title: 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: In 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) alphalgP120 labelling, a lysosome-specific protein, in the perinuclear region of the cell (within 1.5mum of the nucleus) was approximately 4-fold greater than in the sub-plasmalemmal (within 1.5mum of the plasma membrane) and approximately 2-fold greater than in the extra-perinuclear (remainder) regions; (2) RyR3 labelling within the perinuclear region was approximately 4- and approximately 14-fold greater than that in the extra-perinuclear and sub-plasmalemmal regions, and approximately 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 alphalgp120 labelling within the perinuclear region was approximately 2-fold greater for RyR3 than for RyR2 or RyR1; (4) co-localization between alphalgp120 and each RyR subtype declined markedly outside the perinuclear region. Furthermore, selective block of RyR3 and RyR1 with dantrolene (30muM) 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.
authors.affiliation: School of Chemical Sciences and Pharmacy, University of East Anglia, Norwich, NR4 7TJ, Norfolk, UK.
authors.forename: Nicholas P
authors.initials: NP
authors.lastname: Kinnear
authors.forename: Christopher N
authors.initials: CN
authors.lastname: Wyatt
authors.forename: Jill H
authors.initials: JH
authors.lastname: Clark
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Calcraft
authors.forename: Sidney
authors.initials: S
authors.lastname: Fleischer
authors.forename: Loice H
authors.initials: LH
authors.lastname: Jeyakumar
authors.forename: Graeme F
authors.initials: GF
authors.lastname: Nixon
authors.forename: A Mark
authors.initials: AM
authors.lastname: Evans
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling; D002478:Cells, Cultured; D005453:Fluorescence; D006321:Heart / Q000502:physiology; D008168:Lung / Q000166:cytology / Q000378:metabolism*; D008247:Lysosomes / Q000378:metabolism*; D008297:Male; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000378:metabolism*; D009249:NADP / Q000031:analogs & derivatives* / Q000378:metabolism; D020033:Protein Isoforms; D011651:Pulmonary Artery / Q000166:cytology / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D012433:Ryanodine / Q000494:pharmacology; D019837:Ryanodine Receptor Calcium Release Channel / Q000378:metabolism*; D012519:Sarcoplasmic Reticulum / Q000378:metabolism
chemical_list: D020033:Protein Isoforms; D019837:Ryanodine Receptor Calcium Release Channel; D012433:Ryanodine; D009249:NADP; C024376:NAADP; D002118:Calcium