SISTEMAS DE CRESTAS PAPILARES DIGITALES

Expressing COS-7 Cells

Figure 4.2.4.1 shows the plot obtained from hSPCA1d membranes, which were treated with thapsigargin and thus values represent Ca2+-dependent ATPase activity specific to this thapsigargin-insensitive protein. Figure 4.2.4.1 also shows the plot obtained from hSERCA2b membranes without the presence of thapsigargin in their reaction mixtures. Both plots represent mean Ca2+-dependent ATPase activities measured from reactions that lasted for different durations of time. These were required to determine the optimal length of reaction time to use for the assay in order to measure any differences in activity when experimental conditions were altered, such as with the presence of potential inhibitors. From this, 45 minutes was selected as a suitable length of reaction time because it is within the linear range of values obtained for both hSPCA1d and hSERCA2b, which meant 45 minutes was enough time to both produce sufficiently measurable amounts of activity and not reach plateau due to substrate utilisation or product inhibition.

4.2.5. The Effects of Bis-phenol, TBBPA, TFP, 2-APB, BPA, cADPR & NAADP on Ca2+_{-Dependent ATPase Activities of Microsomal Membranes from}

hSPCA1d- & hSERCA2b- Over-Expressing COS-7 Cells

Plots of mean thapsigargin-insensitive Ca2+-dependent ATPase activities against different concentrations of bis-phenol (figure 4.2.5.1), TBBPA (figure 4.2.5.2), TFP (figure 4.2.5.3), 2-APB (figure 4.2.5.4), BPA (figure 4.2.5.5), cADPR (figure 4.2.5.6) and NAADP (figure 4.2.5.7) present in the reaction mixtures with hSPCA1d

membranes are shown. Plots of mean Ca2+-dependant ATPase activities (without thapsigargin treatment) against different concentrations of bis-phenol (figure 4.2.5.1), TBBPA (figure 4.2.5.2) and TFP (figure 4.2.5.3) measured from the present study are also shown for hSERCA2b membranes alongside their hSPCA1d counterparts. The data for 2-APB inhibition of hSERCA2b membranes was obtained from a previous study (Wootton and Michelangeli, 2006) (figure 4.2.5.4).

Figure 4.2.5.1. Effect of Different Bis-phenol Concentrations on Mean Ca2+_{-ATPase Activities From Microsomal Membranes of hSPCA1d- &}

hSERCA2b- Over-Expressing COS-7 Cells

Percentage activity was calculated from making activity measurements in the presence of bis-phenol relative to those of vehicle (DMSO) control. DMSO

content in all assays was 0.1% of their total volume. Mean activities were measured from three replicate assays for each bis-phenol concentration tested. Error bars indicate S.E.M values. Assay mixtures for “hSPCA1d”

membranes contained 1µM thapsigargin and those for “hSERCA2b” membranes did not contain thapsigargin.

Figure 4.2.5.2. Effect of Different TBBPA Concentrations on Mean Ca2+-ATPase Activities From Microsomal Membranes of hSPCA1d-

& hSERCA2b- Over-Expressing COS-7 Cells

Percentage Ca2+-dependent ATPase activities measured from hSPCA1d and hSERCA2b membranes in reaction mixtures that contained TBBPA at different concentrations. Mean activities were measured from three replicate assays for each TBBPA concentration tested and percentage activities calculated in the same way as stated for figure 4.2.5.1. Error bars indicate S.E.M values. Assay mixtures for hSPCA1d membranes contained 1µM thapsigargin and those for hSERCA2b membranes did

Figure 4.2.5.3. Effect of Different TFP Concentrations on Mean Ca2+-ATPase Activities From Microsomal Membranes of hSPCA1d-

& hSERCA2b- Over-Expressing COS-7 Cells

Percentage Ca2+-dependent ATPase activities measured from hSPCA1d and hSERCA2b membranes in reaction mixtures that contained TFP at

different concentrations. Mean activities were measured from three replicate assays for each TFP concentration tested and percentage activities calculated in same way as stated for figure 4.2.5.1. Error bars

indicate S.E.M values. Assay mixtures for hSPCA1d membranes contained 1µM thapsigargin and those for hSERCA2b membranes did

not contain thapsigargin.

Figure 4.2.5.4. Effect of Different 2-APB Concentrations on Mean Ca2+-ATPase Activities From Microsomal Membranes of hSPCA1d-

& hSERCA2b- Over-Expressing COS-7 Cells

Percentage Ca2+-dependent ATPase activities measured from hSPCA1d and hSERCA2b membranes in reaction mixtures that contained 2-APB at

different concentrations. Mean activities were measured from three replicate assays for each 2-APB concentration tested and percentage activities calculated in same way as stated for figure 4.2.5.1. Error bars

indicate S.E.M values. Assay mixtures for hSPCA1d membranes contained 1µM thapsigargin and those for hSERCA2b membranes did

not contain thapsigargin. The data for hSERCA2b membranes was obtained from a previous study (Wootton and Michelangeli, 2006)

Figure 4.2.5.5. Effect of Different BPA Concentrations on Mean Ca2+_{-ATPase Activities From Microsomal Membranes of hSPCA1d-}

Over-Expressing COS-7 Cells

Percentage activity was calculated from making activity measurements in the presence of BPA relative to those of vehicle (DMSO) control in the

same way as stated for figure 4.2.5.1. Mean activities were measured from three replicate assays for each BPA concentration tested. Error bars indicate S.E.M values. 1µM thapsigargin was added to reaction mixtures.

Figure 4.2.5.6. Effect of Different cADPR Concentrations on Mean Ca2+_{-ATPase Activities From Microsomal Membranes of hSPCA1d-}

& hSERCA2b- Over-Expressing COS-7 Cells

Percentage Ca2+-dependent ATPase activities measured from hSPCA1d and hSERCA2b membranes in reaction mixtures that contained cADPR at different concentrations. Mean activities were measured from three replicate assays for each cADPR concentration tested and made relative

to those of vehicle (dH20) controls to calculate percentage activities.

Error bars indicate S.E.M values. Assay mixtures for hSPCA1d membranes contained 1µM thapsigargin and those for hSERCA2b

Figure 4.2.5.7. Effect of Different NAADP Concentrations on Mean Ca2+-ATPase Activities From Microsomal Membranes of hSPCA1d-

Over-Expressing COS-7 Cells

Percentage activities were calculated in the same way as stated for figure 4.2.5.6. Mean activities were measured from three replicate assays for each

NAADP concentration tested. Error bars indicate S.E.M values. 1µM thapsigargin was added to reaction mixtures.

IC

Values of hSPCA1d & SERCA Inhibitors