High levels of xanthine oxidoreductase in rat endothelial, epithelial and connective tissue cells. A relation between localization and function?

The localization of xanthine oxidoreductase activity was investigated in unfixed cryostat sections of various rat tissues by an enzyme histochemical method which specifically demonstrates both the dehydrogenase and oxidase forms of xanthine oxidoreductase. High activity was found in epithelial cells from skin, vagina, uterus, penis, liver, oral and nasal cavities, tongue, esophagus, fore-stomach and small intestine. In addition activity was demonstrated in sinusoidal cells of liver and adrenal cortex, endothelial cells in various organs and connective tissue fibroblasts. Xanthine oxidoreductase produces urate which is a scavenger of oxygen-derived radicals. Because the enzyme is found in epithelial and endothelial cells which are subject to relatively high oxidant stress, it is postulated that in these cells xanthine oxidoreductase is involved in the antioxidant enzyme defense system. In addition, a possible role for the enzyme in proliferation and differentiation processes is discussed.     

Broad expression of fructose-1,6-bisphosphatase and phosphoenolpyruvate carboxykinase provide evidence for gluconeogenesis in human tissues other than liver and kidney

The importance of renal and hepatic gluconeogenesis in glucose homeostasis is well established, but the cellular localization of the key gluconeogenic enzymes liver fructose-1,6-bisphosphatase (FBPase) and cytosolic phosphoenolpyruvate carboxykinase (PEPCK) in these organs and the potential contribution of other tissues in this process has not been investigated in detail. Therefore, we analyzed the human tissue localization and cellular distribution of FBPase and PEPCK immunohistochemically. The localization analysis demonstrated that FBPase was expressed in many tissues that had not been previously reported to contain FBPase activity (e.g., prostate, ovary, suprarenal cortex, stomach, and heart). In some multicellular tissues, this enzyme was detected in specialized areas such as epithelial cells of the small intestine and prostate or lung pneumocytes II. Interestingly, FBPase was also present in pancreas and cortex cells of the adrenal gland, organs that are involved in the control of carbohydrate and lipid metabolism. Although similar results were obtained for PEPCK localization, different expression of this enzyme was observed in pancreas, adrenal gland, and pneumocytes type I. These results show that co-expression of FBPase and PEPCK occurs not only in kidney and liver, but also in a variety of organs such as the small intestine, stomach, adrenal gland, testis, and prostate which might also contribute to gluconeogenesis. Our results are consistent with published data on the expression of glucose-6-phosphatase in the human small intestine, providing evidence that this organ may play an important role in the human glucose homeostasis.     

Localization of xanthine oxidase in crystalline cores of peroxisomes. A cytochemical and biochemical study

The ultrastructural cytochemical localization of xanthine oxidase activity in rat liver was investigated by the cerium technique. The reaction product was found in the cytoplasm of endothelial cells in liver sinusoids and, in addition, in crystalline cores of peroxisomes of liver parenchymal cells. Xanthine oxidase was also present in peroxisomal cores of beef liver and kidney, but not in rat kidney peroxisomes, which lack crystalline cores. The localization in peroxisomal cores of rat liver was confirmed also biochemically using highly purified peroxisomal fractions and subfractions containing exclusively the crystalline cores. Moreover, high levels of molybdenum were found in isolated peroxisomal cores by atomic absorption spectroscopy, thus corroborating the association of the molybdenum-containing enzyme with the cores. Since urate oxidase is also present within the same compartment of peroxisomes, it is possible that the crystalline cores harbor a complex of several enzymes involved in the purine metabolism.     

Ultrastructural localization of glucose-6-phosphatase activity in proximal convoluted tubule cells of rat kidney

Summary The ultrastructural localization of glucose 6-phosphatase activity was investigated in the proximal convoluted tubule cells of the rat kidney. The reaction product for the enzyme activity was present in the endoplasmic reticulum and nuclear envelope, as reported for the hepatic enzyme and others, but was absent from the brush border, plasma membrane and other organelles. The metabolic significance of the association of this enzyme with the endoplasmic reticulum and the role of the enzyme in the active reabsorption and transport of glucose in the renal tubules are discussed.     

