Cadmium uptake by cells of renal origin

We compared the ability of rat glomerular mesangial cells and LLC-PK1 cells to take up Cd2+ from solution. The former are smooth muscle-like cells of mesenchymal origin, the latter an established line of proximal tubular epithelium. Both cells, as well as primary glomerular epithelia, accumulated Cd2+ against a concentration gradient in a time-dependent manner. Uptake by mesangial cells obeyed a Michaelis model with an apparent Km of 19 microM and could be described by an initial rapid step of surface binding followed by rate-limiting internalization. In contrast, uptake by LLC-PK1 cells was non-saturable under accessible concentrations of Cd2+ and internalization was not a necessary consequence of association with the cell surface. In several other cell types, Cd2+ uptake has been shown to be inhibited by blockage of cell-surface sulfhydryl groups. In contrast, uptake by neither mesangial nor LLC-PK1 cells was depressed by N-ethylmaleimide, which actually enhanced the surface binding and to a lesser extent the uptake by the LLC-PK1 cell line. Neither depended on metabolic energy for uptake or utilized Ca2+ channels. The internalization process was temperature dependent and was obliterated at 2 degrees C. In mesangial cells, this allowed direct observation of the internalization event from a presaturated surface pool. The rate of this process was consistent with the Vmax calculated from the Michaelis model. Surface binding and uptake were decreased by binding of Cd2+ to serum proteins and albumin and were much less dependent on the presence of low molecular weight components of serum. Therefore, these cells may be especially sensitive to Cd2+ at concentrations encountered in vivo because of the low protein content of the plasma ultrafiltrate. Surface binding of Cd2+ to mesangial cells was suppressed by competing divalent ions following the order of the Irving-Williams series (Mn less than Co less than Ni less than Cu greater than Zn), although Zn2+ showed the greatest effect on internalization. In LLC-PK1 cells, Zn2+ and Cu2+ were both effective in decreasing Cd2+ uptake. We conclude that Cd2+ uptake by the tubular epithelial cells is rapid and independent of specific cell surface interactions, whereas uptake by rat mesangial cells follows binding to a specific surface ligand saturating at about 1.5 x 10(7) copies/cell. In both types of cells the uptake appears quite specific for Cd2+ and shows some cross-reactivity with other metal cations explicable by competitive ligand binding.     

Stable plasma membrane levels of hCTR1 mediate cellular copper uptake

The human copper transporter 1 (hCtr1), when heterologously overexpressed in insect cells, mediates saturable Cu uptake. In mammalian expression systems, a rapid Cu-dependent internalization of hCtr1 has been reported in cells that overexpress epitope-tagged hCtr1 when exposed to Cu in the external medium. This finding led to the suggestion that such internalization may be a step in the hCtr1 transmembrane Cu transport mechanism. We have demonstrated that preincubation in Cu-containing media of sf9 cells stably expressing hCtr1 has no effect on the initial rate of Cu transport. Furthermore, Western blot analyses of fractionated sf9 cell membranes show no evidence of a regulatory Cu-dependent internalization from the plasma membrane. In similar studies on human embryonic kidney (HEK) 293 cells, we showed that incubation with Cu does not alter the initial rate of Cu uptake mediated by endogenous levels of hCtr1 compared with untreated cells. Confirmation that hCtr1 mediates this transport is provided by specific small interfering RNA-dependent decreases in hCtr1 protein levels and in Cu transport rates. Western blot analysis and confocal microscopy of human embryonic kidney 293 cells showed that the majority of hCtr1 protein is localized at the plasma membrane and no significant internalization is detected upon Cu treatment. We concluded that internalization of hCtr1 is not a required step in the transport pathway; we suggest that oligomeric hCtr1 acts as a conventional transporter providing a permeation pathway for Cu through the membrane and that internalization of endogenous hCtr1 in response to elevated extracellular Cu levels does not play a significant regulatory role in Cu homeostasis.     

