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Introduction
Acid-sensing ionic channels (ASIC) are
H+-gated cation channels, which belong to the degenerin/epithelial sodium
channel superfamily. They are activated quickly as the extracellular pH falls. Until now, 6 ASIC subunits have been
cloned: ASIC1a[1], ASIC1b[2],
ASIC2a[3], ASIC2b[4],
ASIC3[1,2,5], and ASIC4[6]. Functional ASIC are usually tetrameric assemblies of these
ASIC subunits in homomeric or heteromeric
conformation[7].
Different ASIC subunits have different kinetics and biological characteristics. Both the ASIC1a and ASIC2a subunits
have been shown to be abundant in the brain, whereas ASIC2b is present in both brain and sensory
neurons[8,9]. In the rat hippocampal neurons, our studies showed that these 3 subunits could form the function of ASIC
[10]. ASIC4 was found to coexpress with other subunits in many
areas of the brain. ASIC3 is expressed almost exclusively in the sensory neurons in
rats[11]. Different from other subtypes, homomeric ASIC1a can flux
Ca2+ besides Na+ permeability
[12].
Until now, the functions of ASIC were still unclear,
although they were related in acidosis-induced injury. In sensory neurons, ASIC were shown to play an important role in
nociception during tissue acidosis, for instance, in muscle and cardiac
ischemia[13] and in
inflammation[14]. In peripheral sensory neurons, ASIC were implicated in
mechanosensation[15] and the perception of pain during tissue acidosis,
particularly in the ischemic myocardium where ASIC are likely
transducer of anginal pain[16_19]. The presence of ASIC in the
brain, which lacks nociceptors, suggests that these
channels have functions beyond nociception. Now, it has shown
that ASIC1a is involved in synaptic plasticity,
learning/memory, and fear
conditioning[20,21] and participates in
neuronal damage associated with tissue
acidosis[22].
ASIC have been found in malignant
gliomas[23]. Recent studies have revealed that the surface expression of ASIC2
could inhibit the amiloride-sensitive current and migration
of glioma cells[24]. In our former
studies[25], we also found that extracellular acid induced cell death and apoptosis in rat
C6 glioma cells via the ASIC1a mechanism, and reducing
ASIC1a proteins by the stable expression of short hairpin
RNA inhibited apoptosis and increased cell viability
following acid exposure. These studies indicated that ASIC might
have some important functions in the gliomas within acidosis.
However, the underlying mechanism of ASIC1a involved in
acidosis-induced cytotoxicity in rat C6 glioma cells is still
unknown. Due to the function of intracellular calcium
([Ca2+]i) and the high
Ca2+ permeability of ASIC1a, we aimed to explore the relationship between
Ca2+ and ASIC1a during acidosis combined with the newly-developed RNA
interference (RNAi) technique and the calcium imaging experiment
in the present study.
Materials and methods
Materials Fluo-3 acetoxymethyl ester (Fluo-3-AM) was
purchased from Molecular Probes (Eugene, OR, USA).
Anti-ASIC1a was purchased from Cell Signaling Technology
(Beverly, MA, USA). G418, penicillin, L-glutamine,
nimodi-pine, MK801 (dizocilpine maleate),
6-cyano-7-nitroquino-xaline-2,3-dione (CNQX), and all other reagents were
purchased from Sigma (St Louis, MO, USA) unless otherwise
indicated.
Cell culture The rat glioma C6 cell line was derived from
rat neurogliocytoma. The wild-type C6 cells were cultured in
RPMI-1640 medium (Invitrogen, Carlsbad, CA, USA)
supplemented with 10% fetal bovine serum (Invitrogen, USA), 100
units/mL penicillin, 100 µg/mL streptomycin,
L-glutamine
(0.03%, w/v), and sodium bicarbonate (2.2%,
w/v). The ASIC1a-silenced cells and the negative control cells were
cultured with G418 (400 mg/L). The cells were cultured at
37 °C and 5% CO2 in a humidified incubator (310/Thermo,
Forma Scientific, Marietta, OH, USA).
