NOS- and Non-NOS NADPH Diaphorases in the Insular Cortex of the Syrian Golden Hamster

Abstract

We had previously shown NADPH diaphorase activity in fixed tissue slices of the insular cortex of the Syrian golden hamster (Mesocricetus auratus). The objective of this work was to determine the chemical identity of agents responsible for the observed NADPH diaphorase activities. Three different enzymatic NADPH diaphorase activities were distinguished in the insular cortex. (a) The activity seen in endothelial cells was not characterized histochemically, but it co-localized with eNOS-like immunoreactivity. (b) The neuronal Type I activity showed little sensitivity to 10(-5) M dicoumarol, could use either alpha- or beta-NADPH with almost equal facility, and co-localized with nNOS-like immunoreactivity. This activity was primarily attributable to nNOS. (c) The neuronal Type II activity was greatly attenuated by 10(-5) M dicoumarol, had a strong preference for beta-NADPH (rather than alpha-NADPH), and did not co-localize with any NOS-like immunoreactivity. These characteristics also apply to the NADPH diaphorase activity observed in the diffuse blue band in Layers II and III of agranular and dysgranular insular cortex and in the meshwork of cortical fibers. This staining was due primarily to a dicoumarol-sensitive dehydrogenase(s), either an isozyme of DT diaphorase (EC 1.6.99.2), or NADPH dehydrogenase (quinone) (EC 1.6. 99.6), or to a novel dicoumarol-sensitive NADPH dehydrogenase.

Telencephalic Organization in the Spotted African Lungfish, <i>Protopterus dolloi:</i> A New Cytological Model

“…The brains were embedded and postfixed for 2-4 h, and 35-m frozen sections were cut in the three standard anatomical planes. Following the protocol of Scherer-Singer et al [1983], as modified by Marín et al [1998b], free-floating sections were incubated in a solution containing 1 m M ␤ -NADPH, 0.8 m M nitroblue tetrazolium and 0.06% Triton-X 100 (Sigma, St. Louis, Mo., USA) in 0.1 M PB for 1-2 h at 34 ° C. Control sections were preincubated for 2 h with the addition of sodium azide (5 m M ) and/or miconayole (10 -4 M ) which are inhibitors of mitochondrial and cytochrome P450 enzymes [Wehby and Frank, 1999]. The reacted sections were rinsed several times in 0.1 M PB and mounted out of alcoholic gelatin onto gelatin-coated glass slides.…”

Direct single cell determination of nitric oxide synthase related metabolites in identified nitrergic neurons

“…Although these methods present the advantage of relative simplicity and routinely produce preliminary anatomical maps, there remains the possibility that such protocols generate some false positive identifications of the localization of NOS activity. For example, NADPH-d staining has been associated with several different enzymes [57][58][59][60][61][62][63][64][65][66][67], and some published accounts of NADPH-d staining in Lymnaea, including CGC [54] have used lower concentrations of fixative compared to more established protocols [40,52,59,60,[68][69][70] designed to reveal specific fixative resistant NADPH-d activity as a putative marker for NOS activity. Furthermore, current NO-sensitive electrodes are constrained by both their large tip size (2 mm diameter in [55]) and sensitivity [56] such that reliable NO measurements in the low nanomolar range in complex biological tissues, and especially from single neurons, are difficult to interpret.…”

Immunocytochemical Detection of Neuronal Nitric Oxide Synthase (nNOS)-IR in Embryonic Rat Stomach Between Days 13 and 21 of Gestation

“…The peroxidase activity was developed with 0.03% 3,3 Ј -diaminobenzidine tetrahydrochloride (DAB; DAKO). The sections were counterstained with hematoxylin, dehydrated, and mounted in mounting medium (BioGenex; San Ramon, CA) (Wehby and Frank 1999).…”