Contractility of airway smooth muscle is involved in airflow limitation, which is implicated in the pathophysiology of asthma and chronic obstructive pulmonary disease (COPD). Spasmogens act on trimeric G protein-coupled receptors (GPCRs), such as acetylcholine (ACh), histamine, leukotrienes, and prostaglandins, to mediate airway smooth muscle contraction. Airway smooth muscle tone is ultimately regulated by the activation of myosin light chain (MLC); MLC is phosphorylated via myosin light chain kinase (MLCK) and dephosphorylated via myosin phosphatase (MP). Activation of MLCK contracts airway smooth muscle mediated by Ca2+-dependent mechanisms, which is due to increased concentrations of intracellular Ca2+ ([Ca2+]i) via a Ca2+ influx through Ca2+ channels (Ca2+ dynamics). In contrast, inactivation of MP contracts airway smooth muscle by Ca2+-independent mechanisms, which are due to an increase in the sensitivity to Ca2+ via Rho-kinase, a protein affected by RhoA, a monomeric G protein (Ca2+ sensitization) . RhoA/Rho-kinase processes are widely distributed in tissues including the respiratory system and regulated by agonists for GPCRs.
β2-adreneoceptor agonists and muscarinic receptor antagonists counteract spasmogen-induced contraction with reducing [Ca2+]i (antagonizing Ca2+ dynamics). β2-adrenoceptor agonists also suppress airway smooth muscle contraction by reducing sensitivity to Ca2+ (antagonizing Ca2+ sensitization) [1, 2]. Inhibition in both Ca2+ dynamics and Ca2+ sensitization is involved in the effects of β2-adrenoceptor agonists against spasmogen-induced contraction. Moreover, β2-adrenoceptor agonists relax airway smooth muscle via 3'-5'-cyclic adenosine monophosphate (cAMP)-dependent protein kinase (protein kinase A: PKA), leading to inactivation (phosphorylation) of MLCK. Large-conductance Ca2+-activated K+ (KCa, BKCa, Maxi-K+) channels are markedly activated by PKA-induced phosphorylation [3, 4, 5, 6] and Gs-induced action (G, a stimulatory trimeric G protein of adenylyl cyclase) [4, 5, 6, 7]. KCa channels are activated by β2-adrenoceptor agonists via Gs and suppressed by muscarinic receptor agonists via Gi, an inhibitory trimeric G protein of adenylyl cyclase [7, 8]. Since KCa channels have a large conductance of outward currents and exist innumerably on the cell membrane in airway smooth muscle , the opening of these channels also regulates airway smooth muscle tone mediated by membrane potential-dependent Ca2+ influx (Ca2+ dynamics), such as L-type voltage-dependent Ca2+ (VDC) channels . Therefore, not only Ca2+ signaling (Ca2+ dynamics and Ca2+ sensitization) but also KCa channels play a key role in the functional antagonism between β2-adrenoceptors and muscarinic receptors in airway smooth muscle.
Alterations of contractile phenotype, i.e. hyperresponsiveness to contractile agents (airway hyperresponsiveness) or hyporesponsiveness to relaxant agents (β2-adrenergic desensitization), occurs due to intrinsic or extrinsic factors involved in the pathophysiology of asthma. Dysfunctional contractility, which is a characteristic feature of patients with asthma, may depend on Ca2+ signaling (Ca2+ dynamics and Ca2+ sensitization) and KCa channels [1, 6, 11, 12]. Furthermore, airway smooth muscle cells have the ability to change the degree of various functions, such as contractility, proliferation, migration, and synthesis of inflammatory mediators [1, 13, 14]. The plasticity from a contractile phenotype to other phenotypes (proliferation, migration, or secretion of chemical mediators) may enhance airway inflammation, leading to airway remodeling, which is also characterized in asthma. This phenotype change may also be associated with Ca2+ signaling (Ca2+ dynamics and Ca2+ sensitization) and KCa channels.
Ca2+ signaling and KCa channels involved in the regulation of airway smooth muscle tone may be therapeutic targets in asthma and COPD [1, 6, 10, 11, 12, 15]. To elucidate the cause of the pathophysiology in asthma and COPD, and to establish a rational bronchodilator use for these diseases, the mechanisms underlying the regulation of airway smooth muscle tone via β2-adrenergic and muscarinic receptors were examined by using physiological techniques such as single-channel recording in tracheal smooth muscle cells, isometric tension recordings of isolated tracheal smooth muscle and simultaneous recording of isometric tension and F340/F380 in Fura-2–loaded tracheal smooth muscle. In this chapter, the functional characteristics of airway smooth muscle involved in alterations of contractile and proliferative ability are focused on Ca2+ signaling (Ca2+ dynamics and Ca2+ sensitization) mediated by G protein/KCa/VDC linkage and RhoA/Rho-kinase processes.