Building the stable ASIC1a-silenced cell line
To observe the role of
[Ca2+]i in the acidosis and its relationship
with ASIC1a, we built the ASIC1a-silenced C6 cell line. We
designed and prepared the shRNA sequences directly against
ASIC1a, and the control sequence was designed according
to Dharmacon siDESIGN Center software (Dharmacon, Lafayette, CO, USA). The sequence for ASIC1a RNAi was:
5'-GAT CCC GCGTGAATTCTACGACAGATTCAAGAGA-TCTGTCGTAGAATTCACGCTTTTT-3' (forward) and
5'-AGCTAAAAAGCGTGAATTCTACGACAGATCTCTTGAATCT-GTCGTAGAATTCACGCGG-3' (reverse); the sequence for
the control RNAi was: 5'-GATCCCTTCTCCGAACGTGTCA-CGTTTCAAGAGAACGTGACACGTTCGGAGAATTTTT-3'
(forward) and 5'-AGCTAAAAATTCTCCGAACGTGTCA-CGTTCTCTTGAAACGTGACACGTTCGGAGAAGG-3'
(reverse). The shRNA were synthesized in desalted and
purified form by Dharmacon (USA). Then, we constructed
the plasmid vectors with the pGCsi-U6-Neo-GFP (Green
Fluorescent Protein) cloning vector. These C6 cells cotransfected
pGCsi-U6-Neo-GFP with either an ASIC1a-specific RNAi or
a control RNAi expression vector. Stable clones were
selected with G418 and were screened for ASIC1a protein
knockdown. Then we chose 1 clone from the ASIC1a-
specific RNAi (C6-sh1a) and the control RNAi clones
(C6-neg).
RT-PCR Total RNA was purified using RNeasy columns
(Qiagen, Valencia, CA, USA). RT-PCR was performed in 50
µL reactions using 100 ng of RNA, 0.5 µmol/L
of each primer, and an annealing temperature of 53 °C for 25 cycles. All
other PCR conditions and reagents were supplied and
recommended by the manufacturer's protocol for the Titan
one-step system (Roche Applied Science, Indianapolis, IN, USA).
The primer sequences for ASIC1a were:
5'-CGGATCCATGG-AATTGAAGACCGAGGA-3' (forward) and
5'-CGATATCTG-CAGGTAAAGTCCTCAAACG-3' (reverse); for the GAPDH,
the primers were: 5'-GTC AAC GGA TTT GGT CGT ATT G-3'
(forward) and 5'-AGT GAT GGCATG GAC TGT GGT-3' (reverse).
Western blot analysis The Western blot analyses of
ASIC1a were performed using specific antibodies (ADI, San
Antonio, TX, USA)[1]. A cell lysate containing 30 µg protein
was fractionated by SDS-PAGE, and then the proteins were
transferred to a nitrocellulose membrane. The membrane
was first rinsed with TBST [20 mmol/L Tris-HCl (pH 7.4), 0.15
mol/L NaCl, and 0.05% Tween 20] and then blocked with 5%
(w/v) skim milk in TBST for 1 h at room temperature. The
blocked membrane was subsequently probed for 1 h at room
temperature with 1:200_1:1000 dilutions of first antibodies in
blocking buffer. After the membrane had been washed 3
times with TBST, it was incubated for 1 h at room
temperature either with horseradish peroxidase-conjugated
antibodies against rabbit immunoglobulin G. After the membrane
had been washed with TBST, bands of protein on the
membrane were visualized using the Enhanced
chemiluminescence detection system (PerkinElmer Life Sciences, Boston,
MA, USA).
Measurement of [Ca2+]i calcium
The cells were plated into a polylysine-coated coverslip 1 d before each experiment.
After washing 3 times with D-Hanks' balanced salt buffer,
the cells were incubated with 4 µmol/L Fluo-3-AM
(Molecular Probes, USA) for 30 min at 37 °C. The cells were then washed
twice with fresh D-Hanks' buffer and immediately used for
the experiments. The fluorescence of intracellular Fluo-3
was quantitated by confocal laser scanning fluorescence
microscopy (Leica TCS4D, Leica Lasertechnik, Heidelberg,
Germany) using excitation and emission wavelengths of 488
and 525, respectively. Gray scale images with 0_255 steps
were collected at different time points before and up to 10
min and archived as Tiff image files for later analysis. In
order to eliminate the effect of the glutamate receptors and
voltage-gated Ca2+ channels, MK801 (10 µmol/L), CNQX (20
µmol/L), and nimodipine (5 µmol/L) were also added in the
extracellular solutions. Each time, 7_9 cells were measured,
and the mean value of the fluorescence of each cell was
calculated. After recording the basic fluorescence intensity
for approximately 150 s, the acidic solution (hydrochloric
acid), 20 µL of which had been adjusted and could make the
pH value of the extracellular solution (pH 7.4, 1.5 mL)
decrease to pH 6.0, was directly added with a pipettor far
from these recording cells. The recording continued while
adding acidic solution and lasted for approximately 600 s.
