Osmotically induced fluid-phase uptake of fluorescent markers by protoplasts ofChenopodium album

Summary Protoplasts fromChenopodium album suspension culture show large, up to 5-fold, changes in surface area upon hypertonic or hypotonie treatment. These surface area variations cannot be explained by elastic stretching of the plasmalemma. An exchange of membrane material between the plasmalemma and an internal membrane source takes place. Fluid-phase uptake experiments with the fluorescence dyes 5, 6-carboxyfluorescein and Lucifer yellow CH demonstrated that osmotic shrinkage of protoplasts is accompanied by vesicular uptake of the external medium into protoplast cytoplasm. Confocal laser scanning microscopy, as well as conventional fluorescence microscopy, revealed the number, diameter and distribution of the osmocytotic vesicles at different osmotic levels. The rate of osmocytotic vesicle uptake was higher in the presence of calcium chloride than in the presence of EDTA in the external medium. At 6.9 mM calcium chloride we observed a loss of vesicular fluorescence upon returning protoplasts to 0.4 M from 0.8 M sorbitol.     

l-Proline transport into renal OK epithelial cells: a second renal proline transport system is induced by amino acid deprivation

Influx of [³H]-l-proline into renal OK cells revealed that basal transport was mediated by the transporter SIT1. When cells were submitted for 8 h to amino acid deprivation, uptake of l-proline was now dominated by a low-affinity system with an apparent K _m of 4.4 ± 0.6 mM and a V _max of 10.2 ± 0.6 nmol/mg of protein/min operating in addition to the high-affinity SIT1 system with a K _m of 0.12 ± 0.01 mM and a V _max of 0.28 ± 0.04 nmol/mg of protein/min. The low- and high-affinity proline transporting systems were sensitive to inhibitors of JNK and PI-3 kinases, whereas a GSK-3 inhibitor affected only the upregulated transport system. Ion-replacement studies and experiments assessing substrate specificities for both systems provided strong evidence that SNAT2, that showed two- to threefold increased mRNA levels, is the responsible transporter mediating the increased proline influx under conditions of amino acid deprivation.     

Transport of organic cations by a renal epithelial cell line (OK)

The goal of this study was to determine the mechanisms involved in the transport of the organic cation, tetraethylammonium (TEA), across the apical membrane of OK cells. [14C]TEA accumulated in OK cell monolayers reaching equilibrium in 2 h. The uptake of [14C]TEA at equilibrium was dependent upon temperature and was inhibited by sodium azide and by various organic cations, including N1-methylnicotinamide (NMN), mepiperphenidol, and cimetidine but not by the organic anion, p-aminohippuric acid. The initial uptake of [14C]TEA was characterized by a saturable process. The mean +/- S.D. Km was 27.8 +/- 2.6 microM and the Vmax was 414 +/- 26.5 pmol/mg protein/min. Both an accelerated efflux and influx of [14C]TEA in the presence of a trans-gradient of unlabeled TEA and NMN was observed, whereas a deaccelerated influx and efflux was observed in the presence of a trans-gradient of mepiperphenidol. The mechanism of interaction between NMN and TEA was examined. NMN significantly increased the apparent Km (mean +/- S.D.) of TEA to 82.8 +/- 16.4 microM (p less than 0.001), whereas the Vmax (mean +/- S.D.) was only slightly affected (478 +/- 72 pmol/mg protein/min) suggesting a competitive inhibition. The stimulatory effect of trans-gradients of NMN on TEA transport was due to an increase in the Vmax of TEA suggesting that NMN trans-stimulates TEA transport by increasing the turnover rate of the exchanger. In the presence of an inwardly directed proton gradient, the efflux at 30 s of [14C]TEA from the OK cell monolayers was significantly accelerated (p less than 0.05). Studies with the pH-sensitive fluorescent probe, 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein, suggested that TEA could drive the countertransport of protons. In apical membrane vesicles prepared from OK cells, the uptake of [3H]NMN exhibited an apparent "overshoot phenomenon" in the presence of an initial outwardly directed proton gradient. Protons competitively inhibited TEA uptake suggesting that the proton/organic cation and the organic cation/organic cation self exchange mechanism are the same mechanism. This is the first report describing both TEA self-exchange and proton/TEA exchange in the apical membrane of a continuous cell line. OK cells are an excellent model for the study of organic cation transport across the apical membrane.     

