| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
,**,1
* Pharmakologie und Toxikologie, Pharmazeutisches Institut, Universität Tübingen, Tübingen, Germany;
Institut für Pharmakologie für Pharmazeuten, Universitätsklinikum Hamburg-Eppendorf, Hamburg, Germany;
Leibniz Zentrum für Medizin und Biowissenschaften, Borstel, Germany;
Institut für Pharmakologie und Toxikologie, Universität Marburg, Marburg, Germany;
|| Institut für Klinische Chemie und Molekulare Diagnostik, Universität Marburg, Marburg, Germany;
¶ Institut für Pharmakologie und Toxikologie, Technische Universität München, München, Germany;
# Institut für Anatomie, Universität Erlangen-Nürnberg, Erlangen, Germany; and
** Institut für Pharmakologie und Toxikologie, Rheinisch-Westfälische Technische Hochschule Aachen, Aachen, Germany
1Correspondence: Institut für Pharmakologie für Pharmazeuten, Universitätsklinikum Hamburg-Eppendorf, Martinistr. 52, D-20246 Hamburg, Germany. E-mail: korth{at}uke.uni-hamburg.de
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Key Words: knockout mice • BK channel • airway contractility • cyclic GMP/PKG signaling • cholinergic agonists
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
subunit (1
2
3)
. However, the functional role of BK channels in airway smooth muscle has been assessed so far only in in vitro studies using pharmacological blockers of BK channels, such as tetraethylammonium (TEA), charybdotoxin (ChTX), or iberiotoxin (IbTX). Acute pharmacological blockade of BK channels increased baseline contractility and/or enhanced contractions induced by muscarinic agonists or nerve stimulation (4
5
6
7
8
9)
. Airway smooth muscle relaxes in response to drugs which increase intracellular cAMP and cGMP levels and convincing evidence has been presented that BK channels—at least in part—are involved in this process (10)
. Relaxation of tracheal rings induced by ß-adrenoreceptor agonists or NO donors was antagonized by ChTX and IbTX, which both shifted the respective concentration-effect curves to the right (4
5
6
7
8
, 11
, 12)
. Recently, it was shown that cationic proteins, which are implicated in the pathogenesis of bronchial hyperresponsiveness (13)
are potent blockers of BK channels and may thus attenuate relaxation of airway smooth muscle by ß-adrenoceptor agonists (14)
. In another study, estrogen has been shown to prevent cholinergic-induced constriction of asthmatic tracheal rings by activating BK channels via the NO-cGMP-protein kinase G pathway (15)
. This finding can help explain why estrogen can often relieve airway responsiveness in asthma and why a relation exists between the phase of the menstrual cycle and emergency department visits for acute asthma (16)
. In the present study we were especially interested in the long-term consequences that might have occurred in mice without functional BK channels in order to evaluate the possibility whether loss-of-function mutations in the BK channel gene could contribute to obstructive airway diseases in humans. To our surprise, we found a paradoxical phenotype with reduced sensitivity of airways toward bronchoconstrictors and an enhanced sensitivity toward bronchodilators. Both effects are the result of compensatory mechanisms, which become increasingly prominent with increasing physiological integration of the test system used for phenotyping airway function in the BK channel-deficient mice. Our data suggest that membrane hyperpolarization alone is neither necessary nor sufficient for relaxation in airway smooth muscle. Thus a better understanding of agonist evoked relaxation demands a new emphasis on mechanisms other than BK channel activation.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
of both genders with hybrid SV129/C57BL6 background (always F2 generation) were randomly assigned to the experimental procedures with respect to the German legislation on animal protection. (see Supplemental Data for detailed methods)
Immunohistochemistry
BK channel staining was performed on slides with 8 µm-cryostat-slices from nonfixed WT and BK–/– trachea. After tagging the binding of anti-BK(674–1115) (18)
with an Alexa555-conjugated donkey anti-rabbit IgG (Molecular Probes), BK channel expression was analyzed using confocal-laser scanning microscopy (18)
.
