Neuronal NADPH diaphorase is a nitric oxide synthase.

TLDR

NADPH diaphorase histochemistry labels discrete neuronal populations, but its enzymatic role was unclear until it was identified as the 150‑kDa, calcium/calmodulin‑dependent nitric oxide synthase that produces NO from L‑arginine. The study aimed to biochemically characterize neuronal NADPH diaphorase. The enzyme was purified to homogeneity from rat brain using affinity chromatography and anion‑exchange HPLC, and its identity was confirmed by immunoblotting and immunostaining with a specific antibody. The purified 150‑kDa protein exhibited nitric oxide synthase activity, copurified and co‑immunoprecipitated with NADPH diaphorase, was competitively inhibited by nitro blue tetrazolium, confirming that neuronal NADPH diaphorase is a nitric oxide synthase and that its histochemistry marks NO‑producing neurons.

Abstract

NADPH diaphorase histochemistry selectively labels a number of discrete populations of neurons throughout the nervous system. This simple and robust technique has been used in a great many experimental and neuropathological studies; however, the function of this enzyme has remained a matter of speculation. We, therefore, undertook to characterize this enzyme biochemically. With biochemical and immunochemical assays, NADPH diaphorase was purified to apparent homogeneity from rat brain by affinity chromatography and anion-exchange HPLC. Western (immunoblot) transfer and immunostaining with an antibody specific for NADPH diaphorase labeled a single protein of 150 kDa. Nitric oxide synthase was recently shown to be a 150-kDa, NADPH-dependent enzyme in brain. It is responsible for the calcium/calmodulin-dependent synthesis of the guanylyl cyclase activator nitric oxide from L-arginine. We have found that nitric oxide synthase activity and NADPH diaphorase copurify to homogeneity and that both activities could be immunoprecipitated with an antibody recognizing neuronal NADPH diaphorase. Furthermore, nitric oxide synthase was competitively inhibited by the NADPH diaphorase substrate, nitro blue tetrazolium. Thus, neuronal NADPH diaphorase is a nitric oxide synthase, and NADPH diaphorase histochemistry, therefore, provides a specific histochemical marker for neurons producing nitric oxide.