Pathways involved in fluid phase and adsorptive endocytosis in neuroblastoma

The endocytosis of ricin, horseradish peroxidase (HRP), and a conjugate of ricin-HRP by monolayer cultures of murine neuroblastoma was studied using morphological and biochemical techniques. The binding of (125)I-ricin and (125)I-ricin-HRP to cells at 4 degrees C, as a function of ligand concentration, was a saturable process. The apparent affinity constants, determined at equilibrium, were 2.8 X 10(6) M(-1) for ricin and 1 x 10(6) M(-1) for ricin-HRP. The number of binding sites per cell was 8 x 10(7) and 3 x 10(7) for the lectin and the conjugate, respectively. The binding of (125)I-ricin to monolayers as not proportional to cell density. We found reduced binding at higher cell concentrations, suggesting a decrease in the accessibility of the ligand for the receptor site or fewer sites with increasing cell population. Neuroblastoma cells have an acid-phosphatase-positive network of cisternae and vesicles near the Golgi apparatus (GERL). Ricin-HRP undergoes endocytosis in vesicles and cisternae corresponding to GERL, and in residual bodies (dense bodies). The cellular uptake of ricin-HRP was 100-200 times greater than free HRP and there was no stimulation of fluid phase endocytosis by ricin. When monolayers were exposed to concentrations of native HRP 100-fold that of the conjugate, cellular uptake of peroxidase was comparable, but HRP was localized only in residual bodies and never in elements of GERL. These results support the conclusion that GERL is involved in the adsorptive endocytosis of ricin-HRP, while residual bodies are involved in the bulk uptake of HRP. In addition, the binding, uptake, and possible recycling of (125)I- subunit B (the binding subunit) of ricin and of (125)I-ricin was examined by quantitative electron microscope autoradiography. Both ricin and its binding subunit displayed similar autoradiographic grain distributions at 4 degrees C, and there was no evidence of their breakdown or recycling to the plasma membrane during endocytosis for 2 h.     

Transcellular processing of disulfide- and thioether-linked peroxidase--polylysine conjugates in cultured MDCK epithelial cells

Horseradish peroxidase (HRP) was conjugated to nondegradable polycationic poly(D-lysine) (PDL) through either a thioether (HRP-S-PDL) or a disulfide (HRP-SS-PDL) linkage. The binding and transcytosis of these conjugates was studied in Madin-Darby canine kidney (MDCK) cell monolayers grown on 3-microns microporous polycarbonate filters. Conjugation of HRP to PDL with both linkages markedly increased the binding of this protein onto the cell monolayers. However, an enhancement of the transcellular transport of HRP in both apical-to-basal and basal-to-apical directions was observed only in HRP-SS-PDL, but not in HRP-S-PDL. HRP-SS-PDL transport was inhibited by colchicine and by 4 degrees C incubation. The transport of 14C-sucrose was not affected by the presence of conjugates. These results indicate that the transport of the conjugate across the cell monolayers was due to a transcellular process rather than to any leakage of the cell junction caused by polycations. The disulfide linkage between HRP and PDL was cleaved rapidly at the basal and, to a lesser extent, at the apical surface of the cell. Neuraminidase treatment decreased the binding of the conjugates onto the cell surface, but did not decrease the transcellular transport, suggesting that not all surface-bound conjugates were available for transcytosis. These results demonstrate that disulfide linkages can be cleaved during transcytosis in MDCK cells. The cleavage, however, occurs mostly at the binding site on the cell surface, which may prevent the cellular uptake of the intact conjugate.     

