**3. Airway smooth muscle tone regulated by KCa channels**

#### **3.1. Characteristics and physiological roles of KCa channels**

### *3.1.1. Structure of KCa channels*

mediator regulated by protein kinase C [22]. Since MLC activity is sustained, not suppressed, by loss of MLC dephosphorylation via inactivation of MP, airway smooth muscle tone is enhanced without increasing [Ca2+]i (Ca2+-independent contraction: Ca2+ sensitization) [19, 23]. Airway smooth muscle tone is regulated by the degree of MLC phosphorylation mediated by both MLCK and MP activity. Alterations of contractile phenotype, which are due to both Ca2+ dynamics and Ca2+ sensitization, have clinical relevance to airflow limitation, airway hyperresponsiveness, and reduced responsiveness to β2-adrenoceptor agonists (β2-adrenergic desensitization), which are implicated with the pathophysiology of obstructive pulmonary

**SOC**

**MLCK**

**(relaxation)MLC pMLC (contraction)**

**Ca2+/CaM**

**Ca2+**

**Ca2+ dynamics**

**Ca2+ sparks**

**Gs Gq**

**VDC**

**Ca K+ 2+**

**MP**

**Figure 1. Role of Ca2+ dynamics and Ca2+ sensitization in the regulation of airway smooth muscle tone.** Ca2+ signal‐ ing via Ca2+ dynamics and Ca2+ sensitization contributes to the functional antagonism between β2-adreneceptor ago‐ nists and contractile agonists (such as histamine, ACh, LTs, and PGs), acting on GPCRs. MLC phosphorylation (pMLC), which is regulated by a balance between MLCK and MP, is fundamental for controlling contraction in airway smooth muscle. GPCR-related agents cause Ca2+ influx by activating ROC and cause Ca2+ release from SR by producing IP3. The latter process induces Ca2+ influx via activating SOC. An increase in intracellular concentrations of Ca2+ medi‐ ated by these processes enhances the binding of Ca2+ to CaM. A Ca2+−CaM complex (Ca2+/CaM) augments MLCK activ‐ ity, leading to MLC phosphorylation (Ca2+ dynamics: Ca2+-dependent mechanisms). On the other hand, contractile agonists activate RhoA by acting on G-protein–coupled receptors. Rho-kinase activated by GTP-RhoA phosphorylates (inactivates) MP, leading to MLC phosphorylation (Ca2+ sensitization: Ca2+-independent mechanisms). ACh: acetylcho‐ line, LTs: leukotrienes, PGs: prostaglandins, β2: β2-adrenoceptors, GPCRs: G-protein−coupled receptors, AC: adenylyl cyclase, ROC: receptor-operated Ca2+ influx, SOC: store-operated Ca2+ influx, IP3: inositol-1,4,5-triphosphate, SR: sarco‐ plasmic reticulum, PKA: protein kinase A, CaM: calmodulin, MLCK: myosin light chain kinase, MLC: myosin light

**Ca2+**

**IP3R**

**Ca2+ Ca2+**

**GTP-RhoA**

**Ca2+ senstization**

**RhoA**

**GPCR**

**ROC ACh, LTs, PGs etc**

**IP3**

**SR**

**Rho-kinase**

channels, VDC: L-type voltage-dependent

diseases, such as asthma and COPD [1].

**<sup>2</sup> KCa -agonists**

292 Muscle Cell and Tissue

**AC**

**cAMP**

**PKA**

**activation inhibition**

Ca2+ channels Illustrated based on ref. [1]

chain, MP: myosin phosphatase, KCa: large-conductance Ca2+-activated K+

KCa channels are composed of a tetramer formed by pore-forming α-subunits along with accessory β-subunits, and these channels are activated by increased membrane potential and increased [Ca2+]i . The α-subunit is ubiquitously expressed by mammalian tissues and encoded by a single gene (Slo, KCNMA1) [24, 25]. The α-subunit transmembrane domains comprise seven membrane-spanning segments (S0-S6) with extracellular loops and share homology with all voltage-gated K<sup>+</sup> channels with six transmembrane domains (S1-S6) and a pore helix. S1-S4 are arranged in a bundle that forms the voltage-sensing component, and S5-S6 and pore helices contribute to form the pore-forming component and the K+ selective filter [26]. The Cterminal tail confers the Ca2+-sensing ability of the KCa channels, involving a pair of Ca2+-sensing domains that regulate the conductance of K+ (RCK), i.e., RCK1 and RCK2 [27]. Although the Ca2+ sensor of the KCa channels has high specificity for Ca2+, other factors including divalent cations also influence the opening of these channels. Magnesium (Mg2+) enhances activation of these channels via a distinct binding site in the voltage sensor and RCK1 domain [28]. On the other hand, intracellular protons (H<sup>+</sup> ) attenuate the opening of the KCa channels [9, 29]. KCa channels associate with modulatory β-subunits, which are expressed in a cell-specific manner and have unique regulatory actions on these channels. The β-subunits bring about diversity of the KCa channels. There are four distinct β-subunits, β1-4, which are encoded by KCNMB1, KCNMB2, KCNMB3, and KCNMB4. These β-subunits in the KCa channels consist of two transmembrane domains with intracellular N- and C-termini and a long extracellular loop. The β1 subunit was the first β-subunit to be cloned and is primarily expressed in smooth muscle [30].

### *3.1.2. Electrical characteristics of KCa channels*

KCa channels are densely distributed on the cell membrane in airway smooth muscle cells and have a large conductance (about 250 pS in a symmetrical 135-150 mM K<sup>+</sup> medium) [31, 32, 33], as compared to other K+ channels. In freshly isolated human bronchial smooth muscle cells, single currents of the KCa channels were also recorded in cell-attached patches, inside-out patches, and outside-out patches [34, 35]. These channels have a conductance of about 210 pS in symmetrical 140 mM K<sup>+</sup> medium. KCa channels are highly selective for K<sup>+</sup> despite their large conductance [36]. Ca2+ sensitivity may be increased by intracellular Mg2+, as is the case in vascular muscle [37]. Effects of intracellular pH (pH<sup>i</sup> ) on KCa channels have been studied in rabbit tracheal muscle by using inside-out patches [9]. KCa channel activity was markedly inhibited by intracellular acidification, by reducing the sensitivity to Ca2+ and also by short‐ ening the open state of the channel. On the other hand, intracellular alkalization had an opposite effect (increasing Ca2+ sensitivity and lengthening the open state of the channel). Single-channel currents of KCa channels in guinea pig and canine tracheal muscle, studied in outside-out patches, were reversibly blocked by external application of charybdotoxin (ChTX) or iberiotoxin (IbTX), selective antagonists of KCa channels. This effect was not a result of reduced current amplitude; rather, it was caused by reducing the open-state probability (nPo), the fraction of the time during which the channel is open [7, 38]. In bovine trachealis, externally applied tetraethylammonium (TEA, 1 mM) strongly reduced the amplitude of single KCa channel current, different from the effects of ChTX (100 nM) on these channels without affecting current amplitude [32]. The effect of ChTX was also reversible. In contrast, the KCa channels were not affected by 4-aminopyridine (4-AP, 1 mM) applied internally or (2 mM) externally.