Ultrastructural localization of xanthine oxidase activity in the digestive tract of the rat

Summary Precise localization of xanthine oxidase activity might elucidate physiological functions of the enzyme, which have not been established so far. Because xanthine oxidase is sensitive to chemical (aldehyde) fixation, we have localized its activity in unfixed cryostat sections of rat duodenum, oesophagus and tongue mounted on a semipermeable membrane. Previous studies had shown that this procedure enables the exact localization of activities of peroxisomal oxidases with maintenance of acceptable ultrastructure. Moreover, leakage and/or diffusion of enzyme molecules was prevented with this method. The incubation medium to detect xanthine oxidase activity contained hypoxanthine as substrate and cerium ions as capturing agent for hydrogen peroxide. After incubation, reaction product in the sections was either visualized for light microscopy or sections were fixed immediately and processed for electron microscopy. At the ultrastructural level, crystalline reaction product specifically formed by xanthine oxidase activity was found to be present in the cytoplasmic matrix of enterocytes and goblet cells and in mucus of duodenum. Moderate activity was found in the cytoplasm of apical cell layers of epithelia of oesophagus and tongue, with highest activity in the cornified layer. Moreover, large amounts of reaction product were found to surround bacteria present between cell remnants of the cornified layer of the oesophagus. Many bacteria surrounded by the enzyme showed signs of destruction and/or cell death. The intracellular localization of xanthine oxidase activity in the cytoplasm of epithelial cells as well as the extracellular localization suggest that the enzyme plays a role in the lumen of the digestive tract, for instance in the defence against microorganisms.     

Tissue distribution,molecular cloning,and gene expression of cytosolic glutathione peroxidase in Japanese monkey

Cytosolic glutathione peroxidase (GPX-1) is an important antioxidant enzyme that scavange hydrogen peroxide in mammalian cells. The level of GPX-1 activity in Japanese monkey (Macaca fuscata) tissues was determined and it was found to be high in the liver, kidney, and adrenal gland followed by the small intestine. We also cloned the GPX-1 cDNA that included the whole protein-coding region. The active-site selenocysteine was assumed to be encoded by a TGA codon. Compared to the GPX-1s of other mammalian species, essential residues in catalysis were well conserved in monkey GPX-1. Amino acid substitutions were frequent in the N- and C-terminal regions which are less essential in catalysis. Expression of GPX-1 mRNA was found to be high in the liver, kidney, and adrenal gland, in consistence with the tissue distribution of GPX-1 activity.     

Fine structural localization of acetylcholinesterase activity in rat submandibular gland

Using the indirect thiocholine method, the ultrastructural localization of acetylcholinesterase (AChE) activity in the normal rat submandibular gland was studied. Cytochemical demonstration of AChE is based on coupling the hydrolysis of acetylthiocholine iodide to the precipitation of heavy metal salts. AChE-associated reaction product was selectively revealed in the perinuclear space and in the endoplasmic reticulum of the intercalated duct cells, in some cells of granular convoluted tubules, and in the striated duct epithelium, as well as in the myoepithelial cells. Although AChE activity generally occurred inside the cells, electron-dense precipitates were shown in intercellular space and in the stroma of the gland. Fine localization of AChE activity was also found in nerve bundles, predominantly between axons and between axons and Schwann cell. Our observations indicate that AChE is synthesized in the epithelium of the ducts and in the myoepithelial cells of the salivary gland. It is not known yet whether this enzyme is released from the intracytoplasmic membrane system into the extracellular space and then transported to the regions of the gland innervation. Conceivably AChE synthesized in the submandibular gland cells could also be considered an inhibitory modulator of the regulatory functions of biologically active polypeptides.     

