Article

Der histochemische Nachweis der Monoaminooxidase mit Thiazolyl-substituierten Tetrazoliumsalzen in Gegenwart von Metallionen

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Abstract

Es wird eine Methode zum histochemischen Nachweis des Monoaminoxydase-systems beschrieben. Unter Verwendung thiazolyl-substituierter Tetrazoliumsalze erfolgt in Gegenwart von Metallionen und Tryptaminhydrochlorid als Substrat die Bildung eines feingranulren Metallformazankomplexes. Die Gre und Beschaffenheit des Komplexes spricht fr eine intramitochondriale Lokalisation der MAO. Die Lokalisation der MAO in einigen Organen und Geweben wird beschrieben.A method is described for histochemical demonstration of monoamine oxidase system. Using tryptamine hydrochloride as substrate with thiazolyl-substituted tetrazolium salts in presence of metal ions there is formed a fine granular metalformazan complex. The size and quality of the complex suggest an intramito-chondrial localization of MAO. The distribution of MAO in some organs and tissues is described.

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Chapter
Histo- and cytochemical methods locate separately in cells or tissues the chemicals that appear in the same biochemically separated fraction of a tissue. Histochemical methods might distinguish a greater diversity of related compounds in a tissue than can be recognized by biochemical methods. Failure to recognize the same things as identical in the two settings increases the difficulties, giving rise to confusing differences in nomenclature. Inorganic components of tissues might cause difficulty from a histochemical point of view because they occur bound in such ways that they are not reactive, because they occur in water-soluble ionic forms, or because their concentration is below the levels of sensitivity of available methods. Negative results then, as indeed with any material, have limited value in excluding the presence of any of these materials. However, evidence for the existence of copious inorganic materials that are not easily demonstrated by histochemical staining methods is provided by the technique of microincineration that the results obtained might serve as an object lesson in relation to this point.
Article
A transition metal compound that is bound in tissues by any appropriate cytochemical reaction may catalyze the generation of an insoluble osmiophilic polymer from organic monomers such as 3,3'-diaminobenzidine. When the polymers are treated with osmium tetroxide, electron-opaque, insoluble osmium blacks (coordination polymers of osmium) are formed at the sites of the particular macromolecule or enzyme permitting its light, and electron, microscopic localization. This approach represents a distinct advantage over earlier cytochemical methods because the shorter incubation time needed here results in less artifactual deposition of metal ions, and less tendency to crystallize the reaction product. In addition, the shorter incubation times permit longer fixation of tissues and hence less artifact due to enzyme diffusion.
Article
The developmental pattern of H2O2-producing oxidases (OX) was studied in chick kidneys (mesonephros, metanephros), intestine, liver, yolk sac and adrenal glands between embryonic days (ED) 5-20 as well as in chick organs after hatching. Sections from snap frozen tissue fixed in cold cacodylate-buffered 2% glutaraldehyde were processed by cerium-DAB-Co-H2O2 methods for benzylamine OX, diamine OX, histamine OX, alpha-hydroxyacid OX, D-amino acid OX (AAOX) and monoamine OX (MAOX). Prenatally, only activities of AAOX and MAOX could be demonstrated. AAOX appeared primarily in the proximal tubular cells of both types of kidneys. In the metanephros the enzyme was also detected in the thick ascending limbs of Henle's loops. The amount of reaction product in tubular cells increased with their maturation. MAOX activity was detected in immature enterocytes, in smooth muscle cells of large systemic arteries (on ED 5-6) as well as in proximal tubular cells of the mesonephros and adrenal gland. Later the enzyme appeared also in smooth muscle cells of the intestinal wall and in endothelial and smooth muscle cells of arterioles of the mesonephros. In the metanephros MAOX was detected at the same locations with a time delay because of a developmental shift of the kidney. Inhibition tests revealed that MAOX differs in epithelial cells from that in smooth muscle cells. Benzylamine OX, diamine OX and histamine OX were detected postnatally in smooth muscle cells of the arterial media and muscularis externa of the intestinal wall with low activities. It is concluded that MAOX and AAOX activities represent useful markers in the development of renal tubules. In addition, MAOX activity can be considered an indicator of maturation of components of the vascular wall.
Article
The histochemically detectable monoamine oxidase activity in certain organs of young and old rats is compared. Regardless of age, the activity is strong in the liver, faint in the skeletal muscle, and absent in the kidney. In the myocardium, however, the quantity of monoamine oxidase increases strongly with age. Its activity is manifest in the form of granular and diffuse formazan precipitates; both disappear after a preliminary treatment of the animals with a monoamine oxidase inhibitor. This finding indicates that the diffuse as well as the previously identified granular precipitates represent monoamine oxidase.
