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

N. P. Kinnear, C. N. Wyatt, J. H. Clark, P. J. Calcraft, S. Fleischer, L. H. Jeyakumar, G. F. Nixon, A. M. Evans

Research output: Contribution to journalArticlepeer-review

Abstract

In arterial myocytes the Ca2+ mobilizing messenger NAADP evokes spatially restricted Ca2+ bursts from a lysosome-related store that are subsequently amplified into global Ca2+ waves by Ca2+-induced Ca2+-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 extra-perinuclear 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 (30 μM) abolished global Ca2+ waves but not Ca2+ bursts in response to intracellular dialysis of NAADP (10 nM). 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 Ca2+ signalling by NAADP.
Original languageEnglish
Pages (from-to)190-201
Number of pages12
JournalCell Calcium
Volume44
Issue number2
DOIs
Publication statusPublished - Aug 2008

Keywords

  • Lysosomes
  • Ryanodine receptors
  • NAADP

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