The increase in activity of acid hydrolases in muscles of rats after subcutaneous administration of dimethyl-para-phenylene diamine. A combined histochemical and biochemical investigation. I. The histochemical investigation.

The activity of acid hydrolases in skeletal muscles of normal rats and of rats after subcutaneous administration of dimethyl-para-phenylene diamine (DPPD) was studied with a combined histochemical and biochemical investigation. In this communication the histochemical findings are presented. After 4 days of DPPD treatment, coagulation necrosis, fragmentation and disintegration of fibres were seen in the muscles. An inflammatory infiltrate was seen between the muscle fibres. These pathological changes reached maximum intensity after 7 to 9 days. After 11 days the changes became less, despite continued treatment with DPPD. From the histochemical findings it appeared that the activity of acid phosphatase, beta-glucuronidase and E600 resistant non-specific esterase was increased in both a granular and a diffuse pattern in the skeletal muscles of the DPPD rats. The increase in activity of leucine aminopeptidase was much less pronounced and was mainly granular. The increase in the activity of acid hydrolases ran parallel to the severity of the pathological changes and reached a maximum after 7 to 9 days of DPPD treatment. The statistical calculations of the histochemical findings revealed that the increased activity of one acid hydrolase was significantly paralleled by an increased activity of a second hydrolase. There was a moderate probability that the activity of all other histochemically studied acid hydrolases, with the exception of leucine aminopeptidase, was increased. There was no difference in activity and localization of the acid hydrolases studied in aerobic type I and anaerobic type II fibres. The localization of acid phosphatase and beta-glucuronidase activity in muscle fibres and in inflammatory infiltrate mostly coincided. In cases where these enzymes were localized both centrally and in the subsarcolemnal areas of the muscle fibres, the activity of E600 resistant non-specific esterase was usually, and the activity of leucine aminopeptidase was exclusively located in the subsarcolemnal areas. All of the acid hydrolases examined were found to be present in the inflammatory exudate and in the connective tissue.     

Evaluation of histochemical observations of activity of acid hydrolases obtained with semipermeable membrane techniques: A combined histochemical and biochemical investigation

Summary The reliability of enzyme histochemical observations of activities of acid hydrolases was investigated with a combined histochemical and biochemical study. Specimens of m. soleus, m. plantaris, m. gastrocnemius and diaphragm of normal and of vitamin E deficient rabbits were used. For the histochemical investigation, activity and localization of acid phosphatase, -glucuronidase, leucine aminopeptidase and E₆₀₀ resistant non-specific aryl-esterase were examined with semipermeable membrane techniques. For the biochemical investigation, activity of acid phosphatase, -glucuronidase, cathepsin D, acid maltase and neutral maltase was determined.By means of statistical calculations the enzyme activities demonstrated with histochemical techniques were compared with the enzyme activities determined with biochemical techniques.In the present communication the histochemical findings are reported and discussed. From the histochemical findings it appeared that activity of the acid hydrolases investigated is strongly increased in both a granular and a diffuse pattern in skeletal muscle of vitamin E deficient rabbits. The statistical calculations of the histochemical findings clearly reveal that the increased activity of one acid hydrolase was highly significantly paralleled by an increased activity of a second acid hydrolase. Moreover the probability that the activity of all other histochemically studied acid hydrolases was significantly increased was rather high.The increase in activity of the acid hydrolases studied was the same in muscles with an aerobic or an anaerobic metabolism. Moreover there was no difference in activity and localization of the acid hydrolases in aerobic type I and anaerobic type II fibres.The localization of acid phosphatase and -glucuronidase activity in muscle fibres mostly coincided. In cases where these enzymes were localized both centrally and in the subsarcolemnal areas of the muscle fibres, the activity of E₆₀₀ resistant naphtholesterase was usually, and the activity of leucine aminopeptidase was exclusively located in the subsarcolemnal areas. All of the examined acid hydrolases were found to be present in the inflammatory exudate and in the connective tissue.This study was partly extracted from the Ph. D. thesis of D.E. Israël (1977).     

Evaluation of histochemical observations of activity of acid hydrolases obtained with semipermeable membrane techniques: a combined histochemical and biochemical investigation 2. The biochemical investigation and comparison with the histochemical observations.