#### *3.1.3. Physiological role of KCa channels*

Typical action potentials have not been found in airway muscle under physiological condi‐ tions. This lack of action potentials is believed to be due to a marked increase in K+ conductance of the plasma membrane upon depolarization [39]. Thus, when the K+ conductance of the membrane is reduced by blocking K+ channels, one would expect an increase in excitability. In airway smooth muscle that is only weakly excitable, spontaneous phasic contractions can be initiated along with electrical activities by applying K+ channel blocking agents, such as TEA, 4-AP, ChTX and IbTX [40]. Some of these contractions are accompanied by electrical activity. These observations suggest that outward K+ currents passing through KCa channels may be functioning in an important regulatory role in these smooth muscle cells [41].

In excitation-contraction coupling of smooth muscle cells, local increases in Ca2+ concentrations occur due to focal releases of Ca2+ through ryanodine receptors (RyR) from the sarcoplasmic reticulum (SR), termed Ca2+ sparks [42]. Hundreds of KCa channels are opened by the Ca2+ sparks from SR close to the sarcolemma, leading to spontaneous outward currents (STOCs) (Figure 1). The coupling of ryanodine-mediated Ca2+ sparks to KCa channel-mediated STOCs is enhanced by the β<sup>1</sup> subunit, resulting in hyperpolarization of smooth muscle cells and the subsequent reduction of Ca2+ influx and initiation of muscle relaxation. In KCa channel β<sup>1</sup> subunit knockout mice, tracheal contraction induced by carbachol (CCh), a muscarinic receptor agonist, was enhanced as compared to wild-type mice, and not only the single channel activity of KCa channels in an inside-out patch but also STOCs in a whole cell configuration were markedly attenuated in tracheal smooth muscle cells of knockout mice as compared to wild-type mice [43]. IbTX (30 nM) enhances contraction induced by methacholine (MCh), a muscarinic receptor agonist, and verapamil, an inhibitor of VDC, suppresses the effect of IbTX on tension, demonstrating that KCa channel inhibition augments contraction via a Ca2+ influx through VDC channels [10].

#### **3.2. Stimulatory regulation of KCa channels by β2-adrenergic receptor agonists**

#### *3.2.1. cAMP-dependent phosphorylation*

The involvement of cAMP-dependent processes in KCa channel regulation has been examined in rabbit tracheal smooth muscle cells by using single-channel recording. In the presence of cAMP and adenosine triphosphate (ATP, 0.3 mM), application of PKA (10 units/ml) to the cytosolic side of inside-out membrane patches reversibly increased the nPo of KCa channels without changes in the amplitude of single-channel currents, and the recovery from this activation was significantly delayed by okadaic acid, an inhibitor of protein phosphatases [3]. A similar effect was observed with the catalytic subunit of PKA (10 units/ml), indicating that phosphorylation of a KCa channel protein enhances the open state of the channel [3, 4]. External application of isoprenaline (0.2 µM), a β2-adrenoceptor agonist, and okadaic acid (10 µM) also increased the activation of KCa channels in the cell-attached patch-clamp configuration, and the recovery from this activation was also significantly delayed by okadaic acid (Figure 2A) [3]. In Xenopus oocytes, similar results were observed in β-adrenergic action [44]. Moreover, external application of forskolin (10 µM), a direct activator of adenylyl cyclase, increased the KCa channel activity in tracheal smooth muscle cells [45]. These results are in accordance with results obtained in cultured smooth muscle cells of rat aorta using isoprenaline (10 µM), forskolin (10 µM), and dibutyryl cAMP (100 µM) in cell-attached patches and by using PKA (0.5 µM) and cAMP (1 µM) in inside-out patches [46]. These findings demonstrate that β2 adrenoceptor agonists augment KCa channel activity via PKA-mediated phosphorylation in airway smooth muscle.

outside-out patches, were reversibly blocked by external application of charybdotoxin (ChTX) or iberiotoxin (IbTX), selective antagonists of KCa channels. This effect was not a result of reduced current amplitude; rather, it was caused by reducing the open-state probability (nPo), the fraction of the time during which the channel is open [7, 38]. In bovine trachealis, externally applied tetraethylammonium (TEA, 1 mM) strongly reduced the amplitude of single KCa channel current, different from the effects of ChTX (100 nM) on these channels without affecting current amplitude [32]. The effect of ChTX was also reversible. In contrast, the KCa channels were not affected by 4-aminopyridine (4-AP, 1 mM) applied internally or (2 mM)

Typical action potentials have not been found in airway muscle under physiological condi‐ tions. This lack of action potentials is believed to be due to a marked increase in K+ conductance of the plasma membrane upon depolarization [39]. Thus, when the K+ conductance of the

In airway smooth muscle that is only weakly excitable, spontaneous phasic contractions can

TEA, 4-AP, ChTX and IbTX [40]. Some of these contractions are accompanied by electrical

In excitation-contraction coupling of smooth muscle cells, local increases in Ca2+ concentrations occur due to focal releases of Ca2+ through ryanodine receptors (RyR) from the sarcoplasmic reticulum (SR), termed Ca2+ sparks [42]. Hundreds of KCa channels are opened by the Ca2+ sparks from SR close to the sarcolemma, leading to spontaneous outward currents (STOCs) (Figure 1). The coupling of ryanodine-mediated Ca2+ sparks to KCa channel-mediated STOCs is enhanced by the β<sup>1</sup> subunit, resulting in hyperpolarization of smooth muscle cells and the subsequent reduction of Ca2+ influx and initiation of muscle relaxation. In KCa channel β<sup>1</sup> subunit knockout mice, tracheal contraction induced by carbachol (CCh), a muscarinic receptor agonist, was enhanced as compared to wild-type mice, and not only the single channel activity of KCa channels in an inside-out patch but also STOCs in a whole cell configuration were markedly attenuated in tracheal smooth muscle cells of knockout mice as compared to wild-type mice [43]. IbTX (30 nM) enhances contraction induced by methacholine (MCh), a muscarinic receptor agonist, and verapamil, an inhibitor of VDC, suppresses the effect of IbTX on tension, demonstrating that KCa channel inhibition augments contraction via a Ca2+ influx

may be functioning in an important regulatory role in these smooth muscle cells [41].

