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 affnity 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, NADPHdependent 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.The NADPH diaphorase histochemical technique is based on the presence in certain neurons of an enzyme that can catalyze the NADPH-dependent conversion of a soluble tetrazolium salt to an insoluble, visible formazan (1,2). This method has proven useful for the examination of select populations of neurons in both experimental studies and in human neuropathology. In particular, NADPH diaphorase has been shown to be a selective marker for forebrain neurons containing both somatostatin and neuropeptide Y (3) and for the ascending cholinergic reticular system in the mesopontine tegmentum (4). This method has, therefore, been used to examine these neurons in Huntington disease (5), Alzheimer disease (6, 7), progressive supranuclear palsy (8), and ischemia (9, 10). NADPH diaphorase-containing neurons appear relatively resistant to anoxia and excitotoxic damage (11-13), and those in the striatum are selectively spared in Huntington disease (5).Although the NADPH diaphorase activity has been well defined histochemically (14), the function of this enzyme has remained a mystery. Previous attempts to characterize the enzyme biochemically have been hampered by lack of a specific assay (15, 16) because several proteins can exhibit NADPH-dependent diaphorase activity in brain homogenate (17). Therefore, we have used both a biochemical assay and an antibody that specifically recognizes neuronal NADPH diaphorase (18,19) to monitor purification of this enzyme from rat brain. Because NADPH diaphorase is an NADPHdependent enzyme, a purification protocol similar to that recently used for the NADPH-dependent brain enzyme, nitric oxide synthase, was att...
Oscillatory firing patterns are an intrinsic property of some neurons and have an important function in information processing. In some cells, low voltage-activated calcium channels have been proposed to underlie a depolarizing potential that regulates bursting. The sequence of a rat brain calcium channel alpha 1 subunit (rbE-II) was deduced. Although it is structurally related to high voltage-activated calcium channels, the rbE-II channel transiently activated at negative membrane potentials, required a strong hyperpolarization to deinactivate, and was highly sensitive to block by nickel. In situ hybridization showed that rbE-II messenger RNA is expressed in regions throughout the central nervous system. The electrophysiological properties of the rbE-II current are consistent with a type of low voltage-activated calcium channel that requires membrane hyperpolarization for maximal activity, which suggests that rbE-II may be involved in the modulation of firing patterns.
Functional expression of the rat brain alA Ca channel was obtained by nuclear injection of an expression plasmid into Xenopus oocytes. The alA Ca current activated quickly, inactivated slowly, and showed a voltage dependence typical of high voltage-activated Ca channels. The alA current was partially blocked (=23%) by w-agatoxin IVA (200 nM) and substantially blocked by a-conotoxin MVIIC (5 pM blocked "70%). Bay K 8644 (10 pM) or a-conotoxin GVIA (1 IM) had no significant effect on the alA current. Coexpression with rat brain Ca channel 13 subunits increased the alA whole-cell current and shifted the current-voltage relation to more negative values. While the f18b and (3 subunits caused a significant acceleration of the alA inactivation kinetics, the 82. subunit dramatically slowed the inactivation of the alA current to that seen typically for P-type Ca currents. In situ loaliztion with antisense deoxyoligonucleotide and RNA probes showed that alA was widely distributed throughout the rat central nervous system, with moderate to high levels in the olfactory bulb, in the cerebral cortex, and in the CA fields and dentate gyrus of the hippocampus. In the cerebellum, prominent alA expression was detected in Purkuije cells with some labeling also in granule cells. Overall, the results show that aj channels are widely expressed and share some properties with both Qand P-type channels.
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