Clathrin-independent pinocytosis is induced in cells overexpressing a temperature-sensitive mutant of dynamin

A stable HeLa cell line expressing a dynamin mutant, dynts, exhibits a temperature-sensitive defect in endocytic clathrin-coated vesicle formation. Dynts carries a point mutation, G273D, corresponding to the Drosophila shibirets1 allele. The ts-defect in receptor-mediated endocytosis shows a rapid onset (< 5 min) and is readily reversible. At the nonpermissive temperature (38 degrees C) HRP uptake is only partially inhibited. Moreover, when cells are held at the nonpermissive temperature, fluid phase uptake fully recovers to wild-type levels within 30 min, while receptor-mediated endocytosis remains inhibited. The residual HRP uptake early after shift to the nonpermissive temperature and the induced HRP uptake that occurs after recovery are insensitive to cytosol acidification under conditions that potently inhibit receptor-mediated endocytosis of Tfn. Together, these results suggest that a dynamin- and clathrin-independent mechanism contributes to the total constitutive pinocytosis in HeLa cells and that dynts cells rapidly and completely compensate for the loss of clathrin- dependent endocytosis by inducing an alternate endocytic pathway.     

Effects of brefeldin A on endocytosis, transcytosis and transport to the Golgi complex in polarized MDCK cells

We have studied the effects of brefeldin A (BFA) on endocytosis and intracellular traffic in polarized MDCK cells by using the galactose-binding protein toxin ricin as a membrane marker and HRP as a marker of fluid phase transport. We found that BFA treatment rapidly increased apical endocytosis of both ricin and HRP, whereas basolateral endocytosis was unaffected, as was endocytosis of HRP in the poorly polarized carcinoma cell lines HEp-2 and T47D. Tubular endosomes were induced by BFA both apically and basolaterally in some MDCK cells, comparable with those seen in HEp-2 and T47D cells. In addition, in MDCK cells, BFA induced formation of small (< 300 nm) vesicles, labeled both after apical and basolateral uptake of HRP, as well as some very large (> 700 nm) vacuoles, which were only labeled when HRP was present in the apical medium. In contrast, neither in MDCK nor in HEp-2 or T47D cells, did BFA have any effect on lysosomal morphology. Moreover, transcytosis in the basolateral-apical direction was stimulated both for HRP and ricin. Other vesicular transport routes were less affected or unaffected by BFA treatment. Two closely related structural analogues of BFA (B16 and B21), unable to produce the changes in Golgi and endosomal morphology seen after BFA treatment in a number of different cell lines, were also unable to mimic the effects of BFA on MDCK cells.     

Pluronic micelles as a tool for low-molecular compound vector delivery into a cell: effect of Staphylococcus aureus enterotoxin B on cell loading with micelle incorporated fluorescent dye.

Micelles of pluronic P85 (poly(oxyethylene)-poly(oxypropylene) block copolymer) are used as microcontainers for in vitro delivery of fluorescein into Jurkat and MDCK cells. In order to target the fluorescein containing micelles into the cell, Staphylococcus aureus enterotoxin B (SEB) is covalently conjugated with a pluronic molecule and the conjugate is incorporated into the micelle content. The incorporation of SEB capable of receptor-mediated endocytosis results in a drastic enhancement of the efficiency of cell loading with the fluorescent dye. This effect is not observed under the conditions (4 degrees C) when endocytosis is abolished.     

Binding sites and endocytosis of heparin and polylysine are changed when the two molecules are given as a complex to Chinese hamster ovary cells

This paper characterizes the complex formed in vitro between polylysine and heparin in the presence of heparin excess, and investigates the interaction of this complex with the surface of Chinese hamster ovary cells. It examines the kinetics of surface binding and cellular uptake of the complex and shows that both processes can be distinguished from those of free heparin and free polylysine. The view that these three ligands bind to different surface sites is further supported by the fact that their interaction with cells is influenced differently by cell detachment with trypsin, detachment with EGTA or exposure to acid pH. Membrane transport of the complex is a saturable process suggestive of receptor-mediated endocytosis. It is, however, less effective than would be expected on the basis of the binding kinetics. Only 40% of the complex bound at 0 degrees C is internalized during a 2 h reincubation period at 37 degrees C, suggesting some degree of uncoupling between binding and endocytosis. These data confirm prior results obtained with methotrexate-polylysine conjugates. We had shown that the addition of heparin to a medium containing a methotrexate-polylysine conjugate leads unexpectedly to a marked cellular uptake of drug conjugate, which is capable of killing cells that are otherwise resistant to free methotrexate (Shen, W.-C. and Ryser, H.J.-P. (1981) Proc. Natl. Acad. Sci. USA 78, 7589-7593). The polylysine X heparin complex is therefore of interest as a potential carrier for intracellular drug delivery through endocytosis.     