Ezetimibe blocks the internalization of NPC1L1 and cholesterol in mouse small intestine

The multiple transmembrane protein Niemann-Pick C1 like1 (NPC1L1) is essential for intestinal cholesterol absorption. Ezetimibe binds to NPC1L1 and is a clinically used cholesterol absorption inhibitor. Recent studies in cultured cells have shown that NPC1L1 mediates cholesterol uptake through vesicular endocytosis that can be blocked by ezetimibe. However, how NPC1L1 and ezetimibe work in the small intestine is unknown. In this study, we found that NPC1L1 distributed in enterocytes of villi and transit-amplifying cells of crypts. Acyl-CoA cholesterol acyltransferase 2 (ACAT2), another important protein for cholesterol absorption by providing cholesteryl esters to chylomicrons, was mainly presented in the apical cytoplasm of enterocytes. NPC1L1 and ACAT2 were highly expressed in jejunum and ileum. ACAT1 presented in the Paneth cells of crypts and mesenchymal cells of villi. In the absence of cholesterol, NPC1L1 was localized on the brush border of enterocytes. Dietary cholesterol induced the internalization of NPC1L1 to the subapical layer beneath the brush border and became partially colocalized with the endosome marker Rab11. Ezetimibe blocked the internalization of NPC1L1 and cholesterol and caused their retention in the plasma membrane. This study demonstrates that NPC1L1 mediates cholesterol entering enterocytes through vesicular endocytosis and that ezetimibe blocks this step in vivo.     

UPTAKE OF CADMIUM BY FRESHWATER GREEN ALGAE: EFFECTS OF PH AND AQUATIC HUMIC SUBSTANCES1

The effects of humic substances and low pH on short‐term Cd uptake by Pseudokirchneriella subcapitata (Korshikov) Hindak and Chlamydomonas reinhardtii Dang were investigated under defined exposure conditions. The uptake experiments were run in the presence of either a synthetic organic ligand (nitrilotriacetate) or natural organic ligands (Suwannee River fulvic or humic acid). An ion‐exchange method was used to measure the free Cd²⁺ concentrations in the exposure solutions. At pH 5, measured free Cd²⁺ concentrations agreed with estimations made using the geochemical equilibrium model WHAM, but at pH 7 the model overestimated complexation by both Suwannee River fulvic and humic acids compared with the ion‐exchange measurements. Consistent with the metal internalization step being rate limiting for overall short‐term uptake, intracellular Cd uptake was linear for exposure times less than 20 min at pH 5 or pH 7 for both algal species. After taking into account complexation of Cd in solution, Suwannee River humic substances had no additional effects on cadmium uptake at pH 7, as would be predicted by the free ion model. This absence of effects other than complexation persisted at pH 5, where the tendency of humic substances to adsorb to the algal cell surface is favored. Changes in pH strongly influenced Cd uptake, with the intracellular flux of Cd being at least 20 times lower at pH 5 than at pH 7 for P. subcapitata. Our results support models such as the free ion model or the biotic ligand model, in which humic substances act indirectly on Cd uptake by reducing the bioavailability of Cd by complexation in solution.     

Acute waterborne cadmium uptake in rainbow trout is reduced by dietary calcium carbonate

The effects of elevated dietary calcium (as CaCO3) and acute waterborne Cd exposure (50 microg/l) on whole body uptake, tissue uptake, and internal distribution of newly accumulated Cd, Ca2+, and Na+ in juvenile rainbow trout were examined. Fish were fed with three diets (mg Ca2+/g food): 20 (control), 30 and 60 for 7 days before fluxes were measured with radiotracers. The highest dietary Ca2+ elevation reduced waterborne whole body Ca2+ uptake, but did not protect against inhibition of waterborne Ca2+ uptake by waterborne Cd. Both Ca2+-supplemented diets reduced newly accumulated Ca2+ in the gills in relation to the control treatment, but did not prevent the Cd-inhibiting effect against accumulation of new Ca2+ in most compartments. Fish fed with Ca2+-supplemented diets showed markedly lower rates of whole body uptake and internalization (in some tissues) of waterborne Cd, illustrating that, while dietary Ca2+ supplementation did not protect against the impact of waterborne Cd on waterborne Ca2+ uptake, it did protect against the uptake of Cd. Waterborne Cd had no effect on Na+ fluxes, total Cl-, and in most body compartments, newly accumulated Na+ and total Na+ were also not affected. Dietary supplementation with CaCO3 had the same protective effect as demonstrated by dietary supplementation with CaCl2 in an earlier study. Thus, the reduction of waterborne Cd uptake and internalization by dietary Ca2+ was specifically due to Ca2+ and not to the anion.     

