Mj Hardonk

Cytochemical demonstration of phosphatases in the rat liver by a cerium-based method in combination with osmium tetroxide and potassium ferrocyanide postfixation

SummaryAcid phosphatase, alkaline phosphatase, glucose-6-phosphatase, Mg-activated adenosine triphosphatase and 5′ nucleotidase were demonstrated in the rat liver using a cerium-based method. This method can be applied routinely and yields better results than the lead-based method. The tissue was postfixed in osmium tetroxide and potassium ferrocyanide which considerably enhances the membrane contranst in comparison with solely osmium tetroxide postfixation. This facilitates the precize localization of the reaction product.

IMMUNOHISTOCHEMICAL DEMONSTRATION OF DNA-INCORPORATED 5-BROMODEOXYURIDINE IN FROZEN AND PLASTIC EMBEDDED SECTIONS

SummaryThe application of an immunohistochemical method in the detection of replicating cells, that have incorporated 5-bromodeoxyuridine (BrdUrd), was studied on frozen and plastic embedded sections of different rat tissues. Hydrolysis conditions employed in the Feulgen procedure are essential in making the incorporated BrdUrd accessible to the monoclonal anti-BrdUrd antibodies. To demonstrate the incorporated BrdUrd in plastic embedded sections a subsequent etching with xylene and digestion with protease is necessary. Data obtained with this method are completely comparable with those found by the tritiated thymidine method. In comparison with the thymidine method, the BrdUrd method is much less time consuming and does not require precautions in working with radioactivity. The BrdUrd-method enables a more precise localization as is especially shown in the plastic embedded sections.

Formaldehyde treated albumin contains monomeric and polymeric forms that are differently cleared by endothelial and kupffer cells of the liver : evidence for scavenger receptor heterogeneity

Formaldehyde treated albumin (F-HSA) was found to consist of a monomeric and a polymeric fraction. Both fractions were primarily endocytosed by rat liver sinusoidal cells. However, immunohistochemical staining of endocytosed material showed that the relative contribution of the endothelial and Kupffer cells in uptake of the monomer and the polymer differed significantly, with the monomer mainly having an endothelial cell- and the polymer predominantly having a Kupffer cell pattern of distribution. To directly confirm these heterogeneous patterns, we injected in vivo the 125I-labeled F-HSA fractions and isolated the endothelial and Kupffer cells by centrifugal elutriation. 73.7% of the monomeric F-HSA was found in endothelial cells and only 14.9% was found in Kupffer cells. In contrast, the polymeric F-HSA (1500 kD) was mainly endocytosed by Kupffer cells (71%), whereas the endothelial cells contributed only for 24% in hepatic uptake. In vivo studies and isolated perfused rat liver experiments showed that endocytosis of both monomer and polymer was inhibited by co-administration of polyinosinic acid, a well known inhibitor for scavenger receptors, indicating that these receptors on endothelial and Kupffer cells are mainly involved in this uptake process.

Zonal heterogeneity of rat hepatocytes in the in vivo uptake of 17 nm colloidal gold granules

SummaryThe in vivo uptake in hepatocytes of intravenously injected colloidal gold granules with a diameter of 17 nm or 79 nm and coated with bovine serum albumin or with polyvinyl-pyrrolidone was studied. Irrespective of coating only the 17 nm granules were taken up in hepatocytes. Perivenous hepatocytes did take up much more gold granules than periportal hepatocytes. The gold granules were found in lysosomes around bile canaliculi. Two hours after injection hepatocytes contained the maximal amount of granules. At least a portion of the granules was discharged into the bile. The observed zonal gradient in the uptake of 17 nm gold granules might be caused by the greater supply of granules to the perivenous hepatocytes as a combined result of the higher porosity of the endothelial lining and the smaller number of Kupffer cells with a low endocytic activity in this zone.

Histochemical demonstration of enzymes related to NADPH-dependent hydroxylating systems in rat liver after phenobarbital treatment

SummaryMale rats were given 100mg phenobarbital for three days intraperitoneally. Biochemically an increase was found in activity of nitro-anisole demethylation and in the content of cytochrome P-450. Enzymhistochemically an increase in activity was noted for NADPH tetr. red., G6PD, ICD, and Naftol AS-D-esterase; a decrease was seen in G6Pase and glycogen, but no difference was found in NADH tetr. red. From these results it has been suggested that NADPH tetr. red. is directly involved in the hydroxylation chain, while G6PD and ICD are more indirectly involved.

A new method for the histochemical demonstration of steroid producing cells in human tissues.

