IL-1b modulate the Ca2+-activated big-conductance K channels(BK) via reactive oxygen species in cultured rat aorta smoothmuscle cells

Yuan Gao • Ying Yang • Qigang Guan • Xuefeng Pang •

Haishan Zhang • Dingyin Zeng

Received: 10 July 2009 / Accepted: 19 November 2009 / Published online: 1 December 2009

� Springer Science+Business Media, LLC. 2009

Abstract The large conductance Ca2?-activated K?

(BK) channel, abundantly expressed in vascular smooth

muscle cells, plays a critical role in controlling vascular

tone. Activation of BK channels leads to membrane

hyperpolarization and promotes vasorelaxation. BK chan-

nels are activated either by elevation of the intracellular

Ca2? concentration or by membrane depolarization. It is

also regulated by a diversity of vasodilators and vasocon-

strictors. Interleukin-1b (IL-1b) is one of the cytokines that

play important roles in the development and progression of

a variety of cardiovascular diseases. The effects of IL-1bon vascular reactivity are controversial, and little is known

about the modulation of BK channel function by IL-1b. In

this study, we investigated how IL-1b modulates BK

channel function in cultured arterial smooth muscle cells

(ASMCs), and examined the role of H2O2 in the process.

We demonstrated that IL-1b had biphasic effects on BK

channel function and membrane potential of ASMCs, that

is both concentration and time dependent. IL-1b increased

BK channel-dependent K? current and hyperpolarized

ASMCs when applied for 30 min. While long-term (24–

48 h) treatment of IL-1b resulted in decreased expression

of a-subunit of BK channel, suppressed BK channel

activity, decreased BK channel-dependent K? current and

depolarization of the cells. H2O2 scavenger catalase com-

pletely abolished the early effect of IL-1b, while it only

partly diminished the long-term effect of IL-1b. These

results may provide important molecular mechanisms for

therapeutic strategies targeting BK channel in inflammation-

related diseases.

Keywords IL-1b � Reactive oxygen species �Smooth muscle cells � BK channel

Introduction

The large conductance Ca2?-activated K? (BK, also

termed BKCa, Slo or MaxiK) channel, abundantly expres-

sed in vascular smooth muscle cells, plays a critical role in

controlling vascular tone [1, 2]. Activation of BK channels

promotes K? outward current leading to membrane

hyperpolarization and promotes closure of voltage-acti-

vated Ca2? channels (VACC) thereby opposing the role of

VACC in vasoconstriction [3]. Hypertension, atheroscle-

rosis, and diabetes are associated with altered expression,

current amplitude, open probability, and Ca2?-sensing of

BK channels. It has been shown that cardiovascular risk

factors such as chronic cigarette smoking and aging down-

regulated BK channel expression [4, 5]. Therefore, BK

channels have been suggested as the therapeutic targets for

treatment of cardiovascular diseases.

BK channels in vascular SMCs are tetramers of four

pore-forming a-subunits and four accessory b1-subunits

that strongly modify the basic channel properties. Expres-

sion levels of a- and b1-subunits are important for the

modulation of BK channel function and vascular reactivity.

Decreased expressions of both a- and b1-subunits are

Electronic supplementary material The online version of thisarticle (doi:10.1007/s11010-009-0338-4) contains supplementarymaterial, which is available to authorized users.

Y. Gao � Y. Yang � Q. Guan � X. Pang � H. Zhang �D. Zeng (&)

Department of Cardiology, the First Affiliated Hospital, China

Medical University, No 155 Nanjing Bei Street, Heping District,

Shengyang 110001, People’s Republic of China

e-mail: zenghetao@hotmail.com

123

Mol Cell Biochem (2010) 338:59–68

DOI 10.1007/s11010-009-0338-4

associated with increased coronary reactivity in the elderly

[4, 6]. BK channels are activated either by elevation of the

intracellular Ca2? concentration ([Ca2?]i) or by membrane

depolarization [7]. Their function in SMCs is dynamically

regulated by a diversity of vasodilators and vasoconstric-

tors. Smooth muscle dilators, such as b2-adrenoceptor

agonists [8], nitric oxide (NO) [9, 10], prostaglandin I2,

arachidonic acid, and carbon monoxide (CO)-activated BK

channels, while potent vasoconstrictors such asangiotensin

II and thromboxane A2 can inhibit BK activity [11]. The

mechanisms involve diverse signal transduction pathways

that are either cAMP or cGMP dependent or involve G-

proteins and/or a balance between phosphorylation–

dephosphorylation mechanisms [8]. Reactive oxygen spe-

cies (ROS) are also important for modulating BK channel

activity [12].