2. Mechanism of airway smooth muscle tone
Contractile agonists acting on GPCRs cause contraction of airway smooth muscle with increasing [Ca2+]i mediated by Ca2+ influx passing through Ca2+ channels (Ca2+ dynamics). When ligands are connected to the GPCRs, receptor-operated Ca2+ (ROC) influx is activated , and Ca2+ is released from sarcoplasmic reticulum (SR) via the production of inositol-1,4,5-triphosphate (IP3). This Ca2+ release activates store-operated capacitative Ca2+ (SOC) influx (Figure 1) . Moreover, VDC channels are mainly activated by membrane depolarization under the condition of high K+ at the extracellular side. Ca2+ influx passing through VDC channels contributes to high K+-induced contraction. In contrast, VDC is partly involved in the GPCR-mediated Ca2+ influx . An increase in [Ca2+]i enhances the binding of Ca2+ to calmodulin (CaM), a calcium-binding messenger protein. MLCK activity is augmented by a Ca2+-CaM complex (Ca2+/CaM), and MLC is phosphorylated (activated) by MLCK , leading to contraction of airway smooth muscle (Ca2+-dependent contraction: Ca2+ dynamics) [10, 17, 19]. After activated MLC is dephosphorylated (inactivated) by MP, contraction is reversed to relaxation (Figure 1). On the other hand, contractile agonists activate RhoA mediated by stimulating GPCRs. RhoA is activated by binding to GTP (RhoA-GTP: active form of RhoA). Rho-kinase is activated by RhoA-GTP, and MP is phosphorylated by Rho-kinase (MP inactivation) (Figure 1) [20, 21]. MP is also phosphorylated by CPI-17, which is another potential mediator regulated by protein kinase C . 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 diseases, such as asthma and COPD .
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
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+ 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 . The C-terminal 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 . 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 . On the other hand, intracellular protons (H+) 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 .
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+ 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+ medium. KCa channels are highly selective for K+ despite their large conductance . Ca2+ sensitivity may be increased by intracellular Mg2+, as is the case in vascular muscle . Effects of intracellular pH (pHi) on KCa channels have been studied in rabbit tracheal muscle by using inside-out patches . KCa channel activity was markedly inhibited by intracellular acidification, by reducing the sensitivity to Ca2+ and also by shortening 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 . 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 conditions. This lack of action potentials is believed to be due to a marked increase in K+ conductance of the plasma membrane upon depolarization . 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 . 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 .
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 . 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 β1 subunit, resulting in hyperpolarization of smooth muscle cells and the subsequent reduction of Ca2+ influx and initiation of muscle relaxation. In KCa channel β1 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 . 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 .
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 . 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) . In Xenopus oocytes, similar results were observed in β-adrenergic action . Moreover, external application of forskolin (10 μM), a direct activator of adenylyl cyclase, increased the KCa channel activity in tracheal smooth muscle cells . 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 . These findings demonstrate that β2-adrenoceptor agonists augment KCa channel activity via PKA-mediated phosphorylation in airway smooth muscle.
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 . 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 αs (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 . 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) . 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.
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 dose-response 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) . PKA produced an approximately 6-fold stimulation, and addition of αs 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 αs 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 αs (inside-out patches) at the level of channel open-time kinetics . 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 concentration-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 MCh-induced contraction was also attenuated in the presence of ChTX, similar to isoprenaline . 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) . 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) . 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 . 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
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 demonstrate 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) . 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 . 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), MCh-induced contraction was significantly attenuated, and this effect by pertussis toxin was reversed in the presence of ChTX . 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 . 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 . 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)
220.127.116.11. NO, cGMP
Nitric oxide (NO), which is primarily generated by nitric oxide synthase (NOS) in the endothelium, 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 . 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 . 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 . PKG may be cross-activated by cAMP to stimulate KCa channels . Moreover, dual pathways of KCa channel modulation by NO have been demonstrated; these pathways are the PKG-dependent pathway  and the direct activation of NO with the channel protein . 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 .
18.104.22.168. Protein kinase C
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 . In contrast, PKC reversed cAMP-induced activation of these channels . 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 , whereas cSrc-induced phosphorylation augmented these channels in HEK 293 cells .
22.214.171.124. 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 . The redox state influences the gating of KCa channels . 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 . 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 . On the other hand, H2O2 caused contraction of tracheal smooth muscle in a concentration-dependent fashion and elevation of [Ca2+]i . Moreover, peroxynitrite (OONO-), an oxidant generated by the reaction of NO and superoxide, caused contraction of the cerebral artery by inhibiting KCa channel activity .