Western blot analyses
Murine tracheal muscles were homogenized with an Ultra Turrax and centrifuged with 13,000 g rpm at 4°C for 3 min. The supernatant was concentrated to a final volume of 
120 µl using a Vivaspin-30 concentrator (Sartorius, Göttingen, Germany). After SDS-PAGE (110 µg protein per lane), Western blot analysis was performed with antibodies against regulatory subunit RII of PKA (Santa Cruz Biotechnology, Santa Cruz, CA; 1:100), soluble guanylate cyclase ß1 subunit (Cayman Chemical, Denver, CO; 1:300), PKG (19, 1:300), IP3 receptor-associated cGMP kinase substrate IRAG (20, 1:500), MAP kinase (Cell Signaling Technology, Beverly, MA, USA; 1:500). Immunostained proteins were visualized using alkaline phosphatase-conjugated donkey anti-rabbit IgG (Dianova, Hamburg, Germany; 1:5,000) prior to quantification with Biodoc analysis software (Biometra, Göttingen, Germany).
Cyclic GMP determination
Dissected trachea was incubated in buffer for 30 min at 37°C prior to stimulation with or without 1 µM DEA-NO (Alexis, Grünberg, Germany) for 1 min. After washing with PBS, the tissue was snap-frozen and homogenized in 10% trichloroacetic acid. The resulting supernatant was extracted with water-saturated diethyl ether and assayed for cGMP content using a cGMP enzyme immunoassay (EIA) system kit (Cayman Chemical, Ann Arbor, MI, USA).
Electrophysiology on tracheal smooth muscle cells
For tracheal smooth muscle cell (TSMC) isolation, tracheas were digested at 37°C in Ca2+-free physiological saline solution (PSS) containing papain for 30 min and subsequent in PSS containing Ca2+, collagenase type H and hyaluronidase for 10 min. For measuring outward membrane currents (whole-cell mode), the free Ca2+ concentration was adjusted to 300 nM. The holding potential was –10 mV and test pulses of 300-ms duration were applied every 5 s to potentials ranging from –60 to +80 mV. Membrane potentials were measured using the whole-cell perforated-patch technique with 250 µg/ml nystatin in the pipette solution. For recording of macroscopic Ca2+ channel currents, cells were voltage-clamped at a holding potential of –60 mV, and the potential was stepped, for 300 ms every 5 s, in 10-mV increments up to +50 mV. The inward current was measured as peak inward current with reference to zero current. A low-pass filter was set at a cutoff frequency of 1 kHz, and signals were digitized at 5 kHz.
In vitro analysis of airway contractility
Tracheal rings
Tracheal rings were mounted in an organ bath chamber (19)
and equilibrated for 30 min under 5 mN resting tension in tempered and aerated modified Krebs-Henseleit solution with buffer exchanges every 10 min. Isometric tension was continuously recorded. Compounds were applied either as a single dose to induce bronchoconstriction, or cumulatively to obtain dose–effect relationships.
Isolated-perfused lung
Lungs were placed in a thorax chamber (configurated and equipped as described by von Bethmann et al. (21)
and perfused via the pulmonary artery with modified Krebs-Henseleit solution containing 0.1% glucose (Glc) and 0.3% HEPES at a constant flow rate (1 ml/min) in a nonrecirculating manner. Lungs were ventilated with 90 breaths/min and hyperinflation (–25 cm H2O) was performed every 5 min. After an equilibration of 30 min, buffer or methacholine (MCh) was applied cumulatively in a concentration-dependent manner. The airway resistance was calculated from the ratio of MCh-induced resistance to control resistance (before adding MCh).
Precision-cut lung slices
Lungs with tracheal cannulation were instilled with agarose solution (0.75% in minimal essential medium (MEM; Biochrom, Germany); 37.0±0.5°C). After agarose hardening, lung lobes were dissected, embedded in 3% agarose solution, cooled for 5 min on ice, and cut into 250-µm slices. The slices were incubated overnight in MEM containing 100 U/ml penicillin and 100 µg/ml streptomycin. For experiments, a single slice was transferred to an incubation chamber (22)
and equilibrated for 10 min in MEM medium. MCh was cumulatively applied in a concentration-dependent manner. The luminal area of airways was quantified using an image analysis program (Optimas 6.0; Optimas Corporation, Bothell, WA, USA).
Airway responsiveness to MCh by head-out body plethysmography
The airway responsiveness of awake, nonsensitized WT and BK–/– mice to inhaled aerosolized MCh was measured using head-out body plethysmography as described (23
, 24)
. The air flow at 50% VT (tidal volume) during expiration (midexpiratory flow, EF50) was recorded in the absence and presence of aerosolized MCh (25, 50, 75, 100, and 125 mg/ml). Aerosols were delivered for 5 min with 5-min-recovery intervals in between. Data were calculated as percentage of basal EF50 values for each MCh-concentration.
