monensin and oxophenylarsine

The internalization of [3H]propionyl[Met(O2)11]SP(7-11) which binds one binding site and of [3H][Pro9]SP which binds the two binding sites associated with the NK-1 receptor has been examined in CHO cells. The quantity of [3H][Pro9]SP measured inside the cytoplasm in kinetic experiments is fully temperature-dependent. In contrast, [3H]propionyl[Met(O2)11]SP(7-11) internalization reaches the same extent whatever the temperature, although the rate slowed down with lower temperature. The extent of internalization of [3H][Pro(9)]SP relative to the total specific bound is biphasic, when the extent of internalization of [3H]propionyl[Met(O2)11]SP(7-11) remains constant. For [3H][Pro9]SP, a high-affinity high-yield component inhibited in the presence of propionyl[Met(O2)11]SP(7-11) and a low-affinity low-yield component in the internalization process could be determined. Saturation studies show that [3H][Pro9]SP-binding parameters are insensitive to both phenylarsine oxide and monensin treatment, whereas [3H]propionyl[Met(O2)11]SP(7-11) maximal binding is decreased in both cases. Altogether, these data suggest that the two radiolabeled peptides should not follow the same internalization pathway.

Topics: Animals; Arsenicals; CHO Cells; Cricetinae; Endocytosis; Kinetics; Models, Biological; Monensin; Peptide Fragments; Receptors, Neurokinin-1; Substance P; Temperature; Transfection

Angiotensin II (Ang II) uptake and transport across monolayers of bovine brain microvessel endothelial cells (BMECs) was demonstrated. Ang II transport was linear up to 2 h, saturable with a K(m) of 1.7 nM, and tended to be polarized with the apical-to-basolateral transport being greater. [3H]Ang II transport was found to be inhibited by excess unlabeled Ang II, by the Ang II analog sarathrin, and by the endocytic inhibitor phenylarsine oxide. Ang II-(2-8) and-(3-8) were shown to significantly increase the transport of Ang II. These results demonstrate for the first time the receptor-mediated transcytosis of Ang II across brain microvessel endothelium.

Topics: Angiotensin II; Animals; Arsenicals; Biological Transport, Active; Blood-Brain Barrier; Cattle; Cell Membrane Permeability; Cells, Cultured; Cerebrovascular Circulation; Endothelium, Vascular; Enzyme Inhibitors; Microcirculation; Monensin; Receptors, Angiotensin

The pharmacological characteristics, localization and process of internalization of the insulin-like growth factor I and II receptors were studied in rat primary hippocampal cultured neurons grown under serum-free conditions. [125I]insulin-like growth factor-I binding was specific with an apparent affinity (Kd) of 0.1 nM and IC50 values of 0.1, 2.9 and 99.7 nM for insulin-like growth factor-I, insulin-like growth factor-II and insulin, respectively. The competition by insulin suggests the presence of genuine insulin-like growth factor-I receptors and not insulin-like growth factor binding proteins. In contrast, [125I]insulin-like growth factor-II binding showed a Kd of 0.1 nM and IC50 values of 0.2 and 20.5 nM for insulin-like growth factor-II and insulin-like growth factor-I while insulin was inactive, a well established characteristic of the insulin-like growth factor-II receptor. Using emulsion autoradiography, specific binding sites for [125I]insulin-like growth factor-I and -II were over the whole cultured neurons. The use of selective insulin-like growth factor-I and -II receptor antibodies further confirmed the existence of these receptors in rat hippocampal cultured neurons. To investigate the respective internalization profile of [125I]insulin-like growth factor-I and [125I]insulin-like growth factor-II receptor-ligand complexes in neurons, a technique of acid stripping was used. The apparent rate of endocytosis was found to be greater for the insulin-like growth factor-II than for the insulin-like growth factor-I receptor complexes. The internalization of [125I]insulin-like growth factor-I and [125I]insulin-like growth factor-II ligand-receptor complexes was confirmed using phenylarsine oxide which significantly blocked both internalization processes. In order to eliminate possible receptor recycling, monensin was used and shown to have no effect on the internalization of either ligand. Since the insulin-like growth factor-I receptor is coupled to tyrosine kinase activity, tyrphostin 47, a specific tyrosine kinase inhibitor. was used and shown to decrease [125I]insulin-like growth factor-I but not the [125I]insulin-like growth factor-II receptor internalization profile. Accordingly, insulin-like growth factor-I is apparently internalized mostly via the insulin-like growth factor-I tyrosine kinase type receptor, while insulin-like growth factor-II is not. The insulin-like growth factor-II receptor ligand complex is likely internalized via a pathw

