Immunohistochemical localization of glutathione S-transferase-T1 in murine kidney, liver, and lung

 Glutathione S-transferase-mediated metabolism of exogenous compounds usually leads to detoxification, but there are some exceptions. For example, glutathione S-transferase-T1 (GSTT1) can also generate genotoxic metabolites. Studies on the biology of GSTT1 are limited by the lack of specific antibodies recognizing GSTT1 in animal tissues. We localized GSTT1 immunohistochemically in mouse kidney, liver, and lung using a novel antibody targeted against the C-terminus of rat GSTT1 (rGSTT1). The antibody was characterized using immunoblot and shown to specifically recognize rGSTT1 and mouse GSTT1, but not human GSTT1. In kidney, GSTT1 staining was detected only in collecting duct epithelium. In liver, pericentral hepatocytes showed cytoplasmic and nuclear staining. Nuclear staining was also observed in several other hepatocytes without relation to liver zonation. Nuclei and supranuclear cytoplasm of bile duct epithelium and endothelium of interlobular arterioles also reacted strongly. In lung, staining was observed in bronchiolar epithelium and in surrounding muscle cells. Type II pneumocytes and endothelial cells of intrapulmonary capillaries also showed strong positive staining. This report describes the first immunohistochemical localization of GSTT1 in mammalian tissues. The reported location of GSTT1 is consistent with its known metabolic activity toward compounds such as dichloromethane and their metabolism into genotoxic products. Accepted: 11 May 1998     

Tissue-specific expression and subcellular distribution of murine glutathione S-transferase class kappa.

Class kappa glutathione S-transferases are a poorly characterized family of detoxication enzymes whose localization has not been defined. In this study we investigated the tissue, cellular, and subcellular distribution of mouse glutathione S-transferase class kappa 1 (mGSTK1) protein using a variety of immunolocalization techniques. Western blotting analysis of mouse tissue homogenates demonstrated that mGSTK1 is expressed at relatively high levels in liver and stomach. Moderate expression was observed in kidney, heart, large intestine, testis, and lung, whereas sparse or essentially no mGSTK1 protein was detected in small intestine, brain, spleen, and skeletal muscle. Immunohistochemical (IHC) analysis for mGSTK1 revealed granular staining of hepatocytes throughout the liver, consistent with organelle staining. IHC analysis of murine kidney localized GSTK1 to the straight portion of the proximal convoluted tubule (pars recta). Staining was consistent with regions rich in mitochondria. Electron microscopy, using indirect immunocolloidal gold staining, clearly showed that mGSTK1 was localized in mitochondria in both mouse liver and kidney. These results are consistent with a role for mGST K1-1 in detoxification, and the confirmation of the intramitochondrial localization of this enzyme implies a unique role for GST class kappa as an antioxidant enzyme.     

Immunohistochemical localization of pyruvate kinase isoenzymes in chicken tissues.

A method for the localization of pyruvate kinase isoenzymes type L, M2 and M1 in tissue sections is described. Mono-specific antibodies directed against isoenzymes of pyruvate kinase from chicken and the peroxidase antiperoxidase method were used. The following preferential localizations of the isoenzymes in chicken tissues were observed: Pyruvate kinase M1 was found in skeletal muscle. The white muscle fibers were more intensely stained than the red. Some dark muscles (e.g., anterior latissimus dorsi) and the heart muscle showed no reaction with antiserum against pyruvate kinase M1. Pyruvate kinase type L was found in the hepatocytes and in kidney cortex. Pyruvate kinase type M2 was seen in the distal tubules of kidney, in hepatocytes and sinusoidal cells in liver, in lung, adipose tissue, and in the spleen mainly in the bursa dependent areas. Pyruvate kinase type M2 was detected in high concentrations in the granulation tissue of regenerating liver after partial hepatectomy. Liver sections of a hen bearing a pancreatic tumor showed an unusually high content of pyruvate kinase type M2 in some hepatocytes, which were each clustered to spots in the liver parenchyma. Thus, contrary to previous reports, the tissue distribution of isoenzymes in chicken is similar to that of other vertebrates.     

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.     