The intensity of the fluorescence in individual cells was
measured using Leica quantitation software (Germany).
Statistical analysis Data were described using mean±
SEM. Mean values were compared using either Student's
t-test or ANOVA. Two-way ANOVA was used to separate
the effects of 2 variables and determine their interaction.
P<0.05 was considered statistically significant.
Results
Confirming the ASIC1a-silenced C6 cell line
In the rat C6 glioma cells, the mRNA expression of ASIC1a, ASIC1b,
ASIC2a, and ASIC2b was identified with the RT-PCR
experiment and we did not find the expression of ASIC3 and
ASIC4[25]. To observe the role of
[Ca2+]i in the acidosis and its relationship with ASIC1a, we used the ASIC1a-silenced
C6 cell line. The method to build the stable cell line was
described in Materials and methods.
The result of the RT-PCR (Figure 1A) and Western
blotting (Figure 1B) showed that a dramatic decrease in ASIC1a
mRNA level and protein expression occurred in the
ASIC1a-silenced cells (C6-sh1a) compared with the wild-type C6 cells
(C6-neo) and the control RNAi cells (C6-neg). It was confirmed
that the ASIC1a-knockdown C6 glioma cell line was built.
Application of acid increased the
[Ca2+]i transitorily in the wild-type C6 cells, but not in the ASIC1a-silenced cells
It is well-known that
[Ca2+]i plays a critical role in the
regulation of cell death via either the apoptotic or necrotic
pathways[26_30]. ASIC1 has been proposed to play a role in the
pathogenesis of diseases associated with extracellular
acidosis, including seizures and cerebral
ischemia[20_22]. Here, we tested the changes of
[Ca2+]i among the 3 types of cells
applied with extracellular acid. From the confocal micrographs
(Figure 2A) and the representative traces (Figure 2B), the
application of extracellular acid (pH 6.0) significantly
increased the [Ca2+]i in the wild-type C6 cell line (Figure
2A,2B), while the changes of
[Ca2+]i in the C6-sh1a cells were
much lower than those in 2 other types of cell lines (Figure
2C, 2D). After the acid application,
[Ca2+]i recovered near the basal value. Thus, the results showed that ASIC1a
mediated the increase of [Ca2+]i
induced by extracellular acid, which also indicates that cytotoxicity induced by acidosis
might be related with [Ca2+]i
.
Knockdown of ASIC1a decreased
[Ca2+]i during pronged acid exposure
Although the results suggest that ASIC1a mediated the increase of
[Ca2+]i induced by extracellular acid,
it might be only part of the reason for cytotoxicity. After the
acid application, the detectable increase in
[Ca2+]i occurred and lasted for only several minutes (Figure 2B). During
prolonged acid application, ASIC1a currents desensitized, and
[Ca2+]i returned to basal values (Figure 2A, 2B). If the acid
stimulus (pH 6.0) to cells was limited in a few minutes, the
cytotoxicity among the 3 types of cells would not be severe
and distinct (data not shown). So we measured the
[Ca2+]i before and after the 3 h acid stimulus when apoptosis among
the 3 types of cell lines was distinct. Using the Fluo-3
fluorescent Ca2+-imaging technique, we found that
[Ca2+]i in the wild-type C6 cells and C6-neg cells had increased compared
to the C6-sh1a cells (Figure 3). This result was consistent
with acid-induced cytotoxicity, although the detailed
mechanism is still unclear. The result suggests that prolonged acid
exposure induces the changes of
[Ca2+]i via ASIC1a mechanisms.
Discussion
The ASIC were activated by H+ and inactivated rapidly
after activation. This transient current carried
Na+ displays a
Na+/K+ selectivity ratio of 10. At the same time, ASIC
showed some permeability to Ca2+. The relative
Ca2+ permeability was highest in ASIC1a, for which
PNa/PCa permeability ratios of 2.5_17 have been
determined[1]. The PNa/PCa ratio for ASIC3 and ASIC1b was ~100. Thus, we presumed that
the Ca2+ permeability of ASIC1a might be important for its
cellular function. From our studies, the
[Ca2+]i in the cells expressing ASIC1a was higher than that in the
ASIC1a-silenced cells, which indicated that ASIC1a activity enhanced
acidosis-induced injury, not only because the higher
[Ca2+]i contrast to the ASIC1a-silenced cells could evoke the
mechanism of apoptosis and then lead to cell death of some cells,
but also because the overloading of
[Ca2+]i in some cells activated a cascade of cytotoxic events and directly induced
cell death[26_29].
