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.

https://www.pnas.org/content/pnas/88/7/2811.full.pdf

Neuronal NADPH diaphorase is a nitric oxide synthase.

Section I-Peripheral Nerve.- 1 Peripheral Nerve Structure.- 1. Introduction.- 2. Histology and Development.- 3. The Axon.- 3.1. Filaments and Microtubules.- 3.2. Other Organelles and the Axolemma.- 4. Sheaths of Axons.- 4.1. Schwann Cells.- 4.2. Myelin.- 4.3. Function of Schwann Cells and Their Myelin Sheaths.- 4.4. Connective Tissue Sheaths.- 5. References.- 2 The Nerve Impulse.- 1. Introduction.- 2. Passive Electrical Properties.- 3. Voltage-Clamp Analysis of the Ionic Current.- 4. Momentary Current-Voltage Relations.- 5. The Threshold Conditions for Excitation.- 6. Factors Determining Conduction Velocity.- 7. References.- 3 Axoplasmic Transport-Energy Metabolism and Mechanism.- 1. Introduction.- 2. Fast Axoplasmic Transport.- 2.1. Characterization.- 2.2. Mechanism and Energy Supply.- 2.3. Transport and Membrane Function.- 3. Slow Axoplasmic Transport.- 3.1. Characterization.- 3.2. Mechanism.- 4. References.- Section IIA-Junctional Transmission-Structure.- 4 Neuromuscular Junctions and Electric Organs.- 1. Introduction.- 2. The Typical Neuromuscular Junction.- 2.1. Distribution and Location of Nerve Terminals.- 2.2. The Axon.- 2.3. The Synaptic Space.- 2.4. Postjunctional Muscle Fiber.- 3. Variations of Motor End Plates.- 3.1. Variations from Class to Class.- 3.2. Endings on Slow-Twitch and Rapid-Twitch Fibers.- 3.3. Endings on Slow Tonic Muscle Fibers.- 4. Electric Organs.- 4.1. Electrocytes.- 4.2. Innervation and Ultrastructure.- 5. References.- 5 The Peripheral Autonomic System.- 1. Anatomical Considerations: Sympathetic and Parasympathetic Divisions.- 2. Morphological Observations.- 2.1. Preganglionic Neurons.- 2.2. Postganglionic Neurons.- 2.3. Adrenal and Extra-Adrenal Chromaffin Cells.- 3. References.- 6 Ultrastructure of Ganglionic Junctions.- 1. General Considerations.- 2. Sympathetic Ganglia.- 2.1. Amphibia.- 2.2. Reptiles.- 2.3. Mammals.- 2.4. Some Effects of Different Fixatives.- 3. Parasympathetic Ganglia.- 3.1. Ciliary Ganglion.- 3.2. Otic Ganglion.- 3.3. Ganglia of the Enteric Plexuses.- 3.4. Cardiac Ganglion Cells.- 4. Summary and Comment.- 5. References.- Section IIB-Junctional Transmission-Function.- 7(i) Neuromuscular Transmission-Presynaptic Factors.- 1. Synthesis, Storage, and Release of Acetylcholine.- 1.1. Synthesis of ACh.- 1.2. Storage and Release.- 2. The Acceleration of Release by Nerve Impulses.- 2.1. The Role of the Nerve Impulse.- 2.2. The Role of Ca2+.- 2.3. After- Effects of Depolarization-Secretion Coupling.- 3. References.- 7 (ii) Neuromuscular Transmission-The Transmitter-Receptor Combination.- 1. Introduction.- 2. Molecular Basis of Chemoelectric Transduction.- 3. Pharmacology.- 4. Chemical Nature of the Acetylcholine Receptor.- 5. Desensitization.- 6. References.- 7 (iii) Neuromuscular Transmission-Enzymatic Destruction of Acetylcholine.- 1. Location and Measurement of Cholinesterases at the Junction.- 1.1. Histochemical Staining.- 1.2. Microchemical Methods.- 1.3. Assay of External AChE.- 1.4. Radioautographic Methods.- 2. Amounts and Types of Cholinesterase at the Junctions.- 3. Requirement for AChE in Impulse Transmission.- 4. Relation of AChE to ACh- Receptors.- 5. Quantitative Relation of AChE to ACh at the End Plate.- 6. References.- 8 Ganglionic Transmission.- 1. Introduction.- 2. Response of Autonomic Ganglia to Preganglionic Volleys.- 2.1. Response of Normal Ganglia.- 2.2. Response of Curarized Ganglia.- 2.3. Slow Ganglionic Responses and Afterdischarges.- 3. Electrical Constants of Ganglion Cell Membrane.- 4. Action Potentials of Single Ganglion Cells.- 4.1. Response to Antidromic Stimulation.- 4.2. Response to Direct Intracellular Stimulation.- 4.3. Response to Orthodromic Stimulation.- 4.4. Ionic Requirement for Generation of Action Potential.- 5. Nature and Electrogenesis of Postsynaptic Potentials.- 5.1. The "Fast" Excitatory Postsynaptic Potential.- 5.2. The "Slow" Excitatory Postsynaptic Potential.- 5.3. The "Late Slow" Excitatory Postsynaptic Potential.- 5.4. The "Slow" Inhibitory Postsynaptic Potential.- 6. Cholinergic and Adrenergic Receptors at Preganglionic Nerve Terminals.- 6.1. Cholinergic Receptor Site.