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Title: Examination of mitochondrial and cellular response to metabolic stresses : changes in mitochondrial membrane potential by modulators of ischaemic preconditioning and metabolic stress-induced extracellular nucleotide accumulation
Author: Thompson, Christopher
Awarding Body: University of Liverpool
Current Institution: University of Liverpool
Date of Award: 2013
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Cardiac and cerebral ischaemia can induce cellular hypoxia which results in injury potentially via apoptosis. In 2004, this accounted for 12.9 million (21.9%) of the world’s deaths. Brief periods of ischaemia are known to protect cells from a subsequent and more sustained ischaemic event. This action is known as ischaemic preconditioning (IPC). Pharmacological agents can be used to manipulate IPC. Diazoxide is well documented to promote IPC, while 5-hydroxydecanoyl (5-HD) abolishes diazoxide-induced IPC. The mitoKATP channel, the sarcoKATP channel or succinate dehydrogenase are implicated in facilitating IPC. Actions of these agents are likely to converge on mitochondrial function. Also, during hypoxia it is widely reported that extracellular ATP becomes elevated. Whilst the activity of ATP via P2 signalling is well documented along with the activity of adenosine via A receptors, little attention has been paid to extracellular ADP or potential P2Y signalling arising from hypoxia. The aim of this study was to: 1. Establish the influence of diazoxide and 5-HD on mitochondrial membrane potential (m). 2. Investigate the generation of extracellular nucleotides during hypoxia. 3. Generate a cardiomyocyte (CM) model in which IPC and hypoxia-related nucleotide modulation could be characterised. Methods. To measure m, tetramethylrhodamine, ethyl (TMRE) signalling was measured using a microplate reader. Hypoxia was replicated by metabolic poisoning (4 mM cyanide, 10 mM 2-deoxyglucose (2-DG) and 5 μM ionomycin) and the resulting extracellular nucleotide was measured using ATP monitoring reagent (AMR, ViaLight) and a Berthold tube luminometer (LB955). HL-1 cells and primary chick CMs were examined to determine if they could be used as a contractile, cultured myocyte model. Results. The IPC-inducing agent, diazoxide, induced significant depolarisation (F/F0=0.098, auc. 7.86±1.0%) and surprisingly so did the antagonist, 5-HD (F/F0=0.046, auc. 3.13±0.6%). When used in combination, 5-HD negated the substantial diazoxide induced depolarisation (F/F0=0.036, auc. 5.08±0.7%). After metabolic poisoning, the observed extracellular ATP was elevated from 5±1 nM to 60±6 nM. The ADP concentration was much greater than the observed ATP and was also elevated from 259±31 nM to 4202±394 nM during chemical induced hypoxia (CIH). Oxidised ATP (oATP) reduced the extracellular ATP and ADP concentration by 53±16% and 40±32%, respectively. Primary chick CMs offered a more appropriate CM model compared to HL-1 cells. Chick CMs displayed contractile activity and positive CM specific antibody staining. Discussion. The data is consistent with a diazoxide-induced depolarisation arising from potassium channel modulation, while 5-HD appears to modulate m as a metabolic agent rather than as a channel inhibitor. The presence of relatively large concentrations of extracellular ADP after CIH, suggest that P2Y1,6,11 signalling may be significant in hypoxia. The extracellular nucleotides appear to arise not from ecto-enzyme activity or connexin release but via an oATP-inhibited mechanism. This suggests a potential linkage with P2X7 receptors. Isolated chick primary CMs produced a beating myocyte culture expressing cardiomyocyte markers. This model system has the potential to be used with recombinant probes in order to monitor cell and mitochondrial function during hypoxia.
Supervisor: Simpson, Alec Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral