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 ne rvous system. NADPH-diaphorase, reported to be identical to nitric oxi de 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 me asured spectrophotometrically at 585 nm after incubating retinal total homogenates (100-150 mu g protein) with 1 mM NADPH and 0.5 mM nitrobl ue tetrazolium in 50 mM Tris buffer, pH 8.1, at 37 degrees C, NADPH-di aphorase was detected in 14-day old retinas and 53-65% of the enzyme a ctivity was inhibited by 3 mM N-G-nitro-L-arginine (NARG), the arginin e analog. One mM L-N-5-(1-iminoethyl)ornithine (NIO) was the most pote nt inhibitor (63% inhibition) while 3 mM N-G-nitro-L-arginine methyl e ster (NAME) (33% inhibition) and 1 mM N-G-monomethyl-L-arginine acetat e (NMMA) (14% inhibition) were less effective. Enzyme activity was inc reased by 48% by 2 mM calcium chloride, an effect reversed by 1 mM EGT A or EDTA. Basal enzyme levels were also partially inhibited by the ch elators, indicating the presence of calcium-dependent and -independent isoforms of nitric oxide synthase in the retina. The results show tha t the NADPH-diaphorase assay is simple and sensitive and that the diff erent isoforms of nitric oxide synthase expressed in chick retinal cel ls during development can be demonstrated.