Uptake Of Peroxidase Research Articles

Distinct endocytotic pathways in epidermal growth factor-stimulated human carcinoma A431 cells [published erratum appears in J Cell Biol 1990 Mar;110(3):859

Addition of EGF to human epidermoid carcinoma A431 cells increases the rate of fluid-phase pinocytosis 6-10-fold as measured by horseradish peroxidase uptake (Haigler, H.T., J. A. McKanna, and S. Cohen. 1979. J. Cell Biol. 83:82-90). We show here that in the absence of extracellular Na+ or in the presence of amiloride the stimulation of pinocytosis by EGF is substantially reduced. Amiloride had no effect on the endocytosis of EGF itself or of transferrin, demonstrating that the receptor-mediated endocytotic pathway operated normally under conditions that blocked stimulated pinocytosis. Amiloride blocked EGF-stimulated pinocytosis in both HCO3(-)-containing and HCO3(-)-free media. The EGF-stimulated pinocytotic activity can frequently be localized to areas of the cell where membrane spreading and ruffling are taking place. These results demonstrate that (a) EGF induces a distinct amiloride-sensitive endocytotic pathway on A431 cells; (b) occupied EGF receptors do not utilize this pathway for their own entry; (c) endocytosis of occupied EGF receptors is not in itself sufficient to stimulate pinocytosis.

A cytochemical study of horseradish peroxidase uptake in rat junctional epithelium.

The uptake of horseradish peroxidase (HRP) was studied in rat junctional epithelium (JE). Intravenous HRP (12.5 mg, 25 mg, or 50 mg) was only endocytosed by the entire JE (particularly by the coronal portion of the JE). Although HRP uptake was negligible at a concentration of 12.5 mg, marked uptake was observed at concentrations of 25 mg and 50 mg. However, there was no difference in the HRP uptake by the JE between the concentrations of 25 mg and 50 mg. By electron microscopy, HRP was detected in the organellae (micropinocytotic vesicles, intracytoplasmic vacuoles, dense bodies, and multivesicular bodies) in the junctional epithelial cells (JE cells). HRP-positive micropinocytotic vesicles were detected in all JE cells. However, intracytoplasmic vacuoles, which are characteristically observed in JE cells, were not always positive for HRP. Numerous HRP-negative vacuoles were detected in the JE cells. The number of HRP-positive organellae in the coronal portion of the JE was pronounced, compared with those in other portions. These findings provided evidence that the entire JE, especially its coronal portion, has endocytotic ability by micropinocytotic vesicles, rather than intracytoplasmic vacuoles, for foreign substances.

Satellite cells as blood-ganglion cell barrier in autonomic ganglia

In a preliminary study a difference in the uptake and transport of wheat germ agglutinin-horseradish peroxidase (WGA-HRP) between the trigeminal ganglion and the superior cervical ganglion was observed. After injection of WGA-HRP and HRP into the trigeminal ganglion, peroxidase was found in the space between the satellite cell processes and the ganglion cells. The ganglion cells showed pinocytosis and uptake of WGA-HRP and HRP. In the superior cervical ganglion WGA-HRP and HRP were found alongside the satellite cells but were absent in the space between satellite cells and ganglion cells. Intravenous injection revealed the presence of HRP in the space between sensory ganglion cells and their satellite cells of the trigeminal and nodose ganglion whereas HRP was absent in the space between autonomic ganglion cells and their satellite cells of the superior cervical, medial cervical and pterygopalatine ganglion although HRP lined the satellite cell membranes. By means of electron microscopy, satellite cell processes in the superior cervical ganglion were found to enwrap ganglion cells very tightly with a marginal space between both cell types. Satellite cells and their proceses were mutually anchored by numerous tight junctions. In the trigeminal ganglion the extracellular space between ganglion cells and satellite cells was larger and satellite cells were found to be more loosely arranged around the ganglion cells. Satellite cell processes were only occasionally linked by tight junctions. It is concluded that satellite cells in autonomic ganglia comprise an effective barrier for WGA-HRP and HRP and probably large molecules in general. This barrier is absent in sensory ganglia.

