Shedding of transferrin receptor from rat reticulocytes during maturation in vitro: soluble transferrin receptor is derived from receptor shed in vesicles

Measurements of circulating transferrin (Tf) receptor are useful in assessing erythropoiesis; however, steps involved in the generation of soluble Tf receptor from cellular receptor are incompletely understood. To obtain a better understanding of this process, we investigated the loss of Tf receptor during terminal maturation of rat reticulocytes in vitro. Previous studies have identified Tf receptor-containing vesicles in the culture medium of maturing reticulocytes. In the present study, vesicle-free reticulocyte culture medium was found to contain functional and immunoreactive soluble Tf receptor, which increased over time. During a 44-hour incubation, Tf receptor on reticulocytes decreased by approximately 69%, while, of the Tf receptor shed to the medium, 65% was present in vesicles and 35% was in a soluble form. Isolated vesicles reincubated in fresh medium released soluble Tf receptor to the medium. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the isolated soluble receptor protein was mainly 190 Kd and 95 Kd under nonreducing and reducing conditions, respectively, similar in size to the vesicular and cellular receptor. Our studies show that loss of Tf receptor from rat reticulocytes during maturation in vitro involves shedding of cellular Tf receptor in vesicles and release of soluble receptor from these vesicles.     

Inhibition of cellular iron uptake by haem in mouse erythroleukaemia cells

Haemin inhibited iron uptake from transferrin (Tf) by mouse erythroleukaemia cells (MELC) induced for differentiation by hexamethylene bisacetamide (HMBA). The rate of 59Fe internalization was decreased, but the rate and the extent of 125I-Tf endocytosis was unaffected by the addition of haemin. Haemin inhibited 59Fe incorporation into haem by a greater proportion than the overall uptake of 59Fe from Tf. The reduction of total cellular 59Fe uptake was more pronounced at 59Fe-Tf concentrations closer to saturation. Exogenous 5-aminolaevulinic acid stimulated 59Fe utilization for haem synthesis in MELC but did not revert the inhibition induced by haemin. Haem synthesis measured by 14C-glycine incorporation into haem was maintained for at least 1 h without an external transferrin iron source and was inhibited by the addition of haemin equally over the whole range of Tf concentrations studied. Desferrioxamine (DFO) stimulated cellular uptake of 59Fe by the uninduced cells and reverted the inhibition of 59Fe transport into HMBA treated cells caused by haemin. Addition of DFO within a short-term incubation had no effect on haem synthesis measured by 14C-glycine incorporation into haem. No evidence for a direct effect of haem on the transferrin cycle or iron release was found. It was concluded that the reduction of iron uptake by haemin treated MELC is secondary to the decrease in iron utilization for haem synthesis.     

Binding and internalization of transforming growth factor-beta 1 by human hepatoma cells: evidence for receptor recycling.

Cellular processing of 125I-labeled transforming growth factor-beta 1 was investigated in the human hepatoma cell lines Hep G2 and Hep 3B. Binding of 125I-transforming growth factor-beta 1 to cell surface receptors was specific, saturable and calcium-independent. Both cell lines exhibited a single class of high-affinity (Kd = 2.2 x 10(-10) mol/L) binding sites (4.5 x 10(3) for the Hep G2 cell; 1.5 x 10(3) for the Hep 3B cell) for both human and porcine transforming growth factor-beta 1. Binding was temperature dependent, time dependent and pH dependent. Cell-bound 125I-transforming growth factor-beta 1 was removed by brief exposure to acidic medium (pH less than 4) but was converted into an acid-resistant state rapidly after shifting the cells to 37 degrees C. Spontaneous dissociation of bound ligand over a 6 hr period at 4 degrees C was less than 10%. Disuccinimidyl suberate was used to covalently label 125I-transforming growth factor-beta 1 to cell-surface binding sites. Labeling of the ligand/receptor complexes was inhibited by unlabeled transforming growth factor-beta 1 but was unaffected by other growth factors. The radiolabeled complexes showed approximate molecular weights of 280,000, 85,000 and 65,000 when run on reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Cell-bound 125I-transforming growth factor-beta 1 was internalized and degraded at 37 degrees C, and the products were released into the medium as trichloroacetic acid-nonprecipitable radioactivity. The lysosomotropic base chloroquine and the carboxylic ionphore monensin inhibited degradation and release of 125I-labeled products from the cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Transferrin modulation of colony stimulating factor elaboration by adherent blood and bone marrow cells

