Endocytosis Of Horseradish Peroxidase Research Articles

Microfilament network is needed for the endocytosis of water channels and not for apical membrane insertion upon vasopressin action.

In the current study, a novel role for the microfilaments in vasopressin-induced water transport in toad urinary bladders, a popular model for the mammalian collecting duct, was established. Vasopressin-induced water transport was not affected by cytochalasin D (CD, 20 microM) or latrunculin B (Lat B, 0.5-2 microM), microfilament-disrupting reagents, suggesting that the initial trafficking of vesicles containing water channels and insertion of membranes into the apical membrane are microfilament-independent. After the removal of vasopressin, bladders treated with CD or Lat B continued to transport water at least 2-3-fold greater than those that received the vehicle. Furthermore, the enhanced water transport was inhibited by HgCl2 (1 mM), a potent inhibitor of water channel-mediated water flow, suggesting that the enhanced water flow was through water channels. In addition, Lat B and CD inhibited vasopressin-induced endocytosis of horseradish peroxidase (HRP), a fluid endocytotic marker. These results suggested that although microfilaments are not needed for the initial trafficking of water channels to the apical side, the microfilament network is essential for the retrieval of water channels following their insertion into apical membranes.

Bafilomycin A1, a specific inhibitor of V-type H+-ATPases, inhibits the acidification of endocytic structures and inhibits horseradish peroxidase uptake in isolated rat sinusoidal endothelial cells.

The role of vacuolar type H+-ATPases (v-ATPases) and pH gradient between the endocytic compartments and cytoplasm in the endocytosis of horseradish peroxidase, a mannose-terminated glycoprotein, was investigated morphologically in isolated rat sinusoidal endothelial cells. Toward this purpose, a specific inhibitor of v-ATPases, bafilomycin A1, was used to inhibit v-ATPases in the vacuolar system. Uptake of horseradish peroxidase was examined by electron microscopy. Fluorescent staining by acridine orange showed that bafilomycin A1 inhibited the acidification of the endocytic compartments. Horseradish peroxidase was taken up via mannose receptors and was distributed in the endocytic structures in the isolated sinusoidal endothelial cells. Uptake of horseradish peroxidase was significantly inhibited by bafilomycin A1, and this finding was confirmed by morphometrical analysis. These results suggest that: a) v-ATPases are necessary for acidification of the endocytic compartments in the sinusoidal endothelial cells and b) the pH gradient between the endocytic compartments and the cytoplasm that is generated by v-ATPases is necessary for the receptor-mediated endocytosis of a mannose-terminated glycoprotein, horseradish peroxidase.

Endocytosis of horseradish peroxidase by brain microvascular and umbilical vein endothelial cells in culture: an ultrastructural and morphometric study.

The ability to form tight junctions and the paucity of fluid phase endocytosis showed by brain microvacular endothelial cells (BMECs) make up the structural basis of the blood-brain barrier (BBB). Most studies on cultured BMECs focused on intercellular junctions, whereas endocytosis received lesser attention. We studied endocytosis of horseradish peroxidase in primary and passage 1 and 2 BMEC cultures from rat brain as well as in human umbilical vein endothelial cell (HUVEC) culture. Endocytic activity was also analyzed in passage 1 BMECs treated with lipopolysaccharide (LPS, 1 microg/ml for 4 h), which mimics BBB disruption in bacterial meningoencephalitis. The percent of cytoplasmic area occupied by endocytic profiles (vesicles <70 nm and vacuoles >70 nm) and their mean number per cell were significantly lower in primary and passaged BMEC than in HUVEC cultures. The area and number of endocytic profiles significantly increased in BMECs after exposure to LPS. BMECs cultured under standard conditions may be a suitable model for studying the mechanism of increased fluid phase endocytosis in certain diseases and injury states.

Novel method for isolating mammalian cells defective in fluid-phase endocytosis.

A new method for isolating mutants defective in fluid-phase endocytosis has been developed based on the observation that endocytosed horseradish peroxidase can be made lethal to cells. The method was used to isolate a mutant from Chinese hamster ovary cells, termed HRP-1, that was temperature-sensitive for viability and had a 70% reduction in the rate of horseradish peroxidase endocytosis at the restrictive temperature. At high temperature, HRP-1 cells were also defective in the secretory path and their Golgi complex disappeared at the resolution of fluorescence microscopy. These properties are similar to two previously described mutants of CHO cells, DS28-6 and V.24.1. In complementation tests, mutants HRP-1, DS28-6, and V.24.1 all appeared to be in the same complementation group.

