Histochemical model studies of enzyme activity after thermal damage

Summary The reliability of histochemical determinations of the enzyme activity after thermal damage has been studied with the aid of two model systems. Polyacrylamide films and erythrocyte ghosts containing either -glucuronidase or alkaline phosphatase, were submitted to heating and the activities retained were assessed both biochemically and histochemically. For the enzymes studied, the results show that tissue alterations induced by heat can influence histochemical reaction procedures, and that with these model systems, factors which are important for the histochemical quantitation of enzyme activities in thermally damaged tissues can be evaluated quantitatively. Potentialities of these model systems in the study of evaluating thermal damage through histochemical enzyme activity determinations, are discussed.To whom offprint requests should be sent     

Light microscopic demonstration of myoid material in nuclei

Summary In the rabbit and bovine cornea the activity of alkaline phosphatase using histochemical as well as biochemical methods was investigated. Biochemically the enzyme activity was studied in separated corneal layers. In the histochemical investigation the best results were obtained in cryostat sections using the azocoupling method with naphthol AS-MX phosphate and Variamine Blue RT Salt. The enzyme activity was found not only in the epithelium and endothelium (as was described previously) but even in keratocytes. The mutual relation of activities in the epithelium and in keratocytes differed in both species. The overall activity found by histochemical methods is in good agreement with the biochemical determination of alkaline phosphatase (p-nitrophenyl phosphate as the substrate). Besides the histochemical approach shows an uneven distribution of alkaline phosphatase activity in individual cells which cannot be assessed by the biochemical determination.     

Evaluation of the validity of the histochemical lead nitrate technique for alkaline and acid deoxyribonuclease.

The results of several tests and the characteristic morphological distribution of the enzymatic activity appeared to be in favor of the validity and specificity of the histochemical lead nitrate technique for alkaline and acid deoxyribonuclease (DNAse) detection. These tests included thermal inhibition, omission of substrate, use of different chemical inhibitors and the reproduction of histochemical staining on Coujard's slides. Most of these results were in conformity with the biochemical data gathered from literature. Topographically selective inhibition of alkaline or acid DNAse by different factors suggested that there might exist two kinds of alkaline or acid DNase--one cytoplasmic and the other one nuclear. The whole histochemical procedure produced relatively small loss of alkaline and acid DNAse activities as verified by biochemical methods.     

Histochemical characterization of neuronal NADPH-diaphorase

We examined the properties of neuronal NADPH-diaphorase in sections of rat striatum, using histochemical procedures. NADPH-diaphorase histochemistry stained discrete populations of central neurons and provided a Golgi-like image of the neurons exhibiting this activity. The NADPH-diaphorase reaction appeared to be enzyme catalyzed, since it was abolished by pre-treatment with proteases, heat, and acid or alkaline denaturation. Under anaerobic conditions, any tetrazolium salt with a redox potential more positive than NADPH could be reduced by the enzyme. NADPH-diaphorase activity was sensitive to inhibition by sulfhydryl reagents but was unaffected by metal chelators, superoxide dismutase, and catalase. Therefore, the enzyme is unlikely to be a metalloenzyme or to reduce tetrazoliums by producing superoxide anions or hydrogen peroxide. Various analogues of beta-NADPH could be used by the enzyme; however, beta-NADH, which can be used by DT-diaphorase, was ineffective. The enzyme was also resistant to dicumarol, an inhibitor of DT-diaphorase activity. Electron microscopy indicated that the NADPH-diaphorase reaction resulted in staining of various membranous organelles. We conclude that neuronal NADPH-diaphorase is a membrane-bound enzyme distinct from DT-diaphorase and other known enzymes with diaphorase activity. The histochemical characteristics presented here should now enable meaningful biochemical studies of neuronal NADPH-diaphorase to be undertaken.     

The value of enzyme histochemical techniques in the classification of fibre types of human skeletal muscle

Summary In the present investigation the results of a lead salt technique and two calcium salt techniques for the demonstration of the activity of myosin adenosine triphosphatase in sections of both normal and pathological human skeletal muscle specimens are compared. It was seen that the histochemical results obtained by the different techniques are similar, especially with regard to the identification of fibre-types.It can be clearly stated, that the alkaline phosphatase activity present in muscle fibres of diseased skeletal muscles revealed only a very slight activity with the substrate ATP, so the alkaline phosphatase activity in general did not disturb the reliability of the different myosin ATPase techniques.Moreover it was found that the presence of the mitochondrial Ca²⁺-ion activated ATPase with a high pH-optimum in muscle fibres did not give rise to faulty results.From studies with dinitrophenol it can be concluded that this substance activates the myosin ATPase present in type I fibres especially.     

