Aspects of the distribution of Na⁺,K⁺ ATPase in cultured cells with particular reference to the effects of fluid shear stress
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A two-part study was conducted into aspects of the distribution of Na⁺,K⁺ATPase α1 and β-1subunits in HeLa and bovine aortic endothelial cells (BAECs). The primary experimental emphasis was to examine Na⁺,K⁺ATPase distribution in relation to cell type, cell morphology and age using immunocytochemical, Western blot analysis, 3H ouabain assays and various cell permeabilisation techniques. This forms Part I of the study. Part II of the study focussed on the potential up/down regulation in addition to changes in Na⁺,K⁺ATPase distribution throughout the cytoskeleton under the influence of laminar fluid shear stress. Initially aspects of HeLa cell morphology were examined using anthroylouabain, a fluorescence derivative of the cardiac glycoside ouabain. It was discovered that affinity for ouabain in HeLa cells decreases for cells that are actively dividing whereas confluent cells exhibit a 3-fold increase in sensitivity to ouabain. Initial findings by Lamb (1996) showed that the dissociation constant Kd for ouabain binding to the sodium pump of HeLa cells is dependent on cell morphology. Results of this study show that rounder cells take up more anthroylouabain compared to flatter cells. Laser scanning confocal microscopy reveals this was not an artefact of differing depth of cells, but a real difference of ca. 5-fold. Further studies examined BAECs in addition to HeLa cells in order to compare the quantity of ouabain binding between young and old cells. The results demonstrate that both HeLa cells and BAECs used for experimentation at Day 4 after plating have similar levels of ouabain binding, whereas BAECs harvested at Day 14 show less ouabain binding in comparison to younger cells (one-way ANOVA F₂.₅₆=8.60, p<0.001). Different pretreatments were used before BAEC cells were stained for the α -1 subunit of the Na pump and actin. These were permeabilising with SDS (sodium dodecylsulphate) and hypotonically rupturing the cell membrane. The aim was to expose antigenic sites in intra-cellular compartments leading to the augmentation of α -1 subunit staining. Results of this experiment showed α -1 subunits a similar distribution pattern to actin. However it is difficult to state with certainty that there is an actual co-localisation. BAECs were subjected to steady laminar shear stresses using specialised parallel plate chambers of 15 and 50 dyn cm⁻². Immunocytochemical techniques were used to reveal any physical differences in pump distribution after exposure to flow stresses. In addition Western blot analysis was used to explore possible up or down regulation of the α -1 subunit. Results indicate that under a flow stress of 50 dyn cm⁻² for 4 hours there is a marked change in sodium pump distribution from the cell periphery to the cytoplasm. In addition, there are concurrent and specific morphological changes in BAECs i.e. reorganisation and alignment of actin stress fibres with the direction of flow. Protein estimations derived from gel densitometry analysis of ECLs for cells sheared at 15 and 50 cm⁻²over a time course experiment showed no significant difference with either time or flow stress (two-way ANOVA without replication: df=l, F=32.85 p=0.001). Additional experiments were designed to expose cells to the same shear magnitude but then incubate them for 8 hours at 37°C to look for down or up-regulation of sodium pump protein over time. Shearing under a flow stress of 50 cm⁻²produced a characteristic bell-shaped distribution over time. Statistical analysis showed a significant difference with time and flow stress (two-way ANOVA with replication df=7, F=4.47, p=0.033) indicating possible up-regulation of pump protein after incubation.
Thesis, PhD Doctor of Philosopy
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