Article
Methods are presented for the intramitochondrial localization of various diphosphopyridine nucleotide and triphosphopyridine nucleotide-linked dehydrogenases in tissue sections. The cytochemical reactions studied involve the oxidation of the substrates by a specific pyridino-protein. The electron transfer of tetrazolium salt is mediated by the diaphorase system associated with the dehydrogenase. The final electron acceptor was either p-nitrophenyl substituted ditetrazole (nitro-BT) or N-thiazol-2-yl monotetrazole (MTT), the latter giving rise to metal formazan in the presence of cobaltous ions. Mitochondrial localization of the formazan precipitate could be achieved by using hypertonic incubating media containing high concentrations of substrate and co-enzyme. A fast reduction of tetrazolium salt was obtained by chemically blocking the respiratory chain enzymes beyond the flavoproteins. Although diaphorase systems are implicated in the reduction of tetrazolium salts, specific dehydrogenases are solely responsible for the distinct distribution pattern obtained in tissues with various substrates. The present findings in tissue sections are discussed in conjunction with existing biochemical evidence from differential centrifugation experiments.
Article
Die Darstellung der C,N-Diphenyl-N'-thiazolyl-(2)-formazane, die in der 4- bzw. 5-Stellung des Thiazolrings substituiert sind, erfolgte entweder durch Kupplung von diazotiertem Anilin mit Benzaldehydthiazolyl-(2)-hydrazonen oder durch Kupplung von diazotierten 2-Amino-thiazolen mit Benzaldehyd-phenylhydrazon. Der zweite Weg setzt die Diazotierbarkeit der substituierten 2-Amino-thiazole voraus. Die einzelnen Formazane lassen sich durch Cyclodehydrierung mittels N-Bromsuccinimids in die entsprechenden Tetrazoliumsalze überführen, die als Indikatoren für biologische Reduktionsprozesse verwendbar und im Gegensatz zu den bisher bekannten gegen Lichteinwirkung beständig sind. Bei der Einwirkung von Isoamylnitrit auf C,N-Diphenyl-N'-[4-phenyl-thiazolyl-(2)]-formazan entsteht kein Tetrazoliumsalz, sondern eine Nitroso-Verbindung.
Article
Pancreatic cells have been studied in rats under normal and abnormal metabolic conditions by electron microscopy. The epithelium of the ducts is crowned by microvilli. Their morphology resembles the B-cells of the islets. The excretory cells of the pancreas show remarkable variations in the lumen of the labyrinthlike tubes of the ergastoplasma and in the density of the walls of the tubes. Isolated A-cells are found between excretory cells with a direct contact of their cell-membranes. A-cells and B-cells can be identified by the number and location of small granules and by the shape and number of the mitochondria. The A-cells have numerous small granules and little mitochondria, the B-cells have many big mitochondria and fewer granules. Alloxan-injections are followed by hyperglycaemia and necrosis of the ergastoplasma in B-cells and later of the whole B-cell. The excretory epithelium shows disorder of the ergastoplasma. After cobalt-chloride injections vacuoles are found in the excretory cells and widespread degeneration of the mitochondria of the excretory and isletcells is seen as well. After injections of Rastinon and ACTH the number and size of the mitochondria are increased throughout the organ, especially in the B-cells, which show a diminution of granules. The blood-sugar varies, with ACTH above, and with Rastinon below the normal state.
Article
MAO of the brain was investigated histochemically in mice, rats, guinea pigs and rabbits. Fresh frozen sections were subjected to the tryptamine-tetrazolium method by Glenner, Burtner and Brown (1957). MAO activity of the brain of 4 animal species is generally similar with respect to its pattern of distribution. However, the intensity of enzyme action of the brain as a whole differs somewhat in animal species, being highest in guinea pigs, intermediate in rats and lowest in mice and rabbits. The enzyme action occurs mainly in the neuropil of the cerebral grey matter, while weak or negative activity is generally observed in the white matter excepting the tractus retroflexus of Meynert. The marked activity is encountered in the interpeduncular nucleus, locus coeruleus, area postrema, dorsal nucleus of the vagus nerve, hypothalamus, habenular nuclei and midline nuclear group of the thalamus, nucleus of the brachium conjunctivum, and central grey matter. The enzyme activity is weak or negative in the neocortex, striatum, mamillary body, thalamic nuclei (excepting the habenula and midline nuclear group), subthalamic nucleus, substantia nigra, red nucleus and nuclei of the somatic cranial nerves. The possible function and significance of MAO in the brain were discussed particularly by comparing the sites of this enzyme with those of succinic dehydrogenase and cytochrome oxidase, and the inverse relation between these enzymes was suggested.
