each region was calculated. A decrease in this ratio was
interpreted as loss of DYm, whereas an increase in the ratio was
interpreted as gain in DYm.
Detection of mitochondrial cytochrome c and AIF levels
107 astrocytes were washed with ice-cold phosphate-buffered
saline, and cells were resuspended for 15 min on ice in
permeabilization buffer containing 75 mmol/L NaCl, 1 mmol/L
NaH2PO4, 250 mmol/L sucrose, 1 mmol/L phenylmethylsulfonyl
fluoride, additional protease inhibitors, and 0.05% digitonin.
Following a centrifugation step at 800 g at 4C for 10 min, the
supernatant was separated from the pellet consisting of cellular
debris. The crude mitochondrial pellet was collected by centrif-
ugation at 13 000 g at 4C for 10 min. Mitochondrial proteins
(50 lg) were then subjected to immunoblot analysis using a
monoclonal Cytochrome c (1 : 1000) and AIF antibody (1 : 1000)
as described below.
Western blotting
Cells were washed twice with ice-cold PBS and homogenized in
200 lL lysis buffer. After incubation for 20 min on ice, cell
lysates were centrifuged (10 000 g for 10 min at 4C) and protein
concentration in the extracts was determined by the Bradford
assay (Kruger 1994). Proteins in cell extracts were denatured with
sodium dodecyl sulphate sample buffer and separated by 10%
SDS-PAGE. Proteins were transferred to nitrocellulose membranes
 2007 The Authors
Journal Compilation  2007 International Society for Neurochemistry, J. Neurochem. (2007) 103, 569–579
Iptakalim inhibits astrocytic apoptosis 571
using a Bio-Rad miniprotein-III wet transfer unit. The membranes
were incubated with 5% bovine serum albumin dissolved in TBST
(pH 7.5, 10 mmol/L Tris-HCl, 150 mmol/L NaCl, and 0.1%
Tween-20) at 25C for 1 h, washed three times and incubated with
different antibodies [c-Jun NH2-terminal kinase (JNK), phosphor-
JNK, extracellular signal-regulated kinase (ERK), and phosphor-
ERK at 1 : 1000]. The membranes were washed three times with
TBST buffer and incubated with the secondary antibody (1 : 2000)
for 1 h followed by four washings. Signal detection was performed
with an enhanced chemiluminescence kit.
Statistical analysis
All values were expressed as mean ± SEM. The significance of the
difference between control and samples treated with various drugs
was determined by one-way ANOVA followed by the post hoc least
significant difference test. Differences were considered significant at
p < 0.05.
Results
IPT inhibits MPP+-induced astrocytic apoptosis
To explore whether IPT could inhibit MPP+-induced astro-
cytic apoptosis, the nuclear staining with Hoechst 33342 in
the MPP+-treated astrocytes was observed. As showed in
Fig. 1, untreated astrocytes exhibited regular and round-
shaped nuclei. In contrast, the condensation and fragmenta-
tion of nuclei, characteristic of apoptotic cells, was evident in
astrocytes treated with different concentrations of MPP+ for
48 h (Fig. 1a). Pretreatment with 1, 10, or 100 lmol/L IPT
reduced the MPP+-induced astrocytic apoptosis in a concen-
tration-dependent manner. Diazoxide (DZ), a selective mit-
ochondrial KATP (mitoKATP) channel opener, had the similar
effect to IPT with a maximum inhibition at 100 lmol/L.
Pretreatment with selective mitoKATP channel blocker
5-hydroxydecanoate (5-HD, 250 lmol/L) could abolish the
inhibitory effect of IPT (10 lmol/L) or DZ (100 lmol/L) on
MPP+-induced astrocytic apoptosis (Fig. 1b). However,
sarcolemmal KCO P1075 (1, 10, 100 lmol/L) failed to
inhibit MPP+-induced astrocytic apoptosis (p > 0.05) (data
not shown).
