Capillary Electrophoresis Analysis of Nitric Oxide Synthase Related Metabolites in Single Identified Neurons

Intracellular concentrations of L-citrulline (Cit) and its metabolites are related to nitric oxide synthase (NOS) activity, an enzyme producing the intercellular messenger NO in animal tissues including the nervous system. A capillary electrophoresis system using laser-induced fluorescence detection is described, and methods are developed to monitor the levels of L-arginine (Arg), Cit, and related molecules in identified neurons of the marine slugs, Pleurobranchaea californica and Aplysia californica. The limits of detection for Arg, Cit, L-argininosuccinate, L-ornithine, and L-arginine phosphate range from 50 amol to 17 fmol (5 nM to 17 μM in the neurons under study); these detection limits are significantly lower than actual intracellular levels of the metabolites, allowing the direct assay of single cells. The levels of NOS metabolites in individual neurons varied from 6 (Arg) and 4 mM (Cit) in putative NOS-containing neurons down to <1 μM (undetectable) levels in many putative NOS-negative cells. The Arg/Cit ratio is independent of cell volume, correlates with NADPH-diaphorase staining, and appears to be a characteristic parameter for the presence of NOS activity in identified neurons.