Article
Under the light microscope the final reaction products of modern histochemical methods for dehydrogenases, diaphorases, and cytochrome oxidase appear as small dot-like deposits 0.1 to 0.25 μ in diameter situated at regular intervals within the mitochondrial membrane. So far only the dehydrogenase and diaphorase methods have been studied with the electron microscope, with results that suggest that the sites of enzyme activity are localized on the cristae mitochondriales. Correlation of the results of light and electron microscopy is not easy but it is suggested that the dot-like deposits must represent a relatively gross artifact, due to relative over-incubation.
Article
IN an earlier paper1, I have reported that ditetrazolium chloride, a substance introduced by Seligman and Ruthenburg2 for the histochemical detection of succinoxidase in tissue sections, can also be used for the detection of the same enzyme in mitochondria isolated from kidney and liver. This has been recently confirmed by Schiebler3 on the granula isolated from the neurohypophysis by differential sedimentation.
Article
After the subcutaneous injection of l-isonicotinyl-2-isopropyl hydrazine (IIH) into rats and guinea pigs, the monoamine oxidase activity of liver homogenates, liver mitochondria, and brain mitochondria is almost completely inhibited. The probable significance of such an inhibition is indicated.
Article
DIANZANI1 has shown that ditetrazolium can be used for demonstrating the activity of, among other enzymes, tyramine (amine) oxidase in mitochondria isolated from liver and kidney. The essential reaction here is a dehydrogenation2, and hydrogen acceptors other than oxygen may be used in the oxidation of tyramine by amine oxidase3. It is therefore of interest that amine oxidase activity can be demonstrated in frozen sections of the tissue, using a tetrazolium compound as the hydrogen acceptor; even though the method is not entirely satisfactory, it shows the general distribution of the enzyme. Neotetrazolium4 was found to be much more satisfactory than blue (di-)tetrazolium.
Article
Metal compounds were studied for possible effects on the amine oxidase system of rat liver. Some activated, others inhibited this system. Certain compounds exhibited both actions, the former being detectable at the lower concentrations. The data obtained extend the evidence of others for amine oxidase as a sulphydryl enzyme, but other mechanisms for metal inhibition of amine oxidase must also be considered. The actions of sulphydryl compounds on amine oxidase activity were investigated. BAL, thioglycollic acid, cysteine, and cystine were inhibitors, glutathione activated the enzyme system under certain conditions. Intravenous administration to rats of mercuric acetate, cadmium chloride, lead acetate, and manganese sulphate, all being in vitro inhibitors of amine oxidase, did not affect the enzyme activity of brain and liver in acute experiments.
Article
An enzyme system effecting the dehydrogenation of amines has been detected in rat brain and liver suspensions by the use of tetrazolium dyes as terminal electron acceptors. Kinetic data on this system are presented and the evidence for requirement of a cofactor is described. Thus, after washing or dialysis, rat brain suspensions have a considerably lowered tetrazolium reducing activity, which can be restored by addition of boiled extracts of rat liver, rat brain, pig liver, or baker's yeast. The heat-stable cofactor in pig liver and rat brain which is necessary for the activity of the tetrazolium reducing system is dialyzable. Pig liver extracts lose their cofactor activity on ashing. The nature of the electron transporting system active in amine dehydrogenation is discussed and the properties of the tetrazolium reducing and the amine oxidase systems are compared.
Article
1) A method is proposed for the demonstrations of dehydrogenases and diaphorases using the principle of simultaneous chelation of an enzymically produced monoformazan by means of metallic ions. 2) Using 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyl tetrazolium and M/60 cobalt a black metal-formazan complex is produced. Its nature is such that fine intracellular localisation becomes possible. 3) Chelation occurs under histochemical conditions with cobalt, copper, nickel and uranium and it is suggested that the electron opacity so induced may facilitate localisations of dehydrogenases on the electron microscope level.
Article
1 . Data has been presented showing that tetrazolium salts can be used for the histochemical localization of monoamine oxidase, although there are limitations to its application to in vitro studies. These limitations have been discussed. 2. It appears that an indole carbonyl compound is necessary for 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl tetrazolium chloride reduction. A diaphorasetype system may be required to transfer the electrons from the indole carbonyl intermediate to INT.
