**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 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 Ca2+-ATPase, caused an increase in [Ca2+]i and contraction, demonstrating Ca2+ entry through 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 further increases in [Ca2+]i and tension, demonstrating Ca2+ entry independent of SOC and VDC (non-SOC) [17]. 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 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 enhanced MCh-induced contraction with elevation of [Ca2+]i . These effects of IbTX on tension and [Ca2+]i are antagonized by verapamil [10], 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) [102]. 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.
