Wykes, Victoria Sarah Mary; (2004) Functional characteristics of nitric oxide receptor isoforms. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Physiologically, nitric oxide (NO) signal transduction occurs through activation of guanylyl cyclase (GC)-coupled receptors, which catalyse cGMP formation. The accumulation of cyclic guanosine 3'-5' monophosphate (cGMP) engages a number of downstream targets to trigger various biological effects, and is ultimately degraded by phosphodiesterases. Unlike with many neurotransmitter receptors, knowledge of the functional characteristics of the NO receptors is limited. The aim of the present research was to begin to address this deficiency by examining the kinetics of the activation of the native receptor and of heterologously-expressed receptor isoforms in intact cells or cell lysates. In cells from a brain region that is enriched in the NO-cGMP signalling pathway, namely the striatum, NO-evoked cGMP accumulation was located in a neuronal subpopulation and the receptor activity displayed a rapidly-desensitizing profile similar to that found previously in cerebellar astrocytes. To determine if this pattern of activity is peculiar to particular receptors, the two known heterodimeric isoforms, α1β1 and α2β1, were expressed in COS-7 cells. NO was applied in fixed concentrations using a recently-developed method based on balancing NO release from a donor with NO inactivation by red blood cells. The two isoforms were highly sensitive to NO, half-maximal cGMP accumulation occurring at concentrations of about 1 nM in both cases. Furthermore, the NO-evoked activity of both receptors desensitized with very similar kinetics. To circumvent limitations of the method for NO delivery used in the initial experiments, particularly problems of haemolysis, a new technique, in which a chemical NO scavenger was substituted for the red blood cells, was evaluated and found to be superior in several respects. The new method was used to determine the kinetic parameters of the receptor purified from bovine lung and, for comparison, cell lysates containing the two isoforms. Half-maximal activity in all cases required about 1 nM NO. In addition, the Hill slopes were close to 1, questioning a previous conclusion that receptor activation requires binding of more than one NO molecule per receptor. Contrasting with another previous study which suggested that membrane association sensitizes the receptor to NO, comparison of cytosolic and membrane preparations of two different tissues (cerebellum and platelets) revealed that their sensitivities to NO were indistinguishable when studied using fixed concentrations.

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