**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 charac‐ teristic 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) [108], tryptase (trypsin-like neutral serine-class protease) and SLIGKV (non-enzymatic activator of protease-activated receptor 2, PAR2) [112] 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 [113]. Furthermore, oxidative stress and mechanical stress are related to the pathophysiology of not only COPD but also asthma. 8-isoprostaglandin F2α, an isoprostane [114], and hydrogen peroxide (H2O2) [84] 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; contrac‐ tion 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 [115]. Force maintenance is due to Ca2+ sensitization induced by Rho-kinase [116]. PKC, which is an intracellular signal trans‐ duction pathway for GPCR activation, also contracts airway smooth muscle mediated by both Ca2+ dynamics and Ca2+ sensitization [22].

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).

Ca2+ Dynamics and Ca2+ Sensitization in the Regulation of Airway Smooth Muscle Tone http://dx.doi.org/10.5772/59347 307

**Figure 5. Involvement of G proteins/KCa/VDC channel linkage (Ca2+ dynamics) and RhoA/Rho-kinase processes (Ca2+ sensitization) in the pathophysiology of asthma and COPD**. Chronic exposure to lipid mediators, cytokines, and other substances related to asthma and COPD, which are released and synthesized from inflammatory cells and epithelial cells in airways, affects airway smooth muscle functions via the G proteins/KCa/VDC channel linkage due to Ca2+ dynamics and RhoA/Rho-kinase processes due to Ca2+ sensitization. These inflammatory processes cause not only alterations of contractility but also changing to proliferative phonotype in airway smooth muscle, referred to as a phe‐ notype change. The former phenomenon is attributed to airflow limitation, airway hyperresponsiveness, and β2-adre‐ nergic desensitization; the latter phenomenon is attributed to airway remodeling via cell proliferation and migration. Therefore, G proteins/KCa/VDC channel linkage and RhoA/Rho-kinase processes are involved in almost all of the prin‐ cipal mechanisms of asthma and COPD. These pathways involved in Ca2+ dynamics and Ca2+ sensitization are molecu‐ lar targets for therapy of these diseases. VDC: L-type voltage-dependent Ca2+ channels, KCa: large-conductance Ca2+ activated K+ channels. Illustrated based on ref. [1].

#### **6.2. Airway hyperresponsiveness**

Rho-kinase, is used clinically to suppress cerebral vasospasm following subarachnoid hemorrhage [111]. Alteration of contractility of airway smooth muscle regulated by Ca2+ sensitization is also involved in airflow limitation, airway hyperresponsiveness, and β2-

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 charac‐ teristic feature of asthma and COPD (Figure 5). These agonists cause contraction of airway

ROC, and partly VDC. Sphingosine 1-phosphate (S1P: a bioactive lysophospholipid) [108], tryptase (trypsin-like neutral serine-class protease) and SLIGKV (non-enzymatic activator of protease-activated receptor 2, PAR2) [112] released from mast cells induce airway smooth

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 [113]. Furthermore, oxidative stress and mechanical stress are related to the pathophysiology of not only COPD but also asthma. 8-isoprostaglandin F2α, an isoprostane [114], and hydrogen peroxide (H2O2) [84] produced by

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

tion 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 [115]. Force maintenance is due to Ca2+ sensitization induced by Rho-kinase [116]. PKC, which is an intracellular signal trans‐ duction pathway for GPCR activation, also contracts airway smooth muscle mediated by both

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

 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

: contraction due to S1P and tryptase released from mast cells; contrac‐

oxidative stress contract airway smooth muscle by increasing [Ca2+]i

by Ca2+ dynamics via Ca2+ entry passing through SOC,

. Since clinical studies have demonstrated that S1P

.

**6. Role of Ca2+ dynamics and Ca2+ sensitization in airway disorders**

adrenegic desensitization [1].

306 Muscle Cell and Tissue

**6.1. Airflow limitation (contraction)**

smooth muscle with increasing [Ca2+]i

muscle contraction with increasing [Ca2+]i

Ca2+ dynamics and Ca2+ sensitization [22].

(bronchoconstriction) associated with these diseases (Figure 5).