Statistical analysis
The results are presented as mean ± SE. Paired or unpaired Student’s t-tests were used as appropriate to evaluate differences between two groups, and ANOVA was used for multiple groups. SigmaPlot 8.01 was used for statistical analyses. P < 0.05 was considered to indicate statistical significance.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
-subunit (BK–/–) (Fig. 1
A).
|
WT tracheal smooth muscle cells (TSMCs) exhibited pronounced noninactivating BK channel currents, measured as voltage-activated whole-cell outward currents, sensitive to IbTX, a selective blocker of BK channels (Fig. 1B
; BK currents). As further shown in Fig. 1B, BK
channel currents were completely absent in TSMCs from BK channel -subunit knockout mice. Currents shown in Fig. 1B
were elicited from a holding potential of –10 mV by depolarizing the cells every 5 s for 300 ms from –60 to + 80 mV in 10-mV increments. The whole-cell capacitance was not different between WT and BK–/– TSMCs, indicating similar cell sizes (WT: 13.2±3.7 pF, n=6; BK–/–: 14.2±3.1 pF, n=7). We also assessed voltage-gated (IbTX-insensitive) outward currents using the same experimental protocol, as described before. As shown in Fig. 1B
, right, voltage-gated outward current densities of both genotypes were similar at all voltages (non-BK currents).
Next, the influence of BK channels on membrane potential was investigated. Figure 1C
shows a typical recording from a WT TSMC. The recording was obtained in the current-clamp modus by using the nystatin-perforated patch-clamp technique. Similar to findings in other isolated smooth muscle cells, spontaneous transient hyperpolarizations (STHs) with amplitudes of over 20 mV were registered. STHs are induced via Ca2+ sparks by BK channel activation (25)
, and therefore, inhibition of BK channels by IbTX (300 nM) induced membrane potential depolarization due to the suppression of STHs. Calculation of time-averaged membrane potential (illustrated by the continuous line in Fig. 1C
) revealed that STHs caused an average membrane potential change of 11 mV because IbTX depolarized the membrane potential from –48 to –37 mV. In contrast to WT cells, STHs were completely absent in TSMCs from BK–/– mice, and IbTX did not further affect membrane potential (lower trace in Fig. 1C
). As shown by the bar graph in Fig. 1C
, the average membrane potential of TSMCs from BK–/– mice (35.2±1.5 mV, n=8) is 10 mV less negative than the membrane potential of cells from WT mice (44.9±3.0 mV, n=6).
Carbachol elicits phasic contractions in tracheal rings of BK–/– mice
Airway smooth muscle tone is strongly influenced by the parasympathetic nervous system. Postganglionic nerve fibers release acetylcholine (Ach), which binds to muscarinic receptors on airway smooth muscle and leads to its contraction. In the present study, carbachol was used to contract tracheal rings of WT and BK–/– mice. Carbachol produced a concentration-dependent increase of tonic tension in WT preparations (Fig. 2
A). The threshold concentration of carbachol to increase tension varied from 0.03 to 0.1 µM, and the maximum tension was achieved with 10 µM. Tracheal rings from BK–/– mice, however, showed a different response when exposed to carbachol. Application of the muscarinic receptor agonist at concentrations up to 0.3 µM induced a rapid rise of tension, which was followed, after a spontaneous relaxation, by phasic contractions, which continued as long as the agonist was present (Fig. 2A
). With the exception of 0.03 µM carbachol where spontaneous relaxation was complete (not shown), relaxation was only partial, and phasic contractions were superimposed on a tonic contraction component. Phasic activity decreased or disappeared when the concentration of carbachol was increased to 1 µM or higher concentrations. Phasic contractions were sometimes irregular but showed in most cases a more regular pattern. The frequency of phasic activity was also variable and ranged from 4 to 7 contractions per 10 min.
|
The maximal forces that could be generated by 10 µM carbachol in tracheae of WT mice (25.3±0.8 mN; n=9) and BK–/– mice (25.2±1.1 mN; n=10) were not significantly different. When in five tracheae from BK–/– mice, the epithelium was removed, phasic activity was preserved in the presence of carbachol. This finding excludes the involvement of an epithelium-derived factor in initiating phasic contractions.