Topics: Animals; Arsenicals; Endocytosis; Enzyme Inhibitors; Hippocampus; Immunohistochemistry; Iodine Radioisotopes; Ionophores; Ligands; Monensin; Neurons; Nitriles; Phenols; Protein-Tyrosine Kinases; Rats; Rats, Sprague-Dawley; Receptor, IGF Type 1; Receptor, IGF Type 2; Tyrphostins

Internalization of the NK1 receptor (NK1R) and substance P was observed in cells transfected with cDNA encoding the rat NK1R by using anti-receptor antibodies and cyanine 3-labelled substance P (cy3-substance P). After incubation at 4 degrees C, NK1R immunoreactivity and cy3-substance P were confined to the plasma membrane. Within 3 min of incubation at 37 degrees C, NK1R immunoreactivity and cy3-substance P were internalized into small intracellular vesicles located beneath the plasma membrane. Fluorescein isothiocyanate-labelled transferrin and cy3-substance P were internalized into the same vesicles, identifying them as early endosomes. After 60 min at 37 degrees C, NK1R immunoreactivity was detected in larger, perinuclear vesicles. Internalization of 125I-labelled substance P was studied by using an acid wash to dissociate cell-surface label from that which has been internalized. Binding reached equilibrium after incubation for 60 min at 4 degrees C with no detectable internalization. After 10 min incubation at 37 degrees C, 83.5 +/- 1.0% of specifically bound counts were internalized. Hyperosmolar sucrose and phenylarsine oxide, which are inhibitors of endocytosis, prevented internalization of 125I-labelled substance P and accumulation of NK1R immunoreactivity into endosomes. Acidotropic agents caused retention of 125I-labelled substance P within the cell and inhibited degradation of the internalized peptide. Continuous incubation of cells with substance P at 37 degrees C reduced 125I-substance P binding at the cell surface. Therefore, substance P and its receptor are internalized into early endosomes within minutes of binding, and internalized substance P is degraded. Internalization depletes NK1Rs from the cell surface and may down-regulate the response of a cell to substance P.

Topics: Amino Acid Sequence; Ammonium Chloride; Animals; Arsenicals; Cell Line, Transformed; Chloroquine; Colchicine; Endocytosis; Epithelium; Hypertonic Solutions; Immune Sera; Immunohistochemistry; Kinetics; Leupeptins; Microscopy, Fluorescence; Molecular Sequence Data; Monensin; Peptides; Rats; Receptors, Neurokinin-1; Recombinant Proteins; Substance P; Time Factors; Transfection

Binding and internalization of angiotensin II (AII) were studied on bovine adrenal medullary cells in primary culture. Binding of [125I]AII was reversible, saturable, specific and showed high affinity. AII was found to be internalized by bovine adrenal medullary cells. Monensin increased whereas phenylarsine oxide (PhAsO) decreased the internalization. Excess of unlabelled AII or saralasin could block the internalization, indicating a receptor mediated internalization process. The kinetic analysis indicated that, during the first 4 min, about 25% of the membrane bound ligand was internalized per min and the recycling of internalized ligand and receptor initiated around 4 min.

Topics: Adrenal Medulla; Angiotensin II; Animals; Arsenicals; Cattle; Cells, Cultured; Endocytosis; Iodine Radioisotopes; Kinetics; Monensin; Receptors, Angiotensin; Saralasin