Ligation of Cell Surface Heparan Sulfate Proteoglycans by Antibody-Coated Beads Stimulates Phagocytic Uptake into Epithelial Cells: A Model for Cellular Invasion byNeisseria gonorrhoeae

Binding of a particular opacity outer membrane protein (Opa) ofNeisseria gonorrhoeaeto cell surface heparan sulfate proteoglycans (HSPGs) of epithelial cells results in tight bacterial adherence; however, the role of this ligand–receptor interaction in triggering the subsequent bacterial internalization step is uncertain. Here we have used latex beads coated with HSPG-ligating antibodies as anin vitromodel to study the role of HSPGs in gonococcal uptake into epithelial cells. Beads and gonococci showed the same cell line-specified adherence patterns and increase in phagocytic uptake mediated by serum or purified vitronectin (Vn). Heparitinase digestion as well as antibody competition experiments indicate that a critical level of HSPG ligation is necessary and sufficient to trigger phagocytic uptake into epithelial cells. Vn was found to specifically enhance HSPG-dependent phagocytic uptake while phagocytosis resulting from the ligation of other cell surface receptors was unaffected in the presence of Vn. Pharmacologicial studies with PKC inhibitors suggest a role for PKC in phagocytic uptake of HSPG-ligating beads. The use of drugs impairing cytoskeletal functions indicates that HSPG-dependent phagocytosis requires actin polymerization by a process distinct from receptor-mediated endocytosis.     

Metabolic energy-independent mechanism of internalization for the cell penetrating peptide penetratin

Cellular uptake of vector peptides used for internalization of hydrophilic molecules into cells is known to follow two different pathways: direct translocation of the plasma membrane and internalization by endocytosis followed by release into the cytosol. These pathways differ in their energy dependence. The first does not need metabolic energy while the second requires metabolic energy. Herein we used erythrocytes and plasma membrane vesicles to study membrane perturbations induced by the cell penetrating peptide penetratin. The results show that cell penetrating peptides are able to be internalized by two metabolic energy-independent pathways: direct crossing of the plasma membrane and endocytosis-like mechanisms. The last mechanism involves the induction of membrane negative curvature resulting in invaginations that mimic the endosomal uptake in the absence of ATP. This new mechanism called "physical endocytosis" or "self-induced endocytosis" might explain different data concerning the independence or dependence on metabolic energy during cellular uptake and reveals the autonomous capacity of peptides to induce their internalization.     

Cell type-dependent internalization of the Escherichia coli STb enterotoxin

Previous studies have suggested that internalization of the Escherichia coli STb enterotoxin in human and rat intestinal epithelial cells is involved in STb pathogenesis, but toxin uptake in porcine jejunum epithelium, the in vivo target tissue, still remains elusive. Using flow cytometry, we studied the internalization of fluorescein isothiocyanate-labelled STb in porcine intestinal epithelial IPEC-J2 and murine fibroblast NIH-3T3 cell lines. In contrast to the selective pronase resistance of STb in NIH-3T3 cells at 37 °C, but not at 4 °C, indicative of toxin internalization, most of the toxin was pronase-sensitive at both temperatures in IPEC-J2 cells, indicating reduced uptake, but significant cell surface binding. Actin reorganization is required for STb internalization by NIH-3T3 cells, confirming STb endocytosis in these cells. The toxin receptor, sulfatide, could not explain these internalization differences because both cell lines possessed surface sulfatide and internalized antisulfatide antibodies over time at 37 °C. Inhibition of lipid rafts endocytosis, known to contain sulfatide, with methyl-β-cyclodextrin or genistein, did not influence toxin uptake by either cell line. STb internalization is therefore differentially regulated depending on the cell type, possibly by factors other than sulfatide. Although a small STb fraction could be internalized by porcine intestinal epithelial cells, our findings suggest the ability of STb to induce, from the cell surface, intracellular signalling leading to fluid secretion in porcine intestinal epithelium.     

