|
|
|
|
 |
|||
|
|||
ARTICLE |
NOS- and Non-NOS NADPH Diaphorases in the Insular Cortex of the Syrian Golden Hamster
Richard G. Wehbya and Marion E. Franka,ba Program in Neuroscience, Schools of Medicine and Dental Medicine, University of Connecticut Health Center, Farmington, Connecticut
b Department of BioStructure and Function, Schools of Medicine and Dental Medicine, University of Connecticut Health Center, Farmington, Connecticut
Correspondence to: Richard G. Wehby, Dept. of Brain and Cognitive Sciences, MIT, E25-236, Cambridge, MA 02139.
 |
Summary |
|---|
 Top Summary IntroductionMaterials and MethodsResultsDiscussionLiterature Cited |
|---|
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 
- or ß-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 ß-NADPH (rather than -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. (J Histochem Cytochem 47:197–207, 1999)
Key Words: NADPH diaphorase, nitric oxide synthase, dicoumarol, dicumarol, NADPH dehydrogenase
|
Introduction |
|---|
|
TopSummaryIntroductionMaterials and MethodsResultsDiscussionLiterature Cited |
|---|
An enzyme is said to be an NADPH diaphorase if it can transfer electrons from NADPH to a colorless, soluble tetrazolium salt, producing a colored, insoluble formazan derivative (
Nitric oxide (NO) is believed to serve as a neuromodulator or neurotransmitter in a number of different processes, including a model of a mechanism for a type of memory, long-term potentiation (LTP) (for review see
The Syrian golden hamster (Mesocricetus auratus) is a well-established animal model for gustatory studies (for review see
Two experimental approaches were used: (a) a systematic study of the effects of variation in reaction parameters on diaphorase staining to determine which staining responses most closely reflect the known NOS diaphorase responses, and (b) direct determination of the presence of NOS by immunohistochemistry with commercially available antibodies to the isozymes of NOS.
|
Materials and Methods |
|---|
|
TopSummaryIntroductionMaterials and MethodsResultsDiscussionLiterature Cited |
|---|
All experiments were carried out on male Syrian golden hamsters (Charles River Labs; Wilmington, MA) weighing 120–190 g and were approved by the Center for Laboratory Animal Care of the University of Connecticut Health Center. All chemicals were purchased from Sigma (St Louis, MO) unless otherwise indicated. Animals were deeply anesthetized (Nembutal 80 mg/kg, IP; Abbot Laboratories, King of Prussia, PA) and intracardiac perfusion was performed using a constant pressure pump at an approximate flow rate of 2.8 ml/min. Washout (3–5 min) was followed by fixation in 4% formaldehyde (electron microscopic grade; Tousimis Research, Rockville, MD) or freshly depolymerized paraformaldehyde in 0.1 M sodium phosphate buffer, pH 7.4, for 45 min. The brain was removed and postfixed for 2 hr at 4C in the same fixative. The brain was then immersed either in 30% sucrose in 0.1 M sodium phosphate buffer, pH 7.4, until it sank to the bottom of the solution (for frozen sections) or in 0.1 M sodium phosphate buffer, pH 7.4 (for vibratome sections). Sections were cut at 35-µm thickness in the transverse plane and collected serially into cold (4C) 0.1 M sodium phosphate buffer, pH 7.4. To maintain enzymatic activities intact, sections were maintained at 4C in as many steps of the tissue cutting and processing as possible.
NADPH Diaphorase Histochemistry
The standard NADPH diaphorase reaction (
The reaction permitted ready visualization of Type I and II neurons, as well as the diffuse blue band and the background meshwork of fibers (see Results). At 1.5 hr, the reaction typically was not long enough to stain endothelial cells, so the effects on endothelial cell staining were not determined. The effects of several variables were studied.
pH. Preincubation and incubation reactions were run at pH 6.8, 7.4, or 8.5.
Elevated Temperature. Free-floating sections were placed in phosphate buffer in a microcentrifuge tube. The tube was then incubated for 5 min in a water bath at either RT or 55, 65, or 77C. Sections were then removed from the microcentrifuge tube and placed in phosphate buffer at RT. Preincubation and incubation then proceeded as normal.
Co-factors.
Sections were incubated using equimolar amounts of either ß-NADPH, -NADPH, or ß-NADH.
Chelators. Both the preincubation and incubation media included EDTA (1–10 mM) or EGTA (1–10 mM).
Enzyme Inhibitors.
Both the preincubation and incubation media included one of the following (all referenced in
Competitive Electron Acceptor.
Dichlorophenolindophenol (DPIP, 10-4 M) was used to capture electrons enzymatically transferred from NADPH (
Sulfhydryl Inhibitor.
