Immunocytochemical localization of delta 3, delta 2-enoyl-CoA isomerase in rat liver. The effects of di-(2-ethylhexyl)phthalate, a peroxisome proliferator

Immunocytochemical localization of delta 3, delta 2-enoyl-CoA isomerase (isomerase) was investigated in rat liver. Livers of di-(2-ethylhexyl)phthalate (DEHP)-treated or untreated rats were perfusion-fixed and embedded in Epon or Lowicryl K4M. By light microscopy, reaction deposits for the enzyme were present in the cytoplasmic granules of hepatocytes and interlobular bile duct epithelium. Weak staining was noted in sinus-lining cells. After administration of DEHP, the granular staining of the hepatocytes was markedly enhanced, whereas the staining reaction of the sinus-lining cells decreased. The isomerase staining pattern was quite similar to that of long-chain acyl-CoA dehydrogenase (a mitochondrial marker), but different from that of catalase (a peroxisomal marker). Under electron microscopy, gold particles for isomerase were seen to be confined mainly to mitochondria of the hepatocytes, the bile duct epithelial cells and sinus-lining cells. Peroxisomes were weakly labeled. After DEHP administration, the peroxisomes were markedly induced, but the mitochondria were not. Quantitative analysis showed that the induction of the peroxisomal isomerase was only 2-fold whereas the mitochondrial isomerase was enhanced about 5-fold, 40 times as high as the peroxisomal enzyme. The results show that the mitochondria are the main intracellular site for isomerase and the peroxisomes a minor site. The mitochondrial isomerase of the rat liver is markedly induced by peroxisome proliferators, DEHP and clofibrate.     

Immunocytochemical localization of D-amino acid oxidase in the central clear matrix of rat kidney peroxisomes

Light and electron microscopic localizations of D-amino acid oxidase (DAO) in rat kidney was investigated using immunoenzyme and protein A-gold techniques. The enzyme was purified from rat kidney homogenate and its antibody was raised in rabbits. By Ouchterlony double-diffusion analysis and immunoblot analysis with anti-(rat kidney DAO) immunoglobulin, the antibody was confirmed to be monospecific. The tissue sections (200 micron thick) of fixed rat kidney were embedded in Epon or Lowicryl K4M. Semi-thin sections were stained for DAO by the immunoenzyme technique after removal of epoxy resin for LM, and ultra-thin sections of Lowicryl-embedded material were labeled for DAO by the protein A-gold technique for EM. By LM, fine cytoplasmic granules of proximal tubule were stained exclusively. Among three segments of proximal tubules, and S2 and S3 segments were heavily stained but the S1 segment only weakly so. By EM, gold particles indicating the antigenic sites for DAO were exclusively confined to peroxisomes. Within peroxisomes, the gold particles were localized in the central clear matrix but not in the peripheral tubular substructures. The results indicate that D-amino acid oxidase in rat kidney is present exclusively in peroxisomes in the proximal tubule and that within peroxisomes it is found only in central clear matrix and not in the peripheral tubular substructures.     

Immunocytochemical localization of serine: pyruvate aminotransferase in peroxisomes of the human liver parenchymal cells

The localization of serine:pyruvate aminotransferase (SPT) in human liver was investigated by indirect immunoenzyme and protein A-gold techniques. By light microscopy, diaminobenzidine reaction product was present in cytoplasmic granules of the parenchymal cells. By electron microscopy, gold particles indicating the antigenic sites for SPT were exclusively confined to peroxisomes but not to mitochondria. By double labeling technique, both peroxisomal marker enzyme, catalase and SPT were detected in the same peroxisomes. Quantitative analysis of the labeling density showed that SPT is contained only in peroxisomes. The results indicate that in human liver most of SPT is contained in the peroxisomes.     

Immunocytochemical localization of serine: pyruvate aminotransferase in peroxisomes of the human liver parenchymal cells

Summary The localization of serine:pyruvate aminotransferase (SPT) in human liver was investigated by indirect immunoenzyme and protein A-gold techniques. By light microscopy, diaminobenzidine reaction product was present in cytoplasmic granules of the parenchymal cells. By electron microscopy, gold particles indicating the antigenic sites for SPT were exclusively confined to peroxisomes but not to mitochondria. By double labeling technique, both peroxisomal marker enzyme, catalase and SPT were detected in the same peroxisomes. Quantitative analysis of the labeling density showed that SPT is contained only in peroxisomes. The results indicate that in human liver most of SPT is contained in the peroxisomes.     