When added to cultured normal, adherent blood and marrow cells at concentrations of 25-100 micrograms/ml (3 X 10(-7) to 1.3 X 10(-6) M), human transferrin enhanced colony-stimulating factor (CSF) elaboration. Fe saturated and relatively unsaturated Tf were equally effective in increasing CSF release, but soluble ferric nitriloacetate was ineffective. Dose-dependent increases in CSF release by adherent blood and marrow cells occurred in serum-free media and the continuous presence of Tf was necessary for this effect. Using a monoclonal anti-Tf receptor antibody, normal blood mononuclear cells contained no detectable Tf receptor positive cells. However, after 5 d culture in serum-free medium, Tf receptor positive cells were identified among normal adherent blood cells, and the per cent receptor positive cells increased with Tf concentration. We conclude Tf modulates CSF production from normal, adherent blood and marrow cells in vitro. These findings indicate a possible role for Tf in intramedullary regulation of normal granulopoiesis.     

Release of thyrotropin receptor from thyroid plasma membranes: effect of hydrocortisone, propranolol, and adenosine 3',5'-monophosphate

Soluble TSH receptors were released into the medium when bovine thyroid plasma membranes were incubated in 0.01 M Tris-HCl, pH 7.5, at 0 or 20 C. This is a conventional hypotonic medium used in binding assays. The characteristics of binding of bovine [125I]TSH to the released TSH receptor were almost the same as those of binding to TSH receptors solubilized by lithium 3,5-diiodosalicylate or to the original plasma membrane. Released TSH receptor had two binding sites with Ka values of 0.7 x 10(10) and 0.1 x 10(8) M-1. T3, T4, KI, methimazole, and propylthiouracil had no effect on spontaneous TSH receptor release or on bovine [125I]TSH binding to solubilized TSH receptor. Hydrocortisone (10(-5)--10(-3) M) and d,l-propranolol (10(-3) M) inhibited receptor release. cAMP increased the release of TSH receptor. Hydrocortisone, d,l-propranolol, and cAMP had no effect on bovine [125I]TSH binding to solubilized or released TSH receptor. d,l-Propranolol and hydrocortisone may act as membrane-stabilizing agents. cAMP stimulation of release suggests that the release mechanism could depend upon a protein kinase-phosphoprotein system. Although these studies were conducted with membranes in an unphysiological medium, receptor release may occur normally and could be a source of circulating antigen related to production of antireceptor antibody in autoimmune thyroid diseases. Release of receptors during incubation in vitro may affect the results of studies of hormone-receptor interaction.     

Adenosine diphosphate stimulation of cultured hematopoietic cell lines

Adenosine diphosphate (ADP) plays a critical role in platelet activation both by exogenous stimulation and the release of endogenous intracellular stores. As the platelet ADP receptor is not well defined, we have chosen to identify and characterize several cell lines that possess functional receptors for this nucleotide. Rat promegakaryoblasts (RPM), human erythroleukemia cells (HEL), U937, and K562 leukemia cells responded to ADP, as measured by a rapid increase in intracellular calcium. In the case of RPM cells, ADP was the only naturally occurring platelet agonist capable of eliciting this response. Binding studies with [3H]ADP and fixed cells showed 3.99 +/- 1.77 x 10(5) binding sites/cell for RPM cells (apparent dissociation constant [kd] = 7.75 +/- 2.3 x 10(-8) mol/L), 8.19 +/- 3.25 x 10(5) sites/cell for HEL cells (kd = 2.15 +/- 0.84 x 10(-7) mol/L, 1.15 +/- 0.23 x 10(6) sites/cell for U937 cells (kd = 2.20 +/- 0.53 x 10(-7) mol/L) and 5.39 +/- 2.80 x 10(5) sites/cell for K562 cells (kd = 1.37 +/- 0.39 x 10(-7) mol/L), Inhibition studies with unlabeled nucleotides and analogues showed that binding was approximately 85% specific and the inhibitory pattern was similar to that seen with mature platelets. The purine base adenosine resulted in little or no inhibition. These studies indicate that both human and rat hematopoietic cell lines possess intact ADP receptors and may be useful tools in future studies of the structure and function of this important platelet-activation system.     

