**5. Airway smooth muscle tone regulated by Ca2+ sensitization**

#### **5.1. Characteristics and physiological role of RhoA/Rho-kinase**

**4. Airway smooth muscle tone regulated by Ca2+ dynamics**

smooth muscle, various spasmogens including contractile agonists acting on GPCRs augment

(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 path‐ ways. 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

SOC [17]. 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

(non-SOC) [17]. The Ca2+ influx and contraction via non-SOC was inhibited by Y-27632. In

In fura-2–loaded tissues of tracheal smooth muscle, verapamil caused an inhibition of MChinduced 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

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

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+

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

induced by closing VDC channels via hyperpolarization of cell membrane.

are antagonized by verapamil [10], demonstrating that KCa channel inhibition results

is increased by Ca2+ entry via various pathways described earlier (Ca2+ dynamics),

in fura-2-loaded tissues of tracheal

. These effects of IbTX on tension

in a concentration-dependent fashion

and contraction, demonstrating Ca2+ entry through

and tension, demonstrating Ca2+ entry independent of SOC and VDC

**4.1. Membrane potential-independent Ca2+ dynamics**

the tone of airway smooth muscle with elevated [Ca2+]i

**4.2. Membrane potential–dependent Ca2+ dynamics**

enhanced MCh-induced contraction with elevation of [Ca2+]i

dynamics for improving these pathological conditions in the airways.

Ca2+-ATPase, caused an increase in [Ca2+]i

contrast, Y-27632 did not affect SOC.

**4.3. Effects of Ca2+ release from the SR**

further increases in [Ca2+]i

304 Muscle Cell and Tissue

and [Ca2+]i

When [Ca2+]i

[Ca2+]i

In simultaneous recordings of isometric tension and [Ca2+]i

Although an increase in [Ca2+]i plays an important role in the contraction of airway smooth muscle (Figure 1) [18], 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 mono‐ meric 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) reor‐ ganization, 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 [1].

#### **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 [21]. 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 concen‐ tration-dependent manner, but there was no significant decrease in [Ca2+]i [19]. 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 concentra‐ tion-dependent manner [108, 110]. Fasudil hydrochloride (HA-1077), a specific inhibitor of 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 adrenegic desensitization [1].
