**1. Introduction**

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) [1]. 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 spasmogeninduced 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 [9], 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 [10]. 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 desensitiza‐ tion), 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 (prolif‐ eration, 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**

Ca2+ dynamics due to G proteins/KCa/VDC channel linkage and Ca2+ sensitization due to RhoA/Rho-kinase processes are therapeutic targets for these diseases.

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

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) [1]. RhoA/Rho-kinase processes are widely distributed in tissues

β2-adreneoceptor agonists and muscarinic receptor antagonists counteract spasmogeninduced 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,

) channels are markedly activated by PKA-induced phosphorylation [3, 4, 5, 6] and

, an inhibitory trimeric G protein of adenylyl cyclase [7, 8]. Since KCa

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

channels have a large conductance of outward currents and exist innumerably on the cell membrane in airway smooth muscle [9], 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 [10]. 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 desensitiza‐

including the respiratory system and regulated by agonists for GPCRs.

) via a Ca2+ influx through Ca2+ channels (Ca2+ dynamics). In contrast, inactivation

channels, β2-adrenoceptors, G proteins, Rho-kinase, Intrinsic efficacy

**Keywords:** Ca2+-activated K+

**1. Introduction**

290 Muscle Cell and Tissue

Ca2+ ([Ca2+]i

Maxi-K+

receptor agonists via Gi

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 [16], 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) [17]. 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 [10]. 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 [18], 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 inacti‐ vation) (Figure 1) [20, 21]. MP is also phosphorylated by CPI-17, which is another potential mediator regulated by protein kinase C [22]. Since MLC activity is sustained, not suppressed, by loss of MLC dephosphorylation via inactivation of MP, airway smooth muscle tone is enhanced without increasing [Ca2+]i (Ca2+-independent contraction: Ca2+ sensitization) [19, 23]. Airway smooth muscle tone is regulated by the degree of MLC phosphorylation mediated by both MLCK and MP activity. Alterations of contractile phenotype, which are due to both Ca2+ dynamics and Ca2+ sensitization, have clinical relevance to airflow limitation, airway hyperresponsiveness, and reduced responsiveness to β2-adrenoceptor agonists (β2-adrenergic desensitization), which are implicated with the pathophysiology of obstructive pulmonary diseases, such as asthma and COPD [1].

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