The membrane potential of isolated TSMCs is characterized by STHs, which are blocked by IbTX and are absent in cells from BK–/– mice (Fig. 1C
). Spontaneous Ca2+ release (Ca2+ sparks) via ryanodine receptors (RyRs) of the sarcoplasmic reticulum (SR) is thought to be a main determinant of BK channel activity and hence membrane potential in smooth muscle cells (25)
. As shown in Fig. 2B
, treatment of a WT TSMC with 30 µM ryanodine, a blocker of RyRs, resulted in marked inhibition of STHs and in a depolarization of the membrane potential by 9 mV. To investigate the influence of ryanodine on contractile activity, carbachol was added cumulatively to tracheal rings of WT mice pretreated for 30 min with 30 µM ryanodine. Similar to BK–/– mice, carbachol induced at low concentrations phasic contractions superimposed on a tonic component (Fig. 2C
). Phasic activity became smaller at 0.3 µM carbachol and disappeared at higher concentrations (Fig. 2C
). Similar effects as those shown in Fig. 2C
were obtained in nine other preparations, although a great variability in the frequency of phasic activity was seen. The experiments with ryanodine support the findings in BK–/– mice and point to the importance of functional BK channels for the maintenance of stable tonic contractions during parasympathetic activity.
Phasic contractions of BK–/– tracheas depend on L-type Ca2+ channel activation
To get more insight into the nature of phasic contractions, tracheae from BK–/– mice were contracted with 0.1 µM carbachol. When phasic activity was stable, the L-type Ca2+ channel blocker nifedipine (1 µM) was added to the organ bath and, after establishment of a new steady state, the concentration-force relationship for carbachol was completed. As shown by the original force recording in Fig. 3
A, application of nifedipine resulted in an almost complete relaxation of the trachea and eliminated spontaneous activity. Phasic activity did not recur when force was increased with carbachol in a concentration-dependent fashion (0.3 to 100 µM). Similar results were obtained in five additional BK–/– tracheae. The experiments clearly indicate that Ca2+ influx through voltage-gated L-type Ca2+ channels is essential for the generation of phasic contractions.
|
To estimate the contribution of L-type Ca2+ current to carbachol-induced contractions, tracheae from WT and BK–/– mice were exposed to cumulatively increasing concentrations of carbachol in the presence of 1 µM nifedipine. The resulting concentration-force relationships are compared in Fig. 3B
with nifedipine-free control curves. As shown in Fig. 3B
, left, concentration-force curves in the absence of nifedipine were superimposable in WT and BK–/– tracheae The respective EC50 values were 0.18 ± 0.02 µM (WT; n=9) and 0.17 ± 0.02 µM (BK–/–; n=10). In the presence of nifedipine, however, there was a more pronounced rightward shift of the concentration-force curve obtained with BK–/– tracheal rings, indicating that the Ca2+ channel blocker inhibited the carbachol-induced contraction more effectively in BK–/– than in WT tracheae (Fig. 3B
, right). The respective EC50 values for carbachol were 0.51 ± 0.1 (WT; n=7) and 1.7 ± 0.2 µM (BK–/–; n=6). The maximal forces generated by 100 µM carbachol in nifedipine-pretreated tracheae of WT mice (25.1±3.4 µM; n=7) and BK–/– mice (24.7±1.7 µM; n=6) were not significantly different. The higher sensitivity of BK–/– tracheae toward nifedipine could have been due to an unexpected up-regulation of L-type Ca2+ channels in this genotype. Experimental evidence, however, excluded this possibility because current densities obtained from nifedipine-sensitive L-type Ca2+ channels were not significantly different in BK–/– and WT TSMCs (Fig. 3C
).
