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The shaping of nitric oxide signals by a cellular sink

Charmaine Griffiths and John Garthwaite

1. The functioning of nitric oxide (NO) as a biological messenger necessitates that there be an inactivation mechanism. Cell suspensions from a rat brain region rich in the NO signalling pathway (cerebellum) were used to investigate the existence of such a mechanism and to determine its properties.
2. The cells consumed NO in a manner that could not be explained by reaction with O₂, superoxide ions or contaminating red blood cells. Functionally, the mechanism was able to convert constant rates of NO formation into low steady-state NO concentrations. For example, with NO produced at 90 nM min^-1, the cells (20 10⁶ ml^-1) held NO at 20 nM. Various other cell types behaved similarly.
3. The influence of NO inactivation on the ability of NO to access its receptor, soluble guanylyl cyclase, was explored by measuring cGMP accumulation in response to the clamped NO concentrations. The extrapolated steady-state EC₅₀ for NO was 2 nM, a concentration readily achieved by low NO release rates, despite inactivation.
4. When confronted by higher NO release rates for several minutes, the clamping mechanism failed, resulting in a progressive rise in NO concentration. While the clamp was maintained, cellular respiration was unaffected but, as it failed, respiration became inhibited by NO. The IC₅₀ was measured to be 120 nM (at 100-140 µM O₂).
5. It is concluded that cerebellar (and other) cells possess a powerful NO inactivation mechanism that, extrapolated to the whole tissue, would impose on NO a half-life of around 100 ms. This and other properties of the device appear ideal for shaping low-level NO signals for activating its receptor, soluble guanylyl cyclase, whilst avoiding adverse effects on mitochondrial function. The exhaustibility of the mechanism provides a scenario for NO to become toxic.

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