Cleavage of transcriptional activator Oct-1 by poliovirus encoded protease 3Cpro

To understand the regulation of receptor-mediated endocytosis in hepatocytes, we have used two specific inhibitors of serine-threonine protein phosphatases (PP), microcystin (MCYST) and okadaic acid (OKA) as probes to alter protein phosphorylation in hepatocytes. We have then examined the impact of these changes on the specific binding and uptake of transferrin (Tf) in hepatocytes. The measurement of PP activity in hepatocyte lysates showed that OKA and MCYST shared a common inhibition of protein phosphatase 2A (PP2A). Our results showed that both OKA (250 nmol/L) and MCYST (500 nmol/L) significantly reduced Tf uptake at steady state (P < or = .05). The measurement of Tf internalization after 15 minutes in protein phosphatase inhibitor-pretreated cells revealed that the initial uptake was also significantly reduced. Binding studies showed that pretreatment with either of the phosphatase inhibitors did not result in significant changes in the K(d) for Tf binding to transferrin receptor (TfR). Additionally, no significant changes in the number of TfR in the plasma membrane were observed in phosphatase inhibitor-pretreated cells. The treatment of hepatocytes with nocodazole (NOC), which results in microtubule disassembly and inhibition of microtubule-based vesicle transport, caused comparable reductions in initial and steady state levels of transferrin accumulation. The changes in transferrin accumulation by both phosphatase inhibitors and nocodazole were accompanied by redistribution of the microtubule-anchored Golgi apparatus and lysosomal network from the perinuclear region to the cell periphery. Our data show that the regulation of Tf uptake by receptor-mediated endocytosis is mediated by PP2A and additionally may occur through regulation of microtubule-based vesicle transport.     

Nanomolar concentrations of nocodazole alter microtubule dynamic instability in vivo and in vitro

Previous studies demonstrated that nanomolar concentrations of nocodazole can block cells in mitosis without net microtubule disassembly and resulted in the hypothesis that this block was due to a nocodazole-induced stabilization of microtubules. We tested this hypothesis by examining the effects of nanomolar concentrations of nocodazole on microtubule dynamic instability in interphase cells and in vitro with purified brain tubulin. Newt lung epithelial cell microtubules were visualized by video-enhanced differential interference contrast microscopy and cells were perfused with solutions of nocodazole ranging in concentration from 4 to 400 nM. Microtubules showed a loss of the two-state behavior typical of dynamic instability as evidenced by the addition of a third state where they exhibited little net change in length (a paused state). Nocodazole perfusion also resulted in slower elongation and shortening velocities, increased catastrophe, and an overall decrease in microtubule turnover. Experiments performed on BSC-1 cells that were microinjected with rhodamine-labeled tubulin, incubated in nocodazole for 1 h, and visualized by using low-light-level fluorescence microscopy showed similar results except that nocodazole-treated BSC-1 cells showed a decrease in catastrophe. To gain insight into possible mechanisms responsible for changes in dynamic instability, we examined the effects of 4 nM to 12 microM nocodazole on the assembly of purified tubulin from axoneme seeds. At both microtubule plus and minus ends, perfusion with nocodazole resulted in a dose-dependent decrease in elongation and shortening velocities, increase in pause duration and catastrophe frequency, and decrease in rescue frequency. These effects, which result in an overall decrease in microtubule turnover after nocodazole treatment, suggest that the mitotic block observed is due to a reduction in microtubule dynamic turnover. In addition, the in vitro results are similar to the effects of increasing concentrations of GDP-tubulin (TuD) subunits on microtubule assembly. Given that nocodazole increases tubulin GTPase activity, we propose that nocodazole acts by generating TuD subunits that then alter dynamic instability.     