N-ethyl maleimide (NEM, 10 mM) was used to irreversibly modify sulfhydryl groups (such as glutathione or thiols on proteins) that could otherwise donate their reducing hydrogens. NEM (10 mM) was also combined with pyruvate (60 mM), because this combination inhibits activity due to both endogenous sulfhydryls and to lactate dehydrogenase (NADPH diaphorase activity produced by this combination is the so-called "nothing dehydrogenase" effect) (
Each histochemical test was assessed in at least three animals, and in each animal a minimum of nine sections were examined. Results were qualitatively consistent for all sections. For each animal, adjacent sections served as controls. Experimental sections were scored relative to their controls, which were preincubated and reacted for the same lengths of time and at the same pH as the experimental sections.
Immunohistochemistry
Washout, fixation, postfixation, and sectioning were the same as for brain sections used for NADPH diaphorase histochemistry. Polyclonal antibodies were preincubated with hamster liver acetone proteins (1% in 0.01 M PBS, pH 7.4, 30 min at RT) (Sandra Hill, personal communication) to quench nonspecific antibodies. Free-floating sections were blocked with 10% heat-inactivated goat serum (HIGS; Boehringer Mannheim, Indianapolis, IN) in PBS + 0.1% NP40 for 2 hr at RT with agitation. The blocking solution was aspirated, and then free-floating sections were incubated with antibodies to human NOS (rabbit polyclonal anti-nNOS, anti-eNOS and anti-macNOS; mouse monoclonal anti-nNOS and anti-eNOS) (Transduction Laboratories; Lexington, KY) all at 1:500 in 10% HIGS in PBS + 0.1% NP40 for 24 hr at 4C. Sections were rinsed with PBS + 0.1% NP40. After a second blocking with 10% HIGS in PBS + 0.1% NP40 (2 hr at RT), sections were incubated with the species-appropriate biotinylated goat anti-IgG (1:100; Boehringer Mannheim) in 10% HIGS in PBS + 0.1% NP40 overnight at 4C. Sections were then rinsed in PBS + 0.1% NP40, followed by further rinsing in 0.1 M sodium phosphate buffer + 0.1% NP40 (pH 7.4), and endogenous peroxidases were quenched with 0.5% H2O2, in 0.1 M phosphate buffer (pH 7.4) for 20 min. Sections were rinsed with 0.1 M sodium phosphate buffer + 0.1% NP40 (pH 7.4), and then antibodies were visualized with an ABC reaction (Vectastain Elite ABC Peroxidase kit; Vector Laboratories, Burlingame, CA) in 0.1 M sodium phosphate buffer, pH 7.4, using DAB histochemistry. Two sets of control experiments were carried out: sections were incubated either without the primary or without the secondary antibody.
Two methods were used to determine if individual neurons stained for both nNOS and NADPH diaphorase. (a) After reaction with DAB, sections were temporarily mounted (on plain, nonsubbed slides) and coverslipped using a 3:1 mixture of glycerol and 0.1 M sodium phosphate buffer, pH 7.4. The location of cells stained with DAB was mapped using a camera lucida. Coverslips were then removed, sections returned to phosphate buffer, and subsequently reacted for NADPH diaphorase as previously described. These sections were then mounted, cleared, and coverslipped with DPX. The sections were then re-drawn, and the location of cells that stain for NADPH diaphorase was drawn. Alternatively, (b) after reaction with DAB, free-floating sections were reacted for NADPH diaphorase, and then mounted but not coverslipped. Drawings were made of the mounted sections, marking the location of cells that stain for NADPH diaphorase. The slides were then treated with a warm (~72C) mixture of alkali (pH 9 glycine–NaOH buffer) and dimethylformamide to remove the blue formazan precipitate (Richard Weinberg, personal communication), so as to reveal the DAB reaction product. Slides were allowed to dry. Some sections were de-fatted in xylene for 4 hr, and then osmicated (0.2% OsO4) to intensify the DAB reaction product. Sections were then cleared and coverslipped with DPX, and the location of the cells reacted with DAB (i.e., cells that contain NOS) was drawn.
Statistical Analysis
For comparison of double- and single-labeled cells in each animal, the number of cell profiles in each category (i.e., NADPHd+/nNOS+, NADPHd+/nNOS-, NADPHd-/nNOS+) was counted in the insular cortex in at least six sections per animal and was expressed as a percent of the total number of labeled cells in each animal. The percentages in each category from three animals were compared with the analysis of variance for repeated measures (StatMost for Windows; DataMost, Sandy, UT). Post hoc comparisons were made using Newman–Keuls' test.