Regulatory effects of gallium on transferrin-independent iron uptake by human leukemic HL60 cells

Gallium, a pharmacologically important metal, resembles iron with respect to transferrin (Tf) binding and Tf receptor-mediated cellular uptake. In the present study, we examined the effect of gallium on Tf- independent iron uptake by HL60 cells. In contrast to the inhibitory effect of Tf-gallium on Tf-iron uptake, gallium nitrate, in a time-, temperature-, and concentration-dependent manner, stimulated Tf- independent uptake of iron-nitrilotriacetic acid (Fe-NTA). Preexposure of cells to gallium followed by removal of gallium also resulted in sustained stimulation of iron uptake. The anti-Tf receptor monoclonal antibody 42/6 blocked Tf-iron uptake, but had no effect on gallium- induced stimulation of Tf-independent iron uptake. Gallium increased the number of cell membrane iron-binding sites, without a change in their affinity for iron. Ferric chloride stimulated Tf-independent gallium uptake. Although gallium nitrate inhibited cell growth in Tf- free medium, cellular proliferation was restored by Fe-NTA. Gallium and iron appear to share the same Tf-independent cellular uptake system in HL60 cells. Exposure of cells to gallium results in the activation of cell membrane non-Tf iron carriers that may play a role in overcoming the Tf-independent growth-inhibitory effects of gallium.     

Transferrin receptor on rat Kupffer cells in primary culture

Kupffer cells may play a role in the turnover of iron in acute viral hepatitis. The transferrin receptor of rat Kupffer cells in primary culture was therefore investigated in this study. Daily specific bindings on 125I-diferric transferrin (Tf) to rat Kupffer cells in primary culture from day 3 to day 6 of culture were 1.64 +/- 0.08%, 4.16 +/- 0.05%, 4.34 +/- 0.07% and 2.63 +/- 0.07%, respectively. The specificity of the Tf binding sites was examined by competition studies showing that galactose (30 mmol x l-1) and ovalbumin (90 mumol x l-1) did not compete for the binding sites, but human lactoferrin (50 mumol x l-1) competed for the binding sites by about 30%. The affinity and capacity of Tf receptor on rat Kupffer cells in 5-day culture were analyzed according to the method of Scatchard. A single class of 125I-diferric Tf binding sites with an affinity constant of 1.65 x 10(7) l x l-1) and a capacity of 6.86 x 10(6) sites/cell was found. After zymosan (500 micrograms/ml) preincubation for 30 min, the binding capacity increased about 1.7-fold, and this increase depended upon the increase of the affinity of Tf receptor. These data suggest that Kupffer cells in the activated state accelerate the removal of elevated serum iron.     

Gamma-interferon modulates human monocyte/macrophage transferrin receptor expression

Although circulating human monocytes do not express transferrin (Tf) receptors, cultured adherent blood cells display high-affinity Tf binding sites. In the present studies, effects of various cytokines and biologically active proteins on human monocyte/macrophage Tf receptors were investigated. After culture, Tf receptor expression by adherent blood cells was time dependent and plateaued by 7 days. The addition of interleukin-1 (IL-1), alpha-interferon (alpha-IFN), granulocyte/macrophage-colony stimulating factor (GM-CSF), or human IgG to macrophages cultured for 4 days did not alter Tf receptor expression. Fe-saturated, human Tf caused a significant, dose-dependent decrease in receptor expression. At a dose of 100 U/mL, gamma- interferon (gamma-IFN) significantly increased Tf receptor expression by macrophages cultured for 4 (230% +/- 51% of control) or 7 days (150% +/- 22%). Scatchard analyses showed increased binding sites but no change in receptor affinity. Northern and slot blot analysis of cellular mRNA from macrophages cultured for 4 to 7 days and exposed to gamma-IFN showed a two- to fivefold increase in Tf receptor mRNA, but less than or equal to 30% increase in beta-actin mRNA. Ferritin content of gamma-IFN-treated macrophages was 47% to 63% of control cells. Net uptake of 59Fe from Tf by gamma-IFN-treated cells was 10% to 17% of control, but uptake of radiolabeled Tf was comparable. When macrophages were labeled with 59Fe and then exposed to gamma-IFN, cell-associated Fe was reduced by 43%, indicating that gamma-IFN caused macrophage Fe release. gamma-IFN specifically modulates Tf receptor display by inducing Fe release and reducing cellular Fe content. Regulation of Tf receptor expression in macrophages is controlled by cellular Fe content and is thus similar to regulatory mechanisms in dividing cells.     

