Mitochondrial-dependent mechanisms are involved in angiotensin II-induced apoptosis in dopaminergic neurons
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Mitochondrial-dependent mechanisms are involved in angiotensin II-induced apoptosis in dopaminergic neurons
Zhou Ou 0
Teng Jiang 0
Qing Gao 0
You-Yong Tian 0
Jun-Shan Zhou 0
Liang Wu 0
Jian-Quan Shi 0
Ying-Dong Zhang 0
0 Department of Neurology, Nanjing First Hospital , PR China
Introduction: We recently demonstrated that angiotensin II (Ang II) was involved in the etiology of Parkinson's disease (PD) via induction of apoptosis of dopaminergic neurons, but the mechanisms are not completely elucidated. Here, we asked whether mitochondrial-dependent mechanisms contributed to the Ang II-induced dopaminergic neuronal apoptosis. Materials and methods: CATH.a cells were incubated with Ang II in combination with mitochondrial permeability transition pore (mPTP) inhibitors or angiotensin receptor antagonists, and apoptosis rate, caspase-3 activity, cytochrome c levels, and mPTP opening were assessed. Results: We showed that Ang II triggered apoptosis via a mitochondrial-dependent pathway, as elevated cytochrome c levels were observed in the cytosol. By employing cyclosporin A and sanglifehrin A, two specific mPTP inhibitors, we revealed that cytochrome c release from mitochondria into cytoplasm was ascribed to mPTP opening. Meanwhile, the aforementioned effects could be abrogated by an AT1 receptor antagonist losartan rather than an AT2 receptor antagonist PD123319. Conclusion: This study demonstrates that Ang II triggers mitochondrial-dependent apoptosis via facilitating mPTP opening through an AT1 receptor-mediated fashion in dopaminergic neurons. These findings give insight into the effect of Ang II in the etiology of PD, and reinforce the application of AT1 receptor antagonists for PD treatment.
Parkinson's disease; renin-angiotensin system; angiotensin II; apoptosis; mitochondrial permeability transition pore; cytochrome C
As the most common type of neurodegenerative
movement disorder, Parkinson’s disease (PD) is characterized
by evident motor symptoms such as rigidity,
bradykinesia, resting tremor, and postural instability.1 The reduction
of dopamine levels within the striatum and substantia
nigra is a well-known feature of PD, which is attributed to
selective dopaminergic neuron loss within basal ganglia
structures and is closely related to the progression of the
aforementioned motor symptoms.2 To date, the molecular
mechanisms underlying the loss of dopaminergic neurons
are still unclear.
In the circulation system, the renin-angiotensin system
(RAS) serves as a critical regulator in the homeostasis of
water and sodium as well as blood pressure through its
major effector, angiotensin II (Ang II). Recently, mounting
evidence has suggested that an independent RAS exists in
most parts of brain,3–5 and is closely related to the etiology
of several neurodegenerative diseases, such as PD.6,7 An
increased level of Ang II within the striatum and substantia
nigra of PD rodent models has been noted, suggesting a
*Zhou Ou, Teng Jiang and Qing Gao are co-first authors.
hyperactivation of brain RAS in PD etiology.8 In addition,
recent studies from our laboratory demonstrated that Ang
II could directly cause dopaminergic neuron loss via
triggering apoptosis,9,10 a specific type of programmed cell
death. Nevertheless, the potential mechanisms which
underlie Ang II-induced apoptosis in dopaminergic
neurons remained largely unexplored.
Apoptosis is closely modulated by two different
signaling pathways: the receptor-dependent apoptotic pathway
and the mitochondrial-mediated apoptotic pathway.11
During the mitochondrial-dependent apoptotic process,
the mitochondrial permeability transition pore (mPTP)
was open in response to the stimulation of pro-apoptotic
factors, and cytochrome c was released from the
mitochondria into the cytosol, subsequently triggering the
downstream apoptosis cascade.12 Interestingly, emerging
evidence suggests that Ang II initiated apoptosis via a
mitochondrial-dependent fashion in peripheral tissues
including lung13 and heart.14 Based on this information, we
hypothesized that Ang II may also trigger the apoptosis of
dopaminergic neurons through a mitochondrial-dependent
mechanism in the central nervous system. In the present
study, we attempted to verify this hypothesis using a
dopaminergic neuronal cell line (CATH.a cells).
