Dimethylsulfoxide as carrier in enzyme cytochemistry

Summary Addition of dimethylsulfoxide (DMSO) to the incubation medium of succinate dehydrogenase in a concentration of 10% enhances the staining reaction in the hyphae of the fungus Cercosporella herpotrichoides after an incubation period of 15 min. Controls without DMSO remain unstained. DMSO causes a rapid penetration of the components of the medium through the mucilage that covers the hyphae.     

A permeability test for the study of mitochondrial injury in in vitro cultured heart muscle and endothelioid cells.

A cytochemical permeability test for the detection of injury to in situ mitochondria of cultured heart cells is presented. The test is based on the increased rate at which injured mitochondria stain for succinate dehydrogenase activity. Whereas an intact inner mitochondrial membrane limits the rate at which Nitro Blue tetrazolium and phenazine methosulphate reach succinate dehydrogenase, injured mitochondria allow these reactants to reach the enzyme more rapidly to form microscopically-observable formazan granules. The extent of staining at fixed durations of incubation with the reactants was assessed on a blind basis with pseudo dark-field microscopy, using a standardized rating scale. Differences in the staining of control and treated cells were analysed statistically by a semi-quantitative method. Treatment of the cultures with either vitamin A or chlorpromazine, resulted in more rapid mitochondrial staining. Brief pre-fixation of the cells with cold acetone also labilized the mitochondria as did a delay in the change of culture medium.     

Enzyme reaction rate studies in electromotor neurons of the weakly electric fish Apteronotus leptorhynchus

A histochemical analysis of reaction rates of a series of enzymes was performed in electromotor neurons of the weakly electric fish Apteronotus leptorhynchus. These neurons were selected because of their functional homogeneity. The high metabolic activity of these cells as well as their large size facilitate cytophotometric analysis in cryostat sections. Sections were incubated for the activity of hexokinase, glucose-6-phosphate dehydrogenase, succinate dehydrogenase, NADPH dehydrogenase, NADPH ferrihaemoprotein reductase and beta-hydroxybutyrate dehydrogenase. All media contained polyvinyl alcohol as tissue stabilizer and Nitro BT as final electron acceptor. Measurements were performed with a Vickers M85a cytophotometer. Linear relationships between the specific formation of formazan (test minus control reaction) and incubation time were obtained for all enzymes although some reactions showed an initial lag phase or an intercept with the ordinate. The relatively high activities of hexokinase, succinate dehydrogenase and the extremely low activity of hydroxybutyrate dehydrogenase indicate that energy is mainly supplied by glycolysis. Glucose-6-phosphate dehydrogenase showed a high activity whereas NADPH reductase and dehydrogenase activity were low in electromotor neurons, indicating that the NADPH generated is largely used for biosynthesis. Despite their synchronous firing pattern activity, electromotor neurons showed a considerable heterogeneity with respect to their metabolic activity.     

Methodological problems in the histochemical demonstration of succinate semialdehyde dehydrogenase activity

Methodological aspects of the histochemical technique for the demonstration of succinate semialdehyde dehydrogenase activity (EC 1.2.1.24) (indicative of the degradative step of gamma-aminobutyric acid catabolism) have been analysed in rat Purkinje neurons, where gamma-aminobutyric acid has been shown to be a neurotransmitter, and in hepatocytes, where it is metabolized. During a histochemical incubation for the enzyme, artefacts of succinate dehydrogenase activity and the 'nothing dehydrogenase' reaction are produced. Inhibition of these artefacts by the addition of two inhibitors, malonate and p-hydroxybenzaldehyde, revealed specific reaction products. Formazan granules, which can be ascribed only to specific succinate semialdehyde dehydrogenase activity, are obtained by adding malonate to the incubation medium in order to inhibit both succinate dehydrogenase activity and nothing dehydrogenase. The formation of these granules is completely inhibited by p-hydroxybenzaldehyde, an inhibitor of succinate semialdehyde dehydrogenase activity. Different levels of succinate semialdehyde dehydrogenase activity were noted in Purkinje neurons. This activity was also found in hepatocytes, mostly in the portal area, but with a lesser degree of intensity and specificity. Indeed, non-specific formazan granules were still produced, because of the 'nothing dehydrogenase' reaction, even in the presence of malonate. Thus, a malonate-insensitive 'nothing dehydrogenase' reaction seems to be present in neural and hepatic tissues.     