Uptake of human eosinophil peroxidase and myeloperoxidase by cells involved in the inflammatory process.

We have recently shown that human neutrophils bind and internalize human eosinophil peroxidase (EPO) but not myeloperoxidase (MPO). In the present work, we studied the interactions of human EPO and MPO with other cells that may be involved in the inflammatory process, i.e., lymphocytes, monocytes, platelets, fibroblasts, and endothelial cells. The results indicate that EPO is bound by all the cell types considered, but is efficiently internalized only by lymphocytes, monocytes, and endothelial cells. Conversely, MPO binds appreciably only to fibroblasts and endothelial cells, although with a lower affinity than EPO, but its internalization by any of the cell types studied is hardly detectable. Furthermore, both peroxidases bind strongly to collagen fibers, whereas only EPO binds to elastin. The results suggest that EPO, owing to its high cytophilia, exerts its biological activity close to the site at which it is released from the eosinophil.

Lectin-ferritin binding on spontaneously diabetic and control rat retinal microvasculature.

We previously reported, in the spontaneously diabetic Bio-Breeding (BB) rat, an increase in horseradish peroxidase (HRP) uptake that was associated with reduction and patching of cationized ferritin (CF) binding to anionic sites on the luminal plasma membrane of the retinal capillary endothelium. To see whether alterations in the negatively charged terminal sugar residues, N-acetyl-glucosamine (NAG) and sialic acid (SA), might contribute to these changes in the diabetic rat retina, we used lectin-ferritin (Fe) conjugates to study the distribution of these sugars on the retinal endothelial luminal membranes. Wheat germ agglutin (WGA, binds to NAG and SA) and Limax flavius (LFA, binds only SA) were used. Plasma membrane WGA-Fe binding was dense and uniform in control animals. Binding sites were also found in coated luminal vesicles, within some uncoated luminal vesicles and on their diaphragms. Unlabeled uncoated luminal vesicles were also seen, suggesting two populations of uncoated vesicles. In diabetic animals, the binding sites were present within the same membrane associated microdomains as in the controls. However, in the majority of outer plexiform layer (OPL) vessels in diabetic animals, WGA-Fe binding was reduced to a single, discontinuous layer of particles (p less than 0.02). In both diabetic and control vessels, WGA-Fe binding was greatly reduced by the addition of competitive sugars. A few particles remained on the plasma membrane, on the diaphragms of some vesicles, and at the edge of vesicles. LFA-Fe binding was similar to that seen with WGA-Fe in the presence of competitive sugars. These results suggest that luminal membranes of retinal capillaries are rich in NAG and contain little SA. The sparse WGA-Fe binding pattern in the diabetic OPL may reflect decreases in number or accessibility of NAG residues, since similar binding patterns are seen in both the control and diabetic animals under conditions specific for SA. Thus, alteration of terminal NAG residues may contribute to decreased luminal surface anionic sites and increased pinocytotic transport in the retinal microvasculature of spontaneously diabetic BB rats.

Comparative investigations on the uptake of phallotoxins, bile acids, bovine lactoperoxidase and horseradish peroxidase into rat hepatocytes in suspension and in cell cultures

Two alternative uptake mechanisms for phallotoxins by liver cells are debated: carrier-mediated uptake and receptor-mediated endocytosis. We have compared the properties of hepatocellular uptake of the phallotoxins, phalloidin and demethylphalloin, with the uptake of cholate as a substrate for carrier-mediated uptake and compared with iodinated bovine lactoperoxidase or iodinated horseradish peroxidase, as the latter are known to be taken up by vesicular endocytosis. Uptake of phallotoxins and [ 14C]cholate uptake into isolated hepatocytes is independent of extracellular calcium but inhibited by A23187 or by monensin. Uptake of bovine lactoperoxidase strictly depends on external Ca 2+, was insensitive to A23197 and was not inhibited by monensin. No mutual uptake inhibition between phalloidin or cholate and peroxidases was seen, indicating independent permeation pathways in hepatocytes. However, high concentrations of cytochalasin B inhibited the uptake of either phalloidin, cholate or bovine lactoperoxidase. Horseradish peroxidase uptake, which was taken as an indicator for fluid pinocytosis, was low in isolated hepatocytes and could not account for the amount of phalloidin or cholate taken up. In cultured rat hepatocytes, uptake of phallotoxins decreased within 1 day to 10% of the uptake seen in freshly isolated hepatocytes. The results indicate different mechanisms for hepatocellular phallotoxin/bile-acid uptake and peroxidase internalization. As monolayer cultures of hepatocytes rapidly lost the carrier-mediated uptake of phallotoxins and bile acids, freshly isolated hepatocytes might be a more suitable experimental model than cultured cells for kinetic studies on this transport system.