**3.2. Stimulatory regulation of KCa channels by β2-adrenergic receptor agonists**

The involvement of cAMP-dependent processes in KCa channel regulation has been examined in rabbit tracheal smooth muscle cells by using single-channel recording. In the presence of cAMP and adenosine triphosphate (ATP, 0.3 mM), application of PKA (10 units/ml) to the

channels, one would expect an increase in excitability.

channel blocking agents, such as

currents passing through KCa channels

externally.

294 Muscle Cell and Tissue

*3.1.3. Physiological role of KCa channels*

membrane is reduced by blocking K+

through VDC channels [10].

*3.2.1. cAMP-dependent phosphorylation*

be initiated along with electrical activities by applying K+

activity. These observations suggest that outward K+

**Fig 2 Figure 2. Stimulation and inhibition of KCa channels by β2-adrenoceptor and muscarinic receptor agonists in singlechannel recording of tracheal smooth muscle cells.** A: A continuous recording of the effects of external application of isoprenaline (0.2 µM) and okadaic acid (10 µM) on KCa channels in a cell-attached configuration held at −40 mV. Iso‐ prenaline increased KCa channel activity, and okadaic acid enhanced the effects of isoprenaline on these channels (up‐

per trace). The time course for washing out the effects of isoprenaline was markedly prolonged in the presence of okadaic acid (middle trace). These results demonstrate that KCa channel activity is regulated by phosphorylation via PKA. Okadaic acid augmented KCa channel activity, demonstrating that phosphatase activity is still intact in this exper‐ imental condition (lower trcae). B: A continuous recording of the effects of PKA and αs\*GTPγs on KCa channels in an inside-out patch held at 0 mV (left panel). PKA (0.5 U/ml) maximally increased KCa channel activity, and addition of the αs\*GTPγs (1 nM) enhanced KCa channel activity prestimulated by PKA (0.5 U/ml), indicating that αs activates KCa channels independent of PKA. Calibration bars, 3 pA and 4 s. Fold stimulation of channel activity are shown under the condition of addition of PKA (0.5 U/ml) and subsequently by addition of αs\*GTPγs (1 nM) (right panel). C: A continu‐ ous recording of the effects of ISO (1 µM) and mACH (10 µM) on KCa channels in an outside-out patch held at 0mV. External application of ISO increased KCa channel activity, whereas, following washout, mACH decreased this channel activity (upper trace), indicating that KCa channels are key molecules for the functional antagonisms between these two receptors. Calibration bars, 3 pA and 10 s. Relationship between nPo and time for an experiment similar to the upper trace with agonists added in reverse order (lower trace). PKA: protein kinase A, αs: α-subunit of Gs, which is stimulato‐ ry G protein of adenylyl cyclase, ISO: isoprenaline, mACH: methacholine, KCa: large-conductance Ca2+-activated K+ channels, nPo: open−state probability, U: unit. Cited from ref. [3, 4, 7].

#### *3.2.2. Membrane-delimited activation by Gs 30 nM*

Activation of KCa channels by isoprenaline is mediated by the α-subunit (αs) of the stimulatory G protein of adenylyl cyclase (Gs), independent of cAMP-dependent protein phosphorylation [4, 7]. In porcine, canine and ferret tracheal muscle cells, isoprenaline increased the activation of KCa channels in outside-out patches when guanosine triphosphate (GTP, 100 µM) was present at the cytosolic side of the patch. A similar increase in KCa channel activity was also observed even when phosphorylation was inhibited by the nonmetabolizable ATP analog, adenosine 5'-[β, γ-imido] diphosphate (ATP [β; γ ΝΗ], AMP-PNP (1 mM)) [4, 7]. In inside-out patch configuration with a patch pipette containing isoprenaline (1 µM), nonhydrolyzable GTP analog guanosine 5'-O-(3-thiotriphosphate) (GTP-γ-S, 100 µM) similarly potentiated the KCa channel activity. The recombinant αs proteins preincubated with GTP-γ-S (αs\*GTPγS, 100-1000 pM) increased the channel activity in a concentration-dependent manner when applied to the cytosolic side of inside-out patches [7]. The maximum effects of αs\*GTPγS were observed at 1000 pM, and the nPo of KCa channels was augmented to approximately 16-fold. On the other hand, αs preincubated with guanosine 5'-O-(2-thio-diphoshate) (GDP-β-S) had no effect on these channels. These results indicate that the KCa channels are directly activated by α<sup>s</sup> (membrane-delimited action) and that cAMP-dependent phosphorylation is not required. A direct action of Gs protein on the KCa channels has also been demonstrated in channels from rat or pig myometrium incorporated into planar lipid bilayers, by using GTP-γ-S and AMP-PNP [47]. β2-adrenoceptor agonists act on smooth muscle without the intracellular signal transduction processes (the cAMP-PKA pathway).

#### *3.2.3. Dual regulation by cAMP-dependent and -independent processes*

To examine whether receptor-channel coupling could occur in β2-adrenergic action on KCa channels, isoprenaline was applied to outside-out patches in the presence of GTP (100 µM) and AMP-PNP (1 mM), the competitive ATP inhibitor, in porcine tracheal smooth muscle cells (Figure 3) [4]. Isoprenaline (1 µM) markedly activated KCa channel activity without an alteration in current amplitude and returned to the control level within 5 min after drug washout. The nPo of the channels was increased to approximately fivefold in the presence of isoprenaline. This result was roughly equivalent to the level of channel stimulation previously reported in outside-out experiments in the absence of ATP, but without AMP-PNP. Consistent with a membrane-delimited, G protein-dependent coupling mechanism, addition of guanine nucleotides to the cytosolic side stimulated KCa channel activity in inside-out patches exposed to isoprenaline on the external side. Internal application of GTP (100 µM) also led to a marked increase in KCa channel activity; the nPo of the channels was increased to an approximately equivalent fold, as compared to the experimental condition when isoprenaline was applied to the outside-out patches in the presence of GTP. Stimulation of channel activity resulted in an apparent shift in the relationship between voltage and nPo by 10-15 mV after the addition of 100 µM GTP.

