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Am J Physiol Lung Cell Mol Physiol 292: L898-L907, 2007. First published December 8, 2006; doi:10.1152/ajplung.00461.2005
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Bradykinin activates calcium-dependent potassium channels in cultured human airway smooth muscle cells

Bo Liu, Anette M. Freyer, and Ian P. Hall

Division of Therapeutics and Molecular Medicine, University of Nottingham, Queens Medical Centre, Nottingham, United Kingdom

Submitted 2 November 2005 ; accepted in final form 1 December 2006


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Bradykinin (BK) is an inflammatory mediator that can cause bronchoconstriction. In this study, we investigated the membrane currents induced by BK in cultured human airway smooth muscle (ASM) cells. Depolarization of the cells induced outward currents, which were inhibited by tetraethylammonium (TEA) in a concentration-dependent manner with an IC50 of 0.33 µM. The currents were increased by elevating intracellular free Ca2+ concentration, suggesting they are calcium-activated potassium channels [IK(Ca)]. Preexposure to inhibitor of IK(Ca) of large conductance (BKCa), iberiotoxin, and small conductance (SKCa), apamin, inhibited the increase of outward current induced by BK. The relative contribution of BKCa was greatest in early passage cells. Both nickel and SKF-96365 (10 µM) inhibited the increase of the IK(Ca) induced by BK; however, the L-type Ca2+ channel blocker, nifedipine, had no effect. Activation of the BK-induced current was inhibited by heparin, indicating dependence on intact inositol 1,4,5-triphosphate (IP3)-sensitive intracellular Ca2+ stores. BK also increased inositol phosphate accumulation and induced a transient Ca2+-activated chloride current (CACC) and a sustained nonselective cation current (ICAT). In summary, BK activates BKCa, SKCa, CACC, and ICAT via IP3-sensitive stores in human ASM.

asthma; airway tone; bronchial relaxation

THE CONTRACTILE STATE OF airway smooth muscle (ASM) is a critical determinant of airway caliber and is dependent on a range of inputs from G protein coupled receptors and ion channels. Ca2+ plays a key role in this process: release from intracellular stores leads to the initial phase of the contractile response to spasmogens, while influx from extracellular sources through a poorly defined pathway contributes to the maintenance of contraction (27). These pathways also are important in both the synthetic and mitogenic responses of airway myocytes and myofibroblasts (44). A range of different ion channels contributes to or modulates these processes. ASM expresses both voltage-dependent and receptor/store-operated Ca2+ channels; however, functional studies demonstrate that inhibition of voltage-dependent Ca2+ pathways do not play a major role in the control of ASM tone (17, 21, 31). In contrast, Ca2+ entry through nonvoltage-dependent pathways following stimulation by agonists such as histamine and bradykinin (BK) appears to be important in the control of the contractile response (30). In previous work, we (6) have shown that ASM expresses a range of transient receptor potential channel homologues, which may contribute to this influx pathway.

Relaxant responses of ASM to agents such as isoprenaline are at least in part dependent on activation of K+ channels (26, 29), although other pathways including altered sensitivity of the contractile apparatus to Ca2+ may also play a role (7). The exact mechanism whereby ASM tone returns to baseline after exposure to agonists, which raise intracellular Ca2+, remains unclear. As well as sequestration and efflux of Ca2+ (20), stimulation of calcium-activated potassium channels [IK(Ca)] by the elevated intracellular Ca2+ is thought to contribute to membrane hyperpolarization and thus relaxation (26, 29).

In ASM ex vivo, the major K+ current is carried by IK(Ca) of large conductance (BKCa) (29, 50), but ASM is also known to express a number of other K+ channels that may contribute to the control of K+ efflux (40, 42) including Ca2+-activated K+ channels of intermediate or small conductance (SKCa), delayed rectifier K+ (KIR) (11, 48), and ATP-sensitive K+ (KATP) (32, 33) channels.

ASM cells are very plastic, and relative channel expression is known to alter once ASM cells are grown in culture (41). Furthermore, ion channel activity is altered in ASM cells from rats with a hyperresponsive phenotype (45), in atrial myocytes grown in an altered extracellular matrix environment (49), in smooth muscle cells exposed to proinflammatory mediators (19), and following exposure to environmental toxins (52). K+ channels other than BKCa may thus assume greater importance in ASM cell function in the asthmatic airway, where the matrix environment is altered and inflammatory mediators abound.

Here we show in cultured ASM, where BKCa activity is partly downregulated but some IK(Ca) activity persists, that BK is able to stimulate KCa activity through release of Ca2+ from internal stores via inositol 1,4,5-triphosphate (IP3)-dependent pathways and Ca2+ influx. BK also induces a large calcium-activated chloride channel (CACC), which, in turn, results in a subsequent influx of Ca2+ from extracellular sources via a current with properties typical of nonselective ion channel current (ICAT).


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Collection and dissociation of ASM. Following informed written patient consent and local ethical committee approval (Ethics Committee of City Hospital, Nottingham, United Kingdom), macroscopically normal proximal bronchi of patients undergoing thoracotomy for lung cancer were obtained immediately after operation and transferred in cold physiological salt solution (PSS) (see Solutions and chemicals) to the laboratory within 20 min. Bronchial tissue was washed three times in HEPES-buffered, calcium and magnesium-free HBSS. ASM was carefully stripped from cartilage of the airway and minced finely in HBSS supplemented with collagenase A (2 mg/ml) and incubated in the same enzymatic solution for 1 h at 37°C with gentle trituration every 15 min. Cells were collected following filtration through a sterile, 100-µm cell strainer and seeded on a 25-mm plastic cell culture plate at a density of 5–10 x 103 cells/cm2 in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FCS, 2 mM glutamine, 50 U/ml penicillin, and 50 µg/ml streptomycin. Cells were grown at 37°C in a humidified incubator under 5% CO2 and exhibited more than 95% smooth muscle {alpha}-actin staining. Cells were also stained for myosin heavy chain. Cells were passaged with 0.05% trypsin and 0.5 mM EDTA. We used cells from four separate donors during passages 1–7.

