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

Nicholas P. Kinnear, Christopher N. Wyatt, Jill H. Clark, Peter J. Calcraft, Sidney Fleischer, Loice H. Jeyakumar, Graeme F. Nixon, A. Mark Evans

Research output: Contribution to journalArticle

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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) alpha lgP120 labelling, a lysosome-specific protein, in the perinuclear region of the cell (within 1.5 mu m of the nucleus) was similar to 4-fold greater than in the sub-plasmalemmal (within 1.5 mu m of the plasma membrane) and similar to 2-fold greater than in the extra-perinuclear (remainder) regions; (2) RyR3 labelling within the perinuclear region was similar to 4- and similar to 14-fold greater than that in the extra-perinuclear and sub-plasmalemmal regions, and similar to 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 alpha lgp120 labelling within the perinuclear region was similar to 2-fold greater for RyR3 than for RyR2 or RyR1; (4) co-localization between alpha lgp120 and each RyR subtype declined markedly outside the perinuclear region. Furthermore, selective block of RyR3 and RyR1 with dantrolene (30 mu M) 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. (C) 2007 Elsevier Ltd. AR rights reserved.

Original languageEnglish
Pages (from-to)190-201
Number of pages12
JournalCell Calcium
Volume44
Issue number2
Early online date11 Jan 2008
DOIs
Publication statusPublished - Aug 2008

Keywords

  • lysosomes
  • ryanodine receptors
  • NAADP
  • CA2+ release channel
  • adenine-dinucleotide phosphate
  • functional-characterization
  • sarcoplasmic-reticulum
  • 2-pool mechanism
  • cells
  • stores

Cite this

Kinnear, N. P., Wyatt, C. N., Clark, J. H., Calcraft, P. J., Fleischer, S., Jeyakumar, L. H., ... Evans, A. M. (2008). Lysosomes co-localize with ryanodine receptor subtype 3 to form a trigger zone for calcium signalling by NAADP in rat pulmonary arterial smooth muscle. Cell Calcium, 44(2), 190-201. https://doi.org/10.1016/j.ceca.2007.11.003

Lysosomes co-localize with ryanodine receptor subtype 3 to form a trigger zone for calcium signalling by NAADP in rat pulmonary arterial smooth muscle. / Kinnear, Nicholas P.; Wyatt, Christopher N.; Clark, Jill H.; Calcraft, Peter J.; Fleischer, Sidney; Jeyakumar, Loice H.; Nixon, Graeme F.; Evans, A. Mark.

In: Cell Calcium, Vol. 44, No. 2, 08.2008, p. 190-201.

Research output: Contribution to journalArticle

Kinnear, Nicholas P. ; Wyatt, Christopher N. ; Clark, Jill H. ; Calcraft, Peter J. ; Fleischer, Sidney ; Jeyakumar, Loice H. ; Nixon, Graeme F. ; Evans, A. Mark. / Lysosomes co-localize with ryanodine receptor subtype 3 to form a trigger zone for calcium signalling by NAADP in rat pulmonary arterial smooth muscle. In: Cell Calcium. 2008 ; Vol. 44, No. 2. pp. 190-201.
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AU - Kinnear, Nicholas P.

AU - Wyatt, Christopher N.

AU - Clark, Jill H.

AU - Calcraft, Peter J.

AU - Fleischer, Sidney

AU - Jeyakumar, Loice H.

AU - Nixon, Graeme F.

AU - Evans, A. Mark

PY - 2008/8

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N2 - 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) alpha lgP120 labelling, a lysosome-specific protein, in the perinuclear region of the cell (within 1.5 mu m of the nucleus) was similar to 4-fold greater than in the sub-plasmalemmal (within 1.5 mu m of the plasma membrane) and similar to 2-fold greater than in the extra-perinuclear (remainder) regions; (2) RyR3 labelling within the perinuclear region was similar to 4- and similar to 14-fold greater than that in the extra-perinuclear and sub-plasmalemmal regions, and similar to 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 alpha lgp120 labelling within the perinuclear region was similar to 2-fold greater for RyR3 than for RyR2 or RyR1; (4) co-localization between alpha lgp120 and each RyR subtype declined markedly outside the perinuclear region. Furthermore, selective block of RyR3 and RyR1 with dantrolene (30 mu M) 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. (C) 2007 Elsevier Ltd. AR rights reserved.

AB - 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) alpha lgP120 labelling, a lysosome-specific protein, in the perinuclear region of the cell (within 1.5 mu m of the nucleus) was similar to 4-fold greater than in the sub-plasmalemmal (within 1.5 mu m of the plasma membrane) and similar to 2-fold greater than in the extra-perinuclear (remainder) regions; (2) RyR3 labelling within the perinuclear region was similar to 4- and similar to 14-fold greater than that in the extra-perinuclear and sub-plasmalemmal regions, and similar to 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 alpha lgp120 labelling within the perinuclear region was similar to 2-fold greater for RyR3 than for RyR2 or RyR1; (4) co-localization between alpha lgp120 and each RyR subtype declined markedly outside the perinuclear region. Furthermore, selective block of RyR3 and RyR1 with dantrolene (30 mu M) 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. (C) 2007 Elsevier Ltd. AR rights reserved.

KW - lysosomes

KW - ryanodine receptors

KW - NAADP

KW - CA2+ release channel

KW - adenine-dinucleotide phosphate

KW - functional-characterization

KW - sarcoplasmic-reticulum

KW - 2-pool mechanism

KW - cells

KW - stores

U2 - 10.1016/j.ceca.2007.11.003

DO - 10.1016/j.ceca.2007.11.003

M3 - Article

VL - 44

SP - 190

EP - 201

JO - Cell Calcium

JF - Cell Calcium

SN - 0143-4160

IS - 2

ER -