LAS SENTENCIAS INTERPRETATIVAS-MANIPULATIVAS DEL TRIBUNAL

The data presented thus far suggest that lysosomes are predominantly localised in the perinuclear region of isolated PASMCs. Labelling of RyR subtypes revealed that RyR3 is the predominant subtype within this region. Furthermore, measurement of the density of co-localisation between αIgp120 and individual RyR subtypes

revealed that the greatest degree of co-localisation was in the following order: RyR3

>> RyR2 ≥ RyR1 suggesting that lysosomes and RyR3 form a trigger zone for Ca2+

signalling by NAADP. To further verify this proposal, the effect on NAADP- mediated Ca2+ release by selectively blocking RyR1 and RyR3 with dantrolene was examined. Dantrolene selectively blocks Ca2+ release via RyR3 and RyR1 but is without effect on RyR2 (Zhao et al., 2001). Thus, the rank order of co-localisation between lysosomes and the 3 subtypes (RyR3 >> RyR2 ≥ RyR1) offered the

opportunity to determine whether or not RyR3 underpinned the amplification of NAADP-mediated Ca2+ bursts into global Ca2+ waves.

The effect of dantrolene on Ca2+ transients triggered by intracellular dialysis from a patch pipette of NAADP (10 nM) was studied in the whole-cell configuration of the patch clamp technique and under current clamp conditions (I = 0), with changes in the intracellular Ca2+ concentration reported by the Fura-2 fluorescence ratio (F340/F380). An exemplar record is shown in Fig. 3.13A. The upper panel shows a

series of pseudocolour images of the Fura-2 fluorescence ratio recorded in an isolated PASMC during the intracellular dialysis of 10 nM NAADP, with the record of the Fura-2 fluorescence ratio against time shown in the bottom panel. Intracellular dialysis of 10 nM NAADP induced spatially restricted Ca2+ bursts (indicated by the white arrow in Fig. 3.13A, Image 2) which either returned to basal levels or proceeded and then triggered a global Ca2+ wave. The mean Fura-2 fluorescence ratio

(± S.E.M.) increased from a basal level of 0.51 ± 0.05 to a peak of 1.42 ± 0.21 before returning to baseline (n = 5).

Fig. 3.13. Dantrolene inhibits the NAADP-evoked global Ca2+ wave in pulmonary arterial smooth muscle cells

A Top panel, series of pseudocolour images of the Fura-2 fluorescence ratio recorded in an isolated PASMC during the intracellular dialysis of 10 nM NAADP. NAADP evokes a Ca2+ burst indicated by the white arrow (image 2) which precedes a global Ca2+ _{wave. Bottom panel, corresponding record of}

the Fura-2 fluorescence ratio against time for the cell imaged in the top panel. Access to the cytosol via the formation of the whole-cell configuration is indicated by the two bars at the beginning of the record. Note, the loss of a GΩ seal is indicated at the end of the record by three bars. B Top panel, series of pseudocolour images of the Fura-2 fluorescence ratio recorded in an isolate PASMC during the intracellular dialysis of 10 nM NAADP following the preincubation (15 min) with 30 µM

dantrolene. Dantrolene inhibited the global Ca2+ _{wave but not the localised Ca}2+ _{bursts (indicated in}

image 3 by the white arrow) in response to NAADP. Bottom panel, corresponding record of the Fura-2 fluorescence ratio against time for the cell imaged in the top panel.

Following preincubation (15 min) of PASMCs with 30 µM dantrolene,

intracellular dialysis of 10 nM NAADP failed to induce a global Ca2+ wave. However, dantrolene did not inhibit NAADP-induced Ca2+ bursts. Fig. 3.13B shows one such record. The upper panel shows a series of pseudocolour images in which the NAADP-induced Ca2+ burst is indicated (white arrow, image 3) with the record of the Fura-2 fluorescence ratio against time shown in the lower panel. In the presence of dantrolene the change in the Fura-2 fluorescence ratio was markedly attenuated, increasing from a basal level of 0.55 ± 0.03 to a peak of only 0.83 ± 0.05 (n = 6). The bar chart in Figure 3.14 shows that the mean change in the Fura-2 fluorescence ratio triggered by NAADP was markedly decreased from 0.91 ± 0.2 in the absence of

dantrolene (n = 5; Appendix 1, Table 3.34) to 0.28 ± 0.03 (n = 6, P < 0.05; Fig 3.13;

Appendix 1, Table 3.35) in the presence of dantrolene (30 µM). The fact that

dantrolene abolished the global Ca2+ wave but not the preceding Ca2+ burst triggered by NAADP is consistent with the effect of blocking all RyRs with ryanodine and of depleting SR Ca2+ stores with thapsigargin (Boittin et al., 2002). When taken together these data therefore support the proposal that RyR3 is a pre-requisite for the amplification of NAADP-induced Ca2+ bursts into global Ca2+ waves. Thus, it is likely that RyR3, but not RyR1 or RyR2 is targeted to lysosome-SR junctions to comprise a trigger zone for Ca2+ _{signalling by NAADP.}

3.3.5.1 Dantrolene is without effect on ionomycin-induced Ca2+ _{influx in pulmonary}

arterial smooth muscle cells

A previous report has suggested that dantrolene may inhibit the fluorescence reporting capability of Fura-2 (Nohmi et al., 1991). Therefore, it was important to determine whether or not dantrolene, when applied to PASMCs, compromised the ability of Fura-2 to reliably report changes in intracellular Ca2+. To this end I investigated the effects of dantrolene on the ability of the ionophore ionomycin to raise the intracellular Ca2+ concentration in PASMCs. Application of ionomycin (1

µM) induced an increase in the Fura-2 fluorescence ratio from 0.43 ± 0.02 to 0.74 ±

0.05 in the absence, and from 0.42 ± 0.01 to 0.78 ± 0.05 in the presence of 30 µM

dantrolene. The bar chart in Fig. 3.14 shows the mean (± S.E.M.) change in the fluorescence ratio in response to ionomycin (1 µM) in the absence (0.31 ± 0.04, n =

11; Appendix 1, Table 3.36) and following pre-incubation (15 minutes) with 30 µM

dantrolene (0.36 ± 0.05, n = 12; Fig. 3.14; Appendix 1, Table 3.36). These data show

quite clearly that dantrolene was without effect on the change in the Fura-2 fluorescence ratio resulting from ionomycin-induced Ca2+ influx, in marked contrast to its inhibitory effect on Ca2+ signalling by NAADP. It is unlikely, therefore, that dantrolene compromises the ability of Fura-2 to report changes in intracellular Ca2+ concentration under the conditions of my experiments.

Fig. 3.14. Dantrolene inhibits the global Ca2+ release in response to NAADP but not ionomycin in pulmonary arterial smooth muscle cells

Bar chart showing the peak change in the Fura-2 fluorescence ratio (mean ± S.E.M.) recorded in

PASMCs in response to either the intracellular dialysis of NAADP (10 nM) or extracellular application of ionomycin (1 mM). The response to NAADP and ionomycin were recorded in either the absence (white box; NAADP n = 5; ionomycin n = 11) or presence of 30 µM dantrolene (grey box; NAADP n

= 6; ionomycin n = 12). * indicates a statistically significant difference (P < 0.05) when compared to

the peak change in fluorescence ratio in response to 10 nM NAADP in the absence of 30 µM

dantrolene.