[Ca2+]i

modest effect on [Ca2+]i

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 respon‐ siveness to muscarinic receptor agonists (ACh and MCh) and histamine. Airway hyperres‐ ponsiveness 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 [117]. In human airway smooth muscle passively sensitized with human asthmatic serum, contraction due to histamine is significantly elevated [118]. 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 [110]. 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 [122]. 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 [124]. These obser‐ vations 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 [125]. 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 pheno‐ typic 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 [128]. On the other hand, Ca2+ dynamics (Ca2+ mobilization) also contributes to altering the contractile phenotype of airway smooth muscle, leading to augmented respon‐ siveness to spasmogens [129]. 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 [130], 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) [131]. PKA-induced phosphor‐ ylation, 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) [132]. On the other hand, βARK-induced phosphorylation, which is pro‐ duced by exposure to a high concentration of β2-adrenoceptor agonists, leads to homologous desensitization (a specific reduced response to β2-adrenoceptor agonist) [133]. These phenom‐ ena 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 [137], growth factors such as transforming growth factor (TGF)-β1 [138] and platelet-derived growth factor (PDGF) [139], lipid mediators such as lysophosphatidylcholine (Lyso-PC), a lysophos‐ pholipid produced by phospholipase A2 [140], and S1P [141], or PAR2 agonists such as tryptase and SLIGKV [112]. Therefore, desensitization of β2-adrenoceptors in airway smooth muscle is an extremely important phenomenon that occurs due to both the treatment and the patho‐ physiology 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).

augmented in porcine tracheal smooth muscle, and this enhanced contraction due to KCl is attenuated by Y-27632 [122]. 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 [124]. These obser‐ vations 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 [125]. 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 pheno‐ typic 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 [128]. On the other hand, Ca2+ dynamics (Ca2+ mobilization) also contributes to altering the contractile phenotype of airway smooth muscle, leading to augmented respon‐ siveness to spasmogens [129]. 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 [130], indicating that both Ca2+ dynamics and Ca2+ sensitization play

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) [131]. PKA-induced phosphor‐ ylation, 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) [132]. On the other hand, βARK-induced phosphorylation, which is pro‐ duced by exposure to a high concentration of β2-adrenoceptor agonists, leads to homologous desensitization (a specific reduced response to β2-adrenoceptor agonist) [133]. These phenom‐ ena 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 [137],

key roles in airway hyperresponsiveness (Figure 5).

**6.3. Desensitization of β2-adrenergic receptors**

308 Muscle Cell and Tissue

**Fig 6 Figure 6. Inhibitory effects of Gs/KCa channel linkage on the β-adrenergic desensitization after repeated exposure to a β-adrenoceptor agonist in isometric tension recording of tracheal smooth muscle.** (A) A typical example of repeat‐ ed application of ISO (0.3 µM) to tissues precontracted by MCh (1 µM) at intervals of 20 min under the following ex‐ perimental conditions: control (upper trace), preincubation with CTX (2 µg/ml) for 6 h (middle trace), and preincubation with CTX and in the presence of IbTX (30 nM) throughout the experiment (lower trace). The Gs/KCa channel stimulatory linkage is involved in the prevention of β2-adrenergic desensitization. (B) A typical example of simultaneously recorded isometric tension (upper trace) and F340/F380 (lower trace) after repeated exposure to MCh (1 µM) with ISO (0.3 µM) in fura-2–loaded tissues of tracheal smooth muscle in guinea pigs. (C) A typical example of simultaneously recorded isometric tension (upper trace) and F340/F380 (lower trace) after repeated exposure to MCh (1

µΜ) with ISO (0.3 µM) in the presence of verapamil (3 µM) in fura-2–loaded tissues similar to (B). Ca2+ dynamics via the KCa/VDC channel linkage are involved in β2-adrenergic desensitization. ISO: isoprenaline, MCh: methacholine, CTX: cholera toxin, IbTX: iberiotoxin, KCa channels: large-conductance Ca2+-activated K+ channels. Cited from ref. [10, 135].

#### *6.3.1. Ca2+ dynamics*

In fura-2–loaded tissues of guinea pig tracheal smooth muscle, the relaxant effect of isopre‐ naline on MCh-induced contraction was gradually attenuated with increasing [Ca2+]ifollowing 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 [10] (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 [139]. 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 TGFβ<sup>1</sup> and PDGF (heterologous desensitiza‐ tion) (Figure 8). In contrast, the relaxant effects of db-cAMP and theophylline are not dimin‐ ished 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 isopre‐ naline against MCh-induced contraction following continuous exposure to Lyso-PC [140] 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 concentrationdependent 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 (ho‐ mologous 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 [112] and S1P [141]. 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 respon‐ siveness 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 Ca2+ Dynamics and Ca2+ Sensitization in the Regulation of Airway Smooth Muscle Tone http://dx.doi.org/10.5772/59347 311