- 6.2. Adrenergic Receptor Site.- 7. References.- 9 Function of Autonomic Ganglia.- 1. Introduction.- 2. Ganglia as Coordinating Centers.- 2.1. The Relay Hypothesis of Ganglionic Function.- 2.2. Development of a Stochastic Hypothesis.- 3. Experimental Evidence.- 3.1. Observed Patterns of Innervation.- 3.2. Ganglionic Activity and Factors Influencing It.- 3.3. Relative Autonomy of Ganglia.- 4. Conclusions.- 5. References.- 10 Peripheral Autonomic Transmission.- 1. Introduction.- 2. Definition of the Autonomic Neuromuscular Junction.- 2.1. Relation of Nerve Fibers to Muscle Effector Bundles.- 2.2. Relation of Nerve Fibers to Individual Smooth Muscle Cells.- 3. Adrenergic Transmission.- 3.1. Introduction.- 3.2. Structure of Adrenergic Neurons and Storage of Noradrenaline.- 3.3. Electrophysiology of Adrenergic Transmission.- 3.4. Ionic Basis of the Action of Catecholamines on the Postjunctional Membrane.- 3.5. Summary.- 4. Cholinergic Transmission.- 4.1. Introduction.- 4.2. Localization of Acetylcholinesterase.- 4.3. Electrophysiology of Cholinergic Transmission.- 4.4. Ionic Basis of the Action of ACh on the Postjunctional Membrane.- 4.5. Summary.- 5. Purinergic Transmission.- 5.1. Introduction.- 5.2. Electrophysiology of Purinergic Transmission.- 5.3. Summary.- 6. Conclusions.- 7. References.- 11 "Trophic" Functions.- 1. Introduction.- 2. Regulation of Taste Buds.- 3. Regulation of Amphibian Limb Regeneration.- 4. Regulation of Physiological and Metabolic Properties of Muscle.- 4.1. Resting Membrane Potential.- 4.2. Acetylcholine Sensitivity.- 4.3. Cholinesterase Activity.- 4.4. The Role of ACh Release.- 4.5. The Dynamic Nature of the Muscle Fiber.- 4.6. Plasticity of the Motor Unit.- 5. Mechanisms of Neural Regulation.- 6. References.- Section III-Receptors-Structure and Function.- 12 Cutaneous Receptors.- 1. Introduction.- 2. Morphology of Cutaneous Nerves.- 2.1. Uniformity of Cutaneous Axons.- 2.2. Relative Numbers of Myelinated and Nonmyelinated Axons.- 3. Morphology of Cutaneous Receptors.- 3.1. Encapsulated Receptors.- 3.2. Unencapsulated Corpuscular Receptors.- 3.3. Noncorpuscular Receptors.- 4. Physiology of Cutaneous Receptors.- 4.1. Cutaneous Mechanoreceptors.- 4.2. Cutaneous Thermoreceptors.- 4.3. Nociceptors.- 5. References.- 13 The Pacinian Corpuscle.- 1. Introduction.- 2. Morphology.- 3. Afferent Responses to Mechanical Stimuli.- 4. Mechanical Properties of the Corpuscle.- 5. Receptor Potentials.- 6. Impulse Activity in the Nerve Terminal.- 7. Distribution of Pacinian Corpuscles.- 8. Central Effects of Impulses from Pacinian Corpuscles.- 9. References.- 14 Receptors in Muscles and Joints.- 1. Introduction.- 2. Joint Receptors.- 3. Tendon Organs.- 4. Muscle Spindles.- 4.1. Reptiles.- 4.2. Amphibia.- 4.3. Birds.- 4.4. Mammals.- 5. Uncertain Origin of Adaptation.- 6. References.- 15 Enteroceptors.- 1. Introduction.- 2. Methods.- 2.1. Histology.- 2.2. Physiology.- 3. Cardiovascular Receptors.- 3.1. Systemic Arterial Baroreceptors.- 3.2. Pulmonary Arterial Baroreceptors.- 3.3. Ventricular Receptors.- 3.4. Atriovenous Receptors.- 4. Respiratory System Receptors.- 4.1. Cough and Irritant Receptors.- 4.2. Pulmonary Stretch Receptors.- 4.3. Type J Receptors.- 4.4. Other Receptors.- 5. Alimentary System Receptors.- 5.1. Muscular Receptors.- 5.2. Serosal Receptors.- 5.3. Muscularis Mucosae Receptors.- 5.4. Chemoreceptors.- 5.5 Hepatic Osmoreceptors.- 6. Urinary Tract Receptors.- 6.1. Bladder.- 6.2. Urethra.- 7. Other Enteroreceptors.- 8. References.- 16 Arterial Chemoreceptors.- 1. Introduction.- 2. Structure.- 2.1. Light Microscopy.- 2.2. Electron Microscopy.- 2.3. Degeneration Studies.- 3. Function.- 3.1. Types of Activity in the Nerve Supply to the Receptor Complex.- 3.2. The Type I Cell.- 4. The Identity of the Receptor.- 4.1. The Received View.- 4.2. A New Hypothesis.- 5. References.- 17 Taste Receptors.- 1. Introduction.- 2. Gustatory Nerve Fiber Response to Chemical Stimuli.- 2.1. Multiple Sensitivity of Single Chorda Tympani Fibers.- 2.2. Neural Code for Quality of Taste and "Across-Fiber Pattern" Theory.- 3. Electrical Responses of Gustatory Cells to Chemical Stimuli.- 3.1. Innervation and Structure of Taste Bud.- 3.2. How Do Gustatory Cells Respond to Chemical Stimuli?.- 4. References.

