Complementary distribution of NADPH-diaphorase and I-arginine in the snail nervous system

Since the interneuronal messenger nitric oxide (NO) can not be stored in neurones, the regulation of the NO-producing enzyme nitric oxide synthase (NOS) is crucial. Neuronal NOS metabolises L-arginine to nitric oxide (NO) and L-Citrulline in a Ca2+-dependent manner. Thus, availability of L-arginine to NOS may modulate NO production. In this study, we examined the cellular distribution 'of reduced nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase, L-arginine and L-citrulline. Using NADPH-diaphorase histochemistry to visualise putative NO-producing cells and immunocytochemistry to localise L-arginine, we showed that the distribution Of L-arginine-immunoreactive neurones correlates well with those of NADPH-diaphorase-positive neurones in cerebral ganglia of the pulmonate Helix pomatia. However, substrate and enzyme were visualised in separate but adjacent neurones. We further examined whether NADPH-diaphorase-labelled cells contain the L-Citrulline. Following elevation of intracellular Ca2+ by the Ca2+ ionophore, ionomycin, or by a high-K+ solution, the number Of L-Citrulline-immunoreactive neurones in mesocerebrum and pedal lobe increased up to tenfold. Preincubation of ganglia with the NOS inhibitor N-G-nitro-L-arginine prevented ionomycin or high-K+ solution-induced L-Citrulline synthesis. Most L-citrulline-immunoreactive neurones contain NADPH-diaphorase activity. In conclusion, these experiments indicate a complementary distribution of NOS and L-arginine and suggest an unknown signalling pathway between neurones to maintain L-arginine and NO homeostasis.