Ilse M. C. Vogels

The value of enzyme leakage for the prediction of necrosis in liver ischemia.

SummaryFollowing the clamping of the afferent vessels of the left lateral and median lobes in rat liver, a considerable part of these lobes show signs of necrosis 24 h after 90 min of ischemia, wheras no necrotic areas can be detected after 30 min interruption of the blood flow. The purpose of this study was to examine the value of an analysis of the leakage of enzymes from the liver parenchyma in the early phase after restoration of the blood flow after ichemia for a prediction of the occurrence of necrosis. Leakage of the enzymes GPT, GOT and LDH can be detected in the blood plasma with a maximum activity between 1 and 5 h both following 30 and 90 min of ischemia; a considerable difference in clearance is observed, however, in the period afterwards, the normal situation being reached after 24 h with the 30-min ischemic period, but not following the 90-min period. With use of an enzyme histochemical reaction, in situ a depletion of LDH-activity in the hepatocytes could be detected within a short period of time after 30 min temporary ischemia and a restoration during the following period of 24 h; the decrease in LDH-activity persisted during 24 h with a 90-min period of ischemia. Electronmicroscopically cytoplasmic blebs arosen from hepatocytes are observed in the lumen of sinusoids immediately after 30 min of ischemia, whereas after 90 min of ischemia actual leakage of cytoplasmic material takes place through the damaged surface of the hepatocytes. Enzyme leakage probably takes place via these both types of shedding of cytoplasm. It is concluded that the enzyme leakage as such cannot be used as a discriminating test between reversible and irreversible damage of the liver parenchyma.

Multidrug-resistance P-glycoprotein (MDR1) secretes platelet-activating factor

The human multidrug-resistance (MDR1) P-glycoprotein (Pgp) is an ATP-binding-cassette transporter (ABCB1) that is ubiquitously expressed. Often its concentration is high in the plasma membrane of cancer cells, where it causes multidrug resistance by pumping lipophilic drugs out of the cell. In addition, MDR1 Pgp can transport analogues of membrane lipids with shortened acyl chains across the plasma membrane. We studied a role for MDR1 Pgp in transport to the cell surface of the signal-transduction molecule platelet-activating factor (PAF). PAF is the natural short-chain phospholipid 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine. [(14)C]PAF synthesized intracellularly from exogenous alkylacetylglycerol and [(14)C]choline became accessible to albumin in the extracellular medium of pig kidney epithelial LLC-PK1 cells in the absence of vesicular transport. Its translocation across the apical membrane was greatly stimulated by the expression of MDR1 Pgp, and inhibited by the MDR1 inhibitors PSC833 and cyclosporin A. Basolateral translocation was not stimulated by expression of the basolateral drug transporter MRP1 (ABCC1). It was insensitive to the MRP1 inhibitor indomethacin and to depletion of GSH which is required for MRP1 activity. While efficient transport of PAF across the apical plasma membrane may be physiologically relevant in MDR1-expressing epithelia, PAF secretion in multidrug-resistant tumours may stimulate angiogenesis and thereby tumour growth.

CD34 marks angiogenic tip cells in human vascular endothelial cell cultures

The functional shift of quiescent endothelial cells into tip cells that migrate and stalk cells that proliferate is a key event during sprouting angiogenesis. We previously showed that the sialomucin CD34 is expressed in a small subset of cultured endothelial cells and that these cells extend filopodia: a hallmark of tip cells in vivo. In the present study, we characterized endothelial cells expressing CD34 in endothelial monolayers in vitro. We found that CD34-positive human umbilical vein endothelial cells show low proliferation activity and increased mRNA expression of all known tip cell markers, as compared to CD34-negative cells. Genome-wide mRNA profiling analysis of CD34-positive endothelial cells demonstrated enrichment for biological functions related to angiogenesis and migration, whereas CD34-negative cells were enriched for functions related to proliferation. In addition, we found an increase or decrease of CD34-positive cells in vitro upon exposure to stimuli that enhance or limit the number of tip cells in vivo, respectively. Our findings suggest cells with virtually all known properties of tip cells are present in vascular endothelial cell cultures and that they can be isolated based on expression of CD34. This novel strategy may open alternative avenues for future studies of molecular processes and functions in tip cells in angiogenesis.