Evidence for succinate production by reduction of fumarate during hypoxia in isolated adult rat heart cells

It has been demonstrated that perfusion of myocardium with glutamic acid or tricarboxylic acid cycle intermediates during hypoxia or ischemia, improves cardiac function, increases ATP levels, and stimulates succinate production. In this study isolated adult rat heart cells were used to investigate the mechanism of anaerobic succinate formation and examine beneficial effects attributed to ATP generated by this pathway. Myocytes incubated for 60 min under hypoxic conditions showed a slight loss of ATP from an initial value of 21 +/- 1 nmol/mg protein, a decline of CP from 42 to 17 nmol/mg protein and a fourfold increase in lactic acid production to 1.8 +/- 0.2 mumol/mg protein/h. These metabolite contents were not altered by the addition of malate and 2-oxoglutarate to the incubation medium nor were differences in cell viability observed; however, succinate release was substantially accelerated to 241 +/- 53 nmol/mg protein. Incubation of cells with [U-14C]malate or [2-U-14C]oxoglutarate indicates that succinate is formed directly from malate but not from 2-oxoglutarate. Moreover, anaerobic succinate formation was rotenone sensitive. We conclude that malate reduction to succinate occurs via the reverse action of succinate dehydrogenase in a coupled reaction where NADH is oxidized (and FAD reduced) and ADP is phosphorylated. Furthermore, by transaminating with aspartate to produce oxaloacetate, 2-oxoglutarate stimulates cytosolic malic dehydrogenase activity, whereby malate is formed and NADH is oxidized. In the form of malate, reducing equivalents and substrate are transported into the mitochondria where they are utilized for succinate synthesis.     

Microphotometric measurement of initial maximum reaction rates in quantitative enzyme histochemistryin situ

Summary Final reaction product formation was recorded microphotometrically for succinate dehydrogenase in cross-sectioned muscle fibres at initial rate conditions and during prolonged incubations. Incubations with gel films and aqueous reaction medium both showed a decline of reaction rates. Maximum reaction rates could only be determined at initial rate conditions during the first minute of the incubation. Reaction rates recorded in different areas of the same tissue section were found to change with time to different degrees. From these results it was concluded that quantitative histochemical measurements of enzyme reactionsin situ can only be valid if measured under initial maximum velocity conditions.     

Localization of dihydroorotate oxidase in myocardium and kidney cortex of the rat

Biochemical studies have demonstrated that dihydroorotate dehydrogenase (DHOdehase; EC 1.3.3.1 or 1.3.99.11) is the sole enzyme of de novo pyrimidine synthesis in mitochondria, whereas the rest of the pathway takes place in the cytosol. The dehydrogenation of dihydroorotate to orotate is linked to the respiratory chain via ubiquinone. In this study, we show for the first time the ultrastructural localization of DHOdehase. Since the purified enzyme was found to act both as dehydrogenase and as oxidase, the cerium capture technique for detecting enzymatically generated hydrogen peroxide could be applied to pin-point the in situ activity of DHOdehase oxidase in mitochondria of rat heart and kidney cortex. Cerium perhydroxide as the final reaction product was detected predominantly in the matrix with some focal condensation along the inner membrane, but not in the intermembrane space. From this pattern of localization, it is concluded that the active site of the membrane-bound enzyme could face the mitochondrial matrix similar to succinate dehydrogenase. The reliability of the applied method for the demonstration of DHOdehase oxidase was demonstrated by the addition of Brequinar sodium to the incubation medium. This quinoline-carboxylic acid derivative is a potent inhibitor of DHOdehase and has proven anti-proliferative activity. The present observations do not ascertain whether the oxidase is permanently active as a constant portion of the enzyme in vivo, similar to xanthine oxidase/dehydrogenase. However, DHOdehase should be considered as a source of radical oxygen species under pathophysiological conditions.     