Quantification of neutral cysteine protease bleomycin hydrolase and its localization in rat tissues

A neutral cysteine protease, bleomycin hydrolase (BH), was found to be present in the range 3.7-131.1 ng per mg of rat tissues by enzyme-lined immunosorbent assay (ELISA). Newborn rat skin contained the highest amount of BH, and relatively high levels of BH were detected in the kidney and liver of 6-week-old male rats. The tissue distribution of BH in female rats was similar to that in male rats. Moreover, BH was detected in the extracts of erythrocytes and leukocyte-rich cells as well as in those of rat hemo-lymphocytic lineage cell lines by Western blotting. The BH level was increased at 6 weeks after birth and then slightly decreased. By immunohistochemistry, BH was localized as granular staining in the distal and proximal tubular cells of the kidney, and it was also detected in hepatocytes of the liver, in the red pulpy region of the spleen and in neurons of the brain. An immunoelectron microscopic study showed that BH-immunoreactivity was essentially located in the cytoplasm and at the outer membrane of the rough endoplasmic reticulum of epithelial cells of the kidney, as well as in that of hepatocytes of the liver. These results suggest that BH may play ubiquitous and unique roles in rat tissues.     

Detection,tissue distribution and (sub)cellular localization of fatty acid-binding protein types

Summary This overview of recent work on FABP types is focussed on their detection and expression in various tissues, their cellular and subcellular distribution and their binding properties. Besides the 3 well-known liver, heart and intestinal types, new types as the adipose tissue, myelin and (rat) renal FABPs have been described. Recent observations suggest the occurrence of more tissue-specific types, e.g. in placenta and adrenals. Heart FABP is widely distributed and present in skeletal muscles, kidney, lung, brain and endothelial cells. The cellular distribution of FABP types appears to be related to the function of the cells in liver, muscle and kidney. The presence of FABP in cellular organelles requires more evidence. The functional significance of the occurrence of more FABP types is unclear, in spite of the observed differences in their ligand-protein interaction.Abbreviations FABP(s) Fatty Acid-Binding Protein(s)     

Immunohistochemical localization of pyruvate kinase isoenzymes in chicken tissues

Summary A method for the localization of pyruvate kinase isoenzymes type L, M₂ and M₁ in tissue sections is described. Mono-specific antibodies directed against isoenzymes of pyruvate kinase from chicken and the peroxidase antiperoxidase method were used. The following preferential localizations of the isoenzymes in chicken tissues were observed: Pyruvate kinase M₁ was found in skeletal muscle. The white muscle fibers were more intensely stained than the red. Some dark muscles (e.g., anterior latissimus dorsi) and the heart muscle showed no reaction with antiserum against pyruvate kinase M₁. Pyruvate kinase type L was found in the hepatocytes and in kidney cortex. Pyruvate kinase type M₂ was seen in the distal tubules of kidney, in hepatocytes and sinusoidal cells in liver, in lung, adipose tissue, and in the spleen mainly in the bursa dependent areas. Pyruvate kinase type M₂ was detected in high concentrations in the granulation tissue of type M₂ was detected in high concentrations in the granulation tissue of regenerating liver after partial hepatectomy. Liver sections of a hen bearing a pancreatic tumor showed an unusually high content of pyruvate kinase type M₂ in some hepatocytes, which were each clustered to spots in the liver parenchyma. Thus, contrary to previous reports, the tissue distribution of isoenzymes in chicken is similar to that of other vertebrates.     

Differential expression of ACAT1 and ACAT2 among cells within liver, intestine, kidney, and adrenal of nonhuman primates

Two closely related enzymes with more than 50% sequence identity have been identified that catalyze the esterification of cholesterol using acyl-CoA substrates, namely acyl-CoA:cholesterol acyltransferase 1 (ACAT1) and ACAT2. Both are membrane-spanning proteins believed to reside in the endoplasmic reticulum of cells. ACAT2 has been hypothesized to be associated with lipoprotein particle secretion whereas ACAT1 is ubiquitous and may serve a more general role in cellular cholesterol homeostasis. We have prepared and affinity purified rabbit polyclonal antibodies unique to either ACAT enzyme to identify their cellular localization in liver and intestine, the two main lipoprotein-secreting tissues of the body, and for comparison, kidney and adrenal. In the liver, ACAT2 was identified in the rough endoplasmic reticulum of essentially all hepatocytes whereas ACAT1 was confined to cells lining the intercellular spaces among hepatocytes in a pattern typical of Kupffer cells. In the intestine, ACAT2 signal was strongly present in the apical third of the mucosal cells, whereas ACAT1 staining was diffuse throughout the mucosal cell, but with strong signal in goblet cells, Paneth cells, and villus macrophages. In the kidney, ACAT1 immunostaining was specific for the distal tubules and podocytes within the glomerulus. In the adrenal, ACAT1 signal was strongly present in the cells of the cortex, and absent from other adrenal cell types. No ACAT2 signal was identified in the kidney or adrenal.We conclude that only the cells of the liver and intestine that secrete apolipoprotein B-containing lipoproteins contain ACAT2, whereas ACAT1 is present in numerous other cell types. The data clearly suggest separate functions for these two closely related enzymes, with ACAT2 being most closely associated with plasma cholesterol levels.     