**Fig. 7** 

**Figure 7. The effects of Ca2+ sensitization mediated by RhoA/Rho-kinase on β-adrenergic desensitization in tracheal smooth muscle.** A: A typical example of simultaneously recorded isometric tension (upper trace) and F340/F380 ratio (lower trace) induced by MCh (1 µM) with ISO (0.3 µM) inhibition before and after exposure to Lyso-PC (10 M) for 15 min. Pretreatment with Lyso-PC attenuates ISO-induced relaxation without elevating [Ca2+]i , indicating that Ca2+ sensi‐ tization is involved in β2-adrenergic desensitization. B: A typical example of the inhibitory effects of ISO (0.3 µM) on MCh-induced contraction (1 µM) before and after exposure to Lyso-PC (10 µM) for 15 min in the absence (upper trace) and presence (lower trace) of Y-27632 (10 µM) throughout the experiments. Y-27632 inhibits β2-adrenergic desensitiza‐ tion induced by Lyso-PC, indicating that Ca2+ sensitization via RhoA/Rho-kinase processes is involved in this phenom‐ enon. MCh: methacholine, ISO: isoprenaline, Lyso-PC: lysophosphatidylcholine. Cited from ref. [140].

to β2-adrenoceptor agonists occurs due to Ca2+ sensitization via the RhoA/Rho-kinase proc‐ esses, 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*

µΜ) with ISO (0.3 µM) in the presence of verapamil (3 µM) in fura-2–loaded tissues similar to (B). Ca2+ dynamics via the KCa/VDC channel linkage are involved in β2-adrenergic desensitization. ISO: isoprenaline, MCh: methacholine,

In fura-2–loaded tissues of guinea pig tracheal smooth muscle, the relaxant effect of isopre‐ naline on MCh-induced contraction was gradually attenuated with increasing [Ca2+]ifollowing 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

exposure to forskolin, db-cAMP and theophylline, the relaxant effect of these cAMP-related

Furthermore, after exposure to PDGF for 15 min, the relaxant effect of isoprenaline against

responsiveness to isoprenaline was reversed by verapamil [139]. 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 TGFβ<sup>1</sup> and PDGF (heterologous desensitiza‐ tion) (Figure 8). In contrast, the relaxant effects of db-cAMP and theophylline are not dimin‐ ished 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

In fura-2–loaded tissues of guinea pig tracheal smooth muscle, the inhibitory effect of isopre‐ naline against MCh-induced contraction following continuous exposure to Lyso-PC [140] 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 concentrationdependent 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 (ho‐ mologous 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 [112] and S1P [141]. 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 respon‐ siveness 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

MCh-induced contraction was markedly attenuated with increasing [Ca2+]i

channels. Cited from ref. [10,

, and this reduced

[10] (Figure 6C). In contrast, after repeated

(homologous desensitization) [10, 135].

CTX: cholera toxin, IbTX: iberiotoxin, KCa channels: large-conductance Ca2+-activated K+

the addition of verapamil with no change in [Ca2+]i

Gs/KCa channel stimulatory linkage (Figures 5, 8).

agents was not diminished with no change in [Ca2+]i

135].

310 Muscle Cell and Tissue

*6.3.1. Ca2+ dynamics*

*6.3.2. Ca2+ sensitization*

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

**Figure 8. Role of Ca2+ dynamics and Ca2+ sensitization in the desensitization of β2-adrenoceptors in airway smooth muscle.** Phosphorylation of β2-adrenoceptors is essential for reduced responsiveness to their agonists. There are two pathways in the mechanisms of β2-adrenergic desensitization: 1) cAMP-independent phosphorylation of their recep‐ tors via members of the GRK family such as βARK (homologous desensitization), and 2) cAMP-dependent phosphory‐ lation of their receptors via PKA (heterologous desensitization). Inactivation of Gs, which is linked to β2-adrenoceptors, is involved in desensitization of the receptors mediated by Ca2+ dynamics and Ca2+ sensitization. Impairment of the stimulatory linkage between Gs/PKA and KCa channels causes an increase in the membrane potential, leading to Ca2+ influx passing through VDC channels (Ca2+ dynamics: Ca2+-dependent mechanisms). On the other hand, impairment of the inhibitory correlation between Gs/PKA and RhoA/Rho-kinase processes causes an increase in Rho-kinase activity, leading to a reduced MP activity (Ca2+ sensitization: Ca2+-independent mechanisms). β2: β2-adreneceptors, AC: adenyl‐ yl cyclase, GRK: G protein-receptor kinase, βARK: β-adrenoceptor kinase, PKA: protein kinase A, MLCK: myosin light chain kinase, MLC: myosin light chain, MP: myosin phosphatase, KCa: large-conductance Ca2+-activated K+ channels, VDC: L-type voltage-dependent Ca2+ channels. Illustrated based on ref. [1, 2, 10, 112, 134, 135, 136, 138, 139, 140, 141, 142, 145, 146].

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) [145]. 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: isopre‐ naline = adrenaline > indacaterol, formoterol, procaterol > salbutamol > salmeterol > tulobu‐ terol [97, 145] (Table 1); these efficacies are similar to the values measured by changing the level of intracellular cAMP [144]. 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 [146].


