mechanisms
Direct evidence in support of the proposal that an acidic Ca2+ store is the source of NAADP-mediated Ca2+ signals in vascular smooth muscle was gained through further experiments on the effect of Bafilomycin A1 on Ca2+ signals evoked by NAADP. Given the similarity in Ca2+ signals evoked by NAADP and Bafilomycin A1, in association with Dr. F.X. Boittin, I examined whether or not depletion of Bafilomycin A1-sensitive Ca2+ stores attenuated Ca2+ signals in response to NAADP. Strikingly, preincubation of isolated pulmonary artery smooth muscle cells with Bafilomycin A1 was seen to completely abolish Ca2+ signals in response to the intracellular dialysis of NAADP. Consistent with previous studies which have shown Bafilomycin A1 to be selective for vacuolar proton pumps (Bowman, et al., 1988), preincubation of cells with Bafilomycin A1 was without effect on the mobilisation of SR Ca2+ stores by the activation of RyRs via caffeine, or the activation of IP3Rs by intracellular dialysis of IP3. These positive controls
(caffeine and IP3) indicate that depletion of Bafilomycin A1-sensitive Ca2+
stores selectively disrupts Ca2+ mobilisation by NAADP, without any discernable effect on Ca2+ uptake via SERCA pumps and subsequent SR Ca2+ release via RyRs and IP3Rs. These findings are consistent with findings in sea
urchin eggs (Churchill, et al., 2002), pancreatic acinar cells (Yamasaki, et al., 2004), the clonal pancreatic cell line (MIN6; Yamasaki, et al., 2004) and cortical neurons (Brailoiu, et al., 2005), in which NAADP has been suggested to initiate Ca2+ signals from acidic organelles only in the presence of a proton gradient generated by the actions of the vacuolar proton pump.
Although NAADP appears to mobilise an acidic Ca2+ store in both sea urchin eggs and pulmonary artery smooth muscle cells, there appears to be a
significant difference between the responses to Bafilomycin A1 within these cells. Thus, Bafilomycin A1 did not deplete the NAADP-sensitive Ca2+store in sea urchin eggs, as application of NAADP induced a robust Ca2+signal in the presence of Bafilomycin A1. However, Bafilomycin A1 prevented refilling of this store, because there was no Ca2+ signal in response to a further challenge by NAADP (Churchill et al., 2002). This is in marked contrast to the observations described here in pulmonary artery smooth muscle cells, where Bafilomycin A1 abolished Ca2+signals in response to the initial application of NAADP. It is conceivable that this difference in response may be due to the existence of different NAADP receptors in the two preparations. Whereas the NAADP receptor in sea urchin eggs may remain in a closed state until activation by NAADP, the NAADP receptor expressed in vascular smooth muscle may be tonically active at rest. This could render the lysosomal Ca2+ stores in this tissue ‘leaky’, with a constant turnover of Ca2+. If this were the case, it suggests that the blockade of Ca2+uptake as a result of Bafilomycin A1 treatment alters the equilibrium between Ca2+uptake and Ca2+ release in these stores, causing Ca2+ release which ultimately results in the depletion of lysosomal Ca2+ stores in much the same manner as thapsigargin depletes SR Ca2+stores. This may explain why Bafilomycin A1 induced global Ca2+ waves in pulmonary artery smooth muscle cells, but elicited no Ca2+ signals in sea urchin eggs (Churchillet al., 2002). Consistent with this proposal, disruption of reserve granules in the sea urchin egg by osmotic lysis via the actions of GPN was, unlike Bafilomycin A1, shown to elicit spatially restricted Ca2+ signals (Churchillet al., 2002). Furthermore, a previous investigation in cultured bone- marrow-derived macrophages showed a decrease in the luminal Ca2+ concentration within lysosomes in response to Bafilomycin A1 (Christensen, et al., 2002), whilst a ‘non-leaky’ acidic Ca2+ store similar to that described previously by Churchill et al. (2002) may also be present in fibroblasts where the Ca2+ concentration within end stage lysosomes remains unaffected by treatment of cells with Bafilomycin A1 (Gerasimenko, et al., 1998). It is entirely possible, therefore, that the nature of Ca2+ cycling via NAADP receptors may vary in a cell- and function-specific manner.
held opinion within the field. Indeed a number of groups have proposed that NAADP mobilises Ca2+ from ER/SR stores by directly activating RyRs. In isolated nuclear preparations from pancreatic acinar cells it has been shown that NAADP- (and cADPR-) mediated Ca2+ signals were blocked by the RyR inhibitors ryanodine and ruthenium red, but not by caffeine (Gerasimenko, et al., 2003), whilst IP3 mediated-Ca2+release was seen to be inhibited by
caffeine, but not by ruthenium red (Gerasimenko, et al., 2003). Gerasimenkoet al.concluded that NAADP, like cADPR, mobilises Ca2+from nuclear envelope Ca2+stores through direct activation of RyRs. NAADP has also been proposed to directly activate RyR subtype 1 purified from skeletal muscle and reconstituted into lipid bilayers (Hohenegger, et al., 2002). Also, it has been suggested that NAADP directly activates RyR subtype 2 isolated from cardiac muscle and reconstituted into lipid bilayers (Mojzisova, et al., 2001). However, there has been a contradictory report which suggests that NAADP is without affect on all three RyR subtypes, as examined in lipid bilayer recordings of RyR1- and RyR3-subtypes isolated from rabbit skeletal muscle and bovine diaphragm muscle, respectively, or on crude microsome preparations from canine heart ventricles containing RyR2 receptors (Copello, et al., 2001). Furthermore, a more recent study carried out in Jurkat T-cells utilising mRNA technology to knockdown the expression of RyRs showed that NAADP mediated Ca2+ signalling in these cells was inhibited but not abolished (Langhorst, et al., 2004).
Therefore, there are clearly contradictory reports in the literature regarding NAADP regulation of RyRs. Whilst some studies show evidence to support direct activation of RyRs by NAADP, others provide clear evidence that RyR subtypes are not regulated by NAADP. These differences in opinion may arise from the fact that it is extremely difficult to separate ER/SR compartments with RyRs from closely associated lysosomes, as has been shown in the sea urchin egg where cADPR- and IP3-sensitive ER fractions
were contaminated with NAADP-sensitive reserve granule stores following separation on a Percoll density gradient (Lee and Aarhus, 1995). Furthermore, it may be extremely difficult to resolve Ca2+ signals generated via NAADP receptors following blockade of RyRs. However, the findings presented here clearly demonstrate that NAADP elicits spatially restricted Ca2+ signals from
bafilomycin-sensitive, lysosome-related organelles that are subsequently amplified by CICR via RyRs located on the SR in pulmonary artery smooth muscle cells.
3.3.3 Lysosomes form discrete clusters in pulmonary artery smooth muscle