Nitrite and nitrate levels in individual molluscan neurons: Single-cell capillary electrophoresis analysis

Abstract

Cell and tissue concentrations of NO2 and NO3 are important indicators of nitric oxide synthase activity and crucial in the regulation of many metabolic functions, as well as in nonenzymatic nitric oxide release. We adapted the capillary electrophoresis technique to quantify NO2- and NO3- levels in single identified buccal neurons and ganglia in the opisthobranch mollusc Pleurobranchaea californica, a model system for the study of the chemistry of neuron function. Neurons were injected into a 75-μm separation capillary and the NO2- and NO3- were separated electrophoretically from other anions and detected by direct ultraviolet absorbance. The limits of detection for NO2- and NO3- were <200 fmol (<4 μM in the neurons under study). The NO2- and NO3- levels in individual neurons varied from 2 mM (NO2-) and 12 mM (NO3-) in neurons histochemically positive for NADPH- diaphorase activity down to undetectable levels in many NADPH-diaphorase- negative cells. These results affirm the correspondence of histochemical NADPH-diaphorase activity and nitric oxide synthase in molluscan neurons. NO2- was not detected in whole ganglion homogenates or in hemolymph, whereas hemolymph NO3- averaged 1.8 ± 0.2 x 10-3 M. Hemolymph NO3- in Pleurobranchaea was appreciably higher than values measured for the freshwater pulmonate Lymnaea stagnails (3.2 ± 0.2 x 10-5 M) and for another opisthobranch, Aplysia californica (3.6 ± 0.7 x 10-4 M). Capillary electrophoresis methods provide utility and convenience for monitoring NO2- /NO3- levels in single cells and small amounts of tissue.