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Title: Dynamics of NO-cGMP signalling in brain tissue
Author: Griffiths, Charmaine Helen
Awarding Body: University of London
Current Institution: University College London (University of London)
Date of Award: 2001
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Nitric oxide (NO) is established as a signalling molecule in the CNS. The kinetics of production and the mechanism of inactivation of NO have received surprisingly little attention despite their importance for determining the concentration and, consequently, the actions of this molecule. In this study, the activity of neuronal nitric oxide synthase (nNOS) and the inactivation of NO were investigated in various brain tissue preparations. Activation of the glutamate-NO-cGMP pathway was monitored by measuring cGMP levels. In brain slices NOS activity was responsible for increased cGMP production following NMDA application, and in a model of ischaemia. The profile of cGMP during simulated ischaemia and reperfusion resembled closely that found in vivo and the changes were attenuated by inhibition of cellular proteins implicated in neurodegeneration, namely nNOS, NMDA receptors and voltage-gated Na+ channels. cGMP was also observed in the brain slice incubation medium, suggesting that this intracellular second messenger may also have extracellular functions. To quantify the NO produced during simulated reperfusion, nNOS activity and the accumulation of NO breakdown products were measured. However, NO production was not detected. Similarly, NMDA application evoked large cGMP increases in the absence of detectable NO. From these data it was estimated that less than 3 % of the cerebellar slice nNOS content became active during this stimulation. The consumption of NO by brain tissue was then addressed. Using cerebellar cell suspensions and NO donors, two functionally distinct 'NO sinks' were observed. The first translated a constant rate of NO production into a rapidly-achieved steady-state NO concentration sufficiently low to engage physiological signalling events such as sGC activation, without causing potentially pathological effects such as respiratory chain inhibition. The second sink slowed the rise of NO concentration in the face of continuous NO production. Additionally NO inactivation in different perfused rat tissue homogenates varied with total haem content, implicating cellular haem in bioinactivation of NO. The interplay between the rates of NO synthesis and breakdown is likely to be a critical determinant of whether NO acts as a signalling molecule or a toxin.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available