Polarity in the transcytotic processing of apical and basal membrane-bound peroxidase-polylysine conjugates in MDCK cells.

A conjugate of horseradish peroxidase (HRP) to poly(L-lysine) (PLL) was used to characterize a non-lysosomal proteolytic compartment in the MDCK Strain I epithelial cell line. This compartment is expressed in a polar fashion, and is capable of degradation of the PLL moiety in the conjugate followed by release of HRP via a basal-to-apical, but not apical-to-basal, transcytotic pathway. This uptake, cleavage, and transport process appears to require approximately 2 hr, as there is a 2 hr lag-time between conjugate administration to the basal surface and HRP release to the apical medium. Monensin (10 microM) failed to inhibit this process, indicating that participation of the trans-Golgi network (TGN) in the trafficking of internalized conjugate is not the rate-determining step. Inhibition of HRP transport was found to be elicited by 50 micrograms/ml leupeptin, but only when applied to the basal surface. Brief trypsinization of either the basal or apical surfaces of cells preloaded with HRP conjugate showed no appreciable inhibitory effect on the apical release of HRP, indicating that an intracellular compartment rather than surface-bound enzymes is responsible for the degradation of the PLL moiety in the conjugate. Our results demonstrate the presence of an intracellular proteolytic compartment which is accessible in the basal-to-apical, but not apical-to-basal, transport pathway; and this compartment can be exploited for the transcytosis of membrane-bound molecules.     

Visualization of the binding, endocytosis, and transcytosis of low- density lipoprotein in the arterial endothelium in situ

We investigated the interaction and transport of low-density lipoprotein (LDL) through the arterial endothelium in rat aorta and coronary artery, by perfusing in situ native, untagged human, and rat LDL. The latter was rendered electron-opaque after it interacted with the endothelial cell and was subsequently fixed within tissue. We achieved LDL electron-opacity by an improved fixation procedure using 3,3'-diaminobenzidine, and mordanting with tannic acid. The unequivocal identification of LDL was implemented by reacting immunocytochemically the perfused LDL with anti LDL-horseradish peroxidase conjugate. Results indicate that LDL is taken up and internalized through two parallel compartmented routes. (a) A relatively small amount of LDL is taken up by endocytosis via: (i) a receptor-mediated process (adsorptive endocytosis) that involved coated pits/vesicles, and endosomes, and, probably, (ii) a receptor-independent process (fluid endocytosis) carried out by a fraction of plasmalemmal vesicles. Both mechanisms bringing LDL to lysosomes supply cholesterol to the endothelial cell itself. (b) Most circulating LDL is transported across the endothelial cell by transcytosis via plasmalemmal vesicles which deliver LDL to the other cells of the vessel wall. Endocytosis is not enhanced by increasing LDL concentration, but the receptor-mediated internalization decreases at low temperature. Transcytosis is less modified by low temperature but is remarkably augmented at high concentration of LDL. While the endocytosis of homologous (rat) LDL is markedly more pronounced than that of heterologous (human) LDL, both types of LDL are similarly transported by transcytosis. These results indicate that the arterial endothelium possesses a dual mechanism for handling circulating LDL: by a high affinity process, endocytosis secures the endothelial cells' need for cholesterol; by a low-affinity nonsaturable uptake process, transcytosis supplies cholesterol to the other cells of the vascular wall, and can monitor an excessive accumulation of plasma LDL. Since in most of our experiments we used LDL concentrations above those found in normal rats, we presume that at low LDL concentrations saturable high-affinity uptake would be enhanced in relation to nonsaturable pathways.     