Inflammation plays an important role in the develop-

ment and progression of a variety of cardiovascular con-

ditions, most notably coronary atherosclerosis and

congestive heart failure [13]. Interleukin-1b (IL-1b) is one

of the key mediators implicated in these processes. In terms

of vascular reactivity, the effects of IL-1b are controver-

sial. Some studies suggest that IL-1b renders vessels hyp-

oresponsive to vasoconstrictors by NO-dependent, and NO-

independent mechanisms [14–17]. Activation of ATP-

sensitive K? channels is proposed to be partly responsible

for the NO-independent mechanism [14]. However, other

studies showed that IL-1b increases the responsiveness of

arteries to vasoconstrictors [18, 19] and causes constrictive

remodeling and vasospasm in coronary arteries [20, 21].

IL-1b is known to suppress the voltage-gated K (KV)

channel activity in neurons [22, 23], pheochromocytoma-

12 (PC12) cells [24, 25] and carotid body glomus cells

[25]. However, little is known about the modulation of BK

channel function by IL-1b.

In this study, we investigated how IL-1b modulates

BK channel function in arterial smooth muscle cells

(ASMCs). Since ROS is an important mediator of IL-1bsignaling pathway, we also examined the role of H2O2 in

the process. We demonstrated that IL-1b had biphasic

effects on BK channel function and membrane potential

of ASMCs, that is both concentration and time depen-

dent. Early effect (30 min) of IL-1b was to increase BK

channel-dependent K? current and hyperpolarize the

cells, while long-term (24–48 h) treatment of IL-1bresulted in decreased expression of a-subunit of BK

channel, decreased BK channel activity, and depolariza-

tion of the cells. H2O2 scavenger catalase completely

abolished the early effect of IL-1b, while it only partly

diminished the long-term effect of IL-1b. These results

may provide important molecular mechanisms for thera-

peutic strategies targeting BK channel in inflammation-

related diseases.

Materials and methods

Reagents and solutions

IL-1b, IBTX (Iberiotoxin), NS1619, 4-aminopyridine (4-

AP), catalase, and anti-a-subunit of BK channel antibody

were purchased from Sigma (USA). DiBAC4 (3) was

obtained from Invitrogen. DMEM was purchased from

GIBCO. Fetus bovine blood serum was obtained from

HYCLONE. The external solution for patch-clamp exper-

iments was composed of 40 mM K-Asp, 100 mM KCl,

1 mM CaCl2, and 10 mM Hepes, pH 7.2–7.4. The pipette

solution contained 100 mM K-Asp, 40 mM KCl, 10 mM

Hepes, and 2 mM EGTA, pH 7.2–7.4. Incubation solution

contained 124 mM NaCl, 5 mM KCl, 1.2 mM KH2PO4,

1.3 mM MgSO4, 2.4 mM CaCl2, 26 mM NaHCO3, and

10 mM glucose.

Cell culture

Rat aorta smooth muscle cells (ASMCs) were purchased

from ATCC (No.: A-10) and were cultured with 5% CO2 at

37�C in Dulbecco’s minimal essential medium (DMEM)

containing 10% fetal bovine serum, 100 IU/ml penicillin,

100 lg/ml streptomycin sulfate. ASMCs between passages

5 and8 were used for all the experiments. In order to

measure the membrane currents, the cells from the stock

culture were plated onto glass coverslips, and used for

Patch-Clamp experiments 2–3 days after plating. The

coverslips were transferred to a chamber (1 ml) mounted

on the stage of an Olympus confocal microscope

(FV1000S-IX81, Japan) for the experiments.

Measurement of membrane potential

We used DiBAC4 (3) to detect the membrane potential.

DiBAC4 (3) are oxonol derivatives that are lipophilic and

negatively charged with excitation maxima at approxi-

mately 490 nm. Hyperpolarization results in extrusion of

the dye and then a decrease in cellular fluorescence,

whereas depolarization in enhanced fluorescence intensity

[26, 27]. ASMCs were loaded with 5 lM DiBAC4 (3) for

30 min at room temperature in the dark. After loading,

ASMCs were washed three times with HEPES-buffered

Hank’s solution. The fluorescence intensity was detected

using an Olympus confocal microscope with the excitation

and emission wavelength of 488 nm and 525 nm, respec-

tively. Fluorescent images were scanned after 30 min’s

application of IL-1b to detect the early effect. For longer

treatment, cells were preexposed to IL-1b for 24 or 48 h

prior to loading the cells with DiBAC4 (3). The cells were

pretreated with 200 U/ml catalase for 40 min before the

application of IL-1b. IBTX (100 nM) or NS1619 (30 lM)

60 Mol Cell Biochem (2010) 338:59–68

123

was applied to the cells before the detection of membrane

potential. Quantification of relative fluorescence intensity

was performed using Image J software. Relative fluores-

cence intensity was determined by the subtraction of

background value. Data were shown as the average of three

independent experiments with five repeats for each sample.