Because a major limitation of ODN (oligodeoxynucleotide) use is inefficient cellular uptake, methods to improve ODN uptake could have important implications in the investigational and possibly therapeutic use of ODNs. In this study, antisense c-myc ODN cellular uptake in elevated extracellular calcium was increased up to 48-fold in the four cell lines examined. The role of calcium in ODN cellular uptake was examined using a 21-base ODN complementary to the c-myc proto-oncogene and the Rauscher cells. Cells were pretreated with uptake inhibitors in either 1.8 (physiologic) or 5.4 mM calcium prior to addition of (32P) labelled ODN. In physiologic calcium conditions, ODN cellular uptake was partially dependent on cellular energy and a trypsin-sensitive surface protein. In contrast, in the presence of elevated (5.4 mM) extracellular calcium, trypsinization and metabolic inhibition had a reduced and no effect, respectively, on uptake. Endocytosis and lysosomotropic inhibitors did not decrease uptake in either calcium concentrations. Therefore, the mechanism of ODN uptake may depend on the level of extracellular calcium. Furthermore, surface binding accounted for approximately 60% of total uptake in both physiologic and elevated calcium concentrations, suggesting that the increased uptake was not due exclusively to increased surface binding. Thus, the predominant mechanism of ODN uptake may depend on the extracellular calcium concentration.

Topics: Animals; Arsenicals; Base Sequence; Biological Transport; Calcium; Cell Line; Chloroquine; Deoxyglucose; Genes, myc; Humans; Kinetics; Leukemia, Erythroblastic, Acute; Macrophages; Molecular Sequence Data; Monensin; Oligonucleotides, Antisense; Proto-Oncogene Mas; Trypsin; Tumor Cells, Cultured

It has been suggested that there are multiple pathways for the cellular internalization of insulin. To investigate these pathways we have examined the effects of three perturbations of endocytosis on the insulin internalization process and have compared these effects with those obtained using an asialoglycoprotein, asialofetuin (Afet), and epidermal growth factor (EGF). Freshly isolated hepatocytes were incubated with radiolabeled ligands and internalization measured under conditions of anoxia to deplete cellular ATP, in the presence of phenylarsine oxide (PAO) to inhibit endocytosis, and in the presence of monensin to interfere with endosomal acidification. Afet internalization essentially was blocked by all three treatment processes, while insulin internalization was inhibited approximately 40% in the presence of anoxia, and 54% in the presence of PAO. Monensin exhibited differential effects on internalization of high and low insulin concentrations. The effects of the treatment processes on EGF internalization were intermediate to those seen with Afet and insulin. These results suggest that insulin and EGF utilize routes of internalization exhibiting different energy requirements that may correspond to coated pit, non-coated pit, and fluid-phase internalization pathways. The observations with Afet internalization remain consistent with utilization of the coated pit pathway.

Topics: Adenosine Triphosphate; alpha-Fetoproteins; Animals; Arsenicals; Asialoglycoproteins; Cell Hypoxia; Endocytosis; Epidermal Growth Factor; Fetuins; Insulin; Liver; Male; Monensin; Rats; Rats, Inbred Strains

We have studied the mechanisms of angiotensin II (A-II) transport through a cultured arterial endothelial cell monolayer. The transport of 125I-labeled A-II was inhibited by excess unlabeled A-II (50 microM) and [Sar1, Ile8]-A-II (50 microM), but was not inhibited by bradykinin (50 microM). The transport process was shown to be temperature dependent and was inhibited by 10 mM NaN3 plus 50 mM 2-deoxyglucose. Monensin (50 microM), an inhibitor of endocytotic trafficking, reduced the rate of transport of 125I-A-II. It is also shown that the specific pathway for A-II transport was unidirectional from the apical to the basolateral surface of the endothelial cell monolayer.

Topics: Adenosine Triphosphate; Ammonium Chloride; Angiotensin II; Animals; Arsenicals; Binding, Competitive; Biological Transport; Chromatography, Gel; Endothelium, Vascular; In Vitro Techniques; Iodine Radioisotopes; Monensin; Temperature

A glycine-HCl buffer (glycine, 50 mM/NaCl, 0.15 M/HCl, pH 3.5) was used to strip insulin bound to adipocyte cell surfaces. Adipocytes retained their integrity in the glycine buffer and their binding capacity for [125I]iodoinsulin could be completely recovered on transfer of the cells to physiological media. At 37 degrees C, [125I]iodoinsulin binds rapidly to plasma membrane receptors; maximal binding occurs within 10 min. At this temperature, the initial binding is followed by rapid internalization, degradation of the hormone and subsequent loss of label. Insulin treatment, at 37 degrees C, induced internalization of 37% of the plasma membrane insulin receptors. Phenylarsine oxide (PAO), a confirmed inhibitor of protein internalization, allowed insulin binding but completely inhibited degradation of the hormone. Monensin, a carboxylic ionophore which impairs uncoupling hormone-receptor complexes, effectively restricted insulin degradation over short time periods (less than 30 min). Addition of monensin to insulin-stimulated cells did not impair D-glucose uptake. It has previously been reported that PAO inhibits hexose transport through the direct interaction with the glucose transporters and low concentrations of PAO (1 microM) transiently inhibit insulin-stimulated glucose uptake. This recovery phenomenon was again observed when PAO was added to insulin-stimulated, monensin-treated adipocytes. The data suggests that lysosomal degradation of insulin is not requisite for signal transduction.