Materials and methods
Reagents and cell culture
Ang II, cyclosporin A (CsA, a specific mPTP inhibitor),
sanglifehrin A (SfA, another specific mPTP inhibitor),
losartan (an Ang II type 1 (AT1) receptor antagonist) and
PD123319 (an Ang II type 2 (AT2) receptor antagonist)
were purchased from Sigma-Aldrich Inc. Mouse CATH.a
cells were provided by American Tissue Cell Collection,
and maintained in Roswell Park Memorial Institute
(RPMI) 1640 medium containing 10% fetal bovine serum,
and 1% penicillin-streptomycin at 37°C with 5% CO2
according to the methods described previously.9,10 This
dopaminergic neuronal cell line stably expresses AT1 and
AT2 receptors, and the density ratio of AT1 to AT2 receptor
is about 2.07:1.
Flow cytometry analysis
Quantitative evaluation of cell apoptosis was performed
using a flow cytometer with a fluorescein isothiocyanate
(FITC) Annexin V and propidium iodide (PI) Double
Labeling Apoptosis Detection Kit (BD Biosciences)
according to previously reported procedure.9,10,15 Briefly,
cultured CATH.a cells were subjected to different
treatments, and washed with phosphate-buffered saline (PBS).
Trypsin was then utilized until the cells detached, and
4°C PBS was adopted to scatter the cells before
centrifugation. Afterwards, cells were collected and stained with
FITC Annexin V solution and/or PI solution at 37°C in the
dark for 15 min. Following that step, the fluorescence of
10,000 cells was measured with a FACSCalibur System
(BD Biosciences) within 1 h. Opening of mPTP in CATH.a
cells was measured with flow cytometric analysis
following the CoCl2-calcein fluorescence quenching assay.16 In
brief, after co-incubation of cells with Calcein-AM (1 μM,
Molecular Probes, Life Technologies) for 30 min at 37°C
in the dark, CoCl2 (1 mM, Sigma) was added and cells
incubated for additional 10 min. Afterwards, the
fluorescence of 10,000 cells was evaluated with a flow cytometer
(FACSCalibur) at the excitation wavelength of 530 nm.
The data of above-mentioned two assay were processed
with FSC express software (De Novo Software).
Colorimetric assay for caspase-3 activity
Colorimetric assay was employed to evaluate the
caspase-3 activity in CATH.a cells as described elsewhere.17
Briefly, cultured CATH.a cells were subjected to indicated
treatment. Then, cells were washed, harvested and lysed in
extraction buffer. The caspase-3 activity was assessed
according to the instructions of manufacturer for a
colorimetric assay kit (Abcam).
Assessment of cytosolic cytochrome c levels
Cytochrome c levels in the cytosolic fractions were
measured as described by Li and colleagues.18 In brief,
cultured CATH.a cells were subjected to indicated treatment.