Localization of xanthine oxidoreductase activity using the tissue protectant polyvinyl alcohol and final electron acceptor Tetranitro BT

We have detected xanthine oxidoreductase activity in unfixed cryostat sections of rat and chicken liver, rat duodenum, and bovine mammary gland using the tissue protectant polyvinyl alcohol, the electron carrier 1-methoxyphenazine methosulfate, the final electron acceptor Tetranitro BT, and hypoxanthine as a substrate. Enzyme activity was localized in rat duodenum at lateral membranes and brush borders of enterocytes and in goblet cells and mucus. Hepatocytes in pericentral areas and especially sinusoidal cells showed high activity in rat liver. Xanthine oxidoreductase was also detected in epithelial cells and milk lipid globules of lactating bovine mammary gland, which is known to contain large quantities of the oxidase form of the enzyme. Chicken liver, which contains an inconvertible dehydrogenase form, also showed high activity in sinusoidal cells. Therefore, we conclude that the tetrazolium reaction demonstrates both the dehydrogenase and the oxidase form of xanthine oxidoreductase. Control activity, in the absence of hypoxanthine or in the presence of the competitive inhibitor allopurinol, was low in all tissues studied. Addition of O2 or NAD to the incubation medium did not change the specific reaction in bovine mammary gland or chicken liver, implying that the dehydrogenase and the oxidase form are not dependent on their natural electron acceptors in this tetrazolium salt reaction. We conclude that the present light microscopic method gives specific and precise localization of xanthine oxidoreductase activity in situ.     

THE USE OF BEEF LIVER CATALASE AS A PROTEIN TRACER FOR ELECTRON MICROSCOPY

Beef liver catalase was injected intravenously into mice, and its distribution in the kidney, myocardium, and liver was studied with the electron microscope. A specific and relatively sensitive method was developed for its ultrastructural localization, based on the peroxidatic activity of catalase and employing a modified Graham and Karnovsky incubation medium. The main features of the medium were a higher concentration of diaminobenzidine, barium peroxide as the source of peroxide, and pH of 8.5. Ultrastructurally, the enzyme was seen to permeate the endothelial fenestrae and basement membranes of tubular and glomerular capillaries of the kidney. The urinary space and tubular lumina contained no reaction product. In the myocardial capillaries, the tracer filled the pinocytotic vesicles but did not diffuse across the intercellular clefts of the endothelium. In liver, uptake of catalase was seen both in hepatocytes and in Kupffer cells.     

Angiotensin converting enzyme from sheep mammary, lingual and other tissues

Occurrence of angiotensin converting enzyme (ACE) in mammary gland and tongue taste epithelium was demonstrated for the first time. Six times higher ACE activity in lactating mammary gland, than non-lactating mammary gland, suggested pregnancy and lactation hormonal dependent expression of ACE in female mammals. ACE activity was highest in choroid plexus, less in spinal cord and moderate in cerebrum, medulla, cerebellum and pons. Distribution of ACE in different regions of skin, kidney and among other tissues was different. Presence of ACE in adrenal glands, pancreas, bone marrow and thyroid gland indicated functions other than blood pressure homeostasis for this enzyme.     

Localization of type 8 17beta-hydroxysteroid dehydrogenase mRNA in mouse tissues as studied by in situ hybridization.