126.96.36.199. 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 . On the other hand, 20-HETE is a vasoconstrictor. KCa channel activity was inhibited by 20-HETE, and this phenomenon is mediated by PKC . The vasoconstriction induced by 20-HETE was also attenuated by increasing KCa channel activity . 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 .
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 . In the human airway, muscarinic contraction is more resistant to β2-adrenoceptor–induced relaxation than that induced by other contractile agonists . 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 . 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 . These findings may explain why combined inhalation of a muscarinic antagonist and a β2-adreneceptor agonist causes greater bronchodilation than monotherapy . Furthermore, characteristic interactions between muscarinic receptors and β2-adrenoceptors are involved in prejunctional modulation of ACh release from parasympathetic nerve endings  and intracellular signaling cross-talk at the adenylyl cyclase/PKA level , 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 glycopyrronium bromide was significantly augmented (Figure 4A) . 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 glycopyrronium (3-30 nM) . 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 . 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) . 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 .
4. Airway smooth muscle tone regulated by Ca2+ dynamics
4.1. Membrane potential-independent Ca2+ dynamics
In simultaneous recordings of isometric tension and [Ca2+]i in fura-2-loaded tissues of tracheal smooth muscle, various spasmogens including contractile agonists acting on GPCRs augment the tone of airway smooth muscle with elevated [Ca2+]i in a concentration-dependent fashion (Figure 1) [19, 101]. However, even though contraction fully occurs, these agents cause a modest depolarization of the cell membrane in a microelectrode experiment, indicating that airway smooth muscle contracts by Ca2+ influx via membrane potential–independent pathways. These Ca2+ dynamics with a modest depolarization are involved in Ca2+ influx through SOC and ROC [16, 17]. Depletion of the SR Ca2+ stores by thapsigargin, an inhibitor of the SR Ca2+-ATPase, caused an increase in [Ca2+]i and contraction, demonstrating Ca2+ entry through SOC . Because SOC was not inhibited by nifedipine, an inhibitor of VDC, VDC is not involved in SOC. Under the condition that SOC is fully activated, MCh and histamine caused further increases in [Ca2+]i and tension, demonstrating Ca2+ entry independent of SOC and VDC (non-SOC) . The Ca2+ influx and contraction via non-SOC was inhibited by Y-27632. In contrast, Y-27632 did not affect SOC.
4.2. Membrane potential–dependent Ca2+ dynamics
In fura-2–loaded tissues of tracheal smooth muscle, verapamil caused an inhibition of MCh-induced contraction with reduced [Ca2+]I; however, relaxant effects of verapamil are not so dramatic, indicating that VDC is partly involved in contraction mediated by GPCRs. IbTX enhanced MCh-induced contraction with elevation of [Ca2+]i. These effects of IbTX on tension and [Ca2+]i are antagonized by verapamil , demonstrating that KCa channel inhibition results in contraction with elevation of [Ca2+]i induced by opening VDC channels via depolarization of the cell membrane, whereas channel activation results in relaxation with reduction of [Ca2+]i induced by closing VDC channels via hyperpolarization of cell membrane.
When [Ca2+]i is increased by Ca2+ entry via various pathways described earlier (Ca2+ dynamics), the activity of MLCK is enhanced via CaM, leading to contraction via phosphorylation of MLC (see Section 2). In airway smooth muscle, alteration of contractility regulated by Ca2+ dynamics is involved in the pathophysiology implicated in asthma and COPD, such as airway limitation, airway hyperresponsiveness, and β2-adrenergic desensitization. It is useful to suppress Ca2+ dynamics for improving these pathological conditions in the airways.
4.3. Effects of Ca2+ release from the SR
KCa channels were activated by ACh (30 μM), substance P (0.1 μM) or IP3 (2.4-20 μM), as well as by caffeine (5 mM), suggesting that the activity was due to Ca2+ released from intracellular stores. These activations with the agonists and IP3 were markedly and reversibly reduced by heparin (50-100 μg/ml), which inhibits IP3 binding to its receptors in the SR. Furthermore, in cultured human bronchial smooth bradykinin (0.01-1 μM), an inflammatory mediator caused bronchoconstriction and activated KCa channels in a concentration-dependent manner; the augmented currents were inhibited by heparin (10 μg/ml) . Ca2+ release from the SR via stimulation of IP3 receptors causes an increase in the activation of KCa channels in smooth muscle including airways and vessels. Two pathways participate in Ca2+ release from the SR, the RyR pathway and the IP3 receptor pathway. In smooth muscle cells, the IP3 receptor is more abundant than the ryanodine receptor and reacts to IP3, which is generated from the activation of GPCRs and phospholipase C.