The effect of botulinum toxin type D on the triggered and constitutive exocytosis/endocytosis cycles in cultures of bovine adrenal medullary cells

The extracellular fluid phase marker, horseradish peroxidase, enters chromaffin cells when triggered to secrete catecholamine. This triggered uptake, like secretion, is abolished in cells pre-incubated with botulinum toxin. Endocytosis of horseradish peroxidase into unstimulated cells is unaffected by botulinum toxin but is inhibited when the temperature is reduced. Once internalised by the unstimulated cells, horseradish peroxidase is released back into the extracellular fluid, the rate of release being temperature sensitive but unaffected by carbamylcholine or botulinum toxin. These results suggest that triggered exocytosis is a necessary event to precede triggered endocytosis, and that botulinum toxin may affect only the triggered exocytosis/endocytosis cycle and not the constitutive cycle.

Antigen Recognition in the Female Reproductive Tract. II. Endocytosis of Horseradish Peroxidase by Langerhans Cells in Murine Vaginal Epithelium1

Previous studies have shown that dendritic cells in the murine vaginal epithelium at diestrus and metestrus can endocytose intravaginally administered soluble protein tracers, but the identity of the dendritic cells was not established. In the investigation reported here, we used a combination of histochemistry and transmission electron microscopy to study the endocytosis of exogenous horseradish peroxidase by vaginal dendritic cells and to identify these cells as Langerhans' cells on the basis of their cellular associations, ultrastructural morphology, and the presence of Langerhans' cell granules.

A method for flat embedding thick cryostat tissue sections in plastic resin.

In many histochemical procedures tissues are often sectioned at 30-50 nm (thick sections) to maximize penetration of the substrate into the tissues. After the histochemical reaction the sections can be embedded in plastic resins for thin sectioning and transmission electron microscopy study. Recently, we have used one such procedure to study endocytosis of horseradish peroxidase into Langerhans cells in the murine vaginal epithelium (Parr and Parr 1990). This enzyme has been used extensively as a protein tracer in biologic studies because it can be localized by a simple histochemical procedure for both light and electron microscopy (Graham and Karnovsky 1966). In carrying out the histochemical procedure we encountered problems embedding the thick cryostat sections because they became coiled and twisted during processing. In this report we describe a simple technique for flat embedding thick cryostat sections in plastic resin.

Two threshold values of low pH block endocytosis at different stages.

The influence of low extracellular pH on endocytosis was studied in baby hamster kidney cells. When the extracellular medium was adjusted to pH 5.7, the intracellular pH decreased within 2 min to pH 6.2 and the endocytosis of horseradish peroxidase (HRP) in the fluid phase dropped to an undetectable level. With an external pH of 6.3, the internal pH dropped to pH 6.8 and HRP was internalized at a normal rate for 5 min but accumulation during longer incubation times did not occur. Morphologically, HRP was visualized in the lumen of a subpopulation of tubular and vesicular endosomes. These observations were confirmed by subcellular fractionation studies using free flow electrophoresis. Low extracellular pH also had an effect on the endocytosis of the membrane-spanning glycoprotein G of vesicular stomatitis virus which was implanted into the plasma membrane. The internalization of G-protein was quantitated by a surface fluoroimmunoassay. The endocytosis of G-protein was not affected when the external pH was dropped to 6.3, but was reduced at an external pH of 5.7. The intracellular ATP was not depleted and the reduction of endocytosis was reversible upon return to physiological pH. Clathrin coated pits were detected by electron microscopy at the plasma membrane of the low-pH-treated cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Newcastle disease virus and two influenza viruses: differing effects of acid and temperature on the uptake of infectious virus into bovine and canine kidney cell lines.