In vitro transcription of Myxococcus xanthus genes with RNA polymerase containing sigmaA, the major sigma factor in growing cells

Long-term incubation of proteins with glucose leads to advanced glycation end products (AGEs) with fluorescence and a brown color. We recently demonstrated immunologically the intracellular AGE accumulation in smooth muscle cell (SMC)-derived foam cells in advanced atherosclerotic lesions. To understand the mechanism of AGE accumulation in these foam cells, we have now characterized the interaction of AGE proteins with rabbit-cultured arterial SMCs. In experiments at 4 degrees C, 125I-labeled AGE-bovine serum albumin (AGE-BSA) showed a dose-dependent saturable binding to SMCs with an apparent dissociation constant (Kd) of 4.0 microg/ml. In experiments at 37 degrees C, AGE-BSA underwent receptor-mediated endocytosis and subsequent lysosomal degradation. The endocytic uptake of 125I-AGE-BSA was effectively inhibited by unlabeled AGE proteins such as AGE-BSA and AGE-hemoglobin, but not by acetylated LDL and oxidized LDL, well-known ligands for the macrophage scavenger receptor (MSR). Moreover, the binding of 125I-AGE-BSA to SMCs was affected neither by amphoterin, a ligand for one type of the AGE receptor, named RAGE, nor by 2-(2-furoyl)-4(5)-(2-furanyl)-1H-imidazole-hexanoic acid-BSA, a ligand for the other AGE receptors, p60 and p90. This indicates that the endocytic uptake of AGE proteins by SMCs is mediated by an AGE receptor distinct from MSR, RAGE, p60, and p90. To examine the functional role of this AGE receptor, the migratory effects of AGE-BSA on these SMCs were tested. Incubation with 1-50 microg/ml of AGE-BSA for 14 h resulted in significant dose-dependent cell migration. The AGE-BSA-induced SMC migration was chemotactic in nature and was significantly inhibited (approximately 80%) by an antibody against transforming growth factor-beta (TGF-beta), and the amount of TGF-beta secreted into the culture medium from SMC by AGE-BSA was sevenfold higher than that of control, indicating that TGF-beta is involved in the AGE-induced SMC chemotaxis. These data suggest that AGE may play a role in SMC migration in advanced atherosclerotic lesions.     

Inhibition of insulin-degrading enzyme increases translocation of insulin to the nucleus in H35 rat hepatoma cells: evidence of a cytosolic pathway

We previously demonstrated the translocation of insulin to the nucleus in several cell types and partially characterized the uptake mechanisms and pathways in H35 rat hepatoma cells. Nuclear accumulation of insulin was energy independent, time and temperature dependent, and apparently was not saturable at insulin concentrations which resulted in full receptor occupancy. We also have shown insulin could be internalized by both receptor-mediated and fluid-phase endocytosis. This study investigated subsequent steps involved in the nuclear accumulation of insulin following internalization. We examined the effects of inhibiting insulin degrading enzyme (IDE) with 1,10-phenanthroline on the nuclear accumulation of insulin in H35 cells. 1,10-phenanthroline (2 mM) which markedly inhibited insulin degradation, significantly increased nuclear accumulation of insulin without having any effects on total cell-associated and intracellular insulin. This reagent increased 125I-insulin on the cellular membrane and decreased 125iodine (125I-insulin and 125I-insulin degradation products) in the cytosolic fractions. Chemical extraction and Sephadex G-50 chromatography revealed the insulin associated with the nucleus in 1,10-phenanthroline-treated cells formed the same complex(es) with the nuclear matrix as in control cells. These results suggested that inhibition of cytosolic IDE activity resulted in increased insulin translocation from the cytosol to the nucleus. Furthermore, when IDE activity was inhibited by high cytosolic insulin concentrations, the amount of 125I-insulin in the nucleus was significantly increased. Our study suggests internalized insulin is probably released from endosomes into the cytosol where modulation of IDE activity could have significant effects on the accumulation of insulin, or insulin-cytoplasmic protein complexes, in nuclei. The IDE regulatory mechanism, by controlling the translocation of insulin to the cell nucleus, could play a crucial role in insulin's regulation of gene expression and cell proliferation.     

Stepwise reconstitution of interphase microtubule dynamics in permeabilized cells and comparison to dynamic mechanisms in intact cells