The enzyme type 8 17beta-hydroxysteroid dehydrogenase (17beta-HSD) selectively catalyzes the conversion of estradiol (E2) to estrone (E1). To obtain detailed information on the sites of action of type 8 17beta-HSD, we have studied the cellular localization of type 8 17beta-HSD mRNA in mouse tissues using in situ hybridization. In the ovary, hybridization signal was detected in granulosa cells of growing follicles and luteal cells. In the uterus, type 8 17beta-HSD mRNA was found in the epithelial (luminal and glandular) and stromal cells. In the female mammary gland, the enzyme mRNA was seen in ductal epithelial cells and stromal cells. In the testis, hybridization signal was observed in the seminiferous tubule. In the prostate, type 8 17beta-HSD was detected in the epithelial cells of the acini and stromal cells. In the clitoral and preputial glands, labeling was detected in the epithelial cells of acini and small ducts. The three lobes of the pituitary gland were labeled. In the adrenal gland, hybridization signal was observed in the three zones of the cortex, the medulla being unlabeled. In the kidney, the enzyme mRNA was found to be expressed in the epithelial cells of proximal convoluted tubules. In the liver, all the hepatocytes exhibited a positive signal. In the lung, type 8 17beta-HSD mRNA was detected in bronchial epithelial cells and walls of pulmonary arteries. The present data suggest that type 8 17beta-HSD can exert its action to downregulate E2 levels in a large variety of tissues.     

Neuropeptide W is expressed in the noradrenalin-containing cells in the rat adrenal medulla

Neuropeptide W (NPW) is an endogenous ligand for GPR7, a member of the G-protein-coupled receptor family. NPW plays an important role in the regulation of both feeding and energy metabolism, and is also implicated in modulating responses to an acute inflammatory pain through activation of the hypothalamus-pituitary-adrenal axis. GPR7 mRNA has been shown to be expressed in the hypothalamus, pituitary gland and adrenal cortex. Similarly, NPW expression has been demonstrated in the brain and pituitary gland. However, the precise distribution of NPW-producing cells in the adrenal gland remains unknown. The aim of this study was to explore the distribution and localization of NPW immunoreactivity in the rat adrenal gland. Total RNA was prepared from the hypothalamus, pituitary gland and adrenal gland. RT-PCR revealed the expression of NPW mRNA in these tissues, while in situ hybridization demonstrated the presence of NPW mRNA in the adrenal medulla. When immunohistochemistry was performed on sections of adrenal gland, NPW-like immunoreactivity (NPW-LI) was observed in the medulla but not in the cortex. Moreover, NPW-LI was found to be co-localized in cells which expressed dopamine beta hydroxylase but not phenylethanolamine-N-methyltransferase. The finding that NPW is expressed in noradrenalin-containing cells in the adrenal medulla suggests that it may play an important role in endocrine function in the adrenal gland.     

Preliminary observations on a glucose-6-phosphate-hydrolyzing enzyme in secretory cells: a light microscopic study

Synopsis A glucose-6-phosphate-hydrolyzing enzyme was localized histochemically in a variety of secretory cells of the rat. Cells exhibiting enzyme activity include thyroid and parafollicular cells, parathyroid and secretory epithelium of the trachea, bronchi and bronchioles. Clusters of ganglion cells underlying these organs are also heavily reactive. In its cytoplasmic staining pattern and its ability to hydrolyze glucose-6-phosphate, the enzyme activity localized in these secretory cells appears similar to glucose-6-phosphatase found in liver and kidney.     

Tissue- and Cell-Specific Co-localization of Intracellular Gelatinolytic Activity and Matrix Metalloproteinase 2

Matrix metalloproteinase 2 (MMP-2) is a proteolytic enzyme that degrades extracellular matrix proteins. Recent studies indicate that MMP-2 also has a role in intracellular proteolysis during various pathological conditions, such as ischemic injuries in heart and brain and in tumor growth. The present study was performed to map the distribution of intracellular MMP-2 activity in various mouse tissues and cells under physiological conditions. Samples from normal brain, heart, lung, liver, spleen, pancreas, kidney, adrenal gland, thyroid gland, gonads, oral mucosa, salivary glands, esophagus, intestines, and skin were subjected to high-resolution in situ gelatin zymography and immunohistochemical staining. In hepatocytes, cardiac myocytes, kidney tubuli cells, epithelial cells in the oral mucosa as well as in excretory ducts of salivary glands, and adrenal cortical cells, we found strong intracellular gelatinolytic activity that was significantly reduced by the metalloprotease inhibitor EDTA but not by the cysteine protease inhibitor E-64. Furthermore, the gelatinolytic activity was co-localized with MMP-2. Western blotting and electron microscopy combined with immunogold labeling revealed the presence of MMP-2 in different intracellular compartments of isolated hepatocytes. Our results indicate that MMP-2 takes part in intracellular proteolysis in specific tissues and cells during physiological conditions.     