Following that step, cells were collected and homogenized
in a buffer containing 20 mM hydroxyethylpiperazine
ethane sulfonic acid (HEPES)-KOH (pH 7.0), 250 mM
sucrose, 10 mM KCl, 2 mM MgCl2, 1 mM ethylene
glycol tetraacetic acid (EGTA), 1 mM ethylene diamine
tetraacetic acid (EDTA), 1 mM dithiothreitol (DTT),
1 mM phenylmethyl sulphonyl fluoride (PMSF) and
the protease inhibitors cocktail. Nuclei were removed
through centrifugation at 1000× g for 10 min at 4°C. Post
nuclear supernatant was centrifuged at 100,000× g for
1 h, and the supernatant (cytosolic fraction) were then
collected. Cytochrome c levels were assessed with an
enzyme-linked immunosorbent assay (ELISA) kit (R&D
CoCl2-calcein fluorescence quenching assay
The opening of mPTP was detected with a CoCl2-calcein
fluorescence quenching assay.16 Under physiological
conditions, calcein-acetomethoxy (AM) can freely cross cellular
membranes, and the cytosolic esterase cleave the AM group
to yield the fluorescent calcein. Co-incubation of cells with
CoCl2 quenches the fluorescence in the cells, except in
mitochondria, since CoCl2 cannot pass through
mitochondrial membrane. However, calcein in mitochondria is also
quenched by CoCl2 once the mPTP opening, leading to
reduced fluorescence. Images of cells were taken at 488 nm
excitation and 525 nm emissions according to
Data were expressed as mean±standard deviation (SD)
Comparisons among groups were carried out with
oneway analysis of variance (ANOVA) followed by Turkey’s
post-hoc test using SPSS software. Values of p<0.05 were
considered to be statistically significant.
Ang II induces CATH.a cells apoptosis via a
Firstly, we tested the actions of Ang II on CATH.a cell
apoptosis. CATH.a cells were exposed to various concentrations
of Ang II (5 nM, 50 nM and 500 nM) for 24 h. Afterwards,
flow cytometry was applied to assess the percentage of
apoptotic CATH.a cells. When compared with vehicle, 50
nM and 500 nM Ang II elevated the percentage of apoptotic
cells from 3.0% to 16.6% and 22.8% (p<0.05), respectively
(Figure 1(a) and (b)). To confirm the association of Ang II
with apoptosis, we further detected activity of the caspase-3,
the key apoptosis executioner,19 by means of colorimetric
assay. Figure 1(c) shows that 50 nM and 500 nM Ang II
markedly increased the caspase-3 activity by 2.9-fold and
4.6-fold (p<0.05), respectively. Mitochondrial release of
apoptogenic cytochrome c is an indispensable step to
initiate mitochondria-dependent apoptosis. To determine
whether the mitochondria-dependent pathway participated
in the apoptosis caused by Ang II, the cytosolic levels of
cytochrome c was then detected using an ELISA kit. Figure
1(d) reveals that the cytochrome c levels in 50 nM and 500
nM Ang II-treated groups was higher than the
vehicletreated group by 1.7-fold and 2.5-fold (p<0.05),
respectively. These findings indicated that Ang II induces CATH.a
cells apoptosis via a mitochondria-dependent pathway.
Opening of mPTP is involved in the
mitochondria-dependent apoptosis induced by
Ang II in CATH.a cells
To investigate possible mechanisms underlying the
mitochondria-dependent apoptosis triggered by Ang II,
we then focused on the mPTP opening. The mPTP
opening in CATH.a cells after Ang II treatment was measured
with a CoCl2-calcein fluorescence quenching assay. As
shown by Figure 2(a) and (b), 50 nM and 500 nM Ang II
significantly reduced the signal of calcein fluorescence
in CATH.a cells by 34.9% and 60.4% (p<0.05),
respectively, indicating an increase in mPTP opening. This
result was further confirmed using flow cytometry
During mitochondrial apoptotic cascade, the opening
of mPTP is believed to cause apoptosis via facilitating
cytochrome c release. To determine whether the opening
of mPTP led to the release of cytochrome c and
subsequent cell apoptosis in this scenario, we co-incubated
CATH.a cells with Ang II (50 nM) and mPTP inhibitor
CsA (20 μ M) or SfA (1 μ M), for 24 h. As shown by Figure
3(a), the reduction of calcein fluorescence signal caused
by Ang II in CATH.a cells was fully reversed by CsA or
SfA, indicating that the mPTP opening was markedly
inhibited. Meanwhile, the increase in cytochrome c levels
induced by Ang II was abolished by CsA or SfA,
suggesting that mitochondrial release of cytochrome c was also
attenuated by inhibition of mPTP opening (Figure 3(b)).