**Fig. 3**

per trace). The time course for washing out the effects of isoprenaline was markedly prolonged in the presence of okadaic acid (middle trace). These results demonstrate that KCa channel activity is regulated by phosphorylation via PKA. Okadaic acid augmented KCa channel activity, demonstrating that phosphatase activity is still intact in this exper‐ imental condition (lower trcae). B: A continuous recording of the effects of PKA and αs\*GTPγs on KCa channels in an inside-out patch held at 0 mV (left panel). PKA (0.5 U/ml) maximally increased KCa channel activity, and addition of the αs\*GTPγs (1 nM) enhanced KCa channel activity prestimulated by PKA (0.5 U/ml), indicating that αs activates KCa channels independent of PKA. Calibration bars, 3 pA and 4 s. Fold stimulation of channel activity are shown under the condition of addition of PKA (0.5 U/ml) and subsequently by addition of αs\*GTPγs (1 nM) (right panel). C: A continu‐ ous recording of the effects of ISO (1 µM) and mACH (10 µM) on KCa channels in an outside-out patch held at 0mV. External application of ISO increased KCa channel activity, whereas, following washout, mACH decreased this channel activity (upper trace), indicating that KCa channels are key molecules for the functional antagonisms between these two receptors. Calibration bars, 3 pA and 10 s. Relationship between nPo and time for an experiment similar to the upper trace with agonists added in reverse order (lower trace). PKA: protein kinase A, αs: α-subunit of Gs, which is stimulato‐ ry G protein of adenylyl cyclase, ISO: isoprenaline, mACH: methacholine, KCa: large-conductance Ca2+-activated K+

Activation of KCa channels by isoprenaline is mediated by the α-subunit (αs) of the stimulatory G protein of adenylyl cyclase (Gs), independent of cAMP-dependent protein phosphorylation [4, 7]. In porcine, canine and ferret tracheal muscle cells, isoprenaline increased the activation of KCa channels in outside-out patches when guanosine triphosphate (GTP, 100 µM) was present at the cytosolic side of the patch. A similar increase in KCa channel activity was also observed even when phosphorylation was inhibited by the nonmetabolizable ATP analog, adenosine 5'-[β, γ-imido] diphosphate (ATP [β; γ ΝΗ], AMP-PNP (1 mM)) [4, 7]. In inside-out patch configuration with a patch pipette containing isoprenaline (1 µM), nonhydrolyzable GTP analog guanosine 5'-O-(3-thiotriphosphate) (GTP-γ-S, 100 µM) similarly potentiated the KCa channel activity. The recombinant αs proteins preincubated with GTP-γ-S (αs\*GTPγS, 100-1000 pM) increased the channel activity in a concentration-dependent manner when applied to the cytosolic side of inside-out patches [7]. The maximum effects of αs\*GTPγS were observed at 1000 pM, and the nPo of KCa channels was augmented to approximately 16-fold. On the other hand, αs preincubated with guanosine 5'-O-(2-thio-diphoshate) (GDP-β-S) had no effect on these channels. These results indicate that the KCa channels are directly activated by α<sup>s</sup> (membrane-delimited action) and that cAMP-dependent phosphorylation is not required. A direct action of Gs protein on the KCa channels has also been demonstrated in channels from rat or pig myometrium incorporated into planar lipid bilayers, by using GTP-γ-S and AMP-PNP [47]. β2-adrenoceptor agonists act on smooth muscle without the intracellular signal

To examine whether receptor-channel coupling could occur in β2-adrenergic action on KCa channels, isoprenaline was applied to outside-out patches in the presence of GTP (100 µM) and AMP-PNP (1 mM), the competitive ATP inhibitor, in porcine tracheal smooth muscle cells (Figure 3) [4]. Isoprenaline (1 µM) markedly activated KCa channel activity without an alteration in current amplitude and returned to the control level within 5 min after drug washout. The nPo of the channels was increased to approximately fivefold in the presence of isoprenaline. This result was roughly equivalent to the level of channel stimulation previously

channels, nPo: open−state probability, U: unit. Cited from ref. [3, 4, 7].

*3.2.2. Membrane-delimited activation by Gs 30 nM*

296 Muscle Cell and Tissue

transduction processes (the cAMP-PKA pathway).

*3.2.3. Dual regulation by cAMP-dependent and -independent processes*

**Figure 3. Dual pathway and dual regulation of KCa channels in the functional antagonisms between β2-adrenocep‐ tors and muscarinic receptors.** At least two mechanisms are involved in activation of KCa channels following the β2 adrenoceptor activation; one is mediated through cAMP-dependent channel phosphorylation and the other through direct, cAMP-independent regulation by Gs protein (dual pathway). In contrast, in the muscarinic suppression of KCa channels, Gi proteins connected to M2 receptors are involved (dual regulation). The relationship between G proteins and KCa channels, i.e. the Gs/KCa stimulatory linkage and the Gi /KCa inhibitory linkage, may play a key role in the func‐ tional antagonisms (relaxation, contraction) between β2-adrenoceptors and muscarinic receptors in airway smooth muscle. β2: β2-adrenoceptors, M2: M2 muscarinc receptors, AC: adenylyl cyclase, Gi : inhibitory G protein of adenylyl cyclase, Gs: stimulatory G protein of adenylyl cyclase, PKA: protein kinase A, KCa: large-conductance Ca2+-activated K+ channels. Illustrated based on ref. [3, 4, 7, 53].

Stimulation of KCa channels by the catalytic subunit of cAMP-dependent PKA was examined in inside-out patches. KCa channel activity was progressively stimulated by a cumulative doseresponse protocol (PKA between 0.0005 and 5.0 units/ml). The maximum level of KCa channel stimulation by PKA was observed at either 0.5 or 5.0 units/ml (approximately sevenfold stimulation). At peak effect, the mean stimulation was approximately 7-fold, which was substantially less than the approximately 16-fold stimulation previously observed for 1 nM αs\*GTPγS. To examine the dual pathway of β2-adrenoceptor/channel coupling, inside-out patches were stimulated to near maximum with PKA (0.5 unit/ml). This concentration was chosen since it provided near maximal stimulation in all patches and a stable stimulation of channel activity over time. Following incubation with PKA for 5 min, αs\*GTPγS (1 nM) was added. KCa channels were potently activated by the addition of recombinant αs protein after stimulation by the near maximally effective concentration of PKA (Figure 2B) [4]. PKA produced an approximately 6-fold stimulation, and addition of α<sup>s</sup> produced an approximately 15-fold increase over baseline channel activity (Figure 2B). The fold stimulation produced during the condition of combined PKA and αs application was more than twice as great as the maximal fold stimulation that could be produced by PKA alone, suggesting that PKA and α<sup>s</sup> affect the channels independently.