5. Airway smooth muscle tone regulated by Ca2+ sensitization
5.1. Characteristics and physiological role of RhoA/Rho-kinase
Although an increase in [Ca2+]i plays an important role in the contraction of airway smooth muscle (Figure 1) , it is generally considered that muscarinic receptor agonists and histamine increase tension at a constant [Ca2+]i. This phenomenon is referred to as Ca2+ sensitization [103, 104] and is mediated by a G protein-coupled mechanism. Rho is a monomeric G protein that belongs to the Ras superfamily. The Rho family makes up a major branch that contains Rho, Rac, and CdC42. Rho has isoforms of A-G; however, most of the function is described based on studies of RhoA. RhoA exhibits both GDP/GTP binding activity and GTPase activity, and it acts as a molecular switch between a GDP-bound inactive state (GDP-RhoA) and a GTP-bound active state (GTP-RhoA). When cells are stimulated with G protein–coupled receptor agonists, receptor tyrosine kinases and higher concentrations of potassium chloride (KCl), GDP-RhoA is converted to GTP-RhoA. RhoA and Rho-kinase are widely distributed to many organs, including the respiratory system. Rho-kinase (160 kDa) is an effector molecule of RhoA [105, 106]. Rho-kinase activated by GTP-RhoA interacts with MP and hinders MP activity by phosphorylating threonine 696 and 853 of myosin phosphatase targeting subunit 1 (MYPT1), a myosin-binding subunit [107, 108]. Rho-kinase has effects on contraction due to Ca2+ sensitization, stress fiber formation due to actin (cytoskeletal) reorganization, cell migration, and cell proliferation [20, 109]. These processes are implicated in the major pathophysiological characteristics of asthma and COPD, such as airflow limitation, airway hyperresponsiveness, β2-adrenergic desensitization, eosinophil recruitment and airway remodeling .
5.2. Role of RhoA/Rho-kinase on contraction
Y-27632, a pyridine derivative, was developed as a specific Rho-kinase inhibitor. Y-27632 suppresses Ca2+ sensitization and relaxes vascular smooth muscle to treat hypertension in rats . The effects of Y-27632 on MCh-induced contraction were analyzed by using strips of guinea pig airway smooth muscle treated with fura-2. Y-227632 suppressed contraction induced by agonists, such as MCh, histamine, prostaglandins, and leukotrienes, in a concentration-dependent manner, but there was no significant decrease in [Ca2+]i . Recently, it has been demonstrated that MYPT1 is an effective protein for Rho-kinase action on MP in airway smooth muscle cells and that Y-27632 inhibits the phosphorylation of MYPT1 in a concentration-dependent manner [108, 110]. Fasudil hydrochloride (HA-1077), a specific inhibitor of Rho-kinase, is used clinically to suppress cerebral vasospasm following subarachnoid hemorrhage . Alteration of contractility of airway smooth muscle regulated by Ca2+ sensitization is also involved in airflow limitation, airway hyperresponsiveness, and β2-adrenegic desensitization .
6. Role of Ca2+ dynamics and Ca2+ sensitization in airway disorders
6.1. Airflow limitation (contraction)
Airway smooth muscle contraction due to muscarinic receptor agonists (ACh, MCh and CCh), histamine, prostaglandins or leukotrienes is involved in airflow limitation, which is a characteristic feature of asthma and COPD (Figure 5). These agonists cause contraction of airway smooth muscle with increasing [Ca2+]i by Ca2+ dynamics via Ca2+ entry passing through SOC, ROC, and partly VDC. Sphingosine 1-phosphate (S1P: a bioactive lysophospholipid) , tryptase (trypsin-like neutral serine-class protease) and SLIGKV (non-enzymatic activator of protease-activated receptor 2, PAR2)  released from mast cells induce airway smooth muscle contraction with increasing [Ca2+]i. Since clinical studies have demonstrated that S1P and tryptase may be involved in the pathophysiology of asthma, these substances have been examined as novel mediators. ATP is released from injured airway epithelium during the inflammatory processes implicated in asthma. Extracellular ATP also causes contraction of airway smooth muscle with increasing [Ca2+]i . Furthermore, oxidative stress and mechanical stress are related to the pathophysiology of not only COPD but also asthma. 8-iso-prostaglandin F2α, an isoprostane , and hydrogen peroxide (H2O2)  produced by oxidative stress contract airway smooth muscle by increasing [Ca2+]i.
