Biochemical characterization of NADPH-diaphorase in the chick embryo retina: stimulation by calcium ions and inhibition by arginine analogs.

Nitric oxide is an important intercellular messenger in the central nervous system. NADPH-diaphorase, reported to be identical to nitric oxide synthase, is present in specific groups of cells in several neural tissues, including the retina. We determined NADPH-diaphorase activity in homogenates of the chick embryo retina. The enzyme activity was measured spectrophotometrically at 585 nm after incubating retinal total homogenates (100-150 micrograms protein) with 1 mM NADPH and 0.5 mM nitroblue tetrazolium in 50 mM Tris buffer, pH 8.1, at 37 degrees C. NADPH-diaphorase was detected in 14-day old retinas and 53-65% of the enzyme activity was inhibited by 3 mM NG-nitro-L-arginine (NARG), the arginine analog. One mM L-N5-(1-iminoethyl)ornithine (NIO) was the most potent inhibitor (63% inhibition) while 3 mM NG-nitro-L-arginine methyl ester (NAME) (33% inhibition) and 1 mM NG-monomethyl-L-arginine acetate (NMMA) (14% inhibition) were less effective. Enzyme activity was increased by 48% by 2 mM calcium chloride, an effect reversed by 1 mM EGTA or EDTA. Basal enzyme levels were also partially inhibited by the chelators, indicating the presence of calcium-dependent and -independent isoforms of nitric oxide synthase in the retina. The results show that the NADPH-diaphorase assay is simple and sensitive and that the different isoforms of nitric oxide synthase expressed in chick retinal cells during development can be demonstrated.