The reliability of enzyme histochemical semipermeable membrane techniques for the demonstration of acid hydrolases was investigated with a combined histochemical and biochemical study. In part 1 the histochemical findings were presented. In this communication the biochemical findings are reported and compared with the histochemical findings. In m. soleus, m. plantaris, m. gastrocnemius and diaphragm of vitamin E deficient rabbits the activity of the lysosomal acid hydrolases, cathepsin D, acid maltase, acid phosphatase and beta-glucuronidase is significantly increased. This increase in activity of the investigated acid hydrolases was equal for muscles with an aerobic or an anaerobic metabolism. By means of statistical calculations the activity of the enzymes demonstrated with histochemical techniques was compared with the enzyme activity determined with biochemical techniques. From the results of this investigation it can be concluded that the histochemical semipermeable membrane techniques for the demonstration of activity of acid hydrolases are very reliable. Considering the fact that these techniques are also tissue-saving, they are therefore extremely suitable for the study of catabolic wasting processes in skeletal muscle tissues of patients with inherited or acquired muscular diseases.     

Evaluation of enzyme histochemical observations for metabolic studies. A combined histochemical and biochemical investigation of experimental induced muscle-changes

Summary The degree of conformity between enzyme histochemical (part I) and biochemical observations was studied in a variable model. Rabbit skeletal muscle altered by reinnervation was used. The most important feature of the biochemical part of this investigation is the constancy of the activity ratio of the enzymes phosphofructokinase (PFK), which is ratelimiting for the glycolysis and l-glycerol-3-phosphate: acceptor oxidoreductase (GPOX) in spite of the marked metabolic changes after cross-reinnervation. In consequence it is most likely that in case of pathologically altered tissue one may also rely on the histochemical GPOX-reaction as an indication of the PFK-activity, which cannot be demonstrated histochemically to any reliable degree.In order to evaluate the indicator significance of the GPOX-reaction, the histochemical estimations (part I) were compared with the biochemical results. This comparison disclosed a striking parallellism per type of muscle in discerning the presence or the absence of metabolic changes after cross-reinnervation or auto-reinnervation. Quantitatively though there were some variations especially for the m. soleus. Consequently the histochemical impressions of the investigated enzymes are applicable in predicting the biochemical results within this model, but cannot be used as a substitute. It was concluded that where only small amounts of tissue are available histochemical examination offers a reliable screening method for muscle diseases and can indicate a more appropriate biochemical investigation to confirm the diagnosis.This study was taken from the Ph. D. thesis of A. C. Jöbsis (1971).     

Evaluation of enzyme histochemical observations for metabolic studies. A combined histochemical and biochemical investigation of experimentally induced skeletal muscle-changes

Summary The reliability of enzyme histochemical observations for metabolic studies on skeletal muscle tissue was investigated with a combined histochemical and biochemical study. Specimens of musculus soleus with a predominantly aerobic metabolism and of musculus flexor digitorum longus with a predominantly anaerobic metabolism of rabbits in which both muscles were surgically cross-reinnervated or auto-reinnervated were used. For the histochemical investigation activities and localisations of succinate dehydrogenase, l-glycerol-3-phosphate: acceptor oxidoreductase, nicotinamide adenine dinucleotide: tetrazolium oxidoreductase and of -glucan phosphorylase were examined. For the biochemical investigation maximal activity of phosphofructokinase, the rate limiting enzyme for the regulation of the glycolysis was measured. In addition the activities of succinate dehydrogenase and l-glycerol-3-phosphate: acceptor oxidoreductase to characterize the aerobic metabolism and the key role in gearing energy requirements to glycolysis respectively were biochemically determined. For further information about metabolic aspects the isoenzyme ratio of lactate dehydrogenase was established. In the present paper the histochemical findings are reported and discussed.Part of this study was taken from the Ph. D. thesis of A. C. Jöbsis (1971).     

Peroxisomes of the rat cardiac and soleus muscles increase after starvation. A biochemical and immunocytochemical study

We have investigated the change of catalase activity in the homogenates of rat cardiac and skeletal muscles. After 7 days' starvation, the catalase activity of heart increased about 3-fold and that of soleus muscle enhanced 2-fold higher than that of control rats. Immunoblot analysis of catalase showed a single band in the homogenates of cardiac and soleus muscles and increase of catalase antigen after starvation. Light microscopic immunoenzyme staining showed that after starvation catalase positive granules markedly increased in both the cardiac and soleus muscle. Quantitative analysis of the staining showed that number of the granules per 100 microns 2 of tissue section was about 1.4-fold in the soleus muscle and 1.7-fold in the cardiac muscle after starvation. By electron microscopy of alkaline DAB staining, we confirmed that the granules were peroxisomes, which increased in both number and size. Furthermore, we stained the peroxisomes for catalase by a protein A-gold technique. Labeling density (gold particles/micron 2) of the cardiac and soleus muscles from the starved rat increased approximately 1.4 times as much as that of normal animal. When the numerical density is multiplied by the labeling density, the values are largely consistent with the enhancement of catalase activity. These results show that increase in the catalase activity of the muscle tissue after starvation is caused by increase in number and size of peroxisomes.     

