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11. CADENA DE CUSTODIA
NG and HG cells loaded with Fluo-3 Ca2+-sensitive fluorescent dye were viewed using fluorescence microscopy and video recordings of their Ca2+-mediated responses
to AVP in the absence and presence of thapsigargin were analysed. Changes in fluorescence were measured from 10 cells for each experiment and four experiments were done for each AVP treatment (i.e. in the absence and presence of thapsigargin) and glucose culture condition. Four averaged plots (one for each experiment) of relative changes in fluorescence over time, for each agonist treatment and glucose culture condition, are presented in figures 3.2.4.1-3.2.4.3.
Analysis of Ca2+ transients produced by thapsigargin in NG and HG cells shown in figure 3.2.4.1 indicate there was no major difference observed between the cells from the two glucose culture conditions tested, since the general average response profile from all experiments is near the same and mean peak heights of change in F/F0 were 0.41 ± 0.06 for NG cells and 0.40 ± 0.05 for HG cells. Student’s
t-test of these values confirm no statistical significance in the difference observed for these values between NG and HG cells from the four experiments for each glucose culture condition (P>0.05). The same lack of difference was also observed from NG and HG cells that were treated with AVP in the absence the thapsigargin, as shown in figure 3.2.4.2. Again, the response profiles for all experiments in the latter case were, on average, the same as each other and resembled typical profiles observed previously (Byron and Taylor, 1995). Furthermore, the mean peak heights of change in F/F0 were
NG
HG
Figure 3.2.4.1. Thapsigarigin-Stimulated Ca2+ Transients in A7r5 Cells Cultured in Normal & High Glucose Media
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Figure 3.2.4.1. Thapsigarigin-Stimulated Ca2+ Transients in A7r5 Cells Cultured in Normal & High Glucose Media
Changes in [Ca2+] within A7r5 cells grown in either normal (“NG”) or high (“HG”) glucose-supplemented media were detected by monitoring Fluo-3 fluorescence levels in dye-loaded cells. Each trace represents mean values
calculated from 10 cells. Arrows indicate the time-point at which 1µM thapsigargin was added.
HG
NG
Figure 3.2.4.2. AVP-Stimulated Ca2+ Transients in A7r5 Cells Cultured in Normal & High Glucose Media (No Thapsigargin Pre-Treatment)
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Figure 3.2.4.2. AVP-Stimulated Ca2+ Transients in A7r5 Cells Cultured in Normal & High Glucose Media (No Thapsigargin Pre-
Treatment)
Changes in [Ca2+] within A7r5 cells grown in either normal (“NG”) or high (“HG”) glucose-supplemented media were detected by monitoring Fluo-3 fluorescence levels in dye-loaded cells. Each trace represents mean values calculated from 10 cells. Arrows indicate the time-point at which 10nM
HG
NG
Figure 3.2.4.3. AVP-Stimulated Ca2+ Transients in A7r5 Cells Cultured in Normal & High Glucose Media (Pre-Treatment with Thapsigargin)
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↑
Figure 3.2.4.3. AVP-Stimulated Ca2+ Transients in A7r5 Cells Cultured in Normal & High Glucose Media (Pre-Treatment with
Thapsigargin)
Changes in [Ca2+] within A7r5 cells grown in either normal (“NG”) or high (“HG”) glucose-supplemented media were detected by monitoring Fluo-3 fluorescence levels in dye-loaded cells. Each trace represents mean values calculated from 10 cells. Arrows indicate the time-point at which 10nM
AVP was added. Cells were pre-treated with 1µM thapsigargin for 5 minutes before the addition of AVP. The thapsigargin response was recorded to ensure this pre-treatment was effective on the cells (but profiles
1.14 ± 0.13 for NG cells and 0.98 ± 0.20 for HG cells, which were found to not be statistically significant in their difference to each other according to Student’s T-test analysis (P>0.05).
On the other hand, figure 3.2.4.3 does show a difference in mean Ca2+- mediated responses to AVP after pre-treatment with thapsigargin between NG and HG cells, whereby their associated response profiles indicate both faster rise and fall phases of change in intracellular [Ca2+] were recorded following AVP stimulation in HG cells compared to NG cells. This difference was further analysed by measuring the kinetics of both phases from the response profiles shown in figure 3.2.4.3. Both the time taken to reach half-maximal response and half-life of decay in response to AVP were measured from each of the four mean response profiles and the mean values of these were calculated for each glucose culture condition, which are shown in table 3.2.4.4 with their S.E.M values. Student’s T-test analysis showed the
difference between NG and HG cells for both kinetics measurements were statistically significant (P<0.05).
The mean peak heights of change in F/F0 for these Ca2+-mediated AVP
responses were not found to be statistically significant (P>0.05), whereby values of 0.32 ± 0.06 and 0.39 ± 0.04 were obtained for NG and HG cells, respectively. However, by making the mean peak heights of change in F/F0 from thapsigargin pre-
treated cells relative to those from cells that were stimulated with AVP in the absence of thapsigargin, it can be shown that, on average, 28% and 40% of Ca2+-mediated response to this agonist is insensitive to thapsigargin treatment in NG and HG cells, respectively. These differences between the two AVP treatments were found to be statistically significant from Student’s T-test calculation for both NG (P<0.05) and HG (P<0.01) cells.
Two types of control experiments were done to further investigate where the AVP-stimulated Ca2+ release originated from following thapsigargin treatment. Only HG cells were used for these. All control experiments lasted 10 minutes each and, like the experiments discussed above, involved thapsigargin treatment 1 minute after initiating their recording. One experiment involved treating the cells again with 1µM thapsigargin four minutes after the first treatment, instead of AVP, in the absence of extracellular Ca2+. This showed whether or not thapsigargin-sensitive Ca2+ release was still possible after one treatment of A7r5 cells with 1µM of this SERCA inhibitor.