Cytophotometric analysis of reaction rates of succinate and lactate dehydrogenase activity in rat liver, heart muscle and tracheal epithelium

Reaction rates of succinate and lactate dehydrogenase activity in cryostat sections of rat liver, tracheal epithelium and heart muscle were monitored by continuous measurement of formazan formation by cytophotometry at room temperature. Incubation media contained polyvinyl alcohol as tissue protectant and Tetranitro BT as final electron acceptor. Control media lacked either substrate or substrate and coenzyme. Controls were also performed by adding malonate (a competitive inhibitor of succinate dehydrogenase), pyruvate (a non-competitive inhibitor of lactate dehydrogenase), oxalate (a competitive inhibitor of lactate dehydrogenase) or N-ethylmaleimide (a blocker of SH groups). A specific malonate-sensitive linear test minus control response for succinate dehydrogenase activity was obtained in liver (1.6 mumol H2cm-3 min-1) and tracheal epithelium (0.8 mumol H2cm-3 min-1) but not in heart muscle. All variations in the incubation conditions tested did not result in a linear test minus control response in the latter tissue. Because the reaction was sensitive to malonate, it was concluded that the initial reaction rate was the specific rate of succinate dehydrogenase activity in heart muscle (9.1 mumol H2 cm-3 min-1). Test minus control reactions for lactate dehydrogenase activity were distinctly non-linear for all tissues tested. This appeared to be due to product inhibition by pyruvate generated during the reaction and therefore it was concluded that the appropriate control reaction was the test reaction in the presence of 20 mM pyruvate. The initial rate of the test minus this control was the true rate of lactate dehydrogenase activity. The lactate dehydrogenase activity thus found in liver parenchyma was 5.0 mumol of H2 generated per cm3 liver tissue per min.     

Hepatic organelle interaction. IV. Mechanism of succinate enhancement of formaldehyde accumulation from endoplasmic reticulum N-dealkylations

Further evidence for organelle interaction during drug metabolism by the liver is presented. The apparent stimulation by succinate of formaldehyde accumulation in the medium, which was reported to occur with liver slices and homogenates as well as with mitochondria plus microsomes, has been shown to be the result of succinate inhibition of mitochondrial aldehyde dehydrogenase. The mechanism of succinate inhibition is shown to be by reverse electron transport, and an increase in the NADH to NAD+ ratio in the mitochondria; the aldehyde dehydrogenase requires the oxidized form of the pyridine nucleotide as its cofactor. Studies on in vitro N-demethylation by liver microsomes and endoplasmic reticulum segments which cosediment with the mitochondria indicate that formaldehyde produced by the mixed function oxidase is handled differently from formaldehyde added to the medium. The latter is mainly retained in the medium containing 5 mM semicarbazide, while the generated formaldehyde is more than 50% consumed by the mitochondria. Electron microscopy has indicated that the microsomes and the endoplasmic reticulum fragments have a tendency to align themselves close to the mitochondria when present in the same medium. Consequently, it is possible that formaldehyde released to the medium adjacent to the mitochondria, as by N-demethylation, would be exposed to semicarbazide for shorter periods than that added directly to the medium. In agreement with this suggestion, complexing of formaldehyde with semicarbazide was observed spectroscopically not to be an extremely rapid reaction even at 37 degrees C. This is believed to be the reason for the greater extent of consumption of formaldehyde generated by the endoplasmic reticulum.     

Preparation of rat enterocyte mitochondria.