Receptor-mediated endocytosis of alpha 2-macroglobulin and transferrin in rat caput epididymal epithelial cells in vitro

The endocytic activity of epithelial cells from the rat epididymis in vitro has been examined by following the uptake of tracer compounds conjugated to proteins. Transferrin-gold and alpha 2-macroglobulin-gold were taken up initially in coated pits, internalized and sequestered into tubular-vesicular structures, multivesicular bodies and, in the case of alpha 2-macroglobulin, into lysosomes. Uptake could be prevented by an excess of unlabeled protein. Studies using 125I-alpha 2-macroglobulin and 125I-transferrin also showed that the uptake of these proteins was specific and could be displaced with increasing amounts of unlabeled protein. In addition, binding of 125I-transferrin to cells was saturable at 4 degrees C. These studies indicate that transferrin and alpha 2-macroglobulin are taken up by receptor-mediated endocytosis. In contrast, a fluid phase marker, bovine serum albumin-gold (BSA-gold), was initially taken up predominantly in uncoated caveolae rather than coated pits, and could not be displaced with excess BSA. By virtue of their charge, polycationized ferritin and unlabeled colloidal gold were taken up and internalized by adsorptive endocytosis, a pathway which is similar to fluid phase endocytosis. The uptake and internalization of alpha 2-macroglobulin and transferrin differed in a number of respects. Uptake and internalization of alpha 2-macroglobulin but not of transferrin was dependent on extracellular calcium. Only alpha 2-macroglobulin was transferred into lysosomes, whereas transferrin was recycled to the cell surface. Although the proton ionophore, monensin, and the transglutaminase inhibitor, dansylcadaverine, did not stop uptake and internalization of either alpha 2-macroglobulin or transferrin, they did prevent the transfer of alpha 2-macroglobulin to lysosomes.     

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°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.     

Endocytosis, intracellular transport, and turnover of anionic and cationic proteins in cultured mouse peritoneal macrophages

It was previously shown that cultured mouse peritoneal macrophages ingest anionic derivatives of horseradish peroxidase (HRP) and ferritin by fluid-phase endocytosis and accumulate them in lysosomes. Cationic derivatives were taken up by adsorptive endocytosis and transported to lysosomes but subsequently appeared also in stacked cisternae, tubules, and vesicles of the Golgi complex. In the present investigation, the effect of molecular net charge on the rate of cellular inactivation of a protein was studied. The results demonstrate that anionized HRP was inactivated at a higher initial rate than cationized HRP. This is in agreement with the finding that the cationic protein partly escaped from the digestive compartment of the cells, that means the lysosomes. The effects of phorbol myristate acetate (PMA)--a diterpene ester and a tumor promoter--and monensin--a carboxylic ionophore and a perturbant of the Golgi complex--on fluid-phase endocytosis of HRP and intracellular transport of cationized ferritin (CF) were also studied. PMA stimulated fluid-phase endocytosis of HRP but did not interfere with transport of CF to the Golgi complex. Contrarily, monensin inhibited uptake of HRP and almost totally blocked transport of CF to the Golgi complex. The findings support the idea that membrane and content of endocytic vesicles are treated separately. The content is emptied into lysosomes where macromolecular material normally is degraded. The membrane becomes part of the lysosomal envelope in connection with endocytic vesicle-lysosome fusion. Subsequently, membrane patches are detached from the lysosomes and reutilized. This is at least partly mediated via the Golgi complex and particularly its tubular and vesicular parts. Since the cationic tracers do not bind to the membrane in a stable way, it is not possible to extend the conclusions to individual membrane constituents.     