Immunocytochemical localization of cathepsins B and H in rat liver

Summary Light and electron microscopic localization of cathepsins B and H in rat liver was investigated by immunoenzyme and protein A-gold techniques. For light microscopy (LM), semi-thin sections of the Epon-embedded material were stained by the immunoenzyme technique after removal of epoxy resin. For electron microscopy (EM), ultrathin sections of the Lowicryl K4M-embedded material were stained by the protein A-gold technique. By LM, reaction deposits for cathepsins B and H were present in the cytoplasmic granules of parenchymal cells and endothelial cells, and Kupffer cells. The sinus-lining cells and the parenchymal cells showed the similar staining intensity. By EM, gold particles were present exclusively in lysosomes of all the cell types cited above. The same results were obtained from quantitative analysis. In addition, Golgi complexes themselves were mostly negative but some small vesicles on the trans side of them were labeled for these proteinases. The results indicate that cathepsins B and H are present in the lysosomes of rat liver and that these enzymes seem to be transported by small vesicles from endoplasmic reticulum to lysosomes via tubuloreticular network of the trans Golgi region.     

Immunocytochemical localization of cathepsins B and H in rat liver

Light and electron microscopic localization of cathepsins B and H in rat liver was investigated by immunoenzyme and protein A-gold techniques. For light microscopy (LM), semi-thin sections of the Epon-embedded material were stained by the immunoenzyme technique after removal of epoxy resin. For electron microscopy (EM), ultra-thin sections of the Lowicryl K4M-embedded material were stained by the protein A-gold technique. By LM, reaction deposits for cathepsins B and H were present in the cytoplasmic granules of parenchymal cells and endothelial cells, and Kupffer cells. The sinus-lining cells and the parenchymal cells showed the similar staining intensity. By EM, gold particles were present exclusively in lysosomes of all the cell types cited above. The same results were obtained from quantitative analysis. In addition, Golgi complexes themselves were mostly negative but some small vesicles on the trans side of them were labeled for these proteinases. The results indicate that cathepsins B and H are present in the lysosomes of rat liver and that these enzymes seem to be transported by small vesicles from endoplasmic reticulum to lysosomes via tubuloreticular network of the trans Golgi region.     

Immunocytochemical localization of acid phosphatase in rat liver

Localization of acid phosphatase (ACPase) in rat liver was investigated by immunocytochemical techniques. Rat liver was fixed by perfusion and cut into thick tissue slices, which were embedded in Epon or Lowicryl K4M. For light microscopy (LM), semithin Epon sections were stained for the enzyme ACPase by an indirect immunoenzyme technique. For electron microscopy (EM), ultra-thin Lowicryl K4M sections were stained by a protein A-gold technique. By means of LM, granular reaction deposits were observed in hepatocytes and sinus-lining cells. Stained granules were present in the juxtanuclear cytoplasm, but they did not correspond to a typical staining pattern for the Golgi complex. EM revealed that gold particles indicating ACPase antigens were present on lysosomes and on some vesicles locating in the trans Golgi region. Endosomelike vesicles were strongly positive for the labeling. Golgi cisterna were mostly negative, but weak signals were noted in dilated sacules. The plasma membranes on the sinusoidal and bile canalicular sides were labeled by a few gold particles. The results indicate that ACPase is present in endosomes and in a restricted area of plasma membrane, as well as in the lysosomal system.     