Rat enterocyte mitochondria were prepared with respiratory control ratios of 4 or 5 and occasionally 6. When EGTA was excluded from the mitochondrial incubation medium the calculated P/O ratios were high, especially those based on the first addition of ADP. These ratios were lowered by increasing the EGTA concentration from 1 mM to 2 mM in the mitochondrial preparation medium and including 1 mM-EGTA in the incubation medium. The use of EDTA in the enterocyte isolation medium led to the mitochondria requiring added cytochrome c. Substituting EGTA for EDTA abolished this requirement. The mitochondrial fraction consisted of two components, an upper cream-coloured layer rich in DNA and a lower brown-coloured layer poor in DNA. Both components were capable of oxidative phosphorylation with succinate or the glutamate/malate couple as substrates. The mitochondrial yield was assessed by assaying succinate dehydrogenase activity, and the contamination of the mitochondrial fraction by other cell organelles was assessed by assays for appropriate marker enzymes.     

Enzyme cytochemistry of unfixed leukocytes and bone marrow cells using polyvinyl alcohol for the diagnosis of leukemia

Cytochemical methods for the demonstration of enzyme activities in blood and bone marrow cells were systematically improved by the addition of an inert polymer, polyvinyl alcohol (PVA), to the incubation medium and by using optimized reaction media. The methods investigated were tetrazolium salt methods for lactate, glucose-6-phosphate, succinate and glutamate dehydrogenase, the indoxyl-tetrazolium salt method for alkaline phosphatase, the diaminobenzidine method for peroxidase, and diazonium salt methods for chloroacetate esterase, beta-glucosaminidase, beta-glucuronidase, acid phosphatase, and dipeptidylpeptidase II and IV. PVA in the media preserved the morphology of cells very well and prevented leakage of large molecules such as enzymes from the cells. Therefore, fixation or long periods of air-drying prior to incubation leading to substantial loss of enzyme activity could be avoided. A brief period of drying (2 min at 37 degrees C) of the cell preparations just before the incubation was sufficient for making the cells permeable. Localization of enzyme activities was very precise and precipitation of the final reaction product was confined to sites which are known to contain the enzyme under study (granules, mitochondria, lysosomes). These advantages advocate the use of PVA in haematological enzyme cytochemistry and especially for diagnosis of leukemia.     

Microphotometric determination of structure-bound oxidoreductase activities avoiding nothing-dehydrogenase artefacts: succinate dehydrogenase

 A tetrazolium-based microphotometric method has been devised for the determination of structure-bound dehydrogenase activities with correction for nothing-dehydrogenase artefacts. The method is based on the microphotometric recording of maximum reaction rates in a simple incubation chamber and consists of two successive measurements on the same section, the first in the absence and the second in the presence of the substrate. Following the first measurement, the substrate-free medium is quickly exchanged with the substrate-containing medium and a second measurement is taken. Subtraction of the first from the second reaction rate yields the enzyme activity corrected for nothing-dehydrogenase. Measurements of succinate dehydrogenase (SDH) in skeletal muscle fibres, liver, cardiac atrium and ventricle demonstrate the feasibility of the method. Measurements on the extensor digitorum longus muscle of rat reveal a range of up to fivefold differences in SDH activity within the fibre population of this muscle. Accepted: 11 April 1997     

Histochemical studies of Ca-ATPase, succinate and NAD+-dependent isocitrate dehydrogenases in the shell gland of laying Japanese quails: with special reference to calcium-transporting cells

In order to elucidate the problem of which cells are involved in calcium transport and to estimate the role of mitochondria in calcium transport in the avian shell gland, the fine structure and the Ca-ATPase, succinate dehydrogenase (SDH) and nicotinamide adenine dinucleotide (NAD+)-dependent isocitrate dehydrogenase (NAD+-ICDH) activity of the shell gland of egg-laying Japanese quails were examined. The surface epithelial cells, consisting of ciliated cells with cilia and microvilli and non-ciliated cells with microvilli, had many large and electron-dense granules. The tubular-gland cells occupied the proprial layer and lacked secretory granules. When an egg was in the shell gland, the well-developed mitochondria of tubular-gland cells characteristically tended to accumulate in the apical cytoplasm, while they were scattered throughout the cytoplasm when an egg was not in the shell gland. Intense Ca-ATPase activity was found on the microvilli of tubular-gland cells, and moderate activity was found on the lateral-cell surface. In the surface epithelial cells, the basolateral cell surface showed moderate enzymatic activity. Both SDH and NAD+-ICDH activity were found in tubular-gland cells when an egg was in the shell gland. These results strongly suggest that calcium for eggshell calcification is actively transported by the tubular-gland (depending on Ca-ATPase activity) and that the mitochondria of gland cells may play an important role in this process as an energy source.     

