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

• Published in: Analytical chemistry (Washington) Vol. 70; no. 11; pp. 2243 - 2247
• Main Authors: Floyd, Philip D, Moroz, Leonid L, Gillette, Rhanor, Sweedler, Jonathan V
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 01.06.1998
• Subjects: Analytical chemistry; Analytical, structural and metabolic biochemistry; Animals; Aplysia; Arginine - analogs & derivatives; Arginine - analysis; Argininosuccinic Acid - analysis; Biochemistry; Biological and medical sciences; Chemistry; Citrulline - analysis; Electrophoresis, Capillary; Enzymes and enzyme inhibitors; Exact sciences and technology; Fundamental and applied biological sciences. Psychology; General, instrumentation; Mollusca; Nerve Tissue Proteins - metabolism; Neurons; Neurons - metabolism; Nitric Oxide Synthase - metabolism; Nitric Oxide Synthase Type I; Organophosphorus Compounds - analysis; Ornithine - analysis; Oxidoreductases; Aplysia californica; Enzyme; Capillary electrophoresis; Metabolite; Central nervous system; Gastropoda; Nitric-oxide synthase; Microassay; Neuron; Enzymatic activity; Analytical method; Isolated cell; Oxidoreductases; Invertebrata; Mollusca; Quantitative analysis
• ISSN: 0003-2700; 1520-6882
• DOI: 10.1021/ac9713013

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.