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

Abstract

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.

Mitochondrial ROS-PKCε signaling axis is uniquely involved in hypoxic increase in [Ca2+]i in pulmonary artery smooth muscle cells

“…Although there are many reports to suggest a pre-eminent role for inhibition of K + channels by hypoxia, with subsequent membrane depolarization and Ca 2+ entry via voltage-dependent Ca 2+ channels [1], others have provided evidence for capacitative Ca 2+ entry through store-operated channels [8; 29]. Intracellular Ca 2+ release is also a critical event for the elevation of [Ca 2+ ] i and constriction in PASMCs [2][3][4][5][6][7]. PKC and other protein kinases are implicated in hypoxic responses [30].…”

“…Freshly isolated mouse resistance pulmonary and mesenteric artery SMCs (PASMCs and MASMCs) and tissues were prepared as described previously [7]. Briefly, mice were euthanized by sodium pentobarbital (150 mg/kg, i.p.).…”

“…[Ca 2+ ] i were measured by a dual excitation wavelength fluorescence method using the IonOptix fluorescence photometric system (Milton, MA), as described previously [7]. Freshly isolated cells were loaded with 5 μM fura-2/AM.…”

“…Fura-2/AM, and 5,6-chloromethyl-2,7-dichlorodihydrofluorescein diacetate (CM-DCF-DA) were obtained from Molecular Probes (Eugene, OR); PKCɛ translocation peptide inhibitor from Calbiochem (La Jolla, CA); monoclonal antibodies to PKCɛ, PKC inhibitor, Protein Gcoated beads from Santa Cruz (Santa Cruz, CA); and rotenone, myxothiazol, hydrogen peroxide, phorbol 12-myristate 13-acetate (PMA), GÖ6976, myeline basic protein (4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14), phosphatidyl serine, diolein, histone IIIs from Sigma (St. Louis, MO).…”

“…In contrast, SMCs from systemic arteries display decreased [Ca 2+ ] i and relax in response to hypoxia. The response of PASMCs to acute hypoxia involves calcium entry through voltagedependent and store-operated Ca 2+ channels, as well as Ca 2+ release from the sarcoplasmic reticulum [1][2][3][4][5][6][7][8]. Hypoxia-dependent changes in reactive oxygen species (ROS) concentration have been proposed to mediate HPV by several laboratories, although the details of this hypothesis differ greatly [9; 10].…”