The role of phosphatidic acid in astrocyte intracellular signalling

Jakobson, Katherine (1991). The role of phosphatidic acid in astrocyte intracellular signalling. PhD thesis The Open University.



1. Astrocyte-enriched cultures prepared from the neonatal rat cerebral cortex were used to assess the effects of putative mitogens on astroglial proliferation. Subconfluent cultures, 11 div, were deprived of serum, thus halting cell division, and then exposed to these agents. 10% v/v foetal 3 calf serum elicited increases in [ H]thymidine incorporation into astrocyte DNA ranging from 245% to 434% over control. Insulin elicited a dose-dependent increase in [3H]thymidine incorporation (EC50 = 16y(Uml-1 ). Platelet-derived growth factor and phosphatidic acid had a biphasic effect on 3 [ H]thymidine incorporation apparently due to the presence of high concentrations of their respective vehicles, ethanediol and CHCl3:MeOH (1:2 v/v), at the higher mitogen concentrations tested.
2. The effects of platelet-derived growth factor and 3 phosphatidic acid on [3H]thymidine incorporation were dependent upon the age of the cultures used. For example, at 5 div platelet-derived growth factor reduced incorporation whereas phosphatidic acid increased incorporation compared to control.
3. The phosphatidic acid mitogenic signal could be amplified from a 13% to 43% increase over control by increasing the time of serum depletion from 24h to 48h. The mitogenic effect of phosphatidic acid was dependent upon the presence of long chain fatty acids.
4. Prolonged phorbol ester pretreatment, abolished the mitogenic action of phosphatidic acid on astrocytes and also reduced that of 10% v/v foetal calf serum by 41%.
5. In order to determine whether the mitogenic effects of phosphatidic acid involved phosphoinositide turnover cultures prelabelled with [3H]inositol were exposed to phosphatidic acid and inositol phosphates accumulation and inositol phospholipid labelling monitored. Phosphatidic acid elicited a dose- and time-dependent increase in the accumulation of total and individual labelled inositol phosphates which was dependent upon a low extracellular calcium concentration and phosphatidic acid containing long chain fatty acids.
6. Phosphatidic acid stimulated changes in inositol phospholipid labelling and 45Ca++ efflux and both effects were dependent on phosphatidic acid containing long chain fatty acids.
7. Phosphatidic acid had no detectable effect on phosphoinositide turnover but did stimulate 45Ca++ efflux from fibroblasts.
8. The possible release of phosphatidic acid from agonist-stimulated astrocytes was studied by exposing [3H]arachidonic acid prelabelled cultures to carbachol, ATP and noradrenaline. All three agonists increased the formation of labelled phosphatidic acid but they did not evoke phosphatidic acid release from astrocytes.
9. R59 022, a diacylglycerol kinase inhibitor, inhibited carbachol-stimulated phosphatidic acid production and 45Ca++ efflux but had no effect on carbachol-induced phosphoinositide turnover.

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