Our previous studies on isolated pulmonary artery smooth muscle cells have shown that inositol 1,4,5-trisphosphate (IP₃), cyclic adenosine diphosphate-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP) mobilise Ca²⁺ from intracellular stores (Boittin et al. 2002, 2003). Our data are consistent with the view that intracellular dialysis of IP₃ and cADPR mobilises Ca²⁺ from the sarcoplasmic reticulum (SR) by activating IP₃ receptors and ryanodine receptors, respectively. In contrast, however, we found that NAADP triggered spatially restricted ‘bursts’ of Ca²⁺ release from a thapsigargin-insensitive intracellular store, and subsequent global Ca²⁺ waves by Ca²⁺-induced Ca²⁺ release via ryanodine receptors in the SR. Significantly, Ca²⁺ bursts took two forms (1) a synchronous increase in Ca²⁺ around the entire perimeter of the cell or (2) a spatially restricted increase in Ca²⁺ between 2 and 10 µm in diameter (Boittin et al. 2002). We therefore investigated the nature of the Ca²⁺ store accessed by NAADP.

Pulmonary artery smooth muscle cells were isolated from male Wistar rats (150-200 g) that were humanely killed. Using Fura-2 fluorescence imaging as described previously (Boittin et al. 2002), we have now shown that both initial Ca²⁺ bursts and global Ca²⁺ waves induced by intracellular dialysis of NAADP from a patch pipette are abolished after depletion of lysosome-related Ca²⁺ stores by a vacuolar proton pump inhibitor, bafilomycin A1. Thus, under control conditions, 10 nM NAADP increased the Fura-2 fluorescence ratio (F₃₄₀/F₃₈₀) by 130 ± 34 % (mean ± S.E.M., n = 5), at the peak of the Ca²⁺ wave. In marked contrast, 10 nM NAADP was without effect in paired cells that had been pre-incubated (40 min) with 100 nM bafilomycin. Importantly, bafilomycin (100 nM) was without effect on Ca²⁺ waves induced via IP₃ receptor activation by intracellular dialysis of IP₃ (n = 4), or via ryanodine receptor activation by extracellular application of caffeine (10 mM; n = 4). Furthermore, deconvolved images of lysosomes labelled with lysotracker red (excitation 568 nm, emission 590 nm), revealed dense clusters. Consistent with the spatial distribution of the two forms of Ca²⁺ burst initiated by NAADP (Boittin et al. 2002), the lysosomes clustered in either (1) a ring around the perimeter of the cell, or (2) a tight, spatially restricted unit approximately 2-6 µm in diameter. We propose, therefore, that NAADP triggers Ca²⁺ bursts by mobilising lysosome-related Ca²⁺ stores in pulmonary artery smooth muscle, in a manner consistent with previous studies on the sea urchin egg (Churchill et al. 2002).