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|Title:||Molecular mechanisms of lithium action on phosphoinositide signalling.|
|Presented at:||University of Leicester|
|Abstract:||The work described in this thesis examines the phosphoinositide (PI) signalling system and its disruption by the anti-manic agent lithium. The effects of lithium upon the accumulation of labelled and unlabelled inositol (poly) phosphates in muscarinic cholinoceptor-stimulated rat brain slices and Chinese hamster ovary (CHO) cells expressing the human M1-muscarinic receptor subtype (CHO-Ml) were examined. Similarly, the effects of this ion on other intermediates of this second messenger signalling system were examined in order to give an overall picture of the action of lithium. These included the accumulation of CMP-phosphatidic acid (CMP-PA), a precursor of the (poly) phosphoinositide lipids, and the agonist-stimulated levels of the (poly) phosphoinositide lipids. Initial experiments examined phosphoinositide metabolism in cortex, hippocampus and striatum to determine whether there were regional variations in both this signalling system and the effects which lithium had upon it. Both cortical and hippocampal PI metabolism were similar, however, striatum was significantly different such that in the continued presence of agonist this region was unable to maintain the initial elevated levels of Ins(l,3,4,S)P4, unlike the other regions examined. Lithium appeared to have a similar disruptive effect on PI metabolism in all regions, with statistically similar EC50 values for the accumulation of InsP1 in all regions. The effects of lithium upon PI metabolism stimulated by a variety of different agonists was examined to determine whether the action of lithium was agonist dependent Lithium appeared to have a similar disruptive effect upon PI metabolism stimulated by these various agonists. Studies examining the effects of lithium upon the carbachol-stimulated accumulation of the inositol (poly) phosphate isomers revealed the presence of a lithium-sensitive accumulation of the inositol bisphosphate Ins(4,5)P2. This study was unable to determine the source of this isomer, however, the formation of this isomer in both cerebral cortex slices and CHO cells suggests the possibility that Ins(4,5)P2 may be a dephosphorylation product of Ins(l,4,5)P3. The lithium-sensitivity of the accumulation of this isomer also suggests that a novel Uthium- sensitive 4- or 5-phosphatase activity may be present in these preparations. The effects of lithium on phosphoinositide metabolism in CHO cells expressing the human M1-muscarinic receptor subtype was also examined to determine whether this cell line would represent a suitable model of cerebral PI metabolism. It was hoped that the use of this cell-line would result in clearer more interpretable data. Indeed, a definitive analysis of PI metabolism in this cell line clearly demonstrated that the addition of lithium to agonist-stimulated cells resulted in a decrease in the intracellular myo-inositol reserves within the cell which resulted in a decrease in the (poly)phosphoinositide precursor of Ins(l,4,5)P3, PtdIns(4,5)P2. In turn this resulted in a time-dependent decrease in the agonist-stimulated levels of Ins(l,4,5)P3 after a lag period of 5-10 min, similar to that observed in cerebral cortex slices. The data demonstrate that the CHO-Ml cell-line is a valuable tool in elucidating the actions of lithium upon PI signalling. In conclusion, the results described in this thesis clearly demonstrate the profound effects that this monovalent ion has on phosphoinositide signalling in the preparations examined. The main action of lithium appears to be the inhibition of the inositol monophosphatase, however, this agent may also have other more subtle effects upon this complex system. These possibilities and their implications are also discussed.|
|Rights:||Copyright © the author. All rights reserved.|
|Appears in Collections:||Theses, Dept. of Biochemistry|
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