Acta Poloniae Pharmaceutica ñ Drug Research, Vol. 73 No. 4 pp. 895ñ902, 2016
ISSN 0001-6837
Polish Pharmaceutical Society
DRUG BIOCHEMISTRY
BLOCKADE OF LARGE CONDUCTANCE CA2+ ACTIVATED K+ CHANNEL
MAY PROTECT NEURONAL CELLS FROM HYPOXIA MIMETIC INSULT
AND OXIDATIVE STRESS
JIN ZHANG
School of Pharmacy and Pharmaceutical Science, Cardiff University, 2.33 Redwood Building,
King Edward VII Ave., Cathays, Cardiff, Wales, CF10 3NB, U.K.
Abstract: Previous studies have linked neuronal cell death with changes of intracellular Ca2+ ([Ca2+]i) homeostasis. Such changes of [Ca2+]i has been noticed in various neurodegenerative models. It has also been suggested that K+ channel, such as large conductance Ca2+ activated K+ channel (BK), might present a neuronal protective effect. Hence, this study has established two cell insult models, oxidative stress induced by H2O2 and
hypoxia mimetic induced by CoCl2, on a human neuronal cell line SH-SY5Y, since both insults are related to
neurodegeneration and are able to increase the [Ca2+]i. MTS assays were used to test the possible effect of BK
activators and blockers. According to the results, BK activators, NS1619 and isopimaric acid (IPA), would
potentiate toxicity to the cells under both of the two insults. But the K+ channel blockers, tetraethylammonium
(TEA) and tetrandrine, have presented the neuronal protective effect against CoCl2 insult. Considering that
[Ca2+]i, which would activate the BK channel, is the key issue in neurodegeneration, it would be suggested by
the results from this study that K+ channel blocker, rather than activator, would potentially present the neuronal
protective effect.
Keywords: oxidative stress, hypoxia mimetic, BK, MTS assay, neuroprotection
and Ca2+ influx through NMDA receptor (2). In
ALS, the Ca2+ content in motor neurones is significantly increased as well (2), and such an increase
has been linked with a copper (Cu)/zinc (Zn)-superoxide dismutase (SOD) mutation and with the death
of motor neurones (3). In AD, Ca2+ entering through
putative Ca2+ channels formed by β-amyloid (Aβ)
stemming from amyloid precursor protein (APP)
(4), and this certain Ca2+ current through an Aβ
channel has been recorded with electrophysiological
techniques previously (5). In PD, the death of
dopaminergic neurones is contributed by mitochondrial stress, with a possible role of perturbed [Ca2+]i
homeostasis downstream of the mitochondrial alterations (6). In HD, a polyglutamine expansion would
modify hunting to mutant huntingtin fragmentation,
and the latter could be switched to huntingtin
oligomers through polyglutamine expansion and/or
the increasing of hydrogen peroxide (H2O2). And the
huntingtin oligomers may then increase [Ca2+]i and
cause cell damage or death (7).