Reduced bronchial contractility in BK–/– mice exposed to methacholine
The experiments described so far on isolated TSMCs and tracheal rings are intriguing, but they do not support the hypothesis that ablation of the pore-forming BK channel -subunit by gene targeting leads to an enhanced airway contractility in mice as one would expect from the deletion of a K+ channel, which plays important roles in setting membrane potential and limiting responses to excitatory stimuli. To further analyze airway responsiveness under more physiological conditions, we used two in vitro mouse models with maintained pulmonary microanatomy, the precision-cut lung slice (PCLS), which allows the direct measurement of airway area and the isolated-perfused lung (IPL), in which airway resistance of the whole organ can be studied. Finally, in an in vivo model, total mouse lung capacity was assessed by body plethysmography. Initial experiments showed that baseline airway area and airway resistance were not significantly different in WT and BK–/– mice (data not shown). We next investigated the responses of PCLSs and IPLs to methacholine, a stable analog of Ach and a potent bronchoconstrictor. Airways in mouse PCLSs responded to methacholine with an immediate contraction. This effect was concentration dependent with an almost complete bronchoconstriction at 10 µM methacholine in WT mice. Most surprising, airway contraction induced by methacholine was significantly attenuated in slices from BK–/– mice (Fig. 4
A). Airway area was decreased at 0.5 µM methacholine to 56.3 ± 13.1% in BK–/– (n=6) and to 29.5 ± 5.3% of control in WT PCLSs (n=8). Perfusion of methacholine (0.1 to 100 µM) through the pulmonary artery of IPLs resulted in a rapid and concentration-dependent increase in pulmonary resistance (Fig. 4B
). In accordance with the results in PCLSs, methacholine was significantly less effective to increase airway resistance in BK–/– mice. At 100 µM methacholine, airway resistance in BK–/– mice was only 50% that of WT mice (0.13±0.02 in four BK–/– and 0.26±0.04 cm H2O·s·ml–1 in four WT IPLs; Fig. 4B
). In vivo experiments using body plethysmography showed an even more surprising result. Whereas aerosolized methacholine inhaled by WT mice reduced the respiratory capacity in a dose-dependent way, there was almost no response to methacholine up to 125 mg/ml in BK–/–mice (88.8±5.8% of basal respiratory capacity in eight BK–/– mice; Fig. 5
). Taken together, all three lung models showed unequivocally that the sensitivity of the mouse airways toward the bronchoconstrictor methacholine is strongly attenuated in BK–/– as compared to WT mice.
|
|
Increased ß-adrenoreceptor-mediated bronchodilation in BK–/– mice
Our in vitro and in vivo findings so far indicate a reduced cholinergic responsiveness of BK–/– airway smooth muscle. Besides the parasympathetic input, airway tone is also influenced by circulating catecholamines, which stimulate ß-adrenoceptors (ß2-ARs) that are prominently expressed in bronchial smooth muscle but also in other cell types of the lung (26)
. ß2-AR agonists are effective bronchodilators and are therefore the most widely prescribed drugs for the acute treatment of bronchoconstriction in asthma attack. To evaluate the effectiveness of the ß-adrenoreceptor agonist isoprenaline in WT and BK–/– mice, we used carbachol-contracted tracheal rings. The precontraction induced by 3 µM carbachol was not significantly different in BK–/– (17.4±1.0 mN; n=7) and WT preparations (18.5±1.7 mN; n=7). As illustrated in Fig. 6
, isoprenaline, which acts via the cAMP/PKA signaling cascade reversed the carbachol-induced constriction in a concentration-dependent manner in both genotypes. In BK–/– tracheal rings, however, the isoprenaline effect was significantly more pronounced than in WT. This observation seems to be paradoxical and suggests that BK channel activation by bronchodilators seems to be an epiphenomenon rather than a causal event (27)
, but on the other hand, it could also be due to a compensatory up-regulation of cAMP/PKA signaling in BK–/– trachea.
|
Enhanced cGMP/PKG signaling in BK–/– tracheae
Our findings described so far are completely unexpected, considering the well-established role of BK channels as negative feedback regulators of cellular excitability. It is, however, possible that BK channels play such an important role in airway physiology that long-term adaptation mechanisms compensate for the loss of functional channels. To evaluate possible underlying compensatory mechanisms resulting from long-term BK channel -subunit deletion, we focused on the protein expression of cyclic nucleotide-dependent pathways. We found significant increases in the expression of soluble guanylyl cyclase (sGC; 1.44±014-fold; n=4), cGMP-dependent protein kinase I (PKG; 2.30±0.23-fold; n=4) and IP3R-associated cGMP kinase substrate (IRAG; 3.62±1.06-fold; n=4) in tracheae of BK–/– mice when compared to WT (Fig. 7
A). Interestingly, we did not notice any significant alteration in the expression level of a regulatory subunit of cAMP-dependent protein kinase (PKA RII), indicating that the cAMP/PKA signaling cascade was probably not part of the compensatory mechanism. There was a small but significantly higher basal cGMP level in tracheae from BK–/– mice (33.5±2.4 in four BK–/–- and 27.5±1.4 pmol mg–1 wet wt in four wt preparations, P < 0.05), and only in tracheae from BK–/– mice did NO (1 µM) stimulate basal cGMP levels (45.6±1.9 pmol mg–1 wet wt; n=4; P < 0.01; Fig. 7B
). This finding indicates that the increased expression of sGC was functionally effective. Taken together, the results suggest that the amplification of cGMP signaling proteins could be an important mechanism counterbalancing the otherwise increased contractility of BK–/– airways.