Patch-clamp electrophysiology. Conventional whole cell configuration patch-clamp technique was employed to record from cultured human ASM. The cells were placed directly in the cell chamber mounted on the stage of an inverted microscope (Nikon Eclipse 200), allowed to settle, and then washed with PSS at a constant speed (6 ml/min). Pipettes were drawn from borosilicate glass on a pipette puller PIP5 (HEKA, Lambrecht, Germany) and had resistances of 4–7 M{Omega} when filled with electrolyte. Automatic series resistance compensation was performed routinely and monitored continuously. Recordings were terminated if the access resistance changed by more than 25% during the recording period.

Voltage pulses were delivered through an EPC-9 amplifier. Data were filtered at 1 kHz (–3 dB down) and sampled at 2 kHz using Pulse software (v8.53, HEKA). Cell resting membrane potentials were recorded in current-clamp mode immediately after a stable electrical access had been established. Following this, the cells was depolarized from a holding potential of –60 mV ranging from –100 mV to +100 mV in 10-mV steps for a duration of 400 ms for recording of the outward currents. For recording of inward chloride currents, the cells were voltage-clamped at –60 mV. Reagents containing experimental drugs were delivered through a 1-µm-diameter puffer pipette connected to a pressure injection device controlled electronically (DAD system). The pipette was positioned about 50–60 µm from the cell. The application of the drug into cell was triggered by an offset sent by the amplifier to the command voltage signal. All experiments were carried out at room temperature (20–24°C).

Additional experiments were performed in current clamp mode. Changes in cell membrane potentials were continuously monitored in current clamp mode when stable whole cell recording had been achieved.

[3H]inositol phosphate accumulation. Medium was aspirated from confluent monolayers of primary human ASM cells grown in a 24-well plate and replaced with 300 µl of inositol-free DMEM containing [3H]myo-inositol at 2 µCi/ml for 24 h. After this time, the medium was again removed and cells washed twice with 1 ml of Hanks'/HEPES buffer. Cells were kept at 37°C while 300 µl of Hanks'/HEPES containing 10 mM of LiCl was added to each well for 15 min and agonist added for the final 10 min as indicated. Reactions were stopped by removing the medium and adding 1 ml of methanol/0.12 M HCl (1:1 vol/vol), which had been stored at –20°C. Samples were stored at –20°C for at least 30 min. An aliquot (800 µl) from each well was neutralized to pH 7 with an appropriate volume (typically 4.8 ml) of buffer (25 mM Tris/0.5 M NaOH/H2O at 0.238/0.025/0.737 vol/vol/vol). [3H]inositol phosphates were separated from free [3H]myo-inositol by anion exchange chromatography on Dowex-Cl columns as per Daykin et al. (8).

Solutions and chemicals. PSS consisted of NaCl (135 mM), KCl (5 mM), MgCl2 (1 mM), CaCl2 (1 mM), and HEPES (10 mM) and was pH-adjusted to 7.2 with NaOH. Intracellular solution, consisting of KCl (140 mM), MgCl2 (1 mM), EGTA (0.01 mM), Mg(ATP)2 (2 mM), HEPES (10 mM), pH adjusted to 7.2 with KOH, was used to record the outward currents. For recording of inward currents, KCl was replaced by cesium: CsCl (140 mM), MgCl2 (1 mM), CaCl2 (1 mM), EGTA (3 mM), Mg(ATP)2 (2 mM), HEPES (10 mM), pH adjusted to 7.2 with CsOH, and 10 mM tetraethylammonium (TEA) was included in the bath solution.

Collagenase A, Igepal, BSA, BK, iberiotoxin (Ibt), TEA, nifedipine, SKF-96365, lanthanum, nickel, cobalt, de-Arg10-Hoe 140 and N{alpha}-adamantaneacetyl-D-Arg-[Hyp3, Thi5,8, D-Phe7]-BK were purchased from Sigma (Poole, Dorset, United Kingdom). Monoclonal mouse anti-smooth muscle {alpha}-actin antibody and myosin heavy chain antibody was purchased from Sigma and a goat-anti-mouse immunoglobulin (Ig) G from Dako (Glostrup, Denmark).

Data and statistical analysis. Data acquisition and analysis were performed with the Pulse (v8.53) software and Microsoft Excel (Microsoft, Redmond, WA). Results are expressed as the means ± SE of n observations. Statistically significant differences were evaluated by using ANOVA or paired or unpaired two-tailed Student's t-test as appropriate. A P value of <0.05 was considered to be significant.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Basal electro-physiological properties of human ASM. The resting membrane potentials of ASM was –36.98 ± 2.89 mV and the membrane capacitance 140.08 ± 6.02 pF (both n = 29). Step depolarizations of ASM from –100 mV to +100 mV from a holding potential of –60 mV led to the activation of slowly activating outward currents. Pharmacological dissection of the currents with 1, 10, and 20 mM TEA reduced mean outward currents at +100 mV by 25.2 ± 1.6% (P > 0.05), 56.2 ± 4.6% (P < 0.05), and 63.4 ± 2.9% (P < 0.01; n = 12, respectively) in a concentration-dependent manner. Steady-state I-V curve analysis demonstrated depolarization-induced outward currents with delayed rectifier properties (Fig. 1).


Figure 1
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  		Fig. 1. A–F: Effect of different concentrations of tetraethylammonium (TEA) on membrane currents (I) induced by bradykinin (BK; 1 µM) in cells depolarized from –100 to +100 mV [holding potential (hp) = –60 mV) with 10 mV steps, each with a duration of 400 ms. Application of TEA inhibited the induced currents in a concentration-dependent manner. Vm, membrane voltage.