Photographs
Photographs were taken using a Leitz microscope (Leica; Deerfield, IL) and Ektachrome 160T color slide film (Kodak; Rochester, NY). Developed photographic slides were scanned (Microtek Lab; Redondo Beach, CA) and then cropped, arranged, and labeled in Photoshop 4.0 (Adobe Systems; San Jose, CA).
|
Results |
|---|
|
TopSummaryIntroductionMaterials and MethodsResultsDiscussionLiterature Cited |
|---|
Control Results
Details of the control results for the NADPH diaphorase activity in the insular cortex of the Syrian golden hamster will be presented elsewhere (unpublished observations). Briefly, after 1.5 hr of incubation at RT, pH 8.5, the following types of activities were observed.
Neuronal Type I. These cells resembled Golgi-impregnated neurons, with dark blue reaction product appearing to fill the perikarya, dendrites, and axons (Figure 1a). The reaction product was confined to the cytoplasm, because when the plane of section revealed the nucleus the nucleus was unstained. Type I cells were scattered, with most present in the deep layers (unpublished observations).
|
Neuronal Type II. These cells also had a nonstaining nucleus, but the histochemical reaction only stippled the perikarya and the most proximal regions of dendrites (Figure 1b). This stippling did not reflect an incomplete reaction because the NADPH diaphorase reaction never filled in these cells, even when the reaction was allowed to proceed for over 12 hr. Type II neurons were almost exclusively located in an accumulation of many such cells in Layers II and III of dysgranular and agranular insular cortex (Figure 1c and Figure 1d).
Neuronal Fibers. In transverse sections, stained fibers could be seen running in all directions and in all layers throughout the insular cortex (Figure 1a and Figure 4c). Some were quite fine and followed tortuous paths in their travel. Many had swellings or varicosities which were either globular or elongated. Although fine fibers twisted around neurons, none formed clearly identifiable pericellular nests.
Diffuse Blue Band. There was a diffuse blue band of staining in Layers II and III of agranular and dysgranular insular cortex (Figure 1b–d) and extending along the rostrocaudal length of the rhinal fissure (unpublished observations). Nonstained cell profiles were visible within this band, as was the accumulation of neuronal Type II cells described above (Figure 1b). The combination of many Type II cells and the diffuse blue band easily marked this region by the rhinal fissure so that it was visible even to the naked eye (Figure 1c).
In addition, although not typically seen after 1.5 hr, after the reaction had proceeded for several hours the endothelial cells lining the cerebral blood vessels also stained with NADPH diaphorase activity (Figure 1a).
Histochemical Experiments for NADPH Diaphorase Activity
Effects of pH 6.8–8.5 (n = 4 animals).
Within this range, a change in pH affected the background color of the sections. They were very pale lavender at pH 8.5, light blue at 7.4, and dark blue at 6.8. Although it appeared that the neuronal Type II response was abolished at pH 6.8, close examination revealed that the response was still present but was masked by the dark blue color of the background at this pH. It was not possible to determine from visual inspection if either the diffuse blue band or the fiber meshwork was present at pH 6.8. The neuronal Type I response, far more robust than the Type II, was easy to see at all three pHs. All subsequent qualitative tests were run at both pH 7.4 and 8.5, and the only discernible effect was to change the background color of the reaction, irrespective of the test (compare Figure 1d and Figure 3a).
Effect of Pre-exposure to Elevated Temperatures (n = 3 animals). Exposure of sections to elevated temperature for 5 min diminished (65C) or abolished (77C) both neuronal Type I and Type II responses, as well as that of the diffuse blue band. Sections exposed to RT and 55C were unaffected in their staining. This sensitivity to elevated temperatures indicates that these responses are enzyme-mediated (Figure 2a and Figure 2b).
Effect of Different Co-factors.
Neuronal Type I cells were stained when either -NADPH (n = 5 animals) or ß-NADPH (n = 10), was used (Figure 3a and Figure 3b). At the standard incubation time of 1.5 hr, however, the response with -NADPH was slightly less robust than with ß-NADPH. Somata were more weakly stained and dendrites were not stained for long distances (Figure 3b). Type II neurons and the diffuse blue band, however, were virtually absent when -NADPH was used as substrate for the standard incubation time of 1.5 hr (Figure 3b). This was not an absolute inability to use -NADPH, however, because when the reaction was allowed to proceed for many hours (10–20 hr) the response intensity resembled the normal response (data not shown). Interestingly, much of the meshwork of fine cortical fibers seen with ß-NADPH was also absent at 1.5-hr incubation with -NADPH and yet was present after extended incubation. nNOS has been reported to use both - and ß-NADPH (- or ß-NADPH or of ß-NADH (n = 4 animals) resulted in no staining (data not shown), so the reactions required a co-enzyme.