AT1 receptors mediate the release of prostaglandins in porcine smooth muscle cells and rat astrocytes.

Angiotensin II (AII) can release arachidonic acid metabolites such as prostacyclin (PGI2) and PGE2 from cells in cultures. It has recently been reported that the AT1 selective nonpeptide AII receptor antagonist losartan had similar effects. The present study was undertaken to further evaluate the effects of AII and losartan on cells which synthesize prostaglandins, including vascular smooth muscle, endothelial, and glial cells. Inhibition of specific [125I]AII binding was demonstrated in porcine smooth muscle cell (PSMC) suspensions with unlabeled AII and losartan. The IC50 values were 1.3 x 10(-9) mol/L and 7.7 x 10(-9) mol/L, respectively. PD123177 (an AT2 selective antagonist) had no effect on binding. AII produced a concentration-related increase in calcium mobilization (fura-2 fluorescence) which was blocked by losartan (IC50 = 8.4 x 10(-8) mol/L) but not by PD123177 (10(-6) mol/L). AII (10(-7) to 10(-5) mol/L) stimulated the basal release of PGI2 by 100%. This response was blocked by losartan (10(-6) to 10(-5) mol/L) but not by PD123177 (10(-6) to 10(-5) mol/L) and neither agent stimulated basal release in PSMC. Similar effects of AII and antagonists were observed upon receptor binding and PGE2 release in primary rat astrocyte (RA) cultures. AII did not release PGI2 from porcine endothelial cells, bovine pulmonary arterial endothelial cells, or rat C6 glioma cells. Losartan had no significant effect at 10(-5) mol/L. By contrast, bradykinin or the calcium ionophore A23187 dramatically increased PGI2 release in each of these cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of the truncation of luteinizing hormone receptors at the plasma membrane is different in rat and mouse Leydig cells.

Regulation of the truncation of LH receptors was investigated in two types of mouse tumor Leydig cells (MA10 and MLTC-1), rat testis Leydig cells (RTL), and a rat tumor Leydig cell (R2C). Receptor numbers were measured by binding [125I]hCG to the cells cultured in monolayers. Addition of 3.3 nM LH for 2 h at 34 C had no detectable effect on binding sites in RTL or R2C cells, but in MA10 and MLTC-1 cells it caused a loss in binding sites. The effect on MA10 and MLTC-1 cells could be mimicked by inhibiting receptor internalization with 5 mM NaN3 and prevented by the addition of protease inhibitors. Incubating RTL and R2C cells with protease inhibitors caused a 2- to 3-fold increase in binding sites and a 2- to 3-fold increase in LH (0.033 and 0.33 nM)-stimulated cAMP production. When RTL and MA10 cells were incubated in the presence of [125I]hCG, a radioactive protein complex with an approximate mol wt of 80,000-90,000 was released into the incubation medium. We conclude that LH receptors are regulated by proteolysis at the plasma membrane in both mouse and rat Leydig cells. Furthermore, truncation of the LH receptor in the mouse Leydig cells is involved in down-regulation, whereas in the rat it is a continuous process.     

Release of growth hormone binding protein from IM-9 lymphocytes by endopeptidase is dependent on sulfhydryl group inactivation

