In situ localization of transketolase activity in epithelial cells of different rat tissues and subcellularly in liver parenchymal cells.

Metabolic mapping of enzyme activities (enzyme histochemistry) is an important tool to understand (patho)physiological functions of enzymes. A new enzyme histochemical method has been developed to detect transketolase activity in situ in various rat tissues and its ultrastructural localization in individual cells. In situ detection of transketolase is important because this multifunctional enzyme has been related with diseases such as cancer, diabetes, Alzheimer's disease, and Wernicke-Korsakoff's syndrome. The proposed method is based on the tetrazolium salt method applied to unfixed cryostat sections in the presence of polyvinyl alcohol. The method appeared to be specific for transketolase activity when the proper control reaction is performed and showed a linear increase of the amount of final reaction product with incubation time. Transketolase activity was studied in liver, small intestine, trachea, tongue, kidney, adrenal gland, and eye. Activity was found in liver parenchyma, epithelium of small intestine, trachea, tongue, proximal tubules of kidney and cornea, and ganglion cells in medulla of adrenal gland. To demonstrate transketolase activity ultrastructurally in liver parenchymal cells, the cupper iron method was used. It was shown that transketolase activity was present in peroxisomes and at membranes of granular endoplasmic reticulum. This ultrastructural localization is similar to that of glucose-6-phosphate dehydrogenase activity, suggesting activity of the pentose phosphate pathway at these sites. It is concluded that the method developed for in situ localization of transketolase activity for light and electron microscopy is specific and allows further investigation of the role of transketolase in (proliferation of) cancer cells and other pathophysiological processes.     

Loss of peroxisomes causes oxygen insensitivity of the histochemical assay of glucose-6-phosphate dehydrogenase activity to detect cancer cells.

Oxygen insensitivity of carcinoma cells and oxygen sensitivity of non-cancer cells in the histochemical assay of glucose-6-phosphate dehydrogenase (G6PD) enables detection of carcinoma cells in unfixed cell smears or cryostat sections of biopsies. The metabolic background of oxygen insensitivity is still not understood completely. In the present study, rat hepatocytes, rat hepatoma cells (FTO-2B), and human colon carcinoma cells (HT29) were used to elucidate these backgrounds. The residual activity in oxygen was 0%, 55%, and 80% in hepatocytes, hepatoma cells, and colon carcinoma cells, respectively. N-ethylmaleimide (NEM), a blocker of SH-groups, did not affect G6PD activity in both carcinoma cell types but reduced G6PD activity in hepatocytes by 40%. Ultrastructural localization of G6PD activity was exclusively in the cytoplasm of carcinoma cells, but in hepatocytes both in cytoplasm and peroxisomes. NEM abolished peroxisomal G6PD activity only. Histochemical assay of catalase activity demonstrated absence of peroxisomes in both carcinoma cell lines. It is concluded that absence of SH-sensitive G6PD activity in peroxisomes in cancer cells is responsible for the oxygen-insensitivity phenomenon.     

LIVER PARENCHYMAL CELL INJURY : I. Initial Alterations of the Cell Following Poisoning with Carbon Tetrachloride

The structure of the endoplasmic reticulum, plasma membrane, mitochondria, and Golgi apparatus of the liver parenchymal cell is strikingly altered within 1 hour following the administration of a single oral dose of carbon tetrachloride to rats. Progressive loss of glucose-6-phosphatase activity accompanies dispersal of the ergastoplasm. Electron microscopy reveals that these changes are associated with vacuolization of the cisternae of the granular endoplasmic reticulum, degranulation of its membranes, and the appearance of increased number of free ribosomes in the adjacent cytoplasmic matrix. Concomitantly, calcium enters the liver parenchymal cell and is sequestered by mitochondria. First increased at 30 minutes, calcium content is maximal at 1 hour and returns to normal at 2 hours. Although succinic and glutamic dehydrogenase activity patterns within the liver lobule are unaffected, liver cell mitochondria enlarge and some appear to fuse or assume cup-like configurations. Microvilli lining the space of Disse become irregularly indistinct and increasingly pleomorphic by 30 minutes when the plasma membrane becomes increasingly permeable to calcium. Golgi vesicles swell and discharge their granules during the period of poisoning studied. Although all the changes observed may be the result of direct interaction of carbon tetrachloride with the membranes of the cytoplasmic constituents of the liver parenchymal cell, the possibility that the irreversible changes observed in the granular endoplasmic reticulum may be due to the chemical interaction between the poison and this system is discussed.     