Immunocytochemical localization of Δ3, Δ2-enoyl-CoA isomerase and NADPH-dependent-2,4-dienoyl-CoA reductase in rat kidney

Localization of ³, ²-enoyl-CoA isomerase (ECI) and NADPH-dependent-2,4-dienoyl-CoA reductase (DCR) in the rat kidney was investigated by immunocytochemical techniques. The kidneys were perfusion-fixed and embedded in Epon or LR White. For light microscopy, semi-thin sections of Epon-embedded materials were stained by the immunoenzyme technique after the epoxy resin was removed by treatment with sodium ethoxide. For electron microscopy, ultra-thin sections of LR White-embedded materials were stained by the protein A-gold technique. By light microscopy, the S1 segment of the proximal tubule was most heavily stained for ECI and DCR whilst S2 and S3 segments showed intermediate staining. A weak staining reaction was observed in the distal tubule and the medullary collecting tubule. In the cortical collecting tubule, heavily stained cells were present between weakly stained cells. By electron microscopy, gold particles showing the antigenic sites for ECI were confined mainly to the mitochondria, but few particles were observed in the peroxisomes. Gold labeling for DCR was localized both in the mitochondria and the peroxisomes. The labeling intensity of the peroxisomes was much higher than that of the mitochondria. The results suggest that metabolism of unsaturated fatty acids occurs mainly in the mitochondria and the peroxisomes of the proximal tubule in the kidney.     

Localization of cathepsin L in rat kidney revealed by immunoenzyme and immunogold techniques

Localization of cathepsin L in rat kidney was investigated by immunocytochemical techniques. Kidneys were fixed by perfusion and embedded in Epon or Lowicryl K4M without postosmication. For light microscopy (LM), semi-thin sections of the Epon-embedded material were stained by the immunoenzyme technique after removal of epoxy resin. For electron microscopy (EM), ultra-thin sections of Lowicryl K4M-embedded material were stained by the protein A-gold technique. By LM, reaction deposits for cathepsin L were present in the cytoplasmic granules of proximal tubule cells, but little or no reaction product was noted in distal tubule, collecting tubule, and most of urinary tubules in the medulla. By EM, heavy gold label for cathepsin L was confined exclusively to lysosomes of the proximal tubule cells, but little or no label to those of the other segments. In immunocytochemical control sections, no reaction was observed. These results indicate that a main container of cathepsin L is lysosomes of the proximal tubule and suggest that the enzyme plays a role in the degradation of endocytosed proteins.     

Localization of cathepsin L in rat kidney revealed by immunoenzyme and immunogold techniques

Summary Localization of cathepsin L in rat kidney was investigated by immunocytochemical techniques. Kidneys were fixed by perfusion and embedded in Epon or Lowicryl K4M without postomication. For light microscopy (LM), semi-thin sections of the Epon-embedded material were stained by the immunoenzyme technique after removal of epoxy resin. For electron microscopy (EM), ultra-thin sections of Lowicryl K4M-embedded material were stained by the protein A-gold technique. By LM, reaction deposits for cathepsin L were present in the cytoplasmic granules of proximal tubule cells, but little or no reaction product was noted in distal tubule, collecting tubule, and most of urinary tubules in the medulla. By EM, heavy gold label for cathepsin L was confined exclusively to lysosomes of the proximal tubule cells, but little or no label to those of the other segments. In immunocytochemical control sections, no reaction was observed. These results indicate that a main container of cathepsin L is lysosomes of the proximal tubule and suggest that the enzyme plays a role in the degradation of endocytosed proteins.     

Immunocytochemical localization of cytosol 5'-nucleotidase in chicken liver

We investigated the localization of cytosol 5'-nucleotidase in chicken liver by use of a pre-embedding immunoenzyme technique. Cytosol 5'-nucleotidase was purified from chicken liver and a monospecific antibody to this enzyme was raised in a rabbit. Fab fragments of the antibody were conjugated with horseradish peroxidase. Tissue sections of the fixed chicken liver were incubated with the peroxidase-Fab fragments, followed by DAB reaction for peroxidase. By light microscopy, dark-brown staining was present in the cytoplasm of parenchymal cells, Kupffer cells, and endothelial cells. The latter two types of cells were stained more strongly than the former. By electron microscopy, reaction deposits were present in the cytoplasmic matrix but not in cell organelles, such as mitochondria, endoplasmic reticulum, and peroxisomes, or in nuclei. In control sections incubated with peroxidase-conjugated Fab fragments from non-immunized rabbit, no specific reaction was noted. The results indicate that cytosol 5'-nucleotidase is contained more in the sinus-lining cells and less in the parenchymal cells, and that the enzyme is present in the cytoplasmic matrix of these cells.     