Denervation and reinnervation of fast and slow muscles. A histochemical study in rats.

A histochemical study, using myosin-adenosine triphosphatase activity at pH 9.4, was conducted in soleus and plantaris muscles of adult rats, after bilateral crushing of the sciatic nerve at the sciatic notch. The changes in fiber diameter and per cent composition of type I and type II fibers plus muscle weights were evaluated along the course of denervation-reinnervation curve at 1, 2, 3, 4 and 6 weeks postnerve crush. The study revealed that in the early denervation phase (up to 2 weeks postcrush) both the slow and fast muscles, soleus and plantaris, resepctively, atrophied similarly in muscle mass. Soleus increased in the number of type II fibers, which may be attributed to "disuse" effect. During the same period, the type I fibers of soleus atrophied as much or slightly more than the type II fibers; whereas the type II fibers of plantaris atrophied significantly more than the type I fibers, reflecting that the process of denervation, in its early stages, may affect the two fiber types differentially in the slow and fast muscles. It was deduced that the type I fibers of plantaris may be essentially different in the slow (soleus) and fast (plantaris) muscles under study. The onset of reinnervation, as determined by the increase in muscle weight and fiber diameter of the major fiber type, occurred in soleus and plantaris at 2 and 3 weeks postcrush, respectively, which confirms the earlier hypotheses that the slow muscles are reinnervated sooner than the fast muscles. It is suggested that the reinnervation of muscle after crush injury may be specific to the muscle type or its predominant fiber type.     

Alkaline phosphatase in cholestatic and cirrhotic rats. A biochemical and histochemical study

Alkaline phosphatase (ALP) was studied by enzyme histochemical methods and by biochemical quantitations in rat livers with chronic bile duct obstruction and experimental cirrhosis. The most evident ALP increase was histochemically found in portal tracts of rats with bile duct obstruction and localized to the walls of proliferating blood vessels. Furthermore, a slight canalicular membrane enzyme increase was histochemically found in both groups, most evident in cirrhosis, whereas the biochemical assay of ALP in serum and liver from both pathological groups showed 3 times higher values compared to controls. The portal tracts did not seem to contribute to the serum increase, since the rise of serum ALP was similar in chronic bile duct obstruction and in experimental cirrhosis without changes of the portal tracts. It is concluded that the increase ALP activity in serum from rats with bile duct obstruction and cirrhosis mainly has a hepatocytic origin.     

The activity of kidney lysosomal hydrolases and renin in hypertensive rats.

In renal cortices of hypertensive rats the activity of renin and β-glucuronidase is significantly enhanced, while the activity of acid phosphatase remains pratically unchanged. A significant decrease in protein content accompanies the rise in enzyme activity. The distribution pattern of renin, β-glucuronidase and acid phosphatase after isopycnic centrifugation of cortical homogenates from hypertensive animals is different from that of controls.     

Stimulation of hepatic and renal diamine oxidase activity after acute ethanol administration

The effect of a single administration of ethanol (2 g/kg body weight) on hepatic and renal diamine oxidase activity was studied in fasted rats. Diamine oxidase activity significantly increased in liver and kidney 6 h after ethanol intubation. Pyrazole (an inhibitor of alcohol dehydrogenase), cycloheximide or actinomycin D (inhibitors of macromolecular syntheses), as well as prior adrenalectomy, prevented the ethanol-induced stimulation of diamine oxidase in the liver, but not in the kidney. The results demonstrated that the enhancement of diamine oxidase activity in the liver was due to an enzyme induction mediated by alcohol metabolism as well as by adrenals. In contrast, the stimulation of diamine oxidase activity in the kidney did not depend on synthesis of new enzyme molecules and was not mediated by ethanol metabolism or adrenal hormones.     

Combined histochemical and biochemical investigation to the reliability of the demonstration of arylsulphatase activity in cryostat sections.