Succinate dehydrogenase activity in the wall of rabbit aorta

Summary An investigation of succinate dehydrogenase activity in the wall of rabbit aorta was carried out. The level of succinate dehydrogenase per se in the smooth muscle cells was found to be fairly high, while the mitochondrial level of carrier CoQ was low. The latter may explain the low level or lack of activity of succinate dehydrogenase in these cells as noticed by previous authors.A reliable image of the actual level of succinate dehydrogenase was obtained only by adding CoQ₁₀ to the incubation system. PMS should be avoided, as it induced a Nothing dehydrogenase reaction even at low concentrations.     

Effect of amphotericin B on the enzyme system of Candida albicans]

The mechanism of amphotericin B action was studied with the aid of cytochemical methods providing determination of the activity of the 4 main enzymes characterizing the cell energetics, i. e. succinate dehydrogenase, lactate dehydrogenase, alcohol dehydrogenase and glucose-6-phosphate dehydrogenase inside the cell. With an increase in the concentration of amphotericin B in the medium the activity of all the 4 enzymes decreased, the percentage of the inactive cells being regularly increased. Changes in the fermentative activity of C. albicans as dependent on the incubation time with the antibiotic were studied. Only the respiration activity decreased in 2 hours. As a result of a 4-hour exposure to the polyen in the cells of C. albicans the activity of the lactic acid fermentation, respiration through succinate dehydrogenase and activity of the pentose shunt decreased 1.5--2 times. In 24 hours of incubation the activity of the above decreased 80--90 per cent as compared to the activity of the initial culture.     

Effect of polyene antibiotics on the isolated kidney mitochondria of dogs

The effect of amphotericin B and nystatin on mitochondria isolated from the dog kidneys was studied. It was shown that incubation of the isolated kidney mitocondria with amphotericin B or nystatin at a wide range of their concentrations, i.e. from 0.1 to 100 gamma per 0.5 mg of the mitochondrial protein did not affect the activity of succinate dehydrogenase of these cell microstructures. The absence of changes in the succinate dehydrogenase activity of the mitochondria under the effect of the polyenic antibiotics is significant from the viewpoint of elucidating their molecular mechanisms of the nephrotoxic effect.     

Schistosoma mansoni and Biomphalaria glabrata: Ultrastructural localization of enzymes with diaminobenzidine in larvae and host digestive glands.

All mitochondria contained reaction product when daughter sporocysts of Schistosoma mansoni and digestive glands of the snail host, Biomphalaria glabrata, were cytochemically incubated for 45 or 60 min with alkaline 3, 3′-diaminobenzidine (DAB) at pH 7.4 and 9.0. The pigment marked the presence of cytochrome c-cytochrome oxidase activity, and was not found in parasite or gland tissues incubated with DAB and KCN at pH 7.4, 9.0, and 9.8.After incubation for 45 min in the pH 7.4 DAB medium, tegumental mitochondria in young intrasporocyst cercariae showed DAB reaction product, but little or none of the pigment was found in tegumental mitochondria of older, glycocalyx-covered cercariae. In contrast, mitochondria of subtegumental cells were strongly DAB positive at all stages of intrasporocyst cercarial development. No differences in DAB reactivity were detected in mitochondria of sporocysts, or of infected and uninfected host gland cells.Reaction product was found in certain vacuoles of digestive cells incubated in the pH 9.8 DAB medium with KCN, but not in the pH 9.8 DAB medium with amino triazole, or in the pH 7.4 DAB medium. No peroxisomes or microperoxisomes were found in the tissues studied.     