Detection of alkaline phosphatase activity after conventional isoelectric focusing by an indoxyl-tetrazolium salt technique

Alkaline phosphatase was solubilized from human and rat tissues using papain in the presence of TRITON X-100 and subjected to isoelectric focusing (IEF) in polyacrylamide or agarose gels. Up till now, usually 1- and 2-naphthylphosphates have been used as substrates in order to specifically stain molecular forms of this enzyme by the azo-dye technique. In this paper, the use of another histochemical substrate, 5-bromo-4-chloro-3-indoxyl phosphate, in combination with tetrazolium salts [McGadey, J. (1970) Histochemie 23, 180-184] is presented. After hydrolysis, the released indoxyl moieties reduce tetrazolium salts to insoluble formazans at the zones of alkaline phosphatase activity. Zymogrammes showing molecular forms of alkaline phosphatase from 20 rat organs and the application of this staining technique for the detection of alkaline phosphatase activity in non-dialyzed human plasma after IEF are presented.     

Histochemical analysis of molluscan stomach and intestinal alkaline phosphatase: A sialoglycoprotein

Summary Though sialoprotein nature of alkaline phosphatase of certain mammalian organs has been suggested by biochemical investigations, no histochemical techniques have yet been applied to elucidate this concept. With this view, the alkaline phosphatase of stomach and intestine of a mollusc—Semperula maculata—was analysed histochemically to elucidate its sialoglycoprotein nature. The localisation of alkaline phosphatase and sialic acid was investigated by employing well known and standard histochemical techniques.Alkaline phosphatase was localised selectively in the brush border of the mucosa of stomach and intestine, it was Mg⁺⁺ nonsensitive but showed a structure-linked sensitivity to phenylalanine. The sialomucins were selectively localised in the brush border, whereas the goblet cells contained both the sialomucins and sulfomucins, and the connective tissue of lamina propria contained sulfomucins. The localisation of alkaline phosphatase and sialomucins in the brush border uniquely coincided with each other. The alkaline phosphatase activity in the brush border was completely lost after neuraminidase treatment at 37.5° C for 16 h. Such effect of neuraminidase on alkaline phosphatase activity was pH dependent and controlled by velocity of reaction. Heat-inactivated neuraminidase showed no effect on alkaline phosphatase activity.These histochemical results have been interpreted as suggesting a sialoglycoprotein nature of alkaline phosphatase in the brush border, and sialic acid somehow seems to be essential for enzyme activity. These results, thus, indicate necessity of visualising some of the sialo-glycoproteins as macromolecules with catalytic activity.     

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.     

Modulation of alkaline phosphatase activity in alveolar type II like cells

Alveolar type II like cells (ALT II) represent a small subpopulation of alveolar type II cells, which is able to proliferate, can be passaged and possess many characteristics of differentiated adult type II cells. A correlation was found between the growth and development of ALT II cells in culture and their alkaline phosphatase activity. Unlike alveolar type II cells, which lose the activity in culture, ALT II cells regain the activity and maintain it for a long culture period. Quantitative histochemical analysis of the stained cells indicate that 80% of the cells at days 15-20 in culture are alkaline phosphatase positive. Inhibition studies indicate that alkaline phosphatase from ALT II cells and freshly isolated type II cells were similar. The inhibition of ALT II alkaline phosphatase by L-levamisole and its heat stability are similar to that of the bone enzyme and differ from the intestinal enzyme. Alkaline phosphatase expression is considered part of the differentiated phenotype of these cells. Therefore, the presence of this enzyme in ALT II cells adds support to the notion that these cells maintain many aspects of mature alveolar type II cells.     

Alkaline inorganic pyrophosphatase activity of mammalian-cell alkaline phosphatase

Alkaline phosphatase prepared from mammalian cell cultures was found to have alkaline inorganic pyrophosphatase activity. Both of these activities appear to be associated with a single protein, as demonstrated by: (1) concomitant purification of alkaline phosphatase and alkaline inorganic pyrophosphatase; (2) proportional precipitation of alkaline phosphatase and inorganic pyrophosphatase activities by titrating constant amounts of an enzyme preparation with increasing concentration of antibody; (3) immune electrophoresis, which showed that precipitin bands that have alkaline phosphatase activity also have pyrophosphatase activity; (4) inhibition of pyrophosphatase activity by cysteine, an inhibitor of alkaline phosphatase activity; (5) similar subcellular localization of the two enzyme activities as demonstrated by histochemical methods; (6) hormonal and substrate induction of alkaline phosphatase activity in mammalian cell cultures, which produced a nearly parallel rise in inorganic pyrophosphatase activity.     