Uptake of endocytic markers into mitotic yeast cells

[3H]alpha-Factor and Lucifer yellow were used to measure receptor mediated and fluid-phase endocytosis in the yeast Saccharomyces cerevisiae, arrested in mitosis by depolymerization of the microtubules or due to a mutation preventing nuclear division (cdc16). Both processes continued at roughly the same level as during interphase. This shows that in yeast endocytosis is not interrupted during mitosis like in mammalian cells.     

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.     

Alpha-2-adrenergic modulation of the parathyroid hormone-inhibition of phosphate uptake in cultured renal (OK) cells

Parathyroid hormone enhances the formation of cAMP and decreases the Na+-dependent uptake of phosphate in cultured renal cells derived from the American opossum (OK cells). Epinephrine, acting as an alpha 2-adrenergic agonist, inhibits the PTH-induced synthesis of cAMP by a pertussis toxin-sensitive mechanism and blunts the inhibition of phosphate transport by PTH. Na+-dependent alpha-methylglucoside and Na+ uptakes by the cells are unaffected by PTH and epinephrine. These findings suggest that alpha 2-adrenergic agonists may selectively modulate PTH-sensitive phosphate transport in the renal proximal tubule.     

Effects of ammonium chloride and chloroquine on endocytic uptake of liposomes by Kupffer cells in vitro

In this study we investigated the interaction of liposomes with rat Kupffer cells in maintenance culture by using the lysosomotropic amines ammonium chloride and chloroquine as inhibitors of intralysosomal degradation. The liposomes (large unilamellar vesicles) contained either the metabolically inert ³H-labeled inulin or the degradable ¹²⁵I-labeled bovine serum albumin. In control incubations, the cells released nearly all accumulated protein label and about 30% of the lipid label when they were incubated in the absence of liposomes, after an initial uptake period of 1 h in the presence of liposomes. This release of label was, for the greater part, suppressed in the presence of ammonia or chloroquine. When the inhibitors were present during the initial uptake period, a several-fold increase in the amount of protein label accumulating in the cells and a smaller, but still marked, increase in lipid label accumulation were observed. The effect of ammonia when present during uptake was readily reversible in contrast to that of chloroquine. Experiments with encapsulated inulin revealed that both lysosomotropic agents also affected the uptake process per se to some extent, probably as a result of impaired membrane/receptor recycling. Labeled liposomes adsorbed to the cells at 4°C were effectively internalized and processed intracellulary after shifting the temperature to 37°C, even when a 500-fold excess of unlabeled liposomes was present in the medium during the 37°C incubation. The observed effects of ammonia and chloroquine indicate that, after uptake, the liposomes are degraded within lysosomes, thus confirming our previous conclusion that endocytosis is the major uptake mechanism at 37°C. From the temperature-change experiments we conclude that, at 4°C, the liposomes are bound with high affinity to the cells, remaining firmly attached to the cell-surface structures which initiate their internalization when the temperature is raised to 37°C.     

Subtypes of thymic epithelial cells defined by neuroendocrine markers

The study attempted to define characteristics of thymic epithelial cells within rat thymus based on the expression of neuroendocrine markers. Using an immunohistochemical approach, the following markers were localised: protein gene product 9.5 (PGP 9.5), neuron-specific enolase (NSE) and chromogranin A (ChA). It was shown that cells displaying immunostaining typical for individual markers reside in distinct regions of the thymus and represent subtypes within various populations of thymic epithelial cells. An immunoreactivity for PGP 9.5 was found exclusively in a subtype of cortical epithelial cells, located mostly within the inner zone of the cortex. On the other hand, NSE represented a marker of most epithelial cells located in the medulla. Few such cells which were negative for NSE proved positive for ChA. Among the cells with a strong reaction for NSE some cells also manifested a positive reaction for ChA. While the pattern of neuroendocrine marker distribution may reflect functional properties of thymic epithelial cells which might be different within distinct areas of the thymus, the differential expression of individual markers seems to reflect biological activity of the cells and/or distinct stages of their differentiation.     