|
To assess the functional importance of an up-regulated cGMP/PKG signaling in airways directly, we incubated WT and BK–/– tracheal rings with increasing concentrations of carbachol in the presence of the PKG inhibitor Rp-8-Br-cGMPS (100 µM). To abolish phasic contractions which persisted in the presence of the PKG inhibitor, the Ca2+ channel blocker nifedipine (1 µM) was also present. As shown by the EC50 values in Fig. 7C
, the PKG inhibitor shifted the carbachol concentration-effect curves of WT and BK–/– tracheae to the left. However, this shift was much more pronounced in BK–/– than in WT preparations. The respective EC50 values shifted from 1.38 ± 0.13 (n=4–6) to 0.41 ± 0.17 µM (n=4–6) in BK–/– and from 0.39 ± 0.03 (n=4–7) to 0.15 ± 0.02 µM (n=4–7) in WT tracheae. These findings point to a functionally operative PKG, which is compensatorily up-regulated in BK–/– trachea. The observed up-regulation of PKG expression may also contribute to the enhanced response of BK–/– trachea toward isoprenaline, since cAMP-dependent cross-activation of PKG seems to be a possible mechanism at high concentrations of cyclic nucleotides (28)
.
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
-subunit of the BK channel permitted the identification of a paradoxical phenotype in mouse airways, which was strongly influenced by compensatory mechanisms. One of these mechanisms, up-regulation of the components of the cGMP/PKG signaling system could be identified in the present study. In addition, we found that isolated tracheae from BK–/– mice responded to carbachol with slow phasic contractions, indicating that BK channels play an important role in stabilizing tonic contractions induced by muscarinic receptor agonists.
Airway smooth muscle behaves as an electrically quiescent tissue that exhibits a stable membrane potential and depolarizes gradually without prominent spike depolarizations when exposed to excitatory agonists such as ACh. In contrast to multicellular tissues, membrane potential of isolated single airway smooth muscle cells is characterized by spontaneous transient membrane potential hyperpolarizations (STHs), which are responsible for voltage fluctuations of 20 mV and more. STHs are due to local intracellular Ca2+ transients (Ca2+ sparks) caused by the coordinated opening of ryanodine-sensitive Ca2+ channels of the sarcoplasmic reticulum of smooth muscle cells, including airway smooth muscle (29)
. BK channels are activated both by depolarization and micromolar Ca2+ concentrations, and it has been estimated that Ca2+ released by Ca2+ sparks into the diffusion-limited space between outer membrane and sarcoplasmic reticulum can reach sufficiently high concentrations to account for BK channel activation and STHs (30)
. Our findings that STHs are completely absent in tracheal cells from BK–/– mice and membrane potential is depolarized by 10 mV in comparison to WT cells, indicate that BK channels are dynamic regulators of membrane potential. Measurement of membrane potential by perforated-patch recordings resulted in a time-averaged mean potential of –45 mV in WT cells, which was close to the mean membrane potential of –47 mV determined in intact bovine tracheal tissue (31)
. Whether the membrane depolarization induced by the lack of BK channels was sufficient to activate Ca2+ influx via voltage-operated Ca2+ channels is not known, but our finding that baseline airway resistance and baseline pulmonary compliance was not significantly different between BK–/– and WT mice argue against this possibility. This view is further supported by the observation that BK channel blockers failed to increase basal tone of the mouse trachea (32)
. There may, however, be differences between species because BK channel blockers were found to increase basal tone of human bronchi, an effect that was reversed by the Ca2+ channel blocker verapamil (8)
.
