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-arginino-succinate, L-ornithine, and L-arginine phosphate range from 50 amol to 17 fmol (5 nM to 17 microM 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 form 6 (Arg) and 4 mM (Cit) in putative NOS-containing neurons down to < 1 microM (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.
Floyd
P D
PD
Department of Chemistry, University of Illinois, Urbana 61801, USA.
Moroz
L L
LL
Gillette
R
R
Sweedler
J V
JV
D000818:Animals; D001048:Aplysia; D001120:Arginine / Q000031:analogs & derivatives / Q000032:analysis*; D001125:Argininosuccinic Acid / Q000032:analysis; D002956:Citrulline / Q000032:analysis*; D019075:Electrophoresis, Capillary; D008974:Mollusca; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D009474:Neurons / Q000378:metabolism*; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D052248:Nitric Oxide Synthase Type I; D009943:Organophosphorus Compounds / Q000032:analysis; D009952:Ornithine / Q000032:analysis
D009419:Nerve Tissue Proteins; D009943:Organophosphorus Compounds; C015441:phospho-L-arginine; D001125:Argininosuccinic Acid; D002956:Citrulline; D001120:Arginine; D009952:Ornithine; D019001:Nitric Oxide Synthase; D052248:Nitric Oxide Synthase Type I