Genetic evidence for functional role of ryanodine receptor 1 in pulmonary artery smooth muscle cells

Abstract

Ryanodine receptor 1 (RyR1) is well-known to be expressed in systemic and pulmonary vascular smooth muscle cells (SMCs); however, its functional roles remain largely unknown. In the present study, we attempted to determine the potential importance of RyR1 in membrane depolarization-, neurotransmitter-, and hypoxia-induced Ca2+ release and contraction in pulmonary artery SMCs (PASMCs) using RyR1 homozygous and heterozygous gene deletion (RyR1−/− and RyR1+/−) mice. Our results indicate that spontaneous local Ca2+ release and caffeine-induced global Ca2+ release are significantly reduced in embryonic RyR1−/− and adult RyR+/− cells. An increase in [Ca2+]i following membrane depolarization with high K+ is markedly attenuated in RyR1−/− and RyR1+/− PASMCs in normal Ca2+ or Ca2+-free extracellular solution. Similarly, muscle contraction evoked by membrane depolarization is reduced in RyR1+/− pulmonary arteries in the presence or absence of extracellular Ca2+. Neurotransmitter receptor agonists and inositol 1,4,5-triphosphate elicit a much smaller increase in [Ca2+]i in both RyR1−/− and RyR1+/− cells. We have also found that neurotransmitter-evoked muscle contraction is significantly inhibited in RyR1+/− pulmonary arteries. Hypoxia-induced increase in [Ca2+]i and contraction are largely blocked in RyR1−/− and/or RyR1+/− PASMCs. Collectively, our findings provide genetic evidence for the functional importance of RyR1 in spontaneous local Ca2+ release, and membrane depolarization-, neurotransmitter-, as well as hypoxia-induced global Ca2+ release and attendant contraction in PASMCs.

Hypoxia Induces Intracellular Ca2+ Release by Causing Reactive Oxygen Species-Mediated Dissociation of FK506-Binding Protein 12.6 from Ryanodine Receptor 2 in Pulmonary Artery Myocytes

“…Interestingly, membrane depolarization does not trigger normal Ca 2þ release from the SR in RyR2 À=þ airway SMCs (9). We have previously reported that both RyR1 and RyR3 gene deletion greatly inhibit the hypoxic increase in [Ca 2þ ] i in PASMCs (7,32). These results, together with the finding that hypoxia is all but incapable of inducing an increase in [Ca 2þ ] i in RyR2…”

“…This time period was chosen, because our previous studies have shown that maximal cellular responses occur before or at 5 min after acute hypoxic exposure (7,17,18,23,(30)(31)(32). In control experiments, tissues or cells were identically treated but exposed to a normoxic PSS.…”

“…HPV may result from an increase in intracellular Ca 2þ concentration ([Ca 2þ ] i ) in PA smooth muscle cells (PASMCs). We and other investigators have shown that Ca 2þ release from the sarcoplasmic reticulum (SR) through ryanodine receptors (RyRs) plays an important role in the hypoxic increase in [Ca 2þ ] i in PASMCs and HPV (3,4,7,12,13,19,25,31,32). The importance of RyRs in hypoxic responses in PASMCs is reinforced by findings that Ca 2þ release from the SR is likely to inhibit voltage-dependent K þ channels (15,22) and to open storeoperated Ca 2þ channels (13,14), which cause extracellular Ca 2þ influx, thus providing a positive feedback mechanism to enhance the hypoxic increase in [Ca 2þ ] i and contraction.…”

Up-regulation of ryanodine receptor expression increases the calcium-induced calcium release and spontaneous calcium signals in cerebral arteries from hindlimb unloaded rats

“…The increase in RyR1 in HU animals could explain why the increase of CICR was induced by P2X1 activation (near 40 %). This result could be correlated with the spatial distribution and/or coupling of P2X1, VDCC and RyR subtypes in VSMC [10,11,23,43].…”

Important role of PLC-γ1 in hypoxic increase in intracellular calcium in pulmonary arterial smooth muscle cells

“…All animal experiments were approved by the Institutional Animal Care and Use Committee of Albany Medical College. PAs and PASMCs were prepared from Swiss-Webster mice (Taconic, Germantown, NY), as we described previously (11,12,18,19,27,33,34). Briefly, the heart and lungs were carefully removed from mice and placed in ice-cold physiological saline solution (PSS) containing (in mM): 130 NaCl, 1 MgSO4, 1.8 CaCl2, 5.4 KCl, 10 HEPES [4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid], and 10 glucose (pH 7.4).…”