The parasympathetic nerves provide the dominant autonomic control of airway smooth muscle. They release ACh, which binds to muscarinic receptors and causes contraction and bronchoconstriction (33)
. Both M2 and M3 muscarinic receptors are present on airway smooth muscle (34)
, and functional studies with M3-receptor knockout mice have shown that in contrast to other smooth muscle tissues, e.g., urinary bladder, both receptor subtypes are needed for maximal tracheal contractility (35)
. Airway smooth muscles contract tonically in response to muscarinic stimulation, and this contraction is associated with a biphasic change in intracellular Ca2+ concentration. Stimulation of muscarinic receptors activates G proteins that stimulate the phospholipase C, which degrades phosphatidylinositol 4,5-bisphosphate into IP3 and diacylglycerol (DAG). IP3 in turn induces an initial burst of Ca2+ release from intracellular stores by binding to the IP3 receptor/channel in the sarcoplasmic reticulum membrane. After this transient rise of intracellular Ca2+, an influx of extracellular Ca2+ is required to maintain the sustained contraction produced during prolonged muscarinic receptor stimulation. Muscarinic receptor agonists induce membrane depolarization (36)
, probably by activation of Cl– and nonselective cation currents (37)
and by suppression of K+ currents, and may thus open voltage-operated Ca2+ channels. Agonist-induced contractions in airway smooth muscle, however, are relatively insensitive to dihydropyridine Ca2+ channel blockers (38)
at concentrations sufficient to completely block voltage-operated Ca2+ currents (39)
. This fact may explain why Ca2+ channel blockers have no beneficial clinical effects in the management of most obstructive lung diseases (40)
. Our experiments on tracheae from WT mice confirm the weak inhibitory effect of the Ca2+ channel blocker nifedipine. In tracheae from BK–/– mice, however, we found that nifedipine was an effective inhibitor of carbachol-induced contractions. The increased sensitivity to nifedipine was most likely due to the more pronounced membrane depolarization induced by carbachol in BK–/– than in WT tracheal cells. A dihydropyridine-sensitive "window current" of steady-state Ca2+ influx with a peak current at approximately –30 mV has been described in equine tracheal myocytes (41)
, and it is conceivable that this current is activated in BK–/– tracheae and contributed to contraction. Our results indicate that the coupling between muscarinic receptor stimulation and airway smooth muscle contraction switched in BK channel-deficient preparations from the pharmacomechanical mode to a mode dependent on membrane potential, labeled earlier as electromechanical coupling (42)
.
In contrast to WT preparations, tracheae from BK–/– mice exhibited slow phasic contractions, which were prominent at low and moderate carbachol concentrations and disappeared at concentrations of 1 µM or higher. Nifedipine completely abolished phasic contractions, indicating that Ca2+ influx through L-type Ca2+ channels was responsible for the instable contractile activity. The plant alkaloid ryanodine has received considerable interest in muscle pharmacology as a specific tool for inhibiting the function of the sarcoplasmic reticulum by causing the Ca2+ release channels to remain in a subconducting state with subsequent leak of Ca2+ from the store (43)
. In smooth muscle cells, ryanodine inhibits Ca2+ sparks (25)
and as a consequence, it abolished STHs and depolarized the membrane potential of isolated tracheal smooth muscle cells from WT mice to a similar extent as IbTX. Ryanodine alone did not increase basal tone of tracheal rings from WT mice, but carbachol in the presence of ryanodine induced phasic contractions similar to those in tracheae from BK–/– mice. This observation confirms again that functional BK channels are necessary for stable tonic contractions in airway smooth muscle. The mechanism underlying phasic contractions is not known. The fact, however, that rhythmic activity is abolished by nifedipine argues strongly in favor of a role for L-type Ca2+ channels in the initiation and maintenance of phasic contractions.
Considering the function of BK channels as negative feedback regulators, we expected that carbachol enhanced airway contraction more effectively in BK–/– than in WT preparations. However, there was neither a shift to the left of the carbachol concentration-effect curve nor was there a higher level of developed force at any carbachol concentration even when the respective maximum of phasic contractions was evaluated. In contrast, when the EC50 values of carbachol were compared in the presence of nifedipine, it became apparent that tracheae lacking BK channels were considerably less sensitive to carbachol than WT preparations, indicating that an enhanced Ca2+ influx through L-type Ca2+ channels compensated for the reduced effectiveness of carbachol in the absence of nifedipine.
To further analyze airway function in BK–/– mice, we used precision-cut lung slices (PCLSs) as a useful model to study, by means of videomicroscopy, contraction of individual airways of various sizes (44)
. In addition, we used the isolated-perfused lung (IPL) model, which allowed the assessment of airway resistance as part of the integrated responses of the whole organ. To our surprise, we found in both models that the response to muscarinc receptor stimulation was significantly attenuated when BK channels were lacking in the bronchial muscle. An even more striking effect was observed in in vivo experiments, in which the respiratory capacity was analyzed by whole body plethysmography. Whereas respiratory capacity was decreased concentration-dependently in WT mice challenged with aerosols generated from methacholine solutions, there was almost no response to the muscarinic receptor agonist in BK–/– mice. Comparison of the three different lung models reveals that the effects induced by stimulation of muscarinic receptors became increasingly more attenuated, the more the lung model was physiologically integrated.