As described earlier, Y-27632 inhibited the contraction induced by spasmogens such as MCh, histamine, prostaglandins, and leukotrienes, which are involved in the pathophysiology of asthma and COPD, in a concentration-dependent manner, with no significant decrease in [Ca2+]i in strips of guinea pig airway smooth muscle treated with fura-2. Furthermore, Y-27632 also inhibited the following types of contraction in a concentration-dependent manner with a modest effect on [Ca2+]i: contraction due to S1P and tryptase released from mast cells; contraction due to isoprostanes and Hydrogen peroxide (H2O2) produced by oxidative stress; and contraction due to ATP synthesized in injured airway epithelium. Spasmogens, which are implicated in the pathophysiology of asthma and COPD, cause force generation in airway smooth muscle via both Ca2+ influx and Ca2+ sensitization . Force maintenance is due to Ca2+ sensitization induced by Rho-kinase . PKC, which is an intracellular signal transduction pathway for GPCR activation, also contracts airway smooth muscle mediated by both Ca2+ dynamics and Ca2+ sensitization .
These findings indicate that a contractile phenotype in airway smooth muscle cells is altered by the inflammatory processes related to obstructive pulmonary diseases, such as asthma and COPD, via both Ca2+ dynamics and Ca2+ sensitization, leading to the airflow limitation (bronchoconstriction) associated with these diseases (Figure 5).
6.2. Airway hyperresponsiveness
Airway hyperresponsiveness is a characteristic feature of asthma, and it is essential for the diagnosis and severity assessment of asthma. Airway hyperresponsiveness is also observed in some patients with COPD. This airway disorder is clinically defined as increased responsiveness to muscarinic receptor agonists (ACh and MCh) and histamine. Airway hyperresponsiveness is mediated by various inflammatory stimulations involved in the pathophysiology of asthma, such as antigens, chemical mediators, cytokines, and eicosanoids. In a postmortem study of airway smooth muscle strips of patients with asthma, the response to histamine and ACh was greater than in healthy individuals . In human airway smooth muscle passively sensitized with human asthmatic serum, contraction due to histamine is significantly elevated . When airway smooth muscle is exposed for an extended period of time to interleukin (IL)-5, IL-13, IL-17, or tumor necrosis factor (TNF)α, which are released from inflammatory cells and epithelial cells in airways, contraction due to muscarinic receptor agonists and KCl is significantly increased [119, 120, 121]. This enhancement of contraction induced by TNFα may be involved in Ca2+ sensitization via RhoA/Rho-kinase . In the presence of a lower concentration of leukotriene C4, KCl-induced contraction is markedly augmented in porcine tracheal smooth muscle, and this enhanced contraction due to KCl is attenuated by Y-27632 . When airway smooth muscle is exposed to S1P released from mast cells or ATP released from damaged epithelial cells, contraction in response to MCh is markedly increased after exposure to S1P or ATP, and its augmented contraction is suppressed by Y-27632 in a concentration-dependent manner [108, 113, 123]. Furthermore, pre-treatment of 8-iso-prostaglandin E2, an isoprostane, causes an increased response to CCh in airway smooth muscle, and its augmented contraction is suppressed by Y-27632 . These observations indicate that airway hyperresponsiveness is caused by direct interactions among inflammatory cells, airway epithelial cells and airway smooth muscle cells and that Ca2+ sensitization based on Rho-kinase–induced MYPT1 phosphorylation contributes to the airway hyperreactivity [107, 108]. Suppression of geranylgeranyltransferase, which is involved in the activation of RhoA, also reduces hyperresponsiveness in mouse bronchus . Alterations of Ca2+ regulatory mechanisms in airway smooth muscle may play a key role in this phenomenon. Therefore, the pathophysiology of asthma (inflammatory processes involved in this disease) and alterations in the mechanical properties directly affect the function of airway smooth muscle cells via the RhoA/Rho-kinase processes. In airway smooth muscle cells, this phenotypic change for contractility induced by not only Ca2+ sensitization but also cytoskeleton reorganization (cell stiffness) may cause an augmented response to spasmogens [1, 126, 127]. Lung resistance in response to MCh was increased in mice sensitized by allergen challenges, as compared with control mice (airway hyperresponsiveness). Fasudil hydrochloride (HA-1077), an inhibitor of Rho-kinase, suppressed the augmented response to MCh by allergen challenges . On the other hand, Ca2+ dynamics (Ca2+ mobilization) also contributes to altering the contractile phenotype of airway smooth muscle, leading to augmented responsiveness to spasmogens . Moreover, acidification of esophageal lumen increases the contractile response to ACh and KCl in guinea pig trachealis mediated by activation of VDC channels and Rho-kinase , indicating that both Ca2+ dynamics and Ca2+ sensitization play key roles in airway hyperresponsiveness (Figure 5).