 
BK-induced currents in human ASM. Application of different concentrations of BK (0.01–10 µM) caused an increase in both inward and outward currents in a dose-dependent manner (1 µM maximally increased current 3.2 ± 0.37-fold cf. baseline at 100 mV, EC50 = 0.33 µM, n = 14). The increase of current occurred over a wide voltage range (–100 mV to +100 mV) (Fig. 2, A–G). BK (1 M) caused a marked depolarization of these cells (Fig. 2H), which was almost completely abolished by the addition of 100 µM niflumic acid (data not shown).


Figure 2
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  		Fig. 2. A–G: Effect of BK on IK induced by depolarizing cells from –100 to +100 mV (hp = –60 mV) in 10-mV steps, each with a duration of 400 ms. BK caused a robust increase of the current in a concentration-dependent manner (0.01–1 µM). H: depolarization of membrane potentials induced by BK in the current clamp mode.

 
The outward current was dependent on intracellular calcium, as increasing estimated intracellular free calcium from 133 nM (n = 9) to 937 nM (n = 12) increased the current by 80–90% (P < 0.05) between +20 to +60 mV (data not shown).

To study the inward current further, the main charge carrier in the pipette was changed to cesium, and the cells voltage-clamped at –60 mV. Addition of BK (1 µM) induced a biphasic current: a transient inward current of large amplitude and a sustained current of small amplitude, characteristics suggestive of CACC and ICAT respectively (data not shown) (24).

Role of IK(Ca) of large and small conductance in BK-induced currents. To attempt to define the channels underlying the BK-induced current, we studied early and late passage ASM cells from four different donors. In early passage (passages 2–3) cells, preincubation of cells with two concentrations of Ibt (100 nM), an inhibitor of BKCa, inhibited BK-induced currents by 90 ± 15% (n = 6; Fig. 3). The magnitude of BK-induced IK(Ca) at –90 mV in these early passage cells was 8.4 ± 2.5 pA/pF. The BK-induced current in later passage (passages 6–7) cells was reduced in magnitude (4.7 ± 1.4 pA/pF), and Ibt was less effective at blocking IK(Ca) in these cells (64 ± 12% inhibition, n = 6), suggesting that, in early passage cells, the majority of IK(Ca) represents residual BKCa, whereas, in the later passage cells, a greater component of IK(Ca) is carried by SKCa. In keeping with this observation, apamin (100 nM), which blocks SKCa and had no effect on the basal currents induced by depolarization, partially abrogated currents induced by BK (1 µM) in late passage cells (Fig. 4). Similarly, inward currents induced by histamine, a well-characterized contractile agonist, were also inhibited in later passage cells by apamin. Apamin decreased histamine-induced outward current density at 100 mV by 41% (n = 3; P < 0.01).


Figure 3
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  		Fig. 3. Effect of iberiotoxin (Ibt; 100 nM) on BK-induced outward currents in cells of different passages. Currents were expressed as drug-sensitive currents by digitally subtracting the leak currents before drug application. A and B: the effect of Ibt (n = 7) on BK-induced potassium currents in cells at early passage (passages 2–3) and late passage (passages 6–7), respectively. In cells of early passages, BK (1 µM) induced a bigger response than that in late passages, and inhibitory effect of Ibt was also more obvious.

 

Figure 4
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  		Fig. 4. A–F: Effect of apamin (100 nM) on BK-induced IK. Steady-state I-V curves were obtained by applying depolarizing pulses ranging –100 to +100 mV from an hp of –60 mV in 10-mV increments for 400-ms durations. Currents were compared between control, apamin, BK only, and BK plus apamin. BK greatly inhibited the outward current induced in human airway smooth muscle, when the cells were preincubated with apamin in late passage cells (passage 6).

 
Effects of Ca channel blockers on BK-induced currents. As these findings highlight a central role of calcium in depolarization and BK-induced signaling, we set out to characterize currents induced by BK further. The effects of cobalt (Co2+, 100 µM), nickel (Ni2+, 100 µM), lanthanum (La3+, 100 µM), and SKF-96365 (10 µM, a partially selective blocker of receptor-operated Ca2+ entry pathways) were tested on BK-induced inward and outward currents in ASM cells. Figure 5 shows a typical biphasic inward membrane current response when 1 µM BK was applied directly to the cells voltage-clamped at –60 mV. Applying BK to the cell for 35 s, a transient, rapidly inactivating, low-noise inward current was evoked after initial delay of about 5 s, followed by noisy, sustained, small-amplitude currents. The mean peak amplitude of the transient inward currents from nine cells was –1,040.2 ± 160.54 pA and that of the sustained inward currents was –245.55 ± 54.22 pA (measured at the end of BK application). These currents are akin to those identified in ASM cells in response to acetylcholine: the transient rapidly inactivating inward CACC and the sustained ICAT (24). Treatment of cells with Ca2+ channel blockers, Co2+ (100 µM), Ni2+ (100 µM), La3+ (100 µM), and SKF-96365 (10 µM), for 10 min decreased CACC and ICAT by 59.05% (P < 0.05; n = 6) and 18.65% (P > 0.05; n = 6) (Fig. 5, A and E), 47.59% (P < 0.05; n = 6) and 12.65% (P > 0.05; n = 6) (Fig. 5, B and F), 82.57% (P < 0.01; n = 8) and 61.11% (P < 0.05; n = 6) (Fig. 5, C and G), and 64.42% (P < 0.01; n = 12) and 53.82% (P < 0.05; n = 12) (Fig. 5, D and H), respectively. To confirm that the inhibition of the inward currents translated into inhibition of the outward current, we studied the effect of the Ca2+ channel blockers on the K+ current. Co2+ (100 µM), Ni2+ (100 µM), and SKF-96365 (10 µM) inhibited the BK-induced amplification by 10.13% (P > 0.05; n = 6), 26.08% and 34.54% (both P < 0.05; n = 6), respectively (Fig. 6). Nifedipine, a voltage-gated calcium channel antagonist, did not alter BK-induced inward or outward currents significantly (Fig. 7).


