Hepatoma Tissue Culture (HTC) cells grown in the presence of 60µM arachidonic acid for 24, 36 and 48 hours became progressively more thermosensitive than control cells. However, this difference in thermal sensitivity was only detectable with the clonogenic assay and not with the colorimetric assay. Attempts were also made to manipulate cellular cholesterol levels. Firstly, some cells were incubated with phosphatidylcholine liposomes to deplete the plasma membrane of cholesterol: Secondly, another group of cells were treated with 25 hydroxycholesterol, an inhibitor of cholesterol synthesis, to lower cholesterol levels: Finally, a third group of cells were supplemented with cholesterol hemisuccinate, a hydrophilic ester of cholesterol. The first two approaches did not enhance the thermal sensitivity of HTC cells. Supplementation with cholesterol hemisuccinate, which was predicted to partition in to the plasma membrane and reduce membrane fluidity, resulted in increased thermal sensitivity of the cells. Thus, the thermal sensitivity of HTC cells could be enhanced by supplementation with either arachidonate or cholesterol hemisuccinate. A rapid plasma membrane isolation procedure was developed which generated plasma membranes in relatively high yield and purity. The plasma membrane- enriched fraction was also assayed for contaminating intracellular membranes by determining marker enzyme activities associated with these membranes. Using this method, plasma membranes were prepared from HTC cells grown m 60µM arachidonic acid for 36 hours and from cells grown in normal medium. Analysis of the plasma membrane showed that the arachidonic acid content of the phospholipid fatty acyl groups had been significantly increased in cells grown in the presence of this fatty acid. There was no change in the cholesterol/phospholipid molar ratio or cholesterol concentration relative to amount of protein in the plasma membranes from the two cell populations. The measurement of fluidity using DPH fluorescence polarisation revealed that the increase in the arachidomc acid content of the plasma membrane phospholipid acyl groups was associated with enhanced plasma membrane fluidity when compared to control plasma membranes. This increase in plasma membrane fluidity correlated with the enhanced thermal sensitivity of the cells grown in arachidonic acid-containing medium when compared to cells grown in normal medium. Furthermore, the thermal sensitivity of Na(^+), K(^+) –ATPase and alkaline phosphodiesterase I were assessed in plasma membranes derived from arachidonic acid-supplemented and control cells. The enhanced fluidity of plasma membranes derived from arachidonate-supplemented cells also correlated with increased thermosensitivity of alkaline phosphodiesterase I.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:316356 |
| Date | January 1990 |
| Creators | Ladha, Shabirali |
| Publisher | Durham University |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://etheses.dur.ac.uk/6197/ |
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