The results of flowcytometry further confirmed the
anti-apoptotic effects of IPT on MPP+-induced astrocytic
apoptosis (Fig. 1c). Annexin-V binding and PI uptake were
detected in astrocytes. The portion of annexin-V positive
cells increased from 9% in control cells to 29% in the cells
treated with MPP+ for 48 h. The treatment of IPT (10 lmol/L)
or DZ (100 lmol/L) 30 min prior to MPP+ addition induced a
decrease in the portion of apoptotic cells from 29% in cells
treated with MPP+ to 11% and 10%, respectively. And these
effects were abolished by mitoKATP channel blocker 5-HD
(250 lmol/L), suggesting that IPT could inhibit MPP+-
induced astrocytic apoptosis through opening mitoKATP
channels.
572 S. Zhang et al.
(a)
Con. MPP+ 50µmol/L MPP+ 100µmol/L
MPP+ 200µmol/L MPP+ 400µmol/L MPP+ 800µmol/L
(b)
(c)
Fig. 1 Iptakalim (IPT) inhibits MPP+-induced astrocytic apoptosis.
The morphological features of apoptosis were monitored by fluores-
cence microscopy after staining with Hoechst 33342. Scale bar:
50 lm. (a) MPP+ (50–800 lm) induced astrocytic apoptosis in a
concentration-dependent manner when incubated for 48 h. (b) Pre-
treatment with IPT (10 lmol/L) or diazoxide (DZ) (100 lmol/L) for
30 min inhibited MPP+ (200 lmol/L)-induced astrocytic apoptosis,
which was abolished by 5-hydroxydecanoate (5-HD; 250 lmol/L). (c)
And the apoptosis rate was measured by flowcytometry analysis after
staining with Annexin V-FITC and propidium iodide (PI). Necrotic cells
IPT suppresses MPP+-induced endogenous glutathione
depletion in astrocytes
GSH is known to protect proteins from oxidation by
conjugating with oxidized thiol groups, and depletion of
GSH may be responsible for inhibition of complex I activity
via thiol oxidation (Jha et al. 2000). To search into the
relationship between endogenous glutathione and MPP+-
induced cell damage, the GSx level of astrocyte was
measured. As demonstrated in Fig. 2a, the GSx level was
significantly increased by 35%, 67%, and 69% of control
after exposure to IPT 1, 10, and 100 lmol/L respectively.
 2007 The Authors
Journal Compilation  2007 International Society for Neurochemistry, J. Neurochem. (2007) 103, 569–579
were identified as single PI-positive, early apoptotic cells were Annexin
V-FITC-positive only, and cells in late apoptosis were recognized as
double-positive for Annexin V-FITC and PI. Cells that stained negative
for both Annexin V-FITC and PI were classified as live cells. The data
presented as mean ± SEM of four individual experiments. *p £ 0.05;
**p £ 0.01, compared with untreated group. #p £ 0.01; ##p £ 0.001,
compared with MPP+-treated group. +p £ 0.05; ++p £ 0.01, compared
with both IPT and MPP+-treated group. $p £ 0.05; $$p £ 0.01, com-
pared with both DZ and MPP+-treated group.
MitoKCO DZ also increased the GSx level to 115%, 124%,
152%, and 154% of control at the concentration of 10, 50,
100, and 200 lmol/L in astrocytes (Fig. 2b). The GSx level
was decreased to 40% of control after incubation of the
astrocytes with MPP+ (200 lmol/L) for 48 h. But MPP+
failed to decrease the GSx level at low concentration
(<25 lmol/L) (Fig. 2c). Either IPT (10 lmol/L) or DZ
(100 lmol/L) could suppress MPP+-induced endogenous
GSx depletion and increase GSx level by 49% and 43% of
control respectively, which was abolished by mitoKATP
channel blocker 5-HD (250 lmol/L) (Fig. 2d). These results

(a)
(c)
Fig. 2 Iptakalim (IPT) or diazoxide (DZ) suppresses MPP+-induced
endogenous glutathione depletion in astrocytes. (a) Incubation with
increasing concentration of IPT (0.01–100 lmol/L) or (b) DZ (1–
200 lmol/L) for 48 h increased the intracellular GSx levels in astro-
cytes. (c) Exposure to MPP+ (5–200 lmol/L) for 48 h decreased
intracellular GSx levels in a concentration-dependent manner in ast-
rocytes. (d) Astrocytes were treated with IPT (10 lmol/L) or DZ
demonstrate that IPT can inhibit MPP+-induced endogenous
GSx depletion through opening mitoKATP channels.