The entry of 2 influenza viruses was compared to the entry of the Ulster strain of Newcastle disease virus (NDV) by measuring the escape rate of preadsorbed virus from neutralization using a microwell assay. The 2-minute entry of Ulster into Madin-Darby bovine kidney (MDBK) cells increased exponentially over the temperature range for fusion of 30-37 degrees C and was prevented by pH 4.6 which corresponded to the pH within endosomes. The 2-hour entry of both influenza viruses into MDBK cells increased exponentially over the temperature range of 0-30 degrees C with Q10 values similar to Q10 for the endocytosis of horse radish peroxidase and their entry times were reduced to less than 10 minutes at pH 4.6. When Madin-Darby canine kidney (MDCK) cells were used the entry of one influenza virus, NIB-8, took 2 minutes whereas the other, A/Jap/Bel, took 2 hours. The reason for this was unknown. NIB-8 contains genes 1, 2, 3, 7, 8 from A/Puerto Rico/8/34 (H1N1) virus and genes 4, 5, 6 from A/Bangkok/1/79 (H3N2) virus. A/Jap/Bel is a triple reassortant which shares genes 3 and 7 with NIB-8 whereas 1, 2, 4, 5 are from A/Japan/305/57 (H2N2) and genes 6, 8 are from A/Bel/42 (H1N1).

Endocytosis in the retinal and choroidal microcirculation.

The endocytosis of horseradish peroxidase (HRP) by the vascular cells of retinal and choroidal blood vessels was compared in immersion and perfusion fixed eyes from individual rats. The mechanisms of endocytosis of HRP appeared identical in both retinal and choroidal vessels. The bulk of internalised tracer occurred in macropinosomes 300-400 nm in diameter. Tracer was localised to a 20-30 nm layer on the internal aspect of the limiting membrane. This layer was coincident with the glycocalyx of the luminal plasma membrane as revealed by ruthenium redosmium tetroxide staining. Horseradish peroxidase was also internalised by a small scattered population of vesicles (100-130 nm in diameter). The size of these vesicles suggested that they may have arisen from clathrin coated regions of the plasma membrane. It is suggested that the endocytosis of HRP in retinal and choroidal vascular endothelium occurs as a function of plasma membrane recycling. Horseradish peroxidase may also be internalised as a 'contaminant' of the glycocalyx in coated pits involved in receptor mediated endocytosis. The smooth 80 nm plasmalemmal caveolae of the retinal and choroidal vascular endothelial cells did not appear to participate either in absorptive endocytosis or vesicular transport.

Phorbol myristate acetate induces endocytosis as well as exocytosis and hydroosmosis in toad urinary bladder

The induction of the hydroosmotic response in the toad urinary bladder is considered to be associated with membrane addition mediated by exocytosis at the affected luminal membrane and reversed by endocytic retrieval at that surface. The permeability, exocytosis and endocytosis are initiated by antidiuretic hormone (ADH) receptor interaction on the basolateral membrane. In other hormone responsive systems, phorbol ester (phorbol myristate acetate, PMA), a tumor promoter, has been implicated in the regulation of various transport processes through the activation of protein kinase C and cytoskeletal protein phosphorylation. We found that addition of 10 −6 M PMA to the mucosa induces an hydroosmotic response which is gradual and which reaches a maximum within 60 min, equal to about 1 3 the maximal ADH response. Morphologically, PMA causes rapid exocytosis of the granules, endocytosis of horseradish peroxidase from the mucosal medium into tubules and multivesicular bodies and elongation of apical microvilli. Controls treated with mucosal 0.1% dimethylsulfoxide (DMSO) or an inactive PMA isomer on the mucosal surface, or PMA on the serosal surface lack the hydroosmotic, exocytic, endocytic and cytoskeletal changes. Addition of serosal ADH to PMA-treated bladders results in a precocious hydroosmotic and exocytic ADH response, but a lowering of the maximal response. Also pretreatment of bladders with PMA prevented the ADH-induced increase in transepithelial potential difference. Thus, apical events mediating the PMA hydroosmotic response are correlated with exo- and endocytosis and elongation of apical microvilli.

Differences between the endocytosis of horseradish peroxidase and its conjugate with wheat germ agglutinin by cultured fibroblasts.

A covalent conjugate of wheat germ agglutinin (WGA) with horseradish peroxidase (HRP) was used for a morphologic study of its adsorptive endocytosis by cultured human fibroblasts. Initial binding at 4 degrees C of the conjugate was observed over the entire plasma membrane, including "coated" and smooth pits. Endocytosis of HRP and the WGA-HRP conjugate was observed in lysosomes, but only the conjugate was seen in a cisterna of the Golgi apparatus (GERL), and in adjacent coated vesicles.

