Nitric oxide (NO) is an intercellular messenger in the brain, where it stimulates guanylyl cyclase, producing cGMP. The NO-cGMP pathway is involved in many physiological functions but at high levels, and through other mechanisms, NO can also be toxic. The regulation of the NO concentration is, therefore, critical. Whilst NO formation through NO synthases is now well established, it has only recently become apparent that brain (and other tissue) also possesses a powerful NO consuming activity (Griffiths & Garthwaite, 2001). This study aimed to determine the kinetic properties of the NO inactivating pathway in brain tissue.
The experiments used either dispersed cells or intact slices from the cerebellum of 8-day-old rats that had been killed humanely in accordance with Home Office regulations. In the dispersed cells, application of 0-250 µM diethylaminetriamine/NO adduct led to differing steady-state NO concentrations, from which the inactivation rates were determined. These followed Michaelis-Menten kinetics, yielding a Km of 62 nM and a Vmax of 0.43 nmol (mg protein s)-1.
Experiments were carried out on cerebellar slices to evaluate the rate of NO inactivation in more physiological brain tissue. The steady-state levels of cGMP provided an index of the penetration of NO into the slices when they were bathed in solutions containing various fixed NO concentrations (generated using appropriate donors). As predicted should the inward diffusion of NO be limited by NO consumption, half-maximal cGMP accumulation required an external NO concentration of 1.0 µM, which is 500-fold higher than that required in dispersed cerebellar cells. Also as predicted, cGMP immunocytochemistry indicated that, at lower external NO concentrations, marked gradients of NO existed across the slice thickness at steady state. This approach further suggested that the rate of inactivation of NO was grossly similar in different regions of the cerebellum. Application of a simplified diffusion model to the data suggested that the Vmax value for intact cerebellar tissue is at least 1 µM s-1, which is about 2-fold higher than the value predicted from the isolated cells after correcting for the different protein concentrations.
The kinetic properties of the NO inactivation pathway suggest that, when NO release rates are low, the mechanism serves to translate those release rates rapidly (under 40 ms time scale) into proportionate NO concentrations constrained to target guanylyl cyclase (0.5-20 nM). At higher NO release rates, the inactivation pathway would be instrumental in preventing toxic NO concentrations (> 100 nM) being attained under normal conditions.
This work was supported by The Wellcome Trust and the Sir Jules Thorn Charitable Trust.