Our previous studies on isolated pulmonary artery smooth muscle cells (PASMC) have shown that nicotinic acid dinucleotide phosphate (NAADP) triggers Ca²⁺ release from intracellular stores (Boittin et al. 2002) and that these stores are lysosome-related (Kinnear et al. 2003). Pulmonary vasoconstrictors mediate their actions, in part, by altering intracellular Ca²⁺. In this study we have investigated the effect of selectively depleting the lysosomal Ca²⁺store on the actions of two vasoconstrictors: prostaglandin-F2 alpha (PGF_2α) and endothelin-1 (ET-1) and the effect these vasoconstrictors have on NAADP levels. PASMC were isolated from adult male Wistar rats (150-300g) that had been killed by cervical dislocation. Changes in intracellular Ca²⁺ were detected using Fura-2 fluorescence imaging, as described previously (Boittin et al. 2002). PGF_2α(2 μM)caused an increase in the f340/f380 fluorescence ratio from 0.54±0.04 to 1.29±0.18 (n=5, mean±S.E.M.). The vacuolar proton pump inhibitor bafilomycin-A1 (100nM, 50min) had no effect on this rise in Ca²⁺ (n=5).In contrast to this, the Ca²⁺ signals observed with ET-1 were abolished by incubation with bafilomycin A1. ET-1 (100 nM) caused an increase in f340/f380 fluorescence ratio from 0.56±0.09 to 1.17±0.02 (n=4) in the absence of bafilomycin A1. This increase was inhibited by 98.4% after incubation with bafilomycin A1 (100nM, 50min). Incubation of the cells with thapsigargin (1μM)prevented ET-1 (100nM) from inducing global Ca²⁺ waves and contraction. However, small spatially restricted Ca²⁺ bursts were still observed in 5 out of 9 cells causing an increase in f340/f380 ratio of 17±4% within a given region of interest in the cell. Similarly, after incubation of cells with ryanodine (20μM), spatially restricted Ca²⁺ bursts were also detected in 3 out of 8 cells with the increase in f340/f380 ratio measuring 24±0.1% within a region of interest. Using a competitive radioreceptor binding assay (Masgrau et al. 2003) the effect of ET-1 on NAADP levels in endothelium denuded pulmonary arteries was investigated. A 30s exposure of ET-1 (1μM) increased levels of NAADP from 0.21±0.04 to 1.33±0.2 pmol/mg protein (n=12). Exposure of arteries without endothelium to PGF_2α (2 μM) caused no significant increase in NAADP levels (n=3). These data suggest that ET-1 but not PGF_2α mobilises Ca²⁺ from a thapsigargin and ryanodine-insensitive store via NAADP.