Figure 5
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  		Fig. 5. Effect of cations and SKF-96365 on BK-induced inward currents. BK (1 µM) applied directly to the cells voltage-clamped at –60 mV for 35 s evoked transient, rapidly inactivating, low-noise inward current (CACC) followed by noisy, sustained, small amplitude currents (ICAT) at the end of BK application. Treatment of cells with Co2+ (100 µM), Ni2+ (100 µM), La3+ (100 µM), and SKF-96365 (10 µM) all inhibited CACC, whereas Ni2+, La3+, and SKF-96365 inhibited ICAT, while Co2+ had no obvious effect on ICAT. A and E: Co2+; B and F: Ni2+; C and G: La3+; D and H: SKF-96365. *P < 0.05; **P < 0.01 compared with control.

 

Figure 6
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  		Fig. 6. Effect of Ni2+, Co2+, and SKF-96365 on BK-induced Ca2+-activated K+ channels of intermediate or small conductance (SKCa). Peak current densities at –100 mV were obtained by applying depolarizing pulses ranging from –100 to +100 mV with a duration of 400 ms. Both Ni2+ (100 µM) and SKF-96365 (SKF; 10 µM) inhibited BK-induced SKCa, whereas Co2+ had no obvious effects.

 

Figure 7
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  		Fig. 7. A–F: The L-type Ca2+ channel antagonist, nifedipine (Nif), does not inhibit BK-induced potassium currents. Steady-state I-V curves of outward currents were induced by applying depolarizing pulses from –100 to +100 mV (hp = –60 mV) in 10-mV increments for a duration of 400 ms. Nifedipine (1 µM) had no effects on outward currents induced by BK (1 µM).

 
Depletion of intracellular stores inhibits both inward and outward currents induced by BK. As these data suggested a role for sarco-endoplasmic reticulum Ca2+-ATPase (SERCA)-regulated Ca2+ stores in BK-induced CACC and ICAT, we investigated the effect of thapsigargin, a selective inhibitor of SERCA of intracellular organelles. Cells were dialyzed with electrode solution containing thapsigargin (2 µM) for 5-min rupture of the membrane. BK was then applied directly to the cell voltage-clamped at –60 mV. In a series of experiments, predepletion of intracellular stores by thapsigargin reduced CACC and ICAT by 92% (P < 0.001; n = 11) and 77% (P < 0.05; n = 11), respectively, and K+ currents by 60.81% (P < 0.01; n = 7) when cells were held at 100 mV compared with control group (Fig. 8).


Figure 8
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  		Fig. 8. Predepletion of intracellular stores blocks the BK-induced CACC and SKCa. Intracellular electrode solution containing thapsigargin (Thaps; 2 µM) was used to dialyze the cells, which were voltage-clamped at –60 mV. BK (1 µM) was delivered through a puffer pipette directly to the cells. CACC induced by BK were continuously recorded. SKCa were induced by depolarizing the cells from –100 to +100 mV from an hp of –60 mV. A: control; B: following application of Thaps for 5 min prior to BK; C: summary of effects of BK on CACC; D: effect of Thaps on BK induced SKCa. **P < 0.01 compared with BK only.

 
Role of IP3 and ryanodine receptors in BK-induced inward and outward currents. To confirm the importance of internal calcium stores in BK-induced calcium signaling, we studied the effect of 10 mg/ml heparin (which inhibits IP3 receptor-mediated Ca2+ release) and 50 µM ruthenium red (a ryanodine receptor antagonist) on BK-induced currents. Figure 9 shows typical examples of these experiments. CACC in the heparin group (n = 8) was decreased by 88% (P < 0.001) compared with control (n = 12), ICAT by 27% (P > 0.05), and K+ efflux by 82.04% (P < 0.01; n = 7), while the negative control de-N-heparin (n = 8) had no significant effect. Furthermore, 10 µM 2-aminoethoxydiphenyl borate (n = 10) decreased BK-induced Ca2+ influx by 78% (P < 0.001) and K+ efflux by 48% (P < 0.01). Ruthenium red (n = 7) had no effect on CACC, had variable effects on the K+ currents, and increased ICAT in cultured ASM (results not shown).


Figure 9
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  		Fig. 9. Effect of blocking of inositol 1,4,5-triphosphate (IP3) receptors or ryanodine receptors on BK-induced CACC and SKCa. BK (1 µM) was applied directly onto the cells using a puffer pipette. The cells were dialyzed with low molecular weight heparin (10 mg/ml), de-N-heparin (10 mg/ml), or ruthenium red (RR; 50 µM) for 5 min. CACC were induced by voltage-clamping the cells at –60 mV, and SKCa were induced by depolarizing the cells from –100 to +100 mV from an hp of –60 mM. A: control; B: de-N-heparin; C: heparin; D: ruthenium red; E: effect of heparin (10 mg/ml) on BK-induced SKCa compared with de-N-heparin; F: effect of ruthenium red (50 µM) on BK-induced SKCa.

 
BK induces inositol phosphate production. We sought support for the hypothesis that internal calcium stores were central to BK-mediated signaling by measuring inositol phosphate formation in response to BK. BK (1 µM) maximally induced a 33-fold increase in total [3H]inositol phosphate accumulation (EC50 = 8.95 nM, 95% confidence intervals 4.58–17.47 nM, n = 4) in human ASM cells.