Effects of Chelators (n = 3 animals). In the concentration range tested, EDTA and EGTA had no effect. Therefore, none of the responses required Ca2+ or Mg2+ (data not shown).
Effect of Enzyme Inhibitors.
The inhibitors were selected on the basis of their effect on common enzymes that are known to produce a response in the NADPH diaphorase reaction (-NADPH) with 
10-5 M dicoumarol (n = 5 animals) (Figure 4a–d). The same concentrations of dicoumarol, however, greatly attenuated the neuronal Type II response seen in the standard 1.5-hr incubation (n = 5 animals) (Figure 4a and Figure 4b). Similarly, the same concentrations of dicoumarol greatly attenuated the NADPH diaphorase activities of the diffuse blue band and much of the meshwork of cortical fibers (n = 5 animals) (Figure 4a–d). Dicoumarol is known to be a specific and potent inhibitor of two diaphorases, DT diaphorase (E.C. 1.6.99.2) (
Effect of Electron Acceptor.
DPIP greatly attenuated all responses: neuronal Types I and II, the diffuse blue band; and the meshwork of cortical fibers (n = 3 animals; data not shown). This indicates that the NADPH diaphorase activities correspond to enzymes at the correct redox potential difference to transfer electrons from NADPH to DPIP instead of to NBT. Failure to transfer electrons to NBT results in an absence of colored formazan reaction product. The NADPH diaphorase activities of both NOS and dehydrogenases are known to transfer electrons from NADPH to DPIP (
Effect of Sulfhydryl Inhibitors.
NEM markedly attenuated neuronal Type I responses and abolished neuronal Type II and diffuse blue band responses (n = 3 animals; data not shown). Therefore, the reaction mechanism for these reactions required sulfhydryl groups. The mechanisms for NOS and dehydrogenase NADPH diaphorases are known to proceed via binding to sulfhydryl groups (
These histochemical tests showed that all NADPH diaphorase activities were abolished by elevated temperature and were attenuated by DPIP and NEM. The NADPH diaphorase activity of Type I neurons could use either - or ß-NADPH. In contrast, the NADPH diaphorase activities of Type II neurons and of the diffuse blue band had a strong preference for ß-NADPH over -NADPH and were greatly attenuated by dicoumarol (Table 1). Taken together, these results indicate that the bulk of the neuronal Type I response was most likely produced by NOS (
|
Antibody Experiments
Preliminary dilution series experiments established 1:500 as the most effective concentration for specific labeling by polyclonal anti-nNOS and monoclonal anti-eNOS in the hamster insular cortex. There was slightly less background in vibratome than in frozen sections (data not shown). The other antibodies (polyclonal anti-macNOS and anti-eNOS, monoclonal anti-nNOS) were also tested with dilution series but did not show any specific labeling.
Antibodies against the nNOS isozyme labeled scattered single cells. Cell somata and principal dendrites were labeled, as were a few scattered fibers running through the cortex (Figure 5a). Neither fine dendrites nor dendritic spines were seen. Similarly, the rich meshwork of cortical fibers visible with NADPH diaphorase staining was not seen. Occasionally a cell was labeled in Layers II–III of dysgranular and agranular insular cortex, but this label resembled that of Type I neurons. No label resembling the accumulation of Type II neurons or the diffuse blue band was observed.
Antibodies against the eNOS isozyme labeled only cortical endothelial cells (Figure 5b). Nothing resembling Type I or II neurons was labeled, and there was no label corresponding to the diffuse blue band.
In double label experiments (NADPH diaphorase and nNOS) in three animals, the number of labeled cell profiles in the insular cortex was scored in one of three categories: NADPHd+/nNOS+, NADPHd+/nNOS-, or NADPHd-/nNOS+ (Figure 6a and Figure 6b; total number of cells counted was 484). Most cells were double labeled (92 ± 1%), some cells were labeled by NADPH diaphorase but not anti-nNOS (3 ± 2%), and others were labeled by anti-nNOS but not NADPH diaphorase (5 ± 2%) [F(2,4) = 659.88; p<0.001] (Figure 7). Subsequent analysis showed that the number of double labeled cells was significantly different from that of singly labeled cells (p<0.05), but that the number of singly labeled cells was not different from each other. These results showed that under the present experimental conditions the neuronal Type I response was reliably associated with the presence of the nNOS isozyme.
|
No antibody label was ever seen to correspond to the diffuse blue band, despite repeated attempts with prolonged incubations, permeabilizing agents, frozen and vibratome tissues, and use of commercially available mono- and polyclonal antibodies to nNOS, eNOS, and macNOS.
|
Discussion |
|---|
|
TopSummaryIntroductionMaterials and MethodsResultsDiscussionLiterature Cited |
|---|
Methodological Considerations
The preservation of activity (both enzymatic and immunologic) in the tissue was central to the results. This was facilitated in two ways: by fixation with 4% formaldehyde and by maintaining the tissues at 4C for as many steps of the processing as possible.