Slow internalization of human chorionic gonadotropin by cultured granulosa cells

Kinetic studies were performed on two-day cultures of rat ovarian granulosa cells to follow the fate of surface-bound 125I-labeled human chorionic gonadotropin (125I-hCG). Low pH was used to release hCG from its surface receptor, allowing us to distinguish between surface-bound and internalized hormone. Because our results indicated that hormone is lost from the cell surface by dissociation as well as internalization, equations were derived to determine independent rate constants for each process. We calculate that if hormone binding were irreversible, the t 1/2 for internalization would be 8.5 hour. Morphometric studies on the uptake of horseradish peroxidase indicate that the t 1/2 for internalization of bulk membrane in granulosa cells is 55 to 77 minutes. Thus, the rate of uptake of surface-bound hCG appears to be seven to nine times slower than the rate of uptake of bulk plasma membrane, which suggests that the LH/hCG receptor may be selectively excluded from the endocytic vesicles of granulosa cells.     

Changes in 45Ca and 109Cd uptake, membrane potential and cell pH in Saccharomyces cerevisiae provoked by Cd2+

The effect of Cd2+ poisoning of Saccharomyces cerevisiae on 45Ca, 109Cd and [14C]tetraphenylphosphonium (TPP) uptake and cell pH was examined. At Cd2+ concentrations that produced substantial K+ efflux the rates of uptake of 45Ca, 109Cd and [14C]TPP increased progressively during incubation of the cells with Cd2+, and the cell pH was lowered concomitantly. The initial rates of uptake of the divalent cations and of TPP were increased in cells pre-loaded with Cd2+, which shows that stimulation of the ion fluxes was exerted by the Cd2+ that accumulated in the cells. The distribution ratio of TPP between cells and medium, however, was decreased by Cd2+. Although hyperpolarization of the cell membrane by Cd2+ cannot be excluded, it is argued that Cd2+ primarily stimulated divalent cation uptake by increasing the cation permeability of the cell membrane allowing the cations to enter the cells more easily.     

Characteristics of Cd uptake, translocation and accumulation in a novel Cd-accumulating tree, star fruit (Averrhoa carambola L., Oxalidaceae)

Cadmium (Cd) accumulation by terrestrial higher plants is an intriguing phenomenon that may be exploited for phytoextraction of Cd-contaminated soils. Characterizing the physiological processes responsible for elevated concentrations of Cd in shoots is a first step towards a comprehensive understanding of the mechanisms underlying Cd accumulation in plants and may eventually improve the efficiency of phytoextraction. Woody species that can accumulate Cd have been recently recommended as good candidates for phytoextraction of Cd-contaminated soils. However, little is known about the mechanisms of Cd accumulation by woody species. In an attempt to understand the physiological processes contributing to Cd accumulation in woody species, Cd uptake and translocation by a novel tropical Cd-accumulating tree, star fruit (Averrhoa carambola) were characterized and compared with those of a non-Cd-accumulating tree (Clausena lansium). Our results showed that A. carambola had higher Cd uptake and root-to-shoot translocation efficiencies than C. lansium, which might account for its greater Cd-accumulating capacity. Furthermore, Cd accumulation by A. carambola was not significantly affected by zinc (Zn), whereas Zn accumulation was greatly lowered by Cd. This phenomenon could not be fully explained by a simple competition between Cd²⁺ and Zn²⁺, implying the existence of a transport system with a preference for Cd over Zn. Collectively, our results indicate that A. carambola has noteworthy physiological traits associated with accumulation of Cd to high levels.     

Albumin endocytosis via megalin in astrocytes is caveola- and Dab-1 dependent and is required for the synthesis of the neurotrophic factor oleic acid