In addition, inhibiting mPTP rescued the Ang II-induced
apoptosis in CATH.a cells, as the elevation in apoptosis
rate and caspase-3 activity were remarkably attenuated by
CsA or SfA (Figure 3(c) and (d)). Taken together, these
results suggested that opening of mPTP participated in the
mitochondria-dependent apoptosis triggered by Ang II in
AT1 receptor is implicated in the mPTP opening
and mitochondria-dependent apoptosis caused
by Ang II
To ascertain which type of receptors mediated the
aforementioned actions of Ang II, we co-treated CATH.a cells
with 50 nM Ang II and 1 μ M losartan or 1 μ M PD123319
for 24 h. The dose for PD123319 (1 μ M) was selected
according our previous findings that PD123319 at this
dose could effectively block most of the effects mediated
by the AT2 receptor in CATH.a cells.15 Figure 4(a) reveals
that co-incubation with losartan completely reversed the
Ang II-induced decline of calcein fluorescence signal in
CATH.a cells, indicating that the mPTP opening was
significantly inhibited. Meanwhile, the increase in cytosolic
cytochrome c levels induced by Ang II was blunted by
losartan, suggesting that mitochondrial release of
cytochrome c was also ameliorated (Figure 4(b)).
Moreover, losartan rescued the Ang II-induced apoptosis
in CATH.a cells, since the elevation in apoptosis rate and
caspase-3 activity were remarkably attenuated (Figure 4(c)
and (d)). As opposed to losartan, no significant effect of
PD123319 on Ang II-induced increase in mPTP opening
and the subsequent release of cytochrome c was noted,
since the calcein fluorescence signal as well as the
cytosolic cytochrome c levels in CATH.a cells was unaffected
by PD123319 co-treatment. In addition, PD123319 cannot
Figure 1. Angiotensin II (Ang II) induces apoptosis in CATH.a cells via a mitochondria-dependent pathway. CATH.a cells were
treated with different concentrations of Ang II (5, 50, and 500 nM) for 24 h. In (a) and (b) cell apoptosis was measured by flow
cytometry using a commercial apoptosis detection kit. The apoptosis rate=(annexin V+PI+ cells+annexin V+PI− cells)/total cells×100
%. (c) The activity of caspase-3 was directly evaluated using a colorimetric assay kit. (d) ELISA was performed with a commercial
ELISA kit to valuate cytosolic cytochrome c. All figures are representative of three independent experiments, performed in
triplicate. Data were analyzed by one-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test. Columns represent
mean±standard deviation (SD). *p<0.05 versus control group.
ELISA: enzyme-linked immunosorbent assay; FITC: fluorescein isothiocyanate; PI: propidium iodide.
ameliorate the apoptosis caused by Ang II, since the
increase in apoptosis rate as well as the caspase-3 activity
stayed unchanged following PD123319 co-treatment in
CATH.a cells. Taken together, these results highlighted
that AT1 receptor is closely relevant to the mPTP opening
and mitochondria-dependent apoptosis caused by Ang II.