There has been much debate as to the proper fixation conditions for histo- and immunohistochemical demonstration of NOS. Sensitivity to fixation by NOS was reported by
The histochemical experiments were subject to certain limitations. First, they were qualitative, not quantitative. The experiments were run for a period of time sufficient to see a qualitative difference, if one existed, on gross or microscopic visual inspection. For our purposes, 1.5 hr at RT was sufficient to see a difference between control and experimental conditions. Results that are more subtle than this would not be detected and would unknowingly be false-negatives.
Second, the conclusions from the inhibitor experiments depended on correct identification of the action of inhibitors. All inhibitor effects and working concentrations were selected from the literature. If an inhibitor has an undocumented effect, this will result in a false identification. Alternatively, if insufficient time was allotted to permit the inhibitor to penetrate into the appropriate cell compartment, a false-negative will be reported. This latter possibility was minimized by preincubating for 2 hr in the presence of 0.3% Triton X-100, far longer than the usual preincubation time of 10–20 min.
Third, most inhibitors were presented singly. Therefore, if several different enzymes contribute to the overall staining, inhibition of one or a few may not be sufficient to diminish the total staining. Two combinations were tested, (a) sodium azide and miconazole and (b) NEM and pyruvate. Sodium azide (5 mM) and miconazole (10-4 M) were presented simultaneously to see if a combination of mitochondrial and cytochrome P450 enzymes was contributing to the staining. This combination was found to have no detectable effect on the observed staining. NEM (10 mM) and pyruvate (60 mM) were also tested, because this combination inhibits the "nothing dehydrogenase" effect. This effect, first noted in the 1950s, occurred when no substrate was present in an enzymatic control reaction, but a colored reaction product was observed (
Fourth, experiments with inhibitors work only in a negative sense. If a reaction is blocked by the inhibitor, then (assuming there are no unknown inhibitor effects) the enzyme is scored as "present." If nothing inhibits the reaction, though, there potentially remains something else, undetected, that may contribute to the stain. Therefore, although most of the neuronal Type I response appears to be attributable to nNOS, perhaps some other enzyme, thus far undetected, is contributing to the total staining.
Antibody experiments have other possible pitfalls. A major problem is the possible failure of the antibodies to penetrate into a compartment where the target antigen is located, resulting in false-negatives. This was minimized by including a detergent, 0.1% NP40, throughout the procedure. Unless a target antigen was located within multiply membraned organelles, e.g., in the matrix of the mitochondria, it is likely that permeabilization was not a major problem because two different antibodies, those to nNOS and eNOS, specifically bound to target antigens.
Another potential source of false-negatives is the slight differences between the immunogen (human nNOS and eNOS and mouse macNOS) and the endogenous antigen, such that the endogenous target may not be recognized by the antibody. An attempt was made to minimize this possibility by the use of polyclonal antibodies, which included antibodies raised against a variety of immunogenic sites. This strategy was not entirely successful. Although the polyclonal anti-nNOS worked and the monoclonal anti-nNOS did not, the polyclonal anti-eNOS did not work but the monoclonal anti-eNOS did.
Yet another source of potential false-negatives is if the antigen is present in tissue at levels below the detectability of the secondary system. A commercial ABC kit was used to visualize the primary antibodies because this struck a balance between high sensitivity and ease of use. It remains possible, however, that some signal went undetected because of insufficient amplification by the secondary antibody.
A potential source of false-positive responses is the endogenous cellular peroxidases. This possibility was minimized by quenching the peroxidases by pretreating with excess H2O2.
A further potential source of a false-positive response is the presence in the tissue of an immunoreactive substance that is recognized by the antibody but that is not NOS. This possibility exists with any antibody and cannot be escaped. For this reason, all positive responses to NOS antibodies presented in this work are more precisely termed "NOS-like immunoreactivity."
