Quantitative succinate dehydrogenase histochemistry in the hippocampus of aged rats

Quantitative histochemical methods (microphotometric kinetic and end-point measurements, and morphometric analyses of reactive areas) were used to investigate the levels of succinate dehydrogenase (SDH) in the hippocampus of young adult (3-6 months old) and aged male rats (24-27 months old). Methodological studies concerning the demonstration of SDH activity, which were performed using hippocampi of young animals, revealed a linear relationship between the reaction time and the amount of reaction product for up to 20 min; kinetic (continuous) and end-point measurements provided the same results. In a number of experiments, it was established that an incubation medium consisting of 100 mM succinate, 10 mM sodium azide, 3 mM nitro blue tetrazolium chloride, 0.25 mM phenazine methosulfate, and 7.5% polyvinylalcohol in 0.05 M Hepes buffer (final pH 7.5) was optimal for quantitative SDH histochemistry in the hippocampus. Comparative quantitative investigations of SDH activity in rat hippocampi showed that, in most regions and layers of the hippocampus of both young and aged rats, the levels of SDH activity increased along the rostrocaudal axis of the hippocampus, i.e., higher levels were present in the caudal than in the rostral pole. In both groups, the highest SDH levels were observed in the molecular layer of the cornu ammonis (CA)-1, the CA-3, and the fascia dentata (middle and outer thirds), most of which are termination fields of the excitatory perforant path arising from the regio ento-rhinalis. Furthermore, in almost all of the investigated layers, the older animals exhibited lower SDH levels than young animals. These differences were statistically significant in the molecular layer of the fascia dentata and in most layers of the CA-3. The lower SDH levels in aged animals are discussed in relation to the reduced capacity for energy metabolism in the aging brain.     

Quantitative succinate dehydrogenase histochemistry in the hippocampus of aged rats

Summary Quantitative histochemical methods (microphotometric kinetic and end-point measurements, and morphometric analyses of reactive areas) were used to investigate the levels of succinate dehydrogenase (SDH) in the hippocampus of young adult (3–6 months old) and aged male rats (24–27 months old). Methodological studies concerning the demonstration of SDH activity, which were performed using hippocampi of young animals, revealed a linear relationship between the reaction time and the amount of reaction product for up to 20 min; kinetic (continuous) and end-point measurements provided the same results. In a number of experiments, it was established that an incubation medium consisting of 100 mM succinate, 10 mM sodium azide, 3 mM nitro blue tetrazolium chloride, 0.25 mM phenazine methosulfate, and 7.5% polyvinylalcohol in 0.05M Hepes buffer (final pH 7.5) was optimal for quantitative SDH histochemistry in the hippocampus. Comparative quantitative investigations of SDH activity in rat hippocampi showed that, in most regions and layers of the hippocampus of both young and aged rats, the levels of SDH activity increased along the rostrocaudal axis of the hippocampus, i.e., higher levels were present in the caudal than in the rostral pole. In both groups, the highest SDH levels were observed in the molecular layer of the cornu ammonis (CA)-1 the CA-3, and the fascia dentata (middle and outer thirds), most of which are termination fields of the excitatory perforant path arising from the regio entorhinalis. Furthermore, in almost all of the investigated layers, the older animals exhibited lower SDH levels than young animals. These differences were statistically significant in the molecular layer of the fascia dentata and in most layers of the CA-3. The lower SDH levels in aged animals are discussed in relation to the reduced capacity for energy metabolism in the aging brain.Dedicated to Professor Dr. T.H. Schiebler on the occasion of his 65th birthdaySupported by the Deutsche Forschungsmeinschaft (Ku 541/2-1)     

Clycerolphosphate oxidase and succinate dehydrogenase activities in IIA and IIB fibres of mouse and rabbit tibialis anterior muscles

Summary Quantitative microphotometric measurements of two mitochondrial flavoproteins, glycerolphosphate oxidase (GP-OX) and succinate dehydrogenase (SDH), were performed on serial sections of mouse and rabbit tibialis anterior (TA) muscles in order to study the distribution of these two enzymes and their activity ratios in IIA and IIB fibres. The measurements showed a large scatter of the two enzyme activities in these two myosin-based fibre types. In rabbit TA, IIA and IIB fibres have similar GP-OX activities, whereas generally IIA fibres have higher SDH activities than IIB fibres. An inverse distribution of the two enzymes exists in mouse muscle. Generally, IIA fibres of mouse TA display low SDH and IIB fibres high SDH activities. The mean activity of GP-OX is slightly higher in IIA than in IIB fibres of mouse TA. Since measurements of both enzymes were taken in the same fibres, the ratio of their activities in each fibre could be evaluated. The SDH/ GP-OX activity ratios vary significantly between the two fibre populations both in rabbit and in mouse. The ratio is high in IIA and low in IIB fibres of rabbit TA, whereas it is low in IIA and high in IIB fibres of mouse TA.     

Glycerolphosphate oxidase and succinate dehydrogenase activities in IIA and IIB fibres of mouse and rabbit tibialis anterior muscles

Quantitative microphotometric measurements of two mitochondrial flavoproteins, glycerolphosphate oxidase (GP-OX) and succinate dehydrogenase (SDH), were performed on serial sections of mouse and rabbit tibialis anterior (TA) muscles in order to study the distribution of these two enzymes and their activity ratios in IIA and IIB fibres. The measurements showed a large scatter of the two enzyme activities in these two myosin-based fibre types. In rabbit TA, IIA and IIB fibres have similar GP-OX activities, whereas generally IIA fibres have higher SDH activities than IIB fibres. An inverse distribution of the two enzymes exists in mouse muscle. Generally, IIA fibres of mouse TA display low SDH and IIB fibres high SDH activities. The mean activity of GP-OX is slightly higher in IIA than in IIB fibres of mouse TA. Since measurements of both enzymes were taken in the same fibres, the ratio of their activities in each fibre could be evaluated. The SDH/GP-OX activity ratios vary significantly between the two fibre populations both in rabbit and in mouse. The ratio is high in IIA and low in IIB fibres of rabbit TA, whereas it is low in IIA and high in IIB fibres of mouse TA.     

Studies on the optimalisation and standardisation of the light microscopical succinate dehydrogenase histochemistry

The present investigation was undertaken in order to establish an optimal tissue pretreatment and an optimal incubation medium for the histochemical demonstration of succinate dehydrogenase (E.C. 1.3.99.1). The investigations were performed on steroid producing (testicle, adrenal gland) and steroid dependent (Fallopian tube) tissues. We studied the influences fo formalin fixation, acetone, magnesium ions, cyanides, electron carries (phenazine methosulfate, menadione coenzyme Q10), osmolarity, substrate concentration and inhibitors (oxalacetate, oxalate, malonate, 4-chloromercuribenzoic acid). The following procedure yields blameless morphological integrity and enzyme localization as well as optimal SDH-activity: Freezing of tissue cubes (diameter less than 5 mm) in propane cooled with liquid nitrogen or in melting freon. Incubation of 5 micrometer cryostat sections in narrow jars in the following medium (38.5 ml):--10 ml of 0.2 M sodium phosphate buffer pH 7.6 (52 mM).--18 mg tetranitro-BT in 0.5 ml dimethylformamide and aqua bidest. ad 10 ml (0.5 mM).--2.6 mg KCN in 16 ml aqua bidest. (1 mM).--540 mg succinate (disodium salt, hexahydrate) in 2 ml aqua bidest. (52 mM).--3 mg PMS (phenazine methosulfate) in 0.5 ml aqua bidest. (0.25 mM). The incubation medium has an osmolarity of 440 mosm. The incubation is carried out for 10 min at 37 degree C in darkness. To avoid non specific formazan deposits in lipid containing tissues a preincubation of the cryostat sections in 100% acetone at--22 degree C or--40 degree C for 7--10 min and an incubation time of 20--30 min is recommended. Control incubations adduced proof at the specificity of the SDH demonstration. Parallel incubation without PMS in order to determine indirectly the content of endogenous CoQ10 is further recommended.     

Relationship between myoglobin and succinate dehydrogenase in mouse soleus and plantaris muscle fibres

Summary This report describes a quantitative histochemical study of myoglobin in skeletal muscle fibres. The muscle fibres were classified as fast or slow on the basis of their quantitative myofibrillar ATPase histochemistry. A large range of myoglobin absorbance values was found among fast skeletal muscle fibres. This range was relatively small among slow fibres. The concentrations of myoglobin and the activities of succinate dehydrogenase in individual muscle fibres in serial sections are weakly correlated in both the mouse soleus and plantaris muscle. The myoglobin concentration is higher in fast and slow oxidative soleus muscle fibres and the succinate dehydrogenase activity in these fibres is lower than in oxidative plantaris muscle fibres in the same range of cross-sectional area.     

Classification of muscle fiber types based on succinic dehydrogenase and myofibrillar ATPase reactions in normal and randomly reinnervated rat skeletal muscles.

In the normal and randomly reinnervated plantaris muscle of rat staining for succinic dehydrogenase (SDH) activity differentiates three fiber types (A, B and C), staining for myofibrillar adenosine triphosphatase (ATPase) differentiates three fiber types (alpha, beta and alpha beta). Here we present our finding type A corresponds to alpha beta fibers, B to beta or alpha beta, C to alpha or alpha beta. In normal soleus muscle both classifications were found to be compatible and B fibers correspond to beta and C to alpha fibers. An exception is the small percent of alpha beta fibers which correspond to B type. In randomly reinnervated soleus muscle changes in ATPase activity are not followed by changes in SDH staining and B fibers correspond to alpha, beta or alpha beta types.     

Myofibrillar ATPase histochemistry of rat skeletal muscles: a "two-dimensional" quantitative approach

In histochemical investigations of skeletal muscle, the fibers are commonly classified into three types according to their staining for myofibrillar ATPase (mATPase). In serial sections of skeletal muscles from normal Wistar rats, we compared two common staining methods for mATPase: (a) an ac-ATPase technique, with pre-incubation at pH 4.7, and (b) a fixed alk-ATPase technique, using treatment with 5% paraformaldehyde followed by pre-incubation at pH 10.4. In addition, the same fibers were stained in subsequent serial sections for succinate dehydrogenase (SDH) activity. Staining intensities were objectively evaluated by microphotometric measurements of optical density. Combining both mATPase methods in consecutive serial sections ("two-dimensional approach") led to the identification of four distinct clusters of fibers: Types I, IIA, and two subgroups of Type IIB, as separated by their staining densities for fixed alk-ATPase (IIBd dark, IIBm moderate). The mean intensity of SDH staining per fiber type, as measured in the central core of the fibers, was ranked such that IIA greater than I greater than IIBd greater than IIBm. The analyzed muscles (tibialis anterior, biceps brachii) were markedly heterogeneous with respect to the topographic distribution of different fiber types. In comparison to other muscle portions, the regions containing Type I fibers ("red" portions) showed a higher IIBd vs IIBm ratio and more intense SDH staining for either subtype of the IIB fibers. The IIBd fibers probably correspond to the Type 2X fibers of Schiaffino et al.     

Quantitative relationship between protonophoric and uncoupling activities of substituted phenols

The protonophoric activity through liposomal membranes was measured and compared with the uncoupling activity with the oxidative phosphorylation of rat-liver mitochondria for 19 substituted phenols. Quantitative analyses of the protonophoric activity of the phenols in terms of physicochemical molecular parameters showed that the activity was mostly decided by two factors: the partition coefficient between the liposome and aqueous buffer phases and the acid dissociation constant. Correlation was excellent between protonophoric and uncoupling activities when the difference in the effect of acidity of phenols between liposomal and mitochondrial membranes was taken into account. The results were further evidence for the shuttle-type of mechanism of weakly acidic uncouplers based on the Mitchell chemiosmotic hypothesis.     

Association between the alpha-ketoglutarate dehydrogenase complex and succinate thiokinase

The kinetic parameters of the individual reaction of pig heart alpha-ketoglutarate dehydrogenase complex, succinate thiokinase and the alpha-ketoglutarate dehydrogenase complex-succinate thiokinase coupled system were studied. The KCoAm of alpha-ketoglutarate dehydrogenase complex and the K-succinyl CoAm of succinate thiokinase decreased in the coupled system when compared to those of the individual enzyme reactions. This phenomenon can be explained by the interaction between the alpha-ketoglutarate dehydrogenase complex and succinate thiokinase. By means of poly(ethylene glycol) precipitation, ultracentrifugation and gel chromatography we were able to detect a physical interaction between the alpha-ketoglutarate dehydrogenase complex and succinate thiokinase. Of the seven investigated proteins only succinate thiokinase showed association with alpha-ketoglutarate dehydrogenase complex. On the other hand, succinate thiokinase did not associate with other high molecular weight mitochondrial enzymes such as pyruvate dehydrogenase complex and glutamate dehydrogenase. On this basis, the interaction between succinate thiokinase and alpha-ketoglutarate dehydrogenase complex was assumed to be specific. These in vitro data raise the possibility that a portion of the citric acid cycle enzymes exists as a large multienzyme complex in the mitochondrial matrix.     