Intravital multi-photon microscopy reveals several levels of heterogeneity in endocytic uptake by mouse renal proximal tubules

Understanding renal function requires one to integrate the structural complexity of kidney nephrons and the dynamic nature of their cellular processes. Multi-photon fluorescence microscopy is a state-of-the-art imaging technique for in vivo analysis of kidney tubules structure and function in real time. This study presents visual evidence for several levels of heterogeneity of proximal tubular endocytic uptake in the superficial renal mouse cortex and illustrates the potential of multi-photon microscopy for providing a comprehensive and dynamic portrayal of renal function.     

Uptake of endocytic markers by rice cells: variations related to the growth phase

Endocytosis is now considered a basic cellular process common to plant cells. Although both non-specific and receptor-mediated endocytosis appear to take place in plant cells, the physiological role of the latter remains unclear. We have investigated the endocytic process in rice cell suspensions using two biotinylated proteins, peroxidase and bovine serum albumin (bHRP and bBSA), as markers. First, we show that markers are internalized by rice cells and appear in intracellular membranes. The uptake of the two markers is temperature dependent, saturable with time and markers dose and it is competed by free biotin. Thus, it shows the properties of a receptor-mediated process. We also show that uptake of markers is strongly influenced by growth phase as optimal uptake occurs during the lag phase, but the initiation of the exponential growth phase decreases uptake drastically. Arrest of the cell cycle by starvation of either a nutrient (phosphate) or a growth regulator (2,4-dichlorophenoxyacetic acid), both components of the culture medium, does not modify the rate of bBSA uptake. Subsequent readdition of these components results in growth recovery and a dramatic decrease in bBSA uptake. On the other hand, nocodazole treatment, a method to arrest the cell cycle by microtubule depolymerization, inhibited bBSA uptake. The possible causes for this arrest of endocytosis are discussed.     

Riboflavin uptake by human-derived colonic epithelial NCM460 cells

Normal microflora ofthe large intestine synthesize a number of water-soluble vitaminsincluding riboflavin (RF). Recent studies have shown that colonicepithelial cells posses an efficient carrier-mediated mechanism forabsorbing some of these micronutrients. The aim of the present studywas to determine whether colonic cells also posses a carrier-mediatedmechanism for RF uptake and, if so, to characterize this mechanism andstudy its cellular regulation. Confluent monolayers of thehuman-derived nontransformed colonic epithelial cells NCM460 and[³H]RF were used in the study. Uptake of RF wasfound to be 1) appreciable and temperature and energydependent; 2) Na⁺ independent; 3) saturableas a function of concentration with an apparent K_mof 0.14 µM and V_max of 3.29 pmol · mgprotein¹ · 3 min¹; 4) inhibited by the structural analogslumiflavin and lumichrome (K_i of 1.8 and 14.1 µM,respectively) but not by the unrelated biotin; 5) inhibited ina competitive manner by the membrane transport inhibitor amiloride(K_i = 0.86 mM) but not by furosemide, DIDS, orprobenecid; 6) adaptively regulated by extracellular RF levels with a significant and specific upregulation and downregulation in RFuptake in RF-deficient and oversupplemented conditions, respectively;and 7) modulated by an intracellularCa²⁺/calmodulin-mediated pathway. These studies demonstratefor the first time the existence of a specialized carrier-mediatedmechanism for RF uptake in an in vitro cellular model system of humancolonocytes. This mechanism appears to be regulated by extracellularsubstrate level and by an intracellularCa²⁺/calmodulin-mediated pathway. It is suggested that theidentified transport system may be involved in the absorption ofbacterially synthesized RF in the large intestine and that this sourceof RF may contribute toward RF homeostasis, especially that of colonocytes.

