NITRITE AND NITRATE LEVELS IN INDIVIDUAL MOLLUSCAN NEURONS - SINGLE-CELL CAPILLARY ELECTROPHORESIS ANALYSIS

Cell and tissue concentrations of NO2- and NO3- are important indicato rs of nitric oxide synthase activity and crucial in the regulation of many metabolic functions, as well as in nonenzymatic nitric oxide rele ase. We adapted the capillary electrophoresis technique to quantify NO 2- 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 inject ed into a 75-mu m separation capillary and the NO2- and NO3- were sepa rated electrophoretically from other anions and detected by direct ult raviolet absorbance. The limits of detection for NO2- and NO3- were <2 00 fmol (<4 mu M in the neurons under study). The NO2- and NO3- levels in individual neurons varied from 2 mM (NO2-) and 12 mM (NO3-) in neu rons histochemically positive for NADPH-diaphorase activity down to un detectable levels in many NADPH-diaphorase-negative cells. These resul ts affirm the correspondence of histochemical NADPH-diaphorase activit y and nitric oxide synthase in molluscan neurons. NO2- was not detecte d in whole ganglion homogenates or in hemolymph, whereas hemolymph NO3 - averaged 1.8 +/- 0.2 x 10(-3) M. Hemolymph NO3- in Pleurobranchaea w as appreciably higher than values measured for the freshwater pulmonat e Lymnaea stagnalis (3.2 +/- 0.2 x 10(-5) M) and for another opisthobr anch, Aplysia californica (3.6 +/- 0.7 x 10(-4) M). Capillary electrop horesis methods provide utility and convenience for monitoring NO2-/NO 3- levels in single cells and small amounts of tissue.