Pinocytosis in eyes of a snail, Helix aspersa

Extirpated eyes of the snail Helix aspersa , from which lenses were removed surgically, were incubated in horseradish peroxidase (HRP) and prepared cytochemically for electron microscopy to determine the positions of HRP. There was some internalization of tracer by pinocytic vesicles and tubules in Type I sensory cells and much uptake by Type II sensory cells and by supportive cells. Eyes of dark-adapted snails exhibited more endocytosis of HRP than eyes of light-adapted specimens. There was no uptake of tracer by the distal ends of retinal cells in eyes incubated in HRP but with lenses in place. Pinocytosis occurred basally, however, where the cells rest on the capsule of the eye. New features described include: pinocytic tubules, fusion of photic vesicles to form endoplasmic reticulum (ER), origin of microvillar membrane from ER, development of autophagic vacuoles, and degeneration of microvilli. Turnover of photoreceptoral membranes of sensory cell microvilli is discussed.

Biology of the scrotum. III. Effects of abdominal temperature upon the epithelial cells of the rat cauda epididymidis.

Abdominal temperature suppresses the sperm storage function of the epididymis as well as transepithelial ion and water transport by the caudal epithelium, but has no obvious effect on its ultrastructure. Thus,thecellular basis of that temperature effect has been uncertain. When the scrotal cauda epididymidis was subjected to a 20 min perfusion with horseradish peroxidase (HRP) in lactated Ringer’s saline (which has a higher Na4 contentthan,and is mildly hyposmolar to, normal caudal fluid), the principal cells swelled substantially and intercellular spaces indicative of fluid imbibition appeared in the epithelium. Morphometric analysis indicated that after like perfusion of the 16-day cryptic (abdominal) cauda maintained in continuity with a normal scrotal testis, organdIes constituted a smaller proportion of the volume of the principal cells, and intercellular spaces were found to be absent. In neither the scrotal nor cryptic situation did the clear or basal cells display any swelling reaction or other structural modification that might distinguish them from these cell types in the normal nonperfused scrotal cauda. Furthermore, deep body temperature did not affect the normal differential pattern of HRP uptake by the epithelium, i.e., heavy by clear cells and light by principal cells. An increased incidence of necrotic cells of unknown origin was also observed in the epithelium of the 16-day cryptic cauda. These and related studies suggest that in the epithehium of the cauda epididymidis, 1) it is the water and ion transport function of the principal cells that are inhibited by deep body temperature, 2) neither HRP endocytosis nor any other ultrastructural feature of the clear cells is noticeably affected by deep body temperature and/or perfusion, and 3) HRP uptake is not a reliable indicator of fluid resorption by the caudal epithelium. The observations point to passage directly across the apical plasznalemma of the normal principal cell, by a nonvesicular mechanism, as an important route of water absorption. This may account in large part for the abundant stereociia that serve to enlarge the luminal surface area of these particular cells.

Endocytosis in absorptive cells of cultured human small-intestinal tissue: effect of cytochalasin B and D.

The effect of cytochalasin B (CB) and cytochalasin D (CD) on the endocytotic uptake of horseradish peroxidase (HRP) by intestinal absorptive cells was investigated by morphometric methods. The results showed that CD inhibited endocytosis considerably, and without any detrimental side-effects. CB had hardly any effect on the endocytosis of HRP, but caused a significant decrease in the number of apical vesicles and tubules involved in the transport of cell-coat glycoproteins from the Golgi apparatus to the brush border. Electron-microscopic autoradiographic analysis of the effect of CD showed that although endocytosis is inhibited significantly by the drug, the amount of radiolabelled cell-coat material entering the lysosome-like bodies was unaltered compared with control cultures. These observations support our hypothesis that the cell-coat glycoproteins of the absorptive cells enter the lysosome-like bodies by a crinophagic rather than by an exocytotic-endocytotic mechanism.

The effect of cytochalasin B and D on the endocytosis of horseradish peroxidase and transport of cell-coat glyco-proteins in absorptive cells of cultured human small-intestinal tissue

The effect of cytochalasin B and D on the endocytosis of horseradish peroxidase and transport of cell-coat glyco-proteins in absorptive cells of cultured human small-intestinal tissue

Endocytosis of horseradish peroxidase and lactoperoxidase by absorptive cells of cultured human small-intestinal tissue

Endocytosis of horseradish peroxidase and lactoperoxidase by absorptive cells of cultured human small-intestinal tissue

Effects of colchicine on endocytosis of horseradish peroxidase by rat peritoneal macrophages.