Herein, we demonstrated that Ang II triggered CATH.a cells
apoptosis, as flow cytometry analysis revealed that the cell
apoptosis rate was significantly elevated after Ang II
incubation. Moreover, this was further supported by an increased
caspase-3 activity, the key apoptosis executioner,19 in the
cytoplasm of Ang II-treated CATH.a cells. These results
were consistent with our recent observations that Ang II
caused apoptosis in dopaminergic neurons.9,10
There has been wide agreement that apoptosis is
closely regulated by two different signaling pathways:
the receptor-dependent apoptotic pathway and the
mitochondrial-mediated apoptotic pathway.11 During the
mitochondrial-dependent apoptotic process, cytochrome c
is released from mitochondria into cytosol in response to
the stimulation of pro-apoptotic factors, subsequently
triggering the downstream apoptosis cascade.20 In this study,
we showed that the level of cytochrome c in cytosol of
CATH.a cells was significantly elevated after Ang II
administration, suggesting that Ang II initiated apoptosis
via a mitochondrial-mediated fashion. This was in
according with prior findings revealing that Ang II facilitated
cytochrome c release and thus triggered
mitochondrialdependent apoptosis in primary lung endothelial cells,13
atrial myocytes21 as well as mouse calvaria osteoblast.18
mPTP is comprised of the matrix protein cyclophilin D
(CypD), the inner mitochondrial membrane protein adenine
nucleotide translocator, and the outer mitochondrial
membrane protein voltage dependent anion channel.22 During
the process of mitochondria-dependent apoptosis, the
opening of mPTP resulted in the release of several pro-apoptotic
factors, such as cytochrome c, from the mitochondria into
the cytosol.12 Based on this information, we hypothesized
that the cytochrome c release in this scenario might be
ascribed to the opening of mPTP caused by Ang II. To test
this hypothesis, a specific mPTP inhibitor CsA was used in
our experiment. As expected, inhibition of mPTP by CsA
attenuated cytochrome c release and ameliorated cell
apoptosis caused by Ang II. Meanwhile, this result was
confirmed by SfA, another specific mPTP inhibitor, further
indicating that the opening of mPTP was involved in the
cytochrome c release as well as the subsequent apoptosis
caused by Ang II. This was compatible with previous
findings from Ricci and colleagues, which revealed that Ang II
facilitated the release of cytochrome c via opening of mPTP
and subsequently triggered apoptosis in isolated neonatal
Moreover, we tried to investigate which type of
receptors mediated the aforementioned effects of Ang II. We
showed that the opening of mPTP, release of cytochrome c
and subsequent apoptosis in Ang II-treated CATH.a cells
were completely abolished by losartan, an AT1 receptor
blocker. On the contrary, an AT2 receptor blocker
PD123319 had no impact on these actions triggered by
Ang II. Therefore, we ascertained that Ang II induced
mitochondrial-mediated apoptosis was dependent on AT1
receptor in this scenario. Our findings were consistent with
a previous study from Zhao and colleagues, which showed
that Ang II triggered mitochondrial-dependent apoptosis in
cardiomyocytes by interacting with the AT1 receptor.14
However, in contrast to our findings, Lv and colleagues
reported that Ang II induced apoptosis in cardiomyocytes
Herein, we reveal that Ang II facilitates the release of
cytochrome c by opening of mPTP and thus triggers
mitochondria-dependent apoptosis in CATH.a cells. We also
show that the aforementioned impacts of Ang II on CATH.a
cells are dependent on AT1 receptors. These results give
more insight into the effects of Ang II in the etiology of
PD, and reinforce the application of AT1 receptor blockers
for the therapies of this neurodegenerative disease. In
future, animal models of PD should be employed to
validate these findings, and further studies are warranted to
investigate other potential mechanisms involved in Ang
II-induced apoptosis of dopaminergic neurons.
Figure 4. The mitochondrial-dependent apoptosis induced by angiotensin II (Ang II) is mediated by AT1 receptor instead of AT2
receptor in CATH.a cells. CATH.a cells were co-incubated with Ang II (50 nM), an AT1 receptor antagonist losartan (1 µM), and
an AT2 receptor antagonist PD123319 (1 µM) for 24 h, and (a) the calcein fluorescence was evaluated by flow cytometric analysis.
(b) ELISA was performed using a commercial ELISA kit to valuate cytosolic cytochrome c in CATH.a cells. (c) Cell apoptosis
was measured by flow cytometry using a commercial apoptosis detection kit. (d) The activity of caspase-3 was measured by a
commercial detection kit. All figures are representative of three independent experiments, performed in triplicate. Data were
analyzed by one-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test. Columns represent mean±standard
deviation (SD). *p<0.05 versus control group, #p<0.05 versus Ang II-treated group. AT1: Ang II type 1; AT2: Ang II type 2;
ELISA: enzyme-linked immunosorbent assay.
Declaration of conflicting interests
The author(s) disclosed receipt of the following financial support
for the research, authorship, and/or publication of this article:
This work was supported by National Natural Science Foundation
of China (81271418) and Natural Science Foundation of Jiangsu
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