Microtubules in permeabilized cells are devoid of dynamic activity and are insensitive to depolymerizing drugs such as nocodazole. Using this model system we have established conditions for stepwise reconstitution of microtubule dynamics in permeabilized interphase cells when supplemented with various cell extracts. When permeabilized cells are supplemented with mammalian cell extracts in the presence of protein phosphatase inhibitors, microtubules become sensitive to nocodazole. Depolymerization induced by nocodazole proceeds from microtubule plus ends, whereas microtubule minus ends remain inactive. Such nocodazole-sensitive microtubules do not exhibit subunit turnover. By contrast, when permeabilized cells are supplemented with Xenopus egg extracts, microtubules actively turn over. This involves continuous creation of free microtubule minus ends through microtubule fragmentation. Newly created minus ends apparently serve as sites of microtubule depolymerization, while net microtubule polymerization occurs at microtubule plus ends. We provide evidence that similar microtubule fragmentation and minus end-directed disassembly occur at the whole-cell level in intact cells. These data suggest that microtubule dynamics resembling dynamics observed in vivo can be reconstituted in permeabilized cells. This model system should provide means for in vitro assays to identify molecules important in regulating microtubule dynamics. Furthermore, our data support recent work suggesting that microtubule treadmilling is an important mechanism of microtubule turnover.     

Tubulin polymerization by paclitaxel (taxol) phosphate prodrugs after metabolic activation with alkaline phosphatase

Paclitaxel (taxol) phosphate derivatives BMY46366, BMY-46489, BMS180661 and BMS180820 were used to determine the ability of alkaline phosphatase to convert these water-soluble potential prodrugs to tubulin-polymerizing metabolites (i.e., paclitaxel). Compounds were treated up to 180 min with an in vitro metabolic activation system composed of 10% bovine alkaline phosphatase in 0.2 M tris, pH 7.4, or in 0.2 M glycine, pH 8.8, plus 0.05 M MgCl2. Samples were tested (either by direct addition or after methylene chloride extraction/dimethyl-sulfoxide resuspension) in spectrophotometric tubulin polymerization assays utilizing bovine-derived microtubule protein. Pretreatment of 2'- and 7-phosphonoxyphenylpropionate prodrugs BMS180661 and BMS180820 with alkaline phosphatase for 30 to 120 min yielded relative initial slopes of about 20 to 100% at test concentrations equimolar to paclitaxel. High-performance liquid chromatography/mass spectrometry of BMS180661 treated with alkaline phosphatase confirmed the production of paclitaxel from the prodrug. In contrast, 2'- and 7-phosphate analogs BMY46366 and BMY46489 treated with alkaline phosphatase were not active in tubulin assays. None of the paclitaxel phosphate prodrugs polymerized tubulin in the absence of metabolic activation. The differences in tubulin polymerization with metabolic activation may be related both to accessibility of the phosphate group to the enzyme and to anionic charge effects. These results demonstrate that certain paclitaxel phosphate prodrugs can be metabolized by alkaline phosphatase to yield effective tubulin polymerization.     

Cholesterol derivative of poly(ethylene glycol) inhibits clathrin-independent, but not clathrin-dependent endocytosis

The effect of poly(ethylene glycol) cholesteryl ethers (PEG(n)-Chols) with two different numbers of units (n = 50 and 200) in the hydrophilic PEG moiety on cellular endocytic activity was studied on HT-1080 cells. The amphipathic molecules were soluble in aqueous solution. When fluorescein derivatives of PEG-Chols (one fluorescein at the distal end of PEG) were incubated with the cells in culture, the cellular fluorescence was localized at the plasma membrane level and in intracellular vesicles. Fluorescence quantification indicated that for the same external concentration, twice more FPEG(50)-Chol than FPEG(200)-Chol was associated with the cells under the same conditions. Regardless of the length of PEG moiety, PEG-Chols' interaction with cells reduced the endocytic internalization of a fluid phase marker, horseradish peroxidase (HRP) depending on the cell-associated amount. In contrast, internalization of 125I-labeled epidermal growth factor (EGF) through receptor-mediated endocytosis did not change upon incubation with PEG(50)-Chol. The effect of PEG(200)-Chol was also small, since EGF internalization showed a reduction of 10-20%, while at the same concentration as much as 80% of HRP uptake was inhibited. PEG(50)-Chol did not influence the internalization of a larger ligand, 125I-transferrin (Tfn). However, in the presence of PEG(200)-Chol, the uptake of 125I-Tfn decreased remarkably, and yet, PEG(200)-Chol has no influence on the binding and internalization of a monoclonal antibody directed toward the ectodomain of the Tfn-receptor. These results suggested that incorporation of PEG-Chols in the outer monolayer of the plasma membrane specifically inhibited clathrin-independent, but not clathrin-dependent endocytosis.     