Quantitative immunocytochemical studies on differential induction of serine:pyruvate aminotransferase in mitochondria and peroxisomes of rat liver cells by administration of glucagon or di-(2-ethylhexyl)phthalate

Differential induction of serine: pyruvate amino-transferase (SPT) in rat liver parenchymal cells by administration of glucagon or di-(2-ethylhexyl)phthalate (DEHP) was studied using post-embedding immunocytochemical techniques and morphometric methods. Two groups of rats were fasted for 5 days and daily received peritoneal injection of glucagon (300 micrograms/100 g) or physiological saline. Another two groups of rats were fed on laboratory chow with or without 2% DEHP for 2 weeks. Livers were perfusion-fixed, cut into tissue sections (50-100 micron), and processed to cytochemistry for catalase, immunocytochemistry for SPT, and conventional procedures for electron microscopy. The morphometric analysis showed that glucagon injection has negligible effect on the volume and numerical density and mean diameter of peroxisomes, whereas volume density of mitochondria was decreased by 25%. By DEHP administration peroxisomes were about 3-fold increased in the volume and numerical density. Mitochondria was increased about 40% in the numerical density, but unchanged in the volume density. Light and electron microscopic immunocytochemistry demonstrated that glucagon injection exclusively enhanced mitochondrial SPT, whereas DEHP administration exclusively induced in peroxisomal SPT. Quantitative analysis showed that by the glucagon injection, the labeling density of mitochondria was increased about 4-fold, but that of peroxisomes was 1.6 times as much as control, while by DEHP administration, the labeling density of peroxisomes was enhanced about 3-fold but that of mitochondria was decreased by 13%. The results clearly indicate that glucagon induces mitochondrial SPT, whereas peroxisome proliferator, DEHP induces peroxisomal SPT.     

Immunocytochemical demonstration of serine:pyruvate aminotransferase in peroxisomes and mitochondria of rat kidney

Summary The light- and electron-microscopic localization of serine:pyruvate aminotransferase (SPT) in rat kidney was studied using immunoenzyme and protein A-gold techniques. Rat kidneys were fixed by perfusion through the abdominal aorta and small tissue slices were embedded in Epon, Lowicryl K4M, or LR Gold. The Epon was removed from the semithin sections, which were then stained using the immunoenzyme technique. Ultrathin sections of Lowicryl K4M- or LR gold-embedded materials were labeled using the protein A-gold technique. At light microscopy, discrete granular reaction deposits were exclusively present in the proximal tubule, all of whose segments were positive for SPT. A weakly positive reaction was observed in the distal tubules. At electron microscopy, gold particles indicating the antigenic sites for SPT were confined to the peroxisomes and mitochondria. The labeling intensity of both organelles was dependent on the embedding resins used. The labeling of Lowicryl K4M-embedded material was weaker than that of LR gold-embedded material; Quantitative analysis confirmed this result. Our results indicate that, in rat kidney, the main intracellular sites for SPT are peroxisomes and mitochondria of the proximal tubule.     

Immunocytochemical demonstration of serine: pyruvate amino-transferase in peroxisomes and mitochondria of rat kidney

The light- and electron-microscopic localization of serine: pyruvate aminotransferase (SPT) in rat kidney was studied using immunoenzyme and protein A-gold techniques. Rat kidneys were fixed by perfusion through the abdominal aorta and small tissue slices were embedded in Epon, Lowicryl K4M, or LR Gold. The Epon was removed from the semithin sections, which were then stained using the immunoenzyme technique. Ultrathin sections of Lowicryl K4M- or LR gold-embedded materials were labeled using the protein A-gold technique. At light microscopy, discrete granular reaction deposits were exclusively present in the proximal tubule, all of whose segments were positive for SPT. A weakly positive reaction was observed in the distal tubules. At electron microscopy, gold particles indicating the antigenic sites for SPT were confined to the peroxisomes and mitochondria. The labeling intensity of both organelles was dependent on the embedding resins used. The labeling of Lowicryl K4M-embedded material was weaker than that of LR gold-embedded material; Quantitative analysis confirmed this result. Our results indicate that, in rat kidney, the main intracellular sites for SPT are peroxisomes and mitochondria of the proximal tubule.     