6.3. Desensitization of β2-adrenergic receptors
After β2-adrenoceptors are excessively activated, responsiveness to an agonist is attenuated. This phenomenon is referred to as desensitization of β2-adrenoceptors. The phosphorylation of β2-adrenoceptors, which leads to desensitization via uncoupling Gs from the receptors, is mediated by two types of protein kinases, cAMP-dependent PKA and cAMP-independent protein kinases such as β2-adrenergic receptor kinase (βARK) . PKA-induced phosphorylation, which is produced by exposure to a low concentration of β2-adrenoceptor agonists, leads to heterologous desensitization (a nonspecific reduced response to other agonists involving cAMP) . On the other hand, βARK-induced phosphorylation, which is produced by exposure to a high concentration of β2-adrenoceptor agonists, leads to homologous desensitization (a specific reduced response to β2-adrenoceptor agonist) . These phenomena also occur in tracheal smooth muscle, including human tissues [10, 134, 135, 136]. β2-adrenergic desensitization occurs after continuous [134, 135, 136] or repetitive administration [10, 135, 136] of β2-adrenoceptor agonists or after exposure to substances related to the inflammatory processes in asthma, including inflammatory cytokines such as IL-1β , growth factors such as transforming growth factor (TGF)-β1  and platelet-derived growth factor (PDGF) , lipid mediators such as lysophosphatidylcholine (Lyso-PC), a lysophospholipid produced by phospholipase A2 , and S1P , or PAR2 agonists such as tryptase and SLIGKV . Therefore, desensitization of β2-adrenoceptors in airway smooth muscle is an extremely important phenomenon that occurs due to both the treatment and the pathophysiology of asthma. Reduced responsiveness to β2-adrenoceptor agonists after excessive or repeated exposure to these agonists was prevented when Gs linked to β2-adrenoceptors was irreversibly activated by pre-treating airway smooth muscle with cholera toxin (2 μg/ml) for 6 h [134, 135, 142, 143] (Figure 6A). On the other hand, in the presence of ChTX or IbTX, this β2-adrenergic desensitization was markedly enhanced [134, 135]. Inactivation of the Gs/KCa channel linkage plays an important role in β2-adrenergic desensitization (Figures 5, 8).
6.3.1. Ca2+ dynamics
In fura-2–loaded tissues of guinea pig tracheal smooth muscle, the relaxant effect of isoprenaline on MCh-induced contraction was gradually attenuated with increasing [Ca2+]i following repeated exposure to isoprenaline with MCh for 10 min every 30 min [10, 135] (Figure 6B), and this reduced responsiveness to isoprenaline was avoided by pre-exposure to cholera toxin or the addition of verapamil with no change in [Ca2+]i  (Figure 6C). In contrast, after repeated exposure to forskolin, db-cAMP and theophylline, the relaxant effect of these cAMP-related agents was not diminished with no change in [Ca2+]i (homologous desensitization) [10, 135]. Furthermore, after exposure to PDGF for 15 min, the relaxant effect of isoprenaline against MCh-induced contraction was markedly attenuated with increasing [Ca2+]i, and this reduced responsiveness to isoprenaline was reversed by verapamil . The relaxant effects of not only β2-adrenoceptor agonists but also forskolin are markedly attenuated with elevated [Ca2+]i after exposure to growth factors, such as TGFβ1 and PDGF (heterologous desensitization) (Figure 8). In contrast, the relaxant effects of db-cAMP and theophylline are not diminished after exposure to TGFβ1 and PDGF. These results indicate that β2-adrenergic desensitization occurs via dysfunction of the receptor/Gs/adenylyl cyclase processes in airway smooth muscle and that the cAMP-independent pathway is involved in this phenomenon [3, 4. 7, 8]. These results indicate that the Ca2+ influx passing through VDC is involved in β2-adrenergic desensitization and that VDC activity may be augmented by dysfunction of the Gs/KCa channel stimulatory linkage (Figures 5, 8).
6.3.2. Ca2+ sensitization
In fura-2–loaded tissues of guinea pig tracheal smooth muscle, the inhibitory effect of isoprenaline against MCh-induced contraction following continuous exposure to Lyso-PC  was markedly attenuated with no changes in [Ca2+]i (Figure 7A). This reduced responsiveness to isoprenaline was reversed to the control response by application of Y-27632 in a concentration-dependent manner (Figure 7B). In contrast, the relaxant effect of cAMP-related agents such as forskolin, theophylline, and db-cAMP, was not diminished after exposure to Lyso-PC (homologous desensitization). Similar to Lyso-PC, reduced responsiveness to isoprenaline was observed with no changes in [Ca2+]i after the exposure of tracheal smooth muscle to tryptase and SLIGKV  and S1P . The relaxant effects of forskolin were not attenuated after exposure to tryptase and SLIGKV; in contrast, the relaxant effects were markedly diminished after exposure to S1P, indicating that the receptor/Gs/ adenylyl cyclase process is also involved in the dysfunction of β2-adrenoceptors in airway smooth muscle. cAMP activity may still be intact under this condition of excessive stimulation of β2-adrenoceptors. Furthermore, in the presence of bisindolylmaleimide, a membrane-permeable inhibitor of PKC, reduced responsiveness to isoprenaline is not prevented after exposure to an agonist [134, 135, 140]. These observations indicate that after exposure to these lipid mediators and PAR 2 agonists, tolerance to β2-adrenoceptor agonists occurs due to Ca2+ sensitization via the RhoA/Rho-kinase processes, not via PKC. This β2-adrenergic desensitization is caused by elevated sensitization to intracellular Ca2+ based on Gs inactivation and Rho-kinase activation, although little is known about the functional relationship between Gs and RhoA/Rho-kinase (Figures 5, 8).
