Lysosomes Co-Localize with Ryanodine Receptor Subtype 3 to Form a Trigger Zone for Calcium Signalling by NAADP in Rat Pulmonary Arterial Smooth Muscle

Authors

Nicholas P. Kinnear
Christopher N. Wyatt, Wright State University - Main CampusFollow
Jill H. Clark
Peter J. Calcraft
Sidney Fleischer
Loice H. Jeyakumar
Graeme F. Nixon
A. Mark EvansFollow

Document Type

Article

Publication Date

8-2008

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.

Repository Citation

Kinnear, N. P., Wyatt, C. N., Clark, J. H., Calcraft, P. J., Fleischer, S., Jeyakumar, L. H., Nixon, G. F., & 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://corescholar.libraries.wright.edu/ncbp/201

DOI

10.1016/j.ceca.2007.11.003