IPT prevents MPP+-induced DYm loss and pro-apoptotic
factor release in astrocytes
As DYm loss, a consequence of mitochondrial membrane
permeability, is detected in several models of apoptosis,
molecular probe JC-1 was used to detect the effect of IPT on
MPP+-induced astrocyte DYm change. Astrocytes exposed to
MPP+ (200 lmol/L) for 6 h displayed a loss of DYm
(Fig. 3a), indicated by fluorescence of JC-1 shifted from
red-orange to greenish yellow. Pretreatment with 10 lmol/L
IPT or 100 lmol/L DZ could prevent mitochondria from loss
of DYm induced by MPP+, which was abolished by addition
of 5-HD (250 lmol/L) (Fig. 3b).
The effect of IPT on MPP+-induced change of astrocyte
mitochondrial membrane permeability was assessed by
measuring mitochondrial pro-apoptotic factors (cytochrome
c and AIF) levels. Pretreatment with MPP+ (200 lmol/L)
markedly decreased mitochondrial cytochrome c and AIF
levels in astrocytes, implying that MPP+ increased the release
of cytochrome c and AIF from mitochondria and subse-
quently increased the AIF nuclear levels and cytochrome c
cytosolic levels, respectively. Cytochrome c release occurred
as early as 3 h after MPP+ exposure, whereas AIF release
occurred in a delayed fashion (Fig. 3c). Either IPT (10 lmol/
L) or DZ (100 lmol/L) significantly depressed the MPP+-
induced the decrease of mitochondrial cytochrome c and AIF
 2007 The Authors
Journal Compilation  2007 International Society for Neurochemistry, J. Neurochem. (2007) 103, 569–579
Iptakalim inhibits astrocytic apoptosis 573
(b)
(d)
(100 lmol/L) for 30 min prior to MPP+ (200 lmol/L) exposure for 48 h.
The endogenous glutathione depletion induced by MPP+ was sup-
pressed. The data presented as mean ± SEM of four individual
experiments. *p £ 0.05; **p £ 0.01, compared with untreated group.
#
p £ 0.01, compared with MPP+-treated group. +p £ 0.05, compared
with both IPT and MPP+-treated group. $p £ 0.05, compared with both
DZ and MPP+-treated group.
levels, and these effects were abolished by mitoKATP
channel blocker 5-HD (250 lmol/L) (Fig. 3d). These data
indicate that IPT can prevent MPP+-induced DYm loss and
subsequent releasing pro-apoptotic factor in astrocytes by
opening mitoKATP channels.
IPT inhibits MPP+-induced JNK phosphorylation in
astrocytes
Increasing evidence shows that JNK plays an important role in
apoptosis (Lotharius et al. 2005). So we investigated whether
IPT could affect MPP+-induced JNK phosphorylation in
astrocytes. Treatment with MPP+ (200 lmol/L) led to a rapid
and transient phosphorylation of JNK with the peak levels of
phospho-JNK occurring at 30 min, indicating that JNK
signaling pathway is activated in response to MPP+ treatment
in astrocytes (Fig. 4a). Next, we detected whether IPT could
regulate MPP+-induced JNK phosphorylation at 30 min time
point. As shown in Fig. 4b, pretreatment with IPT (10 lmol/L)
or DZ (100 lmol/L) reduced MPP+-induced increase of
phospho-JNK by over 40%. And these effects were abolished
by mitoKATP channel blocker 5-HD (250 lmol/L). These
results indicate that IPT can inhibit MPP+-induced JNK
phosphorylation via opening mitoKATP channels.