SummarySteroid producing cells in human tissues can be demonstrated histochemically by the use of isopropanol as substrate. The Leydig cells of the testis, the theca cells of the ovary and the zona reticularis of the adrenal are stained by this method. A possible relation of this ‚secondary alcohol dehydrogenase” with the enzyme that catalyses the oxidative cleavage of the cholesterol side-chain is discussed.

A HISTOCHEMICAL-STUDY ABOUT THE ZONAL DISTRIBUTION OF THE GALACTOSE-BINDING PROTEIN IN RAT-LIVER

Summary1.Dog intestinal alkaline phosphatase (IAP), an asialoglycoprotein, appeared to be a good marker for the histochemical detection of the galactose specific binding protein in cryostat sections of rat liver.2.Binding of IAP to the receptor is optimal at neutral or slightly alkaline pH values. The binding could be inhibited by galactose and galactose containing sugars, whereas glucose and mannose did not show any effect. In contrast to fetuin itself desialylated fetuin completely inhibited IAP binding. Pretreatment of sections with phospholipase C or with trypsin inhibited IAP binding; collagenase did not show any influence.3.The presence of the galactose-binding protein showed a distinct zonal distribution. In the area around the central vein (zone 3) the highest IAP binding capacity was found.

Simultaneous immunohistochemical demonstration of antigen expression and 5-bromodeoxyuridine incorporation in plastic embedded sections

SummaryIn this study a double immunohistochemical staining procedure is described for the simultaneous demonstration of antigen expressing cells and replicating cells in rat thymus. As markers for cell surface antigen expression a monoclonal antibody against Ia-expressing cells (His 19) and a monoclonal antibody against cells of the monocyte-macrophage lineage (ED2) were used. Replicating cells were demonstrated by the incorporation of 5-bromodeoxyuridine (BrdUrd). Tissue pieces were fixed in a periodate-lysine-paraformaldehyde fixative and embedded in glycol methacrylate. To demonstrate Ia-expressing cells or ED2-positive macrophages in plastic embedded sections a digestion with trypsin is necessary. The staining procedure was applied sequentially and was performed with a peroxidase and an alkaline phosphatase labeled reagent yielding respectively a brown and a blue reaction product. Results with this staining procedure on plastic embedded sections of rat thymus, an organ with a high DNA synthesizing capacity, showed incorporation of BrdUrd predominantly in the cortex. ED2-positive macrophages were only found in the cortex. The la-positive epithelial reticular cells demonstrated extremely well their stellate form.

The role of the liver in clearance of glycoproteins from the general circulation, with special reference to intestinal alkaline phosphatase.

Glycoproteins represent a wide variety of macromolecules with important physiological functions. Characteristic variations in carbohydrate composition and plasma concentration of these proteins may occur during pathological conditions. Steady-state plasma concentrations are determined by release from normal or diseased tissues and simultaneous clearance from the general circulation.The liver occupies a central position in the production but also clearance and catabolism of such glycoproteins. A number of specialized receptor-mediated transport processes for different types of glycoproteins in this organ is reviewed. Membrane recognition is generally followed by absorptive endocytosis and vesicle transport to lysosomes, Golgi system and/or bile canaliculis. The charge of the protein, the nature of the terminal sugar residue or complex formation with other glycoproteins may determine the extent of uptake in the various cell types of the liver.By means of these transport processes the liver is able to remove potentially dangerous macromolecules such as denatured proteins, aggressive enzymes and immunocomplexes from the general circulation. Drugs can bind to some of these proteins or may interact with the hepatic transport or catabolism processes. Special attention is paid to the hepatic clearance of asialoglycoproteins with terminal galactose groups. Intestinal alkaline phosphatase is used as a model compound to characterize the pharmacokinetic profiles of hepatic uptake and biliary excretion in the ratin vivo and isolated perfused rat livers. Histochemical and electron-microscopic studies demonstrated a galactose-specific, receptor-mediated endocytotic process, mainly but not exclusively localized in centrolobular hepatocytes. Drug interactions with these processes will be the subject of further investigations.

The use of phenazinemethosulphate as an electron carrier in the histochemical demonstration of dehydrogenases.

SummaryThe use of phenazine methosulfate (PMS) has been investigated in the histochemical demonstration of dehydrogenase in some organs of the rat. The demonstration of nicotinamid-adenine-dinucleotide (NAD) linked dehydrogenases, which in the conventional methods without PMS is dependent upon the localisation of reduced NAD (NADH)-diaphorase, is greatly hindered by PMS. This inhibition is caused by the inactivation of the diaphorase by PMS. However in tissues or cells lacking the diaphorase, the nucleotide-linked dehydrogenases can be made visible by the addition of PMS to the conventional dehydrogenase reagents. PMS strongly activates the nucleotide-independent dehydrogenases such as succinate dehydrogenase.