On the nature of the ‘nothing dehydrogenase’ reaction

SummaryThe biochemical mechanism underlying the ‘nothing dehydrogenase’ reaction during the histochemical demonstration of dehydrogenases using tetranitro BT as the final electron acceptor has been investigated in unfixed, frozen rat liver sections. The reaction is stronger with NAD+ than either with NADP+ or in the absence of coenzyme. As much as 50% of the reaction is due to lactate dehydrogenase converting endogenous lactate and is largely inhibited by pyruvate. No NAD+-dependent alcohol dehydrogenase activity was detected at pH 7.45, the pH used for the incubations. The coenzyme-independent activity may be caused by SH-groups present in proteins and compounds like glutathione and cysteine and can be inhibited byN-ethylmaleimide andp-chloromercuribenzoic acid. It was also found that the ‘nothing dehydrogenase’ reaction mainly occurs during the first few minutes of incubation, levelling off quickly to a slow rate. When studying the kinetics of dehydrogenase reactions with tetrazolium salts, it should be realized that the ‘nothing dehydrogenase’ reaction, which as a whole is nonlinear with time, can interfere seriously with the dehydrogenase reaction to be analysed and may yield initial reaction rates that are too high. The findings of the present study reveal the nature of the reactions used for detection of necrosis in tissues with tetrazolium salts.

Altered expression of genes related to blood―retina barrier disruption in streptozotocin-induced diabetes

Disruption of the blood-retina barrier (BRB) is an early phenomenon in preclinical diabetic retinopathy (PCDR). Two vascular permeability pathways may be affected, the paracellular pathway involving endothelial cell tight junctions, and the endothelial transcellular pathway mediated by endocytotic vesicles (caveolae). The relative contribution of both pathways to vascular permeability in PCDR is unknown. We compared transcription levels in entire rat retina of genes related to these pathways between control conditions and after 6 and 12 weeks of streptozotocin-induced diabetes, as well as in bovine retinal endothelial cells (BRECs) exposed to VEGF and bovine retinal pericytes (BRPCs), using real-time quantitative RT-PCR. To confirm endothelial-specificity, immunohistochemical staining was performed in rat retina, and mRNA transcript levels were compared between BRECs and BRPCs. mRNA and protein of most paracellular transport-related genes were specifically expressed by retinal endothelial cells, whereas vesicle transport-related mRNA and proteins were present in various retinal cell types, including endothelial cells. Expression of selected endothelial cell tight junction genes and particularly that of occludin and claudin-5 was reduced in the diabetic retina and in BRECs after exposure to VEGF. Expression of 6 out of 11 vesicular transport-related genes was upregulated after induction of diabetes. Of these, only plasmalemma vesicle-associated protein (PV-1) was exclusively expressed in BRECs and not in BRPCs. PV-1 transcription was markedly induced in diabetic retina and by VEGF in BRECs. Caveolin-1 immunostaining was primarily found in the retinal vasculature, and its mRNA levels in BRECs were highly abundant and VEGF-inducible. Whereas the endothelial tight junction genes occludin and claudin-5 showed a transient downregulation, we observed long-term upregulation in diabetic retina and VEGF-induced expression in BRECs of the vesicular transport-related genes caveolin-1 and PV-1. The altered gene expression profiles observed in this study suggest a transient induction of the paracellular pathway and prolonged involvement of transcellular endothelial transport mechanisms in the increased permeability of retinal capillaries in PCDR.

Enzyme histochemical reactions in unfixed and undecalcified cryostat sections of mouse knee joints with special reference to arthritic lesions

SummaryThe use of unfixed and undecalcified cryostat sections of mouse knee joints is described for the study of enzyme histochemical reactions. Non-inflamed knee joints and knee joints of mice with antigen induced arthritis have been used. Joints were embedded in gelatin and subsequently cut at low speed with a motor-driven cryostat fitted with a tungsten carbide knife at an obtuse angle (10°). The sections were attached to transparent tape to keep the integrity of the tissue intact. The following histochemical reactions were carried out succesfully: the tetrazolium salt reaction for dehydrogenase and reductase activity, the post-azocoupling method for acid phosphatase and cathepsin B activity and the simultaneous azo-coupling method for esterase activity. In all cases the morphology and integrity of the sections were well kept and serial sections were obtained without any difficulty. Nonspecific staining of the tape did not occur. The localization of the final reaction product was meeting criteria for specific and precise histochemical methods with the exception of the metal salt method because of nonspecific staining of undecalcified bone. Cytophotometry of the final reaction product appeared to be reproducible and valid as demonstrated by reaction for glucose-6-phosphate dehydrogenase activity in synoviocytes from knee joints with induced arthritis. End point measurements as well as kinetic measurements of the formazan production were performed and linear relationships were found between the specific formazan formation and section thickness or incubation time, respectively. It is concluded that cryostat sections attached to transparent tape are an excellent tool for the study of the metabolism in tissues adjacent to bone matrix. Changes of enzyme activities in synoviocytes, chondrocytes and osteoclasts during induced arthritis are discussed.