Some observations on the fine structure of light and dark cells in the gall bladder epithelium of the mouse

Summary Electron microscopic observations have been made of the two epithelial cell types, light barrel-shaped and dark rod-shaped cells in the gall bladder of the mouse.The light cells have a voluminous cytoplasm of low electron opacity in which cell organelles such as mitochondria, elements of granular endoplasmic reticulum, and free ribosomes undergo more or less degenerative changes. However, there are a relatively abundant Golgi apparatus and numerous lysosomal dense bodies. The ultrastructural features of the light cells suggest that they are an aged, degenerative cell type with declining functional activity and a high degree of hydration.The dark cells are characterized by a high concentration of mitochondria and free ribosomes, more or less distinctive elements of granular endoplasmic reticulum, and well developed components of the Golgi apparatus. Such ultrastructural characteristics indicate that the dark cell type has a high synthetic activity.What has been observed in the present study can well be correlated with the results of previous studies on the same cells by methods of light microscopic histochemistry.     

NADPH production by the pentose phosphate pathway in the zona fasciculata of rat adrenal gland.

Biosynthesis of steroid hormones in the cortex of the adrenal gland takes place in smooth endoplasmic reticulum and mitochondria and requires NADPH. Four enzymes produce NADPH: glucose-6-phosphate dehydrogenase (G6PD), the key regulatory enzyme of the pentose phosphate pathway, phosphogluconate dehydrogenase (PGD), the third enzyme of that pathway, malate dehydrogenase (MDH), and isocitrate dehydrogenase (ICDH). However, the contribution of each enzyme to NADPH production in the cortex of adrenal gland has not been established. Therefore, activity of G6PD, PGD, MDH, and ICDH was localized and quantified in rat adrenocortical tissue using metabolic mapping, image analysis, and electron microscopy. The four enzymes have similar localization patterns in adrenal gland with highest activities in the zona fasciculata of the cortex. G6PD activity was strongest, PGD, MDH, and ICDH activity was approximately 60%, 15%, and 7% of G6PD activity, respectively. The K(m) value of G6PD for glucose-6-phosphate was two times higher than the K(m) value of PGD for phosphogluconate. As a consequence, virtual flux rates through G6PD and PGD are largely similar. It is concluded that G6PD and PGD provide the major part of NADPH in adrenocortical cells. Their activity is localized in the cytoplasm associated with free ribosomes and membranes of the smooth endoplasmic reticulum, indicating that NADPH-demanding processes related to biosynthesis of steroid hormones take place at these sites. Complete inhibition of G6PD by androsterones suggests that there is feedback regulation of steroid hormone biosynthesis via G6PD.     

Electron microscopical demonstration of glucose-6-phosphatase in native cryostat sections fixed with glutaraldehyde through semipermeable membranes.

The cytochemical demonstration of glucose-6-phosphatase (G6Pase) activity in native cryostat sections fixed with glutaraldehyde through semipermeable membranes is superior to conventional methods with regard to exact localization and lack of inactivation and diffusion of the enzyme, together with simultaneous excellent preservation of the tissue fine structure. In rat liver not only hepatocytes but also many bile duct epithelia and endothelia of arterioles and venules show a marked G6Pase activity in the membranes of the endoplasmic reticulum including the nuclear envelope.     

