Direct single cell determination of nitric oxide synthase related metabolites in identified nitrergic neurons

The biochemical characterization of individual nitrergic (NO releasing) neurons is a non-trivial task both in vertebrate and invertebrate preparations. In spite of numerous efforts, there are limited data related to intracellular concentrations of essential metabolites involved in NO synthesis and degradation. This situation creates controversies in both identification of nitrergic neurons and the selection of reliable reporters of NOS activity in heterogeneous cell populations. We take advantage of identified neurons from the pulmonate mollusc Lymnaea stagnalis to perform direct single cell microanalysis of intracellular concentrations of the major nitric oxide synthase (NOS) related metabolites such as arginine, citrulline, argininosuccinate, NO(2)(-),and NO(3)(-). Capillary electrophoresis protocols have been developed to quantitate levels of these metabolites in single identified neurons from the buccal, cerebral, and pedal ganglia using laser-induced fluorescence and conductivity detection. The limits of detection (LODs) for arginine (Arg) and citrulline (Cit) are 84 amol (11nM) and 110 amol (15 nM), respectively, and LODs for NO(2)(-)and NO(3)(-) are <200 amol (<10nM) each. We report that intracellular concentrations of NOS related metabolites are in the millimolar range and less than 1% of a single cell is required for microchemical analysis. From four cell types tested, only the esophageal motoneuron B2 contains active NOS, and they also contain surprisingly high nitrite levels (up to 5mM) compared to other neurons tested (peptidergic B4, dopaminergic RPeD1, and serotonergic CGC). These B2 neurons also exhibit an Arg/Cit ratio susceptible to the selective NOS inhibitor l-iminoethyl-N-ornithine whereas others neurons do not even though they all may contain NOS transcripts. On the contrary, we found that absolute concentrations of other NOS related metabolites including nitrates are not reliable markers of NOS activity and demonstrate the need for multiple assays for NOS activity.