The Peripheral Nervous System

Publisher Summary This chapter presents a procedure for the preparation of γ- glutamyl transpeptidase In the assay of γ- glutamyl transpeptidase the most widely used substrate is L-y-glutamyl-p-nitroanilide p -Nitroaniline, released during transpeptidation and hydrolysis, is readily determined from the increase in absorbance at 410 nm A variety of other procedures has been used to determine the activity of γ-glutamyl transpeptidase Other chromogenic substrates include L-γ- glutamylanilide and L-γ-glutamylnaphthylamides Assay procedures in which glutathione is used are complicated by its oxidation to glutathione disulfide; oxidation can be retarded by including ethylenediaminetetraacetic acid in the reaction mixtures Convenient spectrophotometric assays involving the use of S -substituted glutathione derivatives are also available Such derivatives include S -pyruvoylglutathione and S -acetophenoneglutathione  For purification procedures, various methods of solubilization are employed including the treatment of the particulate enzyme with detergents, organic solvents, and proteinases The rat kidney enzyme can be purified following its solubilization with either proteinases (eg, papain and bromelain) or detergents (eg, Triton X-100 and Lubrol WX)

Gamma-glutamyl transpeptidase.

This progress report is concerned primarily with problems relating to the structure and reactions of hydroxamic acids and N-hydroxyimides, and also surveys some of the biological activities of these compounds. Particular significance attaches to the Lossen rearrangement of O-acylated hydroxamic acids, which leads to isocyanates or their reaction products.