Expression of the aspartate/glutamate mitochondrial carriers aralar1 and citrin during development and in adult rat tissues.

Aralar1 and citrin are members of the subfamily of calcium-binding mitochondrial carriers and correspond to two isoforms of the mitochondrial aspartate/glutamate carrier (AGC). These proteins are activated by Ca2+ acting on the external side of the inner mitochondrial membrane. Although it is known that aralar1 is expressed mainly in skeletal muscle, heart and brain, whereas citrin is present in liver, kidney and heart, the precise tissue distribution of the two proteins in embryonic and adult tissues is largely unknown. We investigated the pattern of expression of aralar1 and citrin in murine embryonic and adult tissues at the mRNA and protein levels. In situ hybridization analysis indicates that both isoforms are expressed strongly in the branchial arches, dermomyotome, limb and tail buds at early embryonic stages. However, citrin was more abundant in the ectodermal components of these structures whereas aralarl had a predominantly mesenchymal localization. The strong expression of citrin in the liver was acquired postnatally, whereas the characteristic expression of aralar1 in skeletal muscle was detected at E18 and that in the heart began early in development (E11) and was preferentially localized to auricular myocardium in late embryonic stages. Aralar1 was also expressed in bone marrow, T-lymphocytes and macrophages, including Kupffer cells in the liver, indicating that this is the major AGC isoform present in the hematopoietic system. Both aralar1 and citrin were expressed in fetal gut and adult stomach, ovary, testis, and pancreas, but only aralar1 is enriched in lung and insulin-secreting beta cells. These results show that aralar1 is expressed in many more tissues than originally believed and is absent from hepatocytes, where citrin is the only AGC isoform present. This explains why citrin deficiency in humans (type II citrullinemia) only affects the liver and suggests that aralar1 may compensate for the lack of citrin in other tissues.     

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.     

Placental transfer of halogenated benzenes (pentachloro-, pentachloronitro-, and hexabromo-) in rats.

The present study was carried out to provide information on the placental transfer of three organohalogens of environmental concern. Pentachloro-, pentachloronitro-, and hexabromobenzene were administered per os to rats daily on days 6 through 15 of gestation at level of 40, 100, and 200 mg/kg body weight. On day 22, the dams were killed and fetuses removed by caesarean section. Maternal brain, heart, kidney, liver, spleen and adipose tissue as well as whole fetus, fetal liver and fetal brain were analyzed for organohalogen residue by GLC. Pentachlorobenzene accumulated in the fetus to a greater extent than hexabromobenzene. In maternal tissues pentachlorobenzene accumulated to the greatest extent in adipose tissue, followed by liver, spleen, brain, heart and kidney. With hexabromobenzene, the greatest accumulation was observed in adipose tissue, followed by spleen, liver, heart, kidney and brain. Pentachloronitrobenzene was not detected (0.05 p.p.m.) in any maternal or fetal tissue.     

Tissue distribution and immunohistochemical localization of the collagen-binding heat-shock protein gp46 in neonatal rats

Summary Collagen-binding heat-shock proteins ofM _r 46–47 kDa have been postulated to function as putative molecular chaperones in the biosynthesis of collagen in several species. The rat homologue of this family of heat-shock proteins is called gp46. In the present study, we employed Western blotting and immunohistochemical methods to determine the tissue distribution and cellular localization of gp46 in the thoracic aorta, heart, kidney, liver and lung of eight-day-old Wistar rats. Highest levels of gp46 were detected in the thoracic aorta and lung, followed by the kidney and heart. Gp46 levels were low to undetectable by Western blot analysis in the liver. Immunohistochemistry revealed that gp46 labelling was observed almost exclusively in three distinct cell types: fibroblasts, muscle cells, and some epithelial cells. Gp46 was detected in the fibroblasts of the hepatic triad, in the interstitium of the alveolar wall and in the tunica adventitia of blood vessels in the majority of tissues examined, in atrial and ventricular cardiomyocytes, in vascular smooth muscle cells of the abluminal portion of the tunica media, in parietal epithelial cells and mesangial cells of the glomerulus, in epithelial cells of the distal tubules and collecting ducts in the kidney and clusters of immature renal tubules, in epithelial cells of the bile duct, and in mesodermal cells surrounding the liver. These results demonstrate that gp46 is present in collagen producing cells and cells undergoing rapid growth and development, suggesting that gp46 may play a significant role in these processes.     