ERK activation is involved in the anti-apoptotic effects of
IPT on astrocytes
Extracellular signal-regulated kinase/mitogen-activated pro-
tein kinases (MAPK) pathway has been demonstrated to be
574 S. Zhang et al.
(a)
(b)
(c)
(d)
involved in anti-apoptotic pathways (De Girolamo and Billett
2006; Yue et al. 2000). In present study, ERK1/2 inhibitor
PD98059 (50 lmol/L) had no effect on the normal condition
and MPP+-induced astrocytic apoptosis. However, in the
presence of PD98059, the protective effects of IPT (10 lmol/
L) and DZ (100 lmol/L) on MPP+-induced astrocytic
apoptosis were inhibited (Fig. 5a). These results suggest that
ERK/MAPK pathway might be involved in the anti-
apoptotic effects of IPT on astrocytes. To confirm it, we
performed the western blot analysis of phospho-ERK1/2 in
 2007 The Authors
Journal Compilation  2007 International Society for Neurochemistry, J. Neurochem. (2007) 103, 569–579
Fig. 3 Iptakalim (IPT) alleviates MPP+-in-
duced mitochondrial membrane potential
loss and pro-apoptotic factor release in
astrocytes. Quantification of mitochondrial
membrane potential expressed as a ratio of
J-aggregate to JC-1 monomer (red : green)
fluorescence intensity. Scale bar: 100 lm.
(a) Astrocytes were treated for various
periods of time up to 8 h with MPP+
(200 lmol/L), and the mitochondrial mem-
brane potential was measured. (b) Pre-
treatment with IPT (10 lmol/L) or diazoxide
(DZ; 100 lmol/L) for 30 min prior to MPP+
(200 lmol/L) exposure for 6 h suppressed
MPP+-induced mitochondrial depolariza-
tion. And 5-hydroxydecanoate (5-HD;
250 lmol/L) blocked these effects. (c)
Western blot analysis of mitochondrial AIF
and cytochrome c level. Astrocytes were
exposed to MPP+ (200 lmol/L) for various
periods of time up to 24 h. *p £ 0.05, com-
pared with AIF control groups. #p £ 0.05;
##
p £ 0.01, compared with cytochrome c
control groups. (d) IPT (10 lmol/L) or DZ
(100 lmol/L) inhibited MPP+-induced de-
crease of cytochrome c and AIF levels in
astrocytic mitochondria. Western blots at
the top of each panel are from a typical
experiment. Bar graphs are the quantified
results expressed as mean ± SEM of rel-
ative cytochrome c or AIF levels from four
independent experiments. Results are rep-
resentative of three separate experiments
and each bar indicates the mean ± SEM.
*p £ 0.05; **p £ 0.01, compared with un-
treated group. #p £ 0.05; ##p £ 0.01, com-
pared with MPP+-treated group. +p £ 0.05,
compared with both IPT and MPP+-treated
group. $p £ 0.05, compared with both DZ
and MPP+-treated group.
astrocytes exposed to KCOs and /or MPP+. Enhanced level
of phospho-ERK1/2 was detected 15 min after exposure to
IPT (10 lmol/L) or DZ (100 lmol/L), which sustained for at
least 4 h (Fig. 5b). Exposure of astrocytes to MPP+ alone
also increased phospho-ERK1/2 level within 15 min, but it
subsided to control level at 2 h and decreased to below
control level at 4 h (Fig. 5c). However, both IPT and DZ
could maintain MPP+-induced increase of phospho-ERK1/2
levels for at least 4 h, which was completely abolished by the
ERK1/2 inhibitor PD98059 (Fig. 5d). These results show

(a)
(b)
Fig. 4 Iptakalim (IPT) or diazoxide (DZ) inhibits MPP+-induced c-Jun
NH2-terminal kinase (JNK) phosphorylation in astrocytes. (a) Astro-
cytes were exposed to MPP+ (200 lmol/L) for the indicated times. (b)
Astrocytes were pretreated with IPT (10 lmol/L) or DZ (100 lmol/L)
for 30 min and then exposed to MPP+ (200 lmol/L) for another
30 min. Western blots at the top of each panel are from a typical
experiment. Bar graphs are the quantified results expressed as
mean ± SEM of relative phospho-JNK levels from four independent
experiments. *p £ 0.05; **p £ 0.01, compared with untreated group.
#
p £ 0.01, compared with MPP+-treated group. +p £ 0.05, compared
with both IPT and MPP+-treated group. $p £ 0.05, compared with both
DZ and MPP+-treated group.
that IPT inhibits MPP+-induced astrocytic apoptosis via
activating ERK/MAPK signal pathway.
Discussion
Although much of the work on neuroprotection has focused
on improving the survival of neurons, a prominent concom-
itant effect of brain insults is the damage of glia, in particular
astrocytes. Increasing evidence implies that astrocytic apop-
tosis participates in many neurodegenerative disorders
(Benjelloun et al. 1998; Franz et al. 2002; Szydlowska et al.