Histochemical studies of Ca-ATPase, succinate and NAD+-dependent isocitrate dehydrogenases in the shell gland of laying Japanese quails: with special reference to calcium-transporting cells

Summary In order to elucidate the problem of which cells are involved in calcium transport and to estimate the role of mitochondria in calcium transport in the avian shell gland, the fine structure and the Ca-ATPase, succinate dehydrogenase (SDH) and nicotinamide adenine dinucleotide (NAD⁺)-dependent isocitrate dehydrogenase (NAD⁺-ICDH) activity of the shell gland of egg-laying Japanese quails were examined. The surface epithelial cells, consisting of ciliated cells with cilia and microvilli and non-ciliated cells with microvilli, had many large and electron-dense granules. The tubular-gland cells occupied the proprial layer and lacked secretory granules. When an egg was in the shell gland, the well-developed mitochondria of tubular-gland cells characteristically tended to accumulate in the apical cytoplasm, while they were scattered throughout the cytoplasm when an egg was not in the shell gland. Intense Ca-ATPase activity was found on the microvilli of tubular-gland cells, and moderate activity was found on the lateral-cell surface. In the surface epithelial cells, the basolateral cell surface showed moderate enzymatic activity. Both SDH and NAD⁺-ICDH activity were found in tubular-gland cells when an egg was in the shell gland. These results strongly suggest that calcium for eggshell calcification is actively transported by the tubular-gland (depending on Ca-ATPase activity) and that the mitochondria of gland cells may play an important role in this process as an energy source.     

Studies in succinate dehydrogenase histochemistry

Summary The activity of succinate tetrazolium reductase was investigated in liver and kidney from the rat and mouse. The results obtained were related to the cellular level of succinate dehydrogenase (SDH) as well as to the level of CoQ.It was concluded that the low activity in centrolobular areas of the liver lobules compared with the perilobular areas, exclusively is due to a naturally deprivation of CoQ.The level of SDH as well as of CoQ was very high in renal cortical tubules rich in mitochondria (proximal and distal convoluted tubules, the ascending thick limb of Henle). This was indicated by the facts that the initial reaction rate was high and no enhancement was obtained by the addition of CoQ₁₀.In all experiments the activity of fresh frozen sections were compared with the activity of sections from briefly prefixed tissue. The influence of different fixatives, variation in Nitro BT concentration, cryoprotection (dimethyl sulfoxide, DMSO) and osmolar protection (sucrose) was investigated and discussed. Further, the substrate-carrying effect of DMSO was investigated and discussed.Brief (5 min) fixation at 0–4° C—especially with 1% buffered (pH=7.2) methanol-free formaldehyde (from paraformaldehyde) gave excellent preservation of morphology and caused no inhibition of SDH activity. Furthermore, the fixation caused an enhancement of Nitro BT penetration into the tissue and an enhancement of formazan substantivity.The incubation time needed for the appearance of both the red and blue formazan was recorded in order to follow the initial reaction rate. This procedure proved to be a sensitive indicator, when the influence of components added (CoQ₁₀, DMSO, sucrose etc.) was studied.     

Assay of succinate dehydrogenase activity by the tetrazolium method: evaluation of an improved technique in skeletal muscle fractions

An improved spectrophotometric method for measuring succinate dehydrogenase (EC 1.3.99.1) activity with the use of 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyltetrazolium chloride (INT) is described. The procedure has been evaluated in mitochondrial fractions and homogenates of frog skeletal muscle. For mitochondrial suspensions, extraction of formazan with alcohol was found to be superior to extraction with ethyl acetate. For homogenates, complete extraction of formazan required sequential treatment with alcohol and ethyl acetate; the generally employed procedure of extracting once with ethyl acetate alone led to serious underestimation of the amount of formazan in the tissue. Observations of mitochondrial suspension incubated with various concentrations of INT led to the selection of 0.8 mM INT for optimal results. Higher concentrations, although commonly used, can exert undesirable inhibitory effects on succinate dehydrogenase activity, especially at low concentrations of mitochondria and after longer periods of incubation. The problem of instability of succinate dehydrogenase was solved by the addition of buffer at pH 7.5.