The hydrophilic GH-binding protein of serum is a derivative of the GH receptor. Little is known how this GH binding protein is released from the receptor which is firmly anchored in the plasma membrane. The IM-9 lymphocytes provide a useful laboratory model for studying this process because they are richly endowed with GH receptors and, under special conditions, are able to shed these receptors during incubation. Incubation of IM-9 cells for 90 min at 30 C did not result in the appearance of significant [125I]hGH binding in conditioned medium as determined with an ultrogel AcA 44 minicolumn. When iodoacetamide, 20 mM, or N-ethylmaleimide, 5 mM, was added during incubation, the conditioned medium bound 20-35% of [125I]human(h)GH. p-Chloromercuriphenyl sulfonic acid was less effective in promoting shedding of GH-binding protein. In contrast, aprotinin, phenylmethylsulfonylfluoride (PMSF), bacitracin, leupeptin, pepstatin, phosphoramidon, or chloroquine did not promote release of GH binding protein and did not affect iodoacetamide-induced release. Release was not inhibited by the addition of serum lacking GH binding protein. GH binding protein release was markedly temperature sensitive and practically ceased at 4 C. GH binding protein incubated with [125I] hGH was cross-linked with disuccinimidyl suberate. After sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence of dithiothreitol the complex migrated with an estimated molecular weight of 100,000 whereas [125I]hGH cross-linked to the membrane-bound GH receptor of the IM-9 cells migrated with an estimated molecular weight of 135,000. The smaller size of the binding protein is consistent with its derivation from the extracellular domain of the GH receptor. Because the release of this GH binding is greatly augmented by iodoacetamide and N-ethylmaleimide, two known sulfhydryl reactive reagents, we suggest that a free sulfhydryl group, either on the GH receptor or on a neighboring protein normally maintains the integrity of the receptor. The loss of this sulfhydryl group destabilizes the receptor and permits a membrane endopeptidase to release the GH binding protein. Cleavage is not dependent on lysosomal action and is not inhibited by protease inhibitors.     

A low-affinity human granulocyte-macrophage colony-stimulating factor/murine erythropoietin hybrid receptor functions in murine cell lines

To identify domains in hematopoietic growth factor receptors that are important for signal transduction, a hybrid receptor (GMER) was constructed by splicing the DNA of the entire extracellular and transmembrane domains of the human granulocyte-macrophage colony- stimulating factor (GM-CSF) receptor alpha 2 subunit (GMR) to the cytoplasmic domain of the murine erythropoietin receptor (mEpoR). The hybrid receptor was introduced into the interleukin-3 factor-dependent murine hematopoietic cell line Ba/F3. Cells that expressed high receptor numbers were selected by cell sorting using phycoerythrin- labeled human GM-CSF. Immunoprecipitation of GMER from Ba/F3 cells showed a band with an Mr of 105,000 daltons. Human GM-CSF binding to Ba/F3 cells that expressed GMER showed a kd of 3.0 nmol/L and 475 binding sites/cell, while the same cells that expressed GMR had 300 sites/cell and a kd of 3.5 nmol/L. The proliferative response to GM-CSF of Ba/F3 cells that expressed GMER showed 1/2 maximal cell growth (as measured by 3H-thymidine incorporation) at a GM-CSF concentration of 2.5 x 10(-8) mol/L. When cultured in human GM-CSF, Ba/F3-GMER cells expressed cell surface glycophorin. Similar results were obtained with Ba/F3 cells transfected with the mEpoR and cultured in erythropoietin. Expression of GMR plus the human GM-CSF receptor beta chain in the same cell line also resulted in human GM-CSF stimulated proliferation; however, cell surface glycophorin was not detected. These data show that a low-affinity GM-CSF/Epo hybrid receptor can promote GM-CSF- dependent proliferation and can induce the expression of glycophorin, an erythroid-specific protein.     

Increase in luteinizing hormone content occurs in cultured human fetal pituitary cells exposed to gonadotropin-releasing hormone

To investigate the mechanisms by which GnRH regulates LH production during intrauterine life, dispersed pituitary cells from second trimester human fetuses were cultured on extracellular matrix-coated plates for 48 h. Exposure of cells to 3 x 10(-10) mol/L GnRH for 1-48 h significantly increased cumulative LH secretion compared to that in respective controls (P less than 0.01). The rate of GnRH-stimulated LH release was accelerated during the first 6 h, after which it declined to a level similar to that of basal release. This phenomenon was associated with a decrease in the GnRH concentration of the medium. Exposure of cells to GnRH (3 x 10(-10) to 10(-6) mol/L) for 48 h induced a dose-dependent elevation of total LH which correlated with an increase in releasable, but not cellular, LH. Desensitization to GnRH (10(-7) mol/L) occurred when cells were cultured with pharmacological amounts of GnRH for 48 h. These results indicate that GnRH induces the increase in total and releasable LH in human fetal pituitary cells. These cells also appear to inactivate GnRH. Thus, GnRH may increase LH production in the human fetal pituitary and the pituitary receptor mechanism may be involved in GnRH action on LH release during intrauterine life.     

