Nitric oxide-mediated cGMP signal transduction in the central nervous system

Nitric oxide (NO) functions as a signalling molecule throughout the brain where, via the intracellular generation of cGMP, it participates in many functions, such as in synaptic plasticity. The initial experiments were based on the finding that, in optic nerve, NO released from blood vessels tonically depolarises axons. The aim was to test the hypothesis that the tonic NO production is maintained by phosphorylation of endothelial NO synthase (eNOS). The results from extracellular recordings of changes in the axonal membrane potential suggested that PI3 kinase-mediated eNOS phosphorylation is partially responsible. The subsequent aim was to determine if blood vessel-neuron communication may be more widespread, by investigating if this mechanism accounts for basal NO production in the developing rat hippocampus. For this purpose, measurements of cGMP were chosen as a sensitive index of the local NO concentration. Contrary to expectations, no clear evidence for a dominant role of either eNOS or the neuronal NO synthase emerged, although the data suggested that NO formation was calcium-dependent. The next step was to characterise the target cells of endogenous and exogenous NO in the hippocampus, particularly in the light of findings that, with a better tool for inhibiting the dominant phosphodiesterase activity (phosphodiesterase-2), much higher cGMP levels could be evoked than previously. Accordingly, instead of a predominant location in astrocytes, cGMP immunocytochemistry showed widespread staining of neuronal elements (somata, dendrites, neuropil) throughout the tissue. The final objective was to begin to analyse NO transduction in cells in real-time, using a newly developed fluorescent cGMP sensor. Cell lines expressing various levels of guanylyl cyclase and phosphodiesterase were selected for study. Cellular responsiveness to extremely low NO concentrations (down to 3 pM) could be detected. Moreover, the findings illustrated how the interplay between guanylyl cyclase and phosphodiesterase activities serves to generate distinct cellular cGMP profiles