Intracellular delivery of an antisense?oligonucleotide via endocytosis of a G protein-coupled receptor

Gastrin-releasing peptide receptor (GRPR), a member of the G protein-coupled receptor superfamily, has been utilized for receptor-mediated targeting of imaging and therapeutic agents; here we extend its use to oligonucleotide delivery. A splice-shifting antisense oligonucleotide was conjugated to a bombesin (BBN) peptide, and its intracellular delivery was tested in GRPR expressing PC3 cells stably transfected with a luciferase gene interrupted by an abnormally spliced intron. The BBN-conjugate produced significantly higher luciferase expression compared to unmodified oligonucleotide, and this increase was reversed by excess BBN peptide. Kinetic studies revealed a combination of saturable, receptor-mediated endocytosis and non-saturable pinocytosis for uptake of the conjugate. The K_m value for saturable uptake was similar to the EC₅₀ value for the pharmacological response, indicating that receptor-mediated endocytosis was a primary contributor to the response. Use of pharmacological and molecular inhibitors of endocytosis showed that the conjugate utilized a clathrin-, actin- and dynamin-dependent pathway to enter PC3 cells. The BBN-conjugate partially localized in endomembrane vesicles that were associated with Rab7 or Rab9, demonstrating that it was transported to late endosomes and the trans-golgi network. These observations suggest that the BBN-oligonucleotide conjugate enters cells via a process of GRPR mediated endocytosis followed by trafficking to deep endomembrane compartments.     

Effects of prolonged exposure to ammonia on fluid-phase,receptor-mediated,and adsorptive (non specific) endocytosis in cultured neuroblastoma cells

Summary The effect of prolonged exposure to ammonia on fluid-phase, receptor-mediated, and adsorptive (non specific) endocytosis in cultured neuroblastoma (Neuro-2a) cells were studied using fluorescein-labeled dextran, concanavalin A conjugated with fluorescein isothiocyanate, and cationized ferritin as tracers. Ammonia treatment increased the rate of endocytosis of cationized ferritin as well as the number of cell elements involved in the process. Moreover, the number of cytoplasmic components containing acid phosphatase activity was also found to increase following ammonia treatment. In contrast, flow-cytometric analyses showed that, under experimental conditions, exposure to ammonia did not alter the intralysosomal pH and had little effect on the fluid-phase and receptor-mediated endocytosis of fluorescein-labeled dextran and concanavalin-A fluorocrome, respectively.     

Enhanced intracellular delivery of methotrexate by a receptor-mediated process

In the present investigation we have described a method of enhancing the uptake of methotrexate by macrophages. This enhanced uptake was mediated by endocytosis through the "scavenger receptor" system which recognized maleylated bovine serum albumin. Experimental evidence showed that macrophages internalized methotrexate coupled to maleylated bovine serum albumin through a saturable process at 37 degrees C leading to an eightfold higher concentration of cell-associated methotrexate compared to the free drug. Following uptake, the drug conjugate was degraded in the lysosomes leading to intracellular release of a pharmacologically active form of methotrexate. When administered to macrophages infected with Leishmania mexicana amazonensis, the drug conjugate could eliminate the intracellular amastigotes more efficiently than the free drug. The leishmanicidal effect of the drug conjugate was inhibited in the presence of excess maleylated bovine serum albumin and lysosomal inhibitors such as chloroquine and monensin. Addition of folinic acid to the medium also prevented the elimination of the amastigotes by the drug conjugate. These results suggested that the scavenger receptor-mediated endocytosis of the drug conjugate led to enhanced transport and intracellular release of a pharmacologically active form of methotrexate resulting in more efficient killing of the amastigotes compared to the free drug. This modality of delivering drugs selectively to macrophages might have utility in the chemotherapy of macrophage-associated disorders in general.     