Cellular and synaptic morphology of a feeding motor circuit in Aplysia californica.

The cellular and synaptic morphology of a component of the feeding motor circuit in Aplysia californica was examined with light and electron microscopic techniques. The circuit consists of a pair of inhibitory premotor interneurons, B4 and B5, as well as two motoneurons, B15 and B16, which innervate the accessory radula closer muscle. The neurons have wide, varicose arborizations in the buccal ganglion neuropil. All four of these neurons are cholinergic, and in addition, B15 contains immunoreactivity to sera raised against small cardioactive peptide B. Varicose processes in the accessory radula closer muscle are immunoreactive with antisera against several neuropeptides. We identified specific neuromuscular junctions by visualizing horseradish peroxidase uptake in recycled synaptic vesicles. Direct innervation of the accessory radula closer muscle by B15 and B16 is demonstrated by experiments in which horseradish peroxidase is transported from motoneuronal soma to the terminals on muscle fibers. In addition, specific synaptic contacts between B4 and B5 and each of the motoneurons are observed in the buccal ganglion neuropil. Finally, multiple contacts consistent with peptidergic, serotoninergic, and cholinergic synapses are made onto the neurons, suggesting that a variety of transmitters modulate motor output at each level of the hierarchical circuit. These results support the physiological evidence suggesting the involvement of neuropeptides as well as "classical" transmitters in the modulation of circuitry governing feeding behavior in Aplysia.

Two types of very long visual fibers found in the optic lobe of the flesh-fly, Boettcherisca peregrina

Horseradish peroxidase (HRP) uptake (through a Corneal incision) in photoreceptor cells of the compound eye of Boettcherisca peregrina, resulted in the labeling of two types of very long visual fibers. One of them (the long fiber, If) penetrates through the lamina and medulla, and directly terminates within the lobula. The other (the bypass fiber, bpf) terminates in the medulla, like the axons of R7 and R8 photoreceptor cells, but the fibers run a considerably roundabout course. The photoreceptor cells with these very long fibers are region-dependent within the retina. Both the If and bpf are found almost exclusively in the male fly.

Endogenous peroxidase activity in human cutaneous and adenoidal mast cells.

We have studied peroxidase activity in human cutaneous and adenoidal mast cells using different methods, in order to determine the optimal technical conditions for its demonstration. In 1.25% glutaraldehyde-fixed cells, no peroxidase activity was seen. On the contrary, in tannic acid-aldehyde-fixed cells or in unfixed cells peroxidase activity was revealed independently of the DAB concentration or the incubation time in DAB medium. The reaction product was localized in perinuclear cisternae and endoplasmic reticulum. Granules were always unreactive with all techniques employed. Golgi apparatus was generally negative and only occasional cells exhibited one or two positive peripheral cisternae. This activity appears sensitive to fixation by glutaraldehyde and is inhibited by 3-amino-1,2,4-triazole (AMT) and by lack of H2O2 or DAB in the incubation medium, but not by potassium cyanide, sodium azide, or sodium pyruvate, at the concentrations used. The peroxidase activity described in this report is an endogenous peroxidase and is not related to uptake of exogenous peroxidase by mast cells. It can therefore be considered as an ultracytochemical marker of human mast cells.