There Are at Least Three NADPH Diaphorase Activities in the Insular Cortex of the Hamster
The results lead to the conclusion that there are at least three different NADPH diaphorase activities in the insular cortex of the hamster. The neuronal Type I response was co-localized in neurons with immunoreactivity to nNOS, could use either - or ß-NADPH (but showed a slight response decrement with -NADPH), and was not inhibited by any of the histochemical tests (except for a slight decrement with dicoumarol). This response pattern is consistent with the activity reported for what was subsequently identified as nNOS in tissue fixed with 4% formaldehyde (-NADPH, they used PLP fixative rather than just 4% formaldehyde. Given the apparent sensitivity of NOS to fixation conditions, as discussed above, it appears best to compare the present results with those seen by others who used the same fixative (4% formaldehyde), i.e.,
The neuronal Type I response showed a slight diminution of the intensity of the response when either dicoumarol or -NADPH was used. This can be interpreted in at least two ways: (a) nNOS has a sensitivity to dicoumarol and a slight preference for ß-NADPH, at least in the hamster, or (b) the total staining in Type I neurons also contains a component of staining contributed by non-NOS diaphorases that are sensitive to dicoumarol and show a preference for ß-NADPH (e.g., such as the neuronal Type II response). The second interpretation appears more likely because (a)
A third interpretation is that Type I neurons differ from Type II neurons only in having a greater concentration of non-NOS diaphorases. This interpretation would then hold that the dicoumarol has the same attenuating effect on neuronal Type I and II activities but that there is such a great concentration of enzyme in the Type I neuron that reaction product can accumulate and be visible. This interpretation appears unlikely given our demonstrated co-localization of neuronal Type I NADPH diaphorase activity with nNOS-like immunoreactivity [which has a very vigorous NADPH diaphorase activity (
A second NADPH diaphorase activity was co-localized in endothelial cells with immunoreactivity to eNOS. The typical 1.5-hr incubation was occasionally but not usually long enough to see this activity using the NADPH diaphorase reaction, so its histochemical characteristics were not systematically determined. However, this endothelial cell diaphorase activity was usually visible if the reaction was allowed to proceed longer (Figure 1a).
A third NADPH diaphorase activity was found in the Type II neurons, the diffuse blue band, and in much of the cortical fiber meshwork. This third activity did not co-localize with any NOS-like immunoreactivity (see Results), had a strong preference for ß-NADPH relative to -NAPDH, and was greatly attenuated by 10-5 M dicoumarol. The strong preference for ß- versus -NADPH identified the enzyme as a dehydrogenase (
Modification of NADPH Diaphorase Reaction for NOS
The fixation protocol for NADPH diaphorase used 4% formaldehyde for a minimum of 45 min during transcardial perfusion, followed by an additional 1–4 hr of postfixation in 4% formaldehyde. In accordance with the results of
In the present work dicoumarol permitted easy discrimination between the diaphorase activities due primarily to nNOS and those due primarily to other enzymes. For other tissues, which may contain other enzymes, either a cocktail of inhibitors for the most likely enzymes in that tissue should be added (e.g., a mixture of miconazole, dicoumarol, sodium azide and levamisole) or a series of control experiments with individual inhibitors must be carried out. Alternatively, the presence of NOS-containing cells could be confirmed with antibodies directed against NOS. This last method may produce false-negatives, however, if the antibody is ineffective in the particular tissue preparation. When there is NADPH diaphorase staining but no labeling by anti-NOS antibody (e.g., as with the diffuse blue band, in that no antibody binding in that region was detected) and to avoid false-negatives, it is necessary to test a number of inhibitors and/or stereoisomers of the co-factor to see if the staining is attenuated and therefore is due to something other than NOS. Ideally, a specific inhibitor of the diaphorase activity of NOS could be used to test for the specificity of the NOS diaphorase response, but no such inhibitor exists.
Dicoumarol-sensitive, NADPH-dependent Enzymes
The work reported here shows that a dicoumarol-sensitive dehydrogenase was likely responsible for the bulk of the staining of the Type II neurons, the diffuse blue band, and the meshwork of cortical fibers, as well as some of the staining seen in Type I neurons. There are three enzymes that are known to be inhibited by dicoumarol (
"DT diaphorase" was named for its ability to use both DPNH (reduced diphosphopyridine nucleotide, now called NADH) and TPNH (reduced triphosphopyridine nucleotide, now called NADPH) (
NADPH dehydrogenase (quinone) (EC 1.6.99.6) is a flavoprotein, but other than the original purification and characterization report by Koli et al. in 1969, nothing else is known about this enzyme. It is not as sensitive to dicoumarol as DT diaphorase. Inhibition in cell-free extracts occurs in the range 10-5 M (
NADPH:quinone reductase (EC 1.6.5.5) has substrate specificity for certain quinones, and therefore is not likely to be responsible for the observed NADPH diaphorase activity.
