Roy, B; (2008) Regulation of the nitric oxide receptor. Doctoral thesis , UCL (University College London).

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Abstract

The guanylyl cyclase-coupled nitric oxide receptor (GC) acts like a classical neurotransmitter receptor, binding the ligand NO and forming the second messenger cGMP. This thesis investigated the regulation of the NO receptor by endogenous regulators and two groups of pharmacological activators. Haem-mimetic compounds activate the NO-insensitive, haem-free form of the receptor. BAY58-2667 activated the haem-free receptor with half the efficacy of NO activating the haem-reduced receptor. These findings prompt reassessment of physiological and pathophysiological roles of the haem-free receptor, and contradict reports that haem mimetics also activate the haem-oxidised receptor. The second group of compounds activates GC by inhibiting receptor deactivation. BAY41-2272 activated purified GC with EC50= 43 23 nM in the presence of maximally stimulating NO concentrations, and this activation was prevented by NO scavengers. BAY41-2272 renders GC the most potent known NO detector (EC50 = 47 3 pM), confirming earlier theoretical predictions (Garthwaite, 2005). Recently a dual-site model for NO-stimulation of GC was proposed (Cary et al., 2005). Predictions of the Cary model were tested on rat cerebellar cells and platelets, using a new technique for delivering repetitive pulses of NO. The findings suggest that the proposed model is of doubtful relevance, and support the existing one site, two states model. The simple model is further refined by incorporation of regulation by nucleotides and Ca2+. In this new model both ATP and substrate GTP act as allosteric regulators. Inhibition by Ca2+ proved rather complex, involving two inhibitory sites that predominately affected NO-stimulated GC activity and also inhibited receptor deactivation. The new model also partially reconciles the different behaviour of GC when purified from its cellular environment. While this new model appears kinetically robust, further investigation is required to properly incorporate Ca2+ into the scheme and also to link these regulatory changes to the underlying structural modifications.

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