The reliability of the enzyme histochemical technique, for the demonstration of arylsulphatase activity, using 6-bromo-2-naphthylsulphate as a substrate, is biochemically tested by using partly purified lysosome and microsome preparations from fresh human placenta tissue. Microsomes from frozen placenta with an arylsulphatase deficiency and lysosomes from rat liver, are also investigated. For the biochemical test methods, 6-bromo-2-naphthylsulphate and p-nitrocatecholsulphate are used as substrates. Under similar reaction conditions, varying the pH of the incubation medium and adding inhibitors or activators, the histochemical and biochemical reactions are compared. The results of this study show that the enzyme histochemical technique--except for some limitations--is suitable for the demonstration of microsomal arylsulphatase in cryostat sections.     

The pentose phosphate pathway in skeletal muscle under patho-physiological conditions. A combined histochemical and biochemical study

Over the last 30 years, research into the neuromuscular apparatus, has expanded greatly. Multidisciplinary investigations have rapidly advanced our understanding both of diseases and of the basic neuromuscular mechanisms. The mode of pathological reaction of the neuromuscular apparatus is now quite well understood. The most notable aspect of the reaction of the injured neuromuscular apparatus is the remarkably stereotyped character of the resulting pathological changes as demonstrated by a wide variety of harmful causes, producing surprisingly similar effects. The findings of our combined histochemical and biochemical investigations presented in this monograph, are in complete harmony with the stereotyped character of the pathological changes. For example, it is particularly striking that many affected muscle fibres of patients with muscular dystrophies, congenital myopathies, inflammatory myopathies, metabolic myopathies, endocrine myopathies, or with diseases of the lower motor neuron, display an enhanced activity of both oxidative enzymes of the pentose phosphate pathway. Likewise, we found that experimental animals with disordered skeletal muscles, provoked by different types of agents or treatments, reveal the same marked rise in activity of GPDH and PGDH in the muscle fibres, with a positive correlation between the activity of both enzymes. Other findings of our investigations point to a positive correlation between the activity of GPDH and PGDH on the one hand and that of the non-oxidative enzymes of the pentose phosphate pathway, the enzymes TA, TK, RPI and RPE on the other hand. The rise in activity of PGDH and, in particular, of GPDH is regulated by two different mechanisms. The first represents a rapid control mechanism based on the stimulation of both oxidative enzymes of the pentose phosphate pathway by NADP+ and on their inhibition by NADPH. The other mechanism represents a long-term effect directed at the synthesis of the enzymes. It is this type of mechanism which is responsible for the rise in activity of GPDH and PGDH we observed. The findings obtained with the applied enzyme histochemical techniques clearly demonstrated that the rise in activity of both enzymes is not homogeneously distributed in the disordered skeletal muscles of man and experimental animals. For that reason, in order to obtain reliable quantitative information about enzyme activities in the muscle fibres themselves, the application of biochemical assays on a micro-scale was indispensable. The biochemical assay of enzyme activities was performed on histologically and histochemically selected dissected muscle specimens.(ABSTRACT TRUNCATED AT 400 WORDS)     

Decreased lysosomal protease content of skeletal muscles from streptozotocin-induced diabetic rats: a biochemical and histochemical study

Summary The activity of four lysosomal proteases in soleus and extensor digitorum longus muscles was studied in streptozotocin-induced diabetic rats using newly developed fluorescence histochemical and biochemical techniques. The results indicate that the content of lysosomal protease in skeletal muscle cells was decreased three weeks after the induction of diabetes. The reduction was most pronounced in the extensor digitorum longus for all the proteases tested, but in the soleus only cathepsin B and dipeptidyl peptidase II showed a decrease. Biochemical assays on total muscle homogenates and muscle extracts confirmed the histochemical observations that protease activity was significantly lower in diabetic muscles. This decrease in activity varied with the duration of diabetes beginning as early as 48 h for the soleus. In conclusion, myofibre-specific decreases in lysosomal proteases occur following diabetes.     

Combined histochemical and biochemical investigation to the reliability of the demonstration of arylsulphatase activity in cryostat sections

Summary The reliability of the enzyme histochemical technique, for the demonstration of arylsulphatase activity, using 6-bromo-2-naphthylsulphate as a substrate, is biochemically tested by using partly purified lysosome and microsome preparations from fresh human placenta tissue. Microsomes from frozen placenta with an arylsulphatase deficiency and lysosomes from rat liver, are also investigated. For the biochemical test methods, 6-bromo-2-naphthylsulphate and p-nitrocatecholsulphate are used as substrates. Under similar reaction conditions, varying the pH of the incubation medium and adding inhibitors or activators, the histochemical and biochemical reactions are compared. The results of this study whow that the enzyme histochemical technique — except for some limitations — is suitable for the demonstration of microsomal arylsulphatase in cryostat sections.