The synthesis and release of the neurotrophic factor oleic acid requires internalization of albumin into the astrocyte, which is mediated by megalin. In this study, we show that the binding and internalization of albumin involve its interaction with megalin, caveolin-1, caveolin-2 and cavin, but not with clathrin in astrocytes from primary culture. Electron microscopy analyses revealed albumin-gold complexes localized in caveolae, but not in clathrin-coated vesicles. Neither chlorpromazine nor silencing clathrin expression modified albumin uptake. Silencing caveolin-1 strongly reduced the binding and internalization of albumin and the distribution of megalin in the plasma membrane. However, silencing caveolin-2 only decreased albumin internalization, suggesting that caveolin-1 is responsible for megalin recruitment to the caveolae and that caveolin-2 participates in caveolae internalization. In most tissues, the cytosolic adaptor protein disabled (Dab)-2 connects megalin to clathrin, astrocytes lack Dab-2; instead, they express Dab-1, which interacts with caveolin-1 and megalin and is required for albumin internalization. The transcytosis of albumin in astrocytes, including the passage through the endoplasmic reticulum, which is a compulsory step for oleic acid synthesis, was confirmed by electron microscopy analyses. Thus, whereas silencing clathrin did not modify the synthesis and release of oleic acid, the knock-down of caveolin-1, caveolin-2 and Dab-1 strongly reduced the synthesis and release of this neurotrophic factor. In conclusion, caveola-mediated endocytosis of albumin requires megalin and the adaptor protein Dab-1 in cultured astrocytes. Albumin endocytosis may be a key step in brain development because it stimulates the synthesis of oleic acid, which in turn promotes neuronal differentiation.     

The effects of chronic ethanol administration on the rates of internalization of various ligands during hepatic endocytosis.

The purpose of the present study was to further characterize the ethanol-induced impairments in hepatic endocytosis. Specifically, we examined the effects of ethanol treatment on receptor-ligand internalization via the coated and noncoated pit pathways. Insulin, epidermal growth factor (EGF) and asialoorosomucoid (ASOR) were used as model ligands to study internalization by isolated hepatocytes. ASOR and EGF are thought to be internalized strictly in coated pit regions of the cell membrane, while insulin may be internalized in both coated and uncoated membrane regions. Ethanol administration for 5-7 weeks decreased internalization of ASOR and EGF while internalization of insulin was unchanged during a single round of endocytosis of surface-bound ligand. Similarly, a more quantitative measure of endocytosis, the endocytic rate constant, was decreased for EGF and ASOR but not for insulin in livers of experimental rats. When endocytosis of Lucifer yellow, a fluorescent dye known to be internalized in the cell by fluid-phase endocytosis was examined, the initial rates of dye uptake were not significantly altered by alcohol administration. These results indicate that ethanol may selectively impair internalization occurring by coated pits while it has a minimal effect on initial uptake of molecules which are internalized by noncoated membrane regions.     

Entropic Control of Receptor Recycling Using Engineered Ligands

Receptor internalization by endocytosis regulates diverse cellular processes, from the rate of nutrient uptake to the timescale of essential signaling events. The established view is that internalization is tightly controlled by specific protein-binding interactions. However, recent work suggests that physical aspects of receptors influence the process in ways that cannot be explained by biochemistry alone. Specifically, work from several groups suggests that increasing the steric bulk of receptors may inhibit their uptake by multiple types of trafficking vesicles. How do biochemical and biophysical factors work together to control internalization? Here, we show that receptor uptake is well described by a thermodynamic trade-off between receptor-vesicle binding energy and the entropic cost of confining receptors within endocytic vesicles. Specifically, using large ligands to acutely increase the size of engineered variants of the transferrin receptor, we demonstrate that an increase in the steric bulk of a receptor dramatically decreases its probability of uptake by clathrin-coated structures. Further, in agreement with a simple thermodynamic analysis, all data collapse onto a single trend relating fractional occupancy of the endocytic structure to fractional occupancy of the surrounding plasma membrane, independent of receptor size. This fundamental scaling law provides a simple tool for predicting the impact of receptor expression level, steric bulk, and the size of endocytic structures on receptor uptake. More broadly, this work suggests that bulky ligands could be used to drive the accumulation of specific receptors at the plasma membrane surface, providing a biophysical tool for targeted modulation of signaling and metabolism from outside the cell.     

Uptake and efflux of copper-64 in Menkes''-disease and normal continuous lymphoid cell lines.

The accumulation of copper over 2 h by normal lymphoid cells and those from Menkes'-disease patients (Menkes' cells) was found to be biphasic, with an initial phase of rapid uptake, an approach to steady state at around 40-60 min, followed by a further accumulation phase. The accumulation of copper was not diminished by the addition of a variety of metabolic inhibitors, suggesting that copper uptake is not an active process. The presence of carbonyl cyanide m-chlorophenylhydrazone in the culture medium stimulated the uptake and accumulation of copper in both normal and Menkes' cells to the same absolute level. This effect appeared to be specific for copper, since the accumulation of Zn and Cd was unaffected. Menkes' cells did not differ from normal in their initial rate of copper uptake. Analysis of the uptake curve suggested that the membrane transport of copper involves both passive and facilitated diffusion. Initial rate of efflux from the cells was approximated by two methods. Menkes' cells did not appear to be affected in this function. It seems likely that the basic defect in Menkes' disease involves a step in intracellular copper transport rather than the membrane transport of copper.     