Mesurement of H2O2 concentration

H2O2 concentration was measured using Hydrogen Per-

oxide Assay Kit (Beyotime Institute of Biotechnology,

China) according to the manufacturer’s instructions. The

absorbance was measured at 560 nm, and H2O2 concen-

tration was calculated using standard curve method. Data

were shown as the average of three independent experi-

ments with three repeats for each sample.

Patch-clamp experiments

We used PC-10 puller (RWD Life Science, CN) to draw

patch electrodes, and the electrode resistance was 3–5 MXwhen filled with the pipette solution. In whole cell

recording, when the high-resistance seal was formed, the

cell membrane under the patch pipette was ruptured to

form the whole cell patch configuration. The seal resistance

was measured before and after forming whole cell patch

recording. The duration of patch-clamp experiments was

10 min and no channel run-down was observed. In order to

detect the current density of whole cell K ? currents, we

clamped the cell membrane potential from 0 mV to

?60 mV by a 200-m-long depolarizing step pulse in

10 mV increments. Current density was obtained by

dividing the peak outward K? current with cell capacitance

(pA/pF). With the use of inside-out patch, we clamped the

cell membrane potential at ?50 mV (depolarization) that

activates BK channels and studied BK channel activity

with the same holding potential in the presence of 5 mM

4-AP. An Axopatch 700B amplifier (Axon Instruments,

CA) was used for the recording. The signal was filtered at

1 kHz by digitada 1314 (Axon Ins.). Data were collected at

sample rate of 5 kHz and were analyzed with software

Axon patch 10.0. Channel activity was defined as NPo that

was calculated from data samples of 120 s duration in the

steady state as follows: NPo =

Pt1þ2 t2þ...iti

T where ti is the

fractional open time spent at each of the observed current

levels, T is total recording time.

Whole cell extracts and western blotting analysis

In order to prepare whole cell extracts, ASMCs were

washed with phosphate-buffered saline (PBS), collected

by centrifugation, and lysed in high-salt buffer (50 mM

Tris-HCl pH 7.4, 500 mM NaCl, 1% NP-40 and 20%

glycerol) supplemented with 0.5 mM PMSF, 5 mM b-

mercaptoethanol and protease inhibitor mix (Complete-

Mini; Roche Biochemicals). Lysates were cleared by

centrifugation for 30 min at 14,000 rpm at 4�C. Whole

cell extracts were separated by SDS-PAGE and blotted

onto polyvinylidene difluoride membranes. Blocking was

performed at room temperature for 2 h in TBS (15 mM

Tris, 150 mM NaCl, pH 7.4) with 5% non-fat milk, fol-

lowed by incubation for 2 h at room temperature with

anti-a-subunit of BK channel antibody (LifeSpan Bio-

Sciences, USA; 1:500) or anti-b-actin antibody (Santa

Cruz; USA; 1:1000) diluted in TBS with 1% non-fat milk.

After several washes with TBS containing 0.05% Tween-

20 the membranes were incubated with anti-rabbit IgG-

horseradish peroxidase conjugate at 1:2000 dilution

(DAKO, Ely, UK) for 1 h in TBS with 1% non-fat milk.

Following several washes, proteins were visualized using

enhanced chemiluminescence (Amersham Biosciences,

Sweden) according to the manufacturer’s recommenda-

tions. Quantification of the western blot signal was per-

formed using Image J software. The expression level of

BK channel a-subunit was normalized by b-actin

expression level. Data were shown as the average of three

independent experiments.

Data analysis

Data were presented as means ± standard error of the

mean (SEM). The changes of membrane potential and K?

or BK currents induced by different concentrations of

IL-1b versus control were compared statistically by an

analysis of variance (ANOVA) followed by Student–

Newman–Keuls (S–N–K) post-hoc test. In order to esti-

mate the frequency- and time-dependent effects of IL-1b,

the analysis of covariance and the Student t-test were

used. The threshold of significance was P \ 0.05 or

P \ 0.01.

