Intracellular Ca²⁺ release through ryanodine receptors (RyRs) plays important roles in smooth muscle excitation-contraction coupling, but the underlying regulatory mechanisms are poorly understood. Here we show that FK506 binding protein of 12.6 kDa (FKBP12.6) associates with and regulates type 2 RyRs (RyR2) in tracheal smooth muscle. FKBP12.6 binds to RyR2 but not other RyR or inositol 1,4,5-trisphosphate receptors, and FKBP12, known to bind to and modulate skeletal RyRs, does not associate with RyR2. When dialyzed into tracheal myocytes, cyclic ADP-ribose (cADPR) alters spontaneous Ca²⁺ release at lower concentrations and produces macroscopic Ca²⁺ release at higher concentrations; neurotransmitter-evoked Ca²⁺ release is also augmented by cADPR. These actions are mediated through FKBP12.6 because they are inhibited by molar excess of recombinant FKBP12.6 and are not observed in myocytes from FKBP12.6-knockout mice. We also report that force development in FKBP12.6-null mice, observed as a decrease in the concentration/tension relationship of isolated trachealis segments, is impaired. Taken together, these findings point to an important role of the FKBP12.6/RyR2 complex in stochastic (spontaneous) and receptor-mediated Ca²⁺ release in smooth muscle.

In this study we examined the expression of RyR subtypes and the role of RyRs in neurotransmitter- and hypoxia-induced Ca²⁺ release and contraction in pulmonary artery smooth muscle cells (PASMCs). Under perforated patch clamp conditions, maximal activation of RyRs with caffeine or inositol triphosphate receptors (IP₃Rs) with noradrenaline induced equivalent increases in [Ca²⁺]_i and Ca²⁺-activated Cl⁻ currents in freshly isolated rat PASMCs. Following maximal IP₃-induced Ca²⁺ release, neither caffeine nor chloro-m-cresol induced a response, whereas prior application of caffeine or chloro-m-cresol blocked IP₃-induced Ca²⁺ release. In cultured human PASMCs, which lack functional expression of RyRs, caffeine failed to affect ATP-induced increases in [Ca²⁺]_i in the presence and absence of extracellular Ca²⁺. The RyR antagonists ruthenium red, ryanodine, tetracaine, and dantrolene greatly inhibited submaximal noradrenaline– and hypoxia-induced Ca²⁺ release and contraction in freshly isolated rat PASMCs, but did not affect ATP-induced Ca²⁺ release in cultured human PASMCs. Real-time quantitative RT-PCR and immunofluorescence staining indicated similar expression of all three RyR subtypes (RyR1, RyR2, and RyR3) in freshly isolated rat PASMCs. In freshly isolated PASMCs from RyR3 knockout (RyR3^−/−) mice, hypoxia-induced, but not submaximal noradrenaline–induced, Ca²⁺ release and contraction were significantly reduced. Ruthenium red and tetracaine can further inhibit hypoxic increase in [Ca²⁺]_i in RyR3^−/− mouse PASMCs. Collectively, our data suggest that (a) RyRs play an important role in submaximal noradrenaline– and hypoxia-induced Ca²⁺ release and contraction; (b) all three subtype RyRs are expressed; and (c) RyR3 gene knockout significantly inhibits hypoxia-, but not submaximal noradrenaline–induced Ca²⁺ and contractile responses in PASMCs.

Hypoxic exposure causes pulmonary vasoconstriction, which serves as a critical physiologic process that ensures regional alveolar ventilation and pulmonary perfusion in the lungs, but may become an essential pathologic factor leading to pulmonary hypertension. Although the molecular mechanisms underlying hypoxic pulmonary vasoconstriction and associated pulmonary hypertension are uncertain, increasing evidence indicates that hypoxia can result in a significant increase in intracellular reactive oxygen species concentration ([ROS]_i) through the mitochondrial electron-transport chain in pulmonary artery smooth muscle cells (PASMCs). The increased mitochondrial ROS subsequently activate protein kinase C-ɛ (PKCɛ) and NADPH oxidase (Nox), providing positive mechanisms that further increase [ROS]_i. ROS may directly cause extracellular Ca²⁺ influx by inhibiting voltage-dependent K⁺ (K_V) channels and opening of store-operated Ca²⁺ (SOC) channels, as well as intracellular Ca²⁺ release by activating ryanodine receptors (RyRs), leading to an increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) and associated contraction. In concert with ROS, PKCɛ may also affect K_V channels, SOC channels, and RyRs, contributing to hypoxic Ca²⁺ and contractile responses in PASMCs. Antioxid. Redox Signal. 11, 611–623.

Cerebral vascular dysfunction and associated diseases often occur in type-1 diabetes, but the underlying mechanisms are largely unknown. In this study, we sought to determine whether big-conductance, Ca²⁺-activated K⁺ (BK) channels were impaired in vascular (cerebral artery) smooth muscle cells (CASMCs) from streptozotocin-induced type-1 diabetic mice using patch clamp, molecular biologic, and genetic approaches. Our data indicate that the frequency and amplitude of spontaneous transient outward currents (STOCs) are significantly decreased, whereas the activity of spontaneous Ca²⁺ sparks is increased, in diabetic CASMCs. The sensitivity of BK channels to voltage, Ca²⁺, and the specific inhibitor iberiotoxin are all reduced in diabetic myocytes. Diabetic mice show increased myogenic tone and decreased contraction in response to iberiotoxin in cerebral arteries and elevated blood pressure. The expression of the BK channel β1, but not α-subunit protein, is markedly decreased in diabetic cerebral arteries. Diabetic impairment of BK channel activity is lost in CASMCs from BK channel β1-subunit gene deletion mice. In conclusion, the BK channel β1-subunit is impaired in type-1 diabetic vascular SMCs, resulting in increased vasoconstriction and elevated blood pressure, thereby contributing to vascular diseases in type-1 diabetes.