2006), and astrocytic dysfunction may further contribute to
neuronal loss and the overall pathology (Seifert et al. 2006).
Inhibition of astrocytic apoptosis is an essential neuropro-
tective strategy (Takuma et al. 2004). Therefore, the drugs
that can inhibit astrocytic apoptosis may offer protective
effects on neurodegenerative disorders such as PD.
 2007 The Authors
Journal Compilation  2007 International Society for Neurochemistry, J. Neurochem. (2007) 103, 569–579
Iptakalim inhibits astrocytic apoptosis 575
The present study showed that, as a novel KCO, IPT
possessed similar protective effects to classic mitoKCO DZ
on MPP+-induced astrocytic apoptosis in vitro. And this anti-
apoptotic effect of IPT could be abolished by selective
mitoKATP channel blocker 5-HD. The protective effect of IPT
on MPP+-induced astrocytic apoptosis was further confirmed
by flow cytometry analysis. However, sarcolemmal KCO
P1075 failed to protect against MPP+-induced astrocytic
apoptosis. These findings suggest that IPT inhibit astrocytic
apoptosis through opening mitoKATP channels in astrocytes.
KCOs, especial mitochondria KCOs, can provide protective
effects for neurons and neuroblast against cell damage
induced by ischemia, trauma, and toxic reagents (such as
rotenone and MPP+) (Hu et al. 2005; Nakagawa et al. 2005;
Yang et al. 2006a). Moreover, Bajgar demonstrated that the
amount of mitoKATP channels located in brain cells is at least
six fold higher than that in heart cells (Bajgar et al. 2001),
indicating that mitoKATP channels play essential role in the
functions of central nervous system. Thus, mitoKATP chan-
nels may be an important molecular target for the regulation
of astrocytic functions. It was found that IPT exhibited
marked neuroprotective effect on rotenone-induced PD
model (Yang et al. 2005, 2006b). However, opening the
KATP channels, which were composed of Kir6.2 and SUR1
subunits, in dopamine neurons exhibited an unexpected role
in promoting neurodegenerative process of PD (Deutch and
Winder 2006; Liss et al. 2005). Our previous study found
that IPT failed to open KATP channels in rat substantia nigra
pars compacta DA neurons (Wu et al. 2006). Thus, IPT may
offer protective effects on PD model through opening KATP
channels composed of Kir6.1 and SUR1/SUR2 subunits in
astrocytes (Thomzig et al. 2001).
The involvement of oxidative stress in MPP+-induced
dopaminergic neuronal apoptosis, as demonstrated by an
increase in the product of reactive oxygen species and/or a
decrease in antioxidants, has been described (Chun et al.
2001; Kaul et al. 2003). And it has been demonstrated that
oxidative stress might originate in astrocytes rather than in
neurons (Makarov et al. 2006; Sagara et al. 1993). However,
whether a similar cytotoxic effect of MPP+ occurs in
astrocytes is unclear. The present study showed that MPP+
could decrease endogenous glutathione level in astrocytes.
Glutathione, the major antioxidant in cells, has been shown
to be neuroprotective both in vivo and in vitro (Cho et al.
2003). Astrocytes surrounding dopaminergic neurons in the
brain may have a close relation with the selective vulnerab-
ility of these neurons by scavenging reactive oxygen species
and releasing CysGly, which is the precursor of GSH
synthesis in neurons. Hence, alteration in astrocytic gluta-
thione level may be an important contributor to the
pathogenesis of neurodegenerative disease such as PD
(Jenner and Olanow 1998). The results of present study
reveal that treatment with IPT alone could significantly
increase the endogenous glutathione level in astrocytes. IPT
576 S. Zhang et al.
(a)
(b)
(c)
also inhibited the glutathione depletion induced by MPP+,
which was abolished by mitoKATP channel blocker 5-HD.