Phosphorylation of Bcl-2 is a marker of M phase events and not a determinant of apoptosis

Phosphorylation of Bcl-2 protein is a post-translational modification of unclear functional consequences. We studied the correlation between Bcl-2 phosphorylation, mitotic arrest, and apoptosis induced by the anti-tubulin agent paclitaxel. Continuous exposure of human cervical carcinoma HeLa cells to 50 ng/ml paclitaxel resulted in mitotic arrest with a symmetrical bell-shaped curve over time. The number of mitotic cells was highest at 24 h (82%), then declined as arrested cells progressed into apoptosis, and barely no mitotic cells were present at 48-60 h. The time curves of paclitaxel-induced cyclin B1 accumulation and stimulation of Cdc2/cyclin B1 kinase activity were identical and superimposable to that of M phase arrest. In contrast, apoptosis was first detected at 12 h and steadily increased thereafter until the termination of the experiments at 48-60 h, when about 80-96% of cells were apoptotic. Bcl-2 phosphorylation was closely associated in time with M phase arrest, accumulation of cyclin B1, and activation of Cdc2/cyclin B1 kinase, but not with apoptosis. At 24 h, when about 82% of the cells were in mitosis, almost all Bcl-2 protein was phosphorylated, whereas at 48 h, when 70-90% of the cells were apoptotic, all Bcl-2 protein was unphosphorylated. Similar results were obtained with SKOV3 cells, indicating that the association of paclitaxel-induced M phase arrest and Bcl-2 phosphorylation is not restricted to HeLa cells. We used short exposure to nocodazole and double thymidine to synchronize HeLa cells and investigate the association of Bcl-2 phosphorylation with mitosis. These studies demonstrated that Bcl-2 phosphorylation occurs in tight association with the number of mitotic cells in experimental conditions that do not lead to apoptosis. However, a continuous exposure to nocodazole resulted in a pattern of Bcl-2 phosphorylation, M phase arrest, and apoptosis similar to that observed with paclitaxel. The phosphatase inhibitor okadaic acid was found to inhibit the dephosphorylation of phosphorylated Bcl-2 and to delay the progression of nocodazole M phase-arrested cells into interphase. In contrast, the serine/threonine kinase inhibitor staurosporine, but not the tyrosine kinase inhibitor genistein, led to rapid dephosphorylation of phosphorylated Bcl-2 and accelerated the progression of nocodazole M phase-arrested cells into interphase. Immune complex kinase assays in cell-free systems demonstrated that Bcl-2 protein can be a substrate of Cdc2/cyclin B1 kinase isolated from paclitaxel-treated cells arrested in M phase. Taken together, these studies suggest that Bcl-2 phosphorylation is tightly associated with mitotic arrest and fail to demonstrate that it is a determinant of progression into apoptosis after mitotic arrest induced by anti-tubulin agents.     

Activation of the metaphase checkpoint and an apoptosis programme in the early zebrafish embryo, by treatment with the spindle-destabilising agent nocodazole

We have studied the developmental activation of the metaphase checkpoint, and the consequences of activating this checkpoint, in the zebrafish embryo. (1) Treatment with nocodazole (a microtubule destabiliser) before mid-blastula transition (MBT) produces complete destruction of all nuclei in the deep cell layer of the embryo. In contrast, nocodazole treatment after MBT efficiently produces metaphase arrest in this cell layer. Thus, the metaphase checkpoint becomes activated at MBT. (2) Although a metaphase arrest is induced by nocodazole, it is not induced by paclitaxel (a microtubule stabiliser). Thus the metaphase checkpoint appears to sense a destabilisation, but not a stabilisation, of spindle microtubules. (3) Metaphase-arrested cells (in nocodazole) can be driven into the next interphase by adding the Ca2+-specific ionophore A23187. Thus, a Ca2+-signalling pathway lies downstream of, or parallel to, the metaphase checkpoint. (4) After mid-gastrula stage, treatment with nocodazole produces DNA fragmentation in all three cell layers. In the enveloping epithelial monolayer (EVL), this is associated with a classical apoptotic phenotype. In the deep layer, it is associated with an unusual, highly condensed nuclear state that is entered directly from metaphase arrest. Thus, after the mid-gastrula stage, the embryo responds to nocodazle by undergoing apoptosis. (5) Nocodazole-induced apoptosis in the deep cell layer can be blocked by the caspase-1,4,5 inhibitors Ac-YVAD-CHO and Ac-YVAD-CMK. This suggests that a homologue of the C. elegans ced-9-ced-4-ced-3 pathway is involved in control over apoptosis in the early zebrafish embryo.     