The gating kinetics of KCa channels were quantitatively examined by monitoring the effects of stimulation of channel activity by isoprenaline (outside-out patches) and by PKA and α<sup>s</sup> (inside-out patches) at the level of channel open-time kinetics [4]. KCa channel open-times were well fit by the sum of two exponentials of mean duration τ1 and τ2, similar to previous reports [8, 9, 48]. The effect of αs on open-time kinetics was remarkably similar to that produced by isoprenaline on open-time kinetics; that is, αs did not alter the mean lifetimes, but increased the proportion of long open-time events. In contrast, the major kinetic effect of PKA was on open-state time constants, resulting in an increase in the mean duration of the long openings. The effect of PKA on channel kinetics was distinct from that of αs, consistent with distinct or independent modulatory effects at the channel protein.

#### *3.2.4. Role in relaxation by β2-adrenergic receptor agonists*

Airway smooth muscle relaxation produced by β-adrenoceptor activation is generally accompanied by membrane hyperpolarization, observed with intracellular microelectrodes in guinea pig, dog, and human tracheal muscles [49, 50], for which activation of KCa channels is thought to be responsible for the relaxation, as described earlier. This idea is supported by the observations in guinea pig and human trachealis that the relaxation by noradrenaline (1 µM) against CCh-induced contraction was nearly blocked by ChTX (50 nM) and that the concen‐ tration-relaxation curves to β2-adrenoceptor agonists, such as isoprenaline and salbutamol, were selectively shifted to the right by ChTX [51, 52]. The relaxant effect of forskolin on MChinduced contraction was also attenuated in the presence of ChTX, similar to isoprenaline [45]. Therefore, an increase in KCa channel activity may contribute to airway smooth muscle relaxation induced by β2-adrenoceptor agonists and cAMP-related agents. After Gs activity was irreversibly augmented by incubation with cholera toxin (2 µg/ml) for 6 h in guinea pig trachea, MCh-induced contraction was significantly attenuated, and this effect by Gs was reversed in the presence of ChTX (100 nM) [53]. The Gs/KCa stimulatory linkage may also be involved in β-adrenergic relaxation in airway smooth muscle.

#### **3.3. Inhibitory regulation of KCa channels by muscarinic receptor agonists**

#### *3.3.1. Membrane-delimited inhibition by Gi*

When MCh (50 µM) was applied to outside-out patches of porcine or canine tracheal muscle cells, the nPo of the KCa channel was markedly decreased without changes in the amplitude of single-channel currents [8, 54]. The decreased nPo is due to a reduction in channel open times, probably reflecting a decrease in the Ca2+ sensitivity of the channel. The muscarinic inhibition of KCa channels, similar to that found in airway smooth muscle, has been reported for the circular muscle of canine colon. The inhibition of KCa channels through muscarinic activation in guinea pig and swine tracheal muscle cells may be partly responsible for the prolonged suppression by ACh of STOCs following a transient increase [55, 56]. This suppression has been observed in longitudinal muscle cells of the rabbit jejunum. As discussed by Saunders and Farley, this inhibition is difficult to explain by the depletion of intracellular Ca2+ stores, because it occurs even with elevated Ca2+ concentrations. In the porcine and canine trachealis, the inhibition of KCa channels produced by muscarinic stimulation was potentiated by cytosolic application of GTP (100 µM), and strong, irreversible, potentiation was obtained with GTP-γ-S (100 µM) [8]. On the other hand, when GDP-β-S (1 mM) was applied to the cytosolic side, muscarinic inhibition was not observed. Incubation (4-6 h) of airway smooth muscle cells with pertussis toxin (0.1-1.0 µg/ml), which blocks signal transduction through ADP ribosylation of Gi , the inhibitory G protein of adenylyl cyclase, abolished the channel inhibition by MCh, without reducing channel activity in the control state [8]. The Gi /KCa inhibitory linkage may be involved in the muscarinic action in airway smooth muscle.

#### *3.3.2. Dual regulation by Gs and Gi*

stimulation by the near maximally effective concentration of PKA (Figure 2B) [4]. PKA produced an approximately 6-fold stimulation, and addition of α<sup>s</sup> produced an approximately 15-fold increase over baseline channel activity (Figure 2B). The fold stimulation produced during the condition of combined PKA and αs application was more than twice as great as the maximal fold stimulation that could be produced by PKA alone, suggesting that PKA and α<sup>s</sup>

The gating kinetics of KCa channels were quantitatively examined by monitoring the effects of stimulation of channel activity by isoprenaline (outside-out patches) and by PKA and α<sup>s</sup> (inside-out patches) at the level of channel open-time kinetics [4]. KCa channel open-times were well fit by the sum of two exponentials of mean duration τ1 and τ2, similar to previous reports [8, 9, 48]. The effect of αs on open-time kinetics was remarkably similar to that produced by isoprenaline on open-time kinetics; that is, αs did not alter the mean lifetimes, but increased the proportion of long open-time events. In contrast, the major kinetic effect of PKA was on open-state time constants, resulting in an increase in the mean duration of the long openings. The effect of PKA on channel kinetics was distinct from that of αs, consistent with distinct or

Airway smooth muscle relaxation produced by β-adrenoceptor activation is generally accompanied by membrane hyperpolarization, observed with intracellular microelectrodes in guinea pig, dog, and human tracheal muscles [49, 50], for which activation of KCa channels is thought to be responsible for the relaxation, as described earlier. This idea is supported by the observations in guinea pig and human trachealis that the relaxation by noradrenaline (1 µM) against CCh-induced contraction was nearly blocked by ChTX (50 nM) and that the concen‐ tration-relaxation curves to β2-adrenoceptor agonists, such as isoprenaline and salbutamol, were selectively shifted to the right by ChTX [51, 52]. The relaxant effect of forskolin on MChinduced contraction was also attenuated in the presence of ChTX, similar to isoprenaline [45]. Therefore, an increase in KCa channel activity may contribute to airway smooth muscle relaxation induced by β2-adrenoceptor agonists and cAMP-related agents. After Gs activity was irreversibly augmented by incubation with cholera toxin (2 µg/ml) for 6 h in guinea pig trachea, MCh-induced contraction was significantly attenuated, and this effect by Gs was reversed in the presence of ChTX (100 nM) [53]. The Gs/KCa stimulatory linkage may also be

affect the channels independently.

298 Muscle Cell and Tissue

independent modulatory effects at the channel protein.

*3.2.4. Role in relaxation by β2-adrenergic receptor agonists*

involved in β-adrenergic relaxation in airway smooth muscle.