A quantitative analysis of the endocytic pathway in baby hamster kidney cells

A morphological analysis of the compartments of the endocytic pathway in baby hamster kidney (BHK) cells has been made using the fluid-phase marker horseradish peroxidase (HRP). The endocytic structures labeled after increasing times of endocytosis have been identified and their volume and surface densities measured. In the first 2 min of HRP uptake the volume density of the labeled structures increased rapidly and thereafter remained constant for the next 13-18 min. This plateau represents the volume density of endosome organelles and accounts for 0.65% of the cytoplasmic volume (or 6.8 microns 3 per cell). The labeled structures consist of tubular-cisternal elements which are frequently observed in continuity with 300-400 nm vesicles. After 15-20 min of internalization the volume density of HRP-labeled structures again increased rapidly and reached a second plateau between 30 and 60 min of labeling. This second increase corresponded to detectable levels of HRP reaching later, acid phosphatase (AcPase)-reactive compartments. These structures, comprising the prelysosomes and lysosomes, were mostly vesicular and collectively accounted for 3.5% of the cytoplasmic volume (or 37 microns 3 per cell). The absolute peripheral surface areas of the two classes of organelles (endosomes and prelysosomes/lysosomes) were estimated to be 430 and 370 microns 2 per cell, respectively. The volume of fluid internalized in the first 2 min of uptake was five- to sevenfold less than the volume of the compartment labeled in this time. To account for these results we propose that, after uptake from the cell surface, HRP is delivered to, and diluted in, endosomes that are preexisting organelles initially devoid of the marker. With increasing times of endocytosis the concentration of HRP in the early endosomes increases, as more of the marker enters this compartment. An elevation in HRP concentration in endosomes during the early time points was shown directly using anti- HRP antibodies and colloidal gold on cryosections. The stereological values given in the present study, in combination with earlier studies, provide a minimum estimate for both the total surface area of membranes and the rate of membrane synthesis in a BHK cell.     

Effects of prolonged exposure to ammonia on fluid-phase, receptor-mediated, and adsorptive (non specific) endocytosis in cultured neuroblastoma cells. A flow-cytometry and cytochemical study

The effect of prolonged exposure to ammonia on fluid-phase, receptor-mediated, and adsorptive (non specific) endocytosis in cultured neuroblastoma (Neuro-2a) cells were studied using fluorescein-labeled dextran, concanavalin A conjugated with fluorescein isothiocyanate, and cationized ferritin as tracers. Ammonia treatment increased the rate of endocytosis of cationized ferritin as well as the number of cell elements involved in the process. Moreover, the number of cytoplasmic components containing acid phosphatase activity was also found to increase following ammonia treatment. In contrast, flow-cytometric analyses showed that, under experimental conditions, exposure to ammonia did not alter the intralysosomal pH and had little effect on the fluid-phase and receptor-mediated endocytosis of fluorescein-labeled dextran and concanavalin-A fluorocrome, respectively.     

Exchange kinetics and composition of endocytic membranes in terms of plasma membrane constituents: a morphometric study in macrophages

Intracellular membrane traffic, during endocytosis in mouse bone marrow-derived macrophages, was studied quantitatively by morphometric and kinetic analysis. Three functionally different markers were used: Horseradish peroxidase (HRP) served as a fluid-phase (FP) marker (1000 micrograms HRP/ml in the presence of mannan) or as a receptor-mediated (RM) membrane marker (25 micrograms HRP/ml) and, third, plasma membrane (PM) glycoconjugates, enzymatically labeled with [3H]galactose at the cell surface, served as a covalent membrane marker. The cell surface was labeled with [3H]galactose, followed by either FP or by RM uptake of HRP. The kinetics of the intracellular appearance of the markers were measured as the membrane area stained by HRP-reaction product and as the number of autoradiographic grains associated with these membranes. The following compartments were distinguished: PM, coated vesicles (VI), pinosomes or endosomes (VII), secondary lysosomes (VIII), and HRP-negative vesicles (EV). Tubular structures of VII became labeled with HRP only during RM uptake. The markers flowed first into VI and VII, and after 5 min into VIII. EV became labeled with the covalent membrane marker starting from 5 min. The ratio of autoradiographic grain number to HRP-stained membrane area remained constant with time although substantially different for the various compartments, viz. 100% (VI), 50% (VII and EV) and 30% (VIII) as compared to the PM (100%). This indicated that endosomes were only partially derived from internalized PM and that secondary lysosomes contained a substantial pool of PM constituents. The observed kinetics suggested that once every 30 to 40 min the entire PM was internalized, the bulk of which was recycled after 4 min from a prelysosomal compartment(s) leaving only 12 to 20% for recycling via membranes of secondary lysosomes after a residence time of 24 to 33 min.     