The time-course of appearance and net accumulation of horseradish peroxidase (HRP) presented orally to rainbow trout Salmo gairdneri (Richardson)

1. 1. A sensitive enzyme-linked immunosorbent assay (ELISA) technique was used in order to determine horseradish peroxidase (HRP) uptake from the rainbow trout gut. 2. 2. HRP was detected in blood plasma and various tissues within 15 min of oral intubation. 3. 3. The time-course of net accumulation (uptake-degradatation) over a 75 min period was recorded. 4. 4. The presence of HRP reached a maximum in the body tissues approximately 45 min after intubation and on a ng/g wet weight basis the order of accumulation within the tissues was liver > spleen = kidney > plasma > heart. 5. 5. The total organ accumulation (net) was in the order liver > plasma > kidney > spleen > heart.

Effects of cholesterol and lipoproteins on endocytosis by a monocyte-like cell line

The human monocyte/macrophage-like cell line U937 is a cholesterol auxotroph. Incubation of these cells in the growth medium in which delipidated fetal calf serum has been substituted for fetal calf serum depletes cellular cholesterol and inhibits growth. The cholesterol requirement of these cells for growth can be satisfied by human low-density lipoprotein (LDL), and very-low-density lipoprotein (VLDL), but not by high-density lipoprotein (HDL). U937 cells can bind and degrade LDL via a high-affinity site and this recognition is altered by acetylation of LDL. This indicates that these cells express relatively high LDL receptor activity and low levels of the acetyl-LDL receptor. The cells were used to study the role of cholesterol in lectin-mediated and fluid-phase endocytosis. Growth of the cells in the medium containing delipidated fetal calf serum results in impairment of both concanavalin A-mediated endocytosis of horseradish peroxidase and concanavalin A-independent endocytosis of Lucifer Yellow. Supplementation of the medium with cholesterol prevents cellular cholesterol depletion, supports growth and stimulates Lucifer Yellow endocytosis but fails to restore horseradish peroxidase endocytosis. However, if the cells are incubated in the presence of no less than 40 μg LDL protein/ml to maintain normal cell cholesterol levels, concanavalin A-mediated endocytosis of horseradish peroxidase is activated. The effect of LDL is specific since neither VLDL nor HDL 3 at the same protein concentration activates horseradish peroxidase uptake by the cells. Furthermore, the activation of endocytosis by LDL is not inhibited by the inclusion of heparin or acetylation of the LDL indicating that binding of LDL to the LDL receptor is not required for these effects. The mediation of activation of horseradish peroxidase endocytosis by the lectin is presumed to involve binding of LDL to concanavalin A associated with the cell surface which in turn stimulates horseradish peroxidase binding and uptake by adsorptive endocytosis. The rate of fluid endocytosis and endosome formation seems to depend on cellular cholesterol content presumably because cholesterol is involved in maintaining the appropriate plasma membrane structure and fluidity.

Biochemical and ultrastructural effects of chloroquine on horseradish peroxidase uptake and lysosomal enzyme activities in innervated and denervated mouse skeletal muscle

The effects of chloroquine treatment on horseradish peroxidase (HRP) uptake and lysosomal enzyme activities in innervated and denervated mouse skeletal muscle have been studied using biochemical, histochemical and ultrastructural techniques. Chloroquine treatment caused a large (59–101%) increase in the activity of cathepsin D in both innervated and denervated muscle. The activity of N-acetyl-β- d-glucosaminidase also increased slightly in denervated muscle. No effect was observed on acid phosphatase activity. The in vivo uptake of HRP in innervated and denervated muscle was unaffected by chloroquine treatment. The results show that the activities of certain lysosomal enzymes may increase in skeletal muscle without an increase in endocytic activity. This is discussed in comparison to what is seen in denervated and dystrophic muscle. Histochemical and ultrastructural studies showed the HRP uptake to occur segmentally in denervated muscle fibres from untreated as well as chloroquine-treated animals. Ultrastructurally the peroxidase-positive phagosomes occurring in these segments were found to contain increased levels of undegraded material after chloroquine treatment suggesting that these phagosomes are of a lysosomal nature and also participate in autophagic processes.

