Multiple ryanodine receptor subtypes and heterogeneous ryanodine receptor-gated Ca2+ stores in pulmonary arterial smooth muscle cells

Abstract

Ryanodine receptors (RyRs) of pulmonary arterial smooth muscle cells (PASMCs) play important roles in major physiological processes such as hypoxic pulmonary vasoconstriction and perinatal pulmonary vasodilatation. Recent studies show that three subtypes of RyRs are coexpressed and RyR-gated Ca2+ stores are distributed heterogeneously in systemic vascular myocytes. However, the molecular identity and subcellular distribution of RyRs have not been examined in PASMCs. In this study we detected mRNA and proteins of all three subtypes in rat intralobar PASMCs using RT-PCR and Western blot. Quantitative real-time RT-PCR showed that RyR2 mRNA was most abundant, approximately 15-20 times more than the other two subtypes. Confocal fluorescence microscopy revealed that RyRs labeled with BODIPY TR-X ryanodine were localized in the peripheral and perinuclear regions and were colocalized with sarcoplasmic reticulum labeled with Fluo-5N. Immunostaining showed that the subsarcolemmal regions exhibited clear signals of RyR1 and RyR2, whereas the perinuclear compartments contained mainly RyR1 and RyR3. Ca2+ sparks were recorded in both regions, and their activities were enhanced by a subthreshold concentration of caffeine or by endothelin-1, indicating functional RyR-gated Ca2+ stores. Moreover, 18% of the perinuclear sparks were prolonged [full duration/half-maximum (FDHM) = 193.3 +/- 22.6 ms] with noninactivating kinetics, in sharp contrast to the typical fast inactivating Ca2+ sparks (FDHM = 44.6 +/- 3.2 ms) recorded in the same PASMCs. In conclusion, multiple RyR subtypes are expressed differentially in peripheral and perinuclear RyR-gated Ca2+ stores; the molecular complexity and spatial heterogeneity of RyRs may facilitate specific Ca2+ regulation of cellular functions in PASMCs.