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Title: Regulation of inositol polyphosphate metabolism in airways smooth muscle
Author: Lynch, Barbara Jane
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 1995
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Agonist-induced contraction of airways smooth muscle is mediated by phosphoinositide hydrolysis and the production of the second messenger inositol 1,45-trisphosphate (Ins(1,4,5)P3). Muscarinic receptor-stimulation of bovine tracheal smooth muscle (BTSM) results in a transient increase in Ins(1,4,5)P3 mass despite a sustained, non-desensitising hydrolysis of its precursor phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2). Hence the rapid metabolism of Ins(1,4,5)P3 appears to be the major regulator of Ins(1,4,5)P3 levels under agonist-stimulated conditions. A model system has been developed to facilitate detailed study of the pathways involved in this complex metabolism. BTSM slices were labelled to equilibrium with myo-[3H]inositol in the presence of agonist and subsequent carbachol (CCh)- or histamine (Hist)-stimulations carried out in the presence of lithium ions to block InsP1 breakdown. A delayed accumulation of [3H]Ins1/3P and [3H]Ins4P was observed under agonist-stimulated conditions. Moreover, there was no demonstrable phosphoinositide hydrolysis either following membrane-depolarisation, or secondary to a physiologically relevant increase in intracellular calcium in this thesis. The model therefore provides an appropriate system for the study of receptor-stimulated PtdIns(4,5)P2-derived Ins(1,4,5)P3 metabolism. Cell-free experiments confirmed that Ins(1,4,5)P3 is metabolised primarily by two different pathways - a 3-kinase and a 5-phosphatase pathway - which yield mutually exclusive products. H.P.L.C. separation of the individual [3H]inositol polyphosphate (InsPP) isomers accumulating in BTSM slices enabled the 3-kinase and 5-phosphatase metabolites to be quantified, and facilitated the determination of flux of the inositol headgroup through these two pathways. The pattern of Ins(1,4,5)P3 metabolism varies during the lifetime of the agonist-stimulated response. The 5-phosphatase enzyme is highly dominant especially at early time-points following agonist-stimulation, whilst the 3-kinase becomes increasingly important at later time-points.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available