Solubilization of luteinizing hormone receptor from human corpora lutea in a stable form and identification of the hormone-binding unit by ligand blotting

LH receptors were solubilized from human corpora lutea in phosphate-buffered saline containing 1% Triton X-100, 20% glycerol, and protease inhibitors. The presence of 20% (vol/vol) glycerol was necessary for quantitative preservation of [125I]hCG-binding activity in detergent solution. The solubilized receptors were stable for several weeks at -20 C and at -80 C and for at least 18 h at 4 C. Binding of [125I]hCG to the soluble LH receptors was time and temperature dependent and varied linearly with the amount of soluble protein. Equilibrium binding studies revealed a single class of high affinity [125I]hCG-binding sites with an equilibrium dissociation constant (Kd) of 4.3 x 10(-10) mol/L (at 20 C). The molecular size of the human LH receptors was analyzed by ligand blotting. Solubilized receptors were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions and transferred to nitrocellulose. Incubation of the protein blot with [125I]hCG to the 85/90K mol wt species was inhibited by unlabeled hCG. These results indicate that LH receptors can be solubilized in nonionic detergent while maintaining their hormone-binding activity and demonstrate that the receptors contain 85/90K hormone-binding species.     

Retroendocytosis of insulin in rat adipocytes

A variety of ligands internalized by receptor-mediated endocytosis follow a short circuit pathway that does not lead to degradation but results in rapid exocytosis of intact ligand, a process termed retroendocytosis. We studied the time course of [125I]iodoinsulin processing and retroendocytosis after internalization in isolated rat adipocytes. After steady state binding and internalization, surface receptor-bound insulin was removed by exposing cells to a low pH at low temperatures. The cells containing internalized [125I]iodoinsulin were reincubated in fresh medium; subsequently, the radioactivity remaining within the cells and released into the medium were analyzed at various times by trichloroacetic acid (TCA) precipitation, Sephadex G-50 gel filtration, and reverse phase HPLC. Cell-associated radioactivity progressively decreased after reincubation in 37 C buffer, with 50% released in 9 min and 85% by 45 min. In the media, TCA-precipitable material appeared quickly, with a t1/2 of 2 min, and plateaued by 10 min. TCA-soluble material was released continually throughout the 45-min period. The release of both TCA-precipitable and TCA-soluble material was temperature and energy dependent. Sephadex G-50 chromatography demonstrated the loss of insulin from the intracellular pool and its appearance in the medium with a time course similar to that of TCA-precipitable material. Reverse phase HPLC demonstrated that the intracellular and medium radioactivity eluting in peak II (insulin peak) on Sephadex G-50 was composed of both intact insulin and intermediates. In conclusion, these studies demonstrated that after the internalization of insulin, rat adipocytes release not only small mol wt degradation products of insulin, but also insulin intermediates and intact insulin. The rate of retroendocytosis reported here is almost identical to the rate of insulin receptor recycling in rat adipocytes. Therefore, retroendocytosis may serve as an excellent in vitro reflection of the extent and rate of insulin receptor recycling.     

Expression of transcobalamin II receptors by human leukemia K562 and HL-60 cells

Plasma membrane receptors for the serum cobalamin-binding protein transcobalamin II (TCII) were identified on human leukemia K562 and HL-60 cells using immunoaffinity-purified human TCII labeled with [57Co]cyanocobalamin. The Bmax values for TCII receptors on proliferating K562 and HL-60 cells were 4,500 and 2,700 per cell, respectively. Corresponding dissociation constants (kd) were 8.0 x 10(-11) mol/L and 9.0 x 10(-11) mol/L. Rabbit TCII also bound to K562 and HL-60 cells but with slightly reduced affinities. Calcium was required for the binding of transcobalamin II to K562 cells. Brief treatment of these cells with trypsin resulted in almost total loss of surface binding activity. After removal of trypsin, surface receptors for TCII slowly reappeared, reaching pretrypsin treatment densities only after 24 hours. Reappearance of receptors was blocked by cycloheximide. TCII receptor densities on K562 and HL-60 cells correlated inversely with the concentration of cobalamin in the culture medium. This suggests that intracellular stores of cobalamin may affect the expression of transcobalamin receptors. Nonproliferating stationary-phase K562 cells had low TCII receptor densities (less than 1,200 receptors/cell). However, the density of TCII receptors increased substantially when cells were subcultured in fresh medium. Up-regulation of receptor expression coincided with increased 3H-thymidine incorporation, which preceded the resumption of cellular proliferation as measured by cell density. In the presence of cytosine arabinoside, which induces erythroid differentiation, K562 cells down-regulated expression of TCII receptors. When HL-60 cells were subcultured in fresh medium containing dimethysulfoxide to induce granulocytic differentiation, the up-regulation of TCII receptors was suppressed. This event occurred well before a diminution of 3H-thymidine incorporation and cessation of proliferation. Thus, changes in the regulation of expression of TCII receptors correlate with both the proliferative and differentiation status of cells.     