Previous studies have suggested the large conductance Ca2+ activated K+ channel (BK) as a neu-
Neurodegenerative diseases, including Alzheimerís disease (AD), Parkinsonís disease (PD),
ischemic stroke, Huntingdonís disease (HD), amyotrophic lateral sclerosis (ALS) and others are widely spread nowadays. Regarding the possible mechanism of neurodegenerative neuronal death, the overload of intracellular calcium (Ca2+) concentration is
a widely accepted hypothesis. Neurones must maintain ionic gradients and a membrane potential to
function properly in their signalling role, and even
small decrements in membrane potential can alter
firing properties and lead to significant brain dysfunction. Further, neurones need to maintain the
intracellular Ca2+ concentration ([Ca2+]i) low (~ 100
nM), hence Ca2+ entering into neurones from any
pathway could be a cause of neuronal death. In
stroke, compromised blood flow in vascular damage
results in a massive release of glutamate which can
activate the N-methyl-D-asparate (NMDA) receptor. The activation of NMDA receptor can cause a
rise in [Ca2+]i (1). In addition, membrane associated
oxidative stress can impair the function of glutamate
transporters, and promote membrane depolarization
* Corresponding author: e-mail: [email protected]; phone: +1-902-4032378
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JIN ZHANG
ronal protective target (8, 9). The increasing of
[Ca2+]i would open the BK channel and cause the K+
releasing from the cells. Such K+ release would
change the membrane potential, and would spike
narrowing and thus limited Ca2+ entry through voltage gated Ca2+ channel (Cav) (10, 11). BK channel is
composed by 1 α subunit with 0ñ4 different β subunit(s). The channel area is located in α subunit, but
the modulator binding area might be located on
either α or some particular β subunit, and the later
would be related to the selectivity of BK modulators. For example, the BK channel composed with
β4 subunit would be resistant to iberiotoxin (IbTX)
which is a BK blocker (12).
Therefore, a human neuroblastoma cell line,
SH-SY5Y, which can be differentiated to human
neurone has been selected here. Whether the BK
channel is existing in neuronal cell line has been
studied first with polymerase chain reaction (PCR).
Then, the possible effect of BK channel in neuronal
cell death has been studied by testing BK activators,
NS1619 and isopimaric acid (IPA), and K+ channel
blockers, tetraethylammonium (TEA) and tetrandrine, in the cell proliferation and MTS assays with
two cell insult models. One is the oxidative stress
induced by hydrogen peroxide (H2O2) since previous studies have linked it with neurodegeneration
and suggested that H2O2 would increase the [Ca2+]i
(13). And the other one is hypoxia mimetic induced
by cobalt chloride (CoCl2) because it was related to
some neurodegenerative disease as well and was
proved to increase the generation of Aβ (14).
MATERIALS AND METHODS
Materials
SH-SY5Y cell line is from Sigma. PCR kit,
reverse transcription kit, and MTS [3-(4,5dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt]
are from Promega. Fetal bovine serum (FBS), penicillin ñ streptomycin mixture (P/S), phosphatebuffered saline (PBS, pH = 7.4), TRIzol and
primers (designed on Primer III webpage) are from
Invitrogen. Minimum essential Eagle medium (EMEM), nutrient mixture F12 ham (F12), non
essential amino acid (NEAA), glutamine, retinoic
acid, CoCl2, H2O2, phenazine methosulfate (PMS),
chloroform (CHCl3), ethylenediaminetetraacetic
acid (EDTA), trypsin and nuclease free water
(H2O) were from Sigma-Aldrich. Ethanol
(C2H5OH) and isopropyl alcohol [(CH3)2CHOH]
were from Fisher. CoCl2 was dissolved in PBS as
10 mM for stock, and H2O2 and TEA were dis-
solved in PBS as 100 mM for stock, IbTX was dissolved in PBS as 100 µM for stock, NS1619 is dissolved in C2H5OH as 10 mM for stock, and isopimaric acid (IPA) and tetrandrine were dissolved in
DMSO as 10 mM for stock.
Cell culture
The cells are cultured in culture medium in
flasks (25 cm2 or 75 cm2) in an incubator kept at
37OC and 5% carbon dioxide (CO2). The prescription for SH-SY5Y culture is: E-MEM / F12 1 : 1,
plus 15% FBS, 2 mM glutamine, 1 ◊ NEAA and 100
u P/S. SH-SY5Y cells can be differentiated to
human neurones after 7 days of retinoic acid treatment, and the differentiation medium is the culture
medium plus 10 µM retinoic acid. The SH-SY5Y
cells with passage numbers higher than 22 were not
used in this study.
Reverse transcription ñ PCR (RT-PCR)
RNA was extracted from a confluent 75 cm2
flask of alive cells with TRIzol. The TRIzol was
removed with CHCl3 and the RNA sample was
washed twice with (CH3)2CHOH and C2H5OH,
respectively. The final RNA sample was dissolved
in H2O and its concentration was measured with a
UV spectrophotometer.