In situ detection of spontaneous superoxide anion and singlet oxygen production by mitochondria in rat liver and small intestine

In the present study, the endogenous formation of reactive oxygen species was localized in rat liver and small intestine. The 3,3′-diaminobenzidine (DAB)-Mn2+ technique in which cobalt ions were included in the incubation medium was applied to unfixed cryostat sections of intact tissues. Addition of manganese ions to the DAB-Co2+- containing medium greatly increased the amounts of final reaction product formed compared with incubations with only DAB and cobalt ions. In liver, a blue final reaction product was deposited, particularly in hepatocytes surrounding portal tracts. In the small intestine, the DAB--cobalt complex was mainly found at the basal side of enterocytes. Goblet cells remained unstained. Electron microscopical images revealed that an electron-dense reaction product was exclusively present at both inner and outer membranes and at the intermembrane space in mitochondria of liver parenchymal cells and duodenal enterocytes. It was shown that the spontaneous formation of final reaction product was enzymatic and dependent on the presence of oxygen in the medium. Sulphide decreased the reaction, which may indicate that cytochrome c oxidase was partially involved. Benzoquinone and histidine, which are scavengers of superoxide anions and singlet oxygen respectively, reduced the amount of final reaction product considerably. Furthermore, the formation of final reaction product was sensitive to specific inhibitors of NADH:coenzyme Q reductase and aldehydeoxidase, indicating that these enzymes were at least partly responsible for the generation of superoxide anions and singlet oxygen and for the formation of the DAB--cobalt complex.

Cytophotometry of glucose-6-phosphate dehydrogenase activity in individual cells.

SummaryWith the aid of thin films of polyacrylamide gel containing purified glucose-6-phosphate dehydrogenase subjected to cytochemical procedures for the enzyme using tetranitro blue tetrazolium, arbitrary units of integrated absorbance obtained with a Barr & Stroud GN5 cytophotometer were converted into units of enzyme activity. This conversion enabled cytochemical data to be compared directly with biochemical values. The conversion was applied to the cytochemical estimation of glucose-6-phosphate dehydrogenase activity in isolated rat hepatocytes, mouse oocytes, rabbit thymocytes, human granulocytes and human fibroblasts. Several control procedures were performed to confirm the admissibility of this conversion, such as: the estimation of the absorption characteristics of the formazans of tetranitro blue tetrazolium both in solution and precipitated in biological specimens; the linearity of the relationship between the increase of absorbance and incubation time; and the effect of different incubation conditions on the amount of specific formazan production.

Localization of cathepsin B activity in fibroblasts and chondrocytes by continuous monitoring of the formation of a final fluorescent reaction product using 5-nitrosalicylaldehyde

SummaryThe histochemical fluorescence method using 5-nitrosalicylaldehyde for the demonstration of cathepsin B activity has been used. Precipitation of the fluorescent final reaction product was analysed continuously during incubation for cathepsin B activity. Unfixed cultured human fibroblasts as well as cryostat sections of mouse metacarpal bone explants were used. Continuous monitoring of the formation of the fluorescent reaction product showed that after a certain lag phase, depending on the enzyme activity in the tissue, discrete granules appeared which became increasingly fluorescent with incubation time. Subsequently, recrystallization and redistribution of the final reaction product started to occur. It is concluded that the coupling reaction with 5-nitrosalicylaldehyde is sufficiently fast for a proper localization of proteinase activity and can be used for ‘kinetic’ analysis of enzyme activity. The method provides indications of relative amounts of cathepsin B activity in different cell types within a tissue section. It appeared from the study on metacarpal bone explants that fibroblasts in perichondrium and periosteum contained a relatively high cathepsin B activity whereas chondrocytes showed a low but distinct activity. This observation suggests that cysteine proteinases are not only involved in collagen degradation by fibroblasts but that they also play a role in the intracellular digestion of collagen by chondrocytes.

A sensitive cytochemical staining method for glucose-6-phosphate dehydrogenase activity in individual erythrocytes

SummaryA sensitive cytochemical staining method for glucose-6-phosphate dehydrogenase activity in individual human erythrocytes is described. This staining method can be used for the rapid routine discrimination of patients with a deficiency of the enzyme in its homozygote or heterozygote form, but also for quantitative localization of its activity in individual erythrocytes. The staining procedure in its optimal form consists of a treatment of the erythrocytes with sodium nitrite, then a “fixation” in 0.025% glutaraldehyde (under NADP+ protection of the active site of the enzyme), followed by incubation of the cells in suspension in the presence of tetranitro BT, 1-methoxyphenazine methosulphate and polyvinyl alcohol. Using this new technique, a sharp localization is obtained of the glucose-6-phosphate dehydrogenase activity, which enables discrimination between red cells with different levels of enzyme activity, as a consequence of enzyme deficiencies or age changes.