ß2-Adrenoceptor agonists are potent bronchodilators and are widely used clinically to reverse bronchoconstriction in asthma and other obstructive airway diseases. There is convincing evidence that relaxation induced by this class of drugs is, at least in part, mediated by BK channels, which are activated by phosphorylation of PKA or phosphorylation-independently through the -subunit of the stimulatory G protein (Gs) (45)
. Contrary to what has been reported in earlier studies with BK channel blockers, the ß-adrenoceptor agonist isoprenaline proved to be a more potent relaxant of precontracted tracheal rings from BK–/– than from WT mice. This surprising finding prompted us to consider the possibility that the long-term lack of BK channels could have been compensated for by an adaptive up-regulation of the cAMP/PKA signaling cascade. What we found, however, was an up-regulation of the cGMP/PKG signaling system in airways of BK–/– mice without significant changes of the cAMP/PKA pathway. NO appears to play an important role in regulating several biological functions in the lung, including relaxation of airway smooth muscle (46
, 47)
. NO can be synthesized in a number of cell types of the lung, including macrophages, neutrophils, mast cells, nonadrenergic noncholinergic inhibitory neurons, vascular smooth muscle cells, and airway epithelial cells (47)
. Liberated NO leads to the accumulation of cGMP by stimulating soluble guanylyl cyclase, and activates PKG. Possible targets of PKG that induce airway relaxation include IRAG, a protein modulating Ca2+ release from IP3-sensitive stores (48)
, myosin light-chain phosphatase (49)
, and BK channels (50
, 51)
. Immunoblot analysis in airways from BK–/– mice showed with respect to WT animals a significant increase of soluble guanylyl cyclase, which could be stimulated with NO, a 2.3-fold increase of PKG and an almost 4-fold increase of IRAG. Up-regulation of the components of NO/cGMP/PKG signaling should have profound effects on airway contractility. More specifically, enhanced phosphorylation of up-regulated IRAG, which is a substrate for PKG, should effectively inhibit IP3-mediated Ca2+ release from intracellular stores during cholinergic stimulation. This is in line with our observation that carbachol-induced contractions were considerably weaker in BK–/– than in WT tracheae when Ca2+ influx through voltage-gated Ca2+ channels was blocked with nifedipine. Moreover, the PKG inhibitor Rp-8-Br-cGMPS shifted the EC50 values of carbachol in tracheae from BK–/– more effectively to the left than in WT preparations, indicating that PKG played a prominent role in airway contractility of mice lacking BK channels. Finally, the enhanced relaxation by ß-adrenoreceptor agonists in tracheae from BK–/– mice can be explained by cross-activation of up-regulated PKG by cAMP (52)
.
In summary, mice with a deleted BK channel -subunit exhibit an unexpected phenotype, which is characterized by a reduced sensitivity toward cholinergic stimulation. We were able to disclose an adaptive up-regulation of the cGMP/PKG signaling pathway as an underlying mechanism, compensating for an enhanced Ca2+ influx through voltage-operated Ca2+ channels due to membrane depolarization. Other compensatory mechanisms cannot be excluded at present. The fact that the reduced sensitivity to cholinergic stimulation was particularly prominent in lung models with higher physiological integration indicates that compensatory mechanisms in airway contractility are not confined to tracheal smooth muscle.
|
ACKNOWLEDGMENTS |
|---|
Received for publication August 17, 2006. Accepted for publication October 25, 2006.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
. Science 86,1583-1587This article has been cited by other articles:
|
|
 |
|
  K. D. Huebner, D. S. Jassal, O. Halevy, M. Pines, and J. E. Anderson Functional resolution of fibrosis in mdx mouse dystrophic heart and skeletal muscle by halofuginone Am J Physiol Heart Circ Physiol, April 1, 2008; 294(4): H1550 - H1561. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
) between methacholine-induced resistance and control resistance (before methacholine was added). Each methacholine dose was applied for 20 min; n = 4 per genotype. *P < 0.05.