Seasonal ultrastructural variations in pinealocytes of the woodchuck,Marmota monax

The ultrastructure of the pinealocyte in the woodchuck, Marmota monax, was studied during the four seasons of the year. Fall cells have a fairly uniform cytoplasmic density, organelles consistent with synthetic and/or secretory activity and rather extensive pericapillary and intercellular spaces. Many winter pinealocytes are nearly devoid of ribosomes and granular endoplasmic reticulum but contain lipid droplets associated with mitochondria. Pericapillary and intercellular spaces are minimal. Spring glands have the greatest variation in cytoplasmic density with intercellular and pericapillary spaces similar to that seen in fall glands. Cells containing electron dense cytoplasm have Golgi zone associated, secretory granules, free ribosomes, short sections of granular endoplasmic reticulum and dense bodies. Cells with a more electron lucent cytoplasm are similar to the most frequently observed summer pinealocytes which have numerous Golgi zones but few associated secretory granules. Microtubules are prominent in the cytoplasm of these cells, the plasma membranes are smooth and intercellular and pericapillary spaces are minimal. A yearly rhythm or cyclic activity of the pinealocyte is suggested.     

Electron microscopical demonstration of glucose-6-phosphatase in native cryostat sections fixed with glutaraldehyde through semipermeable membranes

Summary The cytochemical demonstration of glucose-6-phosphatase (G6Pase) activity in native cryostat sections fixed with glutaraldehyde through semipermeable membranes is superior to conventional methods with regard to exact localization and lack of inactivation and diffusion of the enzyme, together with simultaneous excellent preservation of the tissue fine structure. In rat liver not only hepatocytes but also many bile duct epithelia and endothelia of arterioles and venules show a marked G6Pase activity in the membranes of the endoplasmic reticulum including the nuclear envelope. This work was kindly supported by the Deutsche Forschungsgemeinschaft An erratum to this article is available at .     

The primordial germ cells in early mouse embryos: light and electron microscopic studies

The migrating primordial germ cells in mouse embryos aged between 8.5 and 11 days were identified under the light and electron microscopes by means of localizing alkaline phosphatase activity, and their ultrastructure was studied. Most of the migrating primordial germ cells were round with smooth contour; they were invariably in close contact with irregularly shaped surrounding cells. The ultrastructural characteristics of early primordial germ cells included: prominent nucleoli, cytoplasmic protrusions into the nuclei, Golgi complexes with alkaline phosphatase activity, disappearance of granular endoplasmic reticulum with increasing age of the embryos, dense cytoplasm with extremely abundant ribosomes, and cytoplasmic dense granules. The significance of these findings is discussed in relation to the origin and migration of mouse primordial germ cells.     

Isolation and characterization of infected and uninfected cells from soybean nodules : role of uninfected cells in ureide synthesis

The distribution of organelles and associated enzymes between cells containing bacteroids and uninfected cells from nodules of Glycine max L. Merr. cv Amsoy 71 was investigated by separation of protoplasts on a sucrose step-gradient. Infected protoplasts were much larger, irregular in shape, and more dense than uninfected protoplasts. The peroxisomal enzymes, uricase and catalase, were present at much higher specific activity in the uninfected cell fraction. Allantoinase, an enzyme of the endoplasmic reticulum, had a greater specific activity in the uninfected cell fraction. Several enzymes whose products are required for purine biosynthesis, including phosphoglycerate dehydrogenase, aspartate aminotransferase, 6-phosphogluconate dehydrogenase, and glucose-6-phosphate dehydrogenase, exhibited a higher specific activity in the uninfected cell fraction. Isozymes of aspartate aminotransferase were separated on native gels and located by an activity stain. The soluble isozyme was predominantly found in the uninfected cell fraction. These data suggest that peroxisomes, containing uricase and catalase for conversion of uric acid to allantoin, are present only in the uninfected cells of soybean nodules. The uninfected cells also appear to be the site of the allantoinase reaction.     

