Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.649739
Title: Catecholamine release from isolated chromaffin cells under conditions of anoxia or metabolic inhibition
Author: Dry, Katherine L.
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 1990
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Abstract:
A significant release of catecholamines within the heart has been observed during myocardial ischaemia. Ischaemia-induced catecholamine release can be markedly inhibited by desipramine and other amine uptake blocking agents, allowing investigation of the importance of such release for arrhythmia production. The mechanism of this release appears to occur by a carrier-mediated efflux from neurones, which is not operative under normal conditions. The aim of the project has been to study this release process in chromaffin cells isolated from the bovine adrenal medulla, which are recognised as a model system for studying the sympathetic nervous system. Understanding this process of catecholamine release may lead to new methods of protecting the heart against ischaemia-induced arrhythmias. Isolated chromaffin cells could be maintained in primary culture for up to 7 days and retained all their normal secretory responses. Conditions designed to mimic ischaemia, that is, anoxia or metabolic inhibition, resulted in a significant release of catecholamines. This release was shown to be independent of extracellular calcium but, in contrast to the release observed in ischaemic hearts, it was not inhibited by uptake1 blockers. One of the main criteria for exocytosis is the co-release of other secretory granule components. Polyacrylamide gel electrophoresis and Western blotting techniques were utilised to examine this following metabolic inhbition. Significant levels of the granule proteins chromogranin A, neuropeptide Y and ATP were measured following metabolic inhibition, indicative of an exocytotic mechanism. Furthermore, there was no release of the cytosolic protein lactate dehydrogenase, indicating that there was no breakdown of the cell membrane during metabolic inhibition. Over the first 10 minutes of metabolic inhibition there was a marked uptake of 22Na+ by the cells. It is suggested that this Na+ influx triggers the catecholamine release by affecting the cytosolic Ca2+ concentration through a direct effect on intracellular stores. Intracellular Ca2+ mobilisation was measured using the calcium-sensitive fluorescent probe Fura-2. It was found that cytosolic free calcium levels were increased in response to metabolic inhibition. The conditions requird to evoke carrier-mediated efflux were also examined. Cytosolic levels of catecholarmines could be artificially raised following treatment with reserpine. Cytoplasmic catecholamine levels were measured following permeabilisation with the detergent digitonin which renders the plasma membrane leaky. Conditions designed to reverse the uptake carrier and cause carier-mediated efflux in the presence of raised cytoplasmic catecholamines such as removal of extracellular sodium, did not evoke any catecholamine overflow. These studies suggest the chromaffin cell uptake1 carrier is not reversible and may be gated in some way. This work has, therefore, raised questions concerning the suitability of chromaffin cells as a conventional model for sympathetic nerve terminals.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.649739  DOI: Not available
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