Reverse transcription has been taken to transfer
the extracted RNA into cDNA for PCR. Reverse
transcription has been done with reverse transcriptase and RNAsin kit and the same amount (1 µg) of
RNA has been used in each reaction of reverse transcription to make sure the PCR or Q-PCR results are
comparable. Negative control was run in every reaction and its prescription was the same as with the
sample only except the reverse transcriptase.
PCR has been done with Go Taq enzyme kit
and the PCR of β-actin, which is a housekeeping
gene, was applied as the positive control. The
primers for BK subunits are shown in Table 1.
The optimized PCR conditions are: 58OC as the
annealing temperature, 30 cycles.
Cell proliferation and MTS assay
Cells were taken out from a confluent 25 cm2
flask with EDTA/trypsin and seeded at a density of
15,000 per well in 100 µL of relevant culture medium without drug treatment. After the cells were
incubated at 37OC overnight, medium was aspirated
and 100 µL of insult medium (culture medium without FBS) containing insult drugs (such as H2O2 or
CoCl2) and/or modulators was added into each well.
Then, cells were incubated at 37OC for 24 h. After
the insult, 20 µL of MTS/PMS (20 : 1) were added
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Blockade of large conductance Ca2+ activated K+ channel...
into each well. Then, cells were incubated at 37OC
for another 2 to 4 h. Finally, absorbance data were
acquired at a wavelength of 490 nm with a plate
reader.
Blank and control wells were run alongside
treatment wells. The blank is the absorbance of the
same treatments but without any cells, and the control is the absorbance of cells cultured in medium
without any drug. The absorbance of relevant blank
was subtracted from the relevant data during analysis. The final result of each sample was compared
with the one from control, and can present the relative cell number to control. Each cell proliferation
was at least in triplicate for statistics.
Statistics
In each group of results, outlier is determined
by both G test and Q test. If a datum is suggested as
an outlier by both of the two tests, it would be
removed from further statistics. T test was used for
two groupsí comparison, and ANOVA with posthoc test (Tukey, Bonferroni or Dunnett was used
under different circumstances) was used for the
comparison of three groups or more. The significance level was set as p < 0.05.
RESULTS
PCR
The PCR result (Fig. 1) has shown that all of
the 5 subunits are existing in both undifferentiated
and differentiated SH-SY5Y cells.
H2O2 insult to SH-SY5Y cell line and BK activatorís effect
A range of concentrations (100ñ600 µM) of
H2O2 have been applied on both undifferentiated and
differentiated SH-SY5Y cells in cell proliferation
and MTS assays to demonstrate the oxidative stress
insult to the cell line. Then, NS1619, which is a BK
activator targeting on α subunit (12), was combined
with the same range of H2O2 insult on both undifferentiated and differentiated SH-SY5Y cells to see
whether it would protect the cells or not. Since 3ñ30
µM of NS1619 was reported to active the BK channel in previous studies (12), the concentration 10
µM was used in this study. The results are shown in
Figure 2.
From the result above, it could be noticed that
the differentiated SH-SY5Y cells, which should be
the human neurones, were more sensitive to H2O2
Figure 1. PCR of BK subunits in SH-SY5Y cell line
Table 1. PCR primers for BK subunits.
