Monensin inhibits receptor-mediated endocytosis of asialoglycoproteins in rat hepatocytes

Isolated rat liver parenchymal cells incubated in the presence of monensin exhibited a reduced uptake of ¹²⁵I-asialofetuin (¹²⁵I-AF). Binding studies indicated that the effect was due to a rapid reduction in the number of active surface receptors for the asialoglycoprotein. Monensin had no effect on receptor internalization, but apparently interrupted the recycling of receptors back to the cell surface. Monensin also inhibited the degradation of ¹²⁵I-AF previously bound to the cells; this inhibition was probably not due to a direct effect on intralysosomal proteolysis, as no lysosomal accumulation of undegraded ligand could be demonstrated in subcellular fractionation studies by means of sucrose gradients. It is more likely that monensin inhibits transfer of the labelled ligand from endocytic vesicles to lysosomes, as indicated by the accumulation of radioactivity in the former and by the ability of monensin to prevent the normally observed time-dependent increase in the buoyant density of endocytic vesicles. Whereas the effect of monensin on binding and uptake of asialofetuin was reversible, the effect on asialofetuin degradation could not be reversed.     

Intracellular segregation of asialoglycoproteins and their receptor: a prelysosomal event subsequent to dissociation of the ligand-receptor complex

Rat hepatocytes in monolayer culture rapidly internalized asialoglycoproteins and the receptors to which they are bound. Subsequent to endocytosis, the receptor-ligand complex is dissociated within an acidic endosome (Harford, J., K. Bridges, G. Ashwell, and R. D. Klausner, 1983, J. Biol. Chem. 258:3191-3197; Harford, J., A. W. Wolkoff, G. Ashwell, and R. D. Klausner, 1983, J. Cell Biol. 96:1824- 1828). Here we show that addition of the proton ionophore monensin to the cells after dissociation has occurred results in intracellular rebinding of ligand molecules. With increasing time inside the cell, the ability of ligand to reassociate with receptor progressively decreases consistent with a segregation of receptor and ligand. The combination of colchicine and cytochalasin B appears to retard the process of segregation. In contrast, removal of sodium from the medium, while inhibiting degradation of ligand, does not affect the decrease in monensin-mediated rebinding. Nonetheless, both sodium deprivation and treatment with colchicine plus cytochalasin B result in the ligand remaining in a low density, nonlysosomal subcellular fraction. Thus, segregation, like dissociation, appears to occur in a pre-lysosomal endocytic compartment. Perturbation of the endocytic pathway by reduced temperature (18 degrees C) was also explored. Our data are consistent with two temperature-sensitive steps: receptor-ligand dissociation is inhibited and there is an independent temperature-sensitive step involved in delivery of ligand to lysosomes. This second effect was localized as being beyond the point in the pathway sensitive to sodium deprivation.     

Monensin inhibits ligand dissociation only transiently and partially and distinguishes two galactosyl receptor pathways in isolated rat hepatocytes

Monensin has been shown to inhibit the dissociation of internalized asialoorosomucoid (ASOR) from galactosyl (Gal) receptors in hepatocytes (Harford et al., J. Cell. Biol., 96:1824, 1983). Examination of the long-term kinetics of dissociation of a single round of surface-bound 125I-ASOR in the presence of monensin revealed, however, that dissociation resumed after a lag of 30-40 min. Dissociation proceeded slowly with apparent first order kinetics (k = 0.006-0.022 min-1) and reached a plateau after 4 h, both in freshly isolated cells in suspension and in cells cultured for 24 h. Only a portion of the ligand bound to surface Gal receptors was capable of dissociating. The degree of dissociation was correlated with the expression of a subpopulation of receptors we have recently designated as state 1 Gal receptors (Weigel et al., Biochem. Biophys. Res. Commun. 140:43, 1986). The recovery and dissociation of a portion of 125I-ASOR-receptor complexes after the lag period is not due to a depletion of monensin, since a second addition of the drug has no affect once dissociation resumes. Furthermore, as assessed by the accumulation of the fluorescent dye acridine orange, cells have not recovered the ability to acidify intracellular compartments during the time that dissociation occurs. The results support a model for the hepatic Gal receptor system, in which there are two functionally different receptor populations, recycling pathways, and ligand processing pathways. Monensin blocks dissociation of 125I-ASOR from receptors in the major pathway completely. In the minor pathway dissociation proceeds to completion only after a lag. In this minor pathway monensin appears to temporarily delay a maturation or translocation process that must occur prior to dissociation. We conclude that the observed dissociation in the presence of monensin cannot be mediated by low pH, or by pH or pNa gradients.     