*3.3.1. Membrane-delimited inhibition by Gi*

**3.3. Inhibitory regulation of KCa channels by muscarinic receptor agonists**

When MCh (50 µM) was applied to outside-out patches of porcine or canine tracheal muscle cells, the nPo of the KCa channel was markedly decreased without changes in the amplitude of single-channel currents [8, 54]. The decreased nPo is due to a reduction in channel open times,

As described earlier, KCa channels are markedly activated by β2-adrenoceptor agonists; in contrast, KCa channels are markedly suppressed by muscarinic receptor agonists via G proteins (Figure 3). The activation process is mediated by the stimulatory G protein, Gs; in contrast, the suppression process is mediated by the inhibitory G protein, Gi (dual regulation). To demon‐ strate the functional antagonistic, hormone-linked stimulatory and inhibitory regulation of KCa channels by G proteins at the single-channel level, isoprenaline and MCh were sequentially applied to identical outside-out patches under the condition of physiologic Ca2+ concentration and GTP (100 µM) [7]. External application of isoprenaline (1 µM) markedly increased KCa channel activity, and following drug washout this channel activity reversed to baseline; then, external application of MCh (10 µM) markedly decreased this channel activity (Figure 2C). Receptor-linked stimulatory and inhibitory modulation of KCa channels was not sequentially dependent as shown by an experiment in which this channel activity was inhibited by MCh and then activated by isoprenaline. Consistent with these outside-out experiments, internal addition of guanine nucleotides stimulated KCa channels when isoprenaline was present at the extracellular side in inside-out patches, and conversely, guanine nucleotides suppressed the channel activity when MCh was present at the extracellular side in inside-out patches [7]. These results indicate that the functional antagonism between β2-adrenergic and muscarinic action converges on a single KCa channel current. Therefore, KCa channel activity plays a key role in the regulation of airway smooth muscle tone.

#### *3.3.3. Role in contraction by muscarinic receptor agonists*

After incubation of tracheal smooth muscle with pertussis toxin (1.0 µg/ml for 6 h), MChinduced contraction was significantly attenuated, and this effect by pertussis toxin was reversed in the presence of ChTX [53]. The Gi /KCa inhibitory linkage may be involved in the muscarinic-induced contraction in airway smooth muscle. From a functional point of view, it would be favorable to reduce the K+ conductance of the plasma membrane to produce excitation by agonists such as ACh. Gi protein couples with the M2 subtype of muscarinic receptors, leading to an inhibition in cAMP. These M2 receptors exist on the surface of airway smooth muscle cells. A selective M2 receptor antagonist (AF-DX 116, a benzodiazepine derivative) suppressed MCh-induced contraction in a concentration-dependent manner and potentiated relaxation induced by isoprenaline and forskolin in MCh-precontracted tracheal muscle [53]. AF-DX116 had no effect on isoprenaline-induced relaxation when the preparation was precontracted with histamine. The functional antagonism between isoprenaline (or forskolin) and M2 receptor stimulation may not only be simply mediated by inhibition of adenylyl cyclase through the M2 receptors but also be exerted by the direct inhibition of KCa channels by pertussis toxin–sensitive Gi protein through activation of muscarinic receptors, since there is evidence that the activation of KCa channels is involved in the relaxation induced by forskolin and isoprenaline. Furthermore, M2 receptors inhibited the activity of KCa channels via dual pathways of a direct membrane-delimited interaction of Gβγ and activation of phospholipase C/protein kinase C [57]. In KCa channel β1 subunit knockout mice, CCh-induced contraction and membrane depolarization in tracheal smooth muscle were enhanced as compared to wild-type mice, and these augmented effects of CCh were inhibited in the presence of AF-DX116 [43, 58]. These results indicate that the KCa channel β1 subunit plays a functional role in opposing M2 muscarinic receptor signaling.

#### *3.3.4. Regulation of KCa channels by other factors (cGMP, protein kinase C)*

#### *3.3.4.1. NO, cGMP*

Nitric oxide (NO), which is primarily generated by nitric oxide synthase (NOS) in the endo‐ thelium, causes relaxation of vascular smooth muscle cells via hyperpolarization of the cell membrane [59, 60]. NO also augmented the KCa channel activity in vascular smooth muscles, and NO-induced vasodilation was attenuated by blockade of the KCa channel activity [61]. The NO/3'-5'-cyclic guanosine monophosphate (cGMP) pathway plays an important role in relaxation of smooth muscle including vessels and airways. KCa channels were markedly enhanced by cGMP-mediated processes, suggesting that activation of these channels leads to cGMP-induced relaxation of smooth muscle [62, 63]. The KCa channel α-subunit null mice had increased vascular smooth contraction as compared to wild-type mice [64]. This phenomenon was due to an impaired response to cGMP-dependent vasorelaxation, indicating that the KCa channel is an important effector for cGMP-mediated action. Protein kinase G (PKG) was involved in this activation of KCa channels via the NO/cGMP pathway [65, 66]. Activation of KCa channels via dopamine receptors occurs through PKG and mediates relaxation in coronary and renal arteries [67]. PKG may be cross-activated by cAMP to stimulate KCa channels [68]. Moreover, dual pathways of KCa channel modulation by NO have been demonstrated; these pathways are the PKG-dependent pathway [69] and the direct activation of NO with the channel protein [70]. Since the stimulatory effect of NO on KCa channels was abolished by knockdown of the β-subunit with antisense, the β-subunit acts as a mediator of NO [71].

#### *3.3.4.2. Protein kinase C*

reversed in the presence of ChTX [53]. The Gi

would be favorable to reduce the K+

300 Muscle Cell and Tissue

excitation by agonists such as ACh. Gi

channels by pertussis toxin–sensitive Gi

*3.3.4.1. NO, cGMP*

functional role in opposing M2 muscarinic receptor signaling.

*3.3.4. Regulation of KCa channels by other factors (cGMP, protein kinase C)*

/KCa inhibitory linkage may be involved in the

conductance of the plasma membrane to produce

protein couples with the M2 subtype of muscarinic

protein through activation of muscarinic receptors,

muscarinic-induced contraction in airway smooth muscle. From a functional point of view, it

receptors, leading to an inhibition in cAMP. These M2 receptors exist on the surface of airway smooth muscle cells. A selective M2 receptor antagonist (AF-DX 116, a benzodiazepine derivative) suppressed MCh-induced contraction in a concentration-dependent manner and potentiated relaxation induced by isoprenaline and forskolin in MCh-precontracted tracheal muscle [53]. AF-DX116 had no effect on isoprenaline-induced relaxation when the preparation was precontracted with histamine. The functional antagonism between isoprenaline (or forskolin) and M2 receptor stimulation may not only be simply mediated by inhibition of adenylyl cyclase through the M2 receptors but also be exerted by the direct inhibition of KCa

since there is evidence that the activation of KCa channels is involved in the relaxation induced by forskolin and isoprenaline. Furthermore, M2 receptors inhibited the activity of KCa channels via dual pathways of a direct membrane-delimited interaction of Gβγ and activation of phospholipase C/protein kinase C [57]. In KCa channel β1 subunit knockout mice, CCh-induced contraction and membrane depolarization in tracheal smooth muscle were enhanced as compared to wild-type mice, and these augmented effects of CCh were inhibited in the presence of AF-DX116 [43, 58]. These results indicate that the KCa channel β1 subunit plays a