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     

Separation of NADH-fumarate reductase and succinate dehydrogenase activities in Trypanosoma cruzi

A recent review suggested that the activity of NADH-fumarate reductase from trypanosomatids could be catalyzed by succinate dehydrogenase working in reverse (Tielens and van Hellemond, Parasitol. Today 14, 265-271, 1999). The results reported in this study demonstrate that the two activities can easily be separated without any loss in either activity, suggesting that fumarate reductase and succinate dehydrogenase are separate enzymes.     

Effect of ruthenium complexes on the activities of succinate dehydrogenase and cytochrome oxidase

In this article, we report the effects of acute administration of ruthenium complexes, trans-[RuCl(2)(nic)(4)] (nic=3-pyridinecarboxylic acid) 180.7 micromol/kg (complex I), trans-[RuCl(2)(i-nic)(4)] (i-nic=4-pyridinecarboxylic acid) 13.6 micromol/kg (complex II), trans-[RuCl(2)(dinic)(4)] (dinic=3,5-pyridinedicarboxylic acid) 180.7 micromol/kg (complex III) and trans-[RuCl(2)(i-dinic)(4)]Cl (i-dinic=3,4-pyridinedicarboxylic acid) 180.7 micromol/kg (complex IV) on succinate dehydrogenase (SDH) and cytochrome oxidase (COX) activities in brain (hippocampus, striatum and cerebral cortex), heart, skeletal muscle, liver and kidney of rats. Our results showed that complex I inhibited SDH activity in hippocampus, cerebral cortex, heart and liver; and inhibited COX in heart and kidney. Complex II inhibited SDH in heart and hippocampus; COX was inhibited in hippocampus, heart, liver and kidney. SDH activity was inhibited by complex III in heart, muscle, liver and kidney. However, COX activity was increased in hippocampus, striatum, cerebral cortex and kidney. Complex IV inhibited SDH activity in muscle and liver; COX activity was inhibited in kidney and increased in hippocampus, striatum and cerebral cortex. In a general manner, the complexes tested in this work decrease the activities of SDH and COX in heart, skeletal muscle, liver and kidney. In brain, complexes I and II were shown to be inhibitors and complexes III and IV activators of these enzymes. In vitro studies showed that the ruthenium complexes III and IV did not alter COX activity in kidney, but activated the enzyme in hippocampus, striatum and cerebral cortex, suggesting that these complexes present a direct action on COX in brain.     

Thyroid hormone regulation of MHC isoform composition and myofibrillar ATPase activity in rat skeletal muscles.

Myosin heavy chain (MHC) isoform composition and Ca2+ Mg2+ dependent ATPase activity were determined in myofibrils prepared from skeletal muscles (diaphragm, soleus, plantaris and tibialis anterior) of euthyroid (C), hypothyroid (Tx) and hyperthyroid (T3) rats. Direct comparison between T3 and Tx gave an indication of the maximal effect of thyroid hormones. Significant differences in MHC-1 and MHC-2B proportions and in ATPase activity were found in all muscles. The difference in MHC-2A/X proportion was significant only in soleus, diaphragm and plantaris. When T3 and C were compared, significant variations in MHC isoform composition were found only in plantaris and diaphragm. The comparison between Tx and C showed significant differences in MHC isoform distribution and in ATPase activity in most muscles. The differences in ATPase activity among muscles and among thyroid states were consistent with those in MHC isoform distribution. From the correlations between ATPase activity and MHC isoform distribution the enzymatic activities of individual MHC isoforms were calculated. The results indicate that MHC isoform distribution is controlled by thyroid state in all skeletal muscles and that changes in MHC isoforms distribution are accompanied by proportional changes in ATPase activity.     