Sulfurylation of deoxycorticosterone in human fetal tissues

We determined the specific activity of 21-hydroxysteroid sulfotransferase in a number of human fetal tissues and in tissues of a prepubertal boy (5 years of age). In fetal tissues, the highest specific activities of this enzyme were found in adrenal gland, liver, kidney, intestine, aorta, and testis. In the tissues of the prepubertal boy, 21-hydroxysteroid sulfotransferase activity was demonstrable only in adrenal and liver. Thus, 21-hydroxysteroid sulfotransferase activity is present in some fetal tissues in which DOC may be formed by 21-hydroxylation of progesterone, as steroid 21-hydroxylase activity has been demonstrated previously in adrenal, kidney, and testis. We speculate that sulfurylation of DOC in some tissue sites of DOC formation and action may regulate the action of this mineralocorticosteroid.     

Subcellular distribution of OM cytochrome b-mediated NADH-semidehydroascorbate reductase activity in rat liver

Tissue, cellular, and subcellular distributions of OM cytochrome b-mediated NADH-semidehydroascorbate (SDA) reductase activity were investigated in rat. NADH-SDA reductase activity was found in the post-nuclear particulate fractions of liver, kidney, adrenal gland, heart, brain, lung, and spleen of rat. Liver, kidney, and adrenal gland had higher NADH-SDA reductase activity than other tissues, and OM cytochrome b-dependent activity was 60-70% of the total activity. On the other hand, almost all of the reductase activity of heart and brain cells was mediated by OM cytochrome b. The ratio of the OM cytochrome b-mediated activities of NADH-SDA reductase to rotenone-insensitive NADH-cytochrome c reductase varied among these tissues. OM cytochrome b-mediated NADH-SDA reductase and rotenone-insensitive NADH-cytochrome c reductase activities were mainly present in the parenchymal cells of rat liver. The localization of the cytochrome-mediated reductase activities in the outer mitochondrial membrane was confirmed by subfractionation of liver mitochondria. Among the submicrosomal fractions, OM cytochrome b-mediated NADH-SDA reductase activity was highest in the cis-Golgi membrane fraction, in which monoamine oxidase activity was also highest. On the other hand, OM cytochrome b-mediated rotenone-insensitive NADH-cytochrome c reductase activity showed a slightly different distribution pattern from the NADH-SDA reductase activity. Thenoyltrifluoroacetone (TTFA), a metal chelator, effectively inhibited the NADH-SDA reductase activity, though other metal chelators did not affect the activity. TTFA failed to inhibit rotenone-insensitive NADH-cytochrome c reductase activity at the concentration which gave complete inhibition of NADH-SDA reductase activity.     

Digitonin reaction in electron microscopy

Summary The ultrastructural localization of cholesterol (3-hydroxysterols) has been studied by the digitonin reaction adapted to the electron microscope. The reaction was undertaken in the adrenal gland, liver, and seminiferous tubules of adult albino rats. The digitonincholesterol crystals formed in the reaction proved to be an osmiophilous cylindrical structure made up of coaxial lamellae.     