Inflammatory cytokines and vascular endothelial growth factor stimulate the release of soluble tie receptor from human endothelial cells via metalloprotease activation

Activation of endothelial cells, important in processes such as angiogenesis, is regulated by cell surface receptors, including those in the tyrosine kinase (RTK) family. Receptor activity, in turn, can be modulated by phosphorylation, turnover, or proteolytic release of a soluble extracellular domain. Previously, we demonstrated that release of soluble tie-1 receptor from endothelial cells by phorbol myristate acetate (PMA) is mediated through protein kinase C and a Ca2+-dependent protease. In this study, the release of soluble tie-1 was shown to be stimulated by inflammatory cytokines and vascular endothelial growth factor (VEGF), but not by growth factors such as basic fibroblast growth factor (bFGF) or transforming growth factor alpha (TGFalpha). Release of soluble tie by tumor necrosis factor alpha (TNFalpha) or VEGF occurred within 10 minutes of stimulation and reached maximal levels within 60 minutes. Specificity was shown by fluorescence-activated cell sorting (FACS) analysis; endothelial cells exhibited a significant decrease in cell surface tie-1 expression in response to TNF, whereas expression of epidermal growth factor receptor (EGF-R) and CD31 was stable. In contrast, tie-1 expression on megakaryoblastic UT-7 cells was unaffected by PMA or TNFalpha. Sequence analysis of the cleaved receptor indicated that tie-1 was proteolyzed at the E749/S750 peptide bond in the proximal transmembrane domain. Moreover, the hydroxamic acid derivative BB-24 demonstrated dose-dependent inhibition of cytokine-, PMA-, and VEGF-stimulated shedding, suggesting that the tie-1 protease was a metalloprotease. Protease activity in a tie-1 peptide cleavage assay was (1) associated with endothelial cell membranes, (2) specifically activated in TNFalpha-treated cells, and (3) inhibited by BB-24. Additionally, proliferation of endothelial cells in response to VEGF, but not bFGF, was inhibited by BB-24, suggesting that the release of soluble tie-1 receptor plays a role in VEGF-mediated proliferation. This study demonstrated that the release of soluble tie-1 from endothelial cells is stimulated by inflammatory cytokines and VEGF through the activation of an endothelial membrane-associated metalloprotease.     

The role of diferric transferrin in iron absorption and transferrin concentration in rat pancreatic juice and milk.

Transferrin (Tf) was found immunologically in pancreatic juice of normal rats at a concentration of 0.28 +/- 0.10 mg/ml but was found to be approximately 4-times higher in iron-deficient rats. Iron saturation of pancreatic Tf of normal rats was 40% and similar to that of serum Tf. Approximately 27% of a dose of iron from 59Fe-diferric Tf was absorbed through the ligated segments of proximal intestine in normal rats. The iron absorption ratio of 59Fe-diferric Tf was higher from the duodenal and jejunal segments than the ileum and significantly inhibited by monodansylcadaverine (MDC) or 20-times excess of unlabeled diferric Tf. The presence of Tf receptors was demonstrated by specific binding of 125I-diferric Tf to the brush border membrane vesicles of the small intestine in normal rats. The binding was not inhibited by the presence of albumin or IgG. The association constant (Ka) was 1.03 +/- 0.50 x 10(8) M-1 and number of binding sites was 5.10 +/- 0.96 x 10(12) sites/mg protein in the proximal intestine. The number of binding sites for Tf was higher in the proximal intestine than in the distal one. These results suggested that some iron was absorbed as diferric Tf into enterocytes through receptor-mediated endocytosis.