Results

IL-1b exerts biphasic effect on the membrane potential

of ASMC

In order to investigate if IL-1b affects the membrane

potential of ASMCs, we treated ASMCs with 1, 10, or

50 ng/ml IL-1b for 30 min, 24 h, or 48 h. The membrane

potential was determined by the intensity of DiBAC4 (3)

detected under confocal microscope. DiBAC4 (3) is a

voltage-sensitive fluorescent dye that is lipophilic and

negatively charged. Hyperpolarization results in extrusion

of the dye and then a decrease in cellular fluorescence

Mol Cell Biochem (2010) 338:59–68 61

123

intensity [26, 27]. To our surprise, we found an interesting

biphasic effect of IL-1b on membrane potential of ASMCs.

As shown in Fig. 1, while 30 min application of IL-1bcaused hyperpolarization of ASMCs indicated by a

decrease in fluorescence intensity, long-term treatment of

IL-1b for 24 or 48 h led to depolarization of ASMCs

indicated by an increase in fluorescence intensity. Inter-

estingly, both effects were concentration dependent

(Fig. 1b). 1 or 10 ng/ml IL-1b had no effect when applied

for 30 min, but 50 ng/ml IL-1b almost doubled the mem-

brane potential. After longer treatment for 24 and 48 h, 1

and10 ng/ml IL-1b started to depolarize the cells and

50 ng/ml IL-1b had the maximum effect at 48 h that

decreased the membrane potential to almost half of the

control. These data indicate that IL-1b has biphasic effects

on the membrane potential of ASMCs that is both con-

centration- and time- dependent.

Hydrogen peroxide and BK channel mediate the

modulation of membrane potential by IL-1b

Since there are increasing evidence suggesting that ROS

including hydrogen peroxide (H2O2) are important medi-

ators in interleukin 1 signaling pathway [28], we wanted to

study whether the biphasic effects of IL-1b on membrane

potential was also mediated by H2O2. To this end, we

treated ASMCs with saline or 50 ng/ml IL-1b for 30 min

or 48 h in the absence or presence of 200 U/ml catalase, a

scavenger of H2O2 (Fig. 2). Catalase was applied 40 min

prior to the application of IL-1b. The membrane potential

of ASMCs were detected with DiBAC4 (3), and the

quantifications of the fluorescence intensity were summa-

rized. Compared with saline-treated cells, catalase did not

affect the membrane potential by itself. We observed that

50 ng/ml IL-1b remarkably hyperpolarized ASMCs when

applied for 30 min. The fluorescence intensity of DiBAC4

(3) was reduced to almost half of the control. Interestingly,

this effect was almost completely abolished by H2O2

scavenger catalase (Fig. 2a). A 48-h treatment of 50 ng/ml

IL-1b almost doubled the fluorescence intensity of Di-

BAC4 (3) indicating that IL-1b depolarized ASMCs in this

situation. This effect was partially abolished by H2O2

scavenger catalase (Fig. 2b). Furthermore, the intracellular

H2O2 concentration was increased dramatically by 50 ng/

ml IL-1b treatment both for 30 min and 48 h, and the

effect was significantly diminished by catalase (Fig. 3).

These results suggested that H2O2 mediated the hyperpo-

larization of ASMCs by IL-1b when applied for 30 min,

and H2O2 also played an important role in depolarization of

ASMCs induced by long-term treatment of IL-1b.

It is well known that BK channels have high channel

conductance and high density in VSMCs. It is conceivable

that BK channels may mediate the modulation of mem-

brane potential by IL-1b in ASMCs. Therefore, we

examined the effect of IL-1b on membrane potential in the

presence of 100 nM IBTX, an inhibitor of BK channel, or

30 lM NS1619, an opener of BK channel. We found that

while inhibition of BK channel by IBTX completely

abolished the hyperpolarization effect of IL-1b when

applied for 30 min, BK channel opener NS1619 only

slightly reduced the depolarization effect of IL-1b when

applied for 48 h (Fig. 2a, b). There was no synergistic

effect when catalase in combination with IBTX or NS1619

was applied. These data indicated that when applied for

30 min IL-1b hyperpolarizes ASMCs by activating BK

channel. Suppression of BK channel also plays an impor-

tant role in the depolarization effect of IL-1b when applied

for longer period. However, since opening of BK channel

by NS1619 only slightly rescued the depolarization effect

of IL-1b, there must be some other mechanisms contrib-

uting to the effect of IL-1b. We, therefore, investigated

Fig. 1 Effect of IL-1b on membrane potential in ASMCs. ASMCs

were incubated with 1, 10, or 50 ng/ml IL-1b for 30 min, 24, or 48 h.

The cells treated with saline were used as control. Membrane

potential was measured with DiBAC4(3) using confocal microscopy.

a Representative confocal images showing the effect of IL-1b on

membrane potential; b Quantification of relative fluorescence inten-

sity. The fluorescence intensity of control cells was considered 100%.

