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Title: Regulation of Ca2+ efflux from the intracellular stores of permeabilized hepatocytes
Author: Beecroft, M. D.
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 1998
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I have used rat hepatocytes to examine the characteristics of Ca2+ uptake into intracellular stores and the leak from them, the mechanism underlying Ins(1,4,5)P3-induced quantal Ca2+ release, and interactions between synthetic ligands and Ins(1,4,5)P3 receptors. At 37°C, inhibition of ATP-dependent Ca2+ uptake caused a monoexponential decline in Ca2+ content of the stores but failed to reduce it to the level observed before uptake occurred. Reducing the temperature decreased the rate, but not the extent, of passive Ca2+ efflux. Passive efflux was not regulated by ATP or cytosolic Ca2+; nor mediated by either the Ca2+ pump or the Ins(1,4,5)P3 receptor; but it did appear to be mediated by an ion channel. I conclude that luminal Ca2+ regulates the rate of passive efflux from intracellular stores, terminating the leak when the luminal Ca2+ concentration falls below a critical level. Ins(1,4,5)P3 induced quantal Ca2+ release at both 37°C and 2°C, albeit more slowly and at lower concentrations of Ins(1,4,5)P3 at 2°C. At 2°C, comparable amounts of Ca2+ were released whether Ins(1,4,5)P3 was added in two sequential submaximal doses, or as a single addition of the sum of the doses. Quantal responses to Ins(1,4,5)P3 were more rapidly completed from stores that had been partially depleted of Ca2+. I conclude that quantal Ca2+ release is due to an all-or-none emptying of discrete Ca2+ stores with heterogeneous sensitivities to Ins(1,4,5)P3. In 45Ca2+ flux experiments, adenophostin was shown to be a potent agonist of hepatic Ins(1,4,5)P3 receptors, with an affinity ˜ 10-fold greater than Ins(1,4,5)P3. Analysis of the effect of a range of synthetic adenophostin analogues in both functional and radioligand binding assays, suggests that the adenine moiety of adenophostin may contribute to its enhanced affinity. The EC50/Kd ratios of Ins(1,4,5)P3 and all synthetic analogues calculated from radioligand binding (pH 8.3, 2°C), and 45Ca2+ flux (pH 7.0, 37°C) experiments were similar, with the exception of the compound (2S)-9-{1-(α-D-glucopyranosyl 3,4-bisphosphate)-2-monophosphate-prop-3-yl}-adenine (acyclophostin), which displayed a ratio ˜ 4-fold greater than Ins(1,4,5)P3.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available