The effect of 12-myristate 13-acetate phorbol ester on human sperm hyperactivation

Although several dextran-coated iron oxide preparations are in preclinical and clinical use, little is known about the mechanism of uptake into cells. As these particles have been shown to accumulate in macrophages and tumor cells, we performed cellular uptake and inhibition studies with a prototypical monocrystalline iron oxide nanoparticle (MION). MION particles were labeled with fluorescein isothiocyanate or radioiodinated and purified by gel permeation chromatography. Two preparations of MION particles were used in cell experiments: nontreated MION and plasma-opsonized MION purified by gradient density purification. As determined by immunoblotting, opsonization resulted in C3, vitronectin, and fibronectin association with MION. Incubation of cells with fluorescent MION showed active uptake of particles in macrophages both before and after opsonization. In C6 tumor cells, however, intracellular MION was only detectable in dividing cells. Quantitatively, 125I-labeled MION was internalized into cells with uptake values ranging from 17 ng (in 9L gliosarcoma) to 970 ng iron per million cells for peritoneal macrophages. Opsonization increased MION uptake into macrophages sixfold, whereas it increased the uptake in C6 tumor cells only twofold. Results from uptake inhibition assay suggest that cellular uptake of nonopsonized (dextran-coated) MION particles is mediated by fluid-phase endocytosis, whereas receptor-mediated endocytosis is presumably responsible for the uptake of opsonized (protein-coated) particles.     

A possible association between paternal longevity and major depression among elderly men

Platinum-based chemotherapy is the standard treatment for advanced ovarian cancer, with response rates of 40-60%. In patients who fail platinum treatment, paclitaxel has resulted in response rates of 10-48%. Docetaxel has partial non-cross-resistance with and is twice as potent in vitro as paclitaxel in inhibiting microtubule disaggregation. The combination of docetaxel and cyclophosphamide is synergistic in pre-clinical studies and clinically active in breast cancer. We present the case of a patient with platinum and paclitaxel refractory ovarian cancer who achieved a remission with docetaxel and cyclophosphamide.     

The effect of a free radical scavenger and platelet-activating factor antagonist on FFA accumulation in post-ischemic canine brain

The effects of the platelet-activating factor antagonist BN 50739 and a free radical scavenger dimethyl sulfoxide on the accumulation of free fatty acids in post-ischemic canine brain are reported. Following 14 min of complete normothermic ischemia and 60 min of reperfusion, the total brain FFAs were approximately 150% higher than in the control group (p < 0.05). Perfusion with the platelet-activating factor antagonist BN50739 in its diluent dimethyl sulfoxide during 60 min of post-ischemic reoxygenation resulted in a 61.8% (p < 0.01) reduction in the total brain free fatty acid accumulation. Palmitic, stearic, oleic, linoleic, and arachidonic acids decreased by 53.8%, 63.5%, 69.0%, 47.4%, and 57.2%, respectively. Although dimethyl sulfoxide alone caused stearic and arachidonic acids to return to the normal concentration range, BN 50739 had a significant influence on recovery of palmitic, oleic, and linoleic acids and was previously shown to provide significant therapeutic protection against damage to brain mitochondria following an ischemic episode. Because free fatty acid accumulation is one of the early phenomena in cerebral ischemia, this study provides evidence to support the hypothesis that both platelet-activating factor and free radicals are involved in initiating cerebral ischemic injury.     

Effect of bafilomycin A1 and nocodazole on endocytic transport in HeLa cells: implications for viral uncoating and infection