     

Localisation of Alanine uptake by cultured renal epithelial cells (LLC-PK1) to the basolateral membrane

Alanine uptake by LLC-PK¹ cells has previously been demonstrated to be almost exclusively sodium dependent. We here confirm that when the cells are grown on an impermeable substratum there is a marked fall in uptake as confluence is reached. By applying an autoradiographic technique to visualize transported alanine, it is clear, however, that even in subconfluent cultures there is marked cellular inhomogeneity with regard to uptake, which takes place predominantly in those cells at the periphery of growing islands and not those at the interior. In contrast, when cells are grown on permeable substrata, a uniform distribution of silver grains is found. In two other types of experiment, we found that when confluent cell monolayers on an impermeable support were treated briefly with a chelating agent or suspended by mechanical treatment, there was a marked increase per cell in sodium-dependent alanine uptake and in ouabain-sensitive potassium uptake. We conclude that the apparent fall in alanine uptake as cells reach confluence on an impermeable support is due to masking of transport sites, which are predominantly, if not exclusively, located at the basolateral membrane.     

Uridine uptake by isolated intestinal epithelial cells of guinea pig

Uptake of uridine was studied in isolated intestinal epithelial cells of guinea pig. Uptake was not severely influenced by metabolism. Free uridine was accumulated within cells 13-fold. Uptake was saturable with an apparent K_m value of 46 μM and a V_max of 0.9 nmol/mg protein per min. Uracil inhibited uptake only slightly; adenosine, guanosine and cytosine inhibited strongly. Antimycin A and ouabain inhibited almost 90%. If the extracellular Na⁺ concentration was decreased to 5 mM, the rate of uptake decreased 6.5-fold. The stimulatory effect of Na⁺ was related to the transmembraneous Na⁺-gradient. Cells from jejunum transported about 30% faster than cells from ileum. In conclusion, isolated enterocytes of guinea pig posses an active transport system for uridine.     

Uridine uptake by isolated intestinal epithelial cells of guinea pig

Uptake of uridine was studied in isolated intestinal epithelial cells of guinea pig. Uptake was not severely influenced by metabolism. Free uridine was accumulated within cells 13-fold. Uptake was saturable with an apparent Km value of 46 microM and a Vmax of 0.9 nmol/mg protein per min. Uracil inhibited uptake only slightly; adenosine, guanosine and cytosine inhibited strongly. Antimycin A and ouabain inhibited almost 90%. If the extracellular Na+ concentration was decreased to 5 mM, the rate of uptake decreased 6.5-fold. The stimulatory effect of Na+ was related to the transmembraneous Na+-gradient. Cells from jejunum transported about 30% faster than cells from ileum. In conclusion, isolated enterocytes of guinea pig posses an active transport system for uridine.     

Exit of intracellular Porphyromonas gingivalis from gingival epithelial cells is mediated by endocytic recycling pathway

Gingival epithelial cells function as an innate host defence system to prevent intrusion by periodontal bacteria. Nevertheless, Porphyromonas gingivalis, the most well‐known periodontal pathogen, can enter gingival epithelial cells and pass through the epithelial barrier into deeper tissues. However, it is poorly understood how this pathogen exits from infected cells for further transcellular spreading. The present study was performed to elucidate the cellular machinery exploited by P. gingivalis to exit from immortalized human gingival epithelial cells. P. gingivalis was shown to be internalized with early endosomes positive for the FYVE domain of EEA1 and transferrin receptor, and about half of the intracellular bacteria were then sorted to lytic compartments, including autolysosomes and late endosomes/lysosomes, while a considerable number of the remaining organisms were sorted to Rab11‐ and RalA‐positive recycling endosomes. Inhibition experiments revealed that bacterial exit was dependent on actin polymerization, lipid rafts and microtubule assembly. Dominant negative forms and RNAi knockdown of Rab11, RalA and exocyst complex subunits (Sec5, Sec6 and Exo84) significantly disturbed the exit of P. gingivalis. These results strongly suggest that the recycling pathway is exploited by intracellular P. gingivalis to exit from infected cells to neighbouring cells as a mechanism of cell‐to‐cell spreading.     