5′-Nucleotidase

Summary 5′-Nucleotidase and alkaline phosphatase activity was investigated in the developing kidney of the mouse by histochemical and electrophoretic methods. The growth of the kidneys was studied by determining the incorporation of radioactive thymidine by autoradiography. During development the isoenzyme patterns of 5′-nucleotidase and alkaline phosphatase behaved in a different way. In correlating the histochemical and electrophoretic changes, it has been found that the 5′-nucleotidase isoenzymes as well as the alkaline phosphatase isoenzymes are located in different parts of the kidney. In the convoluted part of the proximal tubule 5′-nucleotidase isoenzyme 3 and alkaline phosphatase isoenzyme 5 are present, while in the straight part of this tubule 5′-nucleotidase isoenzyme 5 and — upto three weeks — alkaline phosphatase isoenzyme 3 are located. So in tissue structures having different functional capacities, different isoenzymes of 5′-nucleotidase and alkaline phosphatase are found.     

Cytochemical and biochemical characterization of testicular peritubular myoid cells

Testicular peritubular myoid cells secrete a paracrine factor that is a potent modulator of Sertoli cell functions involved in the maintenance of spermatogenesis. These cells also play an integral role in maintaining the structural integrity of the seminiferous tubule. To better understand this important testicular cell type, studies were initiated to characterize cultured peritubular cells using biochemical and histochemical techniques. The electrophoretic pattern of radiolabeled secreted proteins was similar for primary and subcultured peritubular cells and was unique from that of Sertoli cells. Morphologic differences between Sertoli cells and peritubular cells were noted and extended with histochemical staining techniques. Desmin cytoskeletal filaments were demonstrated immunocytochemically in peritubular cells, both in culture and in tissue sections, but were not detected in Sertoli cells. Desmin is proposed to be a marker for peritubular cell differentiation as well as a marker for peritubular cell contamination in Sertoli cell cultures. Peritubular cells and Sertoli cells were also stained histochemically for the presence of alkaline phosphatase. Staining for the alkaline phosphatase enzyme was associated with peritubular cells but not with Sertoli cells. Alkaline phosphatase is therefore an additional histochemical marker for peritubular cells. Biochemical characterization of peritubular cells relied on cell-specific enzymatic activities. Creatine phosphokinase activity, a marker for contractile cells, was found to be associated with peritubular cells, while negligible activity was associated with Sertoli cells. Alkaline phosphatase activity assayed spectrophotometrically was found to be a useful biochemical marker for peritubular cell function and was utilized to determine the responsiveness of primary and subcultured cells to regulatory agents. Testosterone stimulated alkaline phosphatase activity associated with primary cultures of peritubular cells, thus supporting the observation that peritubular cells provide a site of androgen action in the testis. Retinol increased alkaline phosphatase activity in subcultured peritubular cells. Alkaline phosphatase activity increased in response to dibutyryl cyclic adenosine monophosphate (AMP) in both primary and subcultured peritubular cell cultures. Observations indicate that the ability of androgens and retinoids to regulate testicular function may be mediated, in part, through their effects on peritubular cells. This provides additional support for the proposal that the mesenchymal-epithelial cell interactions between peritubular cells and Sertoli cells are important for the maintenance and control of testicular function. Results imply that the endocrine regulation of tissue function may be mediated in part through alterations in mesenchymal-epithelial cell interactions.     

An Embedding Method for Histochemical Studies of Undecalcified Skeletal Growth Plate

We have used glycol methacrylate to study undecalcified skeletal growth plate and subchondral bone. Minor modifications of the original technique including dehydration in glycol methacrylate vacuum infiltration and polymerization in the cold make it quite suitable for embedding of such tissues. Moreover, specimens can be processed quickly and the morphologic and biochemical integrity of the tissue retained so that histochemical procedures can be readily applied. Collagen, glycosaminoglycan, glycogen, lipid, calcium and the activity of alkaline and acid phosphatase were localized. This technique appears to be very useful for studying skeletal tissues.     

Organ specificity of the structural organization and fine distribution of lymphatic capillary networks: histochemical study

Histochemical studies of the microcirculatory system were reviewed with regard to the organ specificity of the structural organization and fine distribution of the lymphatic capillary network. The lymphatics and blood vessels are characterized by an enzyme-histochemical method using 5'-nucleotidase (5'-Nase), alkaline phosphatase (ALPase) and/or diaminopeptidase (DAPase) staining in addition to an immunohistochemical method. The 5'-Nase-positive lymphatic vessels can be distinguished histochemically from arterial and venous vessels based on ALPase and DAPase activity, respectively. The specificity and localization of the enzyme reactions were confirmed by comparative histochemical studies of the same specimen with light microscopy and scanning or transmission electron microscopy. These histochemical methods are discussed in relation to their ability to demonstrate the organ specificity of vascular networks under normal and pathological conditions.     

