Lysosomes and ryanodine receptor subtype 3 colocalise to form a trigger zone for calcium signalling by NAADP in rat pulmonary artery smooth muscle

N.P. Kinnear1, C.N. Wyatt1, L.H. Jeyakumar2, S. Fleischer2, G.F. Nixon3 and A.M. Evans1 (S.B. Guild) 1School of Biology, University of St Andrews, UK, 2Department of Medicine and Biological Sciences, Vanderbilt University, USA, 3Biomodeical Sciences, University of Aberdeen, UK.

Our previous studies have shown that lysosomes colocalise with a sub-population of ryanodine receptors (RyRs) to form a trigger zone for Ca2+ signalling by NAADP in pulmonary artery smooth muscle cells (PASMC; Kinnear et al. 2004). We therefore sought to determine which of the three RyR subtypes comprise the trigger zone.

PASMC were isolated from adult male Wistar rats (150-300g) that were humanely killed (Kinnear et al., 2004). RyRs and lysosomes were identified using affinity-purified rabbit anti-RyR1, 2 and 3 antibodies (Morton-Jones et al., 2006) and mouse antibodies against the lysosomal membrane protein αlgp120 (Grimaldi, et al., 1987). Primary antibody binding was visualised by means of fluorescently tagged secondary antibodies (Evans et al., 2005). Fluorescently labelled cells were visualised using a DeltaVision imaging system (Applied Precision) and captured images were analysed using Volocity software (Improvision).

For analysis, cells were divided into perinuclear and non-perinuclear regions. The perinuclear region was defined as the volume of the cell located within 2.5 μm of the nucleus. The majority of αlgp120 labelling and, therefore, lysosomal clusters were located within the perinuclear region of cells (61 ± 3 %, mean ± SEM, n = 40). Examination of the labelling of the 3 RyR subtypes showed that there was a significantly greater labelling of RyR3 within the perinuclear region of cells (0.07 ± 0.01, n = 12) than was observed for labelling of RyR1 (0.03 ± 0.01, n = 14, P = 0.005, F = 11.15, one-way ANOVA test, data considered significant if P = <0.05). There was no statistical difference between the labelling of RyR3 and RyR2 (0.04 ± 0.01, n = 12, P = 0.1, F = 2.98) in the perinuclear region.

Examination of the colocalisation between RyR subtype- and lysosomal-labelling indicated that within the perinuclear region RyR3 (0.0068 ± 0.0016 μm3 per μm3 of region, n = 14) colocalised with αlgp120 labelling to a significantly greater extent than either RyR2 (0.0016 ± 0.0005 μm3 per μm3 of region, n = 12, P = 0.007 F = 8.57) or RyR1 (0.0016 ± 0.0007 μm3 per μm3 of region, n = 14, P = 0.008 F = 8.52). Colocalisation between lysosomal and RyR3 labelling outwith the perinuclear region was markedly lower (0.0007 ± 0.0003 μm3 per μm3 of region, n = 14) than that observed in the perinuclear region. There was no significant difference between colocalisation of lysosomal and RyR3 labelling with either lysosomal and RyR1 labelling (0.0006 ± 0.0003 μm3 per μm3 of region, n = 14, P = 0.74 F = 0.11) or lysosomal and RyR2 labelling (0.0007 ± 0.0002 μm3 per μm3 of region, n = 12, P = 0.872 F = 0.03) outwith the perinuclear region of cells.

In conclusion, we propose that the trigger zone for Ca2+ signalling by NAADP in PASMC is formed between lysosomes and RyR3 and in the perinuclear region of the cell proximal to the nucleus.

Evans et al., (2005) J. Biol. Chem. 280, 41504 – 41511
Grimaldi et al., (1987) Biochem. J. 245, 557 – 566
Kinnear et al., (2004) J. Biol. Chem. 279, 54319 – 54326
Morton-Jones et al., (2006) Neurosci.137, 275 – 286

This work was supported by the Wellcome Trust and the BBSRC, the authors would like to thank Dr. J.P. Luzio ( University of Cambridge) for his kind gift of anti-αlgp120 antibodies.