Activation of both inward and outward currents by BK is mediated through B2 receptors. To verify that the BK effect was specific, we used the B1 antagonist, de-Arg10-Hoe 140, and the B2 receptor antagonist, N{alpha}-adamantaneacetyl-D-Arg-[Hyp3, Thi5,8, D-Phe7]-BK in an attempt to inhibit BK-induced currents. N{alpha}-adamantaneacetyl-D-Arg-[Hyp3, Thi5,8, D-Phe7]-BK inhibited the BK-induced outward potassium currents in a concentration-dependent manner, whereas de-Arg10-Hoe 140 produced no significant effect on BK-induced CACC or ICAT (Fig. 10).


Figure 10
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  		Fig. 10. Effect of BK receptor antagonists on BK-induced SKCa and CACC. The cells were preincubated with either N{alpha}-adamantaneacetyl-D-Arg-[Hyp3, Thi5,8, D-Phe7]-BK, a B2-selective antagonist, or de-Arg10-Hoe 140, a B1-selective antagonist, for 5 min. BK-induced SKCa were either induced by depolarizing the cells from –100 to +100 mV (hp = –60 mV, duration 400 ms, and 10-mV steps) or by holding the cells at –60 mV for induction of CACC. A: steady-state I-V curve showing inhibitory effects of B2 antagonist (1 µM) on SKCa; B: preincubation with the B2 antagonist inhibits the BK-induced CACC in a concentration-dependent manner (0.001–1 µM); C: B1 antagonist (1 µM) failed to reverse BK-induced CACC.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Asthma is a common disease characterized by airway inflammation, hyperresponsiveness, and airway remodeling. In asthmatic patients, increased BK and its related kinins have been detected (35). Kinins are produced endogenously in many tissues and are implicated in both physiological and pathophysiological conditions. In the airways, BK exerts multiple effects, including bronchoconstriction, vasodilatation, plasma extravasation, and cough, by acting directly on ASM or indirectly due to the release of other inflammatory mediators. It has long been recognized that asthmatic individuals are more sensitive to the bronchoconstriction properties of BK than nonasthmatic individuals (12). More recent studies have determined that inflammation upregulates BK receptor expression in ASM cells (38, 53) and potentiates calcium signaling in response to BK (1, 2). Potential underlying reasons for these effects include the observations that inflammatory cytokines upregulate G protein expression (18) and increase phospholipase activity and inositol turnover (51). However, less is known about the relaxant mechanisms that follow the bronchoconstrictor response. As K+ channels are thought to be important in the regulation of both basal smooth muscle cell tone and agonist-induced relaxation (5, 15, 22, 25, 29, 46), we decided to investigate the effect of BK on K+ channel activity.

Cultured human ASM are a widely used and validated model for the study of ASM cell physiology and pharmacology as they retain many of their ex vivo characteristics but show important differences to ASM from other species (14). This present study thus extends observations previously made in animal ASM cells. Initial characterization of cultured human ASM cells at baseline confirmed the presence of a Ca2+-dependent K+ current with delayed rectifier properties (Fig. 1) in keeping with earlier findings in ferret cells (11, 42).

Early passage cells retained some BKCa activity, which has been demonstrated in acutely dissociated cells (Fig. 3; Refs. 23, 28, 41, 42), although the magnitude of this current was reduced compared with those previously described in acutely dissociated cells, and in later passage cells, BKCa declined further, probably because of progressive loss of expression of this channel in culture (41). BK increased K+ efflux in a concentration-dependent manner (Fig. 2). In early passage cells, the majority of this increase was Ibt sensitive, and in later passage cells, an element of BK-induced BKCa remained; however, in these later passage cells, apamin also reduced BK-induced current, suggesting a contribution from BK activation of SKCa, which is known to be expressed in both fresh and cultured ASM cells (41). The progressive reduction in BK-mediated activation of BKCa (47) is probably because BKCa channel expression is gradually lost following prolonged cell culture. The mechanism for the transition from predominant K+ channels of large conductance identified in acutely dissociated cells to that of small conductance in later passage cultured human ASM (41) is not very clear. ASM isolated from bronchial tissues undergo morphological and physiological transformation in vitro, that is from a contractile phenotype to a synthetic one, which is thought to mirror some of the in vivo changes during airway remodeling (13). This raises the interesting possibility that during remodeling and ASM phenotype transformation BKCa may be downregulated, leaving SKCa as a major mechanism in regulating and counteracting effects of raised cytosolic Ca2+ concentrations.

The type of calcium signal that activates IK(Ca) appears to depend on the cell type (43). The signaling pathway underlying BK-induced currents in ASM cells is still uncertain. Several studies have shown that BK is a Gq/G11-coupled receptor agonist with the potential to couple to phosphatidylinositide-specific phospholipase C (PI-PLC) (45), phosphatidylcholine phospholipase C (PC-PLC) (37), or phospholipase D (36). Stimulation of PI-PLC will promote the production of the second messenger, IP3, which releases Ca2+ from intracellular stores to the cytoplasm, and 1,2-diacylglycerol, which activates PKC. In addition to this Ca2+ signaling pathway, Hyvelin and colleagues have described functional ryanodine/caffeine-sensitive Ca2+-release channels in ASM cells, which appear to modulate BK-induced Ca2+ signaling (18a). In the current study, we used heparin, an antagonist of IP3 receptors on the intracellular stores, and ruthenium red, a relatively specific antagonist of ryanodine receptors (RYR), and found that heparin abolished the BK-induced currents, while ruthenium red had little effect (Fig. 8). We deduced, therefore, that in this system, BK-induced currents were activated in an IP3-dependent manner but RYR receptors are probably not involved. The observation that BK activates phospholipase C in these cells leading to inositol phosphate accumulation adds weight to this conclusion. The central role of intracellular calcium stores was confirmed by the observation that pretreatment with thapsigargin, which depletes these stores, also inhibited BK-induced K+ currents (Fig. 7).