Monensin inhibits the binding of 3H-flunitrazepam to and reveals the intracellular passage of GABAA/benzodiazepine receptor.

Effects of monensin were examined on the intracellular processing of the GABAA/benzodiazepine receptor (GABAA/BZDR) in neuron cultures derived from embryonic chicken brain, using 3H-flunitrazepam as the probe for the benzodiazepine modulator site on the receptor. Incubation of cultures with 0.1 or 1 microM monensin for 3 h blocked the binding of 3H-flunitrazepam by about 18%. Loss of ligand binding was due to a reduction in the number of binding sites, with no significant changes in receptor affinity. The general cellular protein synthesis and glycosylation in the cells were inhibited by 26% and 56%, respectively, in the presence of 1 microM monensin, as detected by assaying the incorporation of 3H-leucine and 3H-galactose. In contrast, an increase was observed for mannose incorporation by the cultures in the presence of the drug. Moreover, the results from in situ trypsinization of the cultures following monensin treatment showed that monensin did not alter the distribution of intracellular and surface receptors. The data suggest that monensin induces the down-regulation of GABAA/BZDR by generating abnormal glycosylation of the receptor and interrupting its transport within the Golgi apparatus, as well as from the Golgi apparatus to the intracellular pool and cell membrane. The galactosylation of receptor proteins may be important for the maturation of the receptor.     

Monensin and FCCP inhibit the intracellular transport of alphavirus membrane glycoproteins

Temperature-sensitive mutants of semliki forest virus (SFV) and sindbis virus (SIN) were used to study the intracellular transport of virus membrane glycoproteins in infected chicken embryo fibroblasts. When antisera against purified glycoproteins and (125)I- labeled protein A from staphylococcus aureus were used only small amounts of virus glycoproteins were detected at the surface of SFV ts-1 and SIN Ts-10 infected cells incubated at the restrictive temperature (39 degrees C). When the mutant-infected cells were shifted to the permissive temperature (28 degrees C), in the presence of cycloheximide, increasing amounts of virus glycoproteins appeared at the cell surface from 20 to 80 min after the shift. Both monensin (10muM) and carbonylcyanide-p- trifluoromethoxyphenylhydrazone (FCCP; 10-20 muM) inhibited the appearance of virus membrane glycoproteins at the cell surface. Vinblastine sulfate (10 μg/ml) inhibited the transport by approximately 50 percent, whereas cytochalasin B (1 μg/ml) had only a marginal effect. Intracellular distribution of virus glycoproteins in the mutant-infected cells was visualized in double-fluorescence studies using lectins as markers for endoplasmic reticulum and Golgi apparatus. At 39 degrees C, the virus membrane glycoproteins were located at the endoplasmic reticulum, whereas after shift to 28 degrees C, a bright juxtanuclear reticular fluorescence was seen in the location of the Golgi apparatus. In the presence of monensin, the virus glycoproteins could migrate to the Golgi apparatus, although transport to the cell surface did not take place. When the shift was carried out in the presence of FCCP, negligible fluorescence was seen in the Golgi apparatus and the glycoproteins apparently remained in the rough endoplasmic reticulum. A rapid inhibition in the accumulation of virus glycoproteins at the cell surface was obtained when FCCP was added during the active transport period, whereas with monensin there was a delay of approximately 10 min. These results suggest a similar intracellular pathway in the maturation of both plasma membrane and secretory glycoproteins.     

The relationship between autophagy and the intracellular degradation of asialoglycoproteins in cultured rat hepatocytes

The relationship between autophagy and the intracellular distribution of endocytosed asialoorosomucoid was studied in cultured rat hepatocytes. Overt autophagy was induced by shifting the cells to a minimal salt medium. Incubation in minimal salt medium led to the formation of buoyant lysosomes at the expense of denser lysosomes manifested as a dual distribution of these organelles in Nycodenz gradients. Asialoorosomucoid was labeled with 125I-tyramine cellobiose. The labeled degradation products formed from this ligand are trapped at the site of degradation and may therefore serve as markers for the subgroup of lysosomes involved in the degradation. In control cells the degradation of the ligand was initiated in a light prelysosomal compartment and continued in denser lysosomes. In cells with high autophagic activity, the degradation of labeled asialoorosomucoid took place exclusively in a buoyant group of lysosomes. These results suggest that degradation of endocytosed ligand takes place in the same secondary lysosomes as substrate sequestered by autophagic mechanisms. These light lysosomes represent a subgroup of active lysosomes which are gradually recruited from dense bodies. Data are also presented that indicate that insulin may prevent the change in buoyant density brought about by incubation in deficient medium.     