Quantitative immunocytochemical studies on differential induction of serine

Summary Differential induction of serine: pyruvate aminotransferase (SPT) in rat liver parenchymal cells by administration of glucagon or di-(2-ethylhexyl)phthalate (DEHP) was studied using post-embedding immunocytochemical techniques and morphometric methods. Two groups of rats were fasted for 5 days and daily received peritoneal injection of glucagon (300 g/100 g) or physiological saline. Another two groups of rats were fed on laboratory chow with or without 2% DEHP for 2 weeks. Livers were perfusionfixed, cut into tissue sections (50–100 ), and processed to cytochemistry for catalase, immunocytochemistry for SPT, and conventional procedures for electron microscopy. The morphometric analysis showed that glucagon injection has negligible effect on the volume and numerical density and mean diameter of peroxisomes, whereas volume density of mitochondria was decreased by 25%. By DEHP administration peroxisomes were about 3-fold increased in the volume and numerical density. Mitochondria was increased about 40% in the numerical density, but unchanged in the volume density. Light and electron microscopic immunocytochemistry demonstrated that glucagon injection exclusively enhanced mitochondrial SPT, whereas DEHP administration exclusively induced in peroxisomal SPT. Quantitative analysis showed that by the glucagon injection, the labeling density of mitochondria was increased about 4-fold, but that of peroxisomes was 1.6 times as much as control, while by DEHP administration, the labeling density of peroxisomes was enhanced about 3-fold but that of mitochondria was decreased by 13%. The results clearly indicate that glucagon induces mitochondrial SPT, whereas peroxisome proliferator, DEHP induces peroxisomal SPT.     

Localization of cathepsin D in rat liver

Summary Light and electron microscopic localization of cathepsin D in rat liver was investigated by post-embedding immunoenzyme and protein A-gold techniques. By light microscopy, cytoplasmic granules of parenchymal cells and Kupffer cells were stained for cathepsin D. Weak staining was also noted in sinusoidal endothelial cells. In the parenchymal cells many of positive granules located around bile canaliculi. In the Kupffer cells and the endothelial cells, diffuse staining was noted in the cytoplasm in addition to granular staining. By electron microscopy, gold particles representing the antigenic sites for cathepsin D were seen in typical secondary lysosomes and some multivesicular bodies of the parenchymal cells and Kupffer cells. The lysosomes of the endothelial cells and fat-storing cells were weakly labeled. Quantitative analysis of the labeling density in the lysosomes of these three types of cells demonstrated that the lysosomes of parenchymal cells and Kupffer cells are main containers of cathepsin D in rat liver. The results suggest that cathepsin D functions in the intracellular digestive system of parenchymal cells and Kupffer cells but not so much in that of the endothelial cells.     

Localization of cathepsin D in rat liver. Immunocytochemical study using post-embedding immunoenzyme and protein A-gold techniques

Light and electron microscopic localization of cathepsin D in rat liver was investigated by post-embedding immunoenzyme and protein A-gold techniques. By light microscopy, cytoplasmic granules of parenchymal cells and Kupffer cells were stained for cathepsin D. Weak staining was also noted in sinusoidal endothelial cells. In the parenchymal cells many of positive granules located around bile canaliculi. In the Kupffer cells and the endothelial cells, diffuse staining was noted in the cytoplasm in addition to granular staining. By electron microscopy, gold particles representing the antigenic sites for cathepsin D were seen in typical secondary lysosomes and some multivesicular bodies of the parenchymal cells and Kupffer cells. The lysosomes of the endothelial cells and fat-storing cells were weakly labeled. Quantitative analysis of the labeling density in the lysosomes of these three types of cells demonstrated that the lysosomes of parenchymal cells and Kupffer cells are main containers of cathepsin D in rat liver. The results suggest that cathepsin D functions in the intracellular digestive system of parenchymal cells and Kupffer cells but not so much in that of the endothelial cells.     

Immunoelectron microscopical localization of ornithine transcarbamylase in hepatic parenchymal cells of the rat

Summary The electron microscopical localization of ornithine transcarbamylase in rat liver was investigated by a protein A—gold technique applied to thin sections of Lowicryl K4M- or LR gold-embedded materials and to ultracryosections. Gold particles were exclusively confined to mitochondria of the parenchymal cells but not of sinus-lining cells. In mitochondria, gold particles were present in the matrix and closely associated with the inner membrane. The most intensive labelling was obtained from ultracryosections, while weaker labelling was noted in sections of materials embedded in both Lowicryl K4M and LR gold. The association of the enzyme with the inner membrane was confirmed by quantitative analysis of distribution pattern.     

