Type-3 Ryanodine Receptors Mediate Hypoxia-, but Not Neurotransmitter-induced Calcium Release and Contraction in Pulmonary Artery Smooth Muscle Cells

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.