Hall, C.N.; (2005) Nitric oxide inactivation in the brain. Doctoral thesis , University of London.

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Abstract

Nitric oxide (NO) is a signalling molecule in the central nervous system and other tissues. NO synthesis and its immediate targets are well-characterised moleculaiiy, but little is known about how the NO signal is terminated. Recent work has suggested the existence of a biological sink for NO in dispersed brain tissue. This study aimed to discover the kinetic properties of NO inactivation in brain and to elucidate the mechanism(s) underlying this process. Measurements of cGMP accumulation in brain slices from cerebellum indicated that intact brain inactivates NO. When analysed using a model of NO diffusion and inactivation, the experimental data were consistent with the maximum rate of inactivation being fast (over 1 uM/s). It was inferred from the kinetics that biological inactivation of NO would predominantly affect NO signalling when several sources of NO are concurrently active. The mechanism of NO consumption initially studied in dispersed brain tissue has since been shown to be the reaction of NO with lipid peroxyls, a process that may have relevance to pathophysiology. Pharmacological and other tests showed that the breakdown of NO by cerebellar slices was, however, independent of this mechanism. Further manipulations also eliminated other potential routes of NO metabolism (reaction with red blood cells or superoxide, or autoxidation) as underlying causes. Lipid peroxide-independent NO inactivation was also found in acute and cultured cerebellar cells. The reaction exhibited marked oxygen- dependence. In dilute preparations, NO metabolism was largely lost on cell lysis, but was recoverable by addition of the electron donor NADPH. This activity resided in the membrane fraction following high speed centrifugation. Inactivation of NO in NADPH-treated lysates or membrane fractions, and in intact cells, was partially inhibited by cyanide. This evidence suggests the involvement of membrane-bound haem and flavoproteins. In concentrated tissue preparations and intact brain slices, however, an alternative process becomes dominant. Which of these mechanisms is most important physiologically requires further investigation. Their relative distributions with respect to the sites of NO release may be critical.

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