Alternatively, the neuronal Type II response could be produced by a novel or incompletely characterized dehydrogenase with high sensitivity to dicoumarol, or by a group of electron transport enzymes, one of which has sensitivity to dicoumarol. The latter possibility appears unlikely, given the failure of sodium azide (which blocks the last step of the electron transport chain) to affect NADPH diaphorase staining. Given the uncertainty regarding the identity of the enzyme, it will be referred to here as "dicoumarol-sensitive NADPH dehydrogenase." This enzyme was found along almost the entire length of the rhinal fissure (unpublished observations). If extensive spatial distribution is a criterion of functional importance, it is likely to be an important enzyme for the brain. More work is needed to determine the precise identity and role(s) of the dicoumarol-sensitive NADPH dehydrogenase.
|
Acknowledgments |
|---|
Supported by a grant (5P50DC00168) and by a training grant (5T33DC00025) from the National Institutes of Deafness and Communication Disorders.
We thank Sandra Hill and Richard Weinberg for helpful suggestions on the techniques and Lawrence Savoy for excellent technical assistance. We thank Michael Barry, Thomas Hettinger, Michael Huerta, Konrad Talbot, and William Shoemaker for helpful comments on the manuscript.
Received for publication October 2, 1998; accepted October 6, 1998.
|
Literature Cited |
|---|
|
TopSummaryIntroductionMaterials and MethodsResultsDiscussionLiterature Cited |
|---|
Bairoch A (1996) The ENZYME data bank in 1995. Nucleic Acids Res 24:221–222, accessed via "ENZYME: Enzyme nomenclature database" (release 23.0; includes updates to 10 September 1998), ExPASy Server for Molecular Biology, Swiss Institute of Bioinformatics, <http://expasy.hcuge.ch/sprot/enzyme.html (accessed 14 September 1998)
Bredt DS (1995) Molecular characterization of nitric oxide synthase. In Vincent SR, ed. Nitric Oxide in the Nervous System. San Diego, Academic Press, 1-20
Bredt DS, Hwang PM, Snyder S (1990) Localization of nitric oxide synthase indicating a neural role for nitric oxide. Nature 347:768-770[Medline]
Dawson TM, Bredt DS, Fotuhi M, Hwang PM, Snyder SH (1991) Nitric oxide synthase and neuronal NADPH diaphorase are identical in brain and peripheral tissues. Proc Natl Acad Sci USA 88:7797-7801
Dixon M (1979) The Enzymes. 3rd Ed. New York, Academic Press
Ernster L, Danielson L, Ljungren M (1962) DT Diaphorase. I. Purification from the soluble fraction of rat-liver cytoplasm, and properties. Biochim Biophys Acta 58:171-188[Medline]
Hope BT, Michael GJ, Knigge KM, Vincent SR (1991) Neuronal NADPH diaphorase is a nitric oxide synthase. Proc Natl Acad Sci USA 88:2811-2814
Hope BT, Vincent SR (1989) Histochemical characterization of neuronal NADPH-diaphorase. J Histochem Cytochem 37:653-661[Abstract]
Koli AK, Yearby C, Scott W, Donaldson KO (1969) Purification and properties of three separate menadione reductases from hog liver. J Biol Chem 24:621-629
Levine W, Giuditta A, Englard S, Streckers HJ (1960) Brain diaphorases. J Neurochem 6:28-36
Li R, Bianchet MA, Talalay P, Amzel LM (1995) The three-dimensional structure of NAD(P)H:quinone reductase, a flavoprotein involved in cancer chemoprotection and chemotherapy: mechanism of the two-electron reduction. Proc Natl Acad Sci USA 92:8846-8850
Lind C, Cadenas E, Hochstein P, Ernster L (1990) DT-diaphorase: purification, properties, function. Methods Enzymol 186:287-301[Medline]
Matsumoto T, Nakane M, Pollock JS, Kuk JE, Förstermann U (1993) A correlation between soluble brain nitric oxide synthase and NADPH-diaphorase activity is only seen after exposure of the tissue to fixative. Neurosci Lett 155:61-64[Medline]
Murphy TH, De Long MJ, Coyle JT (1991) Enhanced NAD(P)H: quinone reductase activity prevents glutamate toxicity produced by oxidative stress. J Neurochem 56:990-999[Medline]
