Phosphoinositide signalling in a neuroblastoma cell line.

In the studies described in this thesis, the ability of muscarinic agonists to initiate phosphoinositide metabolism, subsequently leading to the mobilisation of Ca2+ from intracellular stores was examined in permeabilised human neuroblastoma SH-SY5Y cells. Muscarinic receptors, determined as being of the M3 subtype, were found to possess different levels of receptor reserve for the production of the second messenger, inositol 1,4,5-trisphosphate (Ins(l,4,5)P3) and the mobilisation of Ca2+ in this permeabilised cell preparation. The effects of agonist pretreatment of intact cells with the muscarinic agonist carbachol has also been examined. Such pretreatment attenuated the ability of muscarinic agonists to elicit Ca2+ mobilisation in permeabilised cells. The rate of desensitisation was dependent on the dose of agonist used, the temperature at which pretreatment was performed and was affected by the extracellular Ca2+ concentration. The mechanism of such receptor-mediated desensitisation was studied. The structure-activity relationships of a number of inositol phosphates and inositol phosphate analogues have also been studied with regards their interactions with the Ca2+-mobilising Ins(l,4,5)P3 receptor, Ins(l,4,5)P3 5-phosphatase and Ins(l,4,5)P3 3-kinase. This work has identified partial agonists at the Ins(l,4,5)P3 receptor, an inhibitor of Ins(l,4,5)P3 3-kinase which interacts poorly with the Ins(l,4,5)P3 receptor, and a highly potent and selective inhibitor of Ins(l,4,5)P3 5-phosphatase. In conclusion, the results obtained characterise a permeabilised cell preparation in which receptor coupling to phospholipase C is maintained, leading to the formation of Ins(l,4,5)P3 and mobilisation of Ca2+ from intracellular stores, and identifies this as a useful model in which this coupling can be modulated by cell membrane-impermeant agents.