* P \ 0.01, compared with control

62 Mol Cell Biochem (2010) 338:59–68

123

whether voltage-gated K (Kv) channels also mediate the

effect of long-term IL-1b on the membrane potential. We

studied the effect of 48-h treatment of 50 ng/ml IL-1b on

membrane potential in the presence of a Kv channel

inhibitor, 5 mM 4-AP or IBTX. As shown in Fig. 2c,

blockage of Kv channel by 4-AP increased the fluorescence

intensity of DiBAC4 (3) indicating 4-AP depolarized

ASMCs. However in ASMCs treated with 50 ng/ml IL-1bfor 48 h, 4-AP alone or co-application of 4-AP and IBTX

didn’t significantly affect the fluorescence intensity of

DiBAC4 (3), suggesting that both BK channel and Kv

channel were suppressed by 48-h treatment of 50 ng/ml

IL-1b. Interestingly, 48-h treatment of 50 ng/ml IL-1bresulted in an even higher fluorescence intensity of Di-

BAC4 (3) than 4-AP treatment, implying the depolarization

effect of 50 ng/ml IL-1b was mediated to a large extent by

the blockage of BK channels and to a less extent by the

blockage of Kv channels.

Biphasic effect of IL-1b on BK channel-dependent K?

current in ASMCs

In order to investigate whether IL-1b affects the whole cell

K? current and what percentage BK channels account for,

we examined whole cell steady-state outward K? currents

in single ASMC. We performed whole cell patch clamping

in ASMCs treated with 50 ng/ml IL-1b for 30 min or for

48 h in the absence or presence of BK channel inhibitor

IBTX (100 nM) in the external solution. The cells treated

with saline instead of IL-1b were used as control. The cell

membrane potential was clamped from 0 mV to ?60 mV

Fig. 2 IL-1b modulates membrane potential by acting on H2O2 and

BK channel. ASMCs were treated with 50 ng/ml IL-1b for 30 min (a)

or for 48 h (b and c) in the presence or absence of H2O2 scavenger

catalase (200 U/ml), BK channel inhibitor IBTX (100 nM), BK

channel opener NS1619 (30 lM), or Kv channel inhibitor 4-AP

(5 mM). The cells treated with saline were used as control. Membrane

potential of ASMCs was measured with DiBAC4 (3) using confocal

microscopy. Data were shown as quantifications of the relative

fluorescence intensity. The fluorescence intensity of control cells was

considered 100%. * P \ 0.01, compared with control. # P \ 0.01,

compared with IL-1b treament alone

Fig. 3 IL-1b increased intracellular H2O2 concentration in ASMCs.

ASMCs were treated with 50 ng/ml IL-1b for 30 min or 48 h in the

presence or absence of H2O2 scavenger catalase (200 U/ml). Catalase

was applied to the cells 40 min prior to the application of IL-1b. The

cells treated with saline were used as control. H2O2 concentration was

determined using standard curve method. * P \ 0.01, compared with

control. # P \ 0.05, compared with IL-1b treament alone

Mol Cell Biochem (2010) 338:59–68 63

123

by a 200-m-long depolarizing step pulse in 10 mV incre-

ments. Whole cell recordings were shown in Fig. 4a.

Current density was obtained by dividing the peak outward

K? current with cell capacitance (pA/pF) and plotted

against the membrane voltage (Fig. 4b).

In whole cell recordings (Fig. 4), we found that 30 min

application of 50 ng/ml IL-1b increased the whole cell

K ? channel current, which was completely abolished by

BK channel inhibitor IBTX. These results suggested that

transient treatment of IL-1b increased BK channel-depen-

dent K? current that led to the hyperpolarization of

ASMCs. However, when ASMCs were treated with 50 ng/

ml IL-1b for 48 h, the whole cell K? channel current was

dramatically decreased and co-treatment with BK channel

inhibitor IBTX had no further effect. These results implied

that 48-h treatment of IL-1b decreased BK channel-

dependent whole cell K? current that led to the depolar-

ization of ASMCs. Interestingly, we also found that when

membrane voltage is 60 mv, K? current density was sig-

nificantly lower in cells treated with IL-1b for 48 h than in

control when BK channels were inhibited by IBTX. This

indicates that to some extent 48-h IL-1b treatment also

suppressed K? current from other K ? channels such as

KV channels. Taken together with the results shown in

Fig. 2c, these results suggest that the suppression of Kv

channels also contributed to the depolarization effect of IL-

1b when applied for 48 h.