Topics: Adipose Tissue; Animals; Arsenicals; Glucose; In Vitro Techniques; Insulin; Male; Monensin; Rats; Rats, Inbred Strains; Receptor, Insulin; Signal Transduction

The effects of agents that inhibit receptor-mediated endocytosis on type I (slow or high-affinity) and type II (fast or low-affinity) NGF binding have been examined in rat PC12 cells. Compounds interfering with endocytosis eliminate type I NGF binding; those interfering with acidification of endosomal vesicles cause increased type I binding at the expense of type II binding. Measurement of NGF binding during and after treatment with inhibitors indicates that NGF receptors rapidly cycle from the cell surface into an undefined endocytotic compartment and back to the surface with little degradation of receptor or NGF, consistent with a model in which NGF receptors are rapidly and reversibly endocytosed or sequestered; those receptors free on the surface represent type II NGF receptors, while those in the process of endocytosis represent type I NGF receptors. The type I and type II NGF receptor species can be interconverted by agents that can manipulate the position of the receptor in the internalization cycle.

Topics: Animals; Arsenicals; Chloroquine; Digitonin; Endocytosis; Monensin; Nerve Growth Factors; Pheochromocytoma; Rats; Receptors, Cell Surface; Receptors, Nerve Growth Factor; Tumor Cells, Cultured

Exposure of cultured embryonic chicken bone cells to the PTH agonists bovine (b) PTH-(1-34) and [8Nle, 18Nle, 34Tyr]bPTH-(1-34)amide [bPTH-(1-34)A] reduces the subsequent cAMP response to the hormone and decreases the specific binding of 125I-labeled PTH to these cultures. To determine whether PTH receptor down-regulation in cultured bone cells is mediated by cellular internalization of PTH-receptor complexes, we measured the uptake of [125I]bPTH-(1-34) into an acid-resistant compartment. Uptake of radioactivity into this compartment was inhibited by incubating cells at 4 C with phenylarsineoxide and unlabeled bPTH-(1-34). Tracer uptake into the acid-resistant compartment at any time was directly proportional to total cell binding at 22 C. Thus, it is likely that PTH-receptor complexes are internalized by bone cells. This mechanism may explain the loss of cell surface receptors after PTH pretreatment. To determine whether internalized PTH-receptor complexes are reinserted into the plasma membrane, we measured PTH binding and PTH stimulation of cAMP production after cells were exposed to monensin, a known inhibitor of receptor recycling. Monensin (25 microM) had no effect on PTH receptor number or affinity and did not alter PTH-stimulated cAMP accumulation. However, monensin (25 microM) incubated with cells pretreated with various concentrations of bPTH-(1-34) for 1 h potentiated the effect of the hormone to reduce subsequent [125I]bPTH-(1-34) binding and PTH-stimulated cAMP accumulation by more than 2 orders of magnitude. Chloroquine also potentiated PTH-induced down-regulation of PTH receptors. By contrast, neither agent influenced PTH binding or PTH-stimulated cAMP production in cells pretreated with the antagonist bPTH-(3-34)A. Thus, monensin potentiated PTH receptor loss only in cells pretreated with PTH agonists, indicating that antagonist-occupied receptors may be processed differently from agonist-occupied receptors in bone cells. The data further suggest that the attenuation of PTH stimulation of cAMP production in treated bone cells may be, at least in part, due to receptor-mediated endocytosis of the hormone.

Topics: Animals; Arsenicals; Bone and Bones; Cell Membrane; Cells, Cultured; Chick Embryo; Chloroquine; Cyclic AMP; Drug Synergism; Monensin; Parathyroid Hormone; Peptide Fragments; Receptors, Cell Surface; Receptors, Parathyroid Hormone