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Title: Analysis of the mechanisms responsible for the generation and decoding of intracellular calcium signals
Author: Bradley, J.
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 1997
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In many cells, including hepatocytes, receptors coupled to phosphoinositide hydrolysis stimulate oscillatory changes in cytosolic [Ca2+] ([Ca2+]i). I have used two methods to examine the means whereby such [Ca2+]i transients are linked to the stimulation of glycogenolysis. By perifusing rat hepatocytes prelabelled with [3H]-glucose, I showed that phenylephrine increases the rate of [3H]-glucose release by 2-3 fold, and confirmed this effect using a colorimetric assay for glucose. When cultured hepatocytes were loaded with Fura-2 and incubated with thapsigargin in a Ca2+-free medium, restoration of external Ca2+ caused Ca2+ entry. Brief pulses (10s or 30s) of extracellular medium containing Ca2+ (5mM), produced [Ca2+]i transients comparable to those observed in single hepatocytes treated with Areg8-vasopressin (duration = 1.0 -1.5min). Using a similar protocol to manipulate [Ca2+]i in hepatocytes prelabelled with [3H]-glucose, I demonstrated that single [Ca2+]i transients stimulate a 2-fold increase in [3H]-glucose release (n=4). Moreover, stimulated glucose release outlasted the transient elevation of [Ca2+]i having a duration of 7.4 ± 0.6min (n=4). In conclusion, stimulation of glucose release by imposed transients of [Ca2+]i in a population of hepatocytes indicates that the discontinuous signal of [Ca2+]i transients can be decoded to produce a continuous response. Further characterisation of this decoding process requires single cell measurements of glucose and [Ca2+]i. Such measurements are not yet possible with the presently available techniques for enzyme immobilisation on glucose microbiosensors. Overexpression of type I and type III InsP3 receptors results in cell death, the mechanism of which requires further study. It is possible, however, to induce the expression of the type I InsP3 receptor and the effects of increasing InsP3 receptor level on elementary Ca2+ release events should provide insight into the mechanism by which [Ca2+]i waves are generated.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available