In our previous study, we observed that acidosis-induced
cytotoxicity could be obviously inhibited by targeting the
ASIC1a mRNA with short hairpin RNA[25]. In the present
study, we further studied the underlying mechanism involved
in this process. It was found that the activation of ASIC1a
could induce glutamate-independent and VGCC
(voltage-gated calcium channels)-independent
Ca2+ entry in the wild-type C6 cells, but not in the stably ASIC1a-silenced C6 cell
line. Ca2+ performed a very important role in cytotoxicity.
Evidence has revealed that cytotoxicity could be directly
induced by the increase of
[Ca2+]i[24_28]. When
Ca2+ has a 20% increase in the total calcium content, cell death can take
place[28]. According to the cytotoxicity of
[Ca2+]i, it indicated that the increase of
[Ca2+]i was most likely to be
respon-sible for acidosis-induced cytotoxicity. Thus, these results
provided evidence that the change of
[Ca2+]i during extracellular acidosis might be responsible for the cytotoxicity
via the ASIC1a mechanism. The results indicated that
[Ca2+]i was one of the mechanism for cytotoxicity via the ASIC1a
pathway.
ASIC1a is a quick transient channel that can only induce
short depolarization or calcium entry in the cell. Even if the
acidic stimulus lasts, ASIC1a becomes inactive. The reason
why [Ca2+]i was lower in the ASIC1a silenced cells than in the
longer exposure to acid remains unclear, and further research
was still needed to explain the mechanism in detail.
We utilized the highly specific technology of RNAi to
directly demonstrate that extracellular acid induces
cytotoxicity by activating ASIC1a with the resultant toxicity of
[Ca2+]i . Using ASIC1a-deficient mice is limited and difficult, thus,
the use of ASIC1a shRNA will greatly facilitate the precise
identification of the ASIC1a gene function and its mechanism.
RNAi mediated by shRNA is a powerful technology that
allows the silencing of genes with great specificity and
potency. Through our studies, we are sure that RNAi will be
more widely used in future pharmacological research.
References
1 Waldmann R, Champigny G, Bassilana F, Heurteaux C, Lazdunski
M. A proton-gated cation channel involved in acid-sensing.
Nature 1997; 386: 173_7.
2 Waldmann R, Bassilana F, de Weille J, Champigny G, Heurteaux
C, Lazdunski M. Molecular cloning of a non-inactivating
proton-gated Na+ channel specific for sensory neurons. J Biol Chem
1997; 272: 20975_8.
3 Waldmann R, Lazdunski M. H+-gated cation channels: neuronal
acid sensors in the NaC/DEG family of ion channels. Curr Opin
Neurobiol 1998; 8: 18_24.
4 Lingueglia E, de Weille JR, Bassilana F, Heurteaux C, Sakai H,
Waldmann R, et al. A modulatory subunit of acid sensing ion
channels in brain and dorsal root ganglion cells. J Biol Chem
1997; 272: 29 778_83.
5 Babinski K, Le KT, Seguela P. Molecular cloning and regional
distribution of a human proton receptor subunit with biphasic
functional properties. J Neurochem 1999; 72: 51_7.
6 Akopian AN, Chen CC, Ding Y, Cesare P, Wood JN. A new
member of the acid-sensing ion channel family. Neuroreport
2000; 11: 2217_22.
7 Waldmann R, Champigny G, Lingueglia E, De Weille JR, Heurteaux
C, Lazdunski M. H+-gated cation channels. Ann N Y Acad Sci
1999; 868: 67_76.
8 Price MP, Snyder PM, Welsh MJ. Cloning and expression of a
novel human brain Na+ channel. J Biol Chem 1996; 271: 7879_82.
9 Bassilana F, Champigny G, Waldmann R, de Weille JR, Heurteaux
C, Lazdunski M. The acid-sensitive ionic channel subunit ASIC
and the mammalian degenerin MDEG form a heteromultimeric
H+-gated Na+ channel with novel properties. J Biol Chem 1997;
272: 28 819_22.