The CEACAM1 transmembrane domain, but not the cytoplasmic domain, directs internalization of human pathogens via membrane microdomains

Several bacterial pathogens exploit carcinoembryonic antigen-related cell adhesion molecules (CEACAMs) to promote attachment and uptake into eukaryotic host cells. The widely expressed isoform CEACAM1 is involved in cell–cell adhesion, regulation of cell proliferation, insulin homeostasis, and neo-angiogenesis, processes that depend on the cytoplasmic domain of CEACAM1. By analysing the molecular requirements for CEACAM1-mediated internalization of bacteria, we surprisingly find that the CEACAM1 cytoplasmic domain is completely obsolete for bacterial uptake. Accordingly, CEACAM1-4L as well as a CEACAM1 mutant with a complete deletion of the cytoplasmic domain (CEACAM1 ΔCT) promote equivalent internalization of several human pathogens. CEACAM1-4L- and CEACAM1 ΔCT-mediated uptake proceeds in the presence of inhibitors of actin microfilament dynamics, which is in contrast to CEACAM3-mediated internalization. Bacteria-engaged CEACAM1-4L and CEACAM1 ΔCT, but not CEACAM3, localize to a gangliosid GM1- and GPI-anchored protein-containing portion of the plasma membrane. In addition, interference with cholesterol-rich membrane microdomains severely blocks bacterial uptake via CEACAM1-4L and CEACAM1 ΔCT, but not CEACAM3. Similar to GPI-anchored CEACAM6, both CEACAM1-4L as well as CEACAM1 ΔCT partition into a low-density, Triton-insoluble membrane fraction upon receptor clustering, whereas CEACAM3 is not detected in this fraction. Bacterial uptake by truncated CEACAM1 or chimeric CEACAM1/CEACAM3 molecules reveals that the transmembrane domain of CEACAM1 is responsible for its association with membrane microdomains. Together, these data argue for a functional role of lipid rafts in CEACAM1-mediated endocytosis that is promoted by the transmembrane domain of the receptor and that might be relevant for CEACAM1 function in physiologic settings.     

Gene delivery by dendrimers operates via a cholesterol dependent pathway

Understanding the cellular uptake and intracellular trafficking of dendrimer–DNA complexes is an important prerequisite for improving the transfection efficiency of non-viral vector-mediated gene delivery. Dendrimers are synthetic polymers used for gene transfer. Although these cationic molecules show promise as versatile DNA carriers, very little is known about the mechanism of gene delivery. This paper investigates how the uptake occurs, using an endothelial cell line as model, and evaluates whether the internalization of dendriplexes takes place randomly on the cell surface or at preferential sites such as membrane rafts. Following extraction of plasma membrane cholesterol, the transfection efficiency of the gene delivered by dendrimers was drastically decreased. Replenishment of membrane cholesterol restored the gene expression. The binding and especially internalization of dendriplexes was strongly reduced by cholesterol depletion before transfection. However, cholesterol removal after transfection did not inhibit expression of the delivered gene. Fluorescent dendriplexes co-localize with the ganglioside GM1 present into membrane rafts in both an immunoprecipitation assay and confocal microscopy studies. These data strongly suggest that membrane cholesterol and raft integrity are physiologically relevant for the cellular uptake of dendrimer–DNA complexes. Hence these findings provide evidence that membrane rafts are important for the internalization of non-viral vectors in gene therapy.     