Article
1. By the use of tryptamine as substrate in the presence of tetrazolium salts in a solution buffered to pH 7.6 histochemical localization of monoamine oxidase activity is achieved during a 45 minute incubation of tissue sections at 37°C. 2. Inhibition experiments implicate the postulated acetaldehyde product of monoamine oxidase action on tryptamine and serotonin in the reduction of the tetrazoles. It was not determined whether this reduction is nonenzymatic or mediated through a flavoprotein enzyme system. 3. Localization of the formazan to autonomic ganglia and fibers, chief cells of the gastric fundus, mucosal epithelium of the duodenum and certain specific zones of the renal tubules by means of the tryptamine-tetrazolium technique was confirmed as indicating monoamine oxidase activity by identical localization using the hydrazone precipitation technique of Koelle and Valk.
Article
Studies on the role of purines as cofactors in the enzymatic reduction of tetrazolium salts by monoamines have led to the following results: (1) With whole rat liver extracts as the source of enzymes, several purines exhibit cofactor activity either as the free base or as the corresponding riboside and ribotide derivatives. (2) In contrast to this, mitochondrial material from rat liver is active only if adenylic acid or one of several ribotidic derivatives containing an adenylyl or similar moiety is used as cofactor. (3) Mitochondrial material utilizes hypoxanthine as cofactor for the amine/tetrazolium system only in combination with the supernatant obtained by centrifugation of tissue homogenates at 20,000 g. The additional factor present in this supernatant portion is heat-labile and nondialyzable. The possibility that this additional factor is an enzyme or enzymes converting the free base to the ribotide is discussed.Inhibition studies have revealed that the amine/tetrazolium enzyme system is sensitive to several metal-binding agents, but no direct evidence for the role of a metal in the enzymatic reaction could be obtained. It was also found that nicotinamide and adenine, neither of which exhibits cofactor activity, are potent inhibitors of the enzyme system studied.
Methods are presented for the intramitochondrial localization of various diphosphopyridine nucleotide and triphosphopyridine nucleotide-linked dehydrogenases in tissue sections. The cytochemical reactions studied involve the oxidation of the substrates by a specific pyridino-protein. The electron transfer of tetrazolium salt is mediated by the diaphorase system associated with the dehydrogenase. The final electron acceptor was either p-nitrophenyl substituted ditetrazole (nitro-BT) or N-thiazol-2-yl monotetrazole (MTT), the latter giving rise to metal formazan in the presence of cobaltous ions. Mitochondrial localization of the formazan precipitate could be achieved by using hypertonic incubating media containing high concentrations of substrate and co-enzyme. A fast reduction of tetrazolium salt was obtained by chemically blocking the respiratory chain enzymes beyond the flavoproteins. Although diaphorase systems are implicated in the reduction of tetrazolium salts, specific dehydrogenases are solely responsible for the distinct distribution pattern obtained in tissues with various substrates. The present findings in tissue sections are discussed in conjunction with existing biochemical evidence from differential centrifugation experiments.
Article
A number of enzymatic histochemical staining reactions were applied to the livers of rats that had been given dl-ethionine for periods up to 330 days. The ethionine lesion is characterized by early central hepatocellular necrosis, proliferation of oval cells, and degenerative and regenerative alterations in the liver cells leading to cirrhosis and areas of adenofibrosis. Finally, liver cell carcinoma develops. The zonal differences of various enzymatic staining reactions in the liver lobules characteristic for the normal liver disappear in regenerating liver lobules. Most enzymes are demonstrable in these regenerating cells, as for instance succinic dehydrogenase, nonspecific esterase, glucose-6-phosphatase, diphosphopyridine nucleotide diaphorase and others. Hepatic carcinoma cells likewise retain many of the staining reactions. There is, however, a complete lack of bile canalicular activity, which is best seen with the ATPase technique. Such a deficiency occurs also in some regenerating nodules without obvious evidence of malignancy. Thus, the lack of bile canalicular staining in cirrhotic liver lobules may indicate impending carcinomatous transformation. Oval "ductular" cells showed a lack of various enzymatic staining reactions, but did show moderate DPN diaphorase and nonspecific esterase activity. The origin of these cells from liver cells, although not proven, is definitely suggested. In areas of oval cell proliferation, there was marked increase in the intensity of sinusoidal staining with various phosphatase techniques. Fibrous tissue, particularly in areas of adenofibrosis, showed areas of marked adenosine triphosphatase activity. On the other hand, 5-nucleotidase activity was increased strongly in areas where reticulum fibers had been deposited. Increase in nonspecific esterase and acid phosphatase in Kupffer cells indicated apparently a marked stimulation of the reticuloendothelial system.
Article
(1) The monoamine oxidase activity of rat liver cells is localized predominantly, and perhaps exclusively, in the mitochondria. (2) In this respect it conforms to a precedent since other aerobic processes have been shown to have a mitochondrial localization.
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