ASM cells express voltage-gated and nonvoltage-gated Ca2+ channels (both receptor- and store-operated) (30, 44). As Ni2+ and SKF-96365, although not Co2+ or nifedipine (Figs. 5 and 6), inhibited BK-induced K+ efflux, this suggests that opening of the BK-induced K+ channels relies on nonvoltage-dependent Ca2+ channel activity. These findings are in keeping with published characterizations of BK-induced calcium influx (30, 36, 39), although the molecular identity of the Ca2+ influx channels remains to be identified. We have also recently demonstrated a key contribution from STIM1 in signaling Ca2+ influx following store depletion in these cells, although the molecular identity of the channel(s) through which this occurs remains unclear (34). Thus BK activates IK(Ca) and causes release of Ca2+ from intracellular stores and activation of capacitative Ca2+ channels in an IP3-dependent manner and is probably reliant on Ca2+-activated chloride channels as BK-induced depolarization is inhibited by niflumic acid (Fig. 2; Ref. 30). Kinins act through specific receptors that are widespread and belong to two major categories, B1 and B2. B3 receptors are also expressed in cultured ASM cells, although less is known about their function (10). B1 receptor expression is inducible, has been shown to be modulated by interleukins, and predominantly mediates the rapid acute response of asthma (smooth muscle contraction or relaxation) (3, 4). B2 receptors are responsible for most of the biological effects of kinins, including arterial vasodilatation, plasma extravasations, vasoconstriction, activation of sensory fibers, and stimulation of the release of prostaglandins, endothelium-dependent relaxing factor (from endothelia), noradrenalin (from nerve terminals and adrenals), and other endogenous agents (16). The pharmacological characteristics of the B2 receptor sites mediating this array of biological effects show differences between species. This study suggests that BK-induced currents are mediated through B2 receptors rather than B1 receptors, although we did not directly examine the possibility that atypical or B3 receptors are involved in BK-induced Ca2+ and K+ signaling.

In conclusion, we have shown that BK stimulates a biphasic Ca2+ response: release from IP3-sensitive stores and influx through store-operated cation channels, which then activates a K+ efflux through Ca2+-activated K+ channels involving both BKCa and SKCa.


   GRANTS
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was funded in part by Asthma UK Grant 02/010.


   ACKNOWLEDGMENTS
 
We thank the cardiothoracic surgeons and pathologists at the Nottingham City Hospital for their help in obtaining tissue samples.