Name
BKα, 3,537 bp mRNA
BKfl1, 1,518 bp mRNA
BKfl2, 2,543 bp mRNA
BKfl3, 1,835 bp mRNA
BKfl4, 1,631 bp mRNA
Sequence
Forward
5'-ACGCAATCTGCCTCGCAGAGTTG-3'
Reverse
5'-CATCATGACAGGCCTTGCAG-3'
Forward
5'-CTGTACCACACGGAGGACACT-3'
Reverse
5'-GTAGAGGCGCTGGAATAGGAC-3'
Forward
5'-CATGTCCCTGGTGAATGTTG-3'
Reverse
5'-TTGATCCGTTGGATCCTCTC-3'
Forward
5'-AACCCCCTTTTCATGCTTCT-3'
Reverse
5'-TCTTCCTTTGCTCCTCCTCA-3'
Forward
5'-GTTCGAGTGCACCTTCACCT-3'
Reverse
5'-TAAATGGCTGGGAACCAATC-3'
Location
1,639 - 2,047 bp
668 - 865 bp
808 - 1,044 bp
1,404 - 1,680 bp
648 - 892 bp
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JIN ZHANG
Figure 2. H2O2 insult to both undifferentiated and differentiated SH-SY5Y cells, and the effect of NS1619 against the oxidative stress on
both cells. *** p < 0.001, H2O2 insult between undifferentiated and differentiated cells; # p < 0.05, NS1619 effect to H2O2 insult on differentiated cells; n = 3
Figure 3. IPA effect to H2O2 insult on SH-SY5Y cell line (n = 3). a: on undifferentiated cells; b: on differentiated cells
insult. The difference of relative cell number at 300
µM was significant (p < 0.001) between undifferentiated and differentiated cells. It could also be found
that 10 µM NS1619 has failed to protect neither
cells from H2O2 insult, and somehow even would
aggravate the insult of oxidative stress on differentiated SH-SY5Y cells. On the differentiated cells,
10 µM of NS1619 has significantly decreased the
relative cell number from H2O2 insult at 150 µM (p
< 0.05).
Besides NS1619, the effect of IPA was tested
on the H2O2 insult model on both undifferentiated
and differentiated SH-SY5Y cells as well. IPA is
another BK activator which can active BK channel
consisting with only α subunit with EC50 at about 3
µM (12). The results are shown in Figure 3.
From Figure 3, it could be suggested that IPA
has not increased the relative cell numbers from
H2O2 insult on neither undifferentiated nor differentiated cells. On the differentiated cells, 10 µM IPA
would even present the trend to decrease the relative
cell number under high concentrations of H2O2 (the
difference was not significant in post-hoc of
ANOVA, but was significant in t-test at 450 µM, p
< 0.01). These results suggested that IPA has failed
to protect the cells from H2O2 insult neither, and
even has presented a trend to potentiate the toxic
effect on the differentiated cells.
CoCl2 insult to SH-SY5Y cell line
A range of concentrations (100 nM to 1 mM)
of CoCl2 insults have been taken on both undiffer-
Blockade of large conductance Ca2+ activated K+ channel...
entiated and differentiated SH-SY5Y cells to estimate the ED50. After the relative cell numbers under
different CoCl2 concentrations have been tested,
trend line was estimated, and the ED50 was calculated according to the trend line. The sample sizes are
3 to 9 according to different CoCl2 concentrations.
Figure 4 has demonstrated the dosage-effect curve
of CoCl2 insult to both undifferentiated and differentiated SH-SY5Y cells.
BK activators effect to CoCl2 insult on SH-SY5Y
cells
Based on the CoCl2 insult result above, BK
modulators, NS1619 and IPA as activators, as well
as K+ channel blockers, TEA and tetrandrine, have
been tested on the insult model on both undifferentiated and differentiated SH-SY5Y cells to find out
the effect of BK channel in hypoxia mimetic insult
on neurones.
Both NS1619 and IPA have been tested against
CoCl2 insult on both undifferentiated and differentiated SH-SY5Y cells. Figure 5 presents the effect of
BK activators.
From Figure 5, it would be noticed that both
NS1619 and IPA themselves would increase the cell
proliferation in both undifferentiated and differentiated SH-SY5Y cells, however both NS1619 and
IPA have aggravated the toxic effect of CoCl2.