Different strategies for formation of pegylated EGF-conjugated PEI/DNA complexes for targeted gene delivery.

With the aim of generating gene delivery systems for tumor targeting, we have synthesized a conjugate consisting of polyethylenimine (PEI) covalently modified with epidermal growth factor (EGF) peptides. Transfection efficiency of the conjugate was evaluated and compared to native PEI in three tumor cell lines: KB epidermoid carcinoma cells, CMT-93 rectum carcinoma cells, and Renca-EGFR renal carcinoma cells. Depending on the tumor cell line, incorporation of EGF resulted in an up to 300-fold increased transfection efficiency. This ligand-mediated enhancement and competition with free EGF strongly suggested uptake of the complexes through the EGF receptor-mediated endocytosis pathway. Shielded particles being crucial for systemic gene delivery, we studied the effect of covalent surface modification of EGF-PEI/DNA complexes with a poly(ethylene glycol) (PEG) derivative. An alternative way for the formation of PEGylated EGF-containing complexes was also evaluated where EGF was projected away from PEI/DNA core complexes through a PEG linker. Both strategies led to shielded particles still able to efficiently transfect tumor cells in a receptor-dependent fashion. These PEGylated EGF-containing complexes were 10- to 100-fold more efficient than PEGylated complexes without EGF.     

Testosterone induces a rapid stimulation of endocytosis,amino acid and hexose transport in mouse kidney cortex

Testosterone induced a rapid (<1 min) stimulation of endocytosis, amino acid and hexose transport, measured by the temperature-sensitive uptake of HRP, ¹⁴C-AIB and ³H-DG, in mouse kidney cortex slices. The hormonal increment in uptake persisted for at least 60–120 min, showed time-, energy-, and Na⁺-dependence, and varied with substrate and testosterone concentration. Testosterone was maximally effective at 10^?8 to 10^?7 M. Peroxidase histochemistry indicated that the hormonal increase in HRP uptake is restricted to proximal tubules. Testosterone was more effective than DHT, whereas cyproterone acetate, androsterone and dexamethasone had little or no stimulating effect on this uptake. Kidney slices from androgen-insensitive $\text{tfm}\text{Y}$ mice did not respond to testosterone. The rapid increase in endocytosis, amino acid and hexose transport may represent a direct, receptor-mediated response of the surface membrane of target cells to testosterone.     

Neoglycoproteins as carriers for receptor-mediated drug targeting in the treatment of experimental visceral leishmaniasis

Methotrexate (MTX) coupled to mannosyl bovine serum albumin (BSA) was taken up efficiently through the mannosyl receptors present on macrophages. Binding experiments indicate that conjugation does not decrease the affinity of the neoglycoprotein for its cell surface receptor. The drug conjugate eliminated intracellular amastigotes of Leishmania donovani in mouse peritoneal macrophages about 100 times more efficiently than free drug on the basis of 50% inhibitory dose. Inhibitory effect of the conjugate was directly proportional to the density of sugar on the neoglycoprotein carrier. Colchicine and monensin, inhibitors of receptor-mediated endocytosis, can prevent the leishmanicidal effect of the conjugate. Antileishmanial effect of the conjugate can be competitively inhibited by mannose-BSA and mannan. In a murine model of experimental visceral leishmaniasis the drug conjugate reduced the spleen parasite burden by more than 85% in a 30-day model whereas the same concentration of free drug caused little effect. These results indicate that MTX-neoglycoprotein conjugate binds specifically to macrophages, and is internalized and degraded in lysosomes releasing the active drug to act on Leishmania parasites. These results also represent the potential for a general approach to intracellular targeting of clinical agents for macrophage-associated disorders.