In acutely isolated pulmonary arterial smooth muscle cells (PASMCs) the Ca2+ mobilizing messenger nicotinic acid adenine dinucleaotide phosphate (NAADP) triggers bursts of Ca2+ release from lysosomes (Boittin et al., 2002; Kinnear et al., 2004) by activating the Two Pore Domain Channel subtype 2 (TPC2;Calcraft et al., 2009). These Ca2+ bursts initiate a propagating wave by subsequently triggering Ca2+-induced Ca2+ release (CICR) from the SR via ryanodine receptors (RyRs). A large proportion of lysosomes form tight clusters within the perinuclear region of PASMCs and in a manner consistent with the spatially restricted nature of NAADP-dependent Ca2+ bursts. Moreover, these lysosomal clusters are closely associated with a sub-population of RyRs (Kinnear et al., 2004). In fact, lysosomes may be separated from closely apposed RyRs by a narrow junction or cleft (<100 nm) that is beyond the resolution of light microscopy. Thus, lysosome-sarcoplasmic reticulum (SR) junctions may form a highly organised “trigger zone” for Ca2+ signaling in response to NAADP. This may constitute a novel nanodomain with unique functional characteristics, in that the presence of such a junction offers an explanation for the fact that NAADP-dependent Ca2+ bursts induce propagating Ca2+ waves by triggering CICR from the SR via RyRs in an all-or-none manner, and as such these proposed junctions may provide a “margin of safety” with respect to the initiation of vasoconstriction by NAADP. The precise mechanisms involved appear yet more complex, because all three RyR sub-types are expressed in arterial smooth muscle and some of these lysosome clusters may selectively couple to RyR3 (Kinnear et al., 2008). Determining factors in this respect could be the relative sensitivity of each RyR sub-type to CICR, the maximum gain in response to Ca2+ and the relative sensitivity of each receptor sub-type to inactivation by Ca2+. RyR3 would provide for a higher “margin of safety” with respect to the all-or-none amplification of Ca2+ bursts at the putative lysosome-SR junction. Inclusion of RyR3 alone in this model cannot, however, explain the subsequent generation of a propagating Ca2+ wave as RyR3 labeling declines markedly outside the perinuclear region of PASMCs. However, labeling for RyR2 increases markedly as we move from the perinuclear to the extraperinuclear region. This suggests that RyR2 may function to receive Ca2+ from RyR3 at the interface of lysosome-SR junctions and thereby allow for further propagation of the Ca2+ signal by CICR. Importantly, our recent studies have also shown that SERCA2a is almost entirely restricted to the perinuclear SR of PASMCs (Clark et al., 2010) and thus sits proximal to RyR3. Therefore we have the necessary transporter to support Ca2+ uptake into the SR within the region of lysosome-SR junctions. In marked contrast to SERCA2a, however, SERCA2b is targeted to the SR proximal to the plasma membrane (PM). Moreover it is this region of the SR to which RyR1 may be preferentially targeted. The combination of limited support for signal propagation by RyR1 with the removal of Ca2+ from the cytoplasm by SERCA2b may effectively segregate the PM-SR space in these cells (Clark et al., 2010). That this may be the case is supported by functional data. Thus, adenylyl cyclase coupled receptors have been shown to elicit Ca2+ release proximal to the PM via RyRs and thereby open Ca2+-activated K+ channels in the plasma membrane. This initiates membrane hyperpolarization which will facilitate Ca2+ removal from the proposed junctional space between the SR and the plasma membrane, leading ultimately to vasodilation. In this respect kinetic information may be used to formulate hypotheses, given that SERCA2a and SERCA2b exhibit quite different kinetics. SERCA2b has a higher affinity for Ca2+ but lower Vmax than SERCA2a. SERCA2b may therefore be dominant at rest and function to maintain resting levels of Ca2+ in the vicinity of the contractile apparatus. However, its low Vmax may lead to saturation of this SERCA with Ca2+ upon release of Ca2+ from the bulk SR, via RyR3 and RyR2, in response to stimuli that elicit vasoconstriction. Thereby, SERCA2b would allow the Ca2+ concentration to rise in the vicinity of the contractile apparatus, until such time as vasodilation is promoted by, for example, activation of adenylyl cyclase coupled receptors that may: (1) increase the activity of SERCA2b by PKA-dependent phosphorylation and facilitate the removal of Ca2+ from the bulk cytoplasm, and (2) trigger PKA-dependent Ca2+ release from the peripheral SR store via RyR1, to promote PM hyperpolarization and Ca2+ removal from the proposed PM-SR nanojunction (Clark et al. 2010). It seems unlikely that such co-ordinated regulation of opposing cellular functions could occur without the presence of membrane-membrane junctions, as these are required to support the locks and the keys required for the initiation of Ca2+ signals with the desired spatiotemporal pattern. All procedures accorded with current UK legislation