Monensin inhibits the first cellular movements in early chick embryo

Summary In early chick blastoderm at stage XIII, the interaction of the hypoblast with the epiblast triggers on the epiblast the first extensive cellular migrations, which result in formation of the primitive streak, the source of the axial mesoderm. During this period, extracellular material (ECM) is secreted and assembled into an organized network in the extracellular spaces and is implicated in regulating the behaviour of the cells that contact it. The first cellular migrations and inductions are inhibited when early chick blastoderm is treated with the glycosylation-perturbing ionophore monensin. The difference in amount and in organization of ECM between monensin-treated embryos and control embryos is striking. Even blastoderms at stage X, which are essentially free of ECM, show extensive ECM after monensin treatment. Monensin produces a substantial change in the polypeptide pattern with the induction or marked accentuation of multiple charged species (isoforms) of polypeptides different from those present in the control embryos. The interference of monensin with the migration and induction mechanisms is permanent in embryos before the primitive streak (PS) stage, and it seems that the respective signals or the sensitivity of the epiblast/hypoblast cells to them must be very stage specific. Monensin-treated embryos probably secrete abnormal ECM that does not provide the proper conditions for the hypoblast to interact with the epiblast cells.     

Antibody-induced receptor loss. Different fates for asialoglycoproteins and the asialoglycoprotein receptor in HepG2 cells

The human asialoglycoprotein receptor (ASGP-R) is a membrane glycoprotein which participates in receptor-mediated endocytosis and delivery of its ligands to lysosomes for degradation. In order to examine the pathways and mechanisms responsible for the turnover and degradation of the ASGP-R we have followed the fate of the ASGP-R in HepG2 cells during exposure to anti-receptor antibody as well as inhibitors of lysosomal processing and receptor recycling. Incubation of cells at 37 degrees C with anti-ASGP-R antibody results in the rapid (t 1/2 30 min) loss of mature 46,000-Da ASGP-R (control, t 1/2 20 h). This process requires whole IgG, since Fab fragments do not induce loss of receptor. Furthermore, this antibody-induced loss is specific, since incubation with antibody to the transferrin receptor does not alter cellular ASGP-R content. Of note, weak bases (e.g. primaquine) abrogate this antibody-induced loss of ASGP-R. Inhibitors of lysosomal proteases (EC64 and leupeptin) do not alter this antibody-mediated loss. Furthermore, this effect occurs at 18 degrees C, a temperature at which delivery of ligand to the lysosome is blocked. Thus, the present observations suggest a unique pathway for antibody-induced ASGP-R loss which is distinct from the pathway of lysosomal delivery of ligand.     

The large intracellular pool of asialoglycoprotein receptors functions during the endocytosis of asialoglycoproteins by isolated rat hepatocytes

The function of intracellular asialoglycoprotein receptors during the endocytosis of asialo-orosomucoid in isolated hepatocytes was assessed by following changes in the occupancy of intracellular receptors. Unoccupied total cellular (inside and surface) or surface receptors were quantified at 0 degrees C by the binding of 125I-asialo-orosomucoid in the presence or absence, respectively, of digitonin. Freshly isolated cells had about 17% of their total receptors on the surface. After incubation at 37 degrees C, the receptor distribution changed to 25 to 50% on the cell surface and 50 to 75% inside the cell. At 37 degrees C, the average total number of receptors/cell was 4.5 x 10(5). Dissociation constants, determined from equilibrium binding studies in the presence or absence of digitonin to assess total or surface receptors, were identical (5.4 +/- 1.4 and 5.6 +/- 1.1 x 10(-9) M, respectively). In the presence of asialo-orosomucoid at 37 degrees C, there was both a time- and a concentration-dependent decrease in surface and intracellular receptor activity. This receptor activity decrease was reversed by removing asialo-orosomucoid from the medium or by washing the digitonin-permeabilized cells with ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid prior to quantification of receptor activity. Within 1 to 2 h in the presence of excess asialo-orosomucoid, a steady state was attained in which approximately 70% of the intracellular receptors were occupied. The kinetics of receptor activity recovery on the cell surface after internalization of a pulse of ligand is different than the rate of recovery of internal receptor activity. The results suggest that all of the internal asialoglycoprotein receptors are functional and participate during endocytosis. Internal receptors may be functionally equivalent to those on the surface or they may serve a reservoir or routing function for internalized ligand.     

Monensin inhibits the processing of herpes simplex virus glycoproteins, their transport to the cell surface, and the egress of virions from infected cells.