6.3.3. Intrinsic efficacy
The potency of a β2-adrenoceptor agonist depends on its receptor affinity and intrinsic efficacy. Intrinsic efficacy (intrinsic activity) refers to the ability of an agent to activate its receptors without regard for their concentration. Some agonists completely activate receptors, but others only partially activate them. The former are referred to as full agonists, and the latter are referred to as partial agonists. Moreover, partial agonists are subclassified as weak partial agonists, which have lower efficacy, and strong partial agonists, which have higher efficacy [144, 145]. Intrinsic efficacy was measured indirectly as a physiological response (changes in smooth muscle relaxation determined by isometric tension recording in vitro) . The ratio of the intrinsic efficacy of any two β2-agonists is expressed as a fraction between 0 and 1 by concentration-inhibition curves, taking that of adrenaline as 1. The order of efficacy (the maximal percent relaxation against 10 μM MCh-induced contraction) was as follows: isoprenaline = adrenaline > indacaterol, formoterol, procaterol > salbutamol > salmeterol > tulobuterol [97, 145] (Table 1); these efficacies are similar to the values measured by changing the level of intracellular cAMP . Isoprenaline behaves as a full agonist, and other agonists behave as partial agonists. Isoprenaline caused β2-aderergic desensitization greater than that of other agonists, indicating that excessive activation of a full agonist leads to reduced responsiveness to β2-adrenoceptor agonists in airway smooth muscle [134, 135, 136, 142, 145]. In contrast, tulobuterol, which is the weakest partial agonist, caused a modest reduction in response to an agonist, even in cases of excessive exposure to tulobuterol .
6.4. Airway remodeling
Airway inflammatory reactions involving activated eosinophils act on the epithelium, subepithelium, and smooth muscle layers and bring about characteristic structural changes in the airways. Subepithelial fibrosis results from the deposition of collagen fibers and proteoglycans under the basement membrane (thickening of the airway wall). This phenomenon is known as airway remodeling, which is thought to be related to asthma severity. Airway smooth muscle contributes to airway remodeling by mass formation via cell proliferation and migration [147, 148]. Unlike normal cells, increased airway smooth muscle cell proliferation in patients with asthma is not suppressed by glucocorticosteroids because of CCAAT/enhancer-binding protein (C/EBP)-α deficiency in airway smooth muscle cells .
6.4.1. Cell proliferation
Factors facilitating the proliferation of airway smooth muscle cells are roughly divided into the following two groups: 1) ligands (polypeptide growth factors) of tyrosine kinase receptors (RTKs), such as epidermal growth factor (EGF) and PDGF, and 2) ligands (contractile agents) of GPCRs, such as leukotriene D4, thromboxane A2 and endothelin. When ligands bind to growth factor receptors, tyrosine kinase is first activated, followed by Ras and extracellular regulated kinase (ERK)1/2, to transmit information to the nucleus . Next, via cyclin D1 activation, DNA synthesis and cell proliferation occur . In addition to this main pathway for smooth muscle proliferation, cross-talk between RTKs and GPCRs is mediated by phosphatidylinositol 3-kinase (PI3K), p70S6 kinase, and glycogen synthase kinase-3 (GSK-3) [150, 152]. The involvement of the Rho family (RhoA, Rac and Cdc42) in the control mechanisms of airway smooth muscle cell proliferation has not been sufficiently clarified. EGF- and PDGF-induced cell proliferation is not suppressed by inactivation of RhoA/Rho-kinase signaling ; in contrast, the activation of RhoA, not Rac or cdc42, causes the proliferation of human bronchial smooth muscle cells that have been stimulated with serum. This proliferative reaction is suppressed by Y-27632, C3 exoenzyme, and simvastatin, a HMG-CoA reductase inhibitor, which attenuate proliferation via the geranylgeranylation of RhoA . Another factor, M2 muscarinic receptor, facilitates the proliferation of airway smooth muscle cells [154, 155]. A recent clinical trial has demonstrated that an antagonist of VDC channels inhibits airway remodeling in patients with severe asthma . Therefore, Ca2+ influx via VDC channels is enhanced since KCa channel activity is attenuated by Gi when MCh is applied to airway smooth muscle [7, 8]. These results indicate that both Ca2+ dynamics and Ca2+ sensitization contribute to the proliferation of airway smooth muscle cells (Figure 5).