Fast turnover of clear vesicle membrane in frog sympathetic ganglion

The results of three independent experiments are presented. In the first experiment, the afferent fibers of the right side IX and X sympathetic ganglia of five frogs were stimulated at 100 Hz and control unstimulated ganglia were taken from five other frogs. In the second experiment, the right IX and X ganglia from eight other frogs were stimulated at 10 Hz; the left side ganglia of the same eight frogs served as controls. A third experiment was performed in the presence of extracellular horseradish peroxidase, the sympathetic ganglia of three frogs were stimulated at 10 Hz, those of three other frogs were stimulated at 100 Hz and control ganglia exposed to horseradish peroxidase, but not stimulated were taken from another three frogs. Postganglionic compound action potentials were recorded in each experiment to verify the effectiveness of the stimulation. Following stimulation, the ganglia were fixed and prepared for electron microscopy. Between 10 and 20 synaptic profiles were photographed for each ganglia. The numerical density on area (i.e. number of vesicles per μm 2) was calculated separately for the clear vesicles and for the large dense-core vesicles of each profile. Statistical tests indicated no significant difference in the numerical density on area of the clear vesicles, or of the dense-core vesicles between the control and the ganglia stimulated at either 10 or 100 Hz. However, in the experiment done in presence of horseradish peroxidase, a significant increase of the percentage of vesicles labeled with horseradish peroxidase was obtained following stimulation at either 10 or 100 Hz. This indicates that a rapid recycling of the vesicular membrane is occurring in this system without any significant change in the numerical density of the clear vesicles. Horseradish peroxidase uptake is therefore a better indicator of synaptic activity than the reduction of the numerical density of vesicles. These results also indicate that the numerical density on area of large dense-core vesicles, presumably containing peptides, is not modified by repeated stimulation as indicated by a few earlier investigations at other synapses.

The time-course of appearance and net accumulation of horseradish peroxidase (HRP) presented orally to juvenile carp Cyprinus carpio (L.)

1. 1. A sensitive enzyme-linked immunosorbent assay (ELISA) technique was used in order to determine horseradish peroxidase (HRP) uptake from the carp gut. 2. 2. HRP was detected in blood plasma and various tissues within 15 min of oral intubation. 3. 3. The time-course of net accumulation (uptake-degradation) over a 2 hr period was recorded. 4. 4. The presence of HRP reached a maximum in the body tissues approximately 1 hr after intubation and on a μg/g wet weight basis the order of accumulation within the tissues was spleen > kidney > liver. 5. 5. The total organ accumulation (net) was in the order liver > kidney > spleen.

Serological determinants of fluorescent granular perithelial cells along small cerebral blood vessels in rodent

As reported previously, the perivascular cells laden with fluorescent granules (FGP cell) are situated in Virchow-Robin space and show marked uptake capacity for intraventricularly administered substances. The phagocytic FGP cells are derived developmentally form leptomeningeal cells and are recognizable in various kinds of animals, including humans. In the present paper, the FGP cells of rodents are studied by immunological techniques. It is clearly demonstrated that rat FGP cells express the antigenic determinant for Ia antibody at about 7 days after birth, and also that mouse FGP cells show a positive reaction against mouse Fc and splenic macrophage antibodies at about the same developmental stage. The specific determinants of FGP cells appear concurrently with the initiation of horseradish peroxidase uptake. On the other hand, Ia antigen is also shown in subarachnoid macrophages, but not in immature FGP cells, endothelium and pericyte. Based on these findings, it seems reasonable to consider that the FGP cells are indigenous cerebral macrophages and significant for the local immune response in a cerebral tissue. Further, in this paper, to prevent confusion of the FGP cell with the other perivascular cells, the authors propose designating the FGP cell as Mato cell.

Lack of relationship between the direction of operant turning and horseradish peroxidase uptake by crossed nigro-striatal projections

We examined the effect of training rats to perform an operant turning response on the crossed nigrostriata) projections. Rats were reinforced for circling behavior with water. After 7 days of reinforced circling, the animals were implanted with horseradish peroxidase in the striatum either ipsilateral or contralateral to the direction in which they had been trained to turn. Subsequently, the substantia nigra, ventral tegmental area, and retrorubral area in both hemispheres were examined for labeled cells. This analysis failed to reveal any relationship between the direction of reinforced circling and efferents from these areas to the striatum.