IL-1b modulates BK channel activity through H2O2

Since the above results suggested IL-1b modulated mem-

brane potential by acting on BK channel, we wanted to

further investigate whether and how IL-1b affects BK

channel activity of ASMCs. For this purpose, we measured

single BK channel activity of ASMCs using inside-out

patch clamping. We added 5 mM 4-AP into the bath

solution to inhibit the Kv channel activity and held the

membrane potential at ?50 mV. The channels recorded

were confirmed to be BK channels because they could be

blocked by addition of IBTX (Supplemental Fig. 1). BK

channel activity was defined by NPo as described in

‘‘Materials and Methods’’. No channel run-down was

observed during the experiment period. As shown in Fig. 5,

addition of 200 U/ml catalase to the external solution

during inside-out clamping had no effect on single BK

channel activity when ASMCs were treated for 30 min

or for 48 h. An addition of 50 ng/ml IL-1b increased

BK channel activity when applied for 30 min. Interest-

ingly, H2O2 scavenger catalase completely abolished the

enhanced BK channel activity by IL-1b. Together with

the previous findings that catalase completely abolished the

hyperpolarization effect of IL-1b, these data showed that

50 ng/ml IL-1b applied for a short period enhanced BK

channel activity resulting in the hyperpolarization of

ASMCs, and this process was mediated by H2O2.

In order to investigate whether long-term treatment of

IL-1b affects BK channel activity, we treated ASMCs with

saline or 50 ng/ml IL-1b for 48 h in the presence or

absence of 200 U/ml catalase. Catalase was applied 40 min

prior to the application of saline or IL-1b. Single BK

channel activity was then analyzed using inside-out

clamping. We found that BK channel activity was signifi-

cantly decreased by 48-h treatment of 50 ng/ml IL-1b that

could be partially rescued by H2O2 scavenger catalase

Fig. 4 IL-1b modulates BK channel-dependent K? current in

ASMCs. a Whole cell recordings show the effect of 50 ng/ml IL-

1b on whole cell K? current when applied for 30 min or 48 h in the

absence or presence of 100 nM IBTX. The cells treated with saline

were used as control. IBTX was applied to the cells prior to the patch-

clamp experiments. The currents were elicited by incremental 10 mV

depolarizing steps from 0mv to ?60 mV. b Current–voltage

relationships for whole cell K? current in ASMCs. Current density

was obtained by dividing the peak outward K? current with cell

capacitance (pA/pF) and plotted against the memrane voltage.

* P \ 0.05, compared with control. # P \ 0.01, compared with IL-

1b treament alone

64 Mol Cell Biochem (2010) 338:59–68

123

(Fig. 5). Suppression of BK channel activity by longer

treatment of IL-1b may explain, at least, partially its

depolarization effect as shown above. Catalase partially

rescued the suppressed BK channel activity by IL-1bsuggesting that H2O2 also played an important role in this

process.

Long-term treatment of IL-1b inhibits the expression of

BK channel a-subunit

Since the protein expression level of BK channel plays an

important role in regulating BK channel function, we

investigated whether IL-1b affects the expression level of

the a-subunit of BK channel. These experiments were

performed using Western blotting analysis. We found that

30-min application of IL-1b did not change the expression

level of the a-subunit of BK channel (data not shown).

However, long-term treatment (48 h) of 10 and 50 ng/ml

IL-1b significantly down-regulated the expression of the

BK channel a-subunit in a dose-responsive manner, and

this effect was significantly diminished by catalase

(Fig. 6). The down-regulated expression level of a-subunit

of BK channel accounted for the decreased BK channel-

dependent K? current induced by IL-1b and explained why

the depolarization effect of IL-1b could only be partially

abolished by BK channel opener NS1619.

Discussion

In this study, we demonstrated that IL-1b had biphasic

effects on BK channel function and membrane potential of

ASMCs, both being concentration and time dependent. IL-

1b increased BK channel-dependent K? current and hy-

perpolarized ASMCs when applied for 30 min. However,

long-term (24–48 h) treatment of IL-1b resulted in

decreased expression of a-subunit of BK channel, sup-

pressed BK channel activity, decreased BK channel-

dependent K? current, and depolarization of the cells.