HEp-2 cells or Vero cells infected with herpes simplex virus type 1 were exposed to the ionophore monensin, which is thought to block the transit of membrane vesicles from the Golgi apparatus to the cell surface. We found that yields of extracellular virus were reduced to less than 0.5% of control values by 0.2 microM monensin under conditions that permitted accumulation of cell-associated infectious virus at about 20% of control values. Viral protein synthesis was not inhibited by monensin, whereas late stages in the post-translational processing of the viral glycoproteins were blocked. The transport of viral glycoproteins to the cell surface was also blocked by monensin. Although the assembly of nucleocapsids appeared to be somewhat inhibited in monensin-treated cells, electron microscopy revealed that nucleocapsids were enveloped to yield virions, and electrophoretic analyses showed that the isolated virions contained immature forms of the envelope glycoproteins. Most of the virions which were assembled in monensin-treated cells accumulated in large intracytoplasmic vacuoles, whereas most of the virions produced by and associated with untreated cells were found attached to the cell surface. Our results implicate the Golgi apparatus in the egress of herpes simplex virus from infected cells and also suggest that complete processing of the viral envelope glycoproteins is not essential for nucleocapsid envelopment or for virion infectivity.     

Monensin inhibits synthesis of proteoglycan, but not of hyaluronate, in chondrocytes

Monensin (10nm-1mum) inhibited the incorporation of [(35)S]sulphate and [(3)H]glucosamine into proteoglycans by rat chondrosarcoma cells, but the incorporation of [(3)H]glucosamine into hyaluronate was unaffected. The results suggest that hyaluronate synthesis occurs in a cell compartment separate from chondroitin sulphate synthesis.     

Molecular size of the hepatic receptor for asialoglycoproteins determined in situ by radiation inactivation

Radiation inactivation was used to determine the functional molecular size of the rat liver membrane protein which binds desialylated glycoproteins. Purified plasma membranes from rat liver were irradiated with high energy electrons from a linear accelerator and then assayed for 125I-asialo-orosomucoid binding activity. Target size analysis of the data revealed that increasing doses of ionizing radiation from 1-48 megarads resulted in a monoexponential decay in binding activity due to a decrease in the number of available binding sites; dissociation and binding affinity were unaffected. The molecular weight of the rat binding protein, determined in situ by target analysis, was 104,000 +/- 17,000; that of the rabbit binding protein was 109,000 +/- 5,000. Comparison of the value obtained by irradiation of the intact rat plasma membrane with that of the purified receptor revealed the latter to have an apparent molecular weight of 148,000 +/- 16,000. Evidence is presented to indicate that the observed increase in target size was a response to the presence of Triton X-100 used in the solubilization and assay procedure. In contrast to the size of the ligand binding functional unit, the antireceptor antibody binding site was estimated to be 30,000 +/- 2,000.     

Monensin inhibits the conversion of proalbumin to serum albumin in cultured rat hepatocytes

Effects of monensin, a carboxylic ionophore, on intracellular transport of albumin were studied in primary cultured rat hepatocytes. The lag time after which newly synthesized albumin first appeared in medium was 10 min in the control cells, while it was prolonged to 40 min in the monensin-treated cells. In addition, this inhibition of secretion by monensin was accompanied by an intracellular accumulation of proalbumin. The results strongly suggest that monensin arrests the intracellular transport of proalbumin before the site where its conversion takes place.     

Monensin inhibits recycling of macrophage mannose-glycoprotein receptors and ligand delivery to lysosomes.

Binding studies with cells that had been permeabilized with saponin indicate that alveolar macrophages have an intracellular pool of mannose-specific binding sites which is about 4-fold greater than the cell surface pool. Monensin, a carboxylic ionophore which mediates proton movement across membranes, has no effect on binding of ligand to macrophages but blocks receptor-mediated uptake of 125I-labelled beta-glucuronidase. Inhibition of uptake was concentration- and time-dependent. Internalization of receptor-bound ligand, after warming to 37 degrees C, was unaffected by monensin. Moreover, internalization of ligand in the presence of monensin resulted in an intracellular accumulation of receptor-ligand complexes. The monensin effect was not dependent on the presence of ligand, since incubation of macrophages with monensin at 37 degrees C without ligand resulted in a substantial decrease in cell-surface binding activity. However, total binding activity, measured in the presence of saponin, was much less affected by monensin treatment. Removal of monensin followed by a brief incubation at pH 6.0 and 37 degrees C, restored both cell-surface binding and uptake activity. Fractionation experiments indicate that ligands enter a low-density (endosomal) fraction within the first few minutes of uptake, and within 20 min transfer to the lysosomal fraction has occurred. Monensin blocks the transfer from endosomal to lysosomal fraction. Lysosomal pH, as measured by the fluorescein-dextran method, was increased by monensin in the same concentration range that blocked ligand uptake. The results indicate that monensin blockade of receptor-mediated endocytosis of mannose-terminated ligands by macrophages is due to entrapment of receptor-ligand complexes and probably receptors in the pre-lysosomal compartment. The inhibition is linked with an increase in the pH of acid intracellular vesicles.     

