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Title: Functional consequences of inositol 1,4,5-trisphosphate receptor overexpression on the phosphoinositide signalling cascade
Author: Davis, Richard Jon
Awarding Body: University of Leicester
Current Institution: University of Leicester
Date of Award: 1999
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The ubiquitously expressed inositol 1,4,5-trisphosphate receptor (InsP3R) is a crucial component in the process of signal transduction, encoding an extracellular stimulus into an intracellular Ca2+ signal. In this study, mouse L-fibroblast cells, transfected with either type 1 (InsP3R) cDNA (L15) or the vector control (Lvec) were used to investigate the functional consequences of increased InsP3R density and isoform specificity on phosphoinositide-linked receptor signalling. L15 cells study overexpress ~8 fold the levels of the type 1 InsP3R, than Lvec cells, which endogenously express essentially only the type 1 InsP3R protein. L-cells were found to endogenously express H1-histamine receptors, LPA receptors and P2Y(2) -nucleotide receptors. P2Y(2) receptors were coupled via a PTX-insensitive G-protein to phosphoinositidase C activation, resulting in identical time- and concentration-dependent accumulation of Ins(1,4,5)P3 in both Lvec and L15 cells. These data suggest changes in InsP3R number do not influence upstream components of this signalling cascade. However the consequent release of Ca2+ from intracellular stores was substantially augmented in L15 cells at all comparable agonist concentrations. Single cell microfluorimetry revealed that L-cells produced complex sinusoidal cytosolic Ca2+ oscillations in response to submaximal UTP concentrations. InsP3R overexpression reduced the threshold of receptor-mediated Ca2+ signalling and modulated the amplitude, frequency and spatial orientation of cytosolic Ca2+ oscillations. Intact and permeabilised cell studies revealed that L15 cells exhibited a substantially larger InsP3-sensitive Ca2+ store and enhanced the potency of Ca2+ mobilisation for a wide range of InsP3 analogues by 2-4 fold. Stop-flow kinetic measurements revealed the rate and extent of bi-directional InsP3-induced Ca2+ release was substantially increased (~2 fold). Taken together, these data could contribute to the amplification of Ca2+ signalling exhibited by InsP3R overexpression. Sustainable oscillatory responses were dependent on extracellular Ca2+ entry, although this variable was not influenced by InsP3R overexpression.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available