Tissue distribution of Diablo/Smac revealed by monoclonal antibodies

Diablo/Smac is a mammalian pro-apoptotic protein that can antagonize the inhibitor of apoptosis proteins (IAPs). We have produced monoclonal antibodies specific for Diablo and have used these to examine its tissue distribution and subcellular localization in healthy and apoptotic cells. Diablo could be detected in a wide range of mouse tissues including liver, kidney, lung, intestine, pancreas and testes by Western blot analysis. Immunohistochemical analysis found Diablo to be most abundant in the germinal cells of the testes, the parenchymal cells of the liver and the tubule cells of the kidney. In support of previous subcellular localization analysis, Diablo was present within the mitochondria of healthy cells, but released into the cytosol following the induction of apoptosis by UV.     

Tissue and cellular distribution of alpha-L-iduronidase in the pig

We investigated the alpha-L-iduronidase activity of various pig tissues. Furthermore, we examined the tissues using antibody, enzyme immunoassay (EIA), and immunohistochemical methods. The amounts of enzyme measured by the EIA method in the various tissues were proportional to their enzyme activities and also to their immunohistochemical characteristics. The tissues could thus be classified into three groups: a high enzyme activity group composed of the liver, kidney, and spleen; a moderate activity group comprising the lung, lymph nodes, stomach, ileum, colon, and pancreas; and a low activity group consisting of the heart, diaphragm, iliopsoas muscle, cerebrum, cerebellum, and skin. The molecular weight of the enzyme in each tissue did not reveal any heterogeneity, having two components of 70 KD and 62 KD by Western blot analysis. Immunohistochemically, alpha-L-iduronidase was strongly detected in the lysosomal membranes of cells of the mononuclear phagocyte system, epithelial cells of the proximal tubules in the kidney, and some blastic cells, whereas hepatocytes revealed weak positive reactions. The tissue and cellular distribution of the enzyme appeared to have a close relation to tissues that manifest or are affected by alpha-L-iduronidase deficiency.     

Fhit protein is preferentially expressed in the nucleus of monocyte-derived cells and its possible biological significance

The FHIT gene encompassing the most active common human fragile region, FRA3B, has been proposed as a tumour suppressor gene for important common human carcinomas. The mechanism in which Fhit protein exerts its tumour suppressor activity is still obscure. To further understand the Fhit function associated with its intracellular localization we have investigated its cellular localization and distribution in human normal and cancerous tissues. Data of 1500 samples from immunohistochemistry showed that Fhit protein was preferentially and stably expressed in the nucleus of monocyte-derived or histiocytic lineage cells including monocytes of the circulating blood cells, macrophages of the connective tissue, Kupffer cells of the liver, alveolar macrophages or dust cells of the lung, osteoclasts of bone, microglia of the brain, epithelioid cells under chronic inflammatory conditions, foreign-body giant cells, Langerhans cells of the epidermis and dendritic cells of various kinds of human tissue, although the protein could also be infrequently observed in the nucleus of some quiescent epithelial cells. In active cells other than histiocytes, Fhit protein was detected either in cytoplasm or was negative. Neurons expressed Fhit strongly and neuroglial cells did so moderately but only in the cytoplasm. There was no Fhit protein detected in the neutrophils, lymphocytes, plasma cells and lipocytes. The present data showes that the stable nuclear localization of Fhit is not only a special marker for histiocytes with various morphologies but also may suggest the other function concerning Fhit as a signaling molecule related to anti-proliferation function. The detailed biological function related to nuclear localization of Fhit protein in the histiocytes remains to be further studied.     

Immunohistochemical analysis of paraoxonases-1, 2, and 3 expression in normal mouse tissues

The paraoxonase (PON) enzyme family, comprising PON1, PON2, and PON3, are antioxidant enzymes that degrade oxidised phospholipids. We describe the immunohistochemical localisation of the PON proteins in the normal mouse. Antibodies were obtained by inoculating rabbits with peptides derived from specific sequences of mature PONs. PON1 and PON3 were detected in the skin external epithelium, acini of the sebaceous glands, tongue epithelium, acini of the submandibular gland, surface epithelia of the stomach and the intestine, hepatocytes, exocrine pancreas acini, fibre tracts of the encephalon and the spinal cord, skeletal and cardiac muscle, eye lens epithelium and retinal layers, adipocytes, chondrocytes, epithelial cells of the trachea and bronchiole, ovary follicular fluid, seminiferous tubules, spermatozoa, and kidney proximal tubules. PON2 expression was weaker than that of PON1 and PON3, and was absent in some of the tissues studied, such as submandibular gland, nerve cells, and adipocytes. In muscle cells, PON2 expression was restricted to the endomysium. Apolipoprotein A-I did not colocalise with PONs, suggesting local synthesis. This study provides an experimental model to investigate the role played by these enzymes as antioxidants and their relationship with the development of a variety of diseases.