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

Abstract

Recent studies show that three subtypes of RyRs are coexpressed and RyR-gated Ca 2ϩ 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, ϳ15-20 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. Ca 2ϩ 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 Ca 2ϩ stores. Moreover, 18% of the perinuclear sparks were prolonged [full duration/half-maximum (FDHM) ϭ 193.3 Ϯ 22.6 ms] with noninactivating kinetics, in sharp contrast to the typical fast inactivating Ca 2ϩ sparks (FDHM ϭ 44.6 Ϯ 3.2 ms) recorded in the same PASMCs. In conclusion, multiple RyR subtypes are expressed differentially in peripheral and perinuclear RyR-gated Ca 2ϩ

RyR1-specific requirement for depolarization-induced Ca2+ sparks in urinary bladder smooth muscle

“…This seducing scheme is also supported by Yang et al showing that caffeine and endothelin may activate different Ca 2+ sparks in pulmonary artery myocytes (Yang et al, 2005), and by ZhuGe et al who reported multiple Ca 2+ sparks sites in smooth muscle cells (ZhuGe et al, 2004).…”

“…RyR2 has been suggested to be the primary contributor of Ca 2+ sparks in urinary bladder myocytes based on the increase in the number of spontaneous and triggered Ca 2+ sparks in mice that do not express FKBP12.6 (FKBP12.6 -/-mice) (Ji et al, 2004). However, all three isoforms of RyR are expressed in mouse detrusor muscle (Ji et al, 2004) and several studies suggested a role for RyR1 in smooth muscle Ca 2+ waves or Ca 2+ sparks (Coussin et al, 2000;Yang et al, 2005;Du et al, 2005;Du et al, 2006).…”

An Ryr1 I4895T mutation abolishes Ca 2+ release channel function and delays development in homozygous offspring of a mutant mouse line

“…Type 1 ryanodine receptor (RyR1) predominates in skeletal muscle but is expressed at lower levels in other tissues (3)(4)(5)(6)(7)(8)(9). In skeletal muscle, RyR1 is located in the junctional terminal cisternae of the SR, where it assembles in ordered orthogonal arrays.…”

Proteomic analysis of protein nitration in rat cerebellum: effect of biological aging

“…The tandem MS spectra of the nitrated peptides of these proteins are shown in Figs 5-7, respectively. The ryanodine receptor is the primary calcium release protein in the sarco/endoplasmic reticulum and plays a major role in muscle contraction (Yang et al 2005b). The physiological consequences of nitration of the similar to ryanodine receptor 3 protein are currently unknown; however, the age-dependent increase in cytosolic calcium (Zhu et al 2005) may be caused, in part, by a dysfunctional ryanodine receptor.…”