Identification of Functionally Segregated Sarcoplasmic Reticulum Calcium Stores in Pulmonary Arterial Smooth Muscle

Abstract

In pulmonary arterial smooth muscle, Ca2+ release from the sarcoplasmic reticulum (SR) via ryanodine receptors (RyRs) may induce constriction and dilation in a manner that is not mutually exclusive. We show here that the targeting of different sarcoplasmic/endoplasmic reticulum Ca2+-ATPases (SERCA) and RyR subtypes to discrete SR regions explains this paradox. Western blots identified protein bands for SERCA2a and SERCA2b, whereas immunofluorescence labeling of isolated pulmonary arterial smooth muscle cells revealed striking differences in the spatial distribution of SERCA2a and SERCA2b and RyR1, RyR2, and RyR3, respectively. Almost all SERCA2a and RyR3 labeling was restricted to a region within 1.5 μm of the nucleus. In marked contrast, SERCA2b labeling was primarily found within 1.5 μm of the plasma membrane, where labeling for RyR1 was maximal. The majority of labeling for RyR2 lay in between these two regions of the cell. Application of the vasoconstrictor endothelin-1 induced global Ca2+ waves in pulmonary arterial smooth muscle cells, which were markedly attenuated upon depletion of SR Ca2+ stores by preincubation of cells with the SERCA inhibitor thapsigargin but remained unaffected after preincubation of cells with a second SERCA antagonist, cyclopiazonic acid. We conclude that functionally segregated SR Ca2+ stores exist within pulmonary arterial smooth muscle cells. One sits proximal to the plasma membrane, receives Ca2+ via SERCA2b, and likely releases Ca2+ via RyR1 to mediate vasodilation. The other is located centrally, receives Ca2+ via SERCA2a, and likely releases Ca2+ via RyR3 and RyR2 to initiate vasoconstriction.

Bidirectional Coupling between Ryanodine Receptors and Ca2+ Release-activated Ca2+ (CRAC) Channel Machinery Sustains Store-operated Ca2+ Entry in Human T Lymphocytes

“…It is possible that within the puncta, the RyR, STIM1, and CRAC channels localize in close proximity to each other but distant from SERCA. This hypothesis is supported by previous reports that RyR and SERCA may be segregated in different ER subcompartments (57,58). Spatial co-localization of RyR, STIM1, and CRAC channels would facilitate RyR activation by SOCE via the CICR mechanism and subsequent formation of discrete microdomains of low [Ca 2ϩ ] ER ("cold spots") in the vicinity of STIM1.…”

An RYR1 mutation associated with malignant hyperthermia is also associated with bleeding abnormalities

“…Cells in mouse aorta and tail artery express the RYR1 transcript, though as expected, to a much lower extent than skeletal muscle ( Figure 2A) and the presence of the RYR1 Y522S mutation in the heterozygous state does not affect the expression of RYR1 ( Figure 2B) no of RYR2 and RYR3 (Supplementary Figure 3). RT-PCR on mRNA isolated from tail RyR1-mediated calcium release from subplasmalemmal stores causes activation of BK channels resulting in cellular hyperpolarization leading to vasodilation (35,36).…”

Regulation of Cellular Communication by Signaling Microdomains in the Blood Vessel Wall

“…The released calcium further activates release of calcium from the SR via RyR3 found at SR-lysosomes nanojunctions in a CICR manner (Kinnear et al, 2004(Kinnear et al, , 2008. After activation of RyR3, the RyR2 isoform is activated in a CICR manner, and the calcium released from the SR is propagated as a wave in the cytoplasm to activate contraction of SMCs (Kinnear et al, 2004(Kinnear et al, , 2008Clark et al, 2010). By use of a lysosome marker and labeled ryanodine, Kinnear et al (2008) demonstrated a close proximity between the lysosomes and ryanodine receptors.…”