   FOOTNOTES
 

Address for reprint requests and other correspondence: I. P. Hall, Div. of Therapeutics and Molecular Medicine, University Hospital of Nottingham, Nottingham NG7 2UH, UK (e-mail: ian.hall{at}nottingham.ac.uk)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Amrani Y, Bronner C. Tumor necrosis factor alpha potentiates the increase in cytosolic free calcium induced by bradykinin in guinea-pig tracheal smooth muscle cells. C R Acad Sci III 316: 1489–1494, 1993.[Medline]
  2. Amrani Y, Martinet N, Bronner C. Potentiation by tumour necrosis factor-alpha of calcium signals induced by bradykinin and carbachol in human tracheal smooth muscle cells. Br J Pharmacol 114: 4–5, 1995.[ISI][Medline]
  3. Bryborn M, Adner M, Cardell LO. Interleukin-4 increases murine airway response to kinins, via up-regulation of bradykinin B1-receptors and altered signalling along mitogen-activated protein kinase pathways. Clin Exp Allergy 34: 1291–1298, 2004.[CrossRef][ISI][Medline]
  4. Christiansen SC, Eddleston J, Woessner KM, Chambers SS, Ye R, Pan ZK, Zuraw BL. Up-regulation of functional kinin B1 receptors in allergic airway inflammation. J Immunol 169: 2054–2060, 2002.[Abstract/Free Full Text]
  5. Corompt E, Bessard G, Lantuejoul S, Naline E, Advenier C, Devillier P. Inhibitory effects of large Ca2+-activated K+ channel blockers on beta-adrenergic- and NO-donor-mediated relaxations of human and guinea-pig airway smooth muscles. Naunyn Schmiedebergs Arch Pharmacol 357: 77–86, 1998.[CrossRef][ISI][Medline]
  6. Corteling RL, Li S, Giddings J, Westwick J, Poll C, Hall IP. Expression of transient receptor potential C6 and related transient receptor potential family members in human airway smooth muscle and lung tissue. Am J Respir Cell Mol Biol 30: 145–154, 2004.[Abstract/Free Full Text]
  7. Croxton TL, Lande B, Hirshman CA. Role of G proteins in agonist-induced Ca2+ sensitization of tracheal smooth muscle. Am J Physiol Lung Cell Mol Physiol 275: L748–L755, 1998.[Abstract/Free Full Text]
  8. Daykin K, Widdop S, Hall IP. Control of histamine induced inositol phospholipid hydrolysis in cultured human tracheal smooth muscle cells. Eur J Pharmacol 246: 135–140, 1993.[CrossRef][ISI][Medline]
  9. Deshpande DA, Walseth TF, Panettieri RA, Kannan MS. CD38/cyclic ADP-ribose-mediated Ca2+ signaling contributes to airway smooth muscle hyper-responsiveness. FASEB J 17: 452–454, 2003.[Abstract/Free Full Text]
  10. Farmer SG, Ensor JE, Burch RM. Evidence that cultured airway smooth muscle cells contain bradykinin B2 and B3 receptors. Am J Respir Cell Mol Biol 4: 273–277, 1991.[ISI][Medline]
  11. Fleischmann BK, Washabau RJ, Kotlikoff MI. Control of resting membrane potential by delayed rectifier potassium currents in ferret airway smooth muscle cells. J Physiol 469: 625–638, 1993.[Abstract/Free Full Text]
  12. Glynn AA, Michaels L. Bronchial biopsy in chronic bronchitis and asthma. Istanbul Tip Fak Mecmuasi 15: 142–153, 1960.[Medline]
  13. Halayko AJ, Amrani Y. Mechanisms of inflammation-mediated airway smooth muscle plasticity and airways remodeling in asthma. Respir Physiol Neurobiol 137: 209–222, 2003.[CrossRef][ISI][Medline]
  14. Hall IP, Kotlikoff MI. Use of cultured airway myocytes for study of airway smooth muscle. Am J Physiol Lung Cell Mol Physiol 268: L1–L11, 1995.[Abstract/Free Full Text]
  15. Hiramatsu T, Kume H, Kotlikoff MI, Takagi K. Role of calcium-activated potassium channels in the relaxation of tracheal smooth muscles by forskolin. Clin Exp Pharmacol Physiol 21: 367–375, 1994.[ISI][Medline]
  16. Hirayama Y, Miyayasu K, Yamagami K, Imai T, Ohkubo Y, Mutoh S. Effect of FK3657, a non-peptide bradykinin B2 receptor antagonist, on allergic airway disease models. Eur J Pharmacol 467: 197–203, 2003.[CrossRef][ISI][Medline]
  17. Hirota K, Hashiba E, Yoshioka H, Kabara S, Matsuki A. Effects of three different L-type Ca2+ entry blockers on airway constriction induced by muscarinic receptor stimulation. Br J Anaesth 90: 671–675, 2003.[Abstract/Free Full Text]
  18. Hotta K, Emala CW, Hirshman CA. TNF-{alpha} upregulates Gi{alpha} and Gq{alpha} protein expression and function in human airway smooth muscle cells. Am J Physiol Lung Cell Mol Physiol 276: L405–L411, 1999.[Abstract/Free Full Text]
  19. Hyvelin JM, Martin C, Roux E, Marthan R, Savineau JP. Human isolated bronchial smooth muscle contains functional ryanodine/caffeine-sensitive Ca-release channels. Am J Respir Crit Care Med 162: 687–694, 2000.[Abstract/Free Full Text]
  20. Ichinose M, Miura M, Takahashi T, Yamauchi H, Kageyama N, Tomaki M, Endoh N, Sakurai E, Watanabe T, Shirato K. Allergic airway response and potassium channels: histamine release and airway inflammation. Methods Find Exp Clin Pharmacol 17, Suppl C: 36–39, 1995.[ISI][Medline]
  21. Janssen LJ. Ionic mechanisms and Ca2+ regulation in airway smooth muscle contraction: do the data contradict dogma? Am J Physiol Lung Cell Mol Physiol 282: L1161–L1178, 2002.[Abstract/Free Full Text]
  22. Janssen LJ, Walters DK, Wattie J. Regulation of [Ca2+]i in canine airway smooth muscle by Ca2+-ATPase and Na+/Ca2+ exchange mechanisms. Am J Physiol Lung Cell Mol Physiol 273: L322–L330, 1997.[Abstract/Free Full Text]
  23. Koga Y, Sodeyama N, Satoh S, Teshigawara T, Yanagisawa T, Taira N, Endoh M. Relaxation of airway smooth muscle induced by potassium in the presence of Ca-antagonists. Jpn J Pharmacol 50: 387–396, 1989.[Medline]
  24. Kotlikoff MI. Potassium currents in canine airway smooth muscle cells. Am J Physiol Lung Cell Mol Physiol 259: L384–L395, 1990.[Abstract/Free Full Text]
  25. Kotlikoff MI, Wang YX. Calcium release and calcium-activated chloride channels in airway smooth muscle cells. Am J Respir Crit Care Med 158: S109–S114, 1998.[Abstract/Free Full Text]
  26. Kume H, Hall IP, Washabau RJ, Takagi K, Kotlikoff MI. Beta-adrenergic agonists regulate KCa channels in airway smooth muscle by cAMP-dependent and -independent mechanisms. J Clin Invest 93: 371–379, 1994.[ISI][Medline]
