The Role of Arachidonic Acid and Its Metabolites in Controlling Inositol Lipid Metabolism in Swiss 3T3 Cells

Newlands, Christine A (1988) The Role of Arachidonic Acid and Its Metabolites in Controlling Inositol Lipid Metabolism in Swiss 3T3 Cells. PhD thesis, University of Glasgow.

Abstract

Swiss 3T3 fibroblasts were chosen as a model system to study the role of arachidonic acid and/or its metabolites in controlling sermuM- and PDGF-induced inositol lipid metabolism. (0.01 - 10 muM) caused a rapid stimulation of inositol phospholipid hydrolysis which was maximal within 1-2 minutes of stimulation with the agonist. The level of Ptdlns was not significantly affected by PGF2a. The time course of [3H]-InsP accmuMulation suggests that InsP was not produced directly from Ptdlns, but was formed by the dephosphorylation of InsP2 and InsP3 at least within 1 minute after the PGF2a addition. 1.6 muM PGF2a caused a two-three fold increase in [3H] -PtdA levels, but did not affect the levels of any other major phospholipid measured. Having shown that PGF2a stimulated inositol lipid metabolism in 3T3 cells, it was postulated that the formation of prostaglandins from AA might be intermediatory in the action of PDGF on inositol phospholipid hydrolysis. In order to prove this, it was necessary to be able to inhibit their formation. Surprisingly, the cyclooxygenase inhibitors tested, (flurbiprofen and indomethacin), on their own caused a stimulation of inositol lipid metabolism. This was observed as an increase in [3H]-InsP levels and in [3H]-PtdA levels. The inhibitors had no effect on the levels of any other major phospholipid. The effect of flurbiprofen on inositol lipid metabolism was shown to be dose-related, with 100 muM flurbiprofen producing the maximmuM stimulation. By inhibiting cyclooxygenase activity, it seemed possible that flurbiprofen might have re-directed free AA towards the lipoxygenase pathway and thus the possibility that a stimulatory lipoxygenase metabolite was responsible for the increase in inositol phospholipid hydrolysis was investigated. BW755C, an inhibitor of both cyclooxygenase and lipoxygenase also caused a stimulation of inositol lipid metabolism, in a dose-related manner. The effect was maximal with 100 muM BW755C. Although it remains possible that BW755C did not inhibit all the 1.o. enzymes, it seemed more likely that the stimulation of inositol phospholipid hydrolysis was due to AA itself. Further evidence which suggested this to be the case was that flurbiprofen and BW755C, in a dose-dependent manner caused an increase in the level of free [3H]-AA in the medimuM surrounding the cells. The drugs also caused an increase in the level of [3H] -arachidonyl PtdA. Exogenously applied AA increased the level of [3H]-InsP in a dose-related manner which was maximal with 100 muM AA. This concentration of AA was thought too high to be of physiological significance. However, one contributing factor here was that the majority of the exogenously applied AA bound to albmuMin in the BSS surrounding the cells. In albmuMin-free BSS 0.1 muM AA gave almost an equivalent increase in InsP formation as 100 muM AA in cells surrounded by albmuMin-containing BSS. Also, in experiments where the effects of BW755C and indomethacin on [3H]-AA release and on [3H]-arachidonyl PtdA formation were studied, the results were consistent with the idea that endogenously released AA was binding to albmuMin in the BSS surrounding the cells. Further support for the theory that AA itself stimulated inositol lipid metabolism in 3T3 cells was that flurbiprofen and BW755C were not additive with exogenously applied AA in stimulating inositol phospholipid hydrolysis. This suggests that they have the same pathway of action. Studies were then carried out to investigate the pathway by which AA was liberated. Melittin, a PLA2 activator, stimulated both AA release and inositol lipid metabolism in a dose-related manner. Dexamethasone, an indirect inhibitor of PLA2 abolished the BW755C- and flurbiprofen-induced increase in inositol lipid metabolism in a dose-related manner. These results suggest that the pathway is involved in the liberation of AA from the membrane phospholipids in 3T3 cells. (Abstract shortened by ProQuest.).

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Keywords: Pharmacology
• Date of Award: 1988
• Depositing User: Enlighten Team
• Unique ID: glathesis:1988-77630
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 14 Jan 2020 11:53
• Last Modified: 14 Jan 2020 11:53
• URI: https://theses.gla.ac.uk/id/eprint/77630