Demonstration of glucose-6-phosphate dehydrogenase in rat Kupffer cells by a newly-developed ultrastructural enzyme-cytochemistry

Although various tissue macrophages possess high glucose-6-phosphate dehydrogenase (G6PD) activity, which is reported to be closely associated with their phagocytotic/bactericidal function, the fine subcellular localization of this enzyme in liver resident macrophages (Kupffer cells) has not been determined. We have investigated the subcellular localization of G6PD in Kupffer cells in rat liver, using a newly developed enzyme-cytochemical (copper-ferrocyanide) method. Electron-dense precipitates indicating G6PD activity were clearly visible in the cytoplasm and on the cytosolic side of the endoplasmic reticulum of Kupffer cells. Cytochemical controls ensured specific detection of the enzymatic activity. Rat Kupffer cells abundantly possessed enzyme-cytochemically detectable G6PD activity. Kupffer cell G6PD may play a role in liver defense by delivering NADPH to NADPH-dependent enzymes. G6PD enzyme-cytochemistry may be a useful tool for the study of Kupffer cell functions.     

Dehydrogenases of the pentose phosphate pathway in rat liver peroxisomes

Subcellular distribution of pentose-phosphate cycle enzymes in rat liver was investigated, using differential and isopycnic centrifugation. The activities of the NADP+-dependent dehydrogenases of the pentose-phosphate pathway (glucose-6-phosphate dehydrogenase and phosphogluconate dehydrogenase) were detected in the purified peroxisomal fraction as well as in the cytosol. Both dehydrogenases were localized in the peroxisomal matrix. Chronic administration of the hypolipidemic drug clofibrate (ethyl-alpha-p-chlorophenoxyisobutyrate) caused a 1.5-2.5-fold increase in the amount of glucose-6-phosphate and phosphogluconate dehydrogenases in the purified peroxisomes. Clofibrate decreased the phosphogluconate dehydrogenase, but did not alter glucose-6-phosphate dehydrogenase activity in the cytosolic fraction. The results obtained indicate that the enzymes of the non-oxidative segment of the pentose cycle (transketolase, transaldolase, triosephosphate isomerase and glucose-phosphate isomerase) are present only in a soluble form in the cytosol, but not in the peroxisomes or other particles, and that ionogenic interaction of the enzymes with the mitochondrial and other membranes takes place during homogenization of the tissue in 0.25 M sucrose. Similar to catalase, glucose-6-phosphate dehydrogenase and phosphogluconate dehydrogenase are present in the intact peroxisomes in a latent form. The enzymes have Km values for their substrates in the millimolar range (0.2 mM for glucose-6-phosphate and 0.10-0.12 mM for 6-phosphogluconate). NADP+, but not NAD+, serves as a coenzyme for both enzymes. Glucose-6-phosphate dehydrogenase was inhibited by palmitoyl-CoA, and to a lesser extent by NADPH. Peroxisomal glucose-6-phosphate and phosphogluconate dehydrogenases have molecular mass of 280 kDa and 96 kDa, respectively. The putative functional role of pentose-phosphate cycle dehydrogenases in rat liver peroxisomes is discussed.     

Ultrastructure of the haemocytes of Tetrodontophora bielanensis Waga (Collembola)

Five types of haemocytes: prohaemocytes, plasmatocytes, granular haemocytes, spherule cells and phagocytes, have been distinguished on the basis of ultrastructural studies. Prohaemocytes are ovoid cells with a simple structural organization. Plasmatocytes are larger; their cytoplasm contains well-developed rough endoplasmic reticulum, numerous mitochondria and free ribosomes. Granular haemocytes are the most numerous of the blood cells, characterized by the presence of electron-dense granules. The cytoplasm of spherule cells contains many spherules made up of filamentous material of medium electron density. Rough endoplasmic reticulum, free ribosomes and mitochondria are also found in the cytoplasm. Phagocytes are the largest haemocytes. Their cytoplasm contains an abundance of lysosomes and myelin structures. In addition to haemocytes, cells intermediate between plasmatocytes and granular haemocytes have been observed, which indicates that the granular haemocytes are derived from plasmatocytes.     