  27. Kume H, Mikawa K, Takagi K, Kotlikoff MI. Role of G proteins and KCa channels in the muscarinic and beta-adrenergic regulation of airway smooth muscle. Am J Physiol Lung Cell Mol Physiol 268: L221–L229, 1995.[Abstract/Free Full Text]
  28. Kuo KH, Dai J, Seow CY, Lee CH, van Breemen C. Relationship between asynchronous Ca2+ waves and force development in intact smooth muscle bundles of the porcine trachea. Am J Physiol Lung Cell Mol Physiol 285: L1345–L1353, 2003.[Abstract/Free Full Text]
  29. McCann JD, Welsh MJ. Calcium-activated potassium channels in canine airway smooth muscle. J Physiol 372: 113–127, 1986.[Abstract/Free Full Text]
  30. Mikawa K, Kume H, Takagi K. Effects of BKCa channels on the reduction of cytosolic Ca2+ in cGMP-induced relaxation of guinea-pig trachea. Clin Exp Pharmacol Physiol 24: 175–181, 1997.[ISI][Medline]
  31. Murray RK, Kotlikoff MI. Receptor-activated calcium influx in human airway smooth muscle cells. J Physiol 435: 123–144, 1991.[Abstract/Free Full Text]
  32. Nara M, Sasaki T, Shimura S, Oshiro T, Irokawa T, Kakuta Y, Shirato K. Effects of histamine and endothelin-1 on membrane potentials and ion currents in bovine tracheal smooth-muscle cells. Am J Respir Cell Mol Biol 19: 805–811, 1998.[Abstract/Free Full Text]
  33. Nielsen-Kudsk JE. Potassium channel modulation: a new drug principle for regulation of smooth muscle contractility. Studies on isolated airways and arteries. Dan Med Bull 43: 429–447, 1996.[ISI][Medline]
  34. Nuttle LC, Farley JM. Muscarinic receptors inhibit ATP-sensitive K+ channels in swine tracheal smooth muscle. Am J Physiol Lung Cell Mol Physiol 273: L478–L484, 1997.[Abstract/Free Full Text]
  35. Peel SE, Liu B, Hall IP. A key role for STIM1 in store operated calcium channel activation in airway smooth muscle. Respir Res 7: 119, 2006.[CrossRef][Medline]
  36. Proud D, Togias A, Naclerio RM, Crush SA, Norman PS, Lichtenstein LM. Kinins are generated in vivo following nasal airway challenge of allergic individuals with allergen. J Clin Invest 72: 1678–1685, 1983.[ISI][Medline]
  37. Pyne S, Pyne NJ. Bradykinin stimulates phospholipase D in primary cultures of guinea-pig tracheal smooth muscle. Biochem Pharmacol 45: 593–603, 1993.[CrossRef][ISI][Medline]
  38. Pyne S, Pyne NJ. Bradykinin-stimulated phosphatidylcholine hydrolysis in airway smooth muscle: the role of Ca2+ and protein kinase C. Biochem J 311: 637–642, 1995.[ISI][Medline]
  39. Schmidlin F, Scherrer D, Daeffler L, Bertrand C, Landry Y, Gies JP. Interleukin-1beta induces bradykinin B2 receptor gene expression through a prostanoid cyclic AMP-dependent pathway in human bronchial smooth muscle cells. Mol Pharmacol 53: 1009–1015, 1998.[Abstract/Free Full Text]
  40. Smith JJ, McCann JD, Welsh MJ. Bradykinin stimulates airway epithelial Cl– secretion via two second messenger pathways. Am J Physiol Lung Cell Mol Physiol 258: L369–L377, 1990.[Abstract/Free Full Text]
  41. Snetkov VA, Hirst SJ, Twort CH, Ward JP. Potassium currents in human freshly isolated bronchial smooth muscle cells. Br J Pharmacol 115: 1117–1125, 1995.[ISI]
  42. Snetkov VA, Hirst SJ, Ward JP. Ion channels in freshly isolated and cultured human bronchial smooth muscle cells. Exp Physiol 81: 791–804, 1996.[Abstract]
  43. Snetkov VA, Ward JP. Ion currents in smooth muscle cells from human small bronchioles: presence of an inward rectifier K+ current and three types of large conductance K+ channel. Exp Physiol 84: 835–8460, 1999.[Abstract]
  44. Stocker M. Ca2+-activated K+ channels: molecular determinants and function of the SK family. Nat Rev Neurosci 5: 758–770, 2004.[CrossRef][ISI][Medline]
  45. Sweeney M, McDaniel SS, Platoshyn O, Zhang S, Yu Y, Lapp BR, Zhao Y, Thistlethwaite PA, Yuan JX. Role of capacitative Ca2+ entry in bronchial contraction and remodeling. J Appl Physiol 92: 1594–1602, 2002.[Abstract/Free Full Text]
  46. Tao FC, Shah S, Pradhan AA, Tolloczko B, Martin JG. Enhanced calcium signaling to bradykinin in airway smooth muscle from hyperresponsive inbred rats. Am J Physiol Lung Cell Mol Physiol 284: L90–L99, 2003.[Abstract/Free Full Text]
  47. Thirstrup S, Nielsen-Kudsk JE, Mellemkjaer S. Involvement of K+ channels in the relaxant effect of vasoactive intestinal peptide and atrial natriuretic peptide in isolated guinea-pig trachea. Eur J Pharmacol 319: 253–259, 1997.[CrossRef][ISI][Medline]
  48. Tomasic M, Boyle JP, Worley JF 3rd, Kotlikoff MI. Contractile agonists activate voltage-dependent calcium channels in airway smooth muscle cells. Am J Physiol Cell Physiol 263: C106–C113, 1992.[Abstract/Free Full Text]
  49. Waldron GJ, Sigurdsson SB, Aiello EA, Halayko AJ, Stephens NL, Cole WC. Delayed rectifier K+ current of dog bronchial myocytes: effect of pollen sensitization and PKC activation. Am J Physiol Lung Cell Mol Physiol 275: L336–L347, 1998.[Abstract/Free Full Text]
  50. Wang YG, Samarel AM, Lipsius SL. Laminin binding to beta1-integrins selectively alters beta1- and beta2-adrenoceptor signalling in cat atrial myocytes. J Physiol 527: 3–9, 2000.[Abstract/Free Full Text]
  51. Wang YX, Fleischmann BK, Kotlikoff MI. Modulation of maxi-K+ channels by voltage-dependent Ca2+ channels and methacholine in single airway myocytes. Am J Physiol Cell Physiol 272: C1151–C1159, 1997.[Abstract/Free Full Text]
  52. Yang CM, Chien CS, Wang CC, Hsu YM, Chiu CT, Lin CC, Luo SF, Hsiao LD. Interleukin-1beta enhances bradykinin-induced phosphoinositide hydrolysis and Ca2+ mobilization in canine tracheal smooth-muscle cells: involvement of the Ras/Raf/mitogen-activated protein kinase (MAPK) kinase (MEK)/MAPK pathway. Biochem J 354: 439–446, 2001.[CrossRef][ISI][Medline]
  53. Ye H, Ma WL, Yang ML, Liu SY, Wang DX. Effect of chronic cigarette smoking on large-conductance calcium-activated potassium channel and Kv1.5 expression in bronchial smooth muscle cells of rats. Sheng Li Xue Bao 56: 573–578, 2004.[Medline]
  54. Zhang Y, Adner M, Cardell LO. Up-regulation of bradykinin receptors in a murine in-vitro model of chronic airway inflammation. Eur J Pharmacol 489: 117–126, 2004.[CrossRef][ISI][Medline]



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