The Membrane potential is largely determined by K?

efflux. The two types of K? channels that dominate arterial

smooth muscle K? conductance are BK channels and

voltage-gated K (Kv) channels. IL-1b has been known to

suppress KV channel activity in various cells or tissues

[22–25]. However, little is known about the modulation of

Fig. 6 Expression of BK channel a-subunit is downregulated by

long-term treatment of IL-1b. a Representative western blots show

the expression level of BK channel a-subunit (BK-a) and b-actin in

whole cell extracts of ASMCs treated with saline or IL-1b (1, 10 or

50 ng/ml) for 48 h in the absence or presence of catalase (200 U/ml).

b Quantification of the expression level of BK-a was normalized by

b-actin expression level

Fig. 5 H2O2 mediates the modulation of BK channel channel activity

by IL-1b. a Single channel recordings show the BK channel activity

after 30-min or 48-h treatment by saline or IL-1b (50 ng/ml) in the

absence or presence of H2O2 scavenger catalase (200 U/ml).

b Quantification of BK channel activity (NPo) shown in a.

* P \ 0.01, compared with saline-treated cells. # P \ 0.05, compared

with IL-1b treatment alone

Mol Cell Biochem (2010) 338:59–68 65

123

BK channel function by IL-1b and whether IL-1b also

suppressed KV channel activity in smooth muscle cells. We

found in this study that the hyperpolarization effect of IL-

1b was solely mediated by the increased function of BK

channels. However, the inhibition of both BK channels and

Kv channels mediated the depolarization effect of long-

term IL-1b treatment. In this study, we investigated in

detail how IL-1b modulates the function of BK channels.

Reactive oxygen species has been suggested to be

important mediators in IL signaling pathway [28]. In vas-

cular smooth muscle cells, a major source of ROS is

NAD(P)H oxidase which generates H2O2 [29]. We found

in this study that IL-1b treatment indeed increased intra-

cellular H2O2 level and the effect could be abolished by the

application of catalase, which is a common enzyme to

catalyze the decomposition of hydrogen peroxide (H2O2) to

water and oxygen. The hyperpolarization of ASMCs by a

30-min treatment of IL-1b was almost completely abol-

ished by BK channel blocker IBTX and catalase, indicating

that IL-1b hyperpolarized ASMCs by activating BK

channel and this was mediated by H2O2. H2O2 scavenger

catalase partially abolished the suppressed single BK

channel activity, the reduced expression of BK channel

a-subunit, and the depolarization effect induced by IL-1b;

furthermore, there were no synergistic effect between cat-

alase and BK channel opener NS1619 on depolarization of

ASMCs. These results implied that the suppression of BK

channel function and depolarization of ASMCs induced by

long-term treatment of IL-1b were, at least, partially

mediated by H2O2. Therefore, our results suggest that H2O2

mediated both the stimulatory effect of 30-min treatment of

IL-1b and the inhibitory effect of long-term treatment of

IL-1b on BK channels.

According to the literature, H2O2 may either activate

[30–36] or inhibit [37–41] BK channels. The discrepancy is

likely caused by the different experimental conditions.

First, it seems that the response of BK channel to H2O2 is

dependent on the cell types studied. Different cell types

may have unique cellular environment such as redox state

and uniquely activated signaling pathways, and these dif-

ference may have impact on the regulation of ROS on BK

channel function. For example, all the studies using smooth

muscle cells show excitatory responses of BK channel to

1 lM-1 mM H2O2 [30–32], and that 10 lM-1 mM H2O2

produced concentration-dependent relaxation of endothe-

lium-denuded coronary artery ring within 5-30 min of

administration [30, 32, 34]. Common effects of H2O2

applied to heterologously expressed BK channels in

HEK293 cells and Xenopus oocytes are inhibitory [37–40].

In two studies using cultured human umbilical vein endo-

thelial cells (HUVECs), 100-200 lM or 0.5-1.0 mM

exogenous H2O2 or induction of endogenous H2O2 by

glucose oxidase stimulated BK channels [35, 36].

However, in endothelial cells isolated from porcine renal

arteries, 1 lM-10 mM H2O2 inhibited BK channels in

inside-out patches [41]. Second, it seems that the effects of

H2O2 on BK channels are dependent on the concentration

and duration of H2O2 treatment. All the studies in smooth

muscle cells and HUVECs used lower concentration of

H2O2 (\1 mM) and have stimulatory effect within

5-30 min [30–32, 35, 36]. However, higher concentration

of H2O2 ([1 mM) was used in most of the studies showing

inhibitory effect of H2O2 on BK channel [37–39]. Indeed,

the inhibitory effect of H2O2 on BK channel is concen-

tration and time dependent. As shown in Soto’s study, the

decrease in BK channel activity occurred abruptly after a

lag time that was H2O2 concentration dependent. With

8 mM H2O2, the lag time was about 10 min but was absent

when 23 mM H2O2 was used. When H2O2 concentration

was 4 mM, the channel activity decreased after lag periods

longer than 30 min [38].