An embedding method for histochemical studies of undecalcified skeletal growth plate

We have used glycol methacrylate to study undecalcified skeletal growth plate and subchondral bone. Minor modifications of the original technique including dehydration in glycol methacrylate vacuum infiltration and polymerization in the cold make it quite suitable for embedding of such tisssues. Moreover, specimens can be processed quickly and the morphologic and biochemical integrity of the tissue retained so that histochemical procedures can be readily applied. Collagen, glycosaminoglycan, glycogen, lipid, calcium and the activity of alkaline and acid phosphatase were localized. This technique appears to be very useful for studying skeletal tissues.     

Histochemical changes in acid and alkaline phosphatase activities in the growing follicles and corpora lutea of the rat ovary

Changes in acid and alkaline phosphatase activities are studied histochemically in different components of the growing follicles and corpora lutea of different generations. Theca cells of the growing follicles showed strong acid phosphatase activity as compared to that observed in the oocyte and granulosa cells. Alkaline phosphatase activity is localized only in the theca interna cells of growing follicles. However, after ovulation with the formation of corpora lutea, the granulosa lutein cells also showed this activity. Changes in the histochemical distribution of the acid and alkaline phosphatases in the follicles and corpora lutea are discussed in relation to folliculogenesis and corpus luteum and regression.     

Demonstration of amyloid with Mesitol WLS-Congo Red: Application of a textile auxiliary to histochemistry

Summary Previous histochemical investigations demonstrated similarities in the binding of Congo Red and other direct cotton dyes by amyloid and cellulose. It seemed threfore of interest to determine whether or not the cellulose-like reactivity of amyloid extends also to dye solutions containing an anionic reserving agent. These reagents are used in the dyeing of wool-cellulose (Halbwolle) fabrics to prevent binding of direct cotton dyes by proteins. Mesitol WLS-Congo Red solutions stained amyloid selectively; other tissue structures, except some hyaline deposits in arterioles, remained unstained. The cause of this non-specific reaction could not be determined with certainty. Therefore, the alkaline Congo Red method is recommended for histochemical identification of amyloid. However, the Mesitol WLS-Congo Red technic was very useful for demonstration of amyloid after prolonged storage of tissues in formalin; amyloid in such material showed little or no reactivity with the alkaline Congo Red or the Sirius dye methods. This pilot study indicates that anionic reserving agents can be effectively employed under conditions of histochemical technics.     

Induction of enhanced alkaline phosphatase activity in the melano-macrophage centres of Oreochromis aureus (Steindachner) through starvation and vaccination

Employing histochemical methods, alkaline phosphatase activity was studied in the melano-macrophage centres of the spleen of the cichlid fish Oreochromis aureus (Steindachner). Enzyme activity was observed to be very low in normal fish. Prolonged starvation induced an enhanced enzyme response. Starvation followed by antigenic stimulation through an intraperitoneal injection of a bacterial vaccine further elevated the levels of alkaline phosphatase activity. The marked response of the pigment-bearing macrophages to bacterial antigen provides further evidence of the lymphoreticular nature of these pigmented cell aggregates. The association of alkaline phosphatase activity and lipofuscin (the most common pigment in fish melano-macrophage centres) with phagocytic cells has been documented in higher animals including     

Detecting Enzymatic Activity in Cells Using Fluorogenic Substrates

Fluorogenic substrates can detect enzymatic activity associated with cells. It is difficult, however, to detect activity within a single cell or in an organelle since hydrolytic substrates yield products that rapidly leak from the cell. Several new solutions are presented including trapping the fluorescent product in membranes, in cell organelles, or as a glutathione conjugate. Novel substrates also are described that directly yield highly fluorescent precipitates at the site of enzymatic activity. These can be used for detecting endogenous activity in cells or for enzyme-amplified histochemical detection. Some of these substrates can be used in live cells.     

Histochemical characteristics of a tonic smooth muscle.

It is suggested that ABRM, smooth muscle of Mytilus edulis L. and Mytilus galloprovincialis Lmk. (Mollusca Pelecypoda), is composed of one histochemical fibre type. The fibres are characterized by a low myofibrillar ATPase activity. Succinic and nicotinamide adenine dinucleotide oxidoreductase activities are distributed in a reverse pattern than that of the ATPase activity. Glycogen phosphorylase is richly represented in ABRM fibres and this detection is in opposition with the negative detection of alkaline phosphatase activity. These preliminary histochemical observations are similar to those found in some vertebrate smooth muscles. Mitochondrial glycerol-3-phosphate, 6-phosphogluconate, lactate and octopine dehydrogenases are not detected in muscle fibres whereas glio-interstitial tissues show weak but distinct reactivity. These last results especially characterize Mytilus catch fibres and are briefly discussed in relationship with previous physiological, biochemical and morphological observations.