We found that both IPT and DZ could increase astrocytic
glutamate uptake (Zhang and Hu 2004). And MPP+ inhibited
glutamate uptake and subsequently increased extracellular
glutamate levels in astrocytes, which was reversed by IPT
(Hu et al. 2005). MPP+ could also decrease glutathione level
in astrocytes (McNaught and Jenner 2000). As glutamate is a
precursor for glutathione, the inhibitory effect of MPP+ on
glutamate uptake may lead to the reduction of glutathione
level in astrocytes. Thus, we inferred that IPT-induced the
increase of glutamate uptake could result in enhanced
production of glutathione. In addition, the current explan-
ation for glutamate-mediated toxicity is due to oxidative
glutamate toxicity besides excitotoxicity. This mechanism
involves inhibition of cystine transport by the cystine-
 2007 The Authors
Journal Compilation  2007 International Society for Neurochemistry, J. Neurochem. (2007) 103, 569–579
Fig. 5 Extracellular signal-regulated kinase
(ERK) pathway mediates the anti-apoptotic
effect of iptakalim (IPT) on astrocytes. (a)
Pretreatment with PD98059 (50 lmol/L) for
30 min inhibited the anti-apoptotic effects of
IPT (10 lmol/L) or diazoxide (DZ;
100 lmol/L) on astrocytes. (b) Western blot
analysis of astrocytes stained for phospho-
ERK showed that IPT (10 lmol/L) or DZ
(100 lmol/L) quickly induced a sustained
activation of ERK1/2, lasting at least 4 h. (c)
MPP+ (200 lmol/L) alone activated ERK1/2
for only the first 2 h after exposure. (d) IPT
(10 lmol/L) or DZ (100 lmol/L) maintained
the increased phospho-ERK1/2 levels for at
least 4 h after MPP+ exposure, and this
effect was abolished by PD98059 (50 lmol/
L). The data presented as mean ± SEM of
four individual experiments. *p £ 0.05;
**p £ 0.01, compared with untreated group.
#
p £ 0.05; ##p £ 0.01, compared with
MPP+-treated group. +p £ 0.01, compared
with both IPT and MPP+ treated group.
$
p £ 0.01, compared with both DZ and
MPP+ treated group.
glutamate anti-porter Xc-, leading to glutathione depletion
(Cho and Bannai 1990). Because IPT and MPP+ share a
common mechanism at regulating astrocytic glutamate
uptake, we proposed that IPT might reverse the inhibitory
effect of MPP+ on glutathione level through enhancing the
glutamate uptake, reducing extracellular glutamate levels and
subsequently alleviating the inhibitory effect on cystine-
glutamate anti-porter in astrocytes. As glutathione partici-
pates in the regulation of apoptosis (Hall 1999), IPT may
prevent astrocytic apoptosis through increasing intracellular
glutathione level.
Furthermore, the anti-apoptotic mechanisms of IPT were
explored. GSH depletion is directly responsible for the noted
decreases in complex I activity and the subsequent mito-
chondria dysfunction (Jha et al. 2000). Mitochondrial dys-
function is a prominent feature in apoptosis, and release of

pro-apoptotic proteins (e.g., cytochrome c, an apoptosis-
inducing factor, and Endo G etc) from the mitochondrial
intermembrane space has been considered to be a critical
event that occurs during apoptosis (Green and Reed 1998).
Both cytochrome c and AIF are important for cell viability
when they are located in mitochondria, but when either is
released from the mitochondria, it activates death programs.
Mitochondrial release of cytochrome c into the cytoplasm
induces the formation of an oligomeric complex containing
cytochrome c and Apaf-1. This complex, called the apopto-
some, supports the catalytic activation of caspase-9, which
further cleaves and activates the effector caspase-3 resulting
in the subsequent degradation of cellular death substrates.
Thus, release of cytochrome c is a key step in the initiation of
caspase-dependent apoptosis (Li et al. 1997). AIF induces
caspase-independent cell death primarily. Following AIF
translocation from the mitochondria to the nucleus, classic
apoptotic features, such as phosphatidylserine exposure,
partial chromatin condensation and nuclear condensation,
occur in the absence of caspase activation. AIF appears to
play an important role in the acute neurotoxicity induced by
trauma, hypoglycemia, transient ischemia, and chronic
neurodegenerative diseases (Zhang et al. 2002; Zhu et al.