The Chemistry of Hydroxamic Acids and N-Hydroxyimides

http://directory.umm.ac.id/Data%20Elmu/jurnal/A/Aquaculture/Vol182.Issue3-4.Feb2000/61079.pdf

Distribution of intestinal enzyme activities along the intestinal tract of cultured Nile tilapia, Oreochromis niloticus L.

Some important principles in 3,3'-diaminobenzidine ultrastructural cytochemistry.

Monotetrazolium salts were designed and prepared which incorporate a phthalhydrazide moiety to make them lipophobic and a benzothiazole moiety to make them react with osmium tetroxide after they are reduced to the corresponding formazans. The tetrazolium salts themselves do not react with osmium tetroxide under the conditions used for the cytochemical demonstration of enzyme activities. Although all of the new formazans, when dissolved in dimethylformamide, were reoxidized to the tetrazolium salts by osmium tetroxide, they were not reoxidized by osmium tetroxide when they were dissolved in tetrahydrofuran or precipitated by reduction in tissue, but gave dark complexes. One of the tetrazolium salts, 2-(2’-benzothiazolyl)-5-styryl-3-(4’-phthalhydrazidyl) tetrazolium chloride (BSPT), was readily reduced by succinic dehydrogenase activity, gave a formazan which produced a dark osmium complex relatively rapidly and gave good localization of succinic dehydrogenase activity on the membranes of mitochondria of rat myocardial cells. Although this tetrazoliu.m salt (BSPT) was not photoreduced by fresh cells of Elodea to stain chloroplasts as seen with nitroblue tetrazolium or distyryl nitroblue tetrazolium, the 5-p-nitrophenyl derivative of BSPT was photoreduced by chloroplasts. The preparation of BSPT and its use in demonstrating succinic dehydrogenase activity ultracytochemically is given here. The preparation and ultracytochemical use of the 5-p-nitrophenyl derivative in chioroplasts will be published later.

Nonosmiophilic tetrazolium salts that yield osmiophilic, lipophobic formazans for ultrastructural localization of dehydrogenase activity

When 4-aminophthalhydrazide is freshly diazotized and used as the coupling agent in demonstrating aminopeptidase activity or γ-glutamyl transpeptidase activity, with substrates that yield 4-methoxy-2-naphthylamine or even 2-naphthylamine, azo dyes are produced which are readily osmiophilic. Although the distribution of the reddish azo dyes is similar to that noted with earlier methods in light microscopy, the distribution of the deposits in electron microscopic preparations is distinctly membraneous in location in contrast to the droplet distribution of previous methods. We attribute membraneous localization of enzymatic activity with the new methods presented here to the lipophobic properties of the azo dyes provided by their 4-aminophthalhydrazide (4-APH) moiety. The new agent 4-aminophthalhydrazide was designed with this in mind.

MEMBRANEOUS ULTRASTRUCTURAL DEMONSTRATION OF AMINOPEPTIDASE AND γ-GLUTAMYL TRANSPEPTIDASE ACTIVITIES WITH A NEW DIAZONIUM SALT THAT YIELDS A LIPOPHOBIC, OSMIOPHILIC AZO DYE:

Ultraviolet Absorption Spectra of Some Hydroxamic Acids and Hydroxamic Acid Derivatives

The preparation of a new osmiophilic ditetrazolium salt (DS-NBT) containing two styryl groups, but retaining the essential structural features of nitro-BT, is given. DS-NBT may be used to demonstrate succinic dehydrogenase activity (SDH) faster than with nitro-BT and fully as rapidly as with TC-NBT. DS-NBT is more stable than TC-NBT and it is more reliably and more readily osmicated than TC-NBT. The ultrastructural sites of SDH and NADH2 diaphorase activity are similar to those shown for cytochrome oxidase activity with diaminobenzidine in rat heart. The sites of lactic dehydrogenase activity are found in the sarcoplasmic reticulum and mitochondria of rat skeletal muscle fixed in formaldehyde for 3 hr. Tissue penetration of the doubly charged ditetrazolium salt (DS-NBT) was facilitated by 40-sec sonication and by 5% dimethyl sulfoxide. Illustrative examples and controls are included.

An osmiophilic distyryl ditetrazolium salt (ds-nbt) for ultrastructural dehydrogenase activity

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