Horseradish peroxidase (HRP) was used as an exogenous marker to study the effects of microtubule-disruptive drugs on endocytosis in cultures of thioglycollate-elicited rat peritoneal macrophages. Colchicine and vinblastine, but not lumicolchicine or cytochalasin B, reduced HRP uptake by about 30-40%. However, as determined by stereological measurements, the size of the HRP-containing compartment within the cells remained unaltered. In both control cells and cells treated with colchicine or vinblastine the HRP-reactive vesicles were preferentially located close to the dictyosomes (stacks of cisternae) despite the fact that the Golgi complex was disorganized in the treated cells. These results suggest that intact cytoplasmic complex was disorganized in the treated cells. These results suggest that intact cytoplasmic microtubules are required to maintain a normal rate of fluid phase endocytosis in macrophages. On the other hand, it seems as if microtubules are not necessary for the translocation of newly formed endocytic vesicles/lysosomes to the dictyosomes, from which they probably are supplied with digestive enzymes.

Endocytosis in chronically denervated mouse skeletal muscle. A biochemical and ultrastructural study with horseradish peroxidase

Endocytosis of horseradish peroxidase was studied in vivo in innervated mouse extensor digitorum longus and soleus muscles and in the same muscles 6 days after surgical denervation, by a combination of biochemical and morphological techniques. Biochemical determination of peroxidase activity in extensor digitorum longus and soleus muscles at different times after a single intravenous injection of horseradish peroxidase followed by extensive washing of the muscles, showed that peak levels of horseradish peroxidase in muscle tissue were obtained 2 h after injection. The uptake of horseradish peroxidase in denervated extensor digitorum longus and soleus muscles was approximately three times greater than in innervated control muscles. Light and electron microscopic examination of denervated and innervated extensor digitorum longus muscles, demonstrated that horseradish peroxidase was present in the extracellular space, i.e. between muscle fibers and in transverse tubules already 30 min after an intravenous horseradish peroxidase injection. Light-microscopic examination, 2–4 h after an intravenous injection of horseradish peroxidase, revealed that a population of denervated muscle fibers contained horseradish peroxidase in bodies or vacuoles restricted to segments of the fibers. Such bodies, containing horseradish peroxidase, were not observed in innervated muscle fibers. Ultrastructural examination showed that these horseradish peroxidase-bodies were limited by a single membrane and located adjacent to transverse tubules between myofibrils showing early signs of degeneration. Coated or uncoated vesicles of about 50–100 nm appeared to originate from horseradish peroxidase-labelled transverse tubules in denervated muscle fibers. These findings suggest that endocytosis (micropinocytosis) occurs from transverse tubules in segments of denervated muscle fibers where signs of degeneration can be observed. Following the entry of horseradish peroxidase, the tracer accumulates in heterophagosomes and secondary lysosomes of diverse appearance.

Increased endocytosis with lysosomal activation in skeletal muscle of dystrophic mouse.

Endocytosis in dystrophic muscles was studied by a combination of biochemical, radiochemical, and light and electron microscopic techniques. It was observed that the uptake of horseradish peroxidase (HRP) and 3H-Inulin in vitro was increased in leg skeletal muscles from dystrophic mice compared with littermate controls. Endocytosis of HRP in vivo was also increased in dystrophic muscles. When HRP was administered intravenously, light microscopic examination of the muscles showed that the macromolecular tracer was present not only in the extracellular space but also as intracellular deposits in several dystropic muscle fibers. Ultrastructural examination of these fibers showed HRP to be present in membrane limited bodies of variable size, some of which likely represented secondary lysosomes, located preferentially close to the A-I junction. HRP was also found inside vacuoles which were sometimes in close vicinity to autophagic vacuoles. Primary uptake vesicles containing HRP appeared to originate from the sarcolemma and the transverse tubules. Biochemical determination of lysosomal enzyme activities revealed elevated levels of both cathepsin D and N-acetylglucosaminidase in dystrophic muscles as compared with controls. The results suggest an increased endocytic activity in dystrophic muscles with distribution of exogenous marcromolecular tracers into endocytic vesicles and lysosomal structures. The hypothesis is put forward that endocytic activity constitutes an important mechanism of lysosomal activation in dystrophic muscles.