2003; Wang et al. 2003). Therefore, therapeutic strategies
targeting both caspase-dependent and independent pathways
may be more protective against some toxic insults. The
present study demonstrated that IPT significantly inhibited
MPP+-induced loss of DYm and subsequent release of pro-
apoptotic protein (cytochrome c and AIF) from mitochondria
in primary cultured astrocytes. These findings suggest that
IPT may protect against astrocytic apoptosis by inhibiting
caspase-dependent and independent apoptosis pathways.
JNK is stress-activated MAPK and has been implicated in
various stress-mediated apoptosis such as nerve growth
factor withdrawal, excitotoxic stress and oxidative stress
(Davis 2000; Dickens et al. 1997). And the level of phospho-
JNK also increases in substantia nigra of MPTP-treated mice
(Saporito et al. 2000). In present study, treatment with MPP+
led to a rapid and transient phosphorylation of JNK,
indicating that JNK signaling pathway is activated in
response to MPP+ treatment in astrocytes. And IPT could
suppress MPP+-induced increase of phospho-JNK level,
which was also abolished by mitoKATP channel blocker 5-
HD. Thus, the results in the present study demonstrate that
IPT may exert anti-apoptotic effect via suppressing mito-
chondria and JNK/MAPK apoptotic pathways.
Potential drugs that prevent apoptosis are mainly divided
into two groups: inhibitors of apoptotic signal pathways and
stimulators of survival signal pathways (Takuma et al. 2004).
The ERK/MAPK signal pathway is required for survival
signaling in response to growth factors in non-cardiomyo-
cytic cells (Parrizas et al. 1997). However, several recent
studies have suggested that the ERK/MAPK signal pathway
participates in the regulation of cell apoptosis (De Girolamo
 2007 The Authors
Journal Compilation  2007 International Society for Neurochemistry, J. Neurochem. (2007) 103, 569–579
Iptakalim inhibits astrocytic apoptosis 577
and Billett 2006; Yue et al. 2000). To demonstrate the role
of ERK/MAPK signal pathway in the protect effect of IPT
on MPP+-induced astrocytic apoptosis, ERK1/2 inhibitor
PD98059 was used in this study. Inhibition of ERK/MAPK
pathway by PD98059 failed to induce astrocytic apoptosis in
the normal condition, suggesting that this pathway does not
play a major role in astrocytic survival. However, PD98059
could inhibit the anti-apoptotic effect of IPT. Western
blotting analyses demonstrated that IPT could activate
ERK/MAPK and maintain increased phospho-ERK1/2 level
after MPP+ exposure, suggesting that ERK/MAPK pathway
may be the downstream signal pathway of mitoKATP
channels in astrocytes. These results demonstrate that IPT
can protect against MPP+-induced astrocytic apoptosis
through activating ERK/MAPK survival signal pathway.
Our results also showed that the selective mitoKCO DZ
had the similar anti-apoptotic effects to IPT. DZ was
originally developed as an antihypertensive agent, but was
found to induce hyperglycemia by reducing insulin secretion.
The clinical use of DZ has been hampered by its lack of
potency and selectivity giving rise to side effects (Hansen
2006). In contrast, IPT possesses several advantages, such as
free penetration through the blood-brain barrier and low-
toxic side-effects during systemic administration (Wu et al.
2005). Therefore, IPT is a promising neuroprotectants for
treating neurodegenerative disease.
In conclusion, the present study reveals for the first time
that IPT protects against MPP+-induced astrocytic apoptosis
via inhibition of mitochondria apoptotic pathway and regu-
lating the MAPK signal transduction pathways (such as JNK,
EERK/MAPK) by opening mitoKATP channels in astrocytes.
These findings indicate that IPT, a novel and blood-brain
barrier permeable KCO, is a promising anti-apoptotic agent
for protection of astrocytes. And targeting mitoKATP channels
expressed in astrocytes may offer a novel therapeutic strategy
for neurodegenerative diseases such as PD.
Acknowledgements
These studies were supported in part by grants from the National
Natural Science Foundation of China (No.30625038 and No.
30572172), the Key Project of Natural Science Foundation of
Jiangsu Educational Department (No. 05KJA31014 and No.
06KJA31029), the Key Project of Jiangsu Health Department
(No.K200501), and Specialized Research Fund for the Doctoral
Program of Higher Education of China (No. 20040312004).
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