10 Weng XC, Zheng JQ, Gai XD, Li J, Xiao WB. Two types of acid
sensing ion channels expressed in rat hippocampal neurons.
Neurosci Res 2004; 50: 493_9.
11 Olson TH, Riedl MS, Vulchanova L, Ortiz-Gonzalez XR, Elde R.
An acid sensing ion channel (ASIC) localizes to small primary
afferent neurons in rats. Neuroreport 1998; 9: 1109_13.
12 Yermolaieva O, Leonard AS, Schnizler MK, Abboud FM, Welsh
MJ. Extracellular acidosis increases neuronal cell calcium by
activating acid-sensing ion channel 1a. Proc Natl Acad Sci USA
2004; 101: 6752_7.
13 Reeh PW, Steen KH. Tissue acidosis in nociception and pain.
Prog Brain Res 1996; 113: 143_51.
14 Price MP, Lewin GR, McIlwrath SL, Cheng C, Xie J, Heppenstall
PA, et al. The mammalian sodium channel BNC1 is required for
normal touch sensation. Nature 2000; 407: 1007_11.
15 Bevan S, Yeats J. Protons activate a cation conductance in a
sub-population of rat dorsal root ganglion neurons. J Physiol 1991;
433: 145_61.
16 Ugawa S, Ueda T, Ishida Y, Nishigaki M, Shibata Y, Shimada S.
Amiloride-blockable acid-sensing ion channels are leading acid
sensors expressed in human nociceptors. J Clin Invest 2002;
110: 1185_90.
17 Sluka KA, Price MP, Breese NM, Stucky CL, Wemmie JA, Welsh
MJ. Chronic hyperalgesia induced by repeated acid injections in
muscle is abolished by the loss of ASIC3 but not ASIC1. Pain
2003; 106: 229_39.
18 Chen CC, Zimmer A, Sun WH, Hall J, Brownstein MJ, Zimmer A.
A role for ASIC3 in the modulation of high-intensity pain stimuli.
Proc Natl Acad Sci USA 2002; 99: 8992_7.
19 Benson CJ, Eckert SP, McCleskey EW. A possible mediator of
myocardial ischemic sensation. Circ Res 1999; 84: 921_8.
20 Wemmie JA, Askwith CC, Lamani E, Cassell MD, Freeman JHJ,
Welsh MJ. Acid-sensing ion channel 1 is localized in brain
regions with high synaptic density and contributes to fear
conditioning. J Neurosci 2003; 23: 5496_502.
21 Wemmie JA, Chen J, Askwith CC, Hruska-Hageman AM, Price
MP, Nolan BC. The acid-activated ion channel ASIC contributes
to synaptic plasticity, leaning, and memory. Neuron 2002; 34:
463_77.
22 Xiong ZG, Zhu XM, Chu XP, Minami M, Hey J, Wei WL,
et al. Neuroprotection in ischemia: blocking calcium-permeable
acid-sensing ion channels. Cell 2004; 118: 687_98.
23 Berdiev BK, Xia J, McLean LA, Markert JM, Gillespie GY,
Mapstone TB, et al. Acid-sensing ion channels in malignant
gliomas. J Biol Chem 2003; 278: 15 023_34.
24 Vila-Carriles WH, Kovacs GG, Jovov B, Zhou ZH, Pahwa AK,
Colby G, et al. Surface expression of ASIC2 inhibits the
amiloride-sensitive current and migration of glioma cells. J Biol Chem
2006; 281: 19 220_32.
25 Weng XC, Zheng JQ, Jin QE, Ma XY. Involvement of ASIC1a in
apoptosis and cell death induced by extracellular acidosis. Acta
Pharmacol Sin 2006; 1 (Suppl 1): 186.
26 Choi DWW. Glutamate neurotoxicity in cortical cell culture is
calcium dependent. Neurosci Lett 1985; 58: 293_7.
27 Mattson MPP, Chan SL. Calcium orchestrates apoptosis. Nat
Cell Biol 2003; 5: 1041_3.
28 Leist M, Nicotera P. Calcium and neuronal death. Rev Physiol
Biochem Pharmacol 1998; 132: 79_125.
29 Sattler R, Sattler M, Tymianski Y. Molecular mechanisms of
calcium-dependent excitotoxicity. J Mol Med 2000; 78: 3_13.
30 Kristián T, Siesjö BK. Calcium in ischemic cell death. Stroke
1998; 29: 705_18.
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