Interleukin 10 promotes macrophage uptake of HDL and LDL by stimulating fluid-phase endocytosis

ObjectiveHighly elevated plasma levels of interleukin-10 (IL-10) are causally associated with “Disappearing HDL Syndrome” and low plasma LDL-cholesterol, but the underlying mechanism is poorly understood. Fluid-phase endocytosis, a process highly dependent on actin dynamics, enables cells to internalize relatively high amounts of extracellular fluids and solutes. We sought to investigate whether IL-10 induces lipoprotein uptake by fluid-phase endocytosis in macrophages.Methods and resultsMacrophages (RAW264.7, Kupffer and human) were incubated with vehicle (PBS) or IL-10 (20 ng/ml) for 7 days. Uptake of HDL, LDL, and/or fluid-phase endocytosis probes (albumin-Alexa680®, 70 kDa FITC-Dextran and Lucifer Yellow, LY) was evaluated by FACS. Intracellular cofilin and phosphorylated cofilin (p-cofilin) levels were determined by immunoblotting.Macrophage uptake of lipoproteins and probes was non-saturable and increased after IL-10 incubation (p < 0.0001). Furthermore, pre-incubation with fluid-phase endocytosis inhibitors (LY294002, Latrunculin A, and Amiloride) significantly reduced uptake (p < 0.05). IL-10 increased the cofilin/p-cofilin ratio (p = 0.021), signifying increased cofilin activation and hence filamentous actin. Consistently, phalloidin staining revealed increased filamentous actin in macrophages after IL-10 treatment (p = 0.0018). Finally, RNA-seq analysis demonstrated enrichment of gene sets related to actin filament dynamics, membrane ruffle formation and endocytosis in IL-10-treated macrophages (p < 0.05). IL-10 did not alter mRNA levels of Ldlr, Vldlr, Scarb1, Cd36 or Lrp1. In primary human monocyte-derived macrophages and murine Kupffer cells, IL-10 incubation also increased uptake of lipoproteins, albumin and LY (p < 0.01).ConclusionsInterleukin-10 induces the uptake of HDL and LDL by fluid-phase endocytosis by increasing actin-filament rearrangement in macrophages, thus providing a plausible mechanism contributing to “Disappearing HDL Syndrome”.     

Phosphate uptake by kidney epithelial (LLC-PK1) cells

Phosphate uptake by the cultured kidney epithelial cell (LLC-PK1) was studied. The uptake was Na+ dependent, saturable with respect to phosphate and Na+, and energy dependent. The characteristics of the cell uptake system resembled the properties of phosphate transport in the kidney. Parathyroid hormone, dibutyryl cyclic AMP, and forskolin decreased Na+-dependent phosphate uptake. These agonists did not affect Na+-dependent alpha-methylglucoside uptake. Vasopressin and isoproterenol, which do not affect renal phosphate transport, did not inhibit phosphate uptake by the cell. These findings suggest that the cultured cell system may be a useful experimental model for studies of renal phosphate transport and its regulation.     

Calcium uptake by duodenal epithelial cells is increased during lactation

It is known that duodenal absorption of calcium is increased during lactation, and, more specifically, it has been shown that duodenal active transport of calcium is increased. The energy-requiring step for this active transport process is thought to occur at the basolateral membrane of the duodenal epithelial cells, where calcium is pumped from the cell into the extracellular fluid. The present experiments were carried out to determine whether calcium uptake into duodenal epithelial cells is also altered during lactation. Uptake of 45Ca into isolated duodenal epithelial cells from lactating animals showed markedly enhanced calcium uptake when compared with cells from control animals. Uptake was calcium concentration dependent for both groups. Since duodenal epithelial cells are known to contain increased calbindin9k, a calcium-binding protein, during lactation, calcium uptake was also measured in cells that were preincubated in medium containing calcium. In these experiments, the total 45Ca uptake was reduced, but the difference between lactating and control remained. These experiments show that the uptake of calcium into duodenal epithelial cells is among the components of the transcellular calcium transport process which is enhanced during lactation.