Endocytosis in adipokinetic hormone (AKH)-producing cells of the migratory locust, [formula omitted], in rest and during flight

Intrinsic gland cells of the corpus cardiacum of Locusta synthesize AKH, and release it into the haemolymph shortly after onset of flight. AKH stimulates the release of diacylglycerol — the fuel for the flight muscles — from the fat body. The peak of haemolymph AKH is reached after about 30 min flight. Release of AKH mainly occurs from short processes of the gland cells, by means of exocytosis. It may be expected that this release is followed by compensatory re-uptake of membrane material by means of endocytosis near the site of exocytosis. To get information on the location of endocytotic activity and on the intracellular endocytotic pathway, the uptake of the tracer horse-radish peroxidase (HRP) by the gland cells in flying and in resting locusts was studied electron microscopically

Endocytosis, proteolysis, and exocytosis of exogenous proteins by cultured myotubes.

The endocytic activity of chick myotubes in cultures was investigated. Differentiated myotubes internalized the fluid-phase marker horseradish peroxidase in membrane-bound particles which typically displayed reaction product at the inner surface of the vesicle. Pulse-chase experiments demonstrated a rapid decrease in the number of horseradish peroxidase-containing vesicles and a redistribution from a uniform to a perinuclear pattern. Horseradish peroxidase uptake was extensively inhibited by incubation at 0-4 degrees C consistent with an endocytic mechanism. To further characterize the process, the fate of labeled protein was investigated. Following uptake [3H] hemoglobin A was extensively degraded (40-50%) to acid-soluble products within 10 h. Degradation displayed a biphasic pattern with a rapid early phase followed by a much slower second phase. The decreasing rate of proteolysis can be accounted for, in part, by a simultaneous exocytosis of a substantial fraction (25-30%) of acid-insoluble label from myotubes. The lysosomotropic agents methylamine, monensin, and chloroquine significantly inhibited (23-75%) proteolysis, indicating a lysosomal site of degradation. Part of the inhibitory effect results from an increase in exocytosis in the presence of these agents. Degradation of endocytosed [3H]hemoglobin A was not inhibited by insulin. In contrast degradation of endogenous myotube proteins was inhibited (40%) by insulin and blocked by methylamine. These results suggest that cultured myotubes possess a coupled endocytic/exocytic pathway for macromolecules and that a fraction of the internalized substrate is degraded by an insulin-insensitive lysosomal pathway.

Rapid effects of insulin on the cycling of the insulin receptor in a human monocyte cell line (U-937).

The insulin receptor and its regulation by insulin was studied in U-937 monocytes, a human cell line with properties similar to those of normal peripheral blood monocytes. Treatment of this cell with insulin for 8-16 h produced an overall loss in the insulin receptor, i.e., a loss of receptors from the cell surface and internal pools. In contrast, short-term insulin treatment (15-30 min) caused a reduction in cell surface receptors but an increase in the internal receptors, as judged by pronase treatment at 4 degrees C to distinguish receptor location. After the removal of insulin and pronase, the internalized receptors were rapidly reinserted back into the cell surface after warming to 37 degrees C. Further studies showed an insulin-mediated increase in fluid-phase pinocytosis as measured by horseradish peroxidase (HRP) uptake. The amount of HRP accumulation and the time course for this stimulation were similar to those for receptor internalization. These features plus other results suggest that the insulin-stimulated internalization of insulin receptors may require an acceleration in the rate of pinocytic vesicle formation.

Fluid phase and mannose receptor-mediated uptake of horseradish peroxidase in mouse bone marrow-derived macrophages. Biochemical and ultrastructural study.

A detailed study of horseradish peroxidase (HRP) uptake by in vitro cultured bone marrow-derived macrophages was undertaken. Biochemical quantitations performed over a wide range of HRP concentrations, in the absence or presence of yeast mannan, showed that these macrophages pinocytose HRP by both fluid phase and mannose receptor-mediated uptake. The relative contribution of these two types of endocytosis varied with the concentration of enzyme in the extracellular medium. A morphological study at the light and electron microscope levels conducted in parallel confirmed the biochemical data.