Lam, Amanda; (2004) Tetrahydrobiopterin and nitric oxide metabolism. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Tetrahydrobiopterin (BH4) is an essential cofactor for all isoforms of nitric oxide synthase (NOS). This enzyme catalyses the conversion of arginine to citrulline and in the process produces nitric oxide (NO). Increased NO formation and altered BH4 levels have been implicated in a number of disorders such as neurodegeneration and sepsis. In this thesis the inter-relationship between BH4 and NO metabolism was studied, in addition to examination of the downstream consequences of a partial BH4 deficiency on reduced glutathione (GSH) status and mitochondrial electron transport chain (ETC) activity. It was found that in cerebrospinal fluid and in cultured rat astrocytes, NO could decrease BH4 availability, possibly leading to impaired NO formation. To examine whether a partial decrease in BH4 lead to decreased NO formation in vivo, a mouse model of partial BH4 deficiency (hph-1 mouse) was used. This mouse was found to display a partial deficiency in BH4 in brain (-58%), liver (-71%) and kidney (-71%) compared to wildtype mice, in addition to a 61% decrease in plasma NO metabolites. Impaired NO formation persisted in the hph-1 mouse upon induction of NO synthesis in vivo, with an intraperitoneal injection of lipopolysaccharide (LPS). Plasma NO metabolites were 52% and 42% lower in hph-1 mice than in wildtype mice at 4.5 and 9 hours post LPS administration. In astrocytes cultured from hph-1 mice, BH4 levels were significantly lower than in cultured wildtype mouse astrocytes. In addition, GSH levels were 56% greater and NADH: ubiquinone oxidoreductase (complex I) was 40% lower in hph-1 mouse cultured astrocytes than wildtype mouse astrocytes. Following stimulation of NO synthesis, levels of GSH increased in wildtype and hph-1 astrocytes, and increased to a greater extent in hph-1 astrocytes (2.1-fold) than wildtype astrocytes (1.4-fold). The results from this thesis suggest that increased NO formation can lead to decreased BH4 availability. This in turn may limit NO formation and lead to alterations in GSH status and impaired complex I activity.

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