Localization of cytosolic NADP-dependent isocitrate dehydrogenase in the peroxisomes of rat liver cells: biochemical and immunocytochemical studies.

Two types of NADP-dependent isocitrate dehydrogenases (ICDs) have been reported: mitochondrial (ICD1) and cytosolic (ICD2). The C-terminal amino acid sequence of ICD2 has a tripeptide peroxisome targeting signal 1 sequence (PTS1). After differential centrifugation of the postnuclear fraction of rat liver homogenate, approximately 75% of ICD activity was found in the cytosolic fraction. To elucidate the true localization of ICD2 in rat hepatocytes, we analyzed the distribution of ICD activity and immunoreactivity in fractions isolated by Nycodenz gradient centrifugation and immunocytochemical localization of ICD2 antigenic sites in the cells. On Nycodenz gradient centrifugation of the light mitochondrial fraction, ICD2 activity was distributed in the fractions in which activity of catalase, a peroxisomal marker, was also detected, but a low level of activity was also detected in the fractions containing activity for succinate cytochrome C reductase (a mitochondrial marker) and acid phosphatase (a lysosomal marker). We have purified ICD2 from rat liver homogenate and raised a specific antibody to the enzyme. On SDS-PAGE, a single band with a molecular mass of 47 kD was observed, and on immunoblotting analysis of rat liver homogenate a single signal was detected. Double staining of catalase and ICD2 in rat liver revealed co-localization of both enzymes in the same cytoplasmic granules. Immunoelectron microscopy revealed gold particles with antigenic sites of ICD2 present mainly in peroxisomes. The results clearly indicated that ICD2 is a peroxisomal enzyme in rat hepatocytes. ICD2 has been regarded as a cytosolic enzyme, probably because the enzyme easily leaks out of peroxisomes during homogenization. (J Histochem Cytochem 49:1123-1131, 2001)     

Hepatic subcellular distribution of short-chain beta-ketoacyl coenzyme A reductase and trans-2-enoyl coenzyme A hydratase: 25- to 50-fold stimulation of microsomal activities by the peroxisome proliferator, di-(2-ethylhexyl)phthalate

The present study demonstrates unequivocally the existence of short-chain trans-2-enoyl coenzyme A (CoA) hydratase and beta-ketoacyl CoA reductase activities in the endoplasmic reticulum of rat liver. Subcellular fractionation indicated that all four fractions, namely, mitochondrial, peroxisomal, microsomal, and cytosolic contained significant hydratase activity when crotonyl CoA was employed as the substrate. In the untreated rat, based on marker enzymes and heat treatment, the hydratase activity, expressed as mumol/min/g liver, wet weight, in each fraction was: mitochondria, 684; peroxisomes, 108; microsomes, 36; and cytosol, 60. Following di-(2-ethylhexyl)phthalate (DEHP) treatment (2% (v/w) for 8 days), there was only a 20% increase in mitochondrial activity; in contrast, peroxisomal hydratase activity was stimulated 33-fold, while microsomal and cytosolic activities were enhanced 58- and 14-fold respectively. A portion of the cytosolic hydratase activity can be attributed to the component of the fatty acid synthase complex. Although more than 70% of the total hydratase activity was associated with the mitochondrial fraction in the untreated rat, DEHP treatment markedly altered this pattern; only 11% of the total hydratase activity was present in the mitochondrial fraction, while 49 and 29% resided in the peroxisomal and microsomal fractions, respectively. In addition, all four subcellular fractions contained the short-chain NADH-specific beta-ketoacyl CoA (acetoacetyl CoA) reductase activity. Again, in the untreated animal, reductase activity was predominant in the mitochondrial fraction; following DEHP treatment, there was marked stimulation in the peroxisomal, microsomal, and cytosolic fractions, while the activity in the mitochondrial fraction increased by only 39%. Hence, it can be concluded that both reductase and hydratase activities exist in the endoplasmic reticulum in addition to mitochondria, peroxisomes, and soluble cytoplasm.