6.4.2. Cell migration
Cell migration is a characteristic function of inflammatory cells, fibroblasts and smooth muscle cells, and it plays an important role in various pathophysiological environments, such as inflammatory cell infiltration and airway smooth muscle hyperplasia . Migration of airway smooth muscle cells is enhanced by the extracellular matrix . Cell migration occurs due to contraction involving actin, myosin reactions and actin reorganization. Since RhoA/Rho-kinase signaling is the most important factor controlling the cytoskeleton of airway smooth muscle cells and other cells , this pathway may control the migration of airway smooth muscle cells via changes in the cytoskeleton. Hence, RhoA/Rho-kinase may be involved in airway remodeling mediated not only by cell proliferation but also by cell migration. Urokinase, PDGF, leukotriene and lysophosphatidic acid facilitate the migration of human airway smooth muscle cells [160, 161, 162, 163]. Moreover, heat shock protein, PI3K, p38 mitogen-activated protein kinase, prostaglandin D2, and IL-13 facilitate airway smooth muscle migration [160, 164, 165]. Y-27632 significantly suppresses the increased migration of airway smooth muscle cells, due to PDGF or leukotriene stimulation [161, 162], indicating that RhoA/Rho-kinase signaling (Ca2+ sensitization) plays an important role in controlling cell migration (Figure 5). On the other hand, Ca2+ dynamics regulate the migration of airway smooth muscle cells and inflammatory cells. Ca2+ influx via SOC channels contributes to PDGF-induced cell migration of airway smooth muscle , and increasing [Ca2+]i via other mechanisms also causes substance P–induced cell migration of airway smooth muscle  (Figure 5). Since IL-13 enhances Ca2+ oscillation in airway smooth muscle cells, cell migration induced by IL-13 may be regulated by Ca2+ dynamics .
6.4.3. Interaction between airway smooth muscle and inflammatory cells
As described earlier, contractility of airway smooth muscle is altered by tryptase and S1P, which are released from mast cells, and Lyso-PC, which is synthesized in the membrane of various inflammatory cells [108, 112, 140, 141]. Ca2+ sensitization by RhoA/Rho-kinase processes contributes to this phenomenon. When sensitized mice are subjected to allergen challenges, eosinophil infiltration is markedly increased in the airways. In allergen-challenged mice, pretreatment with Rho-kinase inhibitors such as Y-27632 or fasudil hydrochloride (HA-1077) markedly suppressed an increase in eosinophil recruitment in the airway in a dose-dependent manner . The actions of Lyso-PC are mediated by RhoA/Rho-kinase, leading to β2-aderenergic desensitization , and administration of Lyso-PC to guinea pigs enhances eosinophil recruitment and resistance in the airways . The effects of S1P are also mediated by RhoA/Rho-kinase processes, leading to airway hyperresponsiveness  and remodeling . S1P increased mRNA and protein expression of vascular cell adhesion molecule (VCAM)-1 when S1P is applied to pulmonary endothelial cells, leading to eosinophil infiltration to the airways, and this upregulation of VCAM-1 is attenuated by C3 exoenzyme and Y-27632 . Y-27632 reduces not only the number of eosinophils but also macrophages and neutrophils in an animal model of allergic asthma . Ca2+ sensitization via RhoA/Rho-kinase processes contributes to recruitment of inflammatory cells to the airways.
Therefore, Ca2+ sensitization by RhoA/Rho-kinase processes [1, 173, 174, 175] and Ca2+ dynamics by ion channels including VDC and SOC [6, 11, 12, 176] may be a therapeutic target for obstructive pulmonary diseases including asthma.
Ca2+ signaling, which is due to Ca2+ dynamics and Ca2+ sensitization, contributes to alterations of contractility that lead to airway disorders (airflow limitation, airway hyperresponsiveness, and β2-adrenergic desensitization), which are characteristic features of asthma and COPD. Ca2+ dynamics and Ca2+ sensitization also facilitate the proliferation and migration of airway smooth muscle via changing to proliferative phenotype. A recent report has indicated that bitter taste receptor stimulation causes relaxation of airway smooth muscle via activation of KCa channels . Hence, Ca2+ dynamics due to G proteins/KCa/VDC channels and Ca2+ sensitization due to RhoA/Rho-kinase processes may be therapeutic targets for asthma and COPD, and research in these areas may provide novel strategies in the development of bronchodilators for these diseases.