Bafilomycin A1 (baf), a specific inhibitor of vacuolar proton ATPases, is commonly employed to demonstrate the requirement of low endosomal pH for viral uncoating. However, in certain cell types baf also affects the transport of endocytosed material from early to late endocytic compartments. To characterize the endocytic route in HeLa cells that are frequently used to study early events in viral infection, we used 35S-labeled human rhinovirus serotype 2 (HRV2) together with various fluid-phase markers. These virions are taken up via receptor-mediated endocytosis and undergo a conformational change to C-antigenic particles at a pH of <5.6, resulting in release of the genomic RNA and ultimately in infection (E. Prchla, E. Kuechler, D. Blaas, and R. Fuchs, J. Virol. 68:3713-3723, 1994). As revealed by fluorescence microscopy and subcellular fractionation of microsomes by free-flow electrophoresis (FFE), baf arrests the transport of all markers in early endosomes. In contrast, the microtubule-disrupting agent nocodazole was found to inhibit transport by accumulating marker in endosomal carrier vesicles (ECV), a compartment intermediate between early and late endosomes. Accordingly, lysosomal degradation of HRV2 was suppressed, whereas its conformational change and infectivity remained unaffected by this drug. Analysis of the subcellular distribution of HRV2 and fluid-phase markers in the presence of nocodazole by FFE revealed no difference from the control incubation in the absence of nocodazole. ECV and late endosomes thus have identical electrophoretic mobilities, and intraluminal pHs of <5.6 and allow uncoating of HRV2. As bafilomycin not only dissipates the low endosomal pH but also blocks transport from early to late endosomes in HeLa cells, its inhibitory effect on viral infection could in part also be attributed to trapping of virus in early endosomes which might lack components essential for uncoating. Consequently, inhibition of viral uncoating by bafilomycin cannot be taken to indicate a low pH requirement only.     

Synthesis, cancericidal and antimicrotubule activities of 3-(haloacetamido)-benzoylureas

The four title compounds (not hitherto reported) were synthesized from 3-aminobenzoic acid through its trifluoroacetic acid-acid chloride derivative, reaction with urea and aminolytic deprotection to yield 3-aminobenzoylurea, followed by unconventional haloacetylation. Three key factors were found essential for antitumor activity: (i) the cytotoxic nature of the halogen: I > Br > Cl > F (ID90 0.014->10 microM); (ii) the position of the halogen: only the 3-position (meta) expressed relevant activity; and (iii) the presence of the urea group (1-position). The selectivity of the bromo and iodo compounds were higher than those of vinblastine and paclitaxel in terms of cytotoxicity (ID50 ratios in nonmalignant myocardial fibroblasts and CEM leukemia cells) and therapeutic indices (P338 leukemia bearing mice). Relevant mechanisms of bioactivity were mitotic arrest and apoptosis. Complete inhibition of microtubule assembly occurred in cell-free systems (at 2.8 versus 2.1 microM for vinblastine); in contrast to paclitaxel, the target compounds did not interfere with microtubule disassembly. The strong cancericidal and antimicrotubular activities of the bromine and iodine compounds justify further exploration of their potential in antineoplastic chemotherapy.     

Nerve growth factor processing and trafficking events following TrkA-mediated endocytosis

We expressed the high affinity nerve growth factor receptor TrkA in Chinese hamster ovary (CHO) fibroblasts to study nerve growth factor (NGF) trafficking and processing events following receptor-mediated ligand internalization in a nonneuronal and p75 minus cell line. These stable clonal cell lines express approximately 2.5 x 10(5) TrkA receptors and bind 125I-NGF with high affinity (Kd = 4 x 10(-10) M). The TrkA receptors are autophosphorylated on tyrosine residues upon NGF stimulation and are capable of tyrosine phosphorylating downstream signaling molecules. The t1/2 of 125I-NGF internalization is 5 min, and the probability of an occupied TrkA receptor internalizing within 1 min at 37 C is 9.8%. By 2 h following endocytosis, less than 10% of internalized 125I-NGF is degraded, as determined by TCA precipitation. Thirty minutes following ligand endocytosis, endocytosed 125I-NGF is delivered back to the cell surface and released by the cell (retroendocytosis), possibly by remaining associated with recycling TrkA receptors. We measured the effect of acidification on 125I-NGF-TrkA association and found that, at pH 6, 40% of 125I-NGF remains bound. Thus, NGF may remain associated with the TrkA receptor at low pH conditions in the endosome and can thereby be targeted back to the plasma membrane for release by the cell. In conclusion: 1) TrkA, in the absence of p75, is fully capable of mediating 125I-NGF endocytosis; 2) internalized 125I-NGF is slowly and inefficiently degraded; 3) following internalization, 125I-NGF is retroendocytosed; and 4) the ability of 125I-NGF to remain receptor-associated during acidic conditions may provide a mechanism for its retroendocytosis via recycling TrkA vesicles.     