Pearse AGE (1972a) Oxidoreductases II. In Pearse AGE, ed. Histochemistry: Theoretical and Applied. 3rd Ed. Baltimore, Williams & Wilkins, 921-961
Pearse AGE (1972b) Principles of oxidoreductase histochemisty. In Pearse AGE, ed. Histochemistry: Theoretical and Applied. 3rd Ed. Baltimore, Williams & Wilkins, 880-920
Racker E (1955) Mechanism of action and properties of pyridine nucleotide-linked enzymes. Physiol Rev 35:1-56
Schmidt HHHW, Gagne GD, Nakane M, Pollock JS, Miller MF, Murad F (1992) Mapping of neural nitric oxide synthase in the rat suggests frequent co-localization with NADPH diaphorase but not with soluble guanylyl cyclase, and novel paraneural functions for nitrinergic signal transduction. J Histochem Cytochem 40:1439-1456[Abstract]
Schultzberg M, Segura–Aguilar J, Lind C (1988) Distribution of DT diaphorase in the rat brain: biochemical and immunohistochemical studies. Neuroscience 27:763-776[Medline]
Schuman EM (1995) Nitric oxide signaling, long-term potentiation and long-term depression. In Vincent SR, ed. Nitric Oxide in the Nervous System. San Diego, Academic Press, 1-20
Smith DV, Frank ME (1993) Sensory coding by peripheral taste fibers. In Simon SA, Roper SD, eds. Mechanism of Taste Transduction. Boca Raton, FL, CRC Press, 295-338
Spessert R, Layes E (1994) Fixation conditions affect the intensity but not the pattern of NADPH-diaphorase staining as a marker for neuronal nitric oxide in rat olfactory bulb. J Histochem Cytochem 42:1309-1315[Abstract]
Spessert R, Wohlgemuth C, Reuss S, Layes E (1994) NADPH-diaphorase activity of nitric oxide synthase in the olfactory bulb: co-factor specificity and characterization regarding the interrelation to NO formation. J Histochem Cytochem 42:569-575[Abstract]
Stoward PJ, Altman FP, Seidler E (1991a) Principles of oxidoreductase histochemistry. In Stoward PJ, Pearse AGE, eds. Histochemistry: Theoretical and Applied. Vol 3. 4th Ed. Edinburgh, Churchill Livingstone, 1-25
Stoward PJ, Meijer AEFH, Seidler E, Wohlrab F (1991b) Dehydrogenases. In Stoward PJ, Pearse AGE, eds. Histochemistry: Theoretical and Applied. Vol 3. 4th Ed. Edinburgh, Churchill Livingstone, 27-71
Stoward PJ, Pearse AGE, eds. (1991) Histochemistry: Theoretical and Applied. 4th ed. Edinburgh, Churchill Livingstone
Tracey WR, Nakane M, Pollock JS, Förstermann U (1993) Nitric oxide synthases in neuronal cells, macrophages and endothelium are NADPH diaphorases, but represent only a fraction of total cellular NADPH diaphorase activity. Biochem Biophys Res Commun 195:1035-1040[Medline]
Valtschanoff JG, Weinberg RJ, Kharazia VN, Nakane M, Schmidt HHHW (1993a) Neurons in rat hippocampus that synthesize nitric oxide. J Comp Neurol 331:111-121[Medline]
Valtschanoff JG, Weinberg RJ, Kharazia VN, Schmidt HHHW, Nakane M, Rustioni A (1993b) Neurons in rat cerebral cortex that synthesize nitric oxide: NADPH diaphorase histochemistry, NOS immunocytochemistry, colocalization with GABA. Neuroscience Lett 157:157-161[Medline]
Van Noorden CJF, Kooij A, Vogels IMC, Fredriks WM (1985) On the nature of the `nothing dehydrogenase' reaction. Histochem J 17:1111-1118[Medline]
Vincent SR (1995) Localization of nitric oxide neurons in the central nervous system. In Vincent SR, ed. Nitric Oxide in the Nervous System. New York, Academic Press, 83-102
Wenland B, Schweizer FE, Tyan TA, Nakane M, Murad F, Scheller RH, Tsien RW (1994) Existence of nitric oxide synthase in rat hippocampal pyramidal cells. Proc Natl Acad Sci USA 91:2151-2155
Yamamoto T, Shimura T, Sako N, Yasoshima Y, Sakai N (1994) Neural substrates for conditioned taste aversion in the rat. Behav Brain Res 65:123-137[Medline]
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  Z. Bayram, M. Asar, S. Cayli, and R. Demir Immunocytochemical Detection of Neuronal Nitric Oxide Synthase (nNOS)-IR in Embryonic Rat Stomach Between Days 13 and 21 of Gestation J. Histochem. Cytochem., May 1, 2002; 50(5): 671 - 680. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| Guidelines | Subscriptions | About | exPRESS - Current - |