Nitric oxide (NO), which is primarily generated by nitric oxide synthase (NOS) in the endo‐ thelium, causes relaxation of vascular smooth muscle cells via hyperpolarization of the cell membrane [59, 60]. NO also augmented the KCa channel activity in vascular smooth muscles, and NO-induced vasodilation was attenuated by blockade of the KCa channel activity [61]. The NO/3'-5'-cyclic guanosine monophosphate (cGMP) pathway plays an important role in relaxation of smooth muscle including vessels and airways. KCa channels were markedly enhanced by cGMP-mediated processes, suggesting that activation of these channels leads to cGMP-induced relaxation of smooth muscle [62, 63]. The KCa channel α-subunit null mice had increased vascular smooth contraction as compared to wild-type mice [64]. This phenomenon was due to an impaired response to cGMP-dependent vasorelaxation, indicating that the KCa channel is an important effector for cGMP-mediated action. Protein kinase G (PKG) was involved in this activation of KCa channels via the NO/cGMP pathway [65, 66]. Activation of KCa channels via dopamine receptors occurs through PKG and mediates relaxation in coronary and renal arteries [67]. PKG may be cross-activated by cAMP to stimulate KCa channels [68]. Moreover, dual pathways of KCa channel modulation by NO have been demonstrated; these pathways are the PKG-dependent pathway [69] and the direct activation of NO with the

KCa channels are activated via phosphorylation of their channels by PKA and PKG, as described earlier. However, the effects of protein kinase C (PKC) on these channels are still controversial. PKC enhanced the activity of KCa channels in rat pulmonary arterial smooth muscle [72]. In contrast, PKC reversed cAMP-induced activation of these channels [73]. The phosphorylation by PKC acts on KCa channels via direct inhibition and also acts as a switch to influence the effects of PKA and PKG [74, 75]. In addition to these pathways, c-Src and tyrosine kinase suppressed the activity of the KCa channels in coronary and aortic myocytes [76], whereas cSrcinduced phosphorylation augmented these channels in HEK 293 cells [77].

#### *3.3.4.3. Redox and ROS*

Reactive oxygen species (ROS) synthesized in endothelial and smooth muscle cells exert physiological and pathophysiological effects on smooth muscle via altering intracellular reduction and/or oxygen (redox) status [78]. The redox state influences the gating of KCa channels [79]. However, the effects of redox are complex. Preferential oxidation of methionine increased the activity of KCa channels, whereas oxidation of cysteines reduced the channel activity [80, 81]. KCa channel activity was enhanced by hydrogen peroxide (H2O2) in pulmonary arterial smooth muscle, resulting in vasodilation mediated by membrane hyperpolarization [82]. Hydrogen peroxide (H2O2) may directly bind to KCa channels to regulate them, or it may activate these channels via the phospholipase A2-arachidonic acid pathway and metabolites of lipoxygenase [83]. On the other hand, H2O2 caused contraction of tracheal smooth muscle in a concentration-dependent fashion and elevation of [Ca2+]i [84]. Moreover, peroxynitrite (OONO-), an oxidant generated by the reaction of NO and superoxide, caused contraction of the cerebral artery by inhibiting KCa channel activity [85].

#### *3.3.4.4. Arachidonic acid*

Arachidonic acid and its metabolites such as 20-hydroxyeicosatetraenoic acid (20-HETE) and epoxyeicosatrienoic acids (EETs) play an important role in the regulation of vascular smooth muscle tone. Arachidonic acid and EETs caused vasodilation mediated by increasing KCa channel activity [86, 87]. In airway smooth muscle, 20-HETE also caused relaxation with membrane hyperpolarization via activation of KCa channels [88]. On the other hand, 20-HETE is a vasoconstrictor. KCa channel activity was inhibited by 20-HETE, and this phenomenon is mediated by PKC [89]. The vasoconstriction induced by 20-HETE was also attenuated by increasing KCa channel activity [89]. Acute hypoxia reduced the generation of 20-HETE, and subsequently the inhibitory action of 20-HETE on KCa channels was removed in cerebral arterial smooth muscle cells [90].

### **3.4. Synergistic effects between muscarinic and β2-adrenergic receptors**

The combination of muscarinic receptor antagonists with β2-adrenoceptor agonists has pharmacological rationale as a bronchodilator therapy for COPD [91]. In the human airway, muscarinic contraction is more resistant to β2-adrenoceptor–induced relaxation than that induced by other contractile agonists [92]. Muscarinic receptors and β2-adrenoceptors are unevenly distributed in the human airways. β2-adrenoceptors were increased in the distal airways: segmental bronchus < subsegmental bronchus < lung parenchyma [93]. M3 receptors are expressed more exclusively in segmental than subsegmental bronchus; in contrast, the M2 subtype is widely distributed throughout the airways, while the M1 subtype is found only in parenchyma [93]. These findings may explain why combined inhalation of a muscarinic antagonist and a β2-adreneceptor agonist causes greater bronchodilation than monotherapy [94]. Furthermore, characteristic interactions between muscarinic receptors and β2-adreno‐ ceptors are involved in prejunctional modulation of ACh release from parasympathetic nerve endings [95] and intracellular signaling cross-talk at the adenylyl cyclase/PKA level [96], resulting in synergistic effects on relaxation of airway smooth muscle. KCa channel activity may contribute to these interactions between these two receptors; however, little is known about the detailed underlying mechanisms.