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K+ channel blocker effect against CoCl2 insult on
SH-SY5Y cells
Based on the results above which suggested the
potentiated toxic effect shown by BK activators, this
study has also tested some blockers of K+ channel to
see whether they would present any effect on the
opposite direction. TEA, which would inhibit K+
channel in the high-micromolar or millimolar range,
and IbTX, which would inhibit the BK channel with
IC50 at about 10 nM (12) have been tested. Besides,
a herbal source alkaloid, tetrandrine, which would
induce a flicker block to BK channel (15) has been
tested as well. Figure 6 has shown the effects of
those blockers.
From Figure 6, it could be noticed that both
TEA and tetrandrine, which are K+ channel blockers,
would protect the differentiated SH-SY5Y cells
from CoCl2 insult. The protective effect of TEA was
not significant on undifferentiated cells. But, BK
channel blocker, IbTX, has failed to present any protective effect on neither undifferentiated nor differentiated cells.
DISCUSSION
Cell insults
From Figures 3 and 4, it would be noticed that
the differentiated SH-SY5Y cells are more sensitive
Figure 4. Dosage-effect curve of CoCl2 insult to both undifferentiated and differentiated SH-SY5Y cells. It can be suggested that the differentiated cells are more sensitive to CoCl2 insult, and the differences are significant at 10 and 20 µM. The LD50 of CoCl2 to undifferentiated and differentiated cells were estimated at around 40 and 30 µM, respectively. * p < 0.05; n = 3
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JIN ZHANG
than the undifferentiated ones to both oxidative
stress induced by H2O2 and hypoxia mimetic
induced by CoCl2. In H2O2 insult, the relative cell
number of differentiated cells was significantly
lower than the counterpart of undifferentiated cells
at 300 µM. In CoCl2 insult, the relative cell numbers
of differentiated cells were significantly lower as
well at 10 and 20 µM.
Since it is believed that both of the two
insults would increase the [Ca2+]i, according to
previous studies, it would be suggested that the
differentiated SH-SY5Y cells are more sensitive
to the [Ca2+]i. This kind of results would potentially suggest that some of the Ca2+ modulators in the
cells, such as some of the Ca2+ release channel on
the endoplasmic reticulum (ER) like ryanodine
receptor (RyR) or inositol trisphosphate (IP3),
would possibly have a higher expression in the
differentiated than in the undifferentiated SHSY5Y cells .
BK activator may aggravate the insults
From Figures 3 and 5, it could be noticed that
BK activators, NS1619 and IPA, have failed to presented any protective effect against neither oxidative
stress induced by H2O2 nor hypoxia mimetic
induced by CoCl2. And, except the negative results
shown by NS1619 and IPA on undifferentiated SHSY5Y cells with H2O2 insult, all of the other results
have indicated that the BK activators have even
aggravated the insult by significantly reduced the
relative cell numbers from insults.
Figure 5. BK activators effect to CoCl2 insult on SH-SY5Y cell line. a: NS1619 effect to CoCl2 insult on undifferentiated cells; b: IPA
effect to CoCl2 insult on undifferentiated cells; c: NS1619 effect to CoCl2 insult on differentiated cells; d: IPA effect to CoCl2 insult on differentiated cells. * p < 0.05; ** p < 0.01; ***: p < 0.001; n = 6 for control and IPA in b, and n = 3 for all the other groups
Blockade of large conductance Ca2+ activated K+ channel...
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Figure 6. K+ channel blockers effect against CoCl2 insult on SH-SY5Y cell line. a: TEA effect on differentiated cells; b: IbTX effect on
differentiated cells; c: tetrandrine effect on differentiated cells; d: TEA effects on undifferentiated cells; e: IbTX effect on undifferentiated cells. * p < 0.05; ** p < 0.01; *** p < 0.001; n = 3
Actually, as reported, BK channel is activated
by intracellular Ca2+. Both of the two insults, H2O2
and CoCl2, would increase the [Ca2+]i and cause the
cell death, according to previous studies. In that case,
the BK channel on the cells should have been activated already when the cells were treated with
insults. Hence, if the BK channel were further activated with extra activators, the K+ efflux, and cell
shrinkage and death thereafter, might be caused (16).
