To determine whether or not Bafilomycin A1 mobilised the NAADP- sensitive Ca2+ store in pulmonary artery smooth muscle, I then examined the pharmacology of Bafilomycin A1-mediated Ca2+ release. As mentioned previously, recent work from this laboratory has shown that preincubation (20 min) of isolated pulmonary artery smooth muscle cells with thapsigargin (1
M) significantly attenuated Ca2+signals generated by the intracellular dialysis of NAADP (10 nM; Boittin, et al., 2002). In these cells, however, spatially restricted Ca2+ bursts were still detected in all the cells examined (Boittin, et al., 2002). In a similar fashion, preincubation (20 min) of isolated pulmonary artery smooth muscle cells with thapsigargin (1 M) eliminated global Ca2+ signals in response to the extracellular application of Bafilomycin A1 (100 – 300 nM). However, small spatially restricted and asynchronus Ca2+ transients were observed in 8 out of 11 cells (Fig. 3.4, Appendix 1, Table 3.3). In these 8 cells a region of interest (ROI) was drawn around the area covered by this spatially localised release event (Fig. 3.4, image 7). The increase in Fura-2 fluorescence ratio within this ROI was then determined. On average, these spatially restricted increases in the Fura-2 fluorescence ratio were seen to rise from 0.72 ± 0.03 to 0.91 ± 0.02 (Appendix 1, Table 3.3, results indicated with an asterisk (*)), an increase in the intracellular Ca2+ concentration from ~210 nM to ~310 nM (Fig 3.4). In the remaining 3 cells of the 11 examined, the
increase in Fura-2 was seen as a non-uniform increase in Fura-2 fluorescence ratio across the entire cell. As the increase in Fura-2 fluorescence ratio within these 3 cells did not occur in a spatially restricted fashion, an ROI was drawn around the entire cell and the increase in Fura-2 fluorescence measured. The
Fig. 3.4:Thapsigargin abolishes global Ca2+waves in response to Bafilomycin A1, but not localized Ca2+ release events: Upper panel: shows a pseudocolour representation of the changes in Fura-2 fluorescence ratio within an isolated pulmonary artery smooth muscle cell after preincubation (20 min) with thapsigargin (1 M) in response to the extracellular application of Bafilomycin A1 (300 nM). Note, the spatially restricted Ca2+burst indicated by the ROI in pseudocolour image 7.Lower panel: shows the average Fura-2 fluorescence ratio against time within the entire cell shown in the upper panel, the insert shows the Fura-2 fluorescence ratio against time on an expanded scale for the ROI indicated in pseudocolour representation in image 7.
Fig. 3.5: Ryanodine abolishes global Ca2+waves in response to Bafilomycin A1, but not localized Ca2+ release events. Upper panel: shows a pseudocolour representation of the changes in Fura-2 fluorescence ratio within an isolated pulmonary artery smooth muscle cell after preincubation (20 min) with ryanodine (20 M) in response to the extracellular application of Bafilomycin A1 (100 nM). Note, the spatially restricted Ca2+ release event indicated by an ROI in pseudocolour image 8. Lower panel: shows the average Fura-2 fluorescence ratio against time in the entire cell shown in the upper panel. The insert shows the Fura-2 fluorescence ratio against time on an expanded scale for the ROI indicated in pseudocolour representation 8.
average rise in Fura-2 fluorescence ratio in all 11 cells examined was from 0.64 ± 0.05 to 0.79 ± 0.06 (n = 11, Appendix 1, Table 3.3), an increase in intracellular Ca2+ from ~200 nM to ~250 nM. Statistical comparison of the increase in Fura-2 fluorescence ratio observed in response to the application of Bafilomycin A1 following preincubation of cells with thapsigargin (0.16 ±
0.02, n = 11; Appendix 1, Table 3.4) against the increase in Fura-2 fluorescence in cells to the application of Bafilomycin A1 in the absence if thapsigargin (0.42 ± 0.04, n = 20; Appendix 1, Table 3.4), by means of a one- way ANOVA test, showed there was a significant difference between the test and control groups (P = <0.0001; Appendix 1, Table. 3.4). The results of experiments in isolated pulmonary artery smooth muscle cells preincubated with thapsigargin suggest, therefore, that Bafilomycin A1, like NAADP, causes an initial phase of Ca2+release from a non-SR Ca2+store, that is amplified into a global Ca2+signal by SR Ca2+release.
Given that amplification of NAADP-mediated Ca2+ bursts into global Ca2+ waves requires amplification via CICR from RyRs on the SR, I next sought to determine whether or not CICR via RyRs was required for the generation of global Ca2+ signals by Bafilomycin A1. When isolated pulmonary artery smooth muscle cells were preincubated with ryanodine (20
M, 20 min), the extracellular application of Bafilomycin A1 (100 – 300 nM) failed to induce global Ca2+ waves. However, in 5 out of 8 cells examined under these conditions localized Ca2+release events were observed (Fig. 3.5). ROI’s were drawn to encompass localised Ca2+release events and the increase in Fura-2 fluorescence ratio was measured. Within a given ROI in cells preincubated with ryanodine, Bafilomycin A1-induced Ca2+ release events increased the Fura-2 fluorescence ratio from 0.66 ± 0.06 to 0.88 ± 0.09 (Fig 3.5, Appendix 1, Table 3.5, results marked with asterisk (*)). This equated to an increase in the cytoplasmic Ca2+ concentration within these regions from ~180 nM to ~300 nM. In the remaining 3 cells the increase in Fura-2 fluorescence ratio was observed as a non-uniform increase across the entire cell. As there was no clear spatial definition to these rises, ROI’s were drawn to encompass the entire cell. Following this the increase in Fura-2 fluorescence ratio was determined. On average, the Fura-2 fluorescence ratio was seen to increase from 0.61 ± 0.05 to 0.79 ± 0.08 across all the cells examined, corresponding to an increase in intracellular Ca2+concentration from ~160 nM to ~300 nM. As was the case in cells preincubated with thapsigargin, the inhibition by ryanodine of the increase in Fura-2 fluorescence ratio in response to the application of Bafilomycin A1 (0.18 ± 0.03, n = 8; Appendix 1, Table
3.6) was significantly lower than the increases in Fura-2 fluorescence ratio within control cells exposed to Bafilomycin A1 in the absence of ryanodine (0.42 ± 0.04, Appendix 1, Table 3.6) using a one-way ANOVA test (P = < 0.0001, Appendix 1, Table 3.6).
Fig. 3.6:Bar chart showing the similarity in the pharmacology underlying Ca2+signalling in isolated pulmonary artery smooth muscle cells in response to NAADP (10 nM) and Bafilomycin A1 (100 – 300 nM).
Fig. 3.6 compares the pharmacological profile of Ca2+signals generated by NAADP and Bafilomycin A1, respectively. On the basis of the similarities in this pharmacology, I conclude that both NAADP and Bafilomycin A1 generate Ca2+ bursts from a thapsigargin- and ryanodine-insensitive Ca2+store, that are then amplified into global Ca2+ waves by CICR via RyRs located on the SR (Fig. 3.6).
3.2.5 Bafilomycin A1 abolishes Ca2+ release mediated by NAADP without