In isometric tension recordings of guinea pig tracheal smooth muscle, indacaterol (1 nM), a long-acting β2-adrenoceptor agonist, modestly inhibited MCh-induced contraction (1 µM) (Figure 4A). When glycopyrronium bromide (10 nM), a long-acting muscarinic receptor antagonist, was applied in the presence of indacaterol (1 nM), the relaxant effect of glycopyr‐ ronium bromide was significantly augmented (Figure 4A) [97]. The value of percent relaxation for the combination of indacaterol with glycopyrronium bromide was more than the sum of that for each agent. Similar results were observed between indacaterol (1 nM) and glycopyr‐ ronium (3-30 nM) [97]. Moreover, similar results were also observed between other β2 adreneceptor agonists, such as salbutamol and procaterol, and other muscarinic receptor antagonists, such as atropine and tiotropium (our unpublished observation). These results indicate that the combination of muscarinic receptor antagonists with β2-adrenoceptor agonists causes a synergistic inhibition against muscarinic contraction. This phenomenon was observed in isolated human bronchus [98]. This synergistic effect was enhanced after exposure to pertussis toxin (1 µg/ml) or cholera toxin (2 µg/ml) for 6 h; in contrast, the effect was attenuated in the presence ChTX (100 nM) or IbTX (30 nM). A reduction in this synergistic effect induced by ChTX or IbTX was reversed to the control response in the presence of verapamil (Figure 4B) [99]. Inactivation of the Gi /KCa inhibitory linkage and activation of the Gs/KCa stimulatory linkage are involved in this synergistic effect between muscarinic receptor antagonists and β2-adrenoceptor agonists in airway smooth muscle (Figure 3) [4, 7, 53]. Moreover, the KCa/VDC channel linkage is also involved in this synergistic effect. On the other hand, synergistic effects did not occur between β2-adrenoceptor agonists and theophylline in airway smooth muscle (our unpublished observation). Although the clinical relevance of this result is still unknown, this result may provide evidence that combination therapy between muscarinic receptor antagonists and β2-adrenoceptor agonists is an effective bronchodilator therapy for COPD [100].

Ca2+ Dynamics and Ca2+ Sensitization in the Regulation of Airway Smooth Muscle Tone http://dx.doi.org/10.5772/59347 303

**3.4. Synergistic effects between muscarinic and β2-adrenergic receptors**

the detailed underlying mechanisms.

302 Muscle Cell and Tissue

verapamil (Figure 4B) [99]. Inactivation of the Gi

therapy for COPD [100].

The combination of muscarinic receptor antagonists with β2-adrenoceptor agonists has pharmacological rationale as a bronchodilator therapy for COPD [91]. In the human airway, muscarinic contraction is more resistant to β2-adrenoceptor–induced relaxation than that induced by other contractile agonists [92]. Muscarinic receptors and β2-adrenoceptors are unevenly distributed in the human airways. β2-adrenoceptors were increased in the distal airways: segmental bronchus < subsegmental bronchus < lung parenchyma [93]. M3 receptors are expressed more exclusively in segmental than subsegmental bronchus; in contrast, the M2 subtype is widely distributed throughout the airways, while the M1 subtype is found only in parenchyma [93]. These findings may explain why combined inhalation of a muscarinic antagonist and a β2-adreneceptor agonist causes greater bronchodilation than monotherapy [94]. Furthermore, characteristic interactions between muscarinic receptors and β2-adreno‐ ceptors are involved in prejunctional modulation of ACh release from parasympathetic nerve endings [95] and intracellular signaling cross-talk at the adenylyl cyclase/PKA level [96], resulting in synergistic effects on relaxation of airway smooth muscle. KCa channel activity may contribute to these interactions between these two receptors; however, little is known about

In isometric tension recordings of guinea pig tracheal smooth muscle, indacaterol (1 nM), a long-acting β2-adrenoceptor agonist, modestly inhibited MCh-induced contraction (1 µM) (Figure 4A). When glycopyrronium bromide (10 nM), a long-acting muscarinic receptor antagonist, was applied in the presence of indacaterol (1 nM), the relaxant effect of glycopyr‐ ronium bromide was significantly augmented (Figure 4A) [97]. The value of percent relaxation for the combination of indacaterol with glycopyrronium bromide was more than the sum of that for each agent. Similar results were observed between indacaterol (1 nM) and glycopyr‐ ronium (3-30 nM) [97]. Moreover, similar results were also observed between other β2 adreneceptor agonists, such as salbutamol and procaterol, and other muscarinic receptor antagonists, such as atropine and tiotropium (our unpublished observation). These results indicate that the combination of muscarinic receptor antagonists with β2-adrenoceptor agonists causes a synergistic inhibition against muscarinic contraction. This phenomenon was observed in isolated human bronchus [98]. This synergistic effect was enhanced after exposure to pertussis toxin (1 µg/ml) or cholera toxin (2 µg/ml) for 6 h; in contrast, the effect was attenuated in the presence ChTX (100 nM) or IbTX (30 nM). A reduction in this synergistic effect induced by ChTX or IbTX was reversed to the control response in the presence of

Gs/KCa stimulatory linkage are involved in this synergistic effect between muscarinic receptor antagonists and β2-adrenoceptor agonists in airway smooth muscle (Figure 3) [4, 7, 53]. Moreover, the KCa/VDC channel linkage is also involved in this synergistic effect. On the other hand, synergistic effects did not occur between β2-adrenoceptor agonists and theophylline in airway smooth muscle (our unpublished observation). Although the clinical relevance of this result is still unknown, this result may provide evidence that combination therapy between muscarinic receptor antagonists and β2-adrenoceptor agonists is an effective bronchodilator

/KCa inhibitory linkage and activation of the

**Figure 4. Synergistic action in combination of β2-adrenocetor agonists with muscarinic receptor antagonists against tracheal smooth muscle contraction.** A: Left panel: A typical example of the inhibitory effects of GB (10 nM), a longacting muscarinic receptor antagonist (LAMA) against MCh (1 µM)-induced contraction (upper trace). A typical exam‐ ple of the inhibitory effects of equimolar amounts of GB in the presence of indacaterol (1 nM), a long-acting β2 adrenoceptor agonist (LABA) against MCh-induced contraction (1 µM) (lower trace). Right panel: Percent inhibition of combining GB with indacaterol is greater than the sum of each agent, demonstrating synergistic action between β2 adrenoceptor agonists and muscarinic receptor antagonists. B: The percent inhibition of GB (10 nM) with indacaterol (1 nM) against MCh-induced contraction (1 µM) was markedly augmented after incubation with PTX (1 µg/ml) and CTX (2 µg/ml) for 6 h. In contrast, the percent inhibition was significantly attenuated in the presence of ChTX (100 nM), and this ChTX-induced effect was reversed to the control level by addition of Vera (1 µM). These results indicate that the G proteins (Gi , Gs)/KCa channel linkage and the KCa/VDC channel linkage contributed to this synergistic action, similar to the mechanisms shown in Figures 1, 3. GB: glycopyrronium bromide, MCh: methacholine, PTX: pertussis toxin, CTX: cholera toxin, ChTX: charybdotoxin, Vera: verapamil, KCa: large-conductance Ca2+-activated K+ channels, VDC: L-type voltage-dependent Ca2+ channels. Cited from ref. [97, 99].

**Fig. 4** 