Sepsis increases endocytosis of endotoxin into hepatocytes

BACKGROUND: Chylomicrons bind endotoxins and accelerate their clearance from plasma to the liver. This results in reduced mortality from septic shock in a rodent model. We hypothesized that the clearance of the LPS-chylomicron (LPS-CM) complex by hepatocytes is due to receptor-mediated endocytosis and that sepsis up-regulates this process. METHODS: Three groups of Sprague-Dawley rats; (1) control; (2) pretreated with 10 micrograms/kg LPS 24 hours before treatment; and (3) pretreated with 17-alpha-ethinyl estradiol (EE, 5 mg/kg subcutaneously for 3 days), were infused with labeled I125-LPS alone or with I125-LPS bound to chylomicron. Livers were removed 2.5, 15, and 30 minutes after LPS injection, and hepatic endosomes were isolated from the liver homogenates by serial ultracentrifugation in sucrose gradients. RESULTS: The injection of I125-LPS-CM complexes resulted in higher levels of endosomal I125-LPS in all groups, as compared with I125-LPS alone. In addition, the endosomal uptake of I125-LPS was markedly increased by both LPS and EE pretreatments. CONCLUSIONS: These data strongly suggest a primary role for receptor-mediated endocytosis in the increased clearance of LPS when bound to chylomicron. In addition, exposure to LPS appears to increase the accumulation of LPS in endosomes by a mechanism similar to that of EE, which is known to up-regulate receptor-mediated lipoprotein uptake. This endogenous pathway for the catabolism of endotoxins may provide a teleological explanation for the hypertriglyceridemia observed during sepsis.     

Internalization of activated platelet-derived growth factor receptor-phosphatidylinositol-3' kinase complexes: potential interactions with the microtubule cytoskeleton

Phosphatidylinositol (PI)-3' kinase catalyzes the formation of PI 3,4-diphosphate and PI 3,4,5-triphosphate in response to stimulation of cells by platelet-derived growth factor (PDGF). Here we report that tyrosine-phosphorylated PDGF receptors, the p85 subunit of PI-3' kinase (p85), and activated PI-3' kinase are found in isolated clathrin-coated vesicles within 2 min of exposure of cells to PDGF, indicating that both receptor and activated PI-3' kinase enter the endocytic pathway. Immunofluorescence analysis of p85 in serum-starved cells revealed a punctate/reticular staining pattern, concentrated in the perinuclear region and displaying high focal concentration at the centrosome. In addition, partial coalignment of p85 with microtubules was observed after optical sectioning microscopy and image reconstruction. The association of p85 with the microtubule network was further evidenced by the microtubule-depolymerizing drug nocodazole, which caused a redistribution of p85 from the perinuclear region to the cell periphery. Interestingly, the most significant effect of PDGF on the distribution of p85 was an increase in the staining intensity of this protein in the perinuclear region, and this effect was eliminated by prior treatment of cells with nocodazole. These results suggest that PDGF receptor-p85 complexes internalize and transit in association with the microtubule cytoskeleton. In addition, the high concentration of p85 in intracellular structures in the absence of PDGF stimulation suggests additional roles for this protein independent of its association with receptor tyrosine kinases.     

Scattered Golgi elements during microtubule disruption are initially enriched in trans-Golgi proteins

