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Neuronal NADPH-diaphorase is a nitric oxide synthase Hope, Bruce Thomas


The enzyme responsible for the neuronal NADPH-diaphorase histochemical reaction was identified in rat brain by employing a variety of histochemical and biochemical techniques. The histochemical reaction catalyzes the NADPH-dependent reduction of tetrazolium dyes to colored insoluble formazans. Although the histochemical reaction has been widely employed in neuroanatomical and neuropathological studies, the identity of the enzyme responsible for the reaction has been unknown. Previous attempts to determine the identity of the enzyme have failed due to the lack of a specific biochemical assay. Some biochemical characteristics of the histochemical reaction in rat striatum were determined in order to develop a specific biochemical assay for neuronal NADPH-diaphorase. The histochemical reaction used several different analogs of NADPH but did not use β-NADH. All tetrazolium analogs with redox potentials above that for β-NADPH were reduced, although the reduction of some tetrazoliums was oxygen-sensitive. The reaction appeared not to require metal ions, flavins, peroxides, or superoxide anions as all methods to remove these factors did not influence staining. The DT-diaphorase activator, menadione, and the inhibitor, dicumarol, did not affect neuronal NADPH-diaphorase. Electron microscopic results suggested neuronal NADPH-diaphorase was membrane-bound, particularly with the endoplasmic reticulum. These results correlate with no known enzymes, including those previously proposed as neuronal NADPH-diaphorase. Employing an antiserum which specifically detected neuronal NADPH-diaphorase, we found the enzyme to be nitric oxide (NO) synthase. NO synthase produces the membrane-permeable second messenger NO. Immunoreactive NADPH-diaphorase activity was copurified to apparent homogeneity with NO synthase activity. The antiserum specifically immunoprecipitated both NO synthase and NADPH-diaphorase activities and specifically labelled a 150 kD band on Western blots, similar to NO synthase from previous reports. The NADPH-diaphorase substrate, NBT, competed with the NO synthase substrate, arginine, for electrons from NADPH. As expected, immunoreactivity for citrulline was found only in NADPH-diaphorase neurons. Citrulline is produced along with NO from the substrate, arginine. NADPH-diaphorase activity was weak to moderate in the cerebellum even though this region contains high levels of NO synthase. The cerebellum also had no citrulline- or NADPH-diaphorase immunoreactivity. We suggest the NO synthase in the cerebellum is a different form from that in the rest of the brain. We conclude that the neuronal NADPH-diaphorase histochemical reaction is due to a form of NO synthase and therefore NADPH-diaphorase is a histochemical marker of NO synthase in the brain. The product NO is the endogenous activator of soluble gu any late cyclase in the brain. This allows a discussion of the anatomical and functional relationships between NO synthase/NADPH-diaphorase and guanylate cyclase to determine the possible functions of these enzymes and their second messenger products in the brain.

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