Lysosomes co-localize with ryanodine receptor subtype 3 to form a trigger zone for calcium signalling by NAADP in rat pulmonary arterial smooth muscle

Abstract

SummaryIn arterial myocytes the Ca 2+ mobilizing messenger NAADP evokes spatially restricted Ca 2+ bursts from a lysosome-related store that are subsequently amplified into global Ca 2+ waves by Ca 2+ -induced Ca 2+ -release from the sarcoplasmic reticulum (SR) via ryanodine receptors (RyRs). Lysosomes facilitate this process by forming clusters that co-localize with a subpopulation of RyRs on the SR. We determine here whether RyR subtypes 1, 2 or 3 selectively co-localize with lysosomal clusters in pulmonary arterial myocytes using affinity purified specific antibodies. The density of: (1) αlgP120 labelling, a lysosome-specific protein, in the perinuclear region of the cell (within 1.5 μm of the nucleus) was ~4-fold greater than in the sub-plasmalemmal (within 1.5 μm of the plasma membrane) and ~2-fold greater than in the extra-perinuclear (remainder) regions; (2) RyR3 labelling within the perinuclear region was ~4-and ~14-fold greater than that in the extraperinuclear and sub-plasmalemmal regions, and ~2-fold greater than that for either RyR1 or RyR2; (3) despite there being no difference in the overall densities of fluorescent labelling of lysosomes and RyR subtypes between cells, co-localization with αlgp120 labelling within the perinuclear region was ~2-fold greater for RyR3 than for RyR2 or RyR1; (4) co-localization between αlgp120 and each RyR subtype declined markedly outside the perinuclear region. Furthermore, selective block of RyR3 and RyR1 with dantrolene (30μM) abolished global Ca 2+ waves but not Ca 2+ bursts in response to intracellular dialysis of NAADP (10nM). We conclude that a subpopulation of lysosomes cluster in the perinuclear region of the cell and form junctions with SR containing a high density of RyR3 to comprise a trigger zone for Ca 2+ signalling by NAADP.

CD38 Mediates Angiotensin II–Induced Intracellular Ca2+ Release in Rat Pulmonary Arterial Smooth Muscle Cells

“…Previous work by our and other laboratories have identified all three RyR subtypes in PASMCs, with RyR2 being the most abundant subtype (23,39). RyR1 and RyR2 are associated mainly with SR in the subsarcolemmal region, whereas RyR3 is localized predominantly in the perinuclear SR (23,40 There is substantial evidence suggesting that Ang II enhances ROS production through activation of NOX, contributing to Ca 21 response in VSMCs (17). Several studies have shown that CD38 activity is regulated by redox state and oxidative stress (19,20,22,27,43).…”

Lysosomes shape Ins(1,4,5)P3-evoked Ca2+ signals by selectively sequestering Ca2+ released from the endoplasmic reticulum

“…7; supplementary material Movie 1), allowing lysosomes selectively to accumulate Ca 2+ released from the ER. The reciprocal relationship is also important because in many cells NAADP-evoked Ca 2+ release from lysosomes triggers Ca 2+ release from the ER by Ca 2+ -induced Ca 2+ release via Ins(1,4,5)P 3 R (Calcraft et al, 2009) or RyR (Brailoiu et al, 2010;Cancela et al, 1999;Kinnear et al, 2008;Lee et al, 1997). Lysosome-ER junctions are reminiscent of those between mitochondria and ER (Fig.…”

“…8). Ca 2+ release via TPC2 can trigger Ca 2+ release via RyR or Ins(1,4,5)P 3 R in the ER (Brailoiu et al, 2010;Calcraft et al, 2009;Kinnear et al, 2008), and Ca 2+ release via Ins(1,4,5)P 3 R is selectively accumulated by lysosomes. The latter may regulate the behaviour of lysosomes by increasing lysosomal pH; by priming TPC2, which appears to be stimulated by luminal Ca 2+ (Pitt et al, 2010), to respond to NAADP; and it may regulate endolysosomal trafficking (Luzio et al, 2010).…”

Calcium signals regulated by NAADP and two-pore channels - their role in development, differentiation and cancer

“…These findings have led to the 'NAADP trigger hypothesis', whereby Ca 2+ released by the action of NAADP can recruit IP 3 R and RyR-mediated signalling pathways via CICR (Churchill and Galione 2001). Differences in the extent of ER store involvement in different cell types is likely to be a consequence of different patterns of localisation of the stores and receptors, with recruitment occurring when NAADP-mediated Ca 2+ release occurs in close proximity to ER channels (Kinnear et al, 2004, Kinnear et al, 2008, Morgan et al, 2011 (Morgan et al, 2013). This effect may be dependent on local microdomains of high [Ca 2+ ]i at junctions between the ER and acidic organelles.…”