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