Simultaneous demonstration of acid phosphatase and glucose-6-phosphate dehydrogenase in mouse hepatocytes. A novel electron-microscopic dual staining enzyme-cytochemistry

Acid phosphatase (ACPase) and glucose-6-phosphate dehydrogenase (G6PD) play important roles in cell biology/disease pathophysiology in various organs including the liver. The purpose of the present report is to introduce a new enzymecytochemical method to simultaneously demonstrate the subcellular localization of ACPase and G6PD within the same hepatocyte in the mouse liver. The ultrastructural localization of ACPase and G6PD were demonstrated, with concomitant use of the cerium method and the copper-ferrocyanide method, respectively. ACPase labelings were localized in the lysosomes, and G6PD labelings were visible in the cytoplasm and on the cytosolic side of the endoplasmic reticulum of the hepatocyte. This novel double staining procedure may be a useful histochemical tool for the study of liver functions in both physiological and pathological conditions.     

The detection of sorbitol dehydrogenase in the cytoplasm of liver cells in birds and its relation to alcohol dehydrogenase and glucose-6-phosphate dehydrogenase

Comparative studies have been made in the specific activity of sorbitol dehydrogenase, glucose-6-phosphate and alcohol dehydrogenases in the cytoplasm from the liver of wild and domestic ducks, hen and pheasant. High activity of all the three enzymes was found in ducks indicating the effective sorbitol (polyol) metabolism of glucose. The activity of glucose-6-phosphate dehydrogenase is an order lower as compared with the activity of sorbitol and alcohol dehydrogenases in the cytoplasm of hen liver. The same relationship was found for the activity of sorbitol dehydrogenase in the cytoplasm of pheasant liver.     

Destructive and restorative processes of the liver in rats under intensive physical loading

The ultrastructure of rat liver cells after running exercise was investigated. When rats were trained for a month and sacrificed immediately after the last exercise it was revealed that the number of liver cells mitochondria increased, but many of them had alterations: mitochondria became swollen, had lucid matrix. There were some variations in degree of alterations between different mitochondria: a) in the same hepatocyte, b) in different hepatocytes of the same animal, that was connected with individual sensitivity of organelles on the levels of the cell and of the organ. Rough endoplasmic reticulum bore few ribosomes. Glycogen was absent. There were abundant vesicles of smooth endoplasmic reticulum, autophagic vacuoles and peroxisomes in the liver cell cytoplasm. Adaptation of rat liver to the exercise programme becomes evident by 1.5 month of exercise. Mitochondria and rough endoplasmic reticulum were numerous and of normal structure. There were many peroxisomes and glycogen granules in the cytoplasm of hepatocyte. The presence of large autophagic vacuoles in the cytoplasm of some hepatocytes were obviously connected with more rapid destruction of some organelles, than in control.     

Glucose-6-phosphate dehydrogenase and mouse Kupffer cell activation: an ultrastructural dual staining enzyme-cytochemical study

Glucose-6-phosphate dehydrogenase (G6PD) plays an important role in Kupffer cell function, especially in phagocytosis activity. Although it was suggested that Kupffer G6PD may be upregulated in Kupffer phagocytosis/activation, direct morphological evidence has been lacking. Acid phosphatase (ACP), a representative lysosomal enzyme, can be used as a cytochemical marker for phagocyte activation. Using an ultrastructural enzyme-cytochemical dual staining method, I simultaneously localized G6PD and ACP activity in mouse Kupffer cells on a cell-by-cell basis, and examined whether or not cytochemically detectable G6PD activity increases in phagocytosing/activated mouse Kupffer cells. Glucose-6-phosphate dehydrogenase labelings were observed in the cytoplasm and on the cytosolic side of the endoplasmic reticulum, and ACP labelings were seen in the lysosomes. In phagocytosing Kupffer cells, in which ACP deposits were observed not only in the lysosomes but also on the phagosomal membranes and phagosomal contents, G6PD labelings were denser than dormant Kupffer cells. Enzyme-cytochemically detectable G6PD activity increases in phagocytosing/activated mouse Kupffer cells. Kupffer cell G6PD, activated in phagocytosing Kupffer cells, may play an important role not only in liver defense but also in liver disease pathogenesis/pathophysiology.     

