| Popis: |
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 |