**Table 1. Intrinsic efficacy of β2-adrenoceptor agonists.** Values of intrinsic efficacy of β2-adrenoceptor agonists were measured as a physiologic response in airway smooth muscle. The values of intrinsic efficacy were expressed as the maximum percent inhibition for each β2-adrenoceptor agonist against MCh-induced contraction (1 and 10 µM) in guinea pig tracheal smooth muscle. MCh: methacholine. Cited from ref. [1, 97, 145].

#### **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 proteo‐ glycans 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 [149].

#### *6.4.1. Cell proliferation*

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) [145]. 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: isopre‐ naline = adrenaline > indacaterol, formoterol, procaterol > salbutamol > salmeterol > tulobu‐ terol [97, 145] (Table 1); these efficacies are similar to the values measured by changing the level of intracellular cAMP [144]. 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

**2**

**receptor phosphorylation**

**desensitization**

**GRKs**

**homologous pathway**

142, 145, 146].

312 Muscle Cell and Tissue

**agonists**

**Gs AC**

**cAMP**

**K+**

**Rho-kinase**

**Ca2+ dynamics**

**RhoA**

**inhibition activation**

**Figure 8. Role of Ca2+ dynamics and Ca2+ sensitization in the desensitization of β2-adrenoceptors in airway smooth muscle.** Phosphorylation of β2-adrenoceptors is essential for reduced responsiveness to their agonists. There are two pathways in the mechanisms of β2-adrenergic desensitization: 1) cAMP-independent phosphorylation of their recep‐ tors via members of the GRK family such as βARK (homologous desensitization), and 2) cAMP-dependent phosphory‐ lation of their receptors via PKA (heterologous desensitization). Inactivation of Gs, which is linked to β2-adrenoceptors, is involved in desensitization of the receptors mediated by Ca2+ dynamics and Ca2+ sensitization. Impairment of the stimulatory linkage between Gs/PKA and KCa channels causes an increase in the membrane potential, leading to Ca2+ influx passing through VDC channels (Ca2+ dynamics: Ca2+-dependent mechanisms). On the other hand, impairment of the inhibitory correlation between Gs/PKA and RhoA/Rho-kinase processes causes an increase in Rho-kinase activity, leading to a reduced MP activity (Ca2+ sensitization: Ca2+-independent mechanisms). β2: β2-adreneceptors, AC: adenyl‐ yl cyclase, GRK: G protein-receptor kinase, βARK: β-adrenoceptor kinase, PKA: protein kinase A, MLCK: myosin light chain kinase, MLC: myosin light chain, MP: myosin phosphatase, KCa: large-conductance Ca2+-activated K+ channels, VDC: L-type voltage-dependent Ca2+ channels. Illustrated based on ref. [1, 2, 10, 112, 134, 135, 136, 138, 139, 140, 141,

**(KCa) (VDC)**

**Ca2+**

**MP MLC MLCK**

**desensitization**

**Ca2+ dynamics**

**Ca2+ senstization**

**Ca2+/CaM**

**PKA**

**heterologous pathway**

response to an agonist, even in cases of excessive exposure to tulobuterol [146].

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 [150]. Next, via cyclin D1 activation, DNA synthesis and cell proliferation occur [151]. In addition to this main pathway for smooth muscle proliferation, cross-talk between RTKs and GPCRs is mediated by phos‐ phatidylinositol 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 PDGFinduced cell proliferation is not suppressed by inactivation of RhoA/Rho-kinase signaling [126]; 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 [153]. 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 [156]. 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+ sensiti‐ zation 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 [157]. Migration of airway smooth muscle cells is enhanced by the extracellular matrix [158]. 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 [159], 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 PDGFinduced cell migration of airway smooth muscle [166], and increasing [Ca2+]i via other mechanisms also causes substance P–induced cell migration of airway smooth muscle [167] (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 [168].

#### *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 dosedependent manner [128]. The actions of Lyso-PC are mediated by RhoA/Rho-kinase, leading to β2-aderenergic desensitization [140], and administration of Lyso-PC to guinea pigs enhances eosinophil recruitment and resistance in the airways [169]. The effects of S1P are also mediated by RhoA/Rho-kinase processes, leading to airway hyperresponsiveness [108] and remodeling [170]. S1P increased mRNA and protein expression of vascular cell adhesion molecule (VCAM)-1 when S1P is applied to pulmonary endothelial cells, leading to eosinophil infiltra‐ tion to the airways, and this upregulation of VCAM-1 is attenuated by C3 exoenzyme and Y-27632 [171]. Y-27632 reduces not only the number of eosinophils but also macrophages and neutrophils in an animal model of allergic asthma [172]. Ca2+ sensitization via RhoA/Rhokinase 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.