Biogenesis of peroxisomes: immunocytochemical investigation of peroxisomal membrane proteins in proliferating rat liver peroxisomes and in catalase-negative membrane loops

Treatment of rats with a new hypocholesterolemic drug BM 15766 induces proliferation of peroxisomes in pericentral regions of the liver lobule with distinct alterations of the peroxisomal membrane (Baumgart, E., K. Stegmeier, F. H. Schmidt, and H. D. Fahimi. 1987. Lab. Invest. 56:554-564). We have used ultrastructural cytochemistry in conjunction with immunoblotting and immunoelectron microscopy to investigate the effects of this drug on peroxisomal membranes. Highly purified peroxisomal fractions were obtained by Metrizamide gradient centrifugation from control and treated rats. Immunoblots prepared from such peroxisomal fractions incubated with antibodies to 22-, 26-, and 70-kD peroxisomal membrane proteins revealed that the treatment with BM 15766 induced only the 70-kD protein. In sections of normal liver embedded in Lowicryl K4M, all three membrane proteins of peroxisomes could be localized by the postembedding technique. The strongest labeling was obtained with the 22-kD antibody followed by the 70-kD and 26-kD antibodies. In treated animals, double-membraned loops with negative catalase reaction in their lumen, resembling smooth endoplasmic reticulum segments as well as myelin-like figures, were noted in the proximity of some peroxisomes. Serial sectioning revealed that the loops seen at some distance from peroxisomes in the cytoplasm were always continuous with the peroxisomal membranes. The double-membraned loops were consistently negative for glucose-6-phosphatase, a marker for endoplasmic reticulum, but were distinctly labeled with antibodies to peroxisomal membrane proteins. Our observations indicate that these membranous structures are part of the peroxisomal membrane system. They could provide a membrane reservoir for the proliferation of peroxisomes and the expansion of this intracellular compartment.     

Altered redox state of luminal pyridine nucleotides facilitates the sensitivity towards oxidative injury and leads to endoplasmic reticulum stress dependent autophagy in HepG2 cells

Maintenance of the reduced state of luminal pyridine nucleotides in the endoplasmic reticulum – an important pro-survival factor in the cell – is ensured by the concerted action of glucose-6-phosphate transporter and hexose-6-phosphate dehydrogenase. The mechanism by which the redox imbalance leads to cell death was investigated in HepG2 cells. The chemical inhibition of the glucose-6-phosphate transporter, the silencing of hexose-6-phosphate dehydrogenase and/or the glucose-6-phosphate transporter, or the oxidation of luminal NADPH by themselves did not cause a significant loss of cell viability. However, these treatments caused ER calcium store depletion. If these treatments were supplemented with the administration of a subliminal dose of the oxidizing agent menadione, endoplasmic reticulum vacuolization and a loss of viability were observed. Combined treatments resulted in the activation of ATF6 and procaspase-4, and in the induction of Grp78 and CHOP. In spite of the presence of UPR markers and proapoptotic signaling the effector caspases – caspase-3 and caspase-7 – were not active. On the other hand, an elevation of the autophagy marker LC3B was observed. Immunohistochemistry revealed a punctuated distribution of LC3B II, coinciding with the vacuolization of the endoplasmic reticulum. The results suggest that altered redox state of endoplasmic reticulum luminal pyridine nucleotides sensitizes the cell to autophagy.     

Microsomal hexose-6-phosphate and 6-phosphogluconate dehydrogenases in extrahepatic tissues: human placenta and pig kidney cortex

An oxidative pathway of glucose-6-phosphate was found in the microsomal fraction of two extra-hepatic tissues: human placenta and pig kidney cortex. Oxidation activity in microsomes, measured by the formation of 14CO2 from [1-14C] glucose-6-phosphate, was observed only after Triton X-100 treatment and in the presence of methylene blue and NADP. Hexose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase were present in a latent form and required treatment with detergent for full activation. Our results suggest that these enzymes are located in the luminal space of placental and kidney microsomes, and that, as in the liver, they generate NADPH on the inner side of the endoplasmic reticulum when G6P and NADP are available.