In this study, we showed that IL-1b treatment induced

progressive H2O2 generation in smooth muscles. About

80 lM and 120 lM endogenous H2O2 were induced by 30-

min and 48-h treatment of 50 ng/ml IL-1b, respectively.

The early stimulative effects are in agreement with the

published data showing that in smooth muscle cells lower

concentration of H2O2 increases BK channel activity

within 20–30 min of application in both cell-attached patch

clamp and inside out patch clamp [30–32]. H2O2 is

reported to either directly or indirectly activate BK chan-

nels in smooth muscle cells. The indirect mechanisms

involve Ca2? sparks [33], phospholipase A2 (PLA2)/ara-

chidonic acid signaling cascades [30, 31], and the cGMP-

dependent signaling pathway [32]. The mechanisms by

which H2O2 mediates the stimulative effect of 30-min

treatment of IL-1b on BK channel were not addressed in

this study, and need to be further investigated.

It has been shown that the inhibitory effects of H2O2 on

BK channels result from the decreased number of channels

available to open [38, 39] and the decreased open proba-

bility due to the oxidation of cysteine residue 911 of BK

channel a-subunit and the decreased [37, 40]. If tissue has a

high superoxide level, increased NO release induced by

H2O2 could interact with superoxide to form peroxynitrite

which inhibits BK channels in vascular tissue and causes

vasoconstriction [42, 43]. All these processes are time

consuming. In contrast to stimulative effect of H2O2 on BK

channel which occurs shortly after IL-1b treatment, the

inhibitory effect of H2O2 on BK channel may involve

procedures that take longer time to occur. Indeed, our

results demonstrated that H2O2 partially mediated the

inhibitory effect on both BK channel activity and a-subunit

expression induced by long-term application of IL-1b. It is

likely that in the early stage of IL-1b treatment, low level

of H2O2 is generated resulting in the activation of BK

66 Mol Cell Biochem (2010) 338:59–68

123

channels. However, after long-term treatment of IL-1b, the

inhibitory effect of H2O2 involving more complex mech-

anisms dominates and leads to the inhibition of BK chan-

nels. Since H2O2 scavenger only partially abolished the

inhibitory effect of IL-1b, other mechanisms that mediate

the intermediate and late events in IL-1 signaling may also

be involved, e.g., NF-jB signaling pathway [28].

IL-1b is one of the cytokines that play important roles in

the development and progression of a variety of cardiovas-

cular diseases, most notably coronary atherosclerosis and

congestive heart failure [13]. The effects of IL-1b on vas-

cular reactivity are controversial. Some studies suggest that

IL-1b renders vessels hyporesponsive to vasoconstrictors by

NO-dependent [44–46] and NO-independent mechanisms

[14–17]. However, in vivo treatment with IL-1b increases

the responsiveness of mesenteric arteries to vasoconstrictors

[18] and causes constrictive remodeling and vasospasm in

coronary arteries [20, 21]. Incubation of temporal arteries

with IL-1b for 2 days causes increased vessel responsive-

ness to endothelin B receptor agonists [19]. The discrepancy

may be caused by different experimental conditions, in

which the time course of IL-1b treatment is important. As

reviewed by Prabhu S. D., the responses to cytokines involve

early response that is dependent on the ambient physiologic

milieu and relative contributions of the underlying signaling

pathways that are activated, such as NO and Ca2? transient.

The late response lasting from hours to days depends on the

production of secondary mediators and the combined

influence of NO, ROS, and other signaling pathways [47].

The results from this study showed that IL-1b indeed had

biphasic effects on BK channel and membrane potential.

The early effect of IL-1b release during inflammation may

cause vasorelaxation due to the increased function of BK

channel in smooth muscle cells. However, longer period

exposure to IL-1b may result in vasoconstriction due to the

decreased BK channel open probability and reduced

expression level of the alpha subunit of BK channel, as well

as the decreased function of Kv channel.

In conclusion, we found that IL-1b had biphasic effects

on BK channel function and membrane potential of

ASMCs, both being concentration and time dependent. IL-

1b increased BK channel function and hyperpolarized

ASMCs when applied for 30 min, while long-term (24–

48 h) treatment of IL-1b resulted in decreased expression

of a-subunit of BK channel, decreased BK channel activity,

suppressed Kv channel function, and depolarization of the

cells. The early effect of IL-1b to activate BK channel is

mediated by H2O2. The long-term effect of IL-1b to inhibit

BK channel was partially mediated by H2O2. Other sig-

naling pathways may also be involved. These results may

provide important molecular mechanisms for therapeutic

strategies targeting BK channel in inflammation-related

diseases.

Acknowledgements This study was supported by the National

Basic Research Program (‘‘973’’program) of China (2005CB523310).

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