Age and changes in succinate dehydrogenase activity in functionally different muscles of the young rat

The antigravitational m. triceps brachii, its antagonist m. brachialis and the muscle having a universal functional specialization--m. serratus ventralis--have been studied in 35 male rats of Wistar strain, 60-105-day-old. Succinate dehydrogenase activity is determined in muscle fibers. Changes in the muscle fibers continue after the rats reach their sex maturation. Certain stageness of the process is observed, but the division of the period into separate steps either is absent (m. brachials), or their number is not great as compared to those during the 1st--60th days after birth. The borders of the periods in the muscles studied coinside (the 70th--74th day). Although it is possible to reveal the periods and separate steps in the changes occurring in the muscle fibers and in the muscles of the animals having reached their sex maturation, nevertheless, the borders between them are not distinct, the reconstruction during this age proceeds slower and more smoothely than before the sex maturation. It is possible that in young rats after sex maturation differentiation of the muscle fibers continues and that, in its turn, stimulates further specialization of the muscles as organs. The changes in the muscle fibers revealed histochemically occur most slowly under a low static loading, and when loading of various modality (kinetic and static) are combined, they are mostly pronounced.     

Catalytic enzyme histochemistry and biochemical analysis of dihydroorotate dehydrogenase/oxidase and succinate dehydrogenase in mammalian tissues,cells and mitochondria

Dihydroorotate dehydrogenase (EC 1.3.3.1 or EC 1.3.99.11) catalyzes the fourth sequential step in the de novo synthesis of uridine monophosphate. In eukaryotes it is located in the inner mitochondrial membrane, with ubiquinone as the proximal and cytochrome oxidase as the ultimate electron transfer system, whereas the rest of pyrimidine biosynthesis takes place in the cytosol. Here, the distribution of dihydroorotate dehydrogenase activity in cryostat sections of various rat tissues, and tissue samples of human skin and kidney, was visualized by light microscopy using the nitroblue tetrazolium technique. In addition, a hydrogen peroxide-producing oxidase side-reactivity of dihydroorotate dehydrogenase could be visualized by trapping the peroxide with cerium-diaminobenzidine. The pattern of activity was similar to that of succinate dehydrogenase, but revealed a less intensive staining. High activities of dihydroorotate dehydrogenase were found in tissues with known proliferative, regenerative, absorptive or excretory activities, e.g., mucosal cells of the ileum and colon crypts in the gastro-intestinal tract, cultured Ehrlich ascites tumor cells, and proximal tubules of the kidney cortex, whilst lower activities were present in the periportal area of the liver, testis and spermatozoa, prostate and other glands, and skeletal muscle. Dihydroorotate dehydrogenase and succinate dehydrogenase activity in Ehrlich ascites tumor cells grown in suspension culture were quantified by application of nitroblue tetrazolium or cyanotolyl tetrazolium and subsequent extraction of the insoluble formazans with organic solvents. The ratio of dihydroorotate dehydrogenase to succinate dehydrogenase activity was 14. This was in accordance with that of 15 obtained from oxygen consumption measurement of isolated mitochondria on addition of dihydroorotate or succinate. The ratio determined with mitochondria from animal tissues was up to 115 (rat liver, bovine heart). The application of the enzyme inhibitors brequinar sodium and toltrazuril verified the specificity of the histochemical and biochemical methods applied.     

Fibre-optic spectrophotometry of immature bovine skeletal muscles and the cellular distribution of myoglobin and succinate dehydrogenase

Summary Samples of diaphragm and pectoralis profundus were taken from nine calves with a range of blood haemoglobin levels of 4 to 8.5 g/100 ml. In both muscles, fibres with strong succinate dehydrogenase activity contained myoglobin, but in the pectoralis there were many fibres with strong alkaline ATPase activity and weak succinate dehydrogenase activity that had low or undetected levels of myoglobin. The whole cross-sectional area of individual fibres was scanned to map the distribution of succinate dehydrogenase activity. Among fibres with similar levels of ATPase activity, those from the diaphragm had greater succinate dehydrogenase activity than those from the pectoralis. Subsarcolemmal succinate dehydrogenase activity was greater than the axial succinate dehydrogenase activity, and radial gradients of succinate dehydrogenase activity were steepest in the diaphragm. For pectoralis fibres with weak ATPase, the mean and the axial succinate dehydrogenase activities were correlated with blood haemoglobin levels (r=0.62 and r=0.61, respectively;P<0.05 with a Student'st-test). Muscle colour was measured directly by fibre-optic spectrophotometry and correlations of absorbance with succinate dehydrogenase activity were obtained. Absorbance at 620 nm 24 h post-mortem was correlated with succinate dehydrogenase activity in pectoralis fibres with weak ATPase (r=0.81;P<0.005).