Nevertheless, this result is not going against
with previous studies which suggested the neuronal
protective role of BK channel (9). Those studies
have pointed out that the activation of BK channel
would block the Ca2+ entry from extracellular sauce
by reducing the NMDA receptor mediated Ca2+
overload (11). However, obviously, the entry of
extracellular Ca2+ would not be the only cause to
increase the [Ca2+]i. Some of the intracellular Ca2+
modulators, such as RyR or IP3 on the ER, have
been proved to be involved in the anti-neurodegenerative treatment as well (17).
K+ blockers potentially have neuronal protective
effect
Since the BK activators would possibly aggravate the insults, it is reasonable to suggest that the K+
channel blocker, which acts in the opposite direction, might protect the cells from insult by avoiding
the possible K+ efflux. Actually, the data shown in
Figure 6a and 6c have suggested that both TEA and
tetrandrine, K+ channel blockers, would protect the
differentiated SH-SY5Y cells from CoCl2 insult by
significantly increasing the relative cell number.
This result consists with the findings from previous
studies. One study in 1998 has suggested that 100
µM to 5 mM of TEA would significantly reduce the
neuronal death induced by Aβ25-35 in 24ñ48 h (18).
For the negative result demonstrated by IbTX,
it is possible to be contributed by the existence of β4
subunit. Previous studies have suggested that β1
enhances the toxin binding but complexes of β4
with BK are resistant to IbTX (12). From the PCR
result shown in Fig. 1, it could be noticed that both
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JIN ZHANG
β1 and β4 messages exist, which might be the reason for the negative results of IbTX in this study.
Society and Henry Lester Trust. Besides, I much
appreciate Prof. Kenneth T. Wann for his idea and
discussion on this study.
CONCLUSION AND FUTURE WORKS
REFERENCES
To summarize all of the results above, it could
be suggested that BK channel is existing and functional in SH-SY5Y cell line and the differentiated
SH-SY5Y cells are more sensitive to both oxidative
stress induced by H2O2 and hypoxia mimetic
induced by CoCl2.
More importantly, BK activators, NS1619 and
IPA, have failed to show any neuronal protective
effect on neither oxidative stress nor hypoxia
mimetic, and would somehow aggravate the insults
by reducing the relative cell numbers. That could be
explained by the overactivation of BK channel and
the K+ efflux caused thereafter.
In the contrast, K+ channel blockers, TEA and
tetrandrine, have demonstrated some protective
effect on differentiated SH-SY5Y cells against the
hypoxia mimetic induced by CoCl2. That consists
with previous studies and has suggested that the
blockade of K+ channel might be a new strategy for
neuronal protection. And the negative result shown
by IbTX, which could be contributed by the β4 subunit, has suggested the possible selectivity of BK
blockers. If BK channel in different organs would
have different β subunits expression, it would be
possible to find some selective BK blockers which
might target on the neurones only rather than some
other irrelative organs. This could possibly be the
future strategy of neurodegeneration treatment.
The future works would be to investigate the
expression of intracellular Ca2+ channels, such as
RyR and IP3, to see whether they would be higher
expressed in the differentiated SH-SY5Y cells. The
possible effects of K+ blockers, for example TEA
and tetrandrine, are going to be tested on the neuronal cell models with oxidative stress to see
whether they would present any protection or not. If
the K+ blockers would be neuroprotective, the β
subunits of BK channel in neurones will be studied
to develop selective BK blockers as the strategy of
neurodegeneration treatment.
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Acknowledgments
Received: 19. 06. 2015
This study is supported by School of Pharmacy
and Pharmaceutical Science, Great Britain ñ China