Theoretical study on temperature dependence of cellular uptake of QDs nanoparticles

Cellular uptake kinetics of nanoparticles is one of the key issues determining the design and application of the particles. Models describing nanoparticles intrusion into the cell mostly take the endocytosis process into consideration, and the influences of electrical charges, sizes, concentrations of the particles have been investigated. In this paper, the temperature effect on the cellular uptake of Quantum Dots (QDs) is studied experimentally. QDs are incubated with the SPCA-1 human lung tumor cells, and the nanoparticles on the cell membrane and inside the cell are quantified according to the fluorescence intensities recorded. It is found that the amounts of nanoparticles attached onto the cell membrane and inside the cell both increase with temperature. Based on the experimental results, a model is proposed to describe the cellular uptake dynamic process of nanoparticles. The process consists of two steps: nanoparticles adsorption onto the cell membrane and the internalization. The dynamic parameters are obtained through curve fitting. The simulated results show that the internalization process can be categorized into different phases. The temperature dependent internalization rate constant is very small when below 14?°C. It increases distinctly when temperature rises from 14?°C to 22?°C, but there is no evident increase as temperature further increases above 22?°C. Results show that by incorporating a temperature-independent internalization factor, the model predictions well fit the experimental results.     

Inhibition of pinocytosis in rat embryo fibroblasts treated with monensin

Rat embryo fibroblasts cultured in the presence of monensin exhibited an inhibited uptake of horseradish peroxidase. The inhibition was detected after 3 h, after which time the cells became increasingly vacuolated; the concentration of monensin required to inhibit pinocytosis (0.4 microM for half-maximum inhibition at 18 h) was similar to that found by others to inhibit secretion. Both the exchange of 5'-nucleotidase between the membranes of cytoplasmic organelles and the cell surface and the internalization of anti-5'-nucleotidase bound to the cell surface were inhibited by approximately 90% in monensin- treated cells. The effects of monensin were reversible: cells cultured first with monensin, and then in fresh medium, exhibited control levels of horseradish peroxidase uptake, exchange of 5'-nucleotidase, and internalization of anti-5'-nucleotidase bound to the cell surface. After monensin treatment, the median density of both galactosyl transferase and 5'-nucleotidase increased from 1.128 to 1.148, and the median density of both N-acetyl-beta-glucosaminidase and horseradish peroxidase taken up by endocytosis decreased from 1.194 to 1.160. The results indicate that monensin is a reversible inhibitor of pinocytosis and, presumably, therefore, of membrane recycling. They suggest that the inhibition of membrane recycling occurs at a step other than the fusion of pinocytic vesicles with lysosomes and is perhaps a consequence of an effect of the ionophore on the Golgi complex.     

Specific binding and uptake of 131I-MIBG and 111In-octreotide in metastatic paraganglioma--tools for choice of radionuclide therapy

Tumor-specific uptake of the radiolabeled nor-epinephrine analogue meta-iodobenzylguanidine via norepinephrine transporter or radiolabeled somatostatin analogues octreotide/octreotate via somatostatin receptors offers possibilities to diagnose and treat metastatic pheochromocytoma/paraganglioma. High uptake of 123I-meta-iodobenzylguanidine is dependent on high expression of vesicular monoamine transporters responsible for mediating uptake of biogenic amines into dense core granules. A patient with metastatic paraganglioma (liver and bone metastases) underwent surgical removal of the primary after injection of 131I-meta-iodobenzylguanidine and 111In-octreotide. Radioactivity was determined in biopsies from tumor and normal tissue biopsies. The tumor/blood concentration value was high: 180 for 131I-meta-iodobenzylguanidine 3 h after injection and 590 for 111In-octreotide 27 h after injection. Studies of primary tumor cell cultures demonstrated increased cell membrane binding and internalization over time for 131I-meta-iodobenzylguanidine. The vesicular monoamine transporter antagonist reserpine and the norepinephrine transporter inhibitor clomipramine reduced internalization by 90% and 70%, respectively, after 46 h of incubation. The results demonstrated increased cell membrane binding and internalization over time also for 111In-octreotide. Internalization was highest for a low concentration of 111In-octreotide. Excess of octreotide reduced internalization of 111In-octreotide with 75% after 46 h of incubation. In conclusion, uptake and tumor/blood concentration values of radiolabeled meta-iodobenzylguanidine and somatostatin analogues can be determined for metastatic pheochromocytoma/paraganglioma to evaluate the possibility to use one or both agents for therapy. For this patient, the high tumor/blood values clearly demonstrated that therapy using both radiopharmaceuticals would be most beneficial. In vitro studies verified specific cell-membrane binding and internalization in tumor cells of both radiopharmaceuticals.