Monensin inhibits the expression of sucrase-isomaltase in Caco-2 cells at the mRNA level

Using L-[35S]methionine labeling, SDS-PAGE and Northern blot analysis of sucrase-isomaltase mRNA, two different concentrations of monensin were used to delineate in Caco-2 cells the effect of the drug on the conversion of the high mannose to the complex form of sucrase-isomaltase from its dual effect on the biosynthesis of the enzyme and on the rate of glucose consumption. At 0.1 microM the drug has no effect on the rate of glucose consumption and, although it inhibits the conversion of the high mannose to the complex form of the enzyme, it has no effect on the level of sucrase-isomaltase mRNA and on the amount of neosynthesized enzyme. At 1 microM, in addition to its inhibiting effect on the maturation of the enzyme, monensin provokes concomitantly an increase in the rate of glucose consumption and a decrease in the level of sucrase-isomaltase mRNA and in the amount of neosynthesized enzyme. All these effects are reversible within 48 h after removal of the drug.     

Monensin and bile protein secretion. A tool to investigate hepatocyte intracellular vesicular polarity

Bile derived from monensin treated bile-fistula rats was analysed by SDS-polyacrylamide gels. Differences in proteins were observed for bands presumptively corresponding to transferrin, secretory-IgA and free secretory components after treatment with the ionophore. The results show that the secretion of proteins in bile is differentiated and asyncronous, confirming a polarity of the hepatocyte subcellular compartments.     

Hydroxyurea inhibits intracellular Toxoplasma gondii multiplication

Tachyzoites of Toxoplasma gondii multiply within the parasitophorous vacuole (PV) until the lysis of the host cell. This study was undertaken to evaluate the effect of hydroxyurea (a specific drug that arrests cell division at G1/S phase) on the multiplication of T. gondii tachyzoites in infected Vero cells. Infected host cells were treated with hydroxyurea for periods varying from 5 to 48 h, and the survival and morphology of the parasite were determined. Hydroxyurea arrested intracellular T. gondii multiplication in all periods tested. After 48 h of incubation with hydroxyurea, intracellular parasites were not easily observed in Vero cells. Ultrastructural observations showed that infected host cells treated with hydroxyurea for 24 h or more presented disrupted intracellular parasites within the PV. However, the host cells exhibited a normal morphology. Our observations suggest that hydroxyurea was able to interfere with the cycle of the intracellular parasite, leading to the complete destruction of the T. gondii without affecting the host cells.     

Pigment epithelium-derived factor inhibits angiogenesis via regulated intracellular proteolysis of vascular endothelial growth factor receptor 1

Pigment epithelium-derived factor (PEDF) has been identified as one of the most potent of endogenous negative regulators of blood vessel growth in the body. Here we report that PEDF is able to inhibit growth factor-induced angiogenesis in microvascular endothelial cells through a novel pathway requiring cleavage and intracellular translocation of the transmembrane domain of the VEGFR-1. Analysis of the subcellular distribution of VEGFR-1 revealed the appearance of an 80-kDa C-terminal domain in the cytosol of cells treated with VEGF and PEDF that correlated with a decrease of the full-length receptor in the nuclear and cytoskeletal fractions. This regulated intramembrane proteolysis is dependent on gamma-secretase because inhibition of gamma-secretase abolished the inhibitory effect of PEDF on VEGF-induced angiogenesis as well as VEGFR-1 cleavage. The addition of PEDF to microvascular endothelial cells significantly increases gamma-secretase activity even in the absence of VEGF, showing that VEGF binding to VEGF-R1 is essential for substrate availability. This increase in activity was associated with translocation of presenilin 1 from the perinuclear region to the cell membrane. PEDF was also able to inhibit VEGF-induced phosphorylation of VEGFR-1. Taken together we have identified two novel pathways by which PEDF inhibits VEGF-induced angiogenesis: regulated intramembrane proteolysis and inhibition of phosphorylation. This confirms the importance of PEDF and VEGFR-1 in the negative regulation of angiogenesis.