Carbonic anhydrase in the rat salivary glands: distribution and ultrastructural localization

Rat salivary glands were studied by Hanson's method to specify the ultrastructural localization of carbonic anhydrase (CA). Two different procedures were used: 1) The embedding of the tissues in water-soluble resins, followed by the incubation of the resin sections on the medium. 2) The embedding in epon-araldite of previously incubated frozen sections. Light and electron microscopy were used to observe the distribution and the ultrastructural localization of the cobalt precipitate. In parotid and mandibular glands, CA was localized in the secretion granules and the hyaloplasma of the secretory endpieces. The enzyme was also detected on the basal and lateral membranes of the striated duct cells in the three glands. In the convoluted granular duct cells of the mandibular gland CA was found in the hyaloplasma only. In the sublingual gland, CA was localized in the hyaloplasma of the serous crescents and no activity was detected in the mucous tubules. As regards the localization of the enzyme in the granules of the secretory endpieces of parotid and mandibular glands, it appears that CA has to be considered as a secretory product of these cells; this localization is consistent with the presence of the enzyme in rat saliva.     

Quantitation and immunohistochemical localization of cathepsins E and D in rat tissues and blood cells

The distribution of cathepsins E and D in various rat tissues and blood cells was determined by immunoprecipitation and by immunohistochemistry with discriminative antibodies specific for each enzyme. While cathepsin D was detected in all of the tissues and blood cells tested (except for erythrocytes), cathepsin E had a relatively limited distribution. The cathepsin E content was highest in the stomach and was succeeded in the following order by the urinary bladder, thymus, spleen, cervical lymph node and bone marrow. Significant amounts of cathepsin E were also found in the colon, rectum, jejunum, skin, lung, kidney and submandibular gland. The other tissues tested had little or no detectable cathepsin E content. Of the blood cells tested, lymphocytes and peritoneal neutrophils contained high levels of cathepsin E. Erythrocytes had cathepsin E only as aspartic proteinases. When the subcellular localization of cathepsin E in the neutrophils was investigated by fractionation of the postnuclear supernatants, the enzyme behaved as a soluble cytosolic enzyme. In contrast, cathepsin D was mainly associated with the granular fraction. The immunohistochemical localization of cathepsins E and D was clearly different in the stomach, large intestines, kidney and urinary bladder, but was similar in the lymph node and spleen. The tissue-fixed macrophages, which were notable in the skeletal and cardiac muscle tissues, submucosal layers of the gastrointestinal tracts, salivary gland, lung and trachea, also exhibited similar intense immunoreactivities demonstrative of both cathepsins E and D.     

Immunochemical distribution and immunohistochemical localization of 20β-hydroxysteroid dehydrogenase in neonatal pig tissues

Immunochemical distribution of 20β-hydroxysteroid dehydrogenase (HSD) in neonatal pig tissues was investigated by Western blot analysis of the proteins reacting with anti-20β-HSD antibody. 20β-HSD was present in all organs investigated: brain, lung, thymus, submandibular gland, heart, liver, kidney, spleen, adrenal gland, testis, epididymis, prostate, vas deferens and seminal vesicle. In particular, high concentrations of 20β-HSD were detected in the testis, followed by the kidney and liver, by the [¹²⁵I]-protein A binding method. Immunohistochemical localization of the enzyme was achieved in paraffin sections of the testis, kidney, liver, epididymis, and vas deferens by the streptoavidin-biotin complex method. In the testis, very strong immunostaining was found only in interstitial Leydig cells, whereas the cells in seminiferous tubules, such as Sertoli cells and spermatogenic cells, were entirely negative. In the kidney, strong immunostaining was detected in epithelial cells of Henle's loop. The immunoreactive proteins were also localized in the hepatic lobules of the liver, tall columnar cells of the ductus epididymidis of the epididymis, and mucosal epithelium cells and muscularis of the vas deferens. These observations indicate that tissue distribution of 20β-HSD is similar to that of carbonyl reductase in the human and rat. However, the specific and abundant expression of 20β-HSD in testicular Leydig cells of the neonatal pig, which are concerned with the synthesis of androgens, suggests that 20β-HSD has a very important physiological role in testicular function during the neonatal stage.