We have addressed the question of whether or not Golgi fragmentation, as exemplified by that occurring during drug-induced microtubule depolymerization, is accompanied by the separation of Golgi subcompartments one from another. Scattering kinetics of Golgi subcompartments during microtubule disassembly and reassembly following reversible nocodazole exposure was inferred from multimarker analysis of protein distribution. Stably expressed alpha-2,6-sialyltransferase and N-acetylglucosaminyltransferase-I (NAGT-I), both C-terminally tagged with the myc epitope, provided markers for the trans-Golgi/trans-Golgi network (TGN) and medial-Golgi, respectively, in Vero cells. Using immunogold labeling, the chimeric proteins were polarized within the Golgi stack. Total cellular distributions of recombinant proteins were assessed by immunofluorescence (anti-myc monoclonal antibody) with respect to the endogenous protein, beta-1,4-galactosyltransferase (GalT, trans-Golgi/TGN, polyclonal antibody). ERGIC-53 served as a marker for the intermediate compartment). In HeLa cells, distribution of endogenous GalT was compared with transfected rat alpha-mannosidase II (medial-Golgi, polyclonal antibody). After a 1-h nocodazole treatment, Vero alpha-2,6-sialyltransferase and GalT were found in scattered cytoplasmic patches that increased in number over time. Initially these structures were often negative for NAGT-I, but over a two- to threefold slower time course, NAGT-I colocalized with alpha-2,6-sialyltransferase and GalT. Scattered Golgi elements were located in proximity to ERGIC-53-positive structures. Similar trans-first scattering kinetics was seen with the HeLa GalT/alpha-mannosidase II pairing. Following nocodazole removal, all cisternal markers accumulated at the same rate in a juxtanuclear Golgi. Accumulation of cisternal proteins in scattered Golgi elements was not blocked by microinjected GTPgammaS at a concentration sufficient to inhibit secretory processes. Redistribution of Golgi proteins from endoplasmic reticulum to scattered structures following brefeldin A removal in the presence of nocodazole was not blocked by GTPgammaS. We conclude that Golgi subcompartments can separate one from the other. We discuss how direct trafficking of Golgi proteins from the TGN/trans-Golgi to endoplasmic reticulum may explain the observed trans-first scattering of Golgi transferases in response to microtubule depolymerization.     

Enterocolitis

Human monocyte-derived dendritic cells were differentiated in vitro for 7 days with granulocyte macrophage-colony stimulating factor and interleukin-13. These cultured dendritic cells are at an immature stage of differentiation and exhert high endocytic activity via surface mannose receptor and via fluid-phase macropinocytosis. We have investigated the modulation of endocytosis by interleukin-10 in these cells. When added during the last 24 h of the 7-day culture, interleukin-10 significantly stimulated the uptake of fluorescein-labelled dextran (39 +/- 16% increase, mean +/- SD of 6 experiments), a sugar binding to the mannose receptor. This effect was dose dependent and correlated with the length of exposure to interleukin-10, with a maximal effect (more than seven-fold increase) when the cytokine was added at the beginning of the culture (day 0). The interleukin-10-increased fluorescein-labelled-dextran endocytosis was mostly mediated via the mannose receptor, as unlabelled mannose and specific antimannose receptor monoclonal antibody inhibited most of the uptake. Moreover, interleukin-10-treated cells expressed increased levels (up to four-fold) of mannose receptor. Interleukin-10 also increased, although to a lesser extent, the fluid-phase endocytosis (macropinocytosis) of fluorescein-labelled albumin. Interleukin-10 had the opposite effect on the differentiation and functional activity of monocyte-derived dendritic cells; cells having a very low stimulatory capacity and reduced expression of MHC class II and CD1a after a 7-day exposure. Thus interleukin-10 had a strong immunosuppressive effect on the differentiation and functional activity of monocyte-derived dendritic cells and yet strongly stimulated endocytosis in these cells. We speculate that an increased endocytic activity would eventually result in a decreased availability of antigens in the external milieu, thus contributing to the immunosuppressive and tolerogenic activity of interleukin-10.     

Myocardial digoxin uptake: dissociation between digitalis-induced inotropism and myocardial loss of potassium

The time course of myocardial uptake of digoxin, of increase in inotropic effect and of changes in myocardial potassium content were studied following a single intravenous dose of digoxin. Nineteen dogs with intact circulation were investigated by the use of a biopsy technique which allowed samplings before and 10, 30, 60, and 90 min after administration of digoxin. The myocardial concentration of digoxin was 196 X 10(-9) mol/kg 10 min after administration of digoxin. Uptake continued at a slower rate, maximum concentration being 293 X 10(-9) mol/kg at 60 minutes. The inotropic effect increased parallel with the uptake of digoxin; 10 min after digoxin, contractility was 127% of the control value and this increased to 139% at 90 minutes. Myocardial potassium content was slightly increased 10 min after digoxin, suggesting an initial stimulation of membrane Na+-K+ ATPase. A subsequent significant fall in the myocardial potassium content probably reflects ATPase inhibition. The temporal dissociation between the early onset of the positive inotropic effect and the delayed inhibition of membrane Na+-K+ ATPase indicates that inotropism of digitalis glycosides is not mediated by the same binding site as that responsible for inhibition of Na+-K+ ATPase.